Magnetic nanoparticles for targeted vascular delivery

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Life, 63(8): 613–620, August 2011

Critical Review Magnetic Nanoparticles for Targeted Vascular Delivery Michael Chorny, Ilia Fishbein, Scott Forbes and Ivan Alferiev Division of Cardiology Research, The Children’s Hospital of Philadelphia, Philadelphia, PA

Summary Magnetic targeting has shown promise to improve the efficacy and safety of different classes of therapeutic agents by enabling their active guidance to the site of disease and minimizing dissemination to nontarget tissues. However, its translation into clinic has proven difficult because of inherent limitations of traditional approaches inapplicable for deep tissue targeting in human subjects and a need for developing well-characterized and fully biocompatible magnetic carrier formulations. A novel magnetic targeting scheme based on the magnetizing effect of deep-penetrating uniform fields is presented as an example of a strategy providing a potentially clinically viable solution for preventing injury-triggered reobstruction of stented blood vessels (in-stent restenosis). The design of optimized magnetic carrier formulations and experimental results showing the feasibility of uniform field-controlled targeting for site-specific vascular delivery of small-molecule pharmaceuticals, biotherapeutics, and cells are discussed in the context of antirestenotic therapy. The versatility of this approach applicable to different classes of therapeutic agents exerting their antirestenotic effects through distinct mechanisms prompts exploring the utility of uniform field-mediated magnetic stent targeting for combination therapies with enhanced efficiencies and improved safety profiles. Additional improvements in terms of site specificity and protracted carrier retention at the site of injury may be expected from the development and use of magnetic carriers exhibiting affinity for arterial wall-specific antigens. Ó 2011 IUBMB IUBMB Life, 63(8): 613–620, 2011 Keywords

magnetic targeting; biodegradable nanoparticle; drug delivery; gene therapy; vascular disease; stent angioplasty; restenosis.


MRI, magnetic resonance imaging; MNP, magnetic nanoparticle; PTX, paclitaxel; PLA, polylactide; PLGA, polylactide-co-glycolide; CAR, Coxsackie adenovirus receptor; Ad, adenovirus.

Received 29 March 2011; accepted 30 March 2011 Address correspondence to: Michael Chorny, PhD, The Children’s Hospital of Philadelphia, Abramson Research Building, Suite 702 G, 3615 Civic Center Boulevard, Philadelphia, PA 19104-4318. Tel.: 1215-590-3063. E-mail: [email protected] ISSN 1521-6543 print/ISSN 1521-6551 online DOI: 10.1002/iub.00479

EXPERIMENTAL STRATEGIES FOR MAGNETIC VASCULAR TARGETING—CONCEPTS AND CHALLENGES Targeted delivery strategies have shown promise to increase efficacy and minimize side effects of different classes of pharmaceuticals and are now emerging as an essential element of novel therapies with improved safety profiles. Magnetic guidance is among the most intensively explored targeting approaches because of its potential to reduce systemic drug exposure and make a local therapeutic effect achievable at significantly lower drug doses. It is unique in its ability to actively guide and concentrate therapeutic agents formulated in magnetically responsive particles at specific sites through the application of a long-range magnetic force, unsimilar to other approaches that do not involve active external guidance but rather rely on preferential retention or site-specific activation of drugs in their target region. In its simplest form, magnetic targeting is based on two primary elements: a magnetic field source and magnetically responsive drug carrier particles. The magnetic force acting on the particle is directly proportionate to the strength of the field and to its gradient (1). It is the strongest in the vicinity of the field source and decreases with distance from it, as both the field strength and its gradient fade out. Thus, the longest distance from the source at which the magnetic force is still strong enough to counteract the hydrodynamic force and/or thermal motion of particles defines the region within which a resultative particle capture can take place (2, 3). The ability to effectively extend this distance and maximize the fraction of the captured particles by using increasingly strong permanent magnets has provided the basis for a number of preclinical and clinical studies that demonstrated the utility of this targeting scheme for magnetically guided particle delivery to superficially located sites (4, 5). However, the essential requirement for a strong magnetic field and a field gradient simultaneously existent at the target site poses critical limitations to the applicability of this approach with respect to deep tissue targeting including major blood vessels. Although magnetic fields generated by externally applied magnets can reach depths up to several centimeters (6, 7), such permanent magnets create relatively weak magnetic field gradients insufficient to attract the magnetic



carrier particles. On the other hand, considerably stronger local field gradients can be achieved by using an alternative strategy that employs magnetic implants as the field source as recently published by several groups (8–10). However, the magnetic field generated by the implant decays rapidly and thus may become the limiting factor in this case as the majority of the particles fail to be magnetized sufficiently for their effective magnetic guidance (2). Permanent magnetic implants also pose safety concerns and may need to be surgically removed, which makes their use as a platform for magnetically targeted delivery less attractive for clinical applications. Although the ongoing search for improved magnetic field source configurations enabling targeting of nonsuperficially located tissues has shown some promise (11–15), the virtual impossibility of focusing the magnetic force at a distance from the sole magnetic field source as theoretically shown by Grief and Richardson (1) still remains a major limitation of this one-source magnetic targeting strategy (16).

Two-Source Strategy for Magnetically Targeted Delivery of Therapeutic Agents to Stented Blood Vessels The use of a two-source strategy that combines a strong and deep-penetrating magnetizing field with high magnetic field gradients at the target site has been recently proposed as a potentially more efficient alternative for specific therapeutic applications (2, 17), and its feasibility was shown in several experimental studies (3, 18, 19). As opposed to the traditional approach, this targeting scheme is based on three components: magnetically responsive particles, a magnetizable implant, and an externally generated uniform magnetic field. As mentioned previously, no translational force can be exerted on a particle placed in a uniform field devoid of field gradients. However, strong uniform fields, such as those available from MRI scanners and magnetic catheter navigation systems (20), can be used to both magnetize the carrier particles and induce high field gradients on a magnetizable implant (shown schematically in Fig. 1), thereby creating a region of extreme field nonuniformity in its vicinity, which results in a considerably extended particle capture zone (3). Thus, the limitations of the restricted field penetration depth or inadequate field gradients can be effectively addressed by exploiting an additional latent field source (a magnetizable implant) activated and controlled externally with devices in current clinical use. Stents made of steel types possessing paramagnetic properties are a most common example of vascular implants that as part of a two-source configuration can provide a platform for magnetically targeted therapy aimed at preventing arterial reobstruction (restenosis) as discussed below. Notably, neither the implant nor the particles need to be permanently magnetic in this uniform field-controlled targeting approach and can be made of materials with negligible magnetic remanence whose clinical safety has been well established, such as 304 grade stainless steel and nanocrystalline iron oxides, respectively (21, 22). Although not providing a universally applicable solution to magnetically guided delivery to non-

Figure 1. Schematically shown stent-targeted delivery of magnetic carriers controlled by an externally applied uniform field. The uniform field magnetizes the carrier particles and the stent creating a particle capture region spatially restricted to the stented arterial segment. superficially located sites in the body, this strategy applied for vascular targeting in the setting of stent angioplasty represents an attractive therapeutic modality with potential to significantly improve outcomes in patients with complex vascular disease.

THE DESIGN OF NANOPARTICLES FOR MAGNETICALLY TARGETED DELIVERY OF THERAPEUTIC AGENTS The properties of a magnetically targeted drug carrier need to be optimized with respect to a specific clinical application, and, thus, no uniform guidelines exist for creating an ideal magnetic particle; however, a number of general considerations apply to all magnetic carrier formulations. A particle and all its components have to be nontoxic, fully biodegradable, or bioeliminable, and the particle size should be compatible with parenteral administration, preferably below 1 lm (23). In addition to these requirements applying to any intravascularly administered particulate carrier intended for cardiovascular disease applications, a requirement for negligible magnetic remanence (i.e., superparamagnetism) applies to magnetic particle formulations (24). As embolization of blood vessels is an obvious safety concern, the absence of retained magnetization is necessary for ensuring that the magnetic exposure does not cause irreversible particle aggregation, i.e., the particles remain colloidally stable with their size unchanged upon removal of a magnetic field. To be efficient as targeted drug delivery carriers, particles should exhibit strong magnetization in the presence of a magnetizing field, enabling their efficient capture at the site of interest using a magnetic setup of a clinically applicable design. The magnetic responsiveness of an individual particle is dependent on the type and amount of the magnetizable component in its


structure. Nanocrystalline iron oxides are typically used in magnetic carrier formulations because of their history of safe clinical use, although other materials exhibiting a stronger saturation magnetization, such as metallic iron or substituted ferrites, are being investigated for magnetic targeting applications and may also be used if proven biocompatible (25, 26). At a given magnetic field strength, the magnetic responsiveness of a particle is a direct function of the amount of the magnetizable substance in its composition and, thus, can be controlled by changing the iron oxide loading and/or the size of a particle. Thus, whereas the upper size limit of a magnetic carrier is dictated by safety considerations mentioned above, the lower size threshold is defined by the minimal magnetic responsiveness required for its effective capture at the target site. The magnetic guidance capacity of a carrier, while essential, is not the only determinant of its efficiency. As a therapeutic agent released from the particle can no longer be influenced by the magnetic force, its release kinetics from the particle formulation should be sufficiently slow compared with the rate of the particle capture to ensure that most of the drug payload is targeted to the region of interest in association with the carrier (27). Further adjustments of the drug release behavior may be necessary depending on its mode of action and the critical period of the disease (i.e., facilitated vs. sustained release). Specific examples of magnetic carriers designed for stent-targeted delivery of small-molecule pharmaceuticals, gene delivery vectors, and therapeutic enzymes are discussed below.

RESTENOSIS THERAPY AND A RATIONALE FOR EXPLORING MAGNETICALLY TARGETED DELIVERY STRATEGIES Stent angioplasty is extensively used to relieve vessel narrowing in patients with obstructive vascular disease. Stents initially used as mere metallic scaffolds physically preventing reocclusion of the vessel have been later reengineered to provide local delivery of antiproliferative drugs inhibiting the growth and migration of vascular smooth muscle cells triggered by arterial injury in the course of stent angioplasty. Although the therapeutic efficacy of drugs released to the arterial wall from the surface of a stent has been well established in patients with coronary artery disease or noncomplex vascular lesions, this approach has not been equally effective in the noncoronary vasculature and in complex settings with the observed frequency of recurrent stenosis often higher than 10% (28, 29). Recent studies suggest that the suboptimal performance of drugeluting stents in these clinical settings is likely due to the inability to adjust the pharmacological effect of the drug to the timescale of the disease progression (30), which may vary considerably between different vessel types and as a result of additional risk factors, e.g., in diabetic patients. Furthermore, even when the drug payload in the stent coating is far from exhausted, its release may become too slow for maintaining therapeutically adequate drug levels at later time points. In practice,


it has been shown that as much as 90% of the drug can remain permanently bound to the polymeric stent coating (31), a result that could not have been expected from previously conducted in vitro release studies (32). These findings provide a rationale for developing alternative therapeutic strategies that would be more flexible with respect to the drug dosing and provide a better control over the local levels of the therapeutic agent at the site of arterial injury. Magnetically targeted delivery of drugs formulated in biodegradable nanoparticles to magnetizable stents has been proposed as an approach that potentially enables efficient localization of the drug to the stented arterial region (3). In contrast to drugeluting stents whose drug-loading capacity is limited and the drug payload cannot be renewed at later times, the magnetic targeting strategy may be compatible with multiple drug dosing, which is of particular importance for the effective use of stent angioplasty for the treatment of noncoronary atherosclerotic vascular disease. The possibility of individually optimized drug dose regimens and the use of drug combinations applied in the form of magnetically responsive nanoparticles (MNPs) are also important potential advantages of this experimental approach that can enable a safe and efficient, site-specific pharmacological intervention for inhibiting in-stent restenosis.

MAGNETIC NANOPARTICLES FOR UNIFORM FIELD-CONTROLLED TARGETING OF PACLITAXEL TO STENTED ARTERIES Paclitaxel (PTX) is a small-molecule pharmaceutical extensively used clinically as an anticancer drug and more recently as a therapeutic component of drug-eluting stents because of its potent antiproliferative effect on dividing cells. By binding to b-tubulin and causing the assembly of abnormally stable microtubules PTX prevents normal cell division and arrests the cell cycle at the G2/M phase (33). Stent-targeted delivery of PTX formulated in MNP can potentially provide protracted presence of the drug in the stented arterial segment at therapeutically adequate levels without exposing a patient to potentially toxic drug doses. Its formulation in MNP made of biodegradable polyesters, polylactide (PLA), or polylactide-co-glycolide (PLGA), with inclusion of nanocrystalline magnetite, has been accomplished using polymer precipitation methods, such as nanoprecipitation (34) or emulsification-solvent evaporation (3). Our group recently described the formulation and characterization of PLA-based MNP loaded with PTX and investigated their use for uniform field-controlled magnetic targeting to 304 type stainless steel stents in the rat carotid stenting model of restenosis (3). The formulation of MNP was carried out in two steps. First, crystalline magnetite was obtained by modified alkaline precipitation and coated with oleic acid, a lipophilic surfactant promoting the formation of stable colloids in organic media. In the next step, the dispersion of magnetite in chloroform was used to dissolve PLA and PTX, and the obtained organic phase was emulsified by sonication in an aqueous solution of albumin



Figure 2. Characterization of PTX-loaded MNP formulated for magnetically guided delivery to stented arteries. Near-spherical polymeric MNP formed with inclusion of nanocrystalline magnetite (transmission electron microscopy, A) exhibited a uniform size distribution (photon correlation spectroscopy, B) and strong saturation magnetization with negligible remanence (hysteresis loop measurements using an alternating gradient magnetometer, C). The PTX release kinetics from MNP was measured under sink conditions by an external sink method with UV-spectrophotometric detection (D). Reproduced from Chorny et al. (3). acting as a colloidal stabilizer. Albumin was chosen as a surfactant based on its biocompatibility and excellent surface-stabilizing properties (35). Solid MNPs were formed after the organic solvent elimination under reduced pressure with a narrow size distribution and an average size of 263 6 7 nm (Figs. 2A and 2B). Transmission electron microscopy showed near-spherical particles containing a large amount of magnetite in the form of nanocrystallites embedded in the polymeric matrix (Fig. 2A). This MNP structure was consistent with the strong magnetic responsiveness in the absence of retained magnetization exhibited by the formulation (Fig. 2C). An external sink method was adapted from Chorny et al. (36) for measuring the release kinetics of PTX incorporated in MNP. This method addresses the discrepancy between the apparent slow release exhibited by poorly water-soluble compounds in aqueous media and their actual release kinetics under sink conditions, which are reasonably satisfied when the drug concentration in the release medium is maintained below 10% of the drug solubility. Minute amounts of sparingly water-soluble compounds can effectively saturate traditionally employed release media, such as aqueous buffers, which rapidly establishes an equilibrium between the release and the reverse association of the drug with the carrier (37). Once an equilibrium is established, no increase in the amount of free drug in the acceptor phase can be determined, similar to a situation where a particle is surrounded by an unstirred layer preventing the diffusion of the released drug away from the carrier. However, in a biological environment with a constant turnover of amphiphilic molecules capable of binding and rapidly transporting hydrophobic compounds, the release typically occurs at near-infinite dilution and is practically irreversible (37, 38). The external sink in vitro release method uses an acceptor phase that is immiscible with water, a nonsolvent for the particle-forming polymer and a good solvent for the drug. A sufficient solubility of the drug in the release medium (a 1:1 mixture of heptane and

1-octanol) was verified prior to the release studies, and the free drug partitioning into the sink sampled at predetermined time points was quantified spectrophotometrically. The release of PTX followed a biphasic pattern with a rapid phase (about 60% of the drug released after 8 hr) followed by slower release observed over the next 40 hr (Fig. 2D). These results are consistent with diffusion through the polymeric matrix of the particles as the dominant release mechanism (37). PTX-loaded MNP efficiently inhibited growth of cultured rat aortic smooth muscle cells after a 5-min exposure to a high-gradient magnetic field (3) in contrast to a significantly lower inhibitory effect of free PTX or PTX-loaded MNP applied without a magnetic exposure. The antiproliferative effect of magnetically guided PTX-loaded MNP in cell culture correlated with an enhanced cell uptake of the carrier and a fluorescent PTX analog incorporated in MNP as a tracer. Local administration of MNP applied to stented rat carotid arteries in the presence of a uniform magnetizing field resulted in a fourfold higher initial amount of MNP associated with the stented region compared with nonmagnetic control conditions. As mentioned above, after the removal of the uniform field the stent and MNP lose their magnetic moments. In the absence of the magnetic interaction, targeted MNP redistributed from the stented arterial segment over time with complex kinetics. Notably, the amounts of MNP determined in the arteries of magnetically treated animals remained 5.5- to 9.5-fold higher than in the nonmagnetic control group up to 5 days post-treatment. Despite the redistribution of a sizable fraction of initially captured MNP, a significant reduction in the neointima to media ratio was revealed 14 days postsurgery in animals treated with PTX-loaded particles in the presence of the uniform field but not in control animals. Importantly, a therapeutic effect was achieved with a single dose of MNP-encapsulated PTX three orders of magnitude lower than the reported maximal tolerated dose of PTX in small animals (39). Further improvements in the antirestenotic efficacy may



be anticipated after optimization of the original strategy presented in this study. The formulation can hypothetically be redesigned to (a) prevent the escape of MNP from the target region after the removal of the magnetizing field and (b) achieve more protracted drug release kinetics. This can be accomplished, for example, by functionalizing the surface of MNP in order to impart affinity for the injured arterial tissue (40, 41) and through the use of slow-eluting covalent drug conjugates as described by Chan et al. (41). Site-specific drug administration using the uniform field-controlled magnetic targeting strategy can also be repeated to replenish the amount of the drug at the target site as discussed above, which is likely to improve the therapeutic efficacy of the MNP-mediated therapy.

Magnetically Targeted Delivery of Biotherapeutics: The Design of MNP as Carriers for Gene Vectors and Therapeutic Proteins Formulation of MNP for magnetically targeted delivery of biotherapeutics, such as gene vectors or therapeutic proteins, poses several unique challenges. In contrast to small-molecule pharmaceuticals, the integrity and functionality of biotherapeutics can be significantly compromised by conditions used by traditional particle formulation methods (42). High-shear homogenization and organic solvents used as part of the formulation process have been shown to irreversibly damage biological macromolecules through denaturation and/or aggregation (43– 45). Their physical and functional stability can also be affected by the acidic environment created inside the particle matrix because of the autocatalytic degradation of the polymer (46, 47). Thus, different formulation strategies where the exposure to these factors is either minimized or avoided have to be developed and implemented. The association of gene vectors with preformed magnetic microparticles and nanoparticles has been investigated by several groups as an alternative with a minimal effect on the vector activity (48–51). The formation of complexes between gene vectors and surface-modified biodegradable polymer-based MNP was recently explored by our group (52, 53). The ability to control the essential properties of the complex-forming MNP, including their size and magnetic responsiveness, by adjusting variables in the formulation process as shown in (52) is an important advantage of this approach. The association of plasmid DNA with PLA-based MNP was achieved using ion-pair complexation. MNPs were prepared by a modification of the emulsification-solvent diffusion method in the presence of in situ-formed surface-active polyethyleneimine oleate. The size of MNP was controlled by adjusting the volume ratio between the water-miscible and water-immiscible solvent in the organic phase (tetrahydrofuran and chloroform, respectively). The use of polyethyleneimine in its fatty acid salt form allowed for its stable association with MNP and subsequent efficient complexation of plasmid DNA with the particle surface (Fig. 3). MNP surface-associated DNA was shown to be protected from degradation by DNase I and

Figure 3. The design of MNP for magnetically guided nonviral gene delivery. The association of plasmid DNA with the surface of preformed PLA-based MNP modified with an ion-pair polyethyleneimine oleate complex prevents nuclease-mediated degradation and enables magnetically driven transfection under high serum conditions. Adapted from Chorny et al. (52).

nucleases in serum enabling magnetically driven transfection of vascular cells under high serum conditions. A more specific complexation approach using a MNP surfaceassociated affinity adaptor molecule, recombinant D1 domain of the Coxsackie adenovirus receptor (CAR), was applied for creating affinity complexes between MNP and replication-deficient adenovirus (Ad) (53). The utility of Ad for cardiovascular gene therapeutic applications is hampered by the low permissiveness of vascular cells to adenoviral transduction necessitating the use of high Ad titers (54), which in turn can cause serious systemic side effects as a result of the narrow therapeutic window of the vector. Adenoviral gene delivery using stent-targeted MNP-Ad complexes can potentially address this limitation by enabling a kinetically more favorable vector processing uncoupled from the CAR receptor, which is poorly expressed by vascular cells (55) in combination with magnetic guidance confining the vector to its target site (53). D1-functionalized MNP prepared using a modified emulsification-solvent evaporation approach exhibited multivalency with respect to Ad with several viral particles associated with the surface of an individual MNP (Fig. 4). Cellular uptake of MNP-Ad affinity complexes by cultured vascular cells and the resultant transduction efficiency was determined after cell treatment with or without a high-gradient



energy is required during the nonpolymeric MNP formation step. The incorporation of the cargo in the particle matrix rather than its surface complexation to preformed MNP makes it possible to derivatize the carrier with affinity ligands to achieve more efficient and specific targeting to the site of disease.

Figure 4. An affinity complex of Ad with preformed PLAbased MNP surface modified with the recombinant D1 domain of Coxsackie adenovirus receptor. The morphology of the MNP-Ad affinity complexes was examined by transmission electron microscopy. Reproduced from Chorny et al. (53).

magnetic field in comparison to free Ad or Ad applied with MNP coated with nonimmune IgG used as a control. The internalization of MNP by arterial smooth muscle and endothelial cells was significantly enhanced by an exposure to a high-gradient field compared with nonmagnetic conditions. However, an increase in the gene transfer efficiency in comparison to free vector was only observed with D1-functionalized MNP, suggesting that the formation of stable MNP-Ad complexes is critical for magnetically guided adenoviral gene transfer. Regression analysis of the reporter gene expression mediated by MNP-Ad affinity complexes formulated with Ad encoding green fluorescent protein in the two cell types as a function of the respective MNP and Ad formulation amounts confirmed that the gene transfer was essentially dependent on the interaction between MNP and Ad and was driven primarily by the magnetically responsive complexes. The feasibility of a different formulation strategy developed for encapsulation of biotherapeutics in MNP under mild conditions without the use of organic solvents was recently demonstrated with the antioxidant enzymes, catalase and superoxide dismutase, and the protective efficiency of MNP-encapsulated catalase was shown in cultured endothelial cells challenged with hydrogen peroxide (56). This formulation method, termed controlled aggregation/precipitation, is potentially applicable to both proteins and gene vectors and is based on the coentrapment of a macromolecule and nanocrystalline magnetite in the matrix of MNP formed by precipitation of calcium or zinc salts of oleic acid in the presence of a surface stabilizer, such as Poloxamer 407. Notably, the precipitation of oleate in the form of its waterinsoluble salt is a spontaneous process; therefore, no external

Stent-Targeted Cell Delivery Using MNP Endothelial denudation in the course of an angioplasty procedure increases the risk of thrombotic events and restenosis (57, 58). Rapid restoration of a continuous and functional endothelial layer can, thus, be essential for preventing thrombosis and inhibiting the progression of vessel renarrowing, which makes approaches aimed at facilitating the endothelium regeneration a potentially important therapeutic component of the obstructive vascular disease treatment (59, 60). In the context of stent angioplasty, direct seeding of endothelial cells on vascular stents as a means to promote reendothelialization has been investigated over the last 20 years (61–64). However, the feasibility of this approach has not been conclusively demonstrated in vivo, likely because of the low survival rates of cells exposed to the high pressure required for stent deployment. Cell delivery post-stenting, on the other hand, suffers from low rates of cell homing because of the inability to effectively retain the cells at the stented arterial segment in the presence of blood flow (10). The use of magnetic forces for site-specific delivery of cells is a relatively new concept that has recently been explored by several groups as a means for improving cell homing efficiency. Notably, the magnetic force in these animal studies was generated using either externally applied permanent magnets (13, 65, 66) or permanently magnetized nickel-coated stents (10). Targeted delivery of magnetically responsive cells to stented arteries in the presence of a uniform magnetizing field was investigated by our group as a potentially more clinically viable alternative (18). Fluorescentlabeled MNPs were prepared by emulsification-solvent evaporation and applied to endothelial cells in the presence of a highgradient magnetic field. Endothelial cell loading with MNP was accomplished without significantly compromising the cell viability, and MNP-loaded cells were shown to acquire a strong magnetic responsiveness with minimal retained magnetization in accordance with the magnetic properties of PLA-based MNP. After stent implantation, uniform field-controlled delivery of cells expressing the luciferase reporter protein was carried out in the rat carotid stenting model either in temporarily isolated arterial segments or under uninterrupted blood flow via the aortic arch, and the cell localization efficiency was compared to that in nonmagnetic control animals by bioluminescence imaging. A significantly higher cell-associated luminescent signal measured in the magnetic delivery animal group corresponded to a notably larger number of fluorescent MNP-impregnated cells detected on the stent surface after magnetic targeting. Although the MNP formulation used for the cell modification in this study was not designed to act as a therapeutic agent carrier,


the therapeutic efficacy of this magnetic cell targeting approach can potentially be enhanced by the high levels of transgene expression achievable with MNP formulated with gene vectors as mentioned above by enabling a combined gene/cell therapy. Further studies are required for evaluating the therapeutic potential of this MNP-based approach for stent-targeted delivery of genetically modified cells. In summary, site-specific delivery of therapeutic agents through the utilization of magnetic targeting strategies has considerable potential in vascular disease therapy. The successful translation of magnetically controlled vascular targeting into clinic is expected to be realized through the design of new, pharmaceutically acceptable and efficient magnetic carrier formulations, progress in developing conceptually novel guidance approaches potentially applicable to deep tissue targeting, and integration of affinity targeting strategies for achieving protracted retention of magnetic carriers at their site of action.

REFERENCES 1. Grief, A. D. and Richardson, G. (2005) Mathematical modelling of magnetically targeted drug delivery. J. Magn. Magn. Mater. 293, 455– 463. 2. Yellen, B. B., Forbes, Z. G., Halverson, D. S., Fridman, G., Barbee, K. A., Chorny, M., Levy, R., and Friedman, G. (2005) Targeted drug delivery to magnetic implants for therapeutic applications. J. Magn. Magn. Mater. 293, 647–654. 3. Chorny, M., Fishbein, I., Yellen, B. B., Alferiev, I. S., Bakay, M., Ganta, S., Adamo, R., Amiji, M., Friedman, G., and Levy, R. J. (2010) Targeting stents with local delivery of paclitaxel-loaded magnetic nanoparticles using uniform fields. Proc. Natl. Acad. Sci. USA 107, 8346–8351. 4. Lubbe, A. S., Alexiou, C., and Bergemann, C. (2001) Clinical applications of magnetic drug targeting. J. Surg. Res. 95, 200–206. 5. Alexiou, C., Schmid, R. J., Jurgons, R., Kremer, M., Wanner, G., Bergemann, C., Huenges, E., Nawroth, T., Arnold, W., and Parak, F. G. (2006) Targeting cancer cells: magnetic nanoparticles as drug carriers. Eur. Biophys. J. 35, 446–450. 6. Goodwin, S., Peterson, C., Hoh, C., and Bittner, C. (1999) Targeting and retention of magnetic targeted carriers (MTCs) enhancing intra-arterial chemotherapy. J. Magn. Magn. Mater. 194, 132–139. 7. Neuberger, T., Scho¨pf, B., Hofmann, H., Hofmann, M., and von Rechenberg, B. (2005) Superparamagnetic nanoparticles for biomedical applications: possibilities and limitations of a new drug delivery system. J. Magn. Magn. Mater. 293, 483–496. 8. Kubo, T., Sugita, T., Shimose, S., Nitta, Y., Ikuta, Y., and Murakami, T. (2001) Targeted systemic chemotherapy using magnetic liposomes with incorporated adriamycin for osteosarcoma in hamsters. Int. J. Oncol. 18, 121–125. 9. Pislaru, S. V., Harbuzariu, A., Agarwal, G., Witt, T., Gulati, R., Sandhu, N. P., Mueske, C., Kalra, M., Simari, R. D., and Sandhu, G. S. (2006) Magnetic forces enable rapid endothelialization of synthetic vascular grafts. Circulation 114, I314–I318. 10. Pislaru, S. V., Harbuzariu, A., Gulati, R., Witt, T., Sandhu, N. P., Simari, R. D., and Sandhu, G. S. (2006) Magnetically targeted endothelial cell localization in stented vessels. J. Am. Coll. Cardiol. 48, 1839– 1845. 11. Asmatulu, R., Zalich, M. A., Claus, R. O., and Riffle, J. S. (2005) Synthesis, characterization and targeting of biodegradable magnetic nanocomposite particles by external magnetic fields. J. Magn. Magn. Mater. 292, 108–119.


12. Mathieu, J. B. and Martel, S. (2007) Magnetic microparticle steering within the constraints of an MRI system: proof of concept of a novel targeting approach. Biomed. Microdevices 9, 801–808. 13. Kyrtatos, P. G., Lehtolainen, P., Junemann-Ramirez, M., Garcia-Prieto, A., Price, A. N., Martin, J. F., Gadian, D. G., Pankhurst, Q. A., and Lythgoe, M. F. (2009) Magnetic tagging increases delivery of circulating progenitors in vascular injury. JACC Cardiovasc. Interv. 2, 794– 802. 14. Mathieu, J. B. and Martel, S. (2010) Steering of aggregating magnetic microparticles using propulsion gradients coils in an MRI Scanner. Magn. Reson. Med. 63, 1336–1345. 15. Riegler, J., Wells, J. A., Kyrtatos, P. G., Price, A. N., Pankhurst, Q. A., and Lythgoe, M. F. (2010) Targeted magnetic delivery and tracking of cells using a magnetic resonance imaging system. Biomaterials 31, 5366–5371. 16. Amirfazli, A. (2007) Nanomedicine: magnetic nanoparticles hit the target. Nat. Nanotechnol. 2, 467–468. 17. Avile´s, M. O., Ebner, A. D., and Ritter, J. A. (2008) Implant assistedmagnetic drug targeting: comparison of in vitro experiments with theory. J. Magn. Magn. Mater. 320, 2704–2713. 18. Polyak, B., Fishbein, I., Chorny, M., Alferiev, I., Williams, D., Yellen, B., Friedman, G., and Levy, R. J. (2008) High field gradient targeting of magnetic nanoparticle-loaded endothelial cells to the surfaces of steel stents. Proc. Natl. Acad. Sci. USA 105, 698–703. 19. Kempe, H. and Kempe, M. (2010) The use of magnetite nanoparticles for implant-assisted magnetic drug targeting in thrombolytic therapy. Biomaterials 31, 9499–9510. 20. Kiemeneij, F., Patterson, M. S., Amoroso, G., Laarman, G., and Slagboom, T. (2008) Use of the Stereotaxis Niobe magnetic navigation system for percutaneous coronary intervention: results from 350 consecutive patients. Catheter. Cardiovasc. Interv. 71, 510–516. 21. Weissleder, R., Stark, D. D., Engelstad, B. L., Bacon, B. R., Compton, C. C., White, D. L., Jacobs, P., and Lewis, J. (1989) Superparamagnetic iron oxide: pharmacokinetics and toxicity. AJR Am. J. Roentgenol. 152, 167–173. 22. Hiramoto, J. S., Reilly, L. M., Schneider, D. B., Skorobogaty, H., Rapp, J., and Chuter, T. A. (2007) The effect of magnetic resonance imaging on stainless-steel Z-stent-based abdominal aortic prosthesis. J. Vasc. Surg. 45, 472–474. 23. Chorny, M., Cohen-Sacks, H., Fishbein, I., Danenberg, H. D., and Golomb, G. (2004) Biodegradable nanoparticles as drug delivery systems for parenteral administration. In Tissue Engineering and Novel Delivery Systems (Yaszemsky, M. J., Trantolo, D. J., Lewandrowski, K.-U., Hasirci, V., Altobelli, D. E., and Wise, D. L., eds.). pp. 393–422, Marcel Dekker, New York. 24. Gupta, A. K. and Gupta, M. (2005) Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications. Biomaterials 26, 3995–4021. 25. Giri, J., Pradhan, P., Somani, V., Chelawat, H., Chhatre, S., Banerjee, R., and Bahadur, D. (2008) Synthesis and characterizations of waterbased ferrofluids of substituted ferrites [Fe1-xBxFe2O4, B5Mn, Co (x50-1)] for biomedical applications. J. Magn. Magn. Mater. 320, 724– 730. 26. Hadjipanayis, C. G., Bonder, M. J., Balakrishnan, S., Wang, X., Mao, H., and Hadjipanayis, G. C. (2008) Metallic iron nanoparticles for MRI contrast enhancement and local hyperthermia. Small 4, 1925–1929. 27. Dobson, J. (2006) Magnetic nanoparticles for drug delivery. Drug Dev. Res. 67, 55–60. 28. Nakazawa, G., Ladich, E., Finn, A. V., and Virmani, R. (2008) Pathophysiology of vascular healing and stent mediated arterial injury. EuroIntervention 4(Suppl C), C7–C10. 29. Aminian, A., Kabir, T., and Eeckhout, E. (2009) Treatment of drug-eluting stent restenosis: an emerging challenge. Catheter. Cardiovasc. Interv. 74, 108–116.



30. Umashankar, P. R., Hari, P. R., and Sreenivasan, K. (2009) Effect of blood flow on drug release from DES: an experimental study. Int. J. Cardiol. 131, 415–417. 31. van Beusekom, H. M., Schoemaker, R., Roks, A. J., Zijlstra, F., and van der Giessen, W. J. (2007) Coronary stent healing, endothelialisation and the role of co-medication. Neth. Heart J. 15, 395–396. 32. Kamath, K. R., Barry, J. J., and Miller, K. M. (2006) The Taxus drugeluting stent: a new paradigm in controlled drug delivery. Adv. Drug Deliv. Rev. 58, 412–436. 33. Farb, A., Heller, P. F., Shroff, S., Cheng, L., Kolodgie, F. D., Carter, A. J., Scott, D. S., Froehlich, J., and Virmani, R. (2001) Pathological analysis of local delivery of paclitaxel via a polymer-coated stent. Circulation 104, 473–479. 34. Za´visova´, V., Koneracka´, M., Mu´ckova´, M., Kopcansky´, P., Tomasovicova´, N., Lancz, G., Timko, M., Pa¨toprsta´, B., Bartos, P., and Fabia´n, M. (2009) Synthesis and characterization of polymeric nanospheres loaded with the anticancer drug paclitaxel and magnetic particles. J. Magn. Magn. Mater. 321, 1613–1616. 35. Verrecchia, T., Spenlehauer, G., Bazile, D. V., Murry-Brelier, A., Archimbaud, Y., and Veillard, M. (1995) Non-stealth (poly(lactic acid/ albumin)) and stealth (poly(lactic acid-polyethylene glycol)) nanoparticles as injectable drug carriers. J. Control. Release 36, 49–61. 36. Chorny, M., Fishbein, I., Danenberg, H. D., and Golomb, G. (2002) Study of the drug release mechanism from tyrphostin AG-1295-loaded nanospheres by in situ and external sink methods. J. Control. Release 83, 401–414. 37. Washington, C. (1990) Drug release from microdisperse systems: a critical review. Int. J. Pharm. 58, 1–12. 38. Washington, C. (1989) Evaluation of non-sink dialysis methods for the measurement of drug release from colloids: effects of drug partition. Int. J. Pharm. 56, 71–74. 39. Vassileva, V., Grant, J., De Souza, R., Allen, C., and Piquette-Miller, M. (2007) Novel biocompatible intraperitoneal drug delivery system increases tolerability and therapeutic efficacy of paclitaxel in a human ovarian cancer xenograft model. Cancer Chemother. Pharmacol. 60, 907–914. 40. Song, C., Labhasetwar, V., Cui, X., Underwood, T., and Levy, R. J. (1998) Arterial uptake of biodegradable nanoparticles for intravascular local drug delivery: results with an acute dog model. J. Control. Release 54, 201–211. 41. Chan, J. M., Zhang, L., Tong, R., Ghosh, D., Gao, W., Liao, G., Yuet, K. P., Gray, D., Rhee, J. W., Cheng, J., Golomb, G., Libby, P., Langer, R., and Farokhzad, O. C. (2010) Spatiotemporal controlled delivery of nanoparticles to injured vasculature. Proc. Natl. Acad. Sci. USA 107, 2213–2218. 42. Maa, Y. F. and Hsu, C. C. (1997) Protein denaturation by combined effect of shear and air-liquid interface. Biotechnol. Bioeng. 54, 503–512. 43. Maa, Y. F. and Hsu, C. C. (1996) Effect of high shear on proteins. Biotechnol. Bioeng. 51, 458–465. 44. Zambaux, M. F., Bonneaux, F., Gref, R., Dellacherie, E., and Vigneron, C. (1999) Preparation and characterization of protein C-loaded PLA nanoparticles. J. Control. Release 60, 179–188. 45. Mok, H., Park, J. W., and Park, T. G. (2007) Microencapsulation of PEGylated adenovirus within PLGA microspheres for enhanced stability and gene transfection efficiency. Pharm. Res. 24, 2263–2269. 46. Schwendeman, S. P. (2002) Recent advances in the stabilization of proteins encapsulated in injectable PLGA delivery systems. Crit. Rev. Ther. Drug Carrier Syst. 19, 73–98. 47. Rexroad, J., Evans, R. K., and Middaugh, C. R. (2006) Effect of pH and ionic strength on the physical stability of adenovirus type 5. J. Pharm. Sci. 95, 237–247. 48. Mah, C., Fraites, T. J. Jr., Zolotukhin, I., Song, S., Flotte, T. R., Dobson, J., Batich, C., and Byrne, B. J. (2002) Improved method of recombinant AAV2 delivery for systemic targeted gene therapy. Mol. Ther. 6, 106–112.

49. Kadota, S., Kanayama, T., Miyajima, N., Takeuchi, K., and Nagata, K. (2005) Enhancing of measles virus infection by magnetofection. J. Virol. Methods 128, 61–66. 50. Morishita, N., Nakagami, H., Morishita, R., Takeda, S., Mishima, F., Terazono, B., Nishijima, S., Kaneda, Y., and Tanaka, N. (2005) Magnetic nanoparticles with surface modification enhanced gene delivery of HVJ-E vector. Biochem. Biophys. Res. Commun. 334, 1121–1126. 51. Tresilwised, N., Pithayanukul, P., Mykhaylyk, O., Holm, P. S., Holzmuller, R., Anton, M., Thalhammer, S., Adiguzel, D., Doblinger, M., and Plank, C. (2010) Boosting oncolytic adenovirus potency with magnetic nanoparticles and magnetic force. Mol. Pharm. 7, 1069–1089. 52. Chorny, M., Polyak, B., Alferiev, I. S., Walsh, K., Friedman, G., and Levy, R. J. (2007) Magnetically driven plasmid DNA delivery with biodegradable polymeric nanoparticles. FASEB J. 21, 2510–2519. 53. Chorny, M., Fishbein, I., Alferiev, I., and Levy, R. J. (2009) Magnetically responsive biodegradable nanoparticles enhance adenoviral gene transfer in cultured smooth muscle and endothelial cells. Mol. Pharm. 6, 1380–1387. 54. Baker, A. H. (2004) Designing gene delivery vectors for cardiovascular gene therapy. Prog. Biophys. Mol. Biol. 84, 279–299. 55. Chorny, M., Fishbein, I., Alferiev, I. S., Nyanguile, O., Gaster, R., and Levy, R. J. (2006) Adenoviral gene vector tethering to nanoparticle surfaces results in receptor-independent cell entry and increased transgene expression. Mol. Ther. 14, 382–391. 56. Chorny, M., Hood, E., Levy, R. J., and Muzykantov, V. R. (2010) Endothelial delivery of antioxidant enzymes loaded into non-polymeric magnetic nanoparticles. J. Control. Release 146, 144–151. 57. Parikh, S. A. and Edelman, E. R. (2000) Endothelial cell delivery for cardiovascular therapy. Adv. Drug Deliv. Rev. 42, 139–161. 58. Kipshidze, N., Dangas, G., Tsapenko, M., Moses, J., Leon, M. B., Kutryk, M., and Serruys, P. (2004) Role of the endothelium in modulating neointimal formation: vasculoprotective approaches to attenuate restenosis after percutaneous coronary interventions. J. Am. Coll. Cardiol. 44, 733–739. 59. Gulati, R. and Simari, R. D. (2004) Autologous cell-based therapies for vascular disease. Trends Cardiovasc. Med. 14, 262–267. 60. Gulati, R. and Simari, R. D. (2009) Defining the potential for cell therapy for vascular disease using animal models. Dis. Model. Mech. 2, 130–137. 61. Dichek, D. A., Neville, R. F., Zwiebel, J. A., Freeman, S. M., Leon, M. B., and Anderson, W. F. (1989) Seeding of intravascular stents with genetically engineered endothelial cells. Circulation 80, 1347–1353. 62. Koren, B., Weisz, A., Fischer, L., Gluzman, Z., Preis, M., Avramovitch, N., Cohen, T., Cosset, F. L., Lewis, B. S., and Flugelman, M. Y. (2006) Efficient transduction and seeding of human endothelial cells onto metallic stents using bicistronic pseudo-typed retroviral vectors encoding vascular endothelial growth factor. Cardiovasc. Revasc. Med. 7, 173–178. 63. Patel, H. J., Su, S. H., Patterson, C., and Nguyen, K. T. (2006) A combined strategy to reduce restenosis for vascular tissue engineering applications. Biotechnol. Prog. 22, 38–44. 64. Zhao, Q., Wei, M., and Zhao, B. (2010) Fabrication of stents with endothelial function in vitro. Scand. Cardiovasc. J. 44, 76–81. 65. Consigny, P. M., Silverberg, D. A., and Vitali, N. J. (1999) Use of endothelial cells containing superparamagnetic microspheres to improve endothelial cell delivery to arterial surfaces after angioplasty. J. Vasc. Interv. Radiol. 10, 155–163. 66. Hofmann, A., Wenzel, D., Becher, U. M., Freitag, D. F., Klein, A. M., Eberbeck, D., Schulte, M., Zimmermann, K., Bergemann, C., Gleich, B., Roell, W., Weyh, T., Trahms, L., Nickenig, G., Fleischmann, B. K., and Pfeifer, A. (2009) Combined targeting of lentiviral vectors and positioning of transduced cells by magnetic nanoparticles. Proc. Natl. Acad. Sci. USA 106, 44–49.

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