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A Novel Concept for Hyperthermia Induced Local Chemotherapy

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A Novel Concept for Hyperthermia Induced Local Chemotherapy A Novel Concept For Hyperthermia Induced Local Chemotherapy Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) Submitted to the Department of Biology, Chemistry and Pharmacy of Freie Universität Berlin by Jiang Gao from Xi´an, China March, 2011 The work for this PhD thesis was performed from August 2008 to March 2011 in the laboratory of Prof. Volker A. Erdmann in the Institute of Chemistry and Biochemistry at the Free University Berlin. Hereby I declare that I have written this thesis by myself, marked the sources of any quotations or content obtained otherwise. Diese Arbeit wurde in der Zeit von August 2008 bis März 2011 unter der Aufsicht von Professor Dr. Volker A. Erdmann am Institut für Biochemie der Freie Universität Berlin angefertigt. Die Verfasserin versichert, die Arbeit eigenständig durchgeführt und alle Hilfsmittel angegeben zuhaben. Gutachter: Prof. Dr. Volker A. Erdmann Prof. Dr. Rudolf Tauber Date of defence: 13.09.2011 Meinen Eltern Index Index 1 Introduction 1 1.1 Nucleic Acids..................................................................................................1 1.1.1 Basic Knowledge.....................................................................................1 1.1.2 Melting Temperature of Nucleic Acids…….............................................4 1.1.3 Nucleic Acids Synthesis and Nucleic Acids Analogues..........................5 1.1.4 Hammerhead Ribozyme….................................................................6 1.2 Nanoparticles………….......................................................................................9 1.2.1 Basic Properties of Nanoparticles…......................................................9 1.2.2 Magnetic Nanoparticles and Superparamagnetism.............................10 1.2.3 Hyperthermia and Magnetic Fluid Hyperthermia...................................11 1.2.4 NanoTherm® Therapy……………………………………….....................14 1.2.5 Nanoparticles and Drug Delivery…………..……………………………16 1.3 Crosslinker and Bioconjugation.......................................................................19 1.3.1 Zero-Length Crosslinkers......................................................................19 1.3.1.1 Carbodiimides…………………………………………………….19 1.3.1.2 N,N´-Carbonyldiimidazole………………………………………...22 1.3.2 Homobifunctional Crosslinkers…..........................................................24 1.3.3 Heterobifunctional Crosslinkers…….....................................................25 2 Task………….….................................................................................28 3 Methods.............................................................................................29 3.1 Analysis of Nucleic Acids........................................................................29 3.1.1 Gel Electrophoresis............................................................................29 3.1.1.1 Agarose Gel Electrophoresis......................................................29 3.1.1.2 Polyacrylamide Gel Electrophoresis (PAGE)…..........................30 3.1.1.2.1 Denaturing PAGE............................................................30 3.1.1.2.2 Native PAGE………………..............................................31 3.1.2 Detection of Nucleic Acids on Gels....................................................31 3.1.2.1 Ethidium Bromide Staining.........................................................31 3.1.2.2 SYBR®Gold Staining…………………………............................31 I Index 3.1.2.3 UV Shadowing…………………………………............................32 3.1.2.4 Phosphor Imaging…….………………………............................32 3.1.2.5 Fluorescence Scanning………………………............................32 3.1.3 Determination of Nucleic Acid Concentration…...............................33 3.1.3.1 UV Spectrophotometry……........................................................33 3.1.3.2 Fluorescence Labeling………………………............................33 3.1.3.3 Quant-iTTM OliGreen®ssDNA Reagent.……............................34 3.1.4 Purification of Nucleic Acids ……………………...............................35 3.1.4.1 Purification by Silica-Based Resins............................................35 3.1.4.2 Microcon Centrifugal Filter Devices..………............................36 3.1.4.3 Gel Filtration Chromatography.............………............................36 3.1.4.4 Ion-Exchange Chromatography.…..........................................37 3.1.5 Nucleic Acids 5´-End Labeling…………………...............................37 3.2 Bioconjugate Chemistry….......................................................................38 3.2.1 Coupling of Nanoparticle to Nucleic Acids with Sulfo-SMCC….........38 3.2.2 Coupling of Nanoparticle to Nucleic Acids with Sulfo-GMBS….........39 3.2.3 Coupling of Nanoparticle to Nucleic Acids with Sulfo-EDC…............40 3.2.4 Coupling of Nanoparticle to Nucleic Acids with EDC/Sulfo-NHS…....41 3.2.5 Coupling of Nanoparticle to Nucleic Acids with PDITC………...........42 3.2.6 Coupling of Nanoparticle to Nucleic Acids with Epoxide NPs…........42 3.2.7 Coupling of Nanoparticle to Nucleic Acids with gold-coated NP…….43 3.2.8 Coupling of Cytostatic drug to Nucleic Acids……………….............43 3.2.8.1 Coupling of Chlorambucil to DNA via EDC….............................43 3.2.8.2 Coupling of Chlorambucil to DNA via CDI….............................44 3.2.8.3 Coupling of Chlorambucil to DNA via DCC/NHS…….................44 3.2.8.4 Coupling of Methotrexate to DNA via DCC/NHS………..........45 3.3 Biochemical and Biophysical Methods....................................................46 3.3.1 Serum Stability Assay……………………….....................................46 3.3.2 MALDI-TOF-MS…………………......................................................46 3.3.3 DNA Melting Curves……….............................................................46 3.3.4 Determination of Release Curves…...................................................48 3.3.5 Release Experiment in the Applicator MFH®12-TS A…....................49 3.3.6 Atomic Absorption Spectroscopy…....................................................49 3.3.7 TEM Images of Cells……………......................................................50 II Index 3.3.8 Thin Layer Chromatogrphy…………………………..…....................50 3.3.9 MTT Assay…………………………………..........................................50 3.4 Nucleic Acids Dependent Drug-Delivery System....................................51 3.4.1 dsDNA-Nanoparticle-Drug Delivery System……...............................51 3.4.2 Ribozyme-Nanoparticle-Drug Delivery System..................................51 4 Results……........................................................................................53 4.1 dsDNA-Nanoparticle Drug Delivery System…..............................................53 4.1.1 Immobilization of Oligonucleotides to Nanoparticles..............................54 4.1.2 Comparison of Melting and Release Curves of dsDNA.........................59 4.1.3 Choice of the Sequence of the dsDNA Linker……………………………62 4.1.4 Release Experiment in the Magnetic Field Applicator MFH®-12-TSA…64 4.1.5 dsDNA-NP-Doxorubicin Release Experiment…………………………..66 4.2 Ribozyme-Nanoparticle-Drug-Delivery System…….......................................69 4.2.1 Choice of the Ribozyme, Substrate and Inhibitor...................................70 4.2.2 Activity of Mirror-Image Ribozymes........................................................72 4.2.3. Stability of Mirror-image Nucleic acids…………………………………73 4.2.4. Release Experiments in Buffer…………………………………………….74 4.2.5. Release Experiments in Human Serum…………………………………..77 4.2.6. Stability of Nanoparticle-Nucleic Acid-Conjugates……………………..79 4.2.7. Cellular Uptake of Nanoparticle-Nucleic Acid-Conjugates……………...79 4.2.8. Intracellular Distribution of Nanoparticle-Nucleic Acid Conjugates…81 4.3 Cytostatic Drug Coupling with Nucleic Acids…………………………………84 4.3.1 Coupling of Chlorambucil……….……………………………………….84 4.3.2. Cytotoxicity of Chlorambucil Conjugates………………………………..92 4.3.3. Coupling of Methotrexate…………………………………………….…….93 4.3.4. Cytotoxicity of Methotrexate Conjugates………………………………....96 5 Discussion.........................................................................................98 5.1 The Hybrid Approach ……………………………...............................................98 5.2 The Ribozyme Approch………………………………………………………….103 5.3 Conjugation of Cytotoxic Drugs to Oligonucleotides…………………………108 5.4 Prospects of the Novel-Drug Delivery System……………………………..…109 III Index 6 Summary………...............................................................................112 7 Zusammenfassung.........................................................................114 8 References………............................................................................116 9 Appendix………...............................................................................124 9.1 Materials……………………............................................................................124 9.1.1 Chemicals.............................................................................................124 9.1.2 Enzyme…………………………………………………............................125 9.1.3 Nanoparticles………………………………………………………………125 9.1.4 Nucleic Acids………………………………………………………………..126 9.1.5 Buffers………………………………………………………………...……..126 9.1.6 Devices and Consumption Materials....................................................127 9.2 Abbreviations…….........................................................................................129 9.3 Acknowledgements……………………………..………………………………..133
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