ANALYSIS OF POLYETHYLENIMINE BY ELECTROSPRAY IONIZATION MASS SPECTROMETRY AND SIZE EXCLUSION CHROMATOGRAPHY By Jianghong Gu Submitted to the Faculty of the College of Arts and Sciences of American University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy In Chemi /Dr. Monika Konakli'onaklieva Dean 0 1 the College of Arts and Sciences Dr. Milena Shahu Date 2005 American University Washington, D.C. 20016 AMERICAN UNIVERSITY LIBRARY Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 3187219 Copyright 2005 by Gu, Jianghong All rights reserved. INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. ® UMI UMI Microform 3187219 Copyright 2006 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. © COPYRIGHT by Jianghong Gu 2005 ALL RIGHTS RESERVED Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ANALYSIS OF POLYETHYLENIMINE BY ELECTROSPRAY IONIZATION MASS SPECTROMETRY AND SIZE EXCLUSION CHROMATOGRAPHY By Jianghong Gu ABSTRACT The efficient delivery of therapeutic genes into target cells or tissues is a critical goal in gene therapy. Polyethylenimine (PEI) has been widely proven to effectively transfer genes both in vitro and in vivo for various diseases. Studies have reported that PEFs efficiency as a gene delivery vehicle depends on its molecular weight and structure. However, commercial PEI has not been thoroughly characterized. Therefore the goal of this research is to develop methods to analyze commercial PEI, both linear and branched, through the use of electrospray ionization mass spectrometry (ESI-MS), size exclusion chromatography (SEC), and a combination of these two techniques. The method development involved the measurement of molecular weight distribution (MWD), the analysis of ion attachment, the determination of end groups, and the assessment of the degree of branching. The preliminary analysis of molecular weight distribution by ESI-MS showed that the MWD of each sample was found to be very similar. PEI analyzed by electrospray shows preferential ion attachments to most molecules. An ESI study with mono and ii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. divalent cations suggests the lithium cations appear to be the optimum candidate for cationization of ESI technique. The universal calibration concept was applied to analysis of molecular weight distribution by SEC. The result demonstrated the polymer structure played a dominant role in this study. Coupling ESI-MS with SEC technique enables the separation and characterization of complex polymer community. This study demonstrated the existence of two types of PEI molecules (non-cyclic and cyclic molecules). The end groups for these two types of PEI oligomers were obtained, respectively. To assess branching, imine derivatives of the PEI were prepared by reacting 7.5 equivalents of benzaldehyde and 4-fluorobenzaldehyde with PEI, respectively. ESI-MS spectra of the imine derivatives were compared and discussed. Analysis of PEI by ESI, SEC, and the combination of both techniques can provide valuable data, but the data is limited. For analysis of large PEI molecules, such as PEI- 1800 or higher, the MALDI-TOF-MS technique may be needed to overcome the detection limit of ESI-MS technique. 111 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS I would like to first thank and acknowledge a few persons who helped and supported me in my studies in the chemistry department of American University. I am greatly indebted to my advisor, Professor James E. Girard, for his patient instruction, greatest help, continuous support and encouragement in the past five years. I am also grateful to Dr. Monika Konaklieva and Dr. Milena Shahu for serving as members on my dissertation committee. Thank you, Dr. Konaklieva, for greatly supporting and helping me finish my research. Dr. Shahu, I thank you for always offering me encouragement and assistance. To the faculty, staff, and students in chemistry department, I would like to acknowledge the support, help, and friendship shown to me. Thanks to all of you for making our chemistry department a warm family. I also extend my appreciation to Robert Classon and Christopher Gilles at Shimadzu Scientific Instruments, Inc. They always provided me with very helpful suggestions and assistance related to my research whenever possible. Finally, I want to dedicate this dissertation to my family. Without their invaluable love, care, and support I couldn’t realize my ideal. Countless thanks to my parents, who keep encouraging and guiding me whenever I fall down. Special thanks to Liwei for his constant care and support. iv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS ABSTRACT.................................................................................................................................ii ACKNOWLEDGEMENTS......................................................................................................iv LIST OF ILLUSTRATIONS....................................................................................................ix LIST OF TABLES...................................................................................................................xiii Chapter 1. INTRODUCTION................................................................................................................. 1 Polyethylenimine (PEI) ..............................................................................................6 Synthesis.................................................................................................... 6 Aziridine .............................................................................................8 Branched P E I .................................................................................. 10 Linear PE I ........................................................................................15 Chemical Properties................................................................................ 18 2. PEI TRANSFECTION CONSIDERATIONS..................................................................21 PEI/DNA Complexes ...............................................................................................21 Influencing Factors on PEI Transfection Efficiency ............................................ 26 Mechanism................................................................................................................31 3. PEI CHARACTERIZATION.............................................................................................33 Polymer Characterization ........................................................................................ 33 Previous Characterization of Polyethylenimine ....................................................35 v Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Statement of Dissertation ........................................................................................ 36 4. PRELIMINARY ANALYSIS OF PEI BY MASS SPECTROMETRY........................ 38 Introduction ...............................................................................................................38 MALDI-TOF MS....................................................................................40 Electrospray Ionization Mass Spectrometry ........................................42 Preliminary Determination of Molecular Weight Distribution ...........................45 Introduction ............................................................................................. 45 Experimental ........................................................................................... 45 Samples and Reagents ....................................................................45 Instrumentation ...............................................................................46 Procedures ............................................................................. 46 Results and Discussion ........................................................................... 46 Ion Attachment Analysis ......................................................................................... 48 Introduction ............................................................................................. 48 Experimental ........................................................................................... 56 Samples and Reagents ....................................................................56 Instrumentation ...............................................................................56