트리페닐포스포니움-폴리(입실론-카프로락톤) 결합체를 이용한 미토콘드리아 표적 유전자 전달 시스템 = Mitochondria-targeting triphenylphosphonium-conjugated poly(ε-caprolactone) based gene delivery systems

다국어 초록 (Multilingual Abstract)

The mitochondria having their own DNA like the nucleus play a key role regulating calcium homeostasis, apoptosis and production of ATP in the living cell. Mitochondria dysfunction induces many critical human disorders such as inherited mitochondrial diseases, cancer, neurodegenerative diseases, and neuromuscular diseases and could be treated by mitochondria targeting gene therapy.
For mitochondria targeting gene delivery, triphenylphosphonium-b-poly(ε-caprolactone)-triphenylphosphonium (TPP-PCL-TPP) polymer was designed using PCL having a molecular weight of 1.25 kDa (PCL1.25kDa) and TPP and called as TPCL1 polymer in the previous study. TPCL1/pDNA polyplexes prepared with C/A (cation/anion ratio) above 0.5, size less than 150 nm, and zeta-potentials of 40 mV. With increasing C/A values of TPCL1/pDNA polyplexes, their transfection efficiencies were increased and reached to a saturated level and especially, showed comparable to that of bPEI25kDa/pDNA polyplexes (N/P 5) in HepG2 and MCF7 cells. Also, sustained gene expression of TPCL1/pDNA polyplexes were observed through time-dependent transfection for at least 10 ~ 14 days in HepG2 and MCF7 cells. Moreover, the mitochondrial uptakes of TPCL1/pDNA polyplexes were more dramatically increasing with increasing C/A values than their cellular uptake and nuclear uptake. Importantly, the mitochondrial preference to the nucleus of TPCL1/pDNA complexes were about 3-fold higher than that of bPEI25kDa/pDNA complexes (N/P 5). In conclusion, this study may indicate that TPCL polymeric gene carriers have potentials in sustained gene delivery as well as mitochondria-targeting gene therapy.

목차 (Table of Contents)

• CONTENTS
• LIST OF TABLES AND FIGURES ···············································Ⅵ
• LIST OF ABBREVIATIONS ·····················································Ⅷ
• ENGLISH ABSTRACT ····························································Ⅹ
• 1. INTRODUCTION ·······································································1
• 2. MATERIALS AND METHODS ···························································5
• 2.1 Materials ··················································································5
• 2.2 Cells and cell culture····································································5
• 2.3 Preparation and physicochemical characterization of self-assembled TPCL nanoparticles (NPs) ··········································································6
• 2.4 In vitro cytotoxicity of TPCL NPs····················································6
• 2.5 Preparation of TPCL1-pDNA complexes···········································7
• 2.6 Particle size and zeta potential of polyplexes ·······································8
• 2.7 Gel electrophoresis for gene condensation assay ··································8
• 2.8 Dye-quenching assay for compactness of polyplexes ····························8
• 2.9 Heparin-induced decomplexation assay ···········································9
• 2.10 Transfection efficiency assay ·····················································9
• 2.11 Cellular viability of polyplexes ·····················································10
• 2.12 Intracellular localization of polyplexes ································11
• 2.13 Cellular uptake, nuclear uptake, mitochondrial uptake of TPCL1/pDNA polyplexes ············································································11
• 2.14 Cellular internalization pathways ··················································13
• 3. RESULTS AND DISCUSSION ····················································14
• 3.1 In vitro cytotoxicity of TPCL NPs ·············································14
• 3.2 Preparation and physicochemical characteristics of TPCL1/pDNA polyplexes ·················································································15
• 3.3 Gel electrophoresis for gene condensation assay······························16
• 3.4 Dye-quenching assay for compactness of polyplexes·······················17
• 3.5 Heparin-induced decomplexation assay ·······································18
• 3.6 Transfection efficiency assay··················································20
• 3.7 Cellular viability of polyplexes ················································22
• 3.8 Intracellular localization of polyplexes ········································24
• 3.9 Cellular uptake, nuclear uptake, mitochondrial uptake of TPCL1/pDNA polyplexes ···········································································29
• 3.10 Cellular internalization pathways ············································32
• 4. CONCLUSION ·······························································36
• 5. REFERENCES ······························································37
• KOREAN ABSTRACT························································47