Synthesis, Antibacterial Activity and Effects on Molecular Loading of Poly(vinyl benzoate)

300 Pages·2015·8.75 MB·English

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USF Tampa Graduate Theses and Dissertations USF Graduate Theses and Dissertations
7-6-2012
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Ryan Cormier
University of South Florida, [email protected]
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Cormier, Ryan, "N-Acyl Ciprofloxacins: Synthesis, Antibacterial Activity and Effects on Molecular Loading
of Poly(vinyl benzoate) Nanoparticles" (2012). USF Tampa Graduate Theses and Dissertations.
https://digitalcommons.usf.edu/etd/4018
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N-Acyl Ciprofloxacins: Synthesis, Antibacterial Activity and Effects on Molecular

Loading of Poly(vinyl benzoate) Nanoparticles

by

Ryan Cormier

A dissertation submitted in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy
Department of Chemistry
College of Arts and Sciences
University of South Florida

Major Professor: Edward Turos, Ph.D.
Jon Antilla, Ph.D.
Bill Baker, Ph.D.
Roman Manetsch, Ph.D.

Date of Approval:
July 6, 2012

Keywords: PVB, MRSA, Ciprofloxacin, Bartonella, Staphylococcus aureus

Copyright © 2012, Ryan Cormier
TABLE OF CONTENTS

LIST OF TABLES ...............................................................................................................v

LIST OF FIGURES .......................................................................................................... vii

LIST OF ABBREVIATIONS .......................................................................................... xiii

ABSTRACT .......................................................................................................................xv

CHAPTER ONE: THE USE OF POLYMER NANOPARTICLES AS
ANTIBIOTIC DELIVERY SYSTEMS OR AS ANTIBACTERIAL
MATERIALS

1.1 Introduction ..................................................................................................1
1.1.1 Preparation of Nanoparticles ............................................................1
1.1.1.1 Emulsion Evaporation Technique .....................................2
1.1.1.2 Nanoprecipitation ..............................................................3
1.1.1.3 Emulsion Polymerization ..................................................3
1.1.1.4 Ionotropic Gelation ...........................................................4
1.1.2 Types of Drug Loading ....................................................................5
1.2 Poly(lactic-co-glycolic acid) (PLGA) Nanoparticles ...................................7
1.2.1 Aminoglycosides/Glycopeptides .....................................................8
1.2.1.1 Gentamicin Loaded PLGA Nanoparticles ........................8
1.2.1.2 Streptomycin and Vancomycin Loaded PLGA
Nanoparticles ..................................................................10
1.2.1.3 Teicoplanin Loaded PLGA Nanoparticles ......................13
1.2.2 β-Lactams .......................................................................................15
1.2.2.1 Nafcillin Loaded PLGA Nanoparticles ...........................15
1.2.3 Fluoroquinolones ...........................................................................17
1.2.3.1 Ciprofloxacin Loaded PLGA Nanoparticles ...................17
1.2.3.2 Levofloxacin Loaded PLGA Nanoparticles ....................20
1.2.4 Macrolides......................................................................................22
1.2.4.1 Azithromycin Loaded PLGA Nanoparticles ...................22
1.2.4.2 Clarithromycin Loaded PLGA Nanoparticles ................25
1.2.5 Rifamycins .....................................................................................28
1.2.5.1 Rifampin/Rifampicin Loaded PLGA
Nanoparticles ..................................................................28
1.2.6 Other Antibiotics ............................................................................32
1.2.6.1 Carvacrol Loaded PLGA Nanoparticles .........................32
1.2.6.2 Chloramphenicol Loaded PLGA Nanoparticles .............33
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1.2.6.3 Violacein Loaded PLGA Nanoparticles .........................35
1.3 Polyacrylate Nanoparticles ........................................................................37
1.3.1 Poly(alkyl cyanoacrylate) Nanoparticles .......................................37
1.3.1.1 Aminoglycosides/Polypeptides .......................................38
1.3.1.1.1 Actinomycin D Loaded PACA
Nanoparticles ..................................................38
1.3.1.1.2 Gentamicin Loaded PACA
Nanoparticles ..................................................39
1.3.1.1.3 Netilmicin Loaded PACA
Nanoparticles ..................................................41
1.3.1.2 β-Lactams ........................................................................43
1.3.1.2.1 Amoxicillin Loaded PACA
Nanoparticles ..................................................43
1.3.1.2.2 Ampicillin Loaded PACA
Nanoparticles ..................................................45
1.3.1.3 Fluoroquinolones ............................................................52
1.3.1.3.1 Ciprofloxacin Loaded PACA
Nanoparticles ..................................................52
1.3.1.3.2 Moxifloxacin Loaded PACA
Nanoparticles ..................................................56
1.3.2 Poly(styrene-co-butyl acrylate) Nanoparticles ..............................57
1.3.2.1 β-Lactams ........................................................................57
1.3.2.1.1 N-Thiolated β-Lactam Loaded
Polyacrylate Nanoparticles .............................57
1.3.2.1.2 Penicillin Loaded Polyacrylate
Nanoparticles ..................................................61
1.3.2.3 Carbohydrate Derivatized Penicillin,
Ciprofloxacin, and N-Thiolated β-Lactam Loaded
Polyacrylate Nanoparticles .............................................64
1.4 Chitosan Based Nanoparticles ...................................................................66
1.4.1 β-Lactams .......................................................................................67
1.4.1.1 Amoxicillin Loaded Chitosan Nanoparticles ..................67
1.4.1.2 Ceftriaxone Loaded Chitosan Nanoparticles ..................71
1.4.2 Fluoroquinolones ...........................................................................73
1.4.2.1 Ciprofloacin Loaded Chitosan Nanoparticles .................73
1.4.3 Poly/Glycopeptides ........................................................................74
1.4.3.1 Nisin Loaded Chitosan Nanoparticles ............................74
1.4.3.2 Vancomycin Loaded Chitosan Nanoparticles .................75
1.5 Conclusions ................................................................................................76

CHAPTER 2: SYNTHESIS AND ANTIBACTERIAL ACTIVITY OF N-ACYL
CIPROFLOXACINS

2.1 Introduction ................................................................................................77
2.2 Synthesis ....................................................................................................79
2.3 Antibacterial Activity .................................................................................83
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2.3.1 N-Acyl Ciprofloxacin Antibacterial Activity Against
Gram-Positive Bacteria ..................................................................83
2.3.1.1 Staphyloccocus aureus ....................................................84
2.3.1.2 Bacillus anthracis and Enterococcus faecalis ................87
2.3.2 N-Acyl Ciprofloxacin Antibacterial Activity Against
Gram-Negative Bacteria ................................................................92
2.3.2.1 Bartonella .......................................................................92
2.3.2.2 Escherichia coli ............................................................103
2.3.3 N-Acyl Ciprofloxacins and Eukaryotic Cells ..............................105
2.3.4 Mechanism of Action of N-Acyl Ciprofloxacins.........................106
2.4 Conclusions and Future Directions ..........................................................108

CHAPTER 3: THE USE OF N-ACYL CIPROFLOXACINS IN STUDYING
THE EFFECTS OF LIPOPHILICITY ON MOLECULAR
CARRIER PROPERTIES OF POLY(VINYL BENZOATE)
NANOPARTICLES

3.1 Introduction ..............................................................................................112
3.1.1 Synthesis of PVB Nanoparticles ..................................................114
3.1.2 Physical Characteristics of PVB Nanoparticles ...........................115
3.1.3 Molecular Loading and In vitro Molecular Release Studies .......117
3.1.4 Studies on the Biodegradation Properties of PVB
Nanoparticles In vitro ..................................................................119
3.1.5 Nanoparticle Stability in Rat Blood Serum .................................120
3.2 In vitro Cytotoxicity .................................................................................121
3.3 Use of N-Acyl Ciprofloxacins to Determine the Effects of Varying
Lipophilicity on Drug Loading and Drug Release ...................................122
3.3.1 Synthesis of N-Acyl Ciprofloxacin Loaded Nanoparticles .........122
3.3.2 Physical Characteristics of N-Acyl Ciprofloxacin Loaded
Nanoparticles ...............................................................................124
3.3.3 N-Acyl Ciprofloxacin Drug Loading ...........................................128
3.3.4 In vitro Release of N-Acyl Ciprofloxacins From PVB
Nanoparticles ...............................................................................132
3.3.5 Antimicrobial Activity .................................................................134
3.4 Conclusions and Future Directions ..........................................................135

CHAPTER 4: CONCLUSIONS ......................................................................................137

CHAPTER 5: MATERIALS AND METHODS

5.1 Synthetic Procedures for N-Acyl Ciprofloxacins ....................................141
5.1.1 Materials ......................................................................................141
5.1.2 General Methods for the Synthesis of N-Acyl
Ciprofloxacins ..............................................................................141
5.2 Antimicrobial Testing for N-Acyl Ciprofloxacins ...................................151
5.2.1 Bacterial Growth Conditions .......................................................151
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5.2.2 Disk Diffusion Assays .................................................................151
5.2.3 Minimum Inhibitory Concentration Assays.................................152
5.2.4 Spontaneous Mutation Frequencies .............................................153
5.2.5 Sequence Analysis of Quinolone Binding Domains ....................153
5.2.6 Intracellular Activity Assay .........................................................154
5.2.7 DNA Gyrase Assay ......................................................................154
5.3 Preparation and Characterization of Poly(vinyl benzoate)
Nanoparticles ...........................................................................................155
5.3.1 Materials ......................................................................................155
5.3.2 Preparation of Nanoparticles ........................................................155
5.3.3 Determination of Particle Size and Zeta Potential .......................156
5.3.4 Determination of Drug Loading ...................................................156
5.3.5 In Vitro Drug Release Study ........................................................156

REFERENCES ................................................................................................................158

APPENDIX 1: ................................................................................................................166

APPENDIX 2: .................................................................................................................275
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LIST OF TABLES

Table (1.1): In vivo assays determining efficacy of streptomycin loaded
nanoparticles against M. tuberculosis ........................................................12

Table (1.2): In vivo assays determining efficacy of vancomycin loaded
nanoparticles (NP1, NP2, NP3) against S. pneumonia. .............................13

Table (1.3): Range of MIC values for ciprofloxacin loaded nanoparticles, in
µg/ml. .........................................................................................................18

Table (1.4): Drug loading results shown as % of drug in 100 mg of
nanoparticle. ...............................................................................................23

Table (1.5) Encapsulation efficiencies and particle sizes of nanoparticle
formulations used. ......................................................................................24

Table (1.6): Effects of varying PEG on particle size and amoxicillin association
of nanoparticles ..........................................................................................44

Table (1.7): In vivo mice study evaluating the efficacy of ampicillin loaded
nanoparticles ..............................................................................................44

Table (1.8): Particle sizes of PIBCA and PIHCA nanoparticles ...................................47

Table (1.9): Particle size and ampicillin loading for liposomes and PIHCA
nanoparticles ..............................................................................................48

Table (1.10): Particle size and drug loading for chitosan-glutamate and fucose-
conjugated chitosan-glutamate nanoparticles ............................................68

Table (1.11): Number of viable colonies and in vivo clearance rate of mice
infected with H. pylori. ..............................................................................70

Table (1.12): Encapsulation efficiencies and particle sizes of ceftriaxone loaded
chitosan nanoparticles ................................................................................72

Table (2.1): Zones of inhibition for N-acyl ciprofloxacins against MRSA ...................84

Table (2.2): Minimum inhibition concentrations against MRSA and MSSA ...............86

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Table (2.3): Spontaneous mutation frequency results for N-acyl ciprofloxacins ..........86

Table (2.4): Zones of inhibition for N-acyl ciprofloxacins against B. anthracis ...........88

Table (2.5): Zones of inhibition for N-acyl ciprofloxacins against E. faecalis .............90

Table (2.6): Zones of inhibition for N-acyl ciprofloxacins against B. henselae ............93

Table (2.7): Zones of inhibition for N-acyl ciprofloxacins against B. quintana ...........95

Table (2.8): Zones of inhibition for N-acyl ciprofloxacins against B.
elizabethae .................................................................................................97

Table (2.9): Zones of inhibition for N-acyl ciprofloxacins against B. vinsonii .............99

Table (2.10): Minimum inhibition concentrations for N-acyl ciprofloxacins
against B. henselae ...................................................................................101

Table (2.11): Zones of inhibition for N-acyl ciprofloxacins against E. coli ..................104

Table (2.12): Zones of inhibition for N-acyl ciprofloxacins against S. cerevisiae ........106

Table (3.1): Average sizes of PVB nanoparticles ........................................................115

Table (3.2): Zeta potentials for PVB nanoparticles .....................................................117

Table (3.3): Encapsulation of fluorophores in PVB nanoparticles ..............................118

Table (3.4): Sizes of N-acyl ciprofloxacin loaded nanoparticles .................................125

Table (3.5): Zeta potentials of N-acyl ciprofloxacin loaded nanoparticles .................126

Table (3.6): N-acyl ciprofloxacins loading into PVB nanoparticles ...........................135

Table (A1): HPLC retention times for N-acyl ciprofloxacins .....................................215

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LIST OF FIGURES

Figure (1.1): Visual representations of oil in water (o/w) and water in oil in
water (w/o/w) emulsions ..............................................................................2

Figure (1.2): Visual representation of emulsion polymerization process .........................4

Figure (1.3): Ionic crosslinking with TPP and coacervation to prepare
nanoparticles ................................................................................................4

Figure (1.4): Adsorption onto the surface (left) and molecules loaded inside of
the nanoparticle ............................................................................................5

Figure (1.5): Covalently bound drug (left) versus encapsulated drug (right) ...................5

Figure (1.6): Graphical representation of burst release .....................................................6

Figure (1.7): PLGA (left) and PEG (right) ........................................................................7

Figure (1.8): Lidocaine......................................................................................................8

Figure (1.9): Gentamicin ...................................................................................................9

Figure (1.10): Bis(2-ethylhexyl) sulfosuccinate sodium salt ............................................10

Figure (1.11): Streptomycin (left) and Vancomycin (right) ..............................................11

Figure (1.12): Teicoplanin ................................................................................................14

Figure (1.13): Synthesis of mPEG-PLGA diblock copolymer .........................................14

Figure (1.14): Nafcillin .....................................................................................................15

Figure (1.15): Nanoparticle size distributions ...................................................................16

Figure (1.16): Eudragit® product line used in nanoparticle synthesis, and
ciprofloxacin ..............................................................................................17

Figure (1.17): Size distribution of nanoparticles with varying amounts of polymer
and drug. ....................................................................................................19

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Figure (1.18): Levofloxacin ..............................................................................................20

Figure (1.19): Poly caprolactone (PCL) ............................................................................20

Figure (1.20): Pluronic F-68 .............................................................................................21

Figure (1.21): Various molecules associated with PLGA nanoparticles ..........................23

Figure (1.22): Minimum inhibitory concentrations of nanoparticles compared to
free drug against S. typhi ............................................................................25

Figure (1.23): Clarithromycin ...........................................................................................26

Figure (1.24): Encapsulation efficiencies for clarithromycin loaded PLGA
nanoparticles ..............................................................................................26

Figure (1.25): Particle sizes for clarithromycin loaded PLGA nanoparticles ...................27

Figure (1.26): Minimum inhibitory concentrations of clarithromycin loaded
nanoparticles against S. aureus ..................................................................28

Figure (1.27): Rifampicin..................................................................................................29

Figure (1.28): Preparation of rifampicin loaded PLGA nanoparticles in mannitol
microspheres ..............................................................................................30

Figure (1.29): L-leucine ....................................................................................................31

Figure (1.30): Carvacrol ....................................................................................................33

Figure (1.31): Chloramphenicol ........................................................................................34

Figure (1.32): PVA (left) and PS-80 (right) ......................................................................34

Figure (1.33): Violacein ....................................................................................................35

Figure (1.34): Minimum inhibitory concentrations for violacein loaded PLGA
nanoparticles ..............................................................................................36

Figure (1.35): Poly(alkyl cyanoacrylate) polymers ..........................................................37

Figure (1.36): Actinomycin D ...........................................................................................38

Figure (1.37): Concentrations of drug in various tissues ..................................................39

Figure (1.38): Particle sizes of nanoparticle formulations ................................................40
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