Magnetic Field-Assisted Gene Delivery: Achievements and Therapeutic Potential

The discovery in the early 2000's that magnetic nanoparticles (MNPs) complexed to nonviral or viral vectors can, in the presence of an external magnetic field, greatly enhance gene transfer into cells has raised much interest. This technique, called magnetofection, was initially developed mainly to improve gene transfer in cell cultures, a simpler and more easily controllable scenario than in vivo models. These studies provided evidence for some unique capabilities of magnetofection. Progressively, the interest in magnetofection expanded to its application in animal models and led to the association of this technique with another technology, magnetic drug targeting (MDT). This combination offers the possibility to develop more efficient and less invasive gene therapy strategies for a number of major pathologies like cancer, neurodegeneration and myocardial infarction. The goal of MDT is to concentrate MNPs functionalized with therapeutic drugs, in target areas of the body by means of properly focused external magnetic fields. The availability of stable, nontoxic MNP-gene vector complexes now offers the opportunity to develop magnetic gene targeting (MGT), a variant of MDT in which the gene coding for a therapeutic molecule, rather than the molecule itself, is delivered to a therapeutic target area in the body. This article will first outline the principle of magnetofection, subsequently describing the properties of the magnetic fields and MNPs used in this technique. Next, it will review the results achieved by magnetofection in cell cultures. Last, the potential of MGT for implementing minimally invasive gene therapy will be discussed.

represents a variant of MDT in which the gene coding for a therapeutic molecule, rather than the molecule itself, is delivered to a therapeutic target area in the body. The advantage of MGT over MDT lies in the fact that in the former, when a vector complex unit transduces a target cell it generates large numbers of therapeutic molecules (amplification effect) for an extended period of time. If these are secreted molecules they will be released into the intercellular space.
This article will first outline the principle of magnetofection, subsequently describing the properties of the magnetic fields and MNPs used in this technique. Next, it will review the results achieved by magnetofection in cell cultures. Last, the potential of MGT for implementing minimally invasive gene therapy will be discussed. For a highly comprehensive review on magnetically-enhanced nucleic acid delivery the reader is referred to a recent article by Plank e. al (5).

MAGNETOFECTION
As indicated above, magnetofection is a methodology based on the association of MNPs with gene vectors in order to enhance gene transfer in the presence of a magnetic field. It was developed by Christian Plank and collaborators for gene transfer in cell cultures and in vivo using MNP-naked DNA complexes or MNP-viral vector complexes [4]. In this context the principle of magnetofection in cells was assumed to be simple (Fig. 1): the MNP-DNA complex is added to a culture of adherent cells and a magnet, placed close below the bottom of the flask or plate, attracts the magnetic complexes to the bottom where they come in close contact with the cells and are physically internalized, without any particular effect of the magnetic force on the endocytic uptake mechanism [6]. For MNP-viral vector complexes it was thought that the magnetic field brought the complexes close to the cells thus favoring their internalization through viral receptor-mediated mechanisms. This results in a transduction improvement that in some cases is remarkable. For instance, in HEK293 cell cultures exposed to MNP-adenoviral vector complexes, magnetofection may induce over a 50-fold increase in transduction levels (Fig. 2). For MNP-adenoviral vector complexes, the internalization mechanism outlined above does not hold, as suggested by the fact that certain cell lines (e.g., NIH3T3, K562 and primary human peripheral blood lymphocytes) which express little or no coxsackie virus and adenovirus (CAR) receptors and are therefore refractory to adenovectors, can be successfully transduced by magnetofection using MNP-adenovector complexes [4]. Furthermore, ultrastructural analysis of MNP-recombinant adenovirus (RAd) complexes by electron and atomic force microscopy showed structurally intact adenoviruses fully surrounded by magnetic particles that occasionally bridged several virus particles [7]. Since this configuration would prevent virions from binding to their cell receptors, a still unknown internalization mechanism is likely to be involved. Also, kinetic studies with gold/iron oxide-based MNP-RAd complexes in adenovirus resistant cell lines provided additional evidence for a non receptor-mediated internalization mechanism for RAd-MNP complexes [8].

Regardless of the mechanisms by which magnetofection enhances gene transfer, over the years this
technique has demonstrated to be highly effective in cell cultures (see below) and constitutes a promising tool for the implementation of MGT in vivo (see below). The commercial availability of magnetofection reagents has made this methodolgy readily accessible to nonspecialist researchers.
Besides a gene vector, two other key components are necessary to implement magnetofection namely, a suitable magnetic field applicator and properly formulated MNPs.

PHYSICAL PROPERTIES OF MAGNETS AND MNP
For any biomedical application using MNP-based vectors, the magnetic component of these complexes (i.e., the magnetic core) needs to be specifically designed and engineered regarding its chemical and magnetic properties, so that the magnetic interaction can be maximized. The underlying physical interaction is related to the force generated on the magnetic core of any MNP-based complex, when a magnetic particle with magnetic (dipole) moment µ is placed in a non-uniform magnetic field B. In such a non-uniform magnetic field the force F exerted on a magnetic dipole with value (µ is related to the spatial variation, assumed in the x-direction) of B through its spatial derivatives (equation 1) Figure 3 shows the magnetic field B numerically simulated for a disk-shaped permanent magnet (NdFeSm) having 2 cm in diameter and 1 cm in height . The B profile was obtained applying a finite element method (FEM) on the corresponding Maxwell equations and boundary conditions. It can be seen that the field B decreases from a maximum value at the surface to a 25% of this value just 1 cm away from the magnet surface. Moreover the magnetic force, proportional to the derivative dB/dx, drops similarly within the same 1 cm distance, making it difficult to apply this simple method for obtaining constant forces within any practical working volume. emu/cm 3 ) and low toxicity. Although from the production point of view magnetite is cheap and relatively easy to obtain in highly purified form, the manufacture of MNPs featuring magnetically ordered cores of few nm in diameter is a major challenge because the high surface/volume ratio causes superficial disorder effects to become dominant.
In magnetofection, the magnetic field is applied to move the MNP-gene vector complexes towards the target site. In practice, this means that the target site ought to be subject to a magnetic flux density which is sufficient to cause saturation magnetization of the magnetic complex and ought to be subject

Biocompatibility. MNPs have to show low or negligible toxic effects on both cell cultures and in vivo.
Different kinds of viability assays are to be performed before a given MNP is considered as non-toxic.
Dispersion stability. MNPs should be available as monodisperse (i.e., nonaggregated) particles suspended in suitable physiological fluids. Sample preparation should ensure stability against particle precipitation, aggregation and/or self assembly phenomena.
High magnetic response, in order to induce magnetic complex migration towards and concentration in the target area under the effect of an external magnetic field. Proper magnetic field profiles are also needed; they are usually designed by numerical simulation of magnet configurations. These calculations are in principle capable of engineering efficient magnetic field applicators particularly for in vivo use.

SYNTHESIS OF MNPs
Magnetic nanoparticles can be produced by a number of physical and chemical routes that differ in the final properties of the products. For an overview on synthesis procedures and characteristics of nanoparticles suitable for gene delivery see (11). A broad classification scheme can be made based on the physical state of the starting materials. In the top-down strategy, the starting bulk material is reduced to nanometric scale in one (thin films), two (nanowires) or three (nanoparticles, or quantum dots) dimensions. This route is often based in physical processes like mechanical alloying, laser machining, laser chemical etching, reactive ion etching, among others. On the contrary, the bottom-up approach uses atomic or molecular units as starting materials to grow larger, nanometric structures.
Bottom up techniques include chemical vapor deposition (CVD), reactive sputtering, plasma enhanced CVD, pulsed laser deposition (PLD), molecular beam epitaxy (MBE), and also wet routes like sol-gel and microemulsion thechniques. Most of the above techniques have attained good control of physical parameters of the products such as phase purity, particle shape, crystalline order and the attainable range of particle sizes, although tailoring all of these parameters in a single product remains a challenging task. Two main approaches for MNP synthesis can be considered.

Thermal decomposition from organic precursors
Monodisperse iron-oxide nanoparticles of different sizes ranging from 2 to 20 nm can be obtained by PEI. In the first approach, the MNPs are coated during the synthesis, while the post-synthesis coating method consists on grafting the polymer or surfactant onto the magnetic particles once synthesized.

MAGNETOFECTION IN CELLS
As already stated, magnetofection was initially developed mainly to enhance gene transfer in cell culture, a simpler and more easily controllable scenario than in vivo models. Magnetofection in cell lines not only facilitated the optimization of protocols and MNP formulations but it also provided evidence for some unique capabilities of this approach. Progressively, an increasing number of publications combining magnetofection in cell culture and in experimental animals are beginning to emerge. This section will review studies exclusively dealing with magnetofection in cells, leaving for the next section the consideration of reports documenting in vivo studies. In tissue engineering a major challenge comes from insufficient formation of blood vessels in implanted tissues. One approach to overcome this problem has been the production of angiogenic cell sheets using a combination of two techniques namely, magnetic cell accumulation and magnetofection . This technique has also been employed to target cytotoxic drugs to brain tumors which are particularly difficult targets due to the fact that the drug must cross the blood brain barrier (BBB). It was reported that microparticles 1-2 μm in diameter could be concentrated in an intracerebral rat glioma [44]. Although the concentration of the particles in the tumor was low it was significantly higher than in controls injected with nonmagnetic particles. Better results were achieved in these tumors employing 10-20 nm MNP particles [45]. Electron microscopic analysis revealed the presence of MNPs in the interstitial space of the tumors but not in normal brain tissue where MNPs were only found in the vasculature. In another study, MDT in rat brain tumors achieved some degree of success only when the BBB was disrupted immediately prior to particle injection [46].

Neuronal and glial cells
There have been a few trials of MDT in humans although none of them has been followed up and  (Fig. 5). To reach the third ventricle from the cisterna magna the magnetic complexes need to travel counterflow.
Since CSF flow velocities are over ten times lower than arterial blood flow velocities (0.4 cm/s [70] versus 5 cm/s [71], respectively), the strength of the magnetic field to be applied in order to ovecome CSF counterflow force remains within the capacity of cylindrically or conically shaped Nd-Fe-B permanent magnets. If successful, this proof-of-concept approach could be extended to other regions of the brain.

Myocardial Infarction
Heart failure remains as one of the major causes of morbidity and mortality throughout the world, In spite of the promise these experimental studies offer, it is important to mention that in human patients, an external magnet placed over the chest would need to generate a very strong magnetic field in order to achieve in the heart, field gradients high enough as to prevent the arterial blood flow from washing away the MNP-vector complexes.
An alternative strategy to improve magnetic force is to insert a magnetizable coronary stent at the target site. Under the influence of an external magnetic field, the stent will create locally a highgradient magnetic field. This procedure is termed implant-assisted magnetic drug targeting [87][88][89].
The feasibility of this approach was suggested by a study in an isolated swine heart ventricle perfusion model carrying an intra-arterial stent coil fabricated from ferromagnetic stainless steel 430 wire and used to capture 100-nm diameter magnetite particles that mimicked magnetic drug carrier particles The above studies are encouraging and suggest that MGT to the cardiovascular system could be a rewarding research avenue and that it merits to be explored further.

CONCLUDING REMARKS
During the past two decades the biomedical applications of magnetic fields and MNPs have expanded remarkably due to the possibilities they open for noninvasive diagnostic and therapeutic approaches.
In this context, the discovery that MNP-gene vector complexes can, in the presence of a magnetic field, greatly enhance gene transfer into cells and eventually allow the development of minimally invasive gene delivery approaches in vivo, is raising much interest in this emerging technology. Many of the studies reviewed here constitute important landmarks in the path towards a mature MGT technology.
In his seminal book, Engines of Creation [97], KE Drexler defined nanotechnology as a manufacturing methodology based on the manipulation of individual atoms and molecules in order to construct complex structures, specified at the atomic level. In practice, Drexlerian nanotechnology remains as an embryonic discipline, with its practical implementation lying in the future. What is generally known as nanotechnology should be called applied nanoscience which is a discipline in active development.
Drexlerian theorists imagine a medical branch of nanotechnology called nanomedicine [98]. This medical specialty will be based on the use of intelligent nanoinstruments or nanobots which after being injected into the bloodstream will survey the body searching for faulty cells, repairing them or destroying those beyond repair (Fig. 6, left). These nanobots will be wirelessly controlled by external computers. Figure 6 right, diagrammatically represents a current MNP-adenovector complex. It could be considered as a gene delivery nanoinstrument. Its central component, the viral vector, has the capability to recognize and enter its target cells and deliver to them its therapeutic gene(s). To a certain extent it can also be wirelessly controlled, not by a computer, but by a magnetic field. Therefore, if Drexlerian nanomedicine becomes a reality in the future, perhaps these magnetic complexes will be considered as predecessors of therapeutic nanobots.    Right panel: Induction field profile as a function of distance to the surface, along x-and y-directions.
Inset: values of the spatial derivative dB/dx (proportional to the magnetic force) along x-and ydirections taken from the same simulations.