Liposome encapsulated antimicrobial metal ions and essential oils

273 Pages·2012·5.48 MB·English

by  Wan Li Low

L E
IPOSOME NCAPSULATED
A M I
NTIMICROBIAL ETAL ONS
E O
AND SSENTIAL ILS

Thesis submitted for the

Degree of Doctor of Philosophy

WAN LI LOW, BSC.(HONS), MSB, MAPS
Liposome Encapsulated
Antimicrobial Metal Ions and
Essential Oils

A thesis submitted in fulfilment of the requirements of the
University of Wolverhampton for the award of the degree of
Doctor of Philosophy (PhD)

JANUARY 2012

This work or any part thereof has not previously been submitted in any form
to the University or to any other body whether for the purpose of assessment,
publication or for any other purpose. Save for any express
acknowledgements, references and/or bibliographies cited in the work, I
confirm that the intellectual content of the work is the result of my own efforts
and no other person. The right of Wan Li Low to be identified as author of this
work is asserted in accordance with ss. 77 and 78 of Copyright, Designs and
Patents Act 1988. At this date copyright is owned by the author.

W.L.Low
Signature:
Date : 26th JANUARY 2012
Abstract
This study investigates the feasibility of using TTO and Ag+ alone and
in combination either as free or liposome encapsulated agents. Based on the
minimum lethal concentration (MLC), the fractional lethal concentration index
(FLCI) showed that treatment with unencapsulated combinations of TTO and
Ag+ exerted a synergistic effect against P. aeruginosa (FLCI = 0.263) and
indifferent effects against S. aureus and C. albicans (0.663 and 0.880,
respectively). Using polyvinyl alcohol (PVA) emulsified agents in combination,
showed synergistic effects against P. aeruginosa and S. aureus (FLCI =
0.325 and 0.375, respectively), but C. albicans remained indifferent (FLCI =
0.733). Time kill experiments revealed that the combined agent
concentrations and elimination time (to the lowest limit of detection, LOD) are
as follows: C. albicans: 0.12%v/v :2.5x10-4 :1.5hrs, P. aeruginosa:
TTO Ag+
1%v/v :3.2x10-4 :15mins and S. aureus: 1.2%v/v :3.2x10-4 :30mins.
TTO Ag+ TTO Ag+
Repeating these experiments with emulsified TTO encapsulated in liposomes
(lipo-TTO:PVA ) against P. aeruginosa and S. aureus reduced the
30-70kDa
effective amount of TTO required (compared to free TTO). However, this was
not observed in C. albicans. The required effective concentration of Ag+ from
liposome encapsulated Ag+ (lipo-Ag+) was shown to remain the same as free
Ag+. The effective concentration and elimination time of liposomal agents in
combination are as follows: C. albicans: 0.05%v/v :8.9x10-5 :2.0hrs,
TTO:PVA Ag:PVA
P. aeruginosa: 0.25%v/v :3.2x10-4 :30mins and S. aureus:
TTO:PVA Ag:PVA
0.05%v/v :6.0x10-4 :1.5hrs. These results showed the potential of
TTO:PVA Ag:PVA
using TTO and Ag+ in combination, along with liposome delivery systems to
effectively lower the MLC.
Scanning electron micrographs of microorganisms exposed to Ag+
showed a reduction in cell size when compared to untreated cells.
Transmission electron micrograph of C. albicans showed the cell surface
damaging potential of Ag+. Furthermore, this investigation also demonstrated
the feasibility of using chitosan hydrogels as an alternative delivery system
for TTO and/or Ag+. The development of these controlled release systems to
deliver alternative antimicrobial agents may allow sustained targeted delivery
at microbiocidal concentrations.
CONTENTS
List of tables 1
List of figures 3
List of photomicrographs 5
List of appendices 5
Acknowledgement 7
Dedication 9
Abbreviation 10

CHAPTER 1 12
1. Introduction 13
1.1. Benefits and problems associated with microorganisms 14
1.2. Microorganisms and wound management 15
1.2.1. Wound healing mechanism 18
1.2.2. Common problems 26
1.2.3. Topical drug/antimicrobial agents and delivery
systems 27
1.3. Alternative antimicrobial agents 36
1.4. Plant products as antimicrobial agent 38
1.5. Tea tree essential oil as an antimicrobial 51
1.5.1. History, background of use and production of tea
tree oil 51
1.5.2. Mode of action 52
1.5.3. Applications 54
1.5.4. Wound healing benefits 55
1.5.5. Resistance 56
1.5.6. Toxicity concerns 57
1.6. Heavy metals as antimicrobial agents 58
1.7. Silver as an antimicrobial 63
1.7.1. History and background of use 63
1.7.2. Mode of action 66
1.7.3. Applications 71
1.7.4. Wound healing benefits 73
1.7.5. Resistance 75
1.7.6. Toxicity concerns 78
1.8. Controlled release delivery systems 79
1.8.1. Background 79
1.8.2. Liposomes 81
1.8.3. Chitosan hydrogels 88
1.8.4. Poly vinyl alcohol 90
1.9. Combined antimicrobial agent preparations (TTO and Ag+)
with controlled release delivery systems (liposomes)
applied to wound management 91
1.10. Aims and objective 94

CHAPTER 2 95
2. Methods and materials 96
2.1. Preparation of microbial growth media 96
2.2. Preparation of microbial cultures 96
2.3. Preparation of silver nitrate (AgNO ) solution and TTO 97
3
2.4. Preparation of TTO:PVA emulsion and Ag+:PVA
30-70kDa 30-
solution 97
70kDa
2.5. Viable counts 98
2.6. Minimum inhibitory concentration (MIC) and minimum
lethal concentration (MLC) 98
2.7. Chequerboard assay 100
2.8. Preparation of liposome encapsulated agents via Reverse-
phase Evaporation Vesicle (REV) method 102
2.9. Time kill studies using free and liposome encapsulated
agent(s) singly and in combination 104
2.10. Hydrogel formulation 105
2.11. Inductively Coupled Plasma (ICP) analysis to determine
the amount of Ag+ 111
2.12. Vanillin assay for the determination of monoterpene
release from TTO 112
2.13. Gas chromatography-mass spectrometry (GC-MS) 113
2.14. Chitosan assay (Ninhydrin assay) 114
2.15. Fluorescent microscopy, SEM and TEM 116
2.15.1. Fluorescence microscopy 116
2.15.2. Scanning electron microscopy (SEM) 117
2.15.3. Transmission electron microscopy (TEM) 118

CHAPTER 3 120
3. Results 121
3.1. MIC/MLC 121
3.2. Chequerboard assay 124
3.2.1. Isobolograms 124
3.2.2. Determination of synergistic combinations of Ag+
with TTO and TTO:PVA with Ag+:PVA 128
30-70kDa 30-70kDa
3.2.3. Fractional Lethal Concentration Index (FLCI) 129
3.3. Time kill studies 131
3.4. Time kill using emulsified and liposome-encapsulated
agents 135
3.5. Quantification of silver ions (Ag+) and TTO (monoterpenes) 140
3.5.1. ICP analysis for the quantification of silver ions
(Ag+). 140
3.5.2. Vanillin assay for monoterpene release 146
3.5.3. Release of agents from lipo-TTO:PVA and
30-70kDa
lipo-Ag+ 150
3.5.4. GCMS analysis of whole TTO 152
3.6. Fluorescence microscopy, SEM and TEM 154
3.7. TTO and/or Ag+ Hydrogels 166
3.7.1. Appearance of ionically crosslinked chitosan
hydrogels 166
3.7.2. Cumulative chitosan and monoterpene release after
24 hours incubation 170
3.7.3. Well diffusion assay of hydrogels containing TTO
and/or Ag+ 176
CHAPTER 4 188
4. Discussion 189

CHAPTER 5 209
5. Conclusion 210

CHAPTER 6 212
6. Future work 213

CHAPTER 7 216
7. Appendices 217

CHAPTER 8 231
8. List of conferences attended, publications and posters 232

CHAPTER 9 236
9. References 237
List of tables
Table 1: Common topical antimicrobial and their uses ............................... 29
Table 2: Types of wound dressing ............................................................. 33
Table 3: Summary of the major classes of active antimicrobial
compounds from plants ................................................................ 40
Table 4: Common essential oils extracted from plants and their
medicinal and/or antimicrobial activity .......................................... 45
Table 5: Examples of heavy metals with antimicrobial properties .............. 60
Table 6: Examples of antimicrobial compounds that show enhanced
activity in the presence of various metal ions ............................... 61
Table 7: Applications of heavy metals with lower toxicity to human
cells .............................................................................................. 62
Table 8: Applications of antimicrobial silver in industry ............................. 72
Table 9: Modifications of liposomes that affect function ............................. 85
Table 10: Chequerboard assay detailing the concentration of TTO and
AgNO used against the microorganisms .................................. 100
3
Table 11: Chequerboard assay detailing the concentration of
TTO:PVA and Ag+:PVA used against the
30-70kDa 30-70kDa
microorganisms .......................................................................... 101
Table 12: Formulations used to prepare lipo-TTO-Ag+-PVA for
30-70kDa
experiments against the named microorganism ......................... 104
Table 13: Composition ratio of hydrogel formulations ................................ 106
Table 14: Formulation details of hydrogels containing TTO and/or Ag+ ..... 108
Table 15: MIC/MLC of TTO and Ag+ against the microorganisms ............. 121
Table 16: MLC of emulsified TTO:PVA against the microorganisms ......... 122
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