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Camptothecins are potent topoisomerase I inhibitors used to treat high-risk pediatric solid tumors, but they often show poor efficacy due to intrinsic or acquired chemoresistance. Here, we developed a multivalent, polymer-based prodrug of a structurally optimized camptothecin (SN22) designed to overcome key chemoresistance mechanisms. The ability of SN22 vs. SN38 (the active form of irinotecan/CPT-11) to overcome efflux pump-driven drug resistance was tested. Tumor uptake and biodistribution of SN22 as a polymer-based prodrug (PEG-[SN22]4) compared with SN38 was determined. The therapeutic efficacy of PEG-[SN22]4 to CPT-11 was compared in: (i) spontaneous neuroblastomas (NB) in transgenic TH-MYCN mice; (ii) orthotopic xenografts of a drug-resistant NB line SK-N-BE(2)C (mutated TP53); (iii) flank xenografts of a drug-resistant NB-PDX; and (iv) xenografts of Ewing sarcoma and rhabdomyosarcoma. Unlike SN38, SN22 inhibited NB cell growth regardless of ABCG2 expression levels. SN22 prodrug delivery resulted in sustained intratumoral drug concentrations, dramatically higher than those of SN38 at all time points. CPT-11/SN38 treatment had only marginal effects on tumors in transgenic mice, but PEG-[SN22]4 treatment caused complete tumor regression lasting over 6 months (tumor free at necropsy). PEG-[SN22]4 also markedly extended survival of mice with drug-resistant, orthotopic NB and it caused long-term (6+ months) remissions in 80% to 100% of NB and sarcoma xenografts. SN22 administered as a multivalent polymeric prodrug resulted in increased and protracted tumor drug exposure compared with CPT-11, leading to long-term “cures” in NB models of intrinsic or acquired drug resistance, and models of high-risk sarcomas, warranting its further development for clinical trials.Significance:SN22 is an effective and curative multivalent macromolecular agent in multiple solid tumor mouse models, overcoming common mechanisms of drug resistance with the potential to elicit fewer toxicities than most cancer therapeutics.

BackgroundHypercholesterolemia (HC) has previously been shown to augment restenotic response in several animal models and humans. However, the mechanistic aspects of in-stent restenosis (ISR) on a hypercholesterolemic background, including potential augmentation of systemic and local inflammation precipitated by HC are not completely understood. CD47 is a transmembrane protein known to abort crucial inflammatory pathways. Our present studies have examined the interrelation between HC, inflammation, and ISR and investigated the therapeutic potential of stents coated with a CD47-derived peptide (pepCD47) in the hypercholesterolemic rabbit model.Methods and ResultsPepCD47 was immobilized on metal foil coupons and stents using polybisphosphonate coordination chemistry and pyridyldithio/thiol conjugation. The relative abundance of the surface-associated cells on bare metal (BM) and pepCD47 foils exposed to whole rabbit blood showed a 40% inhibition of cell attachment on pepCD47-modified surfaces. Likewise, cytokine expression analyzed in buffy coat-derived cells cultured over the BM and pepCD47-derivatized foils demonstrated a M2/M1 increase with pepCD47 coating. Hypercholesterolemic and normocholesterolemic rabbit cohorts underwent bilateral implantation of BM and pepCD47 stents in the iliac location. Hypercholesterolemia increased neointimal growth in comparison with normocholesterolemic animals at 4 weeks post-stenting. These untoward outcomes were mitigated in the arteries of hypercholesterolemic rabbits treated with pepCD47-derivatized stents. Compared to NC animals, inflammatory cytokine immunopositivity and macrophage infiltration of peri-strut areas increased in HC group animals, and was attenuated in the arteries of hypercholesterolemic rabbits treated with pepCD47 stents.ConclusionsAugmented inflammatory responses triggered by HC underlie severe ISR morphology in hypercholesterolemic rabbits. Blockage of initial platelet and leukocyte attachment to stent struts through CD47 functionalization of stents mitigates pro-restenotic effects of HC.

Aliphatic polyesters are among materials most extensively used for producing biodegradable polymeric nanoparticles currently in development as delivery carriers and imaging agents for a range of biomedical applications. Their clinical translation requires robust particle labeling methodologies that allow reliably monitoring the fate of these formulations in complex biological environments. In the present study, a practical and versatile synthetic strategy providing conjugates of poly(D,L-lactide) representative of this class of polymers with BODIPY fluorophores varying in functional groups and excitation/emission maxima was investigated as a tool for making traceable nanoparticles. Polymer-probe conjugation was accomplished by carbodiimide-induced and 4-(dimethylamino)pyridinium 4-toluenesulfonate-catalyzed esterification of the polymer’s terminal hydroxyl group, either directly with a carboxy-functionalized fluorophore or with amine-protected amino acids (Boc-glycine or Boc-6-aminohexanoic acid). In the latter case, the amino acid-derivatized polymeric precursors were reacted with amine-reactive BODIPY dyes after the removal of the protective group. Unlike nanoparticles encapsulating a strongly hydrophobic BODIPY(505/515) (logP(o/w) = 4.3), nanoparticles labeled covalently with its carboxy-functionalized analogue (BODIPY FL) demonstrated stable particle-tracer association under perfect sink conditions. Furthermore, in contrast to the encapsulated dye rapidly partitioning from particles onto cell membranes but not stably retained by cultured cells, the internalization of the covalently attached probe was an irreversible process requiring the presence of serum, consistent with active nanoparticle uptake by endocytosis. In conclusion, the conjugation of particle-forming polymers with BODIPY fluorophores offers an effective and accessible labeling strategy for making traceable polyester-based biodegradable nanoparticles and is expected to facilitate their development and optimization as therapeutic carriers and diagnostic agents.

Percutaneous coronary interventions (PCI) are the mainstay for treatment of advanced coronary disease. A majority of PCI involve deployment of a stent in the affected vascular segment. This chapter introduces the concept of using stents as a platform for delivering gene therapies to the vasculature with the overarching aim of mitigating in-stent restenosis (ISR), late stent thrombosis (LST), and neoatherosclerosis (NA), a triad of delayed complications that reduce the overall success rate of PCI. The chapter provides a detailed methodology for coatless reversible attachment of adenoviral (Ad) and adeno-associated viral (AAV) vectors to the metal stent struts along with representative in vitro and in vivo results.

Camptothecins are potent topoisomerase I inhibitors used to treat high-risk pediatric solid tumors, but they often show poor efficacy due to intrinsic or acquired chemoresistance. Here, we developed a multivalent, polymer-based prodrug of a structurally optimized camptothecin (SN22) designed to overcome key chemoresistance mechanisms. The ability of SN22 vs. SN38 (the active form of irinotecan/CPT-11) to overcome efflux pump-driven drug resistance was tested. Tumor uptake and biodistribution of SN22 as a polymer-based prodrug (PEG-[SN22]4) compared with SN38 was determined. The therapeutic efficacy of PEG-[SN22]4 to CPT-11 was compared in: (i) spontaneous neuroblastomas (NB) in transgenic TH-MYCN mice; (ii) orthotopic xenografts of a drug-resistant NB line SK-N-BE(2)C (mutated TP53); (iii) flank xenografts of a drug-resistant NB-PDX; and (iv) xenografts of Ewing sarcoma and rhabdomyosarcoma. Unlike SN38, SN22 inhibited NB cell growth regardless of ABCG2 expression levels. SN22 prodrug delivery resulted in sustained intratumoral drug concentrations, dramatically higher than those of SN38 at all time points. CPT-11/SN38 treatment had only marginal effects on tumors in transgenic mice, but PEG-[SN22]4 treatment caused complete tumor regression lasting over 6 months (tumor free at necropsy). PEG-[SN22]4 also markedly extended survival of mice with drug-resistant, orthotopic NB and it caused long-term (6+ months) remissions in 80% to 100% of NB and sarcoma xenografts. SN22 administered as a multivalent polymeric prodrug resulted in increased and protracted tumor drug exposure compared with CPT-11, leading to long-term “cures” in NB models of intrinsic or acquired drug resistance, and models of high-risk sarcomas, warranting its further development for clinical trials. Significance: SN22 is an effective and curative multivalent macromolecular agent in multiple solid tumor mouse models, overcoming common mechanisms of drug resistance with the potential to elicit fewer toxicities than most cancer therapeutics.

Magnetic guidance shows promise as a strategy for improving the delivery and performance of cell therapeutics. However, clinical translation of magnetically guided cell therapy requires cell functionalization protocols that provide adequate magnetic properties in balance with unaltered cell viability and biological function. Existing methodologies for characterizing cells functionalized with magnetic nanoparticles (MNP) produce aggregate results, both distorted and unable to reflect variability in either magnetic or biological properties within a preparation. In the present study, we developed an inverted-plate assay allowing determination of these characteristics using a single-platform approach, and applied this method for a comparative analysis of two loading protocols providing highly uniform vs. uneven MNP distribution across cells. MNP uptake patterns remarkably different between the two protocols were first shown by fluorimetry carried out in a well-scan mode on endothelial cells (EC) loaded with BODIPY558/568-labeled MNP. Using the inverted-plate assay we next demonstrated that, in stark contrast to unevenly loaded cells, more than 50% of uniformly functionalized EC were captured within 5 min over a broad range of MNP doses. Furthermore, magnetically captured cells exhibited unaltered viability, substrate attachment, and proliferation rates. Conducted in parallel, magnetophoretic mobility studies corroborated the markedly superior guidance capacity of uniformly functionalized cells, confirming substantially faster cell capture kinetics on a clinically relevant time scale. Taken together, these results emphasize the importance of optimizing cell preparation protocols with regard to loading uniformity as key to efficient site-specific delivery, engraftment, and expansion of the functionalized cells, essential for both improving performance and facilitating translation of targeted cell therapeutics.

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.

Despite the use of intensive multimodality therapy, the majority of high-risk neuroblastoma (NB) patients do not survive. Without significant improvements in delivery strategies, anticancer agents used as a first-line treatment for high-risk tumors often fail to provide clinically meaningful results in the settings of disseminated, recurrent, or refractory disease. By enhancing pharmacological selectivity, favorably shifting biodistribution, strengthening tumor cell killing potency, and overcoming drug resistance, nanocarrier-mediated delivery of topoisomerase I inhibitors of the camptothecin family has the potential to dramatically improve treatment efficacy and minimize side effects. In this study, a structurally enhanced camptothecin analog, SN22, reversibly coupled with a redox-silent tocol derivative (tocopheryl oxamate) to allow its optimally stable encapsulation and controlled release from PEGylated sub-100 nm nanoparticles (NP), exhibited strong NB cell growth inhibitory activity, translating into rapid regression and durably suppressed regrowth of orthotopic, MYCN-amplified NB tumors. The robust antitumor effects and markedly extended survival achieved in preclinical models recapitulating different phases of high-risk disease (at diagnosis vs. at relapse with an acquired loss of p53 function after intensive multiagent chemotherapy) demonstrate remarkable potential of SN22 delivered in the form of a hydrolytically cleavable superhydrophobic prodrug encapsulated in biodegradable nanocarriers as an experimental strategy for treating refractory solid tumors in high-risk cancer patients.

High-risk solid tumors continue to pose a tremendous therapeutic challenge due to multidrug resistance. Biological mechanisms driving chemoresistance in high-risk primary and recurrent disease are distinct: in newly diagnosed patients, non-response to therapy is often associated with a higher level of tumor "stemness" paralleled by overexpression of the ABCG2 drug efflux pump, whereas in tumors relapsing after non-curative therapy, poor drug sensitivity is most commonly linked to the dysfunction of the tumor suppressor protein, p53. In this study, we used preclinical models of aggressive neuroblastoma featuring these characteristic mechanisms of primary and acquired drug resistance to experimentally evaluate a macromolecular prodrug of a structurally enhanced camptothecin analog, SN22, resisting ABCG2-mediated export, and glucuronidation. Together with extended tumor exposure to therapeutically effective drug levels via reversible conjugation to Pluronic F-108 (PF108), these features translated into rapid tumor regression and long-term survival in models of both ABCG2-overexpressing and p53-mutant high-risk neuroblastomas, in contrast to a marginal effect of the clinically used camptothecin derivative, irinotecan. Our results demonstrate that pharmacophore enhancement, increased tumor uptake, and optimally stable carrier-drug association integrated into the design of the hydrolytically activatable PF108-[SN22]

Spatially and temporally controlled delivery of biologicals, including gene vectors, represents an unmet need for regenerative medicine and gene therapy applications. Here we describe a method of reversible attachment of serotype 2 adeno-associated viral vectors (AAV2) to metal surfaces. This technique enables localized delivery of the vector to the target cell population in vitro and in vivo with the subsequent effective transduction of cells adjacent to the metal substrate. The underlying bioengineering approach employs coordination chemistry between the bisphosphonic groups of polyallylamine bisphosphonates and the metal atoms on the surface of metallic samples. Formation of a stable polybisphosphonate monolayer with plentiful allyl-derived amines allows for further chemical modification to consecutively append thiol-modified protein G, an anti-AAV2 antibody, and AAV2 particles. Herein we present a detailed protocols for the metal substrate modification, for the visualization of the metal surface-immobilized vector using direct and indirect fluorescent AAV2 labeling and scanning electron microscopy, for quantification of the surface-immobilized vector load with RT-PCR, and for the localized vector transduction in vitro and in vivo.

Introduction: Bioprosthetic heart valves (BHV) fabricated from glutaraldehyde treated bovine pericardium (BP) are widely used to treat heart valve disease. BHV dysfunction from structural valve degeneration (SVD) develops over time, often necessitating device replacement. SVD is associated with both calcification and the accumulation of advanced glycation end products (AGE) and serum proteins within BHV leaflets. Here, we introduce a new strategy for mitigating AGE-associated SVD by modifying BP with a poly(2-methyl-2-oxazoline) (POZ). Hypothesis: Attached POZ will prevent BP deterioration by mitigating serum protein and pro-inflammatory AGE uptake. Methods: Covalently modified BP-POZ was studied for its effects on serum albumin and AGE entry in vitro via a mass-uptake assay and immunostaining. Resistance of BP-POZ to oxidation was assessed in comparison to poly(ethylene glycol) (PEG). Macrophage studies assessed the inflammatory properties of BP-POZ. A juvenile rat subdermal model was used to study serum protein and AGE uptake and calcification within BP-POZ, with or without ethanol pretreatment. A heart valve pulse duplicator was used to assess the protective effects of BP-POZ on BHV hydrodynamics following accelerated glycation. Results: POZ mitigated the accumulation of serum albumin and AGE in vitro . After exposure to AGE and macrophages, POZ preserved BP collagen structure while TNFα levels remained at baseline. Under oxidation conditions, POZ retained its protective function, unlike PEG. In the rat model, POZ reduced BP AGE (p Conclusions: SVD due to AGE and serum proteins can be effectively mitigated through POZ modification, that does not interfere with ethanol pretreatment mitigation of BP calcification.

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

Background: Hypercholesterolemia (HC) has previously been shown to augment restenotic response in several animal models. However, the mechanistic aspects of in-stent restenosis (ISR) on an HC background are not fully understood. Methods: HC was induced in 5 NZW rabbits by hypercholesterolemic diet (HCD) fed for 4 weeks prior to bilateral implantation of stainless steel stents in the iliac location. The diet was continued until sacrifice. In parallel, stents were deployed in the iliac arteries of 5 normocholesterolemic (NC) rabbits. All animals were euthanized 4 weeks after stenting. Harvested arteries were formalin-fixed. The stent struts were dissolved in a mixture of nitric and hydrofluoric acids. The destented arteries were paraffin-embedded, sectioned, stained according to the Verhoeff-vanGieson method, and the lumen area, neointimal thickness, neointimal area, neointima-to-media ratio, and percent of luminal stenosis were determined morphometrically. A semiquantitative scale was used to assess the intensity and spread of TNFa expression by immunohistochemistry (IHC). The prevalence of peri-strut macrophages (MΦ) was determined by IHC as a percentage of a strut circumference infiltrated with MΦ. Results: HC diet drastically increased severity of ISR (Fig). The corresponding values of the lumen area, neointimal thickness, neointimal area, neointima-to-media ratio and percent of luminal stenosis for the groups of NC and HC animals were 1.86±0.44 vs 1.48±0.54 (p Conclusions: The inflammatory response to stent deployment is intensified in HC metabolic conditions, leading to the augmented neointimal expansion and ISR.

[Image: see text] Conventional treatment approaches fail to provide durable control over aggressive malignancies due to intrinsic or acquired drug resistance characteristic of high-risk disease. SN-38, a potent camptothecin analog specifically targeting DNA topoisomerase I cleavage complexes, has shown promise in preclinical studies against aggressive solid tumors. However, its clinical utility is limited by inadequate solubility in pharmaceutically acceptable vehicles and by poor chemical and metabolic stability. Micelles formulated from amphiphilic invertible polymers (AIPs) can address these issues by concomitantly enabling solubilization of water-insoluble molecular cargoes and by protecting chemically labile agents from inactivation. Furthermore, the inversion of the AIP and disruption of the carrier–drug complexes triggered by contact with cell membranes makes it possible to deliver the therapeutic payload into the cell interior without compromising its biological activity. In the present study, we characterized a novel AIP-based micellar formulation of SN-38 and evaluated its growth inhibitory effect on neuroblastoma (NB) cells derived either at diagnosis or at relapse after intensive chemoradiotherapy. Colloidally stable, drug-loaded micellar assemblies with a uniform

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, improving the biocompatibility of metal stents remains a significant challenge. The goal of this protocol is to describe a robust technique of metal surface modification by biologically active peptides to increase biocompatibility of blood contacting medical implants, including endovascular stents. CD47 is an immunological species-specific marker of self and has anti-inflammatory properties. Studies have shown that a 22 amino acid peptide corresponding to the Ig domain of CD47 in the extracellular region (pepCD47), has anti-inflammatory properties similar to the full-length protein. In vivo studies in rats, and ex vivo studies in rabbit and human blood experimental systems from our lab have demonstrated that pepCD47 immobilization on metals improves their biocompatibility by preventing inflammatory cell attachment and activation. This paper describes the step-by step protocol for the functionalization of metal surfaces and peptide attachment. The metal surfaces are modified using polyallylamine bisphosphate with latent thiol groups (PABT) followed by deprotection of thiols and amplification of thiol-reactive sites via reaction with polyethyleneimine installed with pyridyldithio groups (PEI-PDT). Finally, pepCD47, incorporating terminal cysteine residues connected to the core peptide sequence through a dual 8-amino-3,6-dioxa-octanoyl spacer, are attached to the metal surface via disulfide bonds. This methodology of peptide attachment to metal surface is efficient and relatively inexpensive and thus can be applied to improve biocompatibility of several metallic biomaterials.

In-stent restenosis remains an important clinical problem in the era of drug eluting stents. Development of clinical gene therapy protocols for the prevention and treatment of in-stent restenosis is hampered by the lack of adequate local delivery systems. Herein we describe a novel stent-based gene delivery platform capable of providing local arterial gene transfer with adeno-associated viral (AAV) vectors. This system exploits the natural affinity of protein G (PrG) to bind to the Fc region of mammalian IgG, making PrG a universal adaptor for surface immobilization of vector-capturing antibodies (Ab). Our results: 1) demonstrate the feasibility of reversible immobilization of AAV2 vectors using vector tethering by AAV2-specific Ab appended to the stent surface through covalently attached PrG, 2) show sustained release kinetics of PrG/Ab-immobilized AAV2 vector particles into simulated physiological medium in vitro and site-specific transduction of cultured cells, 3) provide evidence of long-term (12 weeks) arterial expression of luciferase with PrG/Ab-tethered AAV2Luc, and 4) show anti-proliferative activity and anti-restenotic efficacy of stent-immobilized AAV2iNOS in the rat carotid artery model of stent angioplasty.

Increased susceptibility to thrombosis, neoatherosclerosis, and restenosis due to incomplete regrowth of the protective endothelial layer remains a critical limitation of the interventional strategies currently used clinically to relieve atherosclerotic obstruction. Rapid recovery of endothelium holds promise for both preventing the thrombotic events and reducing post-angioplasty restenosis, providing the rationale for developing cell delivery strategies for accelerating arterial reendothelialization. The successful translation of experimental cell therapies into clinically viable treatment modalities for restoring vascular endothelium critically depends on identifying strategies for enhancing the functionality of endothelial cells (EC) derived from high cardiovascular risk patients, the target group for the majority of angioplasty procedures. Enhancing EC-associated nitric oxide (NO) synthesis by inducing overexpression of NO synthase (NOS) has shown promise as a way of increasing paracrine activity and restoring function of EC. In the present study, we developed a direct contact co-culture approach compatible with highly labile effectors, such as NO, and applied it for determining the effect of EC functionalization via NOS gene transfer on the growth of co-cultured arterial smooth muscle cells (A10 cell line) exhibiting the defining characteristics of neointimal cells. Bovine aortic endothelial cells magnetically transduced with inducible NOS-encoding adenovirus (Ad) formulated in zinc oleate-based magnetic nanoparticles (MNP[(iNOS)Ad]) strongly suppressed growth of proliferating A10 and attenuated the stimulatory effect of a potent mitogen, platelet-derived growth factor (PDGF-BB), whereas EC functionalization with free (iNOS)Ad or MNP formulated with a different isoform of the enzyme, endothelial NOS, was associated with lower levels of NO synthesis and less pronounced antiproliferative activity toward co-cultured A10 cells. These results show feasibility of applying magnetically facilitated gene transfer to potentiate therapeutically relevant effects of EC for targeted cell therapy of restenosis. The direct contact co-culture methodology provides a sensitive and reliable tool with potential utility for a variety of biomedical applications.

Morphometric analysis of explanted arteries remains the gold standard in assessing pathological mechanisms of arterial disease, and the efficacy of therapeutic interventions. Software currently used for morphometric analysis (ImageJ, ImagePro, etc.) requires manual tracing of each region of interest by the user to obtain direct and derived data needed for analysis. Manual segmentation of borders between differently stained arterial compartments is time-consuming and prone to bias, even when the operator is blind to the treatment type. Here, we report newly designed morphometric software, floodSeg, which greatly aids analysis through semiautomated segmentation, followed by automated computation and data entry. The program includes tools for both rapid component selection, and precision user-controlled contour correction. In practice, segmentation occurs following the selection of seed points and corresponding threshold values, for each desired component. The flood fill algorithm is used to map out components, followed by Sklansky’s convex-hull algorithm to obtain the outer contours. If necessary, convexity defects can be overcome through manual point placement on top of existing points, and regeneration of the contour. floodSeg was tested using a set of non-uniformly stained stented rat arteries, and compared against manually obtained results. The accuracy of the resulting measurements was within the expected limit based on repeated manual measurements by the same operator, and did not exceed 3%. Most notably, the duration for data acquisition using floodSeg was less than 20% of the time required for manual measurements by an experienced operator. Thus, our contribution is an improvement on widely used software, with significant potential for application in a multitude of areas of pathology practice.

In-stent restenosis (ISR) and late stent thrombosis are the major complications associated with the use of metal stents and drug eluting stents respectively. Our lab previously investigated the use of peptide CD47 in improving biocompatibility of bare metal stents in a rat carotid stent model and our results demonstrated a significant reduction in platelet deposition and ISR. However, this study did not characterize the stability of the pepCD47 on metal surfaces post storage, sterilization and deployment. Thus, the objective of the present study was 1) to test the stability of the peptide post - storage, sterilization, exposure to shear and mechanical stress and 2) to begin to expand our current knowledge of pepCD47 coated metal surfaces into the preclinical large animal rabbit model. Our results show that the maximum immobilization density of pepCD47 on metal surfaces is approximately 350 ng/cm2. 100% of the pepCD47 was retained on the metal surface post 24 weeks of storage at 4 °C, exposure to physiological shear stress, and mechanical stress of stent expansion. The bioactivity of the pepCD47 was found to be intact post 24 weeks of storage and ethylene oxide sterilization. Finally our ex vivo studies demonstrated that compared to bare metal the rabbit pepCD47 coated surfaces showed - 45% reduced platelet adhesion, a 10-fold decrease in platelet activation, and 93% endothelial cell retention. Thus, our data suggests that pepCD47 coating on metal surfaces is stable and rabbit pepCD47 shows promising preliminary results in preventing thrombosis and not inhibiting the growth of endothelial cells. Statement of significance Biocompatibility of bare metal stents is a major challenge owing to the significantly high rates of in-stent restenosis. Previously we demonstrated that peptide CD47 functionalization improves the biocompatibility of bare metal stents in rat model. A similar trend was observed in our ex vivo studies where rabbit blood was perfused over the rabbit pepCD47 functionalized surfaces. These results provide valuable proof of concept data for future in vivo rabbit model studies. In addition, we investigated stability of the pepCD47 on metal surface and observed that pepCD47 coating is stable over time and resistant to industrially relevant pragmatic challenges.

Chondrosarcoma is the second most common primary bone sarcoma. Treatment of chondrosarcoma is limited to surgery due to radiation and chemotherapy resistance of this cancer. An ideal treatment for chondrosarcoma would be a well-tolerated, minimally invasive local or systemic treatment modality to halt or slow tumor growth prior to resection of local, unresectable local, or metastatic disease. Palovarotene, an agonist of nuclear retinoic acid receptor γ (RARγ) has shown therapeutic action for treatment of heterotopic ossification and osteochondroma without serious adverse effects in animal models. We hypothesized that selective agonists of RARγ would have an inhibitory effect on chondrosarcoma. All human chondrosarcoma specimens expressed RARγ as determined by immunohistochemical staining. The ΗCS-2/8 chondrosarcoma cell line, established from low-grade human chondrosarcoma, was used to examine the actions of RARγ agonists. In ΗCS2/8 pellet cultures, RARγ agonist treatment reduced the mass size and significantly decreased total glycosaminoglycan, protein amounts, and gene expression levels of cartilage matrix molecules when compared with control groups. Systemic treatment with RARγ agonists significantly inhibited the growth of ΗCS-2/8 cell transplants in vivo. Furthermore, local injection of RARγ agonist-loaded poly-lactic acid nanoparticles induced regression of the mass size of the transplants. Histologic analysis demonstrated that RARγ agonist treatment inhibited cell proliferation activity and stimulated encapsulation of the tumor. These findings indicate that RARγ agonists, including palovarotene, may have an anti-tumor effect on low-grade chondrosarcomas. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:1045-1051, 2020.

The blood compatibility of various intravascular (IV) devices (e.g., catheters, sensors, etc.) is compromised by activation of platelets that can cause thrombus formation and device failure. Such devices also carry a high risk of microbial infection. Recently, nitric oxide (NO) releasing polymers/devices have been proposed to reduce these clinical problems. CD47, a ubiquitously expressed transmembrane protein with proven anti-inflammation/anti-platelet properties when immobilized on polymeric surfaces, is a good candidate to complement NO release in both effectiveness and longevity. In this work, we successfully appended CD47 peptides (pepCD47) to the surface of biomedical grade polyurethane (PU) copolymers. SIRPα binding and THP-1 cell attachment experiments strongly suggested that the pepCD47 retains its biological properties when bound to PU films. In spite of the potentially high reactivity of NO toward various amino acid residues in CD47, the efficacy of surface-immobilized pepCD47 to prevent inflammatory cell attachment was not inhibited after being subjected to a high flux of NO for three days, demonstrating excellent compatibility of the two species. We further constructed a CD47 surface immobilized silicone tubing filled with NO releasing S-nitrosoglutathione/ascorbic acid (GSNO/AA) solution for synergistic biocompatibility evaluation. Via an ex vivo Chandler loop model, we demonstrate for the first time that NO release and CD47 modification could function synergistically at the blood/material interface and produce greatly enhanced anti-inflammatory/anti-platelet effects. This concept should be readily implementable to create a new generation of thromboresistant/antimicrobial implantable devices.

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.

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.

Purpose: Currently, Experimental Design: SN38, the active metabolite of irinotecan (CPT-11), is a potent therapeutic agent that is readily encapsulated in polymeric nanoparticles. Tocopherol oxyacetate (TOA) is a hydrophobic mitocan that was linked to SN38 to significantly increase hydrophobicity and enhance nanoparticle retention. We treated neuroblastomas with SN38-TOA nanoparticles and compared the efficacy with the parent prodrug CPT-11 using a mouse xenograft model. Results: Nanoparticle treatment induced prolonged event-free survival (EFS) in most mice, compared with CPT-11. This was shown for both SH-SY5Y and IMR-32 neuroblastoma xenografts. Enhanced efficacy was likely due to increased and sustained drug levels of SN38 in the tumor compared with conventional CPT-11 delivery. Interestingly, when recurrent CPT-11–treated tumors were re-treated with SN38-TOA nanoparticles, the tumors transformed from undifferentiated neuroblastomas to maturing ganglioneuroblastomas. Furthermore, these tumors were infiltrated with Schwann cells of mouse origin, which may have contributed to the differentiated histology. Conclusions: Nanoparticle delivery of SN38-TOA produced increased drug delivery and prolonged EFS compared to conventional delivery of CPT-11. Also, lower total dose and drug entrapment in nanoparticles during circulation should decrease toxicity. We propose that nanoparticle-based delivery of a rationally designed prodrug is an attractive approach to enhance chemotherapeutic efficacy in pediatric and adult tumors. Clin Cancer Res; 24(11); 2585–93. ©2018 AACR.

Bioprosthetic heart valves (BHV) fabricated from glutaraldehyde pretreated heterograft materials, porcine aortic valves or bovine pericardium (BP), are widely used in cardiac surgery. BHV progressively fail in clinical use due to structural degeneration. Previously we reported that dityrosine, an oxidized amino acid, was present in failed clinical BP-BHV explants; unimplanted BP had no detectable dityrosine. In the same studies BP were demonstrated in vitro to be susceptible to oxidative damage, that could be mitigated with BP covalently modified with the antioxidant, 3-(4-hydroxy-3,5-di-tert-butylphenyl)propyl amine (DBP). The present studies compared in rat subdermal implants glutaraldehyde pretreated BP to BP modified with either DBP or the chemical reactions used to link DBP. All BP explants regardless of DBP demonstrated reduced hydroxyproline and increased digestibility by collagenase. However, the DBP-BP explants showed significant inhibition of reduced explant shrink temperatures (an index of crosslinking) as compared with control BP. Significant mitigation of calcification was observed in both the BP-DBP and chemically modified explants as compared with BP. Dityrosine was not detectable in the 90 day explants. It is concluded that rat subdermal BP implants undergo both calcific and noncalcific structural degeneration, but without the formation of dityrosine, unlike clinical BP explants.

Oxidative stress and inflammation are intertwined contributors to numerous acute vascular pathologies. A novel dual bioactive nanoparticle with antioxidant/anti-inflammatory properties was developed based on the interactions of tocopherol phosphate and the manganese porphyrin SOD mimetic, MnTMPyP. The size and drug incorporation efficiency were shown to be dependent on the amount of MnTMPyP added as well as the choice of surfactant. MnTMPyP was shown to retain its SOD-like activity while in intact particles and to release in a slow and controlled manner. Conjugation of anti-PECAM antibody to the nanoparticles provided endothelial targeting and potentiated nanoparticle-mediated suppression of inflammatory activation of these cells manifested by expression of VCAM, E-selectin, and IL-8. This nanoparticle technology may find applicability with drug combinations relevant for other pathologies.

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.

The clinical use of bioprosthetic heart valves (BHV) is limited due to device failure caused by structural degeneration of BHV leaflets. In this study we investigated the hypothesis that oxidative stress contributes to this process. Fifteen clinical BHV that had been removed for device failure were analyzed for oxidized amino acids using mass spectrometry. Significantly increased levels of ortho-tyrosine, meta-tyrosine and dityrosine were present in clinical BHV explants as compared to the non-implanted BHV material glutaraldehyde treated bovine pericardium (BP). BP was exposed in vitro to oxidizing conditions (FeSO4/H2O2) to assess the effects of oxidation on structural degeneration. Exposure to oxidizing conditions resulted in significant collagen deterioration, loss of glutaraldehyde cross-links, and increased susceptibility to collagenase degradation. BP modified through covalent attachment of the oxidant scavenger 3-(4-hydroxy-3,5-di-tert-butylphenyl) propyl amine (DBP) was resistant to all of the monitored parameters of structural damage induced by oxidation. These results indicate that oxidative stress, particularly via hydroxyl radical and tyrosyl radical mediated pathways, may be involved in the structural degeneration of BHV, and that this mechanism may be attenuated through local delivery of antioxidants such as DBP.

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.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.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.

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.

Studying the morphology of the arterial response to endovascular stent implantation requires embedding the explanted stented artery in rigid materials such as poly(methyl methacrylate) to enable sectioning through both the in situ stent and the arterial wall, thus maintaining the proper anatomic relationships. This is a laborious, time-consuming process. Moreover, the technical quality of stained plastic sections is typically suboptimal and, in some cases, precludes immunohistochemical analysis. Here we describe a novel technique for dissolution of metallic and plastic stents that is compatible with subsequent embedding of "destented" arteries in paraffin, fine sectioning, major staining protocols, and immunohistochemistry.

CD47 is a ubiquitously expressed transmembrane protein that, through signaling mechanisms mediated by signal regulatory protein alpha (SIRPα1), functions as a biological marker of 'self-recognition'. We showed previously that inflammatory cell attachment to polymeric surfaces is inhibited by the attachment of biotinylated recombinant CD47 (CD47B). We test herein the hypothesis that CD47 modified blood conduits can reduce platelet and neutrophil activation under clinically relevant conditions. We appended a poly-lysine tag to the C-terminus of recombinant CD47 (CD47L) allowing for covalent linkage to the polymer. SIRPα1 expression was confirmed in isolated platelets. We then compared biocompatibility between CD47B and CD47L functionalized polyvinyl chloride (PVC) surfaces and unmodified control PVC surfaces. Quantitative and Qualitative analysis of blood cell attachment to CD47B and CD47L surfaces, via scanning electron microscopy, showed strikingly fewer platelets attached to CD47 modified surfaces compared to control. Flow cytometry analysis showed that activation markers for neutrophils (CD62L) and platelets (CD62P) exposed to CD47 modified PVC were equivalent to freshly acquired control blood, while significantly elevated in the unmodified PVC tubing. In addition, ethylene oxide gas sterilization did not inhibit the efficacy of the CD47 modification. In conclusion, CD47 modified PVC inhibits both the adhesion and activation of platelets and neutrophils.

Background One of the most important factors responsible for the calcific failure of bioprosthetic heart valves is glutaraldehyde crosslinking. Ethanol (EtOH) incubation after glutaraldehyde crosslinking has previously been reported to confer anticalcification efficacy for bioprostheses. The present studies investigated the anticalcification efficacy in vivo of the novel crosslinking agent, triglycidyl amine (TGA), with or without EtOH incubation, in comparison with glutaraldehyde. Methods The TGA crosslinking (± EtOH) was used to prepare porcine aortic valves for both rat subdermal implants and sheep mitral valve replacements, for comparisons with glutaraldehyde-fixed controls. Thermal denaturation temperature, an index of crosslinking, cholesterol extraction, and hydrodynamic properties were quantified. Explant endpoints included quantitative and morphologic assessment of calcification. Results Thermal denaturation temperatures after TGA were intermediate between unfixed and glutaraldehyde-fixed. EtOH incubation resulted in almost complete extraction of cholesterol from TGA or glutaraldehyde-fixed cusps. Rat subdermal explants (90 days) demonstrated that TGA-EtOH resulted in a significantly greater level of inhibition of calcification than other conditions. Thus, TGA-ethanol stent mounted porcine aortic valve bioprostheses were fabricated for comparisons with glutaraldehyde-pretreated controls. In hydrodynamic studies, TGA-EtOH bioprostheses had lower pressure gradients than glutaraldehyde-fixed. The TGA-ethanol bioprostheses used as mitral valve replacements in juvenile sheep (150 days) demonstrated significantly lower calcium levels in both explanted porcine aortic cusp and aortic wall samples compared with glutaraldehyde-fixed controls. However, TGA-EtOH sheep explants also demonstrated isolated calcific nodules and intracuspal hematomas. Conclusions The TGA-EtOH pretreatment of porcine aortic valves confers significant calcification resistance in both rat subdermal and sheep circulatory implants, but with associated structural instability.

The uniform alignment of endothelial cells inside small-diameter synthetic grafts can be directed by surface topographies such as microgrooves and microfibers to recapitulate the flow-induced elongation and alignment of natural endothelium. These surface micropatterns may also promote directional migration and potentially improve anastomotic ingrowth of endothelial cells inside the synthetic grafts. In this paper, we developed electrospinning and spin casting techniques to pattern the luminal surface of small-diameter polyurethane (PU) grafts with microfibers and microgrooves, respectively, and evaluated endothelial cell orientation on these surface micropatterns. Tracks of circumferentially oriented microfibers were generated by electrospinning PU onto a mandrel rotated at high velocity, whereas longitudinal tracks of microgrooves were generated by spin casting PU over a rotating poly(dimethylsiloxane) mold. We found that both PU grafts possessed longitudinal Young’s moduli in the range of 0.43 ± 0.04 to 2.00 ± 0.40 MPa, comparable with values obtained from native artery. Endothelial cells seeded onto the grafts formed confluent monolayers with individual cells exhibiting elongated morphology parallel to the micropatterns. The cells were phenotypically similar to natural endothelium as assessed by the expression of the endothelial cell-specific marker, vascular endothelial cell cadherin. In addition, the cells were also responsive to stimulation with the pro-inflammatory cytokine tumor necrosis factor-α as assessed by the inducible expression of intercellular adhesion molecule-1. These results demonstrate that our micropatterned PU grafts possessed longitudinal Young’s moduli in the same range as native vascular tissue and were capable of promoting the formation of aligned and cytokine-responsive endothelial monolayers.

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°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.

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.

Targeting gene therapy remains a challenge. The use of magnetic force to achieve this was investigated in the present study. It was hypothesized that nanoparticles with both controllable particle size and magnetic properties would enable magnetically driven gene delivery. We investigated this hypothesis by creating a family of novel biodegradable polymeric superparamagnetic nanoparticle (MNP) formulations. Polylactide MNP were formulated using a modified emulsification-solvent evaporation methodology with both the incorporation of oleate-coated iron oxide and a polyethylenimine (PEI) oleate ion-pair surface modification for DNA binding. MNP size could be controlled by varying the proportion of the tetrahydrofuran cosolvent. Magnetically driven MNP-mediated gene transfer was studied using a green fluorescent protein reporter plasmid in cultured arterial smooth muscle cells and endothelial cells. MNP-DNA internalization and trafficking were examined by confocal microscopy. Cell growth inhibition after MNP-mediated adiponectin plasmid transfection was studied as an example of a therapeutic end point. MNP-DNA complexes protected DNA from degradation and efficiently transfected quiescent cells under both low and high serum conditions after a 15 min exposure to a magnetic field (500 G). There was negligible transfection with MNP in the absence of a magnetic field. Larger sized MNP (375 nm diameter) exhibited higher transfection rates compared with 185 nm- and 240 nm-sized MNP. Internalized larger sized MNP escaped lysosomal localization and released DNA in the perinuclear zone. Adiponectin plasmid DNA delivery using MNP resulted in a dose-dependent growth inhibition of cultured arterial smooth muscle cells. It is concluded that magnetically driven plasmid DNA delivery can be achieved using biodegradable MNP containing oleate-coated magnetite and surface modified with PEI oleate ion-pair complexes that enable DNA binding.

Heart valve replacements fabricated from glutaraldehyde (Glut)-crosslinked heterograft materials, porcine aortic valves or bovine pericardium, have been widely used in cardiac surgery to treat heart valve disease. However, these bioprosthetic heart valves often fail in long-term clinical implants due to pathologic calcification of the bioprosthetic leaflets, and for stentless porcine aortic valve bioprostheses, bioprosthetic aortic wall calcification also typically occurs. Previous use of the epoxide-based crosslinker, Triglycidyl amine (TGA), on cardiac bioprosthetic valve materials demonstrated superior biocompatibility, mechanics, and calcification resistance for porcine aortic valve cusps (but not porcine aortic wall) and bovine pericardium, versus Glut-prepared controls. However, TGA preparation did not completely prevent long-term calcification of cusps or pericardium. Herein we report further mechanistic investigations of an added therapeutic component to this system, 2-Mercaptoethylidene-1,1-bisphosphonic acid (MABP), a custom synthesized thiol bisphosphonate, which has previously been shown in a preliminary report to prevent bioprosthetic heterograft biomaterial calcification when used in combination with initial TGA crosslinking for 7 days. In the present studies we have further investigated the effectiveness of MABP in experiments that examined: 1) The use of MABP after optimal TGA crosslinking, in order to avoid any competitive interference of MABP-reactions with TGA during crosslinking; 2) Furthermore, recognizing the importance of alkaline phosphatase in the formation of dystrophic calcific nodules, we have investigated the hypothesis that the mechanism by which MABP primarily functions is through the reduction of alkaline phosphatase activity. Results from cell-free model systems, cell culture studies, and rat subcutaneous implants, show that materials functionalized with MABP after TGA crosslinking have reduced alkaline phosphatase activity, and in vivo have no significant calcification in long term implant studies. It is concluded that bioprosthetic heart valves prepared in this fashion are compelling alternatives for Glut-prepared bioprostheses.

The effectiveness of endovascular stents is hindered by in-stent restenosis (ISR), a secondary re-obstruction of treated arteries due to unresolved inflammation and activation of smooth muscle cells in the arterial wall. We previously demonstrated that immobilized CD47, a ubiquitously expressed transmembrane protein with an established role in immune evasion, can confer biocompatibility when appended to polymeric surfaces. In present studies, we test the hypothesis that CD47 immobilized onto metallic surfaces of stents can effectively inhibit the inflammatory response thus mitigating ISR. Recombinant CD47 (recCD47) or a peptide sequence corresponding to the Ig domain of CD47 (pepCD47), were attached to the surfaces of both 316L-grade stainless steel foils and stents using bisphosphonate coordination chemistry and thiol-based conjugation reactions to assess the anti-inflammatory properties of CD47-functionalized surfaces. Initial in vitro and ex vivo analysis demonstrated that both recCD47 and pepCD47 significantly reduced inflammatory cell attachment to steel surfaces without impeding on endothelial cell retention and expansion. Using a rat carotid stent model, we showed that pepCD47-functionalized stents prevented fibrin and platelet thrombus deposition, inhibited inflammatory cell attachment, and reduced restenosis by 30%. It is concluded that CD47-modified stent surfaces mitigate platelet and inflammatory cell attachment, thereby disrupting ISR pathophysiology.

The present studies investigated the hypothesis that affinity immobilization of replication-defective adenoviruses (Ad) on the surfaces of biodegradable nanoparticles (NP) can improve transduction through uncoupling cellular uptake from the coxsackie-adenovirus receptor (CAR). Ad was tethered to the surfaces of polylactide-based NP that were surface-activated using a photoreactive polyallylamine-benzophenone-pyridyldithiocarboxylate polymer, which enabled (via thiol chemistry) the covalent attachment of Ad-binding proteins, either the recombinant D1 domain of CAR or an adenoviral knob-specific monoclonal antibody. Gene transfer by NP-Ad complexes was studied in relation to cellular uptake as a function of cell type and the character of NP-Ad binding. NP-Ad complexes, but not Ad applied with or without control nonimmune IgG-modified NP, significantly increased green fluorescent protein reporter expression in endothelioma and endothelial and arterial smooth muscle cells (SMC) in direct correlation to the extent of NP-Ad internalization. CAR-independent uptake of NP-Ad was confirmed by demonstrating inhibition of free Ad- but not NP-Ad complex-mediated transduction by knob protein. Complexes formulated with an Ad encoding inducible nitric oxide synthase inhibited growth of cultured SMC to a significantly greater extent than those with (GFP)Ad or (NULL)Ad or free vector. It is concluded that Ad-specific affinity tethering to biodegradable NP can significantly increase the level of gene expression via a CAR-independent uptake mechanism.

Polyurethane (PU) components of cardiovascular devices are subjected to oxidation-initiated surface degradation, which leads to cracking and ultimately device failure. In the present study, we investigated a novel bromoalkylation chemical strategy to covalently attach the antioxidant, di-tert-butylphenol (DBP), and/or cholesterol (Chol) to the PU urethane nitrogen groups to hypothetically prevent oxidative degradation. These experiments compared PU, PU-DBP, PU-Chol, and PU-Chol-DBP. A series of comparative oxidative degradation studies involved exposing PU samples (modified and unmodified) to H2O2-CoCl2 for 15 days at 37 degrees C, to cause accelerated oxidative degradation. The extent and effects of degradation were assessed by attenuated total reflectance Fourier transformation infrared spectroscopy (FTIR), scanning electron microscopy (SEM), surface contact angle measurements, and mechanical testing. Both the Chol and DBP modification conferred significant resistance to oxidation related changes compared to unmodified PU per FTIR and SEM results. SEM demonstrated cavitation only in unmodified PU. However, contact angle analysis showed significant oxidation-induced changes only in the Chol-modified PU formulations. Most importantly, uniaxial stress-strain testing revealed that only PU-DBP demonstrated bulk elastomeric properties that were minimally affected by oxidation; PU, PU-Chol, PU-Chol-DBP showed marked deterioration of their stress-strain properties following oxidation. In conclusion, these results demonstrate that derivatizing PU with DBP confers significant resistance to oxidative degradation compared with unmodified PU.