Effects of surface charge density of membranes on the irreversible electroporation induced rate constant of pore formation of vesicles

Abstract

In this study, the effects of surface charge density of membranes on the irreversible electroporation (IRE)-induced rate constant of pore formation in the membranes of giant unilamellar vesicles (GUVs) was investigated. GUVs were synthesized by a mixture of anionic lipid dioleoylphosphatidylglycerol (DOPG) and neutral lipid dioleoylphosphatidylcholine (DOPC) in a physiological buffer (10 mM PIPES, pH 7.0, 150 mM NaCl and 1 mM EGTA) using the natural swelling method. The IRE signal (pulsating direct current) of frequency 1.1 kHz was applied through the gold coated electrode system. By changing the DOPG mole fraction X of values 0.10, 0.20, 0.40 and 0.60, the corresponding surface charge densities of membranes were determined. For all values of X, the rate constant increased with the increase of constant electric tension due to IRE signal. The rate constant found higher at lower tension due to the increase of X. From the fitting of tension-dependent rate constant data, the line tensions were found 10.1 ± 0.1, 11.2 ± 0.3, 12.1 ± 0.1 and 13.1 ± 0.3 pN for X = 0.10, 0.20, 0.40 and 0.60, respectively. The influence of X on the critical tension of electroporation in GUVs was also investigated. The critical tension decreased from 9.0 ± 0.3 to 6.0 ± 0.2 mN/m with the increase of X from 0 to 0.60 in GUVs. The theoretical equation was fitted to the X dependent normalized critical tension and the binding constant of lipid-ion interaction was obtained 0.75 M-1. The decreased in energy barrier for the formation of a prepore, due to the increased of X, was the main factor explaining the decreased of critical tension and increased of rate constant of pore formation in GUVs.

Contents Abstract vi Contents vii List of Figures x List of Tables xvii List of Abbreviations xix

CHAPTER 1: INTRODUCTION 1

1.1 Preface 1 1.2 Objective of the Study 5 1.3 Outline of the Thesis 5

CHAPTER 2: LITERATURE REVIEW AND THEORETICAL ASPECTS 6

2.1 Literature Review 6 2.2 Cell Membrane Physiology 14 2.3 Lipid and Lipid Bilayer Structure 16 2.4 Model Membrane 18 2.5 Vesicles 19 2.6 Vesicles and its Classification 20 2.7 Phospholipids 21 2.8 Membrane Electrical Model and Equation 22 2.8.1 The mechanism of charges of surfaces in liquid 22 2.8.2 Charged surfaces in water without added electrolyte 23 2.8.3 The Poisson-Boltzmann (PB) equation 24 2.8.4 Charged surface in electrolyte solution 25 2.8.5 The Grahame equation 27 2.8.6 Surface charge of isolated surfaces 27 2.8.7 Debye length 28

CHAPTER 3: MATERIALS AND METHODS 29

3.1 Chemicals and Regents 29 3.2 Synthesis of Lipid Membranes of GUVs 30 3.3 Preperation of Sample 31 3.4 Purification Method of Lipid Membrane 32 3.5 Observations of Lipid Membrane of GUVs 34 3.5.1 Suspension of GUVs in microchamber 34 3.5.2 Microscopy 34 3.6 Description of the IRE Technique 35

CHAPTER 4: RESULTS AND DISCUSSION 38

4.1 Pore Formation in 20%DOPG/80%DOPC-GUVs at 5.75 mN/m 38 4.2 Probability of Pore Formation in 20%DOPG/80%DOPC-GUVs at 5.75 mN/m 39 4.3 Rate Constant Pore Formation in 20%DOPG/80%DOPC-GUVs at 5.75 mN/m 41 4.4 Pore Formation in 20%DOPG/80%DOPC-GUVs at 6.5 mN/m 42 4.5 Probability of Pore Formation in 20%DOPG/80%DOPC-GUVs at 6.5 mN/m 43 4.6 Rate Constant Pore Formation in 20%DOPG/80%DOPC-GUVs at 6.5 mN/m 45 4.7 Pore Formation in 20%DOPG/80%DOPC-GUVs at 7.25 mN/m 46 4.8 Probability of Pore Formation in 20%DOPG/80%DOPC-GUVs at 7.25 mN/m 47 4.9 Rate Constant Pore Formation in 20%DOPG/80%DOPC-GUVs at 7.25 mN/m 48 4.10 Theoretical Aspect of the Probability of Pore Formation and the Rate Constant of Pore Formation 49 4.11 Dependence of Ppore (60 s) on c for 20%DOPG/80%DOPC-GUVs 50 4.12 Dependence of Rate Constant of Pore Formation on c for 20%DOPG/80%DOPC-GUVs 51 4.13 Pore Formation in 60%DOPG/40%DOPC-GUVs at 4.5 mN/m 53 4.14 Probability of Pore Formation in 60%DOPG/40%DOPC-GUVs at 4.5 mN/m 54 4.15 Rate Constant Pore Formation in 60%DOPG/40%DOPC-GUVs at 4.5 mN/m 56 4.16 Pore Formation in 60%DOPG/40%DOPC-GUVs at 5.5 mN/m 57 4.17 Probability of Pore Formation in 60%DOPG/40%DOPC-GUVs at 5.5 mN/m 58 4.18 Rate Constant Pore Formation in 60%DOPG/40%DOPC-GUVs at 5.5 mN/m 59 4.19 Pore Formation in 60%DOPG/40%DOPC-GUVs at 6.0 mN/m 60 4.20 Probability of Pore Formation in 60%DOPG/40%DOPC-GUVs at 6.0 mN/m 61 4.21 Rate Constant Pore Formation in 60%DOPG/40%DOPC-GUVs at 6.0 mN/m 62 4.22 Dependence of Ppore (60 s) on c for 60%DOPG/40%DOPC-GUVs 63 4.23 Dependence of Rate Constant of Pore Formation on c for 60%DOPG/40%DOPC-GUVs 64 4.24 Dependence of Ppore (60 s) on c at Various DOPG/DOPC- GUVs 65 4.25 Dependence of Rate Constant on c for Various Surface Charge Density 66 4.26 Determination of Critical Tension by Changing Mole Fraction and Its Theoretical Aspects 68 4.27 Theoretical Aspect of Critical Tension 72