An Ultra-Thin Polymer Coating for the Tethering of Adenoviral Vector to the Surface of Coronary Stents

Our group has previously demonstrated stent-based gene delivery with either viral or plasmid vectors. However, these previous studies utilized bulky PLGA or collagen stent coatings, known to cause inflammatory reactions in stented arteries. In the present experiments we successfully attached adenoviruses either directly, or via anti-adenovirus antibodies to the steel surface of stents using chemical coordination with biphosphonates

Biphosphonate-Mediated Gene Vector Delivery from the Metal Surfaces of Stents

The clinical use of metallic expandable intravascular stents has resulted in imporved therapeutic outcomes for coronary artery disease. However, arterial reobstruction after stenting, in-stent restenosis, remains an important problem. Gene therapy to treat in-stent restenosis by using gene vector delivery from the metallic stent surfaces has never been demonstrated. The present studies investigated the hypothesis that metal-biphosphonate binding can enable site-specific gene vector delivery from metal surfaces. Polyallylamine biphosphonate (PAA-BP) was synthesized by using Michael addition methodology. Exposure to aqueous solutions of PAA-BP resulted in the formation of a monomolecular biphosphonate later on metal alloy surfaces (steel, nitinol, and cobalt-chromium), as demonstrated by x-ray photoelectron spectroscopy. Surface-bound PAA-BP enabled adenoviral (Ad) tethering due to covalent thiol-binding of either anti-Ad antibody or a recombinant Ad-receptor protein, D1. In arterial smooth muscle cell cultures, alloy samples configured with surface-tethered Ad were demonstrated to achieve site-specific transduction with a reporter gene, (GFP). Rat carotid stent angioplasties using metal stents exposed to aqueous PAA-BP and derivatized with anti-knob antibody or D1 resulted in extensive localized Ad-GFP expression in the arterial wall. In a separate study with a model therapeutic vector, Ad-inducible nitric oxide synthase (iNOS) attached to the biphosphonate-treated metal stent surface via D1, significant inhibition of restenosis was demonstrated (neointimal/media ration 1.68 ± 0.27 and 3.4 ± 0.35; Ad-iNOS vs. control, P \u3c 0.01). Is is concluded that effective gene vector delivery from metallic stent surfaces can be achieved using this approach

Magnetically Responsive Biodegradable Nanoparticles Enhance Adenoviral Gene Transfer in Cultured Smooth Muscle and Endothelial Cells

Nema sumnje da je terorizam jedan od trenutno najozbiljnijih problema savremenog društva. Veoma je važno da nacionalni pravni sistemi budu efikasni i dobro pripremljeni za izazove globalnog terorizma. Efikasnost, ali na pogrešan način, može dovesti do inkriminacija u krivičnom zakonodavstvu demokratskog društva koje ukazuju na autoritativne težnje. Zakonski okviri variraju od države do države što otežava opsežnu uporednu studiju, ali, ipak uočavamo razlike između zemalja koje prate međunarodne tendencije i onih koje smatraju da je njihovo postojeće represivno zakonodavstvo prilagođeno suzbijanju terorizma. Cilj ovog rada je da ukaže na to da su nacionalne jurisdikcije, na čelu s krivičnopravnim odredbama, ključna karika u suzbijanju terorizma. Ovom analizom ispitaćemo, takođe, trenutne zakonske strategije i trendove u borbi protiv terorizma i proveriti da li su tačne tvrdnje da nove zakonske odredbe u oblasti borbe protiv terorizma predstavljaju deo same logike terorizma i da negiraju pravnu državu.There is no doubt that terrorism is one of the most serious problems of contemporary society. It is very important for national legal systems to be efficient and well prepared to meet the global terrorism challenges. Legal frameworks vary widely from state to state, making a comprehensive comparative study difficult. However, there are some differences between those countries which follow international tendencies and those which believe that their existing repressive legislation is appropriate for fight against terrorism. The aim of this paper is to show that national jurisdictions, with their criminal law provisions, remain critical bodies on the first line of defence against terrorism. The paper deals with legal strategies and trends in fighting terrorism, and we will try to answer whether new legal provisions really follow the logic of terrorism and deny the rule of law

Stent-mediated gene delivery for site-specific transgene administration to the airway epithelium and management of tracheobronchial tumors.

BACKGROUND: Gene therapy is currently under investigation as a means of managing a variety of pulmonary diseases. Unfortunately, gene transfer to bronchial epithelium has been hampered by the lack of stable and efficient transduction. Recent studies have shown that gene vectors could be tethered to the metallic surfaces of intra-arterial stents. This approach enables efficacious and site-specific adenoviral gene delivery to the vascular endothelium. OBJECTIVES: We hypothesized that airway mesh stents impregnated with viral gene vectors could be used for local gene delivery to benign and malignant bronchial epithelium. METHODS: Serotype 5 adenoviral vectors (Ad5, E1-/E3-) containing the reporter genes green fluorescent protein (Ad.GFP) or β-galactoside/LacZ (Ad.LacZ), or a therapeutic gene, Ad.INF-β, were coupled to either metallic mesh disks or stents via anti-Ad knob antibodies. These platforms were assessed for their ability to transfect bronchial epithelial cells from both rats and humans, as well as murine (L1C2) and human (A549) lung cancer cell lines. Gene transfer was quantified by fluorescent microscopy, scanning fluorimetry for Ad.GFP, and light microscopy studies assessing β-galactosidase staining for Ad.LacZ. Metallic mesh and stent-mediated gene transfer was also performed in a murine flank tumor model and in a rat endotracheal tumor model in order to evaluate the therapeutic potential. RESULTS: In these studies, murine and human non-small cell lung cancer (NSCLC) cells were successfully transfected with reporter genes in vitro. Ad.LacZ-complexed mesh successfully transfected reporter genes into established murine flank NSCLC tumors. In addition, Ad.LacZ-tethered stents could effectively transfect both tracheobronchial epithelium and submucosal glands in rats. Similar epithelial transfection was achieved in ex vivo human bronchial epithelium. Pilot in vivo experimentation provided data supporting the concept that therapeutic genes could also be delivered with this technology. In additional pilot in vivo experiments, the growth of murine flank tumors was inhibited by placement of mesh disks coupled with Ad.muINF-β, and rats bearing endotracheal tumors demonstrated a trend towards prolonged survival with insertion of Ad.ratINF-β-tethered stents. CONCLUSIONS: Stent-mediated gene delivery successfully enabled site-specific vector administration to target rat and human airway cells in cell culture, organ culture and in vivo. Local tracheobronchial gene delivery via stents could provide a viable clinical solution for overcoming the difficulties encountered with vector delivery within the lungs, in particular by lowering requisite vector titers and by directing desired vectors to areas of interest. This strategy may prove valuable for treating tumors involving the tracheobronchial tree, as well as other nonmalignant tracheobronchial disorders

Local delivery of gene vectors from bare-metal stents by use of a biodegradable synthetic complex inhibits in-stent restenosis in rat carotid arteries

BACKGROUND: Local drug delivery from polymer-coated stents has demonstrated efficacy for preventing in-stent restenosis; however, both the inflammatory effects of polymer coatings and concerns about late outcomes of drug-eluting stent use indicate the need to investigate innovative approaches, such as combining localized gene therapy with stent angioplasty. Thus, we investigated the hypothesis that adenoviral vectors (Ad) could be delivered from the bare-metal surfaces of stents with a synthetic complex for reversible vector binding. METHODS AND RESULTS: We synthesized the 3 components of a gene vector binding complex: (1) A polyallylamine bisphosphonate with latent thiol groups (PABT), (2) a polyethyleneimine (PEI) with pyridyldithio groups for amplification of attachment sites [PEI(PDT)], and (3) a bifunctional (amine- and thiol-reactive) cross-linker with a labile ester bond (HL). HL-modified Ad attached to PABT/PEI(PDT)-treated steel surfaces demonstrated both sustained release in vitro over 30 days and localized green fluorescent protein expression in rat arterial smooth muscle cell cultures, which were not sensitive to either inhibition by neutralizing anti-Ad antibodies or inactivation after storage at 37 degrees C. In rat carotid studies, deployment of steel stents configured with PABT/PEI(PDT)/HL-tethered adenoviral vectors demonstrated both site-specific arterial Ad(GFP) expression and adenovirus-luciferase transgene activity per optical imaging. Rat carotid stent delivery of adenovirus encoding inducible nitric oxide synthase resulted in significant inhibition of restenosis. CONCLUSIONS: Reversible immobilization of adenovirus vectors on the bare-metal surfaces of endovascular stents via a synthetic complex represents an efficient, tunable method for sustained release of gene vectors to the vasculature

Nanocarrier Design for Dual-Targeted Therapy of In-Stent Restenosis

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