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NANOSTRUCTURE MEDIATED ENHANCEMENT OF
ANTIBACTERIAL POTENTIAL OF SELECTED ANTIBIOTICS
BY
Mr. SHUJAT ALI
Thesis submitted to the Department of Chemistry,
University of Malakand for the partial fulfillment of the
requirement for the degree of
DOCTOR OF PHILOSPHY (PhD)
IN
CHEMISTRY
DEPARTMENT OF CHEMISTRY
UNIVERSITY OF MALAKAND
2016
NANOSTRUCTURE MEDIATED ENHANCEMENT OF
ANTIBACTERIAL POTENTIAL OF SELECTED ANTIBIOTICS
BY
Mr. SHUJAT ALI
DOCTOR OF PHILOSOPHY (PhD)
IN
CHEMISTRY
DEPARTMENT OF CHEMISTRY
UNIVERSITY OF MALAKAND
2016
Declaration I declare that the thesis “NANOSTRUCTURE MEDIATED ENHANCEMENT OF
ANTIBACTERIAL POTENTIAL OF SELECTED ANTIBIOTICS” is my original work and
has never been presented for the award of any degree at any other University before and that all
the information sources I have used and or quoted have been acknowledged with proper citations.
November 2016
Shujat Ali
Certificate It is recommended that this thesis submitted by Mr. SHUJAT ALI entitled
“NANOSTRUCTURE MEDIATED ENHANCEMENT OF ANTIBACTERIAL
POTENTIAL OF SELECTED ANTIBIOTICS” be accepted as fulfilling this part of
the requirement for the degree of Doctor of philosophy (PhD) in Chemistry.
______________________ ______________________
SUPERVISOR CO-SUPERVISOR
Dr. Mumtaz Ali Dr. Muhammad Raza Shah,T.I.
Assistant Professor Associate Professor
Department of Chemistry HEJ research institute of chemistry
University of Malakand University of Karachi,
K.P.K. Pakistan Karachi-75270, Pakistan
______________________ ______________________
EXTERNAL EXAMINER CHAIRMAN
Department of Chemistry
University of Malakand
Dedicated
To
My Loving Parents
For their constant support and infinite love to my pursuit of dreams and happiness.
Contents
List of Contents
Acknowledgments .......................................................................................................................... i
List of Figures ................................................................................................................................ ii
Abbreviations ............................................................................................................................... ix
Abstract .......................................................................................................................................... x
CHAPTER-1 .................................................................................................................................. 1
INTRODUCTION ........................................................................................................................ 1
1.1 Nanotechnology .................................................................................................................... 1
1.1.1. History of Nanotechnology ........................................................................................... 2
1.2 Nanomaterials........................................................................................................................ 4
1.3 Metals nanoparticles .............................................................................................................. 5
1.3.1 Silver nanoparticles ........................................................................................................ 6
1.3.2 Gold Nanoparticles ......................................................................................................... 7
1.4 Synthesis of nanomaterials .................................................................................................... 7
1.4.1 Chemical reduction ......................................................................................................... 9
1.4.2 Solvent antisolvent precipitation .................................................................................. 11
1.4.3 Hydrothermal Method .................................................................................................. 12
1.4.4 Pyrolysis ....................................................................................................................... 12
1.4.5 Chemical vapor deposition ........................................................................................... 13
1.4.6 Bio-based Protocols ...................................................................................................... 13
1.4.7 Sol-gel process .............................................................................................................. 14
1.4.8 Reverse micelle method................................................................................................ 14
1.4.9 Chemical precipitation .................................................................................................. 15
1.4.10 Green Chemistry approaches ...................................................................................... 15
1.4.11 Microwave irradiation ................................................................................................ 16
1.5 Characterization of nanoparticles ........................................................................................ 17
1.6 Applications of nanoparticles .............................................................................................. 18
1.6.1 Electronics .................................................................................................................... 19
1.6.2 Catalysis........................................................................................................................ 20
1.6.3 Plasmonics .................................................................................................................... 20
1.6.4 Waste water treatment .................................................................................................. 21
Contents
1.6.5 Agriculture .................................................................................................................... 22
1.6.6 Health and Medicines ................................................................................................... 22
1.7 Nanoparticles as antimicrobial ............................................................................................ 23
1.7.1 Methods used for antimicrobial evaluation .................................................................. 26
1.8 Beta-lactam antibiotics ........................................................................................................ 27
1.8.1 Cephalosporins ............................................................................................................. 27
1.8.1.1 First generation cephalosporins ............................................................................. 28
1.8.1.2 Second generation cephalosporins ......................................................................... 28
1.8.1.3 Third generation cephalosporins ............................................................................ 29
1.8.1.4 Fourth generation cephalosporins .......................................................................... 29
1.8.1.5 Fifth generation cephalosporins ............................................................................. 29
1.9 Spectrum of activity of B-lactam antibiotics....................................................................... 30
1.10 Resistance of bacteria towards antibiotics ........................................................................ 31
1.11 Approaches to Combat Resistant Bacteria ........................................................................ 33
CHAPTER-2 ................................................................................................................................ 35
MATERIALS AND METHODS ............................................................................................... 35
2.1 Materials .............................................................................................................................. 35
2.2 General Procedure ............................................................................................................... 35
2.3 Modification of Ceftriaxone via conjugation with Ag nanoparticles .................................. 35
2.4 Modification of Ceftriaxone via conjugation with Au nanoparticles .................................. 36
2.5 Modification of Cefadroxil via conjugation with Ag nanoparticles ................................... 37
2.6 Modification of Cefadroxil via conjugation with Au nanoparticles ................................... 37
2.7 Modification of Cephradine via conjugation with Ag nanoparticles .................................. 38
2.8 Modification of Cephradine via conjugation with Au nanoparticles .................................. 38
2.9 Modification of Ampicillin via conjugation with Ag nanoparticles ................................... 39
2.10 Modification of Ampicillin via conjugation with Au nanoparticles ................................. 39
2.11 Modification of Cefixime via conjugation with Ag nanoparticles .................................... 39
2.12 Modification of Cefixime via conjugation with Au nanoparticles .................................... 40
2.13 Synthesis of Polymer-Encapsulated Ceftriaxone Nanoparticles ....................................... 40
2.13.1 Drug entrapment efficiency ........................................................................................ 40
2.13.2 In vitro release studies ................................................................................................ 41
Contents
2.14 Synthesis of Polymer-Encapsulated Cefixime Nanoparticles ........................................... 41
2.14.1 Drug entrapment efficiency ........................................................................................ 42
2.14.2 In vitro release studies ................................................................................................ 42
2.15 Characterization ................................................................................................................ 43
2.16 Stability of the nanoparticles ............................................................................................. 43
2.17 Evaluation of antibacterial activity ................................................................................... 43
CHAPTER-3 ................................................................................................................................ 45
RESULTS AND DISCUSSION ................................................................................................. 45
3.1 Modification of Ceftriaxone via conjugation with Ag and Au nanoparticles ..................... 45
3.1.1 Synthesis of AgNPs stabilized with Ceftriaxone .......................................................... 45
3.1.2 Characterization of Cef-AgNPs .................................................................................... 46
3.1.3 Stability of the silver nanoparticles stabilized with Ceftriaxone .................................. 48
3.1.3.1 Thermal stability .................................................................................................... 48
3.1.3.2 Salt Stability ........................................................................................................... 49
3.1.3.3 PH stability............................................................................................................. 50
3.1.4 Synthesis of AuNPs stabilized with Ceftriaxone .......................................................... 51
3.1.5 Characterization of Cef-AuNPs .................................................................................... 51
3.1.6 Stability of the silver nanoparticles stabilized with Ceftriaxone .................................. 54
3.1.6.1 Thermal stability .................................................................................................... 54
3.1.6.2 Salt Stability ........................................................................................................... 55
3.1.6.3 PH stability............................................................................................................. 55
3.1.7 Evaluation of antibacterial potential of Cef-AgNPs and Cef-AuNPs .......................... 56
3.2 Modification of Cefadroxil via conjugation with Ag and Au nanoparticles ....................... 62
3.2.1 Synthesis of AgNPs stabilized with Cefadroxil ........................................................... 62
3.2.2 Characterization of Cefd-AgNPs .................................................................................. 62
3.2.3 Stability of the Cefd-AgNPs ......................................................................................... 65
3.2.3.1 Thermal stability .................................................................................................... 65
3.2.3.2 Salt Stability ........................................................................................................... 66
3.2.3.3 PH stability............................................................................................................. 66
3.2.4 Synthesis of AuNPs stabilized with Cefadroxil ........................................................... 67
3.2.5 Characterization of Cefd-AuNPs .................................................................................. 67
Contents
3.2.6 Stability of Cefd-AuNPs ............................................................................................... 70
3.2.6.1 Thermal stability .................................................................................................... 70
3.2.6.2 Salt Stability ........................................................................................................... 70
3.2.6.3 PH stability............................................................................................................. 71
3.2.7 Evaluation of antibacterial potential of Cefd-AgNPs and Cefd-AuNPs ...................... 72
3.3 Modification of Cephradine via conjugation with Ag and Au nanoparticles ..................... 78
3.3.1 Synthesis of AgNPs stabilized with Cephradine .......................................................... 78
3.3.2 Characterization of Cpn-AgNPs ................................................................................... 79
3.3.3 Stability of Cpn-AgNPs ................................................................................................ 81
3.3.3.1 Thermal stability .................................................................................................... 81
3.3.3.2 Salt Stability ........................................................................................................... 82
3.3.3.3 PH stability............................................................................................................. 83
3.3.4 Synthesis of AuNPs stabilized with Cephradine .......................................................... 84
3.3.5 Characterization of Cpn-AuNPs ................................................................................... 84
3.3.6 Stability of Cpn-AuNPs ................................................................................................ 86
3.3.6.1 Thermal stability .................................................................................................... 86
3.3.6.2 Salt Stability ........................................................................................................... 87
3.3.6.3 PH stability............................................................................................................. 88
3.3.7 Evaluation of antibacterial potential of Cpn-AgNPs and Cpn-AuNPs ......................... 89
3.4 Modification of Ampicillin via conjugation with Ag and Au nanoparticles ....................... 95
3.4.1 Synthesis of AgNPs stabilized with Ampicillin ........................................................... 95
3.4.2 Characterization of Mpn-AgNPs .................................................................................. 96
3.4.3 Stability of Mpn-AgNPs ............................................................................................... 98
3.4.3.1 Thermal stability .................................................................................................... 98
3.4.3.2 Salt Stability ........................................................................................................... 99
3.4.3.3 PH stability........................................................................................................... 100
3.4.4 Synthesis of AuNPs stabilized with Ampicillin ......................................................... 100
3.4.5 Characterization of Mpn-AuNPs ................................................................................ 101
3.4.6 Stability of Mpn-AuNPs ............................................................................................. 103
3.4.6.1 Thermal stability .................................................................................................. 103
3.4.6.2. Salt Stability ........................................................................................................ 104
Contents
3.4.6.3 PH stability........................................................................................................... 105
3.4.7 Evaluation of antibacterial potential of Mpn-AgNPs and Mpn-AuNPs ..................... 106
3.5 Modification of Cefixime via conjugation with Ag and Au nanoparticles ....................... 113
3.5.1 Synthesis of AgNPs stabilized with Cefixime ............................................................ 113
3.5.2 Characterization of Cfm-AgNPs ................................................................................ 113
3.5.3 Stability of Cfm-AgNPs ............................................................................................. 116
3.5.3.1 Thermal stability .................................................................................................. 116
3.5.3.2 Salt Stability ......................................................................................................... 116
3.5.3.3 PH stability........................................................................................................... 117
3.5.4 Synthesis of AuNPs stabilized with Cefixime ............................................................ 118
3.5.5 Characterization of Cfm-AuNPs ................................................................................ 118
3.5.6 Stability of Cfm-AuNPs ............................................................................................. 121
3.5.6.1 Thermal stability .................................................................................................. 121
3.5.6.2 Salt Stability ......................................................................................................... 122
3.5.6.3 PH stability........................................................................................................... 122
3.5.7 Evaluation of antibacterial potential of Cfm-AgNPs and Cfm-AuNPs ...................... 123
3.6 Modification of Ceftriaxone via encapsulation with Polymer .......................................... 130
3.6.1 Synthesis of Polymer-Encapsulated Ceftriaxone Nanoparticles ................................ 130
3.6.2 Characterization of Cef-PEG ...................................................................................... 130
3.6.3 Drug entrapment efficiency ........................................................................................ 132
3.6.4 In-vitro release study .................................................................................................. 132
3.6.5 Antibacterial study of Polymer-Encapsulated Ceftriaxone Nanoparticles ................. 133
3.7 Modification of Cefixime via encapsulation with Polymer .............................................. 134
3.7.1 Synthesis of Polymer-Encapsulated Cefixime Nanoparticles .................................... 134
3.7.2 Characterization of Cfx-PEG ...................................................................................... 135
3.7.3 Drug entrapment efficiency ........................................................................................ 136
3.7.4 In-vitro release study .................................................................................................. 136
3.7.5 Antibacterial study of Polymer-Encapsulated Cefixime Nanoparticles ..................... 137
REFERENCES .......................................................................................................................... 138
LIST OF PUBLICATIONS ..................................................................................................... 165
Acknowledgments
i
Acknowledgments
I wish to start my litany of gratitude by thanking ALLAH for having given me health, strength
and wisdom for the auspicious accomplishment of this work. I am grateful to so many people who
extended their possible help, in their own way for this research work to be conducted. I extend my
sincere gratitude to my supervisor Dr. Mumtaz Ali, assistant Professor, Department of Chemistry,
University of Malakand, who provided me an opportunity to work in his group and for his invaluable
guidance during the course of this thesis. I am grateful to my Co-Supervisor Dr. Muhammad Raza
Shah who not only gave me the opportunity to explore and innovate in his laboratory and opened up
new areas of interest, but also provided constant support, guidance and encouragement.
I am also indebted to Prof. Dr. Rashid Ahmad, Chairman, Department of Chemistry, University
of Malakand for his administrative assistance and guidance during my PhD studies. My sincere thanks
are to all the esteemed faculty members of Department of Chemistry, University of Malakand for their
help and valuable suggestions during my PhD study. I would like to thank Dr. Shah Zeb and Dr. Wadood
Ali, Department of Pharmacy, University of Malakand for introducing me to polymeric nanoparticles
research and constantly providing advice and encouragement.
I would like to express my gratitude and thanks to Miss. Samina Perveen, IAC, HEJ Karachi for
her help in bioassay studies and Dr. Massimo F. Bertino of Virginia Commonwealth University USA for
theoretical assistance. I would also like to convey my appreciation to all my fellows especially to Dr.
Adnan, Dr. Hanif Ahmad, Dr. Shujaat Ahmad, Mr. Misal Bacha, Mr. Hafiz Kamran, Mr. Zarif Gul,
Mr. Umar Ali, Mr. Nasib Khan, Mr. Ibrahim, Mr. Faizan Ur Rehman, Mr. Idrees Khan, and Mr. Alam
Khan, Mr. Tariq Shah for having made the department feel like home.
I am also thankful to Dr. Kiramat, Dr. Burhan, Dr. Ateeq, Dr. Saif Afridi, Mr. Farid Ahmad,
Mr. Farman Ali, Mr. Anwer Shamim, Mr. Shafi Ullah and Mr. Imran for their nice company in
Karachi. It was a wonderful time with them.
Special thanks to my friends Dr. Ajmal Iqbal, Nazim Hassan, Mr. Sher Ali Khan, Mr. Sultan
Muhammad, and Mr. Khurshid Iqbal for their encouragement and fruitful suggestions.
I would like to thank those whom I am dedicating this work, to my family and My parents
especially to my father (Mr. Rahim Bakhsh) for their support and patience and for the most precious
thing they have given me, their unconditional love, my sisters and my brothers (Mr. Amjad Ali, Mr.
Azmat Ali and Mr. Iftikhar Ali) whose infinite prayers, unwavering support kept my morale high during
difficult times. This work would have never been possible without their constant loving support. We
earned this together, and now I am ready for our next wonderful journey!
Shujat Ali
List of Figures
ii
List of Figures
Figure 1. 1: Comparative size of nano-materials and biological components . ............................................ 2
Figure 1. 2: History and Development of nanotechnology . ......................................................................... 3
Figure 1. 3: Oscillation of free electrons under the effect of an electromagnetic wave. ............................... 5
Figure 1. 4: Scheme of Bottom-Up and Top-Down approaches for the synthesis of NPs ........................... 9
Figure 1. 5: Schematic representation of the steps involved in the reduction method ................................ 10
Figure 1. 6: Schematic sketches of the different approaches for the synthesis of nanomaterials ............... 17
Figure 1. 7: Applications of nanocomposites in various fields . ................................................................. 19
Figure 1. 8: Schematic representation of various classes of β-lactam antibiotics ....................................... 30
Figure 3. 1: UV-Visible spectrum of Ceftriaxone stabilized silver nanoparticles ...................................... 46
Figure 3. 2: IR spectra of Ceftriaxone (black) and Cef-AgNPs (red) ......................................................... 47
Figure 3. 3: AFM analysis of Cef-AgNPs. Topography (A) and Particles size distribution (B) ................ 48
Figure 3. 4: Thermal stability of Ceftriaxone stabilized silver nanoparticles ............................................. 49
Figure 3. 5: Stability of Cef-AgNPs against various concentration of salt ................................................. 50
Figure 3. 6: Stability of Cef-AgNPs against pH ......................................................................................... 50
Figure 3. 7: UV-Visible spectrum of Ceftriaxone stabilized gold nanoparticles ........................................ 51
Figure 3. 8: IR spectra of Ceftriaxone (black) and Cef-AuNPs (red) ......................................................... 53
Figure 3. 9: AFM analysis of Cef-AuNPs. Topography (A) and Particles size distribution (B) ................ 53
Figure 3. 10: Thermal stability of Ceftriaxone stabilized gold nanoparticles ............................................. 54
List of Figures
iii
Figure 3. 11: Stability of Cef-AuNPs against various concentration of salt ............................................... 55
Figure 3. 12: Stability of Cef-AuNPs against pH ....................................................................................... 56
Figure 3. 13: MIC of Ceftriaxone (1), Cef-AgNPs (2), Cef-AuNPs (3), bare AgNPs (4) and bare AuNPs
(5). ........................................................................................................................................... 57
Figure 3. 14: AFM images of Escherichia coli ATCC 8739 before treatment. .......................................... 58
Figure 3. 15: E. coli treated with 1 mg Ceftriaxone (a), 1 mg Cef-AgNPs (b) and 1 mg Cef-AuNPs (c) for
2 hrs ......................................................................................................................................... 59
Figure 3. 16: E. coli treated with 1 mg Cef-AgNPs (a) and 1 mg Cef-AuNPs (b) for 1 hr........................ 60
Figure 3. 17: E. coli treated with 5 mg Ceftriaxone (a), 5 mg Cef-AgNPs (b) and 5 mg Cef-AuNPs (c) for
2 hrs ......................................................................................................................................... 60
Figure 3. 18: E. coli treated with 5 mg Ceftriaxone (a), 5 mg bare AgNPs (b) and 5 mg bare AuNPs (c) for
8 hrs ......................................................................................................................................... 61
Figure 3. 19: UV-Visible spectrum of Cefadroxil stabilized silver nanoparticles ...................................... 63
Figure 3. 20: IR spectra of Cefadroxil (black) and Cefd-AgNPs (red) ....................................................... 64
Figure 3. 21: AFM analysis of Cefd-AgNPs. Topography (A) and Particles size distribution (B) ............ 64
Figure 3. 22: Thermal stability of Cefadroxil stabilized silver nanoparticles ............................................. 65
Figure 3. 23: Stability of Cefd-AgNPs against various concentration of salt ............................................. 66
Figure 3. 24: Stability of Cefd-AgNPs against pH ..................................................................................... 67
Figure 3. 25: UV-Visible spectrum of Cefadroxil stabilized gold nanoparticles ........................................ 68
Figure 3. 26: IR spectra of Cefadroxil (black) and Cefd-AuNPs (red) ....................................................... 69
Figure 3. 27: AFM analysis of Cefd-AuNPs. Topography (A) and Particles size distribution (B) ............ 69
List of Figures
iv
Figure 3. 28: Thermal stability of Cefadroxil stabilized gold nanoparticles ............................................... 70
Figure 3. 29: Stability of Cefd-AuNPs against various concentration of salt ............................................. 71
Figure 3. 30: Stability of Cefd-AuNPs against pH ..................................................................................... 72
Figure 3. 31: MICs of Cefadroxil (1), Cefd-AgNPs (2), Cefd-AuNPs (3), bare AgNPs (4) and bare AuNPs
(5). ........................................................................................................................................... 73
Figure 3. 32: AFM images of Staphylococcus aureus ATCC 11632 before treatment ............................. 74
Figure 3. 33: AFM images of S. aureus treated with Cefadroxil (A), Cefd-AgNPs (B) and Cefd-AuNPs
(C) for 1 hr ............................................................................................................................... 74
Figure 3. 34: AFM images of S. aureus treated with Cefadroxil (A), Cefd-AgNPs (B) and Cefd-AuNPs
(C) for 2 hrs. ............................................................................................................................ 75
Figure 3. 35: AFM images of S. aureus treated with Cefadroxil (A), Cefd-AgNPs (B) and Cefd-AuNPs
(C) for 4 hrs ............................................................................................................................. 76
Figure 3. 36: AFM images of S. aureus treated with Cefadroxil (A), Bare AgNPs (B) and Bare AuNPs (C)
for 8 hrs ................................................................................................................................... 77
Figure 3. 37: UV-Visible spectrum of Cephradine stabilized silver nanoparticles ..................................... 79
Figure 3. 38: IR spectra of Cephradine (black) and Cpn-AgNPs (red) ....................................................... 80
Figure 3. 39: AFM analysis of Cpn-AgNPs. Topography (A) and Particles size distribution (B) ............. 81
Figure 3. 40: Thermal stability of Cephradine stabilized silver nanoparticles ............................................ 82
Figure 3. 41: Stability of Cpn-AgNPs against various concentration of salt .............................................. 83
Figure 3. 42: Stability of Cpn-AgNPs against pH ...................................................................................... 83
Figure 3. 43: UV-Visible spectrum of Cephradine stabilized gold nanoparticles ...................................... 84
Figure 3. 44: IR spectra of Cephradine (black) and Cpn-AuNPs (red) ....................................................... 85
List of Figures
v
Figure 3. 45: AFM analysis of Cpn-AuNPs. Topography (A) and Particles size distribution (B) ............. 86
Figure 3. 46: Thermal stability of Cephradine stabilized gold nanoparticles ............................................. 87
Figure 3. 47: Stability of Cpn-AuNPs against various concentration of salt .............................................. 88
Figure 3. 48: Stability of Cpn-AuNPs against pH ...................................................................................... 88
Figure 3. 49: MICs of Cephradine (1), Cpn-AgNPs (2), Cpn-AuNPs (3), bare AgNPs (4) and bare AuNPs
(5) ............................................................................................................................................ 90
Figure 3. 50: AFM images of S. aureus ATCC 25923 before treatment, Tophography (A) and 3D (B) ... 90
Figure 3. 51: AFM images of S. aureus treated with Cephradine (A), Cpn-AgNPs (B) and Cpn-AuNPs
(C) for 1 hr ............................................................................................................................... 91
Figure 3. 52: AFM images of S. aureus treated with Cephradine (A), Cpn-AgNPs (B) and Cpn-AuNPs
(C) for 2 hrs ............................................................................................................................. 92
Figure 3. 53: AFM images of S. aureus treated with Cephradine (A), Cpn-AgNPs (B) and Cpn-AuNPs
(C) for 4 hrs ............................................................................................................................. 93
Figure 3. 54: AFM images of S. aureus treated with Cephradine (A), bare AgNPs (B) and bare AuNPs (C)
for 8 hrs ................................................................................................................................... 94
Figure 3. 55: UV-Visible spectrum of Ampicillin stabilized silver nanoparticles ...................................... 96
Figure 3. 56: IR spectra of Ampicillin (black) and Mpn-AgNPs (red) ....................................................... 97
Figure 3. 57: AFM analysis of Mpn-AgNPs. Topography (A) and Particles size distribution (B) ............ 98
Figure 3. 58: Thermal stability of Ampicillin stabilized silver nanoparticles ............................................. 99
Figure 3. 59: Stability of Mpn-AgNPs against various concentration of salt ............................................. 99
Figure 3. 60: Stability of Mpn-AgNPs against pH .................................................................................... 100
Figure 3. 61: UV-Visible spectrum of Ampicillin stabilized gold nanoparticles ...................................... 101
List of Figures
vi
Figure 3. 62: IR spectra of Ampicillin (black) and Mpn-AuNPs (red) ..................................................... 102
Figure 3. 63: AFM analysis of Mpn-AuNPs. Topography (A) and Particles size distribution (B) .......... 103
Figure 3. 64: Thermal stability of Ampicillin stabilized gold nanoparticles ............................................ 104
Figure 3. 65: Stability of Mpn-AuNPs against various concentration of salt ........................................... 105
Figure 3. 66: Stability of Mpn-AuNPs against pH .................................................................................... 105
Figure 3. 67: MICs of Ampicillin (1), Mpn-AgNPs (2), Mpn-AuNPs (3), bare AgNPs (4) and bare AuNPs
(5). ......................................................................................................................................... 107
Figure 3. 68: AFM images of S. aureus ATCC 11632 before treatment, Tophography (A) and 3D (B) .. 108
Figure 3. 69: AFM images of S. aureus treated with Ampicillin (A), Mpn-AgNPs (B), Mpn-AuNPs for 1
hr ............................................................................................................................................ 109
Figure 3. 70: AFM images of S. aureus treated with Ampicillin (A), Mpn-AgNPs (B), Mpn-AuNPs for 2
hrs .......................................................................................................................................... 110
Figure 3. 71: AFM images of S. aureus treated with Ampicillin (A), Mpn-AgNPs (B) and Mpn-AuNPs
(C) for 4 hrs ........................................................................................................................... 111
Figure 3. 72: AFM images of S. aureus treated with Ampicillin (A), bare AgNPs (B) and bare AuNPs (C)
for 8 hrs ................................................................................................................................. 112
Figure 3. 73: UV-Visible spectrum of Ceftriaxone stabilized silver nanoparticles .................................. 114
Figure 3. 74: IR spectra of Ceftriaxone (black) and Cef-AgNPs (red) ..................................................... 115
Figure 3. 75: AFM analysis of Cfm-AgNPs. Topography (A) and Particles size distribution (B) ........... 115
Figure 3. 76: Thermal stability of Cefixime stabilized silver nanoparticles ............................................. 116
Figure 3. 77: Stability of Cfm-AgNPs against various concentration of salt ............................................ 117
Figure 3. 78: Stability of Cfm-AgNPs against pH .................................................................................... 117
List of Figures
vii
Figure 3. 79: UV-Visible spectrum of Cefixime stabilized gold nanoparticles ........................................ 119
Figure 3. 80: IR spectra of Cefixime (black) and Cfm-AuNPs (red) ........................................................ 119
Figure 3. 81: AFM analysis of Cfm-AgNPs. Topography (A) and Particles size distribution (B) ........... 120
Figure 3. 82: Thermal stability of Cefixime stabilized gold nanoparticles ............................................... 121
Figure 3. 83: Stability of Cfm-AuNPs against various concentration of salt ............................................ 122
Figure 3. 84: Stability of Cfm-AuNPs against pH .................................................................................... 123
Figure 3. 85: MICs of Cefixime (1), Cfm-AgNPs (2), Cfm-AuNPs (3), bare AgNPs (4) and bare AuNPs
(5). ......................................................................................................................................... 124
Figure 3. 86: AFM images of S. aureus ATCC 25923 before treatment, Tophography (A) and 3D (B) . 125
Figure 3. 87: AFM images of S. aureus treated with Ampicillin (A), Mpn-AgNPs (B) and Mpn-AuNPs
(C) for 1 hr ............................................................................................................................. 126
Figure 3. 88: AFM images of S. aureus treated with Ampicillin (A), Mpn-AgNPs (B) and Mpn-AuNPs
(C) for 2 hrs ........................................................................................................................... 127
Figure 3. 89: AFM images of S. aureus treated with Ampicillin (A), Mpn-AgNPs (B) and Mpn-AuNPs
(C) for 4 hrs ........................................................................................................................... 128
Figure 3. 90: AFM images of S. aureus treated with Ampicillin (A), bare AgNPs (B) and bare AuNPs (C)
for 8 hrs ................................................................................................................................. 129
Figure 3. 91: IR spectra of Ceftriaxone (black) and Cef-PEG (red) ......................................................... 131
Figure 3. 92: AFM analysis of Cef-PEG. Topography (A) and Particles size distribution (B) ................ 132
Figure 3. 93: In-vitro drug release study of Cef-PEG (pH 7.4) at 37°C ................................................... 133
Figure 3. 94: MICs of Cefrtiaxone against E.coli (1), Cef-PEG against E. coli (2), Cefrtiaxone against S.
aureus (3) and Cef-PEG against S. aureus (4) ...................................................................... 134
List of Figures
viii
Figure 3. 95: IR spectra of Cefixime (black) and Cfx-PEG (red) ............................................................. 135
Figure 3. 96: AFM analysis of Cfx-PEG. Topography (A) and Particles size distribution (B) ................ 136
Figure 3. 97: In-vitro drug release study of Cfx-PEG (pH 7.4) at 37°C ................................................... 137
Figure 3. 98: MICs of Cefixime S. aureus (1), Cfx-PEG against S. aureus (2), Cefixime against E. coli (3)
and Cfx-PEG E. coli (4) ........................................................................................................ 137
Abbreviations
ix
Abbreviations
AFM
Atomic Force Microscope
AgNO3 Silver Nitrate
AgNPs
AuNPs
Silver nanoparticles
Gold nanoparticles
DCM Dichloromethane
FT- IR
MIC
Cfx-PEG
Cef-PEG
Fourier Transform Infra-Red
Minimum inhibitory concentration
Cefixime encapsulated with polyethylene glycol
Ceftriaxone encapsulated with polyethylene glycol
HAuCl4 Tetrachloroauric acid
MeOH Methanol
TEA Trimethylamines
UV
SPB
PEG
PVA
S. aureus
E. coli
NPs
3D
KBr
EE
Ultra Violet
Surface Plasmon band
Polyethylene glycol
Polyvinyl alcohol
Staphylococcus aureus
Escherichia coli
Nanoparticles
Three dimensional
Potassium bromide
Entrapment efficiency
Abstract
x
Abstract
This PhD dissertation focusses on antibiotics coated silver and gold nanoparticles (NPs), analysis
of their photo-physical and enhanced antibacterial properties.
The drug resistant bacteria are increasing day by day due to irrational use of antibiotics. Bacterial
resistance towards the existing antibiotics is a global health issue and these drugs are at high
risks in this regard. To overcome this problem new methodologies and measurements are
dreadfully needed. In this context, the present study was designed to modify some selected
antibacterial drugs through nanochemical approach to enhance their antibacterial potential. The
beta-lactam antibiotics are most commonly used for the treatment of bacterial infections. Silver
and gold NPs stabilized with these antibiotics were successfully synthesized though chemical
reduction method. The NPs were characterized with Ultra-Violet visible spectrophotometry,
Fourier transform infra-red spectroscopy (FTIR) and atomic force microscopy (AFM). The
analysis confirmed the formation of poly-dispersed NPs of size less than 100 nm. The NPs were
found stable at high temperature (up to 100oC), at various pH range and in different salt
concentrations. The antibacterial potential of conjugated antibiotics were compared with pure
antibiotics and unconjugated gold and silver NPs using AFM and conventional techniques such
as the agar well diffusion method. Analysis of bacterial cells surface topography was recorded
under AFM before and after treating with the antibiotics conjugated with Ag and Au NPs, free
antibiotics and bare Ag and Au NPs. Conjugation to AgNPs enhanced the antibacterial activity
of Ceftriaxone by 2 times, and conjugation to AuNPs by 6 times. The antibacterial potential of
Cefadroxil was enhanced up to 2 and 3 times on conjugation with AgNPs and AuNPs,
respectively. Similarly, the antibacterial potential of Cephradine was enhanced up to about 2
times on conjugation with AgNPs and conjugation to AuNPs by about 6 times. It was found that
Ampicillin conjugated to Ag and Au NPs are about 5 and 10 times more active than pure
Abstract
xi
Ampicillin, respectively. Similarly, Cefixime conjugated to Ag and Au NPs are about 3 and 8
times more active than pure Cefixime, respectively. The study also explored the improved
kinetics of the antibiotics as the drugs coated with the NPs destroyed bacteria more timely than
the free drugs. The antibiotics were also encapsulated with polymers to create nanoscale
materials. Ceftriaxone and Cefixime were successfully encapsulated with polyethylene glycol
(PEG). The polymeric nanosized Ceftriaxone and Cefixime were found more active than their
respective free drugs.
Chapter-1 Introduction
1
CHAPTER-1
INTRODUCTION
1.1 Nanotechnology
Nanotechnology is the research to design, manufacture and manipulate small particles which
have dimension smaller than 100 nm. In other words nanotechnology deals with synthesis of
nanomaterials (range from 1 to 100 nm) of variable shapes and their applications in different
fields. The word nano is from Greek, it means “dwarf”. A nanometer is one billionth of a meter,
or approximately the length of three atoms linked side by side. The length of C-C bond is from
0.12 to 0.15 nm, a DNA double helix has diameter about 2 nm (Figure 1.1). Nanotechnology is a
vast field and is associated with different areas of science as organic chemistry, surface science,
physics, molecular biology and microfabrication, thus providing a connected area of research and
applications [1]. It is a fascinating and fast emergent field of science which presents materials
exhibiting structural features among those of atoms and bulk materials with a minimum of one
dimension in the nano-scale [2]. Nanotechnology creates a foremost attention in the development
of nanomaterials and is an attractive research area in terms of exploitation of nanoparticles due to
their particular physicochemical uniqueness [3]. The dimensions of nanomaterials is much closed
to biological components such as proteins and DNA as shown in figure 1.1. The various tools
developed through nanotechnology are used for diagnosis and treatment of several diseases at the
molecular scale [4, 5]. Nanotechnology creating new biological and chemical nanostructures,
explore their novel characteristics and discover how to assemble these structures into complex
functional devices. It is the field of nanotechnology that imparts a mark in research and building
an impact in the spheres of life.
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2
Figure 1. 1: Comparative size of nano-materials and biological components [6].
1.1.1. History of Nanotechnology
Nanoparticles is considered a discovery of modern science era but actually it has a very long
history. Its emergence was marked when in 1959 R. Feyman delivered his lecture “There is
Plenty of Room at the Bottom” at the annual meeting of the “American Physical Society” [7].
This narrated the probability of manipulation of matter at atomic level. The word
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3
nanotechnology was coined in the year 1974 by Prof. Norio Taniguchi for precision
manufacturing of material at the nanoscale [8]. The discovery of scanning tunneling microscope
(STM) in 1981 offered extraordinary imagining and was effectively used as a suitable tool for
the analysis of nanometarials [9]. The discovery of fullerenes in 1985 explored the conceptual
framework of nanotechnology [10]. Commercial use of nanomaterials started in early 2000 but
relatively limited to the bulk application of nanomaterials, rather than the transformative
applications anticipated by the field. In the last few decades, nanotechnology developed
extensively and rising as a cutting edge technology interdisciplinary with material science,
chemistry and biology. Similarly, bio-nanotechnology and bio-nanoscience offered research that
operates at the edge of medicine, chemistry, biology, engineering and materials sciences (Figure
1.2). The integration of nanotechnology is expected to accelerate in the next decade with
information technology, biotechnology and cognitive science.
Figure 1. 2: History and Development of nanotechnology [11].
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4
1.2 Nanomaterials
Material can be broadly classified on the basis of their size into Macroscopic and Mesoscopic
materials. Macroscopic particles are visible to the naked eye while Mesoscopic can be seen with
optical microscopes. In the gap between the mesoscopic and microscopic there is another
category of matter, the nanoscale particles or nanoscopic particles [12]. According to
nanotechnology, a particle is a tiny object that behave as a whole unit. In broad sense particles
are classified in the following three categories according to their diameter.
• Coarse particles have size range from 2,500 nm to 10,000 nm.
• Fine particles have size from 100 nm to 2,500 nm.
• Ultrafine particles, or nanoparticles show structural features in the size less than 100 nm.
Nanomaterials are objects that have structural constituents smaller than 1 µm in at least one
dimension. While building blocks (atomic and molecular) of matter are considered
nanomaterials, as a bulk crystal with lattice spacing of nanometers but overall dimensions, are
usually excluded [6]. Nanoparticle can be defined as any object (conductor, insulator or
semiconductor) which is synthesized controllably in the size-range of about 1 to 100 nm [13]. In
other words nanoparticle is a small entity that behaves as a whole unit and is the bridge between
bulk materials and atomic or molecular structures. At this dimension and size range nanoparticles
achieve size dependent properties different from their respective bulk material or an atomic
cluster. Nanotechnology has engineered different nanomaterial such as nanotubes [14], nanorods
[15], fullerene [16], nanoelectronics [17], nanoionics [18] and nanopillars [19]. Agglomerates of
nanoclusters or nanoparticles are termed as nanopowders. Crystals of nanometer-sized or
ultrafine particles are commonly called nanocrystals. Nanocomposites are multiphase solid
materials which have at least one of the phases in nanoscale (less than 100 nm).
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1.3 Metals nanoparticles
Metal based nanomaterials are usually called metal nanoparticles. Metallic nanoparticles are
coming up with great imputes, because of their promising properties and unique characters like
high surface to volume ratio and greater surface energy [20]. Several methods have been adopted
for the manufacturing of metal nanoparticles. Commonly used reducing agents for the production
of metal nanoparticles comprised; sodium borohydride, polyols, hydrazine, N,N-
dimethyformamide and sodium citrate [21]. Due to the magnetic interaction and Vander Waals
forces, metal nanoparticles synthesized by chemical approaches usually undergo accumulation.
This agglomeration decreasing the interfacial free energy and specific surface area, lead to
reduce particles reactivity. To prevent agglomeration the surfaces of the particles are coated with
suitable stabilizers. The metallic nanoparticles of bare zinc, titanium, copper, gold, magnesium
and silver have been synthesized and used for various purposes [22, 23]. The nanoparticles of
noble metals are considered non-toxic and have high thermal stability; thus, adding value to their
medical applications [24, 25]. Metal nanoparticles showed wonderful antimicrobial potential,
and used in burn treatment, dental materials, water treatment and as antimicrobial pigments [20].
Metal nanoparticles have coordinated specific electronic structures and the capability to
accumulate excess electrons. These electronic cloud in the metal nanoparticles are in oscillation
relative to the metal core (Figure 1.3) in response to the electromagnetic field providing the base
for the surface-plasmon-resonance [26].
Figure 1. 3: Oscillation of free electrons under the effect of an electromagnetic wave.
Chapter-1 Introduction
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1.3.1 Silver nanoparticles
In ancient time metallic silver has been used for different purposes, i.e. jewelry, coins, silver
vessels for preservation of food stuffs and water to avoid bacterial growth as well as lot of other
applications in the field of health and medicines [27]. M. C. Lea reported the creation of a citrate
stabilized silver nanomaterials for the first time in 1889 [28]. In the modern era, silver
nanoparticles have engrossed much interest of researchers and technologists to design silver-
based nanomaterials for various applications [29]. Similarly a large number of approaches have
been adopted for the synthesis of silver nanoparticles (AgNPs) including physical, chemical and
biological approaches [29, 30]. The unique behavior of AgNPs improved technological methods
as well as present electronics, sensors and optical devices. It could be reflected from publications
on their synthesis and applications in reputed journals. AgNPs are found the most frequently
exploited nanomaterials in medical sciences and they are also reported as major part of the
commercial products. AgNPs have been the focus of study in modern era owing to their
distinctive chemical, physical, and biological characteristics and magnificent applications in drug
delivery, catalysis, biosensing and nanodevices fabrication [31]. Studies on AgNPs demonstrated
that their optical, catalytic and electromagnetic properties are intensely swayed by their size and
shape, which can be assorted using various synthetic approaches, reaction conditions, stabilizers
and reducing agents. Hence, various morphological nanomaterials obtained from different
techniques. The cherished optical properties of AgNPs lead to new approaches in imaging and
sensing applications, providing an extensive range of detection modes, such as scattering,
colorimetric, and SER (surface enhanced Raman) scattering procedures, at very low detection
limits [32].
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1.3.2 Gold Nanoparticles
Among the noble metals gold has been used since ancient time in scientific research. Michael
Faraday worked on gold in 1850s and changed the chemistry of gold into a fascinating scientific
work [33]. Gold nanoparticles (AuNPs) hold a significant position in nanotechnology due to their
interesting shape and size dependent characteristics, non-toxic behavior, ease of synthesis and
wide spread applications. AuNPs exhibit special advantages in nanotehnology due to their small
size, greater surface area and distinctive behavior. AuNPs can be synthesized in different shapes
as gold nanorods, nanospheres, nanocages, nanobelts, nanostars and nanoprism [34]. Various
approaches have been established for the production of gold nanoparticles including chemical,
physical and biological methods [35]. Revolutionary changes have been molded by AuNPs in the
field of medicines and is being used in diagnosis, therapeutics, targeted-drug-delivery and
imaging. These properties are developed owing to their stability, inert nature, high dispersity,
biocompatibility and non-cytotoxicity [36, 37].
1.4 Synthesis of nanomaterials
Due to the unique characteristics of nanoparticles, such as catalytic activity, magnetic properties,
electronic properties, optical properties and antibacterial properties, they are getting the interest
ofresearchers for their novel procedures of synthesis. Metal nanoparticles are generally
synthesized through chemical, physical, biological and mechanical approaches [38-42],
electrochemical techniques [43], photochemical reactions in reverse micelles [44],one phase
synthesis in organic solvents [45], two-phase synthesis [46] and by green chemistry process [47].
Comprehensively nanostructures in various forms such as nanocubes [48], nanowires [39, 50],
nanorods [51], nanoplates [52], nanotadpoles [53] and nanobelts [54] have been synthesized by
chemical, physical and biological approaches. They demonstrate significant different optical
Chapter-1 Introduction
8
characteristics as the gold nanorods show two absorption peaks, whereas the sphere-shaped
display only a single surface plasmon resonances peak [55].
In broad sense two main approaches are used for the synthesis of nanoparticles.
Top-down approach
In this method nano-sized objects are synthesized from bulk material without atomic-level
control. This approach involves starting with a bulk material and renovating or refining it down
to the desired size and shape [56]. The principle behind this approach is the modification of a
bulk piece of the substantial into the desired nanoparticles and following stabilization of the
subsequent nanomaterials by the addition of suitable protecting agents. The frequently used top
down methods are milling, grinding and drilling etc. (Figure 1.4).
Bottom-up approach
In this method nanomaterials are shaped from molecular components which collect themselves
chemically and arrange into more complex structures [57]. In this method nanometerials are
build-up from the 'bottom', i.e. starting from atom, molecule or cluster (Figure 1.4). The bottom-
up approach is a better way to synthesize nanoparticles of desired shape and size, homogeneous
chemical composition, better optical and electronic properties [58].
Chapter-1 Introduction
9
Figure 1. 4: Scheme of Bottom-Up and Top-Down approaches for the synthesis of NPs [59]
Comprehensively nano-materials are synthesized using various chemical, physical and biological
approaches (Figure 1.6) some of them are as under.
1.4.1 Chemical reduction
It is the most commonly used process for the synthesis of nanoparticles as stable, colloidal
dispersions in organic solvents or water (Figure 1.5). This method includes the use of reducing
agents like citrate, triethylamine, borohydride and elemental hydrogen. Reduction of metal ions
generally creates colloidal metal with particle diameters in nanometers. Synthesis of metal NPs is
established on a two-step reduction method, in which a strong reducing agent is used to generate
small metal particles, followed by further reduction with a weaker reducing agent to enlarge the
particle size [60]. However, the initial sol was not consistent and specific apparatus are required,
therefore, the synthesis of nanoparticles by chemical reduction method is most often carried in
the existence of stabilizers or capping agents in order to prevent useless agglomeration [61]. In
chemical approaches, metal ions are reduced to neutral atoms and then stabilized with a suitable
capping agents. It is more suitable way to obtain significantly small nanoparticles of desired size
Chapter-1 Introduction
10
and uniform shapes. Flocculation is a hurdle in obtaining small sized and uniform nanoparticles.
Therefore appropriate stabilizer or capping agents are added during the reduction of the metal for
the neutralization of the electrostatic repulsive force. Various compounds that have reactive
groups like amine, thiol, thiosulfate, sulphide, isocyanide, xanthate, and selenide have been used
to stabilize different metal NPs including gold (Au), silver (Ag), copper (Cu), platinium (Pt),
palladium (Pd), and nickle (Ni) by self-assembly [62-64]. For example, derivatives of thiol have
been used as good capping agent/ stabilizers, as they have potent chemical attraction to
functionalize the nanoparticles by creation of layer on the surface [65]. Amines including simple
primary amines [66], multifunctional amino polymers [67] and amino acids [68, 69] have been
used as reducing and stabilizing agents in the synthesis of nanoparticles.
Figure 1. 5: Schematic representation of the steps involved in the reduction method
Chapter-1 Introduction
11
1.4.2 Solvent antisolvent precipitation
In this procedure clear solution of substance is injected with a specific rate into the water
containing a suitable stabilizer under stirring. Precipitation of solid nanoparticles occurs upon
mixing at room temperature. The suspension formed is centrifuged and the resultant
nanoparticles are washed with purified water to remove the unwanted materials. Stabilizers such
as Sodium dodecyl sulfate (SLS), Hydroxypropyl methylcellulose (HPMC), Polyvinyl alcohol
(PVA), Polyvinyl acetate (PVAc) and Polyethylene glycol PEG are used for this purpose [70].
Different types of protocols are used under this technique [71] as the evaporative precipitation of
nanosuspension (EPN) and antisolvent precipitation with a syringe pump (APSP). For the APSP
method, the solution of the substance is introduced at a fixed flow rate into the antisolvent of a
specific amount under stirring. Different ratios of the solution to antisolvent are used to optimize
the reaction; nanoparticles synthesized are filtered and dried. In the EPN scheme, the solution of
the substance is prepared in a specific solvent and then a nano-suspension is produced by
addition an antisolvent. Nnanoparticles in the nano-suspension are obtained by evaporation of
the antisolvent and solvent [72].
New methods such as “supercritical antisolvent with enhanced mass transfer” (SAS-EM), is a
modified type of the usual supercritical antisolvent (SAS) method. In this technique supercritical
carbon dioxide is used as an antisolvent. The adaptation in the SAS-EM method is that it atomize
the solution jet into microdroplets by using a surface, vibrating at an ultrasonic frequency.
Furthermore, the ultrasound field also improves turbulence and mixing within the supercritical
phase which cause high mass transfer between the antisolvent and solution. These effects provide
particles about tenfold smaller than those synthesized by the usual SAS process [73].
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12
1.4.3 Hydrothermal Method
Hydrothermal synthesis can be defined as crystal growth or crystal production under elevated
temperature and pressure from materials which are insoluble in normal temperature and pressure
[74]. In this technique material are dissolved in aqueous medium at elevated temperature and
pressure, followed by crystallization of the dissolved substances from the fluid. At elevated
temperatures, water plays an important role in the transformation of originator material. The
characteristics of the reactants such as their solubility and reactivity also alter at elevated
temperatures. These changes offer more factors to synthesis high quality nanoparticles and
nanotubes, which cannot be obtained at low temperature. In the synthesis of nanocrystals, factors
such as temperature, pressure, reaction time and the corresponding precursor-product system, are
adjusted to retain narrow particle size distribution. Different types of nanostructures such as
nanowires and nanorods have been successfully produced by this technique [75, 76].
1.4.4 Pyrolysis
In this method chemical precursors decompose into solid compounds and the undesirable waste
constituents evaporate [77]. Generally the pyrolysis leads to the production of powders with size
distribution in the micrometer scale. Preparation system and reaction conditions like decreasing
of the reaction rate or decomposition of the precursor in an inert solvent are adjusted to develop a
uniform nanoscale material. This technique can be used to prepare carbon nanotubes, composite
materials and several types of nanoparticles including metals, metal oxides and semiconductors
[78-80]. Pyrolysis of the organic precursors provides a way of manufacturing nanotubes of
different kinds such as aligned and Y-junction carbon nanotubes. When organometallics are used
as precursors, carbon nanotubes obtained are further used to prepare other important
nanostructures [81].
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1.4.5 Chemical vapor deposition
In this method vaporized precursors are put into a chemical vapor deposition (CVD) reactor and
adsorb onto a material held at high temperature. These deposited molecules decompose
thermally or react with other vapors to form crystals [82]. Usually the CVD technique contains
three steps, first step is the transfer of reactants to the growth surface, the second step is chemical
reactions on the growth surface and the last step is the removal of the gas phase reaction by-
products from the surface. CVD permits the development of multi-component nanoparticles by
employing multiple precursors. Techniques such as inert gas condensation, ion sputtering, pulsed
laser ablation, flame synthesis and thermal plasma synthesis fall under the vapor phase process
and are commonly used to synthesize variety of nanoparticles [83].
1.4.6 Bio-based Protocols
Biological systems such as microbes [84, 85] fungi [86], enzyme [87], polysaccharide [88] and
plant extracts [89] have been used to synthesize metal nanoparticles of great interest. Extracts
from living sources may act both as capping as well as reducing agents in NPs synthesis. The
reduction of metal ions by these bio-molecules such as polysaccharides, proteins, enzymes,
amino acids and vitamins is environment friendly and termed as green synthesis of nanoparticles.
These bio-inorganic constituents can be exceptionally complex in both structure and function,
and also display attractive hierarchical sorting from the nanometer to the macroscopic length
scale, which cannot be achieved in laboratory. The biomedically valid gold nanoparticles have
been produced using marine brown algae Turbinaria conoides which showed antibacterial
activity especially against Streptococcus species [90]. Fungus like Curvularia inaequalis is a
novel potential candidate for unconventional bio-synthesis of silver nanoparticles with
antimicrobial activity [91]. The exploitation of microorganisms, such as bacteria [92, 93], yeast
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14
and fungi [94] in the production of nanoparticles is a comparatively new area of research in the
field of nanotechnology.
1.4.7 Sol-gel process
In sol gel technique actually inorganic polymerization reactions occur [95]. This method
comprises four steps, namely hydrolysis, poly-condensation, drying and thermal-decomposition.
Size of the sol particles depends on the condition such as solution composition, temperature and
pH. Size of the particles can be adjusted by adjusting these factors. This technique has been
applied for the synthesis of metal oxide nanostructures [96, 97]. A simple way of sol-gel
procedure is applied to produce uniform sized SnO2 nanoparticles using PEG as stabilizing
reagent [98].
1.4.8 Reverse micelle method
Reverse micelles are spherical aggregates shaped by the self-assembly of surfactants in a
nonpolar solvents, while micelles are spherical aggregates produced by the self-assembly of
surfactants in water [99]. The size of reverse micelles can be modulated in the nanometer range
by different factors. Most important among these is the water-surfactant molar ratio
(water/surfactant). Droplet reverse micelles are generally not stable and a dynamic exchange
phenomenon occurs among the colliding droplets. The definite adsorption of surfactants on
inorganic substances offer the use of reverse micelles as successful nano-reactors for the
production of nanomaterials with desired shape, size, composition, and structure. Several
modified procedures are used for the production nanoparticles through reverse micelle method.
Usually micro-emulsions are prepared from metal salts which contained metal ions and a
precipitating agent (NaOH etc) is added for the production of nanoparticles. Standard solution of
metal salt is prepared in double distilled water to get metal ions. Known volume of this solution
Chapter-1 Introduction
15
is taken and mixed with specific amount of surfactant (Cyclohexane and Tergitol NP-9 etc) and
co-surfactant (n-octanol). These micro-emulsions convert into a transparent solution after stirring
overnight at room temperature. Usually the solution is heated to get a precipitate which is
washed with acetone, centrifuged and then dried. The powder thus obtained is further heated in
air to get pure powders. It is necessary to maintain the stoichiometry in different micro-emulsion
systems according to the molecular formula. Mono-phasic, homo-geneous nanostructures of
LaCaMnO3, LaSrMnO3 and LaMnO3 have been synthesized using this technique [100].
1.4.9 Chemical precipitation
This technique uses the kinetics of nucleation and particle development in uniform solutions,
which can be adapted by the meticulous release of cations and anions. Mono-disperse
nanoparticles can be produced by carefully controlling the kinetics of the precipitation, pH,
temperature, use of suitable surfactant and the concentration of the reactants and ions. These are
the factors which determine the precipitation process. Therefore, it is essential to control these
factors. By regulating these influences nanoparticles with desired size distributions can be
produced [101]. Synthesis of Ferric Chloride Doped Zinc Sulphide nanoparticles have been
synthesized via this method [102].
1.4.10 Green Chemistry approaches
Chemical and physical methods are the most admired approaches for the synthesis of
nanomaterials. Though, several chemical approaches cannot evade the utilization of hazardous
substances in the synthesis of nanoparticles. An emergent need is to design ecofriendly methods
of nanoparticles synthesis that do not use hazardous materials. Nanoparticles of noble metals
such as silver, platinum and gold are commonly applied to human communicating areas. The use
of environmentally benevolent resources like plant extract [103-105] glucose [47], chitosan
Chapter-1 Introduction
16
[106], soluble starch [107], microorganisms [108] and fungus [109, 110] have been used as
substitute reducing, stabilizing and capping agents. This offers frequent advantages of eco
friendliness and compatibility for biomedical and pharmaceutical uses as they do not utilize toxic
materials in the synthesis approaches [101].
1.4.11 Microwave irradiation
In this method metal salt solution are prepared and small amount of a base is added dropwise
with magnetic stirring to get a colloid system, which was maintained at room temperature. Then,
the reaction mixture is transferred into a microwave heating instrument and heated at selected
temperature for a definite time. Then, the reaction mixture is cooled to ambient temperature. The
precipitate is collected and washed to remove the unwanted material [111, 112]. Simple and one
step microwave irradiation technique can be used for the synthesis of nanoparticles using
specific reducing agent. Metal salt solution mixed with citric acid as reducing agent is stirred and
then heated in a microwave oven. Color of the solution changes, which indicates the formation of
nanoparticles. The reactions take place under microwave irradiation in short duration and can be
usefully exploited for the generation of nanostructures [130].
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17
Figure 1. 6: Schematic sketches of the different approaches for the synthesis of nanomaterials
1.5 Characterization of nanoparticles
Nanoparticles synthesized through any method must be characterized in order to know their
fundamental properties such as size, the net charge, mono-dispersity, adsorption to biomolecules,
aggregation, stability and flocculation in various media. This provides fundamental information
in terms of use of these nanoparticles. Characterization of the synthesized nanoparticles is carried
out by using a variety of different procedures. The common techniques used are UV-visible
spectrophotometry, FT-IR spectroscopy, Atomic Force Microscopy (AFM), Transmission
Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), X-rays Diffraction (XRD),
Nanoparticle Surface Area Monitor (NSAM), Time of Flight Mass Spectroscopy (ATFMS),
Photon Correlation Spectroscopy (PCS), Scanning Mobility Particle Sizer (SMPS), Aerosol
Chapter-1 Introduction
18
Condensation Particle Counter (CPC), Differential Mobility Analyzer (DMA), Aerosol Particle
Mass Analyzer (APM) and Nanoparticle Tracking Analysis (NTA) [113-115].
1.6 Applications of nanoparticles
Nanotechnology is one of the most composite and brainstorming specialized area of research that
combines the efforts of professional chemists, material scientists, physicists, mathematicians,
physicians and several others. Nowadays nanotechnology is one of the most attractive areas of
scientific research, due to its broader applications in different interdisciplinary fields. Shape and
size-controlled nanoparticles and their assemblies possess application in various fields [116].
Among these, noble metals nanoparticles are getting much attention owing to their use in various
fields of science and technology.
The applications of nanoparticles is outlined in figure 1.7. Among the well-known applications
of NPs, some examples are catalytic compounds, microelectronics, synthetic rubber, inks and
pigments, scientific instruments, photographic supplies, ultrafine polishing compounds, surface
disinfectants,dental materials, coatings and adhesives, synthetic bone, optical fiber cladding,
cosmetics and UV absorbers for sun screens, pharmaceutics and drug delivery systems. Few
significant applications of nanomaterials are describe below.
Chapter-1 Introduction
19
Figure 1. 7: Applications of nanocomposites in various fields [116].
1.6.1 Electronics
Nanoparticles enhance local electric fields and this lead to their application as Surface Enhanced
Raman Scattering (SERS) [117]. The enhancement enables the recognition of individual
molecules absorbed on metal particles [118, 119]. Electronics based nanoparticles can be
exploited to produce digital displays which are brighter in color as well as cheaper [120]. Silver
Nanoparticles are valuable for production of high conductivity devices for printed electronics.
These nanoparticles convert readily at low temperatures to very conductive silver elements
Chapter-1 Introduction
20
suitable for cheaper, printed electronic applications [121]. Gold nanoparticles can be used as
links in resistors, conductors and electronic chips. Low resistance printable gold nanoparticles
serve as conductors for flexible electronics ranging from printable inks to electronic chips [122].
1.6.2 Catalysis
Metals in the form of nanoparticles are catalytically active, gold is considered to be un-reactive
in the bulk form. Though, clusters of gold are established to be catalytically active [123]. Metal
nanoparticles are suitable for catalyzing the redox reactions, which can be monitored through
electro analytical approaches [124]. The increasing percentage of surface atoms with lessening
particle size makes the nanoparticles very reactive and is used effectively in heterogeneous
catalysis [125].
The application of nanoparticles in catalysis is used in fuel cell, catalytic converters,
photocatalytic devices and for organic synthesis [126]. Catalytic degradation of hazardous
organic dyes such as methylene blue, methyl orange, and eosin Y can be carried in the presence
of silver nanoparticles [127]. Silver nanoparticle catalytically affect the electron transfer
reduction of [Co(NH3)5Cl](NO3)2 by iron (II), the rate is significantly influenced in presence of
nanoparticles [128].
1.6.3 Plasmonics
Plasmonics is the localization, directing and exploitation of electromagnetic waves beyond the
diffraction limit and down to the nanoscale [129]. The localized surface plasmon resonance of a
nanoparticle is accountable for its capability to absorb and disperse light at definite wavelengths
this property of silver can be used for plasmonic applications [130]. In plasmonics, metal
nanoparticles act as antennas to change light into localized electric fields or to route light into
desired location with nanometer exactness. These uses are made feasible through a strong
Chapter-1 Introduction
21
interaction between incident light and free electrons in the nanoparticles. Plasmonic structures
can be used in the detection of molecules as the silver and gold nanoparticles act as biosensor
[131]. The chemically functionalized films bind to a target molecule like DNA strand or protein
and the dielectric environment near the surface of the metal film is distorted. Consequently, the
change in coupling geometry is used to monitor binding between the metal film and the
excitation source necessary to create propagating surface Plasmon (PSP) [132].
1.6.4 Waste water treatment
Biological and chemical contaminants have threatened the quality of drinking water. Variety of
approaches are used for the purification of drinking water. Substantial progress has been made to
use nanoparticles for waste water purification [133]. Silver nanoparticles and graphene oxide
nanosheets composites are used as a germicidal agent for water cleaning. Micron-scale
nanosheets facilitate them to be simply placed on porous ceramic sheaths making them a
potential biocidal material for water disinfection [134]. Heavy metal ions can cause direct effect
on human health and lead to lethal human illnesses. Advanced techniques for the recognition of
metal ions not only can offer perception into the physiological action of the heavy metal ions but
also in vast claim for waste management and drinking water safety. Numerous techniques have
been used to notice metal ions existing in environmental or biological specimens, though,
compatibility with aqueous environments and easiness for sampling remain considerable
challenges for many of these approaches. Nanotechnology has led to the development of novel
recognition mechanisms for heavy metal ions [135]. Heavy metals, drugs and dyes in wastewater
are main environmental problems as they are usually resistant to degradation by biological
management techniques. Thus, research has focused to optimize adsorption and development of
Chapter-1 Introduction
22
novel substitute adsorbents with high adsorptive capacity and cheapness. Consequently, much
concentration has currently been paid to nanotechnological methods [136].
1.6.5 Agriculture
Nanotechnology can improve agricultural production, and if it in formulate agrochemicals for
applying fertilizers and pesticides for high crop production, nanosensors for identification of
diseases, recognition of residues of agrochemicals, detection of herbicides and nano strategies for
the genetic manipulation of plants [137, 138]. The effect of Zinc oxide NPs have studied on
peanuts and found that it promote seed germination, seedling vigor, plant growth and
enlargement of stem and root in peanuts [139]. Nanoparticles can also be used as nano-fertilizers,
these nanofertilizers enahance the yield of crops [140].
1.6.6 Health and Medicines
Nanotechnology is a fast emergent field of science, providing nanomaterials that possess unique
physical and chemical characteristics and considered to have broad applications in new
therapeutic and diagnostic conception in all areas of medicine [141, 142] including drug
delivery, detection of biomolecules and prevention of disease [143, 144]. Nanoparticles have
vast biomedical uses based on their size to combat against a variety of harmful attackers within
the human body. They can combat against bacteria and viruses in the similar way as immune
system’s cells effort in the body. They can be equipped with cameras and sensors that move in
the blood vessels and find the site affected of the cancer [145, 146]. Gold nanoparticles release
heat when excited by light of specific wavelength. This phenomenon can be used to kill the
tumor cells in treatment called hyperthermia therapy [147]. Nanoparticles can also be associated
with fluorescent substances so that they can be visualized both on optical imaging instruments
and MRI [147, 148]. It is shown that silver and gold nanoparticles conjugated with heparin
Chapter-1 Introduction
23
derivative have effect in pathological angiogenesis accelerated diseases such as cancer and
inflammatory disorders [149]. Boronic acid-capped silver NPs are being used for diagnosing and
determiation of blood sugar level. The contact of glucose and boronic acid capped silver NPs
result in the accumulation of the nanoparticles and this cause a change in the plasmon peak of
silver NPs from 397 nm to 640 nm [150]. Nanparticles are considerd to absorb more light as
compare to dye, therefore metal nanoparticles are about 1 million fold more potent to absorb
light in IR region and convert it into heat energy. As a consequence, these nanoparticles have
been used in optical imaging and thermal therapy of tumors [151, 152]. Gold nanoparticles have
photothermal characters which could be exploited for localized heating, drug release and
enhancing their activity [153]. Diseases like tuberculosis (TB) have always had an enormous
effect on human health. Silver and gold nanoparticles have shown potential in the treatment of
TB [154].
1.7 Nanoparticles as antimicrobial
Emerging transmissible diseases and the increasing incidences of antibiotics resistance among
pathogenic bacteria have created difficulties in the treatment of contagious diseases. Due to the
raising rates of infections with developing multidrug resistance a very little choice left for the
doctors to treat illness. To handle this problem, researcher are working to enhance the
antibacterial potential of the existing drugs or to develop the next class of medicines or agents
which can fight against these multidrug resistant bacteria. Nanoparticles have unique
physiochemical characteristics which can be manipulated properly for desired applications [155].
This has led to the increase in the research on nanoparticles and their prospective uses as
antimicrobials. In the existing condition, nanoparticles are found the most capable and new
therapeutic agents [156]. The potential applications of nanoparticles as the most capable
Chapter-1 Introduction
24
antimicrobials is gaining importance in medical devices, therapeutics, prophylaxis, textile fabrics
and food industry. The small size and large surface area of the nanoparticles enhance their
contact with the microbes to carry out a wide range of possible antimicrobial actions [25]. Gold
has a long history of use in the world as nervine (a substance that could revitalize people
suffering from nervous conditions) [157]. In the 16th century gold was suggested for the cure of
epilepsy [157]. In the start of the 19th century gold was recommended in the treatment of
syphilis (a common venereal disease) [158]. Gold based therapy for tuberculosis was established
in 1920s when the bacteriostatic action of gold cyanide towards the Tubercle bacillus was
discovered by Robert Koch [159]. Gold particles are predominantly and broadly exploited in
living things due to their biocompatibility. On near infrared irradiation the nanomaterials, with
typical NIR absorption can devastate bacteria and cancer cells through photothermal heating.
Gold nanoparticles can be conjugated with photo-sensitizers for photodynamic antimicrobial
chemotherapy [160]. Gold nanorods combined with photosensitizers can kill methicillin-resistant
Staphylococcus aureus (MRSA) bacteria by photo-dynamic anti-microbial chemotherapy and
NIR photothermal radiation [161, 162]. Gold nanoparticles that absorb light can be used in
conjugation with particular antibodies to photothermally kill Staphylococcus aureus by means of
laser [163]. The efficiency of the antimicrobial action of gold nanoparticles can be improved by
the addition of antibiotics [164]. The antibactrial activity of the vancomycin was improved on
capping with gold nanoparticle against vancomycin resistant enterococci (VRE) [165]. The
coating of antibiotics with gold nanoparticles has an antimicrobial effect on a variety of Gram-
negative and Gram-positive bacteria [166, 167]. Cefaclor reduced gold NPs have powerful anti-
microbial action on both Gram-negative and Gram positive bacteria related to bared cefaclor and
gold nanoparticles. The gold nanoparticles produce holes in the cell wall, causing the outflow of
Chapter-1 Introduction
25
cell contents and cell death. It is also assumed that gold nanoparticles attach to the DNA of
bacterial cell and hinder the transcription and uncoiling of DNA [168].
Silver compounds and its various salts are used since ancient time to treat microbial infections
[169]. Modern study hve reported that silver in the nano-sized demonstrated antimicrobial
characteristics [25]. To elucidate the inhibitory effect of silver nanoparticles on bacterial cell
numerous mechanisms have been proposed. It is believed that the high attraction of silver
towards sulfur and phosphorus is the most important aspect of the antimicrobial outcome.
Bacterial cell membrane have sulfur-containing proteins, silver NPs react with these proteins
outside or inside the cell membrane, which in turn disturbs bacterial cell capability. Moreover it
is proposed that nanoparticles released silver ions and can react with phosphorus group in DNA,
causing in inactivation of DNA replication, or can leading to the inhibition of enzyme functions
by reacting with sulfur containing proteins [170, 171]. It has been currently established that
silver nanoparticles of small size make minute openings on the bacterial cell walls. The
cytoplasmic substance is released to the surrounding through these holes, and thus bacterial cell
death occurs without affecting the extracellular and intracellular proteins as well as nucleic acids
[172]. Electron microscopy and optical imaging results showed that silver nanoparticles pierce
the membranes of the Gram negative bacteria, with some nanoparticles found inside the cell
[173]. Even though the comprehensive mechanism by which nanoparticles can penetrate and
disturb the membranes remains to be clarified. Small sized nanostructures exhibit higher
antibacterial activity than large particles. This can be owing to high penetration when these
particles have smaller in size. The antibacterial activity is associated to the total surface area of
the nanoparticles. Particles of small size with larger surface to volume ratio have better
antibacterial properties [174]. Silver nanoparticles synthesized through a single step modified
Chapter-1 Introduction
26
Tollens procedure was evaluated for antimicrobial action against drug resistant microorganisms
[175]. These are important results, especially when antibiotic resistance among pathogens is
raising at an alarming rate and very little alternative choices are on hand to tackle the issue. A
similar effort has been made to enhance the antimicrobial action of the existing drug via
conjugation with silver and gold NPs and compiled in this dissertation.
1.7.1 Methods used for antimicrobial evaluation
Various procedures have been applied for the evaluation of antibacterial action of NPs such as
minimum inhibitory concentration (MIC) [176], minimum bactericidal concentration (MBC)
[177], disc diffusion method [178], growth inhibition method, colony-counting procedure [179],
agar or broth dilution technique [180], microdilution method [181] and turbidity assay [182].
Microscopy such as AFM, SET and TEM [87].
AFM is a very fascinating kind of microscopy, with established resolution on the order of
fractions of a nanometer [183]. Atomic force microscopy (AFM) is a powerful device for
microbiological research [184-187]. This versatile system showed cellular structures at high
resolution and also determines many shapes of basic infrastructure in the molecular or cellular
size range [188-190]. The most interesting usefulness of AFM over the other nanoscale imaging
tools is its capacity of examining live cells in actual time. Usually, a liquid cell is used to
maintain live micro-organisms in buffer solutions. This offered the way for undeviating in-vitro
study of microbial incidences [191]. The AFM can operate in fluid, ambient, and gas situations
and can analyze physical characteristics including hardness, adhesion, elasticity, friction and
chemical functionality [192]. Here in this study the comparative antimicrobial action of the
synthesized NPs related to their respective parent drugs were evaluated through MIC followed
Chapter-1 Introduction
27
by AFM. AFM clearly showed the changes in bacterial cells and the action mechanism of the
synthesized NPs.
1.8 Beta-lactam antibiotics
These are bactericidal drugs. They inhibit in the synthesis of peptidoglycan thereby inhibiting
bacterial cell wall formation. Principally, the action of beta-lactams is generally expressed
against reproducing bacteria that are making their cell wall intensively. However, beta-lactam
antibiotics could not be used against microorganisms without the peptidoglycan comprising cell
wall (mycoplasmata, mycobacteria, chlamydiae, rickettsiae). Βeta-lactam rings compose of four-
membered cyclic amide. This name is given because the β-carbon (relative to the carbonyl
group) has a nitrogen atom attached to it. β-lactam antibiotics are a broad class of antibacterial
drugs comprising of all antibiotic agents that have a β-lactam ring in their molecular
constructions. Some classes of b-lactame antibiotics aregiven below.
1.8.1 Cephalosporins
Cephalosporins is a class of β-lactam antibiotics and was derived for the first time from the
fungus Cephalosporium acremonium. This class was formerly called as "Cephalosporium”. It
was isolated for the first time in 1948 by Giuseppe Brotzu from cultures [193]. These substances
were found effective in the treatment of typhoid fever caused by Salmonella typhi.
Cephalosporin molecule consist of two ring systems which contain a dihydrothiazine ring and a
β- lactam ring. The main structure can be named as 7-ACA (7-aminocephalosporanic acid)
which could be produced by hydrolysis of its natural compound cephalosporin C. Analogous of
7-ACA are reasonably stable to acid hydrolysis and tolerant to β-lactamases. The side-chain of
Cephalosporin C is derived from D-aminoadipic acid.
Chapter-1 Introduction
28
Based on the antimicrobial activities, cephalosporins are classified into different generations
(Figure 1.8).
Structure of Cephalosporins
1.8.1.1 First generation cephalosporins
The first available cephalosporins in the market was named as the first generation. The members
of this class have strong antimicrobial action against gram-positive microbes but they have
limited activities against gram-negative species. The activity of the drugs against anaerobes is
due to the similarity with penicillin. They have simple structures in which a Small and non-polar
specie (methyl group) is attached at position C-3. Ampicillin, Cephradine and Cefadroxil are the
members of first generation.
1.8.1.2 Second generation cephalosporins
The basic structure of early second generation is similar to the first generation cephalosporins.
The important modification in the structure of second generation was observed after the
introduction of α-iminomethoxy group to the side chain at C-7. This side chain was incorporated
for the first time in the structure of cefuroxime which is a second generation antibiotics. This
side chain led to the enhancement in the resistance to β-lactamase enzymes, this is owing to the
stereo-chemical hindrance of the beta-lactam ring. The incorporation of aminothiazole ring to the
Chapter-1 Introduction
29
C-3 side chain is an additional significant change which improved its potential intensely.
Cefazolin, cefametazole and cefuroxime are the members of second generation.
1.8.1.3 Third generation cephalosporins
This generation have the aminothiazole group at C-7 and different other groups like 7-α-
iminohydroxy and 7-α-iminomethoxy are also at 7-α-position. 7-α-ethylidene group is present in
Ceftibuten which has given to it a maximum resistance against β-lactamase enzymes. These
drugs are used for treatment of severe infections produced by gram-negative bacteria as they
have a broad spectrum of action and better activity against gram-negative bacteria. They may be
mostly useful in handling hospital-acquired infections, while developing levels of extended
spectrum beta lactamases are decreasing the clinical value of this class of antibiotics [194].
Ceftriaxone, Cefixime and cefaclor are the members of third generation.
1.8.1.4 Fourth generation cephalosporins
This generation has a broad spectrum summarizing the previous generations. They can fight
against some potent beta lactamases. The members of this class have more resistance against
gram-negative microbes as compared to the 2nd and 3rd generation cephalosporins. The
zwitterion effect of these compounds is believed to be responsible for this property [195]. Due to
the presence of quaternary nitrogen (positively charged) in the side chain at position C-3,
members of this class easily penetrate through the cell membrane of gram-negative species.
Cefepime, cefozopran and cefirome.
1.8.1.5 Fifth generation cephalosporins
Currently this class has ceftobiprole, ceftaroline and ceftolozane drugs. Ceftaroline is came from
a fourth generation cephalosporin (Cefozopran), it contain alkoxyimino group at position C-7
and has additional resistance to β-lactamase. It is considered that ceftaroline is a fifth generation
Chapter-1 Introduction
30
cephalosporin, although it doesn’t have the anti-psedomonal property of ceftobiorole [196].
Ceftolozane is considered to be the emerging choice for the management of complex urinary
tract and intra-abdominal infections. These are the only active β-lactam antibacterial drugs
against MRSA. Detail classification of β-lactam antibiotics is given in figure 1.8.
Figure 1. 8: Schematic representation of various classes of β-lactam antibiotics
1.9 Spectrum of activity of B-lactam antibiotics
First generation antibiotics possess strong action against gram positive bacterial species while
less active against gram negative bacteria. They have good activity against gram positive bacteria
with a little deficiencies and are less active against gram positive strands. Fourth generation
cephalosporins are active against both gram positive and gram negative bacteria.
Chapter-1 Introduction
31
1.10 Resistance of bacteria towards antibiotics
Antibiotics are lifesaving drugs, however, some bacterial strains are becoming resistant to
generally used antibacterial medicines. Bacteria that are able to live and even reproduce in the
presence of an antibiotic are called antibiotic-resistant-bacteria. Multi-drug resistant bacteria are
those that are resistant to many antibiotics.
The ignorance and irrational use of antibiotics led to a huge bacterial resistance. An ignorant
individual may under dose or overdose himself, thus expose the microorganisms to a non-lethal
amount of the medicine and make them resistant. The pathogens develop resistance and make the
drug less effective against that type of bacteria. Generally the effectiveness and easy approach to
antibiotics led to their overuse, particularly in live-stock, promoting bacteria to develop
resistance. The Common forms of antibiotic misuse include;
Failure of medical experts to recommend the accurate dosage of antibacterial drugs.
Failure to take the whole recommended course of the antibiotic.
Improper dosage and management.
Failure to rest for adequate recovery.
Irrational antibiotic treatment.
Unnecessary use of antibiotics.
A report on respiratory tract diseases established that "physicians were more likely to prescribe
antibiotics to patients who appeared to expect them" [197]. Antibiotics should only be utilized
when required and only when prescribed by the concerned physicians. The five rights that a
physician should adhere to drug administration are; the right patient, the right time, the right
drug, the right dose and the right route [198].
Chapter-1 Introduction
32
The “European Centre for Disease Prevention and Control (ECDC)” states that antibiotic
resistance endures to be a severe public health risk globally. In a declaration issued in November
19th, 2012, the ECDC said that “an estimated 25,000 people die each year in the European Union
from antibiotic-resistant bacterial infections” [199]. A report of WHO released in April 2014
stated that “this serious threat is no longer a prediction for the future; it is happening right now in
every region of the world and has the potential to affect anyone, of any age, in any country [200].
As bacteria change day by day through mutation, hence existing antibiotics no longer worked to
treat infections and is a major threat to public health”. Antibiotic resistance is not decently
mapped all over the world, but affected the developing countries with already loose healthcare
systems [201]. Many strains of S. aureus have developed resistance to the action of antibiotics
[202]. If an infected person took antibiotics, the drug kill the non-resistant strains only and
leaving the resistant strains un-effected. These bacteria may then reproduce, and in case of
infection, it is more difficult to treat [203].
Bacterial infectious illnesses are global health issue that has drawn the public attention as a
human health risk, which spreads to economic and social problems. Increased epidemics and
infections of pathogenic strains, appearance of bacterial mutations, development of bacterial
resistance, lack of appropriate vaccine in underdeveloped nations, and hospital associated
infections, are worldwide health threats to human being. Hence, developing novel bactericidal
agents has become a crucial demand.
The greatest thoughtful distress with antibiotic-resistance is that some bacteria have developed
resistant to nearly all of the certainly available antibacterial drugs. This is a major public-health
issue because these bacteria cause serious infections. Important examples of these bacteria are
methicillin-resistant-Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus
Chapter-1 Introduction
33
(VRE), Klebsiella pneumoniae carbapenemase-producing bacteria (KPC), multi-drug-resistant
Mycobacterium tuberculosis (MDR-TB) and multidrug-resistant A. baumannii (MRAB) [204].
Among the bacterial strains E. coli and S. aureus are the most common bacteria and are selected
for our study.
1.11 Approaches to Combat Resistant Bacteria
Disease caused by multi-drug resistant bacteria is a worldwide health issue. Health professionals
are trying to address such life threating issue. To prevent irrational and over-use of antibiotics,
several health-care organizations have launched programs to advocate for careful use of all
antibiotics and keep our existing, but limited options active for as long as possible. Combination
therapy is one of the effective strategies for the handling of multi-drug-resistant bacterial-
infections. This include combination of antibiotics, and the application of adjuvants that may
instantaneously target resistance mechanisms like the β-lactamase-enzymes inhibition, or target
the resistance indirectly by interfering of the bacterial reaction to antibiotics by directing
bacterial signaling pathways such as two-component systems. Antibiotic-adjuvant groupings are
an fascinating tactic to treat infections and to develop novel therapeutics to handle multidrug-
resistant bacterial infections [205].
The need for new antimicrobials agents may be never-ending and researchers are adopting a
variety of approaches for the development of novel antimicrobials.
Natural-product research is an effective means for obtaining potent antimicrobials as
Ravu et al. found a strain of Bacillus amyloliquefaciens that exhibited potent activity
against MRSA [206].
Chapter-1 Introduction
34
Some investigators work to design novel derivatives of existing antibacterials. Chen et al.
synthesized derivatives of chlorantraniliprole, in order to discover new insecticide
substitutes [207].
Complexes exhibited antimicrobial potential and researchers look for antimicrobial
complexes. Shrestha et al. developed kanamycin complex (a novel antifungal) called K20
[208].
Repurposing present medicines. Wang et al. established that antimony potassium tartrate
(an antiparasitic drug), had good antitumor activity-blocking angiogenesis [209].
High throughput-screening (HTS) provide a targeted methodology, in which scientists
pick a definite protein typical to a microbe and discover prospective inhibitors. Park et al.
studied that some anti-tuberculosis drugs were frequently targeting the same bacterial
processes as TB gained antibiotic-resistance, and examined for inhibitors of a various
cellular-mechanism [210].
Nanotechnology-Application of metallic NPs as a powerful nano-weapon against
multidrug resistant bacteria. The Ag NPs of some antibiotics like penicillin, vancomycin
and amoxicillin, exhibited enhanced antibacterial activity against S. aureus and E. coli
[211].
There are lot of bacterial strains which showed drug resistance but commonly E. coli and S.
aureus are the most resistant strains. To overcome this problem the present study is designed to
enhance the antibacterial potential of already existing antibacterial drugs. The enhancement was
achieved via modification of these drugs into nano-sized materials.
Chapter-2 Materials and Methods
35
CHAPTER-2
MATERIALS AND METHODS
2.1 Materials
Tetrachloroauric acid trihydrate (HAuCl4.3H2O) and silver salt (AgNO3) were bought from
Merck, triethylamine from Scharlau. The antibiotics were supplied by Pharmagen Limited,
Lahore, Pakistan. Bacterial strains (E. coli ATCC 8739, S. aureus ATCC 11632 and S. aureus
ATCC 25923) were obtained from IAC, ICCBS, University of Karachi, Pakistan.
2.2 General Procedure
Synthesis of nanoparticles were carried out through a standard approach via reduction of gold or
silver salt [212]. One mM Solution of metal salt (AgNO3/HAuCl4.3H2O) and a one mM solution
of each drug were prepared separately in deionized water. The stock solutions (drug solution and
metal salt solution) were mixed using different mole ratio in order to optimize the reaction. After
stirring for 30 min, reducing agent (Trimethylamine/NaBH4) was added dropwise to the reaction
mixture. Color change of the reaction mixture was examined followed by UV-visible
spectroscopy. Optimized ratio which have good result in respect of color and UV-Spectrum were
selected for bulk synthesis of nanoparticles. After completion of the reaction the suspensions
were freeze-dried (SP Scientific Lyophilizer/Freez dryer, Model 650 F X S 1000 - SS 25C) to get
the NPs. Nanoparticles were washed repeatedly to remove reaction by-products and unreacted
precursors. Glass-wares used throughout the research work were washed thoroughly in deionized
water.
2.3 Modification of Ceftriaxone via conjugation with Ag nanoparticles
Silver nitrate solution (1 mM) and Ceftriaxone solution (1 mM) were prepared in deionized
water. These two solutions were mixed using different mole ratio of Ag and Ceftriaxone. These
Chapter-2 Materials and Methods
36
solutions were mixed in ratios like 1:1, 2:1, 3:1, 4:1….. 20:1 and 1:1, 1:2, 1:3, 1:4….. 1:20
(Ceftriaxone : Silver Nitrate). After stirring the reaction mixture for 30 min, 0.1 mL of
trimethylamine (reducing agent) was added to the reaction mixture. The reaction mixtures were
immediately became colored (i.e. dark brown); UV-visible spectra were obtained after stirring
for 2 hours. Under same conditions color change and UV-visible spectroscopy analysis of the
reaction mixtures was monitored carefully. The reaction of 8:1 (Ag : Ceftraixone) ratio was
found to have good result in respect of color change and UV-visible spectrum. This was taken as
the optimized ratio for further synthesis. For bulk synthesis the reaction was carried in a 250 mL
round bottom flask using the optimized ratio of Ag and Ceftriaxone. Then, the suspension was
freeze-dried and the nanoparticles were collected and washed repeatedly with water to remove
reaction by-products and un-reacted precursors.
2.4 Modification of Ceftriaxone via conjugation with Au nanoparticles
1 mM solution of HAuCl4.3H2O and a 1 mM solution of Ceftriaxone were prepared in deionized
water. These two solutions were mixed in different mole ratio of Au and Ceftriaxone. These
solutions were mixed in ratios like 1:1, 2:1, 3:1, 4:1….. 20:1 and 1:1, 1:2, 1:3, 1:4…….1:20
(Ceftriaxone : HAuCl4.3H2O solution). After stirring for 30 min, 0.1 mL of triethylamine was
added to the reaction mixture. Some of the colorless reaction mixtures turned dark brown; after
stirring for 2 hours UV-visible spectra were obtained. Under same conditions color change and
UV-visible spectroscopy analysis of the reaction mixtures was monitored carefully. The reaction
of 15:1 Au : Ceftriaxone mole ratio was found to have good result in respect of color change and
UV-visible spectrum. This was taken as the optimized ratio for further synthesis. For bulk
synthesis the reaction was carried in a 250 mL round bottom flask using the optimized ratio of
Chapter-2 Materials and Methods
37
Au and Ceftriaxone. The nanoparticles were collected through freeze-drying and washed
repeatedly to eliminate reaction by-products and un-reacted precursors.
2.5 Modification of Cefadroxil via conjugation with Ag nanoparticles
Cefadroxil was dissolved in deionized water and 1 mM solution was prepared. 1 mM solution of
silver salt (AgNO3) was prepared. The solutions were marked as stock solutions. These two
solutions were mixed in ratios like 1:1, 2:1, 3:1, 4:1….. 20:1 and 1:1, 1:2, 1:3, 1:4….. 1:20
(Cefadroxil : Silver Nitrate solution). After stirring for 30 minutes, 0.1 mL of triethylamine was
added to the reaction mixture. Some of the colorless reaction mixtures immediately turned
yellowish red; color change and UV-visible spectroscopy analysis of the reaction mixtures was
monitored carefully. The reaction of 12:1 Ag : Cefadroxil mole ratio was found to have good
result in respect of color change and UV-visible spectrum. This was taken as the optimized ratio
for further synthesis. For bulk synthesis the reaction was carried in a 250 mL round bottom flask
using the optimized ratio of Ag and Cefadroxil. The reaction mixture was then freeze-dried and
the nanoparticles were collected. Nanoparticles were washed repeatedly to eliminate reaction by-
products and unreacted precursors.
2.6 Modification of Cefadroxil via conjugation with Au nanoparticles
A 1 mM solution of Cefadroxil and a 1 mM solution of HAuCl4.3H2O were mixed in ratios like
1:1, 2:1, 3:1, 4:1….. 20:1 and 1:1, 1:2, 1:3, 1:4….. 1:20 (HAuCl4.3H2O : Cefadroxil). After
stirring for 30 minutes, 0.1 mL of triethylamine was added to the reaction mixture. Some of the
colourless reaction mixures turned into dark brown; color change and UV-visible spectroscopy
analysis of the reaction mixtures was monitored carefully. The reaction of 10:1 Au : Cefadroxil
mole ratio was found to have good result in respect of color change and UV-visible spectrum.
This was taken as the optimized ratio for further synthesis. The synthesized nanoparticles were
Chapter-2 Materials and Methods
38
then freeze-dried to collect the nanoparticles. Nanoparticles were washed repeatedly to remove
reaction by-products and unreacted precursors.
2.7 Modification of Cephradine via conjugation with Ag nanoparticles
1 mM solution of Cephradine and a 1 mM solution of AgNO3 were prepared and were marked as
stock solutions. Cephradine solution was added to AgNO3 solution under stirring and
triethylamine (0.1 mL) was added to the reaction mixture. The reaction was adjusted at 7:1 ratio
of AgNO3 solution to Cephradine solution. The reaction mixture turned from colorless to
yellowish red; UV-visible spectra were obtained after completion of the reaction. The
suspensions were freeze-dried and the nanoparticles were collected, washed repeatedly, to
eliminate unreacted precursors and reaction by-products.
2.8 Modification of Cephradine via conjugation with Au nanoparticles
One mM solution of Cephradine and a 1 mM solution of HAuCl4.3H2O were mixed in ratios like
1:1, 2:1, 3:1, 4:1….. 20:1 and 1:1, 1:2, 1:3, 1:4….. 1:20 (HAuCl4.3H2O : Cephradine). After
stirring for 30 minutes, 0.1 mL of triethylamine was added to the reaction mixtures. Some of the
colourless reaction mixures turned into dark brown; color change and UV-visible spectroscopy
analysis of the reaction mixtures was monitored carefully. The reaction of 9:1 Au : Cephradine
mole ratio was found to have good result in respect of color change and UV-visible spectrum.
This was taken as the optimized ratio for further synthesis. The synthesized nanoparticles were
then freeze-dried to collect the nanoparticles. Nanoparticles were washed repeatedly to remove
reaction by-products and unreacted precursors.
Chapter-2 Materials and Methods
39
2.9 Modification of Ampicillin via conjugation with Ag nanoparticles
Ampicillin solution was added to AgNO3 solution under stirring followed by addition of 0.1 mL
of triethylamine. The solutions were mixed in ratios like 1:1, 2:1, 3:1, 4:1….. 20:1 and 1:1, 1:2,
1:3, 1:4….. 1:20 (AgNO3 : Ampicillin). The reaction was optimized at 9:1 ratio of AgNO3
solution to Ampicillin solution. The reaction mixture turned from colorless to yellowish red; UV-
visible spectra were obtained after stirring for 2 h. Then, the suspensions were freeze-dried and
the collected nanoparticles were washed repeatedly to wipe away unreacted precursors and
reaction by-products.
2.10 Modification of Ampicillin via conjugation with Au nanoparticles
One mM solution of Ampicillin and a 1 mM solution of HAuCl4.3H2O were mixed in ratios like
1:1, 2:1, 3:1, 4:1….. 20:1 and 1:1, 1:2, 1:3, 1:4….. 1:20 (HAuCl4.3H2O : Cephradine). After
stirring for 30 minutes, 0.1 mL of triethylamine was added to the reaction mixtures. The reaction
of 9:1 Au : Ampicillin mole ratio was found to have good result in respect of color change and
UV-visible spectrum. The optimized ratio of gold solution and Ampicillin (9:1) was used in bulk
synthesis. The suspension was freeze dried to collect the nanoparticles and then washed to
remove the un-reacted precursors and reaction by-products.
2.11 Modification of Cefixime via conjugation with Ag nanoparticles
Cefixime solution was added to AgNO3 under stirring followed by addition of 0.1 mL of
triethylamine. The solution were mixed in different ratios and the reaction was optimized at 10:1
ratio of AgNO3 solution to Cefixime solution. The reaction mixture turned from colorless to
yellowish red; UV-visible spectra were obtained after stirring for 2 h. Then, the suspensions were
freeze-dried and the collected nanoparticles were repeatedly washed to remove unreacted
precursors and reaction by-products.
Chapter-2 Materials and Methods
40
2.12 Modification of Cefixime via conjugation with Au nanoparticles
Cefixime solution (1 mM) was added dropwise into HAuCl4 solution under stirring. Different
ratios of Cefixime and gold solutions were used and a 13:1 Au : Cefixime mole ratio was found
to have good result in respect of color change and UV-visible absorbance. The optimized ratio of
gold solution and Cefixime (13:1) was used in bulk synthesis. The suspension was freeze dried to
collect the nanoparticles and then washed to remove the unreacted precursors and reaction by-
products.
2.13 Synthesis of Polymer-Encapsulated Ceftriaxone Nanoparticles
Supersaturation solution (400mg/mL) of Ceftriaxone in water was prepared and was filtered
through a 0.4 lm nylon membrane to obtain a clear Ceftriaxone solution. The drug solution was
then introduced with a speed of 100 µl/s into the ethanol having 0.1 % PEG stabilizer (1 mg/mL)
under stirring at 2,000 rpm. The stabilizers were taken in different concentrations like 0.001,
0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 % w/v. Different drug and stabilizers
volume ratios were tried to obtain the nanoparticles. Drug and PEG (0.1%) volume ratios of 3:1
(PEG : Drug) was found to be appropriate ratio for the synthesis of nanoparticles. This
formulation was used for the bulk synthesis of Cef-PEG. Precipitation was observed immediately
upon mixing at room temperature. The obtained suspension was centrifuged at 15,000 rpm for 20
min and the collected nanoparticles were washed three times with purified water to remove
unreacted precursors.
2.13.1 Drug entrapment efficiency
Cef-PEG suspension (2 mL) was taken and were centrifuged at 15000 rpm for 30 minutes.
Supernatant containing free drug was removed. Phosphate buffer (2 mL) was added to the pellets
containing entrapped drug and was again centrifuged at the same speed for the same time. The
Chapter-2 Materials and Methods
41
process was done three times. The washed nanosuspension pellets containing only entrapped
drug was disrupted with methanol and diluted properly, followed by spectroscopic analysis at
260 nm for quantification of Ceftriaxone, using following formula [213].
Drug entrapment efficiency (EE %) = (amount of Ceftriaxone entrapped/total amount of
Ceftriaxone in the formulation) × 100
2.13.2 In vitro release studies
Cef-PEG formulation containing approximately 2 mg of Ceftriaxone was taken in phosphate
buffer (pH 7.4, 4 mL), filled into the dialysis membrane (12000 KDa) and located in a beaker
(100 mL) having 50 mL phosphate buffer media (pH 7.4). The beaker was positioned in a shaker
at 100 rpm at ambient temperature. Media (2 mL) was removed at specified time intervals. After
each withdrawal, fresh media (2 mL) was introduced to avoid drug saturation. The samples were
diluted properly and Ceftriaxone absorbance was evaluated at 260 nm using UV
spectrophotometer. The Ceftriaxone released from Cef-PEG was investigated up to 24 hours.
Calibration curve was constructed by running Ceftriaxone in a concentration range of 9-400
µg/mL.
2.14 Synthesis of Polymer-Encapsulated Cefixime Nanoparticles
PEG encapsulated Cefixime nanoparticles (Cfx-PEG) were synthesized by solvent antisolvent
precipitation technique. The drug is soluble in methanol and was used as water miscible solvent.
The solution (30 mg/mL) of Cefixime in methanol was filtrated through a 0.4 lm nylon
membrane to obtain a clear Cefixime solution. The acquired solution was then added with a
speed of 100 µl/s into the aqueous solution of 0.1 % PEG stabilizer (1 mg/mL) under stirring at
2,000 rpm. The polymer were taken in different concentrations like 0.001, 0.01, 0.05, 0.1, 0.2,
0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 % w/v. Different drug and stabilizers volume ratios were
Chapter-2 Materials and Methods
42
tried to obtain the nanoparticles. Drug and PEG (0.1%) volume ratios of 1:2 (PEG : Drug) was
found to be appropriate ratio for the synthesis of nanoparticles. This formulation was used in the
bulk synthesis of Cfx-PEG. Precipitation was observed instantly upon mixing at room
temperature. The acquired suspension was centrifuged at 15,000 rpm for 20 min and the
collected nanoparticles were washed three times with purified water to remove unreacted
precursors.
2.14.1 Drug entrapment efficiency
Cfx-PEG suspension (2 mL) was taken and were centrifuged at 15000 rpm for 30 minutes.
Supernatant containing free drug was removed. 2 mL phosphate buffer was added to the pellets
containing entrapped drug and was again centrifuged at the same speed for the same time. The
process was repeated three times. The washed pellets containing only entrapped drug was
disrupted with methanol and diluted properly, followed by spectroscopic analysis at 289 nm for
quantification of Cefixime, using following formula [213, 214].
Drug entrapment efficiency (EE %) = (amount of Cefixime entrapped/total amount of Cefixime
in the formulation) × 100
2.14.2 In vitro release studies
Cfx-PEG formulation having 2 mg of Cefixime was taken in phosphate buffer (pH 7.4, 4 mL)
and filled into the dialysis membrane with 12000 KDa, placed in a beaker (100 mL) containing
50 mL phosphate buffer media (pH 7.4). The beaker was positioned in a shaker with stirring
speed of 100 rpm at ambient temperature. Media (2 mL) was removed at specific time intervals.
After each withdrawal fresh phosphate buffer was introduced to avoid drug saturation. The
samples were diluted up to 20 mL and analyzed with a Thermo Scientific Evolution 300
spectrophotometer to measure the absorbance at 289 nm. The Cefixime released from Cfx-PEG
Chapter-2 Materials and Methods
43
was followed till 24 hours with samples at 0.30, 1, 2, 4, 6, 8, 10, 12 and 24 hours to determine its
persistent release study.
2.15 Characterization
“Thermo Scientific Evolution 300 spectrophotometer” was used to collect the UV-visible
spectra. FT-IR spectra were obtained with a “Bruker Victor 22 spectrophotometer” using KBr
pellets. Deionized water was used for the synthesis of NPs and further study. Atomic force
microscope “AFM, Agilent Technologies 5500, USA in the ACAFM mode” was used to
characterize and evaluated the antibacterial potential of nanoparticles. The images are recorded
with a high frequency Si cantilever of force constant 42 N m-1, 125 mm length and resonance
frequency of 330 kHz. Similar conditions were provided for the analysis of the entire samples.
2.16 Stability of the nanoparticles
To conclude whether the NPs would be stable under physiological conditions the suspension was
studied for temperature, salinity and pH stability. Coagulation of NPs generally resulted in
shifting of the surface plasmon towards longer wavelengths [215], meanwhile UV-visible
spectroscopy was used to describe the stability of the NPs. pH stability was studied by using a
pH meter “model-510 Oakton, Eutech having glass-electrode and Ag/AgCl electrode as a
reference”.
2.17 Evaluation of antibacterial activity
For the calculation of MIC agar well diffusion method was used. The determination of the MIC
for the drugs samples was calculated with and without its Ag and Au nanoparticles. In detail, we
used nutrient agar as medium to grow bacteria at the concentration of 106 cells per mL and
duplicate dilution was used to determine the MIC. The 60 mm well was made using a borer. A
specific volume of the drugs and its Ag and Au NPs were used separately to avoid a nonspecific
Chapter-2 Materials and Methods
44
merged zone of inhibition. In separate wells the samples solution were added in various
concentrations and the plate were incubated at normal temperature for 2 h to let the diffusion
process to occur before it was incubated for 24-48 hrs at 370C. The zones of inhibition were
quantified using a millimeter scale.
Antimicrobial potential and action mechanism of the synthesized nanoparticles were further
evaluated under AFM. Bacteria were grown on tryptic soy agar (Oxoid UK) at 37oC for 24 hours
in static conditions and made noticeable as stock bacterial culture. 10 µL drop(s) of polylysine
were added on mica surface (freshly cleaved) and placed to dry, in the meanwhile, a newly
incubated culture of bacteria on tryptic soy agar (Oxoid UK) was inoculated in distilled water
(sterilized) to make 106 cfu of bacteria and few (5-10 µL) droplets of this solution were shifted
onto a freshly cleaved mica surface and kept to dry under controlled environment. Subsequently,
it was analyzed by atomic force microscope to check the morphology. Quantified concentrations
(MICs) of drugs samples and its Ag and Au NPs were taken into vials and incubated for 1 hours
at 37oC. After incubation 5-10 µL drops of each samples was transferred separately onto freshly
cleaved mica coated with polylysine and left to dry before being analyzed by AFM. Similar
manner was applied for 2, 4, 6 and 8 hours to determine the kinetically controlled destruction of
bacterial cells at the same temperature. Bare silver and gold NPs were also treated in a similar
way to record the effect of bare silver and gold nanoparticles on bacteria, which were considered
as a negative control. In this manner we recorded control (before treatment), treated with drugs,
treated with bare Ag and Au NPs (negative control) and drug stabilized Ag and Au nanoparticles
images of bacteria in similar conditions using AFM. Si cantilever of 125 mm length, force
constant 42 Nm-1, resonance frequency 330 kHz and with a spring-constant value of 0.01-0.01
N/m made by Veeco model MLCT-AUHW was used throughout the study.
Chapter-3 Results and Discussion
45
CHAPTER-3
RESULTS AND DISCUSSION
Beta lactam antibiotics namely Ceftriaxone, Cefadroxil, Cephradine, Ampicillin and Cefixime
were conjugated to Ag and Au nanoparticles (NPs). Ceftriaxone and Cefixime antibiotics were
also encapsulated with polymers to create nanoscale materials. The nanoparticles were
characterized through Ultra violet (UV) visible, Fourier transform-Infra red (FT-IR)
spectroscopic techniques and atomic force microscopy (AFM). The antibacterial potential of the
conjugates against bacteria (Escherichia coli and Staphylococcus aurous) was compared to that
of pure antibiotics and of unconjugated nanoparticles (NPs) using AFM and more formal
procedures such as the agar well diffusion method. Conjugation to Ag and Au nanoparticles
enhanced the antibacterial activity of the antibiotics, significantly. Conjugation also performed to
increase the kinetics of the antibiotics. Thus, for example, Ag and Au conjugates severely
damaged membranes and completely disrupted the cell morphology more timely than their
respective free antibiotics. The details of the modification of the selected drugs and their
antimicrobial evaluation is given below.
3.1 Modification of Ceftriaxone via conjugation with Ag and Au nanoparticles
3.1.1 Synthesis of AgNPs stabilized with Ceftriaxone
Silver nanoparticles stabilized with Ceftriaxone (Cef-AgNPs) were synthesized in one pot by
mixing solutions of AgNO3 and Ceftriaxone. Triethylamine was used as reducing agent. Upon
mixing the color turned to dark brown, designated the formation of Ag nanoparticles. The
formation of Cef-AgNPs was confirmed by color change, UV-visible spectroscopy, FT-IR and
AFM.
Chapter-3 Results and Discussion
46
For the quantification of Ceftriaxone in Cef-AgNPs, the nanoparticles were completely removed
from the suspension by centrifugation. The supernatant obtained after the removal of
nanoparticles was freeze-dried. The weight of the dried materials was noted. The amount of
Ceftriaxone was calculated, to be 26 % by weight for Cef-AgNPs conjugates [216].
3.1.2 Characterization of Cef-AgNPs
The characterization and optical characteristics of NPs are commonly studied by UV-Visible
spectrophotometry. The appearances of the surface-plasmon absorption-bands (SPB) are known
to be displaying the morphological behaviors of NPs [217].
The UV-visible spectrum of Cef-AgNPs exhibited SPB at 408 nm (Figure 3.1), which can be
reconciled with the characteristic plasmonic absorption of AgNPs [218].
Figure 3. 1: UV-Visible spectrum of Ceftriaxone stabilized silver nanoparticles
The absorbance intensity was maximum at 8:1 (Ag: Ceftraixone) ratio indicated complete
reduction of silver ions. Further increase in the Ceftriaxone amount caused the reduction in the
Chapter-3 Results and Discussion
47
intensity of the absorbance band, this is due to the aggregation [219], thereby decreasing the
number of nanoparticles.
The conjugation Ceftriaxone was furthermore established by FT-IR spectroscopy. Significant
absorption bands noted in the spectrum of Ceftriaxone (Figure 3.2) at 3426 cm-1 and 3264 cm-1,
might be assigned to the stretching vibrations of N-H and O-H groups, respectively. The band at
2935 cm-1 was correlated to the stretching vibrations of C-H groups. Carbonyl groups (C=O) was
distinguished by bands in the region of 1742 cm-1 and 1649 cm-1, while the band at 1537 cm-1
was related with the aromatic ring. The bands at 1399 cm-1 and 1033 cm-1 could be allocated to
the stretching vibrations of C-N and C-O respectively.
Figure 3. 2: IR spectra of Ceftriaxone (black) and Cef-AgNPs (red)
The formation Cef-AgNPs resulted in the reduction of absorbance intensities and merging of
bands of C=O (1742 and 1650 cm-1), N-H (3426 cm-1) and O-H (3264 cm-1) stretching [220].
The changes in IR spectrum clearly indicated the conjugation of ceftriaxone to silver NPs.
Chapter-3 Results and Discussion
48
The formation of Cef-AgNPs was also characterized by AFM. The study exposed the formation
of spherical and poly-dispersed silver NPs stabilized by Ceftriaxone. The images showed the
presence of various size nanoparticles of size less than 100 nm with a maximum particles in the
size range of 25-35 nm (Figure 3.3).
A B
Figure 3. 3: AFM analysis of Cef-AgNPs. Topography (A) and Particles size distribution (B)
3.1.3 Stability of the silver nanoparticles stabilized with Ceftriaxone
3.1.3.1 Thermal stability
The stability of Cef-AgNPs was characterized by UV-Visible spectroscopy. Coagulation of NPs
generally resulted in shifting of the surface plasmon towards longer wavelengths [215]. At room
temperature, the synthesized conjugates were found stable for several days and no change was
observed in the SPB. For further thermal stability the suspension was heated up to 100oC and
monitored through UV-Visible spectroscopy, which did not show any shifting in the SPB
(Figure 3.4). A little decrease in the SPB was observed without any precipitation, this may be
due to the dominant electronic dephasing mechanism which involves electron-electron
Chapter-3 Results and Discussion
49
interactions as the electron-surface and electron-defect scattering increase at high temperature.
The velocity of an electron is related to its state-energy and temperature. It is known that at
elevated temperature increased velocity of the electrons leads to a more damping and faster
dephasing, which resulted reduction in the absorbance of plasmon band [221].
Figure 3. 4: Thermal stability of Ceftriaxone stabilized silver nanoparticles
3.1.3.2 Salt Stability
The effect of different concentrations of aqueous solution of salt (NaCl) was studied on the Cef-
AgNPs and the suspensions were found stable for NaCl concentrations up to 2 M (Figure 3.5).
However, higher concentration of NaCl resulted reduction in the SPB due to the aggregation
promoted by Cl-1 ions [222].
Chapter-3 Results and Discussion
50
Figure 3. 5: Stability of Cef-AgNPs against various concentration of salt
3.1.3.3 PH stability
pH stability of the suspensions were measured to determine the behavior of Cef-AgNPs under
different pH conditions. The suspensions were found stable for a pH between 6 and 12 as the
aggregation was not observed in this range (Figure 3.6).
Figure 3. 6: Stability of Cef-AgNPs against pH
Chapter-3 Results and Discussion
51
3.1.4 Synthesis of AuNPs stabilized with Ceftriaxone
The synthesis of gold nanoparticles stabilized with Ceftriaxone (Cef-AuNPs) was carried out
through a single step reduction method by mixing solutions of HAuCl4 and Ceftriaxone in the
presence of triethylamine as reducing agent. The formation of Cef-AuNPs was monitored
carefully by observing color change and recording UV-visible spectra. The color of the reaction
mixture changed to dark brown, indicated the formation of Au nanoparticles. The formation of
Cef-AuNPs were further confirmed by FT-IR and AFM.
The quantity of Ceftriaxone in Cef-AuNPs was determined by centrifuging the suspension. The
precipitated Cef-AgNPs were collected. The supernatant was centrifuged three times to remove
the nanoparticles completely. Then, the supernatant was freeze-dried and the residues was
weighed. The amount of Ceftriaxone was found 8 % by weight in Cef-AuNPs [216].
3.1.5 Characterization of Cef-AuNPs
Cef-AuNPs were characterized by UV-visible spectroscopy. The spectrum exhibited SPB at 538
nm (Figure 3.7), which can be reconciled with the characteristic plasmonic absorption of AuNPs
[223].
Figure 3. 7: UV-Visible spectrum of Ceftriaxone stabilized gold nanoparticles
Chapter-3 Results and Discussion
52
The absorbance intensity was maximum at 15:1 (Au:Ceftraixone) ratio indicated complete
reduction of Au ions at this ratio. Further increase in the Ceftriaxone amount caused the decrease
in the intensity of the absorbance band, this is due to the aggregation [219] thereby decreasing
the number of nanoparticles.
Conjugation of Ceftriaxone was also established by FT-IR spectroscopy (Figure 3.8). Distinctive
absorption bands seen in the FT-IR spectrum of Ceftriaxone were described below.
Band at 3426 cm-1 was correlated to the stretching vibrations of N-H group and a band at 3264
cm-1 of about the same intensity could be allotted to O-H groups. Similarly the band at 2935 cm-1
was associated to stretching vibrations of C-H groups and bands for carbonyl groups (C=O)
were found at 1742 cm-1 and 1649 cm-1. Furthermore the band related with the torsional
vibrations of aromatic ring was present at 1537 cm-1. The bands at 1399 cm-1 and 1033 cm-1
might be associated to the of C-N and C-O respectively. The changes in the spectrum, especially
the lessening in absorbance intensities and integration of bands of C=O (1742 cm-1 and 1650 cm-
1), N-H (3426 cm-1) and O-H (3264 cm-1) stretching clearly indicated the formation of
ceftriaxone conjugated gold NPs [220].
Chapter-3 Results and Discussion
53
Figure 3. 8: IR spectra of Ceftriaxone (black) and Cef-AuNPs (red)
Highly sophisticated equipment, AFM was used for the characterization and size determination
of Cef-AuNPs. The images reported in Figure 3.9 showed the spherical and poly dispersed Cef-
AuNPs. The morphology of the nanoparticles disclosed that particles have diameter in the
preferred range (from 10-40 nm with maximum particles of size range from 20-30 nm) as
presented in the histogram (Figure 3.9 B).
A B
Figure 3. 9: AFM analysis of Cef-AuNPs. Topography (A) and Particles size distribution (B)
Chapter-3 Results and Discussion
54
3.1.6 Stability of the silver nanoparticles stabilized with Ceftriaxone
3.1.6.1 Thermal stability
At room temperature the Cef-AuNPs were found stable for several days and no change was
observed in the SPB. For thermal stability the suspension was heated and monitored through
UV-Visible spectroscopy, as the nanoparticles coagulated they change the surface plasmon to
longer wavelengths [215]. The analysis did not show any shifting in the SPB, indicated the
stability of the nanoparticles up to 100oC (Figure 3.10).
Figure 3. 10: Thermal stability of Ceftriaxone stabilized gold nanoparticles
Upon heating no precipitation was observed however a slight decrease in the absorbance peak
was due to the dominant electronic dephasing mechanism at high temperature [221].
Chapter-3 Results and Discussion
55
3.1.6.2 Salt Stability
The effect of different concentrations of NaCl aqueous solution was studied on the Cef-AuNPs
and were found stable at salt concentrations up to 1 M (Figure 3.11). However further increase
in concentration of NaCl resulted reduction in the SPB which is due to the aggregation
stimulated by Cl-1 ions [222].
Figure 3. 11: Stability of Cef-AuNPs against various concentration of salt
3.1.6.3 PH stability
Cef-AuNPs were kept in different pH medium to study its stability against various pH. After 24
hours Uv-visible spectra of the samples were recorded. The characteristics band of Cef-AuNPs at
538 nm was correlated with spectra. The NPs were analyzed in different pH and were found
stable at pH between 6 and 12 as the aggregation was not observed in this pH range (Figure
3.12).
Chapter-3 Results and Discussion
56
Figure 3. 12: Stability of Cef-AuNPs against pH
3.1.7 Evaluation of antibacterial potential of Cef-AgNPs and Cef-AuNPs
Ceftriaxone was modified via conjugation with silver and gold NPs. The Cef-AgNPs and Cef-
AuNPs were evaluated for their antibacterial potential in comparison with unconjugated
ceftriaxone and NPs.
The enhancement of antibacterial activity has been hot study in different era. Anacona et al.
reported Ceftriaxone transition metal complexes and its enhanced antibacterial activities [224]
and Junejo et al. reported silver nanoparticles coated with Ceftriaxone and studied its catalytic
activity [225].
We report here the enhancement of antibacterial potential via new and emerging techniques as
well as conventional methods. The morphological study of bacterial cell and effect of
antibacterial agents was studied under fascinating equipment AFM. For instance E. coli was
selected for the study as it has been one of the emerging resistance strains of bacteria against
ceftriaxone [226].
Chapter-3 Results and Discussion
57
For the calculation of minimum inhibitory concentration (MIC) zone of inhibition method was
used [227]. The MIC of pure Ceftriaxone, Cef-AgNPs and Cef-AuNPs were established to be 3.1
± 0.71 µg mL-1, 4.1 ± 0.32 µg mL-1 and 4.3 ± 0.57 µg mL-1 respectively. While the MIC of un-
conjugated Ag and Au NPs were found to be 48 ± 1.5 µg mL-1and 73 ± 1.9 µg mL-1, respectively
(Figure 3.13).
Figure 3. 13: MIC of Ceftriaxone (1), Cef-AgNPs (2), Cef-AuNPs (3), bare AgNPs (4) and bare
AuNPs (5).
The MIC of pure Ceftriaxone was about 2 times less as related to its Ag and Au nanoparticles,
while unconjugated Ag and Au NPs exhibited very poor MIC values when compared with pure
Ceftriaxone and its Ag and Au conjugates. As Ceftriaxone signified a small weight fraction of
the nanoparticles (26 wt% for AgNPs and 8 wt% for AuNPs), the results indicated that
nanoparticles have a 2-6 times enhanced activity than pure Ceftriaxone. AFM study confirmed
the results of the zone of inhibition investigation. Untreated E. coli cultures displayed cells with
Chapter-3 Results and Discussion
58
regular shapes and smooth membranes with mean height of 0.3 mm, mean width of 0.95 mm,
and mean length of 1.5 mm (Figure 3.14).
Figure 3. 14: AFM images of Escherichia coli ATCC 8739 before treatment.
Cultures were then treated with Ceftriaxone (two different doses, 1 mg and 5 mg) and their effect
on bacterial cell morphology was analyzed carefully. The 1 mg sample showed irregular surfaces
after treatment for 2 hours (Figure 3.14a). Further morphology disintegration was observed with
increasing the duration of treatment, after 8 h the cells were melted and absolutely destructed.
Culture treated with 5 mg dose displayed a faster disintegration, as presented in Figure 3.17a
and 3.18a. Cef-AgNPs were found to destroy cells more rapidly than pure Ceftriaxone.
Ceftriaxone conjugated with Ag nanoparticles of a 1mg concentration (corresponded to about
0.25 mg pure Ceftriaxone) caused cell rupture by 1 hour (Figure 3.16a) and a 5 mg
concentration resulted in the complete destruction in 2 hours (Figure 3.17b).
Chapter-3 Results and Discussion
59
Figure 3. 15: E. coli treated with 1 mg Ceftriaxone (a), 1 mg Cef-AgNPs (b) and 1 mg Cef-
AuNPs (c) for 2 hrs
Cef-AuNPs also degraded cells faster than pure Ceftriaxone, as shown in Figure 3.15c, 3.16b
and 3.17c. Significantly, unconjugated Au and Ag NPs at 5 mg induced only marginal
morphological changes even after long incubation time (i.e. 8 hours), as presented in figure
3.18b and c.
Chapter-3 Results and Discussion
60
Figure 3. 16: E. coli treated with 1 mg Cef-AgNPs (a) and 1 mg Cef-AuNPs (b) for 1 hr
Figure 3. 17: E. coli treated with 5 mg Ceftriaxone (a), 5 mg Cef-AgNPs (b) and 5 mg Cef-
AuNPs (c) for 2 hrs
Chapter-3 Results and Discussion
61
Figure 3. 18: E. coli treated with 5 mg Ceftriaxone (a), 5 mg bare AgNPs (b) and 5 mg bare
AuNPs (c) for 8 hrs
The enhanced activity of Cef-AgNPs and Cef-AuNPs could be explained as nanoparticles can
simply enter into the bacterial cell by binding to proteins in the bacterial cell membrane due to
the high chemical attractiveness of metal (Ag and Au) for sulfur. Within the cell the
nanoparticles interact with DNA and protein; it can interrupt vital functions and eventually
destroy the cell [228]. The anti-microbial action of nanoparticles has been previously studied by
high resolution imaging systems. Morones et al. recorded the antibacterial activity of silver NPs
with transmission electron microscopy (TEM) shown ultrastructural measures containing the
cellular organelles of E. coli [144] however it merely imagined in the time when all E. coli were
not alive. In the current work AFM made a three dimensional view of living E. coli by revealing
Chapter-3 Results and Discussion
62
a comprehensive topographic pictures of surface and shape; phase imaging that visualized
examination of boundary stiffness, width, length and height.
3.2 Modification of Cefadroxil via conjugation with Ag and Au nanoparticles
3.2.1 Synthesis of AgNPs stabilized with Cefadroxil
Silver nanoparticles stabilized with Cefadroxil (Cefd-AgNPs) were synthesized in one pot by
mixing Cefadroxil with ionic solutions of Ag in 12:1 (Ag:Cefadroxil) volume ratio in the
presence of triethylamine as reducing agent. Upon mixing the reaction mixture turned to
yellowish red; indicated the formation of Cefadroxil coated silver nanoparticles. The amount of
conjugated Cefadroxil was measured by centrifuging out Cefd-AgNPs suspension and the
precipitated NPs were collected. The supernatant was repeatedly centrifuged to remove the
synthesized nanoparticles. The supernatant containing the free Cefadroxil was then freeze-dried
and determined the weight of the unreacted drug. It was calculated that Cefd-AgNPs have 11.39
wt% Cefadroxil [216].
3.2.2 Characterization of Cefd-AgNPs
The formation of Cefd-AgNPs was monitored by UV-visible spectroscopy and a strong SPB was
observed at 386 nm (Figure 3.19) which can be correlated with the characteristic plasmonic
absorption of AgNPs [218].
Chapter-3 Results and Discussion
63
Figure 3. 19: UV-Visible spectrum of Cefadroxil stabilized silver nanoparticles
Conjugation of Cefadroxil to AgNPs was further confirmed by FT-IR spectroscopy (Figure
3.20). The characteristic absorbance peaks associated to Cefadroxil were found in region 3199
cm-1 and 3510 cm-1 which could be allocated to the stretching vibrations of O-H and N-H groups,
respectively. The band at 2910 cm-1 was assigned to the C-H stretching vibrations and carbonyl
group of the lactam ring showed the stretching vibration at 1757 cm-1. The amide carbonyl band
was observed at 1685 cm-1. The band at .1562 cm-1 was attributed to the torsional vibrations of
aromatic ring. The band at 1398 cm-1 can be correlated to the C-N stretching vibrations of the
thiazole and lactam ring. Cefadroxil conjugated with Ag metal resulted in the decrease in
absorbance intensities and merging of bands of O-H (3510 cm-1), N-H (3199 cm-1) and C=O
(1757 and 1685 cm-1) stretching [229].
Chapter-3 Results and Discussion
64
Figure 3. 20: IR spectra of Cefadroxil (black) and Cefd-AgNPs (red)
The size and morphology of the Cefd-AgNPs was determined by AFM. Analysis showed that
Cefd-AgNPs were spherical in shape and their size range from 10-50 nm (with a maximum
particle size distribution ranges of 20-30 nm) (Figure 3.21).
A B
Figure 3. 21: AFM analysis of Cefd-AgNPs. Topography (A) and Particles size distribution (B)
Chapter-3 Results and Discussion
65
3.2.3 Stability of the Cefd-AgNPs
3.2.3.1 Thermal stability
Cefd-AgNPs were kept at room temperature for several days and monitored its color and UV-
visible absorption. No change was observed in the color and SPB of the NPs suspension which
indicated the stability of the NPs for several days at ambient temperature. For thermal stability
the suspension was heated gradually and monitored its color and UV-Visible spectra. Usually
coagulation of NPs in a suspension are noticed by a shift in the surface plasmon towards longer
wavelengths [215]. The analysis did not show any color change and shifting in the SPB,
indicated the stability of the NPs up to 100oC (Figure 3.22).
Figure 3. 22: Thermal stability of Cefadroxil stabilized silver nanoparticles
At high temperature a little reduction in the absorbance peak was observed because as the
temperature raised it increased the speed of electrons that caused to a more damping constant and
faster dephasing. Therefore it resulted reduction of SPB [221].
Chapter-3 Results and Discussion
66
3.2.3.2 Salt Stability
The effect of different concentrations of NaCl aqueous solution on the stability of Cefd-AgNPs
was studied and it was found that these nanoparticles were stable up to 100 mM (Figure 3.23).
However, further increase in concentration of NaCl resulted reduction in the SPB indicated the
instability of Cefd-AgNPs at higher concentration. This is due to the aggregation promoted by
Cl-1 ions [222].
Figure 3. 23: Stability of Cefd-AgNPs against various concentration of salt
3.2.3.3 PH stability
pH stability of the Cefd-AgNPs were measured by changing the pH of the suspension. The NPs
were left in different pH medium for 24 hours and UV-Visible spectra were recorded. Analysis
showed that Cefd-AgNPs were stable at a pH between 4 and 12 as the aggregation was not
observed in this range (Figure 3.24).
Chapter-3 Results and Discussion
67
Figure 3. 24: Stability of Cefd-AgNPs against pH
3.2.4 Synthesis of AuNPs stabilized with Cefadroxil
Gold nanoparticles stabilized with Cefadroxil (Cefd-AuNPs) were synthesized through one-step
reduction method. Cefadroxil solution was mixed with ionic solutions of Au in 10:1
(Au:Cefadroxil) ratio in the presence of triethylamine as reducing agent. The color of the
reaction mixture turned to dark brown; indicated the formation nanoparticles. The amount of
conjugated Cefadroxil was measured by centrifuging out Cefd-AuNPs. The supernatant was
repeatedly centrifuged to remove the nanoparticles. The supernatant containing the unreacted
Cefadroxil was determined. Calculation exposed that Cefd-AuNPs containing 6.6 wt % of
Cefadroxil [216].
3.2.5 Characterization of Cefd-AuNPs
The reaction mixture was monitored by UV-visible spectroscopy to notice the formation of Cefd-
AuNPs and a strong SPB was observed at 560 nm (Figure 3.25), which can be reconciled with
the characteristic plasmonic absorption of AuNPs [223].
Chapter-3 Results and Discussion
68
Figure 3. 25: UV-Visible spectrum of Cefadroxil stabilized gold nanoparticles
Formation of Cefd-AuNPs was further confirmed by FT-IR spectroscopy (Figure 3.26), where
the characteristic absorbance bands related to Cefadroxil were found in region 3199 cm-1, 3510
cm-1 which could be assigned to the stretching vibrations of O-H and N-H groups, respectively.
The band at 2910 cm-1 was associated to the C-H stretching vibrations, carbonyl group of the
lactam ring showed the stretching vibration at 1757 cm-1 and the carbonyl group of amide was
observed at 1685 cm-1, whereas the torsional vibrations of aromatic ring gave a band at 1562 cm-
1. The band at 1398 cm-1 could be due to the stretching vibrations of C-N of the thiazole and
lactam ring. Cefadroxil conjugation with AuNPs consequence in integration of bands and the
decrease in absorbance intensities of O-H (3510 cm-1), N-H (3199 cm-1) and C=O (1757 and
1685 cm-1) stretching [229].
Chapter-3 Results and Discussion
69
Figure 3. 26: IR spectra of Cefadroxil (black) and Cefd-AuNPs (red)
To determine the size and morphology of the Cefd-AuNPs it were visualized under deter AFM.
The images showed round shape Cefd-AuNPs of size range of 20-80 nm with a maximum
particles in the size of 40-60 Nm (Figure 3.27).
A B
Figure 3. 27: AFM analysis of Cefd-AuNPs. Topography (A) and Particles size distribution (B)
Chapter-3 Results and Discussion
70
3.2.6 Stability of Cefd-AuNPs
3.2.6.1 Thermal stability
Cefd-AuNPs were found stable at room temperature for several days. For thermal stability the
suspension was heated and monitored through UV-visible spectroscopy. Coagulation of NPs
have been considered to be accompanied by a red shift in the surface plasmon band [215]. Cefd-
AuNPs were found to be stable upto 80oC (Figure 3.28). Further increase in temperature caused
reduction in the SPB, and the absorbance band completely disappeared at 100oC.
Figure 3. 28: Thermal stability of Cefadroxil stabilized gold nanoparticles
3.2.6.2 Salt Stability
NaCl aqueous solution was used to study the effect of salt on the stability of Cefd-AuNPs. The
suspension of Cefd-AuNPs were mixed with the salt solutions of different concentrations and
kept at room temperature for 24 hours. UV-visible spectra were recorded and it was found that
Cefd-AuNPs were stable up to 100 mM salt concentration (Figure 3.29). However further
Chapter-3 Results and Discussion
71
increase in concentration resulted reduction in the SPB which may be due to the aggregation
promoted by Cl-1 ions [222].
Figure 3. 29: Stability of Cefd-AuNPs against various concentration of salt
3.2.6.3 PH stability
PH stability of the Cefd-AuNPs were measured by changing the pH of its suspension. The NPs
were left in different pH medium for 24 hours followed by UV-visible spectroscopy. Analysis
showed that Cefd-AuNPs were stable for a pH between 4 and 12 as the aggregation was not
observed in this range (Figure 3.30).
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72
Figure 3. 30: Stability of Cefd-AuNPs against pH
3.2.7 Evaluation of antibacterial potential of Cefd-AgNPs and Cefd-AuNPs
Cefadroxil is a 1st generation beta-lactam antibiotic, and has strong action (both in-vivo and in-
vitro) against different microbes [230]. Several studies have been carried to enhance the
antibacterial potential and kinetics of Cefadroxil in past. Sarac et al. synthesized Cefadroxil
derivatives of and examined its anti-microbial potential [231] and Bukhari et al. reported the
antimicrobial study of schiff base transition metal complexes derived from Cefadroxil [232].
Here in this study Cefadroxil was conjugated to Ag and Au nanoparticles to enhance their
antimicrobial potential. For instance S. aureus was selected for the subject study as it has been
reported the most emerging resistant strain against Cefadroxil [233, 234].
Minimum inhibitory concentration (MIC) of Cefadroxil, Cefd-AgNPs and Cefd-AuNPs was
calculated through a zone of inhibition [227]. The MIC of pure Cefadroxil, Cefd-AgNPs and
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73
Cefd-AuNPs were found to be 10 ± 0.2 µg mL-1, 40 ± 0.2 µg mL-1 (corresponds to a 4.5 µg
Cefadroxil) and 50 ± 0.2 µg mL-1 (correspond to a 3.3 µg Cefadroxil), respectively. While the
MIC of bare Ag and Au NPs were calculated to be 85 µg mL-1 and 100 µg mL-1, respectively
(Figure 3.31). Although pure Cefadroxil had less MIC value as compared to conjugated, but the
amount of Cefadroxil present in the nano-conjugates is very less (11.395% for Cefd-AgNPs and
6.6% for Cefd-AuNPs), this indicated that conjugated Cefadroxil has a 2-3 times greater activity
than Cefadroxil alone.
Figure 3. 31: MICs of Cefadroxil (1), Cefd-AgNPs (2), Cefd-AuNPs (3), bare AgNPs (4) and
bare AuNPs (5).
AFM study confirmed the magnitudes of the zone of inhibition scrutiny. S. aureus displayed
cells with smooth membranes and regular shapes (mean length of 1.052 µm, mean height of
0.104 µm, mean width of 1.082 µm and with a maximum height of 0.719 µm) as shown in
(Figure 3.32).
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74
Figure 3. 32: AFM images of Staphylococcus aureus ATCC 11632 before treatment
Figure 3. 33: AFM images of S. aureus treated with Cefadroxil (A), Cefd-AgNPs (B) and Cefd-
AuNPs (C) for 1 hr
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75
Bacterial cells were then treated with pure Cefadroxil, Cefd-AgNPs, Cefd-AuNPs and Bare Ag
& Au NPs to examine the comparative efficacy and kinetics under AFM. Bacteria treated with
10 µg (MIC) of unconjugated Cefadroxil for 1 hour showed slight effect and only lesion on
bacterial cell surface was observed (Figure 3.33a). Morphological degradation of cells increased
with time as in a 2 hours treatment further affected bacterial cells were noticed (Figure 3.34a).
Figure 3. 34: AFM images of S. aureus treated with Cefadroxil (A), Cefd-AgNPs (B) and Cefd-
AuNPs (C) for 2 hrs.
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76
Furthermore, a 4 hours treatment produced considerable damages in cell bodies (Figure 3.35a)
while after 8 hours the cell morphology were distorted completely (Figure 3.36a). We assume
that this altering of the bacterial forms is due to destruction of the cell wall, followed by
spreading out of peptidoglycan on the mica surface. On the other hand the culture treated with 40
µg (MIC) of Cefd-AgNPs for 1 hour showed relatively more effect as compared to pure
Cefadroxil (Figure 3.33b) and further effect was exposed in 2 hours treatment (Figure 3.34b)
while after 4 hours it led to complete destruction and melted bacterial cells were observed
(Figure 3.35b).
Figure 3. 35: AFM images of S. aureus treated with Cefadroxil (A), Cefd-AgNPs (B) and Cefd-AuNPs
(C) for 4 hrs
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Similarly the culture treated with 50 µg (MIC) of Cefd-AuNPs for 1 and 2 hours exhibited
relatively more influence on the bacterial cells morphology (Figure 3.33c and 3.34c), while in 4
hours treatment it completely damaged the cells and melted S. aureus were observed (Figure
3.35c). Unconjugated Ag and Au NPs of 85 µg and 100 µg (MICs) respectively cannot showed
distinctive effect but only minimal morphological changes and slight influence was observed
even after treatment for 8 hours (Figure 3.36b, c).
Figure 3. 36: AFM images of S. aureus treated with Cefadroxil (A), Bare AgNPs (B) and Bare
AuNPs (C) for 8 hrs
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78
The interaction of NPs with a bacterial cell still need further exploration. Many studies have
shown that the NPs can simply enter into the bacterial cell by sticking to proteins in the cell
membrane due to the chemical attraction of metal (Ag and Au present in the conjugates) towards
sulfur [235]. After that, NPs get inside into the cell causing perforation and leads to the release of
the cellular matrix [144, 236, 237]. Here in this case Cefadroxil reacted with the outer layer of S.
aureus thereby enhancing the membrane’s permeability. Subsequently the NPs get into the cell
through the membrane and may be attached to the bacterial DNA and protein; thus, cause death
of the cell by disturbing metabolism and vital functions [25, 172, 228]. Consequently, the mutual
action of Cefadroxil and Ag or Au NPs led to enhanced antibacterial potential [168]. In this
study AFM explored noticeable investigation of alive S. aureus by offering detailed topographic
demonstration of surface, shape and phase imaging morphology that let analysis of height, width,
length and boundary stiffness.
3.3 Modification of Cephradine via conjugation with Ag and Au nanoparticles
3.3.1 Synthesis of AgNPs stabilized with Cephradine
The synthesis of silver nanoparticles stabilized with Cephradine (Cpn-AgNPs) was carried out
through one-step reduction method. Cpn-AgNPs was successfully synthesized by mixing
solutions of AgNO3 and Cephradine in the presence of triethylamine as reducing agent. Upon
mixing the color of the reaction mixture changed to yellowish red, indicated the formation of
Cpn-AgNPs. The amount of conjugated Cephradine was calculated by centrifuging out Cpn-
AgNPs from the suspension. For the complete removal of Cpn-AgNPs the suspension was
centrifuged three times. The unreacted cephradine present in the supernatant was determined.
The Cephradine was quantified by this method to be 18.14% by weight for Cpn-AgNPs
conjugates [216].
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79
3.3.2 Characterization of Cpn-AgNPs
The formation of Cpn-AgNPs was examined by UV-visible spectroscopy. The spectrum of the
Cpn-AgNPs exhibited peak at 394 nm (Figure 3.37), which can be correlated with the
distinguishing plasmonic absorption of AgNPs [218].
Figure 3. 37: UV-Visible spectrum of Cephradine stabilized silver nanoparticles
The absorbance intensity was maximum at 7:1 (Ag:Cephradine) ratio indicating complete
reduction of silver ions. Further increase in the Cephradine amount resulted reduction in the
intensity of the absorbance band, this is due to the aggregation of NPs [219].
The conjugation of Cephradine was also confirmed by FT-IR spectroscopy (Figure 3.38).
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80
Figure 3. 38: IR spectra of Cephradine (black) and Cpn-AgNPs (red)
Significant absorption bands noted in the FT-IR spectrum of Cephradine were 3440 cm-1 and
3300 cm-1 which can be allotted to the N-H and O-H groups respectively and the band for C-H
groups stretching vibrations is present at 3025 cm-1. Band at 1760 cm-1 can be associated with the
stretching vibration of carbonyl group of the lactam ring and the amide carbonyl shown band at
1680 cm-1. The bands at 1355 cm-1 might be allocated to the stretching vibrations of C-N of the
thiazole and lactame ring. On the other hand the spectrum of conjugated Cephradine have shown
integration of bands and the lessening in absorbance intensities of C=O (1760 cm-1 and 1680 cm-
1), N-H (3440cm-1) and O-H (3300 cm-1) stretching [220].
Cpn-AgNPs formation was further established by AFM analysis. The images exposed the
formation of spherical and poly dispersed Cpn-AgNPs. The images showed the presence of
various size NPs in the size range of 20-80 nm with a maximum particles of diameter range from
35-50 nm as shown in the size distribution histogram (Figure 3.39).
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A B
Figure 3. 39: AFM analysis of Cpn-AgNPs. Topography (A) and Particles size distribution (B)
3.3.3 Stability of Cpn-AgNPs
3.3.3.1 Thermal stability
At room temperature the synthesized conjugates were found stable for several days and no
change was observed in the SPB. For further thermal stability the Cpn-AgNPs suspension was
heated up to 100oC and monitored through UV-visible spectroscopy, which did not show any
shifting or withdrawal of the SPB (Figure 3.40). This indicated that the NPs are stable up to
100oC [215].
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Figure 3. 40: Thermal stability of Cephradine stabilized silver nanoparticles
A little decrease in the absorbance peak was observed without any precipitation, this may be due
to the electronic dephasing mechanism which involves electron-electron interactions as the
electron-surface and electron-defect scattering increase at high temperature. The velocity of an
electron depend on its state energy and temperature. As at elevated temperature increased
velocity of the electrons leads to a more damping constant and so to a faster dephasing and
therefore subsequent decrease of absorbance of plasmon band occur [221].
3.3.3.2 Salt Stability
The effect of different concentrations of NaCl aqueous solution was studied on Cpn-AgNPs and
the suspensions were found to be stable for NaCl concentrations up to 100 mM (Figure 3.41).
However higher concentration of NaCl results reduction in the SPB which is due to the
accumulation of NPs stimulated by Cl-1 ions [221].
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Figure 3. 41: Stability of Cpn-AgNPs against various concentration of salt
3.3.3.3 PH stability
pH stability of the suspensions were measured to determine whether the Cpn-AgNPs would be
stable under physiological conditions. The suspensions were found stable for a pH between 4 and
12 as the aggregation was not observed in this range (Figure 3.42).
Figure 3. 42: Stability of Cpn-AgNPs against pH
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84
3.3.4 Synthesis of AuNPs stabilized with Cephradine
The gold NPs stabilized with Cephradine (Cpn-AuNPs) were synthesized through a single-step
reduction method by mixing solutions of HAuCl4 and Cephradine in the presence of
triethylamine as reducing agent. The reaction mixture turned to violet, indicated the formation of
Cpn-AuNPs. The amount of conjugated Cephradine was determined by centrifuging Cpn-AuNPs
from the suspension. The precipitated NPs were collected. The supernatant was centrifuged three
times then was freeze-dried to determine the amount of unreacted Cephradine. The Cephradine
was quqntified, using this manner, to be 6.35 % by weight for Cpn-AuNPs [216].
3.3.5 Characterization of Cpn-AuNPs
UV-visible spectrum of the Cpn-AuNPs demonstrated peak at 532 nm (Figure 3.43), which can
be resolved with the distinctive plasmonic absorption of AuNPs [223].
Figure 3. 43: UV-Visible spectrum of Cephradine stabilized gold nanoparticles
Maximum absorbance intensity of SPB was found at 9:1 (Au:Cephradine) ratio indicated
complete reduction of Au ions at this ratio. Further increase in the Cephradine amount caused the
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85
decrease in the intensity of the absorbance which may be due to aggregation and hence, reducing
the number of nanoparticles [219].
The conjugation of Cephradine was also established by FT-IR spectroscopy (Figure 3.44).
Distinctive absorption bands appeared in the Cephradine FT-IR spectrum at 3440 cm-1 and 3300
cm-1 which might be allocated to the stretching vibrations of N-H and O-H groups respectively.
The band for the C-H groups stretching vibrations was observed at 3025 cm-1. The stretching of
C=O of the lactame ring was detected at 1760 cm-1 and the amide C=O shown a band at 1680
cm-1. The band at 1588 cm-1 could be related to the torsional vibrations of aromatic ring. The
bands at 1355 cm-1 might be allocated to the stretching of C-N of the thiazole and lactame ring.
The conjugation of Cephradine with AuNPs caused in integration of bands and the lessening in
absorbance intensities of C=O (1760 and 1680 cm-1), N-H (3440 cm-1) and O-H (3300 cm-1)
stretching [220].
Figure 3. 44: IR spectra of Cephradine (black) and Cpn-AuNPs (red)
AFM was used for the size determination of Cpn-AuNPs. The analysis revealed the formation of
spherical and poly dispersed NPs. The images showed the presence of narrow sized nanoparticles
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86
in the size range of 20-35 nm with a maximum particles of diameter range from 10-20 nm as
shown in the Figure 3.45.
A B
Figure 3. 45: AFM analysis of Cpn-AuNPs. Topography (A) and Particles size distribution (B)
3.3.6 Stability of Cpn-AuNPs
3.3.6.1 Thermal stability
Thermal stability of the Cpn-AuNPs was determined by UV-visible spectroscopy because
aggregation of NPs is usually escorted by a change in the SPB [215]. At ambient temperature the
synthesized conjugates were found stable for several days and no change was observed in the
SPB. For further stability the Cpn-AuNPs suspension was heated and monitored through UV-
Visible spectroscopy. Heating up to 100oC did not show precipitation and shift in the SPB
(Figure 3.46) indicated the stability of Cpn-AuNPs up to 100oC.
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Figure 3. 46: Thermal stability of Cephradine stabilized gold nanoparticles
A little decrease in the absorbance peak was observed, this may be due to the dominant
electronic dephasing mechanism which involves electron-electron interactions as the electron-
surface and electron-defect scattering increase at high temperature. The velocity of an electron is
related to its state energy and temperature. As at elevated temperature increased speed of the
electrons leads to a greater damping constant and hence to a faster dephasing and therefore
subsequent reduction of absorbance of plasmon band [221].
3.3.6.2 Salt Stability
NaCl aqueous solution was used to study the effect of its diverse concentrations on the Cpn-
AuNPs stability. Cpn-AuNPs were found stable for NaCl concentrations up to 50 mM (Figure
3.47). However increased concentration of NaCl resulted in more Cl-1 which caused the
aggregation of Cpn-AuNPs. This led to the reduction of SPB of Cpn-AuNP [222].
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88
Figure 3. 47: Stability of Cpn-AuNPs against various concentration of salt
3.3.6.3 PH stability
PH stability of the suspensions was measured to determine whether the Cpn-AuNPs would be
stable under physiological conditions. The suspensions were found stable for a pH between 1 and
12 as the aggregation was not observed in this range (Figure 3.48).
Figure 3. 48: Stability of Cpn-AuNPs against pH
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89
3.3.7 Evaluation of antibacterial potential of Cpn-AgNPs and Cpn-AuNPs
In order to enhance the antibacterial potential and kinetics of Cephradine, it was modified into
nano-sized metal based materials (Cpn-AgNPs and Cpn-AuNPs). Sultana et al. have synthesized
Cephradine metal complexes and studied its antimicrobial activity [238], Choudry et al. have
also synthesized and studied antimicrobial behavior of Cephradine metal complexes [239] and
Zhong et al. synthesized nanosized Cephradine with a small size and greater surface area [240].
Chohan et al. synthesized Cephradine based metal complexes and screen these for antimicrobial
activity [241].
In the current study effort was made to enhance the antibacterial potential of the cephradine via
modification into nanosized material. Antimicrobial action of the modified Cephradine was
evaluated with conventional biological methods like minimum inhibitory concentration (MIC)
and fascinating equipments like AFM. MICs were evaluated through a zone of inhibition [227].
The MIC of pure Cephradine, Cpn-AgNPs and Cpn-AuNPs were established to be 10 ± 0.2 µg
mL-1, 25 ± 0.3 µg mL-1 (corresponds to a 4.59 µg Cephradine) and 25 ± 0.2 µg mL-1 (correspond
to a 1.58 µg Cephradine), respectively. While the MIC of bare Ag and Au NPs were calculated
to be 50 µg mL-1 and 60 µg mL-1, respectively (Figure 3.49). The MIC of pure Cephradine was
less as compared to its Ag & Au conjugates, while unconjugated Ag and Au NPs exhibited
relatively high minimum inhibitory values when compared with the pure Cephradine and its Ag
and Au conjugates. As Cephradine was present in quite less amount in the NPs (18.395% for
Cpn-AgNPs and 6.35% for Cpn-AuNPs), this indicated that conjugated cphradine had a 2-6
times higher antibacterial potential than pure Cephradine.
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Figure 3. 49: MICs of Cephradine (1), Cpn-AgNPs (2), Cpn-AuNPs (3), bare AgNPs (4) and
bare AuNPs (5)
The MIC values are also validated by the AFM study. Freshly grown cells of S. aureus ATCC
25923 showed spherical cells with healthy membranes of mean length of 1.45 mm, mean height
of 0.35 mm and mean width of 0.95 mm as presented in figure 3.50.
Figure 3. 50: AFM images of S. aureus ATCC 25923 before treatment, Tophography (A) and 3D
(B)
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91
Bacterial cells were then treated with minimum killing concentration of Cephradine, and cell
morphology was analyzed as a function of incubation time.
Figure 3. 51: AFM images of S. aureus treated with Cephradine (A), Cpn-AgNPs (B) and Cpn-
AuNPs (C) for 1 hr
Cephradine like other β-lactam antibacterial drugs, acts by precluding the synthesis of the
peptidoglycan layer of bacterial cell walls which is essential for cell wall structural integrity.
Bacteria treated with MIC dose of unconjugated Cephradine for 1 hour showed slight effect and
only lesion on bacterial cell surface was observed (Figure 3.51a).
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Figure 3. 52: AFM images of S. aureus treated with Cephradine (A), Cpn-AgNPs (B) and Cpn-
AuNPs (C) for 2 hrs
Cell Morphological degradation increased with time and a 2 hours treatment have further
affected bacterial cells as size reduction and rough surfaces of the cells was noticed (Figure
3.52a). After 4 hours treatment, destruction of cell bodies were observed and besides spherical
shaped S. aurious, cells with tending to oval shape were detected (Figure 3.53a) while after 8
hours the cell morphology were degraded and completely distorted (Figure 3.54a). We assume
that this altering of the bacterial forms is due to destruction of the cell wall, followed by
spreading out of peptidoglycan on the mica surface.
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Figure 3. 53: AFM images of S. aureus treated with Cephradine (A), Cpn-AgNPs (B) and Cpn-
AuNPs (C) for 4 hrs
Conjugated Cephradine were found to degrade the cells faster than pure Cephradine due its
strong bactericidal effect in nano-size range.
The culture treated with the MIC dose of Cpn-AgNPs for 1 hour were found to effect the cell
more than respective pure Cephradine as the bacterial cells shape distortion and surface
roughness is more noticeable (Figure 3.51b) and a further more effect was seen in 2 hours
treatment where the cell walls rupturing is more prominent (Figure 3.52b) while after 4 hours it
led to complete destruction of bacterial cells (Figure 3.53b). Similarly Cpn-AuNPs of MIC dose
have showed relatively more effect on the bacterial cells in 1 hour and 2 hours treatment (Figure
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94
3.51c and 3.52c), while treatment for the same amount in 4 hours led to the complete destruction
of S. aureus cells and melted cells were observed (Figure 3.53c).
Figure 3. 54: AFM images of S. aureus treated with Cephradine (A), bare AgNPs (B) and bare
AuNPs (C) for 8 hrs
Unconjugated Ag and Au NPs of MIC doses cannot showed distinctive effect but only minimal
morphological changes and slight influence was observed even after treatment for 8 hours
(Figure 3.54b and c).
The high affinities of Ag and Au towards sulfur aid the nanoparticles to adhere to proteins in the
cell membrane and penetrate into the cell. Inside the cell the NPs interact with DNA, protein, and
Chapter-3 Results and Discussion
95
phosphorus thus, it can interrupt vital cellular actions and eventually lead to the cell death. Here
in this case Cephradine reacts with the outer layer of S. aureus thereby enhancing the
membrane’s permeability. NPs precipitated on the bacterial cell or gather in the cytoplasm or in
the periplasm region disturb the cellular proceedings, resulting in membranes disruption and
disorder. Subsequently the NPs get into the cell through the membrane and may be attached to
the bacterial DNA and protein; thus, cause death of the cell by disturbing metabolism and vital
functions [25, 172, 228]. Consequently, the mutual action of Cephradine and Ag or Au NPs leads
to enhanced antibacterial potential [168].
3.4 Modification of Ampicillin via conjugation with Ag and Au nanoparticles
3.4.1 Synthesis of AgNPs stabilized with Ampicillin
Ampicillin was capped with silver nanoparticles (Mpn-AgNPs) by mixing its aqueous solution
with ionic solutions of Ag in the presence of triethylamine as a reducing agent. Different volume
ratios of the Ag salt solution and Ampicillin solution was reacted and a 9:1 (Ag:Ampicillin) mole
ratio was found to have best result in respect of color and SPB. The reaction mixture was turned
to yellowish red; the reaction was monitored through UV-visible spectroscopy to observe the
formation of Mpn-AgNPs. Further increase in the Ampicillin amount resulted reduction in the
intensity of the absorbance band, this is due to the aggregation thereby decreasing the number of
nanoparticles [219]. The amount of conjugated Ampicillin was determined by centrifuging out
Mpn-AgNPs. The supernatant was repeatedly centrifuged to remove the Mpn-AgNPs
completely. The amount of unreacted ampicillin was determined and finally it was calculated
that Mpn-AgNPs have 18 wt% of ampicillin [216].
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3.4.2 Characterization of Mpn-AgNPs
The UV-visible spectrum of the Mpn-AgNPs exhibited peak at 396 nm (Figure 3.55) which can
be correlated with the typical plasmonic absorption of AgNPs [218].
Figure 3. 55: UV-Visible spectrum of Ampicillin stabilized silver nanoparticles
The formation of Mpn-AgNPs was further confirmed by FT-IR spectroscopy (Figure 3.56). The
FT-IR spectrum of Ampicillin exhibited absorption bands in region 3512 cm-1 and 3205 cm-1
which might be associated to the stretching of O-.H and N-H groups. The band at 2968 cm-1 can
be allocated to the stretching of C-H groups, carbonyl group of lactam ring showed the stretching
vibration at 1774 cm-1 and the carbonyl group of amide exhibited band at 1688 cm-1. The band at
1372 cm-1 could be allocated to the stretching vibrations of C-N of the thiazole and lactame. The
conjugation of Ampicillin with AgNPs result in the decrease in absorbance intensities and
merging of bands of O-H (3512 cm-1), N-H (3205 cm-1) and C=O (1774 and 1688 cm-1)
stretching [229].
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Figure 3. 56: IR spectra of Ampicillin (black) and Mpn-AgNPs (red)
AFM was used for the size determination of Ag and nanoparticles. The analysis revealed the
formation of spherical and poly dispersed Mpn-AgNPs. According to the AFM images their size
ranges from 15-50 nm with maximum particles of size range from 25-40 nm as shown in the size
distribution histogram (Figure 3.57).
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98
A B
Figure 3. 57: AFM analysis of Mpn-AgNPs. Topography (A) and Particles size distribution (B)
3.4.3 Stability of Mpn-AgNPs
3.4.3.1 Thermal stability
UV-Visible spectroscopy was used for the description ofstability of the Mpn-AgNPs because
aggregation of NPs is usually accompanied by a change in the SPB [215]. At ambient the
synthesized conjugates were found stable for several days and no change was observed in the
SPB. For further thermal stability the Mpn-AgNPs suspension was heated and monitored through
UV-Visible spectroscopy. It was found that Mpn-AgNPs were stable up to 100oC because any
decrease or shifting in the SPB was not observed (Figure 3.58).
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Figure 3. 58: Thermal stability of Ampicillin stabilized silver nanoparticles
3.4.3.2 Salt Stability
The effect of NaCl aqueous solution was studied on Mpn-AgNPs stability. The Mpn-AgNPs
were found stable for NaCl concentrations up to 100 mM (Figure 3.59). However, higher
concentration of NaCl resulted the aggregation of Mpn-AgNPs [222] as the SPB intensity was
reduced.
Figure 3. 59: Stability of Mpn-AgNPs against various concentration of salt
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3.4.3.3 PH stability
The PH stability of Mpn-AgNPs were examined to determine whether the conjugates would be
stable under physiological conditions. The suspensions were found to be stable for a pH between
3 and 12 as the aggregation was not observed in this range (Figure 3.60).
Figure 3. 60: Stability of Mpn-AgNPs against pH
3.4.4 Synthesis of AuNPs stabilized with Ampicillin
Gold nanoparticles stabilized with Ampicillin (Mpn-AuNPs) were synthesized through single
step reduction method by mixing solutions of HAuCl4 and Ampicillin in the presence of
triethylamine as reducing agent. The solutions were reacted in different volume ratios and a 12:1
(Au:Ampicillin) ratio was found to be suitable ratio for the synthesis of Mpn-AuNPs. Upon
mixing the color of the reaction mixture turned to purple red, indicated the formation of Mpn-
AuNPs.
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For the quantification of ampicillin in Mpn-AuNPs, the nanoparticles were completely removed
from the suspension by centrifugation. The supernatant obtained after the removal of
nanoparticles was freeze-dried. The weight of the dried materials was noted. The amount of
Ceftriaxone was calculated, to be 6.03 % by weight for Mpn-AuNPs [216].
3.4.5 Characterization of Mpn-AuNPs
The reaction was periodically examined by UV-visible spectroscopy to check the formation of
Mpn-AuNPs. The UV-visible spectrum of the Mpn-AuNPs demonstrated peak at 540 nm
(Figure 3.61), which can be related with the representative plasmonic absorption of AuNPs
[223].
Figure 3. 61: UV-Visible spectrum of Ampicillin stabilized gold nanoparticles
Different volumes ratios of HAuCl4 and Ampicillin solution were reacted and maximum
absorbance intensity of SPB was found at a 12:1 (Au:Ampicillin) ratio indicated complete
reduction of Au ions at this ratio. Further increase in the Ampicillin amount caused the decrease
in the intensity of the absorbance which may be due to aggregation of Mpn-AgNPs and hence,
reducing the number of nanoparticles [219].
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The conjugation of Ampicillin with gold NPs was further established by FT-IR spectroscopy
(Figure 3.62). The FT-IR spectrum of Ampicillin exhibited characteristics absorption bands in
region 3512 cm-1 and 3205 cm-1 which could be associated to the stretching vibrations of O-H
and N-H groups, respectively. The band at 2968 cm-1 can be consigned to the stretching
vibrations of C-H groups, lactam ring carbonyl group showed the stretching vibration at 1774
cm-1 and that of amide exhibited band at 1688 cm-1. The band at 1372 cm-1 can be allocated to
the stretching vibrations of C-N of the thiazole and lactame. The conjugation of Ampicillin with
AuNPs resulted in the decrease in absorbance intensities and merging of bands of O-H (3512 cm-
1), N-H (3205 cm-1) and C=O (1774, 1688 cm-1) stretching [229].
Figure 3. 62: IR spectra of Ampicillin (black) and Mpn-AuNPs (red)
The size and shape of Mpn-AuNPs was determined by AFM. The images are reported in figure
3.63. Mpn-AuNP possess spherical shape with average size ranges from 15-50 nm with
maximum particles in the range of 20-30 nm.
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A B
Figure 3. 63: AFM analysis of Mpn-AuNPs. Topography (A) and Particles size distribution (B)
3.4.6 Stability of Mpn-AuNPs
3.4.6.1 Thermal stability
The stability of Cef-AgNPs was characterized by UV-visible spectroscopy. Coagulation of NPs
generally resulted in shifting of the surface plasmon towards longer wavelengths [215]. At
ambient temperature the synthesized conjugates were found to be stable for several days and no
change was observed in the SPB. For further thermal stability the Mpn-AuNPs suspension was
heated and monitored through UV-Visible spectroscopy. Heating up to 50oC did not show
precipitation and shifting in the SPB (Figure 3.64).
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Figure 3. 64: Thermal stability of Ampicillin stabilized gold nanoparticles
A little decrease in the absorbance peak was observed, this may be due to the dominant
electronic dephasing mechanism which involves electron-electron interactions as the electron-
surface and electron-defect scattering increase at high temperature. The velocity of an electron is
related to its state energy and with the temperature. As at elevated temperature increased velocity
of the electrons leads to a more damping constant and therefore to a faster dephasing and hence
subsequent reduction of absorbance of plasmon band occur [221].
3.4.6.2. Salt Stability
Aqueous solution of NaCl was used to study the effect on the stability of Mpn-AuNPs.
Suspension of Mpn-AuNPs were found to be stable for NaCl concentrations up to 50 mM
(Figure 3.65). However high concentration of NaCl resulted in the reduction of SPB which may
be due to the aggregation of Mpn-AuNPs promoted by Cl-1 ions [222].
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105
Figure 3. 65: Stability of Mpn-AuNPs against various concentration of salt
3.4.6.3 PH stability
The PH stability of Mpn-AuNPs were measured to determine whether the Mpn-AuNPs would be
stable under physiological conditions. The suspension were found to be stable for a pH between
3 and 12 as the aggregation was not observed in this range (Figure 3.66).
Figure 3. 66: Stability of Mpn-AuNPs against pH
Chapter-3 Results and Discussion
106
3.4.7 Evaluation of antibacterial potential of Mpn-AgNPs and Mpn-AuNPs
The morphological analysis and mechanism of action of the antimicrobial activity of Ampicillin
conjugated with AgNPs (Mpn-AgNPs) and AuNPs (Mpn-AuNPs) against Staphylococcus aureus
ATCC 11632 using AFM was explored in this study. S. aureus is a sensitive strain of bacteria
that infect human and can cause respiratory disease, food poisoning and skin infections [242]. S.
aureus is notorious for its capability to develop resistance to antibiotics and has created a
worldwide problem in clinical treatment [233].
The study was carried out to examine the boosted antibacterial action and kinetics of the
modified Ampicillin through AFM against S. aureus, which is not yet explored. Nikiyan et al.
studied the membranolytic properties in the mechanisms of action of the antibiotics Ampicillin,
magainin and human platelets extract by using Bacillus cereus and Escherichia coli [243], Saha
et al. have synthesized chitosan NPs of Ampicillin trihydrate and claimed to be capable of
sustained delivery of Ampicillin [244] and Brown et al. have functionalized Ampicillin with Ag
and Au NPs and study their antimicrobial activity against different bacterial strains by
determining their minimum bactericidal concentration (MBC) [245].
The analysis offering the description on visualization of the effect of Ampicillin and its Ag and
Au NPs on S. aureus by AFM. The minimum inhibitory concentration (MIC) of Ampicillin and
its Au and Ag NPs was determined through zone of inhibition [227]. The MIC of pure
Ampicillin and conjugated Ampicillin were found to be 50 ± 0.1 µg mL-1, 60 ± 0.3 µg mL-1
(corresponds to a 10.8 µg Ampicillin) and 75 ± 0.3 µg mL-1 (correspond to a 4.52 µg
Ampicillin), respectively. While the MIC of bare Ag and Au NPs were calculated to be 85 ± 0.3
µg mL-1 100 ± 0.2 µg mL-1, respectively (Figure 3.67).
Chapter-3 Results and Discussion
107
Figure 3. 67: MICs of Ampicillin (1), Mpn-AgNPs (2), Mpn-AuNPs (3), bare AgNPs (4) and
bare AuNPs (5).
The MIC of unconjugated Ampicillin is in agreement with a former study [246]. Although the
MIC of Ag and Au conjugates was more than pure Ampicillin, but the conjugates contain a small
weight fraction of the Ampicillin (18 % for Mpn-AgNPs and 6.03% for Mpn-AuNPs), this
specified that Ampicillin conjugated to Ag and Au NPs is about 5 and 10 times more active than
pure Ampicillin respectively.
The MIC values were supported by AFM which explored the more persuasive and rapid action
of the conjugates. Morphological characterization of the control S. aureus samples showed
typically round cells with smooth membranes and spherical shapes with a mean length of 1.052
µm, mean width of 1.082 µm and mean height of 0.104 µm and with a maximum height of 0.719
µm, as shown in figure 3.68.
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Figure 3. 68: AFM images of S. aureus ATCC 11632 before treatment, Tophography (A) and 3D (B)
Bacterial cultures were then treated with pure Ampicillin, Mpn-AgNPs, Mpn-AuNPs, Bare Ag
and Au NPs to study the comparative action and kinetics under AFM. Bacteria treated with MIC
dose of unconjugated Ampicillin for 1 hour showed slight effect and only lesion on bacterial cell
surface was observed (Figure 3.69).
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109
Figure 3. 69: AFM images of S. aureus treated with Ampicillin (A), Mpn-AgNPs (B), Mpn-
AuNPs for 1 hr
Cell Morphological degradation increased with time as a 2 hours treatment have further affected
bacterial cells and after 4 hours considerable damages of cell bodies were observed (Figure
3.70a and 3.71a).
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110
Figure 3. 70: AFM images of S. aureus treated with Ampicillin (A), Mpn-AgNPs (B), Mpn-
AuNPs for 2 hrs
While after 8 hours the cell morphology were degraded and completely distorted (Figure 3.72a).
On the other hand the culture treated with the MIC dose of Mpn-AgNPs for 1 hour were found to
effect the cell more than respective pure Ampicillin (Figure 3.69b) and a further more effect
was seen in 2 hours treatment (Figure 3.70b) while after 4 hours it led to complete destruction of
bacterial cells (Figure 3.71b). Similarly Mpn-AuNPs of MIC dose have showed relatively more
effect on the bacterial cells in 1 hour and 2 hours treatment (Figure 3.69c and 3.70c), while
treatment for the same amount in 4 hours led to the complete destruction of S. aureus cells
(Figure 3.71c).
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Figure 3. 71: AFM images of S. aureus treated with Ampicillin (A), Mpn-AgNPs (B) and Mpn-
AuNPs (C) for 4 hrs
Unconjugated Ag and Au NPs of MIC doses cannot showed distinctive effect but only minimal
morphological changes and slight influence was observed even after treatment for 8 hours
(Figure 3.72b and c).
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112
Figure 3. 72: AFM images of S. aureus treated with Ampicillin (A), bare AgNPs (B) and bare
AuNPs (C) for 8 hrs
The interaction of NPs with a bacterial cell still need further exploration, however many studies
have shown that first metal NPs adsorb to surface of a microorganism due to resultant
electrostatic pressure and high affinity of metals towards sulphur in the proteins [235]. After that,
NPs get inside into the cell causing perforation and leads to the release of the cellular matrix
[144, 236, 237]. Here in this case Ampicillin react with the outer peptidoglycan layer of S.
aureus thereby enhancing the membrane’s permeability. Subsequently the NPs get into the cell
through the membrane and may be attached to the bacterial DNA and protein; thus, caused death
Chapter-3 Results and Discussion
113
of the cell by disturbing metabolism and vital functions [25, 172, 228]. Consequently, the mutual
action of Ampicillin and Ag or Au NPs resulted the enhancement of antibacterial potential [168].
In this study AFM explored noticeable investigation of living bacterial cells by offering thorough
topographic demonstration of surface, shape and phase imaging morphology that let analysis of
height, width, length and boundary stiffness.
3.5 Modification of Cefixime via conjugation with Ag and Au nanoparticles
3.5.1 Synthesis of AgNPs stabilized with Cefixime
Cefixime was coated with AgNPs (Cfm-AgNPs) by mixing its aqueous solution with ionic
solutions of Ag in the presence of triethylamine as reducing agent. Different volumetric ratios of
the Ag salt solution and Cefixime solutions have reacted and a 10:1 (Ag:Cefixime) mole ratio
was found to have best results in respect of color and SPB. The reaction mixture was turned to
yellowish red indicated the formation Cfm-AgNPs. The amount of conjugated Cefixime was
determined by centrifuging out Cfm-AgNPs from the suspension. The supernatant was
repeatedly centrifuged to remove the synthesized Cfm-AgNPs. The supernatant was then freeze-
dried and the amount of unreacted drug was calculated. Using this method the Cefixime was
quantified, to be 20.85 wt% for Cfm-AgNPs [216].
3.5.2 Characterization of Cfm-AgNPs
The reaction was monitored through UV-visible spectroscopy to monitor the formation of Cfm-
AgNPs. UV-visible spectrum of the Cfm-AgNPs exhibited plasmonic peak at 397 nm (Figure
3.73), which are in agreement with the distinctive plasmonic absorption bands of AgNPs [218].
Chapter-3 Results and Discussion
114
Figure 3. 73: UV-Visible spectrum of Ceftriaxone stabilized silver nanoparticles
Further increase in the Cefixime amount caused the decrease in the intensity of the absorbance
band, this is due to the aggregation thereby decreasing the number of NPs [219].
The conjugation of Cefixime with the AgNPs was also established by FT-IR spectroscopy and
the resultant FT-IR spectra are shown in figure 3.74. The spectrum of Cefixime showed
characteristics absorption bands at 3559 cm-1, 3296 cm-1, 1771 cm-1 and 1669 cm-1 which could
be correlated to the stretching vibrations of OH, NH, lactame carbonyl and amide carbonyl
groups respectively [247]. In the case of nanoparticles, these distinguishing peaks were altered
(broadening, reduction, frequency shifts and/or disappearance) which could be attributed to the
conjugation of Cefixime to the NPs [229].
Chapter-3 Results and Discussion
115
Figure 3. 74: IR spectra of Ceftriaxone (black) and Cef-AgNPs (red)
Cfm-AgNPs were visualized under AFM. The analysis exposed the formation of spherical and
poly dispersed Cfm-AgNPs. The images showed the presence of various size nanoparticles in the
size range from 10-50 nm with a maximum particles range of 20-30 nm as shown in the size
distribution histogram (Figure 3.75).
A B
Figure 3. 75: AFM analysis of Cfm-AgNPs. Topography (A) and Particles size distribution (B)
Chapter-3 Results and Discussion
116
3.5.3 Stability of Cfm-AgNPs
3.5.3.1 Thermal stability
At ambient temperature the Cfm-AgNPs were found stable for several days and no change was
observed in the SPB. For further thermal stability study, the Cfm-AgNPs suspension was heated
and monitored through UV-visible spectroscopy, as aggregation of NPs is usually noticed by a
change in the SPB [215]. Heating up to 100oC did not showed any precipitation and shift in the
SPB (Figure 3.76) indicated the stability of Cfm-AgNPs up to this temperature.
Figure 3. 76: Thermal stability of Cefixime stabilized silver nanoparticles
3.5.3.2 Salt Stability
Cfm-AgNPs were mixed with salt (NaCl) solutions of different concentrations and kept at room
temperature for 24 hous. UV-visible spectra were recorded to study the effect of salt on the
stability of Cfm-AgNPs. Cfm-AgNPs were found stable for salt concentrations up to 200 mM
(Figure 3.77). However increased concentration of NaCl resulted in the aggregation and
reduction of SPB [222].
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117
Figure 3. 77: Stability of Cfm-AgNPs against various concentration of salt
3.5.3.3 PH stability
Cfm-AgNPs were stored for 24 hours in different pH medium. The samples were carefully
analyzed, Cfm-AgNPs were found stable for a pH between 3 and 12 as the aggregation was not
observed in this range (Figure 3.78).
Figure 3. 78: Stability of Cfm-AgNPs against pH
Chapter-3 Results and Discussion
118
3.5.4 Synthesis of AuNPs stabilized with Cefixime
Gold nanoparticles stabilized with Cefixime (Cfm-AuNPs) were synthesized through single step
reduction method by mixing solutions of Cefixime and HAuCl4 in the presence of triethylamine
as reducing agent. Upon reacting in a specific volume the reaction mixture turned to violet color,
indicated the formation of Cfm-AuNPs. The amount of Cefixime was determined by centrifuging
out Cfm-AuNPs from the suspension. The precipitated nanoparticles were collected and the
process was repeated three times to collect Cfm-AuNPs completely. The supernatant containing
unreacted Cefixime was then freeze-dried. The Cefixime was quantified, using this manner, to be
7.2 % by weight for Cfm-AuNPs [216].
3.5.5 Characterization of Cfm-AuNPs
The formation of Cfm-AuNPs was checked by UV-visible spectroscopy. The spectrum of the
Cfm-AuNPs showed peak at 532 nm (Figure 3.79), which is in agreement with the
distinguishing plasmonic absorption of AuNPs [223]. Different volumes ratios of HAuCl4 and
Cefixime solutions were reacted and maximum absorbance intensity of SPB was found at a 13:1
(Au: Cefixime) ratio indicated complete reduction of Au ions at this ratio. Further increase in the
Cefixime amount caused the reduction in the intensity of the absorbance band which may be due
to aggregation followed by lessening the number of Cfm-AuNPs [219].
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119
Figure 3. 79: UV-Visible spectrum of Cefixime stabilized gold nanoparticles
The formation of Cfm-AuNPs was also established by FT-IR spectroscopy and the resultant FT-
IR spectra are shown in figure 3.80. The spectrum of Cefixime showed characteristics
absorption bands at 3559 cm-1, 3296 cm-1, 1771 cm-1 and 1669 cm-1 which could be correlated to
the stretching vibrations of OH, NH, lactame carbonyl and amide carbonyl groups, respectively
[247]. In the case of Cfm-AuNPs, these distinguishing peaks were altered (broadening,
reduction, frequency shifts and/or disappearance) that could be attributed to the conjugation of
Cefixime to the NPs [229].
Figure 3. 80: IR spectra of Cefixime (black) and Cfm-AuNPs (red)
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120
Cfm-AuNPs were visualized under AFM. The images are reported in figure 3.81 which exposed
the formation of spherical and poly dispersed NPs. The analysis showed the presence of various
size NPs with diameter range of 10-50 nm with a maximum particles in the size ranges from 30-
40 nm as presented in the size distribution histogram.
A B
Figure 3. 81: AFM analysis of Cfm-AuNPs. Topography (A) and Particles size distribution (B)
Chapter-3 Results and Discussion
121
3.5.6 Stability of Cfm-AuNPs
3.5.6.1 Thermal stability
At ambient temperature the synthesized conjugates were found stable for several days and no
change was observed in the SPB. For further thermal stability the Cpn-AgNPs suspension was
heated up to 100oC and monitor through UV-Visible spectroscopy, which did not show any
shifting or withdrawal of the SPB (Figure 3.40). This indicated that the NPs are stable up to
100oC [215] as heating up to 100oC did not showed precipitation and shift in the SPB (Figure
3.82).
Figure 3. 82: Thermal stability of Cefixime stabilized gold nanoparticles
The speed of an electron is related to its state energy and therefore with the temperature. Due to
the electron-electron interactions at high temperature, as the electron-surface and electron-defect
scattering increase at high temperature, hence, a slight decrease in the SPB occurred. It is known
Chapter-3 Results and Discussion
122
that at elevated temperature increased velocity of the electrons leads to a more damping constant
and therefore to a faster dephasing and hence subsequent reduction of SPB [221].
3.5.6.2 Salt Stability
Salt stability of Cfm-AuNPs was examined by mixing it with NaCl aqueous solution (different
concentration). Cfm-AuNPs were found stable for salt concentration up to 500 mM (Figure
3.83). However increased concentration of NaCl resulted in the reduction of SPB which may be
due to the aggregation of Cfm-AuNPs promoted by Cl-1 ions [222].
Figure 3. 83: Stability of Cfm-AuNPs against various concentration of salt
3.5.6.3 PH stability
PH stability of Cfm-AuNPs were measured by changing the pH of the Cfm-AuNPs suspension.
The Cfm-AuNPs were found stable for a pH between 3 and 12 as the aggregation was not
observed in this range (Figure 3.84).
Chapter-3 Results and Discussion
123
Figure 3. 84: Stability of Cfm-AuNPs against pH
3.5.7 Evaluation of antibacterial potential of Cfm-AgNPs and Cfm-AuNPs
The antibacterial potential and action mechanism of Cefixime and its Ag and Au conjugates
against S. aurous was studied by using a fascinating tool (AFM). The results showed the
enhancement in the antibacterial activity of the drug. Many efforts have been made to enhance
the antibacterial potential of Cefixime as Pillai et al. have synthesized Cefixime metal complexes
and studied their antibacterial activities [248], Kuang et al. synthesized amorphous Cefixime NPs
by antisolvent precipitation method and suggested that the dissolution and solubility of the drug
were considerably improved [247], Anacona et al. evaluated the antibacterial action of transition
metals complexes of Cefixime [249], Danish et al. syntheized metal complexes and organo-tin
compounds of Cefixime and studied their antioxidant and enzyme-inhibition potential [250],
Hussein et al. synthesized Cefixime nanocrystals by anti-solvent precipitation method using PVP
stabilizer [251] and Nazari et al. evaluated the combined effects of gold nanoparticles and gold
ions with 14 different antibiotics including Cefixime against Pseudomonas aeruginosa bacteria
[252].
Chapter-3 Results and Discussion
124
Similar effort have been made to enhance the antibacterial potential of cefixime. Cefixime was
successfully conjugated with silver and gold NPs. The comparative antimicrobial action of Cfm-
AgNPs and Cfm-AuNPs was studied by AFM and other usual biological methods such as the
agar well diffusion method [227]. The MIC of pure Cefixime, Cfm-AgNPs and Cfm-AuNPs
were found to be 25 ± 0.32 µg mL-1, 35 ± 0.13 µg mL-1 (corresponds to a 7.29 µg Cefixime) and
45 ± 0.12 µg mL-1 (correspond to a 3.24 µg Cefixime), respectively. While the MIC of bare Ag
and Au NPs were calculated to be 50 ± 0.31 µg mL-1, 60 ± 0.52 µg mL-1, respectively (Figure
3.85). Although the MIC of Ag and Au conjugates was higher than pure Cefixime, but the
conjugates contain a small weight fraction of the Cefimixe (20.85 % for Cfm-AgNPs and 7.20 %
for Cfm-AuNPs), this specified that Cefixime conjugated to Ag and Au NPs is about 3 and 8
times more active than pure Cefixime, respectively.
Figure 3. 85: MICs of Cefixime (1), Cfm-AgNPs (2), Cfm-AuNPs (3), bare AgNPs (4) and bare
AuNPs (5).
Further confirmation of the MIC values was supported by AFM which explored the more
persuasive and rapid action of the conjugates. Morphological characterization of the control S.
aureus ATCC 25923 samples showed typically round cells with smooth membranes spherical
Chapter-3 Results and Discussion
125
shape and with a mean length of 1.052 µm, mean width of 1.082 µm and mean height of 0.104
µm and with a maximum height of 0.719 µm, as shown in figure 3.86.
Figure 3. 86: AFM images of S. aureus ATCC 25923 before treatment, Tophography (A) and 3D
(B)
Bacterial culture were then treated with pure Cefixime, Cfm-AgNPs, Cfm-AuNPs, Bare AgNPs
and AuNPs to study the comparative action and kinetics under AFM. Bacteria treated with MIC
dose of unconjugated Cefixime for 1 hour showed slight effect and only lesion on bacterial cell
surface was observed (Figure 3.87a).
Chapter-3 Results and Discussion
126
Figure 3. 87: AFM images of S. aureus treated with Cefixime (A), Cfm-AgNPs (B) and Cfm-
AuNPs (C) for 1 hr
Morphological degradation increased with time as a 2 hours treatment have affected bacterial
cells with increased roughness related to the smooth surfaces of the cells before treatment. The
change can be easily detected in images shown in figure 3.88a. In a 4 hours treatment, the effect
on the bacterial cells was further enhanced and considerable damages of cell bodies were
observed (Figure 3.89a), while after 8 hours the cell morphology were degraded and completely
distorted (Figure 3.90a).
Chapter-3 Results and Discussion
127
Figure 3. 88: AFM images of S. aureus treated with Cefixime (A), Cfm-AgNPs (B) and Cfm-
AuNPs (C) for 2 hrs
On the other hand the culture treated with the MIC dose of Cfm-AgNPs for 1 hour was affected
the cell more than respective pure Cefixime which may be owing to the changes produced in the
membrane by Cfm-AgNPs that may lead to alterations in cell osmolarity without the incidence of
lysis of cells in the observable images (Figure 3.87b). Furthermore influence was seen in 2
hours treatment where lysis of the membranes initiated (Figure 3.88b) while after 4 hours it led
to complete destruction of bacterial cells (Figure 3.89b). Similarly, Cfm-AuNPs of MIC dose
Chapter-3 Results and Discussion
128
have showed relatively potent effect on the bacterial cells in 1 hour and 2 hours treatment as
compare to free Cefixime (Figure 3.87c and 3.88c).
Figure 3. 89: AFM images of S. aureus treated with Cefixime (A), Cfm-AgNPs (B) and Cfm-
AuNPs (C) for 4 hrs
Treatment with the same amount for 4 hours led to the complete destruction of S. aureus cells
(Figure 3.89c). Unconjugated Ag and Au NPs of MIC doses did not show distinctive effect but
only minimal morphological changes and slight influence was observed even after treatment for
8 hours (Figure 3.90b and c).
Chapter-3 Results and Discussion
129
Figure 3. 90: AFM images of S. aureus treated with Cfm (A), bare AgNPs (B) and bare AuNPs
(C) for 8 hrs
Many studies have shown that first metal NPs adsorb to surface of a microorganism due to
resultant electrostatic pressure and high affinity of metals towards sulphur in the proteins [235].
After that, NPs get inside the cells causing perforation and leads to the release of the cellular
matrix [144, 236, 237]. Here in this case Cefixime reacted with the outer peptidoglycan layer of
S. aureus membrane thereby enhancing the membrane’s permeability. Subsequently the NPs get
into the cell through the membrane and may be attached to the bacterial DNA and protein; thus,
causing death of the cell by disturbing metabolism and vital functions [25, 172, 228].
Consequently, the nanosized Cefixime led to enhanced antibacterial potential [168]. In this
Chapter-3 Results and Discussion
130
study, AFM explored noticeable investigation of living bacterial cells by presenting complete
topographic demonstration of surface, shape and phase imaging morphology that let analysis of
height, width, length and boundary stiffness.
3.6 Modification of Ceftriaxone via encapsulation with Polymer
3.6.1 Synthesis of Polymer-Encapsulated Ceftriaxone Nanoparticles
Polymer encapsulated Ceftriaxone nanoparticles (Cef-PEG) were synthesized by antisolvent
precipitation process [70, 253]. The supersaturated solution of the drug was introduced to PEG
solution at controlled rate. This high supersaturation led to fast nucleation ratio and produced an
enormous number of nuclei that decreased the solute mass for successive growth. The NPs were
produced as the nucleation was halted by the stabilizer through electrostatic and steric
mechanism [242, 254]. Generally, a suitable stabilizer is required which must possess
respectable affinity for drug particles and dynamic adsorption onto the nanosized drug surface in
the solvent-water mixture [255]. The effect of PEG was studied on the precipitation of
Ceftriaxone. Drug and PEG volume ratios of 3:1 (PEG:Drug) was found to be appropriate ratio
for the synthesis of nanoparticles. Precipitation was observed immediately upon mixing at room
temperature. The obtained suspension was centrifuged at 15,000 rpm for 20 min and the
collected nanoparticles were washed three times to remove unreacted precursors.
3.6.2 Characterization of Cef-PEG
Cef-PEG were characterized and chemical structure of Ceftriaxone was evaluated with FT-IR
spectroscopy. The characteristic bands that were seen in the Ceftriaxone FT-IR spectrum were
3426 cm-1 and 3264 cm-1, which could be consigned to the stretching vibrations of N-H and O-H
groups. The band at 2935 cm-1 was allocated to C-H stretching and the stretching of carbonyl
groups (C=O) appeared at 1742 cm-1 and 1649 cm-1. The band at 1537 cm-1 might be related with
Chapter-3 Results and Discussion
131
the aromatic ring. The bands at 1033 cm-1 is be allocated to the stretching vibrations of C-O
group (Figure 3.91).
Figure 3. 91: IR spectra of Ceftriaxone (black) and Cef-PEG (red)
The spectra showed that raw Ceftriaxone and polymer encapsulated Ceftriaxone exhibited same
IR spectrum. It is clear that the chemical structure of the ceftriaxone is not altered during the
nanoparticles formation.
AFM results shown in figure 3.92 exposed the formation of spherical and poly dispersed Cef-
PEG nanoparticles. The morphology of the nanoparticles disclosed that particles have diameter
in the preferred range (from 10-80 nm with maximum particles of size range from 40-60 nm) as
presented in the histogram.
Chapter-3 Results and Discussion
132
A B
Figure 3. 92: AFM analysis of Cef-PEG. Topography (A) and Particles size distribution (B)
3.6.3 Drug entrapment efficiency
Maximum drug entrapment efficiency was observed in 3:1 (PEG:Drug) formulation. It exhibited
average of 61.25% entrapment efficiency and was found higher as compared to other
formulations. Entrapment efficiency depend on the nature of polymer and drug. Apart from this,
amount of polymer also affect the drug entrapment, as a specific concentration can entrap more
amount of drug. When the amount of polymer was further increased, this resulted reduction in
the entrapment, which might be owing to greater thickness of the polymeric solution that hamper
dispersion of drug in the polymer [94].
3.6.4 In-vitro release study
The in-vitro release behavior of Cef-PEG was studied at neutral pH (7.4). The cumulative
percentage of Ceftriaxone released from Cef-PEG at different time intervals is shown in figure
3.93. Cef-PEG released 50 percent of the Ceftriaxone within initial 4 hours. After this the NPs
Chapter-3 Results and Discussion
133
have exhibited considerable sustained release of the drug as 84 percent release was detected till
24 hours.
The initial burst release may be due to the presence of adsorbed drug on the polymer surface and
the later sustained release of the drug form Cef-PEG is owing to the slow diffusion of drug to the
polymer surface through the polymer [213].
Figure 3. 93: In-vitro drug release study of Cef-PEG (pH 7.4) at 37°C
3.6.5 Antibacterial study of Polymer-Encapsulated Ceftriaxone Nanoparticles
The synthesis of Cef-PEG were carried out to enhance the antibacterial potential of the drug
against E. coli. The MIC was evaluated through a zone of inhibition [227] using Ceftriaxone and
Cef-PEG. The MIC of pure Ceftriaxone and Cef-PEG were found 2.8 ± 0.63 µg mL-1 and 1.7 ±
0.22 µg mL-1, respectively against E. coli and 43 ± 0.53 µg mL-1 and 35 ± 0.72 µg mL-1,
respectively against S. aureus (Figure 3.94). This indicated that polymer encapsulated
Ceftriaxone had a 1.6 times higher activity than pure Ceftriaxone.
Chapter-3 Results and Discussion
134
Figure 3. 94: MICs of Cefrtiaxone against E.coli (1), Cef-PEG against E. coli (2), Cefrtiaxone
against S. aureus (3) and Cef-PEG against S. aureus (4)
3.7 Modification of Cefixime via encapsulation with Polymer
3.7.1 Synthesis of Polymer-Encapsulated Cefixime Nanoparticles
Polymer encapsulated Cefixime nanoparticles (Cfx-PEG) were synthesized by antisolvent
precipitation process [70, 253]. The supersaturated solution of the drug was introduced to PEG
solutions at controlled rate. This high supersaturation led to fast nucleation ratio and produced an
enormous number of nuclei that decreased the solute mass for successive growth. Thus
Nanoparticles were produced as the nucleation was halted by the PEG through electrostatic and
steric mechanism [242, 254]. Generally, a suitable stabilizer is required which must possess
respectable affinity for drug and dynamic adsorption onto the nanosized drug surface in the
solvent-water mixture [255]. Drug and PEG volume ratios of 1:2 (PEG:Drug) was found
appropriate ratio for the synthesis of nanoparticles. Precipitation was observed immediately upon
mixing at room temperature. The reaction mixture was centrifuged at 15,000 rpm for 20 min and
the collected NPs were washed three times to remove unreacted precursors.
Chapter-3 Results and Discussion
135
3.7.2 Characterization of Cfx-PEG
The FT-IR spectrum of Cefixime (Figure 3.95) showed characteristic absorption bands at 3559
cm-1, 3296 cm-1, 1771 cm-1 and 1669 cm-1 which could be correlated to the stretching vibrations
of OH, NH, lactame carbonyl and amide carbonyl groups respectively.
The analysis showed that raw Cefixime and polymer encapsulated Cefixime exhibited same IR
spectrum, thus determined that the chemical nature of the Cefixime is not changed during
nanoparticles formation.
Figure 3. 95: IR spectra of Cefixime (black) and Cfx-PEG (red)
AFM was used for the size and shape determination of Cfx-PEG. The images exposed the
formation of spherical and poly dispersed nanoparticles. The analysis showed the presence of
various size nanoparticles with their sizes range of 10-80 nm with a maximum particles of 45-65
nm as shown in the size distribution histogram (Figure 3.96).
Chapter-3 Results and Discussion
136
A B
Figure 3. 96: AFM analysis of Cfx-PEG. Topography (A) and Particles size distribution (B)
3.7.3 Drug entrapment efficiency
Maximum drug entrapment efficiency was observed in 1:2 (PEG:Drug) formulation. It exhibited
average of 75.00% entrapment efficiency and was found higher as compared to other
formulations. Entrapment efficiency depend on the nature of polymer and drug. Apart from this,
amount of polymer also affect the drug entrapment, as a specific concentration can entrap more
amount of drug. When the amount of polymer is further increased, this resulted reduction in the
entrapment, which might be owing to greater thickness of the polymeric solution that hamper
dispersion of drug in the polymer [94].
3.7.4 In-vitro release study
The in-vitro release behavior of Cfx-PEG was studied at neutral pH (7.4). The cumulative
percentage of Cefixime released from Cfx-PEG at different time intervals is shown in figure
3.97. Cfx-PEG released 50 percent of the Ceftriaxone within initial 4 hours. After this, the NPs
has exhibited considerable sustained release of the drug as 83 percent release was detected till 24
hours. This proposes that in vitro drug release showed initial fast release followed by sustained
release. The initial fast release may be due to the presence of adsorbed drug on the polymer
surface and the later sustained release of the drug form Cfx-PEG is owing to the slow diffusion
of drug to the polymer surface through the polymer [213].
Chapter-3 Results and Discussion
137
Figure 3. 97: In-vitro drug release study of Cfx-PEG (pH 7.4) at 37°C
3.7.5 Antibacterial study of Polymer-Encapsulated Cefixime Nanoparticles
The antibacterial activity of the Cfx-PEG was evaluated relative to the free drug. The MIC was
calculated through a zone of inhibition [227] using Cefixime and Cfx-PEG. MICs of cefixime
and Cfx-PEG were found 50 ± 0.82 µg mL-1 and 35 ± 0.35 µg mL-1, respectively against S.
aureus while 4 ± 0.65 µg mL-1 and 3.2 ± 0.85 µg mL-1, respectively against E. coli (Figure
3.98). This indicated that polymer encapsulated Cefixime had higher activity than pure Cefixime.
Figure 3. 98: MICs of Cefixime against S. aureus (1), Cfx-PEG against S. aureus (2), Cefixime
against E. coli (3) and Cfx-PEG E. coli (4)
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List of Publications
165
LIST OF PUBLICATIONS
1. Muhammad Raza Shah, Shujat Ali, Muhammad Ateeq, Samina Perveen, Shakil Ahmed,
Massimo F. Bertino and Mumtaz Ali “Morphological analysis of the antimicrobial action
of silver and gold nanoparticles stabilized with ceftriaxone on Escherichia coli using
atomic force microscopy” New. J. Chem., 38, 5633 (2014).
2. Muhammad Raza Shah, Shujat Ali, Samina Perveen, Mumtaz Ali and Shakil Ahmed
“Nanostructure Mediated Enhancement of Antibacterial Potential of Cefadroxil: Step
towards lead anti MRSA agents” (Submitted).
3. Shujat Ali, Muhammad Raza Shah, Samina Perveen, Mumtaz Ali and Shakil Ahmed
“Enhancement of antibacterial potential of Cephradine via conjugation with Ag and Au
nanoparticles and their assessment as a boosted antibacterial against S. aureus ATCC
25923 under AFM” (Submitted).
4. Shujat Ali, Muhammad Raza Shah, Samina Perveen, Mumtaz Ali and Shakil Ahmed
“Nanostructure mediated enhancement of antibacterial potential of Ampicillin and
investigation of their mode of action against Staphylococcus aureus ATCC 11632 using
AFM” (Submitted).
5. Shujat Ali, Muhammad Raza Shah, Samina Perveen, Mumtaz Ali and Shakil Ahmed
“Enhancement of antimicrobial activity of Cefixime against Staphylococcus aureus
ATCC 25923; a mechanistic approach (Submitted).