NL2029638B1

NL2029638B1 - Modified hyaluronic acid hydrogel loaded with akkermansia muciniphila and preparation method and application thereof

Info

Publication number: NL2029638B1
Application number: NL2029638A
Authority: NL
Inventors: Zhang Wei; Fu Yacheng
Original assignee: Xiangya Hospital Central South Univ
Priority date: 2021-11-04
Filing date: 2021-11-04
Publication date: 2022-10-20
Also published as: NL2029638A

Abstract

The present invention relates to the technical field of slow release of drugs, and more particularly to a modified hyaluronic acid hydrogel loaded with akkermansia muciniphila and a preparation method and an application thereof. The present 5 invention can achieve the purpose of treating and preventing enteritis through targeted delivery of AKK, and the toxicity can be ignored. (+ Fig. l)

Description

P775/NLpd

MODIFIED HYALURONIC ACID HYDROGEL LOADED WITH AKKERMANSIA MUCINIPHILA AND PREPARATION METHOD AND APPLICATION THEREOF

TECHNICAL FIELD The present invention relates to the technical field of slow release of drugs, and more particularly to a modified hyaluronic acid hydrogel loaded with akkermansia muciniphila and a prepara- tion method and an application thereof.

BACKGROUND ART Hyaluronic acid has rich functional groups, including carbox- yl and hydroxyl, and can be adopted for chemical modification. Due to inherent properties, such as repeated disaccharide units and a high viscosity, the hyaluronic acid is beneficial to self- crosslinking to form a supramolecular hydrogel.

However, an additional cross-linking agent is used in a con- ventional hyaluronic acid encapsulating system, which consequently causes a problem of exogenous biotoxicity.

SUMMARY The present invention aims to provide a modified hyaluronic acid hydrogel loaded with mucinophilic-akkermansia muciniphila and a preparation method and an application thereof. By adopting the preparation method, an additional cross-linking agent is not used, a self-crosslinking reaction of hyaluronic acid can be achieved, and a problem of exogenous biotoxicity is not caused.

According to the present invention, after a hydrogel is ob- tained through self-crosslinking of thiolated hyaluronic acid, ed- dy mixing and culture are sequentially carried out sequentially in an alkaline buffer to carry out oxidation-reduction treatment, and after akkermansia muciniphila (AKK) is embedded into the finally prepared hyaluronic acid, the purpose of treating and preventing enteritis can be achieved through targeted delivery of AKK, and the toxicity can be ignored. By adopting the method, direct con-

tact of AKK with the environment is successfully prevented, and the survival rate of AKK in resisting to acidity and bile salt in the delivery process in gastrointestinal tracts is increased. Af- ter reaching a target site, a carrier triggered by oxidation re- duction is rapidly degraded through a reversible disulfide bond in small intestines to release prebiotics cells, then AKK is dis- charged from the body of a host, to achieve the purpose of treat- ing inflammations, and no additional cross-linking agent is used.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a 1H NMR spectrum of sodium hyaluronate and thi- olated hyaluronic acid prepared in Example 1; FIG. 2 shows an SEM image of thiolated hyaluronic acid pre- pared in Example 1; FIG. 3 shows an SEM image of a modified hyaluronic acid hy- drogel loaded with akkermansia muciniphila prepared in Example 1.

DETAILED DESCRIPTION OF THE EMBODIMENTS According to the present invention, sodium hyaluronate, N- hydroxy succinimide, l-ethyl-3-(3-dimethyl amino propyl) car- bodiimide hydrochloride and water are mixed; and after an ami- dation reaction, L-cysteine methyl ester hydrochloride is subse- quently added to carry out a thiolation reaction, to obtain thio- lated hyaluronic acid.

According to the present invention, the mass ratio of the so- dium hyalurcnate to N-hydroxy succinimide to 1-ethy1-3-{(3-dimethyl amino propyl) carbodiimide hydrochloride is preferably (0.2-0.8): (0.3-0.9): (0.6-1.5), more preferably (0.3-0.86): (0.5-0.8): (0.9-

1.2), and most preferably 0.4: 0.575: 0.958. According to the pre- sent invention, the mass ratio of the sodium hyaluronate to water is (0.2-0.8): 100, more preferably (0.3-0.6): 100, and most pref- erably 0.4: 100. According to the present invention, the water is preferably deionized water.

According to the present invention, the amidation reaction is preferably carried out under a condition of room temperature; and the time of the amidation reaction is preferably 0.5-5 hours, more preferably 1-3 hours, and most preferably 1 hour. According to the present invention, the purpose of the amidation reaction is to sufficiently activate carboxyl in the sodium hyaluronate.

According to the present invention, the chemical formula of the amidation reaction is as shown in Formula 1: Rag i. oo fa a oo a ree 5 bd 3 p= tm en od J A re | ar J A RE w an EE | vw En J Formula 1; According to the present invention, the mass ratio of the so- dium hyalurcnate to L-cysteine methyl ester hydrochloride is (0.2-

0.8): (0.6-1.2), more preferably (0.3-0.6): {(0.8-0.9), and most preferably 0.4: 0.855.

According to the present invention, the thiclation reaction is preferably carried out under conditions of pH=4-6 and photopro- tection; the pH value is more preferably 4.8; the wavelength of the photoprotection is preferably 254 nm; and a light intensity is preferably 20 mW/cm®. According to the present invention, the pH value is preferably achieved by adding 1.0 mol/L sodium hydroxide or 1.0 mol/L hydrochloric acid for adjustment into a thiolation reaction system.

According to the present invention, the chemical formula of the thiolation reaction is shown in Formula 2:

SN . Da EN = w SP vo A mA oo Sw” ug et \ . Lode LY rider Le » on has os oN mt FE

HANHS HASH Formula 2; After the thiolation reaction is completed, the preparation method preferably further includes carrying out post-treatment on a product system obtained after the thiolation reaction; the post- treatment preferably includes mixing the obtained product system with hydrochloric acid with a pH value of 3.5, and molecular in- terception of dialysis is1000. After the dialysis is completed, the preparation method preferably further includes freeze-drying.

According to the present invention, the wavelength of ultra- violet radiation is preferably 365 nm, the light intensity is preferably 30 mW/cm®, and the time is preferably 120 seconds. Ac- cording to the present invention, the ultraviolet radiation is preferably carried out in a mold.

According to the present invention, the pH value of the alka- line buffer is preferably 8; and the time for eddy mixing is pref- erably 10 seconds. According to the present invention, the culture temperature is preferably 37°C. After the culture is completed, a complete gel is formed in a uniform shaking mode preferably in the preparation method.

According to the present invention, the viscosity of a vis- cous flow state is preferably 20-100mPa.s, and more preferably 80- 100 mPa.s.

According to the present invention, the concentration of an akkermansia muciniphila suspension is preferably 10°-10° CFU/mL.

According to the present invention, the preparation method of the akkermansia muciniphila suspension preferably includes the following steps:

After culturing the AKK in an MRS liguid medium, collecting cells, washing the collected cells with buffered saline, then re- suspending the cell microspheres with sterile PBS, to obtain the akkermansia muciniphila suspension. According to the present in- 5 vention, the AKK is preferably a commercially available product well known by a person skilled in the art. Components and propor- tions of the MRS liquid medium are not specially restricted in the present invention, and compositions well known by a person skilled in the art can be used.

According to the present invention, the culture preferably lasts overnight in a double-chamber oxygen-free air box of 37 °C; and the atmosphere in the double-chamber oxygen-free air box in- cludes 88 wvol% of N;, 10 vols of CO: and 2% of Hs.

According to the present invention, the temperature for col- lecting cells is preferably 4 °C; the mode for collecting the cells is preferably centrifugation; the rotation speed of the centrifu- gation is preferably 5000 rpm/min; and the time is preferably 5 minutes.

According to the present invention, the pH value of the ster- ile PBS is preferably 7.4.

According to the present invention, the dose ratio of the ox- idation-reduction sensitive hydrogel to the akkermansia muciniphi- la suspension is preferably (5-10) g: (1-4) ml, more preferably (6-8) g: (2-3) ml, and most preferably 7g: 2 ml.

According to the present invention, the temperature of cool- ing is preferably -10 °C to -30 °C.

The present invention further provides a modified hyaluronic acid hydrogel loaded with the akkermansia muciniphila prepared by using the preparation method of the foregoing technical solution.

The modified hyaluronic acid hydrogel includes an oxidation- reduction sensitive hyaluronic acid hydrogel and akkermansia mu- ciniphila encapsulated in the oxidation-reduction sensitive hyalu- ronic acid hydrogel.

The present invention further provides an application of the modified hyaluronic acid hydrogel loaded with the akkermansia mu- ciniphila of the technical solution in preparing a drug for treat-

ing enteritis. Example 1

0.4 g of sodium hyaluronate, 0.575 g (5 mmol) of N-hydroxy succinimide and 0.958 g (bmmol) of 1-ethy1-3-{3-dimethyl amino propyl) carbodiimide hydrochloride were fully dissolved into 100 mL of deionized water; after reaction at room temperature for 1 hour, 0.855 g (5 mmol) of L-cysteine methyl ester hydrochloride was added, and stirred for 24 hours under photoprotection (the wavelength of 254 nm; and the intensity of 20 mW/cm®); in the whole process, the pH value of a solution was maintained at 4.8 by add- ing a 1.0 mol/L sodium hydroxide solution or a 1.0 mol/L hydro- chloric acid solution; after the reaction was completed, the ob- tained solution was mixed with a hydrochloric acid solution with a pH value of 3.5, and dialysis was carried out, with a molecular interception of the dialysis of 1000, and freeze-dried, to obtain thiolated hyaluronic acid; ultraviolet radiation was carried out on the thiolated hyalu- ronic acid under conditions of a wavelength of 365 nm and a light intensity of 30 mW/cm® for 120s, to obtain a hyaluronic acid self- crosslinked hydrogel; 16 mg of the hyaluronic acid self-crosslinked hydrogel was dissolved into a buffer with a pH value of 8, after eddy mixing for 10 seconds, cultured at 37 °C, and shaken uniformly, to obtain an oxidation-reduction sensitive hyaluronic acid hydrogel; the AKK was cultured in an MRS liquid medium in a double- chamber oxygen-free air box of 37 °C (88 vol% of N:, 10 vol% of CC., and 2 vol% of H,), and centrifuged for 5 minutes at 5000 rpm/min at 4 °C; cells were collected; the collected cells were washed with buffered saline twice; cell microspheres were resus- pended with 200 pL of sterile PBS, to obtain a 10° CFU/mL akkerman- sia muciniphila suspension; and 70 g of the oxidation-reduction sensitive hyaluronic acid hy- drogel was heated to a viscous flow state (the viscosity of 80 mPa.s), mixed with 20 mL of the akkermansia muciniphila suspen- sion, and cooled at minus 30 °C, to obtain a modified hyaluronic acid hydrogel loaded with the akkermansia muciniphila.

A 1H NMR spectrum test was carried out on sodium hyaluronate and the thiolated hyaluronic acid prepared in Example 1. Test re- sults were shown in FIG. 1, a characteristic peak occurring at

2.01 ppm was attributed to protons of N-acetyl in an HA polymer, thus verifying existence of an HA main chain in a modified sample. Compared with a spectrum of natural hemagglutinin, the peak of he- magglutinin occured at 3.35 ppm, which indicated the cysteine de- rivative contained a -COCH:- part. Existence of a methylene proton (-CH:-SH) at about 2.85 ppm also supplemented a thiol group to be successfully grafted to an HA polymer. The results proved success- ful synthesis of HA-SH.

An SEM test was carried out on the thiolated hyaluronic acid prepared in Example 1 and the modified hyaluronic acid hydrogel loaded with the akkermansia muciniphila. Test results were shown in FIG. 2 and FIG. 3.FIG. 2 and FIG. 3 showed that the hydrogel was of an intercommunicated porous structure after AKK was encap- sulated, and pores were extremely non-uniform in size and may have high nutrient permeability, so that an appropriate habitat was provided for the AKK. As shown in FIG. 3, local magnified imaging of the hydrogel showed that probiotics of a surface morphology were not remarkably changed by encapsulation, this may be due to the fact that glucan and heteropolysaccharides have existed on cell walls of gram positive bacteria, which indicated that the bi- ocompatibile environment of the HA-based hydrogel supported growth of the AKK.

Test examples Cytotoxicity test: 8 g of an oxidation-reduction sensitive hyaluronic acid hy- drogel was soaked into cell mediums in identical growth states (the cell type is 293T) for 12 hours, 24 hours, 36 hours and 48 hours respectively, to obtain 4 samples; After three generations of passage were carried out on the 4 samples respectively, the samples were respectively inoculated on a 96-well plate (the density of 2*10%/well), and cultured over- night; 100 pL of a fresh RPMI 1640 medium (500 pg/mL) containing thiazolyl blue was added into each well; 4 hours later, a generat- ed crystal was dissolved with 100 pL dimethyl sulfoxide substitut- ing a supernate; absorbancy values of each group of cells were measured at 570 nm, which were respectively 0.812, 0.810, 0.811 and 0.810; g of trehalose, 10 g of skim milk powder, 0.5 g of glycer- inum, and 5 g of soya protein were added into 100 mL of deionized water, and dissolved and mixed uniformly; transglutaminase was added and uniformly mixed and stirred for 10 minutes; the tempera- 10 ture was kept at 37 °C for 40 minutes in a water bath®°C; cell cul- ture was carried out for 24 hours with the obtained solution sub- stituting a medium; 100 pL of the fresh RPMI 1640 medium (500 ng/mL) containing thiazolyl blue was added into each well; 4 hours later, the generated crystal was dissolved with 100 pL of sulfox- ide substituting the supernate; and an absorbancy value of cells (that is, untreated absorbancy values) was measured, which was

0.811.

The cytotoxicity was expressed by a ratio (cell survival rate) of an absorbancy value of a cell treated with the oxidation- reduction sensitive hyaluronic acid hydrogel to an absorbancy val- ue of an untreated cell, that is, survival rates of cells treated with the oxidation-reduction sensitive hyaluronic acid hydrogel for 12 hours, 24 hours, 36 hours and 48 hours were respectively

100.12%, 99.88%, 100% and 99.88%.

Hemolysis test: Mouse erythrocytes (mRBC)were freshly extracted, diluted with normal saline to 2 wt%, mixed with the oxidation-reduction sensi- tive hyaluronic acid hydrogel of an equal volume, incubated in an incubator of 37 °C for 2 hours, and centrifuged for 10 minutes at a speed of 10000 rpm; an absorbance test was carried out on a super- nate at 545 nm; and mRBC suspension treated with 0.1% (v/v) Tri- ton-X as a negative control (100% bacteriolysis), and untreated Mrbcs were treated with normal saline for comparison; Test results: hemolysis of groups of the oxidation-reduction sensitive hyaluronic acid hydrogel was almost not observed by na- ked eyes, and was compared with an untreated control group. It in-

dicated that the acute toxicity and good metabolic activity of the oxidation-reduction sensitive hyaluronic acid hydrogel can be ne- glected, and this provided an up-and-coming biological material for providing live probiotics and preventing side effects. Test on whether the hydrogel can be used as a drug delivery platform of an oxidation-reduction reaction:

0.3 g of a freshly prepared oxidation-reduction sensitive hy- aluronic acid hydrogel was soaked into 5 mL of 5 mol/L dithio- threitol and 10 mol/L dithiothreitol respectively; a PBS buffer (pH=7.4) was taken as a control; a sample was suck-dried with blotting paper; a wet weight (W.) of the sample was tested 40 minutes later; the oxidation-reduction sensitivity of the oxida- tion-reduction sensitive hyaluronic acid hydrogel was expressed by a wet weight residue. The lower the wet weight residue was, the better a delivery effect was; wet weight residue=W./Wyx100%, in the formula, W, was the weight of the hydrogel before soaked; W, was the weight of the hydrogel after soaked and suck-dried; and the weight of the oxidation-reduction sensitive hyaluronic acid hydro- gel was reduced to 60% within 40 minutes.

200 pL of a mixed solution of sodium sulfide (5 mM) and PBS was injected into a methyl red dyed oxidation-reduction sensitive hyaluronic acid hydrogel column mold, then the glass bottle was inversely placed; culture was carried out for 40 minutes at a room temperature; the weight thereof was measured; and the weight of the oxidation-reduction sensitive hyaluronic acid hydrogel was slightly increased due to water expansion.

The result showed that the oxidation-reduction sensitive hya- luronic acid hydrogel was poor in oxidation-reduction sensitivity, and can be used as the drug delivery platform of the oxidation- reduction reaction.

Test on release of the AKK in the oxidation-reduction sensi- tive hyaluronic acid hydrogel: 10 mL of the modified hyaluronic acid hydrogel loaded with the akkermansia muciniphila prepared in Example 1 was soaked into 50 mL of a PBS buffer, a 5 mM DTT solution and a 10 mM DTT solu- tion respectively, and continuously oscillated; at a time interval of 40 minutes, an optical density (OD, the larger the optical den-

sity was, the higher the release rate was) of 100 pL of an ambient medium was measured at 600 nm by using a microplate reader; and the OD values were respectively 0.434 and 0.678. Thus, it indicated that the AKK was continuously and rapidly released under stimulation of a high-concentration DTT solution; the release amount was approximate to 100%; the hydrogel in the modified hyaluronic acid hydrogel loaded with the akkermansia mu- ciniphila was completely dissolved within 40 minutes; no leakage of massive substance was caused by the PBS medium; and the results showed that a vulcanized hyaluronic acid self-crosslinked hydrogel was capable of rapidly responding to reduction stimulation, and through release of substances, it provided a significant basis for targeted delivery of probiotics according to intestinal bacteria and microenvironments.

Claims

CONCLUSIONS

A method for preparing a modified hyaluronic acid hydrogel loaded with akkermansia muciniphila, comprising the steps of: mixing sodium hyaluronate, N-hydroxysuccinimide, 1-ethyl-3-{(3-dimethylaminopropyl)carbodiimide hydrochloride and water, and after an amidation reaction, adding L-cysteine methyl ester hydrochloride to carry out a thiolation reaction, to obtain thiolated hyaluronic acid; performing ultraviolet irradiation on the thiolated hyaluronic acid, to obtain a hyaluronic acid self-crosslinked hydrogel; dissolving the hyaluronic acid self-crosslinked hydrogel in an alkaline buffer, performing eddy mixing and culturing, to obtain an oxidation-reduction sensitive hyaluronic acid hydrogel; heating the oxidation-reduction sensitive hyaluronic acid hydrogel to a viscous flow state, adding the akkermansia muciniphila suspension for mixing, cooling, to obtain a modified hyaluronic acid hydrogel loaded with the akkermansia muciniphila.

The method of claim 1, wherein the mass ratio of the sodium hyaluronate to N-hydroxysuccinimide to 1-ethyl-3-{(3-dimethylaminopropyl)carbodiimide hydrochloride is (0.2-0.8): (0.3-0 .9): (0.6-1.5), the mass ratio of the sodium hyaluronate to L-cysteine methyl ester hydrochloride is (0.2-0.8): (0.6-1.2).

The method according to claim 1, wherein the thiolation reaction is carried out under conditions of pH = 4-6 and light protection.

Use of the modified hyaluronic acid hydrogel loaded with akkermansia muciniphila and prepared according to the method of claims 1 to 3 in the preparation of a medicament for the treatment of enteritis.