An injectable hydrogel and its preparation method and application

Abstract

This invention relates to an injectable hydrogel, its preparation method, and its applications. The composite gel comprises biodegradable polyester microspheres containing milk exosomes that provide nutrients. A cross-linked hyaluronic acid gel is prepared by cross-linking hyaluronic acid, DMTMM, and L-lysine ethyl ester dihydrochloride. PLLA microspheres containing milk exosomes are then added and mixed to form the injectable hydrogel. This composite gel possesses ideal mechanical properties, and its degradation rate meets the requirements for injectable fillers. The PLLA microspheres are loaded with active nutrients, providing a slow, long-term release, while the gel provides short-term release and injection performance, achieving a short-term plus long-term repair effect. The bioactive function of milk exosomes, the collagen regeneration function of PLLA microspheres, and the immediate filling function of hyaluronic acid gel are organically integrated into the same system, enabling immediate filling, long-term regeneration, and bioactive regulation.

Description

Technical Field

[0001] This invention belongs to the field of medical aesthetic preparation technology, and in particular relates to an injectable hydrogel, its preparation method and application. Background Technology

[0002] Hyaluronic acid (HA), also known as hyaluronic acid, is a high-molecular-weight linear polysaccharide formed by the repeated alternation of N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA) disaccharide units. Its molecular weight varies greatly, with the molecular formula (C14H20NO11Na)n. The molecular weight of the disaccharide unit is 401.3. It is an acidic mucopolysaccharide that, when combined with water molecules, can form a viscoelastic substance. HA is mainly found in the skin and connective tissue in the human body, serving as an extracellular matrix substance for cell embedding. Besides providing a certain volume of extracellular matrix for cells, it can also affect tissue stability, cohesion, and viscoelasticity. Whether abroad or in major Chinese cities, injectable cosmetic techniques are currently a popular plastic surgery technique. Subcutaneous injection of fillers can quickly eliminate skin wrinkles. Hyaluronic acid is one of the most mainstream facial fillers. Due to its good biocompatibility, biodegradability and immediate effects, it is widely used in wrinkle removal, shaping and skin moisturizing.

[0003] Hyaluronic acid must be cross-linked before it can be used as a filler, and chemical cross-linking is an effective method to enhance the structural stability of hyaluronic acid. Currently, this is mainly achieved by introducing cross-linked structures into hyaluronic acid molecules using bifunctional cross-linking agents. Most bifunctional cross-linking agents are highly reactive chemical reagents. The carboxyl groups of hyaluronic acid are activated under the action of bifunctional cross-linking agents, leading to a cross-linking reaction. Cross-linked hyaluronic acid exhibits higher biostability. However, bifunctional cross-linking agents themselves have a certain degree of toxicity, and the biocompatibility and solubility of hyaluronic acid polymers linked by cross-linking agents are reduced, thus limiting the application of this method. Furthermore, currently available hyaluronic acid gels as mainstream dermal fillers, while possessing good moisturizing and filling effects, typically lack active nutritional repair functions. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides an injectable hydrogel, its preparation method, and its applications.

[0005] The technical solution adopted in this invention is: a method for preparing injectable hydrogel, which involves mixing PLLA microspheres containing milk exosomes with sodium hyaluronate crosslinked gel, and uniformly dispersing the microspheres in the gel to form an injectable hydrogel.

[0006] Preferably, the steps for preparing PLLA microspheres containing milk exosomes are as follows:

[0007] Step 1: Mix milk exosomes with trehalose, freeze-dry to obtain milk exosome freeze-dried powder;

[0008] Step 2: Dissolve poly-L-lactic acid (PLLA) in an organic solvent, add milk exosome lyophilized powder to the poly-L-lactic acid solution, and disperse to obtain an S / O suspension;

[0009] Step 3: Prepare a polyvinyl alcohol aqueous solution. Under stirring, add the S / O suspension to the polyvinyl alcohol aqueous solution and stir until homogeneous to form an S / O / W double emulsion solution.

[0010] Step 4: Leave the S / O / W double emulsion solution open to evaporate the organic solvent, add pure water and stir to obtain microspheres, purify and collect to obtain PLLA microspheres containing milk exosomes.

[0011] Preferably, in step 1, milk exosomes and trehalose are mixed at a mass ratio of 1:1-5; in step 2, the amount of lyophilized exosome powder used is 5-20% of the mass of PLLA; in step 3, the volume ratio of S / O suspension to polyvinyl alcohol aqueous solution is 1:3-1:10.

[0012] Preferably, after pre-freezing at 80°C, the product undergoes a first drying process at -20°C to -10°C, followed by a second drying process at 20-25°C.

[0013] Preferably, the particle size of the milk exosome-loaded PLLA microspheres is 40-75 μm.

[0014] Preferably, sodium hyaluronate crosslinked gel is constructed using lysine ethyl ester as a crosslinking agent and DMTMM as a crosslinking activator.

[0015] Preferably, the specific steps are as follows:

[0016] Step 1: Prepare a 1-5% (w / v) lysine ethyl ester solution using water as the solvent;

[0017] Step 2: Dissolve sodium hyaluronate in lysine ethyl ester solution. The weight-average molecular weight of sodium hyaluronate is 100W-150WDa, and the concentration of sodium hyaluronate is 6.0-11.0% (w / v). Let it stand at 4℃ for 8-24 hours to allow it to swell.

[0018] Step 3: Slowly add DMTMM under stirring conditions. The amount of DMTMM should be 0.3-1.5 times the molar number of sodium carboxyl groups in sodium hyaluronate.

[0019] Step 4: Continue stirring the reaction at room temperature for 10-30 minutes to form a transparent gel; this is lysine ethyl ester crosslinked sodium hyaluronate gel.

[0020] Preferably, the mass ratio of milk exosome-loaded PLLA microspheres to gel is 1:5-100.

[0021] Injectable hydrogels with nutritional repair functions were prepared by an injectable hydrogel preparation method.

[0022] Application of injectable hydrogels with nutritional repair functions in the preparation of injection filler materials.

[0023] The advantages and positive effects of this invention are: it organically integrates the bioactive function of milk exosomes, the collagen regeneration function of PLLA microspheres, and the instant filling function of hyaluronic acid gel into the same system, achieving a triple synergistic effect of "instant filling + long-term regeneration + bioactive regulation".

[0024] The S / O / W method was used to disperse milk exosomes in the oil phase as solid powder, avoiding direct contact between exosomes and water, and eliminating membrane structure damage and leakage of active ingredients caused by the oil-water interface. Trehalose, as a freeze-drying protectant, formed hydrogen bonds with the surface of milk exosome membranes during freeze-drying through a "water substitution" mechanism, maintaining the natural conformation of exosome membrane structure and internal active molecules, and preventing freeze-drying damage. Attached Figure Description

[0025] Figure 1 TEM image of PLLA microspheres containing milk exosomes in Example 1;

[0026] Figure 2 TEM image of the composite gel prepared in Example 1;

[0027] Figure 3 TEM image of PLLA microspheres containing milk exosomes in Comparative Example 1;

[0028] Figure 4 TEM image of the gel prepared in Comparative Example 2. Detailed Implementation

[0029] The embodiments of the present invention will now be described with reference to the accompanying drawings.

[0030] This invention relates to an injectable hydrogel, its preparation method, and its application. The composite gel comprises biodegradable polyester microspheres containing milk exosomes that provide nutrients. The PLLA microspheres containing milk exosomes are cross-linked with hyaluronic acid, DMTMM, and L-lysine ethyl ester dihydrochloride to prepare an L-lysine dihydrochloride cross-linked hyaluronic acid gel. A solution of hyaluronic acid is then added to form the injectable hydrogel. This composite gel possesses ideal mechanical properties, and its degradation rate meets the requirements for injectable fillers. The PLLA microspheres are loaded with active nutrients, providing a slow, long-term release, while the gel offers both short-term release and injection performance, achieving a short-term and long-term repair effect.

[0031] A sodium hyaluronate crosslinked gel was constructed using lysine ethyl ester as a crosslinking agent and DMTMM as a crosslinking activator. The specific preparation method is as follows:

[0032] Step 1: Dissolve lysine ethyl ester dihydrochloride in water to prepare a 1-5% (w / v) lysine ethyl ester solution, and adjust the pH to 6.5-7.4 with 0.1-1.0 M NaOH; the preferred concentration of the lysine ethyl ester solution is 4.63% (w / v).

[0033] Step 2: Dissolve sodium hyaluronate in lysine ethyl ester solution to prepare a sodium hyaluronate solution with a concentration of 6.0-11.0% (w / v). The weight average molecular weight of sodium hyaluronate is 100W-150WDa. Let it stand at 4℃ for 8-24 hours to swell. The preferred concentration of sodium hyaluronate solution is 9.0-10.0% (w / v).

[0034] Step 3: Slowly add DMTMM under stirring conditions. The amount of DMTMM is 0.3-1.5 times the molar number of sodium carboxyl groups in hyaluronic acid; preferably, the amount of DMTMM is 1.2 times the molar number of sodium carboxyl groups in hyaluronic acid. Step 4: Continue stirring at room temperature for 10-30 minutes. The viscosity of the system gradually increases, forming a transparent gel. Place the gel in a dialysis bag and dialyze with PBS buffer for 54-72 hours to remove unreacted DMTMM and byproducts, obtaining lysine ethyl ester crosslinked sodium hyaluronic acid gel.

[0035] A sodium hyaluronate crosslinked gel was constructed using lysine ethyl ester as a crosslinking agent and DMTMM as a crosslinking activator. Lysine ethyl ester is a derivative of lysine, an essential amino acid, and can be metabolized and utilized by the body. The byproduct N-methylmorpholine of DMTMM can be effectively removed by dialysis, without any concerns about residual toxicity. The crosslinking reaction was carried out at room temperature and neutral pH, avoiding damage to the active ingredients caused by harsh conditions such as high temperature, strong acid, and strong alkali. By adjusting the amount of lysine ethyl ester and DMTMM, the degree of crosslinking and gel mechanical properties can be precisely controlled to meet different clinical needs.

[0036] PLLA microspheres loaded with milk exosomes were prepared by a solid / oil / water (S / O / W) emulsion solvent evaporation method, the specific preparation method of which is as follows:

[0037] Step 1: Mix milk exosomes and trehalose at a mass ratio of 1:1-5, preferably 1:2; add PBS buffer to adjust the pH to 6.5-7.4; freeze-dry the mixture, with a pre-freezing temperature below -80℃ for 2-4 hours, a first drying temperature of -20℃ to -10℃ for 24-48 hours, a second drying temperature of 20-25℃ for 12-24 hours; after freeze-drying, obtain a loose milk exosome freeze-dried powder, grind it through a 200-mesh sieve.

[0038] Step 2: Dissolve poly-L-lactic acid (PLLA) in ethyl acetate to prepare a PLLA solution with a concentration of 8-15% (w / v); add milk exosome lyophilized powder to the PLLA solution, with the amount of exosome lyophilized powder being 5-20% of the mass of PLLA; perform ultrasonic dispersion or high-speed shear dispersion under ice bath conditions to uniformly disperse the exosome lyophilized powder in the PLLA solution, obtaining an S / O suspension.

[0039] Step 3: Prepare the external aqueous phase. Prepare a 0.5-2.0% (w / v) polyvinyl alcohol (PVA) aqueous solution, pre-saturated with ethyl acetate. Under mechanical stirring at 200-600 rpm, slowly add the prepared S / O suspension dropwise into the external aqueous phase. The volume ratio of the oil phase to the external aqueous phase is 1:3-1:10. The preferred stirring speed is 250-350 rpm, and the preferred volume ratio of the oil phase to the external aqueous phase is 1:4-1:6. Continue stirring for 5-20 minutes to form an S / O / W double emulsion solution.

[0040] Step 4: Place the S / O / W double emulsion solution in an open container and stir at 100-300 rpm for 2-4 hours at room temperature to allow the ethyl acetate to evaporate. Add an equal volume of pure water and continue stirring at 50-200 rpm for 6-12 hours. Collect the microspheres by centrifugation or filtration, and wash them 1-4 times with pure water to remove residual PVA and unencapsulated substances. Freeze-dry to obtain PLLA microspheres loaded with milk exosomes. In some embodiments of the present invention, the particle size of the PLLA microspheres loaded with milk exosomes is controlled at 40-75 μm.

[0041] A dual protection strategy of "trehalose co-freeze-drying + S / O / W emulsification" fundamentally solves the interfacial denaturation problem that exists in the traditional W / O / W double emulsification method when encapsulating protein or vesicle active ingredients. Trehalose, as a freeze-drying protectant, forms hydrogen bonds with the surface of milk exosome membranes during freeze-drying through a "water substitution" mechanism, maintaining the natural conformation of the exosome membrane structure and internal active molecules, and preventing freeze-drying damage. The S / O / W method disperses milk exosomes in the oil phase as solid powder, avoiding direct contact between exosomes and water, thereby eliminating membrane structure damage and active ingredient leakage caused by the oil-water interface. The prepared milk exosome-loaded PLLA microspheres achieve an exosome activity retention rate of over 85% and an encapsulation rate of over 90%, which is significantly better than the traditional W / O / W method.

[0042] PLLA microspheres loaded with milk exosomes are uniformly dispersed in a lysine ethyl ester-DMTMM crosslinked sodium hyaluronate gel network. Specifically, PLLA microspheres loaded with milk exosomes are added to the lysine ethyl ester-DMTMM crosslinked sodium hyaluronate gel at a mass ratio of 1:5-100, preferably 1:5-10. Under aseptic conditions, a PBS solution of sodium hyaluronate is added, and the microspheres are physically mixed to uniformly disperse them in the gel, resulting in a composite injectable gel. The microspheres can participate in the formation of multiple soft elastic interlocking structures, thereby achieving better dispersibility and encapsulation of the microspheres within the gel. This effectively protects the microspheres during moist heat sterilization, reducing their degradation and prolonging the degradation time after implantation, thus extending the effect of the microspheres in stimulating collagen regeneration. The PLLA microspheres, loaded with active nutrients, release them slowly over a long period, while the sodium hyaluronate gel provides short-term release and injection properties, achieving a short-term effect combined with a long-term repair effect. In addition, adding sodium hyaluronate solution again during the mixing stage can reduce the viscosity of the mixed system and give the product injectability; it can also serve as a dispersion medium to promote the uniform dispersion of PLLA microspheres.

[0043] The particle size of the milk exosome-loaded PLLA microspheres is controlled within the range of 40-75 μm, allowing them to pass smoothly through 27G injection needles without affecting the convenience of clinical operation. The large-diameter microspheres are not easily phagocytosed and cleared by macrophages, allowing them to remain at the injection site for a long time and exert a continuous regenerative stimulation effect. By optimizing the microsphere particle size distribution and the uniform dispersion of the gel matrix, the risk of nodule formation caused by microsphere aggregation is effectively avoided. The microspheres are dispersed in cross-linked hyaluronic acid gel, and the three-dimensional network structure of the gel is used to physically fix the microspheres, preventing sedimentation and aggregation of microspheres during storage and injection.

[0044] The composite injectable gel comprises poly-L-lactic acid (PLLA) microspheres loaded with milk exosomes, hyaluronic acid, lysine ethyl ester dihydrochloride, and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM). Milk extract is rich in whey protein, lactoferrin, growth factors, and amino acids, which nourish the skin, promote collagen regeneration, and brighten the complexion. DMTMM is an aqueous condensing agent commonly used in polysaccharide chemical modification in recent years. Compared with EDC / NHS, it has higher reaction efficiency, easier removal of byproducts, and can directly activate carboxyl groups to form amide bonds under mild conditions, exhibiting extremely low toxicity. Lysine ethyl ester, as a diamino small molecule, can act as a "bridging agent" to react with the carboxyl groups on HA (activated by DMTMM) to form a cross-linked network. It is also a derivative of an essential amino acid, exhibiting good cell compatibility. Poly-L-lactic acid (PLLA) microspheres can achieve long-lasting filling by stimulating collagen regeneration, but lack immediate filling effect when used alone. By organically combining the advantages of these three factors, a composite system with immediate filling, long-lasting regeneration, and bioactivity is constructed. The resulting composite injectable gel has the functions of immediate filling, long-lasting regeneration, and bioactivity regulation, and is highly safe with good stability of active ingredients. It has significant clinical application value and market prospects.

[0045] This system organically integrates the bioactive functions of milk exosomes, the collagen regeneration function of PLLA microspheres, and the immediate filling function of hyaluronic acid gel into a single system, achieving a triple synergistic effect of "immediate filling + long-lasting regeneration + bioactive regulation." Hyaluronic acid gel provides immediate physical filling, meeting immediate shaping needs; PLLA microspheres, after slow degradation, stimulate autologous collagen regeneration, achieving long-lasting effects (6-12 months); milk exosomes continuously release various bioactive molecules (miRNA, proteins, lipids), exerting anti-inflammatory, antioxidant, angiogenesis-promoting, and tissue repair biological functions, fundamentally improving skin condition. The three components complement each other in function and duration: hyaluronic acid gel has the fastest onset of action (immediate), PLLA microspheres have a slower onset but a longer duration (months to years), and milk exosomes are continuously released during microsphere degradation, achieving full-process bioactive regulation.

[0046] The present invention will now be described with reference to the accompanying drawings. Experimental methods not specifically described in terms of operation steps are performed in accordance with the corresponding product manuals. Unless otherwise specified, the instruments, reagents, and consumables used in the embodiments can be purchased from commercial companies.

[0047] Example 1:

[0048] Take 10 mL of milk exosome solution (200 μg / mL), add 40 mg of trehalose (exosome to trehalose mass ratio 1:2), adjust the pH to 7.0, stir to dissolve, and dispense into lyophilization bottles. Lyophilization procedure: pre-freezing temperature -80℃, pre-freezing time 3 hours; primary drying temperature -20℃, primary drying time 48 hours; secondary drying temperature 25℃, secondary drying time 12 hours. A loose, white lyophilized powder is obtained, which is then ground and passed through a 200-mesh sieve for later use.

[0049] 1.2 g of poly-L-lactic acid (PLLA) was dissolved in 10 mL of ethyl acetate. The solution was stirred and heated magnetically at 40 °C to aid dissolution. After cooling to room temperature, a 12% (w / v) PLLA solution was obtained. 120 mg of the above-mentioned lyophilized milk exosome powder (10% of the PLLA mass) was added to the PLLA solution. The mixture was emulsified for 20 min at 2200 rpm under ice bath conditions using an emulsifier to uniformly disperse the lyophilized exosome powder in the PLLA solution, resulting in an S / O suspension.

[0050] Dissolve 1.0 g of polyvinyl alcohol (PVA) in 100 mL of pure water. After heating to dissolve, cool to room temperature and presaturate with ethyl acetate (add 5 mL of ethyl acetate, stir for 2 hours, allow to stand and separate into layers, and collect the aqueous phase). Under mechanical stirring at 300 rpm, slowly add the prepared S / O suspension dropwise to the external aqueous phase, with a volume ratio of oil phase to external aqueous phase of 1:5. Then, emulsify using an emulsifier for 10 min at a speed of 2200 rpm to form a homogeneous S / O / W double emulsion.

[0051] The above S / O / W double emulsion was transferred to an open beaker and stirred at 200 rpm for 3 hours at room temperature. 100 mL of pure water was added, and stirring was continued at 150 rpm for 8 hours to allow complete evaporation of the ethyl acetate. The solidified microsphere suspension was passed through a 200-mesh sieve, and the microspheres on the sieve were collected. The microspheres were washed three times with pure water, then washed once with anhydrous ethanol, and filtered under vacuum. The washed microspheres were freeze-dried (using the same procedure as for milk exosomes) to obtain milk exosome-loaded PLLA microspheres, whose microstructure is shown in the figure. Figure 1 As shown.

[0052] 46 mg of lysine ethyl ester dihydrochloride was dissolved in 1 mL of purified water, and the pH was adjusted to 7.0 with 0.1 M NaOH to obtain a lysine ethyl ester solution. 0.5 g of sodium hyaluronate (molecular weight 120 WDa) was dissolved in 10 mL of lysine ethyl ester solution (pH 7.0), and allowed to swell at 4°C for 12 hours to obtain a 5% (w / v) sodium hyaluronate solution. 150 mg of DMTMM (equivalent to 1.2 times the molar number of carboxyl groups in sodium hyaluronate) was slowly added with stirring, and the reaction was continued at room temperature for 30 min. The viscosity of the system gradually increased, forming a transparent gel. The gel was placed in a dialysis bag (molecular weight cutoff 3500 Da) and dialyzed against PBS buffer for 72 hours to remove unreacted DMTMM and the byproduct N-methylmorpholine, yielding a lysine ethyl ester crosslinked sodium hyaluronate gel. Homogenization was performed to obtain sodium hyaluronate gel particles.

[0053] 0.15 g of the prepared milk-loaded exosome PLLA microspheres were added to 1.0 g of the prepared lysine ethyl-DMTMM crosslinked sodium hyaluronate gel particles. The sodium hyaluronate gel network can physically adsorb the microspheres. Under aseptic conditions, the microspheres were uniformly dispersed by stirring at 210 rpm / min with controlled stirring speed and time. During stirring, sodium hyaluronate (molecular weight 30 WDa) prepared with PBS solution was added, and the sodium hyaluronate solution concentration was 7% (m / v). The mixture was stirred for 3 h. The composite gel was then filled into 1 mL pre-filled syringes and sterilized by moist heat (F0=15) and stored at 4℃.

[0054] The prepared composite gel was examined by transmission electron microscopy, and its microstructure was as follows: Figure 2 As shown.

[0055] Example 2:

[0056] Take 10 mL of milk exosome solution (200 μg / mL), add 40 mg of trehalose (mass ratio of exosomes:trehalose = 1:2), adjust the pH to 7.0, stir to dissolve, and dispense into lyophilization bottles. Lyophilization procedure: pre-freezing temperature -80℃, pre-freezing time 3 hours; primary drying temperature -20℃, primary drying time 48 hours; secondary drying temperature 25℃, secondary drying time 12 hours. A loose, white lyophilized powder is obtained, which is then ground and passed through a 200-mesh sieve for later use.

[0057] 1.2 g of poly-L-lactic acid (PLLA) was dissolved in 10 mL of ethyl acetate. The solution was stirred and heated magnetically at 40 °C to aid dissolution. After cooling to room temperature, a 12% (w / v) PLLA solution was obtained. 120 mg of the above-mentioned lyophilized milk exosome powder (10% of the PLLA mass) was added to the PLLA solution. The mixture was emulsified for 20 min at 2200 rpm / min under ice bath conditions using an emulsifier to uniformly disperse the lyophilized exosome powder in the PLLA solution, resulting in an S / O suspension.

[0058] Dissolve 1.0 g of polyvinyl alcohol (PVA) in 100 mL of pure water. After heating to dissolve, cool to room temperature and presaturate with ethyl acetate (add 5 mL of ethyl acetate, stir for 2 hours, allow to stand and separate into layers, and collect the aqueous phase). Under mechanical stirring at 300 rpm, slowly add the prepared S / O suspension dropwise to the external aqueous phase, with a volume ratio of oil phase to external aqueous phase of 1:5. Then, emulsify using an emulsifier for 10 min at a speed of 2200 rpm to form a homogeneous S / O / W double emulsion.

[0059] The above S / O / W double emulsion was transferred to an open beaker and stirred at 200 rpm for 3 hours at room temperature. 100 mL of pure water was added, and stirring was continued at 150 rpm for 8 hours to allow complete evaporation of the ethyl acetate. The solidified microsphere suspension was passed through a 200-mesh sieve, and the microspheres on the sieve were collected. The microspheres were washed three times with pure water, then washed once with anhydrous ethanol, and filtered under vacuum. The washed microspheres were then freeze-dried (using the same procedure as for milk exosomes) to obtain milk exosome-loaded PLLA microspheres.

[0060] 46 mg of lysine ethyl ester dihydrochloride was dissolved in 1 mL of purified water, and the pH was adjusted to 7.0 with 0.1 M NaOH to obtain a lysine ethyl ester solution. 0.5 g of sodium hyaluronate (molecular weight 120 WDa) was dissolved in 10 mL of lysine ethyl ester solution (pH 7.0), and allowed to swell at 4°C for 12 hours to obtain a 5% (w / v) sodium hyaluronate solution. 125 mg of DMTMM (equivalent to 1.0 times the molar number of carboxyl groups in sodium hyaluronate) was slowly added with stirring, and the reaction was continued at room temperature for 30 min. The viscosity of the system gradually increased, forming a transparent gel. The gel was placed in a dialysis bag (molecular weight cutoff 3500 Da) and dialyzed against PBS buffer for 72 hours to remove unreacted DMTMM and the byproduct N-methylmorpholine, yielding a lysine ethyl ester crosslinked sodium hyaluronate gel. Homogenization yielded sodium hyaluronate gel particles.

[0061] 0.15 g of the prepared milk-loaded exosome PLLA microspheres were added to 1.0 g of the prepared lysine ethyl-DMTMM crosslinked sodium hyaluronate gel particles. Under aseptic conditions, the mixture was stirred at 210 rpm / min. During stirring, sodium hyaluronate (molecular weight 30 WDa) dissolved in PBS solution was added, with a sodium hyaluronate solution concentration of 7% (m / v). The mixture was stirred for 3 h. The composite gel was then filled into 1 mL pre-filled syringes and sterilized by moist heat (F0=15) and stored at 4℃.

[0062] Example 3:

[0063] Take 10 mL of milk exosome solution (200 μg / mL), add 40 mg of trehalose (mass ratio of exosomes to trehalose = 1:2), adjust the pH to 7.0, stir to dissolve, and dispense into lyophilization bottles. Lyophilization procedure: pre-freezing temperature -80℃, pre-freezing time 3 hours; primary drying temperature -20℃, primary drying time 48 hours; secondary drying temperature 25℃, secondary drying time 12 hours. A loose, white lyophilized powder is obtained, which is then ground and passed through a 200-mesh sieve for later use.

[0064] 1.2 g of poly-L-lactic acid (PLLA) was dissolved in 10 mL of ethyl acetate. The solution was stirred and heated magnetically at 40 °C to aid dissolution. After cooling to room temperature, a 12% (w / v) PLLA solution was obtained. 120 mg of the above-mentioned lyophilized milk exosome powder (10% of the PLLA mass) was added to the PLLA solution. The mixture was emulsified for 20 min at 2200 rpm / min under ice bath conditions using an emulsifier to uniformly disperse the lyophilized exosome powder in the PLLA solution, resulting in an S / O suspension.

[0065] Dissolve 1.0 g of polyvinyl alcohol (PVA) in 100 mL of pure water. After heating to dissolve, cool to room temperature and presaturate with ethyl acetate (add 5 mL of ethyl acetate, stir for 2 hours, allow to stand and separate into layers, and collect the aqueous phase). Under mechanical stirring at 300 rpm, slowly add the prepared S / O suspension dropwise to the external aqueous phase, with a volume ratio of oil phase to external aqueous phase of 1:5. Then, emulsify using an emulsifier for 10 min at a speed of 2200 rpm to form a homogeneous S / O / W double emulsion.

[0066] The above S / O / W double emulsion was transferred to an open beaker and stirred at 200 rpm for 3 hours at room temperature. 100 mL of pure water was added, and stirring was continued at 150 rpm for 8 hours to allow complete evaporation of the ethyl acetate. The solidified microsphere suspension was passed through a 200-mesh sieve, and the microspheres on the sieve were collected. The microspheres were washed three times with pure water, then washed once with anhydrous ethanol, and filtered under vacuum. The washed microspheres were then freeze-dried (using the same procedure as for milk exosomes) to obtain milk exosome-loaded PLLA microspheres.

[0067] Dissolve 46 mg of lysine ethyl ester dihydrochloride in 1 mL of purified water, and adjust the pH to 7.0 with 0.1 M NaOH to obtain a lysine ethyl ester solution. Dissolve 0.5 g of sodium hyaluronate (molecular weight 120 WDa) in 10 mL of lysine ethyl ester solution (pH 7.0), and allow to swell at 4°C for 12 hours to obtain a 5% (w / v) sodium hyaluronate solution. Slowly add 100 mg of DMTMM (equivalent to 0.8 times the molar number of carboxyl groups in sodium hyaluronate) with stirring, and continue stirring at room temperature for 30 min. The viscosity of the system gradually increases, forming a transparent gel. Place the gel into a dialysis bag (molecular weight cutoff 3500 Da), and dialyze with PBS buffer for 72 hours to remove unreacted DMTMM and the byproduct N-methylmorpholine, obtaining a lysine ethyl ester crosslinked sodium hyaluronate gel.

[0068] 0.15 g of the prepared milk-loaded exosome PLLA microspheres were added to 1.0 g of the prepared lysine ethyl ester-DMTMM crosslinked sodium hyaluronate gel. Under aseptic conditions, the mixture was stirred at 210 rpm / min. During stirring, sodium hyaluronate (molecular weight 30 WDa) dissolved in PBS solution was added, with a sodium hyaluronate solution concentration of 7% (m / v). The mixture was stirred for 3 h. The composite gel was then filled into 1 mL pre-filled syringes and sterilized by moist heat (F0=15) and stored at 4℃.

[0069] Comparative Example 1:

[0070] The difference between this and the milk exosome PLLA microspheres of Example 1 is that this one was prepared using the traditional W / O / W double emulsion method, with the specific steps as follows:

[0071] Milk exosome solution (protein concentration 200 μg / ml) was mixed with trehalose at a ratio of 1:2. 1.2 g of poly-L-lactic acid (PLLA) was dissolved in 10 mL of ethyl acetate and heated with magnetic stirring at 40°C to aid dissolution. After cooling to room temperature, a 12% (w / v) PLLA solution was obtained. 120 mg of the above milk exosome trehalose solution (10% of the PLLA mass) was added to the PLLA solution. Emulsification was performed for 20 min at 2200 rpm / min under ice bath conditions using an emulsifier to ensure uniform dispersion of the exosomes in the PLLA solution, yielding a w / o colostrum.

[0072] Dissolve 1.0 g of polyvinyl alcohol (PVA) in 100 mL of pure water. After heating to dissolve, cool to room temperature and presaturate with ethyl acetate (add 5 mL of ethyl acetate, stir for 2 hours, allow to stand and separate into layers, and collect the aqueous phase). Under mechanical stirring at 300 rpm, slowly add the prepared W / O primary emulsion dropwise to the external aqueous phase, with a volume ratio of oil phase to external aqueous phase of 1:5. Then, emulsify using an emulsifier for 10 min at a speed of 2200 rpm to form a homogeneous W / O / W double emulsion.

[0073] The above W / O / W double emulsion was transferred to an open beaker and stirred at 200 rpm for 3 hours at room temperature. 100 mL of pure water was added, and stirring was continued at 150 rpm for 8 hours to allow complete evaporation of ethyl acetate. The solidified microsphere suspension was passed through a 200-mesh sieve, and the microspheres on the sieve were collected. The microspheres were washed three times with pure water, then washed once with anhydrous ethanol, and filtered under vacuum. The washed microspheres were freeze-dried (using the same procedure as for milk exosomes) to obtain milk exosome-loaded PLLA microspheres, whose microstructure is shown in the figure. Figure 3 As shown, compared to the smooth-surfaced microspheres prepared in Example 1, these microspheres have depressions on their surface.

[0074] Comparative Example 2:

[0075] The difference from Example 1 is that sodium hyaluronate gel was prepared using the traditional BDDE crosslinking method. The specific steps are as follows:

[0076] 0.5 g of sodium hyaluronate (molecular weight 120 WDa) was dissolved in 10 mL of 1% sodium hydroxide solution to obtain a 5% (w / v) sodium hyaluronate solution. BDDE (1.2 times the molar number of carboxyl groups in sodium hyaluronate) was slowly added with stirring, and the reaction was continued at room temperature for 30 min. The viscosity of the system gradually increased, forming a transparent gel. The gel was placed in a dialysis bag (molecular weight cutoff 3500 Da) and dialyzed with PBS buffer for 72 hours to remove unreacted BDDE crosslinking agent, yielding a crosslinked sodium hyaluronate gel.

[0077] The prepared composite gel was examined by transmission electron microscopy, and its microstructure was as follows: Figure 4 As shown.

[0078] Example 4: Hydrogel performance testing

[0079] The swelling degree of the composite gels prepared in Examples 1-3 was tested respectively, and the extrusion force was tested by a syringe thrust tester (TL15810-D). The results are shown in Table 1. The microsphere gel prepared in Example 1 has better elasticity, exhibits a denser degree of cross-linking, has a more stable gel state, better immediate filling effect, and a longer maintenance time.

[0080] Table 1

[0081]

[0082] The encapsulation efficiency and activity retention rate of the composite gels prepared in Comparative Example 1 and Comparative Example 1 were tested respectively.

[0083] The encapsulation efficiency was determined using the BCA method, and the specific steps are as follows: The gels from Example 1 and Comparative Example 1 were lysed using acetonitrile, an organic reagent, and centrifuged at 10000 g / min for 10 min. The supernatant was collected. The working standard curve solution was prepared according to the BCA protein quantification kit instructions. 20 µL of the working standard curve solution AF was transferred to each well of a 96-well plate, with two replicates for each concentration point. 20 µL of the sample solution was transferred to each well of a 96-well plate, with at least two replicates for each sample. 200 µL of BCA working reagent was added to all the above samples. The plate was sealed with adhesive aluminum foil and linearly mixed at 493 cpm for 30 s in a microplate reader, then incubated at room temperature for 5 min. The absorbance was measured at 480 nm using the microplate reader platform. The protein concentration in the sample was calculated using the standard curve method.

[0084] Calculation formula: Encapsulation efficiency % = (Actual amount of exosome protein measured in the sample / Total amount of exosome protein added during preparation) * 100% The results are shown in Table 2. The encapsulation efficiency of the preparation method in Example 1 reached 90.5%, which was significantly higher than that of the comparative method.

[0085] The active ingredient in the composite gel is EGF protein, and the retention rate of the active ingredient EGF protein was detected by ELISA.

[0086] Reagent preparation: Washing buffer preparation: Dilute the concentrated washing buffer with deionized water to 1× working solution; Standard preparation: Take the lyophilized standard, add the specified volume of standard diluent according to the instructions, let stand at room temperature for 15 minutes to fully dissolve, and then perform serial dilution; Antibody working solution: Dilute with antibody diluent at the specified ratio (1:1000); HRP working solution: Dilute with HRP diluent at the specified ratio (1:500).

[0087] Detection Procedure: Completely decompose the gels from Example 1 (S / O / W method) and Comparative Example 1 (W / O / W method) using acetonitrile, an organic reagent. Centrifuge at 10000 g / min for 10 min and collect the supernatant. Add sample / standard: Add the standard, blank control, and test sample to the corresponding wells, creating two sub-wells, 100 μL / well; incubate at 37°C for 90 min. Add detection antibody: Discard the liquid in the wells (no washing required), immediately add biotinylated detection antibody working solution, 100 μL / well; incubate at 37°C for 1 hour. Wash (3 times): Discard the liquid, add 350 μL of washing buffer to each well, soak for 1 minute, discard, pat dry on absorbent paper, repeat washing 3 times with 350 μL / well. Add HRP conjugate: Add HRP working solution, 100 μL / well; incubate at 37°C for 30 min. Wash (5 times): Same washing steps as above, 350 μL / well, repeat 5 times. Color development: Add 90 μL of TMB substrate to each well and incubate at 37°C in the dark for 15 minutes. Termination: Add 50 μL of stop solution to each well (in the same order as substrate addition) and read the reading immediately. Reading: Measure the 450 nm OD value using a microplate reader and calculate the EGF content.

[0088] The results are shown in Table 2. The exosome-PLLA microsphere composite system prepared in Example 1 not only has a high encapsulation rate, but also a good activity retention rate and can be stably preserved.

[0089] Furthermore, by observing the morphology of the microspheres in the composite gel using transmission electron microscopy, it was found that the microspheres in the composite gel of Example 1 had regular shapes and smooth surfaces; while some of the microspheres in the composite gel prepared in Comparative Example 1 had concave surfaces.

[0090] Table 2

[0091]

[0092] The residual crosslinking agent in Example 1 and Comparative Example 2 were tested respectively.

[0093] DMTMM residues in the composite gel of Example 1 were detected by HPLC. A Waterssymmetry C18 column (4.6 x 250 mm, 5 μm) was used. Mobile phase A was 0.1% trifluoroacetic acid aqueous solution, and mobile phase B was acetonitrile. The flow rate was 1 mL / min, the column temperature was 30 °C, the injection volume was 80 μL, and the detection wavelength was 230 nm. The elution program is shown in Table 3.

[0094] Table 3

[0095]

[0096] DMTMM standard solution (2 μg / ml): Accurately weigh an appropriate amount of DMTMM reference standard and place it in a suitable volumetric flask. Dissolve and dilute with water, mix well, and prepare a solution with a final concentration of 2 μg / ml. 1500 U / ml hyaluronidase solution (HAse): Weigh an appropriate amount of hyaluronidase and dissolve it in purified water to prepare a 1500 U / ml hyaluronidase solution. Prepare fresh before use. Sample solution: Accurately weigh approximately 1 g of the sample from Example 1 and place it in a suitable centrifuge tube. Add 0.8 ml of HAse solution, then add 0.2 ml of water. Incubate at 37°C for 48 hours until the sample is completely dissolved. Centrifuge at 10000 g / min for 5 min and collect the supernatant to obtain the sample solution.

[0097] Inject the sample for analysis and record the chromatogram. Calculate the residual DMTMM content in the sample using the external standard method.

[0098] GC method was used to detect BDDE residues in the composite gel of Comparative Example 2.

[0099] The chromatographic column used was HP-5, 30 m x 0.32 mm x 0.25 μm; the initial column temperature was 200℃, held for 5 min, then increased to 280℃ at 20℃ / min, held for 5 min; the detector was FID, the carrier gas was nitrogen, the injection port temperature was 240℃, the detector temperature was 280℃, the carrier gas flow rate was 1.5 mL / min, the injection volume was 2 μL, and the split ratio was 1:1. BDDE standard solution (2 μg / ml): Accurately prepare a BDDE standard solution with a final concentration of 2 μg / ml. Sample solution: Accurately weigh approximately 2 g of the comparative example 2 sample, place it in a 10 ml test tube, add 0.8 ml of HAse solution, then add 0.2 ml of water, and place in a 37℃ oven for enzymatic hydrolysis for 48 hours until the sample is completely dissolved. Then add 2 ml of ethyl acetate, mix well, centrifuge at 10000 g / min for 5 min, and collect the supernatant to obtain the sample solution.

[0100] Inject the sample for analysis and record the chromatogram. Calculate the residual BDDE content in the sample using the external standard method.

[0101] The results showed that no cross-linking agent residue was detected in the composite gel of Example 1, while 1.7 ppm of cross-linking agent BDDE was detected in Comparative Example 2.

[0102] Further comparison was made of the anti-inflammatory capabilities of the composite gels from Example 1 and Comparative Example 2.

[0103] L929 (mouse fibroblast) cells were seeded into 96-well plates (10,000 cells / well); after adhesion, gels from Example 1 and Comparative Example 2 were added, and the cells were cultured for 48 hours; excess gel residue was washed away with buffer, and MTT solution (final concentration 0.5 mg / mL) was added to each well, and the cells were cultured for 4 hours. The supernatant was carefully aspirated, and DMSO solution was added; the absorbance (OD value) was measured at 570 nm using a microplate reader.

[0104] The results are shown in Table 4. In the experimental wells containing the composite gel of Example 1, the cells showed mild inflammation and higher cell survival rate.

[0105] Table 4

[0106] Example 1: The composite gel formulation exhibits excellent biocompatibility. There is no risk of toxic cross-linking agent residues. It possesses suitable mechanical properties, making it suitable for dermal filler applications.

[0107] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. A method for preparing an injectable hydrogel, characterized in that: PLLA microspheres containing milk exosomes were mixed with sodium hyaluronate cross-linked gel, and the microspheres were uniformly dispersed in the gel to form an injectable hydrogel.

2. The method for preparing injectable hydrogel according to claim 1, characterized in that: The steps for preparing PLLA microspheres containing milk exosomes are as follows: Step 1: Mix milk exosomes with trehalose, freeze-dry to obtain milk exosome freeze-dried powder; Step 2: Dissolve poly-L-lactic acid (PLLA) in an organic solvent, add milk exosome lyophilized powder to the poly-L-lactic acid solution, and disperse to obtain an S / O suspension; Step 3: Prepare a polyvinyl alcohol aqueous solution. Under stirring, add the S / O suspension to the polyvinyl alcohol aqueous solution and stir until homogeneous to form an S / O / W double emulsion solution. Step 4: Leave the S / O / W double emulsion solution open to evaporate the organic solvent, add pure water and stir to obtain microspheres, purify and collect to obtain PLLA microspheres containing milk exosomes.

3. The method for preparing injectable hydrogel according to claim 2, characterized in that: In step 1, milk exosomes and trehalose are mixed at a mass ratio of 1:1-5; in step 2, the amount of lyophilized exosome powder used is 5-20% of the mass of PLLA; in step 3, the volume ratio of S / O suspension to polyvinyl alcohol aqueous solution is 1:3-1:

10.

4. The method for preparing injectable hydrogel according to claim 2, characterized in that: After pre-freezing at 80℃, it undergoes a first drying process at -20℃ to -10℃, followed by a second drying process at 20-25℃.

5. The method for preparing injectable hydrogel according to claim 2, characterized in that: The particle size of the milk exosome-loaded PLLA microspheres is 40-75 μm.

6. The method for preparing injectable hydrogels according to any one of claims 1-5, characterized in that: Sodium hyaluronate crosslinked gel was constructed using lysine ethyl ester as a crosslinking agent and DMTMM as a crosslinking activator.

7. The method for preparing injectable hydrogel according to claim 6, characterized in that: The specific steps are as follows: Step 1: Prepare a 1-5% (w / v) lysine ethyl ester solution using water as the solvent; Step 2: Dissolve sodium hyaluronate in lysine ethyl ester solution. The weight-average molecular weight of sodium hyaluronate is 100W-150WDa, and the concentration of sodium hyaluronate is 6.0-11.0% (w / v). Let it stand at 4℃ for 8-24 hours to allow it to swell. Step 3: Slowly add DMTMM under stirring conditions. The amount of DMTMM should be 0.3-1.5 times the molar number of sodium carboxyl groups in sodium hyaluronate. Step 4: Continue stirring the reaction at room temperature for 10-30 minutes to form a transparent gel; this is lysine ethyl ester crosslinked sodium hyaluronate gel.

8. The method for preparing injectable hydrogel according to claim 6, characterized in that: The mass ratio of milk exosome-loaded PLLA microspheres to gel was 1:5-100.

9. An injectable hydrogel with nutritional repair function prepared by any of the injectable hydrogel preparation methods according to claims 1-8.

10. The application of the injectable hydrogel with nutritional repair function as described in claim 9 in the preparation of injectable filler materials.

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