| Hyaluronan (Hyaluronic Acid, HA) is a high-molecular mass polysaccharide which is composed of D-glucuronic acid (GlcUA) and N-acetyl-D-glucosamine (GlcNAc) units linked through altering β-(1→4) and P-(1→3) glycosidic bonds. As a main component of extracellular matrix in the cell, HA is an important constituent of connective tissue, such as skin, vitreous, umbilical cord, cartilage and synovial fluid. HA has unique physiological functions such as moisturizing effect, lubrication, tissue repair and prevent damage. It is reported that HA plays an important role in maintaining tissue integrity, as well as in developing and remodeling tissues during in inflammation, wound repair, and embryonic development. It is more and more widely applied in the field of cosmetics, food and medicine.As the deepening of the HA application in medical field, it was found that HA can be degraded easily by hyaluronidase in vivo, therefore the retention time inside the body is relatively short. The researchers hope to moderate HA and obtain HA derivatives which can resistance to enzymatic hydrolysis, and have longer retention time in the body. The commercial crosslinked HA has a good resistance to enzymatic hydrolysis, but lacks of certain lubricity, and should not be used for the joint injection. Thus the HA derivatives which have resistance to enzymatic hydrolysis and lubricity are studied more and more widely.The moderated sites of HA include the carboxyl, hydroxyl, amino and acetyl reduction ends. The hydroxyl alkylation hyaluronic acid can be obtaind by the modification of hydroxyl group of hyaluronic acid (HA) with epoxy alkane compounds. In this study, hydroxypropyl hyaluronic acid (HHA) is obtained by the modification of HA with epoxy propane. We found that HHA can not only resist to the enzyme hydrolysis but also has good lubricity. In this work, we studied many aspects of HHA, including the preparation method, the physical and chemical stability, the resisted characteristics of HHA to HAase degradation. Besides, the cell toxicity and the structure of HHA were also investigated. we also established its quality detection methods. The research includes the following contents.(1) The study of HHA preparation processThe preparation method of HHA was set up through investigation of the usage amounts of epoxy propane, NaOH, reaction temperature. HHA prepared by this method has obvious resistance to HAase and higher molecular weight, besides, it also has low degree of substitution.(2) HHA physical-chemical stability studyWe also determined the characteristics of HHA using HA which has close relative molecular mass (Mr) as control. The results showed that the thermal stability, acid and alkali stability and oxidation stability of HHA and HA did not show significant difference.(3) The study of degradation-resisting by hyaluronidaseThe property of resistance to hyaluronidase degradation was studied by comparing the rangeability of the sample solution’s viscosity which is treated with hyaluronidase. The result showed that HHA has a good resistance to hyaluronidase degradation compared with HA.(4)The research of HHA’s cytotoxicityUsing HA with similar relative molecular mass as reference substance, the cytotoxicity of HHA was evaluated. The results showed that the proliferation rate and cells form are not obvious changed compared with HA. Their totoxicity has no significant difference.(5)The establishment of quality standards of HHAReferencing the determination method of hydroxypropyl starch’s substitution degree, HHA’s substitution degree detection method by spectrophotometry was established. The analytical methods of the key indicators were also determined such as the the property of resistance to hyaluronidase degradation, dry weightlessness, kinematic viscosity, pH, and microorganisms.etc.HHA has the excellent lubricity and liquidity, It also has obvious resistance to hyaluronidase degradation. It can be used to develop joint injections or fillers for subcutaneous injection used in hairdressing. It is expected to prolong retention time in the body and the effect of long-term treatment. |
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