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Theses and Dissertations

2-1-2020

PEGylated chitosan / doxorubicin nanoparticles and conjugated PEGylated chitosan / doxorubicin nanoparticles and conjugated

with monoclonal antibodies for breast cancer therapy with monoclonal antibodies for breast cancer therapy

Omar Helmi Zidan

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i

School of Sciences and Engineering

PEGylated Chitosan / Doxorubicin Nanoparticles

and conjugated with Monoclonal Antibodies for Breast

Cancer Therapy

A Thesis Submitted to

The Nanotechnology Master's Program

In partial fulfilment of the requirements for

The degree of Master of Science

By:

Omar Helmi Zidan

Under the supervision of:

Dr. Wael Mamdouh

Associate Professor, Department of Chemistry, The American University in

Cairo

18th December, 2019

ii

The American University in Cairo

PEGylated Chitosan / Doxorubicin Nanoparticles

and conjugated with Monoclonal Antibodies for Breast Cancer Therapy

A Thesis Submitted by

Omar Helmi Zidan

To the Nanotechnology Graduate Program

18th December 2019

In partial fulfillment of the requirements for

The degree of Master of Science

Has been approved by

Thesis Committee Supervisor/Chair

_______________________________________________

Affiliation ______________________________________

Thesis Committee Reader/Examiner

______________________________________________

Affiliation _____________________________________

Thesis Committee Reader/Examiner

______________________________________________

Affiliation ______________________________________

Thesis Committee Reader/External Examiner

_______________________________________

Affiliation_______________________________________

________________ _____________ _________________ _______________

Dept. Chair/Director Date Dean Date

iii

Acknowledgement

I would like than to God for giving me the opportunity to accomplish this work with unlimited

amounts of blessings. Right now, it’s good moments to thank all people who helped me a lot

with guidance and advices. I have received a lot of support and motivation. First and foremost,

I deeply thank my beloved supervisor Dr. Wael Mamdouh for his great patience and support.

I relay appreciate everything he has done for me; I have learnt a lot from him. He has a lot of

contribution to my career life whether inside or outside the AUC. He was always available

during his supervision and he gave me and my colleagues a lot of his time. I would like, also,

to thank all professors in the chemistry department. I have worked as TA with most of them

and I have learnt a lot from all of them; Dr. Adham Ramadan, Dr. Hassan Azzazy, Dr. Anwar

Abdel Nasser, Dr Hatem Talima, Dr. Mohamed Farag, Dr, Shahinaz Abdelrahman, Dr Nahed

Yakoub and Dr Tamer shoeib

Special thanks to my family members who did a lot for me. Of course, It’s hard to put into

words exactly how grateful I am for all your help and support. Special thanks to my father, my

mother, my sister, my brothers and my dear lovely nephews and nieces. Special thanks to all

my dearest colleagues in the AUC for their unlimited support and their help in the lab; Yasmin

el Kashef, Jailan Essam, Sarar Omar, Fatma El Shishiny, Amro Sheta, Sherif Galal,

Mariam Gamal, Khadija sadek, Mohga Essam, Nouran Sharaf, Hend El Khouly, James

Kegere, Ahmed Emad I need also to thank my dear friends who left the AUC Marwan Rezk,

Ahmed Baracat, RashaEssam, Omar Hamed, Sherouk Nasrat, Mona Tarek. I never forget

times we spent together, and I wish you the best in your lives.

I would like to extend my sincere to my dear technicians and all people in the chemistry

department; Mr. Mahmoud Abdel Moezz, Ahmed Momaia, Samir Shamma and Mr.

Emad Rafat and Hend Mashhour. Their help is really appreciated, and I can’t forget many

days they have a great contribution and facilitated many experiments in my research work.

iv

Abstract

The potential of nanobiomedical field for developing a promising therapeutic nano-sized drug

delivery system is seen to be a great pharmaceutical trend for encapsulation and release of

various antineoplastic drugs. In this context, the current work is targeting preparation of

biodegradable chitosan nanoparticles (CSNP) that have been intended for selective and

sustained release of doxorubicin (DOX) within breast tumor microenvironment. Surface

modification of these CSNP with Polyethylene glycol (PEG) was performed in order for

enhancing its blood circulation time without being opsonized or captured by immunogenic

reticuloendothelial system (RES). PEG has maintained high particles stability profile and

enhanced its surface positive charge for the sake of intracellular attachment via electrostatic

attachment with negatively charged tumor cell membrane. Advanced tumor selectivity has

been achieved through functionalization of two different types of breast cancer specific

monoclonal antibodies (mAb); anti-human mammaglobin (Anti-hMAM) and anti-human

epidermal growth factor (Anti-HER2) in two different separate nano formulations. This

functionalization has the potential of evading systemic side effects of parenteral free DOX and

promoting cancerous endocytosis through receptor mediated interaction. In-vitro cytotoxicity

effects of PEGylated DOX loaded CSNP, free DOX, Anti-HER2 PEGylated DOX loaded

CSNP and Anti-hMAM PEGylated DOX loaded CSNP were tested against breast cancer cell

line (MCF7) and normal fibroblast cell line (L929). Notably, Anti-hMAM PEGylated DOX

loaded CSNP and Anti-HER2 PEGylated DOX loaded CSNP formulations were the most

cytotoxic against MCF7 cancer cells than L929 normal cells compared to free DOX.

Confirmatory bright filed images of the two cell lines have exhibited cancerous cellular damage

after 24 hours exposure time to these nano formulations. Finally, we believe that dose

dependent system toxicity of freely ingested DOX can be hindered with such targeted nano

formulated drug delivery system.

Scheme 1: Graphical diagram of DOX encapsulation and release within tumor cell

v

Table of Contents:

Acknowledgement ................................................................................................................... iii

Abstract .................................................................................................................................... iv

List of Abbreviations .............................................................................................................. vii

List of Figures .........................................................................................................................viii

List of Tables ........................................................................................................................... xii

Thesis scope and objectives ...................................................................................................... 1

Chapter 1: Introduction and Literature Review .................................................................. 4

1.1.Cancer ................................................................................................................................. 5

1.2. Breast Cancer ..................................................................................................................... 7

1.3. Types of Breast Cancer ...................................................................................................... 8

1.3.1. Pre-Invasive Breast Cancer .............................................................................. 8

1.3.1.1. Ductal Carcinoma In-Situe (DCIS) ..................................................... 9

1.3.1.2. Lobular Carcinoma In-Situe (LCIS) ................................................... 9

1.3.2. Invasive Breast Cancer .......................................................................... 10

1.3.2.1. Invasive Ductal Carcinoma ..................................................... 11

1.3.2.2. Invasive Lobular Carcinoma ................................................... 11

1.3.3. Molecular subtypes of Breast Cancer ..................................................... 11

1.3.3.1. Estrogen Receptor mediated Breast Cancer ............................ 12

1.3.3.2. Progesterone Receptor mediated Breast Cancer ..................... 13

1.3.3.3. Human Epidermal Growth factor receptors Breast Cancer ... 13

1.3.4. Triple Negative Breast Cancer ............................................................... 15

1.4. Pathological Stages of Breast Cancer ................................................................... 17

1.5. Breast Cancer Current Therapy ........................................................................................ 19

1.5.1. Surgery ........................................................................................................... 19

1.5.2. 1.5.2. Radiotherapy ........................................................................................ 20

1.5.3. 1.5.3. Hormonal Therapy .............................................................................. 22

1.5.4. 1.5.4. Chemotherapy ...................................................................................... 23

1.5.4.1. Doxorubicin ..................................................................................... 24

1.5.4.2. Mechanism of Action of Doxorubicin ............................................. 25

1.5.4.2.1. Limitations of Doxorubicin ........................................... 27

1.6. Targeted Drug Delivery System ........................................................................... 29

1.7. Nanotechnology................................................................................................................ 29

1.8. Nanotechnology Enhanced pH Manipulation ............................................................. 31

1.9.Nanotechnology Enhanced Surface Charge Manipulation .................................... 33

1.10. Nanotechnology Enhanced Biomarker Attachment......................................... 33

1.10.1. Passive Targeting .......................................................................................... 34

1.10.2. Active Targeting ............................................................................................ 34

1.11.Nanotechnology Enhanced Prolonged Biological Circulation .............................. 36

1.12.Chitosan as Nanoparticle Platform ........................................................................ 36

1.13.Polyethylene Glycol ............................................................................................... 39

1.14.Anti-Human Mammaglobin Antibody ................................................................... 40

vi

Chapter 2: Material and Methods ........................................................................................ 42

2.1. Materials ................................................................................................................... 43

2.2. Preparation of chitosan nanoparticles ....................................................................... 43

2.3. Stability of chitosan nanoparticles ............................................................................ 46

2.4. Coating of chitosan nanoparticles with PEG ............................................................ 46

2.5.Encapsulation of doxorubicin into PEGylated CSNP ................................................ 46

2.6.Conjugation of monoclonal Antibodies .................................................................... 52

2.7.Characterization of nanoparticles ............................................................................. 53

2.7.1. Dynamic light scattering .............................................................................. 53

2.7.2. Electrophoretic Light Scattering .................................................................. 53

2.7.3. Fourier Transmission Infra-Red Spectroscopy ............................................ 54

2.7.4. X-Ray Diffraction ........................................................................................ 54

2.7.5. Scanning Electron Microscope .................................................................... 54

2.7.6. Transmission Electron Microscope.............................................................. 55

2.7.7. Ultra-Violet / Visible light Spectroscopy .................................................... 55

2.8. Drug Loading, Release Profile, Swelling test and Stability of nanoparticles ........... 55

2.8.1. Drug Loading......................................................................................................... 55

2.8.2. Swelling Test .......................................................................................................... 56

2.8.3. Stability of Nanoparticles ....................................................................................... 56

2.8.4. Drug Release Profile ............................................................................................... 57

2.9. Cell Culture Analysis Profile .................................................................................... 57

2.9.1. Cell line Maintenance ............................................................................................. 57

2.9.2. Direct Cytotoxicity ................................................................................................. 57

2.10. Theoretical Background ................................................................................... 58

Chapter 3: Results and Discussion ....................................................................................... 70

3.1. Preparation of CSNP ............................................................................................ 71

3.1.1. Ionotropic gelation ................................................................................. 71

3.1.2. Factors affecting size of CSNP .............................................................. 72

3.1.3. Stability of the selected CSNP ............................................................... 79

3.2. PEGylation of CSNP ............................................................................................ 76

3.3. Encapsulation of Doxorubicin .............................................................................. 81

3.3.1. Standard Curve of Doxorubicin ............................................................. 81

3.3.2. Doxorubicin loading capacity and encapsulation efficiency ................. 82

3.3.3. UV-VIS of Doxorubicin loaded PEGylated CSNP ............................... 84

3.3.4. FTIR Analysis ........................................................................................ 85

3.3.5. Swelling studies of DOX loaded PEGylated CSNP .............................. 87

3.3.6. In-Vitro Release Profile ......................................................................... 88

3.3.7. In-Vitro Release Kinetics ....................................................................... 90

3.4. Functionalization of monoclonal antibodies ......................................................... 91

3.5. Cytotoxicity Analysis............................................................................................ 94

3.6 Size Increment Profile .......................................................................................... 102

Appendix (Supporting Information) ................................................................................. 104

Chapter 4: Conclusion and Future Perspectives................................................................... 119

References: ........................................................................................................................... 122

vii

List of Abbreviations

Anti-HER2: Anti-Human Epidermal Growth Factor Receptor antibody

Anti-hMAM: Anti-Mammaglobin antibody

Cs: Chitosan

CSNP: Chitosan Nanoparticles

DLS: Dynamic Light Scattering

DOX: Doxorubicin

EDC: 1-Ethyl-3- (3-Dimethylaminoprobyl) Carbodiimide

EE: Encapsulation Efficiency

EPR: Enhanced Permeability Retention

FTIR: Fourier Transmission Infra-Red Spectroscopy

HNMR: Proton Nuclear Magnetic resonance

IPN: Inter Penetrating Network

LC: Loading Capacity

mAb: Monoclonal Antibody

PBS: Phosphate Buffer Solution

PDI: Poly Dispersity Index

PEG: Polyethylene Glycol

RES: Reticuloendothelial System

SEM: Scanning electron microscope

TEM: Transmission Electron Microscope

TPP: Tri-Poly Phosphate

UV-VIS: Ultraviolet-Visible Spectroscopy

XRD: X-ray Diffraction

Z average: Zeta potential average

viii

List of Figures:

Chapter 1:

Scheme 1: Graphical diagram of DOX encapsulation and release within tumor cell ............ iv

Scheme 2: Full schematic diagram of different work activities in this study ......................... 3

Figure 1.1: Initiation of cancerous tumor by the action of genetic alteration that results from

various environmental factors. ................................................................................................. 5

Figure 1.2: The angiogenesis pathway of a cancerous tumor with overexperession of VEGF

glycoprotein results in initiation of new microblood vessels that feed and carry nutrients and

oxygen to tumor area so that, the tumor cells can divide and grow easily ............................... 6

Figure 1.3: (A) Total cancer prevalence rate in 2018 including various types of cancer with

various physiological sites in human body, with total estimated number of 18,078,957 cancer

patients. (B) estimated rate of death cases because of cancer all ages and all types in by the end

of 2018 that reached around 9.6 million worldwide ................................................................. 7

Figure 1.4: (A) Different types of cancer with variable death rates including breast cancer

(6.6%) death rate in 2018, (B) the incidence rate of Breast cancer in different world zones for

2018 ........................................................................................................................................... 7

Figure 1.5: Schematic representation of breast tissue anatomy ............................................... 8

Figure 1.6: Schematic illustration of ductal carcinoma in situ ................................................ 9

Figure 1.7: Schematic illustration of lobular carcinoma in situ (LCIS) ................................ 10

Figure 1.8: The difference between invasive tumor (A), and non- invasive or in-situ tumor (B)

.................................................................................................................................................. 10

Figure 1.9: Illustrative figure shows the difference between invasive ductal carcinoma and

invasive lobular carcinoma ..................................................................................................... 11

Figure 1.10: Three basic types of receptors within breast tumor cell .................................... 12

Figure 1.11: The effect of anti-estrogenic drug Tamoxifen up on placebo and tamoxifen

treated breast cancer patients ................................................................................................... 12

Figure 1.12: The anti-progesterone action with a breast cell mediated progesterone ............ 13

Figure 1.13: Difference between HER2 positive and HER2 negative breast tumor cell ....... 14

Figure 1.14: HER2 signaling pathway with a cascade manner that ultimately target the final

breast tumorigenesis pathway ................................................................................................. 14

Figure 1.15: The Incidence rates of molecular types of breast cancer (A) and subtypes of

TNBC (B) ................................................................................................................................ 15

Figure 1.16: Pathogenesis of breast cancer including both molecular and histologic types . 16

ix

Figure 1.17: (A) Removal of sentinel lymph nodes with blue colored drainage, (B) removal of

axillary lymph nodes ............................................................................................................... 19

Figure 1.18: Different types of radiation therapy based on the affected area and the conducted

surgery ..................................................................................................................................... 21

Figure 1.19: Different Toxicity and severity rates with different breast sized patients ......... 21

Figure 1.20: (A) Radiotherapy related breast skin fibrosis, (B) Moist desquamation of a

radiotherapy affected breast .................................................................................................... 22

Figure 1.21: Inhibition of estrogen action through Hormonal therapy .................................. 23

Figure 1.22: (A) Chemical formula of DOX, (B) DOX bacterial strain of Streptomyces

Peucetius ................................................................................................................................. 25

Figure 1.23: Programmed cell death of cancerous cell with DOX (anthracycline) ............... 26

Figure 1.24: Simplified illustration of a nano-based drug delivery system ............................ 29

Figure 1.25: Nanometer scale with relative sizes of various biological objects .................... 30

Figure 1.26: TEM images of various shapes of nanostructures; (A) nanoparticles, (B)

nanocubes and (C)

Nanorods-shapes ..................................................................................................................... 30

Figure 1.27: Five routes of administration for nano based drug delivery systems ................. 31

Figure 1.28: Schematic representation of mitochondrial phosphorylation and glycolysis .... 32

Figure 1.29: pH dependency scale relative to cancerous tumor size ..................................... 32

Figure 1.30: Electrostatic interaction between nanoparticles (+ve) and cancerous cell wall (-

ve) has developed

instant drug release (192) ......................................................................................................... 33

Figure 1.31: Schematic illustration that differentiate between active (A) and passive

targeting (B) ............................................................................................................................ 34

Figure 1.32: preparation of chitosan by deacetylation of chitin ............................................ 37

Figure 1.33: chemical formula of Sodium tripolyphosphate ................................................. 39

Figure 1.34: Chemical structure of polyethylene glycol ........................................................ 40

Chapter 2:

Figure 2.1: Cross-linked chitosan with TPP .......................................................................... 44

Figure 2.2: General scheme for preparation of CS NPs ......................................................... 45

Figure 2.3: General scheme for preparation of PEGylated CS NPs ...................................... 48

Figure 2.4: Chemical scheme for preparation of PEGylated cross-linked CS NPs ............... 49

Figure 2.5: illustrative scheme for encapsulation of DOX inside PEGylated CS NPs ........... 51

Figure 2.6: Systematic setup of the basic principle for dynamic light scattering with

detecting different

scattering angles of the laser beam ......................................................................................... 58

x

Figure 2.7: Illustrative description of different components of dynamic light scattering

instrument ............................................................................................................................... 59

Figure 2.8: Schematic diagram showing the principle of electrophoretic light scattering with

a multiple layer of

illustrative charged layers (A), and the shape of the sample cell kit (B) ................................ 60

Figure 2.9: schematic representation of the FTIR working principle .................................... 61

Figure 2.10: illustrative scheme of X-ray diffraction with diffraction angle θ (Bragg’s law)

.................................................................................................................................................. 62

Figure 2.11: X-ray beam bombarding the Inner shell electrons ............................................ 63

Figure 2.12: The electronic excitation by absorbing high energy UV light spectrum ........... 64

Figure 2.13: The transmittance of UV light beam through a sample ..................................... 65

Figure 2.14: schematic representation of UV-VIS spectrometer principle ............................ 66

Figure 2.15: Multi pin sample’s holder carrying different aluminum foils with spread

samples for TEM imaging

procedure ................................................................................................................................. 67

Figure 2.16: Illustrative description of SEM unite with variable .......................................... 68

Figure 2.17: Schematic representation of the different components of TEM ....................... 69

Chapter 3:

Figure 3.1: Effect of processing pH (A) and deacetylation degree (B) of CS on the size of

CSNP........................................................................................................................................ 72

Figure 3.2: Effect of TPP concentration on the final size of CSNP ....................................... 74

Figure 3.3: Stability profile of the selected CSNP sample; S8 for three weeks at pH 7.4 ..... 75

Figure 3.4: stability profile of PEGylated and non-Pegylated CSNP ..................................... 77

Figure 3.5: XRD pattern of Chitosan powder, CSNP and PEGylated CSNP ......................... 79

Figure 3.6: TEM image of Pegylated CSNP (B and D), non-PEGylated CSNP (A and C) and

schematic

illustration of the PEG layer that surround CSNP (E) ............................................................. 81

Figure 3.7: standard calibration curve of DOX in PBS .......................................................... 82

Figure 3.8: TEM images of DOX loaded PEGylated CSNP (A, B), and schematic diagram of

DOX loaded

Figure 3.9: UV-Vis spectra of DOX loaded CSNP, non-loaded CSNP and free DOX .......... 84

Figure 3.10: FTIR spectrum of CS powder, CSNP, DOX, Pegylated CSNP and DOX loaded

PEGylated CSNP ..................................................................................................................... 85

Figure 3.11: TEM images of DOX loaded PEGylated CSNP at pH 7.4 (a), pH 6.5 (b) after

24 hrs incubation and schematic illustration of the difference between non-swelled (pH7.4)

and swelled nanoparticles (pH 6.6) (c)

.................................................................................................................................................. 86

Figure 3.12: DOX release profile of PEGylated DOX loaded CSNP at pH 6.6 and pH 7.4 .. 87

Figure 3.13: Illustrative diagram of DOX release mechanism .............................................. 88

xi

Figure 3.14: Schematic illustration of the final mAb functionalized DOX loaded PEGylated

CSNP........................................................................................................................................ 89

Figure 3.15: TEM images of Anti-hMAM DOX loaded PEGylated CSNP (A, B) and Anti-

HER2 DOX loaded PEGylated CSNP (C, D) .......................................................................... 91

Figure 3.16: HNMR spectrum of Anti-hMAM / DOX loaded PEGylated CSNP (A) and

Anti-HER2 / DOX loaded PEGylated CSNP (B) .................................................................... 92

Figure 3.17: FTIR spectrum of mAbs /DOX loaded PEGylated CSNP and non-

functionalized DOX loaded PEGylated CSNPs ...................................................................... 93

Figure 3.18: Colorimetric MTT assay of DOX loaded PEGylated CSNP (1.25, 2.5, 5 and 10

Ug/ml) (A), Free DOX concentrations (1.25, 2.5, 5 and 1 Ug/ml) (B) and Anti-HER2 and

Anti-hMAM functionalized DOX loaded PEGylated CSNP. ................................................. 94

Figure 3.19: Bright field images of L929 cells with white arrows referring to living cells (A),

and MCF7 cells with white arrows referring to dead cells (B) after exposure to DOX –

PEGylated CSNP (0.5 Ug/ml) ................................................................................................. 95

Figure 3.20: Bright field images of L929 cells with white arrows referring to dead cells (A),

and MCF7 cells with white arrows referring to living cells (B), after exposure to Free DOX

.................................................................................................................................................. 97

Figure 3.21: Bright field images of L929 cell line with white arrows referring to living cells

(A), and MCF7 cell line with white arrows referring to dead cells (B) cells after exposure to

Anti-HER2 functionalized DOX loaded PEGylated CSNP..................................................... 98

Figure 3.22: Bright field images of L929 cells with white arrows referring to living cells (A)

and MCF7 with white arrows referring to dead cells (B) cells after exposure to Anti-hMAM

functionalized DOX loaded PEGylated CSNP ........................................................................ 99

Figure 3.23: TEM images and the relevant DLS histograms of non-PEGylated CSNP (A, B)

and PEGylated

................................................................................................................................................ 100

Figure 3.24: TEM images and their relevant DLS histograms of DOX loaded PEGylated

CSNP (E, F), Anti-HER2 DOX loaded PEGylated CSNP (G, H), and Anti-hMAM DOX

loaded PEGylated CSNP (I, J)

................................................................................................................................................ 102

xii

List of Tables

Chapter 1:

Table1.1: Various subcategories of TNBC with their relative genetic characteristic behaviors

.................................................................................................................................................. 15

Table 1.2: TNM grading system for different stages of breast cancer ................................... 17

Table 1.3: Five years survival rates of different stages of breast cancer ............................... 19

Table 1.4: Various adverse effects of using chemotherapy after one-month treatment ........ 24

Table 1.5: Examples of generic pharmaceutical market products (147, 148) ......................... 26

Table 1.6: Drug-drug interaction with resulted adverse reactions of doxorubicin vs some

frequently

administered types of drugs for a cancer patient ..................................................................... 28

Table 1.7: Example of different nanostructured drug delivery formulas that are assisted with

various

biological targeting moieties. .................................................................................................. 35

Table 1.8: Some CSNP formulations used for breast cancer drug delivery .......................... 38

Chapter 2:

Table 2.1: Optimum conditions for the applied factors in preparation of CS NPs ................ 45

Table 2.2: Sample variations relative to the polymeric concentrations of PEG while keeping

TPP and Cs concentrations fixed ............................................................................................ 47

Table 2.3: Sample variation relative to DOX concentrations while keeping the polymeric

concentrations for Cs, TPP and PEG fixed. ............................................................................. 50

Table 2.4: comparison between TEM and SEM .................................................................... 69

Chapter 3:

Table 3.1: Variation of sample formulas according to chitosan and TPP concentrations ...... 71

Table 3.2: Particle size and distribution of different samples with various concentrations of

TPP and chitosan polymer ...................................................................................................... 72

Table 3.3: Size, PDI and zeta potential of PEGylated CSNPs ................................................ 76

Table 3.4: DLS analysis of DOX loaded and unloaded CSNP ............................................... 83

Table 3.5: Resulted LC% and EE% of different DOX-PEGylated CSNP at different drug

concentrations .......................................................................................................................... 83

xiii

Table 3.6: Release kinetics of the released DOX according to different systems equations

with various resulted values .................................................................................................... 90

1

Thesis Scope and Objectives

2

Thesis Scope and Objectives:

The current study is an indicative attempt to apply readily available and naturally existent

polymers; chitosan (CS), in order to enhance the pharmacotherapeutic efficacy of an anti-

cancer drug; Doxorubicin (DOX) and its smart delivery system against breast cancer cell line.

The main objective here is to design a nano-formulation platform that will be able to travel

inside the human physiological blood circulatory system. This circulatory journey is oriented

to be from a certain site of administration till reaching the selected site of action by the aid of

cell specific ligand(s) known as smart antibodies. This partially invasive drug delivery system

aims to deliver, localize, and prolong the release of the loaded DOX. Besides, it is an efficient

system to minimize the undesirable immunogenic uptake of this entire therapeutic platform by

the effect of mono-nuclear phagocyte system (MPS) that is known, also, as reticuloendothelial

system (RES). Thereby, facilitating the accumulation of effective doses of DOX within the

tumor environment without harming the other non-cancerous organs. Thus, minimizing its

severe side effects like heart failure, bone marrow suppression, mucositis and alopecia (1).

The referred nanoparticles conjugated system will be based on a biodegradable and

biocompatible polymeric chitosan nanoparticles (CSNP) which will be functionalized with

Polyethylene Glycol (PEG), DOX and biological marker(s) over the different chapters and

activities of this study. In activity 1, the CSNP will be prepared within a bottom-up approach

using ionotropic gelation method (2). The resultant nanoparticles will be characterized using

different characterization analysis in order to confirm the consistency of their crosslinking,

size, shape, distribution, morphology and surface charge. Various characterization techniques

have been exploited for this purpose such as Dynamic Light Scattering (DLS), Scanning

Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), X-Ray Diffraction

(XRD), Ultraviolet-Visible spectrophotometry (UV-VIS) and Infrared spectroscopy using

Fourier Transform Infrared Spectroscopy (FTIR). In Activity 2, the prepared CSNPs have been

loaded with a polymeric Polyethylene Glycol (PEG). The main purpose behind adding this

polymer has been to ensure a prolonged and extended circulation of the nanoparticles till

reaching the breast tumor area without being uptake by RES. These PEGylated CSNP have

been characterized, also, using the same characterization analysis as activity 1. (3)

3

Encapsulation of DOX inside the PEGylated CSNP has been done as the third activity. This

step has been done with very minute concentrations of DOX. These minute concentrations

have been tested in terms of encapsulation efficiency (EE%), loading capacity (LC%) and cell

cytotoxicity. The dye compound 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromidefor colorimetric assay (MTT assay) has been an indicative test of the most effective

DOX concentration that can kill the highest percentage of MCF-7 breast tumor cells (3). The

concentration of choice has been tested for the drug release behavior in order to monitor and

expect its behavior inside the tumor microenvironment. Besides cell cytotoxicity test, this

activity has been characterized also with FTIR, TEM, Zeta-sizer, Zeta potential and UV-Vis

spectroscopy. These characterization analyses have confirmed the successful encapsulation

and stability of DOX inside the PEGylated CSNP. Achieving such dose reduction with

maintaining good stability of this nano-formula in normal saline pH is an outstanding step for

obtaining efficient delivery process of highly effective doses of DOX. Therefore, the

integration of such multidisciplinary nanosystem into pharmaceutical DOX drug delivery

process has empowered its activity against breast tumor cells.

Scheme 2: Full schematic diagram of different work activities in this study

4

Chapter 1

General Introduction

&

Literature Review

5

1.1. Cancer

For many decades, cancer has been recognized as one of the top unresolved threatening health

problems that represents the second leading cause of death worldwide according to some

contemporary reports by World Health Organization (WHO). Typically, cancer can start within

any organ in the body at any time for many identified and unidentified etiologies as shown in

Figure (1.1) (4). The main problem with cancer is the fast cellular growth of a group of mutated

cells that are known as tumor cells. Basically, Cancer starts with one normal healthy cell that

develops a damage or unexpected change in its Deoxyribonucleic Acid (DNA) molecule. This

change, which may be inherited or developed by many factors such as radiation, oxidative

stress, infection, smoking and hormonal disturbances can cause gene mutation and rather

interfere with the normal cell division instructions. Therefore, it turns from a normal cell to a

cancerous cell that grows out of control. Tumor is developed when this cancer cell continues

to grow and divide.

Figure 1.1: Initiation of cancerous tumor by the action of genetic alteration that results from various

environmental factors. (5)

6

Tumor cells don’t have any specific physiological function in the human body, and they can

easily avoid the immune system. Besides, they ignore the physiological signals that aim to

control or stop cell division. The tumor grows and gets bigger by the flood of oxygen and

different types of nutrients using blood supply through cancerous angiogenesis formation as

illustrated in Figure (1.2) (6).

Figure 1.2: The angiogenesis pathway of a cancerous tumor with overexperession of VEGF glycoprotein results

in initiation of new microblood vessels that feed and carry nutrients and oxygen to tumor area so that, the tumor

cells can divide and grow easily (7)

The increased numbers of cancer diseased cases worldwide are tremendously growing every

day, the mortality rate at 2018 has been 9.6 million deaths (5). In Aisa, the highest mortality

rate, the estimated number of death cases has been more than 5 million. While the remaining

amount of death cases has been localized in different areas worldwide including Europe,

Africa, Oceania and North America as shown in Figure (1.3) (B). On the other hand, the

estimated economic expenses that has been accompanied with cancer treatment in US is about

$1,16 Trillion per year. Besides, more than 300,000 new cancer cases among the age of 0-19

years are being annually diagnosed. Therefore, it has been proposed by some WHO guidelines

that patient awareness and early screening of a tumor can improve the treatment access (8).

7

(A) (B)

Figure 1.3: (A) Total cancer prevalence rate in 2018 including various types of cancer with various

physiological sites in human body, with total estimated number of 18,078,957 cancer patients. (B) estimated rate

of death cases because of cancer all ages and all types in by the end of 2018 that reached around 9.6 million

worldwide (8)

1.2. Breast cancer

Breast cancer is the highest comon cancer type within female population, and it has been ranked

worldwide as one of the top leading causes of high death rates among the same demographic

population section. Being one of the top international public health issues over the century,

breast cancer has a direct influence on different dimensions of human lives including

physiological, psychological, mental and social aspects. Meanwhile, it has an incidence rate of

more than 2.1 million new cases every year as shown in Figure (1.3 A). In 2018, the mortality

rates of breast cancer have been 627,000 deaths as shown in Figure (1.4 A).

(A) (B)

Figure 1.4: (A) Different types of cancer with variable death rates including breast cancer (6.6%) death rate in

2018, (B) the incidence rate of Breast cancer in different world zones for 2018 (8)

8

Anatomical picture of a female’s breast tissue is composed of a group of lobes that are

estimated as 10-20 lobes (9). Each of these lobes is made up of many smaller lobules which

function as glands that produce and carry the breast milk. These lobules are connected together

by ducts that act as a passing canal for milk drainage from the lobules to the nipples. The second

part of breast tissue is called stroma which is a supporting tissue that integrates the surrounding

connective tissue and fatty tissue. Typically, breast cancer originates at the cells of either the

lobules (lobular carcinoma) or ducts (ductal carcinoma), or any type of these supporting tissues

as shown in Figure (1.5).

Figure 1.5: Schematic representation of breast tissue anatomy (10)

1.3. Types of breast cancer:

1.3.1. Preinvasive breast cancer:

It is a less frequently dominated cancer type which originates only and locally inside either the

milk ducts or milk lobules. It doesn’t spread outside these specific two sites. They can be

considered as the early stage or precancerous states of breast cancer. Typically, this type of

cancer can easily respond to early treatment. It represents around 25% of all known types and

subtypes of breast cancer. For instance, in United Kingdome, around 7400 women get such

preinvasive breast cancer every year which may originate in breast duct known as ductal

9

carcinoma in situ (DCIS), or in breast lobule known as lobular carcinoma in situ (LCIS) (11).

A Full detailed description of the different types of breast cancer is illustrated in Figure (1.16).

1.3.1.1. Ductal Carcinoma in Situ (DCIS):

Ductal carcinoma can be diagnosed as cancerous cells that grow locally inside the walls of

ducts and don’t invade the external surrounding breast tissues. Approximately, all women with

such type of tumor can be cured easily and the early detection of such type is, also, another

helpful factor for efficient treatment. Because such tumor type is confined locally within the

ductal tissue only, DCIS is also known as intraductal or non-invasive cancer. It is not a life-

threatening subtype, but in rare cases it can develop a more sever invasive type. Usually,

women develop such breast cancer subtype after the age of 48 yrs (12). DCIS can be

subcategorized into three types; low grade, intermediate grade and high grade. The last two

types can be screened as a thick mass accompanied with some nipple discharge.

Figure 1.6: Schematic illustration of ductal carcinoma in situ (13)

1.3.1.2. Lobular Carcinoma in Situ (LCIS):

This type of cancer tumor is another confined and localized tumor. The cells grow exclusively

inside the lining lobules only without harming the adjacent walls and tissues or cells. Although

it is physiologically confined, it can however be diagnosed hardly using such advanced

screening as mammograms. Some studies have proposed that such type may not be considered

as a real type of breast cancer, but they consider it as a marker for future tumor. It is detected

as a change in the cellular structure in a biopsied tissue. It differs from DCIS as if it was left

without treatment, it won’t proceed to such aggressive invasive subtype (13, 14).

10

Figure 1.7: Schematic illustration of lobular carcinoma in situ (LCIS) (13)

1.3.2. Invasive breast cancer:

This is a non-confined breast cancer tumor in which the cells are growing out of control and

spread outside the ductal and lobular tissues. The tumor grows outside the confined areas as

shown in figure (1.8) (A). It represents around 75% of breast cancer tumor types and has been

estimated that the US is developing 180,000 invasive breast cancer cases every year (14). The

majority of this number is diagnosed with invasive ductal carcinoma. It can affect women

population randomly at any stage of their ages. Besides, invasive breast cancer can affect men

population as well.

(A) (B)

Figure 1.8: The difference between invasive tumor (A), and non- invasive or in-situ tumor (B) (15)

11

1.3.2.1. Invasive ductal carcinoma:

This type of aggressive breast cancer represents around > 80% of the invasive carcinoma. It

initiates mainly in the ductal lining and spread out within the nearby breast tissues as shown in

figure (1.9) (A). Such tumors use physiological blood stream and lymphatic pathways in order

to move outside the breast tissue which is known as metastasis. Its main diagnosis starts with

screening of a lump or mass that the physician can identify. The use of X-ray supported

Mammogram is highly considered (16).

1.3.2.2. Invasive Lobular carcinoma:

It is the second most common type of aggressive breast tumors that accounts for around 15%

of the breast tumor cases. It spreads out of the lobular tissue through the fatty and connective

tissues as shown in figure (1.9) (B). Usually, the lobular carcinoma in situ (LCIS) is the main

precursor of this type. Its metastasis is mainly located within the abdominal area such as colon,

uterus, and ovary.

Figure 1.9: Illustrative figure shows the difference between invasive ductal carcinoma and invasive lobular

carcinoma (17)

1.3.3. Molecular subtypes (Receptor mediated breast cancer):

Clinically, breast cancer tumors have been categorized according to the presence of certain

hormonal known receptors and the status of these receptors. Histologically, there are three

types of cellular receptors that are attached to breast tumor cells; estrogen receptors (ER),

progesterone receptors (PR) and human epidermal growth factor receptor 2(HER2) as shown

in Figure (1.10) (18).

12

Figure 1.10: Three basic types of receptors within breast tumor cell (19)

1.3.3.1. Estrogen receptor mediated breast cancer:

Around 65 % of breast cancer tumors are estrogen receptors positive (ER+) meaning that these

tumor cells have estrogen receptors in the outer surface of their cellular membranes.

Physiologically, estrogen receptors are functioning as stabilizers for the effect of the

endogenous estrogen and, in some cases, responding to other therapeutic agents (20). Only 15-

20 % are considered to be estrogen receptors negative (ER-). This type of breast cancer is

riskier than ER+. Patients with ER+ tumors are typically treated with hormonal therapy,

immunotherapy and/or chemotherapy. They respond well to such types of therapies as shown

in Figure (1.11).

Figure 1.11: The effect of anti-estrogenic drug Tamoxifen up on placebo and tamoxifen treated breast cancer

patients. (21)

13

1.3.3.2. Progesterone receptor mediated breast cancer:

Progesterone (PR) is one of the endogenous steroidal hormones that is involved in different

physiological processes such as embryogenesis, pregnancy and menstruation cycles. It has

been stated that progesterone is involved, also, in breast cancer tumor progress.

Physiologically, PR is functioning as an activator for rapid activation of protein kinase that is

involved in some modification processes of proteins. Additionally, it acts as a mediator for

signals transduction pathways within the breast tissues. Therefore, it has been found that the

presence of such PR receptors can play a role with the development, growth and even the

treatment of the breast cancer. Likewise, in ER mediated tumors, some patients have been

found to efficiently respond to hormonal treatment. Those are patients with PR+, respond to

anti-progesterone drugs as shown in Figure (1.12) While others with PR- don’t respond well

to hormonal therapy and they respond, majorly, to chemotherapy and immunotherapy (22).

Figure 1.12: The anti-progesterone action with a breast cell mediated progesterone (23)

1.3.3.3. human epidermal growth factor receptor 2(HER2) breast cancer:

HER2 is an over expressed oncogene that is naturally existent on the external surface of normal

breast and tumor breast cells. Normally, in a healthy woman, HER2 is responsible for cell

growth, division and self-repair. Basically, HER2 has been discovered during early 1980s, and

it has been stated to be one of the key factors that participate in the development of breast tumor

within 15 – 20 % of the total patient population. This oncogene is responsible for encoding

various types of fundamental proteins that are responsible for maintaining persistent tumors

14

growth. The overexpression of HER2 gene in breast cancer patients is originated by its cellular

mutation related over amplification, and this results in initiation of too many HER2 receptors

as shown in figure 1.13. Theses receptors are responsible for uncontrolled growth and division

of tumor cells (24).

Figure 1.13: Difference between HER2 positive and HER2 negative breast tumor cell (25)

HER2 (+ve) tumor cells provide their own tumor growth through a proliferative, angiogenic

and cell progressive pathway as shown in figure (1.14). This pathway is enzymatic based

network that is supported with many different signals that develop a cascade manner which

ends up with uncontrolled growth of the tumor (26).

Figure 1.14: HER2 signaling pathway with a cascade manner that ultimately target the final breast

tumorigenesis (26)

15

1.3.4. Triple Negative Breast Cancer (TNBC):

This is a major heterogeneous challenging aggressive type of breast carcinoma that occurs in

young women. TNBC is mainly characterized by the absence of the most important three types

of receptors that feed the tumor cells; ER, PR and HER2 receptors. This represents a distinct

challenge in front of so many trials for controlling the growth of such type. The distinct

aggressive nature associated with this type is related to the lack of targeted therapy besides its

critical pathological nature. It has been estimated that TNBC represent around 20% of all

worldwide invasive breast cancer patients as shown in figure (1.15) (A) (27).

(A) (B)

Figure 1.15: The Incidence rates of molecular types of breast cancer (A) and subtypes of TNBC (B) (28)

TNBC has many different molecular subtypes as shown in Figure (1.15) (B) and Table (1.1);

Luminal A, Luminal B, Immunomodulatory (IM), mesenchymal stem-like (MSL),

mesenchymal (M), basal like 1 and 2 (BL1, BL2), luminal androgen receptors (LAR) and

normal tumors (29). The molecular profile for TNBC entails that the majority of infected

patients are basal like category. This category is characterized by overexpression of CK5 and

CK14 genes, which are used immunohistochemically for diagnosis of solid breast carcinomas

like intraductal papilloma with usual ductal hyperplasia (IPUDH).

Table1.1: Various subcategories of TNBC with their relative genetic characteristic behaviors (30)

16

Figure 1.16: Pathogenesis of breast cancer including both molecular and histologic types (31)

17

1.4. Stages of breast cancer:

Many worldwide health and treatment organizations that are concerned with the global public

health have proposed and classified different stages of breast cancer according to many factors.

For instance, size of the tumor, hormonal receptors overexpression, type of the tumor, how

deep the tumor is within the healthy unaffected breast tissues, metastatic tendency to nearby

organs and spreading to the nearby lymph nodes. However, the most often applied system for

staging has been the American Joint Committee on Cancer (AJCC) (32). This system is called

TNM system that stands for (Tumor Node Metastasis), and it classifies breast cancer according

to two main sub-classifications; clinical and pathological (Table 1.2)

Table 1.2: TNM grading system for different stages of breast cancer (33)

TNM Description Stage patients (%)

Tumor (T)

T0 Tumor is not formed 0 NA

T1 Tumor < 2 cm 1 6.8%

T2 Tumor (2 cm – 5 cm) 2 19.7%

T3 Tumor ≥ 5 cm 3 28.4%

T4 Tumor of any size 4 45.4%

Lymph node (N)

N0 No tumor in lymph nodes 0 20.9%

N1 Non movable tumor 1 24.9%

N2 Movable Tumor 2 31.7%

N3 Tumor in 4 – 10 lymph

nodes

3 22.5%

N4 NA 4 NA

Metastasis (M)

M0 No metastasis 0,1,2,3 83%

M1 Distant metastasis 4 17%

18

o Stage 0:

This is the earliest stage of breast cancer that is characterized by a noninvasive nature of

the tumor, and sometimes called in-situ carcinoma. The cancerous and non-cancerous cells

are separated from each other and there is no evidence of any cancerous invasion. The

prognosis and treatment possibilities in this stage are high, since there are separate

boundaries between the growing cancerous cells and healthy cells. DCIS can be considered

as example of such stage.

o Stage 1:

This is the secondary stage that provides a descriptive picture of invasive carcinoma.

Sometimes microscopic examination of such invasion is a rigid evidence of this clinical

grade. Through this examination, physicians can define the general size of the tumor and

the involvement of the nearby lymph nodes. Some studies differentiate between stage 1A

and 1B (33). In stage 1A, the tumor’s size can reach 2 cm while keeping the adjacent lymph

nodes unaffected. While in stage 1B, the tumor’s size may reach 0.2 mm while the adjacent

lymph nodes are affected and involved in the tumor growth.

o Stage 2:

The size of the tumor in this stage is < 5 cm and having the adjacent lymph nodes affected

with the tumor. Likewise, stage 1, this stage is classified into two sub classes; 2A which

has a tumor growing in the axillary or sentinel lymph nodes, while the breast tissue has no

tumor. On the other hand, in 2B subclass, the tumor is large and reaches to 5 cm, though it

doesn’t reach the adjacent lymph nodes.

o Stage 3:

This is one of the advanced stages of breast tumor in which the axillary lymph nodes are

highly involved with having a large tumor size of 5 cm. It can be sub-classified into three

stages; A, B and C. Breast tumor of stage 3A, tumor is found to be between 5 – 9 axillary

lymph nodes. In stage 3B, Tumor is found in 9 axillary or sentinel lymph nodes. This type

is called inflammatory breast cancer since it has a swelling of the affected skin area. In

stage 3C, tumor is found in more than 10 lymph nodes.

o Stage 4:

This stage of breast cancer is known as metastasis, and characterized by the spread of tumor

cells to the nearby organs including distant organs such as liver, bone, colon and brain and

19

distant lymph nodes. Physicians may call this tumor type ‘De Novo” and it has some

possibility at this stage that the tumor is a recurrence of a previous breast cancer.

Table 1.3: Five years survival rates of different stages of breast cancer (34)

Stage Description 5 years

survival rate

Stages 0 and 1 Primary diagnosed cancer (high curability rate) 90 %

Stage 2 Early stage breast tumor (intermediate curability rate) 70%

Stage 3 Advanced carcinoma (augmented therapy is required) 48%

Stage 4 Late stage metastatic breast carcinoma (very low

curability rate)

20%

1.5. Breast cancer current therapy:

1.5.1. Surgery

Surgery is the main traditional management strategy that has a prominent role in evading breast

tumor. This strategy is prioritized over many other treatment techniques for those patients

whose tumors are localized and did not metastasize to further areas of the nearby body organs

(35). Additionally, surgery is highly considered, also, for such patients who are at late complex

stages of breast cancer. In these stages, the removal of axillary or sentinel lymph nodes and

other breast lesions are the main targets for performing a surgery as showed in Figure (1.17).

(A) (B)

Figure 1.17: (A) Removal of sentinel lymph nodes with blue colored drainage, (B) removal of axillary lymph

nodes (35)

20

o Mastectomy:

This is one of the well-known breast surgeries that entails the removal of the whole breast

tissue including the outer skin tissue and axillary lymph nodes. According to many studies,

this is one of the options that are offered for early stages patients such as primary stages;

1,2 and 3 and non-invasive DCIS and LCIS patients. Some preventive procedures, also,

may recommend such surgery for patients who are believed to be at high risk of having

later breast carcinoma (36).

o Lumpectomy:

It is a partial surgical procedure that is less inclusive than mastectomy. It entails the removal

of a benign part of the breast. In other words, lumpectomy is a partial mastectomy that is

highly recommended for patents whose malignant tumors are surrounded by healthy non-

cancerous tissues including lymph nodes. Notably, most surgeons and patients as well

recommend lumpectomy than mastectomy owing to their high concern with losing breast-

based asexuality (37).

1.5.2. Radiotherapy:

It is one of the highly considered treatment methodologies that proved its effectiveness in

evading and control many breasts malignant tumors’ growth. Radiotherapy could be a

completely curative treatment for some breast cancer cases. A combination therapy that

combines radiotherapy with many other therapeutic techniques may enhance the survival

benefits in many breast carcinomas. Wang et al has recently conducted a rigid conclusion of

minimizing the breast tumor recurrence rates of 50% within 10 years survivals when radiation

therapy has been combined with breast tumor surgery (38). Moreover, the death rate, also, has

been decreased by 20% for 15 years.

21

Figure 1.18: Different types of radiation therapy based on the affected area and the conducted surgery (39)

Clinically, many randomized and retrospective studies have reviewed the acute and chronic

toxicity profiles associated with adjuvant radiotherapy on breast cancer patients. The size of

the breast is a one indicative factor that may enhance or diminish the radiotherapy toxicity as

shown in figure (1.19).

Figure 1.19: Different Toxicity and severity rates with different breast sized patients (40)

Harsolia et al. has demonstrated a strong relationship between the breast edema, dermatitis and

chronic hyperpigmentation relative to the intensity of the breast size. Besides, Shah et al. has

concluded the same sever rates of these aforementioned adverse effects. Moist desquamation,

subcutaneous fibrosis and breast shrinkage have been reported by Pignol et al. for 5 years

simple radiation therapy as shown in Figure (1.20) (41).

22

(A) (B)

Figure 1.20: (A) Radiotherapy related breast skin fibrosis, (B) Moist desquamation of a radiotherapy affected

breast (42)

1.5.3. Hormonal therapy:

In premenopausal patients, the prevention of estrogen action is achieved through two major

techniques; ovarian ablation or the use of therapeutic estrogen analogues such as Luteinizing

hormone releasing hormone (LHRH) like goserelin (Zoladex©). In the same context, selective

estrogen receptor modulators (SERM), like tamoxifen and raloxifene, is frequently used as

adjuvant treatment for ovarian cancer and breast cancer. It has been reported by a collaborative

study that tamoxifen has shown a significant reduction of the tumor recurrence rate by 74%, it

could also minimize the death rate by 26% (43). Another adjuvant hormonal treatment that has

attracted some oncological interest is selective estrogen receptor degrader (SERD) like

fulvestrant. This type of therapy is used to damage or degrade the estrogen receptors. It is often

applied for post-menopausal women and sometimes it can be combined with goserelin in order

for augmenting their antagonizing effect against breast tumor.

Postmenopausal breast cancer patients, who represents quiet higher rates than premenopausal

patients, have 80% of their breast tumors ER+. Physiologically, estrogen in postmenopausal

women is produced in minute quantities through some organs such as breast tissues, liver and

adrenal glands. That is because their ovaries are no longer producing sufficient estrogen. It has

been shown according to Czick et al. that some hormonal treatment, such as tamoxifen and

aromatase inhibitors (AIs), can effectively stop the conversion of androgens hormones to

estrogens through inhibition of the aromatase enzyme as shown in Figure (1.21) (44).

23

Figure 1.21: Inhibition of estrogen action through Hormonal therapy (45)

1.5.4. Chemotherapy:

Anthracyclines (a subcategory of chemotherapy) has been reported by many studies as the most

potent class of chemotherapeutics that can interfere with such enzymatic associated cell growth

(46). The initiation of redox reactions by anthracyclines has a powerful role on generating

reactive oxygen species (ROS). These ROS causes oxidative stress and cancerous cell death.

In the same context, anthracyclines interfere with topoisomerase II enzyme whom inhibition

arrest the cancerous cell growth. Therefore, the use of chemotherapy is highly recommended

in sever cancer types, such as TNBC and HER2+, including stages 3and 4. These late stages

have characteristic inclusion of metastasis and lymphatic carcinomas with independence of

size of the tumor, patient age and nodal status (47).

On the other hand, having various therapeutic alternatives is always recommended owing the

wide adverse effects that can be clinically decisive in shifting between different treatment

options. Chemotherapy has been listed by WHO as one of the top recommended treatment

plans of breast carcinoma. Chemotherapy has been reported as a source of generating ROS,

this caused a programed cardiotoxicity for patients who receive various chemotherapeutic

agents. It’s been for many years with a great conflict of the imbalance between the risks and

24

befits of using chemotherapy and the evoked cardiotoxicity. Besides, the significant adverse

effect of hair loss associated with chemotherapy has been discussed with many retrospective

studies (48). The other mild to severe adverse reactions of chemotherapy have been reported,

also, by many clinical studies are listed in Table (1.4). They include nausea, vomiting, fever,

abdominal pain, photosensitivity, constipation, mucositis, azoospermia, co-enzyme Q10

deficiency, alopecia, injection site reaction, rash, pruritis and infection (49).

Table 1.4: Various adverse effects of using chemotherapy after one-month treatment (50)

1.5.4.1. Doxorubicin:

It is one of the chemotherapeutic anthracycline type of antibiotics that is obtained from a

specific bacterium species called Streptomyces Peucetius as shown in figure (1.22). DOX has

been used for the curing of solid based cancerous tumors in all ages; children and adults. It has

been used, also, for effective treatment of soft tissues tumors such as ovarian cancer, bladder

cancer, neuroblastoma, thyroid cancer, soft tissue sarcoma and breast cancer. It has been

approved by FDA at 1974 as one of the safe medicines for cancer treatment. Additionally, it

has been managed to be one of the tops WHO lists of the most effective drugs against cancer

by many health practitioners (52).

25

(A) (B)

Figure 1.22: (A) Chemical formula of DOX, (B) DOX bacterial strain of Streptomyces Peucetius (51)

1.5.4.2. Mechanism of action of DOX:

DOX have been elucidated with its intense ability to confine and limit the cancerous tumor’s

growth with various modes of actions.

o First, generating stressful ROSs. DOX and anthracyclines, in general, contain

hydroxyquinone which is chelating structure for elements like iron. By the aid of some

mitochondrial mediators such as NADH dehydrogenase and cytochrome P450

reductase, iron-DOX complex enhances the conversion of electrons from glutathione

to oxygen and its derivatives. This results in accumulation of ROS and production of

free radicals as shown in Figure (1.23) (53).

o Hydroxyl radicals, single oxygen, superoxide ions and hydrogen peroxide are

accumulated inside the cell and can’t be detoxified. This causes DNA damage and

gradually trigger cancerous cell apoptosis. The cell membrane lipids are exposed to

peroxidation, and this causes rabid destruction of cell membrane. Loss of energy and

activated lipid metabolism are other findings for such the oxidative stresses (54).

26

Figure 1.23: Programmed cell death of cancerous cell with DOX (anthracycline) (54)

o Second, it interferes with the actions of Topoisomerase-II (TOP II) enzyme which is

fundamental enzyme for the completion of DNA structural stabilization. Therefore,

DOX arrests the relegation of the small DNA breaks and, thereby, block the cellular

growth. Then, finally, it enhances gradual apoptosis.

o Third, DOX has an intercalation activity between the double helical base pairs of the

cancerous DNA. Therefore, it interferes with the synthesis of DNA and RNA, and this

in turn inhibit cancerous cell division (55)

o Finally, it has been reported by Chapner et al. that DOX interferes with the function of

vascular nitric oxide synthase, thus disrupt the vascular tone of the angiogenic blood

vessels that transfer blood and nutrients to the tumor area (56). It is effective, also, to

kill the disseminated cancerous cells that are resistant to hormonal therapy or are

metastatic. Various generic pharmaceutical products are listed in table (1.5).

Table 1.5: Examples of generic pharmaceutical market products (57, 58)

Generic drug Conc. Administration company Year

Adriamycin® 2mg/mL Intravenous West-Ward Pharmaceutical

Corps

1996

DOX HCL® 2mg/mL Intravenous Fresenius Kabi 2000

27

DOX HCL® 2mg/mL Intravenous Pfizer Inc. 2011

DOX HCL® 2mg/mL Intravenous Sun Pharmaceuticals 2012

DOX HCL® 2mg/mL Intravenous Actavis Pharma 2014

DOX HCL® 2mg/mL Intravenous Athenex Pharmaceutical

Division

2017

DOX HCL® 2mg/mL Intravenous Dr. Reddy's Laboratories Inc

2018

1.5.4.2.1. Limitations of non-targeted DOX:

The main hindrance to DOX clinical anticancerous application has been its non-selective

pharmacological activity against healthy and tumor cells. This poor selectivity generates dose

dependent toxicity and severe side effects within normal cells. One of the widely conducted

sever toxicity is its known intense cardiotoxicity. The second considerable hindrance is the

elevated levels of DOX cellular resistance. These two main reasons besides many other adverse

drug reactions are the top restricting clinical effects that cause treatment failure (59).

It has been reported that 11% of patients receiving DOX are experiencing these adverse

reactions within the first few days of treatment. Furthermore, Oikonomou et al. has reported

that cardiac arrhythmias have been manifested in 26% of DOX receiving breast cancer patients

during the first 5 years of treatment. Another clinical study, Luu et al., has revealed a 50%

mortality within the first year of DOX administration being manifested with acute

cardiotoxicity and congestive heart failure (CHF) (60). Some other drug-drug interactions are

summarized in table (1.6).

Another drawback for the conventional use of DOX is the elevated drug resistance levels owing

to the multiple frequent dosing system. The severity and clinical grade of the tumor are the two

main reasons for such multiple doses. This elevated resistance is associated with many reasons;

first, the overexpression of multiple drug resistance -1 gene (MDR1) and multi drug resistance

protein (MRP). Second, impaired DNA repairing, which improve the tumor cell apoptotic

resistance. Third, mutation and enzymatic degradations of Topoisomerase II, which change the

expression level of topoisomerase II. Finally, alteration of the cellular membrane fatty acid

composition that deteriorate some cellular repair mechanisms (61).

28

Table 1.6: Drug-drug interaction with resulted adverse reactions of doxorubicin vs some frequently administered types of drugs for a cancer patient

Co-administered drug Drug-drug interaction Mechanism Solution Ref.

N-nitrosouria

(streptozocine)

Increased DOX toxicity and

bone marrow suppression

Prolong the half-life elimination

of DOX

Decreased doses of DOX (62)

Digoxin Decreased blood serum level of

digoxin

Reduced intestinal absorption of

digoxin

Changing the does regimen of

digoxin

(63)

Quinolone antibiotics I. Increased DOX toxicity

II. Decreased antibacterial

activity of quinolones

I. Inhibited cytochrome P450

3A4 and cytochrome P450

1A2; responsible for dox

metabolism

II. Decreased systemic

absorption of quinolones

Caution and dose monitoring (64, 65)

Cyclophosphamide Added cardiotoxicity Reduced metabolism of DOX Avoided (66)

Barbiturates

(phenobarbital)

Decreased DOX efficacy increased plasma clearance of

DOX

One of them should be stopped

based on a risk-benefit ratio

evaluation.

(67)

Cyclosporine Increased DOX related

cardiotoxixity with a high

serum level of more than 50%

Cyclosporines interfere with P-

glycoprotein and reduce DOX

metabolism by reducing CYT

P450 enzyme.

Caution with regular checking (68)

Antiviral (stavudine and

zidovudine)

Minimize the antiviral activity Decreased intracellular

activation

Avoided (69)

29

1.6. Targeted drug delivery:

In the same sense and over the past decade, researchers have combined the use of

pharmaceutical sciences to nanoscience as a trial for achieving highly effective targeting

treatment. This combination has brought different optimized formulas for the targeted delivery

of wide variety of components such as proteins, antibodies, narrow therapeutic indices drugs,

poorly water-soluble drugs, organic based drugs and polymeric drugs. Besides, the implication

of nanoscience has provided as new controlled release drug delivery systems which is

therapeutically outstanding and cost effective as well. Nanoparticles could effectively prove its

durability and efficacy as a successful nano-based platform for the synthesis of promising

anticancer treatment. The scientific concept behind this system is shown in Figure (1.24). The

nanoparticles act as a protective carrier that carries an entrapped drug. This drug, whether water

soluble or insoluble, is carried either inside it or upon the particle’s surface. The loaded amount

of drug is released in a controllable manner over certain time. The nanoparticle is marked with

certain biological marker in order for targeted and specific delivery of the entrapped drug. This

biological marker can be antibody, protein or DNA molecule (70).

Figure 1.24: Simplified illustration of a nano-based drug delivery system (70)

1.7. Nanotechnology:

Nanotechnology has been shown to the light of medicinal and bio-medicinal applications for

many years. It could open the door in front of fabrication of various nanosized material

formulations that have been used within many various applications. Various Nano formulated

shaped material have been reported in many studies including dendrimers, polymeric

nanoparticles, quantum dots, core-shell nanoparticles and carbon nanotubes (figure 1.25). All

these nano- formulations have been fabricated with high control over their chemical, electrical,

physical and biological safety applications (71). Generally, a material within the nanoscale

30

behave as if it is completely new material in terms of optical, magnetic, chemical and physical

performance, especially when its intended application is size dependent.

Figure 1.25: Nanometer scale with relative sizes of various biological objects (72)

Synthesis of nanoparticles can be engineered for performing various surface and core

modifications within the nanoparticles. Two main approaches for manufacturing nanoparticles;

bottom-up and top-down approaches. During both methodologies, nanoparticles can be

conjugated with different types of ligands including functional chemical groups, biological

molecules, permeation enhancers, targeting moieties, magnetic dyes or surface cross-linkers.

Furthermore, the beauty of nanotechnology, in general, is that there is a good control over the

resulted final geometrical shape of the nanostructure. For instance, nano-rod, nano-spheres,

nano-cubes, nanofibers and nanowires as shown in Figure (1.26).

(A) (B) (C)

Figure 1.26: TEM images of various shapes of nanostructures; (A) nanoparticles, (B) nanocubes and (C)

nanorods (73)

31

Biologically, nanotechnology develop another spectacular manipulation of some serious

specifications such as stability in different pH, surface charges, and drug release. These

properties are extremely critical whenever the intended application has been biological or

therapeutic. Therefore, there are five available routes of administration for any nano-based

structures as illustrated in Figure (1.27). That is because there are many variables included to

affect the intended nanoparticles such as immune response, enhanced permeability retention

(EPR), lymphatic drainage and the pH of the diseased or affected organ area. All these factors

can strengthen or weaken the pharmacological action of the loaded drug. Breast cancer has

some characteristic pathological features for which nanotechnology can be a considerable

solution for such problematic issues.

Figure 1.27: Five routes of administration for nano based drug delivery systems

1.8. Nanotechnology enhanced pH manipulation:

First, breast tumor environment is generally acidic in nature. Some reports have reported that

the microenvironment of the breast tumor is ranging between pH (6.5 – 6.9). While the normal

blood stream pH value is neutral to slightly alkaline; ranging between pH=7.2 – 7.5 (75). The

reason for this pH difference is known as Warburg effect (76). This phenomenon differentiates

between healthy and cancerous cells. Normally, mitochondrial phosphorylation is the main

source of energy for the cell to obtain ATP. While cancerous cell uses metabolic glycolysis to

obtain its energy. This glycolysis ends up with accumulated levels of pyruvate and lactate. This

32

occurs within anerobic conditions that are attained by the action of tumor acidosis which

minimize the amount of oxygen that is required for keeping the organ area healthy as shown in

Figure (1.28).

Figure 1.28: Schematic representation of mitochondrial phosphorylation and glycolysis (78)

Furthermore, DOX has been reported by Fukamachi et al. as one of the chemotherapeutic drugs

whose therapeutic efficacy is not changed between the acidic and alkaline environments.

Indeed, it rather showed more cytotoxic efficacy within highly acidic and larger tumor sizes

than smaller less acidic. These results are agreed, also, with Hiroshi et al. (79). In other words,

the larger the tumor size, the higher therapeutic efficacy that DOX has Figure (1.29).

Figure 1.29: pH dependency scale relative to cancerous tumor size (79)

33

1.9. Nanotechnology enhanced surface charge manipulation:

Second, and on the same context, cancerous cell membrane of the tumor cell is negatively

charged. That is because the accumulated lactate anions tend to remove the positive ions and

leaving the cancerous cell membrane with negative charges. The poor blood perfusion is a

supporting factor for this cellular reaction. Therefore, the fabricated nanoparticles that are

intended to work in such environment should have certain specifications. They are preferred to

have a net positive charge upon their surfaces, so that an electrostatic interaction can maximize

the internalization rate of the positively charged nanoparticles. Besides, it has been developed

by Osaka et al. that the accumulation rates of nanoparticles would be greater when electrostatic

interaction between the nanoparticles and cancer cell membrane takes place. This would

provide better drug diffusion with a controlled release behavior as shown in Figure (1.30) (80).

Figure 1.30: Electrostatic interaction between nanoparticles (+ve) and cancerous cell wall (-ve) has developed

instant drug release (80)

1.10. Nanotechnology enhanced biomarker attachment:

One of the main effective advantages that entails the great role of nanotechnology in

therapeutics is the ability of attaching targeting moieties to the surfaces of the drug carriers.

These moieties are acting as a guiding monitor that direct the nanoparticle or nanocarrier from

the site of drug introduction to the site of action which is breast area. Attaching such targeting

moiety has been a great next step for a new era in designing effective drug delivery systems.

Nanotechnology enables many researchers to conduct various types of biomarker attached

nano-formulations that are directed for the treatment of various diseases. Some examples for

various nano-based drug delivery systems are shown in Table (1.7). The guidance of such nano

formulations is going to be routed in one of two targeting pathways; active and passive

targeting Figure (1.31) (81).

34

1.10.1. Passive targeting:

Passive targeting entails the accumulation of the injected nano formulations within the tumor

tissue with enhanced extravasation out of the tumor. Theses targeted nano formulations could

efficiently penetrate the tumor through some factors such as their small nano-size, leaky

vasculature, disorganized permeable blood vessels. The main reasons for such leaky nature of

these blood vessels are hypoxia, inflammation and lack of nutrients due to high metabolic rate.

This leakage tendency is referred as enhance permeability retention (EPR). Within these leaky

permeable vessels, larger pores are formulated and allow transfer of particles with size up to

400 nm (82). Some studies have assumed a much broader range of these pores’ sizes of (380 –

780) nm (53).

1.10.2. Active targeting:

Active targeting is highly efficient ligand-receptor interaction that promote a targeted and

precise drug release within the tumor environment. This interaction is taken place between the

overexpressed surface epitopes of the tumor cell and the attached ligand biomarkers. It is more

efficient than passive targeting (83). The entrapped drug is guaranteed to be released and

diffuse inside the cell with active targeting. Once the ligand receptor conjugation takes place,

plasma membrane close so that an internalization endosome is formulated.

Figure 1.31: Schematic illustration that differentiate between active (A) and passive targeting (B) (84)

35

Table 1.7: Example of different nanostructured drug delivery formulas that are assisted with various biological targeting moieties.

Nanostructure Monitored

disease

Targeting

moiety

Target Entrapped drug Mode of

action

Ref.

Multiwalled carbon

nanotube

Colon cancer Hyaloronic acid CD44 receptors that are over expressed on

colon tumor cells

Gemcitabine

(chemotherapeutic

anticancer)

(Gemzar ®)

Intracellular (85)

Liposomes

Non-small

cell lung

tumor

GE11 peptide Epidermal growth factor receptors

(EGFRs)

DOX

(chemotherapeutic

anticancer)

Intracellular (86)

Dendrimers

Ovarian

cancer

Primary antibody

(mAbK1)

Mesothelin, a glycosyl-

phosphatidyl inositol (GPI) protein

Paclitaxel

(chemotherapeutic

anticancer)

Intracellular (87)

Polymeric micelles

Pancreatic

cancer

anti-tissue factor

(TF) antibody

(clone 1894

Pancreatic overexpressed tissue factors

antigen receptors.

Oxaliplatin (platinum

derived

chemotherapy)

(Eloxatin ®)

Intracellular (88)

36

1.11. Nanotechnology enhanced prolonged biological circulation:

One of the critical factors that affect the therapeutic efficacy of the intended Nano formulated

drug is its rapid biological clearance. During this biological journey, this nano-formulation

encounter many biological defending mechanisms that slow down their migration and, in turns,

speed up their elimination through lymphatic drainage. Immunologic reticuloendothelial

system (RES) and mononuclear phagocyte system (MPS) are phagocytic systems that is

majorly existent in the liver (89). This system is capable of capturing the nanocarrier and

deteriorate its action through immunological phagocytosis.

Generally, in order for a drug carrier to be therapeutically effective, it must have some criteria

available upon its biological ingestion. It must be (1) non captured and escape from the RES

and MPS, (2) of high stability profile along over its biological journey till reaching the tumor

area, (3) circulate freely within the systemic blood circulation without being encountered by

various immunological responses and (4) accumulate within the tumor area so that a

therapeutic concentration of the drug is released (90). For this purpose, some polymers are

being suggested to be used so that we ensure high versatility and solubility of the nanocarrier.

Hao et al. have suggested the use of polyethylene glycol (PEG) and poly (lactic co-glycolic

acid) (PLGA) as hydrophobic co polymers in order for solubilization of water insoluble drugs.

Owing to the biological performance, this study has reported the preference of coated

nanoparticles than uncoated nanoparticles. Another study, Nadeem et al., has developed the

use of poly-vinyl alcohol (PVA) as a coating material for iron oxide nanoparticles-based drug

delivery system (91).

1.12. Chitosan as a nanoparticle platform:

CS has been therapeutically used as a polymer based nano drug platform (s) for the delivery of

various drug types that are intended for the treatment of many diseases like breast cancer.

Different nano-formulations of CS have been suggested to be used for such biomedical

application such as CS nanoparticles, nano fibers, nanoliposomes and nanogels (92). CS

possesses a prolonged circulation behavior and low immune clearance rates. Besies, it has what

is called shielding effect against RES system that retard its detection (93). Furthermore, CS has

the ability to efficiently penetrate the tight junction of the epithelial membranes, therefore, it

has an improved permeation through this junction.

37

Chitosan nanoparticles (CSNP) have been used as a successful drug carrier of many anticancer

agents for breast cancer therapy. Many studies have conducted a better antitumor activity for

CSNP loaded with various anticancer drugs compared to these free unloaded drugs. Table (1.8)

is showing the potential of some CSNP to be used as efficient drug delivery systems in breast

cancer therapy. CSNP can be synthesized with many applicable preparation techniques such as

ionotropic gelation, emulsification-crosslinking, co-precipitation, reverse micellization,

microemulsion, desolation, solvent evaporation and coacervation (94).

Figure 1.32: preparation of CS by deacetylation of chitin (95)

Ionotropic gelation is a simple and direct preparation technique that allow synthesis of

crosslinked CSNP within aqueous conditions. It represents an electrostatic bridging between

positively charged CS (through amine NH2 group) and negatively charged polyanionic polymer

such as tripolyphosphate (TPP), genipen and glutraldehyde. The result of such bridging is

formation of a complex gel like structure that represent crosslinked CSNP. Therefore,

ionotropic gelation is more preferred than other techniques. Besides, it doesn’t require the use

of extensive preparation conditions such as high-tech procedures and the burdens of using

organic solvents (96).

38

Table 1.8: Some CSNP formulations used for breast cancer drug delivery

Nano-

formulations

Coating

agent

Particles size Synthesis Drug Target

cancer

Route of

administration

Mechanism of

action

Ref

CSNP +

PLGA

PLGA (90 – 105) nm Ionotropic

gelation

DOX Breast cancer Topical

(implant)

Passive targeting

(EPR,

extracellular)

(97)

CSNP Uncoated (100 – 150) nm solvent

evaporation

Tamoxifen Breast cancer Systemic

(intravenous)

pH responsive

(extracellular)

(98)

CSNP

(micellar

form)

α-tocopherol

succinate

81.4 ± 10.5 nm

coupling

reaction

Paclitaxel and

Docetaxel

Breast cancer Systemic

(intravenous)

Charge affinity

(intracellular)

(99)

CSNP (core-

shell)

PEG (34 – 48) nm Copolymer

grafting

Anti-HER2 drug

(trastuzumab) +

DOX

Breast cancer Systemic

(intravenous)

Targeting ligand (100)

CSNP Thiolation (200 – 700) nm Self-assembly DOX Breast cancer Systemic

(intravenous)

Targeting ligand

enhanced

antibody-antigen

interaction

(101)

CSNP Uncoated 473 ± 41 nm

Ionotropic

gelation

DOX General

cancer

therapy

Systemic

(intravenous)

EPR (102)

39

TPP has been shown to be more biologically safe than other polyanionic gelation polymers as a

cross-linker for synthesizing CSNP. It is one of the considerable factors that influence many

specifications of the resulted nanoparticulate system such as size average, surface charge, and

particles distribution (see appendix SI). Moreover, the mixing technique of TPP into acidic CS

aqueous solution affect these parameters as well. For instance, dropwise addition, one-shot

addition and dilution techniques are three different mixing procedures that lead to variation of size,

distribution and potential of the resulted CSNP (103).

Figure 1.33: chemical formula of Sodium tripolyphosphate (104)

1.13. Polyethylene glycol (PEG):

PEG is a synthetic hydrophilic based polymer that has been used in numerous pharmacotherapeutic

applications such as wound dressing and drug delivery. It has unique chemical properties such as

biocompatibility, solubility, neutrality, non-irritating and odorless. It is composed of a linear chain

of polyether with hydrophilic terminal groups of OHs as shown in figure (1.34). This hydroxyl

group is responsible for its hydrophilicity, and it acts as electron donor. This electronic donation

is responsible for many chemical characteristics of PEG including its good compatibility with

organic solvents through weak hydrogen bonding interaction. This allow rapid release of the

entrapped pharmaceutical drug. PEG has low melting temperature and fast solidification rate;

therefore, it can be used preparation of chemical and organic dispersions (105). It has been

approved as biodegradable polymer with non-immunogenicity and nonantigenicity effect for safe

biological applications by FDA (106). For these reasons, it is widely used as encapsulating or

coating agents with various nanoparticles-based drug delivery systems

40

Figure 1.34: Chemical structure of polyethylene glycol (107)

1.14. Anti-Mammaglobin antibody (Anti-hMAM):

MAM has been demonstrated as a protein-based biomarker antigen that is overexpressed on the

cellular surfaces of all breast tumor cancer types. Many studies have encountered the analysis of

MAM as breast cancer biomarker. The results recommend the independent use of MAM as a

diagnostic and therapeutic biomarker. These promising results have put MAM as one of the

targeting ligands that entails an antigen-antibody interaction upon the membrane surface of breast

tumor cells (108).

In this context, in breast tumor cells, the membrane associated MAM interaction can play a very

important role in designing an actively targeting drug delivery system for minimizing the growth

of such tumor tissue. By conjugating MAM to a nanoparticulate carrier that entrap antineoplastic

drug will promote its therapeutic activity within the acidic based microenvironment of breast tumor

area (109). This open the door in front of nanoscience and biopharmaceutical science for

improving new methodologies of breast cancer curing. However, the number of reports that have

been conducted on the use of this biomarker for active targeted breast cancer drug delivery systems

are yet low. The potential of anticipated frequent application of such biomarker is still growing.

Lian et al. has conducted a nano-based drug delivery of doxorubicin to the breast tumor area. Anti-

hMAM has been used as a specific targeting ligand for specific targeting of breast tumor tissue

(110). The results of this study came promising with efficient specific membrane targeting and

functional drug carrying signaling system.

MAM has been discovered first in 1994 by Watson-Fleming within breast tissue cells. They could

identify MAM-A and MAM-B. The overexpression of MAM-A receptors is restricted to breast

41

cancer patients only, while MAM-B is related to various tumor organs such as ovary, thyroid,

uterus and breast (110, 111). Leygue et al. has experimentally concluded the diagnostic capability

of MAM-A for diagnosis of breast cancer in a clinical study. The study has applied reverse

transcriptase polymerase chain reaction (RT-PCR) to test the over-expression of the MAM-A

coding RNA in 20 breast tumor affected females with advanced lymph nodes tumors. The results

have confirmed its over expression with a recommendation of using MAM-A as a diagnostic target

in breast cancer.

42

Chapter 2

Materials & Methods

43

2.1 Materials:

The materials used for the current experimental work were purchased from different local and

international sources. CS polymer of low molecular weight (LMW) with 89.9% deacetylation

degree and 65 cp viscosity was purchased from Priemex ehf, Chitoclear®, Iceland. Polyethylene

glycol (PEG), acetic acid 100%, potassium bromide (KBr) (FTIR grade, ≥99% trace metals basis),

and Dulbecco’s modified Eagle’s medium (DMEM), were purchased from Sigma Aldrich, Germany.

DEMEM reagent was supplemented with 4500 mg/L glucose, L-glutamine, sodium pyruvate,

sodium bicarbonate, 10% FBS, and 5% penicillin/streptomycin. Anhydrous sodium tri-poly

phosphate (TPP) (technical grade 85%) was purchased from Sigma Aldrich®, Missouri, USA.

Doxorubicin hydrochloride (molecular weight of 579.96 g/mol) was purchased from Sigma

Aldrich®, Switzerland (Suisse). Anti HER2 and anti Mammoglobin antibodies (originated from

rabbit source) were purchased from Sigma Aldrich®, Steinheim, Germany. Phosphate buffered

saline (PBS) was purchased from BioWhittaker® Reagents, Lonza, Walkersville, USA. For

cleaning and sterilization purposes, acetone and Ethanol 99.9% were purchased from Medical Pure

Life®, Europe and Alamia®. Egypt, respectively. 1-Ethyle-3-(3-dimethylaminopropyl)

carbodiimide (EDC) was purchased from Sigma Aldrich®, Switzerland (Suisse). MTT reagent, 3-

[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide and dimethyl sulfoxide (DMSO),

were purchased from Serva Electrophoresis, Heidelberg, Germany. Fetal bovine serum (FBS),

Trypsin EDTA, Penicillin/Streptomycin, were obtained from Lonza, Switzerland. CO2 incubator

(Heracell incubator, Thermo Scientific, USA). Most of the other aforementioned chemicals and

materials were stored in a well-controlled storage conditions including ventilation, humidity and

temperature.

2.2. Preparation of Chitosan nanoparticles (CSNPs):

Chitosan nanoparticles (CSNPs) have been fabricated using Ionotropic gelation technique as

referred by many different studies with some modifications including various processing factors

as described in the supporting information appendix; part 1 (SI.1) (112). Ionotropic gelation

method is a soft and straightforward technique in which CS NPs can simply be formulated via

electrostatic interaction between acidic phase and alkaline phase. In other words, whitish clouds

44

of nanoparticles were spontaneously prepared upon addition of TPP (alkaline phase) as a

negatively charged cross-linking agent, to the positively charged poly-cationic chitosan solutions

(acidic phase) as shown in Figure (2.1). Various concentrations of acidic chitosan solutions

(0.05%, 0.25%, 0.5% and 1%) were used against various concentrations of TPP aqueous solution

(0.1%, 0.4%, 0.5% and 0.7%) as illustrated in the appendix (SI.2.1). The chitosan acidic solution

and TPP solution were filtered first with syringe filters of 0.4 and 0.2 µm pores sized filters

respectively before the crosslinking addition (113).

Figure 2.1: Cross-linked CS with TPP (113)

TPP was dropped to chitosan acidic solution in a dropwise manner within a moderate stirring

speed. The mixture was left for proper mixing while keeping the applied mixing technique, time

and rate within acceptable ranges Figure (2.2). The stirring time was adjusted to 30 min with low

stirring speed of 250 rpm at room temperature. By comparing various mixing techniques such as

hand stirring, magnetic stirring, homogenization or sonication, the magnetic stirring was

45

efficiently applied over other stated methods. It is simple and possesses a strong effect on reducing

the sizes of CS NPs (114).

Figure 2.2: General scheme for preparation of CS NPs

In order for ensuring high stability of the formulated CS NPs, some factors such as polymers’

concentrations, pH, molecular weights of chitosan, stirring time and stirring speed were studied in

a more detailed manner as shown in the appendix (SI.2). All these factors have a direct influence

on size, particle charge and stability of the resulted nano-system. Each factor was experimented

and studied independently, and the resulted recommended parameters that were applied for our

current study are listed in Table (2.1).

Table 2.1: Optimum conditions for the applied factors in preparation of CS NPs

Preparation Factors Applied parameters

Molecular weight of chitosan Low molecular weight chitosan, DA 89.9%

Concentration of chitosan 1% Acetic acid aqueous solution (1%)

Concentration of TPP 0.4% Aqueous solution

pH 5

Stirring time 30 minutes

Stirring speed 250 RPM

Temperature Ambient room temperature

46

The resulted samples of the synthesized CS NPs were representing a mélange of various

concentrations of chitosan and TPP (see the appendix, SI.2 and Table S2). After successful

formation of whitish clouds of nanoparticles, the mixtures were centrifuged for 30 min at 10,000

rpm. These specific centrifugation parameters were enough to separate the formulated

nanoparticles (115). The resulted biphasic system, after centrifugation, was divided into two parts

as shown in the appendix (SI.1.3 and table 1). The first part was the supernatant, which was

discarded since it contained the unreacted polymers and acetic acid residues. The second part

separated nanoparticles pellet was re-suspended in fresh distilled water to be washed from any

excess un-reacted chitosan and TPP. The washing process was done twice at 10 min centrifuging

time at 5000 rpm, and this part was divided according to the sampling process for further

characterization experiments (116).

2.3. Stability of CS NPs:

Stability of the formulated CS NPs as a candidate for the following anticancer tests was crucial

step. For investigating the stability of the surface of the resulted nanoparticles, their mean particles

diameter size, surface zeta potential and poly distribution index (PDI) were analyzed by using

Malvern Zeta sizer (NANO ZS, Malvern, UK) (see appendix SI.3, and Table 6) (117). An

indicative stability profile was developed, and the samples were incubated in fresh distilled water

with pH of 7.2 for three weeks at room temperature, and the required data of the size, distribution

and surface zeta potential were collected over this stated period.

2.4. Coating of CS NPs with Polyethylene Glycol (PEG):

CS NPs showed different stability profiles with different conditions, resulted in some variations

for successful delivery of the entrapped material inside these nanoparticles. Some specifications

within the physical or chemical structure of the resulted CS NPs could be modified or tailored so

that they could fit within the desired intended application. For instance, the poor water solubility

of sole chitosan in water revealed some drawbacks with some chitosan-based drug delivery

systems of some hydrophilic drugs (118). Increasing hydrophilicity of CS NPs and enhancing the

47

stability of an entrapped hydrophilic drug are two examples of specific properties that could be,

fortunately, modified with a hydrophilic polymer like polyethylene glycol (PEG) (119). The

modification adjusted by grafting this copolymer to the basic structure of CS NPs was reported in

the literature as successful platform of maintaining high nanoparticles’ stability. This process is

known as “PEGylation” and the resulted PEGylated CS NPs are called “stealth” (120).

Table 2.2: Sample variations relative to the polymeric concentrations of PEG while keeping TPP and Cs

concentrations fixed

Sample Cs (%) TPP (%) PEG (%)

1 0.4 1 5 10

1 √ √ √

2 √ √ √

3 √ √ √

PEGylated CS NPs were prepared following Calvo et al. with some modifications (121, 122). This

aims to provide high particles stability profile and enhance application durability on the final

selected PEG concentration. Three different aqueous solutions of PEG (1%, 5% and 10%) were

prepared as shown in table 2.2 by using a simple stirring speed around 500 rpm for 30 min at room

temperature. All other preparation parameters were kept constant, so that the optimal functioning

conditions can provide a sole explanation of the physical and chemical properties of the resulted

nanoparticles. After dissolving PEG in distilled water, we could identify three different PEG

concentrations whose viscosities varied according to the weighted amount of PEG powder. The

dissolution of PEG didn’t require addition of any excipients like acetic acid or any additives. That’s

because it is readily water-soluble polymer and has an FDA (Food and Drug Administration)

approval for safe pharmaceutical and food applications (123).

48

Figure 2.3: General scheme for preparation of PEGylated CS NPs

The three aforementioned prepared PEG concentrations were added drop-wisely to the chitosan

acidic solution (CS 1%). Then, the three mixtures were left for mixing for 30 min with stirring

speed of 250 rpm. This provided three homogenous mixtures of both polymers; chitosan and PEG.

The PEGylated CS NPs were formulated upon drop wise addition of TPP solution to chitosan

acidic solutions containing the previously stated PEG concentrations as shown in Figure (2.3) and

(2.4). Ionotropic gelation process started upon cross-linking of PEGylated chitosan solutions with

the polyanion TPP. This process took place immediately, and the three mixtures were left on the

magnetic stirrers for 30 min at low stirring speed of 250 rpm at the ambient temperature. Then, the

PEGylated CS NPs were centrifuged and separated from the excess unreacted chitosan, TPP, PEG

and acetic acid residues. The separation was performed by centrifuging at 10,000 rpm for 30 min

at 24°C using HERMILE Labotechnik 36 HK centrifuge (Germany) (see Appendix SI.4.4). The

resulted pellet of PEGylated CS NPs was later divided into three parts like those non-PEGylated

CS NPs that were discussed in the appendix (SI. 1.3 and table 1) (124).

49

Figure 2.4: Chemical scheme for preparation of PEGylated cross-linked CS NPs (112)

Some vital parameters were identified such as size, external morphology, surface zeta potential

and PDI of the resulted PEGylated CS NPs. The effect of various concentrations of PEG on these

parameters was analyzed. It was confirmed that change in PEG concentration could affect such

parameters of these nanoparticles. This was confirmed before by some previously published

reports (125). The effects of PEGylation process on the external positive amino groups and hence

the stability of PEGylated CSNPs were studied also in accordance with another previously

published study (112).

2.5. Encapsulation of Doxorubicin (DOX) within CS NPs:

The capability of PEGylated CS NPs to effectively entrap anticancer DOX drug was evaluated in

this phase. After successful preparation of stable, biologically durable and biocompatible

PEGylated CS NPs, we have studied different protocols that were intended for prosperous

encapsulation of a hydrophilic drug inside the core of PEGylated CS NPs’ structure (127). These

50

protocols have identified the use of different drugs including DOX. PEGylated CS NPs were the

same nanostructured matrix that was used as the platform to deliver these drugs. This study is

mainly dependent on the efficient use and modification of DOX’s pharmacological action. Many

reasons that were related to the physical and chemical properties of the intended drugs were

analyzed for the specific use of DOX over other anticancer drugs as will be discussed in more

details in the next chapter (128).

Integrating DOX within such nan-formulation was expected to provide many effective

pharmacological and non-pharmacological indications over sole non encapsulated DOX.

Furthermore, PEGylated CS NPs were expected to entrap such hydrophilic DOX and travel inside

the bloodstream without leaking the loaded drug or being captured by the immunologic responding

reticuloendothelial system (129). All preparation parameters were fixed in order to investigate the

effect of different concentrations of DOX (table 2.3) on the encapsulation efficiency. Additionally,

the preparation procedure was performed in light protected environment due to the photo-

degradation nature of DOX (130).

Table 2.3: Sample variation relative to DOX concentrations while keeping the polymeric concentrations for Cs,

TPP and PEG fixed.

Sample Cs (%) TPP (%) PEG (%) DOX (µg/ml)

1 0.4 5 1.25 2.5 5 10

1 √ √ √ √

2 √ √ √ √

3 √ √ √ √

4 √ √ √ √

After dissolving PEG in acidic chitosan solution, it was left to be mixed for 30 min as shown in

Figure (2.5) with stirring speed of 250 rpm. DOX was added to the CS-PEG mixture while

maintaining light protective performing conditions. Four different concentrations of DOX were

used; 1.25 µg/mL, 2.5 µg/mL, 5 µg/mL and 10 µg/ml. Four different drug concentrations were

obtained by the end of this step. The color of the four solutions were varied with red color intensity

according to their corresponding concentration. The drug-copolymers were left for homogenous

51

mixing in order to achieve complete chemical interaction between the positive amino NH2 group

of DOX and negative polyanion of TPP, likewise the noncovalent encapsulation between DOX

and of CS / PEG polymers. They were left for 1 hour within a mild stirring speed of 250 rpm. This

period of time was quiet enough to allow efficient interaction capability between DOX, TPP and

the polymers.

Figure 2.5: illustrative scheme for encapsulation of DOX inside PEGylated CS NPs

Finally, the polyanion TPP was added to the four solutions in order to allow sufficient cross-linking

between DOX and the other polymers. The solutions were kept mixing under mild stirring

conditions within a speed 250 rpm for 30 min. The formation of white turbid clouds inside the

three solutions appeared instantaneously upon drop-wise addition of TPP under these mild stirring

conditions. These whitish clouds, simply, represent aggregates of DOX loaded PEGylated CS NPs.

These clouds were kept in light protected conditions to avoid photo-degradation of any remaining

DOX.

The formulated CS NPs were directly separated by using centrifuging parameters at 10,000 rpm

for 30 min using HERMLE Labotechnic 36 HK (Germany) in order to be ready for further analysis

and testing. The sedimented NPs required considerable multiple washing in order to ensure

collecting pure DOX loaded CS NPs without any residue or unreacted chemicals. The unused

supernatant was analyzed each time for measuring the amount of non-encapsulated DOX. CARY

500 SCAN varian (Hi-tech, NJ, USA) UV-VIS spectrophotometer was used for characterizing the

52

presence of DOX in the produced supernatants. The instrumental response for the supernatant at

each round was plotted against the previously and basically issued regression equation of

calibration curve of DOX.

The fresh isolated pellets of DOX loaded CS NPs were divided into two main parts. The first part

had vigorous mixing or redispersion of these formulated nanoparticles in a fresh media, like

distilled water, using ultrasonic probe sonicator; Ploytron (Thermo Fisher, USA). The applied

sonication parameters were 50% Amplitude for 3-5 min. The sonicated part of NPs was, then,

diluted with distilled water so as to provide a liquid nano-system with a concentration of 1mg/ml.

This DOX loaded CS NPs suspension was characterized later for particles’ surface morphology,

shape, size, distribution and zeta potential using TEM and DLS. The second part was characterized

for the chemical interaction between DOX with the applied polymers using FTIR (Thermo Fisher

Scientific Nicolet 380 Spectrophotometer, USA). For this purpose, the second part of the NPs

pellet was freeze-dried using BIOBASE freeze drier (Jinan, China) (see appendix SI 4.2).

2.6. Conjugation of Monoclonal Antibodies (mAbs):

Attaching biological ligands to the surfaces of the nanoparticles was expected in enhance the

specificity and sensitivity of the entire nanocarrier. Anti-Human Epidermal Growth Factor

Receptors (Anti-HER2) and Anti Human Mammaglobin antibody (Anti-hMAM) have been

suggested to be used as the targeting moieties. Such conjugation my promote the cancerous

endocytosis and cancerous sell uptake. In turn, it may minimize the normal cells attachment. This

has been expected to decrease the systemic adverse drug reactions that are of the entrapped DOX

drug and minimize its oxidative cardiotoxicity. The conjugation of the mAbs has been done

according to collected procedure using some previous reports (130, 131).

In this sense, after selecting the nano-formula with 5 Ug/ml Loaded DOX, the nanoparticles have

been freeze dried. EDC has been used to activate the COOH gp on the surfaces of DOX loaded

PEGylated CSNP. After around 15 minutes slight mixing, centrifugation of the solution has

resulted in a pellet of nanoparticles that have activated COOH group on the surface. BPS has been

used for washing the nanoparticles to remove the excess EDC. On the other side, mAbs has been

53

shacked with micropipette and then was introduced to the nanoparticles mixed with EDC. EDC is

extremely hygroscopic; therefore, its storage should be highly considered (132).

Then mAbs have been mixed to the COOH activated nanoparticles. They have been left on the

orbital shaker with slight speed of 150 RPM for 2 hours. Then the two samples with two separates

different mAbs have been centrifuged at 20,000 RPM for 30 minutes. Then PBS has been used for

washing for 2 times at least with 5 minutes centrifuging at 8000 RPM. The resulted pellet has

been resuspended in fresh PBS with slight shaking in order to resuspend the nanoparticles with

surface conjugated mAbs. The ratios between the chemicals were different. For instance, the ratio

between EDC to the nanoparticles was 5:1 and the ratio between nanoparticles and mAbs was

1:50. HNMR spectrum and FTIR spectrum have been conducted to ensure the surface conjugation

of these mAbs. Besides, TEM imaging have been conducted as well in order for confirming the

surface decoration and spherical shape of the resulted nano-systems.

Degree of substitution (DS) = 𝑀𝑜𝑑(COOH)

𝑇𝑜𝑡 (COOH) × 100 ……. Eq. (2.1) (246)

2.7. Characterization of CSNPs, PEGylated CSNPs and DOX loaded

PEGylated CSNPs:

2.7.1. Dynamic light scattering (DLS):

The main application of criteria of the NPs were the smallest size, least aggregation tendency and

smallest PDI value as measured by Dynamic light scattering (DLS) enhanced Zeta sizer

instrument. The concentration of all different samples was kept constant at 1mg/ml.

2.7.2. Electrophoretic light scattering:

In the current study, we mainly used zeta potential analysis during different phases of the study in

order for investigating the surface charges of the resulted CS NPs, PEGylated CS NPs and DOX

loaded CSNPs. Any variation in the surface zeta potential indicated a change in the surface

structure, which was a good indicator of surface attachment and hence the charge density would

54

be affected with that attachment and the stability rate of the prepared NPs. The higher the value of

zeta potential, whether positive or negative, the higher the tendency of the colloidal dispersion to

resist particles attraction or aggregation. The information obtained from zeta potential analysis

have had a good directionality of which sample will be more stable and more efficient to be a

platform for DOX drug delivery.

2.7.3. Fourier transform Infrared (FTIR) spectroscopy:

FTIR spectroscopy was applied using FTIR (Thermo Fisher Scientific, Nicolet 380)

spectrophotometer to develop a graphical fingerprint of the chemical structures of the control

nanoparticulate samples (CS, TPP and PEG), to be compared with the prepared nano-formulations

including CS NPs, PEGylated CS NPs and DOX loaded CS NPs. The samples were lyophilized

for at least 72 hours under -60 C temperature. Then KBr was used with all samples to prepare the

sample pellet. KBr was lyophilized first in order not to affect the sample absorbance since KBr is

optically transparent, and acts as inactive sample carrier within the IR region (133).

2.7.4. X-Ray Diffraction Spectroscopy:

XRD spectroscopy analysis were carried out using Bruker D8 (MS, USA) in order to investigate

the internal crystalline structures of all samples including the cross-linked CS NPs, and PEGylated

CS NPs. The linkage of some polymers like TPP and PEG would provide some effect on the entire

crystalline structure of CS. Therefore, the entrapment efficiency of these polymers would be shown

with XRD analysis.

2.7.5. Scanning Electron Microscopy (SEM):

The particle’s morphology and size of the produced NPs with attachment of PEG were

characterized by using SEM. The primary characterization of CSNP and PEGylated CSNP was

done with an oven dried sample. These samples were diluted as 1mg/mL, and then moved to a

spread aluminum sheet, over which it was dried at 60C temperature for 10 min. After drying, the

sample was gold sputtered for 15 min so that the surface of the sample become conductive and,

hence, interact with the bombarded electrons and also to enhance the resolution and quality of the

images.

55

2.7.6. Transmission Electron Microscopy (TEM):

TEM analysis were performed using Ultra-High Resolution schotty Transmission Electron

Microscope SU 7000. The preparation of TEM sample was done with diluting the prepared

solutions to 0.02 mg/mL. Three different types of samples; CS NPs, PEGylated CS NPs and DOX

loaded PEGylated CS NPs were prepared. The provided information by TEM images was related

to the morphology, topography, size and particle distribution was helpful to show the successful

attachment of PEG and DOX. One drop of the diluted sample was dried and dyed with

phosphotungestin dye to keep the surface of the nanoparticles conductive; and therefore, the

electrons can be captured easily resulting in better image resolution.

2.7.7. UV-Vis Spectroscopy:

UV-Vis spectroscopy analysis were performed using CARY500 SCAN Varian UV-VIS

spectrometer (Hi-tech, NJ, USA) for different characterizations analysis that were related to DOX

concentrations. First, a calibration curve of 6 different concentration of DOX aqueous solution

using deionized water was plotted. Then, the absorbance of the unloaded free DOX that was

measured in the supernatant of DOX loaded CSNP preparation was determined at wavelength of

480 nm using clear quartz processing cell. This allowed the determination of the loading capacity

of such NPs relative to very minute concentrations of DOX that were reaching few micro liters.

Besides, the release profile of DOX loaded CS NPs has been detected with the same UV

spectrophotometer. PEGylated CS NPs was used as a black. The scattering of UV-Vis light beam

that was done by such PEGylated CSNP solution was excluded. Moreover, the UV-Vis absorption

of many different samples with various concentrations that were related to various time slots were

analyzed in order to reveal the releasing behavior of the nano formulations.

2.8. Drug Loading, Release Profile and Stability of NPs:

2.8.1. Drug loading:

Loading of DOX to PEGylated CS NPs was performed using different weights of DOX; 0.125 mg,

0.25 mg, 0.5 mg and 1 mg in order to define the most suitable drug concentration that could achieve

the best loading capacity. DOX loaded PEGylated CS NPs were dissolved in PBS medium and

kept on stirrer for 24 hours at 100 rpm. This was done within light protected environment under

56

ambient temperature. Thereafter, the DOX loaded NPs were centrifuged at 20,000 RPM for 30

min under 4 ºC temperature. The concentration of free DOX drug in the supernatant layer was

detected using UV-Vis spectroscopic analysis at wavelength of 482 nm. Drug loading capacity has

been detected using the given equation (Eq. 2.2).

Loading capacity (LC%) = (𝑇𝑜𝑡𝑎𝑙 𝑑𝑟𝑢𝑔 𝑎𝑑𝑑𝑒𝑑−𝑑𝑟𝑢𝑔 𝑖𝑛 𝑠𝑢𝑝𝑒𝑟𝑛𝑎𝑡𝑎𝑛𝑡)

𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑛𝑎𝑛𝑜𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒𝑠 × 100 …... Eq. (2.2) (134)

Encapsulation efficiency =(𝑇𝑜𝑡𝑎𝑙 𝑑𝑟𝑢𝑔 𝑎𝑑𝑑𝑒𝑑−𝑑𝑟𝑢𝑔 𝑖𝑛 𝑠𝑢𝑝𝑒𝑟𝑛𝑎𝑡𝑎𝑛𝑡)

𝑇𝑜𝑡𝑎𝑙 𝑑𝑟𝑢𝑔 𝑎𝑑𝑑𝑒𝑑 × 100 …….Eq. (2.3) (134)

2.8.2. Swelling test:

CSNP has been shown by many studies to have a swelling tendency which may differ according

to the pH of the dispersive media (135). Swelling test in the current study has been performed in

order to assess the influence of various pH levels on the swelling of the DOX loaded PEGylated

CSNP and predict their effect on the entrapped drug. Therefore, swelling indices have been

calculated for the nanoparticles in two different pH media; 6.6 and 7.4. These two specific pH

media are two model values that are related to the breast tumor pH and normal blood pH levels

respectively (136). Swelling index has been obtained using the given equation Eq. (2.4), where Sw

is the swelling of the nanoparticles after time t, Wo is the weight of nanoparticles before incubation

or in a dry form and Wt is the weight of nanoparticles after time t or after incubation time.

% Sw =Wt −W0

W0 × 100 ……. Eq. )2.4) (137)

2.8.3. Stability of DOX PEGylated CS NPs:

The highest LC% of DOX was determined from the previous experiments. Thereafter, the stability

profile of the highest DOX loaded sample was investigated. The stability of the samples was

confirmed after incubation in BPS of pH 7.4 for two weeks at 37 ºC. UV-Vis spectroscopy was

applied to detect the release profile of DOX by measuring its concentration at wavelength of 480

nm.

57

2.8.4. Drug Release Profile:

Release profile of the highest DOX loaded PEGylated CS NPs was studied. The sample was tested

in two different pH media. The first one mimicking the normal blood circulatory pH 7.4, while the

second one was mimicking the tumor microenvironment pH 6.6. The samples were kept inside the

incubator for around 72 hours at 37 ºC temperature. Within various time intervals, the

concentration of the released DOX was determined by using UV-Vis spectroscopy as well at

wavelength of 480 nm. The applied time slots were (1, 3, 6, 9, 12, 18. 24, 36, 48 and 72) hours.

Re-compensation for the withdrawn amount with another fresh media was carried out.

2.9. Cell culture analysis:

2.9.1. Cell line maintenance:

The anticancer activity of the fabricated CS NPs, CS NPs-PEG, and CS NPs/DOX was analysed

in-vitro against human isolated breast cancer cell line (MCF-7) and a normal mouse fibroblast cell

line (L929). The cell lines were propagated as a monolayer separately in DMEM. They were

passaged three times per week in 75 cm2 cell culture flasks and safely incubated at 37°C in 5%

CO2 incubator. Regular cell detachment was proceeded before passaging times and the specified

MTT assays using trypsin (0.25%) containing 0.1% EDTA. Trypan blue has been applied in cell

counting using hemocytometer.

2.9.2. Direct Cytotoxicity (MTT assay):

The anticancer property of DOX before and after encapsulation inside CS NPs against MCF-7

cells and the cyto-compatibility of fabricated samples against L-929 cells were investigated based

on ISO10993-5 standard procedure (138). Colorimetric 3-(4,5-Dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide dye analysis (MTT cell viability analysis) has been used to detect the

viability profile of both types of cells with 24 hrs incubation in a 96 well plate. In order to attain a

semi-confluent cell growth after 24 hrs; MCF-7 cells seeded at density of 25 × 103 cells/well, while

L-929 cells seeded at density of 3 × 103 cells/ well in 96 well plate. Following that, a direct addition

of 50 ul/well from previously prepared serial dilutions of CS NPs, CS NPs-PEG, and CS NPs/DOX

(1.25, 2.5, 5 and 10) µg/ mL. After 24 hrs, MTT reagent has been used to replace the media with

1 mg/ml conc. The replacing volume was 100 µl that has been incubated for 4 hours. PBS was

used for washing and removal of MTT medium. This washing process was done tow times. 100

58

µl of 100% DMSO was applied in order to dissolve insoluble Formazan crystalline products, that

are expected to be cytotoxic and damaging to the cells. A microplate reader (SPECTROstar Nano,

BMG LABTECH, Germany) has been used at Abs of 570 nm to detect the cell viability. Control

sample has showed 100% viability, while the viability of the remaining wells has been identified

using Eq. (2.5)

Cell Viability Rate =Sample ABS

Control ABS × 100 ……. Eq. (2.5) (139)

2.10. Theoretical background:

2.10.1. Zeta-Sizer and Zeta-Potential (MALVERN) :

This is a main dual systematic instrument that includes both dynamic light scattering (DLS) that

is used to assess the nanoparticle’s sizes and distribution, and electrophoretic light scattering (ELS)

that is used to determine the surface charge of a particles within a solution medium.

Figure 2.6: Systematic setup of the basic principle for dynamic light scattering with detecting different scattering

angles of the laser beam (140)

The basic principle of DLS is simple as a monochromatic laser beam is directed towards the sample

cuvette. When the incident laser beam illuminates the particles in the cuvette, it gets scattered in

all directions. A photon detector is used to receive the scattered light beam at a certain scattering

angle . In another words, when the particles scatter the laser beam, they provide information about

their size, motion and distribution. Physically, the scattering is related to the hydrodynamic radius

59

(Rh) of the particles (141). The correlation of the laser scattering intensity is done through software

analysis or the digital correlator as shown in the DLS scheme in Figure (2.7).

Figure 2.7: Illustrative description of different components of dynamic light scattering instrument (141)

By defining the diffusion parameters from Eq. (2.6), we can calculate the hydrodynamic radius Rh

that corresponds to the particles size. D represents diffusion coefficient, kB represents Boltzmann

constant, while T is the temperature and n is the sample viscosity of the sample. By defining such

parameters, we can calculate the. The concentration of the sample should be very low, because

this equation is valid for single scattered light. When sample’s concentration is too high, multiple

scattering occur and this equation become invalid.

……… Eq. (2.6) (143)

The surface charge of any nanoparticle is a key indicator of the aggregation or stability tendency.

Zeta potential represent the electro-kinetic potential of nanoparticles or any other particles in

general within a colloidal system. The higher the charge value on the particles surface, the more

stability suspension or colloidal system.

The electrophoretic light scattering instrument is processing with an electric field is applied the

sample cell. This electric filed cause the particles to move because of the interaction between them.

Through this interaction between the particles and the applied field, they acquire some charge.

This charge affects the speed and direction of particles movement. Besides, the strength of the

60

applied electrical field and the nature of the colloidal medium are two important factors also that

affect them (144).

(A) (B)

Figure 2.8: Schematic diagram showing the principle of electrophoretic light scattering with a multiple layer of

illustrative charged layers (A), and the shape of the sample cell kit (B) (144)

2.10.2. Fourier Transform Infrared (FTIR) Spectroscopy:

It is one of the well-known analytical techniques that is mainly used to characterize various

materials types including organic, in-organic and polymeric chemical materials. The change in the

surface structure or surface functional group(s) can be monitored and analyzed by FTIR. This

allow us to understand the significant change of the physico-chemical properties of the material

after addition of certain polymer or chemical substance to the surface or the core of the main

material. Besides, the durability and structural stability of a material can be another feature that

can be easily monitored through FTIR (145).

Some characteristic structural moieties of a chemical material can be represented in a specific

pattern within a certain range of infra-red absorbance value. Analytically, the ability of these

moieties to absorb the incident infra-red spectrum in the range of 400 to 4000 provide a graphical

analysis of the chemical information of the applied sample. through FTIR, we can also release a

chemical comparison between the polymerized material and the original or the standard ones

through the intensity, position and behavior of the absorption peaks. It can be used, also, to identify

material’s decomposition, oxidation, deterioration or any undesired contaminants. Therefore,

61

FTIR can provide a rigid justification that supports a successful change in the chemical and

physical properties of the applied material (146).

Figure 2.9: schematic representation of the FTIR working principle (147)

The working principle of FTIR is mainly focusing on the interaction between the sample material

and a light source within the infrared region in a surrounding electromagnetic field. Once the

sample disc is subjected to the IR light, it absorbs the light and get excited. The inner structural

molecules are excited also and moving to a higher vibrational level at a certain frequency. The

energy difference between the ground energy level and excitation level is basically equal to the

light energy that the molecules have absorbed.

Light is sent to the sample through an instrumental device called interferometer. The IR radiation

is sent to the sample material through a beam splitter. As a result, the radiation light is divided into

two halves. The first one is going to a fixed mirror, while the second half is going to a continuously

moving mirror. Then the two split beams recombine again at the detector and pass through the

sample again. In this case, the sample absorb all characteristic wavelengths, in an interference

pattern, from the interferogram. The energy variation is detected through the detector which read

it versus time. Through Fourier Transform mathematical technique, a software generated

procedure provides a relationship between time and frequency. This relationship is identified later

62

with against referenced libraries that develop the spectrum information of the sample that is

considered as a fingerprint for such specific material (148).

2.10.3. X-ray diffraction:

X-ray diffraction is advanced new characterization techniques that is used to analyze a crystalline

material and studying its atomic spacing as well. Besides three-dimension structural analysis, XRD

is efficient also to study a unit cell dimensions. XRD is mainly based on the interaction between

X-rays and a crystalline sample. the main source of X-rays emission is a cathode ray tube that is

directed to the sample with a monochromatic radiation pattern. When the X-rays hit the powdered

sample, it diffracts with a diffraction angle (149). This constructive interference and the

relationship between the angle of diffraction and the electromagnetic radiation wavelength is

expressed as Bragg’s law that is shown in (figure 2.10) and Eq.2.

nλ=2d sin θ. …… Eq. (2.7) (150)

Where θ is the scattering angle or the angle between the incident X-ray beam and the lattice plane,

is the wavelength of the incident electromagnetic waves, d is the interplanar distance (Figure

2.10). n represents the positive integer factor or the order of reflection.

Figure 2.10: illustrative scheme of X-ray diffraction with diffraction angle θ (Bragg’s law) (151)

The X-ray cathode tube produces an X-ray beam by heating the filament of the tube that produces

a series of electrons. These electrons are directed towards the sample material and accelerated by

63

applying voltage. These electrons are going to bombard the sample and expel its inner shell

electrons as shown in (figure 2.11). In order to do that, the incident electrons must have sufficient

high energy. By dislodging the inner shell electrons, X-ray is emitted. A crystal monochromator

is used to produce monochromatic rays. As the sample and X-ray detector are rotated, the

diffracted emissions are recorded by the processing detector. When the dimensional arrangement

of the incident rays satisfies Bragg’s law, the detector record the signal and convert it to

mathematical counts that is illustrated through a computer monitor (152).

Figure 2.11: X-ray beam bombarding the Inner shell electrons (152)

2.10.4. Ultra-Violet / Visible Spectroscopy:

UV-Vis spectroscopy is a frequently used characterization analyses that mainly target the optical

properties of different samples. Through this analysis technique, we can develop qualitative and

quantitative analysis, detect the presence of undesired impurities and detect the presence or

absence of certain compositional functional groups. Generally, the electromagnetic spectrum of

sunlight consists of a range of radiation regions including the UV-Vis region which is located at

the range of 210-900 nm. The interaction of the sample to light in this region is the principle

source of UV-Vis spectroscopic analysis. When light hit the sample’s molecules, the atomic

electronic transition takes place immediately between different energy levels or, typically, from

the higher occupied molecular orbital (HUMO) or ground state to lower unoccupied molecular

orbital (LUMO) or excited state as shown in (figure 2.12). This intermolecular transition is

accompanied by absorption of electromagnetic radiation in this region.

64

Figure 2.12: The electronic excitation by absorbing high energy UV light spectrum (153)

HUMO and LUMO orbitals are called also bonding shells and antibonding shells, and the

difference between them is known as band gap. In order for absorption to take place, the UV light

photons must have energy amount that matches to this band gap. In this concern, different

molecules will definitely have different band gap energies and, therefore, will have different

absorption spectra. Once the sample was hit with a beam of UV light, it absorbs part of it and the

intensity of the released beam will exponentially decreased as shown in (figure 2.13) and

absorbance equation Eq. (6). However, the transmittance has the same relationship between the

incident light (Iº) and the transmitted light (I), but absorbance is the counteractive principle of

transmittance.

𝑇 = 𝐼/𝐼° …….. Eq. (2.8) (154)

𝐴 = log 10 𝐼°

𝐼 ……… Eq. (2.9) (154)

65

Figure 2.13: The transmittance of UV light beam through a sample (154)

UV-VIS spectroscopy is mainly based on the ability of measuring the spectrum of a certain sample

containing chromophore or light absorbing molecules. This allow us to obtain a graphical

relationship between the wavelength or frequency at which light is absorbed, and the absorption

rate that is detected by the amount of chromophore exist in the sample. therefore, the higher the

amount of this chromophore in the applied sample, the more dense the amount of the absorbed

light. This relationship reveals another direct relationship between the sample concentration and

wavelength or frequency of the emitted UV light. Beer’s Lambart law has exploited this optical

relationship in order to develop a linear relationship between concentration and absorbance as

shown in Eq (7).

𝐴 = ℰ 𝑐 𝑙 ……… Eq. (2.10) (155)

A: the absorbance

ℰ: absorption coefficient

c: molar concentration of the solute

l: path length

This equation (Beer’s Lambart law) can be used analytically to relates the concentration of a

substance in a sample to the color intensity of the same sample. when we have large number of

samples and different concentration in each sample, we can use the developed linear relationship

that this law provides. However, we need first to issue a calibration curve. This calibration or

66

regression curve is based on plotting 3 to 4 concentrations of the sample and getting their

corresponding absorbance values. This plot will work as a reference from which we can obtain the

concentration of any unknown concentration of the same sample (155).

Figure 2.14: schematic representation of UV-VIS spectrometer principle (154)

2.10.5. Scanning Electron Microscopy (SEM):

SEM is a powerful and reliable analysis techniques that is used for imaging samples. This

technique is much advanced than simple light microscope since it uses electrons to image the

sample unlike light microscope that use light. The wavelength of electrons is much smaller than

that of light, therefore the magnification and resolution of SEM is more preferred than traditional

light microscope. SEM provides such electron-oriented magnification through the use of a set of

coils that scan the electrons bombarded from the sample (156).

67

Figure 2.15: Multi pin sample’s holder carrying different aluminum foils with spread samples for TEM imaging

(157)

As shown in (figure 2.16), the raster pattern of the coils is used to scan the sample through the

generated electrons for the electron source at the top of the instrument. Basically, these electrons

are produced when their thermal energy exceed that of the main electron sources. The role of the

positive anode that is fixed below the electron source is to attract and accelerate the speed of the

emitted electrons under protective vacuum. This vacuum has a critical indirect effect on the overall

quality of the released images by protecting the electrons from interacting with any external atoms,

molecules, noise or contamination (158). On the other hand, the use of such condenser lens and

objective lens is mainly applied for adjusting the electron path, and this ends up with controlling

the resolution. The integration of apertures may be considered in some SEM systems in order to

control the size of the beam and control the current as well. This may give a good opportunity for

controlling the resolution of the resulted images (159). Typically, the main use of SEM is to

analyze different types of samples such as insulating, thin organic, biological and vacuum sensitive

samples with satisfying resolution and magnification power.

68

Figure 2.16: Illustrative description of SEM unite with variable (160)

2.10.6. Transmission Electron Microscopy (TEM):

TEM is a powerful and versatile tool for imaging of many different types of samples. It is widely

used in biological fields, forensic analysis, material sciences, medical and nanotechnology

applications. The main use of TEM is to provide some information about the crystalline structure,

size, particles distribution, morphological, chemical structure, structural defects and topographical

properties. Similar to SEM, the source of these information is basically dependent on the

interaction of the sample to a very high energy electron beam that is much stronger than SEM. The

electron beam is accelerated with applying voltage approximately more than 200Kv. The electrons

are accelerated to a very high speed near the speed of light. While the wavelength of the electrons

in the electron beam is million times shorter than normal electromagnetic wavelength of light, the

spatial resolution and magnification quality of TEM images is much higher than normal light

microscopy (161).

TEM is operated in a similar process to the simple light microscope. However, TEM uses a stream

of electrons produced by an electron gun (figure 2.17) instead of using light source. The electron

beam is directed towards the sample in a very high speed that is enough to pass through the sample.

A primary condenser lens is used to focus the emitted electrons into a thin and coherent electron

beam. Once the electrons could pass through the sample, they are scattered. These scattered

electrons are then focused by an electromagnetic lens called objective lens. This lens focuses the

received electrons into an image that is transmitted through a projector lens. This projector lens is

69

used mainly to expand the electron beam into a monitor or camera supported projector so that the

image is seen by the user (162).

Figure 2.17: Schematic representation of the different components of TEM (163)

Table 2.4: comparison between TEM and SEM (162-164)

Item TEM SEM

Sample Very thin Thick and conductive

Resolution Very high, ~ 0.5 Aº High, ~ 0.5 nm

Electron beam Pass through the sample Surface analysis

Sample preparation Extensive Simple

Surface modification Dyeing Gold sputtering

Voltage Very high Medium

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Chapter 3

Results and Discussion

Synthesis and Characterization of

PEGylated Chitosan /

Doxorubicin Nanoparticles

and Conjugated with Monoclonal

Antibodies for Breast Cancer Therapy

71

3.1. Preparation of CSNP:

In order to successfully prepare a conjugated drug delivery system that is effective against breast

cancer, CSNP have been used to encapsulate anticancer drug like DOX, and the nano-system was

further conjugated with monoclonal antibodies. Several parameters have been considered and

found to have considerable effects on the preparation of the final formulations such as: particles

size, size distribution, surface charge, stability in different pH media, surface functionalization,

physiological circulation time, biocompatibility, biodegradability, swelling matrix, and drug

diffusion rate. Ionotropic gelation method was used to prepare CSNP due to its efficiency and the

resulted high particles’ stability for longer periods (165).

3.1.1. Ionotropic Gelation:

As shown in Table (3.1), the integration of different concentrations of CS polymer and TPP, as a

cross-linker, for preparation of CSNP. TPP has been shown to be biologically safe, non-toxic and

non-carcinogenic owing to its hydrolyzed phosphate ions (166). The resulted CSNP in Table (3.2)

had various sizes and PDI values. Moreover, the final appearance of the formulated CSNP was

slightly different from each other. Some samples have shown whitish uniform colloidal systems

of suspended CSNP. While others have shown high tendencies of particles aggregation with

whitish turbid solution (112).

Table 3.1: Variation of sample formulas according to chitosan and TPP concentrations

TPP conc

(%)

Chitosan concentration (%)

0.05 0.1 0.5 1

0.1 S1 S2 S3 S4

0.3 S5 S6 S7 S8

0.5 S9 S10 S11 S12

0.7 S13 S14 S15 S16

The polymeric matrix of CSNP, by ionic gelation technique, is controlled and tunable through an

electrostatic interaction between (+vely) charged NH2+ from CS and (-vely) charged polyanions

from TPP (166). This gelling formation interaction has been found to be affected by the

72

concentrations of the two polymers, and the resulted particles sizes are, also, affected as shown in

Table (3.2).

Table 3.2: Particle size and distribution of different samples with various concentrations of TPP and chitosan

By demonstrating all of these previous results, sample 8 has been the sample of choice. It has

demonstrated the smallest size with acceptable poly dispersity index. The applied CS:TPP ratio has

been close to 3:1. The size of this sample has been 190 ± 5.6 with a good particle’s distribution of PDI

0.51 ± 0.002. Biologically, this size can develop many pharmacokinetic and therapeutic benefits

compared to larger sized drug carriers (112). Many factors have been shown to affect the size and PDI

of the resulted CSNP such as processing pH and deacetylation degree of CS (167). Therefore, the effect

of these parameters has been explored on this sample.

3.1.2. Factors affecting size of CSNP:

Figure 3.1: Effect of processing pH (A) and deacetylation degree (B) of CS on the size of CSNP

Sample Particle size (nm) PDI S1 3668.6 ± 967.6 0.31 ± 0.17 S2 4986 ± 1300 0.64 ± 0.07 S3 1607 ± 63.66 0.43 ± 0.01 S4 518.76 ± 28.28 0.86 ± 0.05 S5 1594.5 ± 195.8 0.86 ± 0.02 S6 3894 ± 376.7 0.69 ± 0.26 S7 1582.5 ± 228.3 1.37 ± 0.36 S8 190 ± 5.6 0.15 ± 0.02 S9 1979.5 ± 64.3 0.22 ± 0.05 S10 1895 ± 161.3 0.41 ± 0.07 S11 678.66 ± 15.02 0.85 ± 0.06 S12 690.9 ± 28.42 0.24 ± 0.02 S13 5645.3 ± 685.5 0.45 ± 0.11 S14 2362 ± 678.34 0.24 ± 0.06 S15 2265.6 ± 141.78 1.33 ± 0.31 S16 1300.6 ± 664.8 0.66 ± 0.32

73

o Processing pH:

Size has been shown to be decreased with decreasing the processing pH media. That’s may be

because of the high protonation rate at highly acidic media. It increases the reactivity of the

terminal NH2 groups of chitosan and improve the ionic strength of the dissolution media (168).

Therefore, the lower the pH value, the lower the size we have as shown in Figure (3.1A). However,

the distribution of the resulted particles has been non uniform with high PDI value of the resulted

CSNP. Consequently, lowest pH media are not always the best solution, the current study

recommends adjusting pH of the processing medium to 5 (169).

o Deacetylation degree:

On the other hand, degree of deacetylation is another critical factor that has been discussed with

many published reports (170). The results of the current work have revealed that sizes of CSNP

decrease as the degree of deacetylation of CS increases as shown in Figure (3.1B). This may be

related to the amount of deacetylated amine groups. In another words, high deacetylation degree

provides high density of positive NH2+ charged groups. This high positive charge density enhances

higher crosslinking interaction with TPP. Although, this may contradict with Vivek et al. that

proposed that the surface charge of CSNP is -35 (171), however, Phaechamud et al. and Ling et al

agreed on this (172). Therefore, CS with high deacetylation degree has been used in the current

study.

o CS: TPP ratio:

The relationship between CSNP’s size increment and TPP concentration has been demonstrated

in Figure (3.2). Whenever the concentration of TPP is increased or decreased than the selected

concentration (0.3%), the sizes and PDI of the resulted CSNP are going to massively increase with

having some of them reaching micro sized ranges.

When TPP concentration is over increased, the cross-linking rate of CS is increased. That’s may

be due to the accumulation of the excess TPP molecules upon the outer surfaces of the already

crosslinked CSNP. This may enhance more intramolecular interaction, and higher occupation rate

of free NH2+ groups of chitosan by TPP. This can provide what is called oversaturation of CSNP’s

74

surface and become more reactive to proceed another cross-linking process. Therefore, the size

becomes greater

Figure 3.2: Effect of TPP concentration on the final size of CSNP

On the other side, at very lower TPP concentrations, the size of the crosslinked CSNP tends to

increase, and whitish turbid clouds are seen in the preparation media. At very high conc of CS

relative to lower TPP, neutralization of the protonated cationic NH2+ groups of CS by TPP is

decreased, and this led to higher sedimentation rate of chitosan polymer, especially, when chitosan

acidic solution is rich with acetic acid (113, 173). Therefore, the current work may conclude that

there is an optimum ration of CS: TPP which is expected to be near 3:1 (168).

3.1.3. Stability of the selected CSNP sample:

Surface charge of CSNP is a remarkable factor that is considered as an indicative factor of the

particle’s stability and their tendency to form aggregates. The colloidal suspending appearance of

CSNP is highly affected by the charge on their surfaces. This charge is influenced by the same

previous parameters (174, 175). In order to make sure of the surface stability of the selected CSNP

sample; S8, a three weeks stability profile has been developed as shown in Figure (3.3).

75

Figure 3.3: Stability profile of the selected CSNP sample; S8 for three weeks at pH 7.4

Furthermore, size stability profile has been confirmed with calculating size increment percentage,

which is an indication of size variation along the stability period of time. It has been calculated

according to the given equation (Eq.3.1). Owing to the high charge surface positive zeta potential

value of the selected CSNP sample with Zeta value of 42.8 mEv, the particles have been shown to

have a small size increment percentage as 8.9%. That because of a direct relationship between

having high zeta potential value and showing a good stability profile for longer time (166).

Size increment % =Size at the biginning −Size after 22 days

Size at the beginning × 100 ……. Eq. (166)

Finally, the size of the developed CSNP as drug carrier is a substantial factor that needs to be

studied very well before drug encapsulation or PEGylation process. This has a strong influence on

the efficiency of the developed drug delivery system. As the size of the nanoparticles decreases,

the drug cellular uptake increases. Besides, a direct relationship between nanoparticle size and

pharmacokinetic drug distribution is considered. Nanoparticles’ sizes below 100 nm have 6-fold

more cellular uptake rate than micro sized particles (177). Furthermore, CSNP within a size range

less than 200 nm provided more potent dosage forms than larger micro range particles. The

advantages of nanoparticles in this size ranges are enhanced muco-adhesion, biocompatibility,

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increased therapeutic index, enhanced stability and less carcinogenicity. This has been consistent

with Pawar et al, that could achieve > 95% drug entrapment efficiency within a size range (100 –

200) nm (178). Therefore, the recommended size of the prepared CSNP in the current study has

been less than 200 nm.

3.2. PEGylation of CSNP:

Coating of the prepared CSNP’s surface with PEG has been reported in many studies showing

high NP surface stability and solubilization. The increased blood circulation time and

immunological RES resistance of CSNP that are coated with PEG is a guiding factor for seeking

PEGylation process. PEG aims to protect CSNP by blocking their surface positive charges that

can be included in the opsonization process by RES (179, 180).

Table 3.3: Size, PDI and zeta potential of PEGylated CSNPs

Samples Size PDI Zeta Pot.

Control (CSNP) 192 ± 4.1 0.19 ± 0.01 +43 ev ± 0.4

CSNP-PEG 1% 175 ±4.8 0.25 ± 0.03 + 41.8 ev ± 0.6

CSNP-PEG 5% 169 ± 5.8 0.15 ±0.01 + 35.6 ev ± 1.1

CSNP-PEG 10% 173 ± 2.9 0.21 ± 0.02 + 39 ev± 1.2

Table (3.3) provides the results of PEGylation process using various concentrations of PEG; 1%,

5% and 10%. The resulted PEGylated CSNP have had various sizes, PDI and zeta potential values.

The surprising observation about the PEGylated CSNP is that their sizes were found to be smaller

than those free non-PEGylated CSNP by 8.7%, 12% and 9.8% respectively. This can be

demonstrated by the effect of PEG surface brushes acting as colloidal stabilizer on CSNP surfaces.

Moreover, the polymeric layer of PEG has provided “stearic stability” upon conjugation to the

surface of CSNP. This has affected their size, PDI and surface zeta potential values (181).

PEG forms an internal structural network with chitosan which is known as semi interpenetrating

network (IPN) (112). This network has been proposed to be formulated during crosslinking of

chitosan. It can increase the stabilization and compactness of the surfaces of CSNP. Therefore,

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their sizes and surfaces charges decrease after PEGylation. Besides, zeta potential value of CSNP

has decreased because of the attribution of blocking surface terminal NH2+ groups of chitosan and,

thereby, decreasing their zeta potential values (182).

The recommended PEG surface coverage with PEG conjugation has been attributed to 5% PEG

concentration. The resulted nanoparticles have had lower size than 1% and 10% PEG

concentrations. Besides, the particles distribution and surface charge values of the selected PEG

concentration have pointed out its tendency to be stable. Assuming that further increase in PEG

concentration can destabilize the nanoparticles due to the repulsive behaviors of the neighboring

PEG chains (183). Further stability profile of the three PEG concentrations has been conducted in

the current study as illustrated in Figure (3.4). PEGylated CSNP that have 5% PEG has been the

most stable as referred by the blue arrow. While those with 10% PEG and 1% PEG were less

stable.

Figure 3.4: stability profile of PEGylated and non-Pegylated CSNP

Basically, it is very rational to give high concern to surface stability issue of the PEGylated CSNP.

Verreechia et al. has indicated its pharmacokinetic preference towards PEGylated CSNP with

78

stable surface conjugated PEG. The study has reported that PEGylated CSNP have higher blood

plasma level with lower hepatic execration rates compared to non-PEGylated ones (184).

The developed system in the current work tend to be positively charged, that aims to enhance its

later electrostatic interaction to further tumor cells in the coming stages of the study. Generally,

positive nano-carrier systems are not biologically favored, because they are easy to be captured by

phagocytes and RES. Besides, they initiate electrostatic interaction with negative blood proteins

(185). Furthermore, thrombogenic embolization and blood hemolysis are two adverse reactions

that are expected to be initiated by positive systems.

Therefore, grafting PEG can promote the biological safety and circulatory retention time of CSNP

without casing such toxic adverse blood reactions. Furthermore, PEGylation improves the

uniqueness of CSNP as an ideal nano carrier for later chemotherapeutic DOX encapsulation

process. For instance, Clin et al. has reported higher liposomal accumulation of PEGylated DOX

liposomes within a tumor area than non-PEGylated ones (186). This may affect the cancerous

cellular drug uptake.

79

Figure 3.5: XRD pattern of Chitosan powder, CSNP and PEGylated CSNP

The conjugation reaction or PEGylation of CSNP could be confirmed through XRD patterns of

CS, CSNP, PEG, PEGylated CSNP as shown in Figure (3.5). Basically, this conjugation has been

formulated between the amino group NH2+ group from chitosan and C-O-C of PEG (187). This is

in agreement with Calvo et al. that assumes, also, inter and intra molecular H-bonding between CS

and PEG (112). The patterns show some representative peaks which indicates the crystalline

behaviors of the tested structures. Chitosan powder, for instance, has two characteristic peaks at

the angles of 21º and 10º as shown in the diffractogram, this demonstrated a high degree of

crystallinity (188).

On the other side, the diffractogram of CSNP shows no peaks, which is an indication of the

destroyed crystalline structure of chitosan by TPP entry. This is related to the amorphous structure

of CSNP which is composed of dense of cross linked counterions (188). In the diffractogram of

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PEGylated CSNP, the peak at 2θ of 10 º has been diminished. This is related to the decreased

crystallinity of CSNP by the entry of PEG. The pattern of PEG has surpassed that of chitosan with

its characteristic peaks at 19.12 º and 23.1 º, and this resulted in peaks shifting (189). Therefore,

there is a good evidence for the efficient PEGylation of CSNP.

CSNP could maintain their spherical morphological shape after grafting of PEG on their surfaces.

The PEGylation layer have demonstrated a brush like structure that surrounded CSNP. This has

been confirmed with TEM pictures of both PEGylated; Figure (3.2 B and D) and non-PEGylated

CSNP; Figure (3.6 A and C).

B A

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Figure 3.6: TEM image of Pegylated CSNP (B and D), non-PEGylated CSNP (A and C) and schematic illustration

of the PEG layer that surround CSNP (E)

3.3. Encapsulation of Doxorubicin:

Doxorubicin has been selected as effective chemotherapeutic agent against all different types of

breast cancer including both solid and soft tumors (190). DOX has been encapsulated successfully

within the PEGylated CSNP via a pre-crosslinking entrapment. In other words, DOX has been

introduced into CS-PEG solution before drop wise addition of TPP solution. Achieving good

encapsulation with lower drug leakage has been fairly expected by the presence of PEG stabilizer.

This enhances initiation of intramolecular hydrogen bonding between DOX and the reacting

polymers (191).

Slight mixing has been required besides pH adjustment from 5 to 6.5. This is assumed to enhance

the entrapment of DOX inside the polymeric matrix. Both factors; the deprotonation pka of

chitosan at 6.6, besides the hydrophilic nature of DOX promote more intracellular hydrogen

interactions (192). Coinciding with that, PEG brushes are being maintained in order for enhancing

the system biocompatibility (193). Non-covalent interaction is therapeutically more preferred than

covenant entrapment (194). covalent interaction in a biological drug delivery system is more

difficult to manage or predict

3.3.1. DOX Standard Curve:

Experimentally, variable concentrations of DOX have been fed in order to evaluate the highest

loading and entrapment content of DOX; 1.25 µg/mL, 2.5 µg/mL, 5 µg/mL and 10 µg/mL. This

has required a prior quantification of DOX, that has been issued by plotting its calibration curve.

E

82

UV-VIS spectrophotometer has been used to detect the absorbance values of the prepared known

concentrations of DOX. The plotted concentrations have been as following; 1 µg/mL, 10 µg/mL,

30 µg/mL, 50 µg/mL, 70 µg/mL and 90 µg/mL. The linear pattern of the graph has been translated

into a fitting regression equation as shown in Figure (3.3) and Eq 3.2.

Y= 8.05 X – 0.0245 ……. Eq. (3.2)

Figure 3.7: standard calibration curve of DOX in PBS

3.3.2. DOX Loading Capacity (LC%) and Encapsulation Efficiency (EE%):

On the other hand, the resulted DOX loaded PEGylated CSNP have been analyzed with DLS in

order for detecting the modification effect of DOX entrapment on the size, PDI and zeta potential

values of the nanoparticles. Table (3.4) shows the results of all prepared formulations. The sizes

of the resulted formulations have been slightly smaller than the PEGylated CSNP. Besides, the

surface net charge values have been slightly lower also. Based on this two information, this slight

change in size and surface charge may be attributed to the entrapped amount of DOX (97).

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Table 3.4: DLS analysis of DOX loaded and unloaded CSNP

Nano-formulation Size PDI Zeta Pot.

CSNP 195.4 ± 3.8 0.22 ± 0.005 + 42.5 mV± 1.3

CSNP - PEG 188.6 ± 0.5 0.19 ± 0.002 + 38.4 mV± 3.8

CSNP- PEG - DOX 1.25 µg/ml

mg

177.8 ± 2.6 0.18 ± 0.002 + 35 mV ± 1.6

CSNP-PEG - DOX 2.45 µg/ml 180.6 ± 4.3 0.20 ± 0.008 + 33.4 mV ± 0.9

CSNP-PEG – DOX 5 µg/ml 178.5 ± 4.7 0.21 ± 0.006 + 34.6 mV ± 2.6

CSNP-PEG – DOX 10 µg/ml 176 ± 5.7 0.22 ± 0.005 + 33.6 mV± 3.59

The LC% and EE% are two main important parameters that define the efficacy of the entire drug

delivery nano-carrier system. The results are given in averages of triplicate experimented values.

As showed in Table (3.5), the LC% and EE% are independent from each other. They continued to

increase gradually with increasing the fed DOX concentration, till certain concentration (10 µg/ml)

then decreased. At this point the internal chitosan network has been saturated with DOX, besides,

the drug has reached its saturation solubility (102). Therefore, higher drug concentrations are

expected to be eluted in the loading supernatant media (196).

Table 3.5: Resulted LC% and EE% of different DOX-PEGylated CSNP at different drug concentrations

Drug formula LC% EE%

1.25 µg/ml 0. 033 % 71.44 %

2.45 µg/ml 0.08 % 77.5 %

5 µg/ml 0.114 % 80.4 %

10 µg/ml 0. 09 % 78.5 %

The potentials of PEGylated CSNP to load such DOX concentrations are proposed to be dependent

on its internal spacing structure besides capillary forces (197). When the initial fed DOX,

concentrations have been compared to the calculated loaded amounts, 5 Ug/ml concentration has

been selected as the highest LC% and EE%, for the following experiments (101, 198).

Furthermore, the cell viability tests have confirmed the lowest viable rate of breast tumor cells

(MCF-7) with 24 hours incubation after ingestion of 5 µg/ml nano-drug formulation using 96 well

plate. This will be discussed in more details in cell cytotoxicity part. The selected sample of DOX

conjugated to CSNP has been confirmed with spherical morphology through TEM images as

shown in Figure (3.8).

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Figure 3.8: TEM images of DOX loaded PEGylated CSNP (A, B), and schematic diagram of DOX loaded

PEGylated CSNP

3.3.3. UV-VIS spectrum of DOX loaded PEGylated CSNP:

In order to confirm the efficiency of the prepared nano-system to effectively encapsulate DOX,

the UV-Vis spectra of free DOX, unloaded CSNP and DOX loaded PEGylated CSNP are shown

in Figure (3.9). The nano particulate samples have been incubated in slightly acidic pH media for

24 hours in the ambient conditions before centrifuging. Then, the resultant supernatant has been

matched to the UV spectrum of free DOX which has a characteristic absorption peak at wavelength

of (481 nm) (199). The unloaded CSNP have revealed a broad absorption band at the area of 200-

300 nm. This band is thought to be related to C=O group.

63 nm

DOX

C

85

Figure 3.9: UV-Vis spectra of DOX loaded CSNP, non-loaded CSNP and free DOX

While the supernatant of DOX loaded PEGylated CSNP showed stronger peak on the spectrum

with a characteristic peak at 480 nm wavelength, which is thought to be related to DOX. In fact,

CSNP has a characteristic swelling behavior that can be generally noted in acidic pH media. That’s

because the amino group related pKa value of chitosan is around 6.5. Therefore, the crosslinked

framework of CSNP start degrading in this acidic pH. The protonated NH2 groups of CSNP in

acidic pH enhances its solubility. This can improve the release of the entrapped DOX (200).

3.3.4. FTIR Spectroscopy analysis:

FTIR spectroscopy tests of CS powder, CSNP, DOX, PEGylated CSNP and DOX loaded

PEGylated CSNP are shown in Figure (3.10). In chitosan powder, broad and strong peak at 3446

cm-1 might be related to O-H stretching mode. This peak has been shifted to lower wavenumber at

3422.3 cm-1 and 3411 cm-1 in CSNP and PEGylated CSNP respectively. This probably means that

the more polymeric cross linkage with TPP and PEG, the lower peak shifting wavenumber. This

refer to the enhancement of H-bonding in the two nano formulations (201). Therefore, the bonds

in PEGylated CSNP is stronger than those in CSNP (96).

86

Figure 3.10: FTIR spectrum of CS powder, CSNP, DOX, Pegylated CSNP and DOX loaded PEGylated CSNP

The peak at 1600 cm-1 in chitosan represents bending vibration amide I bond N-H. In CSNP, it has

been shifted to 1540 cm-1. Likewise, the peak at 1650 cm-1 amide II stretching in chitosan powder

has been shifted to 1612 cm-1 in nanoparticulate systems. This supports the assumption of

successful TPP crosslinking of CSNP (202). Amide III bond peak of N-O has appeared in

PEGylated CSNP at 1380 cm-1 region. (131). Furthermore, a characteristic P=O peak has been

observed at 1155 cm-1 region of the formulated nanoparticulate systems, this peak can be related

to the linkage between NH2 of chitosan and phosphoric of TPP.

A peak at 2888.3 cm-1 wavenumber has appeared in PEGylated CSNP can be attributed to -CH2

stretching vibration. This may enhance the assumption of successful PEGylation of CSNP (204).

Another PEGylation specific peak is vibrational C-O-C peak that appeared with high intensity at

1101 cm-1 (205). After introduction of DOX to the system, two characteristic peaks at 1590 cm-1

and 1651 cm-1 have appeared in the IR spectrum compared to the spectrum of free DOX. These

two peaks are assumed to be related to stretching C=C of DOX inside the PEGylated CSNP matrix.

Moreover, OH stretching band has been shifted to 3430 cm-1 with more intensity and broader

87

appearance, which may represent increased strength of its hydrogen bonding $. Another peak at

1021.3 in PEGylated DOX loaded CSNP may refer to C—O (25).

3.3.5. Swelling Studies of DOX loaded PEGylated CSNP

After 24 hours of incubation, the samples have been weighed and their swelling indices have been

determined in two different pH media; 6.6 and 7.4. DOX loaded PEGylated CSNP have had higher

tendency for swelling at acidic 6.5 pH media than slightly neutral 7.4 pH. Swelling indices at 6.6

pH and 7.4 pH were 62.5 % and 7.89 % respectively. The difference in swelling behavior has been

investigated, also, with a TEM image as shown in Figure (3.11). The nanoparticles in 7.4 pH could

maintain their polymeric spherical shapes as referred by the blue arrows. While in 6.6 pH, the

polymeric matrix of the nanoparticles has been destroyed (206).

Figure 3.11: TEM images of DOX loaded PEGylated CSNP at pH 7.4 (a), pH 6.5 (b) after 24 hrs incubation and

schematic illustration of the difference between non-swelled (pH7.4) and swelled nanoparticles (pH 6.6) (c)

(c)

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There is relationship between having high swelling rate and release of the entrapped drug from

CSNP, which may be due to the pores formed in the matrix network of the nanosystems. Swelling

has been shown to be affected by some parameters such temperature, swelling time, crosslinking

degree and pH. By maintaining all these parameters fixed and changing the pH of the media, the

effect of pH has been demonstrated. This variation between pH 6.6 and pH 7.4 may be related to

the hydrophilicity of CSNP and its polymeric chains. The presence of some active groups such as

—COOH, —OH and NH2 — may have another contribution (206).

Besides, the protonation of chitosan (pKa 6.6) at lower pH levels is increasing and its positive

amino groups induce its swelling. While at higher pH levels, chitosan is less protonated and

uncharged, thus its swelling and solubility decrease (207). Another rationale is that, in higher pH

levels, water tend to form a hydrogen bonds with functional active groups such as —COOH, —

OH and NH2 —groups. These H-bonds tend to fill the spaces inside the nano matrix and, hence,

minimize swelling. Furthermore, the rheological behavior of DOX loaded CSNP and sizes of

counter anions at these pH levels are participating in this swelling behavior (208). In conclusion,

DOX loaded CSNP demonstrates a pH dependent swelling, and this has a direct effect on the

release of the entrapped drug.

3.3.6. In-vitro Release Profile:

Figure 3.12: DOX release profile of PEGylated DOX loaded CSNP at pH 6.6 and pH 7.4

89

The in-vitro release behavior in our study has exhibited a selective pH dependent DOX release

rate. This has been investigated with two aforementioned pH levels; 7.4 and 6.6, that mimic the

physiological and cancerous conditions at 37°C as shown in Figure (3.12). The retention of DOX

inside PEGylated CSNP should be equilibrated between its stability in normal noncancerous

environment versus its targeted release at cancerous acidic conditions (209). In nanosystems, the

release profile can be demonstrated in three main phases; desorption, swelling then erosion as

shown in Figure (3.13). The In-vitro release behavior of DOX loaded nano-system in acidic 6.6

pH has showed basic burst release that has been followed by controlled release of DOX. The burst

release has been related to the diffusion of DOX out of the nano matrices. This is known as burst

effect that entails a combination of polymeric CSNP degradation and drug desorption (210).

Figure 3.13: Illustrative diagram of DOX release mechanism

The burst release of DOX has been identified during the first 3 hours with around 45% at pH 6.6

as shown in Figure (3.12). While less than 14% of the drug has been released at the pH 7.4. This

can be a good predicting indication of the polymeric stability in normal blood pH compared to

acidic tumor environment. This can be related to the pH media in a way that the protonation of the

amino NH2 groups of both CS and DOX is increasing in acidic media compared to neutral one.

Such protonation can be a main causative factor for the polymeric diffusion and erosion as we

90

mentioned before. After 3 hours of burst drug release, the drug has exhibited a slow sustained

release over 72 hours incubation time. This controlled release may be related to simultaneous

occurrence of minute molecules diffusion and the degradation of the remaining polymeric

structures (97). Our results suggest that DOX release is affected with pH level of the ingestion

media. The drug release rate has decreased by increasing the processing pH of the media. The

breast tumor environment is acidic because of the high anerobic glycolysis that enhance the acidity

levels. Besides, the endocytosis uptake pathway of DOX loaded PEGylated CSNP is expected at

such slightly acidic pH levels; however, it has been reported that its release decreases with

extremely acidic conditions (211). The massive oxidative stress at the tumor area enhances such

extreme acidity. Therefore, it is assumed that the tumor pH is a preferable media for the

nanoparticles to release the entrapped DOX compared to normal physiological conditions.

3.3.7. In-vitro release kinetics:

The releasing kinetics of DOX loaded PEGylated CSNP have been demonstrated in order to

predict physiological performance of the released drug. The relationship between the drug

concentration and release rate has been described with different mathematical models; Zero-order,

First order, Hixson crowell, Higuchi and Korsemeyer Peppas (212). According to the results, DOX

release may fit with Higuch model at the acidic 6.6 pH. This system hypothesized that DOX

particles are smaller than the nano-system thickness Additionally, this model entails that the

release rate is related to the pH and the nano carrier matrix has a good solubility rate. On the other

side, at pH 7.4, the release rate follows Korsemeyer Peppas model that assume one dimensional

drug release relative to the drug dosage form (213).

Table 3.6: Release kinetics of the released DOX according to different systems equations

91

3.4. Functionalization of Monoclonal Antibodies:

Figure 3.14: Schematic illustration of the final mAb functionalized DOX loaded PEGylated CSNP

The attachment of mAb to the surface of DOX loaded PEGylated CSNP has been suggested for

enhancing the intracellular uptake and endocytosis. This may be achieved through specific surface

receptors attachment of mAb to cancer cells. The functionalization of Anti-HER2 (Anti Human

Epidermal Growth Factor II) mAb and Anti-hMAM (Anti Human Mammaglobin) mAb to the

surfaces of the nanoparticles has slightly increased their sizes as shown by the TEM images in

Figure (3.14) and (3.15).

Each mAb has been immobilized to the surface of the nanoparticles separately by EDC

crosslinking. Supported activation of COOH terminal groups on the PEGylated surfaces of DOX

loaded CSNP has prepared the surface of these nanoparticles for further attachments of the mAbs.

In other words, this may facilitate the entry of mAb for successful surface conjugation. Therefore,

ratio between EDC to the mAbs was 5:1. This may increase the opportunities of surface satisfaction

of the nanoparticles with the antibodies separately. This could be confirmed with FTIR spectrum.

Furthermore, the size of the nanoparticles is greater than the mAbs, therefore the ratio of

nanoparticles to the mAbs was high (214).

The presence of PEG layer besides a stronger EDC supported linkage, may together help the

nanocarriers to proceed their way without detachment of the surface mAbs (146). This is from a

92

biological point of view. Furthermore, high consideration has been given to the use of PBS for

frequent washing in order to eliminate the presence of the unfavorable attached EDC molecules.

This may grasp further circulating molecules (216).

Figure 3.15: TEM images of Anti-hMAM DOX loaded PEGylated CSNP (A, B) and Anti-HER2 DOX loaded

PEGylated CSNP (C, D)

In order to explore the surface chemistry of the particles surfaces that are functionalized with

mAbs, HNMR spectrum has been conducted as shown in Figure (3.16). Peaks at 2.4, 2.6 and 2.7

ppm are responding of H protons of CH2 groups of mAb functionalized nanoparticles. Besides, the

appearance of triplicate peaks at 3.2 and 3.3 ppm are representing amino NH2 groups, which may

93

confirm the conjugation of the antibodies to the PEGylated surface. The increased amount of

nitrogen within the surface of nanoparticles can be illustrated with the increased immobilization

of the mAbs. Moreover, FTIR spectrum has been conducted as well, and it confirm this assumption

of surface coverage with mAbs related NH2 groups (217).

Figure 3.16: HNMR spectrum of Anti-hMAM / DOX loaded PEGylated CSNP (A) and Anti-HER2 / DOX loaded

PEGylated CSNP (B)

94

The FTIR spectrum of Anti-HER2 DOX loaded PEGylated CSNP and Anti-hMAM DOX loaded

PEGylated CSNP has been explored to define the change in the absorption peaks relative the non-

functionalized DOX loaded PEGylated CSNP. This is shown in Figure (3.17). The shifting of OH

peak from 3430 cm-1 to 3300 is an indication of the pronounced COOH group to the surface of the

CSNP. Moreover, the increased intensity of the peak at 1600 cm-1 can be attributed to the increased

surface coverage with NH2 groups. These groups may be related to the immobilized mAbs (216).

The disappearance of the characteristic peak of PEG at 1094 cm-1 indicates, also, the conjugation

of the mAbs to the surfaces.

Figure 3.17: FTIR spectrum of mAbs /DOX loaded PEGylated CSNP and non-functionalized DOX loaded

PEGylated CSNPs

3.5. Cytotoxicity Analysis:

The cytotoxic effect of different nano-formulated systems on MCF7 cells have been more

prominent than various free DOX drug concentrations. L929 cells were less responsive to the

different nano-formulations as shown in Figure (3.18) (102).

Tra

nsm

itta

nce

(%

)

Wavenumber (cm-1)

95

Figure 3.18: Colorimetric MTT assay of DOX loaded PEGylated CSNP (1.25, 2.5, 5 and 10 Ug/ml) (A), Free DOX

concentrations (1.25, 2.5, 5 and 1 Ug/ml) (B) and Anti-HER2 and Anti-hMAM functionalized DOX loaded

PEGylated CSNP.

A

B

C

96

Various nano-formulations have been screened including unloaded CSNP, PEGylated CSNP,

DOX loaded PEGylated CSNP (variable concentrations), free DOX, Anti-HER2 functionalized

PEGylated DOX loaded CSNP and Anti hMAM functionalized PEGylated DOX loaded CSNP.

The cytotoxicity results suggest that nanoformulated DOX systems have shown greater toxicity on

cancerous MCF7 cells. As shown in Figure (3.14A), PEGylated DOX loaded CSNP with drug

concentrations of 1.25, 2.5, 5 and 10 µg/ml could achieve MCF7 cell viability rates of 24.7± 4.2

%, 18.66 ±2.3%, 14.98± 1.5% and 39.44± 3.1% respectively. Normal L929 cells have been less

affected by these nano-formulated drug concentrations and less responsive to the released drug,

although both types of cells had identical same incubation conditions of time, type of media and

drug interval concentrations. The released drug could achieve successful endocytosis in case of

MCF7 and killed more than 50% of the seeded cells. (130).

The nanoparticles encapsulating 5 µg/ml DOX has shown the highest cytotoxicity effect with

14.89% viable MCF7 cells. Le et al. has reported the minimal cytotoxic effect of the released DOX

when the drug concentration is increasing than proposed concentrations in this study (218). On the

other hand, the effect of the same 5Ug/ml nano-DOX formula on normal L929 has been the least

compared with other concentrations. This may put some confirmatory assets on its therapeutic

durability and biological safety (102).

Free DOX concentrations have demonstrated higher viability rates of MCF7 cells with 30 ± 1.98%,

34 ± 1.45%, 38 ± 4.31% and 45 ± 2.21% viability rates for similar free drug concentrations of the

nano formulated DOX. Therefore, their cytotoxic effect on cancerous cells is lower than nano

DOX formulations. We have demonstrated their partial dose dependent cytotoxicity on MCF7 and

L929. The most efficient free DOX concentration could revel around 30 ± 1.98 % viability rate.

While in nano formulated DOX, the lowest viability rate has been 14.89 ± 1.5%. This suggests a

good indication that our PEGylated CSNP system has improved the cellular uptake and

intracellular delivery of DOX with one-time higher rate, under the same exposure time relative to

free DOX (53). Confirmatory bright filed images of MCF7 and L929 after exposure to different

drug formulations are shown in Figures (3.19) -(3.22).

97

Figure 3.19: Bright field images of L929 cells with white arrows referring to living cells (A), and MCF7 cells with

white arrows referring to dead cells (B) after exposure to DOX – PEGylated CSNP (0.5 µg/ml)

98

Figure 3.20: Bright field images of L929 cells with white arrows referring to dead cells (A), and MCF7 cells with

white arrows referring to living cells (B), after exposure to Free DOX

99

Figure 3.21: Bright field images of L929 cell line with white arrows referring to living cells (A), and MCF7 cell line

with white arrows referring to dead cells (B) cells after exposure to Anti-HER2 functionalized DOX loaded

PEGylated CSNP

100

Figure 3.22: Bright field images of L929 cells with white arrows referring to living cells (A) and MCF7 with white

arrows referring to dead cells (B) cells after exposure to Anti-hMAM functionalized DOX loaded PEGylated CSNP

101

Functionalization of monoclonal antibodies to the surfaces of DOX loaded PEGylated CSNP as a

targeting moiety was assumed to be a productive therapeutic conjugate in terms of cancer cell

cytotoxicity. This has empowered the cellular uptake and cell viability rates of these nano

formulations. In this sense, Anti HER2 and Anti hMAM antibodies have been used for

functionalization of DOX loaded PEGylated CSNP separately. This could enhance the tumor cell

susceptibility and higher intracellular accumulation of the released DOX. Our results have showed

the potential of the nano formulated DOX functionalized with these monoclonal antibodies to

increase its cytotoxic effect in MCF7 three times as free DOX, while maintaining higher viability

rates of normal L929 cells (131).

Anti-HER2 and Anti-hMAM functionalized nano formulations have shown MCF7 viability rates

of 1.79 ± 0.06% and 2.99 ± 0.023% respectively. While Normal L929 have been less responsive

and less affected by the same formulations. This may be a good marking point that MCF7 are more

affected by these antibodies functionalized nano formulations, while normal L929 cells are less

susceptible and less affected. Specific antigen-antibody mediated drug accumulation my

developed more efficient cellular endocytosis and, hence, higher drug efficacy on MCF7. On the

other hand, L929 normal cells lack such tumor cell specific receptors like HER2, for instance.

Therefore, such intracellular drug uptake is not expected, and receptor mediated coupling

mechanism hiders drug release or accumulation with normal cells (219). The bright field images

of the cells after identical exposure to antibodies functionalized DOX loaded PEGylated CSNP

have demonstrated the viability profile. The difference in cellular shape and uniformity responses

of both types of cells may exhibit their different susceptibilities.

The MTT test has been performed in CO2 incubator under processing temperature of 37 °C. The

temperature has been proposed to affect the intracellular action of DOX and affect its diffusion. In

this sense, Miller’s law of diffusion entails a strong relationship between the temperature and drug

diffusion rates (220). Therefore, the diffusion and cytotoxicity profiles of all our experimented

formulations, including loaded and unloaded DOX, are less likely to be affected by this

relationship. That’s because the experimental processing temperature was 37°C, which is higher

than the intermolecular self-aggregation temperature of DOX. In this temperature, intermolecular

102

π – interaction may be formulated specially less than 4°C, which minimize the diffusion of DOX.

However, our study has been performed at physiological 37°C temperature (221).

3.6. Size increment profile:

Over the different activities of our study, variable sizes of CSNP have been obtained. The size

increment over all the activities didn’t exceed 10%. This is a good indication for the surface

stability. The size increment after PEGylation of CSNP, encapsulation of DOX to PEGylated

CSNP, functionalization of Anti-HER2 to DOX loaded PEGylated CSNP and Functionalization

of Anti-hMAM to DOX loaded PEGylated CSNP have been 10%, 7.23%, 4.3% and 4.72% (222).

Figure 3.23: TEM images and the relevant DLS histograms of non-PEGylated CSNP (A, B) and

PEGylated CSNP (C, D)

103

Figure 3.24: TEM images and their relevant DLS histograms of DOX loaded PEGylated CSNP (E, F), Anti-

HER2 DOX loaded PEGylated CSNP (G, H), and Anti-hMAM DOX loaded PEGylated CSNP (I, J)

104

Appendix

(supporting Information)

105

Supporting Information:

SI. 1. Synthesis of chitosan nanoparticles.

SI. 1.1. Dissolving Chitosan:

Chitosan acidic solution has been prepared simply through dissolving LMW chitosan powder with

different weights including 1 gm, 0.5 gm, 0.25 gm and 0.05 gm in 1% acetic acid. The pH has

been adjusted to 5.0 using 0.1N NaOH. These prepared solutions have been left to be mixed and

dissolve for 24 hours under magnetic stirring at 750 RPM within the ambient temperature. The

resulted four solutions have showed different viscosities and some dissolved traces have been

removed out using 0.4 Um syringe filter mesh. All preparation parameters and conditions have

been controlled within this step-in order to obtain homogenous chitosan acidic solutions. The

stirring beakers have been covered with protective par-film sheets in order for avoiding the

entrance of any contaminants, and suitable sizes of magnetic bars have been used for the mixing

process.

SI. 1.2. Dissolving Sodium tri-polyphosphate:

Sodium tri-polyphosphate is a chemical cross linker that has been used in this work in order to

induce the ionic gelation process through which, chitosan nanoparticles would be formulated. TPP

has been dissolved in purified distilled water within four different concentrations; 0.1%, 0.4%,

0.5% and 0.7%. Suitable sizes of magnetic bars have been used for stirring process. The solutions

have been rapidly mixed within 35 minutes at 500 RPM within the ambient conditions.

SI. 1.3. Preparation of Crosslinked chitosan nanoparticles:

After formation of homogenous chitosan and TPP solutions, they have been mixed together.

Whitish clouds of chitosan nanoparticles have been spontaneously formed upon addition of the

two polymers together. In order for separating these nanoparticles, we have applied efficient

centrifuging forces. Therefore, the reacted polymers part, that formulated crosslinked chitosan

nanoparticles, could be separated from the unreacted eluted polymers. The resulted pellet of

106

chitosan nanoparticles has been used for further investigative characterization techniques. For this

purpose, the pellet has been divided into three sections as shown in figure SI. 1 and table SI. 1.

Table SI. 1: variable conditions for characterizing chitosan nanoparticles

Nanoparticles pellet Action Characterization Purpose

First part (minor) Oven drying Scanning electron microscopy (SEM),

Second part (moderate) Sonication + dilution

(distilled water)

Dynamic light scattering (DLS), Ultraviolet-Visible

spectrophotometry (UV-VIS) and Transmission electron

microscopy (TEM)

Third part (major) Freeze drying Fourier transmission infra-red spectroscopy (FTIR), X-ray

Diffraction (XRD)

Figure SI. 1: Divided pellet undergoes further investigation techniques with a prior sample preparation technique

such as oven drying, freeze drying and resuspending in distilled water.

SI. 2. Factors affecting chitosan nanoparticles:

The morphological chemical properties of chitosan nanoparticles are significantly affected with

various parameters and factors. This is a part of the reason of why chitosan is a remarkable

biopolymer and possess attractive advantages for various biological and non-biological

applications. Changing some parameters such as polymers concentration, mixing rate and mixing

time can obviously change the size and shape of the formulated nanoparticles. It has a simple

107

analogy like tailoring a specific nano-carrier or nanosystem with providing certain specific resulted

features and properties, by playing with these stated parameters.

SI. 2.1. Effect of polymers concentrations:

The particle size, shape, distribution and surface charge of the prepared chitosan nanoparticles can

be influenced by the concentrations of chitosan and TPP. In order to investigate this influence,

various concentrations of both polymers have been applied with keeping all other preparation

parameters constant. Identical volumes of both polymer solutions have been applied, while their

concentrations have been varied. Four different concentrations of chitosan acidic solutions have

been used; 0.05%, 0.25%, 0.5% and 1% (w/v).

On the other hand, sodium tri-poly phosphate concentrations were 0.1%, 0.4%, 0.5% and 0.7%

(w/v). Sixteen samples of whitish clouds of chitosan nanoparticles have been formulated up on

drop wise addition of sodium tri-polyphosphate to chitosan acidic solutions with a dropping

syringe. Based on the number of concentrations of both polymers, the optimization of the

nanoparticles has taken place in a quadruple manner as shown in table 2.

Table SI. 2: General scheme for the applied polymers concentrations; Cs and TPP

Sample Cs (w/v) % TPP (w/v) % 0.05 0.1 0.5 1 0.1 0.4 0.5 0.7

1

2

3

4

5

6

7

8

9

10

11

108

12

13

14

15

16

The mixture of chitosan solution and TPP has been left for mixing on the magnetic stirring under

the previously stated preparation parameters in terms of mild speed and time within protected

ambient conditions. The resulted sixteen samples of chitosan nanoparticles have been visually

analyzed as turbid suspensions of undissolved white clouds of nanoparticles. After using particles

size analyzer which has been based on dynamic light scattering physical principle (Malvern

Zetasizer®), sample 8 has been the sample of choice in terms of size and distribution.

SI. 2.2. The effect of Stirring speed:

Another significant factor that has a direct influence on the resulted chitosan nanoparticles is

related to the identified mixing technique. Stirring speed has been reported to directly affect the

NP’s size and distribution (223). Although it has reported by Oliviera et. al., that stirring time and

speed have low influence on the size and distribution of the formulated nanoparticles (224). We

have investigated that this contradict with our study. Four main different stirring speeds have been

investigated and analyzed to produce various sizes and distributions; (250, 500, 750 and 1000)

RPM. All other parameters have been constant including polymers concentrations. The sample of

choice, sample 8, has been investigated in these four different stirring speeds as shown in table 2.

Table SI. 3: Various stirring speed provide variation of Cs nanoparticle’s size and distribution.

Stirring speed Particle size PDI

250 RPM 182 ± 5.3 0.37 ± 0.004

500 RPM 345.9 ± 1.3 0.517 ± 0.003

750 RPM 357.2 ± 4.45 0.512 ± 0.041

1000 RPM 391.4 ± 2.2 0.491 ± 0.007

SI. 2.3. The effect of stirring time:

109

Stirring time is one of the main influential factors that affect the size and distribution uniformity

of the formulated chitosan nanoparticles. In order to investigate that notion, we have performed

this preparation procedure with keeping all other preparation factors constant. The polymers

concentrations, stirring speeds and temperature have been kept constant with changing the

performing stirring time. The influence of changing stirring time on particle size and distribution

has been studied with three different time slots; (15, 30 and 60) minutes as shown in figure 3.

According to the resulted smallest particles sizes and smallest PDI, the optimized procedure has

been selected. (225, 226)

Table SI. 4: Variation of the stirring time provides variation of particles size and distribution.

Stirring time Particle size PDI

15 min 245 ± 7.6 0.67 ± 0.004

30 min 182.2 ± 5.45 0.304 ± 0.009

60 min 209.3 ± 25.45 0.609 ± 0.082

SI. 2.4. The effect of chitosan molecular weight:

Molecular weight of raw chitosan powder is another effective factor that influence the size of the

resulted chitosan nanoparticles (227). It has been reported by many studies that there is a direct

relationship between the molecular weight of chitosan relative to particles size and distribution of

the resulted chitosan nanoparticles (228). Considerably, the effect of varying the molecular

weights of chitosan on the resulted nanoparticles has been studied with holding the initial chitosan

and TPP constant. The molecular weights of chitosan have been changed within three different

preparations; Low molecular weight chitosan (LMWC), medium molecular weight chitosan

(MMWC) and high molecular weight chitosan (HMWC). However, all other preparation

parameters and factors have been kept constant, and the optimized molecular weight has been

selected based on the resulted particle size and distribution.

Table SI. 5: Variation of chitosan molecular weight provides variation of particles size and distribution

110

Chitosan Molecular weight Degree of Deacetylation particles size PDI

LMWC (110) 89.9 % 187 ± 2.7 0.282 ± 0.015

MMWC (400) 84.8 % 357 ± 80.5 0.81 ± 0.14

HMWC (800) 76 % 444.2 ± 106.9 0.52 ± 0.07

SI. 2.5. The effect of pH:

The external morphology mean sizes and uniform distribution of chitosan nanoparticles can be

affected by different pH of chitosan solution. The relation between the crosslinking process of

chitosan/TPP and changing the pH of chitosan solution has been reported by some studies (229).

For investigating this effect, we have adjusted the pH of chitosan solution from 2.6 to 5.5 (2.6, 3.1,

3.5, 4.5, 5 and 5.5). The protonation degree of chitosan polymer can greatly control the cross-

linking process of CSNP. On the other hand, this protonation degree can be affected with chitosan

solution pH, therefore we need to investigate this factor and whether it needs to be controlled

within specific range that doesn’t harm the intended physiological environment in which the

nanoparticle would deliver certain drug or not.

Table SI. 6: various processing pH provide variation of particles size and distribution

pH Size PDI

5.5 196 ± 4.1 0.42 ± 0.06

5 182 ±3.7 0.22 ± 0.005

4.5 180.8 ± 4.4 0.34 ± 0.003

3.5 162 ± 4.6 0.35 ± 0.008

3.1 147 ±1.9 0.41 ± 0.05

2.6 136 ± 6.1 0.47 ± 0.005

SI. 3. Stability of chitosan nanoparticles:

Throughout all the optimization procedure and manipulation of the different preparation factors of

the resulted chitosan nanoparticles, we could identify the optimum procedure through which we

can obtain chitosan nanoparticles with satisfying physical and chemical properties. The main

characteristic features of the nano-system that we have investigated are particle size, poly

distribution and zeta potential value as shown in table 6.

111

Table SI. 7: Stability profile for the prepared chitosan nanoparticles along over three weeks

Zeta potential is an indicative parameter to the surface charge and hence the aggregation tendency

or size stability in the intended suspending medium as shown in table 7 (230). This is a very

important parameter whenever the formulated nano-system would be intended to be injected in a

biological tissue within biomedical applications. In other words, the size stability of the in-vivo

colloidal nanoparticles system can be assessed with zeta potential value.

Table SI. 8: Various ranges of Zeta potential values with their corresponding aggregation tendencies (231)

Time Particle size PDI

1 day 183.7 ± 5.8 0.20

2 days 187 ± 3.9 0192

4 days 187.6 ± 4.6 0.223

6 days 195 ± 2.12 0.253

8 days 198.5 ± 3.5 0.262

10 days 193.4 ± 6.1 0.292

12 days 184.5 ± 5.7 0.239

14 days 192.2 ± 8.4 0.209

16 days 200.1 ± 2.1 0.190

18 days 196 ± 3.4 0.242

20 days 204.4 ± 4.6 0.240

22 days 201.1 ± 5.2 0.189

112

SI. 4. Theoretical and Instrumental background:

SI. 4.1. Ultrasonic Probe Sonicator (Polytron, Thermo-fisher:

Ultrasonic probe sonicator is an instrument that is used for mixing and extraction of various

chemical or polymeric compounds. This device is working by generating powerful energy in the

form of sound waves that are enough to agitate or suspend micro and nano sized particles in a

solution (232). These sound waves are ultrasonic waves (frequency > 20 kHz). They provide rapid

and violent vibration of the probe tip, and this creates a kind of cavitation that collapse within the

solution or medium as shown in (Figure 2).

Figure SI. 2: controlled compression and expansion energy that bring particles to larger sizes which later

breakdown into much smaller ones (232)

This cavitation intensity is controlled by the setting agitation time and amplitude settings. It creates

what is called cavitation bubbles that grow in size until it reaches a critical size that can

aggressively disrupt the intermolecular covalent bonding of the applied medium as shown in

(figure 2). Therefore, the greater the frequency, the stronger the waves, the higher cavitation

intensity and hence the stronger particles agitation. It has been recommended by many studies that

ultrasonic probe sonicator is a recommended high-speed mixing technique and put it as the first

technique of choice for mixing (233)

113

Ultrasonic probe sonicator is mainly composed of three main parts; generator, converter and probe

tip (figure 3). Generator is the processing unite through which, we can control the sonication

parameters. It provides high voltage pulses of electrical energy that is produced basically form

normal alternating current and convert into such powerful pulses through that generator unite

(234).

Figure SI. 3: The three main Instrumental parts of a Probe-Sonicator (234)

Converter is a piezoelectric unite of the ultrasonic instrument. It is typically controlled through the

high voltage pulses that are created by the generator. These pulses provide such enough powerful

energy at a certain un-fluctuated frequency of 20 kHz. The main function of the converter is to

convert the produced electrical energy into mechanical energy. Therefor there is a high voltage

cable that connect between the converter and the generator. The outer shape of converter, as shown

in figure X, is cylindrical. The third part of the ultrasonic sonicator is the probe or horn. This the

instrumental part of the device the is put in direct contact to the applied sample. It has threaded

ends whose longitudinal tip expands and contract during processing (235).

SI. 4.2. Freeze drying (BIOBASE, Jinan, China):

Freeze drying is a dehydration process or removal of solvent and it is called also lyophilization. It

is a frequently used technique in many pharmaceutical industries, different scientific laboratories

and food industries. Basically, freeze drying is applied for drying heat labile samples so that it

becomes more stable in a powdered form without presence of moisture any solvent after being

frozen (236). Samples like proteins, tissues, plasma, dead cells and pharmaceuticals are extremely

114

heat labile and can’t be dried by oven drying technique, therefore we can use freeze drying for

keeping them in more a stable state. The basic scientific concept behind freeze drying is related to

sublimation phenomenon. This phenomenon states that solid iced material can undergo phase

transition to become gaseous vapor without being exposed to the liquid state. This occurs by

applying low pressure and low temperature that allow the frozen water or solvent to sublimate as

shown in (figure 4). This diagram, also, represents water phase diagram. In other words, it

represents the physical state of water under different conditions including temperature and

pressure. Through the diagrams, sublimation occurs when pressure is lowered, and heat is applied

to the frozen sample. Sublimation rate increases more with vacuum. This is a simple explanation

of the freeze-drying process and its scientific concept.

Figure SI. 4: Theoretical illustration of freeze-drying (237)

Sublimation is the main target for a freeze drier unite in order to completely dry the sample and

evaporate the remaining excess water by breaking the bonds between the sample and water. Three

main phases for the freeze drier to pass through as shown in (figure 5); freezing, primary drying

(vacuum sublimation) and secondary drying (adsorption) (238).

115

Figure SI. 5: Graphical illustration for different stages of freeze-drying process (238)

SI. 4.2.1. Freezing:

In this phase, the sample is completely frozen in a vial that is done by freezer or even a chilled

bath. The sample is cooled down to reach below the triple point in order to ensure that the sample

will not undergo melting but rater with expose to sublimation.

SI. 4.2.2. Primary drying (vacuum sublimation):

This is the stage in which around 95% of the entire drying process takes place. The application of

vacuum in this stage is a reason of rapid sublimation. It is a slow phase in which preassure is

lowerd and moderate heat is applied because excessive heat may cause breakdown or change in

the structure of the sample. The released water vapor is condensed and solidified within the

condenser.

SI. 4.2.3. secondary drying:

It is called, also, adsorption phase in which the ionic bonds between water and the applied material

is broken and removed. This breakage is owing to the increased temperature in the freeze drier to

a degree higher than the primary drying phase so that the remaining 1-5 % of the residual moisture

is removed. The dried samples can be preserved within dry un-moisturized environment such as

silica jar or refrigerator so that they can be prepared for later phases of the study.

The main purpose of using freeze drier during the current study has been to provide a stable form

of free unloaded CSNP, PEGylated CSNP and DOX loaded CSNP. The dried forms of these

nanoparticles have been used for further characterization analytical techniques such as FTIR and

116

XRD. Additionally, these dried forms have had more stability profile and longer shelf life as a

powered form. They can be used anytime with a definite amount of micro and milligrams of the

nanoparticles. During drug loading and release studies, we have used to weigh certain amppunts

of nanoparticles in order to investigate some critical analytical tests such as release, loading and

cell viability percentages.

SI. 4.3. Magnetic stirrer (ISG®, China)

It is a laboratory device that is frequently used in most chemistry, physic and biology laboratories

for mixing of different fluids. It is a simple device in which a magnetic bar is allowed to stir at the

bottom of certain sample as shown in figure 6 This rotating bar is stirring within a rotating

magnetic field. The sited bar is immersed inside the vessel of liquid sample, and it must be of

suitable size relative to the applied vessel.

Figure 6: Magnetic stirrers (239)

In case of glass vessel, it can be broken if the size of the stirring bar is quite large and strong.

Another important factor that affect the stirring process is the viscosity of the liquid. When the

applied liquid has high volum or high viscosity, additional mechanical stirring is required. The

main controllers with magnetic stirrer device are rotating speed, that is defined as round per minute

(RPM), time that is defined in minutes and temperature, that is defined by Celsius C (240). The

size of the stirring bar is another considerable factor that variably affect mixing process. As shown

in figure 7, there is a variety of sizes and shapes of the used magnetic bars. The selection of a

specific size or shape is basically related to the purpose of mixing process and the size of the

container in which we perform mixing of the sample.

117

Figure 7: Different shapes of magnetic mixing bars (241)

SI. 4.4. Centrifugator (HERMLE Labotechnik 36 HK, Germany)

Centrifugation is one of the fast separation techniques that involves sedimentation of suspended

particles to the base of a liquid medium. This sedimentation can take place naturally without any

external interference, but this may take very long time. Many factors affect the particles

sedimentation or separation including particles shape, density and size, in addition to the viscosity

and temperature of the suspending medium. it has wide application in most biological and chemical

experiments such as DNA separation and proteins purification (242).

It is a simple instrument in which the sample is placed in a vessel called centrifuging tube. This

tube is allowed to rotate in a rotor whose speed is adjusted before the sedimentation process. As

the rotor speed gets higher, a centrifugal force is moved and each particle in the medium will sense

that force as shown in (figure 8). This result in immediate sedimentation of these particles. The

sedimentation rate is directly proportional to the difference between the density of the medium and

the density of its suspended particles. Additionally, the viscosity and some other physical and

chemical properties influence the sedimentation rate. If we fixed the viscosity of the medium and

centrifugal forces, the molecular weight of the dissolved particles will be directly proportional to

the sedimentation rate (243).

118

Figure 8: Illustrative description of centrifuging process (244)

SI. 4.5. Oven drying (TITANOX, Italy):

Drying by oven is the process of removing any solvent that is mixed with the applied sample, and

this sample must be non-heat labile in order to avoid its destruction. It is applicable in small scales

like laboratories and larger industrial scale as well. The main purpose of oven drying remains the

same at both scales is to remove the mixed solvent and avoiding moisture. It is completely made

of stainless steel and have a key and lock door as shown in (figure 9). During our experiment,

drying oven has been used to dry PEGylated and non-PEGylated chitosan nanoparticles so as to

be ready for SEM analysis. Each sample has been spreaded over an aluminum foil that has been

fixed on a simple watch glass, then entered to the oven that has been adjusted at 60C for 10

minutes in order to evaporate the solvent. In few times, the drying oven has been used to sterilize

and dry the applicable used glassware. (245)

119

Chapter 4

Conclusion and Future

Perspectives

Figure 5.5: Microscopic images

120

Conclusion:

The current study has developed a therapeutic assumption of developing a selective targeted drug

delivery system for the treatment of breast cancer. The proposed system has intended chitosan

nanoparticles in order to entrap DOX inside, with maintaining the external surface of the

nanoparticles decorated with PEG polymer. In order to enhance its specificity and sensitivity, the

nano-system has been functionalized with two different types of breast cancer specific monoclonal

antibodies; Anti-HER2 and Anti-hMAM. Along over the study, some characterization techniques

have been applied as confirmatory procedure to ensure the durability of the final therapeutic cargo.

Examples of which, DLS, TEM, XRD, FTIR, UV-VIS and HNMR. In this sense, primary size

adjustment of blank unloaded CSNP has been performed to put some control over the size, surface

charges and stability in different pH media. Then PEG has been grafted on the external surface of

CSNP in order to enhance its stability against immunogenic responses and prevent its lymphatic

uptake or RES uptake. Three PEG concentrations have been demonstrated; 5% PEG had higher

stability compared with other proposed concentrations with the lowest particle size. This is because

the final size of the nano-system has been supposed to be less than 200 nm. That is because the

RES capturing size limit is known to be hindered for size range less than 200nm. After confirming

stability of the PEGylated CSNP and its size stability below that RES opsonization range, DOX

has been encapsulated in different concentrations with various nano-formulations. 5Ug/ml

concentration formula has been the most effective one in terms of loading capacity and surface

stability. Therefore, it has been selected for the functionalization of mAbs. Each one of the mAbs

has been functionalized separately; having two final types of nano-functionalized formulations.

The cytotoxicity analysis has been conducted against MCF7 breast cancer cells and L929

fibroblasts has been conducted. Various nano-formulations have been tested against the effect of

free DOX on the same two types of cells with maintaining fixed temperature, drug concentration

and incubation period. The Anti-HER2 and Anti-hMAM functionalization could promote the

MCF7 cytotoxicity, with achieving less than 3% cell viability. While, L929 cells were less reactive

and less responsive than MCF7. Therefore, the assumption of encapsulating DOX into a selective

targeted nano-system has enhanced its in-vitro therapeutic effect with promoted cancer cell

endocytosis. Bright filed images have exhibited cancerous and normal cell responses to the

developed formulations. Therefore, the current study recommend the application of polymeric

PEGylated chitosan nanocarrier with mAbs functionalization as a novel therapeutic system for

121

selective release of the entrapped DOX. This may provide one of the nanoscience based biomedical

solutions for breast cancer treatment.

Future Perspectives:

During this study, some tests and experiments were required to be implemented in order to give a

clear understanding of the structural and therapeutic efficiency of the developed nano-

formulations. The structural stability of the developed nano-formulations can be empowered with

the following:

o Body fluid analysis me give a better prediction of the biological behavior of the developed

formulation. This will detect the efficacy of some parameters that have been already tested

in this study. Particles stability along over the its site of introduction till reaching its site of

action can be demonstrated with this test. Besides, the convenience of the nanoparticles’

sizes developed in this study can be confirmed with this test.

o Animal testing may improve the pharmacokinetic and pharmacodynamic profiles of the

developed nano-formulations. Detecting some parameters such as plasma level and

elimination rate of the injected drug may promote a better understanding about the assigned

dose of the final drug. Besides, monitoring In-vivo blood release of the drug can be another

confirmatory procedure that is achieved with such animal testing. It illustrates a clear

relationship between the release rate and different pH leves.

122

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