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ACKNOWLEDGEMENTS
First of all, praise is to Allah, the Mighty, and the Merciful for all
of His graces and for the completion of this work. I would have never
ever done anything without His support and guidance. I am asking Allah
to make this thesis, with all the time and effort invested, helpful to
others.
I wish to express my sincere gratitude to my supervisor, Dr. Nihal
saad lecturer of biophysics (Biophysics department, Faculty of Science,
Cairo University) for her Supervision, valuable comments, patience,
encouragement, endless discussions and guidance during the whole
work. I have no words to describe my deep and sincere appreciation to
her. I had the privilege to work with her .
Many thanks to Dr Mohamed (Teaching Assistant, Egyptian
atomic energy authority) for his help in providing us with the prepared
magnetic nanoparticles.

Contents
Abstract ……………………………………………………………...…………….1
Introduction……...………………………………………………………………...2
1.1 Cancer……….…………………………………………………………………2
1.1.1 Cancer therapy………………………………………………………………2
1.1.1.1 Biological therapy………………………………………………………….2
1.1.1.2 Hormonal therapy………………………………………………………….3
1.1.1.3 Surgery……………………………………………………………………..3
1.1.1.4 Chemotherapy……………………………………………………………...4
1.1.1.5 Radiotherapy……………………………………………………………….4
1.1.1.6 Supportive therapy…………………………………………………………5
1.1.1.7 Hyperthermia………………………………………….…………………...5
1.1.1.8 Photodynamic Therapy (PDT)…………………………………………….5
1.2 Nanotechnology………………………………………………………………..7
1.2.1 Nanomedicine………………………………………………………………..7
1.2.2 Nanoparticles………………………………………………………………..7
1.2.2.1 Properties of nanoparticles………………………………………………8
1.2.2.2 Synthesis of nanoparticles……………………………………………….10

1.2.2.3 Characterization of nanoparticles…………………………..……………11
1.2.2.3.a Transmission electron microscopy (TEM)…………………………….12
2.2.3.b scanning electron microscope (SEM)……………………………………15
2.2.3.c Dynamic light scattering…………………………………………………18
2.2.3.d Atomic Force Microscopy (AFM)………………………………………21
1.2.2.4 Functionalization…………………………………………………………25
1.2.2.4.1 Surface coating for biological applications……………………………25
1.3 Magnetic nanoparticles………………………………………………………26
1.3.1 Types of magnetic nanoparticles…………………………………………..27
1.3.1.1 Oxides……………………………………………………………………..27
1.3.1.2 Metallic nanoparticles……………………………………………………27
1.3.2 Synthesis of magnetic nanoparticles………………………………………28
1.3.2.1 Co-precipitation…………………………………………………………..28
1.3.2.2 Thermal decomposition………………………………………………..…28
1.3.2.3 Microemulsion……………………………………………………………28
1.3.2.4 Flame spray synthesis…………………………………………………….29
1.3.3 Cancer diagnosis and therapy using magnetic nanoparticles…………….29
1.3.3.1 Drug Delivery using magnetic nanoparticles……………………………29

1.3.3.2 Targeting of magnetic nanoparticles…………………………………….31
1.3.3.3 Magnetic hyperthermia…………………………………………..………32
1.3.3.4 Magnetic Nanoparticles: Suitable For Cancer Therapy…………..……34
2.1 Materials…………………………………………………….………………..38
2.2 Methods……………………………………………………………………….39
2.2.1 Sample preparation…………………………………………………...…….39
2.2.2 Characterization of Fe3O4 nanoparticles…………………………………41
2.2.2.1 Transmission electron microscope……………………………………….41
2.2.2.2 Dynamic light scattering………………………………………………….41
2.2.2.3 Atomic Force Microscopy (AFM)………………………………………..43
2.2.3 In-vitro cytotoxicity test…………………………………………………….43
2.2.4 Inoculation of mice with tumor cells………………………………………44
2.2.4 Organs biodistribution……………………………………………………...46
Results and Discussion…………………………………………………………...47
3.1 Characterization of Fe3O4 nanoparticles…………………………………...47
3.1.1 Transmission electron microscope micrographs………………………….47
3.1.2Dynamic light scattering measurements……………………………………49
3.1.3 Atomic force microscopy………………………………………….………..51
3.2 In-vitro cytotoxicity assay……………………………………………..……...53

3.3 Organ biodistribution of Fe3O4 nanoparticles……..………………….……56
Conclusion………………………………………………………………………58

Abstract
1
Abstract
In the event of cancer treatment, hyperthermia therapy has met successful
cancer cells damage with highly reduced toxicity to normal cells. One of the
most interesting nanoparticles that could have a revolution in the field of cancer
hyperthermia therapy is magnetic nanoparticles. Magnetic nanoparticles are a
class of nanoparticle which can be manipulated using magnetic field. This can
drive them to the target organs for gene or drug delivery. Such particles
commonly consist of magnetic elements such as iron, nickel and cobalt and their
chemical compounds. While nanoparticles are smaller than 1 micrometer in
diameter (typically 5–500 nanometers), the larger microbeads are 0.5–500
micrometer in diameter. The magnetic nanoparticles have been the focus of
much research recently because they possess attractive properties which could
see potential use in catalysis, biomedicine, magnetic resonance imaging,
magnetic particle imaging, data storage, environmental remediation, ,
nanofluids, and optical filters.
In this study, the prepared samples have been characterized by using
transmission electron microscope (TEM), dynamic light scattering, atomic force
microscope. In-vivo organ biodistribution of Fe3O4 nanoparticles accumulated
in tumor bearing female balb mice was monitored by measuring the
concentration of iron in different organs as a function of time using atomic
absorption spectroscopy. Resultant heating and therapeutic efficacy were
assessed by assessing the cellular damage.
The results revealed that Fe3O4 nanoparticles have a spherical shape with
size distribution of 30 ± 5 nm. The biodistribution data revealed that maximum
accumulation of iron was in liver and spleen due to their relatively small size.
Cytotoxicity test revealed that there is small amount of necrotic cells after the
interaction of Fe3O4 nanoparticles with the magnetic field.

IntroductionChapter I
2
1.1 Cancer
Cancer is a class of diseases characterized by out-of-control cell growth.
There are over 100 different types of cancer, and each is classified by the type of
cell that is initially affected.
Cancer harms the body when damaged cells divide uncontrollably to form
lumps or masses of tissue called tumors (except in the case of leukemia where
cancer prohibits normal blood function by abnormal cell division in the blood
stream). Tumors can grow and interfere with the digestive, nervous, and
circulatory systems and they can release hormones that alter body function.
Tumors that stay in one spot and demonstrate limited growth are generally
considered to be benign1
.
More dangerous, or malignant, tumors form when two things occur:
 a cancerous cell manages to move throughout the body using the blood or
lymph systems, destroying healthy tissue in a process called invasion that
cell manages to divide and grow, making new blood vessels to feed itself
in a process called angiogenesis.
 When a tumor successfully spreads to other parts of the body and grows,
invading and destroying other healthy tissues, it is said to have
metastasized. This process itself is called metastasis, and the result is a
serious condition that is very difficult to treat2
.
In 2007, cancer claimed the lives of about 7.6 million people in the world.
Physicians and researchers who specialize in the study, diagnosis, treatment, and
prevention of cancer are called oncologists.
1.1.1 Cancer therapy
1.1.1.1 Biological therapy

3
Biological therapies use substances that occur naturally in the body to
destroy cancer cells. They include monoclonal antibodies, cancer growth
inhibitors, vaccines and gene therapy.
Drawbacks of Biological therapy
 Some chemical compounds do not readily degrade by biological
treatment.
 Biocides used in the manufacturing environment greatly inhibit biological
reactions.
 Digestion rates are slow, in general days or weeks, requiring large storage
tanks.
 Changing from one metal removal fluid to another can greatly reduce or
eliminate effective treatment.
 Bacteria must be fed constantly, or they will die off. If they die off, re-
acclimating the bacteria to the waste stream may take several days1
.
1.1.1.2 Hormonal therapy
Hormonal therapies alter the way hormones which occur naturally in the
body affect cancer cells. They're most commonly used to treat breast and
prostate cancer.
1.1.1.3 Surgery
Surgery aims at removing the tumor and assesses its severity and extension
in the body.
Drawbacks of surgery:

4
That requires hospitalization and because treatment may damage healthy
cells and tissues, unwanted side effects are common. These side effects depend
on many factors, including the location of the tumor and the type and extent of
the treatment. Side effects may not be the same for each person, and they may
even change from one treatment session to the next3
.
1.1.1.4 Chemotherapy
There are more than 50 different chemotherapy drugs that may be used
alone or in combination. Different drugs cause different side effects and may be
given in a variety of ways.
Drawbacks of chemotherapy:
Long after you complete your treatment, you might discover another form
of cancer such as leukemia or Hodgkin's and non-Hodgkin's lymphoma. They
are cancers of white blood cells called lymphocytes. Or there could be damage
to liver, kidneys, heart, nervous system and brain. Sometimes, there might be
blood in the urine. Chemotherapy can damage sperm. Getting pregnant with
damaged sperm can increase the risk of producing genetically defective babies2
.
1.1.1.5 Radiotherapy
Radiotherapy is the use of high energy rays to destroy cancer cells. It may
be used to cure some cancers, to reduce the chance of recurrence or for symptom
relief.
Drawbacks of Radiation therapy:
Radiation therapy can cause many side effects. Some are minor and
diminish after therapy is stopped. The side effects include fatigue, skin
inflammation in the treated areas, frequent or uncomfortable urination and rectal
bleeding or irritation4
.

5
1.1.1.6 Supportive therapy
Supportive therapy can be given in addition to or as part of your
main treatment. They include steroids, blood or platelet transfusions and
bisphosphonates.
1.1.1.7 Hyperthermia
The idea of using heat to treat cancer has been around for some time, but
early attempts had mixed results. Today, newer tools allow more precise
delivery of heat, and hyperthermia is being studied for use against many types of
cancer 5,6
.
Drawbacks of hyperthermia:
For both human and veterinary tumors remains the inability to adequately
maintain uniform temperatures to the tumors.
The heat can affect the other cells which are nearby to the cancer affected
region. Regional hyperthermia does not kill the cancer cells directly, but it
assists the other conventional cancer treatments to function against cancer in a
better way.
1.1.1.8 Photodynamic Therapy (PDT)
Photodynamic therapy or PDT is a treatment that uses special drugs,
called photosensitizing agents, along with light to kill cancer cells. The drugs
only work after they have been activated or "turned on" by certain kinds of light
.
drawbacks of PDT:
 PDT can cause burns, swelling, pain, and scarring in nearby healthy
tissue.

6
 Porfimer sodium makes the skin and eyes sensitive to light for
approximately 6 weeks after treatment, thus, patients are advised to avoid
direct sunlight and bright indoor light for at least 6 weeks 4
.

7
1.2 Nanotechnology
A basic definition: Nanotechnology is the engineering of functional systems
at the molecular scale. This covers both current work and concepts that are more
advanced.
In its original sense, 'nanotechnology' refers to the projected ability to
construct items from the bottom up, using techniques and tools being developed
today to make complete, high performance products.
1.2.1 Nanomedicine
Nanomedicine is the medical application of nanotechnology.
Nanomedicine ranges from the medical applications of nanomaterials, to
nanoelectronic biosensors, and even possible future applications of molecular
nanotechnology. Current problems for nanomedicine involve understanding the
issues related to toxicity and environmental impact of nanoscale materials. One
nanometer is one-millionth of a millimeter 8
.
1.2.2 Nanoparticles
In nanotechnology, a particle is defined as a small object that behaves as a
whole unit in terms of its transport and properties. Particles are further classified
according to size: in terms of diameter, coarse particles cover a range between
10,000 and 2,500 nanometers. Fine particles are sized between 2,500 and 100
nanometers. Ultrafine particles or nanoparticles are sized between 100 and 1
nanometers.
Nanoparticles may or may not exhibit size-related properties that differ
significantly from those observed in fine particles or bulk materials. Although
the size of most molecules would fit into the above outline, individual molecules
are usually not referred to as nanoparticles.

8
Nanoparticle research is currently an area of intense scientific interest due
to a wide variety of potential applications in biomedical, optical and electronic
fields 8
.
1.2.2.1 Properties of nanoparticles
Nanoparticles are of great scientific interest as they are effectively a bridge
between bulk materials and atomic or molecular structures. A bulk material
should have constant physical properties regardless of its size, but at the nano-
scale size-dependent properties are often observed. Thus, the properties of
materials change as their size approaches the nanoscale and as the percentage of
atoms at the surface of a material becomes significant. For bulk materials larger
than one micrometer (or micron), the percentage of atoms at the surface is
insignificant in relation to the number of atoms in the bulk of the material. The
interesting and sometimes unexpected properties of nanoparticles are therefore
largely due to the large surface area of the material, which dominates the
contributions made by the small bulk of the material.
Nanoparticles often possess unexpected optical properties as they are small
enough to confine their electrons and produce quantum effects. For example
gold nanoparticles appear deep red to black in solution. Nanoparticles of usually
yellow gold and gray silicon are red in color. Gold nanoparticles melt at much
lower temperatures (~300 °C for 2.5 nm size) than the gold slabs (1064 °C). And
absorption of solar radiation in photovoltaic cells is much higher in materials
composed of nanoparticles than it is in thin films of continuous sheets of
material, i.e, the smaller the particles, the greater the solar absorption.
Other size-dependent property changes include quantum confinement in
semiconductor particles, surface plasmon resonance in some metal particles and
super-paramagnetism in magnetic materials. Ironically, the changes in physical
properties are not always desirable. Ferromagnetic materials smaller than 10 nm

9
can switch their magnetisation direction using room temperature thermal energy,
thus making them unsuitable for memory storage.
Suspensions of nanoparticles are possible since the interaction of the
particle surface with the solvent is strong enough to overcome density
differences, which otherwise usually result in a material either sinking or
floating in a liquid.
The high surface area to volume ratio of nanoparticles provides a
tremendous driving force for diffusion, especially at elevated temperatures.
Sintering can take place at lower temperatures, over shorter time scales than for
larger particles. This theoretically does not affect the density of the final product,
though flow difficulties and the tendency of nanoparticles to agglomerate
complicates matters. Moreover, nanoparticles have been found to impart some
extra properties to various day to day products. For example the presence of
titanium dioxide nanoparticles imparts what we call the self-cleaning effect, and
the size being nano-range, the particles can not be observed. Zinc oxide particles
have been found to have superior UV blocking properties compared to its bulk
substitute. This is one of the reasons why it is often used in the preparation of
sunscreen lotions, and is completely photostable.
Clay nanoparticles when incorporated into polymer matrices increase
reinforcement, leading to stronger plastics, verifiable by a higher glass transition
temperature and other mechanical property tests. These nanoparticles are hard,
and impart their properties to the polymer (plastic). Nanoparticles have also been
attached to textile fibers in order to create smart and functional clothing.
Metal, dielectric, and semiconductor nanoparticles have been formed, as
well as hybrid structures (e.g., core-shell nanoparticles). Nanoparticles made of
semiconducting material may also be labeled quantum dots if they are small
enough (typically sub 10 nm) that quantization of electronic energy levels

01
occurs. Such nanoscale particles are used in biomedical applications as drug
carriers or imaging agents 9
.
Semi-solid and soft nanoparticles have been manufactured. A prototype
nanoparticle of semi-solid nature is the liposome. Various types of liposome
nanoparticles are currently used clinically as delivery systems for anticancer
drugs and vaccines.
Nanoparticles with one half hydrophilic and the other half hydrophobic
are termed Janus particles and are particularly effective for stabilizing
emulsions. They can self-assemble at water/oil interfaces and act as solid
surfactants 8
.
1.2.2.2 Synthesis of nanoparticles
There are several methods for creating nanoparticles, including both
attrition and pyrolysis. In attrition, macro or micro scale particles are ground in a
ball mill, a planetary ball mill, or other size reducing mechanism. The resulting
particles are air classified to recover nanoparticles. In pyrolysis, a vaporous
precursor (liquid or gas) is forced through an orifice at high pressure and burned.
The resulting solid (a version of soot) is air classified to recover oxide particles
from by-product gases. Pyrolysis often results in aggregates and agglomerates
rather than single primary particles 8
.
Thermal plasma can also deliver the energy necessary to cause evaporation
of small micrometer size particles. The thermal plasma temperatures are in the
order of 10,000 K, so that solid powder easily evaporates. Nanoparticles are
formed upon cooling while exiting the plasma region. The main types of the
thermal plasma torches used to produce nanoparticles are dc plasma jet, dc arc
plasma and radio frequency (RF) induction plasmas. In the arc plasma reactors,
the energy necessary for evaporation and reaction is provided by an electric arc
which is formed between the anode and the cathode. For example, silica sand

00
can be vaporized with an arc plasma at atmospheric pressure. The resulting
mixture of plasma gas and silica vapor can be rapidly cooled by quenching with
oxygen, thus ensuring the quality of the fumed silica produced.
In RF induction plasma torches, energy coupling to the plasma is
accomplished through the electromagnetic field generated by the induction coil.
The plasma gas does not come in contact with electrodes, thus eliminating
possible sources of contamination and allowing the operation of such plasma
torches with a wide range of gases including inert, reducing, oxidizing and other
corrosive atmospheres. The working frequency is typically between 200 kHz
and 40 MHz. Laboratory units run at power levels in the order of 30–50 kW
while the large scale industrial units have been tested at power levels up to 1
MW. As the residence time of the injected feed droplets in the plasma is very
short it is important that the droplet sizes are small enough in order to obtain
complete evaporation. The RF plasma method has been used to synthesize
different nanoparticle materials, for example synthesis of various ceramic
nanoparticles such as oxides, carbours/carbides and nitrides of Ti and Si (see
Induction plasma technology)10
.
Inert-gas condensation is frequently used to make nanoparticles from
metals with low melting points. The metal is vaporized in a vacuum chamber
and then super-cooled with an inert gas stream. The super-cooled metal vapor
condenses into nanometer-sized particles, which can be entrained in the inert gas
stream and deposited on a substrate or studied in situ.
1.2.2.3 Characterization of nanoparticles
Nanoparticle characterization is necessary to establish understanding and
control of nanoparticle synthesis and applications. Characterization is done by
using a variety of different techniques, mainly drawn from materials science.
Common techniques are electron microscopy (TEM, SEM), atomic force

02
microscopy (AFM), dynamic light scattering (DLS), x-ray photoelectron
spectroscopy (XPS), powder X-ray diffraction (XRD), Fourier transform
infrared spectroscopy (FTIR), matrix-assisted laser desorption/ionization time-
of-flight mass spectrometry (MALDI-TOF), ultraviolet-visible spectroscopy,
dual polarization interferometry and nuclear magnetic resonance (NMR).
Whilst the theory has been known for over a century , the technology for
Nanoparticle tracking analysis (NTA) allows direct tracking of the Brownian
motion and this method therefore allows the sizing of individual nanoparticles in
solution 8-10
.
1.2.2.3.a Transmission electron microscopy (TEM)
Transmission electron microscopy (TEM) is a microscopy technique
whereby a beam of electrons is transmitted through an ultra thin specimen,
interacting with the specimen as it passes through. An image is formed from the
interaction of the electrons transmitted through the specimen; the image is
magnified and focused onto an imaging device, such as a fluorescent screen, on
a layer of photographic film, or to be detected by a sensor such as a CCD camera
(Figure1.1).
TEMs are capable of imaging at a significantly higher resolution than light
microscopes, owing to the small de Broglie wavelength of electrons. This
enables the instrument's user to examine fine detail—even as small as a single
column of atoms, which is tens of thousands times smaller than the smallest
resolvable object in a light microscope. TEM forms a major analysis method in a
range of scientific fields, in both physical and biological sciences. TEMs find
application in cancer research, virology, materials science as well
as pollution, nanotechnology, and semiconductor research.
At smaller magnifications TEM image contrast is due to absorption of
electrons in the material, due to the thickness and composition of the material.

03
At higher magnifications complex wave interactions modulate the intensity of
the image, requiring expert analysis of observed images. Alternate modes of use
allow for the TEM to observe modulations in chemical identity, crystal
orientation, electronic structure and sample induced electron phase shift as well
as the regular absorption based imaging 11
.

04
Figure (1.1) Transmission electron microscope

05
2.2.3.b scanning electron microscope (SEM)
Scanning electron microscope (SEM) is a type of electron microscope that
images a sample by scanning it with a beam of electrons in a raster scan pattern.
The electrons interact with the atoms that make up the sample producing signals
that contain information about the sample's surface topography, composition,
and other properties such as electrical conductivity (Figure 1.2).
In a typical SEM, an electron beam is thermionic-ally emitted from
an electron gun fitted with a tungsten filament cathode. Tungsten is normally
used in thermionic electron guns because it has the highest melting point and
lowest vapor pressure of all metals, thereby allowing it to be heated for electron
emission, and because of its low cost. Other types of electron emitters
include lanthanum hexaboride (LaB6) cathodes, which can be used in a standard
tungsten filament SEM if the vacuum system is upgraded and field emission
guns (FEG), which may be of the cold-cathode type using tungsten single crystal
emitters or the thermally assisted Scotty type, using emitters of zirconium oxide.
The electron beam, which typically has an energy ranging from 0.2 keV to
40 keV, is focused by one or two condenser lenses to a spot about 0.4 nm to
5 nm in diameter. The beam passes through pairs of scanning coils or pairs of
deflector plates in the electron column, typically in the final lens, which deflect
the beam in the x and y axes so that it scans in a raster fashion over a rectangular
area of the sample surface.
When the primary electron beam interacts with the sample, the electrons
lose energy by repeated random scattering and absorption within a teardrop-
shaped volume of the specimen known as the interaction volume, which extends
from less than 100 nm to around 5 µm into the surface. The size of the
interaction volume depends on the electron's landing energy, the atomic number
of the specimen and the specimen's density. The energy exchange between the

06
electron beam and the sample results in the reflection of high-energy electrons
by elastic scattering, emission of secondary electrons by inelastic scattering and
the emission of electromagnetic radiation, each of which can be detected by
specialized detectors. The beam current absorbed by the specimen can also be
detected and used to create images of the distribution of specimen
current. Electronic amplifiers of various types are used to amplify the signals,
which are displayed as variations in brightness on a computer monitor (or, for
vintage models, on a cathode ray tube). Each pixel of computer video memory is
synchronized with the position of the beam on the specimen in the microscope,
and the resulting image is therefore a distribution map of the intensity of the
signal being emitted from the scanned area of the specimen. In older
microscopes image may be captured by photography from a high-resolution
cathode ray tube, but in modern machines image is saved to a computer data
storage 12
.

07
Figure (1.2) Schematic diagram of an SEM.

08
2.2.3.c Dynamic light scattering
Dynamic light scattering (also known as photon correlation
spectroscopy or quasi-elastic light scattering) is a technique in physics that can
be used to determine the size distribution profile of
small particles in suspension or polymers in solution. It can also be used to
probe the behavior of complex fluids such as concentrated polymer solutions.
When light hits small particles, the light scatters in all directions (Rayleigh
scattering) as long as the particles are small compared to the wavelength (below
250 nm). If the light source is a laser, and thus is monochromatic and coherent,
then one observes a time-dependent fluctuation in the scattering intensity. This
fluctuation is due to the fact that the small molecules in solutions are
undergoing Brownian motion, and so the distance between the scatters in the
solution is constantly changing with time. This scattered light then undergoes
either constructive or destructive interference by the surrounding particles, and
within this intensity fluctuation, information is contained about the time scale of
movement of the scatters. Sample preparation either by filtration or
centrifugation is critical to remove dust and artifacts from the solution (Figure
1.3).
The dynamic information of the particles is derived from an autocorrelation
of the intensity trace recorded during the experiment. The second order
autocorrelation curve is generated from the intensity trace as follows:
where is the autocorrelation function at a particular wave vector, ,
and delay time, , and is the intensity. At short time delays, the correlation
is high because the particles do not have a chance to move to a great extent
from the initial state that they were in. The two signals are thus essentially

09
Figure (1.3) Hypothetical dynamic light scattering of two samples:
Larger particles on the top and smaller particle on the bottom

21
unchanged when compared after only a very short time interval. As the time
delays become longer, the correlation decays exponentially, meaning that,
after a long time period has elapsed, there is no correlation between the
scattered intensity of the initial and final states. This exponential decay is
related to the motion of the particles, specifically to the diffusion coefficient.
To fit the decay (i.e., the autocorrelation function), numerical methods are
used, based on calculations of assumed distributions. If the sample
is monodisperse then the decay is simply a single exponential. The Siegert
equation relates the second-order autocorrelation function with the first-order
autocorrelation function as follows:
where the parameter β is a correction factor that depends on the geometry and
alignment of the laser beam in the light scattering setup. It is roughly equal to
the inverse of the number of speckle from which light is collected. The most
important use of the autocorrelation function is its use for size determination.
DLS is used to characterize size of various particles including proteins,
polymers, micelles, carbohydrates, and nanoparticles. If the system is
monodisperse, the mean effective diameter of the particles can be determined.
This measurement depends on the size of the particle core, the size of surface
structures, particle concentration, and the type of ions in the medium.
Since DLS essentially measures fluctuations in scattered light intensity due
to diffusing particles, the diffusion coefficient of the particles can be determined.
DLS software of commercial instruments typically displays the particle
population at different diameters. If the system is monodisperse, there should
only be one population, whereas a polydisperse system would show multiple
particle populations. If there is more than one size population present in a

20
sample then CONTIN analysis must be applied. For more than two populations
CONTIN analysis at several scattering angels is required.
Stability studies can be done conveniently using DLS. Periodical DLS
measurements of a sample can show whether the particles aggregate over time
by seeing whether the hydrodynamic radius of the particle increases. If particles
aggregate, there will be a larger population of particles with a larger radius.
Additionally, in certain DLS machines, stability depending on temperature can
be analyzed by controlling the temperature in situ 13
.
2.2.3.d Atomic Force Microscopy (AFM)
The remarkable feature of Scanning Probe Microscopes (SPM) is their
ability to “view” details at the atomic and molecular level, thus increasing our
understanding of how systems work and leading to new discoveries in many
fields. These include life science, materials science, electrochemistry, polymer
science, biophysics, nanotechnology and biotechnology.
Atomic force microscopy is currently applied to various environments (air,
liquid, vacuum) and types of materials such as metal semiconductors, soft
biological samples, conductive and non-conductive materials. With this
technique size measurements or even manipulations of nano-objects may be
performed (Figure 1.4).
In all SPM techniques a tip interacts with the sample surface through a
physical phenomenon. Measuring a “local” physical quantity related with the
interaction, allows constructing an image of the studied surface. All the data are
transferred to a PC, where, with the use of the appropriate software, an image of
the surface is created.
The scanning tunneling microscope (STM) is the ancestor of all scanning
probe

22
Figure (1.4) Principle of AFM

23
microscopes. It was invented in 1982 by Gerd Binning and Heinrich Rohrer at
IBM Zurich. Five years later they were awarded the Nobel Prize in Physics for
their invention.
The atomic force microscope (AFM) was also invented by Binning et al. in
1986. While the STM measures the tunneling current (conducting surface), the
AFM measures the forces acting between a fine tip and a sample. The tip is
attached to the free end of a cantilever and is brought very close to a surface.
Attractive or repulsive forces resulting from interactions between the tip and the
surface will cause a positive or negative bending of the cantilever.
The bending is detected by means of a laser beam, which is reflected from
the back side of the cantilever. The deflection of the probe is typically measure
by a “beam bounce” method. A semiconductor diode laser is bounced off the
back of the cantilever onto a position sensitive photodiode detector. This
detector measures the bending of cantilever during the tip is scanned over the
sample. The measured cantilever deflections are used to generate a map of the
surface topography.
The dominant interactions at short probe-sample distances in the AFM are
Van der Waals (VdW) interactions. However long-range interactions (i.e.
capillary, electrostatic, magnetic) are significant further away from the surface.
These are important in other SPM methods of analysis. During contact with the
sample, the probe predominately experiences repulsive Van der Waals forces
(contact mode). This leads to the tip deflection described previously. As the tip
moves further away from the surface attractive Van der Waals forces are
dominant (non-contact mode) (Figure 1.5) 14
.

24
Figure (1.5) Schematic of AFM instrument showing “beam bounce”
method of detection using a laser and position sensitive photodiode
detector.

25
1.2.2.4 Functionalization
The surface coating of nanoparticles is crucial to determining their
properties. In particular, the surface coating can regulate stability, solubility and
targeting. A coating that is multivalent or polymeric confers high stability15
.
1.2.2.4.1 Surface coating for biological applications
For biological applications, the surface coating should be polar to give
high aqueous solubility and prevent nanoparticle aggregation. In serum or on the
cell surface, highly charged coatings promote non-specific binding, while
polyethylene glycol linked to terminal hydroxyl or methoxy groups repel non-
specific interactions. Nanoparticles can be linked to biological molecules which
can act as address tags, to direct the nanoparticles to specific sites within the
body, specific organelles within the cell, or to follow specifically the movement
of individual protein or RNA molecules in living cells. Common address tags
are monoclonal antibodies, aptamers, streptavidin or peptides. These targeting
agents should ideally be covalently linked to the nanoparticle and should be
present in a controlled number per nanoparticle. Multivalent nanoparticles,
bearing multiple targeting groups, can cluster receptors, which can activate
cellular signaling pathways, and give stronger anchoring. Monovalent
nanoparticles, bearing a single binding site, avoid clustering and so are
preferable for tracking the behavior of individual proteins16
.

26
1.3 Magnetic nanoparticles
In biotechnology, the essential features of nanoparticles are their nano-scale
dimensions, their magnetic properties and their capability of carrying active
biomolecules for specific tasks. In order to be easily localized/targeted inside the
human body, the nano-scale dimensions of particles allow them not only to pass
through the narrowest blood vessels but also penetrate through cell membranes
when necessary.
Magnetic nanoparticles are a class of nanoparticle which can be
manipulated using magnetic field. This can drive them to the target organs for
gene or drug delivery. Such particles commonly consist of magnetic elements
such as iron, nickel and cobalt and their chemical compounds. While
nanoparticles are smaller than 1 micrometer in diameter (typically 5–500
nanometers), the larger microbeads are 0.5–500 micrometer in diameter. The
magnetic nanoparticles have been the focus of much research recently because
they possess attractive properties which could see potential use in catalysis,
biomedicine, magnetic resonance imaging, magnetic particle imaging, data
storage, environmental remediation, , nanofluids, and optical filters17
.
The active biomolecules bound to the surface of these nanoparticles can
then be released. As a result, a functional magnetic nanoparticle consists of a
number of components; the magnetic core, the protective coating, and the
surface functionality. For biomedical applications, magnetic nanoparticles
should also have active biomolecules according to the specific applications.
Other entities may also be included for multifunctional particles such as hybrid
fluorescent/magnetic particles. The challenge in this area is to put all these
components together in a small, nanometer-scale space.

27
1.3.1 Types of magnetic nanoparticles
Currently, three different kinds of magnetic nanoparticles are being
produced and used:
1.3.1.1 Oxides:
Ferrite nanoparticles are the most explored magnetic nanoparticles up to
date. Once the ferrite nanoparticles become smaller than 128 nm. They become
superparamagnetic which prevents self-agglomeration since they exhibit their
magnetic behavior only when an external magnetic field is applied. With the
external magnetic field switched off, the remanence falls back to zero. Just like
non-magnetic oxide nanoparticles, the surface of ferrite nanoparticles is often
modified by surfactants, silicones or phosphoric acid derivatives to increase their
stability in solution18
.
1.3.1.2 Metallic nanoparticles
Metallic nanoparticles have the great disadvantage of being pyrophoric and
reactive to oxidizing agents to various degrees. Making their handling difficult
and enabling unwanted side reactions17-19
.
1.3.1.3 Metallic with a shell
The metallic core of magnetic nanoparticles may be passivated by gentle
oxidation, surfactants, polymers and precious metals. In an oxygen environment,
Co nanoparticles form an anti-ferromagnetic CoO layer on the surface of the Co
nanoparticle. Recently, work has explored the synthesis and exchange bias effect
in these Co core CoO shell nanoparticles with a gold outer shell. Nanoparticles
with a magnetic core consisting either of elementary Iron or Cobalt with a
nonreactive shell made of graphene have been synthesized recently. The
advantages compared to ferrite or elemental nanoparticles are:

28
1-Higher magnetization.
2-Higher stability in acidic and basic solution as well as organic solvents.
3-Chemistry on the graphene surface via methods already known for carbon
nanotubes.
1.3.2 Synthesis of magnetic nanoparticles
The established methods of magnetic nanoparticle synthesis include:
1.3.2.1 Co-precipitation
Co-precipitation is a facile and convenient way to synthesize iron oxides
(either Fe3O4 or γ-Fe2O3) from aqueous Fe2+/Fe3+ salt solutions by the
addition of a base under inert atmosphere at room temperature or at elevated
temperature. The size, shape, and composition of the magnetic nanoparticles
very much depends on the type of salts used (e.g.chlorides, sulfates, nitrates), the
Fe2+/Fe3+ ratio, the reaction temperature, the pH value and ionic strength of the
media. In recent years, co-precipitation approach has been used extensively to
produce ferritenanoparticles of controlled sizes and magnetic properties.
1.3.2.2 Thermal decomposition
Monodisperse magnetic nanocrystals with smaller size can essentially be
synthesized through the thermal decomposition of organometallic compounds in
high-boiling organic solvents containing stabilizing surfactants.
1.3.2.3 Microemulsion
Using the microemulsion technique, metallic cobalt, cobalt/platinum
alloys, and gold-coated cobalt/platinum nanoparticles have been synthesized in
reverse micelles of cetyltrimethlyammonium bromide, using 1-butanol as the
cosurfactant and octane as the oil phase.

29
1.3.2.4 Flame spray synthesis
Using flame spray pyrolysis and varying the reaction conditions, oxides,
metal or carbon coated nanoparticles are produced at a rate of > 30 g/h17-20
.
1.3.3 Cancer diagnosis and therapy using magnetic nanoparticles
1.3.3.1 Drug Delivery using magnetic nanoparticles
The concept of using magnetic nanoparticles for drug delivery was proposed
in the late 1970s by Widder, Senyi and colleagues. The basic premise is that
therapeutic agents are attached to, or encapsulated within, a magnetic micro- or
nanoparticle. These particles may have magnetic cores with a polymer or metal
coating which can be functionalized, or may consist of porous polymers that
contain magnetic nanoparticles precipitated within the pores. By functionalizing
the polymer or metal coating it is possible to attach, for example, cytotoxic
drugs for targeted chemotherapy or therapeutic DNA to correct a genetic
defect17
.
In the past few years, considerable interest has been devoted towards the
design of new drug-delivery systems with the aim to target the drug to a specific
site, such that the drug is released at a controlled rate and at the desired time. .
Drug targeting has emerged as one of the modern technologies for drug delivery.
Targeting specific sites in the body simplifies drug administration procedures,
reduces the quantity of drug required to reach therapeutic levels, decreases the
drug concentration at on-target sites (possibly reducing side effects) and,
essentially, increases the concentration of the drug at target sites18
.

31
Figure (1.6) possible mechanisms for drug delivery using magnetic nanoparticles

30
1.3.3.2 Targeting of magnetic nanoparticles:
Magnetically targeted drug-delivery systems are a promising method of
targeted drug delivery that allow delivery of particles to the desired target area
and fix them at a local site away from the reticuloendothelial system (RES), with
the aid of a magnetic field as shown in figure (2). Naturally, the ability of
magnetic particles (MPs) to concentrate will depend on both the blood flow rate
and the intensity of the magnetic field, so the chance of efficient drug
accumulation in smaller blood vessels with lower blood flow rate is higher than
in central vessels (aorta) with a very fast blood flow. Typically, the intended
drug and a suitable magnetically active component are formulated in a
pharmacologically active, stable formulation.
A wide variety of polymeric carriers have been devised for controlling
drug release. Special attention has been paid to the biodegradability of the
polymer and the compatibility of drug and polymer.
Suitable biodegradable polymers, such as chitosan, polylactides, poly(ε-
caprolactone), poly(alkylcyanoacrylate), polyglycolides, poly(lactide-co-
glycolides), polyanhydrides or polyorthoesters are usually chosen to make drug-
carrying particles. These natural or synthetic polymers are incorporated with
MPs and drugs using various techniques.
The biodegradable polymeric nanoparticles (NPs) have attracted
considerable attention because of their ability to avoid being quickly taken up by
the RES, which prolongs circulation time in the blood and their ability to target
drugs to specific sites and reduce side effects 20
.

32
1.3.3.3 Magnetic hyperthermia
Magnetic nanoparticles are used in an experimental cancer treatment called
magnetic hyperthermia in which the fact that nanoparticles heat when they are
placed in an alternative magnetic field is used (Figure 1.7). Magnetic
Hyperthermia is selective (localized) heating of tumor cells using magnetic
nanoparticles which are targeted and attached onto the cancer regions. Typically
uses ~100 KHz ac magnetic field. Heating to ~41 to 45o
C for ~30 minutes can
preferentially damages tumor cells.
When cells in the body are exposed to higher than normal temperatures,
changes take place inside the cells. These changes can make the cells more
likely to be affected by radiation therapy or chemotherapy. Very high
temperatures can kill cancer cells outright, but they also can injure or kill normal
cells and tissues.
This is why hyperthermia must be carefully controlled and should be done
by doctors with experience in using it.
The idea of using heat to treat cancer has been around for some time, but
early attempts had mixed results. For instance, it was hard to maintain the right
temperature in the right area while limiting the effects on other parts of the body.
But today, newer tools allow better control and more precise delivery of heat,
and hyperthermia is being studied for use against many types of cancer.
Hyperthermia is a promising approach to cancer therapy, , and various
methods inducing hyperthermia, such as the use of hot water, capacitive heating
and induction heating, among others, have been employed. Some researchers
have proposed the concept of 'intracellular' hyperthermia and have have
developed submicron MPs for inducing hyperthermia.

33
Figure (1.7) Magnetic drug targeting; magnetic drug carriers
disintegrate in the target zone and release the drug.

34
The inevitable technical problem with hyperthermia is the difficult issue of
heating only the local tumor region to the intended temperature without
damaging the surrounding healthy tissue. MNPs have been used for
hyperthermia treatment in an attempt to overcome this obstacle.
Magnetic fluid hyperthermia involves the introduction of ferromagnetic or
superparamagnetic particles into the tumor tissue; under the alternating magnetic
field, the MPs can generate heat by hysteresis loss (Figure 3). The particles
transform the energy of the alternating magnetic field into heat by several
physical mechanisms, and transformation efficiency strongly depends on the
frequency of the external field as well as the nature of the particles, including
magnetism and surface modification. Owing to the strong magnetic property and
low toxicity, the application of Fe3O4 in biotechnology and medicine has
attracted significant attention18
.
1.3.3.4 Magnetic Nanoparticles: Suitable For Cancer Therapy
Magnetic nanoparticles (with a size of some few to several hundred
nanometres) are a new, promising means of fighting cancer. The particles serve
as a carrier for drugs: "loaded" with the drugs, the nanoparticles are released into
the blood stream, where they move until they come under the influence of a
targeting magnetic field which holds them on to the tumour - until the drug has
released its active agent. Besides this pharmaceutical effect, also a physical
action can be applied: an electromagnetic a.c. field heats up the accumulated
particles so much that they destroy the tumour. Both therapeutic concepts have
the advantage of largely avoiding undesired side effects on the healthy tissue
(Figure 1.8).
These procedures have already been successfully been applied in the animal
model and have, in part, already been tested on patients. Here it is important to
know before application whether the particles tend to aggregate and thus might

35
occlude blood vessels. Information about this can be gained by
magnetorelaxometry developed at the PTB. In this procedure, the particles are
shortly magnetised by a strong magnetic field in order to measure their
relaxation after the switch-off of the field by means of superconducting quantum
interferometers, so-called "SQUIDs"(Figure 1.9).
Conclusions on their aggregation behaviour in these media can be drawn
from measurements of suspensions of nanoparticles in the serum or in whole
blood. As an example, it could be shown in this way that certain nanoparticles in
the blood serum form clusters with a diameter of up to 200 nm - a clear
indication of aggregation, so that these nanoparticles do not appear to be suitable
for therapy.
At present, the high technical effort connected with the use of helium-
cooled magnetic field sensors is still standing in the way of using this method
routinely in practice. In a joint project with Braunschweig Technical University
supported by the Ministry of Education and Research (BMBF), the procedure is
currently being transferred to a simpler technology based on fluxgate
magnetometers.
Another measuring facility is currently being set up in the PTB which will
allow emissivity measurements to be performed under vacuum conditions in an
extended temperature and wavelength range - in particular for space
applications.

36
Figure (1.8) Therapeutic strategy using magnetic particles. Functionalized
magnetic nanoparticles accumulate in the tumor tissues via the DDS.
Magnetic nanoparticles can be used as a tool for cancer diagnosis by MRI.
DDS: Drug-delivery system; MI: Magnetoimpedance.(62)

37
Figure (1.9) An oriented structure of magnetic nanoparticles in
hyperthermia treatment of cancer, compared to the well-known case of an
ordinary magnets. The schematic illustrations show (a) the needle of a
magnetic compass oriented in the direction of the Earth’s magnetic field, and
(b) ferromagnetic nanoparticles under irradiation with a high frequency
magnetic field of weaker intensity than the anisotropic magnetic field, in
which the nanoparticles align in planes perpendicular to the magnetic field.

Chapter II
Materials &
Methods

thodsMaterials & MeChapter II
83
Materials and Methods
2.1 Materials
The polyamide 6 (PA6, Mn = 25,000) was purchased from Toray
Industries, Inc. The polystyrene (PS Mn = 100,000) was purchased from
Shanghai Petrochemical Co. The polystyrene with terminal maleic anhydride
groups on the PS chain (designated as FPS, Mn = 71,000; maleic anhydride
group = approximately 1.3 wt.%) was used as a reactive compatibilizer for the
PS/PA6 blends. Fe(acac)3
, phenyl ether, 1, 2-hexadecanediol, oleic acid,
oleylamine and ethanol were purchased from Sigma , Aldrich.

83
2.2 Methods
2.2.1 Sample preparation
Two millimole of Fe(acac)3
was mixed in 20 mL of phenyl ether with 10
mmol of 1, 2-hexadecanediol, 6 mmol of oleic acid, and 6 mmol oleylamine.
The mixture was kept under nitrogen and was refluxed at 260 °C for 30 min.
The mixture was then cooled to room temperature and a darkbrown substance
was produced that was treated with ethanol under air. This resulted in the
precipitation of a dark-brown particulate material from the solution. The
particles were then dissolved in hexane, oleic acid, and oleylamine and
reprecipitated with ethanol to give colloidal Fe3O4 nanoparticles.
polystyrene , polyamide 6, Fe3O4 nanoparticles and FPS were
simultaneously Introduced to Hakke and mixed at 240 ᴼC for 10 min at a
rotational speed of 100 rpm. Prior to compounding, these raw materials were
dried overnight in a vacuum oven at 80ᴼC. The mixtures were then pressed at
260 ᴼC for 15 min. In order to investigate the PA6 domain size and the
distribution of Fe3O4, sox let extraction of the PS matrix was carried out using
methylbenzene for 2 h (Figure 2.1).

04
Figure (2.1) Fe3O4 nanoparticles preparation steps

04
2.2.2 Characterization of Fe3O4 nanoparticles
2.2.2.1 Transmission electron microscope
The synthesized Fe3O4 nanoparticles was imaged using JEM 1230
transmission electron microscopy (TEM) (Jeol, LTD, Tokyo, Japan) operating at
200 kV. Samples were prepared by placing a drop of solution on a carbon coated
copper grid and allowing the grid to dry on filter paper. The average size of
Fe3O4 nanoparticles was determined from the TEM images using the Image J
software.
2.2.2.2 Dynamic light scattering
The hydrodynamic mean diameter and size distribution of nanoparticles
were determined by dynamic light scattering (NICOMPTM 380 ZLS, Santa
Barbara, California, USA) (figure2.2) at the scattering angle of 90º after diluting
samples with DI. The technique was based on the scattering of incident laser
light due to the random Brownian motion of the nanoparticles. Sample volume
used for the analysis was kept constant i.e. 2 ml to nullify the effect of stray
radiations from sample to sample. Characterization of nanoscale particles can be
weighted by number, intensity, volume or any other property of the particle
being measured. In this study, the size distribution of nanoparticles was
measured in terms of number weight. Prior to sample loading, glass vessels were
thoroughly cleaned, washed with DI water, and dried. The measurements were
repeated at least three times at room temperature and the standard deviation (SD)
was calculated using origin 6.0 software. Measurements were made in the
central lab of Egyptian atomic energy authority.

04
Figure (2.2) dynamic light scattering apparatus

08
2.2.2.3 Atomic Force Microscopy (AFM)
Atomic force microscopy (AFM) was used to study the surface morphology,
three-dimensional organization and size distribution of the NPs. The NPs
suspension was diluted 10-fold with Millipore water, and a drop was deposited
on freshly cleaved mica. The sample was air-dried at room temperature and
mounted on the microscope scanner. The AFM images were collected with
Agilent 5500 AFM (USA) operating in noncontact mode. Measurements were
made in the central lab of Egyptian atomic energy authority.
2.2.3 In-vitro cytotoxicity test
Cell culture assays are used to assess the biocompatibility of a material
or extract through the use of isolated cells in vitro. These techniques are useful
in evaluating the toxicity or irritancy potential of materials and chemicals. They
provide an excellent way to screen materials prior to in vivo tests.
The predictive value of in vitro cytotoxicity tests is based on the idea of
‘basal’ cytotoxicity – that toxic chemicals affect basic functions of cells which
are common to all cells, and that the toxicity can be measured by assessing
cellular damage. The development of in vitro cytotoxicity assays has been driven
by the need to rapidly evaluate the potential toxicity of large numbers of
compounds, to limit animal experimentation whenever possible, and to carry out
tests with small quantities of compound. Evidence for the utility of in vitro
cytotoxicity tests has led many pharmaceutical companies to screen compound
libraries to remove potentially toxic compounds early in the drug discovery
process. Early identification of toxic effects can help project teams prioritize
between chemical series and identify Structure-Toxicity Relationships to reduce
cost downstream.

00
The hela cell line, were maintained in RPMI 1640 containing
penicillin/streptomycin/glutamine and 10% fetal bovine serum (Life
Technologies, Gaithersburg, MD) in a humidified atmosphere of 5% CO2 at
37°C. Cells at 70–80% confluency were trypsinized (0.25% trypsin with 1 mM
EDTA), washed, resuspended in growth medium, and plated in 96-well plates
with 0.2 ml of the 104 cell/ml cell suspension seeded in each well. After
overnight incubation, cells were divided into groups control, treated with
magnetic nanoparticles , treated with magnetic field only and group treated with
magnetic nanoparticles and magnetic field.
The cell number was determined using a modified MTT assay, the CellTiter
96 Aqueous Non-RadioactiveCell Proliferation Assay (Promega Corp., Madison,
WI) containing MTS and PMS . The MTS/PMS solution (20 ml) was added to
each well, the cells were incubated for 3 h, and the absorbance was measured at
490 nm on an MRX microplate reader (Dynex Technologies, Inc., Chantilly,
VA) (Figure 2.3). The percentage of survival was calculated as the absorbance
ratio of treated to untreated cells. Each experimental treatment was performed in
quadruplicate in three independent experiments.
2.2.4 Inoculation of mice with tumor cells
Ehrlich ascites tumor was chosen as a rapidly growing experimental tumor
model where various experimental designs for anticancer agents can be applied
(Klein and Revesz, 1953; Dasyukevich and Solyanikn , 2007).
Ehrlich ascites carcinomas cells (1x106
cells), obtained from National
Cancer Institute “NCI” Cairo University, were intraperitoneally injected into
female balb mice. Ascites fluid was collected on the 7th day after injection. The
Ehrlich cells were washed twice and then re-suspended in 5 ml saline (5x106
viable cells). Female balb mice with 22-25 g body weight and 6-8 weeks old
(obtained from the animal house of NCI) were then injected subcutaneously in
their right flanks with Ehrlich ascites, the tumors were developed in a single and

04
solid form. Tumor growth was monitored post-inoculation until the desired
volume was reached. All animal procedures and care were performed using

04
Figure (2.3) cell line consists of 96 well , group one contain MNP with different
concentration And exposed to MF300 Gaws (50Hz) for 30 min.

04
guidelines for the care and use of laboratory animals, and approved by the
animal Ethics Committee at Cairo University (National research council, 1996).
2.2.5 Organs biodistribution
In vivo stability and pharmacokinetics properties of magnetic nanoparticles in
aqueous solution were assessed by measuring the percentage of iron content in
various organs using Atomic Absorption Spectrometry (AAS).
Freshly synthesized 5mgm magnetic nanoparticles solutions were dissolved in 1
ml deionized water for intravenous injection. Fe3O4 nanoparticles solution was
intravenously administered to tumor bearing via tail . For each time point, 3
animals injected with magnetic nanoparticles were sacrificed at times
3h,48h,72h following injection. Various organs and tissues (heart, tumor, brain,
liver, spleen, kidneys, lungs) were excised and weighed for analysis. 1g of
tissue (±5%) was digested with 1 mL of trace metal concentrated nitric acid
(HNO3) in teflon vials for 12 hours at 85 0C in an oven. Cooled samples were
diluted to 10 mL with DI water.
Liver, kidney, spleen, tumor, brain , heart and lungs were analyzed by
flame atomic absorption using a standard curve of 0-5 mg/L (corresponding to
tissue iron levels of 2-500 μg/g). The estimated concentration of Fe3O4
nanoparticles in different tissue was expressed in terms of percent injected dose
(%ID).

Chapter III
Results &
Discussion

Results & DiscussionIIChapter I
74
Results and Discussion
3.1 Characterization of Fe3O4 nanoparticles
The success of hyper thermal therapy requires a well prepared Fe3O4
nanoparticles having specific physical and chemical properties. In order to
obtain a complete picture of the geometry, optical response, surface charge and
size distribution of the prepared nanoparticles, a combination of TEM, AFM and
dynamic light scattering (DLS) should be performed.
3.1.1 Transmission electron microscope micrographs
Transmission electron microscopy (TEM) is often used to characterize the
morphology of synthesized Fe3O4 nanoparticles. TEM images also reveal a
nearly spherical shape of the two samples. TEM image of a PS/PA6/Fe3O4
mixture with a composition ratio of 60/40/20 (by weight). In order to fully
visualize the dispersion of Fe3O4, the PA6 domains were not stained; however,
they could still be recognized as small bright areas, corresponding to areas of
PA6, surrounded by the dark PS/PA6 interface. It is clear that the PA6 domains
are dispersed in the PS matrix and that most of the magnetic nanoparticles are
trapped within the PA6 domains.

74
Figure (3.1) TEM images of a PS/PA6/Fe3O4 mixture with a
composition ratio of 60/40/20 a,b,c respectively.

74
3.1.2Dynamic light scattering measurements
A somewhat more complete picture of the entire size distribution of gold
nanoshells can be obtained using dynamic light scattering (DLS). The
measurement is based on the fact that particles, emulsions and molecules in
suspension undergo Brownian motion. This is the motion induced by the
bombardment by solvent molecules that themselves are moving due to their
thermal energy. If the particles or molecules are illuminated with a laser, the
intensity of the scattered light fluctuates at a rate that is dependent upon the size
of the particles as smaller particles are ‘‘kicked’’ further by the solvent
molecules and move more rapidly.
Size distribution is a crucial parameter for the characterization of nanoscale
particles and can be weighted by number, intensity, volume or any other
property of the particle being measured. The size also affects the biodistribution
of the particles in different tissue organs.
In this work, the size distribution of Fe3O4 nanoparticles was measured in
terms of intensity weight. Fe3O4 nanoparticles were found to have an average
size about 30 nm ± 8 nm (Figure 3.2).

05
Figure (3.2) Gaussian size distribution obtained by DLS measurement
of Fe3O4 nanoparticles.

05
3.1.3 Atomic force microscopy
AFM provides a 3D profile of the surface on a nanoscale, by measuring forces
between a sharp probe (<10 nm) and surface at very short distance (0.2-10 nm
probe-sample separation). The probe is supported on a flexible cantilever. The
AFM tip “gently” touches the surface and records the small force between the
probe and the surface.
Transmission Electron microscopy (TEM) analysis and Atomic force
microscopy (AFM) for confirmed the presence of nanoparticles and provided
morphological information about them. A 3D profile of the Fe3O4 nanoparticles
surface shown in Figure 3.3.

05
Figure (3.3): AFM image of Fe3O4 nanoparticles.

05
3.2 In-vitro cytotoxicity assay
Cell culture assays are used to assess the biocompatibility of a material
or extract through the use of isolated cells in vitro. These techniques are useful
in evaluating the toxicity or irritancy potential of materials and chemicals. They
provide an excellent way to screen materials prior to in vivo tests.
There are 3 basic parameters upon which these measurements are based.
The first assay type is the measurement of cellular metabolic activity. An early
indication of cellular damage is a reduction in metabolic activity. Tests which
can measure metabolic function measure cellular ATP levels or mitochondrial
activity (via MTS metabolism). Another parameter often tested is the
measurement of membrane integrity. The cell membrane forms a functional
barrier around the cell, and traffic into and out of the cell is highly regulated by
transporters, receptors and secretion pathways. When cells are damaged, they
become ‘leaky’ and this forms the basis for the second type of assay. Membrane
integrity is determined by measuring lactate dehyrogenase (LDH) in the
extracellular medium. This enzyme is normally present in the cytosol, and
cannot be measured extracellularly unless cell damage has occurred. Other
assays measure the uptake of fluorescent dye (ethidium DI) normally excluded
from intact cells. It has been shown that changes in metabolic activity are better
indicators of early cell injury, and that effects on membrane integrity are
indicative of more serious injury, leading to cell death. The third type of assay is
the direct measure of cell number, since dead cells normally detach from a
culture plate, and are washed away in the medium. Cell number can be measured
by direct cell counting, or by the measurement of total cell protein or DNA,
which are proportional to the number of cells.
As shown in figure 3.4, at concentration 50 mgm of Fe3O4 nanoparticles there is
no cell death and 100% of the hela cells remain viable. As the concentration of
the drug increase the percent of cells survey decrease. The magnetic field (blue

07
Column) decreases the percent of cell survival to ~ 85%. The combination of
Fe3O4 nanoparticles and magnetic field decrease the percent of cell survival to ~
80%. These indicate that the heat produce during the interaction of Fe3O4
nanoparticles with the magnetic field was inefficient to destroy the cells.

00
0 100 200
0
10
20
30
40
50
60
70
80
90
100
110
%ofsurvivingcells
nanoparticles concentration
magnet+nanoparticles
nanoparticles only
magnetic field
Figure (3.4) histogram indicate the relation between the percent of
cell survival and magnetic nanoparticles concentration

05
3.3 Organs biodistribution of Fe3O4 nanoparticles
After in vivo administration of colloidal nanoparticles, the in vivo
distribution of the particles largely depends on their particle size and surface
properties such as surface charge and surface hydrophobicity. The influence of
these physico-chemical characteristics on the uptake of particles by the
mononuclear phagocyte system (MPS) comprising mainly the macrophages of
the liver and the spleen after intravenous administration. MPS has a major role
in removing foreign materials from the blood circulation. Advances in prolonged
circulation time, selective drug deposition and reduced MPS uptake have been
achieved by modification of the surface characteristics of particles determining
the interaction with the MPS. A potential therapeutic application necessitates
biodegradability and biocompatibility of the nanoparticulate carrier system and
surface modifications which are stable under in vivo conditions.
In our study, 30 nm magnetic nanoparticles showed wide spread
concentration of iron in tissues. After 3 hr, The highest concentration was
observed in the spleen, followed by liver and kidney. After 48 hr, the highest
concentration was observed in the spleen , followed by liver, brain ,tumor and
lung. After 72 hr, the highest concentration was observed in the lung , followed
by tumor , heart and spleen (Figure 3.5). It’s expected that if the particles size
increase the accumulation of particles will decrease in liver , spleen and increase
in the tumor due to the enhanced permeability and retention of tumor
vasculature.

04
Figure (3.5) organs biodistribution of Fe3O4 nanoparticles
brain tumor lung liver spleen heart kidney
0
100
200
300
400
500
µgFe/goftissue
Control
3 hours
48 hours
72 hours

Conclusion
In this work magnetic nanoparticles of diameter ~ 30 nm were
prepared and found to be reproducible under restricted preparation
conditions. It is important to characterize the physicochemical properties
of the prepared nanoparticles in order to successfully perform the
hyperthermia therapy. In-vivo organs biodistribution were performed
using atomic absorption spectroscopy. Revealed that maximum
accumulation of the magnetic nanoparticles were in liver , spleen and
tumor. Moreover, the percentage cell survival and cellular damage were
assessed using cytotoxicity assay.

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