Sodium hyaluronate composite filler for injection, preparation method and application thereof

Abstract

The present invention relates to the field of biomedical materials technology, specifically disclosing an injectable sodium hyaluronate composite filler, its preparation method, and its application. The composition comprises methoxypolyethylene glycol-poly-L-lactic acid nanoparticles, sodium hyaluronate, and a dispersion medium. This composite filler enables precise placement and filling of the injection site, exhibits excellent hydrophilicity and biocompatibility, reduces inflammatory reactions after implantation, and promotes earlier and more effective collagen regeneration. Animal studies have demonstrated its safety and effectiveness.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and particularly relates to a sodium hyaluronate composite filler for injection, and a preparation method and application thereof. Background Art

[0002] In recent years, injectable fillers for cosmetic and medical applications have rapidly developed. They have garnered widespread attention for their ability to provide support and fill the injection site. Sodium hyaluronate, an acidic mucopolysaccharide widely present in human connective tissue, stabilizes the extracellular matrix. Its strong water absorption capacity helps maintain skin elasticity and retain moisture, making it widely used in injectable micro-plastic surgery. However, while it possesses excellent biocompatibility and degradability, it also rapidly degrades in the body, a problem often addressed through cross-linking with diepoxides. However, increasing the degree of cross-linking increases the granularity of hyaluronic acid and the resulting inflammatory response, even leading to the risk of granulomas. Therefore, simply increasing the duration of hyaluronic acid's effect through cross-linking carries significant risks. One approach to avoiding the risk of inflammation and prolonging its effectiveness is to add other ingredients to hyaluronic acid gels. Among these, synthetic polymers are increasingly favored by beauty enthusiasts due to their reduced risk of infection and safety compared to animal-derived materials. However, synthetic polymers must exhibit good biocompatibility before they can be used in mixed gels. However, not all materials are suitable as additives to hyaluronic acid gel. In order to be evenly mixed in the hyaluronic acid gel and not easily aggregate after injection into the human body, the material must be both lipophilic and hydrophilic, that is, amphiphilic.

[0003] However, traditional polylactic acid microsphere injection products are not easy to redissolve due to their poor hydrophilicity, and after redissolution, the polylactic acid microspheres have poor dispersibility and are prone to aggregation and deposition, which can easily cause needle blockage during intradermal injection, subcutaneous nodules after injection, and thicker needles used during injection, which can increase the patient's pain. Sodium carboxymethyl cellulose (CMC) is sometimes used as a suspending agent to improve the above-mentioned disadvantages, such as the listed products Avilan and Sculptra. However, CMC is not an endogenous substance in the human body and its biocompatibility is poor. In addition, polylactic acid microsphere products using CMC as a suspending agent generally require a long period of hydration before they can be used for intradermal injection, which brings inconvenience to the application.

[0004] In 2021, a facial filler called Rubai Angel was launched. It is a cross-linked sodium hyaluronate gel containing L-lactic acid-ethylene glycol copolymer microspheres. The L-lactic acid-ethylene glycol copolymer microspheres have a particle size of 20 to 45 μm. It is easy to inject and can be injected immediately without reconstitution. The cross-linked hyaluronic acid compound has an immediate filling effect, and the poly-L-lactic acid subsequently stimulates collagen regeneration. However, all polylactic acid microsphere products, including Rubai Angel, have a relatively slow onset of effect, with the actual effect not being seen until two weeks after injection and reaching its peak three to six months later. Summary of the Invention

[0005] The purpose of the present invention is to provide an injectable sodium hyaluronate composite filler and its preparation method and application, specifically an injectable sodium hyaluronate composite filler containing methoxypolyethylene glycol-poly-L-lactic acid nanoparticles and its preparation method and application, to address the shortcomings of the existing technology.

[0006] In a first aspect, the present invention provides an injectable sodium hyaluronate composite filler comprising methoxy polyethylene glycol-poly-L-lactic acid nanoparticles, sodium hyaluronate, and a dispersion medium; the concentration of the methoxy polyethylene glycol-poly-L-lactic acid nanoparticles in the dispersion medium is 5 mg / mL to 300 mg / mL, for example, 5 mg / mL to 50 mg / mL, 50 mg / mL to 100 mg / mL, 100 mg / mL to 150 mg / mL, 150 mg / mL to 200 mg / mL, or 200 mg / mL to 300 mg / mL.

[0007] The concentration of sodium hyaluronate in the dispersion medium is 5 mg / mL to 30 mg / mL, for example, 5 mg / mL to 10 mg / mL, 10 mg / mL to 15 mg / mL, 15 mg / mL to 20 mg / mL, or 20 mg / mL to 30 mg / mL.

[0008] The dispersion medium is water, physiological saline, glucose solution, or phosphate buffer; the average particle size of the methoxypolyethylene glycol-poly-L-lactic acid nanoparticles is 10 to 500 nm, for example, 10 to 50 nm, 50 to 100 nm, 100 to 200 nm, 200 to 300 nm, 300 to 400 nm, or 400 to 500 nm; and the polydispersity index (PDI) of the methoxypolyethylene glycol-poly-L-lactic acid nanoparticles is 0.05 to 0.4, for example, 0.05 to 0.1, 0.1 to 0.2, 0.2 to 0.3, or 0.3 to 0.4.

[0009] The sodium hyaluronate composite filler is in a gel state and does not need to be re-dissolved during use and can be directly injected.

[0010] In the sodium hyaluronate composite filler containing methoxy polyethylene glycol-poly-L-lactic acid nanoparticles, the molecular weight of sodium hyaluronate is 2×105 ~2.5×10 6 Dalton. For example, 6×10 5 ~2.5×10 6 Dalton.

[0011] In the sodium hyaluronate composite filler containing methoxy polyethylene glycol-poly-L-lactic acid nanoparticles, the molecular weight of the methoxy polyethylene glycol block in the methoxy polyethylene glycol-poly-L-lactic acid polymer is 500-10,000 Daltons, preferably 1,000-5,000 Daltons, for example, 1,000-1,500 Daltons, 1,500-3,000 Daltons, or 3,000-5,000 Daltons; the molecular weight of the poly-L-lactic acid block in the methoxy polyethylene glycol-poly-L-lactic acid polymer is 500-20,000 Daltons, preferably 1,000-10,000 Daltons, for example, 1,000-1,500 Daltons, 1,500-3,000 Daltons, 3,000-5,000 Daltons, or 5,000-10,000 Daltons.

[0012] The second aspect of the present invention provides a preparation method of the injectable sodium hyaluronate composite filler, the specific steps of which are: 1) mixing an organic solvent with a methoxy polyethylene glycol-poly-L-lactic acid polymer to obtain an oil phase, wherein the mass fraction of the methoxy polyethylene glycol-poly-L-lactic acid polymer in the oil phase is 0 to 20%, excluding 0, for example 1%, 3%, 5%, 10%, 12%, 15%, 18% or 20%.

[0013] 2) After removing the organic solvent, distilled water is added for hydration (the amount of distilled water added is determined by the amount of methoxy polyethylene glycol-poly-L-lactic acid polymer, generally 5 to 20 times the mass of the polymer), and the mixture is allowed to stand for 15 to 60 minutes (e.g., 15 to 30 minutes, 30 to 60 minutes) to obtain a methoxy polyethylene glycol-poly-L-lactic acid nanoparticle solution. Specifically, the oil phase is subjected to rotary evaporation under vacuum conditions for 10 to 120 minutes to remove the organic solvent. Then, an appropriate amount of distilled water is added for hydration for 10 to 60 minutes to obtain an aqueous solution of methoxy polyethylene glycol-poly-L-lactic acid nanoparticles having a concentration of 1 mg / mL to 100 mg / mL. The concentration may be 1 mg / mL to 5 mg / mL, 5 mg / mL to 20 mg / mL, 20 mg / mL to 50 mg / mL, or 50 mg / mL to 100 mg / mL.

[0014] 3) Freeze-dry to obtain methoxypolyethylene glycol-poly-L-lactic acid nanoparticle freeze-dried powder. There are no specific freeze-drying conditions; virtually any freeze-drying apparatus can be used. The freeze-drying temperature is -50°C to -40°C, and the drying time is 12 to 72 hours, depending on the specific needs.

[0015] 4) providing a hyaluronic acid solution that is completely swollen in a dispersion medium;

[0016] 5) re-dissolving the methoxypolyethylene glycol-poly-L-lactic acid nanoparticle freeze-dried powder prepared in step 3) with a dispersion medium to obtain a nanoparticle solution;

[0017] 6) The nanoparticle solution of step 5) is added to the hyaluronic acid solution of step 4) to obtain a sodium hyaluronate composite filler containing methoxy polyethylene glycol and poly-L-lactic acid nanoparticles.

[0018] The third aspect of the present invention is the use of the sodium hyaluronate composite filler described above, or the sodium hyaluronate composite filler prepared by any of the preparation methods described above, in tissue repair materials and / or filling biomaterials.

[0019] The beneficial effects of the present invention are at least:

[0020] 1. The composite filler of the present invention has high biocompatibility. The polymer nanoparticles, due to their hydrophilic surface, are more easily dispersed and thus help reduce the appearance of subcutaneous nodules without clogging blood vessels. Furthermore, due to their loose structure, the nanoparticles have good biodegradability and can stimulate collagen regeneration faster and more effectively.

[0021] 2. The sodium hyaluronate composite filler product containing methoxypolyethylene glycol-poly-L-lactic acid nanoparticles for injection provided by the present invention is uniform and fine, has good needle permeability, is easy to inject, does not require reconstitution, and is simple to operate;

[0022] 3. It has no irritating effect on the skin, has a long local retention time, good plasticity, and few side effects. It can stimulate collagen regeneration faster and better, thus significantly eliminating wrinkles.

[0023] 4. The preparation process conditions of the present invention are easy to control, the operation steps are few, and the product quality is stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a particle size diagram of methoxy polyethylene glycol-poly (L-lactic acid) nanoparticles prepared in Example 1.

[0025] Figure 2 State diagrams of composite fillers prepared with sodium hyaluronate of different molecular weights prepared in Examples 1, 2, and 3, where a is a top view, b is a front view, 1) is the composite filler prepared in Example 1, 2) is the composite filler prepared in Example 2, and 3) is the composite filler prepared in Example 3.

[0026] Figure 3 State diagrams of the composite fillers containing different concentrations of methoxypolyethylene glycol-poly-L-lactic acid nanoparticles prepared in Examples 4 and 5, where a is a top view, b is a front view, 1) is the composite filler prepared in Example 4, and 2) is the composite filler prepared in Example 5.

[0027] Figure 4 State diagrams of the composite fillers containing different concentrations of methoxypolyethylene glycol-poly-L-lactic acid nanoparticles prepared in Examples 6 to 10, 1) is the composite filler prepared in Example 6, 2) is the composite filler prepared in Example 7, 3) is the composite filler prepared in Example 8, 4) is the composite filler prepared in Example 9, and 5) is the composite filler prepared in Example 10.

[0028] Figure 5 The collagen volume ratio test results in test example 4 are shown in Figure 1. Group 1 is the normal saline group, and Group 2 is the commercially available moist white angel. TM Facial fillers: Group 3: 100 mg / mL nanoparticles / high molecular weight sodium hyaluronate, and Group 4: 200 mg / mL nanoparticles / high molecular weight sodium hyaluronate.

[0029] Figure 6 The graph shows the test results of the percentage of newly formed type III collagen fibers in Test Example 4. Group 1 is the normal saline group, and Group 2 is the commercially available moist white angel. TM Facial fillers: Group 3: 100 mg / mL nanoparticles / high molecular weight sodium hyaluronate, and Group 4: 200 mg / mL nanoparticles / high molecular weight sodium hyaluronate. DETAILED DESCRIPTION

[0030] The following examples are intended to illustrate the present invention but are not intended to further limit the present invention. The present invention is further described in detail below with reference to the examples, but the present invention is not limited to these examples and the preparation methods used. Moreover, those skilled in the art may make equivalent substitutions, combinations, improvements or modifications to the present invention based on the description of the present invention, but these are all within the scope of the present invention.

[0031] Example 1

[0032] 1. Preparation of methoxy polyethylene glycol-poly (L-lactic acid) nanoparticles.

[0033] 1) In a round bottom flask, add the polymer methoxy polyethylene glycol 2000 -Poly-L-lactic acid 1300 (mPEG 2000 -b-PLLA 1300 ) 100 mg was dissolved in 10 mL of acetonitrile to obtain an oily phase;

[0034] 2) Use a rotary evaporator to evaporate the acetonitrile in a 60°C water bath to form a uniform film of the polymer on the wall, then hydrate with 10 mL of deionized water and let it stand for 30 minutes to fully self-assemble to form a nanoparticle solution, such as Figure 1As shown in FIG, the particle size of the methoxy polyethylene glycol-poly-L-lactic acid nanoparticles measured by dynamic light scattering method is 98.53 nm, and the polydispersity index (PDI) is 0.228, indicating that the obtained nanoparticles have a small particle size and a uniform particle size distribution.

[0035] 3) freeze-drying the obtained nanoparticle solution for 48 hours to obtain methoxy polyethylene glycol-poly (L-lactic acid) nanoparticle freeze-dried powder.

[0036] 2. Prepare sodium hyaluronate composite filler containing methoxy polyethylene glycol-poly (L-lactic acid) nanoparticles.

[0037] 4) Weigh 213 mg of a solution with a molecular weight of 2.3×10 6 Dalton's hyaluronic acid (Shandong Zhongshan Biotechnology Co., Ltd.) was added to a 10 mL volumetric flask and distilled water was added. After 48 hours, it was completely swollen (the amount of distilled water added was sufficient to ensure complete swelling of the hyaluronic acid). The volume was then fixed to obtain hyaluronic acid solution A. The density of the solution was measured to be 0.99 g / mL.

[0038] 5) Redissolve 1 g of methoxypolyethylene glycol-poly-L-lactic acid nanoparticle freeze-dried powder with 1 mL of distilled water to obtain a nanoparticle solution with a concentration of 1 g / mL.

[0039] 6) Weigh 7.9 g of hyaluronic acid solution A (actually containing 170 mg of hyaluronic acid) into a 10 mL volumetric flask, add 1 mL of the above nanoparticle solution, dilute to 10 mL with distilled water, and mix thoroughly to obtain methoxy polyethylene glycol-poly-L-lactic acid nanoparticle sodium hyaluronate composite filler A. The concentration of methoxy polyethylene glycol-poly-L-lactic acid nanoparticles is 100 mg / mL, and the concentration of sodium hyaluronate is 17 mg / mL.

[0040] Example 2

[0041] The difference from Example 1 is that in step 4), the molecular weight of hyaluronic acid is 1.15×10 6 Daltons, hyaluronic acid solution B was obtained. The density of the solution was measured to be 1.0 g / mL. In step 6), 7.98 g of hyaluronic acid solution A (actually containing 170 mg of hyaluronic acid) was weighed into a 10 mL volumetric flask. Finally, methoxypolyethylene glycol-poly-L-lactic acid nanoparticle sodium hyaluronate composite filler B was obtained.

[0042] Example 3

[0043] The difference from Example 1 is that in step 4), the molecular weight of hyaluronic acid is 6 to 7×10 5Daltons, hyaluronic acid solution C was obtained. The density of the solution was measured to be 1.0 g / mL. In step 6), 7.98 g of hyaluronic acid solution A (actually containing 170 mg of hyaluronic acid) was weighed into a 10 mL volumetric flask. Finally, the sodium hyaluronate composite filler C containing methoxypolyethylene glycol-poly-L-lactic acid nanoparticles was obtained.

[0044] The state diagram of composite fillers prepared with sodium hyaluronate of different molecular weights is shown in the figure Figure 2 ,Depend on Figure 2 It can be seen that the composite fillers prepared with sodium hyaluronate of different molecular weights are all milky white viscous gels. When injected superficially, they can avoid the possible light transmittance and Tyndall phenomenon of transparent hyaluronic acid, and the filling effect is more realistic and natural.

[0045] Example 4

[0046] The difference from Example 1 is that

[0047] In step 5), 2 g of methoxypolyethylene glycol-poly-L-lactic acid nanoparticle freeze-dried powder was reconstituted with 1 mL of distilled water to obtain a nanoparticle solution with a concentration of 2 g / mL.

[0048] In step 6), 7.9 g of hyaluronic acid solution A (actually containing 170 mg of hyaluronic acid) was weighed into a 10 mL volumetric flask, 0.5 mL of the nanoparticle solution from step 5) was added, and the volume was adjusted to 10 mL with distilled water and mixed uniformly to obtain a sodium hyaluronate composite filler D containing 100 mg / mL methoxypolyethylene glycol-poly-L-lactic acid nanoparticles, with a sodium hyaluronate concentration of 17 mg / mL.

[0049] Example 5

[0050] The difference from Example 1 is that

[0051] In step 5), 2 g of methoxypolyethylene glycol-poly-L-lactic acid nanoparticle freeze-dried powder was reconstituted with 1 mL of distilled water to obtain a nanoparticle solution with a concentration of 2 g / mL.

[0052] In step 6), 7.9 g of hyaluronic acid solution A (actually containing 170 mg of hyaluronic acid) was weighed into a 10 mL volumetric flask, 1 mL of the above-mentioned nanoparticle solution was added, and the volume was adjusted to 10 mL with distilled water and mixed uniformly to obtain a sodium hyaluronate composite filler D containing 200 mg / mL methoxypolyethylene glycol-poly-L-lactic acid nanoparticles, with a sodium hyaluronate concentration of 17 mg / mL.

[0053] The state diagram of the composite filler containing different concentrations of methoxy polyethylene glycol-poly (L-lactic acid) nanoparticles is shown in Figure 2. Figure 3 ,Depend on Figure 3It can be seen that the composite fillers containing different concentrations of methoxypolyethylene glycol-poly-L-lactic acid nanoparticles are all milky white viscous gels. When injected superficially, they can avoid the possible light transmittance and Tyndall phenomenon of transparent hyaluronic acid, and the filling effect is more realistic and natural.

[0054] Examples 6 to 10

[0055] This group of examples prepares composite fillers containing different concentrations of methoxy polyethylene glycol-poly (L-lactic acid) nanoparticles. The difference from Example 1 is that:

[0056] Step 4) Weigh 213 mg of hyaluronic acid (molecular weight 6×10 5 Daltons) in a 10 mL volumetric flask, add an appropriate amount of distilled water, wait for 48 hours until it swells completely, and then adjust the volume to obtain a hyaluronic acid solution.

[0057] Step 5) 1 g of methoxypolyethylene glycol-poly-L-lactic acid nanoparticle freeze-dried powder was reconstituted with 1 mL of distilled water to obtain a nanoparticle solution with a concentration of 1 g / mL.

[0058] Step 6) Weigh 2.32 g of the five hyaluronic acid solutions (actually containing 50 mg of hyaluronic acid) into five different 10 mL volumetric flasks, add 0.05 mL, 0.1 mL, 0.2 mL, 0.3 mL, and 0.5 mL of the nanoparticle solution, respectively, dilute to 10 mL with distilled water, and mix well to obtain sodium hyaluronate composite fillers containing 5 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, and 50 mg / mL of methoxypolyethylene glycol-poly-L-lactic acid nanoparticles, respectively.

[0059] Test Example 1

[0060] With different molecular weights (2.3×10 6 Dalton, 1.15×10 6 Dalton, molecular weight is 6~7×10 5 Daltons) sodium hyaluronate solution (concentration of 17 mg / mL) was used as a control, and the needle permeability of a total of 7 composite fillers prepared in Examples 4 to 10 was investigated. The results are shown in Table 1.

[0061] Table 1

[0062]

[0063] It can be seen that the needle permeability of the 7 tested products is good.

[0064] Test Example 2

[0065] The states of the various composite fillers prepared in Examples 6 to 10 were examined, and the results are shown in Table 2.

[0066] Table 2

[0067]

[0068] As can be seen, the fillers in various formulations all appear milky white. When the nanoparticle content is low, the composite filler exhibits excellent transparency. As the nanoparticle concentration increases, the transparency decreases to opaque. Because the milky white composite gel effectively reduces light transmission in the contoured area, the result is more realistic and natural.

[0069] Test Example 3

[0070] The composite filler samples containing methoxypolyethylene glycol-poly-L-lactic acid nanoparticles prepared in Examples 4 to 10 were used to verify their skin irritation and displacement at the implantation site as dermal fillers. The specific experimental method is as follows:

[0071] Samples to be tested: two samples of composite fillers containing different concentrations of methoxy polyethylene glycol-poly (L-lactic acid) nanoparticles prepared in Examples 4 and 5, labeled as 100 mg / mL nanoparticles / high molecular weight sodium hyaluronate and 200 mg / mL nanoparticles / high molecular weight sodium hyaluronate, respectively; five samples of composite fillers containing different concentrations of methoxy polyethylene glycol-poly (L-lactic acid) nanoparticles prepared in Examples 6 to 10, labeled as 5 mg / mL nanoparticles / low molecular weight sodium hyaluronate, 10 mg / mL nanoparticles / low molecular weight sodium hyaluronate, 20 mg / mL nanoparticles / low molecular weight sodium hyaluronate, 30 mg / mL nanoparticles / low molecular weight sodium hyaluronate, and 50 mg / mL nanoparticles / low molecular weight sodium hyaluronate, respectively; commercially available moist white angel TM Facial fillers served as the positive control group, and normal saline served as the blank control group.

[0072] Experimental animals: Purebred New Zealand white rabbits aged 4 to 6 months, weighing 210 to 215 g, were divided into three groups (corresponding to three time points), with 9 rabbits in each group;

[0073] Injection: On the day of implantation, the rabbit's back was shaved and routinely disinfected with iodine and ethanol. Six subcutaneous injections (0.5 mL / point) were made on either side of the spine, 3 cm apart. Each symmetrical injection site constituted a test sample. The injection sites were then marked with Coomassie Brilliant Blue. The injection sites were re-colored every two weeks to prevent fading due to metabolic factors.

[0074] Detection method:

[0075] The skin reaction at the implantation site was observed daily for one month after implantation to check for redness, swelling, or bulges. The injected tissue was palpated for nodules, granulations, or other tissue proliferations. The animals were not sacrificed. The experimental results are shown in Table 2.

[0076] The experimental animals were killed at three time points after implantation (1 month, 3 months, and 6 months), with 9 animals killed at each time point. The subcutaneous tissue including the implant material was excised and placed in a 10% formaldehyde solution. After fixation for 48 hours, the sections were routinely dehydrated, transparent, waxed, embedded, and sectioned. The thickness of the sections was 3 μm. After the paraffin sections were dewaxed to water, the sections were immersed in hematoxylin solution for 5 minutes, washed and separated, and then immersed in eosin solution for 2 minutes. After dehydration and sealing, the sections were examined under a microscope. The results are shown in Table 3.

[0077] Table 3

[0078]

[0079] / : Indicates not applicable here.

[0080] As shown in Table 3, compared with the marketed products, the seven tested samples showed no adverse skin appearance reactions after being implanted subcutaneously in the back of rabbits, and there was no obvious inflammatory reaction or risk of displacement, indicating that the tested samples have good biocompatibility, high safety, and can achieve precise positioning and filling of the injection site.

[0081] Test Example 4

[0082] The composite filler samples containing methoxypolyethylene glycol-poly-L-lactic acid nanoparticles prepared in Examples 4 and 5 were used to verify their ability to stimulate collagen regeneration as dermal fillers. The specific experimental method was as follows:

[0083] Test samples: two samples of composite fillers containing different concentrations of methoxypolyethylene glycol-poly-L-lactic acid nanoparticles prepared in Examples 4 and 5, labeled as 200 mg / mL nanoparticles / high molecular weight sodium hyaluronate (Group 4) and 100 mg / mL nanoparticles / high molecular weight sodium hyaluronate (Group 3), commercially available moist white angel TM The facial filler served as the positive control group (Group 2), and normal saline served as the blank control group (Group 1).

[0084] Experimental animals: Purebred New Zealand white rabbits aged 4 to 6 months, weighing 210 to 215 g, were divided into three groups (corresponding to three time points), with three rabbits in each group;

[0085] Injection: On the day of implantation, the rabbit's back was shaved and routinely disinfected with iodine and ethanol. Eight subcutaneous injections (0.5 mL / point) were made on either side of the spine, 2 cm apart. Each symmetrical injection site represented one group of test samples. From top to bottom, the injection sites were group 4, group 3, group 2, and group 1. The injection sites were then marked with Coomassie Brilliant Blue. The injection sites were re-pigmented every two weeks to prevent fading due to metabolic factors.

[0086] Detection method:

[0087] The experimental animals were sacrificed at three time points after implantation (1 month, 3 months, and 6 months), with three animals sacrificed at each time point. The subcutaneous tissue, including the implanted material, was excised and placed in a 10% formaldehyde solution. After fixation for 48 hours, the tissue was routinely dehydrated, transparent, wax-impregnated, embedded, and sectioned. The sections were 3 μm thick and stained with Masson and Sirius red.

[0088] Masson staining: After paraffin sections were dewaxed and hydrated, the sections were immersed in potassium dichromate overnight, then stained with iron hematoxylin for 1 minute, with ponceau acid fuchsin for 6 minutes, with phosphomolybdic acid for 1 minute, and with aniline blue for 30 seconds. After dehydration and mounting, the sections were examined under a microscope. Three sections filled with cell tissue were randomly selected from each section at 200x magnification. The mean percentage of collagen-positive area in the three sections to the total tissue area was calculated using Image-ProPlus 6.0 software. The results are shown in the attached figure. Figure 5 .

[0089] Sirius red staining: After tissue fixation, Sirius red staining was performed and examined using an optical microscope equipped with a polarizing filter. Three sections filled with cell tissue were randomly selected for each slice under a 200x microscope. The mean percentage of type III collagen in the three sections was calculated using Image-ProPlus 6.0 software. The results are shown in the attached Figure 6 .

[0090] from Figure 4 The collagen volume ratio results show that after 1 month, 3 months and 6 months of injection, the collagen volume ratio of each test group was higher than that of the blank control group. The order of the groups at each time point from high to low is: 200mg / mL nanoparticles / high molecular weight sodium hyaluronate group>100mg / mL nanoparticles / high molecular weight sodium hyaluronate>Rubber Angel TM >Blank control group.

[0091] Depend on Figure 6 The results of the percentage of newly formed type III collagen fibers showed that after 1 month, 3 months and 6 months of injection, the content of newly formed type III collagen fibers in each experimental group was higher than that in the blank control group. The order of the content of type III collagen fibers in each group at each time point was as follows: 200 mg / mL nanoparticles / high molecular weight sodium hyaluronate group > 100 mg / mL nanoparticles / high molecular weight sodium hyaluronate > White Angel TM >Blank control group.

[0092] White Angel TM The content of L-lactic acid-ethylene glycol copolymer microspheres in the 100 mg / mL nanoparticles / high molecular weight sodium hyaluronate group was lower than that in the wet white angel group. TMHowever, the collagen volume ratio and percentage of newly formed type III collagen fibers in the 100 mg / mL nanoparticles / high molecular weight sodium hyaluronate group were higher than those in the wet white angel group at each time point. TM , indicating that the tested samples have better biodegradability, can promote collagen regeneration earlier and more effectively, and have significant advantages compared with similar facial fillers.

Claims

1. A sodium hyaluronate composite filler for injection, characterized in that: It is composed of methoxy polyethylene glycol-poly (L-lactic acid) nanoparticles, sodium hyaluronate and dispersion medium; The concentration of the methoxy polyethylene glycol-poly (L-lactic acid) nanoparticles in the dispersion medium is 10 mg / mL to 200 mg / mL; The concentration of sodium hyaluronate in the dispersion medium is 10 mg / mL to 20 mg / mL; The average particle size of the methoxy polyethylene glycol-poly (L-lactic acid) nanoparticles is 50 nm to 200 nm; The molecular weight of the methoxy polyethylene glycol block in the methoxy polyethylene glycol-poly-L-lactic acid polymer is 1000-5000 Daltons; the molecular weight of the poly-L-lactic acid block in the methoxy polyethylene glycol-poly-L-lactic acid is 1000-10000 Daltons.

2. The sodium hyaluronate composite filler according to claim 1, characterized in that The molecular weight of the sodium hyaluronate is 2×10 5 ~2.5×10 6 Dalton; and / or, the sodium hyaluronate composite filler is in a gel state; And / or, the polydispersity coefficient of the methoxypolyethylene glycol-poly-L-lactic acid nanoparticles is 0.05-0.

4.

3. The sodium hyaluronate composite filler according to claim 1, characterized in that The concentration of the methoxy polyethylene glycol-poly (L-lactic acid) nanoparticles in the dispersion medium is 100 mg / mL to 200 mg / mL; And / or, the molecular weight of the sodium hyaluronate is 6×10 5 ~2.5×10 6 Dalton.

4. The sodium hyaluronate composite filler according to any one of claims 1 to 3, characterized in that The dispersion medium is one or a combination of at least two of water, physiological saline, glucose solution or phosphate buffer.

5. The method for preparing the sodium hyaluronate composite filler for injection according to claim 1, comprising the following steps: 1) mixing an organic solvent with a methoxy polyethylene glycol-poly (L-lactic acid) polymer to obtain an oil phase; 2) After removing the organic solvent, distilled water was added for hydration and the mixture was allowed to stand for 15 to 60 minutes to obtain a methoxy polyethylene glycol-poly-L-lactic acid nanoparticle solution; 3) Freeze-drying to obtain methoxy polyethylene glycol-poly (L-lactic acid) nanoparticle freeze-dried powder; 4) providing a sodium hyaluronate solution that is completely swollen in a dispersion medium; 5) re-dissolving the methoxypolyethylene glycol-poly-L-lactic acid nanoparticle freeze-dried powder prepared in step 3) with a dispersion medium to obtain a nanoparticle solution; 6) adding the nanoparticle solution from step 5) to the sodium hyaluronate solution from step 4) to obtain a sodium hyaluronate composite filler containing methoxypolyethylene glycol and poly-L-lactic acid nanoparticles; Step 6) In the sodium hyaluronate composite filler: The concentration of the methoxy polyethylene glycol-poly (L-lactic acid) nanoparticles is 10 mg / mL to 200 mg / mL; The sodium hyaluronate concentration is 10 mg / mL to 20 mg / mL; The average particle size of the methoxy polyethylene glycol-poly (L-lactic acid) nanoparticles is 50 nm to 200 nm; In step 1), the molecular weight of the methoxy polyethylene glycol block in the methoxy polyethylene glycol-poly-L-lactic acid polymer is 1000-5000 Daltons; the molecular weight of the poly-L-lactic acid block in the methoxy polyethylene glycol-poly-L-lactic acid polymer is 1000-10000 Daltons.

6. The preparation method according to claim 5, characterized in that The mass fraction of methoxy polyethylene glycol-poly-L-lactic acid in the oil phase in step 1) is 0-20%, excluding 0; And / or, in step 3), the freeze-drying temperature is -50°C to -40°C, and the time is 12 to 72 hours; And / or, in step 2), the concentration of the methoxypolyethylene glycol-poly-L-lactic acid nanoparticle solution is 1 mg / mL to 100 mg / mL.

7. The preparation method according to claim 5, characterized in that Step 6) In the sodium hyaluronate composite filler: the molecular weight of the sodium hyaluronate is 2×10 5 ~2.5×10 6 Dalton.

8. The preparation method according to claim 5, characterized in that The organic solvent is selected from at least one of ethanol, methanol, acetonitrile, chloroform or dichloromethane.

9. Use of the sodium hyaluronate composite filler according to any one of claims 1 to 4 in tissue repair materials and / or filling biomaterials.

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