A loaded exosome composite hydrogel and a preparation method thereof

Abstract

The application provides a kind of load exosome composite hydrogel and preparation method thereof, belong to biomaterial and drug delivery technical field;Its preparation method includes: modified chitosan;Modified hyaluronic acid;Preparation of biphasic calcium phosphate nanoparticles;Preparation of composite hydrogel.The application is through nucleophilic ring-opening reaction of 1,2-epoxy butane and the hydroxyl group of chitosan, form hydroxybutyl ether bond, give hydroxybutyl chitosan temperature sensitive characteristics, then, again, after activation 3,4-dihydroxyphenylalanine is condensed with hydroxybutyl chitosan, form amide bond, to graft 3,4-dihydroxyphenylalanine on hydroxybutyl chitosan, after modification of chitosan, 3,4-dihydroxyphenylalanine can form strong adhesion interface with cartilage, synovial membrane and other tissues through hydrogen bond, pi-pi stacking and metal coordination, thereby effectively prolonging the residence time of hydrogel in joint.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomaterials and drug delivery, in particular to a kind of loaded exosome composite hydrogel and preparation method thereof. BACKGROUND

[0002] Osteoarthritis is a chronic degenerative joint disease, characterized by degeneration of articular cartilage in synovial joints. In clinical practice, the commonly used treatment methods include the use of analgesics and anti-inflammatory drugs. Traditional drug delivery methods mainly include oral administration, transdermal administration and local injection administration. However, due to the avascular nature of cartilage tissue, oral administration cannot achieve an effective drug concentration in the joint cavity, which may lead to the need to increase the dosage, thereby increasing the risk of side effects such as gastric bleeding. Transdermal administration can only relieve superficial tissue inflammation and is difficult to penetrate deep tissue. Local injection administration, such as intra-articular injection, can deliver drugs directly to the injury site, reducing systemic side effects. However, the drug is quickly metabolized and eliminated by synovial capillaries and the lymphatic system, resulting in low treatment efficiency and difficulty in maintaining a long-term effective concentration. In order to achieve the desired effect, frequent injections are required, which not only reduces patient compliance and increases treatment costs, but also increases the risk of joint infection.

[0003] Studies have shown that miR-144-3p can reduce the expression of Smurf2 by targeting ONECUT2, and regulate the proliferation, apoptosis and inflammation of osteoarthritis fibroblasts. However, miR-144-3p has poor stability in vivo and is easily degraded by nucleases. Exosomes are small membrane vesicles secreted by cells, which have natural biocompatibility and low immunogenicity. Loading miR-144-3p into exosomes can protect it from degradation. However, exosomes injected directly into the body are difficult to retain locally and are easily dissipated. Hydrogels, due to their unique scaffold effect, lubrication, anti-inflammatory and drug delivery and release effects, can promote the retention of exosomes without being quickly removed or leaked from the joint. Therefore, injectable hydrogels can be used to load exosomes for therapeutic exosome targeted delivery through joint injection.

[0004] Natural substances such as chitosan are considered ideal materials for injectable hydrogels due to their biocompatibility, biodegradability, non-toxicity, biological activity, anti-inflammatory effect and antibacterial activity. However, pure chitosan hydrogel is brittle and prone to fracture under the mechanical stress of daily activities of osteoarthritis patients, leading to destruction of the hydrogel structure, rapid drug release, loss of sustained release effect, and inability to maintain cartilage defect area filling, resulting in accelerated joint wear. In addition, the tissue adhesion ability of chitosan hydrogel is low, which is easily washed away by body fluids or mechanical stress, resulting in a short retention time of the hydrogel in the joint cavity and low bioavailability.

[0005] Active oxygen plays an important role in the occurrence and progression of osteoarthritis, and excessive active oxygen and inflammatory cytokines in chondrocytes can damage DNA, mitochondria and proteins, causing chondrocyte apoptosis and degradation of the extracellular matrix of the cartilage, eventually leading to gradual damage of the extracellular matrix and cartilage, and osteoarthritis inflammation increases the level of active oxygen, which in turn leads to worsening inflammation, thus forming a vicious cycle, therefore, reducing the excessive active oxygen level in chondrocytes is beneficial to enhance the therapeutic effect of the drug delivery system on osteoarthritis.

[0006] Therefore, it is necessary to propose a high-toughness, tissue-adhesive and active oxygen-reducing exosome-loaded composite hydrogel and a preparation method thereof to improve the therapeutic effect on osteoarthritis. SUMMARY

[0007] In view of the deficiencies of the prior art, the purpose of the present application is to provide an exosome-loaded composite hydrogel and a preparation method thereof.

[0008] A preparation method of an exosome-loaded composite hydrogel, comprising the following steps:

[0009] S1: modifying chitosan

[0010] First, hydroxybutyl chitosan is prepared and dissolved to prepare a hydroxybutyl chitosan solution, then 3,4-dihydroxyphenylalanine is dissolved and activated to prepare a 3,4-dihydroxyphenylalanine activation solution, and then the 3,4-dihydroxyphenylalanine activation solution is added to the hydroxybutyl chitosan solution for reaction to obtain modified chitosan;

[0011] S2: modifying hyaluronic acid

[0012] The hyaluronic acid is dissolved and activated, and then L-methionine ethyl ester hydrochloride is added for reaction to obtain modified hyaluronic acid;

[0013] S3: preparing biphasic calcium phosphate nanoparticles

[0014] The calcium nitrate solution and the diammonium hydrogen phosphate solution are mixed in a volume ratio of 1:(1.2-1.3), then 0.1 mol / L sodium hydroxide solution is added to adjust the pH to 10, and after stirring and reaction until no precipitate is generated, filtration, washing and vacuum drying are performed, and then heat treatment is performed at 1120-1130℃ for 1-2h, and after cooling, ball milling is performed to obtain biphasic calcium phosphate nanoparticles;

[0015] S4: preparing a composite hydrogel

[0016] The modified chitosan and the modified hyaluronic acid are dissolved respectively, then the cellulose nanocrystals, the above-mentioned biphasic calcium phosphate nanoparticles and the hyaluronic acid solution are mixed into the modified chitosan solution, and the beta-glycerophosphate sodium solution is added, after being fully mixed, the miR-144-3p exosome loaded is mixed in, to obtain a composite hydrogel.

[0017] Further, S1 specifically comprises the following steps:

[0018] S1.1: chitosan is added into 50% potassium hydroxide solution at a solid-liquid ratio of 1g:(10-20)mL, and stirred for alkalization for 8-10h, after filtration, it is dispersed in 50% isopropyl alcohol aqueous solution at a solid-liquid ratio of 1g:(20-30)mL, and 1,2-epoxybutane is added, and heated and stirred for reaction for 20-24h at 60-70℃, after neutralization, it is dialyzed with deionized water and freeze-dried to obtain hydroxybutyl chitosan, wherein the volume ratio of 1,2-epoxybutane to isopropyl alcohol aqueous solution is 1:(1.6-1.8);

[0019] S1.2: the above-mentioned hydroxybutyl chitosan is added into hydrochloric acid solution with pH of 5 at a solid-liquid ratio of 1g:(50-60)mL, and fully stirred and dissolved to obtain a hydroxybutyl chitosan solution;

[0020] S1.3: 3,4-dihydroxyphenylalanine is dissolved in hydrochloric acid solution with pH of 5 at a solid-liquid ratio of 1g:(20-30)mL, and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide are added, and stirred for activation for 15-25min to obtain a 3,4-dihydroxyphenylalanine activation solution;

[0021] S1.4: the above-mentioned 3,4-dihydroxyphenylalanine activation solution is added dropwise into the above-mentioned hydroxybutyl chitosan solution at a volume ratio of 1:(4-5), and stirred for reaction for 20-24h under the protection of nitrogen, then 0.1mol / L glycine solution is added to terminate the reaction, and dialyzed and freeze-dried to obtain modified chitosan.

[0022] Further, S2 specifically comprises the following steps:

[0023] S2.1: hyaluronic acid is dissolved in deionized water at a mass ratio of 1:(90-100), then 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide are added, and the pH is adjusted to 4-5, and stirred for activation for 12-16h to obtain an activated hyaluronic acid solution;

[0024] S2.2: L-methionine ethyl ester hydrochloride is added to the activated hyaluronic acid solution described above, and the pH is adjusted to 7-8 with 0.1 mol / L sodium hydroxide solution, and after stirring for 20-24 h, the modified hyaluronic acid is obtained by dialysis and freeze-drying, wherein the mass ratio of L-methionine ethyl ester hydrochloride to hyaluronic acid is 1:(2.6-3).

[0025] Further, S4 specifically comprises the following steps:

[0026] S4.1: The modified chitosan prepared in step S1.4 is added to 0.1 ml / L hydrochloric acid solution at a solid-liquid ratio of 1 g:(40-50) mL at 0-4°C, and is fully stirred and dissolved to obtain a modified chitosan solution, and the modified hyaluronic acid prepared in step S2.2 is added to deionized water at a solid-liquid ratio of 1 g:(40-50) mL, and is fully stirred and dissolved to obtain a modified hyaluronic acid solution;

[0027] S4.2: Cellulose nanocrystals and the biphasic calcium phosphate nanoparticles prepared in step S3 are added to the above-mentioned modified chitosan solution, and after ultrasonic dispersion for 20-30 min, the above-mentioned modified hyaluronic acid solution is added, and is fully stirred and mixed uniformly to obtain a pre-mixed solution, wherein the addition amount of cellulose nanocrystals and biphasic calcium phosphate nanoparticles is 6-8% and 3-5% of the mass of the modified chitosan solution;

[0028] S4.3: 60% β-glycerophosphate sodium solution is added to the above-mentioned pre-mixed solution while stirring, and after fully mixing, a blank hydrogel solution is obtained, and the miR-144-3p exosome-loaded solution is added, and is fully stirred and mixed uniformly to obtain a composite hydrogel.

[0029] Further, in step S1, the mass ratio of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride to N-hydroxysuccinimide to 3,4-dihydroxyphenylalanine is 1:(2-3) and 1:(4-6), respectively, and the volume ratio of glycine solution to 3,4-dihydroxyphenylalanine activation solution is 1:(1.8-2.2).

[0030] Further, in step S2, the molar ratio of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride to hyaluronic acid is (350-360):1, and the molar ratio of N-hydroxysulfosuccinimide to 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is 1:1.

[0031] Further, the concentration of the calcium nitrate solution is 0.4 mol / L, and the concentration of the diammonium hydrogen phosphate solution is 0.2 mol / L.

[0032] Further, the volume ratio of the modified hyaluronic acid solution to the modified chitosan solution is (3-5):1, and the volume ratio of the beta-glycerophosphate sodium solution to the premixed solution is 1:(14-18).

[0033] Further, the loading amount of the miR-144-3p exosome is (1000-3000) μg / mL of the blank hydrogel solution, and the preparation steps of the miR-144-3p exosome are as follows: the exosome of HEK293 cells is extracted by ultracentrifugation, and after purification, it is incubated with the miR-144-3p prepared by the in vitro transcription method, and the miR-144-3p is loaded into the purified exosome by ion transfection technology, to obtain the miR-144-3p exosome.

[0034] Further, a loaded exosome composite hydrogel is prepared by the preparation method of the loaded exosome composite hydrogel according to any one of the above.

[0035] Compared with the prior art, the present application has at least the following beneficial effects:

[0036] 1、In the present application, 1, 2-epoxy butane and the hydroxyl group of chitosan are subjected to nucleophilic ring-opening reaction to form a hydroxybutyl ether bond, and the prepared hydroxybutyl chitosan realizes fast temperature-responsive sol-gel transition through hydrogen bond changes within and between molecular chains and hydrophobic interactions between hydroxybutyl groups, thereby endowing the hydroxybutyl chitosan with temperature-sensitive properties, and then, the hydroxybutyl chitosan is subjected to condensation reaction with activated 3, 4-dihydroxyphenylalanine to form an amide bond, so as to graft 3, 4-dihydroxyphenylalanine on the hydroxybutyl chitosan, and after modification of chitosan, the ortho-diphenol group of 3, 4-dihydroxyphenylalanine can form a strong adhesion interface with cartilage, synovial membrane and other tissues through hydrogen bond, pi-pi stacking and metal coordination, and at the same time, the amino side chain of 3, 4-dihydroxyphenylalanine can also enhance the adhesion to the surface of joint tissue through hydrogen bond or electrostatic interaction, thereby effectively prolonging the residence time of the hydrogel in the joint.

[0037] 2、In the present application, the carboxyl group of hyaluronic acid is activated, and then reacted with the amino group of L-methionine ethyl ester hydrochloride to form a stable amide bond, so as to graft L-methionine ethyl ester to the hyaluronic acid main chain, and after the modified hyaluronic acid and the modified chitosan are made into a composite hydrogel, the thioether group of L-methionine ethyl ester can scavenge active oxygen, inhibit inflammatory factors, and trigger the swelling and disintegration of the hydrogel, thereby responsively releasing the loaded miR-144-3p exosome, realizing controlled release of the drug, and improving the treatment effect of the drug on osteoarthritis.

[0038] 3、In the present application, after the cellulose nanocrystals and biphasic calcium phosphate nanoparticles are compounded in the hydrogel, the cellulose nanocrystals can form a nanoscale crosslinked network through hydrogen bonding and mechanical interlocking, disperse stress and delay crack propagation, while the biphasic calcium phosphate nanoparticles fill the pores of the hydrogel matrix to form a "soft-hard" composite structure, and the rigid particles inhibit the overall deformation through the pinning effect, and under the temperature trigger, the hydrogen bonding network of the cellulose nanocrystals and the rigid support of the biphasic calcium phosphate nanoparticles realize uniform stress distribution, so as to achieve the effect of synergistically improving the fracture toughness of the hydrogel. BRIEF DESCRIPTION OF DRAWINGS

[0039] The accompanying drawings, which are incorporated herein and constitute part of the specification, illustrate embodiments of the present disclosure and, together with the description, further serve to explain the principles of the present disclosure and to enable a person skilled in the relevant art to implement and use the present disclosure.

[0040] Figure 1 The preparation method flow chart of the exosome-loaded composite hydrogel used in the embodiments of the present application.

[0041] Figure 2 The cumulative release rate curve of the miR-144-3p exosome-loaded composite hydrogel prepared in Example 1 and Comparative Example 2 of the present application.

[0042] Figure 3 The absorbance histogram of the composite hydrogel prepared in Example 1 and Comparative Example 2 of the present application. DETAILED DESCRIPTION

[0043] A kind of exosome-loaded composite hydrogel and its preparation method provided by the present application are described in detail below in combination with the drawings and specific embodiments.

[0044] Example 1

[0045] A preparation method of an exosome-loaded composite hydrogel, as shown in Figure 1 , comprising the following steps:

[0046] Preparation of miR-144-3p exosome loading:

[0047] The exosomes of HEK293 cells were extracted by ultracentrifugation, and after purification, they were incubated with miR-144-3p prepared by in vitro transcription method, and miR-144-3p was loaded into the purified exosomes by ion transfection technology, to obtain miR-144-3p exosome loading.

[0048] S1: modified chitosan

[0049] S1.1: Chitosan is added into 50% potassium hydroxide solution at a solid-liquid ratio of 1 g: 10 mL, and stirred for alkalization for 8 h, and then filtered, and then dispersed in 50% isopropyl alcohol aqueous solution at a solid-liquid ratio of 1 g: 20 mL, and 1,2-epoxybutane is added, and heated and stirred at 60°C for reaction for 20 h, and then neutralized, and then subjected to dialysis with deionized water and freeze-drying to obtain hydroxybutyl chitosan, wherein the volume ratio of 1,2-epoxybutane to isopropyl alcohol aqueous solution is 1:1.6;

[0050] S1.2: The above hydroxybutyl chitosan is added into hydrochloric acid solution with pH of 5 at a solid-liquid ratio of 1 g: 50 mL, and fully stirred and dissolved to obtain a hydroxybutyl chitosan solution;

[0051] S1.3: 3,4-Dihydroxyphenylalanine is dissolved in hydrochloric acid solution with pH of 5 at a solid-liquid ratio of 1 g: 20 mL, and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide are added, and stirred for activation for 15 min to obtain a 3,4-dihydroxyphenylalanine activation solution, wherein the mass ratio of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride to N-hydroxysuccinimide to 3,4-dihydroxyphenylalanine is 1:2 and 1:4, respectively;

[0052] S1.4: The above 3,4-dihydroxyphenylalanine activation solution is added dropwise into the above hydroxybutyl chitosan solution at a volume ratio of 1:4, and stirred for reaction for 20 h under the protection of nitrogen, and then 0.1 mol / L glycine solution is added to terminate the reaction, and then subjected to dialysis and freeze-drying to obtain modified chitosan, wherein the volume ratio of glycine solution to 3,4-dihydroxyphenylalanine activation solution is 1:1.8;

[0053] S2: Modified hyaluronic acid

[0054] S2.1: Hyaluronic acid is dissolved in deionized water at a mass ratio of 1:90, and then 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide are added, and the pH is adjusted to 4, and stirred for activation for 12 h to obtain an activated hyaluronic acid solution, wherein the molar ratio of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride to hyaluronic acid is 350:1, and the molar ratio of N-hydroxysuccinimide to 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is 1:1;

[0055] S2.2: L-methionine ethyl ester hydrochloride is added into the above activated hyaluronic acid solution, and 0.1 mol / L sodium hydroxide solution is used to adjust the pH to 7, and stirred for reaction for 20 h, and then subjected to dialysis and freeze-drying to obtain modified hyaluronic acid, wherein the mass ratio of L-methionine ethyl ester hydrochloride to hyaluronic acid is 1:2.6;

[0056] S3: Preparation of biphasic calcium phosphate nanoparticles

[0057] A calcium nitrate solution with a concentration of 0.4 mol / L and a diammonium hydrogen phosphate solution with a concentration of 0.2 mol / L were mixed at a volume ratio of 1:1.2, and a 0.1 mol / L sodium hydroxide solution was added to adjust the pH to 10. After stirring and reaction until no precipitate was generated, filtration, washing, and vacuum drying were performed. Then, the sample was heat-treated at 1120°C for 1 h, cooled, and ball-milled to obtain biphasic calcium phosphate nanoparticles.

[0058] S4: Preparation of a composite hydrogel

[0059] S4.1: The modified chitosan prepared in step S1.4 was added to a 0.1 ml / L hydrochloric acid solution at a solid-liquid ratio of 1 g:40 mL, and fully stirred and dissolved to obtain a modified chitosan solution. The modified hyaluronic acid prepared in step S2.2 was added to deionized water at a solid-liquid ratio of 1 g:40 mL, and fully stirred and dissolved to obtain a modified hyaluronic acid solution.

[0060] S4.2: Cellulose nanocrystals and biphasic calcium phosphate nanoparticles prepared in step S3 were added to the above modified chitosan solution, and ultrasonically dispersed for 20 min. Then, the above modified hyaluronic acid solution was added, and fully stirred and mixed to obtain a premix solution. The addition amount of the cellulose nanocrystals and the biphasic calcium phosphate nanoparticles was 6% and 3% of the mass of the modified chitosan solution, respectively. The volume ratio of the modified hyaluronic acid solution to the modified chitosan solution was 3:1.

[0061] S4.3: A 60% β-glycerophosphate sodium solution was added to the above premix solution while stirring, and fully mixed to obtain a blank hydrogel solution. Then, miR-144-3p exosomes were added, and fully stirred and mixed to obtain a composite hydrogel. The volume ratio of the β-glycerophosphate sodium solution to the premix solution was 1:14, and the addition amount of the miR-144-3p exosomes was 1000 μg / mL of the blank hydrogel solution.

[0062] Example 2

[0063] A preparation method of an exosome-loaded composite hydrogel, as shown in Figure 1 , comprises the following steps:

[0064] Preparation of miR-144-3p exosomes:

[0065] HEK293 cell exosomes were extracted by ultracentrifugation, and after purification, were co-incubated with miR-144-3p prepared by an in vitro transcription method. The miR-144-3p was loaded into the purified exosomes by ion transfection technology to obtain miR-144-3p exosomes.

[0066] S1: modified chitosan

[0067] S1.1: chitosan was added into 50% potassium hydroxide solution at a solid-liquid ratio of 1g:15mL, and alkalization was stirred for 8-10h, after filtration, it was dispersed in 50% isopropanol aqueous solution at a solid-liquid ratio of 1g:25mL, and 1,2-epoxybutane was added, and heating stirring reaction was carried out at 65℃ for 22h, after neutralization, deionized water dialysis and freeze drying, hydroxybutyl chitosan was obtained, wherein the volume ratio of 1,2-epoxybutane to isopropanol aqueous solution was 1:1.7;

[0068] S1.2: the above hydroxybutyl chitosan was added into hydrochloric acid solution with pH of 5 at a solid-liquid ratio of 1g:55mL, and was fully stirred to dissolve, to obtain hydroxybutyl chitosan solution;

[0069] S1.3: 3,4-dihydroxyphenylalanine was dissolved in hydrochloric acid solution with pH of 5 at a solid-liquid ratio of 1g:25mL, and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide were added, and stirring activation was carried out for 20min, to obtain 3,4-dihydroxyphenylalanine activation solution, wherein the mass ratio of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide to 3,4-dihydroxyphenylalanine was 1:2.5 and 1:5 respectively;

[0070] S1.4: the above 3,4-dihydroxyphenylalanine activation solution was added dropwise into the above hydroxybutyl chitosan solution at a volume ratio of 1:4.5, and after stirring reaction for 22h under the protection of nitrogen, 0.1mol / L glycine solution was added to terminate the reaction, and dialysis and freeze drying were carried out, to obtain modified chitosan, wherein the volume ratio of glycine solution to 3,4-dihydroxyphenylalanine activation solution was 1:2;

[0071] S2: modified hyaluronic acid

[0072] S2.1: hyaluronic acid was dissolved in deionized water at a mass ratio of 1:95, and then 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide were added, and the pH was adjusted to 4.5, and stirring activation was carried out for 14h, to obtain activated hyaluronic acid solution, wherein the molar ratio of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride to hyaluronic acid was 355:1, and the molar ratio of N-hydroxysuccinimide to 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride was 1:1;

[0073] S2.2: L-methionine ethyl ester hydrochloride was added to the above activated hyaluronic acid solution, and 0.1 mol / L sodium hydroxide solution was used to adjust the pH to 7.5. After stirring for 22 h, the modified hyaluronic acid was obtained by dialysis and freeze-drying, wherein the mass ratio of L-methionine ethyl ester hydrochloride to hyaluronic acid was 1:2.8;

[0074] S3: Preparation of biphasic calcium phosphate nanoparticles

[0075] A calcium nitrate solution with a concentration of 0.4 mol / L and a diammonium hydrogen phosphate solution with a concentration of 0.2 mol / L were mixed at a volume ratio of 1:1.25, and 0.1 mol / L sodium hydroxide solution was added to adjust the pH to 10. After stirring until no precipitate was formed, filtration, washing, and vacuum drying were performed. Then, the sample was heat-treated at 1125°C for 1.5 h, cooled, and ball-milled to obtain biphasic calcium phosphate nanoparticles.

[0076] S4: Preparation of composite hydrogel

[0077] S4.1: The modified chitosan prepared in step S1.4 was added to 0.1 ml / L hydrochloric acid solution at a solid-liquid ratio of 1 g:45 mL at 2°C, and fully stirred and dissolved to obtain a modified chitosan solution. The modified hyaluronic acid prepared in step S2.2 was added to deionized water at a solid-liquid ratio of 1 g:45 mL, and fully stirred and dissolved to obtain a modified hyaluronic acid solution.

[0078] S4.2: Cellulose nanocrystals and biphasic calcium phosphate nanoparticles prepared in step S3 were added to the above modified chitosan solution, and ultrasonic dispersion was performed for 25 min. Then, the above modified hyaluronic acid solution was added, and fully stirred and mixed to obtain a premix solution, wherein the addition amount of cellulose nanocrystals and biphasic calcium phosphate nanoparticles was 7% and 4% of the mass of the modified chitosan solution, and the volume ratio of the modified hyaluronic acid solution to the modified chitosan solution was 4:1.

[0079] S4.3: While stirring, 60% β-glycerophosphate sodium solution was added to the above premix solution, and fully mixed to obtain a blank hydrogel solution. Then, miR-144-3p exosome-loaded hydrogel was obtained by adding and fully stirring and mixing the miR-144-3p exosome, wherein the volume ratio of the β-glycerophosphate sodium solution to the premix solution was 1:16, and the addition amount of the miR-144-3p exosome-loaded hydrogel was 2000 μg / mL of the blank hydrogel solution.

[0080] Example 3

[0081] A preparation method of an exosome-loaded composite hydrogel, as shown in Figure 1 , comprises the following steps:

[0082] Preparation of miR-144-3p exosome-loaded hydrogel:

[0083] Exosomes of HEK293 cells were extracted by ultracentrifugation, and after purification, were incubated with miR-144-3p prepared by in vitro transcription method, and miR-144-3p was loaded into the purified exosomes by ion transfection technology to obtain exosomes loaded with miR-144-3p.

[0084] S1: modified chitosan

[0085] S1.1: chitosan was added to 50% potassium hydroxide solution at a solid-liquid ratio of 1g:20mL, and alkalization was stirred for 10h, and after filtration, it was dispersed in 50% isopropyl alcohol aqueous solution at a solid-liquid ratio of 1g:30mL, and 1,2-epoxybutane was added, and heated and stirred at 70℃ for 24h, and after neutralization, deionized water dialysis and freeze-drying, hydroxybutyl chitosan was obtained, wherein the volume ratio of 1,2-epoxybutane to isopropyl alcohol aqueous solution was 1:1.8;

[0086] S1.2: the above hydroxybutyl chitosan was added to a hydrochloric acid solution with a pH of 5 at a solid-liquid ratio of 1g:60mL, and was fully stirred and dissolved to obtain a hydroxybutyl chitosan solution;

[0087] S1.3: 3,4-dihydroxyphenylalanine was dissolved in a hydrochloric acid solution with a pH of 5 at a solid-liquid ratio of 1g:30mL, and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide were added, and stirred for 25min to obtain a 3,4-dihydroxyphenylalanine activation solution, wherein the mass ratio of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide to 3,4-dihydroxyphenylalanine was 1:3 and 1:6, respectively;

[0088] S1.4: the above 3,4-dihydroxyphenylalanine activation solution was added dropwise to the above hydroxybutyl chitosan solution at a volume ratio of 1:5, and after stirring for 24h under the protection of nitrogen, 0.1mol / L glycine solution was added to terminate the reaction, and after dialysis and freeze-drying, modified chitosan was obtained, wherein the volume ratio of glycine solution to 3,4-dihydroxyphenylalanine activation solution was 1:2.2;

[0089] S2: modified hyaluronic acid

[0090] S2.1: Dissolve hyaluronic acid in deionized water at a mass ratio of 1:100, then add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, and adjust the pH to 5, stir for 16 h to obtain an activated hyaluronic acid solution, wherein the molar ratio of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride to hyaluronic acid is 360:1, and the molar ratio of N-hydroxysuccinimide to 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is 1:1;

[0091] S2.2: Add L-methionine ethyl ester hydrochloride to the above activated hyaluronic acid solution, and adjust the pH to 8 with 0.1 mol / L sodium hydroxide solution, stir for 24 h, then dialyze and freeze-dry to obtain modified hyaluronic acid, wherein the mass ratio of L-methionine ethyl ester hydrochloride to hyaluronic acid is 1:3;

[0092] S3: Preparation of biphasic calcium phosphate nanoparticles

[0093] Mix the calcium nitrate solution with a concentration of 0.4 mol / L and the diammonium hydrogen phosphate solution with a concentration of 0.2 mol / L at a volume ratio of 1:1.3, then adjust the pH to 10 by adding 0.1 mol / L sodium hydroxide solution, stir until no more precipitate is produced, then filter, wash and vacuum dry, and heat treat at 1130°C for 2 h, cool, and ball mill to obtain biphasic calcium phosphate nanoparticles;

[0094] S4: Preparation of composite hydrogel

[0095] S4.1: At 4°C, add the modified chitosan prepared in step S1.4 to 0.1 ml / L hydrochloric acid solution at a solid-liquid ratio of 1g:50mL, stir to dissolve thoroughly to obtain a modified chitosan solution, and add the modified hyaluronic acid prepared in step S2.2 to deionized water at a solid-liquid ratio of 1g:50mL, stir to dissolve thoroughly to obtain a modified hyaluronic acid solution;

[0096] S4.2: Add cellulose nanocrystals and biphasic calcium phosphate nanoparticles prepared in step S3 to the above modified chitosan solution, ultrasonically disperse for 30 min, then add the above modified hyaluronic acid solution, stir thoroughly to mix uniformly to obtain a premix solution, wherein the addition amount of cellulose nanocrystals and biphasic calcium phosphate nanoparticles is 8% and 5% of the mass of the modified chitosan solution, and the volume ratio of the modified hyaluronic acid solution to the modified chitosan solution is 5:1;

[0097] S4.3: while stirring, 60% β-glycerophosphate sodium solution was added to the above premixed solution, after fully mixing, a blank hydrogel solution was obtained, and then the miR-144-3p exosome was added and stirred to obtain a composite hydrogel, wherein the volume ratio of the β-glycerophosphate sodium solution to the premixed solution was 1:18, and the amount of the miR-144-3p exosome added was 3000 μg / mL of the blank hydrogel solution.

[0098] Comparative Example 1

[0099] The difference between the present comparative example 1 and example 1 is that steps S1.2-S1.4 are removed, and the modified chitosan in step S4.1 is replaced with an equal amount of hydroxybutyl chitosan.

[0100] Comparative Example 2

[0101] The difference between the present comparative example 2 and example 1 is that step S2 is removed, and the modified hyaluronic acid in step S4.1 is replaced with an equal amount of hyaluronic acid.

[0102] Comparative Example 3

[0103] The difference between the present comparative example 3 and example 1 is that the cellulose nanocrystals in step S4.2 are replaced with an equal amount of biphasic calcium phosphate nanoparticles.

[0104] Comparative Example 4

[0105] The difference between the present comparative example 4 and example 1 is that the biphasic calcium phosphate nanoparticles in step S4.2 are replaced with an equal amount of cellulose nanocrystals.

[0106] Test Example

[0107] Test 1: The viscosities of the composite hydrogels prepared in examples 1-3 and comparative example 1 were tested using a Malvern rotational rheometer, with the temperature set at 37°C and the shear force set at 0.1 r / s, and the results are shown in Table 1.

[0108] Table 1: Viscosity test results of composite hydrogels

[0109] Viscosity (Pa s) Example 1 2785 Example 2 2812 Example 3 2797 Comparative Example 1 1984

[0110] As shown in Table 1 above, the viscosity of the composite hydrogel prepared in comparative example 1 without modifying the hydroxybutyl chitosan with 3,4-dihydroxyphenylalanine was significantly lower than that of example 1. Therefore, it can be seen that after condensation reaction between the hydroxybutyl chitosan and the activated 3,4-dihydroxyphenylalanine to form an amide bond, and grafting the 3,4-dihydroxyphenylalanine on the hydroxybutyl chitosan, the adhesion of the composite hydrogel to the surface of the joint tissue can be enhanced, thereby effectively prolonging the retention time of the hydrogel in the joint.

[0111] Test 2: In vitro drug release experiment of the composite hydrogel prepared in Example 1 was carried out in phosphate buffer solution (0.1 mol / L, pH 7.4) containing H2O2 (10 mmol / L) and phosphate buffer solution (0.1 mol / L, pH 7.4) not containing H2O2, respectively. The composite hydrogel prepared in Example 1 was placed in two dialysis bags, respectively, 1 mL of release medium (phosphate buffer solution containing H2O2 / phosphate buffer solution not containing H2O2) was added to the dialysis bag, and the dialysis bag was placed in 9 mL of the same release medium. The release system was placed in a water bath shaker at 37°C and rotated at 150 r / min. 1 mL of sample of the release medium outside the dialysis bag was taken at regular time intervals, the amount of miR-144-3p exosome loaded was determined, and 1 mL of blank medium was supplemented to the release system. The cumulative release rate of miR-144-3p exosome loaded was calculated and the cumulative release rate curve was drawn. The cumulative release rate of miR-144-3p exosome loaded in the composite hydrogel prepared in Comparative Example 2 was determined and the cumulative release rate curve was drawn in the same way using phosphate buffer solution (0.1 mol / L, pH 7.4) containing H2O2 (10 mmol / L) as the release medium, and the results are shown in Figure 2 .

[0112] As shown in Figure 2 , the release rate of the composite hydrogel prepared in Example 1 in the presence of H2O2 was significantly faster than that in the absence of H2O2, and the 25h cumulative release rate of miR-144-3p exosome loaded in the composite hydrogel in the presence of H2O2 was significantly higher than that in the absence of H2O2, indicating that the composite hydrogel prepared in Example 1 has the active oxygen response characteristic. In addition, the release rate of the composite hydrogel prepared in Example 1 in the presence of H2O2 was also significantly faster than that of the composite hydrogel prepared in Comparative Example 2 in the presence of H2O2, indicating that after the carboxyl group of hyaluronic acid is activated and then reacted with the amino group of L-methionine ethyl ester hydrochloride, the modified hyaluronic acid is prepared into a composite hydrogel with modified chitosan, which can responsively release miR-144-3p exosome loaded and achieve controlled release of the drug to improve the therapeutic effect of the drug on osteoarthritis.

[0113] Test 3: The composite hydrogels prepared in Example 1 and Comparative Example 2 were added to 5 mM H2O2 solution and shaken in a shaker at 37°C for 6 h. Then 50 μL of supernatant was added to 150 μL of Ti(SO4)2 solution, and after 1 hour of culture, the enzyme label instrument was detected at 405 nm to determine the H2O2 scavenging capacity of the composite hydrogel, and the results are shown in Figure 3 .

[0114] As shown in Figure 3As shown, the absorbance of the composite hydrogel prepared in Comparative Example 2 without modification of hyaluronic acid by L-methionine ethyl ester hydrochloride is higher than that of Example 1, indicating that the composite hydrogel of Example 1 eliminates more H2O2. Therefore, it can be seen that grafting L-methionine ethyl ester to the hyaluronic acid backbone, modifying the hyaluronic acid, and then preparing the composite hydrogel from the modified hyaluronic acid and modified chitosan, L-methionine ethyl ester can eliminate reactive oxygen species and inhibit inflammatory factors.

[0115] Test 4: After the composite hydrogels prepared in Examples 1-3 and Comparative Examples 3-4 were gelled at 37°C, their fracture energies were measured, respectively, and the results are shown in Table 2.

[0116] Table 2: Test results of fracture energy of composite hydrogel

[0117] Energy to break (J / m 2 ) Example 1 898 Example 2 902 Example 3 903 Comparative Example 3 738 Comparative Example 4 824

[0118] As shown in Table 2 above, when only one of biphasic calcium phosphate nanoparticles or cellulose nanocrystals is added in Comparative Example 3 and Comparative Example 4, the fracture energy of the prepared composite hydrogel is lower than that of Example 1. Therefore, it can be seen that under temperature triggering, cellulose nanocrystals and biphasic calcium phosphate nanoparticles can synergistically improve the fracture toughness of the hydrogel.

[0119] The above examples are only illustrative of the principles and effects of the present application, and are not intended to limit the present application. Any person skilled in the art can modify or change the above examples without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and technical ideas disclosed by the present application should be covered by the claims of the present application.

Claims

1. A method for preparing an exosome-loaded composite hydrogel, characterized in that, Includes the following steps: S1: Modified chitosan First, hydroxybutyl chitosan is prepared and dissolved to prepare a hydroxybutyl chitosan solution. Then, 3,4-dihydroxyphenylalanine is dissolved and activated to prepare a 3,4-dihydroxyphenylalanine activated solution. Then, the 3,4-dihydroxyphenylalanine activated solution is added to the hydroxybutyl chitosan solution for reaction to obtain modified chitosan. S2: Modified hyaluronic acid Hyaluronic acid is dissolved and activated, and then L-methionine ethyl ester hydrochloride is added to react and obtain modified hyaluronic acid. S3: Preparation of biphasic calcium phosphate nanoparticles Mix calcium nitrate solution and diammonium hydrogen phosphate solution at a volume ratio of 1:(1.2-1.3), then add... The pH was adjusted to 10 with 0.1 mol / L sodium hydroxide solution, and the reaction was stirred until no more precipitate was produced. The mixture was then filtered, washed, and vacuum dried. After heat treatment at 1120-1130℃ for 1-2 hours, cooled, and ball-milled to obtain biphasic calcium phosphate nanoparticles. S4: Preparation of composite hydrogels The modified chitosan and modified hyaluronic acid were dissolved separately. Then, cellulose nanocrystals, the biphasic calcium phosphate nanoparticles and hyaluronic acid solution were mixed into the modified chitosan solution, and β-glycerophosphate sodium solution was added. After thorough mixing, miR-144-3p-loaded exosomes were mixed in to obtain a composite hydrogel.

2. The method for preparing a loaded exosome composite hydrogel according to claim 1, characterized in that, S1 specifically includes the following steps: S1.1: Chitosan was added to a 50% potassium hydroxide solution at a solid-liquid ratio of 1g:(10-20)mL, stirred and alkalized for 8-10 hours, filtered, and then dispersed in a 50% isopropanol aqueous solution at a solid-liquid ratio of 1g:(20-30)mL. 1,2-Epoxybutane was added, and the mixture was heated and stirred at 60-70℃ for 20-24 hours. After neutralization, the mixture was dialyzed with deionized water and freeze-dried to obtain hydroxybutyl chitosan, wherein the volume ratio of 1,2-epoxybutane to isopropanol aqueous solution was 1:(1.6-1.8). S1.2: Add the above hydroxybutyl chitosan to a hydrochloric acid solution with pH 5 at a solid-liquid ratio of 1g:(50-60)mL, stir thoroughly to dissolve, and obtain a hydroxybutyl chitosan solution; S1.3: Dissolve 3,4-dihydroxyphenylalanine in hydrochloric acid solution with pH 5 at a solid-liquid ratio of 1g:(20-30)mL, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, stir and activate for 15-25min to obtain 3,4-dihydroxyphenylalanine activated solution; S1.4: The above 3,4-dihydroxyphenylalanine activation solution was added dropwise to the above hydroxybutyl chitosan solution at a volume ratio of 1:(4-5). Under nitrogen protection, the reaction was stirred for 20-24 hours. Then, 0.1 mol / L glycine solution was added to terminate the reaction. After dialysis and freeze-drying, modified chitosan was obtained.

3. The method for preparing a loaded exosome composite hydrogel according to claim 2, characterized in that, S2 specifically includes the following steps: S2.1: Dissolve hyaluronic acid in deionized water at a mass ratio of 1:(90-100), then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxythiosuccinimide, adjust the pH to 4-5, stir and activate for 12-16 hours to obtain an activated hyaluronic acid solution. S2.2: Add L-methionine ethyl ester hydrochloride to the above activated hyaluronic acid solution, adjust the pH to 7-8 with 0.1 mol / L sodium hydroxide solution, stir the reaction for 20-24 h, and then dialyze and freeze-dry to obtain modified hyaluronic acid, wherein the mass ratio of L-methionine ethyl ester hydrochloride to hyaluronic acid is 1:

1. (2.6-3)。 4. The method for preparing a loaded exosome composite hydrogel according to claim 3, characterized in that, S4 specifically includes the following steps: S4.1: At 0-4℃, add the modified chitosan obtained in step S1.4 to 0.1ml / L hydrochloric acid solution at a solid-liquid ratio of 1g:(40-50)mL, stir thoroughly to dissolve, and obtain a modified chitosan solution. Then, add the modified hyaluronic acid obtained in step S2.2 to deionized water at a solid-liquid ratio of 1g:(40-50)mL, stir thoroughly to dissolve, and obtain a modified hyaluronic acid solution. S4.2: Add cellulose nanocrystals and biphasic calcium phosphate nanoparticles obtained in step S3 to the above modified chitosan solution, disperse by ultrasonication for 20-30 min, then add the above modified hyaluronic acid solution, stir thoroughly and mix evenly to obtain a premixed solution, wherein the amount of cellulose nanocrystals and biphasic calcium phosphate nanoparticles added is 6-8% and 3-5% of the mass of the modified chitosan solution, respectively. S4.3: While stirring, add 60% sodium β-glycerophosphate solution to the above premixed solution. After thorough mixing, a blank hydrogel solution is obtained. Then, add the loaded miR-144-3p exosomes and stir to mix evenly to obtain a composite hydrogel.

5. The method for preparing a loaded exosome composite hydrogel according to claim 2, characterized in that, In step S1, the mass ratios of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to 3,4-dihydroxyphenylalanine are 1:(2-3) and 1:(4-6), respectively, and the volume ratio of glycine solution to 3,4-dihydroxyphenylalanine activation solution is 1:(1.8-2.2).

6. The method for preparing a loaded exosome composite hydrogel according to claim 3, characterized in that, In step S2, the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to hyaluronic acid is (350-360):1, and the molar ratio of N-hydroxythiosuccinimide to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:

1.

7. The method for preparing a loaded exosome composite hydrogel according to claim 1, characterized in that, The concentration of calcium nitrate solution is 0.4 mol / L, and the concentration of diammonium hydrogen phosphate solution is 0.2 mol / L.

8. The method for preparing a loaded exosome composite hydrogel according to claim 4, characterized in that, The volume ratio of modified hyaluronic acid solution to modified chitosan solution is (3-5):1, and the volume ratio of sodium β-glycerophosphate solution to premixed solution is 1:(14-18).

9. The method for preparing a loaded exosome composite hydrogel according to claim 4, characterized in that, The amount of miR-144-3p-loaded exosomes added was (1000-3000) μg / mL of blank hydrogel solution, and the preparation steps of miR-144-3p-loaded exosomes were as follows: exosomes from HEK293 cells were extracted by ultracentrifugation, purified, and co-incubated with miR-144-3p prepared by in vitro transcription. miR-144-3p was loaded into the purified exosomes by ion transfection technology to obtain miR-144-3p-loaded exosomes.

10. A hydrogel loaded with exosomes, characterized in that, It is prepared by the method for preparing a loaded exosome composite hydrogel according to any one of claims 1-9.

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