In vitro and in vivo investigation of nanoparticles of a novel copolymer for substained and controlled delivery of docetaxel

15 Figure 3: Packaging of docetaxel in commercial formulation Taxotere®.. 17 Figure 5: Molecular structures of parent drug docetaxel and its major metabolites.. 79 Figure 26: FESEM image

IN VITRO AND IN VIVO INVESTIGATION OF NANOPARTICLES OF

A NOVEL BIODEGRADABLE COPOLYMER FOR SUSTAINED AND

CONTROLLED DELIVERY OF DOCETAXEL

GAN CHEE WEE

NATIONAL UNIVERSITY OF SINGAPORE

2010

IN VITRO AND IN VIVO INVESTIGATION OF NANOPARTICLES OF

A NOVEL BIODEGRADABLE COPOLYMER FOR SUSTAINED AND

CONTROLLED DELIVERY OF DOCETAXEL

GAN CHEE WEE

(B.Eng (Hons.), NUS)

A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF ENGINEERING DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING

NATIONAL UNIVERSITY OF SINGAPORE

2010

All the professional officers and lab technologists, Mr Chia Phai Ann, Dr Yuan Ze Liang, Mr Boey Kok Hong, Ms Lee Chai Keng, Ms Chew Su Mei, Ms Samantha Fam, Ms Alyssa Tay, Ms Dinah Tan, Ms Li Xiang, Mdm Priya, Mdm Li Fengmei, and many other staffs from Laboratory Animal Centre (LAC) who have unconditionally helped in various kinds of administrative works as well as experiments and have willingly shared their knowledge and expertise to further enhance my learning process

My dear colleagues, Dr Mei Lin, Dr Sneha Kulkarni, Ms Sun Bingfeng, Mr Prashant, Mr Liu Yutao, Ms Anitha, Ms Anbharasi, Mr Phyo Wai Min, Ms Chaw

Su Yin, Mr Tan Yang Fei and all the final year students for all their kind assistances and supports they provided

Finally, I am very grateful and appreciative of the scholarship provided by NUS

TABLE OF CONTENTS

ACKNOWLEDGEMENTS i

TABLE OF CONTENTS ii

SUMMARY vii

NOMENCLATURE ix

LIST OF TABLES xiii

LIST OF FIGURES xiv

CHAPTER 1: INTRODUCTION 1

1.1 Background 1

1.2 Objectives and Thesis Organization 4

CHAPTER 2: LITERATURE REVIEW 6

2.1 Definition and Facts 6

2.2 Causes of Cancer 6

2.3 Cancer Treatments and Limitations 7

2.3.1 Problems in Chemotherapy 9

2.3.2 Anticancer Drugs 13

2.3.2.1 Taxanes 14

2.3.2.2 Pharmacodynamics 17

2.3.2.3 Pharmacokinetics 18

2.3.2.4 Toxicology 19

2.4 Alternatives of Drug Formulations 21

2.4.1 Liposomes 21

2.4.2 Micelles 24

2.4.3 Dendrimers 27

2.4.4 Prodrugs 29

2.4.5 Nanosphere 32

2.5 Fabrication Methods of Nanosphere 35

2.5.1 Emulsion/Solvent Evaporation 36

2.5.2 Solvent Displacement 38

2.5.3 Salting Out 41

2.5.4 Supercritical Fluid (SCF) Technology 44

2.6 Roles of Surfactants 47

2.6.1 Drug Carriers 47

2.6.2 Stabilization of Emulsion (Emulsifiers) 48

2.6.3 Targeted Cancer Therapy 50

2.7 Vitamin E TPGS 51

2.7.1 Properties of Vitamin E TPGS 51

2.7.2 TPGS as Solubilizer 53

2.7.3 TPGS as Permeability and Bioavailability Enhancer 55

2.7.4 TPGS for Sustained and Controlled Delivery Applications 57

CHAPTER 3: SYNTHESIS AND CHARACTERIZATION OF PLA-TPGS COPOLYMER 61

3.1 Introduction 61

3.2 Materials 62

3.3 Methods 62

3.3.1 Synthesis of PLA-TPGS Copolymer 62

3.3.2 Characterization of PLA-TPGS Copolymer 63

3.3.2.1 1H Nuclear Magnetic Resonance (NMR) Spectroscopy 63

3.3.2.2 Gel Permeation Chromatography (GPC) 64

3.3.2.3 Thermogravimetric Analysis (TGA) 64

3.3.2.4 Fourier Transform Infrared Spectroscopy (FR-IR) 64

3.4 Results and Discussion 65

3.4.1 1H NMR Spectroscopy 65

3.4.2 GPC 67

3.4.3 TGA 68

3.4.4 FT-IR Spectroscopy 68

3.5 Conclusion 70

CHAPTER 4: FABRICATION AND CHARACTERIZATION OF PLA-TPGS NANOPARTICLES 71

4.1 Introduction 71

4.2 Materials 72

4.3 Methods 72

4.3.1 Preparation of PLA-TPGS Nanoparticles 72

4.3.2 Characterization of Drug-loaded PLA-TPGS Nanoparticles 73

4.3.2.1 Particle Size Analysis 73

4.3.2.2 Surface Morphology 73

4.3.2.3 Surface Charge 73

4.3.2.4 Surface Chemistry of Drug-loaded PLA-TPGS NPs 74

4.3.2.5 Thermal Analysis of Drug-loaded and unloaded

PLA-TPGS NPs 74

4.3.2.6 Drug Encapsulation efficiency 74

4.3.2.7 In Vitro Drug Release 75

4.4 Results and Discussion 75

4.4.1 Particle Size and Size Distribution 75

4.4.2 Surface Morphology 78

4.4.3 Surface Charge 80

4.4.4 Surface Chemistry 80

4.4.5 Drug Encapsulation 83

4.4.6 In Vitro Drug Release 85

4.5 Conclusion 86

CHAPTER 5: IN VITRO CELLULAR STUDY OF PLA-TPGS NANOPARTICLES 5.1 Introduction 88

5.2 Materials 88

5.3 Methods 89

5.3.1 Cell Culture 89

5.3.2 Cellular Uptake of Nanoparticles 89

5.3.3 In Vitro Cell Cytotoxicity 90

5.4 Results and Discussion 91

5.4.1 Cellular Uptake 91

5.4.2 Cell Viability 95 5.5 Conclusion

CHAPTER 6: IN VIVO PHARMACOKINETICS AND EX VIVO BIODISTRIBUTION

6.3.2.2 Tissue Collection, Sample Processing and Analysis 103

SUMMARY

Biodegradable polymeric nanoparticle formulation has become an attractive regimen which provides a platform for developing sustainable, controlled and targeted drug delivery system to improve the therapeutic efficacy and reduce the clinical side effects of most antineoplastic drugs In recent years, amphiphilic biodegradable copolymers consisting of hydrophobic and hydrophilic segments have drawn significant attention from researchers due to the enhancement of drug encapsulation capability as a result of a more stable oil-water suspension during nanoparticle fabrication process Meanwhile, it has been reported that copolymers could better induce long-circulating ‘stealth’ effect by conjugating to poly(ethylene glycol) (PEG) which could avoid the binding of opsonins, reduce the recognition and elimination by the reticuloendothelial system (RES) Together with small particle size and enhanced permeability and retention (EPR) effect of leaky vasculature, the efficiency of drug delivery to tumor site is improved D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), an alternative to PEG, is an amphiphilic macromolecule, water-soluble derivative of natural vitamin E It is an effective emulsifier

in nanotechnology for biomedical applications Co-administration of TPGS can enhance

the solubility, cellular internalization, inhibit P-glycoprotein mediated multi-drug efflux

transport system, and increase the oral bioavailability of various anticancer drugs By conjugating TPGS as part of copolymer, its nanoparticle formulation of therapeutic agents can potentially improve the solubility, stability and permeability of drugs Furthermore, polysorbate 80-associated hypersensitivity reaction and other drug-related toxicities such

as cumulative fluid retention, peripheral neuropathy and leucopenia can be reduced

In this study, we synthesize a novel amphiphilic PLA-TPGS copolymer with the PLA to TPGS weight ratio of 89:11 The copolymerization was characterized by 1H NMR, GPC,

TGA and FT-IR Following that, the nanoparticle formulations of PLA-TPGS are prepared by a modified single solvent emulsification/evaporation technique with either PVA or TPGS as emulsifier Characterizations of nanoparticles such as particle size and size distribution, drug encapsulation efficiency (EE), surface morphology, surface charge and drug release profile are done Generally, particle size of TPGS-emulsified NPs was smaller (~ 240 nm), but with higher EE (up to 85%) and stability, than that of PVA-emulsified NPs (~ 270nm with EE ~63%) Drug release profiles of these NPs showed biphasic release with about 5 – 14% initial burst in the first 6 h followed by sustained release of drug up to 79% after 30 days Human breast adenocarcinoma MCF-7 cell line is employed to assess cellular uptake efficiency of the NPs TPGS-emulsified NP formulation achieved higher cellular uptake compared to PVA-emulsified NPs

Cytotoxicity evaluation of the NP formulations in vitro showed an order of IC50:

Taxotere® > PVA-emulsified NP > TPGS-emulsified NP, suggesting a more effective

formulation of TPGS-emulsified NPs In vivo pharmacokinetics and biodistribution

investigation demonstrated longer NPs circulation and therapeutic effect in blood plasma than commercial Taxotere® Tissue sample analysis from rats injected with NP

formulation showed significant decrease of drug accumulation in some important organs, but excluding lungs which is about 2-fold higher than Taxotere® Nevertheless, NP

formulation demonstrated a much better release kinetic with lesser side effects than Taxotere®, thus revolutionizing the way in which cancer is treated while making

controlled and sustained cancer chemotherapy feasible

CLSM confocal laser scanning microscopy

CMC critical micelle concentration

CNS central nervous system

CTAB cetyltrimethylammonium bromide

EPR enhanced permeability and retention

FBS fetal bovine serum

FESEM field emission scanning electron microscopy

FT-IR fourier transform infrared spectroscopy

GAS gas anti-solvent

GI gastro-intestinal

GPC gel permeation chromatography

HCPE hyperbranchedconjugated polyelectrolyte

HIV human immunodeficiency virus

HLB hydrophile-lipophile balance

1

HPLC high performance liquid chromatography

H NMR proton nuclear magnetic resonance

HPMA N-(2-hydroxypropyl)methacrylamide

HVC hydrophobic vacuum cleaner

IC50

LDL low-density lipoprotein

inhibitory concentration at which 50% cell population is suppressed

LLS laser light scattering

mMRI molecular magnetic resonance imaging

xi

MRT mean residence time

MTD maximum tolerated dose

PVA polyvinyl alcohol

PNP PVA-emulsified PLA-TPGS nanoparticles

xii

RES reticuloendothelial system

RESS rapid expansion from supercritical solution

SAR structure-activity relationship

max time to achieve the maximum concentration (Cmax

TGA thermogravimetry analysis

)

THF tetrahydrofuran

TNP TPGS-emulsified PLA-TPGS nanoparticles

TPGS d-α-tocopheryl polyethylene glycol 1000 succinate

Tween 80 polyoxyethylene-20-sorbitan monooleate (or polysorbate 80)

XPS x-ray photoelectron spectroscopy

xiv

LIST OF FIGURES

Figure 2: Chemical structures of paclitaxel and docetaxel 15

Figure 3: Packaging of docetaxel in commercial formulation Taxotere® 16

Figure 4: Molecular structure of polysorbate 80 (or Tween 80) 17

Figure 5: Molecular structures of parent drug docetaxel and its major metabolites 20

Figure 6: Molecular structure of basic unit (phospholipid) of liposome 22

Figure 7: Arrangement of lipid bilayer in liposome 22

Figure 8: Two possible structures of spherical micelles 25

Figure 9: General structure of polyamidoamine (PAMAM) dendritic molecule 27

Figure 10: Metabolic process of levodopa to dopamine 30

Figure 11: Various types of nanocarriers and their respective characteristics 33

Figure 12: Lipid-polymer hybrid system with a hydrophobic core, lipid interlayer and

Figure 13: Solvent displacement as nanosphere fabrication technique ** represent

Figure 15: Roles of PVA in stabilization of nanoemulsion during salting out process

xv

Figure 16: Simplified scheme of gas anti-solvent precipitation by SCF technology 46

Figure 17: Molecular structure of d-α-tocopherol (Vitamin E) 52

Figure 18: Molecular structure and various segments of TPGS 52

Figure 19: Ring-opening polymerization reaction in the synthesis PLA-TPGS 63

Figure 20: 1H-NMR spectra of the TPGS, lactide monomer and PLA-TPGS 66

Figure 21: Gel permeation chromatogram of TPGS monomer and PLA-TPGS

Figure 22: TGA thermogram of TPGS monomer and PLA-TPGS copolymer 68

Figure 23: FT-IR spectra of TPGS, lactide monomer and PLA-TPGS copolymer 69

Figure 24: FESEM images of docetaxel-loaded TPGS-emulsified PLA-TPGS NPs 78

Figure 25: FESEM images of docetaxel-loaded PVA-emulsified PLA-TPGS NPs 79

Figure 26: FESEM images of coumarin 6-loaded TPGS-emulsified (left) and

Figure 27: XPS C1s envelope of PLA-TPGS copolymer and unloaded PLA-TPGS NPs

Figure 28: XPS wide scan spectra of docetaxel-loaded TPGS-emulsified (TNP) and

Figure 29: DSC curves of pure docetaxel, docetaxel recovered from emulsification, docetaxel-loaded PLA-TPGS NPs, unloaded PLA-TPGS NPs and a mixture of

Figure 30: In vitro drug release profiles of docetaxel-loaded PLA-TPGS NPs using

TPGS and PVA as emulsifier Data represent mean ± SD (n=3) 86

xvi

Figure 31: MCF-7 cell uptake efficiency of TPGS-emulsified (TNP) and emulsified (PNP) coumarin 6-loaded PLA-TPGS NPs at 100, 250 and 500 µg/ml incubated at 37°C Data represent mean ± SD (n=6) 92 Figure 32: Confocal laser scanning microscopy (CLSM) of MCF-7 cells after 2 h incubation with 250 µg/ml coumarin-6-loaded TPGS-emulsified NPs (Row A), PVA-emulsified NPs (Row B) and free coumarin-6 (Row C) at 37.0 °C The cells were stained by propidium iodide (Red channel, column 2) and the coumarin-6-loaded PLA-TPGS NPs are green in color (Green channel, column 1) 94 Figure 33: Viability of MCF-7 breast cancer cells incubated with docetaxel-loaded TPGS- or PVA-emulsified PLA-TPGS NPs in comparison with that of Taxotere® at different docetaxel concentrations after 24, 48 and 72 h Data represent mean ± SD

Figure 34: In vivo pharmacokinetics profiles of plasma drug concentration versus time

after i.v administration of Taxotere® and TPGS-emulsified PLA-TPGS nanoparticles formulation using SpD rats (n=5) at the same docetaxel dose of 10 mg/kg 105 Figure 35: Biodistribution of docetaxel delivered by commercial Taxotere® and PLA-TPGS NPs to SpD rats at 1, 5, 10 and 24 h after i.v administration at the same

CHAPTER 1: INTRODUCTION

There has been a sustained interest during recent years in developing localized and sustained treatment for cancer and other fatal diseases such as cardiovascular restenosis Biodegradable polymeric carriers have become a promising platform for sustained, controlled and targeted drug delivery to improve the therapeutic effects and reduce the side effects of the otherwise unprotected drug (Kataoka et al., 2001; Farokhzad and Langer, 2006; van Vlerken et al., 2007) The challenge lies in the polymeric materials selection and the engineering of the nanoparticulate systems that are specifically taken up

by targeted cancer cells and subsequently release their drug payload at a plasma concentration within the therapeutic window of the drug for a prolonged period in order to achieve anti-tumor response (Gref et al., 1994; Langer, 2001; Ferrari, 2005) Efficient chemotherapy requires that the anticancer drug concentration in the blood be maintained between the minimum effective level and the maximum tolerable level for a sufficiently long period It has been reported in the literature that ‘stealth’ nanoparticles with surface modification by poly(ethylene glycol) (PEG) could avoid being recognized and eliminated

by the reticuloendothelial system (RES) and thus remain longer in the blood circulation system (Gref et al., 1994; Bazile et al., 1995; Feng et al., 2007; Terada et al., 2007) Nanoparticles of biodegradable polymers are made up of natural or synthetic macromolecules, which are compatible with human body (biocompatibility) and degradable in physiological condition into harmless byproducts While delivering the therapeutic agent to the diseased cells, the polymeric matrix degrades and is eventually

metabolized and eliminated from the body The degradation rate depends on the physicochemical properties of the polymers, which are determined and adjustable by their compositions, molecular structures as well as molecular weights Hence, nanoparticle formulation of therapeutic agents can improve their solubility, permeability, stability and therapeutic effects with reduced side effects (Torchilin, 2006)

A wide range of U.S FDA-approved biodegradable polymers such as poly(lactide) (PLA), poly(d,l-lactide-co-glycolide) (PLGA) and poly(caprolactone) (PCL) polyesters are initially designed for application in textile grafts, surgical stents or implants Although they are biocompatible, their strong mechanical strength, extremely slow degradation rate and difficulty in further modification due to hydrophobic nature have limited their use as drug delivery devices for cancer therapy Moreover, nanoparticles made up of those polymers are limited to be directly conjugated to hydrophilic molecular probes for targeting, in which amphiphilic linker molecules are usually needed, causing complications for the targeting procedures (Debotton et al., 2008) Generally, two strategies have been developed to solve this problem One is to coat the nanoparticles by amphiphilic polymers and another is to synthesize copolymers to incorporate hydrophilic elements into the hydrophobic chains so that the system will be more stable thermodynamically in oil-water suspension during nanoparticle fabrication process (Kataoka et al., 2001; Feng et al., 2006) D-α-tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS or simply, TPGS) is one of the potential candidates TPGS is

an amphiphilic macromolecule with hydrophile-lipophile balance (HLB) 13 The chemical structure of TPGS is similar to that of other amphiphiles, comprising lipophilic alkyl tail and hydrophilic polar head portion Its bulky structure and large surface area make it an

effective emulsifier in the nanoparticle technology for biomedical applications, which can result in high drug encapsulation efficiency and high cellular internalization (Mu and Feng, 2003; Win and Feng, 2006) Moreover, it has been found that co-administration of vitamin E TPGS could enhance the therapeutic effects, inhibit P-glycoprotein mediated multi-drug resistance, and increase the oral bioavailability of anticancer drugs (Amass et al., 1998; Soppimath et al., 2001)

Docetaxel is a poorly water-soluble semi-synthetic taxane analogue commonly used in the treatment of breast cancer, ovarian cancer, small and non-small cell lung cancer, prostate cancer, etc Pre-clinical studies demonstrated that docetaxel had several advantages over paclitaxel (Jones, 2006) Compared with paclitaxel, docetaxel showed wider cell-cycle bioactivity, greater affinity for the β-tubulin binding site and greater uptake with slower efflux from the tumor cells, resulting in longer intracellular retention time and higher intracellular concentrations (Riou et al., 1992; Riou et al., 1994; Brunsvig et al., 2007).It was reported that docetaxel exhibited 12-fold cytotoxic activity than paclitaxel and docetaxel showed higher growth inhibition in human epidermal growth receptor (HER2) positive cells compared to paclitaxel (Riou et al., 1992; Hanauske et al., 1994; Lavelle et al., 1995).In clinical trials, docetaxel demonstrated superior efficacy versus paclitaxel in a randomized Phase III study, which directly compares docetaxel and paclitaxel at approved dose and schedule (Jones et al., 2005).Its commercial formulation Taxotere® is formulated

in high concentration of Polysobate 80 (Tween 80), such as 40mg/ml which has been found to be associated with severe side effects including hypersensitivity reactions, cumulative fluid retention, nausea, mouth sores, hair loss, peripheral neuropathy, fatigue and anemia and has shown incompatibility with the common PVC intravenous

administration sets (Gelderblom et al., 2001; Immordino et al., 2003; Baker et al., 2004).Therefore, to avoid the application of Tween 80-based adjuvant and to increase the drug solubility, alternative formulations have been attempted, which include liposomes (Immordino et al., 2003), nanoparticles (Musumeci et al., 2006; Cheng et al., 2007),docetaxel-fibrinogen-coated olive oil droplets (Engels et al., 2007),nanoparticle-aptamer bioconjugates (Farokhzad et al., 2006) Among them, the nanoparticle formulation showed advantages such as greater stability than others during storage Furthermore, such a colloidal system is able to extravasate solid tumors into the inflamed or infected site, where the capillary endothelium is defective (Barratt, 2000; Brigger et al., 2002).Nanoparticles could also reduce the multi-drug resistance faced by many anticancer drugs, including docetaxel, by internalization mechanism of drug-loaded nanoparticles such as endocytic process (Panyam and Labhasetwar, 2003; Bareford and Swaan, 2007) Meanwhile, they also reduce drug efflux from cells mediated by the P-glycoprotein (Brigger et al., 2002) This motivates us to combine the advantages from TPGS by synthesizing PLA-TPGS copolymers for various potential biomedical applications, including formulation of imaging agents for cellular and molecular imaging and targeted drug therapy (Zhang et al., 2007; Pan and Feng, 2009)

1.2 Objectives and Thesis Organization

In this thesis, we focus on the formulation of PLA-TPGS nanoparticles encapsulating anticancer drug docetaxel for prolonged chemotherapy treatment At the same time, the effect of different emulsifiers such as TPGS and PVA on characteristics of PLA-TPGS nanoparticles is studied Other than that, a series of cell works involving cancer cell lines

as well as animal models are included to evaluate the formulation before it is tested in clinical trials

The first chapter of this thesis is to provide a general background and concepts of developing nanoscale device for cancer chemotherapy Next, Chapter 2 provides a detailed review on the current progress in related fields of drug delivery Some examples and results from journals are cited for the benefit of the readers The rationale behind the strategies of is also clearly explained in this chapter Then, Chapter 3 presents the synthesis and characterization of PLA-TPGS amphiphilic copolymer of the optimized 89:11 PLA:TPGS component ratio Following that, Chapter 4 includes the nanoparticle preparation and characterization The docetaxel-loaded PLA-TPGS NPs are prepared by a modified single emulsion solvent evaporation/extraction technique with either PVA or TPGS as emulsifier, which are then characterized in such aspects as particle size and size distribution, drug encapsulation efficiency, surface morphology, surface charge and drug

release profile In vitro cellular study is reported in Chapter 5 Human breast

adenocarcinoma MCF-7 and human colon cancer HT-29 cell lines are employed to assess cellular uptake of the NPs as well as to evaluate the cell viability of the NP formulations, which is done in close comparison with Taxotere® In Chapter 6, in vivo pharmacokinetics

and biodistribution using Sprague-Dawley (SpD) rats is investigated to further confirm the advantages of the PLA-TPGS NP formulation versus the pristine drug Finally, conclusion and suggestions for future work are provided in Chapter 7, following by Chapter 8 which contains all the reference papers cited in this thesis

CHAPTER 2: LITERATURE REVIEW

2.1 Definition and Facts

Cancer is the leading cause of death globally According to US National Cancer Institute, cancer is defined as diseases in which abnormal cells undergo uncontrolled growth (or mitosis) and have to ability to invade other tissues of the body through the blood circulation and lymphatic systems (http://www.cancer.gov/cancertopics/what-is-cancer) One among three people will be diagnosed with cancer during their lifetime, and new cases of cancer are increasing at a rate of 1% per year (http://news.bbc.co.uk/2/hi/health/3444635.stm) Currently, more than 200 types of cancer have been discovered, with probability of getting cancer being distinct in different types of tissues or organs, even within the same individuals

119#genetic) However, extrinsic factors play an even more essential role in determining the development of cancer Extrinsic factors consist of a wide variety of causes, ranging from environmental factors to the personal daily lifestyle practiced by the individuals Diet that we consume everyday directly influences the risk of getting cancer Preservatives such as nitrosamine, nitrosamide and sulphites as well as colorings which are usually added during food processing can potentially accumulate in the body and cause cancer (http://www.cfsan.fda.gov/~dms/fdpreser.html;http://www.nswcc.org.au/editorial.asp?pageid=2345) Concerns are equally given to genetically-modified (GM) food as well as food rich in methyl donors as some research reports show that too much such food may potentially trigger genetic mutations, causing tumor growth (Watters, 2006; http://www.independent.co.uk/life-style/health-and-wellbeing/health-news/suppressed-report-shows-cancer-link-to-gm-potatoes-436673.html) On the other hand, about 70% of cancer deaths took place in low to middle income nations, where there is lack of knowledge and resource on how to prevent and diagnose cancer, as pointed out by World Health Organization (http://www.who.int/cancer/modules/en/) In addition to that, some habits such as smoking, drinking, unhealthy work-life balance are major factors causing cancers For instance, more than 38,000 people are diagnosed with lung cancer every year, with almost 90% of deaths from lung cancer are due to tobacco (http://info.cancerresearchuk.org/cancerstats/types/lung/?a=5441)

2.3 Cancer Treatments and Limitations

Some of the common treatments available to cancer are surgery, chemotherapy, radiation therapy, immunotherapy, monoclonal antibody therapy and gene therapy Each method

has its advantages and disadvantages, and depends on the physiology of the individuals as

an effective treatment strategy in one person may fail in another

Surgical removal of tumors from cancer patients is usually the first consideration in cancer treatment This is especially the case when the tumor size is large and starts to damage the functionality of the tissues or organs surrounding it Unfortunately, surgery has a few drawbacks Firstly, surgery is an invasive method of cancer treatment with potential wound infection And, it can only be done when the tumor is sufficiently large to be removed Secondly, for patients with medical history such as haemophilia, it may not be advisable to undergo such procedure Thirdly, surgery can sometimes trigger the metastasis of tumor, even it is successfully removed (Weiss and DeVita, 1979)

Radiotherapy is also another primary treatment modality in which ionizing radiation is used to destroy cancerous tissues However, this method is only applicable to localized tumor such as prostate cancer and recurrence of cancer also occurs in some patients (De Riese et al., 2002) Therefore, a combination of surgery and radiotherapy will usually have immediate local response in terms of tumor cell death But, it is not effective in controlling re-growth and metastatic secondary tumor growth (Camphausen et al., 2001; Chen et al., 2006)

Meanwhile, hormone therapy is restricted to organ-confined cancers such as breast and prostate cancer and long term treatment of metastatic tumor using this method is unlikely (Corral et al., 1996; De Riese et al., 2002) Immunotherapy, by stimulating the immune

system through general or specific immune enhancement, only renders a low success rate

to patients (Chen et al., 2006)

Chemotherapy, often used in combination with other treatment modalities, is the treatment

of diseases or cancers using chemical agents or antineoplastic drugs These chemical agents, which are usually very toxic, can inhibit the tumor growth But they can also kill the normal, healthy cells, and thus bring unwanted side effects Nowadays, various kinds

of anticancer drugs are available in the market Some examples include paclitaxel, chlorambucil, fluorouracil, methotrexate and doxorubicin The cytotoxic mechanisms of chemotherapeutic agents differ from each other, depending on the nature of the drugs, the molecular structure, physicochemical properties and the sites of actions in the body

2.3.1 Problems in Chemotherapy

The common problem with most antineoplastic drugs is their poor solubility in aqueous phase Paclitaxel, for example, is highly hydrophobic with a solubility of less than 0.5 mg/L in water (Feng and Chien, 2003; Hennenfent and Govindan, 2006) This is not desirable because the drug has to be dissolved in blood, with water as the major component, in order to be transported to the cancer cells Therefore, solubilizers or adjuvants are necessary to increase the solubility of anticancer drugs It is also this reason why most of the current commercial drug formulations are only able to be administered intravenously (infusion) Routes of administration are thus limited In Taxol®, the

commercial formulation for paclitaxel, Cremophor EL is applied as the adjuvant (Hennenfent and Govindan, 2006; Xie et al., 2007) Cremophor EL, a nonionic surfactant,

consists of polyethoxylated castor oil and dehydrated ethanol (1:1 v/v) Although

Cremophor EL is a vehicle for various hydrophobic pharmaceutical agents including cyclosporine and diazepam, it has been found to cause serious adverse effects to patients Biologic effects such as hypersensitivity, nephrotoxicity and peripheral neuropathies are believed to have associated with the use of Cremophor EL in the formulation (Theis et al., 1995; Gelderblom et al., 2001; Hennenfent and Govindan, 2006; Feng et al., 2007) The molecular structure of Cremophor EL is shown below (Aliabadi et al., 2005):

Figure 1: Molecular structure of Cremophor EL

Secondly, human body will normally treat most anticancer drugs as foreign substances which the body cannot recognize As a result, the native drugs administered into the body will greatly be subjected to the degradation by some endogenous enzymes or macromolecules which are considered as part of the body natural defense mechanism and immune system The first-pass metabolism is an important process that takes place in liver and intestine before the drugs are absorbed into the circulatory system (Feng et al., 2007)

It is the physiological barrier to be crossed before the drugs can be distributed to other parts of the body The most common kind of enzyme involved in this degradation of drugs is cytochrome P450 (or CYP), mainly located in liver and intestine CYP is a large family of hemoproteins which consists of 18 families and 43 subfamilies and it contributes

to nearly 75% of total metabolic process in human body (Nelson et al., 1993; Danielson,

2002; Guengerich, 2008) It is found on the membrane of endoplasmic reticulum as well

as mitochondria However, most members from CYP1, CYP2 and CYP3 families take part in drug metabolism (Guengerich, 2008) For instance, almost 80% of administered docetaxel, an anticancer drug popular for its efficacy towards various types of cancer, is metabolized by CYP3A4 through hepatic transformation (Baker et al., 2006; Bradshaw-Pierce et al., 2007)

Besides that, there are other systems which act as barriers to hamper the effective absorption of drug in the body Protein such as P-glycoprotein (or P-gp) is a ATP-binding

cassette (ABC) transporter encoded by MDR1 gene and is well known for its drug efflux

mechanism (Ling, 1997; Béduneau et al., 2007) Because it has the capability of removing various toxic substances from cells over-expressing P-gp in such organs as liver, kidney, and small intestine, cellular multi-drug resistance (MDR) is developed (Thiebaut et al.,

1987) In addition to CYP, it is the presence of P-gp in the lower gastro-intestinal (GI) tract and other multidrug resistance proteins (MRP) , such as MRP 1-5 and breast cancer resistance protein (BCRP), that usually cause the low oral bioavailability of most antineoplastic drugs (Malingré et al., 2001; Schinkel and Jonker, 2003; Varma et al., 2003; Varma and Panchagnula, 2005) Moreover, it has been reported that the synergistic effect between P-gp and CYP3A4 could further speed up the first-pass elimination of drugs in intestinal enterocytes (Schuetz et al., 1996; Lown et al., 1997; van Asperen, 1997; Varma

et al., 2004) Therefore, oral chemotherapy at home is still not feasible until a very novel, stable and sustained drug formulation emerges Also, P-gp is greatly over-expressed in capillary endothelium of blood vessels lining the central nervous system (CNS), which together make up the blood-brain barrier (BBB), leading to the failure of chemotherapy to

brain cancer due to restricted permeability of drugs to tumor sites (Béduneau et al., 2007;

Pardridge, 2007)

Another reason causing the clearance of drugs once they are present in physiological system is the high probability of binding to endogenous proteins in the circulatory system The high-binding affinity of most commercial formulations to plasma proteins reduces the amount of free drug required for the treatment at the targeted sites (Rawat et al., 2006) In fact, this protein-binding process, especially for hydrophobic drugs, is spontaneous and is part of the opsonization process In this case, the exogenous drugs will be considered as a foreign material (antigen), which promotes the binding of opsonins (immunoglobulins, laminin and C-reactive proteins, for example) and will eventually be recognized and taken

up by phagocytes This mononuclear phagocyte system (MPS), which involves macrophages (located in tissues and organs such as liver, spleen, lung and lymph nodes) and monocytes (found in blood stream), is also classified as the reticulo-endothelial system (RES) of the immune mechanism (Müller et al., 1997; Hume, 2006; Owen and Peppas, 2006) As a result, sustainability of the drugs is affected

For a drug formulation to be effective, solubility, stability and permeability of drugs are the three basic criteria that must be fulfilled in order to achieve successful chemotherapy Unfortunately, sudden exposure of the body to certain level of drug dosage for certain time interval is usually an effective way in classical chemotherapy for cancer treatment However, we must also consider the severe side effects due to the abrupt increase in concentration of cytotoxic drugs in the blood plasma because most commercial drugs not only kill cancer cells, but also the healthy cells Low amount of cisplastin, a

chemotherapeutic agent that cross-links DNA to retard its replication in tumor, can cause serious systemic toxicity to patients if the dosage administered is not properly monitored (Sumer and Gao, 2008) Hence, the drugs must not only reach the desired site of action and remain accumulated at the site for sufficient period of time, the desired rate of drugs being exposed to the patients at certain time must be considered In fact, controlled release and specificity of drugs has become the major factors in designing novel formulations for cancer therapy using state-of-the-art bio- and nano-technology

2.3.2 Anticancer Drugs

There are various kinds of drugs commercially available in the market for cancer chemotherapy In generally, all these anticancer drugs are categorized into few groups, depending on the way or mechanism by which the drugs act on the cancer cells Some of them include alkylating-like agents, anti-metabolites, anthracyclines and alkaloids

Cisplatin, an alkylating-like agent with a structure of cis-Pt(NH3)2Cl2, is used to treat

cancers such as small cell lung cancer, colon cancer, ovarian cancer and sarcomas It contains platinum in the molecular structure The cytotoxic effect of cisplatin is mainly contributed from the platinum complexes which can bind and interact with the basic sites

of DNA, resulting in DNA crosslinking (Lippert, 1999) When the DNA is unable to replicate, apoptosis is induced leading to cell death However, low water solubility, low lipophilicity, serious toxicity and rapid inactivation restrict its clinical application (Chupin

et al., 2004) Chlorambucil, another alkylating-like agent which can be taken orally, is often used for treatment of chronic lymphocytic leukemia

Examples of anthracyclines are daunorubicin and doxorubicin which have been the effective chemotherapeutic agents for breast cancer, leukemic cells, myeloma cells and so

on It is naturally produced by Streptomyces strain of bacteria (Lomovskaya et al., 1999) This type of drug is believed to intercalate into DNA, thus preventing the growth of cancer cells due to the inhibition of enzymes helicase and topoisomerase II which are essential in DNA transcription and cell mitosis (Fornari et al., 1994) Another mechanism of action is the generation of oxygen free radicals that damage the cell membrane The main side effects occur especially to the heart include congestive heart failure and arrhythmias

Alkaloid is a general group of natural compounds which contain basic nitrogen atoms in the molecular structure Two sub-groups of alkaloids that have the antitumor capability are vinca alkaloids and taxanes The mechanism of action of these drugs is to interfere with the microtubule function in a cell cycle (Cutts, 1961; Kruczynski et al., 1998) While

vinca alkaloids such as vindesine and vinorelbine can inhibit the assembly of microtubule

by reducing the rate of tubulin addition, taxanes have the opposite effect, inhibiting the disassembly of microtubules during mitosis

2.3.2.1 Taxanes

In the past few decades, research has shown that taxanes could be promising chemotherapeutic agents because of effective single-agent activity such as high response and patient survival rates in a broad spectrum of advanced carcinoma (Bunn and Kelly, 1998) And taxanes are currently being widely used in oncology The most common

taxanes are paclitaxel and docetaxel They are diterpenes and their molecular structures are different only at a few side chains as shown in Figure 2

Figure 2: Chemical structures of paclitaxel and docetaxel

(Source: Mortier et al., 2005)

Taxanes are originally isolated from natural source of plants of genus Taxus For instance, paclitaxel is derived from the bark of Pacific yew tree (Taxus brevifolia) While it is not

feasible to synthesize paclitaxel from economic point of view, semi-synthetic analogue is one of the possible solutions to the limited availability of yew trees (Feng and Chien, 2003) Docetaxel is a new generation of and an alternative to paclitaxel It is a semi-synthetic form of taxane which is derived from a renewable non-cytotoxic compound, 10-

deacetyl baccatin III, extracted from the needles of European yew tree (Taxus baccata)

(Ringel and Horwitz, 1991; Denis et al., 1998)

In commercial formulation, paclitaxel developed by Bristol-Myers Squibb Company is packaged under the trade name Taxol®, usually in a product concentration of 6mg/ml with

Cremophor EL as the adjuvant (Figure 1)

Meanwhile, docetaxel in its commercial formulation Taxotere® is developed by the

pharmaceutical company Sanofi-Aventis The packaging of Taxotere® is shown in Figure

3 The concentration approved is 40mg docetaxel per mL of polyoxyethylene-20-sorbitan

monooleate (polysorbate 80 or Tween 80) (Figure 4) This high drug concentration is to be mixed with 13% ethanol in saline solution and is further diluted with 250 mL of 0.9% sodium chloride (or 5% glucose) before clinical administration through infusion

Figure 3: Packaging of docetaxel in commercial formulation Taxotere®

Both paclitaxel and docetaxel has been proven by U.S Food and Drug Administration (FDA) to be clinically effective in the treatment of a wide range of local or metastatic malignancies such as ovarian, breast, head and neck and non-small-cell lung cancers (NSCLC) (Eisenhauer and Vermorken, 1998) On the other hand, they are also effective

against melanoma, Kaposi’s sarcoma (KS) and some digestive system-related cancers (Eisenhauer and Vermorken, 1998; Dubois et al., 2003)

Figure 4: Molecular structure of polysorbate 80 (or Tween 80)

Similar to paclitaxel, the cytotoxic nature of docetaxel is due to the ability to perturb the cell mitosis Microtubules, a component of cytoskeleton, have a function of correctly segregating chromosomes during cell division When the binding and stabilization of microtubules by docetaxel happens, microtubules are unable to depolymerize or disassemble into free tubulin As a result, late G2 and early M phases of the cell cycle are

blocked and division fails (Gelmon, 1994; Huizing et al., 1995) Eventually, apoptosis takes place

Although docetaxel is the analogue to paclitaxel, there is significant difference between the pharmacodynamics and pharmacokinetics of the two drugs

2.3.2.2 Pharmacodynamics

At molecular level of pharmacodynamics, docetaxel has shown about 1.9-fold greater binding affinity to ß-tubulin Docetaxel also has a wider cell cycle bioactivity It has been

reported that docetaxel exerts its cytotoxic effect on cells undergoing S, G2 and M phases

of a cell cycle, compared to only G2 and M phases in the case of paclitaxel (Gligorov and

Lotz, 2004) And, because cell apoptosis induced by docetaxel is through the phosphorylation of bcl2, a protein required to inhibit cell death, apoptotic pathway is activated with 100-fold lesser drug concentration than that of paclitaxel (Haldar et al., 1997) Moreover, docetaxel has greater cellular uptake and slower drug efflux from tumor cells than paclitaxel, thus leading to higher efficacy and longer drug retention time at tumor sites (Riou et al., 1994)

2.3.2.3 Pharmacokinetics

As mentioned earlier, both taxanes are mainly distributed and metabolized in the liver, especially by CYP3A4 and CYP3A5 isoenzymes (Royer et al., 1996; Baker et al., 2006; Bradshaw-Pierce et al., 2007) A major fraction of drugs are also distributed to spleen, intestine and plasma proteins Meanwhile, about 80% of the dose is excreted through feces and about 6% is eliminated renally (Marlard et al., 1993) However, if compared to paclitaxel, docetaxel demonstrates a linear pharmacokinetics and elimination half-life behaviors over 1 hour after drug administration This would imply that any adjustment on the dosage given to patients could give a proportional outcome in terms of area under concentration-time curve (AUC) and peak concentration (Cmax) (Bissery et al., 1991; Gligorov and Lotz, 2004; McGrogan et al., 2008) Hence, unlike non-linear pharmacokinetics of paclitaxel, it becomes easier to predict the various important parameters describing the pharmacokinetics of docetaxel under different treatment schedules

2.3.2.4 Toxicology

Docetaxel shares some common side effectsas paclitaxel such as neutropenia, neuropathy and myalgia (Gligorov and Lotz, 2004; McGrogan et al., 2008) However, there is difference between these side effects in terms of grade of toxicity Toxicities such as neutropenia, leukopenia and fluid retention are most common severe side effects experienced by patients treated with docetaxel, while those for paclitaxel is more dose-dependent (Hurria et al., 2006) On the other hand, heart-related toxicity is milder for docetaxel Cardiac toxicity is more often observed when patients are given paclitaxel/anthracycline combination chemotherapy, probably due to the drug-drug interaction phenomena (Gligorov and Lotz, 2004) This may further highlight one of the advantages of using docetaxel as a single-agent chemotherapeutic drug in cancer therapy

Another obvious difference between paclitaxel and docetaxel is the more serious hypersensitivity and anaphylaxis reactions which are believed to be triggered by the Cremophor EL used as an adjuvant in the Taxol® formulation as mentioned earlier

Polysorbate 80 is thought to have a lower systemic exposure compared to Cremophor EL, therefore potential side effects attributed to polysorbate 80 is expected to be lessened, even though low hypersentivity reaction such as hypotension has been reported (Bissery, 1995; ten Tije et al., 2003; McGrogan et al., 2008) Fortunately, this can be further alleviated by applying anti-histamines and corticosteroids (Schrijvers et al., 1993)

Additionally, the level or grade of toxicity of docetaxel itself differs, relying on the dose and sequence in which the drug is given For instance, a 3-week-schedule therapy of docetaxel often causes fluid retention, myelosuppression, skin and nail disorders

Whereas, weekly dosing results in different toxicity profiles such as less hematologic and neurologic toxicities but with higher level of asthenia (Burstein et al., 2000) Other non-hematologic toxicities commonly associated with docetaxel include diarrhea, dyspnea, hallucination, hair loss and infection (Bissett et al., 1993; Hurria et al., 2006)

Some of the major docetaxel metabolites are shown in Figure 5

Side Chain

Metabolite Parent Drug

A

B

C and D

Figure 5: Molecular structures of parent drug docetaxel and its major metabolites

The parent drug is metabolized by hepatic transformation through oxidative reactions to

form primary alcohol (A), which is subsequently transformed into an oxazolidinedione (B)

as well as two hydroxyoxazolidinones (C and D) (Monegier et al., 1994) In vitro cell

viability has shown that the cytotoxic effect of all the metabolites formed after metabolism

of docetaxel is significantly reduced and negligible in comparison to its parent drug (Sparreboom et al., 1996)

2.4 Alternatives of Drug Formulations

In view of the high rate at which cancer is developed and diagnosed in people around the world each year, various kinds of alternatives to more effectively deliver the chemotherapeutic agents have been discovered and developed since the past few decades Together with the problems faced in traditional ways in which cancer patients are treated, this regimen has even become more and more popular in recent years

The development of new nanoparticulate drug formulations is no longer restricted only to chemistry and pharmacy, but is a multi-disciplinary area involving technologies from material science, life science, biology as well as chemical and biomedical engineering fields This new dimension of delivery system, combined with the nanotechnology, could provide a new hope to better treat the cancer in a more effective and comprehensive way Some of the novel therapeutic devices are described in the following sections

2.4.1 Liposomes

Liposomes are spherical vesicles which are made of one or a few natural phospholipid bilayers Phospholipid bilayer consists of two monolayer lipids with the hydrophobic tails being assembled in a way that is protected and surrounded by outer layers of hydrophilic heads of the same amphiphilic lipid molecules It has a thickness of about 5 – 6nm (http://www.azonano.com/Details.asp?ArticleID=1243) Examples of the molecular structure of phospholipids and liposome are shown in Figure 6 and Figure 7 below

Figure 6: Molecular structure of basic unit (phospholipid) of liposome

(Source: http://www.uic.edu/classes/bios/bios100/lecturesf04am/lect02.htm)

Figure 7: Arrangement of lipid bilayer in liposome

Size of liposome is generally dependent on several factors such as fabrication process conditions and the composition of the lipids forming the layers of liposomes Liposome with single lipid layer (small unilamellar vesicles or SUV) can be as small as 25nm while some types of liposomes with more concentric lipid bilayers such as large unilamellar vesicles (LUV) or multilamellar vesicles (MLV) can have sizes up to several microns (Rawat et al., 2006) However, Straubinger and Balasubramanian have demonstrated that MLV undergoing extended high-energy sonication resulted in drug-loaded liposomes of size 25 – 35nm in diameter (Straubinger and Balasubramanian, 2005)