The molecular mechanisms of the antimicrobial properties of laser processed nano-particles

207 Pages·2017·4.38 MB·English

by  Peri Korshed

The molecular mechanisms of the
antimicrobial properties of laser processed
nano-particles

A thesis submitted to the University of Manchester for the degree of Doctor of
Philosophy in Medicine in the Faculty of Biology, Medicine and Health

2018

Peri Ahmad Korshed

Genomic Medicine Research Centre
Division of Evolution and Genomic Science
School of Biological Science
Contents
List of Figures ....................................................................................................................... 9
List of Tables ...................................................................................................................... 11
List of Abbreviations ......................................................................................................... 12
Abstract ............................................................................................................................... 15
Declaration .......................................................................................................................... 16
Copyright statement........................................................................................................... 17
Dedication ........................................................................................................................... 18
Acknowledgements ............................................................................................................. 19
Rational for submitting in alternative format ................................................................. 20
Thesis outline ...................................................................................................................... 21
Chapter One ....................................................................................................................... 22
1. Introduction ................................................................................................................. 22
1.1. Nanoparticles and their properties ......................................................................... 22
1.2. Nanoparticles as antimicrobials and applications ................................................. 23
1.2.1. Antibacterial activities of silver nanoparticles (Ag NPs) ............................... 25
1.2.2. Antibacterial activities of Titanium dioxide nanoparticles (TiO NPs) ......... 27
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1.2.3. Antibacterial activities of other noble metal nanoparticles ............................ 29
1.2.4. Antibacterial activity of composite nanoparticles .......................................... 32
1.2.5. Antibacterial activity of alloy nanoparticles .................................................. 32
1.2.6. Antibacterial activity of hybrid nanoparticles ................................................ 33
1.2.7. Antibacterial activity of core shell nanoparticles ........................................... 34
1.3. Antiviral activity of nanoparticles ......................................................................... 35
1.4. Antifungal activity of nanoparticles ...................................................................... 36
1.5. Factors influencing the bactericidal effect of nanoparticles .................................. 36
1.5.1. Size ................................................................................................................. 36
1.5.2. Shape .............................................................................................................. 37
1.5.3. Concentration ................................................................................................. 38
1.5.4. Surface charge ................................................................................................ 38
1.6. Mechanisms of nanoparticles activity ................................................................... 39
1.6.1. Adhesion of nanoparticles to bacterial cell envelope ..................................... 43
1.6.2. Interaction with thiol group ............................................................................ 44
1.6.3. ROS formation ............................................................................................... 45

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1.6.4. DNA damage .................................................................................................. 46
1.7. Toxicity of nanoparticles to human cells and environments ................................. 46
1.8. Laser production of nanoparticles ......................................................................... 51
1.8.1. Producing NPs by laser ablation in liquid ...................................................... 53
1.8.2. Factors influencing the size and composition of NPs .................................... 54
1.9. Antibacterial activity of NPs produced by laser process for coating in medical
applications ....................................................................................................................... 54
1.10. Problem statement ................................................................................................. 55
1.11. Project aims and objectives ................................................................................... 55
Chapter Two ....................................................................................................................... 57
2. Materials and Methods ............................................................................................... 57
2.1. Nanoparticles production by pulsed laser ablation ................................................ 57
2.2. UV-Visible absorbent spectrum measurement ...................................................... 58
2.3. Zeta potential measurement ................................................................................... 58
2.4. Silver ion measurement ......................................................................................... 59
2.5. NP’s morphology and elemental composition ...................................................... 59
2.6. Antibacterial test methods ..................................................................................... 59
2.6.1. Bacterial culture ............................................................................................. 59
2.6.2. Determination of antibacterial activity by Well-diffusion method ................ 60
2.7. Detection of ROS production ................................................................................ 60
2.7.1. Detection of ROS production using HPF indicator........................................ 60
2.7.2. Detection of ROS production using DCFH-DA indicator ............................. 61
2.8. Lactate dehydrogenase (LDH) release assay ......................................................... 62
2.9. Protein leakage determination ............................................................................... 62
2.10. Glutathione reductase assay .................................................................................. 63
2.11. Effect of lipid peroxidation (LPO) ........................................................................ 63
2.11.1. TBA solution preparation ............................................................................... 63
2.11.2. Standard MDA solution preparation .............................................................. 64
2.11.3. Testing laser NPs ........................................................................................... 64
2.12. DNA fragmentation ............................................................................................... 64
2.13. Cell culture and cytotoxicity assay ........................................................................ 65
2.13.1. Cell culture ..................................................................................................... 66
2.13.2. Cytotoxicity assay .......................................................................................... 66

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2.14. Transmission Electron Microscopy ....................................................................... 67
2.14.1. TEM imaging of human cells ......................................................................... 67
2.14.2. TEM imaging on bacterial cells ........................................................................ 67
2.14.3. TEM for imaging NPs size and shape ............................................................ 68
2.15. NP’s size separation by sucrose gradient centrifugation ....................................... 68
2.16. Silver Based Wound Dressing ............................................................................... 69
2.17. Conditions used to test the durability of laser Ag NPs antibacterial effect ........... 69
2.18. Statistical analysis ................................................................................................. 70
Chapter Three .................................................................................................................... 71
3. Results .......................................................................................................................... 71
The Molecular Mechanisms of the Antibacterial Effect of Picosecond Laser
Generated Silver Nanoparticles and Their Toxicity to Human Cells ........................... 71
3.1. Abstract ................................................................................................................. 71
3.2. Introduction ........................................................................................................... 72
3.3. Materials and Methods .......................................................................................... 75
3.3.1. Nanoparticles production ............................................................................... 75
3.3.2. Bacteria culture and the determination of antibacterial activities of NPs ...... 75
3.3.3. Detection of ROS generation ......................................................................... 76
3.3.4. Lactate dehydrogenase (LDH) release assay ................................................. 76
3.3.5. Protein leakage determination ........................................................................ 77
3.3.6. Glutathione reductase assay ........................................................................... 77
3.3.7. Effect of lipid peroxidation (LPO) ................................................................. 77
3.3.8. DNA fragmentation ........................................................................................ 78
3.3.9. UV-Visible absorbent spectrum measurement............................................... 78
3.3.10. Zeta potential measurement ........................................................................... 78
3.3.11. Silver ion measurement .................................................................................. 78
3.3.12. Cell culture and cytotoxicity assay ................................................................ 79
3.3.13. Transmission Electron Microscopy ............................................................... 80
3.3.14. Statistical analysis .......................................................................................... 80
3.4. Results ................................................................................................................... 80
3.4.1. Laser generated Ag NPs have strong antibacterial activity against both gram-
negative and gram-positive strains including MRSA ................................................... 80
3.4.2. Laser generated Ag NPs specifically induce the generation of nonhydroxyl
ROS ………………………………………………………………………………83

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3.4.3. Laser generated Ag NPs cause glutathione depletion in bacterial cells ......... 85
3.4.4. Laser generated Ag NPs induce bacterial lipid peroxidation ......................... 86
3.4.5. Laser generated Ag NPs reduce the integrity of the bacterial cell membrane
………………………………………………………………………………87
3.4.6. Laser generated Ag NPs release Ag ion ......................................................... 88
3.4.7. The cytotoxicity of laser generated Ag NPs to human cells .......................... 91
3.5. Discussion ............................................................................................................. 94
3.5.1. Antibacterial activities of laser generated Ag NPs ........................................ 94
3.5.2. The molecular mechanisms for the antibacterial effects by laser generated Ag
NPs ………………………………………………………………………………95
3.5.3. The toxicities of laser Ag NPs to human cells ............................................... 97
3.6. Conclusions ........................................................................................................... 98
Chapter Four .................................................................................................................... 100
4. Results ........................................................................................................................ 100
Antibacterial mechanisms of a novel type picosecond laser-generated silver-titanium
nanoparticles and their toxicity to human cells ............................................................. 100
4.1. Abstract ............................................................................................................... 100
4.2. Introduction ......................................................................................................... 101
4.3. Materials and methods ......................................................................................... 103
4.3.1. NP production .............................................................................................. 103
4.3.2. UV-visible spectrophotometry ..................................................................... 103
4.3.3. Bacteria culture and the determination of antibacterial activities of NPs .... 103
4.3.4. Detection of ROS generation ....................................................................... 104
4.3.5. GSH reductase measurement ....................................................................... 104
4.3.6. LPO analysis ................................................................................................ 105
4.3.7. Lactate dehydrogenase (LDH) release assay ............................................... 105
4.3.8. Bradford assay .............................................................................................. 105
4.3.9. DNA fragmentation ...................................................................................... 106
4.3.10. Cell culture and cytotoxicity assay .............................................................. 106
4.3.11. Transmission electron microscopy (TEM) .................................................. 107
4.3.12. Statistical analysis ........................................................................................ 107
4.4. Results ................................................................................................................. 107
4.4.1. Antibacterial effects of laser-generated Ag-TiO compound NPs ............... 107
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4.4.2. Effect of ROS generation by laser-generated Ag-TiO compound NPs ...... 109
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4.4.3. Effect of laser-generated Ag-TiO compound NPs on cellular GSH reductase
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level ……………………………………………………………………………..110
4.4.4. Effect of laser-generated Ag-TiO compound NPs on LPO ........................ 111
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4.4.5. Impact of laser-generated Ag-TiO compound NPs on the integrity of
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bacterial cell membrane .............................................................................................. 112
4.4.6. Effect of laser-generated Ag-TiO compound NPs on bacterial DNA damage
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…………………………………………………………………………….112
4.4.7. The toxicity of laser-generated Ag-TiO compound NPs to human cells .... 114
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4.5. Discussion ........................................................................................................... 117
4.6. Conclusion ........................................................................................................... 120
Chapter Five ..................................................................................................................... 122
5. Results ........................................................................................................................ 122
Effect of storage condition on the long-term stability of bactericidal effect for laser
generated Ag nanoparticles ............................................................................................. 122
5.1. Abstract ............................................................................................................... 122
5.2. Introductions ........................................................................................................ 122
5.3. Materials and Methods ................................................................................. 124
5.3.1. Nanoparticles production ............................................................................. 124
5.3.2. Bacteria culture and the determination of the antibacterial activities of NPs
…………………………………………………………………………….125
5.3.3. Storage conditions ........................................................................................ 125
5.3.4. NP’s morphology and elemental composition ............................................. 126
5.3.5. Statistical analysis ........................................................................................ 126
5.4. Results ................................................................................................................. 126
5.4.1. Duration of the antibacterial effects of laser generated Ag NPs .................. 126
5.4.3. Comparison of the antibacterial duration between laser generated and
commercial Ag NPs .................................................................................................... 131
5.6. Conclusions ......................................................................................................... 138
Chapter Six ....................................................................................................................... 139
6. Results ........................................................................................................................ 139
Size-dependent antibacterial activity of laser generated Ag and Ag-TiO NPs ......... 139
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6.1. Abstract ............................................................................................................... 139
6.2. Introduction ......................................................................................................... 139
6.3. Material and Methods .......................................................................................... 141
6.3.1. Nanoparticles production ............................................................................. 141

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6.3.2. Preparation of the sucrose with a density gradient....................................... 141
6.3.3. Nanoparticles separation by sucrose gradient centrifugation ....................... 142
6.3.4. Characterization of separated NPs ............................................................... 142
6.3.5. Antibacterial activity examination ............................................................... 142
6.3.6. Bacterial oxidative stress ROS test .............................................................. 143
6.3.7. Human cell cytotoxicity test......................................................................... 143
6.3.8. Transmission Electron Microscopy ............................................................. 143
6.3.9. Statistical analysis ........................................................................................ 144
6.4. Results ................................................................................................................. 144
6.4.1. Separation of laser Ag and Ag-TiO NPs using sucrose gradient
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centrifugation .............................................................................................................. 144
6.4.3. Generation of reactive oxygen species (ROS) ............................................. 149
6.5.1. Nanoparticles separation .............................................................................. 153
6.5.2. Laser Ag and Ag-TiO NPs antibacterial test .............................................. 154
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6.5.3. Oxidative stress ............................................................................................ 154
6.5.4. Toxicity of laser generated NPs ................................................................... 155
6.6. Conclusions ......................................................................................................... 155
Chapter Seven .................................................................................................................. 156
7. Results ........................................................................................................................ 156
A comparative study of the antibacterial activity of laser generated Ag, Ag-TiO NPs
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and commercial silver-based medical dressings ............................................................ 156
7.1. Abstract ............................................................................................................... 156
7.2. Introduction ......................................................................................................... 156
7.3. Material and Methods .......................................................................................... 159
7.3.1. Nanoparticles production ............................................................................. 159
7.3.3. Measurement of antibacterial activities using a direct co-culture method ... 161
7.3.4. Measurement of antibacterial activities using well-diffusion method ......... 162
7.3.5. Transmission Electron Microscopy ............................................................. 162
7.3.6. Statistical analysis ........................................................................................ 162
7.4. Results ................................................................................................................. 162
7.5. Discussion ........................................................................................................... 167
7.6. Conclusion ........................................................................................................... 170
Chapter Eight ................................................................................................................... 171
8. General Discussion .................................................................................................... 171

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8.1. The Antibacterial Effect of Laser Generated Ag and Ag-TiO NPs ................... 171
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8.2. The Molecular Mechanisms of the Antibacterial Effect of Laser Generated Ag
and Ag-TiO NPs ............................................................................................................ 173
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8.3. Toxicity of Laser Generated Ag and Ag-TiO NPs to Human Cells .................. 174
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8.4. Conclusions and perspectives .............................................................................. 175
8.4.1. Conclusions .................................................................................................. 175
8.4.2. Perspectives .................................................................................................. 175
8.5. Future Works ....................................................................................................... 176
References ......................................................................................................................... 178

Word count: 62,969 words

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List of Figures
Figure 1. 1 Bactericidal mechanisms of silver nanoparticles ..................................................... 42
Figure 1. 2 Different bactericidal activity of silver nanoparticles ....................................... 43
Figure 1. 3 Excitation steps of laser ablation in NPs synthesis in liquid media .................. 52
Figure 1. 4 Production of NPs by laser ablation of a solid target in a liquid medium ......... 53

Figure 2. 1 Diagram of nanoparticles producing using laser ablation in liquid ................... 57
Figure 2. 2 The 400 Edge wave picosecond laser machine ................................................. 58

Figure 3. 1The antibacterial activity of laser generated Ag NPs against gram positive and
negative bacteria................................................................................................................... 82
Figure 3. 2 The effect of laser generated Ag NPs on the production of reactive oxygen
species (ROS) in E. coli. ...................................................................................................... 84
Figure 3. 3 Absorbent spectrum of laser generated Ag NPs. ............................................... 85
Figure 3. 4 Changes of cellular glutathione level in laser Ag NPs treated E. ...................... 86
Figure 3. 5 Lipid peroxidation in E. coli by laser generated Ag NPs. ................................. 87
Figure 3. 6 Effect of laser generated Ag NPs on E. coli membrane integrity. ..................... 88
Figure 3. 8 DNA degradation by laser Ag NPs in E. coli. E. ............................................... 91
Figure 3. 9 Cytotoxicity of laser generated Ag NPs to human cells. ................................... 93
Figure 3. 10 TEM images of human lung AC549 cell line treated with Ag NPs. ............... 94

Figure 4. 1The antibacterial activity of laser-generated Ag-TiO NPs against Gram-positive
2
and Gram-negative bacteria. .............................................................................................. 108
Figure 4. 2 The effect of laser-generated Ag-TiO NPs on the production of ROS in
2
Escherichia coli. ................................................................................................................. 110
Figure 4. 3 Changes of cellular glutathione reductase level in laser Ag-TiO NP-treated
2
Escherichia coli. ................................................................................................................. 111
Figure 4. 4 Lipid peroxidation in Escherichia coli by laser-generated Ag-TiO NPs. ...... 111
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Figure 4. 5 Effect of laser-generated Ag-TiO NPs on Escherichia coli membrane integrity
2
............................................................................................................................................ 113
Figure 4. 6 DNA degradation by laser Ag-TiO NPs in Escherichia coli. ........................ 114
2
Figure 4. 7 Cytotoxicity of laser-generated Ag-TiO NPs to human cells......................... 116
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Figure 4. 8 TEM images of human lung A549 cell line treated with laser-generated Ag-
TiO NPs. ........................................................................................................................... 117
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Figure 5. 1 Antibacterial duration of laser generated Ag NPs. Samples of laser generated
Ag NPs were stored under three different conditions: light, dark, and cold. ..................... 127
Figure 5. 2 Impact of air exposure on the duration of antibacterial activities of Ag NPs.. 128
Figure 5. 3 The effect of frequent air exposure of laser generated Ag NPs on its
antibacterial activities under different storage conditions. ................................................ 130
Figure 5. 4 The effect of frequent air exposure on the antibacterial effect of the chemically
produced Ag NPs. .............................................................................................................. 131
Figure 5. 5 EDX spectra of laser generated Ag NPs after storage. .................................... 133

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Figure 5. 6 Surface elemental mapping of laser generated Ag NPs using HAADF-STEM.
............................................................................................................................................ 135

Figure 6. 1 Separation of Ag and Ag-TiO NPs by sucrose gradient centrifugation. ........ 145
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Figure 6. 2 UV adsorption spectra of sucrose fractions for laser generated Ag and Ag-TiO
2
NPs. .................................................................................................................................... 146
Figure 6. 3TEM images of fractions of laser generated Ag and Ag-TiO NPs that were
2
separated by sucrose gradient centrifugation. .................................................................... 147
Figure 6. 4 Size dependent antibacterial effect of laser generated Ag and Ag-TiO NPs. 149
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Figure 6. 5 Nanoparticle size dependent induction of ROS generation by E. coli bacterial
cells. ................................................................................................................................... 150
Figure 6. 6 Human cytotoxicity of different sizes laser Ag and Ag-TiO NPs. ................. 151
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Figure 6. 7 TEM images of human dermal fibroblast cells HDF treated by different sixed
laser Ag and Ag-TiO NPs. ................................................................................................ 152
2

Figure 7. 1 The commercialy available wound dressings. A, Acticoat; Q, Aquacel; C,
Contreet Foam; and U, Urgotul. ......................................................................................... 160
Figure 7. 2 Wound dressing sample preparation. ............................................................... 161
Figure 7. 3 TEM imaging of structures and distributions of the Ag NPs embedded in the 4
types of commercial wound dressings (A, B, and C) and the laser Ag NPs (D).. ............. 163
Figure 7. 4 The antibacterial activity was measured using well-difussion method for the
laser Ag NPs, Ag-TiO NPs, and the four types of commercialy available wound dressings
2
Acticoat, Aquacel, Contreet Foam, and Urgotul. ............................................................... 165
Figure 7. 5 The antibacterial activity of the commercialy available wound dressing
(Acticoat, Aquacel, Contreet Foam, and Urgotul) and laser generated Ag and Ag-TiO
2
NPs using direct bacterial killing method. ......................................................................... 166

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