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Biomedical applications of distally controlled magnetic nanoparticles
Review
Biomedical applications of distally controlled magnetic nanoparticles Jose´ Luis Corchero1,2,3 and Antonio Villaverde2,3,1 1
CIBER de Bioingenierı´a, Biomateriales y Nanomedicina (CIBER-BBN), Bellaterra, 08196, Barcelona, Spain Institute for Biotechnology and Biomedicine, Universitat Auto`noma de Barcelona, Bellaterra, 08193 Barcelona, Spain 3 Department of Genetics and Microbiology, Universitat Auto`noma de Barcelona, Bellaterra, 08193 Barcelona, Spain 2
Nano-sized magnetic particles are increasingly being used across a wide spectrum of biomedical fields. Upon functionalization to enable specific binding, magnetic particles and their targets can be conveniently positioned in vitro and in vivo by the distal application of magnetic fields. Furthermore, such particles can be magnetically heated after reaching their in vivo targets, thus inducing localized cell death that has a considerable therapeutic value in, for instance, cancer therapy. In this context, innovative biomedical research has produced novel applications that have exciting clinical potential. Such applications include magnetically enhanced transfection, magnetically assisted gene therapy, magnetically induced hyperthermia and magnetic-force-based tissue engineering, and the principles and utilities of these applications will be discussed here. Introduction The biomedical applications of magnetic particles can be traced back to the 1950s, when Gilchrist and coworkers [1] treated lymphatic nodes and metastases by injecting metallic particles, which were heated using a magnetic field. Soon afterwards, magnetic particles were progressively incorporated as support materials for enzyme immobilization [2], drug delivery [3] and tools for targeted cell separation [4]. Since then, the success in the synthesis of magnetic particles has empowered a plethora of exciting biotechnological applications. Nanoparticles are amorphous or semicrystalline structures with at least one dimension ranging between 10 and 100 nm. Several of their characteristics, such as size uniformity, surface area, adsorption kinetics, biocompatibility, superparamagnetism and magnetic moment, can be finely tuned during the production process for specific purposes. Aside from the diverse functionalities conferred by the particle size itself (for instance, colloidal dispersion), magnetism offers additional properties that are of great biomedical interest. In particular, the ability to distally control the position of particles in a given media to induce their accumulation or separation from similar structures has found a spectrum of powerful applications in innovative medicines, as discussed below. Most materials with high magnetic moment, such as cobalt and nickel, are toxic, susceptible to oxidation and hence limited in their biomedical applications, but ferrimagnetic magnetite (Fe3O4) and maghemite (g-Fe2O3) are Corresponding author: Villaverde, A. ([email protected])
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suitable for in vivo applications. In the context of the emerging concern about the potential toxicity of nanoparticles [5], it is noteworthy that magnetic iron oxide particles are highly biocompatible, as the iron cell homeostasis is well controlled by uptake, excretion and storage and the iron excess is efficiently cleared from the body. In fact, iron-based particles do not cause oxidative stress or long-term changes in the levels of liver enzymes in rat models [6] (an indicator of biosafety), and indeed good tolerances to high doses of such materials have been reported [7]. Although these iron-based materials are often referred to as ‘magnetic’, the more accurate term ‘superparamagnetic’ designates their ability to become magnetized upon exposure to a magnetic field but have no permanent magnetization (remanence) once the field is turned off. This allows efficient magnetinduced clumping, as well as particle dispersion when the magnet is removed. In addition, the application of inorganic or polymeric coating layers to magnetic particles minimizes hydrophobic interactions, thus enhancing desirable properties, such as colloid dispersion and biocompatibility, and enabling the modification of surfaces with functional groups required for applications based on specific interactions. Strategies for the preparation of magnetic particles and their surface coating have been recently summarized [8–10]. The addition of reactive chemical groups on the particle surface (also designated as activation) allows for attachment of specific ligands or other functional molecules (usually termed decoration or functionalization) (Figure 1a). Nowadays, activated magnetic particles that are ready for customized decorations (Table 1) are commercially available, as are particles that already display specific ligands (Table 2). An interesting alternative to synthetic magnetic materials are bacterial magnetic Fe3O4 nanoparticles (BacMPs, Figure 1b). BacMPs are produced by Magnetospirillum magneticum and related organisms [11] and are surrounded by a lipid bilayer membrane rich in transmembrane proteins [12]. Functional polypeptides with transmembrane domains can be easily embedded in vitro [13,14] (Figure 1b), and plasmid-encoded fusion proteins of natural transmembrane BacMPs (such as Mms13 and Mms16 acting as anchors) or functional peptides (such as the ZZ domain of protein A acting as functional ligands) can be expressed in magnetotactic bacteria, thus allowing direct production of functionalized BacMPs [11]. At present, magnetic particles are widely used for diagnosis in magnetic nuclear resonance (MNR) [15]. However,
0167-7799/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2009.04.003 Available online 27 June 2009
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Figure 1. Architecture of magnetic particles. (a) Synthetic magnetic particles mainly comprise the magnetic core (blue) usually made of ferrimagnetic magnetite, and a polymeric coating layer (green). Depending on the intended function, additional elements can be incorporated, including antibodies (pale blue), drugs (black), imaging agents (yellow) or diverse chemical groups for specific or unspecific ligand binding (red). These different components can also be combined to give rise to multi-functional magnetic particles. (b) In bacterial magnetic particles (BacMPs), the magnetic core (blue) is surrounded by a lipidic bilayer (gray), into which transmembrane bacterial proteins (red) are typically inserted. BacMP-producing magnetotactic bacteria can be transformed with plasmid DNA encoding fusions between natural transmembrane proteins and other functional domains (dark blue), which are then exposed on the particle surface. Alternatively, functional cationic peptides (green string) can also be incorporated in vitro by spontaneous association with purified BacMPs.
the distal arrangement of their spatial distribution through magnetic fields both in vitro and in vivo, and consequently that of any associated entities such as drugs, biomolecules, cells or viruses, has led to the emergence of novel biomedical applications for magnetic nanoparticles (MNPs). Separation of macromolecules or cells The production of recombinant pharmaceuticals or the isolation of proteins and nucleic acids from natural samples requires that these macromolecules are separated from complex samples to be highly purified. Furthermore, emerging medical applications, for example assisted reproductive techniques (ARTs) or autologous bone marrow transplantation, require the isolation of specific cell types from human samples, or alternatively, the depletion of undesired types from clinical material, such as the removal of metastatic cancer cells. These objectives can be straightforwardly achieved both at small and large scales with the use of magnetically controlled particles, thus skipping the multiple-step approaches of conventional separation approaches while achieving the high degree of specificity that is required for clinical uses.
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Protein purification Chromatographic protein purifications provide high-resolution separations, but these methods cannot handle ‘dirty’ samples because colloidal contaminants frequently plug the packed-bed columns. By contrast, functionalized magnetic particles allow for quick and efficient purification from crude cell extracts or from other samples rich in cell debris [16], thereby eliminating the need for most of the pre-treatment steps, including centrifugation, filtration and membrane separation (Figure 2a). To achieve this, magnetic particles can be decorated either with broadspecificity ligands, such as streptavidin or protein A, or with specific recognition groups, including monoclonal or polyclonal antibodies. In one of the most common immobilized metal affinity chromatography (IMAC) applications, six histidine residues (‘6His tag’) are incorporated into the C- or Nterminus of a recombinant protein. This 6His tag binds strongly to a metal chelate, such as the Ni2+–nitrilotriacetate (Ni–NTA) complex, that is immobilized on a resin. Four of the six coordination sites on the octahedral Ni2+ center are occupied by the NTA ligand, and the remaining coordination sites are occupied by two of the six imidazole moieties in the 6His tag. In 1996, Ji and coworkers [17] developed a method to covalently modify superparamagnetic beads with a six-carbon spacer and a Ni–NTA complex for affinity purifications. The covalent attachment of these chelator complexes offered a simple and versatile platform for separation of histidin-tagged proteins consisting of only a few steps: (i) mixing and incubation for specific binding; (ii) magnet-driven immobilization of nanoparticles onto the tube wall; (iii) washing; and (iv) imidazole-mediated recovery of histidinetagged peptides or proteins. Ni–NTA magnetic beads can be reused in principle, but this requires reloading the beads with toxic Ni2+ ions before reuse. In an attempt to overcome this drawback, Frenzel and coworkers [18] developed a novel type of magnetic bead, consisting of a magnetic core and a nickel–silica matrix in which the nickel ions are tightly integrated. Because this avoids the loss of nickel ions in the elution steps, the beads can be directly reused without the need for activation with Ni2+ ions, and hence the handling of toxic Ni2+ salts. Automated high-throughout processes based on Ni–NTA-magnetic-particle-based purifications have recently been described and have been successfully applied to proteomic studies [19,20].
Table 1. Examples of commercially available magnetic particles and their applications Activated surface Amine Carboxyl Sulphonyl ester Aldehyde Hydrazide Glycidyl ether (epoxy)
Application Covalent attachment of proteins or ligands with retention of biological activity Covalent attachment of proteins or ligands with retention of biological activity Covalent attachment of any ligand containing amino or sulfhydryl groups Attachment of ligands containing primary and secondary amino groups Attachment of aldehyde- or ketone-containing ligands Binding of ligands through amine or thiol groups
Representative product xMag-NH2
Distributor a BioChain (www.biochain.com)
Carboxyl-coated TurboBeads1
Turbobeads (www.turbobeads.com)
Dynabeads1 Tosylactivated
Invitrogen Dynal AS (www.invitrogen.com/dynal) Bioclone Inc. (www.bioclon.com)
BcMag1 Aldehyde-Terminated Magnetic Beads BcMag1 Hydrazide-Modified Magnetic Beads Dynabeads1 Epoxy
Bioclone Inc. (www.bioclon.com) Invitrogen Dynal AS (www.invitrogen.com/dynal)
a
The distributor refers specifically to the particles used in the given product. Particles with these activated surfaces can also be obtained from other distributors.
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Table 2. Common specific ligands available for magnetic particles and their applications Coupled ligand Streptavidin
Application Capture of biotinylated molecules
Representative product Sera-Mag1 Magnetic SpeedBeadsTM Streptavidin
Protein G
IgG purification
BioMag1 Plus Protein G Particles
Protein A
IgG purification
Protein A Magnetic Beads
Amylose
MBP fusion proteins isolation
Amylose Magnetic Beads
Glutathione
Purification of GST fusion proteins
GST MagBeads
Oligo-(dT)
mRNA purification
Dynabeads1 mRNA Purification Kit
Nickel
Isolation of polyhistidine-tagged proteins Isolation of polyhistidine-tagged proteins Isolation of polyhistidine-tagged proteins Separation of mannose glycoproteins Selection of cells presenting specific antigens Positive selection or depletion of apoptotic cells
MagneHisTM Protein Purification System
Distributor a Thermo Scientific Seradyn (www.thermo.com) Polysciences, Inc. (www.polysciences.com) New England Biolabs (www.neb.com) New England Biolabs (www.neb.com) GenScript Corporation (www.genscript.com) Invitrogen Dynal AS (www.invitrogen.com/dynal) Promega (www.promega.com)
Histidine Adem-Kit
Ademtech (www.ademtech.com)
Iminodiacetic acid (IDA) Cobalt Concanavalin A Monoclonal antibodies Annexin V
Dynabeads
TM
TALON
1
xMag-ConA Conjugates Mannose Glycoprotein Kit Human Whole Blood CD4 Selection Kit (for positive selection of CD4+ cells) Annexin V MicroBead Kit
Invitrogen Dynal AS (www.invitrogen.com/dynal) BioChain (www.biochain.com) Stem Cell Technologies (www.stemcell.com) Miltenyi Biotec (www.miltenyibiotec.com)
a
The distributor refers specifically to the particles used in the given product. Particles with these ligands can also be obtained from other distributors.
For specialized applications, decoration of the magnetic particles with groups other than Ni–NTA can prove beneficial. For example, phosphopeptides could be enriched with MNPs that are coated with zirconium phosphonate [21] and His-tagged fusion proteins could be directly isolated from bacterial lysates with Cu2+-charged magnetic particles [22]. Magnetic particles have also been decorated with specific dyes for the isolation of particular enzymes to which these molecules bind specifically, including Reactive Red 120 for b-casein [23] and Cibacron Blue F3GA for lysozyme [24]. Such dyes are inexpensive and can be easily immobilized, especially on matrices bearing hydroxyl groups. In drug discovery, the identification of protein ligands through the screening of complex samples, such as cells or botanical extracts, is known as ‘ligand fishing’. In this context, magnetic particles have been successfully used for the identification and selection of ligands that are able to interact with human serum albumin [25] and with heat shock protein 90a [26]. Similarly, magnetic particles have also been employed for phage display, where the positive, binding phage particles are sorted by magnetic particles that are functionalized with the target [14,27,28].
Figure 2. Schematic representation of magnetically driven preparative separations. (a) Target macromolecular species, usually proteins or nucleic acids (blue spheres), can be separated from potentially similar molecular species (green and orange symbols) present in complex samples by magnetic particles (gray spheres) that have been functionalized with specific ligands. Incubation of the sample with magnetic particles leads to specific binding followed by magnetdriven isolation of the nanoparticle–target complexes onto the tube wall. After washing and elution, the target molecule can be recovered. (b) In magnetically assisted cell sorting (MACS), specific cell types are isolated from complex samples. Magnetic particles (gray spheres) are functionalized with ligands of surface receptors present in a single cell type (here in the blue cells), which permits their separation in a chromatographic column to which a magnet has been applied. Two alternative processing steps are possible depending on whether these target (blue) cells are to be isolated or depleted from the sample.
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Nucleic acid applications The isolation of DNA or RNA from original complex samples is required for its detection, cloning, sequencing, amplification and hybridization, methodologies that are involved in diagnosis, forensic science, tissue and blood typing and detection of genetic variations (Figure 2a) [29]. In this general area, magnetic particles have been successfully used to facilitate, for instance, plasmid isolation from crude bacterial lysates [30] or DNA extraction from agarose gels [31,32]. A magnetic-based microfluidic system has been developed for RNA extraction in more specialized applications [33]. More recently, a novel solid-phase single base extension (SBE) protocol that is based on MNPs has been implemented for the multiplexed detection of single nucleotide
Review polymorphisms (SNPs) [34]. This study used the ‘T-to-C’ nucleotide mutation as a proof of principle for the method and used streptavidin-coated magnetic particles that carried biotinylated extension primers as a solid-phase for SBE. The reaction was performed directly on the particle surface in two separate tubes, one containing fluorescent–ddATP to detect the T (wild) allele signals and the other containing fluorescent–ddGTP to detect the C (mutant) allele signals. After completion of SBE, particles from both tubes were spotted onto a glass slide to generate a bead array for genotype discrimination among the three possible genotypes (homozygous wild-type, homozygous mutant and heterozygote). This approach offers a platform for SNP detection that is high-throughput, low-cost and fast, and it also provides analytical flexibility. Furthermore, Willner and coworkers [35] described the use of nucleic acids that were anchored to magnetic particles and functioned as DNAzyme-synthesizing machines for amplified chemiluminescence detection of single-base mutations. The major achievement of this approach was its marked reduction in background signal, as the magnetic particles allowed the DNA machines to be separated from the media components. Cell sorting Magnetic particles linked to specific antibodies can be used to either isolate specific cell types from complex samples or deplete undesired cell types (as well as a combination of both). Tucker and coworkers used a magnetic-assisted cell sorting (MACS) system to prepare viable homogenous cultures of dorsal root ganglion neurons based on defined cell surface markers [36], and Marek and coauthors [37] designed a MACS-based co-isolation method that used CD11b MicroBeads to obtain highly pure microglia and astrocytes from the same mixed-cell starting sample. Furthermore, Qiu and colleagues [38] used the MACS strategy with anti-CD14 and anti-CD16 antibody beads to deplete macrophages and neutrophils from sputum and to enrich the content of bronchial epithelial cells (which are used as diagnostic material for the early detection of lung cancers) from 1.1% in the starting population to 40%. MACS have been also recently employed for ARTs, whose efficiency is often compromised by apoptotic events that reduce sperm quality upon cryopreservation. One such event is the externalization of phosphatidylserine (PS) residues, which are normally present on the inner side of the sperm plasma membrane. This was exploited in an approach in which superparamagnetic particles were conjugated with annexin V before being bound to PS and subsequently used to separate dead and apoptotic spermatozoa from the remaining viable sperm cells [39]. Using a similar concept, cancer cells can be removed from the bone marrow of patients who are about to undergo autologous transplantation; removal can be achieved, for example, by using anti-CD38 antibodies to negatively select and purge myeloma cells from bone marrow samples. The selective ex vivo separation of tumoral cells from bone marrow, or from peripheral blood stem cell preparations, before autologous stem cell reimplantation is increasingly being used as an adjunctary measure for hematopoietic rescue after highdose therapy for refractory cancer therapy [40]. Similarly,
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Wang and coworkers [41] employed MACS and fluorescence-assisted cell sorting (FACS) to enrich and detect gastric cancer cells, which are disseminated in bone marrow in patients with gastric cancer. In a further development of single marker cell separation, Adams and coworkers [42] proposed a multitarget magnetic activated cell sorter (MT-MACS) that utilizes microfluidics technology for the simultaneous sorting of two (and potentially more) cell types in a continuous-flow manner. Notably, MACS approaches comply with good manufacturing practice (GMP) guidelines, which means that any CD4+CD25+ T cells [43] and CD34+ cells [44], as well as other types isolated in this way, would be suitable for clinical trials. In addition, CD3+ T cells can be (at least partially) depleted by MACS, resulting in intact CD56+ natural killer (NK) cells, and this method has entered clinical trials as a procedure to prevent acute graft-versus-host disease (GVHD) in leukemia or transplantation patients [45]. Pathogen detection and molecular diagnosis Magnetic-based immunoseparation of target molecules often surpasses the detection limits of traditional analytical methods, making magnetic particles also useful for diagnosis. For example, magnetic immuno-capture of analytes can be coupled with their detection using an antibody labeled with a reporter molecule. Baldrich and Mun˜oz [46] produced dually labeled magnetic particles, which were functionalized with both an antibody and a reporter enzyme. The capture of Escherichia coli cells with the specific antibody generated a shadowing effect on the particle surface that interfered with the activity of the reporter enzyme in a way that is proportional to the bacterial concentration. Detection of other pathogens, such as Leptospira sp. [47], Mycobacteria [48] or Listeria monocytogenes [49] could also improved via magnetic enrichment from biological samples. Moreover, magnetic particles have also been adapted to the detection of viral particles, as recently demonstrated for dengue [50], avian influenza [51] and hepatitis B [52] viruses. PCR-based diagnosis methods can also be improved with the use of magnetic particles, as shown by Amagliani and colleagues [53], who were able to detect DNA from Listeria monocytogenes in milk samples with a sensitivity of 10 cfu/ml, after the specific DNA, in this case the listeriolysin O gene hlyA, was magnetically captured before PCR amplification. Other magnetic-based analytical PCR strategies have been described elsewhere [11]. Apart from pathogen detection, other biomolecules are also being detected with MNPs, such as in the diagnosis of solid tumors by analysis of the human serum proteome via magnetic peptide separation coupled to MALDI-TOF analysis [54,55]. Similar magnetic-based analytical strategies have been more recently implemented for the screening and detection of diverse sera biomarkers, particularly in cancer medicine [56,57], where they open unprecedented opportunities for early diagnosis and prognosis. Positioning MNPs for in vivo applications Thus far, we have discussed in vitro applications of magnetic particles through the controlled positioning of molecules, cells or viruses to which they are attached. 471
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However, MNPs can be also used in vivo, after their intracellular delivery (usually used in cultured cells) or systemic administration, for the control of the spatial localization of magnetized cells or the biodistribution of therapeutic agents. In these applications, physical forces are used instead of the weaker biological forces, thereby making it possible to achieve desired biodistribution patterns without the need for unequivocally specific, difficultto-identify cell surface ligands. Nucleic acid transfer and gene therapy The introduction of foreign genetic material into cultured cells can be achieved by electroporation or liposomemediated transfer, but both approaches are accompanied by undesired toxic effects and have only limited efficiency. A magnetically enhanced nucleic acid delivery method (commercialized as MagnetofectionTM by chemicell, http://www.chemicell.com) [58], uses magnetic force on nucleic acids attached to magnetic particles to direct them towards and into target cells (by placing a magnet under the cell culture dish or plate). In combination with nonviral DNA carriers, such as polyethylenimine (PEI), lipofectamine or dioleoyl trimethylammonium propane (DOTAP)-cholesterol, magnetofection was shown to increase in vitro transgene expression levels by up to three orders of magnitude over standard vectors [59]. Endothelial cells, which are highly resistant to conventional transfection methods, could also be successfully magnetofected [60]. Moroever, a different study reported that 45% of an embryonic stem cell population could be magnetofected, which is a threefold increase over a 15% gene delivery efficiency achieved with the standard FuGene 6 method [61]. In gene therapy, dramatic side effects of viral vectors have severely impaired this otherwise promising therapeutic strategy [62]. In this regard, combining viral vectors with magnetic particles can be used to optimize conditions for viral-mediated gene delivery by magnetofection. This means that lower viral doses can be used along with shorter periods of viral exposure, and thus undesired effects can be reduced, as shown for adenoviral [63] and retroviral [59] vectors. In non-viral gene delivery, coupling superparamagnetic particles to LipofectamineTM 2000– or cationic lipid–plasmid DNA liposome complexes resulted in an up to 300-fold increase in reporter gene expression in an in vitro cystic fibrosis model [64]. MNPs associating to transferrin [65] or the application of pulsed magnetic fields [66] also resulted in high transgene expression levels. Similarly, Xiang et al. [67] developed a non-viral delivery system based on PEI-coated BacMPs, which were able to bind to, stabilize and deliver DNA with higher efficiency than PEI–DNA complexes in animal models. Drug delivery and targeting Several therapeutic agents, including proteins or nucleic acids, are potentially destroyed, inadequately absorbed or insufficiently distributed when orally administered or injected. However, drugs associated to MNPs might circumvent most of these obstacles and are able to accumulate at the desired locations within the body through magnetic targeting (Figure 3a). Furthermore, magnetic 472
Figure 3. In vivo therapeutic applications of distally controlled magnetic nanoparticles. (a) Magnetic particles (gray spheres) associated to therapeutic molecules (shown as orange spheres) act as vehicles for drug delivery, and after systemic administration they are concentrated to the target organ (O) with the help of a magnet (M). (b) In magnetically mediated hyperthermia (MMH), systemically administered magnetic particles (gray spheres) accumulate in a tumor (T), either through the enhanced permeability and retention (EPR) effect or upon magnetic- or ligand-based targeting. Particles in the tumor are then heated (illustrated by the change to red spheres) through the external application of an alternating magnetic field (AMF), and this results in the death of the tumor cells.
accumulation can reduce the toxicity of the drug and enhance its efficacy. Importantly, magnetic-driven targeting allows ligand-based drug delivery to be bypassed; therefore, there is no need to identify specific cell receptors, which in any case are frequently not entirely specific for the target organ. The inhalation of aerosols is the most straightforward strategy for targeting into lung areas, as recently shown by Dames and colleagues [68], who employed magnetic particles contained in aerosol droplets (nanomagnetosols). Aside from magnetic-driven concentration at desired body locations, nanoparticles preferentially accumulate in tumors through the so-called ‘enhanced permeability and retention’ (EPR) effect (Figure 3b), which is thought to arise from two factors: (i) growing tumors produce vascular endothelial growth factors (VEGFs) that promote angiogenesis; and (ii) many tumors lack an effective lymphatic drainage system, which leads to a subsequent accumulation of nanoparticles. This causes tumor-associated neovasculatures to be highly permeable, allowing the leakage of circulating nanoparticles into the tumor interstitium. Moreover, very recent results showed that exposure to iron nanoparticles increased the endothelial cell permeability via microtubule remodeling, which was modulated by oxidative stress caused by reactive oxygen species [69]. These observations have opened exciting opportunities for MNPs in personalized cancer therapies that make use of additional magnetic-derived capabilities, which will be discussed in the next section. Magnetically mediated hyperthermia Magnetite particles that are exposed to an external alternating magnetic field (AMF) become heated by either
Review hysteresis loss or relaxation loss depending on their size and the specific surface area (SSA) [70]. Because cancer cells are killed at temperatures over 43 8C, whereas normal cells survive at these higher temperatures, magnetically mediated hyperthermia (MMH) induced by AMF can be used to selectively destroy cancer cells in which magnetic particles have been accumulated (Figure 3b). Since the initial observations and therapeutic applications of hyperthermia [1], MMH has evolved into three general sub-classes, arterial embolization hyperthermia (AEH), direct injection hyperthermia (DIH) and intracellular hyperthermia (IH). In AEH, the arterial supply is used to deliver magnetic particles into the tumor tissue, whereas in DIH the particles are directly injected into the tumor. In IH, magnetic particles are modified to facilitate their cellular uptake by the tumor. For example, antibody-conjugated liposomes (immunoliposomes) containing magnetite nanoparticles can efficiently deliver magnetic particles to tumoral cells if the chosen antibody shows sufficient specificity for tumoral antigens that are exposed on the tumor cell surface. Detailed descriptions of MMH and relevant examples of its applications can be found elsewhere [71–73]. Interestingly, several clinical trials (some in phase III) have been completed or are currently in progress in which MMH is used to treat breast cancer, cervical cancer and soft tissue sarcomas, among others [74]. Another interesting application has utilized magnetic lipidic nanoparticles with embedded drugs, which were solid at body temperature but melted at around 45 8C to 55 8C. These were combined with super-paramagnetic gFe2O3 nanoparticles and exposed to AMF, which resulted in the dissipated heat melting of the lipid matrices and the release of the drug in a controlled manner [75]. As a final example, Ito and coworkers [76] combined gene therapy with hyperthermia and used magnetite cationic liposomes (MCLs) as carriers for a plasmid containing a tumor necrosis factor-g (TNF-g) encoding gene that was under the control of the thermoinducible gadd 153 promoter. Exposure of MCLs to AMF led to their heating by hysteresis loss, and in turn to TNF-g expression. In vivo, TNF-g expression alone did not significantly inhibit tumor growth. However, the synergistic effect of TNF-g expression and hyperthermia-induced cell death resulted in a threefold increase of TNF-g gene expression in the tumor area and led to an arrest of tumor growth. Tissue engineering Tissue engineering exploits biology and engineering principles for the development of functional substitutes of lost or damaged tissues. Typically, tissue engineering has been based on the expansion of pluripotent autologous cells in vitro before seeding them onto three-dimensional (3D) biodegradable scaffolds to mimic their native organization and differentiation, followed by the introduction of the colonized scaffold into the cell donor. An emerging tissue engineering strategy, so-called ‘magnetic-forcebased tissue engineering’ (Mag-TE), employs cells that have been magnetically labeled with MCLs (which might also be further modified with integrin-binding peptides, such as RGD [77], to facilitate cellular uptake). The MCL-
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Figure 4. Uses of magnetic particles in tissue engineering. (a) Different cell types, indicated in purple and blue, are separately labeled using magnetite cationic liposomes and sequentially seeded onto an ultra-low attachment plate under which a magnet is placed. This leads to the generation of 3D multilayered heterotypic cell sheets by magnetic-force-based tissue engineering (Mag-TE). Removal of the magnet allows the recovery of the construct for use. (b) Tubular structures can be generated by folding preformed cell layers, obtained as shown in panel (a), around rod-shaped magnetic models. Such tubular constructs are recovered after removal of the magnet.
labeled cells can then be organized by magnetic force, as shown in Figure 4a [78]. In this approach, a magnet is applied to the underside of ultra-low-attachment well plates, which attract and accumulate magnetically labeled cells. In this way, populations of MCL-labeled cells can be sequentially driven to the surface to create 2D patterned or even 3D multilayered structures, as already demonstrated for human umbilical vein endothelial cells [79], retinal pigment epithelial cells [80], keratinocytes [77,78], mesenchymal stem cells [81] and cardiomyocytes [82], among others. Interestingly, MCLs did not compromise mesenchymal stem cell differentiation [81] or electrical connections of cardiomyocytes [82], which is indicative of their lack of toxicity. Furthermore, Mag-TE allowed the in vitro fabrication and harvesting of cell sheets that contained HepG2 as a hepatocyte model, or NIH3T3 cells as a stromal fibroblast model [83], as well as the construction of a heterotypic, layered co-culture system of rat hepatocytes and human aortic endothelial cells (HAECs), providing proof-of-principle for the applicability of this approach to the generation of complex heterogenous tissues [84]. A variation of Mag-TE, so-called ‘Mag-seeding’, specifically facilitates the seeding of cells into the deep internal space of the scaffolds [85], resulting in higher scaffold-seeding efficiencies. Tubular structures have also been created using the Mag-TE method; in particular, urinary tissue has been formed from a monotypic urothelial cell layer, and vascular tissues have been created that consist of heterotypic layers of endothelial cells, smooth muscle cells and fibroblasts [86]. In this 473
Review approach, magnetically labeled cells formed a cell sheet onto which a cylindrical magnet was rolled, which was removed after the tubular structure had been formed (Figure 4b). Using a similar concept, magnetic organoid patterning (MOP) allows the fabrication of 3D tissue constructs in vitro using spherical aggregates of cells (multicellular spheroids) as building blocks [87]. Endothelial progenitor cells (EPCs), which might help to generate new vessels and so have a high potential for revascularization in ischemic sites, have also been explored as a model system in magnetic targeting studies [88]. EPCs labeled with anionic MNPs (AMNPs) and embedded in MatrigelTM (from BD Biosciences, http:// www.bdbiosciences.com) can be remotely guided both in vitro an in vivo by applying a magnetic field. Concluding remarks MNPs have developed into highly promising tools for a wide spectrum of biomedical applications, many of which are based on the ability of magnetic fields to control their distal location or thermal activation. Apart from straightforward preparative separation approaches for large-scale protein or nucleic acid production, the variety of available functionalities makes MNPs particularly suited for smallscale isolation or depletion of cell types from natural samples and for biomarker proteomic analyses, as well as for magnetic-targeted gene therapy, and some of these applications are already in clinical trial phases. Furthermore, the introduction of magnetic particles into mammalian cells enables not only 2D and 3D tissue engineering but also the design of complex biological entities, such as tubular tissues or ordered 3D assemblies consisting of several cell types, which thus far are not achievable by conventional tissue culture. The proven lack of toxicity of MNPs and their progressive development into in vivo applications is expected to provide exciting tools in the near future for in situ manipulations, in which either molecules or cells could be magnetically distributed for precise drug release or tissue engineering. The use of magnetic forces to achieve distal control instead of less specific ligand-based biological targeting might represent an important step towards innovative drug delivery, which is in urgent need of novel vehicles and delivery methods that will increase the local concentration of active compounds and allow therapeutic doses and drug toxicity to be reduced. Acknowledgements We appreciate the financial support for the design and production of recombinant proteins and nanocomplexes for biomedical applications received from Ministerio de Ciencia e Innovacio´n (MICINN) (PETRI 950947.OP.02, BIO2005-23732-E, BIO2007-61194, EUI2008-03610), Age` ncia de Gestio´ d’Ajuts Universitaris i de Recerca (AGAUR) (2005SGR-00956) and CIBER de Bioingenierı´a, Biomateriales y Nanomedicina (CIBER-BBN), Spain. A.V. was also supported by Institucio´ Catalana de Recerca i Estudis Avanc¸ats (ICREA) (Generalitat de Catalunya) through an ICREA ACADEMIA award.
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54 Villanueva, J. et al. (2004) Serum peptide profiling by magnetic particle-assisted, automated sample processing and MALDI-TOF mass spectrometry. Anal. Chem. 76, 1560–1570 55 Villanueva, J. et al. (2005) Correcting common errors in identifying cancer-specific serum peptide signatures. J. Proteome Res. 4, 1060–1072 56 Martorella, A. and Robbins, R. (2007) Serum peptide profiling: identifying novel cancer biomarkers for early disease detection. Acta Biomed 78 (Suppl. 1), 123–128 57 Freed, G.L. et al. (2008) Differential capture of serum proteins for expression profiling and biomarker discovery in pre- and posttreatment head and neck cancer samples. Laryngoscope 118, 61–68 58 Plank, C. et al. (2003) The Magnetofection method: using magnetic force to enhance gene delivery. Biol. Chem 384, 737–747 59 Scherer, F. et al. (2002) Magnetofection: enhancing and targeting gene delivery by magnetic force in vitro and in vivo. Gene Ther. 9, 102–109 60 Krotz, F. et al. (2003) Magnetofection potentiates gene delivery to cultured endothelial cells. J. Vasc. Res. 40, 425–434 61 Lee, C.H. et al. (2008) Simple, Efficient, and reproducible gene transfection of mouse embryonic stem cells by Magnetofection. Stem Cells Dev 17, 133–142 62 Edelstein, M.L. et al. (2007) Gene therapy clinical trials worldwide to 2007 – an update. J. Gene Med. 9, 833–842 63 Bhattarai, S.R. et al. (2008) Laboratory formulated magnetic nanoparticles for enhancement of viral gene expression in suspension cell line. J. Virol. Methods 147, 213–218 64 Xenariou, S. et al. (2006) Using magnetic forces to enhance non-viral gene transfer to airway epithelium in vivo. Gene Ther. 13, 1545–1552 65 Pan, X. et al. (2008) Cationic lipid-coated magnetic nanoparticles associated with transferrin for gene delivery. Int. J. Pharm. 358, 263–270 66 Kamau, S.W. et al. (2006) Enhancement of the efficiency of non-viral gene delivery by application of pulsed magnetic field. Nucleic Acids Res. 34, e40 67 Xiang, L. et al. (2007) Bacterial magnetic particles (BMPs)–PEI as a novel and efficient non-viral gene delivery system. J. Gene Med. 9, 679–690 68 Dames, P. et al. (2007) Targeted delivery of magnetic aerosol droplets to the lung. Nat. Nanotechnol. 2, 495–499 69 Apopa, P.L. et al. (2009) Iron oxide nanoparticles induce human microvascular endothelial cell permeability through reactive oxygen species production and microtubule remodeling. Part. Fibre Toxicol. 6, 1 70 Motoyama, J. et al. (2008) Size dependent heat generation of magnetite nanoparticles under AC magnetic field for cancer therapy. Biomagn. Res. Technol. 6, 4 71 Jordan, A. and Maier-Hauff, K. (2007) Magnetic nanoparticles for intracranial thermotherapy. J. Nanosci. Nanotechnol. 7, 4604–4606 72 Kawai, N. et al. (2008) Effect of heat therapy using magnetic nanoparticles conjugated with cationic liposomes on prostate tumor in bone. Prostate 68, 784–792 73 Takamatsu, S. et al. (2008) Selective induction hyperthermia following transcatheter arterial embolization with a mixture of nano-sized magnetic particles (ferucarbotran) and embolic materials: feasibility study in rabbits. Radiat. Med. 26, 179–187 74 Thiesen, B. and Jordan, A. (2008) Clinical applications of magnetic nanoparticles for hyperthermia. Int. J. Hyperthermia 24, 467–474 75 Hsu, M.H. and Su, Y.C. (2008) Iron-oxide embedded solid lipid nanoparticles for magnetically controlled heating and drug delivery. Biomed. Microdevices 10, 785–793 76 Ito, A. et al. (2001) Heat-inducible TNF-alpha gene therapy combined with hyperthermia using magnetic nanoparticles as a novel tumortargeted therapy. Cancer Gene Ther 8, 649–654 77 Ito, A. et al. (2007) Magnetic force-based cell patterning using Arg-GlyAsp (RGD) peptide-conjugated magnetite cationic liposomes. J. Biosci. Bioeng. 104, 288–293 78 Ito, A. et al. (2004) Construction and harvest of multilayered keratinocyte sheets using magnetite nanoparticles and magnetic force. Tissue Eng. 10, 873–880 79 Akiyama, H. et al. (2009) Fabrication of complex three-dimensional tissue architectures using a magnetic force-based cell patterning technique. Biomed. Microdevices, DOI: 10.1007/s10544-009-9284-x 80 Ito, A. et al. (2005) Construction and delivery of tissue-engineered human retinal pigment epithelial cell sheets, using magnetite nanoparticles and magnetic force. Tissue Eng. 11, 489–496
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Biomedical applications of distally controlled magnetic nanoparticles Jose´ Luis Corchero1,2,3 and Antonio Villaverde2,3,1 1
CIBER de Bioingenierı´a, Biomateriales y Nanomedicina (CIBER-BBN), Bellaterra, 08196, Barcelona, Spain Institute for Biotechnology and Biomedicine, Universitat Auto`noma de Barcelona, Bellaterra, 08193 Barcelona, Spain 3 Department of Genetics and Microbiology, Universitat Auto`noma de Barcelona, Bellaterra, 08193 Barcelona, Spain 2
Nano-sized magnetic particles are increasingly being used across a wide spectrum of biomedical fields. Upon functionalization to enable specific binding, magnetic particles and their targets can be conveniently positioned in vitro and in vivo by the distal application of magnetic fields. Furthermore, such particles can be magnetically heated after reaching their in vivo targets, thus inducing localized cell death that has a considerable therapeutic value in, for instance, cancer therapy. In this context, innovative biomedical research has produced novel applications that have exciting clinical potential. Such applications include magnetically enhanced transfection, magnetically assisted gene therapy, magnetically induced hyperthermia and magnetic-force-based tissue engineering, and the principles and utilities of these applications will be discussed here. Introduction The biomedical applications of magnetic particles can be traced back to the 1950s, when Gilchrist and coworkers [1] treated lymphatic nodes and metastases by injecting metallic particles, which were heated using a magnetic field. Soon afterwards, magnetic particles were progressively incorporated as support materials for enzyme immobilization [2], drug delivery [3] and tools for targeted cell separation [4]. Since then, the success in the synthesis of magnetic particles has empowered a plethora of exciting biotechnological applications. Nanoparticles are amorphous or semicrystalline structures with at least one dimension ranging between 10 and 100 nm. Several of their characteristics, such as size uniformity, surface area, adsorption kinetics, biocompatibility, superparamagnetism and magnetic moment, can be finely tuned during the production process for specific purposes. Aside from the diverse functionalities conferred by the particle size itself (for instance, colloidal dispersion), magnetism offers additional properties that are of great biomedical interest. In particular, the ability to distally control the position of particles in a given media to induce their accumulation or separation from similar structures has found a spectrum of powerful applications in innovative medicines, as discussed below. Most materials with high magnetic moment, such as cobalt and nickel, are toxic, susceptible to oxidation and hence limited in their biomedical applications, but ferrimagnetic magnetite (Fe3O4) and maghemite (g-Fe2O3) are Corresponding author: Villaverde, A. ([email protected])
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suitable for in vivo applications. In the context of the emerging concern about the potential toxicity of nanoparticles [5], it is noteworthy that magnetic iron oxide particles are highly biocompatible, as the iron cell homeostasis is well controlled by uptake, excretion and storage and the iron excess is efficiently cleared from the body. In fact, iron-based particles do not cause oxidative stress or long-term changes in the levels of liver enzymes in rat models [6] (an indicator of biosafety), and indeed good tolerances to high doses of such materials have been reported [7]. Although these iron-based materials are often referred to as ‘magnetic’, the more accurate term ‘superparamagnetic’ designates their ability to become magnetized upon exposure to a magnetic field but have no permanent magnetization (remanence) once the field is turned off. This allows efficient magnetinduced clumping, as well as particle dispersion when the magnet is removed. In addition, the application of inorganic or polymeric coating layers to magnetic particles minimizes hydrophobic interactions, thus enhancing desirable properties, such as colloid dispersion and biocompatibility, and enabling the modification of surfaces with functional groups required for applications based on specific interactions. Strategies for the preparation of magnetic particles and their surface coating have been recently summarized [8–10]. The addition of reactive chemical groups on the particle surface (also designated as activation) allows for attachment of specific ligands or other functional molecules (usually termed decoration or functionalization) (Figure 1a). Nowadays, activated magnetic particles that are ready for customized decorations (Table 1) are commercially available, as are particles that already display specific ligands (Table 2). An interesting alternative to synthetic magnetic materials are bacterial magnetic Fe3O4 nanoparticles (BacMPs, Figure 1b). BacMPs are produced by Magnetospirillum magneticum and related organisms [11] and are surrounded by a lipid bilayer membrane rich in transmembrane proteins [12]. Functional polypeptides with transmembrane domains can be easily embedded in vitro [13,14] (Figure 1b), and plasmid-encoded fusion proteins of natural transmembrane BacMPs (such as Mms13 and Mms16 acting as anchors) or functional peptides (such as the ZZ domain of protein A acting as functional ligands) can be expressed in magnetotactic bacteria, thus allowing direct production of functionalized BacMPs [11]. At present, magnetic particles are widely used for diagnosis in magnetic nuclear resonance (MNR) [15]. However,
0167-7799/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2009.04.003 Available online 27 June 2009
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Figure 1. Architecture of magnetic particles. (a) Synthetic magnetic particles mainly comprise the magnetic core (blue) usually made of ferrimagnetic magnetite, and a polymeric coating layer (green). Depending on the intended function, additional elements can be incorporated, including antibodies (pale blue), drugs (black), imaging agents (yellow) or diverse chemical groups for specific or unspecific ligand binding (red). These different components can also be combined to give rise to multi-functional magnetic particles. (b) In bacterial magnetic particles (BacMPs), the magnetic core (blue) is surrounded by a lipidic bilayer (gray), into which transmembrane bacterial proteins (red) are typically inserted. BacMP-producing magnetotactic bacteria can be transformed with plasmid DNA encoding fusions between natural transmembrane proteins and other functional domains (dark blue), which are then exposed on the particle surface. Alternatively, functional cationic peptides (green string) can also be incorporated in vitro by spontaneous association with purified BacMPs.
the distal arrangement of their spatial distribution through magnetic fields both in vitro and in vivo, and consequently that of any associated entities such as drugs, biomolecules, cells or viruses, has led to the emergence of novel biomedical applications for magnetic nanoparticles (MNPs). Separation of macromolecules or cells The production of recombinant pharmaceuticals or the isolation of proteins and nucleic acids from natural samples requires that these macromolecules are separated from complex samples to be highly purified. Furthermore, emerging medical applications, for example assisted reproductive techniques (ARTs) or autologous bone marrow transplantation, require the isolation of specific cell types from human samples, or alternatively, the depletion of undesired types from clinical material, such as the removal of metastatic cancer cells. These objectives can be straightforwardly achieved both at small and large scales with the use of magnetically controlled particles, thus skipping the multiple-step approaches of conventional separation approaches while achieving the high degree of specificity that is required for clinical uses.
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Protein purification Chromatographic protein purifications provide high-resolution separations, but these methods cannot handle ‘dirty’ samples because colloidal contaminants frequently plug the packed-bed columns. By contrast, functionalized magnetic particles allow for quick and efficient purification from crude cell extracts or from other samples rich in cell debris [16], thereby eliminating the need for most of the pre-treatment steps, including centrifugation, filtration and membrane separation (Figure 2a). To achieve this, magnetic particles can be decorated either with broadspecificity ligands, such as streptavidin or protein A, or with specific recognition groups, including monoclonal or polyclonal antibodies. In one of the most common immobilized metal affinity chromatography (IMAC) applications, six histidine residues (‘6His tag’) are incorporated into the C- or Nterminus of a recombinant protein. This 6His tag binds strongly to a metal chelate, such as the Ni2+–nitrilotriacetate (Ni–NTA) complex, that is immobilized on a resin. Four of the six coordination sites on the octahedral Ni2+ center are occupied by the NTA ligand, and the remaining coordination sites are occupied by two of the six imidazole moieties in the 6His tag. In 1996, Ji and coworkers [17] developed a method to covalently modify superparamagnetic beads with a six-carbon spacer and a Ni–NTA complex for affinity purifications. The covalent attachment of these chelator complexes offered a simple and versatile platform for separation of histidin-tagged proteins consisting of only a few steps: (i) mixing and incubation for specific binding; (ii) magnet-driven immobilization of nanoparticles onto the tube wall; (iii) washing; and (iv) imidazole-mediated recovery of histidinetagged peptides or proteins. Ni–NTA magnetic beads can be reused in principle, but this requires reloading the beads with toxic Ni2+ ions before reuse. In an attempt to overcome this drawback, Frenzel and coworkers [18] developed a novel type of magnetic bead, consisting of a magnetic core and a nickel–silica matrix in which the nickel ions are tightly integrated. Because this avoids the loss of nickel ions in the elution steps, the beads can be directly reused without the need for activation with Ni2+ ions, and hence the handling of toxic Ni2+ salts. Automated high-throughout processes based on Ni–NTA-magnetic-particle-based purifications have recently been described and have been successfully applied to proteomic studies [19,20].
Table 1. Examples of commercially available magnetic particles and their applications Activated surface Amine Carboxyl Sulphonyl ester Aldehyde Hydrazide Glycidyl ether (epoxy)
Application Covalent attachment of proteins or ligands with retention of biological activity Covalent attachment of proteins or ligands with retention of biological activity Covalent attachment of any ligand containing amino or sulfhydryl groups Attachment of ligands containing primary and secondary amino groups Attachment of aldehyde- or ketone-containing ligands Binding of ligands through amine or thiol groups
Representative product xMag-NH2
Distributor a BioChain (www.biochain.com)
Carboxyl-coated TurboBeads1
Turbobeads (www.turbobeads.com)
Dynabeads1 Tosylactivated
Invitrogen Dynal AS (www.invitrogen.com/dynal) Bioclone Inc. (www.bioclon.com)
BcMag1 Aldehyde-Terminated Magnetic Beads BcMag1 Hydrazide-Modified Magnetic Beads Dynabeads1 Epoxy
Bioclone Inc. (www.bioclon.com) Invitrogen Dynal AS (www.invitrogen.com/dynal)
a
The distributor refers specifically to the particles used in the given product. Particles with these activated surfaces can also be obtained from other distributors.
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Table 2. Common specific ligands available for magnetic particles and their applications Coupled ligand Streptavidin
Application Capture of biotinylated molecules
Representative product Sera-Mag1 Magnetic SpeedBeadsTM Streptavidin
Protein G
IgG purification
BioMag1 Plus Protein G Particles
Protein A
IgG purification
Protein A Magnetic Beads
Amylose
MBP fusion proteins isolation
Amylose Magnetic Beads
Glutathione
Purification of GST fusion proteins
GST MagBeads
Oligo-(dT)
mRNA purification
Dynabeads1 mRNA Purification Kit
Nickel
Isolation of polyhistidine-tagged proteins Isolation of polyhistidine-tagged proteins Isolation of polyhistidine-tagged proteins Separation of mannose glycoproteins Selection of cells presenting specific antigens Positive selection or depletion of apoptotic cells
MagneHisTM Protein Purification System
Distributor a Thermo Scientific Seradyn (www.thermo.com) Polysciences, Inc. (www.polysciences.com) New England Biolabs (www.neb.com) New England Biolabs (www.neb.com) GenScript Corporation (www.genscript.com) Invitrogen Dynal AS (www.invitrogen.com/dynal) Promega (www.promega.com)
Histidine Adem-Kit
Ademtech (www.ademtech.com)
Iminodiacetic acid (IDA) Cobalt Concanavalin A Monoclonal antibodies Annexin V
Dynabeads
TM
TALON
1
xMag-ConA Conjugates Mannose Glycoprotein Kit Human Whole Blood CD4 Selection Kit (for positive selection of CD4+ cells) Annexin V MicroBead Kit
Invitrogen Dynal AS (www.invitrogen.com/dynal) BioChain (www.biochain.com) Stem Cell Technologies (www.stemcell.com) Miltenyi Biotec (www.miltenyibiotec.com)
a
The distributor refers specifically to the particles used in the given product. Particles with these ligands can also be obtained from other distributors.
For specialized applications, decoration of the magnetic particles with groups other than Ni–NTA can prove beneficial. For example, phosphopeptides could be enriched with MNPs that are coated with zirconium phosphonate [21] and His-tagged fusion proteins could be directly isolated from bacterial lysates with Cu2+-charged magnetic particles [22]. Magnetic particles have also been decorated with specific dyes for the isolation of particular enzymes to which these molecules bind specifically, including Reactive Red 120 for b-casein [23] and Cibacron Blue F3GA for lysozyme [24]. Such dyes are inexpensive and can be easily immobilized, especially on matrices bearing hydroxyl groups. In drug discovery, the identification of protein ligands through the screening of complex samples, such as cells or botanical extracts, is known as ‘ligand fishing’. In this context, magnetic particles have been successfully used for the identification and selection of ligands that are able to interact with human serum albumin [25] and with heat shock protein 90a [26]. Similarly, magnetic particles have also been employed for phage display, where the positive, binding phage particles are sorted by magnetic particles that are functionalized with the target [14,27,28].
Figure 2. Schematic representation of magnetically driven preparative separations. (a) Target macromolecular species, usually proteins or nucleic acids (blue spheres), can be separated from potentially similar molecular species (green and orange symbols) present in complex samples by magnetic particles (gray spheres) that have been functionalized with specific ligands. Incubation of the sample with magnetic particles leads to specific binding followed by magnetdriven isolation of the nanoparticle–target complexes onto the tube wall. After washing and elution, the target molecule can be recovered. (b) In magnetically assisted cell sorting (MACS), specific cell types are isolated from complex samples. Magnetic particles (gray spheres) are functionalized with ligands of surface receptors present in a single cell type (here in the blue cells), which permits their separation in a chromatographic column to which a magnet has been applied. Two alternative processing steps are possible depending on whether these target (blue) cells are to be isolated or depleted from the sample.
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Nucleic acid applications The isolation of DNA or RNA from original complex samples is required for its detection, cloning, sequencing, amplification and hybridization, methodologies that are involved in diagnosis, forensic science, tissue and blood typing and detection of genetic variations (Figure 2a) [29]. In this general area, magnetic particles have been successfully used to facilitate, for instance, plasmid isolation from crude bacterial lysates [30] or DNA extraction from agarose gels [31,32]. A magnetic-based microfluidic system has been developed for RNA extraction in more specialized applications [33]. More recently, a novel solid-phase single base extension (SBE) protocol that is based on MNPs has been implemented for the multiplexed detection of single nucleotide
Review polymorphisms (SNPs) [34]. This study used the ‘T-to-C’ nucleotide mutation as a proof of principle for the method and used streptavidin-coated magnetic particles that carried biotinylated extension primers as a solid-phase for SBE. The reaction was performed directly on the particle surface in two separate tubes, one containing fluorescent–ddATP to detect the T (wild) allele signals and the other containing fluorescent–ddGTP to detect the C (mutant) allele signals. After completion of SBE, particles from both tubes were spotted onto a glass slide to generate a bead array for genotype discrimination among the three possible genotypes (homozygous wild-type, homozygous mutant and heterozygote). This approach offers a platform for SNP detection that is high-throughput, low-cost and fast, and it also provides analytical flexibility. Furthermore, Willner and coworkers [35] described the use of nucleic acids that were anchored to magnetic particles and functioned as DNAzyme-synthesizing machines for amplified chemiluminescence detection of single-base mutations. The major achievement of this approach was its marked reduction in background signal, as the magnetic particles allowed the DNA machines to be separated from the media components. Cell sorting Magnetic particles linked to specific antibodies can be used to either isolate specific cell types from complex samples or deplete undesired cell types (as well as a combination of both). Tucker and coworkers used a magnetic-assisted cell sorting (MACS) system to prepare viable homogenous cultures of dorsal root ganglion neurons based on defined cell surface markers [36], and Marek and coauthors [37] designed a MACS-based co-isolation method that used CD11b MicroBeads to obtain highly pure microglia and astrocytes from the same mixed-cell starting sample. Furthermore, Qiu and colleagues [38] used the MACS strategy with anti-CD14 and anti-CD16 antibody beads to deplete macrophages and neutrophils from sputum and to enrich the content of bronchial epithelial cells (which are used as diagnostic material for the early detection of lung cancers) from 1.1% in the starting population to 40%. MACS have been also recently employed for ARTs, whose efficiency is often compromised by apoptotic events that reduce sperm quality upon cryopreservation. One such event is the externalization of phosphatidylserine (PS) residues, which are normally present on the inner side of the sperm plasma membrane. This was exploited in an approach in which superparamagnetic particles were conjugated with annexin V before being bound to PS and subsequently used to separate dead and apoptotic spermatozoa from the remaining viable sperm cells [39]. Using a similar concept, cancer cells can be removed from the bone marrow of patients who are about to undergo autologous transplantation; removal can be achieved, for example, by using anti-CD38 antibodies to negatively select and purge myeloma cells from bone marrow samples. The selective ex vivo separation of tumoral cells from bone marrow, or from peripheral blood stem cell preparations, before autologous stem cell reimplantation is increasingly being used as an adjunctary measure for hematopoietic rescue after highdose therapy for refractory cancer therapy [40]. Similarly,
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Wang and coworkers [41] employed MACS and fluorescence-assisted cell sorting (FACS) to enrich and detect gastric cancer cells, which are disseminated in bone marrow in patients with gastric cancer. In a further development of single marker cell separation, Adams and coworkers [42] proposed a multitarget magnetic activated cell sorter (MT-MACS) that utilizes microfluidics technology for the simultaneous sorting of two (and potentially more) cell types in a continuous-flow manner. Notably, MACS approaches comply with good manufacturing practice (GMP) guidelines, which means that any CD4+CD25+ T cells [43] and CD34+ cells [44], as well as other types isolated in this way, would be suitable for clinical trials. In addition, CD3+ T cells can be (at least partially) depleted by MACS, resulting in intact CD56+ natural killer (NK) cells, and this method has entered clinical trials as a procedure to prevent acute graft-versus-host disease (GVHD) in leukemia or transplantation patients [45]. Pathogen detection and molecular diagnosis Magnetic-based immunoseparation of target molecules often surpasses the detection limits of traditional analytical methods, making magnetic particles also useful for diagnosis. For example, magnetic immuno-capture of analytes can be coupled with their detection using an antibody labeled with a reporter molecule. Baldrich and Mun˜oz [46] produced dually labeled magnetic particles, which were functionalized with both an antibody and a reporter enzyme. The capture of Escherichia coli cells with the specific antibody generated a shadowing effect on the particle surface that interfered with the activity of the reporter enzyme in a way that is proportional to the bacterial concentration. Detection of other pathogens, such as Leptospira sp. [47], Mycobacteria [48] or Listeria monocytogenes [49] could also improved via magnetic enrichment from biological samples. Moreover, magnetic particles have also been adapted to the detection of viral particles, as recently demonstrated for dengue [50], avian influenza [51] and hepatitis B [52] viruses. PCR-based diagnosis methods can also be improved with the use of magnetic particles, as shown by Amagliani and colleagues [53], who were able to detect DNA from Listeria monocytogenes in milk samples with a sensitivity of 10 cfu/ml, after the specific DNA, in this case the listeriolysin O gene hlyA, was magnetically captured before PCR amplification. Other magnetic-based analytical PCR strategies have been described elsewhere [11]. Apart from pathogen detection, other biomolecules are also being detected with MNPs, such as in the diagnosis of solid tumors by analysis of the human serum proteome via magnetic peptide separation coupled to MALDI-TOF analysis [54,55]. Similar magnetic-based analytical strategies have been more recently implemented for the screening and detection of diverse sera biomarkers, particularly in cancer medicine [56,57], where they open unprecedented opportunities for early diagnosis and prognosis. Positioning MNPs for in vivo applications Thus far, we have discussed in vitro applications of magnetic particles through the controlled positioning of molecules, cells or viruses to which they are attached. 471
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However, MNPs can be also used in vivo, after their intracellular delivery (usually used in cultured cells) or systemic administration, for the control of the spatial localization of magnetized cells or the biodistribution of therapeutic agents. In these applications, physical forces are used instead of the weaker biological forces, thereby making it possible to achieve desired biodistribution patterns without the need for unequivocally specific, difficultto-identify cell surface ligands. Nucleic acid transfer and gene therapy The introduction of foreign genetic material into cultured cells can be achieved by electroporation or liposomemediated transfer, but both approaches are accompanied by undesired toxic effects and have only limited efficiency. A magnetically enhanced nucleic acid delivery method (commercialized as MagnetofectionTM by chemicell, http://www.chemicell.com) [58], uses magnetic force on nucleic acids attached to magnetic particles to direct them towards and into target cells (by placing a magnet under the cell culture dish or plate). In combination with nonviral DNA carriers, such as polyethylenimine (PEI), lipofectamine or dioleoyl trimethylammonium propane (DOTAP)-cholesterol, magnetofection was shown to increase in vitro transgene expression levels by up to three orders of magnitude over standard vectors [59]. Endothelial cells, which are highly resistant to conventional transfection methods, could also be successfully magnetofected [60]. Moroever, a different study reported that 45% of an embryonic stem cell population could be magnetofected, which is a threefold increase over a 15% gene delivery efficiency achieved with the standard FuGene 6 method [61]. In gene therapy, dramatic side effects of viral vectors have severely impaired this otherwise promising therapeutic strategy [62]. In this regard, combining viral vectors with magnetic particles can be used to optimize conditions for viral-mediated gene delivery by magnetofection. This means that lower viral doses can be used along with shorter periods of viral exposure, and thus undesired effects can be reduced, as shown for adenoviral [63] and retroviral [59] vectors. In non-viral gene delivery, coupling superparamagnetic particles to LipofectamineTM 2000– or cationic lipid–plasmid DNA liposome complexes resulted in an up to 300-fold increase in reporter gene expression in an in vitro cystic fibrosis model [64]. MNPs associating to transferrin [65] or the application of pulsed magnetic fields [66] also resulted in high transgene expression levels. Similarly, Xiang et al. [67] developed a non-viral delivery system based on PEI-coated BacMPs, which were able to bind to, stabilize and deliver DNA with higher efficiency than PEI–DNA complexes in animal models. Drug delivery and targeting Several therapeutic agents, including proteins or nucleic acids, are potentially destroyed, inadequately absorbed or insufficiently distributed when orally administered or injected. However, drugs associated to MNPs might circumvent most of these obstacles and are able to accumulate at the desired locations within the body through magnetic targeting (Figure 3a). Furthermore, magnetic 472
Figure 3. In vivo therapeutic applications of distally controlled magnetic nanoparticles. (a) Magnetic particles (gray spheres) associated to therapeutic molecules (shown as orange spheres) act as vehicles for drug delivery, and after systemic administration they are concentrated to the target organ (O) with the help of a magnet (M). (b) In magnetically mediated hyperthermia (MMH), systemically administered magnetic particles (gray spheres) accumulate in a tumor (T), either through the enhanced permeability and retention (EPR) effect or upon magnetic- or ligand-based targeting. Particles in the tumor are then heated (illustrated by the change to red spheres) through the external application of an alternating magnetic field (AMF), and this results in the death of the tumor cells.
accumulation can reduce the toxicity of the drug and enhance its efficacy. Importantly, magnetic-driven targeting allows ligand-based drug delivery to be bypassed; therefore, there is no need to identify specific cell receptors, which in any case are frequently not entirely specific for the target organ. The inhalation of aerosols is the most straightforward strategy for targeting into lung areas, as recently shown by Dames and colleagues [68], who employed magnetic particles contained in aerosol droplets (nanomagnetosols). Aside from magnetic-driven concentration at desired body locations, nanoparticles preferentially accumulate in tumors through the so-called ‘enhanced permeability and retention’ (EPR) effect (Figure 3b), which is thought to arise from two factors: (i) growing tumors produce vascular endothelial growth factors (VEGFs) that promote angiogenesis; and (ii) many tumors lack an effective lymphatic drainage system, which leads to a subsequent accumulation of nanoparticles. This causes tumor-associated neovasculatures to be highly permeable, allowing the leakage of circulating nanoparticles into the tumor interstitium. Moreover, very recent results showed that exposure to iron nanoparticles increased the endothelial cell permeability via microtubule remodeling, which was modulated by oxidative stress caused by reactive oxygen species [69]. These observations have opened exciting opportunities for MNPs in personalized cancer therapies that make use of additional magnetic-derived capabilities, which will be discussed in the next section. Magnetically mediated hyperthermia Magnetite particles that are exposed to an external alternating magnetic field (AMF) become heated by either
Review hysteresis loss or relaxation loss depending on their size and the specific surface area (SSA) [70]. Because cancer cells are killed at temperatures over 43 8C, whereas normal cells survive at these higher temperatures, magnetically mediated hyperthermia (MMH) induced by AMF can be used to selectively destroy cancer cells in which magnetic particles have been accumulated (Figure 3b). Since the initial observations and therapeutic applications of hyperthermia [1], MMH has evolved into three general sub-classes, arterial embolization hyperthermia (AEH), direct injection hyperthermia (DIH) and intracellular hyperthermia (IH). In AEH, the arterial supply is used to deliver magnetic particles into the tumor tissue, whereas in DIH the particles are directly injected into the tumor. In IH, magnetic particles are modified to facilitate their cellular uptake by the tumor. For example, antibody-conjugated liposomes (immunoliposomes) containing magnetite nanoparticles can efficiently deliver magnetic particles to tumoral cells if the chosen antibody shows sufficient specificity for tumoral antigens that are exposed on the tumor cell surface. Detailed descriptions of MMH and relevant examples of its applications can be found elsewhere [71–73]. Interestingly, several clinical trials (some in phase III) have been completed or are currently in progress in which MMH is used to treat breast cancer, cervical cancer and soft tissue sarcomas, among others [74]. Another interesting application has utilized magnetic lipidic nanoparticles with embedded drugs, which were solid at body temperature but melted at around 45 8C to 55 8C. These were combined with super-paramagnetic gFe2O3 nanoparticles and exposed to AMF, which resulted in the dissipated heat melting of the lipid matrices and the release of the drug in a controlled manner [75]. As a final example, Ito and coworkers [76] combined gene therapy with hyperthermia and used magnetite cationic liposomes (MCLs) as carriers for a plasmid containing a tumor necrosis factor-g (TNF-g) encoding gene that was under the control of the thermoinducible gadd 153 promoter. Exposure of MCLs to AMF led to their heating by hysteresis loss, and in turn to TNF-g expression. In vivo, TNF-g expression alone did not significantly inhibit tumor growth. However, the synergistic effect of TNF-g expression and hyperthermia-induced cell death resulted in a threefold increase of TNF-g gene expression in the tumor area and led to an arrest of tumor growth. Tissue engineering Tissue engineering exploits biology and engineering principles for the development of functional substitutes of lost or damaged tissues. Typically, tissue engineering has been based on the expansion of pluripotent autologous cells in vitro before seeding them onto three-dimensional (3D) biodegradable scaffolds to mimic their native organization and differentiation, followed by the introduction of the colonized scaffold into the cell donor. An emerging tissue engineering strategy, so-called ‘magnetic-forcebased tissue engineering’ (Mag-TE), employs cells that have been magnetically labeled with MCLs (which might also be further modified with integrin-binding peptides, such as RGD [77], to facilitate cellular uptake). The MCL-
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Figure 4. Uses of magnetic particles in tissue engineering. (a) Different cell types, indicated in purple and blue, are separately labeled using magnetite cationic liposomes and sequentially seeded onto an ultra-low attachment plate under which a magnet is placed. This leads to the generation of 3D multilayered heterotypic cell sheets by magnetic-force-based tissue engineering (Mag-TE). Removal of the magnet allows the recovery of the construct for use. (b) Tubular structures can be generated by folding preformed cell layers, obtained as shown in panel (a), around rod-shaped magnetic models. Such tubular constructs are recovered after removal of the magnet.
labeled cells can then be organized by magnetic force, as shown in Figure 4a [78]. In this approach, a magnet is applied to the underside of ultra-low-attachment well plates, which attract and accumulate magnetically labeled cells. In this way, populations of MCL-labeled cells can be sequentially driven to the surface to create 2D patterned or even 3D multilayered structures, as already demonstrated for human umbilical vein endothelial cells [79], retinal pigment epithelial cells [80], keratinocytes [77,78], mesenchymal stem cells [81] and cardiomyocytes [82], among others. Interestingly, MCLs did not compromise mesenchymal stem cell differentiation [81] or electrical connections of cardiomyocytes [82], which is indicative of their lack of toxicity. Furthermore, Mag-TE allowed the in vitro fabrication and harvesting of cell sheets that contained HepG2 as a hepatocyte model, or NIH3T3 cells as a stromal fibroblast model [83], as well as the construction of a heterotypic, layered co-culture system of rat hepatocytes and human aortic endothelial cells (HAECs), providing proof-of-principle for the applicability of this approach to the generation of complex heterogenous tissues [84]. A variation of Mag-TE, so-called ‘Mag-seeding’, specifically facilitates the seeding of cells into the deep internal space of the scaffolds [85], resulting in higher scaffold-seeding efficiencies. Tubular structures have also been created using the Mag-TE method; in particular, urinary tissue has been formed from a monotypic urothelial cell layer, and vascular tissues have been created that consist of heterotypic layers of endothelial cells, smooth muscle cells and fibroblasts [86]. In this 473
Review approach, magnetically labeled cells formed a cell sheet onto which a cylindrical magnet was rolled, which was removed after the tubular structure had been formed (Figure 4b). Using a similar concept, magnetic organoid patterning (MOP) allows the fabrication of 3D tissue constructs in vitro using spherical aggregates of cells (multicellular spheroids) as building blocks [87]. Endothelial progenitor cells (EPCs), which might help to generate new vessels and so have a high potential for revascularization in ischemic sites, have also been explored as a model system in magnetic targeting studies [88]. EPCs labeled with anionic MNPs (AMNPs) and embedded in MatrigelTM (from BD Biosciences, http:// www.bdbiosciences.com) can be remotely guided both in vitro an in vivo by applying a magnetic field. Concluding remarks MNPs have developed into highly promising tools for a wide spectrum of biomedical applications, many of which are based on the ability of magnetic fields to control their distal location or thermal activation. Apart from straightforward preparative separation approaches for large-scale protein or nucleic acid production, the variety of available functionalities makes MNPs particularly suited for smallscale isolation or depletion of cell types from natural samples and for biomarker proteomic analyses, as well as for magnetic-targeted gene therapy, and some of these applications are already in clinical trial phases. Furthermore, the introduction of magnetic particles into mammalian cells enables not only 2D and 3D tissue engineering but also the design of complex biological entities, such as tubular tissues or ordered 3D assemblies consisting of several cell types, which thus far are not achievable by conventional tissue culture. The proven lack of toxicity of MNPs and their progressive development into in vivo applications is expected to provide exciting tools in the near future for in situ manipulations, in which either molecules or cells could be magnetically distributed for precise drug release or tissue engineering. The use of magnetic forces to achieve distal control instead of less specific ligand-based biological targeting might represent an important step towards innovative drug delivery, which is in urgent need of novel vehicles and delivery methods that will increase the local concentration of active compounds and allow therapeutic doses and drug toxicity to be reduced. Acknowledgements We appreciate the financial support for the design and production of recombinant proteins and nanocomplexes for biomedical applications received from Ministerio de Ciencia e Innovacio´n (MICINN) (PETRI 950947.OP.02, BIO2005-23732-E, BIO2007-61194, EUI2008-03610), Age` ncia de Gestio´ d’Ajuts Universitaris i de Recerca (AGAUR) (2005SGR-00956) and CIBER de Bioingenierı´a, Biomateriales y Nanomedicina (CIBER-BBN), Spain. A.V. was also supported by Institucio´ Catalana de Recerca i Estudis Avanc¸ats (ICREA) (Generalitat de Catalunya) through an ICREA ACADEMIA award.
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