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Cysteine-hyaluronic acid conjugates prepared by freeze-drying and thiol-ene click chemistry, and synthetic methods and applications thereof

A technology of hyaluronic acid and cysteine, which is applied in the field of biomedicine, can solve the problems of CD44 receptors not being recognized normally, reducing cell repair damage, and in situ injection, etc., achieving broad biomedical prospects and good rheology chemical properties, increase the effect of cell adhesion

Active Publication Date: 2019-02-19
HAINAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It overcomes the shortcomings of natural hyaluronic acid being cleared quickly in the body and low mechanical strength
However, the cross-linked hydrogel prepared by the carboxyl group of hyaluronic acid may produce some side reactions
Studies have shown that if its carboxyl group is chemically modified, it will reduce the negative charge of hyaluronic acid, which will cause CD44 receptors to not recognize normally
Cellular CD44 receptors are considered to be reduced by the negative charge of hyaluronic acid and hyaluronic acid (Morra M. Engineering of biomaterials surfaces by hyaluronan. [J]. Biomacromolecules, 2005, 6 (3): 1205-1223. The negative charge of hyaluronic acid will affect the interaction between cell CD44 receptors and hyaluronic acid, thereby reducing the ability of cells to repair damage (Herrera MB, Bussolati B, Bruno S, et al. Exogenous mesenchymal stem cells localize to the kidney by means of CD44 following acute tubular injury[J].KidneyInternational, 2007, 72(4):430-441.), so hyaluronic acid has lost its superiority as a biological material
However, the hyaluronic acid hydrogel obtained through the reaction of its hydroxyl group often needs to react with ultraviolet light or use other methods to pre-form the hydrogel, so that in situ injection cannot be achieved.

Method used

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  • Cysteine-hyaluronic acid conjugates prepared by freeze-drying and thiol-ene click chemistry, and synthetic methods and applications thereof
  • Cysteine-hyaluronic acid conjugates prepared by freeze-drying and thiol-ene click chemistry, and synthetic methods and applications thereof
  • Cysteine-hyaluronic acid conjugates prepared by freeze-drying and thiol-ene click chemistry, and synthetic methods and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0101] The preparation method of the cysteine-functionalized hyaluronic acid conjugate prepared by freeze-drying and thiol-ene click chemistry comprises the following steps:

[0102] 1) Take the structural formula as The compound 1 hyaluronic acid with the structural formula is The compound 2 formed by freeze-drying method has the structural formula: Compound 3;

[0103] 2) Take the structural formula as The compound 4 and the structural formula are The compound 5 forms the structural formula as Compound 6;

[0104] 3) Compound 6 undergoes a reduction reaction with a disulfide bond reducing agent to form a structural formula of Compound 7 of

[0105] 4) Taking compound 3 and compound 7, under photoinitiator and ultraviolet light irradiation, thiol-ene click chemistry forms the structural formula as Compound 8;

[0106] 5) Remove the tert-butoxycarbonyl and acetamidomethyl protecting groups on compound 8 respectively to obtain the structural formula: Compound...

Embodiment 1

[0120] 1) Synthesis of compound 3

[0121]

[0122] Synthetic scheme of compound 3 in table 1

[0123]

[0124]Remarks: 1g 100,000 hyaluronic acid contains 2.636mmol primary alcohol hydroxyl groups; allyl glycidyl ether molecular weight: 114.14g / mol density: 0.962g / ml boiling point: 154°C (lit.)

[0125] Take 14g of hyaluronic acid (100kDa), dissolve it in 700ml of ultrapure water in a 1000ml round bottom flask to prepare a hyaluronic acid aqueous solution with a concentration of 2% (w / v), and prepare seven 250ml round bottom flasks numbered 1 to 7 Flasks, fill each flask with 100ml of 2% hyaluronic acid solution for later use, take 9.76ml of allyl glycidyl ether, add ultrapure water to 100ml with a volumetric flask and mix evenly, add the The corresponding volume of allyl glycidyl ether aqueous solution was stirred and mixed evenly at room temperature, transferred to a petri dish, frozen at -80°C, and freeze-dried to obtain a series of modified allyl glycidyl ether hya...

Embodiment 2

[0154] The specific route of the covalently cross-linked hyaluronic acid hydrogel that can be formed in situ by injection mediated by the natural chemical ligation reaction is as follows:

[0155]

[0156] Experimental steps:

[0157] Solution A: Weigh 35 mg of the cysteine-hyaluronic acid conjugate (Cys-HA) with a series of modification ratios synthesized in Example 1 and dissolve it in a 1 ml centrifuge tube with 350 ul of phosphate buffer (pH 7.6).

[0158] Solution B: Weigh 35mg of 10k four-branch polyethylene glycol oxyester conjugate (4-ARM-PEG-S-S) and dissolve it in 350ul of phosphate buffer (pH7.6) in a 1ml centrifuge tube.

[0159] Solution A and solution B were quickly mixed with a vortex mixer and added to the rheometer for testing. Time sweep is performed first, frequency sweep is performed when the modulus curve no longer changes, and finally strain sweep is performed. The parameter settings are as follows.

[0160] The parameters in the rheological test are...

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Abstract

The present invention discloses a cysteine functionalized hyaluronic acid conjugate prepared through freeze drying and thiol-ene click chemistry, and a synthesis method thereof, and discloses applications of the hyaluronic acid conjugate in preparation of an injectable in-situ hydrogel, and applications of the formed injectable in-situ hydrogel in islet cell culture and insulin secretion. According to the present invention, a series of cysteine functionalized hyaluronic acid conjugates having different modification rates and stable ether bonds are obtained through freeze drying and thiol-ene click chemistry, wherein the cysteine functionalized hyaluronic acid conjugate has the thiol active group, such that the further functionalized modification can be performed; and the hyaluronic acid conjugates having different modification rates and the polyethylene glycol conjugate can be subjected to the oxo ester-mediated natural chemical link reaction to obtain the an injectable in-situ formed hydrogel, wherein the obtained hydrogel has the good rheological property, the free thiol group can increase the cell adhesion of the hyaluronic acid and can be adopted as the cell scaffold material so as to be used in the islet cell culture and the insulin secretion stimulation, and the broad biomedical prospects are provided.

Description

technical field [0001] The invention relates to a cysteine-functionalized hyaluronic acid conjugate formed by freeze-drying and thiol-ene click modification and its synthesis method, as well as its application in the preparation of injectable in-situ hydrogel, belonging to the field of biomedicine . Background technique [0002] Bioscaffold materials are key to tissue engineering and regenerative medicine research. Because the scaffold material not only provides structural support for cells, but also acts as a template to guide tissue regeneration and control tissue structure. Among them, hydrogel-based bioscaffolds are particularly widely used in tissue engineering and regenerative medicine. [0003] Hydrogel is a hydrophilic polymer network that can absorb a large amount of water and has good biocompatibility. Although the application of hydrogels in biomedicine has made great progress, however, the development of covalently cross-linked hydrogels that meet the clinical...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08B37/08C08J3/075C08L5/08C08L71/02A61L27/20A61L27/18A61L27/50A61L27/52A61L27/54
CPCA61L27/18A61L27/20A61L27/50A61L27/52A61L27/54A61L2400/06C08B37/0072C08J3/075C08J2305/08C08J2471/02C08L5/08C08L71/02
Inventor 张响胡碧煌李雨欣
Owner HAINAN UNIV
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