Review

Biomedical applications of distally

controlled magnetic nanoparticles
José Luis Corchero1,2,3 and Antonio Villaverde2,3,1
1
CIBER de Bioingenierı́a, Biomateriales y Nanomedicina (CIBER-BBN), Bellaterra, 08196, Barcelona, Spain
2
Institute for Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain
3
Department of Genetics and Microbiology, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain

Nano-sized magnetic particles are increasingly being suitable for in vivo applications. In the context of the
used across a wide spectrum of biomedical fields. Upon emerging concern about the potential toxicity of nanopar-
functionalization to enable specific binding, magnetic ticles [5], it is noteworthy that magnetic iron oxide particles
particles and their targets can be conveniently posi- are highly biocompatible, as the iron cell homeostasis is well
tioned in vitro and in vivo by the distal application of controlled by uptake, excretion and storage and the iron
magnetic fields. Furthermore, such particles can be mag- excess is efficiently cleared from the body. In fact, iron-based
netically heated after reaching their in vivo targets, thus particles do not cause oxidative stress or long-term changes
inducing localized cell death that has a considerable in the levels of liver enzymes in rat models [6] (an indicator
therapeutic value in, for instance, cancer therapy. In this of biosafety), and indeed good tolerances to high doses
context, innovative biomedical research has produced of such materials have been reported [7]. Although these
novel applications that have exciting clinical potential. iron-based materials are often referred to as ‘magnetic’, the
Such applications include magnetically enhanced trans- more accurate term ‘superparamagnetic’ designates their
fection, magnetically assisted gene therapy, magneti- ability to become magnetized upon exposure to a magnetic
cally induced hyperthermia and magnetic-force-based field but have no permanent magnetization (remanence)
tissue engineering, and the principles and utilities of once the field is turned off. This allows efficient magnet-
these applications will be discussed here. induced clumping, as well as particle dispersion when
the magnet is removed. In addition, the application of
Introduction inorganic or polymeric coating layers to magnetic particles
The biomedical applications of magnetic particles can be minimizes hydrophobic interactions, thus enhancing desir-
traced back to the 1950s, when Gilchrist and coworkers [1] able properties, such as colloid dispersion and biocompat-
treated lymphatic nodes and metastases by injecting met- ibility, and enabling the modification of surfaces with
allic particles, which were heated using a magnetic field. functional groups required for applications based on specific
Soon afterwards, magnetic particles were progressively interactions. Strategies for the preparation of magnetic
incorporated as support materials for enzyme immobiliz- particles and their surface coating have been recently sum-
ation [2], drug delivery [3] and tools for targeted cell separ- marized [8–10].
ation [4]. Since then, the success in the synthesis of magnetic The addition of reactive chemical groups on the particle
particles has empowered a plethora of exciting biotechno- surface (also designated as activation) allows for attachment
logical applications. Nanoparticles are amorphous or semi- of specific ligands or other functional molecules (usually
crystalline structures with at least one dimension ranging termed decoration or functionalization) (Figure 1a). Nowa-
between 10 and 100 nm. Several of their characteristics, days, activated magnetic particles that are ready for custo-
such as size uniformity, surface area, adsorption kinetics, mized decorations (Table 1) are commercially available, as
biocompatibility, superparamagnetism and magnetic are particles that already display specific ligands (Table 2).
moment, can be finely tuned during the production process An interesting alternative to synthetic magnetic materials
for specific purposes. Aside from the diverse functionalities are bacterial magnetic Fe3O4 nanoparticles (BacMPs,
conferred by the particle size itself (for instance, colloidal Figure 1b). BacMPs are produced by Magnetospirillum
dispersion), magnetism offers additional properties that are magneticum and related organisms [11] and are surrounded
of great biomedical interest. In particular, the ability to by a lipid bilayer membrane rich in transmembrane
distally control the position of particles in a given media proteins [12]. Functional polypeptides with transmembrane
to induce their accumulation or separation from similar domains can be easily embedded in vitro [13,14] (Figure 1b),
structures has found a spectrum of powerful applications and plasmid-encoded fusion proteins of natural transmem-
in innovative medicines, as discussed below. brane BacMPs (such as Mms13 and Mms16 acting as
Most materials with high magnetic moment, such as anchors) or functional peptides (such as the ZZ domain of
cobalt and nickel, are toxic, susceptible to oxidation and protein A acting as functional ligands) can be expressed in
hence limited in their biomedical applications, but ferrimag- magnetotactic bacteria, thus allowing direct production of
netic magnetite (Fe3O4) and maghemite (g-Fe2O3) are functionalized BacMPs [11].
At present, magnetic particles are widely used for diag-
Corresponding author: Villaverde, A. ([email protected]) nosis in magnetic nuclear resonance (MNR) [15]. However,
468 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
 Review Trends in Biotechnology Vol.27 No.8

Protein purification
Chromatographic protein purifications provide high-resol-
ution separations, but these methods cannot handle ‘dirty’
samples because colloidal contaminants frequently plug
the packed-bed columns. By contrast, functionalized mag-
netic 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 broad-
specificity ligands, such as streptavidin or protein A, or
with specific recognition groups, including monoclonal or
Figure 1. Architecture of magnetic particles. (a) Synthetic magnetic particles polyclonal antibodies.
mainly comprise the magnetic core (blue) usually made of ferrimagnetic In one of the most common immobilized metal affinity
magnetite, and a polymeric coating layer (green). Depending on the intended
function, additional elements can be incorporated, including antibodies (pale chromatography (IMAC) applications, six histidine resi-
blue), drugs (black), imaging agents (yellow) or diverse chemical groups for dues (‘6His tag’) are incorporated into the C- or N-
specific or unspecific ligand binding (red). These different components can also be
terminus of a recombinant protein. This 6His tag binds
combined to give rise to multi-functional magnetic particles. (b) In bacterial
magnetic particles (BacMPs), the magnetic core (blue) is surrounded by a lipidic strongly to a metal chelate, such as the Ni2+–nitrilotria-
bilayer (gray), into which transmembrane bacterial proteins (red) are typically cetate (Ni–NTA) complex, that is immobilized on a resin.
inserted. BacMP-producing magnetotactic bacteria can be transformed with
plasmid DNA encoding fusions between natural transmembrane proteins and
Four of the six coordination sites on the octahedral Ni2+
other functional domains (dark blue), which are then exposed on the particle center are occupied by the NTA ligand, and the remain-
surface. Alternatively, functional cationic peptides (green string) can also be ing coordination sites are occupied by two of the six
incorporated in vitro by spontaneous association with purified BacMPs.
imidazole moieties in the 6His tag. In 1996, Ji and
coworkers [17] developed a method to covalently modify
the distal arrangement of their spatial distribution through superparamagnetic beads with a six-carbon spacer and a
magnetic fields both in vitro and in vivo, and consequently Ni–NTA complex for affinity purifications. The covalent
that of any associated entities such as drugs, biomolecules, attachment of these chelator complexes offered a simple
cells or viruses, has led to the emergence of novel biomedical and versatile platform for separation of histidin-tagged
applications for magnetic nanoparticles (MNPs). proteins consisting of only a few steps: (i) mixing and
incubation for specific binding; (ii) magnet-driven immo-
Separation of macromolecules or cells bilization of nanoparticles onto the tube wall; (iii) wash-
The production of recombinant pharmaceuticals or the ing; and (iv) imidazole-mediated recovery of histidine-
isolation of proteins and nucleic acids from natural tagged peptides or proteins. Ni–NTA magnetic beads can
samples requires that these macromolecules are separated be reused in principle, but this requires reloading the
from complex samples to be highly purified. Furthermore, beads with toxic Ni2+ ions before reuse. In an attempt to
emerging medical applications, for example assisted repro- overcome this drawback, Frenzel and coworkers [18]
ductive techniques (ARTs) or autologous bone marrow developed a novel type of magnetic bead, consisting of
transplantation, require the isolation of specific cell types a magnetic core and a nickel–silica matrix in which the
from human samples, or alternatively, the depletion of nickel ions are tightly integrated. Because this avoids the
undesired types from clinical material, such as the removal loss of nickel ions in the elution steps, the beads can be
of metastatic cancer cells. These objectives can be straight- directly reused without the need for activation with Ni2+
forwardly achieved both at small and large scales with the ions, and hence the handling of toxic Ni2+ salts. Auto-
use of magnetically controlled particles, thus skipping the mated high-throughout processes based on Ni–NTA-mag-
multiple-step approaches of conventional separation netic-particle-based purifications have recently been
approaches while achieving the high degree of specificity described and have been successfully applied to proteo-
that is required for clinical uses. mic studies [19,20].

Table 1. Examples of commercially available magnetic particles and their applications

Activated surface Application Representative product Distributor a
Amine Covalent attachment of proteins or ligands with xMag-NH2 BioChain (www.biochain.com)
retention of biological activity
Carboxyl Covalent attachment of proteins or ligands with Carboxyl-coated TurboBeads1 Turbobeads (www.turbobeads.com)
retention of biological activity
Sulphonyl ester Covalent attachment of any ligand containing Dynabeads1 Tosylactivated Invitrogen Dynal AS
amino or sulfhydryl groups (www.invitrogen.com/dynal)
Aldehyde Attachment of ligands containing primary and BcMag1 Aldehyde-Terminated Bioclone Inc. (www.bioclon.com)
secondary amino groups Magnetic Beads
Hydrazide Attachment of aldehyde- or ketone-containing BcMag1 Hydrazide-Modified Bioclone Inc. (www.bioclon.com)
ligands Magnetic Beads
Glycidyl ether Binding of ligands through amine or thiol groups Dynabeads1 Epoxy Invitrogen Dynal AS
(epoxy) (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.

469
 Review Trends in Biotechnology Vol.27 No.8

Table 2. Common specific ligands available for magnetic particles and their applications
Coupled ligand Application Representative product Distributor a
Streptavidin Capture of biotinylated molecules Sera-Mag1 Magnetic SpeedBeadsTM Streptavidin Thermo Scientific Seradyn
(www.thermo.com)
Protein G IgG purification BioMag1 Plus Protein G Particles Polysciences, Inc.
(www.polysciences.com)
Protein A IgG purification Protein A Magnetic Beads New England Biolabs
(www.neb.com)
Amylose MBP fusion proteins isolation Amylose Magnetic Beads New England Biolabs
(www.neb.com)
Glutathione Purification of GST fusion proteins GST MagBeads GenScript Corporation
(www.genscript.com)
Oligo-(dT) mRNA purification Dynabeads1 mRNA Purification Kit Invitrogen Dynal AS
(www.invitrogen.com/dynal)
Nickel Isolation of polyhistidine-tagged MagneHisTM Protein Purification System Promega (www.promega.com)
proteins
Iminodiacetic Isolation of polyhistidine-tagged Histidine Adem-Kit Ademtech (www.ademtech.com)
acid (IDA) proteins
TM 1
Cobalt Isolation of polyhistidine-tagged Dynabeads TALON Invitrogen Dynal AS
proteins (www.invitrogen.com/dynal)
Concanavalin A Separation of mannose glycoproteins xMag-ConA Conjugates Mannose Glycoprotein Kit BioChain (www.biochain.com)
Monoclonal Selection of cells presenting specific Human Whole Blood CD4 Selection Kit (for positive Stem Cell Technologies
antibodies antigens selection of CD4+ cells) (www.stemcell.com)
Annexin V Positive selection or depletion of Annexin V MicroBead Kit Miltenyi Biotec
apoptotic cells (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 immobil-
ized, 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].

Nucleic acid applications

The isolation of DNA or RNA from original complex samples
is required for its detection, cloning, sequencing, amplifica-
tion and hybridization, methodologies that are involved in
Figure 2. Schematic representation of magnetically driven preparative
diagnosis, forensic science, tissue and blood typing and
separations. (a) Target macromolecular species, usually proteins or nucleic acids detection of genetic variations (Figure 2a) [29]. In this
(blue spheres), can be separated from potentially similar molecular species (green general area, magnetic particles have been successfully
and orange symbols) present in complex samples by magnetic particles (gray
spheres) that have been functionalized with specific ligands. Incubation of the
used to facilitate, for instance, plasmid isolation from crude
sample with magnetic particles leads to specific binding followed by magnet- bacterial lysates [30] or DNA extraction from agarose gels
driven isolation of the nanoparticle–target complexes onto the tube wall. After [31,32].
washing and elution, the target molecule can be recovered. (b) In magnetically
assisted cell sorting (MACS), specific cell types are isolated from complex samples.
A magnetic-based microfluidic system has been devel-
Magnetic particles (gray spheres) are functionalized with ligands of surface oped for RNA extraction in more specialized applications
receptors present in a single cell type (here in the blue cells), which permits their [33]. More recently, a novel solid-phase single base exten-
separation in a chromatographic column to which a magnet has been applied. Two
alternative processing steps are possible depending on whether these target (blue) sion (SBE) protocol that is based on MNPs has been imple-
cells are to be isolated or depleted from the sample. mented for the multiplexed detection of single nucleotide

470
Review Trends in Biotechnology Vol.27 No.8

polymorphisms (SNPs) [34]. This study used the ‘T-to-C’ Wang and coworkers [41] employed MACS and fluor-
nucleotide mutation as a proof of principle for the method escence-assisted cell sorting (FACS) to enrich and detect
and used streptavidin-coated magnetic particles that car- gastric cancer cells, which are disseminated in bone
ried biotinylated extension primers as a solid-phase for SBE. marrow in patients with gastric cancer. In a further de-
The reaction was performed directly on the particle surface velopment of single marker cell separation, Adams and
in two separate tubes, one containing fluorescent–ddATP to coworkers [42] proposed a multitarget magnetic activated
detect the T (wild) allele signals and the other containing cell sorter (MT-MACS) that utilizes microfluidics technol-
fluorescent–ddGTP to detect the C (mutant) allele signals. ogy for the simultaneous sorting of two (and potentially
After completion of SBE, particles from both tubes were more) cell types in a continuous-flow manner. Notably,
spotted onto a glass slide to generate a bead array for MACS approaches comply with good manufacturing prac-
genotype discrimination among the three possible geno- tice (GMP) guidelines, which means that any CD4+CD25+
types (homozygous wild-type, homozygous mutant and het- T cells [43] and CD34+ cells [44], as well as other types
erozygote). This approach offers a platform for SNP isolated in this way, would be suitable for clinical trials. In
detection that is high-throughput, low-cost and fast, and addition, CD3+ T cells can be (at least partially) depleted by
it also provides analytical flexibility. MACS, resulting in intact CD56+ natural killer (NK) cells,
Furthermore, Willner and coworkers [35] described the and this method has entered clinical trials as a procedure
use of nucleic acids that were anchored to magnetic particles to prevent acute graft-versus-host disease (GVHD) in leu-
and functioned as DNAzyme-synthesizing machines for kemia or transplantation patients [45].
amplified chemiluminescence detection of single-base
mutations. The major achievement of this approach was Pathogen detection and molecular diagnosis
its marked reduction in background signal, as the magnetic Magnetic-based immunoseparation of target molecules
particles allowed the DNA machines to be separated from often surpasses the detection limits of traditional analyti-
the media components. cal methods, making magnetic particles also useful for
diagnosis. For example, magnetic immuno-capture of ana-
Cell sorting lytes can be coupled with their detection using an antibody
Magnetic particles linked to specific antibodies can be used labeled with a reporter molecule. Baldrich and Muñoz [46]
to either isolate specific cell types from complex samples or produced dually labeled magnetic particles, which were
deplete undesired cell types (as well as a combination of functionalized with both an antibody and a reporter
both). Tucker and coworkers used a magnetic-assisted cell enzyme. The capture of Escherichia coli cells with the
sorting (MACS) system to prepare viable homogenous specific antibody generated a shadowing effect on the
cultures of dorsal root ganglion neurons based on defined particle surface that interfered with the activity of the
cell surface markers [36], and Marek and coauthors [37] reporter enzyme in a way that is proportional to the
designed a MACS-based co-isolation method that used bacterial concentration. Detection of other pathogens, such
CD11b MicroBeads to obtain highly pure microglia and as Leptospira sp. [47], Mycobacteria [48] or Listeria mono-
astrocytes from the same mixed-cell starting sample. cytogenes [49] could also improved via magnetic enrich-
Furthermore, Qiu and colleagues [38] used the MACS ment from biological samples. Moreover, magnetic
strategy with anti-CD14 and anti-CD16 antibody beads particles have also been adapted to the detection of viral
to deplete macrophages and neutrophils from sputum and particles, as recently demonstrated for dengue [50], avian
to enrich the content of bronchial epithelial cells (which are influenza [51] and hepatitis B [52] viruses. PCR-based
used as diagnostic material for the early detection of lung diagnosis methods can also be improved with the use of
cancers) from 1.1% in the starting population to 40%. magnetic particles, as shown by Amagliani and colleagues
MACS have been also recently employed for ARTs, whose [53], who were able to detect DNA from Listeria mono-
efficiency is often compromised by apoptotic events that cytogenes in milk samples with a sensitivity of 10 cfu/ml,
reduce sperm quality upon cryopreservation. One such after the specific DNA, in this case the listeriolysin O gene
event is the externalization of phosphatidylserine (PS) hlyA, was magnetically captured before PCR amplification.
residues, which are normally present on the inner side Other magnetic-based analytical PCR strategies have been
of the sperm plasma membrane. This was exploited in an described elsewhere [11].
approach in which superparamagnetic particles were con- Apart from pathogen detection, other biomolecules are
jugated with annexin V before being bound to PS and also being detected with MNPs, such as in the diagnosis of
subsequently used to separate dead and apoptotic sperma- solid tumors by analysis of the human serum proteome via
tozoa from the remaining viable sperm cells [39]. Using a magnetic peptide separation coupled to MALDI-TOF
similar concept, cancer cells can be removed from the bone analysis [54,55]. Similar magnetic-based analytical strat-
marrow of patients who are about to undergo autologous egies have been more recently implemented for the screen-
transplantation; removal can be achieved, for example, by ing and detection of diverse sera biomarkers, particularly
using anti-CD38 antibodies to negatively select and purge in cancer medicine [56,57], where they open unprecedented
myeloma cells from bone marrow samples. The selective ex opportunities for early diagnosis and prognosis.
vivo separation of tumoral cells from bone marrow, or from
peripheral blood stem cell preparations, before autologous Positioning MNPs for in vivo applications
stem cell reimplantation is increasingly being used as an Thus far, we have discussed in vitro applications of
adjunctary measure for hematopoietic rescue after high- magnetic particles through the controlled positioning
dose therapy for refractory cancer therapy [40]. Similarly, of molecules, cells or viruses to which they are attached.

471
Review Trends in Biotechnology Vol.27 No.8

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 pat-
terns without the need for unequivocally specific, difficult-
to-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 liposome-
mediated 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 Figure 3. In vivo therapeutic applications of distally controlled magnetic
nanoparticles. (a) Magnetic particles (gray spheres) associated to therapeutic
the cell culture dish or plate). In combination with non- molecules (shown as orange spheres) act as vehicles for drug delivery, and after
viral DNA carriers, such as polyethylenimine (PEI), lipo- systemic administration they are concentrated to the target organ (O) with the help
fectamine or dioleoyl trimethylammonium propane of a magnet (M). (b) In magnetically mediated hyperthermia (MMH), systemically
administered magnetic particles (gray spheres) accumulate in a tumor (T), either
(DOTAP)-cholesterol, magnetofection was shown to through the enhanced permeability and retention (EPR) effect or upon magnetic- or
increase in vitro transgene expression levels by up to three ligand-based targeting. Particles in the tumor are then heated (illustrated by the
orders of magnitude over standard vectors [59]. Endo- change to red spheres) through the external application of an alternating magnetic
field (AMF), and this results in the death of the tumor cells.
thelial cells, which are highly resistant to conventional
transfection methods, could also be successfully magneto-
fected [60]. Moroever, a different study reported that 45% accumulation can reduce the toxicity of the drug and
of an embryonic stem cell population could be magneto- enhance its efficacy. Importantly, magnetic-driven target-
fected, which is a threefold increase over a 15% gene ing allows ligand-based drug delivery to be bypassed;
delivery efficiency achieved with the standard FuGene 6 therefore, there is no need to identify specific cell receptors,
method [61]. which in any case are frequently not entirely specific for the
In gene therapy, dramatic side effects of viral vectors target organ. The inhalation of aerosols is the most
have severely impaired this otherwise promising thera- straightforward strategy for targeting into lung areas, as
peutic strategy [62]. In this regard, combining viral vectors recently shown by Dames and colleagues [68], who
with magnetic particles can be used to optimize conditions employed magnetic particles contained in aerosol droplets
for viral-mediated gene delivery by magnetofection. This (nanomagnetosols).
means that lower viral doses can be used along with Aside from magnetic-driven concentration at desired
shorter periods of viral exposure, and thus undesired body locations, nanoparticles preferentially accumulate in
effects can be reduced, as shown for adenoviral [63] and tumors through the so-called ‘enhanced permeability and
retroviral [59] vectors. In non-viral gene delivery, coupling retention’ (EPR) effect (Figure 3b), which is thought to
superparamagnetic particles to LipofectamineTM 2000– or arise from two factors: (i) growing tumors produce vascu-
cationic lipid–plasmid DNA liposome complexes resulted lar endothelial growth factors (VEGFs) that promote
in an up to 300-fold increase in reporter gene expression in angiogenesis; and (ii) many tumors lack an effective lym-
an in vitro cystic fibrosis model [64]. MNPs associating to phatic drainage system, which leads to a subsequent
transferrin [65] or the application of pulsed magnetic fields accumulation of nanoparticles. This causes tumor-associ-
[66] also resulted in high transgene expression levels. ated neovasculatures to be highly permeable, allowing the
Similarly, Xiang et al. [67] developed a non-viral delivery leakage of circulating nanoparticles into the tumor inter-
system based on PEI-coated BacMPs, which were able to stitium. Moreover, very recent results showed that
bind to, stabilize and deliver DNA with higher efficiency exposure to iron nanoparticles increased the endothelial
than PEI–DNA complexes in animal models. cell permeability via microtubule remodeling, which was
modulated by oxidative stress caused by reactive oxygen
Drug delivery and targeting species [69]. These observations have opened exciting
Several therapeutic agents, including proteins or nucleic opportunities for MNPs in personalized cancer therapies
acids, are potentially destroyed, inadequately absorbed or that make use of additional magnetic-derived capabilities,
insufficiently distributed when orally administered or which will be discussed in the next section.
injected. However, drugs associated to MNPs might cir-
cumvent most of these obstacles and are able to accumu- Magnetically mediated hyperthermia
late at the desired locations within the body through Magnetite particles that are exposed to an external
magnetic targeting (Figure 3a). Furthermore, magnetic alternating magnetic field (AMF) become heated by either

472
Review Trends in Biotechnology Vol.27 No.8

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 nor-
mal cells survive at these higher temperatures, magneti-
cally mediated hyperthermia (MMH) induced by AMF can
be used to selectively destroy cancer cells in which mag-
netic 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 facili-
tate their cellular uptake by the tumor. For example,
antibody-conjugated liposomes (immunoliposomes) con-
taining 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 Figure 4. Uses of magnetic particles in tissue engineering. (a) Different cell types,
indicated in purple and blue, are separately labeled using magnetite cationic
trials (some in phase III) have been completed or are liposomes and sequentially seeded onto an ultra-low attachment plate under
currently in progress in which MMH is used to treat breast which a magnet is placed. This leads to the generation of 3D multilayered
cancer, cervical cancer and soft tissue sarcomas, among 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
others [74]. structures can be generated by folding preformed cell layers, obtained as shown in
Another interesting application has utilized magnetic panel (a), around rod-shaped magnetic models. Such tubular constructs are
lipidic nanoparticles with embedded drugs, which were recovered after removal of the magnet.

solid at body temperature but melted at around 45 8C to

55 8C. These were combined with super-paramagnetic g-
Fe2O3 nanoparticles and exposed to AMF, which resulted labeled cells can then be organized by magnetic force, as
in the dissipated heat melting of the lipid matrices and the shown in Figure 4a [78]. In this approach, a magnet is
release of the drug in a controlled manner [75]. As a final applied to the underside of ultra-low-attachment well
example, Ito and coworkers [76] combined gene therapy plates, which attract and accumulate magnetically
with hyperthermia and used magnetite cationic liposomes labeled cells. In this way, populations of MCL-labeled cells
(MCLs) as carriers for a plasmid containing a tumor can be sequentially driven to the surface to create 2D
necrosis factor-g (TNF-g) encoding gene that was under patterned or even 3D multilayered structures, as already
the control of the thermoinducible gadd 153 promoter. demonstrated for human umbilical vein endothelial cells
Exposure of MCLs to AMF led to their heating by hyster- [79], retinal pigment epithelial cells [80], keratinocytes
esis loss, and in turn to TNF-g expression. In vivo, TNF-g [77,78], mesenchymal stem cells [81] and cardiomyocytes
expression alone did not significantly inhibit tumor [82], among others. Interestingly, MCLs did not compro-
growth. However, the synergistic effect of TNF-g expres- mise mesenchymal stem cell differentiation [81] or elec-
sion and hyperthermia-induced cell death resulted in a trical connections of cardiomyocytes [82], which is
threefold increase of TNF-g gene expression in the tumor indicative of their lack of toxicity. Furthermore, Mag-TE
area and led to an arrest of tumor growth. allowed the in vitro fabrication and harvesting of cell
sheets that contained HepG2 as a hepatocyte model, or
Tissue engineering NIH3T3 cells as a stromal fibroblast model [83], as well as
Tissue engineering exploits biology and engineering prin- the construction of a heterotypic, layered co-culture sys-
ciples for the development of functional substitutes of lost tem of rat hepatocytes and human aortic endothelial cells
or damaged tissues. Typically, tissue engineering has (HAECs), providing proof-of-principle for the applicability
been based on the expansion of pluripotent autologous of this approach to the generation of complex heterogenous
cells in vitro before seeding them onto three-dimensional tissues [84]. A variation of Mag-TE, so-called ‘Mag-seed-
(3D) biodegradable scaffolds to mimic their native organ- ing’, specifically facilitates the seeding of cells into the
ization and differentiation, followed by the introduction of deep internal space of the scaffolds [85], resulting in
the colonized scaffold into the cell donor. An emerging higher scaffold-seeding efficiencies. Tubular structures
tissue engineering strategy, so-called ‘magnetic-force- have also been created using the Mag-TE method; in
based tissue engineering’ (Mag-TE), employs cells that particular, urinary tissue has been formed from a mono-
have been magnetically labeled with MCLs (which might typic urothelial cell layer, and vascular tissues have been
also be further modified with integrin-binding peptides, created that consist of heterotypic layers of endothelial
such as RGD [77], to facilitate cellular uptake). The MCL- cells, smooth muscle cells and fibroblasts [86]. In this

473
Review Trends in Biotechnology Vol.27 No.8

approach, magnetically labeled cells formed a cell sheet 3 Widder, K.J. et al. (1978) Magnetic microspheres: a model system of site
specific drug delivery in vivo. Proc. Soc. Exp. Biol. Med. 158, 141–146
onto which a cylindrical magnet was rolled, which was
4 Widder, K.J. et al. (1979) Magnetic protein A microspheres: a rapid
removed after the tubular structure had been formed method for cell separation. Clin. Immunol. Immunopathol. 14, 395–400
(Figure 4b). Using a similar concept, magnetic organoid 5 Sanvicens, N. and Marco, M.P. (2008) Multifunctional nanoparticles –
patterning (MOP) allows the fabrication of 3D tissue con- properties and prospects for their use in human medicine. Trends
structs in vitro using spherical aggregates of cells (multi- Biotechnol. 26, 425–433
6 Jain, T.K. et al. (2008) Biodistribution, clearance, and biocompatibility
cellular spheroids) as building blocks [87].
of iron oxide magnetic nanoparticles in rats. Mol. Pharm. 5, 316–327
Endothelial progenitor cells (EPCs), which might help to 7 Lubbe, A.S. et al. (1996) Preclinical experiences with magnetic drug
generate new vessels and so have a high potential for targeting: tolerance and efficacy. Cancer Res. 56, 4694–4701
revascularization in ischemic sites, have also been 8 Horak, D. et al. (2007) Preparation and properties of magnetic nano-
explored as a model system in magnetic targeting studies and microsized particles for biological and environmental separations.
J. Sep. Sci. 30, 1751–1772
[88]. EPCs labeled with anionic MNPs (AMNPs) and 9 Laurent, S. et al. (2008) Magnetic iron oxide nanoparticles: synthesis,
embedded in MatrigelTM (from BD Biosciences, http:// stabilization, vectorization, physicochemical characterizations, and
www.bdbiosciences.com) can be remotely guided both in biological applications. Chem. Rev. 108, 2064–2110
vitro an in vivo by applying a magnetic field. 10 Lu, A.H. et al. (2007) Magnetic nanoparticles: synthesis, protection,
functionalization, and application. Angew. Chem. Int. Ed. Engl. 46,
1222–1244
Concluding remarks 11 Arakaki, A. et al. (2008) Formation of magnetite by bacteria and its
MNPs have developed into highly promising tools for a application. J. R. Soc. Interface 5, 977–999
wide spectrum of biomedical applications, many of which 12 Tanaka, M. et al. (2006) Origin of magnetosome membrane: proteomic
are based on the ability of magnetic fields to control their analysis of magnetosome membrane and comparison with cytoplasmic
membrane. Proteomics 6, 5234–5247
distal location or thermal activation. Apart from straight-
13 Tanaka, T. et al. (2004) Spontaneous integration of transmembrane
forward preparative separation approaches for large-scale peptides into a bacterial magnetic particle membrane and its
protein or nucleic acid production, the variety of available application to display of useful proteins. Anal. Chem. 76, 3764–3769
functionalities makes MNPs particularly suited for small- 14 Tanaka, T. et al. (2008) Novel method for selection of antimicrobial
scale isolation or depletion of cell types from natural peptides from a phage display library by use of bacterial magnetic
particles. Appl. Environ. Microbiol. 74, 7600–7606
samples and for biomarker proteomic analyses, as well
15 Peng, X.H. et al. (2008) Targeted magnetic iron oxide nanoparticles for
as for magnetic-targeted gene therapy, and some of these tumor imaging and therapy. Int. J. Nanomedicine 3, 311–321
applications are already in clinical trial phases. Further- 16 Safarik, I. and Safarikova, M. (2004) Magnetic techniques for the
more, the introduction of magnetic particles into mamma- isolation and purification of proteins and peptides. Biomagn. Res.
lian cells enables not only 2D and 3D tissue engineering Technol. 2, 7
17 Ji, Z. et al. (1996) Development of magnetic beads for rapid and
but also the design of complex biological entities, such as efficient metal-chelate affinity purifications. Anal. Biochem. 240,
tubular tissues or ordered 3D assemblies consisting of 197–201
several cell types, which thus far are not achievable by 18 Frenzel, A. et al. (2003) Novel purification system for 6xHis-tagged
conventional tissue culture. The proven lack of toxicity of proteins by magnetic affinity separation. J. Chromatogr. B Analyt.
MNPs and their progressive development into in vivo Technol. Biomed. Life Sci. 793, 325–329
19 Lin, C.T. et al. (2006) Automated purification of recombinant proteins:
applications is expected to provide exciting tools in the combining high-throughput with high yield. Protein Expr. Purif. 47,
near future for in situ manipulations, in which either 16–24
molecules or cells could be magnetically distributed for 20 Lin, C.T. et al. (2009) Automated 96-well purification of hexahistidine-
precise drug release or tissue engineering. The use of tagged recombinant proteins on MagneHis Ni2+-particles. Methods
Mol. Biol. 498, 129–141
magnetic forces to achieve distal control instead of less
21 Wei, J. et al. (2008) Highly efficient enrichment of phosphopeptides by
specific ligand-based biological targeting might represent magnetic nanoparticles coated with zirconium phosphonate for
an important step towards innovative drug delivery, which phosphoproteome analysis. Rapid Commun. Mass Spectrom. 22,
is in urgent need of novel vehicles and delivery methods 1069–1080
that will increase the local concentration of active com- 22 Chiang, C.L. et al. (2008) Purification of recombinant enhanced green
fluorescent protein expressed in Escherichia coli with new immobilized
pounds and allow therapeutic doses and drug toxicity to be
metal ion affinity magnetic absorbents. J. Chromatogr. B Analyt.
reduced. Technol. Biomed. Life Sci. 864, 116–122
23 Akgol, S. et al. (2005) Magnetic dye affinity beads for the adsorption of
Acknowledgements beta-casein. Macromol. Biosci. 5, 786–794
We appreciate the financial support for the design and production of 24 Basar, N. et al. (2007) Lysozyme purification with dye-affinity beads
recombinant proteins and nanocomplexes for biomedical applications under magnetic field. Int. J. Biol. Macromol. 41, 234–242
received from Ministerio de Ciencia e Innovación (MICINN) (PETRI 95- 25 Moaddel, R. et al. (2007) Automated ligand fishing using human serum
0947.OP.02, BIO2005-23732-E, BIO2007-61194, EUI2008-03610), albumin-coated magnetic beads. Anal. Chem. 79, 5414–5417
Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) 26 Marszall, M.P. et al. (2008) Ligand and protein fishing with heat
(2005SGR-00956) and CIBER de Bioingenierı́a, Biomateriales y shock protein 90 coated magnetic beads. Anal. Chem. 80, 7571–
Nanomedicina (CIBER-BBN), Spain. A.V. was also supported by 7575
Institució Catalana de Recerca i Estudis Avançats (ICREA) (Generalitat 27 Matsumoto, S.E. et al. (2008) A rapid and efficient strategy to generate
de Catalunya) through an ICREA ACADEMIA award. antigen-specific human monoclonal antibody by in vitro immunization
and the phage display method. J. Immunol. Methods 332, 2–9
References 28 Xiao, Y. et al. (2007) Selection and identification of human
1 Gilchrist, R.D. et al. (1957) Selective inductive heating of lymph nodes. gonadotropin-releasing hormone promoter binding peptides by
Ann. Surg. 4, 596–606 phage display-CEMSA. J. Mol. Recognit. 20, 51–57
2 Dunnill, P. and Lilly, M.D. (1974) Letter: Purification of enzymes using 29 Berensmeier, S. (2006) Magnetic particles for the separation and
magnetic bio-affinity materials. Biotechnol. Bioeng. 16, 987–990 purification of nucleic acids. Appl. Microbiol. Biotechnol. 73, 495–504

474
Review Trends in Biotechnology Vol.27 No.8

30 Zhang, H.P. et al. (2009) Fabrication of mono-sized magnetic anion 54 Villanueva, J. et al. (2004) Serum peptide profiling by magnetic
exchange beads for plasmid DNA purification. J. Chromatogr. B particle-assisted, automated sample processing and MALDI-TOF
Analyt. Technol. Biomed. Life Sci. 877, 127–133 mass spectrometry. Anal. Chem. 76, 1560–1570
31 Saiyed, Z.M. et al. (2007) Extraction of DNA from agarose gel using 55 Villanueva, J. et al. (2005) Correcting common errors in identifying
magnetic nanoparticles (magnetite or Fe3O4). Anal. Biochem. 363, 288– cancer-specific serum peptide signatures. J. Proteome Res. 4, 1060–1072
290 56 Martorella, A. and Robbins, R. (2007) Serum peptide profiling:
32 Sarkar, T.R. and Irudayaraj, J. (2008) Carboxyl-coated magnetic identifying novel cancer biomarkers for early disease detection. Acta
nanoparticles for mRNA isolation and extraction of supercoiled Biomed 78 (Suppl. 1), 123–128
plasmid DNA. Anal. Biochem. 379, 130–132 57 Freed, G.L. et al. (2008) Differential capture of serum proteins
33 Liu, C.J. et al. (2009) Magnetic-bead-based microfluidic system for for expression profiling and biomarker discovery in pre- and
ribonucleic acid extraction and reverse transcription processes. posttreatment head and neck cancer samples. Laryngoscope 118, 61–68
Biomed. Microdevices 11, 339–350 58 Plank, C. et al. (2003) The Magnetofection method: using magnetic
34 Liu, H. et al. (2009) Multiplex single nucleotide polymorphisms force to enhance gene delivery. Biol. Chem 384, 737–747
genotyping using solid-phase single base extension on magnetic 59 Scherer, F. et al. (2002) Magnetofection: enhancing and targeting gene
nanoparticles. Anal. Biochem. 386, 126–128 delivery by magnetic force in vitro and in vivo. Gene Ther. 9, 102–109
35 Willner, I. et al. (2008) Analysis of DNA and single-base mutations 60 Krotz, F. et al. (2003) Magnetofection potentiates gene delivery to
using magnetic particles for purification, amplification and DNAzyme cultured endothelial cells. J. Vasc. Res. 40, 425–434
detection. Analyst (Lond.) 133, 923–927 61 Lee, C.H. et al. (2008) Simple, Efficient, and reproducible gene
36 Tucker, B.A. et al. (2005) A procedure for selecting and culturing transfection of mouse embryonic stem cells by Magnetofection. Stem
subpopulations of neurons from rat dorsal root ganglia using Cells Dev 17, 133–142
magnetic beads. Brain Research Protocols 16, 50–57 62 Edelstein, M.L. et al. (2007) Gene therapy clinical trials worldwide to
37 Marek, R. et al. (2008) Magnetic cell sorting: a fast and effective method 2007 – an update. J. Gene Med. 9, 833–842
of concurrent isolation of high purity viable astrocytes and microglia 63 Bhattarai, S.R. et al. (2008) Laboratory formulated magnetic
from neonatal mouse brain tissue. J. Neurosci. Methods 175, 108–118 nanoparticles for enhancement of viral gene expression in
38 Qiu, Q. et al. (2008) Magnetic enrichment of bronchial epithelial cells suspension cell line. J. Virol. Methods 147, 213–218
from sputum for lung cancer diagnosis. Cancer 114, 275–283 64 Xenariou, S. et al. (2006) Using magnetic forces to enhance non-viral
39 Dirican, E.K. et al. (2008) Clinical outcome of magnetic activated cell gene transfer to airway epithelium in vivo. Gene Ther. 13, 1545–1552
sorting of non-apoptotic spermatozoa before density gradient 65 Pan, X. et al. (2008) Cationic lipid-coated magnetic nanoparticles
centrifugation for assisted reproduction. J. Assist. Reprod. Genet. 25, associated with transferrin for gene delivery. Int. J. Pharm. 358, 263–270
375–381 66 Kamau, S.W. et al. (2006) Enhancement of the efficiency of non-viral
40 Hardwick, R.A. et al. (1992) Design of large-scale separation systems gene delivery by application of pulsed magnetic field. Nucleic Acids Res.
for positive and negative immunomagnetic selection of cells using 34, e40
superparamagnetic microspheres. J. Hematother. 1, 379–386 67 Xiang, L. et al. (2007) Bacterial magnetic particles (BMPs)–PEI as a
41 Wang, G.Y. et al. (2009) Detection of disseminated tumor cells in bone novel and efficient non-viral gene delivery system. J. Gene Med. 9,
marrow of gastric cancer using magnetic activated cell sorting and 679–690
fluorescent activated cell sorting. J. Gastroenterol. Hepatol. 24, 299–306 68 Dames, P. et al. (2007) Targeted delivery of magnetic aerosol droplets to
42 Adams, J.D. et al. (2008) Multitarget magnetic activated cell sorter. the lung. Nat. Nanotechnol. 2, 495–499
Proc. Natl. Acad. Sci. U. S. A. 105, 18165–18170 69 Apopa, P.L. et al. (2009) Iron oxide nanoparticles induce human
43 Hoffmann, P. et al. (2006) Isolation of CD4+CD25+ regulatory T cells for microvascular endothelial cell permeability through reactive
clinical trials. Biol. Blood Marrow Transplant. 12, 267–274 oxygen species production and microtubule remodeling. Part. Fibre
44 Beelen, D.W. et al. (2000) Transplantation of highly purified HLA- Toxicol. 6, 1
identical sibling donor peripheral blood CD34+ cells without 70 Motoyama, J. et al. (2008) Size dependent heat generation of magnetite
prophylactic post-transplant immunosuppression in adult patients nanoparticles under AC magnetic field for cancer therapy. Biomagn.
with first chronic phase chronic myeloid leukemia: results of a Res. Technol. 6, 4
phase II study. Bone Marrow Transplant. 26, 823–829 71 Jordan, A. and Maier-Hauff, K. (2007) Magnetic nanoparticles for
45 Sao, H. et al. (1999) A new marrow T cell depletion method using anti- intracranial thermotherapy. J. Nanosci. Nanotechnol. 7, 4604–4606
CD6 monoclonal antibody-conjugated magnetic beads and its clinical 72 Kawai, N. et al. (2008) Effect of heat therapy using magnetic
application for prevention of acute graft-vs.-host disease in allogeneic nanoparticles conjugated with cationic liposomes on prostate tumor
bone marrow transplantation: results of a phase I-II trial. Int. J. in bone. Prostate 68, 784–792
Hematol. 69, 27–35 73 Takamatsu, S. et al. (2008) Selective induction hyperthermia following
46 Baldrich, E. and Muñoz, F.X. (2008) Enzyme shadowing: using transcatheter arterial embolization with a mixture of nano-sized
antibody–enzyme dually-labeled magnetic particles for fast bacterial magnetic particles (ferucarbotran) and embolic materials: feasibility
detection. Analyst (Lond.) 133, 1009–1012 study in rabbits. Radiat. Med. 26, 179–187
47 Fernandes, C.P. et al. (2008) An immunomagnetic separation-PCR 74 Thiesen, B. and Jordan, A. (2008) Clinical applications of magnetic
method for detection of pathogenic Leptospira in biological fluids. nanoparticles for hyperthermia. Int. J. Hyperthermia 24, 467–474
Hybridoma (Larchmt) 27, 381–386 75 Hsu, M.H. and Su, Y.C. (2008) Iron-oxide embedded solid lipid
48 Kaittanis, C. et al. (2007) One-step, nanoparticle-mediated bacterial nanoparticles for magnetically controlled heating and drug delivery.
detection with magnetic relaxation. Nano Lett. 7, 380–383 Biomed. Microdevices 10, 785–793
49 Yang, H. et al. (2007) Rapid detection of Listeria monocytogenes by 76 Ito, A. et al. (2001) Heat-inducible TNF-alpha gene therapy combined
nanoparticle-based immunomagnetic separation and real-time PCR. with hyperthermia using magnetic nanoparticles as a novel tumor-
Int. J. Food Microbiol. 118, 132–138 targeted therapy. Cancer Gene Ther 8, 649–654
50 Chang, W.S. et al. (2008) Rapid detection of dengue virus in serum 77 Ito, A. et al. (2007) Magnetic force-based cell patterning using Arg-Gly-
using magnetic separation and fluorescence detection. Analyst (Lond.) Asp (RGD) peptide-conjugated magnetite cationic liposomes. J. Biosci.
133, 233–240 Bioeng. 104, 288–293
51 Sakudo, A. and Ikuta, K. (2008) Efficient capture of infectious H5 avian 78 Ito, A. et al. (2004) Construction and harvest of multilayered
influenza virus utilizing magnetic beads coated with anionic polymer. keratinocyte sheets using magnetite nanoparticles and magnetic
Biochem. Biophys. Res. Commun. 377, 85–88 force. Tissue Eng. 10, 873–880
52 Satoh, K. et al. (2008) A new method of concentrating hepatitis B virus 79 Akiyama, H. et al. (2009) Fabrication of complex three-dimensional
(HBV) DNA and HBV surface antigen: an application of the method to tissue architectures using a magnetic force-based cell patterning
the detection of occult HBV infection. Vox Sang. 95, 174–180 technique. Biomed. Microdevices, DOI: 10.1007/s10544-009-9284-x
53 Amagliani, G. et al. (2006) Development of a magnetic capture 80 Ito, A. et al. (2005) Construction and delivery of tissue-engineered
hybridization-PCR assay for Listeria monocytogenes direct detection human retinal pigment epithelial cell sheets, using magnetite
in milk samples. J. Appl. Microbiol. 100, 375–383 nanoparticles and magnetic force. Tissue Eng. 11, 489–496

475
Review Trends in Biotechnology Vol.27 No.8

81 Shimizu, K. et al. (2007) Bone tissue engineering with human 85 Shimizu, K. et al. (2006) Enhanced cell-seeding into 3D porous scaffolds
mesenchymal stem cell sheets constructed using magnetite by use of magnetite nanoparticles. J. Biomed. Mater. Res. B Appl.
nanoparticles and magnetic force. J. Biomed. Mater. Res. B Appl. Biomater. 77, 265–272
Biomater. 82, 471–480 86 Ito, A. et al. (2005) Novel methodology for fabrication of tissue-
82 Shimizu, K. et al. (2007) Construction of multi-layered cardiomyocyte engineered tubular constructs using magnetite nanoparticles and
sheets using magnetite nanoparticles and magnetic force. Biotechnol. magnetic force. Tissue Eng. 11, 1553–1561
Bioeng. 96, 803–809 87 Lin, R.Z. et al. (2008) Magnetic reconstruction of three-dimensional
83 Ito, A. et al. (2007) Construction of heterotypic cell sheets by magnetic tissues from multicellular spheroids. Tissue Eng Part C. Methods 14,
force-based 3-D coculture of HepG2 and NIH3T3 cells. J. Biosci. 197–205
Bioeng. 104, 371–378 88 Wilhelm, C. et al. (2007) Magnetic control of vascular network
84 Ito, A. et al. (2004) Tissue engineering using magnetite nanoparticles formation with magnetically labeled endothelial progenitor cells.
and magnetic force: heterotypic layers of cocultured hepatocytes and Biomaterials 28, 3797–3806
endothelial cells. Tissue Eng. 10, 833–840

476