Topic talk:Biotechnology

=—'Ahmed Abushrida

A Brief Overview of Iron Oxide Nanoparticles Iron oxide nanoparticles (IONP) have been studied extensively over the past few decades to fabricate various novel contrast agents. Considerable research has been directed towards developing particles with an appropriate size and stability and modifying their properties by coating them with biodegradable polymers, to enhance their application in providing high resolution in magnetic resonance imaging (MRI) and as a good diagnostic agent. Over 50 years ago the application of small IONPs was investigated for in vitro diagnostic tests (Gilchrist et al., 1957). Since then, studies on different types of iron oxides have been carried out in the field of magnetic particles with a small size of about 5–20 nm in diameter. Studies reported to date have dealt with maghemite, γ-Fe2O3, or magnetite, Fe3O4. Of these two, magnetite is a very promising candidate and subsequently its biocompatibility has already been tested (Schwertmann and Cornell, 1991) Review of Iron Oxide Nanoparticles A basic requirement for the use of nanoparticles in different areas in medical applications is a controlled particle size distribution. Superparamagnetic iron oxide nanoparticles (SPIONs) with suitable surface modification can be used for applications such as MRI contrast agents, drug delivery, tissue repair, hyperthermia, detoxification of biological fluids and immunoassay. For all these applications, the particle size must be smaller than 100 nm with a narrow distribution and have high magnetisation values (Gupta and Gupta, 2005). The control of particle size is most important because the properties of the nanocrystals rely greatly on the dimension of the nanoparticles. Many studies have investigated the behaviour of the nanoparticles, and others have focussed on improving their applications, the stability of the particles and the control of surfactants (Laurent et al., 2008). The type of method used for producing nanoparticles is very important. Additionally, the stability of the uncoated nanoparticles should be addressed as the aggregation process due to Van der Waals forces is the principal disadvantage of IONPs. To prevent aggregation the nanoparticle must be coated by degradable nontoxic and biocompatible polymer. There are many polymers which have been used to coat iron oxide particles, such as dextran, poly(ethylene glycol) (PEG), poly(lactide-co-glycolide) (PLGA), and poly(vinylalchohol) (PVA). The nature of surface coatings and their subsequent geometric arrangement on nanoparticles determines not only the overall size of the colloid but also plays a significant role in the biokinetics and biodistribution of nanoparticles in the body. Some authors have reviewed the different factors related to the clearance of the IONPs from blood: particle size, dose, surface charge, coating material, and stability in physiological environment. The IONPs could also bind enzymes, nucleotides and drugs. Moreover, it has been proposed that, by using an external magnetic field, nanoparticles can be directed to a tumour, organ or tissue. (Gupta and Gupta, 2005). Various methods have been developed for the preparation of IONPs (Mornet et al., 2006). The co-precipitation technique is a very cheap and simple method (Qu et al., 1999). Hydrothermal synthesis techniques are an alternative method for the preparation of highly crystalline IONPs (Wang et al., 2005). But there is no direct way to control the size and the shape of the final particles (Figuerola et al., 2010). Over the last ten years, significant advances have been made on the colloidal synthesis of nanocrystals in high-boiling point organic solvents (Park et al., 2004). There are others, such as sol-gel reactions, thermal decomposition method, etc. Characterisation of the nanoparticles, including their physicochemical properties and structure, can be done using techniques such as direct light scattering (DLS), Transmission electron microscopy (TEM), X-ray diffraction and photon correlation spectroscopy. There are many factors affecting the properties of nanoparticles such as the chemical and physical size, surface coating and the thickness of surface coating. The nanoparticles have a large surface area. Furthermore, the attractive forces between the particles make them aggregate. To overcome all these problems, nanoparticles can be coated by polymers. There are two kinds of polymer: natural, like dextran, and synthetic like PVA. The coating must be hydrophilic and biocompatible so that the particles can disperse in biological fluids. Also the surface coating has an effect on the magnetic behaviour of nanoparticles. Agglomeration will occur when the particles have a strong magnetic attraction or the polymers have a short length. On the other hand particles with sufficiently low ferromagnetic effects and polymers with long length can produce sufficient repulsive forces to prevent the agglomeration (Huber, 2005). The copolymers typically have a hydrophobic part that acts as an anchor and a hydrophilic part which forms tails to prevent bridge flocculation between one particle and another particle. Polymers must cover the entire surface of particles in order to overcome this aggregation problem (Storm et al., 1995). The iron oxide core can be coated with polymers during the synthesis process and it must be a long chain. However, the pH and temperature have an effect on the surface bound polymers (Enochs et al., 1993). Covering particles by a dense brush of polymers, e.g. dextran, prevents the particles reacting with blood proteins and receptors (Weissleder et al., 1995a). Properties of Iron Oxide Nanoparticles The size of particles usually refers to the total diameter of the particles including the iron oxide and coating. Because the smallest capillaries are 4 µm in diameter, particles or aggregates larger than this size will be captured and precipitated and  this could cause emboli within the capillary bed of the lungs (Kreuter, 1983). Particles tend to aggregate based on their magnetic energy, thus reducing their surface charge. As a result, this could lead to precipitation that may cause a very high risk if the particles are injected. The surface charge and aggregation behaviour of the particles in the blood must be known (Storm et al., 1995). Nanoparticles were eliminated immediately via macrophages of the mononuclear phagocytic system (MPS) because the defence system in the body recognised them as being alien to the body (Muller et al., 1997). Particle sizes smaller than 4 µm are taken up by the cells of the reticuloendothelial system, mainly in the liver (60–90%) and spleen (3–10%) (Neuberger et al., 2005). While it is more likely that small particles up to 100 nm will be phagocytosed through liver cells, there is a tendency for particles larger than 200 nm to be filtered by the venous sinuses of the spleen (Moghimi et al., 2001). When particles with sizes in the range between 30 and 100 nm are injected intravenously, the larger particles will be eliminated by the liver from the blood faster than the small particles. Therefore, the larger particles are, the shorter is their plasma half-life-period (Chouly et al., 1996b). The uptake can be classified according the particle size, as phagocytosis of all size, or pinocytosis (particles <150 nm) (Muller et al., 1997). Phagocytes recognise and remove the larger particles, while smaller particles can be removed by all types of cell through pinocytosis. An increase in particles size leads to an increase in the phagocytotic activity (Maaßen et al., 1993). For particles smaller than about 15 nm the cooperative phenomenon of ferromagnetism cannot be seen for longer periods and temporary magnetisation does not remain after the particles have been subject to a temporary external magnetic field (Sorensen, 2002). Ferromagnetic particles occur when unpaired electron spins align themselves spontaneously. Then the material is able to exhibit magnetisation without being in a magnetic field. Single atoms cannot exhibit ferromagnetism, but when a number of atoms unite in solid form, ferromagnetic properties arise and particles exhibit permanent magnetisation once the ferromagnetic particles are removed from the field. The ferromagnetic material will initially oppose the field change soon after field reversal, but finally the majority of domains will have turned their magnetisation vectors and the same inverse magnetisation is achieved. However, when the dimensions of ferromagnetic materials is reduced to particle dimensions smaller than a particular domain, the particles are no longer ferromagnetic but exhibit superparamagnetism (Elliott, 1998). In the case of paramagnetic particles, a magnetic field is altered by the magnetic materials present in it. If a particle contains magnetic moments that can be aligned in an external magnetic field, this will amplify the field. Such substances exhibit the property of paramagnetism. In contrast to ferromagnetic materials (ferromagnetism), no permanent magnetization remains in paramagnetic materials when they are removed from the magnetic field. Paramagnetism can be understood by postulating permanent atomic magnetic moments, which can be reoriented in an external field. These moments can be either due to orbiting electrons or due to atomic nuclei. The torque applied by an external magnetic field on these moments will tend to orientate them parallel to the field (Chen, 1986). 1.4	Synthesis of Iron Oxide Nanoparticles Numerous methods can be used to synthesise IONPs for medical applications and especially for medical imaging applications. These methods include thermal hydrolysis, sol-gel syntheses, hydrothermal reactions, microemulsions, sonochemical reactions, flow injection synthesis, electrospray syntheses, hydrolysis and thermolysis of precursors, and the common method co-precipitation, which is widely used for preparation of IONPs. Co-precipitation Method The wet chemical method has many advantages for producing IONPs. It is simple and efficient and has the capability of control over the size, composition and even the shape of the nanoparticles (Gupta and Curtis, 2004a, Gupta and Wells, 2004 and Reimers and Khalafalla, 1972). Iron oxide, either Fe3O4 or γ Fe2O3, can be produced using the co-precipitation of Fe2+ and Fe3+ aqueous salt solutions by addition of a base (Reimers and Khalafalla, 1972). The size, shape and composition of nanoparticles can be controlled according on the type of salts used (sulphates, nitrates, chlorides, etc.), ionic strength of the media, Fe2+ and Fe3+ ratio, and pH (Hadjipanayis and Siegel, 1993 and Sjoegren et al., 1994). In the traditional way, magnetite is prepared by adding a base to an aqueous mixture of Fe2+ and Fe3+ chloride at a 1:2 molar ratio. The precipitated magnetite is black in colour. (Schwertmann and Cornell, 1991 and Cotton, 1988) Aqueous co-precipitation of Fe3+ and Fe2+ at a ratio of 2:1 to prepare the magnetite nanoparticle is usually carried out in the presence of a base at pH 8–14 under anaerobic conditions. Magnetite is sensitive to oxidation and not stable in the air. This could critically affect the physical and chemical properties of the IONPs. Often the particles are coated with organic or inorganic molecules during the precipitation process to prevent the oxidation caused by air and also the formation of aggregates. To overcome the oxidation of Fe3O4 nanoparticles as which depends on oxidation speed and species, the reaction must be under a N2 atmosphere to protect the particles and to reduce the size, when compared with methods without removing the oxygen (Gupta and Curtis, 2004a and Kim et al., 2001). The influence of different wet-chemical synthesis parameters such as iron (II) iron (III) ratio or varying pH and ionic strength on the resulting iron oxide structure have been described by (Jolivet et al., 1992 and Tronc et al., 1992). Electronic or ion transfers are dependent upon the pH of the suspension. In basic medium, the oxidation of magnetite involves the oxidation–reduction of the surface of magnetite. Under anaerobic and acidic conditions, surface Fe2+ ions are desorbed as hexa-aquo complexes in solution (electron and ion transfer). Where A and B stand for tetrahedral and octahedral sites, respectively, and L represents a vacancy. Maghemite nanoparticles, bearing a high positive charge density (σ ≈ 0.3 C m–2 at pH 2, and low ionic strength, 10–2/5.10–2 mol l–1), carry high positive charge density and can easily be dispersed in acidic water, forming cationic sols practically free from aggregation. Maghemite particles could easily be concentrated aqueous dispersions when resulting from oxidation of magnetite (Jolivet and Massart, 1983, Jolivet et al., 1997 and Prene et al., 1993). There is no migration of iron ions towards the interior of particles. Electrons and presumably protons are injected into the particle from the ferrous hydroxide adsorbed layer. This layer crystallises as spinel and the reaction stops when equal populations of Fe3+ and Fe2+ in the octahedral sub-lattice are reached. Similar electron transfers occur during adsorption of ferric ions on magnetite (Belleville et al., 1992). Several parameters can influence the properties of the particles produced. These parameters include: •	Nature of salt •	Concentration of salts •	Volume ratio of the salt phase •	Temperature •	Ionic strength This method has many advantages. It can easily produce a large number of nanoparticles (Laurent et al., 2008). (Babes et al., 1999) have investigated the basics of the co-precipitation process, the influence of different parameters (media composition, iron / iron ratio, injection fluxes, iron, temperature, and oxygen) on magnetic properties, size and morphology. Results by (Gnanaprakash et al., 2007) showed that the initial pH and temperature of the ferrous and ferric salt solution before initiation of the precipitation reaction and the final pH are critical parameters controlling the formation of magnetite and nanoparticles. Gas phase Methods Gas phase methods for preparing nanomaterials is based on thermal decomposition (pyrolysis), reduction and disproportionation, oxidation, or other reactions to precipitate solid products from the gas phase (Pierson, 1999). In the chemical vapour deposition (CVD) process, a carrier gas stream with precursors is delivered continuously by a gas delivery system to a reaction chamber maintained under vacuum at high temperature (> 900 C) (Tavakoli et al. and 2007, Chang et al., 1994a). The products stick together and form aggregates or nanoparticles after the CVD reactions take place in the heated reaction chamber. Growth and agglomeration of the particles are minimised via rapid expansion of the two-phase gas stream at the outlet of the reaction chamber. Then heat treatment of the nano powders in various high-purity gas streams allows all the properties, such as compositional and structural modifications, particle purification and crystallisation, as well as transformation to a desirable size, composition, and morphology, to take place (Tavakoli et al., 2007 and Pierson, 1999). Important factors influencing the production of the nanoparticles are low concentrations of precursor in the carrier gas, as well as rapid expansion and quenching of the nucleated clusters or nanoparticles as they exit from the reactor (Tavakoli et al., 2007 and Chang et al., 1994b). Laser pyrolysis of organometallic precursors is dependent on the resonant interaction between laser photons and one or more gaseous species, reactants or sensitisers. A sensitiser is an energy transfer agent that is excited by absorption of CO¬2 laser radiation and transfers the absorbed energy to the reactants by collision (Dumitrache et al., 2005). The technique includes heating a flowing mixture of gases with an uninterrupted wave CO2 laser to initiate and sustain chemical reaction until a critical concentration of nuclei is reached in the reaction zone, and homogeneous nucleation of particles occurs (Tartaj et al., 2005). The nucleated particles formed during the reaction are entrained by the gas stream and are collected at the exit (Tartaj et al., 2003a). Aerosol / vapour Methods Spray and laser pyrolysis are good direct techniques for producing small particles based on chemical processes to provide a high level of production; by spraying a solution into a series of reactors where the aerosol droplets undergo evaporation of the solvent and solute condensation within the droplet, followed by drying and thermolysis of the precipitated particle at higher temperature (Pecharromán et al., 1995). Very small IONPs with a size about 5 nm are obtained by this method (Veintemillas-Verdaguer et al., 1998). Thermal Decomposition Method This method depends on two main routes to form the ferrites through hydrothermal conditions by oxidation or neutralisation of mixed metal hydroxides through the reaction of different ferrous salts as in the first method (Willard et al., 2004). Small particle sizes between 4–20 nm have been obtained using this method in the presence of alcohol, oleylamine and oleic acid. Particles can be transformed into a hydrophilic form by adding a bipolar solvent (Sun et al., 2004a). A modification of this method was done by Maity et al., (2009) who used solvent–free thermal decomposition just in the presence of the stabilising surfactant and produced small nanoparticles around 5 nm in diameter. Different parameters of the thermal decomposition procedure can be used to obtain high crystallinity and narrow size distribution and particle sizes between 4.9 to 14.1 nm in diameter. Further modification of this method by Ahniyaz et al., (2008) using thermal decomposition of an iron 2-methoxy-ethoxide with surfactant-free conditions together with oleic acid yielded relatively monodisperse iron oxide nanocrystals and resulted in an average size of about 5.6 nm. Polyols Method The polyol process is a chemical method which refers to the use of polyols such as ethylene glycol and diethylene glycol to reduce metal salts to metal particles. Preparation of a variety of inorganic compound with non-aggregated particles has been successful. The polyols in this method often work as high boiling solvents and reducing agents, and also as stabilisers to control the growth of particles and prevent the aggregation of the particles. One of the important advantages of this method is that it can control experimental conditions. Moreover, it is easy to scale up (Fievet et al., 1989b and Claus and Hans-Otto, 2001). In a similar reaction procedure with a variety of liquid polyols, including ethylene glycol, diethylene glycol, triethylene glycol and tetraethylene glycol Fe(acac)3 (acac acetyl cetonate) have  been reduced  to magnetite. The size obtained by this method was 20 nm in diameter and required only a single iron rich precursor and further reducing agent and surfactants (Cai and Wan, 2007). By using hybrid (organo-inorganic) aerosols and the temperature of pyrolysis, the method depended on utilising ethanol/water solutions containing iron inorganic salts and mono- or polysaccharides and produced nanoparticles with sizes from 50 to 400 nm in diameter (Tartaj et al., 2007). Microemulsion Method The aqueous phase, containing Fe3+ (1M) and Fe2+ (0.5 M) in 0.1 M HCL, prevents oxidation with different ratios of microemulsion containing a volume ratio of cyclohexane. The method is based on stirring the mixture under a N2 atmosphere at room temperature for 10 min then stirring again at 50C. Base microemulsion was injected to precipitate the particles and the change of colour to black was evidence of the formation of magnetite particles and their aggregation. Particles were subjected to washing with 0.5% tetramethylammoniun hydroxide and a large amount of acetone to remove the surfactants and flocculate the particles. Particles were dried at 65C for at least 24h (Vidal-Vidal et al., 2006). Chin and Yaacob, (2007) have adopted this method to prepare IONPs at room temperature via water in oil microemulsion, resulting in IONPs which were spherical in shape and less than 10 nm. In the opposite way, Capek, (2004) used one microemulsion. IONPs were obtained in bis(2-ethylhexyl) sulfosuccinate AOT microemulsions with a stopped-flow technique. The processes of nucleation and growth can be observed in the production of nanoparticles that after precipitation and drying showed no aggregates in TEM images with a size less than 4 nm. Flow Injection Method This method is a modified version of manufacturing zinc oxide nanoparticles (Wang and Muhammed, 1999). The idea of this technique is that it consists of continuous injection of reagents into a carrier stream. One reagent injected might be sufficient. The reaction mixture transfers into the capillary reactor at the same time as the reaction takes place. By the ratio between the length of the manifold and the pumping rate, the residence time of the particles in the reaction mixture can easily be controlled. Mechanically, a propulsion unit, injection manifold, capillary reactor, and product collector are connected to the flow injection system. The most important part of the system is the injection manifold, which could be T- or X-shaped, where the reagents mix head-on with high accuracy in time and amount. The manifold function can be provided by an injection valve with segmentation capabilities. The function of the propulsion unit is the transportation of solutions through the system. The time of the reacting mixture residence can be defined by the pumping rate and dimensions of the capillary reactor. The additional features, such as inert atmosphere and temperature control, that could be provided by the injection system have led to the development of this method and nanoparticles have been obtained with a narrow size distribution in the range 2–7 nm in diameter (Salazar-Alvarez et al., 2006). Electrochemical Method This method consists of an electrolytic cell with anode and cathode, using an iron plate and stainless steel thin sheet as anode and cathode respectively where the space between them was 5 mm. The pH was maintained at 10 with concentrated sodium hydroxide solution and the electrolyte was Na2S2O3 with a concentration of 0.02 mol/L. The applied current density was defined at the start by adjusting the voltage, until the change of the colour of the mixture to black occurred during the first 20 min. The electrolysis reaction was directly controlled by imposing the temperature, current density and intense agitation for a specific time. The final product was centrifuged and the residue was removed by deionised water at constant temperature in a hydrogen stream into the reactor for reduction at constant flow rate (2 L/h) for 3 h. The particles were then cooled to room temperature under a hydrogen atmosphere. To modulate the magnetic properties of the composites the reduction temperature was varied between 300 and 400C. IONPs have been synthesised within the pores of mesoporous silica (MS) microspheres by an electrochemical method to produce IONPs with a diameter of 20 nm inside the pore of MS spheres (Wang et al., 2007). Sizes varying from 20 to 30 nm of IONPs have been obtained using electrochemical method (Cabrera et al., 2008). IONPs having an average size of 6.2 nm with a quite narrow distribution were synthesised with new parameters based on electro-precipitation in ethanol medium. The reaction precipitated the Fe(OH)3. The next step was reduction of iron hydroxide to magnetite in presence of hydroxyl ions which are generated at the cathode (Ibrahim et al., 2009). Surface Coating A variety of biodegradable polymers have been used to coat the IONPs. Polymers can be manipulated by modifying with different functional groups to increase the stability of the nanoparticles. These coatings can also protect the nanoparticles in vitro and in vivo. This review will cover both natural organic polymers and synthetic polymers. Dextran Dextran is a polysaccharide (C6H10O5)n, composed exclusively of alpha-D(1-6) linkages with some unusual 1,3 glucosidic linkages at branching points. Dextrans are used to coat IONPs in aqueous solution, resulting in particles with overall sizes between 20–50 nm (Tueng et al., 1993). The first report of the formation of magnetite in the presence of dextran was by Molday and MacKenzie, (1982). Co-precipitation is the most common method to obtain polysaccharide-coated iron oxide particles (Goetze et al., 2002). An average magnetite core size of 7.1 nm was found by X-ray diffraction and that of 8 nm was found by transmission electron microscopy. An average diameter of 25 nm was observed for dextran coated IONPs by scanning electron microscopy and a hydrodynamic diameter of 25–300 nm was obtained by photon correlation spectroscopy. The coated particles showed a weak negative charge in the buffer solutions at pH between 5.5 and 9.5 (Xu et al.,2005). By using the Molday co-precipitation method, ferumoxtran-10 and ferumoxides have been produced (Lee et al., 2002). The effects of the molecular weight of dextran upon the formation and the stability, on size, morphology, coating efficiency and magnetic property have all been investigated. Dextrans with molecular weights of 3000, 10,000, 20,000 and 40,000 have been used. The low molecular weight dextran gave the smaller desired particle size of 77.80 nm, while increasing the molecular weight resulted in a particle size increase to 121.4 nm, 156.2 nm and 192.1 nm respectively (Hong et al., 2009). A comparison has been made between the laser pyrolysis technique and co-precipitation method for preparation of dextran coated IONPs to explore the mechanism of adsorption of the dextran on the surface of IONPs. At 25–500 °C, the mechanism of adsorption is probably hydrogen bonding between dextran hydroxyl groups and the iron oxide particle surface (Carmen Bautista et al., 2005). Dextran provides hydroxyl functional groups that can be substituted, carboxymethyl dextran (CMD) being very common. IONPs coated by CMD using a co-precipitation method with diameters of about 90 nm and 120 nm were produced. The stability of particles against aggregation was investigated over a period of more than one week. A strong agglomeration and increase of particle size was observed after one day (Dutz et al., 2007). Polyethylene Glycol (PEG) Poly(ethylene glycol) (PEG) is non-toxic and non-immunogenic, and provides high steric stabilisation. Its uncharged hydrophilic residues and high surface mobility keep the particles apart and stable in aqueous solution and prevent protein adsorption to the particles and adhesion to cells (Yasugi et al., 1999 and Zhou et al., 2003). Therefore, the presence of covalently immobilised PEG on the surface of IONPs is most likely to enhance the biocompatibility and stabilisation of the nanoparticles. PEG has chains with some terminal functionality and can be coupled with biopolymers to create biodegradable copolymers such as poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) (PCL-PEG-PCL, PCEC) copolymers (Gou et al., 2008). PEG polymers with molecular weights below 100,000 Da are amphiphilic and soluble in water. They are also soluble in many organic solvents, such as methylene chloride, toluene, acetone, chloroform, and ethanol. This allows PEG to be reacted with IONP surfaces using different chemistries that require the use of either aqueous or organic solvents (Veiseh et al., 2010). Various methods of coating were developed to prepare small (60–100 nm) and ultrasmall (20–35 nm) particles without size separation processes (Acar et al., 2005). Iron nanoparticles were coated with PEG diacid via the grafting of aminopropylsilane groups and the coupling of oxidised PEG through the formation of a covalent bond. PEG diacid is covalently coated on the surface of the nanoparticles via at least one of their carboxylic groups, leaving the other COOH group available for further chemical reaction. PEG diacid coated IONPs were investigated by TEM and found to have an approximately spherical shape and an average diameter of 20 nm (Feng et al., 2008). Kumagai et al., (2009) have reported a method to synthesise block copolymer-coated IONPs by chelation between the carboxylic acid groups in poly(ethylene glycol)–poly(aspartic acid) block copolymer (PEG–PAsp) and Fe on the surface of the IONPs. The TEM image showed PEG–PAsp-coated nanoparticles forming clusters with a size range of 100 nm, the hydrodynamic diameter with DLS and shown to be in the range of 100 to 120 nm with a unimodal distribution, for Asp/Fe ratios ranging from 0.02–0.5. In a one-step procedure, IONPs were coated with silanated monomethoxy poly(ethylene glycol) (m-PEG) in aqueous medium. The mean particle sizes of the nanoparticles determined by X-ray diffraction and TEM gave an average size of 20 nm in diameter (Hu et al., 2008). 1.5.3	Poly(D, L-lactic-co-glycolic acid) (PLGA) Poly(dl-lactic-co-glycolic acid) is a biocompatible, biodegradable and non-toxic material used for preparing nanoparticles and microparticles. PLGA is a copolymer of lactic and glycolic acid (Zimmer and Kreuter, 1995 and Bala et al., 2004). Lee et al., (2004) have prepared magnetic nanoparticles of Fe3O4 encapsulated within PLGA for MRI contrast agents by using an emulsification diffusion method. The size was reduced to 120 nm by increasing the homogeniser speed up to 22,000 rpm. Zhao et al., (2009) have produced poly(lactic acid) (PLA)-coated magnetic nanoparticles using uncapped PLA with free carboxylate groups. Magnetic microspheres (MMS) with the matrix material PLGA or PLA were then formed by the emulsion solvent evaporation method. The physical properties of these particles were compared to those of oleate-coated or oleate/sulphonate bilayer coated magnetic particles. PLA coated IONPs using w/o emulsion resulted in particle sizes of 180 ± 50 nm diameter. The polymer shell showed high protection of particles in pH 5.5 to 10.8 sensitivity with respect to the stability of magnetite particles (Gómez-Lopera et al., 2006). 1.5.4	Polyvinyl alcohol PVA is a water-soluble polymer and has isolated hydroxyl functional groups, which can adsorb and complex with metal ions (Jiu et al., 2002). Xu and Teja, (2008) have modified the surface of IONPs with PVA by continuous hydrothermal synthesis. The final polymer coated particles produced at different concentrations of PVA showed different sizes from 7 to 27 nm. The average particle size was shown to increase with temperature and residence time, and is accompanied by morphology changes in some cases. (Mahmoudi et al., 2010) have  reported  synthesis  of IONPs  with PVA  by precipitation of  iron  oxide  salts  in PVA aqueous solution  at various temperatures and at different concentrations. The results suggest that the PVA binds to the surface of the nanoparticles due to the presence of OH groups on the polymer chains and the nanoparticles. Also, using the same method, PVA coated super-paramagnetic iron oxide nanoparticles (SPION) of 6 10 nm core and 30 nm hydrodynamic sizes were obtained. The IONPs were first obtained by classical co-precipitation in water. A thermochemical treatment and centrifugation was then applied to obtain well-dispersed primary nanoparticles. PVA of molecular weight 12,000 was used to coat the particles (Schöpf et al., 2005). Lee et al., (1996) found that crystallinity of the core decreased with increasing concentration of PVA present during the synthesis of iron oxide particles in the range of 4–10 nm through a precipitation reaction to form a stable dispersion. Binding between the surface of nanoparticles and the polymer was found using FTIR absorbance shifts. Alginate Alginate is a linear polymer composed of α-d-mannuronate (M) and α-l-guluronate (G) units linked by α-1,4 and α-1,4 glycosidic bonds. M and G units are organised in MM, GG and MG blocks (Rocher et al., 2010). Alginate-coated IONPs showed good biocompatibility and some magnetic targeting under a magnetic field. The high stability provided by alginate coated IONPs may be attributed to the binding of the carboxyl group of alginate to iron oxide (Ma et al., 2007). Morales et al., (2008) and Finotelli et al., (2004) have prepared alginate-coated particles by dropping commercial sodium alginate solution (3%) into aqueous ferric chloride solution. Two samples were prepared, sample A with 0.01 M and sample B with 0.5 M ferric chloride solution, which produced preformed IONPs encapsulated in Ca-alginate beads. IONPs coated with alginate were Fe3O4 with a core diameter of 5–10 nm and had a hydrodynamic diameter of 193.8 nm. ξ-potential was -65.0 mV with good stability (Ma et al., 2008). Chitosan Chitosan is a hydrophilic, biocompatible, biodegradable,  alkaline, non-toxic natural polymer (Chung et al., 2005). Chitosan-coated IONPs varied from 10 to 80 nm with the different molecular weight of the chitosan (Zhi et al., 2006). Tsai et al., (2010) have reported a simple method to coat IONPs in situ precipitation of ferrous hydroxide by the alkaline treatment of chitosan. Ammonium hydroxide solution was rapidly added to the brown solution under sonication at 50 C. The results revealed that the IONPs coated with chitosan had a hydrodynamic diameter of 87.2 nm. Zhang et al., (2010b) have prepared chitosan coated IONPs via a novel photochemical method in an emulsifier-free aqueous system at room temperature. The results, using scanning electron microscopy (SEM) and TEM showed that the chitosan-coated nanoparticles were a regular shape with a mean diameter of 41 nm. However, the average size was 64 nm when measured by photon correlation spectroscopy. Microspheres composed of SPION with spherical particle size about 15nm were synthesised and embedded in the presence of polyglucosamine (chitosan) by a sonochemical method. The ferrofluid, a solution of SPION-embedded chitosan, was sprayed on the surface of an alkali solution with a nozzle to produce IONPs in chitosan microspheres of 100–150 µm. The SPION-chitosan microspheres showed a strong enhancement of MR image contrast similar to the ferrofluid in vitro (Kim et al., 2007) In another study, IONPs were synthesised by a classic method, co-precipitation resulting in a mean particle size of 14.1 nm. These particles were then dispersed well in two polymers, chitosan or o-carboxymethylchitosan OCMCS. The results revealed the mean particle radius as measured by DLS of 42 nm for chitosan/Fe3O4 nanoparticles, and 38 nm with OCMCS/Fe3O4. Fe3O4 nanoparticles have net positive charge and OCMCS stabilised magnetic Fe3O4 nanoparticles have functional carboxyl groups (Zhu et al., 2008). Chitosan coated IONPs have also been prepared by a crosslinking method. Oleic acid modified IONPs were firstly prepared by co-precipitation, chitosan was then added to coat the surface of the nanoparticles by physical absorption. The average size of such chitosan coated IONPs was approximately 10.5 nm. The chitosan coated IONPs had a remarkable heating effect which has great potential in hyperthermia therapy (Qu et al., 2010). Silica Coating of IONPs with a silica layer provides a chemically inert surface in biological systems for the core of IONPs (Deng et al., 2005). The two main methods for coating IONP with silica, the Stöber method and the reverse micelle method, have both been used for the preparation of different inorganic nanoparticles. The Stöber method is an easy method for coating nanoparticles with a layer of silica simply by mixing the nanoparticles, aqueous solution and alkoxysilane in alcohol. With the sol–gel method, the reaction ends in a short time or at a maximum will take several hours. The parameters which are very important to control the coating thickness are the optimisation of solvent components and volume of alkoxysilane (Narita et al., 2009). The uncoated IONPs with a particle size of 30 nm were prepared by a co-precipitation method which was followed by coating with silica using the Stöber method. The size of the coated nanoparticles was 50 nm using TEM (Khosroshahi and Ghazanfari, 2010). Using another method by Lien and Wu, (2008) to coat IONPs, thermosensitive polymers were grafted onto the surfaces of 6 nm monodisperse IONPs coated with silica. The nanoparticles were synthesised using reverse microemulsions and free radical polymerisation. The nanoparticles produced contained a layer of silica coated onto the surface of nanoparticles in the range from 20 to 30 nm. The poly(N-isopropylacrylamide) (PNIPAM) grafted silica-coated IONPs were produced with different ratios of PNIPAM. The particle sizes of three samples increased by around 3–5 nm compared to that of pure silica coated IONPs as determined by TEM. PNIPAM grafted onto SiO2/Fe3O4 nanoparticles were confirmed by FT-IR spectra. Amine modified silica coated IONPs were prepared using a water-in-oil microemulsion technique, which employed amine-modified silica as the shell and iron oxide as the core of the magnetic nanoparticles. The particle size of the core was 8 nm while the coating increased the particle size up to 40 ±5 nm (Ashtari et al., 2005). Deng et al., (2005) used the sol–gel method for the preparation of silica-coated IONPs. The particle size of coated nanoparticles was 40 nm using TEM. Silica coated IONPs with about 10 nm diameter IONP cores and about 2 nm thick silica shells were prepared using a co-precipitation method, and then the nanoparticles were covalently bonded with amine and carboxyl groups. The presence of the functional groups was proved by FTIR spectra (Jang and Lim, 2010). Gold Gold is another inorganic coating that has been used to coat nanoparticles and act like a coating polymer to enhance the stability. These properties give gold a particularly attractive surface chemistry for IONPs. However, one limitation is that gold-coating can weaken the magnetic properties of IONPs (Dale 2005). The formation of a gold shell on the IONPs was achieved by an iterative reduction method using hydroxylamine as a reductant. The surface of the gold coated IONPs was further functionalised with mercapto-hexadecanoic acid (MHA) to bind the positively charged Arg6-esterase effectively. The gold-coated IONPs had a size distribution of 50–100 nm in diameter with an average size of about 70 nm, while the core IONPs had a size of 8–10 nm (Jeong et al., 2006). Gold was coated onto IONPs using a facile reverse micelle procedure and the effect of water to surfactant molar ratio on the size was investigated. The particles were coated by polyglycerol through capping with thiol followed by polymerisation of glycidol. It was shown that ultrafine nanoparticles 9–16 nm could be synthesised with a thin gold layer of about 2 nm thicknesses (Jafari et al., 2010). Lu et al., (2006) have prepared well dispersed nanoparticles comprised of a citrate stabilized gold shell and Fe oxide core. Results from (TEM) and energy dispersive spectroscopy (EDS) indicated that the particle coated was with gold and had a size larger than 10 nm in diameter. Monosized core/shell, IONPs/gold with magnetic–optic multifunctional properties were synthesised using a modified nanoemulsion method in the presence of poly(vinylpyrrolidone) (PVP) as the surfactant. The iron oxide /gold core–shell were prepared by seeding the iron nanoparticles core and then coating with a gold shell. The particle size of coated IONPs using TEM, was 8.7 nm, while the uncoated IONPs had a size of 3–6 nm diameter, FTIR confirmed that the PVP was bound to the surface of the nanoparticles (Liu et al., 2010). 1.5.9	Other Polymer coated iron oxide nanoparticles IONPs have been coated with many other polymers to improve their stability and protect them in the biological environment. For example, superparamagnetic IONPs were coated with polyethylenimine and then conjugated with DNA. IONPs were prepared by the co-precipitation method. PEI coating of IONPs followed this procedure: the iron oxide suspension was mixed at various ratios with 25 kDa polyethylenimine PEI. An average size was 27±12 nm as obtained by TEM. The complexes were added to cells and exposed to permanent and pulsating magnetic fields. The presence of these magnetic fields significantly increased the transfection efficiency (Steitz et al., 2007). IONPs could be stabilised during the synthesis by oleic acid using the co precipitation method, and then further stabilised by coating them with PEI and co-polymer using direct ligand-exchange reactions. The first layer coating the IONPs was PEI. The second was formed by poly(ethylene oxide) PEI. The hydrodynamic diameter of IONPs was 10 nm and 30 nm with a core size of 10 nm. The nanoparticles coated with PEG-g-PEI showed an average hydrodynamic diameter of 25 nm, however, the nanoparticles coated with PEI had smaller hydrodynamic sizes of 14–15 nm (Duan et al., 2008). Magnetic polymer nanospheres were also prepared using new procedures based on miniemulsion polymerisation. A stable water-based dispersion of sodium dodecyl sulfate and oleic acid coated magnetite aggregates was first synthesised as a bilayer and mixed with monomer styrene miniemulsion. The second step was another encapsulation of magnetite into monomer droplets using miniemulsification. The particle size of the latex-3 particles showed a diameter of 8 nm of superparamagnetic magnetite particles and after encapsulating within the polymer nanosphere the size increased to an average diameter of 80 nm (Zheng et al., 2005). Another study showed that superparamagnetic particles could be stabilised with polyaniline. The polyaniline and IONPs composite was formed by polymerisation in the presence of the ferrofluid. IR spectra confirmed the composition on the surface of the oxide particles (Kryszewski and Jeszka, 1998). Hu et al., (2006) have prepared a magnetite/poly(L-lactic acid) composite using a solvent evaporation/extraction technique in an oil/water emulsion. The average size of the composite was about 200 nm. However, the size of the core was 6.2 ± 0.7 nm in diameter. The composite was then loaded with an anti-cancer drug. A method based on the so-called anionic polymerisation procedure to coat IONPs resulted in a shell of poly(ethyl-2-cyanoacrylate). The particle size of coated IONPs was determined by TEM to be 144 ±15 nm. The images showed the layer coating the nanoparticles, and the thickness of the polymer shell was about 30 nm (Arias et al., 2007). Ma et al., (2009) have also described the coating of IONPs with polyacrylic acid (PAA) to prevent the aggregation of the IONPs using chemical co-precipitation. PAA oligomer of low molecular weight was used as a dispersing agent and coating. The TEM result revealed that the average diameter of uncoated particles was 9.2 ± 2.6 nm while coated nanoparticles size using dynamic light scattering measurement indicated a hydrodynamic diameter of 246 ±11 nm. In drug delivery using ethylcellulose, coated IONPs were synthesised by an emulsion solvent evaporation process. The particles were then used as a drug carrier loaded with diclofenac sodium for arthritis treatment. The particle size was measured using TEM showing an average diameter of 430 ± 40 nm and a spherical shape. The nanoparticles showed good loading, and a slow drug release profile (Arias et al., 2009). Applications of Iron Oxide Nanoparticles IONPs have been used a research tool for a variety of applications over the last decade. IONPs have been applied in various medical fields, and numerous reports have emerged using IONPs as a powerful tool to perform different functions. Currently, IONPs are commercially available and approved. In addition to being a research tool, IONPs hold great promise in MRI, drug delivery, cellular labelling/cell separation, tissue repair, hyperthermia treatment, etc. 1.6.1	Magnetic Resonance Imaging (MRI) Diagnostic medical imaging has been the subject of enormous improvements over the last 35 years with the development of techniques such as MRI. This is one of the most useful non-invasive methods in the application of diagnostic imaging and is characterised by its high resolution of soft-tissues and by its absence of exposure to radiation. MRI, both in preclinical and clinical settings, has many advantages: high speed, clear image and high resolution with relative accessibility and low cost better than other techniques such as positron emission tomography (Wang et al., 2006). To recognise the difference between healthy and pathological tissues, MR imaging uses contrast agents that can be localised to a particular tissue or cellular epitope which will permit the visualisation and characterisation of different disease states (Morawski et al., 2005). Today the main contrast agents are, firstly, paramagnetic gadolinium based contrast agents which shorten the longitudinal relaxation time (T1) and increase the contrast of the image (positive enhancement) (Hanns-Joachim et al., 2003). Secondly, IONPs, which shorten the transverse relaxation time (T2) and provide many advantages as contrast agents, such as improved contrast, carrying high payloads and long circulation times (Cormode et al., 2010). Magnetic resonance imaging relies on the nuclear magnetic resonance of protons in a molecule, and for medical imaging, specifically follows water. It can act in different tissues to give a picture of anatomical structure. The characteristic measure in MRI is the proton relaxation rate (R) or its inverse, the relaxation time (T). There are two types of proton relaxation times, the longitudinal relaxation time, T1, (spin–lattice relaxation) and the transverse relaxation time, T1 (spin–spin relaxation). Moreover, there are two types of contrast agents, positive and negative; positive acts on T1 to give a positive enhancement of the signal appearing bright on the MRI scan. The negative agents provide negative enhancement, appearing as dark spots in the scan (Kubaska et al., 2001). Comparing gadolinium-based contrast agents and superparamagnatic iron oxide nanoparticles in terms of rates, SPION can produce enhanced relaxation in certain organs at lower doses than paramagnetic ions (Corot et al., 2006, Wang et al., 2001). Gadolinium contrast agents are commonly used in MRI but are non-specific with rapid accumulation in the liver, so they only permit a short time-imaging window (Fahlvik et al., 1990). Overall, the transverse relaxation (T1and T2) effect of SPION is mainly used in detection of liver lesions by MR imaging. The shortening of T2 relaxation times in the transverse relaxation occurs when SPION are distributed in reticuloendothelial cells, such as Kupffer cells (KCs), according to phagocytic activity, and because of local field inhomogeneities that produce rapid dephasing of neighbouring proton spins. However, because of the lack of reticuloendothelial cells, liver tumours such as metastatic liver cancer cannot absorb these agents. Therefore, the contrast between tumour tissue and surrounding normal liver tissue is improved because of signal loss in the liver tissue (Saini et al., 1987). Currently, only two SPION preparations have been approved for clinical use especially for liver MR imaging, such as Ferumoxides (i.e. Endorem® in Europe, Feridex® in the USA and Japan, Advanced Magnetics, USA) coated with dextran (Weissleder et al., 1989). Ma et al., (2008) have used MRI to provide information on liver tumour by using IONPs coated with alginate. The nanoparticles were rapidly cleared from serum and accumulated mainly in the liver and spleen with a total percentage of more than 90% after intravenous injection and could have the capability to enhance detection of a liver tumour as contrast agents in MRI. IONPs prepared by laser-induced pyrolysis have been coated with dextran to give a hydrodynamic diameter of 50 nm, their 1H NMR relaxation times and the magnetic properties of these particles showed they are suitable for use as contrast agents for MRI. Two important factors affect the relaxation properties, in particular particle and crystal size for the transverse relaxation rate, R2 (Morales et al., 2003). Patel et al., (2008) have developed contrast agents comprising of PLGA encapsulated SPION, which were applied as a MRI contrast agent in the liver of a rabbit. The change in spin relaxation time occurred because the nanoparticles in the liver interact with the hydrogen nuclei around the nanoparticles. Comparing the images before and after the injection showed the image with high resolution. For this reason, PLGA encapsulated SPIONs (II) might be used as an MRI contrast agent. Tanaka et al., (2008) modified SPIOs with biotin, hemin, and 5-phosphorylated DNA via the ligand exchange method. From the DLS measurements the particle size was 51.2 ± 22.4 nm and these nanoparticles could be expected to be potential applications both as a contrast agent and for cell cycle monitoring. Microbubbles with a polyvinyl alcohol (PVA) outer layer and a poly(DL-lactide) (PLA) inner layer were prepared using a double emulsion solvent evaporation interfacial deposition (water-in-oil-in-water emulsion) process. The PLA and PVA double-layered polymer shell of 50–70 nm thicknesses can let the 12 nm SIONPs be loaded heterogeneously in their shell to significantly enhance magnetic susceptibility (Yang et al., 2009). IONPs coated with non-magnetic precursor microgel, which is made up of polymerised methacrylic acid (MAA) and ethyl acrylate (EA) crosslinked with di allyl phthalate (DAP), have been used for in vivo tracking of stem cells after transplantation by labelling primary endothelial progenitor  stem cells with magnetic particles. They can be tracked by magnetic resonance. The nanoparticles showed hydrodynamic diameters of 87–766 nm. Micro-gel iron oxide particles might be a useful tool for the study of relaxation induced by magnetic particles and cellular tracking by MRI (Lee et al., 2010). C2-coated superparamagnetic IONPs can induce a significant increase in image contrast where the regions have a large number of apoptotic cells. The C2 is a domain of synaptotagmin I which binds to anionic phospholipids in cell membranes. The protein bound with the SPION can be detected by using MRI. The detection of apoptotic cells using this candidate diagnostic agent was illustrated in vitro with isolated apoptotic tumour cells and in vivo in a tumour treated with chemotherapeutic drugs (Blankenberg et al., 1997). IONPs give diagnostic opportunities to detect diseases and understand their mechanisms. Nanoparticles are used widely to give more information on diseases, such as location. Medical imaging has been traditionally based on the use of X- rays and radioactive substances. The newer diagnostic imaging modalities, such as ultrasound and magnetic resonance, are now well-established techniques in patient management.

Diagnostics such as MRI and ultrasound are starting to develop clearer imaging and more accuracy (Perkins, 1998). Organic and inorganic polymers are used as nanoparticles or contrast agents, evenly coating the metal nanoparticles to reduce systemic toxicity and increase drug bioavailability. However, there are other methods of diagnostic imaging, such as X-ray where contrast agents are non-specific materials dependent on the use of dense materials to attenuate the X-ray photons, for example barium orally or iodine by the intravenous route. Polymer based contrast agents are attractive for use as vascular contrast agents in X-ray because they are polymers having a long clearance time in circulation, such as methoxy-polyethylene glycol (MPEG) and incorporate iodine for CT imaging (Perkins, 2002). Contrast agents for X-ray photons such as barium and iodine for the site specific targeting of X-ray contrast agents has not proven to be of any clinical value, mainly because the reporter moieties (barium and iodine) have to be delivered in such large amounts that it is not considered to be a viable possibility (Perkins, 2002). Another important diagnostic imaging modality is nuclear medicine imaging, which relies on physiological processes and on the administration of radiolabelled tracers. This technique is for tissue imaging. There are many materials used for specific organs such as TC-99 m-bone scanning, lipophilic materials for cardiac and brain and biological materials such as pooled human serum albumin and monoclonal antibodies. Viral contamination is the main problem caused by human blood products. As a result, some radiopharmaceuticals cannot be supplied, e.g. serum albumin used for the measurement of plasma volume and macro-aggregates of serum albumin and for lung perfusion imaging (Hindle and Perkins, 1995). Ultrasound contrast agents work based on the production of a backscattered echo within the frequency range between 1 and 20 MHz. Contrast agents for ultrasound rely on an injectable formulation of constant gas-filled microbubbles which produce a strong reflection of the incident sound beam (Schneider et al., 1992). Polymers like polybutyl-2 cyanoacrylate with a wall thickness of 100 nm are enough to protect the enclosed gas bubble from dissolution in the blood stream and sufficiently elastic to oscillate in the ultra-sound field (Perkins, 2002). Drug Delivery Classic pharmaceuticals have many problems with systemic drug administration, including biodistribution throughout the body. There is also the lack of drug specificity for the pathological site and the need for a large dose to give a high concentration in the site, which could lead to side effects. Drug targeting could overcome some of these problems (Torchilin, 2000b). For example, through the targeting of the drug immobilised on magnetic materials under the action of an external magnetic field (Berry and Curtis, 2003), or to improve the target of drug binding with another molecule which recognises the target site, such as protein, hormones, antibodies, lectins, charged molecules and some low molecular weight ligands such as folate (Sudimack and Lee, 2000). In drug delivery, the charge and surface chemistry of magnetic particles and the size have strong effects on blood circulation and the bioavailability of the particles within the body (Chouly et al., 1996a). The first clinical trial in humans with magnetic targeting were reported by Lubbe et al., (1996). These trials used IONPs coated with starch and anionic phosphate, then the cationic binding to the positively charged amino sugars of epirubicin was possible. Successful preliminary animal trials led to human trials, the treatment consisting of intravenous infusion of the chemically bound drug and a course of conventional chemotherapy. During the infusion and for 45 min after, a magnetic field was built up as close to the unsuccessfully pre-treated tumour as possible (Bonadonna et al., 1993). The physiological parameters of the patient, such as body weight, blood volume, cardiac output, peripheral resistance of the circulation system and organ function, all affect the external magnetic field in addition to the problem of placing the magnetic field close to the target location (Lubbe et al., 2001). The size of the nanoparticles plays a very important role in drug delivery which affects the adsorption of the drug and reaching the defective tissue with maximum dose and minimum side effect. This as a result changes the response of the organ to the drug. After injection, large particles with diameters larger than 200 nm are easily removed by the spleen and ultimately eliminated by the cells of the phagocytic system, causing decreased blood circulation times. However, particles with diameters of less than 10 nm are rapidly removed through extravasation and renal clearance. Particles with sizes from 10 to 100 nm in diameter are optimal for intravenous injection and demonstrate the most prolonged blood circulation times. These particles are small enough both to evade RES of the body as well as to penetrate the small capillaries of the body tissues and so could offer the most effective distribution in certain tissues (Pratsinis and Vemury, 1996). After offering all these advantages with the clear route of the nanoparticles in the body, the nanoparticles provide encouragement to use as tools for studies in this field. (Kayal and Ramanujan, 2010) have shown that, after administration of iron oxide nanoparticles coated with PVA containing doxorubicin, up to 45% of the adsorbed drug was released in 80 h, indicating the success of drug delivery via magnetic particles. IONPs were covered by glycerol mono-oleate and used as a drug carrier. The anticancer chemotherapy agents paclitaxel and rapamycin anticancer showed high entrapment efficiency of about 95% and sustained release of encapsulated drug for more weeks under in vitro conditions. These results offer the possibility of improved therapeutic treatments of cancer cells (Dilnawaz et al., 2010). Aminodextran-coated IONPs were synthesised as drug carriers detectable by MRI. Agents were based on a macromolecular backbone with a high density of sites for MRI reporters. This radiopharmaceutical is the first specifically designed anticancer drug carrier. The aim of these nanoparticles is to increase the pharmaceutical potential of the MRI technique (Saboktakin et al., 2010). Gaihre et al., (2009) have reported the coating of IONPs with gelatine containing doxorubicin DXR and evaluated their potential as a carrier system for magnetic drug targeting. To understand their role, doxorubicin in coated iron oxide particles was prepared using adsorption and desolvation/cross-linking methods. Drug loading was carried out in various conditions of pH. The particles showed pH responsive drug release leading to progressive release of the drug at pH 4 compared to pH 7.4. The increase in the surface charge of coated particles after loading led to adsorption of positive doxorubicin to negative coated iron oxide particles, which indicated the loading had occurred. The increase or decrease in encapsulation efficiency was dependent on the surface charge of the coated particles and also confirmed the involvement of drug-to-particle interaction in the drug-loading. Sun et al., (2008) have reported the development of a chlorotoxin (CTX) loaded IONP drug carrier that may potentially be used as an MRI contrast enhancement agent. The CTX immobilised on the surface of nanoparticles via PEG functional groups offers a platform to allow for conjugation of other diagnostic and therapeutic agents to develop further platforms for both improved visualisation and targeting. Drug delivery was demonstrated in vitro in 9L cells and in vivo in mouse tumour models and compared to studies with control nanoprobes. Cellular uptake experiments showed that the uptake of IONPs coated with PEG and CTX conjugates by glioma cells was considerably higher than that of control nanoparticles. MRI contrast in 9L cells cultured with IONPs coated with PEG–CTX at 100 μg Fe/mL for 1 h at 37 °C were visualised with TEM, which shows that PEG–CTX nanoparticles were internalised into the 9L cells cytoplasm. Then they examined the efficacy of CTX-conjugated nanoprobes in targeting glioma cells and providing contrast enhancement for MRI also under the same conditions. Oleic acid coated IONPs in the presence of pluronic F-127 as stabiliser were used in both drug delivery and magnetic resonance imaging MRI in vivo. The drugs, doxorubicin and paclitaxel were incorporated into nanoparticles, either individually or together. This incorporation of drugs affected the physical size and zeta potential and magnetisation properties of the nanoparticles. Both of these drug nanoparticles showed high efficiency, either alone or in combination, for real time monitoring of drug distribution (Jain et al., 2008b). Hyperthermia Hyperthermia is being introduced as a new modality for cancer therapy. This is based on the principle that under an alternating magnetic field (AMF), a magnetic particle can create heat via hysteresis losses (Baker et al., 2006 and Hergt et al., 2004). The applications of IONPs for hyperthermia treatment were envisaged in the similar work of (Jordan et al., 1993). This experiment provided evidence of the capability of superparamagnetic particles to absorb the energy of an oscillating magnetic field and convert it into heat. This can be used in vivo to increase the temperature of the tumour and to damage the cell by hyperthermia (Jordan et al., 1999 and Moroz et al., 2002). IONPs, prepared using the microemulsion method and then coated with a thin hexamethyldisilazane layer used to protect the iron core, replaced the cetyl trimethyl ammonium bromide (CTAB) coating on the particles. In the next step, phosphatidylcholine was coated on the nanoparticles’ surface. These particles showed high magnetisation and increased hysteresis losses, improving its use for hyperthermia when compared with uncoated IONPs (Zhang et al., 2010a). The effect of the particle size on this application was studied by Gonzales-Weimuller et al., (2009) who showed for the first time that, at a given frequency, the heating rates of superparamagnetic particles are dependent on particle size. The results showed that higher heating rates are possible by increasing the size of the particles from 4.6nm to about 12.5 nm. IONPs were coated with oleic acid by a co-precipitation method then coated with chitosan by crosslinking. The saturation magnetisation of the nanoparticles was 30.7 emu/g measured by magnetic measurement, and showed superparamagnetic properties at room temperature. The results indicated that the heating effect was significant and the heating could be adjusted by changing the concentration of nanoparticles (Qu et al., 2010). IONPs were embedded in silica microparticles for treatment of cancer lesions by magnetically induced local hyperthermia. The injectable formulations form gels entrapping magnetic particles into a tumour. The nanoparticles provided the possibility of administration by syringe with a high proportion of IONPs to permit large heating capacities. Hydrogel (poloxamer, chitosan and organogel) were incorporated with nanoparticles and injected into human cancers in mice. Organogel 8% poly(ethylene-vinyl alcohol) in Dimethyl sulfoxide DMSO containing 40% w/v of magnetic nanoparticles showed magnetically induced local hyperthermia of the tumour where hydrogel alone did not show adequate magnetic response to induce hyperthermia (Le Renard et al., 2010). Tissue Repair Tissue repair using IONPs is accomplished either through separating two apposing tissue surfaces then heating the tissues sufficiently to join them, or through welding, where protein or synthetic polymer-coated nanoparticles are acting as a bridge between two tissue surfaces to improve joining of the tissues. Temperature at 55.5ºC  is the best to induce tissue union (Lobel et al., 2000). Stem cells are the body’s master cells and have a unique ability to renew themselvesand give rise to other specialised cell types. These cells, therefore, have the potential to be used for transplantation purposes, for example, to replace degenerated cells or in the repair of destroyed tissue, providing signals so that the stem cells can yield suitable cell types for the development of a tissue (Kiessling-Cooper and Anderson, 2003). Using thrombin, which has been used for topical haemostasis and wound management for more than six decades, IONPs were bound to thrombin to show the efficiency of treatment for wounds and were compared with unbound thrombin using wounded rat skin. The investigation revealed that thrombin bound to IONPs increased the healing of the wound better than the free thrombin with reduced side effects compared to normal surgery (Ziv-Polat et al., 2010). IONPs have also been used in transplant monitoring. For example, IONPs modified with the near-infrared fluorescent CY5.5 dye (MN-NIRF) and covered with dextran. Preliminary MRI on an islet phantom showed the possibility of detecting a signal drop in the analysis. After implantation under kidney capsules, many MRI examinations were implemented over half a year, with no significant change in the T2 relaxation time in the labelled graft. Both implantation under kidney capsules and administration by intraportal infusion into diabetic mice models resulted in restoration of normoglycemia. The ability of the transplanted islets to secrete insulin was confirmed by ex vivo microscopy studies (Evgenov et al., 2006). In comparison between IONPs coated with poly(vinyl pyrrolidone) (PVP) and commercial IONPs (Feridex formulation) for monitoring of pancreatic islets, the PVP coated nanoparticle labelled cells showed no changes in morphology and viability and showed higher iron accumulation because of the small nanoparticle size, which was 5–8 nm in diameter, and because of the biocompatibility of the PVP coating (Huang et al., 2009).