ANALELE STIINTIFICE ALE UNIVERSITATII “AL. I. CUZA” IASI Tomul II, s. Biofizică, Fizică medicală şi Fizica mediului 2006

RECENT ADVANCES IN BIOLOGICAL AND MEDICAL APPLICATIONS OF MAGNETIC FLUIDS

Mihaela Răcuciu1

Key words: magnetic nanoparticles, magnetic fluids, bio-medical applications.

This review is dedicated to the presentation of magnetic fluid utilizations in life sciences. The article is focused on several possible applications in the field of biology and medicine. Potential applications of biocompatible magnetic fluids in various areas of biosciences and biotechnologies are also suggested on the basis of our own studies.

1. INTRODUCTION

As more and more assumed in the last decades, magnetism plays an important

role in living beings metabolism. For example, the haemoglobin in our blood is an iron complex and is magnetic in nature. Magnetite, Fe3O4, is a biocompatible structure and therefore it is one of the most extensively used biomaterials for different biological and medical applications from cell separation and drug delivery to hyperthermia. A large number of magnetic materials, in the form of nanoparticles, has been exploited for a variety of biomedical applications. At present time specialty literature offers tremendous data that enable the reader to put together numerous evidences suggesting that all living organisms, including animals and humans, contain magnetic nanoparticles and act as magnetic receptors [1].

2. MAGNETIC PARTICLES

Magnetic nanoparticles having possible biomedical applications can be prepared usually in the form of magnetite (Fe3O4), greigite (Fe3S4), maghemite (γ-Fe2O3), various types of ferrites with metal ions (CoO·Fe2O3, MnO·Fe2O3, NiO·Fe2O3, etc.), iron, nickel etc. Synthetic magnetic nanomaterials are often available in the form of magnetic fluids (ferrofluids). The first magnetic nanoparticles in the form of a stable magnetic fluid were prepared in the ’60s. At the same time it was found that various species are able to synthesize biogenic magnetite. So, the teeth of the chiton, a sea mollusk, have magnetic iron caps. Discovered in 1962 by Heinz A. Lowenstam [2], it was the first evidence that ferrous minerals (magnetite) can be produced within living bodies. Prior to this discovery, magnetite was thought to form only metamorphic rocks. In 1975 Blakemore discovered magnetotactic bacteria [3-5], which now represent the most studied biomagnetic system. Magnetotactic bacteria represent a 1 “Lucian Blaga” University, Faculty of Sciences, Dr.I.Ratiu Street, No.7-9, 550024, Sibiu, Romania, mracuciu@yahoo.com

MIHAELA RACUCIU

morphologically and physiologically diverse group of motile, Gram-negative prokaryotes, ubiquitous in the aquatic environments which have the ability to synthesize fine (50-100nm) intracellular structures containing bound ferromagnetic crystalline particles of magnetite (Fe3O4) or greigite (Fe3S4) covered with an intracellular phospholipids membrane in the form of so called magnetosomes [6].

Magnetosomes, defined as intracellular, single-magnetic-domain crystals, either of iron oxides or the iron sulfide greigites, enveloped within a membrane or membrane-like structure [7, 8], are the hallmarks of the magnetotactic bacteria and are considered responsible for their behavior in magnetic field gradients. Although most magnetotactic bacteria produce only one mineral type, a rod-shaped magnetotactic bacterium from the Pettaquamscutt Estuary contains both magnetite and greigite [9] while recently, non-magnetic iron sulfides together with greigite have been identified in some organisms [10, 11]. The iron sulfide-type magnetosomes contain particles of greigite [12, 13] or a mixture of greigite and some non-magnetic greigite precursors including mackinawite (tetragonal FeS) and possibly, sphalerite-type cubic FeS [10, 11]. The behavior of various other organisms is also influenced by the local or temporal variations of the magnetic field. Many organisms contain magnetite: it has been found in insects, birds, dolphins, men, and bacteria [3, 14-21]; moreover, it was also found in the human brain (1992): human brain tissue extracts taken from membranes surrounding the brain and spinal chord (including clustering in the region of the brain where the nose joins the skull) were found to contain crystals of magnetite [22, 23]. During the last years literature reports mentioned another type of magneto-sensitive structures in bacterial and archaeal cells [24, 25].

Extracellular production of nanometer magnetite particles by various types of bacteria has also been described [26] - it has been shown that many neurodegenerative diseases are connected with the disruption of normal iron homeostasis in the brain.

Fig.1 - Electronic microscopy image for magnetotactic bacteria

3. MAGNETIC FLUIDS

Magnetic fluids (known also as ferrofluids) represent a special category of

smart nanomaterials, in particular magnetically controllable nanofluids [27]. They are colloidal suspensions composed of single-domain magnetic nanoparticles, such as Fe3O4, γ-Fe2O3, CoFe2O4, Co, Fe or Fe-C, dispersed within adequate carrier liquids (polar or non polar) [28, 29]. Since their yielding in 1965 by Papell [30], magnetic

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fluids have raised a growing interest in the scientific and industrial communities due to their physical properties and applications.

They brought together various branches of science, e.g. magnetism, physics of fluids and disperse systems, physical and chemical colloidal chemistry, hydro and thermo mechanics as well as engineering sciences. The possibility to control their physical properties by external magnetic fields has led to many innovative applications in mechanical, biomedical and optical systems.

Fig.2 - Idealized illustration of an aqueous magnetic fluid

The research field of magnetic fluids needs to be approached as a multi-

disciplinary area. Chemists studied the synthesis and producing of the magnetic fluids, physicists studied their physical properties and proposed theories to explain them, engineers studied their applicability and used them in technological products, biologists and physicians studied their biomedical possibilities and utilized them in medicine as well as in the biological field research.

In recent years, substantial progress has been made in developing technologies in the field of magnetic microspheres, magnetic nanospheres and magnetic fluids. Techniques based on the use of magnetisable solid-phase supports have found application in numerous biological fields viz. diagnostics, drug targeting, molecular biology, cell isolation and purification, radio immuno-assay, hyperthermia (causing agents for cancer therapy), nucleic acid purification, etc [31–33].

One of the most important points in the biomedical applications of magnetic nanoparticles is the encapsulation of the magnetic material, in order to make it not only stable but also biocompatible, and to have the possibility of producing a bio-magnetic fluid. Coating the nanoparticles with a suitable molecule offers the possibility of attaching them to antibodies, proteins, medical drugs etc. Therefore studies on surface adsorption, the functionalizing and/or conjugating of the particles by coating them with bioactive components are recognized as crucial technical issue in the ferrofluid technology. The selection of the magnetic material as well as a detailed knowledge of its magnetic properties play an important role in the use of the nanoparticles in bio-medicine, especially in the effectiveness of the desired application.

Many of the particles currently used are superparamagnetic, meaning that these particles can be easily magnetized with an external magnetic field and redispersed immediately once the magnet is removed. Currently available formats of particles can be broadly classified into unmodified or naked particles, chemically derivative particles with general specificity ligands (streptavidin, protein A, etc.) and

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chemically derivative particles with specific recognition group viz. monoclonal and polyclonal antibodies [34].

For biomedical applications the use of particles that present superparamagnetic behavior at room temperature (no remanence along with a rapidly changing magnetic state) is preferred [35–37]. The magnetic nanoparticles offer the possibility to be directed towards a specific target in the human body and remaining eventually localized, by means of convenient applied magnetic fields.

The colloidal stability of the magnetic fluid in water at neutral pH and physiological salinity will depend first on the dimensions of the particles, which should be sufficiently small so that precipitation due to gravitation forces can be avoided, and second, on the charge and surface chemistry, which give rise to both steric and electrostatics repulsions [38]. Additional restrictions regarding the particles intended used for biomedical applications are related on whether these particles are going to be used for in vivo or in vitro applications.

In vivo applications (like drug delivery [39-47], magnetic resonance imaging [48-51], tumor hyperthermia [52-55]), require not only stable and biocompatible particles but also biodegradable ones. These problems can be solved by adequate coating and embedding of the particles with suitable biocompatible molecular shells.

When magnetic fluids are used as a delivery system for anticancer agents in localized region tumor therapy – known as “magnetic drug targeting”- one attempts to concentrate a pharmacological agent at its site of action in order to minimize undesirable side effects in the organism and to increase its localized effectiveness [56]. Biophysical targeting refers to the magnetic transport of a responsive drug carrier through the endothelium or the use of a temperature-sensitive drug carrier with concurrent regional hyperthermia [57]. The property of magnetic fluids of absorbing electromagnetic energy at a frequency that is different from the frequency at which water absorbs energy allows the local heating up of a localized volume within a living body, where magnetic fluid has been directed.

Magnetic fluids have been investigated as potential hyperthermia causing agents due to their high specific absorption rate. Hyperthermia is a promising approach for cancer treatment that uses AC magnetic fields to heat cancer tissue supplied with magnetic fluids. The results of such experiments have shown that magnetic fluid hyperthermia is able to reduce the viability of cancer cells, thereby indicating the potential of this therapy [55]. Also for cancer therapy there is the possibility of combining the hyperthermia treatment with chemotherapy.

For example the magnetic fluids have been used for the treatment of cranial aneurisms without surgery [58, 63] and for the magnetically guided selective embolization of the renal artery in case of a renal tumor [59], or, as a contrast agent for MRI in the diagnostic evaluation of liver and spleen tumors [60] where they naturally accumulate. A specific cell separation method called “immuno-magnetic cell separation” for the early detection of cancer [61] was designed based on ferrofluids, while these have also been an important subject in the development of an implantable artificial heart [62]. Magnetic fluids actuators are being developed for implantable artificial hearts, which are driven by external magnetic fields. Magnetic fluids have already been used as a tracer of blood flow in noninvasive circulatory measurements. Scientific reports are also focused on the investigation of the dermal effects of

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magnetic fluids impact with human skin [64], the results indicating that the magnetic fluids are highly compatible with skin tissue even after prolonged exposure periods and intensive massage during application. No abrasive effects on the skin could be detected.

New applications are concerning the use of injectable magnetic fluids in the eye tissues since they could be capable of repairing all areas of the damaged retina [65]. The researchers speculate that use of a magnetized version of the conventional silicone fluid technique would facilitate tissue repairs and make the process more precise due to the possibility of the magnetic fluid driving toward those areas of the eye that are more difficult to reach.

Aqueous magnetic fluids have been successfully used to orientate biological assemblies such as the tobacco mosaic virus, enabling new information acquisition regarding the helical structure of the virus [66].

Generally, the biocompatible magnetic fluids are designed as aqueous magnetic colloids based often on magnetite or maghemite as magnetic cores, coated by non-magnetic shells of biocompatible and even biodegradable organic molecules. Aqueous magnetic fluids are the most commonly used injection vehicles [70]. Typical shell materials are polysaccharides such as dextran [67, 68] and starch [71, 72], or polymers [79], for example polyvinyl alcohol [69]. Other coating materials used for stabilization of water based magnetic fluids are citric acid [74-77], gluconic and aspartic acids [78], glutamic acid [78], 2,3-dimercapto-succinic acid [79,80], β-cyclodextrin [73, 80].

Coating the magnetic nanoparticles allows also the possibility of modifying the particle surface by attaching bioactive components, as antibodies, proteins etc. [68], by means of chemical bonding or by means of adsorption phenomena, which broadens their possibilities for biomedical applications.

A detailed picture of the biomedical applications of magnetic fluids reveals strict requirements regarding: chemical composition, size distribution uniformity, cristalline structure, stability of magnetic properties, surface structure, adsorption properties, solubility and low toxicity [83]. Many of these properties depend decisively on particle size, shape, composition and structure, and therefore rigorous control of the synthesis of magnetic fluids is necessary [84-90].

The first utilizations of magnetic fluids in biology are reported since 1992. Thus, the magnetic fluids were supplied to “in vitro” plant tissue cultures of some agricultural plants and species of ornamental interest: Triticum aestivum, Lilium regale, Chrysanthermum hortorum, Mammilaria sp., Ipomoea batatas, Aztekuim ritteri, Mammilaria duwei, Drosera rotundifolia [91-95]. Relatively small number of studies is dedicated to the influence of magnetic fluids on the plant organism: Corneanu et al. (1997) revealed the stimulatory magnetic fluid effect [94] on the starch accumulation in the vegetal cell (TEM investigations) while Godeanu et al. (1998) evidenced some stimulatory effects on the plant growth [95].

The ultrastructural studies carried out on Mammilaria duwei, achieved in culture medium with magnetic fluids, have shown a higher photosynthetic activity. As well, the chloroplasts have a better evolution regarding the granal system while the subepidermical cells present better developed cytoplasma and organites, comparatively with the plants grown on the culture media in the lack of the magnetic fluids. Better

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results can be obtained by using the cumulative effects of two types of magnetic fluids (water based and petroleum based). In vitro cultures, obtained from explants of different species, in the presence of different concentrations of magnetic fluids for every species or explant type, have shown a higher metabolic activity i.e. an improving of the plants development, of the callus production process as well as the root better development. Experimental studies have been designed concerning the orientation effect of the magnetic particles in culture medium using an electromagnetic exposure of Mammilaria duwei (Cactaceae) species [92]. Thus, in a normal geomagnetic field a positive correlation has been obtained between the development rhythm and the concentrations of the water suspension of the magnetic fluid in the lack of the magnetic exposure, while in the plantlets maintained in a near-null geomagnetic field the correlation was negative. Compared to the aqueous magnetic fluid that induced the growth inhibition when in near-null geomagnetic field, the petroleum based magnetic fluid stimulated the growth process. The growth in height of the plantlets developed on culture medium supplied with oily magnetic fluid exhibited positive correlation with the magnetic fluid concentrations in geomagnetic field but negative correlation was revealed in near-null geomagnetic field. It seems that the inhibition of the rooting process in near-null geomagnetic field – up to 50% - can be prevented by supplying the culture medium with petroleum based magnetic fluid.

On the basis of such studies carried out on Fragaria ananassa, Chrysanthermum hortorum and Mammilaria duwei [93] cultivated on culture medium supplemented with both aqueous and oily magnetic fluids, the authors have concluded that the metabolic activity in plant tissues was significantly increased. Thus, the magnetic fluids usage have led to the vitalization of senescent tissues, diminution of necrosis process and acceleration of springing process.

Other authors studied the effects of magnetic fluids on vine species infected with “Grapevine Fan Leaf” virus. This virus is specific for vine where it induces the plant’s destruction. Vine plants infected with “Grapevine Fan Leaf” virus have been studied after being treated with various dilutions of magnetic fluid - concentrations of the order of several magnetic fluid milliliters per liter. The stimulation of the growing process in the samples supplied with magnetic fluid was revealed, in 75% of the plant specimen the inhibition of virus activity being noticed. The experimental observations led to the conclusion that magnetic fluids were able to stimulate the growth and the development of plants, as well as to inhibit the activity of parasites.

Another group reported [96] the positive effect of the magnetic fluid supply on the biosynthesis of assimilatory pigments in Spirulina Plantensis plantlets. Different concentrations of water based magnetic fluid (10-5g/cm3) have been tested, the results evidencing an increase of biomass with up to 17%.

Our research team has also studied the biological effects of magnetic fluids on young plants and microorganisms culture. The stimulatory effect of petroleum and water based magnetic fluid on the division rate in the root meristeme cells was first noticed. Magnetic fluid presence in culture medium seemed to be able to provide chromosomal aberrations in young vegetal plants of pharmaceutical interest, Chelidonium majus and Papaver somniferum [97] – aiming to continue in the frame of future studies with the selection and propagation of some convenient modified features of the plant. Parallel investigations have been done regarding the effects of petroleum

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based magnetic fluid on the cellulase specific activity in Chaetomium globosum [98]. The authors have noticed that the magnetic fluid concentration range is important as well as the culture’s age: small ferrofluid concentrations led to stimulatory effects in younger cultures while higher concentrations had mainly inhibitory effects, especially in older cultures.

Lately the interest for study the biological effects induced by magnetic fluid supplied in the plant and microorganisms culture medium [99-108] as well as in the animal tissues [109] has increased. Our team has further developed experiments focused on the correlated influence of the magnetic fluid on the biosynthesis of assimilatory pigments, nucleic acids and enzymes in different plant species [108]. Thus, we evidenced that the catalase biosynthesis was stimulated by the vegetal cell as response to the increased concentration of magnetic fluid added in the culture medium [110] - the organism of young cereal plant seems to be able to respond this way to the increased amount of hydrogen peroxide yielded by the magnetic fluid influence. Stimulatory effect of biocompatible magnetic fluid (magnetite coated with citric acid) on the photosynthesis in the young plants of maize was observed [111] - the activity from the LHC II system appearing changed by the magnetic fluid addition as suggested by the chlorophyll ratio that was significantly diminished for certain magnetic fluid concentrations [112]. The use of different other biocompatible magnetic fluids coated with different organic compounds, has led to similar results regarding the growth of young plants during their early ontogenetic stages. So, the relatively low concentrations of an aqueous magnetic fluid stabilized with tetramethylammonium hydroxide N(CH3)4OH was able to stimulate the plant proliferation (up to 30%) and to induce a higher level of various types of chromosomal aberrations: micronucleus, inter-chromatin bridges, retarded and expulsed chromosomes, chromosome fragments [113]. Relatively small levels of the same magnetic fluid induced in young maize plantlets the increase of the chlorophyll level - up to 13% as well that the nucleic acid level - up to 10% while relatively high magnetic fluid concentration may have severe disruptive effects, such as the diminution of the chlorophyll biosynthesis or the decrease of the chlorophyll a/chlorophyll b ratio - about 35 % [114]. Such results regarding the positive influence of magnetic fluids upon plant growth might be explained on the basis of iron importance in the plant metabolism [115-117].

Some molecular and cellular phenomena that could be taken as basic statements for the hypothesis that magnetic fluid can stimulate the plant metabolism, are mainly the existence of an efficient mechanism of iron acquisition by graminaceous plants (resulting in the release of iron complex compounds [118] called phyto-siderophores) and the cooperation between plants and microorganisms, the plants being the beneficiaries of the presence of some growth stimulatory bacteria (since under iron-limited conditions [119], these microorganisms can produce bacterial-siderophores - that can be further internalized by plant root cells).

In this context, the peculiar ability of the fungus Rhizopus arrhizus to produce the siderophore named rhizoferrin was investigated by Yehuda et al. who focused on the mechanisms by which some graminae species utilize Fe from phytosiderophores [120]. We mention the results of Sherker et al. [121] who studied the phytosiderophores produced by plants that are excreted directly to the rhizosphere; iron uptake by barley and corn plants grown in nutrient solution was found to run

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parallel to the diurnal rhythms of phytosiderophore releasing via an indirect mechanism of ligand exchange between the ferrated microbial siderophore and phytosiderophores, which are then taken up by the plant.

4. CONCLUSIONS

Biocompatible magnetic materials might be used for a considerable variety of applications in biology and medicine, since they induce biological effects either in plants, animals and microorganisms. Our own investigations focused mainly on the vegetal species suggested that the biotechnological interest in both aqueous and oily magnetic fluids is highly justified.

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