CN113041214A - Modified hyaluronic acid hydrogel loaded with mucinous-Ackermanella tabescens and preparation method and application thereof - Google Patents
Modified hyaluronic acid hydrogel loaded with mucinous-Ackermanella tabescens and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of drug sustained release, in particular to a modified hyaluronic acid hydrogel loaded with mucinous-Ackermansia tabescens, a preparation method and application thereof. According to the invention, after the mercaptanized hyaluronic acid is subjected to self-crosslinking to obtain hydrogel, redox treatment is carried out, so that the finally prepared hyaluronic acid embedded AKK bacteria can realize the purpose of targeted delivery of the AKK bacteria to treat and prevent enteritis, and the toxicity of the AKK bacteria can be ignored. The method successfully prevents AKK bacteria from directly contacting with environment, and improves the acid resistance and cholate survival rate of AKK bacteria in gastrointestinal tract transportation process. After reaching the destination, the redox-triggered carrier is rapidly degraded in the small intestine by reversible disulfide bonds to release probiotic cells, resulting in the excretion of AKK bacteria from the host, with the goal of combating inflammation.
Description
Technical Field
The invention relates to the technical field of drug sustained release, in particular to a modified hyaluronic acid hydrogel loaded with mucinous-Ackermansia tabescens, a preparation method and application thereof.
Background
Pathogenic enteritis is one of the most common health threats worldwide, and is usually accompanied by symptoms such as nausea, hematochezia, diarrhea and the like, and besides, patients with gastroenteritis often have higher complication incidence rates, such as intestinal abscess, liver cancer, even colon cancer and the like, so that the patients have greater worry about human health. Currently, antibacterial drug therapy is used as a main clinical treatment means for bacterial infection, and the method causes patients to have serious drug resistance crisis and toxic and side effects, so that a new treatment method is urgently needed for relieving bacterial enteritis. In fact, the pathogenesis of intestinal pathogens often begins by the adhesion of epithelial cells and tissues. Previous studies have shown that if adhesion does not occur, the pathogen is repelled by physiological mechanical defense mechanisms such as peristalsis and mucus secretion of the host. Weakening bacterial adhesion can therefore promote pathogen clearance by the host immune system, a promising therapeutic approach.
A number of researchers have reported that AKK bacteria play a beneficial role in regulating human health and disease. They can provide protection to the host from gastrointestinal pathogens by producing bacteriocins, modulating immune and non-immune defense mechanisms, improving gut flora, and the like. More importantly, these probiotics can compete with pathogens for nutrients and localization of intestinal epithelium, thereby inhibiting pathogen adhesion and ameliorating bacterial enteritis. Although only when enough live bacteria laboratories (10 or more)6CFU/mL) are able to function when a foothold is obtained in the intestine. Unfortunately, oral administration of most probiotics results in a great deal of their viability and bioactivity, which is associated with the high acidity and bile salt concentrations present in the human gut.
To date, various encapsulation methods have been successfully developed to address these biological challenges encountered when active probiotics are administered orally. Among the numerous carriers, hydrogels with 3D porous networks are distinguished by their ideal hydrophilicity, superior mechanical properties and high permeability to oxygen/nutrients. Hyaluronic acid is a natural linear hydrophilic polysaccharide, has good biocompatibility, and is widely applied to loading and conveying of special goods. In particular, hyaluronic acid has an abundance of functional groups, including carboxyl and hydroxyl groups, useful for chemical modification. The intrinsic properties of hyaluronic acid, such as repeated disaccharide units and high viscosity, facilitate its self-crosslinking to form supramolecular hydrogels.
However, the existing hyaluronic acid-based encapsulation system has additional cross-linking agent, thereby causing the problem of exogenous biotoxicity.
Disclosure of Invention
The invention aims to provide a modified hyaluronic acid hydrogel loaded with mucinous-Ackermansia and a preparation method and application thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of a modified hyaluronic acid hydrogel loaded with mucinous-Ackermansia tabescens, which comprises the following steps:
mixing sodium hyaluronate, N-hydroxysuccinimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and water, carrying out amidation reaction, adding L-cysteine methyl ester hydrochloride, and carrying out thiolation reaction to obtain thiolated hyaluronic acid;
carrying out ultraviolet irradiation on the thiolated hyaluronic acid to obtain hyaluronic acid self-crosslinking hydrogel;
dissolving the hyaluronic acid self-crosslinking hydrogel in an alkaline buffer solution, and sequentially carrying out vortex mixing and culture to obtain a redox sensitive hyaluronic acid hydrogel;
and heating the redox sensitive hyaluronic acid hydrogel to a viscous state, adding the mucinous-Ackermansia adephaga suspension, mixing, and cooling to obtain the modified hyaluronic acid hydrogel loaded with the mucinous-Ackermansia adephaga.
Preferably, the mass ratio of the sodium hyaluronate to the N-hydroxysuccinimide to the 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride is (0.2-0.8): (0.3-0.9): (0.6-1.5).
Preferably, the mass ratio of the sodium hyaluronate to the L-cysteine methyl ester hydrochloride is (0.2-0.8): (0.6-1.2).
Preferably, the thiolation reaction is performed under the conditions of pH 4-6 and light protection.
Preferably, the wavelength of the ultraviolet radiation is 365nm, and the light intensity is 30mW/cm2And the time is 120 s.
Preferably, the pH of the alkaline buffer is 8.
Preferably, the concentration of the mucinous-Ekermansia suspension is 108～109CFU/mL；
The dosage ratio of the redox-sensitive hydrogel to the mucinous-Ackermansia aegypti suspension is (5-10) g: (1-4) mL.
Preferably, the cooling temperature is-10 to-30 ℃.
The invention also provides a modified hyaluronic acid hydrogel loaded with the mucinous-Ackermansia tabescens, which is prepared by the preparation method in the technical scheme and is characterized by comprising the redox-sensitive hyaluronic acid hydrogel and the mucinous-Ackermansia tabescens embedded in the redox-sensitive hyaluronic acid hydrogel.
The invention also provides application of the modified hyaluronic acid hydrogel loaded with the mucinophile-Ackermansia in preparation of medicines for treating enteritis.
The invention provides a preparation method of a modified hyaluronic acid hydrogel loaded with mucinous-Ackermansia tabescens, which comprises the following steps: mixing sodium hyaluronate, N-hydroxysuccinimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and water, carrying out amidation reaction, adding L-cysteine methyl ester hydrochloride, and carrying out thiolation reaction to obtain thiolated hyaluronic acid; carrying out ultraviolet irradiation on the thiolated hyaluronic acid to obtain hyaluronic acid self-crosslinking hydrogel; dissolving the hyaluronic acid self-crosslinking hydrogel in an alkaline buffer solution, and sequentially carrying out vortex mixing and culture to obtain a redox sensitive hyaluronic acid hydrogel; heating the redox sensitive hyaluronic acid hydrogel to a viscous state, adding a mucinous-Ackermansia Aiensis (AKK) suspension, mixing, and cooling to obtain the modified hyaluronic acid hydrogel loaded with the mucinous-Ackermansia aiensis. According to the invention, after the mercaptanized hyaluronic acid is subjected to self-crosslinking to obtain the hydrogel, further, vortex mixing and culture are sequentially carried out in an alkaline buffer solution for redox treatment, so that the purpose of treating and preventing enteritis by targeted delivery of AKK bacteria can be realized after the finally prepared hyaluronic acid is embedded with the AKK bacteria, and the toxicity of the hyaluronic acid can be ignored. The method successfully prevents AKK bacteria from directly contacting with environment, and improves the acid resistance and cholate survival rate of AKK bacteria in gastrointestinal tract transportation process. Upon reaching the destination, the redox-triggered carrier is rapidly degraded in the small intestine by reversible disulfide bonds releasing the probiotic cells, leading to the excretion of AKK bacteria from the host, with the goal of combating inflammation, and in the absence of additional cross-linking agents.
Drawings
FIG. 1 is a 1H NMR spectrum of sodium hyaluronate and thiolated hyaluronic acid prepared in example 1.
FIG. 2 is an SEM photograph of thiolated hyaluronic acid prepared in example 1;
FIG. 3 is an SEM photograph of a mucinous-Ekermansia-loaded modified hyaluronic acid hydrogel prepared in example 1.
Detailed Description
The invention provides a preparation method of a modified hyaluronic acid hydrogel loaded with mucinous-Ackermansia tabescens, which comprises the following steps:
mixing sodium hyaluronate (HA (Na)), N-hydroxysuccinimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and water, carrying out amidation reaction, adding L-cysteine methyl ester hydrochloride, and carrying out thiolation reaction to obtain thiolated hyaluronic acid (HA-SH);
carrying out ultraviolet irradiation on the thiolated hyaluronic acid to obtain hyaluronic acid self-crosslinking hydrogel;
dissolving the hyaluronic acid self-crosslinking hydrogel in an alkaline buffer solution, and sequentially carrying out vortex mixing and culture to obtain a redox sensitive hyaluronic acid hydrogel;
and heating the redox sensitive hyaluronic acid hydrogel to a viscous state, adding the mucinous-Ackermansia adephaga suspension, mixing, and cooling to obtain the modified hyaluronic acid hydrogel loaded with the mucinous-Ackermansia adephaga.
In the present invention, all the starting materials for the preparation are commercially available products known to those skilled in the art unless otherwise specified.
The method comprises the steps of mixing sodium hyaluronate, N-hydroxysuccinimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and water, carrying out amidation reaction, adding L-cysteine methyl ester hydrochloride, and carrying out thiolation reaction to obtain thiolated hyaluronic acid.
In the present invention, the mass ratio of the sodium hyaluronate, the N-hydroxysuccinimide and the 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride is preferably (0.2 to 0.8): (0.3-0.9): (0.6-1.5), more preferably (0.3-0.6): (0.5-0.8): (0.9-1.2), and most preferably 0.4:0.575: 0.958. In the present invention, the mass ratio of the sodium hyaluronate to the water is preferably (0.2 to 0.8): 100, more preferably (0.3 to 0.6): 100, most preferably 0.4: 100. In the present invention, the water is preferably deionized water.
The mixing process is not particularly limited, and may be performed by a method known to those skilled in the art.
In the invention, the amidation reaction is preferably carried out at room temperature, and the time of the amidation reaction is preferably 0.5 to 5 hours, more preferably 1 to 3 hours, and most preferably 1 hour. In the present invention, the purpose of the amidation reaction is to sufficiently activate the carboxyl group in sodium hyaluronate.
In the present invention, the chemical formula of the amidation reaction is shown in formula 1:
in the invention, the mass ratio of the sodium hyaluronate to the L-cysteine methyl ester hydrochloride is preferably (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.
In the invention, the thiolation reaction is preferably carried out under the conditions of pH 4-6 and light protection; more preferably the pH is 4.8; the wavelength of the light protection is 254nm, and the light intensity is 20mW/cm2. In the present invention, the pH is preferably adjusted by adding 1.0mol/L sodium hydroxide or 1.0mol/L hydrochloric acid to the system of the thiolation reaction. In the present invention, the thiolation reaction is preferably carried out under stirring conditions, and the stirring conditions are not particularly limited in the present invention, and may be carried out by a process known to those skilled in the art.
In the present invention, the chemical formula of the thiolation reaction is represented by formula 2:
after the thiolation reaction is finished, the method also preferably comprises the step of carrying out post-treatment on a product system obtained after the thiolation reaction; the post-treatment is preferably carried out by mixing the obtained product system with hydrochloric acid with the pH value of 3.5 and dialyzing; the molecular cut-off for the dialysis was 1000. After the dialysis is completed, the present invention also preferably includes lyophilization; the lyophilization is not particularly limited in the present invention, and may be carried out by a procedure well known to those skilled in the art.
After the thiolated hyaluronic acid is obtained, the thiolated hyaluronic acid is subjected to ultraviolet irradiation to obtain the hyaluronic acid self-crosslinking hydrogel. In the present invention, the wavelength of the ultraviolet radiation is preferably 365nm, and the light intensity is preferably 30mW/cm2The time is preferably 120 s. In the present invention, the ultraviolet irradiation is preferableIs carried out in a mould.
After the hyaluronic acid self-crosslinking hydrogel is obtained, the hyaluronic acid self-crosslinking hydrogel is dissolved in an alkaline buffer solution, and vortex mixing and culture are sequentially performed to obtain the redox sensitive hyaluronic acid hydrogel. In the present invention, the pH of the alkaline buffer is preferably 8; the time of the vortex mixing is preferably 10s, and the conditions of the vortex mixing are not particularly limited in the present invention and may be performed by using conditions well known to those skilled in the art. In the present invention, the temperature of the culture is preferably 37 ℃. After the culture is completed, the present invention preferably forms an intact gel by shaking.
After the redox sensitive hyaluronic acid hydrogel is obtained, the redox sensitive hyaluronic acid hydrogel is heated to a viscous state, then the mucinous-Ackermansia crenata suspension is added for mixing, and the mixture is cooled to obtain the modified hyaluronic acid hydrogel loaded with the mucinous-Ackermansia crenata.
The heating is not particularly limited in the present invention, and the redox-sensitive hyaluronic acid hydrogel can be brought into a viscous flow state by a procedure well known to those skilled in the art. In the invention, the viscosity of the viscous state is preferably 20 to 100mPa.s, and more preferably 80 to 100 mPa.s.
In the present invention, the concentration of the mucinous-Ackermann suspension is preferably 108～109CFU/mL。
In the present invention, the process for preparing the mucin-ackerman suspension preferably comprises the steps of:
and culturing AKK (alkyl ketene dimer) in an MRS (MRS) liquid culture medium, collecting cells, washing the collected cells by using buffered normal saline, and then resuspending cell microspheres by using sterile PBS (phosphate buffer solution) to obtain the mucinophile-Ackermann suspension.
In the present invention, the AKK bacterium is preferably a commercially available product well known to those skilled in the art. The invention has no special limitation on the components and the proportion of the MRS liquid culture medium, and the MRS liquid culture medium can be prepared by adopting the components which are well known by the technical personnel in the field.
In the present invention, the culture is preferably overnight in a 37 ℃ double chamber anaerobic windbox; the atmosphere in the dual-chamber oxygen-free bellows comprises 88 vol% of N210 vol% CO2And 2% of H2。
In the present invention, the temperature of the collected cells is preferably 4 ℃; the manner of collecting the cells is preferably centrifugation; the rotation speed of the centrifugation is preferably 5000rpm/min, and the time is preferably 5 min.
In the present invention, the pH of the sterile PBS is preferably 7.4.
In the present invention, the ratio of the amount of the redox-sensitive hydrogel to the mucinous-Ackermansia suspension is preferably (5 to 10) g: (1-4) mL, more preferably (6-8) g: (2-3) mL, most preferably 7 g: 2 mL.
In the present invention, the temperature of the cooling is preferably-10 to-30 ℃.
The invention also provides a modified hyaluronic acid hydrogel loaded with the mucinous-Ackermansia tabescens, which is prepared by the preparation method in the technical scheme and is characterized by comprising the redox-sensitive hyaluronic acid hydrogel and the mucinous-Ackermansia tabescens embedded in the redox-sensitive hyaluronic acid hydrogel.
The invention also provides application of the modified hyaluronic acid hydrogel loaded with the mucinophile-Ackermansia in preparation of medicines for treating enteritis. The method of the present invention is not particularly limited, and may be carried out by a method known to those skilled in the art.
The modified hyaluronic acid hydrogel loaded with the mucinous-Ackermansia species and the preparation method and application thereof according to the present invention will be described in detail with reference to the following examples, which should not be construed as limiting the scope of the present invention.
Example 1
0.4g of sodium hyaluronate, 0.575g (5mmol) of N-hydroxysuccinimide and 0.958g (5mmol) of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride were sufficiently dissolved in 100mL of deionized water and reacted at room temperature for 1 hour, and then 0.855g (5mmol) of L-cysteine methyl ester was addedHydrochloride, photoprotection (wavelength 254 nm; intensity 20 mW/cm)2) Stirring for 24h, and in the whole process, adding 1.0mol/L sodium hydroxide solution or 1.0mol/L hydrochloric acid solution to ensure that the pH of the solution is 4.8; after the reaction is finished, mixing the obtained solution with hydrochloric acid solution with the pH value of 3.5, dialyzing, wherein the molecular interception amount of dialysis is 1000, and freeze-drying to obtain thiolated hyaluronic acid;
the thiolated hyaluronic acid is subjected to the treatment of the ultraviolet light at the wavelength of 365nm and the light intensity of 30mW/cm2Performing ultraviolet irradiation for 120s under the condition to obtain hyaluronic acid self-crosslinking hydrogel;
dissolving 16mg of the hyaluronic acid self-crosslinking hydrogel in a buffer solution with the pH value of 8, mixing for 10s by vortex, culturing at 37 ℃, and shaking up to obtain a redox-sensitive hyaluronic acid hydrogel;
AKK bacteria were grown in MRS liquid medium and in a dual chamber anaerobic bellows (88 vol% N) at 37 deg.C2,10vol％CO2,2vol％H2) Culturing, centrifuging at 4 deg.C and 5000rpm/min for 5min, collecting cells, washing the collected cells with buffered physiological saline for 2 times, and resuspending the cell microspheres with 200 μ L sterile PBS to obtain 10-concentration cell microspheres8CFU/mL mucinophil-ackerman suspension.
Heating 70g of the redox-sensitive hyaluronic acid hydrogel to a viscous state (the viscosity is 80mPa.s), adding 20mL of a mucinous-Ackermansia adenantha suspension, mixing, and cooling at-30 ℃ to obtain the modified hyaluronic acid hydrogel loaded with the mucinous-Ackermansia adenantha;
when sodium hyaluronate and thiolated hyaluronic acid prepared in example 1 were subjected to 1H NMR spectroscopic measurement, the results are shown in fig. 1, and the characteristic peak appearing at 2.01ppm can be attributed to the proton of N-acetyl group in HA polymer, confirming the presence of HA backbone in the modified sample. The peak of hemagglutinin appeared at 3.35ppm compared to the spectrum of native hemagglutinin, indicating that our cysteine derivatives contained-COCH3-a moiety. Methylene proton (-CH)2-SH) was present at about 2.85ppm, also complementing the successful grafting of thiol groups to the HA polymer. These results all confirm the successful synthesis of HA-SH。
The thiolated hyaluronic acid prepared in example 1 and the modified hyaluronic acid hydrogel loaded with the muciniphilic-Ackermansia species were subjected to SEM tests, and the test results are shown in FIGS. 2 and 3, and it can be seen from FIGS. 2 and 3 that the hydrogel, after encapsulating AKK, exhibits an interconnected porous structure, has a very non-uniform pore size, may have a high nutrient permeability, and provides a suitable habitat for the AKK. As shown in fig. 3, the locally enlarged imaging of the hydrogel encapsulated probiotics without significant changes in surface morphology, probably due to the presence of dextran and heteropolysaccharide already on the cell wall of these gram-positive bacteria, indicating that the biocompatible environment of the HA-based hydrogel supports the growth of AKK bacteria.
Example 2
0.5g of sodium hyaluronate, 0.8g (6.96mmol) of N-hydroxysuccinimide and 1.1g (5.74mmol) of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride were thoroughly dissolved in 100mL of deionized water, reacted at room temperature for 1 hour, and then 0.9g (5.26mmol) of L-cysteine methyl ester hydrochloride was added thereto under light protection (wavelength 254nm, intensity 20 mW/cm)2) Stirring for 24h, and in the whole process, adding 1.0mol/L sodium hydroxide solution or 1.0mol/L hydrochloric acid solution to ensure that the pH of the solution is 4.8; after the reaction is finished, mixing the obtained solution with hydrochloric acid solution with the pH value of 3.5, dialyzing, wherein the molecular interception amount of dialysis is 1000, and freeze-drying to obtain thiolated hyaluronic acid;
the thiolated hyaluronic acid is subjected to the treatment of the ultraviolet light at the wavelength of 365nm and the light intensity of 30mW/cm2Performing ultraviolet irradiation for 120s under the condition to obtain hyaluronic acid self-crosslinking hydrogel;
dissolving 16mg of the hyaluronic acid self-crosslinking hydrogel in a buffer solution with the pH value of 8, mixing for 10s by vortex, culturing at 37 ℃, and shaking up to obtain a redox-sensitive hyaluronic acid hydrogel;
AKK bacteria were grown in MRS liquid medium and in a dual chamber anaerobic bellows (88 vol% N) at 37 deg.C2,1vol％CO2,2vol％H2) Culturing, centrifuging at 4 deg.C and 5000rpm/min for 5min, collecting cells, washing the collected cells with buffered physiological saline for 2 times, and washing with 200 μ L of sterile waterBacterial PBS (phosphate buffered saline) resuspends cell microspheres to obtain the concentration of 108FU/mL of a mucinophil-Ackermann suspension.
Heating 80g of the redox-sensitive hyaluronic acid hydrogel to a viscous state (the viscosity is 91mPa.s), adding 20mL of a mucinous-Ackermansia adenantha suspension, mixing, and cooling at-30 ℃ to obtain the modified hyaluronic acid hydrogel loaded with the mucinous-Ackermansia adenantha;
sodium hyaluronate and thiolated hyaluronic acid prepared in example 1 were subjected to1H NMR spectroscopy, similar to that of example 1;
example 1 the prepared thiolated hyaluronic acid and the modified hyaluronic acid hydrogel supporting akkermansia muciniphila were subjected to SEM test, and the test results were similar to those of example 1.
Example 3
0.6g of sodium hyaluronate, 0.7g (6.09mmol) of N-hydroxysuccinimide and 1.0g (5.22mmol) of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride were thoroughly dissolved in 100mL of deionized water, reacted at room temperature for 1 hour, and then 0.9g (5.26mmol) of L-cysteine methyl ester hydrochloride was added thereto under light protection (wavelength 254nm, intensity 20 mW/cm)2) Stirring for 24h, and in the whole process, adding 1.0mol/L sodium hydroxide solution or 1.0mol/L hydrochloric acid solution to ensure that the pH of the solution is 4.8; after the reaction is finished, mixing the obtained solution with hydrochloric acid solution with the pH value of 3.5, dialyzing, wherein the molecular interception amount of dialysis is 1000, and freeze-drying to obtain thiolated hyaluronic acid;
the thiolated hyaluronic acid is subjected to the treatment of the ultraviolet light at the wavelength of 365nm and the light intensity of 30mW/cm2Performing ultraviolet irradiation for 120s under the condition to obtain hyaluronic acid self-crosslinking hydrogel;
dissolving 16mg of the hyaluronic acid self-crosslinking hydrogel in a buffer solution with the pH value of 8, mixing for 10s by vortex, culturing at 37 ℃, and shaking up to obtain a redox-sensitive hyaluronic acid hydrogel;
AKK bacteria were grown in MRS liquid medium and in a dual chamber oxygen-free bellows (88% N) at 37 deg.C2,10％CO2,2％H2) Medium culture at 4 deg.CCentrifuging at 5000rpm/min for 5min, collecting cells, washing the collected cells with buffered physiological saline for 2 times, and resuspending the cell microspheres with 200 μ L sterile PBS to obtain a 10-concentration cell microsphere8CFU/mL mucinophil-ackerman suspension.
Heating 8.9g of the redox-sensitive hyaluronic acid hydrogel to a viscous state (viscosity is 102Pa.s), adding 30mL of a mucinous-Ackermansia adenantha suspension, mixing, and cooling at-30 ℃ to obtain the modified hyaluronic acid hydrogel loaded with the mucinous-Ackermansia adenantha;
sodium hyaluronate and thiolated hyaluronic acid prepared in example 1 were subjected to1H NMR spectroscopy, similar to that of example 1;
example 1 the prepared thiolated hyaluronic acid and the modified hyaluronic acid hydrogel supporting akkermansia muciniphila were subjected to SEM test, and the test results were similar to those of example 1.
Test example
Cytotoxicity test:
soaking 8g of redox sensitive hyaluronic acid hydrogel in cell culture medium (cell type 293T) with the same growth condition for 12h, 24h, 36h and 48h to obtain 4 samples;
after three passages of the 4 samples, the samples were inoculated in 96-well multi-plates (density 2X 10)4One/well), cultured overnight, 100. mu.L of fresh RPMI 1640 medium (500. mu.g/mL) containing thiazole blue was added to each well for 4 hours, 100. mu.L of dimethyl sulfoxide was used instead of the supernatant to dissolve crystals, and the absorbance values at 570nm of each group of cells were measured to be 0.812, 0.810, 0.811, and 0.810, respectively;
taking 100mL of deionized water, adding 10g of trehalose, 10g of skimmed milk powder, 0.5g of glycerol and 5g of soybean protein, dissolving and mixing uniformly, adding transglutaminase, mixing uniformly and stirring for 10 minutes, keeping the temperature in a water bath kettle at 37 ℃ for 40 minutes, replacing a culture medium with the obtained solution, culturing cells for 24 hours, adding 100 mu L of fresh RPMI 1640 medium (500 mu g/mL) containing thiazole blue into each hole for 4 hours, replacing supernatant with 100 mu L of dimethyl sulfoxide, dissolving to generate crystals, and testing the absorbance value (namely the untreated absorbance value) of the cells, wherein the absorbance value is 0.811;
cytotoxicity was expressed by the ratio of absorbance value at 570nm of cells treated with the redox-sensitive hyaluronic acid hydrogel to that of untreated cells (cell survival rate); that is, the cell viability rates after the redox-sensitive hyaluronic acid hydrogel treatment for 12h, 24h, 36h and 48h were 100.12%, 99.88%, 100% and 99.88%, respectively.
And (3) hemolytic test:
freshly extracting mouse red blood cells (mRBC), diluting to 2 wt% with normal saline, mixing with redox sensitive hyaluronic acid hydrogel with the same volume, incubating for 2h in a 37 ℃ incubator, centrifuging at 10000rpm for 10min, and taking supernatant to perform absorbance determination at 545 nm; a negative control (100% lysis) was made by treating the untreated mRBC with saline using 0.1% (v/v) Triton-X treated red blood cell suspension;
the test results are: hemolysis of the redox-sensitive hyaluronic acid hydrogel groups was hardly observed with the naked eye, also compared to the untreated control group. This indicates that the acute toxicity and good metabolic activity of the redox sensitive hyaluronic acid hydrogel are negligible, which provides a promising biomaterial to provide live probiotics and avoid side effects.
Testing whether it could serve as a drug delivery platform for redox reactions:
respectively soaking 0.3g of freshly prepared redox sensitive hyaluronic acid hydrogel in 5mL of dithiothreitol with the concentration of 5mol/L and 10 mol/L; PBS buffer (pH 7.4) was used as a control; after blotting the sample with blotting paper, the wet weight (W) was measured at 40mint) (ii) a Wherein the redox sensitivity of the redox-sensitive hyaluronic acid hydrogel is represented by wet weight residual, the lower the wet weight residual, the better the transfer effect is proved, and the wet weight residual is Wt/W0X 100% where W0Is the weight of the hydrogel before soaking; soaking and sucking the weight of the dried hydrogel; said redox sensitive transparentThe weight of the hyaluronic acid hydrogel is reduced to 60% within 40 min;
injecting 200 μ L of a mixed solution of sodium sulfide (5mM) and PBS into a cylindrical mold containing methyl red-stained redox-sensitive hyaluronic acid hydrogel, inverting the glass bottle, culturing at room temperature for 40min, and measuring the weight of the glass bottle, wherein the redox-sensitive hyaluronic acid hydrogel slightly increases in weight due to water swelling;
according to the above results, it is demonstrated that the redox-sensitive hyaluronic acid hydrogel has poor redox sensitivity and can be used as a drug delivery platform for redox reaction.
The release behavior of AKK bacteria in the redox sensitive hyaluronic acid hydrogel was tested:
immersing 10mL of the modified hyaluronic acid hydrogel loaded with the mucinous-Ackermanomyces exsiccus prepared in example 1 into 50mL of PBS buffer solution and DTT solutions with the concentrations of 5mM and 10mM respectively, continuously shaking, taking 100 mu L of surrounding medium at a time interval of 40min, and detecting the Optical Density (OD) at 600nm by using an enzyme labeling instrument (the higher the optical density is, the higher the release rate is), wherein the OD values are 0.434 and 0.678 respectively;
therefore, AKK is continuously and rapidly released under the stimulation of a high-concentration DTT solution, the release amount is close to 100%, and the hydrogel in the modified hyaluronic acid hydrogel loaded with the mucinous-Ackermanomyces avermitilis is completely dissolved within 40 min; and the PBS culture medium does not cause a large amount of cargos to leak, and the results show that the vulcanized hyaluronic acid self-crosslinking hydrogel can quickly respond to reduction stimulation, and cargos are unloaded by water inflow, thereby laying an important foundation for targeted delivery of probiotics according to intestinal flora and microenvironment.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A preparation method of a modified hyaluronic acid hydrogel loaded with mucin-Ackermansia sp is characterized by comprising the following steps:
mixing sodium hyaluronate, N-hydroxysuccinimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and water, carrying out amidation reaction, adding L-cysteine methyl ester hydrochloride, and carrying out thiolation reaction to obtain thiolated hyaluronic acid;
carrying out ultraviolet irradiation on the thiolated hyaluronic acid to obtain hyaluronic acid self-crosslinking hydrogel;
dissolving the hyaluronic acid self-crosslinking hydrogel in an alkaline buffer solution, and sequentially carrying out vortex mixing and culture to obtain a redox sensitive hyaluronic acid hydrogel;
and heating the redox sensitive hyaluronic acid hydrogel to a viscous state, adding the mucinous-Ackermansia adephaga suspension, mixing, and cooling to obtain the modified hyaluronic acid hydrogel loaded with the mucinous-Ackermansia adephaga.
2. The method according to claim 1, wherein the mass ratio of sodium hyaluronate, N-hydroxysuccinimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride is (0.2-0.8): (0.3-0.9): (0.6-1.5).
3. The method according to claim 1, wherein the mass ratio of the sodium hyaluronate to the L-cysteine methyl ester hydrochloride is (0.2-0.8) to (0.6-1.2).
4. The preparation method according to claim 1, wherein the thiolation reaction is performed under the conditions of pH 4 to 6 and photo-protection.
5. The method of claim 1, wherein the ultraviolet radiation has a wavelength of 365nm and an intensity of 30mW/cm2And the time is 120 s.
6. The method of claim 1, wherein the pH of the alkaline buffer is 8.
7. The method of claim 1, wherein the concentration of the mucinous-Ekermansia suspension is 108～109CFU/mL；
The dosage ratio of the redox-sensitive hydrogel to the mucinous-Ackermansia aegypti suspension is (5-10) g: (1-4) mL.
8. The method of claim 1, wherein the cooling temperature is from-10 ℃ to-30 ℃.
9. The modified hyaluronic acid hydrogel carrying a mucin-Ackermansia species produced by the production method according to any one of claims 1 to 8, which comprises a redox-sensitive hyaluronic acid hydrogel and a mucin-Ackermansia species embedded in the redox-sensitive hyaluronic acid hydrogel.
10. Use of the modified hyaluronic acid hydrogel loaded with a muciniphilic-Ackermansia species of claim 9 for the preparation of a medicament for the treatment of enteritis.
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