Superparamagnetic nanoparticles for biomedical applications: Possibilities and limitations of a new drug delivery system

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Journal of Magnetism and Magnetic Materials 293 (2005) 483–496 www.elsevier.com/locate/jmmm

Review

Superparamagnetic nanoparticles for biomedical applications: Possibilities and limitations of a new drug delivery system Tobias Neubergera, Bernhard Scho¨pfa, Heinrich Hofmannb, Margarete Hofmannc, Brigitte von Rechenberga, a

Musculoskeletal Research Unit, Equine Hospital, Vetsuisse Faculty Zurich, University of Zurich, Winterthurerstr. 260, 8057 Zurich, Switzerland b Laboratory of Powder Technology, Institute of Materials, Swiss Federal Institute of Technology, EPFL, 1015 Lausanne, Switzerland c MatSearch Pully, Chemin Jean Pavillard, 14, CH-1009 Pully, Switzerland Available online 2 March 2005

Abstract Nanoparticles can be used in biomedical applications, where they facilitate laboratory diagnostics, or in medical drug targeting. They are used for in vivo applications such as contrast agent for magnetic resonance imaging (MRI), for tumor therapy or cardiovascular disease. Very promising nanoparticles for these applications are superparamagnetic nanoparticles based on a core consisting of iron oxides (SPION) that can be targeted through external magnets. SPION are coated with biocompatible materials and can be functionalized with drugs, proteins or plasmids. In this review, the characteristics and applications of SPION in the biomedical sector are introduced and discussed. r 2005 Elsevier B.V. All rights reserved. Keywords: Superparamagnetic iron oxide nanoparticles (SPION); Nanoparticles; Magnetic drug targeting (MDT); Immunomagnetic cell sorting (IMS); Magnetic hyperthermia; Toxicity; Hyperthermia; Review; Contrast agent; Nanoparticle functionalization; Nanoparticle application

1. Introduction Superparamagnetic iron oxide nanoparticles (SPION) are small synthetic g-Fe2O3 or Fe3O4 Corresponding author. Tel.: +41 1 635 84 10; fax: +41 1 635 89 17. E-mail address: [email protected] (B. von Rechenberg).

particles with a core size of o10 nm and an organic or inorganic coating. If the crystal size is small enough the thermal energy kT (where k is Boltzmann’s constant and T is the absolute temperature) may be sufficient to cause fluctuations of the magnetization direction. The term ‘‘superparamagnetism’’ is used to infer an analogy between the behavior of the small magnetic moment of a single paramagnetic atom and that

0304-8853/$ - see front matter r 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jmmm.2005.01.064

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of the much larger magnetic moment of a nanosized magnetic particle which arises from the coupling of many atomic spins [1]. After eliminating the magnetic field, the particles no longer show magnetic interaction; a feature that is important for their usability [2,3]. Particles such as liposomes containing magnetic oxide particles in their cavities are called magnetosomes [4], whereas aggregates consisting of organic compounds (dextran, starch, etc.) in combination with nanoscale iron oxide particles are called superparamagnetic beads or agglomerates. The particles are well dispersed in a liquid, for medical application normally in water, or form composites with organic or inorganic matrices, the so-called beads. Superparamagnetic magnetization is, compared to normal paramagnetic materials, much higher and can reach nearly the magnetization saturation of ferromagnetic iron oxide. This behavior allows the tracking of such particles in a magnetic field gradient without loosing the advantage of a stable colloidal suspension. Additionally, applying an alternating magnetic field, heating of the particles based on the Ne´el relaxation mechanism can be observed. The surface modification by organic molecules has different tasks to fulfill: (i) stabilize the nanoparticles in a biological suspension with a pH around 7.4 and a high salt concentration, (ii) provide functional groups at the surface for further derivatization, and finally (iii) avoid immediate uptake by the reticulendothelial system (RES) [5]. Drug targeting has emerged as one of the modern technologies for drug delivery [6]. SPION in combination with an external magnetic field allow delivering particles to the desired target area and fixing them at the local site while the medication is released and acts locally (Magnetic drug targeting, MDT) [6–10]. Therefore, the dosage of the medication can be reduced and the systemic effect of the drugs kept to a minimum [1,6]. The possibilities of SPION applications have drastically increased in recent years [1,2,4,9,11]. In the clinical area of human medicine, these particles are being used as delivery systems for drugs [12], genes [13,14] and radionuclides [4]. Furthermore,

ferrofluids, as contrast agents in magnetic resonance imaging (MRI), are routinely applied in the field of diagnostic imaging [15–20]. SPION are also attractive for in vitro applications in medical diagnostics, such as research in genetics [14] and technologies based on immune magnetic separation (IMS) of cells, proteins, DNA/RNA, bacteria, virus and other biomolecules [21]. Furthermore, in recent years, advances were made using SPION in clinical studies of cancer therapy in veterinary [22,23] and human medicine [7,24] by attacking solid tumors with magnetically targeted 4-epidoxorubicin, or enhancing intraarterial chemotherapy through targeted retention of particles. Magnetic fluid hyperthermia (MFH) is another field where SPION are applied to create an increase in temperature by applying an oscillating magnetic field to kill the tumor cells [25,26]. This type of therapy is already used in human patients in the field of oncology. One of the promising applications of SPION in the future could be focused on the musculoskeletal system in humans and animals [27]. Many of the diseases in the musculoskeletal system are characterized by local inflammatory processes and currently are treated with systemic non-steroidal antiinflammatory drugs (NSAID’s) [28] or corticosteroids [29–31]. Apart from systemic side effects from these drugs, such as gastric ulcers or bleeding tendencies, local access or maintaining of therapeutic drug concentrations may be a problem. SPION could serve a major purpose through drug delivery to inflammatory sites to maintain appropriate concentrations while at the same time reducing cost, overall dosage and unwanted side effects. The magnets used in conjunction with SPION as drug carriers would allow switching on and off the magnetic field, thus targeting the particles at the local site for time, dosage and elimination. One of the reasons why the use of SPION was limited in the last 2 decades was their insufficient characterization, the inhomogenous morphology and fast elimination through the RES [1]. However, new technologies in synthesis and methods of particle analysis together with more sophisticated coatings optimized properties of SPION and thus, their use has become highly attractive for medical

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applications in diagnostics and therapy. Iron oxide nanoparticles with an average size below 10 nm can generally be obtained by various chemical and physical synthesis methods [1,2]. Their outstanding properties are principally related to their small size, and by this mainly to their narrow size distributions [32,33]. Three different types of magnetic colloids can be prepared through various stabilization methods [34]. The first magnetic colloid is prepared by coating a magnetic core with a suitable surfactant, e.g., sodium oleate (NaOl), Dextran, PVA on SPION. The second stabilization method is based on the formation of nanocomposites consisting of SPION distributed throughout a nonmagnetic coating, composed of a material like polymeric starch [35]. The presence of polymeric starch during SPION formation hinders cluster growth after nucleation. These polymeric networks cover a large number of continuously formed iron oxide monodomains and hold them apart against attracting forces. The third magnetic colloid suspension is stabilized by the formation of liposome-like vesicles filled with SPION. They may consist of either a polymeric matrix (nanospheres) or of a reservoir system in which an oily or aqueous core is surrounded by a thin polymeric wall (nanocapsules) [5]. Polymers suitable for preparing nanoparticles include poly (alkylcyanoacrylates), poly(methylidene malonate and polyesters such as poly(lactic acid), poly (glycolic acid), poly(e-caprolactone), and their copolymers. Methods for the preparation of nanoparticles can start from either a monomer or from a preformed polymer. They are reviewed in Barratt et al. [36]. Nanospheres can also be formed from natural macromolecules such as proteins and polysaccharides, from nonpolar lipids, and from inorganic materials such as metal oxides and silica as shown by Chastellain et al. [37]. The mentioned new technologies using SPION allow the minimization of systemic side effects while maintaining therapeutic concentrations locally, may lead one step closer to the ‘‘Magic Bullet’’, an expression introduced by Paul Ehrlich as early as 1906 [38,39]. Drug targeting involves passive, active or physical targeting. In passive targeting the dis-

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tribution of the drugs within the body occurs through drug and carrier properties that are unchanged (‘‘Prodrugs’’) [13]. Active targeting is achieved with mechanisms that allow direct targeting of drugs and/or carriers to specific cells, tissues or organ systems through specific recognition mechanisms. Physical targeting allows distribution of drugs and carrier systems through external influences, such as magnets in the case of SPION or heat [40,41]. Several strategies are possible for targeting drugs and carrier systems to their locus of interest: For loco-regional therapy the system is delivered as close as possible to the target site (firstorder targeting). Application of drugs and carriers occurs either intravascularly or into a cavity as it is currently done in cancer therapy using cytostatic drugs. Receptor oriented drug/ carrier systems take advantage of the interaction between the coupling of antibodies and/or hormones to the local receptors (second-order targeting) [42]. The use of colloidal systems, where substances with a very fine distribution and particle size between 5 and 500 nm are used as carrier systems, changes the distribution pattern of drugs within the body [43]. Apart from nanoparticles, also microparticles, nano- and microemulsions, liposomes, niosomes, pharmacosomes and tenside conjugates belong to this group. A complete characterization of the particulate system is necessary to make a decision whether the use of a nanocarrier system is appropriate for a specific in vivo application. Nanoparticles can be described with the following physicochemical properties [44–48] depending upon vital factors for their distribution within the body system: size of particles, toxicity, surface charge, capacity for protein adsorption, surface hydrophobicity, rate of loading, release kinetics, stability resp. degeneration of carrier system, hydration behavior, electrophoretic mobility, porosity, specific surface characteristics, density, cristallinity, contact angle and molecular weight. Size of particles, toxicity, surface charge and protein adsorption capacity are the most important features for use in vivo and thus, will be further discussed in more detail.

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2. Properties and characteristics of particles Size of particles: The size of particles usually refers to the total diameter of the particles including the iron core and the coating. Since the smallest diameter of capillaries in the body is 4 mm [49], larger particles will be mainly captured and withheld in the lungs [50]. Particles with larger sizes and/or aggregations of small particles thus may be trapped, causing emboli within the capillary bed of the lungs [45]. Depending on their magnetic energy, most nanoparticles have a tendency to aggregate, thus reducing their surface charge. This may lead to precipitation that could prove dangerous if these particles are injected intravenously. Therefore, it is important to know the surface charge and aggregation behavior of the particles in blood [51]. Most intravenously applied nanoparticles are recognized as ‘‘foreign’’ from the body system and are eliminated immediately through macrophages of the mononuclear phagocytosis system (MPS) [52]. Particles smaller than 4 mm are taken up through cells of the reticuloendothelial system, mainly in the liver (60–90%) and spleen (3–10%) [45,52]. While it is more likely that small particles up to 100 nm will be phagocytosed through liver cells (openings in the endothelium of liver sinusoids are between 100 and 150 nm), there is a tendency for particles larger than 200 nm to be filtered by the venous sinuses of the spleen [53]. If particles between 30 and 100 nm are intravenously applied, the liver eliminates the larger particles faster from the bloodstream compared to the smaller sizes. Thus, the larger the particles are, the shorter is their plasma half-life-period [54]. Depending on particle size, uptake may be subdivided in phagocytosis (all sizes) or pinocytosis (particles o150 nm) [52,55]. Large particles will be only removed by cells capable of phagocytosis, whereas smaller particles can be removed by all types of cells through pinocytosis (all cells are capable of pinocytosis). Phagocytotic activity increases with size of particles [55]. Under physiologic conditions, particles larger than 10 nm cannot penetrate the endothelium [1]. However, this permeability barrier may be increased under pathologic conditions, such as inflammation or tumor infiltration. There, the

penetration threshold can be increased to allow 700 nm particles [53]. This can also be temporarily achieved through the help of medication, immune modulators, heat or radiation [1]. In conclusion, the uptake of nanoparticles is strongly dependent on particle size as it was proven in vitro [56,57] and in vivo [55,58]. Toxicity of particles: All pharmaceutical substances intended for use in humans and animals require extensive testing for toxic side effects. This is also true for SPION. Apart from acute toxicity, the toxicity of degradation products, stimulation of cells with subsequent release of inflammatory mediators [55], and toxic effects through the particulate system have to be seriously considered. A first indication about the toxicity can be obtained by studying tissues from cell cultures histologically after incubation with nanoparticles [27,59–62]. However, the cytotoxicity is usually much higher in vitro compared to in vivo. This may be explained by the fact that degradation products responsible for the toxicity are eliminated continuously from the application site in vivo. Therefore, toxicity tests conducted in vitro may have limited application [55]. If suspensions containing nanoparticles are used in vivo, they should be hydrophilic and their pH should be close to 7.4 [1]. In addition, they should be degraded and eliminated by the body system without residues, otherwise they may accumulate in certain cell compartments, such as liposomes, or tissues from the phagocytosis system (MPS) [55]. One of the feasible toxicity tests in vivo is the intraperitoneal application of nanoparticles in mice [63,64] that allows studying the LD50 dosage, the mitotic index (mutagenicity), and the effect of particles on macrophages and other cells (liver, spleen, kidney, peritoneal cells) by means of histology. The toxicity (acute and subacute toxicity, mutagenicity), and pharmacokinetics (body distribution, metabolism, bioavailability, elimination) of nanoparticles with a median diameter of 80 nm (measured by laser light scattering) was investigated earlier in a large experimental study in dogs as well as mice [65]. The particles tested were planned for later studies centered on MRIdiagnostics of liver problems. Before intravenous application, the particles were functionalized with

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radioactive iron and several parameters were studied: (i) the distribution of radioactivity within different tissues using a special indicator for Feisotopes, (ii) the relaxation time of liver and spleen in MRI, (iii) the capability to treat a previously induced iron deficit anemia, (iv) pathology of several organ systems by means of histology, (v) chemscreen of blood and urine and (vi) the mutagenicity by means of a special test (Ames Salmonella Microsome Reverse Mutation Assay). One hour after injection, 82.6% of the particles could be detected in the liver and 6.2% in the spleen. The concentration of radioactive particles was slowly decreased in the liver (plasma half-life 3 days) and the spleen (4 days) and the radioactive iron was incorporated into the hemoglobin of erythrocytes. The mean detection time (T2) in the MRI was shorter (2 days), since only unchanged particles are capable of inducing enough contrast to be detected. The previously induced anemia, however, was successfully treated within a period of 7 days. No acute or subacute toxic side effects were found in histology or serologic blood tests, although a maximal dosage of 3000 mmol Fe/kg was applied in the dogs and rats. This corresponds to a 150  increase in dosage compared to what is required for diagnostic liver tests using MRI. Further in vivo tests with SPION based on particles of 100–1000 nm with either dextran, anhydroglucose or carbonate coating, and even clinical tests in humans showed excellent biocompatibility [7,24,66,67]. Surface charge: The measured surface charge of SPION is related to the electrical potential at the shear plane of the double layer, the so-called zeta potential, which is measured through the electrophoretic motility [55]. Using electrostatic stabilization of the SPION, a high zeta potential is needed. The zeta potential is dependent on electrolyte concentration of the soluble medium in vitro and additionally on the adsorbing plasma proteins in vivo [68]. If the zeta potential is lower than a given critical value of the particulate system, aggregation and precipitation of the particles occurs. The surface charge also plays an important role during endocytosis. There should be a slower uptake for negatively charged particles due to the negative ‘‘rejection’’ effect of the negatively

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charged cell membrane. However, the endocytosis index in vitro is minimal with a zeta potential close to zero [69]. In contrast, phagocytosis is increased with a higher surface charge independent of whether the charge is negative or positive [52]. The higher the surface charge, the shorter is the residence time of SPION in the circulatory system [54]. Protein adsorption capacity: If nanoparticles are injected intravenously, immediate interaction with plasma proteins occurs. The adsorption of proteins at the particle surface is called opsonization [70]. The amount of adsorbed proteins is based on the size of the molecules and the charge and hydrophobicity of the particle surface. With increasing size, charge and hydrophobicity of the particles, the capacity of protein adsorption increases [52]. Also hydrophobic interactions have an effect on protein adsorption [71] such that dehydration of hydrophobic areas results in entropy gain which in turn facilitates protein adsorption [72]. The adsorbed protein components play an important role in the biodistribution, degradation and elimination of the nanoparticles [52,73–75]. Proteins that encourage phagocytosis are called opsonins (e.g. immunglobulin G (IgG), complement system, fibronectin) [49,76], whereas those inhibiting phagocytosis are called dysopsonins [70,77]. The adsorption of a variety of proteins on nanoparticle systems has been investigated in vitro and in vivo [70,78–81]. The opsonins and dysopsonins as well as other inactive proteins of the blood plasma are responsible for the overall behavior of nanoparticles in vivo. Therefore, it is suboptimal to study the adsorption behavior of single proteins with nanoparticles [70]. It is much more effective to study all plasma proteins present in blood by means of two-dimensional polyacrylamide gel electrophoresis [52,82]. With this method, several thousands of plasma proteins can be separated at the same time according to their isoelectric point and molecular weight. The main challenge encountered with this method is to identify the adsorbed proteins in combination with the particles without creating artifacts [52]. Magnetic particles in contrast may relatively easily be separated from blood plasma by means of a

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magnetic separation technique (see below in vitro application of SPION). Studies elucidating the pattern of protein adsorption on SPION with a diameter of 65 nm and carboxydextran coating revealed a 40% IgG and 20% fibronectin adsorption of the overall adsorbed protein. In addition, IgM, IgD, complement factor C3, apolipoprotein A-1 and three further undefined proteins were detected. These particles were quickly eliminated and phagocytosed by Kupffer cells in the liver.

3. Application of superparamagnetic nanoparticles (SPN)/microspheres The number of possibilities for use of SPION has dramatically increased over the last couple of years [1,2,4,9,11,14]. In the field of clinical medicine, SPION have been propagated for gene transport, MRI, hyperthermia and radiotherapy (Tables 1 and 2) in vivo. Furthermore, SPION are used for separation of cells, proteins, DNA/RNA, bacteria, virus and biomolecules [21]. In vivo degradation of SPION is dependent on the core material as well as the coating. Materials like iron oxide, albumin, dextran, chitosan can be degraded, while ethylcellulose, polystyrene, polymethacrylate, silica and others are not degradable [1]. Lately, nanoparticles consisting of an iron oxide core are preferred because of their excellent magnetic properties and low toxicity for the body system [5]. Commercially available microspheres containing SPION have a diameter between 1 and 5 mm, but also larger particles may be purchased [83]. In most of the cases nanoparticles can be distinguished in three groups: (i) non-modified, uncoated SPION with a maximum of flexibility that later can be modified, (ii) SPION with a specific chemical surface modification, such as dextran, carboxyl- or amine-groups allowing functionalization with other molecules, and (iii) SPION with specific substances or recognition groups, where the bound molecules are pharmaceuticals, antibodies or other medical substances. Several possibilities for use of SPION in vitro and in vivo are outlined below.

Table 1 List of the most commonly used biodegradable magnetic particles Matrix of magnetic particles

Biomedical application

Erythrocytes

Drug targeting Cell separation

Liposomes

Drug targeting

Phospholipids

Immobilization of membranebound enzymes

Albumin

Drug targeting Cell separation

Starch

Drug targeting MRI Radiotherapy

Poly(lactic acid)

Radiotherapy

Dextran

Cell separation Enzyme immobilization MRT Local hyperthermia Drug targeting Immunoenzyme assay

Chitosan

Drug targeting

Polyalkylcyanoacrylate

Drug targeting

Polyethylene imine

Drug targeting

Table 2 List of the most commonly used non-biodegradable magnetic particles Matrix of magnetic particles

Biomedical application

Ethyl-cellulose Synthetic polymers (e.g., polystyrene, polymethylmetacrylate)

Arterial chemo-embolization Magnetic separation of bacteria, virus, parasites mRNA-purification Isolation of specific gene sequences Immunomagnetic assays

3.1. In vitro use of SPION SPION have proven to be very useful tools for magnetic separation techniques in clinical use and have replaced other separation technologies

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[1,2,9,21,84–91]. This is true for immunomagnetic cell separation and purification, as solid phase for immunoassays for isolation, purification and recognition of proteins [9,84,89]. In addition they are used for molecular biology, where they were shown to be useful for the isolation, purification, hybridization, synthesis and as markers for DNA/ RNA [2,9,84,87,89]. The isolation and detection of microorganisms is easily possible using SPION [2,84], as well as efficient gene transfer of nucleotides or gene sequences into cells [14]. In medical and clinical diagnostics, the interaction of antigens and antibodies is routinely used to measure concentrations of biological markers. Traditionally, antibodies and/or antigens are immobilized as solid phases on filters, tubular structures, plastic spheres or plates. The use of SPION as solid phases for these separation techniques has revolutionized and simplified this field of clinical chemistry through the development of more sensitive, highly efficient and automated immunoassays [1,3,9,85,90,91]. Also magnetic beads consisting of macroporous polymer particles containing iron oxide magnetic particles within the pores (microspheres) are successfully used for this technology of IMS [21,87]. This technique is an attractive, cost effective alternative for fluorescent activated cell sorting (FACS), which in contrast to IMS is less efficient, slower, requires a high dilution of the cell suspension and also has problems with sterility [85]. IMS allows detecting very low concentrations of cells (up to 10 cells/ml) and this is an advantage in early diagnostics of cancer in the search of circulating tumor cells in the blood [21,86] or bone marrow [1,21,85,88]. Cells [9,21], proteins, nucleic acids [9], bacteria, virus, parasites [2,85] and fungal agents may be well conserved if separated with IMS, and can be cultured in appropriate culture media afterwards without having to eliminate the particles [2]. Most of the commercially available magnetic separation systems function according to the same principle: the surface of the SPION is labeled with antibodies against epitopes of cells, bacteria or other target antigens [92]. Thereafter, the labeled particles are mixed with the suspension to be tested. After the antigen and antibody reaction, the labeled, immunomagnetic cell or bacteria

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suspension is pipetted in a separation container and a magnetic field is applied. While the immunomagnetic labeled cells remain within the container, all other substances are washed out. Through the development of the so-called ‘‘Microfabricated Flow System’’, the IMS technology was refined to the point where a continuous separation in a one-step procedure and without complicated washings can be achieved [85]. SPION have also demonstrated their efficiency as non-viral gene vectors that facilitate the introduction of plasmids into the nucleus multifold compared to routinely available standard technologies. As in cell separation, the applied magnetic field allows transporting the genes selectively to the desired local cells within cell cultures. This is possible for the investigation of cell differentiating factors in non-gene manipulated neighboring cells within the same cell culture [14]. 3.2. In vivo application of SPION Magnetic resonance imaging: Clinical diagnostics with MRI has become a popular non-invasive method for diagnosing mainly soft tissue or recent cartilage pathologies, because of the different relaxation times of hydrogen atoms [1,93]. SPION were developed as contrast agents for MRI and increase the diagnostic sensitivity and specificity due to modifications of the relaxation time of the protons [1,16,20,94–115]. The first dextran coated SPION were already 10 years ago officially registered as contrast agents for MRI of the liver in Europe [17]. The efficacy of the SPION as contrast agent in various tissues depends on their physicochemical properties, such as size, charge and coating [54], and can be increased through surface modifications by biologically active substances (antibodies, receptor ligands, polysaccharides, proteins, etc.) [1,16,116]. The hydrodynamic diameter of the SPION used with MRI varies between 20 and 3500 nm, although intravenously applied particles are relatively small and range between 20 and 150 nm with, or 5–15 nm without coating [15,95]. Coatings usually are made from derivates of dextran and poly(ethyleneglycol) [95], but also starch, albumin, silica, etc. [15].

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Since most of the particles are ingested from cells of the reticulendothelial system (RES), their distribution is most easily made visible in the liver, spleen, bone marrow [117] and lymphnodes [15,16,18,94,97,104,106,118]. Renal flow can also be visualized by means of SPION [99,119,120]. Dextran stabilized SPION with an overall size of 45 nm and an iron oxide core of 5 nm were functionalized with HIV-Tat proteins and could be introduced into several cell types, such as human hematopoietic CD34+ cells, mouse neural progenitor cells, human CD4+ lymphocytes and mouse splenocytes, in vitro. This was possible using a concentration of 10–30 pg SPION/ cell (0.5–2  107/cell) without loss of cell viability or function [62]. When these cells were injected intravenously in mice, their accumulation could be observed by means of MRI in the bone marrow, liver and spleen of these experimental animals. Even single cells could be detected through MRI. There was no difference in the biodistribution of loaded compared to non-loaded cells. Part of the loaded cells could even be re-harvested effectively by means of the IMS technology. It was also possible to follow the distribution of T-lymphocytes from the bone marrow with MRI in mice using the same SPION [61]. These results have great implications for research in the field of immunology and stem cell biology [61,62]. Furthermore, SPION were used as oral contrast agents for the diagnosis of gastrointestinal tumors [18,19,87,121], or intravenously for the detection of other tumors in the body system [16,17,23,105], infarcts in the cardiovascular system [18,96, 122–126], experimentally in cerebral areas [103,109,127] with increased permeability of the blood brain barrier [16,128]. SPION are also predestined for use as combined carrier systems for drug delivery while at the same time serving as contrast agent [16]. In this way, the kinetics of the pharmaceutical agent could be followed by means of MRI. In addition, distribution of particles can be influenced through the application of an external magnet [23]. Magnetic drug targeting (MDT): One of the major problems in pharmacotherapy is the delivery of drugs to a specific location and maintenance

of its location for the desired length of time. The total concentration of drugs could be reduced drastically and side effects could be avoided. Using an external magnetic field, SPION functionalized with reversibly bound drugs could be delivered to specific locations and localized in place [7,9]. The method of MDT is not only dependent on the physical properties, concentration and amount of applied particles, but also on the type of binding of the drugs. In addition, the geometry, size and duration of external magnet application and route of SPION injection, as well as vascular supply of the targeted tissues will influence their effect. The physiological parameters of the patient, such as body weight, blood volume, cardiac output, peripheral resistance of the circulatory system and organ function will also affect the efficiency of the external magnet apart from the possibility to place the magnet in close vicinity to the location [1,66]. MDT, however, is dependent on this external magnetic field that in most commercially available magnets a penetration depth of a few millimeters into the tissue is achieved. However, newer investigations report about permanent neodymium iron boron magnets in combination with SPION of excellent magnetic properties that increase the depth of the magnetic field up to 10–15 cm [22,67]. A good model to simulate the magnetic properties of SPION/ magnetic system in blood vessels is the ‘‘magnetic field capture of SPION in flowing system’’. With this system consisting of a circuit of rubber tubes, pumps, magnets and gaussmeter measuring the strength of the magnetic field, it is possible to mimic the distance necessary to withhold and accumulate the SPION in a moving fluid system with different viscosities (water, glycerol 30%) [129,130]. But also in vivo SPION were successfully applied intravenously and accumulated at specific locations by means of external magnets [7,9,14,22,23,66]. This is especially attractive for use in cancer therapy, where chemo- or radiotherapy is demonstrating serious, extensive side effects while only having a small therapeutic margin. Different methods were investigated such as thermo- and pH sensitive liposomes containing encapsulated chemotherapeutical agents or tumor specific antibodies [7]. By the late seventies

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magnetic particles comprised of magnetite were developed for transport of chemotherapeutics in the form of magnetic erythrocytes, magnetic albumin and polymer microspheres [131]. Their intravenous application in combination with an external magnet resulted in accumulation of the drug within the tumor area and tumor regression compared to a control group. The magnetic properties, however, were not sufficient in these particles and thus, further development was not pursued [22]. Experimental trials followed these initial studies investigating several tumor types, and localizations in animal species or even in humans using different types of SPION and magnetic microspheres [1]. There it was shown by means of MRI, histology and gammaradiography that a 6–10 times accumulation of 99Tc radioactively marked SPION (Fe3C, 0.5–5 mm) was achieved after intravenous injection under angiographic control in the liver and lung area in pigs [4]. Alexiou et al. were capable of achieving complete remission of experimentally induced VX-2 squamous cell carcinomas after intraarterial injection of starch coated SPION functionalized with methotrexate in the hind limbs of rabbits [23]. Local, reversible gray discoloration and alopecia was noted in the area of the external magnet, as well as rare urine discoloration that was not considered to be irritating. Further interesting experiments were conducted by Lu¨bbe et al. [66] using 100 nm SPION coated with polymeric anhydroglucose partially functionalized with epirubicin. The SPION were injected into the femoral vein of rats under the influence of an external magnetic field (0.2 T for 5, 10 and 30 min). Approximately 5% of the overall blood volume was injected. Groups without a magnetic field served as controls. With this intravital method, it is possible to visualize the microcirculation in the capillary bed of intact cremaster musculature with the help of a microscope. An irreversible thrombus formed within the capillary bed of the muscle after 10 min of magnet application indicating that this method could be used to induce microembolization of tumors. These observations were repeated by other authors in a similar experiment [67]. In further experiments,

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Lu¨bbe et al. reported complete tumor regression of malignant, aggressively metastasizing adenocarcinomas and hypernephromas that were implanted into ears or abdomen of mice. Again, starchcoated SPION partially functionalized with epirubicin were injected into the tail vein while an external magnetic field was applied in the area of the tumors. Regression was achieved with already an SPION amount equal to 0.5% of the total blood volume, whereas tumor embolization could only be achieved with unphysiologically large doses equal to 10% of the total blood volume. Side effects were minimal after SPION application, except if doses of more than 10–20% of the blood volume were injected. Then, lethargy, reduced food intake over 12–24 h and a grayish discoloration of the skin over the tumor area could be observed in some of the animals. However, the skin discoloration disappeared after 7–14 days. After these encouraging results, the SPION functionalized with epirubicin were used in clinical trials in humans [7,24]. In 14 patients with different types, localization and stages of solid tumors, a test dosage of non-functionalized SPION (0.2% of blood volume) and thereafter, 2–3  0.5% of the blood volume with epirubicin functionalized SPION were injected into veins contralaterally to the tumors while an external magnetic field (0.5–0.8 T) was applied over the tumor for 60–160 min. Blood tests were taken in regular intervals and toxicity was assessed according to the WHO guidelines. In addition, MRI of the tumors and the liver were performed after 24 h and 5 weeks after the last treatment. The sizes of the tumors were measured weekly, and in one case a biopsy sample was studied histologically. In 6 patients, particle accumulation could be detected within the tumor tissue. The application of the non-functionalized SPION went without complications, while the side effects of the epirubicin functionalized particles could be kept to a minimum. As in the animal experiments a grayish, reversible discoloration of the skin was noticed in some of the patients for 24–36 h. Transient increases (24–48 h) of iron and ferritin blood values were reported, although without clinical symptoms. Myelodepression in one patient was attributed to side effects of the

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chemotherapy with epirubicin. An accumulation of SPION in the liver was recorded within the first 2 days, whereas no particles could be detected after 60 days confirming the results of previous studies [1,22,65]. Therefore, it was concluded that MDT was safe to be used in cancer therapy [7,24,66]. Carbon coated SPION (0.1–1 mm size) functionalized with carminomycin and rubomycin were injected into the tail vein of rats with tumors [67]. Using MDT, a three-fold higher concentration of chemotherapeutic agents could be measured in the tumor tissues and a 60% healing rate of the tumors could be achieved. Similar experiments in dogs had the same results after injection of SPION in the arterial vessels of the tumor. Microembolization was also detected. These experiments lead to a clinical trial in 150 human patients with far advanced, different type of tumors. Although the authors reported that most of the patients were cured or their conditions significantly improved, these results must be interpreted with care, because only an extended abstract is available in the literature [67]. Apart from grayish discoloration of skin areas above the tumors, discoloration was also noticed in regional lymph nodes indicating that these particles could be helpful in eliminating metastatic tumor spreading. An entirely different use of SPION in MDT is visualized in postoperative prophylaxis of infections around surgical implants and prosthesis [130]. There, the idea is to combine the implants with soft ferromagnetic materials, for example spirals in vascular prostheses, or frames around artificial heart valves. If complications develop after implantation, such as infection, thrombosis, rejection or calcification, appropriate medication could be delivered to the implant site by means of functionalized SPION injected into an adjacent artery and under the influence of an external magnet. The implant itself would act as a magnet and attract the particles that would release the medication on the local site. After successful preliminary tests with the ‘‘magnetic field capture of SPION in flowing system’’ experimental in vivo studies with dogs using carbon coated SPION (0.1–1 mm) were conducted [67]. A 5 cm long prosthesis was used to replace part of the carotid

artery in those dogs. Thereafter, 99mTC marked SPION were injected and particle accumulation was followed with the help of a gamma detector. The majority of the particles could be detected close to the implant [130]. In another canine model thrombi of the carotid arteries were experimentally induced and treated with MDT. Dextran coated SPION (1 mm in size) functionalized with streptokinase, a common thrombolyticum, were injected into the arteries while an external magnet was applied over the thrombus. Although, the dosage of streptokinase was lower as generally used in clinics, all thrombi could be dissolved in contrast to the control groups where no external magnetic field was applied [132]. MDT with SPION was also successfully used in gene therapy (magnetofection) in first experiments [14]. The transfection efficiency of commonly used viral vectors could be increased up to 100-fold through coupling of these vectors to SPION and application of an external magnetic field. The duration of the gene transport could be reduced to a few minutes, the tropism of adenoviral vectors could be enforced and the low titer of retroviral vectors compensated. The efficiency of this system could be reproducibly proven in vitro and in vivo in gene transport to cells of the gastrointestinal tract in rodents as well as in blood vessels of pigs. Most of the authors using magnetotransfection reported a 2–10 fold particle accumulation and at the same time a significant regression of tumors compared to control groups. After removing the external magnetic field, most of the particles could be found in the liver [1,7,22,23]. Hyperthermia with magnetic ferrofluids: Superparamagnetic particles exposed to an alternating magnetic field can be used for heat induction [5,133]. Through the oscillation of the magnetic moment inside the particles the magnetic field energy in the form of heat is liberated and conducted to the tissue environment [1,5]. SPION have a much higher rate of specific absorption compared to larger magnetic particles with several magnetic domains and therefore, are predestinated for use in hyperthermia, where the tissue is heated up to 41–46 1C [25,26,133]. If the temperature exceeds 56 1C, necrosis, coagulation or carbonization of the tissue is the result; a procedure called

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‘‘thermoablation’’ [133,134]. For obvious reasons thermoablation is only of limited value in clinical applications [25]. In contrast, hyperthermia is highly suitable for cancer therapy, since tumor cells are highly susceptible to elevated temperatures [5,135]. If tumor cells are heated up to 41–45 1C, the tissue damage for normal tissue is reversible while the tumor cells are irreversibly damaged. This can be an advantage when used in combination with therapies such as radio- and chemotherapy [26,133,136]. Interestingly, hyperthermia seems to induce modifications of the cell surface receptor molecules and thus, tumor cells are recognized by the immune system (killer cells) more easily [137]. Furthermore, the bloodbrain barrier is reduced for 60 min when temperatures reach 42.5–43 1C promising an improvement in combined chemotherapies of brain tumors [133]. Conventional hyperthermia treatments including microwaves, ultrasound, radiofrequency, and infrared, have already been successfully used. However, their disadvantage is based on their inability to selectively induce heat formation in specific tumor tissue, inhibition of heat conduction through less heat conductive tissue, such as fat and cranial bone, the invasiveness of the methods, and temperature distribution inhomogeneities. In many in vitro tests with different types of tumor cells and SPION, the optimal physical properties of the SPION [138], antibody mediated tumor adhesion [135,139], particle uptake into tumor cells [138], duration and strength of the magnetic field [135,138,139] was studied to achieve the most efficient tumor cell inactivation. They were then confirmed in animal experiments in vivo (mostly in mice) with experimentally induced tumors [25,26,134,136]. The homogenous celltissue inactivation including temperature increase correlated well with the homogenous distribution of SPION within the target tissue. Temperature varied from 47 to 80 1C in these experiments. It was demonstrated that the location of the particles within the cell (intracellular, interstitial, membrane bound) was very important concerning the efficiency of hyperthermia induction. Intracellularly induced hyperthermia increased the efficiency of particles and allowed reducing their

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dosage [25]. Even after mitosis, 50% of the particles were still present within the tumor cells. This is advantageous if repeated hyperthermia is applied. It means that SPION should be slowly degraded from the cells for this type of application, since changes in the physical properties of cells would drastically reduce the absorbed heat energy. SPION functionalized with antibodies against surface receptors can bind to the cell membrane. Induction of hyperthermia will therefore damage the cell membranes locally without changing the environment. Thus, the particle concentration can be reduced [140]. Currently, clinical trials in human patients affected with prostate and brain tumors are conducted using local magnetic hyperthermia in combination with radiotherapy [133]. Apart from cancer therapy, local magnetic hyperthermia could be used for blood coagulation in small vessels [1], for selective temperature increases in virus infected cells (eg. HIV after coupling of CD4 glycoproteins to SPION) [5] and as a drug delivery mechanism, where substances are coupled to magnetic microspheres or SPION [141]. As a summary, the application of SPION for local magnetic hyperthermia is a promising tool for future therapy where selective, efficient and non-invasive methods for heat induction in tissues are warranted.

4. Conclusions and outlook The use of SPION in the medical field opens new avenues for selective treatment of local tissues where efficiency is increased through local concentrations while at the same time general side effects can be avoided. Although their use is still considered experimental except in MRI, new technologies for particle synthesis, coating and functionalization will render them even more attractive for all kinds of medical applications in the near future. Last but not the least, systematic studies about their detection and elimination behavior within the body system will elicit more reliable data about their clinical safety.

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Acknowledgements This work was supported by the European Community, 5th Framework Program, ‘‘Magnanomed’’ G5RD-2000-00375 and the Vetsuisse Research Grant SPION, University of Zu¨rich, Switzerland.

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