A crosslinked gel material containing polymer microspheres, a preparation method thereof, and an injectable filler

Abstract

The present invention provides a crosslinked gel material containing polymer microspheres, a preparation method thereof and an injection filler. The method comprises: 1) dissolving an aliphatic polyester substance in an organic solvent as an oil phase, wherein the concentration of the aliphatic polyester substance is 5-40 wt%; dissolving a surfactant and a biocompatible polymer in water as a water phase; the molecular weight of the biocompatible polymer is 2 million - 40 million; 2) continuously emulsifying the oil phase and the water phase after mixing to obtain an oil-in-water emulsion; 3) simultaneously curing and crosslinking the emulsion and a crosslinking agent under alkaline conditions to obtain a crosslinked gel block containing polymer microspheres; 4) successively subjecting the crosslinked gel block containing polymer microspheres to dialysis, solvent removal, sterilization, etc. to obtain a crosslinked gel material containing polymer microspheres. The present invention simplifies the production process, can improve the production efficiency and save the input cost, and can obtain a filler product with less crosslinking agent residue.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical aesthetics, and specifically relates to a cross-linked gel material containing polymer microspheres, a preparation method thereof, and an injectable filler. Background Art

[0002] It is reported that among the total number of medical aesthetic treatment courses in China in 2020, skin injection type has the largest usage, accounting for more than 60%. The main skin injection filler products include hyaluronic acid, collagen, and polyesters. A skin filler is a type III medical device for injection, and its principle of action is to inject a biocompatible material into the skin tissue to fill the skin tissue or stimulate the tissue to synthesize collagen, aiming to smooth skin wrinkles or correct contour defects.

[0003] The development process of skin fillers includes: Since 1981, the first-generation skin fillers have been animal-derived collagen fillers (such as Zyderm, Cosmoplast, and Sunmex, etc.), with a relatively short postoperative effect duration (2 - 4 months), there are animal source risks, and a skin allergy test needs to be performed one month before the operation, which is inconvenient to use. Since 2003, the second-generation skin fillers have been cross-linked hyaluronic acid fillers (such as Restylane TM , and etc.), which degrade into free hyaluronic acid in the human body. And hyaluronic acid already exists in the skin, so side effects such as skin allergic reactions are significantly reduced, and it is currently the most widely used clinically. However, the retention time of cross-linked hyaluronic acid fillers in the body is still relatively short (6 - 12 months), and repeated injections must be performed every 6 to 12 months, with frequent operations, bringing a relatively high risk of injection infection. Since 2009, the third-generation fillers are synthetic biodegradable polymer fillers (such as ), containing polylactic acid (PLA) or polycaprolactone (PCL) microparticles, which decompose relatively slowly in the human body (1 - 4 years). Compared with the first two generations of fillers, polyester fillers are semi-permanent fillers, but their biocompatibility is not as good as that of cross-linked hyaluronic acid fillers, and there are more side effects.

[0004] To make up for the defects of the third-generation products, multiple technical solutions have been publicly available. They mainly combine polyester microspheres with cross-linked hyaluronic acid. The polyester microspheres are mixed into or encapsulated in the cross-linked hyaluronic acid gel, which not only endows the product with semi-permanent characteristics but also enables it to have good biocompatibility and reduces the side effects caused by subcutaneous injection. For example, in the patent documents such as KR1020160052318, WO2017196051A1, CN110559489A, and CN111184909A, the main technical idea is to mechanically stir the prefabricated PCL microspheres with cross-linked hyaluronic acid so that the PCL microspheres are mixed between the cross-linked hyaluronic acid gels.

[0005] Specifically, there are two key steps in the production process of the above-mentioned publicly available technical solutions. The first step is to prepare polyester microspheres by the emulsification method (see the process flow chart in Figure 1 ). Polycaprolactone is dissolved in dichloromethane as the oil phase (denoted as dissolution B), and polyvinyl alcohol surfactant is dissolved in water as the water phase (denoted as dissolution A). During the process of injecting the oil phase into the water phase, a water-in-oil emulsion is formed by strong stirring or homogenization emulsification. After procedures such as heat preservation and curing of the microspheres (removing the organic solvent), washing (eluting the surfactant), sieving, and irradiation sterilization, polycaprolactone microspheres are obtained.

[0006] The second step is to prepare the cross-linked hyaluronic acid gel (see the process flow chart in Figure 2 ). Dissolve hyaluronic acid powder and NaOH solution in water, add the NaOH solution containing BDDE, and after cross-linking (heat preservation), PBS dialysis (eluting the residual cross-linking agent), sieving (granulation), and moist heat sterilization and other processes, cross-linked hyaluronic acid gel particles are obtained. Hyaluronic acid (HA), also known as hyaluronic acid sodium, is a glycosaminoglycan composed of disaccharide units of D-glucuronic acid and N-acetylglucosamine; HA is widely distributed in various parts of the human body and has good hydrophilicity, viscoelasticity, lubricity, and biocompatibility; usually, hyaluronic acid exists in the form of a salt, so hyaluronic acid can refer to sodium hyaluronate. Cross-linked hyaluronic acid gel: Cross-linked hyaluronic acid is cross-linked by introducing a cross-linking agent on the basis of the hyaluronic acid long-chain molecule to form a network structure and finally exists in the form of a hydrophilic elastomer. BDDE: 1,4-Butanediol diglycidyl ether (abbreviated as BDDE), is a commonly used hyaluronic acid cross-linking agent.

[0007] The above disclosed technical solution requires at least two steps, namely, investing in two production lines for polyester microspheres and cross-linked hyaluronic acid, to obtain a filler product containing a combination of polyester microspheres and cross-linked hyaluronic acid gel. Among them, the polyester microspheres must be processed through a drying process, and there are repetitive operations in multiple processes, such as heat preservation (curing / cross-linking), washing (dialysis), sieving, and sterilization processes. It can be seen that the existing technical solution has a cumbersome process, low production efficiency, and high input costs. In addition, in the dialysis process of cross-linked hyaluronic acid, the number of dialysis times or dialysis time is limited, which is not conducive to further removing the residue of the cross-linking agent. Summary of the Invention

[0008] In view of this, the present invention provides a cross-linked gel material containing polymer microspheres, a preparation method thereof, and an injectable filler, aiming to simplify the production process, improve production efficiency, and save input costs, while obtaining a filler product with less cross-linking agent residue.

[0009] The present invention provides a preparation method of a cross-linked gel material containing polymer microspheres, comprising the following steps:

[0010] 1) Dissolving an aliphatic polyester substance in an organic solvent as the oil phase, and the concentration of the aliphatic polyester substance is 5-40 wt%; dissolving a surfactant and a biocompatible polymer in water as the water phase; the molecular weight of the biocompatible polymer is 200,000-4,000,000;

[0011] 2) Continuously emulsifying the oil phase and the water phase after mixing to obtain an oil-in-water emulsion;

[0012] 3) Simultaneously curing and cross-linking the emulsion and a cross-linking agent under alkaline conditions to obtain a cross-linked gel block containing polymer microspheres;

[0013] 4) Sequentially subjecting the cross-linked gel block containing polymer microspheres to dialysis, solvent removal, sieving, moist heat sterilization or radiation sterilization to obtain a cross-linked gel material containing polymer microspheres.

[0014] The preparation method provided by the present invention has a simple process, low cost, and high efficiency, and can obtain a filler product with less cross-linking agent residue.

[0015] See Figure 3 , Figure 3 which is a schematic process flow diagram for preparing a cross-linked hyaluronic acid gel containing polyester microspheres in some embodiments of the present invention.

[0016] In the embodiments of the present invention, the water phase is prepared through the dissolution A process, and the oil phase is prepared through the dissolution B process. Among them, an appropriate amount of aliphatic polyester and / or its modified product can be weighed and added to an organic solvent, preferably dissolved by magnetic stirring at room temperature and 200±50 rpm to obtain an oily solution with a concentration of 5-40 wt%, which is the oil phase.

[0017] In addition, an appropriate amount of surfactant is weighed and added to water under stirring at a speed of 300 ± 100 rpm, the temperature is raised to 95 ± 5 °C, and it is dissolved for 2 ± 0.5 h, and then the temperature is lowered to 18 - 28 °C for standby; preferably, an appropriate amount of high-molecular-weight hyaluronic acid powder with a molecular weight of 2 million to 4 million is weighed and added in batches under stirring. After dissolving for 30 - 60 min, a solution is formed as the aqueous phase.

[0018] In the embodiments of the present invention, the aliphatic polyester material is a material for forming polyester microspheres, and is selected from at least one of polylactide, polyglycolide, polycaprolactone, poly(trimethylene carbonate), poly(p-dioxanone), or a copolymer obtained by copolymerizing monomers of two or more of the foregoing polymers, or a polymer modified therefrom; specifically, Mw = 0.5 - 300,000. Among them, the polylactide, also known as polylactic acid, includes poly-L-lactide and poly-D,L-lactide.

[0019] Polycaprolactone (PCL) is a safe and non-toxic degradable material. It can be degraded in the body and the end products are degraded into CO2 and H2O; PCL has good biocompatibility and a long degradation period, and is an ideal filling material for medical aesthetic plastic surgery. Poly(trimethylene carbonate) (PTMC) is in a rubber state at body temperature and has a certain elasticity. It is an amorphous polymer without a fixed melting point; it is widely used in degradable ligation devices, drug controlled release materials, in vivo implant materials, in vivo support materials, etc.

[0020] The modified polymer includes, but is not limited to, polyhydroxyalkanoates, and the polyhydroxyalkanoates are one or more of polyhydroxybutyrate, hydroxybutyrate valerate copolymer, hydroxybutyrate hexanoate copolymer, poly(3-hydroxybutyrate / 4-hydroxybutyrate) copolymer.

[0021] In the embodiments of the present invention, the organic solvent is selected from one or more of substituted or unsubstituted hydrocarbons, ketones, tetrahydrofuran (THF), ethyl acetate, and ethyl lactate. The hydrocarbons include aliphatic hydrocarbons and aromatic hydrocarbons. The substituted hydrocarbons are preferably halogen-substituted compounds; the organic solvent is preferably one or more mixtures of dichloromethane, chloroform, toluene, acetone, N-methylpyrrolidone (NMP), methyl ethyl ketone, and ethyl acetate, and a preferred choice is dichloromethane. Moreover, in the prepared oil phase, the concentration of the aliphatic polyester material is 5 - 40 wt%, preferably 10 - 30 wt%.

[0022] In the embodiments of the present invention, a surfactant and a biocompatible polymer are dissolved in water to form an aqueous phase, generally using water for injection. In this technical solution, the proportion of the aqueous solution is relatively large. The surfactant has a certain hydrophilicity, which is beneficial to dissolution and subsequent emulsification processes. Preferably, it is polyvinyl alcohol and / or Tween, and polyvinyl alcohol is preferably used (specifically, Mw = 25,000 - 150,000). As a preference, the concentration of polyvinyl alcohol in the aqueous phase is 0.1% - 2.0%, and a preferred concentration is 0.5 wt%.

[0023] In the embodiments of the present invention, the biocompatible polymer is one or more of hyaluronic acid, collagen, chitosan, carboxymethyl cellulose derivatives, polyamino acids, dextran, and starch, and hyaluronic acid is preferably used. The molecular weight Mw of the biocompatible polymer is preferably 400,000 - 3,000,000; for example, the molecular weight of the above-mentioned large-molecule hyaluronic acid is 2,000,000 to 4,000,000, preferably 2,600,000 - 3,000,000, and hyaluronic acid with a molecular weight of 200,000 - 400,000 and other polymers can also be used. Optionally, a pore-forming agent is added to the polymer raw material in the embodiments of the present invention to obtain porous microspheres; the pore-forming agents involved are inorganic salts such as calcium chloride, and the dosage can be about 1%.

[0024] After the preparation of the aqueous phase and the oil phase is completed, in the preferred embodiments of the present invention, an emulsification process is carried out: heating to 20 - 40 °C, and under the condition that the stirring or homogenization speed is 600 - 2500 pm, the oil phase is injected into the aqueous phase at a rate of 30 - 60 ml / min by an injection pump. After the injection is completed, emulsification is continued for 15 - 30 min to form an oil-in-water (O / W) emulsion. Among them, in the embodiments of the present invention, heating reduces the viscosity of the solution and improves the emulsification effect; and pumping at a certain speed ensures the stable delivery of the water-oil ratio.

[0025] In the embodiments of the present invention, the emulsification method of stirring or homogenization can also be other methods, such as ultrasonic treatment. The rotation speed of the stirring can be 600 - 2500 rpm, and preferably 1200 - 1600 rpm.

[0026] In the preferred embodiments of the present invention, the reaction kettle is set at 35 - 90 °C and the rotation speed is 200 - 400 rpm. Under stirring, an appropriate amount of sodium hydroxide is added in batches to adjust the emulsified solution to be alkaline. The cross-linking agent is added slowly in batches, and at the same time, a protective atmosphere is opened to purge the liquid surface, and stirring is continued at a rotation speed of 200 ± 50 rpm to obtain a viscoelastic block gel containing polyester microspheres.

[0027] The present invention preferably controls the temperature range for heat preservation at 35 - 90 °C, more preferably 35 - 55 °C, and preferably 45 °C. The emulsion and the crosslinking agent are cured / crosslinked under alkaline conditions. Within a certain temperature range, the higher the temperature, the faster the crosslinking reaction rate, the higher the crosslinking degree, and the greater the viscoelasticity of the product. Among them, the added crosslinking agent can be selected from binary epoxy types, mainly butanediol diglycidyl ether (BDDE), and its specific dosage is 10% - 15% of the mass of hyaluronic acid; other crosslinking agents can also be used, such as crosslinking agents containing aldehyde, sulfone, and imine groups (if the crosslinking agent is changed, its dosage, heat preservation time, and pH of the reaction system need to be slightly adjusted). The reasons for using a higher concentration of crosslinking agent in the examples of the present invention include: there is more aqueous phase in the reaction system, resulting in a low concentration of HA and a low solution viscosity, which is beneficial for high-speed emulsification and dispersion to obtain PCL microspheres with a smaller particle size; on the other hand, it will reduce the number of effective crosslinks. Therefore, the concentration of the crosslinking agent (increase the usage amount) is increased to finally obtain a gel with an appropriate crosslinking degree.

[0028] The present invention preferably uses a protective atmosphere to purge the liquid surface, which can accelerate the volatilization of organic solvents; in this process, the microspheres are cured, the organic solvents are volatilized, and the crosslinking reaction proceed simultaneously to obtain a crosslinked gel block containing polymer microspheres. In the examples of the present invention, sodium hydroxide can be added to achieve an alkaline system (such as a pH value of 9 - 14), which can promote the reaction between hyaluronic acid and BDDE. The reaction time for heat preservation can be 6 - 12 h, preferably 6 - 8 h; the reaction time is relatively long, the crosslinking degree is high, and a crosslinked hyaluronic acid gel with higher viscoelasticity is obtained. Of course, other alkaline reagents can be used to adjust its pH, including but not limited to potassium hydroxide (KOH), sodium carbonate (Na2CO3), ammonia-ammonium chloride (NH3·H2O-NH4Cl) alkaline buffer solution, etc.

[0029] In the examples of the present invention, the obtained block-shaped crosslinked gel is cut into pieces to facilitate dialysis to remove excessive surfactants, crosslinking agents, and organic solvents. Specifically, the crosslinked gel block containing polymer microspheres is cut into irregular block-shaped objects with a size of about 0.5 - 2 cm 3 , transferred to a dialysis device filled with injection water, and dialyzed 7 - 11 times, with each dialysis time of 60 - 120 min, to obtain a crosslinked hyaluronic acid gel containing polyester microspheres. In the examples of the present invention, the crosslinked gel block containing polymer microspheres is washed and dialyzed with injection water, and the dosage of injection water is 20 - 30 times the weight of the gel; using injection water instead of phosphoric acid solution as the dialysis solution is to avoid excessive phosphate content and osmotic pressure exceeding the standard in the intermediate product freeze-dried powder.

[0030] In the embodiments of the present invention, it is preferably to perform dialysis 7 to 11 times, and the dialysis time for each time is 60 to 120 minutes, which can ensure that the residual cross-linking agent is less than 0.05 ppm (the industry standard is ≤ 2 ppm), and further reduce the harm caused by the introduction of the cross-linking agent (such as periodic redness, swelling, itching, etc. at the injection site).

[0031] For the purpose of further removing residual dichloromethane organic solvents, etc., in some embodiments of the present invention, the cross-linked hyaluronic acid gel mass containing polyester microspheres obtained by dialysis is transferred to a tray for drying, preferably placed in a freeze dryer for freeze drying to obtain a freeze-dried mass. Exemplary freeze-drying conditions include: pre-freezing at -40 ± 5 °C for 2 - 3 h, sublimation drying at -5 - 30 °C for 8 - 12 h, and analytical drying at 40 ± 2 °C for 6 - 8 h. The embodiments of the present invention preferably use freeze drying, which helps the freeze-dried gel block maintain a loose structure and is convenient for crushing; other drying methods can also be used, such as air drying. In some other embodiments, the solvent is removed by concentration. Both the drying process and the concentration process can remove organic solvents. In addition, the concentration operation can also adjust the water content in the gel and takes less time (the total concentration time is 2 - 4 h).

[0032] The intermediate product of the prior art process route is generally a gel. The gel contains a large amount of water, requires a storage container of a large size and is inconvenient for storage. Cross-linked hyaluronic acid and polyester microspheres are unstable in water and have a certain degree of hydrolysis. The prepared intermediate product gel must enter the next production process as soon as possible, which is not conducive to the regulation of the production plan.

[0033] In the embodiments of the present invention, a production line is used to simultaneously form polyester microspheres and cross-linked hyaluronic acid gel, saving the input costs of equipment and clean areas; and the prepared intermediate product - the composition of polyester microspheres and cross-linked hyaluronic acid is a freeze-dried powder, which is easy to store and convenient for the arrangement of the production plan.

[0034] Moreover, the freeze drying, etc. as described above ensure that the number of dialysis times is not limited by the content of cross-linked hyaluronic acid in the final product; this is because the more the number of dialysis times for obtaining the gel block, the greater the water absorption of cross-linked hyaluronic acid, and its content gradually decreases, unable to reach the expected marked content of the final product (such as 15 mg / ml); while the product after drying such as freeze drying is a powder, and a quantitative amount of phosphate buffer solution, physiological saline, etc. is added before filling for swelling, and an accurate marked amount of cross-linked hyaluronic acid can be obtained. The dialysis process described above in the present invention can be unlimited in terms of the number of times and time, and thus a filler product with less residual cross-linking agent can be obtained, greatly reducing the side effects caused by the cross-linking agent.

[0035] After removing the solvent, the present invention can directly perform sieving, or perform sieving after pulverization. In a preferred embodiment of the present invention, the freeze-dried block is put into a pulverizer for pulverization, and the powder sieved by a multi-stage sieve (80 mesh / 150 mesh / 300 mesh) is collected, and then a biocompatible small molecule powder such as small molecule hyaluronic acid is mixed in. Among them, adding small molecule hyaluronic acid (uncrosslinked) can increase the fluidity of the end product and effectively reduce the pushing force during clinical use.

[0036] In an embodiment of the present invention, the powder sample obtained by mixing biocompatible small molecules after sieving is sub-packed in a sterilized bag for irradiation sterilization. Among them, for example, low molecular weight hyaluronic acid is used, which acts as a lubricant to reduce the resistance during injection. This part of the low molecular weight hyaluronic acid does not undergo a crosslinking reaction; the molecular weight range of the hyaluronic acid used at this time can be 400,000 - 800,000.

[0037] In some embodiments of the present invention, a moist heat sterilization tank can be used for sterilization, such as a sterilization temperature of 121 °C for 15 min to obtain a sterile intermediate product; this sterilization method is more suitable for biocompatible high molecular raw materials with relatively low molecular weights, such as hyaluronic acid raw materials with a molecular weight of 200,000 - 2,000,000. If a moist heat sterilization method is adopted, swelling can be carried out before the sterilization process. The present invention preferably uses irradiation sterilization (including electron beam sterilization or γ-ray sterilization), which is beneficial to ensuring the product structure, etc. The irradiation dose of the irradiation sterilization is preferably 15 - 25 kGy to obtain a sterile intermediate product (such as crosslinked hyaluronic acid powder containing polyester microspheres), and store it at room temperature for standby.

[0038] After irradiation sterilization, some of the crosslinked HA molecular chains are broken, and the in vivo anti-degradation ability decreases. In an embodiment of the present invention, an appropriate starting molecular weight of HA is selected, and its in vivo degradation period is about 2 months after irradiation; the molecular weight of the polyester microspheres also decreases due to irradiation, reaching the expected molecular weight range; this process simultaneously completes the sterilization of the HA crosslinked gel and the polyester microspheres.

[0039] In the existing publicly disclosed technical solutions, the degradation period of the components of the crosslinked hyaluronic acid gel is too long (more than 3 months), which is not conducive to the polyester microspheres stimulating the regeneration of collagen in the skin tissue in the short term (2 months); while the mechanism of action of the better composition is that in the early stage (2 months), wrinkles are smoothed through the physical filling effect of the gel components, and then the polymer microparticles or microspheres continuously stimulate the skin tissue, causing fibroblast proliferation and promoting collagen secretion, thereby playing a role in correcting skin wrinkles.

[0040] In a preferred embodiment of the present invention, the irradiation sterilization method is adopted in the process, which not only ensures the sterility level of the product, but also causes partial degradation of the crosslinked hyaluronic acid through irradiation, achieving the expected purpose of its in vivo degradation period of about 2 months.

[0041] To obtain a pre-filled gel injectable, the sterile intermediate product needs to be swollen first. The buffer solutions involved include phosphate buffer solution, physiological saline, etc. The specific operation examples are as follows: The sterile intermediate product can be added to the phosphate buffer solution in batches, swollen for 30 - 60 min, preferably stirred at a rotation speed of 200 ± 50 rpm for 10 - 20 min, and mixed evenly to obtain a cross-linked gel solution containing polymer microspheres. It can also be swollen with physiological saline (0.9% NaCl), which has little effect on the swelling time and is within 30 - 60 min.

[0042] The present invention provides a cross-linked gel material containing polymer microspheres obtained by the preparation method as described above. Preferably, the residual cross-linking agent is less than 0.05 ppm, which is far lower than the industry standard. The products of this technology application include cross-linked sodium hyaluronate gel containing polycaprolactone microspheres; the application fields involve facial plastic surgery, vocal cord treatment, urinary incontinence, tumor embolization, bone defect repair, etc.

[0043] In the examples of the present invention, under a hundred-level purification environment, the swollen cross-linked hyaluronic acid gel containing polyester microspheres is filled into a sterile pre-filled syringe by a filling machine to obtain a filler of cross-linked hyaluronic acid gel containing polyester microspheres. Specifically, the mass ratio of polyester microspheres is 2% - 50%, preferably 20% - 30%. The filler products are generally 1 ml / branch, 2 ml / branch, etc.; the proportion of polyester microspheres is based on the content of a single branch of the filler (including cross-linked hyaluronic acid, PBS buffer solution, PCL microspheres). The present invention provides an injection filler filled with the cross-linked gel material containing polymer microspheres as described above.

[0044] In summary, the technical solution of the present invention adopts a one-step method, which can prepare materials such as cross-linked hyaluronic acid gel containing polyester microspheres, omits the drying process in the process of preparing polyester microspheres by the emulsification method, and combines multiple processes in the prior art (curing, washing, sieving, sterilization, etc.); generally, this technical solution can shorten the production cycle (2 - 4 d), significantly improve production efficiency, and greatly reduce cost input. Moreover, the cross-linked hyaluronic acid gel containing polyester microspheres and other materials prepared by the present invention have less residual cross-linking agent. When used as an injection filler, they have effects such as a better degradation period. Description of the Drawings

[0045] Figure 1 It is a process flow chart for the preparation of conventional polymer microspheres in the prior art;

[0046] Figure 2 It is a process flow chart for the preparation of cross-linked sodium hyaluronate gel in the prior art;

[0047] Figure 3 It is a schematic diagram of the process flow for preparing cross-linked hyaluronic acid gel containing polyester microspheres in some embodiments of the present invention. Detailed Description of the Invention

[0048] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present application without creative efforts shall fall within the protection scope of the present application.

[0049] To better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0050] Example 1

[0051] S1.1 Weigh 8 g of polyvinyl alcohol with Mw = 30,000. Add 1000 ml of injection water under stirring at a speed of 300 rpm, heat up to 95 °C, stir and dissolve for 2 h, and cool down to 20 °C for standby; weigh 3 g of hyaluronic acid powder with a weight average molecular weight of 2.6 million, and add and dissolve it in batches under stirring to form a solution as the aqueous phase.

[0052] S1.2 Weigh 40 g of polycaprolactone with Mw = 40,000 and add it to 200 ml of dichloromethane, and mix in 1% calcium chloride. Under room temperature conditions and at a speed of 200 rpm, stir magnetically until dissolved as the oil phase.

[0053] S2 Emulsification

[0054] Heat the aqueous phase to 30 °C, stir mechanically at 1300 rpm, inject the oil phase into the aqueous phase with an injection pump at 40 ml / min, and continue emulsifying for 20 min after injection to form an emulsion of oil-in-water droplets.

[0055] S3 Incubation (curing / crosslinking)

[0056] Set the reaction kettle at 45 °C and a speed of 300 rpm, add 8 g of sodium hydroxide in batches to make the reaction system alkaline, slowly inject 0.5 g of BDDE in batches, continuously stir at 200 rpm, and start purging the liquid surface with nitrogen; incubate and crosslink at 45 °C for 8 h to obtain a viscoelastic gel containing polycaprolactone microspheres.

[0057] S4 Dialysis (washing)

[0058] Cut the bulk gel in S3 into irregular lumps with a size of about 0.5 - 2 cm 3 and transfer them to a dialysis device filled with injection water for 7 times of dialysis, with each dialysis time of 120 min, and collect the dialyzed gel lumps.

[0059] S5 Drying

[0060] Transfer the crosslinked hyaluronic acid gel lumps containing polyester microspheres in S4 to a forced-air oven to remove excess water and obtain dry lumps.

[0061] S6 Crushing

[0062] Put the dried lumps in S5 into a crusher for crushing, collect the powder sieved through a multi-stage sieve (80 mesh / 150 mesh / 300 mesh in sequence), and then mix in 1 g of hyaluronic acid with a molecular weight of 600,000.

[0063] S7 Irradiation Sterilization

[0064] Divide the sample in S6 into sterilization bags and conduct irradiation sterilization with an irradiation dose of 25 kGy to obtain a sterile intermediate product for storage and standby.

[0065] S8 Swelling

[0066] Weigh an appropriate amount of the intermediate product in S7, add it to 156 ml of phosphate buffer solution in batches, swell for 30 min, and stir for 15 min to mix evenly to obtain a cross-linked hyaluronic acid gel solution containing polyester microspheres.

[0067] S9 Filling

[0068] Under a hundred-class purification environment, use a filling machine to fill the cross-linked hyaluronic acid gel containing polyester microspheres in S8 into sterile pre-filled syringes to obtain a filler containing cross-linked hyaluronic acid gel with polyester microspheres, which contains 20% polyester microspheres, the cross-linked hyaluronic acid concentration is 15 mg / ml, and the non-cross-linked hyaluronic acid concentration is 5 mg / ml. The filler specifications are 1 ml / branch or 2 ml / branch, and the proportion of each component is fixed.

[0069] Example 2

[0070] S1 Preparation of Aqueous Phase and Oil Phase

[0071] S1.1 Weigh 8 g of polyvinyl alcohol with Mw = 30,000, add 1000 ml of injection water under stirring at 300 rpm, heat up to 95 °C, stir and dissolve for 2 h, and cool down to 20 °C for standby; weigh 3.5 g of hyaluronic acid powder with a molecular weight of 2.6 million, add it in batches under stirring and dissolve for 45 min to form a solution as the aqueous phase;

[0072] S1.2 Weigh 20 g of polycaprolactone with Mw = 20,000 and 20 g of poly(trimethylene carbonate) with Mw = 80,000 and add them to 200 ml of dichloromethane. Stir magnetically at room temperature and 200 rpm until dissolved as the oil phase.

[0073] S2 Emulsification

[0074] Heat the aqueous phase to 30 °C and mechanically stir at 1300 rpm. Inject the oil phase into the aqueous phase with an injection pump at 40 ml / min. After injection, continue emulsification for 20 min to form an oil-in-water droplet emulsion;

[0075] S3 Heat Preservation (Curing / Cross-linking)

[0076] Set the reactor at 45 °C and a rotation speed of 300 rpm. Add 8 g of sodium hydroxide in 3 - 5 batches to make the reaction system alkaline. Slowly inject 0.5 g of BDDE in batches while stirring continuously at 150 rpm, and start purging the liquid surface with nitrogen; maintain the temperature at 45 °C for cross-linking for 8 h to obtain a viscoelastic gel containing polycaprolactone and poly(trimethylene carbonate) microspheres.

[0077] S4 Dialysis (washing)

[0078] Cut the bulk gel in S3 into irregular pieces with a size of about 0.5 - 2 cm 3 and transfer them to a dialysis device filled with injection water. Conduct dialysis 9 times, with each dialysis time being 120 min, and collect the dialyzed gel pieces.

[0079] S5 Freeze-drying

[0080] Transfer the cross-linked hyaluronic acid gel pieces containing polycaprolactone and poly(trimethylene carbonate) microspheres in S4 to a tray, place them in a freeze-dryer, and conduct freeze-drying (-40 °C for pre-freezing for 2 h, -5 - 30 °C for sublimation drying for 8 - 12 h, 40 °C for desorption drying for 6 - 8 h) to obtain freeze-dried pieces.

[0081] S6 Crushing

[0082] Put the freeze-dried pieces in S5 into a crusher for crushing, collect the powder sieved through multi-stage sieves (successively through 80 mesh / 150 mesh / 300 mesh), and then mix in 0.5 g of hyaluronic acid with a molecular weight of 600,000.

[0083] S7 Irradiation sterilization

[0084] Pack the samples in S6 into sterilization bags and conduct irradiation sterilization with an irradiation dose of 25 kGy to obtain a sterile intermediate product for storage and standby.

[0085] S8 Swelling

[0086] Weigh an appropriate amount of the intermediate product in S7, add it to 156 ml of phosphate buffer solution in batches, swell for 30 min, and stir for 15 min to mix evenly to obtain a cross-linked hyaluronic acid gel solution containing polycaprolactone and poly(trimethylene carbonate) microspheres.

[0087] S9 Filling

[0088] Under a hundred - level purification environment, the cross - linked hyaluronic acid gel containing polycaprolactone and poly(trimethylene carbonate) microspheres in S8 is filled into a sterile pre - filled syringe by a filling machine to obtain a filler containing the cross - linked hyaluronic acid gel with polycaprolactone and poly(trimethylene carbonate) microspheres. It contains 20% of polycaprolactone and poly(trimethylene carbonate) microspheres, the cross - linked hyaluronic acid concentration is 17.5 mg / ml, and the non - cross - linked hyaluronic acid concentration is 2.5 mg / ml.

[0089] Example 3

[0090] Preparation of S1 aqueous phase and oil phase

[0091] S1.1 Weigh 8 g of polyvinyl alcohol with Mw = 30,000, add 1000 ml of injection water under stirring at 300 rpm, heat up to 95 °C, stir and dissolve for 2 h, and cool down to 20 °C for standby; weigh 3 g of hyaluronic acid powder with a molecular weight of 2.6 million, add it in batches under stirring and dissolve for 45 min to form a solution, which is used as the aqueous phase;

[0092] S1.2 Weigh 60 g of polycaprolactone with Mw = 20,000 and add it to 300 ml of dichloromethane. Under room - temperature conditions and at a rotation speed of 200 rpm, stir magnetically until dissolved, which is used as the oil phase.

[0093] S2 Emulsification

[0094] Heat up the aqueous phase to 30 °C, stir mechanically at 1300 rpm, inject the oil phase into the aqueous phase at a rate of 40 ml / min with an injection pump, and continue emulsifying for 20 min after injection to form an emulsion of oil - in - water droplets;

[0095] S3 Incubation (curing / cross - linking)

[0096] Set the reaction kettle to 45 °C and a rotation speed of 300 rpm. Add 8 g of sodium hydroxide in 3 - 5 batches to make the reaction system alkaline. Slowly inject 0.5 g of BDDE in batches, continuously stir at 150 rpm, and start purging the liquid surface with nitrogen; incubate and cross - link at 45 °C for 8 h to obtain a viscoelastic gel containing polycaprolactone microspheres.

[0097] S4 Dialysis (washing)

[0098] Cut the massive gel in S3 into irregular lumps with a size of about 0.5 - 2 cm 3 and transfer them to a dialysis device filled with injection water. Conduct 11 times of dialysis, with each dialysis time of 120 min, and collect the dialyzed gel lumps.

[0099] S5 Freeze - drying

[0100] Transfer the crosslinked hyaluronic acid gel mass containing polycaprolactone microspheres in S4 to a tray, place it in a freeze dryer, and perform freeze drying (-40°C pre-freezing for 2 h, -5 - 30°C sublimation drying for 8 - 12 h, 40°C desorption drying for 6 - 8 h) to obtain a freeze-dried mass.

[0101] S6 Crushing

[0102] Put the freeze-dried mass in S5 into a crusher and crush it. Collect the powder sieved through a multi-stage sieve (successively through 80 mesh / 150 mesh / 300 mesh), and then mix in 1 g of hyaluronic acid with a molecular weight of 600,000.

[0103] S7 Irradiation Sterilization

[0104] Dispense the sample in S6 into a sterilized bag and perform irradiation sterilization with an irradiation dose of 25 kGy to obtain a sterile intermediate product, which is stored for later use.

[0105] S8 Swelling

[0106] Weigh an appropriate amount of the intermediate product in S7 and add it to 136 ml of phosphate buffer solution in batches. Swell for 30 min and stir for 15 min to mix evenly to obtain a crosslinked hyaluronic acid gel solution containing polycaprolactone microspheres.

[0107] S9 Filling

[0108] Under a hundred-class purification environment, use a filling machine to fill the crosslinked hyaluronic acid gel containing polycaprolactone microspheres in S8 into a sterile pre-filled syringe to obtain a filler containing crosslinked hyaluronic acid gel with polycaprolactone microspheres, which contains 30% polyester microspheres, a crosslinked hyaluronic acid concentration of 15 mg / ml, and a non-crosslinked hyaluronic acid concentration of 5 mg / ml.

[0109] Example 4

[0110] S1 Preparation of Aqueous Phase and Oil Phase

[0111] Weigh 8 g of polyvinyl alcohol with Mw = 30,000, add 1000 ml of injection water while stirring at a speed of 300 rpm, heat up to 95°C, stir and dissolve for 2 h, and cool down to 20°C for standby. Weigh 3.5 g of hyaluronic acid powder with a molecular weight of 2,600,000 and add it in batches while stirring to dissolve to form a solution as the aqueous phase;

[0112] Weigh 40 g of poly-L-lactic acid with Mw = 100,000 and add it to 200 ml of dichloromethane. Under room temperature conditions and a rotation speed of 200 rpm, stir magnetically until dissolved as the oil phase.

[0113] S2 Emulsification

[0114] Heat the aqueous phase to 30°C and stir mechanically at 1100 rpm. Inject the oil phase into the aqueous phase at a rate of 40 ml / min using a syringe pump. After injection, continue emulsification for 20 min to form droplets.

[0115] S3 Incubation (Curing / Crosslinking)

[0116] Set the reaction kettle to 45°C and a rotation speed of 300 rpm. Add 8 g of sodium hydroxide in 3 - 5 batches to make the reaction system alkaline. Slowly inject 0.5 g of BDDE in batches and stir continuously at 150 rpm. Start purging the liquid surface with nitrogen. Incubate and crosslink at 45°C for 8 h to obtain a viscoelastic gel containing poly(L-lactic acid) microspheres.

[0117] S4 Dialysis (Washing)

[0118] Cut the bulk gel in S3 into irregular pieces approximately 0.5 - 2 cm 3 in size and transfer them to a dialysis device filled with injection water. Perform dialysis 5 times, with each dialysis time being 60 min, and collect the dialyzed gel pieces.

[0119] S5 Freeze - drying

[0120] Transfer the crosslinked hyaluronic acid gel pieces containing poly(L-lactic acid) in S4 to a tray, place them in a freeze - dryer, and perform freeze - drying (-40°C pre - freezing for 2 h, -5 - 30°C sublimation drying for 8 - 12 h, 40°C desorption drying for 6 - 8 h) to obtain freeze - dried pieces.

[0121] S6 Grinding

[0122] Put the freeze - dried pieces in S5 into a grinder and grind them. Sieve the powder through multiple sieves (successively through 80 mesh / 150 mesh / 300 mesh), collect the particles with a size of 40 - 200 μm, and then mix in 0.5 g of hyaluronic acid with a molecular weight of 600,000.

[0123] S7 Swelling

[0124] Weigh an appropriate amount of the intermediate product in S6 and add it to 156 ml of phosphate buffer solution in batches. Swell for 30 min and stir for 15 min to mix evenly to obtain a crosslinked hyaluronic acid gel solution containing poly(L-lactic acid) microspheres.

[0125] S8 Moist Heat Sterilization

[0126] Dispense the sample in S7 into a moist heat sterilization tank and perform moist heat sterilization at a sterilization temperature of 121°C for 15 min to obtain a sterile intermediate product.

[0127] S9 Filling

[0128] Under a Class 100 purification environment, the cross-linked hyaluronic acid gel containing poly-L-lactic acid microspheres in S8 is filled into a sterile pre-filled syringe by a filling machine to obtain a filler containing the cross-linked hyaluronic acid gel with poly-L-lactic acid microspheres, which contains 20% poly-L-lactic acid microspheres, the cross-linked hyaluronic acid concentration is 17.5 mg / ml, and the non-cross-linked hyaluronic acid concentration is 2.5 mg / ml.

[0129] Example 5

[0130] Preparation of S1 aqueous phase and oil phase

[0131] S1.1 Weigh 8 g of polyvinyl alcohol with Mw = 30,000, add 1000 ml of injection water under stirring at 300 rpm, heat up to 95 °C, stir and dissolve for 2 h, and cool down to 20 °C for standby. Weigh 3.5 g of hyaluronic acid powder with a molecular weight of 400,000, and add and dissolve it in batches under stirring to form a solution as the aqueous phase;

[0132] S1.2 Weigh 40 g of poly-L-lactic acid with Mw = 150,000 and add it to 200 ml of dichloromethane. Stir magnetically at room temperature and 200 rpm until dissolved to obtain the oil phase.

[0133] S2 Emulsification

[0134] Heat up the aqueous phase to 30 °C and stir mechanically at 1200 rpm. Inject the oil phase into the aqueous phase at a rate of 40 ml / min with an injection pump. After injection, continue emulsification for 20 min to form droplets;

[0135] S3 Incubation (curing / cross-linking)

[0136] Set the reaction kettle to 45 °C and 300 rpm. Add 8 g of sodium hydroxide in 3 - 5 batches to make the reaction system alkaline. Slowly inject 0.4 g of BDDE in batches and continuously stir at 150 rpm. Start purging the liquid surface with nitrogen; incubate and cross-link at 45 °C for 12 h to obtain a viscoelastic gel containing poly-L-lactic acid microspheres.

[0137] S4 Dialysis (washing)

[0138] Cut the bulk gel in S3 into irregular lumps with a size of about 0.5 - 2 cm 3 and transfer them to a dialysis device filled with buffer solution for 5 times of dialysis, with each dialysis time of 120 min, and collect the dialyzed gel lumps.

[0139] S5 Concentration

[0140] Transfer the S4 gel block to a rotary evaporator for concentration and simultaneously remove the organic solvent, with the final volume being about 200 ml. The concentrated sample is gel-like and does not need to be swollen with water again.

[0141] S6 Sieving

[0142] The concentrated mass in S5 is sieved through multiple sieves (80 mesh / 150 mesh / 300 mesh in sequence), and particles of 40 - 200 μm are collected.

[0143] S7 Moist heat sterilization

[0144] The samples in S6 are subpackaged into a moist heat sterilization tank for moist heat sterilization at a sterilization temperature of 121 °C for 15 min to obtain sterile intermediate products.

[0145] S8 Filling

[0146] Under a hundred - level purification environment, the cross - linked hyaluronic acid gel containing poly - L - lactic acid microspheres in S7 is filled into sterile pre - filled syringes by a filling machine to obtain a filler containing cross - linked hyaluronic acid gel with poly - L - lactic acid microspheres, which contains 20% poly - L - lactic acid microspheres and the concentration of cross - linked hyaluronic acid is 17.5 mg / ml.

[0147] Example 6

[0148] The corresponding samples are prepared according to the methods of Examples 1 - 4, and are respectively denoted as 1#, 2#, 3#, 4#; the test for BDDE residue is carried out according to the method specified in Appendix F of "YY / T 0961 - 2014 Cross - linked Sodium Hyaluronate Gel for Plastic Surgery". The BDDE residue test results are shown in Table 1.

[0149] Table 1. BDDE residue test results

[0150]

[0151] As can be seen from Table 1, the dialysis time of samples 1 - 3# is twice that of sample 4#, and the dialysis times are increased by more than 2 times. The cross - linker residue of the former is one order of magnitude lower than that of the latter. Increasing the dialysis times and prolonging the dialysis time can significantly reduce the cross - linker BDDE residue amount, thereby improving the biological safety of the product; moreover, with the increase of dialysis times, the cross - linker residue amount gradually decreases.

[0152] The test for pushing force is carried out in accordance with "YY / T 0962 - 2014 Cross - linked Sodium Hyaluronate Gel for Plastic Surgery"; the pushing force test results are shown in Table 2.

[0153] Table 2. Pushing force test data

[0154]

[0155] The experimental data in Table 2 show that the increase in the content of small - molecule hyaluronic acid helps to improve the fluidity of the product and significantly reduces the pushing force of the product; on the other hand, with the increase in the content of PCL microspheres, the pushing force increases accordingly.

[0156] As can be seen from the above embodiments, the process method of the present invention includes: 1) dissolving an aliphatic polyester substance in an organic solvent as the oil phase, wherein the concentration of the aliphatic polyester substance is 5-40 wt%; dissolving a surfactant and a biocompatible polymer in water as the water phase; the molecular weight of the biocompatible polymer is 200,000-4,000,000; 2) continuously emulsifying the oil phase and the water phase to obtain an oil-in-water emulsion; 3) simultaneously curing and crosslinking the emulsion and a crosslinking agent under alkaline conditions to obtain a crosslinked gel block containing polymer microspheres; 4) performing dialysis, solvent removal, sterilization, etc. on the crosslinked gel block containing polymer microspheres to obtain a crosslinked gel material containing polymer microspheres. The present invention simplifies the production process, can improve production efficiency and save input costs, and can obtain a filler product with less crosslinking agent residue.

[0157] Specific examples are used in this article to elaborate on the principle and implementation mode of the present invention. The description of the above examples is only used to help understand the method and its core idea of the present invention. The above is only the preferred implementation mode of the present invention. It should be noted that due to the limited nature of written expression and the objectively infinite specific structures, for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements, refinements or changes can be made, or the above technical features can be combined in an appropriate manner; these improvements, refinements, changes or combinations, or directly applying the concept and technical solution of the invention to other occasions without improvement, should all be regarded as the protection scope of the present invention.

Claims

1. A preparation method of a crosslinked gel material containing polymer microspheres, characterized in that, Comprising the following steps: 1) Dissolve an aliphatic polyester substance in an organic solvent to serve as the oil phase, wherein the concentration of the aliphatic polyester substance is 5-40 wt%; dissolve a surfactant and a biocompatible polymer in water to serve as the water phase; the molecular weight of the biocompatible polymer is 200,000-4,000,000; the surfactant is polyvinyl alcohol and / or Tween; the biocompatible polymer is one or more of hyaluronic acid, collagen, carboxymethyl cellulose derivatives, dextran, and starch; the aliphatic polyester substance is selected from at least one of poly(lactic acid), poly(glycolic acid), polycaprolactone, poly(trimethylene carbonate), and poly(p-dioxanone), or a copolymer obtained by copolymerizing monomers of two or more of the aforementioned polymers, or a modified polymer thereof; the organic solvent is selected from one or more of dichloromethane, chloroform, toluene, and ethyl acetate; 2) Continuously emulsify after mixing the oil phase and the water phase to obtain an oil-in-water emulsion; 3) Simultaneously carry out curing and crosslinking of the emulsion and a crosslinking agent under alkaline conditions, wherein the dosage of the crosslinking agent is 10-15% of the mass of the biocompatible polymer, to obtain a crosslinked gel block containing polymer microspheres; 4) Subject the crosslinked gel block containing polymer microspheres to dialysis, solvent removal, sieving, moist heat sterilization, or irradiation sterilization in sequence to obtain a crosslinked gel material containing polymer microspheres.

2. The preparation method according to claim 1, characterized in that, The concentration of the aliphatic polyester substance is 10-30 wt%.

3. The preparation method according to claim 1, characterized in that, The molecular weight of the biocompatible polymer is 400,000-3,000,000.

4. The preparation method according to any one of claims 1 to 3, characterized in that, Specifically, step 2) is: heating to 20-40°C, and adding the oil phase to the water phase under the condition of stirring or homogenizing at a speed of 600-2500 rpm, and continuously emulsifying to obtain an oil-in-water emulsion.

5. The preparation method according to claim 4, characterized in that, In step 3), the curing and crosslinking are carried out simultaneously under the conditions of a temperature of 35-90°C, purging with a protective atmosphere, and stirring.

6. The preparation method according to claim 4, wherein In step 4), wash and dialyze the crosslinked gel block containing polymer microspheres with water for injection.

7. The preparation method according to claim 6, characterized in that, In step 4), subject the crosslinked gel block containing polymer microspheres to dialysis 7-11 times, and the dialysis time for each time is 60-120 min.

8. The preparation method according to any one of claims 1-3, characterized in that, In step 4), after sieving, a biocompatible small molecule is also mixed in to obtain a powder sample; the biocompatible small molecule is a small molecule hyaluronic acid with a molecular weight of 400,000-800,000.

9. The preparation method according to claim 8, characterized in that, In step 4), after obtaining the powder sample, carry out irradiation sterilization with an irradiation dose of 15-25 kGy to obtain a sterile intermediate product.

10. The preparation method according to claim 9, characterized in that, After irradiation sterilization in step 4), it further includes: batchwise adding the sterile intermediate product to a buffer solution and swelling for 30-60 min to obtain a crosslinked gel solution containing polymer microspheres.

11. The crosslinked gel material containing polymer microspheres obtained by the preparation method according to any one of claims 1-10, with a crosslinking agent residue lower than 0.05 ppm.

12. An injection filler filled with the crosslinked gel material containing polymer microspheres according to claim 11.

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