Low molecular weight chitosan nanoparticulate system at low N:P ratio for nontoxic polynucleotide delivery

Chitosan, a natural polymer, is a promising system for the therapeutic delivery of both plasmid DNA and synthetic small interfering RNA. Reports attempting to identify the optimal parameters of chitosan for synthetic small interfering RNA delivery were inconclusive with high molecular weight at high amine-to-phosphate (N:P) ratios apparently required for efficient transfection. Here we show, for the first time, that low molecular weight chitosan (LMW-CS) formulations at low N:P ratios are suitable for the in vitro delivery of small interfering RNA. LMW-CS nanoparticles at low N:P ratios were positively charged (ζ-potential ~20 mV) with an average size below 100 nm as demonstrated by dynamic light scattering and environmental scanning electron microscopy, respectively. Nanoparticles were spherical, a shape promoting decreased cytotoxicity and enhanced cellular uptake. Nanoparticle stability was effective for at least 20 hours at N:P ratios above two in a slightly acidic pH of 6.5. At a higher basic pH of 8, these nanoparticles were unravelled due to chitosan neutralization, exposing their polynucleotide cargo. Cellular uptake ranged from 50% to 95% in six different cell lines as measured by cytometry. Increasing chitosan molecular weight improved nanoparticle stability as well as the ability of nanoparticles to protect the oligonucleotide cargo from nucleases at supraphysiological concentrations. The highest knockdown efficiency was obtained with the specific formulation 92-10-5 that combines sufficient nuclease protection with effective intracellular release. This system attained >70% knockdown of the messenger RNA, similar to commercially available lipoplexes, without apparent cytotoxicity. Contrary to previous reports, our data demonstrate that LMW-CS at low N:P ratios are efficient and nontoxic polynucleotide delivery systems capable of transfecting a plethora of cell lines

Injectable Lyophilized Chitosan-Thrombin-Platelet-Rich Plasma (CS-FIIa-PRP) Implant to Promote Tissue Regeneration: In Vitro and Ex Vivo Solidification Properties

RÉSUMÉ: Freeze-dried chitosan formulations solubilized in platelet-rich plasma (PRP) are currently evaluated as injectable implants with the potential for augmenting the standard of care for tissue repair in different orthopedic conditions. The present study aimed to shorten the solidification time of such implants, leading to an easier application and a facilitated solidification in a wet environment, which were direct demands from orthopedic surgeons. The addition of thrombin to the formulation before lyophilization was explored. The challenge was to find a formulation that coagulated fast enough to be applied in a wet environment but not too fast, which would make handling/injection difficult. Four thrombin concentrations were analyzed (0.0, 0.25, 0.5, and 1.0 NIH/mL) in vitro (using thromboelastography, rheology, indentation, syringe injectability, and thrombin activity tests) as well as ex vivo (by assessing the implant’s adherence to tendon tissue in a wet environment). The biomaterial containing 0.5 NIH/mL of thrombin significantly increased the coagulation speed while being easy to handle up to 6 min after solubilization. Furthermore, the adherence of the biomaterial to tendon tissues was impacted by the biomaterial-tendon contact duration and increased faster when thrombin was present. These results suggest that our biomaterial has great potential for use in regenerative medicine applications

Stability and binding affinity of DNA/chitosan complexes by polyanion competition

The stability of DNA/chitosan complexes upon exposure to hyaluronic acid, chondroitin sulfate, and heparin, was assessed by fluorescence spectroscopy to quantify DNA release. Only the highly charged heparin was found to release DNA from the complexes. Complex stability upon exposure to heparin increased with the degree of deacetylation and molecular weight of chitosan and with the ratio of chitosan amino groups to DNA phosphate groups (N/P ratio) in the complexes. Isothermal titration microcalorimetry revealed that among polyanions tested, only heparin has a binding affinity to chitosan approaching that of DNA and can therefore release DNA from the complexes. These results also indicate that anionic components with sufficiently high charge density can induce extracellular or intracellular release of DNA, the former negatively affecting delivery efficiency while the latter is required for gene transfer to occur. Our findings also suggest that increased N/P ratio of the complexes can play an important role in preventing premature dissociation of DNA/polycation complexes upon interaction with anionic components in extracellular milieu. (C) 2017 Elsevier Ltd. All rights reserved.Peer reviewe

Regioselective chitosan end-group activation: the triskelion approach

Chitosan (CS) end-group conjugation methods are rarely reported in the literature, mainly since the CS terminal aldehyde moiety produced by nitrous acid depolymerization is only present in trace amounts in its reactive form. In a previous study, our group proposed an intermolecular thioacetylation process that allowed terminal conjugation of thiol-reactive species to chitosan with 50% efficiency. However, this reaction is incompatible with acid-labile substituents and the conversion efficiency of CS end-groups could be limited by the size of the thiol-reactive species engaged in the reaction, mainly by steric hindrance since two substituents are required to obtain the stabilized thioacetal derivative. In the present study, we developed a novel CS end-group thioacetylation approach relying on a new regioselective linker that bears three thiol moieties. This trivalent linker, referred to as triskelion here, was specifically designed for activation of the CS 2,5-anhydro-D-mannose (M-Unit) end-group and consists of a thiolhook for efficient aldehyde conjugation through an intramolecular reaction and a thiol-tail that remains available for subsequent end-group functionalization with any thiol-reactive species. The chemical synthesis of this linker provided the desired material with high yields over three steps. The in situ intramolecular thioacetylation process between the triskelion linker and 2,5-anhydro-D-mannose (M-Unit, monomeric) was assessed by semi-quantitative LC-MS studies, revealing that the corresponding intramolecular thioacetal largely predominated (similar to 90%). This regioselective derivatization was also performed onto M-Unit CS aldehydes and the desired CS-b-triskelion conjugates were obtained with functionalization degrees over 85%, as confirmed by NMR spectroscopy (H-1 and DOSY). As a final assessment of the CS-b-triskelion thiol-tail reactivity, these conjugates were successfully engaged with thiol-reactive magnetic beads into disulfide bond displacement with 50% efficiency. The proposed CS terminal activation with the triskelion linker opens new perspectives for biomedical applications, especially brush-like surface modifications and other copolymer formation through disulfide linkages or Michael-type additions

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 (H-1 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-PEG(2) 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

Lysosomal rupture induced by structurally distinct chitosans either promotes a type 1 IFN response or activates the inflammasome in macrophages

Chitosan is a family of glucosamine and N-acetyl glucosamine polysaccharides with poorly understood immune modulating properties. Here, functional U937 macrophage responses were analyzed in response to a novel library of twenty chitosans with controlled degree of deacetylation (DDA, 60-98%), molecular weight (1 to >100 kDa), and acetylation pattern (block vs. random). Specific chitosan preparations (10 or 190 kDa 80% block DDA and 3, 5, or 10 kDa 98% DDA) either induced macrophages to release CXCL10 and IL-1ra at 5-50 mug/mL, or activated the inflammasome to release IL-1beta and PGE2 at 50-150 mug/mL. Chitosan induction of these factors required lysosomal acidification. CXCL10 production was preceded by lysosomal rupture as shown by time-dependent co-localization of galectin-3 and chitosan and slowed autophagy flux, and specifically depended on IFN-beta paracrine activity and STAT-2 activation that could be suppressed by PGE2. Chitosan induced a type I IFN paracrine response or inflammasome response depending on the extent of lysosomal rupture and cytosolic foreign body invasion. This study identifies the structural motifs that lead to chitosan-driven cytokine responses in macrophages and indicates that lysosomal rupture is a key mechanism that determines the endogenous release of either IL-1ra or IL-1beta

Enhanced Gene Delivery Mediated by Low Molecular Weight Chitosan/DNA Complexes: Effect of pH and Serum

This study was designed to systematically evaluate the influence of pH and serum on the transfection process of chitosan-DNA complexes, with the objective of maximizing their efficiency. The hydrodynamic diameter of the complexes, measured by dynamic light scattering (DLS), was found to increase with salt and pH from 243 nm in water to 1244 nm in PBS at pH 7.4 and aggregation in presence of 10% serum. The cellular uptake of complexes into HEK 293 cells assessed by flow cytometry and confocal fluorescent imaging was found to increase at lower pH and serum. Based on these data, new methodology were tested and high levels of transfection (>40%) were achieved when transfection was initiated at pH 6.5 with 10% serum for 8-24 h to maximize uptake and then the media was changed to pH 7.4 with 10% serum for an additional 24-40 h period. Cytotoxicity of chitosan/DNA complexes was also considerably lower than Lipofectamine. Our study demonstrates that the evaluation of the influence of important parameters in the methodology of transfection enables the understanding of crucial physicochemical and biological mechanisms which allows for the design of methodologies maximising transgene expression