Polymeric drug delivery systems for biological antimicrobial agents

239 Pages·2017·12.22 MB·English

by  Vincent Zhu

A Thesis Submitted for the Degree of PhD at the University of Warwick

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Polymeric Drug Delivery Systems for
Biological Antimicrobial Agents

Chongyu Zhu

A thesis submitted in partial fulfilment of the requirements
of the degree of
Doctor of Philosophy in Chemistry

Department of Chemistry

University of Warwick

April 2017
Table of Contents
Table of Contents ........................................................................................................ i
List of Figures ............................................................................................................. x
List of Tables ........................................................................................................ xviii
List of Schemes ........................................................................................................ xix
Abbreviations ........................................................................................................... xx
Acknowledgements ................................................................................................ xxvi
Declaration ............................................................................................................. xxix
Abstract ................................................................................................................... xxx
Introduction .......................................................................................... 1
1.1 Biological Antimicrobial Agents ..................................................................... 1
1.1.1 MDR Gram-negative bacteria infections and colistin ............................ 2
1.1.1.1 Chemical structure and the related activity of colistin..................................... 2
1.1.1.2 Potential toxicity of colistin ............................................................................. 4
1.1.2 Fungal infection and polyene antimycotics............................................ 7
1.1.2.1 Structure and pharmaceutical behaviours of polyene antimycotics ................. 7
1.1.2.2 Current available formulation of polyene drugs to improve their solubility and
reduce toxicity ............................................................................................................. 9
1.2 Polymer-Based Delivery System for Antimicrobial Biological Agents...... 11
1.2.1 Biological agent-polymer conjugates ................................................... 12
1.2.1.1 Poly(ethylene glycol) (PEG) and PEGylation ............................................... 12
1.2.1.2 Controlled radical polymerisation (CRP) and their conjugates with
antimicrobial agents ................................................................................................... 13
1.2.1.3 Polymer conjugation methods and sites ........................................................ 14
1.2.2 Micellar delivery system of poorly soluble antimicrobial agents ........ 16
1.2.2.1 Cross-linked polymer micelle formulation .................................................... 17
1.2.3 Hydrogel formulation for water-soluble antimicrobial agents ............. 18
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1.2.3.1 Water-soluble antimicrobial agents in hydrogel formulations used in wound
care ............................................................................................................................ 20
1.3 References ....................................................................................................... 21
Approaches for the Development of Mono PEGylation on
Polymyxins .............................................................................................................. 31
2.1 Introduction .................................................................................................... 32
2.2 Results and Discussion ................................................................................... 33
2.2.1 Modification of colistin on Dab residues via reductive amination
approach.............................................................................................................. 33
2.2.1.1 Aqueous selectivity and stability analysis of imine bond via a small molecule
model ......................................................................................................................... 34
2.2.1.2 Colistin modification using small molecule aldehyde model via the schiff base
reaction ...................................................................................................................... 36
2.2.1.3 Synthesis and characterisation of the aldehyde-terminated mPEG (mPEG-
FBA) modification of colistin .................................................................................... 39
2.2.2 Antibiotic activity of Dab-modified colistin conjugates ...................... 46
2.2.3 Thiol-acrylate approach for polymyxin analogue (Pol-SH) modification
.............................................................................................................. 47
2.2.3.1 Pol-SH modification using small molecule acrylate model ........................... 48
2.2.3.2 Pol-SH modification using acrylate modified mPEG derivatives (PEGA) ... 51
2.2.4 Antibiotic activity of Pol-SH polymer conjugates and their native form
.............................................................................................................. 53
2.3 Conclusions ..................................................................................................... 54
2.4 Experimental ................................................................................................... 55
2.4.1 Materials ............................................................................................... 55
2.4.1.1 Chemicals ...................................................................................................... 55
2.4.1.2 Bacterial strains ............................................................................................. 56
2.4.2 Instruments ........................................................................................... 56
2.4.2.1 Nuclear magnetic resonance (NMR) spectroscopy ....................................... 56
Chongyu Zhu ii
2.4.2.2 Fourier transform infrared (FTIR) spectrometry ........................................... 56
2.4.2.3 Gel permeation chromatography (GPC) ........................................................ 57
2.4.2.4 Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry
(MALDI-ToF MS) ..................................................................................................... 57
2.4.2.5 Reversed phase HPLC (RP-HPLC) ............................................................... 57
2.4.2.5.1 Analytical HPLC.................................................................................. 57
2.4.2.5.2 Preparative HPLC (PREP HPLC) ...................................................... 58
2.4.2.6 Ion exchange-fast protein liquid chromatography (IE-FPLC) ....................... 59
2.4.3 Synthesis .............................................................................................. 59
2.4.3.1 Synthesis of mPEG-FBA ............................................................................... 59
2.4.3.2 Synthesis of acrylate modified poly(PEGA ) (APPEGA) .......................... 60
480
2.4.3.2.1 Synthesis of poly(PEGA ) with a hydroxyl end group (poly(PEGA )-
480 480
OH) ............................................................................................................. 60
2.4.3.2.2 Synthesis of APPEGA .......................................................................... 61
2.4.3.3 Synthesis of the colistin-FPy schiff base ....................................................... 63
2.4.3.4 Synthesis of colistin conjugates using the imine reduction approach ............ 63
2.4.3.5 Synthesis of Pol-SH conjugates using thiol-acrylate addition approach ....... 64
2.4.4 Methods ................................................................................................ 65
2.4.4.1 The stability test of the imine bond ............................................................... 65
2.4.4.2 Minimum inhibitory concentration (MIC) test .............................................. 65
2.4.4.3 Time-kill test .................................................................................................. 66
2.4.5 Others ................................................................................................... 67
2.4.5.1 MALDI-ToF MS analysis of the hydrolysis product of mPEG-FBA found in
reaction mixture ......................................................................................................... 67
2.4.5.2 1H-NMR spectra of the commerial PEGA and PEGA ......................... 68
2000 5000
2.5 References ....................................................................................................... 68
A Traceless Reversible Polymeric Colistin Prodrug to Combat
Multidrug-resistant (MDR) Gram-negative Bacteria........................................... 70
3.1 Introduction .................................................................................................... 71
3.2 Results and Discussion ................................................................................... 72
3.2.1 Synthesis of reversibly PEGylated colistin .......................................... 72
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3.2.2 Stability and degradation of the PEGylated colistin conjugates .......... 79
3.2.3 In vitro antimicrobial activity test of the PEGylated colistin conjugates.
.............................................................................................................. 82
3.2.4 In vivo antimicrobial activity and toxicity evaluation of the PEGylated
colistin conjugates .............................................................................................. 86
3.3 Conclusions ..................................................................................................... 88
3.4 Experimental ................................................................................................... 89
3.4.1 Materials ............................................................................................... 89
3.4.1.1 Chemicals ...................................................................................................... 89
3.4.1.2 Bacterial strains ............................................................................................. 89
3.4.2 Instruments ........................................................................................... 90
3.4.2.1 MALDI-ToF MS ........................................................................................... 90
3.4.2.2 GPC ............................................................................................................... 90
3.4.2.3 HPLC ............................................................................................................. 91
3.4.2.3.1 Analytical HPLC.................................................................................. 91
3.4.2.3.2 PREP HPLC ........................................................................................ 91
3.4.3 Synthesis .............................................................................................. 92
3.4.3.1 Synthesis of Boc -colistin .............................................................................. 92
5
3.4.3.2 Synthesis of Boc -col-aaPEG (x = 1,2) ........................................................ 93
5 x
3.4.3.3 Synthesis of col-aaPEG (x = 1,2) ................................................................. 93
x
3.4.4 Methods ................................................................................................ 94
3.4.4.1 Protocol for in vitro release of PEGylated colistin prodrugs ......................... 94
3.4.4.2 Disk diffusion assay ....................................................................................... 95
3.4.4.3 MIC test ......................................................................................................... 96
3.4.4.4 Time-kill test .................................................................................................. 96
3.4.4.5 In vivo efficacy study using a neutropenic mouse thigh infection model ..... 97
3.4.4.6 Measurement of nephrotoxicity in mice ........................................................ 98
3.5 References ....................................................................................................... 99
A Poly(PEGA480) Colistin Prodrug for Antimicrobial Treatment ....
............................................................................................................ 101
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4.1 Introduction .................................................................................................. 102
4.2 Results and Discussion ................................................................................. 103
4.2.1 Design of the initiator linker for colistin conjugation ........................ 103
4.2.2 Polymerisation of PEGA480 from colistin intiator .............................. 105
4.2.3 Hydrolysis test of col-PPEGA conjugates ......................................... 109
4.2.4 Disk diffusion assay for in vitro antimicrobial activity evalutaion of the
col-PPEGA conjugates with various DPs ......................................................... 111
4.2.5 Quantitative in vitro antimicrobial activity evaluation of the col-PPEGA
conjugates with various DPs via broth microdilution method ......................... 112
4.2.6 Kinetics of in vitro antimicrobial activity of the col-PPEGA conjugates
with various DPs ............................................................................................... 113
4.3 Conclusions ................................................................................................... 115
4.4 Experimental ................................................................................................. 116
4.4.1 Chemicals ........................................................................................... 116
4.4.2 Bacterial strains .................................................................................. 116
4.4.3 Instruments ......................................................................................... 116
4.4.3.1 NMR ............................................................................................................ 116
4.4.3.2 MALDI-ToF MS ......................................................................................... 116
4.4.3.3 GPC ............................................................................................................. 117
4.4.3.4 HPLC ........................................................................................................... 117
4.4.3.5 FTIR............................................................................................................. 118
4.4.3.6 Electrospray ionisation-mass spectroscopy (ESI-MS) ................................ 118
4.4.4 Methods .............................................................................................. 118
4.4.4.1 Disk diffusion assay ..................................................................................... 118
4.4.4.2 MIC test ....................................................................................................... 119
4.4.4.3 Time-kill test ................................................................................................ 119
4.4.5 Synthesis ............................................................................................ 120
Chongyu Zhu v
4.4.5.1 Synthesis of initiator precursor, 2-((2-bromo-2-methylpropanoyl)oxy)acetic
acid (BMPAA) ......................................................................................................... 120
4.4.5.2 Synthesis of Boc -colistin-ini ..................................................................... 121
5 2
4.4.5.3 Procedure for the polymerisation of MA using colistin initiator Boc -colistin-
5
ini ............................................................................................................................ 121
2
4.4.5.4 Polymerisation of PEGA of different DPs using colistin initiator Boc -
480 5
colistin-ini ............................................................................................................... 122
2
4.4.5.5 Deprotection of Boc groups from Boc -col-PPEGA conjugates ................. 123
5
4.4.5.6 Protocol for in vitro releasability of col-PPEGA prodrugs .......................... 124
4.5 References ..................................................................................................... 124
A Hydrogel Based Localised Release of Colistin for Antimicrobial
Treatment of Burn Wound Infection ................................................................... 126
5.1 Introduction .................................................................................................. 127
5.2 Results and Discussion ................................................................................. 128
5.2.1 Initial attempts to form a colistin/DF-PEG hydrogel ......................... 128
5.2.2 The formation and characterisation of colistin-loaded hydrogels with
different cross-linker ratios ............................................................................... 129
5.2.3 Colistin release from the hydrogels.................................................... 132
5.2.4 In vitro bio-activity evaluation of colistin-loaded hydrogels ............. 134
5.2.5 In vivo animal ‘burn’ infection model test of colistin-loaded hydrogels .
............................................................................................................ 137
5.3 Conclusions ................................................................................................... 140
5.4 Experimental ................................................................................................. 141
5.4.1 Materials ............................................................................................. 141
5.4.1.1 Chemicals .................................................................................................... 141
5.4.1.2 Bacterial strains ........................................................................................... 141
5.4.2 Instruments ......................................................................................... 142
5.4.2.1 NMR ............................................................................................................ 142
Chongyu Zhu vi
5.4.2.2 HPLC ........................................................................................................... 142
5.4.2.3 Rheometer .................................................................................................... 142
5.4.3 Synthesis ............................................................................................ 143
5.4.3.1 Synthesis of difunctionalized PEG (DF-PEG) ............................................. 143
5.4.3.2 Synthesis of colistin-hydrogels .................................................................... 143
5.4.4 Methods .............................................................................................. 144
5.4.4.1 Rheology analysis ........................................................................................ 144
5.4.4.2 In vitro release ............................................................................................. 145
5.4.4.3 Disk diffusion assay ..................................................................................... 146
5.4.4.4 Time kill test ................................................................................................ 147
5.4.4.5 Animal ‘burn’ infection model test .............................................................. 147
5.5 Reference ....................................................................................................... 149
PEGylation Nystatin Prodrug for Antifungal Treatment ............ 150
6.1 Introduction .................................................................................................. 151
6.2 Results and Discussion ................................................................................. 152
6.2.1 Evaluation of the imine bond under physiological conditions through a
small molecule model ....................................................................................... 152
6.2.2 Conjugation of mPEG-FBA onto Nys through a labile imine bond .. 154
6.2.3 The antifungal activity and the potential cytotoxicity of the labile
PEGylated Nys (mPEG-FBA Nys) .................................................................. 161
6.3 Conclusions ................................................................................................... 163
6.4 Experimental ................................................................................................. 164
6.4.1 Materials ............................................................................................. 164
6.4.1.1 Chemicals .................................................................................................... 164
6.4.2 Cell line and fungal species................................................................ 164
6.4.3 Instruments ......................................................................................... 164
6.4.3.1 NMR ............................................................................................................ 164
6.4.3.2 HPLC ........................................................................................................... 165
Chongyu Zhu vii
6.4.3.3 Ultraviolet-visible (UV-Vis) spectroscopy .................................................. 166
6.4.4 Synthesis ............................................................................................ 166
6.4.4.1 Synthesis of mPEG-FBA Nys conjugate ..................................................... 166
6.4.4.2 Synthesis of reduced mPEG-FBA Nys conjugate ....................................... 166
6.4.5 Methods .............................................................................................. 167
6.4.5.1 Small molecule model for 1H-NMR analysis .............................................. 167
6.4.5.2 Cell viability assay ....................................................................................... 168
6.4.5.3 Aqueous solubility of Nys and mPEG-Nys conjugate determined by UV-Vis
................................................................................................................................. 168
6.5 References ..................................................................................................... 169
Micellar Formulation for the Solubility Improvement of Nystatin
and Its Potential Target Delivery.......................................................................... 170
7.1 Introduction .................................................................................................. 171
7.2 Results and Discussion ................................................................................. 172
7.2.1 A small molecule model for the boronate linker through the diol on Nys
............................................................................................................ 172
7.2.2 Synthesis of the block copolymer containing PBA units on the side
chains ............................................................................................................ 173
7.2.2.1 Synthesis of the monomer containing PBA unit for polymerisation ........... 173
7.2.2.2 Synthesis of the PBzA homopolymer using copper-mediated photoinduced
living radical polymerisation (CP-LRP) .................................................................. 175
7.2.2.3 Synthesis of an amphiphilic block copolymer containing PBA units via CP-
LRP .......................................................................................................................... 178
7.2.2.4 Recovery of the boronic acid groups from the PB A PPEGA block
pin 20 x
copolymer ................................................................................................................ 181
7.2.3 UV analysis for the interactions between the amphiphilc block
copolymers and Nys .......................................................................................... 183
7.2.3.1 Investigation on the increased solubility of Nys in the presence of
PB A PPEGA or PBzA PPEGA through the UV spectroscopy ...................... 183
acid 20 x 20 x
Chongyu Zhu viii