A citric acid-based biomaterial and its preparation method and application

Abstract

The present invention relates to a citric acid-based microparticle injection material and its preparation method and application. Citric acid and 1,8-octanediol are melted and heated to form a POC prepolymer, the POC prepolymer is dissolved by an organic solvent to obtain a dissolving solution, and the dissolving solution is slowly poured into ultrapure water for purification multiple times to obtain a purified POC degradable prepolymer; the POC degradable prepolymer is dissolved by a solvent, inorganic salt particles are added, and after a cross-linking reaction, a POC injectable microparticle material is obtained. Drugs, active molecules, and various metal ions are loaded onto the POC by physical compounding or chemical cross-linking to obtain a citric acid-based microparticle injection material. The citric acid-based biomaterial provided by the present invention, as a new type of medical aesthetic raw material and its related microparticle injection filler, can effectively promote the regeneration, proliferation, migration and chemotaxis of skin cells, promote the production of collagen, and achieve medical aesthetic effects. At the same time, it can activate the body's immune response to reduce inflammation. The degradation product is citric acid, which can be metabolized by the body without obvious immunogenicity and toxic side effects.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a citric acid-based biomaterial and a preparation method and application thereof. Background Art

[0002] Skin aging is a health problem that everyone faces. With age and the accumulation of environmental factors, phenomena such as dryness, wrinkles, and loss of elasticity on the skin surface gradually intensify, and may even lead to skin dysfunction, increasing the risk of skin diseases and skin malignancies. In the current pursuit of youth, beauty, and health, skin aging places a psychological and physiological burden on people. Currently, anti-skin aging strategies such as retinoic acid, radiofrequency technology, and dermal fillers aim to stimulate the production of collagen in the dermis to restore the function of aging skin. Skin aging is a health problem that everyone faces, and with age, skin aging becomes more severe, and the apparent symptoms become more obvious, greatly causing people to worry about changes in their appearance.

[0003] Skin aging is closely associated with changes in collagen. Hydroxyproline is the amino acid with the highest concentration in collagen. Compared to collagen in other tissues, hydroxyproline is primarily present in skin collagen. Therefore, it can be used as a marker for assessing skin aging. Hyaluronic acid is an acidic mucopolysaccharide with water-retaining properties, secreted by fibroblasts in the skin. Hyaluronic acid can improve nutrient metabolism, increase elasticity, and protect against skin aging. Its content gradually decreases with age. Halogenated tyrosine increases with age in both photoexposed and photoprotected skin, suggesting that it may be a useful biomarker of skin aging.

[0004] Dermal fillers have long been an important treatment for skin aging. Currently, dermal fillers primarily utilize polymers such as hyaluronic acid and polylactic acid. These materials possess excellent biocompatibility and biodegradability, integrating with human tissue to achieve the purpose of repairing and filling the skin. Furthermore, these materials offer excellent moisturizing and skin rejuvenation properties, improving skin texture and appearance.

[0005] However, existing injectable medical beauty needles have toxic side effects and are prone to a series of problems such as wound infection. Once they accidentally enter the blood, they can easily cause blood clots. Summary of the Invention

[0006] In view of the fact that existing injectable medical aesthetic materials have toxic side effects and are prone to wound infection and other problems, the present invention provides a citric acid-based biomaterial and its preparation method and application.

[0007] The purpose of the present invention can be achieved by the following technical solutions:

[0008] In a first aspect, the present invention provides a method for preparing a POC injectable particulate material, comprising the following steps:

[0009] The POC degradable prepolymer is freeze-dried and then used for standby use;

[0010] The freeze-dried POC degradable prepolymer is dissolved in a solvent, inorganic salt particles are added, cross-linked, and then ground, the inorganic salt is removed, and dried to obtain a POC injectable microparticle material.

[0011] In one embodiment of the present invention, the solvent is selected from one or a combination of dioxane, anhydrous ethanol, acetone or dichloromethane.

[0012] In one embodiment of the present invention, the ratio of the freeze-dried POC degradable prepolymer to the solvent is 10 g: 10-200 mL.

[0013] In one embodiment of the present invention, the inorganic salt particles are selected from one or a combination of NaCl, KCl, MgCl2, etc. The inorganic salt particles serve as pore-forming agents and hardening materials to facilitate subsequent grinding treatment.

[0014] In one embodiment of the present invention, the amount of the inorganic salt particles added is 1-20 times the amount of the freeze-dried POC degradable prepolymer.

[0015] In one embodiment of the present invention, the cross-linking reaction is carried out at a temperature of >50°C, under vacuum or no vacuum, for 12-72 hours. Preferably, the cross-linking reaction is carried out at a temperature of 60-80°C, for 12-72 hours. More preferably, the cross-linking reaction is carried out at a temperature of 80°C, for 72 hours.

[0016] In one embodiment of the present invention, the inorganic salt particles have a size of 50-150 microns before grinding. The POC degradable prepolymer and the inorganic salt undergo a cross-linking reaction, and after grinding, sieve to obtain a POC injectable particulate material. The POC injectable particulate material preferably has a particle size of 30-100 microns or less than 30 microns. The inorganic salts are removed from the sieved POC injectable particulate material by adding purified water to the POC injectable particulate material to dissolve the inorganic salts, and then freeze-drying the POC injectable particulate material.

[0017] In one embodiment of the present invention, the method for preparing the POC degradable prepolymer comprises the following steps:

[0018] The citric acid and 1,8-octanediol are mixed and melted, and the hydroxyl groups of the 1,8-octanediol can be covalently cross-linked with the carboxyl groups of the citric acid. The mixture is then heated and stirred under vacuum conditions to form a POC prepolymer.

[0019] The POC prepolymer is dissolved in an organic solvent to obtain a dissolving solution. Taking advantage of the fact that the prepolymer is immiscible in water, the dissolving solution is slowly poured into ultrapure water for purification multiple times to obtain a purified POC degradable prepolymer.

[0020] In one embodiment of the present invention, the weight ratio of citric acid to 1,8-octanediol is 1:(1-10) to (1-10):1.

[0021] In one embodiment of the present invention, the mixing and melting of citric acid and 1,8-octanediol is carried out under the protection of an inert gas, and the temperature of the mixed dissolution is 120-160° C., preferably 160° C. The inert gas includes nitrogen and the like.

[0022] In one embodiment of the present invention, the stirring reaction under vacuum conditions is carried out at a stirring speed of 300-1000 rpm at 120-140° C. for 1-48 hours, and then the speed is reduced until continuous stirring is no longer possible, and the reaction is stopped. The stirring reaction under vacuum conditions is preferably carried out at a stirring speed of 300 rpm at 140° C. for 12 hours. The operation of reducing the speed can be to first reduce the stirring speed to 150 rpm, and then reduce the stirring speed to 60 rpm until continuous stirring is no longer possible.

[0023] In one embodiment of the present invention, the volume mass ratio of the organic solvent to the POC prepolymer is 1-100:1.

[0024] In one embodiment of the present invention, the organic solvent is selected from one or a combination of dioxane, anhydrous ethanol, acetone or dichloromethane.

[0025] In one embodiment of the present invention, the dissolution of the POC prepolymer by the organic solvent is carried out under stirring conditions, and the stirring conditions are 50-1000 rpm, preferably 300 rpm.

[0026] In one embodiment of the present invention, when the dissolved solution is slowly poured into ultrapure water for purification multiple times, the volume ratio of the dissolved solution to the ultrapure water is 1:10-1000.

[0027] The POC degradable prepolymer can be stored in a refrigerated environment.

[0028] In a second aspect, the present invention provides a POC injectable particulate material prepared by the method described in the first aspect of the present invention.

[0029] The third aspect of the present invention provides a citric acid-based microparticle injection material, comprising the POC injectable microparticle material, and drugs, active molecules, bioglass or metal ions loaded on the POC injectable microparticle material. The metal ions include but are not limited to Mg 2+ ,Ca 2+ ,Zn 2+ ,Cu 2+ wait.

[0030] In one embodiment of the present invention, the drug or active molecule is selected from adenosine triphosphate and glutamine.

[0031] In one embodiment of the present invention, the loading amount of the adenosine triphosphate relative to 1 gram of the POC injectable microparticle material is 5-50 mg, and the loading amount of the glutamine relative to 1 gram of the POC injectable microparticle material is 10-100 mg.

[0032] When the citric acid-based microparticle injection material includes a POC injectable microparticle material, and adenosine triphosphate and glutamine loaded on the POC injectable microparticle material, the citric acid-based microparticle injection material is recorded as ATP-Gln@ / POC.

[0033] In a fourth aspect, the present invention provides a method for preparing a citric acid-based microparticle injection material, wherein a drug or active molecule is combined with the POC injectable microparticle material by incubation or chemical cross-linking.

[0034] The incubation method comprises: dissolving the drug or active molecule and the POC injectable microparticle material in physiological saline or culture medium, incubating them together for 12-48 hours by physical adsorption on a micro-oscillator, and then separating, purifying, and drying the obtained product by multiple centrifugation at 1000-2000 rpm;

[0035] The chemical cross-linking method is that drugs or active molecules are connected to the POC injectable microparticle material through chemical reaction.

[0036] In another embodiment of the present invention, the chemical cross-linking method is: directly heating ATP with glutamine, POC injectable microparticle material and polyol to form a cross-link; or directly heating ATP, glutamine, citric acid and 1,8-pentanediol during the reaction to form a cross-link; or the above-mentioned POC degradable citric acid-based material, in the case of DCC / NHS as a catalyst, is coupled with ATP and glutamine to form a biomimetic microparticle or scaffold.

[0037] A fifth aspect of the present invention provides a citric acid-based biomimetic material comprising the injectable POC particulate material of the second aspect of the present invention and one or a combination of collagen, chitosan, hyaluronic acid, or hydroxyapatite loaded onto the injectable POC particulate material. The loading amount of the collagen, chitosan, hyaluronic acid, and / or hydroxyapatite is 0% to 50% by weight, and is not zero.

[0038] The preparation method of the citric acid-based biomimetic material can be: a combination of POC injectable microparticle material and one or more of collagen, chitosan, hyaluronic acid or hydroxyapatite is used to form biomimetic microparticles or scaffolds by fixing and cross-linking using DCC / DMAP as a catalyst.

[0039] In one embodiment of the present invention, the citric acid-based biomimetic material includes the POC injectable particulate material provided in the second aspect of the present invention, and one or a combination of collagen, chitosan, hyaluronic acid or hydroxyapatite loaded on the POC injectable particulate material, and a drug or active molecule loaded on the POC injectable particulate material.

[0040] In one embodiment of the present invention, the citric acid-based biomimetic material specifically includes the POC injectable particulate material provided in the second aspect of the present invention, and one or more combinations of collagen, chitosan, hyaluronic acid or hydroxyapatite loaded on the POC injectable particulate material, and adenosine triphosphate and glutamine loaded on the POC injectable particulate material.

[0041] The present invention further provides an injection material, which is composed of the following formula components in weight percentage: 1%-50% of the POC injectable microparticle material, 0.5-10% of sodium carboxymethyl cellulose CMC, and 40-95% of sterile physiological saline;

[0042] Alternatively, the injectable material is obtained by dispersing the POC injectable microparticle material with inorganic microparticles such as collagen, hyaluronic acid, bioglass or hydroxyapatite in sterile saline.

[0043] A sixth aspect of the present invention provides the citric acid-based microparticle injection material or the citric acid-based biomimetic material for use in preparing biomedical and aesthetic materials. In particular, it can be used as a biomedical and aesthetic filler material, and more specifically, for skin tissue dermal fillers and breast augmentation implants.

[0044] The present application scheme uses citric acid as the main material. Citric acid is a natural organic acid with good biocompatibility. Therefore, the nanocarrier based on citric acid can reduce the immune rejection reaction of the body when used in vivo. At the same time, it has good drug release performance. Citric acid nanocarriers can be used as drug carriers to improve the efficacy of drugs and reduce side effects by controlling the release rate and release site of drugs in the body. Biomaterials based on citric acid can be obtained through simple chemical synthesis and preparation methods, and the preparation process is relatively simple and low in cost. Citric acid-based biomaterials have good stability and degradability, can stay in the body for a long time and slowly release drugs, and are gradually metabolized and excreted from the body over time.

[0045] Adenosine triphosphate (ATP) is an intracellular energy molecule that provides energy, promoting cell metabolism and growth. During tissue regeneration, ATP promotes cell division and proliferation, accelerating tissue repair and regeneration. ATP also promotes vasodilation, increasing blood flow and boosting blood and oxygen supply to the myocardium, thereby creating a better environment for tissue regeneration.

[0046] Glutamine is an amino acid with multiple functions within cells. During tissue regeneration, glutamine can promote muscle regeneration. It acts as a sensor molecule, controlling the regeneration process. It also promotes protein synthesis and degradation, providing the energy and substances necessary for muscle regeneration.

[0047] Compared with the prior art, the present invention designs a POC injectable microparticle material loaded with ATP and glutamine (Gln) to obtain a citric acid-based microparticle injectable material, which can release bioactive factors (glutamine) and stimulate the production and continuous supply of endogenous ATP in the body's cells, thereby activating epidermal cell regeneration (such as Figure 5 ), proliferation, and significantly promoted the migration of human epidermal fibroblasts HSF (as shown in Figure 6 ) and chemotaxis (as shown) Figure 7 As shown), it promotes the production of collagen and achieves medical beauty effects.

[0048] In the present application, after the POC injectable microparticle material is used to load ATP and glutamine (Gln) to obtain a citric acid-based microparticle injection material, it can also be combined with collagen and / or chitosan and / or hyaluronic acid and / or hydroxyapatite to form a composite bionic microparticle or scaffold material to prolong its degradation time; at the same time, by loading and slowly releasing ATP and / or glutamine, the effect of delaying skin aging and promoting the regeneration of multifunctional human tissues is further enhanced, and it can be applied to medical aesthetic fields such as skin tissue dermal filling and breast augmentation prosthesis.

[0049] The citric acid-based biomaterial provided by this invention, as a novel cosmetic raw material, can promote the regeneration, proliferation, migration, and chemotaxis of skin cells, as well as the production of collagen, achieving cosmetic effects. It can also activate the body's immune response and reduce inflammation. The degradation product, citric acid, is metabolized by the body without significant immunogenicity or toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Development and preparation of ATP-Gln@ / POC injectable medical aesthetic materials combined with hydroxyapatite, chitosan and collagen injectable materials;

[0051] Figure 2 The effects of different concentrations of citric acid on HSF cell proliferation showed that 2000 μM concentration still had a significant promoting effect on HSF cell proliferation;

[0052] Figure 3 The effects of different concentrations of adenosine triphosphate (ATP) on HSF cell proliferation. 10mM concentration can significantly promote HSF cell proliferation.

[0053] Figure 4 Effects of different concentrations of glutamine Gln on HSF cell proliferation. 60mM concentration can significantly promote HSF cell proliferation.

[0054] Figure 5 Detection of aging marker expression;

[0055] Figure 6 Effects of different groups on HSF cell migration;

[0056] Figure 7 Effects of different groups on HSF cell chemotaxis;

[0057] Figure 8 Collagen production. DETAILED DESCRIPTION

[0058] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0059] Example 1

[0060] Preparation and characterization of ATP / Gln@POC injectable material based on citric acid

[0061] 1. First, prepare the POC degradable prepolymer (POC) as follows:

[0062] 20g of citric acid and 15g of 1,8-octanediol were placed in a 50mL round-bottom flask and dissolved at 160°C under nitrogen protection to form a POC prepolymer for 60 minutes. The reaction was stirred at 140°C and 300 rpm for 12 hours under vacuum. The stirring speed was first reduced to 150 rpm and then to 60 rpm until continuous stirring could no longer be achieved. The reaction was stopped by dissolving the prepolymer in 150mL of dioxane reagent under stirring at 300 rpm. The solution was then slowly poured into an appropriate amount of 1.5L of ultrapure water to obtain a purified POC degradable prepolymer. The prepolymer was pre-frozen in a -80°C refrigerator and then freeze-dried in a vacuum for later use.

[0063] 2. Preparation of POC injectable microparticle material, the method is as follows:

[0064] 10 g of purified POC degradable prepolymer was dissolved in 10 mL of dioxane, and then a sufficient amount of ground NaCl inorganic salt particles with a particle size of 50-150 μm was added. The mixture was cross-linked at 80° C. for 72 hours. After grinding, the POC injectable microparticle material was obtained.

[0065] 3. Preparation of POC-loaded ATP / glutamine (ATP-Gln@ / POC) injectable granules by:

[0066] ATP, glutamine, and 1 mol / L ATP / glutamine solutions were prepared and completely dissolved in DMEM high-glucose medium to achieve a concentration of 10 mM (calculated based on the amount of citric acid), 10 mM ATP, and 20 mM Gln for the injectable POC microparticles. The microparticles were then incubated for 24 hours on a micro-oscillator via physical adsorption. Injectable ATP@POC (ATP 10 mM, POC 10 mM), Gln@POC (Gln 20 mM, POC 10 mM), and ATP-Gln@POC (ATP 10 mM, Gln 20 mM, POC 10 mM) were constructed.

[0067] 4. Injectable ATP-Gln@ / POC promotes skin cell proliferation

[0068] Skin cells were co-incubated with injectable ATP@POC, Gln@POC, and ATP-Gln@POC, and cell proliferation was assessed using CCK-8 and flow cytometry. A cellular senescence model was established by stimulating HSF cells with 20 mg / mL of D-galactose for 24 hours.

[0069] The Cellular Senescence Detection Kit-SPiDER-βGal was used to assess the effect of ATP-Gln@ / POC in delaying HSF cell senescence. Bafilomycin A1 working solution (dilute Bafilomycin A1 DMSO stock solution 1,000-fold with culture medium or HBSS), SPiDER-βGal working solution (mix SPiDERβGal DMSO stock solution and Bafilomycin A1 DMSO stock solution and dilute the mixture 1,000-fold with DMEM high-glucose medium), and stock solution (dilute 1 mL of SPiDER-βGal working solution with 1 mL of culture medium or 1 mL of HBSS to 1 mL of SPiDER-βGal working solution) were prepared. After HSF cells were seeded in 35 mm culture dishes, a control group (PBS phosphate buffer treatment group, no active ingredient), a hyaluronic acid (HA, 10 mM) group, a citric acid group (10 mM), a free ATP group (10 mM), a free Gln group (20 mM), ATP@POC (ATP 10 mM, POC 10 mM), Gln@POC (Gln 20 mM, POC 10 mM), and ATP-Gln@ / POC (ATP 10 mM, Gln 20 mM, POC 10 mM) were set up and cultured in culture medium in a 37°C, 5% CO2 incubator overnight; after removing the culture medium, the cells were washed once with 2 ml of culture medium or 2 mL of HBSS; after adding 1 mL of Bafilomycin A1 working solution, the cells were cultured in a 37°C, 5% CO2 incubator for 1 h. After adding 1 mL of SPiDER-βGal working solution, incubate in a 37°C, 5% CO2 incubator for 30 min; remove the supernatant, wash twice with 2 mL of culture medium or 2 mL of HBSS, and observe under a fluorescence microscope.

[0070] 5. ATP-Gln@ / POC injectable filler material (i.e., ATP-Gln@ / POC in the above examples) promotes the migration of skin cells

[0071] Cell migration can be detected by cell scratching combined with live and dead cell fluorescence staining. HSF cells were digested with trypsin, centrifuged and counted, and 1×10 6 Cells were seeded into 6-well plates at 100 cells / well and cultured overnight in an incubator. After the cells attached, three parallel lines were drawn perpendicular to the well wall using a sterile toothpick. PBS was slowly injected along the well wall, rinsed three times, and the drawn cells were removed. A control group, a citric acid group, a free ATP group, a free Gln group, ATP@POC, Gln@POC, and an ATP-Gln@ / POC group were set up. The cells were observed and photographed under a microscope at 0, 24, and 48 hours, and intercellular spacing was analyzed using Image J software.

[0072] 6. Effect of ATP-Gln@ / POC injectable filler material on cell chemotaxis

[0073] At the same time, the effect on cell chemotaxis was detected by Tanswell assay. HSF cells with a confluence of about 90% and in the logarithmic growth phase were selected and cultured overnight in serum-free DMEM medium to starve the cells. The cells were then digested with trypsin and the concentration of the cell suspension was adjusted to 1×10 5 / mL. Complete culture medium and 600μL of prepared culture medium with different substances were added to the 24-well plate respectively, 200μL of cell suspension was added to the Tanswell chamber, and the cells were evenly distributed by shaking gently. Be careful not to generate bubbles at the bottom of the chamber. The Tanswell chambers were placed in 24-well plates respectively and cultured in a cell culture incubator for 24 hours. Discard the liquid in the upper and lower chambers, wash the cells 2-3 times with PBS, add 600μL of 4% paraformaldehyde to the lower chamber to fix the cells for 15 minutes (shaker, 50rpm), remove the cell fixative, add 600μL of crystal violet staining solution to each lower chamber, and stain for 10 minutes. Wash off the excess crystal violet dye with PBS, wipe off the unmigrated cells in the chamber with a cotton swab, and invert the chamber to air dry naturally at room temperature. Observe under a microscope, and take 3 fields of view in each well. Count the cells.

[0074] 7. Effect of ATP-Gln@ / POC injectable filler material on collagen production in HSF cells

[0075] After 24 hours of treatment with the control group, citric acid group, free ATP group, free Gln group, ATP@POC, Gln@POC, and ATP-Gln@ / POC, the cells were washed three times with PBS to remove the cell culture medium, fixed with 600 μL of 4% paraformaldehyde for 15 minutes (shaking incubator, 50 rpm), washed three times with PBS to remove the cell fixative, and stained with Sirius red staining solution for 10-60 minutes; rinsed briefly with running water, washed three times with PBS to remove the surface stain; stained with Mayer's hematoxylin staining solution for 8-10 minutes, washed three times with PBS to remove the surface stain, and examined under a microscope.

[0076] Results and Discussion

[0077] Through the above method, the citric acid-based ATP / Gln@POC injectable material was successfully constructed. Figure 1 As shown in the top picture, it has excellent injectability and stability and can be used for further research. At the same time, it can form composite hydrogels with collagen, hydroxyapatite, chitosan, etc., to realize the development and preparation of injectable hydrogel medical beauty materials.

[0078] By testing the effects of different release and degradation products of ATP-Gln@ / POC on the proliferation of human epidermal fibroblasts HSF, the results showed that 2000μM citric acid, 10mM ATP and 60mM Gln can significantly promote the proliferation of HSF cells. Figure 2-4 The subsequent ATP-Gln@ / POC concentration was determined based on this result.

[0079] in, Figure 2 The figure shows the effect of different concentrations of citric acid on the proliferation of HSF cells. The concentration of 2000 μM can still significantly promote the proliferation of HSF cells.

[0080] Figure 3 Figure 3 is the effect of different concentrations of adenosine triphosphate (ATP) on HSF cell proliferation. 10 mM concentration can significantly promote HSF cell proliferation.

[0081] Figure 4 Figure 3 shows the effects of different concentrations of glutamine Gln on HSF cell proliferation. 60 mM concentration can significantly promote HSF cell proliferation.

[0082] Delaying cell aging

[0083] SA-β-Gal expression increases with cell aging, and the process of cell aging can be monitored by the change in the number of stained cells. Figure 5 ). Senescent cells were observed in the negative control group after D-gal treatment of HSF cells. However, after 24 hours of incubation, the positive group (HA, 10mM) effectively reduced the number of senescent cells by approximately 50% compared to the model group. The number of senescent cells in the experimental group treated with the injectable ATP-Gln@POC medical aesthetic material was not significantly different from that in the positive control group (D-galactose), and was significantly superior to the positive control (HA) group. These results demonstrate that the injectable ATP-Gln@POC medical aesthetic material can effectively delay the aging of HSF cells and has a superior anti-aging effect.

[0084] Effects on HSF cell migration and chemotaxis

[0085] The migration and chemotaxis of HSF cells are of great significance for the regeneration of aging skin. Therefore, the Transwell assay was used to test the effect of injectable ATP-Gln@POC medical aesthetic material on cell migration. Live and dead cell staining was used to observe cell morphology and count the cell scratch distance. Crystal violet can bind to DNA in the cell nucleus, making the cell nucleus appear purple. After cell staining, the counting results are as follows. Figure 6 and Figure 7As shown, compared with the control group and the positive control HA group, the number of cells crossing the chamber in the ATP-Gln@POC experimental group increased significantly, and the number of migrating cells also increased, and was significantly better than the positive control (HA) group. These results indicate that the injectable ATP-Gln@POC medical aesthetic material can effectively promote the migration and chemotaxis of HSF cells.

[0086] Effects on collagen expression in HSF cells

[0087] Many factors cause the aging of skin HSF cells, which ultimately affect the expression of collagen in HSF cells. As HSF cell aging increases, the level of collagen precursors in the cells decreases, further exacerbating aging. Figure 8 As shown in the figure, after D-gal treatment, the expression of collagen precursors was significantly reduced. Sirius red staining was used to explore the changes in collagen fibers. Collagen fibers appear orange-yellow, with higher collagen fibers in young skin and lower collagen fiber ratios in aged skin. After incubation with ATP-Gln@POC, the production of collagen fibers in human skin fibroblasts was effectively promoted, and was significantly better than the positive control (HA) group. In summary, ATP-Gln@POC can stimulate the production of collagen fibers in the aged dermis to reshape the extracellular matrix, thereby achieving the effect of delaying skin aging.

[0088] As can be seen, the present invention develops and designs a POC injectable medical aesthetic material that can load bioactive substances such as ATP, glutamine, or active metal ions through mesoporous adsorption or chemical reaction. It can also be combined with, but not limited to, collagen, chitosan, and hydroxyapatite for synergistic injection. This invention can promote skin tissue regeneration and anti-aging, providing new materials and methods for medical aesthetics, anti-aging, or tissue regeneration.

[0089] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. A citric acid-based microparticle injection material, characterized in that: The invention comprises a POC injectable particulate material, and adenosine triphosphate and glutamine loaded on the POC injectable particulate material. The loading amount of the adenosine triphosphate relative to 1 gram of the POC injectable microparticle material is 5-50 mg, and the loading amount of the glutamine relative to 1 gram of the POC injectable microparticle material is 10-100 mg; The preparation method of the POC injectable microparticle material is: The POC degradable prepolymer is freeze-dried and then used for standby use; The freeze-dried POC degradable prepolymer is dissolved in a solvent, inorganic salt particles are added, cross-linked, ground, the inorganic salt is removed, and dried to obtain a POC injectable microparticle material; The preparation method of the POC degradable prepolymer comprises the following steps: The citric acid and 1,8-octanediol are mixed and melted, and then heated and stirred under vacuum conditions to react to form a POC prepolymer; The POC prepolymer is dissolved in an organic solvent to obtain a dissolving solution, and the dissolving solution is slowly poured into ultrapure water for purification multiple times to obtain a purified POC degradable prepolymer.

2. The citric acid-based microparticle injection material according to claim 1, characterized in that: The solvent is selected from one or a combination of dioxane, anhydrous ethanol, acetone or dichloromethane; The dosage of the freeze-dried POC degradable prepolymer and the solvent is 10 g: 10-200 mL.

3. The citric acid-based microparticle injection material according to claim 1, characterized in that: The inorganic salt particles are selected from one or a combination of NaCl, KCl, and MgCl2; the added amount of the inorganic salt particles is 1-20 times the mass of the freeze-dried POC degradable prepolymer.

4. The citric acid-based microparticle injection material according to claim 1, characterized in that: The cross-linking reaction conditions are: >50° C., cross-linking for 12-72 hours under vacuum or no vacuum conditions.

5. The citric acid-based microparticle injection material according to claim 1, characterized in that: The weight ratio of citric acid to 1,8-octanediol is 1:(1-10) to (1-10):1; the mixing and melting of citric acid and 1,8-octanediol is carried out under the protection of inert gas, and the temperature of the mixing and melting is 160°C; The stirring reaction conditions under vacuum conditions are: 120-140° C., 300-1000 rpm, reaction time of 1-48 hours, then reducing the speed until continuous stirring is impossible, and the reaction is stopped.

6. The citric acid-based microparticle injection material according to claim 1, characterized in that: The volume mass ratio of the organic solvent to the POC prepolymer is 1-100:1; The organic solvent is selected from one or a combination of dioxane, anhydrous ethanol, acetone or dichloromethane; Dissolving the POC prepolymer by an organic solvent is carried out under stirring conditions, wherein the stirring conditions are 50-1000 rpm; When the dissolved solution is slowly poured into ultrapure water for purification multiple times, the volume ratio of the dissolved solution to the ultrapure water is 1:10-1000.

7. The method for preparing a citric acid-based microparticle injection material according to claim 1, wherein: Adenosine triphosphate and glutamine are combined with the POC injectable microparticle material by incubation or chemical cross-linking. The incubation method comprises: dissolving adenosine triphosphate and glutamine and the POC injectable microparticle material in physiological saline or culture medium, incubating them together for 12-48 hours by physical adsorption on a micro-oscillator, and then separating, purifying, and drying them by centrifugation at 1000-2000 rpm for multiple times; The chemical cross-linking method is as follows: adenosine triphosphate and glutamine are connected to the POC injectable microparticle material through chemical reaction.

8. A citric acid-based biomimetic material, characterized in that: The invention comprises the citric acid-based microparticle injection material according to claim 1, and one or more of collagen, chitosan, hyaluronic acid or hydroxyapatite loaded on the citric acid-based microparticle injection material.

9. The citric acid-based biomimetic material according to claim 8, characterized in that: The loading amount of one or a combination of collagen, chitosan, hyaluronic acid or hydroxyapatite is 0%-50 wt %, and is not 0.

10. An injection material, characterized in that: The injection material is composed of the following formula components in weight percentage: 1%-50% of the citric acid-based microparticle injection material according to claim 1, 0.5-10% of sodium carboxymethyl cellulose CMC, and 40-95% of sterile physiological saline; Alternatively, the injectable material is obtained by dispersing the citric acid-based microparticle injection material according to claim 1 with collagen, hyaluronic acid, bioglass or hydroxyapatite microparticles in sterile saline.

11. A use of the citric acid-based microparticle injection material according to claim 1 or the citric acid-based biomimetic material according to claim 8, characterized in that: The citric acid-based microparticle injection material or the citric acid-based biomimetic material is used to prepare biomedical and cosmetic materials.

12. The use according to claim 11, characterized in that The biomedical cosmetic materials include skin tissue dermal filling materials and breast augmentation prosthesis materials.

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