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
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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
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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
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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
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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
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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,
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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.
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