Polylactic acid microcubes, and preparation method and application thereof

Abstract

The application discloses a kind of polylactic acid microcubes and its preparation method and application, belong to the field of pharmaceutical technology.The preparation method of polylactic acid microcube of the application includes: polylactic acid microsphere is filled into the cube micro-groove of mould;The side length of cube micro-groove is 20~100 μm;Then the mould is placed in the closed container with organic solvent, and make polylactic acid microsphere in the mould not contact with the liquid level of organic solvent, polylactic acid is shaped in mould;The mould that completes shaping is demoulded, and polylactic acid microcube is obtained.The second aspect of the application discloses the polylactic acid microcube obtained by the above method and its application in the preparation of drug or material for strengthening cell mitochondria energy metabolism.The application creatively uses micro-groove filling to combine solvent evaporation method, makes levorotatory polylactic acid gradually dissolved and directional shaping, can obtain the polylactic acid microcube of regular appearance, uniform size, so as to better realize the enhancement of mitochondria function.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a polylactic acid microcube, its preparation method, and its application. Background Technology

[0002] Mitochondria, as the energy metabolism center of eukaryotic cells, convert nutrients such as glucose and fatty acids into adenosine triphosphate (ATP) through the oxidative phosphorylation system, providing approximately 90% of the cell's energy needs. Mitochondrial energy metabolism directly determines cell vitality, proliferation, differentiation, and apoptosis, and is also closely related to overall metabolic homeostasis, exercise endurance, the aging process, and the development of various diseases. Numerous studies have shown that mitochondrial dysfunction is one of the core mechanisms underlying pathological conditions such as obesity, type 2 diabetes, non-alcoholic fatty liver disease, cardiovascular disease, neurodegenerative diseases (such as Alzheimer's disease and Parkinson's disease), and sarcopenia. Strengthening mitochondrial energy metabolism is an important strategy for improving these energy imbalance-related diseases and delaying aging. However, currently, drugs or biomaterials that directly target mitochondria remain very limited.

[0003] Polylactic acid (PLLA) is a fully biodegradable polymer material approved by the U.S. Food and Drug Administration (FDA) and widely used in the biomedical field. Its degradation product, L-lactic acid, enters the tricarboxylic acid cycle and is ultimately metabolized into carbon dioxide and water, which are naturally excreted from the body. Due to its non-toxic, non-irritating, and absorbable characteristics, PLLA has been successfully applied in tissue repair fields such as medical aesthetics, and is typically used in microparticle form.

[0004] Existing research has shown that the microstructure of polylactic acid (PLA) microparticles significantly affects their biological functions, especially collagen-inducing efficiency. Currently, commercially available and literature-reported PLA microparticles are mainly divided into two categories: spherical and irregular morphologies. However, both have inherent drawbacks: while spherical particles have good dispersibility, their isotropic surface characteristics result in a limited mode of interaction with cells and a limited depth of regulation; irregular particles, on the other hand, have uncontrollable morphology between batches, making it impossible to reliably reproduce their efficacy. More importantly, current research and applications of PLA microparticles have not addressed their ability to regulate mitochondrial energy metabolism, nor have they established a link between morphology design and mitochondrial function enhancement.

[0005] Based on this, the present invention develops a polylactic acid microparticle with a regular cubic structure and explores its enhancing effect on mitochondrial energy metabolism, thereby opening up new application directions for the treatment of energy metabolism-related diseases and tissue regeneration. Summary of the Invention

[0006] One of the objectives of this invention is to provide a method for preparing polylactic acid microcubes, which is simple to operate and can produce polylactic acid microcubes with micron-sized and regular particle sizes.

[0007] A second objective of this invention is to provide polylactic acid microcubes, which are prepared by the above-described method.

[0008] A third objective of this invention is to provide applications for the polylactic acid microcubes.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention discloses a method for preparing polylactic acid microcubes, which includes the following steps: Step 1. Filling: Polylactic acid microspheres are filled into a mold, the mold including a matrix, the surface of which is provided with a plurality of cubic micro-grooves for filling polylactic acid microspheres; the side length of the cubic micro-grooves is 20~100μm; Step 2. Shaping: Then, the mold is placed in a sealed container containing organic solvent, ensuring that the polylactic acid microspheres inside the mold do not come into contact with the surface of the organic solvent; taking advantage of the volatile nature of the organic solvent and its ability to dissolve polylactic acid, the organic solvent vapor acts on the polylactic acid microspheres, thereby shaping the polylactic acid in the mold; Step 3. Demolding: Demold the mold after shaping to obtain polylactic acid microcubes.

[0010] In some embodiments of the present invention, the method for preparing the polylactic acid microspheres includes the following steps: S1. Preparation of surfactant solution: Prepare a solution containing surfactant using deionized water; S2. Preparation of emulsion: Dissolve L-polylactic acid in a volatile organic solvent, add deionized water and perform ultrasonic emulsification to obtain an emulsion; S3. Formation of microspheres: Under stirring conditions, the emulsion is dropped into the surfactant solution. After the dropping is completed, stirring is continued to obtain a microsphere suspension. S4. Post-processing: The microsphere suspension was allowed to stand, centrifuged, and the precipitate was washed and then freeze-dried to obtain polylactic acid microspheres.

[0011] In some embodiments of the present invention, in S1, the surfactant includes a high molecular weight surfactant and a low molecular weight surfactant; Preferably, the high molecular weight surfactant includes polyvinyl alcohol; the low molecular weight surfactant includes Tween and Span, preferably Tween 80. Preferably, in the surfactant solution, the concentration of the polymeric surfactant is 1-4 wt%, more preferably 2 wt%; the volume ratio of the small molecule surfactant to deionized water is 1:1500-6000, more preferably 1:3000.

[0012] In some embodiments of the present invention, in S2, the volatile solvent is dichloromethane; Preferably, the specific preparation method of the emulsion is as follows: prepare a dichloromethane solution of 2-10 wt%, preferably 5 wt%, of polylactic acid (L-L), and add deionized water for ultrasonic emulsification for 2-10 min; More preferably, the volume ratio of dichloromethane to deionized water is 10 to 40:1, and more preferably 20:1.

[0013] In some embodiments of the present invention, the side length of the cubic micro-groove is 50 ± 5 μm.

[0014] In some embodiments of the present invention, the organic solvent in step 2 includes dichloromethane; Preferably, in step 2, after polylactic acid is molded in the mold, steps 1 and 2 are repeated once or multiple times until the cubic micro-grooves in the mold are completely filled. More preferably, the interval between each filling is 1 to 4 hours, and more preferably 2 hours.

[0015] In some embodiments of the present invention, in step 3, the mold that has been shaped is ultrasonically treated to cause the polylactic acid microcubes to detach from the mold.

[0016] In some embodiments of the present invention, the mold is pretreated with a release agent before filling, and a layer of release agent is coated on the surface of the mold. The release solution is prepared by diluting the release stock solution 4 to 16 times with a diluent; wherein the release solution comprises the following components in the following proportions: 0.5 to 2 g PVA, 6 to 10 ml water, 8 to 12 ml ethanol, and 0.5 to 2 ml glycerin; the diluent comprises the following components in the following proportions: 6 to 10 ml water, 8 to 12 ml ethanol, and 1 to 4 ml glycerin; Preferably, the stripping stock solution comprises the following components in the following proportions: 1g PVA, 8ml water, 10ml ethanol, and 1ml glycerol; the diluent comprises the following components in the following proportions: 8ml water, 10ml ethanol, and 2ml glycerol. Preferably, the pretreatment operation includes: completely immersing the mold in the release liquid, removing it, removing excess liquid from the surface, and drying it until a uniform release layer is formed on the surface of the mold; more preferably, removing it after soaking for 0.5 to 2 hours; more preferably, drying it at a low temperature; and even more preferably, the drying temperature is 30 to 45°C.

[0017] A second aspect of the present invention discloses a polylactic acid microcube, which is prepared by the method described above.

[0018] The third aspect of this invention discloses the application of the polylactic acid microcubes in the preparation of pharmaceuticals or materials for enhancing mitochondrial energy metabolism in cells.

[0019] In this invention, unless otherwise specified, all polylactic acid refers to L-polylactic acid.

[0020] Compared with the prior art, the present invention has the following beneficial effects: This invention is scientifically designed and ingeniously conceived. It creatively employs a microgroove filling method combined with solvent evaporation to gradually dissolve and orient polylactic acid (PLA), resulting in PLA microcubes with regular morphology and uniform size, thereby better enhancing mitochondrial function. Furthermore, this method requires no high temperature or complex equipment, operates under mild conditions, is simple to operate, has good reproducibility, and is easily scalable, showing broad practical prospects.

[0021] Experimental results show that both spherical and cubic polylactic acid microparticles can enhance mitochondrial function at low concentrations, with cubic polylactic acid microparticles exhibiting a superior mitochondrial function enhancement effect compared to spherical microparticles. Attached Figure Description

[0022] Appendix Figure 1 This is a mold diagram for the present invention.

[0023] Appendix Figure 2 Micrograph of polylactic acid microspheres.

[0024] Appendix Figure 3 Micrographs of polylactic acid microspheres filling a cubic mold (left) and the deformation of polylactic acid microspheres in the cubic mold (right).

[0025] Appendix Figure 4 Microscopic image of polylactic acid cubes after demolding.

[0026] Appendix Figure 5 Figure showing the results of polylactic acid cube regulation of 3T3 cells in the Seahorse assay. Figure 5 A represents the oxygen consumption rate (OCR) curves of 3T3 cells over time under different treatment conditions. Figure 5 B is a bar chart comparing the basal respiratory levels of 3T3 cells under different treatment conditions; Figure 5 C is a bar chart comparing the maximum respiratory level of 3T3 cells under different treatment conditions; Figure 5 D is a bar chart comparing the standby respiratory capacity of 3T3 cells under different treatment conditions. Figure 5 The ordinate of A, OCR (pmol / min / norm. unit), represents the oxygen consumption rate (picomoles / minute / normalized unit). Figure 5The ordinate of B~5D is determined by OCR (pmol / min / 10). 4 cells represent the rate of oxygen consumption (picomoles / minute / 10⁻⁶). 4 (cells). Figure 5 In the table, 3T3-WT represents the blank control group; 3T3-5μg / ml-square, 3T3-10μg / ml-square, 3T3-50μg / ml-square, and 3T3-200μg / ml-square represent cubic microparticle test groups with concentrations of 5, 10, 50, and 200μg / ml, respectively; and 3T3-5μg / ml-circle, 3T3-10μg / ml-circle, 3T3-50μg / ml-circle, and 3T3-200μg / ml-circle represent spherical microparticle test groups with concentrations of 5, 10, 50, and 200μg / ml, respectively.

[0027] Appendix Figure 6 Figure showing the results of polylactic acid cube regulation of H9C2 cells in the seahorse assay. Figure 6 A represents the oxygen consumption rate (OCR) curves of H9C2 cells over time under different treatment conditions. Figure 6 B is a bar chart comparing the basal respiratory levels of H9C2 cells under different treatment conditions; Figure 6 C is a bar chart comparing the maximum respiratory level of H9C2 cells under different treatment conditions; Figure 6 D is a bar chart comparing the reserve respiratory capacity of H9C2 cells under different treatment conditions. Figure 6 A~6D ordinate OCR (pmol / min / 10) 4 cells represent the rate of oxygen consumption (picomoles / minute / 10⁻⁶). 4 (cells). Figure 6 In the table, H9C2-WT represents the blank control group; H9C2-5μg / ml-sqμare, H9C2-10μg / ml-square, H9C2-50μg / ml-square, and H9C2-200μg / ml-square represent cubic microparticle test groups with concentrations of 5, 10, 50, and 200μg / ml, respectively; and H9C2-5μg / ml-circle, H9C2-10μg / ml-circle, H9C2-50μg / ml-circle, and H9C2-200μg / ml-circle represent spherical microparticle test groups with concentrations of 5, 10, 50, and 200μg / ml, respectively. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0029] The method for preparing polylactic acid microcubes of the present invention includes the following steps: Step 1. Filling Polylactic acid microspheres are filled into a mold, the mold including a substrate, the surface of which is provided with a plurality of cubic micro-grooves for filling the polylactic acid microspheres; the side length of the cubic micro-grooves is 20~100μm; preferably 50±5μm; The preparation method of the polylactic acid microspheres includes the following steps: S1. Preparation of surfactant solution: Prepare a solution containing surfactant using deionized water; surfactants include high molecular weight surfactants and low molecular weight surfactants; High molecular weight surfactants include polyvinyl alcohol; low molecular weight surfactants include Tween and Span, preferably Tween 80. In the surfactant solution, the concentration of the high molecular weight surfactant is 1-4 wt%, preferably 2 wt%; the volume ratio of the small molecular weight surfactant to deionized water is 1:1500-6000, preferably 1:3000.

[0030] S2. Preparation of emulsion: Dissolve polylactic acid (PLA) in a volatile organic solvent such as dichloromethane to prepare a 2-10 wt%, preferably 5 wt% PLA solution. Add deionized water and ultrasonically emulsify for 2-10 min to obtain the emulsion. The volume ratio of volatile organic solvent to deionized water is 10~40:1, preferably 20:1.

[0031] S3. Formation of microspheres: Under stirring conditions, the emulsion is dropped into the surfactant solution. After the dropping is completed, stirring is continued to obtain a microsphere suspension. S4. Post-processing: The microsphere suspension was allowed to stand, centrifuged, and the precipitate was washed and then freeze-dried to obtain polylactic acid microspheres.

[0032] Step 2. Shaping Then, the mold is placed in a sealed container containing organic solvent, ensuring that the polylactic acid microspheres inside the mold do not come into contact with the surface of the organic solvent. Taking advantage of the volatile nature of the organic solvent and its ability to dissolve polylactic acid, the organic solvent vapor acts on the polylactic acid microspheres, thereby molding the polylactic acid into the mold. The organic solvent in step 2 includes dichloromethane; In step 2, after polylactic acid is molded in the mold, steps 1 and 2 are repeated once or multiple times until the cubic micro-grooves in the mold are completely filled. Preferably, the interval between each filling is 1 to 4 hours, more preferably 2 hours.

[0033] Step 3. Demolding The molded material is immersed in water and ultrasonically treated to demold the micro cubes. The cubes are then separated from the water by centrifugation and dried to obtain polylactic acid micro cubes.

[0034] To ensure that the shape of the microcube is not damaged during demolding, the mold is pretreated with a release agent before filling, that is, a layer of release agent is coated on its surface.

[0035] The release solution is prepared by diluting the release stock solution 4 to 16 times with a diluent; wherein the release solution comprises the following components in the following proportions: 0.5 to 2 g PVA, 6 to 10 ml water, 8 to 12 ml ethanol, and 0.5 to 2 ml glycerin; the diluent comprises the following components in the following proportions: 6 to 10 ml water, 8 to 12 ml ethanol, and 1 to 4 ml glycerin; Preferably, the stripping stock solution comprises the following components in the following proportions: 1g PVA, 8ml water, 10ml ethanol, and 1ml glycerol; the diluent comprises the following components in the following proportions: 8ml water, 10ml ethanol, and 2ml glycerol. The pretreatment operation includes: immersing the mold completely in the release liquid, removing it, removing excess liquid from the surface, and drying it until a uniform release layer is formed on the surface of the mold; more preferably, removing it after soaking for 0.5 to 2 hours; more preferably, drying it at a low temperature, and even more preferably, the drying temperature is 30 to 45°C.

[0036] The present invention also provides polylactic acid microcubes prepared by the above method, with a side length of 20~100μm.

[0037] The present invention further provides the application of the polylactic acid microcubes in the preparation of pharmaceuticals or materials for enhancing mitochondrial energy metabolism in cells.

[0038] The viscosity-average molecular weight / intrinsic viscosity of the L-type polylactic acid described in this embodiment of the invention is 50800 / 0.59.

[0039] Example 1 This embodiment discloses a method for preparing polylactic acid microcubes according to the present invention. The mold used in this embodiment is shown in the attached figure. Figure 1 As shown, it includes a substrate with multiple cubic microgrooves on its surface for filling polylactic acid microspheres; the side length of each cubic microgroove is 50 ± 5 μm. The mold is made of thin-layer stainless steel through laser drilling.

[0040] The preparation method of polylactic acid microcubes in this embodiment includes the following steps: Step (1) Preparation of polylactic acid microspheres S1. Preparation of surfactant solution: After PVA swells with deionized water, heat and stir to fully dissolve the PVA, cool, and prepare a 2wt% PVA solution; then add Tween 80 under stirring and stir evenly to obtain the surfactant solution. The volume ratio of deionized water to Tween 80 is 3000:1.

[0041] S2. Preparation of emulsion: Dissolve L-polylactic acid in dichloromethane to prepare a 5wt% L-polylactic acid solution, add deionized water and sonicate for 5 min to obtain an emulsion; the volume ratio of dichloromethane to deionized water is 20:1.

[0042] S3. Formation of microspheres: Under stirring conditions of 1000 rpm, the emulsion was dropped into the surfactant solution. After the dropping was completed, stirring was continued at 1500 rpm for 6 hours to obtain a microsphere suspension. S4. Post-processing: The microsphere suspension was allowed to stand overnight, centrifuged, the precipitate was washed with deionized water, and freeze-dried to obtain polylactic acid microspheres, the micrograph of which is attached. Figure 2 As shown.

[0043] Step (2) Mold Pretreatment Immerse the mold completely in the release liquid for 1 hour, remove it, remove excess liquid from the surface, and dry it at 37°C to form a uniform release layer on the mold surface. The release solution is prepared by diluting the release stock solution 8 times with a diluent. The composition of the release stock solution is: 1g PVA, 8ml water, 10ml ethanol, and 1ml glycerin; the composition of the diluent is: 8ml water, 10ml ethanol, and 2ml glycerin.

[0044] Step (3) Filling Polylactic acid microspheres were filled into the cubic micro-grooves of the mold; a microscopic image of the polylactic acid microspheres after filling the mold is attached. Figure 3 As shown in the left figure.

[0045] Step (4) Shaping The mold filled in step (3) is placed in a sealed container containing dichloromethane, and the polylactic acid microspheres in the mold are not in contact with the liquid surface of dichloromethane. Taking advantage of the volatile nature of dichloromethane and its ability to dissolve polylactic acid, the dichloromethane vapor acts on the polylactic acid microspheres, thereby molding the polylactic acid in the mold. After polylactic acid is molded into the mold, steps (3) and (4) are repeated multiple times until the cubic micro-grooves in the mold are completely filled; the interval between each filling is 2 hours.

[0046] Micrographs of polylactic acid microspheres after filling the mold are attached. Figure 3 As shown in the left image, a micrograph of polylactic acid microspheres after being shaped in a cubic mold is attached. Figure 3 As shown on the right, the polylactic acid microspheres fused into a microcubic structure compared to before molding.

[0047] Step (5) Demolding The molded microcubes were immersed in water and ultrasonically treated to demold them. The cubes were then separated from the water by centrifugation and dried to obtain polylactic acid microcubes. A micrograph of these micrographs is attached. Figure 4 As shown.

[0048] Example 2 This embodiment discloses a method for preparing polylactic acid microcubes according to the present invention. The mold used in this embodiment is shown in the attached figure. Figure 1 As shown, it includes a substrate, and the surface of the substrate has a plurality of cubic microgrooves for filling polylactic acid microspheres; the side length of the cubic microgrooves is 20 ± 5 μm.

[0049] The preparation method of polylactic acid microcubes in this embodiment includes the following steps: Step (1) Preparation of polylactic acid microspheres S1. Preparation of surfactant solution: After PVA swells with deionized water, heat and stir to fully dissolve the PVA, cool, and prepare a 1wt% PVA solution; then add Tween 80 under stirring and stir evenly to obtain the surfactant solution. The volume ratio of deionized water to Tween 80 is 1500:1.

[0050] S2. Preparation of emulsion: Dissolve L-polylactic acid in dichloromethane to prepare a 10wt% L-polylactic acid solution, add deionized water and ultrasonically emulsify for 10 min to obtain an emulsion; the volume ratio of dichloromethane to deionized water is 40:1.

[0051] S3. Formation of microspheres: Under stirring conditions of 2000 rpm, the emulsion was dropped into the surfactant solution. After the dropping was completed, stirring was continued at 3000 rpm for 4 hours to obtain a microsphere suspension. S4. Post-processing: The microsphere suspension was allowed to stand overnight, centrifuged, the precipitate was washed with deionized water, and freeze-dried to obtain polylactic acid microspheres.

[0052] Step (2) Mold Pretreatment Immerse the mold completely in the release liquid for 2 hours, remove it, remove excess liquid from the surface, and dry it at 45°C to form a uniform release layer on the mold surface. The release solution is prepared by diluting the release stock solution 16 times with a diluent. The composition of the release stock solution is: 2g PVA, 6ml water, 12ml ethanol, and 0.5ml glycerin; the composition of the diluent is: 6ml water, 12ml ethanol, and 4ml glycerin.

[0053] Step (3) Filling Polylactic acid microspheres were filled into the cubic micro-grooves of the mold; Step (4) Shaping The mold filled in step (3) is placed in a sealed container containing dichloromethane, and the polylactic acid microspheres in the mold are not in contact with the liquid surface of dichloromethane. Taking advantage of the volatile nature of dichloromethane and its ability to dissolve polylactic acid, the dichloromethane vapor acts on the polylactic acid microspheres, thereby molding the polylactic acid in the mold. After polylactic acid is molded into the mold, steps (3) and (4) are repeated multiple times until the cubic micro-grooves in the mold are completely filled; the interval between each filling is 1 hour.

[0054] Step (5) Demolding The molded material is immersed in water and ultrasonically treated to demold the micro cubes. The cubes are then separated from the water by centrifugation and dried to obtain polylactic acid micro cubes.

[0055] Example 3 This embodiment discloses a method for preparing polylactic acid microcubes according to the present invention. The mold used in this embodiment is shown in the attached figure. Figure 1 As shown, it includes a substrate, and the surface of the substrate has a plurality of cubic microgrooves for filling polylactic acid microspheres; the side length of the cubic microgrooves is 100 ± 5 μm.

[0056] The preparation method of polylactic acid microcubes in this embodiment includes the following steps: Step (1) Preparation of polylactic acid microspheres S1. Preparation of surfactant solution: After PVA swells with deionized water, heat and stir to fully dissolve the PVA, cool, and prepare a 4wt% PVA solution; then add Tween 80 under stirring and stir evenly to obtain the surfactant solution. The volume ratio of deionized water to Tween 80 is 6000:1.

[0057] S2. Preparation of emulsion: Dissolve L-polylactic acid in dichloromethane to prepare a 2wt% L-polylactic acid solution, add deionized water and sonicate for 2 min to obtain an emulsion; the volume ratio of dichloromethane to deionized water is 10:1.

[0058] S3. Formation of microspheres: Under stirring conditions of 1000 rpm, the emulsion was dropped into the surfactant solution. After the dropping was completed, stirring was continued at 3000 rpm for 2 hours to obtain a microsphere suspension. S4. Post-processing: The microsphere suspension was allowed to stand overnight, centrifuged, the precipitate was washed with deionized water, and freeze-dried to obtain polylactic acid microspheres.

[0059] Step (2) Mold Pretreatment Immerse the mold completely in the release liquid for 4 hours, remove it, remove excess liquid from the surface, and dry it at 30°C to form a uniform release layer on the mold surface. The release solution is prepared by diluting the release stock solution four times with a diluent. The composition of the release stock solution is: 0.5g PVA, 10ml water, 8ml ethanol, and 2ml glycerin; the composition of the diluent is: 10ml water, 8ml ethanol, and 1ml glycerin.

[0060] Step (3) Filling Polylactic acid microspheres were filled into the cubic micro-grooves of the mold; Step (4) Shaping The mold filled in step (3) is placed in a sealed container containing dichloromethane, and the polylactic acid microspheres in the mold are not in contact with the liquid surface of dichloromethane. Taking advantage of the volatile nature of dichloromethane and its ability to dissolve polylactic acid, the dichloromethane vapor acts on the polylactic acid microspheres, thereby molding the polylactic acid in the mold. After polylactic acid is molded into the mold, steps (3) and (4) are repeated multiple times until the cubic micro-grooves in the mold are completely filled; the interval between each filling is 4 hours.

[0061] Step (5) Demolding The molded material is immersed in water and ultrasonically treated to demold the micro cubes. The cubes are then separated from the water by centrifugation and dried to obtain polylactic acid micro cubes.

[0062] Experimental Example 1 This experiment investigated the effects of different morphologies of polylactic acid microparticles with L-type morphology on mitochondrial energy metabolism in 3T3 cells.

[0063] 1. Experimental Groups: Spherical microparticle test group: L-polylactic acid microspheres prepared according to step (1) in Example 1; Cubic microparticle test group: Polylactic acid microcubes prepared according to the method of Example 1; Blank control group: No microparticles added.

[0064] 2. Cells and Culture Conditions: The NIH3T3 mouse embryonic fibroblast cell line was purchased from the Shanghai Cell Resource Center, Chinese Academy of Sciences. Cells were cultured in high-glucose DMEM complete medium containing 10% fetal bovine serum, with medium changed every other day, and passaged every 2-3 days. Cells in the logarithmic growth phase were harvested and cultured at 0.5 × 10⁻⁶ cells / day. 4 The cells were seeded at a density of / wells in Seahorse XF96 plates and incubated overnight in a 37°C, 5% CO2 cell culture incubator. The cell density was approximately 90% when tested the next day.

[0065] 3. Particulate treatment: Experimental group: Four concentrations were set: 5 μg / ml, 10 μg / ml, 50 μg / ml, and 200 μg / ml. The microparticles were prepared using DMEM serum-free culture medium.

[0066] Control group: DMEM serum-free culture medium without microparticles.

[0067] One hour before the test, replace the cell culture medium with serum-free medium containing or without microparticles, and incubate the cells in an incubator for one hour.

[0068] 4. Mitochondrial stress detection: The day before the test, place the hydration solution in a hydration plate, 0.2 mL per well, and hydrate the probe overnight at 37°C in a CO2-free incubator. At the same time, prepare the mitochondrial stress detection medium (containing 2 mM L-glutamine, 1 mM sodium pyruvate, and 10 mM glucose), adjust the pH to 7.4, filter it through a 0.22 μm filter membrane for sterilization, and store it in a refrigerator at 4°C for later use.

[0069] On the day of the assay, after 1 hour of incubation between cells and microparticles, the cell state was observed. The cell culture medium was then replaced with 0.18 ml / well of mitochondrial stress assay medium, and the cells were incubated at 37°C in a CO2-free incubator for 1 hour to equilibrate. Oligomycin, carbonylcyna-nide 4-(trifluoromethoxy)phenylhydrazone (FCCP), and antimycin A & rotenone were prepared in the mitochondrial stress assay medium to final concentrations of 50 μM, 30 μM, and 10 μM, respectively. The prepared drugs were added to the wells of the probe plate, and the assay was immediately performed. Reaction parameters were set, and the probe plate was calibrated. After calibration, the tray popped out. The hydration plate was removed, a cell culture plate was replaced, and the mitochondrial stress assay was run. After completion, the data were analyzed using Wave software.

[0070] 5. Results are attached. Figure 5 As shown: In the mitochondrial stress assay of 3T3 cells, treatment with low concentrations (5, 10, 50 μg / ml) of spherical and cubic polylactic acid (PLA) microparticles resulted in a concentration-dependent increase in basal mitochondrial respiration, maximum respiratory capacity, and respiratory reserve. The increase was significantly greater in the cubic PLA microparticle group than in the spherical PLA microparticle group. Treatment with high concentrations (200 μg / ml) of both spherical and cubic PLA microparticles resulted in a decrease in basal mitochondrial respiration, maximum respiratory capacity, and respiratory reserve, indicating that high concentrations of these microparticles have an inhibitory effect on mitochondrial function. Therefore, a further treatment concentration of around 50 μg / ml is recommended.

[0071] Experimental Example 2 This experiment investigated the effects of different morphologies of polylactic acid microparticles with L-type morphology on mitochondrial energy metabolism in H9C2 cells.

[0072] 1. Experimental Groups: Spherical microparticle test group: L-polylactic acid microspheres prepared according to step (1) in Example 1; Cubic microparticle test group: Polylactic acid microcubes prepared according to the method of Example 1; Blank control group: No microparticles added.

[0073] 2. Cells and Culture Conditions: The H9C2 rat cardiomyocyte line was purchased from the Shanghai Cell Resource Center, Chinese Academy of Sciences. Cells were cultured in low-glucose DMEM complete medium containing 10% fetal bovine serum, with the medium changed every other day, and passaged every 2-3 days. Cells in the logarithmic growth phase were harvested and cultured at 0.5 × 10⁻⁶ cells / day. 4 The cells were seeded at a density of / wells in Seahorse XF96 plates and incubated overnight in a 37°C, 5% CO2 cell culture incubator. The cell density was approximately 90% when tested the next day.

[0074] 3. Particulate treatment: Experimental group: Four concentrations were set: 5 μg / ml, 10 μg / ml, 50 μg / ml, and 200 μg / ml. The microparticles were prepared using DMEM serum-free culture medium.

[0075] Control group: DMEM serum-free culture medium without microparticles.

[0076] One hour before the test, replace the cell culture medium with serum-free medium containing or without microparticles, and incubate the cells in an incubator for one hour.

[0077] 4. Mitochondrial stress detection: The day before the test, place the hydration solution in a hydration plate, 0.2 mL per well, and hydrate the probe overnight at 37°C in a CO2-free incubator. At the same time, prepare the mitochondrial stress detection medium (containing 2 mM L-glutamine, 1 mM sodium pyruvate, and 10 mM glucose), adjust the pH to 7.4, filter it through a 0.22 μm filter membrane for sterilization, and store it in a refrigerator at 4°C for later use.

[0078] On the day of the assay, after 1 hour of incubation between cells and microparticles, the cell state was observed. The cell culture medium was then replaced with 0.18 ml / well of mitochondrial stress assay medium, and the cells were incubated at 37°C in a CO2-free incubator for 1 hour to equilibrate. Oligomycin, carbonylcyna-nide 4-(trifluoromethoxy)phenylhydrazone (FCCP), and antimycin A & rotenone were prepared in the mitochondrial stress assay medium to final concentrations of 50 μM, 30 μM, and 10 μM, respectively. The prepared drugs were added to the wells of the probe plate, and the assay was immediately performed. Reaction parameters were set, and the probe plate was calibrated. After calibration, the tray popped out. The hydration plate was removed, a cell culture plate was replaced, and the mitochondrial stress assay was run. After completion, the data were analyzed using Wave software.

[0079] 5. Results are attached. Figure 6 As shown: In the H9C2 cell mitochondrial stress assay, treatment with low concentrations (5, 10, 50 μg / ml) of spherical and cubic polylactic acid (PLA) microparticles resulted in a concentration-dependent increase in basal mitochondrial respiration, maximum respiratory capacity, and respiratory reserve. The increase was significantly greater in the cubic PLA microparticle group than in the spherical PLA microparticle group. Treatment with high concentrations (200 μg / ml) of both spherical and cubic PLA microparticles significantly decreased basal mitochondrial respiration, maximum respiratory capacity, and respiratory reserve, indicating that high concentrations of microparticles have an inhibitory effect on mitochondrial function. Therefore, a further treatment concentration of 50 μg / ml is recommended as the optimal level. In summary, both spherical and cubic polylactic acid (PLA) microparticles can enhance mitochondrial function at low concentrations (5–50 μg / mL), with cubic PLA microparticles exhibiting a superior mitochondrial function-enhancing effect compared to spherical microparticles. At high concentrations (200 μg / mL), both spherical and cubic PLA microparticles inhibit mitochondrial function, with cubic PLA microparticles showing a milder inhibitory effect than spherical PLA microparticles.

[0080] The above description is merely a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the concept described herein through the above teachings or related technologies or knowledge. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention should be within the protection scope of the appended claims.

Claims

1. A method for preparing polylactic acid microcubes, characterized in that, The steps include the following: Step 1. Filling: Polylactic acid microspheres are filled into a mold, the mold including a matrix, the surface of which is provided with a plurality of cubic micro-grooves for filling polylactic acid microspheres; the side length of the cubic micro-grooves is 20~100μm; Step 2. Shaping: Then place the mold in a sealed container containing organic solvent, ensuring that the polylactic acid microspheres inside the mold do not come into contact with the surface of the organic solvent, thus shaping the polylactic acid in the mold; Step 3. Demolding: Demold the mold after shaping to obtain polylactic acid microcubes.

2. The method for preparing polylactic acid microcubes according to claim 1, characterized in that, The preparation method of the polylactic acid microspheres includes the following steps: S1. Preparation of surfactant solution: Prepare a solution containing surfactant using deionized water; S2. Preparation of emulsion: Dissolve L-polylactic acid in a volatile organic solvent, add deionized water and perform ultrasonic emulsification to obtain an emulsion; S3. Formation of microspheres: Under stirring conditions, the emulsion is dropped into the surfactant solution. After the dropping is completed, stirring is continued to obtain a microsphere suspension. S4. Post-processing: The microsphere suspension was allowed to stand, centrifuged, and the precipitate was washed and then freeze-dried to obtain polylactic acid microspheres.

3. The method for preparing polylactic acid microcubes according to claim 2, characterized in that, In S1, the surfactant includes high molecular weight surfactants and low molecular weight surfactants; Preferably, the high molecular weight surfactant includes polyvinyl alcohol; the low molecular weight surfactant includes Tween and Span, preferably Tween 80. Preferably, in the surfactant solution, the concentration of the high molecular weight surfactant is 1-4 wt%, more preferably 2 wt%; the volume ratio of the small molecular weight surfactant to deionized water is 1:1500-6000, more preferably 1:3000.

4. The method for preparing polylactic acid microcubes according to claim 2, characterized in that, In S2, the most volatile solvent is dichloromethane; Preferably, the specific preparation method of the emulsion is as follows: prepare a dichloromethane solution of 2-10 wt%, preferably 5 wt%, of polylactic acid (L-L), and add deionized water for ultrasonic emulsification for 2-10 min; More preferably, the volume ratio of dichloromethane to deionized water is 10 to 40:1, and more preferably 20:

1.

5. A method for preparing polylactic acid microcubes according to any one of claims 1 to 4, characterized in that, The side length of the cubic micro-groove is 50 ± 5 μm.

6. A method for preparing polylactic acid microcubes according to any one of claims 1 to 4, characterized in that, The organic solvent in step 2 includes dichloromethane; Preferably, in step 2, after polylactic acid is molded in the mold, steps 1 and 2 are repeated once or multiple times until the cubic micro-grooves in the mold are completely filled. More preferably, the interval between each filling is 1 to 4 hours, and more preferably 2 hours.

7. A method for preparing polylactic acid microcubes according to any one of claims 1 to 4, characterized in that, In step 3, the mold that has been shaped is ultrasonically treated to remove the polylactic acid microcubes from the mold.

8. A method for preparing polylactic acid microcubes according to any one of claims 1 to 4, characterized in that, Before filling, the mold is pretreated with a release agent, and a layer of release agent is coated on the surface of the mold. The release solution is prepared by diluting the release stock solution 4 to 16 times with a diluent; wherein the release solution comprises the following components in the following proportions: 0.5 to 2 g PVA, 6 to 10 ml water, 8 to 12 ml ethanol, and 0.5 to 2 ml glycerin; the diluent comprises the following components in the following proportions: 6 to 10 ml water, 8 to 12 ml ethanol, and 1 to 4 ml glycerin; Preferably, the stripping stock solution comprises the following components in the following proportions: 1g PVA, 8ml water, 10ml ethanol, and 1ml glycerol; the diluent comprises the following components in the following proportions: 8ml water, 10ml ethanol, and 2ml glycerol. Preferably, the pretreatment operation includes: completely immersing the mold in the release liquid, removing it, removing excess liquid from the surface, and drying it until a uniform release layer is formed on the surface of the mold; more preferably, removing it after soaking for 0.5 to 2 hours; more preferably, drying it at a low temperature; and even more preferably, the drying temperature is 30 to 45°C.

9. A polylactic acid microcube, characterized in that, It is prepared by the method according to any one of claims 1 to 8; Preferably, the polylactic acid microcubes have a side length of 20~100μm.

10. The application of polylactic acid microcubes according to claim 9, characterized in that, Applications in the preparation of drugs or materials for enhancing mitochondrial energy metabolism in cells.

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