Magnetic nanoparticles for drug delivery

Controlled release of drugs from nanostructured functional materials, especially nanoparticles (NPs), is attracting increasing attention because of the opportunities in cancer therapy and the treatment of other ailments. The potential of magnetic NPs stems from the intrinsic properties of their magnetic cores combined with their drug loading capability and the biochemical properties that can be bestowed on them by means of a suitable coating. Here we review the problems and recent advances in the development of magnetic NPs for drug delivery, focusing particularly on the materials involved.
Manuel Arruebo, Rodrigo Fernndez-Pacheco, M. Ricardo Ibarra, and Jess Santamara*
Nanoscience Institute of Aragon (INA), Pedro Cerbuna 12, University of Zaragoza, 50009 Zaragoza, Spain *E-mail: iqcatal@unizar.es

NPs are submicron moieties (between 1 nm and 100 nm according to the usual definition, although there are examples of NPs several hundreds of nanometers in size) made of inorganic or organic (e.g. polymeric) materials, which may or may not be biodegradable. Their importance relates to the fact that the characteristics of NPs are different from those of bulk materials of the same composition, which is mainly because of size effects, the magnetic and electronic properties, and the role played by surface phenomena as the size is reduced. Preparation methods for NPs generally fall into the category of socalled bottom-up methods, where nanomaterials are fabricated from atoms or molecules in a controlled manner that is thermodynamically regulated by means such as self-assembly1. Some biomedical applications require core-shell magnetic NPs. They consist of a metal or metallic oxide core, encapsulated in an inorganic or a polymeric

coating that renders the particles biocompatible, stable, and may serve as a support for biomolecules. Their magnetic properties enable these particles to be used in numerous applications, belonging to one or more of the following groups: (i) Magnetic contrast agents in magnetic resonance imaging (MRI)2; (ii) Hyperthermia agents, where the magnetic particles are heated selectively by application of an high frequency magnetic field. (e.g. in thermal ablation/hyperthermia of tumors3); and (iii) Magnetic vectors that can be directed by means of a magnetic field gradient towards a certain location, such as in the case of the targeted drug delivery4. The scientific community is seeking to exploit the intrinsic properties of magnetic NPs to obtain medical breakthroughs in diagnosis, and drug delivery. Perhaps the most promising applications relate to the diagnosis and treatment of cancer.

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Even though, according to the American Cancer Society, cancer deaths in the US have dropped for a second straight year, which is attributed to the decrease in smoking rates and to earlier detection and more effective treatment of tumors5, cancer is still one of the leading causes of death in developed countries. Conventional treatments, including surgery, radiation, chemotherapy, and biologic therapies (immunotherapy) are limited by the accessibility to the tumor, the risk of operating on a vital organ, the spread of cancer cells throughout the body, and the lack of selectivity toward tumor cells. Immunotherapy is still relatively recent, and is most likely to be applied to small tumors, since its effectiveness seems to decrease for more advanced stages of cancer. Multimodal therapy that uses radiotherapy, chemotherapy, immunotherapy, and other forms of treatment in combination with surgery provides a better chance of survival6. The potential of drug delivery systems based on the use of nanoand microparticles stems from significant advantages such as: (i) the ability to target specific locations in the body; (ii) the reduction of the quantity of drug needed to attain a particular concentration in the vicinity of the target; and (iii) the reduction of the concentration of the drug at nontarget sites7 minimizing severe side effects. All these benefits justify the exponential growth in the number of publications dealing with NPs for drug delivery applications (Fig. 1). NPs can act at the tissular or cellular level. The latter implies that they can be endocytosed or phagocytosed (i.e. by dendritic cells, macrophages) resulting in internalization of the NP. In this process, the NP can reach beyond the cytoplasmatic membrane and, in some cases, also beyond the nuclear membrane (i.e. transfection applications). Tumor targeting with magnetic NPs may use passive or active strategies. Passive targeting occurs as a result of extravasation of the NPs at the diseased site (tumor) where the microvasculature is hyperpermeable and leaky, a process aided by tumor-limited lymphatic drainage. Combined, these factors lead to the selective accumulation of NPs in tumor tissue, a phenomenon known as enhanced permeation and retention (EPR)8. The majority of solid tumors exhibit a vascular pore cut-off size between 380 nm and 780 nm, although vasculature organization may differ depending on the tumor type, its growth rate, and microenvironment9. Apart from tumors, size-dependent removal of NPs is a common occurrence in healthy capillaries. Table 110 shows the morphological pore sizes contributing to diffusive permeability in the capillaries of the human vascular system. It can be seen that, from the delivery point of view, there is practically no limitation as the diameters of typical NPs are well below that of the narrowest capillaries. Instead, the main limitation concerns the residence time of NPs in the bloodstream. Thus, the use of conventional NPs for drug delivery by passive targeting would be limited to tumors in mononuclear phagocyte system (MPS) organs (liver, spleen, and bone
Fig. 1 Temporal evolution in the number of scientific papers published involving drug delivery using NPs. (Source: ISI Web of Knowledge The Thomson Corporation. Search terms: drug delivery and nanoparticles. Date of search: December 2006.)

Table 1 Relevant sizes regarding particle distribution through and removal from the capillaries of the human vascular system.
Size Particle removal* Tight-junction capillaries Continuous capillaries Fenestrated capillaries Sinusoidal capillaries Arteriole radius Artery radius Venule radius < 1 nm ~ 6 nm ~ 50-60 nm ~ 100-1000 nm 0.005-0.07 mm 0.08-7.5 mm 8-100 m System/Organ Central nervous system, blood-brain barrier Tissues such as muscle, skin, and lung Kidney, intestine, and some endocrine and exocrine glands Liver, spleen, and bone marrow Circulatory system. Particles supplied by intravenous administration. Elimination involves opsonization and removal by monocytes in blood

Particle delivery

*It is noted that this table expresses only the morphological pores contributing to diffusive permeability. Actual transcapillary exchanges are modified by vesicular transports, which are able to internalize particles with sizes up to 20-30 nm. (Adapted with permission from10. 1999 Elsevier.)

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marrow). Addressing other tumoral tissues does not seem feasible without active targeting strategies because of the short circulation times involved and the low concentration of NPs that is achieved in the tumor area (despite the EPR effect), leading to drug concentrations below the therapeutic level11. Active targeting is based on the over or exclusive expression of different epitopes or receptors in tumoral cells, and on specific physical characteristics. Thus, vectors sensitive to physical stimuli (e.g. temperature, pH, electric charge, light, sound, magnetism) have been developed and conjugated to drugs. Alternatively, active targeting may be based on over-expressed species such as low molecular weight ligands (folic acid, thiamine, sugars), peptides (RGD, LHRD), proteins (transferrin, antibodies, lectins), polysaccharides (hyaluronic acid), polyunsaturated fatty acids, peptides, DNA, etc. Different moieties including dendrimers, micelles, emulsions, nanoparticulated drugs, and liposomes are used to target specific areas in the body (Fig. 2)12,13. The NPs must be endowed with the specific characteristics needed to reach a given target, which means attaining a suitable combination of nature, size, way of conjugating the drug to the NP (attached, adsorbed, encapsulated), surface chemistry, hydrophilicity/hydrophobicity, surface functionalization, biodegradability, and physical response properties (temperature, pH, electric charge, light, sound, magnetism). Among these, size is the main

factor affecting the fate of NPs in passive targeting processes using the permeability of the capillary vessels, as discussed above. Here we shall concentrate on the therapeutic applications of magnetic drug targeting using NPs. Hyperthermia/thermal ablation will not be addressed, although the general implications between both therapies based on magnetism can be inferred. Applications in diagnosis, where magnetic NPs are widely used as contrast agents, will not be addressed here either.

Magnetic drug delivery

The development of magnetic drug delivery
Any overview on drug delivery should start with the deserved recognition of Paul Ehrlich (1854-1915), who proposed that if an agent could selectively target a disease-causing organism, then a toxin for that organism could be delivered along with the agent of selectivity. Hence, a magic bullet would be created able to kill the targeted organism exclusively. Ehrlich received the 1908 Nobel Prize in Medicine for his work in the field of immunity, and the magic bullet idea was even used as a script for the 1940 movie Dr. Ehrlichs Magic Bullet. Since then, various strategies have been proposed to deliver a drug to the vicinity of a tumor including, as mentioned above, the use of vectors sensitive to physical stimuli and tumor-recognition moieties conjugated to a drug.

Fig. 2 NP systems for drug delivery applications. (Adapted with permission from119 2005 Elsevier; and from120 2005 PharmaVentures Ltd.)

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Prior to their use for drug delivery, magnetic microparticles were proposed as contrast agents for localized radiation therapy14 and to induce vascular occlusion of the tumors (antiangiogenic therapy)15,16. Freeman et al.17 proposed in 1960 that magnetic NPs could be transported through the vascular system and concentrated in a specific part of the body with the aid of a magnetic field. The use of magnetic micro- and NPs for the delivery of chemotherapeutics has evolved since the 1970s. Zimmermann and Pilwat18 in 1976 used magnetic erythrocytes for the delivery of cytotoxic drugs. Widder et al.19 described the targeting of magnetic albumin microspheres encapsulating an anticancer drug (doxorubicin) in animal models. In the 1980s, several authors developed this strategy to deliver different drugs using magnetic microcapsules and microspheres20-23. In 1994, Hfeli et al.24 prepared biodegradable poly(lactic acid) microspheres that incorporated magnetite and the beta-emitter 90Y for targeted radiotherapy, and successfully applied them to subcutaneous tumors25. However, all these initial approaches were microsized. Magnetic NPs were used for the first time in animal models by Lbbe et al.26. In 1996, the first Phase I clinical trial was carried out by the same group in patients with advanced and unsuccessfully pretreated cancers using magnetic NPs loaded with epirubicin27. However, in that first trial, more than 50% of the NPs ended up in the liver. Since then, a number of groups around the world have synthesized magnetic vectors and shown potential applications. Different start-ups now manufacture magnetic micro- and NPs, which are used in MRI, magnetic fluid hyperthermia, cell sorting and targeting, bioseparation, sensing, enzyme immobilization, immunoassays, and gene transfection and detection systems. FeRx, Inc. (founded in 1997) produced doxorubicin-loaded magnetic NPs consisting of metallic Fe ground together with activated carbon28,29. A Phase II clinical study in patients with primary liver cancer was conducted using these NPs, although the trial was not successful. Chemicell GmbH currently commercializes TargetMAGdoxorubicin NPs involving a multidomain magnetite core and a cross-linked starch matrix with terminal cations that can be reversibly exchanged by the positively charged doxorubicin. The particles have a hydrodynamic diameter of 50 nm and are coated with 3 mg/ml doxorubicin30. These NPs loaded with mitoxantrone have already been used in animal models with successful results31. Chemicell also commercializes FluidMAG for drug delivery applications. Magnetic NP hydro-gel (MagNaGel) from Alnis Biosciences, Inc. is a material comprising chemotherapeutic agents, Fe oxide colloids, and targeting ligands32. In summary, for magnetic targeting, a drug or therapeutic radionuclide is bound to a magnetic compound, introduced in the body, and then concentrated in the target area by means of a magnetic field (using an internally implanted permanent magnet or an externally applied field). Depending on the application, the particles then release

the drug or give rise to a local effect (irradiation from radioactive microspheres or hyperthermia with magnetic NPs)33. Drug release can proceed by simple diffusion or take place through mechanisms requiring enzymatic activity or changes in physiological conditions such as pH, osmolality, or temperature34; drug release can also be magnetically triggered from the drug-conjugated magnetic NPs.

Drug delivery with magnetic NPs

Different organic materials (polymeric NPs, liposomes, micelles) have been investigated as drug delivery nanovectors using passive targeting, active targeting with a recognition moiety (e.g. antibody), or active targeting by a physical stimulus (e.g. magnetism in magnetoliposomes). However, these organic systems still present limited chemical and mechanic stability, swelling, susceptibility to microbiological attack, inadequate control over the drug release rate35, and high cost. Polymer NPs also suffer from the problem of high polydispersity. Synthesis produces particles with a broad size distribution and irregular branching, which could lead to heterogeneous pharmacological properties. One alternative is to use dendrimers, which have a monodisperse character and globular architecture resulting from their stepwise synthesis and can be purified at each step of growth36. Visualization of dendrimers requires tagging with a specific moiety (i.e. a fluorophore or metal). A major drawback of dendrimers and dendritic polymers, however, is their high cost. The preparation of dendritic polymers that circulate in the blood long enough to accumulate at target sites but that can also be removed from the body at a reasonable rate to avoid long-term accumulation also remains a challenge. Passive targeting using drug-conjugated dendrimers and dendritic polymers has been widely studied, mainly using the EPR effect. Therapies based on active targeting, such as antibody-conjugated dendrimers, constitute a promising alternative in view of the potential of antibodies for selective targeting37. Because of the disadvantages of organic NPs for drug delivery, inorganic vectors constitute an interesting option and are the subject of intense research. Some examples of inorganic magnetic NPs will be given below. The main advantages of magnetic (organic or inorganic) NPs are that they can be: (i) visualized (superparamagnetic NPs are used in MRI); (ii) guided or held in place by means of a magnetic field; and (iii) heated in a magnetic field to trigger drug release or to produce hyperthermia/ablation of tissue. It is important to point out that the latter capability is not restricted to magnetic NPs, but also to other particles capable of absorbing near-infrared, microwave, and ultrasound radiation. Depending on the synthesis procedure, magnetic NPs or nanocapsules can be obtained. We refer to NPs when the drug is covalently attached to the surface or entrapped or adsorbed within the pores of the magnetic carrier (polymer, mesoporous silica, etc.). Nanocapsules (reservoirs) designate magnetic vesicular systems where

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Magnetic nanoparticles Therapy Drug delivery Radiotherapy combined with MRI Hyperthermia/ thermal ablation Musculoskeletal system associated diseases In vivo MRI Diagnosis In vitro Sensing Cell sorting Bioseparation Enzyme immobilization Immunoassays Transfection Purification Fig. 3 Biomedical applications of magnetic NPs. *Ferumoxytol (Advanced Magnetics, Inc.) is in Phase III multicenter clinical studies for use as an intravenous Fe replacement therapeutic for patients with anemic chronic kidney disease.

Anemic chronic * kidney disease

the drug is confined to an aqueous or oily cavity, usually prepared by the reverse micelle procedure, and surrounded by an organic membrane (magnetoliposomes) or encapsulated within a hollow inorganic capsule35. The key parameters in the behavior of magnetic NPs are related to surface chemistry, size (magnetic core, hydrodynamic volume, and size distribution), and magnetic properties (magnetic moment, remanence, coercivity). The surface chemistry is especially important to avoid the action of the reticuloendothelial system (RES), which is part of the immune system, and increase the half-life in the blood stream. Coating the NPs with a neutral and hydrophilic compound (i.e. polyethylene glycol (PEG), polysaccharides, dysopsonins (HSA), etc.) increases the circulatory half-life from minutes to hours or days. Another possibility is to reduce the particle size; however, despite all efforts, complete evasion of the RES does not seem feasible38 and unwanted migration to other areas in the body could cause toxicological problems. In addition to cancer treatment, magnetic NPs can also be used in anemic chronic kidney disease and disorders associated with the musculoskeletal system (i.e. local inflammatory processes, side effects) (Fig. 3). For those disorders, superparamagnetic Fe oxide NPs (SPION), in conjunction with external magnetic fields, seem a suitable alternative for drug delivery to inflammatory sites by maintaining appropriate local concentrations while reducing overall dosage and side effects39.

area or which triggers the drug desorption. Permanent Nd-Fe-B magnets in combination with SPION, which have excellent magnetic properties, can reach effective magnetic field depths up to 10-15 cm in the body39. However, it must be noted that the magnetic carriers accumulate not only at the desired site but also throughout the crosssection from the external source to the depth marking the effective field limit. Obviously, the geometry of the magnetic field is extremely important and must be taken into account when designing a magnetic targeting process. As a means to elude the limitations of using external magnetic fields, internal magnets can be located in the vicinity of the target by using minimally invasive surgery40. Several studies have simulated the interaction between a magnetic implant and magnetic NPs, enabling drug delivery6,41,42. In addition, work in several laboratories40,43,44 is addressing targeted drug delivery with magnetic implants. Another limitation relates to the small size of NPs, a requisite for superparamagnetism, which is in turn needed to avoid magnetic agglomeration once the magnetic field is removed (see below). A small size implies a magnetic response of reduced strength, making it difficult to direct particles and keep them in the proximity of the target while withstanding the drag of blood flow45. Targeting is likely to be more effective in regions of slower blood velocity, and particularly when the magnetic field source is close to the target site. As for all biomedical applications, limitations also arise in extrapolating from animal models to humans. There are many physiological parameters to consider, ranging from differences in weight, blood volume, cardiac output, and circulation time to tumor volume/location/blood flow, complicating the extrapolation of data obtained in animal models46,47. Related to this point is the fact that studies on toxicity (not only direct toxicity, but also toxicity

Limitations of magnetic drug delivery

Since the magnetic gradient decreases with the distance to the target, the main limitation of magnetic drug delivery relates to the strength of the external field that can be applied to obtain the necessary magnetic gradient to control the residence time of NPs in the desired

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of the degradation products and induced responses48) and the fate of magnetic carriers are insufficient and, in many cases, there is insufficient characterization. Finally, state-of-the-art magnetic drug delivery seems mainly applicable to well-defined tumors, as treatment of metastatic neoplasms and small tumors in the early stages of their growth still remains a challenge. Treating emerging tumors will involve the development of a new generation of seek-and-destroy NPs, which specifically recognize small clusters of cancer cells and carry the necessary elements (drugs or hyperthermia agents) for their destruction. A strong interest continues in this field given the capability of NPs to access tumors in regions where conventional surgery cannot be applied.

magnetic field, the magnetic moment of entire crystallites aligns with the magnetic field (Fig. 4). In large NPs, energetic considerations favor the formation of domain walls. However, when the particle size decreases below a certain value, the formation of domain walls becomes unfavorable and each particle comprises a single domain. This is the case for superparamagnetic NPs. Superparamagnetism in drug delivery is necessary because once the external magnetic field is removed, magnetization disappears (negligible remanence and coercivity, see Fig. 4), and thus agglomeration (and the possible embolization of capillary vessels) is avoided. Another key requirement is the biodegradability or intact excretion of the magnetic core. Thus, SPION are considered to be biodegradable with Fe being reused/recycled by cells using normal biochemical pathways for Fe metabolism50,51. For nonbiodegradable cores, a specific coating is needed to avoid exposure (and possible leaching) of the magnetic core and to facilitate intact excretion through the kidneys, so that the half-life of the agent in the blood is determined by the glomerular filtration rate (e.g. contrast agents based on gadolinium)51. Coatings on magnetic NPs The coatings on magnetic NPs often serve multiple purposes. Their role in reducing leaching of the cores has already been mentioned. The coating also often facilitates the stabilization of NPs in an environment with a slightly alkaline pH or a significant salt concentration. For instance, the isoelectrical point of SiO2 is reached at pH 2-3, meaning that silica-coated NPs are negatively charged at the pH of blood, inducing electrostatic repulsion that helps avoid aggregate formation. Silica coatings also have additional advantages. On the one hand, the external surface of silica coatings can be functionalized to allow the binding of biomolecules. This is mainly related to the presence of hydroxyl surface groups in significant concentrations that provide intrinsic hydrophilicity and allow surface attachment by covalent

Tailoring magnetic NPs

Essential requisites Magnetic NPs for biomedical applications must be endowed with the specific characteristics required. As mentioned above, the first requirement is often superparamagnetism. Superparamagnetism occurs in magnetic materials composed of very small crystallites (threshold size depends on the nature of the material, for instance, Fe-based NPs become superparamagnetic at sizes <25 nm49). In a paramagnetic material, the thermal energy overcomes the coupling forces between neighboring atoms above the Curie temperature, causing random fluctuations in the magnetization direction that result in a null overall magnetic moment. However, in superparamagnetic materials, the fluctuations affect the direction of magnetization of entire crystallites. The magnetic moments of individual crystallites compensate for each other and the overall magnetic moment becomes null. When an external magnetic field is applied, the behavior is similar to paramagnetism except that, instead of each individual atom being independently influenced by an external

Fig. 4 Hysteresis loops (magnetization versus applied magnetic field) characteristic of ferromagnetic and superparamagnetic NPs. For comparison, para- and diamagnetic behavior are also shown. The figure also indicates the values of the remanence, Mr, and coercive field, Hc.

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linkage of specific biomolecules. On the other hand, the internal porosity of silica can be used to host a specific drug, a feat achievable while avoiding the unwanted physical adsorption of larger molecules. Thus, according to Ambrose and Fritz52, the smallest major protein in serum is serum albumin, with a molecular mass of approximately 66 000 Da and an effective spherical radius of ~40 . This means that serum albumin, and larger molecules, would be excluded from the channels of microporous and mesoporous (with pore sizes <4 nm) coatings (e.g. silica, carbon, zeolites) on magnetic cores. Finally, silica and other microporous inorganic materials are heat resistant, with high surface areas and good mechanical strength. Figs. 5 and 6 show different types of inorganic coatings on metal cores developed in our laboratory. In addition, coatings play an essential role in retarding clearance by the RES. Depending on their size, surface functionalization, and hydrophilicity, a rapid uptake of uncoated NPs by the mononuclear phagocyte system (MPS) is likely after systemic administration, followed by clearance to the liver, spleen, and bone marrow. Different proteins (antibodies) of the blood serum (opsonins) bind to the surface of foreign bodies, accelerating phagocitation of the particles. To avoid this, biodegradable (e.g. dextran) and nonbiodegradable organic and inorganic coatings can be used as a means to retard detection and uptake by the macrophages of the RES. Perhaps the most widely used coating for this purpose is PEG, a linear neutral polyether, whose attachment to NP surfaces provides a stealth shielding effect, delaying the action of the RES53. PEG shows little toxicity and immunogenicity, and intact excretion is possible, either via the kidneys (for PEG <30 kDa) or in the feces (for PEG >20 kDa)54. Avoiding detection by the RES stems from the protein-resistant character of PEGylated surfaces, which has been ascribed to the combination of a low interfacial energy in water and the steric stabilization effect55. Unfortunately, the immunostealthing function provided by PEG is frequently concurrent with the loss of biomolecular targeting capabilities; therefore, it is necessary to optimize the coating. The nature of the coating is also important in those cases where the surface functionalization might cause hydrogen bonding and, therefore, agglomeration of NPs. For such situations, surface modification can reduce the aggregation and increase the stability of NPs in body fluids. A wide variety of molecules has been loaded onto organic and inorganic shells, e.g. by chemical functionalization or physical absorption. The list includes tumor-recognition moieties such as antibodies in smart contrast agents56-58 and cell-penetrating peptides for MRI applications59,60; and enzymes61, toxins62, genes63-68 (transfection), growth factors69,70, radionucleotides4,71-73, folic acid74, and drugs (mitoxantrone4,75, tamoxifen76, cefradine77, doxorubicin78-83, ammonium glycyrrhizinate84, fludarabine85, danorubicin86, cisplatin and gemcitabine87, pingyangmycin88, nonsteroidal anti-inflammatory drugs89, amethopterin90,91, mitomycin92, paclitaxel21,93, diclofenac sodium94,95, and adriamycin96) for drug delivery applications.
Fig. 6 HRTEM photographs of: (a) magnetite NPs encapsulated in a silica matrix; and (b) EFTEM color map of Au NPs encapsulated in a silica matrix. (b) (a) Fig. 5. High resolution transmission electron microscopy (HRTEM) images of (a) magnetite NP encapsulated in silica; (b) magnetite NPs embedded in a zeolitic (aluminosilicate) matrix; (c) Fe NPs encapsulated in silica (energyfiltering TEM, EFTEM, color map); (d) magnetite NP encapsulated in graphite. (c) (d) (a) (b)

Fate
The distribution of NPs and their loads throughout the body depends on numerous physicochemical factors: size of particles, toxicity, surface charge, capacity for protein adsorption, surface hydrophobicity, drug loading and release kinetics, stability, degeneration of carrier systems, hydration behavior, electrophoretic mobility, porosity, specific surface

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characteristics, density, crystallinity, contact angle, and molecular weight39. Nevertheless, the fate (and also the possible toxicity) of magnetic NPs also depends strongly on the dose and administration route (oral or parenteral: including delivery routes such as intravenous, pulmonary, transdermal, and ocular, in addition to less conventional routes, e.g. when used as scaffold coatings). Some of the implications for the three most common ways of administration are now discussed.

spleen (3-10%). Particles larger than 200 nm are usually filtered to the spleen, whose cut-off point extends up to 250 nm104, while particles up to 100 nm are mainly phagocytosed through liver cells. In general, the larger the particles are, the shorter their plasma half-life-period101. This clearance of particles by Kupffer cells can be, on the other hand, useful for the treatment of liver diseases, such as cancer or leishmania105, tuberculosis, listeria, leprosy, etc.; although it is important to consider that it would simultaneously entail the depletion of a significant number of the patients own defense cells.

Intravenous administration
The general rule for magnetic NPs in parenteral applications it that the carrier is nontoxic, nonimmunogenic, and of a size that avoids embolization of capillary ducts. Once a NP enters the bloodstream, opsonization processes activate the RES system response. Circulating mononuclear phagocytes (monocytes) clear the NPs to the liver, spleen, and bone marrow where resident cells (e.g. Kupffer cells in the liver) capture the NPs prior to degradation, if possible. Depending on biodegradability and size, some of the NPs present in the lysosomal vesicles of Kupffer cells may be incorporated into the bile and be removed in the feces. Other NPs will be filtered by the kidneys and incorporated into the urine. In general, smaller NPs are subject to rapid renal elimination, while larger ones show uptake by the liver, spleen, and bone marrow (Fig. 7)97-103. Large particles will be removed by cells capable of phagocytosis (i.e. by macrophages or dendritic cells), while small particles can be removed by cells capable of endocytosis (i.e. by B and T lymphocytes). Finally, if the magnetic NPs are biodegradable, the decomposition products can be taken up by any cell by means of pinocytosis. According to Neuberger et al.39, magnetic particles smaller than 4 m are removed by cells of the RES, mainly in the liver (60-90%) and

Subcutaneous or intratumoral administration

Unlike water-soluble molecules, which are rapidly absorbed through the blood capillary walls and pass into the circulatory system, small particles injected locally infiltrate into the interstitial spaces around the injection site and are gradually absorbed by the lymphatic capillary system11. For this reason, subcutaneously or locally injected NPs can be used for lymphatic targeting, i.e. as a tool for chemotherapy for lymphatic tumors, although this route is rarely used in clinical practice as it is not useful for targeting metastatic tumors. In this case, magnetism is not required but may be a useful property to hold the particles in place or to enable hyperthermia. As a general rule, colloidal carriers aimed at regional lymph nodes through subcutaneous injection need to be small (60 nm or less)106. Another limitation relates to intratumoral pressure gradients caused by the fast cell proliferation in solid tumors.

Oral administration
Several publications describe the use of magnetic NPs coated with an organic shell as oral drug delivery vectors107, although most concern

Monocrystalline iron oxide nanoparticles < 10 nm 101-103

Blood residence time

Ultrasmall superparamagnetic iron oxide nanoparticles 5-20 nm 100 Ferromagnetic particles 200 nm 98

Non-biodegradable inorganic and polymeric 97 micrometric spheres

Large superparamagnetic iron oxide nanoparticles > 40 nm 99 Plasma half life = 2 h Accumulation in lymph nodes. Excreted by urine and feces Removal by liver (80%) and spleen (15%) Opsonization. Removal by liver, spleen, lungs, and bone marrow macrophages

Particle size Fig. 7 Qualitative diagram showing the evolution of blood residence time with particle size.

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magnetic NPs used as MRI contrast agents for the gastrointestinal tract. The main problem here is that oral delivery of peptides and proteins is hampered by their degradation in gastrointestinal acid, low absorption, first-pass metabolism by the liver, and a significant initial increase in drug concentration. Feng et al.108 describe the fate of chemotherapeutical NPs in oral delivery: particles under 5 m can be removed via lymphatic drainage, particles up to 500 nm can cross the membrane of epithelial cells through endocytosis, and particles less than 50 nm can achieve paracellular passage between intestinal epithelial cells.

It is well documented that the large surface-to-volume ratio of all nanosized particles can potentially lead to unfavorable biological responses if they are inhaled and subsequently absorbed via the lung or swallowed and then absorbed across the gastrointestinal tract115. Interestingly, it has also been reported that, in 20-100 mg/ml concentrations, large magnetic particles show higher cytotoxicity than smaller ones even after normalizing for surface area116 despite the lower surface-to-volume ratio, although it is difficult to perform comparable experiments with differently sized particles. In any case, toxicity studies should consider not only acute toxicity but also that of degradation products, the possible stimulation of cells with subsequent release of inflammatory mediators39, and long term toxicity. For a magnetic carrier with potential as a drug delivery vector, it is necessary, at the very least, to analyze its: (i) toxicity (acute, subacute, and chronic toxicity, teratogenicity, and mutagenicity) in cellular and animal models; (ii) hematocompatibility; (iii) biodegradation (whenever possible); (iv) immunogenicity; and (v) pharmacokinetics (body distribution, metabolism, bioavailability, elimination, organspecific toxicity) before the start of preclinical testing. The contrast agents based on magnetic NPs currently on the market, either involving superparamagnetic Fe oxides (i.e. Feridex, Endorem, GastroMARK, Lumirem, Sinerem, or Resovist) or paramagnetic metals encapsulated in a chelating agent (i.e. Magnevist, Dotarem, Gadovist, Teslacan) have satisfied current regulations regarding use in patients. The same applies to the magnetic drug delivery systems that have already been commercialized, MagNaGel32 and FluidMAG and TargetMAG (Table 2). Recently, the US Environmental Protection Agency has started to regulate a large class of products made with Ag NPs using the legislation developed for pesticides. This is the first federal restriction to focus largely on nanotechnology117.

Safety: influence of the magnetic field

Although all the components of the body are either dia-, para-, superpara-, ferri-, or ferromagnetic, the magnetic fields required to produce obvious effect in the body are very large. Even red blood cells, which each contain micrograms of the Fe protein hemoglobin, show a relatively low response to large fields or steep field gradients, although this low value is enough to be used in functional MRI (fMRI). The other natural Fe-containing compounds in the body are hemosiderin, ferritin, transferrin, and the cytochromes. According to Schenck109, the US Food and Drug Administration (FDA) initially considered applications for approval to market MRI scanners on a case-by-case evaluation of the safety and efficacy information provided. Based on positive clinical and safety experience, the FDA classified magnets with field strength of less than 2 T as nonsignificant risk devices in 1987. Further positive experiences led the FDA to increase this threshold to 4 T in 1996 and again in 2003 (for adults) to 8 T. Even though experiments with strong static magnetic fields (8 T) have been shown to reduce the flow rate of human blood by 30% in in vitro tests110 and it has been reported that magnetic fields above 3 T might affect the normal behavior of erythrocytes, recent studies evaluating human subjects for adverse effects in physiological or neurocognitive functions resulting from exposure to static magnetic fields (up to 8 T) from MRI systems have not shown any clinically relevant effects111. According to Kangarlu and Robitaille112, within the decade, human imaging at fields in excess of 10 T will most probably be achieved and such projects are now being planned.

Perspectives and future challenges

In therapy, we are witnessing the early use of magnetic NPs as drug delivery vectors and as tools for hyperthermia/thermal ablation. Magnetic drug delivery constitutes a promising technology to treat cancer, and several products are already on the market. The limitations inherent in the use of external magnetic fields can, in some cases, be circumvented by means of internal magnets located in the proximity of the target by minimally invasive surgery40,43,44,98,118. Magnetic fluid hyperthermia/thermal ablation is also promising and is currently being applied (i.e. MagForce Nanotecnologies AG), but is limited by the fact that the tumor needs to be localized. This route, therefore, cannot be used in preventive medicine, or for treating early-stage tumors. The greatest therapeutic potential is probably associated with applications involving intelligent particles with a magnetic core (to direct the particles to the vicinity of the target and also for hyperthermia or for temperature-enhanced release of the drug), a recognition layer (to which suitable receptors are attached), and a

Toxicity
When discussing the toxicity of NPs, generalization becomes difficult because their toxicity depends on numerous factors including the dose, chemical composition, method of administration, size, biodegradability, solubility, pharmacokinetics, biodistribution, surface chemistry, shape, and structure, to name but a few. With NPs, as with any new biomedical discovery, the risk-benefit trade-off must be considered to assess whether the risks can be justified. In general, the size, surface area, shape, composition, and coating of an NP are the most important characteristics regarding cytotoxicity113, and modifications of the NP surface are a key tool to minimize toxicological effects114.

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Table 2 Some companies involved in the development and production of magnetic micro- and nanoparticles.
Company Bangs Laboratories, Inc. Polysciences, Inc. Micromod Partikeltechnologie GmbH Guerbet SA Ademtech SA Advanced Magnetics, Inc. Invitrogen Corp. Application Cell separation, DNA and RNA purification, immunoassays Magnetic separation, cell sorting, nucleic acid purification, flow cytometry, calibration, immunoassay, fluorescent microscopy, diagnostic assays Drug delivery, biomagnetic separation, nucleic acid purification X-ray and MRI contrast agents Cell sorting, biomagnetic separation Treatment of anemia, MRI contrast agents Immunoassay and nucleic acid in vitro diagnostics, DNA and RNA isolation, protein purification, cell separation and expansion, food and environmental testing Chemiluminescent and radio-immunoassays, cell separation, protein purification, immuno-precipitation, bacteria detection, immunochromatographic assays Hyperthermia Drug delivery, diagnostic assays Cell separation, enzyme immunoassay Drug delivery for anti-cancer and -infective treatments MagNaGel105 and smart contrast agents Hyperthermia Radiation therapy Drug delivery Drug delivery, in vitro diagnostics Drug delivery (FluidMAG), bioseparation, gene transfection and detection Website www.bangslabs.com www.polysciences.com www.micromod.de www.guerbet.com www.ademtech.com www.advancedmagnetics.com www.invitrogen.com

Estapor (Merck & Co. Inc.)

www.estapor.com www.merck.com www.magforce.com www.polymicrospheres.com www.spherotech.com www.alnis.com www.tritonbiosystems.com www.sirtex.com www.biophan.com www.magnamedics.com www.chemicell.com

MagForce Nanotecnologies AG Polymicrospheres (division of Vasmo, Inc.) Spherotech, Inc. Alnis Biosciences, Inc. Triton Biosystems, Inc. Sirtex Medical Ltd. Biophan Technologies, Inc. Magnamedics GmbH Chemicell GmbH

therapeutic load (adsorbed inside the pores or hosted within internal cavities of the particles). The challenges are formidable, especially those related to the development of suitable recognition layers. Not only must useful recognition moieties be identified and attached to the particles, but they must be loaded to a high density while maintaining their desired characteristics. Finally, as already noted, the biomedical uses of magnetic NPs are not restricted to drug delivery, and indeed new applications for magnetic NPs are also likely in MRI, where contrast agents could be tagged with a recognition moiety, cell sorting/targeting, bioseparation,

sensing, enzyme immobilization, immunoassays, and gene transfection/ detection systems.

Acknowledgments
Support from the Spanish Nanoscience Action NAN200409270-C3-1/2 and from the Consolider CSD2006-00012 and Ciber Ingenio 2010 CB06/01/0026 programs, is gratefully acknowledged. MA acknowledges support of a contract from the Juan de la Cierva program (project PPQ2003-04986). The authors also gratefully acknowledge the Serveis Cientificotcnics of the University of Barcelona for the use of the TEM, and Jordi Arbiol i Cobos for the TEM photographs and helpful discussions.

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