Citric acid-based biomaterial, preparation method therefor, and use thereof

Abstract

The present invention relates to a citric acid-based particulate injection material, a preparation method therefor, and the use thereof. Citric acid and 1,8-octanediol are melted and heated to form a POC prepolymer; the POC prepolymer is dissolved by means of an organic solvent to obtain a dissolved solution, and the dissolved solution is slowly poured into ultrapure water multiple times for purification to obtain a purified POC degradable prepolymer; the POC degradable prepolymer is dissolved by means of a solvent, inorganic salt particles are added, and a crosslinking reaction is carried out to obtain a POC injectable particulate material. A drug, an active molecule, and metal ions are loaded onto the POC by means of physical compounding or chemical crosslinking to obtain a citric acid-based particulate injection material. As a novel aesthetic medicine raw material and a related particulate injection filler thereof, the citric acid-based biomaterial provided in the present invention can effectively promote the regeneration, proliferation, migration, and chemotaxis of skin cells, promote the production of collagen, and achieve an aesthetic medicine effect. The present invention can also activate the body's immune response to reduce inflammation, and a product after degradation is citric acid, which can be metabolized by the body with no obvious immunogenicity and toxic side effects.

Description

A citric acid-based biomaterial and a preparation method and application thereof TECHNICAL FIELD

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

[0002] Skin aging is a health problem that everyone has to face. With age and the accumulation of environmental factors, skin surface dryness, wrinkles, loss of elasticity, and other phenomena gradually intensify, and even lead to skin dysfunction, increasing the risk of skin diseases and skin malignancies. In the pursuit of youth, beauty, and health, skin aging brings psychological and physiological burdens to people. Currently, anti-skin aging strategies such as retinoic acid, radiofrequency technology, and dermal filling are used to stimulate the production of collagen in the dermis to restore the function of aging skin. Skin aging is a health problem that everyone has to face, and with increasing age, skin aging becomes more severe, with increasingly apparent symptoms, greatly increasing people's concerns about appearance changes.

[0003] Skin aging is closely related to changes in collagen. The amino acid with the highest content in collagen is hydroxyproline, which mainly exists in collagen in the skin compared to other tissues. Therefore, it can be used as an indicator for evaluating skin aging. Hyaluronic acid is an acidic glycosaminoglycan with water-retaining effects, which is secreted by fibroblasts in the skin. Hyaluronic acid can improve nutrient metabolism, increase elasticity, and prevent skin aging, and its content gradually decreases with age. Halogenated tyrosine increases with age in both light-exposed and light-protected skin, indicating that it can be a useful biomarker for skin aging.

[0004] Dermal filling technology has always been an important means of treating skin aging. Currently, skin filling materials mainly use high molecular materials such as hyaluronic acid and polylactic acid. These materials have good biocompatibility and biodegradability, can integrate with human tissues, and achieve the purpose of repairing and filling the skin. At the same time, these materials also have good moisturizing and skin-smoothing effects, which can improve the texture and appearance of the skin.

[0005] However, existing injectable medical aesthetic needles have toxic side effects and are prone to wound infections and other problems. Once they enter the blood, they can cause thrombosis and other problems. SUMMARY

[0006] Based on the toxic side effects and wound infection-prone problems of existing injectable medical aesthetic materials, the present application provides a citric acid-based biomaterial and a preparation method and application thereof.

[0007] The object of the present application can be achieved by the following technical solutions.

[0008] In the first aspect of the present application, a preparation method of POC injectable microparticle material is provided, comprising the following steps:

[0009] The POC degradable prepolymer is freeze-dried for standby;

[0010] The freeze-dried POC degradable prepolymer is dissolved in a solvent, inorganic salt particles are added, cross-linking reaction is carried out, and after grinding, removing inorganic salt and drying, the POC injectable microparticle material is obtained.

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

[0012] In an embodiment of the present application, the amount of freeze-dried POC degradable prepolymer and solvent is 10g:10-200mL.

[0013] In an embodiment of the present application, the inorganic salt particles are selected from one or a combination of several of NaCl, KCl, MgCl2, etc., and the inorganic salt particles are used as a pore-forming agent and a hardening material to facilitate subsequent grinding treatment.

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

[0015] In an embodiment of the present application, the cross-linking reaction conditions are >50℃, cross-linking for 12-72 hours under vacuum or non-vacuum conditions. Preferably, cross-linking for 12-72 hours at 60-80℃. Further preferably, cross-linking for 72 hours at 80℃.

[0016] In an embodiment of the present application, the size of the inorganic salt particles before grinding is 50-150 microns. After the cross-linking reaction of the POC degradable prepolymer and the inorganic salt, and after grinding, the POC injectable microparticle material is obtained by sieving, and the particle size of the POC injectable microparticle material is preferably 30-100 microns or <30 microns. The method for removing the inorganic salt from the POC injectable microparticle material obtained by sieving is to add pure water to the POC injectable microparticle material to dissolve the inorganic salt, and then freeze-dry the POC injectable microparticle material.

[0017] In an embodiment of the present application, the preparation method of the POC degradable prepolymer comprises the following steps:

[0018] The citric acid and 1,8-octanediol are mixed and melted, the hydroxyl group of 1,8-octanediol is covalently crosslinked with the carboxyl group of citric acid, and then the POC prepolymer is formed by stirring and reacting under vacuum and heating;

[0019] The POC prepolymer is dissolved in an organic solvent to obtain a solution, and the solution is slowly poured into ultrapure water for purification multiple times, and the purified POC degradable prepolymer is obtained by taking advantage of the immiscibility in water.

[0020] In an embodiment of the present application, the amount of citric acid and 1,8-octanediol is in a weight ratio of 1:(1-10) to (1-10):1.

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

[0022] In an embodiment of the present application, the stirring and reaction under vacuum conditions are carried out at 120-140°C, a stirring speed of 300-1000 rpm, and a reaction time of 1-48 hours, and then the stirring speed is reduced until the stirring cannot be continued, and the reaction is stopped. The stirring and reaction under vacuum conditions are preferably carried out at 140°C, a stirring speed of 300 rpm, and a reaction time of 12 hours. The operation of reducing the stirring speed can be first reducing the stirring speed to 150 rpm, and then reducing the stirring speed to 60 rpm until the stirring cannot be continued.

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

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

[0025] In an embodiment of the present application, the dissolving of the POC prepolymer in the organic solvent is carried out under stirring conditions, and the stirring conditions are 50-1000 rpm, preferably 300 rpm.

[0026] In an embodiment of the present application, when the solution is slowly poured into ultrapure water for purification multiple times, the volume ratio of the 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 of the present application, a POC injectable microparticle material prepared by the method of the first aspect of the present application is provided.

[0029] In a third aspect of the present application, a citrate-based injectable microparticle material is provided, comprising the POC injectable microparticle material, and a drug or an active molecule, a bioglass or a metal ion loaded on the POC injectable microparticle material. The metal ion includes but is not limited to Mg2+, Ca2+, Zn2+, Cu2+ and the like.

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

[0031] In an embodiment of the present application, the loading amount of the adenosine triphosphate is 5-50 mg per 1 g of the POC injectable microparticle material, and the loading amount of the glutamine is 10-100 mg per 1 g of the POC injectable microparticle material.

[0032] When the citrate-based injectable microparticle material comprises the POC injectable microparticle material, and the adenosine triphosphate and the glutamine loaded on the POC injectable microparticle material, the citrate-based injectable microparticle material is denoted as ATP-Gln@ / POC.

[0033] In a fourth aspect of the present application, a preparation method of the citrate-based injectable microparticle material is provided, in which a drug or an active molecule and the POC injectable microparticle material are combined together by incubation or chemical crosslinking,

[0034] The incubation method is as follows: the drug or the active molecule and the POC injectable microparticle material are dissolved in physiological saline or culture medium, and are co-incubated by physical adsorption through a micro-vibration incubator for 12-48 hours, and then are separated, purified and dried by centrifugation at 1000-2000 rpm for multiple times to obtain the citrate-based injectable microparticle material.

[0035] The chemical crosslinking method is as follows: the drug or the active molecule is connected to the POC injectable microparticle material by chemical reaction.

[0036] In another embodiment of the present application, the chemical crosslinking method is as follows: adenosine triphosphate is directly heated and crosslinked with glutamine, the POC injectable microparticle material and polyol; or ATP, glutamine, citric acid and 1,8-pentanediol are directly heated to form crosslinking during reaction; or the above POC degradable citrate-based material is coupled with adenosine triphosphate and glutamine to form a biomimetic microparticle or a scaffold under the catalysis of DCC / NHS.

[0037] In a fifth aspect, the present application provides a citrate-based biomimetic material, comprising the POC injectable microparticle material provided in the second aspect of the present application, and one or a combination of collagen, chitosan, hyaluronic acid or hydroxyapatite loaded on the POC injectable microparticle material. The loading amount of collagen, chitosan and / or hyaluronic acid and / or hydroxyapatite is 0-50wt%, and is not 0.

[0038] The preparation method of the citrate-based biomimetic material can be: forming a biomimetic microparticle or scaffold by fixing and cross-linking the POC injectable microparticle material and one or a combination of collagen, chitosan, hyaluronic acid or hydroxyapatite by DCC / DMAP as a catalyst.

[0039] In an embodiment of the present application, the citrate-based biomimetic material comprises the POC injectable microparticle material provided in the second aspect of the present application, and one or a combination of collagen, chitosan, hyaluronic acid or hydroxyapatite loaded on the POC injectable microparticle material, and a drug or active molecule loaded on the POC injectable microparticle material.

[0040] In an embodiment of the present application, the citrate-based biomimetic material specifically comprises the POC injectable microparticle material provided in the second aspect of the present application, and one or a combination of collagen, chitosan, hyaluronic acid or hydroxyapatite loaded on the POC injectable microparticle material, and adenosine triphosphate and glutamine loaded on the POC injectable microparticle material.

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

[0042] Or an injectable material obtained by dispersing the POC injectable microparticle material and one or a combination of collagen, hyaluronic acid, bioglass or hydroxyapatite in sterile physiological saline.

[0043] In a sixth aspect, the present application provides the citrate-based microparticle injection material or the citrate-based biomimetic material for preparing biomedical and medical aesthetic materials. In particular, as a biomedical and medical aesthetic filling material, more specifically, it can be used for skin tissue dermal filling and breast augmentation prosthesis.

[0044] The application scheme takes citric acid as the main material. Citric acid is a natural organic acid with good biocompatibility. Therefore, the nano-carrier based on citric acid can reduce the immune rejection reaction of the body when applied in vivo. At the same time, it has good drug release performance. The citric acid nano-carrier can be used as a drug carrier to improve the efficacy of the drug and reduce side effects by controlling the release rate and release site of the drug in the body. The citric acid-based biomaterial can be obtained by a simple chemical synthesis and preparation method, and the preparation process is relatively simple and low in cost. The citric acid-based biomaterial has good stability and degradability, can stay in the body for a long time and slowly release the drug, and at the same time, it is gradually metabolized and excreted out of the body over time.

[0045] Adenosine triphosphate (ATP) is an energy molecule in cells that can provide energy for cells, promote cell metabolism and growth. During tissue regeneration, ATP can promote cell division and proliferation, accelerate tissue repair and regeneration. ATP can also promote vasodilation, increase blood flow, and increase blood supply and oxygen supply to the myocardium, thereby providing a better environment for tissue regeneration.

[0046] Glutamine is an amino acid with multiple functions in cells. During tissue regeneration, glutamine can promote muscle tissue regeneration. Glutamine can act as a sensor molecule to control the regeneration program of muscle tissue. At the same time, glutamine can also promote protein synthesis and decomposition, providing the energy and substances needed for muscle regeneration.

[0047] Compared with the prior art, the application designs a POC injectable microparticle material loaded with ATP and glutamine (Gln) to obtain a citric acid-based microparticle injection material, which can release bioactive factors (glutamine) and stimulate the production and continuous supply of endogenous ATP in cells, thereby activating epidermal cell regeneration (as shown in FIG. 5), proliferation, significantly promoting the migration (as shown in FIG. 6) and chemotaxis (as shown in FIG. 7) of human epidermal fibroblasts HSF, and promoting the production of collagen, achieving a medical and aesthetic effect.

[0048] In the application, the POC injectable microparticle material is used to load ATP and glutamine (Gln) to obtain a citric acid-based microparticle injection material, which can be combined with collagen and / or chitosan and / or hyaluronic acid and / or hydroxyapatite to form a composite biomimetic microparticle or scaffold material, prolonging the degradation time. At the same time, by loading and slow-releasing ATP and / or glutamine, the effect of delaying skin aging and promoting the regeneration of human multi-functional tissues is further enhanced, which can be applied to skin tissue dermal filling, breast augmentation prosthesis and other medical and aesthetic fields.

[0049] The citric acid-based biomaterial provided by the application can promote the regeneration, proliferation, migration and chemotaxis of skin cells, promote the production of collagen, and achieve medical and beauty effects. Meanwhile, the citric acid-based biomaterial can activate the immune response of the body and reduce inflammation, and the degradation product is citric acid, which can be metabolized by the body and has no obvious immunogenicity and side effects. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 Development and preparation of ATP-Gln@ / POC injectable medical and beauty material combined with hydroxyapatite and chitosan and collagen injection material;

[0051] Figure 2 Effect of different concentrations of citric acid on HSF cell proliferation, and 2000 μM concentration can still significantly promote HSF cell proliferation;

[0052] Figure 3 Effect of different concentrations of adenosine triphosphate ATP on HSF cell proliferation, and 10 mM concentration can significantly promote HSF cell proliferation;

[0053] Figure 4 Effect of different concentrations of glutamine Gln on HSF cell proliferation, and 60 mM concentration can significantly promote HSF cell proliferation;

[0054] Figure 5 Detection of expression of senescence markers;

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

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

[0057] Figure 8 Production of collagen;

[0058] Figure 9 Regeneration of skin tissue promoted by citric acid microspheres, in which A: HE staining, histopathological observation of regenerated tissue morphology and inflammatory reaction; B: immunohistochemical detection of expression level of pro-inflammatory factor TNFα; C: observation of tissue morphology and collagen expression in the tissue by Masson staining; and D: observation of expression levels of type I collagen and type III collagen in the tissue by Sirius red staining. DETAILED DESCRIPTION

[0059] The application will be described in detail below in combination with the drawings and specific examples.

[0060] Example 1

[0061] Preparation and characterization of ATP / Gln@POC injectable material based on citric acid

[0062] 1. First, the preparation of POC degradable prepolymer (POC) is carried out, and the method is as follows:

[0063] Take citric acid 20 g and 1,8-octanediol 15 g in a 50 mL round-bottom flask, 160°C, nitrogen protection conditions, dissolved to form POC prepolymer, 60 minutes. By vacuum conditions, 140°C, 300 rpm stirring reaction 12 hours, first to 150 rpm stirring speed, and then reduce to 60 rpm stirring speed, until the stirring can not continue to stop the reaction. By 150 mL dioxane reagent in 300 rpm stirring. Again, the dissolved solution is slowly poured into an appropriate amount of 1.5 L ultrapure water, obtain purified POC degradable prepolymer, pre-frozen in-80°C refrigerator, vacuum freeze-dried after standby.

[0064] 2, POC injectable microparticle material preparation, the method is as follows:

[0065] Configuration 10 g of purified POC degradable prepolymer is dissolved in 10 mL of dioxane, and enough amount of 50-150 micron size NaCl inorganic salt particles is added, and crosslinked at 80°C for 72 hours. After grinding, POC injectable microparticle material is obtained.

[0066] 3, POC load ATP / glutamine (ATP-Gln@ / POC) injectable particle material preparation, the method is:

[0067] Respectively configure ATP, glutamine, ATP / glutamine 1 mol / L DMEM high-sugar medium solution, so that it is completely dissolved, so that the concentration of POC injectable microparticle material is 10 mM (calculated as the amount of citric acid), ATP concentration is 10 mM, Gln concentration is 20 mM, by physical adsorption, through the micro-vibration incubator for 24 hours. Respectively construct injectable ATP@POC (ATP 10 mM, POC 10 mM), Gln@POC (Gln 20 mM, POC 10 mM), ATP-Gln@POC (ATP 10 mM, Gln 20 mM, POC 10 mM).

[0068] 4, injectable ATP-Gln@ / POC promotes skin cell proliferation

[0069] Respectively through injectable ATP@POC, Gln@POC, ATP-Gln@POC and skin cells co-incubation, through CCK-8 and flow cytometry detection of cell proliferation. D-galactose concentration of 20 mg / mL when stimulating HSF cells for 24 h to establish cell aging model.

[0070] The ATP-Gln@ / POC delayed HSF cell senescence effect was detected by a cell senescence detection kit-SPiDER-βGal. Bafilomycin A1 working solution (dilute Bafilomycin A1 DMSO stock solution 1,000 times with culture medium or HBSS), SPiDER-βGal working solution (mix SPiDERβGal DMSO stock solution and Bafilomycin A1 DMSO stock solution, dilute the mixed solution 1,000 times with DMEM high glucose culture medium), and storage solution (dilute 1 mL of SPiDER-βGal working solution to 1 mL with 1 mL of culture medium or 1 mL of HBSS) were prepared. After HSF cells were inoculated in a 35 mm culture dish, control group (PBS phosphate buffer treatment group, no active ingredients), hyaluronic acid (Hyaluronic Acid, HA, 10 mM) group, citric acid group (10 mM), free ATP group (10 mM), 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 the culture medium was set at 37°C in a 5% CO2 incubator overnight; after removing the culture medium, 2 mL of culture medium or 2 mL of HBSS was used for washing once; 1 mL of Bafilomycin A1 working solution was added, and the culture was incubated at 37°C in a 5% CO2 incubator for 1 h. After adding 1 mL of SPiDER-βGal working solution, the culture was incubated at 37°C in a 5% CO2 incubator for 30 min; after removing the supernatant, 2 mL of culture medium or 2 mL of HBSS was used for washing twice, and then the observation was performed under a fluorescence microscope.

[0071] 5. ATP-Gln@ / POC injectable filling material (i.e. ATP-Gln@ / POC in the above embodiment) promotes the migration of skin cells

[0072] The migration of cells can be detected by cell scratch combined with live cell fluorescent staining. HSF cells were digested with trypsin, counted after centrifugation, and inoculated into a 6-well plate at 1 × 106 cells / well, and incubated in an incubator overnight; after the cells adhered, 3 parallel lines were scratched in the well perpendicular to the well wall with a sterile toothpick; PBS was slowly injected along the well wall, and the scratched cells were removed after 3 times of rinsing; control group, citric acid group, free ATP group, free Gln group, ATP@POC, Gln@POC, and ATP-Gln@ / POC were set up respectively. At 0 h, 24 h, and 48 h, the observation and photography were performed under a microscope, and the cell spacing was analyzed using Image J software.

[0073] 6. The effect of ATP-Gln@ / POC injectable filling material on cell chemotaxis

[0074] Meanwhile, the effect on cell chemotaxis was detected by Tanswell experiment. HSF cells with about 90% confluence and in logarithmic growth phase were selected, and were cultured in serum-free DMEM medium overnight to make the cells in a state of starvation. Then the cells were trypsinized, and the concentration of the cell suspension was adjusted to 1x105 / mL with serum-free medium. In a 24-well plate, 600 μL of complete medium and the prepared medium with different substances were added, respectively. 200 μL of the cell suspension was added to the Tanswell chamber, and the cells were evenly distributed by gently shaking. It was noted that no bubbles were generated at the bottom of the chamber. The Tanswell chamber was placed in the 24-well plate and cultured in a cell incubator for 24 h. The liquid in the upper and lower chambers was discarded, and the cells were washed with PBS for 2-3 times. 600 μL of 4% paraformaldehyde was added to the lower chamber to fix the cells for 15 min (shaker, 50 rpm). The cell fixation liquid was removed, 600 μL of crystal violet staining solution was added to each lower chamber, and the cells were stained for 10 min. The excess crystal violet dye was washed with PBS, and the cells that did not migrate in the chamber were removed with a cotton swab. The chamber was inverted and naturally air-dried at room temperature. Under a microscope, 3 fields of view were taken for each well. The cells were counted.

[0075] 7. The effect of ATP-Gln@ / POC injectable filling material on collagen production of HSF cells

[0076] After 24 hours of treatment of the control group, the citric acid group, the free ATP group, the free Gln group, the ATP@POC group, the Gln@POC group, and the ATP-Gln@ / POC group, the cells were washed with PBS for 3 times to remove the cell culture liquid, 600 μL of 4% paraformaldehyde was added to fix the cells for 15 min (shaker, 50 rpm), and the cell fixation liquid was removed by washing with PBS for 3 times. The Sirius red staining solution was added dropwise and the cells were stained for 10-60 min. The surface staining liquid was removed by washing with running water and PBS for 3 times. The Mayer hematoxylin staining solution was added dropwise and the cells were stained for 8-10 min. After washing with PBS for 3 times to remove the surface staining liquid, the cells were observed under a microscope.

[0077] Result analysis and discussion:

[0078] Through the above method, the ATP / Gln@POC injectable material based on citric acid was successfully constructed as shown in the topmost graph of FIG. 1, which has excellent injectability and stability, and can be used for further research. Meanwhile, the material can be used to form a composite hydrogel with collagen, hydroxyapatite, chitosan, etc., to realize the development and preparation of an injectable hydrogel medical and beauty material.

[0079] The effect of different release and degradation products of ATP-Gln@ / POC on the proliferation of human epidermal fibroblasts HSF was detected. The results showed that 2000 μM of citric acid, 10 mM of ATP and 60 mM of Gln can significantly promote the proliferation of HSF cells, as shown in Figures 2-4. The subsequent concentration of ATP-Gln@ / POC was determined according to the results.

[0080] Figure 2 shows the effect of different concentrations of citric acid on the proliferation of HSF cells. The 2000 μM concentration can still significantly promote the proliferation of HSF cells.

[0081] Figure 3 shows the effect of different concentrations of ATP on the proliferation of HSF cells. The 10 mM concentration can significantly promote the proliferation of HSF cells.

[0082] Figure 4 shows the effect of different concentrations of Gln on the proliferation of HSF cells. The 60 mM concentration can significantly promote the proliferation of HSF cells.

[0083] Effect of delaying cell aging

[0084] SA-β-Gal can increase its expression with cell aging, and the progress of cell aging can be monitored by the change in the number of stained cells. Qualitative and quantitative analysis by SPiDER-βGal kit showed (Figure 5) that the negative control group showed D-gal treated HSF cells presenting stained senescent cells, and the positive group (HA, 10 mM) effectively reduced the number of senescent cells by about 50% after 24 hours of incubation compared with the model group. The number of senescent cells in the injectable ATP-Gln@POC medical material experimental group was not significantly different from the positive control group (D-gal), and was significantly better than the positive control (HA) group. The results showed that the injectable ATP-Gln@POC medical material can effectively delay the aging of HSF cells, and has better anti-aging effect.

[0085] Effect on HSF cell migration and chemotaxis

[0086] The migration and chemotaxis of HSF cells are of great significance for the regeneration of aged skin. Therefore, the Transwell experiment was used to detect the effect of the injectable ATP-Gln@POC medical aesthetic material on cell migration. The cell morphology was observed by live and dead cell staining, and the cell scratch distance was counted. Crystal violet can bind to DNA in the nucleus, making the nucleus appear purple. After cell staining, the counting results are shown in FIGS. 6 and 7. 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 was significantly increased, and the number of migrated cells was also increased, which was significantly better than that of the positive control (HA) group. The results show that the injectable ATP-Gln@POC medical aesthetic material can effectively promote the migration and chemotaxis of HSF cells.

[0087] Effect on collagen expression of HSF cells

[0088] Many factors cause the aging of HSF cells in the skin, which ultimately affects the expression of collagen in HSF cells. As the aging of HSF cells intensifies, the level of collagen precursor in the cells decreases, which further aggravates aging. As shown in FIG. 8, after D-gal treatment, the expression level of collagen precursor is significantly reduced. By using Sirius red staining, the changes of collagen fibers were explored. The collagen fibers appear orange yellow, and the proportion of collagen fibers in young skin is higher, while the proportion of collagen fibers in aged skin is lower. After incubation with ATP-Gln@POC, the production of collagen fibers in human skin fibroblasts is effectively promoted, and it is significantly better than that of the positive control (HA) group. In summary, ATP-Gln@POC can stimulate the generation of collagen fibers in the dermis to remodel the extracellular matrix, thereby achieving the effect of delaying skin aging.

[0089] It can be seen that the POC injectable medical aesthetic material is developed and designed in the present application, which can load biological active substances such as ATP, glutamine or active metal ions by mesoporous adsorption or chemical reaction, and can be injected in combination with collagen, chitosan, hydroxyapatite and the like. The present application can fill skin tissue, promote skin tissue regeneration and anti-aging, and provide new materials and methods for medical anti-aging or tissue regeneration.

[0090] Example of citric acid microspheres promoting skin tissue regeneration and collagen generation

[0091] Referring to the previous research method, citric acid POC (POC injectable microparticle material of step 2 in example 1) and Gln@POC microsphere material (Gln@POC of step 3 in example 1) with particle size of 30-50 μm were synthesized, and Control group (Ctr) and PLLA (poly-L-lactic acid) were used as controls. After 30 days, the histopathology results were shown in FIG. 9. Compared with the results of Control group (Ctr) and PLLA (poly-L-lactic acid) group, citric acid microsphere POC and Gln@POC both had good ability to promote skin tissue regeneration, and had low inflammatory reaction, significantly increased the thickness of rat skin tissue and the levels of type I and type III collagen in the tissue. The above results provided a theoretical basis and scientific basis for the application of citric acid biomaterial microsphere filling material in medical beauty and soft tissue regeneration.

[0092] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. Those skilled in the art can easily make various modifications to these embodiments, and apply the general principles described herein to other embodiments without creative labor. Therefore, the present application is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art according to the disclosure of the present application without departing from the scope of the present application should be within the scope of protection of the present application.

Claims

1. A method of preparing a POC injectable microparticulate material, characterized by, The method comprises the following steps: The POC degradable prepolymers are freeze-dried for standby; The freeze-dried POC degradable prepolymers are dissolved in a solvent, inorganic salt particles are added, and the POC injectable microparticle material is obtained after cross-linking reaction, grinding, removal of inorganic salt and drying.

2. The method of claim 1, wherein the POC-degradable prepolymer is prepared by the reaction of the POC-degradable monomer and the POC-degradable oligomer. The solvent is selected from one or a combination of dioxane, anhydrous ethanol, acetone or dichloromethane; The amount of the freeze-dried POC degradable prepolymers and the solvent is 10g:10-200mL.

3. The method of claim 1, wherein the POC-degradable prepolymer is prepared by the reaction of the POC-degradable monomer and the POC-degradable oligomer. The inorganic salt particles are selected from one or a combination of NaCl, KCl and MgCl2; the amount of the inorganic salt particles added is 1-20 times the mass of the freeze-dried POC degradable prepolymers.

4. The method of claim 1, wherein the POC-degradable prepolymer is prepared by the reaction of the POC-degradable monomer and the POC-degradable oligomer. The cross-linking reaction is carried out at >50℃ under vacuum or non-vacuum conditions for 12-72 hours.

5. The method for preparing a POC degradable prepolymer according to claim 1, characterized in that, The preparation method of the POC degradable prepolymers comprises the following steps: The citric acid and 1,8-octanediol are mixed and melted, and then heated and stirred under vacuum conditions to form POC prepolymers; The POC prepolymers are dissolved in an organic solvent to obtain a dissolved solution, and the dissolved solution is slowly poured into ultrapure water for multiple times to obtain purified POC degradable prepolymers.

6. The method of claim 5, wherein the POC-degradable prepolymer is prepared by the reaction of the compound of formula (1) with the compound of formula (2) in the presence of the compound of formula (3) and the compound of formula (4). The amount of the citric acid and 1,8-octanediol is in a weight ratio of 1:(1-10) to (1-10):1; the mixing and melting of the citric acid and 1,8-octanediol is carried out under inert gas protection, and the temperature of the mixing and melting is 120-160℃; The stirring conditions under vacuum are as follows: the stirring is carried out at 120-140℃ and a stirring speed of 300-1000r / min for 1-48 hours, then the stirring speed is reduced until the stirring cannot be continued, and then the reaction is stopped.

7. The method of claim 5, wherein the POC-degradable prepolymer is prepared by the reaction of the POC-degradable monomer and the POC-degradable oligomer in the presence of the catalyst. The volume-to-mass ratio of the organic solvent to the POC prepolymers is 1-100:1; The organic solvent is selected from one or a combination of dioxane, anhydrous ethanol, acetone or dichloromethane; The dissolution of the POC prepolymers in the organic solvent is carried out under stirring conditions, and the stirring conditions are 50-1000r / min; When the dissolved solution is slowly poured into ultrapure water for multiple times for purification, the volume ratio of the dissolved solution to the ultrapure water is 1:10-1000.

8. A POC injectable microparticle material, characterized by, The POC injectable microparticle material is prepared by the method of any one of claims 1-7.

9. A citrate-based microparticulate injectable material, characterized in that, The POC injectable microparticle material of claim 8, and a drug, an active molecule, a bioglass or a metal ion loaded on the POC injectable microparticle material.

10. A citrate-based injectable material according to claim 9, wherein, The drug or the active molecule is selected from adenosine triphosphate and glutamine.

11. A citrate-based injectable material according to claim 10, wherein The loading amount of the adenosine triphosphate relative to 1g of the POC injectable microparticle material is 5-50mg, and the loading amount of the glutamine relative to 1g of the POC injectable microparticle material is 10-100mg.

12. A process for the preparation of a citrate-based microparticulate injectable material according to claim 9 or 10, characterised in that, The drug or the active molecule and the POC injectable microparticle material are combined together by incubation or chemical cross-linking, The method of incubation is: dissolving the drug or active molecule and the POC injectable microparticle material in physiological saline or culture medium, co-incubating by physical adsorption through a micro-vibrator for 12-48 hours, and then separating, purifying and drying by centrifugation at 1000-2000 rpm for several times to obtain the product. The method of chemical cross-linking is: connecting the drug or active molecule to the POC injectable microparticle material through chemical reaction.

13. A citrate-based biomimetic material, characterized in that, The POC injectable microparticle material of claim 8, and one or several combinations of collagen, chitosan, hyaluronic acid or hydroxyapatite loaded on the POC injectable microparticle material.

14. A citrate-based biomimetic material according to claim 13, wherein, The loading amount of one or several combinations of collagen, chitosan, hyaluronic acid or hydroxyapatite is 0-50wt%, and is not 0.

15. A citrate-based biomimetic material according to claim 13, wherein, The POC injectable microparticle material is further loaded with a drug or active molecule.

16. A citrate-based biomimetic material according to claim 15, wherein, The POC injectable microparticle material of claim 8, and one or several combinations of collagen, chitosan, hyaluronic acid or hydroxyapatite loaded on the POC injectable microparticle material.

17. An injection material, characterized in that The injection material is composed of the following components in percentage by weight: the POC injectable microparticle material of claim 8 1-50%, carboxymethyl cellulose sodium CMC 0.5-10%, and sterile physiological saline 40-95%. Or the POC injectable microparticle material of claim 8 and collagen, hyaluronic acid, bioglass or hydroxyapatite microparticles are dispersed in sterile physiological saline to obtain an injectable material.

18. Use of a citric acid-based microparticulate injectable material as claimed in claim 9 or a citric acid-based biomimetic material as claimed in claim 12, characterized in that, The citric acid-based microparticle injection material or the citric acid-based biomimetic material is used for preparing a biomedical and medical material.

19. The use according to claim 17, characterized in that, The biomedical and medical material includes skin tissue dermal filling material and breast augmentation prosthesis material. The biomedical and medical material includes skin tissue dermal filling material and breast augmentation prosthesis material.

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