SYNTHESIS OF NOBLE METAL BASED NANOPARTICLES AND …
Transcript of 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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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
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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
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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-
GCU Lahore INTRODUCTION
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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
GCU Lahore INTRODUCTION
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
GCU Lahore INTRODUCTION
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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
GCU Lahore INTRODUCTION
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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
GCU Lahore INTRODUCTION
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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
GCU Lahore INTRODUCTION
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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
GCU Lahore INTRODUCTION
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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
GCU Lahore INTRODUCTION
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(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
GCU Lahore INTRODUCTION
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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
GCU Lahore INTRODUCTION
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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
GCU Lahore INTRODUCTION
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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
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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-
GCU Lahore LITERATURE REVIEW
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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-
GCU Lahore LITERATURE REVIEW
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
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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
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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
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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-
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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)
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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
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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
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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).
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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
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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
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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
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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
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(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).
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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
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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
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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
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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
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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-
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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
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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).
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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).
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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.
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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
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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
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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.
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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
GCU Lahore MATERIALS AND METHOD
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
GCU Lahore MATERIALS AND METHOD
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,
GCU Lahore MATERIALS AND METHOD
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
GCU Lahore MATERIALS AND METHOD
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.
GCU Lahore RESULTS AND DISCUSSION
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).
GCU Lahore RESULTS AND DISCUSSION
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
GCU Lahore RESULTS AND DISCUSSION
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
.
GCU Lahore RESULTS AND DISCUSSION
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
GCU Lahore RESULTS AND DISCUSSION
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)
GCU Lahore RESULTS AND DISCUSSION
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
GCU Lahore RESULTS AND DISCUSSION
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)
GCU Lahore RESULTS AND DISCUSSION
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
GCU Lahore RESULTS AND DISCUSSION
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
GCU Lahore RESULTS AND DISCUSSION
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).
GCU Lahore RESULTS AND DISCUSSION
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
GCU Lahore RESULTS AND DISCUSSION
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
GCU Lahore RESULTS AND DISCUSSION
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
GCU Lahore RESULTS AND DISCUSSION
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
GCU Lahore RESULTS AND DISCUSSION
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
GCU Lahore RESULTS AND DISCUSSION
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.
GCU Lahore RESULTS AND DISCUSSION
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
GCU Lahore RESULTS AND DISCUSSION
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).
GCU Lahore RESULTS AND DISCUSSION
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
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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
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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.
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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
GCU Lahore RESULTS AND DISCUSSION
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
GCU Lahore RESULTS AND DISCUSSION
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.
GCU Lahore RESULTS AND DISCUSSION
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
GCU Lahore RESULTS AND DISCUSSION
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.
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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).
GCU Lahore RESULTS AND DISCUSSION
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
GCU Lahore RESULTS AND DISCUSSION
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
GCU Lahore RESULTS AND DISCUSSION
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
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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
GCU Lahore RESULTS AND DISCUSSION
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).
GCU Lahore RESULTS AND DISCUSSION
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).
GCU Lahore RESULTS AND DISCUSSION
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
GCU Lahore RESULTS AND DISCUSSION
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)
GCU Lahore RESULTS AND DISCUSSION
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)
GCU Lahore RESULTS AND DISCUSSION
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
GCU Lahore RESULTS AND DISCUSSION
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.
GCU Lahore RESULTS AND DISCUSSION
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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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)