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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


2.14.2 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


3.1.6.2 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


3.2.3.2 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


3.2.6.2 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


3.3.3.2 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


3.3.6.2 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


3.4.3.2 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


3.5.3.2 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


3.5.6.2 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


3.6.4 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


3.7.4 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


(C) for 2 hrs. ............................................................................................................................ 75


(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


(C) for 2 hrs ............................................................................................................................. 92


(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


(C) for 1 hr ............................................................................................................................. 126


(C) for 2 hrs ........................................................................................................................... 127


(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.


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


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].


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).


5

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.


6

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].


7

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


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].


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


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


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].


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].


13

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


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


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


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].


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


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.


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


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


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


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


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


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


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


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


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.

https://en.wikipedia.org/wiki/%CE%92-lactam


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


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


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.


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].

https://en.wikipedia.org/wiki/Respiratory_tract_infection


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

http://www.medicalnewstoday.com/articles/252956.php

http://www.medicalnewstoday.com/articles/252956.php


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].


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


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


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


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.


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.


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


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


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.


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


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


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.


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


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.


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


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].


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


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


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].


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)


54

3.1.6 Stability of the silver nanoparticles stabilized with Ceftriaxone


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].


55


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


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).


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].


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


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).


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.


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


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


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].


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].


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)


65

3.2.3 Stability of the Cefd-AgNPs


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].


66


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


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).


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].


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].


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)


70

3.2.6 Stability of Cefd-AuNPs


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


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


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


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



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


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).


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


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.


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).


(C) for 4 hrs


77

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


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].


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).


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).


81

A B

Figure 3. 39: AFM analysis of Cpn-AgNPs. Topography (A) and Particles size distribution (B)

3.3.3 Stability of Cpn-AgNPs


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].


82

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].


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].


83

Figure 3. 41: Stability of Cpn-AgNPs against various concentration of salt


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


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


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


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


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.


87

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].


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].


88

Figure 3. 47: Stability of Cpn-AuNPs against various concentration of salt


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


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.


90

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)


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).


92


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.


93


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


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


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].


96

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].


97

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).


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


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).


99

Figure 3. 58: Thermal stability of Ampicillin stabilized silver nanoparticles


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


100


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.


101

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].


102

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.


103

A B

Figure 3. 63: AFM analysis of Mpn-AuNPs. Topography (A) and Particles size distribution (B)

3.4.6 Stability of Mpn-AuNPs


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).


104

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].


105

Figure 3. 65: Stability of Mpn-AuNPs against various concentration of salt


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


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).


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.


108

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).


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).


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).


111

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).


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


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].


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].


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)


116

3.5.3 Stability of Cfm-AgNPs


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


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].


117

Figure 3. 77: Stability of Cfm-AgNPs against various concentration of salt


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


Figure 3. 78: Stability of Cfm-AgNPs against pH


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].


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)


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)


121

3.5.6 Stability of Cfm-AuNPs


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


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].


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


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



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].


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


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).


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).


127


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


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).


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).


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


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


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.


132

A B

Figure 3. 92: AFM analysis of Cef-PEG. Topography (A) and Particles size distribution (B)


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


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.


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.


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).


136

A B

Figure 3. 96: AFM analysis of Cfx-PEG. Topography (A) and Particles size distribution (B)


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].


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)

References

138

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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).


“Nanostructure mediated enhancement of antibacterial potential of Ampicillin and

investigation of their mode of action against Staphylococcus aureus ATCC 11632 using

AFM” (Submitted).


“Enhancement of antimicrobial activity of Cefixime against Staphylococcus aureus

ATCC 25923; a mechanistic approach (Submitted).

nanostructure mediated enhancement of antibacterial ...prr.hec.gov.pk › jspui › bitstream ›...

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