Nanoassemblies of Chitosan-Based Polyelectrolyte Complexes as Nucleic Acid Delivery Systems.

Nucleic acid delivery requires vectorization for protection from nucleases, preventing clearance by the reticuloendothelial system, and targeting to allow cellular uptake. Nanovectors meeting the above specifications should be safe for the patient, simple to manufacture, and display long-term stability. Our nanovectors were obtained via the green process of polyelectrolyte complexation, carried out at 25 °C in water at a low shear rate using chitosan (a polycationic biocompatible polysaccharide of specific molar mass and acetylation degree) and dextran sulfate as a polyanionic biocompatible polysaccharide. These complexes formed nanoassemblies of primary nanoparticles (20-35 nm) and maintained their colloidal stability for over 1 year at 25 °C. They could be steam sterilized, and a model nucleic acid could be either encapsulated or surface adsorbed. A targeting agent was finally bound to their surface. This work serves as a proof of concept of the suitability of chitosan-based polyelectrolyte complexes as nanovectors by sequential multilayered adsorption of various biomacromolecules.

Reducing-End Functionalization of 2,5-Anhydro-d-mannofuranose-Linked Chitooligosaccharides by Dioxyamine: Synthesis and Characterization.

The nitrous acid depolymerization of chitosan enables the synthesis of singular chitosan oligosaccharides (COS) since their reducing-end unit is composed of 2,5-anhydro-d-mannofuranose (amf). In the present study, we describe a chemical method for the reducing-end conjugation of COS-amf by the commercially available dioxyamine O,O'-1,3-propanediylbishydroxylamine in high mass yields. The chemical structure of resulting dioxyamine-linked COS-amf synthesized by both oximation and reductive amination ways were fully characterized by 1H- and 13C-NMR spectroscopies and MALDI-TOF mass spectrometry. The coupling of chemically attractive linkers such as dioxyamines at the reducing end of COS-amf forms a relevant strategy for the development of advanced functional COS-based conjugates.

Highly stretchable hydrogels from complex coacervation of natural polyelectrolytes.

The controlled complex coacervation of oppositely charged hyaluronic acid (Mw ≈ 800-1000 kg mol-1) and chitosan (Mw ≈ 160 kg mol-1, degree of acetylation = 15%) led to hydrogels with controllable properties in terms of elasticity and strength. In this work, we performed desalting by dialysis of high ionic strength solutions of mixed polyelectrolytes and showed that the control of the pH during the polyelectrolyte assembly greatly impacts the mechanical properties of the hydrogel. First, for pHs from 5.5 to 7.5, a slight coacervation was observed due to low chitosan protonation and poor polyelectrolyte associations. Then, for pHs from 3.0 to 5.5, coacervation and syneresis led to free-standing and easy to handle hydrogels. Finally, for pHs from 2.0 to 3.0 (close to the pKa of the hyaluronic acid), we observed the unusual stretchability of these hydrogels that could arise from the pre-folding of hyaluronic acid chains while physical crosslinking was achieved by hyaluronic acid/chitosan polyelectrolyte complexation.

Glycopolymers as Antiadhesives of E. coli Strains Inducing Inflammatory Bowel Diseases.

n-Heptyl α-d-mannose (HM) is a nanomolar antagonist of FimH, a virulence factor of E. coli. Herein we report on the construction of multivalent HM-based glycopolymers as potent antiadhesives of type 1 piliated E. coli. We investigate glycopolymer/FimH and glycopolymer/bacteria interactions and show that HM-based glycopolymers efficiently inhibit bacterial adhesion and disrupt established cell-bacteria interactions in vitro at very low concentration (0.1 µM on a mannose unit basis). On a valency-corrected basis, HM-based glycopolymers are, respectively, 10(2) and 10(6) times more potent than HM and d-mannose for their capacity to disrupt the binding of adherent-invasive E. coli to T84 intestinal epithelial cells. Finally, we demonstrate that the antiadhesive capacities of HM-based glycopolymers are preserved ex vivo in the colonic loop of a transgenic mouse model of Crohn's disease. All together, these results underline the promising scope of HM-based macromolecular ligands for the antiadhesive treatment of E. coli induced inflammatory bowel diseases.

Efficacy of epicardial implantation of acellular chitosan hydrogels in ischemic and nonischemic heart failure: impact of the acetylation degree of chitosan.

This work explores the epicardial implantation of acellular chitosan hydrogels in two murine models of cardiomyopathy, focusing on their potential to restore the functional capacity of the heart. Different chitosan hydrogels were generated using polymers of four degrees of acetylation, ranging from 2.5% to 38%, because the degree of acetylation affects their degradation and biological activity. The hydrogels were adjusted to a 3% final polymer concentration. After complete macromolecular characterization of the chitosans and study of the mechanical properties of the resulting hydrogels, they were sutured onto the surface of the myocardium, first in rat after four-weeks of coronary ligation (n=58) then in mice with cardiomyopathy induced by a cardiac-specific invalidation of serum response factor (n=20). The implantation of the hydrogels was associated with a reversion of cardiac function loss with maximal effects for the acetylation degree of 24%. The extent of fibrosis, the cardiomyocyte length-to-width ratio, as well as the genes involved in fibrosis and stress were repressed after implantation. Our study demonstrated the beneficial effects of chitosan hydrogels, particularly with polymers of high degrees of acetylation, on cardiac remodeling in two cardiomyopathy models. Our findings indicate they have great potential as a reliable therapeutic approach to heart failure.

Reducing-end "clickable" functionalizations of chitosan oligomers for the synthesis of chitosan-based diblock copolymers.

Chitooligosaccharides (COS) produced by nitrous acid depolymerization of chitosan are unique chitosan oligomers due to the presence of the 2,5-anhydro-d-mannofuranose (amf) unit at their reducing end. In this work, we focused on the reductive amination and the oximation of the amf aldehyde group towards various functionalized anilines, hydrazides and O-hydroxylamines. The aim of this work was to synthesize new COS-based building blocks functionalized at their reducing end by different "clickable" chemical groups such as alkene, alkyne, azide, hydrazide and thiol. Targeted functionalized COS were synthesized in excellent mass yields and fully characterized by NMR spectroscopy and MALDI-TOF mass spectrometry. Our results showed these functionalizations are quantitative, versatile and can be easily performed in mild reaction conditions. Finally, these COS-based building blocks could be useful intermediates for the development of advanced functional COS-based conjugates, as illustrated in this work by the synthesis of new COS-poly(ethylene glycol) (PEG) diblock copolymers.

Effects of Chain Length of Chitosan Oligosaccharides on Solution Properties and Complexation with siRNA.

In the context of gene delivery, chitosan has been widely used as a safe and effective polycation to complex DNA, RNA and more recently, siRNA. However, much less attention has been paid to chitosan oligosaccharides (COS) despite their biological properties. This study proposed to carry out a physicochemical study of COS varying in degree of polymerization (DP) from 5 to 50, both from the point of view of the solution properties and the complexing behavior with siRNA. The main parameters studied as a function of DP were the apparent pKa, the solubility versus pH, the binding affinity with siRNA and the colloidal properties of complexes. Some parameters, like the pKa or the binding enthalpy with siRNA, showed a marked transition from DP 5 to DP 13, suggesting that electrostatic properties of COS vary considerably in this range of DP. The colloidal properties of siRNA/COS complexes were affected in a different way by the COS chain length. In particular, COS of relatively high DP (≥50) were required to form small complex particles with good stability.

Processing and antibacterial properties of chitosan-coated alginate fibers.

The preparation of chitosan-coated alginate fibers by a wet spin process is presented and the characterization of the antibacterial activities of these fibers is discussed. Preformed calcium alginate fibers were passed in chitosan acetate solutions. The coagulation method of the coating consisted in the immersion of fibers in a bath of calcium dihydroxide solution (0.1 M). The antibacterial evaluation was achieved by a CFU (Colony-Forming Units) counting method after 6 h of incubation at 37 °C. The incorporation of chitosan on calcium alginate fibers brings antibacterial activities against Staphylococcus epidermidis, Escherichia coli and various Staphylococcus aureus strains namely MSSA (Methicillin Sensitive Staphylococcus aureus), CA-MRSA (Community Associated Methicillin Resistant Staphylococcus aureus) and HA-MRSA (Healthcare Associated Methicillin Resistant Staphylococcus aureus) which make these chitosan-coated fibers potential candidates for wound dressing materials. Developing a wound dressing with the haemostatic and healing properties of alginate combined with antibacterial properties of chitosan is envisioned for fighting against the infections and more particularly nosocomial diseases.

Glycol chitosan-based nanogel as a potential targetable carrier for siRNA.

A self-assembled glycol chitosan nanogel (GC) is synthesized by chemically grafting hydrophobic chains onto a polysaccharide, which is comprehensively characterized. The obtained macromolecular micelle is decorated with folate-conjugated poly(ethylene glycol) (PEG) (GCFA). An average size distribution of 250 and 200 nm is observed, respectively for the GC and GCFA nanogels. Differential cell localization is observed on incubating the materials with HeLa cells. Whereas the GC nanogel is detected on the cell surface, GCFA is localized in the cytoplasm. The cell viability is not compromised by the nanogels. Interestingly, GC nanogel is poorly internalized by bone marrow derived macrophages (BMDMs), and GCFA is not phagocytosed. Given its ability to complex siRNA, the targetable GC nanogel can be a promising vehicle for siRNA delivery.