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Enzymatic catalysis crosslinking reduction-responsive hyaluronic acid microgel and preparation method thereof

A technology of hyaluronic acid and microgel, which is applied in the directions of non-active ingredients, such as medical preparations, pharmaceutical formulations, fermentation, etc., can solve the problems of application limitation, reduced activity, and the three-dimensional network structure does not have reduction responsiveness, and achieves solidification. The effect of fast speed, overcoming toxic side effects, excellent biocompatibility and biodegradability

Inactive Publication Date: 2016-09-28
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the enzyme catalyzes the addition of H in the cross-linking reaction 2 o 2 May cause horseradish peroxidase to form an inert intermediate and reduce its activity (K. J. aynton, J. K. ewtra, N.iswas and K. E. Taylor., Biochim. Biophys. Acta. 1994, 1206, 272), or lead to loaded proteins and cells, etc. Inactivation, and the three-dimensional network structure formed by the cross-linking of phenolic hydroxyl radicals has no reduction responsiveness, which limits the application of this type of hyaluronic acid hydrogel in the biomedical field

Method used

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  • Enzymatic catalysis crosslinking reduction-responsive hyaluronic acid microgel and preparation method thereof
  • Enzymatic catalysis crosslinking reduction-responsive hyaluronic acid microgel and preparation method thereof
  • Enzymatic catalysis crosslinking reduction-responsive hyaluronic acid microgel and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Preparation of thiolated hyaluronic acid macromolecule (HA-SH).

[0026] Prepare 100 mL of sodium hyaluronate aqueous solution with a concentration of 2 wt%, add such N -Hydroxysuccinimide 2.3g and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC .HCL) 3.832 g into the sodium hyaluronate solution, adjust the pH of the solution to about 5.4, stir at room temperature for 1 h, add 3.372 g of cystamine dihydrochloride, stir at room temperature for 48 h, and dialyze the reaction solution with distilled water for 24 h to remove ungrafted Cystamine dihydrochloride. Add 4.6275 g of DL-dithiothreitol (DTT) to the dialysate, adjust the pH of the solution to 8.5, and react for 24 hours in a dark nitrogen atmosphere. After the reaction, adjust the pH of the reaction solution to 5, and dialyze with twice distilled water for 3 days , freeze-dried to obtain a white flocculent product, and the free sulfhydryl content was 6.5% as measured by Ellman's reagent.

Embodiment 2

[0028] Preparation of hyaluronic acid microgels by cross-linking catalyzed by horseradish peroxidase in inverse emulsion method.

[0029] Preparation of the aqueous phase: Weigh 150 mg of the mercaptolated hyaluronic acid synthesized in Example 1, prepare a 0.4% (w / v) aqueous solution with double distilled water, add tyramine hydrochloride to the aqueous solution, and its final concentration to 50 mM, the aqueous solution was adjusted to pH=7 with dilute NaOH solution. Preparation of oil phase: take liquid paraffin as the oil phase according to the water phase / oil phase volume ratio of 1 / 4, and add Span80 / Tween80=88 / 12 (v / v) mixed surface activity at a concentration of 1 wt%. agent. The water phase was slowly added to the oil phase, emulsified at 1000 rpm for 30 min at high speed, and after forming stable colostrum, horseradish peroxidase (1000 units / mL, 0.48 mL) was added, and stirred at 600 rpm for 4 h to solidify the microspheres. After the reaction was completed, they we...

Embodiment 3

[0031] Surface morphology analysis of hyaluronic acid microgels.

[0032] Take an appropriate amount of freeze-dried microgel and disperse it in double-distilled water, and dehydrate it with 20%, 40%, 60%, 80%, 90%, and 100% absolute ethanol, respectively. Use a dropper to take an appropriate amount of ethanol suspension and drop it on a clean silicon wafer, dry it naturally, and use a scanning electron microscope for morphology analysis after spraying gold, as shown in figure 1 As shown, the particle size of the dried microgel is about 10 um.

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Abstract

The invention discloses enzymatic catalysis crosslinking reduction-responsive hyaluronic acid microgel and a preparation method thereof. The hyaluronic acid microgel is prepared by taking sulfhydrylation hyaluronic acid as a raw material, taking horseradish peroxidase as a catalyst, taking tyramine hydrochloride as an enzymatic catalysis reaction substrate and combining the method that horseradish peroxidase is catalytically crosslinked with sulfydryl to generate a disulfide bond through an inverse emulsion method. The disulfide bond crosslinking structure in the microgel can be broken in the presence of reductive substances such as dithiothreitol, reductive glutathione hormone and L-cysteine, and then the good reduction responsiveness is given to the microgel. The hyaluronic acid microgel can achieve controlled release on the reduction responsiveness of loaded doxorubicin hydrochloride and can be applied to controlled release of tumor-targeted drugs. According to the preparation method, a crosslinking agent does not need to be additionally added into an inverse emulsion system to cure the microgel, the toxicity influence of the crosslinking agent is avoided, the biocompatibility of the microgel is guaranteed, the reaction conditions are mild, and the technological processes are simple.

Description

technical field [0001] The invention belongs to the field of biomedical polymer materials. Background technique [0002] Microgel is a kind of hydrogel microsphere composed of three-dimensional cross-linked network structure with a size between nanometer and micrometer scale, which has good dispersion and stability in water. By selecting appropriate polymer composition and cross-linking agent, microgels can be endowed with excellent biocompatibility, excellent structure controllability and stimuli responsiveness, which makes microgels have broad application prospects in the field of controlled drug release. Microgels that respond to environmental stimuli can respond to external environmental stimuli (such as ultrasound, magnetic field, and light) or the microenvironment (pH, temperature, and redox potential) of the lesion area, and can be controlled by changing the volume or three-dimensional network crosslinking structure Release the drug loaded in it to achieve an ideal d...

Claims

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Application Information

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IPC IPC(8): C12P19/26A61K47/36
CPCC12P19/26A61K47/36
Inventor 彭志平佘英奇李义赵玉莹谢标明
Owner NANCHANG UNIV
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