In situ forming supramolecules and chitosan based hydrogels for regenerative medicine

Using targeted nanoparticles to deliver chemotherapeutic agents in cancer therapy offers many advantages to improve drug delivery and to overcome many problems associated with convention

A dissertation for the degree of doctor of philosophy

Stimuli responsive PEGylated nano-assemblies for

cancer-targeted drug delivery

Department of Molecular Science and Technology

The Graduate School of Ajou University

Dai Hai Nguyen

Stimuli responsive PEGylated nano-assemblies for

cancer-targeted drug delivery

Supervisor: Professor Ki Dong Park

A Dissertation Submitted to the Graduate Faculty in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

June 2013 Department of Molecular Science and Technology

The Graduate School of Ajou University

Dai Hai Nguyen

Acknowledgement

I wish to express in this part my gratitude to the scientists, technicians and other people who were directly and indirectly involved in this work, without the help of whom the findings of this thesis surely could not have been done

First and foremost, I would like to extend immeasurable gratitude to Professor Ki Dong Park, for giving me the opportunity to do my PhD thesis under his supervision I greatly appreciated his supervision for teaching, advising and supporting me throughout

my work I am very grateful for his extreme patience and encouragement during the most stressful time when my results were not good He is a respectable mentor who has kindly supported me in the name of family It was an honor to work under his supervisor

I am grateful to my thesis committee members, Professor Sung-Hwa Yoon, Professor Won-Hee Suh at Ajou University, Professor Ji Hoon Jeong at Sungkyunkwan University, Dr In Kwon Jung at Genoss Company for their numerous suggestions and helpful advice This is a good opportunity to express my gratitude to Professors at Ajou University whose teaching and advice helped me to complete my PhD coursework

I would especially like to thank Dr Yoon Ki Joung who has supported for me for about three years He kindly and friendly guided me from laboratory studies to routine life in Korea I also have deep gratitude towards Dr Jin Woo Bae for being a great mentor His scientific comments are always useful in doing experiments, preparing presentation, and writing a scientific paper

I would like to thank my Vietnamese Professors Thi Phuong Thoa Nguyen, Thi Kieu Xuan Huynh, and Huu Khanh Hung Nguyen for giving this opportunity to me, who taught me fundamental knowledge of chemistry at University of Science-HCMC

I especially appreciate all supports of my past and current members in Biomaterial and Tissue Engineering Laboratory: Dr Kyoung Soo Jee, Dr Jin Woo Bae, Dr Dong Hyun Go, Dr Jung Seok Lee, Dr Kyung Min Park, Dr Se Jin Son, Dr Ngoc Quyen Tran,

Dr Eugene Lih, Jong Hoon Choi, Yeo Jin Jun, In Kyu Hwang, Bae Young Kim, Ji Ho Heo, Seung Soo You, Ki Seong Ko, Ji Hye Oh, Seung Mee Hyun, Dong Hwan Oh, Joo Young Son, Yun Ki Lee, Ji Ho Kim, Min Yong Eom, Thi Thai Thanh Hoang, Thi Phuong

Le I hope all members in BT Lab will obtain the outstanding achievement in your dream and get the happiness in their life

I appreciate all help of my Vietnamese best friends in Korea, Minh Dung Truong, Van Thinh Nguyen, Dinh Chuong Pham, Ngoc Hoi Nguyen, Thanh Quy Nguyen, Hung Cuong Dinh, Thi Hiep Nguyen, Chan Khon Huynh, who helped in several experiments such as XRD, AFM, DLS, Confocal, FACS, cell culture, and animal studies Without them this thesis surely would not have been so multifaceted and prolific I also would like

to be thankful to Korean friends in School of Engineering, Medicine School for your help and support me during my stay here Good luck to all of them

Korean life could be some times stressful and tough, with all the competitiveness and perfectionism Luckily, I have had extensive care, support, and help from my family and friends, who shared with me many wonderful and unforgettable moments throughout

my time here I would like to devote this thesis to them with my sincere gratitude

I would like to thank many of my best friends, Hoang Duy Nguyen, Minh Triet Thieu, Hoang Chuong Nguyen, Nhat Nguyen Nguyen, Xuan Huong Ho… With them I shared the first journey to Korea, as well as the sadness of leaving our lovely home and country

All this would not be possible without my loving immediate family For good or for bad, they are the ones who always stand behind me, and let me know that I am not alone Finally, deeply from my heart, I would like to thank my parents who believe and support me at all time

My best regards to all,

Dai Hai Nguyen

i

Abstract

Cancer is one of the leading causes of death worldwide and chemotherapy is a major therapeutic approach for the treatment which may be used alone or combined with other forms of therapy However, conventional chemotherapy has the potential to harm healthy cells in addition to tumor cells Using targeted nanoparticles to deliver chemotherapeutic agents in cancer therapy offers many advantages to improve drug delivery and to overcome many problems associated with conventional chemotherapy This work covers the general areas of responsive nanocarriers and encompassed methods of fabricating nanocarrier-based drug delivery systems for controlled and targeted therapeutic application

Chapter 1 provides general information of cancer and cancer treatment strategies The recently cancer treatment based on nanocarrier were introduced In addition, the special features as well as requirements of nanoparticles for targeted drug delivery were presented This chapter describes overall objectives of this study with the current status of stimuli-responsive self-assembled nanocarriers for cancer chemotherapy In chapter 2, self-assembled nanogels based on reducible heparin-Pluronic copolymer was developed for intracellular protein delivery Heparin was conjugated with cystamine and the terminal hydroxyl groups of Pluronic were activated with the VS group, followed by coupling of VS groups of Pluronic with cystamine of heparin The chemical structure, heparin content and VS group content of the resulting product were determined by 1H

ii

NMR, FT-IR, toluidine blue assay and Ellman's method The HP conjugate showed a critical micelle concentration of approximately 129.35 mg L−1, a spherical shape and the mean diameter of 115.7 nm, which were measured by AFM and DLS The release test demonstrated that HP nanogels were rapidly degraded when treated with glutathione Cytotoxicity results showed a higher viability of drug-free HP nanogel than that of drug-loaded one Cyclo(Arg–Gly–Asp–D-Phe–Cys) (cRGDfC) peptide was efficiently conjugated to VS groups of HP nanogels and exhibited higher cellular uptake than unmodified nanogels In chapter 3, stimuli–responsive Pluronic micelles is developed for targeting cancer chemotherapy In particularly, the role of crosslinking disulfide bond and hydrazone bond in arrangement of environmental stimuli including redox and pH were discussed Specifically, acrylic acid was grafted onto PPO blocks of Pluronic by dispersion/emulsion polymerization and used to introduce thiol groups as well as hydrazine groups DOX was conjugated to the hydrazone groups to achieve the pH-triggered release The micelles were crosslinked by the formation of disulfide bonds due

to the presence of thiol groups on the polymer backbones The physico-chemical

properties of the micelles were characterized In vitro release studies were performed to

investigate pH-dependent release of DOX from the Pluronic micelles FA was conjugated

to the Pluronic polymer for targeting cancer cell FA conjugated micelles were compared with the micelles without FA using confocal laser scanning microscopy (CLSM) and flow cytometry The Pluronic micelles functionalized with FA targeting ligand on the surface showed the enhanced cellular uptake In chapter 4, self-assembled magnetic nanoparticles

iii

(SAMNs) were fabricated from β-cyclodextrins functionalized superparamagnetic iron oxide (SPIO@CD), paclitaxel (PTX), adamantylamine-poly(ethylene glycol)-vinyl sulfone (ADA-PEG-VS), and c(RGDfC) peptide for integrated cancer cell-targeted drug delivery In this approach, PTX and ADA-PEG-VS enabled the host-guest inclusion with SPIO@CD to form PEG-ADA:SPIO@CD:PTX SAMNs Furthermore, cyclo(Arg-Gly-Asp-d-Phe-Cys) (c(RGDfC)) peptide, a targeting ligand, could conjugate onto the VS groups of the PEG arms of SAMNs The architecture of SAMNs were characterized FT-

IR, TEM, and thermo gravimetric analysis (TGA), which confirmed that PEG, CD have been effectively functionalized on the surface of SPIO nanoparticles SAMNs were enabling to be controlled over the sizes, surface chemistry, payloads of supramolecular nanoparticle vector The sizes, drug entrapment efficiency (DEE), drug loading efficiency

(DLE), and SIPO encapsulation of SAMNs could turn by changing its components In

vitro PTX release profile from SAMNs was highly ADA response Cumulative releases of

PTX from SAMNs were 44.1% and 9.6% with and without ADA treatment after 120 h Most importantly, the analyses of vibration sample magnetometer (VSM) verified that the magnetic property of SAMNs was increased under the external magnetic field c(RGDfC)-conjugated SPIO nanocarriers exhibited a higher level of cellular uptake than

unmodified ones in vitro according to flow cytometry and confocal laser scanning

microscopy (CLSM)

iv

Table of Contents

Abstract i

Table of Contents iv

List of Figures viii

List of Tables xv

Chapter 1 General introduction 1

1 Cancer and strategy treatment 2

2 Nanocarrier strategies in cancer chemotherapy 5

3 Self-assembled nanocarrier for drug delivery 6

4 PEGylated nanocarriers for systemic deliver 9

5 Targeted drug delivery systems for cancer therapy .13

5.1 Passive targeting strategies and recent developments 15

5.2 Active targeting strategies and stimuli-triggered ligand presentation 16

6 Stimuli-response for controlled drug delivery 18

6.1 Concepts for designing stimuli-responsive nanoparticles 18

6.2 Previous studies of stimuli-response for controlled drug delivery 26

7 Overall objectives 28

8 References 30

v

Chapter 2 Self-assembled nanogels based on reducible heparin-Pluronic

copolymer for targeted protein delivery 35

1 Introduction 36

2 Materials and methods 40

2.1 Materials 40

2.2 Synthesis of copolymers and preparation of drug loaded nanogels 40

2.3 Polymer characterizations 44

2.4 In vitro release test 47

2.5 Cytotoxicity assay 47

2.6 Intracellular uptake study 47

2.7 Statistical analysis 48

3 Results and Discussion 50

3.1 Characterization of polymers and nanogels 50

3.2 CMC and size distribution of nanogels 51

3.3 In vitro release profiles of RNase A and heparin 55

3.4 Cytotoxicity of RNase A-loaded nanogels 55

3.5 Cellular uptake of HP−RGD nanogels 58

4 Conclusions 61

5 References 62

Chapter 3 pH- and redox-stimuli sensitive Pluronic micelle for targeted

vi

doxorubicin delivery 65

1 Introduction 66

2 Materials and methods 70

2.1 Materials 70

2.2 Synthesis of copolymers and preparation of micelles 70

2.3 Micelle characterizations 73

2.4 In vitro DOX release 73

2.5 Cytotoxicity assay 74

2.6 In vitro intracellular uptake study 74

2.7 In vivo tumor growth inhibition study 75

3 Results and Discussion 77

3.1 Characterization of Pluronic conjugates 77

3.2 Analysis of intracellular uptake 80

3.3 In vitro cytotoxicity 84

3.4 In vivo tumor growth inhibition 88

4 Conclusions 90

5 References 91

Chapter 4 Self-assembled magnetic nanoparticles based on host-guest inclusion for targeted paclitaxel delivery 95

1 Introduction 96

vii

2 Materials and methods 98

2.1 Materials 98

2.2 Synthesis and preparation of SAMNs 99

2.3 Characterizations 103

2.4 In vitro PTX release test 106

2.5 In vitro intracellular uptake study 106

3 Results and Discussion 107

3.1 Characterization 107

3.2 In vitro release test 112

3.3 In vitro cellular uptake 113

4 Conclusions 116

5 References 117

viii

List of Figures

Figure 1.1 Overview of the clinically most relevant drug targeting strategies (A)

Conventional chemotherapy (free drug) (B) passively targeted drug delivery system by virtue of the enhanced permeability and retention (EPR) effect (C) Active drug targeting to internalization-prone cell surface receptors (over)expressed by cancer cells generally intends to improve the cellular uptake of the nanomedicine systems (D) Active drug targeting to receptors (over)expressed by angiogenic endothelial cells aims to reduce blood supply to tumours (E) Stimuli-sensitive nanomedicines (F) Local drug delivery

Figure 1.2 Example of self-assembled nanocarriers for targeted drug delivery: a

Micelles, an aggregate of surfactant molecules dispersed in a liquid colloid where drugs are physically encapsulated in the inner core b Liposomes, a spherically arranged bilayer structure with drug loaded either in the inner aqueous phase or between the lipid bilayers c Oil/water emulsion, a mixture of liquids that are normally immiscible with drug loaded in the inner oil phase d Nanocapsules, a polymeric membrane which encapsulates an inner liquid core e Nanogels, a nanoparticle composed of a hydrogel f Core-shell particles, the location

of nanocrystals at the core with the polymers on the outer layer

ix

Figure 1.3 (a) Nanocarriers (a1) are coated with opsonin proteins (a2) and associate

with macrophages (a3) for transit to the liver (a4) Macrophages stationary in the liver, known as Kupffer cells, also participate in nanoparticle scavenging (b) Nanocarriers coated with PEG coating (b1) prevents this opsonization (b2), resulting in decreased liver accumulation (b3) and increased availability of the NP for imaging or therapy

Figure 1.4 Conceptual representation of nanoparticle tumor-targeting modalities

Passive targeting: Unlike that found in normal tissue, tumor vasculature

is leaky owing to fenestrations and gaps between endothelial cells that result from abnormal angiogenesis NPs in circulation can passively extravasate through these gaps and enter the tumor interstitium Poor lymphatic drainage found in some tissues helps to retain particles in the tumor space Active targeting: Ligands (e.g antibodies, peptides, small molecules, etc.) targeted toward moieties overexpressed or uniquely present on the plasma membrane of tumor cells can be used to actively enhance NP accumulation at the tumor site and can also help to internalize particles into cells via endocytosis

Figure 1.5 Dual and multi-stimuli responsive polymeric nanocarriers as emerging

controlled drug release systems There are two kinds of stimuli, broadly defined, that can be engineered into delivery systems: internal stimuli

x

(i.e., enzymatic reactions, changes in pH, redox, and temperature) and external stimuli (i.e., heat, light, magnetic and electrical fields)

Figure 1.6 Schematic illustration of block copolymer assemblies which can respond

to a range of stimuli characteristic of tumor tissues and intracellular microenvironments, promoting targeted delivery and controlled release

of therapeutic drugs and imaging agents

Figure 2.1 Schematic illustration showing the formation and redox-sensitive

intracellular delivery of a protein-loaded HP nanogel

Figure 2.2 Synthetic route of heparin−SS−Pluronic−VS conjugate

Figure 2.3 1H NMR (D2O) spectra of Pluronic−DVS (A), heparin−Cya (B), and

heparin−Cya−Pluronic−VS (C)

Figure 2.4 FT-IR spectra of cystamine dihydrochloride (a), heparin (b), Pluronic (c),

heparin−Cya (d), Pluronic−DVS (e), heparin−Cya−Pluronic−VS (f) Figure 2.5 The determination of the CMC from the function of fluorescence

intensity ratios of I 441.5 to I 337.5 versus the log concentration of Cya-Pluronic-VS (pH 7.4)

heparin-Figure 2.6 The size distributions (DLS) of nanogels of heparin−Cya−Pluronic−VS

(a) and RNase A-loaded heparin−Cya−Pluronic−VS (b) and the AFM

xi

image of heparin−Cya−Pluronic−VS (c) Figure 2.7 Release profiles of heparin from heparin−Cya−Pluronic−VS nanogel (A)

and RNase A from RNase A-loaded heparin−Cya−Pluronic−VS nanogel (B) Release studies were performed primarily in PBS media ((pH 7.4) and then treated with GSH at final concentration of 5 mM at 37oC Experiments were performed three times and the data indicate mean ± standard deviation

Figure 2.8 Cell viability of NIH3T3 cells incubated with heparin−Cya−Pluronic

−cRGDfC (♦) and RNase A-loaded heparin−Cya−Pluronic−cRGDfC (■) nanogels for 48 h at 37 oC The cell viability was determined by MTT assay and plotted against the polymer concentration in DMEM at 37 oC Experiments were performed four times and significant differences between the treatment means and control values at respective times are

indicated by * P < 0.05

Figure 2.9 Intracellular uptake of HP nanogels Confocal microscopic images

of HeLa cells incubated with FITC-labeled heparin−Cya−Pluronic−VS and FITC-labeled heparin−Cya−Pluronic−cRGDfC nanogels, which were shown as blue (upper-left), green (upper-right) and merged (lower) fluorescence

xii

Figure 3.1 pH- and redox potential-responsive Pluronic micelles for cancer-targeted

chemotherapy

Figure 3.2 A synthetic route to FA-Pluronic-C/H-DOX (i) PNC, TEA, EDA, THF,

(ii) AA, AIBN, APS, (iii) TEMPO, MeOH (iv) FA, EDC, NHS, MES buffer, (v) hydrazine, cystamine dihydrochloride, EDC, NHS, MES buffer, vi) DTT, borate buffer, (vii) DOX, TEA, DMSO

Figure 3.3 1H NMR spectra of AA-Pluronic-NH2 (i) and FA-Pluronic-C/H (ii)

Figure 3.4 Intensity ratio of pyrene as a function of FA-Pluronic-C/H concentration Figure 3.5 Time course changes in average diameter and size distribution of FA-

Pluronic-C/H-DOX micelles after DTT treatment (n = 4)

Figure 3.6 In vitro release behaviors of DOX from micelles at different pH values

DOX conjugated micelles (FA-Pluronic-C/H-DOX) at pH 7.4 (●) and pH 5.2 (■), DOX encapsulated micelles (FA-Pluronic-C/H/DOX) at pH 7.4 (○), or at pH 5.2 (□) (n = 4)

Figure 3.7 Confocal microscopic images (a-f) of HeLa cells treated with various

DOX formulations at a concentration equivalent to 3 μg/mL of DOX Pluronic-C/H-DOX (a, b), FA-Pluronic-C/H-DOX (c, d), and free DOX (e, f) were incubated with HeLa cells for 1 h (a, c, e) and 4 h (b, d, f)

xiii

Flow cytometry histrogram (g) and fluorescence intensity (h) of various DOX formulations internalized into HeLa cells for 4h

Figure 3.8 Dose-dependent cytotoxicity of (a) FA-Pluronic-C/H micelles and (b)

FA-Pluronic-C/H-DOX against HeLa cells after 48 h incubation (n = 4) Free DOX (×), Pluronic-C/H-DOX (○), and FA-Pluronic-C/H-DOX (●) were incubated with cells for 48 h

Figure 3.9 Tumor growth inhibition test and body weight change of s.c breast

MCF-7/ARD xenograft in female BALB/c mice (Mean ± standard error of the mean, n = 5) Mice were dosed i.v with saline (●), FA-Pluronic(SS) (■), free DOX(▲), FA/DOX-Pluronic(SS) (×) (DOX 4.0 mg/kg) by tail vein injection on days 0, 4, 8 The corresponding nanoparticle dose was 27.9

mg micelles/kg (a) Tumor volume changes, (b) body weight changes, and (c) Photos were taken on day 21 of our in vivo study

Figure 4.1 Schematic illustration for the fabrication of self-assembled magnetic

nanoparticles (SAMNs) with targeted drug delivery and magnetic resonance imaging (MRI) contrast enhancement functions from β-cyclodextrins functionalized superparamagnetic iron oxide (SPIO@CD), paclitaxel (PTX), adamantylamine-poly(ethylene glycol)-vinyl sulfone (ADA-PEG-VS), and cyclo(Arg-Gly-Asp-d-Phe-Cys) (c(RGDfC))

xiv

peptide moieties

Figure 4.2 Synthetic routes employed for the preparation of ADA-PEG-VS and

DOPA-CD moieties

Figure 4.3 1H NMR spectra of DOPA-CD (A) and ADA-PEG-VS (B)

Figure 4.4 FT-IR spectra of (a) bare Fe3O4 NPs; (b) DOPA-CD; (c)

ADA-PEG-VS, (d) SPIO@CD, (e) VS-PEG-ADA:SPIO@CD:PTX SAMNS

Figure 4.5 The particles size distribution of SPIO, SPIO@CD, and

VS-PEG-ADA:SPIO@CD:PTX by DLS (a, b, and c) and by TEM (d, e, and f)

Figure 4.6 In vitro characterization: (A) Magnetization curve of SIPO (white) and

SAMNs (black) at 298 oK measured by SQUID exhibiting magnetic saturation, (B) Photograph of magnetic separation of SAMNs by a magnet

Figure 4.7 In vitro PTX release profiles in PBS (pH 7.4) with (●) and without (■)

ADA from SAMNs

Figure 4.8 Confocal microscopic images (A) and fluorescence intensity (B) of HeLa

cells and c(RGDfC) pretreated HeLa celles incubated with PEG-ADA:SPIO@βCD:PTX SAMNs at 37 oC for 4 h

c(RGDfC)-xv

List of Table

Table 1.1 Selected examples of ligands used in active drug targeting

1

Chapter 1

General introduction

2

1 Cancer and strategy treatment

Cancer is one of the leading causes of death worldwide (13%) Each year 12.7 million people worldwide are diagnosed with cancer and there are 7.6 million deaths from the disease in 2008 (WHO).1 It is estimated that there are 24.6 million people alive who have received a diagnosis of cancer in the last five years By 2030, the number of new cancer cases is expected to rise to 21.4 million, with 13.15 million cancer deaths.2 Cancer's total economic impact was estimated at $895 billion in 2008, or 1.5% of the world's gross domestic product This cost did not include direct medical costs, which could potentially double the total economic cost, according to Atlanta-based ACS.3

The cancer treatment during the twentieth century was based on surgery, radiation and chemotherapy Of these modalities, surgery is most effective at an early stage of disease progression However, most cancer operations carry a risk of: pain, infection, loss

of organ function Surgery can also cause cancer cells to spread to different sites Radiation while destroying cancer cells also burns, scars, and damages healthy cells, tissues, and organs Initial treatment with chemotherapy and radiation will often reduce tumor size Radiation can cause cancer cells to mutate and become resistant and difficult

to destroy.4 Chemotherapy is drug therapy that can kill these cells or stop them from multiplying However, it involves poisoning the rapidly growing cancer cells and also destroys rapidly growing healthy cells in the bone marrow, gastro-intestinal tract, etc., and can cause organ damage, like liver, kidney, heart and lungs, and so on Moreover, when the body has too much toxic burden from chemo the immune system is either

is a method of delivering medication to a patient in a manner that increases the concentration of the medication in some parts of the body relative to others The goal of a targeted drug delivery system is to prolong, localize, target and have a protected drug interaction with the diseased tissue The conventional drug delivery system is the absorption of the drug across a biological membrane, whereas the targeted release system

is when the drug is released in a dosage form The clinically most relevant drug targeting strategies were summarized in Figure 1.1 The advantages to the targeted release system

is the reduction in the frequency of the dosages taken by the patient, having a more uniform effect of the drug, reduction of drug side effects, and reduced fluctuation in circulating drug levels

4

Figure 1.1 Overview of the clinically most relevant drug targeting strategies (A) Conventional chemotherapy (free drug) (B) passively targeted drug delivery system by virtue of the enhanced permeability and retention (EPR) effect (C) Active drug targeting

to internalization-prone cell surface receptors (over)expressed by cancer cells generally intends to improve the cellular uptake of the nanomedicine systems (D) Active drug targeting to receptors (over)expressed by angiogenic endothelial cells aims to reduce blood supply to tumours (E) Stimuli-sensitive nanomedicines (F) Local drug delivery

5

2 Nanocarrier strategies in cancer chemotherapy

The use of nanotechnology in medicine and more specifically drug delivery is set to spread rapidly Currently many substances are under investigation for drug delivery and more specifically for cancer therapy are used in the clinic Interestingly pharmaceutical sciences are using nanocarriers to reduce toxicity and side effects of drugs and up to recently did not realize that carrier systems themselves may impose risks to the patient The kind of hazards that are introduced by using nanocarriers for drug delivery are beyond that posed by conventional hazards imposed by chemicals in classical delivery matrices For nanocarriers the knowledge on particle toxicity as obtained in inhalation toxicity shows the way how to investigate the potential hazards of nanocarriers The toxicology of particulate matter differs from toxicology of substances as the composing chemical(s) may or may not be soluble in biological matrices, thus influencing greatly the potential exposure of various internal organs

Appropriately engineered nano-sized delivery systems can achieve finer temporal control over drug release rates due to their large surface area Nanocarriers can also be inherently useful in systems that require a burst release Nanocarriers, unlike bulk drug delivery systems, can enter cells to deliver drugs and can be designed to respond to intracellular cues Further, since nanocarriers can circulate in the body after being injected they have the ability to target diseases at the site of disorder This feature of nanocarriers is especially useful in cancer therapy, where the size of the delivery system

is the key to target cancers through the enhanced permeability and retention effect (EPR)

6

Diseased cells can also be targeted by attaching ligands or antibodies to the surface of nano-drug delivery systems Targeting allows nanocarriers to hone into diseased cells by targeting specific features of a disease phenotype, such as an over expressed protein or enzyme Another important aspect of delivery that is now being given the importance it deserves is the drug encapsulation stability in these carriers This is especially relevant because it is increasingly realized that the thermodynamic parameters like percent (%) loading do not adequately describe how stable the delivery vehicle would be during circulation in blood, since these vehicles could potentially leak out drugs into hydrophobic sites in surrounding tissue and blood components Delivery vehicles, based

on a single platform, which can satisfy all basic requirements of a versatile nanoscopic delivery vehicle, are quite rare These features however are the foundations of a good delivery vehicle and are fundamental design requirements Thus there are key aspects of a delivery vehicle design that was described as the basic anatomy of a drug delivery vehicle

3 Self-assembled nanocarrier for drug delivery

Nanoparticles are now available that are attractive for a wide range of materials and devices, but novel fabrication methods are also required to take full advantage of the interesting properties of nanoparticulates Approaches based on the self-assembly of systems from individual components offer tremendous cost advantages and an almost a magical "ease of manufacture" compared to lithographic methods Self-assembled nanoparticles also give the great opportunity in terms of diversity and functionality in the design for defined drug delivery purposes Self-assembled nanoparticles have many

7

advantages as highly efficient drug delivery vehicles including nanoscale size, controlled composition and capacity to encapsulate a wide range of drug molecules In particular, by using advanced chemistry and precision engineering at a molecular level, these synthetic polymers provide a wide opportunity for functionalization and versatility which impact the physico-chemical properties of self-assembled systems Examples of self-assembled nanocarriers for targeted drug delivery are showed in Figure 1.2

8

Figure 1.2 Example of self-assembled nanocarriers for targeted drug delivery: a Micelles, an aggregate of surfactant molecules dispersed in a liquid colloid where drugs are physically encapsulated in the inner core b Liposomes, a spherically arranged bilayer structure with drug loaded either in the inner aqueous phase or between the lipid bilayers c Oil/water emulsion, a mixture of liquids that are normally immiscible with drug loaded in the inner oil phase d Nanocapsules, a polymeric membrane which encapsulates an inner liquid core e Nanogels, a nanoparticle composed of a hydrogel f Core-shell particles, the location of nanocrystals at the core with the polymers on the outer layer

9

4 PEGylated nanocarriers for systemic delivery

Clearly, particles with longer circulation times have superior ability to reach the tumor site through passive targeting As opsonization is an integral step in the removal of foreign macromolecules by the RES, many efforts for increasing serum stability and extending circulation time have focused on blocking absorption of opsonins onto the nanoparticle surface.5 For passive targeting to be successful, the nanocarriers need to circulate in the blood for extended times so that there will be multiple possibilities for the nanocarriers to pass by the target site Nanoparticulates usually have short circulation half-lives due to natural defense mechanisms of the body to eliminate them after opsonization by the mononuclear phagocytic system (MPS, also known as reticuloendothelial system Therefore, the particle needs to be extended circulation half-lives

Cellular entrapment in macrophages can be avoided by surface modification of the nanocarriers Among many materials used to make or modify pharmaceutical carriers (lipids, natural and synthetic polymers, emulsions, or dendrimers) special attention was paid to polyethylene glycol (PEG, also known as polyethylene oxide (PEO)), which was used both for chemical modification of various drugs (peptide and protein, first of all) to make them more stable and long-circulating and for the decoration of pharmaceutical carriers to improve their pharmacokinetic properties Figure 1.6 illustrates how opsonin proteins associate with foreign bodies and coat its surface As bacteria and viruses have the same negative surface charge as phagocytic cells, opsonins are critical to reducing the charge repulsion between the two systems Next, phagocytic cells engulf the material and

10

transport it to the liver or spleen for degradation and excretion (Figure 1.3 a3–a4) Additional phagocytic macrophages are permanently located in the liver Known as Kupffer cells, these cells serve as a major filter for many types of NPs and are a major interference with long t½ The PEG polymer on a NP surface increases t½ by reducing this opsonization process (Figure 1.3 b2), thus preventing recognition by monocytes and macrophages, allowing the NPs to remain in the blood pool Hydrophobic particles are also more vulnerable to the RES and hydrophilic PEG reduces these complications In addition to NP–RES interactions, poor t½ can also result from NP–NP interactions (i.e., aggregation) NPs aggregate primarily because the attraction between particles is stronger than the attraction for solvent For spherical NPs, the interaction potential is related to the electrostatic repulsive potential and the van der Waals attraction potential PEG decreases the surface energy of NPs and minimizes van der Waals attraction.6

Prior to NP applications, PEG was used as a nontoxic, water-soluble dispersant/stabilizer FDA approved PEG is a highly hydrophilic, flexible polymer which has an inherent long circulating property The array of already available versatile PEG chemistries make it an attractive polymer to be used in modifying pharmaceuticals or surfaces of pharmaceutical carriers to achieve the desired long-circulating property or add convenient functional groups to conjugate ligands for active targeting Early work with PEGylated NPs stemmed mostly from drug delivery.7 One of the first reports on PEGylation was described by Davis and Abuchowski,8 where they covalently attached methoxy-PEGs (mPEGs) of 1900 and 5000 Da to bovine serum albumin and to liver catalase Later, acrylic microspheres functionalized with PEG-modified human serum albumin increased t½ in vivo.9 Li and colleagues found that 75-nm latex particles

11

remained in rat circulation 40-times longer (half-life 20 min vs 13 h) when coated than uncoated with PEG larger than 5000 kDa.7b In the mid-1990s, Doxil® (liposomal delivery vehicle for doxorubicin) and oncospar (PEG-l-asparaginase) became the first FDA-approved NP therapeutics.7c Doxil increases doxorubicin bioavailability nearly 90-fold at 1 week from injection of PEGylated liposomes versus free drug.10 Later, Abraxane® was introduced as an albumin-functionalized NP for delivery of taxane without cremphor to enhance drug efficiency.11

13

5 Targeted drug delivery systems for cancer therapy

Conventional cancer chemotherapies have dose-related side effects owing to nonspecific biodistribution of drugs Targeted nanomedicines are emerging as one of the promising approaches in anticancer treatment and have major advantages Targeting active molecules to specific sites in the body had been pursued actively ever since Ehrlich first envisaged the use of 'magic bullets' for the therapy of various diseases.13 Interest in this concept has increased significantly in recent decades with the innovations of nanomedicine Cancer nanomedicines have the ability to improve the therapeutic index of drugs by preferential localization at target sites, lower distribution in healthy tissues, delivery of hydrophobic drugs and extended release rate Progress in the development of nanomedicines for targeted drug delivery has been reviewed by Moghimi and colleagues.14 Targeted delivery can be achieved passive, active targeting, or their combinative targeting

14

Figure 1.4 Conceptual representation of nanoparticle tumor-targeting modalities Passive targeting: Unlike that found in normal tissue, tumor vasculature is leaky owing to fenestrations and gaps between endothelial cells that result from abnormal angiogenesis NPs in circulation can passively extravasate through these gaps and enter the tumor interstitium Poor lymphatic drainage found in some tissues helps to retain particles in the tumor space Active targeting: Ligands (e.g antibodies, peptides, small molecules, etc.) targeted toward moieties overexpressed or uniquely present on the plasma membrane of tumor cells can be used to actively enhance NP accumulation at the tumor site and can also help to internalize particles into cells via endocytosis.15

15

5.1 Passive targeting strategies and recent developments

Cellular barriers present formidable obstacles in the delivery of therapeutics for cancer treatment Fortunately, certain aspects of cancer physiology can be exploited to achieve passive targeting to tumor sites Rapid growth of tumors leads to aberrant angiogenic vasculature The newly formed blood vessels are often disorganized and discontinuous, resulting in increased permeability to macromolecules Moreover, lymphatic drainage systems are often poorly developed or non-existent in tumor sites, enabling accumulation of therapeutics.16 This phenomenon, called the enhanced permeation and retention (EPR) effect has increased the tumor concentration of anticancer agents up to 70-fold in some cases.17 Since the pioneering work of Couvreur et al.,18 nanoscale systems have been aggressively investigated for their utility in drug delivery applications (Figure 1.4)

Nanoparticle size is known to play a critical role in achieving passive targeting The majority of solid tumors exhibit a vascular pore cutoff size between 380 and 780 nm.19Therefore, particles need to be of a size much smaller than the cutoff pore diameter to reach to the target tumor sites By contrast, normal vasculature is impermeable to drug-associated carriers larger than 2 to 4 nm compared to free, unassociated drug molecules.20

This nanosize window offers the opportunity to increase drug accumulation and local concentration in target sites such as tumor or inflamed sites by extravasation, and significantly to reduce drug distribution and toxicity to normal tissues Nanocarriers above 10 nm in diameter are generally able to avoid filtration by the kidneys, while less well understood, the upper size limit for passively targeted nanocarriers is thought to be

16

approximately 150 nm.21 Extravasation and diffusional barriers limit nanoparticle access

to tumors when particle size is over 200 nm.21b Additionally, previous studies have shown that nanoparticle clearance rate increases with size.22 One such investigation demonstrated that the blood clearance of 80 nm nanocarriers was half as fast as the clearance of 170 and 240 nm particles Presumably, this effect is due to non-specific protein adsorption on the surface of larger nanocarriers, leading to opsonization and subsequent clearance by the RES.22

5.2 Active targeting strategies and stimuli-triggered ligand presentation

Localized diseases such as cancer or inflammation not only have leaky vasculature but also overexpress some epitopes or receptors that can be used as targets Therefore, nanomedicines can also be actively targeted to these sites Ligands that specifically bind

to surface epitopes or receptors, preferentially overexpressed at target sites, have been coupled to the surface of long circulating nanocarriers.23 Ligand-mediated active binding

to sites and cellular uptake are particularly valuable to therapeutics that are not taken up easily by cells and require facilitation by fusion, endocytosis, or other processes to access their cellular active sites.24 Active targeting can also enhance the distribution of nanomedicine within the tumor interstitium More recently, active targeting has been explored to deliver drugs into resistant cancer cells.25 An important consideration when selecting the type of targeting ligand is its immunogenicity For example, whole antibodies that expose their constant regions on the liposomal surface are more susceptible to Fc-receptor-mediated phagocytosis by the mononuclear phagocytic system.26 Examples of targeting ligands and their targets are listed in Table 1.1

17

Table 1.1 Selected examples of ligands used in active drug targeting

and therapy Non-peptidic RGD

mimetic

avβ3 integrin Integrin positive cell

imaging Mimetic of the sialyl

Lewisx

E-selectin Inflammatory

disease imaging Peptides RGD avβ3 integrin Breast cancer

imaging Chlorotoxin MMP-2 Brain tumor imaging

and therapy Synaptotagm in I, C2

domain

Phospholipids Apoptosis imaging

VHSPNKK VCAM-1 Cardiovascular

disease imaging EPPT1 (YCAREPPT

RTFAYWG)

Underglycosylated mucin-1 antigen

Multiple tumor type imaging

Aptamers A10 RNA aptamer Prostate-specific

membrane antigen

Prostate cancer imaging Thrm-A and Thrm-B

DNA aptamers

Human thrombin protein

alpha-Serum protein detection Proteins Annexin V Phosphatidylserine Apoptosis imaging

LHRH LHRH receptor Breast cancer

imaging Transferrin Transferrin receptor Breast cancer

imaging Antibodies Monoclonal antibody

A7

Colorectal carcinoma Colon cancer

imaging Herceptin

(Trastuzumab)

Her2/neu (Breast cancer)

Breast cancer imaging and therapy Rituxan (Rituximab) CD20 antigen Lymphoma imaging

therapy RGD, Arg-Gly-Asp tripeptide: LHRP, luteinizing hormone releasing hormone; Endothelial vascular adhesion molecule-1, VCAM-1

18

6 Stimuli-response for controlled drug delivery

6.1 Concepts for designing stimuli-responsive nanocarriers

Despite the fact that stability of encapsulation in a delivery carrier is necessary during circulation, drug delivery will only be effective if the drug is released once it reaches its

procedures, reduces the quantity of drug required to reach therapeutic levels, decreases the drug concentration at on-target sites (possibly reducing side effects) and, essentially, increases the concentration of the drug at target sites This can be reached by incorporating chemical moieties into the design that make the carrier responsive to stimuli relevant to the disease being targeted

Interest in stimuli-response is steadily gaining increasing momentum especially in the fields of controlled and self-regulated drug delivery Delivery systems based on stimuli-response are developed to closely resemble the normal physiological process of the diseased state ensuring optimum drug release according to the physiological need There are two kinds of stimuli, broadly defined, that can be engineered into delivery systems:

internal stimuli (i.e., enzymatic reactions, changes in pH, redox, and temperature) and external stimuli (i.e., heat, light, magnetic and electrical fields) When drug delivery systems maintain a response interaction they necessarily require a stimuli-response to

19

Figure 1.5 Dual and multi-stimuli responsive polymeric nanocarriers as emerging controlled drug release systems There are two kinds of stimuli, broadly defined, that can

be engineered into delivery systems: internal stimuli (i.e., enzymatic reactions, changes in

pH, redox, and temperature) and external stimuli (i.e., heat, light, magnetic and electrical fields)

20

6.1.1 Internal stimuli

Internal stimuli of chemical and biochemical origin include cellular pH-shift, redox, and ionic microenvironment of the specific tissues, enzyme over-expression in certain pathological states, host–guest recognitions, and antigen–antibody interactions (Figure 1.6).28

pH stimulus

In the pathological state, the normal pH-gradient existing between extra and intracellular environment is greatly affected A well-established fact is that in solid tumors, the extracellular pH can be significantly more acidic (~ 6–7) than systemic pH (7.4) due to poor vasculature and consequent anaerobic conditions prevailing in the malignant cells.29 Besides, the cellular organelles also exhibit sharp pH differences in different locations, for instance, in cytosolic, endosomal, and lysosomal compartments A polymeric nanocarrier with pH-sensitive modality can register such pH-gradients and, as

a response, can facilitate the release of the payload near the target compartment either by destabilization of the nanocarrier itself or by decomposition of the pH-sensitive linking unit that connects the drug to the carrier A number of nanocarrier-mediated gene transfer approaches have already been extensively studied where the destabilization of the internalized nanocarriers are brought about via a “cross-talk” of nanocarrier surface-charge and environmental pH condition, resulting in the release of the genetic materials

to the cells.30

Redox stimulus