Improvement of vascular function by magnetic nanoparticle-assisted circumferential gene transfer into the native endothelium.

Gene therapy is a promising approach for chronic disorders that require continuous treatment such as cardiovascular disease. Overexpression of vasoprotective genes has generated encouraging results in animal models, but not in clinical trials. One major problem in humans is the delivery of sufficient amounts of genetic vectors to the endothelium which is impeded by blood flow, whereas prolonged stop-flow conditions impose the risk of ischemia. In the current study we have therefore developed a strategy for the efficient circumferential lentiviral gene transfer in the native endothelium under constant flow conditions. For that purpose we perfused vessels that were exposed to specially designed magnetic fields with complexes of lentivirus and magnetic nanoparticles thereby enabling overexpression of therapeutic genes such as endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF). This treatment enhanced NO and VEGF production in the transduced endothelium and resulted in a reduction of vascular tone and increased angiogenesis. Thus, the combination of MNPs with magnetic fields is an innovative strategy for site-specific and efficient vascular gene therapy.

Vascular Repair by Circumferential Cell Therapy Using Magnetic Nanoparticles and Tailored Magnets.

Cardiovascular disease is often caused by endothelial cell (EC) dysfunction and atherosclerotic plaque formation at predilection sites. Also surgical procedures of plaque removal cause irreversible damage to the EC layer, inducing impairment of vascular function and restenosis. In the current study we have examined a potentially curative approach by radially symmetric re-endothelialization of vessels after their mechanical denudation. For this purpose a combination of nanotechnology with gene and cell therapy was applied to site-specifically re-endothelialize and restore vascular function. We have used complexes of lentiviral vectors and magnetic nanoparticles (MNPs) to overexpress the vasoprotective gene endothelial nitric oxide synthase (eNOS) in ECs. The MNP-loaded and eNOS-overexpressing cells were magnetic, and by magnetic fields they could be positioned at the vascular wall in a radially symmetric fashion even under flow conditions. We demonstrate that the treated vessels displayed enhanced eNOS expression and activity. Moreover, isometric force measurements revealed that EC replacement with eNOS-overexpressing cells restored endothelial function after vascular injury in eNOS(-/-) mice ex and in vivo. Thus, the combination of MNP-based gene and cell therapy with custom-made magnetic fields enables circumferential re-endothelialization of vessels and improvement of vascular function.

Endothelial microparticle-mediated transfer of MicroRNA-126 promotes vascular endothelial cell repair via SPRED1 and is abrogated in glucose-damaged endothelial microparticles.

BACKGROUND: Repair of the endothelium after vascular injury is crucial for preserving endothelial integrity and preventing the development of vascular disease. The underlying mechanisms of endothelial cell repair are largely unknown. We sought to investigate whether endothelial microparticles (EMPs), released from apoptotic endothelial cells (ECs), influence EC repair. METHODS AND RESULTS: Systemic treatment of mice with EMPs after electric denudation of the endothelium accelerated reendothelialization in vivo. In vitro experiments revealed that EMP uptake in ECs promotes EC migration and proliferation, both critical steps in endothelial repair. To dissect the underlying mechanisms, Taqman microRNA array was performed, and microRNA (miR)-126 was identified as the predominantly expressed miR in EMPs. The following experiments demonstrated that miR-126 was transported into recipient human coronary artery endothelial cells by EMPs and functionally regulated the target protein sprouty-related, EVH1 domain-containing protein 1 (SPRED1). Knockdown of miR-126 in EMPs abrogated EMP-mediated effects on human coronary artery endothelial cell migration and proliferation in vitro and reendothelialization in vivo. Interestingly, after simulating diabetic conditions, EMPs derived from glucose-treated ECs contained significantly lower amounts of miR-126 and showed reduced endothelial repair capacity in vitro and in vivo. Finally, expression analysis of miR-126 in circulating microparticles from 176 patients with stable coronary artery disease with and without diabetes mellitus revealed a significantly reduced miR-126 expression in circulating microparticles from diabetic patients. CONCLUSIONS: Endothelial microparticles promote vascular endothelial repair by delivering functional miR-126 into recipient cells. In pathological hyperglycemic conditions, EMP-mediated miR-126-induced EC repair is altered.

Analysis of trajectories for targeting of magnetic nanoparticles in blood vessels.

The technique of magnetic drug targeting deals with binding drugs or genetic material to superparamagnetic nanoparticles and accumulating these complexes via an external magnetic field in a target region. For a successful approach, it is necessary to know the required magnetic setup as well as the physical properties of the complexes. With the help of computational methods, the complex accumulation and behavior can be predicted. We present a model for vascular targeting with a full three-dimensional analysis of the magnetic and fluidic forces and a subsequent evaluation of the resulting trajectories of the complexes. These trajectories were calculated with respect to the physiological boundary conditions, the magnetic properties of both the external field and the particles as well as the hydrodynamics of the fluid. We paid special regard to modeling input parameters like flow velocity as well as the distribution functions of the hydrodynamic size and magnetic moment of the nanoparticle complexes. We are able to estimate the amount of complexes, as well as the spatial distribution of those complexes. Additionally, we examine the development of the trapping rate for multiple passages of the complexes and compare the influence of several input parameters. Finally, we provide experimental data of an ex vivo flow-loop system which serves as a model for large vessel targeting. In this model, we achieve a deposition of lentivirus/magnetic nanoparticle complexes in a murine aorta and compare our simulation with the experimental results gained by a non-heme-iron assay.

Identification of magnetic nanoparticles for combined positioning and lentiviral transduction of endothelial cells.

PURPOSE: The combination of magnetic nanoparticles (MNPs) with a magnetic field is a powerful approach to enable cell positioning and/or local gene therapy. Because physical requirements for MNPs differ between these two applications we have explored whether the use of different MNPs can provide site-specific positioning combined with efficient viral transduction of endothelial cells (ECs). METHODS: A variety of MNPs was screened for magnetic cell labeling and lentivirus binding. Then two different MNPs were chosen and their combined application was evaluated regarding EC magnetization and transduction efficiency. RESULTS: The combined use of PEI-Mag2 and NDT-Mag1 particles provided both efficient lentiviral transduction and high magnetic responsiveness of ECs that could be even retained within the vascular wall under flow conditions. The use of these MNPs did not affect biological characteristics of ECs like surface marker expression and vascular network formation. Importantly, with this method we could achieve an efficient functional overexpression of endothelial nitric oxide synthase in ECs. CONCLUSIONS: The application of two different MNPs provides optimal results for magnetic labeling of ECs in combination with viral transduction. This novel approach could be very useful for targeted gene therapy ex vivo and site-specific cell replacement in the vascular system.

Optimization of magnetic nanoparticle-assisted lentiviral gene transfer.

PURPOSE: Targeting of specific cells and tissues is of great interest for clinical relevant gene- and cell-based therapies. We use magnetic nanoparticles (MNPs) with a ferrimagnetic core (Fe(3)O(4)) with different coatings to optimize MNP-assisted lentiviral gene transfer with focus on different endothelial cell lines. METHODS: Lentiviral vector (LV)/MNP binding was characterized for various MNPs by different methods (e.g. magnetic responsiveness measurement). Transduced cells were analyzed by flow cytometry, fluorescence microscopy and iron recovery. Cell transduction and cell positioning under physiological flow conditions were performed using different in vitro and ex vivo systems. RESULTS: Analysis of diverse MNPs with different coatings resulted in identification of nanoparticles with improved LV association and enhanced transduction properties of complexes in several endothelial cell lines. The magnetic moments of LV/MNP complexes are high enough to achieve local gene targeting of perfused endothelial cells. Perfusion of a mouse aorta with LV/MNP transduced cells under clinically relevant flow conditions led to local cell attachment at the intima of the vessel. CONCLUSION: MNP-guided lentiviral transduction of endothelial cells can be significantly enhanced and localized by using optimized MNPs.