A method for measuring the in vitro enzymolysis performance of cross-linked hyaluronic acid gel
A technology for cross-linking hyaluronic acid and hyaluronidase, which is applied in the measurement of color/spectral properties, etc., can solve the problems of complicated operation, high cost, long time consumption, etc., and achieves high repeatability, low cost, and short time consumption. Effect
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Embodiment 1
[0031] The kind screening of embodiment 1 enzyme
[0032] ①HA animal enzyme (derived from bovine testis, H3506, Sigma Aldrich), ②commercially available bacterial HA enzyme (56177-10MG, Sigma Aldrich) and ③self-made bacterial HA enzyme (derived from Bacillus CGMCC No. Da Biopharmaceuticals) was prepared with disodium hydrogen phosphate-sodium dihydrogen phosphate-sodium chloride buffer solution (pH 7.0) to prepare 1200IU / mL enzyme solution, and weighed cross-linked HA gel (cross-linking agent BDDE, HA content 20mg / mL mL) 0.4g (weighing amount is equivalent to HA content 8mg), each enzyme was added in duplicate, disodium hydrogen phosphate-sodium dihydrogen phosphate-sodium chloride buffer (pH 7.0) 2mL, vortex mixed, and then Add 2mL of the prepared enzyme solution and vortex for 10s. The mixture was placed in a 42°C water bath. Start timing after being placed in the water bath, sample 50 μL of the test solution every 10 minutes, add 3 mL of disodium hydrogen phosphate-sodium ...
Embodiment 2
[0038] The screening of embodiment 2 enzymolysis medium and pH
[0039] Prepare disodium hydrogen phosphate-sodium dihydrogen phosphate-sodium chloride buffer solution (PBS) of different pH values, the pH is respectively 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, the self-made bacterial HA enzyme (derived from spore Bacillus CGMCC No.5744, Bloomage Freda Biopharmaceuticals) were prepared with the above enzymatic hydrolysis media to prepare 1200IU / mL enzyme solution, and weighed 0.4g of cross-linked HA gel (cross-linking agent BDDE, HA content 20mg / mL) (The weighing amount is equivalent to 8mg of HA content), each enzymatic hydrolysis medium was added in parallel in duplicate, 2mL of the enzymatic hydrolysis medium was added, vortexed and mixed, then 2mL of the prepared enzyme solution was added, and vortexed for 10s. The mixture was placed in a 42°C water bath. Start timing after placing in the water bath, sample 50 μL of test solution every 10 minutes, add 3 mL of enzymatic hydrolys...
Embodiment 3
[0055] Embodiment 3 enzyme hydrolysis temperature screening
[0056] The self-made bacterial HA enzyme (derived from Bacillus CGMCC No.5744, Bloomage Freda Biopharmaceuticals) was prepared with physiological saline to make an enzyme solution of 1200IU / mL. Weigh 0.4 g of cross-linked HA gel (cross-linking agent BDDE, HA content 20 mg / mL) (weighing weight is equivalent to HA content 8 mg), and add 2 mL of normal saline in parallel at each temperature, vortex to mix, and then Add 2mL of the prepared enzyme solution and vortex for 10s. The mixtures were placed in water baths at 37°C, 42°C, and 45°C, respectively. Start timing after placing in the water bath, sample 50 μL of the test solution every 10 minutes, add 3 mL of normal saline to the test solution for dilution, immediately measure the absorbance at 232 nm with a UV spectrophotometer, and use normal saline as a blank control. Record the absorbance at each time point at different temperatures, and the results are shown in ...
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