Fei Zhengzheng

download Fei Zhengzheng

of 183

  • date post

    27-Nov-2014
  • Category

    Documents

  • view

    120
  • download

    3

Embed Size (px)

Transcript of Fei Zhengzheng

Membrane Sandwich Electroporation for In Vitro Gene Delivery

Dissertation

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By Zhengzheng Fei, M.S. Graduate Program in Chemical Engineering

The Ohio State University 2009

Dissertation Committee: Professor L. James Lee. Yang, Advisor Professor Robert J. Lee Professor Jessica Winter

Copyright by Zhengzheng Fei 2009

ABSTRACT

Gene therapy is the delivery of therapeutic genes into cells and tissues with the aim of treating and curing a disease. As an enhanced understanding of the roles of genes in health and disease, gene therapy is showing promise against various diseases such as cancer, diabetes, Parkinson's disease, and several inherited physiological defects. Viral transduction is very efficient, but safety issues, such as immune and inflammatory responses, have hampered their clinical uses in humans. Non-viral methods, including either chemical transfection with cationic lipids/polymers or physical transfection using electroporation/microinjection, are becoming attractive approaches. Electroporation is one of the most popular non-viral gene transfer methods for in vitro cell transfection. Initial studies with electroporation experienced very low transfection efficiencies and cell viability, severely limiting the development of this technology. The emergence of nucleofection (a modified electroporation technology) provided an efficient means for transfecting cells in vitro. However, nucleofection still encounters many limitations such as the large number of cells required (>106) and high cost involved. Moreover, cell viability is still an issue due to the high electric voltage used and the non-uniform electric field strength distribution generated during the process.

-ii-

To address these problems, we propose to develop an electroporation system based on an innovative micro-/nanoengineering technology for in vitro gene delivery. In our approach, electroporation is carried out in a mild and uniform electric field, with potential for a wider process window that can be generated to cover a wide range of cell lines and even primary cells. A new membrane sandwich electroporation (MSE) approach was demonstrated using plasmids GFP and SEAP as model materials. NIH 3T3 fibroblasts were tested and a significant improvement in transgene expression was observed compared to current electroporation techniques. In the MSE method, the focused electric field enhances cell permeabilization at a low electric voltage, leading to high cell viability; more important, the sandwich membrane configuration is able to provide better gene confinement near the cell surface, facilitating gene delivery into the cells. Next, we demonstrated the use of femtosecond laser fabricated micro-nozzle arrays on a gelatin-coated PET membrane for MSE. Using micro-nozzle array enhanced MSE, we observed high and uniform gene transfection, and good cell viability of mouse embryonic stem (ES) cells compared to the bulk electroporation. Since typically cells or tissues from the patients are very limited and therapeutic materials such as plasmids and oligonucleotides are very expensive, the ability to treat a small number of cells (i.e. a

-iii-

hundred) offers great potential to work with hard-to-harvest patient cells for patientspecific ex vivo gene therapy and in vitro pharmaceutical kinetic studies. Numerical calculation of transmembrane potential qualitatively explains the observed differences between MSE and bulk electroporation. Since theres a good correlation between transfection results and transmembrane potential calculations, the simulation process with the threshold experiments can be used to predict the transfection results, and thus largely reduced the trial-and-error window size. Furthermore, we successfully integrated an electrospun nanofiber scaffold as a cell-binding substrate into MSE, called nanofiber based MSE. With a micro-well spacer, the uniform size of mouse ES cell colonies were obtained, and plasmid transfection by electroporation were performed during colony formation. In addition, repeated plasmid SEAP transfection of NIH 3T3 fibroblasts was tested and better cell survival and recovery rate was observed using the electrospun nanofiber scaffold as compared to using micro-porous membrane. Due to its capacity of extend the exposure time with reprogramming factors, nanofiber based MSE demonstrated the potential for efficient induced pluripotent stem (iPS) cell generation by repeated plasmid transfection.

-iv-

ACKNOWLEDGMENTS

I would like to express my sincere gratitude to my advisor Dr. L. James Lee for his patient guidance, constructive advice and continuous support during my PhD study at the Ohio State University. I am indebted to Dr. Yubing Xie, Dr. Shengnian Wang, Dr. Xin Hu, Dr. Hae Woon Choi, Dr. Sadhana Sharma, Mr. Brian Henslee, Mr. Bo Yu, Dr. Yun Wu, and Dr. Weixiong Wang for their technical support, insightful suggestions, and encouragements. I would like to acknowledge Dr. Jingjiao Guan, Dr. Xulang Zhang, Dr. Chee Guan Koh, Dr. Yong Yang, and all former and current group members for their valuable discussions, and helpful comments. Thanks also go to Mr. Shi-Chiung Yu, Dr. Chunghe Zhang, Mr. Daniel Gallego, Ms. Natalia Higuita, Mr. Yong Chae Lim, Mr. Chi Yen, and all the students and staffs at the center for their warm help on my research project. The financial support and technical directions from NSF sponsored Nanoscale Science and Engineering Center for Affordable Polymeric Biological Devices (NSECCAPBD) is appreciated. Finally, I would like to thank my parents for their love and dedications for raising, supporting, and educating me. Great appreciations to my husband, Mr. Ziru Zhang, for his love, support, accompany and understanding through all these years.

-v-

VITA

June, 2001B.S. Chemical and Biochemical Engineering, Zhejiang University, Hangzhou, China March, 2004 M.S. Chemical and Biochemical Engineering, Zhejiang University, Hangzhou, China October 2004 to present.. Graduate Research Fellow, Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH

PUBLICATIONS 1. Fei, Zhengzheng; Wang, Shengnian; Xie, Yubing; Henslee, Brian E.; Koh, Chee Guan; Lee, L. James. Gene transfection of mammalian cells using membrane sandwich electroporation. Analytical Chemistry (2007), 79, 5719. 2. Guan, Yixing; Fei, Zhengzheng; Lou, Man; Yao, Shanjing. Choromatographic refolding of recombinant human interferon gamma by an immobilized sht GroEL191345 column. Journal of Chouromatography A (2006), 1107, 192. 3. Guan, Yixing; Fei, Zhengzheng; Lou, Man; Yao, Shanjing. Production of minichaperone (sht GroEL191-345) and its function in the refolding of recombinant human interferon gamma. Protein & Peptide Letters (2005), 12, 85. -vi-

4. Jin, Ting; Guan, Yixing; Fei, Zhengzheng; Yao, Shanjing. A combined refolding technique for recombinant human interferon-gamma inclusion bodies by ionexchange chouromatography with a urea gradient. World Journal of Microbiology & Biotechnology (2005), 21, 797. 5. Fei, Zhengzheng; Guan, Yixing; Yao, Shanjing. A colorimetric method to assay biological activity of recombinant human IFN-. Weishengwuxue Tongbao (Chinese Edition) (2004), 31, 65. 6. Guan, Yixing; Fei, Zhengzheng; Lou, Man; Yao, Shanjing. Minichaperone (GroEL191-345)-mediated in vitro refolding of recombinant human interferon gamma inclusion body. Shengwu Huaxue Yu Shengwu Wuli Jinzhan (Chinese Edition) (2004), 31, 907. 7. Jin, Ting; Guan, Yixing; Fei, Zhengzheng; Lou, Man; Yao, Shanjing. Renaturation of recombinant human interferon gamma inclusion body by dilution. Huagong Xuebao (Chinese Edition) (2004), 55, 770.

FIELDS OF STUDY Major Field: Chemical Engineering Minor Field: Biochemical Engineering

-vii-

TABLE OF CONTENTS

Page Abstract ............................................................................................................................... ii Acknowledgments............................................................................................................... v Table of contents.............................................................................................................. viii List of Tables .................................................................................................................... xv List of Figures .................................................................................................................. xvi Chapter 1: 1.1 1.2 Introduction................................................................................................. 1

Background ............................................................................................................. 1 Objectives ............................................................................................................... 3 Membrane sandwich electroporation (MSE) .................................................... 3 Micro-nozzle array enhanced MSE .................................................................. 3 Nanofiber based MSE ....................................................................................... 4 Literature review......................................................................................... 6

1.2.1 1.2.2 1.2.3 Chapter 2: 2.1

Gene delivery .......................................................................................................... 6 Viral versus non-viral ....................................................................................... 6 In vivo versus in vitro........................................................................................ 7

2.1.1 2.1.2 2.2