Injectable polylactic acid microspheres with collagen modified surfaces as well as preparation method and application of injectable polylactic acid microspheres

Abstract

The invention provides an injectable polylactic acid microsphere with collagen modified on the surface, the injectable polylactic acid microsphere is of a core-shell structure, the core is a polylactic acid microsphere with polylysine modified on the surface, the shell is recombinant III-type human collagen modified on the surface of the polylactic acid microsphere, and the recombinant III-type human collagen is modified on the surface of the polylactic acid microsphere through a catalytic reaction of PEG modified II-type lysyl oxidase. In order to overcome the problems of insufficient biological activity and poor filling effect of polylactic acid microspheres for injection in the field of medical beauty, the invention provides injectable polylactic acid microspheres with collagen modified on the surface, amino modification is introduced through electrostatic interaction, then PEG modified type II lysyl oxidase (LOXL2) is introduced for catalytic process, and the collagen modified type II lysyl oxidase (LOXL2) is introduced to prepare the injectable polylactic acid microspheres with collagen modified on the surface. And modifying collagen with biological activity on the surfaces of the polylactic acid microspheres. Compared with conventional polylactic acid microspheres, the cell adhesion and proliferation activity of the polylactic acid microspheres of which the surfaces are modified with collagen are remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the field of medical cosmetology technology, and specifically relates to injectable polylactic acid microspheres with surface modified collagen, and a preparation method and application thereof. Background Art

[0002] With the development of the economy and technology, people, especially women, are increasingly interested in anti-aging facial products. Injectable facial filler materials have been extensively researched and developed in recent years. Currently, polylactic acid (PLA) microspheres are highly favored due to their safety and excellent filling effects. PLA is a biodegradable polymer currently approved for use in the human body and exhibits excellent biocompatibility. After injection, PLA degrades into lactic acid in the body and is ultimately metabolized and excreted. However, PLA microspheres have some drawbacks, including poor biocompatibility and poor filling effects.

[0003] Collagen is an important component of the human extracellular matrix (ECM). Collagen has a definite biological activity of mediating cell adhesion, and specific amino acid sequences in the collagen sequence, such as the RGD (arginine-glycine-aspartic acid) sequence, are considered to be typical cell recognition sites. RGD is recognized by integrins, which are 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, causing conformational changes, transmitting signals into cells, triggering intracellular signaling pathways and regulating cell behavior. Polylactic acid microspheres are surface-modified with collagen, providing a biomimetic interface similar to the human extracellular matrix, improving the biocompatibility of the microspheres. In the field of medical cosmetic filling, the formation of a biomimetic interface is an important technical means to promote the repair and orderly growth of the body's own tissues on the surface of medical cosmetic fillers.

[0004] There are some reports on medical aesthetic injection products made of polylactic acid microspheres and collagen:

[0005] CN116271226A discloses a medical sterile collagen product loaded with poly(L-lactic acid) microspheres and a preparation method thereof. The product comprises sterile poly(L-lactic acid) microspheres and recombinant human type III collagen. The sterile poly(L-lactic acid) microspheres sterilized by irradiation are mixed with a gel containing recombinant human type III collagen obtained after irradiation sterilization, and then filled into an ethylene oxide sterilized syringe to form a sterile medical aesthetic product. CN118059307A discloses a method for preparing a poly(L-lactic acid) composite recombinant collagen gel, comprising the following steps: preparing poly(L-lactic acid) microparticles; preparing an alkaline solution of recombinant collagen; cross-linking the recombinant collagen gel; sterilizing and mixing the poly(L-lactic acid) microparticles and recombinant collagen gel; adding sodium hyaluronate, phosphate buffer, and lidocaine hydrochloride and stirring the mixture; sterilizing the syringe with ethylene oxide, and encapsulating the mixture in the syringe to obtain the poly(L-lactic 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, an anesthetic, and an injectable aqueous solution. The preparation method comprises the following steps: dissolving hyaluronic acid-polylactic acid in an injectable aqueous solution, allowing the solution to swell, adding the anesthetic, stirring, sterilizing, adding the collagen, and packaging. However, since the collagen and polylactic acid microspheres are only physically mixed, the chemical and biological properties of the two materials differ significantly, making it difficult to precisely control the degradation, migration, and bioactivity of the microspheres, and thus failing to fully utilize the excellent properties of both materials.

[0006] CN117618651A discloses injectable L-polylactic acid microspheres for accelerating fibroblast adhesion and directional migration, and a preparation method thereof. The method comprises the following steps: using L-polylactic acid and a polypeptide as raw materials, reacting the L-polylactic acid with succinic acid to obtain carboxyl-terminated PLLA; dissolving the carboxyl-terminated PLLA in dichloromethane or chloroform to prepare a carboxyl-terminated PLLA solution; adding the carboxyl-terminated PLLA solution to an aqueous polypeptide solution and mixing; then dropping a mixed solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide into the mixed solution of the carboxyl-terminated PLLA and polypeptide, stirring for reaction, centrifuging, washing the precipitate with deionized water, and freeze-drying to obtain PLLA / polypeptide microspheres with a uniform polypeptide coating on the surface; and finally, uniformly dispersing sodium carboxymethylcellulose, mannitol, and the PLLA / polypeptide microspheres in water and freeze-drying to obtain an injectable PLLA / polygel composite microsphere powder. The polypeptide is selected from bovine collagen, gelatin, recombinant human type III collagen, and fibronectin. This patent uses a chemical crosslinking method to prepare polylactic acid / polypeptide 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 removal of EDC / NHS when used as a chemical crosslinking agent. Summary of the Invention

[0007] To overcome the existing problems of insufficient bioactivity and poor filling effects of injectable polylactic acid microspheres in the field of traditional Chinese medicine aesthetics, the present invention provides injectable polylactic acid microspheres with surface-modified collagen, as well as their preparation method and application. The invention first introduces amino modifications through electrostatic interaction, and then introduces a type II lysyl oxidase (LOXL2) catalytic process to modify the surface of the polylactic acid microspheres with bioactive collagen.

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

[0009] The invention relates to an injectable polylactic acid microsphere with surface modified collagen, which 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 through a PEG-modified type II lysyl oxidase catalytic reaction.

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

[0011] GDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGE RGSPGPGAAGFPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPLGIAGITGARGLAGPPGMPGPRGSPGPQGVKGESGKPG ANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIK GHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPCCGGVGAAAIAAGIGGEKAGGFAPYYGHHHHHH.

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

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

[0014] Furthermore, PEG-modified type II lysyl oxidase is obtained by coupling type II lysyl oxidase (LOXL2) with a polyethylene glycol monomethyl ether activated lipid (mPEG-NHS ester). The weight-average molecular weight of the mPEG-NHS ester is 5500-7000 Da. The molecular weight of the mPEG-NHS ester plays a significant role. A PEG molecular weight that is too low will not increase the molecular weight of LOXL2 and neutralize the surface charge of the enzyme; a PEG molecular weight that is too high will reduce the catalytic activity of LOXL2. Therefore, it is important to control the length and molecular weight of the PEG segment appropriately.

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

[0016] The present invention uses an enzyme-catalyzed method to modify recombinant human type III collagen on the surface of polylactic acid, which does not introduce risky impurities. Compared with chemical condensation, such as the EDC condensation method, it reduces the residue of risky substances. In addition, the present invention uses recombinant human type III collagen independently developed and produced, which has good cell proliferation, cell adsorption effects and low immunogenicity. At the same time, the present invention uses PEG to modify the LOXL2 enzyme, thereby increasing the molecular weight of LOXL2 and neutralizing the surface charge of the enzyme molecule, inhibiting the self-crosslinking side reaction between polylysine molecules, and improving the loading efficiency of collagen. The use of the enzyme-catalyzed method of the present invention and the independently developed collagen has higher cross-linking efficiency, more uniform collagen coverage, better biological activity, and better filling effect for medical beauty filling 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) The hydrophilic treated polylactic acid microspheres are surface-modified with polylysine by electrostatic interaction to obtain polylactic acid microspheres with surface-modified polylysine.

[0019] (S2) Coupling reaction of type II lysyl oxidase (LOXL2) and polyethylene glycol methyl ether activated lipid (mPEG-NHS ester) to obtain the coupling product;

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

[0021] (S4) adding the coupling product obtained in step (S2) to the enzymatic reaction substrate, incubating to form precipitated insoluble microspheres (PLLA-COL), and freeze-drying to obtain injectable polylactic acid microspheres with collagen modified on the surface.

[0022] Furthermore, in step (S1), the average particle size of the polylactic acid microspheres is 40-60 μm, and the weight-average molecular weight of the polylactic acid is 15,000-30,000 g / mol. The polylactic acid microspheres can be purchased commercially or homemade, for example, by preparing the polylactic acid microspheres by an emulsion method well known in the art. Specifically, polylactic acid is dissolved in dichloromethane, added to an aqueous solution containing polyvinyl alcohol, and an emulsion is formed under stirring. After treatment with an ultrasonic homogenizer, stirring is continued, the residual solvent is evaporated, the microspheres are centrifuged, washed, and the obtained polylactic acid microspheres are stored at 4±2°C.

[0023] Furthermore, in step (S1), the polylactic acid microspheres are hydrophilically treated by soaking the polylactic acid in 0.1-0.5M NaOH and / or KOH alkaline solution for 10-40 minutes, such as 20-30 minutes, and then washing with water until the washing solution is neutral; the polylysine is surface-modified by electrostatic action, specifically, the hydrophilically treated polylactic acid microspheres are immersed in a polylysine (PL) solution under light-proof conditions, placed at 0-10°C for 10-30 hours, taken out, washed, and dried.

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

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

[0026] Furthermore, in step (S3), the pH of the buffer solution is 7.5-9, preferably 8-8.5, such as Na2B4O7 buffer; the concentration of the 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; and the incubation is at 40-50°C for 10-30 min.

[0027] Furthermore, in step (S4), the coupling product is added at a mass ratio of the coupling product to the substrate of 1:300-700, preferably the mass ratio of the LOXL2 enzyme to the substrate is 1:400-500; and the incubation is at 40-60°C for 20-30 hours, such as at 50°C for 24 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the affinity chromatography and gel electrophoresis diagram of the recombinant type III human collagen in Preparation Example 3.

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

[0030] Figure 3 Scanning electron micrographs 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 This is the infrared spectrum of collagen-modified polylactic acid microspheres.

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

[0034] Figure 7 Cell viability graphs of PLA microspheres and collagen-modified PLA microspheres. DETAILED DESCRIPTION

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

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

[0037] Preparation Example 1 Preparation of polylactic acid microspheres

[0038] Poly (L-lactic acid) (PLLA) (Mw = 15000 g / mol), poly (vinyl alcohol) (PVA) (Mw = 160000 g / mol) and dichloromethane (DCM) were all analytically pure.

[0039] (1) 1 g of PLLA was dissolved in DCM (10 mL) and stirred for about 1 hour to prepare an organic phase. 0.4 g of PVA was dissolved in water (20 mL) to prepare an aqueous phase; the mixture was stirred at 90°C for 2 hours using a magnetic stirrer to achieve dissolution.

[0040] (2) 1 part by volume of the PLLA-containing oil phase was slowly added to 5 parts by volume of the PVA-containing aqueous phase and continuously mixed using a vortex mixer at room temperature. Due to the high-speed vortex mixing, an emulsion began to form, and the vortex mixing was continued for 10 minutes;

[0041] (3) To reduce the size of PLLA particles, the emulsion was immediately transferred to an ultrasonic homogenizer (Ningbo Xinzhi), after which the emulsion was stirred at room temperature for another 3 h using a magnetic stirrer to allow self-assembly and evaporate the residual organic solvent.

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

[0043] Preparation Example 2 Polylysine modification of polylactic acid microspheres

[0044] (1) Hydrophilic treatment of PLLA microspheres: The PLLA microspheres were immersed in 0.1 M NaOH solution for 30 min at room temperature and then thoroughly washed with distilled water five times to remove the remaining NaOH to obtain hydrophilic-treated PLLA microspheres.

[0045] (2) 10 g of freshly prepared hydrophilic PLLA microspheres were immersed in 100 mL of a 0.25 wt% polylysine (PL) solution at 4°C for 12 h, and then rinsed in distilled water to remove excess solution. During the above immersion process, polylysine (PL) was stably loaded on the surface of the PLLA microspheres, resulting in polylysine-modified polylactic acid microspheres (PLLA-PL). The entire preparation process was completed under light-shielding conditions.

[0046] Preparation Example 3 Preparation of recombinant human type III collagen

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

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

[0049] The protein sequence of the recombinant type III human collagen developed and produced independently is shown in SEQ ID No. 1. The lysine residue at the end of the sequence can participate in LOX2-mediated biological cross-linking, and the histidine at the end of the sequence is conducive to the separation and purification of collagen. Gel electrophoresis analysis of the collagen after affinity chromatography and ion exchange chromatography is shown in Figure 1. Figure 1 and Figure 2 , which proved the accuracy of the purification process and that high-purity recombinant type III human collagen could 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°C for 1 h to obtain the coupling product. The product was washed and concentrated by 30,000 molecular weight ultrafiltration centrifuge tube to remove small molecular impurities.

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

[0055] (3) The coupling product was then 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 formed precipitated insoluble microspheres (PLLA-COL) were washed with water and ethanol solution (1:1, v / v) and freeze-dried to obtain the finished collagen-modified polylactic acid microspheres.

[0056] Scanning electron microscopy observations Figure 3 After collagen modification, the particle size of polylactic acid microspheres did not change significantly, and the surface became looser and rougher.

[0057] Zeta potential analysis Figure 4 , original polylactic acid microspheres ( Figure 4 a) is electronegative and has a narrow charge distribution, which shows the consistency of the preparation of polylactic acid microspheres. After PLLA microspheres are modified with polylysine ( Figure 4 b), significant positively charged particles were observed, indicating that the polylactic acid microspheres were successfully loaded with polylysine short peptides through electrostatic interaction; the main peak on the left showed that some polylactic acid microspheres were not coated, which was 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 electronegative polylactic acid. Figure 4 It can be seen from the c that after the microspheres were mediated by LOXL2 and neutral charged recombinant collagen was introduced into the surface, the ionic charge was close to neutral and the charge distribution was relatively wide, which was related to the charge diversity of the side chains of collagen.

[0058] Infrared spectroscopy analysis Figure 5 From top to bottom, they are polylactic acid microspheres, polylysine adsorbed polylactic acid microspheres and collagen modified polylactic acid microspheres at 1750cm -1 It is the strongest and most characteristic peak of PLA, which is generated by the stretching vibration of ester carbonyl (C=O). -1 The characteristic peak appears at 1651cm, which is the NH bending vibration (δNH) of the primary amine group (-NH) in the lysine molecule, confirming that polylysine was successfully modified onto the surface of PLA microspheres by electrostatic adsorption. -1 and 1548cm -1 Characteristic peaks appeared at , which are amide I band and amide II band, indicating that collagen was successfully modified onto the surface of PLA microspheres.

[0059] Example 2

[0060] Other conditions were the same as those in Example 1, except that the mass ratio of the modified LOXL2 enzyme to the substrate in step (3) was 1:400.

[0061] Example 3

[0062] Other conditions were the same as those in Example 1, except that the mass ratio of the modified LOXL2 enzyme to the substrate in step (3) was 1:600.

[0063] Example 4

[0064] Other conditions were the same as those in Example 1, except that the mass ratio of the modified LOXL2 enzyme to the substrate in step (3) was 1:700.

[0065] Example 5

[0066] Other conditions were the same as those in Example 1, except that in step (1), the molecular weight Mw of mPEG-NHS ester was 7000 Da.

[0067] Example 6

[0068] Other conditions were the same as those in Example 1, except that in step (1), the molecular weight Mw of mPEG-NHS ester was 4000 Da.

[0069] Example 7

[0070] Other conditions were the same as those in Example 1, except that in step (1), the molecular weight Mw of mPEG-NHS ester was 9000 Da.

[0071] Comparative Example 1

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

[0073] Comparative Example 2

[0074] Other conditions were the same as those in Example 1, except that step 1) was omitted and in step 3), type II lysyl oxidase (LOXL2) was directly used instead of the coupling product, and the feeding was performed according to the same LOXL2:substrate mass ratio.

[0075] Application Examples

[0076] Evaluation of Cell Adhesion and Cell Proliferation Activity of Collagen-Modified PLA Microspheres: Mouse L929 fibroblasts were used as the research object, and the MTT colorimetric assay was used to evaluate the effect of collagen modification on cell adhesion and cell proliferation behavior of PLA microspheres. To eliminate interference from cell adhesion to the culture plate surface, the cell growth experiment on the microsphere surface was conducted using Corning Costar low-adhesion microplates. Only when cells adhere normally to the PLA microsphere surface and proliferate under these conditions will MTT blue-purple crystals, which are characteristic of live cells, be formed and detected.

[0077] PLLA-COL microspheres (0.5 mg) were coated with serum-free medium in a 37°C, 5% CO2 incubator with continuous rotation at 15 rpm for 1.5 hours. Immediately following treatment, the microspheres were washed three times with a double-antibody solution (containing 200 U / mL penicillin and 200 μg / mL streptomycin) in double-distilled water. The resulting product was harvested by centrifugation and freeze-dried and stored at 4°C. Activity verification was performed using a low-adsorption plate.

[0078] Specific operations of activity verification test:

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

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

[0081] 3) Culture seeding conditions: 40,000 L929 cells were seeded per well (diluted with 50 μl DMEM), allowed to stand for 1 hour, and then 20 μl of MTT was added and cultured at 37°C overnight.

[0082] 4) After 14 hours, the culture plate was taken and the absorbance at 490 nm was read. Before reading, 150 μl of DMSO was added and the plate was shaken for 10 minutes.

[0083] like Figure 6 As shown in the figure, after collagen modification (A), fibroblasts adhered well to the surface of polylactic acid microspheres and formed a dense cell layer; in the control group blank polylactic acid microspheres test (B), fibroblasts were still able to attach to the hydrophobic polylactic acid surface and form aggregates, but the cell loading per unit microsphere surface was significantly lower than that in the collagen-modified experimental group, indicating that after collagen modification, the cell adhesion and proliferation activity of polylactic acid microspheres were significantly improved.

[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 blank polylactic acid microspheres (B, comparative example 1) test, fibroblasts were still able to attach to the hydrophobic polylactic acid surface and form aggregates, but the cell loading per unit microsphere surface was significantly lower than that of the collagen-modified experimental group, indicating that after collagen modification, the cell adhesion and proliferation activity of polylactic acid microspheres were significantly improved.

[0085] The collagen-modified polylactic acid microspheres obtained in the above examples and comparative example 1 were tested for cell proliferation activity according to the method of the above application examples. The relative cell viability was expressed as 100% (i.e., 40,000 cells per well) at the beginning of the experiment, i.e., the number of cells at hour 0. The relative cell viability was calculated as the ratio of the number of cells tested after culture to the number at hour 0. The results are as follows: Figure 7 shown.

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

[0087] Table 1 Relative cell viability

[0088]

[0089]

Claims

1. An injectable polylactic acid microsphere with surface modified collagen, characterized in that: It is a core-shell structure, the core is a polylactic acid microsphere modified with polylysine on the surface, 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 through a PEG-modified type II lysyl oxidase-catalyzed reaction.

2. The injectable polylactic acid microspheres with surface modified collagen according to claim 1, characterized in that: The sequence of the recombinant human type III collagen 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 average particle size of the polylactic acid microspheres is 40-60 μm, the weight average molecular weight of the polylactic acid is 15000-30000 g / mol; the weight average molecular weight of the polylysine is 20000-50000 Da, preferably 30000-40000 Da.

4. The injectable polylactic acid microspheres with surface modified collagen according to claim 1, characterized in that: The PEG-modified type II lysyl oxidase is obtained by coupling type II lysyl oxidase (LOXL2) with polyethylene glycol methyl ether activated lipid (mPEG-NHS ester), wherein the weight average molecular weight of the polyethylene glycol methyl ether activated lipid is 5500-7000 Da.

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

6. The method for preparing the injectable polylactic acid microspheres with surface modified collagen according to any one of claims 1 to 5, characterized in that: The following steps are involved: (S1) The hydrophilic treated polylactic acid microspheres are surface-modified with polylysine by electrostatic interaction to obtain polylactic acid microspheres with surface-modified polylysine. (S2) Coupling reaction of type II lysyl oxidase (LOXL2) and polyethylene glycol methyl ether activated lipid (mPEG-NHS ester) to obtain the coupling product; (S3) Polylactic acid microspheres modified with polylysine and collagen are dissolved in a buffer solution as substrates for enzymatic reaction and incubated for later use; (S4) adding the coupling product obtained in step (S2) to the enzymatic reaction substrate, incubating to form precipitated insoluble microspheres (PLLA-COL), and freeze-drying to obtain injectable polylactic acid microspheres with collagen modified on the surface.

7. The preparation method according to claim 6, characterized in that In step (S1), the average particle size of the polylactic acid microspheres is 40-60 μm, and the weight average molecular weight of the polylactic acid is 15000-30000 g / mol; Furthermore, in step (S1), the polylactic acid microspheres are subjected to hydrophilic treatment by soaking the polylactic acid in 0.1-0.5M NaOH and / or KOH alkaline solution for 10-40 minutes, and then washing with water until the washing solution is neutral; the polylysine is surface-modified by electrostatic action, specifically, the hydrophilic treated polylactic acid microspheres are immersed in a polylysine (PL) solution under light-proof conditions, placed at 0-10°C for 10-30 hours, taken out, washed, and dried; Furthermore, in the polylysine solution, the molecular weight of polylysine is 20,000-50,000 Da, preferably 30,000-40,000 Da, the concentration of the polylysine solution is 0.1-1 wt %, preferably 0.25-0.5 wt %, and the mass volume ratio of the polylactic acid microspheres to the polylysine solution is 1 g:3-10 mL.

8. The preparation method according to claim 6, characterized in that In step (S2), the molar ratio of type II lysyl oxidase and polyethylene glycol methyl ether activated lipid is 1:3-10, such as 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°C for 1-2 hours, such as shaking at 4°C for 1 hour.

9. The preparation method according to claim 6, characterized in that In step (S3), the pH of the buffer solution is 7.5-9, preferably 8-8.5, such as Na2B4O7 buffer; the concentration of the polylactic acid microspheres modified with polylysine is 10-100 mg / mL, preferably 30-50 mg / mL; the concentration of the recombinant type III human collagen is 5-20 mg / mL, preferably 7-10 mg / mL; and the mass ratio of the polylactic acid microspheres to the recombinant type III human collagen is 4-6:1, such as 5:1; and the incubation is at 40-50°C for 10-30 min; and / or In step (S4), the coupling product is added at a mass ratio of 1:300-700 for the coupling product and substrate, preferably a mass ratio of LOXL2 enzyme and substrate is 1:400-500; the incubation is at 40-60°C for 20-30 hours, for example, at 50°C for 24 hours.

10. A skin injection filler for medical aesthetics, comprising the injectable polylactic acid microspheres with surface-modified collagen according to any one of claims 1 to 5.

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