Study of aptamer for cancer therapeutics

STUDY OF APTAMER FOR CANCER THERAPEUTICS TAN LIHAN NATIONAL UNIVERSITY OF SINGAPORE 2012 STUDY OF APTAMER FOR CANCER THERAPEUTICS TAN LIHAN (B. ENG, NATIONAL UNIVERSITY OF SINGAPORE) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2012 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. Tan Lihan July 2012 ACKNOWLEDGEMENTS I would like to thank my supervisors, Prof. Neoh Koon Gee, Prof. Choe Woo-Seok and Dr. Su Xiaodi, for their prompt replies to my concerns, critical feedbacks on my research, patience with me, and companionship throughout my Ph.D. studies. I learned a lot from them in both academic and personal aspects. Many thanks to my labmates (esp. Dr. Shi Zhilong and Mr. Rusdianto Budiraharjo) and the laboratory officers (esp. Ms. Li Feng Mei and Ms. Li Xiang) from ChBE, NUS, for all the help that I received in the course of my research. In addition, I render my gratitude to my labmates in Sungkyunkwan University, Korea, for helping me in work and personal life. The opportunity to work in Dr. Su’s group in the Institute of Materials Research and Engineering, A*STAR, and the research scholarship provided by NUS were greatly appreciated. I am also grateful to many other friends for their encouragement and understanding throughout my studies. Lastly, heartfelt thanks to my parents and brother for supporting my studies, sending me from one end of the island to the other in the early morning, doing the housework and their unconditional love. This thesis is dedicated to them! I   TABLE OF CONTENTS ACKNOWLEDGEMENTS . I TABLE OF CONTENTS II SUMMARY . VII LIST OF ABBREVIATIONS . VIII LIST OF TABLES X LIST OF FIGURES . XI CHAPTER INTRODUCTION 1.1. Background . 1.2. Objectives and Scopes . 1.3. Outline of the Thesis . CHAPTER LITERATURE REVIEW 2.1. What are Nucleic Acid Aptamers? How are the Aptamers Found? 2.2. Current Methods for Determining Affinity of Selected ssDNA or RNA Aptamer Sequences for Their Targets 12 2.3. AuNP Properties and Usages in Analyte Detection/Affinity Analyses 15 2.4. Aptamers in Clinical Trials . 19 2.5. Aptamers that can Target Extracellular Membrane Protein on Cancer Cells 24 2.6. Importance of Macrophage Evasion in Drug Delivery . 27 2.7. Breast Cancer: Current Treatment Methods and Limitations . 29 2.8. Bladder Cancer: Current Treatment Methods and Limitations . 31 II   CHAPTER AFFINITY ANALYSIS OF DNA APTAMER-PEPTIDE INTERACTIONS USING GOLD NANOPARTICLES 3.1. Introduction . 35 3.2. Experimental Section 37 3.2.1. Materials . 37 3.2.2. Preparation of citrate-coated AuNPs 38 3.2.3. Colorimetric assay procedure . 39 3.2.4. Fluorescence assay procedure 39 3.2.5. Characterization 39 3.3. Results and Discussion 40 3.3.1. Interaction of ssDNA with AuNPs . 40 3.3.2. Interaction of MUC1 peptide with AuNPs . 43 3.3.3. Detection of ssDNA-MUC1 peptide complex formation and measurement of binding affinity using AuNPs 44 3.3.4. Proposed mechanisms for interaction between ssDNA physically adsorbed on AuNPs and MUC1 peptide 50 3.4. Summary . 58 CHAPTER PEGYLATED MUCIN APTAMER-DOXORUBICIN COMPLEX FOR TARGETED DRUG DELIVERY TO MCF7 BREAST CANCER CELLS 4.1. Introduction . 59 4.2. Experimental Section 61 4.2.1. Materials . 61 4.2.2. Cell culture . 61 III   4.2.3. Intercalation of DOX with MUC1-targeting aptamer (APT) . 61 4.2.4. Synthesis of PEG-modified MUC1-targeting aptamer (PEG-APT and PEG-APT-DOX) 62 4.2.5. Agarose gel electrophoresis 62 4.2.6. Cell cytotoxicity assay (MTT) . 63 4.2.7. Fluorescence microscopy . 64 4.2.8. DOX release from PEG-APT-DOX or APT-DOX complex . 64 4.3. Results and Discussion 65 4.3.1. Study of MUC1-targeting aptamer selective interaction with MCF7 cells using fluorescence microscopy 65 4.3.2. Intercalation efficacy of DOX with MUC1-targeting aptamer 65 4.3.3. Cell cytotoxicity of APT, DOX, and APT-DOX complex . 68 4.3.4. Synthesis of PEG-modified MUC1 targeting aptamer (PEG-APT and PEG-APT-DOX), and study of their cell cytotoxicity . 69 4.3.5. DOX release from PEG-APT-DOX or APT-DOX complex . 72 4.4. Summary . 73   CHAPTER DESIGNER TRIDENTATE MUCIN APTAMER FOR TARGETED DRUG DELIVERY 5.1. Introduction . 74 5.2. Experimental Section 75 5.2.1. Materials . 75 5.2.2. Cell culture . 76 5.2.3. Intercalation of DOX with MUC1-targeting aptamers . 76 5.2.4. Cell cytotoxicity assay 76 IV   5.2.5. DOX release from aptamer-DOX complexes 77 5.2.6. Determination of the cellular Kd of aptamers . 77 5.2.7. Fluorescence microscopy . 78 5.3. Results and Discussion 78 5.3.1. Design of modified APT using Mfold program . 78 5.3.2. Intercalation efficacy of DOX with MUC1-targeting aptamers . 79 5.3.3. Cell cytotoxicity of DOX, APT, APT-DOX, L3 and L3-DOX . 81 5.3.4. DOX release from APT-DOX or L3-DOX complex . 84 5.3.5. Study of MUC1-targeting aptamer selective interaction with MCF7 cells using fluorescence microscopy 85 5.3.6. Determination of the cellular Kd of aptamers . 85 5.4. Summary . 89 CHAPTER STUDY OF THE AFFINITY LIGANDS FOR USE IN TARGETED BLADDER CANCER THERAPY 6.1. Introduction . 90 6.2. Experimental Section 92 6.2.1. Materials . 92 6.2.2. Cell culture . 92 6.2.3. Fluorescence microscopy . 93 6.3. Results and Discussion 94 6.3.1. Affinity analysis of MUC1 ssDNA aptamer (CY5-APT) against MGHU3 . 95 6.3.2. Affinity analysis of MUC1 antibody against MGH-U3 and MCF7 . 97 V   6.3.3. Affinity analysis of EGFR RNA aptamer (CY3-CL4) against T24, UMUC-3 and MGH-U3 . 104 6.3.4. Affinity analysis of HER2 RNA aptamer (CY3-mini RNA) against T24, UM-UC-3 and MGH-U3 . 108 6.3.5. Affinity analysis of ανβ3 integrin peptide aptamer (PLZ4) against T24 117 6.4. Summary . 120 CHAPTER CONCLUSIONS 7.1. Summary of Major Achievements 121 7.2. Suggestions for Future Work 125 7.2.1. Optimization of the AuNP-based assay to increase its sensitivity and/or detection limit and functionality 125 7.2.2. Using aptamer-DOX complexes for in vivo drug delivery . 126 7.2.3. Using MUC1 aptamer modified PEGylated nanoparticles with Fe3O4 or AuNP core for 2-in-1 therapy 126 7.2.4. Selection of ligands for targeted bladder cancer therapy via cell SELEX . 127 REFERENCES . 128 APPENDIX I: LIST OF PUBLICATIONS ARISING FROM THE Ph.D. STUDY 154 VI   SUMMARY Targeted drug delivery using aptamers is a new generation therapeutics that holds a great promise for effective treatment of cancer. Oligonucleotide aptamers, singlestranded DNA (ssDNA) or RNA with affinity akin to that of antibodies, can virtually be selected against any target. In this study, the use of aptamers in cancer therapy was investigated in four subprojects. Firstly, a sensitive and facile gold nanoparticle (AuNP) based assay [with a detection limit of nM mucin (MUC1) peptide] was developed for peptide-aptamer affinity analysis. Secondly, MUC1 targeting aptamer was modified with polyethylene glycol (PEG) to avoid macrophage uptake. Thirdly, MUC1 targeting aptamer was modified to form tridentate aptamer to increase specificity toward MUC1 overexpressing breast cancer cells as compared to macrophages. Doxorubicin (DOX) was then intercalated within the modified aptamers, and the drug-aptamer complexes used for targeted drug delivery to breast cancer cells. Through these modifications, around 6-fold increase in macrophage viability (as compared to when free DOX was used) was achieved. Finally, various newly reported affinity ligands were assessed for their potential uses in bladder cancer drug delivery. 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Surface chemistry of ultrathin films of histidine on gold as probed by high-resolution synchrotron photoemission. J. Phys. Chem. B 2004, 109, 884-891. Zuker, M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 2003, 31, 3406-3415. 153   Appendix I   APPENDIX I LIST OF PUBLICATIONS ARISING FROM THE Ph.D. STUDY Tan, L.; Neoh, K. G.; Kang, E.-T.; Choe, W.-S.; Su, X. PEGylated anti-MUC1 aptamer-doxorubicin complex for targeted drug delivery to MCF7 breast cancer cells. Macromol. Biosci. 2011, 11, 1331-1335. Tan, L.; Neoh, K. G.; Kang, E.-T.; Choe, W.-S.; Su, X. Affinity analysis of DNA aptamer-peptide interactions using gold nanoparticles. Anal. Biochem. 2012, 421, 725-731. Tan, L.; Neoh, K. G.; Kang, E.-T.; Choe, W.-S.; Su, X. Designer tridentate mucin aptamer for targeted drug delivery. J. Pharm. Sci. 2012, 101, 1672-1677. 154    [...]... majority of the human adenocarcinomas, including breast cancer and bladder cancer (Ferreira et al., 2006; Jarrard et al., 1998; Taylor-Papadimitriou et al., 1999) While bladder cancer is only the seventh highest in terms of incident occurrence and ninth highest in terms for fatality for men, there is a high rate of reoccurrence and lack of targeting ligands for bladder cancer that can be applicable for. .. targeting of MUC1 protein on cancer cells by MUC1 specific aptamers, and their ability to intercalate and deliver DOX to the cancer cells The MUC1 specific aptamer S1.3 (APT) was modified with PEG to enhance the selectivity of APT for MCF7 (MUC1 overexpressing breast cancer cells) over macrophages 3) To enhance the selectivity of aptamer in situ during aptamer synthesis (one-step process) instead of post-synthesis... proof of concept study that highlights the feasibility of in vivo SELEX as a method to select ligands binding to extracellular cell receptors in their native conditions 2.2 Current Methods for Determining Affinity of Selected ssDNA or RNA Aptamer Sequences for Their Targets There are various methods to determine the bulk affinity of aptamers in between selection rounds and affinity of selected aptamer. .. 13.7% of total deaths) in 2008, exceeded only by cardiovascular diseases (Butler, 2011) For men, lung and bronchus cancer has the highest occurrence and fatality rate worldwide among the cancer incidents; for women, breast cancer holds the top position (American Cancer Society, 2011b) The current treatment for cancer mainly involves surgical removal of tumor, radiotherapy and/or chemotherapy (American Cancer. .. discussion of the study Chapter 7 provides the recap of major conclusions and additional insights obtained from this study, as well as suggestions for future work An overview of the investigations in this study is presented in a flowchart on page 7 In Chapter 3, AuNPs were used as colorimetric probe and fluorescence quencher for affinity analysis of DNA aptamers toward their target MUC1 peptide ssDNA aptamer- coated... 5.5-fold increase of survivability of RAW cells as compared to when free DOX was used These results indicate that aptamer L3 has good potential for targeted drug therapeutics Currently, there is limited information in the literature on targeting ligands for specific bladder cancer drug delivery Hence, in Chapter 6, the properties of several reported targeting ligands for bladder cancer was investigated... cancer that can be applicable for various types of bladder cancers Hence, it is imperative to study the possible ligands to facilitate targeted drug delivery For breast cancer targeted therapy, systemic circulation of therapeutic drugs is the norm of the treatment (American Cancer Society, 2011a) and hence the importance of macrophage avoidance For bladder cancer targeting, the therapeutic drugs will... especially important for monitoring the increase in bulk aptamer affinity as the screening proceeds For the initial rounds, the population of binder aptamers is low and it is ideal to use only a small amount for affinity test such that the rest of the aptamers could be used for the next round of selection to maximize the sequence variation, and this could only be achieved if the sensitivity of the assay is... insufficient for the recovery of the patient, as residual cancer cells may still be present Hence, radiotherapy and/or chemotherapy are often used in parallel However, both radiotherapy and chemotherapy are not cancer cell specific, leading to the killing of both normal and cancer cells This results in unwarranted side effects and lowers the quality of life of patients Hence, the search is on for the “magic... 2   antibiotics and proteins (Stoltenburg et al., 2007) Some uses for the aptamers are purification of compounds, detection of analytes in sensors, and therapeutics The basic principle of SELEX is illustrated in Figure 2.1 (Stoltenburg et al., 2007) An aptamer library consists of 1013 to 1015 different sequences of ssDNA (James, 2000) For ssDNA SELEX processes, the ssDNA library is first incubated with . STUDY OF APTAMER FOR CANCER THERAPEUTICS TAN LIHAN NATIONAL UNIVERSITY OF SINGAPORE 2012 STUDY OF APTAMER FOR CANCER THERAPEUTICS . development of a AuNP based assay to study peptide -aptamer interaction, 2) the modification of aptamer to tailor drug delivery to cancer cells (but not macrophages), and 3) the study of bladder cancer. microscopy 85 5.3.6. Determination of the cellular K d of aptamers 85 5.4. Summary 89 CHAPTER 6 STUDY OF THE AFFINITY LIGANDS FOR USE IN TARGETED BLADDER CANCER THERAPY 6.1. Introduction