Controlled Delivery of Pan-PAD-Inhibitor Cl-Amidine Using Poly(3-Hydroxybutyrate) Microspheres.

This study deals with the process of optimization and synthesis of Poly(3-hydroxybutyrate) microspheres with encapsulated Cl-amidine. Cl-amidine is an inhibitor of peptidylarginine deiminases (PADs), a group of calcium-dependent enzymes, which play critical roles in a number of pathologies, including autoimmune and neurodegenerative diseases, as well as cancer. While Cl-amidine application has been assessed in a number of in vitro and in vivo models; methods of controlled release delivery remain to be investigated. P(3HB) microspheres have proven to be an effective delivery system for several compounds applied in antimicrobial, wound healing, cancer, and cardiovascular and regenerative disease models. In the current study, P(3HB) microspheres with encapsulated Cl-amidine were produced in a size ranging from ~4-5 µm and characterized for surface morphology, porosity, hydrophobicity and protein adsorption, in comparison with empty P(3HB) microspheres. Cl-amidine encapsulation in P(3HB) microspheres was optimized, and these were found to be less hydrophobic, compared with the empty microspheres, and subsequently adsorbed a lower amount of protein on their surface. The release kinetics of Cl-amidine from the microspheres were assessed in vitro and expressed as a function of encapsulation efficiency. There was a burst release of ~50% Cl-amidine in the first 24 h and a zero order release from that point up to 16 days, at which time point ~93% of the drug had been released. As Cl-amidine has been associated with anti-cancer effects, the Cl-amidine encapsulated microspheres were assessed for the inhibition of vascular endothelial growth factor (VEGF) expression in the mammalian breast cancer cell line SK-BR-3, including in the presence of the anti-proliferative drug rapamycin. The cytotoxicity of the combinatorial effect of rapamycin with Cl-amidine encapsulated P(3HB) microspheres was found to be 3.5% more effective within a 24 h period. The cells treated with Cl-amidine encapsulated microspheres alone, were found to have 36.5% reduction in VEGF expression when compared with untreated SK-BR-3 cells. This indicates that controlled release of Cl-amidine from P(3HB) microspheres may be effective in anti-cancer treatment, including in synergy with chemotherapeutic agents. Using controlled drug-delivery of Cl-amidine encapsulated in Poly(3-hydroxybutyrate) microspheres may be a promising novel strategy for application in PAD-associated pathologies.

Enhanced production of biobased, biodegradable, Poly(3-hydroxybutyrate) using an unexplored marine bacterium Pseudohalocynthiibacter aestuariivivens, isolated from highly polluted coastal environment.

The production and disposal of plastics from limited fossil reserves, has prompted research for greener and sustainable alternatives. Polyhydroxyalkanoates (PHAs) are biocompatible, biodegradable, and thermoprocessable polyester produced by microbes. PHAs found several applications but their use is limited due to high production cost and low yields. Herein, for the first time, the isolation and characterization of Pseudohalocynthiibacter aestuariivivens P96, a marine bacterium able to produce surprising amount of PHAs is reported. In the best growth condition P96 was able to reach a maximum production of 4.73 g/L, corresponding to the 87 % of total cell dry-weight. Using scanning and transmission microscopy, lab-scale fermentation, spectroscopic techniques, and genome analysis, the production of thermoprocessable polymer Polyhydroxybutyrate P(3HB), a PHAs class, endowed with mechanical and thermal properties comparable to that of petroleum-based plastics was confirmed. This study represents a milestone toward the use of this unexplored marine bacterium for P(3HB) production.

Bioresorbable and Mechanically Optimized Nerve Guidance Conduit Based on a Naturally Derived Medium Chain Length Polyhydroxyalkanoate and Poly(ε-Caprolactone) Blend.

Severe peripheral nerve injuries represent a large clinical problem with relevant challenges such as the development of successful synthetic scaffolds as substitutes to autologous nerve grafting. Numerous studies have reported the use of polyesters and type I collagen-based nerve guidance conduits (NGCs) to promote nerve regeneration through critical nerve defects while providing protection from external factors. However, none of the commercially available hollow bioresorbable NGCs have demonstrated superior clinical outcomes to an autologous nerve graft. Hence, new materials and NGC geometries have been explored in the literature to mimic the native nerve properties and architecture. Here, we report a novel blend of a natural medium chain length polyhydroxyalkanoate (MCL-PHA) with a synthetic aliphatic polyester, poly(ε-caprolactone) (PCL), suitable for extrusion-based high-throughput manufacturing. The blend was designed to combine the excellent ability of PHAs to support the growth and proliferation of mammalian cells with the good processability of PCL. The material exhibited excellent neuroregenerative properties and a good bioresorption rate, while the extruded porous tubes exhibited similar mechanical properties to the rat sciatic nerve. The NGCs were implanted to treat a 10 mm long sciatic nerve defect in rats, where significant differences were found between thin and thick wall thickness implants, and both electrophysiological and histological data, as well as the number of recovered animals, provided superior outcomes than the well-referenced synthetic Neurolac NGC.

Binary polyhydroxyalkanoate systems for soft tissue engineering.

Progress in tissue engineering is dependent on the availability of suitable biomaterials. In an effort to overcome the brittleness of poly(3-hydroxybutyrate), P(3HB), a natural biodegradable polyester, and widen its biomedical applications, plasticising of P(3HB) with oligomeric substances of related structure has been studied. A biosynthesised medium-chain-length polyhydroxyalkanoate (mcl-PHA) copolymer, the plasticiser precursor, was obtained using vegetable waste frying oil as a sole carbon source. The mcl-PHA was transformed into an oligomeric derivative by acid hydrolysis. The plasticising effect of the oligomeric mcl-PHA on P(3HB) was studied via characterisation of thermal and mechanical properties of the blends in the course of ageing at ambient conditions. Addition of oligomeric mcl-PHA to P(3HB) resulted in softer and more flexible materials based entirely on PHAs. It was shown that the oligomeric mcl-PHA transformed highly crystalline P(3HB) into materials with a dominant amorphous phase when the content of oligomeric mcl-PHA exceeded 10 wt%. In vitro biocompatibility studies of the new binary PHA materials showed high viability and proliferation of C2C12 myoblast cells. Thus, the proposed approach for P(3HB) plasticisation has the potential for the generation of more pliable biomaterials based on P(3HB) which can find application in unique soft tissue engineering applications where a balance between stiffness, tensile strength and ductility is required. STATEMENT OF SIGNIFICANCE: Polyhydroxyalkanoates, a broad family of natural biodegradable and biocompatible polymers, have emerged as highly promising biomaterials both for bulk and biomedical applications. Here we describe an approach to tune the mechanical properties of stiff and brittle poly(3-hydroxybutyrate) and thereby to expand its potential biomedical applications. Plasticisation, a common practice in the plastic industry to modify polymer mechanical properties, has been used very cautiously for biomedical applications due to plasticiser toxicity and migration. We have developed a plasticiser for poly(3-hydroxybutyrate) based on a structurally related but softer and pliable medium chain length polyhydroxyalkanoate. Additives of oligomeric derivatives of this polymer improved ductility of poly(3-hydroxybutyrate), greatly widening the future applicability of this well-established biomaterial. In parallel, the binary polyhydroxyalkanoate materials also exhibited improved cell attachment and proliferation, a highly desirable outcome.

In Vivo Tracking and 1H/19F Magnetic Resonance Imaging of Biodegradable Polyhydroxyalkanoate/Polycaprolactone Blend Scaffolds Seeded with Labeled Cardiac Stem Cells.

Medium-chain length polyhydroxyalkanoates (MCL-PHAs) have demonstrated exceptional properties for cardiac tissue engineering (CTE) applications. Despite prior work on MCL-PHA/polycaprolactone (PCL) blends, optimal scaffold production and use as an alternative delivery route for controlled release of seeded cardiac progenitor cells (CPCs) in CTE applications in vivo has been lacking. We present herein applicability of MCL-PHA/PCL (95/5 wt %) blends fabricated as thin films with an improved performance compared to the neat MCL-PHA. Polymer characterization confirmed the chemical structure and composition of the synthesized scaffolds, while thermal, wettability, and mechanical properties were also investigated and compared in neat and porous counterparts. In vitro cytocompatibility studies were performed using perfluorocrown-ether-nanoparticle-labeled murine CPCs and studied using confocal microscopy and 19F magnetic resonance spectroscopy and magnetic resonance imaging (MRI). Seeded scaffolds were implanted and studied in the postmortem murine heart in situ and in two additional C57BL/6 mice in vivo (using single-layered and double-layered scaffolds) and imaged immediately after and at 7 days postimplantation. Superior MCL-PHA/PCL scaffold performance has been demonstrated compared to MCL-PHA through experimental comparisons of (a) morphological data using scanning electron microscopy and (b) contact angle measurements attesting to improved CPC adhesion, (c) in vitro confocal microscopy showing increased SC proliferative capacity, and (d) mechanical testing that elicited good overall responses. In vitro MRI results justify the increased seeding density, increased in vitro MRI signal, and improved MRI visibility in vivo, in the double-layered compared to the single-layered scaffolds. Histological evaluations [bright-field, cytoplasmic (Atto647) and nuclear (4',6-diamidino-2-phenylindole) stains] performed in conjunction with confocal microscopy imaging attest to CPC binding within the scaffold, subsequent release and migration to the neighboring myocardium, and increased retention in the murine myocardium in the case of the double-layered scaffold. Thus, MCL-PHA/PCL blends possess tremendous potential for controlled delivery of CPCs and for maximizing possible regeneration in myocardial infarction.

Nanofibrous poly(3-hydroxybutyrate)/poly(3-hydroxyoctanoate) scaffolds provide a functional microenvironment for cartilage repair.

Articular cartilage defects, when repaired ineffectively, often lead to further deterioration of the tissue, secondary osteoarthritis and, ultimately, joint replacement. Unfortunately, current surgical procedures are unable to restore normal cartilage function. Tissue engineering of cartilage provides promising strategies for the regeneration of damaged articular cartilage. As yet, there are still significant challenges that need to be overcome to match the long-term mechanical stability and durability of native cartilage. Using electrospinning of different blends of biodegradable poly(3-hydroxybutyrate)/poly(3-hydroxyoctanoate), we produced polymer scaffolds and optimised their structure, stiffness, degradation rates and biocompatibility. Scaffolds with a poly(3-hydroxybutyrate)/poly(3-hydroxyoctanoate) ratio of 1:0.25 exhibit randomly oriented fibres that closely mimic the collagen fibrillar meshwork of native cartilage and match the stiffness of native articular cartilage. Degradation of the scaffolds into products that could be easily removed from the body was indicated by changes in fibre structure, loss of molecular weight and a decrease in scaffold stiffness after one and four months. Histological and immunohistochemical analysis after three weeks of culture with human articular chondrocytes revealed a hyaline-like cartilage matrix. The ability to fine tune the ultrastructure and mechanical properties using different blends of poly(3-hydroxybutyrate)/poly(3-hydroxyoctanoate) allows to produce a cartilage repair kit for clinical use to reduce the risk of developing secondary osteoarthritis. We further suggest the development of a toolbox with tailor-made scaffolds for the repair of other tissues that require a 'guiding' structure to support the body's self-healing process.