A tunable delivery platform to provide local chemotherapy for pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most devastating and painful cancers. It is often highly resistant to therapy owing to inherent chemoresistance and the desmoplastic response that creates a barrier of fibrous tissue preventing transport of chemotherapeutics into the tumor. The growth of the tumor in pancreatic cancer often leads to invasion of other organs and partial or complete biliary obstruction, inducing intense pain for patients and necessitating tumor resection or repeated stenting. Here, we have developed a delivery device to provide enhanced palliative therapy for pancreatic cancer patients by providing high concentrations of chemotherapeutic compounds locally at the tumor site. This treatment could reduce the need for repeated procedures in advanced PDAC patients to debulk the tumor mass or stent the obstructed bile duct. To facilitate clinical translation, we created the device out of currently approved materials and drugs. We engineered an implantable poly(lactic-co-glycolic)-based biodegradable device that is able to linearly release high doses of chemotherapeutic drugs for up to 60 days. We created five patient-derived PDAC cell lines and tested their sensitivity to approved chemotherapeutic compounds. These in vitro experiments showed that paclitaxel was the most effective single agent across all cell lines. We compared the efficacy of systemic and local paclitaxel therapy on the patient-derived cell lines in an orthotopic xenograft model in mice (PDX). In this model, we found up to a 12-fold increase in suppression of tumor growth by local therapy in comparison to systemic administration and reduce retention into off-target organs. Herein, we highlight the efficacy of a local therapeutic approach to overcome PDAC chemoresistance and reduce the need for repeated interventions and biliary obstruction by preventing local tumor growth. Our results underscore the urgent need for an implantable drug-eluting platform to deliver cytotoxic agents directly within the tumor mass as a novel therapeutic strategy for patients with pancreatic cancer.

The role of scaffold microarchitecture in engineering endothelial cell immunomodulation.

The implantation of matrix-embedded endothelial cells (MEECs) has been reported to have great therapeutic potential in controlling the vascular response to injury and maintaining patency in arteriovenous anastomoses. While there is an appreciation of their effectiveness in clinical and animal studies, the mechanisms through which they mediate these powerful effects remain relatively unknown. In this work, we examined the hypothesis that the 3-dimensional microarchitecture of the tissue engineering scaffold was a key regulator of endothelial behavior in MEEC constructs. Notably, we found that ECs in porous collagen scaffold had a markedly altered cytoskeletal structure with oriented actin fibers and rearrangement of the focal adhesion proteins in comparison to cells grown on 2D surfaces. We examined the immunomodulatory capabilities of MEECs and discovered that they were able to reduce the recruitment of monocytes to an inflamed endothelial monolayer by 5-fold compared to EC on 2D surfaces. An analysis of secreted factors from the cells revealed an 8-fold lower release of Monocyte Chemotactic Protein-1 (MCP-1) from MEECs. Differences between 3D and 2D cultured cells were abolished in the presence of inhibitors to the focal adhesion associated signaling molecule Src suggesting that adhesion-mediated signaling is essential in controlling the potent immunomodulatory effects of MEEC.

Heparanase regulates thrombosis in vascular injury and stent-induced flow disturbance.

OBJECTIVES: The purpose of this study was to examine the role of heparanase in controlling thrombosis following vascular injury or endovascular stenting. BACKGROUND: The use of endovascular stents are a common clinical intervention for the treatment of arteries occluded due to vascular disease. Both heparin and heparan sulfate are known to be potent inhibitors of thrombosis. Heparanase is the major enzyme that degrades heparan sulfate in mammalian cells. This study examined the role of heparanase in controlling thrombosis following vascular injury and stent-induced flow disturbance. METHODS: This study used mice overexpressing human heparanase and examined the time to thrombosis using a laser-induced arterial thrombosis model in combination with vascular injury. An ex vivo system was used to examine the formation of thrombus to stent-induced flow disturbance. RESULTS: In the absence of vascular injury, wild type and heparanase overexpressing (HPA Tg) mice had similar times to thrombosis in a laser-induced arterial thrombosis model. However, in the presence of vascular injury, the time to thrombosis was dramatically reduced in HPA Tg mice. An ex vivo system was used to flow blood from wild type and HPA Tg mice over stents and stented arterial segments from both animal types. These studies demonstrate markedly increased thromboses on stents with blood isolated from HPA Tg mice in comparison to blood from wild type animals. We found that blood from HPA Tg animals had markedly increased thrombosis when applied to stented arterial segments from either wild type or HPA Tg mice. CONCLUSIONS: Taken together, this study's results indicate that heparanase is a powerful mediator of thrombosis in the context of vascular injury and stent-induced flow disturbance.

Matrix-embedded endothelial cells are protected from the uremic milieu.

BACKGROUND: Endothelial cells (ECs) embedded in 3D matrices [matrix-embedded endothelial cells (MEECs)] of denatured collagen implanted around vascular access anastomoses preserve luminal patency. MEEC implant efficacy depends on embedded EC health. As the uremic milieu inhibits proliferation and induces apoptosis of ECs, we examined whether uremia might impact MEECs. METHODS: ECs grown on 2D gelatin-coated polystyrene tissue culture plates (gTCPS) or in MEEC were treated with sera pooled from 20 healthy control or uremic patients with end-stage renal disease. EC viability was examined using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide assay, cell counting and Trypan blue exclusion. Media conditioned (CM) with 2 and 3D-supported ECs were examined for its potential to inhibit vascular smooth muscle cell (vSMC) proliferation using (3)[H] thymidine incorporation and cyclin D1 expression. ECs grown on gTCPS were treated with uremic serum filtered through matrices to examine if matrices retain uremic toxins or whether EC effects were cell mediated. RESULTS: Uremic serum significantly reduced viability and number of live, and increased dead ECs when grown on gTCPS, but not in MEECs. EC survival correlated with vSMC inhibition. While CM from ECs grown in gTCPS with uremic serum inhibited vSMC proliferation no better than uremic serum alone (22 versus 27%), MEEC CM inhibited vSMC proliferation by 47% (P = 0.0004). Cyclin D1 expression tracked with indices of vSMC proliferation. There was no significant difference in EC viability between EC treated with matrix-filtered or unfiltered uremic serum. CONCLUSION: The viability, number and efficacy of MEECs were preserved in uremic serum compared to those of ECs on gTCPS. MEECs are protected from uremic toxicity, not from retention of uremic toxins by matrices, but likely from intrinsic changes in EC sensitivity to uremia. MEECs implanted at vascular access sites should inhibit neointimal hyperplasia in uremia. This study underscores the robustness of matrix embedding as a cell protectant, especially in hostile environments like uremia.

Microsphere-integrated drug-eluting stents: PLGA microsphere integration in hydrogel coating for local and prolonged delivery of hydrophilic antirestenosis agents.

The development of a novel generation of drug-eluting stent (DES) relies upon the idea to obtain very flexible platforms able to overcome some issues associated to available devices and widen their field of application, especially to the currently emerging biotech therapeutics. Here, we propose a new concept of DES named microsphere-integrated drug-eluting stent (MIDES) composed of drug eluting biodegradable poly(D,L-lactide-co-glycolide) microspheres--encapsulating an hydrophilic model molecule (dextran)--fully integrated in a poly(2-hydroxy-ethyl-methacrylate) coating. By implementing a modified spray-coating technique, we have been able to achieve a thin (10 µm), smooth, and homogeneous hydrogel surface embedding underneath a population of two different microparticles formulations--Dex502H and Dex506. The amount of drug can be tailored, resulting in a dextran loading as high as 1.4 µg/cm, by simply reiteration of coating layer deposition making the MIDES a custom-made device where the release kinetics can be further modified by opportunely choosing microsphere properties. DES use is nowadays restricted to delivery of hydrophobic pharmaceuticals; release of hydrophilic therapeutics from MIDES can, however, be finely controlled by specifically engineering biodegradable microspheres. By varying polymer resomer, we obtained a tunable release rate in the first month of delivery. Depending on the microspheres properties release profile changes drastically moving from a biphasic release, in the case of Dex502H, with a burst of about 20% in the first day to a more sustained release for Dex506 particles. As proof of principle, we also demonstrated that MIDES approach can allows the delivery of two different agents opening up the way to a multitherapy in restenosis treatment.

Tubular bridges for bronchial epithelial cell migration and communication.

BACKGROUND: Biological processes from embryogenesis to tumorigenesis rely on the coordinated coalescence of cells and synchronized cell-to-cell communication. Intercellular signaling enables cell masses to communicate through endocrine pathways at a distance or by direct contact over shorter dimensions. Cellular bridges, the longest direct connections between cells, facilitate transfer of cellular signals and components over hundreds of microns in vitro and in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Using various cellular imaging techniques on human tissue cultures, we identified two types of tubular, bronchial epithelial (EP) connections, up to a millimeter in length, designated EP bridges. Structurally distinct from other cellular connections, the first type of EP bridge may mediate transport of cellular material between cells, while the second type of EP bridge is functionally distinct from all other cellular connections by mediating migration of epithelial cells between EP masses. Morphological and biochemical interactions with other cell types differentially regulated the nuclear factor-kappaB and cyclooxygenase inflammatory pathways, resulting in increased levels of inflammatory molecules that impeded EP bridge formation. Pharmacologic inhibition of these inflammatory pathways caused increased morphological and mobility changes stimulating the biogenesis of EP bridges, in part through the upregulation of reactive oxygen species pathways. CONCLUSIONS/SIGNIFICANCE: EP bridge formation appears to be a normal response of EP physiology in vitro, which is differentially inhibited by inflammatory cellular pathways depending upon the morphological and biochemical interactions between EP cells and other cell types. These tubular EP conduits may represent an ultra long-range form of direct intercellular communication and a completely new mechanism of tissue-mediated cell migration.

Bioactivated collagen-based scaffolds embedding protein-releasing biodegradable microspheres: tuning of protein release kinetics.

In tissue engineering, the recapitulation of natural sequences of signaling molecules, such as growth factors, as occurring in the native extracellular matrix (ECM), is fundamental to support the stepwise process of tissue regeneration. Among the manifold of tissue engineering strategies, a promising one is based on the creation of the chrono-programmed presentation of different signaling proteins. This approach is based upon the integration of biodegradable microspheres, loaded with suitable protein molecules, within scaffolds made of collagen and, in case, hyaluronic acid, which are two of the fundamental ECM constituents. However, for the design of bioactivated gel-like scaffolds the determination of release kinetics must be performed directly within the tissue engineering template. In this work, biodegradable poly(lactic-co-glycolic)acid (PLGA) microspheres were produced by the multiple emulsion-solvent evaporation technique and loaded with rhodamine-labelled bovine serum albumin (BSA-Rhod), a fluorescent model protein. The microdevices were dispersed in collagen gels and collagen-hyaluronic acid (HA) semi-interpenetrating networks (semi-IPNs). BSA-Rhod release kinetics were studied directly on single microspheres through confocal laser scanning microscopy (CLSM). To thoroughly investigate the mechanisms governing protein release from PLGA microspheres in gels, BSA-Rhod diffusion in gels was determined by fluorescence correlation spectroscopy (FCS), and water transport through the microsphere bulk was determined by dynamic vapor sorption (DVS). Moreover, the decrease of PLGA molecular weight and glass transition temperature (T(g)) were determined by gel permeation chromatography (GPC) and differential scanning calorimetry (DSC), respectively. Results indicate that protein release kinetics and delivery onset strongly depend on the complex interplay between protein transport through the PLGA matrix and in the collagen-based release media, and water sequestration within the scaffolds, related to the scaffold hydrophilicity, which is dictated by HA content. The proper manipulation of all these features may thus allow the obtainment of a fine control over protein sequential delivery and release kinetics within tissue-engineering scaffolds.

Coating process and early stage adhesion evaluation of poly(2-hydroxy-ethyl-methacrylate) hydrogel coating of 316L steel surface for stent applications.

In this study, a spray-coating method has been set up with the aim to control the coating of poly(2-hydroxy-ethyl-methacrylate) (pHEMA), an hydrophilic polymeric hydrogel, onto the complex surface of a 316L steel stent for percutaneous coronary intervention (PCI). By varying process parameters, tuneable thicknesses, from 5 to 20 microm, have been obtained with uniform and homogeneous surface without crack or bridges. Surface characteristics of pHEMA coating onto metal surface have been investigated through FTIR-ATR, contact angle measurement, SEM, EDS and AFM. Moreover, results from Single-Lap-Joint and Pull-Off adhesion tests as well as calorimetric analysis of glass transition temperature suggested that pHEMA deposition is firmly adhered on metallic surface. The pHEMA coating evaluation of roughness, wettability together with its morphological and chemical stability after three cycles of expansion-crimping along with preliminary results after 6 months demonstrates the suitability of the coating for surgical implantation of stent.

Microsphere-integrated collagen scaffolds for tissue engineering: effect of microsphere formulation and scaffold properties on protein release kinetics.

A promising approach to control the time and space distribution of signalling molecules inside tissue engineering scaffolds consists in entrapping biodegradable microspheres releasing the protein locally for long time frames. However, a rational design of microsphere-integrated scaffolds requires the knowledge of protein release profiles directly within the polymeric template. In this work, PLGA microspheres encapsulating rhodamine-labelled bovine serum albumin (BSA-Rhod) as a model protein were produced in different formulation conditions and tested for their release features in solution and in collagen and collagen/hyaluronic acid (HA) scaffolds. BSA-Rhod release profiles from single microspheres in solution and within the scaffold were assessed by using a confocal laser scanning microscopy (CLSM)-assisted method. Results suggest that the same diffusion-erosion process controls BSA-Rhod release from microspheres in solution and collagen. Nonetheless, two main factors contribute protein release within the scaffold, that is water activity in the release environment and transport properties of the protein in the gel. While microsphere formulation mainly controls the induction time necessary to activate protein release, polymer scaffold composition governs the release rate. Thus, the fine regulation of a tissue engineering construct may be obtained by an appropriate combination of microspheres and scaffolds, providing a spatial and temporal control over signalling molecule delivery.