Functional Co-substituted Poly[(amino acid ester)phosphazene]

144 Pages·2017·9.72 MB·English

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Amanda L. Baillargeon, The University of Western Ontario
Supervisor: Dr. Kibret Mequanint, The University of Western Ontario
A thesis submitted in partial fulfillment of the requirements for the Master of Engineering
Science degree in Biomedical Engineering
© Amanda L. Baillargeon 2014
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FUNCTIONAL CO-SUBSTITUTED POLY[(AMINO ACID
ESTER)PHOSPHAZENE] BIOMATERIALS

(Thesis format: Monograph)

by

Amanda Lee Baillargeon

Graduate Program in Biomedical Engineering

A thesis submitted in partial fulfillment
of the requirements for the degree of
Master of Engineering Science

The School of Graduate and Postdoctoral Studies
The University of Western Ontario
London, Ontario, Canada

© Amanda Lee Baillargeon 2014
Abstract

The development of new and improved biomaterials is essential for tissue engineering and
regenerative medicine applications. Amino acid-based polyphosphazenes are being explored
as scaffold materials for tissue engineering applications due to their non-toxic degradation
products and tunable material properties. This work focuses on the synthesis of non-
functional and novel functional poly[(amino acid ester)phosphazene]s using a facile method
of thermal ring opening polymerization followed by one-pot room temperature substitution.
The family of polyphosphazenes developed in this work is based on L-alanine (PP-A’s), L-
phenylalanine (PP-F’s), and L-methionine (PP-M’s) with L-glutamic acid imparting the
functionality. Characterization of these materials demonstrated that the one-pot substitution
was successful in developing mono- and co-substituted poly[(amino acid
ester)phosphazene]s. Cytotoxicity studies on two-dimensional films showed these materials
to be compatible with NIH-3T3 fibroblasts over the five-day study. The PP-F’s also showed
significantly enhanced cell viability over tissue culture polystyrene at day 1 (p<0.001) and
day 3 (p<0.01). As a proof of concept, electrospinning of PP-A blended with
100
poly(caprolactone) (PCL) (50% PP-A /50% PCL) was conducted to fabricate three-
100
dimensional scaffolds. Overall, this study has shown that poly[(amino acid
ester)phosphazene]s can be synthesized with good yields and better reaction conditions,
leading to materials with promising cytocompatibility for use in biomedical applications.

Keywords
Polyphosphazenes, poly[(amino acid ester)phosphazene]s, biomaterials, biodegradable
polymers, tissue engineering, scaffolds, electrospinning

ii
Acknowledgments
I would like to acknowledge my supervisor, Dr. Kibret Mequanint, for his continued support
and guidance throughout my Master’s degree. I really appreciated the amount of time he
designated weekly to meet with his students and ensure that they were progressing well with
their research; it definitely pushed me to always have new findings to share with him. I
would also like to thank the Natural Sciences and Engineering Research Council (NSERC) of
Canada and the Strategic Training Program in Vascular Research supported by the Canadian
Institutes for Health Research (CIHR) for the financial aid and skills training.
I would like to thank Dr. Darryl Knight and Dr. Kalin Panev for their help in synthetic
chemistry problems, experimental setup, and elucidation of NMR spectra. I also would like
to acknowledge Dr. Shigang Lin for his help with the design and implementation of the cell
culture experiments. I would like to thank Ryan Guterman of the Ragogna research group
for his help in running TGA, DSC, and FTIR analyses. Lastly, I would like to thank Somiraa
Said for her guidance with the electrospinning and planning of cell culture studies. All of the
helpful feedback and discussions from other lab members (especially Tierney Deluzio) were
greatly appreciated.
Finally, I would like to thank my mother and grandparents for their continued love and
support throughout my study. I would not have been able to push through these stressful two
years without you by my side.
Table of Contents

Abstract ............................................................................................................................... ii 
Acknowledgments.............................................................................................................. iii 
Table of Contents ............................................................................................................... iv 
List of Tables ................................................................................................................... viii 
List of Figures ..................................................................................................................... x 
List of Appendices ........................................................................................................... xvi 
List of Abbreviations ...................................................................................................... xvii 
Chapter 1 ............................................................................................................................. 1 
1  Scope and Thesis Outline ............................................................................................... 1 
1.1  Scope ....................................................................................................................... 1 
1.2  Thesis Outline ......................................................................................................... 2 
Chapter 2 ............................................................................................................................. 3 
2  Literature Review, Objectives, and Rationale ................................................................ 3 
2.1  Tissue Engineering Methodology ........................................................................... 3 
2.2  Requirements of Tissue Engineering Scaffolds ...................................................... 5 
2.2.1  Natural Polymers ........................................................................................ 7 
2.2.2  Biodegradable Synthetic Polymers ............................................................. 8 
2.2.3  Biomimetic Materials .................................................................................. 9 
2.2.4  Scaffold Fabrication .................................................................................. 10 
2.3  Synthesis of Polyphosphazenes ............................................................................ 11 
2.3.1  Polymerization to Poly[(dichloro)phosphazene] ...................................... 13 
2.3.2  Macromolecular Substitution of Poly[(dichloro)phosphazene] with
Organic Nucleophiles................................................................................ 18 
2.4  Suitability of Polyphosphazene Biomaterials ....................................................... 20 
iv
2.4.1  In Vitro and In Vivo Biocompatibility ...................................................... 21 
2.4.2  Biodegradability ........................................................................................ 23 
2.4.3  Mechanical Properties ............................................................................... 28 
2.5  Objectives and Rationale ...................................................................................... 29 
Chapter 3 ........................................................................................................................... 32 
3  Materials and Methods ................................................................................................. 32 
3.1  Thermal Ring Opening Polymerization (TROP) .................................................. 32 
3.1.1  Materials ................................................................................................... 32 
3.1.2  Equipment ................................................................................................. 32 
3.1.3  Method ...................................................................................................... 33 
3.2  Macromolecular Substitution of Non-Functional Amino Acid Esters ................. 35 
3.2.1  Materials ................................................................................................... 35 
3.2.2  Sample Nomenclature ............................................................................... 35 
3.2.3  Methods..................................................................................................... 35 
3.3  Preparation of Glutamic Acid Ethyl Ester (E*) ..................................................... 40 
3.3.1  Materials ................................................................................................... 40 
3.3.2  Methods..................................................................................................... 41 
3.4  Macromolecular Co-substitution of Non-Functional Amino Acids with Functional
Glutamic Acid ....................................................................................................... 43 
3.4.1  Materials ................................................................................................... 43 
3.4.2  Methods..................................................................................................... 43 
3.5  Conjugation of a Model Compound to the Functional Polyphosphazenes ........... 48 
3.5.1  Materials ................................................................................................... 48 
3.5.2  Methods..................................................................................................... 48 
3.6  Material Characterization ...................................................................................... 49 
3.6.1  Nuclear Magnetic Resonance Spectroscopy (NMR) ................................ 49 
v
3.6.2  Fourier-Transform Infrared Spectroscopy (FTIR) .................................... 49 
3.6.3  Thermogravimetric Analysis (TGA) ......................................................... 50 
3.6.4  Differential Scanning Calorimetry (DSC) ................................................ 50 
3.6.5  Gel Permeation Chromatography (GPC) .................................................. 50 
3.7  Cytotoxicity Studies of Polymeric Films Using MTT Assays.............................. 51 
3.7.1  Thin Film Preparation ............................................................................... 51 
3.7.2  Fibroblast (NIH-3T3) Cell Study .............................................................. 51 
3.7.3  MTT Assay Protocol ................................................................................. 51 
3.8  Cell Morphology and Adhesion Studies on Polymer Films Using Confocal
Microscopy ........................................................................................................... 52 
3.8.1  Thin Film Preparation ............................................................................... 52 
3.8.2  Cell Fixation, Staining, and Imaging ........................................................ 52 
3.9  Electrospinning of Polyphosphazenes and Polyphosphazene/ Polycaprolactone
Blends ................................................................................................................... 53 
3.9.1  Electrospinning Parameters ...................................................................... 53 
3.9.2  Scanning Electron Microscopy (SEM) Imaging of Electrospun Fibers ... 54 
Chapter 4 ........................................................................................................................... 55 
4  Results and Discussion ................................................................................................. 55 
4.1  Optimization of Thermal Ring Opening Polymerization ...................................... 55 
4.2  Comparison of the Three Macromolecular Substitution Methods Using Non-
Functional Amino Acid Esters .............................................................................. 57 
4.2.1  FTIR, 1H NMR, and 31P NMR Characterization ...................................... 58 
4.2.2  TGA, DSC, and GPC Characterization ..................................................... 70 
4.3  Esterification of Fmoc- and Boc-Glu(OBzl)-OH ................................................. 78 
4.4  TFA Deprotection of Boc-Glu(OBzl)-OEt ........................................................... 81 
4.5  Macromolecular Co-Substitution of Functional and Non-Functional Amino Acid
Esters ..................................................................................................................... 82 
4.5.1  FTIR, 1H NMR, and 31P NMR Characterization ...................................... 83 
vi
4.5.2  TGA, DSC, and GPC Characterization ..................................................... 89 
4.6  Hydrogenation of Glutamic Acid Co-Substituted Polyphosphazenes for Model
Compound Conjugation ........................................................................................ 95 
4.7  Cytotoxicity and Cell Proliferation on Non-Functional and Functional Two-
Dimensional Polyphosphazene Films ................................................................... 96 
4.8  Electrospinning of Poly[(amino acid ester)phosphazene]s and PCL Blend
Materials ............................................................................................................. 100 
4.9  Novelty of This Work ......................................................................................... 102 
5  Conclusions and Future Directions ............................................................................ 103 
5.1  Conclusions ......................................................................................................... 103 
5.2  Future Directions ................................................................................................ 105 
References ....................................................................................................................... 106 
Appendices ...................................................................................................................... 117
Curriculum Vitae ............................................................................................................ 122 
vii
List of Tables
Table 2.1: Summary of in vitro degradation studies of poly[(amino acid ester)phosphazene]s
and their co-substituted polyphosphazenes. The ester refers to the chain attached to the
carboxyl terminus of the amino acid. The detailed degradation profiles can be found in
the cited references. .......................................................................................................... 25
Table 4.1: Summary of the time-varied polymerizations of hexachlorocyclotriphosphazene
(trimer) to poly[(dichloro)phosphazene] (linear precursor) with their respective yields and
extent of conversion. ......................................................................................................... 57
Table 4.2: Summary table of the yields, 1H NMR, 31P NMR, and FTIR analyses of PP-A ,
100
PP-F , and PP-M synthesized by the three methods (LT 2S = low temperature two-
100 100
step reaction, 1P RT = one-pot room temperature reaction, and RT 2S = room
temperature two-step reaction). Note: As described in the methods section, the mass of
poly[(dichloro)phosphazene] (PDCP) is approximate since it cannot be dried fully and
therefore the percentage yields shown are underestimated since yield calculations are
based on PDCP as the limiting reagent. ........................................................................... 58
Table 4.3: Summary of thermal data from TGA and DSC analysis for PP-A , PP-F , and
100 100
PP-M materials synthesized via either the 1P RT or RT 2S method. The decomposition
100
temperatures are presented as the peak decomposition temperatures rather than onset,
meaning they are the values of maximal rate of change in weight % for all phases of the
decomposition (i.e. maxima of the derivative of weight % plot)...................................... 70
Table 4.4: GPC analysis of PP-A , PP-F , and PP-M materials using triple detection
100 100 100
and refractive index detection. A (-) indicates that analysis using that detection method
was not possible and the relative molecular weights are calculated based on polystyrene
standards. .......................................................................................................................... 75
Table 4.5: Summary of percent yields, 1H NMR, 31P NMR and FTIR characterization of the
co-substituted poly[(amino acid ester)phosphazene]s. ..................................................... 84
Table 4.6: Summary of thermal data from TGA and DSC analysis for PP-A E* , PP-F E* ,
90 10 90 10
and PP-M E* polymers. The decomposition temperatures are presented as the peak
90 10
viii
decomposition temperatures rather than onset, meaning they are the values of maximal
rate of change in weight % for all phases of the decomposition (i.e. maxima of the
derivative of weight % plot). ............................................................................................ 90
Table 4.7: GPC analysis of PP-A E* , PP-F E* , and PP-M E* materials using triple
90 10 90 10 90 10
detection and refractive index detection. A (-) indicates that analysis using that detection
method was not possible and the relative molecular weights are calculated based on
polystyrene standards. ....................................................................................................... 94

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