Injectable polylactic acid microspheres with surface-modified collagen, their preparation method and application

Abstract

This invention provides injectable polylactic acid (PLA) microspheres with a surface-modified collagen core-shell structure. The core is a PLA microsphere with a surface-modified polylysine, and the shell is recombinant type III human collagen modified on the surface of the PLA microsphere. The recombinant type III human collagen is modified on the surface of the PLA microsphere via a PEG-modified type II lysyl oxidase (LOXL2) catalytic reaction. This invention overcomes the problems of insufficient bioactivity and poor filling effect of injectable PLA microspheres in the medical aesthetics field. It proposes an injectable PLA microsphere with a surface-modified collagen core, first introducing amino modification through electrostatic interaction, and then introducing a PEG-modified type II lysyl oxidase (LOXL2) catalytic process to modify the surface of the PLA microsphere with bioactive collagen. Compared with conventional PLA microspheres, the cell adhesion and proliferation activities of the surface-modified collagen PLA microspheres of this invention are significantly improved.

Description

Technical Field

[0001] This invention belongs to the field of medical aesthetics technology, specifically relating to an injectable polylactic acid microsphere with surface-modified collagen, its preparation method, and its application. Background Technology

[0002] With economic and technological development, people, especially women, have an increasing desire to purchase facial anti-aging products, leading to extensive research and development of facial filler injection materials in recent years. Currently, polylactic acid (PLA) microspheres are highly favored due to their safety and good filling effect. PLA is a biodegradable polymer material approved for use in the human body, exhibiting excellent biocompatibility. After injection, PLA degrades into lactic acid in the body and is eventually metabolized and excreted. However, PLA microspheres do have some drawbacks, including poor biocompatibility and less effective filling.

[0003] Collagen is a crucial component of the human extracellular matrix (ECM). Collagen possesses definite biological activity mediating cell adhesion, and specific amino acid sequences within the collagen sequence, such as the RGD (arginine-glycine-aspartic acid) sequence, are considered typical cell recognition sites. RGD is recognized by integrins, a class of transmembrane receptors that play a key role in cell-matrix interactions. Integrins bind to the RGD motif through their extracellular ligand-binding domains, leading to conformational changes, transmitting signals into the cell, triggering intracellular signaling pathways, and regulating cellular behavior. Polylactic acid microspheres modified with collagen provide a biomimetic interface similar to the human extracellular matrix, improving the microspheres' biocompatibility. In the field of cosmetic fillers, the formation of biomimetic interfaces is an important technical means to promote the repair and orderly growth of human tissue on the surface of cosmetic fillers.

[0004] There are some existing reports on injectable products for medical aesthetics that combine polylactic acid microspheres and collagen:

[0005] CN116271226A discloses a sterile medical collagen product and its preparation method containing polylactic acid (PLA) microspheres. The product includes sterile PLA microspheres and recombinant human type III collagen. The sterile PLA microspheres, after being sterilized by irradiation, are mixed with a gel containing recombinant human type III collagen obtained after irradiation sterilization, and then filled into a syringe sterilized with ethylene oxide to form a sterile medical aesthetic product. CN118059307A discloses a method for preparing a polylactic acid composite recombinant collagen gel, comprising the following steps: preparing PLA microparticles; preparing an alkaline solution of recombinant collagen; cross-linking the recombinant collagen gel; sterilizing and mixing the PLA microparticles and recombinant collagen gel; adding sodium hyaluronate, phosphate buffer, and lidocaine hydrochloride and stirring; sterilizing the syringe with ethylene oxide; and encapsulating the mixture in the syringe to obtain the polylactic acid composite recombinant collagen gel product. CN117085178A discloses an injectable facial filler composition for cosmetic surgery, comprising the following components: hyaluronic acid-polylactic acid gel, collagen, anesthetic, and an aqueous solution for injection. The preparation method of the facial filler composition specifically includes the following steps: dissolving hyaluronic acid-polylactic acid in an aqueous solution for injection, allowing it to stand and swell, adding the anesthetic, stirring, sterilizing, adding collagen, and packaging. However, collagen and polylactic acid microspheres are only physically mixed, and the chemical and biological properties of the two materials differ significantly. The degradation, migration, and bioactivity of the microspheres cannot be precisely controlled, and the excellent properties of both materials cannot be fully utilized.

[0006] CN117618651A discloses an injectable polylactic acid (PLLA) microsphere for accelerating fibroblast adhesion and directional migration, and its preparation method. Using PLLA and polypeptides as raw materials, PLLA is reacted with succinic acid to obtain carboxyl-terminated PLLA. The carboxyl-terminated PLLA is dissolved in dichloromethane or trichloromethane to prepare a carboxyl-terminated PLLA solution. The carboxyl-terminated PLLA solution is added to an aqueous polypeptide solution and mixed. A mixed solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is then added dropwise to the mixed solution of carboxyl-terminated PLLA and polypeptides. The mixture is stirred, centrifuged, and the precipitate is washed with deionized water and freeze-dried to obtain PLLA / polypeptide microspheres with a uniformly coated polypeptide surface. Finally, sodium carboxymethyl cellulose, mannitol, and PLLA / polypeptide microspheres are dispersed uniformly in water and freeze-dried to obtain injectable PLLA / polypeptide composite microsphere powder. The peptides are selected from bovine collagen, gelatin, recombinant human type III collagen, and fibronectin. This patent uses a chemical crosslinking method to prepare polylactic acid / peptide microspheres, which has poorer biocompatibility than enzymatic methods. The article "EDC / NHS crosslinking in tissue engineering: A review of toxicity and residual removal strategies" details the residual cytotoxicity and difficulty in removing EDC / NHS when used as a chemical crosslinking agent. Summary of the Invention

[0007] To overcome the problems of insufficient bioactivity and unsatisfactory filling effect of injectable polylactic acid (PLA) microspheres in the field of traditional Chinese medicine and aesthetics, this invention proposes an injectable PLA microsphere with surface-modified collagen, its preparation method, and its application. This invention first introduces amino modification through electrostatic interaction, and then introduces a type II lysine oxidase (LOXL2) catalytic process to modify the surface of the PLA microspheres with bioactive collagen.

[0008] The present invention solves the above-mentioned technical problems through the following technical solutions:

[0009] An injectable polylactic acid microsphere with surface-modified collagen has a core-shell structure. The core is a polylactic acid microsphere with surface-modified polylysine, and the shell is recombinant type III human collagen modified on the surface of the polylactic acid microsphere. The recombinant type III human collagen is modified on the surface of the polylactic acid microsphere by a PEG-modified type II lysyl oxidase catalytic reaction.

[0010] Furthermore, the recombinant type III human collagen is produced through in-house fermentation, and its sequence is shown in SEQ ID No. 1:

[0011] GDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGE RGSPGPGAAGFPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPLGIAGITGARGLAGPPGMPGPRGSPGPQGVKGESGKPG ANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIK GHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPCCGGVGAAAIAAGIGGEKAGGFAPYYGHHHHHH.

[0012] The collagen mentioned above has a large number of free lysine residues, which can participate in LOX2-mediated enzymatic reactions, and also has good cell proliferation, cell adsorption effect and low immunogenicity.

[0013] Furthermore, the polylactic acid microspheres have an average particle size of 40-60 μm and a weight-average molecular weight of 15,000-30,000 g / mol; the polylysine has a weight molecular weight of 20,000-50,000 Da, preferably 30,000-40,000 Da.

[0014] Furthermore, the PEG-modified type II lysyl oxidase is obtained by the coupling reaction of type II lysyl oxidase (LOXL2) and polyethylene glycol methyl ether activated ester (mPEG-NHS ester), with a weight-average molecular weight of 5500-7000 Da. The molecular weight of the PEG-NHS activated ester has a significant impact; if the PEG molecular weight is too low, it cannot achieve the goal of increasing the molecular weight of LOXL2 and neutralizing the surface charge of the enzyme molecule; if the PEG molecular weight is too high, it will reduce the catalytic activity of LOXL2. Therefore, it is necessary to control the appropriate length and molecular weight of the PEG segment.

[0015] Furthermore, the zeta potential of the surface-modified collagen-injectable polylactic acid microspheres is between -30 mV and 30 mV; and / or the infrared spectrum of the surface-modified collagen-injectable polylactic acid microspheres is at 1650 ± 10 cm⁻¹. -1 There are characteristic peaks nearby.

[0016] This invention employs an enzymatic catalytic method to modify recombinant type III human collagen on the surface of polylactic acid, avoiding the introduction of risky impurities and reducing the residue of hazardous substances compared to chemical condensation methods, such as EDC condensation. Furthermore, this invention uses independently developed and produced recombinant type III human collagen, exhibiting excellent cell proliferation and adsorption effects, as well as low immunogenicity. Simultaneously, this invention uses PEG to modify the LOXL2 enzyme, increasing its molecular weight and neutralizing the surface charge of the enzyme molecules, inhibiting the self-crosslinking side reaction between polylysine molecules, and improving the collagen loading efficiency. Using the enzymatic catalytic method of this invention and the independently developed collagen results in higher crosslinking efficiency, more uniform collagen coverage, and better bioactivity, leading to superior filling effects in cosmetic filler injections.

[0017] The present invention also provides a method for preparing the above-mentioned surface-modified collagen injectable polylactic acid microspheres, comprising the following steps:

[0018] (S1) After hydrophilic treatment, polylactic acid microspheres were surface-modified with polylysine via electrostatic interaction to obtain polylactic acid microspheres with polylysine modified surface.

[0019] (S2) The coupling reaction of type II lysine oxidase (LOXL2) and polyethylene glycol methyl ether activated ester (mPEG-NHS ester) yielded the coupling product;

[0020] (S3) Polylactic acid microspheres with polylysine surface modification and collagen were dissolved in buffer solution as substrates for enzymatic reaction and incubated for later use;

[0021] (S4) Add the coupling product obtained in step (S2) to the enzyme-catalyzed reaction substrate, incubate to form precipitated insoluble microspheres (PLLA-COL), freeze dry to obtain injectable polylactic acid microspheres with collagen modified on the product surface.

[0022] Further, in step (S1), the average particle size of the polylactic acid (PLA) microspheres is 40-60 μm, and the weight-average molecular weight of PLA is 15,000-30,000 g / mol. PLA microspheres can be commercially available or prepared in-house, for example, using the emulsion method well-known in the art. Specifically, PLA is dissolved in dichloromethane and added to an aqueous solution containing polyvinyl alcohol. An emulsion is formed under stirring conditions, treated with an ultrasonic homogenizer, and then stirred continuously. The residual solvent is evaporated, the microspheres are separated by centrifugation, washed, and the resulting PLA microspheres are stored at 4±2 °C.

[0023] Further, in step (S1), the polylactic acid microspheres undergo hydrophilic treatment by immersing polylactic acid in 0.1-0.5M NaOH and / or KOH alkaline solution for 10-40 minutes, for example, 20-30 minutes. After immersion, the microspheres are washed with water until the washing solution is neutral. The surface is then modified with polylysine through electrostatic interaction. Specifically, under light-protected conditions, the hydrophilic-treated polylactic acid microspheres are immersed in a polylysine (PL) solution and placed at 0-10℃ for 10-30 hours. After immersion, the microspheres are removed, washed, and dried.

[0024] Furthermore, in the polylysine solution, the polylysine has a molecular weight of 20,000-50,000 Da, preferably 30,000-40,000 Da, the polylysine solution concentration is 0.1-1 wt%, preferably 0.25-0.5 wt%, and the mass-to-volume ratio of polylactic acid microspheres to polylysine solution is 1 g: 3-10 mL.

[0025] Further, in step (S2), the molar ratio of type II lysyl oxidase to polyethylene glycol methyl ether activated lipid is 1:3-10, for example, 1:4-6, preferably 1:5; the concentration of type II lysyl oxidase is 1-5 mg / mL, and the molecular weight of polyethylene glycol methyl ether activated lipid is 5500-7000 Da; the coupling reaction conditions are shaking at 2-5℃ for 1-2 hours, for example, shaking at 4℃ for 1 hour. After the reaction, small molecule impurities are removed by ultrafiltration concentration. The PEG treatment of LOXL2 in this invention increases the molecular weight of LOXL2 and neutralizes the surface charge of the enzyme molecules, inhibiting the self-crosslinking side reaction between polylysine molecules and improving the collagen loading efficiency.

[0026] Further, in step (S3), the pH of the buffer solution is 7.5-9, preferably 8-8.5, such as Na2B4O7 buffer; the concentration of polylactic acid microspheres modified with polylysine is 10-100 mg / mL, preferably 30-50 mg / mL; the concentration of recombinant type III human collagen is 5-20 mg / mL, preferably 7-10 mg / mL; and the mass ratio of polylactic acid microspheres to recombinant type III human collagen is 4-6:1, such as 5:1; the incubation is carried out at 40-50°C for 10-30 min.

[0027] Further, in step (S4), the coupling product is added at a mass ratio of 1:300-700 to the substrate, preferably 1:400-500 to the LOXL2 enzyme; the incubation is carried out at 40-60°C for 20-30 hours, for example, at 50°C for 24 hours. Attached Figure Description

[0028] Figure 1 Affinity chromatography and gel electrophoresis diagrams for the preparation of recombinant type III human collagen in Example 3.

[0029] Figure 2 The cationic chromatography and gel electrophoresis diagrams for the preparation of recombinant type III human collagen in Example 3 are shown.

[0030] Figure 3 Scanning electron microscope images of polylactic acid microspheres (a) and collagen-modified polylactic acid microspheres (b).

[0031] Figure 4 Zeta potential diagrams of polylactic acid microspheres (a), polylysine-adsorbed polylactic acid microspheres (b), and collagen-modified polylactic acid microspheres (c).

[0032] Figure 5 Infrared spectrum of collagen-modified polylactic acid microspheres.

[0033] Figure 6 Cellular adsorption diagrams of polylactic acid microspheres (B) and collagen-modified polylactic acid microspheres (A).

[0034] Figure 7 Cell viability diagrams of polylactic acid microspheres and collagen-modified polylactic acid microspheres. Detailed Implementation

[0035] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments.

[0036] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0037] Preparation Example 1: Preparation of Polylactic Acid Microspheres

[0038] Polylactic acid (PLLA) (Mw = 15000 g / mol), polyvinyl alcohol (PVA) (Mw = 160000 g / mol), and dichloromethane (DCM) were all of analytical grade.

[0039] (1) Dissolve 1 g PLLA in DCM (10 mL) and stir for about 1 hour to prepare the organic phase. Dissolve 0.4 g PVA in water (20 mL) to prepare the aqueous phase; dissolve by stirring the mixture at 90 °C for 2 hours using a magnetic stirrer.

[0040] (2) Slowly add 1 volume of the oil phase containing PLLA to 5 volume of the aqueous phase containing PVA and continuously mix using a vortex mixer at room temperature. Due to the high-speed vortex mixing, the emulsion begins to form. Continue vortex mixing for 10 minutes.

[0041] (3) In order to reduce the size of PLLA particles, the emulsion was immediately transferred to an ultrasonic homogenizer (Ningbo Xinzhi), and then the emulsion was stirred at room temperature for 3 hours using a magnetic stirrer to allow for self-assembly and evaporation of residual organic solvents.

[0042] (4) The microspheres were then separated by centrifugation (Eppendorf Centrifuge 5810R-6000rpm for 15 minutes) and decantation, followed by washing three times with distilled water to remove PVA, yielding PLLA microspheres with a particle size range of 20-100μm and an average particle size of 43.2μm. The final product, PLLA microspheres, was stored in an aqueous solution at 4°C in a refrigerator.

[0043] Preparation Example 2: Polylysine Modification of Polylactic Acid Microspheres

[0044] (1) Hydrophilic treatment of PLLA microspheres: PLLA microspheres were immersed in 0.1M NaOH solution for 30 minutes at room temperature, and then thoroughly washed 5 times with distilled water to remove the remaining NaOH, thus obtaining hydrophilic treated PLLA microspheres.

[0045] (2) 10g of freshly prepared hydrophilically treated PLLA microspheres were immersed in 100mL of 0.25wt% polylysine (PL) solution and allowed to stand at 4℃ for 12h. The microspheres were then rinsed in distilled water to remove excess solution. During this impregnation process, polylysine (PL) was stably loaded onto the surface of the PLLA microspheres, resulting in polylysine-modified polylactic acid microspheres (PLLA-PL). The entire preparation process was carried out under light-protected conditions.

[0046] Preparation Example 3: Preparation of Recombinant Type III Human Collagen

[0047] (1) Using Pichia pastoris, which has high-density fermentation characteristics, as the chassis strain, recombinant type III human collagen was expressed exogenously using gene recombination technology to prepare a collagen-containing fermentation broth.

[0048] (2) The fermentation broth was centrifuged, and the supernatant after centrifugation was taken and subjected to affinity chromatography and ion exchange chromatography to prepare high-purity recombinant type III human collagen.

[0049] The protein sequence of the independently developed and produced recombinant type III human collagen is shown in SEQ ID No. 1. The lysine residues at the end of this sequence can participate in LOX2-mediated biocrosslinking, while the histidine residues at the end of the sequence facilitate the separation and purification of collagen. Gel electrophoresis analysis of the collagen after affinity chromatography and ion exchange chromatography treatment is shown below. Figure 1 and Figure 2 This proves the accuracy of the purification process and demonstrates that high-purity recombinant type III human collagen can be obtained.

[0050] SEQ ID No. 1:

[0051] GDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGE RGSPGPGAAGFPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPLGIAGITGARGLAGPPGMPGPRGSPGPQGVKGESGKPG ANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIK GHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPCCGGVGAAAIAAGIGGEKAGGFAPYYGHHHHHH.

[0052] Example 1

[0053] (1) PEG treatment of LOXL2: Type II lysyl oxidase LOXL2 (Genway BIO) 3 mg / ml was mixed with mPEG-NHS ester (Mw = 5500 Da) at a molar ratio of 1:5. The coupling reaction was completed by shaking at 4℃ for 1 h to obtain the coupling product. The product was washed and concentrated by a 30,000 molecular weight ultrafiltration centrifuge tube to remove small molecule impurities.

[0054] (2) Dissolve PLLA-PL microspheres (50 mg / ml) and collagen (10 mg / ml) in 50 mM Na2B4O7 buffer solution at pH 8.0 and incubate at 50 °C for 15 minutes as substrates for the enzymatic reaction.

[0055] (3) Then, the coupling product was added at a ratio of 1:500 (w / w) of coupling product to substrate, and the mixture was incubated at 50°C for 24 hours. Finally, the precipitated insoluble microspheres (PLLA-COL) were washed with water and ethanol solution (1:1, v / v), and the finished collagen-modified polylactic acid microspheres were obtained by freeze drying.

[0056] Scanning electron microscopy observations as follows Figure 3 After modification with collagen, the particle size of polylactic acid microspheres did not change significantly, but the surface became more porous and rough.

[0057] Zeta potential analysis, as follows Figure 4 Original polylactic acid microspheres ( Figure 4 a) exhibits electronegativity and a relatively narrow charge distribution, demonstrating the consistency of polylactic acid (PLLA) microsphere preparation. PLLA microspheres modified with polylysine ( Figure 4 (b) The presence of significantly positively charged particles was observed, indicating that the polylactic acid microspheres were successfully loaded with polylysine short peptides via electrostatic interactions. The main peak on the left shows that some polylactic acid microspheres were not coated, consistent with unmodified polylactic acid microspheres. The newly added secondary peak shifted towards 0, indicating that some microspheres were successfully loaded with polylysine, neutralizing the electronegativity of polylactic acid. Figure 4 As can be seen from c, after the microspheres are mediated by LOXL2 and neutral charges are introduced to reorganize collagen on the surface, the ions are nearly neutral and the charge distribution is relatively broad, which is related to the charge diversity of the side chains of collagen.

[0058] Infrared spectroscopy analysis, such as Figure 5 From top to bottom, they are polylactic acid microspheres, polylysine-adsorbed polylactic acid microspheres, and collagen-modified polylactic acid microspheres, at 1750 cm⁻¹. -1 This is the strongest and most characteristic peak of PLA, generated by the stretching vibration of the ester carbonyl group (C=O). After modification with polylysine, polylactic acid microspheres exhibit this peak at 1553 cm⁻¹. -1 The presence of a characteristic peak at 1651 cm⁻¹ indicates the NH bending vibration (δNH) of the primary amine group (-NH) in the lysine molecule, confirming the successful electrostatic adsorption modification of polylysine onto the PLA microsphere surface. Collagen-modified polylactic acid microspheres showed a peak at 1651 cm⁻¹. -1 and 1548cm -1 The presence of characteristic peaks, namely the amide I band and the amide II band, indicates that collagen has been successfully modified onto the surface of PLA microspheres.

[0059] Example 2

[0060] The other conditions are the same as in Example 1, except that the mass ratio of the modified LOXL2 enzyme to the substrate in step (3) is 1:400.

[0061] Example 3

[0062] The other conditions are the same as in Example 1, except that the mass ratio of the modified LOXL2 enzyme to the substrate in step (3) is 1:600.

[0063] Example 4

[0064] The other conditions are the same as in Example 1, except that the mass ratio of the modified LOXL2 enzyme to the substrate in step (3) is 1:700.

[0065] Example 5

[0066] The other conditions are the same as in Example 1, except that in step (1), the molecular weight of mPEG-NHS ester is Mw = 7000 Da.

[0067] Example 6

[0068] The other conditions are the same as in Example 1, except that in step (1), the molecular weight of mPEG-NHS ester is Mw = 4000 Da.

[0069] Example 7

[0070] The other conditions are the same as in Example 1, except that in step (1), the molecular weight of mPEG-NHS ester is Mw = 9000 Da.

[0071] Comparative Example 1

[0072] Polylactic acid microspheres were used directly without any modification.

[0073] Comparative Example 2

[0074] The other conditions are the same as in Example 1, except that steps 1) and 3) are omitted, and type II lysine oxidase (LOXL2) is directly used instead of the coupling product for feeding, and the feeding is carried out according to the same LOXL2:substrate mass ratio.

[0075] Application examples

[0076] Evaluation of Cell Adhesion and Proliferation Activity of Collagen-Modified Polylactic Acid Microspheres: Using mouse L929 fibroblasts as the research subject, the MTT assay was used to evaluate the effect of collagen modification on the cell adhesion and proliferation behavior of polylactic acid microspheres. To eliminate interference from cell adhesion on the culture plate surface, cell growth experiments on the microsphere surface were performed using Corning Costa low-adsorption plates. Only when cells normally adhered to the polylactic acid microsphere surface and proliferated could the characteristic blue-purple MTT crystals of living cells be formed and detected.

[0077] Each PLLA-COL microsphere (0.5 mg) was coated with serum-free medium in a 37°C, 5% CO2 incubator, with continuous rotation at 15 rpm for 1.5 h during the coating process. Immediately after treatment, the microspheres were washed three times with double-distilled water and a double-antibiotic solution (containing 200 U / mL penicillin and 200 μg / mL streptomycin), centrifuged, freeze-dried, and stored at 4°C. Activity validation tests were performed using low-adsorption plates.

[0078] Specific procedures for activity verification tests:

[0079] 1) Add 150 μl of 10% BSA in DMEM to each well as the basic culture system.

[0080] 2) Add 20 μl of polylactic acid microsphere (blank polylactic acid microsphere and collagen-modified microsphere) suspension to each well.

[0081] 3) Culture and seeding conditions: 40,000 L929 cells per well (diluted with 50 μl DMEM), incubate for 1 h, then add 20 μl MTT solution and incubate overnight at 37°C.

[0082] 4) On the 14th hour, take the culture plate and read the absorbance at 490 nm. Before reading, add 150 μl of DMSO and shake for 10 min.

[0083] like Figure 6 As shown in Figure A, after collagen modification, fibroblasts adhered well to the surface of polylactic acid microspheres and grew, forming a dense cell layer. In the control group (B), fibroblasts could still adhere to the hydrophobic polylactic acid surface and form aggregates, but the cell load per unit surface of microsphere was significantly lower than that in the collagen-modified experimental group, indicating that the cell adhesion and proliferation activity of polylactic acid microspheres was significantly improved after collagen modification.

[0084] After collagen modification (A), fibroblasts adhered well to the surface of polylactic acid microspheres and formed a dense cell layer. In the control group (B, comparative example 1), fibroblasts could still adhere to the hydrophobic polylactic acid surface and form aggregates, but the cell load per unit surface of microsphere was significantly lower than that in the collagen-modified experimental group, showing that the cell adhesion and proliferation activity of polylactic acid microspheres was significantly improved after collagen modification.

[0085] The collagen-modified polylactic acid microspheres obtained in the above examples and Comparative Example 1 were subjected to cell proliferation activity tests according to the methods described in the application examples above. Relative cell viability was expressed as the ratio of the cell number at the start of the experiment (hour 0) to the cell number at hour 0 (40,000 cells per well), after culture, to the cell number at hour 0. The results are as follows: Figure 7 As shown.

[0086] The microspheres prepared in the examples and comparative examples were cultured with cells. After 14 hours of culture, the cell viability was measured. The specific data are shown in Table 1.

[0087] Table 1 Relative cell viability

[0088]

[0089]

Claims

1. An injectable polylactic acid microsphere with a surface-modified collagen, characterized in that, It has a core-shell structure, with the core being polylactic acid microspheres with polylysine modified on the surface, and the shell being recombinant type III human collagen modified on the surface of the polylactic acid microspheres. The recombinant type III human collagen is modified on the surface of the polylactic acid microspheres by a PEG-modified type II lysine oxidase catalytic reaction; the PEG-modified type II lysine oxidase is obtained by a coupling reaction of type II lysine oxidase and polyethylene glycol methyl ether activated lipid.

2. The injectable polylactic acid microspheres with surface-modified collagen according to claim 1, characterized in that, The recombinant type III human collagen sequence is shown in SEQ ID No.

1.

3. The injectable polylactic acid microspheres with surface-modified collagen according to claim 1, characterized in that, The polylactic acid microspheres have an average particle size of 40-60 μm and a weight-average molecular weight of 15,000-30,000 g / mol; the polylysine has a weight molecular weight of 20,000-50,000 Da.

4. The injectable polylactic acid microspheres with surface-modified collagen according to claim 3, characterized in that, The heavy molecular weight of polylysine is 30,000-40,000 Da.

5. The injectable polylactic acid microspheres with surface-modified collagen according to claim 1, characterized in that, The weight-average molecular weight of polyethylene glycol methyl ether activated lipids is 5500-7000 Da.

6. The injectable polylactic acid microspheres with surface-modified collagen according to claim 1, characterized in that, The zeta potential of the surface-modified collagen-injectable polylactic acid microspheres is in the range of -30 mV to 30 mV; and / or the infrared spectrum of the surface-modified collagen-injectable polylactic acid microspheres is at 1650 ± 10 cm⁻¹. -1 There are characteristic peaks nearby.

7. The method for preparing surface-modified collagen-injectable polylactic acid microspheres according to any one of claims 1-6, characterized in that, Includes the following steps: (S1) After hydrophilic treatment, polylactic acid microspheres were surface-modified with polylysine via electrostatic interaction to obtain polylactic acid microspheres with polylysine modified surface. (S2) The coupling product was obtained by the coupling reaction of type II lysine oxidase and polyethylene glycol methyl ether activated lipid; (S3) Polylactic acid microspheres with polylysine modified on the surface and collagen were dissolved in buffer solution as substrates for enzymatic reaction and incubated for later use; (S4) Add the coupling product obtained in step (S2) to the enzyme-catalyzed reaction substrate, incubate to form precipitated insoluble microspheres, freeze-dry to obtain injectable polylactic acid microspheres with collagen modified on the product surface.

8. The preparation method according to claim 7, characterized in that, In step (S1), the average particle size of polylactic acid microspheres is 40-60 μm, and the weight-average molecular weight of polylactic acid is 15000-30000 g / mol.

9. The preparation method according to claim 8, characterized in that, In step (S1), the polylactic acid microspheres are hydrophilically treated by immersing polylactic acid in 0.1-0.5M NaOH and / or KOH alkaline solution for 10-40 minutes, followed by washing with water until the washing solution is neutral; the surface is then modified with polylysine through electrostatic interaction, specifically by immersing the hydrophilically treated polylactic acid microspheres in a polylysine (PL) solution under light-protected conditions for 10-30 hours at 0-10℃, followed by removal, washing, and drying.

10. The preparation method according to claim 9, characterized in that, In the polylysine solution, the molecular weight of polylysine is 20,000-50,000 Da, the concentration of polylysine solution is 0.1-1 wt%, and the mass-to-volume ratio of polylactic acid microspheres to polylysine solution is 1 g: 3-10 mL.

11. The preparation method according to claim 7, characterized in that, In step (S2), the molar ratio of type II lysyl oxidase to polyethylene glycol methyl ether activated lipid is 1:3-10; the concentration of type II lysyl oxidase is 1-5 mg / mL, and the molecular weight of polyethylene glycol methyl ether activated lipid is 5500-7000 Da; the coupling reaction conditions are shaking at 2-5℃ for 1-2 hours.

12. The preparation method according to claim 11, characterized in that, The molar ratio of type II lysyl oxidase to polyethylene glycol methyl ether activated lipid is 1:4-6.

13. The preparation method according to claim 11, characterized in that, The molar ratio of type II lysyl oxidase to polyethylene glycol methyl ether activated lipid is 1:

5.

14. The preparation method according to claim 7, characterized in that, In step (S3), the buffer solution pH is 7.5-9; the concentration of polylactic acid microspheres modified with polylysine is 10-100 mg / mL; the concentration of recombinant type III human collagen is 5-20 mg / mL; and the mass ratio of polylactic acid microspheres to recombinant type III human collagen is 4-6:1; incubation is carried out at 40-50℃ for 10-30 min. and / or In step (S4), the coupling product is added at a mass ratio of 1:300-700 to the substrate, and the mass ratio of type II lysine oxidase to substrate is 1:400-500; incubation is carried out at 40-60°C for 20-30 hours.

15. The preparation method according to claim 14, characterized in that, In step (S3), the buffer solution is Na2B4O7 buffer solution.

16. The preparation method according to claim 14, characterized in that, In step (S3), the concentration of recombinant type III human collagen is 7-10 mg / mL.

17. A skin filler for medical aesthetics, comprising injectable polylactic acid microspheres with surface-modified collagen as described in any one of claims 1-6.

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