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
Procedures ...... 57
Results and Discussion ...... 57
5. ANALYSIS OF PEI BY SIZE EXCLUSION CHROMATOGRAPHY...... 65
Introduction ...... 65
Experimental ...... 67
vi
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Samples and Reagents ...... 67
Instrumentation ...... 67
Procedures ...... 68
Results and Discussion ...... 68
6. ANALYSIS OF PEI BY ESI-MS COUPLED WITH SEC...... 85
Introduction ...... 85
Experimental ...... 86
Samples and Reagents ...... 86
Instrumentation ...... 86
Procedures ...... 87
Results and Discussion...... 88
7. FURTHER ANALYSIS OF PEI BY ESI-MS ...... 109
Confirmation of the Existence of Cyclic Molecules ...... 109
Introduction ...... 109
Experimental ...... 109
Samples and Reagents ...... 109
Instrumentation ...... 109
Procedures ...... 110
Results and Discussion ...... 110
Assessment of the Degree of Branching ...... 117
Introduction ...... 117
Experimental ...... 117
vii
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Samples and Reagents ...... 117
Instrumentation ...... 117
Procedures ...... 118
Results and Discussion ...... 118
8. CONCLUSIONS ...... 138
REFERENCES...... 142
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF ILLUSTRATIONS
Figure 1. Structures of some cationic liposomes used for transfection ...... 4-5
Figure 2. Structures of linear PEI and branched PEI ...... 7
Figure 3. Structures of Aziridine Groups ...... 8
Figure 4. Schematic of three general synthetic methods for preparation of aziridine ...... 9
Figure 5. Schematic of production of branched PEI ...... 11-13
Figure 6. Hydrated forms of linear PEI. Anhydrate (a); hemihydrate (b); sesquihydrate (c); dehydrate (d) ...... 14
Figure 7. Double-Stranded helical chains of linear PEI ...... 15
Figure 8. Schematic of propagation of linear PEI at low temperature ...... 16
Figure 9. Schematic of synthesis of linear PEI from polymerization of 2-oxazoline ...... 17
Figure 10. Structure of protonated linear PEI (a). Branched chain structure of PEI (b) ...... 19
Figure 11. Structure of branched PEI at neutral pH: only about 20% of the overall amino nitrogen is protonated ...... 20
Figure 12. Electron micrograph of linear PEI 22/DNA complexes in 0.15 M saline with an N/P ratio of 2 (A) and in 5% glucose with an N/P ratio of 5 (B) ...... 24
Figure 13. Schematic of PEI transfection ...... 32
Figure 14. Schematic of principle of a mass spectrometer ...... 39
Figure 15. Schematic of the TOF analyzer...... 41
Figure 16. Scheme of an electrospray LC-MS interface ...... 44
Figure 17. Mass Spectrum of linear PEI-423...... 49
ix
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 18. Mass Spectrum of branched PEI-600 ...... 50
Figure 19. Mass Spectrum of branched PEI-1200 ...... 51
Figure 20. Mass Spectrum of branched PEI-1800 ...... 52
Figure 21. Mass Spectrum of branched PEI-600 in LiCl solution ...... 59
Figure 22. Mass Spectrum of branched PEI-600 in NaCl solution ...... 60
Figure 23. Mass Spectrum of branched PEI-600 in KC1 solution ...... 61
Figure 24. Mass Spectrum of branched PEI-600 in CsCl solution ...... 62
Figure 25. Mass Spectrum of branched PEI-600 in T1C1 solution ...... 63
Figure 26. Mass Spectrum of branched PEI-600 in MnCh solution ...... 64
Figure 27. Schematic of a size exclusion chromatography column ...... 66
Figure 28. Schematic of the SEC process ...... 67
Figure 29. SEC chromatogram of branched PEI-600 ...... 71
Figure 30. SEC chromatogram of branched PEI-1200 ...... 72
Figure 31. SEC chromatogram of branched PEI-1800 ...... 73
Figure 32. SEC chromatogram of branched PEI-10000 ...... 74
Figure 33. SEC chromatogram of branched PEI-70000 ...... 75
Figure 34. SEC chromatogram of linear PEI-423 ...... 76
Figure 35. SEC chromatogram of PEG-1540 ...... 77
Figure 36. SEC chromatogram of PEG-3400 ...... 78
Figure 37. SEC chromatogram of PEG-10000 ...... 79
Figure 38. SEC chromatogram of PEG-20000 ...... 80
Figure 39. SEC chromatogram of linear PEI-25000 ...... 81
x
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 40. Relationship between molecular weight and elution volume for PEI and PEG ...... 82
Figure 41. SEC calibration curve for branched PEI ...... 83
Figure 42. SEC calibration curve for linear PEI and PEG ...... 84
Figure 43. Schematic diagram of a SEC-ESI MS system ...... 87
Figure 44. SEC chromatogram ...... 88
Figure 45. MS Chromatogram of LPEI-423 ...... 92
Figure 46. Mass spectrum of LPEI-423 (1st fraction)...... 93
Figure 47. Mass spectrum of LPEI-423 (2nd fraction) ...... 94
Figure 48. Mass spectrum of LPEI-423 (3rd fraction) ...... 95
Figure 49. MS Chromatogram of PEI-600 ...... 96
Figure 50. Mass spectrum of BPEI-600 (1st fraction) ...... 97
Figure 51. Mass spectrum of BPEI-600 (2nd fraction) ...... 98
Figure 52. Mass spectrum of BPEI-600 (3rd fraction) ...... 99
Figure 53. MS Chromatogram of BPEI-1200 ...... 100
Figure 54. Mass spectrum of BPEI-1200 (1st fraction) ...... 101
Figure 55. Mass spectrum of BPEI-1200 (2nd fraction) ...... 102
Figure 56. Mass spectrum of BPEI-1200 (3rd fraction) ...... 103
Figure 57. MS Chromatogram of PEI-1800 ...... 104
Figure 58. Mass spectrum of BPEI-1800 (1st fraction) ...... 105
Figure 59. Mass spectrum of BPEI-1800 (2nd fraction) ...... 106
Figure 60. Mass spectrum of BPEI-1800 (3rd fraction) ...... 107
Figure 61. Mass spectrum of linear PEI-423 with LiCl ...... 112 xi
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 62. Mass spectrum of branched PEI-600 with LiCl ...... 113
Figure 63. Mass spectrum of branched PEI-1200 with LiCl ...... 114
Figure 64. Mass spectrum of branched PEI-1800 with LiCl ...... 115
Figure 65. Mass spectrum of imine derivatives from linear PEI-423 reacting with benzaldehyde ...... 123
Figure 66. Mass spectrum of imine derivatives from branched PEI-600 reacting with benzaldehyde...... 124
Figure 67. Mass spectrum of imine derivatives from branched PEI-1200 reacting with benzaldehyde...... 125
Figure 68. Mass spectrum of imine derivatives from branched PEI-1800 reacting with benzaldehyde...... 126
Figure 69. Mass spectrum of imine derivatives from linear PEI-423 reacting with 4-fluorobenzaldehyde ...... 127
Figure 70. Mass spectrum of imine derivatives from branched PEI-600 reacting with 4-fluorobenzaldehyde ...... 128
Figure 71. Mass spectrum of imine derivatives from branched PEI-1200 reacting with 4-fluorobenzaldehyde ...... 129
Figure 72. Mass spectrum of imine derivatives from branched PEI-1800 reacting with 4-fluorobenzaldehyde ...... 130
Figure 73. Molecular formulae of non-cyclic PEI molecules containing certain number of repeat units and primary amino groups ...... 135
xii
with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES
1. Representative theoretical m/z values for parts of PEI molecules using different calculation methods ...... 53
2. Comparison of representative experimental m/z values of most [M+H]+ ions and their relative intensities with calculated values for BPEI-600 ...... 54
3. Comparison of representative experimental m/z values of most [M+H]+ ions and corresponding relative intensities with calculated values for PEIs ...... 55
4. Experimental values for PEIs and PEGs by SEC ...... 69
5. Representative theoretical m/z values of PEI molecules ...... 108
6. Comparison of representative experimental m/z values of PEI oligomers with lithium attachment to those theoretical m/z values...... 116
7. Representative theoretical m/z values of molecules of imine derivatives from non- cyclic PEI reacting with benzaldehyde and 4-fluorobenzaldehyde, respectively.... 121
8. Representative theoretical m/z values of molecules of imine derivative from cyclic PEI reacting with benzaldehyde and 4-fluorobenzaldehyde, respectively.... 122
9. Representative experimental m/z values of molecules of imine derivatives from non-cyclic PEI reacting with benzaldehyde ...... 133
10. Representative experimental m/z values of molecules of imine derivatives from non-cyclic PEI reacting with 4-fluorobenzaldehyde ...... 134
11. Comparison of representative experimental m/z values with theoretical m/z values of molecules of imine derivatives from cyclic branched PEI-1200 reacting with benzaldehyde ...... 136
12. Comparison of representative experimental m/z values with theoretical m/z values of molecules of imine derivatives from cyclic linear PEI-423 creating with 4-fluorobenzaldehyde ...... 137
xiii
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 1
INTRODUCTION
In the middle of the 19th century, Gregor Johann Mendel, the father of genetics,
opened the first page of genetic history. He hypothesized that a basic physical and
functional unit of heredity exists, which we now name a gene.1 His theories of heredity
and related works, i.e., the finding of particular inheritance, dominant and recessive traits,
and genotype and phenotype, built up the fundamentals for modem genetics. Researchers
have paid particular attention to the relation between genes and diseases over the last
decade. As a result, gene therapy bloomed, which is a therapeutic strategy2 that involves
inserting normal genes, removing or replacing defective ones, or manipulating the genetic
composition of cells to synthesize the therapeutic protein molecules for preventing or
curing genetic abnormalities or diverse diseases, such as inherited genetic diseases,3'9
cardiovascular diseases,10 immunodeficiency,11’12 cancer,13'18 and AIDS.19’20
The efficient and specific delivery and expression of therapeutic genes into target
cells or tissues in vitro and in vivo is a critical goal in gene therapy. The process of
introducing foreign DNA into a cell or organism is called transfection.21 A model gene
transfection ‘vehicle’ (called a vector) should be nonimmunogenic, biodegradable, able to
deliver genes into a wide diversity of cells in vitro and in vivo with high efficiency and
low toxicity, and be apt for effective gene expressions.22 There are numerous potential
delivery vectors that could be used for gene therapy. The vectors can be generally
1
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2
divided into two categories: viral and nonviral.
A viral vector is a recombinant virus, wherein the therapeutic gene is covalently
inserted into its genome to replace part or all of its genes.20’22 Generally, viral vectors are
constructed in cells, while synthetic nonviral vectors are chemically produced in
containers.22 Major viral vectors include retrovirus, adenovirus, adeno-associated virus,
and others such as herpes simplex virus (HSV) and vaccinia virus, etc. Although the
most common vectors used for gene delivery so far are viral vectors because of their high
efficiency,23’24 there are several obvious drawbacks related to their practical application.
First, viral vectors can only carry a small amount of genetic material.20’22’25 It is mostly
limited to 6-8kb26 (an adeno-associated virus can take about 2.5 kb27 to 4.5 kb28 while an
adenovirus can accommodate up to 7.5 kb of gene). Secondly, there are serious safety
risks ’ ' posed by viral vectors in their clinical application because of their
toxicity, ’ ’ the latent replication of viruses, ’ and their tumorigenic potential. ’
Furthermore, viral vectors are immunogenic, ’ produce low yields, ’ ’ and require
heavy laboratory infrastructure 43
The limitations of viral vectors have prompted the development of nonviral vectors.
To date, nonviral gene transfection approaches use physical techniques such as
bioballistic bombardment or use of a gene gun;22’44-49 direct injection of naked DNA;50'54
and applications of synthetic and natural nonviral vectors; the latter of which includes, for
example, histones55 and protamines.56’57 Compared to their counterparts, nonviral vectors
appeared somewhat later in the gene transfection repertoire and also normally showed
less transfection efficiency, ’ ’ ’ whereas, they continue to appeal to the public as gene
transfection agents with their own set of advantages. Regularly, nonviral vectors can
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3
carry a large amount of genetic material, which is two to three times the size of what viral
vectors deliver. Moreover, they are less toxic, nonimmunogenic, and convenient, simple,
and less expensive to produce in large quantities.22’26’36’44’59’60
As precursors, Vaheri and Pagano61 applied a diethylaminoethyl (DEAE) dextran-
mediated RNA delivery system in 1965 and Graham et al.62 used a calcium phosphate
method for DNA transfection in 1973. Synthetic nonviral vectors have been growing fast
during the past 10 or so years. Synthetic nonviral vectors are commonly classified as
cationic liposomes and polymers (polycations). Cationic liposomes are amphiphilic
molecules, which contain three parts: (i) a positively charged head group; (ii) a
hydrophobic anchor, which normally contains fatty alkyl, acyl, or alkoxy chains; (iii) a
linker to connect both the hydrophilic head and hydrophobic anchor. ’ ’ Since Feigner
et al.64 first successfully used N-[l-(2,3-dioleyl)propyl]-N,N,N-trimethylammonium
chloride (DOTMA) as a gene transfection vector in 1987, hundreds of cationic liposomes,
usually including double-chain quaternary ammonium lipids and derivatives of
cholesterol, diacyl glycerol, and polyamines, have been developed,20’36 such as 1,2-
dioleoyloxy-3-(trimethylammonio)propane (DOTAP), dimethyldioctadecylammonium
bromide (DDAB), 3p[N-(n’, N’-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol),
and dioctadecylamido-glycyl-spermine (DOGS) (See Figure 1). However, the uses of
cationic liposomes are limited by several practical factors, such as the low transfection
efficacy,20 the inhibition of serum in vivo for gene delivery by intravenous injection,64"66
and that of the pulmonary surfactant in the airways. The low gene transfer efficiency is
due to the relatively low DNA condensation, inefficient entry of the cationic lipid/DNA
complex (lipoplex), lack of selection for different types of cells, and difficulty in
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4
0(\ • endosomal escape. Alternatively, polycations constitute a promising category for gene
therapy. There are lots of polycations to date used for transfection, such as poly(L-
lysine),67 polyarginine,68’69 chitosan,70’71 poly(A-alkyl-4-vinylpyridinium) salts,72 poly(2-
(dimethylamino)ethyl methacrylate),73 polypropylenimine74 and polyamidoamine
(PAMAM) dendrimers,75 polyethylenimine (PEI), and all kinds of modified polymers.76
Among them, PEI was shown to be one of the most distinguished gene transfer vectors
with its unique properties.
Me cr
N-[ 1 -(2,3-dioleyl)propyl]-N,N,N-trimethylammonium chloride
Me.
Mer
Me Cl' O
1,2-dioleoyloxy-3 -(trimethylammonio)propane
Br'
dimethyldioctadecylammonium bromide
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5
Me' H cr O
3 P[N-(n’, N’ -dimethylaminoethane)-carbamoyl]cholesteroI
O
dioctadecylamido-glycyl-spermine
Figure 1. Structures of some cationic liposomes used for transfection. N-[l-(2,3- dioleyl)propyl]-N,N,N-trimethylammoniumchloride (DOTMA); l,2-dioleoyloxy-3- (trimethylammonio)propane (DOTAP); dimethyldioctadecylammonium bromide (DDAB); 3p[N-(n’, N ’-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol); dioctadecylamido-glycyl-spermine (DOGS).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 6
Polyethylenimine (PEP
PEI is a cationic polymer. It has been extensively used in the manufacture of paper,
water purification, mineral extraction, coating, and even textile and shampoo production,
^ A • A A .*7*7 *7A among others, for more than 50 years ’ ’ ' Because of its versatility in industry, there
are many trade names for PEI, for example, Polyaziridine, Polyamin, Montrek, Corcat,
and Polymin P. Compared with other transfection vectors, PEI is innocuous, very stable,
easy to obtain and use, and extremely inexpensive. Moreover, it is one of the most
efficient synthetic vectors currently available.80 In 1995, Boussif et al.78 first
demonstrated the efficiency of PEI to transfer gene and oligonucleotide into cells in vivo.
Since then, commercial PEI has been widely proven to effectively transfer genes as well
as oligonucleotides not only in vitro24;57;63;8°-93 but also in for
various diseases. It has also been reported that PEI showed orders of magnitude higher
7ft transfection in vitro than its peers such as polylysine. Unlike polylysine, PEI can
effectively transfer genes into cells without targeting agents or other endosomal lytic
agents such as chloroquine.57,80 The unique properties of PEI made it a versatile and
effective transfection agent.
Synthesis
The PEIs compose a large family of polyamines. The family contains two main
forms: linear and branched (Figure 2). Commercial PEIs are available in a wide
molecular weight range from 200 to 800,000 daltons (Da) and with various degrees of
branching.78,98 Commercially available branched PEI is synthesized by the ring-opening
polymerization of protonated ethylenimine (aziridine) monomers under normal
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Linear PEI
CH
HN NH
CH- NH CH NH
CH .CH CH CH- CH CH CH NH CH 'CH HN CH- NH
CH CH. NH NH
Branched PEI
77 Figure 2. Structures of linear PEI and branched PEI.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 8
polymerization conditions. Linear PEI was first synthesized by applying ring-opening
polymerization of 2-H-2-oxazoline and subsequent alkaline hydrolysis of the product
poly(iV-formyl ethylenimine) in 1972." Likewise, Litt and his colleagues100 used 2-alkyl
substituted 2-oxazaline to produce linear PEI later. Gembitskii et al.101 also reported that
linear PEI could be produced by a distinctive “head-to-tail” polymerization at low
temperature, which will be discussed later.
Aziridine
Aziridine and its derivatives usually contain three groups, which are 1-unsubstitued
aziridine, 1-substituted basic aziridine, and “activated” aziridine102 (See Figure 3). There
are specific characteristics for each of these groups, even if they are all susceptible to a
ring-opening polymerization. Among these three groups, 1-unsubstituted aziridine is
commonly and efficiently used for synthesis of PEIs. Unsubstituted aziridine was first
synthesized103 in 1888.
Y
1-unsubstituted aziridine 1-substituted aziridine "activated" aziridine (secondary amine) (tertiary amine) (tertiary amide)
1 (Y) Figure 3. Structures of Aziridine Groups.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 9
Figure 4 presents the schematic of three general synthetic methods for preparation of
aziridines. The first commercial aziridine is produced by the Gabriel method, a S#2
reaction, which involves cyclization between 2-haloalkyl amines and base. The Wenker
method involves the preparation of 2-aminoethyl hydrogen sulfate esters from amino
alcohols and a subsequent reaction with base. The Dow method is an animation reaction
of ethylene dichloride with ammonia followed by a cyclization of the produced 2-
chloroethylamine with calcium oxide.103
Gabriel Method
X— CH2 — CH2NH2RX- +2 NaOH +2 NaX+2 H20
Wenker Method
r n h c h 2c h 2o h + h2so 4 RNH2CH2CH20 S 0 3'
OH'
/I R + H20
Dow Method
C1CH2CH2C1 + NH3 + CaO
Figure 4. Schematic of three general synthetic methods for preparation of aziridine.103
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 10
Branched PEI
PEIs are produced by ring-opening polymerization reactions. Under different
reaction conditions one can obtain linear or branched PEIs. Branched PEIs are produced
by cationic polymerization of ethylenimine, which allows having a branched polymer in a
random way. The polymers are usually synthesized in water or in organic solvents at 90-
110°C. The average molecule weight of the product polymers is 10,000-20,000 Da.
Molecules with higher and lower molecular mass can be obtained by adding a
difunctional alkylating agent or a low molecular mass amine during polymerization,
respectively. Commercial branched PEI104 can be prepared in molecular weights ranging
from 300 to more than 1,000,000 Da.
The polymerization mechanism for synthesizing branched PEI is illustrated in
Figure 5. The conversion of ethylenimine to an alkylating agent (initial species) is firstly
carried out by adding a small amount of various cationic reagents such as H+ or R+.
Subsequently, the initial propagation step is a simple ring-opening reaction of the
protonated azirindine by nucleophilic attack of an unprotonated aziridine. Propagation
reaction continuously proceeds between the intermediate aziridine ring and primary,
secondary, and tertiary amine groups in the growing oligomers. In comparison to the
intermolecular propagation reaction, the chain termination in the polymerization of
ethylenimine is a intramolecular macrocyclization, or “back biting”, to form a big cyclic
ring, which is generally larger than six members.77’103’105
By using rational rate constants in a computer simulation, Jones et al. in 1965 found
that there should be about one branch for every three linear nitrogen atoms of branched
PEIs synthesized in aqueous solutions. That is, the ratio102 of primary, secondary, and
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 11
tertiary amino groups in the polymers was 1:2:1. The result was confirmed when Ham
and his colleague Dick105 studied the degree of branching of the produced branched PEI
in methanol in 1970. They pointed out that there was approximately one branch for every
3 to 3.5 linear nitrogen atoms, and moreover, the degree of branching obtained was about
the same for many synthetic polymerizations of polyethylenimine under different
experimental conditions and of various molecular weights.
Branched PEI is water-soluble, however, linear PEI is insoluble at room temperature
and is soluble only in hot water. Commercial PEI is insoluble in most organic solvents
with the exception of alcohols such as methanol and ethanol. Usually, an aqueous PEI
solution contains 40-60% PEI with an alkaline pH 9-10.106 Linear PEI was generally
observed to form highly crystalline hydrate. Three hydrated forms103’107'109 have been
discovered, which are hemihydrate (0.5»H2O), sesquihydrate (1.5«H20), and dihydrate
(2.0*H2O) (See Figure 6). Anhydrous linear PEI forms double-stranded helixes103’107
(See Figure 7).
Initiation
+> .+ :n h + h :n r
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 12
Propagation
H, H . V / 1 HN. + H NH2CH2CH2
n h 3c h 2c h 2 — n :
NH ; n - c h 2c h 2n h c h 2c h 2n h 3
1 ° : n c h 2c h 2n h — c h 2c h 2n h c h 2c h 2n h 3
+ ; n - c h 2c h 2n h c h 2c h 2n h 2
c h 2c h 2n h 2
H or C 2° * Y I X ^ 'N -^CH 2CH2N - jjj- c h 2c h 2n h HorC
( CH2CH2N - ) - c h 2c h 2n h 2 chain
H or C H or C
: n -( c h 2c h 2n -)^-c h 2c h 2n — c h 2c h 2n h 2 - ( c h 2c h 2n )^ - c h 2c h 2n h 2
chain
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Termination
CH
N
H (-NHCH2CH2 ) ns ^ N C
c h 2 c h 2 + H+ NH
NHCH2CH2 7 m
Figure 5. Schematic of production of branched PEI.103
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 14
£*
£
Figure 6. Hydrated forms of linear PEI. Anhydrate (a); hemihydrate (b); sesquihydrate (c); dehydrate (d). (Reproduced from reference 103)
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 15
Figure 7. Double-Stranded helical chains of linear PEI. (Reproduced from reference 103)
Linear PEI
Linear PEI can be obtained in a similar process to that for branched PEI, but by a
distinctive “head to tail” mechanism102’103 at lower temperatures, such as 40°C. The
“head-to-tail” mechanism is a polymerization of the hydrated oligomer, in which the
propagation is a heterophase propagation of the crystalline macromolecular hydrates.
The propagation takes place by either adding oligmers from the aqueous solution or
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 16
attaching the hydrated forms of the polymer molecule, which contains terminal aziridine
rings, to each other. The propagation step is shown in Figure 8.
linear polymer^wru^NH-
solid oligmer hydrate
Figure 8. Schematic of propagation of linear PEI at low temperature.102’103
Alternatively, linear PEI can be produced from cationic polymerization of 2-
oxazoline monomers and followed by hydrolysis of poly[(Ar-acylimino)ethylene] ( See
Figure 9). Several methods103 for synthesizing 2-oxazoline are below. Isomerization of
A-acyl aziridine can form 2-oxazoline under the conditions of heat, acid, or ionic iodide
catalysis. Other approaches include the cyclization of 2-haloethyl amide with base, the
reaction between nitrile and alkanoamine with the addition of cadmium salts, and
commercial cyclodehydration of 2-hydroxyethyl amide by catalysts such as alumina.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2-alkyl (aryl)-l,3-oxazolin-2-ene
O
Base RCNHCH2CH2X -HX
2+ R-CN + NH2CH2CH2OH Cd -NH,
O 2-OXAZOLINE
RCNHCH2CH2OH -H ,0
CATIONIC R NCFLCH A CATALYSIS
2-OXAZOLINE L
H or wOH'
f - n h c h 2c h 2
Figure 9. Schematic of synthesis of linear PEI from polymerization of 2-oxazoline.11
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 18
Chemical Properties
There are several chemical properties of PEI that are favorable for transfection of
cells in vitro and in vivo. One of these properties is PEI’s chemical structure. PEI is
composed of repetitive units of ethylenimine (-NHCH 2CH2-), which has a molecular
weight of 43 g/mol. Figure 2 shows the structures of those two types of PEI : linear and
branched. The latter form of PEI produced by acid-catalyzed polymerization of aziridine
is randomly branched, which contains protonable primary, secondary, and tertiary amino
nitrogen at a ratio about 1:2.T (Figure 10).102;105;11° Moreover, one out of every three
atoms in the PEI backbone is an amino nitrogen that is capable of being protonated.
These characteristics make PEI a macromolecule that has the potential to be highly
charged. In addition, studies have revealed that the protonation of PEI depends on the pH.
PEI is not fully protonated at neutral pH because of the mutual repulsion between like
charges at close proximity. The overall degree of protonation of PEI can increase from
20% to 45% as the pH is lowered from 7 to 5 (Figure ll).24 Only approximately one-fifth
of the amino nitrogens are protonated under physiological conditions.82 Boletta et al.94
further concluded that about one-sixth of the PEI nitrogens would be protonated at
physiological pH 7.4. There will be approximately zero charge111'113 for PEI molecules
at pH 10.8. Therefore, the polymeric network of PEI presents substantial buffering
capacity at almost all pH levels. This high buffering capacity is called the “proton
sponge” effect.78 The protonability of PEI is crucial to PEI effectiveness as a gene
HO delivery vehicle. Kichler et al. demonstrated that the transfection efficiency of PEIs
relied on their considerable buffering capacity, which gave rise to an endosomolytic-like
activity. Additionally, PEIs are suitable for binding and packaging negatively charged
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 19
DNA with their strong positive surface charge.85 In summary, PEI became a promising
transfection vector because of its flexibility, variety, DNA-condensing, and pH-buffering
properties.
© n h c h 2c h2 ------a n
Protonated linear PEI
©NH NH
© N H
b
Protonated branched PEI
Figure 10. Structure of protonated linear PEI (a). Branched chain structure of PEI (b).110
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 20
NH
NH HN
NH •NH •NH HN
NH HN HN NH
NH- NH-
NH NH HN,
NH NH NH-
HN
NH
Figure 11. Structure of branched PEI at neutral pH: only about 20% of the overall amino nitrogen is protonated.24
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER2
PEI TRANSFECTION CONSIDERATIONS
PEI/DNA Complexes
The positively charged cationic liposomes or polymers can easily form complexes
with negative DNA due to charge interaction between the cationic liposomes or
polycations and the negative charges carried by the phosphate backbone of DNA.
Usually, the liposome/DNA complex and the polymer/DNA complex are named as
lipoplex and polyplex, respectively. The ternary complex of liposome/polycation/DNA is
called lipopolyplex, which contains the cationic liposome/polycation/DNA (LPDI) and
anionic liposome/polycation/DNA (LPDII).20’22
The DNA condensation by polycations is found to be a function of the charge ratio
of the cation to anion.44,82,114 In the case of PEI, the charge ratio is defined as the molar
ratio of nitrogen to phosphorus (N/P). The nitrogen originates from PEI amine groups
and the phosphorus from DNA phosphate groups. The N/P ratio is approximately
calculated by the following• equation. ’ S 7 ' 8 0
N/P = (330/43) * (M i/M2) (1)
It is assumed that the positive charge is from the repeat subunit of PEI, which is
NHCH2CH2 with a molecular weight of 43g/mol, and the negative charge from the
phosphate group of a single deoxyribose nucleotide, which has an average molecular
weight of 330 g/mol. Mi is the mass of PEI (pg) and M 2 is the mass of DNA (pg).
21
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 22
According to the result of the analysis of the PEI/DNA complexes by means of
agarose gel retardation assays, studies have reported the N/P ratio that results in complete
neutralization of the DNA, which is termed a stoichiometric composition.84 Generally,
complete neutralization44,84,89 seems to occur at N/P ratios from approximately 1.5 to 3,
although there are slight differences among these studies under various conditions.
Several studies57,90 showed that the stoichiometric composition is above 2 or 3. Recently,
Zhang et al89 found that branched PEIs completely complexed DNA at lower N/P ratio
than linear PEI did, and then they concluded that branched PEI has a higher DNA affinity
in comparison with linear PEI, and the higher the molecular weight, the stronger the
DNA affinity.
Theoretically, only every fifth or sixth amino nitrogen is in fact protonated at
physiological pH according to the pK profile of PEI in the absence of DNA.115 So
complete DNA condensation should occur at a N/P ratio of about 5 or 6. However,
binding of nucleic acids to PEI will shift the protonation profile of PEI.44;82 According to
the preliminary stoichiometric composition results, almost one in two to three of amino
nitrogen atoms in PEI is protonated and able to combine to DNA under the studied
conditions. However, PEI/DNA complex particles usually become large and unstable at
electroneutrality.44,116,90 A higher N/P ratio is generally used in order to obtain efficient
and tight DNA compaction, by which small and soluble polyplex particles with a net
positive surface charge are produced.44;116
Some surface charge measurements such as the measurement of zeta-potential,
has confirmed the results of the agarose gel retardation analysis. The zeta potential was
found to rise from strongly negative to strongly positive with increasing N/P ratio. £ was
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 23
approximately zero at N/P ratios ranging from 2 to 3.84 PEI/DNA complex is like a
neutralized polyplex ‘core’ enclosed by an excess of positive PEI ‘corona’ at high N/P
ratios.117 A high positive zeta potential could be obtained at the N/P ratios usually used
for efficient condensation and tight compaction of DNA. \ leveled off from 30 mV to 35
mV at N/P ratio above 4.44 Zeta potentials were not quite different for various PEI/DNA
complexes.118 In addition, Godbey et al.77 found that the zeta potential decreased from 37
mV to 31.5 mV after DNA was added to a certain sample of branched PEI in solution and
complexed with it at a N/P ratio of 7.5, and ^ lowered down to 29.2 mV once
centrifugating the PEI/DNA complex.
The shape of PEI/DNA complexes have been examined in great detail by different
research groups using electron microscopy. Studies have demonstrated that the formation
of toroidal PEI/DNA complexes predominated.56,98,119'121 Lemkine and Demeneix58 found
that in the presence of either form of PEI formulating PEI/DNA complexes in saline
medium gave large aggregates, but formulating in glucose gave discrete spheres or
toroids (See Figure 12). This is consistent with the idea that different conditions used for
the preparation of the formulations result in different shapes of formed complexes.122
<7 <7 Godbey et al. have pointed out that the morphology of PEI/DNA complexes might be
independent of the used polymers, but dependent on a more general factor such as the 171 kinetics. Dunlap et al. have also revealed that one of the possible reasons to form
spherical PEI/DNA condensates was kinetics.
The evaluation of particle size has been carried out by using several methods (e.g.
dynamic light scattering, electron microscopy, and atomic force microscopy). Kircheis et
al.44 showed dependencies of the particle size and shape on the N/P ratio and the
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 24
Figure 12. Electron micrograph of linear PEI 22/DNA complexes in 0.15 M saline with an N/P ratio of 2 (A) and in 5% glucose with an N/P ratio of 5 (B). (Modified from reference 58)
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 25
experimental parameters during complex formation such as the DNA concentration, the
ionic strength of solvent, and the kinetics of mixing. This group44 also noted that there
are no major differences in the complexes formed when a different order of reagents are
added, even if Boussif and colleagues91 found that in vitro gene transfer was increased
over 10-fold with a dropwise addition of the polymer to DNA as compared to the
addition of the DNA to polymer.
When complexes are prepared in nonsaline or low salt solutions, small complexes
are usually formed and the sizes of particles are quite consistent between polyplexes
made with either form of PEI.44,77 Tang and Szoka98 found the formation of essential
toroidal PEI/DNA complexes with a size range of 40-60 nm in diameter by electron
microscopy, while Ogris and coworkers124 observed those of 50-80 nm using dynamic
laser light scattering, and Dunlap et al.123 even obtained some that were 20-40 nm by
atomic force microscopy. Goula et al.93 revealed that linear PEI 22-kDa (PEI 22) and
DNA could form complex particles with sizes ranging approximately from 30 to 60 nm in
a 5% glucose solution. Additionally, they mentioned that the size of the complexes was
also directly related to the N/P ratio and the size decreased while the N/P ratio increased.
For the same linear PEI 22, particle sizes induced from 100 nm to between 30 and 60 nm
at a N/P ratio of 2 and 10, respectively. Complexes formed in a salt free solution do not
aggregate and are stable for several hours, but aggregate with time in low saline
conditions.43
In physiological conditions, large complex particles (up to fj.m) can be formed.18 The
condensation of the polymer could even be inhibited in the presence of a high saline
concentration.43 Furthermore, complexes formed with different forms of PEI at higher
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 26
ionic strength are fairly unlike.44 The particle size is a function of the initial molecular
weight and geometry of PEI besides the N/P ratio. Generally, the particle size will
increase as the molecular weight does. The linear PEI forms larger complex particles
than the branched PEI does with a similar molecular weight.89 By using dynamic light
scattering, Zhang et al.89 found that small complex particles (70.7 ±31.7 nm) were
formed with branched PEI 25-kDa (PEI 25) in physiological salt buffers such as Hepes-
buffered saline (HBS) at N/P ratio 8, while Linear PEI 25 complexed with DNA formed
large aggregates (782.2 + 275.5 nm). Depending on the N/P ratio during the formation,
PEI/DNA complexes with different sizes are formed at physiological ionic strength.
Complexes with sizes ranging from 50 nm to several hundred nm are generated with
branched PEIs44 such as PEI 25 or PEI 800. Polyplexes prepared in Dulbecco’s modified
eagle medium (DMEM) and culture medium normally show sizes approximately between
200 and 350 nm.125
Influencing Factors on PEI Transfection Efficiency
Factors including reaction conditions and biophysical parameters of the condensed
complexes during transfection,22;26;43;44;58;77;80;82;88'91;94;;97;116 such as the N/P ratio, surface
charge and the size of the complex, the amount of plasmid DNA, pH, molecular weight
and shape of the polymer, compaction medium, and cell linage, were addressed on the
transfection efficacy and toxicity.
Much work has been done in order to evaluate these factors impacting the PEI
transfection and optimize them for expression with limited cell toxicity. The N/P ratio of
PEI nitrogens to DNA phosphates and surface charge of the polyplex is important in
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 27
terms of transfection efficiency and cytotoxicity. These factors result in a dilemma for
researchers. The condensation of DNA by cationic PEI will depend on the N/P
ratio.43,44,116 The large, unstable, and aggregate complexes are usually formed at low N/P
ratios which result in approximately electro-neutral complexes. The small, soluble, and
cationic polyplexes are made at a high N/P ratio. Particle size is crucial to transfection
efficiency in vivo. On one hand, small cationic polyplex particles can diffuse widely
through the tissue. Furthermore, transfection is most efficient when complex particles are
positively charged because the cationic particles can interact directly with the anionic cell
surface molecules like proteoglycans and then be taken up by cells.90;116 On the other
hand, the cationic complexes will also possibly bind to other polyanionic glycans in the
extracellular matrix and be retained. In addition, for in vivo transfection, highly charged
positive complexes will bind to substrates around the injection site.82 But for the anionic
complexes, neither can they be taken up by cell surface nor be retained by anionic
substances.90 Moreover, Godbey et al.77 revealed that cationic polyplexes could activate
the complement, and increasing the N/P ratio would lead to an increase of complement
activation as well as cytotoxicity. Plank et al.126 also demonstrated that electroneutral
polyplexes will poorly activate the complement system at best. For efficient transfection
in vivo, activation of the complement system by the cationic polyplexes comprises a
potential barrier. The circulating proteins can bind to cationic polyplexes,82 whereas N/P
ratios ranging from 5 to 13.5 have been used successfully to solve the protein binding
problem.77,78
Guo et al.80 found that a higher N/P ratio generally led to more effective transfection
due to higher cellular uptake and endosomal lytic power. The optimal N/P ratio is
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 28
usually above 3. Zhang et al.89 pointed out that an excess of PEI, which would result in a
net positive charged polyplex, was required for most in vitro transfection, and the optimal
N/P ratios are often 6 or greater. Boussif et al78 revealed that the N/P ratio from 9 to 13.5
resulted in maximal transfection efficiency in vitro by testing branched PEI with N/P
ratios ranging from 4.5 to 135. Abdallah et a l97 found that the best transfection
efficiency is at a N/P ratio of 9 in vitro. Guo et al.80 also found that the optimal in vitro
transfection was approximately at the N/P ratio of 10, and higher N/P ratios such as 15 or
above could lead to less efficient transfection, which is possibly because of an increase in
cytotoxicity.• Zhang et al. 80 reported the optimal N/P ratio for in vitro PEI transfection
varied according to cell lineage. For the human CF bronchial epithelial cell line IB3-1,
peak yield was observed at a ratio of 20 with Linear PEI 25. But for brain-derived glial
cells and neuronal cells, the peak yield was obtained at N/P of 9 with branched PEI 25
and at N/P of 5 with branched PEI 50-100, respectively. Nguyen et al.84 observed the
optimal transfection activity of branched PEI 25 in vitro was at a N/P ratio of
approximately 4 for transfection of Cos-7 cells. Boletta et al.94 reported the optimal N/P
was 10 in vivo transfections of the kidney with branched PEI 25. Merlin et al.43
summarized that optimal N/P ratio for in vitro tranfection is ranging from 4 to 10 for
different cell lines, and in most cases, optimal delivery was achieved using N/P ratios
higher than 5. Generally, most studies showed the optimal N/P ratio for in vitro
transfection is slightly on the cationic side, which is also useful to in vivo delivery.26’78
c o Lemkine and Demeneix summarized that the optimal N/P ratio, according to
different studies, ranges from 4 to 10 for in vivo gene delivery. Abdallah et a l97 reported
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 29
that polyplexes of N/P ratio of 6 provided the best transfection efficiencies in both the
new bom and adult central nervous systems.
Particle size is also a crucial concern for in vivo transfection efficiency.
The size of the complexes is directly related to the N/P ratio as observed using electronic
transmission microscopy.43,93 The size of polyplex particles is an important factor both
for internalization and for in vivo diffusion. Small particles, usually less than 100 nm,
seem suitable for in vivo DNA delivery in order to pass the vascular system.22
Most studies reported successful gene transfection both in vitro and in vivo by
applying PEIs with a molecular weight between 25 and 800 kDa. Several studies showed
the efficient transfection of PEIs with a lower molecular weight. From most reports, the
optimal molecular weight89 is between 20 and 30 kDa. However, Bieber et al.85 found
that PEI molecules ranging from 500 to 10,000 Da, which were fractionated by molecular
sieve chromatography, provided more effective and less cytotoxic transfection in vitro
than PEIs with larger molecular weight. Godbey et a l86 compared in vitro transfection
efficiencies of PEIs with molecular weights ranging from 600 to 70,000 Da for
endothelial cells. They found the transfection efficiency increased as the molecular
weight of PEI increased, and PEIs of lower molecular weights than 1800 Da showed no
transfection at all. But Abdallah et al.97 showed the contrary results to those of Godbey’s.
Transfection efficiencies were demonstrated to be inversely correlated to molecular
weight, and branched PEI 25 was the most efficient among branched PEI 50 and PEI 800
for gene transfer into the brain of adult mice. Several differences such as targeted object,
experimental condition, reagent, method, and biophysical parameters of PEI/DNA
complexes could possibly explain the discrepancy.77,86
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 30
The morphology of PEIs is one of these factors which have been found to impact on
DNA transfection efficiency. However, polymer shape does not appear to be an
important factor.22 By comparing branched PEI 25 to linear PEI 22, Boletta et al.94 found
that both of them could form small particles with a size from 20 to 40 nm in diameter at a
N/P ratio of 10, and branched PEI 25 proved superior to its linear counterpart PEI 22 in
spite of the extremely similar behavior of these two forms of PEI in vivo transfections of
the kidney. For various cells, different linear and branched PEIs play different roles.
Zhang and co-workers89 found that linear PEI 25 had the highest transfection efficiency
compared with branched PEI 25 and PEI 50-100 for human CF bronchial epithelial cell
line IB3-1, whereas branched PEIs were more effective for the brain-derived cells. This
group also revealed that branched PEI has a higher DNA affinity, and the higher the
molecular weight of PEI, the stronger the DNA affinity.
co As mentioned before, Lemkine and Demeneix found that in the presence of either
form of PEI formulating PEI/DNA complexes in saline medium gave large aggregates
suitable for in vitro transfection, but formulating in glucose gave small discrete spheres or
QT toroids, highly diffusible particles apt to in vivo use. Goula et al. demonstrated that
linear PEI 22/DNA complexes that were formulated in 5% glucose had a much higher
transfection efficiency in vitro and diffusibility of the particles in the tissues as well,
compared to polyplexes formed in 150 mM sodium chloride. So far, studies80’91’124 have
been reported on the transfection activity of PEI polyplexes in the presence of serum.
Guo et al.80 reported that PEI transfection is extremely sensitive to serum in a nonsaline
or lower salt medium. They found that luciferase expression was significantly reduced
by hundreds of times when 10% fetal bovine serum (FBS) was included in regular media
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 31
during transfection. Boussif et al.91 observed that dropwise addition of PEI to DNA to
form polyplex in 150 mM NaCl medium containing serum led to better transfection.
Florea and colleagues125 observed that fetal serum proteins and chloroquine disrupted the
PEI-DNA interactions by either aggregate formation or electrostatic interactions.
By varying pH, volume, and concentration of sodium chloride of the PEI/DNA
compaction medium, Boussif and co-workers78 tested the impact of these factors on PEI
transfection efficiency in vitro. It turned out that none of these factors significantly
influenced DNA delivery by PEI. Godbey et al.77’86 also obtained consistent result in
terms of pH. They found the changing pHs of PEI solutions before mixing with DNA
would not make any significant difference in transfection efficiency.
Mechanism
The mechanism44,43,58’77,89 by which PEI transfects DNA includes four steps such as
DNA condensation, cellular uptake, endosomal release, and nuclear transport and
complex disassembly (See Figure 13). Basing on the electrostatic interaction,
polycationic PEI will spontaneously stick to and compact DNA to form toroidal complex
particles, which then bind to cells also by electrostatic interactions with the negatively
charged sulfated proteoglycans on the cell membrane followed by non-specific adsorptive
endocytosis, pinocytosis, or phagocytosis. The condensation of DNA by PEI enhances
cellular uptake of these complex particles. Subsequently, the DNA/PEI complex particle
is internalized within endosomes. Cellular trafficking normally directs the endocytosed
particles to lysosomes for degradation. However, the unique chemical properties of PEI
underscore its potential as a transfection vector. Only about one-fifth of the amino
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 32
nitrogens in PEI are protonated under physiological conditions. Therefore, PEI is acting
as a “proton sponge” due to its substantial buffering capacity. The protonation of PEI
will most likely trigger a passive chloride ion influx. The buildup of the proton and
chloride ions leads to the influx of water and then osmotic swelling. With subsequent
endosome rupture, the PEI/DNA complex will exit out of the endosome and be released
into the cytosol. Finally, the complex particle will enter the nucleus. However, how it
enters the nucleus remains the subject of much argument and debate. After fast
disassembly of the PEI/DNA complex, the DNA is freed into the cell nucleus and then
transgene expression is realized.
Cell membrane/
Cytoplasm PEI
m . PnlvnlovPolyplex *>' ~*C \ pH=7.3 pH=5 HjO H20 Nucleus
OQ Figure 13. Schematic of PEI transfection.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 3
PEI CHARACTERIZATION
Polymer Characterization
The complexity of structure, composition, and molecular weight distribution (MWD)
of polymers make their characterization challenging. Polymer characterization generally
involves determination of molecular weight, composition, structure, architecture, end
groups, and polymeric physical properties. To date, the methods and instrumentation
used for polymer characterization typically include: chromatography, spectroscopy, mass
spectrometry (MS), osmometry, viscometry, ultracentrifugation, light scattering, field-
flow fractionation (FFF), and microscopy.127’128
Chromatography techniques such as high-performance liquid chromatography
(HPLC) and size exclusion chromatography (SEC), also referred to as gel permeation
chromatography (GPC), have been widely used for the separation, determination of the
average molecular weight distribution, measurement of the degree of long-chain
branching in polymers, and the analysis of copolymer compositions. Other
chromatography methods, such as inverse gas chromatography (IGC), which has been
used for studying the phase and transport properties of polymers since the early 1970s, is
usually applied for the determination of thermodynamic parameters and diffusion
properties of polymers.127
33
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 34
Spectroscopy methods, such as nuclear magnetic resonance (NMR), fourier
transform infrared (FTIR) as well as ultraviolet (UV), are used for structural
characterization of polymers. Especially for polymers with lower molar masses (less
than 104 Da), these methods are widely applied to end group analysis. X-ray
fluorescence spectrometry can be used as a non-destructive method of elemental analysis
for polymers. Raman spectroscopy is utilized to characterize the crystalline polymers.
Mass spectrometry has developed and played an extremely important role in the
measurement of molecular weights of molecules for the past 50 years. MS also
continually provides unique insight into the field of polymer analysis. With its high
sensitivity, selectivity, specificity, and broad dynamic range, MS has been widely used
for mass determination and structural characterization as well.
Both osmometry and dilute solution viscometry have been used to determine
fundamental molecular parameters. Osmometry is an absolute method for molar mass
analysis of polymer. Two most commonly employed methods of osmometry are vapor
197 pressure osmometry (VPO) and membrane osmometry (MO). MO coupled with VPO
can measure polymer molecular weights up to 5 x 10s Da. Dilute solution viscometry are
applied to measure molecular weight distribution of polymers, and study of polymer
branching and physiochemical properties also.
In the early stage of polymer science, sedimentation analysis was used to provide an
absolute measurement of polymer molecular weight. Sedimentation can197 be applied to
determine polymer molecular weights ranging from 200 tolO7 Da, as well as measure
hydrodynamic properties and thermodynamic data of polymers. To date, other efficient
methods are taking the place of sedimentation analysis.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 35
Static light scattering is one of the newer methods, which is being used for polymer
characterization. Low-angle laser light scattering (LALLS), one type of static light
scattering, is effectively acting as an online detector for size exclusion chromatography
for an absolute determination of molecular weight.
Photon correlation spectroscopy (PCS), also named as simply dynamic light
scattering or quasielastic light scattering (QELS), is used to determine the translational
diffusion coefficients of polymers. Information related to the nature of light scattering
particles can be obtained by this technique.
Field-flow fraction (FFF), one kind of technique for separation, is becoming a very
useful complement to SEC technique. Among FFF techniques, thermal FFF (TFFF) and
flow FFF (FFFF) are generally used for natural and synthetic polymers.
Microscopic techniques, which include optical microscopy, scanning electron
microscopy (SEM), and transmission electron microscopy (TEM), are used for
morphological analysis of polymers.
Previous Characterization of Polvethvlenimine
In the early 70s, researchers began to determine the mass-average molecular weight
(Mm) of PEI by applying various methods such as viscometry, osmometry, ultra
centrifugation, and light scattering.110 A uniform linear relationship between log Mm and
log [q] (intrinsic viscosity) could not be found for commercial PEIs with molecular
weight ranging from 10 to 10 . Using gel permeation chromatography, Godbey et al.
characterized the molecular weight distribution of three commercial PEIs with different
nominal molecular weights. The molecular weight for each PEI sample, obtained by
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 36
their experiments, is fairly different from the nominal weight. Moreover, they found that
differences in polydispersity of PEI reagents possibly resulted in different transfection
results.
As previously mentioned, Dick and Ham102,105 were among the first to report the
branching level of PEIs. They reported, under different experimental conditions, that
there is approximately one branch for every 3 to 3.5 nitrogen atoms within a linear chain
for PEIs with different molecular weights, i.e., the ratio of primary, secondary, and
tertiary amino groups in the polymers is about 1:2:1. Rivas et al.106 described that two
different states, the non-ionic and polyelectrolyte states, are characteristic of PEI. Most
commercial PEIs are branched to different degrees, and the ratios of primary, secondary,
and tertiary amino groups in the polymers are between 1:1:1 and 1:2:1 according to the
degree of branching. The other result,104 which was determined by 13C NMR
spectroscopy, showed that the ratio is approximately 3:4:3 for primary, secondary, and
tertiary amino groups, respectively.
Statement of Dissertation
The analysis of polymers is vital for elucidating polymer properties, and it also helps
in the understanding or predicting of the mechanism of polymer activity. Commercial
PEIs, used by molecular biologists for gene therapy and gene regulation, are not
thoroughly characterized to date. Therefore the aim of this research is to develop
methods for characterizing commercial PEIs, both linear and branched, by the use of
mass spectrometry and SEC. The method development involves the measurement of
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 37
molecular weight distribution (MWD), analysis of ion attachments to PEIs, determination
of the end group, and assessment of the degree of branching.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER4
PRELIMINARY ANALYSIS OF PEI BY MASS SPECTROMETRY
Introduction
Mass spectrometry has developed and become an indispensable analytical technique
in both mass determination and structural characterization for a wide variety of inorganic
and organic species, especially for synthetic polymers and biopolymers such as protein,
polysaccharides, and nucleic acids.
With the introduction and development of soft ionization techniques, such as
chemical ionization (Cl), secondary-ion mass spectrometry (SIMS), matrix-assisted laser
desorption/ionization (MALDI), electrospray ionization (ESI), and so on, mass
spectrometry has rapidly had a great impact on the analysis of bio-macromolecules and
synthetic polymers in recent years.128
Modem mass spectrometer is a highly computerized instrument with extraordinary
sensitivity, selectivity, specificity, and broad dynamic range. The typical principle129 of
MS usually comprises sample introduction, ionization, mass analysis, detection of ions,
and data processing (See Figure 14). The sample is introduced by controlled leaks for
liquids or by various direct insertion probes for solids. The analytes can be ionized
basically by electron ionization, chemical ionization, desorption ionization, or
nebulization ionization. Subsequently, the resulting ions are separated or filtered
according to their mass to charge (m/z) ratio by the mass analyzer. There are five
38
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 39
types129 of mass analyzers available currently, which include (magnetic) sector,
quadrupole mass filter, quadrupole ion trap, time-of-flight and Fourier-transform ion-
cyclotron resonance instruments. As a consequence, the ions are detected by an electron
multiplier. Advanced computer programs are applied to process and report the data. The
obtained mass spectrum displays the relationship of the (relative) abundance of the
produced ions vs. the m/z ratio.
In the late 1980s, the fascinating developments of MALDI and ESI provide
significant impetus into the field of polymer analysis. These two ionization techniques,
coupled with various separation methods, have been widely used for most polymer
analyses.
Ionization Mass AnalysisSample Ion Detection Introduction
Data Processing
Figure 14. Schematic of principle of a mass spectrometer.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 40
MALDI-TOF MS
Hillenkamp130 and Tanaka131 led the way to overcome the limited mass range of
traditional mass spectrometry by initiating the MALDI technique, which involves
embedding the analyte into an ultraviolet absorbing solid or liquid matrix. Application of
the MALDI technique tremendously extends the molar mass range of the detected
compounds up to 106 Da and beyond.128;129;132 MALDI has been successfully used to
measure proteins greater than 300, 000 Da and even polystyrene as high as 1,500,000
Da.128
Most often, a sample for MALDI is prepared by mixing the analyte solution with a
proper matrix solution at a high matrix/sample ratio directly on a probe. The matrix co-
crystallizes with the analyte after solvent evaporating. A pulsed laser is applied directly
on the sample. The energy is absorbed by the matrix and then transferred to the analyte,
which is subsequently desorbed and ionized in the gas phase. The matrix functions as an
energy mediator in order to protect the analyte molecules from degradation. This kind of
soft ionization technique mainly generates singly charged quasimolecular ions.128’133 The
pulsed laser produces ions intermittently. In this case, the time of flight (TOF) mass
analyzer is among the best for pulsed ionization techniques with its additional advantage
for measurement of very large molecules. The mechanism128’134 of the TOF analyzer is
that the velocity of an ion is inversely proportional to the square root of its m/z for a fixed
kinetic energy. A schematic of the time-of-flight analyzer is shown in Figure 15. After
being electro-statically transferred into a time-of-flight analyzer, the analyte ions are
separated and individually detected. As a result, ions with small m/z values travel faster
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 41
than those with larger ones and finally reach the detector earlier after traversing a field-
free region.
With its high sensitivity, resolution, and accuracy, state-of-the-art MALDI-TOF MS
becomes one of the most powerful and productive methods for polymer characterization
in the recent years. It provides accurate and rapid determination of molecular weight
distributions and end groups, the sequencing of repeat units, and identification of
composition and structure of synthetic polymers.135 MALDI-TOF mass spectrometry
coupled with separation methods expand its applications to a wide range of activities.
Laser
m
Ion source Detector
acceleration
Acceleration Field-free drift region sphere
Figure 15. Schematic of the TOF analyzer.128
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 42
Electrospray Ionization Mass Spectrometry
The combination of liquid chromatography (LC) and mass spectrometry unites the
advantages of both techniques and extends the realm of their applications. LC-MS
technique has been broadly used in biochemical, chemical, pharmaceutical,
environmental, forensic and other fields. Molecules, with molecular weight from smaller
than 1000 Da to larger than 100,000 Da, can be qualitatively and quantitatively
analyzed.128 Mass spectrometers, such as quadrupole, magnetic/electric sector, fourier
transform, and time-of-flight instruments, are widely used to combine with LC. Various
interfaces136 are available for LC-MS, which mainly contain particle beam, moving belt,
fast-atom bombardment (FAB), thermospray, plasmaspray, electrospray, photospray, and
atmospheric pressure chemical ionization (APCI).
Electrospray ionization mass spectrometry (ESI-MS) is one of those LC-MS 1 T7 techniques which utilize a soft ionization method. Since 1984 Yamashita and Fenn
introduced ESI-MS to the public, the technique has been fast developed into a mature and
important technique and particularly used for biological and biochemical applications.
ESI-MS can be used for a large range of molecules, such as peptides, proteins,
oligonucleotides, carbohydrates, and synthetic polymers.
In ESI,128;132;134;136;138 a sample solution is made by dissolving the interested analyte
in a proper solvent. The sample solution is pumped through a small-diameter (metal,
fused silica, or glass) capillary tube, which has a smoothly cut tip. A high voltage
(typically 3-6 kV or less) is applied to the tip and a counter-electrode is placed a few
millimeters away from the capillary. The sample stream is sprayed by use of a nebulizer
gas (normally nitrogen), which is concentric with and past the end of the capillary tube,
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 43
and then is drawn into a liquid cone and ionized by the high-voltage at atmospheric
pressure and ambient temperature. Once the highly charged droplets are formed, they
undergo desolvation at the aid of nitrogen while they pass towards the counter-electrode.
Much of the excess of solvent vapor is exhausted to waste. The charge repulsion on the
surface of the droplets increases as the size of the droplet decreases. Once the repulsive
forces are strong enough to defeat the cohesive forces of surface tension, a “coulombic
explosion” occurs,134 which results in the generation of smaller high charged droplets.
The explosions continue until the analyte ions are obtained from these droplets. Figure
16 shows a schematic diagram of an electrospray LC-MS interface. After passing a
nozzle and a skimmer, the analyte ions are then transferred into the mass analyzer by
using lens system, which includes a Q-array, skimmer, octapole stack, and lens stack
configuration. However, the mechanism to explain how the gas-phase analyte ions are
produced from these charged droplets remains controversial. There are a few alternative
explanations to date.
ESI offers the transfer of various ions from solution to the gas phase directly, by
which the ionization of analytes is extremely soft with little or no fragmentation. In
many cases only singly-charged ions are observed, but the conditions of ESI still favor
the formation of multiply-charged ions. The solvents used for ESI-MS are polar solvents,
which are generally composed of water, methanol, ethanol, acetone, acetonitrile, and
dimethylsulfoxide. The available ions in solution for ESI-MS usually includes singly-
charged and multiply-charged cations and anions, such as alkali and alkali earth metal
ions, transition metal ions, protonated organic bases, and anions of inorganic and organic
acids.139 The trend for ESI to form multiply charged ions results in the complexity of an
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 44
ESI-MS spectrum, but it is a benefit that allows the measurement of molecules with large
molecular weights. In addition, the solubility problem for polymers adds more
challenges to ESI-MS application, which makes the use of ESI-MS to analyze synthetic
polymers less productive than that of MALDI-MS. However, the characterization of
polymers by coupling ESI-MS with separation methods is promising.
Nozzle Skimmer
Capillary at
Nebulizer gas To mass analyzer
HPLC eluate
Atomospheric pressure Counter electrode To rotary To high pump vacuum pump
► Pressure gradient
Figure 16. Scheme of an electrospray LC-MS interface.134
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 45
Preliminary Determination of Molecular Weight Distribution
Introduction
Polymers are not as simple as small molecules, which have exact molecular weights.
Polymers typically present a distribution of molecular weights, which is known as
polydisperse. There are several terms used for describing molecular weights of
polymers, such as number average molecular weight (Mn), weight average molecular
weight (Mw), and z average molecular weight (Mz). The number average molecular
weight is similar to the common concept of average molecular weight. It is calculated by
dividing the total weight of all molecules by the total number of molecules.127,140 The
weight molecular weight is determined by the following equation,
Mw = (XNjM2) / (XNiMj) (2)
where Ni is the number of molecules with molecular weight Mi. The ratio of the weight
average molecular weight to the number average molecular weight (Mw/Mn) is known as
the polydispersity index (PDI).140 PDI shows how broad the distribution of polymer
molecular weight is. The closer the PDI is to 1, the narrower the distribution of
molecular weight.
Experimental
Samples and Reagents
Linear PEI with Mn of 423 (LPEI-423) was purchased from Aldrich (St. Louis, MO).
Branched PEIs with number average molecular weights of 600,1200, and 1800 (BPEIs-
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 46
600,1200, and 1800) were obtained from Polysciences (Warrington, PA). HPLC grade
methanol was purchased from Fisher Scientific (Fair Lawn, NJ).
Instrumentation
LC/MS analysis was performed on a Shimadzu LCMS-2010A (Columbia, MD) with
an ESI interface in the positive ion mode. The nebulizing gas was ultra high purity
nitrogen gas with a flow of 4.5 L/min. Ion gauge vacuum was 8.7 x 10'4 Pa, detector
voltage was 1.5 kV, and temperatures of block and curved desolvation line (CDL) were
set correspondingly at 200 and 250 °C.
Procedures
Four commercial PEI samples including LPEI-423, BPEI-600, BPEI-1200, and
BPEI-1800 were first prepared by dissolving ~0.04 g of PEI in 5 mL of methanol,
respectively. A 2pL sample from one of the four prepared PEI solutions was directly
injected into the Shimadzu LCMS-2010A. 100% MeOH was used as the mobile phase
and the flow rate of the mobile phase was set at 0.3 mL/min. After obtaining the
chromatogram and mass spectrum for the first PEI sample, the same procedure was
repeated for each of the other three sample solutions, respectively.
Results and Discussion
Four commercial PEI samples including LPEI-423, BPEI-600, BPEI-1200, and BPEI-
1800 were analyzed by ESI-MS. The molecular weight distribution (MWD) of each
sample, which can be seen in Figures 17-20, was found to be very similar. The MWD
obtained for LPEI-423 was very different and presented a “ffame-shift”. The number
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 47
average molecular weights of LPEI-423, BPEI-600, BPEI-1200, and BPEI-1800 were
found to be about 276,448, 535, and 448, respectively. Most likely, the explanation for
this is due to the difficulty of ionization of molecules with high molecular weights or the
formation of multiply-charged molecules. In addition, there was no apparent background
noise in these four spectra, which proved that ESI-MS is a soft ionization technique with
little or no fragmentation during its analysis process. However, more and more small peaks
appeared as the average molecular weight of PEIs increased from 423 to 1800 Da. Two
different series of PEI molecules were seen in all four spectra. In each series, the
differences of m/z (Am/z) between two adjacent peaks were all 43 Da, which is
corresponding to the molecular weight of a PEI repeat unit (-NHCH 2CH2-). The Am/z
between two adjacent peaks, one peak from each series, was 22 Da. After analyzing the
data of the first series, the following Equation (3) was derived for calculation of its m/z
values,
m/z = N*43 + 17+ 1 (3)
where N is the number of the repeat unit (ethylenimine). The number 1 represents the
ionic mass of an attached H+. The number 17 will be discussed later. In order to simplify
the analysis, integers are substituted into the equation. From the second series, Equation
(4) was derived to calculate the m/z values,
m/z = N * 43 + 17 + 23 (4)
where the number 23 is equal to the ionic mass of Na+. As a result, the two series of this
analysis were found to be protonated and sodiated PEI oligomers correspondingly, with
H+ ions showing predominant attachment. The m/z value and mass number are
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 48
numerically equal because both series present single charged ions. Therefore a formula,
H-(NHCH2CH2)n-NH 2, represents the molecules presented in both series of PEI spectra.
Correspondingly, the number 17 is equal to sum of the molar masses of the end groups,
which are H- and -NH2. The -(NHCtkCPk),!- simply represents the repeat unit with
number of n. Table 1 shows the representative values of theoretical m/z for PEI
oligomers in these two series discussed above by using Equation (3) and (4) directly, and
also comparing these values with those calculated by using an isotope distribution
calculator in reference 141. In Table 1, most m/z values obtained by these two methods
are almost identical up to 12 repeat units, after that there is a difference of 1 Da. This is
mostly due to an isotopic contribution as the molecular weight increases. Furthermore,
the isotopic peaks (M* +1) were seen in all those spectra, where M* represents the
quasimolecular ions, i.e., [M+H]+ or [M+Na]+ ions. Tables 2 and 3 show the
experimental m/z values of [M+H]+ ions in the first series were equal to the masses
calculated by an isotope distribution calculator.
Ion Attachment Analysis
Introduction
Acidic and basic polymers can directly ionize to form [M-nH]"' and [M+nH]n+ ions, 118 respectively. Most synthetic polymers, however, cannot ionize easily because they do
not contain acidic or basic ionizable groups. As a result, cationization has been used as a
very helpful and popular way to aid ionization of synthetic polymers in ESI-MS
technique. Although alkali cations, such as Na+ and K+, are usually found to be present
in synthetic polymers as impurities, which possibly come from glassware, reaction
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 49
© SC SC
_©in
00
- ©
00 C'- Figure Figure 17. Mass spectrum of linear PEI-423.
_© O s-
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 0 50VI mtz 7SO 664 643 6 600 578 PEI-600. 557 535 513 491 ofbranched 470 448 "27 spectrum 405 Mass 362 18. 3 3f9 Figure + + [1\I+NaJ [M+HJ ,------, - - .~ 0~-~------~~~L_--~LL~~~~~~ill_UL~~-ill~~u_~~~-ll~~L_~~~~~ lnt 250e3 500e3 CD CD CD CJl CD CD CD CD CD CJl Cil 0 0 0 0"" 0 c c c c ;:::). g c. (') c. ;:::;: (') c. ;:::;: c. ;:::;: :"""' :::J :::J :::J :::J :iE u;· ..., :iE ..., :iE u;· ::::r i5" ::::r ::::r ::::r i5" ::::r ::::r i5" :::0 a 3 a a 3 11 - - - Reproduced with permission - of the copyright owner. Further reproduction prohibited without permission. "0 "0 "0 "0 "0 "0 '< ©- © 00 rt 00 - Figure 19. Mass spectrum of branched PEI-1200. C“v <*o Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 52 Figure Figure 20. Mass spectrum of branched PEI-1800. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 557 470 513 643 729 6 8 6 815 858 901 calculated1* 3 [M+Na]+ 2 1 2341 2 1 2 341 298 298 513 599 600 771 772 m /z 319 448491 470 calculated1* calculated 3 [M+H]+ 190 190 362 362 384 384 276319 276 534 535 556 405491 405 427 427 448 878 879 900 calculated CH2) 2 58 233 233 255 255 4 6 7 9 1 0 1 2 13 577 578 1 1 14 620 621 642 151617 663 706 749 664 707 750 685 728 18 792 793 814 19 835 836 857 2 0 (NHCH Number ofrepeat unit 2 )n-NH 0 6 7 9 2 1 ] 2 1 6 1 5 1 8 2 5 2 0 N N N N,, N N N i N N gN gN 2 3 3 4 3 CH 5 4 8 2 3 sj 7 3 7 8 9 8 1 0 3 2 H H H H H H HggN H gH gH oH 1 2 1 0 1 6 0 1 8 jo 2 2 2 3 3 3 4 methods CgH C C C CnlfjgNg C C C ^24^63^13 C E26H6gN]4 C C C 3H3 I9 C36H93N C32Hg3Ni7 Q for parts o fPEI Assumed formula H-(NHCH Table 1. Representative theoretical m/z values forparts ofPEI molecules using different calculation aThe m/z valuesaThe were directly calculated according to the assumed formula. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 16.1 21.3 31.6 36.8 42.1 calculated isotope ratio 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 17.0 23.9 29.327.7 26.5 40.9 converted experimental [M+H]+ 1 0 0 14.26 13.94 34.83 experimental lelative intensity 3 362 73.27 363448 17.52 536 15.57 277 4.16 708 6.58 47.2 707 calculated m/z 276 276 24.40 277 363 535536 535 56.24 362 449 449 29.34 448 708 622 622 experimental CH2) 2 8 6 1 0 1 2 14 621 621 16 707 Number o f repeat unit (NHCH 2 )n-NH 7 9 11 1 3 2 Nn+l N N N N 3 3 4 3 CH 5 3 6 3 2 H H 5n+3 with calculated values for BPEI-600 H H 1 6 1 2 2 0 2 4 nH C C 2 C C C28H73N15 C32H83N17 C Isotopic M*+lb Isotopic M*+lb Isotopic M*+lb Isotopic M*+lb Isotopic M*+lb Isotopic M*+lb Assumed formula H-(NHCH aThese values were calculated using an isotope distribution calculator from reference 141. Table 2. Comparison o frepresentative experimental m/z values ofmost [M+H] + ions and their relative intensities bM* represents the quasimolecular ions, i.e., [M+H] ions.+ Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. on 100 100 100 100 16.1 26.5 31.6 26.5 (calculated15) Relative intensity 100 (experimental) 448 100 536 28.57 (calculated)b 276 276 100 277 277 15.7 448 (calculated)3 m/z ofthe highest peak [M+H]+ 276 535 534 535 277 536 535 448 449 449 449 29.34 448 448 448 100 449 449 449 31.71 and that oa fcorresponding isotopic peak (experimental) parts parts oPEI f Assumed formla for H-(NHCH2CH2)n-NH2 H-(NHCH2CH2)6-NH2 H-(NHCH2CH2)12-NH2 H-(NHCH2CH2)10-NH2 H-(NHCH2CH2)10-NH2 intensities with calculated values for PEIs PEI LPEI-423 BPEI-600 BPEI-1200 BPEI-1800 Isotopic M*+lc Isotopic M*+l° Isotopic Isotopic M*+lc Isotopic Isotopic M*+lc Isotopic Table 3. Comparison ofrepresentative experimental m/z values ofmost [M+H]ions and+ corresponding relative aThe m/zvaluesaThe were calculated according to the assumed formula. bThese bThese valueswere calculated using an isotope distribution calculator from reference 141. °M* the represents quasimolecular ions, i.e., [M+H]+ ions. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 56 chemicals, or solvents, an appropriate amount (10'5 to 10-4 M )138 of salts must be added in order to obtain an optimum spectra by ESI-MS. Alkali, alkaline earth, and transition metal cations are all used for this purpose. Four alkali metal ion salts, including LiCl, NaCl, KC1, and CsCl, and other two metal salts (T1C1 and MnCL) were used in this study of ion attachment analysis. The optimum metal cation will be chosen to promote the ionization of PEIs by ESI-MS for further analysis. Experimental Samples and Reagents Branched PEI with number average molecular weight of 600 (BPEI-600) was purchased from Polysciences (Warrington, PA). HPLC grade methanol was purchased from Fisher Scientific (Fair Lawn, NJ). LiCl and MnCh were purchased from Baker & Adamson (Morristown, NJ), NaCl and CsCl were purchased from Mallinckrodt, Inc. (Paris, KY), KC1 was obtained from J. T. Baker Chemical Co. (Phillipsburg, NJ), and T1C1 was purchased from Fisher Scientific (New York, NY). Instrumentation The mass spectra were obtained using a mass spectrometer (Shimadzu LCMS- 2010A, Columbia, MD) coupled with an electrospray probe in the positive ion mode. The ultra high purity nitrogen gas was used as nebulizing gas at 4.5 L/min. Ion gauge vacuum was 8.7 x 10'4 Pa, detector voltage was 1.5 kV, and temperatures of block and curved desolvation line (CDL) were correspondingly 200 and 250 °C. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 57 Procedures The Branched PEI sample with average molecular weight of 600 (BPEI-600) was first prepared by dissolving ~0.04 g of PEI in 1 mL of methanol, in which ~0.5 mL of a 5% LiCl solution was added. A 2 pL sample from the prepared solution was directly injected into the Shimadzu LCMS-2010A. 100% MeOH was used as the mobile phase and the flow rate of the mobile phase was set at 0.3 mL/min. After obtaining the chromatogram and mass spectrum for the lithium sample, the same procedure was repeated for each of the other metal salts, respectively. Results and Discussion PEI analyzed by electrospray shows preferential ion attachments to most molecules. All six cations (Li+, Na+, K+, Cs+, Tl+, and Mn2+) were shown to form adducts with PEI. There was no apparent fragment of the oligomer ions observed. The assumed formula, H-(NHCH2CH2)n-NH 2, was proved to match these oligomers with high intensity. The theoretical m/z values were calculated by using the following equation, m/z = (N * 43 + 17 + W) / Z (5) where N is the number of the repeat unit (ethylenimine). W is the atomic mass of a metal cation with a charge number of Z. By comparing the theoretical m/z, different series of oligomer ions are labeled (See Figures 21-26). The singly charged oligomers predominated in these spectra of PEIs when mono-valent cations were attached. Doubly charged adducts were observed when Mn2+ was added, as can be seen in Figure 26. Even when sodium and hydrogen ions were not added to the sample, H+ and Na+ adducts were found in all spectra except one obtained by adding LiCl to PEI. When the metal ions Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 58 were compared, lithium showed the strongest attachment. The attachment of lithium was so strong that it displaced the H+ and Na+ attachment to PEI that was seen in all other examples. This may be due to the fact that lithium is the strongest Lewis acid. Lithium cations appear to be the optimum candidate for cationization in further PEI analyses by ESI-MS. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. VI 59 '-0 z 713 6r - L'· 6 7 solution. I LiCI 5l4 - in II 541 -600 0 l PEl 5 1 497 4 rr II 4~ branched of I 4 4 4 _u I 1 - 1 3 8 Spectrum J I Mass 3 5 21. 2 2 I - - Figure 2 9 2121 196 1r - + 153 I 9 1~ IM+Lil I 1 :l 1 Int. Oel- 250e3 CD CD c c. (') c. ;:::;: :iE :::0 a ~ CD CJl u;· 3 ~ ~ CD CD 0 0 0 6" (') :::J :::J ..., :iE - - :"""' - ~ ~ 11 CD Cil 0"" c c ;:::). g 6" c. ;:::;: :::J ..., a a CD c. ~ CD CJl 0 c 6" ;:::;: :::J :iE u;· 3 - "0 "0 "0 '< Reproduced with permission of the copyright Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 61 Figure Figure 23. Mass Spectrum of branched PEI-600 in solution. KC1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 62 e V) f- sO 00 — sO SO— __ * ni«N — o- ^4. o o o ------VO 00 r-- jCi r- ITi ir>w V) IT) i"" TV) fO- *n ITi irr V j r-O oo V) Tf- _ J C = >» t~- r s - ITi Tf© - T f _ 00 «rv VO- o ro V) <*> r4 o n . fO 00 o s- e V) ~ *3 55 e•3 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 63 ©sO N - o- fS- IT; O Or~ oo- Tf t— 00 ^r- r*-r- ITi •**®- 00- 0 0 ©fS s - + - V j S Z h + + + s s s <*>1 ,f2 "3 5> © Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 64 r^- r-© _ © © © JO © © © _ © -8 t—0 0 r - IT) *G_ ir. , V) IT) MO r + Y - 2 _ *Ti ON- O h~— 00 y) Tf^r- r- fN — ift 00-o %n00------h- ^ — *o- 00 © ON- fN r—fN fN •Ti io fN fN © cT" Figure 26. Mass Spectrum of branched PEI-600 in MnCl2 solution. fN 00-o itj + ^ _ s O - O a = fO JO SC Z 2 Tf- + + + 5 S '<*>4> i "21 © o 5 IT) fN Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 5 ANALYSIS OF PEI BY SIZE EXCLUSION CHROMATOGRAPHY Introduction Liquid chromatography (LC) has been used for more than 50 years as one of the most effective methods for separating organic molecules. Depending on the nature of the stationary phase and the interactions between the analyte, mobile phase or stationary phase, one can divide LC techniques into four types: size exclusion chromatography (SEC), liquid-liquid chromatography (LLC), liquid-solid chromatography (LSC), and ion-exchange chromatography.142 The separation mechanism that operates is size- exclusion, partition, adsorption, or phase separation, respectively.132 Size exclusion chromatography (SEC), also known as gel permeation chromatography (GPC), has been widely used for the separation and determination of the molecular weight distribution (MWD) of polymers. The combination of SEC with other analytical techniques allows the assessment of polymer branching and measurement of the copolymer composition.142 SEC is used to separate polymers according to the size or hydrodynamic volume of these molecules. One or more separation columns, which are packed with porous beads, are used to separate the polymer molecules (See Figure 27). After a small amount of sample solution is injected and pumped through the column(s), small molecules can enter the pores of the beads and are consequently retarded in the column(s). On the other hand, 65 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 66 the hydrodynamic volumes of large molecules are too large to enter the pores. They stay in the mobile phase and then elute rapidly. Therefore the large molecules have shorter retention time than the small molecules. After traveling through the separation column, the eluate is finally sent to a detector, such as a differential refractive index (DRI), UV, viscometer, osmometer, low-angle laser light scattering (LALLS), and small angle neutron scattering (SANS) detector. In order to obtain accurate molecular weight determination for polymers, sometimes, two or more detectors are coupled together for use.128’142’143 The process is shown in Figure 28. SEC is typically operated in an isocratic mobile phase.132 Relationship between molecular weight and elution/retention volume (V r), which is the product of the retention time (Rt) and the volumetric flow rate, is usually used to generate a calibration curve for a SEC analysis. The calibration curve is a graph of log molecular weight vs. elution volume of standard samples, which have narrow molecular weight distributions. In general, molecular weight decreases as elution volume increases. Porous particles Column Small Large molecule molecule Figure 27. Schematic of a size exclusion chromatography column. (Reproduced from reference 143) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 67 INJECTORCOLUMN(S)DETECTOR(S) DATA ACQUISITION AND ANALYSIS Figure 28. Schematic of the SEC process. Experimental Samples and Reagents Linear PEIs (LPEIs) with number average molecular weights of 423 and 25000 were purchased from Aldrich (St. Louis, MO) and Polysciences (Warrington, PA), respectively. Branched PEIs (BPEIs) with number average molecular weights of 600,1200, and 1800, 10000, 70000 and polyethylene glycol (PEG) with number average molecular weights of 1540, 3400,10000, and 20000 (PEG-1540, 3400,10000, and 20000) were obtained from Polysciences (Warrington, PA). HPLC grade methanol was purchased from Fisher Scientific (Fair Lawn, NJ). Deionized water was prepared in our lab. Glacial acetic acid was purchased from Mallinckrodt (Phillipsburg, NJ). Ammonium acetate was bought from Fisher Scientific (Fair Lawn, NJ). Instrumentation A Waters M-45 solvent delivery system was used to introduce the sample through an injector, which connected with one TSK-GEL PWXL guard column and one TSK-GEL G2500 PWXL column (Tosohaas, Japan). The Waters 410 Differential Reffactometer Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 68 was used as a refractive index detector. The scale factor and sensitivity of the Waters 410 were set at 22 and 16, respectively. The maximum pressure was set at 600 psi. Procedures A solution, which contained 0.5 M acetic acid, 0.5 M ammonium acetate, and appropriate amount of deionized water, was prepared and then was filtered through a Nylon 66 filter membrane from Supelco. Eleven commercial PEI and PEG samples, which include BPEI-600, BPEI-1200, BPEI-1800, BPEI-10000, BPEI-70000, LPEI-423, LPEI-25000, PEG-1540, PEG-3400, PEG-10000, and PEG-20000, were first prepared by dissolving -0.01 g of PEI or PEG in 10 mL of previously prepared solution, respectively. A 20 pL sample from one of the eleven prepared PEI or PEG solutions was directly injected into the SEC system described above. The solution used for preparing samples was applied as the mobile phase and the flow rate of the mobile phase was set at 0.5 mL/min. After obtaining the chromatogram and mass spectrum for the first sample, the same procedure was repeated for each of the other ten sample solutions, respectively. Results and Discussion Figures 29-39 show the GPC chromatograms for all PEI and PEG samples. Two peaks are observed in all figures. The first peaks with earlier elution time were found to represent the “solute molecules”, either PEI or PEG species. The second peaks shown at retention time of 19.27 minutes were proved to be “solvent peaks”, which appear as a result of the water solution used to prepare these samples. Table 4 was generated to combine all data obtained from Figures 29-39. According to Table 4, for all branched PEIs the retention time increases as the average molecular weight decreases. The similar Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 69 conclusion could be drawn for all linear samples, both PEIs and PEGs. It seemed the polymer structure plays a dominant role in this case, even if the polymer type also mattered in the others. Table 4. Experimental values for PEIs and PEGs by SEC samp e Rt (mins) VR (mL)* M„ (Da) log (M„) BPEI 14.99 7.495 600 2.778 14.31 7.155 1,200 3.079 Branched 14.15 7.075 1,800 3.255 12.37 6.185 10,000 4.000 12.29 6.145 70,000 4.845 LPEI 15.99 7.995 423 2.626 PEG 15.23 7.615 1540 3.188 14.13 7.065 3400 3.531 Linear 12.68 6.340 10000 4.000 12.54 6.270 20,000 4.301 LPEI 12.51 6.255 25000 4.398 *V r is the product of Rt and the volumetric flow rate, which is equal to 0.5 mL/min. In general, once the molecular weight of a species is beyond its exclusion limit there is no retention. So polymers with greater molecular weights than the exclusion limit elute together and have the similar elution volumes. On the other hand, polymers with smaller molecular weights than the permeation limit can penetrate into the pores of the beads in a column. Therefore, these PEIs can stay long in the column and finally elute together.144 According to Figure 40 BPEI-70000 molecules were too large to be retained. Similarly, both LPEI-25000 and PEG-20000 molecules were also greater than their Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 70 exclusion limit. In contrast, LPEI-423 molecules were smaller than their permeation limit so that they eluted late. These samples are represented by a hollow square or triangles in Figure 40. Because of this, four branched PEI samples were used to generate a standard calibration curve (See Figure 41). The calibration equation was established with a good R-square (R ) value as followed, y = -0.9269x + 9.7454 (6) R2 = 0.9922 where y represented log molecular weight and x corresponded to elution volume. Another standard calibration curve was prepared for linear samples, both linear PEI and PEG, only basing on qualified samples except LPEI-423, LPEI-25000, and PEG-20000 (See Figure 42). The calibration equation was below. y = -0.6377x + 8.0412 (7) R2 = 0.9999 The universal calibration concept is usually applied to analysis of molecular weight distribution. However, in order to obtain high accuracy, either adding more standard samples with qualified molecular weights to the sample pool or adding a “molecular weight sensitive” detector (e.g., a viscometer) to the DRI detector is necessary. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 71 a 'W's a a •Me a •M4> Figure Figure 29. SEC chromatogram of branched PEI-600. DR1 Intensity Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 72 •ma £ aQ> H a o •-C 4»a N a\ Figure Figure 30. SEC chromatogram of branched PEI-1200. DRI Intensity Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 73 a •pn g , •mma H fl #o a ■** (2 N ON *n Figure Figure 31. SEC chromatogram of branched PEI-1800. DRI Intensity Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 74 s I a H a e ‘■S Va £-ta* Figure Figure 32. SEC chromatogram of branched PEI-10000. DRI Intensity Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 75 |a 4* •ms H a e Tta V V PS Figure Figure 33. SEC chromatogram of branched PEI-70000. DR1 Intensity Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 76 a sw sv H a © a V Figure Figure 34. SEC chromatogram of linear PEI-423. DRI Intensity Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 77 |a u i H a o « u0 1 Figure Figure 35. SEC chromatogram of PEG-1540. DRI Intensity Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 78 |a a(U a e •-C a £ Figure Figure 36. SEC chromatogram of PEG-3400. DRI Intensity Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 79 •m*ta s a •p* H a #o ‘■S a -** £ oo SO ri Figure 37. SEC chromatogram of PEG-10000. DR1 Intensity Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 80 fl >3 « ✓ s H fl #o "•{ 3 fl ■**v pH rr *n N Figure 38. SEC chromatogram of PEG-20000. DRI Intensity Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 81 a I aV a 0 ••c va 1 N a\ Figure Figure 39. SEC chromatogram of linear PEI-25000. DRI Intensity Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 00 ■branched ■Linear Elution volume (mL) Elution volume 6.00 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40 7.60 7.80 8.00 Figure 40. Relationship between molecular weight and elution volume forPEI and PEG. 2.70 3.70 2.20 5.20 3.20 4.20 4.70 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. oo u> = 0.9922 2 branched R •trendline y= -0.9269x+ 9.7454 calibration equation 7.80 7.60 7.40 7.00 Elution volume (mL) Elution volume 6.60 6.80 7.20 6.40 Figure 41. SEC calibration curve forbranched PEI. 6.20 6.00 2.60 3.00 2.80 3.60 3.40 3.20 3.80 4.00 4.20 o OD Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. OO R2 = 0.9999 R2 ▲ ▲ Linear y= -0.6377x + 8.0412 ——Trendline Calibration equation 7.50 7.70 6.90 7.10 7.30 Elutionvolume (mL) Figure 42. SEC calibration curve for linear PEI and PEG. 6.30 6.50 6.706.10 3.20 3.00 3.60 3.40 3.80 4.00 4.20 00 o Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPER 6 ANALYSIS OF PEI BY ESI-MS COUPLED WITH SEC Introduction ESI-MS has become a powerful tool for polymer analysis. The tendency of ESI-MS to generate multiply-charged ions makes the spectra complex and difficult to interpret. The problem can be solved either by using ESI-MS coupled with separation techniques to detect the separated analytes in a sample or by using ESI-FTMS, which has high resolution, to simplify the complicated spectra into singly-charged spectra.132 ESI-MS is compatible with most separation techniques because the sample can be introduced at atmospheric pressure. In 1993, Prokai and Simonsick145 first successfully coupled ESI-MS with on-line SEC to analyze a complex synthetic polymer mixture. From then on ESI-MS coupled with SEC has showed great potential for molecular characterization of polymers. The combination reduces the polydispersity complications so that analysis of complex polymers can be realized. The average molecular weight and distributions can be directly determined for polymer by using these hyphenated techniques without an external calibration. 85 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 86 Experimental Samples and Reagents Linear PEI with Mn of 423 (LPEI-423) was purchased from Aldrich (St. Louis, MO). Branched PEIs with number average molecular weights of 600,1200, and 1800 (BPEI- 600,1200, and 1800) were obtained from Polysciences (Warrington, PA). Glacial acetic acid was purchased from Mallinckrodt (Phillipsburg, NJ). Ammonium acetate was bought from Fisher Scientific (Fair Lawn, NJ). Deionized water was prepared in our lab. Instrumentation SEC-ESI MS analysis was performed by coupling SEC columns with a Shimadzu LCMS- 2010A (Columbia, MD) with an ESI interface in the positive ion mode (See Figure 43). The SEC columns consisted of a TSK-GEL PWXL guard column and a TSK-GEL G2500 PWXL column (Tosohaas, Japan). In general, the separation conditions, such as the mobile phase flow rate and pressure, must accommodate ESI-MS operation. A post-column splitter was inserted into the line to reduce the flow rate to match ESI-MS operation. In our analysis, a T-valve was necessary to combine the SEC column with ESI-MS. The eluate flow was split after exiting from the SEC column in order to decrease the pressure of the whole system. The control valve allowed only parts of the stream to continue to flow into the ESI-MS and the left was dumped to waste. The nebulizing gas was ultra high purity nitrogen gas with a flow of 4.5 L/min. Ion gauge vacuum was 8.7 x 10*4 Pa, detector voltage was 1.5 kV, and temperatures of block and curved desolvation line (CDL) were set correspondingly at 200 and 250 °C. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 87 SEC columns T-valve LC Injector pump Guard SEC column column Control valve To Waste Figure 43. Schematic diagram of a SEC-ESI MS system. Procedures A solution, which contained 0.5 M acetic acid, 0.5 M ammonium acetate, and an appropriate amount of deionized water, was prepared and then was filtered through a Nylon 66 filter membrane from Supelco. Four commercial PEI samples, which include LPEI-423, BPEI-600, BPEI-1200, and BPEI-1800, were first prepared by dissolving -0.01 g of PEI in 10 mL of previously prepared solution, respectively. A 20 pL sample from one of the four prepared PEI solutions was directly injected into the SEC-ESI MS system described above. The solution used for preparing samples was applied as the mobile phase and the flow rate of the mobile phase was set at 0.5 mL/min for the SEC part. After obtaining the total ion chromatogram (TIC) for the first sample, the chromatogram peak was divided into three fractions in the order of the retention time. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 88 Therefore, three corresponding mass spectra were generated for each sample. The same procedure was repeated for each of the other three sample solutions, respectively. Results and Discussion In general, the SEC chromatogram is theoretically observed as the following Figure 44. Sample molecules with high molecular weights elute faster than those with low molecular weights due to the SEC separation. Thus, the large molecules could be separated from the small molecules by this technique. B Average MW e % High MW Low MW Time (min) Figure 44. SEC chromatogram. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 89 The chromatograms and mass spectra obtained for LPEI-423, BPEI-600, BPEI-1200, and BPEI-1800 are shown in Figures 45-60. All four MS chromatograms (Figures 45, 49, 53, and 57) were individually divided into three fractions according to the retention time. The 1st, 2nd, and 3rd fractions corresponded to molecules with large, medium, and small molecular weights, respectively. As discussed before, we also observed the singly- charged protonated and sodiated molecules with the assumed formula, H-(NHCH2CH2)n-NH 2, in most spectra. However, as a rule, ESI has the ability to form multiply-charged ions, and usually multiply-charged ions are produced by attachment of cations, such as H+, Na+, and the like. In our case, for small molecules such as LPEI-423 and the 3 rd fractions of branched PEI molecules, singly-charged ions (mostly H+ adducts) were observed to be predominant. However, for big molecules including the 1st fractions of all branched PEI sample molecules, the majority of ions were found to be multiply charged. The MWD showed that PEI molecules became more charged as the molecular weight of the polymer increased. The formation of doubly- and triply-charged ions was apparent. This was proved by measuring the A tn /z values between two adjacent peaks. As can be seen in Figure 54, the m /z difference between any two adjacent peaks was either 21 (or 22 Da) for doubly charged molecules or 14 (or 15 Da) for triply charged molecules. This is approximately equal to 43 Da (A m /z for singly charged ions) divided by a factor, 2 or 3 correspondingly, which is equivalent to the number of charges of these ions. Based on the formula, H-(NHCH 2CH2)n-NH 2, the spectra show that almost all these multiply charged molecules are 2H+ or 3H+ adducts. The theoretical m/z values for Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 90 [M+zH]z+ ions of this kind of molecules were approximately calculated by using the following equation, m/z = (N*43 + 17 + Z )/Z (8) where N is the number of the repeat unit (ethylenimine) of a PEI molecule. Z is the number of H+ attached to the molecule. In addition, we observed two other series of cationic oligomers with lower intensities than those two series mentioned above in most spectra of both linear and branched PEIs. For each series, the A m /z of any adjacent two peaks was 43 Da, which matched the molar mass of the repeat unit (-NHCH2CH2-). The A m /z is 22 Da between the series with relatively higher intensities and the other with lower intensities. These two new series seemed very similar to those two series previously discovered with singly-charged H+ and Na+ ions attached. With a “frame shift” of A m /z of 17 Da toward the lower molecular weight field, the two new series were generated from the two original series. Therefore the m/z values of molecules in these two new series could be correspondingly calculated by directly subtracting 17 Da from those of molecules in the two original series, which fit the assumed formula H-(NHCH 2CH2)n-NH 2. And since the number of 17 is equal to the total molar mass of the two end groups (H- and NH 2-), presumably, we could draw a conclusion that molecules in these two new series were also singly-charged H+ and Na+ adducts, respectively, and they represented one type of cyclic molecules, which lost the two end groups (H- and NH 2-) by intermolecular cyclization. So we could further assign the other formula to these new type of molecules, which was Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 91 -(NHCH2CH2) NHCH2CH2 represents the repeat unit. The following equations were derived for calculating m/z values of the singly-charged cyclic molecules. Equation (9) was for H+ adducts and Equation (10) for Na+ adducts, m/z = N * 43 + 1 (9) m/z = N * 43 + 23 (10) where N is the number of the repeat unit (ethylenimine). The number of 1 represents the ionic mass of H+ and the number of 23 is equal to the ionic mass of Na+. Table 5 was made to compare the theoretical m/z values of previously discovered molecules (non-cyclic molecules) with those of cyclic molecules, which was shown in both linear and branched PEI spectra. Based on both assumed formulae, H-(NHCH2CH2)„-NH2 and -(NHCH2CH2)n-, Equations (3), (4), (9), and (10) were applied to calculate the m/z values, respectively. Different series of oligomer ions are labeled by comparing the experimental m/z values to the theoretical values. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 92 08 ts Figure Figure 45. MS Chromatogram of LPEI-423. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 93 © 00 ■^fin C4- in © © - © 00" vo- <*) >r, V) - h_ m Os fO m r—VO O Tf in fS in Figure 46. Mass spectrum of LPEI-423 fraction). (1st o fS IN© IN :j aa or-' , ■ o A VO-3 m© 'u + ¥ a i n 5 1 + © © '# ' ' ■§■ a 2 m© in© «s f' IN Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 94 S Osi I XJ o o XJ SO X o 00-5 XJ ■’■r ■'Te so~ rri o fOXJ T- fj Os Ovi00 • rn NXJ e9 N XJ e Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. \0 95Vl m/z 556 550 .531. 500 502...... 450 fraction). (3rd 400 LPEI-423 of 350 spectrum 300 Mass 276 ',) 48. 255 250 Figure 233 212 200 190 /" 150 ~c'lclic II' + •, . , i [M+H] [M+Na] 1 - =I = : + ~ 100 Oe~ Int~ 40e3.:: 20eJ:I 60e3: 80e~ 100~ CD CD CD CJl CD CD CD CD CD CJl Cil 0 0 0 0"" 0 c c c c ;:::). g c. (') c. ;:::;: (') c. ;:::;: c. ;:::;: :"""' :::J :::J :::J :::J :iE u;· ..., :iE ..., :iE u;· ::::r i5" ::::r ::::r ::::r i5" ::::r ::::r i5" :::0 a 3 a a 3 11 - - - Reproduced with permission- of the copyright owner. Further reproduction prohibited without permission. "0 "0 "0 "0 "0 "0 '< ea Figure Figure 49. MS Chromatogram of PEI-600. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 97 N a o N 00 oO © 00 fN © o m Ift © 00 -00 - © -00= Figure 50. Mass spectrum of BPEI-600 fraction). (1st © On On - r^ trrr NO-1 © N© 1 ©...... 1 ' 1 ...... , , , , e , , , , o o e ■ ■ 1 • «o o e o © w •n V) in fO *N Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 98 00 : 9 0 r^00 in Ov 00 00 0 0- Figure Figure 51. Mass spectrum of BPEI-600 fraction). (2nd Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 99'-0 '-0 ,I m/z JMD-f' I 1200 1100 J;Vll, I 1000 ?f'l? 7 900 fraction). (3rd O-?D; 800 rz.,.., l:fV ';:t.' 700 ofBPEI-600 600 spectrum 'i"·=n!~r~ll:!jl * Mass J";' 500 .. 4 52. 448 400 405 Figure 362 ~ 13 319 rr1 300 - 2?8 Is I 276 ' I 2!255l·d, 233 L,UIU..,I*!U,Jlfi!!-M!.M1''U·Y•f·!. 200 190 'L! + ': 2 ! ,.._.._c~Liic + LL'.:\ 100 ' ' Reproduced with permission of the copyright owner. Further reproduction"0 prohibited without permission. "0 "0 '< Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 101 Figure Figure 54. Mass spectrum of BPEI-1200 fraction). (1st Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 0 N fraction). (2nd ofBPEI-1200 spectrum •n . Mass 55. Figure 200 +--.:\clic 2+ 3+ 100 :,, [M+2H) [M+3H) i\i [M+H]+ -· Int. CD CD CD CJl CD CD CD CD CD CJl Cil 0 0 0 0"" 0 c c c c ;:::). g c. (') c. ;:::;: (') c. ;:::;: c. ;:::;: :"""' :::J :::J :::J :::J :iE u;· ..., :iE ..., :iE u;· ::::r i5" ::::r ::::r ::::r i5" ::::r ::::r i5" :::0 a 3 a a 3 11 - - - Reproduced with permission - of the copyright owner. Further reproduction prohibited without permission. "0 "0 "0 "0 "0 "0 '< Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 104 ca a e« a S ■X3 Figure Figure 57. MS Chromatogram of PEI-1800. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 105 Figure Figure 58. Mass spectrum of BPEI-1800 fraction). (1st Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 0 1060\ fraction). (2nd ofBPEI-1800 spectrum Mass 59. Figure ;..·\clic 2+ 3+ j ~ i 00 N © o © -O c 0 -4-> 1 00o 00 00 00 o r» o T©T © to © V) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 5. Representative theoretical m/z values of PEI molecules m/z Number of repeat unit non-cyclic molecules cyclic molecules NHCH2CH2 [M+H]+ [M+Na]+ [M+H]+ [M+Na]+ 4 190 212 173 195 5 233 255 216 238 6 276 298 259 281 7 319 341 302 324 8 362 384 345 367 9 405 427 388 410 10 448 470 431 453 11 491 513 474 496 12 534 556 517 539 13 577 599 560 582 14 620 642 603 625 15 663 685 646 668 16 706 728 689 711 17 749 771 732 754 18 792 814 775 797 19 835 857 818 840 20 878 900 861 883 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 7 FURTHER ANALYSIS OF PEI BY ESI-MS Confirmation of the Existence of Cyclic Molecules Introduction Our previous analysis has showed the existence of two types of molecules in PEI polymers, both linear and branched. The assumed formulae for each were H-(NHCH2CH2)n-NH 2 and -(NHCH 2CH2)n-, respectively. As a result of ion attachment analysis, lithium cationization was selected to confirm the existence of both molecules by ESI-MS. Experimental Samples and Reagents Linear PEI with Mn of 423 (LPEI-423) was purchased from Aldrich (St. Louis, MO). Branched PEIs with number average molecular weights of 600,1200, and 1800 (BPEI- 600,1200, and 1800) were obtained from Polysciences (Warrington, PA). HPLC grade methanol was purchased from Fisher Scientific (Fair Lawn, NJ). LiCl was purchased from Baker & Adamson (Morristown, NJ). Instrumentation LC/MS analysis was performed on a Shimadzu LCMS-2010A (Columbia, MD) with an ESI interface in the positive ion mode. The nebulizing gas was ultra high purity 109 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 110 nitrogen gas with a flow of 4.5 L/min. Ion gauge vacuum was 8.7 x KF4 Pa, detector voltage was 1.5 kV, and temperatures of block and curved desolvation line (CDL) were set correspondingly at 200 and 250 °C. Procedures Four commercial PEI samples including LPEI-423, BPEI-600, BPEI-1200, and BPEI-1800 were first prepared by dissolving -0.04 g of PEI in 5 mL of methanol, respectively, in which -0.05 g of LiCl crystal was individually added. A 2pL sample from one of the four prepared solutions was directly injected into the Shimadzu LCMS- 2010A. Methanol was used as the mobile phase and the flow rate of the mobile phase was set at 0.3 mL/min. After obtaining the chromatogram and mass spectrum for the first sample, the same procedure was repeated for each of the other three sample solutions, respectively. Results and Discussion Four spectra for lithium-cationized LPEI-423, BPEI-600,1200, and 1800 were obtained by ESI-MS (See Figures 61-64). Table 6 was created to help interpreting these spectra. The theoretical m/z values of molecules with lithium ion attachment were determined by two different calculation methods, which included an isotope distribution calculator from reference 141 and the following two equations. Equation (11) was used for Li+ adducts of non-cyclic molecules and Equation (12) used for those of cyclic molecules. m/z = N *43 + 17 + 7 (11) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. I l l m/z = N * 43 + 7 (12) In these equations, N is the number of the repeat unit (ethylenimine). The ionic mass of Li+ is 7 Da and 17 Da is equal to the total molar mass of the end groups, H- and NH 2-. Table 6 also compares the experimental data with these calculated values. The experimental and theoretical data were almost identical up to 12 repeat units, after that there is a difference of 1 Da between the experimental data and the calculated values from the assumed formulae. This could be explained by the isotopic contribution as described before. Table 6 reports that all four spectra were labeled with lithium ion. Apparently, there are two series of Li+ PEI adducts in all four spectra. The dominant series represents the non-cyclic molecules with Li+ attachment and the other series corresponds to the cyclic molecules with Li+ ions attached. When these spectra are compared with the regular spectra of PEIs (Figures 17-20), these four spectra were almost simplified by lithium ions. All the protonated and sodiated oligomers (both non-cyclic and cyclic) in branched PEI- 1800 were incompletely replaced by Li+ adducts. There were still a few H+ adducts observed in the spectrum of branched PEI-1800 (Figure 64). Some of these spectra also showed the doubly charged non-cyclic oligomers, which were [M+2Li ] . Consequently, we could derive the conclusion that there were two types of molecules in PEI, non-cyclic and cyclic. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. N 112...... LiCl. with PEI-423 linear of 351 spectrum 325 30R 300 Mass 282 265 61. 239 Figure 196 (non-cyrlic) (cyclic) + + [1\I+Lij [M+Li] Int. CD CJl a· :::J u;· 3 CD 0 c c. ;:::;: :iE ::::r - 0"" ;:::;: ::::r Cil c g c. a· :::J ..., a a CD c ;:::). ::::r 11 CD 0 :"""' :::J :iE ::::r - CD 0 0 :::J ..., ::::r () - a· - CD CD CJl c. ;:::;: "0 :iE u;· ::::r 3 CD c c. :::0 () a Reproduced with permission of the copyright owner. Further"0 reproduction"0 prohibited without permission. "0 '< Reproduced with permission of the copyright owner. Further reproduction"0 prohibited without permission. "0 e \C "' ... \C or, or, ...<:-"' ...or. .... r-- ,,,..,. ..,. ... ,,,G' •r or, ,,, ...., oc c "' '" "' <:- .:: .:: -;:: -;:: "' ;... ;... <:-"" '"{ '"{ c c -::; 0 0 "?• c c "J + + "' ...J 5N " + + + :;; ~ ~ ' -...:= Reproduced with with permission permission of the of copyright the copyright owner. owner.Further reproduction Further reproduction prohibited without prohibited permission. without permission. Figure Figure 64. Mass spectrum of branched PEI-1800 with LiCl. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. os 179 524 8 6 8 351 437 experimental 524 8 6 8 265 265 610696782 610 696 782 calculated1* cyclic molecules 3 867 265 calculated m /z 799 781 282 experimental 196 196 179 179 541 541 523 627713 627 713 609 695 368 368 351 351 454 454 437 437 calculated1* non-cyclic molecules 3 540 798884 799 885 885 282368 454 282 calculated 2 CH 2 8 6 4 196 141618 626 712 1 0 1 2 attachmentto those theoretical m/z value 2 0 NHCH Table 6. Comparison ofrepresentative experimental m/z values ofPEI oligomers with lithium Number ofrepeat unit aThe m/z values were directly calculated according bThe m/z to correspondingvalues were assumedcalculated formulae. using an isotope distribution calculator from reference 141. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 117 Assessment of the Degree of Branching Introduction ESI-MS combined with some chemical reactions can be used for structural characterization of complicated polymers. To assess branching, imine derivatives of the PEI were prepared by reacting different equivalents of benzaldehyde or 4- fluorobenzaldehyde with PEI in benzene at ~110 °C, respectively. By comparing all the reaction results, 7.5 equivalents of benzaldehyde or 4-fluorobenzaldehyde proved to be an optimal stoichiometric amount that led to complete reactions with little unreacted PEI. The optimal reaction conditions were selected for the following assessment of the degree of branching of PEI. Experimental Samples and Reagents Linear PEI with Mn of 423 (LPEI-423) was purchased from Aldrich (St. Louis, MO). Branched PEIs with number average molecular weights of 600, 1200, and 1800 (BPEI- 600,1200, and 1800) were obtained from Polysciences (Warrington, PA). Benzene and 4-fluorobenzaldehyde were purchased from Acros Organics (New Jersey, USA). Benzaldehyde was obtained from Alfa Aesar (Ward Hill, MA). HPLC grade methanol was purchased from Fisher Scientific (Fair Lawn, NJ). Instrumentation LC/MS analysis was performed on a Shimadzu LCMS-2010A (Columbia, MD) with an ESI interface in the positive ion mode. The nebulizing gas was ultra high purity nitrogen gas with a flow of 4.5 L/min. Ion gauge vacuum was 8.7 x 10'4 Pa, detector Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 118 voltage was 1.5 kV, and temperatures of block and curved desolvation line (CDL) were set correspondingly at 200 and 250 °C. Procedures Eight imine derivatives of four commercial PEI samples, which included LPEI-423, BPEI-600, BPEI-1200, and BPEI-1800, were first prepared by reacting 7.5 equivalents of benzaldehyde or 4-fluorobenzaldehyde with PEI in benzene at ~110 °C, respectively. The reaction was allowed to proceed with azeotropic removal of water until evolution of water ceased. The solvent was evaporated from the crude mixture and the solid product was obtained. Samples for ESI-MS were then prepared by dissolving ~0.05 g of PEI in 10 mL of methanol, respectively. A 3 pL sample from one of the eight prepared solutions was directly injected into the Shimadzu LCMS-2010A. 100% MeOH was used as the mobile phase and the flow rate of the mobile phase was set at 0.3 mL/min. After obtaining the chromatogram and mass spectrum for the first sample, the same procedure was repeated for each of the other seven sample solutions, respectively. Results and Discussion The reaction was shown in Equations (13) and (14). The fluorine in 4- fluorobenzaldehyde, which is a good electron-withdrawing species, makes the carbon in the carbonyl group more electrophilic. Therefore the reactions between PEI and 4- fluorobenzaldehyde were theoretically more effective than those of PEI with benzaldehyde. In addition, only the primary amino group can react with benzaldehyde or 4-fluorobenzaldehyde and the secondary or tertiary amino group cannot. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 119 o p / = \ N - c H2- C H 24 (13) a C -H ^ - c - ^ Q c - h 1 + H ,0 / — \ 9 ■> / = \ n - c h 2—c h 2~> FV_/C'H +h^~ H =1^ fV j hc-h +Hj0 (14) In Equations (13) and (14), H 2N-CH2-CH2-J represents PEI with a primary amino group. The molar mass difference between the produced imine derivative molecules and reacting PEI molecules is 88 Da for reactions of PEI with benzaldehyde and it is 106 Da for those reactions of PEI with 4-fluorobenzaldehyde. The following Equations (15)-(18) show how to calculate m/z values of the singly-charged cationic molecules of imine derivatives. For reactions between PEI with benzaldehyde, m/z = 43*N+17+W+P*88 (from non-cyclic PEI) (15) m/z = 43*N+W+P*88 (from cyclic PEI) (16) For reactions between PEI with 4-fluorobenzaldehyde, m/z = 43*N+17+W+P*106 (from non-cyclic PEI) (17) m/z = 43*N+W+P* 106 (from cyclic PEI) (18) where N is the number of the repeat unit (ethylenimine). The number of 17 is equal to the sum of the molar masses of the end groups (H- and NH 2-) for non-cyclic molecules. W correspondingly represents the molar mass of the single-charged cations which attach to Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 120 PEI molecules. P is the number of primary amino group in a given PEI oligomer molecule. By using Equations (15)-(18), Tables 7 and 8 were created to show the representative theoretical m/z values of molecules of imine derivatives from both cyclic and non-cyclic PEI reacting with benzaldehyde and 4-fluorobenzaldehyde, respectively. According to the original spectra of PEI (Figures 17-20), the m/z values of imine derivative molecules and their corresponding numbers of primary amino group were categorized and labeled (See Figures 65-72). Comparison of the experimental results with the theoretical values demonstrated that both were almost identical for imine derivatives with low molecular weight, but there was a difference of 1 Da for large molecules. This is mostly due to an isotopic contribution. ESI-MS spectra of the imine derivatives of the linear PEI-423 showed that it isn’t totally linear and it contains branched molecules. The imine derivatives of the branched PEI presented spectra which confirmed that it is branched. Based on the type of reacting PEI molecules (non-cyclic or cyclic), two kinds of imine derivative molecules (correspondingly non-cyclic or cyclic) were produced. Most spectra illustrated these two kinds of molecules with H+ and Na+ attachment, respectively. Almost every spectrum showed four series of oligomer molecules of imine derivatives, which included two series of non-cyclic molecules and the other two series of cyclic molecules. These two series corresponding to non-cyclic molecules had relatively higher intensities than the other two series corresponding to cyclic molecules. Each series correspondingly represented the singly-charged protonated or sodiated molecules of these imine derivatives. Representative experimental m/z values of molecules of imine derivatives from non- cyclic PEI reacting with benzaldehyde or 4-fluorobenzaldehyde were shown in Tables 9 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 121 Table 7. Representative theoretical m/z values of molecules of imine derivatives from non-cyclic PEI reacting with benzaldehyde and 4-fluorobenzaldehyde, respectively Number of Number of m/z of imine derivative primary repeat unit with 4- with benzaldehyde amine in PEI fluorobenzaldehyde group in NHCH2CH2 [M+H]+ [M+Na]+ [M+H]+ PEI [M +N af 4 366 388 402 424 2 6 452 474 488 510 8 538 560 574 596 4 454 476 508 530 6 540 562 594 616 3 8 626 648 680 702 5 585 607 657 679 6 628 650 700 722 4 7 671 693 743 765 8 714 736 786 808 7 759 781 849 871 8 802 824 892 914 5 9 845 867 935 957 1 0 8 8 8 910 978 1 0 0 0 9 933 955 1041 1063 1 0 976 998 1084 1106 6 1 1 1019 1041 1127 1149 1 2 1062 1084 1170 1192 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 122 Table 8. Representative theoretical m/z values of molecules of imine derivatives from cyclic PEI reacting with benzaldehyde and 4-fluorobenzaldehyde, respectively Number of Number of m/z o f imine derivative primary repeat unit with benzaldehyde with 4-fluorobenzaldehyde amine in PEI group in NHCH2CH2 [M+H]+ [M+Na]+ [M+H]+ [M+Na]+ PEI 4 349 371 385 407 5 392 414 428 450 6 435 457 471 493 2 7 478 500 514 536 8 521 543 557 579 9 564 586 600 622 10 607 629 643 665 6 523 545 577 599 7 566 588 620 642 8 609 631 663 685 3 9 652 674 706 728 10 695 717 749 771 11 738 760 792 814 8 697 719 769 791 9 740 762 812 834 10 783 805 855 877 4 11 826 848 898 920 12 869 891 941 963 13 912 934 984 1006 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 123 „N a ON oo rn rn~ & ~C 5 r - i j o v c Ifi ift ^ ~N© v© 00— N ■ © VC o O j j j 'v© if) vC NO rn 0 0 0 0 f S ---- ■ o - v C rn r - ' If) in fn © mN hffi ,+7rz H” + s s Figure 65. Mass spectrum of imine derivatives from linear PEI-423 reacting with benzaldehyde. i~fO ' 1*101 ,fS a> ©o *r N Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. K) 4*. 1325/8 1300 1300 m/z 1200 1195/7 1100 1063/6 1020/6 1000 977/6 889/5 846/5 803/5 760/5 737 700 800 900 715/4 671/4 628/4 40/3 i 519/3 500 600 454/3 400 345/2 il i 388/2 L 300 Figure 66. Mass spectrum ofimine derivatives frombranched PEI-600 reacting withbenzaldehyde. 200 Int 0e3: 100e3 50e3- 200e3 Reproduced with permission of the copyright owner. Further reproduction prohibited without perm ission. Figure Figure 67. Mass spectrum of imine derivatives from branched PEI-1200 reacting with benzaldehyde. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 126 . . Mass spectrum of imine derivatives from branched PEI-1800 reacting with benzaldehyde. 6 8 Figure Figure Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. m/z 1100 1000 915/5 900 804/4 765/4 XLuSIm!: 722/4 700 800 700/4 657/4 612/3 94/3 4/2 573/3 530/3. 500 600 508/3 400 381/2 300 200 [M+H+HjO] [M+H] (M +N al + Figure 69. Mass spectrum ofimine derivatives from linearPEI-423 reacting with 4-fluorobenzaldehyde. 100 600e3 200e3 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ro OO J,V^...J279/5.1365/5 1107/5 u 1 1000 1100 1200 1300 m/z 1001/5 , 9 3 900 915/5 958/5 IkiU 809/4 89^/f ' ' 87/it 872, 766/4 722/4 616/3 600 700 800 612/3 H 0/ f 94/ f 573/3 679/4 530/3 500 508/3 489/2 , 400 300 [M+H+H20] [M+Na]+ [M+HJ + + [M+HJ Figure 70. Mass spectrum ofimine derivatives from branched PEI-600 reacting with 4-fluorobenzaldehyde. 100 200 0e3 In i 40e3- 20e3 60e3: Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. K> VO ... 1000 1100 1200 m/z 958/5 900 800 722/4 573/3 0/3 532/2 500 600 700 489/2 424/2 381/2 340, 300 400 275/1 200 100 Figure 71. Mass spectrum ofimine derivatives from branched PEI-1200 reacting with 4-fluorobenzaldehyde. 10006 50006 30000 40000 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 0 130w m/z 1400 9/5 1300 4-fluorobenzaldehyde. 12 with 1200 reacting 1100 1000 PEI-1800 900 branched from 800 809/4 I 766/4 ·nq. 21 72f.14 700 derivatives Mll1 659/3 .v imine ( 600 61()/3 11 of :'( . 573/3 -~ ' .. ~· :1 53()/3 500 spectrum Mass + -:- + 72. Ill \I+ I [M+NaJ [M+H] - - - - 0"300 Figure Int.-~ 5000: 10000: 150001 CJl :::J i5" CD c u;· 3 - CD 0 ;:::;: c. ;:::;: :iE ::::r 0"" :::J ::::r a c g c. i5" a CD Cil c ;:::). ..., ::::r 11 CD 0 :"""' :::J :iE - 0 (') ..., ::::r CD 0 :::J i5" ::::r - CD CJl "0 u;· ::::r 3 - CD c (') c. ;:::;: :iE CD Reproducedc. with permission of the copyright owner. Further reproduction"0 prohibited without permission. :::0 a "0 "0 '< and 10, respectively. Data in Tables 9 and 10 shared the similar results for non-cyclic molecules of imine derivatives. Imine derivatives produced from non-cyclic PEI, which contain either 4, 5, or 6 repeat units and 3 primary imine groups or 5 , 6 , or 7 repeat units and 4 primary imine groups, were demonstrated to predominate in all spectra. Non- cyclic imine derivatives originated from non-cyclic branched PEI-600,1200, or 1800, which contains 8 repeat units and 5 primary amino groups, showed to be of high intensity. It seems that the larger the molecules, the more primary amino group they contain. However, for PEI with relatively large molecules such as branched PEI-1200 and 1800, the m/z values of their imine derivatives would easily pass the maximum detection value (usually 2000 Da) by ESI-MS. So the results obtained by ESI-MS, for these large molecules, are incomplete and limited. In addition, both Table 9 and Table 10 apparently showed that some non-cyclic imine derivatives could be produced from non-cyclic PEI, which contains 5 or 7 repeat units and corresponding 4 or 5 primary imine groups. In order to match this result, the only possible formulae for these PEI molecules were shown in Figure 73, respectively. The formulae of these non-cyclic PEI molecules demonstrated that their end groups are H- and NH 2-, respectively, and the H- end is connected with the N terminal of a -NHCH 2CH2- group. This result further confirmed the assumed formula for non-cyclic PEI oligomers, which is H-(NHCH 2CH2)n-NH 2. These two end groups of non-cyclic PEI correspond to 2 primary amino groups. Therefore, the number of branches in non-cyclic PEI molecules can be determined by subtracting the number of primary amino groups by 2. Generally, for non-cyclic branched PEI, there is one branch for molecules containing 4 or 5 repeat units, and there are two branches for those containing 6 or 7 repeat units. For non-cyclic linear PEI-423, there are one or two Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 132 branches for molecules with 5 or 6 repeat units. In conclusion, there is approximately one branch for every 3 to 5 repeat units in branched PEI and one branch for every 3 to 6 repeat units in linear PEI. In these spectra, the maximum number of branches was 6 . For cyclic PEI oligomers, it is difficult to assess their degree of branching in detail because of the low yield of the cyclic imine derivative. According to the assumed formula for cyclic PEI, which is -(NHCH2CH2)n-, the number of branch and the number of primary amino group is identical. This is very different from the rule used for non- cyclic molecules. Two spectra were selected as representative for comparison. One was the spectrum of imine derivatives from branched PEI-1200 reacting with benzaldehyde, and the other was the spectrum of imine derivatives from linear PEI-423 reacting with 4- fluorobenzaldehyde. Tables 11 and 12 were generated to compare characteristic experimental m/z values of imine derivatives with theoretical m/z values, respectively. Both tables show most of the experimental m/z values and theoretical m/z values are identical, but there is a difference of 1 Da for several data due to an isotopic contribution. Table 11 shows that cyclic PEI molecules, which either contain 7 or 8 repeat units and 2 primary amino groups or contain 6 , 7, 8 , or 9 repeat units and 3 primary amino groups, predominated in the spectrum from branched PEI-1200. Therefore, mostly, there are 2 or 3 branches for cyclic PEI-1200 oligomers with 7 or 8 repeat units and 3 branches for molecules with 6 or 9 repeat units. Table 12 shows that cyclic PEI molecules, which contain 7 or 8 repeat units and 2 or 3 primary amino groups, were prevalent in the spectrum from linear PEI-423. As a result, there are mainly 2 or 3 branches for cyclic PEI-423 oligomers with 7 or 8 repeat units. Generally, there is approximately one branch for every 2 to 4 repeat units in cyclic PEI. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LO U) 560 (5.83) 737*(5.74) 825*(3.45) 868*(1.82) 9 1 1*(2.16) 693(12.16) 607 (13.89) 650(23.10) 519(22.53) 476 (24.93) BPEI-1800 from [M+H]+ [M+Naf 366 (9.28) 388 (8.18) 889*(4.23) 671(23.98) 540(15.99) 562(10.15) 628 (51.06) 452(17.02) 474(3.40) 760*(13.44; 782*(4.50) 803*(18.77; 846*(12.04; — 560 (0.56) 538(8.12) 737*(4.21) 715*(14.25; 782*(2.87) 911*(2.64) 650 (12.33) ) BPEI-1200 1 0 0 from 585 (47.55) 607(3.16) 585 (19.43) 452(11.25) 715*(19.70; 497 (24.32) 519(18.62) 497 (22.45) 846*(44.58; 868*(2.85)889*(18.83; [M+Naf [M+H]+ [M+Na]+ 693(9.20) 671(63.20) 693(7.39) 560 (0.97) 538 (0.37) 388 (7.42) 366 (2.70) 388(1.53) 650 (4.65) 628 ( 474 (2.08) 737*(4.11) 825*(2.48)*(48.79; 803 825*(3.55) 519 (42.68) 562 (10.36) 540(15.51) 562 (9.00) 476(10.36) 454 (48.03) 476(14.81) 454 (100) m/z (relative intensity) BPEI-600 from 671(100) 538 (0.38) 452 (2.84) 628 (76.02) 497 (24.45) 803 *(53.85;803 519 (100) 560(1.64) 474 (5.14) 825 *(3.44)825 782*(1.72) 760*(21.35; 782*(1.11)868*(2.42) 760*(30.89; 846*(53.83; 868*(1.96) 737*(7.42) 715*(47.50; 911*(0.76) 889*(31.44; 9 1 1*(1.56) 607(18.37) 585 (9.66) 607(1.16) LPEI-423 from [M+Hf [M+Naf [M+H]+ 538 (2.42) 452 (2.91) 803*(3.18) 671(14.25) 693(17.12) 889*(0.29) 715*(2.76) 497 (54.17 628 (35.09; 650 (44.62) 540(17.92; 562(41.39) 540(14.09) 8 6 5 8 6 7 6 5 585 (24.67; 7 760*(1.99) 8 4 366(1.34) 388 (2.97) 366 (0.93) 4 454(23.39; 476(13.65) 454 (17.29) 9 846*(1.75) 1 0 Number Number of NHCH2CH2 repeat unit repeat in PEI with benzaldehyde 3 2 4 5 Number of group in PEI primary primary amine *The m/z value was larger than the theoretical value in Table 7 by 1, which was due to the isotopic contribution. Table 9. Representative experimental m/z values ofmolecules ofimine derivatives from non-cyclic PEI reacting Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4^ U> *(15.51) *(26.81) 8 573 573 (100) 596 (2.75) 596 510(16.39) 424(18.98) 616(70.29) 1001 ) 4 4 . from BPEI-1800 4 7 ( [M+H]+ [M+Na]+ 402 402 (0.83) 657(1.99) 679 (31.38) 5 7 4 979*(8.73) 551 (19.68) 551 700(23.18) 722 (87.37) 744*(21.60; 766*(82.69) ; 88 . 6 *( 530 (100) 508(12.18) (74.29) 530 809*(9.08) 787*(17.7i; 809*(44.84) 616(14.86) (14.54) 594 1001 from from BPEI-1200 [M+H]+ [M+Na]+ 402(1.21) 424(13.14) 574 (10.50)574 (0.89) 596 488 (19.96) 510(3.99) 488 (3.36) 744*( 14.94; 766*(27.03; 744*( — 722 722 (100) (22.57) 700 (35.17) 722 510(1.12) 424 (0.94) 424 m/z (relative intensity) 530 (62.39)530 (25.22) 508 (49.13)573 (12.48) 551 (46.84) 573 616(11.41) (6.65) 594 679 (47.78) (5.04) 657 679(13.15) 872*(10.13) 850*(5.17) 872*(5.59) 850*(2.95) 872*(9.44) 809*(19.27) 787*(6.72) 766*(75.44) 958*(28.97) 936*(7.20) 958*(9.72) 936*(14.5i; 95 915*(29.09)*(9.04) 893 915*(12.78;*(9.97) 893 915*(25.34) 1001 *(11.23; 1001 979*(5.20) — 657 (5.32) 657 850*(9.41) 979*(7.82) 574(19.75) 508(11.32) (19.97)551 700 (44.08)700 512 (40.05)! 512 936*(19.83; 893 *(21.26; 893 0 ]+. 3 [M+Naf [M+H]+ [M+Na]+ 424(1.81) 616(10.28) 766*( 18.79; 744*(34.86; 766*( )“ 1 0 0 from LPEI-423 from BPEI-600 (27.58; 510(2.81) 488(1.52) [M+H]+ 402 402 (0.47) 612 612 ( 893 *(4.03) 893 915*(7.13) 787*(3.13) 809*(3.49)11.70; 787*( 936*(1.06) 958*(1.71) 700 (42.99;700 (71.87) 722 574 (39.40;574 596(1.42) *88 979*(0.17) 1001*(0.40; 657 (36.08;657 679(21.42) 508 (37.76;508 530(41.94) (56.73;551 (73.73) 573 8 5 8 7 850*(5.24) 872*(2.02) 5 8 7 744*(8.14) 6 9 6 4 6 4 10 in PEI repeat unit Number of with 4-fluorobenzaldehyde 3 5 4 2 Number o f group in PEI primary primary amine *The m/z value was larger than the theoretical value in Table 7 by which1, was due to the isotopic contribution. aThe aThe m/z value was correspondingto [M+H Table 10. Representative experimental m/zvalues ofmolecules ofimine derivatives from non-cyclic PEI reacting Reproduced with permission of the copyright owner. Further reproduction prohibited without perm ission. 135 Non-cyclic PEI molecules containing 5 repeat units and 4 primary amino groups Yh2 lfH2 (JH2 cn2 c h 2 c h 2 h — n h c h 2c h 2- n c h 2c h 2- n c h 2c h 2— n h 2 Non-cyclic PEI molecules containing 7 repeat units and 5 primary amino groups YH2 YH2 YH2 cjh2 cjh2 c h 2 c h 2 c h 2 c h 2 H—NHCH2CH2 - NCH2CH2—NCH2CH2—NCH2CH2—NH2 Figure 73. Molecular formulae of non-cyclic PEI molecules containing certain number of repeat units and primary amino groups. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 136 Table 11. Comparison of representative experimental m/z values with theoretical m/z values of molecules of imine derivatives from cyclic branched PEI-1200 reacting with benzaldehyde Number of Number of m/z of imine derivative primary repeat unit theoretical amine experimental group in PEI in PEI [M+H]+ [M+Na]+ [M+H]+ [M+Na]+ 4 261 283 — — 1 5 304 326 304 (3.26) — 6 347 369 347(10.74) — 7 390 412 390 (4.50) — 5 392 414 — — 6 435 457 435(1.95) 457(3.37) 2 7 478 500 478 (24.72) 500 (0.97) 8 521 543 521 (17.59) 543 (10.14) 6 523 545 523 (21.12) 545 (0.60) 7 566 588 566 (22.14) 588 (4.93) 3 8 609 631 609 (19.51) 631(10.96) 9 652 674 652 (17.30) 674 (8.28) 8 697 719 698*(16.08) 720* (0.85) 4 9 740 762 741*(7.95) 763 *(3.96) 10 783 805 784*(8.71) 806*(2.71) *The m/z value was larger than the corresponding theoretical value by 1, which was due to an isotopic contribution. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 137 Table 12. Comparison of representative experimental m/z values with theoretical m/z values of molecules of imine derivatives from cyclic linear PEI-423 reacting with 4-fluorobenzaldehyde Number o f Number o f m/z of imine derivative primary amine repeat unit theoretical experimental group in PEI in PEI [M+H]+ [M+Na]+ [M+H]+ [M +N af 4 385 407 385 (2.57) 407(1.27) 5 428 450 428 (9.46) 450(1.56) 6 471 493 471 (11.46) 493 (6.63) 2 7 514 536 514 (13.58) 536 (4.29) 8 557 579 557 (36.40) 579 (3.79) 9 600 622 600 (9.32) 622 (2.06) 10 643 665 643 (4.85) 665 (2.26) 6 577 599 577 (29.97) 599 (5.29) 7 620 642 621* (18.35) 643 *(11.29) 8 663 685 664* (10.90) 686 (2.97) 3 9 706 728 707*(9.85) 729*(1.05) 10 749 771 750*(4.22) 772*(0.95) 11 792 814 793 *(2.67) 815*(0.77) 8 769 791 770*(3.88) 792*(1.70) 9 812 834 813*(2.61) 835*(2.19) 10 855 877 856*(0.49) 878*(1.21) 4 11 898 920 899*(0.38) 921 *(0.33) 12 941 963 942 *(0.44) 964*(0.25) 13 984 1006 985*(0.24) 1007*(0.10) *The m/z value was larger than the corresponding theoretical value by 1, which was due to an isotopic contribution. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 8 CONCLUSIONS 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 transfer genes effectively both in vitro and in vivo for various diseases. Studies have reported that PEI’s efficiency as a gene delivery vehicle depends on its molecular weight and shape. However, commercial PEI has not been thoroughly characterized. Therefore the goal of this research is to develop methods to analyze commercial PEI, which includes linear PEI-423 and branched-600,1200, and 1800, through the use of electrospray ionization mass spectrometry (ESI-MS), size exclusion chromatography (SEC), and 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. The MWD obtained for LPEI- 423 was very different and presented a “frame-shift”. The number average molecular weights of LPEI-423, BPEI-600, BPEI-1200, and BPEI-1800 were found to be about 276, 448, 535, and 448, respectively. Most likely, the explanation for this is due to the 138 with permission of the copyright owner. Further reproduction prohibited without permission. 139 difficulty of ionization of molecules with high molecular weights or the formation of multiply-charged molecules. PEI analyzed by electrospray shows preferential ion attachment to most molecules. An ESI study with mono and divalent cations suggests that the lithium cations appear to be the optimum candidate for cationization of ESI technique. This may be due to the fact that lithium is the strongest Lewis acid. 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 our study, even if the polymer type also mattered in others. For molecules with the same structure, the retention time increases as the average molecular weight decreases. Coupling ESI-MS with SEC technique enable the separation and characterization of complex polymer community. The molecular weight distribution of PEI by ESI-MS coupled with SEC showed that PEI molecules became more charged as the molecular weight of the polymer increased. This study demonstrated the existence of two types of PEI molecules (non-cyclic and cyclic molecules). The two end groups for non-cyclic PEI are H- and NH 2-. Four apparent series of molecular weight distribution were seen in all PEI spectra. They are corresponding to protonated or sodiated non-cyclic or cyclic PEI oligomers, respectively. The differences of m/z (Am/z) between two adjacent peaks of each series were all 43 Da, which is corresponding to the molecular weight of a repeat unit (-NHCH 2CH2-). 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. ESI-MS spectra of the Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 140 imine derivatives of the linear PEI polymer show that it contains branched molecules. The imine derivatives of the branched PEI present spectra confirmed that it is branched. For non-cyclic branched PEI, mostly, there is one branch for molecules containing 4 or 5 repeat units, and there are two branches for those containing 6 or 7 repeat units. For non- cyclic linear PEI-423, there are one or two branches for molecules with 5 or 6 repeat units. Generally, there is one branch for every 3 to 5 repeat units in non-cyclic BPEI and one branch for every 3 to 6 repeat units in non-cyclic LPEI. In addition, the maximum number of branches was 6 in these spectra. For cyclic PEI oligomers, it is difficult to assess their degree of branching in detail because of the low yield of the cyclic imine derivative. For two representative samples, BPEI-1200 and LPEI-423, there is approximately one branch for every 2 to 4 repeat units in cyclic PEI. The ESI-MS result used for the assessment of the degree of branching further confirmed the assumed molecular formula for non-cyclic PEI oligomers, which is H-(NHCH 2CH2)n-NH 2. As previously mentioned, Dick and Ham were among the first to report the branching level of PEIs. They reported, under different experimental conditions, there is approximately one branch for every 3 to 3.5 nitrogen atoms within a linear chain for PEIs with different molecular weights, i.e., the ratio of primary, secondary and tertiary amino groups in the polymers is about 1:2:1. Other studies show that most commercial PEIs are branched to different degrees, and the ratios of primary, secondary and tertiary amino groups in the polymers are between 1:1:1 and 1 :2 :1 according to the degree of branching. In other words, from these studies, a conclusion can be drawn that there is one branch for every 3 to 4 repeat units. Our results are consistent with the data reported by Dick and Ham. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 141 Analysis of PEI by ESI, SEC, and the combination of both techniques can provide valuable data. However, there are a few limitations of these analytical techniques. First, the upper limit of the ESI-MS is limited only to m/z of 2000 Da. Second, the presence of sodium ion in all samples greatly complicated ESI spectra. MALDI-TOF-MS may be another MS technique that could be the optimal choice for analysis of large PEI molecules, such as PEI-1800 or higher. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. REFERENCES 1. http://www.mnsu.edu/emuseum/information/biography/klmno/mendel_gregor.html, Feb.08,2005. 2. http://www.sfaf.org/treatment/beta/b32/b32glos.html, Feb.08, 2005. 3. Crystal, R. G.; McElvaney, N. G.; Rosenfeld, M. A.; Chu, C.-S.; Mastrangeli, A.; Hay, J. G.; Broday, S. L.; Jaffe, H. A.; Elissa, N. T.; Danel, C. Nat. Gen. 1994,8, 42-51. 4. Knowles, M. R.; Hohneker, K. W.; Zhou, Z.; Olsen, J. C.; Noah, T. L.; Hu, P.; Leigh, M. W.; Engelhardt, J. R.; Edwards, L. J. et al. New Engl. J. Med. 1995, 333, 823-31. 5. Caplan, N. J.; Alton, E.; Middleton, P. G.; Dorin, J. R.; Stevenson, B. J.; Gao, X.; Durham, S. R.; Jeffery, P. K.; Hodson, M. E. et al. Nat. M ed. 1995,1, 39-46. 6 . Porteous, D.S.; Dorin, J. R.; McLachlan, G.; Davidson-Smith, H.; Davidson, H.; Stevenson, B. J.; Carothers, A. D.; Wallace, A. J.; Moralle, S. et al. Gene Ther. 1997, 4, 210-18. 7. Gill, D. R.; Southern, K. W.; Mofford, K. A.; Seddon, T.; Huang, L.; Sorgi, F.; Thompson, A.; MacVinish, L. J.; Ratcliff, R. et al. Gene Ther. 1997, 4, 199-209. 8 . Zabner, J.; Couture, L. A.; Gregory, R. J.; Graham, S. M.; Smith, A. E.; Welsh, M. J. C ell 1993, 75, 207-16. 9. Ferrari, S.; Pettenazzo, A.; Garbati, N.; Zacchello, F.; Behr, J. P.; Scarpa, M. Biochim. Biophys. Acta 1999,1 4 4 7219-25. , 10. Sinnaeve, P.; Varenne, O.; Collen, D.; Janssens, S. Cardiovasc. Res. 1999, 4 4 ,498- 506. 11. Cavazzana-Calvo, M.; Hacein-Bey, S.; de Saint Basile, G.; Gross, F.; Yvon, E.; Nusbaum, P.; Selz, F.; Hue, C.; Certain, S.; Casanova, J. -L.; Bousso, P.; Le Deist, F.; Fischer, A. Science, 2000, 288, 669-12. 142 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 143 12. Anderson, W. F. Science, 2000, 2 8 8627-29. , 13. Nabel, G. J.; Nabel, E. G.; Yang, Z. Y.; Fox, B. A.; Plautz, G. E.; Gao, X.; Huang, L.; Sh, S.; Gordon, D.; Chang, A. E. Proc. Natl. Acad. Sci. USA 1993, 9 0 ,11307-11. 14. Rubin, J.; Galanis, E.; Pitot, H. C.; Richardson, R. L.; Burch, P. A.; Charboneau, J. W.; Reading, C. C.; Lewis, B. D.; Stahl, S. et al. Gene Ther. 1997, 4, 419-25. 15. Vogelzang, N. J.; Lestingi, T. M.; Sudakoff, G.; Kradjian, S. A. Hum. Gene Ther. 1994, 5 ,1357-70. 16. Vogelzang, N. J.; Sudakoff, G.; Hersh, E. M. Proc. Am. Soc. Clin. Oncol. 1996, 15, 235. 17. McCormick, F. Nat. Rev. Cancer 2001, 1 ,130-40. 18. Kircheis, R.; Kursa, M.; Wightman, L.; Brunner, S.; Ogris, M.; Schuller, S.; Wagner, E. NATO Science Series, Series A: Life Sciences 2000, 3 2 3186-91. , 19. Bunnell, B. A.; Morgan, R. A. Clin. Microbio. Rev. 1998, 1 1 ,42-56. 20. Liang, K. W.; Huang, L. Polymeric Biomaterials, 2 ed.; Marcel Dekker, Inc.: New York, 2002; Chapter 34. 21. http://www.dddmag.com/Glossary.aspx?LETTER=T, Feb.08, 2005. 22. Huang, L.; Viroonchatapan, E. Nonviral Vectors for Gene Therapy, Academic Press: San Diego, 1999; Chapter 1. 23. Thomas, M.; Klibanov, A. M. Proc. Natl. Acad. Sci. USA 2003 , 1 0 0 , 9138-43. 24. Behr, J. P. C him ia 1997, 51, 34-36. 25. Duchler, M.; Pengg, M.; Brunner, S.; Muller, M.; Brem, G.; Wagner, E. J. Gene Med. 2001, 3, 115-24. 26. Abdallah, B.; Goula, D.; Ghorbel, M.; Guissouma, H.; Benoist, C.; Seugnet, I.; Demeneix, B. A. Ann. N. Y. Acad. Sci. 1998, 839, 87-92. 27. Harris, J. D.; Lemoine, N. R. Trends Gen. 1996, 1 2 ,400-05. 28. Miller, N.; Vile, R. FASEB J. 1995, 9, 190-99. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 144 29. Lemarchand, P.; Jaffe, H. A.; Danel, C.; Cid, M. C.; Kleinman, H. K.; Stratford- Perricaudet, L. D.; Perricaudet, M.; Pavirani, A.; Lecocq, J. P.; Crystal, R. G. Proc. Natl. Acad. Sci. USA 1992, 8 96482-86. , 30. Marshall, E. Science 2000, 2 8 62244-45. , 31. Check, E. N ature 2002, 4 2 0 ,116-18. 32. Boyce, N. N ature 2001, 414, 677-78. 33. Andreadis, S. T.; Roth, C. M.; Le Doux, J. M. et al. Biotechnol. Prog. 1999, 1 5 ,1-11. 34. Bueler, H. Bio. Chem. 1999, 3 8 0 ,613-22. 35. Kochanek, S. Hum. Gene Ther. 1999, 1 0 , 2451-59. 36. Duzgune§, N.; Simoes, S.; Pires, P.; Pedroso de Lima M. C. Polymeric Biomaterials, 2 ed.; Marcel Dekker, Inc.: New York, 2002; Chapter 35. 37. Trapnell, B. C.; Gorziglia, M. Curr. Opin. Biotechnol. 1994, 5, 617-25. 38. Templeton, N. S.; Lasic, D. D. Mol. Biotechnol. 1999, 77,175-80. 39. Miller, A. D. Hum. Gene Ther. 1990, 7, 5-14. 40. Bandyopadhyay, P. K.; Watanabe, S.; Temin, H. M. Proc. Natl. Acad. Sci. USA 1984, 81, 3476-80. 41. Ali, M.; Lemoine, N. R. Ring, C. J. Gene Ther. 1994, 7, 367-84. 42. Flotte, T. R.; Carter, B. J. Gene Ther. 1995, 2, 357-62. 43. Merlin, J. -L.; N ’ Doye, A.; Bouriez, T.; Dolivet, G. Drug News Perspect. 2002, 15, 445-51. 44. Kircheis, R.; Wightman, L.; Wagner, E. Adv. Drug Deliv. Rev. 2001, 53, 341-58. 45. Yang, N. S. Burkholder, J.; Roberts, B.; Martinell, B.; McCabe, D. Proc. Natl. Acad. Sci. USA 1990, 87, 9568-72. 46. Sun, W. H.; Burkholder, J. K.; Sun, J.; Culp, J.; Lu, X. G.; Pugh, T. D.; Ershler, W. B.; Yang, N. S. Proc. Natl. Acad. Sci. USA 1995, 9 22889-93. , 47. Wolff, J. A.; Malone, R. W.; Williams, P.; Chong, P.; Acsadi, G.; Jani, A.; Feigner, P. L. Science 1990, 2 4 71465-68. , Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 145 48. Heller, R.; Jarosteski, M.; Atkin, A.; Moradpour, D.; Gilbert, R.; Wands, J.; Nicolau, C. F E B SLett. 1996, 3 8 9225-28. , 49. Mathiesen, I. G ene Ther. 1999, 6, 508-14. 50. Zhang, G.; Vargo, D.; Budker, V.; Armstrong, N.; Knechtle, S.; Wolff, J. A. Hum. G ene Ther. 1997, 8 ,1 7 6 3 -7 2 . 51. Hickman, M. A.; Malone, R. W.; Lehmann-Bruinsma, K.; Sih, T. R.; Knoell, D.; Szoka, F. C.; Walzem, R.; Carlson, D. M. Powell, J. S. Hum. Gene Ther. 1994,5, 1477-83. 52. Song, Y. K.; Liu, F.; Liu, D. G ene Ther. 1998, 5,1531-37. 53. Sikes, M. L.; O’Malley, B. W. Jr.; Finegold, M. J.; Ledley, F. D. Hum. Gene Ther. 1994, 5, 837-44. 54. Vile, R. G.; Hart, I. R. Cancer Res. 1993, 53, 3860-64. 55. Zaitsev, S. V.; Harberland, A.; Otto, A.; Vorobev, V. I. Haller, H.; Bottger, M. Gene Ther. 1997, 4, 586-92. 56. Wagner, E.; Cotten, M.; Foisner, R.; Bimstiel, M. L. Proc. Natl. Acad. Sci. USA 1991, 8 84255-59. , 57. Li, J.; Wang, Q.; Yu, H.; Shen, F.; Wu, P. C hinese J. Biochem. Mol. Biol. 2004 , 20, 234-40. 58. Lemkine, G. F.; Bemeneix, B. A. Curr. Opin. Mol. Ther. 2001, 3 ,178-82. 59. Ledley, F. D. Hum. Gene Ther. 1995, 6, 1129-44. 60. Lollo, C. P.; Banaszczyk, M. G.; Chiou, H. C. Curr. Opin. Mol. Ther. 2000, 2, 136- 42. 61. Vaheri, A.; Pagano, J. S. Virology 1965, 2 7 ,434-46. 62. Graham, F. L.; Van Der Eb, A. J. Virology 1973, 5 2456-67. , 63. Wang, D.; Narang, A. S.; Kotb, M.; Gaber, A. O.; Miller, D. D.; Kim, S. W.; Mahato, R. I. Biomacromolecules 2003 , 3, 1197-1207. 64. Feigner, P. L.; Gadek, T. R.; Holm, M.; Roman, R.; Chan, H. W.; Wenz, M.; Northrop, J. P.; Ringold, G. M.; Danielsen, M. Proc. Natl. Acad. Sci. USA 1987, 84, 7413-17. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 146 65. Yang, J. -P.; Huang, L. G ene Ther. 1998, 5,380-87. 6 6 . Crook, K.; Stevenson, B. J.; Dubouchet, M.; Porteous, D. J. G ene Ther. 1998, 5, 137-43. 67. Wu, G. Y.; Wu, C. H. J. Biol. Chem. 1987, 2 6 24429-32. , 6 8 . Brown, M. D.; Schatzlein, A.; Brownlie, A.; Jack, V.; Wang, W.; Tetley, L.; Gray, A. I.; Uchegbu, I. F. Bioconjug. Chem. 2000, 11, 880-91. 69. Emi, N.; Kidoaki, S.; Yoshikawa, K.; Saito, H. Biochem. Biopys. Res. Commun. 1997, 2 3 1421-24. , 70. Hejazi, R.; Amiji, M. J. Control. Rel. 2003 , 89, 151-65. 71. Erbacher P.; Zou, S.; Bettinger, T.; Steffan, A. M.; Remy, J. S. Pharm. Res. 1998, 1 5 ,1332-39. 72. Kabanov, A. V.; Astafieva, I. V.; Maksimova, I. V.; Lukanidin, E. M.; Georgiev, G. P.; Kabanov, V. A. Bioconjug. Chem. 1993, 4 ,448-54. 73. van de Weteringen, P.; Chemg, J. Y.; Talsma, H.; Crommelin, D. J.; Hennink, W. E. J. Control. Rel. 1998, 5 3 ,145-53. 74. Zinselmeyer, B. H.; Mackay, S. P.; Schatzlein, A. G.; Uchegbu, I. F. Pharm. Res. 2002, 9, 960-67. 75. Haensler J.; Szoka, F. C. Jr. Bioconjug. Chem. 1993, 4, 372-79. 76. Han, S. O.; Mahato, R. I.; Sung, Y. K. Mol. Ther. 2000, 2, 302-17. 77. Godbey, W. T.; Wu, K. K.; Mikos, A. G. J. Control. Rel. 1999, 60, 149-60. 78. Boussif, O.; Lezoualch, F.; Zanta, M. A.; Mergny, M. D.; Scherman, D.; Demeneix, B.; Behr, J. -P. Proc. Natl. Acad. Sci. USA 1995, 92, 7297-7301. 79. Horn, D. Polym. Amines Ammonium Salts, Invited Lect. Contrib. Pap. Int. Symp., Pergamon Press: Oxford, Engl. 1980, p. 353. 80. Guo, W.; Lee, R. J. J. Control. Rel. 2001, 77,131-38. 81. Guerra-Crespo, M.; Charli, J. L.; Rosales-Garcia, V. H.; Pedraza-Alva, G.; Perez- Martinez, L. J. Neurosci. Methods 2003 , 1 2 7179-92. , Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 147 82. Kichler, A.; Behr, J. -P.; Erbacher, P.Nonviral Vectors for Gene Therapy, Academic Press: San Diego, 1999; Chapter 9. 83. Zhao, C.; Wang, K.; Tian, J.; Zheng, S.; Xu, Q. Acta anatomica sinica 2000, 31, 378-79. 84. Nguyen, H. -K.; Lemieux, P.; Vinogradov, S. V.; Gebhart, C. L.; Guerin, N.; Paradis, G.; Bronich, T. K.; Alakhov, V. Y.; Kabanov, A. V. G ene Ther. 2000, 7,126-38. 85. Bieber, T.; Elsasser, H. -P. Biotechniques 2001, 3 0 ,74-81. 8 6 . Godbey, W. T.; Wu, K. K.; Mikos, A. G. J. Biomed. Mater. Res. 1999, 45, 268-75. 87. Wightman, L.; Kircheis, R.; Rossler, V.; Carotta, S.; Ruzicka, R.; Kursa, M.; Wagner, E. J. Gene Med. 2001, 3, 362-72. 8 8 . Zou, S. M.; Behr, J. -P.; Goula, D.; Demeneix, B. Gene Therapy: Therapeutic Mechanisms and Strategies, Marcel Dekker, Inc.: New York, 2000; Chapter 7. 89. Zhang, C.; Yadava, P.; Hughes, J. M ethods 2004 , 3 3 ,144-50. 90. Zanta, M. A.; Boussif, O.; Adib, A.; Behr, J. P. Bioconjug. Chem. 1997, 5, 839-44. 91. Boussif, O.; Zanta, M. A.; Behr, J. P. G ene Ther. 1996, 3,1074-80. 92. Marschall, P.; Malik, N.; Larin, Z. G ene Ther. 1999, 6 ,1634-37. 93. Goula, D.; Remy, J. S.; Erbacher, P.; Wasowicz, M.; Levi, G.; Abdallah, B.; Demeneix, B. A. G ene Ther. 1998, 5, 712-17. 94. Boletta, A.; Benigni, A.; Lutz, J.; Remuzzi, G.; Soria, M. R.; Monaco, L. Hum. Gene Ther. 1997, 8, 1243-51. 95. Bragonzi, A.; Dina, G.; Villa, A.; Calori, G.; Biffi, A.; Bordignon, C.; Assael. B. M.; Conese, M. G ene Ther. 2000, 7,1753-60. 96. Coll, J. L.; Chollet, P.; Brambilla, E.; Desplanques, D.; Behr, J. P; Favrot, M. Hum. Gene Ther. 1999, 10, 1659-66. 97. Abdallah, B.; Hassan, A.; Benoist, C.; Goula, D.; Behr, J. P.; Demeneix, B. A. Hum. G ene Ther. 1996, 7, 1947-54. 98. Tang, M. X.; Szoka, F. C. Gene Ther. 1997, 4, 823-32. 99. Saegusa, T.; Ikeda, H.; Fujii, H. Macromolecules 1972, 5 ,108. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 148 100. Litt, M. H.; Levy, A.; Herz, J. J. M acrom ol. Sci. -C hem .1975, A 9, 703. 101. Gembitskii, P. A.; Chmarin, A. I.; Zhuk, D. S.Izv. Akad. NaukSSSR Ser. Khim. 1972, 8 ,1898. 102. Ham, G. E. Polym. Amines Ammonium Salts, Invited Lect. Contrib. Pap. Int. Symp., Pergamon Press: Oxford, Engl. 1980, pp 1-17. 103. Mark, H. F.; Bikales, N. M.; Overberger, C. G.; Menges, G. Encyclopedia of Polymer Science and Engineering, 2 ed.; John Wiley and Sons: New York, 1985; Vol. 1. 104. Roark, D. N. Industrial Polymers Handbook: Products, Processes, Applications, Wiley-VCH: New York, 2001, p. 1003. 105. Dick, C. R.; Ham, G. E. J. M acrom ol. Sci. -C h em .1970, .44,1301-14. 106. Rivas, B. L.; Geckeler, K. E. Advances in Polymer Science 1992, 102, 171-88. 107. Chatani, Y.; Kobatake, T.; Tadokoro, H.; Tanaka, R. Macromolecules 1982, 15, 170-76. 108. Chatani, Y.; Tadokoro, H.; Saegusa, T.; Ikeda, H. Macromolecules 1981, 14, 315-21. 109. Chatani, Y.; Kobatake, T.; Tadokoro, H. Macromolecules 1983, 1 6 ,199-204. 110. Horn, D. Polym. Amines Ammonium Salts, Invited Lect. Contrib. Pap. Int. Symp. Pergamon Press: Oxford, Engl. 1980, p. 334. 111. Hostetler, R. E.; Swanson, J. W. J. Polymer Sci. Polymer Chem. ED. 1974, 12, 29-43. 112. Lindquist, G. M.; Stratton, R. A. J. C o llo id Interface Sci. 1976, 5 545-59. , 113. Allan, G. G.; Reif, W. M. SvenskPapperstid. 1971, 7 4 ,25-31. 114. Feigner, P. L. Hum. Gene Ther. m i, 8, 511-12. 115. Suh, J.; Paik, H. J.; Hwang, B. K. Bioorg. Chem. 1994, 22, 318-27. 116. Ogris, M.; Wagner, Ernst. Somat. Cell Mol. Genet. 2002,27, 85-95. 117. Kabanov, A. V.; Szoka, F. C.; Seymour, L. W. Self-Assembling Complexes for Gene Delivery. From Laboratory to Clinical Trail. John Wiley: Chichester, 1998, pp 197-218. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 149 118. Kircheis, R.; Wightman, L.; Schreiber, A.; Robitza, B.; Rossler, V.; Kursa, M.; Wagner, E. G ene Ther. 2001,8 ,28-40. 119. Marx, K.; Ruben, G. J. Biomol. Struct. Dyn. 1986, 4 ,23-39. 120. Wolfert, M. A.; Seymour, L. W. Gene Ther. 1996, 3, 269-73. 121. Wolfert, M. A.; Schaht, E. H.; Tonceva, V.; Ulbrich, K.; Nazarova, O.; Seymore, L. W. Hum. G ene Ther. 1996, 7,2123-33. 122. Chattoraj, D. K.; Gosule, L. C.; Schellman, J. A. J. Mol. Biol. 1979, 1 2 1 ,327-37. 123. Dunlap, D. D.; Maggi, A.; Soria, M. R.; Monaco, L. Nucleic Acids Res. 1997, 25, 3095-3101. 124. Ogris, M.; Steinlein, P.; Kursa, M.; Mechtler, K.; Kircheis, R.; Wagner, E. Gene Ther. 1998, 5 ,1425-33. 125. Florea, B.; Meaney, C.; Junginger, H.; Borchard, G.; A APS PharmSci. 2002, 4, article 12. 126. Plank, C.; Mechtler, K.; Szoka, F. C.; Wagner, E. Human Gene Ther. 1996, 7, 1437-46. 127. Barth, H. G.; Mays, J. W. Modern Methods o f Polymer Characterization, John Wiley & Sons: New York, 1991. 128. Pasch, H.; Schrepp, W.MALDI-TOF Mass Spectrometry o f Synthetic Polymers Springer: Berlin, Germany, 2003. 129. Niessen, W. M. A. Liquid Chromatography-Mass Spectrometry, ed.; 2Marcel Dekker: 1999. 130. Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 6 0 ,2299-2301. 131. Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T.; Matsuo, T. Rapid Commun. Mass Spectrom. 1988, 2,151-53. 132. Murgasova, R.; Hercules, D. M. Anal. Bioanal. Chem. 2002, 373, 481-89. 133. Harris, D. C. Quantitative Chemical Analysis, 6 ed.; W. H. Freeman and Company: New York, 2003. 134. Ardrey, B. Liquid Chromatography-Mass Spectrometry: An Introduction, John Wiley & Sons: England, 2003. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 150 135. Peacock, P. M.; McEwen, C. N. Anal. Chem. 2004 , 76, 3417-28. 136. Herbert, C. G.; Johnstone, R. A. W. Mass Spectrometry Basis, CRC press: 2003. 137. Yamashita, M.; Fenn, J. B. J. Phys. Chem. 1984, 88, 4451 & 4671. 138. Montaudo, G.; Lattimer, R. P. Mass Spectrometry o f Polymers, CRC press: 2002. 139. Cole, R. B. Electrospray Ionization Mass Spectrometry, John Wiley & Sons: New York, 1997, Chapter 1. 140. http://en.wikipedia.org/wiki/Polydispersity, Aug. 01,2005. 141. http://www2.sisweb.com/mstools/isotope.htm, Aug. 01, 2005. 142. Cheremisinoff, N. P. Polymer Characterization Laboratory Techniques and A nalysis, Noyes Publications: Westwood, New Jersey, 1996. 143. http://elchem.kaist.ac.kr/vt/chem-ed/sep/lc/size-exc.htm, Aug. 01,2005. 144. Skoog, D. A.; Holler, F. J.; Nieman T. A. Principles of Instrumental Analysis, 5 ed.; Saunders College Publishing: Philadelphia, 1997, Chapter 28. 145. Prokai, L.; Simonsick, W. J. Rapid Commn. Mass Spectrom. 1993, 7, 853-56. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.