Toxicity evaluation of poly(anhydride) nanoparticles as...

Toxicity evaluation of poly(anhydride) nanoparticles

as carriers for oral administration of drugs

Patricia Ojer Ojer

TESIS DOCTORAL

Pamplona, octubre 2013

TESIS DOCTORAL

Facultad de Farmacia Departamento de Farmacia y Tecnología Farmacéutica

Departamento de Farmacología y Toxicología

Toxicity evaluation of poly(anhydride) nanoparticles

as carriers for oral administration of drugs

Trabajo presentado por Dña.Patricia Ojer Ojer para obtener el

Grado de Doctor

Fdo. Patricia Ojer Ojer


El presente trabajo, titulado “Toxicity evaluation of poly(anhydride) nanoparticles as

carriers for oral administration of drugs”, presentado por DÑA. PATRICIA OJER

OJER para optar al grado de Doctor por la Universidad de Navarra en el área de tecnología

Farmacéutica y Toxicología, ha sido realizado bajo nuestra dirección en los Departamentos

de Farmacia y Tecnología Farmacéutica y de Farmacología y Toxicología de la Universidad

de Navarra. Estimamos que puede ser presentado al tribunal que lo ha de juzgar.

Y para que así conste, firman la presente:

Fdo.: Dr. Juan M. Irache Garreta Fdo.: Dra. Adela López de Cerain Salsamendi


Esta tesis doctoral se ha llevado a cabo gracias a las

ayudas predoctorales de Formación (convocatoria 2007) y

de movilidad internacional (convocatoria 2010) dentro del

plan de Formación y de I+D del departamento de

educación del Gobierno de Navarra y a la ayuda para la

formación del personal investigador de la Asociación de

Amigos de la Universidad de Navarra.

Las investigaciones realizadas en el presente trabajo se han

desarrollado dentro del proyecto CAN “Nanotecnología y

medicamentos” (REF 10828).

A mis padres, Inma y Vicente, y a mi hermano, Daniel.

“Lo esencial es invisible para los ojos”

Antoine de Saint-Exupéry

AGRADECIMIENTOS

Deseo expresar mi agradecimiento a todas las personas e instituciones que durante estos últimos años

han contribuido a que la presente tesis doctoral sea una realidad.

En primer lugar, quisiera agradecer especialmente a mis directores, el Dr. Juan Manuel Irache y la

Dra. Adela López de Cerain, la oportunidad de realizar esta tesis doctoral. Gracias por su inestimable

labor de dirección, su estímulo para seguir creciendo intelectualmente y sobre todo, por la confianza

depositada en mí y en mi trabajo.

Gracias a la Universidad de Navarra y en especial a los investigadores, profesores y personal de los

Departamentos de Farmacia y Tecnología Farmacéutica y de Farmacología y Toxicología, gracias por

la ayuda prestada cuando la he necesitado.

A la asociación de Amigos y el Departamento de Educación del Gobierno de Navarra, por la

aportación económica recibida a través de sus programas de ayudas para la realización de tesis

doctorales y estancias en el extranjero.

I am grateful to the Department of Pharmaceutical Technology and Biopharmaceutics of the

University of Vienna, in particular to Prof. Franz Gabor and Prof. Michael Wirth for giving me the

opportunity to join their group. Thanks to those people who I met in Vienna, and especially to Lukas

Neutsch for your friendliness and your “incubation times”.

A mis compañeros y amigos de los laboratorios de Galénica y Toxicología, ha sido un placer poder

compartir con todos vosotros estos años, no sólo el trabajo dentro del laboratorio sino también los

grandes momentos vividos fuera.

A Hugo. Muchísimas gracias por toda tu ayuda en estos años, porque no sólo te has limitado a

realizar tu trabajo sino que además lo has hecho mucho más divertido, aunque la radio ayudaba

bastante. Gracias por quedarte a trabajar horas que no te correspondían y por venir los fines de

semana.

A Irene Esparza. Gracias por tu apoyo incondicional, tu sabiduría, tu infinita paciencia pero sobre

todo gracias por tu amistad, siempre dispuesta a ayudar y a escuchar aunque para ello tuvieses que

sacar al día 25 horas.

A todas las personas que durante estos años han formado parte de mi vida, besonders Camillo, well

er an mich mehr geglaubt hat als ich selbst.

Todo esto no hubiera sido posible sin el cariño de mi familia, y en especial de mis padres. Este es

también vuestro premio.

15

TABLE OF CONTENTS

ABBREVIATIONS.........................................................................................................................................17

CHAPTER 1......................................................................................................................................................21

GENERAL INTRODUCTION

CHAPTER 2 .....................................................................................................................................................79

AIM AND OBJECTIVES

CHAPTER 3 .....................................................................................................................................................83

EXPERIMENTAL DESIGN

CHAPTER 4 .....................................................................................................................................................87

Spray-drying of poly(anhydride) nanoparticles for drug/antigen delivery

CHAPTER 5 ...................................................................................................................................................113

Cytotoxicity and cell interaction studies of bioadhesive poly(anhydride) nanoparticles for oral

antigen/drug delivery

CHAPTER 6 ...................................................................................................................................................143

Toxicity studies of poly(anhydride) nanoparticles as carriers for oral drug delivery

CHAPTER 7 ...................................................................................................................................................169

Hemocompatibility, acute toxic effects and biodistribution of poly(anhydride) nanoparticles following

intravenous administration

CHAPTER 8 ...................................................................................................................................................195

GENERAL DISCUSSION

CHAPTER 9 ...................................................................................................................................................207

CONCLUSIONS/CONCLUSIONES

ABREVIATIONS

17

ABREVIATIONS

%ID/g: Percentage of injected dose per gram 99mTc: 99mTechnetium

ABI-007: Abraxane®

ADA: Adenosine Deaminase

AFM: Atomic Force Microscopy

ALT: Alanine Transaminase

APCs: Antigen-Presenting Cells

AST: Aspartate Transaminase

ATO: Atovaquone

ATP: Adenosine Thriphosphate

BCS: Biopharmaceutics Classification System

BSA: Bovine Serum Albumin

BSA-FITC: Bovine Serum Albumin-Fluorescein Isothiocyanate Conjugate

Bw: Body weight

CdSe: Cadmium Selenide

CdTe: Cadmium Telluride

CLSM: Confocal Laser Scanning Microscopy

CMC: Critical Micelle Concentration

CNTs: Carbon Nanotubes

CT: Computed Tomography

CTB: Cholera Toxin B subunit

CYP: Cytochrome P450 metabolizing enzyme system

DCFA: 2′7′-dichlorofluorescein diacetate

DISS: Digital Image System

DMF: Dimethylformamide

DOX: Doxorubicin

DPBS: Dulbecco´s Phosphate Buffered Saline

DTXL: Docetaxel

DTNB: 5,5-dithio-bis-2-nitrobenzoic acid

EASAC: European Academies Science Advisory Council

ELSD: Evaporative Light Scattering Detection

EMA: European Medicines Agency

EPR: Enhanced Permeability and Retention

ESR: Erythrocyte Sedimentation Rate

FDA: Food and Drug Administration

FP7: Seventh Framework Program

GCSF: Granulocyte Colony-Stimulating Factor

GIT: Gastrointestinal Tract

HGB: Hemoglobin

HCT: Hematocrit

ABREVIATIONS

18

HER2: Human Epidermal growth factor Receptor 2

HGF: Hepatocytes Growth Factor

HIV: Human Immunodeficiency Virus

HPCD: 2-hydroxipropyl-β-cyclodextrin

HPLC: High Performance Liquid Chromatography

HPMA: N-(2-Hydroxypropyl) methacrylamide

HS: Hot Saline antigenic extract

HSA: Human Serum Albumin

i.m.: intramuscular

i.r.: intravitreous

i.t.: intrathecal

i.v.: intravenous

ILSI RF/RSI: International Life Sciences Institute Research Foundation/Risk Science Institute

InAs: Indium Arsenide

InP: Indium Phosphide

IRIV: Immunopotentiating Reconstituted Influenza Virosome

ISO: International Organization for Standardization

ITLC: Instant Thin Layer Chromatography

JRC: Joint Research Centre of the European Commission

LD50: Lethal Dose 50%

LDH: Lactate dehydrogenase

LPS: Lipopolysaccharide

LUVs: Large Unilamellar Vesicles

MCH: Mean corpuscular hemoglobin

MCHC: Mean Corpuscular Hemoglobin Concentration

MCV: Mean Corpuscular Volume

MLVs: Mutilamellar Vesicles

MPS: Mononuclear Phagocyte System

MTS: 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt

MTT: 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide

MWCNT: Multi-Walled Carbon Nanotubes

NCS: Nanotoxicological Classification System

NCTR: National Center of Toxicological Research

NOAEL: No Observed Adverse Effect Level

NNI: National Nanotechnology Initiative

NP: Conventional poly(anhydride) nanoparticles

NP-HPCD: Nanoparticles containing 2-hydroxypropyl-β-cyclodextrin

OECD: Organisation for Economic Co-operation and Development

Omp: Outer membrane proteins

OVA: Ovalbumin

PACA: Poly(akyl cyanocrylates)

ABREVIATIONS

19

PAMAM: Poly(amidoamine)

PBS: Phosphate buffered saline

PCL: Poly(Ɛ-caprolactone)

PCS: Photon Correlation Spectroscopy

PDI: Polydispersity Index

PEG: Polyethylene glycol

PEG-NP: Pegylated poly(anhydride) nanoparticles

P-gp: P-glycoprotein

PLA: Poly(lactic acid)

PLDsis: Phospholipidosis

PLGA: Poly(lactide-co-glycolide)

PLT: Platelet count

QDs: Quantum Dots

RBC: Red Blood Corpuscles count

RES: Reticular Endothelial System

s.c.: subcutaneous

SD: Spray-Drying

SEM: Scanning Electron Microscopy

SLN: Solid Lipid Nanoparticles

SPECT-CT: Single-Photon Emission Computed Tomography

STM: Scanning Tunneling Microscope

SUVs: Small Unilamellar Vesicles or oligolamellar

SWCNT: Single-Walled Carbon Nanotubes

TBA: Tiobarbituric acid

Tg: Glass transition temperature

TNP: Targeted polymeric nanoparticles

TUNEL: Terminal deoxyribonucleotidyl transferase (TDT)-mediated dUTP-digoxigenin nick end labeling

UPLC: Ultra Performance Liquid Chromatography

VEGF: Vascular Endothelial Growth Factor

WBC: White Blood Corpuscles count

WPN: Working Party on Nanotechnology

WPNM: Working Party on Manufactured Nanomaterials

WST-1: 4-[3- (4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate

XTT: 2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide

CHAPTER 1

GENERAL INTRODUCTION

CHAPTER 1: GENERAL INTRODUCTION

23

1. Definitions

1.1. What Is Nanotechnology?

Nanotechnology has emerged as one of the central technologies in the XXI (1). Much more

than in any other scientific field, the development and progress have been made possible through the

convergence of chemistry, biology, physics, mathematics, engineering and others (2-4).

However, although it seems that nanotechnology has recently appeared, this technology exits since

long time before. Already in the V century B.C., colloidal gold was known in Egypt or China and, for

instance, applied in medicine (5). The conceptual origins of nanotechnology were introduced in 1959

by physicist Richard Feymann (Nobel Prize winner in Physics in 1965) who described a process in

which scientists would be able to manipulate and control individual atoms and molecules in his

famous lecture “There is plenty of room at the bottom” (6). Over a decade later, in 1974, Professor

Norio Taniguchi was the first who coined the term “nanotechnology” (7). However, at this time it

was a more philosophical or theoretical problem. It was not until 1981, when nanotechnology

emerged in the sense as we understand it today. This fact was made possible in part by the

development of tools that enabled the understanding of materials far down to the nanoscale as the

scanning tunneling microscope (STM) or the atomic force microscopy (AFM) (8, 9).

On the definition of nanotechnology there is confusion and disagreement among experts.

Thus, the National Nanotechnology Initiative (NNI) defines nanotechnology as the science of

materials and phenomena in the range of 1 to 100 nm in diameter and agencies, including the Food

and Drug Administration (FDA), continue to use this definition. However, this definition not

encompasses three important requirements to be taken into account defining nanotechnology. First,

the size limitation to the 1-100 nm range would exclude numerous materials and devices, especially in

the pharmaceutical area. Moreover, it is important to bear in mind the requirement that the nano-

structure is man-made. Otherwise you would have to include every naturally formed biomolecules

and material particle, in effect redefining much of chemistry and molecular biology as

“nanotechnology”. Finally, the most important requirement to consider in the definition of

nanotechnology is that the nano-structure has special properties that are exclusively due to its

nanoscale proportions. Due to this fact, alternative definitions that are practical and unconstrained by

any arbitrary size limitations have been presented by different authors and agencies. In this context,

Bawa and collaborators proposed a definition of nanotechnology that deal with the three

requirements aforementioned and that it may considers most adequate. They defined nanotechnology

as “the design, characterization, production, and application of structures, devices, and systems by

controlled manipulation of size and shape at the nanometer scale (atomic, molecular, and

macromolecular scale) that produces structures, devices, and systems with at least one novel/superior

characteristic or property” (10). However, it should be kept in mind that there is still no

comprehensive definition that would be generally and internationally accepted and legally binding


24

(11). Figure 1 shows a scale, within the size range of nanotechnology, which compares visually the

size of various biological components.

Red

Blo

odC

ells

DN

A

Glu

cose

Viru

s Cel

ls

1 nm 10 nm 100 nm 1000 nm 10000 nm1 Å

Ato

m

Figure 1. Scale of size. How small is small?

1.2. What Is Nanomedicine?

The convergence of nanotechnology and medicine has resulted in the interdisciplinary field

of nanomedicine. The neologism “nanomedicine” as the exploitation of nanotechnology for human

health, is a relatively new field of science and technology that appeared in the scientific literature at

the end of the last century. The promotion of interdisciplinary research and the discovery of colloidal

mechanisms of drug delivery in the 1960´s and 1970´s led to the development of the earlier

nanomedicines. In this regard, one of the pioneers was Professor Peter Speiser, whose strategy for

the controlled release was the development of miniaturized delivery systems (12, 13).

As occurs with nanotechnology, there is no unique globally accepted definition for

nanomedicine. Thus, The Medical Standing Committee of the European Science Foundation (ESF)

defined nanomedicine as “the science and technology of diagnosing, treating, and preventing disease

and traumatic injury, of relieving pain, and of preserving and improving human health, using

molecular tools and molecular knowledge of the human body”. At the same time, the National

Institutes of Health (NIH) considers the nanomedicine as “an offshoot of nanotechnology, which

refers to highly specific medical interventions at the molecular scale for curing disease or repairing

damaged tissues, such as bone, muscle, or nerve” (14).

Nanomedicine is one of the main and most fascinating fields of nanotechnology and is

heavily supported by public policy and investments due to the potential beneficial properties that it

provides compared with conventional medicine. Applications of nanomedicine include nanostructure

scaffolds for tissue replacement, nanostructures that allow transport across biological barriers, remote

control of nanoprobes, integrated implantable sensory nanoelectronic systems and multifunctional

chemical structures for drug delivery and targeting of disease. However, the majority of current

commercial applications of nanomedicine are geared towards drug delivery to enable modes of


25

action, as well as better targeting and bioavailability of existing medical substances. In this regard, the

use of nanoparticles has been postulated as one of the more promising approaches for medical

purposes, mostly for drug delivery applications.

However, despite the wide and ever growing exploitation of these new nanosystems, toxicity

studies are very sporadic and limited, and toxicity data concerning the release and the accumulation of

potentially noxious by-products, are as scarce as fragmentary. Moreover, there is a lack of predictive

methods and approved protocols to study the toxicity of these nanodevices, in both for in vitro and in

vivo models. So, to provide guidelines and protocols there is a need to deepen understanding of the

nanomedicine in order to establish the critical points that may affect toxicity.

1.3. Nanoparticles in medicine

From the medical point of view, nanoparticles are defined as submicron sized colloidal

particles, with sizes ranging from 1 to 1000 nm in diameter (15, 16). The increasing interest in

nanomedicine is driven in large part by fast pace of innovation and emerging successes of

nanoparticles which have resulted in the clinical approval of a number of nanomedicines (Table I.).

Nanoparticles can be formulated from diverse materials with unique architectures to serve, among

other applications, as diagnostic tools, imaging or as drug-delivery vehicles. Drug delivery is one of

the most important applications due to the great benefits they provide in the pharmaceutical field. In

this regard, nanoparticles may (i) protect the drug from degradation, (ii) enhance drug absorption by

facilitating diffusion through biological barriers, (iii) modify pharmacokinetic and drug tissue

distribution profile, and/or (iv) improve intracellular penetration and distribution (17). However,

although the safety of the carrier and of its eventual by-products seems to be as important as that of

the active drug, studies to assess nanoparticle toxicity are scarce.


26

Table I. Clinically approved nanoparticle-based therapeutics. From reference (18).

Composition Trade name Company Indication Administration Liposomal platforms Liposomal amphotericin B Abelcet Enzon Fungal infections i.v.

Liposomal amphotericin B AmBisome Gilead Sciences Fungal and protozoal infections i.v.

Liposomal cytarabine DepoCyt SkyePharma Malignant lymphomatous meningitis i.t.

Liposomal daunorubicin DaunoXome Gilead Sciences HIV-related Kaposi`s sarcoma i.v.

Liposomal doxorubicin Myocet Zeneus Combination therapy with cyclophosphamide in metastatic breast cancer

i.v.

Liposomal IRIV vaccine Epaxal Berna Biotech Hepatitis A i.m. Liposomal IRIV vaccine Inflexal V Berna Biotech Influenza i.m.

Liposomal morphine DepoDur SkyePharma, Endo Postsurgical analgesia Epidural

Liposomal veterporfin Visudyne QLT, Novartis Age-related macular degeneration, pathologic myopia, ocular histoplasmosis

i.v.

Liposome-PEG doxorubicin Doxil/Caelyx Ortho Biotech, Shering-Plough

HIV-related Kaposi`s sarcoma, metastatic breast cancer, metastatic ovarian cancer

i.m.

Micellular estradiol Estrasorb Novavax Menopausal therapy Topical Polymeric platforms L-Glutamic acid, L-alanine, L-lysine and L-tyrosine copolymer

Copaxone TEVA Pharmaceuticals Multiple sclerosis s.c.

Methoxy-PEG-poly(D,L-lactide) Taxol Genexol-PM Samyang Metastatic breast cancer i.v.

PEG-adenosine deaminase Adagen Enzon

Severe combined immunodeficiency disease associated with ADA deficiency

i.m.

PEG-anti-VEGF aptamer Macugen OSI Pharmaceuticals

Age-related macular degeneration i.r.

PEG-α-interferon 2a Pegasys Nektar, Hoffmann-La Hepatitis B, hepatitis C s.c.

PEG-GCSF Neulasta Amgen Neutropenia associated with cancer chemotherapy s.c.

PEG-HGF Somavert Nektar, Pfizer Acromegaly s.c. PEG-L-asparaginase Oncaspar Enzon Acute lymphoblastic leukemia i.v., i.m. Poly(allylamine hydrochloride) Renagel Genzyme End-stage renal disease Oral

Other platforms

Albumin-bound paclitaxel Abraxane Abraxis BioScience, AstraZeneca

Metastatic breast cancer i.v.

Nanocrystalline aprepitant Emend Elan, Merck Antiemetic Oral Nanocrystalline fenofibrate Tricor Elan, Abbott Anti-hyperlipidemic Oral

Nanocrystalline sirolimus Rapamune Elan, Wyeth Pharmaceuticals Immunosuppressant Oral

ADA: Adenosine Deaminase; GCSF: Granulocyte Colony-Stimulating Factor; HGF: Hepatocytes Growth Factor; HIV: Human Immunodeficiency Virus; i.m.: intramuscular; i.r.: intravitreous; IRIV: Immunopotentiating Reconstituted Influenza Virosome; i.t.: intrathecal; i.v.: intravenous; PEG: Polyethylene Glycol; s.c.: subcutaneous; VEGF: Vascular Endothelial Growth Factor.


27

2. Overview of the different classes of nanomaterials/nanoparticles

A wide range of nanoparticles formulated from diverse materials has been designed to be

applied in medicine. These materials, called nanomaterials can be chemically “simple” molecules such

as metallic, ceramic or carbon nanoparticles (pure elements) or they can be more complex if

composed from different types of materials, for example, core-shell nanoparticles. Other

nanomaterials have organized structures, including systems such as nanoliposomes and

nanoemulsions. Based on the materials of which nanocarriers are mainly comprised, these can be

broadly classified as: inorganic and organic nanomaterials.

2.1. Inorganic nanomaterials

Inorganic nanomaterials form a large and very disperse group that include ceramic, metallic

and magnetic nanoparticles, carbon nanomaterials and quantum dots (QDs) among others. In early

studies, inorganic nanoparticles demonstrated great potential as nanocarriers of therapeutic agents,

vaccines and imaging agents. However, their clinical application is limited by concerns over toxicity,

lack of biodegradability and persistent tissue accumulation. Therefore, relatively few inorganic

nanoparticles have progressed to clinical application.

The most used inorganic nanomaterials in nanomedicine, particularly as drug delivery

systems, are explained in detail below.

2.1.1. Ceramic nanoparticles

Ceramic nanoparticles are inorganic systems with porous characteristics that have emerged as

drug vehicles more than ten years ago (19). These vehicles are biocompatible ceramic nanoparticles

such as silica, titania and alumina. Ceramic nanoparticles can be used for drug delivery, especially

biomacromolecular therapeutics because of their ultra-low size (less than 50 nm) and porous nature.

Additionally, they protect the adsorbed particles against denaturation induced by extreme pH and

temperature (20). However, one of the main concerns is that these particles are non-biodegradable, as

they can accumulate in the body, thus causing undesirable effects (21, 22).

2.1.2. Carbon nanomaterials

Carbon nanomaterials are composed of a distinct molecular form of carbon atoms that give

them unusual thermal, mechanical and electronical properties (23). The two most important materials

include fullerenes and carbon nanotubes (CNTs). Both materials can be used for tissue- or cell-

specific delivery (24). Fullerenes are novel carbon allotrope with polygonal structure made up

exclusively by 60 carbon atoms. These nanoparticles are characterized by having numerous point of

attachment whose surfaces also can be functionalized for tissue binding (25). CNTs have been one of

the most extensively used types of nanoparticles because of their high electrical conductivity and

excellent strength. There are two classes of CNTs: single-walled carbon nanotubes (SWCNT) and


28

multi-walled carbon nanotubes (MWCNT). MWCNT are larger and consist of many single-walled

tubes stacked one inside the other. The nanoscale dimensions of SWCNT and MWCNT, along with

their high surface area have compelled researchers to utilize them for the delivery of imaging and

therapeutic agents and in the transport of DNA molecules into cells. They also have potential for

vaccine delivery as they amplify the immunological response (26). However, the potential toxicity of

fullerenes and CNTs has been postulated by several authors (27, 28). CNTs and fullerenes toxicity

has been attributed to various factors, the ability to penetrate membranes due to the apolar character

of fullerenes, the dispersant used to solubilize the CNTs and their potent reactions with different

biochemical compounds.

2.1.3. Metallic nanoparticles

Metallic nanoparticles are versatile agents with a variety of biomedical applications including

their use in biosensing/imaging and cancer thermotherapy as well as for targeted drug and gene

delivery (29-31). Gold, silver and copper are most commonly used (32, 33). Metal nanoparticles can

easily be synthesized with a range of sizes (1–150 nm), are stable and can be modified by conjugation

with various functional groups (29). Gold nanoparticles are spherical metallic nanoparticles consisting

of a dielectric core covered by a thin metallic shell which is typically gold. These nanoparticles are the

most extensively studied because their properties lend themselves to multiple applications, such as

labeling, delivery, heating and sensing (34).

2.1.4. Magnetic nanoparticles

Magnetic nanoparticles are increasingly being realized as important nanomaterials in the

industrial sector and they are being widely used for biotechnological and biomedical applications.

Iron oxide nanoparticles are the most commonly explored members of the broader class of magnetic

nanoparticles. Magnetite (Fe3O4, ferromagnetic, superparamagnetic when the size is smaller than 15

nm) and maghemite (γ-Fe2O3, ferromagnetic) have proven particularly popular for biomedical

applications because of their great biocompatibility. They have been investigated for use as

biosensors, for imaging and for drug delivery (35, 36). However, it is interesting to note that, with a

few exceptions, metallic and magnetic nanoparticles used in medicine includes almost exclusively

transition elements and their toxicity can be high if they are not adequately detoxified, which

combined with their prolonged retention in tissues, is of significant concern regarding its safety (36).

2.1.5. QDs

QDs are semi-conductor nanoparticles that measure approximately 2–10 nm, usually consist

of 10 to 50 atoms and fluoresce when stimulated by light. They are comprised of an inorganic core,

an inorganic shell and an aqueous coating to which biomolecules can be conjugated. The size of the

core determines the color of light emitted. Although QDs have been used in electronics and optics

for 20 years, they have only recently been applied to nanomedical research. The most commonly used


29

QDs for biomedical applications contain Cadmium Selenide (CdSe) or Cadmium Telluride (CdTe)

(38). QDs containing Indium Phosphide (InP) and Indium Arsenide (InAs) are also frequently used

(39, 40). Biomedical applications of QDs are primarily focused on their use as imaging agents (41).

Moreover, QDs also have sufficient surface area to attach agents or biomarkers for simultaneous

targeted drug delivery and in vivo imaging or for tissue engineering and diagnostic (42). However,

clinical application of QDs is limited by the high toxicity of cadmium and selenium and their

instability to photolysis and oxidative conditions, which could result in dissolution of QDs core and

their slow elimination. Thus, crucial for biological applications, QDs must be covered with other

materials allowing dispersion and preventing leaking of the toxic heavy metals (43).

2.2. Organic Nanomaterials

Organic nanomaterials are carbon based (with the striking exception of fullerenes and

nanotubes that are considered inorganic) which are generally characterized by their biocompatibility

and low toxicity. Organic nanomaterials have advantages such as high versatility to carry diverse

drugs and conjugate to targeting agents which offers the potential for combining treatment and

diagnosis, the so-called “theranostic” purpose.

Organic nanomaterials can be classified into three main groups: lipid-based nanodevices,

polymer therapeutics and polymeric nanoparticles. Figure 2 shows the structure of the most

representative nanodevices from the lipid-based nanocarriers and polymer therapeutics groups.

a. Polymer-drug conjugate b. Polymeric micelle c. Dendrimers

5-20 nm 60-100 nm 60-200 nm

Hydrophilic block (shell)

Hydrophobic block(core)

Drug

d. Liposomes

50 nm-10 μm

SUV

LUV

MLV

Lipid bilayer

Inner aqueous phase

e. Solid-lipid nanoparticles

50 nm-1 μm

Lipidic solid core

Figure 2. Structure of different nanodevices used for drug delivery purposes. Adapted from

reference (44).


30

2.2.1. Lipid-based nanocarriers encompass four main types of carriers: liposomes, nanoemulsions,

lipid nanocapsules and solid lipid nanoparticles (SLN).

a. Liposomes or (phospho)lipid vesicles, are self-assembled microscopic vesicles which posses a

central aqueous cavity surrounded by a lipid membrane formed by concentric bilayer(s) (lamellas)

(45). Liposomes are highly flexible delivery systems capable to incorporate hydrophilic substances (in

the aqueous interior) or hydrophobic substances (in the lipid membrane). The production of

liposomes, as membrane mimicking drug carriers, started long before any nanotechnology (46-48),

with their first market appearance in 1986 in a cosmetic formulation by Dior, and is considered by

some to be the archetypal nanoparticle (49).

Liposomes are classified into three categories on the basis of their size and lamellarity (i.e.

number of bilayers): (i) small unilamellar vesicles or oligolamellar (SUVs), (ii) large unilamellar vesicles

(LUVs) and mutilamellar vesicles (MLVs) (Figure 2d).

The surface of liposomes can be modified by covalent binding of natural (e.g. proteins,

polysaccharides, glycolipids, antibodies, enzymes) or synthetic molecules (e.g. PEGs) (50-52).

Moreover, by using lipids of different fatty-acid-chain lengths, scientists can construct liposomes to

be temperature-sensitive or pH-sensitive, thereby permitting the controlled release of their contents

only when they are exposed to specific environmental conditions (23).

Liposomes are suitable for topical, i.v. and other parenteral administrations, but because they

are susceptible to degradation in the gastrointestinal tract (GIT) they are rarely suitable for oral use.

The components of liposomes are similar to natural human cell membranes; thus, they confer

liposomal drug delivery with several intrinsic benefits for improving efficacy and safety of different

drugs. Therefore, liposomes have been widely developed as drug delivery systems for the

administration of chemotherapeutic agents for cancer and vaccines for immunological protection

(53). Liposomes have been also proposed as carriers of radiopharmaceuticals, for diagnostic and

imaging purposes as well as vehicles for nucleic acid-based medicines for gene therapy (54-57).

However, their application has been slowed somewhat by stability issues, difficult manufacturing

techniques, cost and because complement-mediated hypersensitivity reactions may occur in response

to i.v. liposome administration (58).

b. Lipid nanocapsules are biomimetic carriers consisting of an oil-filled core with a surrounding

polymer shell. (59). They are characterized by a hybrid structure between polymer nanocapsules and

liposomes. In general, they have an oil core of medium-chain triglycerides, which confers them

special use for the loading of hydrophobic drugs, surrounded by a membrane made from a mixture

of a wide variety of polymers/surfactants with hydrophilic segments such as PEGs. These types of

polymers also enhance the intrinsic colloidal stability of the system minimizing their recognition by

the mononuclear phagocyte system (MPS), a major drawback that often arises after i.v. injection of

drug carriers. Moreover, the targeting capacities of these systems can be implemented by surface


31

modification with specific biomolecules (e.g. antibody fragments, folic acid) enhancing the cell-

targeting through molecular recognition processes (60-64).

Their formulation is based on the phase-inversion temperature phenomenon of an emulsion leading

to lipid nanocapsules formation with good monodispersion (65, 66).

c. Nanoemulsions are oil-in-water dispersions of about 20-200 nm oil where the dispersed droplets

are stabilized by an exterior film composed of surfactants and co-surfactants. Drugs are usually

loaded into the dispersed oily. The advantages of nanoemulsions include simplicity, inexpensive

preparation, high stability, capability to solubilize lipophilic substances and to protect them from

degradation. Nanoemulsions are versatile and can be prepared from different aqueous solutions,

surfactant mixtures and oily phases (67, 68).

d. SLN were developed at the beginning of the 1990s as an alternative carrier system to emulsions,

liposomes and polymeric nanoparticles. SLN are composed of solid lipids (i.e. lipids solid at room

temperature and also at body temperature) stabilized by surfactant(s) and dispersed in an aqueous

solution (Figure 2e). Most commonly, the pharmaceutical agent, mainly hydrophobic drugs, is

dissolved or dispersed within the lipid. The production of SLN is carried out by two main

manufacture methods: the high pressure homogenization technique (69) and the microemulsion

technique (70).

SLN exhibit several advantages such as protection of incorporated active compounds against

degradation, excellent physical stability, high flexibility to modulate the release of the loaded

compound and administration through multiple routes (orally, topically and intravenously) (71, 72).

However, the drug loading capacity of conventional SLN is limited by drug solubility in the lipid

melt, lipid matrix structure and the polymorphic state of the lipid matrix. Thus, drug expulsion after

polymorphic transition during storage and relatively high water content of the dispersions have been

observed (73-76). To minimize these drawbacks, lipid drug conjugate nanoparticles and the so-called

nanostructure lipid carriers have been proposed (71, 77).

2.2.2. Polymer therapeutics

Polymeric therapeutics is an “umbrella” term that emerged through the interdisciplinary

research at the interface of polymer chemistry and biomedical sciences (78). Polymer therapeutics

comprise a variety of complex macromolecular systems, their common feature being the presence of

a rationally designed covalent chemical bond between a water-soluble polymeric carrier and the

bioactive molecule(s). Although polymer therapeutics are nano-sized conjugates with sizes between 2-

200 nm, it should be noted that such constructs are quite distinct from polymeric nanoparticles.

These water-soluble hybrid constructs encompasses at least three distinct classes including:

polymer-drug conjugates, polymer-protein conjugates and supramolecular drug-delivery systems (e.g.

polymer micelles, polyplexes and dendrimers).


32

a. Polymer-drug/protein conjugates were firstly described by Ringsdorf in 1975 (79). Later

Duncan and co-workers exploited the biological rationale and the mechanism of polymer-drug

designs to pioneer a field that has remained of great scientific and therapeutic interest (80, 81).

Polymer-drug conjugates are drug delivery technologies in which a drug is covalently bound to a

water-soluble polymer carrier, normally via a biodegradable linker (often a peptidyl or ester linkage)

(Figure 2a). Polymer-drug conjugates can improve drug solubility, circulation time (through the

properties of the polymer carrier), drug targeting (by using appropriate linkers which respond to

changes in physiological conditions such as temperature, pH, and the presence of enzymes) and

decreased drug toxicity. Among others, the most popular polymers or macromolecular carriers are

the followings: N-(2-hydroxypropyl)methacrylamide copolymers, PEGs, poly(glutamates) and

albumin (82).

Protein-polymer conjugates are similar to drug conjugates with the difference that in this case

the biologically active molecule is a protein or macromolecule. PEG has mainly been the polymer of

choice for preparing these polymer-protein conjugates. Abuchowski et al. in 1977 first introduced the

conjugation of PEG to protein drugs (83). In this “pegylation” technology, linear or branched PEG

derivates are coupled to the surface of the protein (84, 85). Binding polymers to therapeutically

relevant proteins imparts several potential advantages including (i) its protection against degradation,

(ii) longer circulation in blood stream and (iii) a decreased immune response. Thus, all of these

properties result in a less-frequent administration to the patient (86, 87).

b. Dendrimers are monodisperse macromolecules combining a number of unique characteristics

including (i) a well defined, regularly hyperbranched and three dimensional architecture, (ii) a

relatively low polydispersity and (iii) a high and tunable functionality (Figure 2c). This molecular

structure confers the molecule properties substantially different than the linear counterparts (88).

The first family of dendrimers was the poly(amidoamine)s (PAMAM) (89). Other families are

the so-called polyamidoamine-organosilicon derivatives, poly(propyleneimine) dendrimers,

multilingual, chiral, amphiphilic, micellar and Frechet-type dendrimers (90). Dendrimers possess three

distinguished architectural components, namely (i) an initiator core; (ii) interior layers (generations)

composed of repeating units, radically attached to the interior core, and (iii) exterior (terminal

functionality) attached to the outermost interior generations (91). The chain-ends can be

functionalized and, thus, modify the solubility, the miscibility and the reactivity of the resulting

macromolecule (92). These highly functionalized materials enable drug incorporation into the core of

the molecule and drug complexation and conjugation on the surface (93), being the ability of the

molecules to enter the ramified tridimensional structure fully related to the size of the dendrimer.

PAMAM dendrimers and other cationic dendrimers can also act as vectors in gene therapy (94).

These macromolecules are terminated in amino groups which can interact with phosphate groups of

nucleic acids ensuring the formation of transfection complexes, which are also known as dendriplexes

(95).


33

While dendrimer-based products are considered safe, a study published recently

demonstrated that the marketed transfection reagent SuperFect®, consisting of partly hydrolyzed

dendrimers interfered with cell signal transduction pathways via and oxidative stress-dependent

mechanism even at doses that do not lead to over cytotoxicity (96). Thus, this study supports the

importance of understand the genomic of biological effects of dendrimers prior to its clinical

application.

c. Polymeric micelles are nanostructures formed by the self-assembly of amphiphilic copolymers in

aqueous media generally as a result of hydrophobic or ion pair interactions between polymer

segments, above the so-called critical micelle concentration (CMC) (97, 98). Polymeric micelles are

composed by internal and external zones named “core” and “shell”, respectively (Figure 2b). The

different types of micelles used for drug delivery including regular micelles, reverse micelles and uni-

molecular micelles. Regular micelles are self-assemblies of amphiphilic copolymers in an aqueous

medium while reverse micelles are self assemblies of amphiphilic copolymers in a non-aqueous

medium. Uni-molecular micelles are made from the block copolymers with several hydrophobic and

hydrophilic regions in one molecule, which enables the self-assembly of one molecule into a micelle.

The most broadly studied amphiphiles are derivatives of poly(ethylene oxide)-poly(propylene oxide)

block copolymers (99). In the last years a profuse number of amphiphilic polymers based on styrene

oxide and butylenes oxide and conjugates of hydrophilic PEG segments with hydrophobic blocks of

phospholipids (100), poly(L-amino acids) (101) and polyesters (102) have been proposed. Reported

advantages of micelles as delivery systems include simple preparation, increased drug solubility,

reduced toxicity, increased circulation time, enhanced tissue penetration and targetability (103).

However, the conventional micelles also have several disadvantages such as system instability over

long periods of time, sustained release only for short periods of time, inadequate suitability for

hydrophilic drugs and the need for system optimization with each drug (98).

d. Polyplexes are synthetic gene carriers systems formulated from both nucleic acids (i.e.

oligonucleotides, DNA or siRNA) and cationic polymers. Their production is regulated by ionic

interactions with the negative charged phosphate groups of naked DNA or other gene material,

resulting in the formation of positively charged complexes. As lipoplexes (complexes between lipids

and DNA), these structures allowing the protection of DNA from degradation and facilitate the entry

into the cell (104). Polyplexes used in gene therapy are based on the hypothesis that the complexes

adsorb more effectively to the anionic plasma membrane of mammalian cells via electrostatic

interactions then resulting in high transfection efficiency although the transport of the DNA into the

nucleous is still not well understood. Different cationic polymers have been proposed as gene carriers

for the transfecting foreign genes into mammalian cells in vitro including diethylaminoethyl-dextran,

poly(2-dimethylaminoethyl methacrylamide), poly-L-lysine, poly(ethyleneimines) and various

derivatives (105).


34

3. Polymer-based nanoparticulate drug delivery systems

Although polymer-based nanoparticles are a group within organic nanomaterials, their

classification in a different section is due to the special interest that this class of organic

nanoparticulate systems represents in this work.

Polymeric nanoparticles have been extensively investigated due to its potential as drug

delivery systems. These potential have been recognized since the 1960´s (106). One of the pioneers in

the development and evaluation of polymeric nanoparticles for drug delivery and for vaccines was

Professor Peter Speiser, whose strategy for the controlled release was the development of

miniaturized delivery systems (12, 13). His research group was focused at first in the development of

polyacrylic microcapsules and in the late 1960s they developed the first nanoparticles. In parallel,

albumin nanoparticles were developed for the first time in the Johns Hopkins Medical Institution in

Baltimore (107). Another pioneer was Professor Patrick Couvreur, who described the production of

biodegradable nanoparticles made of poly(methyl cyanoacrylate) and poly(ethyl cyanoacrylate) (108).

At that time, other types of nanoparticles were also proposed as poly(acrylamide) (109) or poly(lactic

acid) (PLA) nanoparticles (110).

Polymeric nanoparticles are solid colloidal carriers ranging in size from about 10 to 1000 nm

(111), and depending on the preparation method, nanospheres or nanocapsules can be obtained, with

the therapeutic agent dissolved, entrapped, encapsulated or attached to the polymeric matrix (Figure

3). Nanocapsules are vesicular systems, in which the drug is confined to a cavity surrounded by a

polymer layer, while nanospheres are matrix-type systems in which the drug is physically and

uniformly dispersed (112). Besides, the active substance can also be adsorbed in the surface of both

nanoparticles types.

Nowadays, the role of polymeric nanoparticles in drug delivery systems covers multiple

aspects, from the enhancement of the physical-chemical stability of the drug to the regulation of the

drug release profile and targeting (113). Moreover, the modification of the physico-chemical

properties and/or composition of their surface make possible the development of multifunctional

nanoparticles capable of targeting and controlled release of therapeutic and diagnostic agents. Thus,

by using polymeric nanoparticles a wide number of pharmaceutical and medical applications,

impossible to reach with traditional medicines, can be achieved.

Nanosphere

Nanocapsule

Water/oil liquid core

Polymeric core

Figure 3. Schematic representation of the structure of nanospheres and nanocapsules. Adapted from

reference (65).


35

3.1. Polymers used for the preparation of nanoparticles

Different polymers, both synthetic and natural, have been employed to prepare polymeric

nanoparticles. Ideally, all the polymers applied in the biomedical field must be biocompatible, non-

toxic, free of leachable impurities and readily processable (114). Compared to natural polymers which

have a relatively short duration of drug release, synthetic polymers have a sustained release of the

therapeutic agent over a period of days to several weeks. On the contrary, synthetic polymers are

usually limited by the need of organic solvents and harsh formulation conditions (115, 116). As

natural polymers, the most popular are chitosan and albumin. In a similar way, synthetic polymers

such as poly(esters), poly(alkyl cyanocrylates) and poly(anhydrides) are also used.

3.1.1. Natural Polymers

a. Chitosan is a modified natural carbohydrate polymer prepared by the partial N-deacetylation of

chitin, the second most abundant natural polysaccharide in nature, which is derived from the cuticles

of insect species or crustaceans such as crabs and shrimp (117). It is selected for the preparation of

nanoparticles as it possesses some ideal properties such as biocompatibility, biodegradability and non-

toxicity. Chitosan is soluble in weakly acid solutions, resulting in the formation of a cationic polymer

with high charge density, and can therefore form polyelectrolyte complexes with a large variety of

anionic polymers (118). Moreover, due to the presence of highly reactive amino groups along its

structure, chitosan is susceptible to chemical or biological functionalization (119). Besides, it has

excellent bioadhesive properties and the ability to enhance the penetration of large molecules across

the mucosal surfaces, make it a suitable candidate for the design of mucosal drug delivery systems

and vaccine formulations (120-123).

b. Systems based on proteins including gelatin, collagen, casein, albumin and whey protein have

been studied for delivering drugs, nutrients, bioactive peptides and probiotic organisms (124, 125).

Protein-based nanoparticles are particularly interesting as they hold certain advantages such as greater

stability during storage and in vivo, being non-toxic and non-antigenic and their ease to scale up during

manufacture over other drug delivery systems (126-128). Among the available protein-based drug

carrier systems, albumin nanoparticles have demonstrated to be a versatile macromolecular carrier.

Albumin nanoparticles have been shown to be biodegradable and metabolized in vivo to produce

innocuous degradation products, non-toxic and non-immunogenic (129). Besides, they can

incorporate a great variety of drugs (e.g. hydrophilic, lipophilic, peptides, oligonucleotides) in a

relatively non-specific fashion (130, 131). Commercially, albumins are obtained with significant

quantities from egg white (ovalbumin (OVA)), bovine serum albumin (BSA), and human serum

albumin (HSA). It is also possible to find albumins from soybeans, milk, and grains (132).


36

3.1.2. Synthetic Polymers

a. Poly(esters) are thermoplastic polymers with hydrolytically labile aliphatic ester linkages in their

structure. Aliphatic polyesters with short aliphatic chains between ester bonds are the most used

biodegradable polymers as they can degrade in the time required for most of the biomedical

applications (118). Among them, copolymers between lactic and glycolic acids (poly(lactide-co-

glycolide) (PLGA)), which are approved by the main regulatory agencies (e.g. FDA, European

Medicines Agency (EMA)), are widely employed for the preparation of nanoparticles due to their

biocompatibility and biodegradability (133, 134). PLGA is a poly(ester) composed by one or more of

three different hydroxy acid monomers, glycolic acid and/or d-/l-lactic (135). These copolymers

undergo hydrolysis of its ester linkages in the presence of water and the time required for the

degradation is related to the ratio of monomers used in the preparation, since higher the content of

glycolide units, the lower is the time required for its degradation (136). Moreover, these parameters

also determine the encapsulation efficiency and release rate of drugs from the nanoparticles (137).

However, PLGA degrades in the body producing its original monomers of lactic acid and glycolic

acid which are the by-products of various metabolic pathways creating an acidic microenvironment

(138). Thus, despite its obvious interest as nanoparticulate delivery systems, the use of these polymers

as peptide or protein delivery systems may negatively affect the stability of the loaded compound due

to the bulk degradation mechanism of the polymer and the acidic environment generated during this

process (139-141).

b. Poly(Ɛ-caprolactone) (PCL) is a FDA-approved semicrystalline, bioerodible, biodegradable and

biocompatible polymer that can be used for the formulation of nanoparticles (44). Due to its

semicrystallinity and hydrophobicity, the in vivo degradation of PCL is very slow making it more

suitable for long-term delivery systems, extending over a period of more than one year (115, 142). In

addition, PCL particles, unlike polylactides, do not generate an acidic environment that could

negatively affect the loaded drug and it is also compatible with a lot of other polymers (143).

c. Poly(akyl cyanocrylates) (PACA) are biodegradable, bioerodible and biocompatible polymers

that have been used since at least 1966 due to their excellent adhesive properties (144, 145). However

the employment of PACA polymers for the preparation of nanoparticles is more recent. From a

pharmaceutical point of view, PACA nanoparticles have demonstrated good mucoadhesive

properties and they are rapidly degraded by esterases in biological fluids.

d. Poly(anhydrides) Since the present work has been focused on poly(anhydride) nanoparticles, the

main characteristics of both polymer and nanoparticles have been explained in more detail.


37

Poly (methyl vinyl ether -co-maleic anhydride) copolymers

The copolymers between methyl vinyl ether and maleic anhydride (commercialized as

Gantrez® AN from ISP, Corp.) are a good example of the group of poly(anhydrides) (Figure 4).

CH3 CH CH CH

CO

C OO

OCH3

n

Gantrez® AN

Figure 4. Chemical structure of poly (methyl vinyl ether -co- maleic anhydride) or Gantrez® AN.

Those copolymers are widely employed for pharmaceutical and cosmetic applications as

denture adhesives, thickening and suspending agents and as adjuvants for the preparation of

transdermal patches. In addition, the ester derivatives are also employed as film-coating agents.

From a general point of view, Gantrez® AN copolymers are highly soluble in acetone and

display a low solubility in ethanol and isopropanol. On the other hand, these copolymers display a

very low aqueous solubility; however, in contact with water the anhydride group can be hydrolyzed

and dispersed to give uniform slurry. Table II summarizes the main physico-chemical properties of

the four available Gantrez® AN copolymers.

Table II. Physico-chemical characteristics of the main Gantrez AN copolymers.

Commercial name

Molecular weight (a) Brookfield viscosity at 25ºC

(mPa·s; 5-10%) (b) Tg (ºC)

Gantrez® AN 119 213,000 15-35 152

Gantrez® AN 139 1,080,000 40-145 151

Gantrez® AN 149 1,250,000 45-136 153

Gantrez® AN 169 1,980,000 85-1400 154 (a) Brookfield RVT viscometer, spindle 3, 20 rpm. (b) Glass-transition temperature.

The hydratation process of the anhydride form of theses copolymers is a complex

phenomenon. Thus, the process starts with an initial phase in which the polymer hydration occurs in

contact with water. After this hydratation process (about 3 h), the copolymer begins to swell because

of the hydrolysis phenomenon of the anhydride groups. Finally, in the third phase, the copolymer

dissolves rapidly (146).


38

The process of dissolution is influenced by the pH, temperature, molecular weight and the

degree of substitution of the anhydride groups (147). Actually, Gantrez copolymers have a pKa of

about 5.33 (weak acids), thus, the rate of dissolution is highly dependent on the pH of the medium.

In neutral and basic conditions, this rate is significantly higher than in acidic conditions. Similarly, the

temperature can highly affect the dissolution of the copolymer. Therefore, the hydrolysis of 10%

copolymer dispersion in water takes more than 8 h, when the temperature is 40ºC, and only 15 min

when the temperature is 90ºC (International Speciality Product ISP™).

In an aqueous medium, Gantrez® AN can spontaneously react with molecules containing

either hydroxyl (-OH) or primary amine (-NH2) residues to form stable bonds. These reactions can

be useful to modify the surface of a co-polymer matrix and, thus, modify both their physico-chemical

properties (i.e. degradability, stability and mechanical strength) and pharmaceutical characteristics (i.e.

drug distribution, adhesivity and release rate). Nevertheless, in the same aqueous medium, the

anhydride groups can also be hydrolyzed yielding two free carboxyl groups (Gantrez S). The resulting

product shows a high aqueous solubility and cannot react with nucleophilic groups.

Poly(anhydride) nanoparticles

Nanoparticles from these copolymers (Gantrez ® AN 119) have been developed recently.

Poly(anhydride) nanoparticles can be prepared under mild conditions using a solvent displacement

method (148). Poly(anhydride) nanoparticles can incorporate a wide variety of drugs, including

protein macromolecules, peptides and oligonucleotides.

Another interesting feature is that these nanoparticles can be treated with crosslinking agents

to modify the release profiles of the drug and the rate of degradation thereof. This crosslinking

process is performed by direct incubation of preformed nanoparticles and the crosslinking agent

without using traditional agents questionable from a toxicological point of view (glutaraldehyde

derivatives carbodi-imide, etc.).

One of the most important properties of poly(anhydride) nanoparticles is their ability to

develop strong bioadhesive interactions with components of the gut mucosa. In addition, their

surface can be easily modified by simple incubation with different excipients or ligands in order to

modify their in vivo distribution and even to increase its affinity for the intestinal mucosa. Thus, for

non-specific bioadhesion, poly(anhydride) nanoparticles can be coated with proteins (albumins) (148,

149), polysaccharides (e.g. chitosan and cyclodextrins) (150) and with PEGs or derivatives (151, 152).

To develop specific bioadhesive interactions, proposed ligands include lectins (148, 153), flagellin of

Salmonella enteritidis (154), lypopolysaccharides (155), simple molecules such as thiamine,

mannosamine or derivatives of vitamin B12 (156-158).

This versatility of poly(anhydride) nanoparticles makes them suitable carriers for easy

administration of drugs that exhibit limited oral bioavailability as well as good candidates for oral

immunotherapy.


39

3.2. Preparation of polymeric nanoparticles (Bottom-up & Top-down techniques)

Several methods have been developed to prepare polymeric nanoparticles and selection of a

particular one depends on the nature of the polymeric material forming the resulting nanoparticles

and the properties of the employed drug molecules to load into nanoparticles. They can be classified

into two main categories: bottom-up and top-down techniques. The bottom-up techniques are base

on the use of monomers which are polymerized in situ (i.e. emulsion, microemulsion and interfacial

polymerization). These methods based on polymerization reactions present some drawbacks such as

inadequate biodegradability of the product and the presence of toxic residues.

Top-down techniques involves the use of preformed polymers or purified natural

macromolecules. These techniques are more commonly used to prepare biodegradable nanoparticles

since they avoid the problems associated with bottom-up techniques. Among the different top-down

techniques developed, two approaches are conventionally used. The former is based on spontaneous

formation of nanoparticles whereas the latter is related with emulsification-based methods.

Depending on the solubility or gelling properties of a dissolved polymer, nanoparticles can

be formed spontaneously. The general principle of this method consists in the preparation of a

solution of the polymer and then induce a phase separation by adding a non-solvent of the polymer

(159, 160). Besides, nanoparticles from a hydrophilic polymer, such as alginate or chitosan, can be

prepared based on its gelation properties by ionic gelation (161).

Methods based on nano-emulsion templates derive from microparticle preparation

techniques (162). The first step requires the formation of an emulsion and its homogenization, which

can be achieve by high pressure homogenizers, microfluidizers and sonicators. Emulsification-based

methods differ depending on the properties of the polymer and on the miscibility between the

organic solvent and the aqueous phase. Thus, when hydrophobic polymers are used, some methods

involve volatile and water-immiscible solvents that have to be eliminated by simple evaporation,

leading to polymer precipitation (i.e. emulsification-solvent evaporation and double emulsion

methods). In other methods, polymer precipitation occurs as a result of controlled diffusion

processes using partially or fully water miscible organic solvents (salting out and emulsification-

diffusion methods). Table III summarizes some of the main advantages and drawbacks of the

methods used to prepare nanoparticles.


40

Table III. Overview of the main advantages and drawbacks of some of the most used methods for

the preparation of nanoparticles. Adapted from reference (44).

Preparation

method

Advantages Disadvantages Examples of

polymers

Nanoprecipitation Simple, fast and reproducible

Presence of surfactants Poly(esters)

Easy to scale up Use of organic solvents

Coacervation Easy to scale up Coacervate phase needs

consolidation (i.e. cross-

linkage)

Proteins

Ionic Gelation Organic solvent free method

Low stability due to the weakness of ionic interactions

Alginates, Chitosan

Emulsification-solvent evaporation

Encapsulation of hydrophobic and

Organic solvents PLA, PLGA, PCL

Lipophilic drugs Possible coalescence of nanodroplets during evaporation

Amphiphilic copolymers: PEG-PLGA, PEG-PCL, PEG-PACA

Salting out Minimization of the stress to labile drugs

Purification step is critical to remove electrolytes

PLA, PLGA

High loading efficiency

Easy to scale up

PLA: Poly(lactic acid); PLGA: Poly(lactide-co-glycolide); PCL: poly(ε-caprolactone); PEG: Polyethylene glycol; PACA: Poly(akyl cyanocrylates).

Before obtaining nanoparticles ready to be applied in drug delivery or for other purposes,

supplementary steps in the preparative process may be necessary. Normally, these steps may involve

purification process in order to remove residues of organic solvents used during nanoparticle

preparation as well as traces of unreacted polymers or/and molecules that have not been loaded into

nanoparticles. Among purification methods, the most commonly used are tangential flow filtration

(163), centrifugal filtration (164) and ultracentrifugation (159, 165). Furthermore, when nanoparticles

are developed for parenteral or ophthalmic applications they have to be sterile. Normally,

nanoparticles sterilization is carried out either by filtration before the filling process or by gamma-


41

irradiation when filled in the definitive container (164). Finally, nanoparticles are usually presented as

a dry powder in order to improve their stability as well as facilitate their manipulation, shipping and

storage. Usually, the drying process of nanoparticles suspensions are achieved by lyophilization or

spray-drying (165).

3.3. Behavior of polymeric nanoparticles after oral administration

Oral administration is the preferred route for drug delivery because is patient-friendly,

painless and easy for self-medication. Oral bioavailability of drugs is strongly influence by their

properties; solubility and permeability are two important parameters for their absorption via passive

diffusion. Thus, the Biopharmaceutics Classification System (BCS) defines four categories of drugs

based on their solubility and their permeability (166). The main mechanisms involved in the enhanced

drug absorption by polymeric nanoparticles in oral drug delivery are: (i) protection of the drug from

the harsh environment of the GIT, (ii) prolongation of the residence time in the gut by

mucoadhesion, (iii) endocytosis of the particles and/or (iv) permeabilizing effect of the polymer.

Moreover, specific delivery can be achieved by targeted nanoparticles.

When nanoparticles are administered by the oral route, they diffuse into the liquid medium

and reach the mucosal surface during their transit in the . Then, nanoparticles can be immobilized at

the intestinal surface by an adhesion mechanism called “bioadhesion”; although if adhesion occurs on

the mucus layer lining the mucosal surface, the term “mucoadhesion” is employed. These interactions

are driven by hydrogen bonding, Van der Waals interactions, polymer chain interpenetration,

hydrophobic forces and electrostatic/ionic interactions (167). Despite the duration of adhesion is

limited by mucus clearance mechanisms (168), this phenomenon results in (i) a prolonged residence

time and (ii) the establishment of a drug concentration gradient from the adhered nanoparticle till the

surface of the mucosa. To increase residence time, diffusion in the mucus is critical. In this regard,

PEG coating of nanoparticles surfaces has demonstrated to be an effective strategy to ensure rapid

nanoparticle transport in mucus. Although PEG was first used to increase particles stability due to its

chemical properties, PEG makes particle more hydrophilic and hence modulates their bioadhesive

properties (167, 169). In fact, dense coating with PEG effectively minimizes adhesive interactions

between nanoparticles and mucins, allowing penetration of nanoparticles (168, 170). The particle

mobility also seems to be strongly dependent on surface charges. Transport rates were inversely

related to particle surface potentials, with negatively charged particles displaying significantly higher

transport rates than near neutral, or positively charged particles whose transport was severely limited,

likely by particle aggregation and electrostatic adhesive interactions with mucin fibers (171).

Therefore, nanoparticles interaction with the mucus layer is influenced by their surface characteristics.

Once nanoparticles diffuse through the mucus layer they reach the intestinal epithelium,

which is mainly composed of absorptive enterocytes for a large part sprinkled by mucus-producing

Globet cells and M cells. Particle traslocation/absorption through the intestinal epithelium involves


42

both paracellular and transcellular routes (Figure 5). However, paracellular route is limited because it

utilizes less than 1% of the mucosal surface area. Moreover, juctional complexes (tight or adherens

junctions) restrict or completely block the passage of macromolecules larger than approximately 1

nm. Hence, it is generally admitted that polymeric nanoparticles do not diffuse through the intestinal

barrier by paracellular route. However, new potential modulators of the junctional proteins are

developed to reversibly open membranous barriers and improve drug delivery by the paracellular way

(172).

Regarding transcellular route, two main endocytic mechanisms have been described:

phagocytosis, which is restricted to M cells and phagocytic immune cells, and pinocytosis. The

endocytic pathways differ with the size of the endocytic vesicle, the nature of the cargo and the

mechanisms of vesicle formation (173). It is well established that uptake by enterocytes and M cells is

size-dependent (174). Thus, small particles (50–100 nm) can be translocated by endocytosis through

enterocytes while larger particles are more likely taken up and translocated by M cells. In this context,

molecules transport through M cells have been exploited to orally deliver vaccines as these cells are

specialized for antigens sampling in mucosal immunity.

Figure 5. Schematic representation of the different mechanisms of the uptake of nanoparticles in the

intestinal epithelium. From reference (175).


43

The distribution, bioadhesion and absorption of nanoparticles within the GIT can be

modulated by associating different excipients and/or ligands to polymeric nanoparticles. Thus, coating nanoparticles with lectins has been demonstrated to increase the intensity of the bioadhesion

phenomenon (176). On the other hand, the “decoration” of nanoparticles with vitamins (i.e. vitamin

B12 derivatives or thiamine) (158), sugars or bacterial adhesins permits to target specific receptors or

areas within the gut (177). Indeed, grafting or coating nanocarriers with ligands binding specific

receptors can enhance their internalization and transport.


44

4. Specific Applications of Polymeric nanoparticles as delivery systems

4.1. Tumor Targeting

The use of pharmacological agents developed using classical strategies is frequently limited by

pharmacodynamics and pharmacokinetics problems such as low efficacy or lack of selectivity.

Moreover, drug resistance at the target level due to physiological barriers or cellular mechanisms is

also encountered. In addition, many drugs have a poor solubility, low bioavailability and they can be

quickly cleared in the body by the reticular endothelial system (RES). Furthermore, the efficacy of

different drugs such as chemotherapeutical agents is often limited by dose-dependent side effects as

anticancer drugs, which usually have large volume of distribution and are toxic to both normal and

cancer cells. Therefore, precise drug release into highly specified target involves miniaturizing the

delivery systems to become much smaller than their targets. With the use of nanoparticles, targeting

drug molecules to the site of action is becoming a reality resulting in a personalized medicine. In this

regard, the rationale of using nanoparticles for tumor targeting is based in two main approaches.

First, nanoparticles provide the ability to deliver a concentrate dose of drug in the vicinity of the

tumor due to the enhanced permeability and retention (EPR) effect or active targeting by ligands on

the surface of nanoparticles. Secondly, nanoparticles would reduce the drug exposure to health

tissues by limiting drug distribution to the target organ minimizing the drug dose-dependent side

effects.

4.1.1. Passive targeting with polymeric nanoparticles

In general, nanoparticle delivery of anticancer drugs to tumor tissues can be achieved by

passive or active targeting. Passive targeting refers to the preferential accumulation of nanoparticles

(bearing no affinity ligands) at active sites. Passive targeting takes the advantage of the inherent

physicochemical properties of nanoparticles (size, shape, flexibility...) and exploits the unique

anatomical and pathophysiological abnormalities of tumor vasculature, such as EPR effect (178).

Characteristics of the EPR effect include extensive angiogenesis, defective vascular architecture and

impaired lymphatic drainage/recovery system of the tumors. Moreover, most solid tumors have

elevated levels of vascular permeability factors which augment the permeability of tumor tissues in

comparison to normal tissue, thereby also contributing to the EPR effect (179). Thus, the targeting

effect of nanocarriers is achieved as they remain in the bloodstream, where the vessel structure is

normal, but would extravasate through the leaky vasculature at the tumor site. Upon arrival at the

target sites, the nanocarriers release the drug in the vicinity of the tumor cells. The absence of

lymphatic drainage from the tumors contributes to retention of the nanocarriers, ultimately leading to

the accumulation of high concentrations of drug at the tumor site (Figure 6) (180).


45

Figure 6. Schematic representation of passive targeting of nanoparticles by EPR effect. From

reference (181).

In this context, Maeda and collaborators were the first group, which in the 1980`s

demonstrated the principle of passive targeting of colloidal particles to tumors (182, 183). In their

initial studies, significantly higher concentrations of the cytotoxic drug neocarzinostatin was

discovered in tumor tissue post administration of the drug conjugated with poly(styrene-co-maleic

acid)-neocarzinostatin, in comparison to control experiments where the drug was administered in its

free form. This led Maeda and colleagues to postulate that the enhanced accumulation of the colloidal

particles in the tumor was attributed to the structural features of the tumor vasculature, an

observation which was termed the EPR effect (184). Following the revolutionary work of Maeda,

many other groups have greatly exploited this mechanism of passive targeting for the delivery of

chemotherapeutics into polymeric nanoparticles. For example, Bhardwaj et al. synthesized paclitaxel-

loaded PLGA nanoparticles stabilized with cationic surfactants, and applied this formulation to

increase the oral bioavailability of the drug to treat chemical-induced breast cancer in rats (185). Their

studies showed that paclitaxel-loaded nanoparticles improved efficacy in comparison with i.v. free

paclitaxel since nanoparticles exhibited preferential accumulation in tumors; the EPR effect might be

the key prospective explanation. Other report, demonstrated the superiority of doxorubicin (DOX)

loaded in PACA nanoparticles in a murine hepatic metastases model (186). This work showed that

the incorporation of DOX in PACA nanoparticles resulted in a much greater reduction of the

number of metastases compared to free DOX. Recently, in an elegant experiment, Jäger and co-

workers formulated core-shell polymeric nanoparticles to deliver two different anticancer drugs by

passive tumor targeting (187). They prepared polymeric nanoparticles based on the self assembly of

N-(2-Hydroxypropyl) methacrylamide (HPMA) copolymers and aliphatic biodegradable co-polyester

as a vehicle for docetaxel (DTXL) and DOX. In vivo studies carried out in mice bearing EL-4 T cell

lymphoma demonstrated that the use of nanoparticles simultaneously loaded with DTXL and DOX

provided a more efficient suppression of tumor-cell growth when compared to the treatment with

free drugs or with single-loaded nanoparticles (DTXL or DOX separately). Their experiments


46

showed not only the selectivity of nanoparticles to be accumulated in tumors due to the EPR effect

but also that by means of co-encapsulation the efficacy was increased for both drugs.

The extensive efforts in developing nanoparticles for tumor targeting have ultimately resulted

in the clinical translation of a number of passively targeted polymeric nanoparticles. An interesting

example is Abraxane® (ABI-007) which was approved in 2005 by the FDA for the treatment of

metastatic breast cancer (188, 189). ABI-007 is a formulation of albumin nanoparticles containing

paclitaxel obtained by high-pressure homogenization (190). In a phase III clinical trial with metastatic

breast cancer patients, ABI-007 demonstrated significantly higher tumor response rates (33% vs 19%)

and longer times to tumor progression (23.0 vs 16.9 weeks) than the control formulation (Taxol®)

(188). Besides metastatic breast cancer, ABI-007 has also been evaluated in clinical trials involving

many other cancers including non-small-cell lung cancer (phase II trial) and advanced

nonhematologic malignancies (phase I and pharmacokinetics trials) (191, 192).

4.1.2. Passive targeting with pegylated nanoparticles

In order to achieve effective EPR mediated targeting, polymeric nanoparticles must have

long-circulating half-lives that facilitate more opportunities for the passage of nanoparticles from

systemic circulation into the disordered and permeable regions of tumor vasculature. Therefore, their

usefulness is limited by their rapid blood clearance and recognition by the macrophages of the MPS.

In order to overcome this drawback, pegylation emerged over a decade ago as one possible strategy.

In 1994, based on previous work carried out by Illum and collaborators (193, 194), Gref and

colleagues demonstrated that coating of PLA nanoparticles with PEG chains significantly increased

the circulation half-lives of nanoparticles (195). Since then, there have been a myriad of pegylated

polymeric nanoparticles reported in the literature with the benefits of pegylation demonstrated across

a broad range of polymer molecular architectures and macromolecular assemblies (196, 197). In this

context, pegylation has demonstrated to considerably reduce the opsonization of the nanoparticles

and the rate of the complement system activation. Hence, nanoparticles are less recognized by the

macrophages of the MPS and can remain in blood stream and distribute in tumors.

The value that pegylation provides in drug delivery of chemotherapeutics have resulted in the

clinical translation of a number of passively targeted pegylated nanoparticles. In general, these

nanoparticles are pegylated polymeric micelle formulations that form from the self assembly of

amphiphilic polymers at concentrations above the CMC, yielding nanoparticles which can encapsulate

poorly water soluble drugs (198). Among the different formulations currently in clinical trials,

Genexol-PM® is a novel Cremophor EL-free paclitaxel-encapsulated PEG-PLA micelle formulation

developed for various cancers (199-201). Phase II studies have demonstrated the safety and efficacy

of Genexol-PM® with high response rates in patients with metastatic breast cancer (202). Moreover,

Genexol-PM® plus cisplatin combination chemotherapy has shown significant antitumor activity and

allowed the administration of higher doses of paclitaxel compared to the Cremophor EL-based

formulation in patients with advanced non-small-cell lung cancer (203).


47

However, the success of PEG in transforming polymeric nanoparticles drug delivery has not

been without its challenges. For example, the highly hydrophilic surface of pegylated nanoparticles

might not result in optimal endocytic uptake by cancer cells within the tumor hence efficient drug

delivery into tumors cells rather than in the tumor milieu could be hampered. Then, bearing in mind

all previously mentioned, passive targeting strategies are not without limitations and therefore

considerable efforts are now underway to improve the therapeutic effect by more precise control of

the delivery of the drugs to the active sites.

4.1.3. Active targeting with polymeric nanoparticles

Active targeting of nanoparticles is based on molecular recognition processes such as ligand-

receptor or antigen-antibody interaction. Typically, these receptors/antigens are differentially over

expressed on the cells membrane or in the extra-cellular matrix of the disease tissue so targeted

nanoparticles can be utilized in applications where drug release is either intracellular or extracellular.

In active tumor targeting nanoparticles are covalently coupled with the ligands. Then, the ligand

coupled nanoparticles are received by the tumor cells expressing a homologous receptor on their

surfaces. Thus, the specific ligand-receptor binding ensures that the nanoparticles carrying drugs will

get attached specifically to the tumor cells facilitating delivery of drugs only to the tumor cells

expressing receptor and not the normal healthy cells hence avoiding undesirable side effects. Several

ligand-receptor systems have been already studied, such as folic acid receptor, epidermal growth

factor, or metal receptors among others. As an approach, considering that folate receptors are over

expressed on the surface of some human malignant cells and that cell adhesion molecules (i.e.

selectins and integrins) are involved in metastatic events, nanoparticles bearing specific ligands such

as folate may be used to target ovarian carcinoma while specific peptides or carbohydrates may be

used to target integrins and selectins (204, 205).

Another example is the use of galactolipids that bind to the asialoglycoprotein receptor of the

human hepatoma HepG2 cells (206). In some cases, active targeting needs a receptor mediated cell

internalization to occur. Thus it was demonstrated that the benefits of folate ligand coating were to

facilitate tumor cell internalization and retention of gadolinium labelled-nanoparticles in the tumor

tissue (207). Targeting with small ligands appears more likely to succeed since they are easy to handle

and manufacture. Furthermore, it could be advantageous when the active targeting ligands are used in

combination with the long circulating nanoparticles to maximize the likelihood of the success in

active targeting of nanoparticles. One example of this approach is the design of PEG-coated PACA

nanoparticles coupled to folic acid (208). These conjugated nanoparticles displayed a multivalent and

hence stronger interaction with the surface of the malignant cells expressing high amounts of the

folate receptor at their surface.

In another study, nanoparticles based on gelatine and HSA were decorated by covalent

attachment with the biotin binding protein NeutrAvidin®, enabling the binding of biotinylated drug

targeting ligands by avidin–biotin complex formation (209). Using the human epidermal growth


48

factor receptor 2 (HER2) receptor-specific antibody trastuzumab (Herceptin®) conjugated to the

surface of these nanoparticles, a specific targeting to HER2-overexpressing cells, followed by

effective internalization of these nanoparticles, can be obtained. While the potential benefit of ligand-

mediated nanoparticle targeting is clear, there are few clinical validated studies so far. Hrkach and

collaborators demonstrated the clinical benefits of targeted polymeric nanoparticles (TNP) containing

the chemotherapeutic DTXL for the treatment of patients with solid tumors (210). DTXL-TNP were

targeted to prostate-specific membrane tumor antigen which is expressed on prostate cancer cells and

on the neovasculature of most non prostate solid tumors. In previous preclinical studies carried out

in tumor-bearing mice, DTXL-TNP exhibited markedly enhanced tumor accumulation at 12 h and

prolonged tumor growth suppression compared to a solvent-based DTXL formulation (sb-DTXL).

Moreover, initial clinical data in patients with advanced solid tumors indicated that DTXL-TNP

displayed a pharmacological profile differentiated from sb-DTXL, including pharmacokinetics

characteristics consistent with preclinical data and cases of tumor shrinkage at doses below the sb-

DTXL dose typically used in the clinic (210).

4.2. Oral drug delivery

In recent years, significant research has been done using polymeric nanoparticles as oral drug

delivery vehicles for different purposes such as diabetes treatment or chemotherapy. Compared with

other administration routes, oral administration of drugs represents the most comfortable and

convenient route of administration.

4.2.1. Diabetes treatment

Insulin, as a protein drug used to treat diabetes, has been conventionally administered via

subcutaneous injection (211). Many researchers have attempted to administer insulin orally since its

discovery in 1922 (212). However, the oral bioavailability of insulin is severely hampered by its

inherent instability in the GIT and its low permeability across biological membranes in the intestine.

Thus, to overcome these drawbacks, a possible strategy may be the use of polymeric nanoparticles

which among other benefits, allow encapsulation of bioactive molecules, protect them against

enzymatic and hydrolytic degradation and increase residence time in contact with the absorptive

epithelium (213). Hydrophobic and hydrophilic polymers, including PLGA, PLA, PCL and chitosan

have been used to fabricate nanoparticles for oral insulin delivery (175). In a recent study, Jain and

collaborators developed insulin loaded folate coupled pegylated PLGA nanoparticles (214). In

diabetic rats, the formulations with a mean diameter of 260 nm, exhibited a two-fold increase in the

oral bioavailability (double hypoglycemia) without any hypoglycemic shock as compared to

subcutaneously administered standard insulin solution.


49

4.2.2. Oral chemotherapy

Oral chemotherapy is especially appropriate since many anti-cancer therapies are optimally

effective when given chronically for a continuous tumor exposure (215). Moreover, from the

economic standpoint, oral administration is more attractive as it reduces the cost for hospitalization

and infusion equipment supplies (216). However, despite the numerous advantages described above,

there are also some inherent problems associated with the use of oral anticancer drugs. Thus, for

some compounds, the risk of local irritation of the GIT may limit continued oral administration or

result in poor compliance (217). Nevertheless the main limitation of oral administration is the low

and highly variable oral bioavailability which is limited by factors such as rapid degradation, low

solubility, poor membrane permeability or extensive pre-systemic metabolism. It has been widely

demonstrated that the major determinants of oral bioavailability is the efflux by P-glycoprotein (P-gp)

and/or intestinal and hepatic metabolism by cytochrome P450 metabolizing enzyme system (CYP).

In this context, the encapsulation of anticancer drugs in bioadhesive nanoparticles may be an

interesting approach to promote their oral absorption.

Among the different bioadhesive polymeric nanoparticles currently under investigation,

nanoparticles made from the poly(anhydride) poly(methylvinylether-co-maleic anhydride), termed

poly(anhydride) nanoparticles have demonstrated to be a promising strategy for oral drug delivery

(148). Thus, using fluorouridine, the potential of serum albumin-coated poly(anhydride) nanoparticles

were evaluated. These nanoparticles were capable to specifically target the stomach mucosa hence

minimizing their arrival to the small intestine where P-gp and CYP are located (152). From urine

data, the FURD bioavailability was found to be 4-times higher when loaded in albumin-coated

nanoparticles than in conventional ones (79% vs 21%, respectively). For the control oral solution this

parameter was calculated to be only 11% (149). More recently, the combination between

poly(anhydride) nanoparticles and cyclodextrins has been demonstrated effective to significantly

improve the oral bioavailability of paclitaxel (218, 219). Apart to their well reputed activity as

solubilizing agents, cyclodextrins have the capability to disturb and inhibit the activity of the intestinal

P-gp (220). After oral administration of these nanoparticles to animals, paclitaxel plasma levels were

found to be high and prolonged for at least 24 h post-administration. These sustained levels of the

anticancer drug corresponded with a relative oral bioavailability of about 80% (218). This fact would

be due to the combination of both bioadhesive and inhibitory properties of these cyclodextrin-

poly(anhydride) nanoparticles. In fact, the nanoparticles would be capable to approach the paclitaxel–

cyclodextrin complex till the surface of the mucosa where these carriers would remain immobilized in

intimate contact with the absorptive membrane. Then, nanoparticles would progressively release their

content to the gut environment. At this moment, the paclitaxel–cyclodextrin complex would

dissociate yielding the free drug and the oligosaccharide molecules. Under these conditions, the

cyclodextrins would interact with lipidic components of the membrane disturbing the activity of the

efflux pump and CYP whereas the anticancer drug, simultaneously, would be absorbed. The same


50

approach has been found by the pegylation of paclitaxel loaded poly(anhydride) nanoparticles (221).

Pegylated nanoparticles have shown a high ability to cross the mucus layer and reach the surface of

the enterocytes (152). In addition, PEG has shown a moderate inhibitory effect of both P-gp and

CYP3A4 (222). Therefore, after oral administration of pegylated nanoparticles to animals, paclitaxel

plasma concentration was maintained in a high and constant level for at least 48 h post-

administration. More particularly, the relative oral bioavailability of nanoparticles pegylated with

either PEG 2000 or PEG 6000 was calculated to be about 70% and 40% respectively (221).

4.3. Polymeric nanoparticles as adjuvants and delivery systems for oral vaccination

In the past decades, several new approaches in vaccine development have shown to offer

significant advantages over traditional ones (i.e. attenuated vaccines and bacterines). The new

generation of vaccines contains other defined antigens, called “subunit vaccines”. They include the

use of vaccines based in proteins, synthetic peptides, and/or plasmid DNA (223-225). Although they

offer advantages such as reduced toxicity compared to traditional ones, they are poorly immunogenic

when administered alone. Therefore, the rationale for vaccine design against a specific pathogen

requires the use of the right antigens and the appropriate adjuvant to elicit the corresponding

immune and protective response. In this context, polymeric nanosystems have been proposed as new

adjuvants more convenient to use, safer and easy to administer than the classical ones (such as alum)

(226). Moreover, the new adjuvants based on nanoparticles may be susceptible to be used even

through mucosal routes. In this regard, by using polymeric nanoparticles for mucosal vaccination via

oral route, these systems not only may protect the antigens from its stomach degradation and/or act

as adjuvants, but also can enhance the delivery of the loaded antigen to the gut lymphoid cells due to

their ability to be captured and internalized by cells of the gut-associated lymphoid tissue. In fact,

mucosal vaccination using antigens loaded or encapsulated in particles appears to have a sound

scientific rationale based on the protection of an antigen from exposure to extreme pH conditions, bile and pancreatic secretions (153) and provide a depot-effect (227). In addition, the use of nanoparticles offers another advantage such as the possibility to load an immune-enhancer (i.e.,

cholera toxin B subunit (CTB) or lipopolysaccharide (LPS)) with the antigen in order to increase and

induce the more appropriate immune response (228, 229). In this context, poly(anhydride)

nanoparticles have demonstrated to be good candidates for oral vaccination and immunotherapy

treatments since these nanoparticles have shown excellent properties as adjuvants and appropriate

vehicles for the controlled release of antigens (230-233). Thus, Gomez and co-workers found

enhancements in both Th1 and Th2 markers (IgG2a and IgG1, respectively) after oral administration

of these nanoparticles loaded with OVA as allergen model (234). Moreover, these carriers were able

to protect a model of sensitized mice to OVA from anaphylactic shock. In other elegant study,

Salman and collaborators designed poly(anhydride) nanoparticles coated with mannose (recognized

by lectine CLR type) or Salmonella Enteritidis derived flagellin (Toll like receptor 5 agonist) (177). Both


51

coated nanoparticles demonstrated strong bioadhesive capacity to the gut oral mucosal, including

Peyer´s patches post-oral administration (154, 157). Moreover, using OVA as antigen model, systemic

and mucosal immune responses reported after oral administration, indicated that a single dose of

OVA-mannose and OVA-flagellin nanoparticles, elicited higher and balanced systemic specific

antibody responses (IgG1 (Th2-response) and IgG2a (Th1-response)) compared to non-coated ones.

In addition, coated nanoparticles were able to elicit higher levels of intestinal secretory IgA after oral

immunization compared to subcutaneous immunization.


52

5. Nanotoxicology: Towards a safe and successful nanotechnology

5.1. Nano-specific toxic effects?

It is well known that nanotechnology is one of the most innovative areas of research and

development which can find application in almost all essential aspects of our life. Thus, nanomaterials

are steadily increasing and many products furnished with nanotechnology are released to the market.

However, the successes of nanotechnology have not been associated with a parallel development of

the adequate methodology to asses and manage the potential risks for humans. In this regard, in the

“late lessons from early warnings for nanotechnology” it was pointed out that: “a new technology will

only be successful if those promoting it can show is safe, but history is littered with examples of

promising technologies that never fulfilled the true potential and/or caused untold damage because

early warnings were ignored” (235).

It is clearly demonstrated that the application of nanotechnology in health care defined as

nanomedicine, offers new opportunities to improve medical therapies and diagnosis in all relevant

pathological conditions including cancer. However, the same properties (small size, chemical

composition, structure, large surface area and shape), which make nanoparticles so attractive in

medicine, may contribute to the toxicological profile of nanoparticles in biological systems. Hence, to

use the potential of nanoparticles in nanomedicine, full attention must be given to safety and

toxicological issues (236).

In respond to this concern, nanotoxicology has arisen as a multi-interdisciplinary field in

order to properly assess the safety of nanoparticles before exploiting their advantages. So far, most of

the nanotoxicological research has involved a limited group of engineered nanoparticles, mainly

inorganic nanomaterials. The toxicity of liposomes, dendrimers, polymers and emulsions has been

studied to a much more limited degree, although they are the most employed for medical therapies

and diagnosis. This situation is not surprising as there is a considerable database on the safety of its

bulk materials, which are already frequently used in healthcare products. Therefore, these organic

nanoparticles have been considered safe and not much attention has been paid to its toxicity based

on the safety of its bulk material. However, the properties and behavior at the nano level are

completely different from the bulk materials thus the toxicological approach is substantially different

from the classical way to address the adverse health effects.

Thus, to properly assess nanoparticle toxicity two concepts should be highlighted:

(i) Nanoparticles are developed for their unique properties in comparison to bulk materials.

Since surface is the contact layer with the body tissue and crucial determinant of particle response,

these unique properties need to be investigated from a toxicological standpoint. When nanoparticles

are used for their unique reactive characteristics it may be expected that these same characteristics

also have an impact on the toxicity of such particles.


53

(ii) Nanoparticles are attributed qualitatively different physico-chemical characteristics from

micron-sized particles, which may result in changed body distribution, passage of the blood barrier,

and triggering of blood coagulation pathways. In view of these characteristics specific emphasis

should be on investigations in pharmacokinetics and distribution studies of nanoparticles. What is

currently lacking is a basic understanding of the biological behavior of nanoparticles in terms of

distribution in vivo both at the organ and cellular level (2).

Although current tests and procedures in drug and device evaluation may be appropriate to

detect many risks associated with the use of these nanoparticles, development of new protocols and

guidelines to properly detect all potential “nano-risks” are extremely urgent (237).

Specific importance should be devoted to the toxicity of the empty non-drug-loaded

nanoparticles. For nanodevices used in medicine the focus in most papers is mainly on obtained

reduction of toxicity of the incorporated drug, whereas the possible toxicity of the carrier used is not

considered. Particular attention must be taken in the case of slowly degradable or non-degradable

nanoparticles used for drug delivery. After the delivery of the drug/antigen, nanoparticles residual

components may accumulate in the body causing possible adverse effects. Moreover, nanoparticle

size and persistency are two of the main aspects to consider as toxicity risk factors. In this regard,

Müller and collaborators have proposed a nanotoxicological classification system (NCS) based on the

structure of the BCS displayed in Figure 7 (238). The NCS would be useful as a first approach, since

it allows a clear and straightforward justification of possible toxicological risks of nanoparticles used

in medicine. However, as it was mentioned before, other physicochemical properties such as shape or

surface may exert an influence on the overall adversity in cells and tissues (239).

Figure 7. NCS for a precise differentiation of nanotoxicological risks derived from nanoparticulate

carriers in the future. Suggested by Müller and collaborators (238).


54

Therefore, it is necessary to better understand the influence of these parameters on the

potential nanoparticle toxicity. Recent studies by Warheit (240) and Murdock et al. (241), emphasize

the urgent need for a thorough physical and chemical characterization of nanoparticles before

conducting any toxicological study. Moreover, this characterization should be carried out in the

media to be performed subsequent in vitro studies because sometimes, the protein components of the

culture media may cause aggregation of the nanoparticles as well as its surface modification. In this

context, table IV summarizes the more significant nanoparticle physico-chemical properties that may

be controlled in order to improve biocompatibility hence reducing toxic effects.

Consequently, prior to evaluate the toxicity of nanoparticles by in vitro or in vivo studies, it

should be considered in early stages, designing nanoparticles with appropriate physico-chemical

characteristics that take into account not only its potential uses but also its risks.

Table IV. Physico-Chemical Nanoparticle Properties of relevance for Nanotoxicology.

Characteristic Biological Behavior

SIZE

‐ The smaller nanoparticles are, the more the surface area they have per unit mass;

thus more chemical molecules may attach to this surface, which would enhance

its reactivity and result in an increase of toxic effects

‐ Key factor with respect to translocation across cell barriers or along neuronal

pathways

SHAPE

‐ Spherical nanoparticles with identical surface functionalization are generally

more toxic and more efficiently ingested than rod-shape particles

SURFACE

PROPERTIES

‐ Charge: Positively charged nanoparticles have a greater tendency of interacting

with biological components compared with neutral and anionic. In consequence,

cationic nanoparticles are generally defined as more toxic

‐ Chemistry: Reactive groups on particle surface influence their interaction with

biological material. Thus they would certainly modify the biological effects,

affecting its safety mainly by causing oxidative stress

‐ Area: Due to their larger surface area, nanoparticles have greater tendency to

agglomeration/aggregation which may result in undesirable effects

STABILITY ‐ Non-biodegradable nanoparticle may be accumulated unintentionally in different

tissues like the brain , gut, spleen and lymph nodes leading toxicity

CORONA

‐ Once nanoparticles interact with the biological environments in the human body

they are immediately covered by matrix molecules surrounding the particle

thereby determining the impact and potential toxicity


55

5.2. In vitro assessment of nanoparticle toxicity

In vitro toxicological assessment is a vitally important tool for nanotoxicology and is the next

step that should be conduct when addressing the evaluation of nanoparticles toxicity after its physico-

chemical characterization. The interaction, influence and possible toxicity of nanoparticles at cellular

level are an essential focus in understanding nanoparticle compatibility versus toxicity. In addition,

compared to in vivo studies, in vitro studies benefit from being faster, lower cost, allowing greater

control, and minimizing ethical concerns. Consequently, in vitro models provide the possibility to

investigate toxic effects on human cells extensively, which cannot be conducted in vivo. However, the

extension of results from in vitro experiments for the prediction of in vivo toxicity is problematic due

to the in vitro exposure conditions which usually feature much higher concentrations and exposure

times than found in the cellular environment in vivo.

Commonly, in vitro toxicity studies have used monocultures of cells that are specific to organs

of the body. For example, macrophage cells are used as a model to study the effects of nanoparticles

on the human immune system as well as dendritic cells. Additionally, specific cells such as the human

hepatoma cell line HepG2 cells are also used for example for the liver and the human colon

carcinoma cell line Caco-2 for the intestine. However, although monoculture systems provide the

basis for high-throughput analysis for nanotoxicology, they do not represent a realistic model of how

nanoparticles will interact with a specific organ of the body. Thus, it is essential that co-culture

systems mimicking the in vivo system are established and used for the specific organs that are in

danger or interacting with nanoparticles. However, these systems will not be able to be completely

definitive of the in vivo situation.

For in vitro studies, there are a number of different toxicological endpoints that researchers

have used to assess the potential adverse effects that nanoparticles may have on organs of the human

body. These include numerous different biochemical- and molecular-based testing strategies,

investigating the potential for nanoparticles to cause cytotoxicity, inflammation, oxidative stress, cell

proliferation and genotoxicity. The most commonly used in vitro assessment techniques are

summarized in table V.


56

Table V. Overview of common in vitro toxicological techniques used in nanotoxicology. Adapted

from reference (242).

Assay type Category Cellular property/process

probed Assays

Metabolic activity MTT, MTS, XTT, WST-1, ATP,

Alamar Blue Proliferation

DNA synthesis [3H]thymidine incorporation

Necrosis Membrane integrity LDH, Trypan Blue, Neutral Red,

Propidium iodide

Apoptosis Membrane structure Annexin-V

Viability

Live-Dead Esterase activity/membrane

integrity

Calcein acetoxymethyl/ethidium

homodimer

DNA fragmentation Comet, Sister Chromatid ExchangeDNA damage

DNA double-strand breakage TUNEL

Presence of reactive oxygen

species DCFA, Rhodamine123

Lipid peroxidation C11-BODIPY, TBA assay for

malondialdehyde

Presence of lipid

hydroperoxides Amplex Red

Antioxidant depletion DTNB

Superoxide dismutase activity Nitro blue tetrazolium

Mechanistic

Oxidative stress

Superoxide dismutase

expression Immunobloting

MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; MTS: 3-(4,5-dimethylthiazol-2-yl)-5-(3-crboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; WST-1: 4-[3- (4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate. XTT: 2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide; LDH: Lactate dehydrogenase; TUNEL: Terminal deoxyribonucleotidyl transferase (TDT)-mediated dUTP-digoxigenin nick end labeling; DTNB: 5,5-dithio-bis-2-nitrobenzoic acid; DCFA: 2′7′-dichlorofluorescein diacetate; ATP: Adenosine thriphosphate; TBA: Thiobarbituric Acid.


57

In particular, for cytotoxicity studies, it is important to know in advance the sensitivity of

cultured cells to changes in their environment as temperature or pH changes, among others, since the

nanoparticles may be responsible for these changes. It should be noted that nanoparticles frequently

interfere with commonly used cytotoxicity assays that are based on the detection of fluorescence or

absorbance of various probes. In this context, the enzyme LDH (lactate dehydrogenase) can absorb

to the surface of nanoparticles, masking their toxicity and thus providing a false-negative toxic result

(243). Moreover, Worle-Knirsch and collaborators reported that CNTs interact with the MTT

formazan used and provided false-negative toxicity (244). Therefore, the control of the experimental

conditions is crucial to ensure that the cytotoxicity results are due only to the addition of the

nanoparticles rather than to the culture conditions.

Another important aspect to consider when assessing the potential toxicity of any form of

nanoparticles, is to investigate how the particles enter the cells and in which compartments they may

be found within or, which is also possible, if the nanoparticle stay attached to the cell membrane.

This is of great importance, as it has been shown that the uptake and behavior can influence the

cellular response following nanoparticles exposure (245).

To date, there is a lack of consensus in the published literature on in vitro nanoparticle toxicity

studies due to variable methods, materials, and cell lines. Standardization in experimental set up such

as choice of model (monoculture/co-culture systems) and exposure conditions (cell confluence,

exposure duration, nanoparticle-concentration ranges and dosing increments) is necessary in order

for comparisons between studies conducted by different groups to be effective and also to predict in

vivo responses in order to reduce and avoid extensive testing using laboratory animals. Nevertheless,

to evaluate nanoparticle toxicity, in vitro models are insufficient to predict possible hazards to humans

and to carry on successful transition through the clinical trials to the markets (246). Taking this into

account, data obtained for in vitro studies require verification from in vivo experiments to accurately

assess nanotoxicity.

5.3. In vivo assessment of nanoparticle toxicity

In the past years, the majority of nanotoxicity research has focused on cell culture systems;

however, the data from these studies could be misleading and will require verification from in vivo

studies to evaluate the impact of nanoparticle exposure within intact living organisms. In vivo studies

are typically undertaken in rodents, although recently, zebrafish (Danio rerio) has emerged as a

promising in vivo model for nanotoxicity studies (247).

Despite of the fact that in vivo studies bear ethical consideration, due to the use and sacrifice

of animals, these studies allow examination of long-term effects, tissue localization, biodistribution

and retention/excretion of nanoparticles. To carry out adequate in vivo studies is necessary to fully

understand the overall behavior that nanoparticles could exhibit within the organism: (i) nanoparticles

can be administered or can enter into the body via six principal routes: i.v., dermal, subcutaneous,


58

inhalation, intraperitoneal and oral, (ii) adsorption and/or interaction can occur when the

nanostructures first interact with biological components (proteins, cells), (iii) afterward they can be

absorbed and therefore distribute to various organs in the body and may remain the same structurally,

be modified, or metabolized, or excreted (iv) they enter the cells of the organ where nanoparticles can

be degraded or reside in the cells for an unknown amount of time before leaving to move to other

organs or to be excreted.

Currently there are insufficient data to understand the direct relationship between the

physicochemical properties of nanoparticles and their in vivo behavior, thus, understand this

relationship would provide a basis for evaluating the response and toxicity of nanoparticles and could

lead to predictive models for assessing the toxicity of these new derived-nanotechnology materials.

Beyond the simple LD50 (lethal dose for 50% of participating animals) study, in vivo

nanoparticle toxicity studies typically focus on one or more of three major areas: changes in blood

serum chemistry and cell population, changes in tissue morphology, examined using histology, or the

overall nanoparticle biodistribution. Typically, in vivo experiments utilize several of these, and other,

techniques to maximize the toxicity information gained. As with in vitro studies, one must consider

many aspects prior to beginning studies, such as nanoparticle choice and characterization, prior to

exposure, and choice of relevant model system. Additionally, one must also consider issues of

exposure route, nanoparticle dosing and specific tissue/organ or protein/cell type of interest, to

examine histological or serum/hematological changes, respectively. Last but not least, it is also

important when evaluating in vivo nanotoxicity, to conduct systematic studies to investigate chronic

and acute exposure doses of nanoparticles with different physical or chemical variables such as size,

charge, and hydrophobicity.

However, a major concern, not only in nanotoxicology but in general toxicology, is to

conduct animal testing following the principles of the 3 Rs: Replacement, Reduction and Refinement.

So, as for now, the use of animals is necessary to evaluate the safety of nanoparticles in vivo, and there

are currently no validated guidelines, in vivo nanotoxicological studies should be performed, whenever

possible, following the principles of the 3 Rs. In this context, the standard procedures and guidelines

proposed by the Organization for Economic Cooperation and Development (OECD), which are

currently used to test and assess chemicals, are being modified to encompass these principles. Thus,

in assessing in vivo toxicity of nanoparticles, the use of the OECD guidelines may be considered

adequate because these guidelines following the principles of the 3 Rs; besides, it is noteworthy that

several studies have shown that OECD guidelines can be adapted to assess in vivo nanotoxicity

leading to valuable and consistent results (248-250).


59

5.4. Nanotoxicology regulation: Is the current regulation robust enough to handle

risks of nanomaterials?

Usually it is hard to figure out whether the inconsistencies observed in nanotoxicological

studies are due to improper characterization of material itself or due to an inadequate or flawed

design of the study conducted (239). In fact, the vast number of applications and the enormous

amount of variables influencing the characteristics of the nanomaterials make particularly difficult the

elaboration and standardization of appropriate nanotoxicological protocols. Therefore, drugs and

diagnostics based on nanoparticles present challenges for regulatory agencies such as the FDA and

the EMA, which do not have specific guidance documents for nanoparticles per se. Indeed, there is

still no final consensus on which validated toxicological assays are appropriate and meaningful

enough in the case of nanoparticles, how they optionally have to be adapted to this material, or

whether new assays have to be developed and validated.

The existing medical regulations are expected to be applied to nano-enabled drugs and

diagnostics, but additional regulations, which consider their applicability to nanoparticles, may be

required. In United States, the FDA has formed in 2006 the Nanotechnology Task Force to facilitate

the regulation of nanoproducts. In addition, within the FDA´s National Center of Toxicological

Research (NCTR), there are nanotechnology programs which are focused on two main areas: (i)

evaluating the potential of nanomaterials in FDA-regulated products and (ii) modernizing toxicology

approaches to enhance product safety. In this context, The Nanotechnology Characterization

Laboratory has published a set of protocols relevant for the safety assessment of nanomedicines

(251). In EU, within the Seventh Framework Program (FP7) several projects, such as NanoTEST,

have been addressing the standardization of testing strategies for the safety assessment of

nanoparticles for medical applications (252). Furthermore, the European Academies Science

Advisory Council (EASAC) and the Joint Research Centre of the European Commission (JRC)

recently published a report entitled “Impact of Engineered Nanomaterials on Health: Considerations

for Benefit–Risk Assessment” (253, 254). The report seeks to identify gaps in current knowledge and

provides a set of recommendations for further research; the authors emphasize the importance of

‘safety-by-design’ for the successful implementation of the emerging nanotechnologies.

The International Organization for Standardization (ISO) has published several standards

that are relevant for the classification of engineered nanomaterials (255). In addition, the OECD has

launched the programme work named “Safety of Manufactured Nanomaterials” and within it has

established the OECD working Party on Nanotechnology (WPN) to advise upon emerging policy

issues of science, technology, and innovation related to the responsible development of

nanotechnology (256). WPN has published a list of physicochemical properties that should be fully

characterized for the different nanomaterials. However, the discussion about which properties are

essential to fully characterize and describe nanoparticles is not finished yet.


60

Therefore, the implementation of the regulatory mechanisms and standardized protocols to

address the risk associated to nanotechnology in United States and Europe as well as in the other

countries around the world is in progress. However, although numerous efforts have been carried out

in response to specific calls for proposal of nanosafety projects or by independent projects studies in

the various fields of nanotoxicology, there is still a lack of consistent data that need to be bridged. As

illustrated in Figure 8, this requires, as first main step, performing adequate characterization to

identify the potential risks associated to nanomedicines and thereby carry out nanoparticles

toxicological assessment through methods and protocols developed by the interdisciplinary work.

Thus, only a combination of efforts by the subjects with responsibility, researchers and public

authorities can adequately address the challenge of complexity proposed by nanotoxicology.

Integrate variousaproaches into testing

strategies

• Chemical composition

• Particle size and size distribution

• Morphology (crystalline/amorphous, shape)

• Surface chemistry, coating, functionalization

• Degree of agglomeration/aggregation

• Distribution under experimental conditions

(e.g. media with/without proteins)

•Surface reactivity (corona) and/or surface load (zeta potential).

Potential HazardsIdentification

Assess current toolsand their limitations

Nano-enabled productsdevelopment

(Safety by design)

Physico-ChemicalCharacterization

Nanotoxicity evaluation(in vitro/in vivo studies)

Adapt/Design new Protocols/Methods

(Adapt existing OECD guideilnes)

(NPs interference with assay systmes) (Academia, goverments, industry)

Figure 8. Schematic representation of the evaluation process of toxicity of nanoparticles for medical

purposes highlighting the importance of an adequate physic-chemical characterization to identify

potential risks.


61

6. References

1. S. Parveen, R. Misra, and S.K. Sahoo. Nanoparticles: a boon to drug delivery, therapeutics,

diagnostics and imaging. Nanomedicine. 8:147-166 (2011).

2. R. Bawa. Regulating nanomedicine - can the FDA handle it? Curr Drug Deliv. 8:227-234

(2011).

3. B. Godin, J.H. Sakamoto, R.E. Serda, A. Grattoni, A. Bouamrani, and M. Ferrari. Emerging

applications of nanomedicine for the diagnosis and treatment of cardiovascular diseases.

Trends Pharmacol Sci. 31:199-205 (2010).

4. I. Poirot-Mazeres. Legal aspects of the risks raised by nanotechnologies in the field of

medicine. J Int Bioethique. 22:99-118 (2011).

5. P.C. Chen, S.C. Mwakwari, and A.K. Oyelere. Gold nanoparticles: from nanomedicine to

nanosensing. Nanotech Sci Appl. 1:45-66 (2008).

6. R.P. Feynman. There`s plenty of room at the bottom. In: American Physical Society meeting.

Caltech. 23:22-26 (1960).

7. N. Taniguchi. On the basic concept of nanotechnology. In: Proceedings of the international

conference on production engineering, Part II, Japan Society of Precision Engineering.

Tokyo (1974).

8. G. Binnig, C.F. Quate, and C. Gerber. Atomic force microscope. Phys Rev Lett. 56:930-933

(1986).

9. G. Binnig, H. Rohrer, C. Gerber, and E. Weibel. Surface studies studies by scanning

tunneling microscopy. Phys Rev Lett. 49:57-61 (1982).

10. R. Bawa, S.R. Bawa, S.B. Maebius, T. Flynn, and C. Wei. Protecting new ideas and inventions

in nanomedicine with patents. Nanomedicine. 1:150-158 (2005).

11. G.A. Hodge, D.M. Bowman, and A.D. Maynard. International Handbook on Regulating

Nanotechnologies. Ed: Edward Elgar. Cheltenham, UK, (2010).

12. G. Birrenbach. Über Mizellpolymerisate, mögliche Einschlussverbindungen (Nanokapseln)

und deren Eignung als Adjuvantien, Dissertation, PhD Thesis, Nr. 5071, (1973).

13. G. Birrenbachand and P.P. Speiser. Polymerized micelles and their use as adjuvants in

immunology. J Pharm Sci. 65:1763-1766 (1976).

14. T.J. Webster. Nanomedicine: what's in a definition? Int J Nanomedicine. 1:115-116 (2006).

15. S. Parveen and S.K. Sahoo. Polymeric nanoparticles for cancer therapy. J Drug Target.

16:108-123 (2008).

16. S.K. Sahoo and V. Labhasetwar. Nanotech approaches to drug delivery and imaging. Drug

Discov Today. 8:1112-1120 (2003).

17. P. Couvreur and C. Vauthier. Nanotechnology: intelligent design to treat complex disease.

Pharm Res. 23:1417-1450 (2006).


62

18. L. Zhang, F.X. Gu, J.M. Chan, A.Z. Wang, R.S. Langer, and O.C. Farokhzad. Nanoparticles

in medicine: therapeutic applications and developments. Clin Pharmacol Ther. 83:761-769

(2008).

19. A.K. Cherian, A.C. Rana, and S.K. Jain. Self-assembled carbohydrate-stabilized ceramic

nanoparticles for the parenteral delivery of insulin. Drug Dev Ind Pharm. 26:459-463 (2000).

20. M. Manzano and M. Vallet-Regí. Revisiting bioceramics: bone regenerative and local drug

delivery systems. Prog Solid State Ch. 40:17-30 (2012).

21. Y.F. Zhang, Y.F. Zheng, and L. Qin. A comprehensive biological evaluation of ceramic

nanoparticles as wear debris. Nanomedicine. 7:975-982 (2011).

22. B. Fadeel and A.E. Garcia-Bennett. Better safe than sorry: understanding the toxicological

properties of inorganic nanoparticles manufactured for biomedical applications. Adv Drug

Deliv Rev. 62:362-374 (2010).

23. H.K. Sajja, M.P. East, H. Mao, Y.A. Wang, S. Nie, and L. Yang. Development of

multifunctional nanoparticles for targeted drug delivery and noninvasive imaging of

therapeutic effect. Curr Drug Discov Technol. 6:43-51 (2009).

24. M. Prato, K. Kostarelos, and A. Bianco. Functionalized carbon nanotubes in drug design and

discovery. Acc Chem Res. 41:60-68 (2008).

24. S. Bosi, T. Da Ros, G. Spalluto, and M. Prato. Fullerene derivatives: an attractive tool for

biological applications. Eur J Med Chem. 38:913-923 (2003).

26. D. Pantarotto, C.D. Partidos, R. Graff, J. Hoebeke, J.-P. Briand, M. Prato, and A. Bianco.

Synthesis, structural characterization, and immunological properties of carbon nanotubes

functionalized with peptides. J Am Chem Soc. 125:6160-6164 (2003).

27. S.A. Love, M.A. Maurer-Jones, J.W. Thompson, Y.S. Lin, and C.L. Haynes. Assessing

nanoparticle toxicity. Annu Rev Anal Chem (Palo Alto Calif). 5:181-205 (2012).

28. C.P. 3rd. Firme, and P.R. Bandaru. Toxicity issues in the application of carbon nanotubes to

biological systems. Nanomedicine. 6:245-256 (2010).

29. K.K. Jain. Nanodiagnostics: application of nanotechnology in molecular diagnostics. Expert

Rev Mol Diagn. 3:153-161 (2003).

30. A. Fernandez-Fernandez, R. Manchanda, and A.J. McGoron. Theranostic applications of

nanomaterials in cancer: drug delivery, image-guided therapy, and multifunctional platforms.

Appl Biochem Biotechnol. 165:1628-1651 (2011).

31. M.J. Kogan, I. Olmedo, L. Hosta, A.R. Guerrero, L.J. Cruz, and F. Albericio. Peptides and

metallic nanoparticles for biomedical applications. Nanomedicine (Lond). 2:287-306 (2007).

32. I.S. Chekman, Z.R. Ulberg, N.O. Gorchakova, T.Y. Nebesna, T.G. Gruzina, A.O. Priskoka,

A.M. Doroshenko, and P.V. Simonov. The prospects of medical application of metal-based

nanoparticles and nanomaterials. Lik Sprava. 1-2:3-21 (2011).


63

33. P. Ghosh, G. Han, M. De, C.K. Kim, and V.M. Rotello. Gold nanoparticles in delivery

applications. Adv Drug Deliv Rev. 60:1307-1315 (2008).

34. R.A. Sperling, P. Rivera Gil, F. Zhang, M. Zanella, and W.J. Parak. Biological applications of

gold nanoparticles. Chem Soc Rev. 37:1896-1908 (2008).

35. M. Colombo, S. Carregal-Romero, M.F. Casula, L. Gutierrez, M.P. Morales, I.B. Bohm, J.T.

Heverhagen, D. Prosperi, and W.J. Parak. Biological applications of magnetic nanoparticles.

Chem Soc Rev. 41:4306-4334 (2012).

36. J. Dobson. Magnetic micro- and nano-particle-based targeting for drug and gene delivery.

Nanomedicine (Lond). 1:31-37 (2006).

37. Y.F. Li and C. Chen. Fate and toxicity of metallic and metal-containing nanoparticles for

biomedical applications. Small. 7:2965-2980 (2011).

38. D.J. Bharali and S.A. Mousa. Emerging nanomedicines for early cancer detection and

improved treatment: current perspective and future promise. Pharmacol Ther. 128:324-335

(2010).

39. K.T. Yong, H. Ding, I. Roy, W.C. Law, E.J. Bergey, A. Maitra, and P.N. Prasad. Imaging

pancreatic cancer using bioconjugated InP quantum dots. ACS Nano. 3:502-510 (2009).

40. J. Gao, K. Chen, R. Xie, J. Xie, S. Lee, Z. Cheng, X. Peng, and X. Chen. Ultrasmall near-

infrared non-cadmium quantum dots for in vivo tumor imaging. Small. 6:256-261 (2010).

41. L.A. Bentolila, Y. Ebenstein, and S. Weiss. Quantum dots for in vivo small-animal imaging. J

Nucl Med. 50:493-496 (2009).

42. K.C. Halfpenny and D.W. Wright. Nanoparticle detection of respiratory infection. Wiley

Interdiscip Rev Nanomed Nanobiotechnol. 2:277-290 (2010).

43. J. Weng and J. Ren. Luminescent quantum dots: a very attractive and promising tool in

biomedicine. Curr Med Chem. 13:897-909 (2006).

44. J.M. Irache, I. Esparza, C. Gamazo, M. Agueros, and S. Espuelas. Nanomedicine: novel

approaches in human and veterinary therapeutics. Vet Parasitol. 180:47-71 (2011).

45. W.T. Al-Jamal and K. Kostarelos. Liposomes: from a clinically established drug delivery

system to a nanoparticle platform for theranostic nanomedicine. Acc Chem Res. 44:1094-

1104 (2011).

46. A.D. Bangham, M.M. Standish, and J.C. Watkins. Diffusion of univalent ions across the

lamellae of swollen phospholipids. J Mol Biol. 13:238-252 (1965).

47. J. Kreuter. Nanoparticles and nanocapsules-new dosage forms in the nanometer size range.

Pharm Acta Helv. 53:33-39 (1978).

48. J.J. Marty, R.C. Oppenheim, and P. Speiser. Nanoparticles-a new colloidal drug delivery

system. Pharm Acta Helv. 53:17-23 (1978).


64

49. R.H. Muller and C.M. Keck. Challenges and solutions for the delivery of biotech drugs-a

review of drug nanocrystal technology and lipid nanoparticles. J Biotechnol. 113:151-170

(2004).

50. S.L. Huang. Liposomes in ultrasonic drug and gene delivery. Adv Drug Deliv Rev. 60:1167-

1176 (2008).

51. D.D. Lasic. Novel applications of liposomes. Trends Biotechnol. 16:307-321 (1998).

52. V.P. Torchilin. Recent advances with liposomes as pharmaceutical carriers. Nat Rev Drug

Discov. 4:145-160 (2005).

53. R.D. Hofheinz, S.U. Gnad-Vogt, U. Beyer, and A. Hochhaus. Liposomal encapsulated anti-

cancer drugs. Anticancer Drugs. 16:691-707 (2005).

54. I.A. Bakker-Woudenberg. Long-circulating sterically stabilized liposomes as carriers of agents

for treatment of infection or for imaging infectious foci. Int J Antimicrob Agents. 19:299-

311 (2002).

55. E.T. Dams, W.J. Oyen, O.C. Boerman, G. Storm, P. Laverman, P.J. Kok, W.C. Buijs, H.

Bakker, J.W. van der Meer, and F.H. Corstens. 99mTc-PEG liposomes for the scintigraphic

detection of infection and inflammation: clinical evaluation. J Nucl Med. 41:622-630 (2000).

56. S. Dow. Liposome-nucleic acid immunotherapeutics. Expert Opin Drug Deliv. 5:11-24

(2008).

57. N. Weiner, L. Lieb, S. Niemiec, C. Ramachandran, Z. Hu, and K. Egbaria. Liposomes: a

novel topical delivery system for pharmaceutical and cosmetic applications. J Drug Target.

2:405-410 (1994).

58. J. Szebeni, C.R. Alving, L. Rosivall, R. Bunger, L. Baranyi, P. Bedocs, M. Toth, and Y.

Barenholz. Animal models of complement-mediated hypersensitivity reactions to liposomes

and other lipid-based nanoparticles. J Liposome Res. 17:107-117 (2007).

59. N.T. Huynh, C. Passirani, P. Saulnier, and J.P. Benoit. Lipid nanocapsules: a new platform

for nanomedicine. Int J Pharm. 379:201-209 (2009).

60. S. David, P. Resnier, A. Guillot, B. Pitard, J.P. Benoit, and C. Passirani. siRNA LNCs-a novel

platform of lipid nanocapsules for systemic siRNA administration. Eur J Pharm Biopharm.

81:448-452 (2012).

61. M. Morille, C. Passirani, S. Dufort, G. Bastiat, B. Pitard, J.L. Coll, and J.P. Benoit. Tumor

transfection after systemic injection of DNA lipid nanocapsules. Biomaterials. 32:2327-2333

(2011).

62. M. Morille, C. Passirani, E. Letrou-Bonneval, J.P. Benoit, and B. Pitard. Galactosylated DNA

lipid nanocapsules for efficient hepatocyte targeting. Int J Pharm. 379:293-300 (2009).

63. D. Peer, J.M. Karp, S. Hong, O.C. Farokhzad, R. Margalit, and R. Langer. Nanocarriers as an

emerging platform for cancer therapy. Nat Nanotechnol. 2:751-760 (2007).

64. V.P. Torchilin. Multifunctional nanocarriers. Adv Drug Deliv Rev. 58:1532-1555 (2006).


65

65. B. Heurtault, P. Saulnier, B. Pech, J.E. Proust, and J.P. Benoit. A novel phase inversion-

based process for the preparation of lipid nanocarriers. Pharm Res. 19:875-880 (2002).

66. B. Heurtault, P. Saulnier, B. Pech, M.C. Venier-Julienne, J.E. Proust, R. Phan-Tan-Luu, and

J.P. Benoit. The influence of lipid nanocapsule composition on their size distribution. Eur J

Pharm Sci. 18:55-61 (2003).

67. D.K. Sarker. Engineering of nanoemulsions for drug delivery. Curr Drug Deliv. 2:297-310

(2005).

68. Y. Lu, J. Qi, and W. Wu. Absorption, disposition and pharmacokinetics of nanoemulsions.

Curr Drug Metab. 13:396-417 (2012).

69. R.H. Müller and J.S. Lucks. Arzneistoffträger aus festen Lipidteilchen, Feste

Lipidnanosphären (SLN), European Patent Nº 0605497, 1996.

70. M.R. Gasco. Method for producing solid lipid microspheres having a narrow size

distribution, US Patent 5 250 236, 1993.

71. R.H. Müller, K. Mader, and S. Gohla. Solid lipid nanoparticles (SLN) for controlled drug

delivery - a review of the state of the art. Eur J Pharm Biopharm. 50:161-177 (2000).

72. M.D. Joshi and R.H. Muller. Lipid nanoparticles for parenteral delivery of actives. Eur J

Pharm Biopharm. 71:161-172 (2009).

73. H. Bunjes. Lipid nanoparticles for the delivery of poorly water-soluble drugs. J Pharm

Pharmacol. 62:1637-1645 (2010).

74. R. Cavalli, M.R. Gasco, P. Chetoni, S. Burgalassi, and M.F. Saettone. Solid lipid nanoparticles

(SLN) as ocular delivery system for tobramycin. Int J Pharm. 238:241-245 (2002).

75. W. Mehnert and K. Mäder. Solid lipid nanoparticles: production, characterization and

applications. Adv Drug Deliv Rev. 47:165-196 (2001).

76. M.A. Videira, M.F. Botelho, A.C. Santos, L.F. Gouveia, J.J. Pedroso de Lima, and A.n.J.

Almeida. Lymphatic uptake of pulmonary delivered radiolabelled solid lipid nanoparticles. J

Drug Target. 10:607-613 (2002).

77. J. Pardeike, A. Hommoss, and R.H. Muller. Lipid nanoparticles (SLN, NLC) in cosmetic and

pharmaceutical dermal products. Int J Pharm. 366:170-184 (2009).

78. R. Duncan. Polymer therapeutics as nanomedicines: new perspectives. Curr Opin

Biotechnol. 22:492-501 (2011).

79. H. Ringsdorf. Structure and properties of pharmacologically active polymers. J Polym Sci

Symp. 51:135-153 (1975).

80. R. Duncan and J. Kopecek. Soluble synthetic-polymers as potential-drug carriers. Adv Polym

Sci. 57:51-101 (1984).

81. R. Duncan. Drug polymer conjugates-potential for improved chemotherapy. Anti-cancer

Drug. 3:175-210 (1992).


66

82. R. Duncan. The dawning era of polymer therapeutics. Nat Rev Drug Discov. 2:347-360

(2003).

83. A. Abuchowski, J.R. McCoy, N.C. Palczuk, T. van Es, and F.F. Davis. Effect of covalent

attachment of polyethylene glycol on immunogenicity and circulating life of bovine liver

catalase. J Biol Chem. 252:3582-3586 (1977).

84. P. Caliceti and F.M. Veronese. Pharmacokinetic and biodistribution properties of

poly(ethylene glycol)-protein conjugates. Adv Drug Deliv Rev. 55:1261-1277 (2003).

85. J.M. Harris and R.B. Chess. Effect of pegylation on pharmaceuticals. Nat Rev Drug Discov.

2:214-221 (2003).

86. K.L. Heredia and H.D. Maynard. Synthesis of protein-polymer conjugates. Org Biomol

Chem. 5:45-53 (2007).

87. G.N. Grover and H.D. Maynard. Protein-polymer conjugates: synthetic approaches by

controlled radical polymerizations and interesting applications. Curr Opin Chem Biol.

14:818-827 (2010).

88. C.M. Paleos, D. Tsiourvas, Z. Sideratou, and L.A. Tziveleka. Drug delivery using

multifunctional dendrimers and hyperbranched polymers. Expert Opin Drug Deliv. 7:1387-

1398 (2010).

89. B. Klajnert and M. Bryszewska. Dendrimers: properties and applications. Acta Biochim Pol.

48:199-208 (2001).

90. T. Sakthivel and A.T. Florence. Adsorption of amphipathic dendrons on polystyrene

nanoparticles. Int J Pharm. 254:23-26 (2003).

91. Y. Cheng, Z. Xu, M. Ma, and T. Xu. Dendrimers as drug carriers: applications in different

routes of drug administration. J Pharm Sci. 97:123-143 (2008).

92. J.M. Frechet. Functional polymers and dendrimers: reactivity, molecular architecture, and

interfacial energy. Science. 263:1710-1715 (1994).

93. T.F. Vandamme and L. Brobeck. Poly(amidoamine) dendrimers as ophthalmic vehicles for

ocular delivery of pilocarpine nitrate and tropicamide. J Control Release. 102:23-38 (2005).

94. A. Bielinska, J.F. Kukowska-Latallo, J. Johnson, D.A. Tomalia, and J.R. Baker, Jr. Regulation

of in vitro gene expression using antisense oligonucleotides or antisense expression plasmids

transfected using starburst PAMAM dendrimers. Nucleic Acids Res. 24:2176-2182 (1996).

95. M.A. Mintzer and M.W. Grinstaff. Biomedical applications of dendrimers: a tutorial. Chem

Soc Rev. 40:173-190 (2011).

96. S. Akhtar, B. Chandrasekhar, S. Attur, M.H.M. Yousif, and I.F. Benter. On the nanotoxicity

of PAMAM dendrimers: Superfect® stimulates the EGFR–ERK1/2 signal transduction

pathway via an oxidative stress-dependent mechanism in HEK 293 cells. Int J Pharm.

448:239-246 (2013).


67

97. K. Kataoka, A. Harada, and Y. Nagasaki. Block copolymer micelles for drug delivery: design,

characterization and biological significance. Adv Drug Deliv Rev. 47:113-131 (2001).

98. V.P. Torchilin. Micellar nanocarriers: pharmaceutical perspectives. Pharm Res. 24:1-16

(2007).

99. S.M. Moghimi, A.C. Hunter, and J.C. Murray. Long-circulating and target-specific

nanoparticles: theory to practice. Pharmacol Rev. 53:283-318 (2001).

100. R. Vakil and G.S. Kwon. Poly(ethylene glycol)-b-poly(epsilon-caprolactone) and PEG-

phospholipid form stable mixed micelles in aqueous media. Langmuir. 22:9723-9729 (2006).

101. Z. Sezgin, N. Yuksel, and T. Baykara. Preparation and characterization of polymeric micelles

for solubilization of poorly soluble anticancer drugs. Eur J Pharm Biopharm. 64:261-268

(2006).

102. S.K. Agrawal, N. Sanabria-DeLong, J.M. Coburn, G.N. Tew, and S.R. Bhatia. Novel drug

release profiles from micellar solutions of PLA-PEO-PLA triblock copolymers. J Control

Release. 112:64-71 (2006).

103. N. Nishiyama and K. Kataoka. Current state, achievements, and future prospects of

polymeric micelles as nanocarriers for drug and gene delivery. Pharmacol Ther. 112:630-648

(2006).

104. C. Tros de Ilarduya, Y. Sun, and N. Duzgunes. Gene delivery by lipoplexes and polyplexes.

Eur J Pharm Sci. 40:159-170 (2010).

105. H. Mokand, and T.G. Park. Functional polymers for targeted delivery of nucleic acid drugs.

Macromol Biosci. 9:731-743 (2009).

106. J.W. Kleber, J.F. Nash, and C.C. Lee. Synthetic polymers as potential sustained-release

coatings. J Pharm Sci. 53:1519-1521 (1964).

107. I. Zolle, F. Hosain, B.A. Rhodes, and H.N. Wagner. Human serum albumin

millimcrospheres for studies of reticuloendothelial system. J Nucl Med. 11:379-380 (1970).

108. P. Couvreur, B. Kante, M. Roland, P. Guiot, P. Bauduin, and P. Speiser. Polycyanoacrylate

nanocapsules as potential lysosomotropic carriers: preparation, morphological and sorptive

properties. J Pharm Pharmacol. 31:331-332 (1979).

109. B. Ekman, C. Lofter, and I. Sjoholm. Incorporation of macromolecules in microparticles:

preparation and characteristics. Biochemistry. 15:5115-5120 (1976).

110. R. Gurny, N.A. Peppas, D.D. Harrington, and G.S. Banker. Development of biodegradable

and injectable latices for controlled release of potent drugs. Drug Dev Ind Pharm. 7:1-25

(1981).

111. J. Kreuter. Drug targeting with nanoparticles. Eur J Drug Metab Pharmacokinet. 19:253-256

(1994).

112. T. Kubik, K. Bogunia-Kubik, and M. Sugisaka. Nanotechnology on duty in medical

applications. Curr Pharm Biotechnol. 6:17-33 (2005).


68

113. J.K. Vasir, M.K. Reddy, and V.D. Labhasetwar. Nanosystems in drug targeting: opportunities

and challenges. Curr Nanosci. 1:47-64 (2005).

114. F. Chiellini. Perspectives on in vitro evaluation of biomedical polymers. J Bioact Compat Pol.

21:257-271 (2006).

115. A. Kumari, S.K. Yadav, and S.C. Yadav. Biodegradable polymeric nanoparticles based drug

delivery systems. Colloids Surf B Biointerfaces. 75:1-18 (2010).

116. J.H. Park, M. Ye, and K. Park. Biodegradable polymers for microencapsulation of drugs.

Molecules. 10:146-161 (2005).

117. D.H. Lee and I.J. Kang. Drug delivery system using biodegradable nanoparticle carrier.

Kona. 24:159-166 (2006).

118. K.S. Soppimath, T.M. Aminabhavi, A.R. Kulkarni, and W.E. Rudzinski. Biodegradable

polymeric nanoparticles as drug delivery devices. J Control Release. 70:1-20 (2001).

119. R. Jayakumar, N. Nwe, S. Tokura, and H. Tamura. Sulfated chitin and chitosan as novel

biomaterials. Int J Biol Macromol. 40:175-181 (2007).

120. K. Bowman and K.W. Leong. Chitosan nanoparticles for oral drug and gene delivery. Int J

Nanomedicine. 1:117-128 (2006).

121. A. Masotti and G. Ortaggi. Chitosan micro- and nanospheres: fabrication and applications

for drug and DNA delivery. Mini Rev Med Chem. 9:463-469 (2009).

122. K. Nagpal, S.K. Singh, and D.N. Mishra. Chitosan nanoparticles: a promising system in

novel drug delivery. Chem Pharm Bull (Tokyo). 58:1423-1430 (2010).

123. I.M. van der Lubben, J.C. Verhoef, G. Borchard, and H.E. Junginger. Chitosan for mucosal

vaccination. Adv Drug Deliv Rev. 52:139-144 (2001).

124. A.O. Elzoghby, W.S. Abo El-Fotoh, and N.A. Elgindy. Casein-based formulations as

promising controlled release drug delivery systems. J Control Release. 153:206-216 (2011).

125. A.J. Kuijpers, P.B. van Wachem, M.J.A. van Luyn, L.A. Brouwer, G.H.M. Engbers, J.

Krijgsveld, S.A.J. Zaat, J. Dankert, and J. Feijen. In vitro and in vivo evaluation of gelatin-

chondroitin sulphate hydrogels for controlled release of antibacterial proteins. Biomaterials.

21:1763-1772 (2000).

126. S. Kommareddy and M. Amiji. Preparation and evaluation of thiol-modified gelatin

nanoparticles for intracellular DNA delivery in response to glutathione. Bioconjug Chem.

16:1423-1432 (2005).

127. K. Langer, S. Balthasar, V. Vogel, N. Dinauer, H. von Briesen, and D. Schubert.

Optimization of the preparation process for human serum albumin (HSA) nanoparticles. Int

J Pharm. 257:169-180 (2003).

128. O. Rubino, R. Kowalsky, and J. Swarbrick. Albumin microspheres as a drug delivery system:

relation among turbidity ratio, degree of cross-linking, and drug release. Pharm Res. 10:1059-

1065 (1993).


69

129. F. Kratz, I. Fichtner, U. Beyer, P. Schumacher, T. Roth, H.H. Fiebig, and C. Unger.

Antitumour activity of acid labile transferrin and albumin doxorubicin conjugates in in vitro

and in vivo human tumour xenograft models. Eur J Cancer. 33, Supplement 8:S175 (1997).

130. A. Arnedo, J.M. Irache, M. Merodio, and M.S. Espuelas Millan. Albumin nanoparticles

improved the stability, nuclear accumulation and anticytomegaloviral activity of a

phosphodiester oligonucleotide. J Control Release. 94:217-227 (2004).

131. A. Maham, Z. Tang, H. Wu, J. Wang, and Y. Lin. Protein-based nanomedicine platforms for

drug delivery. Small. 5:1706-1721 (2009).

132. A.O. Elzoghby, W.M. Samy, and N.A. Elgindy. Albumin-based nanoparticles as potential

controlled release drug delivery systems. J Controlled Release. 157:168-182 (2012).

133. A.C. Albertsson and I.K. Varma. Aliphatic polyesters: synthesis, properties and applications.

In: Degradable Aliphatic Polyesters.Ed: Springer Berlin Heidelberg. 157:1-40 (2002).

134. R.A. Jain. The manufacturing techniques of various drug loaded biodegradable poly(lactide-

co-glycolide) (PLGA) devices. Biomaterials. 21:2475-2490 (2000).

135. R.C. Mundargi, V.R. Babu, V. Rangaswamy, P. Patel, and T.M. Aminabhavi. Nano/micro

technologies for delivering macromolecular therapeutics using poly(D,L-lactide-co-glycolide)

and its derivatives. J Control Release. 125:193-209 (2008).

136. M.S. Shive and J.M. Anderson. Biodegradation and biocompatibility of PLA and PLGA

microspheres. Adv Drug Deliv Rev. 28:5-24 (1997).

137. H. Tamber, P. Johansen, H.P. Merkle, and B. Gander. Formulation aspects of biodegradable

polymeric microspheres for antigen delivery. Adv Drug Deliv Rev. 57:357-376 (2005).

138. J.M. Lu, X. Wang, C. Marin-Muller, H. Wang, P.H. Lin, Q. Yao, and C. Chen. Current

advances in research and clinical applications of PLGA-based nanotechnology. Expert Rev

Mol Diagn. 9:325-341 (2009).

139. P. Johansen, Y. Men, H.P. Merkle, and B. Gander. Revisiting PLA/PLGA microspheres: an

analysis of their potential in parenteral vaccination. Eur J Pharm Biopharm. 50:129-146

(2000).

140. S.K. Mallapragada and B. Narasimhan. Immunomodulatory biomaterials. Int J Pharm.

364:265-271 (2008).

141. M. Murillo, C. Gamazo, J.M. Irache, and M.M. Goni. Polyester microparticles as a vaccine

delivery system for brucellosis: influence of the polymer on release, phagocytosis and

toxicity. J Drug Target. 10:211-219 (2002).

142. V.R. Sinha, K. Bansal, R. Kaushik, R. Kumria, and A. Trehan. Poly-epsilon-caprolactone

microspheres and nanospheres: an overview. Int J Pharm. 278:1-23 (2004).

143. R. Da Costa Martins, J.M. Irache, J.M. Blasco, M.P. Munoz, C.M. Marin, M. Jesus Grillo, M.

Jesus De Miguel, M. Barberan, and C. Gamazo. Evaluation of particulate acellular vaccines

against Brucella ovis infection in rams. Vaccine. 28:3038-3046 (2010).


70

144. J. Nicolas and P. Couvreur. Synthesis of poly(alkyl cyanoacrylate)-based colloidal

nanomedicines. Wiley Interdiscip Rev Nanomed Nanobiotechnol. 1:111-127 (2009).

145. C. Vauthier, C. Dubernet, C. Chauvierre, I. Brigger, and P. Couvreur. Drug delivery to

resistant tumors: the potential of poly(alkyl cyanoacrylate) nanoparticles. J Control Release.

93:151-160 (2003).

146. Y. Mo, G.E. Peck, A. Heyd, and G.S. Banker. Recording pH method of characterizing

composition and monitoring dissolution profile of an anhydride-acid copolymer and its salt

derivatives. J Pharm Sci. 66:713-717 (1977).

147. L.C. Lappas and W. McKeehan. Polymeric pharmaceutical coating materials. I. preparation

and properties. J Pharm Sci. 54:176-181 (1965).

148. P. Arbos, M. Wirth, M.A. Arangoa, F. Gabor, and J.M. Irache. Gantrez AN as a new

polymer for the preparation of ligand-nanoparticle conjugates. J Control Release. 83:321-330

(2002).

149. P. Arbos, M.A. Campanero, M.A. Arangoa, and J.M. Irache. Nanoparticles with specific

bioadhesive properties to circumvent the pre-systemic degradation of fluorinated

pyrimidines. J Control Release. 96:55-65 (2004).

150. M. Agueros, M.A. Campanero, and J.M. lrache. Simultaneous quantification of different

cyclodextrins and Gantrez by HPLC with evaporative light scattering detection. J Pharm

Biomed Anal. 39:495-502 (2005).

151. K. Yoncheva, S. Gomez, M.A. Campanero, C. Gamazo, and J.M. Irache. Bioadhesive

properties of pegylated nanoparticles. Expert Opin Drug Deliv. 2:205-218 (2005).

152. K. Yoncheva, E. Lizarraga, and J.M. Irache. Pegylated nanoparticles based on poly(methyl

vinyl ether-co-maleic anhydride): preparation and evaluation of their bioadhesive properties.

Eur J Pharm Sci. 24:411-419 (2005).

153. P. Arbos, M.A. Campanero, M.A. Arangoa, M.J. Renedo, and J.M. Irache. Influence of the

surface characteristics of PVM/MA nanoparticles on their bioadhesive properties. J Control

Release. 89:19-30 (2003).

154. H.H. Salman, C. Gamazo, M.A. Campanero, and J.M. Irache. Salmonella-like bioadhesive

nanoparticles. J Control Release. 106:1-13 (2005).

155. S. Gomez, C. Gamazo, B. San Roman, M. Ferrer, M.L. Sanz, S. Espuelas, and J.M. Irache.

Allergen immunotherapy with nanoparticles containing lipopolysaccharide from Brucella ovis.

Eur J Pharm Biopharm. 70:711-717 (2008).

156. H.H. Salman, C. Gamazo, M. Agueros, and J.M. Irache. Bioadhesive capacity and

immunoadjuvant properties of thiamine-coated nanoparticles. Vaccine. 25:8123-8132 (2007).

157. H.H. Salman, C. Gamazo, M.A. Campanero, and J.M. Irache. Bioadhesive mannosylated

nanoparticles for oral drug delivery. J Nanosci Nanotechnol. 6:3203-3209 (2006).


71

158. H.H. Salman, C. Gamazo, P.C. de Smidt, G. Russell-Jones, and J.M. Irache. Evaluation of

bioadhesive capacity and immunoadjuvant properties of vitamin B(12)-Gantrez

nanoparticles. Pharm Res. 25:2859-2868 (2008).

159. H. Fessi, F. Puisieux, J.P. Devissaguet, N. Ammoury, and S. Benita. Nanocapsule formation

by interfacial polymer deposition following solvent displacement. Int J Pharm. 55:R1-R4

(1989).

160. X. Rong, Y. Xie, X. Hao, T. Chen, Y. Wang, and Y. Liu. Applications of polymeric

nanocapsules in field of drug delivery systems. Curr Drug Discov Technol. 8:173-187 (2011).

161. A.C. Lima, P. Sher, and J.F. Mano. Production methodologies of polymeric and hydrogel

particles for drug delivery applications. Expert Opin Drug Deliv. 9:231-248 (2012).

162. N. Anton, J.-P. Benoit, and P. Saulnier. Design and production of nanoparticles formulated

from nano-emulsion templates - a review. J Controlled Release. 128:185-199 (2008).

163. G. Dalwadi and V.B. Sunderland. Purification of PEGylated nanoparticles using tangential

flow filtration (TFF). Drug Dev Ind Pharm. 33:1030-1039 (2007).

164. R. Da Costa Martins, C. Gamazo, and J.M. Irache. Design and influence of gamma-

irradiation on the biopharmaceutical properties of nanoparticles containing an antigenic

complex from Brucella ovis. Eur J Pharm Sci. 37:563-572 (2009).

165. C. Vauthier and K. Bouchemal. Methods for the preparation and manufacture of polymeric

nanoparticles. Pharml Res. 26:1025-1058 (2009).

166. S. Shugarts and L.Z. Benet. The role of transporters in the pharmacokinetics of orally

administered drugs. Pharm Res. 26:2039-2054 (2009).

167. S.K. Lai, Y.-Y. Wang, and J. Hanes. Mucus-penetrating nanoparticles for drug and gene

delivery to mucosal tissues. Adv Drug Deliv Rev. 61:158-171 (2009).

168. B.C. Tang, M. Dawson, S.K. Lai, Y.-Y. Wang, J.S. Suk, M. Yang, P. Zeitlin, M.P. Boyle, J. Fu,

and J. Hanes. Biodegradable polymer nanoparticles that rapidly penetrate the human mucus

barrier. Proc Natl Acad of Sci USA. 106:19268-19273 (2009).

169. Y.B. Huang, W. Leobandung, A. Foss, and N.A. Peppas. Molecular aspects of muco- and

bioadhesion: tethered structures and site-specific surfaces. J Controlled Release. 65:63-71

(2000).

170. Y.-Y. Wang, S.K. Lai, J.S. Suk, A. Pace, R. Cone, and J. Hanes. Addressing the PEG

mucoadhesivity paradox to engineer nanoparticles that "slip" through the human mucus

barrier. Angew Chem Int Ed Engl. 47:9726-9729 (2008).

171. J.S. Crater and R.L. Carrier. Barrier properties of gastrointestinal mucus to nanoparticle

transport. Macromol Biosci. 10:1473-1483 (2010).

172. M.A. Deli. Potential use of tight junction modulators to reversibly open membranous

barriers and improve drug delivery. Biochim Biophys Acta. 1788:892-910 (2009).


72

173. S.D. Conner and S.L. Schmid. Regulated portals of entry into the cell. Nature. 422:37-44

(2003).

174. A. des Rieux, V. Fievez, I. Theate, J. Mast, V. Preat, and Y.-J. Schneider. An improved in vitro

model of human intestinal follicle-associated epithelium to study nanoparticle transport by M

cells. Eur J Pharm Sci. 30:380-391 (2007).

175. M.C. Chen, K. Sonaje, K.J. Chen, and H.W. Sung. A review of the prospects for polymeric

nanoparticle platforms in oral insulin delivery. Biomaterials. 32:9826-9838 (2011).

176. J.M. Irache, C. Durrer, D. Duchene, and G. Ponchel. Preparation and characterization of

lectin-latex conjugates for specific bioadhesion. Biomaterials. 15:899-904 (1994).

177. H.H. Salman, J.M. Irache, and C. Gamazo. Immunoadjuvant capacity of flagellin and

mannosamine-coated poly(anhydride) nanoparticles in oral vaccination. Vaccine. 27:4784-

4790 (2009).

178. H. Maeda, J. Wu, T. Sawa, Y. Matsumura, and K. Hori. Tumor vascular permeability and the

EPR effect in macromolecular therapeutics: a review. J Control Release. 65:271-284 (2000).

179. X. Wang, L. Yang, Z.G. Chen, and D.M. Shin. Application of nanotechnology in cancer

therapy and imaging. CA Cancer J Clin. 58:97-110 (2008).

180. H. Maeda. The enhanced permeability and retention (EPR) effect in tumor vasculature: the

key role of tumor-selective macromolecular drug targeting. Adv Enzyme Regul. 41:189-207

(2001).

181. S. Acharya and S.K. Sahoo. PLGA nanoparticles containing various anticancer agents and

tumour delivery by EPR effect. Adv Drug Deliv Rev. 63:170-183 (2011).

182. H. Maeda, T. Oda, Y. Matsumura, and M. Kimura. Improvement of pharmacological

properties of protein-drugs by tailoring with synthetic polymers. J Bioact Compat Polym.

3:27-43 (1988).

183. H. Maeda, H. Nakamura, and J. Fang. The EPR effect for macromolecular drug delivery to

solid tumors: improvement of tumor uptake, lowering of systemic toxicity, and distinct

tumor imaging in vivo. Adv Drug Deliv Rev. 65:71-79 (2013).

184. Y. Matsumura and H. Maeda. A new concept for macromolecular therapeutics in cancer

chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent

smancs. Cancer Res. 46:6387-6392 (1986).

185. V. Bhardwaj, D.D. Ankola, S.C. Gupta, M. Schneider, C.M. Lehr, and M.N. Kumar. PLGA

nanoparticles stabilized with cationic surfactant: safety studies and application in oral delivery

of paclitaxel to treat chemical-induced breast cancer in rat. Pharm Res. 26:2495-2503 (2009).

186. N. Chiannilkulchai, N. Ammoury, B. Caillou, J.P. Devissaguet, and P. Couvreur. Hepatic

tissue distribution of doxorubicin-loaded nanoparticles after i.v. administration in

reticulosarcoma M 5076 metastasis-bearing mice. Cancer Chemother Pharmacol. 26:122-126

(1990).


73

187. E. Jager, A. Jager, P. Chytil, T. Etrych, B. Rihova, F.C. Giacomelli, P. Stepanek, and K.

Ulbrich. Combination chemotherapy using core-shell nanoparticles through the self-

assembly of HPMA-based copolymers and degradable polyester. J Control Release. 165:153-

161 (2013).

188. W.J. Gradishar, S. Tjulandin, N. Davidson, H. Shaw, N. Desai, P. Bhar, M. Hawkins, and J.

O'Shaughnessy. Phase III trial of nanoparticle albumin-bound paclitaxel compared with

polyethylated castor oil-based paclitaxel in women with breast cancer. J Clin Oncol. 23:7794-

7803 (2005).

189. M.J. Hawkins, P. Soon-Shiong, and N. Desai. Protein nanoparticles as drug carriers in clinical

medicine. Adv Drug Deliv Rev. 60:876-885 (2008).

190. N.K. Ibrahim, N. Desai, S. Legha, P. Soon-Shiong, R.L. Theriault, E. Rivera, B. Esmaeli, S.E.

Ring, A. Bedikian, G.N. Hortobagyi, and J.A. Ellerhorst. Phase I and pharmacokinetic study

of ABI-007, a Cremophor-free, protein-stabilized, nanoparticle formulation of paclitaxel.

Clin Cancer Res. 8:1038-1044 (2002).

191. M.R. Green, G.M. Manikhas, S. Orlov, B. Afanasyev, A.M. Makhson, P. Bhar, and M.J.

Hawkins. Abraxane, a novel Cremophor-free, albumin-bound particle form of paclitaxel for

the treatment of advanced non-small-cell lung cancer. Ann Oncol. 17:1263-1268 (2006).

192. D.W. Nyman, K.J. Campbell, E. Hersh, K. Long, K. Richardson, V. Trieu, N. Desai, M.J.

Hawkins, and D.D. Von Hoff. Phase I and pharmacokinetics trial of ABI-007, a novel

nanoparticle formulation of paclitaxel in patients with advanced nonhematologic

malignancies. J Clin Oncol. 23:7785-7793 (2005).

193. S.L. Illum and S.S. Davis. Effect of the nonionic surfactant poloxamer 338 on the fate and

deposition of polystyrene microspheres following intravenous administration. J Pharm Sci.

72:1086-1089 (1983).

194. L. Illum, S.S. Davis, R.H. Muller, E. Mak, and P. West. The organ distribution and

circulation time of intravenously injected colloidal carriers sterically stabilized with a block

copolymer-poloxamine 908. Life Sci. 40:367-374 (1987).

195. R. Gref, Y. Minamitake, M.T. Peracchia, V. Trubetskoy, V. Torchilin, and R. Langer.

Biodegradable long-circulating polymeric nanospheres. Science. 263:1600-1603 (1994).

196. Y. Cho and R.B. Borgens. Polymer and nano-technology applications for repair and

reconstruction of the central nervous system. Exp Neurol. 233:126-144 (2012).

197. J.V. Jokerst, T. Lobovkina, R.N. Zare, and S.S. Gambhir. Nanoparticle PEGylation for

imaging and therapy. Nanomedicine (Lond). 6:715-728 (2011).

198. M.L. Adams, A. Lavasanifar, and G.S. Kwon. Amphiphilic block copolymers for drug

delivery. J Pharm Sci. 92:1343-1355 (2003).

199. T.Y. Kim, D.W. Kim, J.Y. Chung, S.G. Shin, S.C. Kim, D.S. Heo, N.K. Kim, and Y.J. Bang.

Phase I and pharmacokinetic study of Genexol-PM, a cremophor-free, polymeric micelle-


74

formulated paclitaxel, in patients with advanced malignancies. Clin Cancer Res. 10:3708-3716

(2004).

200. S.R. Park, D.Y. Oh, D.W. Kim, T.Y. Kim, D.S. Heo, Y.J. Bang, N.K. Kim, W.K. Kang, H.T.

Kim, S.A. Im, J.H. Suh, and H.K. Kim. A multi-center, late phase II clinical trial of Genexol

(paclitaxel) and cisplatin for patients with advanced gastric cancer. Oncol Rep. 12:1059-1064

(2004).

201. C. Oerlemans, W. Bult, M. Bos, G. Storm, J.F. Nijsen, and W.E. Hennink. Polymeric

micelles in anticancer therapy: targeting, imaging and triggered release. Pharm Res. 27:2569-

2589 (2010).

202. K.S. Lee, H.C. Chung, S.A. Im, Y.H. Park, C.S. Kim, S.B. Kim, S.Y. Rha, M.Y. Lee, and J.

Ro. Multicenter phase II trial of Genexol-PM, a Cremophor-free, polymeric micelle

formulation of paclitaxel, in patients with metastatic breast cancer. Breast Cancer Res Treat.

108:241-250 (2008).

203. D.W. Kim, S.Y. Kim, H.K. Kim, S.W. Kim, S.W. Shin, J.S. Kim, K. Park, M.Y. Lee, and D.S.

Heo. Multicenter phase II trial of Genexol-PM, a novel Cremophor-free, polymeric micelle

formulation of paclitaxel, with cisplatin in patients with advanced non-small-cell lung cancer.

Ann Oncol. 18:2009-2014 (2007).

204. S.H. Kim, J.H. Jeong, K.W. Chun, and T.G. Park. Target-specific cellular uptake of PLGA

nanoparticles coated with poly(L-lysine)-poly(ethylene glycol)-folate conjugate. Langmuir.

21:8852-8857 (2005).

205. N. Graf, D.R. Bielenberg, N. Kolishetti, C. Muus, J. Banyard, O.C. Farokhzad, and S.J.

Lippard. alpha(V)beta(3) integrin-targeted PLGA-PEG nanoparticles for enhanced anti-

tumor efficacy of a Pt(IV) prodrug. ACS Nano. 6:4530-4539 (2012).

206. H.L. Jiang, J.T. Kwon, Y.K. Kim, E.M. Kim, R. Arote, H.J. Jeong, J.W. Nah, Y.J. Choi, T.

Akaike, M.H. Cho, and C.S. Cho. Galactosylated chitosan-graft-polyethylenimine as a gene

carrier for hepatocyte targeting. Gene Ther. 14:1389-1398 (2007).

207. M.O. Oyewumi, R.A. Yokel, M. Jay, T. Coakley, and R.J. Mumper. Comparison of cell

uptake, biodistribution and tumor retention of folate-coated and PEG-coated gadolinium

nanoparticles in tumor-bearing mice. J Control Release. 95:613-626 (2004).

208. B. Stella, S. Arpicco, M.T. Peracchia, D. Desmaele, J. Hoebeke, M. Renoir, J. D'Angelo, L.

Cattel, and P. Couvreur. Design of folic acid-conjugated nanoparticles for drug targeting. J

Pharm Sci. 89:1452-1464 (2000).

209. H. Wartlick, K. Michaelis, S. Balthasar, K. Strebhardt, J. Kreuter, and K. Langer. Highly

specific HER2-mediated cellular uptake of antibody-modified nanoparticles in tumour cells. J

Drug Target. 12:461-471 (2004).

210. J. Hrkach, D. Von Hoff, M. Mukkaram Ali, E. Andrianova, J. Auer, T. Campbell, D. De

Witt, M. Figa, M. Figueiredo, A. Horhota, S. Low, K. McDonnell, E. Peeke, B. Retnarajan,


75

A. Sabnis, E. Schnipper, J.J. Song, Y.H. Song, J. Summa, D. Tompsett, G. Troiano, T. Van

Geen Hoven, J. Wright, P. LoRusso, P.W. Kantoff, N.H. Bander, C. Sweeney, O.C.

Farokhzad, R. Langer, and S. Zale. Preclinical development and clinical translation of a

PSMA-targeted docetaxel nanoparticle with a differentiated pharmacological profile. Sci

Transl Med. 4:128ra139 (2012).

211. R.N. Brogden and R.C. Heel. Human insulin. A review of its biological activity,

pharmacokinetics and therapeutic use. Drugs. 34:350-371 (1987).

212. H. Iyer, A. Khedkar, and M. Verma. Oral insulin - a review of current status. Diabetes Obes

Metab. 12:179-185 (2010).

213. C.P. Reisand, and C. Damge. Nanotechnology as a promising strategy for alternative routes

of insulin delivery. Methods Enzymol. 508:271-294 (2012).

214. S. Jain, V.V. Rathi, A.K. Jain, M. Das, and C. Godugu. Folate-decorated PLGA nanoparticles

as a rationally designed vehicle for the oral delivery of insulin. Nanomedicine (Lond). 7:1311-

1337 (2012).

215. E. Pasquier, M. Kavallaris, and N. Andre. Metronomic chemotherapy: new rationale for new

directions. Nat Rev Clin Oncol. 7:455-465 (2010).

216. J.M. Terwogt, J.H. Schellens, W.W. Huinink, and J.H. Beijnen. Clinical pharmacology of

anticancer agents in relation to formulations and administration routes. Cancer Treat Rev.

25:83-101 (1999).

217. J.H. Schellens, M.M. Malingre, C.M. Kruijtzer, H.A. Bardelmeijer, O. van Tellingen, A.H.

Schinkel, and J.H. Beijnen. Modulation of oral bioavailability of anticancer drugs: from

mouse to man. Eur J Pharm Sci. 12:103-110 (2000).

218. M. Agueros, V. Zabaleta, S. Espuelas, M.A. Campanero, and J.M. Irache. Increased oral

bioavailability of paclitaxel by its encapsulation through complex formation with

cyclodextrins in poly(anhydride) nanoparticles. J Control Release. 145:2-8 (2010).

219. M. Agueros, S. Espuelas, I. Esparza, P. Calleja, I. Penuelas, G. Ponchel, and J.M. Irache.

Cyclodextrin-poly(anhydride) nanoparticles as new vehicles for oral drug delivery. Expert

Opin Drug Deliv. 8:721-734 (2011).

220. M. Ishikawa, H. Yoshii, and T. Furuta. Interaction of modified cyclodextrins with

cytochrome P-450. Biosci Biotechnol Biochem. 69:246-248 (2005).

221. V. Zabaleta, G. Ponchel, H. Salman, M. Agüeros, C. Vauthier, and J.M. Irache. Oral

administration of paclitaxel with pegylated poly(anhydride) nanoparticles: permeability and

pharmacokinetic study. Eur J Pharm Biopharm. 81:514-523 (2012).

222. S.W. Wang, J. Monagle, C. McNulty, D. Putnam, and H. Chen. Determination of P-

glycoprotein inhibition by excipients and their combinations using an integrated high-

throughput process. J Pharm Sci. 93:2755-2767 (2004).


76

223. G. Boccadifuoco, B. Brunelli, M.G. Pizza, and M.M. Giuliani. A combined approach to

assess the potential coverage of a multicomponent protein-based vaccine. J Prev Med Hyg.

53:56-60 (2012).

224. S.A. Perez, E. von Hofe, N.L. Kallinteris, A.D. Gritzapis, G.E. Peoples, M. Papamichail, and

C.N. Baxevanis. A new era in anticancer peptide vaccines. Cancer. 116:2071-2080 (2010).

225. S. Lu. Immunogenicity of DNA vaccines in humans: it takes two to tango. Hum Vaccin.

4:449-452 (2008).

226. A.C. Rice-Ficht, A.M. Arenas-Gamboa, M.M. Kahl-McDonagh, and T.A. Ficht. Polymeric

particles in vaccine delivery. Curr Opin Microbiol. 13:106-112 (2010).

227. T. Storni, T.M. Kundig, G. Senti, and P. Johansen. Immunity in response to particulate

antigen-delivery systems. Adv Drug Deliv Rev. 57:333-355 (2005).




229. F. Sarti, G. Perera, F. Hintzen, K. Kotti, V. Karageorgiou, O. Kammona, C. Kiparissides,

and A. Bernkop-Schnurch. In vivo evidence of oral vaccination with PLGA nanoparticles

containing the immunostimulant monophosphoryl lipid A. Biomaterials. 32:4052-4057

(2011).

230. A.I. Camacho, R. Da Costa Martins, I. Tamayo, J. de Souza, J.J. Lasarte, C. Mansilla, I.

Esparza, J.M. Irache, and C. Gamazo. Poly(methyl vinyl ether-co-maleic anhydride)

nanoparticles as innate immune system activators. Vaccine. 29:7130-7135 (2011).

231. J.M. Irache, H.H. Salman, S. Gomez, S. Espuelas, and C. Gamazo. Poly(anhydride)

nanoparticles as adjuvants for mucosal vaccination. Front Biosci (Schol Ed). 2:876-890

(2010).

232. S. Reboucas Jde, J.M. Irache, A.I. Camacho, I. Esparza, V. Del Pozo, M.L. Sanz, M. Ferrer,

and C. Gamazo. Development of poly(anhydride) nanoparticles loaded with peanut proteins:

the influence of preparation method on the immunogenic properties. Eur J Pharm

Biopharm. 82:241-249 (2012).

233. I. Tamayo, J.M. Irache, C. Mansilla, J. Ochoa-Reparaz, J.J. Lasarte, and C. Gamazo.

Poly(anhydride) nanoparticles act as active Th1 adjuvants through Toll-like receptor

exploitation. Clin Vaccine Immunol. 17:1356-1362 (2010).

234. S. Gomez, C. Gamazo, B.S. Roman, M. Ferrer, M.L. Sanz, and J.M. Irache. Gantrez AN

nanoparticles as an adjuvant for oral immunotherapy with allergens. Vaccine. 25:5263-5271

(2007).

235. S.F. Hansen, A. Maynard, A. Baun, and J.A. Tickner. Late lessons from early warnings for

nanotechnology. Nat Nanotechnol. 3:444-447 (2008).


77

236. R. Nijharaand, and K. Balakrishnan. Bringing nanomedicines to market: regulatory

challenges, opportunities, and uncertainties. Nanomedicine. 2:127-136 (2006).

237. G. Forloni. Responsible nanotechnology development. J Nanopart Res. 14:1-17 (2012).

238. R.H. Müller, S. Gohla, and C.M. Keck. State of the art of nanocrystals - special features,

production, nanotoxicology aspects and intracellular delivery. Eur J Pharm Biopharm. 78:1-9

(2011).

239. A. Haase, J. Tentschert, and A. Luch. Nanomaterials: a challenge for toxicological risk

assessment EXS. 101:219-250 (2012).

240. D.B. Warheit. How meaningful are the results of nanotoxicity studies in the absence of

adequate material characterization? Toxicol Sci. 101:183-185 (2008).

241. R.C. Murdock, L. Braydich-Stolle, A.M. Schrand, J.J. Schlager, and S.M. Hussain.

Characterization of nanomaterial dispersion in solution prior to in vitro exposure using

dynamic light scattering technique. Toxicol Sci. 101:239-253 (2008).

242. B.J. Marquis, S.A. Love, K.L. Braun, and C.L. Haynes. Analytical methods to assess

nanoparticle toxicity. Analyst. 134:425-439 (2009).

243. M.J. Clift, P. Gehr, and B. Rothen-Rutishauser. Nanotoxicology: a perspective and discussion

of whether or not in vitro testing is a valid alternative. Arch Toxicol. 85:723-731 (2011).

244. J.M. Wörle-Knirsch, K. Pulskamp, and H.F. Krug. Oops they did it again! Carbon nanotubes

hoax scientists in viability assays. Nano Lett. 6:1261-1268 (2006).

245. K. Unfried, C. Albrecht, L.-O. Klotz, A. Von Mikecz, S. Grether-Beck, and R.P.F. Schins.

Cellular responses to nanoparticles: target structures and mechanisms. Nanotoxicology. 1:52-

71 (2007).

246. P. Rivera Gil, G. Oberdorster, A. Elder, V. Puntes, and W.J. Parak. Correlating physico-

chemical with toxicological properties of nanoparticles: the present and the future. ACS

Nano. 4:5527-5531 (2010).

247. J.P. Bohnsack, S. Assemi, J.D. Miller, and D.Y. Furgeson. The primacy of physicochemical

characterization of nanomaterials for reliable toxicity assessment: a review of the zebrafish

nanotoxicology model. Methods Mol Biol. 926:261-316 (2012).

248. P.P. Dandekar, R. Jain, S. Patil, R. Dhumal, D. Tiwari, S. Sharma, G. Vanage, and V.

Patravale. Curcumin-loaded hydrogel nanoparticles: application in anti-malarial therapy and

toxicological evaluation. J Pharm Sci. 99:4992-5010 (2010).

249. K. Volkovova, O. Ulicna, J. Kucharska, R. Handy, M. Staruchova, A. Kebis, J. Pribojova, J.

Tulinska, and M. Dusinska. Health effects of selected nanoparticlesin vivo: liver function and

hepatotoxicity following i.v. injection of titanium dioxide and Na-oleate coated iron oxide

nanoparticles in rodents. Nanotoxicology.doi: 10.3109/17435390.2013.815285 (2013).


78

250. M. Ema, A. Matsuda, N. Kobayashi, M. Naya, and J. Nakanishi. Dermal and ocular irritation

and skin sensitization studies of fullerene C60 nanoparticles. Cutan Ocul Toxicol. 32:128-134

(2013).

251. Nanotechnology Characterization Laboratory (NCL). National Cancer Institute. U.S.

National Institutes of Health. www.ncl.cancer.gov.

252. NanoTEST. Alternative testing strategies for the assessment of the toxicological profile of

nanoparticles used in medical diagnosis. (7th Framework Programme). www.nanotest-pf7.eu.

253. European Academies Science Advisory Council (EASAC). Impact of Engineered

Nanomaterials on Heath: Considerations for Benefit-Risk Assessment. EASAC Policy

Report - JRC Reference Report. http://www.easac.eu/home/reports-and-statements/detail-

view/article/first-joint.html (2011).

254. Joint Research Centre (JRC). Impact of Engineered Nanomaterials on Health:

Considerations for Benefit-Risk Assessment - EASAC Policy Report - JRC Reference

Report. http://publications.jrc.ec.europa.eu/repository/handle/111111111/22610 (2011).

255. International Organization for Standardization (ISO). ISO/TC 229 – Nanotechnologies.

http://www.iso.org/iso/home/store/catalogue_tc/catalogue_tc_browse.htm?commid=381

983&published=on&includesc=true. (2005).

256. Organization for Economic Cooperation and Development (OECD). Safety of

Manufactured Nanomaterials. http://www.oecd.org/env/ehs/nanosafety/. (2006).

CHAPTER 2

AIM AND OBJECTIVES

CHAPTER 2: AIM AND OBJECTIVES

81

AIM & OBJECTIVES

Until today, many polymeric nanoparticles have demonstrated great potential as oral drug

delivery systems. However, its clinical development sometimes is hindered by the limited knowledge

of their possible harmful effects. Therefore, understanding of the interactions of polymeric

nanoparticles with biological systems according to their physico-chemical properties and to assess

their toxicity is clearly required in order to develop effective and safe nanomedicines for humans.

In this context, an extensive research carried out in the department of Pharmacy and

Pharmaceutical Technology, have proven that poly(anhydride) nanoparticles designed from the

copolymer of methyl vinyl ether and maleic anhydride (Gantrez® AN 119) are ideal candidates as

vehicles for oral delivery of drugs and antigens. Thus, it was considered really interesting to study its

toxicity in relation to their physico-chemical characteristics by using appropriate protocols and

guidelines.

The general aim of this research project was to evaluate the toxicity of different

poly(anhydride) nanoparticles intended for oral delivery. For this purpose, this aim was divided in the

following objectives:

1. To develop a scalable spray-drying procedure for the preparation of different types of

poly(anhydride) nanoparticles in order to simplify the method used so far.

2. To evaluate the in vitro toxicity of the poly(anhydride) nanoparticles in HepG2 and Caco-2

cells as well as their capability to interact and be internalized by intestinal cells.

3. To investigate the toxicological profile of these carriers by the oral route through acute and

sub-acute toxicity studies (according to OECD guidelines) and biodistribution studies.

4. To approach the acute toxicity and biodistribution of the poly(anhydride) nanoparticles by

the i.v. route.

CHAPTER 3

EXPERIMENTAL DESIGN

CHAPTER 3: EXPERIMENTAL DESIGN

85

• Acute toxicity:

OECD guideline 425

• Biodistribution:In vivo/ex vivo studies using 99mTcradiolabelled nanoparticles

Nanoparticle formulation design

and method development

• Selection of saccharide and saccharide/polymer ratio selection

• Spray-drying vs Lyophilization

Applicability:Encapsulation of model drugs

[Hot saline antigenic complex (HS) of Brucella ovis and Atovaquone

(ATO)]

Nanoparticles toxicity

assessment

In vitro

• Cytotoxicity evaluation

• Hemocompatibility studies

• Nanoparticles-Cell interaction studies

In vivo,

Oral

• Acute toxicity:

OECD guideline 425 (limit test)

• Sub-chronic toxicity:

OECD guideline 407

• Biodistribution:In vivo/ex vivo studies using 99mTcradiolabelled nanoparticles

In vivo,

Intravenous

CHAPTER 4


89


Patricia Ojer1, Hesham Salman1, Raquel Da Costa Martins1, Javier Calvo2, Adela López de Cerain1,

Carlos Gamazo1, Jose Luis Labandeira2, and Juan M. Irache1

1 University of Navarra, C/Irunlarrea 1, 31080-Pamplona, Spain. 2 Tres Cantos Medicines Development Campus, GlaxoSmithKline, 28760, Tres Cantos, Spain.

ABSTRACT

Gantrez® AN nanoparticles can be obtained by desolvation of the poly(anhydride) with an aqueous

medium, followed by a purification step and freeze-drying. In order to simplify this method, a spray-

drying procedure was evaluated here. For this purpose, suspensions of nanoparticles were mixed with

carbohydrates before their drying. Lactose, in a saccharide/polymer ratio of 2, was found to be the

most suitable excipient regarding the physico-chemical characteristics and stability of the resulting

nanoparticles. These conditions can be successfully applied to the preparation of conventional,

combined cyclodextrin/polyanhydride or pegylated nanoparticles. The capabilities of the new

procedure were also studied by the characterization of nanoparticles loaded with either an antigenic

extract (HS from Brucella ovis) or atovaquone (ATO). In all cases, the new formulations displayed

similar physico-chemical characteristics that those of nanoparticles obtained by lyophilization. In

summary, the spray-drying procedure can be an adequate alternative to lyophilization in the

preparative process of poly(anhydride) nanoparticles.

Published in Journal of Drug Delivery Science and Technology 20(5):353-359 (2010).

CHAPTER 4: Spray-drying of poly(anhydride) nanoparticles for drug/antigen delivery

91

1. INTRODUCTION

Over the few past decades, the technology of particle size reduction to nanometers has

significantly advanced. The use of nanoparticles for drug delivery purposes may permit to achieve a

personalized health care, rational drug design and targeted drug delivery (1). In principle,

pharmaceutical nanoparticles can be of interest to conduct the loaded drug to its site of action or

absorption and, as a consequence, improve the efficacy of the therapy and/or increase the

bioavailability of the active molecule.

In recent years, poly(anhydride) nanoparticles obtained from the copolymer between methyl

vinyl ether and maleic anhydride (Gantrez® AN) have been developed and proposed as carriers with

bioadhesive properties for the mucosal delivery of drugs (2, 3) and antigens (4, 5). In addition, due to

the interesting ability of this polymer to incorporate different compounds, functionalized

nanodevices can be easily produced including nanoparticles containing cyclodextrins(6), mannose-

coated nanoparticles (7) or pegylated nanoparticles (8). In all cases, these carriers have been obtained

by desolvation of the copolymer in a solution of acetone with a mixture of ethanol and water,

followed by a purification step and freeze-drying.

Lyophilization is a well known technique which is usually employed for the preservation of

biodegradable nanoparticles (9, 10). Nevertheless, one important drawback associated with this

technique is related with the long duration of the process. Therefore, alternatives to a lyophilization

step are always welcomed if they can simplify and shorten the preparative process. In this context,

one possible solution can be the spray-drying (SD) technique.

Spray-drying is a manufacturing process commonly used in the industry, which converts a

liquid into a homogeneous dry powder in a one-step process (11). The process involves the

atomization of a liquid feedstock into a spray of droplets and contacting the droplets with hot air in a

drying chamber. It offers a quicker process and has advantages such as its capability to influence and

modulate the physico-chemical properties of the resulting solids (12). In addition, another important

advantage is that this process can be applied to the effective drying of proteins without any significant

degradation (13). On the other hand, spray-drying has been successfully applied in the production of

a number of microdevices for drug delivery purposes, including poly(+/-)lactide microparticles (14),

polycaprolactone microspheres (15) or albumin microspheres (16).

On the contrary, spray-drying has been scarcely used for drying polymer nanoparticles. In

this context, the first attempt was to spray poly(Ɛ-caprolactone) (PCL) nanocapsules in the presence

of silicon dioxide particles as drying adjuvant (17). Thus, the nanoparticle suspensions led to

microparticles presenting different and homogeneous nanocoating after the drying process (18, 19).

Similarly, other soluble supports (i.e. polyvinylpyrrolidone K30 and mannitol) have also been

proposed to dry polyester nanocapsules (20). Nevertheless, large amounts of these excipients

(excipient/nanocapsules ratio of 10 by weight) were needed to obtain adequate reconstitution in

aqueous media (20).


92

The aim of this work was to develop a spray-drying procedure for the preparation of

different types of poly(anhydride) nanoparticles in order to simplify the method used so far for the

production of these carriers. In addition, the capabilities of the new procedure were also studied by

the incorporation and characterization of the nanoparticles loaded with either an antigenic extract

(HS from Brucella ovis) or ATO, used as models of active compounds.


93

2. MATERIALS AND METHODS

2.1. Chemicals, reagents and solutions

Poly (methyl vinyl ether-co-maleic anhydride) or poly(anhydride) (Gantrez® AN 119; Mw

200,000) was kindly gifted by ISP (Barcelona, Spain). Polyethylene glycol 6000 (PEG6000) was

provided by Fluka (Switzerland). 2-hydroxypropyl-β-cyclodextrin (HPCD) was provided by Sigma–

Aldrich (Steinheim, Germany). Acetone was obtained from VWR Prolabo (Fontenay-sous-Bois,

France). Deionized water (18.2MΩ resistivity) was prepared by a water purification system

(Wasserlab, Pamplona, Spain). ATO was obtained from ChemPacific Corp (Baltimore, United

States). The hot saline antigenic extract (HS) from Brucella ovis was obtained following the procedures

detailed later. Mannitol, lactose, glucose and maltose were of Ph. grade and were purchased from

Roig Pharma S.A. (Barcelona, Spain). All other chemicals and solvents used were of analytical grade.

2.2 Extraction and characterization of the HS antigen

The HS was obtained from the strain B. ovis REO 198 by the hot saline method previously

described (21). The total protein was determined by the BCA™ Protein Assay method (22) using

bovine serum albumin (BSA) as standard. The 2-keto-3-deoxyoctonate (KDO, exclusive marker of

lipopolysaccharide (LPS)) analysis, corrected for 2-deoxyaldoses, was performed by the method of

Warren modified by Osborn (23). The batch of antigen used to prepare the vaccine formulation

contained 66.4±10.6% protein and 39.5±3.8% rough lipopolysaccharide (R-LPS).

2.3. Preparation of poly(anhydride) nanoparticles

All types of nanoparticles were prepared by solvent displacement followed by incorporation

of an excipient and, finally, dried by spray-drying. In order to study the influence of the nature and

amount of saccharide on the physico-chemical properties of the nanoparticles, the following

saccharides were studied: lactose, mannitol, glucose and maltose.

2.3.1. Conventional poly(anhydride) nanoparticles (NP-SD)

Briefly, 500 mg of poly(anhydride) were dissolved and stirred in 30 mL acetone. Then, the

desolvation of the polymer was induced by the addition of 15 mL deionized water under magnetic

stirring to the organic phase. In parallel, 1 g of saccharide was dissolved in 10 mL deionized water

and added to the nanoparticle suspension under agitation for 5 min at room temperature. Finally, the

suspension was dried in a Mini Spray-dryer Büchi B191 (Büchi Labortechnik AG, Switzerland). For

this purpose, the following parameters were selected: inlet temperature: 90°C; outlet temperature

60°C; spray-flow 600 L/h, and aspirator at 90% of the maximum capacity. During the process the

nanoparticle suspension was maintained under moderate agitation. The recovered powder was stored

in closed vials at room temperature.


94

For comparisons, nanoparticles prepared in the same way but in absence of any type of

saccharide were used as control. Similarly, nanoparticles obtained by desolvation and dried by

lyophilization (NP-L) were prepared as described previously (24).

2.3.2. Poly(anhydride) nanoparticles containing 2-hydroxypropyl-β-cyclodextrin (NP-HPCD-

SD)

Briefly, 500 mg of poly(anhydride) were dissolved and stirred in 20 mL acetone. Then, 10 mL

acetone containing 125 mg HPCD were added to the polymer solution under magnetic stirring and

incubated for 30 min. Nanoparticles were obtained by the addition of 15 mL deionized water under

magnetic stirring to the organic phase. In parallel, 1 g of lactose was dissolved in 10 mL deionized

water and added to the nanoparticle suspension under agitation for 5 min at room temperature

before drying of this suspension as described in 2.3.1. The recovered powder was stored in closed

vials at room temperature.

For comparisons, nanoparticles obtained by desolvation and dried by lyophilization (NP-

HPCD-L) were prepared as described previously (6).

2.3.3. Pegylated poly(anhydride) nanoparticles (PEG-NP-SD)

In this case, 62.5 mg PEG6000 were dissolved in 15 mL acetone and mixed with 15 mL

acetone containing 500 mg poly(anhydride) and incubated under magnetic stirring for 1 h. Then,

nanoparticles were obtained by the addition of 15 mL deionized water under magnetic stirring to the

organic phase. The solvents were eliminated under reduced pressure (Buchi R-144, Switzerland) and

nanoparticles were purified by double centrifugation in water at 17,000 rpm for 20 min (Sigma 3K30,

Germany). Nanoparticles were then resuspended in 25 mL deionized water containing 1 g of lactose

and, finally, dried as described in 2.3.1. The recovered powder was stored in closed vials at room

temperature.

For comparisons, pegylated nanoparticles obtained by desolvation and dried by lyophilization

(PEG-NP- L) were prepared as described previously (8).

2.4. Preparation of HS-loaded nanoparticles

To obtain conventional HS-loaded nanoparticles (NP-HS-SD), 20 mg of the HS from

Brucella ovis were dispersed in 2 mL deionized water under sonication. Then, this aqueous phase was

added to 30 mL acetone containing 500 mg poly(anhydride). After incubation for 1 h under magnetic

stirring at room temperature, nanoparticles were obtained by the addition of 15 mL deionized water

and incubation for 5 min at room temperature. Then, 10 mL of water containing 1 g of lactose was

added and the suspension was dried in a Mini Spray-dryer Büchi B191 (Büchi Labortechnik AG,

Switzerland) under the experimental conditions described above (see 2.3.1).

For the preparation of pegylated HS-loaded nanoparticles (PEG-NP-HS-SD), 20 mg of

Brucella ovis HS were dispersed in 2 mL deionized water under sonication. Then, this aqueous phase


95

was added to 15 mL acetone containing 500 mg poly(anhydride). The mixture was incubated for 30

min under magnetic stirring at room temperature. On the other hand, 62.5 mg PEG6000 were

dissolved in 15 mL acetone and stirred for 20 min. Then, this PEG solution was added to the HS and

the polymer mixture and maintained under magnetic agitation for 1 h at room temperature. After

incubation, nanoparticles were formed by the addition of 15 mL deionized water and further

incubation at room temperature for 5 min. The solvents were eliminated under reduced pressure

(Buchi R-144, Switzerland) and nanoparticles were purified by double centrifugation at 17,000 rpm

for 20 min (Sigma 3K30, Germany). Nanoparticles were resuspended in 25 mL deionized water

containing 1 g of lactose. Finally, the suspension was dried in a Mini Spray-dryer Büchi B191 (Büchi

Labortechnik AG, Switzerland), under the experimental conditions described above (see 2.3.1).

2.5. Preparation of atovaquone-loaded nanoparticles

This formulation was prepared by encapsulation of ATO complexed with HPCD in

poly(anhydride) nanoparticles (ATO-HPCD-NP-SD). For this purpose, 20 mg of HPCD and 2.5 mg

ATO were dissolved and incubated in 10 mL of ethanol overnight. Then, the ethanol was evaporated

up to 5 mL under reduced pressure.

On the other hand, 500 mg poly(anhydride) were dissolved in 25 mL of acetone. After

dissolution, the ethanolic phase containing ATO-HPCD complexes was added and the mixture was

incubated for 20 min under agitation at room temperature. The nanoparticles were formed after

addition of 15 mL deionized water to the acetone phase under magnetic stirring. Then, 10 mL of

water containing 1 g of lactose was added and the suspension was dried in a Mini Spray-dryer Büchi

B191 (Büchi Labortechnik AG, Switzerland) under the experimental conditions described above (see

2.3.1).

2.6. Characterization of nanoparticles

2.6.1. Physico-chemical characterization

The particle size and the zeta potential of nanoparticles were determined by photon

correlation spectroscopy (PCS) and electrophoretic laser doppler anemometry, respectively, using a

Zetamaster analyzer system (Malvern Instruments, United Kingdom). The samples were diluted in

deionized water and measured at room temperature with a scattering angle of 90°. All measurements

were performed in triplicate.

The morphological examination of the spray-dried powders was obtained by scanning

electron microscopy (SEM) in a Zeiss DSM 940A scanning electron microscope (Oberkochen,

Germany) coupled with a digital image system (DISS) Point Electronic GmBh. Previously, a small

amount of the spray-dried powders was diluted with deionized water and the resulting suspension

was centrifuged at 17,000 rpm (Sigma 3K30, Germany) for 20 min in order to eliminate sugars.

Finally, the pellet was shaded with a 12 nm gold layer in a Hemitech K 550 Sputter-Coater.


96

The spray-drying process yield was determined as the difference between the total amount of

solids contained in nanoparticle suspensions dried and the sum of the powder collected after the

drying process.

2.6.2. Quantification of HPCD in nanoparticles

The amount of HPCD associated to the nanoparticles was estimated by quantification of

HPCD content in the supernatants collected from the purification step, using a high performance

liquid chromatography - evaporative light scattering detection (HPLC-ELSD) (25).

The supernatants recovered during the purification step were diluted in 10 mL in water. A 1

mL aliquot of the supernatants was transferred to auto sampler vials, capped and placed on the

HPLC auto sampler. The apparatus used for the HPLC analysis was an Agilent model 1100 series LC

(Waldbronn, Germany) coupled with an ELSD 2000 (Alltech, Illinois, United States). An ELSD

nitrogen generator (Alltech) was used as the source of the nitrogen gas. Data acquisition and analysis

were performed with a Hewlett-Packard computer using the ChemStation G2171 AA program.

Separation was carried out at 50°C on a reversed-phase ZorbaxEclipse XDB-Phenyl column (2.1 mm

x 150 mm; particle size 5 μm) obtained from Agilent Technologies (Waldbronn, Germany). This

column was protected by a 0.45 μm filter (Teknokroma, Spain). ELSD conditions were optimized in

order to achieve maximum sensitivity: the drift tube temperature was set at 115°C, the nitrogen flow

was maintained at 3.2 L/min and the gain was set to 1. The mobile phase composition was a mixture

of acetonitrile (A) and water (B) in a gradient elution at a flow-rate of 0.25 mL/min. The injection

volume was 5 μL. Under these experimental conditions, retention times for HPCD and

poly(anhydride) were 10.26 ± 0.06 min and 1.08 ± 0.05 min respectively.

For quantification, 30 mg of dry powder were dispersed in 10 mL water. Then, the

suspensions of nanoparticles were centrifuged twice at 17,000 rpm for 20 min at 4°C and the

supernatants recovered to quantify the free HPCD and poly(anhydride).

The amount of HPCD and poly(anhydride) forming the nanoparticles were calculated as the

difference between the initial amount of cyclodextrin and poly(anhydride) added and the amount of

both recovered in the supernatants.

2.6.3. Quantification of PEG

The amount of PEG associated to the nanoparticles, as well as the amount of

poly(anhydride) in pegylated nanoparticles were estimated by HPLC coupled to and ELSD detector

(26). Separation was carried out at 40°C on a PL aquagel-OH 30 column (300 mm×7.5 mm; particle

size 8 μm) obtained from Agilent Technologies (GB, United Kingdom). ELSD conditions were

optimized in order to achieve maximum sensitivity: the drift tube temperature was set at 110 °C, the

nitrogen flow was maintained at 3 L/min and the gain was set to 1. The mobile phase composition

was a mixture of methanol (A) and water (B) in a gradient elution at a flow rate of 1 mL/min. Under


97

these experimental conditions, retention times for PEG 6000 and the polymer were 6.92 ± 0.02 and

4.48 ± 0.06 min, respectively.

The amount of PEG associated to the nanoparticles was estimated by quantification of PEG

content in the supernatants collected from either the nanoparticles purification step (for freeze-dried

nanoparticles) or the dry powder (for the spray-drying nanoparticles), using HPLC-ELSD.

Supernatants recovered during the purification step were diluted with water until a final volume of 10

mL was attained. For spray-dried nanoparticles, 30 mg of powder was dispersed in a total volume of

10 mL of deionized water before centrifugation at 17,000 rpm for 20 min at 4°C. In all cases, 20μl of

these supernatants were injected onto the HPLC column.

The amount of PEG associated to nanoparticles was calculated as the difference between the

initial PEG added and the amount of PEG recovered in the supernatants during the purification step.

Similarly, the amount of poly(anhydride) was estimated by the difference in the same way.

2.6.4. HS loading in the nanoparticles

For the HS quantification, 10 mg of dry powdered nanoparticles, both NP-HS-SD and PEG-

NP-HS-SD, were resuspended in 1 mL of deionized water and centrifuged (17,000 rpm, 20 min,

4°C). Then, the pellet was dissolved in acetone/dimethylformamide (DMF) (3:1) and kept for 1 h at -

80°C. Samples were centrifuged at 17,000 rpm for 20 min at 4°C and the pellets were washed with 1

mL acetone and kept 30 min at -80°C. After centrifugation under the same conditions, pellets were

resuspended in 500 μL of ultrapure water in order to quantify antigenic proteins by the BCA

Protein™ Assay. In any case, each type of nanoparticle was assayed in triplicate. The calculations were

carried out via the application of a calibration curve of free HS obtained after proteins precipitation

with acetone/DMF in ultrapure water (r2 > 0.999). HS loading was expressed as the amount of HS

(in μg) per mg nanoparticles.

2.6.5. Study of the Structural Integrity of the Entrapped HS

To evaluate the effect of the manufacturing process on the HS protein profile and

antigenicity, proteins from spray-dried nanoparticles were extracted with acetone/DMF (3:1) as

described above, and assayed by SDS-PAGE (21).

2.6.6. Quantification of ATO content

The amount of ATO loaded into nanoparticles was calculated by UPLC (Ultra performance

liquid chromatography). Briefly, the apparatus was an Agilent model 1100 series LC and a diode-array

detector set at 254 nm. Data were collected and processed by chromatographic software MassLynx

4.1 (Waters). The chromatographic system was equipped with a reversed – phase 50 x 2.1 mm UPLC

Acquity BEH C18 column (50 mm x 2.1 mm, 1.7 μm; Waters). The gradient elution buffers were A

(Methanol and 0.1 % formic acid) and B (water and 0.1 % formic acid). The column was eluted with

a linear gradient consisted of 90% A over 0.5 min, 90 to 10 % over 0.5 to 4 min, 10% over 4 to 4.5

min, returned to 90 for 0.5 min and kept for a further 1 min before the next injection. Total run was


98

5 min, volume injection was 5 μL and the flow rate of 500 μL/min. The limit of quantification was

calculated to be 0.5 μg/mL with a relative standard deviation lower than 2.5%.

For analysis, 5 mg of dry powder were digested with 1 mL acetonitrile. After filtering

through 0.2 μm PTFE filter (Millipore, MA, United States), the samples were analyzed and quantified

by UPLC. All samples were assayed in triplicate and results were expressed as the amount of ATO (in

μg) per mg nanoparticles.

2.7. Statistical analysis

Values of P<0.05 were considered as a statistically significant difference. All calculations

were performed using SPSS® statistical software program (SPSS® 15.0, Microsoft, United States).


99

3. RESULTS

3.1. Effect of saccharides on the characteristics of poly(anhydride) nanoparticles

(NP-SD)

When nanoparticle suspensions (obtained by desolvation of an acetone solution of the

polymer with water) were directly dried in the spray-drying apparatus, the subsequent dispersion of

the dry powder in an aqueous medium gave always heterogeneous dispersions characterized by the

presence of large aggregates of polymer as well as some microparticles in the aqueous phase with a

size higher than 2.0 μm. In order to solve this drawback our strategy was to incorporate in the

nanoparticle dispersions, pharmaceutical excipients capable to protect nanoparticles against the

drying stress induced in the spray-drying apparatus.

Figure 1 shows the influence of the saccharide/polymer ratio, when either lactose or

mannitol were used, on the mean size of the resulting poly(anhydride) nanoparticles. Interestingly,

similar results were found for both types of excipients. Thus, for a saccharide/polymer ratio of 0.5, a

similar phenomenon to that observed without any type of excipient was found, large and particles

bigger than 2 μm were detected. On the contrary, when the ratio was 1.0 or higher, the dispersion of

the dry powder in water gave homogeneous suspensions of nanoparticles with a mean size lower than

200 nm. Furthermore, by increasing the saccharide/polymer ratio a slightly decrease of the size of the

resulting nanoparticles was observed (Figure 1.).

Table I summarizes the influence of the type of saccharide on the physico-chemical

characteristics of the resulting nanoparticles for a saccharide/polymer ratio of 2. This ratio was the

most suitable in order to obtain the optimal nanoparticle size using the lowest amount of saccharide.

Figure 1. Influence of the

saccharide/poly(anhydride) ratio

on the diameter of the

nanoparticles obtained by spray-

drying and after resuspension in

purified water. Data expressed as

mean ± SD (n=3). Saccharide:

lactose or mannitol.

0 1 2 3 40

50

100

150

2000

Size

(nm

)

Saccharide/Polymer ratio (w/w)

Lactose Mannitol


100

Table I. Influence of the type of saccharide on the physico-chemical characteristics of nanoparticles

obtained by spray-drying. Experimental conditions; saccharide/polymer ratio of 2. Data expressed as

the mean ± SD (n=6). PDI: polydispersity index. Adhesivity: adhesion of the resulting powder to the

cyclone and receptacle glass walls of the spray-drying apparatus: (-) no adhesion, (+) very slight

adhesion, (+++) intensive adhesion, (++++) very intensive adhesion.

Excipient Size

(nm) PDI

Zeta potential

(mV)

Yields

(%) Adhesivity

Lactose 149 ± 1 0.09 ± 0.02 -57.90 ± 0.95 67.5 ± 0.4 +

Mannitol 135 ± 1 0.09 ± 0.03 -59.40 ± 1.99 62.3 ± 0.7 -

Glucose 129 ± 1 0.13 ± 0.05 -60.80 ± 0.33 54.7 ± 1.7 ++++

Maltose 128 ± 1 0.13 ± 0.06 -60.83 ± 0.37 59.1 ± 1.3 +++

In all cases, the mean size and zeta potential of nanoparticles was similar and independent of

the type of saccharide used. Thus, the diameter of nanoparticles was about 130-150 nm and the

surface charge of the resulting carriers was around –60 mV. Similarly, in all cases, the particles

displayed and spherical shape and a smooth surface. Figure 2 shows SEM photograph corresponding

to nanoparticles prepared in the presence of lactose.

2 μm

Figure 2. SEM photograph of poly(anhydride) nanoparticles obtained by spray-drying in the

presence of lactose.


101

Finally, another important aspect was the amount of poly(anhydride) transformed into

nanoparticles after desolvation and the yield of the spray-drying process. In the former, more than

90% of poly(anhydride) was transformed into nanoparticles. In the latter, the yield of the spray-drying

process was calculated to be between 55 and 68%, depending on the type of saccharide used.

Nevertheless, both lactose and mannitol appeared to be more efficient than glucose or

maltose to induce the production of more homogeneous populations of nanoparticles. In fact, the

polydispersity of nanoparticles was lower with lactose or mannitol than when either glucose or

maltose was used. In addition, the use of glucose or maltose resulted in the production of a sticky and

hygroscopic powder, which hampered their recovery and a significantly lower yield.

According to these results, the stability of the nanoparticles formulated with either mannitol

or lactose was evaluated after their storage in close vials at 25°C one year after their preparation.

Thereafter, nanoparticles formulated with lactose maintained all of their original physicochemical

characteristics (size: 145 nm; PDI: 0.1; zeta potential: -60 mV). However, for nanoparticles prepared

in the presence of mannitol, both the size (about 430 nm) and polydispersity (PDI of 0.5) were

dramatically increased (Figure 3).

0

5

10

15

20

0.1 1 10 100 1000 10000

Inte

nsity

(%)

Size (d.nm)

Mannitol Lactose

Inte

nsity

(%)

Size (d.nm)

0

5

10

15

20

0.1 1 10 100 1000 10000

Inte

nsity

(%)

Size (d.nm)

Mannitol Lactose

Inte

nsity

(%)

Size (d.nm)

1)

2)

Figure 3. Particle size distribution of nanoparticles obtained by spray-drying. 1) Size immediately

after preparation, 2) Size one year after reparation of nanoparticle batch.


102

3.2. Preparation and characterization of NP-HPCD-SD and PEG-NP-SD

From these results it was clear that the most appropriate saccharide for the preservation of

the physico-chemical characteristics of nanoparticles appeared to be lactose at an excipient/polymer

ratio of at least 2. Under these experimental conditions, cyclodextrin-poly(anhydride) nanoparticles

(NP-HPCD) and pegylated nanoparticles (PEG-NP) were prepared by spray-drying and compared

with nanoparticles obtained by lyophilization. Table II summarizes these results.

Table II. Influence of the preparative method on the physico-chemical characteristics of

nanoparticles obtained by either spray-drying or lyophilization. NP-HPCD: 2-hydroxypropyl-β-

cyclodextrin-poly(anhydride) nanoparticles, PEG-NP: poly(anhydride) nanoparticles pegylated with

PEG 6000. Data expressed as mean values ± standard deviations (n=6).

Formulations Size (nm)

PDI Zeta potential

(mV) Yields (%)

HPCD associated

(µg/mg NP)

PEG associated

(µg/mg NP)

NP-HPCD

Spray-Drying 168 ± 1.0 0.10 ± 0.03 -52.8 ± 0.5 74.2 ± 0.8 71.9 ± 3.2 -

NP-HPCD

Lyophilization 140 ± 1.2 0.08 ± 0.02 -58.4 ± 1.5 81.8 ± 2.4 68.4 ± 4.3 -

PEG-NP

Spray-Drying 126 ± 0.6 0.17 ± 0.01 -54.1 ± 3.5 76.4 ± 0.5 - 55.6 ± 2.3

PEG-NP

Lyophilization 158 ± 0.9 0.11 ± 0.02 -62.9 ± 1.0 80.9 ± 1.7 - 53.5 ± 0.9

In all cases, little differences in the physico-chemical characteristics of nanoparticles obtained

by either spray-drying of lyophilization were observed. Interestingly the amount of poly(anhydride)

transformed into nanoparticles was also higher than 90% and the amount of PEG6000 or HPCD

associated to nanoparticles was found to be statistically similar when the carriers were produced by

spray-drying or lyophilization (p<0.05). On the contrary, when nanoparticles were produced by

spray-drying, the resulting nanoparticles displayed a slightly higher polydispersity than when obtained

after lyophilization; although this parameter was always above 0.3.

Similarly, batches produced by spray-drying showed always a lower yield than when produced

by lyophilization (about 68% vs 90%).

The morphological analysis by scanning electron microscopy showed that the incorporation

of HPCD in or pegylation of poly(anhydride) nanoparticles clearly modified the aspect of the

resulting nanoparticles. Thus, the presence of the cyclodextrin or PEG6000 produced rough and


103

irregular shaped nanoparticles, whereas a smooth surface is typical for conventional nanoparticles

(Figure 4).

(A) (A.1)

500 nm

(B) (B.1)

1 μm

(C) (C.1)

1 μm

Figure 4. SEM photographs of nanoparticles obtained by spray-drying. (A) NP-SD. (A.1)

Magnification of a section of photograph (A). (B) NP-HPCD-SD. (B.1) Magnification of a section of

photograph (B). (C) PEG-NP-SD. (C.1) Magnification of a section of photograph (C).


104

3.3. Preparation and characterization of nanoparticles encapsulating model drugs

In order to study the capability of the new method to produce drug- or antigen-loaded

nanoparticles, the HS of Brucella ovis and ATO were selected as model of active molecule to

incorporate into poly(anhydride) nanoparticles prepared by spray-drying.

Table III summarizes the main physicochemical characteristics of these formulations. For HS

loaded nanoparticles, all formulations were characterized by a homogeneous size of about 150 nm,

with a low PDI (≈0.2) and a negative zeta potential. On the other hand, the HS loading was found to

be significantly higher for conventional nanoparticles (NP-HS-SD) than for pegylated ones (PEG-

NP-HS-SD) (p<0.05).

Table III. Physico-chemical characteristics of HS-loaded nanoparticles and ATO-loaded

nanoparticles obtained by spray-drying.Data expressed as the mean ± standard deviation (n=6). NP-

HS-SD: Hs-loaded poly(anhydride) nanoparticles; PEG-NP-HS-SD: HS-loaded in pegylated

nanoparticles; ATO-HPCD-NP-SD: atovaquone loaded in hydroxypropyl cyclodextrin-

poly(anhydride) nanoparticles. PDI: polydispersity index.

Formulations Size

(nm) PDI

Zeta potential

(mV) Yields (%)

HS loading

(µg/mg NP)

ATO loading

(µg/mg NP)

NP-HS-SD 141 ± 2 0.18 ± 0.02 - 49.03 ± 0.4 72.3 ± 0.7 34.9 ± 0.4 -

PEG-NP-HS-SD 155 ± 2 0.21 ± 0.01 - 42.3 ± 1.4 74.4 ± 1.2 27.9 ± 2.0 -

ATO-HPCD-NP-SD 305 ± 7 0.12 ± 0.03 - 34.12 ± 3.6 71.8 ± 1.5 - 27.2 ± 0.5

Figure 5 shows the SDS-PAGE of the HS extracted from the two different nanoparticle

formulations. By comparing the apparent molecular weight of the antigenic proteins, it could be seen

that the protein profile and the integrity of the HS extracted from the nanoparticles was similar to

that of free HS and, furthermore, did not reveal the presence of additional protein bands as

consequence of the entrapment and/or spray-drying procedures.

Finally, ATO-loaded nanoparticles displayed a size of about 305 nm with a negative surface

charge. The antiprotozoan was successfully incorporated with values of encapsulation efficiency

higher than 87%.


105

Figure 5. SDS-PAGE and Coomasie blue stain profiles of free and entrapped HS from B. ovis REO

198. Lanes: 1: Molecular Mass Marker Precision Plus ProteinTM KaleidoscopeTM Standards

(BioRad); 2: free HS from B. ovis REO 198; 3: HS extracted from conventional HS-loaded

nanoparticles (NP-HS-SD); 4: HS extracted from pegylated HS-loaded nanoparticles (PEG-NP-HS-

SD).


106

4. DISCUSSION In the present study we have developed a new strategy to facilitate and simplify the

production of poly(anhydride) nanoparticles. These carriers have been currently produced after the

desolvation of the copolymer (dissolved in acetone) with a mixture of ethanol and water and,

subsequent, lyophilization of the resulting nanoparticles (6, 8, 24). Overall a typical production takes

about 50 h. So, in order to simplify and shorten this process we decided to exchange the

lyophilization step by a drying process in a spray-drier apparatus. This strategy is not new and, in the

last years, different authors have been suggested and described it as a real alternative for

lyophilization of microparticles and nanoparticles (11, 20, 27). In fact, the spray-drying method offers

some important advantages such as its rapidity and, in some cases, the possibility to modulate the

physicochemical characteristics of the resulting powder (12).

During the optimization of the spray-drying process of poly(anhydride) nanoparticles, we

noticed two main problems. The former was that after the spray-drying of poly(anhydride)

suspensions, the yield of the process was low and the resulting powders were extremely difficult to

redisperse in water. The latter was that this irreversible process was amplified when ethanol was

present in the nanoparticle suspensions. These observations are in accordance with a previous work

published by Muller and collaborators, in which they observed a similar phenomenon with PCL

nanocapsules (17). They argued the need of an adjuvant (colloidal silicon dioxide 50% w/w) in order

to induce a dilution effect which would reduce the possibilities for nanoparticle interaction and fusion

due to the temperature of the process.

In our case, we decided to incorporate saccharides or polyols in the poly(anhydride)

nanoparticle suspensions. Polyols, mono and disaccharides, are generally considered adequate and

GRAS excipients for a number of pharmaceutical operations, including spray-drying. Moreover, these

excipients may also be interesting in terms of producing dry powders with adequate flow properties,

which can be of use when facilitating the filling of capsules, vials or sachets. In this context, lactose

and maltose (disaccharides), glucose and mannitol (monosaccharides) were chosen for the spray-

drying of poly(anhydride) nanoparticles. In a first approach, it was demonstrated that a

saccharide/poly(anhydride) ratio of at least 2 was necessary to produce a homogeneous reconstitution

of nanoparticles in an aqueous environment. On the other hand, it was also clear that both lactose

and mannitol displayed a higher capacity to protect the spray-dried nanoparticles than glucose or

maltose (Table I). This fact could be related with a combination of the two following factors:

hygroscopicity and glass transition temperature (Tg). In fact, glucose and maltose are very

hygroscopic in their amorphous states and interact strongly with water molecules (12, 28, 29) whereas

lactose and mannitol are considered poorly hygroscopic (28, 30, 31). Therefore, this high ability to

interact with water molecules would be responsible for the production of a more sticky powder when

glucose or maltose rather than mannitol or lactose were used.


107

On the other hand, when either mannitol or lactose were used the resulting powders

demonstrated adequate flow properties. Moreover, nanoparticles produced in the presence of either

lactose or mannitol displayed similar physico-chemical characteristics to those produced by

lyophilization (6, 8, 24). However, when both types of poly(anhydride) nanoparticles were stored for

a long time at room temperature, their behavior after resuspension in water was completely difference

(Figure 3). In fact, nanoparticles produced in the presence of mannitol appeared to aggregate and,

after resuspension in water, displayed a significantly higher size and PDI. This fact may be explained

by the lower Tg of mannitol (of about 13ºC (32)) compared to that of lactose (114ºC (33)). Thus, a

low Tg would promote crystallization during storage and further creation of crystalline bridges (34),

which consequently negatively affected the stability of nanoparticles. In this context, Yoshinari and

collaborators described that the polymorphic transition from δ- to β - form of mannitol is triggered

by moisture with a consequent morphological change in which water molecules would act as a

molecular loosener to facilitate this conversion as a result of multi-nucleation (30). In contrast, the

high Tg of lactose may provide an advantage in the stabilization of dried nanoparticles due to a better

preservation of the amorphous state. Furthermore, taking into account that the poly(anhydride)

nanoparticles are mainly intended for the encapsulation of biologically active molecules, several

studies have suggested that the election of an amorphous carbohydrate as drying excipients may be

more suitable for the preservation of both the structure and activity of therapeutic proteins (35).

Based on these results, lactose was chosen for the preparation of nanoparticle formulations

(Table II). In all cases, nanoparticles prepared by spray-drying can be considered similar to those

produced by lyophilization. Nevertheless, a slight increment on the PDI of nanoparticle batches

produced by spray-drying was noticed.

The applicability of the spray-drying technique was also evaluated with either drug or

antigen-loaded formulations by the incorporation of the HS from Brucella ovis or ATO into

nanoparticles. For HS-loaded nanoparticles, the amount of antigen entrapped was considered high in

spite of pegylation of nanoparticles decreased the antigen loading of the resulting carriers. More

important, the preparative process did affect neither the integrity nor the composition of proteins

included in the HS. In this point, it is interesting to highlight the maintenance of the profile and

integrity of the outer membrane proteins (Omp 31, Omp 25, Omp 22 and L-Omp 19), which are of

particular importance to induce protection against a Brucella infection (21, 36).

On the other hand, ATO is a highly lipophilic drug. For this reason it was necessary to

prepare a drug-cyclodextrin complex in order to increase the drug encapsulation in nanoparticles.

Under these experimental conditions, the drug loading was calculated to be about 27 µg/mg with

encapsulation efficiency close to 90%. These values are similar to those previously reported by

Cauchetier and collaborators related with the encapsulation of this antiprotozoan in PCL

nanocapsules (37).


108

5. CONCLUSIONS

For the preparation of poly(anhydride) nanoparticles by spray-drying procedure, both the

solvent in which these carriers were sprayed as well as the presence of carbohydrates appear to be key

parameters influencing the characteristics of the final product. Thus, for the preparation of

nanoparticles by this procedure the presence of ethanol has to be avoided. On the other hand, lactose

at a saccharide/polymer ratio of 2 appears to provide the best conditions to produce a dry powder

that can be easily reconstituted in a nanoparticle suspension by simple addition of water. More

importantly, the characteristics of nanoparticles produced by spray-drying were similar to those

previously reported and obtained by lyophilization. Finally, the spray-drying procedure can be applied

to the preparation of different types of poly(anhydride) nanoparticles containing sensitive proteins,

antigenic extracts or drugs.

Acknowledgements

This work was supported by grants from the “Instituto de Salud Carlos III” (number:

PI070326; Res. 15/10/2007) and Department of Health of the Government of Navarra (Res.

2118/2007) in Spain. Patricia Ojer was also financially supported by grant from the Education

Department of the Government of Navarra in Spain. We also thanks to Dr. Jon Etxeberria Uranga,

(Materials Department, CEIT, 20018 San Sebastian, Spain) and Prof. Dr. Rafael Jordana (Zoology

and Ecology Department, University of Navarra, 31080 Pamplona, Spain) for SEM photographs.


109

6. References

1. G.A. Hughes. Nanostructure-mediated drug delivery. Nanomedicine. 1:22-30 (2005).





properties of pegylated nanoparticles. Expert Opin Drug Geliv. 2:205-218 (2005).

4. H.H. Salman, C. Gamazo, M.A. Campanero, and J.M. Irache. Salmonella-like bioadhesive

nanoparticles. J Control Release. 106:1-13 (2005).




6. M. Agueros, P. Areses, M.A. Campanero, H. Salman, G. Quincoces, I. Penuelas, and J.M.

Irache. Bioadhesive properties and biodistribution of cyclodextrin-poly(anhydride)

nanoparticles. Eur J Pharm Sci. 37:231-240 (2009).

7. H.H. Salman, C. Gamazo, M.A. Campanero, and J.M. Irache. Bioadhesive mannosylated

nanoparticles for oral drug delivery. J Nanosci Nanotechnol. 6:3203-3209 (2006).



Eur J Pharm Sci. 24:411-419 (2005).

9. B. Magenheim and S. Benita. Nanoparticle characterization: a comprehensive

physicochemical approach. STP Pharma. 1:221-241 (1991).

10. E. Allemann, R. Gurny, and E. Doelker. Drug-loaded nanoparticles - preparation and drug

targeting issues. Eur J Pharm Biopharm. 39:173-191 (1993).

11. C. Freitas and R.H. Mullera. Spray-drying of solid lipid nanoparticles (SLN TM). Eur J

Pharm Biopharm. 46:145-151 (1998).

12. J. Broadhead, S.K.E. Rouan, and C.T. Rhodes. The spray drying of pharmaceuticals. Drug

Dev Ind Pharm. 18:1169-1206 (1992).

13. M. Adler, M. Unger, and G. Lee. Surface composition of spray-dried particles of bovine

serum albumin/trehalose/surfactant. Pharm Res. 17:863-870 (2000).

14. R. Bodmeier and H.G. Chen. Preparation of biodegradable poly(+/-)lactide microparticles

using a spray-drying technique. J Pharm Pharmacol. 40:754-757 (1988).

15. P. Giunchedi, B. Conti, L. Maggi, and U. Conte. Cellulose acetate butyrate and

polycaprolactone for ketoprofen spray-dried microsphere preparation. J Microencapsul.

11:381-393 (1994).


110

16. F. Pavanetto, I. Genta, P. Giunchedi, B. Conti, and U. Conte. Spray-dried albumin

microspheres for the intra-articular delivery of dexamethasone. J Microencapsul. 11:445-454

(1994).

17. C.R. Muller, V.L. Bassani, A.R. Pohlmann, C.B. Michalowski, P.R. Petrovick, and S.S.

Guterres. Preparation and characterization of spray-dried polymeric nanocapsules. Drug Dev

Ind Pharm. 26:343-347 (2000).

18. A. Raffin Pohlmann, V. Weiss, O. Mertins, N. Pesce da Silveira, and S. Staniscuaski Guterres.

Spray-dried indomethacin-loaded polyester nanocapsules and nanospheres: development,

stability evaluation and nanostructure models. Eur J Pharm Sci. 16:305-312 (2002).

19. P. Tewa-Tagne, S. Briancon, and H. Fessi. Spray-dried microparticles containing polymeric

nanocapsules: formulation aspects, liquid phase interactions and particles characteristics. Int J

Pharm. 325:63-74 (2006).

20. P. Tewa-Tagne, S. Briancon, and H. Fessi. Preparation of redispersible dry nanocapsules by

means of spray-drying: development and characterisation. Eur J Pharm Sci. 30:124-135

(2007).

21. R. Da Costa Martins, C. Gamazo, and J.M. Irache. Design and influence of gamma-

irradiation on the biopharmaceutical properties of nanoparticles containing an antigenic

complex from Brucella ovis. Eur J Pharm Sci. 37:563-572 (2009).

22. P.K. Smith, R.I. Krohn, G.T. Hermanson, A.K. Mallia, F.H. Gartner, M.D. Provenzano,

E.K. Fujimoto, N.M. Goeke, B.J. Olson, and D.C. Klenk. Measurement of protein using

bicinchoninic acid. Anal Biochem. 150:76-85 (1985).

23. M.J. Osborn. Studies on the gram-negative cell wall. I. Evidence for the role of 2-Keto-3-

deoxyoctonate in the lipopolysaccharide of Salmonella Thypimurium. Proc Natl Acad Sci USA.

50:499-506 (1963).



(2002).



Biomed Anal. 39:495-502 (2005).

26. V. Zabaleta, M.A. Campanero, and J.M. Irache. An HPLC with evaporative light scattering

detection method for the quantification of PEGs and Gantrez in PEGylated nanoparticles. J

Pharm Biomed Anal. 44:1072-1078 (2007).

27. M.V. Chaubal and C. Popescu. Conversion of nanosuspensions into dry powders by spray

drying: a case study. Pharm Res. 25:2302-2308 (2008).

28. A. Sokolovsky. Effect of humidity on hygroscopic properties of sugars and caramel. J Ind

Eng Chem. 29:1422-1423 (1937).


111

29. S. Jaya and H. Das. Effect of maltodextrin, glycerol monostearate and tricalcium phosphate

on vacuum dried mango powder properties. J Food Eng. 63:125-134 (2004).

30. T. Yoshinari, R.T. Forbes, P. York, and Y. Kawashima. Moisture induced polymorphic

transition of mannitol and its morphological transformation. Int J Pharm. 247:69-77 (2002).

31. P.F. Fox and P.L.H. McSweeney. Advanced Dairy Chemistry. Volume 3: Lactose, water, salts

and minor constituents. Springer Sciences, New York, 2009.

32. L. Yu, D.S. Mishra, and D.R. Rigsbee. Determination of the glass properties of D-mannitol

using sorbitol as an impurity. J Pharm Sci. 87:774-777 (1998).

33. L.S. Taylor and G. Zografi. Sugar-polymer hydrogen bond interactions in lyophilized

amorphous mixtures. J Pharm Sci. 87:1615-1621 (1998).

34. H.K. Chan, A.R. Clark, J.C. Feeley, M.C. Kuo, S.R. Lehrman, K. Pikal-Cleland, D.P. Miller,

R. Vehring, and D. Lechuga-Ballesteros. Physical stability of salmon calcitonin spray-dried

powders for inhalation. J Pharm Sci. 93:792-804 (2004).

35. A. Millqvist-Fureby, M. Malmsten, and B. Bergenstahl. Spray-drying of trypsin - surface

characterisation and activity preservation. Int J Pharm. 188:243-253 (1999).

36. R. Kittelberger, F. Hilbink, M.F. Hansen, G.P. Ross, G.W. de Lisle, A. Cloeckaert, and J. de

Bruyn. Identification and characterization of immunodominant antigens during the course of

infection with Brucella ovis. J Vet Diagn Invest. 7:210-218 (1995).

37. E. Cauchetier, M. Deniau, H. Fessi, A. Astier, and M. Paul. Atovaquone-loaded

nanocapsules: influence of the nature of the polymer on their in vitro characteristics. Int J

Pharm. 250:273-281 (2003).

CHAPTER 5

Cytotoxicity and cell interaction studies of bioadhesive poly(anhydride) nanoparticles for oral antigen/drug

delivery

115

Cytotoxicity and cell interaction studies of bioadhesive poly(anhydride) nanoparticles for oral

antigen/drug delivery

Patricia Ojer 1, Lukas Neutsch 2, Franz Gabor 2, Juan Manuel Irache 1, and Adela López de Cerain 3

1 Department of Pharmacy and Pharmaceutical Technology. University of Navarra, Irunlarrea 1, E-31008

Pamplona, Spain. 2 Department of Pharmaceutical Technology and Biopharmaceutics, University of Vienna, Pharmacy Center,

Althanstrasse 14, A-1090 Vienna, Austria. 3. Department of Pharmacology and Toxicology, University of Navarra, Irunlarrea 1, E-31008 Pamplona, Spain.

ABSTRACT

The use of bioadhesive polymers as nanodevices has emerged as a promising strategy for oral delivery

of therapeutics. In this regard, poly(anhydride) nanoparticles have shown great potential for oral drug

delivery and vaccine purposes. However, despite extensive research into the biomedical and

pharmaceutical applications of poly(anhydride) nanoparticles, there are no studies to evaluate the

interaction of these nanoparticles at a cellular level. Therefore, the main objectives of this study were

to evaluate the cytotoxicity as well as the cell interaction of different poly(anhydride) nanoparticles:

conventional (NP), nanoparticles containing 2-hydroxypropyl-β-cyclodextrin (NP-HPCD) and

nanoparticles coated with polyethylene glycol 6000 (PEG-NP). For this purpose, nanoparticles were

prepared by solvent displacement method and labelled with BSA-FITC (Bovine Serum Albumin-

Fluorescein Isothiocyanate conjugate). Nanoparticles displayed a size about 175 nm with negative

surface charge. Cytotoxicity studies were developed by MTS and LDH assays in HepG2 and Caco-2

cells. Results showed that only in HepG2 cells, NP and NP-HPCD induced significant cytotoxicity at

the highest concentrations (1 and 2 mg/mL) and incubation times (48 and 72 h) tested. Studies to

discriminate between cytoadhesion and cytoinvasion were performed at 4ºC and 37ºC in Caco-2 cell

line as intestinal cell model. Nanoparticles showed cytoadhesion to the cell surface but not

internalization; PEG-NP was the most bioadhesive followed by NP-HPCD and NP as demonstrated

by flow cytometry. Finally, cellular localization of particles by fluorescence confocal microscopy

confirmed the association of these nanoparticles with cells. Thus, this study demonstrated the safety

of NP, NP-HPCD and PEG-NP at cellular level and its bioadhesive properties within cells.

Published in Journal of Biomedical Nanotechnology 9(11):1891-1903 (2013).

CHAPTER 5: Cytotoxicity and cell interaction studies of bioadhesive poly(anhydride) nanoparticles for oral antigen/drug delivery

117

1. INTRODUCTION

Nanotechnology is one of the fastest growing sectors of the high-tech economy. In the last

two decades it has been widely used in the pharmaceutical industry, medicine and engineering

technology (1, 2). However, while the number of nanotechnological products continues to increase,

studies to characterize their effects after exposure and address their potential toxicity are scarce. Due

to the nanometer size of these products, their properties differ substantially from those bulk materials

of the same composition (3). These properties which make materials on a nanometer scale so

attractive, such as small size, chemical composition, structure, large surface area and shape may

contribute to the toxicological profile in biological systems (4). In this regard, the toxicity of metal-

based nanoparticles, ultrafine particles, asbestos fibers and dust particles has been largely studied,

particularly in lung injury (3, 5, 6). The harmful effects observed for these nanoparticles were

correlated with common biological mechanisms such as inflammation and oxidative stress. Thus,

Donalson and collaborators proved that nanoparticulate forms (< 50 nm) of titanium oxide,

aluminum oxide and carbon black increased the pulmonary toxicity inflammation parameters 10

times more than administration of fine particles of the same products (7). On the other hand, Pan

and co-workers demonstrated in four different cell lines that one of the smallest gold nanoparticles

tested has greater toxicity than other gold nanoparticles with higher sizes (8). Furthermore, Hohr and

co-workers observed an increase in pulmonary inflammatory reaction in rats after inhalation of the

nanoparticulate form of TiO2 in comparison with the microparticulate form (9). Taking into account

the above-mentioned, it has been further shown that the toxicological profile and biological response

to the different nanoparticles are closely related with their physicochemical properties.

Regarding to nanomedicine - the application of nanotechnology to healthcare -, exposure to

nanoparticles for medical purposes involves intentional contact or administration; therefore,

understanding the properties of nanoparticles, evaluate their cytotoxicity as well as their behavior in

adequate cell models and perform in vivo toxicity and biodistribution studies are crucial before clinical

use. However, the potential adverse effects of the nanoparticles have not been extensively studied,

possibly because in nanomedicine, mainly biodegradable, biocompatible and/or pharmaceutical

approved materials are used (10, 11).

In this context, Poly(methyl vinyl ether-co-maleic anhydride) [Gantrez® AN] nanoparticles

have been considered promising platforms for drug delivery and other biomedical applications. In the

last years, poly(anhydride) nanoparticles have successfully been developed as oral drug delivery

systems (12), immunization (13) or allergy treatment (14). In addition, surface modification of these

carriers can be easily carried out by simple incubation between the polymer (or the plain

nanoparticles) and molecules showing hydroxyl or amino groups. For instance, coating

poly(anhydride) nanoparticles with hydrophilics compounds such as PEGs is an adequate strategy to

obtain bioadhesive carriers for mucosal delivery and to target the small intestine (15). Furthermore,

cyclodextrin- poly(anhydride) nanoparticles have been proposed as effective carriers to both develop


118

intense bioadhesive interactions between the gut and increase loading capacity of lipophilic drugs

such as paclitaxel (16). However, although there are plenty of studies supporting the efficacy of these

polymeric nanoparticles, the role of nanoparticle physicochemical properties on their in vitro toxicity

and their interaction with cells needed to be investigated thoroughly. Thus, the aim of this work was

to evaluate in vitro toxicity by means of MTS and LDH assays of plain, pegylated and cyclodextrin

poly(anhydride) nanoparticles using two different cell lines: the human hepatoma cell line HepG2 and

the human colon carcinoma cell line Caco-2. In addition, interaction studies between nanoparticles

and Caco-2 cells (single cells and monolayers) were carried out using a combination of flow

cytometry and fluorescence microscopy.


119



Poly(methyl vinyl ether-co-maleic anhydride) or poly(anhydride) (Gantrez® AN 119; Mw

200,000) was provided by ISPcorp. (Waarwijk, The Netherlands). Polyethylene glycol 6000

(PEG6000) was provided by Fluka (Switzerland). 2-hydroxypropyl-β-cyclodextrin (HPCD) was

provided by Sigma–Aldrich (Steinheim, Germany). Acetone was obtained from VWR Prolabo

(Fontenay-sous-Bois, France). Deionized water (18.2MΩ resistivity) was prepared by a water

purification system (Wasserlab, Pamplona, Spain). BSA-FITC and lactose were purchased from

Sigma (St. Louis, MO. United States). All other chemicals and solvents used were of analytical grade.


All types of nanoparticles were prepared by desolvation of the polymer in acetone and dried

in a Mini Spray-dryer Büchi B290 (Büchi Labortechnik AG, Switzerland) as described previously (17).

2.2.1. Conventional poly(anhydride) nanoparticles (NP)

Briefly, 500 mg of poly(anhydride) were dissolved and stirred in 30 mL acetone. Then, the

desolvation of the polymer was induced by the addition of 15 mL purified water under magnetic

stirring to the organic phase. In parallel, 1 g of lactose was dissolved in 10 mL purified water and

added to the nanoparticle suspension under agitation for 5 min at room temperature. Finally, the

suspension was dried in the Mini Spray-dryer Büchi B290. The recovered powder was stored in

closed vials at room temperature.

2.2.2. Poly(anhydride) nanoparticles containing 2-hydroxypropyl-β-cyclodextrin (NP-HPCD)

Briefly, 500 mg of poly(anhydride) were dissolved and stirred in 20 mL acetone. Then, 10 mL

acetone containing 125 mg HPCD were added to the polymer solution under magnetic stirring and

incubated for 30 min. Nanoparticles were obtained by the addition of 15 mL purified water under

magnetic stirring to the organic phase. In parallel, 1 g of lactose was dissolved in 10 mL purified

water and added to the nanoparticle suspension under agitation for 5 min at room temperature.

Finally, the suspension was dried in the Mini Spray-dryer Büchi B290. The recovered powder was

stored in closed vials at room temperature.

2.2.3. Pegylated poly(anhydride) nanoparticles (PEG-NP)

In this case, 62.5 mg PEG 6000 were dissolved in 15 mL acetone and mixed with 15 mL

acetone containing 500 mg poly(anhydride) and incubated under magnetic stirring for 1 h. Then,

nanoparticles were obtained by the addition of 15 mL purified water under magnetic stirring to the

organic phase. The solvents were eliminated under reduced pressure (Büchi R-144, Switzerland) and

nanoparticles were purified by double centrifugation at 17,000 rpm for 20 min (Sigma 3K30,


120

Germany). The pellet was resuspended in 25 mL purified water containing 1 g of lactose. Finally, the

suspension was dried in the Mini Spray-dryer Büchi B290. The recovered powder was stored in

closed vials at room temperature.

2.3. Preparation of BSA-FITC poly(anhydride) nanoparticle conjugates

To assess the association of poly(anhydride) nanoparticles with Caco-2 cells, fluorescently

labelled nanoparticles were prepared by encapsulation of BSA-FITC in the nanoparticles. For this

purpose, 2.5 mg BSA-FITC was incubated with the polymer (and eventually with HPCD or PEG

6000) in acetone for 30 min. at room temperature. Then nanoparticles were obtained and purified as

described above (see section 2.2.).

2.4. Characterization of the nanoparticles

2.4.1. Size and zeta potential

The particle size and the zeta potential of nanoparticles were determined by photon

correlation spectroscopy (PCS) and electrophoretic laser Doppler anemometry, respectively, using a

Zetamaster analyzer system (Malvern Instruments Ltd., Worces-tershire, United Kingdom) and a

ZetaPlus zeta potential analyzer (Brookhaven Instruments Corp., Holtsville, NY). The diameter of

the nanoparticles was determined after dispersion in purified water (1/10) and measured at 25 ºC by

dynamic light scattering angle of 90 ºC. The zeta potential was determined by diluting the samples in

a 0.1 mM KCl solution adjusted to pH 7.4. All measurements were performed in triplicate.

The morphological examination of the nanoparticles was obtained by scanning electron microscopy

(SEM) in a Zeiss DSM 940A scanning electron microscope (Oberkochen, Germany) coupled with a

digital image system (DISS) Point Electronic GmBh. Previously, a small amount of the spray-dried

powders was diluted with purified water and the resulting suspension was centrifuged at 17,000 rpm

(Sigma 3K30, Germany) for 20 min in order to eliminate sugars. Finally, the pellet was shaded with a

12 nm gold layer in a Hemitech K 550 Sputter-Coater.

2.4.2. Quantification of HPCD and PEG6000

The amount of HPCD and PEG6000 associated to the nanoparticles as well as the amount

of poly(anhydride) transformed into nanoparticles were estimated using two different high

performance liquid chromatography (HPLC) methods previously described (18, 19). Specifically the

apparatus used for the HPLC analysis was a model 1100 series Liquid Chromatography, Agilent

(Waldbronn, Germany) coupled with an evaporative light scattering detector (ELSD) 2000 Alltech

(Illinois, United States). An ELSD nitrogen generator Alltech was used as the source for the nitrogen

gas. Data acquisition and analysis were performed with a Hewlett-Packard computer using the

ChemStation G2171 AA program.


121

The amount of HPCD and PEG associated to nanoparticles was calculated as the difference

between the initial HPCD and PEG and the amount of those recovered in the supernatants.

Similarly, the amount of poly(anhydride) was estimated by the difference in the same way.

2.5. Cell Culture

Human hepatoma cell line HepG2 and the colon carcinoma cell line Caco-2 were obtained

from the ATCC collection (Maryland, United States). HepG2 cells were grown in DMEM

(Dulbecco´s modified Eagle´s medium) supplemented with 10% fetal bovine serum and 1%

penicillin-streptomycin. Caco-2 cells were grown in RPMI-1640 with 10% fetal bovine serum, 4 mM

L-glutamine and 150 µg/mL gentamycine. Tissue culture reagents were from Sigma (St. Louis, MO,

United States) and Gibco Life Technologies Ltd. (United Kingdom). Cells were maintained under

standard cell culture conditions (5% CO2, 95% humidity and 37ºC) and subcultured by trypsinization.

2.5.1. Cytotoxicity assays

To test the cytotoxicity induced by the different poly(anhydride) nanoparticles, MTS and

LDH assay were carried out in both HepG2 and Caco-2 cell lines. Cells were cultured in 96-well

plates (Falcon®, United States). The optimum cell concentration as determined by the growth profile

of the cell lines was 2.5 x 104 cells/mL (100 µL of cell solution/well). All tests were performed using

sub-confluent cells in the logarithmic growth phase. In conducting the experiments it was used

culture media without serum to prevent interactions between serum proteins and nanoparticles.

MTS and LDH assays were performed in samples containing nanoparticles and culture

medium only (without cells). The results demonstrated no interaction between nanoparticles and the

different assays components.

The stock solution of nanoparticles was performed in Dulbecco´s Phosphate Buffered Saline

(DPBS) w/o calcium and magnesium. Before the nanoparticles addition, cell monolayers were

washed with DPBS w/o calcium and magnesium. The following concentrations were used: 0.0625,

0.125, 0.25, 0.5, 1 and 2 mg/mL for 4, 24, 48 and 72 h. The concentration range for the nanoparticle

formulations and the times of exposure were selected based on literature information (20, 21) as well

as on preliminary studies performed in our laboratory. Assays results were confirmed by microscopic

observation. Cell morphology was assessed with a Nikon Eclipse TS-100 phase-contrast microscope.

2.5.1.1. Mitochondrial function

Cell viability was evaluated by measuring mitochondrial function with the MTS assay

(CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay) purchased from Promega (Madison,

United States) that is based on the protocol described for the first time by Mossman (22). In brief,

cells were treated with different nanoparticle formulations for the indicated concentrations and

incubation times. The control received culture medium only. After the treatment, the cultured media

containing the nanoparticles were removed, cells were washed with DPBS and 100 µL of fresh media


122

were added to each well. MTS was added to the cultures and the assay was conducted according to

the manufacturer`s instructions (Cell Titer 96® AQueous Non-Radioactive Cell Proliferation Assay

Technical Bulletin #TB169, Promega Corporation). Finally, absorbance readings were carried out at a

wavelength of 490 nm using a microplate spectrophotometer system (Spectra MR™ microplate,

DYNEX). The relative cell viability of each nanoparticle formulation (%), corrected by the

background absorbance of the culture medium, was calculated as follows: [A]sample/[A]control X

100. Where [A]sample is the absorbance of the test sample and [A]control is the absorbance of

control sample. Cell survival values above 70% were considered as non-cytotoxic.

2.5.1.2. LDH leakage

Cytotoxicity of nanoparticles was assessed by measuring membrane integrity with the LDH

assay (Cytotox 96® Non-Radioactive Cytotoxicity Assay) provided by Promega (Madison, United

States). Briefly, after the cells were exposed to nanoparticles (NP, NP-HPCD and PEG-NP) at the

concentrations and incubations times mentioned, the cultured media were collected and the LDH

activity was determined using the LDH assay. In order to calculate LDH max, 10 µL of lysis solution

(10X) was added to the control wells. The absorbance of each sample was spectrophotometrically

measured at 490 nm with a microplate spectrophotometer system (Spectra MR™ microplate,

DYNEX). The relative cytotoxicity of each formulations (% LDH leakage), corrected by the

background absorbance of the culture medium, was calculated as follows: [A]sample/[A]LDH max X

100. Where [A]sample is the absorbance of the test sample and [A]LDH max is the absorbance of the

LDH max.

2.6. Nanoparticle interaction studies

2.6.1. Association assay to Caco-2 monolayers

The interaction of nanoparticle formulations with Caco-2 monolayers was used as a tool to

study the association of the drug delivery systems with an established cell culture model for the

gastrointestinal epithelium.

Cells were cultured for 10-14 days on 96-well plates at a density of 1.7 x 104/well. The

monolayers were washed three times with PBS (Phosphate Buffered Saline) and incubated with the

different fluorescently labelled formulations at a nanoparticle concentration of 0.5 mg/mL (BSA-

FITC labelled NP, NP-HPCD and PEG-NP). After removing the supernatants, non-stably cell-

associated nanoparticles were removed by washing three times with 100 µL PBS each. Then the cell-

associated fluorescence was determined using a microplate fluorescence reader (Spectrafluor

Fluorometer, Tecan, Austria) set to λex= 485 nm and λem= 520 nm.

In order to discriminate between particle association (cytoadhesion and cytoinvasion) and

particle binding to the cell surface (cytoadhesion), experiments were performed at either 37 or 4ºC.


123

2.6.2. Flow cytometry

To evaluate the amount of nanoparticles associated with single cells, 50 µL cell suspension (5

x 106 cells/mL) were mixed with 50 µL nanoparticle suspension and incubated for 1 h at 4ºC.

Unbound and loosely associated particles were removed by centrifugation and washing with 400 µL

PBS (5 min, 1000 rpm, 4ºC). The cell pellet was resuspended in 1000 µL PBS and analyzed on an

EPICS XL-MLC analytical flow cytometer (488/525 nm; Beckman Coulter, Brea, United States)

using a forward versus side scatter gate for inclusion of the single-cell population and exclusion of

debris and cell aggregates. Intact single cells are located in gate A and gate E, whereas cell fragments

are detected in gate B. To verify that cell viability was not compromise by nanoparticles addition, cells

without any addition of nanoparticles (PBS-treated cells) were carried along as a negative control in

every measurement. A minimum of 2000 cells was acquired per run, and triplicate samples were

analyzed for each experiment. The mean channel number of the logarithmic fluorescence intensity of

the individual peaks was used for further calculations.

2.6.3. Fluorescence microscopy

To visualize the binding characteristics of the fluorescent labelled nanoparticles to Caco-2,

images of both, single cells and monolayers were obtained using a Zeiss Axio Observer.Z1

microscopy system using LD Plan-Neofluoar objectives equipped with phase contrast and differential

interference contrast (DIC) equipped with LED illumination system “Colibri”.

2.6.4. Inmunofluorescence

Caco-2 monolayers were grown on glass cover slides placed in 24-well plates and washed

twice with 500 µL PBS prior to the assay. After 1 h incubation with the different fluorescently

labelled nanoparticles at 4ºC in PBS the monolayers were fixed with methanol at -20ºC, cell layers

were washed and rehydrated. The tight junction associated protein ZO-1 was stained for 1 h at room

temperature with a primary antibody (610966; BD Biosciences, San Jose, United States) diluted 1:100

in a 1% solution of BSA in PBS. Upon washing thrice with PBS-BSA (1% v/v) solution, the

monolayers were incubated with a 1:100 dilution of a secondary goat anti-mouse immunoglobulin-

RPE antibody (Dako Denmark A/S, Glostrup, Denmark) and with a dilution 1:20 of Hoechst 33342

nucleic acid stain (Invitrogen), respectively for 30 min at room temperature. Finally cell layers were

washed three times and mounted for microscopy using a drop of FluorSave (FluorsaveTM Reagent,

345789, Calbiochem®, Merck Biosciences) on a slide. Corresponding negative controls were

performed in the same way applying PBS-BSA solution without the primary antibody.

Inmunofluorescence images of labelled nanoparticles and cells were obtained by fluorescence

microscopy.


124


All experiments were performed in triplicate, and the results were presented as mean ±

standard deviation. The experimental data obtained in cytotoxicity studies were analyzed using non-

parametric test "Kruskal Wallis" and "U Mann-Whitney" with Bonferroni adjustment. p-values less

than 0.05 were considered as a statistically significant difference. All calculations were performed

using SPSS® statistical software program (SPSS® 15, Microsoft, United States).


125

3. RESULTS

3.1. Nanoparticles characterization

The main physicochemical characteristics of poly(anhydride) nanoparticles formulations are

summarized in Table I. All formulations exhibited a homogeneous size of about 175 nm, with a

polydispersity index (PDI) less than 0.3 and a negative zeta potential. Nanoparticles size was also

determined in PBS and cell culture medium and no significant change in mean size was observed

(data not shown). This is in agreement with previously obtained results (23).

Table I. Physico-chemical characteristics of nanoparticles. Data expressed as the mean ± standard

deviation (n=6). NP: poly(anhydride) nanoparticles; NP-HPCD: hydroxypropyl cyclodextrin-

poly(anhydride) nanoparticles; PEG-NP: pegylated nanoparticles. PDI: polydispersity index.

The morphological analysis by scanning microscopy (Figure 1.) showed that NP, HPCD-NP

and PEG-NP consisted of a homogeneous population of spherical particles with a smooth surface in

case of NP and in contrast with rough surface in case of HPCD-NP and PEG-NP. For NP-HPCD,

the amount of associated cyclodextrin was calculated to be about 72 µg/mg whereas for PEG-NP the

amount of PEG associated to nanoparticles was about 56 µg/mg. In addition, more than 95% of

poly(anhydride) was transformed into nanoparticles for all the formulations.

Size

(nm) PDI Zeta Potential

(mV)

Np formation yield

(%) µg ligand/mg Np

NP 167 ± 1 0.11 ± 0.04 -44.6 ± 2.5 97 ± 1.1

NP-HPCD 172 ± 3 0.23 ± 0.02 -36.1 ± 1.7 98 ± 1.2 71.9 ± 3.1

PEG-NP 185 ± 2 0.19 ± 0.06 -32.1 ± 2.5 96 ± 2.2 55.6 ± 2.2


126

Figure 1. SEM photographs of nanoparticles. (A) NP. (A.1) Magnification of a section of

photograph (A). (B) NP-HPCD. (B.1) Magnification of a section of photograph (B). (C) PEG-NP.

(C.1) Magnification of a section of photograph (C).


127

3.2. Mitochondrial Function (MTS assay)

The effect of poly(anhydride) nanoparticle formulations in the mitochondrial function and by

extension the relative cell viability of HepG2 and Caco-2 cells is shown in Figure 2. All nanoparticle

formulations showed no significant cytotoxic effect on Caco-2 viability while NP and NP-HPCD

exhibit significant reductions in relative HepG2 viability (<70%). Thus, cell survival in Caco-2 was

over 70% for all the concentration range and incubation times for both, NP, NP-HPCD and PEG-

NP, although at the highest concentrations tested and long incubation times (48 and 72h) there is a

decrease in cell survival. On the contrary, in HepG2, NP and NP-HPCD showed dependent

significant viability decrease at longer incubations times. In fact, cell viability was below 30% for NP

and NP-HPCD following 72 h exposure at the highest concentration tested (2 mg/mL). Moreover

NP-HPCD showed a significant increase in cytotoxicity at 1 mg/mL after 48 h. The results of the

assay also showed a slight viability decrease in cells treated with PEG-NP following 72 h exposure at

2 mg/mL.

After 48 h and 72 h, the cytotoxic effect appeared to be concentration dependent for all the

nanoparticles tested in both cell lines.

3.3. LDH leakage

Figure 3 shows the effect of poly(anhydride) nanoparticles on relative LDH toxicity. Similarly

to the results obtained in the MTS assay not significant increase in LDH release was observed in

Caco-2 cells compared to controls, where the relative LDH release was always around 40%. With

regard to results obtained in HepG2 cells, these also corresponded with results obtained in MTS

assay. Following 48 and 72 h exposure, the LDH leakage gradually increased for NP and NP-HPCD

formulations at the highest concentrations (1-2 mg/mL), reaching approximately 80% of relative

LDH release. Furthermore, a slight increase in LDH leakage was observed with PEG-NP but it was

not significant compared to controls.


128

Figure 2. Effect of nanoparticles on the viability of HepG2 and Caco-2 cells measured by MTS assay

following 4, 24, 48 and 72 h incubation at various nanoparticle concentrations. (A)(D) NP. (B)(E)

NP-HPCD. (C)(F) PEG-NP. Results presented as % viability relative to controls and expressed as

the mean ± SD (n=6). * Statistically significant difference compared with controls (p<0.05).

4 h 24 h 48 h 72 h

0.062

50.1

250.2

50 0.5 1 20

20

40

60

80

100

120

140

mg/mL

Rel

ativ

e ce

ll vi

abili

ty (%

)

C

Hep

G2

0.062

50.1

250.2

50 0.5 1 20

20

40

60

80

100

120

140

mg/mL

Rel

ativ

e ce

ll vi

abili

ty (%

)

A

* *

0.062

50.1

250.2

50 0.5 1 20

20

40

60

80

100

120

140

mg/mL

Rel

ativ

e vi

abili

ty (%

)

* *

B

**

4 h 24 h 48 h 72 h

0.062

50.1

250.2

50 0.5 1 20

20

40

60

80

100

120

140

mg/mL

Rel

ativ

e ce

ll vi

abili

ty (%

)

E0.0

625

0.125

0.250 0.5 1 2

0

20

40

60

80

100

120

140

mg/mL

Rel

ativ

e ce

ll vi

abili

ty (%

)

D

0.062

50.1

250.2

50 0.5 1 20

20

40

60

80

100

120

140

mg/mL

Rel

ativ

e ce

ll vi

abili

ty (%

)

F

Cac

o2


129

Figure 3. Influence of nanoparticles on the membrane integrity of Caco-2 cells, measured by LDH

assay after 4, 24, 48 and 72 h incubation at various nanoparticle concentrations. (D) NP. (E) NP-

HPCD. (F) PEG-NP. * Statistically significant difference compared with controls (p<0.05). Results

presented as % LDH release relative to controls and expressed as the mean ± SD (n=6).

4 h 24 h 48 h 72 h

CONTROL

0.062

50.1

250.2

50 0.5 1 20

20

40

60

80

100

mg/mL

Rel

ativ

e LD

H r

elea

se (%

)

C

CONTROL

0.062

50.1

250.2

50 0.5 1 20

20

40

60

80

100

mg/mL

Rel

ativ

e LD

H r

elea

se (%

)

A*

**

CONTROL

0.062

50.1

250.2

50 0.5 1 20

20

40

60

80

100

mg/mL

Rel

ativ

e LD

H r

elea

se (%

)

B

*

**

*

Hep

G2

4 h 24 h 48 h 72 h

CONTROL

0.062

50.1

250.2

50 0.5 1 20

20

40

60

80

100

mg/mL

Rel

ativ

e LD

H r

elea

se (%

)

ECONTR

OL

0.062

50.1

250.2

50 0.5 1 20

20

40

60

80

100

mg/mL

Rel

ativ

e LD

H r

elea

se (%

)

D

CONTROL

0.062

50.1

250.2

50 0.5 1 20

20

40

60

80

100

mg/mL

Rel

ativ

e LD

H r

elea

se (%

)

F

Cac

o2


130

3.4. Nanoparticle/cell interaction on Caco-2 monolayers

To evaluate the interaction of the nanoparticles with the gastrointestinal epithelium,

fluorescence-labelled nanoparticles were incubated on Caco-2 monolayers to determine their

behavior in an in vitro intestinal epithelium model.

In preliminary tests, BSA-FITC was identified as a suitable marker molecule and the different

fluorescence-labelled formulations were prepared. To ensure that the fluorescence quantified and

visualized in cells was due to BSA-FITC that is stably associated/entrapped with/in nanoparticles,

the release of BSA-FITC from nanoparticles was studied over 4 h in PBS. Since the release was

found to lie well below 10%, it can be assumed that the fluorescence measured was mainly due to the

cell-associated nanoparticles.

For the cytoadhesion studies in Caco-2, nanoparticles concentration of 0.5 mg/mL was used.

The selection was based on the absence of toxicity at this nanoparticle concentration, as determined

by the MTS and LDH assays.

Figure 4 illustrates the association of BSA-FITC labelled NP, NP-HPCD and PEG-NP with

Caco-2 monolayers at either 4ºC or 37ºC following 4 h incubation. According to the results at both

temperatures the cell-associated fluorescence intensity decreased with time for all nanoparticle

formulations. In fact, the fluorescence intensity decreased more markedly after 3 h incubation at

37ºC, especially for NP and NP-HPCD.

In case of NP, a decrease of 40% of the starting values was observed at 37ºC within 3 h

incubation while at 4ºC the decrease was about 13%. Thereafter, the rate of decline was reduced

resulting in a cell-associated fluorescence intensity of 21% at 4ºC and 45% at 37ºC after 4 h. For NP-

HPCD, at 37ºC and 4ºC the decrease in cell associated fluorescence was about 15% for both

temperatures following 3 h incubation and remained constant after 4 h. Similar results were obtained

for PEG-NP but at 37ºC the remarkable decrease in cell associated fluorescence intensity took place

within 2 h incubation (about 20%) whereas at 4ºC the decline occurred during the first 3 h.

To visualize the interaction of nanoparticles in Caco-2 monolayers fluorescence microscopy

images were obtained at the end of the association assay. Figure 4 shows the association of BSA-

FITC labelled NP-HPCD with Caco-2 monolayers at both temperatures, following 4 h incubation

and extensive cells washing. Green fluorescence, arising from BSA-FITC labelled nanoparticles was

found distributed throughout the imaged area of the cell monolayer. The fluorescence appears to

follow the contour of cells on the apical surface.


131

Figure 4. Influence of the temperature on the association of nanoparticles to Caco-2 monolayers

loaded with 0.5 mg/mL of BSA-FITC labelled nanoparticles. (a) NP at 37ºC and (b) at 4ºC following

4 h incubation. Data expressed as the mean ± standard deviation (n=3).

0 60 120 180 2400

300

600

900

NP 37ºC

NP 4ºC

Time (min)

Cel

l-ass

ocia

ted

mea

n flu

ores

cenc

e in

tens

ity

0 60 120 180 2400

300

600

900

HPCD-NP 37ºC

HPCD-NP 4ºC

Time (min)

Cel

l-ass

ocia

ted

mea

n flu

ores

cenc

e in

tens

ity

0 60 120 180 2400

300

600

900

PEG-NP 37ºC

PEG-NP 4ºC

Time (min)

Cel

l-ass

ocia

ted

mea

n flu

ores

cenc

e in

tens

ity

(a)

(b)


132

A: 65.2% B: 3.9% E: 30.3%

C

A: 72.7% B: 4.4% E: 20.2%

A

A: 63.6% B: 4.2% E: 29.1%

D

A: 70.3% B: 5.4% E: 27.8%

B

3.5. Interaction between BSA-FITC nanoparticles and Caco-2 single cells

To clarify whether the surface modification of nanoparticles modify the interaction with

Caco-2 cells, the different formulations of BSA-FITC labelled nanoparticles were examined with

respect to their association to the cells, the viability and the agglomeration of Caco-2 single cells after

binding. For this purpose 0.5 mg/mL of nanoparticles were incubated with single cells for 1 h at 4ºC

followed by flow cytometric analysis. These results are summarized in Figure 5.

Figure 5. Forward versus side scatter histograms of Caco-2 single cells. Control cells (A); cells treated

with BSA-FITC labelled NP (B); NP-HPCD (C) and PEG-NP (D) after 1 h incubation at 4ºC.


133

Regarding cell viability, without nanoparticles addition, 73% of the cells were detected in gate

A (single cells), 20 % in gate E (agglomerated cells) and 4.4% in gate B (debris, Figure 5). These

results were similar to those obtained with nanoparticles. Thus the influence of the different BSA-

FITC labelled nanoparticles on viability of Caco-2 single cells after 1 h exposure, is suggested to be

negligible.

Taking into account the cell-bound fluorescent intensity (refers to gate A), the values

obtained were 4.32 ± 2.14, 7.12 ± 1.85 and 9.28 ± 2.65 for BSA-FITC labelled NP, NP-HPCD and

PEG-NP respectively. Therefore, although no differences were observed between nanoparticles

formulations in Caco-2 monolayer experiments according to quantitative single cell experiments

PEG-NP and NP-HPCD showed higher cell binding than NP.

3.6. Fluorescence Microscopy of Caco-2 single cells

Complementary information on the association of nanoparticles by Caco-2 cells was

collected via fluorescence microscopy. Single cells were incubated with the nanoparticles under the

same experimental conditions mentioned above. As illustrated in Figure 6, only low amounts of NP

(Fig. 6A) were associated with the cell membrane. In contrast, for NP-HPCD and PEG-NP, high

amount of nanoparticles were bound to the cell surface (Figure 6B and C).

3.7. Confocal Laser Scanning Microscopy of Caco-2 Monolayers

To corroborate the results from the cellular association assays and to visualize nanoparticle

binding as well as tight junctions integrity in Caco-2 monolayers, confocal laser scanning microscopy

(CLSM) was used (Figure 7). Cells were double-stained with anti-ZO1 (tight junctions, red) and

Hoechst 33342 (nuclei, blue). Related to tight junctions integrity, a continuous ring of red

fluorescence, arising from ZO-1 staining, at cell-cell contacts is clearly visible in the control cell layer

(Figure 7A). Fluorescence at the point of contact between the cells can also be seen in cell layers

incubated with nanoparticles (Figure 7B-D), therefore, nanoparticles addition to cells did not affect

tight junctions integrity. Regarding nanoparticle-cell interactions, homogenous distribution of green

fluorescence, arising from BSA-FITC labelled nanoparticles, at cell membrane is visible for the three

nanoparticle formulations. However, slight differences are observed. Thus, for NP-HPCD and PEG-

NP the amount of membrane-bound nanoparticles seems to be higher than for NP.


134

A.1 A.2

B.1 B.2

C.1 C.2

Figure 6. Confocal images of Caco-2 single cells after incubation with BSA-FITC labelled NP (A),

NP-HPCD (B), and PEG-NP (B). The left column shows transmission images (A.1, B.1 and C.1) and

the right one represents the fluorescence images (A.2, B.2, and C.2). A 50 µl cell suspension (5 x 106

cells/mL) was incubated for 1 h at 4ºC.


135

Figure 7. Association of BSA-FITC nanoparticles with Caco-2 monolayers. Green: BSA-FITC-

labelled nanoparticles, blue: Hoechst 33342-labelled nuclei and red: tight junction associated protein

ZO-1. (A) Control cell layers not subjected to nanoparticle incubation. (B) NP, (C) NP-HPCD and

(D) PEG-NP.

A

B

C

D


136

4. DISCUSSION

The employment of bioadhesive polymers as nanoparticulate systems has been mostly used

towards application to oral drug delivery. These nanosystems provide some advantages compared to

oral delivery of no encapsulated drugs. They can protect the therapeutic agent from the harsh

environment of the GIT (Gastrointestinal tract) and increase in situ residence time and intimate

contact with intestinal mucosa (24).

In this context, poly (anhydride) nanoparticles have demonstrated great potential to develop

interactions within the GIT. The presence of anhydride residues confers the ability to modify its

surface with different molecules or ligands which allows to vary their biodistribution within the gut

or/and their bioadhesive potential. As a result these nanoparticles may improve the oral

bioavailability of different drugs such as paclitaxel (25), atovaquone (ATO) (26) or fluorouridine (27)

and/or facilitate the interaction and presentation of the loaded antigen or allergen with the antigen-

presenting cells (APCs) (28).

The promising results obtained with these nanoparticles encouraged us to evaluate their

cytotoxicity and cell-interaction. The following types of poly(anhydride)nanoparticles have been

studied: conventional nanoparticles (NP), nanoparticles containing 2-hydroxypropyl-β-cyclodextrin

(NP-HPCD-) and pegylated nanoparticles (PEG-NP).

The present work was performed with nanoparticles of well-defined sizes of about 160 nm

and narrow size distribution (PDI<0.3) with negative surface charge. Nanoparticles morphology was

found to be affected by the association of either HPCD or PEG as shown in Figure 1. This could

mean that formulations containing either HPCD or PEG have larger surface area than conventional

nanoparticles to establish bioadhesive interactions. Hence rougher nanoparticles should interact in a

larger extent than smooth ones.

Assessing nanoparticles toxicity at cellular level is an important issue to consider for

evaluating their potential bioactivity and a first step before testing toxicity in vivo. Furthermore, select

adequate cell models to perform in vitro studies are critical to obtain valuable results that complement

in vivo studies. In this context, human hepatocarcinoma-derived HepG2 cells and human colonic

adenocarcinoma-derived Caco-2 cells were chosen as models of two important targets for

nanoparticles toxicity upon oral exposure like the liver and the gastrointestinal epithelium. The

human-derived Caco-2 cell model has been widely used in in vitro studies of epithelial

transport/interaction processes, due to its homology to human intestinal epithelial absorptive

enterocytes (29, 30). Furthermore, the International Life Sciences Institute Research

Foundation/Risk Science Institute (ILSI RF/RSI) Nanomaterial Toxicity Screening Working Group

has recommended the use of the immortal cell line, Caco-2 (31). This cell line has been extensively

characterized and has been demonstrated to exhibit a faithful representation of both the in vivo

structural characteristics and the molecular level to mirror the differentiation of human intestinal cells

(32).


137

For HepG2 cells, this cell line derived from human liver carcinoma has widely been used in

nanotoxicological studies (33). Moreover, this cell line retains some of the functions of fully

differentiated primary hepatocytes as some metabolic competence and it has been widely used as a

model system for hepatotoxicity studies (34).

The results for the MTS and LDH assays showed a concentration-dependent cytotoxic

effect. However, by comparing the cytotoxicity of nanoparticles to Caco-2 versus HepG2 cells, a

difference in the respective cellular viabilities was noted at equal tested concentrations with both

cytotoxicity assays. HepG2 cells were more sensitive than Caco-2 cells to poly(anhydride)

nanoparticles, in particular at the highest concentrations (1 and 2 mg/mL). In addition, PEG-NP

were less cytotoxic than NP and NP-HPCD in both cell types and assays. It is not possible to

understand the mechanism underlying these observations but some differences in membrane

composition or structure might be the cause. On the other hand, poly(anhydride) is a biocompatible

and biodegradable (bioerodible) polymer and pegylation has been widely used to increase

nanoparticles biocompatibility (35) and this is may account for the lower cytotoxicity of PEG-NP

compared with the conventional and NP-HPCD. In any case, the in vitro safety of these

poly(anhydride) nanoparticles could be assumed, since all the formulations were well tolerated by

both cell lines showing cytotoxicity only at very high concentrations and long incubation times in

HepG2 cells. In fact, thinking on in vivo studies, these concentrations would be too high to be used in

clinical trials.

The association studies carried out in Caco-2 monolayers showed that although the cell-

associated fluorescence intensity obtained at 37ºC was higher than at 4ºC, results were not different

enough to ensure that nanoparticles were taken up by cells. Indeed, it is known that at 4ºC, the

fluidity of the cell membrane and the metabolism of the cell are reduced to a minimum, so cell-

associated fluorescence intensity primarily reflects the binding of nanoparticles to the surface

(cytoadhesion). In contrast, upon incubation at 37ºC cells are metabolically active and energy-

dependent transport processes are initiated. Thus, the detectable amount of cell-associated

nanoparticles refers to a combined effect of binding (cytoadhesion) and uptake (cytoinvasion) (36,

37). Thus, at 37ºC, BSA-FITC that is taken up by cells, would be quenched in the acidic environment

of the lysosome. So, if there was uptake, the curve at 37ºC should show a steeper decrease than the

curve at 4ºC. As this is not the case, because both curves show the same decrease, there was no

substantial energy-dependent uptake of nanoparticles. These outcomes were in agreement with

previous studies developed by Arbos and co-workers where they observed that while Sambucus nigra

agglutinin coated poly(anhydride) nanoparticle were taken up by cells, plain poly(anhydride)

nanoparticles showed only cytoadhesion (23). Hence, considering all above, the different

poly(anhydride) nanoparticles tested were capable to establish bioadhesive interactions but not to be

taken up by cells.


138

These findings were corroborated via flow cytometric analysis using Caco-2 single cells. The

slight increased observed in the ratio of cells in gate A (healthy single cells) may be due to the fact

that binding of nanoparticles to the cell surface changes granularity of the cells and also because the

high bioadhesive properties of the nanoparticles could induce cell aggregation. Furthermore, the

different cell fluorescence intensity observed between NP, NP-HPCD and PEG-NP confirmed that

surface modification confers larger surface area and subsequently leading to higher cell binding.

The above outcomes were confirmed by CLSM observation of the interaction between the

fluorescent nanoparticles and cell monolayers. The images corroborated the high affinity of

poly(anhydride) nanoparticles to Caco-2 cell surface as well as the heterogeneity of the particle

localization over the monolayer. Moreover, comparing to control group, after incubation with

poly(anhydride) nanoparticles, the staining for ZO-1 proteins showed a continuous ring appearance

between contiguous cells. This fact correlated well with cytotoxicity results.

5. CONCLUSIONS

This study is the first to evaluate the in vitro toxicity and cell interaction of NP, NP-HPCD

and PEG-NP. The results for the cytotoxicity studies in HepG2 and Caco-2 cells after exposure to

the different poly(anhydride) nanoparticles revealed that viability starts to decrease only at very high

concentrations and incubations times, highlighting the safety of nanoparticles, independently of their

chemical composition and surface properties. In addition, association studies in Caco-2 cells showed

cytoadhesion to the cell surface but not internalization. Regarding flow cytometry experiments, due

to the rough surface, the highest cytoadhesion was observed for PEG-NP followed by NP-HPCD

and the lowest correspond to NP. Finally, cellular localization of particles by fluorescence confocal

microscopy demonstrated the strong association of the poly(anhydride) nanoparticles with cells

membranes due to their bioadhesive properties.

Acknowledgments

This work was supported by the Ministry of Science and Innovation in Spain (Project

SAF2008-02538) by Caja Navarra Foundation (Grant number 10828), and by the Department of

Education, Government of Navarra, Spain.


139

6. References

1. J. Curtis, M. Greenberg, J. Kester, S. Phillips, and G. Krieger. Nanotechnology and

nanotoxicology: a primer for clinicians. Toxicol Rev. 25:245-260 (2006).

2. M. Kurath and S. Maasen. Toxicology as a nanoscience?-disciplinary identities reconsidered.

Part Fibre Toxicol. 3:6 (2006).

3. A. Nel, T. Xia, L. Madler, and N. Li. Toxic potential of materials at the nanolevel. Science.

311:622-627 (2006).

4. C. Medina, M.J. Santos-Martinez, A. Radomski, O.I. Corrigan, and M.W. Radomski.

Nanoparticles: pharmacological and toxicological significance. Br J Pharmacol. 150:552-558

(2007).

5. V. Stone and K. Donaldson. Nanotoxicology: signs of stress. Nat Nanotechnol. 1:23-24

(2006).

6. J. Panyam and V. Labhasetwar. Biodegradable nanoparticles for drug and gene delivery to

cells and tissue. Adv Drug Deliv Rev. 55:329-347 (2003).

7. K. Donaldson, V. Stone, A. Clouter, L. Renwick, and W. MacNee. Ultrafine particles. Occup

Environ Med. 58:211-216, 199 (2001).

8. Y. Pan, S. Neuss, A. Leifert, M. Fischler, F. Wen, U. Simon, G. Schmid, W. Brandau, and W.

Jahnen-Dechent. Size-dependent cytotoxicity of gold nanoparticles. Small. 3:1941-1949

(2007).

9. D. Hohr, Y. Steinfartz, R.P. Schins, A.M. Knaapen, G. Martra, B. Fubini, and P.J. Borm. The

surface area rather than the surface coating determines the acute inflammatory response after

instillation of fine and ultrafine TiO2 in the rat. Int J Hyg Environ Health. 205:239-244

(2002).

10. R. Duncan and R. Gaspar. Nanomedicine(s) under the microscope. Mol Pharm. 8:2101-2141

(2011).

11. S.E. McNeil. Nanotechnology for the biologist. J Leukoc Biol. 78:585-594 (2005).



Opin Drug Deliv. 8:721-734 (2011).

13. A.I. Camacho, J. de Souza, S. Sanchez-Gomez, M. Pardo-Ros, J.M. Irache, and C. Gamazo.

Mucosal immunization with Shigella flexneri outer membrane vesicles induced protection in

mice. Vaccine. 29:8222-8229 (2011).

14. S. Gomez, C. Gamazo, B. San Roman, A. Grau, S. Espuelas, M. Ferrer, M.L. Sanz, and J.M.

Irache. A novel nanoparticulate adjuvant for immunotherapy with Lolium perenne. J Immunol

Methods. 348:1-8 (2009).


properties of pegylated nanoparticles. Expert Opin Drug Deliv. 2:205-218 (2005).


140

16. M. Agueros, L. Ruiz-Gaton, C. Vauthier, K. Bouchemal, S. Espuelas, G. Ponchel, and J.M.

Irache. Combined hydroxypropyl-beta-cyclodextrin and poly(anhydride) nanoparticles

improve the oral permeability of paclitaxel. Eur J Pharm Sci. 38:405-413 (2009).

17. P. Ojer, H. Salman, R. Da Costa Martins, J. Calvo, A. López de Cerain, C. Gamazo, J.L.

Lavandera, and J.M. Irache. Spray-drying of poly(anhydride) nanoparticles for drug/antigen

delivery. J Drug Del Sci Tech. 20:353-359 (2010).



Biomed Anal. 39:495-502 (2005).




20. N. Lewinski, V. Colvin, and R. Drezek. Cytotoxicity of nanoparticles. Small. 4:26-49 (2008).

21. C.I. Vamanu, M.R. Cimpan, P.J. Hol, S. Sornes, S.A. Lie, and N.R. Gjerdet. Induction of cell

death by TiO2 nanoparticles: studies on a human monoblastoid cell line. Toxicol In Vitro.

22:1689-1696 (2008).

22. T. Mosmann. Rapid colorimetric assay for cellular growth and survival: application to

proliferation and cytotoxicity assays. J Immunol Methods. 65:55-63 (1983).



(2002).

24. J. Haas and C.M. Lehr. Developments in the area of bioadhesive drug delivery systems.

Expert Opin Biol Ther. 2:287-298 (2002).

25. V. Zabaleta, G. Ponchel, H. Salman, M. Agueros, C. Vauthier, and J.M. Irache. Oral



26. J. Calvo, J.L. Lavandera, M. Agueros, and J.M. Irache. Cyclodextrin/poly(anhydride)

nanoparticles as drug carriers for the oral delivery of atovaquone. Biomed Microdevices.

81:1015-1025 (2011).







29. P. Artursson and C. Magnusson. Epithelial transport of drugs in cell culture. II: Effect of

extracellular calcium concentration on the paracellular transport of drugs of different


141

lipophilicities across monolayers of intestinal epithelial (Caco-2) cells. J Pharm Sci. 79:595-

600 (1990).

30. A.R. Hilgers, R.A. Conradi, and P.S. Burton. Caco-2 cell monolayers as a model for drug

transport across the intestinal mucosa. Pharm Res. 7:902-910 (1990).

31. G. Oberdorster, A. Maynard, K. Donaldson, V. Castranova, J. Fitzpatrick, K. Ausman, J.

Carter, B. Karn, W. Kreyling, D. Lai, S. Olin, N. Monteiro-Riviere, D. Warheit, and H. Yang.

Principles for characterizing the potential human health effects from exposure to

nanomaterials: elements of a screening strategy. Part Fibre Toxicol. 2:8 (2005).

32. M.D. Peterson, W.M. Bement, and M.S. Mooseker. An in vitro model for the analysis of

intestinal brush border assembly. II. Changes in expression and localization of brush border

proteins during cell contact-induced brush border assembly in Caco-2BBe cells. J Cell Sci.

105 ( Pt 2):461-472 (1993).

33. Y. Yuan, C. Liu, J. Qian, J. Wang, and Y. Zhang. Size-mediated cytotoxicity and apoptosis of

hydroxyapatite nanoparticles in human hepatoma HepG2 cells. Biomaterials. 31:730-740

(2010).

34. J. Zou, Q. Chen, X. Jin, S. Tang, K. Chen, T. Zhang, and X. Xiao. Olaquindox induces

apoptosis through the mitochondrial pathway in HepG2 cells. Toxicology. 285:104-113

(2011).

35. K. Knop, R. Hoogenboom, D. Fischer, and U.S. Schubert. Poly(ethylene glycol) in drug

delivery: pros and cons as well as potential alternatives. Angew Chem Int Ed Engl. 49:6288-

6308 (2010).

36. M. Wirth, C. Kneuer, C.M. Lehr, and F. Gabor. Lectin-mediated drug delivery:

discrimination between cytoadhesion and cytoinvasion and evidence for lysosomal

accumulation of wheat germ agglutinin in the Caco-2 model. J Drug Target. 10:439-448

(2002).

37. A. Weissenboeck, E. Bogner, M. Wirth, and F. Gabor. Binding and uptake of wheat germ

agglutinin-grafted PLGA-nanospheres by caco-2 monolayers. Pharm Res. 21:1917-1923

(2004).

CHAPTER 6

Toxicity studies of poly(anhydride) nanoparticles as carriers for oral drug delivery

145

Toxicity studies of poly(anhydride) nanoparticles

as carriers for oral drug delivery Patricia Ojer1,2, Adela López de Cerain2, Paloma Areses3, Ivan Peñuelas3, and Juan M. Irache1

1 Department of Pharmacy and Pharmaceutical Technology. University of Navarra, Pamplona, 31008, Spain. 2 Department of Nutrition and Food Sciences, Physiology and Toxicology. University of Navarra, Pamplona, 31008,

Spain. 3 Radiopharmacy Unit. Department of Nuclear Medicine, University Clinic of Navarra, Pamplona, 31008, Spain.

ABSTRACT

In order to evaluate the acute and subacute toxicity of poly(anhydride) nanoparticles as

carriers for oral drug/antigen delivery three types of poly(anhydride) nanoparticles were assayed:

conventional (NP), nanoparticles containing 2-hydroxypropyl-β-cyclodextrin (NP-HPCD) and

nanoparticles coated with polyethylene glycol 6000 (PEG-NP). Nanoparticles were prepared by a

desolvation method and characterized in terms of size, zeta potential and morphology. For in vivo oral

studies, acute and sub-acute toxicity studies were performed in rats in accordance to the OECD 425

and 407 guidelines respectively. Finally, biodistribution studies were carried out after radiolabelling

nanoparticles with 99mTechnetium (99mTc). The results showed that nanoparticle formulations

displayed a homogeneous size of about 180 nm and a negative zeta potential. The lethal dose 50%

(LD50) for all the nanoparticles tested was established to be higher than 2000 mg/kg body weight

(bw). In the sub-chronic oral toxicity studies at two different doses (30 and 300 mg/kg bw), no

evident signs of toxicity were found. Lastly, biodistribution studies demonstrated that these carriers

remained in the gut with no evidences of particle translocation or distribution to other organs. Thus,

poly(anhydride) nanoparticles (either conventional or modified with HPCD or PEG6000) showed no

toxic effects, indicating that these carriers might be a safe strategy for oral delivery of therapeutics.

Published in Pharmaceutical Research 29(9):2615-2627 (2012).

CHAPTER 6: Toxicity studies of poly(anhydride) nanoparticles as carriers for oral drug delivery

147

1. INTRODUCTION

In the last decades, the development of nanoparticles for medical and pharmaceutical

applications has received a great interest (1-3). Among other, polymeric nanoparticles offer

interesting advantages for drug delivery purposes (4-6). As pharmaceutical dosage forms, polymeric

nanoparticles may protect the loaded therapeutic agent against extreme pH conditions and/or

enzymatic degradation. Their surface modification with ligands permits to drive their distribution in

vivo and, thus, improve their targeting properties for a specific tissue or groups of cells within the

body (5, 7). This functionalization of nanoparticles would be useful to promote the arrival of the

encapsulated drug to its ideal site for action or absorption and, thus, reach a particular target inside

the cell or improve its bioavailability (8). Last but not least, it is also important to remember the

capability of polymeric nanoparticles to control the release of the loaded drug (5).

Although numerous efforts have been carried out to exploit desirable properties of polymeric

nanoparticles, attempts to evaluate potentially undesirable effects are limited in comparison (9). The

same properties as its small size and large surface area, which make nanoparticles so attractive for

medical intentions, may provoke undesirable tissue accumulation and subsequent long-term toxicity

(10). Therefore there is a pressing need for careful consideration of nanoparticles toxicity. Hence,

nanotoxicology as a new science is becoming a trending topic.

Nevertheless, to date, the first challenge that nanotoxicology has to face is the lack of special

regulation to deal with potential risks of nanoparticles. In order to solve this problem, different

organizations are developing new regulatory initiatives, such as thus from both the European

Committee for Standardization (CEN/TC 352) and the International Organization for

Standardization (ISO/TC 229), in order to ensure that products derived from nanomedicine are safe

without hindering innovation (11) and, thus, support commercialisation and market development

(12). In parallel, since 2006, activities related to the development or revisions of test guidelines to

assess nanotoxicology have been performing by the Working Party on Manufactured Nanomaterials

(WPNM) within the Organization for Economic Cooperation and Development (OECD) (13). In

this way, WPMN has recommended the guidelines 425 and 407 for the evaluation of oral acute and

sub-acute toxicity of nanomaterials, respectively (14).

In the last years, promising poly(anhydride) based nanoparticles have been developed from

the copolymer of methyl vinyl ether and maleic anhydride. This polymer shows a great potential for

oral drug/antigen delivery due to its well-studied bioadhesive properties when formulated as

nanoparticles (15-17). This capability to establish bioadhesive interactions can be modulated by the

modification of poly(anhydride) nanoparticles with different compounds such as PEGs or

cyclodextrins (17, 18). In this regard, a study conducted by Yoncheva and collaborators demonstrated

that pegylated nanoparticles possessed high affinity to adhere to the small intestine compared to

conventional nanoparticles (19) and these nanoparticles may be suitable carriers for DNA (20). In

another study it was corroborated the synergistic effect of the combination between bioadhesive


148

nanoparticles and cyclodextrins on the oral bioavailability of paclitaxel and other drugs ascribed to

the groups II and IV of the biopharmaceutics classification system (BCS) (21, 22).

The aim of this study was to evaluate the toxicological profile of different types of

poly(anhydride) nanoparticles: conventional (NP), nanoparticles containing 2-hydroxypropyl-β-

cyclodextrin (NP-HPCD) and nanoparticles coated with PEG 6000 (PEG-NP). More particularly, in

this work we report their safety through acute and sub-acute toxicity studies. Additionally, in vivo

biodistribution of these nanoparticles after oral administration was also investigated.


149




200,000) was provided by ISPcorp. (Waarwijk, The Netherlands). Polyethylene glycol 6000



(Fontenay-sous-Bois, France). Deionized water (18.2MΩ resistivity) was prepared by a water

purification system (Wasserlab, Pamplona, Spain). 99Mo-99mTc generator (Drytec; GE Healthcare Bio-

science, United Kingdom) was eluted with 0.9% NaCl following the manufacturer’s instructions.

SnCl2·2H2O and HCl were from Panreac (Barcelona, Spain); 0.9% NaCl was purchased from Braun

(Barcelona, Spain) and NaOH from Fluka (Switzerland). The anaesthetic isoflurane (Isoflo™) was

from Esteve, (Barcelona, Spain) and the euthanasic T-61 from Intervet (Madrid, Spain). All other

chemicals and solvents used were of analytical grade.


Poly(anhydride) nanoparticles were prepared from a dissolution of Gantrez® AN 119 in

acetone by a simple desolvation method previously described (15). The resulting suspensions were

always dried in a Mini Spray-dryer Büchi B290 (Büchi Labortechnik AG, Switzerland) as described

previously (23). Three different types of nanoparticles were evaluated: conventional nanoparticles

(NP), nanoparticles containing HPCD (NP-HPCD) and pegylated nanoparticles with PEG 6000

(PEG-NP).


Briefly, 500 mg of the copolymer of methyl vinyl ether and maleic anhydride (Gantrez® AN

119) were dissolved and stirred in 30 mL acetone. Then, the desolvation of the polymer was induced

by the addition of 15 mL purified water under magnetic stirring to the organic phase. In parallel, 1 g

of lactose was dissolved in 10 mL purified water and added to the nanoparticle suspension under

agitation for 5 min at room temperature. Finally, the suspension was dried in the Mini Spray-dryer

Büchi B290. The recovered powder was stored in closed vials at room temperature.


Briefly, 500 mg of the copolymer of methyl vinyl ether and maleic anhydride were dissolved

and stirred in 20 mL acetone. Then, 10 mL acetone containing 125 mg HPCD were added to the

polymer solution under magnetic stirring and incubated for 30 min. Nanoparticles were obtained by

the addition of 15 mL purified water under magnetic stirring to the organic phase. In parallel, 1 g of

lactose was dissolved in 10 mL purified water and added to the nanoparticle suspension under


150




In this case, 62.5 mg PEG were dissolved in 15 mL acetone and mixed with 15 mL acetone

containing 500 mg of the copolymer of methyl vinyl ether and maleic anhydride and incubated under

magnetic stirring for 1 h. Then, nanoparticles were obtained by the addition of 15 mL purified water

under magnetic stirring to the organic phase. The solvents were eliminated under reduced pressure

(Büchi R-144, Switzerland) and nanoparticles were purified by double centrifugation at 17,000 rpm

for 20 min (Sigma 3K30, Germany). The pellet was resuspended in 25 mL purified water containing 1

g of lactose. Finally, the suspension was dried in the Mini Spray-dryer Büchi B290. The recovered

powder was stored in closed vials at room temperature.



The mean hydrodynamic diameter and the zeta potential of nanoparticles were determined

by photon correlation spectroscopy (PCS) and electrophoretic laser Doppler anemometry,

respectively, using a Zetamaster analyzer system (Malvern Instruments, United Kingdom) and a


the nanoparticles was determined after dispersion in purified water (1/10) and measured at 25 C by



The morphological examination of the nanoparticles was obtained by scanning electron

microscopy (SEM) in a Zeiss DSM 940A scanning electron microscope (Oberkochen, Germany)

coupled with a digital image system (DISS) Point Electronic GmBh. Previously, a small amount of

the spray-dried powders was diluted with purified water and the resulting suspension was centrifuged

at 17,000 rpm (Sigma 3K30, Germany) for 20 min in order to eliminate sugars. Finally, the pellet was

shaded with a 12 nm gold layer in a Hemitech K 550 Sputter-Coater.

2.3.2. Quantification of HPCD and PEG

The amount of HPCD and PEG associated to the nanoparticles as well as the amount of the

polymer transformed into nanoparticles were estimated using two different high performance liquid

chromatography (HPLC) methods previously described (24, 25). Specifically the apparatus used for

the HPLC analysis was a model 1100 series Liquid Chromatography, Agilent (Waldbronn, Germany)

coupled with an evaporative light scattering detector (ELSD) 2000 Alltech (Illinois, United States).

An ELSD nitrogen generator Alltech was used as the source for the nitrogen gas. Data acquisition

and analysis were performed with a Hewlett-Packard computer using the ChemStation G2171 AA

program.


151

On one hand, PEG separation was carried out at 40ºC on a PL aquagel-OH 30 column

(300mm × 7.5 mm; particle size 8 μm) obtained from Agilent Technologies (GB, United Kingdom).

The mobile phase composition was a mixture of methanol (A) and water (B) in a gradient elution at a

flow rate of 1 mL/min. On the other hand, HPCD separation was carried out at 50ºC on a reversed-

phase Zorbax Eclipse XDB-Phenyl column (2.1mm × 150 mm; particle size 5 μm) obtained from

Agilent Technologies (Waldbronn, Germany). This column was protected by a 0.45 μm filter

(Teknokroma, Spain). The mobile phase composition was a mixture of acetonitrile (A) and water (B)

in a gradient elution at a flow-rate of 0.25 mL/min. In all cases, for ELSD quantifications, the drift

tube temperature was set at 115 C, the nitrogen flow was maintained at 3.2 l/min and the gain was

fixed to 1.



Similarly, the amount of the poly(anhydride) copolymer was estimated by difference in the same way.

2.4. Labelling of nanoparticles with 99mTc

Poly(anhydride) nanoparticles were labeled with 99mTc by reduction with tin chloride (26).

Briefly, 1 mCi of freshly eluted 99mTc pertechnetate was reduced with 0.03 mg/mL stannous chloride

and the pH was adjusted to 4 with 0.1N HCl. Then, nanoparticles (NP, NP-HPCD or PEG-NP) in

water were added to the pre-reduced 99mTc mixture. The suspension was vortexed for 30 s and

incubated at room temperature for 10 min. The overall procedure was carried out in helium-purged

vials using helium-purged solutions to minimize oxygen content and avoid oxidation of tin chloride.

The radiochemical purity was examined by a double-solvent instant thin layer chromatography

(ITLC) system using silica gel coated fiber sheets (Polygram® sil N-RH, Macherey-Nagel, Düren,

Germany) with methyl ethyl ketone (first solvent) and 18% sodium acetate (second solvent) as mobile

phases. After labeling, nanoparticles were mixed with non-labelled ones to adjust the required dose

(either 30 mg/kg or 300 mg/kg) and the volume was adjusted to 1 mL.

2.5. Animals

The experimental protocols involving animals were carefully reviewed and approved by the

Ethical Committee for Animal Experimentation of the University of Navarra (Spain). Eight week old

male and female Wistar rats were purchased from Harlan (Horst, The Netherlands) and employed for

acute and sub-acute toxicity studies as well as biodistribution studies. On the day of arrival, the

animals were weighed in order to assure that bw variation did not exceed ± 20% (27, 28). They were

randomly housed in groups in polycarbonate cages with stainless steel covers to allow acclimatization

to the environmental conditions (12 h day/night cycle, temperature 22 ± 2ºC, relative humidity 55 ±

10%, standard diet ‘‘2014 Teklad Global 14% Protein Rodent Maintenance Diet” from Harlan

Iberica Spain and water ad libitum).


152

2.6. Acute-toxicity study

This study was designed according to the OECD guideline 425 for the Testing of Chemicals

(27). The procedure of the limit test was applied because no toxicity was expected after a single oral

dose: a limit dose of 2000 mg/kg bw was selected. Twenty female Wistar rats were divided into four

groups (n=5): Group I (Control), Group II (NP), Group III (NP-HPCD) and Group IV (PEG-NP).

A single dose of 2000 mg/kg bw was administered by gavage to each animal in 2 mL/100 g bw of

purified water, except for the control group, that received purified water only (vehicle). After the

administration each animal was observed for a period of 48 h for signs of toxicity or mortality. If no

such signs were observed after 48 h, then the same dose was administered to animal number 2. This

procedure was sequentially followed until a total of five animals were administered. The animals were

fasted 4 h prior to dosing and 3 h after the nanoparticles were administered. Individual animal

weights were registered on day 0 and weekly during 14 days. Animals were observed individually for

any clinical signs or any symptoms of toxicity at different time intervals after administration (10 min,

30 min, 1 h, 3 h, and 6 h) and daily during 14 days. On completion of the study, on day 14, blood

samples were extracted from the retro-orbital sinus under isoflurane anesthesia. The following

hematological parameters were analyzed: hemoglobin (HGB; g/L), hematocrit (HCT, %), red blood

corpuscles count (RBC; 1012/L), white blood corpuscles count (WBC; 103/µL), absolute erythrocyte

indices and differential WBC. Biochemical analyses of plasma samples were performed with a Hitachi

911™ (Roche Diagnostics) analyzer using the protocols obtained from Roche for the determination

of the standard parameters: total protein (g/dL), albumin (g/dL), glucose (mg/dL), aspartate

transaminase (AST; U/l), alanine transaminase (ALT; U/l), cholesterol (mg/dL), creatinine (mg/dL),

urea (mg/dL) and total bilirrubin (mg/dL).

Thereafter, the animals were sacrificed in CO2 chamber, subjected to necropsy and various

organs were collected and fixed for further histopathological examination.

2.7. Sub-acute toxicity study

This study was designed according to the OECD guideline 407 for the Testing of Chemicals

(28). The dose selected of each nanoparticle type was based on the expected therapeutic dose

according to previous studies (22), 30 mg/kg bw, and ten times the reported value, 300 mg/kg bw.

Animals were randomly divided into seven groups, containing five male and five female rats per

group. Group I served as the control. Groups II and III received NP, 30 mg/kg bw and 300 mg/kg

bw, respectively. Groups IV and V were treated with NP-HPCD, 30 mg/kg bw and 300 mg/kg bw,

respectively. Finally, Groups VI and VII received PEG-NP, 30 mg/kg bw and 300 mg/kg bw,

respectively.

The animals were administered the respective doses for a period of 28 days, once daily, by

oral gavage. During this period they were observed for any clinical signs of toxicity, mortality or

changes in bw. Blood samples were collected before the first administration, on day 15, and at the


153

end of the study from the retro-orbital sinus under anesthesia and hematological and biochemical

parameters were analyzed (see Acute toxicity study). On day 28, animals were sacrificed in CO2

chamber, subjected to necropsy and various organs were collected, weighed and fixed for further

histopathological examination.

2.8. Histopathological studies

Tissue samples from different body organs (including thymus, kidney, liver, pulmonary,

spleen, stomach, liver and intestines) were taken during necropsy, fixed in 4% formaldehyde solution,

dehydrated and embedded in paraffin. Paraffin sections (3 µm) were cut, mounted onto glass slides,

and dewaxed and stained with haematoxylin and eosin (H&E) for the subsequent histopathological

examination.

2.9. In vivo and ex vivo biodistribution studies with radiolabelled nanoparticles

For this study we only used female rats which were divided in groups of three animals each.

The groups were the same as described in the “Sub-acute toxicity study”. The radiolabelled dose (1

mCi) was administered on days 1, 15 and 28 of the study. After the administration of nanoparticles,

animals were anesthetized with 2% isoflurane and placed in prone position on the gammacamera.

The gammagraphic studies were performed in a single-photon emission computed tomography

(SPECT–CT) (Symbia; Siemens Medical System, United States). A high-resolution low-energy

parallel-hole collimator was used. The scan parameters for computed tomography (CT) were 130 kV,

30 mA s, 1 mm slices and Flash 3D iterative reconstruction with a Gaussian filter with a full-width at

half maximum of 8.4.. For image acquisition the gammacamera was programmed to reach 500.000

events with a static program. The images were acquired 8 h after the administration of the

radiolabelled nanoparticles.

For ex vivo studies, 24 h after the administration of nanoparticles, animals were euthanized

with T-61 (after anesthesia with 2% isoflurane gas). Then blood, urine and different organs (lungs,

heart, spleen, pancreas, liver, kidneys, bone, gut and muscle) were collected and the radioactivity of

each organ measured in a gamma counter (Compugamma CS, RIA; LKB Pharmacia, Finland)

calibrated for 99mTc energy. For practical reasons, the GIT (Gastrointestinal tract) of animals was

divided as follows: stomach, small intestine, caecum and rectum. All of these sections were first

washed by careful gentle injection of 0.9% NaCl through the lumen. The washing liquids were

recovered, weighed and also measured in the gamma counter. Finally, results were expressed as the

percentage of injected dose per gram (%ID/g).


Data were expressed as the mean ± SD of at least three experiments. For the nanoparticles

characterization the Student t test was used. Other statistical significance analysis were carried out


154

using the non-parametric Mann-Whitney U test. P values of < 0.05 were considered as statistically

significant. All calculations were performed using SPSS® statistical software program (SPSS® 15.0,

Microsoft, United States).


155

3. RESULTS



summarized in Table I. Overall the different nanoparticle formulations exhibited a homogeneous

mean size of about 170-190 nm, with a low polydispersity index (PDI). Conventional nanoparticles

(NP) displayed a mean size slightly smaller than that observed for NP-HPCD and PEG-NP. These

observations were confirmed by scanning electron microscopy (Figure 1). The morphological analysis

of the three types of nanoparticles revealed that NP and PEG-NP displayed a round shape while NP-

HPCD were characterized by an irregular aspect. In addition, NP-HPCD showed rough surface in

comparison with the smooth shape observed for NP.

Table I. Physico-chemical characteristics of nanoparticles. NP: poly(anhydride) nanoparticles;

NP-HPCD: hydroxypropyl cyclodextrin-poly(anhydride) nanoparticles; PEG-NP: pegylated

nanoparticles. PDI: polydispersity index. Data expressed as the mean ± standard deviation (n=6).

On the other hand, the incorporation of either HPCD or PEG in the nanoparticles

decreased the negative zeta potential of the resulting carriers.

Concerning the yield of the process, in all cases, this parameter was calculated to be very high

and close to 100 %. Finally, the amount of HPCD associated to nanoparticles was calculated to be

about 87 µg/mg. For pegylated nanoparticles, the amount of PEG linked to nanoparticles was 46

µg/mg.

3.2. Acute-toxicity study

Throughout all the observation period, the animals dosed with the nanoparticles displayed

neither any sign of toxicity nor any abnormal behavior. Similarly, in all cases, the hematological and

biochemical parameters analyzed showed normal values that did not differ from the control group

(data not shown). Finally, gross and histological pathological examination of the vital organs did not

exhibit any evidence of toxicity during the animal necropsies (data not shown). Thus, the different

poly(anhydride) nanoparticles were found to be orally safe at the single limit dose of 2000 mg/kg bw.

Formulation Size (nm) PDI Zeta Potential (mV)

Np formation yield (%)

µg ligand/mg Np

NP 170 ± 2 0.15 ± 0.03 -45.3 ± 2.5 98 ± 3.1

NP-HPCD 189 ± 1.6 0.14 ± 0.01 -35.9 ± 3.5 96 ± 4.2 87.7 ± 2.2

PEG-NP 182 ± 2.1 0.25 ± 0.02 -34.6 ± 3.7 97 ± 2.3 45.7 ± 1.9


156

(A)

dayth0 w

eek

st1

week

nd

2 w

eekrd3

wee

kth4

0

50

100

150Group IGroup IIGroup IIIGroup IVGroup VGroup VIGroup VII

Time (weeks)

Bod

y W

eigh

t (%

of i

nitia

l)

(B)

dayth0 w

eek

st1

week

nd

2 w

eek

rd3

wee

kth4

0

50

100

150

Group IGroup IIGroup IIIGroup IVGroup VGroup VIGroup VII

Time (weeks)

Bod

y W

eigh

t (%

of i

nitia

l)

(B) (C)(A)

Figure 1. SEM photographs of nanoparticles. (A) NP. (B) NP-HPCD. (C) PEG-NP.

3.3. Sub-acute toxicity

During the sub-acute toxicity study no mortality was observed in the different treatment

groups. Detailed physical examinations conducted weekly did not demonstrate any unusual change in

behavior and no signs of toxicity were observed throughout the study. Thus, long-term

administration of the poly(anhydride) nanoparticles had no adverse effects on the general health of

animals. No significant differences were observed in bw of the animals of treatment groups

compared with control ones (Figure 2). All the hematological and biochemical values were found to

be within the normal range with no difference between the control and the treatment groups for both

male and female animals (Figures 3 and 4). The relative organ weight of heart, liver, kidneys, adrenals,

thymus, spleen, ovaries and testis were also unaffected by the treatments (data not shown). Regarding

histology, the gross and histopathology of various organs revealed that the natural architecture

remained normal. No dose related toxicity lesions were observed. Histological findings of a female

dosed with the highest dose, 300 mg/kg bw, are presented in Figure 5.

Figure 2. Body weight (bw) registered of female (A) and male rats (B) through sub-acute toxicity

study.


157

HGB (g/L)

HCT (%) /l)12

RBC (x10

/µl)3

WBC (x10

)3

MCV (µm

MCH (pg)

MCHC (g/dL)

Lymphocy

tes (%

)

Monocytes

(%)

Eosinophile

(%)

/µl)

3

PLT (x10

0

20

40

60

80

100

400

600

800

1000


Uni

ts

(A)

HGB (g/L)

HCT (%) /l)12

RBC (x10

/µl)

3

WBC (x10

)3

MCV (µm

MCH (pg)

MCHC (g/dL)

Lymphocy

tes (%

)

Monocytes

(%)

Eosinophile

(%)

/µl)

3

PLT (x10

0

20

40

60

80

100

400

600

800

1000

1200

Group IGroup IIGroup IIIGroup IVGroup VGroup VIGroup VIIU

nits

(B)

Figure 3. Hematological parameters in female (A) and male rats (B) evaluated for sub-acute toxicity.

Figure 4. Biochemical parameters in female (A.I and A.II) and male rats (B.I and B.II).

Total Pro

tein (g

/dL)

Albumin (g/dL)

Creatin

ine (mg/dL)

Total B

ilirubin (m

g/dL)0.00.10.20.30.40.5

4

6

8


Uni

ts

(A.I)

Total Pro

tein (g

/dL)

Albumin (g/dL)

Creatin

ine (mg/dL)

Total B

ilirubin (m

g/dL)0.00.10.20.30.40.5

4

6

8


Uni

ts

(B.I)

ALT (IU/L)

AST (IU/L)

Cholester

ol (mg/dL)

Glucose

(mg/dL)

Urea (m

g/dL)0

50

100

150

200

250 Group IGroup IIGroup IIIGroup IVGroup VGroup VIGroup VII

Units

(A.II)

ALT (IU/L)

AST (IU/L)

Cholester

ol (mg/dL)

Glucose

(mg/dL)

Urea (m

g/dL)0

50

100

150

200

250 Group IGroup IIGroup IIIGroup IVGroup VGroup VIGroup VII

Uni

ts

(B.II)


158

Figure 5. Light photomicrographs of organ tissues after 28 days administration of poly(anhydride)

nanoparticles: (I) NP; (II) NP-HPCD and (III) PEG-NP. The images presented here are from the

females highest dose group, 300 mg/kg bw. The images are from thymus (a-c), stomach (d-f),

intestine (g-i), Peyer patches (j-l), spleen (m-o) and liver (p-r).

3.4. In vivo biodistribution studies

In order to evaluate the biodistribution of orally administered nanoparticles, the different

types of poly(anhydride) nanoparticles were labelled with 99mTc. The labelling yield was always over

90%. In this study, the animals (as in the sub-acute toxicity study) received orally every day a dose of

nanoparticles (either 30 mg/kg or 300 mg/kg bw) during 28 days. On days 1, 15 and 28, the dose of

nanoparticles included 1 mCi of 99mTc labelled carriers. Figure 6 shows the localization of

radiolabelled nanoparticles 8 h post administration of the dose corresponding to day 15. The images

revealed that nanoparticles were located in the stomach and distal parts of the GIT with no evidences


159

of distribution in other organs or nanoparticle translocation. This distribution was found to be similar

for all the formulations and doses tested. Similarly, the distribution patterns of nanoparticles (NP,

NP-HPCD and PEG-NP) within the animals were found to be similar on the images captured on

days 1, 15 or 28 (data not shown).

Figure 6. Localization of 99mTc labeled

nanoparticles (NP) 8 h post administration

of the dose (300 mg/kg) corresponding to

day 28th. Coronal (left) and sagittal (right)

SPECT-CT fused 6 mm-thick images.

Sagittal cut was made following the A-B

line. Color bar indicates relative radioactivity

concentration. Arrow: stomach; arrowhead:

small intestine and caecum.

3.5. Ex vivo biodistribution studies

According to the previously obtained images, the highest accumulation of radioactivity was

found in the stomach, small intestine, caecum and large intestine. The %ID/g of radiolabelled

nanoparticles (NP, NP-HPCD and PEG-NP) in each one of these organs 24 h after oral

administration of the dose corresponding to day 28 is represented in Figure 7. A relative low

concentration of radioactivity was also found in the liver and the kidneys, but this represented less

than 0.05% ID/g in all the cases (data not shown).

Comparing the different types of formulations, the radioactivity in the stomach and the large

intestine was significantly higher for PEG-NP than for NP and NP-HPCD (p<0.05). Similarly, the

A

B


160

radioactivity found in the washing liquids of the GIT of animals was found to be significantly higher

for animals treated with PEG-NP than with NP or NP-HPCD (data not shown).

Despite NP and NP-HPCD showed similar behavior, NP-HPCD seemed to pass slower

through the stomach and small intestine. In fact, slightly higher radioactivity concentration was found

in these organs for NP-HPCD but this difference was not statistically significant.

Stomach

Small In

testin

e

Caecu

m

Large I

ntestin

e0.0

0.5

1.0

1.5

2.0

NP NP-HPCD PEG-NP

Organs

% ID

/g

*

*

Figure 7. Ex vivo biodistribution studies of 99mTc-labelled poly(anhydride) nanoparticles. Animals

were sacrificed 24 h post-administration, organs extracted and radioactivity of each organ measured. *

Statistically significant difference (p<0.05). Results expressed as the mean ± SD (n=3).


161

4. DISCUSSION

From a general point of view, in vivo toxicity studies are more desirable since direct

verification of the effect of nanoparticles toward the human body is achieved. Nevertheless, to date,

the reported toxicity studies have mainly focused on in vitro examinations rather than in in vivo

experiments. This fact can be due to the easy in execution as well as in the control and interpretation

of the experiments compared with in vivo tests.

The copolymers of methyl vinyl ether and maleic anhydride and their ether and salt

derivatives (Gantrez® series) are widely employed in a diverse range of topical and oral

pharmaceutical formulations as thickening and suspending agents, denture adhesives, film-coating

agents and adjuvants for transdermal patches (29, 30). In the last years, these copolymers have also

been employed to prepare nanoparticles with bioadhesive properties as vehicles for oral drug/antigen

delivery (15-17, 21-25). These carriers offer a high versatility derived from the presence of anhydride

residues which can easily react (under mild conditions) with different ligands and excipients and, thus,

yielding “decorated” nanoparticles with improved targeting or controlled release properties. As a

consequence these nanoparticles may facilitate the interaction and presentation of the loaded antigen

or allergen with the antigen-presenting cells (APCs) (16) and/or improve the oral bioavailability of

different drugs such as fluorouridine (15), paclitaxel (21) or atovaquone (ATO) (22). In spite of these

interesting results, no information about the toxicological profile of these nanoparticles was still

known.

Although Gantrez® polymers alone are generally regarded as non-toxic and non-irritant for

oral administration (31), their properties may change completely upon transformation in a

nanoparticulate system. This is because the size, charge and surface modifications of the

nanoparticles often decide their fate in vivo (32, 33). In this context, the main objective of this work

was to evaluate the toxicity of the following types of poly(anhydride) nanoparticles when orally

administered: conventional nanoparticles (NP), nanoparticles containing 2-hydroxypropyl-β-

cyclodextrin (NP-HPCD) and pegylated nanoparticles (PEG-NP).

For this purpose we decided to follow the standard procedures and guidelines proposed by

the OECD which are currently used to test and assess chemicals. For the acute toxicity study, a limit

test based on the oral administration to laboratory animals of a single oral dose of 2 g nanoparticles

per kg bw in water was applied. This dose was around 200-times higher than the usual dose employed

in previous studies with these nanoparticles (16, 18, 21). In all cases, the administered dose had no

toxic effect. In fact, neither any death occurred nor abnormal or toxic responses were observed in the

rats during the 14 day observation period. In addition no macroscopic pathological alterations

attributed to nanoparticles were found in the necropsies. These findings were consistent with

previous results with nanoparticles containing chitosan. Thus, Sonaje and collaborators (34) reported

an apparent absence of toxicity in mice treated with 100 mg/kg chitosan nanoparticles with a mean

size of about 220 nm. In the same way, the DL50 of gold nanoparticles coated with chitosan (10-50


162

nm) was found to be greater than 2000 mg/kg (35). On the contrary other types of nanoparticles,

such as zinc or titanium oxide particles, appear to induce some toxicological problems. Thus, Wang

and collaborators reported that the oral administration to mice of a large dose of 5 g/kg bw of

titanium oxide nanoparticles (25, 80 or 155 nm) showed no obvious acute toxicity. However, animals

treated with very fine nanoparticles (25 or 80 nm) displayed important changes of serum biochemical

parameters associated to liver injury and nephrotoxicity (36). In another interesting report, it was

demonstrated that the oral administration of 5 g/kg bw of zinc microparticles (1080 nm) induced

more severe liver damage than zinc nanoparticles (60 nm), while these small ones could induce

heavier renal damage and anemia (37). This different toxicological profile between polymeric and

metallic particles may be ascribed, at least in part, to the capabilities of chitosan and poly(anhydride)

to yield carriers able to develop sustained adhesive interactions with components of the mucus layer

and/or the membrane epithelia (19, 38, 39). This fact would minimize the “translocation” and entry

into the circulation for these polymeric nanoparticles compared with “non-adhesive” ones such as

metallic nanoparticles.

Interestingly, an absence of toxicological effects were also observed during the sub-acute

toxicity studies with the three types of poly(anhydride) nanoparticles. All the animals survived the

duration of the study, with no significant changes in clinical signs or bw. In these experiments, the

hematological parameters of all the animals were found to be within the normal ranges with no

differences between the control and experimental groups (p>0.05). These results suggested that

poly(anhydride) nanoparticles are nontoxic when administered orally as they did not affect the

circulating red cells, hematopoiesis or leucopoiesis that could otherwise have caused hematological

disorders (40). With regard to biochemical parameters analyzed, which may reflect alterations in

blood enzymes and are used to diagnose organ diseases (i.e. from heart, liver or kidney), no

significant differences (p>0.05) between control and treated groups of both male and female animals

were also observed. Again, this fact confirmed that poly(anhydride) nanoparticles did not generate

cumulative and latent biochemical changes following multiple administrations even using doses 30-

times higher than those reported in other studies (15-21). Moreover, all the outcomes

aforementioned were corroborated with the macroscopic and histological analysis of the target

organs. Indeed, no toxic lesions were evident in any of the organs evaluated and all the organs tested

showed unaffected natural architecture. In spite of the number of sub-acute oral toxicity studies with

nanoparticles is very scarce, the presented results appears to be in line with other previously reported

involving gold nanoparticles or hydrogel nanoparticles. In the former, Dhar and co-workers reported

the absence of any hematological or biochemical abnormalities observed with gellam gum-reduced

gold nanoparticles (14 nm) orally administered daily at a dose of 300 mg/kg during 28 days (41). In

the latter, nanoparticles obtained by the combination of hydroxyl propyl methyl cellulose and

polyvinyl pyrrolidone (100 nm) were 28-day daily administered to rats and none of the treated

animals evidenced toxic effects on vital organs or metabolic abnormalities (42).


163

In order to gain insight about the fate of poly(anhydride) nanoparticles in the body, the

carriers were radiolabelled with 99mTc and orally administered to rats. Neither imaging nor gamma

counter data showed radioactivity concentration in liver, kidneys or spleen, confirming that

nanoparticles were in no way absorbed through the intestine and transported to the blood circulation.

These findings are different to that observed for other types of nanoparticles such as zinc (36) or

silver nanoparticles (43). In this last report, describing a sub-acute study in rats, it was demonstrated

that the toxicity of silver nanoparticles (60 nm) is related with their accumulation in the kidneys after

absorption through the gut (43). Therefore, the safety of poly(anhydride) nanoparticles would be

directly related to the absence of a “translocation” phenomenon.

Comparing the three types of poly(anhydride) nanoparticles, it was observed that the

radioactivity values quantified in the GIT of animals treated with PEG-NP were three times higher

than for animals dosed with NP-HPCD or NP values (Figure 7). This slow transit through the gut for

PEG-NP is consistent with previous observations reported by Yoncheva and collaborators, who

demonstrated that pegylation of nanoparticles increased the bioadhesive capability of the resulting

carriers (18, 19). In these studies it was postulated that pegylated poly(anhydride) nanoparticles would

display a PEG-surface layer in a “brush” conformation. Due to this morphology, pegylated

nanoparticles would be capable of diffuse across the mucus protective layer and reach the surface of

the enterocytes. As a consequence, the residence time of these nanoparticles in the gut would be

longer than for other types of carriers (i.e. NP or NP-HPCD).

5. CONCLUSIONS

Acute and sub-acute toxicity studies of poly(anhydride) nanoparticles administered by the

oral route to rats demonstrated the absence of adverse effects related to either the treatment or sex.

The distribution of the three different nanoparticle formulations appeared to be restricted to the GIT

of animals and no evidences of “traslocation” or absorption of particulates was found. However, the

residence within the gut of PEG-NP was found to be significantly higher than for conventional

nanoparticles or NP-HPCD. Overall, the current findings confirm that poly(anhydride) nanoparticles

are safe for both oral short-term and prolonged administrations.

Acknowledgments

This work was supported by the Ministry of Science and Innovation in Spain (projects

SAF2008-02538) and Caja Navarra Foundation (Grant 10828). Patricia Ojer was also financially

supported by a grant from the Department of Education of the Government of Navarra in Spain.


164

6. References

1. R. Singh and H.S. Nalwa. Medical applications of nanoparticles in biological imaging, cell

labeling, antimicrobial agents, and anticancer nanodrugs. J Biomed Nanotechnol.. 7:489-503

(2011).

2. W.H. De Jong and P.J. Borm. Drug delivery and nanoparticles: applications and hazards. Int

J Nanomed. 3:133-149 (2008).

3. S. Lanone and J. Boczkowski. Biomedical applications and potential health risks of

nanomaterials: molecular mechanisms. Curr Mol Med. 6:651-663 (2006).

4. S.M. Moghimi, A.C. Hunter, J.C. Murray. Nanomedicine: current status and future prospects.

FASEB J. 19:311-330 (2005).

5. C. Vauthier, D. Labarre, and G. Ponchel. Design aspects of poly(alkylcyanoacrylate)

nanoparticles for drug delivery. J Drug Target. 15:641-663 (2007).

6. J.M. Chan, P.M. Valencia, L. Zhang, R. Langer, and O.C. Farokhzad. Polymeric

nanoparticles for drug delivery. Methods Mol Biol. 624:163-175 (2010).

7. A. Kumari, S.K. Yadav, and S.C. Yadav. Biodegradable polymeric nanoparticles based drug

delivery systems. Colloid Surf B: Biointerf. 75:1-18 (2010).

8. A. des Rieux, V. Fievez, M. Garinot, Y.J. Schneider, and V. Préat. Nanoparticles as potential

oral delivery systems of proteins and vaccines: a mechanistic approach. J Control Release.

116:1-27 (2006).

9. C. Medina, M.J. Santos-Martinez, A. Radomski, O.I. Corrigan, and M.W. Radomski.

Nanoparticles: pharmacological and toxicological significance. Br J Pharmacol. 150:552-558

(2007).

10. P. Rivera Gil, G. Oberdörster, A. Elder, V. Puntes, and W.J. Parak. Correlating physico-

chemical with toxicological properties of nanoparticles: the present and the future. ACS

Nano. 4:5527-5531 (2010).

11. G. Rivière. European and international standardisation progress in the field of engineered

nanoparticles. Inhal Toxicol. 21 Suppl 1:2-7 (2009).

12. M.J.D. Clift, P. Gehr, and B. Rothen-Rutishauser. Nanotoxicology: a perspective and

discussion of whether or not in vitro testing is a valid alternative. Arch. Toxicol. 85:723-731

(2011).

13. Organization for Economic Cooperation and Development. Nanosafety at the OECD: the

First Five Years 2006-2010. Paris: OECD Publishing; 2011. Available from:

http://www.oecd.org/dataoecd/6/25/47104296.pdf

14. Organization for Economic Cooperation and Development. Preliminary Review of OECD

Test Guidelines for their Applicability to Manufactured Nanomaterials. Paris: OECD

Publishing; 2009. Vol. No. 15 - ENV/JM/MONO (2009).


165

15. P. Arbós, M.A. Campanero, M.A. Arangoa, and J.M. Irache. Nanoparticles with specific



16. S. Gómez, C. Gamazo, B.S. Roman, M. Ferrer, M.L. Sanz, and J.M. Irache. Gantrez AN

nanoparticles as an adjuvant for oral immunotherapy with allergens. Vaccine. 25:5263-5271

(2007).

17. M. Agüeros, L. Ruiz-Gatón, C. Vauthier, K. Bouchemal, S. Espuelas, G. Ponchel, and J.M.

Irache. Combined hydroxypropyl-beta-cyclodextrin and poly(anhydride) nanoparticles

improve the oral permeability of paclitaxel. Eur J Pharm Sci. 38:405-413 (2009).



Eur J Pharm Sci. 24:411-419 (2005).

19. K. Yoncheva, L. Guembe, M.A. Campanero, and J.M. Irache. Evaluation of bioadhesive

potential and intestinal transport of pegylated poly(anhydride) nanoparticles. Int J Pharm.

334:156-165(2007).

20. K. Yoncheva, M.N. Centelles, and J.M. Irache. Development of bioadhesive amino-pegylated

poly(anhydride) nanoparticles designed for oral DNA delivery. J Microencapsul. 25:82-89

(2008).

21. M. Agüeros, V. Zabaleta, S. Espuelas, M.A. Campanero, and J.M. Irache. Increased oral

bioavailability of paclitaxel by its encapsulation through complex formation with

cyclodextrins in poly(anhydride) nanoparticles. J Control Release. 145:2-8 (2010).

22. J. Calvo, J.L. Lavandera, M. Agüeros, and J.M. Irache. Cyclodextrin/ poly(anhydride)


13:1015-1025 (2011).

23. P. Ojer, H.H. Salman, R. Da Costa Martins, J. Calvo, A. López de Cerain, C. Gamazo, J.L.



24. M. Agüeros, M.A. Campanero, and J.M. lrache. Simultaneous quantification of different


Biomed Anal. 39:495-502 (2005).




26. P. Areses, M.T. Agüeros, G. Quincoces, M. Collantes, J.Á. Richter, L.M. López-Sánchez, M.

Sánchez-Martínez, J.M. Irache, and I. Peñuelas. Molecular imaging techniques to study the

biodistribution of orally administered (99m)Tc-labelled naive and ligand-tagged

nanoparticles. Mol Imaging Biol. 13:1215-1223 (2011).


166

27. Organization for Economic Cooperation and Development. Test No. 425: Acute Oral

Toxicity: Up-and-Down Procedure. Guidelines for the Testing of Chemicals. Paris: OECD

Publishing; 2006.

28. Organization for Economic Cooperation and Development. Test No. 407: Repeated Dose

28-day Oral Toxicity Study in Rodents. Guidelines for the Testing of Chemicals. Paris:

OECD Publishing; 2001.

29. P. Arbós, M. Wirth, M.A. Arangoa, F. Gabor, and J.M. Irache. Gantrez AN as a new


(2002).

30. N.C. Sharma, H.J. Galustians, J. Qaquish, A. Galustians, K.N. Rustogi, M.E. Petrone, P.

Chaknis, L. Garcia, A.R. Volpe, and H.M. Proskin. The clinical effectiveness of a dentifrice

containing triclosan and a copolymer for controlling breath odor measured organoleptically

twelve hours after toothbrushing. J Clin Dent. 10:131-134 (1999).

31. G.P. Andrews and D.S. Jones. Poly(methyl vinyl ether/maleic anhydride). In: Rowe RC,

Sheskey PJ, Quinn ME, editors. Handbook of Pharmaceutical Excipients. 6th ed. Washington

DC: American Pharmacists Association, London: Pharmaceutical Press. 534-535 (2009).

32. G.N. Chiu, M.Y. Wong, L.U. Ling, I.M. Shaikh, K.B. Tan, A. Chaudhury, and B.J. Tan.

Lipid-based nanoparticulate systems for the delivery of anti-cancer drug cocktails:

implications on pharmacokinetics and drug toxicities. Curr Drug Metab. 10:861–74 (2009).

33. T. Kean and M. Thanou. Biodegradation, biodistribution and toxicity of chitosan. Adv Drug

Deliv Rev. 62:3-11 (2010).

34. K. Sonaje, Y.H. Lin, J.H. Juang, S.P. Wey, C.T. Chen, and H.W. Sung. In vivo evaluation of

safety and efficacy of self-assembled nanoparticles for oral insulin delivery. Biomaterials

30:2329-2339 (2009).

35. V. Pokhartar, S. Dhar, D. Bhumkar, V. Mali, S. Bodhankar, and B.L.V. Prasad. Acute and

subacute toxicity studies of chitosan reduced gold nanoparticles: a novel carrier for

therapeutic agents. J Biomed Nanotecnol. 5:233-239 (2009).

36. B. Wang, W.Y. Feng, T.C. Wang, G. Jia, M. Wang, J.W. Shi, F. Zhang, Y.L. Zhao, and Z.F.

Chai. Acute toxicity of nano- and micro-scale zinc powder in healthy adult mice. Toxicol

Let.. 161:115-123 (2006).

37. J. Wang, G. Zhou, C. Chen, H. Yu, T. Wang, Y. Ma, G. Jia, Y. Gao, B. Li, F. Jiao, Y. Zhao,

and Z. Chai. Acute toxicity and biodistribution of different sized titanium dioxide particles in

mice after oral administration. Toxicol Lett. 168:176-185 (2007).

38. H. Takeuchi, H. Yamamoto, T. Niwa, T. Hino, and Y. Kawashima. Enteral absorption of

insulin in rats from mucoadhesive chitosan-coated liposomes. Pharm Res. 13: 896–901

(1996).


167

39. J. das Neves, M.F. Bahia, M.M. Amiji, and B. Sarmento. Mucoadhesive nanomedicines:

characterization and modulation of mucoadhesion at the nanoscale. Expert Opin Drug

Deliv. 8:1085-1104 (2011).

40. A.W. Hayes. Principles and methods of toxicology. 4th ed. Philadelphia: Taylor & Francis;

2001.

41. S. Dhar, V. Mali, S. Bodhankar, A. Shiras, B.L.V. Prasad, and V. Pokharkar. Biocompatible

gellan gum-reduced gold nanoparticles: cellular uptake and subacute oral toxicity studies. J

Appl Toxicol. 31:411-420 (2011).

42. P.P. Dandekar, R. Jain, S. Patil, R. Dhumal, D. Tiwari, Sharma S, G. Vanage, and V.

Patravale. Curcumin-loaded hydrogel nanoparticles: application in anti-malarial therapy and

toxicological evaluation. J Pharm Sci. 99:4992-5010 (2010).

43. W.Y. Kim, J. Kim, J.D. Park, H.Y. Ryu, and I.J. Yu. Histological study of gender differences

in accumulation of silver nanoparticles in kidneys of Fischer 344 rats. J Toxicol Envirom

Health A. 72:1279-1284 (2009).

CHAPTER 7

Hemocompatibility, acute toxic effects and biodistribution of poly(anhydride) nanoparticles

following intravenous administration

171

Hemocompatibility, acute toxic effects and

biodistribution of poly(anhydride) nanoparticles

following intravenous administration Patricia Ojer1,2, Adela López de Cerain2, María Sánchez3, Ivan Peñuelas3, and Juan M. Irache1

1 Department of Pharmacy and Pharmaceutical Technology. University of Navarra, Pamplona, 31008, Spain. 2 Department of Pharmacology and Toxicology. University of Navarra, Pamplona, 31008, Spain. 3 Radiopharmacy Unit. Department of Nuclear Medicine, University Clinic of Navarra, Pamplona, 31008, Spain.

ABSTRACT

Prior to in vivo studies, the hemocompatibility of the different poly(anhydride) nanoparticles

(conventional (NP), nanoparticles containing 2-hydroxypropyl-β-cyclodextrin (NP-HPCD) and

nanoparticles coated with polyethylene glycol 6000 (PEG-NP)) were studied in vitro at three different

concentrations (0.25, 0.5 and 1 mg/mL). To evaluate the acute toxicity of different intravenously

administered poly(anhydride) nanoparticles rats were exposed to two different doses (50 and 150

mg/kg body weight (bw)). Toxic effects were assessed via general behavior, hematological and serum

biochemical parameters and histopathological observation of the animals. Biodistribution were

assessed at a dose of 50 mg/kg bw using radiolabelling nanoparticles with 99mTechnetium (99mTc).

The results showed that the different poly(anhydride) exhibited a homogenous size of about 175 nm

and a negative zeta potential. Nanoparticles did not induce any agglutination effect neither hemolytic

activity in the hemocompatibility studies carried out. In the acute toxicity study animals dosed with

150 mg/kg bw showed acute episodes of respiratory distress. In fact, 2/6 animals dosed with NP and

NP-HPCD died. The histopathological examinations revealed apparent pathological changes

characterized by the presence of foamy cells in the liver, spleen and lungs (Phospholipidosis

(PLDsis)). Finally, biodistribution studies showed that nanoparticles accumulation occurred

predominantly in organs of the reticular endothelial system (RES). Thus, although all nanoparticle

formulations demonstrated to be hemocompatible in vitro and in vivo, the intravenous (i.v.) toxicity

was dose dependent, with the presence of foamy cells in spleen, liver and lungs as the most relevant

finding observed.

CHAPTER 7: Hemocompatibility, acute toxic effects and biodistribution of poly(anhydride) nanoparticles following intravenous administration

173

1. INTRODUCTION

Research conducted in nanomedicine has enabled the development and of new and/or

improved diagnostic and therapeutic methods. In this context, the use of biodegradable polymers

based nanoparticles has been identified as promising candidates in different pharmaceutical fields.

Among the various routes of administration, polymeric nanoparticles administer by i.v. route

have received much attention. Their use offers advantages such as carriers for otherwise insoluble or

poorly soluble drugs, imaging agents and gene delivery purposes (1). Furthermore, as drug delivery

devices, injectable polymeric nanoparticles can be seen to prolong the blood half life of drugs,

increase their efficacy and represent very attractive properties for targeting applications to improve

the selectivity in drug delivery. Their efficacy largely depends on the control of their biodistribution

within the body. Such a distribution after i.v. administration is considerably influenced by their

interactions with the biological environment and their physicochemical properties, including particle

size, surface charge, morphology, and surface hydrophilicity (2). Indeed, following i.v. administration,

nanoparticles can accumulate in different organs through systemic circulation and induce potential

toxicity. This fact has been widely demonstrated in the last years by different authors in relation to

engineered nanomaterials due to its no biodegradability (3, 4). On the contrary, for biodegradable

PCL (5) and polyacrilamide nanoparticles (6) it has been reported absence of toxicity in an acute

toxicity study after i.v. administration. Hence, using biocompatible and biodegradable polymers has

been outlined as a good option to develop nanoparticles for i.v. administration. In this regard, the

most common choices are polymers as polyesters (e.g. PCL and poly(lactide-co-glycolide) (PLGA))

(7, 8) and macromolecules (e.g. albumin and chitosan) (9, 10).

However, although there are a number of studies regarding the evaluation of the efficacy of

polymeric nanoparticles as drug delivery devices, knowledge on i.v. toxicity of these nanoparticles is

scarce. Therefore, evaluation (parallel to the study of its effectiveness) of the potential toxic effects of

these nanoparticles is required, given that specific mechanisms and pathways through which

nanoparticles may exert their toxic effects remain largely unknown.

In the recent years, poly(methyl vinyl ether-co-maleic anhydride) [Gantrez® AN]

nanoparticles have been considered promising platforms for drug delivery and other biomedical

applications. Poly(anhydride) nanoparticles have successfully been developed as oral drug delivery

systems (11-13), immunization (14-16) or allergy-immunotherapy treatment (17, 18). In addition, the

oral toxicity of plain, cyclodextrin and pegylated poly(anhydride) nanoparticles has been evaluated in

vivo confirming its oral safety (19). Particularly, the investigations demonstrated that LD50 for all

nanoparticles tested was higher than 2000 mg/kg bw and the sub-chronic oral toxicity studies at two

different doses (30 and 300 mg/kg bw) showed no evident signs of toxicity.

Given that the efficacy and safety of these nanoparticles has been widely demonstrated by

the oral route, the assessment of their toxicity by the i.v. route has been the objective of this study, in

order to evaluate the possibility of administering such formulations by the i.v. route as a new strategy


174

that may be useful for some specific applications. To this aim, three different types of

poly(anhydride) nanoparticles have been studied: conventional (NP), nanoparticles containing 2-

hydroxypropyl-β-cyclodextrin (NP-HPCD) and nanoparticles coated with PEG6000 (PEG-NP).

Specifically, the influence of these nanoparticles on erythrocyte sedimentation and agglutination as

well as their hemolytic activity was tested in vitro. Then, single dose in vivo toxicity studies of the

different poly(anhydride) nanoparticles were carried out. In addition, in vivo biodistribution after i.v.

administration using radiolabelled nanoparticles to assess qualitative and quantitative tissue

distribution in rat was also investigated.


175


2.1. Chemicals


200,000) was provided by ISP corp. (Waarwijk, The Netherlands). Polyethylene glycol 6000



(Fontenay-sous-Bois, France). Ultrafiltrated water (18.2MΩ resistivity) was prepared by a water

purification system (Wasserlab, Pamplona, Spain). 99Mo-99mTc generator (Drytec; GE Healthcare Bio-

Science, United Kingdom) was eluted with 0.9% NaCl following the manufacturer’s instructions.

SnCl2 and HCl were purchased from Panreac (Barcelona, Spain) and 0.9% NaCl from Braun

(Barcelona, Spain). The anesthetic isoflurane (Isoflo™) was from Esteve (Barcelona, Spain). All other

chemicals and solvents used were of analytical grade.


Poly(anhydride) nanoparticles were prepared from a dissolution of Gantrez® AN 119 in

acetone by a simple desolvation method following by a dried step in a Mini Spray-dryer Büchi B290

(Büchi Labortechnik AG, Switzerland) as described previously (20). Three different types of

nanoparticles were evaluated: conventional nanoparticles (NP), nanoparticles containing HPCD (NP-

HPCD) and pegylated nanoparticles with PEG 6000 (PEG-NP).



and stirred in 30 mL acetone. Then, the desolvation of the polymer was induced by the addition of 15

mL ultrafiltrated water under magnetic stirring to the organic phase. In parallel, 1 g of lactose was

dissolved in 10 mL ultrafiltrated water and added to the nanoparticle suspension under agitation for 5

min at room temperature. Finally, the suspension was dried in the Mini Spray-dryer Büchi B290. The

recovered powder was stored in closed vials at room temperature.



and stirred in 20 mL acetone. Then, 10 mL acetone containing 125 mg HPCD were added to the

polymer solution under magnetic stirring and incubated for 30 min. Nanoparticles were obtained by

the addition of 15 mL ultrafiltrated water under magnetic stirring to the organic phase. In parallel, 1 g

of lactose was dissolved in 10 mL ultrafiltrated water and added to the nanoparticle suspension under




176


In this case, 62.5 mg PEG were dissolved in 15 mL acetone and mixed with 15 mL acetone

containing 500 mg of the copolymer of methyl vinyl ether and maleic anhydride and incubated under

magnetic stirring for 1 h. Then, nanoparticles were obtained by the addition of 15 mL ultrafiltrated

water under magnetic stirring to the organic phase. The solvents were eliminated under reduced

pressure (Büchi R-144, Switzerland) and nanoparticles were ultrafiltrated by double centrifugation at

17,000 rpm for 20 min (Sigma 3K30, Germany). The pellet was resuspended in 25 mL ultrafiltrated

water containing 1 g of lactose. Finally, the suspension was dried in the Mini Spray-dryer Büchi B191.

The recovered powder was stored in closed vials at room temperature.



The mean hydrodynamic diameter and the zeta potential of nanoparticles were determined

by photon correlation spectroscopy (PCS) and electrophoretic laser Doppler anemometry,

respectively, using a Zetamaster analyzer system (Malvern Instruments, United Kingdom) and a


the nanoparticles was determined after dispersion in purified water (1/10) and measured at 25ºC by



The morphology of the nanoparticles was examined using a scanning electron microscope

(Zeiss DSM 940A SEM; Oberkochen, Germany) with a digital imaging capture system (DISS) (Point

Electronic GmBh; Halle, Germany). For this purpose, a small amount of the spray dried powders

was resuspended in ultrapure water and centrifuged at 27,000×g for 20 min at 4°C to eliminate the

sugars. Then, supernatants were rejected and the pellets were resuspended in water and mounted on a

plate adhered with a double-sided adhesive tape onto metal stubs and dried.

2.3.2. Quantification of HPCD and PEG

The amount of HPCD and PEG associated to the nanoparticles as well as the amount of the

polymer transformed into nanoparticles was estimated using two different high performance liquid

chromatography (HPLC) methods previously described (21, 22). Specifically the apparatus used for

the HPLC analysis was a model 1100 series Liquid Chromatography, Agilent (Waldbronn, Germany)

coupled with an evaporative light scattering detector (ELSD) 2000 Alltech (Illinois, United States).

An ELSD nitrogen generator Alltech was used as the source for the nitrogen gas. Data acquisition

and analysis were performed with a Hewlett-Packard computer using the ChemStation G2171 AA

program.



Similarly, the amount of the poly(anhydride) copolymer was estimated by difference in the same way.


177

2.4. Labelling of nanoparticles with 99mTc

Poly(anhydride) nanoparticles were labelled with 99mTc by reduction of 99mTc-Pertecnetate

(99mTcO4-) with stannous chloride as described previously (23). Briefly, 20 µl of a stannous chloride

solution adjusted to pH 1 with 0.1 N HCl with a final concentration of stannous of 0.03 mg/mL

were added to 1 mg of the different nanoparticle formulation (NP, NP-HPCD and PEG-NP)

followed by the addition of 56 MBq of freshly eluted 99mTcO4- in 0.5 mL of NaCl 0.9%. The final

suspension was vortexed for 30 seconds and incubated at room temperature for 10 min and showed

a pH of 4. The overall procedure was carried out in helium-purged vials using helium-purged

solutions to minimize oxygen content and avoid oxidation of tin chloride. Just before administration

to the animals, radiolabelled nanoparticles were mixed with non-labelled ones to adjust the required

dose for administration to animals of 50 mg/kg bw.

2.5. Hemocompatibility studies

2.5.1. Preparation of erythrocytes

Whole blood was collected from three healthy volunteers in heparinised tubes (lithium

heparin). The study was approved by the Committee on Ethics of the Clinic University of Navarra,

and the volunteers signed a written consent. The experiments on human blood were performed on

the day of its collection. Blood from the three volunteers was centrifuged 10 min at 1500 x g and

room temperature separately. The supernatant consisting on peripheral blood mononuclear cells and

a plasma band was discarded with a Pasteur pipette. The pellet consisting on erythrocytes was washed

three times with isosmotic PBS (Phosphate Buffered Saline) w/o calcium and magnesium (1:1 v/v).

A 4% suspension of erythrocytes in PBS was used for experiments. All the experiments were

performed in triplicate.

2.5.2. Erythrocyte sedimentation and agglutination

Different concentrations of poly(anhydride) nanoparticles (0.25, 0.5 and 1 mg/mL) in 100 µL

of erythrocyte suspension were incubated together in eppendorfs (1:1 v/v) for 1 h at 37ºC. A

negative control of erythrocytes treated with PBS and a positive control of erythrocytes treated with

Triton X-100 0.1% were included in all the experiments. Erythrocytes sedimentation and

agglutination was recorded by using a camera.

2.5.3. Hemolysis assay

Hemolysis induced by nanoparticle treatment was assessed photometrically. 100 µL of

nanoparticle suspensions (0.25, 0.5 and 1 mg/mL) were incubated (1:1 v/v) with the erythrocyte

suspension at 37ºC for 1 h. After incubation all samples were centrifuged for 10 min at 1500 x g and

the supernatants were recovered. Free hemoglobin (HGB) was measured photometrically in the

supernatant at 540 nm. The percentage of hemolysis of each nanoparticle formulation, corrected by


178

the spontaneous absorbance or erythrocytes incubated with PBS suspension was calculated as

follows:

[Eq. 1]

Where, [A]sample is the absorbance of the erythrocytes incubated with nanoparticle

suspension and [A]100% hemolysis is the absorbance of the supernatant of the erythrocyte incubated

with Triton X-100 (0.1%) solution in PBS suspension. All samples were analyzed in triplicate.

2.6. Animals

The experimental protocols involving animals were carefully reviewed and approved by the

Ethical Committee for Animal Experimentation of the University of Navarra (Spain) (Protocol

number 044-11). Eight week old female Wistar rats were purchased from Harlan (Horst, The

Netherlands) and employed for both single dose toxicity studies and biodistribution studies. On the

day of arrival, the animals were weighed in order to assure that bw variation did not exceed ± 20%

(24). They were randomly housed in groups in polycarbonate cages with stainless steel covers to allow

acclimatization to the environmental conditions (12 h day/night cycle, temperature 22 ± 2ºC, relative

humidity 55 ± 10%, standard diet ‘‘2014 Teklad Global 14% Protein Rodent Maintenance Diet”

from Harlan Iberica Spain and water ad libitum).

2.7. Single dose toxicity study

The design of the study was based on the Organisation for Economic Co-operation and

Development (OECD) guideline 425 for the Testing of Chemicals (24) with some modifications.

Animals were treated with either saline (control group) or a suspension of nanoparticles in saline. The

dose selected of each nanoparticle formulation was based on the results of a preliminary study carried

out in our laboratory in which there was not apparent toxicity: 50 mg/kg bw and three times the

reported value, 150 mg/kg bw. Animals were injected via tail vein with saline or test suspensions at a

volume of 0.5 mL/100 g bw.

Animals were randomly divided into seven groups, containing six female Wistar rats per

group. First group served as control and for each nanoparticle formulations (NP, NP-HPCD and

PEG-NP) animals received a dose of either 50 mg/kg bw or 150 mg/kg bw. The groups were named

as: NP50, NP150, NP-HPCD50, NP-HPCD150, PEG-NP50 and PEG-NP150 respectively.

Individual animal weights were registered on day 0 and weekly during 14 days. Animals were

observed individually for any clinical signs or any symptoms of toxicity at different time intervals

after administration (10 min, 30 min, 1 h, 3 h and 6 h) and daily during 14 days. On completion of

the study, on day 14, blood samples were extracted from the retro-orbital sinus under isoflurane

[A] sample

[A] 100% hemolysisX 100% Hemolysis =


179

anesthesia. The following hematological parameters were analyzed: hemoglobin (HGB; g/L),

hematocrit (HCT, %), red blood corpuscles count (RBC), white blood corpuscles count (WBC),

absolute erythrocyte indices and differential WBC. Biochemical analyses of plasma samples were

performed with a Hitachi 911™ (Roche Diagnostics) analyzer using the protocols obtained from

Roche for the determination of the standard parameters: total protein (g/dL), albumin (g/dL),

glucose (mg/dL), aspartate transaminase (AST; U/l), alanine transaminase (ALT; U/l), cholesterol

(mg/dL), creatinine (mg/dL), urea (mg/dL) and total bilirrubin (mg/dL).

Thereafter, the animals were sacrificed in CO2 chamber, subjected to necropsy and various

organs (spleen, heart, liver, kidneys, adrenal glands, ovaries and thymus) were collected and fixed for

further histopathological examination.

2.8. Histopathological studies

Tissue samples from different body organs were taken during necropsy, fixed in 4%

formaldehyde solution, dehydrated and embedded in paraffin. Paraffin sections (3 micrometers) were

cut, mounted onto glass slides, and dewaxed and stained with haematoxylin and eosin (H&E) for the

subsequent histopathological examination.

2.9. In vivo and ex vivo biodistribution studies with radiolabelled nanoparticles

For in vivo studies, female rats were anesthetized with 2% isoflurane in 100% O2 and placed

in prone position on a gammacamera (SYMBIA-T2 TruePoint®; Siemens, Munich, Germany). Either

one of the radiolabelled nanoparticle suspensions (99mTc-NP, 99mTc-NP-HPCD and 99mTc-PEG-NP)

or free 99mTcO4- were intravenously administered in the tail vein of the animals (50 mg/kg bw in 0.5

mL/100 g bw, 56 MBq). Five consecutive single-photon emission computed tomography (SPECT-

CT) studies (90 images, 15 seconds/image, 2 detectors, matrix 128 x 128, zoom 1) where acquired.

SPECT-CT data were exported to PMOD program and volumes of interest (VOIs) drawn on

computed tomography (CT) images for quantification.

For ex vivo studies, a second group of animals (n=3) was sacrificed 5 h after the i.v.

administration of the radiolabelled nanoparticle formulations. Then, blood, urine, feces and different

organs (lungs, heart, spleen, pancreas, liver, kidneys, stomach, small intestine, large intestine, caecum,

rectum, brain, bone and muscle) were collected, weighted and the radioactivity of each organ

measured in a gamma counter (1282 Compugamma CS, LKB Pharmacia, Finland) calibrated for 99mTc energy. Finally, results were expressed as the percentage of injected dose per gram (%ID/g).


Data were expressed as the mean ± SD of at least three experiments. For the nanoparticles

characterization the Student t test was used. Other statistical significance analysis were carried out

using the non-parametric Mann-Whitney U test. P values of < 0.05 were considered as statistically


180

significant. All calculations were performed using SPSS® statistical software program (SPSS® 15.0,

Microsoft, United States).


181

3. RESULTS



summarized in Table I. All formulations exhibited a homogeneous size of about 175 nm, with a

polydispersity index (PDI) lower than 0.3 and a negative zeta potential.

Table I. Physico-chemical characteristics of nanoparticles. NP: poly(anhydride) nanoparticles; NP-

HPCD: hydroxypropyl cyclodextrin-poly(anhydride) nanoparticles; PEG-NP: pegylated

nanoparticles. PDI: polydispersity index. Data expressed as the mean ± standard deviation (n=6).

The morphological analysis by scanning microscopy (Figure 1) revealed that NP, HPCD-NP

and PEG-NP consisted of spherical particles with a smooth surface in case of NP. In contrast

HPCD-NP and PEG-NP displayed a rough surface. For NP-HPCD, the amount of associated

cyclodextrin was calculated to be about 75 µg/mg whereas for pegylated nanoparticles, the amount of

PEG linked to nanoparticles was 53 µg/mg. Finally, concerning the yield of the process, in all cases,

this parameter was calculated to be very high and close to 100 %.

Figure 1. SEM photographs of nanoparticles. (A) NP. (A.1) Magnification of a section of

photograph (A). (B) NP-HPCD. (B.1) Magnification of a section of photograph (B). (C) PEG-NP.

(C.1) Magnification of a section of photograph (C).

Formulation Size (nm) PDI Zeta Potential (mV)

Nanoparticle formation yield (%)

µg ligand/mg Nanoparticle

NP 159 ± 1.2 0.17 ± 0.05 -42.9 ± 1.5 97 ± 2.1

NP-HPCD 180 ± 2.3 0.21 ± 0.03 -38.2 ± 3.3 98 ± 3.3 74.6 ± 2.2

PEG-NP 183 ± 2 0.23 ± 0.04 -31.7 ± 2.3 97 ± 3.1 53.4 ± 2.8


182

3.2. Erythrocyte sedimentation and agglutination

Erythrocyte sedimentation rate (ESR) was determined by the observation of the sediment

height in a given time interval, usually 1 h. Compared to control, the different poly(anhydride)

nanoparticles did not induce any agglutination effect, which suggests that the integrity of the

erythrocytes membrane remained intact (data not shown).

3.3. Hemolysis study

The hemolytic activity of different poly(anhydride) nanoparticles was tested after incubation

with erythrocyte suspension 1 h at 37ºC at different concentrations. The addition of the nanoparticles

at different concentrations did not cause hemolysis (Figure 2). Indeed the percentage of hemolysis

obtained for the treatments and by adding PBS (spontaneous HGB release) were all about 8%.

Triton X-100 (0.1%) PBS NP NP-HPCD PEG-NP0

20

40

60

80

100

120

0.25 mg/mL0.5 mg/mL1 mg/mL

% H

emol

ysis

Figure 2. Hemolytic activity of poly(anhydride) nanoparticles at concentrations of 0.25, 0.5 and 1

mg/mL. Total cell lysis and spontaneous HGB release were achieved by application of Triton X-100

(1%) and PBS respectively. Data expressed as mean ± SD (n=3).

3.4. Single dose toxicity study

Throughout all the observation period, poly(anhydride) nanoparticles at a dose of 50 mg/kg

bw did not produce the death of animals. Moreover, no abnormal clinical signs or behaviors were

detected neither in treated nor in control groups.

On the contrary, all animals dosed with 150 mg/kg bw showed signs of passivity,

piloerection and respiratory distress (Table II). These symptoms were more severe in animals treated

with NP and NP-HPCD and continued 3 h after dosing. Indeed, 2/6 animals died after dosing 150

mg/kg bw of NP and NP-HPCD respectively. In both cases the deaths occurred about 3 h after

administration. However, in animals treated with PEG-NP these symptoms were milder than the

observed in animals treated with NP and NP-HPCD and disappeared 1 h after administration.

Indeed, PEG-NP at a dose of 150 mg/kg bw did not cause any deaths. In all cases, the reported toxic


183

effects returned to normal gradually the next day after administration and mortality was not observed

during the study.

Table II. Mortality rate and symptoms scored after single i.v. administration of vehicle and

nanoparticle suspensions doses of 50 mg/kg bw or 150 mg/kg bw. Saline: Control, NP50/150:

poly(anhydride) nanoparticles; NP-HPCD50/150: hydroxypropyl cyclodextrin-poly(anhydride)

nanoparticles; PEG-NP50/150: pegylated nanoparticles.

Severity of the symptoms: (-) no symptoms; (+) weak; (++) moderate and (+++) strong.

According to weight register, no significant differences were observed in bw of the animals

of treatment groups compared with control ones. Furthermore, all the hematological and biochemical

values obtained on day 14th were found to be within the normal range with no differences between

the control and the treatment groups (data not shown).

The relative organ weight of heart, kidneys, adrenals, thymus and ovaries were also

unaffected by the nanoparticles. However, the i.v. administration of the different poly(anhydride)

nanoparticles induced an increase in the relative weight of spleens (p<0.05) (Figure 3). Furthermore,

PEG-NP induced a significant increase of the liver relative weight at both doses compared with the

control group (p<0.05).

Treatment Piloerection Passivity Respiratory distress

Mortality

Saline - - - 0/6

NP50 - - - 0/6

NP-HPCD50 - - - 0/6

PEG-NP50 - - - 0/6

NP150 ++ +++ +++ 2/6

NP-HPCD150 ++ +++ +++ 2/6

PEG-NP150 + ++ + 0/6


184

Spleen Liver0.00.20.40.60.81.0

5

10

15

CONTROLNP50NP150NP-HPCD50NP-HPCD150PEG-NP50PEG-NP150R

elat

ive

orga

ns w

eigh

t (%

) *

**

* ** *

*

Figure 3. Spleen and liver relative organ weights. Data expressed as mean ± SD (n=6) * (p<0.05)

statistically significant difference compared with control.

3.5. Histopathology

Regarding histology, the gross and histopathology of spleen, liver and lungs revealed

apparent pathological changes for NP, NP-HPCD and PEG-NP at a dose of 150 mg/kg bw (Figure

4). Actually, an intense proliferation of foamy cells located in the red pulp and lymphoid follicles were

observed in the spleen. Moreover, lungs showed normal areas with moderate inflammatory

infiltration in the alveolar septa, highlighting the presence of small groups of foamy cells associated.

In the liver, small periportal hemorrhages were found as well as massive and diffuse infiltration of

foamy cells between hepatocytes.

3.6. In vivo biodistribution studies

For all the formulations tested, nanoparticles disappeared rapidly from the blood-stream

during the first 10 min. after their i.v. administration. Then, during the following min, NP was the

first to reach the liver and disappear from the lungs and NP-HPCD and PEG-NP remained longer in

the lungs and reached the liver later. Moreover, the SPECT-CT images obtained 100 min after

administration, clearly showed different biodistribution patterns for the different type of radiolabelled

poly(anhydride) nanoparticles (Figure 5) although nanoparticles accumulation occurred

predominantly in organs of the RES. Thus, while 99mTc-NP was mainly located in the liver, 99mTc-

HPCD-NP and 99mTc-PEG-NP were mainly located in the lungs and also in the kidneys. On the

contrary, free 99mTc-pertechnetate used as control was basically located in the stomach.


185

Figure 4. Histological sections of spleen, liver and lung samples collected two weeks after single i.v.

administration of different poly(anhydride) nanoparticles at a dose of 150 mg/kg bw. Accumulation

of foamy cells was detected (black arrows).

3.7. Ex vivo biodistribution studies

Additional experiments without imaging permitted more detailed quantification of the

amount of radioactivity in the different organs at 5 h post-administration (Figure 6). According to the

results obtained, the highest accumulation of radioactivity was found in the kidneys, following by the

liver, lungs, spleen, urine and blood. A relative low concentration of radioactivity was also found in

other organs as hearth, intestines, stomach and thymus, but in all cases represented less than 1%

ID/g (data not shown).

Comparing the different formulations, the radioactivity in the spleen, kidney, blood and urine

were similar. However, the highest value of radioactivity in the liver corresponded with 99mTc-HPCD-

NP (10.33%±0.92) following by 99mTc-PEG-NP (9.72%±0.95) and 99mTc-NP (7.9%±0.22).

Moreover, the radioactivity measured in the lungs was found to be significantly lower (p<0.05) for

animals treated with NP (1.97±0.54) than for HPCD-NP (8.29±0.65) and PEG-NP (8.66±0.48).

Thus, although 5 h after administration all formulations seemed to be accumulated in the same

organs, 99mTc-NP-HPCD and 99mTc-PEG-NP appeared to remain longer in the lungs.


186

Figure 5. 3D summed SPECT-CT images of the localization 100 min after administration of the

radiolabelled nanoparticles. (A): 99mTc-NP, (B): 99mTc-NP-HPCD, (C): 99mTc-PEG-NP and (D): free 99mTc-pertechnetate.

Liver

Lungs

Spleen

Kidneys

BloodUrin

e0

5

10

15

NPNP-HPCDPEG-NP

Organs

%ID

/g

*

Figure 6. Ex vivo biodistribution studies of 99mTc-labelled poly(anhydride) nanoparticles. Animals

were sacrificed 5 h post-administration, organs extracted and radioactivity of each organ measured.

Results expressed as the mean ± SD (n=3) * (p<0.05) statistically significant difference.


187

4. DISCUSSION

In the last years, the copolymers of methyl vinyl ether and maleic anhydride have been

employed to prepare nanoparticles with bioadhesive properties as vehicles for oral drug/antigen

delivery (25, 26). Recently, a comprehensive oral biodistribution study has been carried out in

conventional poly(anhydride) nanoparticles (NP), nanoparticles containing 2-hydroxypropyl-β-

cyclodextrin (NP-HPCD) and pegylated nanoparticles (PEG-NP) (19). The results showed that these

nanoparticles remained within the gut after their administration and no evidences of particulate

absorption were found. Therefore, as the effects of these nanoparticles in the circulatory system are

still unknown, evaluate their behavior and safety by i.v. route before testing its therapeutic

effectiveness is mandatory since this route of administration represents the worst case scenario of

toxicity among all the different administration routes. Taking this into account, the aim of the present

study was to assess the in vitro hemocompatibility, potential acute toxicity and biodistribution of NP,

NP-HPCD and PEG-NP following a single i.v. injection in rats.

Our results demonstrated the hemocompatibility of the different poly(anhydride)

nanoparticles independently of their concentration or surface coating. Indeed, none of the

formulations caused any adverse effects on erythrocytes, neither agglutination nor hemolysis. This

outcome was consistent with findings of Bender and co-workers who revealed that lipid-core

nanocapsules with sizes about 130 nm did not induce hemolysis when nanoparticles were uncoated

or coated with chitosan (27).

At a dose of 50 mg/kg bw, rats exposed to the different poly(anhydride) formulations by i.v.

route did not showed abnormal signs of behaviors throughout the acute study suggesting that NP,

NP-HPCD and PEG-NP could be well tolerated at this dose level. On the contrary, when

nanoparticles were evaluated at the higher dose (150 mg/kg bw) evident signs of toxicity were

observed. Thus, 30% of the animals treated with the high dose of either NP or NP-HPCD died (see

Table II). However, among animals treated with PEG-NP mortality was not observed. Nevertheless,

in all groups, the toxicity observed were consistent with an acute episode which was characterized by

passivity, piloerection and respiratory distress. However, the acute episode was resolved within 3 h

for NP and NP-HPCD, or 1 h for PEG-NP.

On the other hand, evident histopathological changes were observed in the spleen, lungs and

liver of animals (see Figure 4). These changes were put in evidence by the presence of macrophages

with foamy material in their cytoplasm in the observed organs. This disorder, called PLDsis, appears

to be caused by cationic amphiphilic drugs, which stimulate uptake and/or accumulation of

phospholipids by cells (28). However, PLDsis is usually reversible and after the end of the treatment,

the phospholipids levels return to normal and the ultrastructural changes diminish or disappear (29).

Due to this, there is a prevailing theory that PLDsis is primarily an adaptative response to drug

exposure rather than a toxic response. In this regard, even though poly(anhydride) nanoparticle do

not possess a cationic amphiphilic nature, it seems that following i.v. administration, macrophages


188

located in the spleen, liver and lungs recognized the different types of poly(anhydride) nanoparticles.

As macrophages are involved in uptake and metabolism of foreign molecules and particulates (30),

they could phagocyte and metabolize the internalized nanoparticles adopting a foamy morphology.

Then high dose administered could contribute to the accumulation of foamy cells in the different

organs and this would induce pathological changes as the hepatosplegnomegaly observed.

All of these observations would be directly related with the biodistribution of nanoparticles

after i.v. administration. Thus, after administration, nanoparticles would interact with serum of

plasma proteins in a phenomenon called “opsonization” (31). This fact would occur with the three

types of nanoparticles; although the class of serum proteins adsorb on the surface of nanoparticles

may be different and thus their fate might be different. There is growing evidence which suggest that

opsonization of pegylated or sterically protected carriers occur efficiently. One example is

complement activation by long-circulating liposomes, which result in surface opsonization with C3b

and iC3b (32, 33).

In this way, poly(anhydride) nanoparticles have been found to efficiently interact with

proteins of the complement. Concretely, Gomez and Camacho evaluated nanoparticle-complement

interactions by analyzing of C3a and the opsonin C3b, demonstrating that poly(anhydride)

nanoparticles can be strong complement fixing surfaces (34, 35).

In any case, after i.v. administration of nanoparticles, these would reach the lung capillaries,

in which it would be facilitated, at least in part, the transient formation of nanoparticle aggregates.

This fact has been observed previously by other authors. Thus, Yang and collaborators described a

similar effect in mice after the administration of chitosan-coated PLGA nanoparticles of about 200

nm (36). Nanoparticles accumulation in lungs was also reported by Xie and co-workers for 80 nm

silica nanoparticles after i.v. administration in mice (37).

In our case, this effect would be more pronounced for NP and NP-HPCD than for PEG-

NP, probably by a different protein adsorption pattern on the surface of nanoparticles. These

aggregates may cause transient embolism in the lungs capillaries inducing respiratory distress as

observed in animals. However, the dissociation of aggregates would soon proceed inducing a loss of

accumulated nanoparticles in the lungs and then, the recovery observed in animals. Once the

nanoparticles would leave the lungs they would be captured the monocyte-macrophage system

located in the liver (i.e. Kuppfer cells) and spleen (i.e. red-pulp macrophages). This effect appeared to

be more rapidly for NP than for NP-HPCD or PEG-NP. Thus, 2 h, post-administration, NP

appeared to be located mainly in the liver (data not shown). On the contrary, for NP-HPCD and

PEG-NP, the amount of radioactivity measured in lungs (5-h post administration) was about 4-time

higher than that observed with NP (see Figure 6).

In this regard, different studies have demonstrated that on exposure to blood, nanoparticles

of different surface characteristics, size and morphology attract different arrays of opsonins as well as


189

other plasma proteins which may account for the different pattern in the rate and site of nanoparticle

clearance for the blood-stream (2, 38, 39).

Finally, the fraction of nanoparticles that would evade their capture by macrophages would

reach the kidneys in which they would accumulate or trap in the glomerule. This behavior observed

for poly(anhydride) nanoparticles is similar to the biodistribution pattern found for chitosan (40) and

PLGA nanoparticles (41). Thus, Zhang and co-workers reported that N-octyl-O-sulfate chitosan

nanoparticles intravenously administered were accumulated with time in kidneys. In another study

conducted by Giri and collaborators, surface modified PLGA nanoparticles for hepatitis B treatment

were found to be mainly accumulated in blood, kidneys, liver, spleen and lungs suggesting that this

biodistribution pattern could be due to the combined activity of the circulating blood passing through

organs as well as the particle uptake by the monocyte-macrophage system.

Until the end of the study, animals showed normal behavior and the results of biochemical

and hematological analysis at day 14th were found within the normal values comparing to control.

Therefore, as PLDsis has been described as a reversible process, it may occur that the

histopathological changes finally disappear. However, this hypothesis should be confirmed by

recovery studies.

Furthermore, although pegylated nanoparticles did not result in increased residence time in

blood, in relation to the single dose toxicity studies carried out, PEG-NP demonstrated to be safer

than NP or NP-HPCD.

5. CONCLUSIONS

This study presents a comprehensive evaluation of hemocompatibility, biodistribution and

acute i.v. toxicity of conventional (NP), nanoparticles containing 2-hydroxypropyl-β-cyclodextrin

(NP-HPCD) and nanoparticles coated with PEG6000 (PEG-NP). All nanoparticle formulations

demonstrated to be hemocompatible independent of their surface coating. After i.v. administration of

a dose of 50 mg/kg bw, which revealed no evident toxicity, the different poly(anhydride)

nanoparticles were distributed in the kidneys, liver, spleen and lungs. The single dose toxicity studies

have revealed that the i.v. toxicity of the different poly(anhydride) nanoparticles was dose dependent.

Only the highest dose of 150 mg/kg bw was toxic, with the presence of foamy cells in spleen, liver

and lungs as the most relevant finding observed.

Acknowledgments

This work was supported by the Ministry of Science and Innovation in Spain (projects

SAF2008-02538) and Caja Navarra Foundation (Grant 10828). Patricia Ojer was also financially

supported by a grant from the Department of Education of the Government of Navarra in Spain.


190

6. References

1. M.A. Dobrovolskaia, A.K. Patri, J. Zheng, J.D. Clogston, N. Ayub, P. Aggarwal, B.W. Neun,

J.B. Hall, and S.E. McNeil. Interaction of colloidal gold nanoparticles with human blood:

effects on particle size and analysis of plasma protein binding profiles. Nanomedicine. 5:106-

117 (2009).

2. S.M. Moghimi, A.C. Hunter, and T.L. Andresen. Factors controlling nanoparticle

pharmacokinetics: an integrated analysis and perspective. Annu Rev Pharmacol Toxicol.

52:481-503 (2011).

3. R.A. Yokel, T.C. Au, R. MacPhail, S.S. Hardas, D.A. Butterfield, R. Sultana, M. Goodman,

M.T. Tseng, M. Dan, H. Haghnazar, J.M. Unrine, U.M. Graham, P. Wu, and E.A. Grulke.

Distribution, elimination, and biopersistence to 90 days of a systemically introduced 30 nm

ceria-engineered nanomaterial in rats. Toxicol Sci. 127:256-268 (2012).

4. Y. Xue, S. Zhang, Y. Huang, T. Zhang, X. Liu, Y. Hu, Z. Zhang, and M. Tang. Acute toxic

effects and gender-related biokinetics of silver nanoparticles following an intravenous

injection in mice. J Appl Toxicol. 32:890-899 (2012).

5. Y. Huang, H. Gao, M. Gou, H. Ye, Y. Liu, Y. Gao, F. Peng, Z. Qian, X. Cen, and Y. Zhao.

Acute toxicity and genotoxicity studies on poly(epsilon-caprolactone)-poly(ethylene glycol)-

poly(epsilon-caprolactone) nanomaterials. Mutat Res. 696:101-106 (2010).

6. Y. Wenger, R.J. 2nd Schneider, G.R. Reddy, R. Kopelman, O. Jolliet, and M.A. Philbert.

Tissue distribution and pharmacokinetics of stable polyacrylamide nanoparticles following

intravenous injection in the rat. Toxicol Appl Pharmacol. 251:181-190 (2011).

7. Z. Zhu, Y. Li, X. Li, R. Li, Z. Jia, B. Liu, W. Guo, W. Wu, and X. Jiang. Paclitaxel-loaded

poly(N-vinylpyrrolidone)-b-poly(epsilon-caprolactone) nanoparticles: preparation and

antitumor activity in vivo. J Control Release. 142:438-446 (2010).

8. X. Song, X. Zhao, Y. Zhou, S. Li, and Q. Ma. Pharmacokinetics and disposition of various

drug loaded biodegradable poly(lactide-co-glycolide) (PLGA) nanoparticles. Curr Drug

Metab. 11:859-869 (2010).

9. S. Gao, J. Sun, D. Fu, H. Zhao, M. Lan, and F. Gao. Preparation, characterization and

pharmacokinetic studies of tacrolimus-dimethyl-beta-cyclodextrin inclusion complex-loaded

albumin nanoparticles. Int J Pharm. 427:410-416 (2012).

10. J.L. Arias, L.H. Reddy, and P. Couvreur. Superior preclinical efficacy of gemcitabine

developed as chitosan nanoparticulate system. Biomacromolecules. 12:97-104 (2011).





191



Opin Drug Deliv. 8:721-734 (2011).

13. J. Calvo, J.L. Lavandera, M. Agueros, and J.M. Irache. Cyclodextrin/poly(anhydride)


13:1015-1025 (2011).

14. R. Da Costa Martins, J.M. Irache, J.M. Blasco, M.P. Munoz, C.M. Marin, M. Jesus Grillo, M.

Jesus De Miguel, M. Barberan, and C. Gamazo. Evaluation of particulate acellular vaccines

against Brucella ovis infection in rams. Vaccine. 28:3038-3046 (2010).



4790 (2009).







18. S. Gomez, C. Gamazo, B. San Roman, A. Grau, S. Espuelas, M. Ferrer, M.L. Sanz, and J.M.

Irache. A novel nanoparticulate adjuvant for immunotherapy with Lolium perenne. J Immunol

Methods. 348:1-8 (2009).

19. P. Ojer, A.L. de Cerain, P. Areses, I. Penuelas, and J.M. Irache. Toxicity studies of

poly(anhydride) nanoparticles as carriers for oral drug delivery. Pharm Res. 29:2615-2627

(2012).

20. P. Ojer, H.H. Salman, R. Da Costa Martins, J. Calvo, A. López de Cerain, C. Gamazo, J.L.





Biomed Anal. 39:495-502 (2005).




23. M. Agueros, P. Areses, M.A. Campanero, H. Salman, G. Quincoces, I. Penuelas, and J.M.

Irache. Bioadhesive properties and biodistribution of cyclodextrin-poly(anhydride)

nanoparticles. Eur J Pharm Sci. 37:231-240 (2009).


192



Publishing; 2006.

25. J.D. Reboucas, J.M. Irache, A.I. Camacho, I. Esparza, V. Del Pozo, M.L. Sanz, M. Ferrer,

and C. Gamazo. Development of poly(anhydride) nanoparticles loaded with peanut proteins:

the influence of preparation method on the immunogenic properties. Eur J Pharm Biopharm

(2012).




27. E.A. Bender, M.D. Adorne, L.M. Colome, D.S. Abdalla, S.S. Guterres, and A.R. Pohlmann.

Hemocompatibility of poly(varepsilon-caprolactone) lipid-core nanocapsules stabilized with

polysorbate 80-lecithin and uncoated or coated with chitosan. Int J Pharm. 426:271-279

(2012).

28. M.J. Reasor, K.L. Hastings, and R.G. Ulrich. Drug-induced phospholipidosis: issues and

future directions. Expert Opin Drug Saf. 5:567-583 (2006).

29. L.A. Chatman, D. Morton, T.O. Johnson, and S.D. Anway. A strategy for risk management

of drug-induced phospholipidosis. Toxicol Pathol. 37:997-1005 (2009).

30. T.M. Saba. Physiology and physiopathology of the reticuloendothelial system. Arch Intern

Med. 126:1031-1052 (1970).

31. D.E. 3rd Owens, and N.A. Peppas. Opsonization, biodistribution, and pharmacokinetics of

polymeric nanoparticles. Int J Pharm. 307:93-102 (2006).

32. J. Szebeni, L. Baranyi, S. Savay, J. Milosevits, R. Bunger, P. Laverman, J.M. Metselaar, G.

Storm, A. Chanan-Khan, L. Liebes, F.M. Muggia, R. Cohen, Y. Barenholz, and C.R. Alving.

Role of complement activation in hypersensitivity reactions to doxil and hynic PEG

liposomes: experimental and clinical studies. J Liposome Res. 12:165-172 (2002).

33. S.M. Moghimi and J. Szebeni. Stealth liposomes and long circulating nanoparticles: critical

issues in pharmacokinetics, opsonization and protein-binding properties. Prog Lipid Res.

42:463-478 (2003).

34. S. Gomerz, C. Gamazo, B. San Roman, C. Vauthier, M. Ferrer and J.M. Irache.

Development of a novel vaccine delivery system based on Gantrez nanoparticles. J. Nansci

Nanotechnol. 6:3283-3289 (2006)





193

36. R. Yang, S.G. Yang, W.S. Shim, F. Cui, G. Cheng, I.W. Kim, D.D. Kim, S.J. Chung, and

C.K. Shim. Lung-specific delivery of paclitaxel by chitosan-modified PLGA nanoparticles via

transient formation of microaggregates. J Pharm Sci. 98:970-984 (2009).

37. G. Xie, J. Sun, G. Zhong, L. Shi, and D. Zhang. Biodistribution and toxicity of intravenously

administered silica nanoparticles in mice. Arch Toxicol. 84:183-190 (2010).

38. F. Alexis, E. Pridgen, L.K. Molnar, and O.C. Farokhzad. Factors affecting the clearance and

biodistribution of polymeric nanoparticles. Mol Pharm. 5:505-515 (2008).

39. H.M. Patel and S.M. Moghimi. Serum-mediated recognition of liposomes by phagocytic cells

of the reticuloendothelial system - the concept of tissue specificity. Adv Drug Deliv Rev.

32:45-60 (1998).39.

40. C. Zhang, G. Qu, Y. Sun, T. Yang, Z. Yao, W. Shen, Z. Shen, Q. Ding, H. Zhou, and Q.

Ping. Biological evaluation of N-octyl-O-sulfate chitosan as a new nano-carrier of

intravenous drugs. Eur J Pharm Sci. 33:415-423 (2008).

41. N. Giri, P. Tomar, V.S. Karwasara, R.S. Pandey, and V.K. Dixit. Targeted novel surface-

modified nanoparticles for interferon delivery for the treatment of hepatitis B. Acta Biochim

Biophys Sin (Shanghai). 43:877-883 (2011).

CHAPTER 8

GENERAL DISCUSSION

CHAPTER 8: GENERAL DISCUSSION

197

GENERAL DISCUSSION

Nanomedicine is a burgeoning field of research with tremendous prospects for the

improvement of the diagnosis and treatment of human diseases. However, the biomedical application

of engineered nanoparticles may be hampered by two concerns. First, the methods for production

and purification of nanoparticles have to be balanced against the actual benefits offered by the new

technology in order to obtain nanomedicines capable of being produced on an industrial scale and

thus get into de market. Secondly, and the most important, the biological behavior and toxicological

properties of new nanomedicines must be carefully assessed.

To date, our understanding of the interactions of nanomaterials with biological systems is

limited and thus it is unclear how nanomedicine-based products might produce harmful biological

responses. In this regard, nanotoxicology has emerged in the last years to recognize and avoid

potential risks associated with nanoparticles. For this to be achieved, detailed physical and chemical

characterization of nanoparticles is imperative as it is known that the physico-chemical properties of

nanoparticles have a direct influence on its behavior and hence on its toxicity. Although certain

relationships between these characteristics and their potential toxicity have been established in

general terms, it is considered that the toxicity of each novel nanoparticulate system has to be

evaluated on a case by case basis, and should not be applicable to other nanosystems. Therefore,

efforts are needed to improve the standardization of assays used for in vitro and in vivo testing of

nanoparticles.

The copolymers of methyl vinyl ether and maleic anhydride (Gantrez®) are widely employed

in a diverse range of topical and oral pharmaceutical formulations due to its adhesive properties and

safety profile. Likewise, these copolymers have demonstrated to be biocompatibles and bioerodibles.

Therefore, a decade ago, our research group employed for the first time these copolymers to prepare

biocompatible and bioerodible nanoparticles with bioadhesive properties as vehicles for oral

drug/antigen delivery. We designed different types of poly(anhydride) nanoparticles as carriers for

oral delivery: conventional nanoparticles (NP), nanoparticles containing 2-hydroxypropyl-β-

cyclodextrin (NP-HPCD) and pegylated nanoparticles (PEG-NP) which have demonstrated high

versatility and efficacy by loading different drugs/antigens. Thus, these nanoparticles could be good

candidates to make the leap to clinical studies. However, before proceeding to clinical studies, design

a scalable manufacturing method and evaluate its safety is undoubtedly necessary.

Bearing in mind that modify the manufacturing process may alter the physicochemical

characteristics of nanoparticles, and hence its toxicity, the first step carried out in the present research

project was to develop a new scalable manufacture method ensuring that the physicochemical

properties of the different poly(anhydride) nanoparticles remain unaltered.

When poly(anhydride) nanoparticles were firstly developed, they were produced by

desolvation of the polymer with a mixture of ethanol and water and subsequent lyophilization.

Although this method of preparation and preservation resulted in nanoparticle formulations with


198

physicochemical characteristics suitable for oral administration, this strategy also had some drawbacks

that hindered its scale-up and subsequent use in clinical studies. Thus, the time necessary to product

and preserve poly(anhydride) nanoparticles was about 50 h. Furthermore, it should be noted that

lyophilization is highly expensive and reserved for products with high added value. On the other

hand, the method of preparation involved the use of two organic solvents such as ethanol and

acetone.

Therefore, to shorten the process we decided to exchange the lyophilization step by a drying

process in a spray-dryer apparatus. During the optimization of the spray-drying process of

poly(anhydride) nanoparticles we noticed that the yield of the process was low and the resulting

powders were extremely difficult to redisperse in water. Moreover, these problems were amplified

when ethanol was present in the nanoparticle suspensions. Thus we decided to incorporate an

adjuvant to induce a dilution effect which may reduce the possibilities for nanoparticle interaction

and fusion due to the process temperature. Furthermore, ethanol was removed, whereby the

preparation process involved acetone and water only.

Carbohydrates, concretely lactose, maltose, glucose and mannitol, were the selected adjuvant

agents that we decided to incorporate in the poly(anhydride) nanoparticle suspension because its well-

establish use as excipients in the pharmaceutical industry. After assessing the ability of the different

carbohydrates to protect the spray-dried nanoparticles, it was demonstrated that a

saccharide/poly(anhydride) ratio of at least 2 was necessary to produce a homogeneous reconstitution

of nanoparticles in an aqueous environment. In addition, among the different carbohydrates

evaluated, lactose was the one that provided the best conditions to produce a dry powder from the

poly(anhydride) nanoparticles suspension.

Compared with nanoparticles produced by lyophilization, the main physico-chemical

characteristics (size, zeta potential and PDI) of the different nanoparticles evaluated were similar to

those prepared by spray-drying when lactose was used as adjuvant, demonstrating that the spray-

drying technique is an adequate alternative to preserve poly(anhydride) nanoparticles as dry powders.

Thus, throughout this research project, by using spray-drying technique, nanoparticles of well-defined

sized about 160-170 nm, narrow size distribution (PDI<0.3) and with negative surface charge were

obtained. Likewise, nanoparticles morphology was similar to those prepared by lyophilization.

The applicability of the spray-drying technique was evaluated with either drug or antigen-

loaded formulation by incorporation of the HS from Brucella ovis or ATO. The results demonstrated

that the preparative process did not affect neither the integrity/composition of the proteins included

in the HS nor the encapsulation efficiency of ATO. Indeed, it has been found that the preparation of

poly(anhydride) nanoparticles by spray-drying not only allows the incorporation of drugs or sensitive

molecules, but could also enhance the ability of these nanoparticles as stimulators of immune

responses. In this context, a study published by our research group, which addresses the application

of these nanoparticles in peanut allergy immunotherapy, demonstrated that spray-dried


199

poly(anhydride) nanoparticles loaded with peanut proteins showed different immunomodulatory

properties than nanoparticles produced by lyophilization (1). Particularly, these differences were due

to the greater stability that spray-dried nanoparticles displayed, which resulted in a slower release rate

of the allergen, and therefore an efficient stimulation of immune responses.

After improving the manufacture method by using the spray-drying technique, we proceeded

to assess their possible harmful effects. When addressing the evaluation of nanoparticles toxicity, the

first step that should be conducted is to evaluate their cytotoxicity and cell interaction since these

studies are the basis for understanding the biocompatibility of the nanoparticles prior to in vivo

studies.

Likewise, it is very important to note that if nanoparticles are highly toxic in vitro, the

modification of physico-chemical particles characteristics should be considered before proceeding

with in vivo toxicity studies as it would not be ethically appropriate to conduct studies involving

animals if it is expected that they are going to suffer due to the high toxicity of the nanoparticles.

In this research project, to evaluate the cytotoxicity of the different poly(anhydride)

nanoparticles, two different cell lines were chosen as models of two important targets for

nanoparticles toxicity upon oral exposure: the human hepatocarcinoma-derived HepG2 cells (as liver

model) and the human colonic adenocarcinoma derived Caco-2 cells (as gastrointestinal epithelium

model). Although the results from the MTS and LDH assays showed a concentration-dependent

cytotoxic effect, this cytotoxicity appeared only at very high concentrations and long incubation times

in HepG2 cells (1-2 mg/mL at 48-72 h). Thus, the in vitro safety of the different poly(anhydride)

nanoparticles was demonstrated. This fact allowed establishing the basis to assess its safety in vivo

because, although it is difficult to extrapolate the results from in vitro to in vivo studies, the absence of

cytotoxicity makes it ethically acceptable to conduct in vivo studies.

The results obtained corroborated the biocompatibility of the different poly(anhydride)

nanoparticles and in turn, its bioadhesive character was clearly confirmed after conducting association

studies in Caco-2 cells, which is directly related to their previously reported oral efficacy (2-4).

Furthermore, this study enabled to establish a direct relation between the physico-chemical

characteristics of the designed nanoparticles and its adhesive properties. In this regard, HPCD-NP

and PEG-NP, which displayed a rough surface on the SEM images, showed higher cell adhesion than

NP with smooth surface. Therefore, for the first time, the specific surface properties of the

poly(anhydride) nanoparticles were demonstrated by in vitro cell interaction studies because their

behavior in cultured cells was well correlated with the assumption that larger surface area of

nanoparticles results in higher cell binding.

Given the efficacy of poly(anhydride) nanoparticles as oral delivery systems and its in vitro

safety, the in vivo toxicity evaluation by this route was considered the adequate next step. For this

purpose, acute and sub-acute toxicity studies were conducted according to the standard procedures


200

and guidelines proposed by the OECD which are recommended at the regulatory level to assess

chemicals safety.

Since so far there are no specific protocols validated for nanoparticles or nanomaterials, we

decided to follow OECD guidelines because they make general recommendations regarding the study

protocols without taking into account the kind of product to be tested. Therefore, these guidelines

could be easily adapted to conduct nanotoxicological studies. The OECD guidelines have been

established in order to obtain reliable and reproducible results and give recommendations regarding

different aspects of the test procedure such as, experimental animal model (species, sex and number

of animals, housing and feeding conditions), dose levels to be tested; observation period; clinical

examination procedure; hematology; blood and urine analytical biochemistry; pathology (gross

necropsy and histopathology). They also include recommendations on the treatment and evaluation

of the results, and the items to be included in a test report. Moreover, OECD guidelines are in

continuous revision and updating in order to incorporate scientific progress in the field and also to

encompass the principles of the 3 Rs (Replacement, Reduction and Refinement) which is an essential

aspect to consider when conducting animal studies.

The results obtained in acute and sub-acute toxicity studies demonstrated the safety of the

different poly(anhydride) nanoparticles by the oral route. The acute toxicity study was performed

according to OECD Guideline 425 “Acute Oral Toxicity: Up-and-Down Procedure” that is one of

the three guidelines that were validated and accepted at the regulatory level as alternatives to the

OECD guideline 401 “Acute Oral Toxicity”, that was deleted on 2001 (5). The procedure of the limit

test was applied and a single dose of 2000 mg/kg bw was administered by gavage. After the

administration of this dose that was approximately 200-times higher than the usual therapeutic dose

employed previously, no toxic effects were reported in the animals during the 14-day study and no

alterations were observed in the necropsies. The sub-acute toxicity studies were carried out according

to Guideline OECD 407 “Repeated dose 28-days Oral Toxicity Study in Rodents” (6). The results

obtained confirmed that poly(anhydride) nanoparticles do not generate cumulative and latent changes

following multiple oral administrations during 28 days, even using doses 30-times higher than those

reported in previous studies. Thus, for the different poly(anhydride) nanoparticles, the NOAEL (No

Observed Adverse Effect Level) after repeated dose 28-day toxicity study was 300 mg/kg bw.

In addition, in the biodistribution studies, where the different nanoparticles were

radiolabelled with 99mTc and orally administered to rats, the safety of poly(anhydride) nanoparticles

was confirmed as no evidences of translocation or absorption of nanoparticles was found. Moreover,

PEG-NP showed longer residence time within the GIT, followed by NP-HPCD and lastly NP.

These differences are consistent with those found in the in vitro studies, and therefore may also be

explained in line with their different surface characteristics.

Despite of the fact that poly(anhydride) nanoparticles were designed for oral delivery, due to

the promising results obtained in terms of efficacy and safety by this route (2-4, 7), it was consider of


201

great interest to evaluate their behavior and safety by i.v. route before assessing their therapeutic

effectiveness.

Following the same strategy that was applied for oral safety assessment of the different

poly(anhydride) nanoparticles, prior to carry out in vivo toxicity studies, nanoparticles

hemocompatibility was tested in vitro. The results demonstrated that all the poly(anhydride)

nanoparticles were hemocompatible as none of the formulations caused any harmful effect on

erythrocytes at any of the concentrations tested (0.25-1 mg/mL). Once the hemocompatibility was

demonstrated in vitro, the in vivo safety of nanoparticles was assessed. For this purpose, acute toxicity

studies, according with OECD guidelines, were performed (5). The acute toxicity study was carried

out at two different doses of 50 and 150 mg/kg bw, being the lower dose based on preliminary

studies developed in our laboratory. Although at a dose of 50 mg/kg bw the animals exposed to

nanoparticles intravenously did not show abnormal signs of behavior throughout the study, at the

higher dose (150 mg/kg bw) evident signs of toxicity were observed in all groups, characterized by

passivity and respiratory distress. In fact, 30% of the animals died because of the administration of

NP and NP-HPCD. It is interesting to note that, in surviving animals, the acute episode was resolved

in 1 h for PEG-NP group and in 3 h for NP and NP-HPCD groups suggesting that, although the

polymer in form of nanoparticles seems to provoke toxicity when it is administered intravenously,

adding PEG to the nanoparticle formulation appears to reduce its toxicity somehow. Thus, once

again, it is clear the relationship between the physico-chemical characteristics of the nanoparticles and

its toxicity, and according to this study, the surface characteristics of the different poly(anhydride)

nanoparticles is a key aspect.

Thus, the i.v. acute toxicity study allowed concluding that the poly(anhydride) nanoparticles

were clearly toxic at the highest dose tested. Furthermore, it is noteworthy that evident

histopathological changes were observed in the spleen, lungs and liver of animals for the three types

of poly(anhydride) nanoparticles, characterized by the presence of macrophages with foamy material

in their cytoplasm. This disorder, called PLDsis is usually dose-dependent and reversible, so there is a

prevailing theory that PLDsis is primary an adaptative response to drug exposure rather than a toxic

response. To date PLDsis has been mainly reported for cationic amphiphilic drugs although some

studies have also reported this disorder for Pluronic F68, cerium and silica oxide nanoparticles,

PAMAM dendrimers and PEG (8-12).

In the present work, if PLDsis had been found in the animals that were treated with PEG-

NP only, the disorder would have been associated with the presence of PEG, which is already

reported to cause PLDsis (8). However, the disorder was observed for the three types of

nanoparticles, so, in this case PLDsis is due not only to the PEG but also to the polymer. Therefore,

this is the first time that PLDsis has been described for polymeric nanoparticles administered by the

i.v. route.


202

Moreover, as it was mention before, for the three nanoparticle formulations evaluated by the

i.v. route, NP and NP-HPCD were found to be the most toxic and PEG-NP the less toxic. This fact

could be due to the adsorption of different blood proteins onto the nanoparticles by a phenomenon

called opsonization. Actually, it is well known that one of the key points to control when performing

i.v. administration is the opsonization phenomenon, and this is directly related to the physico-

chemical characteristics of the nanoparticles. Thus, as our three types of formulations have different

physicochemical properties, the opsonization pattern may be different.

The results obtained in the biodistribution studies support this hypothesis. Thus, it was

observed that after the i.v. administration, although all nanoparticles accumulated in the lungs, NP

disappeared more rapidly than NP-HPCD and PEG-NP, which remained in the lungs for a longer

time. Nanoparticles accumulation in the lungs is consistent with the acute respiratory distress

observed in the animals during the single dose toxicity studies; this symptom was particularly

intensive for NP and NP-HPCD formulations. Therefore, these results clearly demonstrate once

again, that the physico-chemical characteristics lead the biological/toxicological behavior of

nanoparticles. Accordingly, it is not possible to extrapolate the outcomes obtained with one type of

nanoparticles to other types. This study corroborates that the individual characteristics of each system

will determine its toxicity, and as a consequence, any change in these characteristics makes necessary

to re-evaluate their toxicity.

Taking into account the aforementioned, the present work has established a clear relationship

between the physico-chemical characteristics of the poly(anhydride) nanoparticles tested and their

toxicity. In Table I there is an attempt to correlate some physicochemical characteristics with a

nanoparticle biological behavior which has been in some cases demonstrated in this study and, in

other cases it could be expected according to the available data of this and other studies.

Moreover, it is important to note that the physico-chemical characteristics of spray-drying

nanoparticles were similar to those obtained by lyophilization which means that the outcomes

obtained in the present study apply also to the lyophilized nanoparticles, as long as their physico-

chemical characteristics remain similar.


203

Table I. Correlation of poly(anhydride) nanoparticles physicochemical characteristics with their

biological behavior/toxicity.

Characteristic Biological behavior

SIZE 160- 170 nm

(no translocation)

• Nanoparticles are not uptaken by Caco-2 cells so they do not provoke intracellular toxicity.

• After oral administration, poly(anhydride) nanoparticles do not translocate to systemic circulation.

• After i.v. administration, poly(anhydride) nanoparticles do not translocate across neuronal pathways.

CHARGE Negative surface charge

(no membrane disruption)

• Nanoparticles do not disturb cell membranes in HepG2 and Caco-2 cells.

• After oral administration, nanoparticles do not interact with any biological component of the gut.

• After i.v. administration, there is an opsonization phenomenon

STABILITY Nanoparticles are bioerodible

(no accumulation)

• Nanoparticles do not accumulate in tissues following multiple administrations.

SURFACE SHAPE (different bioadhesive

properties)

• NP displaying a smooth surface have less bioadhesive capacity.

• NP-HPCD and PEG-NP displaying a rough surface result in large surface area and therefore greater bioadhesive potential.

SURFACE CHEMISTRY (different opsonization

pattern)

• NP are less hydrophilic and its surface is mainly composed of carboxylic groups highly reactive, thus NP can react easily with molecules containing –OH, -NH or –SH among others.

• NP-HPCD surface is composed of carboxylic groups belonging to the poly(anhydride) and to HPCD, as it was demonstrated that they are located in & out the nanoparticle. Thus NP-HPCD may react with the same kind of molecules that NP, but more efficiently.

• For PEG-NP, several studies have demonstrated that PEG chains form a brush layer surrounding nanoparticle surface. Ethylene groups present in the surface are less reactive, thus this strategy is employed to design more compatible nanoparticles, especially in case of i.v. administration.


204

Therefore, according to the Nanotoxicological Classification System (NCS) (13), as the

poly(anhydride) nanoparticles tested in this work have displayed a size above 100 nm, they are

degradable and are not taken up by cells, NP, NP-HPCD and PEG-NP would be classified as Class I.

This is in agreement with the results obtained for these nanoparticles by the oral route, where the

formulations have demonstrated to be safe and thus good candidates to be used in medicine.

Bearing in mind the design of oral and i.v. study, one may conclude that in order to carry out

an appropriate nanotoxicological study, once the physico-chemical characterization of the

nanoparticles is known, a preliminary in vitro study should be conducted as a first approach, since it

provides a clear and straightforward estimation of possible toxicological risks prior to in vivo studies

and even allows us to give answers to the results obtained from in vivo studies.

Related to in vivo studies, the guidelines followed in this study have been proven to be

suitable to evaluate the safety/toxicity of the poly(anhydride) nanoparticles since it has been

demonstrated that these nanoparticle formulations are safe when they are administered orally but not

intravenously.

Thereby, considering all the above, this research project has permitted to establish the basis

for assessing the toxicity of polymeric nanoparticles intended for oral delivery of drugs and/or

antigens.


205

References

1. J. De Souza Reboucas, J.M. Irache, A.I. Camacho, I. Esparza, V. Del Pozo, M.L. Sanz, M.

Ferrer, and C. Gamazo. Development of poly(anhydride) nanoparticles loaded with peanut

proteins: the influence of preparation method on the immunogenic properties. Eur J Pharm

Biopharm. 82:241-249 (2012).






4790 (2009).






Publishing; 2006.

6. Organization for Economic Cooperation and Development. Test No. 407: Repeated Dose

28-day Oral Toxicity Study in Rodents. Guidelines for the Testing of Chemicals. Paris:

OECD Publishing; 2001.

7. P. Ojer, A.L. de Cerain, P. Areses, I. Peñuelas, and J.M. Irache. Toxicity studies of

poly(anhydride) nanoparticles as carriers for oral drug delivery. Pharm Res. 29:2615-2627

(2012).

8. A. Bendele, J. Seely, C. Richey, G. Sennello, and G. Shopp. Short communication: renal

tubular vacuolation in animals treated with polyethylene-glycol-conjugated proteins. Toxicol

Sci. 42:152-157 (1998).

9. J.Y. Ma, R.R. Mercer, M. Barger, D. Schwegler-Berry, J. Scabilloni, J.K. Ma, and V.

Castranova. Induction of pulmonary fibrosis by cerium oxide nanoparticles. Toxicol Appl

Pharmacol. 262:255-264 (2012).

10. G. Magnusson, T. Olsson, and J.A. Nyberg. Toxicity of Pluronic F-68. Toxicol Lett. 30:203-

207 (1986).

11. S.T. Stern, P.P. Adiseshaiah, and R.M. Crist. Autophagy and lysosomal dysfunction as

emerging mechanisms of nanomaterial toxicity. Part Fibre Toxicol. 9:20 (2012).

12. M.J. Reasor, K.L. Hastings, and R.G. Ulrich. Drug-induced phospholipidosis: issues and

future directions. Expert Opin Drug Saf. 5:567-583 (2006).


206

13. R.H. Muller, S. Gohla, and C.M. Keck. State of the art of nanocrystals-special features,

production, nanotoxicology aspects and intracellular delivery. Eur J Pharm Biopharm. 78:1-9

(2011)

CHAPTER 9

CONCLUSIONS/CONCLUSIONES

CHAPTER 9: CONCLUSIONS/CONCLUSIONES

209

CONCLUSIONS

The experimental work compiled in this research project has been focused on the evaluation of

the oral and i.v. toxicity of poly(anhydride) nanoparticles. All the results obtained for this work have led us

to conclude the following:

1. Preparation of conventional poly(anhydride) nanoparticles, nanoparticles containing 2-

hydroxypropyl-β-cyclodextrin and pegylated nanoparticles by the spray-drying procedure.

1.1. The different poly(anhydride) nanoparticles were easily prepared by the solvent

displacement method using only water and acetone.

1.2. The addition of lactose at a saccharide/polymer ratio of 2 provided the best conditions

to produce a dry powder that were easily reconstituted in a nanoparticle suspension by

simple addition of water and kept their physico-chemical characteristics one year after its

preparation.

1.3. By employing the new industrial scale-up method, the time required to prepare the

nanoparticles was shorten and the physico-chemical characteristics of the nanoparticles

produced were similar to those obtained by lyophilization.

2. Cytotoxicity assessment and in vitro interactions studies in human cell lines.

2.1. Poly (anhydride) nanoparticles showed concentration and incubation time dependent

cytotoxic effects in HepG2 and Caco-2 cells. Thus, only at the highest concentrations and

incubation times (1-2 mg/mL; 48-72 h) tested, conventional and cyclodextrin nanoparticles

proved to be cytotoxic, so poly(anhydride) nanoparticle safety were clearly demonstrated in

vitro.

2.2. The bioadhesive character of poly(anhydride) nanoparticles was confirmed in the

interaction studies carried out in the Caco-2 cell line, and its adhesive properties were directly

related with their surface characteristics. Thus, the highest cytoadhesion was observed for

PEG-NP followed by NP-HPCD and the lowest corresponded to NP

3. Evaluation of acute and sub-chronic oral toxicity according to OECD guidelines and

biodistribution assessment by Technetium radiolabelling.

3.1. Acute and sub-chronic oral toxicity studies, conducted according to the OECD

guidelines 425 and 407, confirmed the oral safety of the different poly(anhydride)

nanoparticles.

3.2. At single dose, the LD50 was greater than 2,000 mg/kg bw in rat by the oral route.


210

3.3. At repeated doses, during 28 days, the NOAEL was 300 mg/kg bw, a dose 30-times higher

than the therapeutic dose.

3.4. Biodistribution studies with 99mTechnetium showed that, following oral administration, the

different poly(anhydride) nanoparticles are located mainly in the intestine and excreted by feces.

3.5. Pegylated-poly(anhydride) nanoparticles shower slower transit through the gut than

conventional or cyclodextrin nanoparticles. This fact confirmed that pegylation of nanoparticles

increase their adhesive properties.

4. Evaluation of hemocompatibility, biodistribution and acute i.v. toxicity of different

poly(anhydride) nanoparticles

4.1. In human erythrocytes, all nanoparticle formulations demonstrated to be hemocompatible

independent of their concentration and physico-chemical characteristics.

4.2. At single dose by i.v. route, poly(anhydride) nanoparticles showed dose dependent toxicity.

Thus, although at a dose of 50 mg/kg bw, the different formulations did not show evident signs

of toxicity, at a dose of 150 mg/kg bw, all formulations revealed to be toxic and its toxicity was

dependent on its physico-chemical characteristics.

4.3. The toxicity of poly(anhydride) nanoparticles were characterized by an respiratory distress

acute episode, being more serious in the case of conventional and cyclodextrin nanoparticles,

where 30% of the animals died.

4.4. In the histopathological examination, the most relevant finding was the presence of foamy

cells in the spleen, liver and lungs. These findings demonstrated, for the first time, that

intravenously administered polymeric nanoparticles could provoke phospholipidosis.

4.5. Intravenously, biodistribution studies showed that, the different poly(anhydride)

nanoparticles are rapidly cleared from the circulation and accumulate in the liver, spleen, lungs

and kidneys. Thus, at short times, the distribution pattern was dependent on the physico-

chemical characteristics of the formulations, which in turn seem to drive the opsonization

pattern of the nanoparticles and therefore its toxicity.


211

CONCLUSIONES

El trabajo experimental recogido en esta memoria se ha enfocado hacia la evaluación de la

toxicidad oral e intravenosa de las nanopartículas de poli(anhídrido). Los resultados obtenidos en esta

tesis doctoral nos han permitido concluir lo siguiente:

1. Obtención de nanopartículas de poli(anhídrido) convencionales, con 2-hidroxipropil-β-

ciclodextrina y poli-etilenglicol 6000 por la técnica del “spray-drying”:

1.1. Las diferentes nanopartículas de poli(anhídrido) se prepararon fácilmente mediante el

método del desplazamiento del disolvente empleando únicamente agua y acetona.

1.2. La adición de lactosa con un ratio sacárido/polímero de 2 y posterior secado por “spray

drying” permitió obtener formulaciones en forma de polvo seco que se reconstituyeron

fácilmente en agua y mantuvieron sus características físico-químicas un año después de su

fabricación.

1.3. Mediante la aplicación del nuevo método escalable a nivel industrial se disminuyó el tiempo

requerido para preparar las nanopartículas obteniéndose formulaciones con características físico-

químicas similares a las nanopartículas preparadas anteriormente por liofilización.

2. Evaluación de la citotoxicidad e interacción in vitro en células humanas:

2.1. Las nanopartículas de poli(anhídrido) mostraron efectos citotóxicos dependientes de la

concentración y el tiempo de incubación en las líneas celulares HepG2 y Caco-2. Así,

únicamente a las concentraciones más altas y mayores tiempos de incubación (1-2 mg/mL; 48-

72 horas) las nanopartículas convencionales y con ciclodextrina demostraron ser citotóxicas, por

lo que su seguridad in vitro quedó claramente demostrada.

2.2. El carácter bioadhesivo de las nanopartículas de poli(anhídrido) quedó confirmado en los

estudios de interacción llevados a cabo en la línea celular Caco-2 y sus propiedades adhesivas

dependieron directamente de sus características de superficie. Así, las nanopartículas de

poli(anhídrido) pegiladas y con ciclodextrina mostraron una mayor adhesión a la superficie de las

células, debido probablemente a su mayor área de superficie en comparación con las

nanopartículas convencionales.

3. Evaluación de la toxicidad aguda y sub-crónica por vía oral según las guías de la OCDE y

evaluación de la biodistribución mediante radiomarcaje con Tecnecio:

3.1. Los estudios de toxicidad oral aguda y sub-crónica, llevados a cabo según las directrices de

las guías de la OCDE 425 y 407, demostraron la seguridad por vía oral de las diferentes

formulaciones de nanopartículas de poli(anhídrido).


212

3.2. A Dosis única, la DL50 fue superior 2.000 mg/kg peso en rata por vía oral.

3.3. A dosis repetidas durante 28 días, se obtuvo una NOAEL de 300 mg/kg peso, que es una

dosis 30 veces superior a la dosis terapéutica.

3.4. Los estudios de biodistribución con Tecnecio, mostraron que tras su administración oral, las

distintas formulaciones se localizan principalmente en el intestino y son eliminadas por heces.

3.5. Las nanopartículas con poli-etilenglicol 6000 mostraron un tránsito más lento en

comparación con las convencionales y con ciclodextrina. Este hecho confirmó que la pegilación

de las nanopartículas de poli(anhídrido) aumenta sus propiedades adhesivas.

4. Evaluación de hemocompatibilidad, toxicidad aguda y biodistribución por vía intravenosa

de las distintas nanopartículas de poli(anhídrido):

4.1. En eritrocitos humanos, todas las formulaciones demostraron ser hemocompatibles

independientemente de la dosis y de sus características físico-químicas.

4.2. A dosis única por vía intravenosa, las nanopartículas de poli(anhídrido) mostraron toxicidad

dependiente de la dosis. Así, si bien a una dosis de 50 mg/kg peso las distintas formulaciones no

produjeron síntomas evidentes de toxicidad, a una dosis de 150 mg/kg peso todas las

formulaciones resultaron ser tóxicas y su toxicidad dependió de sus características físico-

químicas.

4.3. La toxicidad de las nanopartículas de poli(anhídrido) se caracterizó por provocar un

episodio agudo de distrés respiratorio, siendo más grave en el caso de nanopartículas

convencionales y con ciclodextrina donde provocó la muerte del 30% de los animales.

4.4. En el análisis histopatológico, el hallazgo más significativo fue la presencia de células con

aspecto espumoso en el bazo, hígado y pulmón. Estos hallazgos demostraron, por primera vez,

que nanopartículas poliméricas por vía intravenosa pueden producir fosfolipidosis.

4.5. Por vía intravenosa, los estudios de biodistribución demostraron que las diferentes

nanopartículas de poli(anhídrido) desaparecen rápidamente de la circulación sanguínea y se

acumulan principalmente en el hígado, bazo, pulmones y riñones. Así, a tiempos cortos, el

patrón de distribución dependió de las diferentes características físico-químicas de las

formulaciones que a su vez parecen dirigir el patrón de opsonización de las mismas y por lo

tanto su toxicidad.

Toxicity evaluation of poly(anhydride) nanoparticles as...

Documents

Transcript of Toxicity evaluation of poly(anhydride) nanoparticles as...