I

“TO STUDY THE ROLE OF GOLD NANO-

PARTICLES IN DIAGNOSIS AND TREATMENT OF
CANCER”

SUBMITTED BY
MERAB TUFAIL
FATIMA ZAHID
SUPERVISOR
DR. SAMINA TAZAYYEN YOUSAF AZEEMI

DEPARTMENT OF PHYSICS
GOVERNMENT POSTGRADUATE COLLEGE
FOR WOMEN GULBERG LAORE, 2019
LAHORE COLLEGE FOR WOMEN UNIVERSITY,
LAHORE 2019
 II

“TO STUDY THE ROLE OF GOLD NANO-

PARTICLES IN DIAGNOSIS AND TREATMENT OF
CANCER”

A THESIS SUBMITTED TO LAHORE COLLEGE FOR WOMEN UNIVERSITY

IN FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF BS IN
PHYSICS.

BY

MERAB TUFAIL ROLL NO: 30152111

FATIMA ZAHID ROLL NO: 3015219

DEPARTMENT OF PHYSICS GOVERNMENT

POSTGRADUATE COLLEGE FOR WOMEN, GULBERG
LAHORE, 2019
LAHORE COLLEGE FOR WOMEN UNIVERSITY,
LAHORE 2019
 III

GOVERNMENT POSTGRADUATE COLLEGE FOR

WOMEN, GULBERG LAHORE.
DECLARATION FORM:
We Merab Tufail and Fatima Zahid declare that the thesis titled “TO STUDY THE ROLE
OF GOLD NANO-PARTICLES IN DIAGNOSIS AND TRETMENT OF CANCER” is
our own work and is not submitted previously, in whole or in parts, in respect of our any
other academic award.

Signature of student Date

Signature of student Date

I approve the above titled thesis to be submitted for the examination.

Signature of Supervisor Date

 IV

CERTIFICATE
“TO STUDY THE ROLE OF GOLD NANO-PARTICLES IN
DIAGNOSIS AND TREATMENT OF CANCER”
This thesis is submitted by Merab Tufail, Roll No. 30152111 and Fatima Zahid,
Roll No. 3015219 to the department of physics, Lahore College for Women
University, Lahore, Pakistan for the partial fulfillment of the requirement for the
degree of

BS (PHYSICS)
2015-2019

Internal Examiner Date:

External Examiner Date:

Head of Department Date:

Controller of Examinations Date:

GOVT. POSTGRADUATE COLLEGE FOR WOMEN GULBERG, LAHORE

LAHORE COLLEGE FOR WOMEN UNIVERSITY LAHORE

SESSION 2015-2019
 V

Dedication:
“Every challenging work, needs self-effort as well as guidance of
elders especially those who are very close to our heart”
“I dedicate my work to my beloved mother Zubaida a strong
and gentle soul who taught me to trust Allah Almighty,
believe in hard work and encourage me to believe in myself.
Without her support I couldn’t achieve so much. And to my
father Muhammad Tufail. My parents play a vital role in my
success with their encouragement and support I am able to
achieve this success in my life”

Merab Tufail
 VI

Acknowledgement:
First and foremost, thanks to Allah Almighty for all countless gifts and his great blessings
that he has offered me throughout my life, he gave me strength and enable me to complete
this work by opening doors of knowledge to me. Peace and blessing of Allah be upon
THE HOLY PROPHET (P.B.U.H) who exhorted his followers to seek for knowledge
from cradle to grave.

I would like to express my heartfelt gratitude to Dr. Samina, my research work supervisor
for her guidance and critiques of this research work. I am very thankful to Dr. Kusar
Javed Khan, for the inspiration, support, guidance, and making me brave and full of
enthusiasm during my wonderful journey in this college. My special thanks are extended
to all my teachers especially to my honorable teacher Prof. Rabab Kazmi for her
illustrious advices and for the time ma’am spent with me discussing and guiding me in this
research work.

I would like to express my heartfelt gratitude to my siblings without their love and
affection it could not be possible to achieve this success. A special thanks to my loving
sister Tayyaba Sadaf for assistance in technical work.

I am very thankful to my all Friends especially Zainab Aziz for her unfailing moral
support and for sincere suggestions at every step. I am also very grateful to Ftima Zahid
and Amna Arif for being very cooperative in my journey and also for making my life full
of happiness and positivity.

I have no words to describe deep sensation of respect for Mr. Farooq Sabir who provided
me with the facilities being required and conductive conditions for my thesis project.

I like to acknowledge the book which remains a constant inspiration to me which is “THE
SCERET” by RHONDA BYRNE.

MERAB TUFAIL
 VII

Abstract:
Background:

Gold Nano-particles has been used in variety of applications but the use of colloidal gold
as a colorant can be traced at the least to 5th B.C. Since 1857 gold has been recognized as
effective therapeutic agent. The catalytic behavior of gold Nano-particle is used in number
of chemical reactions. Because of controllable surface properties such as surface Plasmon
resonance and high surface to volume ratio make them applicable for use in cancer
diagnosis and treatment.

Objective:

“To study the role of gold Nano-particles in diagnosis and treatment of cancer”

Methodology:

The gold Nano-particles are used in cancer treatment by using them as contrast agent for
diagnosis and also for imaging. The anti-cancer drug is delivered using gold nanoparticles
as drug play load so that the drug might not affect normal cells. Also external radiations
including the visible and near infrared radiations are used to make the gold Nano-particles
more effective in curing cancer.

Result:

Gold nanoparticles are effective tool for the diagnosis, imaging and treatment of cancer as
far as the Au Np’s are properly designed and have surface coating compatible with body
tissue.

Conclusion:

Hence we conclude the use of gold Nano-particle in cancer therapeutics an effective tool
moreover they Au Np’s are helpful tool to bi-pass tradition drug delivery mechanism by
proper characterization.
 VIII

LIST OF CONTENTS
 IX

TABLE OF CONTENTS:

1.1 Introduction to nano-particles: ................................................................................. 1

1.2History of nano-particles: ......................................................................................... 1

1.3General features of nano-particles: ........................................................................... 2

1.4Classification of nano-particles: ............................................................................... 2

1.5 characterization parameter: ...................................................................................... 3

1.6 Nano-particles structure: .......................................................................................... 4

1.7 Approaches to nanotechnology: ............................................................................... 6

1.7.1 Top down approach: .......................................................................................... 7

1.7.2 Bottom up approach: ......................................................................................... 7

1.8 Types of nanoparticles for drug delivery system: .................................................... 7

1.8.1 Advantage of using nano-particles as a drug delivery system: ......................... 7

1.9 Gold and gold (Au) nanoparticles: ......................................................................... 10

1.9.1Physical properties of gold: .............................................................................. 11

1.9.2 Chemical properties of gold............................................................................11

1.10 Structure of gold nanoparticles: ........................................................................... 13

1.11 Different shapes of gold nano-particles: .............................................................. 14

1.12 Properties of gold at nano-scale: .......................................................................... 14

1.13 Optical properties of gold nano-particles:............................................................ 17

1.14 Cancer: ................................................................................................................. 18

1.14.1 Types of cancer: ................................................................................................ 19

1.14.2 Stages of cancer: ............................................................................................... 20

1.14.3 Causes of cancer: .............................................................................................. 22

Chapter no#2 ................................................................................................................ 24

 X

Literature review .......................................................................................................... 24

Chapter#03 ................................................................................................................... 28

Materials Methods: ...................................................................................................... 28

3.1 Cancer treatment using nano-technology: ............................................................. 28

3.2 Cancer drug delivery process using nanoparticles: ................................................ 29

3.3 Gold nano-particles in cancer therapeutics: ........................................................... 31

3.3.1 Imaging: .............................................................................................................. 32

3.3.2 Drug delivery: ..................................................................................................... 41

3.4 Drug targeting using Gold nano-particles: ............................................................. 42

3.4.1 Passive targeting…………………………………………………………….41

3.4.2 Active targeting……………………………………………………………..42

3.5 Cancer therapy using gold nano-particles: ............................................................. 44

3.5.1 Photo-thermal therapy:.................................................................................. 44

3.5.2 Radiation therapy…………………………………………………………...45

Chapter # 04 ................................................................................................................. 49

Results:......................................................................................................................... 49

Discussion: ................................................................................................................... 50

Conclusions:................................................................................................................. 52

Future perspectives: ..................................................................................................... 52

References:……………………………………………………………………………49
XI
 XII

List of figures:

Fig No. Title page No.

1. Various properties of nanoparticles 4
2. Types of nanoparticles. 12
3. Pure gold. 15
4. Impure gold. 15
5. Spherical gold particles. 16
6. Fcc packing of gold nanoparticle. 17
7. Different shapes of gold nanoparticles. 17
8. Tunneling effect of gold nanoparticle. 20
9. Surface Plasmon resonance. 21
10. Cancer in the epithelial layer of skin. 23
11. Healthy blood cells and leukemia blood cells. 24
12. Different stages of cancer. 25
13. Cancer tends to involve multiple mutations 26
14. General mechanism of drug delivery. 33
15. Gold Nano-particle employed in cancer therapeutics. 35
16. X-ray imaging of a hand. 33
17. Pictorial view of CT scanner used in hospitals. 34
18. Surface emission Raman spectroscopy. 36
19. PET images variation with time. 38
20. Protein coating of Nano-particles for protein delivery. 39
21. Mechanism of nucleic acid detection using gold 40
Nano-particles.
22. Nucleic acid imaging using gold Nano-particles. 41
23. Scenario of gene delivery. 43
24. Mechanism of passive targeting. 44
25. Mechanism of active targeting. 44

26. photo-thermal therapy using Au nanos-particles. 46

 XIII

27. Physical mechanism of Au involved in radiation therapy.

47

28. Mechanism of cancer recovery .

51

List of graphs
1. Increase in surface area to volume ratio by reducing the spherical shape of the
gold
Nanoparticle.

List of tables
1. Different Nano-particle system, their structures and characteristics. 11
2 Physical properties of gold. 13
3. Chemical properties of gold. 14
4. Dimensions of different gold-nanoparticles. 16
 XIV

List of abbreviations:

MRI magnetic resonance imaging

CT computed tomography
AFM atomic force microscopy
SEM scanning electron microscopy
TEM transmission electron microscopy.
Np’s nanoparticles
EPR enhance permeation and retention
Au gold
FCC face centered cubic
LED light emitting diode
 XV

Introduction
What is Types of
to nano-
cancer? cancer
particles

Properties of
History of Causes of
goldanno-
nano-particles cancer.
particles

Features of Gold nano- Daignosis of

nano-particles particles cancer.

Classification Treatment of
Structure of cancer using
of nano-
nano-particles
particles Au np's
 XVI

CHAPTER # 01
INTRODUCTION
 1

1.1 Introduction to Nano-particles:

Nano-particles:

The word “Nano” is derived from Latin word “nanus” meaning “dwarf”. Nano-particles
are solid colloidal functional structures with dimensions measured in Nano-meter. 1 Nano-
meter is one billionth of meter i.e.1/109m[1].

Figure 0.1: This figure shows various properties of Nano-particles.

Nanoparticle Nanotechnology is the creation and utilization of materials, devices, and

systems through the control of matter on the nanometer-length scale, i.e., at the level of
atoms, molecules, and supramolecular structures. This term is applied to describe the
construction and utilization of functional structures with at least one characteristic
dimension measured in nanometers (a nanometer is one billionth of a meter (10-9 m).
Nano biotechnology is the application of nanotechnology in life sciences. The most
important applications in healthcare are in diagnostics, drug delivery, and development of
Nano-medicine including Nano-surgical procedures [2].

1.2History of Nano-particles:
Nano- particles have a long history. The historical development of Nano-particles is
described by Paul Ehrlich and Ursula Scheffel and colleagues and the extensive work by
the group of professor Peter Speiser at in the late 1960’s and early 1970’s are described as
from personal point of view.
 2

Special attention is given between 1970 and the early 1980’s.further developments are
made on the Nano-particles for the delivery of drugs.
 The concept of Nano-technology was introduced by physicists Richard P
Feynman at the December 1959 meeting of the American physical society. He
delivered a famous lecture entitled “There is plenty of room at the bottom”. In his
lecture he suggested that twenty four volume of
the encyclopedia Britannica can be written on the
head of pin, and miniaturizing the computer. In
short Richard P Feynman suggested it’s possible
to arrange atoms the way we want [3].
 In 1974 Norio Taniguchi (a Prof. at the Tokyo
University of Science) invented the term Nano-
technology. Concepts given by Feynman was
further developed by E. Drexler in his book
”vehicles of creation: the arrival of new era”

1.3General features of Nano-particles:

 The properties and behavior of particles at the Nano-scale differ significantly when
compared to micro scale .They are structurally unique having high ratio of surface
area to volume ratio.
 Nanoparticles are important scientific tools that have been explored in various
biotechnological, pharmacological and pure technological uses.
 In biological systems and living organisms, Nanoparticles having high quality size
and shape are capable of detecting biochemical changes at single molecular level
in living cells.
 Nano materials are used in clinical imaging technologies such as MRI, CT and
ultrasounds by using advanced contrast agents.

1.4Classification of Nano-particles:
As the Nanoparticles are the mixtures of atom in molecules in the range of 1-100nm so
they can be composed of one or more species of atoms or molecules and can exhibit a
wide range of size dependent-properties. Due to this size range Nano-particles bridge the
 3

gap between small molecules and bulk materials in terms of energy states. They are
generally classified into three classes based on their dimensionality, morphology,
composition, uniformity, and agglomeration.

Figure 1-2 this figure shows different Nano-particles dimensions.

0D 1D 2D 3D
nanoparticles nanoparticles nanoparticles nanoparticles
•They have length, •They have only •They have only •They have all the
bredth and one parameter it length and parameters of
heights that are may be length, breadth for length, breath
confined at a breadth or height example nano and height .for
single point for for example wires nano-tubes, example nano-
example nano- nanofibers,thin dendrimers,fibers cryrals , quantum
sphere. films,surface and fibrils. dots ,colloidsetc.
coatings

1.5 characterization parameter:

Characterization of Nanoparticles is based on the:

 size,
 morphology
 surface charge
Using such advanced microscopic techniques as atomic force microscopy (AFM),
scanning electron microscopy (SEM) and transmission electron microscopy (TEM).
Properties like surface morphology, size and overall shape are determined by electron
Microscopy Techniques.
 4

SURFACE CHARGE

SURFACE
PARTICLE SIZE HYDROPHOBICITY

CHARACTERIZATION
PARAMETERS OF
NANOPARTICLES.

 particle size:
Characterization of Nanoparticles is primarily evaluated by the particle size distribution
.With the use of electron microscopy; it’s now possible to determine size of Nanoparticles.

 surface charge:
Surface charge and intensity determines the interaction of Nanoparticles with the
biological environment as well as their electrostatic interaction with bioactive compounds.
This can be determined through zeta potential of Nanoparticles. Zeta potential is an
indirect measure of the surface charge.

 surface hydrophobicity:
A technique such as x-ray photon correlation spectroscopy not only determine surface
hydrophobicity but also permits the identification of specific chemical groups on surface
of Nano-particles [5].

1.6 Nano-particles structure:

Although it appears that Nano-particles are simple structures yet they are very
complex. Apparently it seems Nano-particles are just small particles like other small
molecules and in reality they behave very differently depending upon different
components of Nano-materials. For example any nanoparticle will have an exceptionally
high surface area to volume ratio; this is one of the reasons for some of their unusual
properties. However, this high surface area also means that the surface of any given
nanoparticle is an important component of the material. So even the simplest nanoparticle
 5

will have a surface chemistry, which is distinctly different from that of the core material.
A nanoparticle can therefore be split into two or three layers;

 a surface that may often be functionalized

 a shell material that may be intentionally added
 the of core material
Often nanoparticles are only referred by their core material because this is the part of the
nanoparticle that results in key properties for most applications.

1.6.1 The surface:

The surface of a nanoparticle may be functionalized with arrange of metal ion Preparing a
nanoparticle with a charged surface is a convenient way to prepare nanoparticles that
disperse in aqueous media. However, many materials do not have convenient surfaces for
stabilization of localized charges. In many of these cases, it is common to use a small
molecule. That will bind to the surface of the particle by a covalent-like bond and also
contain groups capable of carrying a charge. Citrate stabilized gold and silver sols come
under this category [4].

core

shell

surface

Figure 1.3: Schematic representation of Nano-particle core, shell and surface.

1.6.2 The shell:

This is a layer of chemically different material from the core material. The term shell is
usually applied to a second layer that has a completely different structure from that of the
 6

core material. Some excellent examples of these materials are the quantum dots, which
contain a core of one material, such as cadmium selenide, and a shell of another, such as
zinc Supplied.

1.6.3 The core:

This is essentially the center of the nanoparticle and usually used to refer to the
nanoparticle itself. The properties of nanoparticles are generally related to the composition
of their cores, and in some cases the cores and shells. However the exact composition of
the whole nanoparticle is implicit in accurately determining its overall properties [6].

1.7 Approaches to nanotechnology:

The methods of synthesis of nanoparticles are well known for a long time. For the
synthesis of nanoparticles, the processing conditions need to be controlled in such a
manner that the resulting nanoparticles have the following characteristics:

1. Identical size of all particles.

2. Identical shape
3. Identical chemical composition and structure of crystal

Two approaches have been known for preparation of ultrafine particles as follows:

Bottom up approach

Top down approach

–
 7

1.7.1 Top down approach:

In this approach an external force is applied to a solid that leads to break down of material
into smaller particles up to Nano scale structures.

1.7.2 Bottom up approach:

It is also known as build-up approach which means building structures from building
blocks. Here the in this method the Nano-particles are formed from bottom means atom by
atom, molecule by molecule or cluster by cluster. Now from past few years the chemical
methods have been used for the preparation of nanoparticles where a greater control on
size and composition could be achieved [7].

1.8 Types of nanoparticles for drug delivery

system:
Nano carriers have provided a novel platform for target-specific delivery of therapeutic
agents. Cancer drug delivery achieving high therapeutic efficacy and low side effects
requires Nano carriers to tightly retain the drug, efficiently reach the tumor, then quickly
enter the tumor cells and release the drug [8]. Over the past decade, several delivery
vehicles have been designed based on different Nano-materials, such as polymers, [9]
dendrimers, [10] liposomes, [11] nanotubes, [12], and Nano rods [13].

1.8.1 Advantage of using Nano-particles as a drug

delivery system:
Nanoparticles used for drug delivery system possess following advantages:

1. Particle size and surface characteristics of nanoparticles can be easily manipulated

to achieve both passive and active drug targeting after parenteral administration.
2. They control and sustain release of the drug during the transportation and at the
site of localization, altering organ distribution of the drug and subsequent clearance
of the drug so as to achieve increase in drug therapeutic efficiency and reduction in
side effects.
 8

3. Drug loading is relatively high and drugs can be incorporated into the systems
without any chemical reaction; this is an important factor for preserving the drug
activity.
4. Site-specific targeting can be achieved by attaching targeting ligands to surface of
particles or use of magnetic guidance [14].

1.8.2 Types of Nanoparticles for drug delivery system are:

Submicron-sized Nano particles (3-200nm) are:
A. Polymeric Nano-particles
B. Polymeric micells
C. Dendrimer
D. Lipids (liposomes)
E. Viral-based Nano-spheres
F. Carbon Nano tubes

Figure 1- 3 Types of Nano-particles. (Rahman)

 9

Table1.1 Showing system, structure and characteristics of Nano-particles:

System Structure Characteristics

Polymeric Drugs are conjugated to (a) Water-soluble, nontoxic, biodegradable

Np’s the side chain of a (b) Surface modification (pagination)
linear polymer with a (c) Selective accumulation and retention in
Linker cleavable- tumor tissue (EPR effect)
Bond). (d) Specific targeting of cancer cells while
sparing normal cells—receptor-mediated
Targeting with a ligand

Polymeric Amphiphilic block co- (a) Suitable carrier for water-insoluble drug
micells polymers assemble and (b) Biocompatible, self-assembling,
form a micelle with a biodegradable
hydrophobic core and (c) Ease of functional modification
hydrophilic shell. (d) Targeting potential

Dendrimer Radially emerging (a) Bio distribution

hyper branched (b) High structural and chemical
Synthetic polymer with homogeneity
regular pattern and (c) Ease of functionalization, high ligand
repeated units. density
(d) Controlled degradation
(e) Multifunctionality

Liposomes Self-assembling closed (a) Amphiphilic, biocompatible Pegylated

colloidal structures liposomal
Composed of lipid (b) Ease of modification
 10

bilayers. Non-pegylated liposomal

(c) Targeting potential

Viral based Protein cages, which (a) Surface modification by mutagenesis or

Np’s are multivalent, bio conjugation—multiagency
Self-assembled (b) Specific tumor targeting,
structures. multifunctionality
(c) Defined geometry and remarkable
uniformity
(d) Biological compatibility and inert nature

Carbon Carbon cylinders (a) Water-soluble and biocompatible

nanotubes Composed of benzene through
ring. chemical modification
(b) Multifunctionality
This table shows different Nano-particle system, their structures and characteristics
[15].

1.9 Gold and gold (Au) nanoparticles:

Noble metal:
Noble metal nanoparticles are having distinct properties compared to other metallic
nanoparticles due to their optical, electronic and molecular recognition properties. Noble
Nano materials have large optical field enhancements due to the resonant oscillation of
their free electrons in the presence of light. Because of these properties noble metal
nanoparticles are greatly utilized in optical, imaging, sensors, cosmetics, and Cancer
therapy and drug delivery. Among these noble metals, gold and gold nanoparticles are
precious, inert and not easily oxidized when exposing to oxygen or highly acid
environments. Gold nanoparticles exhibit different colors such as red, blue or other colors
depending their size, shape and amount of aggregation. These visible colors reflect the
oscillations of conduction band electrons at appropriate wavelengths. Gold nanoparticles
are highly stable, sensitive and have high level of consistency. Due to these properties they
are greatly admired and utilized for biomedical applications such as drug delivery,
imaging, photo-thermal therapy and immune-chromatographic detection of pathogen in
food and clinical specimen [16].
 11

Gold (Au):
Gold is an element of periodic table of inert group with atomic number 79. It has symbol
Au which is abbreviation of “aurum” derived from Latin language. It’s the first element
recognized by man as metal. The word gold comes from Geolo which means yellow. It is
bright, slightly soft, reddish yellow mineral [17].

Figure 1-4: pure gold Figure 1-5: impure gold

1.9.1Physical properties of gold:

The physical properties of gold are as follows:

Atomic weight 198.97 a.m.u

Atomic number 79
No. of naturally occurring isotopes 1
Melting point 1064 0C
Crystal structure FCC
Density gcm-3 19.3
Thermal conductivity Wm-1k-1 310
Electrical resistivity at 20 0C 0.022
Young’s modulus 79
Hardness 25
Tensile stress 124
 12

1.9.2 Chemical properties of gold:

The chemical properties of gold are as follows [18]:

Chemical formula Au
Chemical activity Gold is chemically inactive, and it is
extremely resistant to chemical actions.
Isotopes It has one stable isotope 197Au.
Alloys Silver and platinum
Reactivity with acids Aqua-Regia: A mixture of nitric acid and
hydrochloric acid can dissolve gold.
Reactivity with non-metals Gold does not react with non-metals
except with halogens with which it forms
alloys.
 13

1.10 Structure of gold nanoparticles:

In the bulk form, gold is a soft, yellow metal, with the face centered cubic crystal
structure, a melting point of 1064°C and excellent electrical conductivity. However, not
one of these ‘facts’ necessarily applies at the Nano-scale. The gold particles are spherical
in shape [19].

Figure 1-6 spherical gold particles.

The packing of gold Nano-particles is Face Centered Cubic as shown below:

Figure 1-7: fcc packing of gold nano-particles. (savka I. Streva)

 14

1.11 Different shapes of gold Nano-particles:

Gold nanoparticles exhibit various sizes ranging from 1 nm to 8 μm and they also exhibit
different shapes such as spherical, Nano- rod, branched, Nano-star, Nano cluster,
Nanotube, Nano sphere Nano shell [20]. Different shapes of gold Nano-particles are
shown below:

Figure 1-8 different shapes of gold Nano-particles.

At Nano-scale the dimensions of gold Nano-particles are mentioned in table below:

Shape of gold Nano-particle: Dimensions (radius)

Silica coated gold Nano-shells 40nm-155nm
Gold Nano-sphere 20nm-80nm
Gold Nano-rod 3.1nm-3.9nm
Table 1.2 showing dimensions of different gold-Nanoparticles [21].

1.12 Properties of gold at Nano-scale:

At Nano-scale there is an enormous change in properties of gold Nano-particles which
includes:

1. Extraordinary surface area to volume ratio.

2. Strange color.
 15

3. Unusual crystal structure.

4. Anomalous melting point.
5. Tunneling electrons, quantized charge and white LEDs.
6. Enhanced strength and toughness.

1. Extraordinary surface area and chemical reactivity

The first and perhaps most important point to note is that, at the Nano-scale, there is a
huge increase in the surface-to-volume ratio of any material [22].

Figure 1-9 increase in surface area to volume ratio by reducing the spherical shape of the
Gold Nano-particles.

2. Strange color:
Bulk gold has a familiar yellow color, caused by a reduction in reflectivity for light [23].
When the particles of gold are small enough their color is ruby red, due to their strong
absorption of green light at about 520 nm, corresponding to the frequency at which a
Plasmon Resonance occurs with the gold. This effect has been used to color glass, even in
Roman times. However, if such tiny particles are allowed to come together in a controlled
fashion, their color can be systematically varied from pink through violet to blue. This is
due to a change in their absorption spectrum on aggregation, caused by increasing
absorbance of the red wavelength of light gold is one of the very few metals noble enough
to survive as a Nano-particle under atmospheric conditions [24].

3. Unusual crystal structures

Bulk gold is face centered cubic (FCC), but at Nano-scale the crystal structure may be
[25]:

1. Cubic

2. Octahedral
 16

3. Rhomb dodecahedral crystal

The FCC structures of gold sustain down to particles of about 10 nm in size (which
contain about 28,000 atoms of gold) but for even smaller sizes the situation is more
complex. The crystal structure of the elements in the bulk state is largely determined by
their electronic configurations, and the element will adopt the packing arrangement that
minimizes its free energy. However, as we have seen, for Nano-particles the surface atoms
become a hugely significant factor. Now the internal energy of the substance must be
minimized with respect to electronic configuration and surface energy and elastic strain
[26].

4. Anomalous melting points

Bulk gold melts at 1064°C. However, not even this property changes at Nano scale. The
melting point of gold Nano-particles is depressed from that of the bulk material. The
reason for this phenomenon is the huge increase in surface area of gold Nano-particles.
The calculations indicate that catalytically active gold particles in the 5 nm size
range(Au3600) would be molten at about 830°C, particles of about 2 nm (about Au200)
would liquefy at 350°C and, by extrapolation, particles of about 1nm(~Au30) diameter
would be molten at 200°C[27].

5. Tunneling electrons, quantized charge and white LEDs:

It is well known that electric current in most (but not all) conductors is associated with the
movement of electrons, and that this can occur in metallic conductors or in semi-
conductors. However, these ‘classical ’observations are not entirely accurate at the Nano
scale [28]. If gold Np’s are positioned between two conductors so that they are close
together but not actually touching, and apply a voltage between them, then there is a
probability that electrons will actually ‘tunnel’ through the gap between them. An electric
current will flow even though there is no physical contact. So at the Nano scale, electric
current may proceed in a kind of jerky way. Nano scale weirdness of this kind has been
used to produce a variety of interesting new electronic devices, such as the resonant
tunneling diode. The exploitation of yet other Nano scale phenomena have led to the
development of the latest generation of colored LEDs, which offer conversion efficiencies
that now compete those of incandescent lighting [29].
 17

Figure 1-10: showing tunneling effect of Au Np’s.

6. Enhanced strength and toughness:

The strength and toughness of bulk gold is greatly enhanced if they are reduced to Nano-
scale. Bulk material on application of stress gets deformed due to dislocation of atoms.
Nano-scale material is too small to cause its dislocation [30].

1.13 Optical properties of gold Nano-particles:

The color is due to the collective oscillation of the electrons in the conduction
band, known as the surface Plasmon Oscillations. The oscillation frequency is usually in
the visible region for gold and giving rise to the strong surface Plasmon resonance
absorption. Therefore, the origins of properties on the Nano scale are different for metal
nanoparticles than for semiconductor nanoparticles. Fig. 1-12 exemplifies the optical
property of gold nanoparticles.

Figure 1-11: The phenomenon of surface Plasmon resonance happens when

electromagnetic radiation falls of surface of gold Nano-particles.
 18

Many applications became possible due to the large enhancement of the surface
electric field on the metal nanoparticles surface. The Plasmon resonance absorption has
absorption coefficient orders of magnitude larger than strongly absorbing dyes. Metal
nanoparticles generate enhanced electromagnetic fields that affect the local environment.
The field is determined by the geometry of the gold nanoparticle and can enhance
fluorescence of the metal itself, the Raman signal of a molecule on the surface, and the
scattering of light. The optical properties of noble metal nanoparticles lead to many uses as
sensing and imaging techniques [31].

1.14 Cancer:
Cancer develops when normal cells in a particular part of the body begin to grow
out of control. There are different types of cancers; all types of cancer cells continue to
grow, divide and re-divide instead of dying and form new abnormal cells. As cells become
more and more abnormal, old or damaged cells survive when they should die, and new
cells form when they are not needed. These extra cells can divide without stopping and
may form growths form tumors.

Forms of cancer:
We have basically two forms of tumors as follows:

Benign tumor:
Characteristics of Benign Tumor:

non- non- slow

capsulated
cancerous invasive growing

Malignant tumor:

Characteristics of malignant tumor:

fast non- invasive

cancerous metastasize
growing capsulated and infiltrte
 19

1.14.1 Types of cancer:

These are major types of cancer as follows

1. Carcinoma
2. Sarcoma
3. Melanoma
4. Lymphoma
5. leukemia

Carcinoma:
Carcinomas are cancers or malignancies that begin in the epithelial cells, which are the cells that
make up the skin, and the tissues that line various internal organs and structures. Some of the most
common carcinomas affect the breast, lung, prostate, and colon.

Figure 1-12: This fig. shows cancer in the epithelial layer of skin.

Sarcoma:
A sarcoma grows in the body’s connective tissue cells, which include fat, blood vessels,
nerves, bones, muscles, deep skin tissues and cartilage

 Soft tissue sarcoma, which forms in soft tissues.

 Bone sarcoma (or osteosarcoma), which develops in bone tissue, cartilage or
bone marrow.
 20

Melanoma:

Melanoma is a form of skin cancer that arises when pigment-producing- cells known as
melanocytes-mutate and become cancerous. The treatment of skin cancer is similar to that of other
cancers, but, unlike many internal cancers, it is easier to access the cancer to remove it completely.
Surgery is the most common treatment for melanoma.

Lymphoma:
Lymphoma is a cancer of the lymphatic system. It affects a type of white blood cells
known as lymphocytes. These help fight disease in the body. They play an important role
in the immune system.

Leukemia:
Leukemia is a cancer which starts in blood-forming tissue, usually the bone marrow. It leads to the over-
production of abnormal white blood cells, the part of the immune system which defends the body against
infection [32].

Figure 1-13: showing healthy blood cells and leukemia blood cells.

1.14.2 Stages of cancer:

Staging helps describe where a cancer is located, if or where it has spread, and whether it
is affecting other parts of the body. Doctors often use diagnostic tests to determine a
cancer’s stage.

Staging can be

 Clinical: clinical staging is based on the results of tests done before surgery, such
as physical examinations and imaging scans.
 21

 Pathological: Pathological staging is based on what is found during surgery.

Stage-0:
This stage describes cancer in situ, which means “in place.” Stage 0 cancers are still located in
the place they started and have not spread to nearby tissues. This stage of cancer is often
highly curable, usually by removing the entire tumor with surgery.

Stage-I:
This stage is usually a small cancer or tumor that has not grown deeply into nearby tissues. It
also has not spread to the lymph nodes or other parts of the body. It is often called early-stage
cancer.

Stage II and Stage III.

In general, these 2 stages indicate larger cancers or tumors that have grown more deeply
into nearby tissue. They may have also spread to lymph nodes but not to other parts of the
body.

Stage IV.
This stage means that the cancer has spread to other organs or parts of the body. It may
also be called advanced or metastatic cancer.

Figure 1-14 figure showing different stages of cancer.

 22

1.14.3 Causes of cancer:

Infectious agents:
The major mechanisms by which infectious agents can promote and maintain tumor
formation can be divided broadly into three main categories. The first is the induction of
chronic inflammation as a result of a continuing immune response to a persistent infection.
This occurs, for example, in the case of hepatitis C virus (HCV) second, oncogenes are
can occur through virus-induced transformation. This is due to the Persistence of the viral
genome in a latent form in an infected cell, either without replication, as with Epstein-Barr
virus (EBV), which infects B lymphocytes .The third mechanism is the chronic
suppression of the immune system by the infectious agent, such as the immunodeficiency
(AIDS) caused by HIV infection.

Mutations:

Mutations in several critical genes can lead to tumors. It is a breakdown of the controls
that regulate cells. That is changes in important genes, changes in the DNA sequence of
chromosomes. The bases (A, T, G, and C) in DNA are altered or lost due to
unrepaired replication errors. For example, the loss of an amino group from cytosine, a
normal base found in DNA, leads to the production of uracil, a base not normally found in
DNA. If this change is not detected and reversed, a mutation can result [33].

Figure 1-15: figure showing how cancer tends to involve multiple mutations.

Diet:

The lowest dietary intake of fruits and vegetables compared to the highest intake has
roughly twice the cancer rate for most types of cancer .using canned food ,genetically
modified food, hydrogenated oils ,white flour ,microwave cooked items etc. is the big
 23

cause of cancer. Nitrosamines are formed from nitrogen oxides present in gas flames or
from other burning. Surprisingly little work has been done on the levels of nitrosamines in
fish or meat cooked in gas ovens or barbecued, considering their mutagenic and
carcinogenic potency.

Alcohol and smoking:

Using tobacco is the major cause of lung cancer in today’s world. Smoking is a severe
oxidative stress, and smoke contains a wide variety of mutagens and rodent carcinogens.
The oxidants in cigarette smoke (mainly nitrogen oxides) deplete the body's antioxidants.
Alcohol has some carcinogen nature that’s why it causes cancer of different types.

Radiation-exposure:

For x-rays or gamma-rays, good evidence of an increase in risk for cancer is shown at
acute doses greater than expected from basic radiobiology, the doses above which
statistically significant risks are seen are somewhat higher for protracted exposures than
for acute exposures specifically evidence of an increase in some cancer risks is shown for
protracted doses greater than 100 mSv, and reasonable evidence for an increase in cancer
risk at protracted doses above 50 mSv. In estimating the lowest dose of x-ray or gamma
ray radiation for evidence of increased cancer risks, it is important to make the distinction
between acute exposures over a very short period (such as the atomic bomb exposures)
and protracted exposures (such as occupational or fractionated exposure). In general,
protracted exposure to x-ray or gamma radiation are associated with lower risks than those
of an acute exposure to the same total dose, both for cancer and other endpoints.

Chapter # 2
Literature review
 24

Chapter no#2

Literature review
Xiaohua Huang, et al. in 2006 has published an article on cancer cell imaging in in this
article they have studied that due to strong electric fields at the surface, the absorption and
scattering of electromagnetic radiation by noble metal nanoparticles are strongly
enhanced. These unique properties provide the potential of designing novel optically
active reagents for simultaneous molecular imaging and photothermal cancer therapy. It is
desirable to use agents that are active in the near-infrared (NIR) region of the radiation
spectrum to minimize the light extinction by intrinsic chromo-phores in native tissue. Gold
Nano-rods with suitable 650 aspect ratios (length divided by width) can absorb and scatter
strongly in the NIR region (-900 nm).In the present work, we provide an in vitro
demonstration of gold Nano-rods as novel contrast agents for both molecular imaging and
photothermal cancer therapy[34].
 25

Dakrong Pissuwan et al. in 2007 had discussed the therapeutic possibilities of plasmonically
heated gold Nano-particles. Nanoparticles of gold, which are in the size range of 10 to 100 nm,
undergo a Plasmon resonance with light. This is a process whereby the electrons of the gold
resonate with incoming radiation causing them to both absorb and scatter light. The effect can be
harnessed to either destroy tissue by local or release payload molecules of therapeutic importance.
Gold nanoparticles can also be conjugated with biologically-active moities, providing possibilities
for targeting to particular tissues. In this paper they review progress in the exploitation of this
Plasmon resonance of gold nanoparticles in photo-thermal therapeutic medicine.

Chae-kyu Kim et al. in 2009 has published an article on Multimodal drug delivery using gold
nanoparticles. In this article they have reviewed that Gold nanoparticles (AuNPs) are
promising Nano carriers for therapeutics due to their facile synthesis, ease of
functionalization, biocompatibility, and inherent non-toxicity. The unique chemical and
physical properties of AuNP monolayers provide versatility in delivery method and
tunability of surface properties. In this paper they have discussed several strategies to
utilize the properties of AuNPs for drug delivery. Including cellular uptake of gold Nano-
particles, monolayer encapsulation of therapeutics and light regulated release of drug using gold
Nano-particles.

Xu Zang et al., in 2012 reviewed the application of nanotechnology in medicine, known

as Nano medicine, has introduced a plethora of nanoparticles of variable chemistry and
design considerations for cancer diagnosis and treatment. One of the most important field
is the design and development of pharmaceutical drugs, based on targeted drug delivery
system (TDDS). Being inspired by physio-chemical properties of nanoparticles, TDDS are
designed.To safely reach their targets and specifically release their cargo at the site of
disease for enhanced therapeutic effects, thereby increasing the drug tissue bioavailability.
Nanoparticles have the advantage of targeting cancer by simply being accumulated and
entrapped in cancer cells. However, even after rapid growth of nanotechnology in Nano
medicine, designing an effective targeted drug delivery system is still a challenging task.
In this review, we reveal the recent advances in drug delivery approach with a particular
focus on gold nanoparticles. We seek to expound on how these Nano-material
communicate in the complex environment. To reach the target site, and how to design the
effective TDDS for complex environments and simultaneously monitor the toxicity on the
basis of designing such delivery complexes. Hence, this review will shed light on the
research, opportunities and challenges for engineering nanomaterial’s with cancer biology
and medicine to develop effective TDDS for treatment of cancer [35].
 26

AK Khan et al. in 2014 have studied the use of gold Nano-particles in targeted drug
delivery of cancer. They focused essentially on the synthesis and applications of gold
nanoparticles in the field of medicine and targeted drug delivery. Nanotechnology has
become one of the most interesting and advanced areas of research in this field. Among
nanoparticles, gold nanoparticles demonstrate special advantages in this field due to their
unique properties, small size and high surface area-to-volume ratio. These particles have
been widely used in various biomedical applications and drug delivery systems due to
their inert nature, stability, high dispersity, non-cytotoxicity and biocompatibility [36].

Nardine S. Abadeer et al, in 2016 had publishes a journal article on use of gold Nano-
particles in cancer therapy .In recent years; there has been a great deal of interest in the
preparation and application of nanoparticles for cancer therapy. Gold nanoparticles are
especially suited to thermal destruction of cancer due to their ease of surface
functionalization and photothermal heating ability. Here, we review recent progress in
gold nanoparticle-mediated thermal cancer therapies. We begin with an introduction to the
properties of gold nanoparticles and heat-generating mechanisms which have been
established. The pioneering work in photothermal therapy is discussed along with the
effects of photothermal heating on cells in vitro. Additionally, radiofrequency-mediated
thermal therapy is reviewed. We focus our discussion on the developments and progress in
nanoparticle design for photothermal cancer therapy since 2010. This includes in vitro and
in vivo studies and the recent progression of gold nanoparticle photothermal therapy
toward clinical cancer treatment [37].

Fen-Ying Kong et al, in 2017 has published a review paper on “Unique Roles of Gold
Nanoparticles in Drug Delivery, Targeting and Imaging Applications” they had reviewed
that the Nanotechnology has become more and more potentially useable in diagnosis and
treatment of diseases. Advances in nanotechnology have led to new and improved
Nanomaterial’s in biomedical applications. Common Nanomaterial’s applicable in
biomedical applications include liposomes, polymeric micelles, graphene, carbon
nanotubes, quantum do, gold nanoparticles (Au NPs), and so on. Among them, Au NPs
have been considered as the most interesting nanomaterial because of its unique optical,
electronic, sensing and biochemical properties. Au NPs have been potentially applied for
medical imaging, drug delivery, and tumor therapy in the early detection, diagnosis, and
treatment of diseases. This review focuses on some recent advances in the use of Au NPs
as drug carriers for the intracellular delivery of therapeutics and as molecular Nano-probes
for the detection and monitoring of target molecules.

Bindeshwar Sah et al. in 2019 have studied the “Effect of size on gold nanoparticles in
radiation therapy: Uptake and survival effects” .Radiation therapy is one of the most
 27

commonly used techniques for the treatment for cancer. A major goal of radiation therapy
is to damage cancer cells, while simultaneously imparting as small a radiation dose as
possible to nearby healthy cells. Due to a high atomic number and the Auger effect, gold
nanoparticles can significantly enhance doses of ionizing radiation. The amount of
enhancement due to gold nanoparticles strongly depends upon several parameters, such as
cellular uptake of nanoparticles, nanoparticles size, concentration, intracellular location
and radiation energy.

Existing literature shows that nanoparticle size can affect the amount of uptake and radio-
sensitization. In this review article, we describe the effect of nanoparticle size on the gold
nanoparticle-mediated effect, touching on both of these clinically important variables. The
results suggest that non-targeted gold nanoparticles see maximum uptake and maximum
radiation therapy enhancement around 50 nm [38].

CHAPTER # 3
METHODOLOGY
 28

Chapter#03

Materials Methods:

3.1 Cancer treatment using Nano-technology:

Cancer drug delivery achieving high therapeutic efficacy and low side effects requires a
Nano carrier to tightly retain the drug, efficiently reach the tumor, then quickly enter the
tumor cells and release the drug. Furthermore, the Nano carrier intended for clinical
applications should use materials safe as pharmaceutical excipients and its formulation
(Nano medicine) should have good manufacture processes with scale-up ability. Thus, the
challenge is to design safe, approvable, and easily scaled-up Nano carriers that
simultaneously meet the two pairs of requirements of ‘drug retention in circulation versus
intracellular release’ and ‘stealthy in circulation versus sticky (cell-binding) in tumor’ at
the right places in order to deliver a cytosolic drug dose lethal to cancer cells with
minimized side effects[39].
 29

Cancer nanotechnology has the potential to dramatically improve current approaches to cancer
detection, diagnosis, imaging, and therapy while reducing toxicity associated with traditional
cancer therapy [40]. A wide variety of nanomaterial’s are currently under investigation and
development for application relative to cancer nanotechnology. These include polymers,
dendrimers, lipids, organometallic, and carbon based materials.

Properties of Nano-particles:
Four unique properties of Nano-particles that distinguish them from other cancer
therapeutics are:

1. The nanoparticle, which can itself have therapeutic or diagnostic properties, can be
designed to carry a large therapeutic “payload”.

2. Nano particles can be attached to multivalent targeting ligands which yield high affinity
and specificity for target cells.

3. Can be made to accommodate multiple drug molecules that simultaneously allow

combinatorial cancer therapy.

4. And nanoparticles can bypass traditional drug resistance mechanisms.

3.2 Cancer drug delivery process using

nanoparticles:

Cancer drug delivery is a process using Nano carriers with appropriate sizes
(usually between several nanometers and 200 nm) and stealth properties to preferentially
carry drugs to tumor tissues via the enhanced permeability and retention (EPR) effect.
However, despite the improved pharmacokinetic properties and the reduced adverse
effects, currently cancer drug delivery has only achieved modest therapeutic benefits [41].

Thus, the design of Nano carriers with more efficient drug delivery and thus higher
therapeutic efficacy is still a pressing need. The cancer drug delivery process can be
divided into three stages, shown in figure below:
 30

Figure 3-1 Mechanism of drug delivery using gold Nano-particles.

 Initially, the drug-loaded Nano carriers circulate in the blood compartments,

including the liver and the spleen. When passing through tumor blood vessels,
some carriers may fall into the pores in the blood vessel wall and diffuse into the
tumor tissue (EPR effect).
 Next, they may further penetrate the tumor tissue, which is non-trivial because of
the high cell density and high interstitial osmotic pressure (B).
 Upon sticking to the surrounding cancer-cell membrane (C), the carrier is expected
to enter the cells via one or several possible pathways, and finally traverse the
crowded intracellular structures and viscous cytosol to the targeted subcellular
sites and release the carried drug cargo [42].

Thus, in order to achieve efficient drug delivery from injecting Nano based drug to the
target in the tumor cells, the drug must simultaneously meet two pairs of challenges:

 The Nano carrier must carry drug very tightly, and should only release at target to
exert its therapeutic action. It must not release during transportation in blood
compartments.
 31

 The Nano carriers must be “slippery” or “stealthy” while in the blood

compartments to effectively travel towards target and then it must also be taken up
by tumor cell i.e. after specific time drug must become “sticky” or cell binding.
 A Nano carriers capable of performing opposite requirements at the same time that
is,
a) Drug must be retained in blood circulation versus targeting in tumor cells
b) Stealthy in blood versus Sticky in tumor
Nano-carriers will accurately deliver drug. Thus increasing efficiency of drug and
minimizing the side effects.

This is how Nano-particles can be employed for cancer therapeutics.

 Nanoparticles can be made to contain multiple targeting ligands that can provide
multivalent binding to cell surface receptors
 Can be made sufficiently large to accommodate multiple types of drug molecules
 Can bypass traditional means of drug-resistance mechanisms that involve cell
surface protein pumps, because they enter the cell through endocytosis.
 Can carry a large number of “effector” molecules that have no effect on
pharmacokinetics or biodistribution [43].

3.3 Gold Nano-particles in cancer therapeutics:

Study of various chemical and physical properties of gold Nano-particles shows that gold
Nano-particles are very helpful in various medical applications including diagnosis and
treatment of cancer. Gold itself can act as contrast in imaging and in delivery of anti-
cancer drug to the site of cancer.
 32

Figure 3-2 showing detailed view of gold Nano-particle employed in cancer therapeutics.

3.3.1 Imaging:
Imaging application of gold-nanoparticles:

1. x-ray imaging/CT
2. SERS
3. Photoacoustic
4. Optical imaging

 x-ray imaging:
X-ray imaging was invented by Wilhelm Rontgen in 1895. It utilizes high-energy
electromagnetic radiation to create images of internal structures. Today it is the most
widely used method of medical imaging, accounting for 50–75% of all medical
imaging done [44]. X-ray imaging is considered safe and cost-effective if the radiation
dose is monitored and limited over the lifetime of the patient.

In addition to being effective in scattering visible light, gold has a high X-ray
attenuation coefficient at the energy levels utilized for clinical X-ray and CT [45].
Research-use only gold nanoparticle formulations are available commercially as X-ray
contrast agent AuroVist™ from Nano probes at sizes of 1.9 and 15 nm. Additionally, a
number of innovative formulations of gold nanoparticles are under development for
 33

varied physiological applications to achieve targeted, high contrast X-ray images for
diseases diagnosis [46].

Contrast in X-ray: imaging is derived from the difference in mass attenuation

between two tissues. Materials with a high atomic number or density produced x-rays
which are more absorbed by bones. Lowering the energy of the X-ray beam creates a
higher contrast between two tissue types because photoelectric events predominate at
lower energy (<50 kVp) [47]. Contrast agents play an important role in allowing
higher energy, safer scans with high contrast by introducing high atomic number
media into the body [48].

Fig 3-3: x-ray imaging of a hand.

 CT–scan:

An important breakthrough in 1973 resulted in the development of CT [49]. Computed

tomography (CT) is among the most popular medical imaging modalities due to its high
resolution images, fast scan time, low cost, and compatibility with all patients. CT scans of
soft tissues require the localization of imaging contrast agents (CA) to create contrast,
revealing anatomic information. Gold nanoparticles (Au NP’s) have attracted interest
recently for their use as CT contrast agent due to their high X-ray attenuation, simple surface
chemistry, and biocompatibility and surface hydrophobicity, potential. Targeting molecules
may be attached to the particles to allow for the targeting of specific cell types and disease
states. AuNP can also be readily designed to incorporate other imaging contrast agents such
as rare earth metals and dyes. CT allows for 3-dimensional (3D) reconstructions of X-ray
images by rotating the detector and the X-ray source about the imaged body.
 34

Figure 3-4 pictorial view of CT scanner used in hospitals.

Cancer detection with actively targeted CT contrast agents takes advantage of the overexpression
of specific surface receptors on cancer cells and of the ability to create nanoparticles that can
specifically home to these receptor [50] .An important advantage of the active targeting approach
is the specificity of the findings; however, since this approach is based on the existence and degree
of overexpression of specific tissue biomarkers, it can be applicable only under particular
biological conditions. However, in vivo CT cancer detection through application of high atomic
number contrast agents remains challenging due to the large amount of gold that must be delivered
and accumulated on the tumor in order to induce sufficient signal to noise ratio in CT. This key
factor – the total amount of gold per voxel (three-dimensional pixel) – is determined mainly by:

(1) Nanoparticle size and their cellular labeling efficiency.

(2) The number of overexpressed receptors on the cancer cell’s surface.

Preparation of contrast agent:

As targeted contrast agents, 30 nm GNPs were prepared using sodium citrate, according to the
methodology described by Enüstun and Turkevich [54]. Particle size, shape, and uniformity were
measured using transmission electron microscopy and proved to be 30 nm diameter spheres with
narrow size distribution (10%). A protective layer of PEG was absorbed on the surface of the
GNPs in order to reduce nonspecific interactions and to prolong circulation time of the
nanoparticles in the blood stream. The PEG layer consisted of a mixture of thiol-polyethylene-
glycol (mPEG-SH) (~85%, MW ~5 kDa) and a hetero functional thiol-PEG-acid (SH-PEG-
COOH) (~15%, MW ~3.4 kDa).
 35

 Optical imaging

Gold is a non-reactive metal but its properties changed at nanometer scale due to drastic
changes in electron behavior at length scale.

The applications of nanoparticles in three aspects for biological imaging are discussed:

1. Direct visualization of AuNPs in biological system e.g. Dark field microscopy

(DF), photothermal imaging.
2. Monitoring of biomolecular events and physiological processes such as SERS,
detection of nuclei acids and protein.
3. In vivo-deep tissue imaging using, Photoacoustic imaging and optical coherence
tomography (OCT) [51-54].

 Surface enhanced Raman scattering:

Surface-enhanced Raman spectroscopy or surface-enhanced Raman
scattering (SERS) is a surface-sensitive technique that enhances Raman
scattering by molecules adsorbed on rough metal surfaces or by nanostructures
such as plasmonic-magnetic silica nanotubes. (Xu) The enhancement factor can be
as much as 1010 to 1011 which mean the technique may detect single molecules
[55].

Figure 3-6 Surface Emission Reman spectroscopy.

Raman scattering is a vibrational spectroscopic technique relying on the inelastic collision between
an incoming source of light and an analyte of interest. This inelastic collision is responsible for the
 36

scatter of a lower energy radiation that serves as a fingerprint and provides information regarding
structure, interaction or environment of the analyte [56].

.SERS substrates are used to detect the presence of low abundance biomolecules, and can therefore
detect proteins in body fluids [57] .Early detection of pancreatic cancer biomarkers was
accomplished using SERS-based immunoassay approach.

A SERS-base multiplex protein biomarker detection platform in a microfluidic chip is used to

detect several protein biomarkers to predict the type of disease and critical biomarkers and increase
the chance of diagnosis between diseases with similar biomarkers [58]. This technology has been
utilized to detect urea and blood plasma label free in human serum and may become the next
generation in cancer detection and screening [59].

The ability of SERS at Nano scale is beneficial for environmental analysis, pharmaceuticals,
material sciences, art and archeological research, forensic science, drug and explosives detection,
food quality analysis [60].

SERS can be used to target specific DNA and RNA sequences using a combination of gold and
silver nanoparticles and Raman-active dyes.

Photoacoustic imaging:

Biomedical Photoacoustic tomography (PAT), also called optoacoustic tomography or

thermoacoustic tomography, is based on the Photoacoustic (PA) effect, which was first described
in 1880 by Alexander Graham Bell.

The combination of Photoacoustic imaging with nanotechnology holds promise for determining the
structural and functional properties of tissues with enhanced sensitivity and specificity and for
monitoring the treatment of diseases.

Photoacoustic imaging is a relatively new method that takes advantage of the same optical
properties as fluorescence imaging. Tissues are irradiated by visible or near-infrared light resulting
in adiabatic expansion. This creates pressure waves, which are in turn measured and used to
reconstruct an image. The modality depends upon the optical and thermal properties of the tissues.
Contrast agents are utilized in cases where depth of penetration is low or there is a lack of natural
contrast between tissues [61].
 37

Particles were added to cells in vitro and irradiated using a tunable laser. Untreated cells showed a
linear photoacoustic response, while treated cells displayed a time variant signal, indicating
particle uptake and particle contrast ability [62].

Gold nanoparticles have become a prime candidate for PAT due to their unusual optical properties
and inherent biocompatibility. Gold nanoparticles served as contrast agent for in vivo tumor
imaging. PEGylated gold nanoparticles (50 nm) (200 μL, 10 mg/mL) were administered via tail
vein and serial PA images were made of tumors following systemic administration. The
accumulation of untargeted PEGylated Nanoparticles inside tumors following systemic
administration is expected and has been demonstrated to occur via a process called “enhanced
permeability and retention [63].

Figure 3-7: PAT images of tumor following tail vein injection of gold nanoparticles at 5 min
following injection (a) and 5 h following injection (b). The color scale (right) represents
optical absorption of tissue (arbitrary units).

Gold Nanorods conjugated with an antibody were also used for targeting cancer cells and for PA
imaging of a single layer of cells. [64]The study validated that gold Nanorods produce high
contrast between targeted tissue and non-targeted tissue for PAT imaging in an in vitro
experiment. The study also showed that Nanorods-based PA imaging appear to be attractive for
early detection of prostate cancer.
 38

Sensing applications of gold nanoparticles:

These are main methods for the surface modification/functionalization of AuNPs

1. Noninvasive detection of serum biomarkers including circulating proteins and nucleic

acids in biological fluids is of central importance for early disease diagnosis, screening and
staging of diseases, as well as the evaluation of response to therapy.
2. Adsorption-based, where the interaction between the ligand and the AuNP surface is held
by electrostatic or hydrophobic interaction.
3. Covalent bonding, where the ligand is linked to the AuNP surface through a thiol group,
either direct conjugation of sulfur containing molecule or through a bi-functional linker (a
thiol group at one extremity that binds to the AuNP and another functional group at the
other extremity, where other biomolecules can be attached).
4. Affinity-based, where the AuNP surface is functionalized with moieties that provide
affinity sites for the coupling of biomolecules. AuNPs are a very versatile scaffold for the
development of sensing platforms. Herein, the most prevalently used bio receptors that
allow AuNPs to have specific biomolecular recognition abilities will be described.

1. Protein detection:

Serum protein marker screening lacks sensitivity and specificity, and their role in early
diagnosis and treatment only [65].
 39

Figure 3-8: Protein coating of Nano-particles for protein delivery.

Proteins are another class of biopolymers that can be used as bio receptors in bio sensing
platforms. Antibodies (Ab) are one of the most used protein bio receptors for bio sensing
due their specificity towards their respective antigen. Peptides and enzymes are also
versatile bio receptors for AuNP-based sensing as they can be functionalized to the surface
of AuNP without losing their bio recognition capabilities [66]. For instance, the high local
concentration of immobilized enzymes at the surface of AuNP coupled to their catalytic
specificity can be used for signal enhancement in LFA platforms, due to a higher local
concentration of the colored product (revelator). This principle was applied for the detection
of human IgG in an LFA format. [67] Peptides on the other hand, can serve as binding
partners of an interaction event or be a substrate for specific reactions.

2. Nuclei acid detection:

Detection of circulating nucleic acids including DNA and RNA in the plasma and serum of
cancer patients, which has genetic characteristics identical to those of the primary tumor, is
an emerging field for noninvasive molecular diagnosis of cancers and is attracting
considerable interest [68]. DNA and RNA characterization for diagnostics purposes relies
on the hybridization of a probe to a given target exploring the strand complementarity
resulting from specific and stable Watson–Crick pairing. DNA/RNA detection schemes are
based on the remarkable optical properties of AuNPs supplementary to the easiness of
chemical functionalization through thiol ligands (e.g., thiol modified oligonucleotides,
antibodies, and other biomolecules).

Figure 3-9: mechanism of nucleic acid detection using gold Nano-particles.

 40

According to the present data, it might be possible to detect over 80% of patients with
cancers using a combination of appropriate circulating RNA and DNA markers. Most of the
detection of cancer biomarkers focused on the detection of proteins or nucleic acids
independently. Research has demonstrated that the combination of protein and nucleic acid
markers could be useful for the diagnosis of early-stage diseases and improving the
sensitivity and specificity of diagnosis [69].

Mirkin et al. first described the use of thiolate oligonucleotides (short single strand DNA,
ssDNA) as a capping agent for AuNP in 1996 [70]. This ssDNA-modified AuNP, through a
precise temperature control, was able to discriminate between a base pair mismatch from a
fully complementary target [71] .Since then, there have been several studies demonstrating
the vast capability of AuNPs functionalized with short nucleic acid sequences to sense a
wide range of clinically relevant nucleic acid sequences. Recognition events occur at the
Nano scale, i.e., in a one-to-one interaction between analyte and the Nano scale structures
that act as signal transducers, allowing for increased sensitivity at lower costs.

Figure 3.10: showing nucleic acid imaging using gold Nano-particles.

 Colorimetric sensing depending on inter particle distance, which represents these best
developed approach, especially for nucleic acid detection;
 Fluorescence quenching/enhancement properties of AuNPs.
 Plasmonic and light scattering properties, including detection based on surface-enhanced
Raman (SERS) and LSPR spectroscopies;
 41

 Piezoelectric sensor using AuNPs to increase sensitivity of detection based on mass

increase
 Electrochemical detection methods based on electrical signal enhancement or generation
provided by AuNPs, using lateral flow devices.

3.3.2 Drug delivery:

1. Gold Nano-particles used for cancer gene delivery:
Following three properties of gold Nano-particle make it applicable for gene therapy:

A. Inertness.
B. Ease of functionalization with thiol linkages.
C. Plasmon resonance.
Gene therapy and the methods of gene delivery:
Gene therapy refers to a mode of treatment that involves introducing new genes
into cells, repairing or replacing existing abnormal genes, or regulating the expression of
particular genes [72]. There are two types of gene therapy, namely somatic gene therapy
and germline gene therapy. In brief, somatic gene therapy involves treating diseases by
genetically modifying somatic cells such that the changes made are only limited to the
patient [73]. Germline gene therapy, on the other hand, aims to correct genetic disorders
in either reproductive cells such as ova and sperm or early embryos, and therefore, any
genetic alterations introduced via this type of gene therapy are inheritable [74].

Method of gene therapy:

Generally, it is possible to accomplish gene therapy by introducing naked DNA

into target cells; however, some nucleic acid-based medicines are not able to cross the
cellular membrane by simple passive diffusion methods because of the negative charge of
large DNA molecules and the negative nature of the cellular membrane. Therefore, it is
important to use a vector to assist the progress of transferring DNA molecules into the
cell. Both somatic gene therapy and germline gene therapy, whether in vitro or in vivo,
require vectors in order to insert genes into cells [75].
 42

Figure 3-11 figure illustrating scenario of gene delivery.

3.3.2 Drug targeting using Gold Nano-particles:

Gold Nano-particles can be employed for the drug targeting at tumor sites. For that
purpose two main conditions must be fulfilled as discussed earlier one the gold particles
must be stealthy during their passage from blood vessels secondly on reaching the
cancerous sites the drug must be revealed from the particle and destroy the cancerous cell.
A gold nanoparticle with 2 nm core diameter could be, in principle, conjugated with ~100
molecules to available ligands (n=~108) in the monolayer [76]. Zubarev et al. have
recently succeeded in coupling of ~70 molecules of paclitaxel, a chemotherapeutic drug,
to a GNP with 2 nm core diameters [77].

Studies shows there are two main forms of drug targeting using gold Nano-particles that
are:

1. Passive targeting

2. Active targeting

Passive targeting:
Passive targeting of nanoparticles relies on abnormal gap junctions (100–600 nm) in the
endothelium of tumor blood vessels for accumulation of gold nanoparticles in tumors. In
order to achieve passive targeting of gold nanoparticles, engineering of particles with
long-circulation half-lives [such as coating particles with hydrophilic polymer such as
 43

polyethylene glycol (PEG)] is most desirable and this type of construct favors passive
accumulation of

Particles inside tumors [78]. Passive tumor targeting takes advantage of the irregularity
and leakiness of tumor vasculature to allow nanoparticle accumulation in the tumor
(caused by

the enhanced permeability and retention effect) [79].

Figure 3-12 figure showing mechanism of passive targeting.

Active targeting:

In the active mode, molecular ligands such as antibodies, peptides, or small molecules are
used to recognize specific receptors on the tumor cell surface, often followed by receptor-
mediated endocytosis and gold nanoparticle internalization [80].
 44

Figure 3-13: figure illustrating mechanism of active drug delivery.

3.3.3 Cancer therapy using gold Nano-particles:

Photo-thermal therapy:
Noble metal nanoparticles, on account of the phenomenon of surface Plasmon resonance, possess
strongly enhanced visible and near-infrared light absorption; several orders of magnitude more
intense compared to conventional laser phototherapy agents. The use of plasmonic nanoparticles as
highly enhanced photo absorbing agents has thus introduced a much more selective and efficient
cancer therapy strategy, also called plasmonic photothermal therapy (PPTT).
Plasmonic gold nanostructures make the PPTT (plasmonic photo-thermal therapy) method
furthermore promising. The development of the PPTT method with special emphasis on the recent
in vitro and in vivo success using gold Nano-spheres coupled with visible lasers and gold Nano-
rods and silica–gold Nano-shells coupled with near-infrared lasers is very useful from the point of
the view of cancer therapeutics, noble metal nanoparticles become very useful as agents for PTT
on account of their enhanced absorption cross sections, which are four to five orders of magnitude
larger than those offered by conventional photo absorbing dyes. This strong absorption ensures
effective laser therapy at relatively lower energies rendering the therapy method minimally
invasive. Additionally, metal nanostructures have higher photo-stability, and they do not suffer
from photo-bleaching. Currently, gold Nano-spheres [81]–[82], gold Nano-rods[83], gold Nano-
 45

shells,[84] gold Nano-cages,[85] are the chief nanostructures that have been demonstrated in
photothermal therapeutics due to their strongly enhanced absorption in the visible and NIR regions
on account of their surface Plasmon resonance (SPR) oscillations. Of these structures, the first
three nanostructures are especially promising because of their ease of preparation, ready multi-
functionalization, and tunable optical properties.

Figure 3-14: figure illustrating photo-thermal therapy using Au Nano-particles.

Photo-thermal therapy mechanism of action:

When the PTT agents absorb light, electrons make transitions from the ground state to the
excited state. The electronic excitation energy subsequently de-excites through non-
radiative decay channels. This results in the increase in the kinetic energy leading to the
overheating of

Local environment around the light absorbing species. The heat produced can be
employed for local cell or tissue destruction [86].

Radiation therapy:
Ionizing radiation can directly or indirectly damage DNA and disrupt the atomic
structure of biomolecules [87]. DNA repair mechanisms may fail leading cells to stop
dividing, die or be mis-repaired, thus acquiring mutations that can result in malignant
transformation.[91] Therefore, avoiding normal tissue is of significant importance in
reducing secondary side effects of radiotherapy. This is done by the strong photoelectric
absorption and secondary electron caused by gamma or X-ray irradiation that can
accelerate DNA strand breakage [88].
 46

Gold nanoparticles (GNPs) possess a number of unique properties that make them
ideal candidates as radio sensitizers on the basis of their strong photoelectric absorption
coefficient and ease of synthesis and bio-compatibility. The differential response between
tumor and normal cells to external radiation is termed therapeutic ratio, can be increased
through the introduction of high-atomic number (Z) material into the target such as Gold
(Z=79) that is a promising radiosensitizer in this regard due to its high atomic number and
mass energy coefficient relative to soft tissue. The mass energy coefficient of gold is 100–
150 times greater than that of soft tissue in the Kev energy range [89]. Consequently, there
is an increased probability of photoelectric interaction at lower energy levels, resulting in
increased energy deposition at the target site. The physical and biological mechanism of
gold Nano-particles as radiotherapy agents is as follows:

Physical mechanism:
The main physical mechanisms through which radiation interacts with nanoparticles in the
Kev range are the:

1. Compton effect

2. Photoelectric effect

In photo-electric effect an incident photon can either be partially or fully absorbed by an

electron from the atom, causing its ejection [90]. The Photoelectric effect electrons are
ejected preferably from an inner atomic orbital. The vacancy left can then be filled by
another outer shell electron that falls to its place, further releasing low-energy photons
promoting a cascade release of secondary electrons [91]. This process is called the Auger
cascade and it is the major contribution to the production of low-energy electrons that
have a range of few micrometers and cause highly localized ionizing events. [92] High-Z
elements such as iodine, gadolinium and gold have been shown to have the ability to
image and radiosensitizer tumors [93].
 47

Figure 3-15: this figure shows the physical mechanism of Au involved in radiation
therapy.

Biological mechanism:
The main mechanisms identified as being involved in the biological response of cells

To gold nanoparticle in radio sensitization are the production of ROS and oxidative stress.

Oxidative stress:
Due to large surface to volume ratio the gold Nano-particles surface is electronically
active thus it catalyzes chemical reaction and promotes ROS and oxidative stress. One of
the identified mechanisms as a possible reason for cytotoxicity is through the interaction
of the NP surface with O2. In this process, donor electrons are transferred from the surface
of the NPs to oxygen molecules generating superoxide, which can lead to ROS production
through dismutation. This has been identified for single-component materials and for
transition metals on the nanoparticle surface [94]. Oxidative stress causes damage to cell
membranes, DNA and protein being identified so far as one of the major causes of NP
cytotoxicity [95]. A mechanism has been established significantly contributing to radio
sensitization, using 1.9-nm thiol-coated GNPs. Irradiation in the presence of GNPs led to
an interaction between GNPs and the cell membrane protein disulfide isomerase (PDI),
resulting in the disruption of thiol balance within the cell, thus causing cellular redox
imbalance and ultimately oxidative stress. This leads to significant increases in cell killing,
causing the GNPs to act as radiosensitizer [96].
 48

CHAPTER # 04
RESULTS AND DISCUSSION
 49

Chapter # 04

Results:
Gold nanoparticles have, in some ways, revolutionized the field of medicine because of its
wide spread applications in targeted drug delivery, imaging, diagnosis and therapeutics
due to their extremely small size, high surface area, stability, non-cytotoxicity and tunable
optical, physical and chemical properties. Functionalized gold nanoparticles with various
biomolecules such as proteins, DNA, amino acids and carboxylic acids have been used in
cancer therapy and provide excellent drug delivery system. Targeted delivery and
programmed release of therapeutic drugs to the specific site is achieved by using gold
nanoparticles because they can bear high drug load and release it to the specific site
through various administration routes and can interact with cancerous cell. Side effects of
conventional drugs have been minimized by conjugation with gold nanoparticles and they
increase the quality life of patients.

Gold nanoparticles have great potential as contrast agents in a variety of imaging

modalities. AuNP size and reactivity also allow their accumulation in certain organ
systems and tissues, especially cancerous tumors. Long circulating, nontoxic contrast
agents that can reveal anatomical and disease information across multiple imaging types
could ease clinical imaging burdens and simplify scanning procedures

The properties of gold at Nano scale vary significantly. For instance the color of gold
changed from bright yellow to reddish purple at Nano-scale gold. Gold is one of those few
metallic elements that are able to be used in Nano-scale devices and systems because it
does not oxidize easily. Another interesting phenomenon is electrical conductivity of gold
decreases and it becomes semi-conductor at Nano-scale. The melting point decreases
whereas the strength of gold nanoparticles increases. The crystal structure is not
necessarily FCC at Nano-scale it may be cubic, octahedral or any other depends upon
Nano-scale dimension. These properties and strange behavior of gold find its applications
in new commercial and scientific opportunities.

Gold nanoparticles have emerged as a promising platform for drug, gene and protein
delivery for the treatment of cancer. The property of gold Nano-particles in combination
of low inherent toxicity, high surface area and tunable stability provides them with unique
attributes that enable new delivery strategies.
 50

Discussion:
Using gold in treatment of cancer there are many strategies. Depending upon type of
cancer and at the same time keeping in concern the side effects of that treatment the
strategies are applied which are as follows:

photo
thermal
therapy
active and
radiation
paasive
therpay
targetting
methods of
using Au Np's
in drug
delivery and
therapy.

Gold Nano-particles possess unique physical and chemical properties that facilitate their
use in cancer diagnosis and treatment. Gold Nano-particles are widely used in cancer drug
delivery which includes active and passive targeting of drug to the cancer sites. Gold
Nano-particles can be attached with targeting ligands which yields specified drug
targeting. As gold Nano-particles have high surface to volume ratio their surface reactivity
is very important moreover a unique property of gold Nano-particles known as surface
Plasmon resonance make their use in photo thermal therapy in which the gold particles are
accelerated from external radiation source of visible and near infrared radiation. When
radiation falls on Au nanoparticles the electrons are accelerated giving rise to three
important phenomenon photoelectric effect, auger effect and Compton-effect. Gold Nano-
particles are also used as drug play load where the any anti -cancer drug can be loaded
inside a gold nanoparticle capsule which is then released inside a tumor. Gene delivery is a
type of cancer treatment in which normal gene is delivered to the cancerous cell which
works by replacing or repairing the effect gene. The inertness and easy thiol linkage of
gold Nano make them effective in gene delivery.

AuNP for use as X-ray contrast agents are currently available commercially, although
they are for research use only. In order to gain clinical acceptance, further in vivo human
research still exist as to their biodistribution, circulation times, and targeting ability. Still,
AuNP remain an excellent platform for X-ray contrast agents. Improvements in their
 51

ability to effectively circulate and localize at a desired area of interest will allow for their
use in clinical settings.

Figure 4-1: showing mechanism of curing cancer.

Nanoparticle drug delivery systems seems to be a viable and promising strategy for the
biopharmaceutical industry. The have advantages over conventional drug delivery system.
They can increase the bioavailability, solubility and permeability of many potent drugs
which are other difficult to deliver orally. Nano particulate drug delivery systems will also
reduce the drug dosage frequency and will increase the patent compliance.in near future
Nano particulate drug delivery systems can be used for exploiting many biological drugs
which have poor aqueous solubility, permeability and less availability. Nanoparticles can
minimize sum of these drugs unique drugs by safeguarding stability and preventing their
structure.in addition, nanoparticles provide ingenious treatment by enabling targeted
delivery and controlled release.

We discuss that it is not a cost effective method and it needs to be approachable to every
patient.
 52

Conclusions:
The properties of gold at Nano scale vary significantly. For instance the color of gold
changed from bright yellow to reddish purple at Nano-scale gold. Gold is one of those few
metallic elements that are able to be used in Nano-scale devices and systems because it
does not oxidize easily. Another interesting phenomenon is electrical conductivity of gold
decreases and it becomes semi-conductor at Nano-scale. The melting point decreases
whereas the strength of gold nanoparticles increases. The crystal structure is not
necessarily FCC at Nano-scale it may be cubic, octahedral or any other depends upon
Nano-scale dimension. These properties and strange behavior of gold find its applications
in new commercial and scientific opportunities. Gold nanoparticles have emerged as a
promising platform for drug, gene and protein delivery for the treatment of cancer. The
property of gold Nano-particles in combination of low inherent toxicity, high surface area
and tunable stability provides them with unique attributes that enable new delivery
strategies.

Future perspectives:
In future strategies should be developed to use gold Nano-particles in combinational drug
targeting the active and passive targeting combined in such a way that the gold Nano-
particles with proper thiol linkage not only not only link to the target receptors like in
passive targeting but also the gold nanoparticles must accommodate inside the tumor long
enough to destroy the cellular structure in single process. Despite many advances in the
field, there is a still a significant requirement for new technologies that will allow for the
earlier treatment of diseases such as cancer, particularly if they also offer greater
specificity and cost-effectiveness.
 53

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