Helicobacter pylori (H. pylori)

169 Pages·2014·8.01 MB·English

UC San Diego
UC San Diego Electronic Theses and Dissertations
Title
Antimicrobial Nanotherapeutics Against Helicobacter pylori Infection /
Permalink
https://escholarship.org/uc/item/03p0w0n8
Author
Thamphiwatana, Soracha
Publication Date
2014

Peer reviewed|Thesis/dissertation
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University of California
UNIVERSITY OF CALIFORNIA, SAN DIEGO

Antimicrobial Nanotherapeutics Against Helicobacter pylori Infection

A dissertation submitted in partial satisfaction of the requirements for the degree
Doctor of Philosophy

in

Nanoengineering

by

Soracha Thamphiwatana

Committee in charge:
Professor Liangfang Zhang, Chair
Professor Michael J. Heller, Co-Chair
Professor Xiaohua Huang
Professor Marygorret Obonyo
Professor Donald J. Sirbuly

2014
©
Soracha Thamphiwatana, 2014
All rights reserved
The Dissertation of Soracha Thamphiwatana is approved, and it is acceptable in
quality and form for publication on microfilm and electronically:

Co-Chair

Chair

University of California, San Diego

2014

iii
DEDICATION

This dissertation is dedicated to my mother, Suninath Thamphiwatana, who has made
countless sacrifices so I could freely pursue my dreams and always support me in
every possible way. My sister, Jinna Thamphiwatana, is always there for me. This
work has been possible because of their unconditional love and never-ending support.

iv
EPIGRAPH

“Imagination is more important than knowledge. Knowledge is limited. Imagination
encircles the world.”

Albert Einstein

v
TABLE OF CONTENTS

Signature page .............................................................................................................. iii
Dedication ..................................................................................................................... iv
Epigraph ........................................................................................................................ v
Table of Contents ......................................................................................................... vi
List of Figures ............................................................................................................... ix
List of Tables .............................................................................................................. xiii
Acknowledgements .................................................................................................... xiv
Vita ............................................................................................................................ xvii
Abstract of the Dissertation ...................................................................................... xviii
Chapter 1 Introduction ................................................................................................... 1
1.1 Targeted Antibiotic Delivery ....................................................................... 4
1.2 Environmentally Responsive Antibiotic Delivery ....................................... 7
1.3 Combinatorial Antibiotic Delivery ............................................................ 10
1.4 Nanoparticle-Enabled Antibacterial Vaccination ...................................... 14
1.5 Nanoparticle-Based Bacterial Detection ................................................... 19
1.6 Conclusions ............................................................................................... 22
1.7 References ................................................................................................. 24

Chapter 2 Antibacterial Activities of Liposomal Linolenic acids (LipoLLA)
Against Antibiotic-Resistant Helicobacter pylori (H. pylori) strains ......... 40
2.1 Introduction ............................................................................................... 41
2.2 Experimental Methods ............................................................................... 44
2.2.1 Materials ..................................................................................... 44
2.2.2 H. pylori Strains and Bacterial Culture ...................................... 44
2.2.3 Preparation of Solutions Containing LLA and LipoLLA ........... 45
2.2.4 Bactericidal Activity against Spiral form of H. pylori SS1 ........ 46
2.2.5 Bactericidal Activity against Coccoid form of H. pylori SS1 .... 47
2.2.6 LipoLLA–H. pylori SS1 Fusion Study ....................................... 48
2.2.7 Scanning Electron Microscope (SEM) Imaging of H. pylori ..... 49
2.2.8 Bactericidal Activity against Antibiotics-Resistant H. pylori .... 49
2.2.9 Resistance Acquisition of H. pylori SS1 upon Treatments ........ 50
2.3 Results and Discussion .............................................................................. 50
2.4 Conclusions ............................................................................................... 64
2.5 References ................................................................................................. 66

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Chapter 3 H. pylori Treatment with Liposomal Linolenic Acid in vivo ..................... 70
3.1 Introduction ............................................................................................... 71
3.2 Experimental Methods ............................................................................... 73
3.2.1 Materials ..................................................................................... 73
3.2.2 Preparation and characterization of LipoLLA ............................ 74
3.2.3 H. pylori culture and LipoLLA in vitro activity ......................... 75
3.2.4 LipoLLA fusion with H. pylori .................................................. 76
3.2.5 LipoLLA in vitro cytotoxicity study .......................................... 76
3.2.6 Gastric retention of LipoLLA ..................................................... 77
3.2.7 Anti-H. pylori efficacy in vivo .................................................... 78
3.2.8 Quantification of inflammatory cytokines .................................. 79
3.2.9 In vivo toxicity study .................................................................. 80
3.3 Results ....................................................................................................... 81
3.3.1 LipoLLA formulation and in vitro characterization ................... 81
3.3.2 Retention and distribution of LipoLLA in mouse stomach ........ 84
3.3.3 Anti-H. pylori efficacy in vivo .................................................... 86
3.3.4 Proinflammatory response to LipoLLA treatment ..................... 88
3.3.5 LipoLLA toxicity evaluation in vivo .......................................... 90
3.4 Discussion .................................................................................................. 91
3.5 Conclusions ............................................................................................... 94
3.6 References ................................................................................................. 96

Chapter 4 Stimuli-Responsive Liposomes ................................................................ 100
4.1 pH-Responsive Liposomes ...................................................................... 101
4.1.1 Introduction .............................................................................. 101
4.1.2 Experimental Methods .............................................................. 104
4.1.2.1 Materials .................................................................... 104
4.1.2.2 Chitosan-modified gold nanoparticles (AuChi)
preparation and characterization ............................................ 105
4.1.2.3 Liposome preparation ................................................ 105
4.1.2.4 AuChi-liposome formulation ..................................... 106
4.1.2.5 AuChi-liposome fusion with H. pylori ...................... 107
4.1.2.6 Drug release study ..................................................... 108
4.1.2.7 Antibacterial activity of AuChi-liposome ................. 108
4.1.3 Results and Discussion ............................................................. 109
4.1.4 Conclusions .............................................................................. 117
4.1.5 References ................................................................................ 119
4.2 Virulence Factor-Responsive Liposomes ................................................ 122
4.2.1 Introduction .............................................................................. 122
4.2.2 Experimental Methods .............................................................. 125
4.2.2.1 Materials .................................................................... 125
4.2.2.2 Preparation of AuChi ................................................. 126
4.2.2.3 Preparation of AuChi-liposome ................................. 126
4.2.2.4 H. pylori bacterial culture .......................................... 127
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4.2.2.5 Liposome stability assay ............................................ 128
4.2.2.6 Effect of phospholipase A (PLA ) on drug release
2 2
from AuChi-liposomes .............................................. 128
4.2.2.7 AuChi-liposome drug release in H. pylori culture .... 129
2.2.2.8 Antibacterial activity measurements ......................... 129
4.2.3 Results and Discussion ............................................................. 130
4.2.4 Conclusions .............................................................................. 138
4.2.5 References ................................................................................ 140

Chapter 5 Conclusions ............................................................................................... 144
5.1 LipoLLA Against Antibiotic-Resistant H. pylori strains ........................ 145
5.2 H. pylori Treatment with LipoLLA in vivo ............................................. 146
5.3 pH-Responsive Liposomes ...................................................................... 147
5.4 Virulence Factor-Responsive Liposomes ................................................ 148

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LIST OF FIGURES

Figure 1.1………..……………………………………….……………………………3
Major nanoparticle-based delivery platforms for treating bacterial infections: (A)
liposome, (B) polymeric nanoparticle, (C) dendrimer, and (D) inorganic nanoparticle.

Figure 1.2……..........………………………..………….……………………………10
Schematic illustration of a phospholipid liposome stabilized by charged gold
nanoparticles and its drug release in response to pH change or the presence of bacterial
toxin.

Figure 1.3…..…….………………………………...………………………………...16
Schematic preparation of nanoparticle-detained toxins, denoted ‘nanotoxoid’,
consisting of substrate-supported RBC membranes into which pore-forming toxins can
spontaneously incorporate.

Figure 1.4.…..………………………………….………………..…………………...18
Schematic illustration of interbilayer-crosslinked multilamellar vesicles (ICMVs) for
vaccine delivery: (A) OVA-loaded ICMVs with MPLA only on the external surface,
and (B) OVA-loaded ICMVs with MPLA throughout the lipid multilayers.

Figure 1.5.…..…………………………….………………………..………………...21
Magneto-DNA assay for the detection of bacterial 16S rRNA. Total RNA is extracted
from the specimen, and the 16S rRNA is amplified by asymmetric RT-PCR. ….
Samples are subsequently analyzed using a miniaturized micro-NMR (µNMR) system.

Figure 2.1…..…….……………….………………………………..……………...…52
Schematic drawing shows the molecular structure of LLA and the structure of
LipoLLA composed of phospholipid, cholesterol, and LLA.

Figure 2.2………..….…....……….………………….……………..………………..53
In vitro bactericidal activity of (A) LLA and (B) LipoLLA at different drug
concentrations against H. pylori SS1. All concentrations refer to LLA concentration,
regardless of the formulation.

Figure 2.3....……..……...........…………………………………..………………......55
FRET measurements of the fusion between LipoLLA and H. pylori SS1. …… A rise
in emission intensity of C NBD (donor) at 520 nm was observed with the increase of
6
bacterial concentrations, indicating the occurrence of fusion.
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