Richard F. Adamo

Targeting stents with local delivery of paclitaxel-loaded magnetic nanoparticles using uniform fields

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.

Epicardially derived fibroblasts preferentially contribute to the parietal leaflets of the atrioventricular valves in the murine heart.

The importance of the epicardium for myocardial and valvuloseptal development has been well established; perturbation of epicardial development results in cardiac abnormalities, including thinning of the ventricular myocardial wall and malformations of the atrioventricular valvuloseptal complex. To determine the spatiotemporal contribution of epicardially derived cells to the developing fibroblast population in the heart, we have used a mWt1/IRES/GFP-Cre mouse to trace the fate of EPDCs from embryonic day (ED)10 until birth. EPDCs begin to populate the compact ventricular myocardium around ED12. The migration of epicardially derived fibroblasts toward the interface between compact and trabecular myocardium is completed around ED14. Remarkably, epicardially derived fibroblasts do not migrate into the trabecular myocardium until after ED17. Migration of EPDCs into the atrioventricular cushion mesenchyme commences around ED12. As development progresses, the number of EPDCs increases significantly, specifically in the leaflets which derive from the lateral atrioventricular cushions. In these developing leaflets the epicardially derived fibroblasts eventually largely replace the endocardially derived cells. Importantly, the contribution of EPDCs to the leaflets derived from the major AV cushions is very limited. The differential contribution of EPDCs to the various leaflets of the atrioventricular valves provides a new paradigm in valve development and could lead to new insights into the pathogenesis of abnormalities that preferentially affect individual components of this region of the heart. The notion that there is a significant difference in the contribution of epicardially and endocardially derived cells to the individual leaflets of the atrioventricular valves has also important pragmatic consequences for the use of endocardial and epicardial cre-mouse models in studies of heart development.

Site-specific gene delivery to stented arteries using magnetically guided zinc oleate-based nanoparticles loaded with adenoviral vectors

Gene therapeutic strategies have shown promise in treating vascular disease. However, their translation into clinical use requires pharmaceutical carriers enabling effective, site‐specific delivery as well as providing sustained transgene expression in blood vessels. While replication‐deficient adenovirus (Ad) offers several important advantages as a vector for vascular gene therapy, its clinical applicability is limited by rapid inactivation, suboptimal transduction efficiency in vascular cells, and serious systemic adverse effects. We hypothesized that novel zinc oleate‐based magnetic nanoparticles (MNPs) loaded with Ad would enable effective arterial cell transduction by shifting vector processing to an alternative pathway, protect Ad from inactivation by neutralizing factors, and allow site‐specific gene transfer to arteries treated with stent angioplasty using a 2‐source magnetic guidance strategy. Ad‐loaded MNPs effectively transduced cultured endothelial and smooth muscle cells under magnetic conditions compared to controls and retained capacity for gene transfer after exposure to neutralizing antibodies and lithium iodide, a lytic agent causing disruption of free Ad. Localized arterial gene expression significantly stronger than in control animal groups was demonstrated after magnetically guided MNP delivery in a rat stenting model 2 and 9 d post‐treatment, confirming feasibility of using Ad‐loaded MNPs to achieve site‐specific transduction in stented blood vessels. In conclusion, Ad‐loaded MNPs formed by controlled precipitation of zinc oleate represent a novel delivery system, well‐suited for efficient, magnetically targeted vascular gene transfer.—Chorny, M., Fishbein, I., Tengood, J. E., Adamo, R. F., Alferiev, I. S., Levy, R. J. Site‐specific gene delivery to stented arteries using magnetically guided zinc oleate‐based nanoparticles loaded with adenoviral vectors. FASEB J. 27, 2198–2206 (2013). www.fasebj.org

Alteration of Differentiation Potentials by Modulating GATA Transcription Factors in Murine Embryonic Stem Cells

Background. Mouse embryonic stem (ES) cells can be differentiated in vitro by aggregation and/or retinoic acid (RA) treatment. The principal differentiation lineage in vitro is extraembryonic primitive endoderm. Dab2, Laminin, GATA4, GATA5, and GATA6 are expressed in embryonic primitive endoderm and play critical roles in its lineage commitment. Results. We found that in the absence of GATA4 or GATA5, RA-induced primitive endoderm differentiation of ES cells was reduced. GATA4 (−/−) ES cells express higher level of GATA5, GATA6, and hepatocyte nuclear factor 4 alpha marker of visceral endoderm lineage. GATA5 (−/−) ES cells express higher level of alpha fetoprotein marker of early liver development. GATA6 (−/−) ES cells express higher level of GATA5 as well as mesoderm and cardiomyocyte markers which are collagen III alpha-1 and tropomyosin1 alpha. Thus, deletion of GATA6 precluded endoderm differentiation but promoted mesoderm lineages. Conclusions. GATA4, GATA5, and GATA6 each convey a unique gene expression pattern and influences ES cell differentiation. We showed that ES cells can be directed to avoid differentiating into primitive endoderm and to adopt unique lineages in vitro by modulating GATA factors. The finding offers a potential approach to produce desirable cell types from ES cells, useful for regenerative cell therapy.

Modulation of NO and ROS production by AdiNOS transduced vascular cells through supplementation with L-Arg and BH4: Implications for gene therapy of restenosis

OBJECTIVE Gene therapy with viral vectors encoding for NOS enzymes has been recognized as a potential therapeutic approach for the prevention of restenosis. Optimal activity of iNOS is dependent on the intracellular availability of L-Arg and BH4 via prevention of NOS decoupling and subsequent ROS formation. Herein, we investigated the effects of separate and combined L-Arg and BH4 supplementation on the production of NO and ROS in cultured rat arterial smooth muscle and endothelial cells transduced with AdiNOS, and their impact on the antirestenotic effectiveness of AdiNOS delivery to balloon-injured rat carotid arteries. METHODS AND RESULTS Supplementation of AdiNOS transduced endothelial and vascular smooth muscle cells with L-Arg (3.0 mM), BH4 (10 μM) and especially their combination resulted in a significant increase in NO production as measured by nitrite formation in media. Formation of ROS was dose-dependently increased following transduction with increasing MOIs of AdiNOS. Exposure of RASMC to AdiNOS tethered to meshes via a hydrolyzable cross-linker, modeling viral delivery from stents, resulted in increased ROS production, which was decreased by supplementation with BH4 but not L-Arg or L-Arg/BH4. Enhanced cell death, caused by AdiNOS transduction, was also preventable with BH4 supplementation. In the rat carotid model of balloon injury, intraluminal delivery of AdiNOS in BH4-, L-Arg-, and especially in BH4 and L-Arg supplemented animals was found to significantly enhance the antirestenotic effects of AdiNOS-mediated gene therapy. CONCLUSIONS Fine-tuning of iNOS function by L-Arg and BH4 supplementation in the transduced vasculature augments the therapeutic potential of gene therapy with iNOS for the prevention of restenosis.

Magnetically enhanced cell delivery for accelerating recovery of the endothelium in injured arteries.

Arterial injury and disruption of the endothelial layer are an inevitable consequence of interventional procedures used for treating obstructive vascular disease. The slow and often incomplete endothelium regrowth after injury is the primary cause of serious short- and long-term complications, including thrombosis, restenosis and neoatherosclerosis. Rapid endothelium restoration has the potential to prevent these sequelae, providing a rationale for developing strategies aimed at accelerating the reendothelialization process. The present studies focused on magnetically guided delivery of endothelial cells (EC) functionalized with biodegradable magnetic nanoparticles (MNP) as an experimental approach for achieving rapid and stable cell homing and expansion in stented arteries. EC laden with polylactide-based MNP exhibited strong magnetic responsiveness, capacity for cryopreservation and rapid expansion, and the ability to disintegrate internalized MNP in both proliferating and contact-inhibited states. Intracellular decomposition of BODIPY558/568-labeled MNP monitored non-invasively based on assembly state-dependent changes in the emission spectrum demonstrated cell proliferation rate-dependent kinetics (average disassembly rates: 6.6±0.8% and 3.6±0.4% per day in dividing and contact-inhibited EC, respectively). With magnetic guidance using a transient exposure to a uniform 1-kOe field, stable localization and subsequent propagation of MNP-functionalized EC, markedly enhanced in comparison to non-magnetic delivery conditions, were observed in stented rat carotid arteries. In conclusion, magnetically guided delivery is a promising experimental strategy for accelerating endothelial cell repopulation of stented blood vessels after angioplasty.

Nanoparticle-mediated delivery of a rapidly activatable prodrug of SN-38 for neuroblastoma therapy

Nanomedicine-based strategies have the potential to improve therapeutic performance of a wide range of anticancer agents. However, the successful implementation of nanoparticulate delivery systems requires the development of adequately sized nanocarriers delivering their therapeutic cargo to the target in a protected, pharmacologically active form. The present studies focused on a novel nanocarrier-based formulation strategy for SN-38, a topoisomerase I inhibitor with proven anticancer potential, whose clinical application is compromised by toxicity, poor stability and incompatibility with conventional delivery vehicles. SN-38 encapsulated in biodegradable sub-100 nm sized nanoparticles (NP) in the form of its rapidly activatable prodrug derivative with tocopherol succinate potently inhibited the growth of neuroblastoma cells in a dose- and exposure time-dependent manner, exhibiting a delayed response pattern distinct from that of free SN-38. In a xenograft model of neuroblastoma, prodrug-loaded NP caused rapid regression of established large tumors, significantly delayed tumor regrowth after treatment cessation and markedly extended animal survival. The NP formulation strategy enabled by a reversible chemical modification of the drug molecule offers a viable means for SN-38 delivery achieving sustained intratumoral drug levels and contributing to the potency and extended duration of antitumor activity, both prerequisites for effective treatment of neuroblastoma and other cancers.

Adenoviral vector tethering to metal surfaces via hydrolyzable cross-linkers for the modulation of vector release and transduction.

The use of arterial stents and other medical implants as a delivery platform for surface immobilized gene vectors allows for safe and efficient localized expression of therapeutic transgenes. In this study we investigate the use of hydrolyzable cross-linkers with distinct kinetics of hydrolysis for delivery of gene vectors from polyallylamine bisphosphonate-modified metal surfaces. Three cross-linkers with the estimated t1/2 of ester bonds hydrolysis of 5, 12 and 50 days demonstrated a cumulative 20%, 39% and 45% vector release, respectively, after 30 days exposure to physiological buffer at 37 °C. Transgene expression in endothelial and smooth muscles cells transduced with substrate immobilized adenovirus resulted in significantly different expression profiles for each individual cross-linker. Furthermore, immobilization of adenoviral vectors effectively extended their transduction effectiveness beyond the initial phase of release. Transgene expression driven by adenovirus-tethered stents in rat carotid arteries demonstrated that a faster rate of cross-linker hydrolysis resulted in higher expression levels at day 1, which declined by day 8 after stent implantation, while inversely, slower hydrolysis was associated with increased arterial expression at day 8 in comparison with day 1. In conclusion, adjustable release of transduction-competent adenoviral vectors from metallic surfaces can be achieved, both in vitro and in vivo, through surface immobilization of adenoviral vectors using hydrolyzable cross-linkers with structure-specific release kinetics.

Endovascular Gene Delivery from a Stent Platform: Gene- Eluting Stents.

A synergistic impact of research in the fields of post-angioplasty restenosis, drug-eluting stents and vascular gene therapy over the past 15 years has shaped the concept of gene-eluting stents. Gene-eluting stents hold promise of overcoming some biological and technical problems inherent to drug-eluting stent technology. As the field of gene-eluting stents matures it becomes evident that all three main design modules of a gene-eluting stent: a therapeutic transgene, a vector and a delivery system are equally important for accomplishing sustained inhibition of neointimal formation in arteries treated with gene delivery stents. This review summarizes prior work on stent-based gene delivery and discusses the main optimization strategies required to move the field of gene-eluting stents to clinical translation.

174. Post-Deployment Modifications of Stent with Endothelial Cells

Accelerated restoration of a functional endothelial layer in the injured arteries following stent angioplasty has been pursued over the past 20 years as an approach for preventing uncontrolled expansion of neointimal tissue and subsequent in-stent restenosis. To achieve enhanced re-growth of endothelium over the denuded arterial segments, strategies based on the pre-procedure seeding of stents with syngeneic/autologous endothelial cells (EC) and protocols involving stent surface modification to expedite capture and proliferation of circulating endothelial progenitor cells (EPC) have been explored. The main hurdle for the clinical translation of these “pro-healing” stents is the impediment in establishing an integral EC/EPC monolayer arrangement immediately after stent deployment, which is paramount for aborting platelet attachment to the exposed sub-endothelium and preventing inflammatory cell recruitment. The current work investigates a novel practical way to attain coverage of stent struts with syngeneic/autologous EC simultaneously with stent implantation. Stainless steel surfaces of tubing and stents were consecutively modified with 1% polyallylamine bisphosphonate, 4 mM sulfo NHS-LC-LC-biotin and 100 µg/ml avidin. Control samples were prepared omitting avidin attachment step. Rat aortic endothelial cells (RAEC) were surface modified in suspension with 1 mM sulfo NHS-LC-LC-biotin for 10 min, followed by pelleting and resuspension in PBS. Stainless steel tubing samples (avidin-coated or control; n=3 for both) were placed in succession inside 2 separate loops of polypropylene tubing that were mounted on a Chandler loop apparatus that models continuous blood flow. RAEC were resuspended in 20 ml of DMEM (2.5×105 EC/ml) and 10 ml of EC suspension was used to fill each loop. After 45 min of biotinylated RAEC recirculation through the avidin-modified and control steel samples (25 dynes/cm2, 37°C), the samples were removed, fixed in 4% PFA, inverted and stained with Hoechst 33258. The density of fluorescent nuclei associated with avidin-modified and control stainless steel surface was determined by fluorescence microscopy, demonstrating a 50-fold higher attachment density of biotinylated EC on avidin-modified surfaces. Firefly luciferase expressing RAEC (via lentiviral transduction) were surface modified with 1 mM sulfo NHS-LC-LC-biotin and intraluminally delivered (2×105 cells) into temporally isolated segments of rat carotid arteries immediately after deployment of PAB/sulfo NHS-LC-LC-biotin/avidin modified (N=4) or PAB only modified control stents (N=4). The animals underwent bioluminescent imaging 2 days post-stent deployment showing a 14.5-fold higher luminescent signal in animals treated with avidin-modified, than with control stents, demonstrating preferential attachment of biotinylated EC to the avidin-modified steel substrate and attesting to the viability of the delivered cells. Post-deployment binding of biotinylated EC to the surface of the avidin-modified stent can present a viable method of stent re-endothelialization and may have a therapeutic value in the prevention of in-stent restenosis and thrombosis.