Amorphization Of Chitin Induced By Superfine Grinding And Its Application

Chitin is the most abundant organic natural polysaccharide on earth after cellulose,and possesses non-toxicity,biodegradability and good biocompatibility,thus having a wide range of applications.However,the highly ordered crystalline structure and the large number of intramolecular and intermolecular hydrogen bonds of chitin make it insoluble in common aquatic and organic solvents,and difficult to be processed.The exploitation of chitin will have huge scientific and economic value,and also be beneficial to environmental protection.In this paper,chitin extracted from the shell of Alaska snow crab was used as the raw material to prepare samples of various crystallinity by destroying its crystalline structure and intermolecular hydrogen bond through ultrafine grinding treatment.Chitin with crystallinity as low as 8.39%was obtained after 60 mins of superfine griding treatment,and the amorphous chitin became readily dissolved in 10%NaOH solution after one round of freezing-thawing process.In the ultrafine grinding,it is supposed that there are two main forces exerted by chitin:shearing force and kneading force.The shearing force provided the major force responsible for the destruction of chitin,while the kneading force caused chitin deformation and reconstruction.Small chitin particles may aggregate again under the activation of kneading force.With competition between various forces,weak forces would be first destroyed,so hydrogen bonding was first destroyed in the process of grinding.followed by destruction of glycosidic bonds.The amorphization of chitin reduced the processing difficulty of chitin and was conducive to the preparation of various functional materials by dissolving and reforming.The chemical reactivity of chitin was significantly improved with the decrease of crystallinity.In this paper,the effects of crystallinity on the acid hydrolyzation.carboxymethylation and deacetylation of chitin were studied.(1)Chitin with different degree of crystallinity was acid hydrolyzed at different temperature.and it was found that the hydrolysis rate of chitin increased and the activation energy decreased greatly with the decrease of the crystallinity.The calculated activation energy was lowered from 218.710 kJ/mol of the raw material to 114.789 kJ/mol of the chitin with crystallinity of 8.39%.The decrease of crystallinity can not only accelerate the penetration of solvent and enhance the capacity of the combination of glycosidic bond with hydrogen ions,but also reduce the space resistance of the formation of carbenium ion,thus facilitating the break of glycosidic bonds.(2)The carboxymethylation of chitin was also affected by crystallinity.With the decrease of the crystallinity,the degree of carboxymethylation of chitin was improved and the substitution site was more selective.For the chitin with crystallinity of 8.39%,the content of carboxymethyl substituted at C6-OH position was 2.89 times that at C3-OH position.(3)Deacetylation of chitin with different crystallinity was also studied,and it was found that the rate increased and the activation energy decreased with the crystallinity.The deacetylation activation energy was reduced from 58.216 kJ/mol of the raw chitin to 31.753 kJ/mol of the chitin with crystallinity of 8.39%.The deacetylation of chitin was SN2 reaction,and the decrease of crystallinity was conducive to the diffusion of sodium hydroxide to chitin molecular,and could reduce the peeling resistance of acetyl groups,accelerate the diffusion of low-molecular electrolytes into the solution,and reduce the formation of solvated structure.In a word,the amorphization of chitin facilitates the diffusion of solvents into the molecules,reduces the activation energy of various reactions,and shows obvious advantages in the preparation of various chitin derivatives.Carboxymethyl chitosan with good water solubility was prepared by deacetylation and carboxymethylation of chitin,and was combined with oxidized hyaluronic acid(OHA)to prepare injectable hydrogel.The gelation time of hydrogel decreased with the concentration of carboxymethyl chitosan(CMCS)and OHA,and increased first and then decreased with the oxidation degree(Dox)of OHA.Hydrogel(OHA-2/CMCS)with a suitable gelation time(?70 s)and mechanical properties(76.0 kPa compression stress at strain of 80%)for wound healing was obtained when OHA with a Dox of 38.1 and a concentration of 4%was combined with CMCS with a concentration of 2%,and was selected as the carrier of blueberry anthocyanin(BA)for wound healing.The in vitro release experiment showed that BA was released from the BA-loaded hydrogel(BA/OHA-2/CMCS)in a short time,thus providing sufficient BA during the initial period of wound healing,and then the release of BA slowed down lasted for about 54 h.The primary skin irritation assays demonstrated that the BA/OHA/CMCS hydrogel had no irritation to skin,showing good biocompatibility.The wound healing experiments showed that OHA/CMCS and BA/OHA/CMCS hydrogel significantly accelerated wound healing and promoted epithelial and tissue regeneration.Compared with OHA/CMCS hydrogel,BA/OHA/CMCS hydrogel showed significant anti-inflammatory effects.promoting collagen deposition and angiogenesis.Western Blot experiment was used to study the mechanism of hydrogel in promoting wound healing.It was found that OHA/CMCS hydrogel could inhibit iNOS protein expression and promote IL-10 protein production,maintaining a low NO level to avoid cell damage,and having a certain anti-inflammatory effect in the early stage of wound healing.BA/OHA-2/CMCS hydrogel could significantly upregulate the expression of VEGF,JAK2 and IL-10 proteins and downregulate NF-?B and iNOS proteins,thus promoting angiogenesis,inhibiting the expression of pro-inflammatory factor,and increasing anti-inflammatory proteins.In the middle stage of wound healing,OHA-2/CMCS hydrogel showed stronger angiogenesis promoting ability,while BA/OHA-2/CMCS hydrogels showed stronger anti-inflammatory activity.These results indicate that OHA-2/CMCS hydrogel and hydrogels loaded with active materials are promising wound dressings.