i

CELL-PENETRATING PEPTIDES; CHEMICAL MODIFICATION,

MECHANISM OF UPTAKE AND FORMULATION DEVELOP-

MENT

Kariem Ezzat

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Cell-penetrating peptides; chemical

modification, mechanism of uptake

and formulation development

Kariem Ezzat

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©Kariem Ezzat, Stockholm 2012

ISBN 978-91-7447-464-0

Printed in Sweden by Universitetetsservice US-AB, Stockholm 2012

Distributor: Department of Neurochemistry, Stockholm University

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To my family

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List of publications

This thesis is based on the following publications referred to in text as

paper I, paper II, paper III and paper IV.

I. Lehto, T., Simonson, O. E., Mäger, I., Ezzat, K., Sork, H.,

Copolovici, D. M., Viola, J. R., Zaghloul, E. M., Lundin, P.,

Moreno, P. M., Mäe, M., Oskolkov, N., Suhorutsenko, J., Smith,

C. I., and El Andaloussi, S. (2011) A peptide-based vector for

efficient gene transfer in vitro and in vivo. Mol. Ther., 19,

1457-1467.

II. Ezzat, K., El Andaloussi, S., Zaghloul, E. M., Lehto, T., Lind-

berg, S., Moreno, P. M., Viola, J. R., Magdy, T., Abdo, R., Guter-

stam, P., Sillard, R., Hammond, S. M., Wood, M. J., Arzumanov,

A. A., Gait, M. J., Smith, C. I., Hällbrink, M., and Langel, Ü.

(2011) PepFect 14, a novel cell-penetrating peptide for oligo-

nucleotide delivery in solution and as solid formulation. Nu-

cleic Acids Res., 39, 5284-5298.

III. Ezzat,K., Helmfors,H., Tudoran,O., Juks,K., Lindberg,S.,

Padari,K., El Andaloussi,S., Pooga,M., and Langel,Ü. (2012)

Scavenger receptor-mediated uptake of cell-penetrating pep-

tide nanoparticles with oligonucleotides. FASEB J., 3, 1172-80.

IV. Ezzat, K., Zaghloul, E. M., El Andaloussi, S., Lehto, T., Hilal

R., Magdy, T., Smith,C. I., Langel,Ü. Solid formulation of cell-

penetrating peptide nanoparticles with siRNA and their sta-

bility in simulated gastric conditions. Resubmitted to J. Con-

trol. Release.

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Additional publications

1. Lehto,T., Abes,R., Oskolkov,N., Suhorutsenko,J., Copolovi-

ci,D.M., Mäger,I., Viola,J.R., Simonson,O.E., Ezzat,K., Gu-

terstam,P., et al. (2010) Delivery of nucleic acids with a stea-

rylated (RxR)4 peptide using a non-covalent co-incubation

strategy. J. Control. Release, 141, 42-51.

2. El Andaloussi,S., Lehto,T., Mäger,I., Rosenthal-Aizman,K.,

Oprea,I.I., Simonson,O.E., Sork,H., Ezzat,K., Copolovi-

ci,D.M., Kurrikoff,K., et al. (2011) Design of a peptide-based

vector, PepFect6, for efficient delivery of siRNA in cell cul-

ture and systemically in vivo. Nucleic Acids Res., 39, 3972-

3987.

3. Ezzat,K., El Andaloussi,S., Abdo,R., and Langel,Ü. (2010)

Peptide-based matrices as drug delivery vehicles. Curr.

Pharm. Des., 16, 1167-1178. Review.

4. Lehto,T., Ezzat.K., and Langel,Ü. (2011) Peptide nanoparti-

cles for oligonucleotide delivery. Prog. Mol. Biol. Transl.

Sci., 104, 397-426. Book chapter.

Patent application

Kariem Ezzat Ahmed, El Andaloussi,S., Guterstam,P., Häll-

brink, M., Johansson,H., Langel,Ü, Lehto,T., Lindgren,M.,

Mäger, I., Sillard, R., Rosenthal-Aizman,K., Tedebark,U and

Lundin,P. (2010) Chemically modified cell-penetrating pep-

tides for improved delivery of gene modulating com-

pounds. WO2010039088.

ix

Abstract

Gene therapy holds the promise of revolutionizing the way we treat

diseases. By using recombinant DNA and oligonucleotides (ONs), gene

functions can be restored, altered or silenced according to the therapeutic

need. However, gene therapy approaches require the delivery of large

and charged nucleic-acid based molecules to their intracellular targets

across the plasma membrane, which is inherently impermeable to such

molecules. In this thesis, two chemically modified cell-penetrating pep-

tides (CPPs) that have superior delivery properties for several nucleic

acid-based therapeutics are developed. These CPPs can spontaneously

form nanoparticles upon non-covalent complexation with the nucleic acid

cargo, and the formed nanoparticles mediate efficient cellular transfec-

tion. In paper I, we show that an N-terminally stearic acid-modified ver-

sion of transportan-10 (PF3) can efficiently transfect different cell types

with plasmid DNA and mediates efficient gene delivery in-vivo when

administrated intra muscularly (i.m.) or intradermaly (i.d.). In paper II, a

new peptide with ornithine modification, PF14, is shown to efficiently

deliver splice-switching oligonucleotides (SSOs) in different cell models

including mdx mouse myotubes; a cell culture model of Duchenne’s

muscular dystrophy (DMD). Additionally, we describe a method for in-

corporating the PF14-SSO nanoparticles into a solid formulation that is

active and stable even when stored at elevated temperatures for several

weeks. In paper III, we demonstrate the involvement of class-A scaven-

ger receptor subtypes (SCARA3 & SCARA5) in the uptake of PF14-SSO

nanoparticles, which possess negative surface charge, and suggest for the

first time that some CPP-based systems function through scavenger re-

ceptors. In paper IV, the ability of PF14 to deliver small interfering RNA

(siRNA) to different cell lines is shown and their stability in simulated

gastric acidic conditions is highlighted.

Taken together, these results demonstrate that certain chemical

modifications can drastically enhance the activity and stability of CPPs

for delivering nucleic acids after spontaneous nanoparticle formation

upon non-covalent complexation. Moreover, we show that CPP-based

nanoparticles can be formulated into convenient and stable solid formula-

tions that can be suitable for several therapeutic applications. Important-

ly, the involvement of scavenger receptors in the uptake of such nanopar-

ticles is presented in this thesis, which could yield novel possibilities to

understand and improve the transfection by CPPs and other gene therapy

nanoparticles.

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Contents

1. Introduction .................................................................................. 1 1.1. Gene therapy ............................................................................ 2

1.1.1. Gene delivery .................................................................. 2 1.1.2. Silencing of disease causing genes .................................. 3 1.1.3. Modification of gene function ......................................... 5

1.2. Nanoparticles for non-viral gene delivery ............................... 6 1.2.1. Small molecules vs. nanoparticles; the uptake paradox .. 6 1.2.2. Nanoparticle-based vectors in gene-therapy ................... 9 1.2.3. Pharmacokinetics of nanoparticle-based systems for nucleic acid delivery .................................................................. 12

1.3. Cell-penetrating peptides (CPPs) ........................................... 16 1.3.1. History ........................................................................... 17 1.3.2. CPPs as drug delivery vehicles ..................................... 18 1.3.3. Uptake mechanism ........................................................ 22

1.4. Scavenger receptors (SRs) ..................................................... 26 1.4.1. Class A scavenger receptors (SCARAs) and nucleic acid binding…….. ............................................................................. 27

1.5. Pharmaceutical formulation ................................................... 29

2. Aims of the study ....................................................................... 31

3. Methodological considerations .................................................. 32 3.1. Solid phase peptide synthesis and peptide design .................. 32 3.2. Cell cultures ............................................................................. 33 3.3. Plasmid delivery (Paper I) ....................................................... 34 3.4. SSOs delivery (Paper II and III) .............................................. 34 3.5. siRNA delivery (Paper IV) ...................................................... 35 3.6. Toxicity ................................................................................... 35 3.7. Dynamic light scattering (DLS), nanoparticle tracking analysis (NTA) and zeta-potential ............................................................... 35 3.8. Solid dispersion and gastric simulation ................................... 36 3.9. Immunofluorescence and transmission electron microscopy (TEM) ............................................................................................. 37 3.10. Animal Experiments .............................................................. 38

4. Results and discussion ............................................................... 39 4.1. Delivery of plasmids via stearyl-TP10 (PF3) in-vitro and in-vivo (Paper I) .................................................................................. 39 4.2. Delivery of SSOs via PF14 and solid formulation development (Paper II) ................................................................... 40

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4.3. Scavenger receptor-mediated uptake of CPP nanoparticles with ONs (Paper III) ....................................................................... 42 4.4. Activity and solid formulation of PF14-siRNA nanoparticles and their stability in simulated gastric conditions (Paper IV) ........ 44

5. Conclusions ................................................................................ 47

6. Acknowledgements .................................................................... 48

7. References .................................................................................. 50

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Abbreviations

AMP

AntimiR BBB

CPP CQ CR DLS DMD GAPDH HPLC HPRT1 i.d i.m MBHA miRNA MR NTA ON

PAMPs PRRs

PCR PF

Poly I Poly G

qPCR RISC

RNAi RT-PCR SCARA

SGF SIF

siRNA SR

SSOs SSPS TFA TIS

Antimicrobial peptide Anti microRNA

Blood-brain barrier Cell-penetrating peptide Chloroquine Charge ratio Dynamic light scattering Duchenne’s muscular dystrophy Glyceraldehyde 3-phosphate dehydrogenase High performance liquid chromatography Hypoxanthine-guaninephosphoribosyl transferase Intradermal Intramuscular p-Methylbenzylhydralamine Micro RNA Molar ratio Nanoparticle tracking analysis Oligonucleotide Pathogen associated molecular patterns Pattern recognition receptor Polymerase chain reaction PepFect

Polyinosinic acid Polyguanilic acid Quantitative real-time PCR RNA-induced silencing complex RNA interference Reverse transcriptase PCR

Class A scavenger receptor Simulated gastric fluid Simulated intestinal fluid

Small interfering RNA Scavenger receptor

Splice-switching oligonucleotides Solid-phase peptide synthesis Trifluoroacetic acid Triisopropylsilane

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1

1. Introduction

One of the major biomedical milestones in history was the com-

plete sequencing of the human genome (1), which has significantly

deepened our knowledge about the genetic causes of diseases. This led

to the emergence of several methods to interfere with disease patho-

physiology on the molecular genetics level, a field that can collective-

ly be called “gene therapy”. Using recombinant DNA and oligonucle-

otides (ONs), gene functions can be restored, altered or silenced ac-

cording to the therapeutic need. However, gene therapy approaches

require the delivery of extremely large and charged nucleic-acid based

molecules to their intracellular targets across the plasma membrane,

which is inherently impermeable to such molecules. Viral and non-

viral vectors have been developed to deliver gene therapies to their

target organs, tissues and subcellular compartments. Due to the limita-

tions and hazards of viral vectors, several non-viral drug delivery

technologies were developed in recent years mainly utilizing nanopar-

ticles as delivery vehicles (2). Despite a tremendous research invest-

ment, so far, no therapeutics based on such nanoparticles have reached

the market. Among the reasons for such a poor translational outcome

are:

1. The poor understanding of the molecular mechanisms of uptake

and biodistribution of nanoparticles.

2. The complex nature of the nanoparticle-based delivery systems,

which presents a manufactural and cost barrier for pharmaceutical

companies interested in their clinical development on large scale.

In this thesis, chemically modified cell-penetrating peptides

(CPPs) were developed, which are able to form nanoparticles with

plasmids, small interfering RNAs (siRNAs) and splice-switching ONs

(SSOs) upon non-covalent complexation and efficiently mediate their

cellular delivery. Importantly, special focus of this thesis work was

dedicated to the identification of the mechanism of uptake of such

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nanoparticles and enhancing their translational potential via pharma-

ceutical formulation techniques.

1.1. Gene therapy

According to the American Society of Gene & Cell Therapy

(ASGCT), gene therapy is defined as “the introduction or alteration of

genetic material within a cell or organism with the intention of curing

or treating a disease” (3). Based on this definition gene therapy ap-

proaches can be roughly divided into 3 types according to their phar-

macological effect (Figure 1.):

Restoration of lost gene function by gene delivery via viral

vectors or plasmids (gene delivery). Silencing of disease causing genes by antigene, antisense or

RNAi (RNA interference) approaches. Modification of gene function by interfering with the splicing

machinery via splice-switching oligonucleotides (SSOs) or anti microRNAs (antimiRs)

1.1.1. Gene delivery

Loss of gene function is the cause of several heritable diseases

and cancers (4, 5). Thus, the delivery of functional genes that could

restore normal phenotype has been the ultimate goal of gene therapy

for decades (6). Viral vectors have been extensively utilized for gene

delivery; however, early promising results have been shadowed by

reports showing that viral insertional mutagenesis might lead to severe

leukemogenic side effects (7, 8, 9). Also, humoral immunity directed

against the viral vector particle is generally observed (10). Recently,

much progress has been accomplished in achieving clinical benefits

with viral vectors without serious side effects (11). However, safer

and efficient non-viral alternatives are still needed for the vast applica-

tions of gene delivery. Non-viral restoration of gene function can be

achieved by the delivery of plasmids carrying the desired gene. When

the plasmid enters the cell, it is transcribed and translated by the cellu-

lar machinery without the need of genome integration (Figure 1.).

Many nanoparticle-based vectors have been designed for this purpose

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and this approach will be discussed in more detail in the following

sections.

1.1.2. Silencing of disease-causing genes

Another promising field of gene therapy is the ability to silence

disease-causing genes by utilizing ONs targeting complementary se-

quences on the DNA or the RNA levels. It can be achieved by differ-

ent methods including:

Non-catalytic blocking of certain DNA or RNA sequences

antigene and antisense. This approach relies on ONs that bind

complementary DNA (antigene) or RNA (antisense) sequenc-

es, sterically blocking the transcription or translation machin-

ery or mediating the degradation of the formed duplex via

RNase H.

Catalytic degradation of target mRNA siRNA. This ap-

proach relies on the recruitment of cellular machinery to cata-

lyze sequence-specific degradation of the target mRNA via the

RNA-induced silencing complex (RISC) complex.

1.1.2.1. Antigene and antisense

When the double-stranded DNA or genes situated in the nucleus

are targeted, the approach is called the antigene strategy (12). Howev-

er, when mRNA is the target; this is called the antisense strategy. An-

tisense activity can be achieved either by blocking the binding sites

for the 40S ribosomal subunit or by the formation of DNA/RNA du-

plexes that renders the RNA susceptible for RNase H digestion (12)

(Figure 1.). Despite the recruitment of RNase H, this strategy is not

catalytic because the antisense strand is also degraded in this reaction.

Natural DNA and RNA have been used for antigene and antisense

approaches together with several chemically modified analogues that

offer better annealing with target sequences and possess enhanced

serum stability. Examples of chemically modified ONs include: phos-

phorothioate DNA, 2’-O-methyl RNA (2’-OMe), locked nucleic acid

(LNA), peptide nucleic acid (PNA) and phosphorodiamidate morpho-

lino oligo (PMO) (13).

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1.1.2.2. RNAi

RNAi is a fundamental pathway in eukaryotic cells, where long

pieces of double stranded RNA are cleaved by an enzyme called Dicer

into shorter fragments called siRNAs that mediate cleavage of com-

plementary mRNA sequences by the help of RISC (Figure 1.) (14,

15). The proof-of-principle study in 2001 demonstrating that synthetic

siRNA could mediate sequence-specific gene knockdown in mamma-

lian cells marked the birth of siRNA therapeutics (16). What makes

the siRNA approach more appealing than other silencing approaches

is that it cleaves target mRNA in a catalytic manner, i.e. the antisense

strand (guide strand) can recruit the RISC complex to degrade several

mRNA molecules having the complementary sequence without being

degraded itself. Thus, lower doses are required to achieve gene

knockdown compared to the conventional antisense approaches. That

is why intensive research has been carried out in the last decade to

develop delivery vectors for siRNA therapeutics (14).

Figure 1. Different gene therapy approaches. a. Viral delivery and ge-

nome integration. b. Plasmid delivery. c. Antisense steric block of trans-

lation d. Antisense DNA/RNA hybrid and RNase H degradation. e.

Antigene. f. Splice-switching ONs. g. siRNA. h. antimiR.

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1.1.3. Modification of gene function

One of the most powerful applications of ON-based therapeutics

is their ability to modulate gene function. This can be achieved by

interfering with the splicing machinery (splice-switching) or by inter-

fering with the microRNA (miRNA) machinery using antimiRs.

1.1.3.1. Splice-switching therapeutics

Recent studies using high-throughput sequencing indicate that

95–100% of human pre-mRNAs have alternative splice forms (17).

Mutations that affect alternative pre-mRNA splicing have been linked

to a variety of cancers and genetic diseases, and SSOs can be used to

silence mutations that cause aberrant splicing, thus restoring correct

splicing and function of the defective gene (Figure 1.) (18, 19). SSOs

are antisense ONs ranging from 15 to 25 bases in length that do not

activate RNase H, which would destroy the pre-mRNA target before it

could be spliced (18, 19). One example of genetic diseases amenable

for SSO therapy that will be addressed in this thesis is Duchenne’s

muscular dystrophy (DMD). DMD is a neuromuscular genetic disor-

der that affects 1 in 3500 young boys worldwide (20). It is caused

mainly by nonsense or frame-shift mutations in the dystrophin gene.

SSOs are used to induce targeted ‘exon skipping’ in order to correct

the reading frame of mutated dystrophin pre-mRNA such that shorter,

partially-functional dystrophin forms are produced (21). SSOs target-

ing exon 51 are currently in human clinical trials in various parts of

Europe to treat DMD (22, 23). However, translating the promising

results of SSOs into drug products requires optimization of many pa-

rameters ranging from enhancement of cellular uptake and biodistribu-

tion to pharmaceutical formulation and long term stability.

1.1.3.2. AntimiRs

MiRNAs are a large family of short RNAs (~21 nucleotides)

that play a key role in post-transcriptional gene regulation (24). They

are predicted to control the activity of ~50% of all protein-coding

genes and dysfunction of individual miRNAs or entire miRNA fami-

lies was shown to be associated with several human diseases, such as

cancer and CNS disorders (24, 25). ONs designed to silence specific

miRNA, antimiRs, are used either to probe their functions or for the

development of novel miRNA-based therapeutics, an approach that

6

has demonstrated promising results in models for atherosclerosis and

cancer (25). Recently, very short (8-mer) LNAs, termed tiny LNAs,

targeting the seed region of specific miRNA families sharing the same

seeding region have been shown to directly up-regulate targets with

negligible off-target effects (26). This demonstrates the wide applica-

bility of this class of ON therapeutics.

1.2. Nanoparticles for non-viral gene delivery

Nanotechnology has attracted a lot of research interest in the

drug delivery field as a very promising method to solve drug delivery problems, especially in the field of gene therapy. This thesis is based on CPP-based vectors that form nanoparticles for delivery of several nucleic acid cargos. This section will give a brief introduction to the concept of nanoparticle gene delivery in general and highlight exam-ples of its use in the field of gene therapy and its unique pharmacoki-netic profile.

1.2.1. Small molecules vs. nanoparticles; the uptake paradox

The plasma membrane forms a barrier that separates the intra-

cellular environment from the outer environment and strictly controls

the translocation of molecules in and out of the cell. Some molecules

with certain physicochemical properties can passively diffuse across

the plasma membrane, and hence represent good drug candidates.

These physicochemical properties are called “drug-likeness”, which

describe how likely a chemical compound to be able to transfer the

intestinal membrane without the need of a carrier or active transport

processes (27, 28). They are summarized in the famous Lipinski rule

of 5 (29):

Molecular weight is less than 500 Da.

Lipophilicity, expressed as logP (the logarithm of the partition

coefficient between water and 1-octanol), is less than 5.

The number of hydrogen bond donators (usually hydroxyl and

amine groups) is less than 5.

The number of hydrogen bond acceptors (estimated by the

number of oxygen and nitrogen atoms) is less than 10.

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Figure 2. The uptake paradox.

The rule does not apply to drugs that utilize specific transporters (ac-

tive transport via carrier proteins), which has gained extensive re-

search focus in recent years (30, 31, 32). Additionally, several refine-

ments have been introduced to the rule of 5 since its publication,

probably the most recent and prominent has been the QED (quantita-

tive estimate of drug-likeness), which is a method to quantify drug-

likeness by a score from 0 to 1 (27). Despite its limitations, the rule of

5 possesses a great predictive power and is used by pharmaceutical

companies to screen chemical libraries for drug candidates.

Looking at the gene therapy approaches discussed earlier, we

see that all of them are nucleic-acid based molecules, which are nega-

tively charged in most cases (except for PNA and PMO) and ranging

between 8 nucleotides (tiny-LNAs) up-to several kilo base-pairs

(plasmids). Applying the rules of drug-likeness to these molecules

clearly shows that they cannot cross the plasma membrane by passive

diffusion. Paradoxically, the solution to this problem was to make

these molecules even larger by incorporating them in nanoparticle-

based delivery systems, and such nanoparticles demonstrated success-

ful delivery in several in-vitro and in-vivo models. This paradox can

be explained by understanding the diverse mechanisms utilized by the

cell to control transportation across the cell membrane. Figure. 2 rep-

8

resents a rough estimate of the size ranges where molecules and parti-

cles can penetrate into the cells. According to the rule of 5, molecules

less than 500 Da, mostly less than 3 nm, possess high permeability

through the cell membrane. Exceeding 500 Da, the passive permeabil-

ity drops dramatically, and this is the range where most impermeable

molecules lie, e.g. proteins and nucleic acids. However, when such

molecules are incorporated in nanoparticles, they form a new entity of

about 10 – 500 nm in size. At this size, they are still out of the scope

of passive diffusion or interaction with small molecule transporters,

however, in the scope of endocytosis, which is the cell’s natural way

of dealing with particulate matter. Thus, the power of nanoparticle-

based delivery system lies in their capability of utilizing the cell natu-

ral endocytic mechanisms as a means of delivering their therapeutic

cargo. The endocytic process is naturally performed by all cell types

as it is crucial for cell survival. It is used to internalize nutrients and

cell-surface receptors controlling the conveyance and extent of signal-

ing (33). Additionally, while it is hijacked by bacteria (34) and viruses

(35) to cause infection, it is also used for immune responses and clear-

ance of bacteria, viruses and apoptotic cells (36). Furthermore, it is

used by the cells to internalize naturally circulating nanoparticles like

lipoproteins (LDL, HDL, etc.) (37) and exosomes (38), that are re-

sponsible for cholesterol, protein and RNA trafficking. This extensive

utilization of the endocytic processes for cellular uptake of large parti-

cles explains the success of nanoparticles in delivering therapeutics to

various cell types by exploiting various endocytic mechanisms (39).

Consequently, the process of endocytosis is the main determinant of

the activity and toxicity of the nanoparticles.

Endocytosis can be specific receptor-mediated or unspecific

non-receptor mediated with different mechanisms of internalization,

mainly: clathrin-mediated, caveolae-mediated, macropinocytosis and

phagocytosis (40) (Figure. 3). The size of the nanoparticle, its shape,

surface charge and the presence of surface antigens, all affect the

mode of interaction with the cell surface and the subsequent endoso-

mal pathway which ultimately decides the fate of the therapeutic cargo

(41). Thus, the study of the specific endocytic mechanism is very im-

portant in the design and development of nanoparticle-based delivery

systems.

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1.2.2. Nanoparticle-based vectors in gene-therapy

For gene therapy applications, various types of inorganic and

organic nanoparticles have been investigated. Generally, the inorganic nanoparticles are synthesized then functionalized with the nucleic acid cargo. On the other hand, organic nanoparticles are usually formed using polymers that self-assemble into nanoparticles upon interaction with nucleic acids. The vast majority of inorganic and organic nano-particles used for gene therapy rely on the presence of a polycationic domain that is able to interact non-covalently with the negatively charged backbone of nucleic acids mediating its compaction and com-plexation (42). This complexation attaches the nucleic acid cargo to the nanoparticle and protects it form degradation by serum nucleases (43). That is why nanoparticles lacking polycationic domain are first functionalized with cationic groups or polycationic polymers on the surface to enable compaction and attachment of DNA (44, 44, 45, 46, 47). Table 1 demonstrates examples of the different nanoparticle-based systems used for nucleic acid delivery via non-covalent com-plexation.

Despite the vast differences in the chemistry of the nanoparticles

mentioned in Table. 1; they all have demonstrated activity in deliver-ing various gene therapeutic agents. The common features shared amongst them are:

Figure.3. Classification of endocytosis and its major mechanisms.

10

Nanoparticle-based system Cargo Ref

I. Inorganic nanoparticles

a. Ca-Mg phosphate Plasmid (48) b. Gold nanoparticles Plasmid (49) c. Carbon nanotubes & fullerenes Plasmid (47, 50) d. Quantum dots siRNA (51) e. Magnetic nanoparticles Plasmid (52) f. Silica nanoparticles ONs (53)

II. Organic nanoparticles

a. Lipid-based

Liposomes Plasmid (54) SNALPs (stable nucleic acid

lipid particles) siRNA (55)

Lipidoids siRNA (56) b. Carbohydrate-based

Dextran Plasmid (57) Cyclodextran Plasmid (58)

Chitosan Plasmid (59)

Hyaluronan Plasmid (60)

Schizophyllan ONs (61)

Pullulan Plasmid (62)

c. Peptide-based

CPPs Will be discussed in de-

tail in coming sections

Poly L-lysines siRNA (63)

Protamines ONs (64)

Histones Plasmid (65)

d. Other

Polyethylenimine (PEI) siRNA (66)

Poly(dl-lactide-co-glycolide)

(PLGA)

Plasmid (67)

Table 1. Various nanoparticle-based approaches for gene therapy and examples for the delivery of different cargos.

11

Size larger than 10 nm in most cases. The presence of a polycationic domain that is respon-

sible for nucleic acid complexation.

In this thesis (Paper III) we describe the role of the negative sur-

face charge in the uptake of CPP nanoparticles with ONs via the inter-

action with scavenger receptors that recognize nucleic acids. The

same receptors were also implicated in the uptake of oligonucleotide-

functionalized gold nanoparticles (68). Interestingly, many gene ther-

apy nanoparticles (Table 1.) are superficially functionalized by a poly-

cationic domain and consequently the complexed, negatively charged

nucleic acid is attached to the surface. Having these results in mind, it

is tempting to speculate that the nucleic acid cargo, can contribute to

the cellular uptake of the nanoparticles by interacting with certain cell

surface receptors. This interaction requires a negatively charged nucle-

ic acid in a complexed form and a certain size for endocytic uptake,

and these are the most common features among the gene therapy na-

noparticles. In this case, nucleic acids that present the delivery prob-

lem that needs to be solved, might also present a part of the solution.

This is clear in the case of scavenger receptor-mediated uptake of nu-

cleic acids, where aggregation of ONs or polyribonuceotides is a per-

quisite for receptor interaction and subsequent uptake (discussed in

detail in section 1.4.1.). If such a mechanism is general, it can explain

why systems as simple as calcium phosphate-DNA nanoprecipitates,

the first chemical transfection method ever, mediate gene transfection

(48, 69). However, this is a hypothesis and definitely needs experi-

mental verification for each delivery system separately.

Furthermore, the diversity shown in Table 1. sheds light on oth-

er problems in the field of nanoparticle-based gene delivery including:

Most of the research focus has been devoted to the development of

new systems; however, not as much focus has been given to deter-

mine the molecular mechanism of uptake of the existing systems.

Comparisons between systems from different chemistries are

scarce (groups working in certain chemistries compare systems of

the same chemistry).

12

Figure 4. Unbalanced research focus between the development of

new nanoparticle-based delivery systems, basic research and trans-

lational approaches.

Translational assessment regarding the feasibility of incorporation

into different dosage forms and the requirements for cost-effective

mass production by pharmaceutical companies are not given the

same attention.

The imbalance in research focus between the development of

new systems, molecular mechanisms and translational development is

presented schematically in Figure 4. In this thesis, work was done in

both directions trying to elucidate the molecular mechanisms behind

the uptake of CPP-based gene therapy nanoparticles and to enhance

the translational potential of such systems via pharmaceutical formula-

tion. This will be discussed in detail in the coming sections.

1.2.3. Pharmacokinetics of nanoparticle-based systems for

nucleic acid delivery

Nanoparticles are non-conventional xenobiotics with a unique

pharmacokinetic profile. It is very important to understand the phar-

13

macokinetic profile of nanoparticles intended for gene therapy, like the CPP based systems discussed in this thesis, to be able to under-stand their pharmacodynamic behavior, biodistribution and toxicity. This will help us to answer questions like: why most of the delivered cargo resides in the liver, is it possible to administer them through other routes, orally or respiratory, how can we enhance the biodistri-bution, etc.?

Any administered drug goes through 4 phases in the body that

are famously abbreviated ADME (Absorption, Distribution, Metabo-lism and Excretion). Figure 5 demonstrates a schematic representation of the pharmacokinetics of nanoparticles and their ADME phases.

Like any other xenobiotic that is not intended to act locally, na-

noparticles have to reach the blood circulation to reach its target or-gans. This can be achieved by direct intravenous or intra-arterial injec-tion. If taken via any other route of administration, they have to find their way to the blood circulation (A= Absorption). For oral admin-istration, after intestinal absorption, xenobiotics can take the hepatic pathway through the intestinal capillaries that relay to the portal vain, or the lymphatic pathway via the lymphatic capillaries that relay di-rectly to the blood circulation through the jugular vein avoiding first pass metabolism in the liver (70). The endothelial fenestrations of lymphatic capillaries are larger than those of the blood capillaries and thus more permeable to macromolecules up to several hundred na-nometers (71). Consequently, it is more likely that orally administered nanoparticles would take the lymphatic pathway. This has been shown by Aouadi et al. using glucan-encapsulated siRNA particles adminis-tered orally targeting M cells in intestinal wall Peyer’s patches to transfer micrometre-sized glucan particles to the gut-associated lym-phatic tissue (72). These orally-administered particles mediated effi-cient RNAi response in the peritoneum, spleen, liver and lung, and lowered serum TNF-αlevels. This demonstrates a proof of principle of how the administration route and the consequent pharmacokinetic profile can be utilized to achieve particular therapeutic goals.

After reaching the systemic circulation, the nanoparticles have

to distribute to the different organs and tissues (D = Distribution). Dis-tribution is highly dependent on the particle size, as nanoparticles can only extravasate in tissues with a discontinuous capillary endothelium. Liver, spleen, bone marrow, solid tumors and inflamed or infected sites harbor capillaries with the largest endothelial fenestrations

14

Fig

ure 5

. A sch

ematic rep

resentatio

n o

f the p

harm

acokin

etics of n

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articles.

15

(pores) (73), which explains why these tissues are easily targeted by nanoparticles. Specifically, hepatic accumulation of nanoparticles ap-pears to be the most prominent due to its high blood perfusion, fenes-trated capillary endothelium (sinusoids) that reaches up to 280 nm and the abundance of hepatic macrophages (Kupffer cells) which are situ-ated within the sinusoids and can ingest and detoxify nanoparticles at high rates (71, 73). Notably, the uptake of nanoparticles (74) and nu-cleic acids (75) by Kupffer cells is largely mediated by the scavenger receptors, a process that is facilitated by adsorption of serum proteins on the nanoparticle surface (opsonization) (76). For other tissues, the size and type (diaphragmed or non-diaphragmed) of capillary endothe-lial fenestrations determine the size of nanoparticles that can reach this organ (71). This biodistribution profile represents a problem that needs a solution for gene therapy nanoparticles to be able to target different tissues instead of just accumulating in organs with wide en-dothelial fenestrations. One solution is to add hydrophilic moieties (polyethylene glycol for example) to the surface to repel serum pro-teins that opsonize the particles for macrophages (41). Although this approach might solve the opsonization problem enhancing the circula-tory half-life of (77), it doesn’t solve the initial problem of accumula-tion in certain tissues due to extravasation. Another solution is not to depend on passive distribution but rather on active distribution by add-ing ligands that promote carrier-mediated transfer across different delivery barriers. Many studies use ligands for cellular targets to en-hance the activity of nanoparticles (78). Despite showing promise, this approach assumes homogenous distribution of the particles in all tis-sues and accessibility of the particles to the target cells. However, the particles have to pass the capillary endothelial barrier first to gain ac-cess to their cellular targets and this is strictly dependent on the size of endothelial fenestrations in this tissue. Thus, the size of the nanoparti-cles has to be carefully adjusted to enhance the distribution to differ-ent tissues or the nanoparticles should carry double ligands; one for transendothelial transport and one cell-type specific. A recent exam-ple of this approach is demonstrated by Baodo et al. by adding pep-tidomimetic monoclonal antibody (MAb) components to gene deliv-ery liposomes. These MAbs bind to specific receptors located on both the blood-brain barrier (BBB) and on brain cellular membranes (insu-lin receptor and transferrin receptor) mediating receptor-mediated transcytosis through the BBB and endocytosis into brain cells (79).

After reaching the target tissue, nanoparticles interact with the

cell membranes and gain access to the cell interior through the process of endocytosis as discussed earlier. In the endosome, two processes need to occur for a pharmacological response to take place: a. escape

16

from the endosome before being degraded in the lysosomes down-stream in the pathway, b. dissociation of the nucleic acid cargo from the carrier nanoparticle to be able to reach their target. This step also involves decomplexation of the nucleic acid cargo. Although these processes are extremely important for the activity of nucleic-acid based therapeutics, they are still poorly understood.

The final step in this long journey of the nanoparticle delivery

system is metabolism (M=Metabolism). Actually, metabolism starts from the point of administration of the nanoparticle, either at the ad-ministration site or in the circulation. However, condensation of the nucleic acid cargo with polycationic domains of the nanoparticles pro-tects it to a great extent in the circulation until it dissociates inside the cell (43). Very recently, Zuckerman et al. have demonstrated a mech-anism responsible for rapid clearance of siRNA delivery nanoparticles through the kidneys (80). They have shown that the glomerular base-ment membrane (GBM) can disassemble cationic cyclodextrin-containing polymer (CDP)-based siRNA nanoparticles facilitating their rapid elimination from circulation. However, more studies are needed focusing on the detailed metabolism of different nanoparticle-based systems.

Understanding the pharmacokinetic parameters of nanoparticles

in general is necessary to understand the properties of the CPP-based gene delivery nanoparticles that are the main focus of this thesis. The next section will be discussing CPP-based delivery in detail.

1.3. Cell-penetrating peptides (CPPs)

CPPs are polybasic and/or amphipathic peptides, usually less

than 30 amino acids in length, that possess the ability to penetrate

cells (Cell-Penetrating Peptides) or transduce (Protein Transduction

Domains) over cellular plasma membranes (81, 82). CPPs have at-

tracted much interest in recent years as promising vectors for the de-

livery of a wide variety of therapeutics ranging from small molecules

up to nanoparticles. This section will discuss CPP-based delivery in

general and focus on non-covalent nanoparticles of CPPs with nucleic

acids and their utilization in gene therapy.

17

1.3.1. History

Many peptides and proteins have desirable therapeutic effects,

but being large and often charged molecules, they have always been

thought incapable of bypassing the plasma membrane. This view was

challenged in the year 1988, when two groups independently pub-

lished results in the same issue of CELL showing that both the recom-

binant and the chemically synthesized 86 amino acids long Tat protein

are rapidly taken up by cells in tissue culture (83, 84). Few years later,

the 60 amino acid homeodomain of the Antennapedia protein in Dro-

sophila was also shown to penetrate cells (85). A very important ad-

vancement in the field came by showing that the cell penetration ca-

pability is imparted by relatively short peptide sequences. The 16-mer

peptide derived from the third helix of the homeodomain of Anten-

napedia termed penetratin (86), the 11-mer peptide derived from Tat

protein (87), the 27-mer chimeric peptide termed transportan (88) and

even simple polyarginines (R8) (89) were all shown to traverse the

plasma membrane. These discoveries marked the birth of the field of

CPPs. Since then, many CPPs have been discovered and studied as

potential drug delivery vehicles, some of which are presented in Table

2.

Table 2. Selection of CPPs and their sequences.

CPP Sequence Ref

Tat (48-60) GRKKRRQRRRPPQ (87)

Penetratin RQIKIWFQNRRMKWKK-amide (86)

pVEC LLIILRRRIRKQAHAHSK-amide (90)

bPrPp MVKSKIGSWILVLFVAMWSDVGLCKKRPKP-

amide

(91)

Transportan GWTLNSAGYLLGKINLKALAALAKKIL-amide (88)

TP10 AGYLLGKINLKALAALAKKIL-amide (92)

MAP KLALKLALKALKAALKLA-amide (93)

Pep-1 KETWWETWWTEWSQPKKKRKV- cysteamide (94)

MPG GALFLGWLGAAGSTMGAPKKKRKV-cysteamide

(95)

Poly Arg (RRR)n (89)

18

1.3.2. CPPs as drug delivery vehicles

CPPs have been shown to be efficient vectors for intracellular

delivery of various therapeutic cargos, especially for proteins and nu-

cleic acids. Two main methods have been utilized to attach the CPP

to its cargo, either via a covalent linkage or through the formation of

non-covalent complexes.

1.3.2.1. Covalent linkage to cargo

CPPs have been coupled to various cargos including proteins,

peptides, small molecules and nucleic acid analogues via covalent

attachment. One of the early applications was demonstrating their

ability to transduce full-length proteins, where the TAT peptide was

extensively studied. TAT-p27 fusion protein was shown to transduce

into cells and mediate biological function inducing cell migration (96).

Importantly, TAT fusion proteins were shown to be delivered in-vivo

to all tissues in mice, including the brain (97). This approach has been

extensively utilized to deliver a myriad of therapeutic proteins. Using

TAT-mediated transduction, Bcl-x(L), GDNF and HSP70 have been

delivered to the brain across the BBB mediating neuroprotecion in

several models (98, 99, 100). In cancer treatment, a CPP derived from

the fibroblast growth factor was conjugated to an anti-Akt single chain

Fv antibody and administrated in vivo with subsequent reduction in

tumor volume (101). Another interesting example is using undeca-

arginine (R11) expressed recombinantly with the four transcription

factors, Oct4, Klf4, Sox2, and c-Myc, to generate induced pulripotent

stem cells (iPS cells) (102). This set-up was almost 10 times more

efficient as compared to other approaches in generating iPS colonies

without the risk of chromosomal integration associated with viral vec-

tors. For peptide delivery, TAT and Penetratin (Table 1) have been

used to deliver peptides derived from the tumor suppressor p53, or

peptides that modulate p53 activity, for reduction of tumor growth and

induction of apoptosis (103, 104).

For gene therapy, covalent coupling of CPPs, which are posi-

tively charged, to negatively-charged nucleic acids has not been very

easy to achieve. Therefore, in most gene therapy approaches using the

covalent coupling strategy, neutral PNA or PMO were used instead of

natural nucleic acids. Also, they can be directly attached to the CPP

utilizing the solid phase peptide synthesis chemistry. Successful deliv-

19

ery of antisense ONs in-vivo using CPPs was for the first time demon-

strated by our group with an antisense PNA complementary to human

galanin receptor 1 (GalR1) mRNA coupled to transportan or pene-

tratin that specifically down-regulated these receptors in rat brains

(105). A number of endogenous proteins including dystrophin for

splice-switching in DMD (106, 107, 108), CD45, and the interleukin-2

(IL-2) receptor (109), have been targeted with PMOs using CPPs as

well. However, the limitation of using only PNA or PMO has led the

development of the other strategy of cargo conjugation to CPPs; non-

covalent complexation.

Small molecules, like antineoplastic agents and antibiotics, have

been coupled to CPPs to enhance biodistribution and cellular uptake.

For example, SynB peptide was utilized for the delivery of benzylpen-

icillin (B-Pc) to the brain and it was found that the brain uptake of

coupled B-Pc was increased eight-fold on average compared to free B-

Pc (110). An example of successful vectorization of chemostatics

comes from our group where two different CPPs, YTA2 and YTA4,

were utilized for the delivery of methotrexate (MTX) into MTX- re-

sistant breast cancer cells (111).

1.3.2.2. Non-covalent complexation and chemical modification of CPPs

It was the group of Gilles Divita who first showed that CPPs can

be used to deliver ONs after non-covalent complexation using the

MPG peptide (95). Having net positive charge, MPG was shown to

form nanoparticles with negatively charged single- and double-

stranded ONs, which are efficiently internalized by cells. This strategy

has drastically expanded the range of therapeutic cargos that can be

delivered via CPPs as it avoids laborious chemical conjugation and

has almost no limitation on the size of the cargo. Since the initial pub-

lication, several CPPs have been developed that can form nanoparti-

cles with various nucleic acids and efficiently mediate their delivery in

several in-vitro and in-vivo settings (112, 113, 114). However, certain

chemical modifications of the CPPs were needed to improve the na-

noparticle formation capability and enhance membrane interaction

upon non-covalent complexation (Table 3). C-terminal cysteamide

modification was shown to be crucial for CPP-mediated siRNA deliv-

20

ery using the MPG, PEP and CADY peptide families by increasing

membrane association and stabilizing particle formation by the for-

mation of peptide dimers (115, 116, 117, 118). Acetylation of these

peptides was also required to enhance stability (117). Addition of hy-

drophobic moieties to CPPs has been shown to be an efficient mean to

increase the efficiency of the nanoparticles. Cholesteryl modification

of polyarginines and MPG-8 was demonstrated to enhance their ac-

tivity for delivery of siRNA in-vivo (119). The group of Shiroh

Futaki at Kyoto university was first to demonstrate that stearylation of

polyarginines could enhance their transfection efficiency by 100-fold

(120). In recent years, our group applied stearylation and other chemi-

cal modifications to the TP10 backbone leading to the development of

the PepFect family of peptides (PFs) (121). We have shown that PF3

and PF14 are very efficient in the delivery of various nucleic acid car-

gos in different settings; both of which will be discussed in detail in

this thesis. We have also shown that PF6 is very efficient in delivering

siRNA to various cell-lines and in-vivo after intravenous administra-

tion (122). PF6 is N-terminally modified TP10 with a trifluorome-

thyl-quinoline moiety attached to one of the lysines in the backbone

via a lysine tree. The trifluoromethyl-quinoline moiety enhances the

escape of the nanoparticles from endosomes after internalization me-

diating a higher RNAi response. Furthermore, we have demonstrated

that stearylation of the (RxR)4 peptide can drastically enhance its abil-

ity to deliver plasmids and SSOs after non-covalent complexation

(123). This peptide was more efficient than stearyl-R9 for plasmid

transfection and mediated splice-switching at much lower doses than

the potent (RxR)4-PMO conjugate.

Another strategy to enhance CPP association with siRNA was to

generate TAT fusion protein with double-stranded RNA-binding do-

main (DRBD) (124). The DRBD binds siRNA with high affinity, and

thereby masks its negative charge allowing TAT-mediated cellular

uptake. This system was shown to be very efficient in delivering siR-

NA to primary cell-lines with no cytotoxicity or induction of innate

immune responses.

21

CPPs Sequence Modification Ref.

MPG peptides

MPG Ac-

GALFLGWLGAAGSTMGAP-

KKKRKV-Cya

Acetylation,

cysteamidation

(95)

MPGΔNLS

Ac-

GALFLAFLAAALSLMGLWSQPKKKR

KV-Cya

Acetylation,

cysteamidation

(115)

MPG-8 β-AFLGWLGAWGTMGWSPKKKRK-

Cya

Cysteamidation (125)

Chol-MPG-8 chol-β-

AFLGWLGAWGTMGWSPKKKRK-

Cya

Cholesterol,

cysteamidation

(125)

Pep and CADY peptides

Pep-2 Ac-KETWFETWFTEWSQPKKKRKV-

Cya

Acetylation,

cysteamidation

(126)

Pep-3 Ac-KWFETWFTEWPKKRK-Cya Acetylation,

cysteamidation

(127)

PEG-Pep-3 PEG- KWFETWFTEWPKKRK-Cya PEGylation,

cysteamidation

(127)

CADY Ac-GLWRALWRLLRSLWRLLWRA-

Cya

Acetylation,

cysteamidation

(118)

Polyarginine peptides

Stearyl-Arg8 Stearyl-RRRRRRRR-NH2 Stearylation (128)

Chol-Arg9 Chol-RRRRRRRRR-NH2 Cholesterol (119)

Stearyl-(RxR)4 Stearyl-RXRRXRRXRRXR-NH2 Stearylation (123)

Tat-DRBD

TAT-DRBD TAT fusion protein with double-stranded

RNA-binding domain

- (124)

PepFects

PF3 Stearyl-

AGYLLGKINLKALAALAKKIL-NH2

Stearylation (129)

PF6 Stearyl-

AGYLLGKaINLKALAALAKKIL-NH2

Stearylation, atrifluoromethyl-

quinoline

(122)

PF14 Stearyl-

AGYLLGKLLOOLAAAALOOLL-NH2

Stearylation (130)

NickFect1 Stearyl-AGY(PO3)LLGKTNLKALAALAKKIL

-NH2

Stearylation, phosphorylation

(131)

Table 3. Examples of CPPs and chemical modifications compatible with non-

covalent delivery of nucleic acids (adapted from our review (123)).

22

1.3.3. Uptake mechanism

Although CPPs have been discovered more than 15 years ago,

the uptake mechanism has been a matter of controversy. The mecha-

nism is thought to be receptor-free and dependent on the interaction

between the cationic residues of CPPs and the negatively charged

plasma membrane. Whether such an interaction leads to direct pene-

tration into the cell or endocytic uptake is still a matter of debate.

In the early days of CPPs, the uptake was mostly studied using

high concentrations of fluorophore-labeled CPPs with cell fixation,

and these studies led to the conclusion that CPPs directly penetrate

through cellular membranes in an energy-independent manner. Sever-

al models were suggested for such interaction, including the carpet

model, the pore formation model and the inverted micelle-mediated

model among others (132, 133). However, the view of direct translo-

cation was challenged by several studies in the late 1990’s and early

2000’s. One of the most prominent studies was a paper published by

Bernard Lebleue’s laboratory in 2003 where they demonstrated that

cell fixation, even at mild conditions, leads to the artifactual uptake of

TAT and R9 (134). Furthermore, they showed that peptide uptake was

inhibited by incubation at low temperature and cellular ATP pool de-

pletion, which strongly suggested the involvement of endocytosis in

the cellular internalization. Additionally, using fluorescence micros-

copy on living cells, they demonstrated a punctuated distribution pat-

tern of the internalized CPPs that was consistent with the distribution

of endocytic vesicles and CPPs co-localized with common endocytic

markers. Since then, endocytosis has been shown to be the main

mechanism of uptake for various CPPs, especially at low doses and

when coupled to a large molecular weight cargo (135, 136). Specifi-

cally for CPP-based nanoparticles with nucleic acids, endocytosis has

been implicated for most of the available platforms like stearyl-Arg8,

Tat-DRBD and PepFects (122, 124, 128, 130, 137). Several endocytic

pathways have been suggested for the uptake of CPPs and their cargo

including classical clathrin mediated endocytosis, caveolae-mediated

endocytosis and macropinocytosis (138). Consequently, several ap-

proaches have been devised to enhance the escape of CPPs and their

cargos out of the endosomes to avoid degradation in the lysosomal

pathway and reach their target subcellular compartments. One exam-

ple is the development of a histidine-containing endosomolytic α-

helical penetratin analogue, EB1, which was able to form complexes

23

with siRNA and promote endosomal escape (139). Other methods

utilized fusogenic peptides, like HA2-peptide, conjugated to CPPs in

order to facilitate release from endosomes (140). Recently, our group

utilized several chemical modifications to enhance endosomal escape

including stearylation and addition of endosomolytic moieties such as

trifluoromethyl-quinoline (122). Nevertheless, this has not ruled out

the fact that many CPPs are still able to gain access to the cell via di-

rect translocation mechanism in well controlled studies and without

fixation artifacts (141). Recently, Verdurmen et al. studied the uptake

mechanism of some arginine rich peptides and found that their direct

penetration depends on a CPP-induced translocation of acid sphingo-

myelinase (ASMase) to the outer leaflet of the plasma membrane and

ceramide formation (142).

1.3.3.1. Physicochemical properties and the uptake mechanism

In my opinion, the differences in the uptake mechanism of CPPs

should not be considered controversial. Controversy comes from the

notion that all CPPs are expected to have a single uptake mechanism

whatever cargos they carry, whatever concentrations are used and

whatever model they are tested in. However, such a universal mecha-

nism cannot exist because all the aforementioned factors drastically

affect the physicochemical properties of the CPP ultimately changing

the way it interacts with cells. CPP-cargo, CPP-medium and CPP-CPP

interactions can vary dramatically depending on the type of cargo and

the way it is attached to the CPP (covalent or non-covalent), the model

system (synthetic vesicles or live cells), the medium (buffer, serum-

free, serum containing) and the concentration used. These interactions

would lead to new entities with different physicochemical properties

on the molecular level and consequently different cellular interactions.

This section will discuss some examples.

An example that spans from the CPP field to their closest rela-

tives, the antimicrobial peptides (AMPs), shows how different concen-

trations can affect the cell entry mechanism and the subsequent phar-

macological effect. Many CPPs at sufficiently high concentrations are

lytic and toxic to both mammalian and bacterial cells (143, 144),

while several AMPs can penetrate into the cells without perforation or

lysis, similar to CPPs (145). Such an analogy led some researchers to

even question the reasons for the distinction between the two classes

(146). This can be understood in light of a physicochemical property

24

called the partition constant. To be able to cause lytic antimicrobial

activity, the peptide should be in a sufficiently high concentration,

called the threshold concentration, enabling the partition from the so-

lution to the membrane, increasing the local concentration and causing

perforation (147). However, at lower concentrations, when this parti-

tioning and localization is not achieved, the peptide can penetrate the

cells via other mechanisms including endocytosis. A very good exam-

ple is the SV13-PV peptide, which has been shown to enter the cell via

direct penetration at high concentrations and via endocytosis at lower

concentrations in the same study (148). Interestingly, at low concen-

trations, SV13-PV forms spherical nanoparticle-like structures upon

association with cell surface glycosaminoglycans (GAGs) on the

plasma membrane, which promote endocytic uptake (149). GAG

clustering prior endocytic internalization has been recently demon-

strated for several other CPPs as well (150, 151, 152).This example

clearly shows how physicochemical properties like the partitioning

from solution or formation of nanoparticles on the cell surface can

change the mechanism of uptake according to the used concentration.

For CPP nanoparticles with nucleic acids, the physicochemical

changes to the CPP are more prominent. The CPP condenses the nu-

cleic acid cargo forming a new entity (i.e. nanoparticle), which pos-

sesses new physicochemical properties. While nucleic acid condensa-

tion with CPPs has been shown experimentally (123), it is not known

if a reciprocal condensation occurs to the CPP as well. Furthermore,

the structure of the nanoparticle is not well characterized i.e. the dis-

tribution of components between the core and the surface is not

known, however, it is always assumed that the peptide should be on

the surface to mediate cellular delivery. Since, the surface of the na-

noparticle is the interaction plane with the cells, it is very important to

characterize its physicochemical properties, especially the surface

charge (zeta potential), which will determine the mode of interaction

with the plasma membrane. Also, surface charge determination can

shed light on the molecules that are superficially expressed.

The zeta potential is a function of the surface charge of the par-

ticle or any adsorbed layer at the interface (153). Each charged parti-

cle in a solution containing ions is surrounded by an electrical

25

double layer of ions and counter-ions (Figure 6.) (154). The first layer

of strongly bound counter-ions is commonly called Stern layer. Since

it is strongly attached, this layer moves together with the particle in

the medium. The second layer contains ions and counter-ions and is

less strongly bound to the suspended particle, thus called “diffusion

layer”. This layer is in direct contact with the bulk of the suspending

liquid, and in contrast to the stern layer, ions of this layer do not move

with the particle while moving in the medium. The potential differ-

ence across the double layer, i.e. between the edge of the Stern layer

and the edge of the diffusion layer (bulk of suspending liquid) is

called zeta potential (154, 155). Zeta potential is usually of the same

sign as the potential at the particle surface (153), however, it is greatly

affected by the dispersion medium, especially the pH and ion concen-

tration. This medium-dependence highlights the importance of meas-

uring the zeta potential of CPP nanoparticles in biorelevant media as

this will eventually affect the surface charge and hence the subsequent

membrane interaction and uptake mechanism. In this thesis (paper

III), we found that certain CPP-ON nanoparticles have negative zeta

potential when dispersed in biorelevant media. In contrary to the ex-

Figure 6. Different layers forming the zeta potential of a particle.

26

pected mode of action of CPPs, this negative charge, probably caused

by ON condensation on the surface, was responsible for the cellular

uptake by interaction with cell surface receptors called the scavenger

receptors (156), which will be discussed later in detail. This is another

example of how the cargo and the medium can drastically change the

physicochemical properties of CPPs driving them into cells by distinct

mechanisms.

1.4. Scavenger receptors (SRs)

SRs were discovered by the Nobel laureates Michael Brown and Joseph Goldstein in 1979 (157, 158). Having discovered the low-density lipoprotein receptor (LDL receptor) a few years earlier, Brown and Goldstein found that acetyl-LDL can still accumulate in macro-phages in atherosclerotic plaques of patients with familial hypercho-lesterolaemia who lack LDL receptors. This implied the presence of other receptors, which were called the scavenger receptors for their presumed role in scavenging modified forms of LDL (159). Now, there are 8 members of scavenger receptor family (A-H), all having the common feature of binding with low specificity to polyanionic ligands including chemically modified LDL despite the structural and functional differences (160, 161, 162). Unlike other receptors, SRs

Scavenger receptors

Modified LDL

Nucleic acids

Nanoparticles

Sulfated polysacharides

Amyloid-beta aggregates and

prions

Viruses

Bacteria

Apoptotic cells

Figure 7. Ligands recognized and endocytosed via scavenger receptors.

27

bind a wide range of polyanionic ligands, and thus they are sometimes referred to as promiscuous receptors (162) or cellular fly-paper (163). Besides their role in LDL metabolism, they are also pattern recogni-tion receptors (PRRs) that play an important role in innate immunity by the identification and endocytosis of wide variety of pathogen-associated molecular patterns (PAMPs). That is why they are highly expressed in macrophages and involved in several immune responses. Staphylococcus aureus bacteria (164), HCV virus (165), lipopolysac-charide (LPS) (166), PrP106–126 prion protein (167) and viral RNA (168) are examples of PAMPs recognized by scavenger receptors (Figure 7). Importantly, SRs are involved in the clearance and phago-cytosis of apoptotic cells by macrophages (159). They are also in-volved in the pathogenesis of Alzheimer’s disease by the uptake of amyloid beta aggregates via microglia (169). Furthermore, they rec-ognize several sulfated polysaccharides (dextran sulfate and fucoidan but not chondroitin sulfate) and polyribonucleotides (polyinosinic (poly I) and polyguanilic (poly G) acid but not polyadenilic nor poly-cytidylic acid (170). Interestingly, they are involved in the uptake of synthetic nanoparticles including crocidolite asbestos (171), silica (172), polystyrene (173), iron oxide (174) and gold nanoparticles ei-ther naked (175) or when functionalized with ONs (68). Therefore, they are thought to play an important role for the accumulation of as-bestos in lung macrophages after excessive inhalation (industrial worker intoxication) leading to asbestosis (171) and also for the ac-cumulation of therapeutic nanoparticles in the liver macrophages (Kupffer cells) after intravenous injection (176) helped by the extrava-sation effect.

1.4.1. Class A scavenger receptors (SCARAs) and nucleic acid

binding

SCARAs are the most well studied subgroup of scavenger re-

ceptors. To date, five different subtypes have been identified: SR-A I/II/III (SCARA1), MARCO (SCARA2), SCARA3 (CSR1 and CSR2), SCARA4 (SRCLI and SRCLII) and SCARA5 (168, 177, 178, 179, 180, 181). They share similar structural features being a trimeric type II transmembrane glycoprotein consisting of a cytoplasmic tail, transmembrane domain, spacer region and collagenous domain (177). However, some differences exist. A helical coiled-coil domain is pre-sent in SCARA1, 3 and 5 (161, 179). Additionally, while a cysteine- rich domain is present in MARCO, SR-AI and SCARA5, it is lacking in SR-AII and SCARA3 and replaced with a lectin-like domain in

28

SCARA4 (161, 177, 179). Although initially discovered in macro-phages and thought to be restricted to this type of cells, later studies showed that SCARAs possess a broader expression profile. Different SCARA subtypes were detected in endothelial cells, smooth muscle cells, splenic dendritic cells, fibroblasts and epithelial cells (168, 177). Interestingly, they are also expressed at the endothelial cells of cere-bral capillaries that form the blood-brain barrier (182). Having such a broad expression profile, their role in the uptake of nanoparticles in non-macrophage cell types might have been overlooked.

The ability of SCARAs to bind nucleic acids is of particular im-

portance to this thesis. Soon after discovery, they were shown to be involved in the uptake of polyribonuclides such as poly I and poly G (183). Poly I and poly G are known to form aggregates in solution (184, 185) and this aggregation was shown to be necessary for SCARA binding (186). Importantly, SCARAs are involved in the recognition and uptake of ON sequences containing unmethylated CG dinucleotides (CpG) from bacterial or viral DNA, which are very well known immunostimulators (187, 188, 189). CpG aggregation is also a perquisite for the receptor recognition (187, 190), a process that is enhanced by oligo G flanking (189, 191) or using phosphorothioate-based CpG which can form aggregates easier due to the more hydro-phobic backbone (190, 191, 192, 193). Double stranded RNA (168, 194, 195) and ON-functionalized gold nanoparticles (68) were also shown to be internalized via SCARA. One of the papers described in this thesis (paper III) demonstrated the role of SCARA in the uptake of CPP-nanoparticles with ONs (156).

The necessity of aggregation of nucleic acids prior to the uptake

via SCARA and the receptors’ role in the uptake of ONs complexed with CPPs or ONs displayed on the surface of gold nanoparticle high-lights the role of nucleic acid condensation in cellular delivery. The success of polycationic carriers despite their great diversity might be due to their shared ability to condense nucleic acids and to present them to certain cellular receptors that recognize particulate patterns (as discussed in sections 1.2.1. & 1.2.2.). This hypothesis can gain further support from the fact that several gene therapy nanoparticles have the polycationic domain on the surface and consequently the condensed nucleic acid cargo is also superficial (42, 43). Furthermore, many gene therapy nanoparticles have negative zeta potential especially when measured in biorelevant conditions (77, 196, 197, 198, 199). Striking-ly, even before the discovery of SCARAs, aggregation of polynucleo-tides enhanced their binding to fibroblasts by 30-fold (200) and such aggregates were used to enhance the transport albumin into sarcoma

29

cells by 75-fold (201). Unfortunately, the latter paper that is boldly titled “Polynucleotide aggregates enhance the transport of protein at the surface of cultured mammalian cells (201)” was largely ignored, having only 10 citations in more than 37 years.

Despite being rationally appealing, solid claims about this nucle-

ic acid-mediated delivery hypothesis can only be made for the systems tested so far, and to extend it to other systems thorough experimental testing should be performed. Nevertheless, the hypothesis might be useful to attract the attention of the research community to the puta-tive role of the cargo in the delivery of the vector and the role of PRRs in the delivery of gene therapy vectors.

1.5. Pharmaceutical formulation

Pharmaceutical formulation is the process of transformation of

the active drug substance into a final medicinal product, which is an

indispensable process before the drug can reach the market and be

used by patients. It has a great effect on enhancing the efficiency and

stability of active pharmaceutical ingredients. Additionally, specific

formulations have to be tailored for different therapeutic approaches.

Thus, it is of extreme importance to apply pharmaceutical formulation

techniques to gene-therapy nanoparticles to be able to enhance their

pharmacological properties and translate them into usable drug prod-

ucts. Unfortunately, as discussed in section 1.2.2., most of the re-

search in the field is focused on the development of new systems

while little attention is given to pharmaceutical formulation. However,

formulation techniques can be very useful in solving fundamental de-

livery problems in fairly simple ways, besides its consideration for

patient use and compliance that adds new dimensions to the drug de-

velopment process. Also, the development of cheap and applicable

methods to formulate nanoparticles will encourage pharmaceutical

companies to invest in the technology.

Among the different pharmaceutical forms present solid formu-

lations remain the most widely used. It is the form used in tablets,

capsules, powders for inhalation and even powders for injection. This

is because they are the most convenient dosage forms to use and also

very stable upon storage and transportation. That is why in this thesis

a method was developed to incorporate CPP nanoparticles in a solid

30

form that could be used in various therapeutic applications. For the

first time we applied the concept of solid dispersion to CPP-based

nanoparticles. Solid dispersion was invented to increase the solubility

of poorly water-soluble drugs by dispersing them over hydrophilic

solid matrices, thus decreasing the particle size and enhancing the

wettability (see details in methods) (202). Although CPP-ON nanopar-

ticles disperse well in water, this technique was still useful to obtain a

uniform distribution of the nanoparticles over a solid matrix.

Furthermore, there are several in-vitro tests that are assigned by

the different pharmacopeias to assess the suitability of a drug product

for a particular route of administration. For oral delivery for example,

there are standard in-vitro methods to test the drug product’s stability

in bench-top experiments that simulate the gastric or the intestinal

conditions. Special buffers are prepared with pH 1.2 for gastric simu-

lation and 6.8 for intestinal simulation. Hydrolytic enzymes can also

be added to these buffers to further assess the stability; pepsin for gas-

tric simulation and pancreatin for intestinal simulation. The drug

product to be tested is incubated in the simulated gastric fluid (SGF)

or the simulated intestinal fluid (SIF) with or without enzymes for

certain periods of time at 37ºC, then samples are taken and analyzed.

In this thesis (paper IV), we used a biological endpoint (RNAi re-

sponse) to test the stability of our formulation. Having a biological

endpoint is more representative of the real residual activity, and to our

knowledge, it is the first time to be applied to RNAi delivery systems.

31

2. Aims of the study

This thesis is aiming to study the activity and the properties of

two new chemically-modified CPPs; PF3 and PF14. Their activity in

the delivery of plasmids, SSOs and siRNA is assessed in different

models. Special focus was given to the elucidation of the uptake

mechanism of PF14-SSO nanoparticles. Furthermore, the thesis de-

scribes methods for formulating such nanoparticles into solid formula-

tions and testing them for the feasibility of oral administration in sim-

ulated gastric conditions.

Paper I: Studying the activity of stearic acid-modified TP10

(PF3) to deliver plasmid DNA to various cell types in-vitro and in-

vivo upon i.m. and i.d. injection.

Paper II: In this paper, ornithine, a non-standard amino acid, is

used to chemically modify a TP-10 analogue together with N-terminal

stearylation. The new peptide, named PF14, is tested in induction of

splice-switching activity in several models and a method for develop-

ing solid formulations utilizing PF14-SSO nanoparticles is decribed.

Paper III: The role of class A scavenger receptors in the uptake

of PF14-SSO nanoparticles is investigated utilizing pharmacological

inhibitors, siRNA knockdown of the receptors, immunofluorescence

and transmission electron microscopy.

Paper IV: The activity of PF14 in delivering siRNA to various

cell types is studied. Additionally, the stability of PF14-siRNA nano-

particles in solid formulation and simulated gastric conditions is in-

vestigated.

32

3. Methodological considerations

The methods used in this thesis are described in detail in the

contributing papers. This part will briefly discuss the main methods

utilized with some theoretical aspects. The selections below are valid

for all papers when nothing else is stated.

3.1. Solid-phase peptide synthesis and peptide design

All peptides in this thesis were synthesized by solid phase pep-

tide synthesis (SPPS), which was pioneered by Bruce Merrifield in

1963 (203). SPPS is based on anchoring the growing peptide chain

onto an insoluble solid matrix followed by adding an N-terminally

protected amino acid then deprotection and adding a new amino acid

in repeated cycles. This method enabled the synthesis of not only

peptide chains but also other polymers including DNA and RNA revo-

lutionizing entire fields of science.

All peptides used in this thesis were synthesized utilizing

methylbenzyl hydrylamine (MBHA) as an anchoring resin to produce

C-terminal amidated peptides, and Fmoc protection chemistry was

used. Peptides were thereafter cleaved from the resin using 95 % tri-

fluoroacetic acid (TFA), 2.5 % triisopropylsilane (TIS) and 2.5 %

H2O. Following cleavage and extraction, peptides were subsequently

freeze-dried. Crude peptides products were purified using semi-

preparative reversed-phase high performance liquid chromatography

(HPLC) and analyzed using matrix-assisted laser desorp-

tion/ionization-time of flight (MALDI-TOF) mass spectrometer. The

main peptides used in this thesis are presented in Table 4.

33

Table 4. The main peptides used in this thesis.

Stearyl-TP10 (PF3) is based on stearic acid modification of

TP10, which is a truncated version of one of the very first CPPs;

Transportan (Table 2.). Both Transportan and TP10 have earlier been

shown to be able to translocate covalently bound cargos across the

plasma membrane (88, 204). N-terminal stearic acid was added to

enhance the ability of TP10 to form non-covalent nanoparticles with

nucleic acids (137). This approach was first developed by Futaki’s

group to enhance the transfection efficiency of polyarginines (120).

On the other hand, the design of PF14 was based on switching lysines

for ornithines as the source of positive charge in the CPP together with

sequence modifications inspired by leucine zippers to enhance nucleic

acid binding. Earlier reports demonstrated that poly-L-ornithine

demonstrated superior transfection efficiency (up to 10-fold) com-

pared to equivalent poly-L-lysine-based systems (205). The superior

efficiency of poly-L-ornithines was related to the higher affinity for

DNA and the ability to make more stable complexes at lower charge

ratios (205). Furthermore, we hypothesized that ornithine, being a

nonstandard amino acid, would be less prone to serum proteases, and

thus could retain the activity in serum conditions.

3.2. Cell cultures

The first propagation of a human cell line in-vitro was the HeLa

cell-line obtained from a cervical cancer patient named Henrietta

Lacks and propagated by George O. Gey in the 1950’s. Since then, in-

vitro cell culture models are serving as indispensable tools in as-

sessing the activity of newly developed therapeutics before testing

them on animal and human subjects. Two types of cell cultures exist,

primary cultures that are obtained directly from the animal and can be

kept for a limited period of time, and permanent cultures, which are

usually of cancerous origin, and can be propagated indefinitely. Sev-

Name Sequence

Stearyl-TP10 (PF3) Stearyl-AGYLLGKINLKALAALAKKIL-NH2

PF14 Stearyl-AGYLLGKLLOOLAAAALOOLL-NH2

34

eral cell-lines were used in this thesis including primary and perma-

nent cell lines. All cell-lines were grown at 37°C, 5% CO2 in humidi-

fied environment with the appropriate culture media supplemented

with nutrients and antibiotics.

3.3. Plasmid delivery (Paper I)

Non-covalent complexation strategy between CPPs and nucleic

acids is the main strategy utilized in this thesis. pGL3 and pEGFP-C1

plasmids, expressing luciferase or EGFP respectively were used.

Plasmids were mixed with CPPs at different peptide/plasmid charge

ratios (CRs), which were calculated theoretically, taking into account

the positive charges of the peptide and negative charges of the plas-

mid. Complexes were formed for 1 h at room temperature then added

to the cells. By measuring luciferase or EGFP expression, the efficien-

cy of delivery for different peptides and CRs can be assessed for dif-

ferent cell-lines.

3.4. SSOs delivery (Paper II and III)

Two cell lines were utilized in this study, HeLa pLuc 705 and

mdx mouse myotubes. The HeLa pLuc705 cell-line is stably transfect-

ed with a luciferase-encoding gene interrupted by a mutated β-globin

intron 2. This mutation causes aberrant splicing of luciferase pre-

mRNA resulting in the synthesis of non-functional luciferase (206).

Masking the aberrant splice site with SSOs redirects splicing towards

the correct mRNA and consequently restores luciferase activity, which

can be quantified by adding luciferin substrate to the cell lysate and

measuring luminescence. Mdx mouse myotubes were obtained from

confluent H2K mdx cells, which is a myogenic cell-line derived from

transgenic mice carrying a point mutation in exon 23 of the dystrophin

gene (207). This cell-line serves as the leading cell model system for

development of drugs and drug delivery systems for treatment of

DMD. Masking the point mutation in exon 23 with SSOs leads to the

production of a shorter, partially functional dystrophin mRNA that can

be quantified with reverse-transcription PCR (RT-PCR). SSOs were

non-covalently complexed with PF14 at different MRs in water.

35

Splice-switching efficiency was calculated as the percentage of the

corrected band (exon skipping %) to the sum of corrected and aberrant

bands.

3.5. siRNA delivery (Paper IV)

In this paper, the nanoparticles were formed by mixing PF14

and siRNA in water at different molar ratios (MRs). Reporter cell-

lines stably expressing luciferase were transfected with PF14 com-

plexed with luciferase siRNA, and the decrease in luciferase expres-

sion was used to quantify the transfection efficiency. In addition, we

investigated the targeting of an endogenous gene, hypoxanthine phos-

phoribosyl transferase 1 (HPRT1), in HUH7 cell-line by measuring

mRNA levels using qPCR (real time quantitative polymerase chain

reaction). HPRT1 was chosen due to the long cellular half-life of the

protein (around 48 h) and thereby minimal impact on the vitality of

the transfected cell and limited off-target effects of the specific

HPRT1 siRNA sequence (208). In order to accurately determine the

degree of down-regulation, glyceraldehyde 3-phosphate dehydrogen-

ase (GAPDH) was used as an internal standard for quantification.

3.6. Toxicity

To exclude that the activity of the peptides in-vitro is associated

with toxicity, WST-1 assay was used. This assay measures cell viabil-

ity as a function of mitochondrial metabolic activity. It measures the

activity of the mitochondrial dehydrogenases to convert tetrazolium

salts to formazan and cell viability and proliferation is directly corre-

lated to the amount of formazan dye formed, which is quantified spec-

trophotometrically.

3.7. Dynamic light scattering (DLS), nanoparticle tracking

analysis (NTA) and zeta potential

To investigate the physicochemical characteristics of the nano-

particles formed upon complexation of the peptides and nucleic acids,

36

DLS, NTA and zeta-potential measurements were carried out. The

nanoparticles in solution after complexation can be regarded as a col-

loidal dispersion system. The DLS concept is based on shining laser

light on the dispersion system where the Brownian motion of the dis-

persed particles causes fluctuations in light-scattering intensity as a

function of time, which can be correlated to the size of the scattering

particles (155, 209). Despite being very useful, the intensity of the

scattered light is proportional to the sixth power of the particle diame-

ter makes this technique very sensitive to the presence of large parti-

cles (210). This limitation has caused differences between the particle

size measured by DLS and that seen in electron microscopy images in

paper III. In paper IV, we used the NTA system, which is dependent

on the refractive index of the nanoparticles and combines laser light

scattering microscopy with a charge-coupled device (CCD) camera

(210). This technique is more accurate than DLS and also enables

visualization of the nanoparticles in solution.

Zeta potential is determined by electrophoresis of the sample

and measuring the velocity of the particles using laser Doppler veloc-

imetry (154). Since zeta potential is highly sensitive to the type and

amount of ions in the suspension and its pH, we measured it together

with particle size in different media including: water, isotonic NaCl

solution, serum-free medium and serum-containing medium. The

measurements in biorelevant conditions help to elucidate the physico-

chemical properties that the nanoparticle possesses in the media where

it interacts with the cell surface.

3.8. Solid dispersion and gastric simulation

In paper II and IV, to test the feasibility of formulating PF14-

ON nanoparticles into solid formulation, solid dispersion technique

was utilized. The technique was developed to enhance the solubility

and wettability of poorly-water-soluble drugs, since dissolution in

gastric fluids is a perquisite for drug absorption. Otherwise, the drug

product will not be available for absorption by intestinal villi. Classi-

cally, the water-insoluble drug and a hydrophilic carrier are dissolved

in a common organic solvent that is then evaporated leaving a molecu-

lar dispersion of the drug on the hydrophilic solid matrix (202). That

is why it is sometimes called “solid solution” and it was shown to en-

37

hance the pharmacokinetic profile of many drugs that had unfavorable

dissolution properties (202). In our case, PF14-ON nanoparticles have

good dispersibility in water; however, the technique was useful to ob-

tain uniform distribution of the particles over the desired solid matri-

ces. The suspension of nanoparticles was first mixed with different

excipient solutions at different concentrations. For solvent evaporation

(in this case water), we chose speed drying under elevated temperature

(55-60ºC) and reduced pressure. Despite being a relatively stressful

drying method, it is more relevant to the widely used, cost-effective,

heat-based pharmaceutical drying techniques.

For gastric simulation experiments, the SGF was prepared accord-

ing to the US pharmacopoeia (0.2% NaCl (w/v) and 0.7% HCl (v/v))

at pH 1.2 with or without pepsin (0.32% (w/v)). The nanoparticles in

solution or in solid formulation were incubated for 30 minutes in the

fluid before addition to the cells. The residual activity was measured

as RNAi response in luciferase expressing cells.

3.9. Immunofluorescence and transmission electron microscopy

(TEM)

In paper III, besides pharmacological inhibitors and siRNA, we

used immunofluorescence and transmission electron microscopy to

further demonstrate the role of SCARA in the uptake of PF-SSO na-

noparticles. For immunofluorescence, cells were incubated with nano-

particles of Cy5-labeled (red) SSOs with PF14 for 30 min at 4 °C or

37 °C. Incubation at 4 °C drastically slows down the endocytic pro-

cesses enabling us to detect colocalization occurring at the cell surface

before internalization. The cells were fixed and permeabilized then

treated with anti-SCARA3 and anti-SCARA5 antibodies and subse-

quently labeled with fluorescently-labeled secondary antibodies. For

TEM, nanogold-labeled SSOs were complexed with PF14 to enable

TEM visualization. Cells were pretreated with SCARA inhibitors or

controls then treated with the nanoparticles for 1h. Afterwards, the

cells were washed and fixed according to standard procedure (as de-

scribed in (149)). Subsequently, ultra-thin sections were cut in parallel

with the coverslip and contrasted with 2% uranyl acetate in 50% etha-

nol for 2 min and in standard lead citrate staining solution for 2 min

and examined with the transmission electron microscope. Sections

38

from the cell surface and cell interior were compared for detection of

binding and internalization of the nanoparticles.

3.10. Animal Experiments

In-vivo animal experiments were carried out in paper I. All ani-

mal experiments were approved by The Swedish Local Board for La-

boratory Animals, and were designed to minimize the suffering and

pain of the animals. PF3-plasmid nanoparticles were injected i.d. or

i.m. into M. tibialis anterior. Gene expression was assessed by imag-

ing of the reporter gene (firefly luciferase) expression. Clinical chem-

istry parameters (alanine transaminase/aspartate transaminase, C-

reactive protein, and creatinine levels) in serum were analyzed after 24

hours post-treatments using standardized techniques. For histopatho-

logical analysis, organs were dissected after 24 hours and fixed in

formalin, embedded in paraffin, and stained with eosin and hematoxy-

lin before taking images.

39

4. Results and discussion

The four papers in this thesis cover two newly developed chem-

ically modified CPPs used for the delivery of different nucleic acid-

based therapeutics.

4.1. Delivery of plasmids via stearyl-TP10 (PF3) in-vitro and in-

vivo (Paper I)

Here, we demonstrate that stearylation of the TP10 peptide (Ta-

ble 4.) has a significant impact on plasmid delivery in different cell-

lines including Chinese hamster ovary (CHO), human embryonic kid-

ney (HEK293), human glioblastoma (U87), human osteosarcoma

(U2OS) and the hard-to-transfect primary mouse embryonal fibro-

blasts (MEF). PF3 transfection efficiency was in parity with the com-

mercial lipid-based transfection reagent Lipofectamine-2000™, with

less toxicity. In addition, PF3 was able to transfect entire cell popula-

tion in a ubiquitous manner. However, the transfection efficiency was

dependent on the CR between the peptide and the plasmid. Compared

to the unstearylated version, PF3 resulted in around 4 orders of magni-

tude increase in luciferase expression levels as compared to plasmid-

treated cells. Clearly, stearic acid renders the peptide more hydropho-

bic and presumably hydrophobicity plays an important role in particle

formation, as it enables more pronounced plasmid DNA condensation

and the formation of small, stable particles. This protects plasmid

DNA, making it more stable against the degrading capacity of serum

nucleases. The drastic increase in activity of PF3 compared to TP10

could also be a result of increased endosomal escape.

Importantly, PF3-plasmid nanoparticles facilitated efficient

gene delivery in muscle and skin, after i.m. or i.d. injections, respec-

tively. In both cases, luciferase activity was increased around 1 log as

40

compared to the background levels and these effects were shown to be

dose-dependent. We also confirmed that the gene delivery did not

trigger any immune response in-vivo and that these treatments were

not associated with any systemic toxicity. Interestingly, these effects

were only seen with the nanoparticles formed at CR1, while at other

CRs, no effect on luciferase activity was seen. The critical dependency

on certain CRs possibly emanates from the avidity of PF3 towards the

plasmid and, therefore, the stability of these nanoparticles. Probably,

at higher CRs, the release of the plasmid from the complexes is per-

turbed and the affinity of PF3 towards it is too great for in-vivo ap-

plicability. These differences between the avidity at different CRs

were confirmed by other assays in the paper.

4.2. Delivery of SSOs via PF14 and solid formulation

development (Paper II)

In this paper we present a new chemically modified CPP, PF14

(Table 4.). Ornithines were utilized as the main source of positive

charges instead of lysines (see peptide design, section 3.1). PF14 was

tested in HeLa 705 and mdx mouse myotubes cell-lines utilizing dif-

ferent MRs. Robust splice-switching was observed in both cell lines in

a dose-dependent manner. This was confirmed on the protein and

mRNA levels by measuring the amount of functional luciferase en-

zyme produced in the HeLa 705 cell-line and by measuring the inten-

sity of corrected mRNA bands via RT-PCR in the mdx cell-line re-

spectively. The activity was not significantly affected by serum condi-

tions, and at certain MRs significantly exceeded the activity of

Lipofectamine2000® without associated toxicity. In addition, PF14

elicited a fast onset of its splice-switching activity as early as 8 hours

and was able to transfect entire cell population without being very

sensitive to increasing cell densities. Co-localization with endocytosis

markers demonstrated that endocytosis is responsible for the uptake of

PF14-SSO nanoparticles and this was further corroborated by the en-

hanced splice-switching activity upon addition of CQ.

Finally, we wanted to take our delivery system a step further

by developing a suitable formulation for administration. Although

CPPs have been extensively exploited in recent years for the delivery

of various therapeutics (113), to our knowledge, formulation of CPPs

41

into different pharmaceutical forms has never been thoroughly stud-

ied. Therefore, to expand the range of therapeutic applications of CPP-

based therapeutics, there is a need for studies exploring the possibility

of incorporating CPPs in different pharmaceutical forms; especially

the solid form which is widely used in several pharmaceuticals. We

applied the solid dispersion technique by mixing the nanoparticles in

suspension with excipient solutions then drying at relatively high tem-

peratures (55 - 60 ⁰C) under vacuum. Mannitol, lactose and PEG 6000

were used as excipients at different concentrations. It was clear that

different excipients and concentrations thereof had a big impact on the

activity of the formulation, with lactose at a concentration of 3.33%

demonstrating splice-switching activity nearly identical to the freshly

prepared nanoparticles in solution. On the contrary, Lipofec-

tamine2000® almost lost its entire activity upon application of this

drying and dispersion procedure. In order to assess that the presence

of intact nanoparticles after the formulation procedure, DLS studies

were performed comparing the freshly prepared nanoparticles with the

solid formulations. We found that the particles size and particle-size

distribution are highly affected by the type of excipient used. The

formulation which mediated the highest splice-switching activity, also

had DLS profile most similar to the freshly prepared nanoparticles.

Upon measuring the zeta potential of the nanoparticles, we found that

they are negatively charged, either freshly prepared or as a solid for-

mulation.

Next, we subjected the best performing formulation (lactose at

3.33%) to stressful stability testing conditions at elevated temperatures

for 2 months. Storage at high temperatures increases the rate of degra-

dation reactions that could take years to occur (211). PF14 formula-

tions demonstrated an excellent stability profile under such conditions,

where no statistically significant loss in transfection efficiencies at any

time-point except for the 60 ⁰C-8-weeks point where the transfection

efficiency decreased to 70% of the initial value. Compared to lyophi-

lized lipoplexes (212, 213), PF14 formulations are remarkably stable

without further additives or special storage conditions.

42

4.3. Scavenger receptor-mediated uptake of CPP nanoparticles

with ONs (Paper III)

As discussed earlier in section 1.3.3., the uptake mechanism of

CPPs cannot be universal for all cargos. The CPP cargo interaction forms a new entity whose properties could be different from the naked CPP. Also, different media can affect the physicochemical properties of CPPs and their cargos, consequently affecting the uptake mecha-nism. These effects were clear when we were assessing the physico-chemical properties of PF14-SSO nanoparticles. In contrary to our expectations, the net surface charge (zeta potential) of the particles was negative and highly dependent on the dispersion medium. In wa-ter, zeta potential was dependent on the peptide/ON ratio, being nega-tive at MR3, almost neutral at MR5 and positive at MR10. However, when the dispersion medium was changed to biorelevant conditions such as isotonic NaCl, serum-free medium or serum-containing medi-um, zeta potential switched to high negative values at all MRs. This shift occurred even in the absence of serum indicating that the salt concentration and pH are mainly responsible for the change rather than serum proteins.

The effect of pH and salt concentration on zeta potential is a

very well documented for colloidal dispersions (214); however, the net negative charge of the particles was surprising. It is expected for a CPP-based vector to have a net positive charge to enable direct inter-action with the negatively-charged cell membrane or integrated GAGs. Importantly, this superficial negative charge is formed in the transfection media, which means that this negative surface is what actually interacts with the cell membrane in transfection conditions. This charge implies that the particles should repel from the cell sur-face rather than directly interact. Thus, we suspected a role of a par-ticular receptor that can mediate such interaction and act as an adapter between the nanoparticle and the cell membrane. SCARAs were very plausible candidates for being the putative receptors as they are well known for binding with low specificity to polyanionic ligands and negatively charged particles (section 1.4). A simple experiment was to study the effect of preincubation with specific SCARA ligands on the splice-switching activity of the nanoparticles. Strikingly, we found that at very low concentrations of poly I, dextran sulfate or fucoidan, the activity of the nanoparticles was abolished completely and in a dose-dependent fashion. For control, we used chemically related mol-ecules that are not SCARA ligands such as polycytidylic acid, chon-droitin sulfate and galactose, and all did not affect the activity except

43

for chondroitin sulfate which had a slight effect at higher doses. Upon pretreatment with fetuin, which is known to mediate uptake of nano-particles via SCARA (215), the activity was increased by more than 50%.

The next step was to determine which SCARA subtypes are ex-

pressed in the HeLa 705 cell-line. Using RT-PCR, we found that it

expresses SCARA3 (with its 2 splice variants) and SCARA5. SiRNA

knock-down of these receptors yielded in more than 50 % decrease in

activity. Furthermore, immunofluorescence and TEM studies were

performed to directly assess the role of SCARAs in the uptake pro-

cess. Using antibodies against SCARA3 and SCARA5, Cy-labeled

PF14-SSO nanoparticles cololocalized with the receptors both on the

cell-surface and after internalization. In the TEM images, it was clear

that the presence of the specific inhibitory ligands, such as dextran

sulfate, prevented both the binding and uptake of the nanoparticles,

while chondroitin sulfate did not. The TEM images are particularly

interesting as they demonstrate the initial concept behind this study

i.e. when the receptors are not available (in this case blocked), the

nanoparticles are not attached to the cell membrane due to electrostat-

ic repulsion.

Despite the different methods used to prove SCARA involve-

ment, the question remains about what is causing the negative surface

charge. We speculated that at least a portion of the SSO cargo is dis-

played and condensed on the surface mediating this interaction (Fig-

ure 8.). To indirectly prove the role of the SSOs, we ran transfection

experiments using the naked SSO at different concentrations. At low-

er concentrations (200 nM) they did not mediate any splice-switching

activity, however, at a much higher dose (5 μM), significant splice-

switching was observed, indicating that the SSOs can be taken by the

cells at high doses where they start to form aggregates. This necessity

of ON aggregation for SCARA mediated uptake is well documented

(section 1.4.1.). Additionally, we performed colocalization experi-

ments using naked Cy-labeled SSOs at different concentrations. Simi-

larly, at higher concentration (1μM), profound colocalization is ob-

served, while much less is observed at 200 nM. Despite not being a

direct proof, these results indicate that the activity of the peptide might

be due to efficient condensation and display of SSOs on the surface.

PF14 has a lipid tail that can form a hydrophobic core leaving the hy-

drophilic cationic residues on the surface to interact and condense the

44

SSO cargo (Figure 8.). However, direct evidence for such particle or-

ganization is still needed and cryo-electron microscopy might be a

good method to shed light on that.

4.4. Activity and solid formulation of PF14-siRNA nanoparticles

and their stability in simulated gastric conditions (Paper IV)

In paper II, we studied the activity of PF14 for SSO delivery

and developed a method for formulating PF14-SSO nanoparticles into

a solid form which preserves the activity for several weeks and can

have several applications. Here, we wanted to extend the PF14 deliv-

ery platform to siRNA. Starting with reporter cell-lines, we found that

PF14 can mediate high rates of RNAi in serum-free and serum-

containing medium in a dose-dependent matter. Around 90 % knock-

down was achieved at doses as low as 50 nM in serum-free conditions

and 100 nM in the presence of serum. We used qPCR to determine the

EC50 and the kinetic profile of PF14-siRNA nanoparticles on the

mRNA level. HPRT1 was the target gene while GAPDH was used as

Figure 8. Hypothetical representation of PF14-

SSO nanoparticle. Grey: PF14, Red: SSO.

45

a control. PF14-siRNA nanoparticles demonstrated EC50 of less than

15 nM in both serum and serum-free media. In kinetic studies, more

than 70% knock-down was observed in the first two hours, signifi-

cantly surpassing Lipofectamine RNAimax®, which the most opti-

mized commercial vector. Interestingly, the RNAi effect was main-

tained up to 4 days confirming sustainable activity of the nanoparti-

cles. Gene knock-down was also demonstrated in the hard-to-transfect

Jurkat suspension cell-line.

Next, we used the solid dispersion method developed in paper II

to formulate the siRNA nanoparticles in to a solid form. The solid

formulations based on mannitol displayed RNAi activity similar to the

freshly-prepared nanoparticles. We used a new technique called the

nanoparticle tracking analysis (NTA) to characterize the particles and

determine the effect of formulation and different media on the particle

size. This method enables tracking of individual particles and thus

overcoming the low resolution problem of DLS. In the NTA meas-

urements, the mean particle size was not significantly different be-

tween the freshly prepared formulation and after solid dispersion ei-

ther in water or serum-containing medium demonstrating that the na-

noparticles remain intact in different media and withstand the drying

and dispersion conditions. Zeta potential measurements revealed a

similar pattern to PF14-SSO complexes; i.e. positive in water and

negative in biorelevant serum-free or serum-containing media. This

indicates that SCARAs might be responsible for the uptake of PF14-

siRNA nanoparticles as well. This was supported by preliminary re-

sults of the SCARA ligands decreasing the RNAi activity of the nano-

particles (unpublished data); however, this needs to be confirmed

more thoroughly.

The most obvious use of a solid formulation is for oral delivery,

and it would be very convenient for patients if it is feasible to deliver

siRNA therapeutics in tablets and capsules. However, the extremely

acidic gastric environment will degrade the siRNA as is soon it reach-

es the stomach via acid hydrolysis. Thus, we wanted to test if the

complexation of siRNA in PF14 nanoparticles can protect it from gas-

tric acidity and whether the nanoparticles can survive gastric proteo-

lytic enzymes. To do that, we incubated the nanoparticles either fresh-

ly-prepared or in solid formulation with SGF for 30 min in the pres-

ence or absence of pepsin. To determine the residual activity, we add-

ed the nanoparticles to the cells after SGF incubation and measured

46

the RNAi response. This method is much more informative than other

analytical methods such as gel electrophoresis or mass spectroscopy

because it shows the residual biological response, which is most im-

portant for any formulation. Interestingly, the nanoparticles were able

to protect the siRNA in these conditions and mediated RNAi response

close to normal. However, when incubated for more than 30 min, pep-

sin started to deteriorate the activity (data not shown), which indicates

the requirement of further protection. Nevertheless, the ability of a

simple, one-step system like PF14 to protect siRNA from acidic deg-

radation is remarkable. This might be due to efficient complexation

and condensation of the siRNA cargo in a confirmation that protects it

from hydrolysis. To confirm the integrity of the particles in SGF,

NTA measurements were performed. The nanoparticles remained in-

tact in SGF with/without pepsin despite displaying a slightly larger

size compared to incubation in water or transfection media. These

results open the door for testing oral CPP-based siRNA therapeutics in

animal models, towards developing convenient drug products for this

new class of therapeutics.

47

5. Conclusions

This thesis describes two new chemically modified CPPs that

were used to deliver nucleic-acid based molecules in various gene

therapy approaches. We clearly demonstrate that these peptides form

stable nanoparticles with the negatively charged nucleic acid cargos

and are efficiently internalized in several cell-types. The nanoparticles

were also active in solid formulation and in simulated gastric condi-

tions and thus can be suitable for different routes of drug administra-

tion. Importantly, we demonstrated that PF14-SSO nanoparticles pos-

sess negative surface charge at biorelevant conditions and that

SCARA3 and SCARA5 are involved in their uptake, suggesting a

putative role of the nucleic acid cargo in the uptake process of such

nanoparticles.

Paper I: stearyl-TP10 (PF3) is an efficient vector for the deliv-

ery of plasmids in-vitro in various cell-lines and in-vivo after i.m. and

i.d. injection.

Paper II: Describes a new CPP, PF14, for delivery of SSOs in

different cell models including mdx mouse cell-line, an in-vitro model

for Duchenne’s muscular dystrophy. A method to formulate PF14-

SSO nanoparticles into a solid form was also developed.

Paper III: Highlights the effect of medium conditions on the

surface charge of PF14-SSO nanoparticles and demonstrates the role

of class A scavenger receptors in their uptake.

Paper IV: Demonstrates the activity of PF14 to deliver siRNA

to various cell types in solution and in solid formulation. Furthermore,

PF14-siRNA nanoparticles are shown to be stable in simulated gastric

conditions.

48

6. Acknowledgements

- First of any acknowledgements goes to God (Allah) for His in-numerable blessings and His guidance for me to the right path.

- To Ülo, my supervisor who has given me the chance to do sci-

ence and kindly and patiently helped me to figure out my way. Thank you so much.

- To my mother and father who have lived their entire lives for

me and my brother to be happy. I owe everything to them. And my brother from whom I learn endurance and perseverance.

- To my soul mate, my most beloved person, my dear wife. She

has been my friend, sister and wife since I met her, and I could never imagine my life without her beautiful face or her loving heart. We now have Yasser, our son, who has given me some-thing to work for the rest of my life.

- To Samir EL-Andaloussi, for teaching me everything from us-

ing a pipette to writing a paper. For involving me in many projects and believing in me at times I didn’t believe in myself. For his help with everything and for being a spectacular friend. Thanks a lot.

- To my mother in law, for her trust in me and her love and help.

She’s done a lot for the success of our marriage and still doing a lot to keep us happy. Thank you.

- To Dr. Hussain El Sawalhy, who made me understand phar-

maceutics and biopharmaceutics. Without his teaching I would have never been able to make this thesis. Thank you.

- To Johan Runesson, for his continuous help and cheerful face.

49

- To Staffan Lindberg, my sincere friend and colleague, we spent very nice time together and we have done nice science. I hope we can continue with that.

- To Andrés Muñoz-Alarcón, who is silent sometimes, funny all

the time and a true friend. Shokran Al Azeem Al Fattah Al Bab.

- To Henrik Helmfors, for all the nice discussions that we had and all the nice experiments we did together. I wish you the best of luck.

- To all Ülo’s group members that were here or still here includ-

ing: Peter Guterstam, Kristin Karlson and Jonas Eriksson for the team spirit and continuous help.

- To Prof. Edvard Smith for the great opportunity of collabora-tion and his help and advice.

- To Eman Zaghloul, for the work we’ve done together in two papers of this thesis and her great efforts. I hope we can con-tinue to collaborate even when we go back to Egypt.

- To all my collaborators, thanks a lot, this work couldn’t have

seen light without your crucial contributions.

- To all the people working in the department of neurochemis-try, thank you for making the best working place ever, and thank you for being part of my everyday delight.

50

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