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A cell delivery strategy was investigated that was hypothesized to enable magnetic targeting of endothelial cells to the steel surfaces of intraarterial stents because of the following mechanisms: (i) preloading cells with biodegradable polymeric superparamagnetic nanoparticles (MNPs), thereby rendering the cells magnetically responsive; and (ii) the induction of both magnetic field gradients around the wires of a steel stent and magnetic moments within MNPs because of a uniform external magnetic field, thereby targeting MNP-laden cells to the stent wires. In vitro studies demonstrated that MNP-loaded bovine aortic endothelial cells (BAECs) could be magnetically targeted to steel stent wires. In vivo MNP-loaded BAECs transduced with adenoviruses expressing luciferase (Luc) were targeted to stents deployed in rat carotid arteries in the presence of a uniform magnetic field with significantly greater Luc expression, detected by in vivo optical imaging, than nonmagnetic controls.

Bioprosthetic heart valves (BHV) fabricated from glutaraldehyde-fixed heterograft tissue, such as bovine pericardium (BP), are widely used for treating heart valve disease, a group of disorders that affects millions. Structural valve degeneration (SVD) of BHV due to both calcification and the accumulation of advanced glycation end products (AGE) with associated serum proteins limits durability. We hypothesized that BP modified with poly-2-methyl-2-oxazoline (POZ) to inhibit protein entry would demonstrate reduced accumulation of AGE and serum proteins, mitigating SVD. In vitro studies of POZ-modified BP demonstrated reduced accumulation of serum albumin and AGE. BP-POZ in vitro maintained collagen microarchitecture per two-photon microscopy despite AGE incubation, and in cell culture studies was associated with no change in tumor necrosis factor-α after exposure to AGE and activated macrophages. Comparing POZ and polyethylene glycol (PEG)–modified BP in vitro, BP-POZ was minimally affected by oxidative conditions, whereas BP-PEG was susceptible to oxidative deterioration. In juvenile rat subdermal implants, BP-POZ demonstrated reduced AGE formation and serum albumin infiltration, while calcification was not inhibited. However, BP-POZ rat subdermal implants with ethanol pretreatment demonstrated inhibition of both AGE accumulation and calcification. Ex vivo laminar flow studies with human blood demonstrated BP-POZ enhanced thromboresistance with reduced white blood cell accumulation. We conclude that SVD associated with AGE and serum protein accumulation can be mitigated through POZ functionalization that both enhances biocompatibility and facilitates ethanol pretreatment inhibition of BP calcification.

The use of stents for vascular disease has resulted in a paradigm shift with significant improvement in therapeutic outcomes. Polymer-coated drug-eluting stents (DES) have also significantly reduced the incidence of reobstruction post stenting, a disorder termed in-stent restenosis. However, the current DESs lack the capacity for adjustment of the drug dose and release kinetics to the disease status of the treated vessel. We hypothesized that these limitations can be addressed by a strategy combining magnetic targeting via a uniform field-induced magnetization effect and a biocompatible magnetic nanoparticle (MNP) formulation designed for efficient entrapment and delivery of paclitaxel (PTX). Magnetic treatment of cultured arterial smooth muscle cells with PTX-loaded MNPs caused significant cell growth inhibition, which was not observed under nonmagnetic conditions. In agreement with the results of mathematical modeling, significantly higher localization rates of locally delivered MNPs to stented arteries were achieved with uniform-field-controlled targeting compared to nonmagnetic controls in the rat carotid stenting model. The arterial tissue levels of stent-targeted MNPs remained 4- to 10-fold higher in magnetically treated animals vs. control over 5 days post delivery. The enhanced retention of MNPs at target sites due to the uniform field-induced magnetization effect resulted in a significant inhibition of in-stent restenosis with a relatively low dose of MNP-encapsulated PTX (7.5 μg PTX/stent). Thus, this study demonstrates the feasibility of site-specific drug delivery to implanted magnetizable stents by uniform field-controlled targeting of MNPs with efficacy for in-stent restenosis.

Impaired endothelialization of endovascular stents has been established as a major cause of in-stent restenosis and late stent thrombosis. Attempts to enhance endothelialization of inner stent surfaces by pre-seeding the stents with endothelial cells in vitro prior to implantation are compromised by cell destruction during high-pressure stent deployment. Herein, we report on the novel stent endothelialization strategy of post-deployment seeding of biotin-modified endothelial cells to avidin-functionalized stents. Acquisition of an avidin monolayer on the stent surface was achieved by consecutive treatments of bare metal stents (BMS) with polyallylamine bisphosphonate, an amine-reactive biotinylation reagent and avidin. Biotin-modified endothelial cells retain growth characteristics of normal endothelium and can express reporter transgenes. Under physiological shear conditions, a 50-fold higher number of recirculating biotinylated cells attached to the avidin-modified metal surfaces compared to bare metal counterparts. Delivery of biotinylated endothelial cells to the carotid arterial segment containing the implanted avidin-modified stent in rats results in immediate cell binding to the stent struts and is associated with a 30% reduction of in-stent restenosis in comparison with BMS.

In-stent restenosis (ISR) complicates revascularization in the coronary and peripheral arteries. Apolipoprotein A1 (apoA1), the principal protein component of HDL possesses inherent anti-atherosclerotic and anti-restenotic properties. These beneficial traits are lost when wild type apoA1(WT) is subjected to oxidative modifications. We investigated whether local delivery of adeno-associated viral (AAV) vectors expressing oxidation-resistant apoA1(4WF) preserves apoA1 functionality. The efflux of 3 H-cholesterol from macrophages to the media conditioned by endogenously produced apoA1(4WF) was 2.1-fold higher than for apoA1(WT) conditioned media in the presence of hypochlorous acid emulating conditions of oxidative stress. The proliferation of apoA1(WT)- and apoA1(4FW)-transduced rat aortic smooth muscle cells (SMC) was inhibited by 66% ± 10% and 65% ± 11%, respectively, in comparison with non-transduced SMC (p < 0.001). Conversely, the proliferation of apoA1(4FW)-transduced, but not apoA1(WT)-transduced rat blood outgrowth endothelial cells (BOEC) was increased 41% ± 5% (p < 0.001). Both apoA1 transduction conditions similarly inhibited basal and TNFα-induced reactive oxygen species in rat aortic endothelial cells (RAEC) and resulted in the reduced rat monocyte attachment to the TNFα-activated endothelium. AAV2-eGFP vectors immobilized reversibly on stainless steel mesh surfaces through the protein G/anti-AAV2 antibody coupling, efficiently transduced cells in culture modeling stent-based delivery. In vivo studies in normal pigs, deploying AAV2 gene delivery stents (GDS) preloaded with AAV2-eGFP in the coronary arteries demonstrated transduction of the stented arteries. However, implantation of GDS formulated with AAV2-apoA1(4WF) failed to prevent in-stent restenosis in the atherosclerotic vasculature of hypercholesterolemic diabetic pigs. It is concluded that stent delivery of AAV2-4WF while feasible, is not effective for mitigation of restenosis in the presence of severe atherosclerotic disease.

Human vascular malformations cause disease as a result of changes in blood flow and vascular hemodynamic forces. Although the genetic mutations that underlie the formation of many human vascular malformations are known, the extent to which abnormal blood flow can subsequently influence the vascular genetic program and natural history is not. Loss of the SH2 domain-containing leukocyte protein of 76 kDa (SLP76) resulted in a vascular malformation that directed blood flow through mesenteric lymphatic vessels after birth in mice. Mesenteric vessels in the position of the congenital lymphatic in mature Slp76-null mice lacked lymphatic identity and expressed a marker of blood vessel identity. Genetic lineage tracing demonstrated that this change in vessel identity was the result of lymphatic endothelial cell reprogramming rather than replacement by blood endothelial cells. Exposure of lymphatic vessels to blood in the absence of significant flow did not alter vessel identity in vivo, but lymphatic endothelial cells exposed to similar levels of shear stress ex vivo rapidly lost expression of PROX1, a lymphatic fate-specifying transcription factor. These findings reveal that blood flow can convert lymphatic vessels to blood vessels, demonstrating that hemodynamic forces may reprogram endothelial and vessel identity in cardiovascular diseases associated with abnormal flow.

The injury-triggered reocclusion (restenosis) of arteries treated with angioplasty to relieve atherosclerotic obstruction remains a challenge due to limitations of existing therapies. A combination of magnetic guidance and affinity-mediated arterial binding can pave the way to a new approach for treating restenosis by enabling efficient site-specific localization of therapeutic agents formulated in magnetizable nanoparticles (MNPs) and by maintaining their presence at the site of arterial injury throughout the vulnerability period of the disease. In these studies, we investigated a dual-targeted antirestenotic strategy using drug-loaded biodegradable MNPs, surface-modified with a fibrin-avid peptide to provide affinity for the injured arterial wall. The MNPs were characterized with regard to their magnetic properties, efficiency of surface functionalization, disassembly kinetics, and interaction with fibrin-coated substrates. The antiproliferative effects of MNPs formulated with paclitaxel were studied in vitro using a fetal cell line (A10) exhibiting the defining characteristics of neointimal smooth muscle cells. Animal studies examined the efficiency of combined (physical/affinity) MNP targeting to stented arteries in Sprague Dawley rats using fluorimetric analysis and fluorescent in vivo imaging. The antirestenotic effect of the dual-targeted therapy was determined in a rat model of in-stent restenosis 28 days post-treatment. The results showed that MNPs can be efficiently functionalized to exhibit a strong binding affinity using a simple two-step chemical process, without adversely affecting their size distribution, magnetic properties, or antiproliferative potency. Dual-targeted delivery strongly enhanced the localization and retention of MNPs in stented carotid arteries up to 7 days post-treatment, while minimizing redistribution of the carrier particles to peripheral tissues. Of the two targeting elements, the effect of magnetic guidance was shown to dominate arterial localization (p = 0.004 vs. 0.084 for magnetic targeting and peptide modification, respectively), consistent with the magnetically driven MNP accumulation step defining the extent of the ultimate affinity-mediated arterial binding and subsequent retention of the carrier particles. The enhanced arterial uptake and sustained presence of paclitaxel-loaded MNPs at the site of stent deployment were associated with a strong inhibition of restenosis in the rat carotid stenting model, with both the neointima-to-media ratio (N/M) and % stenosis markedly reduced in the dual-targeted treatment group (1.62 ± 0.2 and 21 ± 3 vs. 2.17 ± 0.40 and 29 ± 6 in the control animals; p < 0.05). We conclude that the dual-targeted delivery of antirestenotic agents formulated in fibrin-avid MNPs can provide a new platform for the safe and effective treatment of in-stent restenosis.

The fate of nanoparticle (NP) formulations in the multifaceted biological environment is a key determinant of their biocompatibility and therapeutic performance. An understanding of the degradation patterns of different types of clinically used and experimental NP formulations is currently incomplete, posing an unmet need for novel analytical tools providing unbiased quantitative measurements of NP disassembly directly in the medium of interest and in conditions relevant to specific therapeutic/diagnostic applications. In the present study, this challenge was addressed with an approach enabling real-time in situ monitoring of the integrity status of NPs in cells and biomimetic media using Förster resonance energy transfer (FRET). Disassembly of polylactide-based magnetic NPs (MNPs) was investigated in a range of model biomimetic media and in cultured vascular cells using an experimentally established quantitative correlation between particle integrity and FRET efficiency controlled through adjustments in the spectral overlap between two custom-synthesized polylactide—fluorophore (boron dipyrromethene) conjugates incorporated in MNPs. The results suggest particle disassembly governed by diffusion—reaction processes with kinetics strongly dependent on conditions promoting release of oligomeric fragments from the particle matrix. Thus, incubation in gels simulating the extracellular environment and in protein-rich serum resulted in notably lower and higher MNP decomposition rates, respectively, compared with nonviscous liquid buffers. The diffusion—reaction mechanism also is consistent with a significant cell growth-dependent acceleration of MNP processing in dividing vs. contact-inhibited vascular cells. The FRET-based analytical strategy and experimental results reported herein may facilitate the development and inform optimization of biodegradable nanocarriers for cell and drug delivery applications.

To study the uptake of biodegradable microparticles in Caco-2 cells. Biodegradable microparticles of polylactic polyglycolic acid co-polymer (PLGA 50:50) of mean diameters 0.1 micron, 1 micron, and 10 microns containing bovine serum albumin as a model protein and 6-coumarin as a fluorescent marker were formulated by a multiple emulsion technique. The Caco-2 cell monolayers were incubated with each diameter microparticles (100 micrograms/ml) for two hours. The microparticle uptake in Caco-2 cells was studied by confocal microscopy and also by quantitating the 6-coumarin content of the microparticles taken up by the cells. The effects of microparticle concentration, and incubation time and temperature on microparticle cell uptake were also studied. The study demonstrated that the Caco-2 cell microparticle uptake significantly depends upon the microparticle diameter. The 0.1 micron diameter microparticles had 2.5 fold greater uptake on the weight basis than the 1 micron and 6 fold greater than the 10 microns diameter microparticles. Similarly in terms of number the uptake of 0.1 micron diameter microparticles was 2.7 x 10(3) fold greater than the 1 micron and 6.7 x 10(6) greater than the 10 microns diameter microparticles. The efficiency of uptake of 0.1 micron diameter microparticles at 100 micrograms/ml concentration was 41% compared to 15% and 6% for the 1 micron and the 10 microns diameter microparticles, respectively. The Caco-2 cell microparticle (0.1 micron) uptake increased with concentration in the range of 100 micrograms/ml to 500 micrograms/ml which then reached a plateau at higher concentration. The uptake of microparticles increased with incubation time, reaching a steady state at two hours. The uptake was greater at an incubation temperature of 37 degrees C compared to at 4 degrees C. The Caco-2 cell microparticle uptake was microparticle diameter, concentration, and incubation time and temperature dependent. The small diameter microparticles (0.1 micron) had significantly greater uptake compared to larger diameter microparticles. The results thus suggest that the mechanism of uptake of microparticles in Caco-2 cell is particle diameter dependent. Caco-2 cells are used as an in vitro model for gastrointestinal uptake, and therefore the results obtained in these studies could be of significant importance in optimizing the microparticle-based oral drug delivery systems.

The need for right ventricular outflow tract reconstruction is common and growing in congenital heart surgery given expanding indications for the repair of congenital as well as acquired heart disease. Various valved conduit options currently exist including homografts, xenograft pulmonary valved conduits (Contegra™), and porcine valved conduits. The major limitation for all conduits is implant durability, which requires reoperation. Currently, cryopreserved homografts are often used given their superiority shown in long-term data. Significant limitations remain in the cost and availability of the graft, particularly for smaller sizes. Contegra conduits are available in a variety of sizes. Nonetheless, the data regarding long-term durability are less robust and studies comparing durability with homografts have been conflicting. Additionally, there is concern for increased rates of late endocarditis in this conduit. Porcine valved conduits offer a reliable option but are limited by structural valve degeneration associated with all types of bioprosthetic heart valve replacements. New developments in the field of tissue engineering have produced promising bio-restorative valved conduits that may overcome many of the limitations of previous conduit technologies. These remain in the early stages of clinical testing. This review summarizes the clinical data surrounding the conduits used most commonly in clinical practice today and explores emerging technologies that may bring us closer to developing the ideal conduit.