Retinol-loaded sustained-release microspheres and preparation method thereof

Abstract

The invention relates to a retinol loaded sustained release microsphere and a preparation method thereof. According to the preparation method, methacrylic acid esterified hyaluronic acid HAMA, acylated tranexamic acid Ac-TXA and retinol are cross-linked to obtain the retinol loaded sustained release microsphere. According to the invention, the stability of retinol can be improved, slow release is realized, and the transdermal absorption capability is optimized, so that the anti-aging effect is enhanced. The methacrylic acid esterified hyaluronic acid HAMA has good biocompatibility, a stable microsphere structure is formed through photo-crosslinking, the light stability of retinol is enhanced, degradation of retinol under the action of ultraviolet rays and oxygen is reduced, and long-acting release of active ingredients in skin is ensured. Meanwhile, the high hydration capacity of the methacrylic acid esterification hyaluronic acid HAMA can maintain skin moisture balance, and the skin barrier function is further improved.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-skin aging, and particularly relates to a retinol-loaded sustained-release microsphere and a preparation method thereof. Background Art

[0002] Ultraviolet radiation, as the main external factor of skin aging, has always been a research hotspot in the field of beauty and skin care. It can directly damage the DNA of skin cells, trigger oxidative stress, and thus lead to the aging and death of skin cells. Long-term exposure to ultraviolet rays will not only cause wrinkles, sagging and pigmentation on the skin, but also increase the risk of skin cancer. Anti-aging is to delay the process of skin aging, physiological function degradation and tissue damage by intervening in endogenous and exogenous factors. Skin aging is mainly caused by mechanisms such as free radical damage, degradation of collagen by matrix metalloproteinases (MMPs), telomere shortening, glycation and inflammatory aging.

[0003] Current anti-aging strategies mainly include antioxidants, collagen synthesis promoters, DNA repair technologies, glycation inhibitors, as well as stem cells and biomaterials. Among them, antioxidants such as retinol, vitamin C (VC) and vitamin E (VE) can scavenge free radicals and reduce oxidative stress damage; hexapeptide and vitamin C can stimulate collagen synthesis and improve skin elasticity; telomerase activation and DNA repair enzyme technologies are used to protect DNA and delay cell aging; tranexamic acid (TXA), metformin (Metformin), etc. can inhibit glycation and reduce the damage of AGEs to dermal proteins. In addition, stem cells and exosomes (EVs) show good application prospects in promoting tissue regeneration and repairing damaged cells.

[0004] The retinol delivery system is one of the important research directions in skin anti-aging. Technologies such as nanoemulsions, liposomes and solid lipid nanoparticles (SLN) can improve the stability of retinol to a certain extent and enhance its transdermal absorption ability. However, there are still many limitations in the existing technologies, which are specifically as follows:

[0005] Nanoemulsions can increase the water solubility of retinol through surfactants and improve its permeability in the skin. However, the use of surfactants may cause skin irritation, and long-term use may damage the skin barrier. In addition, the stability of retinol in the nanoemulsion system is relatively low, and it is easily degraded by ultraviolet rays and oxygen, resulting in reduced activity.

[0006] Liposome technology uses a phospholipid bilayer structure to encapsulate retinol, improve its bioavailability, and reduce degradation to a certain extent. However, the structure of liposomes is not stable enough, and it is easy to aggregate or disintegrate, affecting the sustained-release effect of retinol. Moreover, the preparation cost of phospholipids is relatively high, making large-scale production have economic problems.

[0007] Solid lipid nanoparticles (SLNs) encapsulate retinol with a solid lipid matrix, enhancing its permeability through the skin barrier and providing a certain degree of protection. However, the lipid matrix of SLNs may form a greasy feeling on the skin surface, affecting the user experience. Moreover, the release rate of SLNs is relatively fast, making it difficult to achieve long-term sustained release, which limits its application in highly effective anti-aging treatments.

[0008] Hydrogel technology shows good application prospects in the field of skin drug delivery. It can act as a carrier to load retinol and provide a certain degree of sustained release effect. However, the mechanical properties and transdermal absorption ability of existing hydrogel systems still need to be optimized. The cross-linked structure of the hydrogel may affect the drug release rate, making it difficult to achieve precise controlled release. Moreover, the biodegradability and skin compatibility of some hydrogel materials still need to be further improved to adapt to different skin types and application requirements.

[0009] Therefore, in view of the deficiencies of the existing technology, it is highly necessary to provide a retinol-loaded sustained-release microsphere and its preparation method to solve the deficiencies of the existing technology. Summary of the Invention

[0010] The first object of the present invention is to provide a preparation method of a retinol-loaded sustained-release microsphere, avoiding the deficiencies of the existing technology. The retinol-loaded sustained-release microsphere prepared by this preparation method can improve the stability of retinol, achieve sustained release, and optimize the transdermal absorption ability, thereby enhancing the anti-aging effect.

[0011] The above object of the present invention is achieved by the following technical measures:

[0012] Provide a preparation method of a retinol-loaded sustained-release microsphere, which is obtained by cross-linking methacrylated hyaluronic acid (HAMA), acylated tranexamic acid (Ac-TXA), and retinol.

[0013] The preparation method of the retinol-loaded sustained-release microsphere of the present invention uses raw materials including methacrylated hyaluronic acid (HAMA), acylated tranexamic acid (Ac-TXA), retinol, a photoinitiator, and water.

[0014] The content of the raw materials in the preparation method of the retinol-loaded sustained-release microsphere of the present invention is as follows:

[0015] The mass fraction of methacrylated hyaluronic acid (HAMA): 2% - 6%;

[0016] The mass fraction of acylated tranexamic acid (Ac-TXA): 0.5% - 5%;

[0017] The concentration of retinol: 25 μg / mL - 1000 μg / mL;

[0018] The mass fraction of the photoinitiator: 0.05% - 0.5%.

[0019] Furthermore, for the preparation method of the retinol-loaded sustained-release microspheres of the present invention, the raw material contents are as follows:

[0020] The mass fraction of methacrylated hyaluronic acid HAMA: 2% - 6%;

[0021] The mass fraction of acylated tranexamic acid Ac-TXA: 1% - 3%;

[0022] The concentration of retinol: 400 μg / mL - 900 μg / mL;

[0023] The mass fraction of photoinitiator: 0.1% - 0.3%.

[0024] Furthermore, for the preparation method of the retinol-loaded sustained-release microspheres of the present invention, the raw material contents are as follows:

[0025] The mass fraction of methacrylated hyaluronic acid HAMA: 4%;

[0026] The mass fraction of acylated tranexamic acid Ac-TXA: 2%;

[0027] The concentration of retinol: 800 μg / mL;

[0028] The mass fraction of photoinitiator: 0.2%.

[0029] In the retinol-loaded sustained-release microspheres, the raw materials further contain absolute ethanol, and the volume fraction of absolute ethanol is 0.15% - 0.40%.

[0030] Preferably, the preparation method of the above methacrylated hyaluronic acid HAMA is to add sodium hyaluronate HA into water, then add methacrylic anhydride to obtain a reaction solution, then add NaOH to adjust the pH to 8.3 - 8.8, stir at room temperature, then dialyze using a cellulose dialysis bag with a cut-off molecular weight of 1.0 kDa - 1.5 kDa, and finally freeze-dry to obtain the methacrylated hyaluronic acid HAMA.

[0031] Preferably, the preparation method of the above acylated tranexamic acid Ac-TXA is to add tranexamic acid TXA into absolute ethanol, add acetyl chloride and triethylamine TEA respectively while controlling the temperature in the range of 5°C to -5°C, stir and react while controlling the temperature in the range of 5°C to -5°C, after the reaction is completed, add dichloromethane and then add water to wash the dichloromethane, and finally remove the organic solvent to obtain the acylated tranexamic acid Ac-TXA.

[0032] Preferably, the preparation method of the above-mentioned methacrylated hyaluronic acid HAMA is to add sodium hyaluronate HA to water, then add methacrylic anhydride to obtain a reaction solution, and then add a 5M NaOH aqueous solution to adjust the pH to 8.5, stir at room temperature, and then dialyze using a cellulose dialysis bag with a molecular weight cut-off of 1.0 kDa to 1.5 kDa, and finally lyophilize to obtain methacrylated hyaluronic acid HAMA, wherein the ratio among the gram amount of sodium hyaluronate HA, the milliliter amount of methacrylic anhydride, and the milliliter amount of water is 1.25: 3 to 5: 50 to 200.

[0033] Preferably, the preparation method of the above-mentioned acylated tranexamic acid Ac-TXA is to add tranexamic acid TXA to absolute ethanol, add acetyl chloride and triethylamine TEA at 0 °C respectively, stir at 0 °C for 3 h to 4 h, after the reaction is completed, add dichloromethane and then add water to wash dichloromethane, and finally remove the organic solvent under reduced pressure to obtain acylated tranexamic acid Ac-TXA, wherein the ratio among the gram amount of tranexamic acid TXA, the milliliter amount of acetyl chloride, the milliliter amount of triethylamine TEA, the milliliter amount of absolute ethanol, the milliliter amount of dichloromethane, and the milliliter amount of water is 1: 0.5 to 1.5: 0.5 to 1.5: 5 to 15: 20 to 50: 20 to 50.

[0034] The second object of the present invention is to provide a retinol-loaded sustained-release microsphere to avoid the deficiencies of the prior art. The retinol-loaded sustained-release microsphere can improve the stability of retinol, achieve sustained release and optimize the transdermal absorption ability, thereby enhancing the anti-aging effect.

[0035] The above object of the present invention is achieved by the following technical measures:

[0036] Provide a retinol-loaded sustained-release microsphere, which is prepared by the preparation method of the above-mentioned retinol-loaded sustained-release microsphere.

[0037] The present invention relates to a retinol-loaded sustained-release microsphere and a preparation method thereof. In the preparation method, methacrylated hyaluronic acid (HAMA), acylated tranexamic acid (Ac-TXA), and retinol are crosslinked to obtain the retinol-loaded sustained-release microsphere. The present invention can improve the stability of retinol, achieve sustained release, and optimize the transdermal absorption ability, thereby enhancing the anti-aging effect. The methacrylated hyaluronic acid (HAMA) used in the present invention has good biocompatibility and forms a stable microsphere structure through photocrosslinking, enhancing the photostability of retinol and reducing its degradation under the action of ultraviolet light and oxygen, ensuring the long-term release of the active ingredient in the skin. At the same time, the high hydration ability of methacrylated hyaluronic acid (HAMA) can maintain the skin moisture balance and further improve the skin barrier function. Moreover, the acylated tranexamic acid (Ac-TXA) of the present invention enhances its lipophilicity and transdermal absorption ability through acylation modification, enabling retinol to more effectively penetrate the stratum corneum and play a role in the dermis. In addition, acylated tranexamic acid (Ac-TXA) itself has anti-inflammatory and whitening effects, can inhibit the release of inflammatory factors, reduce skin irritation, and reduce melanin deposition, thus playing an important role in improving skin texture and skin tone uniformity. Therefore, the retinol-loaded sustained-release microsphere of the present invention provides a new strategy for retinol delivery and has broad prospects in skin repair, anti-aging, and skincare product applications. Description of the Drawings

[0038] The present invention will be further described with reference to the accompanying drawings, but the content in the drawings does not constitute any limitation to the present invention.

[0039] Figure 1 1H NMR spectra of tranexamic acid (TXA) and acylated tranexamic acid (Ac-TXA).

[0040] Figure 2 Fourier transform infrared spectra of tranexamic acid (TXA) and acylated tranexamic acid (Ac-TXA).

[0041] Figure 3 1H NMR spectra of hyaluronic acid MA and methacrylated hyaluronic acid (HAMA).

[0042] Figure 4 Fourier transform infrared spectra of hyaluronic acid MA and methacrylated hyaluronic acid (HAMA).

[0043] Figure 5 Optical microscope images of microdroplets of methacrylated hyaluronic acid (HAMA) at different concentrations.

[0044] Figure 6 Diameter distribution diagrams of microdroplets of methacrylated hyaluronic acid (HAMA) at different concentrations.

[0045] Figure 7It is a scanning electron microscope image of retinol-loaded sustained-release microspheres.

[0046] Figure 8 It is a diameter distribution diagram of retinol-loaded sustained-release microspheres.

[0047] Figure 9 It is an in vitro cumulative release curve of retinol in retinol-loaded sustained-release microspheres. Specific implementation manners

[0048] The technical solution of the present invention will be further described in conjunction with the following embodiments. Unless otherwise specified in the following embodiments, the raw materials used in each embodiment are commercially available products.

[0049] Embodiment 1

[0050] A preparation method of retinol-loaded sustained-release microspheres is obtained by crosslinking methacrylated hyaluronic acid HAMA, acylated tranexamic acid Ac-TXA and retinol.

[0051] The preparation method of the retinol-loaded sustained-release microspheres of the present invention has raw materials including methacrylated hyaluronic acid HAMA, acylated tranexamic acid Ac-TXA, retinol, a photoinitiator, absolute ethanol and water, wherein water is used as a solvent for methacrylated hyaluronic acid HAMA, acylated tranexamic acid Ac-TXA, retinol and the photoinitiator.

[0052] The raw material contents are as follows:

[0053] The mass fraction of methacrylated hyaluronic acid HAMA: 2% - 6%;

[0054] The mass fraction of acylated tranexamic acid Ac-TXA: 0.5% - 5%;

[0055] The concentration of retinol: 25 μg / mL - 1000 μg / mL;

[0056] The mass fraction of the photoinitiator: 0.05% - 0.5%;

[0057] The volume fraction of absolute ethanol is 0.15% - 0.40%.

[0058] It should be noted that the photoinitiator of the present invention can be lithium phenyl-2,4,6-trimethylbenzoylphosphinate LAP, 2,4,6-trimethylbenzoyldiphenylphosphine oxide, 2-hydroxy-2-methylpropiophenone, and can be specifically selected according to the actual situation.

[0059] The preparation method of the retinol-loaded sustained-release microspheres of the present invention is carried out by the following steps:

[0060] S1. Dissolve methacrylated hyaluronic acid (HAMA) in water to obtain solution A, dissolve acylated tranexamic acid (Ac-TXA) and retinol in absolute ethanol to obtain solution B, and then mix solution A and solution B to obtain a dispersion phase. The contents of methacrylated hyaluronic acid (HAMA), acylated tranexamic acid (Ac-TXA), retinol, absolute ethanol, and photoinitiator in the dispersion phase of this example are in accordance with the above raw material contents;

[0061] S2. Place the dispersion phase obtained in S1 into a microfluidic device, with the continuous phase being the microdroplet-forming oil, and control the flow rates of the dispersion phase and the continuous phase to obtain microdroplets;

[0062] S3. Carry out ultraviolet light curing on the microdroplets in S2 to form microgel microspheres, add a demulsifier to remove the microdroplet-forming oil, wash with water, and then centrifuge to obtain the retinol-loaded sustained-release microspheres.

[0063] Among them, the preparation method of the methacrylated hyaluronic acid (HAMA) of the present invention is as follows: add sodium hyaluronate (HA) to water, then add methacrylic anhydride to obtain a reaction solution, then add NaOH to adjust the pH to 8.3 - 8.8, stir at room temperature, then dialyze using a cellulose dialysis bag with a molecular weight cut-off of 1.0 kDa - 1.5 kDa, and finally lyophilize to obtain the methacrylated hyaluronic acid (HAMA).

[0064] The preparation method of the acylated tranexamic acid (Ac-TXA) of the present invention is as follows: add tranexamic acid (TXA) to absolute ethanol, add acetyl chloride and triethylamine (TEA) respectively while controlling the temperature within the range of 5°C to -5°C, stir and react within the range of 5°C to -5°C, after the reaction is completed, add dichloromethane and then add water to wash the dichloromethane, and finally remove the organic solvent to obtain the acylated tranexamic acid (Ac-TXA).

[0065] The retinol-loaded sustained-release microspheres prepared by the preparation method of the retinol-loaded sustained-release microspheres can improve the stability of retinol, achieve sustained release and optimize the transdermal absorption ability, thereby enhancing the anti-aging effect. In the present invention, methacrylated hyaluronic acid HAMA has good biocompatibility, and a stable microsphere structure is formed through photocrosslinking, enhancing the photostability of retinol, reducing its degradation under the action of ultraviolet rays and oxygen, and ensuring the long-term release of the active ingredient in the skin. At the same time, the high hydration ability of methacrylated hyaluronic acid HAMA can maintain the skin moisture balance and further enhance the skin barrier function. Moreover, the acylated tranexamic acid Ac-TXA of the present invention enhances its lipophilicity and transdermal absorption ability through acylation modification, enabling retinol to penetrate the stratum corneum more effectively and play a role deep in the dermis. In addition, acylated tranexamic acid Ac-TXA itself has anti-inflammatory and whitening effects, can inhibit the release of inflammatory factors, reduce skin irritation, and reduce melanin deposition, thus playing an important role in improving skin texture and skin tone uniformity. Therefore, the retinol-loaded sustained-release microspheres of the present invention provide a new strategy for retinol delivery and have broad prospects in skin repair, anti-aging and skin care product applications.

[0066] Example 2

[0067] A preparation method of retinol-loaded sustained-release microspheres, other features are the same as those in Example 1, the difference is that the raw material contents are as follows:

[0068] Mass fraction of methacrylated hyaluronic acid HAMA: 2% - 6%;

[0069] Mass fraction of acylated tranexamic acid Ac-TXA: 1% - 3%;

[0070] Concentration of retinol: 400 μg / mL - 900 μg / mL;

[0071] Mass fraction of photoinitiator: 0.1% - 0.3%;

[0072] Volume fraction of absolute ethanol is 0.20% - 0.30%.

[0073] Among them, the preparation method of methacrylated hyaluronic acid HAMA is to add sodium hyaluronate HA to water, then add methacrylic anhydride to obtain a reaction solution, then add a 5M NaOH aqueous solution to adjust the pH to 8.5, stir at room temperature, then dialyze using a cellulose dialysis bag with a cut-off molecular weight of 1.0 kDa - 1.5 kDa, and finally freeze-dry to obtain methacrylated hyaluronic acid HAMA, where the ratio of the number of grams of sodium hyaluronate HA, the volume in milliliters of methacrylic anhydride, and the volume in milliliters of water is 1.25: 3 - 5: 50 - 200.

[0074] Among them, the preparation method of acylated tranexamic acid Ac-TXA is to add tranexamic acid TXA to absolute ethanol, add acetyl chloride and triethylamine TEA at 0 °C respectively, stir at 0 °C for 3 h to 4 h, add dichloromethane after the reaction is completed, then add water to wash the dichloromethane, and finally remove the organic solvent under reduced pressure to obtain acylated tranexamic acid Ac-TXA. The ratio between the grams of tranexamic acid TXA, the milliliters of acetyl chloride, the milliliters of triethylamine TEA, the milliliters of absolute ethanol, the milliliters of dichloromethane, and the milliliters of water is 1: 0.5 to 1.5: 0.5 to 1.5: 5 to 15: 20 to 50: 20 to 50.

[0075] Specifically, in S2, in the microfluidic device, the flow rate of the dispersed phase is controlled to be 0.05 mL / h to 0.2 mL / h, and the flow rate of the continuous phase is controlled to be 0.5 mL / h to 2 mL / h. In this example, the contents of methacrylated hyaluronic acid HAMA, acylated tranexamic acid Ac-TXA, retinol, absolute ethanol, and photoinitiator in the dispersed phase are in accordance with the above raw material contents.

[0076] Example 3

[0077] A preparation method of retinol-loaded sustained-release microspheres, other features are the same as those in Example 1, the difference is that the raw material contents are as follows:

[0078] Mass fraction of methacrylated hyaluronic acid HAMA: 2%;

[0079] Mass fraction of acylated tranexamic acid Ac-TXA: 2%;

[0080] Concentration of retinol: 25 μg / mL;

[0081] Mass fraction of photoinitiator: 0.2%;

[0082] Volume fraction of absolute ethanol is 0.25%.

[0083] Specifically, in S2, in the microfluidic device, the flow rate of the dispersed phase is controlled to be 0.1 mL / h, and the flow rate of the continuous phase is controlled to be 1 mL / h. The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate LAP. In this example, the contents of methacrylated hyaluronic acid HAMA, acylated tranexamic acid Ac-TXA, retinol, absolute ethanol, and photoinitiator in the dispersed phase are in accordance with the above raw material contents.

[0084] Among them, the preparation method of methacrylated hyaluronic acid HAMA is to add sodium hyaluronate HA into water, then add methacrylic anhydride to obtain a reaction solution, and then add a 5M NaOH aqueous solution to adjust the pH to 8.5, stir at room temperature, then dialyze using a cellulose dialysis bag with a molecular weight cut-off of 1.0 kDa - 1.5 kDa, and finally lyophilize to obtain methacrylated hyaluronic acid HAMA. The ratio among the grams of sodium hyaluronate HA, the milliliters of methacrylic anhydride, and the milliliters of water is 1.25:3:100. Taking 1.25 grams of sodium hyaluronate HA as an example, methacrylic anhydride is 3 mL and water is 100 mL.

[0085] Among them, the preparation method of acylated tranexamic acid Ac-TXA is to add tranexamic acid TXA into absolute ethanol, add acetyl chloride and triethylamine TEA respectively at 0 °C, stir at 0 °C for 3 h - 4 h, after the reaction is completed, add dichloromethane and then add water to wash dichloromethane, and finally remove the organic solvent under reduced pressure to obtain acylated tranexamic acid Ac-TXA. The ratio among the grams of tranexamic acid TXA, the milliliters of acetyl chloride, the milliliters of triethylamine TEA, the milliliters of absolute ethanol, the milliliters of dichloromethane, and the milliliters of water is 1:1:1:10:30:30. Taking 1 gram of tranexamic acid TXA as an example, acetyl chloride is 1 mL, triethylamine TEA is 1 mL, absolute ethanol is 10 mL, dichloromethane is 30 mL, and water is 30 mL.

[0086] Example 4

[0087] A preparation method of retinol-loaded sustained-release microspheres, with other features being the same as those in Example 3, except that the raw material contents are as follows:

[0088] Mass fraction of methacrylated hyaluronic acid HAMA: 4%;

[0089] Mass fraction of acylated tranexamic acid Ac-TXA: 2%;

[0090] Concentration of retinol: 800 μg / mL;

[0091] Mass fraction of photoinitiator: 0.2%;

[0092] Volume fraction of absolute ethanol is 0.20%.

[0093] Example 5

[0094] A preparation method of retinol-loaded sustained-release microspheres, with other features being the same as those in Example 3, except that the raw material contents are as follows:

[0095] Mass fraction of methacrylated hyaluronic acid HAMA: 6%;

[0096] Mass fraction of Acylated Tranexamic Acid (Ac-TXA): 2%;

[0097] Concentration of Retinol: 400 μg / mL;

[0098] Mass fraction of photoinitiator: 0.2%;

[0099] Volume fraction of absolute ethanol is 0.30%.

[0100] Example 6

[0101] A method for preparing retinol-loaded sustained-release microspheres, with other characteristics the same as in Example 3, except that the raw material contents are as follows:

[0102] Mass fraction of Hyaluronic Acid Methacrylate (HAMA): 2%;

[0103] Mass fraction of Acylated Tranexamic Acid (Ac-TXA): 3%;

[0104] Concentration of Retinol: 1000 μg / mL;

[0105] Mass fraction of photoinitiator: 0.2%;

[0106] Volume fraction of absolute ethanol is 0.40%.

[0107] Example 7

[0108] A method for preparing retinol-loaded sustained-release microspheres, with other characteristics the same as in Example 3, except that the raw material contents are as follows:

[0109] Mass fraction of Hyaluronic Acid Methacrylate (HAMA): 4%;

[0110] Mass fraction of Acylated Tranexamic Acid (Ac-TXA): 1%;

[0111] Concentration of Retinol: 800 μg / mL;

[0112] Mass fraction of photoinitiator: 0.2%;

[0113] Volume fraction of absolute ethanol is 0.15%.

[0114] Example 8

[0115] A method for preparing retinol-loaded sustained-release microspheres, with other characteristics the same as in Example 3, except that the raw material contents are as follows:

[0116] Mass fraction of Hyaluronic Acid Methacrylate (HAMA): 4%;

[0117] Mass fraction of Acylated Tranexamic Acid (Ac-TXA): 3%;

[0118] Retinol concentration: 800 μg / mL;

[0119] Mass fraction of photoinitiator: 0.2%;

[0120] Volume fraction of absolute ethanol is 0.30%.

[0121] Example 9

[0122] A preparation method of retinol-loaded sustained-release microspheres, other features are the same as those in Example 3, the difference is that the raw material contents are as follows:

[0123] Mass fraction of methacrylated hyaluronic acid HAMA: 2%;

[0124] Mass fraction of acylated tranexamic acid Ac-TXA: 3%;

[0125] Retinol concentration: 100 μg / mL;

[0126] Mass fraction of photoinitiator: 0.2%;

[0127] Volume fraction of absolute ethanol is 0.25%.

[0128] Example 10

[0129] A preparation method of retinol-loaded sustained-release microspheres, other features are the same as those in Example 3, the difference is that the raw material contents are as follows:

[0130] Mass fraction of methacrylated hyaluronic acid HAMA: 4%;

[0131] Mass fraction of acylated tranexamic acid Ac-TXA: 2%;

[0132] Retinol concentration: 1000 μg / mL;

[0133] Mass fraction of photoinitiator: 0.2%;

[0134] Volume fraction of absolute ethanol is 0.25%.

[0135] Example 11

[0136] A preparation method of retinol-loaded sustained-release microspheres, other features are the same as those in Example 3, the difference is that the raw material contents are as follows:

[0137] Mass fraction of methacrylated hyaluronic acid HAMA: 4%;

[0138] Mass fraction of acylated tranexamic acid Ac-TXA: 0.5%;

[0139] Retinol concentration: 800 μg / mL;

[0140] Mass fraction of photoinitiator: 0.5%;

[0141] Volume fraction of absolute ethanol is 0.25%.

[0142] S2 specifically is in the microfluidic device, controlling the flow rate of the dispersed phase to be 0.05 mL / h and the flow rate of the continuous phase to be 0.5 mL / h.

[0143] The preparation method of methacrylated hyaluronic acid HAMA is to add sodium hyaluronate HA into water, then add methacrylic anhydride to obtain a reaction solution, then add a 5M NaOH aqueous solution to adjust the pH to 8.3, stir at room temperature, then use a cellulose dialysis bag with a cut-off molecular weight of 1.0 kDa - 1.5 kDa for dialysis, and finally lyophilize to obtain methacrylated hyaluronic acid HAMA, where the ratio among the grams of sodium hyaluronate HA, the milliliters of methacrylic anhydride, and the milliliters of water is 1.25:3:50.

[0144] The preparation method of acylated tranexamic acid Ac-TXA is to add tranexamic acid TXA into absolute ethanol, add acetyl chloride and triethylamine TEA at 0 °C respectively, stir at 0 °C for 3 h - 4 h, after the reaction is completed, add dichloromethane and then add water to wash dichloromethane, and finally remove the organic solvent under reduced pressure to obtain acylated tranexamic acid Ac-TXA, where the ratio among the grams of tranexamic acid TXA, the milliliters of acetyl chloride, the milliliters of triethylamine TEA, the milliliters of absolute ethanol, the milliliters of dichloromethane, and the milliliters of water is 1:1.5:1.5:15:50:20.

[0145] Example 12

[0146] A preparation method of retinol-loaded sustained-release microspheres, with other features being the same as in Example 3, the difference being that the raw material contents are as follows:

[0147] Mass fraction of methacrylated hyaluronic acid HAMA: 4%;

[0148] Mass fraction of acylated tranexamic acid Ac-TXA: 5%;

[0149] Concentration of retinol: 800 μg / mL;

[0150] Mass fraction of photoinitiator: 0.2%;

[0151] Volume fraction of absolute ethanol is 0.30%.

[0152] S2 specifically is in the microfluidic device, controlling the flow rate of the dispersed phase to be 0.2 mL / h and the flow rate of the continuous phase to be 2 mL / h.

[0153] The preparation method of methacrylated hyaluronic acid HAMA is to add sodium hyaluronate HA into water, then add methacrylic anhydride to obtain a reaction solution, and then add a 5M NaOH aqueous solution to adjust the pH to 8.8, stir at room temperature, dialyze using a cellulose dialysis bag with a molecular weight cut-off of 1.0 kDa to 1.5 kDa, and finally lyophilize to obtain methacrylated hyaluronic acid HAMA. The ratio among the grams of sodium hyaluronate HA, the milliliters of methacrylic anhydride, and the milliliters of water is 1.25:5:200.

[0154] The preparation method of acylated tranexamic acid Ac-TXA is to add tranexamic acid TXA into absolute ethanol, add acetyl chloride and triethylamine TEA at 0 °C respectively, stir at 0 °C for 3 h to 4 h, add dichloromethane after the reaction is completed, and then add water to wash dichloromethane. Finally, remove the organic solvent under reduced pressure to obtain acylated tranexamic acid Ac-TXA. The ratio among the grams of tranexamic acid TXA, the milliliters of acetyl chloride, the milliliters of triethylamine TEA, the milliliters of absolute ethanol, the milliliters of dichloromethane, and the milliliters of water is 1:0.5:0.5:5:20:50.

[0155] Comparative Example 1

[0156] A preparation method of retinol-loaded sustained-release microspheres, with other features being the same as those in Example 3, except that: retinol is not added, and the method is carried out as follows: S1. Dissolve methacrylated hyaluronic acid HAMA in water to obtain solution A, dissolve acylated tranexamic acid Ac-TXA in absolute ethanol to obtain solution B, and then mix solution A and solution B to obtain a dispersion phase;

[0157] S2. Place the dispersion phase obtained in S1 into a microfluidic device, with the continuous phase being microdroplet-forming oil, and control the flow rates of the dispersion phase and the continuous phase to obtain microdroplets;

[0158] S3. Carry out ultraviolet light curing on the microdroplets in S2 to form microgel microspheres, add a demulsifier to remove the microdroplet-forming oil, wash with water, and then centrifuge to obtain the retinol-loaded sustained-release microspheres.

[0159] Comparative Example 2

[0160] A preparation method of retinol-loaded sustained-release microspheres, with other features being the same as those in Example 3, except that acylated tranexamic acid Ac-TXA is not added, and the method is carried out as follows:

[0161] S1. Dissolve methacrylated hyaluronic acid HAMA in water to obtain solution A, dissolve retinol in absolute ethanol to obtain solution B, and then mix solution A and solution B to obtain a dispersion phase;

[0162] S2. Place the disperse phase obtained in S1 into a microfluidic device. The continuous phase is the oil for generating microdroplets. Control the flow rates of the disperse phase and the continuous phase to obtain microdroplets.

[0163] S3. Carry out ultraviolet curing on the microdroplets in S2 to form microgel microspheres, add a demulsifier to remove the oil for generating microdroplets, and after washing with water and centrifuging, obtain the retinol-loaded sustained-release microspheres.

[0164] Comparative Example 3

[0165] A method for preparing retinol-loaded sustained-release microspheres, having other features the same as those in Example 3, except that no acylated tranexamic acid Ac-TXA and retinol are added, and it is carried out by the following steps:

[0166] S1. Dissolve methacrylated hyaluronic acid HAMA in water to obtain Solution A as the disperse phase.

[0167] S2. Place the disperse phase obtained in S1 into a microfluidic device. The continuous phase is the oil for generating microdroplets. Control the flow rates of the disperse phase and the continuous phase to obtain microdroplets.

[0168] S3. Carry out ultraviolet curing on the microdroplets in S2 to form microgel microspheres, add a demulsifier to remove the oil for generating microdroplets, and after washing with water and centrifuging, obtain the retinol-loaded sustained-release microspheres.

[0169] Comparative Example 4

[0170] A method for preparing retinol-loaded sustained-release microspheres, having other features the same as those in Example 3, except that no methacrylated hyaluronic acid HAMA is added and no acylated tranexamic acid Ac-TXA is added, and it is carried out by the following steps:

[0171] S1. Dissolve retinol in absolute ethanol to obtain Solution B as the disperse phase.

[0172] S2. Place the disperse phase obtained in S1 into a microfluidic device. The continuous phase is the oil for generating microdroplets. Control the flow rates of the disperse phase and the continuous phase to obtain microdroplets. Control the flow rate of the disperse phase to be 0.1 mL / h and the flow rate of the continuous phase to be 1 mL / h, and irradiate with ultraviolet light to obtain a sample.

[0173] Comparative Example 5

[0174] A method for preparing retinol-loaded sustained-release microspheres, having other features the same as those in Example 3, except that: use hyaluronic acid MA to replace methacrylated hyaluronic acid HAMA, and use tranexamic acid to replace acylated tranexamic acid Ac-TXA.

[0175] Effect Example

[0176] 1. Analysis of antioxidant effect (DPPH)

[0177] The samples were prepared according to the instructions of the DPPH free radical scavenging ability detection kit, which was purchased from Macklin with the product number T931095. The samples of Examples 3 - 12 and Comparative Examples 1 - 4 were added to the DPPH solution, and the sample usage amount was 18 mg. After incubating in the dark for 30 minutes, the absorbance at 515 nm was measured with a UV-visible spectrophotometer, and the free radical scavenging rate was calculated to obtain Table 1.

[0178] Table 1. DPPH free radical scavenging rate

[0179]

[0180] Note: Microspheres cannot be formed in Comparative Example 4, so it is not tested.

[0181] From the results in Table 1, it can be seen that the methacrylated hyaluronic acid HAMA of the present invention, as the base material part of the microspheres, basically does not have antioxidant ability. Acyl tranexamic acid Ac-TXA itself is not mainly characterized by antioxidant ability. For the retinol-loaded sustained-release microspheres of the present invention, the antioxidant effect of acyl tranexamic acid Ac-TXA is not obvious. Retinol in the present invention has a significant antioxidant effect, which is mainly attributed to the hydroxyl (-OH) functional group in the molecular structure of retinol. This hydroxyl group can provide hydrogen atoms to react with free radicals, reduce them to stable molecules, inhibit oxidative stress and reduce cell damage.

[0182] 2. Evaluation of skin roughness index

[0183] An aging model was established in rats, and skin aging was simulated by ultraviolet irradiation. The steps for establishing the photoaging model in rats are as follows: S1. Select healthy SD rats (6 - 8 weeks old) and adaptively raise them for one week. The breeding environment is maintained at a temperature of 22 - 24 °C, a humidity of 50 - 60%, and a 12-hour light / dark cycle, and they are allowed to freely eat and drink. Before the experiment, the rats were anesthetized with 1.5% isoflurane, and the hair on the back of the rats was shaved to ensure the integrity of the skin in the irradiation area. S2. The rats were randomly grouped and irradiated with a UVB lamp. The UVB radiation wavelength was set at 280 - 320 nm, and the irradiation intensity was 100 - 200 mJ / cm 2 , irradiated once a day for 10 - 30 minutes each time, and continuously irradiated for 6 - 8 weeks to induce the establishment of the photoaging model. During the irradiation process, the rats were fixed to prevent them from moving and avoid unnecessary radiation to other parts. During the experiment, the appearance changes of the rats' skin were regularly observed and recorded to evaluate the establishment of the photoaging model.

[0184] After successfully establishing the photoaging model, the rats treated with the samples of Examples 3 - 12 and Comparative Examples 1 - 4 were used as the dosing group, and the corresponding samples were applied to specific areas of the rats' backs at a dose of 4 mg / (cm 2 ·day). Then, a skin surface analyzer (such as Visioscan) was used to measure the skin roughness of the rats. First, the equipment was calibrated using the standard reference slides provided by the manufacturer to ensure parameters such as system brightness, contrast, and color. Then, the area on the rats' backs that had been treated with ultraviolet irradiation was selected as the measurement area, with an area of approximately 2×2 cm, ensuring the consistency of the measurement site each time. The image resolution was set to 1024×768 pixels or higher, and a magnification of 20 to 50 times was used to fully capture the fine texture of the skin surface. At the same time, the built-in ultraviolet light source of Visioscan should maintain a fixed light intensity and an irradiation angle perpendicular to the skin surface to ensure stable lighting conditions. Finally, the probe of the equipment was brought into contact with the skin surface, and the equipment was started to perform a skin scan, recording the skin roughness index (Ra value). A higher Ra value indicates an increase in skin roughness, while a lower value indicates smoother skin, obtaining Table 2.

[0185] Table 2. Skin Roughness Index

[0186] The present invention Ra value (μm) Comparative example Ra value (μm) Example 3 2.51±1.36 Comparative example 1 2.75±0.26 Example 4 0.85±0.56 Comparative example 2 0.956±1.15 Example 5 1.25±1.15 Comparative example 3 2.89±1.69％ Example 6 0.36±0.81 Comparative example 4 0.81±0.59 Example 7 1.05±0.59 Comparative example 5 2.72±1.55％ Example 8 0.52±1.39 --- --- Example 9 1.85±0.96 --- --- Example 10 0.65±0.15 --- --- Example 11 1.21±0.32 --- --- Example 12 0.33±0.78 --- ---

[0187] As can be seen from Table 2, acylated tranexamic acid Ac-TXA has strong antioxidant and anti-inflammatory effects, can help enhance the skin barrier function, reduce water loss, and prevent skin damage caused by ultraviolet rays and pollutants, further improving the smoothness and elasticity of the skin. Retinol, as a potent anti-aging ingredient, promotes collagen synthesis, stimulates skin cell renewal, reduces fine lines and wrinkles, improves skin texture, and directly reduces the skin roughness index. By combining the moisturizing effect of methacrylated hyaluronic acid HAMA, the repair effect of acylated tranexamic acid Ac-TXA, and the anti-aging effect of retinol, the present invention not only enhances the elasticity and firmness of the skin but also has a mild exfoliating effect, helping to remove dead skin cells and improve the smoothness of the skin surface. The above data show that after treatment with the retinol-loaded sustained-release microspheres of the present invention, the skin roughness is significantly reduced, indicating that the present invention indeed has good effects in improving skin roughness, moisturizing, and anti-aging.

[0188] 3. Pigmentation Evaluation

[0189] An aging model was established in rats using the ultraviolet irradiation method, and the method for establishing the rat photoaging model was the same as that for evaluating the skin roughness index. After successfully establishing the photoaging model, the rats treated with the samples of Examples 3 - 12 and Comparative Examples 1 - 4 were used as the dosing group, and the corresponding samples were applied to specific areas of the rats' backs at a dose of 4 mg / (cm 2· The dose of (days) was applied to a specific area on the back of rats. For the quantitative analysis of the area of pigmented spots, skin images were usually taken using a skin imaging device (such as DermaScan), and the area of the pigmented spots was measured through image processing software (such as ImageJ).

[0190] Table 3, Area of Pigmented Spots

[0191] The present invention <![CDATA[Spotted area (cm 2 )]]> Comparative example <![CDATA[Spotted area (cm 2 )]]> Example 3 1.89±1.10 Comparative example 1 2.15±0.39 Example 4 1.15±1.26 Comparative example 2 1.99±0.17 Example 5 1.36±0.89 Comparative example 3 3.51±1.26 Example 6 0.69±0.47 Comparative example 4 1.65±0.49 Example 7 1.75±0.59 Comparative example 5 2.67±1.08 Example 8 0.89±0.15 --- --- Example 9 1.42±1.16 --- --- Example 10 1.09±0.98 --- --- Example 11 1.85±1.33 --- --- Example 12 0.88±0.14 --- ---

[0192] As can be seen from Table 3, retinol reduces the formation of pigmented spots and effectively fades existing pigmented spots by promoting skin cell renewal and inhibiting melanin synthesis. Acylated tranexamic acid Ac-TXA has antioxidant and anti-inflammatory effects, and can reduce skin inflammation and oxidative stress caused by ultraviolet rays or environmental pollution, thereby reducing the occurrence of pigmentation. Methyl acrylate esterified hyaluronic acid HAMA indirectly helps reduce the generation of pigmented spots by moisturizing and repairing the skin barrier, promoting skin hydration and health.

[0193] Through the sustained release effect of the retinol-loaded sustained release microspheres of the present invention, these components can effectively penetrate deep into the skin, enhance the treatment effect, reduce the expansion of pigmented spots, and accelerate the fading of existing pigmented spots. By measuring the change in the area of pigmented spots using a skin imaging device, the experimental data shows that the retinol-loaded sustained release microspheres can significantly reduce the area of pigmented spots, indicating that it has a significant effect in the treatment of pigmented spots.

[0194] 4. Evaluation of Skin Hydration Effect

[0195] To evaluate the effect of the treatment on skin water content, the method for establishing the rat photoaging model is the same as that for establishing the skin roughness index evaluation. After the photoaging model is successfully established, the rats treated with the samples of Examples 3 - 12 and Comparative Examples 1 - 4 will be used as the dosing group, and the corresponding samples will be applied to a specific area on the back of the rats at a dose of 4 mg / (cm 2 · day), and then the electrical conductivity method will be used to measure the skin hydration.

[0196] This method quantitatively evaluates the skin water content by measuring the relationship between the electrical conductivity of the skin and the water content. The higher the water content, the greater the electrical conductivity of the skin. During the operation, the Corneometer device automatically measures the electrical conductivity and converts it into the percentage of skin moisture by gently contacting the skin surface, usually presented in the form of the percentage of water content. The skin hydration of the rats was measured 4 weeks after the treatment to evaluate the effect of the microspheres on skin hydration.

[0197] Table 4, Skin Water Content

[0198] The present invention Skin moisture content (%) Comparative example Skin moisture content (%) Example 3 65.19 Comparative example 1 25.69 Example 4 85.16 Comparative example 2 32.16 Example 5 86.29 Comparative example 3 22.59 Example 6 67.85 Comparative example 4 52.15 Example 7 80.16 Comparative example 5 50.34 Example 8 88.15 --- --- Example 9 75.16 --- --- Example 10 85.62 --- --- Example 11 84.32 --- --- Example 12 86.03 --- ---

[0199] As can be seen from Table 4, the methacrylated hyaluronic acid HAMA of the present invention enhances skin hydration, improves skin elasticity and firmness by providing strong moisturization. Acylated tranexamic acid Ac-TXA has antioxidant and anti-inflammatory effects, reduces oxidative damage and inflammatory responses, thereby slowing down skin aging and loss of elasticity. Retinol enhances the structural stability of the skin by promoting collagen synthesis and further improves skin elasticity. It can be seen that the retinol-loaded sustained-release microspheres of the present invention can continuously release these active ingredients and deeply penetrate into the skin, acting on the skin surface and deep layer for a long time, enhancing the skin repair function and increasing the elastic recovery rate.

[0200] 5. Structural Characterization of Acylated Tranexamic Acid Ac-TXA

[0201] 1H NMR spectra of tranexamic acid (TXA) and the obtained acylated tranexamic acid (Ac-TXA) of the present invention were characterized (as shown in Figure 1 ) and Fourier transform infrared (FT-IR) spectra (as shown in Figure 2 ).

[0202] In Figure 1 , the characteristic peak of TXA appeared at 2.96 ppm, which originated from the methylene (-CH2) protons in the ring structure of TXA. The absorption peak at 1.58 ppm in Ac-TXA was the acyl proton absorption peak, indicating that the amino group had reacted with acyl chloride.

[0203] In Figure 2 , the results of the mid-infrared spectrum showed that 3327 cm-1 (hydrogen bond stretching vibration), this peak is usually related to the stretching vibration of amino group (-NH2) and / or hydrogen bond, indicating the presence of amino group in the TXA molecule. 1698 cm-1 (acyl stretching vibration), the intensity of this peak increased in Ac-TXA, which was usually due to the introduction of acyl (-COOR) functional group, indicating successful acylation. The introduction of acyl group changed the molecular structure of TXA and affected the intensity of this absorption peak.

[0204] Through Figure 1 and Figure 2 , it was shown that the synthesis of Ac-TXA of the present invention was successful.

[0205] 6. Structural Characterization of Methacrylated Hyaluronic Acid HAMA

[0206] 1H NMR spectra of hyaluronic acid MA and the obtained methacrylated hyaluronic acid (HAMA) of the present invention were characterized (as shown in Figure 3 ) and Fourier transform infrared (FT-IR) spectra (as shown in Figure 4 ).

[0207] In the nuclear magnetic resonance hydrogen spectrum ( Figure 3) Among them, the signals at 5.63 ppm and 6.05 ppm are the characteristic peaks of the hydrogen atoms of the double bonds introduced in the methacrylation reaction, indicating that the methacrylic acid group has been successfully introduced onto the hyaluronic acid molecule. The changes in these signals suggest that the structure of hyaluronic acid has changed after chemical modification. For methacrylated hyaluronic acid HAMA, in the infrared spectrum ( Figure 4 ) shows several obvious changes. The absorption peak at 3300 cm-1 still exists, but due to the methacrylation reaction, the vibration of the hydroxyl group may be weakened or displaced; the absorption peak at 1651 cm-1 still shows the presence of the carboxyl group, but due to the introduction of the esterification reaction, the vibration mode of the carboxyl group may change slightly. More importantly, a new absorption peak appears at 1240 cm-1, representing the stretching vibration of the ester group (C-O), which indicates that the methacrylation reaction has successfully introduced a new group. This new peak is a characteristic signal of HAMA, verifying the successful synthesis of HAMA.

[0208] 7. Morphology of microdroplets of methacrylated hyaluronic acid HAMA at different concentrations under an optical microscope

[0209] Cross-reference Examples 3, 4, and 5 were used to take microscopic photographs of the microdroplets obtained in S2 and to measure the diameter distribution of these microdroplets, obtaining Figure 5 and Figure 6 .

[0210] Through Figure 5 and Figure 6 It can be seen that as the concentration of methacrylated hyaluronic acid HAMA increases, the size of the microdroplets gradually increases. In the case of 2% methacrylated hyaluronic acid HAMA (Example 3), the average diameter of the microdroplets is 77.59 μm ± 9.10 μm, and the droplet morphology is small and uniform, with the diameter mainly concentrated in the range of 60 μm to 80 μm. As the concentration of methacrylated hyaluronic acid HAMA increases to 4%, the average diameter of the microdroplets increases to 111.96 μm ± 6.55 μm, and the droplet size distribution is relatively concentrated between 100 μm and 120 μm. When the concentration is further increased to 6% methacrylated hyaluronic acid HAMA (Example 5), the average diameter of the microdroplets is 156.04 μm ± 13.9 μm, and the droplet size distribution range increases significantly, mainly concentrated between 140 μm and 160 μm. These results indicate that as the concentration of methacrylated hyaluronic acid HAMA increases, the size of the microdroplets gradually increases, providing an important experimental basis for the control of different droplet sizes in microfluidic technology.

[0211] 8. Scanning electron microscope images of the retinol-loaded sustained-release microspheres of the present invention and their diameter distribution

[0212] Scanning electron microscope (SEM) images of methacrylated hyaluronic acid HAMA microspheres and the retinol-loaded sustained-release microspheres (HAT@R) of Example 4 of the present invention were taken to obtain Figure 7 ; and the particle size statistics of methacrylated hyaluronic acid HAMA microspheres and retinol-loaded sustained-release microspheres (HAT@R) were detected to obtain Figure 8 .

[0213] For the preparation of methacrylated hyaluronic acid HAMA microspheres, 0.8 g of HAMA was weighed and dissolved in 2 mL of deionized water containing 0.2% LAP. The dissolved HAMA was ultrasonically dispersed evenly and loaded into a 2.5 mL syringe as the dispersed phase and placed in a microfluidic device. The continuous phase was 2% microdroplet-forming oil (fluorinated oil). The flow rate of the dispersed phase was controlled at 0.1 mL / h, and the flow rate of the continuous phase was 1 mL / h. The generated microdroplets were collected, and microgel microspheres were formed by ultraviolet light curing. After adding a demulsifier to remove the fluorinated oil on the surface of the microspheres, deionized water was added for washing, and clean HAMA microspheres were collected by centrifugation.

[0214] In the Figure 7 SEM images, HAMA microspheres and HAT@R microspheres showed an obvious porous structure, and the surface presented a reticular morphology. In Figure 8 It can be seen that the diameter distribution of the microspheres shows that the diameters of HAMA and HAT@R microspheres are 122.99 μm ± 7.17 μm and 126.46 μm ± 11.07 μm respectively, and the diameter difference between the two is small, remaining within a similar size range. This shows that the loading of retinol has little effect on the size of the microspheres.

[0215] 9. In vitro cumulative release rate of retinol in the retinol-loaded sustained-release microspheres of the present invention

[0216] To better examine the release of retinol from the microspheres, 20 mg of the retinol-loaded sustained-release microspheres of Example 4 were loaded into a dialysis bag and then placed in 2 mL of PBS with pH = 7.4, and cultured in a constant temperature shaking incubator at 37 °C and 100 rpm. At different time points (1, 3, 6, 24, 48, 72, 96, 120, 144, 168, 192 h), 2 mL of the PBS solution was taken and replaced with an equal amount of fresh PBS. The samples were centrifuged at 4000 rpm for 5 min. The absorbance of retinol in the PBS solution was measured using ultraviolet spectrophotometry at an excitation wavelength of 280 nm, so as to detect the in vitro cumulative release rate of retinol in the retinol-loaded sustained-release microspheres to obtain Figure 9 .

[0217] By Figure 9It can be seen that the in vitro cumulative release rate of retinol is shown, and the release process continuously increases and tends to be stable after a certain time, indicating that the microsphere system can achieve the sustained release of retinol, thereby providing a stable antioxidant effect.

[0218] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A preparation method of retinol-loaded sustained-release microspheres, characterized in that: The retinol-loaded sustained-release microspheres are obtained by crosslinking methacrylated hyaluronic acid (HAMA), acylated tranexamic acid (Ac-TXA), and retinol.

2. The preparation method of the retinol-loaded sustained-release microspheres according to claim 1, wherein: In the retinol-loaded sustained-release microspheres, the raw materials include methacrylated hyaluronic acid (HAMA), acylated tranexamic acid (Ac-TXA), retinol, a photoinitiator, and water, and their contents are as follows: Mass fraction of methacrylated hyaluronic acid (HAMA): 2% - 6%; Mass fraction of acylated tranexamic acid (Ac-TXA): 0.5% - 5%; Concentration of retinol: 25 μg / mL - 1000 μg / mL; Mass fraction of the photoinitiator: 0.05% - 0.5%.

3. The preparation method of the retinol-loaded sustained-release microspheres according to claim 2, wherein, In the retinol-loaded sustained-release microspheres, the raw material contents are as follows: Mass fraction of methacrylated hyaluronic acid (HAMA): 2% - 6%; Mass fraction of acylated tranexamic acid (Ac-TXA): 1% - 3%; Concentration of retinol: 400 μg / mL - 900 μg / mL; Mass fraction of the photoinitiator: 0.1% - 0.3%.

4. The preparation method of the retinol-loaded sustained-release microspheres according to any one of claims 1-3, characterized in that, In the retinol-loaded sustained-release microspheres, the raw material contents are as follows: Mass fraction of methacrylated hyaluronic acid (HAMA): 4%; Mass fraction of acylated tranexamic acid (Ac-TXA): 2%; Concentration of retinol: 800 μg / mL; Mass fraction of the photoinitiator: 0.2%.

5. The preparation method of the retinol-loaded sustained-release microspheres according to any one of claims 1-3, characterized in that: In the retinol-loaded sustained-release microspheres, the raw materials also contain absolute ethanol, and the volume fraction of absolute ethanol is 0.15% - 0.40%.

6. The preparation method of the retinol-loaded sustained-release microspheres according to claim 5, characterized in that, It is carried out by the following steps: S1. Dissolve methacrylated hyaluronic acid (HAMA) in water to obtain solution A, dissolve acylated tranexamic acid (Ac-TXA) and retinol in absolute ethanol to obtain solution B, and then mix solution A and solution B to obtain a dispersion phase; S2. Place the dispersion phase obtained in S(1) into a microfluidic device, with the continuous phase being the microdroplet-forming oil, and control the flow rates of the dispersion phase and the continuous phase to obtain microdroplets; S3. Carry out ultraviolet light curing on the microdroplets in S(2) to form microgel microspheres, add a demulsifier to remove the microdroplet-forming oil, wash with water, and then centrifuge to obtain the retinol-loaded sustained-release microspheres.

7. The preparation method of the retinol-loaded sustained-release microspheres according to claim 6, wherein: The specific operation of S(2) is to control the flow rate of the dispersion phase to be 0.05 mL / h - 0.2 mL / h and the flow rate of the continuous phase to be 0.5 mL / h - 2 mL / h in the microfluidic device.

8. The preparation method of the retinol-loaded sustained-release microspheres according to any one of claims 1-3, characterized in that: The preparation method of the methacrylated hyaluronic acid (HAMA) is to add sodium hyaluronate (HA) to water, then add methacrylic anhydride to obtain a reaction solution, then add NaOH to adjust the pH to 8.3 - 8.8, stir at room temperature, and then dialyze using a cellulose dialysis bag with a cut-off molecular weight of 1.0 kDa - 1.5 kDa, and finally lyophilize to obtain the methacrylated hyaluronic acid (HAMA); The preparation method of the acylated tranexamic acid (Ac-TXA) is to add tranexamic acid (TXA) to absolute ethanol, control the temperature within the range of 5°C to -5°C, add acetyl chloride and triethylamine (TEA) respectively, stir and react within the range of 5°C to -5°C, after the reaction is completed, add dichloromethane and then add water to wash the dichloromethane, and finally remove the organic solvent to obtain the acylated tranexamic acid (Ac-TXA).

9. The preparation method of the retinol-loaded sustained-release microspheres according to claim 8, wherein: The preparation method of the methacrylated hyaluronic acid HAMA is as follows: Sodium hyaluronate HA is added to water, and then methacrylic anhydride is added to obtain a reaction solution. Then, a 5M NaOH aqueous solution is added to adjust the pH to 8.5, and the mixture is stirred at room temperature. Next, dialysis is performed using a cellulose dialysis bag with a molecular weight cut-off of 1.0 kDa to 1.5 kDa. Finally, freeze-drying is carried out to obtain methacrylated hyaluronic acid HAMA. The ratio among the grams of sodium hyaluronate HA, the milliliters of methacrylic anhydride, and the milliliters of water is 1.25: 3 to 5: 50 to 200; The preparation method of the acylated tranexamic acid Ac-TXA is as follows: Tranexamic acid TXA is added to anhydrous ethanol. At 0 °C, acetyl chloride and triethylamine TEA are respectively added, and the mixture is stirred at 0 °C for 3 h to 4 h. After the reaction is completed, dichloromethane is added, and then water is added to wash the dichloromethane. Finally, the organic solvent is removed under reduced pressure to obtain acylated tranexamic acid Ac-TXA. The ratio among the grams of tranexamic acid TXA, the milliliters of acetyl chloride, the milliliters of triethylamine TEA, the milliliters of anhydrous ethanol, the milliliters of dichloromethane, and the milliliters of water is 1: 0.5 to 1.5: 0.5 to 1.5: 5 to 15: 20 to 50: 20 to 50.

10. A retinol-loaded sustained-release microsphere, characterized in that: It is prepared by the preparation method of the retinol-loaded sustained-release microspheres according to any one of claims 1 to 9.

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