Injectable chitosan-platelet-rich plasma implants to promote tissue regeneration: in vitro properties, in vivo residence, degradation, cell recruitment and vascularization.

The purpose of this study was to develop freeze-dried chitosan formulations that can be solubilized in platelet-rich plasma (PRP) to form injectable implants for tissue repair. A systematic approach to adjust formulation parameters, including chitosan number average molar mass (Mn ), chitosan concentration and lyoprotectant concentration, was undertaken to identify compositions that would rapidly (< 1 min) and completely solubilize in PRP, would have paste-like handling properties upon solubilization and coagulate rapidly (< 5 min) to form solid chitosan-PRP hybrid implants that are stable and homogenous. Freeze-dried cakes containing calcium chloride, as well as distinct chitosan Mn , chitosan concentration and lyoprotectant concentration, were prepared. PRP was used to solubilize the freeze-dried cakes and assess in vitro and in vivo performance, the latter as dorsal subcutaneous injections into New Zealand White rabbits. Freeze-dried polymer formulations containing low and medium chitosan Mn and concentrations were rapidly and completely solubilized in PRP. The paste-like chitosan-PRP mixtures coagulated quickly to form solid chitosan-PRP hybrids, which retracted much less than PRP-only controls. Homogeneous dispersion of chitosan within the hybrid clots was strongly dependent on chitosan Mn , and occurred only with medium Mn chitosan. Chitosan-PRP hybrid clots were resident subcutaneously in vivo until at least 2 weeks while PRP controls were quickly degraded in one day. Compared to PRP alone, chitosan-PRP hybrids had much greater capacity to induce local cell recruitment accompanied by angiogenesis, suggesting a strong potential for their use in regenerative medicine. Copyright © 2017 John Wiley & Sons, Ltd.

Regioselective thioacetylation of chitosan end-groups for nanoparticle gene delivery systems.

Chitosan (CS) end-group chemistry is a conjugation strategy that has been minimally exploited in the literature to date. Although the open-chain form of the CS reducing extremity bears a reactive aldehyde moiety, the most common method to generate a reactive end-group on CS is nitrous acid depolymerization, which produces a 2,5-anhydro-d-mannose unit (M-Unit) bearing also an aldehyde moiety. However, the availability of the latter might be low, since previous literature suggests that its hydrated and non-reactive form, namely the gem-diol form, is predominant in acidic aqueous conditions. Oxime-click chemistry has been used to react on such aldehydes with various degrees of success, but the use of a co-solvent and additional chemical reagents remain necessary to obtain the desired and stable covalent linkage. In this study, we have assessed the availability of the aldehyde reactive form on chitosan treated with nitrous acid. We have also assessed its reactivity towards thiol-bearing molecules in acidic conditions where CS amino groups are fully protonated and thus unreactive towards aldehyde. LC-MS and NMR spectroscopy methods (1H and DOSY, respectively) confirmed the regioselective thioacetylation of the reactive aldehyde with conversion rates between 55 and 70% depending on the thiol molecule engaged. The stabilization of the hemithioacetal intermediates into the corresponding thioacetals was also found to be facilitated upon freeze-drying of the reaction medium. The PEGylation of the CS M-Unit aldehyde by thioacetylation was also performed as a direct application of the proposed conjugation approach. CS-b-PEG2 block copolymers were successfully synthesized and were used to prepare block ionomer complexes with plasmid DNA, as revealed by their spherical morphology vs. the rod-like/globular/toroidal morphology observed for polyplexes prepared using native unmodified chitosan. This novel aqueous thiol-based conjugation strategy constitutes an alternative to the oxime-click pathway; it could be applicable to other polymers.

Chitosan-plasmid nanoparticle formulations for IM and SC delivery of recombinant FGF-2 and PDGF-BB or generation of antibodies.

Growth factor therapy is an emerging treatment modality that enhances tissue vascularization, promotes healing and regeneration and can treat a variety of inflammatory diseases. Both recombinant human growth factor proteins and their gene therapy are in human clinical trials to heal chronic wounds. As platelet-derived growth factor-bb (PDGF-BB) and fibroblast growth factor-2 (FGF-2) are known to induce chemotaxis, proliferation, differentiation, and matrix synthesis, we investigated a non-viral means for gene delivery of these factors using the cationic polysaccharide chitosan. Chitosan is a polymer of glucosamine and N-acetyl-glucosamine, in which the percentage of the residues that are glucosamine is called the degree of deacetylation (DDA). The purpose of this study was to express PDGF-BB and FGF-2 genes in mice using chitosan-plasmid DNA nanoparticles for the controlled delivery of genetic material in a specific, efficient, and safe manner. PDGF-BB and FGF-2 genes were amplified from human tissues by RT-PCR. To increase the secretion of FGF-2, a recombinant 4sFGF-2 was constructed bearing eight amino-acid residues of the signal peptide of FGF-4. PCR products were inserted into the expression vector pVax1 to produce recombinant plasmids pVax1-4sFGF2 and pVax1-PDGF-BB, which were then injected into BALB/C mice in the format of polyelectrolyte nanocomplexes with specific chitosans of controlled DDA and molecular weight, including 92-10, 80-10, and 80-80 (DDA-number average molecular weight or M(n) in kDa). ELISA assays on mice sera showed that recombinant FGF-2 and PDGF-BB proteins were efficiently expressed and specific antibodies to these proteins could be identified in sera of injected mice, but with levels that were clearly dependent on the specific chitosan used. We found high DDA low molecular weight chitosans to be efficient protein expressors with minimal or no generation of neutralizing antibodies, while lowering DDA resulted in greater antibody levels and correspondingly lower levels of detected recombinant protein. Histological analyses corroborated these results by revealing greater inflammatory infiltrates in lower DDA chitosans, which produced higher antibody titers. We found, in general, a more efficient delivery of the plasmids by subcutaneous than by intramuscular injection. Specific chitosan carriers were identified to be either efficient non-toxic therapeutic protein delivery systems or vectors for DNA vaccines.

A validated 1H NMR method for the determination of the degree of deacetylation of chitosan.

A method for the determination of the degree of deacetylation (DDA) of chitosan by 1H NMR spectroscopy has been formally validated. Chitosans with DDA ranging from 48 to 100% have been used for the validation. The method is found to be simple, rapid and more precise than other known techniques like IR or titration for %DDA measurements. The precision, ruggedness, robustness, specificity, stability and accuracy of the technique are discussed in this paper.