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- 8558Faculty of science,University of Colombo,Colombo 03. Sri Lanka.

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.

2

2

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.

3

3

Table of contents

Acknowledgement iiAbstract iiiTable of contents ivList of abbreviations v

Chapter 01: Introduction 1Chaptre 02: Synthesis of MIONPs 2Chaptre 03: biological applications of MIONPs 7Chapter 04:conclution

Referances 10

4

4

List of abbreviations

MIONP magnetic iron oxide nano particleMNP magnetic nano particleMabs monoclonal antibodies

5

5

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.

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.

6

6

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

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 via hyperthermia treatment. Most of these promising applications require well-defined and controllable interactions between the MNPs and living cells

Chapter 02Synthesis 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).

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

7

7

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

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 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

8

8

MNPs allowing a fine surface tuning in further steps (chapter 2.4) to construct MNPs with diverse functions.

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

9

9

Fig 2.1 an efficient RNA delivery system, A magnetite loaded cationic liposome, Third generation MIONP

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.

10

10

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

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.

2.5 characterizations of synthesized nano particles

11

11

Fig 2.3. Conversion to water-soluble magnetic nanoparticles using silane ligand exchange with amino, carboxylic acid, and poly(ethylene glycol) end

Fig 2.4. A Multi functional third generation nano particle

Characterization of first, second and advanced generation nanoparticles is done using modern microscopic techniques these include

1. Energy Filtered Transmission Electron Microscopy2. Field Emission Transmission Electron Microscopy3. 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).

12

12

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

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

13

13

example, this principle is used with cytotoxic drugs in cancer treatments. Tissue targeted delivery can be further improved by fabricating the particle surface with tissue specific antibody or ligands and using bio degradable polymers.

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.

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

14

14

Drug particles

Magnetite particles

Fig 3.2-Targeted drug delivery systems-with encapsulated MIONPs

Fig 3.3-Magnetic hyperthermia heating MNP vibrations

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.

15

15

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.

16

16