A porous microsphere for injection, a method for preparing the same, and use thereof

Abstract

The application belongs to the technical field of biological medicine, and discloses a porous microsphere for injection, a preparation method and application thereof. The porous microsphere for injection is prepared by combining heparin-binding peptide III and PLLA; the amino acid sequence of the heparin-binding peptide III is FHRRIKA. The PLLA heparin-binding peptide III microsphere prepared by the application has no cytotoxicity, good cell adhesion capacity, and can inhibit the production of inflammatory factors. The microsphere preparation method provided by the application is simple, does not need large-scale instruments and equipment, has low production cost, and is suitable for large-scale preparation. The microsphere preparation method adopted by the application does not need cross-linking, does not use a cross-linking agent, and has no cross-linking agent residue.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a porous microsphere for injection, its preparation method, and its application. Background Technology

[0002] Poly-L-lactic acid (PLA, trade name: Scuiptra) was approved in Europe in 1999 for the treatment of skin wrinkles, scars, and signs of aging. In August 2004, it received expedited approval from the US Food and Drug Administration for the treatment of facial lipoatrophy associated with human immunodeficiency virus (HIV). Injectable poly-L-lactic acid is derived from a biocompatible and biodegradable synthetic polymer in the α-hydroxy acid family and is widely used in tissue engineering. Poly-L-lactic acid (PLLA) belongs to the PLA family and possesses excellent physical and mechanical properties and biodegradability. Compared to PDLA (polymerized from D-lactide), it has higher crystallinity, chemical stability, and resistance to enzyme degradation. Furthermore, PLLA degrades to L-lactic acid, which is harmless to the human body. In addition, the production process and sources of PLLA are environmentally friendly. Therefore, PLLA is a popular material for tissue engineering.

[0003] However, PLLA has relatively poor biocompatibility and requires modification to enhance its cellular responsiveness. Current technology obtains pure PLLA microspheres through emulsion-solvent evaporation, followed by modification with grafted collagen, gelatin, and other materials. However, PLLA's strong hydrophobicity, poor cell adhesion, and the formation of lactic acid degradation products can lead to mild facial inflammation in the initial stages of PLLA product injection. Adverse reactions caused by PLLA fillers include bruising, edema, nodules, and inflammation. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a porous microsphere for injection, its preparation method and application.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a porous microsphere for injection, which is prepared by combining heparin-binding peptide III and PLLA; wherein the amino acid sequence of the heparin-binding peptide III is FHRRIKA.

[0007] To address the adverse reactions associated with poly-L-lactic acid in injection filling, this invention provides a novel porous microsphere prepared using heparin-binding peptide III. The amino acid sequence of heparin-binding peptide III is FHRRIKA. The porous microspheres of this invention are low-cost, contain non-animal-derived peptides that promote cell adhesion, and compared to production materials such as gelatin and collagen, have the advantages of low immunogenicity and simple preparation process. Furthermore, they are non-cytotoxic, exhibit good cell adhesion ability, and can inhibit the production of inflammatory factors.

[0008] In a preferred embodiment of the injectable porous microspheres of the present invention, the mass ratio of heparin-binding peptide III to PLLA is 1:2 to 1:10.

[0009] In a preferred embodiment of the porous microspheres for injection described in this invention, the particle size of the porous microspheres is 50 μm to 100 μm.

[0010] Secondly, the present invention provides a method for preparing porous microspheres for injection, comprising the following steps:

[0011] (1) Dissolve heparin-binding peptide III in water to obtain a heparin-binding peptide III solution.

[0012] (2) Dissolve PLLA in dichloromethane to obtain a PLLA dichloromethane solution;

[0013] (3) The heparin-binding peptide III solution was added dropwise to the PLLA dichloromethane solution, homogenized, and a suspension was obtained;

[0014] (4) The suspension was added dropwise to a 0.1% polyvinyl alcohol solution, and the dichloromethane was evaporated by stirring to obtain microspheres;

[0015] (5) Remove the aqueous phase, wash, dry, sterilize, and obtain porous microspheres for injection.

[0016] The microsphere preparation method provided by this invention is simple, requires no large-scale instruments or equipment, has low production costs, and is suitable for large-scale production. The microsphere preparation method used in this invention does not involve crosslinking, does not use crosslinking agents, and leaves no crosslinking agent residue.

[0017] In a preferred embodiment of the injectable porous microspheres of the present invention, the mass ratio of heparin-binding peptide III to PLLA is 1:2 to 1:10.

[0018] In a preferred embodiment of the injectable porous microspheres of the present invention, in step (1), the amino acid sequence of the heparin-binding peptide III is FHRRIKA.

[0019] As a preferred embodiment of the injectable porous microspheres of the present invention, in step (1), the heparin-binding peptide III is dissolved in deionized water at a mass-to-volume ratio of 1g:(40-7)mL.

[0020] As a preferred embodiment of the porous microspheres for injection described in this invention, in step (2), the PLLA is dissolved in dichloromethane at a mass-to-volume ratio of 1g:(10-30)mL.

[0021] In a preferred embodiment of the porous microspheres for injection described in this invention, in step (4), the dropping is performed at a rate of 0.5 mL / min to 1.5 mL / min.

[0022] Thirdly, the present invention applies the aforementioned porous microspheres for injection and the aforementioned preparation method to tissue engineering or facial filling.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] (1) This invention provides a novel porous microsphere prepared by binding heparin-binding peptide III. The amino acid sequence of heparin-binding peptide III is FHRRIKA. The porous microspheres of this invention are low in cost, contain non-animal-derived peptides that promote cell adhesion, and have the advantages of low immunogenicity and simple preparation process compared with production materials such as gelatin and collagen.

[0025] (2) The PLLA heparin-binding peptide III microspheres prepared by this invention are non-cytotoxic, have good cell adhesion ability, and can inhibit the production of inflammatory factors. The microsphere preparation method provided by this invention is simple, does not require large-scale instruments and equipment, has low production cost, and is suitable for large-scale preparation. The microsphere preparation method used in this invention does not use cross-linking, does not use cross-linking agents, and leaves no cross-linking agent residue. Attached Figure Description

[0026] Figure 1 SEM image of porous microspheres;

[0027] Figure 2 A statistical graph showing the effect of different concentrations of heparin-binding peptide III on the adhesion of 3T3-L1 cells;

[0028] Figure 3 This is a statistical graph showing the cytotoxicity of porous microspheres to HDF cells.

[0029] Figure 4 A statistical graph showing the cytotoxicity of porous microspheres to HaCaT cells;

[0030] Figure 5 To observe the adsorption of porous microspheres on 3T3-L1 cells using a light microscope;

[0031] Figure 6 HE staining image of tissue at porous microinjection sites. Detailed Implementation

[0032] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0033] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified. The amino acid sequence of the heparin-binding peptide III involved in the examples is: FHRRIKA.

[0034] Example 1:

[0035] (1) Dissolve 250 mg of heparin-binding peptide III in 1.75 mL of deionized water;

[0036] (2) Dissolve 500 mg PLLA in 12 mL of dichloromethane;

[0037] (3) Add the heparin-binding peptide III solution dropwise into the PLLA dichloromethane solution and homogenize it at a speed of 12000 rpm for 5 min.

[0038] (4) The homogenized suspension was added dropwise to 500 mL of 0.1% polyvinyl alcohol (PVA) solution with a stirring speed of 500 rpm at a rate of 1 mL / min, and stirred for 12 h.

[0039] (5) The aqueous phase was removed by vacuum filtration pump, the microspheres were washed three times with deionized water, freeze-dried and stored, and sterilized by irradiation to obtain PLLA heparin-binding peptide III microspheres. The freeze-drying procedure was as follows: pre-freezing at -40℃ for 2 hours, drying at -15℃ for 16 hours, and drying at 25℃ for 4 hours.

[0040] Example 2:

[0041] (1) Dissolve 100 mg of heparin-binding peptide III in 1.75 mL of deionized water;

[0042] (2) Dissolve 500 mg PLLA in 12 mL of dichloromethane;

[0043] (3) Add the heparin-binding peptide III solution dropwise into the PLLA dichloromethane solution and homogenize it at a speed of 12000 rpm for 5 min.

[0044] (4) The homogenized suspension was added dropwise to 500 mL of 0.1% PVA solution with a stirring speed of 500 rpm at a rate of 1 mL / min, and stirred for 12 h.

[0045] (5) The aqueous phase was removed by vacuum filtration pump, the microspheres were washed three times with deionized water, freeze-dried and stored, and sterilized by irradiation to obtain PLLA heparin-binding peptide III microspheres. The freeze-drying procedure was as follows: pre-freezing at -40℃ for 2 hours, drying at -15℃ for 16 hours, and drying at 25℃ for 4 hours.

[0046] Example 3:

[0047] (1) Dissolve 50 mg of heparin-binding peptide III in 1.75 mL of deionized water;

[0048] (2) Dissolve 500 mg PLLA in 12 mL of dichloromethane;

[0049] (3) Add the heparin-binding peptide III solution dropwise into the PLLA dichloromethane solution and homogenize it at a speed of 12000 rpm for 5 min.

[0050] (4) The homogenized suspension was added dropwise to 500 mL of 0.1% PVA solution with a stirring speed of 500 rpm at a rate of 1 mL / min, and stirred for 12 h.

[0051] (5) The aqueous phase was removed by vacuum filtration pump, the microspheres were washed three times with deionized water, freeze-dried and stored, and sterilized by irradiation to obtain PLLA heparin-binding peptide III microspheres. The freeze-drying procedure was as follows: pre-freezing at -40℃ for 2 hours, drying at -15℃ for 16 hours, and drying at 25℃ for 4 hours.

[0052] Example 4:

[0053] (1) Dissolve 500 mg PLLA in 12 mL of dichloromethane;

[0054] (2) Add the PLLA dichloromethane solution dropwise to 500 mL of 0.1% PVA solution at a stirring speed of 500 rpm at a rate of 1 mL / min, and stir for 12 h;

[0055] (3) The aqueous phase was removed by vacuum filtration pump, the microspheres were washed three times with deionized water, freeze-dried and stored, and sterilized by irradiation to obtain PLLA microspheres. The freeze-drying procedure was as follows: pre-freezing at -40℃ for 2 hours, drying at -15℃ for 16 hours, and drying at 25℃ for 4 hours.

[0056] Example 5:

[0057] (1) Dissolve 25 mg of heparin-binding peptide III in 1.75 mL of deionized water;

[0058] (2) Dissolve 500 mg PLLA in 12 mL of dichloromethane;

[0059] (3) Add the heparin-binding peptide III solution dropwise into the PLLA dichloromethane solution and homogenize it at a speed of 12000 rpm for 5 min.

[0060] (4) The homogenized suspension was added dropwise to 500 mL of 0.1% PVA solution with a stirring speed of 500 rpm at a rate of 1 mL / min, and stirred for 12 h.

[0061] (5) The aqueous phase was removed by vacuum filtration pump, the microspheres were washed three times with deionized water, freeze-dried and stored, and sterilized by irradiation to obtain PLLA heparin-binding peptide III microspheres. The freeze-drying procedure was as follows: pre-freezing at -40℃ for 2 hours, drying at -15℃ for 16 hours, and drying at 25℃ for 4 hours.

[0062] Example 6:

[0063] (1) Dissolve 300 mg of heparin-binding peptide III in 1.75 mL of deionized water;

[0064] (2) Dissolve 500 mg PLLA in 12 mL of dichloromethane;

[0065] (3) Add the heparin-binding peptide III solution dropwise into the PLLA dichloromethane solution and homogenize it at a speed of 12000 rpm for 5 min.

[0066] (4) The homogenized suspension was added dropwise to 500 mL of 0.1% PVA solution with a stirring speed of 500 rpm at a rate of 1 mL / min, and stirred for 12 h.

[0067] (5) The aqueous phase was removed by vacuum filtration pump, the microspheres were washed three times with deionized water, freeze-dried and stored, and sterilized by irradiation to obtain PLLA heparin-binding peptide III microspheres. The freeze-drying procedure was as follows: pre-freezing at -40℃ for 2 hours, drying at -15℃ for 16 hours, and drying at 25℃ for 4 hours.

[0068] Experimental Example 1: SEM imaging of microspheres

[0069] The conductive tape was attached to the sample stage, and then the PLLA heparin-binding peptide III microsphere samples of Examples 1-6, which were dried under vacuum, were evenly sprinkled on the conductive tape. The unattached samples were then blown away with a bulb syringe, and a gold sputtering machine was used for coating treatment. The samples were observed under low vacuum conditions.

[0070] like Figure 1As shown, the microspheres have a particle size of approximately 100 μm and a porous structure. When the content of PLLA added is 500 mg, the content of heparin-binding peptide III is no higher than 250 mg, and the resulting microspheres are uniformly spherical (see Examples 1, 2, 3, 4, 5). When the content of heparin-binding peptide III reaches 300 mg, the resulting microspheres are wrinkled and the porous structure is not clear (see Example 6).

[0071] Experimental Example 2: Adhesion of Heparin-binding peptide III to 3T3-L1 cells

[0072] Add 100 μL of 1×PBS solution containing heparin-binding peptide III at different concentrations (1.95 μg / mL to 1000 μg / mL) to a 96-well plate and incubate at 37°C for 2 h. Remove the supernatant, add 1% albumin in 1×PBS solution, incubate at 37°C for 1 h, remove the supernatant, and wash three times with 1×PBS. Mouse embryonic fibroblasts (3T3-L1 cells) were cultured to 80%–90% confluence, digested with 0.25% trypsin for 1 minute, and after terminating digestion with complete culture medium, cell counting was performed at a rate of 2×10⁻⁶ cells / well. 3 Each well was plated and added to the aforementioned 96-well plate. Serum-free culture medium was added to a final volume of 100 μL. The plate was incubated at 37°C and 5% CO2 for 1 h. The plate was then sealed, inverted, and centrifuged at 350 × g for 5 min. The supernatant was removed, and CCK8 working solution was added. The plate was incubated at 37°C for 2 h. The readings were then taken using a microplate reader at a wavelength of 450 nm.

[0073] like Figure 2 As shown, heparin-binding peptide III can promote the adhesion of 3T3-L1 cells, especially at concentrations of 250 μg / mL to 1000 μg / mL, which have a stronger effect on the adhesion of 3T3-L1 cells.

[0074] Experimental Example 3: Cytotoxicity Experiment of Microspheres

[0075] Weigh 50 mg of the PLLA heparin-binding peptide III microspheres from Examples 1-6, soak them in 5 mL of DMEM basal medium for 24 h, centrifuge at 5000 × rpm for 10 min, and collect the supernatant as the microsphere extract. Culture HDF cells and HaCaT cells to 80%–90% confluence, digest with 0.25% trypsin for 1–3 minutes, terminate digestion with complete medium, and then count the cells at a ratio of 2 × 10⁻⁶. 3To determine the cell density per well, human dermal fibroblasts (HDF cells) and human immortalized epidermal cells (HaCaT cells) were seeded into 96-well plates, respectively. Complete culture medium was added to 100 μL, and the plates were incubated at 37°C and 5% CO2 for 24 h. Then, the microsphere extract was added, and the plates were incubated at 37°C and 5% CO2 for 24 h. Finally, CCK8 working solution was added, and the plates were incubated at 37°C and 5% CO2 for 2 h. The readings were then taken using a microplate reader at a wavelength of 450 nm.

[0076] The results are as follows Figure 3 and 4 As shown, the microsphere samples in Examples 1-6 showed no cytotoxicity to human dermal fibroblasts (HDF cells) and human immortalized epidermal cells (HaCaT cells).

[0077] Experiment 4: Cell Culture Experiment with Microspheres

[0078] Weigh 10g of agarose and dissolve it in 50mL of 1×PBS. Autoclave the solution and mix it with DMEM basal medium before cooling. Pour the mixture into a six-well plate to prepare a low-adsorption six-well plate. Allow it to solidify before use.

[0079] 3T3-L1 cells were cultured to 80%–90% confluence, digested with 0.25% trypsin for 1 minute, and the digestion was terminated with complete culture medium. Cell counting was then performed. 50 mg of the PLLA heparin-binding peptide III microsphere sample from Examples 1–6 was weighed and processed at a ratio of 2 × 10⁻⁶. 4 Cells: Mix cells with microspheres at a ratio of 3 mg / well and add to a low-adsorption six-well plate, 1 × 10⁻⁶ cells / well. 5 Cells were seeded and incubated at 37°C and 5% CO2 for 24 hours. Then, the cells were stained with the AM / PI kit according to the instructions, and the cell adsorption was observed using a fluorescence microscope.

[0080] like Figure 5 As shown, within a suitable range of heparin-binding peptide III content (as in Examples 1, 2, 3, and 6), the microspheres can support the normal adhesion and proliferation of dermal fibroblasts (HDF). When heparin-binding peptide is not added or its content is too low (as in Examples 4 and 5), cells cannot adhere to the microspheres for growth.

[0081] Experimental Example 5: Animal Experiments with Microspheres

[0082] Nine female Kunming mice aged 5-8 weeks were purchased and divided into groups of three. Each mouse received injections at six sites: experimental sites (PLLA heparin-binding peptide III microspheres from Examples 1-3 + 0.4% sodium hyaluronate), negative sites (pure PLLA microspheres from Examples 4-6 + 0.4% sodium hyaluronate), and blank sites (0.4% sodium hyaluronate sites). 40 mg of medium molecular weight sodium hyaluronate was weighed and prepared into 10 mL of 0.4% aqueous solution. After autoclaving, 100 mg of microspheres was added to prepare a 10 mg / mL microsphere suspension. The suspension was then subcutaneously injected into the mice at a dose of 100 μL per site. Mice were sacrificed on days 7, 14, and 28 post-injection, and tissue samples from the injection sites were collected for HE staining.

[0083] like Figure 6 As shown, PLLA microspheres containing heparin-binding peptide III can aggregate under the skin of mice and play a supporting role. Microspheres prepared with a specific ratio of heparin-binding peptide III and PLLA are relatively smooth in shape (Examples 1, 2, 3), and the inflammatory response is lower than that of microspheres prepared without heparin-binding peptide III or with a low content (Examples 4, 5) and microspheres with a wrinkled shape (Example 6).

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A porous microsphere for injection, characterized in that, It is prepared by combining heparin-binding peptide III and PLLA; the amino acid sequence of heparin-binding peptide III is FHRRIKA; the mass ratio of heparin-binding peptide III to PLLA is 1:2 to 1:

10.

2. The porous microspheres for injection according to claim 1, characterized in that, The porous microspheres have a particle size of 50 μm to 100 μm.

3. A method for preparing porous microspheres for injection as described in claim 1 or 2, characterized in that, Includes the following steps: (1) Dissolve heparin-binding peptide III in water to obtain a heparin-binding peptide III solution; The amino acid sequence of the heparin-binding peptide III is FHRRIKA. (2) Dissolve PLLA in dichloromethane to obtain a PLLA dichloromethane solution; (3) The heparin-binding peptide III solution was added dropwise to the PLLA dichloromethane solution and homogenized to obtain a suspension; The mass ratio of heparin-binding peptide III to PLLA is 1:2 to 1:10; (4) The suspension was added dropwise to a 0.1% polyvinyl alcohol solution, and the dichloromethane was evaporated by stirring to obtain microspheres; (5) Remove the aqueous phase, wash, dry, sterilize, and obtain porous microspheres for injection.

4. The preparation method according to claim 3, characterized in that, In step (1), the heparin-binding peptide III is dissolved in deionized water at a mass-volume ratio of 1 g:(40~7) mL.

5. The preparation method according to claim 3, characterized in that, In step (2), the PLLA is dissolved in dichloromethane at a mass-to-volume ratio of 1 g:(10~30) mL.

6. The preparation method according to claim 3, characterized in that, In step (4), the dripping is performed at a rate of 0.5 mL / min to 1.5 mL / min.

7. The use of the injectable porous microspheres according to claim 1 or 2, and the preparation method according to any one of claims 3 to 6, in the preparation of tissue engineering or facial filler materials.

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