Durham E-Theses The development of anti-cancer drug delivery systems ZONG, JINGYI How to cite: ZONG, JINGYI (2016) The development of anti-cancer drug delivery systems, Durham theses, Durham University. Available at Durham E-Theses Online: http://etheses.dur.ac.uk/11927/ Use policy Thefull-textmaybeusedand/orreproduced,andgiventothirdpartiesinanyformatormedium,withoutpriorpermissionor charge,forpersonalresearchorstudy,educational,ornot-for-pro(cid:28)tpurposesprovidedthat: • afullbibliographicreferenceismadetotheoriginalsource • alinkismadetothemetadatarecordinDurhamE-Theses • thefull-textisnotchangedinanyway Thefull-textmustnotbesoldinanyformatormediumwithouttheformalpermissionofthecopyrightholders. PleaseconsultthefullDurhamE-Thesespolicyforfurtherdetails. AcademicSupportO(cid:30)ce,DurhamUniversity,UniversityO(cid:30)ce,OldElvet,DurhamDH13HP e-mail: [email protected]: +4401913346107 http://etheses.dur.ac.uk The Development of Anti-Cancer Drug Delivery Systems Jingyi Zong A thesis submitted for the degree of Doctor of Philosophy Department of Chemistry University of Durham October, 2016 Abstract Cancer is undoubtedly one of the main threats to global human health and as a result, despite significant advances in the field, new and improved cancer treatments are still in great need. Although chemotherapy (in combination with other therapies) is widely used to suppress the growth of tumours, many of the current anti-cancer drugs suffer from poor selectivity and consequently severe toxicity. In order to conquer these limitations, targeted drug delivery systems have been designed and studied with the primary aim of improving the accuracy of transporting anti-cancer drugs into cancer cells and tissue areas. The overall aim of the work presented in this thesis is to design new anti-cancer drug delivery systems using three different strategies. In Chapter 2, intelligent stimulus-responsive short elastin-like peptides (ELPs) and elastin-based side chain polymers (ESPs) were synthesised. The conformation and aggregation properties of these ELPs and ESPs were studied in different aqueous buffers (varying pH also) using ultraviolet-visible (UV-Vis) spectroscopy and circular dichroism (CD). Of the ELPs investigated, peptide 10 (N-acetylated VPGVG) was found to have the lowest transition temperature at pH 7 (i.e. 45oC). Amongst all the ESPs, PF100-GABA(VPGVG) (29) was proven to have the lowest transition temperature (47oC) which was most likely due to the fact that it had the highest molecular weight. In Chapter 3, gold nanoparticles (GNPs) were synthesised and functionalised with biomolecules including elastin-like peptides (ELPs), elastin-based side chain polymers (ESPs) and the pro-apoptotic peptide -(KLAKLAK) (KLA). The hybrids materials, D 2 ELP-GNPs and ESP-GNPs were characterized by UV-Vis, CD and transmission electron microscopy (TEM). The hybrids showed the same temperature sensitive properties as the free ELPs and ESPs previously studied, confirming the successful functionalization of GNPs. The KLA-GNPs were found to have increased anti-cancer activity against HeLa cells compared to the free KLA. In Chapter 4, the pro-apoptotic KLA peptide was conjugated to a series of cell penetrating peptoids (CPPos) to prepare peptoid-peptide hybrids (CPPos-KLA). The anti-cancer, antimicrobial and cell penetrating properties of these peptoid-peptide hybrids were investigated. The results demonstrated an increasing trend in anti-cancer ability of CPPos-KLA hybrids (compared to free KLA) and KLA-CPPo6 (57) gave the lowest IC value (ca.8 μM) against HeLa cells. 50 i Acknowledgements First and foremost, I would like to thank my academic supervisors, Professor Neil Cameron and Dr. Steven Cobb, for their kind support and guidance over the four years. With their patience and unrelenting help, I have learned not only knowledge and experience, but also an attitude of doing research which will benefit me for life. I would like to thank Dr. Beth Bromley for her kindly advice in CD experiments and letting me use her lab machines. I would like to thank the staff in the mass spectrometry services (Chemistry Department), in particular Mr Peter Stokes for his advice and help. I would like to thank Dr Aileen Congreve for her kind help and advice on HPLC experiments. I would like to thank Mr. Douglas Carswell for conducting the TGA measurements of my samples and for his support. I would also like to thank Ms Helen Grindley and Mrs Christine Richardson from the Biological and Biomedical Sciences Department for helping me with the preparation of TEM samples and measurements. I would like to express my grateful thanks to Dr. Wong Ka-Leung and his group in Hong Kong Baptist University, especially Lijun Jiang, for their collaboration on all the experiments of anti-cancer screen of KLA peptide and peptide-peptoid hybrids. Many thanks to all the Cameron and Cobb group members, past and present, for making my research much more enjoyable: Dr. Sarah Hehir, Dr. Fanny Joubert, Dr. Arturas Kubilis, Dr. Ahmed Eissa, Dr. Adam Hayward, Dr. Paul Thornton, Dr. Matt Disbury, Dr. David Johnson, Ali Abdulkarim, Maria Andela Caballo Gonzalez, Caitlin Langford, Dr Anica Dose, Maria Czyzewska, Dr. Lara Small, Dr. Asahi Cano-Marqués, Dr. Bnar Ahmed, Dr Chris Coxon, Dr Gabriella Eggimann, Alex Webster, Mark Laws, Ambrose Crofton, Caitlin Mooney, Sam Lear, Hannah Bolt, Beatriz Martínez, Diana Giménez and Dr Alex Hudson. Last but not least, thanks to the Durham University and China Scholarship Council (CSC) for funding my research. I am also very grateful to the Henry Lester and Great Britain China Education awards, for their financial support in my writing-up days. To conclude, I would like to dedicate this Doctoral Thesis to my family, who I will never be able to give back all the support they have given to me as it is immeasurable. Thanks to my mum, dad, my mother-in-law, father-in-law and my lovely sister, tian tian. To my beloved husband, Xilin, who stands by me and always comforts and cares for me. I am so blessed to have you. ii Declaration This work was conducted in the Department of Chemistry at Durham University between September 2012 and September 2016. The work has not been submitted for a degree in this, or any other university. It is my own work, unless otherwise indicated. Copyright The copyright of this thesis rests with the author. No quotation from it should be published without prior consent and information derived from it should be acknowledged. iii Contents ABSTRACT I ACKNOWLEDGEMENTS II DECLARATION III COPYRIGHT III CONTENTS IV LIST OF FIGURES X LIST OF TABLES XVIII LIST OF APPENDICES X LIST OF ABBREVIATION XXI CHAPTER 1: INTRODUCTION 1 1.1 Background ....................................................................................................... 1 1.1.1 Cancer cases and global health .................................................................. 1 1.1.2 Cancer treatment options ........................................................................... 3 1.1.2.1 Surgical resection ............................................................................ 3 1.1.2.2 Radiotherapy ................................................................................... 4 1.1.2.3 Chemotherapy ................................................................................. 6 1.2 Chemotherapy: Advantages and disadvantages ............................................ 9 1.3 Anti-cancer drug delivery systems ................................................................. 10 1.3.1 Polymers .................................................................................................. 10 1.3.1.1 Polymer-drug conjugates .............................................................. 12 1.3.1.2 Polymer micelles ........................................................................... 14 1.3.1.3 Polymer-protein Conjugate ........................................................... 15 iv 1.3.2 Nanoparticles ........................................................................................... 17 1.3.2.1 Polymeric nanoparticles ................................................................ 18 1.3.2.2 Gold nanoparticles (GNPs) ........................................................... 20 1.3.3 Cell penetrating peptides ......................................................................... 22 1.4 Stimuli responsive drug delivery .................................................................... 24 1.4.1 Temperature sensitive targeting .............................................................. 25 1.4.2 pH targeting ............................................................................................. 28 1.4.3 Light activation ........................................................................................ 30 1.5 Summary and project aims ............................................................................. 30 1.6 References ......................................................................................................... 31 CHAPTER 2: SYNTHESIS OF ELASTIN-LIKE PEPTIDE AND ELASTIN-BASED SIDE CHAIN POLYMERS 37 2.1 Introduction ...................................................................................................... 37 2.1.1 Properties of elastin-like polypeptides .................................................... 37 2.1.2 Synthesis of elastin-like polypeptides ..................................................... 40 2.1.2.1 Elastin biosynthesis ....................................................................... 40 2.1.2.2 Solid phase peptide synthesis (SPPS) ........................................... 41 2.1.3 Applications of elastin-like polypeptides (ELPs) .................................... 42 2.1.4 Chapter aims ............................................................................................ 46 2.2 Synthesis of short elastin-like peptides .......................................................... 47 2.2.1 Synthesis of peptide H-VPGVG-NH (9) ............................................... 47 2 2.2.2 Synthesis of acetylated peptide Ac-VPGVG-NH2 (10) .......................... 50 2.2.3 Synthesis of thiol-functionalised peptide HS-VPGVG-NH (11) ........... 52 2 2.3 Lower critical solution temperature (LCST) and circular dichroism (CD) studies of short elastin-like peptides..................................................................... 55 2.3.1 LCST behaviour of short elastin-like peptides ........................................ 55 2.3.1.1 LCST behaviour at different concentrations ................................. 55 2.3.1.2 LCST behaviour at different pH ................................................... 56 2.3.1.3 Summary of LCST behaviours of Rink Amide peptides .............. 57 v 2.3.2 CD of short elastin-like peptides ............................................................. 59 2.4 Synthesis of elastin-based side chain polymers ............................................. 64 2.4.1 Synthesis of pentafluorophenyl acrylate (14) monomer ......................... 64 2.4.2 Synthesis of poly(pentafluorophenyl acrylate) polymers ........................ 67 2.4.3 Synthesis of elastin-like peptides for coupling to P(PFPA) .................... 70 2.4.4 Synthesis of elastin-based side chain polymers ...................................... 74 2.5 LCST and CD experiments of elastin-based side chain polymers .............. 77 2.5.1 LCST behaviour of elastin-based side chain polymers (ESPs) ............... 77 2.5.1.1 Chain length and molecular weight............................................... 77 2.5.1.2 Effect of concentration on polymer 28 and 29 .............................. 80 2.5.1.3 Effect of added sodium chloride on 25 and 26 ............................. 80 2.5.1.4 Summary of LCST behaviour of ESPs ......................................... 81 2.5.2 CD analysis of elastin-based side chain polymers .................................. 82 2.6 Conclusions ....................................................................................................... 84 2.7 References ......................................................................................................... 85 CHAPTER 3: PEPTIDE-FUNCTIONALISED GOLD NANOPARTICLES 88 3.1 Introduction ...................................................................................................... 88 3.1.1 Gold nanoparticles (GNPs) ..................................................................... 88 3.1.2 Preparation of peptide functionalised GNPs ........................................... 89 3.1.2.1 Ligand exchange method .............................................................. 89 3.1.2.2 Chemical reduction ....................................................................... 90 3.1.2.3 Chemical conjugation method....................................................... 91 3.1.3 Applications of peptide functionalised GNPs ......................................... 92 3.1.3.1 Plasmonic biosensors .................................................................... 92 3.1.3.2 Targeted drug delivery and cellular uptake ................................... 94 3.1.3.3 Anti-cancer applications................................................................ 97 3.1.4 Aim of this chapter .................................................................................. 99 3.2 Synthesis of GNPs .......................................................................................... 100 vi 3.3 Preparation and Characterization of Short ELP-GNPs ............................ 103 3.3.1 Synthesis of ELP-GNPs ........................................................................ 103 3.3.2 Properties of ELP-GNPs ....................................................................... 103 3.4 Preparation and Characterization of Elastin based side chain polymer-GNPs (ESP-GNPs) ............................................................................... 109 3.4.1 Synthesis of ESP-GNPs ......................................................................... 109 3.4.2 Properties of ESP-GNPs ........................................................................ 110 3.5 Preparation and Characterization of Synthesis KLA-GNPs ..................... 115 3.5.1 Synthesis of thiol-TTDS-KLA peptide (38) .......................................... 115 3.5.2 Synthesis of KLA-GNPs (39) ................................................................ 119 3.5.3 Cytotoxicity of KLA-GNPs (39) ........................................................... 122 3.6 Preparation and Characterization of KLA-ESP -GNPs ......................... 125 100 3.6.1 Synthesis of KLA-ESP -GNPs (40) ................................................... 125 100 3.6.2 Properties of KLA-ESP -GNPs (40) .................................................. 126 100 3.6.3 Evaluation of the anti-cancer properties of KLA-ESP -GNPs (40) ... 128 100 3.7 Conclusions ..................................................................................................... 131 3.8 References ....................................................................................................... 133 CHAPTER 4: CELL-PENETRATING PEPTOIDS AS A DELIVERY METHOD FOR APOPTOTIC PEPTIDE KLA 135 4.1 Introduction .................................................................................................... 135 4.1.1 Antimicrobial peptides as a new class of anti-cancer agents ................ 135 4.1.2 Anti-cancer activity of KLA peptide ..................................................... 138 4.1.3 Cell penetrating peptoids (CPPos) ........................................................ 140 4.1.4 Chapter aims .......................................................................................... 143 4.2 Synthesis of KLA peptide .............................................................................. 144 4.3 Synthesis of peptoid hybrids ......................................................................... 147 4.3.1 CPPos .................................................................................................... 147 4.3.2 KLA-CPPos ........................................................................................... 150 vii 4.3.3 Rhod-KLA-CPPo6 (59) ......................................................................... 152 4.4 Biological evaluation of peptide-peptoid hybrids ....................................... 156 4.4.1 Cytotoxicity of KLA and KLA-CPPos ................................................. 156 4.4.2 Cellular uptake and in vitro imaging ..................................................... 159 4.4.3 Antibacterial screening .......................................................................... 160 4.5 Conclusions ..................................................................................................... 163 4.6 References ....................................................................................................... 164 CHAPTER 5: CONCLUSIONS AND FUTURE WORK 167 CHAPTER 6: EXPERIMENTAL PROCEDURES 172 6.1 General experimental .................................................................................... 172 6.1.1 Materials ................................................................................................ 172 6.1.2 Instruments ............................................................................................ 173 6.1.2.1 UV-Vis measurement .................................................................. 173 6.1.2.2 DLS measurement ....................................................................... 173 6.1.2.3 TEM measurement ...................................................................... 173 6.1.2.4 CD measurement ......................................................................... 174 6.1.2.5 HPLC purification ....................................................................... 174 6.1.2.6 NMR spectroscopy ...................................................................... 175 6.1.2.7 IR spectroscopy ........................................................................... 175 6.1.2.8 Mass spectrometry ...................................................................... 175 6.1.3 General experimental procedures .......................................................... 175 6.1.3.1 Manual Fmoc-SPPS .................................................................... 175 6.1.3.2 Peptide cleavage .......................................................................... 175 6.1.3.3 Synthesis of peptoids .................................................................. 176 6.1.4 Anti-cancer screening and anti-bacterial screening by collaborators .... 176 6.1.4.1 Cell culture .................................................................................. 176 6.1.4.2 MTT assay ................................................................................... 177 6.1.4.3 Flow cytometry analysis on cellular uptake ................................ 177 6.1.4.4 Confocal microscopic imaging ................................................... 177 viii