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.

Hypercholesterolemia exacerbates in-stent restenosis in rabbits: Studies of the mitigating effect of stent surface modification with a CD47-derived peptide.

BACKGROUND AND AIMS: Hypercholesterolemia (HC) has previously been shown to augment the restenotic response in 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 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: PepCD47 was immobilized on metal foils and stents using polybisphosphonate coordination chemistry and pyridyldithio/thiol conjugation. Cytokine expression in buffy coat-derived cells cultured over bare metal (BM) and pepCD47-derivatized foils demonstrated an M2/M1 macrophage shift with pepCD47 coating. HC and normocholesterolemic (NC) rabbit cohorts underwent bilateral implantation of BM and pepCD47 stents (HC) or BM stents only (NC) in the iliac location. RESULTS: A 40 % inhibition of cell attachment to pepCD47-modified compared to BM surfaces was observed. HC increased neointimal growth at 4 weeks post BM stenting. These untoward outcomes were mitigated in hypercholesterolemic rabbits treated with pepCD47-derivatized stents. Compared to NC animals, inflammatory cytokine immunopositivity and macrophage infiltration of peri-strut areas increased in HC animals and were attenuated in HC rabbits treated with pepCD47 stents. CONCLUSIONS: Augmented inflammatory responses underlie severe ISR morphology in hypercholesterolemic rabbits. Blockage of initial platelet and leukocyte attachment to stent struts through CD47 functionalization of stents mitigates the pro-restenotic effects of hypercholesterolemia.

Intraprocedural endothelial cell seeding of arterial stents via biotin/avidin targeting mitigates in-stent restenosis.

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

Robust Chemical Strategy for Stably Labeling Polyester-Based Nanoparticles with BODIPY Fluorophores.

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 BODIPY505/515 (logPo/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.

Stent-Based Gene Delivery for Coronary Disease.

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.

Selective Retinoic Acid Receptor γ Antagonist 7C is a Potent Enhancer of BMP-Induced Ectopic Endochondral Bone Formation.

Bone morphogenetic proteins (BMPs) have been clinically applied for induction of bone formation in musculoskeletal disorders such as critical-sized bone defects, nonunions, and spinal fusion surgeries. However, the use of supraphysiological doses of BMP caused adverse events, which were sometimes life-threatening. Therefore, safer treatment strategies for bone regeneration have been sought for decades. Systemic administration of a potent selective antagonist of retinoic acid nuclear receptor gamma (RARγ) (7C) stimulated BMP-induced ectopic bone formation. In this study, we developed 7C-loaded poly lactic nanoparticles (7C-NPs) and examined whether local application of 7C enhances BMP-induced bone regeneration. The collagen sponge discs that absorbed recombinant human (rh) BMP-2 were implanted into the dorsal fascia of young adult mice to induce ectopic bone. The combination of rhBMP-2 and 7C-NP markedly increased the total bone volume and thickness of the bone shell of the ectopic bone in a dose-dependent manner compared to those with rhBMP-2 only. 7C stimulated sulfated proteoglycan production, expression of chondrogenic marker genes, and Sox9 reporter activity in both chondrogenic cells and MSCs. The findings suggest that selective RARγ antagonist 7C or the related compounds potentiate the bone inductive ability of rhBMP-2, as well as support any future research to improve the BMP-2 based bone regeneration procedures in a safe and efficient manner.

Stent-based delivery of AAV2 vectors encoding oxidation-resistant apoA1.

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 3H-cholesterol from macrophages to the media conditioned by endogenously produced apoA1(4WF) was 2.1-fold higher than for apoA1(WT) conditioned media in the presence of hypochlorous acid emulating conditions of oxidative stress. The proliferation of apoA1(WT)- and apoA1(4FW)-transduced rat aortic smooth muscle cells (SMC) was inhibited by 66% ± 10% and 65% ± 11%, respectively, in comparison with non-transduced SMC (p < 0.001). Conversely, the proliferation of apoA1(4FW)-transduced, but not apoA1(WT)-transduced rat blood outgrowth endothelial cells (BOEC) was increased 41% ± 5% (p < 0.001). Both apoA1 transduction conditions similarly inhibited basal and TNFα-induced reactive oxygen species in rat aortic endothelial cells (RAEC) and resulted in the reduced rat monocyte attachment to the TNFα-activated endothelium. AAV2-eGFP vectors immobilized reversibly on stainless steel mesh surfaces through the protein G/anti-AAV2 antibody coupling, efficiently transduced cells in culture modeling stent-based delivery. In vivo studies in normal pigs, deploying AAV2 gene delivery stents (GDS) preloaded with AAV2-eGFP in the coronary arteries demonstrated transduction of the stented arteries. However, implantation of GDS formulated with AAV2-apoA1(4WF) failed to prevent in-stent restenosis in the atherosclerotic vasculature of hypercholesterolemic diabetic pigs. It is concluded that stent delivery of AAV2-4WF while feasible, is not effective for mitigation of restenosis in the presence of severe atherosclerotic disease.

Poloxamer-linked prodrug of a topoisomerase I inhibitor SN22 shows efficacy in models of high-risk neuroblastoma with primary and acquired chemoresistance.

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]2  have the potential to effectively combat multiple mechanisms governing chemoresistance in newly diagnosed (chemo-naïve) and recurrent forms of aggressive malignancies. As a macromolecular carrier-based delivery system exhibiting remarkable efficacy against two particularly challenging forms of high-risk neuroblastoma, PF108-[SN22]2 can pave the way to a robust and clinically viable therapeutic strategy urgently needed for patients with multidrug-resistant disease presently lacking effective treatment options.

Poly-2-methyl-2-oxazoline-modified bioprosthetic heart valve leaflets have enhanced biocompatibility and resist structural degeneration.

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.

Nanocarrier-Based Delivery of SN22 as a Tocopheryl Oxamate Prodrug Achieves Rapid Tumor Regression and Extends Survival in High-Risk Neuroblastoma Models.

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.

Adeno-Associated Viral Vector Immobilization and Local Delivery from Bare Metal Surfaces.

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.

Environment-Sensitive Polymeric Micelles Encapsulating SN-38 Potently Suppress Growth of Neuroblastoma Cells Exhibiting Intrinsic and Acquired Drug Resistance.

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 <100 nm size were prepared using an AIP consisting of alternating blocks of poly(ethylene glycol) and polytetrahydrofuran (PEG600-PTHF650). The micellar drug applied in a low nanomolar range (10-50 nM) completely suppressed the growth of chemo-naïve NB cells even after a brief (10 min) exposure. Furthermore, extending the exposure to 24 h resulted in a profound and lasting inhibitory effect of the micellar formulation on the growth of NB cells exhibiting an acquired loss of p53 function. These results suggest that micelle-mediated delivery of SN-38 can potentially offer a new and effective strategy for treating different phases of high-risk disease, including those showing poor response to conventional therapies.

Mitigation of Blood Borne Cell Attachment to Metal Implants through CD47-Derived Peptide Immobilization.

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 like 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.

Experimental Single-Platform Approach to Enhance the Functionalization of Magnetically Targetable Cells.

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.

Structural Optimization and Enhanced Prodrug-Mediated Delivery Overcomes Camptothecin Resistance in High-Risk Solid Tumors.

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.

Studies of combined NO-eluting/CD47-modified polyurethane surfaces for synergistic enhancement of biocompatibility.

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.

Stability and bioactivity of pepCD47 attachment on stainless steel surfaces.

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.

Selective Agonists of Nuclear Retinoic Acid Receptor Gamma Inhibit Growth of HCS-2/8 Chondrosarcoma Cells.

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.

Enhanced Intratumoral Delivery of SN38 as a Tocopherol Oxyacetate Prodrug Using Nanoparticles in a Neuroblastoma Xenograft Model.

Purpose: Currently, <50% of high-risk pediatric solid tumors like neuroblastoma can be cured, and many survivors experience serious or life-threatening toxicities, so more effective, less toxic therapy is needed. One approach is to target drugs to tumors using nanoparticles, which take advantage of the enhanced permeability of tumor vasculature.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.

Optimizing endothelial cell functionalization for cell therapy of vascular proliferative disease using a direct contact co-culture system.

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[iNOSAd]) 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 iNOSAd 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.