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

IN VITRO AND IN VIVO INVESTIGATION OF NANOPARTICLES OF A NOVEL BIODEGRADABLE COPOLYMER FOR SUSTAINED AND CONTROLLED DELIVERY OF DOCETAXEL GAN CHEE WEE NATIONAL UNIVERSITY OF SINGAPORE 2010 IN VITRO AND IN VIVO INVESTIGATION OF NANOPARTICLES OF A NOVEL BIODEGRADABLE COPOLYMER FOR SUSTAINED AND CONTROLLED DELIVERY OF DOCETAXEL GAN CHEE WEE (B.Eng. (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF ENGINEERING DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2010 ACKNOWLEDGEMENTS First of all, I would like to express my deep appreciation and gratitude towards the following people who have helped me to complete the thesis. A profound thank to my research project supervisor, Professor Feng Si-Shen, for offering an opportunity to me to be a part of his Chemotherapeutic Engineering research group. I want to thank him for his invaluable support, either physically or morally, and all the guidance throughout the course of study. All the professional officers and lab technologists, Mr. Chia Phai Ann, Dr. Yuan Ze Liang, Mr. Boey Kok Hong, Ms. Lee Chai Keng, Ms. Chew Su Mei, Ms. Samantha Fam, Ms. Alyssa Tay, Ms. Dinah Tan, Ms. Li Xiang, Mdm. Priya, Mdm. Li Fengmei, and many other staffs from Laboratory Animal Centre (LAC) who have unconditionally helped in various kinds of administrative works as well as experiments and have willingly shared their knowledge and expertise to further enhance my learning process. My dear colleagues, Dr. Mei Lin, Dr. Sneha Kulkarni, Ms. Sun Bingfeng, Mr. Prashant, Mr. Liu Yutao, Ms. Anitha, Ms. Anbharasi, Mr. Phyo Wai Min, Ms. Chaw Su Yin, Mr. Tan Yang Fei and all the final year students for all their kind assistances and supports they provided. Finally, I am very grateful and appreciative of the scholarship provided by NUS. i TABLE OF CONTENTS ACKNOWLEDGEMENTS i TABLE OF CONTENTS ii SUMMARY vii NOMENCLATURE ix LIST OF TABLES xiii LIST OF FIGURES xiv CHAPTER 1: INTRODUCTION 1 1.1 Background 1 1.2 Objectives and Thesis Organization 4 CHAPTER 2: LITERATURE REVIEW 6 2.1 Definition and Facts 6 2.2 Causes of Cancer 6 2.3 Cancer Treatments and Limitations 7 2.3.1 Problems in Chemotherapy 9 2.3.2 Anticancer Drugs 13 2.3.2.1 Taxanes 14 2.3.2.2 Pharmacodynamics 17 2.3.2.3 Pharmacokinetics 18 2.3.2.4 Toxicology 19 Alternatives of Drug Formulations 21 2.4.1 21 2.4 Liposomes ii 2.5 2.6 2.7 2.4.2 Micelles 24 2.4.3 Dendrimers 27 2.4.4 Prodrugs 29 2.4.5 Nanosphere 32 Fabrication Methods of Nanosphere 35 2.5.1 Emulsion/Solvent Evaporation 36 2.5.2 Solvent Displacement 38 2.5.3 Salting Out 41 2.5.4 Supercritical Fluid (SCF) Technology 44 Roles of Surfactants 47 2.6.1 Drug Carriers 47 2.6.2 Stabilization of Emulsion (Emulsifiers) 48 2.6.3 Targeted Cancer Therapy 50 Vitamin E TPGS 51 2.7.1 Properties of Vitamin E TPGS 51 2.7.2 TPGS as Solubilizer 53 2.7.3 TPGS as Permeability and Bioavailability Enhancer 55 2.7.4 TPGS for Sustained and Controlled Delivery Applications 57 CHAPTER 3: SYNTHESIS AND CHARACTERIZATION OF PLA-TPGS COPOLYMER 61 3.1 Introduction 61 3.2 Materials 62 3.3 Methods 62 iii 3.4 3.5 3.3.1 Synthesis of PLA-TPGS Copolymer 62 3.3.2 Characterization of PLA-TPGS Copolymer 63 3.3.2.1 1H Nuclear Magnetic Resonance (NMR) Spectroscopy 63 3.3.2.2 Gel Permeation Chromatography (GPC) 64 3.3.2.3 Thermogravimetric Analysis (TGA) 64 3.3.2.4 Fourier Transform Infrared Spectroscopy (FR-IR) 64 Results and Discussion 65 3.4.1 1 3.4.2 GPC 67 3.4.3 TGA 68 3.4.4 FT-IR Spectroscopy 68 H NMR Spectroscopy Conclusion 65 70 CHAPTER 4: FABRICATION AND CHARACTERIZATION OF PLA-TPGS NANOPARTICLES 71 4.1 Introduction 71 4.2 Materials 72 4.3 Methods 72 4.3.1 Preparation of PLA-TPGS Nanoparticles 72 4.3.2 Characterization of Drug-loaded PLA-TPGS Nanoparticles 73 4.3.2.1 Particle Size Analysis 73 4.3.2.2 Surface Morphology 73 4.3.2.3 Surface Charge 73 4.3.2.4 Surface Chemistry of Drug-loaded PLA-TPGS NPs 74 iv 4.4 4.5 4.3.2.5 Thermal Analysis of Drug-loaded and unloaded PLA-TPGS NPs 74 4.3.2.6 Drug Encapsulation efficiency 74 4.3.2.7 In Vitro Drug Release 75 Results and Discussion 75 4.4.1 Particle Size and Size Distribution 75 4.4.2 Surface Morphology 78 4.4.3 Surface Charge 80 4.4.4 Surface Chemistry 80 4.4.5 Drug Encapsulation 83 4.4.6 In Vitro Drug Release 85 Conclusion 86 CHAPTER 5: IN VITRO CELLULAR STUDY OF PLA-TPGS NANOPARTICLES 5.1 Introduction 88 5.2 Materials 88 5.3 Methods 89 5.3.1 Cell Culture 89 5.3.2 Cellular Uptake of Nanoparticles 89 5.3.3 In Vitro Cell Cytotoxicity 90 5.4 5.5 Results and Discussion 91 5.4.1 Cellular Uptake 91 5.4.2 Cell Viability 95 Conclusion v CHAPTER 6: IN VIVO PHARMACOKINETICS AND EX VIVO BIODISTRIBUTION 6.1 Introduction 100 6.2 Materials 100 6.3 Methods 101 6.3.1 In Vivo Pharmacokinetics (PK) 101 6.3.1.1 Injection of Drugs 101 6.3.1.2 Blood Collection, Sample Processing and Analysis 102 Biodistribution (BD) 103 6.3.2.1 Injection of Drugs 103 6.3.2.2 Tissue Collection, Sample Processing and Analysis 103 6.3.2 6.4 6.5 Results and Discussion 104 6.4.1 Pharmacokinetics 104 6.4.2 Biodistribution 108 Conclusion CHAPTER 7: CONCLUSION AND FUTURE WORKS 112 113 7.1 Conclusion 113 7.2 Future Works 115 REFERENCES 116 vi SUMMARY Biodegradable polymeric nanoparticle formulation has become an attractive regimen which provides a platform for developing sustainable, controlled and targeted drug delivery system to improve the therapeutic efficacy and reduce the clinical side effects of most antineoplastic drugs. In recent years, amphiphilic biodegradable copolymers consisting of hydrophobic and hydrophilic segments have drawn significant attention from researchers due to the enhancement of drug encapsulation capability as a result of a more stable oil-water suspension during nanoparticle fabrication process. Meanwhile, it has been reported that copolymers could better induce long-circulating ‘stealth’ effect by conjugating to poly(ethylene glycol) (PEG) which could avoid the binding of opsonins, reduce the recognition and elimination by the reticuloendothelial system (RES). Together with small particle size and enhanced permeability and retention (EPR) effect of leaky vasculature, the efficiency of drug delivery to tumor site is improved. D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), an alternative to PEG, is an amphiphilic macromolecule, water-soluble derivative of natural vitamin E. It is an effective emulsifier in nanotechnology for biomedical applications. Co-administration of TPGS can enhance the solubility, cellular internalization, inhibit P-glycoprotein mediated multi-drug efflux transport system, and increase the oral bioavailability of various anticancer drugs. By conjugating TPGS as part of copolymer, its nanoparticle formulation of therapeutic agents can potentially improve the solubility, stability and permeability of drugs. Furthermore, polysorbate 80-associated hypersensitivity reaction and other drug-related toxicities such as cumulative fluid retention, peripheral neuropathy and leucopenia can be reduced. vii In this study, we synthesize a novel amphiphilic PLA-TPGS copolymer with the PLA to TPGS weight ratio of 89:11. The copolymerization was characterized by 1H NMR, GPC, TGA and FT-IR. Following that, the nanoparticle formulations of PLA-TPGS are prepared by a modified single solvent emulsification/evaporation technique with either PVA or TPGS as emulsifier. Characterizations of nanoparticles such as particle size and size distribution, drug encapsulation efficiency (EE), surface morphology, surface charge and drug release profile are done. Generally, particle size of TPGS-emulsified NPs was smaller (~ 240 nm), but with higher EE (up to 85%) and stability, than that of PVAemulsified NPs (~ 270nm with EE ~63%). Drug release profiles of these NPs showed biphasic release with about 5 – 14% initial burst in the first 6 h followed by sustained release of drug up to 79% after 30 days. Human breast adenocarcinoma MCF-7 cell line is employed to assess cellular uptake efficiency of the NPs. TPGS-emulsified NP formulation achieved higher cellular uptake compared to PVA-emulsified NPs. Cytotoxicity evaluation of the NP formulations in vitro showed an order of IC50: Taxotere® > PVA-emulsified NP > TPGS-emulsified NP, suggesting a more effective formulation of TPGS-emulsified NPs. In vivo pharmacokinetics and biodistribution investigation demonstrated longer NPs circulation and therapeutic effect in blood plasma than commercial Taxotere®. Tissue sample analysis from rats injected with NP formulation showed significant decrease of drug accumulation in some important organs, but excluding lungs which is about 2-fold higher than Taxotere®. Nevertheless, NP formulation demonstrated a much better release kinetic with lesser side effects than Taxotere®, thus revolutionizing the way in which cancer is treated while making controlled and sustained cancer chemotherapy feasible. viii NOMENCLATURE ABC ATP-binding cassette ACN acetonitrile ADME absorption, distribution, metabolism and excretion APO E apolipoprotein E AUC area under concentration-time curve BBB blood-brain barrier BCRP breast cancer resistance protein BD biodistribution BEHP-PPV poly[2-(20,50-bis(200-ethylhexyloxy)phenyl)-1,4-phenylene vinylene] C max peak concentration CL plasma clearance CLSM confocal laser scanning microscopy CMC critical micelle concentration CNS central nervous system CTAB cetyltrimethylammonium bromide CyA cyclosporine A CYP cytochrome P450 DCM dichloromethane DMEM Dulbecco’s Modified Eagle Medium DMF N,N’-dimethyl formamide DPPC dipalmitoylphosphatidylcholine ix DSC Differential scanning calorimetry DSPE distearoylphosphatidylethanolamine DTX docetaxel EE encapsulation efficiency EPR enhanced permeability and retention FBS fetal bovine serum FESEM field emission scanning electron microscopy FT-IR fourier transform infrared spectroscopy GAS gas anti-solvent GI gastro-intestinal GPC gel permeation chromatography HCPE hyperbranchedconjugated polyelectrolyte HIV human immunodeficiency virus HLB hydrophile-lipophile balance 1 proton nuclear magnetic resonance H NMR HPLC high performance liquid chromatography HPMA N-(2-hydroxypropyl)methacrylamide HVC hydrophobic vacuum cleaner IC 50 inhibitory concentration at which 50% cell population is suppressed LDL low-density lipoprotein LLS laser light scattering mMRI molecular magnetic resonance imaging x MRT mean residence time MTD maximum tolerated dose MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MPS mononuclear phagocyte system MDR multi-drug resistance NP nanoparticle NSCLC non-small-cell lung cancers PBCA polybutylcyanoacrylate PBD PEG-b-polybutadiene PBS phosphate buffer saline PCL poly(caprolactone) PDI polydispersity index PEE PEG-b-polyethylethylene PEG polyethylene glycol P-gp P-glycoprotein PI propidium iodide PLA poly(lactide) PLGA poly(d,l-lactide-co-glycolide) Ptdlns phosphatidylinositol PVA polyvinyl alcohol PNP PVA-emulsified PLA-TPGS nanoparticles QD quantum dots xi RES reticuloendothelial system RESS rapid expansion from supercritical solution SAR structure-activity relationship SD standard deviation SDS sodium dodecyl sulphate SpD Sprague-Dawley t 1/2 half-life t max time to achieve the maximum concentration (C max) TGA thermogravimetry analysis THF tetrahydrofuran TNP TPGS-emulsified PLA-TPGS nanoparticles TPGS d-α-tocopheryl polyethylene glycol 1000 succinate Tween 80 polyoxyethylene-20-sorbitan monooleate (or polysorbate 80) XPS x-ray photoelectron spectroscopy xii LIST OF TABLES Table 1: Particle size, size distribution, encapsulation efficiency, surface charge of docetaxel-loaded and coumarin 6-loaded PLA-TPGS NPs 76 Table 2: IC50 of MCF-7 cells after 24, 48, 72 h incubation with docetaxel formulated in Taxotere®, PVA- and TPGS-emulsified PLA-TPGS concentrations NPs at various drug 98 Table 3: Mean non-compartmental pharmacokinetic parameters of SpD rats for intravenous administration of Taxotere® and TPGS-emulsified docetaxel-loaded PLATPGS NPs at a dose of 10 mg/kg 107 xiii LIST OF FIGURES Figure 1: Molecular structure of Cremophor EL. 10 Figure 2: Chemical structures of paclitaxel and docetaxel. 15 Figure 3: Packaging of docetaxel in commercial formulation Taxotere®. 16 Figure 4: Molecular structure of polysorbate 80 (or Tween 80). 17 Figure 5: Molecular structures of parent drug docetaxel and its major metabolites. 20 Figure 6: Molecular structure of basic unit (phospholipid) of liposome. 22 Figure 7: Arrangement of lipid bilayer in liposome. 22 Figure 8: Two possible structures of spherical micelles. 25 Figure 9: General structure of polyamidoamine (PAMAM) dendritic molecule. 27 Figure 10: Metabolic process of levodopa to dopamine. 30 Figure 11: Various types of nanocarriers and their respective characteristics. 33 Figure 12: Lipid-polymer hybrid system with a hydrophobic core, lipid interlayer and hydrophilic PEG shell. 34 Figure 13: Solvent displacement as nanosphere fabrication technique. ** represent optional; *** represents only for nanocapsules. 39 Figure 14: Diagram of salting out technique. 42 Figure 15: Roles of PVA in stabilization of nanoemulsion during salting out process for NPs preparation. 43 xiv Figure 16: Simplified scheme of gas anti-solvent precipitation by SCF technology. 46 Figure 17: Molecular structure of d-α-tocopherol (Vitamin E). 52 Figure 18: Molecular structure and various segments of TPGS. 52 Figure 19: Ring-opening polymerization reaction in the synthesis PLA-TPGS. 63 Figure 20: 1H-NMR spectra of the TPGS, lactide monomer and PLA-TPGS. 66 Figure 21: Gel permeation chromatogram of TPGS monomer and PLA-TPGS copolymer. 67 Figure 22: TGA thermogram of TPGS monomer and PLA-TPGS copolymer. 68 Figure 23: FT-IR spectra of TPGS, lactide monomer and PLA-TPGS copolymer. 69 Figure 24: FESEM images of docetaxel-loaded TPGS-emulsified PLA-TPGS NPs. 78 Figure 25: FESEM images of docetaxel-loaded PVA-emulsified PLA-TPGS NPs. 79 Figure 26: FESEM images of coumarin 6-loaded TPGS-emulsified (left) and PVAemulsified (right) PLA-TPGS NPs. 79 Figure 27: XPS C1s envelope of PLA-TPGS copolymer and unloaded PLA-TPGS NPs (without using emulsifier). 81 Figure 28: XPS wide scan spectra of docetaxel-loaded TPGS-emulsified (TNP) and PVA-emulsified (PNP) PLA-TPGS NPs. 82 Figure 29: DSC curves of pure docetaxel, docetaxel recovered from emulsification, docetaxel-loaded PLA-TPGS NPs, unloaded PLA-TPGS NPs and a mixture of docetaxel/unloaded NPs. 83 Figure 30: In vitro drug release profiles of docetaxel-loaded PLA-TPGS NPs using TPGS and PVA as emulsifier. Data represent mean ± SD (n=3). 86 xv Figure 31: MCF-7 cell uptake efficiency of TPGS-emulsified (TNP) and PVAemulsified (PNP) coumarin 6-loaded PLA-TPGS NPs at 100, 250 and 500 µg/ml incubated at 37°C. Data represent mean ± SD (n=6). 92 Figure 32: Confocal laser scanning microscopy (CLSM) of MCF-7 cells after 2 h incubation with 250 µg/ml coumarin-6-loaded TPGS-emulsified NPs (Row A), PVAemulsified NPs (Row B) and free coumarin-6 (Row C) at 37.0 °C. The cells were stained by propidium iodide (Red channel, column 2) and the coumarin-6-loaded PLATPGS NPs are green in color (Green channel, column 1). 94 Figure 33: Viability of MCF-7 breast cancer cells incubated with docetaxel-loaded TPGS- or PVA-emulsified PLA-TPGS NPs in comparison with that of Taxotere® at different docetaxel concentrations after 24, 48 and 72 h. Data represent mean ± SD (n=6). 97 Figure 34: In vivo pharmacokinetics profiles of plasma drug concentration versus time after i.v. administration of Taxotere® and TPGS-emulsified PLA-TPGS nanoparticles formulation using SpD rats (n=5) at the same docetaxel dose of 10 mg/kg. 105 Figure 35: Biodistribution of docetaxel delivered by commercial Taxotere® and PLATPGS NPs to SpD rats at 1, 5, 10 and 24 h after i.v. administration at the same docetaxel dose of 10 mg/kg (n=3). 109 Figure 36: Biodistribution of docetaxel delivered to the brain by commercial Taxotere® and PLA-TPGS NPs to SpD rats at 1, 5, 10 and 24 h after i.v. administration at the same docetaxel dose of 10 mg/kg (n=3). 111 xvi CHAPTER 1: INTRODUCTION 1.1 Background There has been a sustained interest during recent years in developing localized and sustained treatment for cancer and other fatal diseases such as cardiovascular restenosis. Biodegradable polymeric carriers have become a promising platform for sustained, controlled and targeted drug delivery to improve the therapeutic effects and reduce the side effects of the otherwise unprotected drug (Kataoka et al., 2001; Farokhzad and Langer, 2006; van Vlerken et al., 2007). The challenge lies in the polymeric materials selection and the engineering of the nanoparticulate systems that are specifically taken up by targeted cancer cells and subsequently release their drug payload at a plasma concentration within the therapeutic window of the drug for a prolonged period in order to achieve anti-tumor response (Gref et al., 1994; Langer, 2001; Ferrari, 2005). Efficient chemotherapy requires that the anticancer drug concentration in the blood be maintained between the minimum effective level and the maximum tolerable level for a sufficiently long period. It has been reported in the literature that ‘stealth’ nanoparticles with surface modification by poly(ethylene glycol) (PEG) could avoid being recognized and eliminated by the reticuloendothelial system (RES) and thus remain longer in the blood circulation system (Gref et al., 1994; Bazile et al., 1995; Feng et al., 2007; Terada et al., 2007). Nanoparticles of biodegradable polymers are made up of natural or synthetic macromolecules, which are compatible with human body (biocompatibility) and degradable in physiological condition into harmless byproducts. While delivering the therapeutic agent to the diseased cells, the polymeric matrix degrades and is eventually 1 metabolized and eliminated from the body. The degradation rate depends on the physicochemical properties of the polymers, which are determined and adjustable by their compositions, molecular structures as well as molecular weights. Hence, nanoparticle formulation of therapeutic agents can improve their solubility, permeability, stability and therapeutic effects with reduced side effects (Torchilin, 2006). A wide range of U.S. FDA-approved biodegradable polymers such as poly(lactide) (PLA), poly(d,l-lactide-co-glycolide) (PLGA) and poly(caprolactone) (PCL) polyesters are initially designed for application in textile grafts, surgical stents or implants. Although they are biocompatible, their strong mechanical strength, extremely slow degradation rate and difficulty in further modification due to hydrophobic nature have limited their use as drug delivery devices for cancer therapy. Moreover, nanoparticles made up of those polymers are limited to be directly conjugated to hydrophilic molecular probes for targeting, in which amphiphilic linker molecules are usually needed, causing complications for the targeting procedures (Debotton et al., 2008). Generally, two strategies have been developed to solve this problem. One is to coat the nanoparticles by amphiphilic polymers and another is to synthesize copolymers to incorporate hydrophilic elements into the hydrophobic chains so that the system will be more stable thermodynamically in oil-water suspension during nanoparticle fabrication process (Kataoka et al., 2001; Feng et al., 2006). D-α-tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS or simply, TPGS) is one of the potential candidates. TPGS is an amphiphilic macromolecule with hydrophile-lipophile balance (HLB) 13. The chemical structure of TPGS is similar to that of other amphiphiles, comprising lipophilic alkyl tail and hydrophilic polar head portion. Its bulky structure and large surface area make it an 2 effective emulsifier in the nanoparticle technology for biomedical applications, which can result in high drug encapsulation efficiency and high cellular internalization (Mu and Feng, 2003; Win and Feng, 2006). Moreover, it has been found that co-administration of vitamin E TPGS could enhance the therapeutic effects, inhibit P-glycoprotein mediated multi-drug resistance, and increase the oral bioavailability of anticancer drugs (Amass et al., 1998; Soppimath et al., 2001). Docetaxel is a poorly water-soluble semi-synthetic taxane analogue commonly used in the treatment of breast cancer, ovarian cancer, small and non-small cell lung cancer, prostate cancer, etc. Pre-clinical studies demonstrated that docetaxel had several advantages over paclitaxel (Jones, 2006). Compared with paclitaxel, docetaxel showed wider cell-cycle bioactivity, greater affinity for the β-tubulin binding site and greater uptake with slower efflux from the tumor cells, resulting in longer intracellular retention time and higher intracellular concentrations (Riou et al., 1992; Riou et al., 1994; Brunsvig et al., 2007). It was reported that docetaxel exhibited 12-fold cytotoxic activity than paclitaxel and docetaxel showed higher growth inhibition in human epidermal growth receptor (HER2) positive cells compared to paclitaxel (Riou et al., 1992; Hanauske et al., 1994; Lavelle et al., 1995). In clinical trials, docetaxel demonstrated superior efficacy versus paclitaxel in a randomized Phase III study, which directly compares docetaxel and paclitaxel at approved dose and schedule (Jones et al., 2005). Its commercial formulation Taxotere® is formulated in high concentration of Polysobate 80 (Tween 80), such as 40mg/ml which has been found to be associated with severe side effects including hypersensitivity reactions, cumulative fluid retention, nausea, mouth sores, hair loss, peripheral neuropathy, fatigue and anemia and has shown incompatibility with the common PVC intravenous 3 administration sets (Gelderblom et al., 2001; Immordino et al., 2003; Baker et al., 2004). Therefore, to avoid the application of Tween 80-based adjuvant and to increase the drug solubility, alternative formulations have been attempted, which include liposomes (Immordino et al., 2003), nanoparticles (Musumeci et al., 2006; Cheng et al., 2007), docetaxel-fibrinogen-coated olive oil droplets (Engels et al., 2007), nanoparticle-aptamer bioconjugates (Farokhzad et al., 2006). Among them, the nanoparticle formulation showed advantages such as greater stability than others during storage. Furthermore, such a colloidal system is able to extravasate solid tumors into the inflamed or infected site, where the capillary endothelium is defective (Barratt, 2000; Brigger et al., 2002). Nanoparticles could also reduce the multi-drug resistance faced by many anticancer drugs, including docetaxel, by internalization mechanism of drug-loaded nanoparticles such as endocytic process (Panyam and Labhasetwar, 2003; Bareford and Swaan, 2007). Meanwhile, they also reduce drug efflux from cells mediated by the P-glycoprotein (Brigger et al., 2002). This motivates us to combine the advantages from TPGS by synthesizing PLA-TPGS copolymers for various potential biomedical applications, including formulation of imaging agents for cellular and molecular imaging and targeted drug therapy (Zhang et al., 2007; Pan and Feng, 2009). 1.2 Objectives and Thesis Organization In this thesis, we focus on the formulation of PLA-TPGS nanoparticles encapsulating anticancer drug docetaxel for prolonged chemotherapy treatment. At the same time, the effect of different emulsifiers such as TPGS and PVA on characteristics of PLA-TPGS nanoparticles is studied. Other than that, a series of cell works involving cancer cell lines 4 as well as animal models are included to evaluate the formulation before it is tested in clinical trials. The first chapter of this thesis is to provide a general background and concepts of developing nanoscale device for cancer chemotherapy. Next, Chapter 2 provides a detailed review on the current progress in related fields of drug delivery. Some examples and results from journals are cited for the benefit of the readers. The rationale behind the strategies of is also clearly explained in this chapter. Then, Chapter 3 presents the synthesis and characterization of PLA-TPGS amphiphilic copolymer of the optimized 89:11 PLA:TPGS component ratio. Following that, Chapter 4 includes the nanoparticle preparation and characterization. The docetaxel-loaded PLA-TPGS NPs are prepared by a modified single emulsion solvent evaporation/extraction technique with either PVA or TPGS as emulsifier, which are then characterized in such aspects as particle size and size distribution, drug encapsulation efficiency, surface morphology, surface charge and drug release profile. In vitro cellular study is reported in Chapter 5. Human breast adenocarcinoma MCF-7 and human colon cancer HT-29 cell lines are employed to assess cellular uptake of the NPs as well as to evaluate the cell viability of the NP formulations, which is done in close comparison with Taxotere®. In Chapter 6, in vivo pharmacokinetics and biodistribution using Sprague-Dawley (SpD) rats is investigated to further confirm the advantages of the PLA-TPGS NP formulation versus the pristine drug. Finally, conclusion and suggestions for future work are provided in Chapter 7, following by Chapter 8 which contains all the reference papers cited in this thesis. 5 CHAPTER 2: LITERATURE REVIEW 2.1 Definition and Facts Cancer is the leading cause of death globally. According to US National Cancer Institute, cancer is defined as diseases in which abnormal cells undergo uncontrolled growth (or mitosis) and have to ability to invade other tissues of the body through the blood circulation and lymphatic systems (http://www.cancer.gov/cancertopics/what-is-cancer). One among three people will be diagnosed with cancer during their lifetime, and new cases of cancer are increasing at a rate of 1% per year (http://news.bbc.co.uk/2/hi/health/3444635.stm). Currently, more than 200 types of cancer have been discovered, with probability of getting cancer being distinct in different types of tissues or organs, even within the same individuals. 2.2 Causes of Cancer There are many causes for cancer. Generally, they can be subdivided into two categories, namely intrinsic and extrinsic factors. Intrinsic factors mainly include the genetic make up of the body, which cannot be controlled by the individuals. For example, one may experience a number of genetic mutations, which ultimately lead to cancer, once he or she is born. These mutations are basically inherited from previous generations, abnormal fertilization or improper fetal growth during pregnancy. Although mutations may not always result in cancer, research has shown that women with genetic predisposition in breast cancer genes such as BRCA1 and BRCA2 can have a very high risk of developing breast cancer in their lifetime (http://www.cancerhelp.org.uk/help/default.asp?page= 6 119#genetic). However, extrinsic factors play an even more essential role in determining the development of cancer. Extrinsic factors consist of a wide variety of causes, ranging from environmental factors to the personal daily lifestyle practiced by the individuals. Diet that we consume everyday directly influences the risk of getting cancer. Preservatives such as nitrosamine, nitrosamide and sulphites as well as colorings which are usually added during food processing can potentially accumulate in the body and cause cancer (http://www.cfsan.fda.gov/~dms/fdpreser.html;http://www.nswcc.org.au/editorial.asp?pag eid=2345). Concerns are equally given to genetically-modified (GM) food as well as food rich in methyl donors as some research reports show that too much such food may potentially trigger genetic mutations, causing tumor growth (Watters, 2006; http://www.independent.co.uk/life-style/health-and-wellbeing/health-news/suppressedreport-shows-cancer-link-to-gm-potatoes-436673.html). On the other hand, about 70% of cancer deaths took place in low to middle income nations, where there is lack of knowledge and resource on how to prevent and diagnose cancer, as pointed out by World Health Organization (http://www.who.int/cancer/modules/en/). In addition to that, some habits such as smoking, drinking, unhealthy work-life balance are major factors causing cancers. For instance, more than 38,000 people are diagnosed with lung cancer every year, with almost 90% of deaths from lung cancer are due to tobacco (http://info.cancerresearchuk.org/cancerstats/types/lung/?a=5441). 2.3 Cancer Treatments and Limitations Some of the common treatments available to cancer are surgery, chemotherapy, radiation therapy, immunotherapy, monoclonal antibody therapy and gene therapy. Each method 7 has its advantages and disadvantages, and depends on the physiology of the individuals as an effective treatment strategy in one person may fail in another. Surgical removal of tumors from cancer patients is usually the first consideration in cancer treatment. This is especially the case when the tumor size is large and starts to damage the functionality of the tissues or organs surrounding it. Unfortunately, surgery has a few drawbacks. Firstly, surgery is an invasive method of cancer treatment with potential wound infection. And, it can only be done when the tumor is sufficiently large to be removed. Secondly, for patients with medical history such as haemophilia, it may not be advisable to undergo such procedure. Thirdly, surgery can sometimes trigger the metastasis of tumor, even it is successfully removed (Weiss and DeVita, 1979). Radiotherapy is also another primary treatment modality in which ionizing radiation is used to destroy cancerous tissues. However, this method is only applicable to localized tumor such as prostate cancer and recurrence of cancer also occurs in some patients (De Riese et al., 2002). Therefore, a combination of surgery and radiotherapy will usually have immediate local response in terms of tumor cell death. But, it is not effective in controlling re-growth and metastatic secondary tumor growth (Camphausen et al., 2001; Chen et al., 2006). Meanwhile, hormone therapy is restricted to organ-confined cancers such as breast and prostate cancer and long term treatment of metastatic tumor using this method is unlikely (Corral et al., 1996; De Riese et al., 2002). Immunotherapy, by stimulating the immune 8 system through general or specific immune enhancement, only renders a low success rate to patients (Chen et al., 2006). Chemotherapy, often used in combination with other treatment modalities, is the treatment of diseases or cancers using chemical agents or antineoplastic drugs. These chemical agents, which are usually very toxic, can inhibit the tumor growth. But they can also kill the normal, healthy cells, and thus bring unwanted side effects. Nowadays, various kinds of anticancer drugs are available in the market. Some examples include paclitaxel, chlorambucil, fluorouracil, methotrexate and doxorubicin. The cytotoxic mechanisms of chemotherapeutic agents differ from each other, depending on the nature of the drugs, the molecular structure, physicochemical properties and the sites of actions in the body. 2.3.1 Problems in Chemotherapy The common problem with most antineoplastic drugs is their poor solubility in aqueous phase. Paclitaxel, for example, is highly hydrophobic with a solubility of less than 0.5 mg/L in water (Feng and Chien, 2003; Hennenfent and Govindan, 2006). This is not desirable because the drug has to be dissolved in blood, with water as the major component, in order to be transported to the cancer cells. Therefore, solubilizers or adjuvants are necessary to increase the solubility of anticancer drugs. It is also this reason why most of the current commercial drug formulations are only able to be administered intravenously (infusion). Routes of administration are thus limited. In Taxol®, the commercial formulation for paclitaxel, Cremophor EL is applied as the adjuvant (Hennenfent and Govindan, 2006; Xie et al., 2007). Cremophor EL, a nonionic surfactant, consists of polyethoxylated castor oil and dehydrated ethanol (1:1 v/v). Although 9 Cremophor EL is a vehicle for various hydrophobic pharmaceutical agents including cyclosporine and diazepam, it has been found to cause serious adverse effects to patients. Biologic effects such as hypersensitivity, nephrotoxicity and peripheral neuropathies are believed to have associated with the use of Cremophor EL in the formulation (Theis et al., 1995; Gelderblom et al., 2001; Hennenfent and Govindan, 2006; Feng et al., 2007). The molecular structure of Cremophor EL is shown below (Aliabadi et al., 2005): Figure 1: Molecular structure of Cremophor EL. Secondly, human body will normally treat most anticancer drugs as foreign substances which the body cannot recognize. As a result, the native drugs administered into the body will greatly be subjected to the degradation by some endogenous enzymes or macromolecules which are considered as part of the body natural defense mechanism and immune system. The first-pass metabolism is an important process that takes place in liver and intestine before the drugs are absorbed into the circulatory system (Feng et al., 2007). It is the physiological barrier to be crossed before the drugs can be distributed to other parts of the body. The most common kind of enzyme involved in this degradation of drugs is cytochrome P450 (or CYP), mainly located in liver and intestine. CYP is a large family of hemoproteins which consists of 18 families and 43 subfamilies and it contributes to nearly 75% of total metabolic process in human body (Nelson et al., 1993; Danielson, 10 2002; Guengerich, 2008). It is found on the membrane of endoplasmic reticulum as well as mitochondria. However, most members from CYP1, CYP2 and CYP3 families take part in drug metabolism (Guengerich, 2008). For instance, almost 80% of administered docetaxel, an anticancer drug popular for its efficacy towards various types of cancer, is metabolized by CYP3A4 through hepatic transformation (Baker et al., 2006; BradshawPierce et al., 2007). Besides that, there are other systems which act as barriers to hamper the effective absorption of drug in the body. Protein such as P-glycoprotein (or P-gp) is a ATP-binding cassette (ABC) transporter encoded by MDR1 gene and is well known for its drug efflux mechanism (Ling, 1997; Béduneau et al., 2007). Because it has the capability of removing various toxic substances from cells over-expressing P-gp in such organs as liver, kidney, and small intestine, cellular multi-drug resistance (MDR) is developed (Thiebaut et al., 1987). In addition to CYP, it is the presence of P-gp in the lower gastro-intestinal (GI) tract and other multidrug resistance proteins (MRP) , such as MRP 1-5 and breast cancer resistance protein (BCRP), that usually cause the low oral bioavailability of most antineoplastic drugs (Malingré et al., 2001; Schinkel and Jonker, 2003; Varma et al., 2003; Varma and Panchagnula, 2005). Moreover, it has been reported that the synergistic effect between P-gp and CYP3A4 could further speed up the first-pass elimination of drugs in intestinal enterocytes (Schuetz et al., 1996; Lown et al., 1997; van Asperen, 1997; Varma et al., 2004). Therefore, oral chemotherapy at home is still not feasible until a very novel, stable and sustained drug formulation emerges. Also, P-gp is greatly over-expressed in capillary endothelium of blood vessels lining the central nervous system (CNS), which together make up the blood-brain barrier (BBB), leading to the failure of chemotherapy to 11 brain cancer due to restricted permeability of drugs to tumor sites (Béduneau et al., 2007; Pardridge, 2007). Another reason causing the clearance of drugs once they are present in physiological system is the high probability of binding to endogenous proteins in the circulatory system. The high-binding affinity of most commercial formulations to plasma proteins reduces the amount of free drug required for the treatment at the targeted sites (Rawat et al., 2006). In fact, this protein-binding process, especially for hydrophobic drugs, is spontaneous and is part of the opsonization process. In this case, the exogenous drugs will be considered as a foreign material (antigen), which promotes the binding of opsonins (immunoglobulins, laminin and C-reactive proteins, for example) and will eventually be recognized and taken up by phagocytes. This mononuclear phagocyte system (MPS), which involves macrophages (located in tissues and organs such as liver, spleen, lung and lymph nodes) and monocytes (found in blood stream), is also classified as the reticulo-endothelial system (RES) of the immune mechanism (Müller et al., 1997; Hume, 2006; Owen and Peppas, 2006). As a result, sustainability of the drugs is affected. For a drug formulation to be effective, solubility, stability and permeability of drugs are the three basic criteria that must be fulfilled in order to achieve successful chemotherapy. Unfortunately, sudden exposure of the body to certain level of drug dosage for certain time interval is usually an effective way in classical chemotherapy for cancer treatment. However, we must also consider the severe side effects due to the abrupt increase in concentration of cytotoxic drugs in the blood plasma because most commercial drugs not only kill cancer cells, but also the healthy cells. Low amount of cisplastin, a 12 chemotherapeutic agent that cross-links DNA to retard its replication in tumor, can cause serious systemic toxicity to patients if the dosage administered is not properly monitored (Sumer and Gao, 2008). Hence, the drugs must not only reach the desired site of action and remain accumulated at the site for sufficient period of time, the desired rate of drugs being exposed to the patients at certain time must be considered. In fact, controlled release and specificity of drugs has become the major factors in designing novel formulations for cancer therapy using state-of-the-art bio- and nano-technology. 2.3.2 Anticancer Drugs There are various kinds of drugs commercially available in the market for cancer chemotherapy. In generally, all these anticancer drugs are categorized into few groups, depending on the way or mechanism by which the drugs act on the cancer cells. Some of them include alkylating-like agents, anti-metabolites, anthracyclines and alkaloids. Cisplatin, an alkylating-like agent with a structure of cis-Pt(NH3)2Cl2, is used to treat cancers such as small cell lung cancer, colon cancer, ovarian cancer and sarcomas. It contains platinum in the molecular structure. The cytotoxic effect of cisplatin is mainly contributed from the platinum complexes which can bind and interact with the basic sites of DNA, resulting in DNA crosslinking (Lippert, 1999). When the DNA is unable to replicate, apoptosis is induced leading to cell death. However, low water solubility, low lipophilicity, serious toxicity and rapid inactivation restrict its clinical application (Chupin et al., 2004). Chlorambucil, another alkylating-like agent which can be taken orally, is often used for treatment of chronic lymphocytic leukemia. 13 Examples of anthracyclines are daunorubicin and doxorubicin which have been the effective chemotherapeutic agents for breast cancer, leukemic cells, myeloma cells and so on. It is naturally produced by Streptomyces strain of bacteria (Lomovskaya et al., 1999). This type of drug is believed to intercalate into DNA, thus preventing the growth of cancer cells due to the inhibition of enzymes helicase and topoisomerase II which are essential in DNA transcription and cell mitosis (Fornari et al., 1994). Another mechanism of action is the generation of oxygen free radicals that damage the cell membrane. The main side effects occur especially to the heart include congestive heart failure and arrhythmias. Alkaloid is a general group of natural compounds which contain basic nitrogen atoms in the molecular structure. Two sub-groups of alkaloids that have the antitumor capability are vinca alkaloids and taxanes. The mechanism of action of these drugs is to interfere with the microtubule function in a cell cycle (Cutts, 1961; Kruczynski et al., 1998). While vinca alkaloids such as vindesine and vinorelbine can inhibit the assembly of microtubule by reducing the rate of tubulin addition, taxanes have the opposite effect, inhibiting the disassembly of microtubules during mitosis. 2.3.2.1 Taxanes In the past few decades, research has shown that taxanes could be promising chemotherapeutic agents because of effective single-agent activity such as high response and patient survival rates in a broad spectrum of advanced carcinoma (Bunn and Kelly, 1998). And taxanes are currently being widely used in oncology. The most common taxanes are paclitaxel and docetaxel. They are diterpenes and their molecular structures are different only at a few side chains as shown in Figure 2. 14 Figure 2: Chemical structures of paclitaxel and docetaxel. (Source: Mortier et al., 2005) Taxanes are originally isolated from natural source of plants of genus Taxus. For instance, paclitaxel is derived from the bark of Pacific yew tree (Taxus brevifolia). While it is not feasible to synthesize paclitaxel from economic point of view, semi-synthetic analogue is one of the possible solutions to the limited availability of yew trees (Feng and Chien, 2003). Docetaxel is a new generation of and an alternative to paclitaxel. It is a semisynthetic form of taxane which is derived from a renewable non-cytotoxic compound, 10deacetyl baccatin III, extracted from the needles of European yew tree (Taxus baccata) (Ringel and Horwitz, 1991; Denis et al., 1998). 15 In commercial formulation, paclitaxel developed by Bristol-Myers Squibb Company is packaged under the trade name Taxol®, usually in a product concentration of 6mg/ml with Cremophor EL as the adjuvant (Figure 1). Meanwhile, docetaxel in its commercial formulation Taxotere® is developed by the pharmaceutical company Sanofi-Aventis. The packaging of Taxotere® is shown in Figure 3. The concentration approved is 40mg docetaxel per mL of polyoxyethylene-20-sorbitan monooleate (polysorbate 80 or Tween 80) (Figure 4). This high drug concentration is to be mixed with 13% ethanol in saline solution and is further diluted with 250 mL of 0.9% sodium chloride (or 5% glucose) before clinical administration through infusion. Figure 3: Packaging of docetaxel in commercial formulation Taxotere®. Both paclitaxel and docetaxel has been proven by U.S. Food and Drug Administration (FDA) to be clinically effective in the treatment of a wide range of local or metastatic malignancies such as ovarian, breast, head and neck and non-small-cell lung cancers (NSCLC) (Eisenhauer and Vermorken, 1998). On the other hand, they are also effective 16 against melanoma, Kaposi’s sarcoma (KS) and some digestive system-related cancers (Eisenhauer and Vermorken, 1998; Dubois et al., 2003). Figure 4: Molecular structure of polysorbate 80 (or Tween 80). Similar to paclitaxel, the cytotoxic nature of docetaxel is due to the ability to perturb the cell mitosis. Microtubules, a component of cytoskeleton, have a function of correctly segregating chromosomes during cell division. When the binding and stabilization of microtubules by docetaxel happens, microtubules are unable to depolymerize or disassemble into free tubulin. As a result, late G2 and early M phases of the cell cycle are blocked and division fails (Gelmon, 1994; Huizing et al., 1995). Eventually, apoptosis takes place. Although docetaxel is the analogue to paclitaxel, there is significant difference between the pharmacodynamics and pharmacokinetics of the two drugs. 2.3.2.2 Pharmacodynamics At molecular level of pharmacodynamics, docetaxel has shown about 1.9-fold greater binding affinity to ß-tubulin. Docetaxel also has a wider cell cycle bioactivity. It has been 17 reported that docetaxel exerts its cytotoxic effect on cells undergoing S, G2 and M phases of a cell cycle, compared to only G2 and M phases in the case of paclitaxel (Gligorov and Lotz, 2004). And, because cell apoptosis induced by docetaxel is through the phosphorylation of bcl2, a protein required to inhibit cell death, apoptotic pathway is activated with 100-fold lesser drug concentration than that of paclitaxel (Haldar et al., 1997). Moreover, docetaxel has greater cellular uptake and slower drug efflux from tumor cells than paclitaxel, thus leading to higher efficacy and longer drug retention time at tumor sites (Riou et al., 1994). 2.3.2.3 Pharmacokinetics As mentioned earlier, both taxanes are mainly distributed and metabolized in the liver, especially by CYP3A4 and CYP3A5 isoenzymes (Royer et al., 1996; Baker et al., 2006; Bradshaw-Pierce et al., 2007). A major fraction of drugs are also distributed to spleen, intestine and plasma proteins. Meanwhile, about 80% of the dose is excreted through feces and about 6% is eliminated renally (Marlard et al., 1993). However, if compared to paclitaxel, docetaxel demonstrates a linear pharmacokinetics and elimination half-life behaviors over 1 hour after drug administration. This would imply that any adjustment on the dosage given to patients could give a proportional outcome in terms of area under concentration-time curve (AUC) and peak concentration (Cmax) (Bissery et al., 1991; Gligorov and Lotz, 2004; McGrogan et al., 2008). Hence, unlike non-linear pharmacokinetics of paclitaxel, it becomes easier to predict the various important parameters describing the pharmacokinetics of docetaxel under different treatment schedules. 18 2.3.2.4 Toxicology Docetaxel shares some common side effects as paclitaxel such as neutropenia, neuropathy and myalgia (Gligorov and Lotz, 2004; McGrogan et al., 2008). However, there is difference between these side effects in terms of grade of toxicity. Toxicities such as neutropenia, leukopenia and fluid retention are most common severe side effects experienced by patients treated with docetaxel, while those for paclitaxel is more dosedependent (Hurria et al., 2006). On the other hand, heart-related toxicity is milder for docetaxel. Cardiac toxicity is more often observed when patients are given paclitaxel/anthracycline combination chemotherapy, probably due to the drug-drug interaction phenomena (Gligorov and Lotz, 2004). This may further highlight one of the advantages of using docetaxel as a single-agent chemotherapeutic drug in cancer therapy. Another obvious difference between paclitaxel and docetaxel is the more serious hypersensitivity and anaphylaxis reactions which are believed to be triggered by the Cremophor EL used as an adjuvant in the Taxol® formulation as mentioned earlier. Polysorbate 80 is thought to have a lower systemic exposure compared to Cremophor EL, therefore potential side effects attributed to polysorbate 80 is expected to be lessened, even though low hypersentivity reaction such as hypotension has been reported (Bissery, 1995; ten Tije et al., 2003; McGrogan et al., 2008). Fortunately, this can be further alleviated by applying anti-histamines and corticosteroids (Schrijvers et al., 1993). Additionally, the level or grade of toxicity of docetaxel itself differs, relying on the dose and sequence in which the drug is given. For instance, a 3-week-schedule therapy of docetaxel often causes fluid retention, myelosuppression, skin and nail disorders. 19 Whereas, weekly dosing results in different toxicity profiles such as less hematologic and neurologic toxicities but with higher level of asthenia (Burstein et al., 2000). Other nonhematologic toxicities commonly associated with docetaxel include diarrhea, dyspnea, hallucination, hair loss and infection (Bissett et al., 1993; Hurria et al., 2006). Some of the major docetaxel metabolites are shown in Figure 5. Metabolite Parent Drug Side Chain A B C and D Figure 5: Molecular structures of parent drug docetaxel and its major metabolites. The parent drug is metabolized by hepatic transformation through oxidative reactions to form primary alcohol (A), which is subsequently transformed into an oxazolidinedione (B) as well as two hydroxyoxazolidinones (C and D) (Monegier et al., 1994). In vitro cell viability has shown that the cytotoxic effect of all the metabolites formed after metabolism 20 of docetaxel is significantly reduced and negligible in comparison to its parent drug (Sparreboom et al., 1996). 2.4 Alternatives of Drug Formulations In view of the high rate at which cancer is developed and diagnosed in people around the world each year, various kinds of alternatives to more effectively deliver the chemotherapeutic agents have been discovered and developed since the past few decades. Together with the problems faced in traditional ways in which cancer patients are treated, this regimen has even become more and more popular in recent years. The development of new nanoparticulate drug formulations is no longer restricted only to chemistry and pharmacy, but is a multi-disciplinary area involving technologies from material science, life science, biology as well as chemical and biomedical engineering fields. This new dimension of delivery system, combined with the nanotechnology, could provide a new hope to better treat the cancer in a more effective and comprehensive way. Some of the novel therapeutic devices are described in the following sections. 2.4.1 Liposomes Liposomes are spherical vesicles which are made of one or a few natural phospholipid bilayers. Phospholipid bilayer consists of two monolayer lipids with the hydrophobic tails being assembled in a way that is protected and surrounded by outer layers of hydrophilic heads of the same amphiphilic lipid molecules. It has a thickness of about 5 – 6nm (http://www.azonano.com/Details.asp?ArticleID=1243). Examples of the molecular structure of phospholipids and liposome are shown in Figure 6 and Figure 7 below. 21 Figure 6: Molecular structure of basic unit (phospholipid) of liposome. (Source: http://www.uic.edu/classes/bios/bios100/lecturesf04am/lect02.htm) Figure 7: Arrangement of lipid bilayer in liposome. Size of liposome is generally dependent on several factors such as fabrication process conditions and the composition of the lipids forming the layers of liposomes. Liposome with single lipid layer (small unilamellar vesicles or SUV) can be as small as 25nm while some types of liposomes with more concentric lipid bilayers such as large unilamellar vesicles (LUV) or multilamellar vesicles (MLV) can have sizes up to several microns (Rawat et al., 2006). However, Straubinger and Balasubramanian have demonstrated that MLV undergoing extended high-energy sonication resulted in drug-loaded liposomes of size 25 – 35nm in diameter (Straubinger and Balasubramanian, 2005). 22 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 phospoholipids, low immunogenicity and reversal of multidrug resistance are other advantages of using liposomes as promising drug delivery devices (Thierry et al., 1992; Warren et al., 1992; Torchilin, 2005). Also, liposomes as carriers offer some interesting properties. It can be customized to encapsulate different kinds of agents to suit certain applications. For hydrophilic compounds such as quinine dihydrochloride which is commonly used for the treatment of celebral malaria, the drug can be entrapped within the aqueous core of liposome. At the same time, for most hydrophobic chemotherapeutic drugs, they can be located at the inner membrane of lipid bilayer. In this way, the agents can be protected from the harsh physiological conditions after administration. Liposome can be a long-acting formulation to the conventional free drug. Example of FDA-approved liposomal formulation Doxil®, which entraps doxorubicin, has been clinically used since 1995 for the treatment of refractory Kaposi’s sarcoma, ovarian and breast cancers (Torchilin, 2005; Farokhzad and Langer, 2006). The promising future of liposome has even encouraged formulation such as vincristine-encapsulated liposome to advance into Phase III clinical trials, before the formulation is approved by U.S. FDA for non-Hodgkin’s lymphoma treatment (Waterhouse et al., 2005). Unfortunately, there are a few drawbacks which could limit the application of plain liposomes. These include premature drug leakage, relatively low drug loading compared to other types of nanocarriers, instability in blood circulatory system and inconsistent 23 reproducibility of liposomal characteristics (Gabizon et al., 2003; Immordino et al., 2003; Torchilin, 2003). This leads to serious problem in predicting the behaviors and potential outcomes of liposomal treatment, thus amplifying the difficulty in studying the efficacy of the formulation. More advanced types of liposomes are under research to further target the liposomes to tumor site. For 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). Further, polymersome, an analogue to liposome, was developed. Instead of the natural lipid bilayer, diblock copolymers such as PEG-bpolybutadiene (PBD) and PEG-b-polyethylethylene (PEE) are used to establish the polymer bilayer of polymersome (Discher et al., 1999; Bermudez et al., 2002). Despite having a higher PEG surface density, which contributes to longer blood circulation time than its counterpart liposome, some of these polymers are extremely slow in biodegradation (Photos et al., 2003). 2.4.2 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 lipophilic regions (tail group) of the surfactant will arrange in such a way that the core of the 24 micelles containing the drug is excluded and protected from the surrounding aqueous phase by the hydrophilic head group. This arrangement is termed oil-in-water micelle. In contrast, colloidal structures entrapping water-soluble drugs are referred to as water-in-oil micelles (or inverse micelles). They are illustrated in Figure 8 below: Figure 8: Two possible structures of spherical micelles. In recent years, micelles formed from amphiphilic diblock or triblock copolymers have gained popular attention in nanomedicine research. The reason is that physiologicalfriendly building blocks of copolymers can be chosen to construct 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). Another advantage of polymeric micelles is the narrow size distribution. This is because the self-assembled 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 (Lawrence, 1994; Jones and 25 Leroux, 1999; 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 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 (Hubbard, 2006p; Rawat et al., 2006). One creative solution from Prabaharan et al. is to synthesize unimolecular micelles inheriting the characteristic of dendrimers by using hyperbranched 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 26 trigger dissociation and the release of adriamycin from polymeric micelles, thus increasing the probability of cytoplasmic toxicity to cancerous cells (Bae et al., 2005). 2.4.3 Dendrimers Another unique type of nanoscale device is dendrimers, which impart important changes over the approaches applied to solving current problems in cancer diagnosis and therapy. The macromolecular and three-dimensional globular structures of dendrimers are due to branching of repeating units (generations) around an inner central core (Figure 9). And, the synthesis and development of dendrimers were reported to be able to start from inner core outwards by Tomalia’s divergent method or from outer branch inwards by Fréchet’s convergent method (Tomalia et al., 1986; Hawker and Fréchet, 1990). Dendrimers based on monomers such as ethylene glycol, melamine, propyleneimine and lysine are some of the common types. Figure 9: General structure of polyamidoamine (PAMAM) dendritic molecule. 27 Like other nanocarriers, dendrimers have been found to carry certain essential biomedical values in oncology. The interstitial space of the central core allows the containment of anti-neoplastic drugs, stimuli-responsive or imaging agents. Meanwhile, the therapeutic and diagnostic agents can also be simultaneously physically adsorbed or chemically conjugated to the peripherals expressing multiple functional groups, hence realizing the potential of dendrimer as multifunctional delivery device. Furthermore, different surfacemodified termini are able to attach to various kinds of targeting moieties to increase the probability of active diffusion through specific tumor binding (Muthu and Singh, 2009). This enables the dendrimers to be used as antiviral agents by inhibiting gp120 proteins, for example, of human immunodeficiency virus (HIV), which otherwise binds to normal healthy cells through CD4 receptors (McCarthy et al., 2005). Monodispersity of size and shape is another important feature of dendrimers. It brings not only convenience to the study of effect of parameters such as size, composition and surface properties on the pharmacodynamics and pharmacokinetics of the formulation, but also consistency in tuning the dendrimers to obtain the quality required for more personalized treatment (Wolinsky and Grinstaff, 2008). For example, structurallycustomized dendrimer by Lee et al. bearing pH-sensitive linkage with 10 wt% doxorubicin molecules on one side of dendritic hemisphere and PEG chains on another shows tumor regression and 100% survival rate over a period of two months (Lee et al., 2006). With this feature, therefore, various optimizations and modifications can be done to fine-tune targeted delivery of drugs. 28 Nevertheless, there is concern about the interaction between dendrimers and the cell membrane. Relying on the active functional end groups on the surface of dendrimers, positively-charged surface has potential disruptive effect of hole formation to the phospholipid membrane of cells compared to negatively-charged or neutral dendrimers. Besides that, size (generations) of the dendrimers is also a key factor in determining the biocompatibility of the dendritic devices (Wolinsky and Grinstaff, 2008). Large and positively-charged amine-coated melamine dendrimers, for instance, are found to induce in vivo hemolytic toxicity compared to non-cytotoxic neutral PEGylated melamine dendrimers, thus raising the questions on suitability of dendrimers as delivery devices (Chen et al., 2004). Therefore, the mechanistic and chemical understandings of the dendrimer’s architecture have to be well-studied when designing more biocompatible and versatile dendritic systems. 2.4.4 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 (Vyas et al., 1993; Nielsen et al., 1994; Testa and Caldwell, 1996; Stella et al., 1998). 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. 29 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, 4-dihydroxy-phenylalanine) (Figure 10), 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 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 10: Metabolic process of levodopa to dopamine. 30 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 (Kopecěk, 1990; Veronese et al., 2005; Duncan et al., 2006). 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 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). 31 2.4.5 Nanosphere Nanosphere is often categorized as one type of the nanoparticle family, with another being nanocapsules. The main difference between the two members of this group of nanocarriers lies in the core structure and architecture of the nanoparticles. It is determined by the method of preparation. For nanospheres, the colloidal particles have phase-separated solid matrix core where the dissolved encapsulants are dispersed or bound within the dense matrix caused by the mutual interaction, intercalation and folding between the polymeric chains (Gref et al., 1994; Gref et al., 1995; Soppimath et al., 2001; Amellar et al., 2003; Rawat et al., 2006). On another hand, nanocapsules consist of an oily central cavity, in which the drugs are entrapped, with the cavity surrounded by single layer of polymeric shell (Allémann et al., 1993; Puglisi et al., 1995; Muthu and Singh, 2009). Some examples of the oil phase partitioned into the core include benzyl benzoate, ethyl oleate, medium chain triglycerides (MCT), soybean oil (Fessi et al., 1989; Ammoury et al., 1990; Ammoury et al., 1991; Santos Magalhaes et al., 1995; Quintanar-Guerrero et al., 1998). A brief summary of the some nanocarriers and their characteristics is shown in Figure 11. Most of the time, the nanoparticle sizes of nanospheres are relatively larger than micelles with higher polydispersity, because the colloidal suspension formed is also strongly dependent on various parameters during fabrication process rather than the thermodynamic of the system alone (Kwon, 1998). However, polymeric nanospheres are usually not significantly affected by the dissociation effect which is a major concern for the micellar formulation as the rapid dilution in blood could cause micelles to dissociate into their free molecular chains. Hence, this feature of nanospheres allows a greater control over the pharmacokinetic and biodistribution of the formulation. 32 Figure 11: Various types of nanocarriers and their respective characteristics. (Adapted from Letchford et al., 2007) In recent years, extensive research on nanospheres has been done in view of their promising abilities in delivering therapeutic or diagnostic agents. As a breakthrough step towards more advanced cancer therapy, diblock or even triblock copolymers consisting of hydrophilic PEG segment were developed to solve some, if not all, of the problems faced in conventional chemotherapy such as poor solubility, rapid elimination due to various physiological barriers (e.g. first-pass metabolism, low intestinal absorption, blood-brain 33 barrier) and untargeted delivery. MePEG-b-PLA, MePEG-PCL, MePEG-polyanhydride, PEG-PIBCA, Tween 80-PLA multiblock copolymers were synthesized to encapsulate drugs such as paclitaxel, insulin, vaccines, oligonucleotides and so on (Allen, 1994; Gref et al., 1994; Peracchia et al., 1997; Peracchia et al., 1997a; Zhang and Feng, 2006). Modifications of nanospheres through combining the characteristic of nanospheres and liposomes have also been demonstrated. For instance, lipid-polymer hybrid nanoparticles such as PLGA/DSPE-PEG/DLPC-PEG blends and PLGA/lecithin/DSPE-PEG blends containing certain surface-active functional groups for targeting purpose were reported to enhance delivery of drug to the targeted sites of action (Chan et al., 2009; SalvadorMorales et al., 2009; Liu et al., 2010). An example of the orientation and architecture of the hybrid nanoparticles is illustrated in Figure 12. Figure 12: Lipid-polymer hybrid system with a hydrophobic core, lipid interlayer and hydrophilic PEG shell. (Adapted from Chan et al., 2009; Salvador-Morales et al., 2009) Furthermore, application of nanospheres in cancer diagnosis and imaging has shown a promising future. One significant improvement in molecular magnetic resonance imaging 34 (mMRI) is the use of polymer-coated iron oxide nanoparticles to replace conventional gadolinium-based T1 contrast agent. By coating iron oxide nanoparticles with polymeric materials, iron oxide nanoparticles becomes highly water-solubilized, with the potential to target the tumor sites using targeting moieties to locate specific cell type for imaging, diagnostic as well as local treatment through hyperthermia (Okassa et al., 2007; Chen et al., 2009; Pouponneau et al., 2009; Shi et al., 2009). Other than using inorganic substances as imaging agents, more novel nanospheres comprising organic conjugated polymers, such as hyperbranchedconjugated polyelectrolyte (HCPE) and poly[2-(20,50-bis(200- ethylhexyloxy)phenyl)-1,4-phenylene vinylene] (BEHP-PPV), which are semi-conductors with photo- and electroluminescent properties have been synthesized for live cell and even in vivo fluorescent imaging (Howes et al., 2009; Pu et al., 2009). This can be another alternative to replace the encapsulation of contrast agents or quantum dots (QD) which can potentially release toxic elements such as cadmium ions and free radicals (Derfus et al., 2004; Hoshino et al., 2004). 2.5 Fabrication Methods of Nanosphere The type of nanoparticles formed and the methods of fabrication depend mainly on the block copolymer composition as well as their respective block length which makes up the whole amphiphilic block copolymers. Taking copolymer with the same molecular weight of hydrophobic and hydrophilic chains as an example, the type of nanoparticles most likely to be obtained is micelles. If it is the case, methods such as direct dissolution, dialysis or film casting can be employed, relying on the physicochemical properties of the copolymer and the features of final nanoparticles required (Letchford and Burt, 2007). The same concept applies to nanospheres in which the polymeric chains are dominated by 35 hydrophobic blocks. The following sections will introduce some of the methods of developing nanospheres containing the therapeutic drugs required for certain treatment purposes. 2.5.1 Emulsion/Solvent Evaporation Also interchangeably termed solvent extraction/evaporation, this is a simple and common method of encapsulating most hydrophobic or water-insoluble chemotherapeutic agents such as aclarubicin and paclitaxel (Wada et al., 1988; Xie et al., 2007). In brief, the polymeric material and drugs are often dissolved in one or a mixture of water-immiscible organic solvents. One example of the organic oil phase is made up of dichloromethane, which has a strong dissolution power for many hydrophobic drugs while a low solubility in water aqueous phase (approximately 1 – 2% (m/m) at 25°C). Other common solvents are ethyl acetate and chloroform. When the organic oil phase is dispersed in the aqueous phase, oil-in-water (o/w) emulsion is formed. The drugs are then dispersed and encapsulated within the polymeric chains, which could be hydrophobic polymer such as PLGA polyester or amphiphilic diblock copolymer such as PLA-PEG. These polymers are partitioned and self-assembled between the o/w interface to stabilize the oil droplets. Most of the time, surfactants are also applied to further stabilize the emulsion and prevent coalescence so as to achieve minimal free energy state of the system. When the organic solvent is removed by extraction to a continuous phase or evaporation into a gas phase, the polymer-drug suspension will subsequently harden, forming solid particles (Vrancken and Claeys, 1970; Wang and Schwendeman, 1999; Albayrak, 2005). Modified emulsion/solvent evaporation has also been done by many studies through coupling of ultrasonication technique to generate ultra-fine, nanoscale droplets to reduce the size of 36 nanospheres to about 100 – 200 nm for the application in nanomedicine (Li et al., 2003; Sahoo et al., 2004; Xu et al., 2005). With the same principle, hydrophilic or ionic drugs such as peptides and proteins can also be encapsulated in polymeric materials using water/oil/water (w/o/w) double emulsion method (Thies, 1991; Crotts and Park, 1995; Lee et al., 2007). In this technique, watersoluble compounds to be encapsulated are firstly dispersed in an organic phase containing the polymers. The primary emulsion is subsequently dispersed in another aqueous phase to create secondary emulsion. The hardened nanoparticles are formed after the same solvent removal step as for single w/o technique. Although emulsion technique is widely used in drug-encapsulated micro- or nanosphere fabrication, the insight of the overall system is complicated and dependent on various experimental conditions and parameters, ranging from the polymer concentration as well as amount of surfactants and stirring speed to the high power of sonication. Among the various important parameters, solvent removal rate has been found to have significant influence on final physicochemical characteristics of nanospheres prepared by solvent evaporation method (Arshady, 1991; Crotts and Park, 1995; Li et al., 1995; Jeyanthi et al., 1996). For instance, Crotts and Park (1995) demonstrated that the faster the rate of removal of DCM from the PLGA polymeric layer solution into continuous phase as well as the larger mean particle diameter obtained was related to the more porous surface of the formed microspheres when a higher inner aqueous volume was included during w/o/w procedure. Meanwhile, for a highly porous surface morphology, the loading efficiency of the entrapped drug is usually found to be lower, most probably as a result of drug loss 37 through these pores during the processing steps. Hence, by controlling emulsion viscosity and hardening speed of polymer through changing the dispersed phase-to-continuous phase ratio in a system, the physicochemical properties such as pore size, drug loading, diameter, and degradation rate can be modified (Li et al., 1995). It is worth taking note that when various parameters in emulsion/solvent evaporation come into play, the control and optimization of the nanoparticle fabrication process become sophisticated. This renders the challenges in high reproducibility of final particle qualities and scale-up process for large scale production (Wang and Schwendeman, 1999). 2.5.2 Solvent Displacement Although nanoparticle is often referred to as a broad category of nanoparticulate system comprising of nanospheres and nanocapsules, they have different structures and spatial distribution of materials, as already discussed in previous section. While interfacial deposition of polymers is only applied to form nanocapsules, solvent displacement, also known as nanoprecipitation, is a common fabrication method of nanosphere and nanocapsules (Letchford and Burt, 2007). In contrast to using water-insoluble solvent in emulsion method, nanoprecipitation is done by dissolving the polymer and hydrophobic drug in a water-miscible organic solvent, which is then mixed with aqueous phase (Figure 13). 38 Figure 13: Solvent displacement as nanosphere fabrication technique. ** represent optional; *** represents only for nanocapsules. (Adapted from Reis et al., 2006) When the solvent containing the polymer and drug is added, often in a drop-wise manner, to the large volume of aqueous phase (non-solvent), the solvent will immediately diffuse out and mix with the water phase. The diffusion then induces interfacial turbulence and subsequently the deposition of the polymer onto the solvent/water interface, thus causing the precipitation of the polymeric chains with the drug being entrapped within the matrix of the nanospheres. In order to speed up the formation of the colloidal suspension, moderate stirring together with a small amount of surfactant are usually required during mixing. Nanoprecipitation has been widely used to obtain various common drug-loaded polymeric nanospheres. These include nanospheres of PLA and PCL homopolymers, PLGA as well as MPEG-PLA diblock and PLGA-PEG-Aptamer (Apt) triblock copolymers (Molpeceres et al., 1996; Némati et al., 1996; Barichello et al., 1999; Dong and Feng, 2007; Gu et al., 2008). One of the advantages of nanoprecipitation is the ability to achieve small particle sizes of 200 nm or less (Fonseca et al., 2002; Reis et al., 2006). In some cases, a size of as 39 small as 80 nm with a narrow size distribution and a moderate encapsulation efficiency of 22-48% can be obtained (Dong and Feng, 2004). The very small size and distribution not only render consistency over the manipulation of nanoparticle behavior, but also enable vascular extravasation and accumulation of drugs at the tumor sites (Yuan et al., 1994; Monsky et al., 1999; Hao et al., 2005; Torchilin, 2006). Ease of preparation without the need of high energy of sonication is another simplification of this technique. The most difficult part of this technique is the optimization of the solvent/non-solvent as well as the polymer/drug combination. Essentially, the solvent in which the polymer and drug are dissolved has to be miscible with the aqueous phase so that mutual diffusion of the solvent/non-solvent can take place. As a result, this technique is only limited to the encapsulation of lipophilic drugs such as cyclosporine and indomethacin (Allémann et al., 1998; Barichello et al., 1999). Other works using nanoprecipitation method for poorly water-soluble drugs involved antifungal bifonazole and clotrimazole (Memişoğlu et al., 2003; Reis et al., 2006). On the other hand, poor entrapment efficiency of more hydrophilic drugs valproic acid (5.6%), phenobarbital (9.4%) and vancomycin (12,1%) due to drug leakage during PLGA nanoparticle formation process has also been reported, with most of the drug being found adsorbed on the surface of the particles (Barichello et al., 1999). Important parameters such as polymer concentration, type of solvent, solvent/water ratio and drug loading have also been comprehensively studied by some researchers. For example, the higher the concentration of polymer in the solvent, the larger the particle size because the increase of organic phase viscosity slows down the rapid diffusion rate of 40 solvent into the aqueous phase, thus creating a resistance to the precipitation of polymeric chains (Mosqueira et al., 2000; Roy Boehm et al., 2000; Chorny et al., 2002). However, the encapsulation efficiency was found enhanced (Dong and Feng, 2004). Apart from that, the effect of solvent type on particle size was studied by Cheng et al. (Cheng et al., 2007). They reported that when a solvent is more miscible with water, such as N,N’-dimethyl formamide (DMF) and acetone, the Hildebrand solubility parameters difference (Δδ) between the two is relatively smaller than those of acetonitrile and tetrahydrofuran (THF), resulting in smaller particle size due to the readily dispersed solvent/polymer solution system in water. 2.5.3 Salting Out Initially, salting out is a process which is extensively used in chemical or biological industry to purify industrial products. It is a separation step, especially for proteins, when high temperature processing is not feasible for heat-sensitive compounds. In recent years, salting out has found its applications in nanotechnological fields to produce nanoparticles for biomedical applications. In salting out technique as shown in Figure 14, the polymer and drug pre-dissolved in an organic phase is dispersed into a saturated electrolyte (calcium chloride and magnesium chloride) or non-electrolyte (sucrose) solution containing a stabilizer such as PVA or polyvinylpyrrolidone (Galindo-Rodriguez et al., 2004; Reis et al., 2006). By using the high concentration salt solution, the solvent is inhibited from diffusion due to the high affinity of the salt ions to attract more water molecules than the solvent (salting out effect) (Galindo-Rodriguez et al., 2004; Wischke and Schwendeman, 2008). The emulsified 41 polymer/drug solution in the concentrated salt solution is then diluted with an excess of water. During the mixing step, the water-miscible organic phase is extracted into the aqueous phase. The water-insoluble polymer and the encapsulants are unable to be extracted, thus forming precipitates as solid nanoparticles. Figure 14: Diagram of salting out technique. (Adapted from Reis et al., 2006) As in other nanoparticle fabrication methods discussed above, various factors can influence the outcome of the nanoparticles. These include the type and concentration of salting agent used to create emulsion, concentration of stabilizer, polymer concentration, organic solvent and the solvent/water ratio. Taking salting agent concentration as an example, the presence of higher amount of salt encourages the intermolecular interaction of the stabilizer as well as stabilizer-polymer interaction, hence increasing the viscosity of the organic solution. Together with the dissociation of the salt ions into a strong ioncounterion system, which induces short-range repulsion formed by the counterions, the enhanced hydrodynamic stability and reduced interfacial tension can protect the nanodroplets from coalescence, then producing smaller nanospheres (Yahya et al., 1996; Ivanov and Kralchevsky, 1997; Ivanov et al., 1999; Yang et al., 2001). 42 On the other hand, the concentration of stabilizer plays another important role in determining the particle size. As reported by Galindo-Rodriguez et al., when the amount of PVA in aqueous phase was increased from 7% w/w to 21% w/w, the average nanoparticle size reduced from 441 nm to 123 nm, in an exponential manner, with narrower size distribution (Galindo-Rodriguez et al., 2004). It was believed that PVA stabilized the overall emulsion system through two general pathways, namely by minimizing the droplet interfacial tension and by controlling the hydrodynamic stability of the external aqueous phase. These processes can be summarized in the schematic diagram in Figure 15 below. Figure 15: Role of PVA in stabilization of nanoemulsion during salting out process for NPs preparation. (Adapted from Galindo-Rodriguez et al., 2004) 43 One of the motivations of salting out is the avoidance of the use of hazardous substances or toxic organic solvents in the fabrication process (Allémann et al., 1993a; Soppimath et al., 2001). Other than easier scale-up of this technique than emulsification with ultrasonication, this method reduces the possibility of denaturation of temperaturesensitive protein encapsulants or bond cleavages of important drugs due to extreme high sonication power or heating processes (Quintanar-Guerrero et al., 1998; Lambert et al., 2001; Wischke and Schwendeman, 2008). Unfortunately, this technique is by far only effective for encapsulating hydrophobic substances while extensive post-washing steps is needed to get rid of the high amount of salt and emulsifier (Couvreur et al., 1995; Reis et al., 2006). 2.5.4 Supercritical Fluid (SCF) Technology As environment-friendly as the salting out method described above, supercritical fluid technology in particle fabrication has become another attractive technique. This is mainly due to the stricter requirement and control by various governing authorities towards the quality and safety of the chemical additives and reagents involved in preparing any potential medical formulation for biotechnological or clinical applications. In recent years, this technology was employed as one alternative to produce drug-loaded submicron particles in view of the influence of nanoparticle size on drug distribution and bioavailability for effective cancer therapy. Some of the methods used included rapid expansion from supercritical solution (RESS) and gas anti-solvent precipitation (GAS) (Randolph et al., 1993; Kim et al., 1996; Soppimath et al., 2001). 44 In RESS method, the drug and polymer are dissolved in a SCF. Gases, such as carbon dioxide CO2, ammonia, ethane and propane, above their critical temperature and pressure demonstrate flow properties of most common gases, but the solubility powers are close to their otherwise liquid solution states. When the drug/polymer in SCF flows through a nozzle, the fluid is expanded into a different pressure or temperature environment. Sudden change in either temperature or pressure or both thus reduces the solvent power of the SCF carrying the drug and polymer, as a result of the sensitivity of SCF properties towards density change near the critical condition (Williams et al., 2002; Wischke and Schwendeman, 2008). Subsequently, the polymer starts precipitating, forming particles with the drug being dispersed within the polymeric matrix. One drawback of this method is that it is limited to low molecular weight of polymer (< 10,000 for PLA as an example) as high molecular mass of polymers often show very low solubility in most SCF even with the addition of low percentage of cosolvent acetone in CO2 (Tom and Debenedetti , 1991; Soppimath et al., 2001; Wischke and Schwendeman, 2008). This creates a problem for those novel, highly customized long polymeric chains which are needed for developing long-circulating, controlled drug delivery devices. On the contrary, in GAS method, the SCF and the saturated drug/polymer solution are separately introduced into a reactor through a nozzle (Figure 16). 45 Figure 16: Simplified scheme of gas anti-solvent precipitation by SCF technology. (Adapted from Soppimath et al., 2001) As opposed to RESS method, the high pressure in the reactor ensures that the anti-solvent is extracted into the SCF, causing the polymers and drugs, which are insoluble in the SCF, to precipitate into nanoparticles. After a certain period, the trapped nanoparticles in the reactor can be collected while the SCF containing the anti-solvent is sent through an expansion vessel for condensation, separation and regeneration of SCF and anti-solvent. The GAS method has been utilized in various studies to produce submicron particles as small as 100 to 500 nm, provided the overall system is carefully optimized by using suitable solvent/cosolvent mixture, temperature and pressure which strongly associates with the mass transfer of the solutes of concern (Dixon et al., 1993; Randolph et al., 1993; Mawson et al., 1995; Williams et al., 2002). In summary, SCF technology not only avoids the use of toxic solvents or additives, it is another technique to encapsulate heat-sensitive proteins, peptides or genes in polymeric 46 nanoparticles as some of the gases used as SCF have a critical temperature well below the denaturation point of proteins. The intermediate properties of SCF between liquid and gas allow more degree of freedom in control and optimization of nanoparticle fabrication process to obtain more homogeneous particle size distribution. However, specially customized equipments and high energy resources for heating and compressing purposes are the barriers to large scale production using this technology. Meanwhile, precise control system to maintain reproducibility and stable operating conditions may further increase the marginal cost at a rate much faster than the benefits. 2.6 Roles of Surfactants Surfactants are made up of amphiphilic molecules or ions which can spontaneously partition to segregate the hydrophobic region from the hydrophilic ones in aqueous or non-aqueous phase (Lawrence, 1994). They can be classified into ionic and non-ionic surfactants, with non-ionic surfactants being relatively less toxic to cell membrane and greater in dissolving poorly soluble drugs (Davis et al., 1970; Rege et al., 2002). In fact, there are many essential roles played by surfactants in development of drug delivery devices. Some of their roles include carriers or vehicles for pharmaceutical drugs, stabilization of the emulsion to achieve better quality of nanoparticulate system, and platform for targeted cancer therapy. 2.6.1 Drug Carriers The most common application of surfactants is the use as self-emulsifying vehicles to transport more drugs throughout the body for enhanced action of the drugs. From another perspective, surfactants are able to solubilize most hydrophobic drugs by forming 47 aggregates which are called micelles. The classical example of surfactant as drug carrier is the hydrophilic non-ionic polysorbate 80 used as an adjuvant to increase the solubility as well as absorption of docetaxel during chemotherapy. As mentioned in previous section, concentration of adjuvant in drug formulation is usually very high. And due to the extreme concentration of adjuvant in the clinical formulation with a CMC value of 0.012mM, the anticancer drug will be transported in the micellar form of certain morphology and size once the formulation is being administered into the blood stream (Chou et al., 2005; Aizawa, 2009). The same concept is also applied to Taxol®, in which Cremophor EL is the carrier of paclitaxel. Unfortunately, these surfactants are very toxic and often result in immunological reactions as described in previous section. Hence, abundance of new polymeric materials has been reported over the years. There are not only biodegradable and more biocompatible, their CMC values are also a few orders lower than those of current surfactants used as adjuvants in commercial formulations. This would mean that nanocarriers formed by these new generation of polymers are more stable and suitable, in comparison with conventional surfactants, to be carriers for drugs in physiological environments where the drugs are very susceptible to the degradation as a result of the reaction induced by plasma proteins or enzymes (La et al., 1996; Béduneau et al., 2007). 2.6.2 Stabilization of Emulsion (Emulsifiers) In nanoparticles fabrication process, surfactants of either low or high molecular weight are often used. They act as emulsifiers, which have great impact on the physicochemical properties of the polymeric nanoparticles. 48 The main reason for using emulsifier as an additive during fabrication of nanoparticles is the ability of emulsifier to stabilize the emulsion formed when an oil-based organic solvent, in which polymer and hydrophobic drugs are dissolved, is mixed with an aqueous phase. Because of the amphiphilic nature of the emulsifier, the molecules will assemble along the oil/water interface to achieve lowest free energy state of the system when extremely fine emulsion droplets are generated during the fabrication procedure (Jones and Leroux, 1999). As a result, the stabilization of the emulsion prevents the flocculation of the oil droplets in water, allowing nanoscale particles encapsulating the hydrophobic drug to be formed when the polymeric molecules solidify. Zhao et al. has recently shown that conjugation between vitamin D3 (cholecalciferol) and methoxy-PEG (mPEG) formed amphiphilic cholecalciferol polyethylene glycol succinate (CPGS) which was added to reduce the size of PLGA nanoparticles while improving the encapsulation efficiency of doxorubicin up to 93% compared to that of 57% without using any emulsifier (Zhao et al., 2007). At the same time, Vitamin D was also found to have significant function in cancer prevention as well as treatment towards certain cancer cell line such as prostate cancer (Grant and Garland, 2004; Gavrilov et al., 2005). This complementary and synergistic effect further motivates the search for more natural but effective emulsifiers for pharmaceutical application. Another feature of surfactant as an emulsifier is often the smaller nanoparticle size. As reported by some researchers, there is an optimum range in which surfactant can give the most favorable effect. Too less surfactant may cause larger particle size while too much surfactant may result in lower drug release and cellular uptake or even decreased drug loading although a relatively smaller particle size is achieved (Scholes et al., 1993; Feng 49 and Huang, 2001; Sahoo et al., 2002; Mu et al., 2004). Nevertheless, parameters such as chemical bonds, structures and HLB of emulsifiers should also be taken into consideration when optimizing the amount of emulsifier required (Jalil and Nixon, 1990; Feng and MacDonald, 1995; Feng and Huang, 2001). 2.6.3 Targeted Cancer Therapy Another unique role of surfactants is that they can serve as a platform for attaching targeting moieties so that the nanocarriers can be directed to the desired site of action. Some examples are the use of lipids and non-ionic surfactants to modify the surface of the nanoparticles (Lawrence, 1994). Phospholipids such as dipalmitoylphosphatidylcholine (DPPC), phosphatidylinositol (Ptdlns), phosphatidylcholine (lecithin) and distearoylphosphatidylethanolamine (DSPE) were some of the common, natural surfactants studied as emulsifiers for controlled and prolonged release of nanospheres and were found to have much greater emulsification efficiency compared to traditional synthetic emulsifier poly(vinyl alcohol) (Feng and Huang, 2001; Shabbits et al., 2002). They serve not only as an additional monolayer coating on polymeric nanoparticles to increase their longevity in blood stream in vivo by inhibition of P-gp (Thierry et al., 1992; Warren et al., 1992), they can also be chemically modified with specific functional groups such carboxylic, amine and methoxyl groups for conjugation with ligand peptides, aptamers or antibodies (Chan et al., 2009; Salvador-Morales et al., 2009). By this way, targeting is achieved without altering the original properties of the core containing drug dispersed in biodegradable polymeric central sphere. 50 Besides that, some members of polysorbate and poloxamer family are found to be capable of interacting with brain endothelium when they are coated on outer surface of the nanoparticles (Kreuter et al., 1997). The adsorption of the plasma protein apolipoprotein E (APO E) on polysorbate-coated nanoparticles are believed to cross the BBB through low-density lipoprotein (LDL) receptors overexpressed in brain capillary endothelial cells, and even higher in brain tumor such as glioblastomas (Murakami et al., 1988; Dehouck et al., 1994; Kreuter et al., 1995). Although Olivier et al. suggested that nanocarriers such as polysorbate 80 coated-polybutylcyanoacrylate (PBCA) nanoparticles cross the BBB is most likely due to the impairment of tight junctions of BBB by the toxicity of polysorbate 80 as well as PBCA polymer (Olivier et al., 1999), coating of nanoparticles with some hydrophilic surfactants has proven to be a possible alternative for efficient delivery of various drugs to the brain (Gulyaev et al., 1999; Goppert and Muller, 2003; Sun et al., 2004). 2.7 Vitamin E TPGS 2.7.1 Properties of Vitamin E TPGS D-α-tocopheryl polyethylene glycol 1000 succinate (Vitamin E TPGS) was synthesized in 1950s and commercially sold by Eastman Chemical Company located in the U.S state of Tennessee. The Vitamin E TPGS (or simply TPGS) is the water-soluble form of the natural, oil-soluble α-tocopherol Vitamin E antioxidant found in food. TPGS is synthesized by the esterification reaction between d-α-tocopheryl acid succinate and polyethylene glycol (PEG) 1000 (http://www.eastman.com/Pages/ProductHome.aspx ?product=71014033). Figure 17 and Figure 18 show the molecular structure of vitamin E and TPGS, respectively. 51 Figure 17: Molecular structure of d-α-tocopherol (Vitamin E). polyethylene glycol acid succinate lipophilic d-α-tocopherol form of Vitamin E Figure 18: Molecular structure and various segments of TPGS. Physically, TPGS is a waxy solid with white or slightly yellowish color and with molecular weight of approximately 1513. TPGS is stable in air with a melting point of about 37°C – 41°C and oxidative thermal degradation temperature of 199°C (http://www.eastman.com/Pages/ProductHome.aspx?product=71014033). Some repeated thermal tests performed by Eastman also suggest that TPGS is quite stable at high temperature, rendering it good thermal properties for various industrial processing applications in which heating may sometimes be required. 52 In addition to that, TPGS is fully soluble in water. Because of the incorporation of the hydrophilic PEG chain, TPGS is an amphiphilic surfactant with a hydrophile-lipophile balance (HLB) of about 13 at room temperature. HLB is a parameter often used to estimate the emulsification stability of a non-ionic surfactant, with the higher value usually implies higher hydrophilic moiety in the molecular structure and enhanced emulsifier function (Griffin, 1954; Kunieda and Shinoda, 1985; Schott, 1989; Zhao et al., 2007). On the other hand, TPGS can form micelles upon dissolving in water at a concentration above its CMC value of 0.02 wt%, and other liquid-like crystalline phases when concentration keeps increasing but well below 20 wt% (Ismailos et al., 1994; Ke et al., 2005). Non-newtonian or gel-like behavior is observed for solution containing 20 wt% or more TPGS. 2.7.2 TPGS as Solubilizer Vitamin E TPGS has a wide variety of functions particularly in biomedical applications. Since 1990s, TPGS has been found to be a good solubilizer for most of the poorly watersoluble pharmaceutical drugs which have high values in disease treatment. For instance, an early work by Ismailos et al. showed that 0.5mM of TPGS could increase the solubility of cyclosporine A (CyA) up to 2-fold at 37°C compared to that at 20°C (Ismailos et al., 1991). The increase was due to the bigger size of non-ionic TPGS micelles encapsulating more CyA (Sokol et al., 1991; Ismailos et al., 1994). This is of significant importance because CyA is required by transplant patients to prevent organ rejection. By coadministering TPGS with CyA, the dose needed to achieve therapeutic blood concentration of CyA in patients can be reduced up to 72% in two months (Sokol et al., 1991). This also lowers the probability of transplant patients suffering from cholestasis 53 caused by the high dose of CyA, thus minimizing any possible medical complication (Stone et al., 1987; Boudreaux et al., 1993). Amprenavir, also known as VX-478, is a potent viral protease inhibitor used to treat HIV infection. It was approved by Food and Drug Administration (FDA) in 1999 and manufactured by GlaxoSmithKline. Although 90% amprenavir binds to plasma protein, the drug-protein interaction is very weak and reversible. Furthermore, its hepatic metabolism is limited as it is able to inhibit, to certain extent, CYP3A4 and CYP2C19 isoenzymes, rendering it a drug formulation that can be taken orally (Livingston et al., 1995; Singh et al., 1996; Adkins and Faulds, 1998). Because amprenavir is poorly water soluble and is a substrate for P-glycoprotein, massive oral dose is often required for effective treatment, thus creating severe adverse effects. However, TPGS was found to improve the solubility of amprenavir in buffer solution at physiological temperature with the solubility being proportional to TPGS concentration above its CMC value (Roy and Tillman, 1997; Yu et al., 1999). Similarly, TPGS has been shown to enhance the solubility of the potent anticancer drugs such as taxanes. Varma et al. reported that above the CMC value of TPGS, the solubility of paclitaxel was linearly increased with higher TPGS concentration, which was due to the micellar solubilization by the surface-active agent TPGS (Varma and Panchagnula, 2005). This is in agreement with increased solubility of lipophilic drug estradiol in various aqueous alcohol solutions with the presence of TPGS for topical drug delivery (Sheu et al., 2003). Another attractive approach, such as conjugation of drugs to TPGS to produce prodrug, is a possible alternative to increase the drug solubility so that these formulations can be used for oral delivery (Amidon et al., 1995; Stella et al., 1998; Cao and Feng, 2008). 54 2.7.3 TPGS as Permeability and Bioavailability Enhancer In general, the most fundamental governing factors of the oral bioavailability of drugs in the body are their solubility and permeability (Amidon et al., 1995; Varma et al., 2004; Varma and Panchagnula, 2005). Apart from the role as a solubilizer, TPGS also enhances intestinal permeability of important pharmaceutical drugs. Co-administration of TPGS has been proven to increase the oral absorption of CyA in animal studies (Singh et al., 1996; Fischer et al., 2002). Same results indicating that TPGS enhanced permeability of CyA and HIV-protease inhibitor were also reported by a few clinical studies involving humans (Sokol et al., 1991; Boudreaux et al., 1993; Chang et al., 1996). All these point to the fact that TPGS is capable of interacting with efflux transporters, thus modulating the intestinal drug absorption as well as metabolism. Similar to some non-ionic surfactants such as unsaturated fatty acids, Solutol, Spans, Tweens, Pluronics and Cremophors, TPGS induces the inhibition of membrane transporter, specifically P-gp (Woodcock et al., 1992; Koga et al., 2000; Rege et al., 2002). Generally, P-gp inhibition could be triggered by three ways, namely, blocking the drug binding sites on P-gp, interfering the energy-dependent efflux process by inhibiting ATP hydrolysis, and disturbing the P-gp function by altering the membrane fluidity using surfactants (Drori et al., 1995; Shapiro and Ling, 1997; Varma et al., 2003). In fact, a few studies demonstrated that non-ionic surfactants, including TPGS, enhanced permeability and reversal of multidrug resistance by fluidizing membrane lipid bilayer (Dudeja et al., 1995; Nerurkar et al., 1997; Rege et al., 2001; Rege et al., 2002). Although the exact mechanism of TPGS in decreasing the P-gp efflux is not fully understood, hydrophobic vacuum cleaner (HVC) model is widely accepted to explain the phenomena (Higgins and 55 Gottesman, 1992; Varma et al., 2003). Explaining this model, when the integrity of membrane is altered, the effect results in conformational change of secondary and tertiary structures of P-gp transmembrane. Hence, this interrupts the P-gp ATPase cyclic process, promoting influx of xenobiotic drugs, which are often P-gp substrates, and reducing access to CYP3A (Dudeja et al., 1995; Wacher et al., 1998; Hugger et al., 2002; Hugger et al., 2002a). The interaction between surfactants and membrane bilayer is believed to be in the lipophilic region or polar hydrophilic head group region or both, due to the presence of poly(ethylene oxide) chain which encourages cellular uptake (Traber et al., 1988; Woodcock et al., 1992). Study from Rege et al. revealed that Cremophor EL and Tween 80, below and above their CMC values, only affected the hydrophobic part of the inner membrane, but not the polar head group (Rege et al., 2002).Whereas, cholesterol and TPGS was found to rigidify the hydrophobic region of cell membrane. TPGS was also shown to dramatically decrease the basolateral-to-apical Caco-2 cell permeability of rhodamine 123 (R123), which is a P-gp substrate, even at very low TPGS concentration. Unfortunately, the potential enhanced permeability and bioavailability by TPGS was unclear whether it was due to P-gp inhibition by TPGS monomer or micellar solubilization of P-gp substrate or both (Dintaman and Silverman, 1999; Yu et al., 1999; van Heeswijk et al., 2001; Rege et al., 2002). However, Varma et al. further described that above CMC of TPGS, the slight compromise in intestinal permeability of paclitaxel was attributed to the micellar formation through which free TPGS monomers were limited (Varma and Panchagnula, 2005a). In animal study using Sprague-Dawley rats, the apparent bioavailability of orally administered paclitaxel (25mg/kg) with TPGS (50 mg/kg) was about 30%, a 6.3-fold 56 increase than oral paclitaxel alone (4.7%) (Varma and Panchagnula, 2005). At the same time, the area-under-the-curve (AUC) of concentration-time profile was 1.5-fold higher than that of paclitaxel co-administered with verapamil (25 mg/kg). Meanwhile, cytotoxicity of vinblastine, doxorubicin and colchicines co-administered with 0.001 wt% TPGS were also found to be enhanced by about 27 to 135-fold in human MDR1 cDNAtransfected NIH 3T3 cell line (NIH 3T3-G185), to a level comparable to unmodified NIH 3T3 (Dintaman and Silverman, 1999). Apart from the use of TPGS to treat neuromuscular abnormalities due to vitamin E deficiency, clinical study of infants and children with severe chronic cholestasis also showed vitamin D3 serum level increased when it was given together with TPGS (25 IU/kg). Again, this demonstrates the capability of TPGS to enhance absorption and bioavailability of various hydrophobic drugs or substances which are subjected to P-gp efflux transport in the body (Traber et al., 1986; Traber et al., 1988; Argao et al., 1992). 2.7.4 TPGS for Sustained and Controlled Delivery Applications With quite a number of studies showing promising results of TPGS in improving the drug potency, it is finding increasing use in various pharmaceutical doses and nanoparticulate formulations to boost the absorption and even targeting effect of the drug delivery system in the molecular level. One example is the hot-melt extrusion application of TPGS in matrix solid dispersion to control the release of drug furosemide and improve its solubility by interaction between functional groups of drug and TPGS (Shin, 1979; Repka and McGinity, 2000; Shin and Kim, 2003). TPGS is also a good candidate for nanomedicine application. Since TPGS has been shown to be a drug solubilizer, it was also used as an additive or surface coating material during nanoparticle fabrication process. This can 57 achieve drug encapsulation efficiency more than 90% with smaller particle size due to its higher emulsification property than other emulsifiers such as PVA, which may be required in much greater amount to achieve the same particle properties (Mu and Feng, 2002; Mu and Feng, 2003; Mu et al., 2004; Lee et al., 2007). Alternatively, TPGS could be incorporated to poly(lactic acid) (PLA), poly(D,L-lactic-coglycolic acid) (PLGA) or poly(ε-caprolactone) (PCL) as part of block copolymers for sustained release of anticancer drugs from polymeric nanoparticles for prolonged cancer therapy (Mu and Feng, 2003; Zhang and Feng, 2006; Feng et al., 2007; Zhang et al., 2007). It was believed that TPGS, containing a PEG chain, has the ability to avoid opsonization. Opsonization is the process whereby the nanoparticulate devices are cleared from the body through the mononuclear phagocytic system (MPS) which is often induced after binding of opsonin proteins to the more hydrophobic or charged surface of the nanoacarriers (Frank and Fries, 1991; Müller et al., 1997; Hume, 2006). To increase the blood circulation half-life of the nanocarriers, the opsonization and phagocytosis processes have to be bypassed. This is achieved by the ‘stealth’ effect of the conventional hydrophilic PEG chain which could temporarily avoid the recognition of macrophages such as Kupffer cells, due to the water bound layer blocking the adhesion of opsonin proteins to the nanoparticle surfaces (Gref et al., 1994; Kaul and Amiji, 2002; Amellar et al., 2003; Letchford and Burt, 2007; Gu et al., 2008). To further optimize the effectiveness of PEG in shielding the nanoparticles from protein binding, the influences of PEG chain length on the opsonization, pharmacokinetic and biodistribution of nanoparticles are also extensively reported in literature. Some studies claimed that in order to achieve sufficient bypass from MPS with increased pharmacokinetic half-life of nanoparticles, a molecular 58 weight of PEG larger than 2000 was advisable, most likely because of the more flexible chain and sufficient layer thickness to protect against opsonins (Gref et al., 1994; Leroux et al., 1995; Peracchia et al., 1997). For PEG-containing TPGS, however, structureactivity relationship (SAR) study by Collnot et al. demonstrated that TPGS consisting of PEG molecular weight of 1000 (TPGS 1000) induced highest apical to basolateral absorptive transport as well as lowest basolateral to apical efflux on Caco-2 monolayer (Collnot et al., 2006; Collnot et al., 2007). This suggested that TPGS 1000 possessed the optimal P-gp inhibitory property compared to other analogs with different PEG chain length. As discussed in the previous section, TPGS 1000 can be a good candidate for oral delivery of drug formulation. But it may not be as good as those analogs with PEG more than 2000, which are predicted to be better in reducing the clearance of nanoparticles by MPS once they reach the blood circulation system. From another perspective, TPGS not only improves bioavailability of drugs, it also avoids the use of some first generation P-gp inhibitors such as verapamil, cyclosporine A (CyA), quinidine and tamoxifen which are not originally applied for efflux transport inhibition purpose and often bring clinical side effects to patients (Sokol et al., 1991; Hunter and Hirst, 1997; van Asperen et al., 1998; Malingré et al., 2001). For example, CyA has the potential to cause infection as a result of suppression of body immune system (Feng and Chien, 2003). For chronic treatment, an oral CyA dose of 15 mg/kg may also be associated with toxicity such as renal dysfunction (Meerum Terwogt et al., 1999). On the other hand, verapamil, a cardiovascular drug also used as P-gp inhibitor, has been shown to bring serious side effects such as toxicity and drug-drug complicated interaction (Berg et al., 1995; Tolcher et al., 1996; Feng and Chien, 2003). To date, no case of toxicity in 59 humans was reported for TPGS. Research in animals done by US National Cancer Institute showed TPGS is safe for oral use up to 1,000 mg/kg/day, indicating that TPGS is biocompatible, biodegradable and possesses various essential pharmacological properties for the use in drug delivery system (http://www.eastman.com/Pages/ProductHome.aspx? product=71014033). Recently, some other derivatives of vitamin E, have been found to selectively induce apoptosis in human cancer cells such as lung, breast, prostate, neuroblastoma and malignant mesothelioma cells, without putting severe risk of cytotoxicity on the healthy cells, thus making vitamin E an attractive compound to be extensively studied and investigated for more derivatization for chemotherapy (Anderson et al., 2004; Swettenham et al., 2005; Neuzil et al., 2007; Mahdavian et al., 2009). 60 CHAPTER 3: SYNTHESIS AND CHARACTERIZATION OF PLA-TPGS COPOLYMER 3.1 Introduction TPGS is a water-soluble derivative of vitamin E, with a polymeric chain of PEG of molecular weight 1000. It acts not only as a drug solubilizer, but also enhances the bioavailability of chemotherapeutic drugs by allowing greater absorption and permeability across various physiological barriers such as gastrointestinal (GI) tract and blood-brain barrier (BBB). Therefore, TPGS is a good candidate to be used in polymer conjugation to incorporate the characteristic of TPGS while maintaining the quality of its chemicallyattached counterpart. Recently, a lot of efforts have been spent by researchers on synthesizing block copolymers. Most of the time, ring-opening polymerization is the choice since it offers freedom in controlling the desired molecular weight, composition, tacticity or biodegradability of polymers. Some polymers obtained through ring-opening polymerization reaction are PLA, PLGA, PLA-PEG, PLGA-PEG and PTMC-PEG (Yoo and Park, 2001; Dong and Feng, 2004; Ben-Shabat et al, 2006; Deng et al, 2007; Kaihara et al., 2007). In this chapter, the focus will be on the synthesis of PLA-TPGS diblock amphiphilic copolymer. The reaction involved is catalytic ring-opening bulk polymerization of lactide using TPGS as an initiator. Following that, various characterization techniques such as proton nuclear magnetic resonance (1H NMR), gel permeation chromatography (GPC), thermogravimetry analysis (TGA) and fourier transform infrared spectroscopy (FT-IR) are employed to determine the quality of the copolymer. 61 3.2 Materials Lactide (3, 6-dimethyl-1, 4-dioxane-2, 5-dione, C6H8O4) was purchased from SigmaAldrich (St. Louis, MO, USA). It was recrystallized twice from ethyl acetate (EA, anhydrous) before use. D-α-tocopheryl polyethylene glycol 1000 succinate, C33O5H54 (CH2CH2O)23 (Vitamin E TPGS or TPGS) was purchased from Eastman Chemical Company (Kingsport, TN, USA). It was freeze-dried for at least two days before use. Stannous octoate (Sn(OOCC7H15)2) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Tetrahydrofuran (THF) was purchased from Tedia. All solvents including EA, dichloromethane (DCM), methanol and toluene were of HPLC grade. They were used without further purification. 3.3 Methods 3.3.1 Synthesis of PLA-TPGS Copolymer PLA-TPGS copolymers were synthesized by ring-opening bulk polymerization of lactide monomer with TPGS as an initiator in the presence of stannous octoate catalyst as shown in Figure 19. In brief, weighed amounts of lactide, TPGS and 0.5 wt% stannous octoate in toluene were added into a round-bottom flask. The flask containing the mixture was evacuated in liquid nitrogen for 45 minutes. After that, the mixture was heated to 145°C for 12 h under vacuum and reflux condition. Synthesis must be carried out in an oxygenand moisture-free environment to ensure high molecular weight and low polydispersity. After reaction, the product was cooled down to room temperature and then taken up in DCM under moderate stirring. The polymer was recovered by precipitation in excess cold methanol. Unreacted lactide monomers and TPGS were further removed by 62 recrystallization twice from DCM/methanol. The final product was collected by filtration and vacuum dried at 40 °C for two days until constant product weight was achieved. + Sn(O2C8H15)2 145°C; vacuum Figure 19: Ring-opening polymerization reaction in the synthesis PLA-TPGS. 3.3.2 Characterization of PLA-TPGS Copolymer 3.3.2.1 1H Nuclear Magnetic Resonance (NMR) Spectroscopy The TPGS content and number-averaged molecular weight (Mn) of the copolymer was determined by 1H NMR in CDCl3 on a Bruker AMX-500 NMR spectrometer (Bruker Instruments, Billerica, MA, USA) at a frequency of 500 MHz. 63 3.3.2.2 Gel Permeation Chromatography (GPC) The Mn and polydispersity index (PDI) of the copolymer were determined by gel permeation chromatography (GPC, Waters Corporation, USA) with a Waters 2414 refractive index (RI) detector, Waters 717 plus Autosampler and Jordi Organic GPC column (7.8 × 300 mm × 5 μm). The mobile phase THF was delivered at a flow rate of 1 ml/min. The injection volume was 100 l of standard or sample solution (0.1 % w/v polystyrene or copolymer in mobile phase). The calibration curve was established by using polystyrene standards (SM-105, Shodex) with a molecular weight range of 162 – 55,100 (molecular weights: 5.51×104, 1.39×104, 2.97×103 and 162). 3.3.2.3 Thermogravimetric Analysis (TGA) The composition of copolymer synthesized can further be confirmed by TGA (TGA 2050, USA). A small amount of sample (5-15mg) was heated under nitrogen purge on an alumina pan from 25°C to 600°C. The heating rate was 10°C/min. The percentage of weight loss during the heating process was obtained from thermogram generated by the software provided. 3.3.2.4 Fourier Transform Infrared Spectroscopy (FT-IR) The molecular structure of PLA-TPGS copolymer was investigated by FTIR (Shimadzu, Kyoto, Japan) in transmission mode. The samples for FTIR analysis were prepared by grinding 98 wt% potassium bromide (KBr) with 2 wt% copolymer and then pressing the mixture into a transparent thin film. The mid-IR scan range applied was from 4000 cm-1 to 400 cm-1. 64 3.4 Results and Discussion 3.4.1 1 H NMR Spectroscopy The PLA-TPGS copolymer obtained from ring-opening polymerization reaction was synthesized in this research for nanoparticle formulation of docetaxel. The structure of the synthesized PLA-TPGS copolymer was detected by 1H NMR in deuterated solvent CDCl3. Figure 20 shows a typical 1H NMR spectroscopy of the PLA-TPGS copolymer and its constitutive monomers. The signals at 5.19 and 1.69 ppm were assigned to the methyne (– CH) protons and methyl (−CH3) protons of PLA segment, respectively. The prominent peak at 3.65 ppm was attributed to the methylene (−CH2) protons of poly(ethylene oxide) (PEO) chain of TPGS monomer. The smaller peaks in the TPGS up-field shift (< 3 ppm) belong to various moieties of vitamin E aliphatic tails (Schroder and Netscher, 2001; Birringer et al., 2003; Momot et al., 2003; Neuzil, 2003). Also, there was no peak detected at 5.05 ppm as observed for lactide monomer. Hence, this implied that the precipitation process in purification treatment of the copolymer can remove almost all, if not all, the TPGS and lactide monomers. The molecular weight of the PLA-TPGS was calculated by using the ratio of the peak areas at 5.19 and 3.65 ppm. The number-averaged molecular weight (Mn) of the PLA-TPGS copolymer was determined to be 14,500, with 10.7 wt% of TPGS. 65 Figure 20: 1H-NMR spectra of the TPGS, lactide monomer and PLA-TPGS. 66 3.4.2 GPC The copolymerization between TPGS and lactide was also confirmed by gel permeation chromatography, where the retention time of substances depend on the size of the molecules to be separated. Smaller molecules will be eluted slower than larger ones because of the longer path travelled along certain matrix pores within the column packing. From GPC analysis, the retention time of the copolymer was 24.5 min due to the higher molecular weight of the copolymer than monomer TPGS (28.3 min) (Figure 21). One narrow peak for the copolymer indicated that the product was not a physical mixture of lactide and TPGS. The number-averaged molecular weight calculated was 13,480, close to the value calculated from NMR. The polydispersity index (PDI) of the copolymer was 1.43. Figure 21: Gel permeation chromatogram of TPGS monomer and PLA-TPGS copolymer. 67 3.4.3 TGA Thermogravimetry analysis (TGA) is a technique to measure the thermal behavior of a sample, especially polymer, by mass change when it is heated gradually to a temperature high enough to decompose all the samples. Additionally, the thermogram in Figure 22 showed that the weight loss after 280°C was about 10.9 wt%, which corresponded to the amount of TPGS loss from the copolymer. This was consistent with the result obtained from 1H NMR. Figure 22: TGA thermogram of TPGS monomer and PLA-TPGS copolymer. 3.4.4 FT-IR Spectroscopy Figure 23 demonstrates the FT-IR spectra of the PLA-TPGS copolymer, TPGS and lactide monomers. The absorption band at 3200 – 3600 cm-1 is attributed to the terminal hydroxyl (−OH) group for PLA-TPGS copolymer and TPGS monomer. Also, the peak at the range 68 of 2850 – 2970 cm-1 was attributed to the stretching vibration of aliphatic groups (−CH) of the hydrocarbon chain. Meanwhile, the multiple peaks found in this frequency range of the PLA-TPGS copolymer were probably the result of conjugative interaction between the repeating lactide units which caused an overtone effect in the spectra. However, the peaks found in the same frequency region for lactide monomer may be due to the C-H bond of the aromatic ring. Figure 23: FT-IR spectra of TPGS, lactide monomer and PLA-TPGS copolymer. On the other hand, it can be seen that the reduction of peak at 930 cm-1 (CH-bend) from lactide and the presence of 740 cm-1 (CH-bend) from PLA-TPGS showed the opening of lactide cyclic ring during the polymerization reaction (Kiremitçi-Gümüşderelioğlu and Deniz, 1999). Whereas, the peaks at 1050-1300 cm-1 is due to the C-O stretching. Furthermore, the strong peak observed at 1770 cm-1 in PLA-TPGS, similar to that of 69 lactide, was from the C=O stretch of ester functional groups. This peak intensity was much more prominent in PLA-TPGS than TPGS (1750 cm-1). This suggested that poly(lactide) segment, which contained repeating number of carbonyl ester groups, has been conjugated as part of the copolymer. Furthermore, the peaks observed in the range of 1690 cm-1 to 1760 cm-1 for lactide, TPGS and PLA-TPGS copolymer were from the C-O bond in the ether and ester functional groups. 3.5 Conclusion To conclude, we successfully synthesized PLA-TPGS amphiphilic copolymer with number-averaged molecular weight of about 14,500 and 11 wt% of TPGS content. The mechanism involved ring-opening polymerization reaction with the presence of stannous octoate catalyst and TPGS as the initiator. Various characterizations of the copolymer were done. 1H NMR Spectroscopy and gel permeation chromatography were applied to confirm the conjugation and polymerization as well as the molecular weight and chain distribution of the copolymer. Meanwhile, thermogravimetry analysis determined the weight percent of each constituent. And, fourier transform infrared spectroscopy further confirmed the copolymerization by ring-opening mechanism. 70 CHAPTER 4: FABRICATION AND CHARACTERIZATION OF PLA-TPGS NANOPARTICLES 4.1 Introduction Nanoparticles of biodegradable polymers have been an alternative to solve some of the problems experienced by traditional chemotherapy due to the sudden exposure to high native drug concentration and the serious side effects of the adjuvants used to solubilize the drug. By encapsulating the anticancer drug in the polymeric matrix core of nanoparticles, drug can be released at a minimum effective concentration over a certain period of time. The controlled and sustainable release of drug avoids some of the drug- or adjuvant-related toxicities. The following sections will emphasize on the fabrication and characterization of drug-loaded nanoparticles. Firstly, solvent emulsification/evaporation technique with ultrasonication is applied to generate nano-emulsion which leads to the forming of sub-micron particles (Suh et al., 1998; Mei et al., 2007; Gelperina et al., 2009). The reasons of using this fabrication method are the simple experimental set-up and shorter processing time due to relatively faster evaporation of water-insoluble, volatile organic solvent from the colloidal suspension, in comparison to dialysis method which required much longer time. Furthermore, when compared to nanoprecipitation method, solvent emulsification/evaporation technique often results in better drug encapsulation efficiency and reproducibility, although the particle size is usually larger. Secondly, nanoparticles obtained with two different emulsifiers (TPGS and PVA) during fabrication process are characterized for the morphology (field emission scanning electron microscopy, FESEM), particle size and size distribution (laser light scattering, LLS), zeta 71 potential (Zeta Analyzer), surface properties (X-ray photoelectron spectroscopy, XPS) and encapculation efficiency (EE) and in vitro drug release kinetic. 4.2 Materials Docetaxel of purity 99.56% was purchased from Jinhe Bio-Technology Co. Ltd (Shanghai, China). Polyvinyl alcohol (PVA) (average Mw = 31,000 – 50,000), Coumarin 6 and mannitol were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cellulose membrane with MWCO 1,000 was from Spectra/Por® (Spectrum Laboratories Inc., Houston, USA). All chemicals such as acetonitrile, DCM and methanol were of HPLC grade. They were used without further purification. Millipore water was prepared by a Milli-Q Plus System (Millipore Corporation, Breford, USA). 4.3 Methods 4.3.1 Preparation of PLA-TPGS Nanoparticles Docetaxel-loaded PLA-TPGS nanoparticles were fabricated by a modified solvent emulsification/evaporation method. A given amount of docetaxel and 100 mg PLA-TPGS copolymer were dissolved in 8 ml DCM (organic phase). The formed solution was poured into 120 ml Millipore water (aqueous phase) containing 0.03 wt% TPGS or 0.2 wt% PVA as emulsifier. The emulsion was rapidly stirred for another 15 seconds. Then, the emulsion was sonicated using a sonicator probe (XL2000, Misonix Inc., USA) for 120 s at 22 W. The organic solvent in the suspension was then evaporated overnight under reduced pressure. The colloid suspension was centrifuged at 11,000 rpm for 20 min and then washed two times to remove the unloaded drug and excess emulsifiers. The particles were resuspended in 10 ml water containing 5% mannitol before freeze-drying for two days. 72 The fluorescent coumarin 6-loaded PLA-TPGS NPs were prepared in the same way except 0.05 (w/v)% coumarin 6 with respect to DCM was encapsulated instead of docetaxel. 4.3.2 Characterization of Drug-loaded PLA-TPGS Nanoparticles 4.3.2.1 Particle Size Analysis Size and size distribution of the docetaxel-loaded PLA-TPGS NPs were measured by LLS (90-PLUS Analyzer, Brookhaven Instruments Corporation, USA). The samples were prepared by diluting the nanoparticle suspension with deionized water to a count rate of 100 – 300 kcps. 4.3.2.2 Surface Morphology The NPs were imaged by FESEM (JSM-6700F, JEOL, Tokyo, Japan) at an accelerating voltage of 5.0 kV. To prepare samples for FESEM, a droplet of particle suspension was transferred to the copper tape adhered to the stub. The droplet was allowed to dry and then coated with a platinum layer by JFC-1300 automatic fine platinum coater (JEOL, Tokyo, Japan) at 30 mA for 30 s. 4.3.2.3 Surface Charge Zeta potential of the drug-loaded PLA-TPGS NPs was detected by the laser Doppler anemometry (Zeta Plus Analyzer, Brookhaven Corporation, USA). The particles (about 2 mg) were suspended in deionized water before measurement. The data were obtained with the average of five measurements. 73 4.3.2.4 Surface Chemistry of Drug-loaded PLA-TPGS NPs The surface chemistry or composition of docetaxel-loaded PLA-TPGS NPs was analyzed by XPS (AXIS His-165 Ultra, Kratos Analytical, Shimadzu Corporation, Kyoto, Japan). A hemispherical analyzer with a pass energy of 80 eV was used to scan a full spectrum of binding energy with a range of 0 – 1,100 eV. Data analysis was done using XPS Peak 4.1 software provided by the manufacturer. 4.3.2.5 Thermal analysis of Drug-loaded and unloaded PLA-TPGS NPs Differential scanning calorimetry (DSC, Mettler-Toledo) was applied to study the state change of docetaxel, docetaxel-loaded or unloaded NPs with respect to temperature. An amount of about 5-15 mg of sample was sealed and heated in alumina pan from 40°C to 240°C at a ramp of 10°C/min under 20 ml/min nitrogen flow. The energy vs. temperature profiles were recorded by the software provided by the manufacturer. 4.3.2.6 Drug Encapsulation Efficiency The docetaxel entrapped in PLA-TPGS nanoparticles was measured by HPLC (Agilent LC1100, Agilent, Tokyo, Japan). Briefly, 3 mg of nanoparticles were dissolved in 1ml of DCM. After evaporation, the drug was reconstituted in 1.2 ml of mobile phase consisting of acetonitrile, methanol and deionized water (45:5:50, v/v/v). The solution was filtered through 0.45 µm syringe filter before transferring into HPLC vial. The flow rate of mobile phase in the HPLC column (Agilent Eclipse XDB-C18, 4.6 × 250 mm, 5 μm) 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 74 R2 of 0.99. The drug encapsulation efficiency was defined as the ratio between the amount of docetaxel encapsulated in nanoparticles and that added in the fabrication process. For fluorescent-encapsulated NPs, the encapsulation efficiency was determined by the same extraction process described for drug-loaded NPs. The fluorescence was measured by HPLC with a flow rate of 1.3 ml/min mobile phase consisting of acetonitrile/water (60:40 v/v). The excitation and emission wavelength were set at 462 nm and 502 nm, respectively, using a fluorescence detector module. All samples were done in triplicate. 4.3.2.7 In Vitro Drug Release In vitro drug release was evaluated by a dialysis method. In brief, 15 mg nanoparticles were dispersed in 5 ml of phosphate buffer saline solution (PBS 10 mM) of pH 7.4 containing 0.1 (w/v)% Tween 80 to form a suspension. Tween 80 was used to avoid the attachment of docetaxel to the tube wall. The suspension was put into a standard grade cellulose dialysis membrane with MWCO 1,000. Then, the closed bag was put into a centrifuge tube and immersed in 15 ml release medium. The tube was kept in an orbital water bath shaking at 120 rpm at 37.0 °C. At given time intervals, the release medium was drawn out for analysis and replaced with fresh medium. The samples were extracted and analyzed according to the same procedures as EE of coumarin 6. 4.4 Results and Discussion 4.4.1 Particle Size and Size Distribution The characterization of the nanoparticles using TPGS or PVA as emulsifier was summarized in Table 1. The nanoparticles were prepared with either 0.2 (w/v)% PVA or 75 0.03 (w/v)% TPGS as emulsifier. The PVA concentration added in the water phase was usually around or above 1.0 % as cited in literature (Suh et al., 1998; Wang and Schwendeman, 1999; Li et al., 2003; Mu and Feng, 2003; Mei et al, 2007). Considering that the TPGS component in the copolymer has a self-emulsifying function, however, a much lower concentration at 0.2 % PVA was used in this research. This is an advantage of the PLA-TPGS copolymer in nanoparticle formulation, which can reduce the amount of surfactant used or even avoid the side effects of the traditional chemical emulsifier such as PVA in pharmaceutical industry. Table 1: Particle size, size distribution, encapsulation efficiency, surface charge of docetaxel-loaded and coumarin 6-loaded PLA-TPGS NPs. Docetaxel-loaded NPs a b Coumarin 6-loaded NPs Emulsifier (w/v)% 0.03% TPGS 0.20% PVA 0.03% TPGS 0.20% PVA Size a (nm) 240.6 ± 9.0 268.6 ± 3.2 296.7 ± 28.4 341.6 ± 17.8 Polydispersity a 0.166 ± 0.022 0.005 ± 0.011 0.333 ± 0.037 0.292 ± 0.027 EE b,c (%) 85.0 ± 3.9 63.4 ± 2.1 80.1 ± 2.7 69.8 ± 1.4 Zeta potential a,d (mV) -38.46 ± 4.64 -23.56 ± 1.19 -31.35 ± 2.59 -22.02 ± 1.34 n=6 n=3 actual drug loading % in nanoparticles  100% theoretica l drug loading % in nanoparticles d Measurement done in deionized water at pH = 7 c EE= 76 The results indicated that the docetaxel-loaded PLA-TPGS NPs have particle size range from 240 nm to 270 nm. As shown in Table 1, the average size of PVA-emulsified NPs was 269 nm, which was slightly larger than the size of TPGS-emulsified NPs (241 nm) (p 500 nm) would be taken up via the lymphatics and small particles (< 500 nm) can cross the membrane of epithelial cells through endocytosis and those about 100 – 200 nm in diameter can achieve optimum cellular uptake (Lefevre et al., 1978; Savic et al., 2003; Win and Feng, 2005). On the other hand, the coumarin 6-loaded TPGS-emulsified NPs were also smaller than that using PVA even though their sizes (Table 1) were relatively larger than those encapsulating docetaxel. Probably, this was due to the partially water-miscible nature of coumarin 6 with which the interaction between coumarin 6 and the matrix polymer as well as emulsifier was different during the encapsulation process. The more hydrophobic coumarin 6 than docetaxel can, in fact, have different precipitation rate of polymeric chains which greatly affect the size and its uniformity (Wischke and Schwendeman, 2008). 77 4.4.2 Surface Morphology Surface morphology of the PLA-TPGS nanoparticles encapsulating docetaxel and coumarin 6 was examined by FESEM. Figure 24 and Figure 25 showed the FESEM images of the docetaxel-loaded TPGS- and PVA-emulsified PLA-TPGS NPs, respectively while Figure 26 showed coumarin 6-loaded NPs with TPGS or PVA as emulsifier. All the particles were observed to be spherical in shape and seemed to have smooth surface within the FESEM resolution level. The FESEM images confirmed the particle size detected from the LLS. Also, the uniformity of particles was generally better for docetaxel compared to coumarin 6, as demonstrated by the PDI values in Table 1. Figure 24: FESEM images of docetaxel-loaded TPGS-emulsified PLA-TPGS NPs. 78 Figure 25: FESEM images of docetaxel-loaded PVA-emulsified PLA-TPGS NPs. Figure 26: FESEM images of coumarin 6-loaded TPGS-emulsified (left) and PVAemulsified (right) PLA-TPGS NPs. 79 4.4.3 Surface Charge In terms of nanoparticle stability in colloidal suspension, high absolute value of zeta potential (~25 mV or higher) indicated high surface charge of the nanoparticles, resulting in a strong repulsive force between particles to stay dispersed from each other in nanosuspension (Müller, 1991; Musumeci et al., 2006). Compared to PVA-emulsified NPs, the greater zeta potential of TPGS-emulsified NPs suggested a higher electrophoretic mobility and stability. The effect of different surfactants on characteristics of fluorescentloaded NPs was found to be of similar trend to those of docetaxel-loaded NPs. Therefore, the assumption of which drug-loaded NPs can be simulated by fluorescent-loaded NPs used in in vitro cellular uptake and imaging studies in Chapter 5 was reasonable. 4.4.4 Surface Chemistry X-ray photoelectron spectroscopy (XPS) can be applied to determine the elements or components presented on the surface of a compound within a depth range of 1 to 10 nm. Figure 27 shows the curve-fitting of the C1s elemental spectra of PLA-TPGS copolymer and unloaded PLA-TPGS NPs (with no emulsifier used). The envelope ratio at 286.0 eV, which corresponded to the C-O-C from PEO segment of TPGS, increased from 11.38% in PLA-TPGS copolymer to 16.48% in PLA-TPGS NPs (without surfactant). This suggested that more TPGS segment of the copolymer was exposed on the surface when NPs were formed using the copolymer concerned. Apart from that, from the XPS wide scan spectrum of docetaxel-loaded PLA-TPGS NPs shown in Figure 28, it can be seen that no obvious nitrogen peaks were observed for 80 C-C/C-H O-C=O C-O-C=O C-O-C C-C/C-H O-C=O C-O-C=O C-O-C Figure 27: XPS C1s envelope of PLA-TPGS copolymer and unloaded PLA-TPGS NPs (without using emulsifier). 81 TPGS-emulsified PLA-TPGS NPs within the nitrogen scan range for binding energy of 394 to 409 eV. The insert showed the higher resolution of nitrogen element signal for the same binding energy range. This implied that most of the drug, if not all, was encapsulated within the polymeric matrix of the nanoparticles rather than being adsorbed on the outer surface of NPs. However, there was a very small nitrogen element signal from 1s orbital of nitrogen atom (N 1s) contributed by docetaxel found on the surface of PVA-emulsified PLA-TPGS NPs, as shown by the higher resolution scan at about 399.2 eV. This can be attributed to the lower efficiency of the high molecular weight PVA as an emulsifier compared to TPGS, causing some drug molecules to be located close to the NP surface rather than the inner core the polymeric matrix. Figure 28: XPS wide scan spectra of docetaxel-loaded TPGS-emulsified (TNP) and PVAemulsified (PNP) PLA-TPGS NPs. 82 4.4.5 Drug Encapsulation As a complement to the XPS spectra shown in Figure 28 above, size of unloaded NPs and a series of thermal behavior analysis of drug and NPs are some other methods that can be applied to further confirm that the anticancer drug was, in fact, encapsulated within the polymeric NPs rather than a purely physical mixing of drug and NPs. Firstly, the size of the TPGS-emulsified unloaded NPs was determined to be 229.2 ± 9.8 nm, which was relatively smaller compared to that with drug encapsulation. Secondly, thermal analysis using DSC was performed. This method is based upon the behavioral change of a compound in the molecular level when the compound is subjected to certain heating profiles. The results are summarized in Figure 29. DTX (5 months storage) (Form I) DTX (recent) (Form I) Endothermic Unloaded PLA-TPGS NPs DTX + unloaded PLA-TPGS NPs (physical mixing) DTX-loaded PLA-TPGS NPs DTX recovered after emulsification (Form V) 40 90 140 190 240 Temperature (°C) Figure 29: DSC curves of pure docetaxel, docetaxel-loaded PLA-TPGS NPs, unloaded NPs, docetaxel/unloaded NPs mixtures and docetaxel recovered from emulsification. 83 It can be seen from Figure 29 that docetaxel (DTX) was stable under the laboratory storage condition as the peak at about 167°C, which corresponded to the endothermic process of melting, remains the same. For unloaded PLA-TPGS NPs, no peak was observed above a temperature of 90°C. The small peak at the range of about 50-60°C was attributed to the glass transition temperature (Tg) of the polymeric NPs. Meanwhile, for docetaxel-loaded PLA-TPGS NPs prepared by solvent emulsification/evaporation method, no melting peak of drug was observed as well. This suggested that DTX, if encapsulated, was in amorphous state. This can be confirmed by the DSC curve of DTX/unloaded PLATPGS NPs physical blend which showed the presence of DTX melting peak, similar to the DSC curve of pure DTX alone. However, the melting peak was shifted to about 93°C if DTX was pre-treated by undergoing emulsification-ultrasonication process followed by freeze-drying recovery. The shift of the melting point was most likely due to the change of crystalline structure of the polymorphic DTX from original Form I to Form V during the pre-treatment procedures. Thus, it can be concluded from the series of analysis that DTX was indeed encapsulated within the NPs prepared by single solvent emulsification/ evaporation technique, in agree with results obtained from XPS. By comparing the effect of emulsifiers on drug encapsulation efficiency (EE), TPGS was found to be better than PVA. As tabulated in Table 1, EE of TPGS-emulsified NPs was calculated to be 85.0%, higher than that of PVA-emulsified NPs (EE = 63.4%). The trend was consistent even for the encapsulation of coumarin 6. Again, this demonstrated the benefit of involving the amphiphilic TPGS in the fabrication of NPs. Higher EE is often favorable as long as drug delivery is concerned. Some advantages for obtaining a high EE included reduction of drug loss during NPs preparation, production cost-saving and a 84 lower amount of drug carrier required to achieve the desired therapeutic effect. This is of utmost importance when a large quantity of polymeric materials could potentially bring unwanted side effects or toxicities, especially for those advanced new functional polymeric carriers for which their long-term health effects in human body was not fully understood or examined. 4.4.6 In Vitro Drug Release As shown in Figure 30, the docetaxel-loaded PLA-TPGS nanoparticles exhibited a biphasic prolonged release up to 30 days, which is characterized by an initial burst effect followed by sustained Fick’s second law diffusive release (Mohamed and van der Walle, 2008). In the first 6 h, the drug release from the TPGS-emulsified nanoparticles was found to be 14.22 ± 2.46%, which was faster than the PVA-emulsified nanoparticles, which is only 5.02 ± 2.00%. This was because the TPGS-emulsified NPs have relatively smaller size than the PVA-emulsified NPs. Smaller particles have larger specific surface area. This may be the reason for the higher initial burst for TPGS-emulsified NPs. Also, the high molecular weight of PVA can potentially create a resistance to the release of drug from the polymeric matrix of NPs into the release medium initially. However, from Day 2 onwards, the cumulative drug release from the TPGS-emulsified nanoparticles was found to be 74.40 ± 1.77%, which was significantly lower than the PVA-emulsified nanoparticles, which was 78.36 ± 1.60%. One of the reasons could be the hydrophilic nature of PVA which enhanced the uptake of water molecules into the polymeric matrix of the NPs, thus speeding up the swelling process by enlarging the pore sizes of NPs (Bouissou et al., 2004; Wischke and Schwendeman, 2008). Although the cumulative release was lower for TPGS-emulsified NPs, the kinetic of drug release, which was 85 represented by the slope of the release profile, was slightly higher, especially from Day 14 onwards. This could be useful to achieve sustainable release of cytotoxic drug over a certain period of time. Figure 30: In vitro drug release profiles of docetaxel-loaded PLA-TPGS NPs using TPGS and PVA as emulsifier. Data represent mean ± SD (n=3). 4.5 Conclusion PLA-TPGS NPs encapsulating docetaxel as well as coumarin 6 were successfully fabricated by solvent emulsification/evaporation technique. Two emulsifiers, namely TPGS and PVA were used in the preparation process. The particle size of TPGSemulsified and PVA-emulsified was determined by LLS to be 240.6 nm and 268.6 nm, 86 respectively. Meanwhile, TPGS-emulsified coumarin 6-loaded NPs (296.7 nm) were also found to be smaller in diameter than that of PVA-emulsified NPs (341.6 nm), although the size distribution was not as good as that of PVA-emulsified NPs . The surface morphology of the NPs was observed using FESEM and was found to have a smooth surface. In terms of NPs stability in suspension, TPGS-emulsified NPs was much more stable than that of PVA-emulsified NPs either for docetaxel-loaded NPs or coumarin 6-loaded NPs, offering a greater repulsive force between each NP to reduce NP aggregation. Furthermore, EE for TPGS-emulsified NPs was calculated to be 1.34-fold higher than PVA-emulsified NPs, suggesting a more effective carrier for drug delivery. On the other hand, XPS, size of unloaded NPs and DSC were applied in order to prove that the drug was encapsulated in the polymeric matrix of NPs. For the drug release behaviors of the NPs, profiles showed that higher initial burst of drug was observed for TPGS-emulsified NPs, even though the percentage of drug released in Day 30 was lower than PVA-emulsified NPs. The PLATPGS NPs was proved to be a good candidate to achieve sustainable release of drug for extended therapeutic effect. The benefits of controlled and prolong drug release to in vitro cellular study and in vivo drug distribution will be demonstrated in the following chapters with more details. 87 CHAPTER 5: IN VITRO CELLULAR STUDY OF PLA-TPGS NANOPARTICLES 5.1 Introduction The efficacy of the nanoparticle formulation of biodegradable polymers is heavily dependent on the ability of the formulation to bypass the resistances exerted from the cancer cells. One well-known barrier is the multidrug-resistant P-gp transporter expressed by most of the cancer cells (Thiebaut et al., 1987; Warren et al., 1992; Ling, 1997). As a result, physicochemical properties of the nanoparticles such as particle size, surface coating and hydrophobicity are critical factors in determining the affinity of the nanoparticles towards the targeted cells to be destroyed, thus directly influencing the drug efficacy (Müller et al., 1997). In this chapter, the main study involved in vitro systems such as the cellular uptake, cell imaging using confocal laser scanning microscope and cell viability. To compare the effect of different emulsifiers in terms of uptake efficiency and cell cytotoxicity over a period of 3 days, amphiphilic TPGS and the most common surfactant, PVA, were applied. Besides that, commercial formulation Taxotere® was also used in cell cytotoxicity study to evaluate some of the advantages of using equivalent drug-loaded nanoparticle formulation over the clinical formulation. The models of cancer cell lines used in the following studies were human breast adenocarcinoma (MCF-7) and human colon cancer cells (HT-29). 88 5.2 Materials Phosphate buffered saline (PBS), Dulbecco’s Modified Eagle Medium (DMEM), coumarin 6, propidium iodide (PI), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay were purchased from Sigma-Aldrich. Trypsin-EDTA solution, penicillin-streptomycin, fetal bovine serum (FBS) were purchased from Gibco (Life Technologies, Switzerland). Triton® X-100 was provided by Bio-Rad (Bio-Rad Laboratories Inc., USA). Commercial formulation Taxotere® was supplied by Aventis Pharmaceuticals, USA. All solvents such as ethanol, isopropanol and sodium hydroxide (NaOH) were from Sigma-Aldrich. They were used without further purification. 5.3 Methods 5.3.1 Cell Culture In this study, human breast adenocarcinoma MCF-7 and human colon adenocarcinoma HT-29 cell lines (American Type Culture Collection, USA) were cultured in 25 cm2 culture flasks using DMEM 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. 5.3.2 Cellular Uptake of Nanoparticles For quantitative study, MCF-7 cells were seeded into 96 well black plates (Costar, IL, USA) at 1.3 × 104 cells/well. After the cells reached confluence, the cells were incubated with coumarin 6-loaded nanoparticle suspension. The nanoparticles were dispersed in the 89 same culture medium at a concentration of 100, 250 and 500 μg/ml. The plates were incubated at 37 °C for 0.5, 1, 2 and 4 h. After certain time interval, the spent suspension in every sample well was removed. The wells were washed three times with 50 µl cold PBS to eliminate traces of excess nanoparticles and reconstituted with 100 μl fresh culture medium. Wells for positive control were left unwashed. After that, 50 μl of 0.5% Triton X-100 in 0.2 N NaOH was introduced into every well to lyse the cell membrane especially the lysosomes. The fluorescence intensity of each well was measured by microplate reader (GENios, Tecan, Switzerland) with excitation wave length at 430 nm and emission wavelength at 485 nm. Cell uptake efficiency was expressed as the percentage of cellassociated fluorescence versus the fluorescence present in the feed solution (positive control). For the qualitative study, cells were reseeded in the chambered cover glass system (LABTEK®, Nagle Nunc, IL). After the cells were incubated with 250 μg/ml coumarin 6loaded nanoparticles at 37 °C for 2 h, the cells were rinsed with cold PBS for three times and then fixed by 75% ethanol for 20 min. Then, the cells were further washed twice with PBS before the nuclei were subsequently stained with propidium iodide (PI) for 40 min. The cell monolayer was washed three times with PBS and observed by confocal laser scanning microscopy (CLSM) (Nikon C1, Nikon Corporation, Japan) with imaging software, NIS-Element AR 3.0. 5.3.3 In Vitro Cell Cytotoxicity Cancer cell viability of the drug-loaded PLA-TPGS NPs was evaluated by MTT assay. 100 μl of MCF-7 cells were seeded into 96-well plates (Costar, IL, USA) at the density of 90 5×103 viable cells/well and incubated at least overnight to allow cell attachment. The spent medium was discarded and the cells were then incubated with the docetaxel-loaded nanoparticle suspension or Taxotere® at 0.025, 0.25, 2.5, 10 and 25 μg/ml equivalent of docetaxel concentration for 24, 48 and 72 h, respectively. At designated time intervals, the medium was removed and the wells were washed twice with cold PBS. Following that, 100 μl of MTT solution was added to each well of the plate. The plates were further incubated for 3-4 h in the incubator. Finally, 50 μl of isopropanol was added into each well to transformed MTT crystals and the absorbance of the transformed MTT solution in the wells was measured at 450 nm using a microplate reader. Cell viability was calculated by the followed equation: Cell viability (%) = (Abss ÷ Abscontrol) × 100 where Abss is the fluorescence absorbance of the cells incubated with the nanoparticle suspension and Abscontrol is the fluorescence absorbance of the cells incubated with the culture medium only (positive control). IC50, the drug concentration at which 50% of the cell population was destroyed in comparison with that of the control sample, was calculated by regression (curve fitting) of the cell viability data. 5.4 Results and Discussion 5.4.1 Cellular Uptake It is clear that the therapeutic effects of the drug-loaded NPs depend on internalization and retention of the NPs by the malignant tumors. Although, in vitro and in vivo experiment can produce different results due to the different environment to which the NPs are 91 exposed, an in vitro investigation can provide some preliminary evidence to show the advantages of the NP formulation over the pristine drug. Figure 31: MCF-7 cell uptake efficiency of TPGS-emulsified (TNP) and PVA-emulsified (PNP) coumarin 6-loaded PLA-TPGS NPs at 100, 250 and 500 µg/ml incubated at 37°C. Data represent mean ± SD (n=6). We conducted in vitro cellular uptake of the TPGS-emulsified and PVA-emulsified PLATPGS NPs by human breast adenocarcinoma MCF-7 cell line. As shown in Figure 31, the MCF-7 cellular uptake of the coumarin 6-loaded TPGS-emulsified or PVA-emulsified 92 PLA-TPGS NPs was time- and concentration-dependent, which implied the involvement of an active endocytosis process (Hu et al., 2009). In all cases, the cellular uptake efficiency was at least more than 14% and 12% for TPGS-emulsified and PVA-emulsified NPs, respectively, after 0.5 h incubation. The average uptake efficiency of TPGSemulsified NPs was always greater than PVA-emulsified NPs at all three different nanoparticle concentrations. For instance, after 4 h incubation time, 42.8 ± 4.6%, 53.3 ± 8.3% and 69.9 ± 13.8% of TPGS-emulsified PLA-TPGS NPs were internalized by the MCF-7 cells at 100, 250 and 500 μg NP/ml cell culture medium, respectively. Meanwhile, the uptake efficiencies PVA-emulsified PLA-TPGS NPs by MCF-7 were 34.5 ± 9.3%, 38.5 ± 6.6% and 50.4 ± 8.8%, respectively, at the same incubation time and nanoparticle concentrations (p < 0.05). The high internalization of NPs demonstrated the advantage of incorporating TPGS as a copolymer component which can enhance the absorption and permeation across membrane of cancer cells. Further improvement in intracellular uptake was shown by using TPGS as surface-active agent in NP preparation. Confocal laser scanning microscopy (CLSM) images of MCF-7 breast cancer cells were captured after 2 h incubation time with coumarin 6-loaded PLA-TPGS NPs at a concentration of 250 μg NP/ml (Figure 32). The fluorescent-loaded NPs (green color) were shown internalized into the elongated cytoplasm of MCF-7. It can be seen from the figure that the fluorescence of the coumarin 6-loaded NPs was closely located around the nuclei which was stained with propidium iodide (red color). Comparing with nanoparticle formulations, it was obvious that the fluorescence of free coumarin 6 (an equivalent amount to that of TPGS-emulsified NPs) within the cytoplasm was significantly lower, thus, demonstrating a lower endocytic affinity for free or unencapsulated coumarin 6. In 93 other words, the detected fluorescent signals were mainly contributed by the entrapped coumarin 6 in NPs. 1 2 3 A B C Figure 32: Confocal laser scanning microscopy (CLSM) of MCF-7 cells after 2 h incubation with 250 µg/ml coumarin-6-loaded TPGS-emulsified NPs (Row A), PVAemulsified NPs (Row B) and free coumarin-6 (Row C) at 37.0 °C. The cells were stained by propidium iodide (Red channel, column 2) and the coumarin-6-loaded PLA-TPGS NPs are green in color (Green channel, column 1). 94 It should be emphasized that the use of fluorescent markers in NP visualization can lead to misinterpretation of NPs uptake data due to the leaking or dissociation of fluorescent markers from the nanoparticles into the released medium and hence subsequently into the cells by diffusion (Suh et al., 1998a). Besides incubating the cells with free coumarin 6 to study its uptake as shown in the confocal images (Row C), in vitro coumarin 6 release can be another method to ensure that the fluorescent observed was due to the coumarin 6 encapsulated in the NPs. Win and Feng have thus shown that the in vitro release kinetics of the encapsulated fluorescent markers from the polymeric NPs and found that the cumulative coumarin 6 release was only less than 4% over 24 h incubation time, which was considered negligible in comparison with the amount of fluorescence encapsulated in the polymeric NPs (Win and Feng, 2005). On the other hand, Sun et al. has also shown that the cumulative release of coumarin 6-loaded PLGA/MMT NPs was less than 3% in the first 24 h (Sun et al., 2008). Moreover, from our simple study using C18 HPLC column, coumarin 6 was found to be eluted slower (27.87 min) than docetaxel (4.22 min) under the same operating parameters and conditions, hence suggesting that coumarin 6 was much more hydrophobic than docetaxel and less readily diffused into the release medium. It is thus reasonable to assume that most of the coumarin 6 was associated in the NPs and the fluorescence observed from the cellular uptake and confocal images mainly reflected coumarin 6-loaded NPs with only very negligible signal contributed by free fluorescent molecules. 5.4.2 Cell Viability Figure 33 showed the in vitro viability of MCF-7 cells cultured with the drug formulated in Taxotere®, the PVA-emulsified or TPGS-emulsified PLA-TPGS NPs at the same 95 equivalent docetaxel concentration of 0.025, 0.25, 2.5, 10 and 25 µg/ml. Firstly, it was observed that the drug formulated in the PLA-TPGS NPs showed better efficacy against the cancer cells than commercial Taxotere®. For example, the cell cytotoxicity after 3 days of incubation at 25 µg/ml drug concentration for TPGS- and PVA-emulsified NP formulations were 2.05-fold (p < 0.05) and 1.38-fold (p < 0.05) higher than that of Taxotere®, respectively. The higher cytotoxicity of the drug formulated in the two nanoparticle formulations can be explained by the higher cellular uptake as well as the prolonged drug release manner in comparison with Taxotere®, which was believed to be able to induce multidrug resistance (MDR). Secondly, the TPGS-emulsified NPs can achieve relatively better therapeutic effect than the PVA-emulsified NPs particularly after 2 days incubation with drug concentrations of 2.5 µg/ml and above. This was attributed to the higher cellular uptake of the nanoparticles (Figure 31), the faster initial drug release from the NPs (Figure 30) and the TPGS masking effect on P-glycoprotein efflux transport system through membrane fluidization or ATPase inhibition (Collnot et al., 2007). Additionally, to reassure that the cell death was due to the release of docetaxel, placebo TPGS- and PVA-emulsified NPs were incubated with MCF-7 cells. Quantitatively, the cell viability of MCF-7 was found to be 92.44 ± 7.08% and 91.63 ± 6.99% after 3 days for unloaded TPGS- and PVA-emulsified NPs, respectively. In other words, the effect of the polymeric material as well as the surfactant on the cell viability was almost negligible. Thus, cell cytotoxicity of MCF-7 observed was mainly attributed to the sustained release of docetaxel itself from the NPs over the period of study. 96 The in vitro therapeutic effects of a dosage form can be quantitatively evaluated by its IC50, which is defined as the drug concentration at which 50% of the cells in a culture are killed over a designated time period. Table 2 summarizes the IC50 value of MCF-7 cells after 24, 48 and 72 h incubation with docetaxel formulated in Taxotere®, the PVA- or TPGS-emulsified PLA-TPGS NPs at various drug concentrations. From the data, the advantage of the NP formulation versus the pristine drug as well as that of TPGSemulsified NPs over PVA-emulsified NPs formulation can be observed. Figure 33: Viability of MCF-7 breast cancer cells incubated with docetaxel-loaded TPGSor PVA-emulsified PLA-TPGS NPs in comparison with that of Taxotere® at different docetaxel concentrations after 24, 48 and 72 h. Data represent mean ± SD (n=6). 97 Generally, the IC50 values were in the order of TPGS-emulsified NP < PVA-emulsified NP < Taxotere®. For example, TPGS-emulsified NP and PVA-emulsified NP formulation were 2.59-fold and 1.35-fold more effective than Taxotere® based on cell viability on Day 2. Therefore, lesser amount of drug is required for nanoparticle formulations to destroy half of the cancer cell population. Looking from another perspective, unnecessary loss of chemotherapeutic agents as a result of physiological efflux system such as p-glycoprotein can be minimized while more drugs can now permeate the cancer cell membrane through the endocytic mechanism. Table 2: IC50 of MCF-7 cells after 24, 48, 72 h incubation with docetaxel formulated in Taxotere®, PVA- and TPGS-emulsified PLA-TPGS NPs at various drug concentrations. Incubation time (h) 5.5 IC50 (µg/ml) PVA-emulsified NPs TPGS-emulsified NPs Taxotere® 24 7.890 5.850 10.380 48 0.472 0.246 0.636 72 0.059 0.043 0.068 Conclusion In vitro cellular study was performed in this chapter. Cellular uptake using fluorescent coumarin 6-loaded TPGS- emulsified and PVA-emulsified NPs demonstrated that the uptake efficiency of both type of NPs was time- and NP concentration-dependent. This suggested the presence of endocytic uptake mechanism of NPs. In addition to that, uptake efficiency of TPGS-emulsified NPs was generally higher than that of PVA-emulsified 98 NPs, attributing to the circumvention of P-gp MDR effect of cancer cells by the amphiphilic TPGS coated on the NP surface. Confocal microscopy images were also taken to show the cell morphology and compartments through staining by fluorescent dyes. On one hand, the results quanlitatively confirmed the cellular uptake study. On the other hand, the fluorescent intensity of unencapsulated free coumarin 6 incubated with the cells was observed to be negligible compared to NP formulations, thus concluding a low affinity of free coumarin 6 to the cells. Furthermore, the advantage of TPGS for drug delivery was also shown by the higher cell cytotoxicity than PVA-emulsified NPs or commercial Taxotere®. The lower IC50 in the order of TPGS-emulsified NP < PVAemulsified NP < Taxotere® further summarized the higher therapeutic efficacy of NP formulation over the free drug. . 99 CHAPTER 6: IN VIVO PHARMACOKINETICS AND EX VIVO BIODISTRIBUTION 6.1 Introduction Regardless of a new generic drug or nanoparticle formulation for drug delivery, the activity of a formulation tested against in vitro models is sometimes unable to be used to predict its fate in in vivo model, not to mention clinical cancers (Johnson et al., 2001). 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 recent years, fluorescence-labeled or radionuclide-labeled nanocarriers are some alternatives quite commonly used to study the circulation and distribution of nanoparticles in the body over a certain period of time (Rossin et al., 2008; Hu et al., 2009). They have been successfully used to more precisely locate the nanocarriers either by tissue analysis or imaging. In this chapter, in vivo pharmacokinetics was carried out to study the long circulation effect and potential toxicity of nanoparticles in rats. In addition to that, we further analyzed the drug distribution and content in various rat organs through biodistribution to demonstrate how nanoparticles can modify the accumulation of docetaxel in various parts of the body. The preliminary results are particularly useful as they serve as a platform for further modification and improvement of the current nanoparticle formulations. 100 6.2 Materials Male Sprague-Dawley (SpD) 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. 6.3 Methods 6.3.1 In Vivo Pharmacokinetics (PK) 6.3.1.1 Injection of Drugs The animals were randomly distributed into two groups (five rats for each group). Group 1 received an i.v. injection of docetaxel-loaded TPGS-emulsified PLA-TPGS NPs while Group 2 received an i.v. injection of commercial Taxotere®. The docetaxel-loaded nanoparticles and Taxotere® were dispersed or diluted with saline while maintaining the volume to be injected at 5 ml/kg rat/site. The formulation was subsequently administrated through the lateral tail vein at a docetaxel dose of 10 mg/kg body weight. All animals 101 were regularly monitored for their general health condition, clinical signs, stress, movement and activity or mortality. 6.3.1.2 Blood Collection, Sample Processing and Analysis For Group 1, blood samples were collected from tail vein at 0 (pre-dose), 0.5, 3, 5, 8, 10, 24, 48, 72, 121, 170, 241, 313 and 360 h. For Group 2, blood samples were collected at 0 (predose), 0.5, 3, 5, 8, 10, and 24 h after administration of the drug. Plasma samples were harvested by centrifugation at 8,000 rpm for 10 min and stored at -80°C until HPLC analysis. Liquid-liquid extraction using 1.2 ml of ethyl acetate was performed to extract the drug from the plasma. After centrifugation at 10,000 rpm for 15 min, the organic layer was transferred to a new tube for drying. Then, the drug was reconstituted in 100 μl of mobile phase A (acetonitrile:methanol:water 45:5:50 v/v/v). After vortex for 1 min, the solution was centrifuged again at 10,000 rpm for 15 min. 90 μl of the solution was transferred into HPLC insert for analysis. A sample volume of 70 μl was injected into HPLC column (Agilent Eclipse XDB-C18, 4.6 × 250 mm, 5 μm) under a flow rate of 1.0 ml/min using gradient elution. The proportion of mobile phases was decreased from 100% mobile phase B (acetonitrile:methanol:water 40:5:55 v/v/v) to 0% in 50 min. The concentration of drug in the sample was determined from the predetermined calibration curve using blank plasma priming with known docetaxel concentration before extraction. The in vivo PK of Taxotere® and the TPGS-emulsified PLA-TPGS NP formulation was expressed in plots of the plasma drug concentration vs. time. The two key parameters which represented the in vivo therapeutic effects were AUC (area under the curve of plasma drug concentration vs. 102 time) and half-life of the drug in the circulation. Other parameters such as maximum concentration and blood clearance were also included. 6.3.2 Biodistribution (BD) 6.3.2.1 Injection of Drugs The animals were randomly distributed into two groups (twelve rats for each group corresponding to 4 time points). Group 1 received an i.v. injection of docetaxel-loaded TPGS-emulsified PLA-TPGS NPs while Group 2 received an i.v. injection of commercial Taxotere®. The docetaxel-loaded nanoparticles and Taxotere® were dispersed or diluted with saline while maintaining the volume to be injected at 5 ml/kg rat/site. The formulation was subsequently administered through the lateral tail vein at a docetaxel dose of 10 mg/kg body weight. All animals were regularly monitored for their general health condition, clinical signs, stress, movement and activity or mortality. 6.3.2.2 Tissue Collection, Sample Processing and Analysis At designated time points, namely 1, 5, 10 and 24 h, three rats from each group were sacrificed by cardiac puncture method under anesthetic condition. All the organs (liver, spleen, kidney, brain, heart, lungs, intestine and stomach) and blood were extracted and rinsed with cold PBS to remove traces of blood. The samples were stored at -80°C until analysis. The blood was analyzed according to the procedures discussed for pharmacokinetics. For organs, the samples were freeze-dried and homogenized into powder. 50 mg of the powder was dissolved and vortexed in 0.5 ml of PBS. Then, tissue solution was extracted with 1.5 103 ml ethyl acetate. The mixture was vigorously stirred for 90 s. After that, the tissue emulsion was centrifuged at 10,000 rpm for 15 min. The organic layer was transferred into a new tube for drying. Following that, 0.1 ml of acetonitrile/water mixture (50:50 v/v) was added into the dried tube. The tube was further vortexed for another 90 s before centrifuging at 10,000 rpm for 15 min. Finally, 90 μl of the solution was transferred into the HPLC insert for analysis. An injection volume of 70 μl was injected into the system. The parameter setting for HPLC system was the same as that for pharmacokinetics. The concentration of drug in the tissue sample was determined from the predetermined calibration curve using blank tissue priming with known docetaxel concentration before extraction. 6.4 Results and Discussion 6.4.1 Pharmacokinetics In vivo pharmacokinetics of docetaxel-loaded PLA-TPGS NPs or commercial formulation Taxotere® was investigated by using SpD rats through the tail vein at a same dose of 10 mg/kg. The pharmacokinetic profiles of the drug concentration in the plasma for both the formulations over a certain period of time are shown in Figure 34, from which it can be seen that PLA-TPGS NP formulation exhibited significant improvement over the commercial formulation Taxotere®. The drug concentration for the PLA-TPGS NP formulation was slowly decreasing. In contrast, there was a dramatic decline of drug concentration for Taxotere® within 24 h after administering the same dose. It has been reported that the concentration of docetaxel required to reduce cell survival in various types of tumors such as murine and human tumor cell lines by 50% (in vitro IC50 or minimum effective level) ranged from 4 – 35 ng/ml (Hill et al., 1994; Bissery, 1995). 104 Therefore, the NPs can ensure a therapeutic effect (above 35 ng/ml) up to 360 h after the i.v. injection. This implied that one dose can make an effective chemotherapy possible by potentially maintaining the drug level above the minimum therapeutic level over a much longer period of time than the free drug at an equivalent dose. Additionally, in comparison with some studies on commercial Taxol® at the same dose, the concentration of free docetaxel in rats at certain time was generally found to be higher, probably due to its relatively higher solubility and the slower efflux (Riou et al., 1994; Gligorov and Lotz, 2004; Brunsvig et al., 2007; Dong and Feng, 2007). 100000 10000 Taxotere NP minimum effective level maximum tolerated level Concentration (ng/ml)a 100000 1000 100 10 10000 0 5 10 15 20 25 1000 100 10 0 50 100 150 200 250 300 350 Time (h) Figure 34: In vivo pharmacokinetics profiles of plasma drug concentration versus time after i.v. administration of Taxotere® and TPGS-emulsified PLA-TPGS nanoparticles formulation using SpD rats (n=5) at the same docetaxel dose of 10 mg/kg. 105 The pharmacokinetic curves were further analyzed by Kinetica software to obtain the mean non-compartmental pharmacokinetic parameters as shown in Table 3. The total area-under-the-curve (AUC0-∞), which determines the overall therapeutic effect of a formulation, was found to be 3.92×105 ± 9.72×104 ng•h/ml for the PLA-TPGS NP formulation, which was 3.43-fold larger than that of Taxotere® (1.14×105 ± 7.13×104 ng•h/ml). The ratio of AUC of novel formulation to commercial drug was in the same order with the results shown by Senthilkumar et al. where AUC for PLGA-mPEG NP formulation was found to be more than 3-fold compared to free docetaxel solution alone (Senthilkumar et al., 2008). The half-life (t1/2), which is the time at which the drug concentration drops to 50% of its initial value, of the drug formulated in PLA-TPGS NPs was found to be 83.8 ± 9.61 h, compared to that for Taxotere® (4.17 ± 1.92 h). Some reports have demonstrated that the mean half-life of Taxotere® at a dose of 10 mg/kg in SpD rats was 3.07 h (Gao et al., 2008). Also, Bissery has shown that the half-life of Taxotere® in normal tissue of tumorbearing mice ranged from 2.2 to 4.5 h while about 1.2 h in blood plasma (Bissery, 1995). As tumor-bearing mice may have higher metabolism and enhanced permeability and retention (EPR) effect than normal healthy rats, the half-life of the free drug is expected to be much lower as a result of the sink condition induced by the tumor. Also, the longer tmax of NP formulation (3.0 h) compared to Taxotere® (0.5 h) implied that the polymeric matrix of the NP can prevent the exposure of the drug all at one time in the body. Thus, the half-life, as well as the mean residence time, of drug concentration for NP formulation was much longer than Taxotere®, as shown in the Table 3. The longer circulation time was attributed to the much lower plasma clearance for NP formulation (0.024 l/h/kg) compared 106 to Taxotere® (0.112 l/h/kg), due to the bio-enhancing ability of TPGS-emulsified NPs to reduce the elimination rate of the drug from the body. Table 3: Mean non-compartmental pharmacokinetic parameters of SpD rats for intravenous administration of Taxotere® and TPGS-emulsified docetaxel-loaded PLATPGS NPs at a dose of 10 mg/kg. Taxotere® NPs 0.5 3.0 14,990 ± 4,800 7,250 ± 1,120 1.14 ×105 ± 7.13 ×104 3.92 ×105 ± 9.72 ×104 t1/2 (h) 4.17 ± 1.92 83.87 ± 9.61 MRT (h) 3.69 ± 1.42 73.89 ± 10.39 111.51 ± 63.97 23.46 ± 5.84 0.84 0.19 Parameter tmax (h) Cmax (ng/ml) AUC0-∞ (ng•h/ml) CL (ml/h/kg) AUCToxic /AUC0-∞ AUC is area under concentration-time curve t1/2 is the biological half-life MRT stands for mean residence time CL stands for plasma clearance AUCToxic /AUC0-∞ is the probability of experiencing any toxicity On the other hand, the peak at which the drug concentration reached a maximum value in the blood circulation was found to be 14.9 µg/ml and 7.2 µg/ml for Taxotere® and PLATPGS NPs, respectively. The maximum tolerated dose (MTD) for Taxotere® has been reported to be 1.6-fold lower than that of Taxol®, which was estimated to be 5,340 ng/ml (Bissery et al., 1991a; Riou et al., 1992; Liebmann et al., 1993). Although both drug 107 concentration of Taxotere® and NPs formulation could exceed the estimated MTD some time during the first 10 h after drug administration, the Cmax for NP formulation was much lower than that of Taxotere®. If lower NP dose is given, the drug concentration can be maintained within the therapeutic window of docetaxel for a longer period. This implied that nanocarriers have great potential to alleviate, if not completely avoid, such risk as prostration and respiratory distress, systemic and other drug-related toxicity (Engels et al., 2007; Xu et al., 2009). Sometimes, the adjuvant used to solubilize the drug often brings unwanted effects as well. As a result, by using nanocarriers to deliver drug, adverse effects associated with the adjuvant, which is normally experienced by patients undergoing chemotherapy, can also be greatly reduced. Therefore, by comparing the two formulations, it can be deduced that NP formulation can be a more efficient device to deliver docetaxel with prolonged therapeutic effect. 6.4.2 Biodistribution Apart from the efficacy of NP formulation, toxicity is another major concern for nanocarriers. We further evaluated the drug distribution of the formulations to various rat organs. From Figure 35, it can be seen that the drug in the form of Taxotere® was mainly distributed to liver, spleen and kidney, very similar to the trend reported (Senthilkumar et al., 2008). But the average drug accumulation in liver for NP formulation was relatively lower than Taxotere® at 10 h after drug administration. This was due to the ability of NP to reduce the first-pass metabolism of liver. At 24 h, the drug level in liver slightly increased, probably due to continuous release of drug from the prolonged nanoparticle 108 circulation in the body, compared to the significant drop for Taxotere® level in liver at the same time. Figure 35: Biodistribution of docetaxel delivered by commercial Taxotere® and PLATPGS NPs to SpD rats at 1, 5, 10 and 24 h after i.v. administration at the same docetaxel dose of 10 mg/kg (n=3). 109 Meanwhile, the drug levels in kidney, heart, intestine and stomach were comparable at various time intervals after Taxotere® injection. Except for lungs, the drug accumulations in these organs after 1 h were significantly lower (p < 0.05) for NP formulation than Taxotere®. It was observed that the drug level in lungs was much higher for NP formulation than Taxotere®, with the drug content AUC0-24h in lungs about 2.2-fold larger. It may be the drawback of this formulation as it may potentially cause toxicity or other side effects to lungs. The observation can be due to the fact that macrophage-like cells are also found in lung alveoli, which has the function of capturing particles during respiration or gas exchange (Hume, 2006). Thus, this process may have induced higher affinity of the NPs to these macrophages located in lungs compared to the native drug, probably because of more diffusion of NPs across the capillary epithelium to lung alveoli as an extravasation effect of small particle size. On the other hand, as already discussed in Section 6.4.1, the respective peak concentration Cmax for Taxotere® and PLA-TPGS NPs (Figure 35 and Table 3) in the blood circulation were found to have exceeded the maximum tolerated dose (MTD) (5,340 ng/ml) estimated for docetaxel. The sudden increase of drug concentration in blood often contributes not only to common hematological toxicity reported in clinical treatment, but also to nonhematological toxicity such as diarrhea (Hurria et al., 2006) which was observed in rats given Taxotere® in our case. Although the probability of experiencing certain kinds of toxicities (AUCToxic) during the course of study was 4.4-fold lower for NPs (19%) than Taxotere® (84%) (Table 3), the NPs were believed to be able to sustain the drug 110 concentration within the therapeutic window of docetaxel for a longer treatment period with negligible AUCToxic if a lower dosage was administered. Lastly, only very small amount Taxotere® can across the blood brain barrier at 5 h (0.032 µg/g) and 10 h (0.023 µg/g) (Figure 36). No drug was detected at 24 h, indicating fast drug metabolism or elimination of drug by P-glycoprotein efflux pumps in brain. Improvement observed for NP formulation was that the NPs can slightly bypass the barrier to attain a drug level of 0.273 µg/g at 5 h. Even though the level dropped to only 0.087 µg/g after 24 h, it showed the capability of the PLA-TPGS NP formulation to deliver drug to the brain for short period treatment of various brain diseases. Figure 36: Biodistribution of docetaxel delivered to the brain by commercial Taxotere® and PLA-TPGS NPs to SpD rats at 1, 5, 10 and 24 h after i.v. administration at the same docetaxel dose of 10 mg/kg (n=3). 111 6.5 Conclusion In this chapter, in vivo tests using rats were performed to evaluate the pharmacokinetics and biodistribution of the drug delivered in NP formulation as well as commercial formulation Taxotere®. PLA-TPGS NPs showed a long circulation effect in the rat blood circulation up to 360 h while still maintaining the drug concentration level in the therapeutic window of docetaxel. Besides that, the NPs demonstrated a prolonged circulation half-life and slower clearance from the body, thus showing the enhanced bioavailability of drug by encapsulation using amphiphilic copolymer containing TPGS. On the other hand, NP formulation can reduce the potential side effects of the drugs by limiting the exposure of the drug to the body at a certain period of time. This can also be observed through the drug distribution of NPs and Taxotere® in various organs. Drug accumulation was essentially lower in some important organs for NPs in comparison to Taxotere®. However, the drug content was higher in lungs for NPs, suggesting a possible formulation for lung-related cancers. In addition to that, NPs can deliver higher amount of drug, for a short period of time, to the brain. It improved the efficiency of conventional drug formulation which was otherwise unable to reach the brain for effective treatments. 112 CHAPTER 7: CONCLUSION AND FUTURE WORKS 7.1 Conclusion The main objective of this thesis is to develop a novel nanoparticulate system using an amphiphilic copolymer PLA-TPGS for the delivery of anticancer drug docetaxel. From a full series of study, namely the synthesis and characterization of PLA-TPGS copolymer, the fabrication and characterization of nanoparticles, in vitro cellular uptake and viability as well as in vivo pharmacokinetics and biodistribution, PLA-TPGS has been shown as a promising drug carrier for more effective chemotherapy. In Chapter 1, thesis objectives and a general background of the developmental progress of nanomedicine were provided. Then, Chapter 2 highlighted some facts about cancer and their 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 started the main purpose of this thesis with the synthesis of amphiphilic PLA-TPGS copolymer with a PLA:TPGS weigh component ratio of 89:11 and a molecular weight of about 14,500. Various characterization methods such as 1H NMR, GPC, TGA and FT-IR were performed to confirm the successful synthesis of PLATPGS copolymer. Following that, Chapter 4 contained the fabrication and characterization processes of PLA-TPGS NPs. In the same chapter, the quality of nanoparticles prepared using TPGS and PVA as emulsifiers was studied various state-of-the-art analytical instruments. TPGS-emulsified PLA-TPGS NPs was demonstrated to have smaller particle size of about 240 nm than that using PVA (296 nm). The NP stability and drug 113 encapsulation efficiency were also found enhanced when TPGS was used as a surfactant. Overall, all the NPs showed a controlled in vitro drug release over a period of 30 days at pH 7.4 and temperature of 37°C. The NPs were further evaluated in Chapter 5 using human breast cancer cell line MCF-7 as an in vitro model. The time- and concentrationdependent cellular uptake of NPs could be explained by the presence of endocytosis process of the model. Also, TPGS-emulsified NPs showed better uptake efficiency than that using PVA, as shown by CLSM images. Furthermore, the therapeutic efficacy of the NP formulation in killing cancer cells was studied using the same cell line. Compared to its current clinical formulation Taxotere®, NP formulations induced greater cytotoxicity over 3 days. Again, TPGS-emulsified NPs, in some cases, showed even higher efficiency in cytotoxicity than PVA-emulsified NPs. This was confirmed by the IC50 values in the order of TPGS-emulsified NPs < PVA-emulsified NPs < Taxotere®. Lastly, animal study in Chapter 6 demonstrated the ability of NPs to reduce elimination and clearance rates by circumventing the MPS or other efflux transport systems in the body. This helped the NPs to cross, to certain extent, the BBB for improved treatment of brain disorders. In contrast to Taxotere®, the maintenance of drug level within therapeutic window up to 360 h as well as lower drug accumulation in some organs implied lower probability of experiencing serious side effects from NP formulation. By and large, it could be concluded that biodegradable PLA-TPGS NPs serves as potential candidates for delivering therapeutic drugs to the targeted tumor sites over an extended period of time in a sustainable manner while reducing the adverse effects of anticancer drugs to enhance the life quality of patients undergoing chemotherapy. 114 7.2 Future Works The application of nanotechnology for the innovation of new nanoparticulate systems for drug delivery will continue to grow in view of the limitations of the current available clinical formulations in tackling the medical complications caused by cancers. Some of the future works that may improve the current work included the following: (i) Development of xenograft tumor models in mice to further evaluate the antitumor efficacy and possible side effects of the nanoparticle formulations. (ii) Conjugation of specific ligands (endogenous or exogenous) onto the nanoparticle surfaces to drive the devices to targeted tumor sites. (iii) Customization of the nanoparticles to carry genes to cell nuclei for gene delivery or DNA repair to treat cancers at the genetic level. (iv) Modifying the nanoparticles to carry therapeutic and imaging agents for multifunctional purposes such as cancer diagnosis, imaging and treatment at the same time. 115 REFERENCES Adkins, J. C. and D. Faulds. Amprenavir, Drugs, 55(6), pp. 837-842. 1998. Aizawa, H. Morphology of polysorbate 80 (Tween 80) micelles in aqueous 1,4-dioxane solutions, J. Appl. Cryst, 42(4), pp. 592-596. 2009. Albayrak, C. Induced phase transition method for the production of microparticles containing hydrophobic active agents. U.S. Patent 6,899,898. 2005. Aliabadi, H. M., A. Mahmud, A. D. Sharifabadi and A. Lavasanifar. Micelles of methoxy poly(ethylene oxide)-b-poly(ε-caprolactone) as vehicles for the solubilization and controlled delivery of cyclosporine A, J. Control. Release, 104, pp. 301-311. 2005. Allémann, E., R. Gurny and E. Doelker. Drug-loaded nanoparticles – preparation methods and drug targeting issues, Eur. J. Pharm. Biopharm, 39(5), pp. 173-191. 1993. Allémann, E., J.-C. Leroux, R. Gurny and E. Doelker. In Vitro Extended-Release Properties of Drug-Loaded Poly(DL-Lactic Acid) Nanoparticles Produced by a SaltingOut Procedure, Pharm. Res, 10(12), pp. 1732-1737. 1993a. Allémann, E., J.-C. Leroux and R. Gurny. Polymeric nano-microparticles for the oral delivery of peptides and peptidomimetics, Adv. Drug Deliv. Rev, 34(2-3), pp. 171-189. 1998. Allen, M. T. The use of glycolipids and hydrophilic polymers in avoiding rapid uptake of liposomes by the mononuclear phagocyte system, Adv. Drug Deliv. Rev, 13(3), pp. 285309. 1994. Amass, W., A. Amass and B. Tighe. A review of biodegradable polymers: Uses, current development in the synthesis and characterization of biodegradable polyesters, blends of biodegradable polymers and recent advances in biodegradation studies. Polymer Int, 47(2), pp. 89-144. 1998. Amellar, T., V. Marsaud, P. Legrand, R. Gref, G. Barratt and J.-M. Renoir. PolyesterPoly(Ethylene Glycol) Nanoparticles Loaded with the Pure Antoestrogen RU 58668: Physicochemical and Opsonization Properties, Pharm. Res, 20(7), pp. 1063-1070. 2003. 116 Amidon, G. L., H. Lennernäs, V. P. Shah and J. R. Crison. A Theoretical Basis for a Biopharmaceutic Drug Classification: The Correlation of in Vitro Drug Product Dissolution and in Vivo Bioavailability, Pharm. Res, 12(3), pp. 413-420. 1995. Ammoury, N., H. Fessi, J.-P. Devissaguet, F. Puisieux and S. Benita. In vitro release pattern of indomethacin from poly(D,L-lactide) nanocapsules, J. Pharm. Sci, 79(9), pp. 763-767. 1990. Ammoury, N., H. Fessi, J.-P. Devissaguet, M. Dubrasquet and S. Benita. Jejunal Absorption, Pharmacological Activity, and Pharmacokinetic Evaluation of IndomethacinLoaded Poly(Isobutyl-Cyanoacrylate) Nanocapsules in Rats, Pharm. Res, 8(1), pp. 101105. 1991. Anderson, K., M. Simmons-Menchaca, K. A. Lawson, J. Atkinson, B. G. Sanders and K. Kline. Differential Response of Human Ovarian Cancer Cells to Induction of Apoptosis by Vitamin E Succinate and Vitamin E Analogue, α-TEA, Cancer Res, 64(12), pp. 42634269. 2004. Argao, E. A., J. E. Heubi, B. W. Hollis and R. C. Tsang. d-α-Tocopheryl Polyethylene Glycol-1000 Succinate Enhances the Absorption of Vitamin D in Chronic Cholestatic Liver Disease of Infancy and Childhood, Pediatr. Res, 31(2), pp. 146-150. 1992. Arshady, R. Preparation of biodegradable microspheres and microcapsules: 2. Polyactides and related polyesters, J. Control. Release, 17(1), pp. 1-21. 1991. AZoNanotechnology Article. Liposomes – Classification, Processing Technologies, Industry Applications and Risk Assessment. Retrieved on June 18, 2009 from the World Wide Web: http://www.azonano.com/Details.asp?ArticleID=1243 Bae, Y., N. Nishiyama, S. Fukushima, H. Koyama, M. Yasuhiro and K. Kataoka. Preparation and biological characterization of polymeric micelle drug carriers with intracellular pH-triggered drug release property: tumor permeability, controlled subcellular drug distribution, and enhanced in vivo antitumor efficacy, Bioconjug. Chem, 16(1), pp. 122-130. 2005. Baker, S. D., M. Zhao, P. He, M. A. Carducci, J. Verweij and A. Sparreboom. Simultaneous analysis of docetaxel and the formulation vehicle polysorbate 80 in human plasma by liquid chromatography/tandem mass spectrometry, Anal. Biochem, 324(2), pp. 276–284. 2004. Baker, S. D., A. Sparreboom and J. Verweij. Clinical pharmacokinetics of docetaxel: recent developments, Clin. Pharmacokinet, 45, pp. 235-252. 2006. 117 Bareford, L. M. and P. W. Swaan. Endocytic mechanisms for targeted drug delivery, Adv. Drug Deliv. Rev, 59(8), pp. 748-758. 2007. Barichello, J. M., M. Morishita, K. Takayama and T. Nagai. Encapsulation of Hydrophilic and Lipophilic Drugs in PLGA Nanoparticles by the Nanoprecipitation Method, Drug Dev. Ind. Pharm, 25(4), pp. 471-476. 1999. Barratt, G. M. Therapeutic applications of colloidal drug carriers, Pharm. Sci. Technol. Today, 3(5), pp. 163–171. 2000. Bazile, D., C. Prud'homme, M.-T. Bassoullet, M. Marlard, G. Spenlehauer and M. Veillard. Stealth MePEG-PLA nanoparticles avoid uptake by the mononuclear phagocytes system, J. Pharm. Sci, 84(4), pp. 493-498. 1995. BBC News. Cancer: The Facts. Retrieved on March 2, 2009 from the World Wide Web: http://news.bbc.co.uk/2/hi/health/3444635.stm. Béduneau, A., P. Saulnier and J.-P. Benoit. Active targeting of brain tumors using nanocarriers, Biomaterials, 28(33), pp. 4947-4967. 2007. Ben-Shabat, S., N, Kumar and A. J. Domb. PEG-PLA Block Copolymer as Potential Drug CarrierL Preparation and Characterization, Macromol. Biosci, 6(12), pp. 1019-1025. 2006. Berg, S. L., A. Tolcher, J. A. O’Shaughnessy, A. M. Denicoff, M. Noone, F. P. Ognibene, K. H. Cowan and F. M. Balis. Effect of R-verapamil on the pharmacokinetics of paclitaxel in women with breast cancer, J. Clin. Oncol, 13, pp. 2039-2042. 1995. Bermudez, H., A. K. Brannan, D. A. Hammer, F. S. Bates and D. E. Discher. Molecular Weight Dependence of Polymersome Membrane Structure, Elasticity, and Stability, Macromolecules, 35(21), pp. 8203-8208. 2002. Birringer, M., J. H. EyTina, B. A. Salvatore and J. Neuzil. Vitamin E analogues as inducers of apoptosis: structure-function relation, Br. J. Cancer, 88(12), pp. 1948-1955. 2003. Bissery, M.-C., A. Renard, G. Montay, M. Bayssas and F. Lavelle. Taxotere: Antitumor activity and pharmacokinetics in mice, Proc. Am. Assoc. Cancer Res, 32, pp. 401. 1991. 118 Bissery, M.-C., D. Guénard, F. Guéritte-Voegelein and F. Lavelle. Experimental Antitumor Activity of Taxotere (RP 56976, NSC 628503), a Taxol Analogue, Cancer Res, 51(18), pp. 4845-4852. 1991a. Bissery, M.-C. Preclinical Pharmacology of Docetaxel, Eur. J. Cancer, 31A, pp. S1-S6. 1995. Bissett, D., A. Setanoians, J. Cassidy, M. A. Graham, G. A. Chadwick, P. Wilson, V. Auzannet, N. Le Bail, S. B. Kaye and D. J. Kerr. Phase I and Pharmacokinetic Study of Taxotere (RP 56976) Administered as a 24-Hour Infusion, Cancer Res, 53, pp. 523-527. 1993. Boudreaux, J. P., D. H. Hayes, S. Mizrah, P. Maggiore, J. Blazek and D. Dick. Use of water-soluble liquid vitamin E to enhance cyclosporine absorption in children after liver transplantation, Transplant. Proc, 25(2), pp. 1875. 1993. Bouissou, C., U. Potter, H. Altroff, H. Mardon and C. van der Walle. PLA/PLGA nanoparticles for sustained release of docetaxel, J. Control. Release, 95(3), pp. 557–566 2004. Bradshaw-Pierce, E. L., S. Eckhardt and D. L. Gustafson. A Physiologically Based Pharmacokinetic Model of Docetaxel Disposition: from Mouse to Man, Clin. Cancer Res, 13, pp. 2768-2776. 2007. Brigger, I., C. Dubernet and P. Couvreur. Nanoparticles in cancer therapy and diagnosis, Adv. Drug Deliv. Rev, 54(5), pp. 631–651. 2002. Brown, C. The Independent. Suppressed report shows cancer link to GM potatoes. Retrieved on January 05, 2009 from the World Wide Web: http://www.independent.co.uk/life-style/health-and-wellbeing/health-news/suppressedreport-shows-cancer-link-to-gm-potatoes-436673.html. Brunsvig, P. F., A. Anderson, S. Aamdal, V. Kristensen and H. Olsen. Pharmacokinetic analysis of two different docetaxel dose levels in patients with non-small cell lung cancer treated with docetaxel as monotherapy or with concurrent radiotherapy, BMC Cancer, 7, pp. 197. 2007. Bunn, P. A. and K. Kelly. New Chemotherapeutic Agents Prolong Survival and Improve Quality of Life in Non-Small Cell Lung Cancer: A Review of the Literature and Future Directions, Clin. Cancer Res, 5, pp. 1087-1100. 1998. 119 Burstein, H. J., J. Manola, J. Younger, L. M. Parker, C. A. Bunnell, R. Scheib, U. A. Matulonis, J. E. Garber, K. D. Clarke, L. N. Shulman and E. P. Winer. Docetaxel Administered on a Weekly Basis for Metastatic Breast Cancer, J. Clin. Oncol, 18, pp. 1212-1219. 2000. Cancer Research UK. Lung Cancer. Retrieved on March 14, 2009 from the World Wide Web: http://info.cancerresearchuk.org/cancerstats/types/lung/?a=5441. Cancer Research UK. What Causes Cancer? Retrieved on March 14, 2009 from the World Wide Web: http://www.cancerhelp.org.uk/help/default.asp?page=119#genetic. Camphausen, K., M. A. Moses, W.-D. Beecken, M. K. Khan, J. Folkman and M. S. O’Reilly. Radiation therapy to a primary tumor accelerates metastatic growth in mice, Cancer Res, 61, pp. 2207-2211. 2001. Cao, N. and S.-S. Feng. Doxorubicin conjugated to D-α-tocopheryl polyethylene glycol 1000succinate (TPGS): Conjugation chemistry, characterization, in vitro and in vivo evaluation, Biomaterials, 29(28), pp. 3856-3865. 2008. Chan, J. M., L. F. Zhang, K. P. Yuet, G. Liao, J.-W. Rhee, R. Langer and O. C. Farokhzad. PLGA-lecithin-PEG core-shell nanoparticles for controlled drug delivery, Biomaterials, 30(8), pp. 1627-1634. 2009. Chang, T., L. Z. Benet and M. F. Hebert. The effect of water-soluble vitamin E on cyclosporine pharmacokinetics in healthy volunteers, Clin. Pharmacol. Ther, 59(3), pp. 297-303. 1996. Chen, H. T., M. F. Neerman, A. R. Parrish and E. E. Simanek. Cytotoxicity, hemolysis, and acute in vivo toxicity of dendrimers based on melamine, candidate vesicles for drug delivery, J. Am. Chem. Soc, 126(32), pp. 10044-10048. 2004. Chen, W. R., H. Zheng, K. Andrienko, S. Stefanov, R. F. Wolf and L. Hong. Effects of surgery, immunization, and laser immunotherapy on a non-immunogenic metastatic tumor model, Proceedings of SPIE-The International Society for Optical Engineering, 6163, pp. 1-6. 2006. Chen, K., J. Xie, H. Xu, D. Behera, M. H. Michalski, S. Biswal, A. Wang and X. Chen. Triblock copolymer coated iron oxide nanoparticle conjugated for tumor integrin targeting, Biomaterials, 30(36), pp. 6912-6919. 2009. Cheng, J., B. A. Teply, I. Sherifi, J. Sung, G. Luther, F. X. Gu, E. Levy-Nissenbaum, A. F. Radovic-Moreno, R. Langer and O. C. Farokhzad. Formulation of functionalized PLGA120 PEG nanoparticles for in vivo targeted drug delivery, Biomaterials, 28(5), pp. 869-876. 2007. Chorny, M., I. Fishbein, H. D. Danenberg and G. Golomb. Lipophilic drug loaded nanospheres prepared by nanoprecipitation: effect of formulation variables on size, drug recovery and release kinetics, J. Control. Release, 83(3), pp. 389-400. 2002. Chou, D. K., R. Krishnamurthy, T. W. Randolph, J. F. Carpenter and M. C. Manning. Effects of Tween 20 and Tween 80 on the stability of Albutropin during agitation, J. Pharm. Sci, 94(6), pp. 1368-1381. 2005. Chupin, V., A. I. P. M. de Kroon and B. de Kruijff. Molecular Architecture of Nanocapsules, Bilayer-Enclosed Solid Particles of Cisplatin, J. Am. Chem. Soc, 126, pp. 13816-13821. 2004. Collnot, E.-M., C. Baldes, M. F. Wempe, J. Hyatt, L. Navarro, K. J. Edgar, U. F. Schaefer and C.-M. Lehr. Influence of vitamin E TPGS poly(ethylene glycol) chain length on apical efflux transporters in Caco-2 cell monolayer, J. Control. Release, 111(1-2), pp. 35-40. 2006. Collnot, E.-M., C. Baldes, M. F. Wempe, R. Kappl, J. Hüttermann, J. A. Hyatt, K. J. Edgar, U. F. Schaefer and C.-M. Lehr. Mechanism of Inhibition of P-Glycoprotein Mediated Efflux by Vitamin E TPGS: Influence on ATPase Activity and Membrane Fluidity, Mol. Pharm, 4(3), pp. 465-474. 2007. Connors, T. A. and R. J. Knox. Prodrugs in Cancer Chemotherapy, Stem Cells, 13(5), pp. 501-511. 1995. Corral, D. A., L. L. Pisters and A. C. von Eschenbach. Treatment options for localized recurrence of prostate cancer following radiation therapy, Urol. Clin. N. America, 23, pp. 677-684. 1996. Couvreur, P., C. Dubernet and F. Puisieux. Controlled drug delivery with nanoparticles: current possibilities and future trends, Eur. J. Pharm. Biopharm, 41(1), pp. 2-13. 1995. Crotts, G. and T. G. Park. Preparation of porous and nonporous biodegradable polymeric hollow microspheres, J. Control. Release, 35(2-3), pp. 91-105. 1995. Cutts, J. H. The Effect of Vincaleukoblastine on Dividing Cells in Vivo, Cancer Res, 21(2), pp. 168-172. 1961. 121 Danielson, P. B. The Cytochrome P450 Superfamily: Biochemistry, Evolution, and Drug Metabolism in Humans, Curr. Drug Metab, 3, pp. 561-597. 2002. Davis, W. W., R. R. Pfeiffer and J. F. Quay. Normal and promoted gastrointestinal absorption of water-soluble substances. I. Induced rapidly reversible hyperabsorptive state in the canine fundic stomach pouch, J. Pharm Sci, 59(7), pp. 960-963. 1970. Debotton, N., M. Pames, J. Kadouche and S. Benita. Overcoming the formulation obstacles towards targeted chemotherapy: In vitro and in vivo evaluation of cytotoxic drug loaded immunonanoparticles, J. Control. Release, 127(3), pp. 219-230. 2008. Dehouck, B., M. P. Dehouck, J. C. Fruchart and R. Cecchelli. Upregulation of the low density lipoprotein receptor at the blood-brain barrier: intercommunications between brain capillary endothelial cells and astrocytes, J. Cell Biol, 126(2), pp. 465-473. 1994. Deng, C., X. Chen, J. Sun, T. Lu, W. Wang and X. Jing. RGD Peptide Grafted Biodegradable Amphiphilic Triblock Copolymer Poly(glutamic acid)-b-Poly(L-lactide)-bPoly(glutamic acid): Synthesis and Self-Assembly, J. Polym. Sci. Part A: Polym. Chem, 45(15), pp. 3218-3230. 2007. Denis, J. N., A. E. Greene, D. Guenard, F. Gueritte-Voegelein, L. Mangatal and P. Potier. Highly efficient, practical approach to natural taxol, J. Am. Chem. Soc, 110, pp. 59175919. 1998. Derfus, A. M., W. C. W. Chan and S. N. Bhatia. Probing the Cytotoxicity of Semiconductor Quantum Dots, Nano Lett, 4(1), pp. 11-18. 2004. De Riese, W. T. W., T. J. Seipel, D. R. Aronoff, B. A. Sharpe and B. T. Mittemeyer. Cryo and laser surgery as new therapeutic options in patients with organ-confined prostate cancer who failed radiotherapy, Proceedings of SPIE-The International Society for Optical Engineering, 4609, pp. 362-372. 2002. Desai, M. P., V. Labhasetwar, E. Walter, R. J. Levy and G. L. Amidon. The mechanism of uptake of biodegradable microparticles in CaCO-2 cells is size dependant, Pharm. Res, 14(11), pp. 1568–1573. 1997. Dintaman, J. M. and J. A. Silverman. Inhibition of P-glycoprotein by D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), Pharm. Res, 16(10), pp. 1550-1556. 1999. Discher, B. M., Y.-Y. Won, D. S. Ege, J. C.-M. Lee, F. S. Bates, D. E. Discher and D. A. Hammer. Polymersomes: Tough Vesicles Made from Diblock Copolymers, Science, 284(5417), pp. 1143-1146. 1999. 122 Dixon, D. J., R. A. Bodmeier and K. P. Johnston. Polymeric Materials Formed by Precipitation with a Compressed Fluid Antisolvent, AIChE J, 39(1), pp. 127-139. 1993. Dong, Y. and S.-S. Feng. Methoxy poly(ethylene glycol)-poly(lactide) (MPEG-PLA) nanoparticles for controlled delivery of anticancer drugs, Biomaterials, 25(14), pp. 28432849. 2004. Dong, Y. and S.-S. Feng. In vitro and in vivo evaluation of methoxy polyethylene glycolpolylactide (MPEG-PLA) nanoparticles for small-molecule drug chemotherapy, Biomaterials, 28(28), pp. 4154-4160. 2007. Drori, S., G. D. Eytan and Y. G. Assaraf. Potentiation of anticancer-drug cytotoxicity by multidrug-resistance chemosensitizers involves alterations in membrane fluidity leading to increased membrane permeability, Eur. J. Biochem, 228(3), pp. 1020-1029. 1995. Dubois, J., D. Guenard and F. Gueritte. Recent development in antitumour taxoids, Expert. Opin. Ther. Pat, 13, pp. 1809-1823. 2003. Dudeja, P. K., K. M. Anderson, J. S. Harris, L. Buckingham and J. S. Coon. Reversal of Multidrug Resistance Phenotype by Surfactants: Relationship to Membrane Lipid Fluidity, Arch. Biochem. Biophys, 319(1), pp. 309-315. 1995. Duncan, R., H. Ringsdorf and R. Satchi-Fainaro. Polymer therapeutics – polymers as drugs, drug and protein conjugates and gene delivery systems: Past, present and future opportunities, J. Drug Target, 14(6), pp. 337-341. 2006. Eisenhauer, E. A. and J. B. Vermorken. The Taxoids: Comparative Clinical Pharmacology and Therapeutic Potential, Drugs, 55, pp. 5-30. 1998. Engels, F. K., R. A. Mathot and J. Verweij. Alternative drug formulations of docetaxel: a review, Anti-Cancer Drugs, 18(2), pp. 95-103. 2007. Farokhzad, O. C. and R. Langer. Nanomedicine: Developing smarter therapeutic and diagnostic modalities, Adv. Drug Deliv. Rev, 58, pp. 1456-1459. 2006. Farokhzad, O. C., J. Cheng, B. A. Teply, I. Sherifi, S. Jon, P. W. Kantoff, J. P. Richie and R. Langer. Targeted nanoparticle-aptamer bioconjugates for cancer chemotherapy in vivo, Proc. Natl. Acad. Sci, 103(16), pp. 6315-6320. 2006a. 123 Feng, S.-S. and R. C. MacDonald. Effects of Chain Unsaturation on the Equation of State for Lipid Monolayers at the Air-Water Interface, Biophys. J, 69(2), pp. 460-469. 1995. Feng, S.-S. and G. Huang. Effects of emulsifiers on the controlled release of paclitaxel (Taxol®) from nanospheres of biodegradable polymers, J. Control. Release, 71(1), pp. 5369. 2001. Feng, S.-S. and S. Chien. Chemotherapeutic Engineering: Application and further development of chemical engineering principles for chemotherapy of cancer and other diseases, Chem. Eng. Sci, 58(18), pp. 4087-4114. 2003. Feng, S.-S. New-concept chemotherapy by nanoparticles of biodegradable polymers – where are we now? Nanomedicine, 1(3), pp. 297-309. 2006. Feng, S.-S., L. Zhao, Z. Zhang, G. Bhakta, K. Y. Win, Y. Dong and S. Chien. Chemotherapeutic engineering: Vitamin E TPGS-emulsified nanoparticles of biodegradable polymers realized sustainable paclitaxel chemotherapy for 168 h in vivo, Chem. Eng. Sci, 62, pp. 6641-6648. 2007. Ferrari, M. Cancer nanotechnology: opportunities and challenges, Nature Rev. Cancer, 5(3), pp. 161–171. 2005. Fessi, H., F. Puisieux, J. Ph. Devissaguet, N. Ammoury and S. Benita. Nanocapsule formation by interfacial polymer deposition following solvent displacement, Int. J. Pharm, 55(1), pp. R1-R4. 1989. Fonseca, C., S. Simões and R. Gaspar. Paclitaxel-loaded PLGA nanoparticles: preparation, physicochemical characterization and in vitro anti-tumoral activity, J. Control. Release, 83(2), pp. 273-286. 2002. Fornari, F. A., J. K. Randolph, J. C. Yalowich, M. K. Ritke and D. A. Gewirtz. Interference by Doxorubicin with DNA Unwinding in MCF-7 Breast Tumor Cells, Mol. Pharmacol, 45(4), pp. 649-656. 1994. Frank, M. M. and L. F. Fries. The role of complement in inflammation and phagocytosis, Immunol. Today, 12(9), pp. 322-326. 1991. Fischer, J. R., K. R. Harkin and L. C. Freeman. Concurrent administration of watersoluble vitamin E can increase the oral bioavailability of cyclosporine A in healthy dogs, Vet. Ther: Res. Appl. Vet. Med, 3(4), pp. 465-473. 2002. 124 Gabizon, A., H. Shmeeda and Y. Barenholz. Pharmacokinetics of Pegylated Liposomal Doxorubicin: Review of Animal and Human Studies, Clin. Pharmacokinet, 42, pp. 419436. 2003. Galindo-Rodriguez, S., E. Allémann, H. Fessi and E. Doelker. Physicochemical Parameters Associated with Nanoparticle Formation in the Salting-out, EmulsificationDiffusion, and Nanoprecipitation Methods, Pharm. Res, 21(8), pp. 1428-1439. 2004. Gao, Y., L. Chen, W. Gu, Y. Xi, L. Lin and Y. Li. Targeted nanoassembly loaded with docetaxel improves intracellular drug delivery and efficacy in murine breast cancer model, Mol. Pharm, 5(6), pp. 1044–1054. 2008. Gavrilov, V., M. Steiner and S. Shany. The Combined Treatment of 1,25dihydroxyvitamin D3 and a Non-steroid Anti-inflammatory Drug is Highly Effective in Suppressing Prostate Cancer Cell Line (LNCaP) Growth, Anticancer Res, 25, pp. 34253429. 2005. Gelderblom, H., J. Verweij, K. Nooter and A. Sparreboom. Cremophor EL: the drawbacks and advantages of vehicle selection for drug formulation, Eur. J. Cancer, 37(13), pp. 15901598. 2001. Gelmon, K. The taxoids: paclitaxel and docetaxel, Lancet, 344, pp. 1267-1272. 1994. Gelperina, S., O. Maksimenko, A. Khalansky, L. Vanchugova, E. Shipulo, K. Abbasova, R. Berdiev, S. Wohlfart, N. Chepurnova and J. Kreuter. Drug delivery to the brain using surfactant-coated poly(lactide-co-glycolide) nanoparticles: Influence of the formulation parameters, Eur. J. Pharm. Biopharm, In Press. 2009. Gligorov, J. and J. P. Lotz. Preclinical Pharmacology of the Taxanes: Implications of the Differences, Oncol, 9, pp. 3-8. 2004. Goppert, T. M. and R. H. Muller. Plasma Protein Adsorption of Tween 80- and Poloxamer 188-stabilized Solid Lipid Nanoparticles, J. Drug Target, 11(4), pp. 225-231. 2003. Grant, W. B. and C. F. Garland. A Critical Review of Studies on Vitamin D in Relatio to Colorectal Cancer, Nutr. Cancer, 48(2), pp. 115-123. 2004. Gref, R., Y. Minamitake, M. T. Peracchia, V. Trubetskoy, V. Torchilin and R. Langer. Biodegradable long-circulating polymeric nanospheres, Science, 263(5135), pp. 16001603. 1994. 125 Gref, R., A. Domb, P. Quellec, T. Blunk, R. H. Müller, J. M. Verbavatz and R. Langer. The controlled intravenous delivery of drugs using PEG-coated sterically stabilized nanospheres, Adv. Drug Deliv. Rev, 16(2-3), pp. 215-233. 1995. Griffin, W. C. Calculation of HLB values of non-ionic surfactants, J. Soc. Cosmet. Chem, 5, pp. 249-256. 1954. Gu, F., L. Zhang, B. A. Teply, N. Mann, A. Wang, A. F. Radovic-Moreno, R. Langer and O. C. Farokhzad. Precise engineering of targeted nanoparticles by using self-assembled biointegrated block copolymers, Proc. Natl. Acad. Sci. USA, 105(7), pp. 2586-2591. 2008. Guengerich, F. P. Cytochrome p450 and chemical toxicology, Chem. Res. Toxicol, 21, pp. 70-83. 2008. Gulyaev, A. E., S. E. Gelperina, I. N. Skidan, A. S. Antropov, G. Y. Kivman and J. Kreuter. Significant transport of doxorubicin into the brain with polysorbate 80-coated nanoparticles, Pharm. Res, 16(10), pp. 1564-1569. 1999. Haldar, S., A. Basu and C. M. Croce. Bcl2 is the Guardian of Microtubules Integrity, Cancer Res, 57, pp. 229-233. 1997. Hanauske, A.-R., H. Depenbrock, D. Shirvani and J. Rastetter. Effects of the microtubuledisturbing agents docetaxel (Taxotere), vinblastine and vincristine on epidermal growth factor-receptor binding of human breast cancer cell lines in vitro, Eur. J. Cancer, 30(11), pp. 1688-1694. 1994. Hao, Y.-L., Y.-J. Deng, Y. Chen, K.-Z. Wang, A.-J. Hao and Y. Zhang. In-vitro cytotoxicity, in-vivo biodistribution and anti-tumour effect of PEGylated liposomal topotecan, J. Pharm. Pharmacol, 57(10), pp. 1279-1287. 2005. Hawker, C. J. and J. M. J. Fréchet. Preparation of Polymers with Controlled Molecular Architecture. A New Convergent Approach to Dendritic Macromolecules, J. Am. Chem. Soc, 112(21), pp. 7638-7647. 1990. Hennenfent, K. L. and R. Govindan. Novel formulations of taxanes: a review. Old wine in a new bottle? Ann. of Oncol, 17, pp. 735-749. 2006. Higgins, C. F. and M. M. Gottesman. Is the multidrug transporter a flippase? Trends Biochem. Sci, 17(1), pp. 18-21. 1992. 126 Hill, B. T., R. D. H. Whelan, S. A. Shellard, S. McClean and L. K. Hosking. Differential cytotoxic effects of docetaxel in a range of mammalian tumor cell lines and certain drug resistant sublines in vitro, Invest. New Drugs, 12(3), pp. 169-182. 1994. Hoshino, A., K. Fujioka, T. Oku, M. Suga, Y. F. Sasaki, T. Ohta, M. Yasuhara, K. Suzuki and K. Yamamoto. Physicochemical Properties and Cellular Toxicity of Nanocrystal Quantum Dots Depend on Their Surface Modification, Nano Lett, 4(11), pp. 2163-2169. 2004. Howes, P., R. Thorogate, M. Green, S. Jickells and B. Daniel. Synthesis, characterization and intracellular imaging of PEG capped BEHP-PPV nanospheres, Chem. Comm, 18(18), pp. 2490-2492. 2009. Hu, K., J. Li, Y. Shen, W. Lu, X. Gao, Q. Zhang and X. Jiang. Lactoferrin-conjugated PEG-PLA nanoparticles with improved brain delivery: In vitro and in vivo evaluations, J. Control. Release, 134(1), pp. 55-61. 2009. Hubbard, A. T. Encyclopedia of Surface and Colloid Science. New York: CRC Press, 2006. Hugger, E. D., K. L. Audus and R. T. Brochardt. Effects of Poly(ethylene glycol) on Efflux Transporter Activity in Caco-2 Cell Monolayers, J. Pharm. Sci, 91(9), pp. 19801990. 2002. Hugger, E. D., B. L. Novak, P. S. Burton, K. L. Audus and R. T. Brochardt. A Comparison of Commonly Used Polyethoxylated Pharmaceutical Excipients on Their Ability to Inhibit P-glycoprotein In Vitro, J. Pharm. Sci, 91(9), pp. 1991-2002. 2002a. Huizing, M. T., V. H. Sewberath Misser, R. C. Pieters, W. W. ten Bokkel Huinink, C. H. N. Veenhof, J. B. Vermorken, H. M. Pinedo and J. H. Beijnen. Taxanes: A New Class of Antitumor Agents, Cancer Invest, 13, pp. 381-404. 1995. Hume, D. A. The mononuclear phagocyte system, Curr. Opin. Immunol, 18, pp. 49-53. 2006. Hunter, J. and B. H. Hirst. Intestinal secretion of drugs. The role of P-glycoprotein and related drug efflux systems in limiting oral absorption, Adv. Drug Deliv. Rev, 25(2-3), pp. 129-157. 1997. Hurria, A., M. T. Fleming, S. D. Baker, W. K. Kelly, K. Cutchall, K. Panageas, J. Caravelli, H. Yeung, M. G. Kris, J. Gomez, V. A. Miller, G. D’Andrea, H. I. Scher, L. 127 Norton and C. Huddis. Pharmacokinetics and Toxicity of Weekly Docetaxel in Older Patients, Clin. Cancer. Ther, 12, pp. 6100-6105. 2006. Immordino, M. L., P. Brusa, S. Arpicco, B. Stella, F. Dosio and L. Cattel. Preparation, characterization, cytotoxicity and pharmacokinetics of liposomes containing docetaxel, J. Control. Release, 91, pp. 417-429. 2003. Ismailos, G., C. Reppas, J. B. Dressman and P. Macheras. Unusual solubility behaviour of cyclosporine A in aqueous media, J. Pharm. Pharmacol, 43(4), pp. 287-289. 1991. Ismailos, G., C. Reppas and P. Macheras. Enhancement of cyclosporine A solubility by Dalphatocopheryl polyethylene glycol 1000 succinate (TPGS), Eur. J. Pharm. Sci, 1(5), pp. 269-271. 1994. Ivanov, I. B. and P. A. Kralchevsky. Stability of emulsions under equilibrium and dynamic conditions, Colloids Surf. A, 128(1-3), pp. 155-175. 1997. Ivanov, I. B., K. D. Danov and P. A. Kralchevsky. Flocculation and coalescence of micron-size emulsion droplets, Colloids Surf. A, 152(1-2), pp. 161-182. 1999. Jalil, R. and J. R. Nixon. Microencapsulation using poly(L-lactic acid) II: Preparative variables affecting microcapsule properties, J. Microencapsul, 7(1), pp. 25-39. 1990. Jeyanthi, R., R. C. Thanoo, R. C. Metha and P. P. DeLuca. Effect of solvent removal technique on the matrix characteristics of polylactide/glycolide microspheres for peptide delivery, J. Control. Release, 38(2-3), pp. 235-244. 1996. Johnson, J. I., S. Decker, D. Zaharevitz, L. V. Rubinstein, J. M. Venditti, S. Schepartz, S. Kalyandrug, M. Christian, S. Arbuck, M. Hollingshead and E.A. Sausville. Relationships between drug activity in NCI preclinical in vitro and in vivo models and early clinical trials, Br. J. Cancer, 84(10), pp. 1424-1431. 2001. Jones, M.-C. and J.-C. Leroux. Polymeric micelles – a generation of colloidal drug carriers, Eur. J. Pharm. Biopharm, 48(2), pp. 101-111. 1999. Jones, S. E., J. Erban, B. Overmoyer, G. T. Budd, L. Hutchins, E. Lower, L. Laufman, S. Sundaram, W. J. Urba, K. I. Pritchard, R. Mennel, D. Richards, S. Olsen, M. L. Meyers and P. M. Ravdin. Randomized phase III study of docetaxel compared with paclitaxel in metastatic breast cancer, J. Clin. Oncol, 23(24), pp. 5542-5551. 2005. Jones, S. Head-to-head: docetaxel challenges paclitaxel, Eur. J. Cancer, Supp 4(4), pp. 48. 2006. 128 Kaihara, S., S. Matsumura, A. G. Mikos and J. P. Fisher. Synthesis of poly(L-lactide) and polyglycolide by ring-opening polymerization, Nature Protocols, 2(11), pp. 2767-2771. 2007. Kataoka, K., A. Harada and Y. Nagasaki. Block copolymer micelles for drug delivery: design, characterization and biological significance, Adv. Drug Deliv. Rev, 47(1), pp. 113-131. 2001. Kaul, G. and M. Amiji. Long-Circulating Poly(Ethylene Glycol)-Modified Gelatin Nanoparticles for Intracellular Delivery, Pharm. Res, 19(7), pp. 1061-1067. 2002. Ke, W.-T., S.-Y. Lin, H.-O. Ho and M.-T. Sheu. Physical characterizations of microemulsion systems using tocopheryl polyethylene glycol 1000 succinate (TPGS) as a surfactant for oral delivery of protein drugs, J. Control. Release, 102(2), pp. 489-507. 2005. Kim, J.-H., T. E. Paxton and D. L. Tomasko. Microencapsulation of Naproxen Using Rapid Expansion of Supercritical Solutions, Biotechnol. Prog, 12(5), pp. 650-661. 1996. Kiremitçi-Gümüşderelioğlu, M. and G. Deniz. Synthesis, Characterization and in Vitro Degradation of Poly(DL-Lactide)/Poly(DL-Lactide-co-Glycolide) Films, Turk. J. Chem, 23, pp. 153-161. 1998. Koga, K., T. Ohyashiki, M. Murakami and S. Kawashima. Modification of ceftibuten transport by the addition of non-ionic surfactants, Eur. J. Pharm. Biopharm, 49(1), pp. 1725. 2000. Kopecěk, J. The Potential of Water-Soluble Polymeric Carriers in Targeted and Sitespecific Drug Delivery, J. Control. Release, 11(1-3), pp. 279-290. 1990. Kreuter, J., R. N. Alyautdin, D. A. Kharkevich and A. A. Ivanov. Passage of peptides through the blood-brain barrier with colloidal polymer particles (nanoparticles), Brain Res, 674(1), pp. 171-174. 1995. Kreuter, J., V. E. Petrov, D. A. Kharkevich and R. N. Alyautdin. Influence of the type of surfactant on the analgesic effects induced by the peptide dalargin after its delivery across the blood-brain barrier using surfactant-coated nanoparticles, J. Control. Release, 49(1), pp. 81-87. 1997. 129 Kruczynski, A., J.-M. Barret, C. Etievant, F. Colpaert, J. Fahy and B. T. Hill. Antimitotic and Tubulin-Interacting Properties of Vinflunine, a Novel Fluorinated Vinca Alkaloids, Biochem. Pharmacol, 55, pp. 635-648. 1998. Kunieda, H. and K. Shinoda. Evalutation of the Hydrophile-Lipophile Balance (HLB) of Nonionic Surfactants, J. Colloid Interface Sci, 107(1), pp. 107-121. 1985. Kwon, G. S. Diblock Copolymer Nanoparticles for Drug Delivery, Crit. Rev. Ther. Drug Carr. Syst, 15(5), pp. 481-512. 1998. La, S. B., T. Okano and K. Kataoka. Preparation and characterization of the micelleforming polymeric drug indomethacin-incorporated poly(ethylene oxide)-poly(beta-benzyl L-aspartate) block copolymer micelles, J. Pharm. Sci, 85(1), pp. 85-90. 1996. Lambert, G., E. Fattal and P. Couvreur. Nanoparticulate systems for the delivery of antisense oligonucleotides, Adv. Drug Deliv. Rev, 47(1), pp. 99-112. 2001. Langer, R. Drug delivery: Drugs on Target, Science, 293(5527), pp. 58–59. 2001. Lavelle, F., M. C. Bissery and C. Combeau. Preclinical evaluation of docetaxel (Taxotere), Semin. Oncol, 22(Supp 14), pp. 3-16. 1995. Lawrence, M. J. Surfactant Systems: Their Use in Drug Delivery, Chem. Soc. Rev, 23(6), pp. 417-424. 1994. Lee, C. C., E. R. Gillies, M. E. Fox, S. J. Guillaudeu, J. M. Fréchet, E. E. Dy and F. C. Szoka. A single dose of doxorubicin-functionalized bow-tie dendrimer cures mice bearing C-26 colon carcinomas, Proc. Natl. Acad. Sci. U.S.A, 103(45), pp. 16649-16654. 2006. Lee, S. H., Z. Zhang and S.-S. Feng. Nanoparticles of poly(lactide)-tocopheryl polyethylene glycol succinate (PLA-TPGS) copolymers for protein drug delivery, Biomaterials, 28(11), pp. 2041-2050. 2007. Lefevre, M. E., J. W. Vanderhoff and J. A. LaIssue. Accumulation of 2-micron latex particles in mouse Peyer’s patches during chronic latex feeding, Experimentia, 34, pp. 120-122. 1978. Leroux, J.-C., F. De Jaeghere, B. Anner, E. Doelker and R. Gurny. An Investigation on the Role of Plasma and Serum Opsonins on the Internalization of Biodegradable Poly(D,LLactic Acid) Nanoparticles by Human Monocytes, Life Sci, 57(7), pp. 695-703. 1995. 130 Letchford, K. and H. Burt. A review of the formation and classification of amphiphilic block copolymer nanoparticulate structures: micelles, nanospheres, nanocapcules and polymersome, Eur. J. Pharm. Biopharm, 65(3), pp. 259-269. 2007. Li, W.-I., K. W. Anderson, R. C. Mehta and P. P. DeLuca. Prediction of solvent removal profile and effect on properties for peptide-loaded PLGA microspheres prepared by solvent extraction/evaporation method, J. Control. Release, 37(3), pp. 199-214. 1995. Li, Y., M. Ogris, E. Wagner, J. Pelisek and M. Rüffer. Nanoparticles bearing polyethyleneglycol-coupled transferrin as gene carriers: preparation and in vitro evaluation, Int. J. Pharm, 259(1-2), pp. 93-101. 2003. Liebmann, J. E., J. A. Cook, C. Lipschultz, D. Teague, J. Fisher and J. B. Mitchell. Cytotoxic studies of paclitaxel (Taxol®) in human tumour cell lines, Br. J. Cancer, 68(6), pp. 1104-1109. 1993. Ling, V. Multidrug resistance: molecular mechanisms and clinical relevance, Cancer Chemother. Pharmacol, 40(Suppl 1), pp. S3-S8. 1997. Lippert, B. Impact of Cisplatin on the recent development of Pt coordination chemistry: a case study, Coor. Chem. Rev, 182, pp. 263-295. 1999. Liu, Y., K. Li, J. Pan, B. Liu and S.-S. Feng. Folic acid conjugated nanoparticles of mixed lipid monolayer shell and biodegradable polymer core for targeted delivery of Docetaxel, Biomaterials, 31(2), pp. 330-338. 2010. Livingston, D. J., S. Pazhanisamy, D. J. T. Porter, J. A. Partaledis and R. D. Tung. Weak Binding of VX-478 to Human Plasma Proteins and Implications for Anti-Human Immunodeficiency Virus Therapy, J. Infect. Dis, 172(5), pp. 1238-1245. 1995. Lomovskaya, N., S. L. Otten and Y. Doi-Katayama. Doxorubicin Overproduction in Streptomyces peucetius: Cloning and Characterization of the dnrU Ketoreductase and dnrV Genes and the doxA Cytochrome P-450 Hydroxylase Gene, J. Bacteriol, 181, pp. 305-318. 1999. Lown, K. S., R. R. Mayo, A. B. Leichtman, H.-L. Hsiao, D. K. Turgeon, P. SchmiedlinRen, M. B. Brown, W. Guo, S. J. Rossi, L. Z. Benet and P. B. Watkins. Pharmacokinetics and Drug Disposition: Role of intestinal P-glycoprotein (mdr1) in interpatient variation in the oral bioavailability of cyclosporine, Clin. Pharmacol. Ther, 62(3), pp. 248-260. 1997. 131 Maeda, H., L. W. Seymour and Y. Miyamoto. Conjugates of Anti-cancer Agents and Polymers: Advantages of Macromolecular Therapeutics in vivo, Bioconjug. Chem, 3(5), pp. 351-362. 1992. Mahdavian, E., S. Sangsura, G. Landry, J. Eytina and B. A. Salvatore. A novel synthesis of tocopherol amines and amides, Tetrahedron Lett, 50(1), pp. 19-21. 2009. Malingré, M. M., D. J. Richel, J. H. Beijnen, H. Rosing, F. J. Koopman, W. W. T. B. Huinink, M. E. Schot and H. M. Schellens. Coadministration of Cyclosporine Strongly Enhances the Oral Bioavailability of Docetaxel, J. Clin. Oncol, 19, pp. 1160-1166. 2001. Marlard, M., C. Gaillard, G. Sanderink, S. Roberts, P. Joannou and V. Facchini. Kinetics, distribution, metabolism, and excretion of radiolabelled Taxotere in mice and dogs, Proc. Am. Assoc. Cancer Res, 34, pp. 393. 1993. Mawson, S., K. P. Johnston, J. R. Combes and J. M. DeSimone. Formation of Poly(1,1,2,2-tetrahydroperfluorodecyl acrylate) Submicron Fibers and Particles from Supercritical Carbon Dioxide Solutions, Macromolecules, 28(9), pp. 3182-3191. 1995. McCarthy, T. D., P. Karellas, S. A. Henderson, M. Giannis, D. F. O’Keefe, G. Heery, J. R. A. Paull, B. R. Matthews and G. Holan. Dendrimers as Drugs: Discovery and Preclinical and Clinical Development of Dendrimer-Based Microbicides for HIV and STI Prevention, Mol. Pharm, 2(4), pp. 312-318. 2005. McGrogan, B. T., B. Gilmartin, D. N. Carney and A. McCann. Taxanes, microtubules and chemoresistant breast cancer, Biochim. Biophys. Acta, 1785, pp. 96-132. 2008. Meerum Terwogt, J. M., M. M. Malingré, J. H. Beijnen, W. W. ten Bokkel Huinink, H. Rosing, F. J. Koopman, O. van Tellingen, M. Swart and J. H. M. Schellens. Coadministration of Oral Cyclosporine A Enables Oral Therapy with Paclitaxel, Clin. Cancer Res, 5(11), pp. 3379-3384. 1999. Mei, L., H. Sun, X. Jin, D. Zhu, R. Sun, M. Zhang and C. Song. Modified Paclitaxelloaded Nanoparticles for Inhibition of Hyperplasia in a Rabbit Arterial Balloon Injury Model, Pharm. Res, 24(5), pp. 955-962. 2007. Memişoğlu, E., A. Bochot, M. Özalp, M. Şen, D. Duchêne and A. A. Hincal. Direct Formation of Nanospheres from Amphiphilic ß-Cyclodextrin Inclusion Complexes, Pharm. Res, 20(1), pp. 117-125. 2003. Mohamed, F. and C. F. van der Walle. Engineering biodegradable polyester particles with specific drug targeting and drug release properties, J. Pharm. Sci, 97(1), pp. 71–87. 2008. 132 Molpeceres, J., M. Guzman, M. R. Aberturas, M. Chacon and L. Berges. Application of central composite designs to the preparation of polycaprolactone nanoparticles by solvent displacement, J. Pharm. Sci, 85(2), pp. 206-213. 1996. Monegier, B., C. Gaillard, S. Sable and M. Vuilhorgne. Structures of the Major Human Metabolites of Docetaxel, Tetrahedron Lett, 35, pp. 3715-3718. 1994. Momot, K. I., P. W. Kuchel, B. E. Chapman, P. Deo and D. Whittaker. NMR study of the association of propofol with nonionic surfactants, Langmuir, 19(6), pp. 2088-2095. 2003. Monsky, W. L., D. Fukumura, T. Gohongi, M. Ancukiewcz, H. A. Weich, V. P. Torchilin, F. Yuan and R. K. Jain. Augmentation of Transvascular Transport of Macromolecules and Nanoparticles in Tumors Using Vascular Endothelial Growth Factor, Cancer Res, 59(16), pp. 4129-4135. 1999. Mortier, K. A., V. Renard, A. G. Verstraete, A. V. Gussem, S. V. Belle and W. E. Lambert. Development and Validation of a Liquid Chromatography-Tandem Mass Spectrometry Assay for the Quantification of Docetaxel and Paclitaxel in Human Plasma and Oral Fluid, Anal. Chem, 77, pp. 4677-4683. 2005. Mosqueira, V. C. F., P. Legrana, H. Pinto-Alphandary, F. Puisieux and B. Barratt. Poly(D,L-lactide) nanocapsules prepared by a solvent displacement process: Influence of the composition on physicochemical and structural properties, J. Pharm. Sci, 89(5), pp. 614-626. 2000. Mu, L. and S.-S. Feng. Vitamin E TPGS used as emulsifier in the solvent evaporation/extraction technique for fabrication of polymeric nanospheres for controlled release of paclitaxel, J. Control. Release, 80(1-3), pp. 129-144. 2002. Mu, L. and S.-S. Feng. A novel controlled release formulation for the anticancer drug Paclitaxel (Taxol®): PLGA nanoparticles containing vitamin E TPGS, J. Control. Release, 86(1), pp. 33-48. 2003. Mu, L., P.-H. Seow, S.-N. Ang and S.-S. Feng. Study on surfactant coating of polymeric nanoparticles for controlled delivery of anticancer drug, Colloid Polym. Sci, 283(1), pp. 58-65. 2004. Müller, R. H. Charge Determinations. In Colloidal Carriers For Controlled Drug Delivery And Targeting: Modification, Characterization And In Vivo Distribution, ed by R. A. Müller, pp. 57-97. Florida: CRC Press. 1991. 133 Müller, R. H., D. Ruhl, M. Luck and B.-R. Paulke. Influence of fluorescent labeling of polystyrene particles on phagocytic uptake, surface hydrophobicity, and plasma protein adsorption, Pharm. Res, 14(1), pp. 18-27. 1997. Murakami, M., Y. Ushio, Y. Morino, T. Ohta and Y. Matsukado. Immunohistochemical localization of apolipoprotein E in human glial neoplasms, J. Clin. Invest, 82(1), pp. 177188. 1988. Musumeci, T., C. A. Ventura, I. Giannone, B. Ruozi, L. Montenegro, R. Pignatello and G. Puglisi. PLA/PLGA nanoparticles for sustained release of docetaxel, Int. J. Pharm, 325(12), pp. 172-179. 2006. Muthu, M. S. and S. Singh. Targeted nanomedicines: effective treatment modalities for cancer, AIDS and brain disorders, Nanomedicine, 4(1), pp. 105-118. 2009. National Cancer Institute. What is cancer? Retrieved on March 1, 2009 from the World Wide Web: http://www.cancer.gov/cancertopics/what-is-cancer. Nelson, D. R., T. Kamataki, D. J. Waxman, F. P. Guengerich, R. W. Estabrook, R. Feyereisen, F. J. Gonzalez, M. J. Coon, I. C. Gunsalus, O. Gotoh, K. Okuda and D. W. Nebert. The P450 Superfamily: Update on New Sequences, Gene Mapping, Accession Numbers, Early Trivial Names of Enzymes, and Nomenclature, DNA Cell Biol, 12, pp. 151. 1993. Némati, F., C. Dubernet, H. Fessi, A. C. Verdière, M. F. Poupon, F. Puisieux and P. Couvreur. Reversion of multidrug resistance using nanoparticles in vitro: influence of nature of the polymer, Int. J. Pharm, 138(2), pp. 237-246. 1996. Nerurkar, M. M., N. F. H. Ho, P. S. Burton, T. J. Vidmar and R. T. Borchardt. Mechanistic Roles of Neutral Surfactants on Concurrent Polarized and Passive Membrane Transport of a Model Peptide in Caco-2 Cells, J. Pharm. Sci, 86(7), pp. 813-821. 1997. Neuzil, J. Vitamin E succinate and cancer treatment: a vitamin E prototype for selective antitumour activity, Br. J. Cancer, 89(10), pp. 1822-1826. 2003. Neuzil, J., E. Swettenham, X.-F. Wang, L.-F. Dong and M. Stapelberg. α-Tocopheryl succinate inhibits angiogenesis by disrupting paracrine FGF2 signalling, FEBS Lett, 581(24), pp. 4611-4615. 2007. Nielsen, L. S., F. Sløk and H. Bundgaard. N-Alkoxycarbonyl prodrugs of mebendazole with increased water solubility, Int. J. Pharm, 102(1-3), pp. 231-239. 1994. 134 Okassa, L. N., H. Marchais, L. Douziech-Eyrolles, K. Hervé, S. Cohen-Jonathan, E. Munnier, M. Soucé, C. Linassier, P. Dubois and I. Chourpa. Optimization of iron oxide nanoparticles encapsulation within poly(D,L-lactide-co-glycolide) sub-micron particles, Eur. J. Pharm. Biopharm, 67(1), pp. 31-38. 2007. Olivier, J.-C., L. Fenart, R. Chauvet, C. Pariat, R. Cecchelli and W. Couet. Indirect Evidence that Drug Brain Targeting Using Polysorbate 80-Coated Polybutylcyanoacrylate Nanoparticles Is Related to Toxicity, Pharm. Res, 16(12), pp. 1836-1842. 1999. Owen III, D. E. and N. A. Peppas. Opsonization, biodistribution, and pharmacokinetics of polymeric nanoparticles, Int. J. Pharm, 307(1), pp. 93-102. 2006. Pan, J. and S.-S. Feng. Folate-decorated poly (lactide)-vitamin E TPGS nanoparticles for targeted delivery of paclitaxel, Biomaterials, 29(17), pp. 2663-2672. 2008. Panyam, J. and V. Labhasetwa. Biodegradable nanoparticles for drug and gene delivery to cells and tissue, Adv. Drug Deliv. Rev, 55(3), pp. 329-347. 2003. Pardridge, W. M. Blood-brain barrier delivery, Drug Discov. Today, 12, pp. 54-61. 2007. Peracchia, M. T., C. Vauthier, C. Passirani, P. Couvreur and D. Labarre. Complement Consumption by Poly(Ethylene Glycol) in Different Conformations Chemically Coupled to Poly(Isobutyl 2-Cyanoacrylate) Nanoparticles, Life Sci, 61(7), pp. 749-761. 1997. Peracchia, M. T., R. Gref, Y. Minamitake, A. Domb, N. Lotan and R. Langer. PEG-coated nanospheres from amphiphilic diblock and multiblock copolymers: Investigation of their drug encapsulation and release characteristics, J. Control. Release, 46(3), pp. 223-231. 1997a. Pharmaceutical Ingredients - EASTMAN Vitamin E TPGS NF: Applications and Properties. Retrieved on August 10, 2009 from the World Wide Web: http://www.eastman.com/Pages/ProductHome.aspx?product=71014033. Photos, P. J., L. Bacakova, B. Discher, F. S. Bates and D. E. Discher. Polymer vesicles in vivo: correlations with PEG molecular weight, J. Control. Release, 90(3), pp. 323-334. 2003. Pouponneau, P., J.-C. Leroux and S. Martel. Magnetic nanoparticles encapsulated into biodegradable microparticles steered with an upgraded magnetic resonance imaging system for tumor chemoembolization, Biomaterials, 30(31), pp. 6327-6332. 2009. 135 Prabaharan, M., J. J. Grailer, S. Pilla, D. A. Steeber and S. Gong. Folate-conjugated amphiphilic hyperbranched block copolymers based on Boltorn® H40, poly(L-lactide) and poly(ethylene glycol) for tumor-targeted drug delivery, Biomaterials, 30(16), pp. 3009-3019. 2009. Pu, K.-Y., K. Li, J. Shi and B. Liu. Fluorescent Single-Molecular Core-Shell Nanospheres of Hyperbranched Conjugated Polyelectrolyte for Live-Cell Imaging, Chem. Mater, 21(16), pp. 3816-3822. 2009. Puglisi, G., M. Fresta, G. Giammona and C. A. Ventura. Influence of preparation conditions on poly(ethylcyanoacrylate) nanocapsule formation, Int. J. Pharm, 125(2), pp. 283-287. 1995. Quintanar-Guerrero, D., E. Allémann, H. Fessi and E. Doelker. Preparation Techniques and Mechanisms of Formation of Biodegradable Nanoparticles from Preformed Polymers, Drug Dev. Ind. Pharm, 24(12), pp. 1113-1128. 1998. Randolph, T. W., A. D. Randolph, M. Mebes and S. Yeung. Sub-Micrometer-Sized Biodegradable Particles of Poly(L-Lactic Acid) via the Gas Antisolvent Spray Precipitation Process, Biotechnol. Prog, 9(4), pp. 429-435. 1993. Rawat, M., D. Singh, S. Saraf and S. Saraf. Nanocarriers: Promising Vehicle for Bioactive Drugs, Biol. Pharm. Bull, 29, pp. 1790-1798. 2006. Rege, B. D., L. X. Yu, A. S. Hussain and J. E. Polli. Effect of Common Excipients on Caco-2 Transport of Low-Permeability Drugs, J. Pharm. Sci, 90(11), pp. 1776-1786. 2001. Rege, B. D., J. P. Y. Kao and J. E. Polli. Effects of nonionic surfactants on membrane transporters in Caco-2 cell monolayers, Eur. J. Pharm. Sci, 16(4-5), pp. 237-246. 2002. Reis, C. P., R. J. Neufeld, A. J. Ribeiro and F. Veiga. Nanoencapsulation I. Methods for preparation of drug-loaded polymeric nanoparticles, Nanomedicine, 2(1), pp. 8-21. 2006. Repka, M. A. and J. W. McGinity. Influence of Vitamin E TPGS on the properties of hydrophilic films produced by hot-melt extrusion, Int. J. Pharm, 202(1-2), pp. 63-70. 2000. Ringel, I. and S. B. Horwitz. Studies with RP 56976 (Taxotere): A Semisynthetic Analogueof Taxol, J. Natl. Cancer Inst, 83, pp. 288-291. 1991. 136 Riou, J. F., A. Naudin and E. Lavelle. Effects of Taxotere on murine and human tumor cell lines, Biochem. Biophys. Res. Commun, 187(1), pp. 164-170. 1992. Riou, J. F., O. Petitgenet, C. Combeau and F. Lavelle. Cellular Uptake and Efflux of Docetaxel (Taxotere®) and Paclitaxel (Taxol®) in P388 Cell Line, Proc. Am. Assoc. Cancer Res, 35, pp. 385. 1994. Rossin, R., S. Muro, M. J. Welch, V. R. Muzykantov and D. P. Schuster. In Vivo Imaging of 64Cu-Labeled Polymer Nanoparticles Targeted to the Lung Endothelium, J. Nucl. Med, 49(1), pp. 103-111. 2008. Roy, A. K. and L. G. Tillman. Compositions comprising an HIV protease inhibitor such as VX478 and water soluble vitamin E compound such as vitamin E TPGS. WO 97/35587. 1997. Roy Boehm, A. L. L., R. Zerrouk and H. Fessi. Poly ε-caprolactone nanoparticles containing a poorly soluble pesticide: formulation and stability study, J. Microencaps, 17(2), pp. 195-205. 2000. Royer, I., B. Monsarrat, M. Sonnier, M. Wright and T. Cresteil. Metabolism of Docetaxel by Human Cytochromes P450: Interactions with Paclitaxel and Other Antineoplastic Drugs, Cancer Res, 56, pp. 58-65. 1996. Sahoo, S. K., J. Panyam, S. Prabha and V. Labhasetwar. Residual polyvinyl alcohol associated with poly(D,L-lactide-co-glycolide) nanoparticles affects their physical properties and cellular uptake, J. Control. Release, 82(1), pp. 105-114. 2002. Sahoo, S. K., W. Ma and V. Labhasetwar. Efficacy of transferrin-conjugated paclitaxelloaded nanoparticles in a murine model of prostate cancer, Int. J. Cancer, 112(2), pp. 335340. 2004. Salvador-Morales, C., L. Zhang, R. Langer and O. C. Farokhzad. Immunocompatibility properties of lipid-polymer hybrid nanoparticles with heterogenous surface functional groups, Biomaterials, 30(12), pp. 2231-2240. 2009. Santos Magalhaes, N. S., H. Fessi, F. Puisieux, S. Benita and M. Seiller. An in vitro release kinetic examination and comparative evaluation between submicron emulsion and polylactic acid nanocapsules of clofibride, J. Microencapsul, 12(2), pp. 195-205. 1995. Savic, R., L. Luo, A. Eisenberg and D. Maysinger. Micellar nanocontainers distribute to defined cytoplasmic organelles, Science, 300(5619), pp. 615-618. 2003. 137 Schinkel, A. H. and J. W. Jonker. Mammalian drug efflux transporters of the ATP binding cassette (ABC) family: an overview, Adv. Drug Deliv. Rev, 55(1), pp. 3-29. 2003. Scholes, P. D., A. G. A. Coombes, L. Illum, S. S. Davis, M. Vert and M. C. Davies. The preparation of sub-200 nm poly(lactide-co-glycolide) microspheres for site-specific drug delivery, J. Control. Release, 25(1-2), pp. 145-153. 1993. Schott, H. Comments on Hydrophile-Lipophile Balance Systems, J. Colloid Interface Sci, 133(2), pp. 527-529. 1989. Schrijvers, D., J. Wanders, L. Dirix, A. Prove, I. Vonck, A. van Oosterom and S. Kaye. Coping with toxicities of docetaxel (TaxotereTM), Ann. Oncol, 4, pp. 610-611. 1993. Schroder, H. and T. Netscher. Determination of the absolute stereochemistry of vitamin E derived oxa-spiro compounds by NMR spectroscopy, Magn. Reson. Chem, 39(11), pp. 701-708. 2001. Schuetz, E. G., W. T. Beck and J. D. Schuetz. Modulators and Substrates of Pglycoprotein and Cytochrome P4503A Coordinately Up-regulate these Proteins in Human Colon Carcinoma Cells, Mol. Pharmacol, 49(2), pp. 311-318. 1996. Senthilkumar, M., P. Mishra and N. K. Jain. Long circulating PEGylated poly(D,Llactide-co-glycolide) nanoparticulate delivery of docetaxel to solid tumors, J. Drug Target, 16(5), pp. 424-435. 2008. Shabbits, J. A., G. N. C. Chiu and L. D. Mayer. Development of an in vitro drug release assay that accurately predicts in vivo drug retention for liposome-based delivery systems, J. Control. Release, 84(3), pp. 161-170. 2002. Shapiro, A. B. and V. Ling. Effect of Quercetin on Hoechst 33342 Transport by Purified and Reconstituted P-Glycoprotein, Biochem. Pharmacol, 53(4), pp. 587-596. 1997. Sheu, M.-T., S.-Y. Chen, L.-C. Chen and H.-O. Ho. Influence of micelle solubilization by tocopheryl polyethylene glycol succinate (TPGS) on solubility enhancement and percutaneous penetration of estradiol, J. Control. Release, 88(3), pp. 355-368. 2003. Shi, D., H. S. Cho, Y. Chen, H. Xu, H. Gu, J. Lian, W. Wang, G. Liu, C. Huth, L. Wang, R. C. Ewing, S. Budko, G. M. Pauletti and Z. Dong. Fluorescent Polystyrene-Fe3O4 Composite Nanospheres for In Vivo Imaging and Hyperthermia, Adv. Mater, 21(21), pp. 2170-2173. 2009. 138 Shi, N. and W. M. Pardridge. Noninvasive gene targeting to the brain, Proc. Natl. Acad. Sci. USA, 97, pp. 7567-7572. 2000. Shin, S. C. Studies on Hydrophobic Drug-Soluble Carrier Coprecipitates (II): Physicochemical Characteristics of Furosemide-PVP Coprecipitates, Arch. Pharm. Res, 2(1), pp. 49-64. 1979. Shin, S.-C. and J. Kim. Physicochemical characterization of solid dispersion of furosemide with TPGS, Int. J. Pharm, 251(1-2), pp. 79-84. 2003. Singh, R., S. Y. Chang and L. C. E. Taylor. In vitro Metabolism of a Potent HIV-protease Inhibitor (141W94) Using Rat, Monkey and Human Liver S9, Rapid Commun. Mass Spectrom, 10(9), pp. 1019-1026. 1996. Sokol, R. J., K. E. Johnson, F. M. Karrer, M. R. Narkewicz, D. Smith and I. Kam. Improvement of cyclosporine absorption in children after liver transplantation by means of water-soluble vitamin E, Lancet, 338(8761), pp. 212-215. 1991. Soppimath, K. S., T. M. Aminabhavi, A. R. Kulkarni and W. E. Rudzinski. Biodegradable polymeric nanoparticles as drug delivery devices, J. Control. Release, 70(1-2), pp. 1-20. 2001. Sparreboom, A., O. van Tellingen, E. J. Scherrenburg, J. J. B. Boesen, M. T. Huizing, W. J. Nooijen, C. Versluis and J. H. Beijnen. Isolation, Purification and Biological Activity of Major Docetaxel Metabolites from Human Feces, Drug Metab. Dispos, 24, pp. 655-658. 1996. Stella, V. J., S. Martodihardjo, K. Terada and V. M. Rao. Some relationships between the physical properties of various 3-acyloxymethyl prodrugs of phenytoin to structure: Potential in vivo performance implications, J. Pharm. Sci, 87(10), pp. 1235-1241. 1998. Stone, B. G., M. Udami, A. Sanghvi, V. Warty, K. Plocki, C. D. Bedetti and D. H. Van Thiel. Cyclosporine A-induced cholestasis: The mechamism in rat model, Gastroenterology, 93(2), pp. 344-351. 1987. Straubinger, R. M. and S. V. Balasubramanian. Preparation and Characterization of Taxane-Containing Liposomes, Methods Enzymol, 391, pp. 97-117. 2005. Suh, H., B. Jeong, R. Rathi and S. W. Kim. Regulation of smooth muscle cell proliferation using paclitaxel-loaded poly(ethylene oxide)-poly(lactide/glycolide) nanospheres, J. Biomed. Mater. Res, 42(2), pp. 331-338. 1998. 139 Suh, H., B. Jeong, F. Liu and S. W. Kim. Cellular uptake study of biodegradable nanoparticles in vascular smooth muscle cells, Pharm. Res, 15(9), pp. 1495–1498. 1998a. Sumer, B. and J. Gao. Theranostic nanomedicine for cancer, Nanomedicine, 3, pp. 137140. 2008. Sun, B., B. Ranganathan and S.-S. Feng. Multifunctional poly(D,L-lactide-co-glycolide)/ montmorillonite (PLGA/MMT) nanoparticles decorated by Trastuzumab for targeted chemotherapy of breast cancer, Biomaterials, 29(4), pp. 475-486. 2008. Sun, W., C. Xie, H. Wang and Y. Hu. Specific role of polysorbate 80 coating on the targeting of nanoparticles to the brain, Biomaterials, 25(15), pp. 3065-3071. 2004. Swettenham, E., P. K. Witting, B. A. Salvatore and J. Neuzil. α-Tocopheryl succinate selectively induces apoptosis in neuroblastoma cells: potential therapy of malignancies of the nervous system? J. Neurochem, 94(5), pp. 1448-1456. 2005. ten Tije, A. J., W. J. Loos, J. Verweij, S. D. Baker, K. Dinh, W. D. Figg and A. Sparreboom. Disposition of polyoxyethylated excipients in humans: Implications for drug safety and formulation approaches, Clin. Pharmacol. Ther, 74, pp. 509-510. 2003. Terada, T., M. Mizobata, S. Kawakami, F. Yamashita and M. Hashida. Optimization of tumor-selective targeting by basic fibroblast growth factor-binding peptide grafted PEGylated liposomes, J. Control. Release, 119(3), pp. 262-270. 2007. Testa, B. and J. Caldwell. Prodrugs revisited: The “ad hoc” approach as a complement to ligand design, Med. Res. Rev, 16(3), pp. 233-241. 1996. The Cancer Council of New South Wales. Cancer Myths and Facts: Food preservatives and cancer. Retrieved on February 20, 2009 from the World Wide Web: http://www.nswcc.org.au/editorial.asp?pageid=2345. Theis, J. G., M. Liau-Chu, H. S. Chan, J. Doyle, M. L. Greenberg and G. Koren. Anaphylactoid reactions in children receiving high-dose intravenous cyclosporine for reversal of tumour resistance: the causative role of improper dissolution of Cremophor EL, J. Clin. Oncol, 13, pp. 2508-2516. 1995. Thiebaut, F., T. Tsuruo, H. Hamada, M. M. Gottesman, I. Pastan and M. C. Willingham. Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues, Proc. Natl. Acad. Sci. USA, 84(21), pp. 7735-7738. 1987. 140 Thierry, A. R., A. Dritschilo and A. Rahman. Effect of Liposomes on P-glycoprotein Function in Multidrug Resistant Cells, Biochem. Biophys. Res. Commun, 187(2), pp. 1098-1105. 1992. Thies, C. Formation of Degradable Drug-Loaded Microparticles by In-Liquid Drying Processes. In Microcapsules and Nanoparticles in Medicine and Pharmacy, ed by M. Donbrow, pp. 47-71. Ann Arbor: CRC. 1991. Tolcher, A. W., K. H. Cowan, D. Solomon, F. Ognibene, B. Goldspiel, R. Chang, M. H. Noone, A. M. Denicoff, C. S. Barnes, M. R. Gossard, P. A. Fetsch, S. L. Berg, F. M. Balis, D. J. Venzon and J. A. O’Shaughnessy. Phase I crossover study of paclitaxel with rverapamil in patients with metastatic breast cancer, J. Clin. Oncol, 14, pp. 1173-1184. 1996. Tom, J. W. and P. G. Debenedetti. Particle Formation with Supercritical Fluids – A Review, J. Aerosol Sci, 22(5), pp. 555-584. 1991. Tomalia, D. A., H. Baker, J. Dewald, M. Hall, G. Kallos, S. Martin, J. Roeck, J. Ryder and P. Smith. Dendritic Macromolecules: Synthesis of Starburst Dendrimers, Macromolecules, 19(9), pp. 2466-2468. 1986. Torchilin, V. P. Recent Advances with Liposomes as Pharmaceutical Carriers, Nat. Rev. Drug Discov, 4, pp. 145-160. 2005. Torchilin, V. P. Recent Approaches to Intracellular Delivery of Drugs and DNA and Organelle Targeting, Annu. Rev. Biomed. Eng, 8, pp. 343-375. 2006. Traber, M. G., H. J. Kayden, J. B. Green and M. H. Green. Absorption of water-miscible forms of vitamin E in a patient with cholestasis and in thoracic duct-cannulated rats, Am. J. Clin. Nutr, 44, pp. 914-923. 1986. Traber, M. G., C. A. Thellman, M. J. Rindler and H. J. Kayden. Uptake of intact TPGS (dα-tocopheryl polyethylene glycol 1000 succinate) a water-miscible form of vitamin E by human cells in vitro, Am. J. Clin. Nutr, 48, pp. 605-611. 1988. U.S. Food and Drug Administration (FDA). A Fresh Look at Food Preservatives. Retrieved on January 10, 2009 from the World Wide Web: http://www.cfsan.fda.gov/~dms/fdpreser.html. van Asperen, J., O. van Tellingen, A. Sparreboom, A. H. Schinkel, P. Borst, W. J. Nooijen and J. H. Beijnen. Enhanced oral bioavailability of paclitaxel in mice co-treated with the P-glycoprotein blocker SDZ PSC 833, Br. J. Cancer, 76(9), pp. 1181-1183. 1997. 141 van Asperen, J., O. van Tellingen, M. A. van der Valk, M. Rozenhart and J. H. Beijnen. Enhanced Oral Absorption and Decreased Elimination of Paclitaxel in Mice Cotreated with Cyclosporine A, Clin. Cancer Res, 4(10), pp. 2293-2297. 1998. van Heeswijk, R. P., A. Veldkamp, J. W. Mulder, P. L. Meenhorst, J. M. Lange, J. H. Beijnen and R. M. Hoetelmans. Combination of protease inhibitors for the treatment of HIV-1 infected patients: a review of pharmacokinetics and clinical experience, Antivir. Ther, 6(4), pp. 201-229. 2001. van Vlerken L. E., T. K. Vyas and M. M. Amiji. Poly(ethylene glycol)-modified Nanocarriers for Tumor-targeted and Intracellular Delivery, Pharm. Res, 24(8), pp. 14051414. 2007. Varma, M. V. S., Y. Ashokraj, C. S. Dey and R. Panchagnula. P-glycoprotein inhibitors and their screening: a perspective from bioavailability enhancement, Pharmacol. Res, 48(4), pp. 347-359. 2003. Varma, M. V. S., S. Khandavilli, Y. Ashokraj, A. Jain, A. Dhanikula, A. Sood, N. S. Thomas, O. Pillai, P. Sharma, R. Gandhi, S. Agrawal, V. Nair and R. Panchagnula. Biopharmaceutic Classification System: A Scientific Framework for Pharmacokinetic Optimization in Drug Research, Curr. Drug Metab, 5(5), pp. 375-388. 2004. Varma, M. V. S. and R. Panchagnula. Enhanced oral paclitaxel absorption with vitamin ETPGS: Effect on solubility and permeability in vitro, in situ and in vivo, Eur. J. Pharm. Sci, 25(4-5), pp. 445-453. 2005. Varma, M. V. S. and R. Panchagnula. Prediction of In Vivo Intestinal Absorption Enhancement on P-Glycoprotein Inhibition, from Rat In Situ Permeability, J. Pharm. Sci, 94(8), pp. 1694-1704. 2005a. Veronese, F. M., O. Schiavon, G. Pasut, R. Mendichi, L. Andersson, A. Tsirk, J. Ford, G. Wu, S. Kneller, J. Davies and R. Duncan. PEG-Doxorubicin Conjugates: Influence of Polymer Structure on Drug Release, in Vitro Cytotoxicity, Biodistribution, and Antitumor Activity, Bioconjug. Chem, 16(4), pp. 775-784. 2005. Vrancken, M. N. and D. A. Claeys. Process for encapsulating water and compounds in aqueous phase by evaporation. U.S. Patent 3,523,906. 1970. Vyas, D. M., H. Wong, A. R. Crosswell, A. M. Casazza, J. O. Knipe, S. W. Mamber and T. W. Doyle. Synthesis and antitumor evaluation of water soluble taxol phosphates, Bioorg. Med. Chem. Lett, 3(6), pp. 1357-1360. 1993. 142 Wacher, V. J., J. A. Silverman, Y. Zhang and L. Z. Benet. Role of P-Glycoprotein and Cytochrome P450 3A in Limiting Oral Absorption of Peptides and Peptidomimetics, J. Pharm. Sci, 87(11), pp. 1322-1330. 1998. Wada, R., S.-H. Hyon, Y. Ikada, Y. Nakao, H. Yoshikawa and S. Muranishi. Lactic Acid oligomer Microspheres Containing an Anticancer Agent for Selective Lymphatic Delivery: I. In Vitro Studies, J. Bioact. Compat. Polym, 3(2), pp. 126-136. 1988. Wang, J. and S. P. Schwendeman. Mechanisms of Solvent Evaporation Encapsulation Processes: Prediction of Solvent Evaporation Rate, J. Pharm. Sci, 88(10), pp. 1090-1099. 1999. Warren, L., J.-C. Jardillier, A. Malarska and M.-G. Akeli. Increased Accumulation of Drugs in Multidrug-resistant Cells Induced by Liposomes, Cancer Res, 52(11), pp. 32413245. 1992. Waterhouse, D. N., T. D. Madden, P. R. Cullis, M. B. Bally, L. D. Mayer and M. S. Webb. Preparation, Characterization, and Biological Analysis of Liposomal Formulations of Vincristine, Methods Enzymol, 391, pp. 40-57. 2005. Watters, E. DNA Is Not Destiny. Discover magazine, 2006. Weiss, R. B. and V. T. DeVita. Multimodal primary cancer treatment (adjuvant chemotherapy): Current results and future prospects. Annals of Internal Medicines, 91, pp. 251-260. 1979. Williams, J. R., A. A. Clifford and S. H. R. Al-Saidi. Supercritical fluids and their applications in biotechnology and related areas, Mol. Biotechnol, 22(3), pp. 263-286. 2002. Win, K. Y. and S.-S. Feng. Effects of particle size and surface coating on cellular uptake of polymeric nanoparticles for oral delivery of anticancer drugs, Biomaterials, 26(15), pp. 2713-2722. 2005. Win, K. Y. and S.-S. Feng. In vitro and in vivo studies on vitamin E TPGS-emulsified poly(D,L-lactic-co-glycolic acid) nanoparticles for paclitaxel formulation, Biomaterials, 27(10), pp. 2285–2291. 2006. Wischke, C. and S. P. Schwendeman. Principles of encapsulating hydrophobic drugs in PLA/PLGA microparticles, Int. J. Pharm, 364(2), pp. 298-327. 2008. 143 Wolinsky, J. B. and M. W. Grinstaff. Therapeutic and diagnostic applications of dendrimers for cancer treatment, Adv. Drug Deliv. Rev, 60(9), pp. 1037-1055. 2008. Woodcock, D. M., M. E. Linsenmeyer, G. Chojnowski, A. B. Kriegler, V. Nink, L. K. Webster and W. H. Sawyer. Reversal of multidrug resistance by surfactants, Br. J. Cancer, 66(1), pp. 62-68. 1992. World Health Organization. Cancer control: knowledge into action. Retrieved on February 27, 2009 from the World Wide Web: http://www.who.int/cancer/modules/en/. Xie, Z., T. Lu, X. Chen, C. Lu, Y. Zheng and X. Jing. Triblock Poly(lactic acid)-bPoly(ethylene glycol)-b-Poly(lactic acid)/Paclitaxel Conjugates: Synthesis, Micellization, and Cytotoxicity, Journal of Applied Polymer Science, 105, pp. 2271-2279. 2007. Xu, Z., W. Gu, J. Huang, H. Sui, Z. Zhou, Y. Yang, Z. Yan and Y. Li. In vitro and in vivo evaluation of actively targetable nanoparticles for paclitaxel delivery, Int. J. Pharm, 288(2), pp. 361-368. 2005. Xu, Z., L. Chen, W. Gu, Y. Gao, L. Lin, Z. Zhang, Y. Xi and Y. Li. The performance of docetaxel-loaded solid lipid nanoparticles targeted to hepatocellular carcinoma, Biomaterials, 30(2), pp. 226-232. 2009. Yahya, G. O., S. K. A. Ali and E. Z. Hamad. Surface and interfacial activities of hydrophobically modified poly(vinyl alcohol) (PVA), Polymer, 37(7), pp. 1183-1188. 1996. Yang, H., P. Zhu, C. Peng, S. Ma, Q. Zhu and C. Fan. Viscometric study of polyvinyl alcohol in NaCl/water solutions ranged from dilute to extremely dilute concentration, Eur. Polym. J, 37(9), pp. 1939-1942. 2001. Yang, X., Y. Chen, R. Yuan, G. Chen, E. Blanco, J. Gao and X. Shuai. Folate-encoded and Fe3O4-loaded polymeric micelles for dual targeting of cancer, Polymer, 49(16), pp. 3477-3485. 2008. Yoo, H. S. and T. G. Park. Biodegradable polymeric micelles composed of doxorubicin conjugated PLGA-PEG block copolymer, J. Control. Release, 70(1-2), pp. 63-70. 2001. Yu, L., A. Bridgers, J. Polli, A. Vickers, S. Long, A. Roy, R. Winnike and M. Coffin. Vitamin E-TPGS increases absorption flux of an HIV protease inhibitor by enhancing its solubility and permeability, Pharm. Res, 16(12), pp. 1812-1817. 1999. 144 Yuan, F., M. Leuning, S. K. Huang, D. A. Berk, D. Papahadjopoulos and R. K. Jain. Microvascular Permeability and Interstitial Penetration of Sterically Stabilized (Stealth) Liposomes in a Human Tumor Xenograft, Cancer Res, 54(13), pp. 3352-3356. 1994. Xie, M., L. Zhou, T. Hu and M. Yao. Intratumoral delivery of paclitaxel-loaded poly(lactic-co-glycolic acid) microspheres for Hep-2 laryngeal squamous cell carcinoma xenografts, Anti-Cancer Drugs, 18(4), pp. 459-466. 2007. Zhang, Z. and S.-S. Feng. In Vitro Investigation on Poly(lactide)-Tween 80 Copolymer Nanoparticles Fabricated by Dialysis Method for Chemotherapy, Biomacromolecules, 7(4), pp. 1139-1146. 2006. Zhang, Z. and S.-S. Feng. Nanoparticles of poly(lactide)/vitamin E TPGS copolymer for cancer chemotherapy: Synthesis, formulation, characterization and in vitro drug release, Biomaterials, 27(2), pp. 262-270. 2006a. Zhang, Z., S. H. Lee and S.-S. Feng. Folate-decorated poly(lactide-co-glycolide)-vitamin E TPGS nanoparticles for targeted drug delivery, Biomaterials, 28(10), pp. 1889-1899. 2007. Zhao, H. Z., E. C. Tan and L. Y. L. Yung. Potential use of cholecalciferol polyethylene glycol succinate as a novel pharmaceutical additive, J. Biomed. Mater. Res, 84A(4), pp. 954-964. 2007. 145 [...]... combine the advantages from TPGS by synthesizing PLA-TPGS copolymers for various potential biomedical applications, including formulation of imaging agents for cellular and molecular imaging and targeted drug therapy (Zhang et al., 2007; Pan and Feng, 2009) 1.2 Objectives and Thesis Organization In this thesis, we focus on the formulation of PLA-TPGS nanoparticles encapsulating anticancer drug docetaxel. .. 10 and 24 h after i.v administration at the same docetaxel dose of 10 mg/kg (n=3) 111 xvi CHAPTER 1: INTRODUCTION 1.1 Background There has been a sustained interest during recent years in developing localized and sustained treatment for cancer and other fatal diseases such as cardiovascular restenosis Biodegradable polymeric carriers have become a promising platform for sustained, controlled and targeted... activity such as high response and patient survival rates in a broad spectrum of advanced carcinoma (Bunn and Kelly, 1998) And taxanes are currently being widely used in oncology The most common taxanes are paclitaxel and docetaxel They are diterpenes and their molecular structures are different only at a few side chains as shown in Figure 2 14 Figure 2: Chemical structures of paclitaxel and docetaxel (Source:... various drug 98 Table 3: Mean non-compartmental pharmacokinetic parameters of SpD rats for intravenous administration of Taxotere® and TPGS-emulsified docetaxel- loaded PLATPGS NPs at a dose of 10 mg/kg 107 xiii LIST OF FIGURES Figure 1: Molecular structure of Cremophor EL 10 Figure 2: Chemical structures of paclitaxel and docetaxel 15 Figure 3: Packaging of docetaxel in commercial formulation Taxotere® 16... et al., 1996; Baker et al., 2006; Bradshaw-Pierce et al., 2007) A major fraction of drugs are also distributed to spleen, intestine and plasma proteins Meanwhile, about 80% of the dose is excreted through feces and about 6% is eliminated renally (Marlard et al., 1993) However, if compared to paclitaxel, docetaxel demonstrates a linear pharmacokinetics and elimination half-life behaviors over 1 hour after... cycle are blocked and division fails (Gelmon, 1994; Huizing et al., 1995) Eventually, apoptosis takes place Although docetaxel is the analogue to paclitaxel, there is significant difference between the pharmacodynamics and pharmacokinetics of the two drugs 2.3.2.2 Pharmacodynamics At molecular level of pharmacodynamics, docetaxel has shown about 1.9-fold greater binding affinity to ß-tubulin Docetaxel also... 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... 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 13 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... 33: Viability of MCF-7 breast cancer cells incubated with docetaxel- loaded TPGS- or PVA-emulsified PLA-TPGS NPs in comparison with that of Taxotere® at different docetaxel concentrations after 24, 48 and 72 h Data represent mean ± SD (n=6) 97 Figure 34: In vivo pharmacokinetics profiles of plasma drug concentration versus time after i.v administration of Taxotere® and TPGS-emulsified PLA-TPGS nanoparticles. .. encapsulation efficiency, surface morphology, surface charge and drug release profile In vitro cellular study is reported in Chapter 5 Human breast adenocarcinoma MCF-7 and human colon cancer HT-29 cell lines are employed to assess cellular uptake of the NPs as well as to evaluate the cell viability of the NP formulations, which is done in close comparison with Taxotere® In Chapter 6, in vivo pharmacokinetics .. .IN VITRO AND IN VIVO INVESTIGATION OF NANOPARTICLES OF A NOVEL BIODEGRADABLE COPOLYMER FOR SUSTAINED AND CONTROLLED DELIVERY OF DOCETAXEL GAN CHEE WEE (B.Eng (Hons.), NUS) A THESIS SUBMITTED... many anticancer drugs, including docetaxel, by internalization mechanism of drug-loaded nanoparticles such as endocytic process (Panyam and Labhasetwar, 2003; Bareford and Swaan, 2007) Meanwhile,... potential biomedical applications, including formulation of imaging agents for cellular and molecular imaging and targeted drug therapy (Zhang et al., 2007; Pan and Feng, 2009) 1.2 Objectives and