Nanoparticles Of PLGA With Encapsulated InsulinFor Oral Controlled Release For Diabetes Treatment

Item Type text; Electronic Thesis

Authors Abduljawad, Marwan

Publisher The University of Arizona.

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1

NANOPARTICLES OF PLGA WITH ENCAPSULATED INSULIN FOR ORAL CONTROLLED RELEASE FOR DIABETES TREATMENT

By

Marwan Abduljawad

____________________________

A Thesis Submitted to the Faculty of the

DEPARTMENT OF CHEMICAL AND ENVIRONMENTAL ENGINEERING

In Partial Fulfillment of the Requirements

For the Degree of

MASTER OF SCIENCE CHEMICAL ENGINEERING

In the Graduate College

THE UNIVERSITY OF ARIZONA

2015

I

STATEMENT BY AUTHOR

The thesis titled Nanoparticle of the PLGA Encapsulated Insulin for Oral

Controlled Release for Diabetes Treatment prepared by Marwan Abduljawad has been

submitted in partial fulfillment of requirements for a master’s degree at the University

of Arizona and is deposited in the University Library to be made available to borrowers

under rules of the Library.

Brief quotations from this thesis are allowable without special permission,

provided that an accurate acknowledgement of the source is made. Requests for

permission for extended quotation from or reproduction of this manuscript in whole

or in part may be granted by the head of the major department or the Dean of the

Graduate College when in his or her judgment the proposed use of the material is in

the interests of scholarship. In all other instances, however, permission must be

obtained from the author.

SIGNED: Marwan Abduljawad

APPROVAL BY THESIS DIRECTOR

This thesis has been approved on the date shown below:

Defense date

Roberto Guzman 12-10-2015

Professor of Chemical Engineering)

II

ACKNOWLEDGMENTS

It is very hard to acknowledge all the people who tried to support me, especially,

my parents and my wife who would not mind to give up everything for me.

I would like to thank King Abdulaziz City for Science and Technology (KACST),

Dr. Turki bin Saud bin Mohammad Al Saud, the President of (KACST), and Dr.

Hamid Almiqren for offering my the scholarship to get my master degree.

I would like also to express my gratitude to Dr. Guzman who puts a lot of effort to

guide me in this field of research and in classes.

Finally, I would like to thank almost everyone who worked with me in our lab

especially Andre, Abdullah, and David.

III

TABLE OF CONTENTS

LIST OF FIGURES ......................................................................................................... 1

CHAPTER 1. INTRODUCTION ...................................................................................... 5

1.1. Nanotechnology in therapeutics ........................................................................... 6

1.2. Nanoparticles in medicine .................................................................................... 6

1.3. Drug delivery ........................................................................................................ 8

1.4. Mathematical modeling of drug release from biodegradable nanoparticles..........12

1.5. Insulin and diabetes ............................................................................................16

1.6. Thesis description ...............................................................................................30

CHAPTER 2. NANOPARTICLES OF PLGA WITH ENCAPSULATED INSULIN FOR

ORAL CONTROLLED RELEASE FOR DIABETES TREATMENT .............................. 31

2.1. Abstract ...............................................................................................................32

2.2. Introduction .........................................................................................................33

2.3. Materials and Methods ........................................................................................34

2.3.1. Materials .......................................................................................................34

2.3.2. Methodology for INP (Insulin Nanoparticles)Preparation ..............................34

2.3.3. Nanoparticles size ........................................................................................36

2.3.4. Insulin release studies ..................................................................................36

2.4. Results and Discussion .......................................................................................38

2.4.1.Insulin release results ....................................................................................38

2.4.2.Imaging and the size of the particles ..............................................................39

IV

2.4.3. Myoglobin release studies ............................................................................40

2.5. Conclusions ........................................................................................................41

CHAPTER 3. NANOPARTICLES OF PLGA WITH ENCAPSULATED

TRYPSIN:KINETICS AND ENZYME ACTIVITY ........................................................... 43

3.1. Abstract ...............................................................................................................44

3.2. Introduction .........................................................................................................45

3.2.1.Model Enzyme-Trypsin ..................................................................................45

3.2.2.Chemistry and function ..................................................................................45

3.2.3.Enzyme encapsulation ...................................................................................46

3.3. Trypsin encapsulation in PLGA nanoparticles .....................................................49

3.3.1. Materials .......................................................................................................49

3.3.2.Methods .........................................................................................................49

3.3.3.Release studies .............................................................................................50

3.3.4.Enzymatic activity ..........................................................................................50

3.4. Determination of kinetics .....................................................................................51

3.4.1.Materials ........................................................................................................51

3.4.2.Methods .........................................................................................................52

3.5. Results and Discussion .......................................................................................53

3.5.1.Determination of kinetics ................................................................................53

3.5.2.Release study ................................................................................................53

3.6. Conclusions ........................................................................................................57

CHAPTER 4. Conclusions .......................................................................................... 58

REFERENCES ............................................................................................................. 61

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List of figures

Figure 1. Computer-generated image of six insulin molecules assembled in a hexamer (Kyriakou, 1994)………………………………….…………………………16

Figure 2. The insulin secretion mechanism in detail (Novak, 2008)………..……18

Figure 3. The reactions of phosphorylation and dephosphorylation of enzymes, part of almost all biological processes. E=enzyme, P = phosphate (Espinal, 1989)…………………………………………………………………………………….19

Figure 4. The tyrosine kinase receptor indicating the external and internal sites it consists of, as well as the conformational changes undertaken. (Siddle, 1992)…………………………………………………………………………………….20

Figure 5. Total number of injections per day for 300 patients: 40% more than 4. 20.1% 3 injections. 34.7 2 injections. Other, only one injection……………..……22

Figure 6. Survey with diabetes patients prefer to take the insulin orally or by injections. Apparently, 80.5% prefer to take the medicine orally instead of injections………………………………………………………………………………..22

Figure 7. Insulin secretion in B cells and natural mechanism. Insulin is produced at a more or less constant rate regardless of blood glucose levels. And stores inside the gaps to prepare for excretion by the cell output (Novak, 2008)…………………………………………………………………………………….24

Figure 8. Organisms that affected by diabetes (Diabetes association in US)..…25

Figure 9. Statistics about the number of people who has been affected by diabetes around the world (Diabetes association in US)………………….………27

Figure 10. Alternative routes for the delivery of insulin (Owens, 2002).………....28

Figure 11. Summary of the alternative insulin delivery products being developed (Taylor and Shota, 2012).……………………………………………………….……29 Figure 12. Scheme of synthesis of Insulin loaded polymeric nanoparticles using a double emulsification technique…………………...…………………………………35

Figure 13. Insulin release for different concentrations at 37 oC…………………..38

Figure 14. SE SEM images of INP using 4800 FESEM with different magnitude, same working distance 9.0mm and AV was 5 kv. A. 70k B. 80k C.110k………..40

Figure 15. Myoglobin drug release for different concentrations at 37 oC…..……41

2

Figure 16. X-ray crystallographic structure of Trypsin (Kühne,1976)……....…..46

Figure 17. Storage time of a laccase-modified paper is enhanced (Rochefort, 2013)…………………………………………………………………………………….47

Figure 18. Eadie-Hofstee plot…………………………………………………...……51

Figure 19. Table Top Schematic of Lab Workspace…………………..………..…52

Figure 20. Kinetics data for different concentration of trypsin and a constant substrate concentration ………………………………………………………...…….53

Figure 21. Absorbance kinetic activity of released from encapsulated enzyme system ………………….………………………………………………………...…….55

Figure 22. Trypsin release behavior from PLGA nanoparticle…………….……...55

Figure 23. Kinetics of samples that were took after releasing study …………….56

3

Abstract

Insulin, a relatively low molecular weight protein has been used for decades in

the treatment of diabetes; it has well-defined properties and delivery

requirements. Due to the current increase of diabetes in the world improved

insulin delivery systems could significantly influence the treatment of diabetes

and the quality of life of the affected people.

The main objective of this work was to encapsulate insulin in polymer

nanoparticles of Poly (DL-Lactic-Co-Glycolic Acid) (PLGA) and poly vinyl alcohol

(PVA). Preliminary results of these functional therapeutic nanoparticles prepared

with PVA and PLGA by using a double emulsion method (water/oil/water) were

obtained in terms of encapsulation efficiency and effective insulin release frm the

nanoparticles. Assessing the bioactivity of insulin once encapsulated and

released is not trivial, thus an indirect protein assay was developed to effectively

and easily assess the activity of proteins going through these processes. Trypsin,

a proteolitic enzyme was used as model protein to investigate the biological

activity of encapsulated and released biomolecules. The activity of trypsin

towards a synthetic substrate, DL-BAPNA was used to measure the enzyme

kinetics and activity before encapsulation, while encapsulated and after the

enzyme was released from the nanoparticles. Results show that the enzyme

maintained substantial activity while encapsulated and after its release It is

anticipated that the biological activity after being released from the nanoparticles

will remain biologically active, however, biological assays remain to be performed

4

to corroborate this argument. In addition to release experiments with trypsin and

insulin, other proteins were also studied. In all cases the release form the

nanoparticles at 37 oC exhibited a three stage release process, The release

process will be modeled according to developed mathematical models that

consider initial burst of molecules, degradation of polymer and diffusion of

molecules from the nanoparticles.

5

CHAPTER 1. INTRODUCTION

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1.1 Nanotechnology in therapeutics

Nanotechnology is considered now a traditional and relevant multidisciplinary

practical field that includes the engineering of nanoobjects or nanotools of <500

nanometers in size (Cuenca et al., 2006). The understanding of nanotechnology

values has been consistently growing and has been accepted as a form of

control in the construction of matter among atoms and molecules at the

nanoscale. This understanding has been used to produce exclusive nanoscale

systems, and to propose new and enhanced materials for many different

applications. One of those applications is the use of nanotechnology in

therapeutics, where one of the most important applications is associated to

diabetes research. The curiosity and importance of the research in nanomedicine

has received immense attention driven by the need to find new and effective

approaches to solve major public health problems, like diabetes, cancer,

cardiovascular and infectious diseases. Nanomedicine is related to the

application of nanodevices and tools in the action and analysis of many different

diseases. The different nanodevices, for example in drug delivery for diabetic

therapy, are designed principally from polymeric systems (Cuenca et al., 2006; Z.

Liu et al., 2010). These materials could be structured to perform a specific activity

or activities, and also to contain explicit characteristics.

1.2 Nanoparticles in medicine

Nano technology has been used in medical fields over decades and as of now it

is called nanomedicine. In 2006, the European Science and Technology

7

Observatory showed that more than 150 companies are using nano technology

in nanoscale therapeutics, and around 24 applications have been approved to be

used in clinics with total exceeding sales of $5.4 billion (L. Zhang, 2006).

According to king Abdullah center for science and technology, nanotechnology

helped in medical field to disease prevention, diagnosis and treatment of medical

rules. Nowadays, we live in a nanoparticles medical technology era, where the

progress of nanotechnology, for example, could provide new ways to carry

medicine inside the human body (nano-carriers) and be able to target different

cells in the body. Nanotechnology techniques could image the body's cells as

easily as if we take now a common image. Nanomedicine could use many kinds

of nanoparticles in medical applications so that they work as carriers of drug or

tools for imaging inside the body. In Therapy applications, for example, gold

nanoparticles can be used to destroy cancer cells, the total diameter of these

nanoparticles is about 120 nm, a smaller than the size of the cancer cell by 170

times. Target therapy involves specific ligands attached to gold nanoparticles

such that they attach specifically to cancer cells, and then when exposed to a

NIR laser beam the heated gold produces a temperature increase which leads to

the burning of those cells and death. The advantage of this method is its potential

accuracythus decreasing the risk to damage healthy cells. One promising

medical applications of nanotechnology is to use of polymer nano-fibers to make

prosthetics blood vessels and recently applications have been proposed to

produce prosthesis devices made of nanoscale protein fibers in the central

8

nervous system of the human person. Similar polymer nano-fiber are been used

in the treatment of burns and wounds and intervention in the cosmetics industry.

In imaging analysis injection of cadmium selenide nanoparticles (quantum dots)

within the body could accumulate within cancer cells selectively and after

exposure to a target region of ultraviolet light, the particles light up, helping to

determine the site of malignant cells and allowing the possible and effective

removal... Nano scale devices and nanotechnology can potentially offer many

platforms to develop artificial bones and tissues. Nanotechnology could play a

big role in improving tissue engineering and cell therapy for many biomedical

applications. . There are attempts in the literature to use silicon nanocapsules

that could work to stop the body's immune system that recognize foreign cells,

while these capsules could block antibodies produced by the body's immune

system while they launch oa sufficient amount of insulin with the capsules

nanoparticles in the blood.

1.3 Drug Delivery

The delivery of drugs and other therapeutic compounds is a subject of extensive

attention because of the different applications in health-related issues. The

universal market for progressive drug delivery systems is projected to approach

$175.6 billion by 2016, this is a rise of $44 billion over the 2010 market (Santini,

2013). The objective of drug delivery is to provide the accurate amount of drug in

the right place, at the right time and for the right length. Although a development

in the suitability of drug administration is wanted, it cannot come at the expense

9

of security or efficacy (Santini, 2013). The approaches of drug delivery are

diverse, from oral, transdermal, pulmonary, implantable, etc. From all the

approaches of drug delivery, nanoformulations are the ones that have received

more consideration in the last years. Appropriately engineered nanoformulations

as drug transporters (nanocarriers) can rise the contact of cells/tissues/tumors to

therapeutic agents. This can result in improved action effects by prolonging

circulation times, protecting entrapped drugs from degradation, and enhancing

tumor acceptance through the enhanced penetrability and retention effect as well

as receptor-mediated endocytosis. Several therapeutic agents such as

chemotherapy, antiangiogenic, or gene therapy agents can be simultaneously

encapsulated and transported by nanocarriers to tumor sites to enhance the

effectiveness of therapy and as such have received generalized attention (Peer

et al., 2007; Shi et al., 2010). These nanosystems could be tuned to offer several

advantages over free drugs, for example, they could protect the drug from

undesired interactions with other organic tissues, as well as target exact tissues,

increasing the desired contacts. In addition, by using nanosystems for drug

delivery, the drug release from the matrix could be effectively controlled to deliver

only needed amount of drug (Danhier et al., 2012; Makadia and S. J. Siegel,

2011; Mu and Feng, 2003; Peer et al., 2007). The nanosystems should also meet

numerous features, for example, they should be made from biocompatible and

biodegradable resources and processing should not be a difficult task (Mu and

Feng, 2003; Peer et al., 2007). Polymeric nanoparticles are defined as nanoscale

10

drug delivery platforms exemplified by biodegradable polymers, dendrimers, and

micelles (Zolnik et al., 2010). Several polymers can be used to produce

polymeric nanoparticles include: poly(amides), poly(amino acids), poly(esters),

poly(ortho esters), poly(urethanes), and poly(acrylamides), etc. (Jain, 2000). A

summary of pharmaceutically used polymers with respect to their

physicochemical features and factors moving drug delivery capabilities, as well

as the essential drug delivery systems are stated in the literature (Jain, 2000;

Khandare and Haag, 2010; Liechty et al., 2010; Moghimi et al., 2001; Parveen

and Sahoo, 2008). Among the materials used in drug delivery, one of the most

effective is the poly(ester): poly-d,l-lactide-co-glycolide (PLGA), which shows

immense potential as a drug delivery carrier and as support for tissue

engineering (Danhier et al., 2012; Makadia and S. J. Siegel, 2011). PLGA is one

of the most used biodegradable polymers for the development of nanomedicines

because it undergoes hydrolysis in the body to produce the biodegradable

metabolite monomers, lactic acid and glycolic acid, which at the same time are

biodegraded to CO2 and water (Danhier et al., 2012; Jain, 2000; Kumari et al.,

2010). Depending of the ratio of these monomers, PLGA will show different

biodegradation rates, structural and mechanical properties (Danhier et al., 2012;

Jain, 2000). Another benefit of PLGA is that it covers a large number of

reachable carboxyl groups that allow their easy functionalization with other

molecules prior and after nanoparticle preparation. Additionally, PLGA is an

accepted polymer by the Food and Drug Administration (FDA), and the European

11

Medicine Agency (EMA) and as such is already used in a host of therapeutic

devices due to their biodegradability and biocompatibility (Danhier et al., 2012;

Parveen and Sahoo, 2008).

PLGA nanoparticles have been prepared by several methods, the most common

procedure is the emulsification–solvent evaporation technique (Danhier et al.,

2012; Grazia Cascone et al., 2002; Jain, 2000; J. Yang et al., 2007). However,

other approaches are also applied, like emulsification-solvent diffusion (Hideki

Murakami et al., 2000; Sahana et al., 2008), nanoprecipitation (Chorny et al.,

2002; Dhar et al., 2008), etc. Practically, any hydrophobic or hydrophilic drug

could be measured for encapsulation into polymeric nanoparticles. The

encapsulation of cytotoxic chemotherapeutic agents in biodegradable PLGA

nanoparticles may offer advantages over other delivery systems, including

liposomes (Jason Park et al., 2009). Several anticancer drugs and other different

compounds have been encapsulated into PLGA nanoparticles using these

methods for the synthesis of nanoparticles. For example, paclitaxel, doxorubicin,

5-fluorouracil, 9-nitrocamptothecin, cisplatin, triptorelin, dexamethasone, etc.,

have been successfully encapsulated on PLGA nanoparticles (Kumari et al.,

2010).

12

1.4 Mathematical modeling of drug release from biodegradable

nanoparticles

The drug release from biodegradable nanoparticles is a difficult problem that

contains many issues, for instance the temperature, the size and shape, the

molecular weight, pH, the dissolution of oligomers inside the matrix, the drug

solubility, amount and location of the drug, drug-drug interactions, polymer-drug

interactions, etc. For example, the trial release of doxorubicin at different

temperatures from poly(N-isopropylacrylamide-acrylamide-allylamine)-coated

magnetic nanoparticles showed that temperature shows a important part by

growing the release rate with a temperature increase (Rahimi et al., 2010). In the

same regard, the degradation rate of PLGA microspheres was found to rise with

a rise in incubation temperature (Dunne et al., 2000). As temperature, other

many aspects that disturb the drug release has been examined in the literature,

but in general there are only three possible behaviors for drug molecules to be

released from a PLGA-based drug delivery systems: (i) transport through water-

filled pores, (ii) transport through the polymer, and (iii) release due to dissolution

of the encapsulating polymer (which does not require drug transport)

(Fredenberg et al., 2011). The usual shapes of release found from PLGA

nanoparticles rely on all the factors stated above, and could hold one, two or

three phases of release, but frequently at least a biphasic behavior is observed.

These phases of release are straight connected to the three ways mentioned

above for drug molecules to be released from PLGA-based drug delivery

13

systems. The transport though water-filled pores could be defined essentially by

diffusion through the nanoparticle, but also it is related to an initial drug burst

release, a process of interfacial diffusion between the solid sphere surface and

the liquid media, such that the drug release rate is considered relative to the

difference between the concentration of drug in the sphere most external polymer

layers, and the concentration of drug in the close to liquid media at the time of

investigation. This phenomenon is also the result of other issues such as the

concentration of drug on the surface, the surface area, the interphase properties,

the solubility of the drug, and the electrostatic interactions between the drug and

the solid polymer matrix.

The nanoparticle degradation – relaxation phenomenon is related to the

degradation by hydrolysis of the PLGA in the aqueous solution. In this stage, the

nanoparticle interphase with the liquid is the first portion exposed to hydrolysis,

and is related to the surface area, and the molecular weight of the polymer.

Initially, the nanoparticle is organized in a very compact way. As time increases,

the polymer in the nanoparticle surface is disjointed in small oligomers by

degradation, promoting water penetration, and consequently nanoparticle

relaxation. Previous work demonstrates that a linear relationship between the

degradation rate and the particle size exists, with the larger particles degrading

the fastest; this is explained in the sense that larger particles have to deal with

longer paths of diffusion of its oligomers to reach the surface of the particle

compared to small particles (Dunne et al., 2000). As the degradation continues

14

from the nanoparticle surface, new paths for water penetration are created. At

this point, the degradation of the nanoparticle could follow two simultaneous

routes, one reducing the radius of the nanoparticle, and the other one increasing

the porosity of the nanoparticle. As the porosity increases, the nanoparticle gets

more relaxed by the water penetration and the hydrolysis now could continue in

several directions simultaneously. The PLGA nanoparticle degradation –

relaxation effects over the drug release has been explained with a first order

equation for the change in the molecular weight of the polymer with respect to

time (Deng et al., 2008; Mohammad and Reineke, 2013), and also by using a

drug release a relationship known as the Prout-Tompkins equation (Dunne et al.,

2000; Fitzgerald and O. I. Corrigan, 1993), which was initially introduced to

describe the thermal decomposition of potassium permanganate (Prout and

Tompkins, 1944). Drug release kinetics from biodegradable devices have been

described in the literature by combining and using different mechanisms of

release (Fredenberg et al., 2011; Lao et al., 2011). Mathematical models of drug

release include processes that consider first order rates for the initial burst

release combined with a second stage that considers bulk degradation of the

polymer in microparticles (Gallagher and O. I. Corrigan, 2000) and nanoparticles

(Owen I. Corrigan and Xue Li, 2009). In these particular cases the bulk

degradation of the polymer was considered by using also the Prout-Tompkins

equation (Fitzgerald and O. I. Corrigan, 1993; Prout and Tompkins, 1944). In

other mathematical models reported in the literature, the mechanisms of release

15

involve a combination of first order rate initial burst with diffusion of the drug

through polymeric microspheres (Batycky et al., 1997; He et al., 2005; Raman et

al., 2005). Other more complex models of release from biodegradable polymeric

microspheres consider three mechanisms: drug diffusion, drug dissolution, and

polymer erosion and are described by three equations that consider drug

concentration in the liquid phase, a virtual solid phase, and an effective solid

phase (M. Zhang et al., 2003). Similar complex systems model the entire drug

release profile as the summation of the three mechanisms: first order burst

release, first order bulk degradation of the polymer, and diffusional release, but in

consecutive time periods for each release stage (Lao et al., 2009). The work in

this thesis describes the use and adaptation of a mathematical model that

incorporates the simultaneous contribution of three phases of controlled release

and used to effectively describe the release of insulin, myoglobin and trypsin

(Lucero-Acuna, 2013).

16

1.5 Insulin and Diabetes

Insulin is a hormone protein, it consists of 51 amino acids divided into two series

A and B combine them bridges of sulfur, and has molecular weight of 5808 Da.

The Canadian physician Frederick Banting along with medical student Charles H.

Best, isolated insulin from pancreatic extracts of dogs in 1921 at the University of

Toronto in Canada. Banting together with the Scotish physiologist J.J.R. Macleod

were awarded the Nobel Prize in medicine in 1923 for this discovery. In 1969, the

crystal structure of insulin was determined by Dorothy Hodgkin, who was

awarded in 1964 the Nobel Prize in Chemistry.

Insulin is produced by the beta cells in pancreas of Langerhans cells and then

pass directly into the bloodstream to affect the target cells of the liver and muscle

cells and other cells where regulates build of carbohydrates from sugar and

starch. It helps to regulate sugar level in our bodies (100-140 mg/dL). When

regulating the insulin fails by itself, then the patient needs to get insulin for the

treatment. As a consequence, insulin is used to treat some type of diabetes

Figure 1. Computer-generated image of six insulin molecules assembled in a hexamer (Kyriakou, 1994)

17

which is called type 1. People with diabetes do not have enough insulin or suffer

from lack of it (Type 2) entirely must calculate doses of insulin every day.

Injection of insulin is administered under the skin and cannot be taken by mouth

because the stomach juices may digest it. Therefore, these people will use

insulin injections to allow their bodies to use the glucose and to avoid

hyperglycemia, high blood sugar.

How is insulin secreted from the pancreas?

Insulin is concealed from the human pancreas and its discharge is mainly

activated when large concentrations of glucose are sensed in the blood after a

meal. Its leading role is thus the uptake of glucose by increasing the rate of the

glycolytic pathway - the process where glucose is transformed to other

carbohydrates that are used in the urea cycle or for fatty-acids metabolism.

Glucose initially arrives the β-pancreatic cells by the help of carriers known as

GLUT 2 that transport the sugar crossways the cell film, so the act of glucose

appears to be accountable for most of the following changes assumed. At the

opening, a rise in the ATP/ADP ratio is detected (Hales, 1984), the energy

currency involved in all metabolic pathways. This rise selectively touches ATP-

sensitive K+ ion channels by closing them and thus preventing K+ passage

crossways the cell film (Figure 2). This reduces the charge difference that

already exists between inner and outer membrane - the effect known as

depolarization. In the meantime, a growing electrical conductivity is detected;

lashing the opening of voltage-sensitive calcium-ion channels (Hales, 1984) the

18

opening of this channel lets the entrance of Ca2+ ions into the membrane which

originally bind certain proteins such as calmodulins or synaptotagmins. As a

result, this pushes the densely packed vesicles of insulin to open and issue

insulin by evolving over the membrane of the β-pancreatic cells.

How does insulin work?

There are three main spots reflect the insulin's activities, which are the liver,

muscle and adipose tissue. After eating food, the insulin activity starts its action.

However, on starving, glucagon is active instead of insulin. The insulin is

responsible to raise the rate of glucose oxidation in the liver; at the same time the

glucose concentration is converted into glycogen, the form in which glucose can

be stored in the body (Espinal, 1989). These procedures are reinforced by a

growth in the number of glucose transporters to the skin of membranes which

have an important role, as described above, as well as by a diversity of

Figure 2. The insulin secretion mechanism in detail (Novak, 2008)

19

phosphorylation and dephosphorylation procedures (Figure 3 ) for activation and

deactivation of enzymes (biological catalysts).

In order for an enzyme to be phosphorylated, a PO3-2 group is donated to by

adenosine triphosphate, ATP. Finally, the opposite procedure in the liver,

gluconeogenesis, is inhibited because of glucose oxidation is stimulated by

insulin.

At low carbohydrate concentrations, fatty-acid breakdown in the liver is

decreased, whereas synthesis is enabled since triacylglycerols in the adipose

tissue are transformed to long chain fatty acids. This is preferred due to the

larger availability of the responsible synthesizing enzyme, lipoprotein lipase

(Kelly, 1992). Finally, secondary actions of insulin include stimulation of protein

synthesis (Kelly, 1992) as well as increased blood flow, vasodilatation, and

hypotension (Scherrer, 1999).

All the insulin activities are done by insulin receptors, recognized as tyrosine

kinase receptors. These are 2-subunit receptors and consist of both an

extracellular domain for insulin to bind as a ligand as well as an intracellular part,

insulin protein kinase (Siddle, 1992) where all phosphorylation events occur.

Figure 3. The reactions of phosphorylation and dephosphorylation of enzymes, part of almost all biological

processes. E=enzyme, P = phosphate (Espinal, 1989)

20

Such as most of polypeptide hormones, upon binding, conformational changes

are undertaken on the two subunits and a sequence of phosphorylation actions

advance through, leading to more of the activities hormones perform (figure 4 )

In addition, all of these activities and gesturing paths are attended by the creation

of second messengers such as cyclic adenosine phosphate, cAMP. Second

messengers are molecules that scatter material everywhere around tissues. The

reduction in the concentration of cAMP is the reason for insulin's activities and

this is because of insulin overpowering the precursor molecule, adenylate

cyclase, from which the second messenger is synthesized (Kuznetsova, 2003).

There are two types of insulin to treat diabetes, human insulin and Insulin

analogue, which are manufactured industrially. In fact, most types of insulin

available concentrations are dosage forms of 100 units / ml. Insulin is divided into

five types of insulin; this division depends on how fast is its effectiveness in the

body after injection:

Figure 4. The tyrosine kinase receptor indicating the external and internal sites it consists of, as well as the

conformational changes undertaken. (Siddle, 1992)

21

1. Very fast-acting insulin in the cells (immediate).

2. Fast-acting insulin (regular).

3. Average insulin effect.

4. Slow-acting insulin (long-term).

5. Mixed insulin, another type consists of the installation of the previous

types of insulin at different rates to provide fast and long-acting effect at

the same time.

Nowadays, there are many ways to treat diabetes but none of them are as

effective as injections. The injections allow sending the insulin directly to the

bloodstream by taking it under the skin. However, more than 50% of patients who

use insulin find the injections harmful because sometimes the place of injection in

the body is hardened and can causes bleeding most of the time. Moreover,

insulin injections are very expensive and many people die because they cannot

afford to buy it, since many people need more than one injection a day as you

will see in the survey results shown below.

22

In the first chart, you can see that people take more than 4 injections during 1

day, which is of course overwhelming and more hurtful. Also, it is obvious that

many people prefer to take the insulin orally if available, which in principle might

be more convenient and less harmful than injections and of course it should be

cheaper and more affordable by everybody.

Development of Diabetes

The insulin produced by the pancreas is the primary hormone that regulates the

transfer of glucose from the blood to most of the body's cells, especially muscle

cells and fat cells, but not transmitted to the central nervous system cells.

Therefore insulin deficiency leads the body's response to any style of diabetic

patterns. Most of the carbohydrates in the food shift to single-glucose within a

few hours. This mono glucose is the main carbohydrate in the blood which is

used as fuel in the cells. Insulin is secreted into the blood by beta cells in the

pancreas Langerhans Islands in response to high blood glucose levels after

consuming foods. Insulin is used about two-thirds of the body's cells to absorb

glucose from the blood or for use as fuel for a cell manufacturing operations

Figure 5. Total number of injections per day for 300 patients:

40% more than 4. 20.1% 3 injections. 34.7 2 injections. Other

only one injection

Figure 6. When I asked the patients if they prefer to take the

insulin orally or by injections and it appears that 80.5%

prefer to take the medicine orally instead of injections

23

needed to produce other molecules or for storage. Also, it is the main indicator of

insulin to convert glucose into glycogen for storage in the liver or muscle cells.

And low levels of glucose leads to reduction of insulin secretion from Beta cells

and to reverse the conversion to glycogen, who works in the opposite direction to

insulin. Thus retrieves glucose from the liver into the blood; while lacking the

muscle cells convert the stored glycogen into glucose mechanism. Increased

insulin levels lead to increase construction in the body, such as cell growth,

protein synthesis and storage of fat. Insulin is the main indicator to turn the

direction of many metabolic processes bidirectional from demolition to

construction and vice versa. When the blood glucose level is low, it stimulates

the burning of body fat. If the amount of insulin available is not sufficient, the cells

response is weak to insulin (resistance or immunity against insulin), patients will

not properly absorb glucose from the body cells. Thus, the final result is a

continued high level of blood glucose, protein synthesis and the weakness of

some metabolic disorders such as blood acidification.

24

Diagnosed Type I and many cases of Type II diabetes based on the initial

symptoms that appear at the beginning of the disease, such as frequent urination

and excessive thirst which accompanied by loss of weight, and these symptoms

usually evolve over days and weeks. Diabetes is marked by discontinuous or

persistent high blood glucose can be inferred by one of the following values:

1. Measure the level of fasting blood glucose 126 mg / dL (7 mmol / L) or

higher.

2. Measuring the level of blood glucose 200 mg / dl (11.1 mmol / L) or higher

after two hours of eating 75 grams of glucose as follows in the glucose

tolerance test.

3. Random measurement of blood glucose level of 200 mg / dL (11.1 mmol /

L) or higher.

Figure 7. Insulin secretions in B cells and natural mechanism. Insulin is produced at a rate of more or less constant

regardless of blood glucose levels. and stores inside the gaps to prepare for excretion by the cell output. (Novak, 2008)

25

Chronic complications of insulin insufficiency

Figure 8. Organs affected by diabetes (Diabetes association in US)

One of the most relevant problems encounter by diabetic patients is to get

acidification diabetic ketoacidosis blood, a dangerous condition to have. The lack

of insulin to the liver converts fat into ketone bodies used by the brain as fuel. But

high levels of ketone bodies lead to the low pH of the blood which causes most of

the symptoms of the occurrence of acidification blood ketoacidosis. Also, chronic

increase of blood glucose leads to damage of blood vessels which may cause

blindness. In many cases, people cannot afford to buy the medicine and die

irremediably

According to the diabetes association in the United States, risk of death for

people with diabetes is 50% higher than for people without it. This is risky and

dangerous, and it is important to take action to reduce these statistics. Another

26

reason of death because of diabetes is the cost increase of health care for

diabetic patients. The stats showed that medical costs for people with diabetes

are twice higher than the people without it.

Diabetes around the world

In 2014, 9% of adults aged 18 years or more people with suffering from diabetes.

In 2012, diabetes was the direct cause of 1.5 million deaths. It has been found

that 80% of these deaths are in low- and middle-income countries. It is expected

that by the end of 2015, there will be 371 million people around the world with

diabetes, this is in fact a very large number and shows how serious this disease

is. According to these studies, insulin in the present preparations is still quite

expensive and not everybody can afford it. Thus, in this work and research the

goals are to contribute in the development of novel strategies for insulin delivery

platforms, such as oral delivery. (Diabetes association in the US).

27

Figure 9. Statistics about the number of people affected by diabetes around the world (Diabetes

association in US)

28

Methodologies for Insulin delivery

Figure 10. Alternative routes for the delivery of insulin (Owens, 2002).

Figure 10 shows that there are numerous options of routes for the delivery of

insulin into cells. The pneumonic route may be the first of these to be utilized for

clinical consideration of insulin-requiring patients with diabetes. Different

alternatives for the delivery of insulin incorporate b | entire organ pancreas

transplantation, or c | transplantation of segregated islet cells or hereditarily

adjusted undifferentiated organisms. Different routes for the delivery of insulin

incorporate nasal, oral–gastrointestinal, buccal, rectal, vaginal, uterus, visual and

dermal (Owens, 2002).

The electronic age is helping advance the treatment of diabetes, and this is vital

in light of the fact that the difficulties of diabetes stay regardless of the

accessibility of successful remedial systems for delivery of insulin. These

29

improvements concentrate on the need to convey precisely timed and estimated

measurements for anticipated blood glucose levels (Taylor and Shota, 2012).

Electronic sensors and pump systems are very much considered to as far as

business configuration and clinical studies for this type of closed circle. New

innovations could overcome sensor related and transport problems to coordinate

insulin release with blood glucose amounts that so far may be unsafe and where

different methodologies could alleviate these issues. Effective systems may rise

up out of artificially controlled storage configuration, for example, the preparation

of glucose delicate film controlled through gated pores or by hydrogel that

adjustments as far as its porousness to release insulin as a response to the

presence of glucose (Tanna S, 2006; Zhang R, 2006; Taylor and Shota, 2012).

Figure 11. Summary of the alternative insulin delivery products being developed (Taylor and Shota, 2012).

30

1.6 Thesis description

In this research, following new trends in the nanoparticle encapsulation of insulin,

polymer nanoparticles of Poly(DL-Lactic-Co-Glycolic Acid) (PLGA) and poly vinyl

alcohol (PVA) were used to encapsulate insulin. The appeal of using this polymer

is that the surface can be modified with a large number of ligands and antibodies

that can be used to target specific areas in the epithelial gastrointestinal and thus

make it able to cross this barrier. Preliminary results of these functional

therapeutic nanoparticles prepared with PVA and PLGA by using a double

emulsion method (water/oil/water) are presented. Since the effective activity of

insulin once encapsulated and released is difficult to accomplish in a simple form,

an enzymatic approach to assess activity of biomolecules after encapsulation

and release was applied in this work. The enzyme trypsin was used as a tool to

assess biological activity of biomolecules by measuring its enzymatic activity

before encapsulation, during encapsulation and after its release from

nanoparticles.

31

CHAPTER 2. NANOPARTICLES OF PLGA WITH

ENCAPSULATED INSULIN FOR ORAL

CONTROLLED RELEASE FOR DIABETES

TREATMENT

32

2.1 Abstract

In this chapter, it is described the encapsulation of insulin in nanoparticles of

PLGA and PVA and its release behavior at 37oF using a double emulsion

method. Nanoparticles were prepared with a blend of the biodegradable polymer

(PLGA) and poly vinyl alcohol (PVA). Nanoparticles containing insulin once

prepared were characterized with SEM after purification with centrifugation and

lyophilization. Release studies were performed simulating physiological pH and

temperature conditions. It was observed that following this procedure, insulin was

effectively encapsulated and released from the nanoparticles following expected

behavior from what is reported in the literature for encapsulated drugs. In

addition, myoglobin was also encapsulated and release studies were also

performed in this section, in order to corroborate the encapsulation and release

strategies. The results with myoglobin followed a similar path.

33

2.2 Introduction

While parenteral administration is the only way of insulin distribution; other ways

of administration (oral, nasal, rectal, pulmonary and ocular) have been widely

explored (Zinman, 2003). Among them, the oral way appears to be the most

suitable and physiological because insulin undertakes a principal hepatic

sidestep, thus justifiably the main outcome by preventing hepatic glucose

production. However, insulin is strongly degraded by proteolytic enzymes in the

gastrointestinal tract. In addition, the hormone does not absorb well after oral

administration. In order to avoid biodegradation and to increase its intestinal

absorption, insulin has been connected to antiproteases (Zhang, 2003),

hydrogels (Torjman, 2004), or shared with absorption enhancers such as

cyclodextrins (Chermak, 1994), bile salts (Plakogiannis, 2002) and surfactants

(Takada, 2002). In this chapter, nanoparticles were prepared according to the

double emulsion method mostly used for encapsulation of peptides, proteins and

hydrophilic drugs.

34

2.3 Preparation of Nanoparticles

2.3.1 Materials:

Insulin from bovine pancreas and myoglobin from equine heart were purchased

from Sigma-Aldrich. PLGA acid terminated (50/50 DL-lactide/glycolide

copolymer, IV midpoint 0.2 dl/g) was purchased from Purac Biomaterials,

Gorinchem, The Netherlands. PLGA with composition 50:50, acid terminated,

and a molecular weight of 7,000-17,000 a.m.u. was purchased from Sigma-

Aldrich, Inc. (Milwaukee, WI). Dichloromethane (CH2Cl2) was purchased from

Fisher Scientific Inc. (Fair Lawn, NJ)., and poly(vinyl alcohol) (PVA) with an

average molecular weight of ~31,000 a.m.u. and 86.7-88.7 mol% hydrolysis were

obtained from Sigma Aldrich, Inc. (St. Louis, MO). ADD SONICATOR AND

CENTRIFUGES INFORMATION

2.3.2 Methods

The preparation of nanoparticles was carried out by the multiple emulsion

technique described by Hoffart et al. 2002. Briefly, 2 mL of an aqueous solution

of insulin was first emulsified, by sonification for (4 minutes + 40% amplitude), in

DCM (10 mL), containing 60 mg of PLGA. The resulting water-in-oil emulsion

was thereafter poured into 25 mL of a polyvinyl alcohol aqueous solution (3%)

and sonicated for (3 minutes + 40% amplitude), involving the formation of the

second water-in-oil-in-water emulsion. After evaporation of DCM under reduced

pressure, the nanoparticles were separated and isolated by centrifugation

35

(50,000 rpm for 20 min). The nanoparticles were washed three times with

deionized water then centrifuged again and kept in suspension in water until use.

The nanoparticle preparations were lyophilized and stored until further analysis.

These lyophilized nanoparticles were used to carry out release studies under

physiological pH and temperature. The schematic representation of the methods

using double emulsion is shown in Figure 12.

Figure 12. Scheme of synthesis of Insulin loaded polymeric nanoparticles using a double

emulsification technique.

36

2.3.3 Nanoparticle size characterization

Nanoparticles were observed using a scanning electron microscope (Hitachi S-

4800 Field Emission Scanning Electron Microscope, Tokyo, Japan) from the

Spectroscopy and Imaging Facilities at the University of Arizona.

2.3.4 Insulin release studies

The release of insulin from the nanoparticles was determined using a buffer

solution and sodium azide. (Avgoustakis et al., 2002; Liang et al., 2011; Sahana

et al., 2008). Polymeric nanoparticles dispersed in 2.5 ml of 10 mM sodium

phosphate buffer pH 7.4 were placed in a tube, and incubated in 30 ml of the

same buffer at a temperature of 37 oC. At specific time intervals, 1 ml of samples

was withdrawn from the incubation medium and analyzed for the insulin. Each

volume withdrawn was replenished with 1 ml of 10 mM sodium phosphate buffer

pH 7.4 with sodium azide.

The concentration of insulin into the nanoparticles was determined by standard

calibration curves in 10 mM PBS buffer pH 7. Also, the drug loading was

determined as the mass ratio of drug entrapped in the nanoparticles to the mass

of nanoparticles recovered using the following formula (Lei et al., 2011):

        100   %

           

amount of drug innanoparticlesDrug loading

amount of nanoparticlesobtained bythe process

(2.1)

37

The encapsulation efficiency (EE) is determined as the mass ratio of entrapped

drug in nanoparticles to the theoretical maximum loading, which is taken to be

the point where the entire supplied drug is encapsulated in the nanoparticles, this

is given in the following formula (Jason Park et al., 2009):

             100 %

                   

amount of drug per amount of nanoparticlesEE

amount of drug per amount of polymer used in formulation

(2.2)

38

2.4 Results and discussion

2.4.1 Insulin release studies

Insulin release studies with different concentrations of insulin loaded at 37 oC

were obtained and given in Figure 13. The release data follow characteristic

processes similar to release behavior of hydrophilic drugs, an initial burst

release, a slow release due to particle degradation and a third step characteristic

of diffusion transport. Thus a 3-stage mathematical model will be used to

correlate the insulin experimental release data. Myoglobin was studied in a

similar fashion as insulin to corroborate the encapsulation and release

experiments considering that myoglobin has a distinctive absorption maximum at

410 nanometers.

Figure 13. Insulin release for different concentrations at 37 oC

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0 5 10 15 20

Co

nc.

of

Insu

lin

Time (hr)

Insulin release

39

The experimental results of insulin release shown in Figure 13 were followed for

up to 18 hours, where the different shapes of release can be clearly observed.

This short term drug release analysis suggests that in fact the nanoparticles with

the encapsulated insulin can be used effectively in the application of insulin as a

therapeutic delivery system. The release curves show that approximately 7% of

the drug is released within the first two hours, which means that the initial burst

mechanism plays a clearly defined role of controlled fast initial release. Based

on the experimental curve of release, after the initial burst stage, a slower

release similar to a lag phase is observed, where at this point, the polymer

degradation becomes more important and dominant in the release process due

to the degradation - relaxation of the nanoparticle. In the last stage of the

experimental curve of release, a plateau seems to appear, where the porosity of

the nanoparticle increases to a certain extent, and now the diffusion process

becomes the dominant mechanism of drug release. Based in these results, it is

evident that the three mechanisms should be considered to happen

simultaneously, however in each case, a rate controlling step appears evident.

2.4.2 Nanoparticle size characterization

After lyophilization the particles were coated and analyzed using the 4800

FESEM (Hitachi S-4800 Field Emission Scanning Electron Microscope).. The

views at different magnitude can be seen in Figure 14. For this analysis the same

working distance of 9 mm was used. At 110k shows clearly details of

nanoparticles. According to this analysis particles of this size will be perfect

40

platform for their use in insulin delivery systems. The surface appears to be quite

smooth and uniform making them suitable for surface modifications with

appropriate ligands.

2.4.3 Myoglobin release studies

The encapsulation and release studies with myoglobin followed similar trends as

the ones with insulin, as can be observed in Figure 15. The absorbance of

myoglobin was carried it out at 410 nanometers compared to absorbance of

insulin at 280 nanometers. This distinctive absorbance of this color protein will

help elucidate completion studies of both encapsulation and release of

biomolecules.

Figure 14. SEM images of INP using 4800 FESEM with different magnitudes, same working distance

9.0mm and AV was 5 kv. A. 70k B. 80k C.110k

41

Figure 15. Myoglobin release at different encapsulation concentrations all at 37 oC

2.5 Conclusions

PLGA nanoparticles were prepared effectively by an implemented emulsification

–solvent evaporation technique. The encapsulation of the proteins insulin and myoglobin

was carried out by double emulsification–solvent evaporation techniques. In both cases,

the encapsulation was apparently effective and the release behavior quite similar. A drug

release mathematical model that has been used to model hydrophobic small molecular

drugs will be implemented on the results for the proteins in this analysis, since

apparently the stages of release followed similar paths as the drugs used in the model.

In future work encapsulation and release studies will be performed with these two

proteins to assess competition or complementary behavior. The activity or

remaining biological activity of the proteins is at this point unknown since assays

to assess such activity are not trivial. To find out if the activity of the proteins

0

0.001

0.002

0.003

0.004

0.005

0.006

0 1 2 3 4 5 6

release

42

remains after encapsulation and release, trypsin a proteolitic enzyme was also

incorporated in nanoparticles and release studies were performed as well. The

details and results are presented in the following chapter of this thesis.

43

CHAPTER 3. NANOPARTICLES OF PLGA WITH

ENCAPSULATED TRYPSIN: KINETICS AND

ENZYME ACTIVITY

44

3.1 Abstract

The main objective of the work presented in this chapter was to determine the

activity of biomolecules subject to nanoencapsulation and release at

physiological conditions. The main idea is to relate the activity of an enzyme, in

this case trypsin, although indirectly, to the possible biological activity that insulin

or other proteins would have maintained after encapsulation in nanoparticles and

subject to a release process at physiological conditions. Trypsin was

encapsulated and released at the same conditions as insulin and myoglobin.

However, in this case, the enzymatic activity of trypsin towards a synthetic

substrate DL-BAPNA, was measured before encapsulation in free form and

standard curves were obtained at different enzyme concentrations. Kinetics of

substrate degradation were similarly performed after the enzyme was

encapsulated and after its release. Results show that the enzyme remained

active after the processes of encapsulation and release. This implies that the

process of double emulsion for the preparation of nanoparticles with

encapsulated proteins does not degrade or affects considerably the biological

activity of such biomolecules. The activity results from these studies will provide

an indirect assessment of the state of biological activity of the insulin and

myoglobin used in this work.

45

3.2 Introduction

3.2.1 Model Enzyme-Trypsin

Trypsin is a peptidase enzyme that cleaves peptide bonds of proteins by

hydrolysis to form smaller peptides and amino acids. The optimum pH is 8 and

the optimum temperature is 37 ° C. It is a specific enzyme that binds the peptide

in positions carboxyl residue of arginine (Arg) or lysine (Lys) in the chain, both

positively charged amino acids (Kühne, 1976).

Trypsin is produced by the pancreas in the form of trypsinogen (inactivated

enzyme), and is then activated in the intestinal duodenum by enterokinase

trypsin (active enzyme) by proteolytic cleavage (Kühne, 1976).

3.2.2 Chemistry and function:

The enzymatic mechanism of trypsin is the same as other serine proteases: a

catalytic triad makes the nucleophilic serine in the active site. The enzyme

catalyzed reaction is thermodynamically favorable but has a high activation

energy (that is kinetically unfavorable). The aspartate residue (Asp 189) located

in the catalytic region (S1) of trypsins serves to attract and stabilize lysines and

arginines (both positively charged) and therefore it is the moiety responsible for

the specificity of the enzyme. The trypsin structure and active site is seen in

Figure 16. Trypsins are endopeptidases, that is, the enzymatic cut is performed

in the middle of the peptide chain rather than in the terminal residues of the same

(Otlewski, 2004). This mechanism of activation is very common among serine

proteases, and serves to prevent self-digestion in the pancreas. The trypsin

46

activity is not affected by the inhibitor fenil alanil clorometil cetona (TPCK), which

disables chymotrypsin. This is important because, in some applications, such as

in mass spectrometry, the specificity of the amino acid site cut is important

(Kühne,1976).

Figure 16. X-ray crystallographic structure of Trypsin (Kühne,1976).

3.2.3 Enzyme Encapsulation

Enzymes have been utilized all through mankind's history and today the their

applications as catalysts have a quite significant part in the heart of

biotechnology procedures (Rochefort, 2013).

47

Enzyme activity in solution in their natural environment can be assessed quite

easily if a chromophoric substrate is available. This is the case with the model

enzyme trypsin used in this work. The synthetic substrate DL-BAPNA, after

enzymatic cleavage releases a chromophoric moiety, o-nitro phenol which

absorbance can be measured at 420 nanometers. However, in all cases of

enzyme immobilization or encapsulation it is also important to assess enzyme

structure after encapsulation in light of the fact that if the enzyme structure is

modified, its activity might suffer significantly.

Figure 17 Storage time of a laccase-modified paper is enhanced (Rochefort, 2013).

48

In enzyme encapsulations commonly used with sol–gel encapsulation, the

enzymes are just physically caught, without covalent holding, inside of the

hydrophilic watery environment provided by the silica lattice. In this way most

proteins are not denatured by sol–gel exemplification and their synergist

properties are like that of their water-solvent partners. (Ghorbani and Jafari,

2013). Other systems of encapsulating enzymes is found in mesoporous silica

circles where the means of immobilization, is by gathering a natural/inorganic

nanocomposite shell on the molecule surface with high loadings, high enzymatic

movement and solidness, and insurance from proteolysis (Wang and Caruso,

2004).

49

3.3 Trypsin encapsulation in PLGA nano particles

3.3.1 Materials:

Trypsin from bovine pancreas was purchased from Sigma-Aldrich. PLGA acid

terminated (50/50 DL-lactide/glycolide copolymer, IV midpoint 0.2 dl/g) was

purchased from Purac Biomaterials, Gorinchem, The Netherlands. PLGA with

composition 50:50, acid terminated, and a molecular weight of 7,000-17,000

a.m.u. was purchased from Sigma-Aldrich, Inc. (Milwaukee, WI).

Dichloromethane (CH2Cl2) was purchased from Fisher Scientific Inc. (Fair Lawn,

NJ)., and poly(vinyl alcohol) (PVA) with an average molecular weight of ~31,000

a.m.u. and 86.7-88.7 mol% hydrolysis were obtained from Sigma Aldrich, Inc.

(St. Louis, MO).

3.3.2 Methods

The preparation of nanoparticles was carried out using the same technique for

the encapsulation of insulin and muoglobin, that is, the multiple emulsion

technique described by Hoffart et al., 2002. Briefly, 2 mL of an aqueous solution

of Trypsin was first emulsified, by sonification for (4 minutes + 40% amplitude), in

DCM (10 mL), containing 60 mg of PLGA. The resulting water-in-oil emulsion

was thereafter poured into 25 mL of a polyvinyl alcohol aqueous solution (3%)

and sonicated for (3 minutes + 40% amplitude), involving the formation of the

second water-in-oil-in-water emulsion. After evaporation of DCM under reduced

pressure, the nanoparticles were isolated by centrifugation (50,000 rpm for 20

50

min). The nanoparticles were washed three times with deionized water then

centrifuged again and kept in suspension in water until use. Because we need

our particles dry, we freeze them for 24 hours followed by lyophilization for

storage and further analysis.

3.3.3 Release studies

The release of trypsin from the nanoparticles was determined using Tris-buffer

solution and a substrate solution made of DL-BAPNA (obtained from Sigma-

Aldrich) (Avgoustakis et al., 2002; Liang et al., 2011; Sahana et al., 2008).

Polymeric nanoparticles dispersed in 20 ml of Tris-buffer with 2 ml of substrate

solution placed into a tube, 2ml samples were withdrawn from the incubation

medium and analyzed for the trypsin. Each volume withdrawn was returned to

the test tube in order to always obtain the correct enzyme activity.

3.3.4 Trypsin enzymatic activity studies

Determination of enzymatic activity upon nano particle encapsulation:

The enzyme activity of trypsin was obtained by determining the kinetic

parameters of the reaction using an spectrophotometric analysis performed for

the natural free enzyme preparations before encapsulation, during encapsulation

and after being released from the nanoparticles.

The analysis of enzyme activity follows, as is the case for trypsin in this work,

Michaelis - Menten behavior. The kinetics follows the equation

51

Where the variation of the parameters Vmax and KM help detemine the effective

enzyme activity. In future work this will provide a tool to determine quantitatively

(1) evidence of the effectiveness of encapsulation and (2) remaining biological

activity of biomolecules (enzymes or proteins). These results will help determine

indirectly the expected biological activity of insulin upon nanoencapsulation. Vmax

and KM can be obtained for example with the Eadie-Hofstee plot, schematically

showing here the parameter estimation values.

Figure 18. Eadie-Hofstee plot

3.4 Determination of Enzyme Kinetics

3.4.1 Materials

Tris-hydroxymethyl-aminomethane (tris) as the working buffer. Prepared by

dissolving 3.03 gr of tris in 450 ml of DI water, adjust the pH to 8.1 with 3 N HCl

and dilute to 500 ml DI water. The substrate , Nα-Benzoyl-DL-arginine-p-

nitroanilide (DL-BAPNA) was prepared by dissolving 43.5 mg of DL-BAPNA in 10

52

ml of DMSO (Dimethyl Sulfoxide), since it is not quite soluble in water . The

enzyme solution was prepared by dissolving 2.4 mg of trypsin in 10 ml of tris-HCl

buffer, the stock solution.

3.4.2 Methods

Pipette to the reference cell of spectrophotometer 2.5 ml of buffer. To the sample

cell add an aliquot of the substrate solution (250 µl) and dilute with buffer to 2.4

ml. Then, zero the spectrophotometer at 410 nm, and at 25 oC. At this point in

time, add 100 µl of the enzyme solution (Trypsin) to sample cell and at time equal

zero start measuring the increase in absorbance. Repeat these steps for each

100 µl increments of enzyme solution. The working environment is shown

schematically in Figure 19.

Figure 19. Table Top Schematic of Lab Workspace

53

3.5 Results and discussion

3.5.1 Determination of enzyme kinetics

Standard enzyme kinetic curves were obtained at different enzyme

concentrations from a range of 0.25 to 1 mg/ml, and at a constant substrate

concentration. The results are plot in Figure 20. This standard curve will provide

the evidence of enzyme activity for the encapsulated enzyme and for the kinetics

after enzyme release from nanoparticles.

Figure 20 kinetics data for different concentration of trypsin and a constant substrate concentration

3.5.2 Release studies

Released studies were performed in duplicate. First, tris-buffer solution was

added to the particles using the substrate DL-BAPNA and started measuring the

0

0.5

1

1.5

2

2.5

3

3.5

0 5 10 15 20 25 30 35

Ab

sorb

ance

at

41

0

Time (min)

0.25 mg/ml

0.5 mg/ml

0.125 mg/ml

0.0625 mg/ml

0.03125 mg/ml

1 mg/ml

54

absorbance from time equal zero. Samples were taken at different time intervals

and the absorbance measured at 410 nanometers. The sample aliquotes were

returned to the reaction vessel. The process was repeated several times to

measure the activity of the enzyme after encapsulation, that is, while the enzyme

was encapsulated. Similarly, enzyme kinetics was performed after enzyme

release studies where the enzyme was measured at different time intervals upon

release. The results were compared with the enzyme kinetics standard curves

previously prepared. The activity of the enzyme while in the nanoparticles

(encapsulated) was compared with the standard curves. Results show that in fact

the enzyme remains active while encapsulated, this based on the conversion of

substrate added to the reaction vessel, before the enzyme release process.

The enzyme kinetics analysis of the time released enzyme was quantitatively

measured and the results are shown in Figure 21. The values for the kinetics

were taken from the released enzyme aliquots obtained in Figure 22 every hour.

The resulting or enzyme recovery activity after encapsulation and released gave

values corresponding to enzyme concentrations between 0.25 and 0.5 mg/ml.

55

Figure 21. Absorbance kinetic activity of released from encapsulated enzyme system.

Figure 22 Trypsin release behavior from PLGA nanoparticles

From the results shown in Figure 22, it appears that the process of release

behavior is quite similar to the result for insulin and myoglobin described in the

previous chapter. One aspect noticeable is the rates of release suggesting that

0

0.5

1

1.5

2

2.5

3

3.5

4

0 50 100 150 200

abso

rban

ce a

t 4

10

Time (min)

Enzyme release after 150 minutes

0.25 mg/ml

0.5 mg/ml

conc. of trypsin afterdrug release

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0.04

0.045

0.05

0 5 10 15 20

con

c. o

f tr

ypsi

n

Time (hr)

Trypsin release

56

the size of the protein influences the release of the biomolecules. Further studies

will be performed to assess this important aspect.

The kinetics for each sample was followed for 30 minutes at 410, using a

Shimadzu spectrophotometer, and the results are given in Figure 23. According

to the calibration curve (Figure 20), the results show that in fact that the

concentration of trypsin released from the particles is between 0.5 and 0.25

mg/ml.

Figure 24 Kinetics of samples that were took after releasing study

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 5 10 15 20 25 30 35

Ab

sorb

ance

at

41

0

Time (min)

kinetics of trypsin after releasing study

After 1 hr

After 2 hrs

After 3 hrs

After 4 hrs

After 5 hrs

After 18 hrs

57

3.6 Conclusions

PLGA nanoparticles were prepared effectively by an implemented emulsification

–solvent evaporation technique using PLGA and PVA with concentration of 3%. The

encapsulation of the enzyme trypsin was carried out successfully and the enzymatic

activity after encapsulation and after released from the nanoparticles is present at

substantial levels of activity. These results represent a relevant finding since one can

anticipate that the behavior of other biomolecules such as insulin and other relevant

hormones or proteins could behave in a similar fashion. Further work will be performed

with other proteins and with different biological assays.

58

CHAPTER 4. Conclusions

59

4. Conclusions and future work

In this research a variety of nanoparticle drug delivery systems were synthesized

and characterized using as a biodegradable matrix carrier poly(lactic-co-glycolic

acid) (PLGA), and polyvinyl alcohol (PVA). PLGA nanoparticles were prepared

effectively by an implemented emulsification –solvent evaporation technique and

the formulation parameters effects over the nanoparticle properties were

investigated. The encapsulation of proteins such as insulin, myoglobin, and

trypsin, was effectively performed into polymeric nanoparticles using this

process.

In all the cases, the release behavior appears to follow a three stage release

process, similar to the behavior of small drugs. A mathematical model used for

anticancer drugs seems to correlate, at least qualitatively to the processes

observed in this work.

Since the biological activity of insulin, the main hormone protein in this work is

difficult to obtain, an enzymatic analysis was performed by measuring the

enzymatic activity of the enzyme trypsin during the encapsulation process and

after released from nanoparticles. The results show that in fact, one can measure

the activity of the biomolecule following this approach and proved that substantial

activity remains in the enzyme after going through the process of encapsulation

and release. This result also provides confidence that other many biomolecules

could behave in a similar fashion.

60

Future biomolecule encapsulation and release studies will involve measuring the

activity of the biological insulin after encapsulation. Future work will involve

modification of the polymer PLGA or other polymers to obtain targeted

nanoparticles with encapsulated biomolecules for effective targeted and

controlled delivery of pharmaceuticals and for materials for diagnostics and

therapy.

.

61

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