SYNTHESIS OF NOBLE METAL BASED NANOPARTICLES AND …

SYNTHESIS OF NOBLE METAL BASED

NANOPARTICLES AND THEIR BIOMEDICAL

APPLICATIONS

SYED AKIF RAZA KAZMI

REGISTRATION NO. 2013-PHD-CHEM-22

SESSION 2013-2016

DEPARTMENT OF CHEMISTRY

GOVT. COLLEGE UNIVERSITY

LAHORE PAKISTAN

A THESIS TITLED

Synthesis of Noble Metal based Nanoparticles and their

Biomedical Applications

Submitted to GC University Lahore

in partial fulfillment of the requirements

for the award of degree of

Doctor of Philosophy

IN

CHEMISTRY

By

Syed Akif Raza Kazmi

Session 2013-2016

Registration No. 2013-PhD-CHEM-22

DEPARTMENT OF CHEMISTRY

GOVT. COLLEGE UNIVERSITY

LAHORE PAKISTAN

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DECLARATION

I, Mr. Syed Akif Raza Kazmi Registration No. 2013-PhD-CHEM-22 hereby

declare that the matter printed in the thesis titled “Synthesis of Noble Metal

based Nanoparticles and their Biomedical Applications” is my own work

and has not been submitted and shall not be submitted in future as research

work, thesis for the award of similar degree in any University, Research

Institution etc in Pakistan or abroad.

At any time, if my statement is found to be incorrect, even after my

Graduation, the University has the right to withdraw my PhD Degree.

___ _______

Dated: _21-06-2021_ Signatures of Deponent

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v

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PLAGIARISM UNDERTAKING

I, Mr. Syed Akif Raza Kazmi Registration No. 2013-PhD-CHEM-22

solemnly declare that the research work presented in the thesis titled “Synthesis of

Noble Metal based Nanoparticles and their Biomedical Applications” is solely my

research work, with no significant contribution from any other person. Small

contribution/ help wherever taken has been acknowledged and that complete thesis

has been written by me.

I understand the zero tolerance policy of HEC and Government College

University Lahore, towards plagiarism. Therefore, I as an author of the above titled

thesis declare that no portion of my thesis has been plagiarized and any material used

as reference has been properly referred/ cited.

I understand that if I am found guilty of any formal plagiarism in the above

titled thesis, even after the award of PhD Degree, the University reserves the right to

withdraw my PhD Degree and that HEC/ University has the right to publish my name

on HEC/ University website, in the list of culprits of plagiarism.

___ ______

Dated: 21-06-2021 Signatures of Deponent

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DEDICATION

To my

beloved father

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IN THE NAME OF ALLAH

THE MOST GRACIOUS, THE MOST MERCIFUL

First of all I avail this opportunity to bow my head before ALMIGHTY

ALLAH, Who has given me the wisdom and perseverance for completing this piece

of research work. I invoke peace for Holy Prophet Hazrat Muhammad (Peace be

upon him) who is forever torch of guidance and knowledge for humanity as a

whole.

I would like to show my deepest gratitude to my supervisor Prof. Dr.

Muhammad Zahid Qureshi, a respectable, responsible, supportive and cooperative

supervisor, who has given me the guidance and encouragement throughout my PhD

research work.

I pay thanks to Prof. Dr. Ahmad Adnan, Chairperson Department of

Chemistry, Prof. Dr. Islam Ullah Khan, Dean Faculty of Science and Technology

and Dr. Ayoub Rasheed Department of Chemistry, GC University Lahore, for their

cooperation during course and research work regarding official and laboratory

matters.

I extend a great debt of gratitude and cordial thanks to Prof. Dr. Jean-

Francois Masson who provides me the opportunity to do part of my research work

under his kind supervision at University of Montreal Canada. I feel great pleasure to

express my sincere gratitude and appreciation for him who rendered me generous

help, co-operation, suggestions and valuable guidance in completion of my research

work. I feel that he deserves all laurels for every success of mine in this research

work. I am thankful to Higher Education Commission (HEC) Pakistan for awarding

me IRSIP scholarship for University of Montreal Canada.

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I am also very thankful to Dr. Shaukat Ali from Department of Zoology G.C.

University Lahore who guides me throughout my antidiabetic studies. I am also

thankful to Dr. Shazia Khursheed from Department of Chemistry G.C. University

Lahore and Dr. Muhammad Saeed from Department of Chemistry LUMS who

support me greatly regarding the characterization of nanomaterials. I am thankful to

my friends and colleagues for all their efforts, help, and services in completing the

task. The most basic source of my life energy resides: my family including my dear

parents, in laws, sisters and siblings. Their support has been unconditional all these

years; they have cherished with me every great moment and supported me whenever I

needed it. I would like to express my sincere gratitude to my loving and encouraging

father Syed Dilawar Hussain who has provided me through moral and emotional

support in my life.

Thank you all.

Syed Akif Raza Kazmi

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Abstract

Among the metals nanoparticles, gold and silver nanoparticles have some

unique characteristics such as high stability, low toxicity, bio-compatibility and ability

of surface modification which make them an attractive tool for biomedical

applications. In present work, gold and silver nanoparticles have been prepared and

successfully applied mainly in four major areas including pH sensitive drug release,

biocatalysis, biosensor and diabetes management.

pH-sensitive doxycycline gold nanoparticles (doxy-AuNPs) are reported here

to act as an effective drug nanocarrier and as a biocatalyst. The AuNPs were

synthesized with doxy as the reducing and capping agent. Various parameters were

optimized to find the best conditions for synthesis of doxy-AuNPs and these were

characterized with UV-vis., x-ray diffraction (XRD), FT-IR and transmission electron

microscopy (TEM). Doxy-AuNPs were then loaded with the anticancer drug

doxorubicin (DOX) where 70% of the initially available drug was loaded within 24

hours. Furthermore, pH-dependent drug release was measured at 60% with invitro

measurements in phosphate buffer saline (PBS). In addition, the doxy-AuNPs were

applied as a biocatalyst. Oxidation of dopamine was taken as a model reaction to

determine the catalytic activity of doxy-AuNPs. Almost complete oxidation of

dopamine occurred in 5 minutes which indicates the fast response of synthesized

doxy-AuNPs as a biocatalyst.

In clinical chemistry, frequent monitoring of drug levels in patients has gained

considerable importance because of the benefits of drug monitoring on human health,

such as the avoidance of high risk of over dosage or increased therapeutic efficacy. In

present work, an ultra sensitive surface plasmon resonance (SPR) biosensor was

developed for the detection and quantification of doxycycline. SPR analysis revealed

the high sensitivity of doxy-AuNPs towards the detection of free doxycycline. More

specifically, doxy-AuNPs bound with protease activated receptor-1 (PAR-1)

immobilized on the SPR sensing surface yield the response in SPR, which was

enhanced following the addition of free doxy (analyte) to the solution of doxy-

AuNPs. This biosensor allowed for doxycycline detection at concentrations as low as

7 pM. The study also examined the role of colloidal stability and growth of doxy-

AuNPs in relation to the response enhancement strategy based on doxy-AuNPs. Thus,

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the doxy-AuNPs based SPR biosensor is an excellent platform for the detection of

doxycycline and demonstrates a new biosensing scheme where the analyte can

provide enhancement.

Diabetes is a life-threatening disease and chronic diabetes affects liver, kidney

and pancreas in human. The root cause of diabetes is mainly associated with oxidative

stress produced by reactive oxygen species. The minocycline is a polyphenolic drug

with excellent antioxidant activities. The objective of this study was to investigate the

antidiabetic potential of minocycline modified silver nanoparticles (Mino/AgNPs)

against alloxan induced diabetic mice. The Mino/AgNPs were synthesized using

minocycline as reducing and stabilizing agents. UV-vis., FT-IR, XRD and

transmission electron microscopy were applied for the characterization of synthesized

Mino/AgNPs. The DPPH free radical scavenging assay was conducted to compare the

antioxidant potential of Mino/AgNPs with that of minocycline and ascorbic acid. The

Mino/AgNPs showed higher radical scavenging activity (IC50 = 19.7 µg/mL) as

compared to the minocycline (IC50 = 26.0 µg/mL) and ascorbic acid (IC50 = 25.2

µg/mL). Further, these Mino/AgNPs were successfully employed for the treatment of

Alloxan induced diabetic mice. Thirty-two mice were divided into four groups:

normal control group; diabetic group left untreated; diabetic group treated with the

standard drug glibenclamide; diabetic group treated with Mino/AgNPs. The

administration of Mino/AgNPs to the diabetic mice showed higher antidiabetic

potential as compared to the drug glibenclamide. Hematological results showed that

the diabetic mice treated with Mino/AgNPs showed significant decrease in fasting

blood glucose level and lipid profile as compared to the diabetic mice left untreated.

Histopathological examination further confirmed the effectiveness of Mino/AgNPs as

an antidiabetic agent. The liver of diabetic mice showed a distorted central hepatic

vein along with distortion in arrangement of cells around the central vein. The

diabetic kidney showed distorted histo-morphology as compared to the kidney of

normal control mice. The pancreas of diabetic mice showed distorted islet cells.

However the treatment of diabetic mice with Mino/AgNPs showed significant

recovery and revival of histo-morphology of kidney, central vein of liver and islet

cells of pancreas. Hence Mino/AgNPs is an excellent antidiabetic agent to overcome

the diabetic disorders.

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TABLE OF CONTENTS

CHAPTER-1 ........................................................................................................................... 1

INTRODUCTION .................................................................................................................... 1

1.1 Nano Technology ................................................................................................... 1

1.2 Why Nanoparticles? .................................................................................................... 2

1.3 History of Noble Metals Nanoparticles ........................................................................ 3

1.4 Properties of Noble metals Nanoparticles.................................................................... 4

1.4.1 Surface Plasmon Resonance ................................................................................. 5

1.5 Synthesis of Noble Metals Nanoparticles ..................................................................... 5

1.6 Applications of Noble Metals Nanoparticles ................................................................ 7

1.6.1 Application of Gold Nanoparticles as Drug Carrier and as biocatalyst .................... 8

1.6.2 Application of Gold nanoparticles to Fabricate SPR Biosensor for drug detection .. 9

1.6.3 Application of Silver Nanoparticles as Potential Antidiabetic Agent ..................... 12

1.7 Aims & Objectives ..................................................................................................... 17

CHAPTER-2 ......................................................................................................................... 18

LITERATURE REVIEW .......................................................................................................... 18

CHAPTER-3 ......................................................................................................................... 41

MATERIALS AND METHOD ................................................................................................. 41

3.1 Materials ................................................................................................................... 41

3.2 Synthesis of Doxycycline derived Gold Nanoparticles (doxy-AuNPs) ........................... 41

3.3 Synthesis of Minocycline Derived Silver Nanoparticles (Mino/AgNPs) ........................ 42

3.4 Characterization of Nanoparticles.............................................................................. 42

3.4.1 XRD studies ........................................................................................................ 42

3.4.2 TEM Studies ........................................................................................................ 42

3.4.3 DLS Studies ......................................................................................................... 43

3.4.4 FT-IR Studies ....................................................................................................... 43

3.5 Application of doxy-AuNPs as drug Carrier and biocatalyst ........................................ 43

3.5.1 Loading of doxorubicin hydrochloride onto gold nanoparticles ........................... 43

3.5.2 Drug Loading Efficiency ....................................................................................... 44

3.5.3 Drug release study .............................................................................................. 44

3.5.4 Catalytic Oxidation of Dopamine......................................................................... 45

3.6 Application of Gold Nanoparticles in Fabrication of SPR Biosensor............................. 45

3.6.1 Preparation of SAM modified Gold Coated Prism ................................................ 45

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3.6.2 Fabrication of the SPR sensor.............................................................................. 45

3.6.3 Immobilization of receptor on sensor surface ..................................................... 46

3.6.4 Electrolytic Stability of doxy-AuNPs..................................................................... 46

3.6.5 Sequential Analysis for determination of concentration of doxycycline ............... 47

3.7 Application of Silver Nanoparticles as Potential Antidiabetic Agent ........................... 47

3.7.1 Antioxidant study – DPPH assay .......................................................................... 47

3.7.2 Experimental Animals ......................................................................................... 48

3.7.3 Induction of Diabetes ......................................................................................... 48

3.7.4 Experimental Design ........................................................................................... 48

3.7.5 Collection of sample ........................................................................................... 49

3.7.6 Biochemical Assay .............................................................................................. 49

3.7.7 Histopathological Studies.................................................................................... 49

3.7.8 Statistical Analysis .............................................................................................. 49

CHAPTER-4 ......................................................................................................................... 50

RESULTS AND DISCUSSION ................................................................................................. 50

4.1 Gold Nanoparticles as drug carrier ....................................................................... 50

4.1.1 Synthesis of doxy-AuNPs ..................................................................................... 51

4.1.2 Effect of pH on Synthesis of doxy-AuNPs ............................................................. 52

4.1.3 Stability of doxy-AuNPs ....................................................................................... 54

4.1.4 TEM of doxy-AuNPs ............................................................................................ 55

4.1.5 Zeta potentials of doxy-AuNPs ............................................................................ 56

4.1.6 FT-IR of doxy-AuNPs ........................................................................................... 57

4.1.7 X-ray Diffraction of doxy-AuNPs .......................................................................... 58

4.1.8 Drug loading ....................................................................................................... 58

4.1.9 TEM of DOX load doxy-AuNPs ............................................................................. 62

4.1.10 pH Responsive Drug Release kinetics of doxy-AuNPs ......................................... 62

4.2 Gold NPs as artificial enzyme (Biocatalyst) ................................................................. 64

4.3 Gold Nanoparticles based SPR biosensor ................................................................... 66

4.3.1 Strategy of the Assay .......................................................................................... 66

4.3.2 Electrolytic Stability of doxy-AuNPs..................................................................... 66

4.3.3 SPR Analysis for Detection of Doxycycline ........................................................... 67

4.4 Minocycline derived silver nanoparticles (Mino/AgNPs) as potential antidiabetic agent

....................................................................................................................................... 73

4.4.1 Synthesis of Mino/AgNPs .................................................................................... 73

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4.4.2 Colloidal Stability of Mino/AgNPs........................................................................ 74

4.4.3 FT-IR Studies of Mino/AgNPs .............................................................................. 75

4.4.4 TEM of Mino/AgNPs ........................................................................................... 76

4.4.5 X-ray Diffraction of Mino/AgNPs ......................................................................... 77

4.4.6 DPPH Radical Scavenging Assay .......................................................................... 78

4.4.7 Antihyperglycemic activity of Mino/AgNPs in alloxan induced diabetic mice ....... 79

4.4.8 Histology Studies ................................................................................................ 83

CONCLUSION AND PERSPECTIVES ...................................................................................... 87

REFERENCES ....................................................................................................................... 90

List of Publications ........................................................................................................... 119

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LIST OF FIGURES

Figure 1 Lycurgus Cup (Left) red color if the light source comes from inside of the cup

(Right) green color, if the light source comes from outside of the cup .................................... 3

Figure 2. Scheme representing the biomedical applications of Noble metals Nanoparticle ..... 7

Figure 3 custom-built SPR instrument ................................................................................. 46

Figure 4 Scheme representing the synthesis of doxy-AuNPs following after the loading and

release of DOX from doxy-AuNPs ..................................................................................... 51

Figure 5 UV-vis spectrum of doxy-AuNPs .......................................................................... 52

Figure 6 UV-vis spectra with pH effect on synthesis of doxy-AuNPs. All spectra were

acquired after 15 minutes of reaction ................................................................................... 53

Figure 7 (a) UV-vis spectra indicating the stability of doxy-AuNPs (b) Absorbance maximum

vs time spectra of doxy-AuNPs ........................................................................................... 54

Figure 8 TEM images and Histogram of doxy-AuNPs (a & b) (Diluted sample) (c & d)

(Concentrated sample) (acquired at 80 kV, exposure of 1200 ms and magnification of

150,000X. The scale bar represents 50 nm) ......................................................................... 55

Figure 9 Zeta potentials of doxy-AuNPs .............................................................................. 56

Figure 10 FT-IR Spectra of Doxycycline (Red) and doxycycline modified Gold Nanoparticles

(Black) ................................................................................................................................ 57

Figure 11 (a) X-ray diffraction of doxy-AuNPs (Diluted sample) (b) X-ray diffraction of

doxy-AuNPs (Concentrated sample) ................................................................................... 58

Figure 12 (a) UV-vis spectra with absorbance of DOX in supernatant at different reaction

intervals (b) Absorbance vs time spectra for DOX absorbance in supernatant (Native DOX at

484 nm and supernatants at 495 nm) ................................................................................... 59

Figure 13 TEM of DOX loaded doxy-AuNPs ...................................................................... 62

Figure 14 In vitro release profile of DOX from doxy-AuNPs .............................................. 63

Figure 15 (a) [Curve-1 Dopamine, Curve-2 Dopamine with H2O2, Curve 3 Dopamine with

H2O2 and doxy-AuNPs] (b) Absorbance maximum vs time spectra showing the rate of

catalytic oxidation of dopamine........................................................................................... 65

Figure 16 UV-vis spectra showing the electrolytic stability of doxy-AuNPs in the presence of

different concentrations of NaCl.......................................................................................... 67

Figure 17 Effect of sodium chloride (NaCl) concentration on the SPR response of doxy-

AuNPs ................................................................................................................................ 68

Figure 18 Propagating SPR response of doxy-AuNPs (control, red trace) and of doxy-AuNPs

with varying concentrations of doxy (analyte, black trace) ................................................... 69

Figure 19 Scheme illustration of doxycycline effect on overgrowth of doxy-AuNPs ............ 70

Figure 20 (a) UV-vis spectra indicating the effect of addition of doxycycline on growth of

doxy-AuNPs (b) Sequential binding curve presenting a correlation between log of doxy

concentration and SPR response. Error bars indicate standard deviation of triplicate

measurements ..................................................................................................................... 71

Figure 21 Schematic presenting the Synthesis and in vivo Antidiabetic Potential of

Mino/AgNPs ....................................................................................................................... 73

Figure 22 UV-vis spectrum of Mino/AgNPs ........................................................................ 74

Figure 23 UV-vis spectra indicating the stability of Mino/AgNPs ........................................ 75

Figure 24 FT-IR Spectra of Minocycline (Red) and Minocycline modified Silver

Nanoparticles (Blue) ........................................................................................................... 76

Figure 25 TEM and Histogram of Mino/AgNPs .................................................................. 77

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Figure 26 X-ray Diffraction of Mino/AgNPs ....................................................................... 78

Figure 27 DPPH free radical scavenging assay .................................................................... 79

Figure 28 Blood Sugar Level (mg/dl) for various study groups ............................................ 80

Figure 29 Hemoglobin Level (mg/dl) for various study group ............................................. 81

Figure 30 Lipid profile (mg/dl) for various study groups ..................................................... 82

Figure 31 SGPT and SGOT Profile (mg/dl) for various study group .................................... 83

Figure 32 Histology of Islet cells of Pancreatic sections of various study groups (Arrowhead

pointing towards the islet tissue of pancreas) ....................................................................... 84

Figure 33 Histology of kidney sections of various study groups (Arrowhead pointing towards

the glomerulus and urinary space of kidney) ........................................................................ 85

Figure 34 Histology of Liver sections of various study groups (Arrowhead pointing towards

the central hepatic vein of liver) .......................................................................................... 86

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LIST OF TABLES

Table 1 Comparative table of drug loading efficiencies and and pH sensitive release

................................................................................................................... .61

Table 2 Comparison with other Analytical Techniques Developed for Doxycycline

Detection ................................................................................................... ..72

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LIST OF ABBREVIATIONS

Nanoparticles NPs

Silver Ag

Silver nanoparticles AgNPs

Gold nanoparticles AuNPs

Doxycycline doxy

Minocycline mino

Doxorubicin DOX

Sodium borohydride NaBH4

Surface plasmon resonance SPR

Ultraviolet–visible UV–Vis

Scanning electron microscopy SEM

Transmission electron microscopy TEM

X-ray diffraction XRD

Fourier transform Infra-red FT-IR

Energy dispersive x-ray EDX

Dynamic light scattering DLS

N-hydroxysuccinimide NHS

16-mercapto-hexadecanoic acid (16-MHA)

N-ethyl-N‟-(3-dimethylaminopropyl)-carbodiimide EDC

Anticancer drug ACD

Protease Activated Receptor PAR1

Reactive oxygen species ROS

Localized Surface Plasmon Resonance LSPR

Minocycline protected silver nanoparticles Mino/AgNPs

2,2-diphenyl-1-picrylhydrazyl DPPH

GCU Lahore INTRODUCTION

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CHAPTER-1

INTRODUCTION

1.1 Nano Technology

Nanotechnology is the most emerging and rapidly growing era of science in the

late 20th century, yet it is still current in the 21

st century. It has the potential to alter

our perceptions and viewpoints. Nanotechnology has such a broad range of

applicability that it finds its applications in almost every field of science including

mechanics (Shi et al., 2019), electronics (Elemike et al., 2019), environment (Ali et

al., 2017), medicine (Kaszewski et al., 2018), diagnostics (Zhou et al., 2015), imaging

(Xia et al., 2018), agriculture (Gabriel Paulraj et al., 2017), food (King, et al., 2018),

drug delivery (Saeedi et al., 2019), energy sciences (Han et al., 2020), biotechnology

(Verma et al., 2020), cosmetics (Chaki Borrás et al., 2020) and paints (Bellotti et al.,

2015), textiles, optoelectronics (Kumar et al., 2019), sensors (Zhao et al., 2015),

catalysis (Cheng Yang et al., 2019) and so on.

Nanotechnology is the creation and manipulation of substances on a nanometer

length scale that leads to the exciting features and diverse applications of the said

substances. The National Nanotechnology Initiative (NNI) of the United States

defines nanotechnology as the “science, engineering, and technology conducted at the

nanoscale, which is about 1 to 100 nanometers”. The prefix „nano‟ comes from the

Greek word “dwarf” meaning small. The word “Nano” meaning 10-9

meter, is such a

small term that the objects smaller than those in quantum universe can only be

clusters of atoms or molecules. A nanometer (nm) is one thousand millionth of a

meter, 10-9

. To further demonstrate this, human hair is 80000 nm wide while the

diameter of a virus is about 100 nm.

It is admirable that nanotechnology in itself is not a single technology but a

meeting of all traditional sciences such as physics, chemistry, biology and material

science which brings them together to further develop these sciences. All fields of

science which can operate at the nanoscale are connected with this emerging

nanotechnology and are largely impacted by the unique features of nanomaterials. At

https://www.asme.org/engineering-topics/articles/bioengineering/building-a-nanoscale-railroad


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the nanoscale, the properties of materials are entirely different from their bulk

counterparts. For example, bulk gold is inert as it doesn‟t corrode or tarnish whereas

at the nanoscale the gold particles show good reactivity. The bulk gold is yellow

while the nanogold never looks like the bulk gold. It can be red, purple or greenish

depending on the size of the particles (Kumar et al., 2011). The bulk gold would be a

poor material to use as a catalyst as it does not do so much whereas because of high

surface to volume ratio, nanogold has shown good potential as catalyst (Al-mahamad,

2020). The ability of surface modification of nanogold with biomaterials makes them

a valuable candidate in medical applications (Kalimuthu et al., 2020). All these

characteristics that emerge due to bringing the materials down to the nanoscale are of

great interest and demand considerable attention of the researchers to further build in

the field of nanotechnology.

1.2 Why Nanoparticles?

“Small is big!” has been a traditional slogan for technology development since

the last few decades. The nanoparticles are extremely small objects as they possess

only a few atoms to a few thousand atoms in contrast to the particles at a larger scale

that might have billions of atoms. This variation in size causes the nanoparticles to

behave significantly different from their larger counterparts and make them an

interesting candidate for research.

Normally a question arises when dealing with nanoparticles: “How the

nanoparticles are such interesting” as the dealing with these nanoparticles is much

complicated in contrast to their bulk counterparts. The unique properties of these

nanostructures clearly describe the answer to this question. At the nanoscale, the

particles show interesting aspects like the size-dependent optical, electronic and

catalytic properties and their potential use in sensors (Castiello & Tabrizian, 2018),

catalysis (Lu et al., 2020), medicine (Kaszewski et al., 2018) and drug delivery

(Rasoulzadehzali & Namazi, 2018). The possibility of controlling and tuning these

electronic and optical properties will make it possible to use these nanoparticles as

versatile analytical probes and turning out to be key materials and building structures

in the 21st century.


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1.3 History of Noble Metals Nanoparticles

The metal clusters have been in use from ancient days. The Noble metals such

as Gold and silver have attracted mankind with their unique value and artistic appeal

in many ancient civilizations. The dazzling colors of these precious metals have

intrigued man from the beginning of humankind. Gold has been applied as a luxurious

feature in many pieces of art and jewellery to increase the worth of the object. Several

characteristics of gold like their outstanding conductivity and inertness towards

oxygen or water have rendered it incredibly useful over time for mankind.

Furthermore, the gold and silver were widely used in the manufacturing of ruby glass

and to color ceramics by the end of the 16th century. The Lycurgus Cup with its

special color is the best-known example of the usage of gold and silver in ruby glass.

The popular Roman Glass Lycurgus Cup (4th century AD) comprises nanoparticles of

gold and silver with a diameter of around 70 nm. The roman ancient craftsmen were

highly skilled but they never realized that they were working on nanoscale (Freestone

et al., 2008; Lee et al., 2006). The existence of these noble nanoparticles imparted a

fascinating color display for the glass. It looks green in reflected light whereas it looks

red when light is transmitted through the glass. This glass is still on display in the

British Museum.

Figure 1 Lycurgus Cup (Left) red color if the light source comes from inside of the cup (Right) green

color, if the light source comes from outside of the cup

During the 17th century, Johann Kunchel and Andreus Cassius further

developed the method of glass coloring by creating “Cassius Purple” prepared by


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reaction of gold salt with tin (II) chloride added to impart red color to the glass

(Habashi, 2016). These metal clusters have also been used for dying fabrics. They

were commonly used for decorative, cosmetic and medicinal purposes. "Drinkable

gold" was utilized in the middle Ages to treat illnesses such as inflammation,

smallpox, furuncles, cardiac problems, seizures and tumors, dysentery, venereal

disorders, and also to detect syphilis, a process that was in usage until the 20th

century (Huaizhi & Yuantao, 2001; Console, 2013).

Furthermore, the first experimental research on gold nanoparticles is dated

back to Michael Faraday's seminal work. He was the first to present extensive studies

with gold metal and thin films. In 1857, he published a paper entitled “The

Experimental Relation of Gold (and Other Metals) to Light” in which he reported that

the gold was dispersed in a ruby-colored solution in a “finely divided metallic state”.

He synthesized the ruby-colored solution of gold by reduction of aqueous gold

chloride using phosphorus in carbon disulfide. After 150 years of his experiments, the

photographs of Faraday‟s gold solution taken by transmission electron microscope

(TEM) showed that he had actually synthesized gold nanoparticles with an average

size of 6 ± 2 nm (Edwards & Thomas, 2007). Faraday‟s process for the synthesis of

metallic colloids was a boost, even though the value of colloidal gold was not known

at that time.

1.4 Properties of Noble metals Nanoparticles

Noble metals nanoparticles such as gold and silver nanoparticles are the most

stable metal nanoparticles and present unique characteristics that are not found in their

bulk counterparts. The preparation of colloidal gold and silver nanoparticles is easy

and experimental conditions can be varied effectively to control the size and shape of

these nanoparticles (Raza et al., 2017). The small size and larger surface to volume

ratio of these nanoparticles render them as an excellent scaffold for the

immobilization of various functional groups, resulting in fast responses and greater

sensitivity for the desired analyte. Furthermore, gold and silver nanoparticles present

excellent biocompatibility for immobilization of different biomolecules which render

them important candidates for biomedical applications such as for the fabrication of

biosensors, or to develop a platform for targeted delivery of drug (Z. Li et al., 2017).


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1.4.1 Surface Plasmon Resonance

The nanoparticles exhibit unique optical characteristics such as surface

plasmon resonance not found in their bulk counterparts. Certain metal colloids such as

gold, silver and copper show significantly strong bands of absorption in the visible

region and are therefore deeply colored (Hemmati et al., 2019). A strong absorption

band of metallic nanoparticles produced in the UV-visible region as a result of an

interacting electromagnetic field is termed as Surface Plasmon band (SPB). This

phenomenon appears in the absorption spectrum of the nanoparticles due to the

collective coherent oscillation of the free conduction band electrons occupying energy

states just above the Fermi level. Normally Ag, Au and Cu nanoparticles with

diameter below 20 nm have the surface plasmon band near 400 nm, 520 nm and 570

nm respectively (Hemmati et al., 2019; Kazmi et al., 2019; Nikhil Kumar &

Upadhyay, 2016) whereas for bigger particles, this band shifts towards the longer

wavelength (e.g., 529 nm for 50 nm gold nanoparticles). The SPB thus provides

knowledge regarding the band structure production in certain metals and has been

extensively studied.

1.5 Synthesis of Noble Metals Nanoparticles

Normally, gold and silver nanoparticles are preferred in various medical applications

due to the ease of synthesis and biocompatibility. Generally, two approaches, top-

down and bottom-up are employed to synthesize the nanoparticles. In the top-down

strategy, the materials in bulk are broken down gradually to the nanosized materials

using various techniques such as lithographic techniques, chemical etching, ball

milling, and sputtering while in bottom-up strategy, atoms are self-assembled to

molecular structures in the nanometer range (Cerjak, 2009). The chemical reduction

method is a famous example of a bottom-up strategy. The synthesis of nanoparticles

in solution is followed by a two-step mechanism: nucleation and successive growth

(Reverberi et al., 2019). The reduction of precursor salt initiates the nucleation which

is further carried by the collision of gold ions, atoms, and small clusters. The variation

in size and shape of the clusters formed at this stage is because of the attachment and

detachment competition between the gold atoms. Nevertheless, the integration of

atoms dominates the detachment producing clusters that are sufficiently large to

become stable because of energy released by the formation of the new volume. The

collision among the stable clusters causes the nucleation process to cease followed by


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the formation of irreversible seeds. The seed growth in the successive stage of growth

is achieved by the reduction of more gold salt upon them (Shen et al., 2014). The

growth rate and seed morphology influence the final size and shape of the resulting

nanoparticles. Furthermore, since the two-step process for the preparation of

nanoparticles is accomplished in the presence of a protecting ligand, therefore the

interaction between atoms/ions/clusters and protecting ligand is important regarding

the control of size and shape of synthesized nanoparticles.

Nanoparticles have been synthesized using various physical, chemical and

biological procedures. Among the conventional methods, the most famous method for

nanoparticle synthesis is the Turkevitch method. In this method, the aqueous solution

of gold chloride is boiled and sodium citrate is added to it. The citrate ions act as both

reducing and stabilizing agent. The citrate ions reduce the Au (III) ions to Au (0)

atoms and monodisperse citrate capped gold nanoparticles are formed with size

ranging from 10 nm to 150 nm (Kimling et al., 2006). The gold nanoparticles

synthesized by this method are water-soluble because of the polarity of physisorbed

citrate ions. Nevertheless, the weaker ionic interaction between citrate and AuNP core

allows the replacement of former with thiol-containing ligands, which interact with

AuNP core more strongly, resulting in AuNPs soluble in organic solvents.

Brust and Schiffrin developed their two-phase synthesis method for gold

nanoparticles to overcome the difficulties associated with the Turkevich method. In

the first step, the aqueous solution of gold chloride is transferred into the organic

phase (toluene) via tetraoctylammonium bromide, (TOAB) (phase transfer agent).

This is followed by the addition of thiol to the organic phase which reduces gold (III)

salt to the gold (I)-thiol polymeric form. The decolorization of the organic phase at

this stage indicates the reaction between thiol and gold (III) ions resulted in the

formation of the polymeric structure. In the last step, the addition of sodium

borohydride (NaBH4) reduced the gold (I)-thiol to the gold (0) oxidation state. The

size of gold nanoparticles synthesized by this method ranges between is 1-4 nm (Brust

et al., 1994).

Another method used for the synthesis of nanoparticles is the seeding growth

method. In this method, seeds of gold particles are utilized for the growth of gold

nanoparticles in the presence of a weak reducing agent. This method allows the step-


7

by-step growth of particles and is more effective as it prevents secondary nucleation

(Nikoobakht & El-Sayed, 2003).

Even though the above methods are useful for nanoparticle synthesis but the

use of organic solvents in these procedures limits their use for the detection of

biomolecules such as proteins, nucleic acids and saccharides. In addition, toxic

chemicals such as sodium borohydride, citric acid, tetraoctylammonium bromide

(TOAB), polyethylene glycol (PEG), hexadecyltrimethylammonium bromide (CTAB)

used in these procedures render them unsuitable for clinical applications. It is,

therefore, necessary to develop biocompatible, non-toxic, clean and environment

friendly methods for nanoparticles synthesis (Kalimuthu et al., 2020).

Green chemistry approach is an impressive alternate to overcome the

limitations of the above mention methods for the synthesis of nanoparticles. The

synthesis of nanoparticles through the use of safer solvents and environment friendly

reagents such as amino acids, antibiotics, analgesics, etc. will help in the reduction of

toxic waste and consequently improve the quality of the environment. Among these,

the use of antibiotics to synthesize the nanoparticles is one of the simplest, cheaper,

greener and environment friendly approach and has received significant attraction

since the last few years.

1.6 Applications of Noble Metals Nanoparticles

Figure 2. Scheme representing the biomedical applications of Noble metals Nanoparticle


8

1.6.1 Application of Gold Nanoparticles as Drug Carrier and as

biocatalyst

Cancer is the second leading cause of death globally (Heron & Anderson,

2016). Although chemotherapy is suggested for the treatment of cancer patients,

nonspecific cytotoxicity and inefficient delivery of anticancer drugs results in serious

side effects on normal tissues of the body and consequently deteriorates the quality of

life (Hosseini et al., 2018). A major challenge is to achieve selective delivery of the

drug within cancer cells so that side effects would be minimized (de Oliveira et al.,

2016). Selective delivery of the drugs to cancer cells needs to be linked with suitable

drug carrier having a linkage cleavable under cancer specific conditions (T.-Y. et al.,

2019). Despite being a simple concept, difficulties are associated with this approach

and research in this area remains important.

Drugs are often bound to a suitable delivery vehicle, such as polymers

(Imperiale et al., 2018; Jing et al., 2018), dendrimers (Sherje et al., 2018), liposomes

(Moosavian & Sahebkar, 2019) or nanoparticles (Sarkar et al., 2017). Then, it needs

to be released from the delivery agent to maintain therapeutic efficacy. For this

purpose, the linkage should be cleavable or it should be bound through weak

noncovalent interactions, providing a level of control in the release of drugs from a

nanocarrier. Furthermore, cancer specific conditions such as acidic pH, higher levels

of glutathionine, heat or light can be used to trigger the release of drug from nano

carrier (T.-Y. et al., 2019). These parameters act as stimuli for the smart release of the

drug. Among these parameters, pH responsive drug release is of particular interest.

The pH values of tumor cells are lower than normal cells (Ghorbani & Hamishehkar,

2017a). This difference in pH between cancer and normal cells can effectively be used

as a tool for targeted drug delivery and consequently can minimize the side effects in

normal cells. Thus, pH based drug release is an interesting approach for a drug

delivery system and the focus of prior research elsewhere (García Rubia et al., 2018;

Chunyu Yang et al., 2014).

The application of AuNPs for drug delivery is mainly attractive as they can be

synthesized in a range of sizes and can readily be functionalized with a variety of

ligands. To date, various therapeutics including Daunorubicin (Taghdisi et al., 2016a),

Dexamethasone (Fontana et al., 2013), Paclitaxel, Erlotinib (Khuroo et al., 2018),

Rituximab (Bisker et al., 2012) and Doxorubicin (Zhu et al., 2018) have been


9

conjugated with AuNPs to attain their desired therapeutic effects, demonstrating the

efficacy of this approach. Furthermore, AuNPs possess the ability to provide catalytic

activity. Their good catalytic response is due to the high surface area to volume ratio

(Ansar et al., 2017). These days, the use of AuNPs as peroxidase nanomimetics is

gaining considerable attention. They are easily synthesized with good stability for the

colorimetric detection of peroxidase substrate such as dopamine. Dopamine is a vital

neurotransmitter that has an important role in central nervous, hormonal,

cardiovascular and renal systems (Basu & Dasgupta, 2000). Different methods have

been developed for the detection of dopamine, including HPLC (G.E. et al., 2014),

chemiluminescence (W. Gao et al., 2017) and electrochemical analysis (Ma et al.,

2019). These approaches involve complicated sample preparation and instruments for

analysis. Colorimetric detection has been explored as one of the convenient approach

(Ge et al., 2014; Ge et al., 2015) which provides naked eye detection with the

simplicity of the procedure, which would be interesting to detect dopamine in some

circumstances.

1.6.2 Application of Gold nanoparticles to Fabricate SPR Biosensor

for drug detection

It is important in the sensing community to develop sensors for frequent

monitoring of drugs. In particular, clinical diagnosis and therapeutic procedures

demand sensors to assess drug levels in patients. There is a need to obtain accurate

test results in a short time for a large number of samples to decide on the course of

medical treatments (Ronkainen et al., 2010). As such, different laboratory-based

techniques are often employed to measure the drug concentrations in the biofluid of

patients, which serve to adjust medication knowing the active concentration of drugs

in blood. As such, regular monitoring of drug levels in patients can prevent its toxicity

and damage to organs (Cohen, 2000; Booth et al., 2018).

Doxycycline is a wide spectrum drug belonging to the tetracycline family of

antibiotics, which has been a drug of choice for treatment of several types of bacterial

infections (Haddada et al., 2019). According to FDA, safe dose of doxycycline is 200

mg on first day of treatment followed by 100 mg per day. In case of prolonged over

dose, patient may suffer from Gastrointestinal and renal diseases. It is a relatively low

toxicity drug and has been recommended for human use for a long time. However, the

long term use of doxycycline may lead to some side effects. Elzeinová et al. reported


10

the adverse effects of doxycycline on testicular tissue and sperm parameters in CD1

outbred mice (Elzeinová et al., 2013). According to them, the treatment of male mice

with doxycycline in puberty led to long-lasting effects on reproductive organs and

spermatozoa in adult males. They reported that the effect of doxycycline was

concentration-dependent. In addition, antitumor activities of doxycycline against

different types of malignancies have also been reported elsewhere. For example, Sun

et al. and Liu et al. have reported cytotoxicity and anti-metastatic activity of

doxycycline in melanoma and breast carcinomas (T. Sun et al., 2009; S. Liu et al.,

2015). According to Son et al., doxycycline has the potential to show apoptotic

activities in pancreatic cancer cells (K. Son et al., 2009). Duivenvoorden et al. has

reported that doxycycline treatment could be effective to reduce the tumor burden in

bone metastasis mouse model of human breast cancer (Duivenvoorden et al., 2002).

All these considerations suggested that this valuable antibiotic also has the potential to

treat other types of human cancers and thus a candidate anticancer drug of high

research value. It is therefore important to monitor the concentration of doxycycline

(doxy) in blood to optimize the dosage and reducing the side effects.

Currently, methods used for doxycycline detection involve analytical

techniques such as high performance liquid chromatography (HPLC) (Hadad et al.,

2008), sequential injection chromatography (SIC) (Ńatínský et al., 2005) and

potentiometry.(X. X. Sun & Aboul-Enein, 2002) These techniques provide accuracy

and reasonably good detection limits but have disadvantages such as the need for

complicated sample preparation, trained personnel, and sophisticated instruments and

thus cannot provide onsite and fast detection. Therefore, there is a need to develop

alternative methods that can provide onsite, fast and sensitive detection of

doxycycline.

Surface plasmon resonance (SPR) biosensor is an optical technique that

measures the binding events quantitatively in real-time without labeling the

interacting molecules (Abadian et al., 2014). The physical principle of the SPR

technique involves the measurement of changes in the refractive index when the

interaction of molecules takes place at the sensor surface (Couture, Zhao, & Masson,

2013). The benefits of the SPR technique include label-free detection, high sensitivity,

real-time monitoring, and crude sample analysis (C. C. Chang et al., 2010). These

advantages make this SPR technique a reliable and convenient one to examine the


11

binding specificity and interaction of biomolecules, as first reported in 1982 when

Liedberg et al. initially reported the use of an SPR based biosensor for the detection

of biomolecular interaction (Liedberg et al., 1983). Till now, this technique has been

primarily used as an effective tool for biomolecular interaction analysis, but more

recently, clinical analysis is increasingly reported.(Masson, 2017) SPR sensing

proceeds without altering or damaging the composition of original analyte (Yuan et

al., 2018) and is increasingly proposed for clinical diagnostics (L. Gao et al., 2018;

Riedel et al., 2017), drug monitoring,(S. S. Zhao et al., 2015b) environmental

monitoring (Brulé et al., 2017; Fodey et al., 2011), food analysis (Atar et al., 2015;

Vaisocherova-Lisalova et al., 2016) and biochemistry (Graybill & Bailey, 2016).

Despite the many advantages of SPR biosensors, the binding of small

molecules to the sensor surface typically results in small shifts, which constitutes a

limitation of SPR biosensors. Most portable and small SPR instruments are not

sensitive enough to assess such small refractive index changes which make them unfit

to use for ultrasensitive detection of small organic drugs (Y. F. Chang et al., 2018). To

overcome this limitation, different groups have employed various enhancement

strategies in conjugation with SPR, such as enzyme (Goodrich et al., 2004),

polymerase chain reaction (PCR) (Carrascosa et al., 2009) and gold nanoparticles

enhancement methods (Yeom et al., 2013a). Among them, gold nanoparticles based

enhancement strategies have received considerable attention and played a significant

role in response amplification of SPR biosensors (Jianlong Wang & Zhou, 2008). The

ease of synthesis, good stability, biocompatibility, low toxicity and ability of surface

functionalization of AuNPs make them an attractive tool for biomedical applications

(Kazmi et al., 2019). AuNPs support localized surface plasmon resonances (LSPR),

arising from the combined oscillations of electrons present in the conduction band of

the metal (Frederix et al., 2003). The electronic coupling between the LSPR of gold

nanostructures and SPR is often applied as a strategy to amplify the response signals

of biosensors. For example, this strategy has been designed for the detection of

methotrexate (S. S. Zhao et al., 2015b) and testosterone (Yockell-Lelièvre et al.,

2015).

Although AuNPs are extensively applied in SPR biosensors, the effect of the

size of nanoparticles on SPR interactions is still not completely understood.

According to Kelly et al., the size and shape of the nanomaterials could be effectively


12

used to control the plasmonic characteristics as well as the electromagnetic field

amplifications of AuNPs (Kelly et al., 2003). Comparatively larger nanostructures

give higher sensitivity towards changes in refractive index than the smaller

nanostructures. Uludag and Tothill studied the effect of the size of AuNPs over the

SPR sensor response. According to them, increasing the size of AuNPs resulted in

higher sensor response (Uludag & Tothill, 2012). Springer et al. observed that the size

of AuNPs affects the diffusion mass transfer rate as well as the SPR signal and

resulted in optical enhancement of SPR biosensor (Ńpringer et al., 2014). All these

considerations suggested that the size of AuNP is critical and needs more attention

while studying the biomolecular interaction via SPR biosensor.

1.6.3 Application of Silver Nanoparticles as Potential Antidiabetic

Agent

Diabetes mellitus along with its secondary complications continued to be a

major threat to human health all over the world (Z. Zhang et al., 2016). It is one of the

five main causes of death globally (Mohammadi Arvanag et al., 2019). A group of

metabolic disorders occurred as a consequence of hyperglycemia and glucose

intolerance, known as diabetes mellitus (DM). Two types of diabetes mellitus are

conventionally known. Insufficient secretion of the hormone insulin from -cells of

the pancreas is classified as type-1 DM and the development of insulin resistance in

the body is classified as type-2 DM (Hussein et al., 2019). Globally more than 90 %

of the diabetes patients suffer from type-2 DM (Dhas et al., 2016). Alarmingly, the

numbers are increasing at a dreadful rate. According to Veiseh et al. more than 280

million adults are suffering from diabetes mellitus and the high prevalence of DM

may cause the 400 million adults to be affected till 2030 (Veiseh et al., 2014).

The high prevalence of DM is mainly associated with modernization of

lifestyle, lack of physical activity, obesity, ethnicity, older age and genetic

polymorphism (Malapermal et al., 2017; Samadder, 2014). The people with type-2

DM develop insulin resistance in the body; consequently cells unable to take glucose

from the blood which finally resulted in the rise of blood glucose level known as

hyperglycemia. When left untreated, the prolonged hyperglycemia resulted in the

metabolic disorder of many organs like kidney, liver, heart and pancreas. These

metabolic disorders become fatal for life, often resulted in diabetes associated


13

secondary complications such as kidney disease, heart disease, liver disease, blindness

and erectile dysfunction (Thorve et al., 2011; Veiseh et al., 2014).

The root cause of diabetes is mainly associated with oxidative stress produced

by reactive oxygen species (ROS) that induced the β-cells dysfunction, insulin

resistance and impaired glucose tolerance. Excess food and lack of physical activity

contribute to the overload of glucose and fatty acid that leads to the formation of ROS

(Wright et al., 2006). According to Rohdes, the pancreatic -cells are highly sensitive

to physiological and pathological stressors, resulting in loss of insulin, triggered by

apoptotic cell death (Rhodes, 2005). The studies of Volpe et al. also reported the

effect of oxidative stress on pancreatic β-cell death and associated diabetic

complications. According to them, diabetes associated complications that are induced

by the hyperglycemia are mainly because of imbalance between ROS, which leads to

the higher oxidative stress and cellular death (Volpe et al., 2018). Thus, these diabetic

complications can effectively be controlled by down-regulating the generation of

ROS.

The change of lifestyle, diet and oral administration of antidiabetic agents are

the key factors to down-regulate the generation of ROS regarding the treatment of

diabetes (Veiseh et al., 2014; Wright et al., 2006). The primary objective for both

type-1 DM and type-2 DM is maintaining the persistent control of glucose level

within the normal glycaemic range (70-140 mg/dL) (Veiseh et al., 2014). The

selection of a suitable drug is a common problem in the treatment of diabetes. Various

antidiabetic drugs and hypoglycemic agents have been introduced for the treatment of

diabetes such as sulfonylureas and biguanides but these drugs do not provide

persistent control over the blood glucose level. In addition, the prolonged use of these

drugs induces toxicity and undesirable adverse effects such as gastrointestinal

discomfort, hypoglycemia, pancreatic degeneration and liver impairment in the body

which renders them less common (Campbell & Taylor, 2010). Therefore to find new

drugs and hypoglycemic agents with minimal side effects and higher efficacy is

interesting and the focus of prior research elsewhere.

Nanobiotechnology is among the demanding areas of research that make use

of the biological substances at the nanoscale and find their applications in different

fields such as biosensor (Kazmi et al., 2020), diagnostics (Hu et al., 2013), bio-

imaging (Zhou et al., 2018), catalysis (Raza et al., 2017), drug delivery system


14

(Kazmi et al., 2019) and nanomedicine (Wicki et al., 2015). In comparison to the

conventional drug formulations, nanomedicine has retrieved more attention for the

last few years due to their benefits such as more precise diagnosis, a higher

percentage of recovery, and more effective therapies (Kouame et al., 2019).

Especially the silver nanoparticles have become more desirable in the field of

nanomedicine because of their fascinating properties such as ease of synthesis,

colloidal stability, biocompatibility, bioavailability, low toxicity, and ability of

surface modification (Burdușel et al., 2018; Park et al., 2011; Siddiqi & Husen, 2017).

The previous studies have reported the administration route and bioavailability of

AgNPs in animal model. The small size AgNPs could be easily absorbed into

gastrointestinal tract and released into blood stream followed by excretion from body

via feces and urine (Jiménez-Lamana et al., 2014). The Park et al. has reported the

bioavailability and excretion of citrate coated AgNPs with average size 7.9 nm.

According to them, bioavailability of rats administrated orally with 1 mg/kg AgNPs

was 1.2% and 4.2% in the rats exposed to the 10 mg/kg AgNPs (Park et al., 2011).

The AgNPs have the ability to reduce the oxidative stress caused by the

imbalance between reactive oxygen species (ROS). The DPPH free radical

scavenging activity of AgNPs have been reported by many workers (Ahn et al., 2019;

Elemike et al., 2017; Khorrami et al., 2018; Küp et al., 2020; Vijayan et al., 2018).

The H2O2 is an important metabolic signal for glucose stimulated secretion of insulin

from -cells (Pi et al., 2007) whereas excessive generation of H2O2 can be harmful

for the integrity and function of -cells (Kaneto et al., 2007). The studies of Campoy

et al. demonstrated the use of EP/AgNPs for protection of INS-I cells from H2O2

induced oxidative injury. They used the Eysenhardtia polystachya (EP) extract to

synthesize AgNPs. They reported that the cells that were exposed to H2O2 showed

marked inhibition in the insulin secretion whereas the cells that were treated with

EP/AgNPs before exposure to H2O2 showed significant increase in insulin secretion.

They anticipated that the polyphenolic compounds present in Eysenhardtia

polystachya may protect the insulin secreting cells from oxidative stress (Campoy et

al., 2018). The keshari et al. has also reported the strong H2O2 scavenging potential of

AgNPs as compared with standard vitamin C. The study demonstrated that the

antioxidant properties of AgNPs arsis because of functional groups present on the

surface of AgNPs (Keshari et al., 2020). The khorrami et al. proposed that the


15

enhanced antioxidant activities of AgNPs are because of the simultaneous action of

polyphenols as antioxidant and nanoparticles as catalyst (Khorrami et al., 2018). A

number of studies have been published reporting the possible mechanisms for

antioxidant properties of AgNPs. However it is necessary to note that the antioxidant

potential of AgNPs largely depends on the chemical composition of the compound

with which it is modified. The nanoparticles prepared using the extracts rich in

phenolic compounds and flavonides showed high scavenging activities (Ahn et al.,

2019; Bedlovičová et al., 2020).

It is therefore minocycline was selected to synthesize AgNPs. In addition to

antimicrobial properties, minocycline has shown strong antioxidant potential and free

radicals scavenging activities. The minocycline is a semi-synthetic antibiotic from

tetracycline group. It has been used for more than 30 years as a drug of choice for

treatment of diseases related to bacterial infections. Now days, non-antibiotic

characteristics of minocycline such as anti-tumor, anti-inflammatory, and antioxidant

(Pourgholami et al., 2012; Soory, 2008) have dragged the attraction of scientist

towards this second generation antibiotic. The minocycline has a polyphenol structure

with multiple ionizable functional groups. At C4 carbon, Minocycline has a dimethyl

amino group which is mainly responsible for enhanced antioxidant potential of

minocycline (Murakami et al., 2020). The Lee et al. has reported the antioxidant

activities of minocycline against the oxidative stressor (H2O2). According to them, the

flies treated with minocycline showed more resistance to hydrogen peroxide (H2O2)

and died less as compared to the flies which did not receive minocycline treatment (G.

J. Lee et al., 2017). The MURAKAMI et al. has also reported the free radicals

scavenging activity of minocycline. The study demonstrated that the antioxidant

activity of minocycline is 200 to 300 times more potent than that of tetracycline.

According to them, minocycline is a chain breaking antioxidant with antioxidant

activities comparable to that of trolox and α-tocopherol (Murakami et al., 2020). A

number of previous reports have been published demonstrating that the minocycline is

an effective antioxidant with free radical scavenging potency similar to vitamin C and

E (Kraus et al., 2005).

To further build on the use of Noble metals nanoparticles for biomedical

applications, we applied gold nanoparticles as drug carrier and as biocatalyst.

Doxycycline is a broad spectrum drug (an antibiotic from the tetracycline group). It


16

has both capabilities of reducing and stabilizing the AuNPs. It can also play the role

of the linker to attach the anticancer drug doxorubicin. Hence, doxycycline (doxy)

was selected to prepare doxy-AuNPs conjugate. Synthesized doxy-AuNPs were

extensively characterized by UV-Vis, Zeta sizer, transmission electron microscopy

(TEM) and X-ray diffraction (XRD). This doxy-AuNPs conjugate was loaded with

anticancer drug doxorubicin (DOX) and drug release kinetics of doxy-AuNPs

conjugate was observed. Moreover, the performance of these doxy-AuNPs as

biocatalyst was determined via the oxidation of dopamine. For this reaction, these

AuNPs were utilized as biocatalyst and the oxidation kinetics were observed,

demonstrating the potential of this nanotechnology platform for drug loading and

biocatalysis. This is one of the rare examples where a single nanoparticle template

that is easy to produce, is used as a delivery agent and as a biocatalyst.

Furthermore, we successfully developed doxy-AuNPs based SPR biosensor

for fast and sensitive detection of doxycycline. The use of the analyte to trigger AuNP

overgrowth is used as a novel sensing principle, where the signal of the SPR sensor is

proportional to the concentration of doxycycline, in opposition to the usual

competition assays resulting in a reduced response of the SPR sensor with

concentration. Synthesized doxy-AuNPs were characterized by UV-Vis, X-ray

diffraction (XRD), FT-IR and transmission electron microscopy (TEM). SPR analysis

was performed to demonstrate the detection of doxycycline. Various conditions were

optimized to improve the SPR response. In this study, doxy-AuNP containing sodium

chloride (NaCl) was employed as reagent providing a further increase of the biosensor

response. Thus, the doxy-AuNPs based SPR biosensor is an excellent platform for the

ultra-sensitive detection of doxycycline.

In addition, considering the antioxidant potential of minocycline and AgNPs,

we examined the antidiabetic potential of minocycline modified silver nanoparticles

(Mino/AgNPs) against the alloxan induced diabetic mice. The Mino/AgNPs were

synthesized and extensively characterized using UV-vis, X-ray diffraction (XRD),

FT-IR and transmission electron microscopy (TEM). The DPPH free radical

scavenging assay was carried out to compare the antioxidant potential of

Mino/AgNPs with that of minocycline and ascorbic acid. Then, the synthesized


17

Mino/AgNPs were successfully applied to examine theirs in vivo antidiabetic potential

against alloxan-induced diabetic mice.

1.7 Aims & Objectives

The present study was carried out to achieve the following goals:

Synthesis of Gold and Silver nanoparticles in aqueous media using antibiotics,

analgesics or other compounds of biological importance as cheaper and

greener chemicals.

Optimization of various synthetic conditions such as pH, temp, the

concentration of precursor salt, the concentration of capping agent, etc. to

control the size and shape of synthesized nanoparticles.

Characterization of synthesized nanoparticles to investigate new geometrical

changes in them

To apply gold nanoparticles (AuNPs) as a drug carrier to develop a platform

for selective delivery of drug

To investigate the biocatalytic response of gold nanoparticles

To develop Gold Nanoparticles based SPR biosensor for the detection of

biomolecules

To examine in vivo antidiabetic potential of silver nanoparticles (AgNPs)

using Albino mice model

GCU Lahore LITERATURE REVIEW

18

CHAPTER-2

LITERATURE REVIEW

A most important fact regarding the cancer treatment is the delivery of drug to

the tumor region which provides greater therapeutic efficiency. The major concern in

treatment of cancer is higher penetration of drug to the tumor region. In this context,

the traditional nanomedicine ( 50 nm) carries some drawbacks such as the non-

homogeneous distribution of drug to the tumor region. It is mainly because the

tumor‟s interstitial spaces have physiological obstruction which hinders the

homogeneous distribution of drug. To overcome this drawback, Son et al., has

synthesized a pH responsive transformable hybrid nanoparticles(TNPs) containing

PEG-PBAE and gold nanoparticles-doxorubicin conjugate (AuNPs-DOX). The PEG-

PBAE was applied as reservoir that carries the ultrasmall nanoparticles (3 nm) to

release in acidic environment (pH 6.5) of tumor. The DOX-AuNPs were injected

intravenously to the mice having tumor. The successful accumulation and dissociation

of DOX-AuNPs conjugate at extracellular level of tumor resulted in release of free

DOX. The deeper diffusion of nanoparticles in tumor cells resulted in bond cleavage

of pH responsive ester linkage which finally resulted in the release of free DOX from

DOX-AuNPs conjugate. The deeper penetration of DOX in the tumor cells resulted in

more effective suppression of tumor growth which tells the potential of this DOX-

AuNPs conjugate to be applied as nanomedicine for cancer therapy (Son et al., 2018).

Khutale and Casey reported the development of nanoparticle drug carrier

system to enhance the chemotherapeutic performance and cellular uptake of

chemotherapeutic drugs available. The thiolated polyethylene glycol(PEG) protected

spherical AuNPs were synthesized by gold chloride reduction followed by covalent

coupling with polyamidoamine(PAMAM) G4 dendrimer. Furthermore, the

doxorubicin was attached with dendrimer through amide linkage. The confocal laser

scanning microscopy was used for monitoring of intracellular drug release. The

studies showed that the developed nano drug carrier system resulted in enhanced

chemotherapeutic performance of doxorubicin. The doxorubicin release from Au-


19

PEG-PAMAM conjugate was significantly amplified at a weak acidic situation but it

was negligible at physiological pH (Khutale & Casey, 2017b).

The sodium 3-mercapto-1-propane sulfonate (3-MPS) functionalized gold

nanoparticles (AuNP-3MPS) were prepared in aqueous medium using various molar

ratio of Au/thiol. The dexamethasone (DXM), a synthetic glucocorticoid steroid was

selected and a bioconjugate of Au-3-MPS-DXM was synthesized. The spherical Au-

3MPS nanoparticles with average size 7-10 nm showed characteristic UV-vis band at

520 nm. The interaction between AuNP-3MPS and DXM was reported via Au (I)

atom on surface of AuNPs and fluorine atom of DXM. The loading efficiency of

dexamethasone on gold 3-mercapto-1-propane sulfonate depending on the thiol

contents was assessed in the range 70– 80%. Furthermore, the release of drug in 5

days was about 70 %. The loading and release behavior of Au-3MPs was mainly

attributed to their unique properties that include: small size, monodispersity and high

water solubility (Venditti et al., 2014).

Luesakul et al. reported the formulation of a pH responsive drug delivery

system based on synthesis of folic acid-N-trimethyl chitosan (TMC-FA) modified

Selenium nanoparticles (SeNPs) for selective release of anticancer drug doxorubicin.

The use of nanoparticles as drug carrier resulted in enhancement of DOX activity by

10-fold. The DOX-loaded SeNPs when accumulated by the cells, showed higher drug

release under acidic conditions as compared to the physiological conditions. At pH

5.3, the cumulative release of DOX was 95.5 % in 6 hours while at pH 7.4 the

cumulative release of DOX was 42.2 % in 6 hours (Luesakul et al., 2018).

The aim of the study was to synthesize gold nanoparticles with selective

support material for examination of their catalytic response. The monomer of N-

metacryl-amido thiomorpholine with thioether functionality was manufactured. Then

N-metacryl-amido thiomorpholine and acrylamide were polymerized to prepare

hydrogel p(AAm-co-MTM) which were used as support material. The Au (III) ions

were selectively absorbed by this hydrogel and sodium borohydride was used to

reduce these Au (III) ions to gold nanoparticles.TEM, XRD, EDX, and SEM analysis

were used to characterize these hydrogel supported gold nanoparticles. The as-

synthesized p(AAm-co-MTM)-AuNPs conjugate showed higher catalytic response

towards the 4-nitrophenol reduction. The calculated parameters of activation for 4-


20

nitrophenol reduction were reported as ΔH#=36.16kJ/mol, ΔS#=−161.37J/molK and

Ea=38.80kJ/mol (Ilgin et al., 2019).

The valuable management for neoplastic syndromes is still representing a

major challenge, regardless of significant improvement in detection methods and

treatment of specific cancers. In many fields, nanotechnology has been providing a

new outcome for targeted cancer drug delivery as well as promising widely used

knowledge. Current research has revealed that a nanoscale delivery system worked as

a vehicle for selective delivery of the drugs and has the capability to demolish cancer

cells. Chemotherapy in conjugation with nano drug carrier has shown to be more

effective treatment (Xin et al., 2016).

Radmansouri et al. studied the combined effect of chemotherapy and

hyperthermia on B16F10 cell lines of melanoma cancer utilizing the doxorubicin

hydrochloride (DOX) loaded chitosan/cobalt ferrite/titanium oxide nanofibers. The

microwave heating method was applied to prepare cobalt ferrite NPs. The rise in

temperature was controlled by the mixture of cobalt ferrite and titanium oxide NPs.

The vibrating sample magnetometer (VSM), field emission scanning electron

microscopy (FESEM) and X-ray diffraction (XRD) analysis were used to characterize

the synthesized nanoparticles. The efficacy of DOX loading as well as the in vitro

release of drug from prepared NPs conjugate was examined at both acidic and

physiological conditions. At acidic conditions, the quick release of drug was observed

by the alteration of magnetic field. The B16F10 cell lines of melanoma cancer were

utilized to examine the anti-tumor potential of prepared nanofibers. The results

showed that the synthesized nanofibers can be used as an effective tool against

localized cancer treatment (Radmansouri et al., 2018).

Gold Nanoparticles (AuNPs) have been the subject of interest for numerous

biomedical applications. In general surface of AuNPs is coated with inorganic/organic

shells to support chemical conjugation as well as to enhance the stability in biological

fluids. Jang et al. studied the formation of dextran stabilized AuNPs (d-AuNPs) using

dextran as reducing and stabilizing agent. The synthesized d-AuNPs showed excellent

stability at high concentration of salt, high temperature and extreme pH. Furthermore,

the d-AuNPs showed good efficacy as a drug carrier to carry anticancer agent

doxorubicin. In contrast to the free DOX, the conjugate of DOX-d-AuNPs showed 1.1

× 105 times higher inhibitory concentration (EC50) in HeLa cells. Notably, the small


21

AuNPs (diameter 30 and 70 nm) expressed higher EC50 than 170 nm (Jang et al.,

2013).

The prevalence of brain interventions has been gradually growing, with the treatments

helping to improve the life of breast cancer patients, including a large percentage of

metastatic cases. The failure of drugs to enter the central nervous system and circulate

within intra-crane tumor sites is also a major challenge in treatment of these tumors.

Morshed et al. have developed a nanoparticle system which can penetrate in the cell

to enhance the delivery of drugs to the metastatic brain cancer cells. The gold

nanoparticles were modified with TAT peptide followed by loading with anticancer

drug doxorubicin. The resulting formulation helped in the enhancement of

cytotoxicity towards two brain metastatic breast cancer cell lines. The nanoparticles

were given intravenously and the vast quantities were accumulated through diffuse

intracranial metastatic microsatellites. Furthermore, in the xenograft mice model, an

intra-cranial MDA-MB-231-Br cell lines survival rate of these particles has improved

by intratumoral administration. The drug release in the context of brain metastatic

breast cancer has improved by the promising application of AuNPs (Morshed et al.,

2016).

The dicarboxylic acid terminated polyethylene glycol (PEG) AuNPs were

synthesized via one step process, in addition to their supplementary make use of to

form nanostructure surfaces for immobilization of biomolecules. A conjugate of

developed nanoparticles with oligonucleotide was synthesized for evaluation of

intercalation process between doxorubicin and modified nanoparticles by means of

surface-enhanced Raman spectroscopy (Spadavecchia et al., 2016).

A great deal of interest for functionalized gold nanoparticles has been found

because of their wide range of medical scope and interesting optical properties.

Salabat and Mirhoseini have reported new microemulsion method for preparation of

biocompatible monohydroxy thioalkylated PEG protected AuNPs. The various

characterization techniques such as Transmission electron microscopy(TEM),

dynamic light scattering(DLS), UV–vis spectrophotometry and Energy dispersive X-

ray analysis were used to fully characterize the AuNPs. The size of AuNPs reported

was 7-9 nm with no cytotoxicity effect to HeLa cells (Salabat et al., 2018).

Naz et al. reported a green synthetic route to prepare silver nanoparticles from

AgNO3 and different concentrations of the seed extract (Setaria verticillata). To


22

investigate the physiochemical possessions of the resulting seed extract from AgNPs,

XRD, FTIR spectrometry, TEM, and ultraviolet-visible spectrophotometry were used.

Against breast cancer cells AgNPs had a dose-dependent cytotoxic effect which

shown anticancer activity. The in vitro toxicity study of Lumbricina (adult

earthworms) showed good inhibition (p 0.05) calculated statistically. In addition, a

new drug carrier system was developed. The nanoparticles were loaded with

doxorubicin (DOX), daunorubicin (DNR), and these hydrophilic anticancer drugs

(ACD) would help to reduce the adverse effects of the medication used for leukemia

chemotherapy (Naz et al., 2017).

Wan et al. reported the use of AuNPs as drug carrier of Docetaxel-decorated

anticancer drug for liver cancer therapy. Liver metastasis is among the life threatening

diseases that have a detrimental effect on human life. The use of drug delivery

platform not only increases the efficacy of medication but also increases the

bioavailability of medication as well as reduces the adverse effects of medication.

Docetaxel (Dxtl) remains the preferred option of improving the survival for patients

with liver metastasis but many patients suffer the negative impacts of modest

reactions and enormous death. They observed that in human liver cancer cells

(HepG2) Docetaxel stacked gold dopped apatite showed greater cytotoxicity (Wan et

al., 2018).

The therapeutic use of anticancer drugs are limited because of the inadequate

transmission of drugs to the cancer cells as well as the number of pathways of drug

resistance found in the malignant cells. Therefore, the nanoparticles responsive to the

specific stimuli could be used as an effective tool to deal with such drug delivery

issues. Ghorbani and Hamishehkar prepared the novel platform of hybrid

gold/nanogels (Au/NGs) to load 6-mercaptopurine (MP) and doxorubicin (DOX)

simultaneously. The loading capacity for MP was 11 % and for DOX was 23 %. The

trigger responsive capability of Au/NGs to release the drug was evaluated by making

comparison of tumor tissue and physiological environment. The integration of

thermosensitive, pH sensitive polymeric segments and disulfide bonding agents has

given the NGs the ability of reducing the acidic environment. This consequently

supported the release of drug in tumor cells. The results of study showed the excellent

cellular uptake and accumulation of drugs by the NGs developed. The cytotoxicity


23

results described the significant inhibition of tumor in contrast to the free DOX@MP

(Ghorbani & Hamishehkar, 2018).

The study explains the improvement and justification of a microbiological

assay, for the purposed of doxycycline (DOX), applying the turbid metric method. In

addition, to informative, the constancy of doxycycline in medication adjacent to basic

plus acidic hydrolysis, oxidative with photolytic breakdowns, using E. coli ATCC

10536 were used for evaluation of the method. The essay showed admirable results of

precision, robustness, linearity, selectivity, and accuracy by using Escherichia coli

ATCC 10536 which is depend on the inhibitory consequence of doxycycline. The

consequences of the evaluation were treated by one-way ANOVA and were establish

to be linear (r= 0.9986) range from 4.0-9.0μg/mL, exact (97.73%) accurate and

(repeatability R.S.D. = 0.99). To evaluate the specificity of the bioassay, the

doxycycline solution was showing to direct ultra visible light, hydrogen peroxide

which causing oxidation, and alkaline with acid hydrolysis. Differences in results

showed due to the methodologies of bioassay and liquid chromatography. The

consequences demonstrated that bioassay is a legal, easy and helpful substitute

method for the resolve of doxycycline in everyday value organize as compared in the

direction of liquid chromatography (Kogawa et al., 2012).

In fabrication of a suitable drug delivery vehicle, the major challenge is to

control the release of drug. In this context, a number of efforts have been made to

develop multifunctional nanoparticles associated with various triggers. The higher

therapeutic efficacy can be achieved via use of the trigger dependent drug delivery

platform with controlled release of drug. Deshpande et al., reported the use of

polymeric shell and thermo responsive gold core nanoparticles for controlled

doxorubicin release. They selected the radiofrequency (rf) as a trigger due to its good

penetration depth in tissue. They recognized that heating efficacy of AuNPs by rf was

not affected by coating of polymer on AuNPs. The developed drug delivery system

showed thermoresponsive release of doxorubicin. Furthermore, the synthesized

nanoparticles were stable and showed a brust as well as a controlled release of

anticancer drug depending on rf. They observed that the use of Au core in

combination with polymeric nanoparticles induce greater cell death in HeLa cells as

compared to the single nanoparticles. They suggested that the use of multi-


24

nanoparticles can effectively enhance the efficacy of drug delivery systems

(Deshpande et al., 2017).

The doxorubicin (DOX) loaded gold nanoparticles (AuNPs) were synthesized

by green method and their anticancer activity was studied against human cancer cell

lines. These gold nanoparticles were analyzed by FTIR spectroscopy, XRD,

ultraviolet spectrophotometer, TEM, and Zetasizer measurements. A significant

surface plasmon resonance band at 532 nm confirmed the development of AuNPs.

XRD and FTIR spectroscopy were used to analyze the crystalline nature of AuNPs

and the interaction of plant with AuNPs respectively. The Zeta sizer and TEM studies

revealed a zeta potential of −19.13 ± 0.2 along with constituent part size of 74.7 nm.

An in vitro anti-cancer assay of doxorubicin-loaded gold nanoparticles against the

human cancer cell lines was studied. They showed good anticancer activity with

variable response to lung, breast, and prostate cancer cell lines. However, in the cell

viability percentage in opposition to liver, cervical, and pancreatic cancer cell lines,

no noteworthy variation was observed between doxorubicin and doxorubicin-gold

NPs. The results of the in vitro anticancer assay of doxorubicin–gold NPs against

human cancer cell lines proposed their prospective in cancer treatments for in vivo

application (Dhamecha et al., 2015).

Dendrimers with hyper branched 3D structure are described as the unique

polymeric nanostructures. The various functional groups present on the surface of

Dendrimers enhances their applicability, versatility and bio-compatibility. In

comparison to the different nanomaterials, Dendrimers have obtained significant

interest due to their distinctive characteristics such as the available polyvalency,

internal cavities, nano-scale uniform size, water solubility, the high degree of

branching, and convenient synthesis approaches. Furthermore these characteristics

make them useful regarding the drug delivery applications. They can be used as a

carrier in favor of a different healing mediator and established huge consideration

from scientists. Nanomaterials can be used for the enhancement of drug efficacy and

reducing their toxicities. The nearby evaluation provides a complete chart of

dendrimers in the pharmaceutical and biomedical field (Sherje et al., 2018).

It was examined that the porous magnetic nanoparticles (MNPs) have large

surface area to load a chemotherapeutic drug doxorubicin and can be used as an

efficient drug delivery agent. The developed MNPs are efficient near-infrared(NIR)


25

photothermal mediator. The invitro studies were carried out and prostate cancer was

destroyed via combination of photothermal therapy (PTT) and chemotherapy using

conjugate of MNPs-DOX. The cancer cells incubated with MNPs-DOX showed

higher percentage of cell death when irradiated with NIR. In comparison to the

chemotherapy or PTT alone, the combination of both PTT and MNPs-DOX showed

higher therapeutic efficacy (Zhang et al., 2015).

Glutamic acid protected AuNPs were synthesized in one, two, and three-

dimensional (1D, 2D, and 3D) super structures triggered by pH changes. The

nanospheres are fusing into one another to form multidimensional network super-

structures. By means of the unbiased COOH group of glutamic acid molecules, the

main driving force (cross-linking) is made available for the fabrication of these

superstructures. Interestingly, the Br− that present in sodium bromide (NaBr) can

restrain such assembling performance of gold NPs proficiently by change some

glutamic acid molecules from gold NPs surfaces in addition to decline the connecting

consequence of the impartial COOH group of glutamic acid on gold NPs. Besides, by

the induction of pH induced super structures, surface enhanced Raman scattering

substrates with elevated action could be used. For biological detection and biosensors,

GNPs provide the suggestion for the exploitation of the glutamic-NPs system due to

chemical tunability of the interparticle and new approaching interested in the pH

linking interactions of bound glutamic acid (Liu et al., 2015).

A new twofold stimuli responsive polyethylene glycol (PEG) block co-

polymer was manufactured that bring manifold anti cancer drugs like methotrexate,

DOX, and 6-6-mercaptopurine used for the stabilization and decoration of gold

nanoparticles. These drugs were effectively loaded in the polymeric shell of

nanoparticles by disulfide-covalent bond formation (MP) and interacted in the

nanoparticles by ionic- interaction of doxorubicin and methotrexate. Furthermore, the

drug-releasing ability of nanoparticles was prompted by the judgment of tumor tissue

in an environment and simulated physiological responses. The improved effectiveness

of the produced nanoparticles was demonstrated with their targeted performance

through methotrexate decoration on a variety of cancer cell lines through diverse

stages of folate receptors for cell cytotoxicity studies. A recent study was conducted

on polyethylene glycolated gold nanoparticles that could afford gifted for the


26

instantaneous three cytotoxic drug delivery and narrative prospects in the treatment of

cancer (Ghorbani & Hamishehkar 2017).

Gold nanoparticles synthesis was mediated by the effect of acetone which has

been studied on 3-aminopropyltrimethoxysilane in non-polar, polar protic and polar A

protic solvents particularly chloroform, acetone and water. The results exposed that

AuNPs are promoting the development of siloxane polymer in chloroform, acetone,

and water solvents while acetone produced siloxane polymer into both solvents except

water. Consequently, siloxane-Au NPs can be prepared by three methods all the way

through control over the process including 3-APTMS, Au3+

, and acetone assorted at

the same time yielding [(siloxane-Ausim)], polymer. These polymers complete into

thin layer pursued by in order decline of Au3+

in non-homogenous system producing

(gold-siloxanehetero) seq, along by way of polymer made first followed by Au+3

homogenous suspension forming (Au-siloxanehomo) sequence of diverse

morphology. The AuNPs produced by these methods are discrete spherical structure

[(AuNPs) water] and the production of siloxane polymer does not yield. For

heterogeneous catalysis can efficiently promote by “(Au-siloxanehetero) seq”

justifying its attention by the diminution of para-nitrophenol with finely prepared

constancy. On the other hand, for subsequent electro analytical purposes in

cooperation (Au-siloxanehomo) seq as well as (siloxane-Ausim) seq can also be

added on the surfaces of glassy carbon for yielding of electrodes modified-polymer.

The representative consequence of dopamine sensing is described which demonstrates

brilliant biocatalyst on peroxidase mimetic action (Pandey et al., 2017).

A sensitive and fast biosensor based on AuNPs enhanced surface plasmon

resonance (SPR) was developed to detect the ochratoxin A (OTA) present in red wine.

The enhancement of signal was achieved by the conjugation of antibody with AuNPs.

An indirect competitive inhibition immunoassay was carried out for the ultra sensitive

detection of OTA (analyte of low molecular weight). The limit of detection of the

reported biosensor for OTA detection was 0.75 ng mL-1

. The use of AuNPs for signal

enhancement resulted in improvement of limit of detection, by more than one order of

magnitude to 0.068 ng mL-1

. The study also examined the role of AuNPs size as well

as the attraction of the recognition elements influencing the AuNPs based signal

enhancement strategies. In addition, the interference of polyphenolic compounds


27

present in red wine with the antibody modified surface of SPR biosensor was also

observed. To overthrown this drawback, the red wine was pre-treated with

polyvinylpyrrolidone (PVP) (Karczmarczyk et al., 2016).

The higher level of cortisol released from adrenal cortex is mainly responsible

for the acute and chronic stress. Therefore the cortisol detection is highly critical to

prevent the growth of diseases associated with acute and chronic stress. The change of

refractive index due to the binding of nanoparticles with biomolecules present on the

surface of SPR biosensor resulted in change of LSPR wavelength. Using this

principle, a unique, cuvette-type disposable LSPR based biosensor was developed to

detect serum cortisol. The construction of the fabricated nanobiosensor contains an

array of plastic unit sensors with single layer coating of AuNPs, on to which the

bovine serum albumin (BSA) in conjugation with cortisol was immobilized. The

cortisol antibody binded with cortisol-BSA conjugate present on surface of AuNPs

which resulted in red shift of LSPR wavelength. The developed competitive assay

based nanobiosensor allowed for the detection of cortisol in the range from 1 to 1000

ng/mL in 20 minutes in both phosphate buffer saline solution and serum as compared

to the traditional methods such as enzyme linked immunosorbent assay (ELISA) that

demands more than 4 hours as well as complicated sample preparation. Thus an ultra

sensitive and highly reliable nanobiosensor was developed for the cortisol detection in

serum (Jeon et al., 2018).

A novel anodic aluminum oxide (AAO) substrate based ultra sensitive

biosensor design was reported by Yeom et al., (2013). The sensitivity of LSPR

biosensor was increased by using gold NPs (GNP) labeled antibodies. In this study,

the use of AuNPs-labeled antibodies overcame problems that are encountered in such

biosensors due to limited sensitivity. To examine the application of the fabricated

biosensor, C-reactive protein (CRP) (a biomarker) was applied. The concentration of

CRP was varied to check the enhancement in sensitivity. The developed sensor device

was applied for the detection of CRP antigen based on sandwich assay. The

enhancement in sensitivity of biosensor was 1.84 times due to the increased response

for the AuNPs-labeled CRP antibody. The limit of detection for the fabricated

biosensor was 100 ag/ml. This reported biosensor was enabled high sensitivity and

selective immunoassay to be performed over a wide range of concentrations (Yeom et

al., 2013).


28

Surface plasmon resonance imaging (SPRi) is a strong optical technique that

provides real time analysis without labeling the biomolecules. However the

improvement in sensitivity is essential when dealing with low molecular weight

analyte. The sensitivity enhancement strategy based on graphene coating to obtain

high throughput SPRi was reported with quantification of enhancement factor. The

half spots of gold chip were covered with single layer of good quality graphene. The

antibody anti-zearalenone (ZEN) was applied as a model analyte and thin film of

poly-dopamine (PDA) was used as reactive layer for immobilization of probe. The

SPRi signals were quantified on two different sensing spots on a single chip and

compared advantageously followed by the evaluation of enhancement factor. It was

observed that the SPRi signals were enhanced 40 percent with graphene coating on

reflectivity based SPRi setup (Wei et al., 2018).

An ultra sensitive hybrid SPR biosensor based on gold-MoS2-Graphene was

reported for DNA hybridization detection. The parameters such as quality factor,

detection accuracy and sensitivity were examined to check the performance of the

fabricated biosensor. It was observed that the addition of MoS2 in mid of layer of

graphene-on-Au resulted in significant enhancement of sensitivity of proposed

biosensor. The use of MoS2 layer between Au layer (thickness 50 nm) and graphene

layer provided the greater detection accuracy (1.28), good quality factor (17.56) and

larger sensitivity (87.8 deg/RIU). The optical properties and absorption ability of

graphene as well as the greater fluorescence quenching capability of MoS2 were the

main cause of increased performance of proposed biosensor. It was observed that the

bonding of nucleotide between double stranded DNA helix can be detected due to the

change in minimum reflectance and SPR angle of the proposed biosensor. Thus the

fabricated biosensor can successfully be applied for the detection of DNA

hybridization (Rahman et al., 2017).

Karczmarczyk et al. reported the fabrication of a sensitive SPR biosensor to

detect the aflatoxin M1 (AFM1) present in milk. The competitive assay based analysis

was carried out to detect AFM1 in which signal amplification was achieved by the

conjugation of antibody with AuNPs. The interference of milk constituents with SPR

sensor surface was minimized by the use of poly (2-hydroxyethyl methacrylate)

p(HEMA). The activity of both polyethylene glycol-based sensor and p(HEMA)-

based sensor was compared advantageously. The reported biosensor allowed for the


29

AFM1 detection in 55 minutes with limit of detection as low as 18 pgmL-1

(Karczmarczyk et al., 2016).

The DNA-modified gold nanorods (AuNRs) based localized surface plasmon

resonance (LSPR) biosensor was reported for the enhancement in spectral shift in

LSPR biosensing platform. The feasibility of reported biosensor was assessed by

using a model analyte interferon gamma (IFN-). The thermal lithographic process

was used to fabricate the gold nanodots modified LSPR chips followed by

functionalization with IFN- aptamers. During the detection process, the binding with

aptamers was followed by the competition between the IFN- and DNA-modified

AuNRs. The shift in spectra was mainly because of DNA-modified AuNRs. This

approach does not demand the multiple binding sites. According to both, the finite-

difference time-domain(FDTD) simulations and experiments, the placement of

AuNRs close to the surface of LSPR chip is very important regarding the

enhancement of LSPR shift. The results obtained from simulations showed that the

AuNRs arrangement on chip surface influence the plasmon coupling between the

nearby AuNRs and Au nanodots on chip surface (Lin et al., 2016).

The sensitivity enhancement and antibody immobilization on sensing surface

are the important factors regarding the fabrication of a SPR immunosensor. A label-

free detection platform with increased sensitivity was developed by assembling

AuNPs on SPR chip. The AuNPs were distributed uniformly over the surface as

shown by SEM image. The gold binding polypeptides(GBP) were fused genetically to

protein A(ProA) which was used as a crosslinker to properly immobilize the antibody

on sensing surface. Both bare and AuNPs modified SPR chips were immobilized with

that novel fused protein GBP-ProA. Afterwards the human immunoglobulin G(hIgG)

was binded with ProA domain. It was observed that the binding of anti-hIgG and

hIgG with GBP-ProA modified Au chip resulted in increased sensitivity as compared

to the bare chip treated identically. Thus the results showed that the modification of

SPR chip with AuNPs enhances the sensitivity of proposed biosensor. Furthermore

the GBP-ProA could be used as an effective crosslinker to properly immobilize the

antibody on to the sensing surface of SPR chip (Ko et al., 2009).

A narrative technique was accounted which based on gold/silver alloy

nanocomposites amplifying surface Plasmon give you an idea about considerable

response for detecting human IgG antibodies. The distinctiveness of nanocomposites


30

alloy was explained in point by TEM, X-ray, UV–vis absorption and photoelectron

spectroscopy (XPS). Due to high dielectric constant of Au/Ag nanoparticles, a large

shift is formed in resonance wavelength. They enlarge in the width of the sensing

membrane and electromagnetic coupling among alloy nanocomposites, the covalent

immobilization of gold film is formed which in relation to 24 nm diameter of

silver/gold alloy nanocomposites. These were exposed to an acceptable reaction on

silver/gold alloy nanocomposites for individual antibodies IgG in the deliberation

range vary from 0.15–40.00 g m/L, SPR. Although the biosensor depend on gold NPs

is demonstrated a reaction in the absorption range of 0.30–20.00 g m/L, as well as

gold film, explain a response range of 1.25–20.00 g m/L (Wang et al., 2011).

For liquid concentration measurement, optic fiber SPR manufacture that is a

sensing probe which stands on a silver mirror reaction. The narrative chemical

method is resource conservation, more suitable, and low-cost when compared to

traditional physical methods that do not need any complicated equipment. The end

reflection optic fiber surface Plasmon resonance sensor was placed with a liquid

concentration measurement system. Subsequently, the association experimentation

was conducted amid natural light and darkroom environment, considering that the end

result that achieve from ordinary illumination was eradicated. The wavelength of

resonance was acquired for measuring glycerol solutions with different concentrations

range of volume from 0% to 50% shifts. The sensitivity of the sensor was originated

in a variety of 346.7-890.7 nm/% (Zhao et al., 2014).

According to Vachali et al. the SPR biosensor is a strong optical technique

that provides label-free detection of biomolecules with greater selectivity and

sensitivity. It is used to examine the non-covalent binding of biomolecules. They

described the biosensing platform to examine the binding of carotenoid binding

proteins as well as their carotenoid ligands for assessment of their binding specificity,

stoichiometry and kinetics affinity. They reported that such characterizations are vital

as they can help in understanding the transportation and uptake of carotenoid to the

targeted tissue such as the macula of human eye (Vachali et al., 2015).

The present study focuses on use of an aqueous extract of Tephrosia tinctoria

for silver nanoparticles green synthesis. They used FT-IR, UV-vis spectrophotometry,

EDX patterns, transmission electron microscopy, Scanning Electron Microscopy and

X-Ray Diffraction for characterization of Ag nanoparticles. Furthermore, Silver NPs


31

were applied to examine their antidiabetic potential. The results clearly describes the

significant inhibition of alpha-Glucosidase and alpha-Amylase, free radical

scavenging capability, and improvement of Glucose uptake rate (Rajaram et al.,

2015).

Balan et al. reported the preparation of silver nanoparticles utilizing the

Lonicera japonica leaf extract. During the process of synthesis, color of solution was

changed from pale yellow to brownish, which was the first indication of synthesis of

AgNPs. The formation of AgNPs was further confirmed using UV-vis.

spectrophotometry where an absorption band was observed at 435 nm, a

characteristics LSPR band of AgNPs. To further analyzed the synthesized AgNPs, the

techniques such as zeta sizer, HR-TEM, XRD and FT-IR. The size of AgNPs shown

by zeta sizer was 53 nm. The spherical morphology of AgNPs was confirmed by

TEM. The interaction of AgNPs with biomolecules present in the extract was

confirmed by FT-IR. The significant antioxidant activity was shown by the

biologically prepared AgNPs. The strong inhibition activity against the α-glucosidase

and α-amylase justified the antidiabetic potential of biologically prepared AgNPs. The

Dixon and LB plots were used to analyze kinetic mechanism of inhibition. The results

demonstrated the significant antidiabetic potential of synthesized AgNPs towards the

main enzymes of diabetes mellitus (Balan et al., 2016).

Zeng et al. synthesized the selenium nanoparticles stabilized by chitosan

(CTS-SeNPs). The orthogonal experiments were used to find the optimized conditions

for synthesis of CTS-SeNPs. The stability, morphology and size of CTS-SeNPs was

examined using SEM, TEM and DLS. Under the optimized conditions for synthesis,

the average size of CTS-SeNPs obtained was 54 nm. The synthesized CTS-SeNPs

showed good stability up to 60 days at 4 C̊. The hypoglycemic effect of CTS-SeNPs

was studied using streptozotocin (STZ)-induced diabetic mice. The results described

that the higher antidiabetic activity was shown at dose 2mg SeNPs/ Kg bw as

compared to the other doses and other selenium treatments (Zeng et al., 2018).

In recent years, by means of the plants extract and their prospective relevance,

the green synthesis of zinc oxide (ZnO) nanoparticles has received a marvelous

interest. Using Silybum marianum seed extract, Arvanag et al, have reported the

microwave-assisted green method for the preparation of ZnO nanoparticles. The ZnO

nanoparticles chemically produced had size larger than the biosynthesized sample


32

(ZnO/extract). The alloxan induced diabetic rats were treated with ZnO/extract

sample and their effectiveness was compared advantageously with extract, ZnO

chemically prepared and insulin treatment. For this purpose, the total triglyceride,

total cholesterol, high-density lipoprotein, insulin, and levels of blood glucose were

measured before as well as after treating and compared these parameters with each

other and studied samples. Furthermore, to evaluate their antibacterial potential,

against E. coli of both ZnO samples was investigated. As of the results, ZnO/extract

NPs were showed fine antibacterial action and exceptional routine in overcome

diabetic disorders (Arvanag et al., 2019).

The plants with medicinal activities and silver nanoparticles (AgNPs) have

been considered as the most important source for treatment of metabolic disorders of

diabetes mellitus. To examine the in-vivo antidiabetic potential of aqueous extract and

AgNPs, PRABHU et al. prepared the AgNPs using leaf extract of Pouteria sapota (P.

sapota). Hot percolation procedure was applied to synthesize the AgNs under ambient

conditions. The assays such as prohibition of α-amylase, glucose uptake by yeast cell

and non-enzymatic glycosylation of hemoglobin were applied to examine the in-vitro

antidiabetic potential of AgNPs and leaf extract. Moreover, streptozotocin induced

diabetic rats were treated with specific doses of AgNPs and leaf extract and their in-

vivo antidiabetic potential was assessed. The Histopathological and biochemical

analysis of liver and kidney samples showed that the blood sugar level was

significantly reduced in rats administrated with AgNPs biologically synthesized and

leaf extract. This showed that the both the AgNPs and leaf extract have potential to

treat metabolic disorders of diabetes mellitus (Prabhu et al., 2018).

Shamprasad et al. reported a new method based on sunlight for the preparation

of gold nanoparticles utilizing escin (a triterpenoid glycoside). The average size of

escin stabilized gold nanoparticles was in the range of 5–20 nm, characterized by

TEM imaging and their UV-vis. band was observed at 530 nm. In L6 rat skeletal

muscle cells, insulin-dependent glucose uptake was found to be improved by escin-

AuNPs. The glucose uptake was further confirmed by calculating approximately the

accumulated glycogen content. They revealed tremendous free radical scavenging

action so, they may be investigated as potential molecule to test in opposition to

diabetic problems (Shamprasad et al., 2019).


33

Liver plays a crucial role in maintaining the blood glucose level. Zhang et al.

applied the N-(2-hydroxy)-propyl-3trimethylammonium chloride functionalized

chitosan and cholic acid (HTCC-CA) as carrier for targeted delivery of insulin to the

liver. A new methodlogy was formulated for effective loading of insulin. HTCC-CA

and insulin were mixed at pH 2 in 50% ethanol followed by the dialysis using

phosphate buffer (pH 7.4) and water. The average size of insulin loaded HTCC-CA

NPs was 86 nm and 98.7 % loading efficiency was reported for insulin. In contrast to

the free insulin, 466 % enhancement in insulin uptake by cells was observed with

application of NPs. Furthermore, the results of treatment of diabetic rats showed that

the insulin bioavailability was increased up to 475 % due to the NPs application. In

contrast to the free insulin, a sustained hypoglycemic effect was shown for more than

24 h by NPs. The cholic acid functionalized nanoparticles have the ability to target the

liver and the biocompatibility of insulin loaded HTCC-CA NPs can be used to

increase the insulin‟s hypoglycemic effect (Zhang et al., 2016).

The present study has been conceded out for the production of gold NPs by

using aqueous extract of Cassia auriculata L. The shape, size, and elemental

examination were conceded out by means of SEM-EDAX, UV-vis., XRD,

transmission electron spectroscopy, and Fourier transform infrared spectroscopy. C.

auriculata can be used for the synthesis of AuNPs which were triangular, stable, and

spherical crystalline with the distinct magnitude of the normal size of 15–25 nm. The

outcome of pH was also premeditated to corroborate the constancy of Au

nanoparticles. The foremost aim of the exploration using antidiabetic potent medicinal

plant was to synthesize AuNPs using plant with antidiabetic medicinal properties. If

tested additionally becomes constant and reducing molecules of NPs possibly will

encourage anti-hyperglycemic actions (Ganesh Kumar et al., 2011).

Govindappa et al. studied on silver NPs by means of aqueous L. extract of

Calophyllum tomentosum (CtAgNPs) to build up simple and ecological method. They

examined the extract to recognize the possession of anti-tyrosinase, anti-

inflammatory, anti-diabetic, anti-bacterial, and antioxidant action. For

characterization of the Calophyllum tomentosum mediated silver nanoparticles, they

utilized UV–vis spectrophotometer, EDX, XRD, FTIR. The leaf extract of C.

tomentosum carry terpenoids, phenols, coumarins, saponins, flavonoids, alkaloids,

glycosides, and tannins. CtAgNPs have shown considerable antibacterial activity on


34

multi drug resistance bacteria strains as well as they had shown strong antioxidant

actions. Meanwhile, these nanoparticles demonstrated tyrosinase inhibitory and strong

anti-inflammatory action. The Ct silver nanoparticles had also powerfully constrained

the DPPIV and alpha-glucosidase compared to alpha-amylase (Govindappa et al.,

2018).

Kumara et al. worked on the fusion of silver NPs by means of leaves of

Holoptelea integrifolia (HI). The silver nanoparticles were characterized by FTIR,

UV-vis spectroscopy, FESEM, XRD analysis, with electron disperse X-ray (EDX).

The biosynthesized AgNPs exhibited significant anti-inflammatory (binding constant

2.60 ± 0.05×10−4), remarkable anti-diabetic (86.66 ± 5.03%), antioxidant activities

(51.49 ± 3.33, 41.18 ± 2.27, and 74.59 ± 3.08% for the metal chelating, nitric oxide,

and DPPH assay), and antibacterial (MIC from 75 to 150 μl) activities. This is a

novel study on the biosynthesis of AgNPs by using Holoptelea integrifolia HI leaves

extract (Kumar et al., 2019).

The rhizomes of Glycyrrhiza glabra contain active compound glycyrrhizin

which has anti-hyperglycemic effects. In vivo study of NPs overloaded by way of

glycyrrhizin or metformin were estimated in rats against type-II diabetes for their anti-

hyperglycemic potency. The nanoparticles were produced by means of biocompatible

gum arabic and polymers chitosan via ionotropic gelation method. Furthermore, for

twenty-one consecutive days to diabetic rat‟s glycyrrhizin, metformin, and nano

formulations were administrated. Glycyrrhizin loaded nanoparticles had vital anti-

diabetic effects, yet while relative to the pure form they contained just about 1/4 of the

prescribed amount (Rani et al., 2017).

Vinotha et al. studied the antioxidant, antidiabetic and antibiofilm properties

of ZnO nanoparticles synthesized via leaf extract of Costus ingneus. The bioactive

components of plant extract were estimated through Gass chromatography mass

spectrometry(GC-MS). The characterization of Costus ingneus coated ZnO

nanoparticles (Ci-ZnO) was achieved using transmission electron microscopy, Ultra

violet visible spectroscopy, FTIR, XRD, and proton 1H NMR spectroscopy. UV–Vis

was used for authentication of Ci-ZnO NPs and they show evidence of a peak at 365

nm. At a concentration of 100μg/ml, antidiabetic activity of Ci-zinc oxide

nanoparticles (74 % and 82 %, respectively) and antioxidant activity of the

nanoparticles (75%) was measured by using the carbohydrate digestive enzymes


35

assay, and the 2, 2-diphenyl-1-picrylhydrazyl hydrate (DPPH) assay. The Ci- ZnO

NPs showed significant antiseptic and biofilm prohibition action in opposition to the

disease-causing bacteria Proteus vulgaris, Vibrio parahaemolyticus Streptococcus

mutans, and Lysinibacillus fusiformis. Additionally, at a concentration of 200μg/ml,

the Ci-zinc oxide nanoparticles showed biocompatibility effects with mammalian

RBCs with the least amount of hemolytic action (0.633% ± 0.005%) (Vinotha et al.,

2019).

The numerous researchers have paid attention to the biosynthesis of

nanoparticles. Bayrami et al. used the ultrasonic-assisted method for the fabrication of

ZnO nanoparticles by means of whortleberry (Vaccinium arctostaphylos L.) extract.

The structure, crystal size, morphology, optical and thermal characteristics of

biosynthesized ZnO NPs (ZnO/extract) were examined and compared with ZnO

prepared chemically (ZnO/chem). Alloxan induced diabetic rats were treated with

ZnO samples and effectiveness of treatment was examined through the effects on

high-density lipoprotein, cholesterol, total triglyceride, insulin, and fasting blood

glucose levels. As compared to the other treatments including leaf extract, ZnO/chem

and insulin, the ZnO/extract sample showed more effectiveness in health recovery of

diabetic rats. Additionally, the ZnO samples were assessed with the help of photo

catalytic and sono processes for removing rhodamine B against gram +ve and gram -

ve bacteria. The consequences of this study designated to facilitate ZnO samples

when compared with the ZnO/chem sample revealed good efficiency for caring of

diabetic rats, bacterial decontamination and oxidative removal of organic compounds

below the effects of UV irradiations with ultrasound (Bayrami et al., 2019).

Bayrami et al. used the Nasturtium officinale leaf extract to synthesize ZnO

nanoparticles through microwave assisted method. The Ext/ZnO NPs were analyzed

by ultraviolet-Vis DRS analyses, transmission electron microscopy, SEM, TGA, X-

ray diffraction, EDXA, BET, FT-IR, and compared with ZnO chemically prepared.

ZnO, watercress leaf extract, extract/zinc oxide, and insulin treatments were governed

toward care for alloxan diabetic Wister mices. The healing effectiveness consequences

of extract zinc oxide, zinc oxide, watercress L. extract, and insulin were compared to

one another. For diabetic, healthy, and the rats renewed by means of the considered

remedial agent's serum levels of the main diabetic indices were estimated such as

fasting blood glucose, lipid profile and insulin. The watercress extract enriched zinc


36

oxide NPs showed the most excellent results in addition to concealed the diabetic

condition of mices. In addition, mutually zinc oxide samples adequate introverted the

action of S. aureus along with E. coli bacteria. The purpose of Nasturtium officinale

L. extract can powerfully allow zinc oxide NPs on the basis of results. The results of

these extract which described better antibacterial as well as improved antidiabetic

actions (Bayrami et al., 2019).

Piyush et al. studied the free radical scavenging activity and antidiabetic

activities of CuNPs prepared using Dioscorea bulbifera tuber extract (DBTE). CuNPs

synthesized by DBTE were analyzed by energy dispersive spectroscopy, TEM, UV-

visible spectroscopy, and dynamic light scattering. Copper NPs were checked for

circular dichroism spectroscopy, carbohydrates digestive enzymes inhibition, along

with the computational docking and fluorescence spectroscopy. Diphenyl

picrylhydrazyl (DPPH), HNO3 and superoxide radical scavenging activities of Copper

nanoparticles were also calculated. The size of produced NPs was between 12 to 16

nm which grew to turn over an ultimate size of 86 to 126 nm in DLS and transmission

electron microscopy respectively. Bio reduced Copper nanoparticles demonstrated

38.70 ± 1.45% inhibition zone against porcine in addition to 34.72 ± 1.22% inhibition

zone murine pancreatic amylase with docking system these zone of inhibition was

confirmed. By means of Trp remainings, fluorescence spectroscopy established the

relations of Cu nanoparticles to the enzyme. Meanwhile, CD spectra point out the

conformational as well as structural changes in the binding of Copper NPs to the

enzyme. They also showed a zone of inhibition against murine intestinal glucosidase

90.67 ± 0.33% as well as 99.09 ± 0.15% against α-glucosidase correspondingly.

Scavenging activity of CuNPs against nitric oxide, 2, 2-diphenyl-1-picrylhydrazyl

(DPPH), and superoxide radicals was 79.06 ± 1.02%, 40.81 ± 1.44%, and 48.39 ±

1.46% respectively. The mainly quick way to manufacture novel Cu nanoparticles by

tuber extract of D.bulbifera interceded by bioreduction to facilitate illustrated

promising antioxidant and anti-diabetic properties (Piyush More et al., 2015).

The present evaluates worked on the prospective antimicrobial, anticancer,

and antidiabetic actions of Ag and Phyto-synthesized Au NPs. Synergistic features of

metal and plant NPs offers curing possessions that might be the clinical bioequivalent

which shown by Phyto nano therapy to many synthetic drugs by way of minimum

side effects. This could allow therapeutic plant psychotherapy to co-exist with present


37

man-made dealing and provide alternative management for long term diseases that are

efficient to prevail over the drawbacks of synthetic mono therapy. In communicable

and non-communicable diseases, this produces a much-needed pattern change in favor

of supplementary clinical learning‟s (Anand et al., 2017).

Jini and Sharmila studied the antidiabetic activities of silver nanoparticles

prepared using Allium cepa. The chemical composition of AgNPs synthesized was

examined by means of FT-IR spectroscopy, SEM and UV–vis. spectroscopy which

showed with the intention of the produced particles were spherical in shape as well as

nano in size. In vitro antidiabetic actions showed that the NPs have an elevated level

of alpha-amylase and alpha-glucosidase inhibitory actions along with enhanced

antioxidant in addition to fewer cytotoxicity effects. It was accomplished with the

intention cure of diabetes, the silver nanoparticles synthesize green can be used as a

potential phytomedicine (Jini & Sharmila, 2020).

The consequence of nano materials such as Au nanoparticles along with

amalgamation with normal yield has revealed good outcomes, in decreasing glycated

hemoglobin and anti-inflammatory actions. To estimate the antidiabetic result of

functionalized Sambucus nigra L. (SN) extract nanoparticles on an investigational

model of diabetes rats were studied. A on its own intramuscular inoculation of

streptozotocin was used for induction of diabetes in 18 male Wistar rats (n = 6)

according to body weight (30 mg/kg body weight b. w.). For two weeks just the once

vehicle (normal saline), Sambucus nigra L. extract (15 mg/kg b. w.) and nanoparticles

(0.3 mg/kg b. w.) were governed by gavage every morning. Afterward, the liver trials

were in use to evaluate for the estimation of, COX-2, metallo proteinases (MMP)-2

and 9 activities, immunohistochemistry and for NFKB expressions. Muscle and blood

samples were also used to check the antioxidant condition along with alanine amino

transferase (ALAT), cholesterol, aspartate amino transferase (ASAT) and serum

glycemia were besides calculated. When comparison of diabetic group in opposition

to diabetic groups treated with vehicle (p < 0.05) or non-diabetic (p < 0.03), they

improved systemic glutathione disulfide (GSH/GSSH) and the muscle ratio in the

diabetic grouping and decreased malon dialdehyde levels contrast to without diabetic

group (p < 0.05) by the administration of NPs extract. After pre-treatment with

nanoparticles in corresponding with the diminution of Kupffer cells percent (< 0.001),

pro matrix metalloproteinases-2 (MMP-2) movement (p < 0.05), and cyclooxygenase-


38

2 expression (p < 0.0001) was decreased. For supplementary dealing as adjuvants in

diabetic therapy, nanoparticles present an enormous prospective due to reduction of

MMPs activity, the increase of antioxidant defense, and inflammation in liver tissue

(Opris et al., 2017).

Dhas et al. worked on Sargassum swartzii were used for gold nanoparticles

(AuNPs) production and checked its anti-diabetic effects by using male Wistar Albino

rats. These nanoparticles were differentiating by using XRD, HR-TEM, ultraviolet vis

spectroscopy, and FTI spectroscopy. In diabetic treated rats, hemoglobin, fasting

blood glucose levels, serum insulin, and glycosylated hemoglobin levels with AuNPs

were significantly decrease when comparison takes place with the control group. Gold

NPs might extensively progress the insulin fighting and glucose level in diabetic rats

which revealed by the serum insulin and blood glucose levels. A gold nanoparticle

moreover shows a diminution into anti inflammation in diabetic rats including tumor

nacrosis factor–α, high sensitive CRP and IL–6. The data showed that gold

nanoparticles produced by using S. swartzii bring to bear antidiabetic result,

consequently, liver, progress pancreas, and kidney spoil reason by diabetic alloxan

induced rats (Dhas et al., 2016).

Recently in antidiabetic revisions, due to unique properties of nano materials

such as biocompatibility, minute size, and capability to break through cell membranes

in favor of transportation drugs are being used. Herein, Gymnema sylvestre R.

antidiabetic potent plant was used for gold nanoparticles production on Wistar albino

rats has been calculated. Microscopic and spectroscopic analyses are used for the

formation of nanoparticles and to study their morphology respectively. Gold NPs has

exposed a noteworthy decline in anti-inflammatory as well as blood glucose stage on

diabetic rats by approximation the interleukin-6, serum levels of TNF-α, and high-

sensitive CRP (Karthick et al., 2014).

Recently, green biosynthesis and an entire description of Au and core shell

Ag/Au NPs revolutionary work have been available. In this, in streptozotocin-induced

diabetic rats, these NPs are evaluated for their antidiabetic actions. Results revealed

that diabetic rats take care of Au and core shell Ag/Au nanoparticles re establish

average glucose levels. In fastidious, as to compare to the control of normal rats, Au

and core shell Ag/Au NPs were bring into being to significantly encourage a

diminution in blood glucose and re establish both the glucokinase action and high


39

serum insulin intensity. An anti inflammatory consequences in diabetic rats assessed

using inflammatory markers interleukin-α and C- reactive proteins were obtained by

disclosure of results, by the effective role of Ag/Au nanoparticles in falling the lipid

profile. By the histopathology of diabetic rats signifies alteration in a lot of cells in the

order of the pyknotic along with apoptotic nuclei, inflammatory cells, and central

veins were checked. The kidney of diabetic rats come into sight with vacuolation and

some tubules pyknotic nuclei, meanwhile, the liver of diabetic rats treated with silver

and gold nanoparticles demonstrated normal hepatic cells with merely a few

hepatocytes necrosis. In diabetic rats, Ag/Au NPs re-established the amplified

quantity of caspase 3 stained cells in the kidney and liver tissues. The results declared

that in diabetic rats, their condition was observed to improve due to Ag@AuNPs by

restraining, suppressing oxidative stress, extended inflammation, and uplifting the

antioxidant protection organization. They have subsequently evoked the probable

effect of gold nanoparticles as a cost effective remedial treatment in diabetic

management also its hurdles (Shaheen et al., 2016).

In the present investigation, from Cassia auriculata lively biocomponent,

propanoic acid 2-(3-acetoxy-4, 4, 14-trimethylandrost-8-en-17-yl) (PAT) was isolated

and for functionalization production of Au NPs were deliberated in the feature. The

rapid formation of stable gold nanoparticle s was achieved by the reaction of

propanoic acid (2, 3-acetoxy-4, 4, 14-trimethylandrost-8-en-17-yl) along with

aqueous HAuCl4. SEM, EDX, FTIR supported by gas chromatography mass

spectroscopy, and UV–Visible spectroscopy XRD, and TEM were used for

confirmation of gold nanoparticles. Au nanoparticles frequently were sphere-shaped

in shape, size range between 12–41 nm and monodisperse. The administration of

AuNPs synthesized using propanoic acid was given to diabetic male albino rats by

induction of alloxan (150 mg/kg body weight) with different concentrations (1.0,

0.75, 0.50, 25, and mg/kg body weight) for 4 weeks. At a prescribed amount of 0.5

mg/kg b.w in experimental rats treated with Au nanoparticles were considerably (p <

0.001) reduced cholesterol, plasma glucose level, and triglyceride along with

extensively increased plasma insulin levels. The newly synthesize green gold

nanoparticles exhibited remarkable inhibitory action against protein tyrosine

phosphatase 1B (Venkatachalam et al., 2013).


40

The silver nanoparticles have a very vital role in modern therapies of diabetes

and medical science. Malapermal et al. used the aqueous extracts from Ocimum

sanctum L., Ocimum basilicum L. and combination of both to prepare silver

nanoparticles stable and size in range from 3 to 25 nm. The silver nitrate was reduced

using different concentrations of extracts which led to the quick synthesis of silver

nanoparticles. In contrast to the conventional chemical procedures, the rate of

synthesis of silver nanoparticles using extracts was considerably high. The

characterization was carried out using dynamic light scattering, TEM, SEM, EDX,

UV–Visible spectroscopy, and FTIR maintained by gas chromatography mass

spectroscopy (GC–MS) that was used to recognize the kind of capping agents. The

rate of carbohydrate digestion was retarded by the prohibition of α-glucosidase and α-

amylase enzymes. Therefore, these are provided as a less evasive strategy and an

alternative to dropping postprandial hyperglycemia in diabetic patients. The silver

nanoparticles produced from O. basilicum and O. sanctum were demonstrated an

inhibitory outcomes towards the enzyme model Bacillus stearothermophilus a-

glucosidase at 79.74 ± 9.51 % and 89.31 ± 5.32%, correspondingly. They were shown

high biocatalytic prospective compared to their particular control and rudimentary

extracts. Besides, the demand for finding the treatments of dual diabetes is required

due to the emerging rate of infections in diabetic patients. As a result, the silver

nanoparticles produced from bioderived compounds displayed antimicrobial action in

opposition to a different bacterial variety including, E. coli, B. subtilis, P. aeruginosa,

S. aureus, and Salmonella species (Malapermal et al., 2017).

GCU Lahore MATERIALS AND METHOD

41

CHAPTER-3

MATERIALS AND METHOD

3.1 Materials

Gold chloride trihydrate, doxorubicin, doxycycline, hydrogen peroxide,

dopamine, N-hydroxysuccinimide (NHS), 16-mercapto-hexadecanoic acid (16-

MHA), 11-mercapto-1-undecanol, ethanolamine hydrochloride, sodium chloride,

Silver nitrate, Minocycline and Alloxan monohydrate were purchased from Sigma

Aldrich. N-ethyl-N‟-(3-dimethylaminopropyl)-carbodiimide (EDC) and sodium

hydroxide were purchased from Fluka chemicals. Protease Activated Receptor

(PAR1) was purchased from Cedarlane. All remaining chemicals were of analytical

grade and used without further purification.

3.2 Synthesis of Doxycycline derived Gold Nanoparticles (doxy-

AuNPs)

To examine the potential of doxy-AuNPs as drug carrier and biocatalyst,

doxy-AuNPs were synthesized using wet chemical reduction method. Synthesis was

carried out using doxycycline as reducing and capping agent. 2 mL of 0.4 mM gold

chloride was taken in a conical flask. To this, 5 mL of deionized water and 1 mL of

0.8 mM doxycycline were added. This was then followed by the addition of 2 mL of

0.01 M sodium hydroxide with continuous stirring for 3 minutes. A ruby red color

was observed within 3 minutes, corresponding to the plasmon resonance for

doxycycline capped gold nanoparticles (doxy-AuNPs). The synthesis of doxy-AuNPs

was monitored in wavelength range 300-800 nm using double beam UV-vis

spectrophotometer (Model Cary 100 Bio).

Furthermore, to fabricate AuNPs base SPR biosensor, procedure for synthesis

of doxy-AuNPs was little modified. The aqueous solutions of 4 mL gold chloride (0.4

mM) and 2 mL doxycycline (0.8 mM) were added in a conical flask. To the mixture,

3 mL sodium hydroxide (0.01 M) was added. Mixture was continuously stirred for 3

minutes. After 3 minutes, a ruby red color was observed. UV-vis spectrophotometer

(Model Cary 100 Bio) was used to monitor the synthesis of doxy-AuNPs in

wavelength range 300-800 nm.


42

3.3 Synthesis of Minocycline Derived Silver Nanoparticles

(Mino/AgNPs)

Synthesis of AgNPs was carried out using minocycline as reducing and

stabilizing agent. 2 mL silver nitrate (0.8 mM) and 2 mL (0.8 mM) minocycline

solution were taken in a conical flask. To the mixture, sodium hydroxide was added

to accelerate the synthesis process. The resulting mixture was continuously stirred for

4 minute. UV-vis spectrophotometer (Model Cary 100 Bio) was used to monitor the

synthesis of Mino/AgNPs in wavelength range 300-800 nm.

3.4 Characterization of Nanoparticles

3.4.1 XRD studies

X-ray diffraction (XRD) is an instrumental technique used to examine the

crystalline nature of the sample under investigation. The working principle of XRD

involves the irradiation of sample with X-rays. As a consequence of this collision, the

scattering angle and intensities of scattered X-rays that leaves the sample are

measured. In present work, the XRD analysis was carried out to confirm the

crystalline nature of as-synthesized doxy-AuNPs and Mino/AgNPs. To prepare the

samples for XRD studies, colloidal solutions of nanoparticles were centrifuged three

times at 10,000 rpm for 30 min and washed with deionized water each time. Then, the

pellets obtained after centrifugation were left overnight to dry under fume hood.

Powder XRD was performed in the 2θ region, from 0 ̊ to 80 ̊ at scanning rate of

0.02 ̊ per minute utilizing Cu Kα1 radiation with a wavelength (λ) of 1.5406 A˚ at a

tube voltage of 40 kV and a tube current of 40 mA.

3.4.2 TEM Studies

The working principle of transmission electron microscope (TEM) is same as

that of light microscope. However TEM uses high energy electron beam instead of

ordinary light. A beam of electron emitted from heated filament is accelerated under

the vacuum and transmitted through the specimen. The transmitted beam of electron

is focused with the help of objective lens and the image is recorded on fluorescent

screen.

The size and morphology of as-synthesized doxy-AuNPs and Mino/AgNPs

was determined via TEM using a FEI Tecnai t12 running at 80 kV with final emission

around 10 µA. Micrographs were taken using an 2k AMT camera. For each


43

micrograph, samples were prepared by dropping 10 µL of nanoparticles solution on a

copper grid coated with carbon and formvar film and left overnight to dry and then

observed with TEM.

3.4.3 DLS Studies

Particle Size of doxy-AuNPs was also examined by dynamic light scattering

using BT-90 nano laser light particle size analyzer. Surface charge of doxy-AuNPs

was examined with zeta potential measurements using zeta sizer (Malvern

Instruments) at 25 °C.

3.4.4 FT-IR Studies

FT-IR spectroscopy is technique which is used to analyze the interaction

between matter and IR radiations. It provides a way to investigate the presence of

certain functional groups in a molecule. It records broad band near infrared to far

infrared spectra. Upon irradiation of sample with IR radiations, the transmittance or

reflectance of light is measured that allows the structural analysis of a compound. In

present work, the role of doxycycline and minocycline in synthesis of AuNPs and

AgNPs respectively was examined through FT-IR analysis. To prepare the samples

for FT-IR, colloidal nanoparticles solutions were centrifuged three times at 10000 rpm

for 30 min and washed each time with deionized water. The pellets obtained after

centrifugation were left overnight to dry under fume hood. FT-IR analysis was carried

out with Bruker Alpha.

3.5 Application of doxy-AuNPs as drug Carrier and biocatalyst

3.5.1 Loading of doxorubicin hydrochloride onto gold nanoparticles

Firstly, 10% m/v doxorubicin (DOX) solution was prepared in water. Then, 3

mL of DOX was taken from this stock solution. To this were added 2 mL of deionized

water and 1 mL of doxy-AuNPs. This mixture was incubated at room temperature for

24 hours. The absorbance and concentration behavior of DOX was monitored after

the regular intervals of time between 1 hour and 24 hours in wavelength range 400-

800 nm using double beam UV-vis spectrophotometer. Then, this mixture of DOX

and doxy-AuNPs was centrifuged for 15 min at 8000 rpm. After centrifugation, the

pellet obtained was recovered from the supernatant and absorption spectrum of

supernatant solution was recorded. Similarly, the absorption spectrum of a control

sample (pure doxorubicin) was also recorded. For this, 3 mL of DOX was taken from


44

the stock solution. To this was added 3 mL of deionized water and the absorption

spectrum was recorded.

Furthermore, DOX loaded doxy-AuNPs were also examined by TEM to

observe potential morphological changes induced by DOX. For this purpose, the

incubated mixture of DOX loaded doxy-AuNPs was first centrifuged and the residue

obtained was spread over the copper grid coated with carbon and formvar film.

Sample was left to dry overnight and then observed with TEM FEI Tecnai t12 running

at 80 kV with final emission around 10 µA. Micrographs were taken using an 2k

AMT camera.

3.5.2 Drug Loading Efficiency

To determine the drug loading efficiency, an indirect method was applied in

which concentration of DOX in supernatant was measured in wavelength range 400-

800 nm using UV-vis. Concisely, a calibration curve was constructed in the

concentration range 0 to 173 µM. In all experiments of drug loading, unknown

concentration of DOX in supernatants was determined from this calibration curve.

Then, the following equation was applied to calculate the drug loading efficiency.

% Drug Loading efficiency =Amount of DOX added initially −amount of DOX in supernatant

Amount of DOX added initially × 100

3.5.3 Drug release study

In vitro release of doxorubicin was examined in phosphate buffer at pH 4.30

and 7.34. To measure this, 6 mL of the DOX loaded doxy-AuNPs suspension was

first centrifuged at 8000 rpm for 15 minutes. Then, 40 mL phosphate buffer with pH

4.30 was taken in a conical flask and the pellet obtained after centrifugation was

transferred to that buffer. The drug release study was carried out at 37 C with

continuous stirring at 100 rpm. In order to observe the drug release kinetics at pre-

determined time intervals, 2 mL aliquots of sample solution were removed and

replaced by equal volume of fresh phosphate buffer. This was done to maintain the

volume of the dissolution experiment constant. DOX release contents were then

quantified from withdrawn buffers by means of caliberation line and following the

absorption of DOX at 484 nm in UV-Vis spectra. A control for the drug release study

was performed under the same conditions but at pH 7.34. The experiments were

performed in triplicate for each of the samples.


45

3.5.4 Catalytic Oxidation of Dopamine

Biocatalytic response of doxy-AuNPs was explored through oxidation of

dopamine. A 1 mL aliquot of 1.5 mM dopamine was taken in cuvette. To this was

added 1 mL of 0.1 M H2O2 followed by addition of 0.5 mL of doxy-AuNPs. The

absorbance and concentration behavior of dopamine was monitored for 5 minutes in

wavelength range 200-650 nm using UV-vis spectroscopy.

3.6 Application of Gold Nanoparticles in Fabrication of SPR

Biosensor

3.6.1 Preparation of SAM modified Gold Coated Prism

The dove prism was coated with 1 nm Cr and 45 nm Au (ESPI metals)

utilizing a Cressington 308R sputter coater. Afterwards, the gold coated dove prism

was dipped into solution of 0.1 mM 16-mercaptohexadecanoic acid and 0.9 mM 11-

mercapto-1-undecanol and left overnight for the formation of self assembled

monolayer (SAM) of 16-mercaptohexadecanoic acid and 11-mercapto-1-undecanol.

After that, the SAM modified gold coated prism was thoroughly rinsed 3 times with

ethanol and purified water and dried under nitrogen.

3.6.2 Fabrication of the SPR sensor

SPR measurements were performed on a custom-built SPR instrument (Zhao

et al., 2015). A dove prism with a gold film (1 nm Cr and 45 nm Au) was

immobilized with a self-assembled monolayer (SAM) of 16-mercaptohexadecanoic

acid and 11-mercapto-1-undecanol. The SAM has the ability to bind receptor

(Protease Activated Receptor1) PAR1 and is capable for resisting nonspecific

adsorption on sensing surface for quantification of biomolecule (Bolduc et al., 2011).

This modified gold-coated prism with SAM was placed into the chip holder of the

SPR setup. Then, a disposable PDMS flow cell was mounted over the prism and

tighten with a clamp. The reference and sample solutions were injected at different

sensing areas via separate injection ports. In PDMS flow cell, there are two separate

flow channels, one for sample solution and other for reference solution. For the

sample solution, the flow channel is S-shaped and comprises three different sensing

areas, providing analysis of sample in triplicate. The total volume of the channel for

sample analysis is 16 μL. For the reference solution, the flow channel covers the

fourth sensing area with a volume 5 μL. The whole SPR system was connected to


46

custom lab view software via a laptop. Data acquired by SPR system was controlled

by software and minimum finding algorithm based on a second order polynomial fit

was used to integrate SPR signal at each time point. The sensorgrams for all four

sensing areas were recorded in real time.

Figure 3 custom-built SPR instrument

3.6.3 Immobilization of receptor on sensor surface

In all SPR experiments, the SAM modified gold coated prism was inserted in

the SPR instrument. First, Milli-Q water was added into the flow cell and left for 15-

20 min for stabilization. Afterwards, the sensing surface was activated with

EDC/NHS and left for 5 min until the resonant wavelength was constant. Then, the

sensing surface was rinsed with PBS, followed by the injection of the receptor

solution of Protease Activated Receptor-1 (PAR-1) at 5 μg mL-1

and reacted for 15

min. The receptor PAR1 was covalently attached to the SAM through activated

carboxylic acid group from EDC/NHS. Subsequently, non-specific binding sites on

the sensing surfaces were blocked by injecting 1 M ethanolamine hydrochloride (pH

8.0) for 10 min followed by rinsing with PBS to remove non-covalently attached

receptor PAR1. This procedure was repeated for all SPR experiments.

3.6.4 Electrolytic Stability of doxy-AuNPs

To examine the electrolytic stability of doxy-AuNPs, different concentrations

of NaCl (from 50 to 1000 mM) were added in doxy-AuNPs colloidal solutions and

their UV spectra were recorded (Model Cary 100 Bio) in the wavelength range 300-

800 nm. To select the suitable electrolytic condition of doxy-AuNPs which can give


47

larger SPR response, doxy-AuNPs containing varying concentrations of NaCl (from

50 to 1000 mM) were injected in SPR followed by rinsing each time before and after

the each injection with same concentration of NaCl in water to record the refractive

index baseline. A control experiment of doxy-AuNPs without NaCl was also

conducted and sensorgrams were recorded in real time.

3.6.5 Sequential Analysis for determination of concentration of

doxycycline

To examine the effect of doxycycline on the growth of synthesized doxy-

AuNPs, varying concentrations of doxycycline (from 1 nM to 1 mM) were added in

suspensions of doxy-AuNPs and their UV spectra were recorded in the wavelength

range of 300-800 nm. Furthermore, to carry out detection of doxycycline with the

SPR system, varying concentrations of doxycycline (0.1 nM to 10 μM) were added in

the colloidal suspension of doxy-AuNPs and injected sequentially in flow cell at room

temperature for 30 min followed by rinsing each time before and after each injection

with 100 mM NaCl in water for 5 min to record baseline. Interaction between

biological receptor PAR1 and doxy-AuNPs was measured as binding shift by SPR

biosensor in real time. Control experiments were performed by injecting doxy-AuNPs

(containing optimized NaCl concentration) without adding free doxycycline in SPR

system. Origin software was used to process the data utilizing the minimum

wavelength finding algorithm. From the sensorgram, last 50 data points of 100 mM

NaCl steps before and after the doxycycline sensing steps were used to calculate the

binding shift from sensorgram. The logarithm of doxy concentration was plotted

against the binding shift to find the correlation between concentration of doxycycline

and binding shift. Triplicate measurements were carried for all conditions.

Reproducibility was obtained from the triplicate SPR measurments of 100 nM

doxyxycline and measured as a coefficient of variation resulting from the ratio of the

standard deviation and the mean response, in percentage.

3.7 Application of Silver Nanoparticles as Potential Antidiabetic

Agent

3.7.1 Antioxidant study – DPPH assay

The antioxidant potential of Mino, Mino/AgNPs and ascorbic acid were

evaluated through DPPH free radical scavenging assay. Briefly, 100 µL of each Mino,


48

Mino/AgNPs and Ascorbic acid at various concentrations (10, 25, 50 and 100 µg/mL)

were added to the 2.9 ml of 0.1 mM DPPH solution in methanol. The resulting

mixtures were kept in dark for 30 minutes. The DPPH solution (2.9 mL DPPH and

100 µL methanol) was used as control solution. The absorbance of control and

reaction mixtures was measured at 517 nm using UV-vis spectrophotometer

(Shimadzu UV-1700). The DPPH scavenging activity was expressed as percentage

and was calculated by following formula:

𝐷𝑃𝑃𝐻 𝑆𝑐𝑎𝑣𝑒𝑛𝑔𝑖𝑛𝑔 𝐸𝑓𝑓𝑒𝑐𝑡 %

=𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝐶𝑜𝑛𝑡𝑟𝑜𝑙 – 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑆𝑎𝑚𝑝𝑙𝑒

𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝐶𝑜𝑛𝑡𝑟𝑜𝑙 × 100

3.7.2 Experimental Animals

Thirty two albino mice were received from the University of Veterinary and

Animal Sciences . Before any kind of experimentation , the mice were left in the

animal house for two weeks at 25 C̊ with frequent access to water and food. This was

done to acclimatize the mice with new environment. The weight of the body of all

mice was measured before and after the treatment. All the experiments performed

during the in vivo studies were approved by the Bioethical Committee of Government

College University Lahore, Pakistan.

3.7.3 Induction of Diabetes

Alloxan monohydrate is a toxic glucose analog that affects the -cells of the

Pancreas and is frequently used in animal model to induce diabetes. The

intraperitoneal injection of Alloxan monohydrate (100 mg/Kg bodyweight) resulted in

the induction of diabetes to the overnight fasted mice. Subsequently, to protect the

mice from hypoglycaemic effects, they were feeded with glucose solution (10%) for

24 hours along with the normal food. To confirm the induction of diabetes, the fasting

blood sugar level of mice was measured regularly with 3 days interval, up to 14 days.

Those mice were considered diabetic and were selected for further experimentation

that carried the fasting blood sugar more than 250 mg/dL.

3.7.4 Experimental Design

To conduct the antidiabetic studies, four groups were made with eight mice in

each group. Group-I; normal control, Group-II; diabetic left untreated, Group-III;

diabetic treated with drug glibenclamide (5 mg/Kg body weight), Group-IV; diabetic


49

treated with Mino/AgNPs (5 mg/Kg body weight). The mice were treated regularly

for 28 days with Glibenclamide and Mino/AgNPs via oral administration.

3.7.5 Collection of sample

After the successful completion of twenty eight days treatment, the mice were

fasted for 12 hours and subsequently, they were given anaesthesia with chloroform.

The mice were then dissected sacrificially and blood samples were obtained by heart

puncture in three distinct tubes. The kidney, pancreas and liver were dissected

followed by washing with phosphate buffer saline (to clear debris) and placed in a

10% formalin solution for further processing.

3.7.6 Biochemical Assay

Blood sugar level and hemoglobin were measured using commercially

available kits. Serum lipid profiles such as triglycerides and total cholesterol were

estimated using respective kits from BD, Bio-sciences, USA. Serum glutamic

oxaloacetic transaminase (SGOT) and serum glutamate pyruvate transaminase

(SGPT) were determined using a standard International federation of clinical

chemistry (IFCC) kinetic method (BD, Biosciences, USA).

3.7.7 Histopathological Studies

For overnight fixation, Kidney, Liver and Pancreas were added in the 10%

formalin solution. Afterward, the dehydration of slices (3-4 mm) of liver, kidney and

pancreas tissues was performed using ascending grades of alcohol , then cleared

(alcohol was extracted ) with xylene and embedded in paraffin wax (58– 60 ̊ C).

Blocks were made and sectioned of 5 mm thickness with a microtome. The staining of

tissue sections was done with hematoxylin and eosin staining (Fischer et al., 2008).

The light microscope was used for the examination of prepared slides.

3.7.8 Statistical Analysis

The statistical analysis of the data was performed through ANOVA using

Statistix 10 software and the data were presented as means ± standard deviation. The

least significant difference (LSD) test was applied for multiple comparisons among

the mean values. The differences were considered statistically significant at p≤0.05.

The figures were plotted using Origin Pro. 8 software.

GCU Lahore RESULTS AND DISCUSSION

50

Chapter No. 4

RESULTS AND DISCUSSION

Following four major biomedical applications of gold and silver NPs will be discussed in this

chapter

Gold Nanoparticles as drug carrier

Gold NPs as an artificial enzyme (biocatalyst)

Gold nanoparticles based SPR biosensor

Silver Nanoparticles as a potential antidiabetic agent

4.1 Gold Nanoparticles as drug carrier

The chemotherapy treatment of cancer induces side effects in the body.

Selective release of drug in the tumor environment can substantially reduce the side

effects of chemotherapeutic drugs. In particular, gold nanoparticles based approaches

for selective delivery of drugs have received considerable attention since last few

years. In the present work, doxycycline modified gold nanoparticles (doxy-AuNPs)

were synthesized via wet chemical reduction method (Choi et al., 2016). The as-

synthesized doxy-AuNPs were extensively characterized and successfully applied as

drug carrier for pH responsive selective release of drug.


51

4.1.1 Synthesis of doxy-AuNPs

A wet chemical reduction method was used to synthesize the doxy-AuNPs.

The synthesis of doxy-AuNPs was carried out using doxycycline as reducing and

capping agent. Immediately after addition of doxycycline and sodium hydroxide in

gold chloride solution, AuNPs were synthesized in 3 minutes with characteristic ruby

red color (Figure 4 Scheme), similar to another synthesis previously reported for L-

methionine (Raza et al., 2017).

Figure 4 Scheme representing the synthesis of doxy-AuNPs following after the loading and release of

DOX from doxy-AuNPs

The synthesis of doxy-AuNPs was pH responsive. The role of sodium

hydroxide was important regarding the synthesis of AuNPs, as in its absence; there

was no synthesis of AuNPs even after several hours. Whereas with addition of NaOH,

the color of solution rapidly turned to ruby red in just 3 minutes. Synthesis of doxy-

AuNPs was confirmed using UV-vis spectrophotometry. A sharp surface plasmon

resonance was observed at 520 nm (Figure 5) which is a characteristic LSPR in

spherical gold nanoparticles thus confirming the synthesis of doxy-AuNPs (J. J.

Zhang et al., 2009).


52

400 500 600 700 8000.0

0.1

0.2

0.3

0.4

0.5

0.6

Ab

so

rba

nce

Wavelength (nm)

Figure 5 UV-vis spectrum of doxy-AuNPs

4.1.2 Effect of pH on Synthesis of doxy-AuNPs

pH influences the synthesis of doxy-AuNPs, and thus, the size and stability of

the NPs. UV-vis spectroscopy clearly demonstrated that the synthesis of doxy-AuNPs

occurred only at basic pHs. This is shown from the emergence of a strong absorption

bands from pH 8 to pH 11 (Figure 6), characteristic of the localized surface plasmon

resonance (LSPR) in spherical AuNP. We correlated the pKa values of doxycycline

(pKa1 3.02 ± 0.3; pKa2 7.97 ± 0.15; pKa3 9.15 ± 0.3) (AC, LK, & HRN, 2014) to the

synthesis of AuNPs occurring only for pHs above 8. Doxycycline is zwitterionic in

acidic and slightly basic pHs between 3 and 8, thus cannot stabilize the AuNP

electrostatically. Li et al. has also reported that doxycycline can rapidly induce

aggregation of AuNPs in acidic medium (J. Li et al., 2014). This phenomenon was

also observed by Liu et al. for AuNP synthesize with glutamate (Y. Liu et al., 2015),

whereas in alkaline medium, they observed a strong absorption band and TEM images

of AuNPs. It is due to the fact that in alkaline medium, deprotonation of doxycycline

occurs and consequently, its surface gets anionic and hence enables to protect the


53

AuNPs colloidial suspension (Shou et al., 2011). The negative charge also contribute

to increase the reducing strength of doxycycline for the reduction of the Au ions. pH-

dependent synthesis of doxy-AuNPs is therefore expected to be based on the ionic

state of doxycycline on surface of AuNPs (J. F. Li, Huang, & Wu, 2017). The most

blue shifted and sharpest LSPR resonance was obtained at pH 11. This pH was

therefore selected for all further studies.

400 500 600 700 8000.0

0.1

0.2

0.3

0.4

0.5

0.6

Absorb

ance

Wavelength (nm)

pH 3

pH 5

pH 6

pH 7

pH 8

pH 9

pH 10

pH 11

pH = 11

Figure 6 UV-vis spectra with pH effect on synthesis of doxy-AuNPs. All spectra were acquired after 15

minutes of reaction

.


54

4.1.3 Stability of doxy-AuNPs

The stability of doxy-AuNPs was monitored for 2 months from the plasmon

wavelength (λmax) as aggregation causes large red shifts of their spectra. The AuNPs

were stored in a closed container in the presence of the synthesis reagents and in

absence of light for the duration of the experiment. The rate of synthesis was much

faster during the first 24 hours which resulted in rapid growth of particles, as seen

from the red shift from 505 nm to 520 nm of the λmax during first the 24 hours of

formation of doxy-AuNPs (Figure 7a). The doxy-AuNPs were then stable as

observed from the constant plasmon resonance at around 525 nm. The absorption

slowly increased with a very slight red shift until two weeks following the synthesis.

Finally, after two months the λmax was at 524 nm with a slight decrease in intensity. It

indicates the good stability of newly synthesized doxy-AuNPs. The stability of the

doxy-AuNPs was also observed from the small change in absorbance maximum of

doxy-AuNPs with time (Figure 7b). Hence, the doxy-AuNPs showed excellent

stability to colloidal aggregation.

Figure 7 (a) UV-vis spectra indicating the stability of doxy-AuNPs (b) Absorbance maximum vs time

spectra of doxy-AuNPs


55

4.1.4 TEM of doxy-AuNPs

TEM images of synthesized doxy-AuNPs were then acquired for further

supporting the synthesis of doxy-AuNPs. Homogenously distributed spherical gold

nanoparticles were obtained with the doxycycline synthesis method with average

particle size 5 nm (Figure 8). Doxy-AuNPs were homogeneous in size and remained

unaggregated for extensive periods of time.

Figure 8 TEM images and Histogram of doxy-AuNPs (a & b) (Diluted sample) (c & d) (Concentrated

sample) (acquired at 80 kV, exposure of 1200 ms and magnification of 150,000X. The scale bar

represents 50 nm)


56

4.1.5 Zeta potentials of doxy-AuNPs

The particle size of doxy-AuNPs measured by DLS method was at around 6

nm which is in good agreement with particle size measured by TEM. Zeta potentials

of the synthesized doxy-AuNPs was found to be -28.5 mV (Figure 9) which shows

good stability of synthesized doxy-AuNPs.

Figure 9 Zeta potentials of doxy-AuNPs


57

4.1.6 FT-IR of doxy-AuNPs

The interaction of doxycycline in synthesis of AuNPs was studied through FT-

IR (Figure 10). In case of pure doxycycline, the absorption band was observed in the

range of 3400-3200 cm-1

for O-H and N-H bonds but this band completely

disappeared in case of doxy-AuNPs. This observation hints about the involvement of

these moieties in the modification process. Likewise the absorption bands in the range

of 1700-1500 cm-1

were attributed to α,β-unsaturated carbonyl of amide and ketone

functionalities of doxycycline. These signals moved to higher values in case of doxy-

AuNPs, although the intensity of signals is low. The shifting to higher values justified

the involvement of hydroxyl group resulting into disappearance of C=C conjugation

(Siddiqui et al., 2020).

Figure 10 FT-IR Spectra of Doxycycline (Red) and doxycycline modified Gold Nanoparticles (Black)


58

4.1.7 X-ray Diffraction of doxy-AuNPs

The crystalline nature of doxy-AuNPs was examined through XRD analysis.

Strong diffraction peaks at 2θ values of 38.37o, 45.41

o, 65.01

o and 77.55

o (Figure

11a) and 38.5, 46.1, 64.5° and 78.6° (Figure 11b) corresponds to the {1 1 1}, {2 0 0},

{2 2 0} and {3 1 1} signatures of metallic AuNPs. Our results of XRD of doxy-

AuNPs are in accordance with other types of AuNPs (Divakaran, Lakkakula, Thakur,

Kumawat, & Srivastava, 2019).

Figure 11 (a) X-ray diffraction of doxy-AuNPs (Diluted sample) (b) X-ray diffraction of doxy-AuNPs

(Concentrated sample)

4.1.8 Drug loading

Doxycycline-capped AuNPs were successfully synthesized for their

application as drug carrier. In a drug delivery system, drug adsorbed to a carrier

through non-covalent interaction is an effective methodology to restrict any potential

issues related to prodrug strategy. We have selected the anticancer drug DOX for

loading on doxy-AuNPs, as this is a common model with high benefit for treatments.

Loading of DOX was monitored from the absorption spectra of remaining DOX

concentration in the supernatant at different reaction times (Figure 12a). The

absorbance maximum (Native DOX at 484 nm and supernatants at 495 nm) as a


59

function of time was plotted to indicate the loading behavior of DOX with time

(Figure 12b).

Figure 12 (a) UV-vis spectra with absorbance of DOX in supernatant at different reaction intervals (b)

Absorbance vs time spectra for DOX absorbance in supernatant (Native DOX at 484 nm and

supernatants at 495 nm)

The change in the absorption wavelength is due to the interaction of DOX

with the AuNP. A gradual decrease in DOX absorbance was observed due to its

absorption on surface of doxy-AuNPs. A relatively faster decrease in absorbance was

observed for first two hours which slowed down thereafter. This was likely due to the

greater surface area available initially and greater adsorption capacity of doxy-

AuNPs. Eventually after 24 hours, 70% of the DOX available in solution was loaded

on the surface of doxy-AuNPs which compared advantageously to other reports in the

literature (Table 1). This loading corresponded to about an equivalent weight of DOX

(33 mg – measured with UV-Vis) to AuNP (27 mg – measured with atomic

absorption), demonstrating the loading capacity of AuNP.

The exact mechanism of interaction between DOX and doxy-AuNPs is

difficult to predict. However, we anticipate that different binding forces may be

responsible for adsorption of DOX on doxy-AuNPs. First, electrostatic interaction

between negative charges on surface of doxy-AuNPs and positively charged drug


60

molecules can contribute to the adsorption process. This kind of loading mechanism

was also reported by Naz et al. They reported the loading of doxorubicin and

donorubicin on AgNPs. According to them, loading of anticancer drug on AgNPs was

the result of electrostatic attraction between negative charged bio-moieties on surface

of AgNPs and positively charged drug molecule (Naz et al., 2017). Secondly at pH

above 7, it is quite likely that DOX is attached to doxy-AuNPs via H-bond interaction.

The N,N-dimethly amino group of doxycycline may involve in making H-bonding

with hydroxyl group of DOX present at proximal position of amino group on the side

ring.34,

(Clara-rahola et al., 2018).


61

Table 3 Comparative table of drug loading efficiencies and and pH sensitive release

Source

Type of drug

conjugate with

NPs

Drug

Loading

Efficiency%

Drug Release under

physiological conditions

Drug Release in

acidic environment

pH Time

(hrs) % Release pH

Time

(hrs)

%

Release

(Taghdisi

et al.,

2016)

Daunorubicin

loaded on

polyvalent

aptamers

modified

AuNPs

60 7.4 72 21 5.5 72 78

(S. Son

et al.,

2018)

Doxorubicin

loaded on

AuNPs

transformable

hybrid

nanoparticles

(DOX-TNPs)

75 7.4 48 Less than

40 6.5 48

More

than 90

(Golshan

et al.,

2017)

Doxorubicin

loaded on

propylene

modified

AuNPs

36.4 7.4 7 83.1 5.3 4 97.2

(Khutale

& Casey,

2017a)

Doxorubicin

loaded on Au-

PEG-PAMAM

48.75 7.4 96 Negligible

release 4 96 50

This

work

Doxorubicin

loaded on

doxy-AuNPs

70 7.34 24 5 4.30 24 60


62

4.1.9 TEM of DOX load doxy-AuNPs

To provide some level of understanding, morphological changes of DOX

loaded doxy-AuNPs were examined by TEM. DOX molecules showed tendency to

give rise to more complex network of doxy-AuNPs after loading on surface of doxy-

AuNPs (Figure 13). These kind of morphological changes in AuNPs were also

observed by Fontana et al. while loading of dexamethasone on AuNPs (Fontana et al.,

2013). Therefore, DOX molecules created a network of doxy-AuNPs that remains in

suspension in aqueous solutions.

Figure 13 TEM of DOX loaded doxy-AuNPs

4.1.10 pH Responsive Drug Release kinetics of doxy-AuNPs

To determine the suitability of doxy-AuNPs as anticancer drug carrier, in vitro

release of DOX was examined in PBS at 37 C under physiological conditions (pH

7.34) and an acidic conditions (pH 4.30). Release of DOX from doxy-AuNPs was pH

dependent (Figure 14). After 24 hours, cumulative release of drug was 60% at pH

4.30 while no significant release of DOX (about 5%) was observed at pH 7.34 in

otherwise identical conditions. The higher percentage of drug release in acidic

medium can be assigned to the protonation of doxycycline, consequently increasing


63

the repulsive forces with DOX. According to the pKa value of DOX (pKa1 7.34

(phenol); pKa2 8.46 (amine); pKa3 9.46 (estimated) (Raval, 2012), it can easily get

protonated and consequently repulsive forces between protonated drug and protonated

doxy-AuNPs results in the fast release of DOX from doxy-AuNPs whereas at pH

7.34, doxycycline gets deprotonated and electrostatic interaction between protonated

drug and negatively charged bio-moieties on surface of doxy-AuNPs restrict the

release of DOX from doxy-AuNPs (Karimi et al., 2018).

0 4 8 12 16 20 240

20

40

60

80

100

Dru

g R

ele

ase

%

Time (hours)

pH 4.30

pH 7.34

Figure 14 In vitro release profile of DOX from doxy-AuNPs

A pH responsive DOX release profile is favorable regarding tumor treatment

(Luesakul et al., 2018). It is expected that at normal physiological conditions (pH 7.4),

much of the DOX will remain bound to the surface of doxy-AuNPs for a significant

duration of time. No release should therefore occur when NPs injected stay in the

plasma, consequently having the potential of minimizing the side effects to the normal

tissues. We predict that a rapid release of drug will take place when the DOX loaded

doxy-AuNPs should be accumulated by the tumor cells because the pH of tumor cells

range from 4.0 to 6.0 (Carbone, 2017). As a result, an adequate high concentration of

DOX can be released within a reasonably short period of time when the NPs are


64

accumulated by the tumor cell, so that substantially improving the effectiveness of

targeted cancer therapy. The small size and biocompatibility of these doxy-AuNPs

make them highly attractive drug delivery vehicle. Due to small size, these doxy-

AuNPs drug carrier may effectively accumulate at a tumor cells after systemic

administration due to well documented enhanced permeability and retention (EPR)

effect. Acidic environment of tumor will facilitate the release of drug from doxy-

AuNPs because of electrostatic repulsion between protonated drug and protonated

doxycycline.

4.2 Gold NPs as artificial enzyme (Biocatalyst)

Doxy-AuNPs were utilized as an alternate biocatalyst to replace the need of

enzyme with peroxidase like activity such as prostaglandin H synthase required for

the oxidation of dopamine (Hastings, 1995; Muñoz et al., 2012). The peroxidase like

activity of doxy-AuNPs was explored via dopamine oxidation (peroxidase substrate)

in the presence of H2O2. The concentrations used for this catalysis experiments were

0.137 mM AuNPs (i.e 27 ppm), 1.5 mM dopamine and 0.1 M H2O2. As doxy-AuNPs

oxidizes the dopamine in the presence of H2O2, color of solution changed from

transparent to orange (characteristic color of aminochrome) (He et al., 2017). This

color change can be used as an indicator for colorimetric detection of dopamine.

Furthermore, along with the color change, oxidation of dopamine was also monitored

by observing the change in its absorption band at 280 nm via UV-vis. The absorption

peak at 280 nm corresponding to dopamine started disappearing with appearance of

new absorption peak, with increasing intensity at 484 nm corresponding to

aminochrome (oxidation product of dopamine) (Joanna Breczko et al., 2012; He et

al., 2017) (Figure 15a- curve 3). The absorbance maximum at 484 nm as a function

of time was plotted to indicate the rate of catalytic oxidation of dopamine (Figure

15b). It was observed that H2O2 alone could not oxidize dopamine even after several

hours and solution of dopamine remains transparent. Only absorption peak at 280 nm

corresponding to dopamine, changes a little (Figure 15a- curve-2) which show some

direct interaction of dopamine with H2O2. However, upon the addition of doxy-

AuNPs to the mixture of dopamine and H2O2, color of solution changes from

transparent to reddish orange. This shows the active role of doxy-AuNPs for

colorimetric detection of dopamine. Almost complete oxidation of dopamine was

carried out in the presence of H2O2 and doxy-AuNPs in just 5 mins. It describes the


65

fast response of synthesized doxy-AuNPs as biocatalyst. He et al., Pandey et al. and

Vázquez-González et al. also reported the similar kind of non-enzymatic processes for

oxidation of dopamine using NPs as biocatalyst (with intrinsic peroxidase like

activity) (He et al., 2017; Vázquez-González et al., 2017; Pandey et al., 2017).

Figure 15 (a) [Curve-1 Dopamine, Curve-2 Dopamine with H2O2, Curve 3 Dopamine with H2O2 and

doxy-AuNPs] (b) Absorbance maximum vs time spectra showing the rate of catalytic oxidation of

dopamine

Previously, enzymatic processes have also been used for oxidation of

dopamine. For example Hasting et al. have reported oxidation of dopamine in

presence of H2O2 using prostaglandin H synthase as biocatalyst (Hastings, 1995).

However, according to his report, enzyme denatured after few minutes of adding into

solution mixture and afterwards, reaction rate cannot be increased even with more

addition of dopamine or H2O2 whereas in present work gold nanoparticles used as

artificial enzyme gave a sustained response as biocatalyst. Furthermore our gold

nanoparticles were stable and reusable. We have reported in our previous work that by

using ionic liquid, one can recover gold nanoparticles from reaction mixture (Raza et

al., 2017c) and can reuse these gold nanoparticles as biocatalyst for next batch of

experiment. Lastly, the exact mechanism or process of these small Au nanoparticles

involved in catalytic reactions is ambiguous or difficult to predict. However we


66

anticipate that presence of doxy-AuNPs increases the formation of .OH radicals in

solution which is the typical behavior of peroxidase to catalyze dopamine.

4.3 Gold Nanoparticles based SPR biosensor

4.3.1 Strategy of the Assay

Doxycycline is a broad spectrum drug with its antimicrobial as well as anti

tumor activities (Haddada et al., 2019; T. Sun et al., 2009; K. Son et al., 2009;

Duivenvoorden et al., 2002; Zhong et al., 2017). Protease activated receptor-1

(PAR1) is the receptor protein with which doxycycline binds to inhibit the tumor

progression (Zhong et al., 2017). Therefore, SPR analysis for doxycycline detection

mainly depends upon the interaction of doxy-AuNPs with PAR1 immobilized on

sensor surface. The interaction of doxy-AuNPs with PAR1 resulted in change of

refractive index which consequently gives SPR response in the form of wavelength

shift. In this work, doxy-AuNPs containing NaCl were employed as an amplification

element to enhance the SPR response. Furthermore, addition of free doxycycline in

doxy-AuNPs colloidal solution causes overgrowth of doxy-AuNPs which

consequently further enhances the SPR biosensor response. The concentration of doxy

was correlated with biosensor response.

4.3.2 Electrolytic Stability of doxy-AuNPs

To examine the electrolytic stability of synthesized doxy-AuNPs, different

concentrations of NaCl (from 50 to 1000 mM) were added in doxy-AuNPs colloidal

solutions and their UV spectra were recorded. Doxy-AuNPs showed good electrolytic

stability with slight red shift (from 520 nm to 530 nm) and small decrease in intensity

up to 100 mM NaCl whereas in case of higher concentrations from 250 to 1000 mM

NaCl, LSPR band of doxy-AuNPs further red shifted from 530 to 540 nm with

significant broadening and decrease in intensity (Figure 16). It was likely due to the

refractive index shift of high salt solution and screening of surface charge on the

AuNP. The effect of salt has been previously shown to have an impact on the Debye

length and the interaction of ligand-modified AuNP for a methotrexate SPR sensor

(Bukar et al., 2014). As such, the stability of the doxy-AuNP was essential to carry

the SPR measurements.


67

400 500 600 700 8000.0

0.2

0.4

0.6

0.8

1.0A

bso

rba

nce

Wavelength (nm)

w/o NaCl

with 50 mM NaCl

with 100 mM NaCl

with 250 mM NaCl

with 500 mM NaCl

with 1000 mM NaCl

Figure 16 UV-vis spectra showing the electrolytic stability of doxy-AuNPs in the presence of different

concentrations of NaCl

4.3.3 SPR Analysis for Detection of Doxycycline

4.3.3.1 Optimization of Electrolytic Conditions of doxy-AuNPs

A shorter Debye length and, as a consequence, decreased colloidal stability are

required for the molecular interaction of target analyte to occur on a surface-bound

receptor. The presence of NaCl causes the electrostatic screening of surface charges

by dissolved ions and reduces the Debye length, and resulted in the higher SPR

response reported previously for a methotrexate assay (Bukar et al., 2014). To select

the suitable electrolytic condition of doxy-AuNPs which can give higher SPR

response, doxy-AuNPs without NaCl and with varied concentrations of NaCl (from

50 to 1000 mM) were injected in SPR system. In the absence of NaCl, very low and

undetectable SPR response was observed for doxy-AuNPs, whereas significantly

higher SPR response was observed for doxy-AuNPs in the presence of NaCl (Figure

17). Very low SPR response of doxy-AuNPs is mainly attributed to the small size and

larger Debye length of doxy-AuNPs in absence of salt which makes them unfit to


68

enter deep in the binding pocket of receptors bound to the sensor surface (Bukar et al.,

2014; Mitchell et al., 2005). On the contrary, addition of NaCl to doxy-AuNPs causes

the electrostatic screening of surface charges by dissolved ions and reduces the Debye

length, and thus resulted in the largest SPR response (Bukar et al., 2014). The control

with the same NaCl concentration before injection of the doxy-AuNP led to changes

in SPR shifts much smaller (i.e. 1 nm) than with the doxy-AuNP. Doxy-AuNP with

50 mM NaCl led to a SPR shift of 9.5 nm whereas the doxy-AuNP with 100 mM

NaCl provided a 25 nm SPR shift. Since the doxy-AuNPs containing 100 mM NaCl

generated maximum SPR response signal (Figure 17), these conditions were selected

for all further SPR bioassays for detection of doxycycline.

0 2000 4000 6000 8000 10000-10

0

10

20

30

40

50

60

70

doxy-AuNP

+ 1000 mM NaCl

doxy-AuNP

+ 500 mM NaCl

doxy-AuNP

+ 250 mM NaCl

doxy-AuNP

+ 100 mM NaCl

Time (sec)

doxy-AuNP

+ 50 mM NaCl

EDC/

NHS

PAR-1

Ethanolamine

Hydrochloride

doxy-AuNP

Bin

din

g S

hif

t (n

m)

Figure 17 Effect of sodium chloride (NaCl) concentration on the SPR response of doxy-AuNPs

(The SPR response was measured in the Kretschmann configuration and the binding shift refers to the

propagating plasmon of the gold film).


69

4.3.3.2 SPR Bioassay for Doxycycline Detection

Sequential SPR analysis was performed for the detection of doxycycline

(analyte). As the Au salt and doxycycline were reacted in equimolar conditions, it is

hypothesized that the reaction is incomplete and thus, leaves unreacted Au ions in

solution in addition to the AuNPs. Different concentrations of doxycycline (0.1 nM to

10 μM) were added in the colloidal solution of doxy-AuNPs (containing 100 mM

NaCl) and injected sequentially into the flow cell of SPR system. In comparison, a

control sample was run in which doxy-AuNPs (containing 100 mM NaCl) were

injected in flow cell of SPR system. For the control sample, a 25 nm binding shift was

observed with doxy-AuNPs (Figure 18, red trace) without the addition of

doxycycline in solution while an increase in binding shift was observed with

successive addition of increasing concentrations of free doxycycline in the doxy-

AuNPs colloidal solutions (Figure 18, black trace). This enhancement of SPR

response on successive addition of free doxycycline is mainly because increased

concentration of doxycycline resulted in rapid growth of doxy-AuNPs, similar to

reported elsewhere for other tetracyclines (Shen, et al., 2014), which consequently

give higher SPR response.

Figure 18 Propagating SPR response of doxy-AuNPs (control, red trace) and of doxy-AuNPs with

varying concentrations of doxy (analyte, black trace)

0 10000 20000 300000

10

20

30

40

50

60

70

Time (sec)

Bin

din

gsh

ift

(nm

)

Reference

Blank

0.1 nM

10 nM1 nM

100 nM1000 nM

100 µM


70

We conclude that the growth was likely due to the already present doxy-

AuNPs in colloidal solution working as a nuclei for the gold atoms remaining from

the AuNP synthesis and doxycycline (analyte). This caused the rapid growth of seeds

of doxy-AuNPs (Figure 19) which consequently resulted in higher SPR response.

Shen et al. has also reported the similar phenomena for effect of tetracycline addition

to the in situ growth mechanism of AuNPs. According to them, AuNPs seeds (citrate

stabilized) present in the solution work as a nuclei for the conjugation and growth of

gold atoms produced as result of reaction between tetracycline and HAuCl4 (Shen et

al., 2014). Furthermore, a similar effect of size of AuNPs on SPR response was

reported by Bukar et al. According to them, gold nanoparticles with large size and

smaller Debye length on the SPR sensor surface prevail over interaction with surface

bound receptors and lead to a higher SPR response(Bukar et al., 2014).

Figure 19 Scheme illustration of doxycycline effect on overgrowth of doxy-AuNPs


71

Our UV-vis results showing the effect of doxycycline addition in doxy-AuNPs

colloidal solution also supports this overgrowth. On addition of varying

concentrations of doxycycline (from 1 nM to 1 mM) in doxy-AuNPs colloidal

solution, the LSPR of doxy-AuNPs showed slight red shift with increase in intensity

(Figure 20a), a typical feature of nanoparticle growth. Calibration of this SPR sensor

shows linearity over several orders of magnitude and detection of doxycycline from

nM to µM (Figure 20b), demonstrating the applicability of the sensor for

doxycycline.

Figure 20 (a) UV-vis spectra indicating the effect of addition of doxycycline on growth of doxy-AuNPs

(b) Sequential binding curve presenting a correlation between log of doxy concentration and SPR

response. Error bars indicate standard deviation of triplicate measurements

The sensor also showed high reproducibility with a coefficient of variation

around 5 %. Reproducibility was obtained from the triplicate SPR measurments of

100 nM doxyxycline and measured as a coefficient of variation resulting from the

ratio of the standard deviation and the mean response, in percentage. The fabricated

SPR biosensor showed a limit of detection of 7 pM. The limit of detection was

obtained as 3 times the noise over the background. Figure 21 shows that the 0.1 nM

concentration far exceeds the noise level. This low limit of detection compared

favorably to the other analytical techniques developed earlier for doxycycline

detection (Table 2) and therefore highlights the advantages of the SPR sensor for

doxycycline.


72

Table 4 Comparison with other Analytical Techniques Developed for Doxycycline Detection

Ref. Method Limit of Detection (LOD)

As reported in

Paper

Value in Mol/L

(Axisa et al., 2000) HPLC 0.125 µg/mL 2.44 × 10−7

mol/L

(Jiang & Zhang,

2004)

Enzyme-amplified

lanthanide luminescence

1.28× 10−8

mole/L 1.28× 10−8

mol/L

(Sunaric et al.,

2009)

kinetic-spectrophotometric

method based on doxy

degradation by Cu (II)/

H2O2

0.57 µg/mL 1.1× 10−6

mol/L

(Adrian et al., 2012) ELISA 0.1 µg/L 1.95× 10−10

mol/L

This work Surface Plasmon

Resonance Biosensor

0.1 nM 1.0 × 10−10

mol/L


73

4.4 Minocycline derived silver nanoparticles (Mino/AgNPs) as

potential antidiabetic agent

Diabetes mellitus with its chronic metabolic disorders consistently remains a

major threat to life. Down-regulating the generation of reactive oxygen species could

be an alternate to reduce the diabetes associated complications. Minocycline is a

semi-synthetic drug with excellent antioxidant properties similar to Vitamin C.

Furthermore, the nanoparticles could work as catalyst to increase the effectiveness of

such phenolic antioxidants (Khorrami et al., 2018). Thus, in present work, the

Minocycline modified sliver nanoparticles (Mino/AgNPs) were prepared using

minocycline as a reducing and capping agent. The prepared Mino/AgNPs were

subjected to extensive characterization and successfully applied to examine theirs in

vivo antidiabetic potential against alloxan-induced diabetic mice (Figure 21).

Figure 21 Schematic presenting the Synthesis and in vivo Antidiabetic Potential of Mino/AgNPs

4.4.1 Synthesis of Mino/AgNPs

UV-vis spectrophotometer is an important instrument to monitor the synthesis

and stability of metal nanoparticles. The mixture of silver nitrate, minocycline and

sodium hydroxide was continuously stirred for 4 min at room temperature. The color

of solution changed within 4 minute from colorless to yellowish brown which was the

first indication of synthesis of Mino/AgNPs (Hemmati et al., 2019). Furthermore the


74

synthesis of Mino/AgNPs was monitored through UV-vis spectrophotometry in the

wavelength range 300-800nm. A sharp LSPR band was observed at 395 nm which is

characteristic LSPR for AgNPs (Figure 22) (S. H. Lee & Jun, 2019).

300 400 500 600 700 8000.0

0.4

0.8

1.2

1.6

2.0

2.4

Abs

orba

nce

Wavelength (nm)

Figure 22 UV-vis spectrum of Mino/AgNPs

4.4.2 Colloidal Stability of Mino/AgNPs

The stability of as-synthesized colloidal Mino/AgNPs solution was monitored for two

weeks from plasmon wavelength (λmax) as aggregation causes the red shift of their

spectra. No significant shift in wavelength was observed for LSPR of Mino/AgNPs

till two weeks (Figure 23) which describes the good stability of as-synthesized

Mino/AgNPs.


75

300 400 500 600 700 8000.0

0.4

0.8

1.2

1.6

2.0

2.4

Ab

so

rba

nce

Wavelength (nm)

After two weeks

After one week

After 96 hr

After 48 hr

After 24 hr

After 12 hr

After 6 hr

After 3 hr

After 1 hr

Figure 23 UV-vis spectra indicating the stability of Mino/AgNPs

4.4.3 FT-IR Studies of Mino/AgNPs

To examine the role of minocycline in the synthesis of AgNPs, FT-IR spectra

of minocycline and of Mino/AgNPs were compared advantageously (Figure 24). The

IR spectrum of Minocycline (red) was significantly different from IR spectrum of

Mino/AgNPs (black). The spectrum of minocycline demonstrated two nominated

signals at 3478.25 cm-1

for O-H bond stretching and 3340.11 cm-1

for N-H bond

stretching. In addition to these two some other signals were also present in the vicinity

of these signals in the region of 3500-3100 cm-1

due to alkylic and vinylic alcohols.

All of these signals became a single broad signal after the formation of nanoparticles

which showed the involvement of these groups in the reduction of silver ions. The

broadness of signal also showed the presence of stretched N-H bond involved in

stabilization of nanoparticles. The signal at 1580.41 cm-1

was attributed to the

carbonyl of amide group. This low value from 1680-1630 cm-1

for amidic carbonyl


76

may be because of extended conjugation from C=C and NH2. The shifting of this

signal to 1633.65 cm-1

showed the involvement of β-hydroxy group in reduction of

silver ions. The involvement of this hydroxyl group has resulted into disappearance of

C=C conjugation and so the signal for amidic carbonyl appeared in normal range. The

signal for carbonyl of ketone can also be observed in the vicinity of 1580.41 cm-1

.

This signal is also shifted to higher value like that of carbonyl of amide due to similar

reason.

Figure 24 FT-IR Spectra of Minocycline (Red) and Minocycline modified Silver Nanoparticles (Blue)

4.4.4 TEM of Mino/AgNPs

The size and morphology of as-synthesized Mino/AgNPs was examined

through TEM. Homogenously distributed spherical silver nanoparticles were obtained

with this method (Figure 25). Average particle size of Mino/AgNPs calculated was

5.5 nm.


77

Figure 25 TEM and Histogram of Mino/AgNPs

4.4.5 X-ray Diffraction of Mino/AgNPs

Crystalline nature of Mino/AgNPs was examined through XRD analysis. XRD

pattern of Mino/AgNPs showed strong diffraction peaks at 38.3, 44.5, 64.6 and 77.5 ̊

corresponding to (111), (200), (220), (311) which reflects the crystalline nature of

Mino/AgNPs (Figure 26). Our XRD results of Mino/AgNPs are in accordance with

the results reported elsewhere (Hemmati et al., 2019).


78

20 30 40 50 60 70 800

500

1000

1500

2000

2500

3000

(311)(220)

(200)

Inte

nsity

2-Theta (degrees)

(111)

Figure 26 X-ray Diffraction of Mino/AgNPs

4.4.6 DPPH Radical Scavenging Assay

The free radical scavenging activities of minocycline, Mino/AgNPs and

ascorbic acid were evaluated through DPPH assay. The results demonstrated that the

percentage of inhibition was concentration dependent and in general increased with

the increase in concentrations of each analyte (Figure 27). It was observed that the

minocycline showed radical scavenging potency similar to that of ascorbic acid.

Furthermore, the Mino/AgNPs showed higher radical scavenging activity (IC50 =

19.7 µg/mL) as compared to the minocycline (IC50 = 26.0 µg/mL) and ascorbic acid

(IC50 = 25.2 µg/mL). We anticipated that the increased radical scavenging activity of

Mino/AgNPs may be due to the additional effect of AgNPs as nanocatalyst. The

Elemike et al. also reported the catalytic effect of AgNPs to enhance the radical

scavenging activity of Costus afer extract. The study reported that the Costus afer

modified AgNPs (CA-AgNPs) showed higher DPPH radical scavenging activities as

compared to the Costus afer leaf extract. They suggested that the increase in


79

antioxidant potential of CA-AgNPs can be due to the presence of phytochemicals

such as flavonoids (with many hydroxyl groups) on surface of AgNPs that contributed

to the proceeding antioxidant activities through hydrogen atom transfer (HAT) and

single electron transfer (SET) mechanism simultaneously (Elemike et al., 2017).

Figure 27 DPPH free radical scavenging assay

4.4.7 Antihyperglycemic activity of Mino/AgNPs in alloxan induced

diabetic mice

The diabetic mice carry the symptoms of diabetes mellitus such as

hyperglycemia, weight loss, polyuria and decreased insulin level. The administration

of standard drug glibenclamide and Mino/AgNPs to the diabetic mice resulted in a

change of blood sugar level (BSL), cholesterol level, triglyceride level and

hemoglobin level as compared to the diabetic mice. However, the Mino/AgNPs

showed higher antidiabetic potential as compared to the drug glibenclamide. In

diabetic mice, the BSL was notably high as compared to the normal mice. However,

the oral administration of Mino/AgNPs to the diabetic mice resulted in significant

(p≤0.05) lowering of BSL relative to the diabetic mice left untreated (Figure 28). The

hypoglycemic action of Mino/AgNPs can be attributed to its free radical scavenging

activity that resulted in decrease of ROS in blood stream. As a consequence of

reduced oxidative stress, the insulin sensitivity was improved, thereby increasing


80

cellular uptake of glucose from blood stream and thus down-regulate the blood sugar

level in treated mice. The Hurrle and Hsu also reported the similar effect of

antioxidants on oxidative stress and insulin resistance. The study has reported the

effect of ROS on different pathways in insulin receptor signal transduction that

ultimately disrupt the expression of glucose transporter 4 (GULT4), a major glucose

transporter in the cell. This affects the uptake of glucose from the blood into the cell

that causes the insulin resistance. However the use of antioxidants reduces the

oxidative stress that ultimately leads to the down-regulation of BSL in blood stream

by improving insulin sensitivity (Hurrle & Hsu, 2017).

Figure 28 Blood Sugar Level (mg/dl) for various study groups

Furthermore, the weight loss and decrease in total hemoglobin level were also

observed in diabetic mice as compared to the normal mice. In diabetic condition, the

reaction between excess glucose and hemoglobin converts hemoglobin to the

glycosylated hemoglobin as a result of which the level of hemoglobin decreases in

diabetic mice (Dhas et al., 2016). However the treatment of diabetic mice with


81

Mino/AgNPs resulted in Significant (p≤0.05) increase in body weight and

hemoglobin level as compared to the untreated diabetic mice (Figure 29).

Group l Group II Group III Group IV

0

2

4

6

8

10

12

14

mg

/dL

Hemoglobin (Hb)

Figure 29 Hemoglobin Level (mg/dl) for various study group

Lipids have a very crucial part in the progression of DM. In a diabetic

condition, the serum‟s lipids level is generally increased which indicates the risk of

coronary heart disease (Al-Shamaony, Al-Khazraji, & Twaij, 1994).

Hypercholesterolemia and Hypertriglyceridaemia are the main risk factors that can

cause atherosclerosis as well as coronary heart disease, the secondary complications

associated with DM (Ananthan et al., 2003). The use of dietary or drug treatment

seems to be effective in decreasing the lipids level in serum and consequently

minimizing the risk of cardiovascular disease. In the present investigation, the

cholesterol and triglycerides (TG) levels were notably high in diabetic mice.

However, the oral administration of Mino/AgNPs to the diabetic mice resulted in a

significant (p≤0.05) decrease in triglycerides and cholesterol levels as compared to the

untreated diabetic mice (Figure 30). We anticipated that the increased level of ROS


82

interfere with cell function, alter the cholesterol and triglyceride metabolism and thus

resulted in higher TC and TG level. However the Mino/AgNPs treatment resulted in

decreased oxidative stress by scavenging free radicals and ROS, normalization of cell

function and consequently down-regulation of TC and TG levels (Murakami et al.,

2020; Seo, Kang, Choi, Choi, & Jun, 2019; R. L. Yang, Shi, Hao, Li, & Le, 2008).

Figure 30 Lipid profile (mg/dl) for various study groups

Furthermore, the administration of Mino/AgNPs to the diabetic mice also

affected the activity of hepatic marker enzymes in the serum. In diabetic mice, the

levels of SGOT and SGPT were raised. In diabetic conditions, the liver cells are

damaged which cause the microsomal cells of the liver to excrete various enzymes

such as SGOT, SGPT and ALP (Daisy, Eliza, & Ignacimuthu, 2008). However, the

oral administration of Mino/AgNPs to the diabetic mice maintained a significantly

(p≤0.05) lower SGOT and SGPT as compared to the diabetic mice left untreated

(Figure 31).


83

.

Group l Group II Group III Group IV0

20

40

60

80

100

120m

g/d

L

SGPT

SGOT

Figure 31 SGPT and SGOT Profile (mg/dl) for various study group

4.4.8 Histology Studies

In Histopathological studies, pancreas, kidney and liver sections of treated,

untreated and normal control mice were examined. The pancreatic islet tissue of

diabetic mice displayed irregular islet boundaries as well as mass distribution of

cytoplasm relative to the normal mice (Figure 32). The treatment of diabetic mice

with both glibenclamide and Mino/AgNPs displayed good regeneration and recovery

of islet tissue of the pancreas. Nevertheless, the Mino/AgNPs showed more

effectiveness in regeneration and recovery of islet tissue than the glibenclamide. The

-cells mass was significantly higher in mice treated with Mino/AgNPs as compared

to the diabetic mice left untreated (Figure 32). We anticipated that the Mino/AgNPs

protected the -cells of pancreas from ROS and suppressed apoptosis in -cells. The

studies of Kaneto et al. also reported that the apoptosis induced by ROS in -cells of

Pancreas was suppressed by the use antioxidant (Kaneto et al., 1999).


84

Figure 32 Histology of Islet cells of Pancreatic sections of various study groups (Arrowhead pointing

towards the islet tissue of pancreas)

The tissue of the normal kidney section presented the normal architecture. The

kidney section of diabetic mice showed distorted glomerular and dilated urinary space

with Necrosis, vacuolation in the renal epithelial and some tubules with apoptotic

cells (Figure 33). The treatment of diabetic mice with glibenclamide displayed

limited improvement in the morphology of glomerular with some dilated urinary

space whereas the treatment of diabetic mice with Mino/AgNPs displayed higher

recovery and regeneration relative to the histo-morphology of kidney sections of

normal mice. The kidney section of diabetic mice treated with Mino/AgNPs showed


85

improved glomerular with improved urinary space very close to the architecture of

normal mice (Figure 33).

Figure 33 Histology of kidney sections of various study groups (Arrowhead pointing towards the

glomerulus and urinary space of kidney)


86

The hepatic sections of the normal liver showed normal architecture with

intact central hepatic vein and slit-like sinusoids and prominent nuclei (Figure 34).

The liver sections of diabetic mice displayed distorted central vein along with

apoptotic nuclei. The oral administration of both drug and Mino/AgNPs to the

diabetic mice showed significant recovery of the central hepatic vein. However, the

treatment with Mino/AgNPs showed better recovery and revival effect as compared to

the drug glibenclamide (Figure 34).

Figure 34 Histology of Liver sections of various study groups (Arrowhead pointing

towards the central hepatic vein of liver)


87

CONCLUSION AND PERSPECTIVES In conclusion, a gold nanoparticles based pH sensitive platform was

successfully developed for the selective release of anticancer drug doxorubicin. The

highly stable AuNPs were synthesized and successfully binded with anticancer drug

doxorubicin. The protocol is simple as we did not use any additional stabilizer for

AuNPs or we also did not use additional linker to attach drug on surface of AuNPs.

Doxycycline plays the role of reducing agent, capping agent and the linker.

Furthermore, a pH sensitive drug release study was successfully carried out in which a

rapid release of anticancer drug was observed at pH 4.30 whereas no significant

release of drug was observed at normal physiological pH 7.34 which may help to

minimize the side effects of drug to the normal tissues of body. Consequently,

improving the efficacy of drug for targeted cancer therapy. Moreover synthesized

AuNPs show a tremendous biocatalytic response for oxidation of dopamine. It can be

used as efficient biocatalyst for colorimetric detection of dopamine. It is expected that

current doxy-AuNPs have an intrinsic peroxidase-like activity towards many

peroxidase substrates. Peroxidase-like response thus, tells the practical applicability of

newly synthesized doxy-AuNPs to work like an artificial enzyme.

Furthermore, an ultra-sensitive SPR biosensor based on doxy-AuNPs has been

successfully developed for the detection of doxycycline. Variation in size and growth

of doxy-AuNPs affected the biosensor response. To overcome the limitation of low

molecular weight of analyte, doxy-AuNPs (containing 100 mM NaCl) has been

applied as an amplification element to obtain significantly enhanced SPR response.

The reported biosensor allowed for the detection of doxycycline as low as 7 pM. The

high sensitivity, low limit of detection, excellent signal response time (less than half

an hour), good stability and reproducibility make this SPR biosensor an excellent

alternative to the conventional methods for the detection of doxycycline. In future, the

present biosensor can be applied in different fields of medical diagnostics and

environmental monitoring for the detection of doxycycline.

Diabetes mellitus is a life-threatening disease all over the world and it

demands significant efforts to be treated effectively. The antioxidants have shown to

be very effective in many bioprocesses including disorders of diabetes mellitus. The

present work was carried out to examine the antidiabetic potential of newly


88

synthesized Mino/AgNPs against the alloxan induced diabetic mice. The DPPH

inhibitory assay was conducted to compare the antioxidant potential of Mino/AgNPs

with that of minocycline and ascorbic acid. The Mino/AgNPs showed higher radical

scavenging activity (IC50 = 19.7 µg/mL) as compared to the minocycline (IC50 = 26.0

µg/mL) and ascorbic acid (IC50 = 25.2 µg/mL). Further, hematological and

histopathological analysis revealed that the Mino/AgNPs showed greater potential as

an antidiabetic agent than the standard drug glibenclamide. The Mino/AgNPs showed

more effectiveness in reducing Blood sugar, cholesterol and triglycerides levels.

Furthermore, the treatment of diabetic mice with Mino/AgNPs also showed

significant regeneration and revival of histo-morphology of kidney, central vein of

liver and islet cells of the pancreas as compared to the normal control mice. Our

results indicated that the as-synthesized Mino/AgNPs have good potential to reduce

the disorders of diabetes mellitus and can be effectively used to treat diabetic

conditions.


89

FUTURE RECOMMENDATIONS

Further research is required to optimize reaction conditions for the synthesis of

specific Au and Ag-NPs on industrial scale. It is recommended to evaluate the in vivo

drug release behavior of DOX-doxy-AuNPs conjugate. Doxycycline (an antibiotic

from tetracycline group) has anticancer activities reported elsewhere. It is therefore,

recommended to evaluate the anticancer potential of both doxy and DOX through in-

vivo studies using Albino mice model. Further research is required to make doxy-

AuNPs based SPR biosensor applicable at clinical level. It is recommended to

perform SPR experiments with clinical samples to examine the effect of biological

interferences on response of biosensor. Efficiency of antibiotics can be augmented

through different formulations with Ag-NPs. The prepared NPs can be used for

developing more effective drug delivery platform for selective release of drugs in test

animals. The synthesized NPs can also be used to fabricate other types of biosensors

for detection and quantification of medicinal drugs.

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90

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List of Publications

1. Syed Akif Raza Kazmi1,*, Muhammad Zahid Qureshi

1, Shoukat Ali

2 and Jean-

Francois Masson2,*. In vitro Drug Release and Biocatalysis from pH Responsive

Gold Nanoparticles synthesized using Doxycycline. Langmuir. 2019, 35, 49, 16266-

16274. doi: 10.1021/acs.langmuir.9b02420. ISSN: 0743-7463. (IF 2021: 3.557)

2. Syed Akif Raza Kazmi1,*, Muhammad Zahid Qureshi

1 and Jean-Francois

Masson2,*. Drug-based Gold Nanoparticles Overgrowth for Enhanced SPR

Biosensing of Doxycycline. Biosensors 2020, 10, 184. doi:10.3390/bios10110184.

(IF 2021: 3.240)

3. Syed Akif Raza Kazmi1,

*, Muhammad Zahid Qureshi1, Sadia

2, Saleh S.

Alhewairini3, Shaukat Ali

4,*, Shazia Khurshid

1, Muhammad Saeed

5, Shumaila

Mumtaz4, Tafail Akbar Mughal

4. Minocycline Derived Silver Nanoparticles for

Assessment of Their Antidiabetic Potential against Alloxan induced Diabetic Mice.

International Journal of Nanomedicine (Under Review). (IF 2021: 4.471)

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