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.

1
 2

CHAPTER # 1
INTRODUCTION
1
 2

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
 3

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
 4

personal point of view. 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 gap between
 5

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.
 6

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 will have a surface chemistry,
 7

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 core
 8

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

 9

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.
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].
 10

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)

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 linear (b) Surface modification (pagination)
polymer with a (c) Selective accumulation and retention in
Linker cleavable- tumor tissue (EPR effect)
Bond). (d) Specific targeting of cancer cells while
 11

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 hyper (a) Bio distribution

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

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

colloidal structures liposomal
Composed of lipid (b) Ease of modification
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, multifunctionality
structures. (c) Defined geometry and remarkable
uniformity
(d) Biological compatibility and inert nature

Carbon Carbon cylinders (a) Water-soluble and biocompatible through

nanotubes Composed of benzene chemical modification
ring. (b) Multifunctionality

This table shows different Nano-particle system, their structures and characteristics
[15].
 12

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].

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

 13

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

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.
 14

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)

 15

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.
3. Unusual crystal structure.
4. Anomalous melting point.
5. Tunneling electrons, quantized charge and white LEDs.
6. Enhanced strength and toughness.
 16

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
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
 17

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].

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

 18

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.

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].
 19

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

1.14.1 Types of cancer:

These are major types of cancer as follows
1. Carcinoma
2. Sarcoma
3. Melanoma
4. Lymphoma
5. leukemia
 20

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.

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.
 21

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.
 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.
 22

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.

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.
 23

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 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
 24

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.
 25

Chapter # 2
Literature review
 26

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].

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.
 27

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].
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
 28

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
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].
 29

CHAPTER # 3
METHODOLOGY
 30

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].

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.
 31

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:

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).
 32

 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.
 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.
 33

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 varied
 34

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.
 35

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-
 36

polyethylene-glycol (mPEG-SH) (~85%, MW ~5 kDa) and a hetero functional thiol-PEG-

acid (SH-PEG-COOH) (~15%, MW ~3.4 kDa).

 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.

 37

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 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].
 38

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.
 39

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].

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

 40

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.

 41

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.
 42

 Plasmonic and light scattering properties, including detection based on surface-

enhanced Raman (SERS) and LSPR spectroscopies;
 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].
 43

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 polyethylene
glycol (PEG)] is most desirable and this type of construct favors passive accumulation of
 44

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].

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

 45

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-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.
 46

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].
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
 47

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].

Figure 3-15: this figure shows the physical mechanism of Au involved in radiation
therapy.
 48

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].
 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.
 51

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 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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