Chitosan as a starting material for wound healing applications Patrulea, V.; Ostafe, V.; Borchard, G. ...
European journal of pharmaceutics and biopharmaceutics,
November 2015, 2015-Nov, 2015-11-00, 20151101, Letnik:
97, Številka:
Pt B
Journal Article
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Chitosan and its derivatives have attracted great attention due to their properties beneficial for application to wound healing. The main focus of the present review is to summarize ...studies involving chitosan and its derivatives, especially N,N,N-trimethyl-chitosan (TMC), N,O-carboxymethyl-chitosan (CMC) and O-carboxymethyl-N,N,N-trimethyl-chitosan (CMTMC), used to accelerate wound healing. Moreover, formulation strategies for chitosan and its derivatives, as well as their in vitro, in vivo and clinical applications in wound healing are described.
•Improved chitosan N-trimethylation through optimization of reaction time and solvent precipitation.•Improved chitosan O-carboxymethylation up to a degree of 80%, based on O-methyl-free N-trimethyl ...chitosan.•Cytocompatibility of the CMTMC chitosan derivative towards human fibroblasts.
We present here the synthesis of a highly O-carboxymethylated chitosan derivative. First, an improved protocol for the two-step synthesis of N-trimethyl chitosan (TMC) from chitosan was developed, yielding a maximum degree of quaternization (DQ) of up to 46.6%. Successively, the chitosan derivative O-carboxymethyl-N-trimethyl chitosan (CMTMC) was synthesized from the TMC obtained by applying an optimized synthesis pathway. In contrast to previous reports, the optimized protocol was shown to yield very high rates (>85%) of O-carboxymethylation of CMTMC, as shown by 1H NMR and heteronuclear single quantum correlation (1H–13C HSQC). Finally, in vitro cytocompatibility (viability >80%) of the polymer was demonstrated using human fibroblasts.
•A new RGDC-functionalized carboxymethylated chitosan derivative for promoting wound healing was synthesized.•The functionalized chitosan was shown to enhance HDF proliferation by 50% compared to ...CMTMC alone.•The functionalized chitosan showed no toxicity in vitro on HDF.•Increased HDF adhesion and spreading in the presence of RGDC-functionalized chitosan.
RGD peptide sequences are known to regulate cellular activities by interacting with α5β1, αvβ5 and αvβ3 integrin, which contributes to the wound healing process. In this study, RGDC peptide was immobilized onto chitosan derivative 1,6-diaminohexane-O-carboxymethyl-N,N,N-trimethyl chitosan (DAH-CMTMC) to display RGDC-promoting adhesion for enhanced wound healing. The efficiency of N-methylation, O-carboxymethylation and spacer grafting was quantitatively and qualitatively analyzed by 1H NMR and FTIR, yielding 0.38 degree of substitution for N-methylation and >0.85 for O-carboxymethylation. The glass transition temperatures for chitosan derivatives were also studied. Peptide immobilization was achieved through sulfhydryl groups using sulfosuccinimidyl (4-iodoacetyl)amino-benzoate (sulfo-SIAB method).
RGDC immobilized peptide onto DAH-CMTMC was found to be about 15.3μg/mg of chitosan derivative by amino acid analysis (AAA). The significant increase of human dermal fibroblast (HDF) viability in vitro over 7 days suggests that RGDC-functionalized chitosan may lead to enhanced wound healing (viability >140%). Moreover, bio-adhesion and proliferation assays confirmed that coatings of RGDC-functionalized chitosan derivatives exhibit in vitro wound healing properties by enhancing fibroblast proliferation and adhesion. These results showed that RGDC peptide-functionalized chitosan provides an optimal environment for fibroblast adhesion and proliferation.
Chitin (poly-β-(1-4)-2-acetamido-2-deoxy-D-glucopyranose) is one of the most abundant biopolymers in the biosphere but is insoluble in most solvents, including water and thus, difficult to extract ...and use for industrial purposes. Chitin can be hydrolyzed or deacetylated, leading to a more soluble and reactive derivate, chitosan (poly-β-(1-4)-2-amino-D-glucopyranose). The two polymers have many appealing applications in various industries but for the studies of derivatives compounds there is a need for improved methods for the assay of chitin and chitosan. One of the most used methods for the assay of reducing sugars is the dinitrosalicylic acid method and one of the most common method used for the assay of amino groups is the ninhydrin method. The present study proposes improved versions of these classic methods by adapting them to microtiter plates in order to enhance the time- and cost-efficiency. As both reactions need a heating step and the ordinary microtiter plates do not support high temperature, this reaction step was performed in a microwave oven, in a controlled environment. The optimal conditions for the dinitrosalicylic acid method proved to be undergoing microwave system for 4 min (R^sup 2^>96%, RSD <0.026), for the ninhydrin method the time of reaction proved to be 4.5 min and the best pH for the reaction was 6. The products of chitosan hydrolysis were, then, successfully tested using the two methods.
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