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Method for detecting in-vitro enzymolysis of cross-linked hyaluronic acid by utilizing water-phase gel permeation chromatography

A technology of gel permeation chromatography and cross-linked hyaluronic acid, which can be used in measurement devices, instruments, scientific instruments, etc., and can solve problems such as affecting test results and errors.

Active Publication Date: 2014-03-26
IMEIK TECH DEV CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method uses a large block of cross-linked hyaluronic acid gel as a whole. If the cross-linked hyaluronic acid is crushed into tiny particles and used as a product, this method is used to detect the quality of the product. The degree of enzymatic hydrolysis will have great limitations, because tiny particles will affect the detection results and cause considerable errors

Method used

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  • Method for detecting in-vitro enzymolysis of cross-linked hyaluronic acid by utilizing water-phase gel permeation chromatography

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Preparation of hyaluronic acid gel

[0031] Dissolve 0.5g of sodium hydroxide in 20ml of water, add 5g of sodium hyaluronate (produced by Shandong Freda Biotechnology Co., Ltd.), stir for 12-14 hours, stir until the sodium hyaluronate is completely dissolved, then add to the reaction system 5g of 1,2,7,8-diepoxyoctane (DEO), stirred quickly and evenly, and reacted at 25°C for 24 hours. The reaction was terminated with 2mol / L hydrochloric acid, and the pH was adjusted to about 5. At the same time, the water in the reaction system was distilled off at 40°C and a vacuum of 13.3Kpa. When the distilled water reached 50mL, the vacuum distillation was stopped. Soak with 200mL sodium hydroxide solution containing 30% ethanol with pH=8-9 for 3 times to neutralize the hydrochloric acid of the gel. Transfer the neutralized gel into a vacuum drying oven, and dry it at 60° C. and 40 Kpa for 10 hours to solidify and shape the gel. The water absorption rate of the gel is 45 times. T...

Embodiment 2

[0033] Enzymatic hydrolysis experiment of cross-linked hyaluronic acid microparticles and molecular weight detection by simultaneous aqueous gel permeation chromatography

[0034] Take 1 mL of the micro-particle cross-linked hyaluronic acid prepared in Example 1 in a colorimetric tube, add 300 units of hyaluronidase (HAase), dilute to 2 mL with water, and place in a constant temperature water bath shaker at 37 ° C. In the middle, start timing after dilution, and start from the 20th minute, take 50 μL of supernatant with a micro syringe every 20 minutes, place the taken out supernatant in an ice-water bath and cool it down to below 5°C rapidly to inhibit hyaluronic acid Enzyme activity. Take the enzymatic hydrolysis supernatant within 4 hours, and detect the molecular weight of the supernatant at different time periods by aqueous gel permeation chromatography (GPC). When the molecular weight tends to a constant, it can be determined that the enzymatic hydrolysis of the product ...

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Abstract

The invention relates to a method for detecting in-vitro enzymolysis of cross-linked hyaluronic acid by utilizing water-phase gel permeation chromatography. Whether the cross-linked hyaluronic acid completely achieves enzymolysis is determined by detecting changes of molecular weight of the hyaluronic acid after the enzymolysis. The method comprises putting small granulated cross-linked hyaluronic acid products of 1mL into a color comparison tube, adding 300unis of hyaluronidase, diluting to the volume of one time with water into a water bath chader with the constant temperature of 37 DEG C, starting to time after dilution, fetching 50mum L supernate through a micro syringe every twenty minutes from the twentieth minutes, putting the fetched supernate into a refrigerator and quickly cooling the supernate to the temperature of 5 DEG C, fetching enzymolysis supernate for 3-5 hours, detecting the molecular weight of the supernate at different time intervals respectively through the water-phase gel permeation chromatography (GPC), and determining that the enzymolysis of the product is finished when the molecular weight trends to be a constant. The method for detecting the in-vitro enzymolysis of the cross-linked hyaluronic acid by utilizing the water-phase gel permeation chromatography is simple, easy to operate, low in cost, safe and reliable.

Description

technical field [0001] The invention relates to a method for detecting cross-linked hyaluronic acid acidase in vitro by aqueous gel permeation chromatography, in particular to determine whether the cross-linked hyaluronic acid is completely hydrolyzed by detecting the change in molecular weight of hyaluronic acid after enzymatic hydrolysis. Background technique [0002] To develop dermal fillers that can be used clinically, a series of chemical modifications and treatments must be performed on hyaluronic acid. If non-cross-linked hyaluronic acid is used as a dermal filler, the hyaluronidase and free radicals naturally present in the skin will cut off the polymerized hyaluronic acid molecules one by one, rapidly degrading the non-cross-linked hyaluronic acid. uric acid. As a result, the half-life of hyaluronic acid in the tissue is only one to two days, and then it is diluted with water and gradually degraded into water and carbon dioxide in the liver. In this way, the prod...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02
Inventor 简军李睿智
Owner IMEIK TECH DEV CO LTD
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