DRUG DELIVERY SYSTEMS FOR ANTIANGIOGENESIS AND ANTICANCER ACTIVITIES WANG ZHE A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACY NATIONAL UNIVERSITY OF SINGAPORE 2010 ACKNOWLEDGEMENTS I am deeply indebted to my main supervisor Associate Professor Ho Chi Lui, Paul and cosupervisor Associate Professor Chui Wai Keung for their continuous encouragement, kind support, careful nurturing and wise advice throughout the period of my PhD study. Sincere appreciation is also extended to Ms. Ng Swee Eng, Ms. Ng Sek Eng, Ms. Quek Mui Hong and Madam Loy Gek Luan for their technical help and support. I would like to thank the Department of Pharmacy, National University of Singapore, for granting me the graduate scholarship to support my study, and for providing me the premises and facilities to carry out the experiments. Additionally, I would like to express my heartfelt gratitude to my dear friends, who never failed to give me great suggestions in both study and life. They are Dr. Lin Haishu, Dr. Huang Meng, Dr. Wu Jinzhu, Dr. Ong Peishi, Dr. Yang Hong, Dr. Anahita Fathi-Azarbayjani, Dr. Wang Chunxia, Dr. Ling Hui, Mr. Li Fang, Mr. Zhang Yaochun, Ms. Cheong Hanhui, Mr. Tarang Nema, Ms. Choo Qiuyi, Ms. Yang Shili, Mr. Sun Feng, Mr. Sun Lingyi. Last but not least, I would like to extend my heartfelt gratitude to my family for their unfailing love and support. I Table of Contents Acknowledgements……………………………………………………………………………… I Table of Contents………………………………………………………………………………….II Summary……………………………………………………………………………………… VIII List of Publications…………………………………………………………………………….…XI List of Tables…………………………………………………………………………………….XII List of Figures………………………………………………………………………………… XIII List of Abbreviations………………………………………………………………………… .XVI Chapter Introduction………………………………………………………………………… .1 1.1 Cancer 1.1.1 Introduction 1.1.2 Metastasis of cancer . 1.1.3 Angiogenesis of cancer . 1.1.4 Relationship of angiogenesis with tumor growth and metastasis . 1.2 Antiangiogenesis therapy 10 1.2.1 Antiangiogenesis inhibitors 10 1.3 Drug delivery system for vascular targeted antiangiogenesis or antitumor therapy 15 1.3.1 Conjugation of drug with ligand for vascular targeting 15 1.3.2 Vascular targeted nanoparticle drug delivery . 19 1.4 Conclusion . 22 Chapter Targeted nanoparticulate drug delivery system to malignant cancer cells 24 2.1 Introduction . 25 2.2 Materials and methods . 26 II 2.2.1 Materials . 26 2.2.2 Conjugation of doxorubicin to PLGA . 27 2.2.3 Preparation of PLGA-doxorubicin (PLGA-Doxo) nanoparticles . 28 2.2.4 Synthesis of Doxo-PLGA-PEG-cRGD nanoparticles 28 2.2.5 Characterization of nanoparticles . 29 2.2.6 Cytotoxicity assay 29 2.2.7 Cell uptake and binding affinity assays 30 2.2.8 DNA fragmentation assay 30 2.2.9 Western blot analysis 31 2.2.10 In vitro drug release 32 2.3 Results . 32 2.3.1 Conjugation of doxorubicin to PLGA 32 2.3.2 Synthesis and characterization of Doxo-PLGA-PEG-cRGD nanoparticle . 33 2.3.3 Cytotoxicity of NPs to various malignant cancer cells . 35 2.3.4 Cell affinity assay . 36 2.3.5 In vitro drug release 37 2.3.6 Cell apoptosis induced by Doxo-PLGA-PEG-cRGD NPs . 37 2.4 Discussion . 39 2.5 Conclusion . 43 Chapter In vitro evaluation of vascular targeted nanocapsule with sequential drug delivery for temporal antiendothelial and anticancer activities 44 3.1 Introduction . 45 III 3.2 Materials and methods . 46 3.2.1. Preparation of Paclitaxel loaded PLGA core . 46 3.2.2. Preparation of core-shell nanocapsule . 47 3.2.3. Conjugation of RGDfK peptide to the nanocapsule 48 3.2.4. In vitro drug release from the core-shell nanocapsule . 48 3.2.5. Cell apoptosis assay . 48 3.2.6. Immunofluorescence staining of tubulin disruption 49 3.2.7. Cellular uptake of the nanocapsule 49 3.2.8. Confocal laser scanning microscope (CLSM) observation of the fluorophores labeled nanocapsule . 50 3.2.9 Co-culture assay . 50 3.3 Results and discussion . 51 3.4 Conclusion . 61 Chapter Targeted nanoparticle drug delivery for combined antiangiogenesis and antitumor activities…………………………………………………………………………… .62 4.1 Introduction . 63 4.2 Materials and methods . 64 4.2.1 Material 64 4.2.1 Preparation of drug loaded nanoparticles . 65 4.2.2 Characterization of nanoparticles . 66 4.2.3 Cellular uptake of NPs and their distribution in cells . 67 4.2.4 Western blot to detect cellular apoptosis induced by PTX NPs . 68 IV 4.2.5 Immnunofluorescence staining of tubulin 68 4.2.6 In vivo antitumor studies 69 4.2.7 Immunohistochemical staining . 70 4.3 Results . 70 4.3.1 Preparation and characterization of targeted nanoparticles 70 4.3.2 Cellular uptake of nanoparticles . 72 4.3.3 In vitro evaluation of efficacy of nanoparticles 75 4.3.4 Antitumor evaluation of different formulations . 77 4.3.5 Histological evaluation of different formulation treatment 78 4.4 Discussion . 80 4.5 Conclusion . 84 Chapter Sequential drug delivery by nanocapsule for targeted disruption of tumor vasculature to enhance anticancer and antimetastatic activities…………………………… 85 5.1 Introduction . 86 5.2 Materials and methods . 87 5.2.1Material . 87 5.2.2 Cell lines and animals. 88 5.3.3 Drug conjugation and nanocapsule preparation method 88 5.2.4 Characterization of nanocapsule . 89 5.2.5 Uptake of nanocapsule by HUVECs and intracellular distribution in cells . 89 5.2.6 Annexin V/propidium iodide apoptosis staining assay 90 5.2.7 Scratch assay 90 V 5.2.8 Tube formation assay . 91 5.2.9 Body distribution and toxicity studies of nanocapsule . 91 5.2.10 In vivo Matrigel plug assay . 92 5.2.11 In vivo primary tumor experiment 92 5.2.12 Liver metastasis prevention experiment . 93 5.2.13 Immunohistochemistry evaluation of angiogenesis, proliferation and apoptosis in tumor . 93 5.3 Results . 93 5.3.1 Physicochemical characterization of nanocapsule 93 5.3.2 Nanocapsule uptake by endothelial cells through endocytosis . 95 5.3.3 Sustained release of conjugated paclitaxel from nanocapsule induced apoptosis in cancer cells 95 5.3.4 The potency of CA4 loaded nanocapsule in inhibiting HUVEC proliferation and initiating vascular disruption effect in vitro 98 5.3.5 Biodistribution, tumor accumulation and tissue toxicity studies of nanocapsule . 99 5.3.6 Antiangiogenesis effect of nanocapsule in the Matrigel® plug model. . 101 5.3.7 Antivasculature and primary tumor growth inhibition effects of nanocapsule 101 5.3. Liver metastasis prevention of nanocapsule. . 107 5.4 Discussion . 108 5.5 Conclusion . 111 Chapter Conclusion and Future Direction…………………………………………………112 6.1 Conclusion . 113 VI 6.2 Future Direction 116 BIBLIOGRAPY……………………………………………………………………… .…… .117 VII Summary Angiogenesis is one of the vital events for organ development and differentiation during embryogenesis as well as wound healing and reproductive functions in adults. Angiogenesis also contributes significantly to tumor growth and metastasis. Hence, search for effective antiangiogenesis agents and therapy is an emerging field in the past decades. From the existing experience, the mono-antiangiogenesis therapy, however, is hampered by the fact that cancer cells could evade the single antiangiogenesis treatment designated to control tumor endothelial cell growth and tumor cell survival. Therefore, combinatorial therapy in which antiangiogenetic agent and chemotherapeutic drug administrated in a scheduled manner could result in a much more favorable therapeutic effect. In this thesis, we designed and fabricated several multifunctional and/or combinatorial nanoparticulated delivery systems aiming to disrupt tumor endothelial cells and induce apoptosis in tumor cells simultaneously for antiangiogenesis and anticancer therapy. To realize the specific tumor site targeting ability, we used a RGD (Arg-Gly-Asp) sequence containing peptide as the targeting ligand, which could target not only to the universally available endothelial cells in tumor microenvironment, but also to some specific types of malignant cancer cells via the overexpressed integrin αvβ3 and/or αvβ5 on cell membrane. In our first study, we investigated the potency of our designed doxorubicin conjugated nanoparticle decorated with PEG (poly(ethylene glycol)) and RGD peptide on surface for drug delivery and apoptosis induction in three malignant cancer cells, DU145, MDA-MB-231, and B16F10. All the three cancer cells were reported to express integrin αvβ3 and/or αvβ5 on cell membrane in different extents. Results showed that this drug conjugated targeted delivery system could be taken up by malignant cells efficiently; and its apoptosis induction ability on particular cancer cells was also effective, even though the overall VIII therapeutic efficacy of the conjugated anticancer drug was, to some extent, compromised after conjugation. In addition, the drug release profile of doxorubin from the matrix was in a sustained release manner (zero-order, r2 > 0.97). In our second study, we prepared a RGD modified core-shell nanocapsule delivery system to investigate the capacity of temporal release of antiangiogenetic drug and anticancer drug in vitro. We found that the drug release profiles of the encapsulated drugs, paclitaxel (PTX) and combretastatin (CA4) could be well-controlled by changing some preparation parameters, such as the density of phospholipid or matrix materials fed. We also found that the uptake of this nanocapsule by the endothelial cells was efficient, and the endothelial cellular cytoskeleton could be effectively disrupted by the antiangiogenetic agent, CA4. The PTX loaded in the nanoparticle core still demonstrated effective therapeutic effect to cancer cells. What is more important, our results indicated that the sequential release of CA4 and PTX from the nanocapsule could temporally diminish endothelial cells before eliminating the cancer cells with the potential to shut down the tumor microvasculature and trap the anticancer drug inside the tumor. Thus, it was evidenced that antiangiogenesis and anticancer activities could be successfully realized with appropriate formulation design and material choice. In our third study, we employed a xenograft tumor bearing mouse model to evaluate our nanoparticulate delivery system for combinatorial therapy. PTX and CA4 were co-formulated into the RGD decorated nanoparticle in a robust nanoprecipitation method. Results from the in vivo model showed that malignant tumor growth was significantly suppressed with this combinatorial method, and histological examination revealed that the mechanism underlining this encouraging therapeutic efficacy was the disruption of tumor vasculature and tumor apoptosis induction by the combined drugs. To establish the hypothesis on the advantage of having temporal delivery of antiangiogenesis and anticancer drugs with our nanoparticulate delivery system, we further designed a micelle delivery system without RGD peptide decorated on the IX polymer is a critical factor in determining the release and efficacy of polymer conjugated drugs (83, 194). The large molecular weight of PLGA (61kDa) used by Sengupta et al. may delay the hydrolysis of the polymer-drug conjugate, thus leading to compromised anticancer drug efficacy. The molecular weight of PLA used for our nanoparticle was optimized at KDa. It could lead to efficient hydrolysis of the conjugate in the cytoplasm. In addition, the conjugation of PLGA to the 3’-NH2 group of doxorubicin via amide bond in their study did not release doxorubicin in the original drug form but as the Doxo-3’-lactamide prodrug (195), leading to compromised therapeutic effect when used alone. In contrast, in our nanocapsule, the 2’-OH of paclitaxel was conjugated to the carboxyl end of PLA via ester bond that could be easily cleaved by hydrolysis upon cellular uptake (177). By choosing a suitable polymer and optimizing the molecular weight of the build-up polyester and the synthetic route for the polymer-drug conjugation, effective drug loading and sequential release of drugs were achieved with our novel nanocapsule. 5.5 Conclusion We have fabricated a novel combinatorial drug delivery nanocapsule with sequential delivery feature for effective antivasculature and anticancer activities in vitro and in vivo. The findings in this study could encourage further investigation of other combinatorial treatments administered in the same or similar delivery systems for effective malignant cancer therapy. In addition, this novel nanocapsule delivery system could be employed as a template for other applications, such as by adding iron oxide nanoparticle in the core of the system for the simultaneous non-invasive in vivo imaging. 111 Chapter Conclusion and Future Direction 112 6.1 Conclusion In this thesis, we investigated combinatorial drug delivery by nanoparticulated particle for simultaneous antiangiogenesis and anticancer therapy. With the rapid advancing of nanotechnology, nanoparticulated drug delivery systems play important roles in biomedical applications, including cancer therapy. Nanoparticles loaded with therapeutic agents could extend the circulation time of drug in human body, accumulate anticancer drug at specific tumor sites, and alleviate the potential side-effects of drugs. Therefore, many investigations have focused on the novel drug delivery systems from synthesizing basic materials components to delivery system fabrication methods. Nevertheless, few efforts have been paid on the therapeutic effects of drug delivery systems on the malignant metastatic cancers. In the metastatic progress of tumor cells, angiogenesis in tumor microenvironment is a key player for tumor malignance and widespreading in the entire body. Thus, the antiangiogenesis therapy was popularly explored, ever since this concept was proposed by Folkman in 1971 (40). Along with the better understanding of biological process of angiogenesis and corresponding discovery and development of antiangiogenic agents, numerous lines of evidences have proved that antiangiogenic agents as monotherapeutics have limited, if any, discernable efficacy, especially in the treatment of advanced malignant cancers. On the other hand, the conventional chemotherapy can result in the well-known drug resistance effect after long-term administration. This drug resistance sets a barrier for better treatment of cancer. In addition, due to the plasticity, tumors are likely to evade single targeted therapeutic method designated to control the proliferation and survival of tumor cells. Although the developed drug delivery systems could, to some extent, alleviated the drug resistance effects of many anticancer drugs and deliver drug in high pay-load to tumor sites, as well as diminishing the side-effects, more effective combination therapy drug delivery systems with simultaneous antiangiogenesis and anticancer activities are urgently desired. 113 Herein, we hypothesize that drug delivery system under controllable drug release features with combined antiangiogenesis and anticancer agents could more efficiently and effectively suppress the growth of malignant tumor masses, decrease the angiogenesis progress, and prevent the metastasis of such tumors in cancer therapy. In chapter of this thesis, a small peptides containing Arg-Gly-Asp (RGD) sequence was selected as the targeting moiety. This RGD sequence was reported as a highly affinitive ligand to αvβ3 and αvβ5 receptors overexpressed on many cancerous cells (83) and highly proliferating tumor endothelial cells (151). In this chapter, we first investigated the potency of RGD surface modified nanoparticle with tethering PEG for delivery of conjugated doxorubicin to different types of malignant metastatic cells. Experiments had demonstrated that the synthesized NPs in this study were able to effectively target conjugated doxorubicin to malignant integrin expression cancer cells, and showed profound apoptotic induction effect to those cells. Although the drug efficacy to cancer cells was compromised after conjugation with PLGA, the resultant sustained release behavior and the property of selective uptake of the NPs by cancer cells could still make Doxo-PLGA-PEG-cRGD NPs a good candidate for anticancer therapy. In chapter 3, we explored the endothelial cell targeting ability of RGD surface tagged nanocapsule with temporal antiangiogenesis and anticancer drug release feature by using a robust self-assembly nanoprecipitation method. The combinatorial nanocapsule preserved the high cytotoxic effect of the anticancer drug encapsulated in the core, and could deliver the drugs in a controlled sustained manner to disrupt tumor vasculature and ablate tumor cells sequentially with the assistance of RGD targeting ligand. Results from chapter and chapter evidenced that these targeted nanoparticulated delivery systems hold the ability for effective anticancer or simultaneous antiangiogenesis and anticancer therapy. 114 In chapter 4, we encapsulated two drugs, PTX and CA4 for the respective anticancer and antiangiogenesis effects, into a RGD targeted nanoparticle, the matrix of which was composed of biodegradable PLGA polyester. The cellular uptake of the prepared nanoparticle was efficient. The nanoparticle loaded with PTX could efficiently induce apoptosis in cancer cells, and disrupt the cytoskeleton when loaded with CA4, respectively. Xenograft tumor model confirmed the effective therapeutic effects of targeted dual-drug loaded nanoparticle, and histological tests also established the mechanism of efficacy by inducing cancer cell apoptosis and tumor endothelial cell death. Therefore, we envision that this robust targeted nanoparticle with combined therapy for antiangiogenesis and anticancer activities may show a formidable candidate for cancer therapy. In chapter 5, we further explored our concept and applied with a more advanced and a welldefined micellar delivery system, in which the polyester was conjugated to PTX to form a core for the controlled and sustained anticancer drug delivery, The polyester-PTX conjugate was further used to physically encapsulate CA4 for the antiangiogenesis function. Interestingly, we found that without RGD targeting function, micelles with appropriate particle size and PEG surface tethering could be uptaken by endothelial cells efficiently, and induce cytoskeleton disruption effectively upon fast CA4 releasing. The drug release profile could also be adjusted accordingly by conjugating the drug to the polyester with different molecular weights, and by altering the encapsulation parameters. The biodistribution experiments demonstrated the long circulation of nanocapsule in body fluid and the preferential accumulation of nanocapsule in tumor. Both in vivo artificial pro-angiogenesis and tumor xenograft assays demonstrated the promising therapeutic effect of the nanocapsule on tumor vasculature disruption, tumor cell proliferation inhibition and tumor cell apoptosis induction. The intrasplenic liver metastasis experiment also confirmed the liver metastatic prevention capacity of this novel nanocapsule. In summary, the findings indicated that this novel dual drug loaded micelle with sequential drug 115 delivery capacity is a promising candidate in combinatorial therapy in fighting against cancer, and may open an avenue for cancer therapy and diagnosis. In summary, this thesis demonstrated the in vitro and/or in vivo efficacy of several prototypes of nanoparticulated delivery systems for combinatorial temporal drug delivery for antiangiogenesis and anticancer therapy. The fabrication of these systems is robust and simple and can be easily modified for the other combinatorial temporal drug delivery. Last but not least, we hope that our study could, to some degree, contribute to the advance of cancer research for better healthcare. 6.2 Future Direction For the future study of this project, there are still some important issues to be addressed: firstly, the different pharmaceutical formulations developed in this study need to be further optimized to enhance the drug loading efficiency and encapsulation efficiency and provide high nanoparticle stability; secondly, other promising tumor vasculature and/or cancerous cell surface receptor targeting ligands such as antibodies or small molecules other than the RGD sequence containing peptide widely used in this study could be utilized for the active targeting purpose; thirdly, some imaging contrast agents could be co-encapsulated in the appropriate nanoparticle formulation for simultaneous bioimaging to monitor the therapeutic function arising from the drugs; fourthly, other tumor animal models could be used to further evaluate the therapeutic efficacy of these delivery systems in cancer treatment. 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Ring-Opening Polymerization-Mediated Controlled Formulation of Polylactide-Drug Nanoparticles. Journal of the American Chemical Society 2009;131:4744-54. 126 [...]... Pharmaceutical Research (In Press) 6 Wang Z., Ho PC A nanocapsular combinatorial sequential drug delivery system for antiangiogenesis and anticancer activities Biomaterials 2010; 31(27), 7115-7123 7 Wang Z., Ho PC Core-shell nanocapsule targeting to the tumor vasculature with sequential drug delivery for antivasculature and anticancer activities Small 2010; 6(22), 2576-2583 8 Wang Z., Lee T.Y., Ho PC cRGD-dextran-oleate... KP CdSe/AsS Core-Shell Quantum Dots: Preparation and Two-Photon Fluorescence J Am Chem Soc 2009; 131(32), 1130011301 4 Wang Z., Chui WK., Ho PC Integrin targeted drug and gene delivery Expert opinion on drug delivery 2010; 7(2), 159-171 5 Wang Z., Chui WK Ho PC Nanoparticulate delivery system targeted to tumor neovasculature for combined anticancer and antiangiogenesis therapy Pharmaceutical Research... these characteristics make integrin a good targeting ligand for both tumor and antiangiogenesis targeting therapy In recent years, Jain and co-workers have proposed that certain antiangiogenetic agents can transiently normalize the abnormal structure and function of the tumor vasculature, and make it more efficient for oxygen, nutrients and drug delivery (67) Thus, combination of vascular 15 targeting... survival time and much less, if not at all, metastasis spots in animal liver when compared with other controls In conclusion, our results supported the hypothesis that the nanoparticulated combinatorial drug delivery system for simultaneous antiangiogenesis and anticancer activities could improve the therapeutic outcome The nanoparticle formulations described in this thesis were all simple, robust and easy... occurrence Table 1-2 lists some of antiangiogenesis inhibitors in different phases of clinical trials 1.3 Drug delivery system for vascular targeted antiangiogenesis or antitumor therapy 1.3.1 Conjugation of drug with ligand for vascular targeting Integrins are important extracellular molecules that regulate angiogenesis process They are transmembrane glycoproteins, and can be internalized by cells upon... nanoparticle for targeted chemotherapeutic delivery of paclitaxel (Submitted for review) 9 Wang Z., Liew G F., Ho PC Double targeting cRGD-HA-DSPE self-assembled PLGA core-shell nanoparticle for the delivery of paclitaxel to breast cancer cells (Submitted for review) XI List of Tables Table 1-1 List of the integrin subunit combinations, their distribution, and RGD recognition 13 1-2 Selected antiangiogenesis. .. according to their environment and interactions with adjacent cells (29) Each step is essential to the formation of tumor associated neovasculature and represents a potential site of therapeutic manipulation 8 Tumor associated angiogenesis often develops in a hazard and poorly regulated manner The resultant vessels tend to form right-angle turns and corkscrew spirals, and fail to diminish progressively... proteins Bcl-2 and Bcl-XL and the proapoptotic protein Bax (46) Another widely accepted method for antiangiogenesis therapy is the interference of angiogenetic growth factor such as VEGF, PDGF or their receptors Typically, VEGF plays a vital role in the tumor development and angiogenesis Hence, the blockage of VEGF function could effectively inhibit the angiogenesis and subsequent tumor growth and metastasis... of VEGF receptor cellular signaling (50), etc Intervention with endothelial cell adhesion and migration: Because interaction between endothelial cell and extracellular matrix is highly involved in the angiogenesis progress for endothelial cell adhesion and migration, many efforts have been paid on this step for antiangiogenesis purpose Interferon (IFN), the first identified endogenous angiogenesis inhibitor,... remodeling and morphogenesis These enzymes are capable of degrading all components of the extracellular matrix (64) Increased activity of these enzymes has been observed in tumor formation, and therefore inhibitors of MMPs represent an attractive approach to treat cancer Batimastat, marimastat, and prinomastat are all potent broad14 spectrum MMPs inhibitors, and can prevent or reduce growth, angiogenesis and . DRUG DELIVERY SYSTEMS FOR ANTIANGIOGENESIS AND ANTICANCER ACTIVITIES WANG ZHE A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY. nanoparticulated combinatorial drug delivery system for simultaneous antiangiogenesis and anticancer activities could improve the therapeutic outcome. The nanoparticle formulations described in. 1.3.1 Conjugation of drug with ligand for vascular targeting 15 1.3.2 Vascular targeted nanoparticle drug delivery 19 1.4 Conclusion 22 Chapter 2 Targeted nanoparticulate drug delivery system to