IN VITRO AND IN VIVO STUDY OF VITAMIN E TPGS COATED IMMUNOLIPOSOMES FOR SUSTAINED AND TARGETED DELIVERY OF DOCETAXEL ANANDHKUMAR RAJU (B.TECH, ANNA UNIVERSITY, INDIA) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF ENGINEERING DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2012 ii    ACKNOWLEDGEMENTS First of all, I would like to take this opportunity to express my deepest gratitude and appreciation to my supervisor Professor Feng Si-Shen, for his invaluable advice, encouragement, guidance and support throughout my course of study. I wish to express my sincere thanks to our visiting scientist Dr. M.S. Muthu, for his kind support, extended help and advice in my experimental work. I like to thank all the professional lab officers and lab technologists, Mr Chia Phai Ann, Dr. Yuan Ze Liang, Mr. Boey Kok Hong, Ms. Samantha Fam, Mdm. Li Fengmei, Ms. Lee Chai Keng, Ms Li Xiang, Mr. Ang Wee Siong and Ms. Dinah Tan, for their technical assistance and administrative works. I would also like to express my warmest thanks to all my colleagues, Dr Li Yutao, Mr Prashant Chandrasekharan, Dr Sneha, Mr Gan Chee Wee, Mr Wai Min, Ms Chaw Su Yin, Mr Tan Yang Fei, Mr Mi Yu, Ms Zhao Jing, for their cooperation and kind support. My special thanks to my family and friends, who have always been there for me through the toughest of all times. iii    iv    TABLE OF CONTENTS ACKNOWLEDGEMENTS ........................................................................................................... iii TABLE OF CONTENTS................................................................................................................ v SUMMARY ................................................................................................................................... xi NOMENCLATURE .................................................................................................................... xiii LIST OF TABLES ...................................................................................................................... xvii LIST OF FIGURES ..................................................................................................................... xix CHAPTER 1: INTRODUCTION .................................................................................................. 1 1.1. Background .......................................................................................................................... 1 1.2. Objectives and Thesis Organization ..................................................................................... 5 CHAPTER 2: LITERATURE REVIEW ....................................................................................... 7 2.1. Cancer and its facts .............................................................................................................. 7 2.2. Cancer prevalence and its causes ......................................................................................... 7 2.3. Cancer Treatments and their Limitations ........................................................................... 11 2.3.1. Problems in Chemotherapy ......................................................................................... 12 2.4. Anticancer Drugs................................................................................................................ 16 2.4.1. Taxanes ........................................................................................................................ 17 2.4.2. Limitations of Taxane formulations ............................................................................ 20 2.4.2.1. Toxicity of vehicles............................................................................................... 20 2.4.2.2. Influence of vehicles of pharmacokinetic on taxanes ........................................... 21 2.4.2.3. Impact of vehicles on efficacy of taxane .............................................................. 21 v    2.5. Drug carrier vehicles in chemotherapeutic engineering ..................................................... 22 2.5.1. Liposomes.................................................................................................................... 22 2.5.2. Micelles ....................................................................................................................... 24 2.5.3. Polymeric nanoparticles .............................................................................................. 26 2.5.4. Prodrugs ....................................................................................................................... 28 2.6. Liposomes: Preparation methods and their types ............................................................... 30 2.6.1. Multilamellar Liposomes (MLV) ................................................................................ 31 2.6.1.1. Lipid Hydration Method ....................................................................................... 31 2.6.1.2. Solvent Spherule Evaporation Method ................................................................. 33 2.6.2. Small Unilamellar Liposomes (SUV).......................................................................... 33 2.6.2.1. Sonication Method ................................................................................................ 33 2.6.2.2. French Pressure Cell Method ................................................................................ 34 2.6.2.3. Support based hydration method .......................................................................... 34 2.6.3. Large Unilamellar Liposomes (LUV) ......................................................................... 35 2.6.3.1. Solvent Injection Methods .................................................................................... 35 2.6.3.2. Detergent Removal Methods ................................................................................ 36 2.6.3.3. Reverse Phase Evaporation Method ..................................................................... 37 2.7. Vitamin E TPGS, an amphiphilic polymer ........................................................................ 38 2.7.1. Structure and Properties............................................................................................. 38 2.7.2. Absorption/Bioavailability Enhancer ........................................................................ 39 2.7.3. Emulsifier and Solubilizer ......................................................................................... 40 vi    2.7.4. Agent for Controlled Delivery Applications ............................................................... 41 2.7.5. TPGS – an anti-neoplastic agent ................................................................................. 42 2.8. Herceptin ............................................................................................................................ 43 2.8.1. Structure and functional aspects of Herceptin ............................................................. 43 2.8.2. Structure and functional aspects of HER2 receptors ................................................... 43 2.8.3. Mechanism of action of Herceptin on HER2 .............................................................. 45 CHAPTER 3: PREPARATION AND CHARACTERIZATION OF VITAMIN E TPGS COATED AND HERCEPTIN CONJUGATED LIPOSOMES ................................................... 47 3.1. Introduction ........................................................................................................................ 47 3.2. Materials ............................................................................................................................. 48 3.3. Methods .............................................................................................................................. 48 3.3.1. Preparation of succinoylated TPGS ............................................................................. 48 3.3.2. Preparation of docetaxel or coumarin-6 loaded liposomes ......................................... 49 3.3.3. Preparation of Herceptin conjugated Liposomes......................................................... 50 3.3.4. Characterization of liposome formulations ................................................................. 50 3.3.4.1. Particle size, polydispersity, zeta potential ........................................................... 50 3.3.4.2. Surface morphology .............................................................................................. 51 3.3.4.3. Surface chemistry.................................................................................................. 52 3.3.4.4. FTIR spectroscopy ................................................................................................ 52 3.3.4.5. Differential Scanning Calorimetry ........................................................................ 52 3.3.4.6. Drug/dye encapsulation efficiency ....................................................................... 53 vii    3.3.4.7. Invitro drug release ............................................................................................... 54 3.4. Results and Discussion ....................................................................................................... 54 3.4.1. Particle size, Polydispersity and zeta potential analysis .............................................. 54 3.4.2. Encapsulation efficiency.............................................................................................. 56 3.4.3. Surface morphology .................................................................................................... 57 3.4.4. Surface chemistry ........................................................................................................ 59 3.4.5. FTIR and DSC studies ................................................................................................. 61 3.4.6. In vitro drug release ..................................................................................................... 63 3.5. Conclusion.......................................................................................................................... 65 CHAPTER 4: IN VITRO CELLULAR STUDY OF VITAMIN E TPGS COATED AND HERCEPTIN CONJUGATED LIPOSOMES .............................................................................. 67 4.1. Introduction ........................................................................................................................ 67 4.2. Materials ............................................................................................................................. 68 4.3. Methods .............................................................................................................................. 68 4.3.1. Cell culture .................................................................................................................. 68 4.3.2. Cellular uptake of liposomes ....................................................................................... 68 4.3.3. Cytotoxicity of liposomal formulations ....................................................................... 70 4.4. Results and Discussion ....................................................................................................... 71 4.4.1. Cellular Uptake ............................................................................................................ 71 4.4.2. Cell Viability ............................................................................................................... 73 4.5. Conclusion.......................................................................................................................... 75 viii    CHAPTER 5: IN VIVO PHARMACOKINETICS ....................................................................... 77 5.1. Introduction ........................................................................................................................ 77 5.2. Materials ............................................................................................................................. 78 5.3. Methods .............................................................................................................................. 78 5.4. Results and Discussion ....................................................................................................... 79 5.5. Conclusion.......................................................................................................................... 82 CHAPTER 6: CONCLUSION AND FUTURE WORKS ............................................................ 83 6.1. Conclusion.......................................................................................................................... 83 6.2. Future works....................................................................................................................... 84 REFERENCES ............................................................................................................................. 87 ix    x    SUMMARY The clinical utility of most cancer therapies is limited either by the inability to deliver therapeutic drug concentrations to the target cancer cells or by severe and harmful toxic effects on normal organs and tissues. Different approaches have been attempted to overcome these problems by providing “site-specific” delivery of drugs to the affected area using various pharmaceutical carriers. Among the different types of nanocarriers used in the delivery of anticancer drugs, liposomes have received the most attention. Liposomes are phospholipid bilayer vesicles and considered as biocompatible, cause very little or no antigenic, pyrogenic, allergic and toxic reactions inside the host. 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. Docetaxel (DTX) has been known to have excellent therapeutic effects for a wide spectrum of cancers such as breast cancer, ovarian cancer and head and neck cancer. Herceptin is a monoclonal antibody which targets and binds with HER2 receptors on the surface of the breast cancer cells. In this study we have prepared docetaxel loaded herceptin conjugated liposomes with TPGS (d-alpha tocopheryl polyethylene glycol 1000 succinate) coating and compared their effect with non-conjugated TPGS coated liposomes and Taxotere® for targeted chemotherapy on breast cancer cells. To facilitate the conjugation of herceptin, carboxyl group terminated TPGS has been synthesized and used in the preparation of herceptin conjugated liposomes. Docetaxel or Coumarin-6 loaded liposomes were prepared by solvent injection method and characterized for their size and size distribution, surface charge, surface chemistry and drug/dye encapsulation xi    efficiency and in vitro drug release profile. SKBR-3 cells were employed as an in vitro model for HER2 positive breast cancer and assessed for their cellular uptake and cytotoxicity of the coumarin-6 and docetaxel loaded immunoliposomes respectively. The particle size of these liposomes ranged between 140-220 nm. High resolution field emission transmission electron microscopy (FETEM) was used to visualize the morphology and surface coating of TPGS on the liposomes. X-ray photoelectron spectroscopy (XPS) and FTIR data confirmed the presence of herceptin conjugated on the surface of liposomes. Differential scanning Calorimetry was used to investigate the molecular arrangement of TPGS-COOH and docetaxel with the lipid bilayer. In vitro cellular uptake was studied by confocal microscopy and higher uptake was observed with immunoliposomes. The IC50 value, which is the drug concentration needed to kill 50 % cells in a designated time period, was found to be 20.23 ± 1.95, 3.74 ± 0.98, 0.08 ± 0.4 μg/ml for the Taxotere®, TPGS coated liposomes and herceptin conjugated liposomes respectively after 24 h incubation with SKBR-3 cells. In vivo PK experiments showed that i.v. administration of herceptin conjugated liposomes achieves 1.9 and 10 times longer half-life respectively than PEG coated liposomes and Taxotere®. The relative bioavailability of docetaxel was increased by 3.47 fold by the herceptin conjugated liposomes. Thus the herceptin conjugated Vitamin E TPGS coated liposomes showed greater potential for sustained and targeted chemotherapy in the treatment of HER2 over expressing breast cancer. xii    NOMENCLATURE ABC ATP binding cassette ACN acetonitrile AUC area under concentration-time curve BD biodistribution Ctot total clearance rate CLSM confocal laser scanning microscopy CMC critical micelle concentration CTAB cetyltrimethylammonium bromide CYP cytochrome P450 DCM dichloromethane DMEM Dulbecco‘s Modified Eagle Medium DPPC dipalmitoylphosphatidylcholine DSPE distearoylphosphatidylethanolamine EE encapsulation efficiency EPR enhanced permeability and retention FBS fetal bovine serum FESEM field emission scanning electron microscopy FETEM field emission transmission electron microscopy FT-IR Fourier transform infrared spectroscopy xiii    HIV human immunodeficiency virus HLB hydrophile-lipophile balance 1 proton nuclear magnetic resonance HPLC high performance liquid chromatography HPMA N-(2-hydroxypropyl) methacrylamide IC50 inhibitory concentration at which 50% cell population is suppressed LLS laser light scattering MRT mean residence time MTT 3-(4,5--2-yl)-2,5-diphenyltetrazolium bromide Dimethylthiazol MPS mononuclear phagocyte system NP nanoparticle NSCLC non-small-cell lung cancers PBS phosphate buffer saline PC phosphatidylcholine PCL poly (caprolactone) PDI polydispersity index PEG polyethylene glycol P-gp P-glycoprotein H NMR xiv    PI propidium iodide PLA poly (lactide) PLGA poly (d,l-lactide-co-glycolide) PVA polyvinyl alcohol RES reticuloendothelial system RESS rapid expansion from supercritical solution SD Sprague-Dawley SDS sodium dodecyl sulphate T1/2 half-life THF tetrahydrofuran TPGS d-α-tocopheryl polyethylene glycol 1000 succinate Tween 80 polyoxyethylene-20-sorbitan monooleate (or polysorbate 80) VSS volume of distribution at steady state XPS x-ray photoelectron spectroscopy xv    xvi    LIST OF TABLES Table 3.1: Formulation of liposomes 49 Table 3.2: Particle size, polydispersity, zeta potential and encapsulation efficiency of liposomes 55 Table 4.1: IC50 values of docetaxel formulated as liposomes or Taxotere® for SK-BR-3 cells (n=5) 74 Table 5.1: Pharmacokinetic parameters of Taxotere®, Herceptin conjugated liposomes and PEG coated liposomes after i.v injection at an equivalent dose of 7 mg/kg 81 xvii    xviii    LIST OF FIGURES Figure 2.1: Chemical structure of Docetaxel 18 Figure 2.2: Basic Structure of a liposome 23 Figure 2.3: A simple diagram showing (A) micelles, (B) reverse micelles 25 Figure 2.4: A representation of Polymeric nanosphere and nanocapsule 27 Figure 2.5: A simple representation of Prodrug concept 29 Figure 2.6: A schematic presentation for the steps involved in lipid hydration method 32 Figure 1.7: A schematic presentation of tip and bath sonicator 33 Figure 2.8: A schematic presentation of ethanol injection method 35 Figure 2.9: Chemical structure of Vitamin E TPGS 38 Figure 2.10: Structure of Herceptin 43 Figure 2.11: Types and functions of HER2 receptors 44 Figure 2.12: Mechanism of action of Herceptin 45 Figure 3.1:  Schematic diagram of (A) Placebo liposome (B) Docetaxel loaded liposome (C) TPGS-COOH coated liposome (D) Herceptin conjugated liposome 51 Figure 3.2A: AFM 2D image of TPGS-COOH coated liposomes showing a particle with 200 nm scale 58 Figure 3.2B: AFM 3D image of TPGS-COOH coated liposomes showing a particle with 200 nm scale 58 Figure 3.3: TEM image of (A) non-coated liposomes showing particles with 200 nm scale (B) TPGS-COOH coated liposomes showing a particle with 200 nm scale 59 Figure 3.4: XPS spectra for the herceptin conjugated and TPGS-COOH coated liposomes. Herceptin presence was confirmed by the presence of N 1s spectra at 397.8eV for conjugated liposomes. TPGS-COOH coating of liposomes was also confirmed by the presence of O1s 60 xix    Figure 3.5: C 1s core level spectra of (A) DTX-TP-COOH liposomes (B) DTX-TP-HER liposomes 61 Figure 3.6: FTIR spectra for the TPGS-COOH coated liposomes and herceptin conjugated liposomes 62 Figure 3.7: DSC thermogram of liposomes prepared with (a) DPPC and cholesterol (b) DPPC, cholesterol, docetaxel and TPGS-COOH 63 Figure 3.8: In vitro drug release from docetaxel loaded liposomes in phosphate buffered saline (pH 7.4). Bar represent ± S.D (n = 3) 64 Figure 4.1: Cellular uptake efficiency of coumarin-6 loaded liposomes on SK-BR-3 cells after 0.5, 1 and 3 h incubation 72 Figure 4.2: Confocal laser scanning microscopy images using SKBR-3 cells after 3 h incubation with the fluorescent coumarin-6 loaded (A) TPGS coated liposomes and (B) Herceptin conjugated liposomes. Fluorescein isothiocyanate (FITC) channel showing the green fluorescence from liposomes distributed in cytoplasm in first column. PI channels showing the red fluorescence from propidium iodide stained nuclei in second column and merged channels of FITC and PI in third column. Scale bar = 10 μm 72 Figure 4.3: Cytotoxicity of docetaxel loaded liposomes on SK-BR-3 cells for 24 h at 37 °C. Cell viability is studied in comparison with commercial formulation Taxotere® 73 Figure 5.1: Pharmacokinetic profiles of Taxotere®, PEG coated liposomes and Herceptin conjugated liposomes after intravenous injection in male SD rats at a single equivalent dose of 7 mg/kg. (n=4) 80 xx    CHAPTER 1: INTRODUCTION 1.1. Background Chemotherapy is one of the most important treatments currently available for the different types of cancer. Since the discovery of chemotherapeutic drugs for cancer, many challenges have been raised due to the systemic toxicity and adverse side effects caused by these drugs (Feng and Chien, 2003). To address this issue many novel drug carrier systems which have the ability of controlled and targeted/site-specific drug delivery to the cancer cells have been developed. The platform for these carrier systems was provided by the nanotechnology concept and consists of wide variety of particles such as micelles, liposomes, solid lipid nanoparticles and nanoparticles of biodegradable polymers (Muthu and Feng, 2010). Liposomes are the lipid bilayer vesicles, composed of either a single bilayer (SLV) or multiple layered vesicles (MLV) and discovered 40 years ago by Bangham et al. in 1965 (Bangham et al., 1965). Since then they have become very versatile and has been used as drug carriers for various diseases including cancer (Sharma et al., 2006). Liposomes can be seen as the simplest artificial biological cells, which have great potential applications in drug delivery, gene therapy, molecular imaging and artificial blood as well as to be used as a model biological cell and cell membrane (Muthu and Feng, 2010). Liposomes have various advantages over other drug carrier systems. They have no biocompatibility problem and provide desired adhesion to the biological cells. Liposomes used in drug delivery applications may vary in size for about less than 200 nm and composed of phospholipids, which are amphiphilic in nature to encapsulate water soluble hydrophilic drugs in the core and hydrophobic drugs in their bilayer region. One of the main problems associated with these liposomes is their poor stability and cholesterol is incorporated into the liposomal 1    membrane to increase their mechanical strength (Torchilin, 2008) and control the release of the encapsulated drugs. With the help of such drug delivery systems, the chemotherapeutic drugs can be reached to the cancer cells through 1) Passive targeting by enhanced permeation and retention (EPR) effect of the leaky vasculature of tumors which allows these nanocarriers to be accumulated in the tumor and 2) Active targeting with ligand conjugation towards particular cell surface markers on the cancer cells (Danhier et al., 2010). Active targeting has always proven to be promising approach for better therapeutic efficacy than passive targeting. Antibody conjugated liposomes or immunoliposomes efficiently targets the drug towards desired tissue or organ. These targeted liposomes along with surface attached ligand were capable of recognizing and binding towards specific molecular targets on the cancer cells. Herceptin® (trastuzumab) is a humanized IgG1 monoclonal antibody approved by US FDA for the treatment of human epidermal growth factor receptor type 2 (HER2) positive metastatic breast cancers (Liu et al., 2010). HER2 is a member of the EGF receptor (EGFR) family, which is a receptor tyrosine specific protein kinase family consisting of four semi homologous receptors EGFR, HER2, HER3 and HER4. These receptors interact with several ligand and generate intracellular signals either by homodimerization or forming heterodimer pairs. The EGFR family is thought to play a primary role in the control of epithelial cell proliferation and mutations affecting EGFR activity can result in cancer (Sun and Feng, 2009). It is known that HER2 is amplified at 20-30% in human invasive breast cancer (Steinhauser et al., 2006). Therefore targeting HER2 as a potential receptor is the key therapeutic strategy for HER2-overexpressing breast cancer cells. Conjugation of herceptin on drug loaded liposomes will elicit synergistic antitumor effects. 2    D-alpha-tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS or simply TPGS) is a PEGylated vitamin E, which has greatly improved the pharmaceutical properties of vitamin E and thus has been widely applied in the food and drug industry. TPGS was prepared from the esterification of D-alpha-tocopheryl acid succinate and PEG 1000. It is an amphiphilic vitamin E and quite stable under normal conditions without hydrolysis. Owing to its hydrophilic-liphophilic balance (HLB) value of about 13, TPGS has excellent water solubility and it is suitable to serve as an effective surfactant which can emulsify hydrophobic molecules (Varma and Panchagnula, 2005b; Wu and Hopkins, 1999). The co-administration of TPGS has been shown to enhance the solubility, inhibit P-glycoprotein mediated multi-drug resistance, and increase the oral bioavailability of anti-cancer drugs (Boudreaux et al., 1993; Dintaman and Silverman, 1999; Mu et al., 2005). We have reported TPGS-doxorubicin conjugate as a novel prodrug which enhanced the therapeutic potential and reduced the systemic side effects of the drug (Anbharasi et al., 2010; Cao and Feng, 2008). Additionally, we studied TPGS as an emulsifier in the preparation of poly (D, L, lactide-co-glycolide) (PLGA) nanoparticles (Mu and Feng, 2003b), and as a component of new biodegradable copolymer polylactide-TPGS (PLA-TPGS) for nanoparticle formulation of anti-cancer drugs(Zhang and Feng, 2006). As an effective emulsifier, TPGS has greatly enhanced the performance of nanoparticles, resulting in much higher emulsification efficiency (67 times higher than polyvinyl alcohol), drug encapsulation efficiency (up to 100%) (Mu and Feng, 2002), cellular uptake, and in vitro cancer cell cytotoxicity, and more desirable in vivo pharmacokinetics (up to 360 h effective treatment for one shot i.v. administration) (Win and Feng, 2006). Also, we have creatively recognized the marvelous advantageous of TPGS derivative (TPGS2000) as an effective composition of docetaxel loaded micelles for synergistic effect (Mi et al., 2011; Mu et al., 2005). 3    We used Docetaxel (N-debenzoyl-N-tert-butoxycarbonyl-10-deacetyl-paclitaxel) as a model anticancer drug in this study. It is a semi-synthetic derivative of the taxoid family of antineoplastic agents (Bissery et al., 1991). 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 (Brunsvig et al., 2007; Riou et al., 1992; Riou et al., 1994). 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 (Hanauske et al., 1994; Lavelle et al., 1995). TPGS coated liposomes was first introduced by Wang et al. in 2005 (Wang, 2005). Doxorubicin loaded TPGS coated liposomes has been developed and the pharmacokinetic results revealed that these liposomes have 24 h longer circulation time than PEG coated liposomes. It has been recently studied that TPGS containing liposomes showed improvement in the permeation of dextran through Caco-2 cells (Transwell® model) without any cytotoxicity effects (Parmentier et al., 2011; Parmentier et al., 2010). They focused on the oral drug delivery for better permeability and stability across the gastro-intestinal tract. We have showed recently that TPGS coated liposomes has better physicochemical properties for tumor targeting compared to PEG coated liposomes (Muthu et al., 2011). Thus it leads to a long way to characterize and decipher the potential advantages of the TPGS coated liposomes on the targeted drug delivery for cancer therapy. 4    1.2. Objectives and Thesis Organization Our aim is to prepare docetaxel loaded herceptin conjugated liposomes via TPGS coating and to compare the effect with the clinical Taxotere® (Docetaxel formulated in polysorbate 80), which causes side effects inspite of higher patient response than Taxol® (Paclitaxel formulated in Cremophor EL). We have already used carboxyl group activated TPGS (TPGS-COOH) for the conjugation of herceptin with polymeric nanoparticles (Sun and Feng, 2009). In this study we used one similar for the preparation of herceptin conjugated liposomes. Following the preparation and characterization of these liposomes, 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. There are six chapters which formed the framework of this thesis. The first chapter gave a general background and concepts of developing liposomal nanocarriers for targeted cancer chemotherapy. In Chapter 2, a detailed literature review on cancer and its causes, current treatments available, problems faced in conventional chemotherapy and the concept of different drug delivery formulations were provided. Then, Chapter 3 presents the preparation and characterization vitamin E TPGS coated and herceptin conjugated liposomes. The liposomes were prepared by solvent injection method and characterized by various state-of-art analytical instruments. Following that, Chapter 4 includes the in vitro cellular study for the liposomes formulations. Human breast adenocarcinoma SK-BR-3 cell lines which over expresses HER2 receptors was employed to assess cellular uptake as well as to evaluate the cell viability of the liposomes formulations which is done in close comparison with Taxotere®. In Chapter 5, in vivo pharmacokinetics using Sprague-Dawley (SD) rats is investigated to further confirm the 5    advantages of the herceptin conjugated liposomes versus the commercial drug. Finally, conclusion and suggestions for future work are provided in Chapter 6, followed by the reference papers cited in this thesis. 6    CHAPTER 2: LITERATURE REVIEW 2.1. Cancer and its facts Among most of the diseases, the leading cause of death globally is cancer. According to US National Cancer Institute, cancer is defined as diseases in which abnormal cells undergo uncontrolled growth (or mitosis) and have the ability to invade other tissues of the body through blood circulation and lymphatic systems.1 Unlike normal cells, cancer cells do not stop reproducing after they have doubled 50 or 60 times. This means that a cancer cell will go on and on and on doubling. The cancer cells may be able to stop themselves self destructing. Or they may self destruct more slowly than they reproduce, so that their numbers continue to increase.2 Eventually a tumour is formed that is made up of billions of copies of the original cancerous cell. 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.3 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. 2.2. Cancer prevalence and its causes Cancer may affect people at all ages but in most cases the number of cancer patient increases with age. All cancers are almost caused by the abnormalities in the genetic material of the transformed cells. These genetic abnormalities in cancer affect 2 types of genes namely Tumor suppressor genes and oncogenes. ___________________________________________ 1 http://www.cancer.gov/cancertopics/cancerlibrary/what-is-cancer http://cancerhelp.cancerresearchuk.org/about-cancer/what-is-cancer/cells/the-cancer-cell 3 http://news.bbc.co.uk/2/hi/health/3444635.stm 2 7    In cancer, the oncogenes are activated and the tumor suppressor genes are inactivated. Here, the oncogenes are responsible for the hyperactive growth and division of the cancer cells, to adjust in different environments and cause programmed cell death. Now the Tumor suppressor genes are responsible for the loss in control over the cell cycle, adhesion with other tissues and interaction with the immune cells. The two wide factors that cause the cancerous cells are the external factors and the internal factors. The external factors include  Tobacco smoking  Chemicals  Radiation  Infections  Alcohol  Poor diet  Lack of physical activity or overweight The internal factors include  Inherited mutations  Hormones  Growing older  Immune conditions These factors are said to be the most common risk factors for cancer. Many of these risk factors can be avoided and several of these factors may act together to cause normal cells to become cancerous. The chemicals that cause cancer are called carcinogens and those chemicals that cause cancer through mutations in DNA are called mutagens. All mutagens are carcinogens, but all carcinogens are not mutagens. They cause rapid rates of mitosis of the cells and thus 8    inactivate the enzyme that does the DNA repair. One of the most important carcinogens is tobacco. Smoking and its related disease remains the world’s most preventable cause of death and so is the cancer also. According to National Cancer Institute (NCI), each year, more than 180,000 Americans die from cancer that is related to tobacco use. Tobacco smoking accounts for at least 30 % of all cancer deaths and 87 % of lung cancer deaths. The risk of developing lung cancer is about 23 times higher in male smokers and 13 times higher in female smokers compared to non- smokers.4 Also, quitting smoking substantially decreases the risk of cancer. Prolonged exposure of radiation such as ultra violet radiation from the sun, sun lamps and tanning booths causes early ageing of the skin and skin damage that can lead to skin cancer. Ionizing radiation usually causes cell damage that leads to cancer. This kind of radiation comes from the rays that enter the earth’s atmosphere from outer space, radioactive fallout, radon gas, x-rays and other sources. The radioactive fallout can come from accidents at nuclear power plants or from the production, testing or use of atomic weapons. People exposed to fallout may have an increased risk of cancer, especially leukemia and cancer of thyroid, breast, lung and stomach. Radon is a radioactive gas that we cannot see, smell or taste. People who work in mines may be exposed to radon. People exposed to radon are at increased risk of lung cancer. The risk of cancer from low dose x-rays is very small and that from the radiation therapy is slightly higher. Being infected with certain viruses or bacteria may increase the risk of developing cancer. HPV (Human papillomavirus) infection is the main cause of cervical cancer. It also may be a risk factor for other types of cancer. Hepatitis B and Hepatitis C viruses can cause liver cancer after many years of infection. ___________________________________________ 4 http://www.cancer.org/downloads/STT/2008CAFFfinalsecured.pdf 9    Infection with HTLV-1 (Human T-cell leukemia/lymphoma virus) increases a person’s risk of developing lymphoma and leukemia. HIV (Human Immunodeficiency Virus) is the virus that causes AIDS. People who possess HIV have a greater risk of having cancer such as lymphoma and a rare cancer called ‘Kaposi’s sarcoma’. EBV (Epstein – Barr virus) infection can cause lymphoma. Human herpes virus 8 (HHV8) is a risk factor for Kaposi’s sarcoma. Helicobacter pylori bacteria can cause stomach ulcers. It can also cause stomach cancer and lymphoma in stomach lining. The viruses are responsible for about 15% of the cancers worldwide. The hormonal imbalance causes cancer due to the hormones acting in the same manner as the nonmutagenic carcinogens. Hormones may increase the risk of breast cancer, heart attack, stroke or blood clot. Diethylsilbestrol (DES), a form of estrogen, was given to pregnant woman in the United States between about 1940 and 1971. Woman who took DES during their pregnancy may have a slightly higher risk of developing breast cancer. Their daughters have an increased risk of developing a rare type of cancer of cervix. The effects on their sons are under study. The immune system malfunction also causes cancer to a greater extent and heredity causes cancer as well. Most cancers develop because of changes (mutations) in genes. A normal cell may become a cancer cell after a series of gene changes occur. Tobacco use, certain viruses, or other factors in a person's lifestyle or environment can cause such changes in certain types of cells. Some gene changes that increase the risk of cancer are passed from parent to child. These changes are present at birth in all cells of the body. It is uncommon for cancer to run in a family. However, certain types of cancer do occur more often in some families than in the rest of the population. For example, melanoma and cancers of the breast, ovary, prostate, and colon sometimes run in families. Several cases of the same cancer type in a family may be linked to inherited gene changes, which may increase the chance of developing cancers. However, environmental factors 10    may also be involved. Most of the time, multiple cases of cancer in a family are just a matter of chance. Having more than two drinks each day for many years may increase the chance of developing cancers of the mouth, throat, esophagus, larynx, liver, and breast. The risk increases with the amount of alcohol that a person drinks. For most of these cancers, the risk is higher for a drinker who uses tobacco. People who have a poor diet, do not have enough physical activity, or are overweight may be at increased risk of several types of cancer. For example, studies suggest that people whose diet is high in fat have an increased risk of cancers of the colon, uterus, and prostate. Lack of physical activity and being overweight are risk factors for cancers of the breast, colon, esophagus, kidney, and uterus. 2.3. Cancer Treatments and their 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 11    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. 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 12    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 (Feng et al., 2007; Gelderblom et al., 2001; Theis et al., 1995). 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 (Danielson, 2002; Guengerich, 2008; Nelson et al., 1993). 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. 13    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 (Béduneau et al., 2007; Ling, 1997). 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 gastrointestinal (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, 2005a). 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; Van Asperen et al., 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 14    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 reticuloendothelial system (RES) of the immune mechanism (Hume, 2006; Müller et al., 1997; 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 cisplatin, 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. 15    2.4. 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 cross linking (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 16    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. 2.4.1. Taxanes The taxanes have emerged as a potent class of chemotherapeutic agents in the treatment of various malignancies over the past two decades. Taxanes include paclitaxel and docetaxel, both of which are known to have antineoplastic activity against a wide variety of cancer cells. Both compounds were approved by the U.S. Food and Drug Administration (FDA) for the treatment of several carcinomas including breast, advanced ovarian, non small cell lung, head and neck, colon, and AIDS-related Kaposi′s sarcoma. The taxanes are a unique class of hydrophobic antineoplastic agents and exhibit their action against tumor cells by altering the microtubule dynamics, which causes cell cycle arrest during mitosis (Schiff et al., 1979). Paclitaxel was first discovered in the early 1960s as a part of National Cancer Institute screening study to identify natural compounds with anti-cancer properties. Paclitaxel was isolated as a crude extract from the bark of the North American pacific yew tree, Taxus brevifolia, and was found to possess excellent cytotoxic effects in the preclinical studies against many tumors (Wani et al., 1971). Because of the scarcity of the drug, the difficulties in its isolation, extraction and formulation, a second taxane drug, Docetaxel was extracted in 1986 from the needles of the European Yew Taxus baccata. It is more readily available because of the regenerating capacity of the source and slightly better solubility, thus having rapid development than that of paclitaxel 17    (Gelmon, 1994). Docetaxel differs from paclitaxel in the 10-position on the baccatin ring and in the 3'-position of the lateral chain, and has a chemical formula of C43H53NO14 and a molecular weight of 807.9 (Figure 2.1). It is trademarked as Taxotere® (807g/mol) by Rhone Poulenc Rorer, is a complex diterpenoid which has a rigid taxane ring and a flexible side chain. It is insoluble in water, but soluble in 0.1 N hydrochloric acid, chloroform, dimethyl formamide, 95%-96% v/v ethanol, 0.1 N sodium hydroxide and methanol. The formulation used in the most recent clinical studies consists of 100% polysorbate 80. Figure 2.1: Chemical structure of Docetaxel Microtubules are among the most strategic subcellular targets of anticancer agents. Like DNA, microtubules are ubiquitous to all eukaryotic cells. They are composed of tubulin dimers consisting of an α and a β-subunit protein that polymerize and, with numerous microtubuleassociated proteins (MAPs), decorate the exterior wall of the hollow micro tubule structure (Correia, 1991). There is a continuous dynamic equilibrium between tubulin dimers and microtubules, i.e., a continuous balance between polymerization and depolymerization. addition to being an essential component of the In mitotic spindle, and required for the maintenance of cell shape, microtubules are involved in a wide variety of cellular activities 18    such as cell motility and transport between organelles within the cell (Crossin and Carney, 1981; Edelman, 1976). Furthermore, they may also have a role in modulating the interactions of growth factors with cell-surface receptors and the proliferative transmembrane signals produced by these interactions. Many of the unique pharmacologic interactions of drugs with microtubules are caused by a dynamic equilibrium between microtubules and tubulin dimers (Rowinsky et al., 1990). Any disruption of the equilibrium, within the microtubule system, would be expected to disrupt the cell division and normal cellular activities in which the microtubules are involved. Taxanes bind preferentially and reversibly to the β- subunit of tubulin in the microtubules rather than to tubulin dimers. The binding site to tubulin differs from the one of vinca-alkaloids and podophyllotoxins. While vincas inhibit polymerization and increase microtubule disassembly, the binding of taxanes enhances polymerization of the tubulin into stable microtubules and further inhibits microtubule depolymerization, thereby inducing the formation of stable microtubule bundles. This disruption of the normal equilibrium ultimately leads to cell death. As an inhibitor of microtubule depolymerization, docetaxel is approximately twice as potent as paclitaxel. In addition, docetaxel generates tubulin polymers that differ structurally from those generated by paclitaxel and does not alter the number of protofilaments in the microtubules, while paclitaxel does. One of the limitations with the clinical use of docetaxel is that it shows very low water solubility, and the only available formulation for clinical use consists of a solution (40 mg/mL) in a vehicle containing a high concentration of Tween 80. Unfortunately, this vehicle has been associated with several hypersensitivity reactions such as nephrotoxicity, neurotoxicity, and cardiotoxicity. In order to overcome the problems faced by Tween 80-based vehicle and in the attempt to increase the drug solubility, alternative dosage forms have been suggested, e.g., liposomal 19    formulations for controlled and targeted delivery of the drug (Fernández-Botello et al., 2008). 2.4.2. Limitations of Taxane formulations 2.4.2.1. Toxicity of vehicles A high incidence of acute hypersensitivity reactions characterized by respiratory distress, hypotension, angioedema, generalized urticaria and rash were observed with paclitaxel administration. It is generally felt that cremophor EL contributes significantly to these hypersensitivity reactions (Weiss et al., 1990; Wiernik et al., 1987). These reactions increased with increasing rate of infusion. Docetaxel has also known to cause infusion related reactions in the absence of pre medication (Bernstein, 2000). But these reactions occurred at a decreased frequency when compared with paclitaxel and effectively managed by pre medication. Agents formulated with cremophor EL cause peripheral neurotoxicity. The oral formulation never induced these adverse side effects. This shows that cremophor EL is not absorbed through the gastrointestinal tract. Furthermore, cremophor EL plasma concentrations achieved after i.v (intravenous) administration have been noted to cause axonal swelling, vesicular degeneration and demyelination in rat dorsal root ganglion neurons exposed to the formulation vehicle (Windebank et al., 1994). Recent evidences suggest that ethoxylated derivatives of castor oil account for this neuronal damage. Polysorbate 80 is also capable of producing vesicular degeneration. Sensory neuropathy has also been associated with docetaxel administration but the incidences are much lower when compared with paclitaxel. However polysorbate 60 containing epipodophyllotoxin etoposide is not a known neurotoxin, suggesting that the mechanism of taxane-induced neuropathy may be multi factorial, atleast in part contributed by vehicle formulation (ten Tije et al., 2003). 20    2.4.2.2. Influence of vehicles of pharmacokinetic on taxanes Both cremophor EL and polysorbate 80 have demonstrated to alter the disposition of intravenously administered Paclitaxel and Docetaxel. Pharmacokinetic studies conducted in mouse models and humans have proved that the non-linear pharmacokinetics of Paclitaxel was due to Cremophor EL (Sparreboom et al., 1996; van Tellingen et al., 1999). This altered pharmacokinetics is a resulted of the micellar entrapment of Paclitaxel by cremophor EL in plasma. It has been shown that the percentage of Paclitaxel entrapped in micelles increases disproportionately with the administration of higher doses of cremophor EL thereby making it less available for the tumor tissue distribution, metabolism and biliary excretion (Sparreboom et al., 1999). Diminished clearance and prolonged exposure to high concentrations of chemotherapeutic agent place patients at higher risk of systemic toxicities. An additional problem linked to the cremophor EL solvent is the leaching of plasticizers from polyvinylchloride (PVC) bags and infusion sets used routinely in clinical practice. Consequently paclitaxel must be prepared and administered in either glass bottles or non-PVC infusion systems with in-line filtration. Polysorbate 80 is thought to be rapidly degraded in plasma and does not interfere with kinetics of docetaxel (van Tellingen et al., 1999), but recent evidence suggest that this vehicle may influence the binding of docetaxel in plasma in concentration-dependent manner (Loos et al., 2003). 2.4.2.3. Impact of vehicles on efficacy of taxane Some in vitro models have demonstrated that cremophor EL and polysorbate 8- may enhance cytotoxic activity by modulating P-glycoproteins (P-gp) and inhibiting multi drug resistance gene expression (Friche et al., 1990; Schuurhuis et al., 1990; Woodcock et al., 1990). However 21    this cytotoxic activity was absent in vivo studies due to low volume of distribution of cremophor EL and rapid degradation of polysorbate 80 in plasma. Several reports suggest that these formulation vehicles may have anti tumor activity on their own. The cytotoxic activity of cremophor EL is thought to result from free radical formation by poly unsaturated fatty acids and in polysorbate 80, the cytotoxic activity is linked to the release of oleic acid, a fatty acid known to interfere with malignant cell proliferation and inhibition of angiogenesis (Chajes et al., 1995; Kimura, 2002; Liebmann et al., 1994). 2.5. Drug carrier vehicles in chemotherapeutic engineering 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. Some of the most common drug delivery nanocarriers that are currently developed or under development are liposomes, micelles, polymeric nanoparticles and produrgs. The aims of these delivery systems are to minimize drug degradation upon administration, prevent undesirable side-effects, and increase drug bioavailability and the fraction of the drug accumulated in the pathological area. 2.5.1. Liposomes Liposomes are drug delivery vehicles which are composed of amphiphilic phospholipids and 22    cholesterol that self-associate into bilayers encapsulating an aqueous interior. These may be formulated into small structures (80-100 nm in size) that encapsulate either hydrophilic drugs in the aqueous interior or hydrophobic drugs within the bilayer. Liposome can also be formed by using biocompatible and biodegradable synthetic lipids or natural/synthetic lipid blends, depending on the characteristics required. Other than the non-toxic effects of phospholipids, low immunogenicity and reversal of multidrug resistance are other advantages of using liposomes as promising drug delivery devices (Thierry et al., 1992; Torchilin, 2005; Warren et al., 1992). Encapsulation of drugs is achieved using a variety of loading methods, most notably the pH gradient method used for loading vincristine (Waterhouse and Madden, 2005) or the ammonium sulfate method for loading doxorubicin.   Figure 2.2: Basic Structure of a liposome Additionally, the liposome surface can be engineered to improve its properties (Allen and Sapra, 2002; Sapra and Allen, 2003). So far, the most noteworthy surface modification is the incorporation of polyethylene glycol (PEG) which serves as a barrier, preventing interactions with plasma proteins and thus retarding recognition by the reticuloendothelial system (RES) (Gabizon and Shmeeda, 2003) and enhancing the liposome circulation lifetime. More advanced types of liposomes are under research to further target the liposomes to tumor site. For 23    instance, Shi and Pardridge have chemically modified PEGylated liposome with mouse monoclonal antibody OX-26 to form immunoliposomes for targeted and prolong drug or gene delivery for brain disorder such as Parkinson’s disease (Shi and Pardridge, 2000). However, despite this versatility, there have been major drawbacks to the use of liposomes for targeted drug delivery, most notably, poor control over release of the drug from the liposome (i.e. the potential for leakage of the drug into the blood), coupled with low encapsulation efficiency, manufacturability at the industrial scale and poor stability during storage (Soppimath and Aminabhavi, 2001). 2.5.2. Micelles Similar to liposomes, micelles are usually made up of surfactant molecules comprising two regions of chains with different hydrophilicity, namely the head and tail groups. Hence, they are also capable of encapsulating hydrophobic as well as hydrophilic drugs, thereby increasing the solubility and bioavailability. This is one of the attractive properties of micelles as an alternative of pharmaceutical nanocarriers. Depending on the nature of the agents to be delivered, different processing strategies can be applied to fabricate micelles. If a very hydrophobic drug such as paclitaxel is considered, the liphophilic regions (tail group) of the surfactant will arrange in such a way that the core of the micelles containing the drug is excluded and protected from the surrounding aqueous phase by the hydrophilic head group. This arrangement is termed oil-inwater micelle. In contrast, colloidal structures entrapping water-soluble drugs are referred to as water-in-oil micelles (or inverse micelles). In recent years, micelles formed from amphiphilic diblock or triblock copolymers have gained popular attention in nanomedicine research. The reason is that physiological- friendly building blocks of copolymers can be chosen to construct 24    micelles which have increased half-life and controlled drug release in the body. On the other hand, the size of micelles often ranges from 5 – 100 nm, which is the favorable range for passive targeting through enhanced permeation and retention (EPR) effect of the leaky vascular and lymphatic system at tumor sites (Hao et al., 2005; Torchilin, 2006).   Figure 2.3: A simple diagram showing (A) micelles, (B) reverse micelles Another advantage of polymeric micelles is the narrow size distribution. This is because the selfassembled core-shell structure of the micelles mainly relies on the thermodynamic equilibrium achievable under the particular reaction condition during fabrication process. As a result, micellization are also very sensitive to the critical micelle concentration (CMC) of the polymeric molecules constituting the micelles (Jones and Leroux, 1999; Lawrence, 1994; Letchford and Burt, 2007). This is perhaps the major concern when designing the materials to be used to develop micelles as drug delivery devices. If the concentration drops below the CMC value, micelles will tend to dissociate into free chains due to thermodynamic instability. Therefore, this becomes a serious issue when it is administered intravenously for treatment. The sudden dilution of micelles by the blood and physiological fluids could potentially cause immediate release of the encapsulated drug, which could result in severe toxicity (Lawrence, 1994). It happens especially to micelles made of conventional surfactants such as ionic surfactants (hexadecyltrimethylammonium bromide, cetyltrimethylammonium bromide (CTAB) and sodium 25    dodecyl sulphate (SDS)) or nonionic surfactants (poloxamers and polysorbates), of which CMC values are orders higher compared to those of novel, high molecular weight amphiphilic polymeric molecules (Rawat et al., 2006). One creative solution from Prabaharan et al. is to synthesize unimolecular micelles inheriting the characteristic of dendrimers by using hyper branched polyester as the core structure to make the micelles relatively more solid (Prabaharan et al., 2009). For this reason, multifunctional micellar system has been developed for various purposes. Simultaneous drug delivery and diagnostic imaging were made possible from the research done by Yang et al (Yang et al., 2008). In their formulation, iron oxide nanoparticles and doxorubicin were encapsulated in 75 nm micelles formed by poly(ethylene glycol)/poly(εcaprolactone) (PEG/PCL) diblock copolymer which has been conjugated to folate for specific targeting to cancer cells. Other work such as pH-sensitive poly(ethylene glycol)- poly(aspartate hydrazone adriamycin) micelles has also been studied by Bae et al. where the acidic condition of intracellular endosomes as well as tumor microenvironment can trigger dissociation and the release of adriamycin from polymeric micelles, thus increasing the probability of cytoplasmic toxicity to cancerous cells (Bae et al., 2005). 2.5.3. Polymeric nanoparticles Polymeric nanoparticles are solid and spherical structure with size ranging from 10 to 1000 nm, in which drugs are encapsulated within the polymer matrix (Danhier et al., 2010; Muthu and Singh, 2009; Torchilin, 2006). The term nanoparticles can be divided into nanospheres and nanocapsules. In nanospheres, the drug is dispersed throughout the particles whereas in nanocapsules, the drug is entrapped in a cavity surrounded by a unique polymeric membrane (Allémann et al., 1993; Hillaireau and Couvreur, 2009). Polymeric nanoparticles are 26    generally synthesized from biodegradable polymers - like the poly (lactic acid) (PLA) and poly (lactic-co-glycolic acid) (PLGA) polyesters or the poly (alkylcyanoacrylates) (PACA) or natural polymers, like albumin. Currently, nanoparticles smaller than 200 nm are extensively investigated to achieve efficient passive targeting through EPR effect, avoid complement system activation and escape from splenic filtration. Moreover, PEGylation and active targeting with antibodies, peptides or cell specific ligands has become essential for efficient drug delivery systems (Danhier et al., 2010). PEGylation refers to the conjugation of polyethylene glycol to drug delivery systems in order to enhance both the stability and circulation time of drug delivery systems (Davis, 2002). The effect of PEGylation can also be achieved in drug delivery systems prepared by using diblock or triblock copolymers consisting of hydrophilic PEG segment.   Figure 2.4: A representation of Polymeric nanosphere and nanocapsule (Bei et al., 2010) For example, MPEG-PLGA, MPEG-PLA, MPEG-PCL and PLA-TPGS copolymers were synthesized and used to fabricate drug nanocarriers to achieve PEGylation effect (Aliabadi et al., 2005; Dong and Feng, 2004; Suh et al., 1998; Zhang and Feng, 2006). An example of nanoparticles approved by US FDA as a nanomedicine is albuminbound paclitaxel (Nab™paclitaxel), used for treating metastatic breast cancer. This nanoparticle formulation does not involve the use of toxic adjuvant Cremophor EL unlike traditional paclitaxel formulation. Due to 27    the absence of toxic solvents and the albumin receptor mediated delivery, Nab™-paclitaxel tends to provide more advantages over traditional solvent-based paclitaxel such as decreased toxicity and increased antitumor activity (Stinchcombe, 2007). 2.5.4. Prodrugs Prodrug is a formulation whereby the chemical agents are manipulated at the molecular level with the hope to increase their biological activities. It is a common but essential strategy applied in drug modification. This strategy usually involves direct chemical modification of bioactive agents to enhance their solubility, stability and permeability in physiological conditions to optimize absorption, distribution, metabolism and excretion (ADME) properties of the original drug compounds (Nielsen et al., 1994; Stella et al., 1998; Testa and Caldwell, 1996; Vyas et al., 1993). To achieve this objective, modification of functional group or conjugation of the drugs to polymeric or targeting molecules is often done to convert the drugs to its inactive form. Thus, when the modified inactive drugs are administered into the body, they can bypass some of the various physiological barriers such as gastro-intestinal (GI) tract or RES. After undergoing the metabolic process in vivo, the breakdown of the molecule into the corresponding constituents then restores the original bioactive form of the drug. In other words, prodrug exhibits the potential to increase the bioavailability of poorly soluble drugs as well as to enhance their biological effects in the body by prolonging the duration of action. One classical example of functional group-modified prodrug is the pharmaceutical compound called levodopa (L-3, 4dihydroxy-phenylalanine), used in clinical treatment of Parkinson’s disease which is characterized by severe depletion of neurotransmitter dopamine in brain. Unlike dopamine, levodopa is capable of crossing the blood-brain barrier (BBB) through neutral amino acid 28    transporter. After BBB permeation of levodopa, decarboxylase enzymes, mostly found in brain tissues, converts levodopa to dopamine, thereby increasing the dopamine concentration in brain. Figure 2.5: A simple representation of Prodrug concept (Jayant Khandare, 2006) In recent years, some polymers serving as a backbone for therapeutic agents as well as targeting moieties to be chemically attached have attracted significant attention in biomedical fields. For example, N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer conjugated with anticancer drug daunomycin or doxorubicin and targeting ligands such as monosaccharides and antibody demonstrated decreased toxicity to normal tissues and low immunogenicity in vivo (Kopecěk, 1990). Meanwhile, polymer-anti cancer drug conjugates such as HPMA/doxorubicin and PEG-camptothecin conjugate have also shown promising therapeutic effect and has gone further into clinical trial due to its synthetic, non-immunogenic, biocompatible and water-soluble properties which could make oral drug delivery feasible (Duncan et al., 2006; Veronese et al., 2005). Furthermore, Cao et al. showed that doxorubicin conjugated to d-α-tocopheryl polyethylene glycol 1000 succinate (or Vitamin E TPGS) exhibited significant cellular uptake of cancer cells in vitro, more than 3-fold longer circulation half-life in rats as well as much lower 29    drug accumulation in heart and intestine compared to the free drug (Cao and Feng, 2008). This implies that polymer-drug conjugate could greatly change the pharmacokinetic, pharmacodynamic and improve therapeutic index of the pristine drug (Maeda et al., 1992). However, some limitations of this formulation include the cytotoxicity of the polymer used, the limited site of polymer for drug conjugation and the possibility of denaturing the bioactivity of the drug molecule when certain functional groups are permanently modified during chemical reaction. Lack of activating enzymes in certain human tumors may also be another drawback for prodrug therapy (Connors and Knox, 1995). 2.6. Liposomes: Preparation methods and their types An important parameter to consider when addressing the formation process of liposomes is the rigidity of the bilayer. As previously mentioned liposomes are composed of phospholipids and the thermal characteristic of these phospholipids dictates the fluidity of the bilayer. They undergo a phase transition at temperatures lower than the melting point (Tm) known as the crystalline transition temperature (Tc). At this temperature the lipidic bilayer loses much of its ordered packing and can be in a liquid-crystalline (‘fluid’) state or in a gel state. The presence of branched chains and unsaturated acyl chains in phospholipids lowers the Tc. Liposomes made up of pure phospholipids will not form at temperatures below the Tc of phospholipids and to reduce this temperature dependency, cholesterol is being added in the formation of liposomes. The preparation method should be carried out well above the Tc of the vesicles so as to ensure that all the phospholipids are dissolved in the suspension medium homogeneously and have sufficient flexibility to align themselves in the structure of lipid vesicles (Mozafari, 2010). Therefore the transition temperature is considered as an important factor in the preparation of liposomes. The 30    Tc of a bilayer depends on: 1. Acyl chain length. 2. Degree of saturation. 3. Polar head group. 4. Nature and ionic strength of the suspension medium Based on their preparation methods, the liposomes are classified into the following three different types. 2.6.1. Multilamellar Liposomes (MLV) 2.6.1.1. Lipid Hydration Method This is the most widely used method for the preparation of MLV. The method involves drying a solution of lipids so that a thin film is formed at the bottom of a round bottomed flask and then hydrating the film by adding aqueous buffer and vortexing the dispersion for some time. The hydration step is done at a temperature above the gel-liquid crystalline transition temperature Tc of the lipid or above the Tc of the highest melting component in the lipid mixture. The compounds to be encapsulated are added either to aqueous buffer or to organic solvent containing lipids depending upon their solubilities. Multilamellar vesicles can be easily prepared by this method and a variety of substances can be encapsulated in these liposomes. The drawbacks of the method are low internal volume, low encapsulation efficiency and the size distribution is heterogeneous (Bangham et al., 1974; Bangham et al., 1965). 31      Figure 2.6: A schematic presentation for the steps involved in lipid hydration method 5 Many different variations have been developed for the user’s suitability which varies with respect to the organic solvent used, possible addition of inert glass or Teflon beads, way of drying lipid film and parameters of agitation such as time, intensity, mode and temperature. On the other hand, MLVs with high encapsulation efficiency can be prepared by hydrating the lipids in the presence of an immiscible organic solvent (petroleum ether, diethyl ether). The contents are emulsified by vigorous vortexing or sonication. The organic solvent is removed by passing a stream of nitrogen gas over the mixture. MLVs are formed immediately in the aqueous phase after the removal of organic solvent (Gruner et al., 1985; Papahadjopoulos and Watkins, 1967). The main drawback of this method is the exposure of the materials to be encapsulated to organic solvent and to sonication. A schematic diagram for this method was shown in Figure 2.6. ________________________________________ 5 http://avantilipids.com/images/PrepOfLiposome/PrepOfLip_2.gif 32    2.6.1.2. Solvent Spherule Evaporation Method A method for the preparation of MLVs of homogeneous size distribution was proposed by Kim et al. (Kim et al., 1985). The process involved dispersing in aqueous solution the small spherules of volatile hydrophobic solvent in which lipids had been dissolved. MLVs were formed when controlled evaporation of organic solvent occurred in a water bath. 2.6.2. Small Unilamellar Liposomes (SUV) 2.6.2.1. Sonication Method Here MLVs are sonicated either with a bath type sonicator or a probe sonicator under an inert atmosphere. The former method has the advantage that there is no direct contact of liposomes with the source of ultrasound waves but the energy density of typical bath sonicators are in general too low to produce homogeneous and small unilamellar vesicles. Direct tip sonication is still probably the most widely used method for the preparation of SUV’s, at least in small scales.   Figure 2.7: A schematic presentation of tip and bath sonicator (Lasic, 1993) Apart from these, the main drawbacks of this method are very low internal volume/encapsulation 33    efficiency, possibly degradation of phospholipids and compounds to be encapsulated, exclusion of large molecules, metal contamination from probe tip and presence of MLV alongwith SUV. Oezden and Hasirci (Oezden and Hasirci, 1991) have prepared a polymer coated liposomes by this method. 2.6.2.2. French Pressure Cell Method The method involves the extrusion of MLV at 20,000 psi at 4°C through a small orifice. The method has several advantages over sonication method. The method is simple, rapid and reproducible and involves gentle handling of unstable materials (Hamilton and Guo, 1984). The resulting liposomes are somewhat larger than sonicated SUVs. Normally the working volumes are upto 50 ml and after several extrusions rather small and unilamellar vesicles are produced. The drawbacks of this method are low maximal concentrations and not too well defined size distribution of vesicles produced. It is not easy to operate at higher temperature in this method and is basically used for lipids with Tc < 20 ̊ C. 2.6.2.3. Support based hydration method This method was given by Lasic et al. (Lasic et al., 1987) and used for the preparation of SUV. They deposited egg phosphatidylcholine mixed with 1.5 %w/v of cetyl tetramethylammonium bromide (a detergent) in CHCI3/CH3OH on various supports for example silica gel powder, zeolite X, zeolite ZSM5. After the removal of organic phase, the system was resuspended by shaking or stirring in distilled water or 5 mM NaCl. There was some loss of phospholipid (about 10-20%) due to adsorption on the supports. The loss was 70% and 95% in the case of silica gel and zeolite ZSM5 respectively. An homogenous population of vesicle with average diameter of 21.5 nm was obtained when zeolite X (particle size of 0.4 mm) was used as a support. 34    2.6.3. Large Unilamellar Liposomes (LUV) They have high internal volume/encapsulation efficiency and are now days being used for the encapsulation of drugs and macromolecules. 2.6.3.1. Solvent Injection Methods (a) Ethanol Injection Method A lipid solution of ethanol is rapidly injected to a vast excess of buffer or aqueous solution. The LUVs are immediately formed. The method is rather simple to scale up and has also found applications in large scale production methods. The drawbacks of the method are that the population is heterogeneous (30-110 nm), liposomes are very dilute, it is difficult to remove all ethanol because it forms azeotrope with water and the possibility of various biologically active macromolecules to inactivation in the presence of even low amounts of ethanol (Batzri and Korn, 1973).   Figure 2.8: A schematic presentation of ethanol injection method (Lasic, 1993) 35    (b) Ether Infusion Method This method is an analogue to the ethanol injection method with the exception of the fact that the solvent is not miscible with water. A solution of lipids dissolved in diethyl ether or ether/methanol mixture is slowly injected to an aqueous solution of the material to be encapsulated at 55-65°C or under reduced pressure. The subsequent removal of ether under vacuum leads to the formation of liposomes. The main drawbacks of the method are that the population is heterogeneous (70-190 nm) and the exposure of compounds to be encapsulated to organic solvents or high temperature (Deamer and Bangham, 1976; Schieren et al., 1987). 2.6.3.2. Detergent Removal Methods This method is considered a very mild treatment and even many sensitive proteins, whose activity cannot survive other physical or chemical treatments, can be incorporated into liposomes unchanged if the proper non denaturating detergent can be used. The detergents at their critical micelles concentrations have been used to solubilize lipids. As the detergent is removed the micelles become progressively richer in phospholipid and finally combine to form LUVs. The detergents were removed by dialysis (Alpes et al., 1986; Kagawa and Racker, 1971; Milsmann et al., 1987). The advantages of detergent dialysis method are excellent reproducibility and production of liposome populations which are homogenous in size. The main drawback of the method is the retention of traces of detergent(s) within the liposomes. A commercial device called LIPOPREP (Diachema AG, Switzerland) which is a version of dialysis system is available for the removal of detergents. Other techniques have been used for the removal of detergents: (a) by using Gel Chromatography involving a column of Sephadex G-25, (b) by adsorption or binding of Triton X-100 (a detergent) to Bio-Beads SM-2. (c) by binding of octyl glucoside (a 36    detergent) to Amberlite XAD-2 beads. 2.6.3.3. Reverse Phase Evaporation Method Reverse phase evaporation method, besides sonication and detergent removal techniques, is the most widely used in liposomes research. In this method, water in oil emulsion is formed by brief sonication of a two phase system containing phospholipids in organic solvent (diethylether or isopropylether or mixture of isopropyl ether and chloroform) and aqueous buffer. The organic solvents are removed under reduced pressure, resulting in the formation of a viscous gel. The liposomes are formed when residual solvent is removed by continued rotary evaporation under reduced pressure. With this method high encapsulation efficiency up to 65% can be obtained in a medium of low ionic strength, for example 0.01 M NaCl. The method has been used to encapsulate small, large and macromolecules. The main disadvantage of the method is the exposure of the materials to be encapsulated to organic solvents and to brief periods of sonication. These conditions may possibly result in the denaturation of some proteins or breakage of DNA strands (Szoka and Papahadjopoulos, 1978). We get a heterogeneous sized dispersion of vesicles by this method. Modified Reverse Phase Evaporation method was presented by Handa et al. (Handa et al., 1987) and the main advantage of the method is that the liposomes had high encapsulation efficiency (about 80%). The Reverse Phase Evaporation method of Szoka and Papahadjopoulos (1978) has also been modified to entrap plasmids without damaging DNA strands. 37    2.7. Vitamin E TPGS, an amphiphilic polymer 2.7.1. Structure and Properties Vitamin E TPGS (d-α-tocopheryl polyethylene glycol 1000 succinate or TPGS) is a water soluble derivative of natural vitamin E and prepared by esterification of d-α-tocopheryl acid succinate with polyethylene glycol 1000. It is an amphiphilic macromolecule comprising of hydrophilic polar head and a lipophilic alkyl tail. Its molecular weight is approximately 1542 Da.   Figure 2.9: Chemical structure of Vitamin E TPGS 6 The hydrophile/lipophile balance (HLB) of TPGS is ~13. It is basically a waxy solid appearing white to light brown in color with a melting point approximately 37-41°C. It is stable in air as well. It is used an effective emulsifier as well as a good solubilizer due to its bulky nature and larger surface area (Fisher et al., 2002). The chemical structure of TPGS was shown in Figure 2.9. TPGS has found wide utility in pharmaceutical formulations as follows.  Improving drug bioavailability  Surfactant properties enhance solubilization of poorly water soluble drugs _______________________________________ 6 www.tpgs.com 38     Stabilization of the amorphous drug form  Enhances drug permeation by P-glycoprotein efflux inhibition  Emulsion vehicle  Functional ingredient in self-emulsifying formulations  Thermal binder in melt granulation/extrusion processing  Reducing drug sensitivity on skin or tissues  Carrier for wound care and treatment  Water-soluble source of vitamin E 2.7.2. Absorption/Bioavailability Enhancer TPGS has received increased attention in the literature for its ability to enhance the absorption of several drugs that have otherwise poor bioavailability. Sokol et al. in 1991 clinically demonstrated that TPGS can enhance absorption of the highly lipophilic drug cyclosporin, which is used for immunosuppressive therapy to manage rejection of transplanted organs. This is a crucial finding for organ transplant recipients. Due to the impaired absorption of cyclosporine, massive doses are required to achieve therapeutic blood plasma concentrations. The study showed that TPGS provides a substantial improvement of cyclosporine absorption and a significant reduction of the high cost of immunosuppressive therapy. While Sokol originally suggested that the increased bioavailability was due to micelle formation enhancing the solubility, others have since provided evidence supporting enhanced permeability due to Pglycoprotein (P-gp) inhibition (Croockewit and Koopmans, 1996; Dintaman and Silverman, 1999). While many of the examples of TPGS use are poorly water soluble drugs there are also 39    examples of using TPGS with poorly permeable drugs that are water soluble (Prasad and Puthli, 2003). Many studies have been conducted to evaluate the mechanism by which TPGS affects bioavailability. Its action is attributable to its ability to improve solubility through micelle formation and through enhancing permeability across cell membranes by inhibition of multi-drug efflux pump P-gp. For oral delivery, TPGS enhances drug efficacy by improving the solubilization or emulsification of the drug in the finished dosage form and through formation of a self-emulsifying drug delivery system in the stomach which may be due to TPGS, which improves the permeability of a drug across cell membranes by inhibiting P-glycoprotein and thus enhance absorption of a drug through intestinal wall and into the bloodstream. It can act as a reversal agent of P-gp mediated multidrug resistance and inhibit P-gp substrate drugs transport (Dintaman and Silverman, 1999). TPGS is more effective P-gp inhibitor than many related excipients with surfactant properties such as cremophor EL, Tween 80, Pluronic P85 and PEG 300. However, it is significantly less potent than other clinically tested pharmacologically active compounds such as cyclosporine, tariquidar and zosuquidar (Dantzig and Law, 2001; Mistry and Stewart, 2001). 2.7.3. Emulsifier and Solubilizer It is estimated that over 40% of new drug entities are poorly water soluble. Some drug delivery systems that can utilize the solubilizing ability of TPGS are solid dispersions, self-emulsifying drug delivery systems, self-microemulsifying drug delivery systems, spray drying and others. Much work has been done to investigate the effect of TPGS on the aqueous solubility of poorly water soluble drugs. It was shown that TPGS can enhance the solubility and bioavailability of poorly absorbed drugs by acting as a carrier in drug delivery systems, thus providing an effective 40    way to improve the therapeutic efficiency and reduce the side effects of the anticancer drugs (Fisher et al., 2002; Youk and Lee, 2005). One of the early discoveries on the solubilizing potential of TPGS is attributable to the work of Ismailos et al. who followed up on the discovery that TPGS can be co-administered with cyclosporine - A resulting in dramatic decrease in the dosage required of this costly drug (Ismailos, 1994). Also, it was found that TPGS improves the solubility of the poorly water soluble drug amprenavir (Yu and Bridgers, 1999). Below the critical micelle concentration, there is no increase in solution. A more recent application involves taxoids which, while important for their chemotherapeutic action, are poorly water soluble and difficult to administer in oral formulation. TPGS is one of the best excipients in which taxoids are soluble. It shows excellent solubilization properties for oral formulation containing paclitaxel and TPGS (Varma and Panchagnula, 2005a). 2.7.4. Agent for Controlled Delivery Applications TPGS can be a good emulsifier or surfactant in fabricating nano/microparticles. TPGS emulsified PLGA nanoparticles fabricated by a modified solvent extraction/evaporation method have narrow polydispersity range from 0.005-0.045 and size around 300-800 nm. TPGS can also achieve emulsification efficiency as the amount of TPGS needed in the fabrication process was only 0.015% (w/w), which was far less than 1% for PVA needed in similar process (Mu and Feng, 2002; Mu and Feng, 2003a). Mu and Feng also found that TPGS could be a good component of the polymeric matrix material in fabrication of PLGA nanoparticles. Feng et al found that nanoparticles coated with TPGS eliminated the side effects caused by human intestinal epithelia cells and cancer cell mortality (Feng and Mu, 2004). The polymeric nanoparticles, in which active agent is dissolved, entrapped, encapsulated, adsorbed, attached or 41    chemically coupled, are an exciting new area of research. Here the co-polymerization of TPGS with a polymer such as PLZ, PCL or PLGA can improve the emulsification efficiency, drug encapsulation efficiency and enhance the cellular uptake of the nanoparticles, thereby increasing the therapeutic effect. These have been demonstrated on microencapsulated paclitaxel (Zhang and Feng, 2006). TPGS is also said to be a more effective and safer emulsifier than PVA with easier usage in fabrication and characterization of polymeric nanospheres for drug delivery (Mu and Feng, 2002). 2.7.5. TPGS – an anti-neoplastic agent TPGS is PEG 1000- conjugates to derivative of α-tocopheryl succinate (TOS) while TOS is a succinyl derivative of vitamin E and has been found to have anticancer properties against leukemia, melanomas, breast, colorectal, malignant brain, lung and prostate cancers (Neuzil and Weber, 2001; Yu and Liao, 2001). TOS differs from other vitamin E derivatives in that TOS itself does not act as an antioxidant (Neuzil, 2002). In xenograft experiments, TOS suppressed tumor growth, both alone and in combination with other anticancer agents (Barnett and Fokum, 2002; Weber and Lu, 2002). The anticancer activity of TOS is mediated by its unique apoptosisinducing properties which appear to be mediated through diverse mechanisms involving the generation of reactive oxygen species (ROS) (Wang and Witting, 2005). These ROS can damage DNA, proteins and fatty acids in cells resulting in apoptotic cell death depending on the strength and duration of ROS generation. It has poor water solubility but its conjugation to PEG makes it water soluble. 42    2.8. Herceptin 2.8.1. Structure and functional aspects of Herceptin Herceptin® (Trastuzumab, HER) is a humanized, monoclonal antibody targeted against the extracellular domain of transmembrane receptor protein (c-erB2/HER2/neu), which is overexpressed in certain types of breast cancer (Nahta and Esteva, 2006; Pohlmann et al., 2009). It has been approved as therapeutic agent of breast cancer by US FDA. The model structure of herceptin is given in Figure 2.10.   Figure 2.10: Structure of Herceptin It consists of two antigen-specific sites that bind to the juxtamembrane portion of the extracellular domain of the HER2 receptor and that prevent the activation of its intracellular receptor tyrosine kinase. The remainder of the antibody is a human IgG with a conserved Fc portion (Cho et al., 2003). Herceptin is presented as a white to pale yellow lyophilized powder and this concentrate has been diluted in solution for infusion. It is being indicated for the treatment of patients with metastatic breast cancer where HER2 is over expressed. 2.8.2. Structure and functional aspects of HER2 receptors HER2 is a 185-kDa transmembrane oncoprotein encoded by the HER2/neu gene and overexpressed in approximately 20 to 25% of invasive breast cancers (Pohlmann et al., 2009). It is a member of the epidermal growth factor receptor (EGFR; also known as ErbB) family of receptor tyrosine kinases, which in humans includes HER1 (EGFR, ERBB1), HER2, HER3 (ERBB3) and 43    HER4 (ERBB4). These receptors comprises of an extracellular region of about 630 amino acids that contains four domains (I/L1, II/CR1, III/L2 and IV/CR2) arranged as a tandem repeat of a two-domain unit, a single membrane-spanning region and a cytoplasmic tyrosine kinase (Hudis, 2007). Binding of ligand to the extracellular region induces receptor dimerization and activation of the cytoplasmic kinase (receptor tyrosine kinase, RTK), which in turn leads to the autophosphorylation and initiation of downstream signaling events. Among all the ErbB receptor family, HER2 has the unusual ability to transform into oncogenic in a ligand – independent manner when overexpressed. The downstream signaling events of this receptor family are shown in Figure 2.11 (Arteaga et al., 2011).   Figure 2.11: Types and functions of HER2 receptors (Arteaga et al., 2011) The signaling cascade following the phosphorylation of RTKs leads to the activation of the PI3K/Akt and the Ras/Raf/MEK/MAP kinase pathways thereby regulating the cell growth, survival and differentiation (Valabrega et al., 2007). Thus at any instance overexpression of 44    HER2 receptor induces oncogenesis in patients characterized by HER2 – positive breast cancer. 2.8.3. Mechanism of action of Herceptin on HER2 Various in vitro and in vivo experimental models have been carried out and observed for the possible effects of herceptin on the regression and inhibition of HER2 overexpressing tumors. The following are some of the proposed mechanism of action of herceptin and an overall scheme was shown in Figure 2.12 (Nahta and Esteva, 2006).   Figure 2.12: Mechanism of action of Herceptin (Nahta and Esteva, 2006) As already mentioned, HER2 activates multiple signaling pathways such as PI3 kinase and MAP kinase cascades leading to cell growth and proliferation. Binding of herceptin on HER2 blocks the ligand binding and disrupts the receptor activation by either homodimerization or heterodimerization. This inhibits further downstream signaling pathways and eventually promotes cell cycle arrest and apoptosis (programmed cell death). The G1 phase of cell cycle is 45    being arrested by reducing the expression of proteins which maintains the sequestration of cyclin-dependent kinase inhibitor p27. This paved the way for p27 to bind and inhibit cyclin E/cdk2 complexes, blocking the cell cycle (Nahta and Esteva, 2006). It was reported that treatment with herceptin reduced the angiogenesis caused by the overexpression of HER2 in human tumor cells. In vitro results showed that there has been reduced endothelial cell migration. This phenomenon was attributed to the increased expression of anti-angiogenic factors and reduced expression of pro-angiogenic factors. In vivo breast cancer models and clinical trials have demonstrated that herceptin is not only cytostatic but also cytotoxic. This ability was related in part to the activation of antibody – dependent cellular cytotoxicity (ADCC). ADCC is mainly due to the activation of natural killer cells (NK) of the immune system, expressing the Fc gamma receptor, which can be bound by the Fc domain of herceptin (Arteaga et al., 2011; Nahta et al., 2006; Valabrega et al., 2007). It was reported that herceptin has the ability to inhibit DNA repair and promotes DNA strand breaks specifically in HER2 overexpressing BT474 and SKBR3 cells. Also it has been shown that herceptin is synergistic with various chemotherapeutic drugs, especially with the DNA damaging drugs via herceptin – mediated inhibition of DNA repair. 46    CHAPTER 3: PREPARATION AND CHARACTERIZATION OF VITAMIN E TPGS COATED AND HERCEPTIN CONJUGATED LIPOSOMES 3.1. Introduction Liposomes are biocompatible and can be used as model cell membranes for elucidating the interactions and functions of proteins with cell membrane. Dipalmitoylphosphatidylcholine (DPPC), a phospholipid and one of the major constituent for biological cell membranes was used to prepare liposomes. Vitamin E TPGS (d-alpha tocopheryl polyethylene glycol 1000 succinate), an amphillic copolymer was incorporated into the lipid bilayer for three main reasons such as 1) to improve the mechanical stability of the bilayer, 2) to enhance the bioavailability and circulation time, 3) as a linker for conjugating herceptin on the surface of liposomes. In this chapter, the focus will be on the preparation methods and characterization of TPGS coated liposomes and herceptin conjugated liposomes. For the latter case, TPGS was reacted with succinic anhydride by the ring opening polymerization mechanism in the presence of DMAP to form succinoylated TPGS, containing carboxyl end groups. All the liposomal formulations used in this study were prepared using solvent injection method. These liposomes are then characterized for their particle size and size distribution (photon correlation spectroscopy, PCS), zeta potential (Zetasizer), surface morphology (transmission electron microscopy, TEM and atomic force microscopy, AFM), surface properties (x-ray photoelectron spectroscopy, XPS), encapsulation efficiency (EE) and in vitro drug release profile. Further, the conjugation of herceptin and the molecular arrangement of succinoylated TPGS inside the lipid bilayer was studied by Fourier transform infrared spectroscopy (FTIR) and differential scanning Calorimetry (DSC) respectively. 47    3.2. Materials Docetaxel of purity 99.56% was purchased from Jinhe Bio-Technology Co. Ltd (Shanghai, China). 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) was a generous gift from Lipoid GmbH (Ludwigshafen, Germany). D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) C33O5H54 (CH2CH2O)23 was from Eastman chemical company (Kingsport, TN, USA). Herceptin (20 mg in 0.95 ml) was offered by Singapore General Hospital, Singapore. Cholesterol, acetone, methanol, ethanol, phosphate buffer saline (PBS), Dichloromethane (DCM), Dimethylaminopyridine (DMAP), SA, Coumarin-6, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Tween-80 was from ICN Biomedicals, Inc. (OH, USA). Clinical formulation Taxotere® was supplied by Aventis Pharmaceuticals, USA. All solvents such as acetonitrile, methanol and ethanol were of high performance liquid chromatography (HPLC) grade. All chemicals were used without further purification. Millipore water was prepared by a Milli-Q Plus System (Millipore Corporation, Bredford, USA). 3.3. Methods 3.3.1. Preparation of succinoylated TPGS Succinoylated TPGS was synthesized by the ring-opening polymerization mechanism in the presence of DMAP as described in our earlier work (Anbharasi et al., 2010). In brief, TPGS (0.77 g), SA (0.10 g) and DMAP (0.12 g) were mixed and allowed to react at 100 ̊ C under nitrogen atmosphere for 24 h. The mixture was cooled to room temperature and taken up in 5.0 ml cold DCM. It is then filtered to remove excessive SA and precipitated in 100 ml diethyl ether 48    at ˗10 ̊ C overnight. The white precipitate was filtered and dried in vacuum to obtain succinoylated TPGS (TPGS-COOH). 3.3.2. Preparation of docetaxel or coumarin-6 loaded liposomes Conventional (non-coated), TPGS and TPGS-COOH coated liposomes were prepared according to the solvent injection method (Muthu et al., 2011). In brief, docetaxel/coumarin-6, DPPC, cholesterol and TPGS or TPGS-COOH (added only in the preparation of coated liposomes) were dissolved in 0.3 ml of ethanol at 60° C, according to the formulae (Table 3.1). Table 3.1: Formulation of liposomes Molar ratio Docetaxel Coumarin-6 (mg) (mg) Batches Compositions DTX-C DPPC:Cholesterol 8:7.7 1.5 - DTX-TP DPPC:Cholesterol:TPGS 8:7.7:1 1.5 - DTX-TPCOOH DPPC:Cholesterol:TPGSCOOH 8:7.7:1 1.5 - CM6-C DPPC:Cholesterol 8:7.7 - 0.3 CM6-TP DPPC:Cholesterol:TPGS 8:7.7:1 - 0.3 CM6-TPCOOH DPPC:Cholesterol:TPGSCOOH 8:7.7:1 - 0.3 DTX: Docetaxel loaded CM6: Coumarin-6 loaded TP: Liposomes prepared with TPGS TP-COOH: Liposomes prepared with TPGS-COOH TP-HER: Herceptin conjugated liposomes prepared with TPGS-COOH 49    The mixtures were injected into 2.7 ml of 1mM phosphate buffered saline (PBS), pH 7.4 isothermally at 60° C. The suspensions formed were kept at 60° C under stirring for 60 min to form multilamellar vesicles (MLV). The small unilamellar vesicles (SUV) were prepared from the MLV suspensions by ultrasonication (Vibra Cell™, 130w, 20 kHz) for 5 min followed by filtration through 0.22 µm filter while keeping the lipids above the phase transition temperature (Tm). Finally, liposomes (SUV) were centrifuged at 11,000 rpm for 15 min to remove the excess non-incorporated drug and stored at 4° C. Liposomes formulae were established to get higher drug encapsulation, controlled drug release and stability (Figure 3.1C and 3.1D). 3.3.3. Preparation of Herceptin conjugated Liposomes Herceptin was diluted in PBS (pH 7.9) to acquire 5 mg/ml concentration as stock solution. Antibody conjugated liposomes were prepared according to a procedure similar to that described by Jun Liu et al (Liu et al., 2007). Briefly TPGS-COOH coated liposomes (0.3ml, 2.5 mg/ml with respect to TPGS-COOH) was diluted in PBS (pH 5.5) and incubated with 200 μl of 100 mM EDC and 200 μl of 25 mM of NHS for 30 min under room temperature with gentle stirring. The activated particles were incubated with 0.5 ml of Herceptin solution for another 30 min at room temperature. The resulting antibody-liposome conjugates were dialyzed in saturated solution of docetaxel/coumarin-6 to make non-sink condition of docetaxel/coumarin-6 while removing the by-products. The size of the dialysis membrane used was 1000 Da. 3.3.4. Characterization of liposome formulations 3.3.4.1. Particle size, polydispersity, zeta potential Size, polydispersity and zeta potential of the liposomes were measured by photon correlation spectroscopy (PCS) using Zetasizer (Nano ZS, Malvern Instruments, Malvern, UK). The 50    liposome samples were analyzed after 50 times dilution with deionized water to a count rate of 100-300 kcps.   Figure 3.1: Schematic diagram of (A) Placebo liposome (B) Docetaxel loaded liposome (C) TPGS-COOH coated liposome (D) Herceptin conjugated liposome 3.3.4.2. Surface morphology The surface properties of TPGS-COOH coated liposomes were visualized by an atomic force microscope (AFM) (Digital 3000 Nanoscope, Santa Barbara, California, USA) under normal atmospheric conditions. Explorer atomic force microscope was set in tapping mode, using high resonant frequency (F0 = 346 kHz) pyramidal cantilevers with silicon probes having force constants of 41 N/m. Scan speed was set at 1 Hz. The sample was diluted 50 times with distilled 51    water and then dropped onto glass slides, followed by vacuum drying during 24 h at 25 °C. The measurements were obtained using AFM image analysis software (Nanoscope 5.30). Surface morphology was also studied by a field emission transmission electron microscopy (FETEM) system (JEM 2010F, JOEL, Japan). Samples of conventional non-coated and TPGSCOOH coated liposomes were prepared by placing one drop on a copper grid and dried under vacuum pressure. The dried liposomes being stained with 1% phosphotungstic acid for 30 s were finally examined. 3.3.4.3. Surface chemistry X-ray photoelectron spectroscopy (Kratos Ultra DLD, Shimadzu, Japan) was used to investigate the surface chemistry of the targeted and non-targeted liposomes. The fixed transmission mode was utilized with a passing energy of 80eV and the binding energy spectrum was analyzed from 0 to 1100 eV. 3.3.4.4. FTIR spectroscopy The Fourier transform infrared (FTIR) spectra for TPGS-COOH coated liposomes and herceptin conjugated liposomes were obtained from the FTIR Spectrophotometer (FTIR 8400S, Shimadzu) for characterizing the surface modification of herceptin on the liposomes. The scanning range was 400-4000 cm-1 and the resolution used was 2 cm-1. 3.3.4.5. Differential Scanning Calorimetry DSC experiments were performed using a Mettler Toledo DSC822 differential scanning calorimeter. Hermetically aluminum sealed pan (40 μl) was filled with approximately 10 mg of sample solution, while an empty pan was used as the reference. The data were collected from -10 52    to 60 °C at a scan rate of 2.5 °C/min. The molecular arrangement of TPGS-COOH and docetaxel within the bilayer was assessed in comparison with the conventional liposomes prepared without TPGS-COOH and docetaxel. 3.3.4.6. Drug/dye encapsulation efficiency The docetaxel encapsulated in the liposomes was measured by HPLC (Agilent LC1100, Agilent, Tokyo, Japan). A reverse-phase HPLC column (Agilent Eclipse XDB-C18, 4.6 × 250 mm, 5 μm) was used. Briefly, 20 μl of drug loaded liposomes were mixed with 80 μl of methanol for liposomes disruption and dissolved in mobile phase consisting of acetonitrile and deionized water (50:50, v/v) to 1 ml. The solution was filtered through 0.45 μm syringe filter before transferred into HPLC vial. The flow rate of mobile phase was set at 1.0 ml/min. The column effluent was detected with a UV/VIS detector at 230 nm. The calibration curve was linear in the range of 50–50,000 ng/ml with a correlation coefficient of R2 = 0.999. The drug encapsulation efficiency was defined as the ratio between the amount of docetaxel encapsulated in the liposomes and that added in the liposomes preparation process. For fluorescent liposomes, the coumarin-6 encapsulation efficiency was determined by the same dilution process as described for the drug-loaded liposomes. The fluorescence was measured by HPLC with a flow rate of 1.3 ml/min mobile phase consisting of acetonitrile/deionized water (60:40 v/v). The excitation and emission wavelength were set at 462 nm and 502 nm, respectively, using a fluorescence detector module. 53    3.3.4.7. Invitro drug release The dialysis bag diffusion technique was used to study the in vitro drug release from the docetaxel loaded liposomes (Muthu et al., 2009). The drug loaded liposomes of a volume equivalent to 25 μg of docetaxel were placed in the dialysis bag (cellulose membrane, molecular weight cut off 1,000 Da), hermetically sealed and immersed into 20 ml of phosphate buffered saline (pH 7.4) containing 0.1% w/v Tween 80. The entire system was kept at 37±0.5 °C with continuous shaking at 100 rpm/min. Samples were withdrawn from the receptor compartment at predetermined time intervals and replaced by fresh medium. Docetaxel has low solubility in phosphate buffered saline (pH 7.4). Therefore, sink conditions were maintained for release studies by adding 0.1% w/v Tween 80 in the release medium. The samples were filtered through 0.45 μm syringe filter before transferred into HPLC vial. The drug content in the samples was determined by HPLC (Agilent LC1100, Agilent, Tokyo, Japan) as described in the drug encapsulation efficiency determination. The drug release profiles were calculated. 3.4. Results and Discussion 3.4.1. Particle size, Polydispersity and zeta potential analysis The mean particle size and polydispersity of docetaxel loaded liposomes were shown in Table 3.2. PCS measurements were undertaken in multimodal analysis to get a true reflection of particle size distribution (Muthu and Singh, 2008). The particle size distribution curves for all the samples were unimodal. The sizes of docetaxel loaded liposomes prepared by the solvent injection method and its population standard deviation were 139.5 ± 1.5, 171.5 ± 1.7, 169.8 ± 0.44 and 191.7 ± 0.6 nm for batches of the drug loaded, conventional liposomes (DTX-C), TPGS 54    coated liposomes (DTX-TP), the TPGS-COOH coated liposomes (DTX-TP-COOH) and the herceptin conjugated liposomes (DTX-TP-HER) respectively. Table 3.2: Particle size, polydispersity, zeta potential and encapsulation efficiency of liposomes. Encapsulation Particle size (nm) Polydispersity Batches (mean ± S.D ) a Zeta potential (mV) efficiency (%) a (mean ± S.Da) (mean ± S.D ) (mean ± S.Da,b) DTX-C 139.5 ± 1.5 0.022 ± 0.005 -2.37 ± 0.28 40.21 ± 0.33 DTX-TP 171.5 ± 1.7 0.232 ± 0.013 -4.88 ± 0.50 44.65 ± 1.1 DTX-TPCOOH 169.8 ± 0.44 0.125 ± 0.008 -27.4 ± 0.50 49.10 ± 0.55 DTX-TP-HER 191.7 ± 0.6 0.161 ± 0.005 14.3 ± 0.5 47.86 ± 0.6 CM6-C 130.4 ± 0.99 0.170 ± 0.003 -3.85 ± 0.960 38.14 ± 0.46 CM6-TP 162.5 ± 0.4 0.203 ± 0.021 -5.22 ± 0.27 49.95 ± 0.43 CM6-TPCOOH 155.1 ± 0.60 0.200 ± 0.006 -15.06 ± 1.1 55.16 ± 0.71 CM6-TP-HER 185.6 ± 2.4 0.216 ± 0.029 7.26 ± 0.08 53.34 ± 0.22 a n=3 b Encapsulation efficiency (%) = (amount of drug or dye loaded in liposomes / amount of drug or dye added during fabrication) x 100 S.D: standard deviation DTX: Docetaxel loaded CM6: Coumarin-6 loaded TP: Liposomes prepared with TPGS TP-COOH: Liposomes prepared with TPGS-COOH TP-HER: Herceptin conjugated liposomes 55    The size of the corresponding coumarin-6 loaded liposomes prepared by the solvent injection method and its population standard deviation were 130.4 ± 0.99, 162.5±0.4, 155.1±0.60 and 185.6±2.4 nm for batches of the coumarin-6 loaded, conventional liposomes (CM6-C), TPGS coated liposomes (CM6-TP), the TPGS-COOH coated liposomes (CM6-TP-COOH) and the herceptin conjugation liposomes (CM6-TP-HER), respectively (Table 3.2). The polydispersity of all the liposomes showed quite narrow size distribution, which is nearer to 0.2. The particle size results indicate that the herceptin conjugation leads to increase the liposome size as compared to TPGS-COOH coated liposomes. This is evident for the conjugation of herceptin over liposomal surface. The zeta potential of all the liposomes were found to be negatively charged except for the herceptin conjugated liposomes. Here, stability of the TPGS-COOH coated liposomes was found to be higher than that of the TPGS coated liposomes owing to the better packing of hydrophobic group of TPGS-COOH into the bilayer of liposomes. Also the negative charge on the TPGSCOOH coated liposomes was due to the presence of carboxylic end groups on the surface (Kocbek et al., 2007). On the other hand, the herceptin conjugated liposomes also has good stability with a positive zeta potential which is attributed to the reduction in the carboxylic group of TPGS-COOH and the positive charge of herceptin, thus confirming the conjugation of herceptin on the surface. 3.4.2. Encapsulation efficiency The drug encapsulation efficiency (EE) of the docetaxel loaded liposomes were 44.65±1.1, 49.10±0.55 and 47.86±0.6% for the DTX-TP, DTX-TP-COOH and DTX-TP-HER liposomes, respectively. Similarly, dye encapsulation efficiency of the coumarin-6 loaded liposomes were 56    49.95±0.43, 55.16±0.71 and 53.36±0.30 % for the CM6-TP, CM6-TP-COOH and CM6-HER liposomes, respectively (Table 3.2). The inclusion of cholesterol to the preparation of liposomes provides a condensed effect with the phospholipids improving the stability and the amount of docetaxel/dye that could be loaded (Zhao and Feng, 2006). This leads to the reduction in the hydrocarbon chain fluidity of the bilayer, keeping the loaded drug/dye intact with them. Coating of TPGS and TPGS-COOH further enhances this effect and especially in the later case, where the stability increased and may be due to better interaction and packing ability of TPGS-COOH with DPPC. This is visualized from the significant increase in the drug/dye encapsulation efficiency of TPGS-COOH coated liposomes when compared with the TPGS coated liposomes. Surface functionalization should not affect the encapsulation efficiency in general as it would decrease the drug diffusion from the liposomes (Sun and Feng, 2009). However the observed EE for the herceptin conjugated liposomes was decreased by 2% of the TPGS-COOH liposomes and considered not a significant loss. This may occur during the conjugation step. 3.4.3. Surface morphology The AFM image of individual DTX-TP-COOH liposome was shown in Figure 3.2A and 3.2B. A smooth surface without any noticeable pinholes or cracks was seen. AFM also revealed that the TPGS-COOH coated liposome was spherical in shape of size below 200 nm, which agrees quite well with the result measured by PCS. Figure 3.3 shows field-emission transmission electron microscope (FETEM) of (A) non-coated liposomes in 200 nm scale, (B) an individual TPGS-COOH coated liposome in 200 nm scale, which revealed that both the conventional liposomes and TPGS-COOH coated liposome (DTXTP-COOH) were spherical in shape (Figure. 3A, 3B). The liposome size as observed by FETEM 57    correlated well with that measured by PCS. Additionally, FETEM images also confirmed the presence of TPGS-COOH coating on the liposomes surface.   Figure 3.2A: AFM 2D image of TPGS-COOH coated liposomes showing a particle with 200 nm scale   Figure 3.2B: AFM 3D image of TPGS-COOH coated liposomes showing a particle with 200 nm scale 58    (A) (B) Figure 3.3: TEM image of (A) non-coated liposomes showing particles with 200 nm scale (B) TPGSCOOH coated liposomes showing a particle with 200 nm scale 3.4.4. Surface chemistry X-ray photoelectron spectroscopy was applied to study the surface chemistry of the docetaxel loaded TPGS-COOH coated liposomes with and without herceptin conjugation. The depth to which the X-ray can penetrate under the surface is about 5-10 nm and thus the peak obtained is for the chemical nature of the surface (Prashant et al., 2010). Figure 3.4 shows the XPS wide scan spectrum of the DTX-TP-COOH and DTX-TP-HER liposomes. In the XPS spectrum of DTX-TP-COOH, there occurred a high intensity of O 1s signal peak at 285.5 eV binding energy position compared to the DTX-TP-HER, which confirmed the presence of oxygen rich carboxyl groups on the surface of the liposomes. This clearly depicts the coating of TPGS-COOH on the liposomes. Also TPGS-COOH does not contain any nitrogen atoms, while herceptin has such atoms in their peptide bond. Therefore nitrogen could be used as a marker to detect the presence of herceptin conjugated on the surface of liposomes. From the XPS spectrum of DTX-TP-HER 59    that there occurred an N 1s signal peak at the 398.7 eV binding energy position and must have come from herceptin (Sun and Feng, 2009), which confirmed their successful conjugation on the liposomal surface.   Figure 3.4: XPS spectra for the herceptin conjugated and TPGS-COOH coated liposomes. Herceptin presence on the surface was confirmed by the presence of N 1s spectra at 397.8eV for conjugated liposomes. The TPGS-COOH coating of liposomes was also confirmed by the presence of O 1s The C 1s core level spectra of DTX-TP-COOH and DTX-TP-HER liposomes were compared in Figure 3.5. There is a prominent O-C=O peak at 288.4 eV for the DTX-TP-COOH liposomes which corresponds to the terminal carboxyl groups present on their surface. This peak decreased considerably for the DTX-TP-HER liposomes and the N-C=O peak at 286.6 eV was enhanced which contributes for the formation of new amide linkages during herceptin conjugation (Wuang et al., 2008). 60      Figure 3.5: C 1s core level spectra of (A) DTX-TP-COOH liposomes (B) DTX-TP-HER liposomes 3.4.5. FTIR and DSC studies The FTIR study was used to establish the chemical modifications occurred on the surface of liposomes due to herceptin conjugation. Figure 3.6 shows the FTIR spectra of DTX-TP-COOH and DTX-TP-HER liposomes. There appeared two characteristic peak at 3300 cm-1 and 1630 cm-1 for the herceptin conjugated liposomes (DTX-TP-HER) which corresponds to the bonded 61    amide stretching vibrations (Arya et al., 2011), thus confirming the successful conjugation of herceptin to the surface of liposome.   Figure 3.6: FTIR spectra for the TPGS-COOH coated liposomes and herceptin conjugated liposomes DSC studies were performed for investigating the effect of incorporation of TPGS-COOH and docetaxel into the lipid bilayer, which consists of DPPC and cholesterol. Figure 3.7 shows the DSC endotherm of DPPC vesicles with cholesterol and DPPC vesicles with cholesterol, docetaxel and TPGS-COOH. There occurred a main phase transition temperature (Tm) at 38.2 ̊ C for the DPPC+cholesterol vesicles which was well agreed with the literature (Zhao et al., 2007). The half height width (HHW) of this peak slightly broadens due to the incorporation of cholesterol into the DPPC vesicles. On the other hand there is a strong shift in the Tm around 27 ̊ C for the vesicles with docetaxel and TPGS-COOH, with the peak being broadened. In principle, 62    the HHW of the main phase transition peak will increase with increasing strength of the molecular arrangement within the bilayer (Chieng and Chen, 2009). With the addition of TPGSCOOH and drug, these molecules act as spacers in the lipid bilayer and lower the attractive forces between lipid hydrocarbon chains, resulting in a destabilization effect on DPPC vesicles. Therefore the hydrocarbon chains can be disturbed more easily by the thermal energy, reaching the liquid crystalline phase at a lower temperature (Chieng and Chen, 2009). This confirms the incorporation of TPGS-COOH and docetaxel into the DPPC bilayer.   Figure 3.7: DSC thermogram of liposomes prepared with (a) DPPC and cholesterol (b) DPPC, cholesterol, docetaxel and TPGS-COOH 3.4.6. In vitro drug release Figure 3.8 shows the accumulated percentage release of docetaxel from the drug loaded TPGScoated liposomes (DTX-TP), the herceptin conjugated liposomes (DTX-TP-HER) and Taxotere® in phosphate buffered saline (pH 7.4) containing 0.1% w/v Tween 80. There is an 63    initial burst release observed for Taxotere within the first 3 hrs, releasing 80% of the drug. The DTX-TP and DTX-TP-HER liposomes however showed exponential release kinetics for more than 1 day without any burst release.   Figure 3.8: In vitro drug release from docetaxel loaded liposomes in phosphate buffered saline (pH 7.4). Bar represent ± S.D (n = 3) After 24 h of dialysis in phosphate buffered saline (pH 7.4), the percentage of docetaxel released from the liposomes were 74, 81.25 and 98.97 % for the DTX-TP-HER, DTX-TP and Taxotere® respectively (Figure 3.8). The effect of surface modification on the drug release was studied. Although there is not much difference in the drug release kinetics of DTX-TP-HER and DTXTP, conjugation of herceptin somehow sustained the release of drug from the liposomes. This may be due to the better interaction and packing of TPGS-COOH with bilayer and further enhanced by the covalent modification by herceptin on the surface. The t50%, at which 50% of the 64    drug encapsulated in the liposomes has released in the phosphate buffered saline (pH 7.4) was about 5.32, 3.48 and 1.55 h for the DTX-TP-HER, DTX-TP liposomes and Taxotere® respectively. The faster rate of drug releases obtained from the TPGS coated liposomes may be explained by the influence of hydrophilic and solubilization property of TPGS on docetaxel (Mu et al., 2005; Sheu et al., 2003). 3.5. Conclusion The vitamin E TPGS coated and herceptin conjugated liposomes were prepared by solvent injection method and characterized for their size and size distribution, surface charge, surface morphology, surface chemistry, drug/dye encapsulation efficiency and in vitro drug release profile. Conjugation of herceptin on the liposome surface was confirmed by Fourier transform infrared spectroscopy. Molecular arrangement of TPGS-COOH and docetaxel with the lipid bilayer was studied using Differential Scanning Calorimetry. The TPGS-COOH coated liposomes showed maximum stability and encapsulation efficiency when compared to TPGS coated liposomes. In vitro drug release study showed better controlled drug release profile for the herceptin conjugated liposomes when compared to non conjugated liposomes and Taxotere®. 65    66    CHAPTER 4: IN VITRO CELLULAR STUDY OF VITAMIN E TPGS COATED AND HERCEPTIN CONJUGATED LIPOSOMES 4.1. Introduction Site specific drug delivery to tumor cells achieves better pharmacokinetics profiles, improved specificity to certain cancer cell type, increased cellular internalization and intracellular delivery and lower systemic toxicity. The key factors involved in tumor targeting by any nanocarriers are the use of proper targeting ligands which targets specific cellular markers on the tumor cells, delivery of proper therapeutic drug for effective treatment and the mode to deliver the drug to the targeted site. Active targeting by ligand conjugation leads to deliver the drug payload specifically to tumors cells through enhanced permeation of their blood capillaries and receptor mediated endocytosis. In this chapter, in vitro cellular uptake, cell imaging using confocal laser scanning microscope and cell viability were studied for herceptin conjugated liposomes and nonconjugated liposomes. Active targeting effect was compared in terms of uptake efficiency and cell viability. In addition, commercial formulation Taxotere® was also used in cell cytotoxicity study to evaluate some of the advantages of using equivalent drug-loaded liposome formulation over the clinical formulation. Human breast adenocarcinoma (SK-BR-3) cell line which over expresses HER2/c-erb-2 gene product was used as a cancer cell model. 67    4.2. Materials Phosphate buffered saline (PBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), penicillin–streptomycin solution, trypsin-EDTA solution and propidium iodide (PI) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Triton X100 was provided by USB Corporation (OH, USA), Fetal bovine serum (FBS) and RPMI media 1640 were purchased from Gibco Life Technologies (AG, Switzerland). SK-BR-3 cells were provided by American Type Culture Collection. Clinical formulation Taxotere® was supplied by Aventis Pharmaceuticals, USA. All solvents such as ethanol, isopropanol and sodium hydroxide (NaOH) were from Sigma-Aldrich. All chemicals were used without further purification. Millipore water was prepared by a Milli-Q Plus System (Millipore Corporation, Bredford, USA). 4.3. Methods 4.3.1. Cell culture In this study, human breast adenocarcinoma SK-BR-3 cell lines (American Type Culture Collection, USA) was cultured in 25 cm2 culture flasks using RPMI supplemented with 10% FBS and 1% penicillin-streptomycin and incubated in SANYO CO2 incubator at 37 °C in a humidified environment of 5% CO2. The medium was changed every 1 - 2 days until confluence was reached. The cells were washed twice with PBS, detached using 0.125% of Trypsin-EDTA solution and centrifuged at 2000 rpm for 5 min to recover the cells for further growth or experiments. 4.3.2. Cellular uptake of liposomes For quantitative study, confluent SK-BR-3 breast cancer cell line (American Type Culture Collection) were harvested with 0.125% Gibco Trypsin-EDTA solution (Invitrogen) and seeded 68    into 96 well assay plates (Corning Incorporated) at 6000 viable cells/well. After the cells reached confluence, the cells were incubated with 100 μl of 5 μg/ml coumarin 6-loaded liposomes (targeted and non targeted) in the RPMI 1640 supplemented with 10% Hyclone fetal bovine serum (FBS, Thermo Scientific) and 1% Gibco penicillin-streptomycin (Invitrogen) at 37 °C for 30 min, 1h and 2 h. At designated time period, the suspension was removed and the wells were washed three times with 50 μl cold PBS. After that, 50 μl of 0.5% Triton X-100 in 0.2 N NaOH was introduced into each well for cell lysis. The fluorescence intensity of each sample well was measured by Tecan microplate reader (GENios) with excitation wavelength at 430 nm and emission wavelength at 485 nm. Cellular uptake efficiency was expressed as the percentage of cells-associated fluorescence after washing versus the fluorescence present in the feed suspension. For the qualitative study, confluent SK-BR-3 breast cancer cells were harvested with 0.125% Gibco Trypsin-EDTA solution (Invitrogen) and seeded in LABTEK® cover glass chambers (Nagle Nunc®) having DMEM at a concentration of 6000 viable cells/chamber. The cells were incubated overnight and were subsequently incubated with coumarin 6-loaded liposomes (targeted and non-targeted) in the RPMI 1640 (concentration of 5 μg/ml) at 37 °C. After 3 h, the cells were washed 3 times with cold PBS and fixed by 75% ethanol for 20 min. Then, the cells were washed twice with cold PBS. The nuclei were stained by incubating with propidium iodide (20 μg/ml) for another 10 min. The cell monolayer was washed three times with PBS and observed by confocal laser scanning microscopy (CLSM) (Nikon A1, Nikon Corporation, Japan) with imaging software, NIS-Element AR 3.0. 69    4.3.3. Cytotoxicity of liposomal formulations Cytotoxicity of docetaxel formulated in the drug-loaded liposomes was investigated by the MTT assay. 100 μl of SK-BR-3 cells were seeded into 96 well plates (Costar, IL, USA) at the density of 5000 viable cells/well with RPMI 1640 and incubated at least overnight to allow cell attachment. The spent medium was discarded and the cells were incubated with docetaxel loaded targeted and non-targeted liposomes and their cytotoxicity was assessed in comparison with Taxotere® at 0.025, 0.25, 2.5 and 25 μg/ml equivalent drug concentration for 24 h. For the competitive effect of herceptin, an excess of free herceptin were incubated with the cells 1 hr prior to the incubation of liposomal formulations. After 24 h, the medium was removed and the wells were washed twice with cold PBS. Following that, 100 μl of MTT solution (0.5mg/ml) prepared in the RPMI 1640 was added to each well of the plate. The plates were further incubated for 3–4 h in the incubator. Finally, MTT in medium was removed and 50 μl of DMSO was added into each well of transformed MTT crystals and the absorbance of the transformed MTT solution in the wells was measured at 450 nm wavelength using a microplate reader. Cell viability was calculated by the followed equation: Cell viability (%) = (ABS of sample/ABS of control) x 100 where ABS of sample is the absorbance of the transformed MTT in cells incubated with the liposomes while the ABS of control is the absorbance of transformed MTT in cells incubated with the culture medium only (positive control). IC50, the drug concentration at which 50% of the cell population in a designated period was destroyed in comparison with that of the control sample, was calculated by regression (curve fitting) of the cell viability data. 70    4.4. Results and Discussion 4.4.1. Cellular Uptake The quantitative cellular uptake by SK-BR-3 breast cancer cells after incubation with the coumarin-6 loaded TPGS coated liposomes (CM6-TP) – non-targeted, herceptin conjugated liposomes (CM6-TP-HER) - targeted and conventional liposomes (CM6-C) - control were shown in Figure 4.1. It can be noticed from Figure 8 that there was no trend of general increase of liposomes uptake by the cells with the incubation time. The absence of time-dependent behavior could be explained by presence of active and maximum endocytosis process within the system on or before 30 min (Gupta et al., 2007). Among the three liposome formulations studied, it was clearly shown that the cellular uptake of the herceptin conjugated liposomes (CM6-TPHER) was significantly higher when compared to the non-targeted liposomes and the control formulations (P < 0.05). It was due to the targeting effect of CM6-TP-HER liposomes, which enhanced the absorption of the liposomes. The confocal images SK-BR-3 cells after incubation with the coumarin-6 loaded TPGS coated liposomes (CM6-TP) and the herceptin conjugated liposomes (CM6-TP-HER) were shown in Figure 4.2. To better compare the intensity of fluorescence among the cells treated with the two types of liposomes, the images were taken under the same imaging parameters such as sensitivity, gain, offset, and laser power constant throughout the cell imaging process. It can be observed that the green fluorescence in the SK-BR-3 cells from the herceptin conjugated liposomes (CM6-TP-HER), was stronger than that of the non-targeted liposomes (the first column). More regions in the cytoplasm were stained in green, implying enhanced uptake of the targeted liposomes (Row 2) (Figure 4.2). 71      Figure 4.1: Cellular uptake efficiency of coumarin-6 loaded liposomes on SK-BR-3 cells after 0.5, 1 and 3 h incubation   Figure 4.2: Confocal laser scanning microscopy images using SKBR-3 cells after 3 h incubation with the fluorescent coumarin-6 loaded (A) TPGS coated liposomes and (B) Herceptin conjugated liposomes. Fluorescein isothiocyanate (FITC) channel showing the green fluorescence from liposomes distributed in cytoplasm in first column. PI channels showing the red fluorescence from propidium iodide stained nuclei in second column and merged channels of FITC and PI in third column. Scale bar = 10 μm 72    4.4.2. Cell Viability In vitro cytotoxicity of the docetaxel formulated in the TPGS coated liposomes (DTX-TP) and the herceptin conjugated liposomes (DTX-TP-HER) were investigated in comparison with Taxotere® at the same equivalent drug concentration on SK-BR-3 breast cells after 24 h incubation at 37 °C and the results are shown in Figure 4.3. It is worthy to note that the docetaxel loaded liposomes achieved the higher cytotoxicity when compared with commercial Taxotere® in all equivalent drug concentration levels applied. This could be due to the effect of controlled docetaxel release from the liposomes (Figure 4.3 and Table 4.1). The docetaxel loaded herceptin conjugated liposomes (DTX-TP-HER) showed higher cytotoxicity compared with the TPGS coated liposomes (Table 4.1). Also, increase in the drug concentration from 0.025 µg/ml to 25 µg/ml resulted in higher cytotoxicity in presence of endocytic mechanism (Wu et al., 2006).   Figure 4.3: Cytotoxicity of docetaxel loaded liposomes on SK-BR-3 cells for 24 h at 37 °C. Cell viability is studied in comparison with commercial formulation Taxotere® 73    Table 4.1: IC50 values of docetaxel formulated as liposomes or Taxotere® for SK-BR-3 cells (n=5) Incubation time (h) 24 Taxotere® 20.23 ± 1.95 IC50 (µg/ml) SK-BR-3 cells DTX-HER + DTX-TP HER 3.74 ± 0.98 0.94 ± 0.37 DTX-HER 0.08 ± 0.4 DTX-TP: Docetaxel loaded liposomes prepared with TPGS (non-targeted) DTX-TP-HER: Docetaxel loaded Herceptin conjugated liposomes (targeted) In order to demonstrate the advantages of the docetaxel loaded liposomes, a quantitative index of inhibitory concentration, IC50, which is the drug concentration required to induce the death of 50% cells incubated in a designated period, was determined. For instance, after 24 h incubation, IC50 for each of the two types of liposomes, namely the DTX-TP-HER, DTX-TP liposomes vs Taxotere® were determined from the cytotoxicity data to be 0.08±0.4, 3.74±0.98, vs. 20.23 ± 1.95 μg/ml, respectively, which imply that the targeted and non-targeted liposomal formulations of docetaxel are 99.65, 81.51 % more efficient than Taxotere® after 24 h treatment, respectively. This indicates that in order to kill the same number of cancer cells, the herceptin conjugated liposomes (DTX-TP-HER) require much lower drug concentration in comparison with the TPGS coated liposomes (Table 4.1). Furthermore the IC50 value for DTX-TP-HER on the cells preincubated with free herceptin was 0.94 ± 0.37 μg/ml which is less cytotoxic when compared to DTX-TP-HER. This was possibly due to the competitive effect of herceptin, which reduced the binding sites available for the herceptin conjugated liposomes by inhibiting the active receptor mediated endocytosis (Gan and Feng, 2010; Wu et al., 2006). However, the cytotoxic effect was more when compared to non-targeted formulation (DTX-TP) due to its synergetic effect with 74    free herceptin. Thus the enhanced cytotoxicity of DTX-TP-HER liposomes can be attributed to their targeting effect towards the HER-2 receptor on the SK-BR-3 breast cancer cells. Most importantly, the herceptin conjugated liposomes could bring high concentration of drug to breast cancer cells and reduce the systemic side effects as efficient and cost-effective drug delivery system for clinical application. 4.5. Conclusion Cellular uptake and cytotoxicity of the herceptin conjugated liposomes formulated with coumarin-6 and docetaxel were investigated in comparison with TPGS coated liposomes and clinical Taxotere®. The uptake efficiency of herceptin conjugated liposomes was higher when compared to non-conjugated liposomes depicting the targeting effect of former. These quantitative results can be confirmed with the images from confocal microscopy. From the IC50 it was observed that the docetaxel loaded herceptin conjugated liposomes could be 97% more efficient than the non-conjugated liposomes after 24 hrs incubation with SK-BR-3 breast cancer cells. The lower IC50 in the order of herceptin conjugated liposomes < non-conjugated TPGS coated liposomes < Taxotere® further summarized the higher therapeutic efficacy of liposome formulations over the commercial drug. 75    76    CHAPTER 5: IN VIVO PHARMACOKINETICS 5.1. Introduction The effect and activity of drug loaded liposomes or a commercial drug tested against in vitro models may sometimes unable to predict its fate in in vivo systems, not to mention clinical cancers. This is due to the much more sophisticated mechanisms that take place within the body compared to the simplified in vitro cell line models. Therefore, research involving animals has been essential to evaluate the formulation for efficacy and safety before proceeding to human trials. In this chapter, in vivo pharmacokinetics for the herceptin conjugated liposomes and clinical Taxotere® was carried out on male SD rats to study the long circulation effect and hence the drug retention effect in the plasma. Generally the TPGS coated liposomes and PEG coated liposomes are said to possess the ‘stealth effect’ which prevents them from capturing by RES cells. Here we have used the PEG coated liposomes from our previous work and compared their drug half-life and clearance rate with herceptin conjugated liposomes and Taxotere®. 77    5.2. Materials Male Sprague-Dawley (SD) rats of 150-200 gm (or 4-5 weeks old) were supplied by the Laboratory Animals Centre of Singapore. They were maintained at the Animal Holding Unit of National University of Singapore. The animal caring, handling, husbandry and the protocols were approved by the Institutional Animal Care and Use Committee (IACUC), Office of Life Sciences, National University of Singapore. The animals were acclimatized at a temperature of 25 ± 2 ºC and a relative humidity of 50 – 60 % under natural light/dark conditions for 4 – 5 days before experiments. Saline 0.9% w/v sodium chloride (NaCl) solution was obtained from B.Braun (B.Braun Corporation, Germany). Rats anaesthesia (Ketamine 24 mg/ml; Medetomidine 0.16 mg/ml) was prescribed by Animal Holding Unit. All solvents including acetonitrile, methanol and ethyl acetate were of HPLC grade. They were used without further purification. 5.3. Methods The animals were randomly distributed into three groups. Group 1 receiving an intravenous (i.v) injection of Taxotere® (n=4). Group 2 receiving an i.v injection of Herceptin conjugated liposomes (n=4). Group 3 receiving an i.v injection of PEG coated liposomes (n=4) and this formulation was used from our previous study (Muthu et al., 2011) for comparing the long circulation characteristics of PEG coated liposomes and herceptin conjugated liposomes. The liposome formulations were dispersed in, and Taxotere® was diluted with saline, and administered at the same Docetaxel dose of 7 mg/kg body weight. All animals were observed for their general condition, clinical signs, and mortality. For Group 1 blood samples were collected at 0.5, 2, 4, 8, 12, and 24 and 48h after administration of Taxotere®. For Groups 2 and 3 blood 78    samples were collected at 1, 2, 4, 8, 12, 24, 48h after administration of the liposomal drug formulations. Plasma samples were harvested by centrifugation at 11,000 rpm for 10 min and stored at - 20 ̊ C for HPLC analysis. Docetaxel in the plasma samples was extracted into 1 ml of diethyl ether and was allowed to evaporate in separate tubes. 100 ml of HPLC mobile phase B (acetonitrile/methanol/water = 40/5/55 v/v/v) was added to the dried tubes, then vortexed and centrifuged at 10 500rpm for 15min. 90 ml of the supernatant was transferred to HPLC vial inserts and 50 ml was injected into the column. An elution gradient was applied by increasing the proportion of A (acetonitrile/methanol/water = 45/5/50 v/v/v) from 0 to 100% in 50 min and then the system was brought to initial condition of 100% B and equilibrated for 4 min by holding at 100% B. The flow rate was 1 ml/min and the total run time was 55 min. The Docetaxel concentration in plasma was determined using the standard curve obtained for known concentrations of Docetaxel in plasma processed similarly. 5.4. Results and Discussion Figure 5.1 shows the concentration of docetaxel in the plasma versus time curve obtained after i.v. injection of Taxotere® and Docetaxel formulated in the herceptin conjugated liposomes (DTX-TP-HER) and PEG coated liposomes (DTX-mPEG) to SD rats at the same Docetaxel concentration of 7 mg/kg (n=4). The key PK parameters were analyzed using Kinetica 5.0 software and the results are listed in Table 5.1, which include AUCtot (ng h/ml) – the total area under the curve which represents the in vivo therapeutic effects, T1/2 (h) – the half-life of the drug in the plasma, i.e. the time at which the drug concentration reached 50% of its peak value in the plasma, MRT (h) – the mean residence time of the drug in the plasma, CLtot (L/h/kg) – the total clearance of the drug from plasma, Vss (L/kg) - volume of distribution at steady state and relative 79    bioavailability (Fr) –which is defined as the ratio of the AUC values between the drug formulation and the commercially available drug injected at the same dose. It was observed from the Figure 5.1 that Taxotere® remains in the plasma with shorter circulation time because of its short half life. On the other hand, both the herceptin conjugated and PEG coated liposomes showed a much longer circulation time. This was due to the so called ‘stealth’ effect of the TPGS-COOH and PEG coating respectively. The long circulation characteristics of the liposomes coated with polyethylene glycol has been reported umpteen number of times in the literature. Docetaxel Concentration in plasma (ng/ml) 100000 Taxotere® DTX‐TP‐HER 10000 DTX‐mPEG 1000 100 10 0 10 20 30 40 50 60 Time (h)   Figure 5.1: Pharmacokinetic profiles of Taxotere®, PEG coated liposomes and Herceptin conjugated liposomes after intravenous injection in male SD rats at a single equivalent dose of 7 mg/kg. (n=4) It can be seen that the half life of DTX-TP-HER liposomes was 43.16 ± 6.26 h and is 1.9 times longer than DTX-mPEG liposomes which has 22.62 ± 3.97 h and 10 times longer than Taxotere® with 4.74 ± 2.15 h. Also the mean residence time for DTX-TP-HER liposomes was 80    50.56 ± 8.13 h which is 2 times and 15 times longer than PEG coated liposomes and Taxotere® respectively. This increased plasma circulation time will provide the herceptin conjugated liposomes with a positive and increased targeting effect (Anbharasi et al., 2010). Moreover, the total AUC for DTX-TP-HER liposomes was 97740 ng.h/ml, which is 2 fold of DTX-mPEG liposomes (48657 ng.h/ml) and 3.5 fold of Taxotere® (28152.1 ng.h/ml) at the same 7mg/kg dose of docetaxel. The total clearance of the DTX-TP-HER liposomes was 0.071 ± 0.009 L/h/kg, which is at a slower rate as compared with PEG coated liposomes and Taxotere®. This was probably due to the effect of TPGS-COOH coating that has slowed down the elimination rate of DTX-TP-HER liposomes and prolonged their circulation in the blood (Win and Feng, 2006). Table 5.1: Pharmacokinetic parameters of Taxotere®, Herceptin conjugated liposomes and PEG coated liposomes after i.v injection at an equivalent dose of 7 mg/kg. PK Parameters Taxotere® DTX-mPEG DTX-TP-HER T1/2 (h) 4.74 ± 2.15 22.62 ± 3.97 43.16 ± 6.26 MRT (h) 3.48 ± 1.88 25.15 ± 4.33 50.56 ± 8.13 AUC 0-inf (ng.h/ml) 28,152.1 ± 2,650 48,657 ± 3,980 97,740 ± 4,220 CLtot (L/h/kg) 0.2486 ± 0.09 0.143 ± 0.023 0.071 ± 0.009 Vss (L/kg) 0.866 ± 0.062 ˗ 3.618 ± 0.98 3.621 ± 1.25 1.72 fold 3.47 fold Relative Bioavailability The volume of distribution at steady state was found to be 3.621 ± 1.25 L/kg for DTX-TP-HER liposomes which is almost same as the PEG coated liposomes (3.618 ± 0.98 L/kg) and 4 times greater than Taxotere® (0.866 ± 0.062 L/kg). The prolonged blood circulation of the herceptin conjugated liposomes and PEG coated liposomes as seen from the mean residence time, allows them to extravasate from the blood compartment and in turn increases their volume of distribution (Dadashzadeh et al., 2008). It has been showed from the table that the relative bioavailability of the docetaxel was increased by 3.47 fold by the DTX-TP-HER liposomes and 81    1.72 fold by the DTX-mPEG liposomes. These results have shown that herceptin conjugated liposomes has better pharmacokinetic parameters than the PEG coated liposomes and Taxotere® and thus provides sustainable and targeted drug delivery. 5.5. Conclusion In this chapter, in vivo pharmacokinetics was studied in male SD rats to evaluate and compare the circulation half-life of the drug from the herceptin conjugated liposomes, PEG coated liposomes and Taxotere®. It was observed that herceptin conjugated liposomes showed longer circulation effect and better pharmacokinetic parameters when compared to the commercial drug and PEG coated liposomes. Thus the TPGS coating on the liposomes has a prolonged circulation half-life over the PEG coating. 82    CHAPTER 6: CONCLUSION AND FUTURE WORKS 6.1. Conclusion The main objective of this thesis is to develop a novel vitamin E TPGS coated liposome formulation conjugated with herceptin for the targeted and sustained delivery of anticancer drug docetaxel. From the results obtained with a series of studies, namely the preparation and characterization of liposomes, in vitro cellular uptake and viability as well as in vivo pharmacokinetics, herceptin conjugated liposomes has been shown as a promising drug carrier for more effective and sustained chemotherapy. In Chapter 1, thesis objectives and a general background of the developmental progress of liposomes and targeted chemotherapy were provided. Then, Chapter 2 highlighted some facts about cancer and its causes, their treatments and problems faced in conventional treatments. In the same chapter, the review on various strategies and nanotechnology applied in developing advanced nanocarrier system were also provided. Chapter 3 contained the preparation and characterization of vitamin E coated and herceptin conjugated liposomes. The size, shape and chemical nature of the liposomes were studied in detail using various state-of-art analytical instruments. Conjugation of herceptin on the liposome surface was confirmed by Fourier transform infrared spectroscopy and x-ray photoelectron spectroscopy. Molecular arrangement of TPGS-COOH and docetaxel with the lipid bilayer was studied using Differential Scanning Calorimetry. The TPGS-COOH coated liposomes showed maximum stability and encapsulation efficiency when compared to TPGS coated liposomes. In vitro drug release study showed better controlled drug release profile for the herceptin conjugated liposomes when compared to non conjugated liposomes and Taxotere®. 83    In Chapter 4, the liposomes were further evaluated using human breast cancer cell line SK-BR-3 as an in vitro model. The uptake efficiency of herceptin conjugated liposomes was higher when compared to non-conjugated liposomes depicting the targeting effect of former. These quantitative results can be confirmed with the images from confocal microscopy. From the IC50 it was observed that the docetaxel loaded herceptin conjugated liposomes could be 97% more efficient than the non-conjugated liposomes after 24 hrs incubation with SK-BR-3 breast cancer cells. The lower IC50 in the order of herceptin conjugated liposomes < non-conjugated TPGS coated liposomes < Taxotere® further summarized the higher therapeutic efficacy of liposome formulations over the commercial drug. Lastly, in vivo pharmacokinetics in Chapter 6 demonstrated the ability of herceptin conjugated liposomes to reduce elimination and clearance rates by circumventing the MPS or other efflux transport systems in the body. Also the TPGS coating proved much better than the PEG coating over the liposomes for enhancing the circulation half-life of the drug. Thus herceptin conjugated liposomes showed greater potential and will satisfy the need for sustained and targeted drug delivery against human breast cancer. 6.2. Future works Further, some of the future works that may improve the current work were included in the following:  Development of xenograft tumor models in mice to further evaluate the anti- tumor efficacy, side effects and clearance rate of the herceptin conjugated liposomes. 84     Loading the liposomes with both therapeutic and imaging agents for multi-functional use such as cancer diagnosis, imaging and treatment at the same time. 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International journal of pharmaceutics 338, 258-266. 101    [...]... administration of herceptin conjugated liposomes achieves 1.9 and 10 times longer half-life respectively than PEG coated liposomes and Taxotere® The relative bioavailability of docetaxel was increased by 3.47 fold by the herceptin conjugated liposomes Thus the herceptin conjugated Vitamin E TPGS coated liposomes showed greater potential for sustained and targeted chemotherapy in the treatment of HER2... non-conjugated TPGS coated liposomes and Taxotere® for targeted chemotherapy on breast cancer cells To facilitate the conjugation of herceptin, carboxyl group terminated TPGS has been synthesized and used in the preparation of herceptin conjugated liposomes Docetaxel or Coumarin-6 loaded liposomes were prepared by solvent injection method and characterized for their size and size distribution, surface charge,... developing liposomal nanocarriers for targeted cancer chemotherapy In Chapter 2, a detailed literature review on cancer and its causes, current treatments available, problems faced in conventional chemotherapy and the concept of different drug delivery formulations were provided Then, Chapter 3 presents the preparation and characterization vitamin E TPGS coated and herceptin conjugated liposomes The... currently available for the different types of cancer Since the discovery of chemotherapeutic drugs for cancer, many challenges have been raised due to the systemic toxicity and adverse side effects caused by these drugs (Feng and Chien, 2003) To address this issue many novel drug carrier systems which have the ability of controlled and targeted/ site-specific drug delivery to the cancer cells have been... diagnosed with cancer during their lifetime, and new cases of cancer are increasing at a rate of 1% per year.3 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 2.2 Cancer prevalence and its causes Cancer may affect people at all ages but in most cases the number of cancer... to a greater extent and heredity causes cancer as well Most cancers develop because of changes (mutations) in genes A normal cell may become a cancer cell after a series of gene changes occur Tobacco use, certain viruses, or other factors in a person's lifestyle or environment can cause such changes in certain types of cells Some gene changes that increase the risk of cancer are passed from parent to... These changes are present at birth in all cells of the body It is uncommon for cancer to run in a family However, certain types of cancer do occur more often in some families than in the rest of the population For example, melanoma and cancers of the breast, ovary, prostate, and colon sometimes run in families Several cases of the same cancer type in a family may be linked to inherited gene changes,... charge, surface chemistry and drug/dye encapsulation xi    efficiency and in vitro drug release profile SKBR-3 cells were employed as an in vitro model for HER2 positive breast cancer and assessed for their cellular uptake and cytotoxicity of the coumarin-6 and docetaxel loaded immunoliposomes respectively The particle size of these liposomes ranged between 140-220 nm High resolution field emission transmission... receptor type 2 (HER2) positive metastatic breast cancers (Liu et al., 2010) HER2 is a member of the EGF receptor (EGFR) family, which is a receptor tyrosine specific protein kinase family consisting of four semi homologous receptors EGFR, HER2, HER3 and HER4 These receptors interact with several ligand and generate intracellular signals either by homodimerization or forming heterodimer pairs The EGFR... similar for the preparation of herceptin conjugated liposomes Following the preparation and characterization of these liposomes, 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 There are six chapters which formed the framework of this thesis The first chapter gave a general background and concepts of ... the herceptin conjugated liposomes Thus the herceptin conjugated Vitamin E TPGS coated liposomes showed greater potential for sustained and targeted chemotherapy in the treatment of HER2 over expressing... 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. .. formulations were provided Then, Chapter presents the preparation and characterization vitamin E TPGS coated and herceptin conjugated liposomes The liposomes were prepared by solvent injection method and