MAGNETIC IRON OXIDE NANO PARTICLES SYNTHESIS

AND BIOLOGICAL USE.

By

N.H.K.S.Senathilake.

August 2009

This report is submitted in partial fulfillment of BC 3006

course unit in the BSc (General)
Degree program.

Student Index number- 8558

Faculty of science,
University of Colombo,
Colombo 03.
Sri Lanka.
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ACKNOWLEDGEMENTS

I am grateful and express my heartiest thanks to my supervisor Dr. W.R.M. De Silva for
her valuable guidance, encouragement and support throughout the study.

I wish to express my special thanks to the coordinator of the course unit

BC3006, Dr. Udayangani Ratnayake guiding and coordinating me in finding success
through out the study.

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ABSTRACT

Nano technology is an emerging multi disciplinary technology branched in virtually all

natural sciences and technologies. Generally nanotechnology deals with structures of the
size 100 nanometers or smaller, and involves developing materials or devices within that
size. Nanotechnology is very diverse, ranging from extensions of conventional device
physics, to completely new approaches based upon molecular self-assembly, to
developing new materials with dimensions on the nanoscale, even to speculation on
whether we can directly control matter on the atomic scale.

Magnetic nanoparticles are a class of nanoparticle which can be manipulated using

magnetic field. Such particles commonly consist of magnetic elements such as iron,
nickel and cobalt and their chemical compounds. These particles have been the focus of
much research recently because they possess attractive properties allowing a magnetic
manipulation without a physical interaction.

When it comes to biomedical applications Iron oxide nano particles is the major interest
as they are less toxic and bio-compatible magnetic particles with shorter relaxation times
allowing a good magnetic manipulation in biological systems. This study covers the
current synthetic methods and biomedical applications of magnetic ion oxide nano
particles in brief.

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Table of contents

Acknowledgement ii
Abstract iii
Table of contents iv
List of abbreviations v

Chapter 01: Introduction 1

Chaptre 02: Synthesis of MIONPs 2
Chaptre 03: biological applications of MIONPs 7
Chapter 04:conclution

Referances 10

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List of abbreviations

MIONP magnetic iron oxide nano particle

MNP magnetic nano particle
Mabs monoclonal antibodies

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Chapter 01
Introduction

Any nano material including iron oxide nano particles are developed in a sequential
manner starting from tiny pure metal, metal oxide, organic polymer particles up to
functionalized advanced generation particles. Though few protocols show slight
exceptions a generalized scheme can be illustrated.

First generation nano particles developed by

nucleation and further growing of pure material
E.g. - pure Fe3O4 nanoparticles

Second generation nano particles developed by

further coating with other substances to have
chemically active surface

Advanced generation particles developed by

further coating with active components which
leads the function-now called functionalized
particles

Though advanced generation particles may further be self assembled to yield a complex
tow dimensional and three dimensional structures, In general biomedical function of
MIONPS deals with advanced generation particles-functionalized with bio-active
components. In few cases it is second generation particles.

Magnetic nanoparticle which can be manipulated using magnetic field,

commonly consist of iron. Iron oxides (magnetite) show faster relaxation over other
magnetically active metals Thus faster and accurate manipulation of the MIONP is
possible. Ability of MIONP to acquire proper function depends on particle size, dispersed
nature of particles, nature of functional coating and the external magnetic program which
controls the particle. Many methods are available to synthesize MIONPs among them, a
novel and straight forward coating method was recently developed ( will be discussed in
detail in a forth coming chapter), enabling stable particle functionalization. Furthermore,
this functionalization method allows the introduction of a tunable coating that can be
specifically adapted according to the application envisioned, thereby ensuring more
controllable cellular interactions, and paving the way to a bright future in medicine.

Functionalized MIONPs can be used in a wide variety of biomedical applications ranging

from contrast agents for magnetic resonance imaging to the deterioration of cancer cells

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via hyperthermia treatment. Most of these promising applications require well-defined

and controllable interactions between the MNPs and living cells

Chapter 02
Synthesis of MIONPs

2.1 Challenges

There are two main challenges to make all biomedical applications of MIONPs come true:
1) a good synthesis route for manufacturing first generation monodisperse MNPs with
diameters <20nm; and 2) a good method to functionalize the surface of this nanoparticles.
The latter determines the ability of the MNPs to interact in a well-defined and controllable
manner with living cells. Such an interaction is mainly achieved by coating the
nanoparticles with biological ligands specific for certain receptors on the cell surface (i.e.,
receptor-mediated interaction). However, in some cases, a chemical functionality can also
be "attractive" for a cell surface (i.e., nonspecific interaction). Once bound to the cell
surface, the nanoparticle can stay there or a mechanism of cellular uptake can be triggered
by which the nanoparticle is engulfed through the cellular membrane and brought into the
cell body ( figure 2.5).

Fig 2.5
Possible
interactions
between (bio-
active)
magnetic
nanoparticles
and living

Magnetic nanoparticles for biomedical applications have to be uniform in size and

monodisperse so that each nanoparticle has nearly identical physical and chemical
properties. Most synthesis routes for monodisperse MNPs are based on the general
principle of a short nucleation step, followed by a slower growth process on the existing
nuclei. A major difficulty encountered during the synthesis of these MNPs is to keep them

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stable in solution without showing any sign of nanoparticle aggregation. As for all
nanoparticles, aggregation is also commonly observed for MNPs due to their extremely
large surface-to-volume ratio and the large surface energy they express. Furthermore, they
could magnetically interact with each other when not properly stabilized.

To circumvent the aggregation problem, a strong repulsive force must be created to

counteract the magnetic and surface-related attractions. Such a repulsive force can be
achieved by electrostatic or by steric repulsion. The first method uses ionic compounds to
coat the particles; the second approach which offers a more efficient stabilization coats
the particles with large molecules such as polymers, or surfactants containing long-chain
hydrocarbons.

2.2 Synthesis of first generation MIONPs

Theory behind producing first generation nano particles is simply a nucleation process as
occur in chemical precipitations there the reaction yields iron oxides and it forms tiny
nuclei suspension initially, followed by aggregating further produced iron oxides on these
nuclei to yield particles that form the precipitation. In nano technology particle diameter
and dispersed nature of the particle is controlled by using different matrix and conditions.
Thus giving rise to deferent techniques in nanoparticle synthesis such as micro-emulsion,
co-precipitation and Thermal decomposition.

2.3 Protocols

Many protocols available today for the synthesis of MIONPs based on micro-emulsion,
co-precipitation, and other water-based methods.

Micro emulsion

Controlling the very low interfacial tension in precipitation matrix (~10-3 mN/m) through
the addition of a co surfactant (e.g., an alcohol of intermediate chain length), these micro
emulsions are produced spontaneously without the need for significant mechanical
agitation. The technique is useful for large-scale production of nanoparticles using
relatively simple and inexpensive hardware (Higgins 1997).

Co precipitation

Metal oxide precipitation and a polymerization reaction is carried out at the same time so
that the developing particles are trapped inside the tiny polymer beads.

The disadvantages of these water-based methods are that the size uniformity and
crystallinity of the MNPs are rather poor, and nanoparticle aggregation is commonly
observed. A Recently developed protocol (Sun et al)utilize thermal decomposition giving
us a simple synthetic procedure that enables the synthesis of very monodisperse and
highly crystalline MNPs with sizes between 3nm and 20nm without showing any sign of
nanoparticle aggregation. Further this method is very useful in producing diverse range of
MNPs allowing a fine surface tuning in further steps (chapter 2.4) to construct MNPs with
diverse functions.

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Thermal decomposition (Sun method)

A typical Thermal decomposition protocol involves the high-temperature decomposition

(>220°C) of an organic iron precursor in the presence of hydrophobic ligands such as
oleic acid .These hydrophobic ligands form a dense coating around the nanoparticles,
thereby avoiding their aggregation. This method has been further adapted by other
researchers to synthesize all types of MNPs containing different materials such as cobalt,
manganese, nickel, platinum, etc. Although this thermal-decomposition method has the
advantage of producing very monodisperse and highly crystalline particles, a major
disadvantage is that the resulting nanoparticles are soluble only in nonpolar solvents due
to their coating with hydrophobic ligands. Hence, to make MNPs suitable for biological
applications, the hydrophobic ligand coating needs to be replaced by a hydrophilic
polymer coating (Fig 2.2) before functionalize biologically, or direct coating with
biocompatible functional coating that allows controlled interaction with different types of
biological species, such as cells, proteins, or DNA. How ewer particles yield directly from
this method can easily be trapped in to hydro phobic particles such as liposomes (Fig 2.1).
Hydrophobic MIONPs

Fig 2.1 an efficient RNA

delivery system, A
magnetite loaded cationic
liposome, Third generation
MIONP

Fig 2.2. Coating over oleic

acid to make the particle
water soluble, a second
generation water soluble
MIONP. Out word directed
functional groups may
further be utilized to
specifically functionalize
the particle

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2.4 Coating to yield second generation MIONP

Based on its expertise in silane self-assembled monolayers for biosensor applications , A

procedure has been developed recently to coat MNPs (via the thermal-decomposition
method) with silane monolayers(Fig 2.3). The advantage of this approach is that it enables
stable, water-soluble monodisperse MNPs bearing a huge variety of functional end
groups. This capability makes it possible to tune the surface functionality of the
nanoparticles, optimizing it for every specific application.

A systematic study of silane ligand exchange, screening nine commercially available

silane monolayers, has shown that the ligand exchange is very effective. The original
hydrophobic ligand was fully replaced by the silane self-assembled monolayer, forming a
dense organic layer with the functional end groups presented to the surrounding liquid.

Furthermore, it was shown that the presence of these end groups strongly determines the
water dispersibility of the nanoparticles. Out of these nine different silanes, only the
amino, carboxylic acid, and poly(ethylene glycol) end functions were found to render the
nanoparticles perfectly soluble in aqueous solutions over a wide pH range. Enhanced
long-term stability and increased resistance against mild acid and alkaline environments
were observed compared to those of other ligands commonly used to stabilize MNPs.
These characteristics are due to the strong covalent linkage of the silane layers onto the
nanoparticles' surface. Combined with Sun's method for synthesizing MNPs, the
functionalization procedure described above results in monodisperse, water-soluble
(thus biocompatible) MNPs with properties that can easily be tuned to the requirements of
the specific application.

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Fig 2.3. Conversion to

water-soluble magnetic
nanoparticles using silane
ligand exchange with
amino, carboxylic acid, and
poly(ethylene glycol) end

2.4 Bio-active third generation MIONPs

Chemically active nano particle can then be coated with bio-active components such as
ligands, antibodies using the knowledge of surface chemistry of the particle(Fig 2.4), or
these particles can be trapped (by co precipitation) in a nano beads like liposomes, bio
degradable polymers which already have active components such as Drugs. Therapeutic
gene, RNA.

Fig 2.4. A Multi functional

third generation nano
particle

2.5 characterizations of synthesized nano particles

Characterization of first, second and advanced generation nanoparticles is done using

modern microscopic techniques these include

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1. Energy Filtered Transmission Electron Microscopy

2. Field Emission Transmission Electron Microscopy
3. Energy Dispersive X-ray spectroscopy
4. X-ray diffraction (XRD) analysis
Images from these techniques clearly shows the particle diameter, dispersed nature and
many other properties which may lead to develop improved protocols in synthesizing
nanoparticles.

Chapter 03

Biological applications of MIONPs

Magnetic nanoparticles (MNPs) have variety of Biological and Medical uses. Ranging
from contrast agents for magnetic resonance imaging to bio-separation, hyperthermia
treatment for cancer. ( figure 3.1).

Fig 3.1. Biomedical applications based on the controlled interactions between

living cells and biologically activated magnetic nanoparticles.

3.1 Diagnostic applications and bio separation

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3.1.1 Contrast enhancers in MRI

MRI. A well-known application in the field of diagnosis is the use of MIONPs as

contrast agents for magnetic resonance imaging (MRI), which is used to better
differentiate healthy and pathological tissues and to visualize various biological events
inside the body. Due to their low toxicity, iron oxide MNPs has received US Food and
Drug Administration approval to be used as MRI signal enhancers. Further it lowers the
hydrogen relaxation time to increase the contrast.

3.1.2 Magnetic labeling for diagnosis and separation.

Another application is the selective magnetic labeling all kinds of biological entities,
such as cells, DNA, and proteins biological entities, via surface coated antibodies.
Advantages compared to conventional labels such as enzymes, fluorescent dyes,
chemiluminescent molecules, and radioisotopes are that MNP labeling enables us to
detect (using MRI), transport and purify biological components at the same time. Further
multi functional MNPs may have many different functions on particular entity,
Labeling with these MNPs detects a particular therapeutic process. Main applications
using monoclonal antibody mediated MNP (biologically activated using Mabs) labels
include.
1.) Selective bio-separation and cell isolation using an external magnetic filed (in vitro
and in vivo) the attraction between an external magnet and the MNPs enables separation
of a wide variety of biological entities. Examples are the isolation of cancer cells in blood
samples or stem cells in bone marrow to allow for improved diagnosis and the removal of
toxins from the human body or blood (Hemoperfusion). Furthermore, MNPs can be
biologically activated to allow the uptake of cells via endocytotic pathways, thereby
allowing certain cellular compartments to be specifically addressed. Once taken up, the
desired cellular compartments can be magnetically isolated and accurately studied using
proteomic analysis.
2.) Labeling of stem cells to noninvasive monitor the distribution and fate of
transplanted stem cells in the human body.
3.) Magnetic labels in biosensing is used in magnetic micro arrays and magnetic
immunoassays. For example, magnetically labeled cancer cells can be purified,
transported, and detected on a single chip surface, enabling simple and cost-effective
cancer screening in a lab-on-a-chip approach.

3.2 Therapeutic use

3.2.1 Targeted drug delivery and Controlled drug release

As thy are small and low toxic to humans, MNPs can be transported in blood stream
through an external magnetic field gradient, to the targeted site in the body and
penetrating deep into the human tissue. In this way, controlled transport of drugs to target
sites can be achieved. The latter usage is realized by attaching a drug to a biocompatible
MNP carrier, injecting the ferro fluid into the bloodstream, and applying an external
magnetic field to concentrate the drug/carrier complexes at the target site. As one
example, this principle is used with cytotoxic drugs in cancer treatments. Tissue targeted

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delivery can be further improved by fabricating the particle surface with tissue specific
antibody or ligands and using bio degradable polymers.

Drug
particles

Magnetite
particles

Fig 3.2-Targeted drug delivery

systems-with encapsulated MIONPs

3.2.2 Hyperthermia-Targeted Magnetic Nanoparticles Heat Tumors to

Death

. Another interesting therapy is based on the ability of MNPs to be heated when a time-
varying magnetic field is applied. This characteristic is used to burn away cancer cells
(hyperthermia), often in combination with chemotherapy. MIONPs are biologically
functionalized to target the tumor. It is in fact known that cancer cells are more sensitive
to temperatures in excess of 41°C than their normal counterparts. This confirms that the
surrounding normal cells are well safeguarded during the process.

Fig 3.3-Magnetic hyperthermia

heating MNP vibrations

Investigators use folic acid to target magnetic nanoparticles to tumor cells. As tumor cells
express folic acid receptors in higher levels, MIONPs functionalizes to have folic
acids(ligand) on the surface. This MIONPs binds to folic acid receptors facilitating the
receptor mediated uptake in to malignant cells. Once the tumor cells engulfed the

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nanoparticles, the researchers then heated the nanoparticles with a rapidly oscillating
magnetic field. Preliminary data from these experiments suggest that the nanoparticles
should be able to heat up cells beyond 43 °C – a known lethal temperature – after being in
the oscillating magnetic field for 20 minutes. Using electron microscopy and cells
growing in culture, the researchers were able to document that only cancer cells bearing
folic acid receptors on their surfaces were able to take up the magnetic nanoparticles.

Chapter 4

Conclusion

Magnetic iron oxide nanoparticles are bio compatible, less toxic and can be manipulated
very effectively in vivo and ex vivo to get many tasks done, including processors which
cannot be achieved or difficult to achieve using older and conventional methods. Much
research focus has to be on MIONPs biomedical uses and It seeks further developments.

References

1. Biomedical applications using magnetic nanoparticles - Els Parton at el

2. Q.A. Pankhurst, at el "Applications of Magnetic Nanoparticles in Biomedicine,"

Journal of Physics 36, pp. R167–R181, 2003.

3. C. Xu, S. Sun, "Monodisperse Magnetic Nanoparticles for Biomedical Applications,"

Polymer International, 56, pp. 821–826, 2007

4. T. Hyeon, "Chemical Synthesis of Magnetic Nanoparticles," Chemical Communication,

pp. 927–934, 2007.

5. S. Sun at el -Nanoparticles," Journal of the American Chemical Society, 126, pp. 273–
279, 2004.

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6. R. De Palma et al., "Silane Ligand Exchange to Make Hydrophobic Super-

paramagnetic Nanoparticles Water-dispersible," Chemistry of Materials, 19, pp. 1821–
1831, 2007.

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