Composite microspheres, a preparation method thereof, a composite microsphere preparation containing the same, and application of the composite microspheres in preparing a product for preventing and treating hair loss and / or promoting hair regeneration

Abstract

The application relates to the technical field of biomedical materials, in particular to a composite microsphere, a preparation method thereof, a composite microsphere preparation containing the same and application of the composite microsphere in preparation of a product for preventing and treating hair loss and / or promoting hair regeneration. The composite microsphere comprises a first microsphere and a second microsphere; the first microsphere comprises poly-DL-lactic acid; and the second microsphere comprises vitamin D3 and a carrier. The composite microsphere is implanted subcutaneously, can effectively cover the complete time window of hair follicle regeneration, and significantly improves the length and quality of regenerated hair.

Description

Technical Field

[0001] This application relates to the field of biomedical materials technology, specifically to composite microspheres and their preparation methods, composite microsphere formulations containing the same, and their application in the preparation of products for preventing and treating hair loss and / or promoting hair regeneration. Background Technology

[0002] The hair follicle cycle includes the anagen (growth) phase, catagen (regression) phase, and telogen (resting) phase. During the critical window of transition from the telogen to the anagen phase, effective hair regeneration requires a strict time sequence: weeks 0-4 need initiation signals to activate hair follicle stem cell mobilization; weeks 4-12 require continuous proliferation signals to maintain stem cell proliferation and differentiation; and weeks 12-24 and beyond require enhanced protective signals to prolong the anagen phase by inhibiting apoptosis and oxidative stress.

[0003] Traditional methods typically involve physically mixing active ingredients such as growth factors and polynucleotides (PNs) with microspheres, or encapsulating them inside the microspheres. For example, patent CN111150836B describes a porous microsphere loaded with growth factors, its preparation method, and its application. This study uses porous microspheres (PHM) as a carrier and prepares KGF-2-PHM microspheres by loading keratinocyte growth factor 2 (KGF-2) through an impregnation process. The growth factor KGF-2 is loaded into the capillary micropores of the microspheres to promote hair follicle growth.

[0004] However, existing microsphere systems are difficult to precisely match the phased needs of early activation, mid-term continuous proliferation, and late-term protection of hair follicles, which restricts the improvement of hair regeneration efficacy. Summary of the Invention

[0005] Based on this, one or more embodiments of this application provide composite microspheres and their preparation methods, composite microsphere formulations containing the same, and their application in the preparation of products for preventing and treating hair loss and / or promoting hair regeneration. The technical solutions include the following:

[0006] One or more embodiments of this application provide composite microspheres, the composite microspheres comprising a first microsphere and a second microsphere; the first microsphere comprises polyracemic lactic acid; the second microsphere comprises vitamin D3 and a carrier.

[0007] In some embodiments of this application, the carrier includes one or more of PLGA, PCL, and liposomes.

[0008] In some embodiments of this application, the mass fraction of polyracemic lactic acid in the first microsphere is ≥99%; and the mass fraction of vitamin D3 in the second microsphere is 2.5%~3%.

[0009] The mass ratio of the first microsphere to the second microsphere is (5~20):1.

[0010] In some embodiments of this application, the polyracemic lactic acid has a weight-average molecular weight of 80,000 Da to 120,000 Da and a polydispersity index of 1.5 to 2.0.

[0011] In some embodiments of this application, the first microsphere satisfies one or more of the following conditions (A1) to (A3):

[0012] (A1) The average particle size is 25 μm to 50 μm;

[0013] (A2) Particle size distribution width Span < 1.5;

[0014] (A3) The glass transition temperature is 40℃~44℃; and / or,

[0015] The average particle size of the second microsphere is 15 μm to 35 μm.

[0016] One or more embodiments of this application provide a method for preparing the composite microspheres, comprising the following steps:

[0017] The first microsphere and the second microsphere are mixed evenly to prepare composite microspheres.

[0018] In some embodiments of this application, the preparation method satisfies one or more of the following conditions (B1) to (B3):

[0019] (B1) The first microsphere is prepared by the following method:

[0020] The polyracemic lactic acid is dissolved in an oil phase solvent to prepare an oil phase; the oil phase and aqueous phase solvent are stirred in step I to prepare an emulsion; and the emulsion is stirred in step II, centrifuged, washed, frozen and dried to prepare the first microspheres.

[0021] Optionally, the polyracemic lactic acid has a mass fraction of 3% to 7% in the oil phase solvent;

[0022] Optionally, the drying includes sublimation drying and desorption drying. The temperature for sublimation drying is -40°C to -20°C, and the temperature for desorption drying is 10°C to 30°C. The total drying time is 22 h to 26 h.

[0023] (B2) The second microsphere is prepared by the following method:

[0024] Vitamin D3 and a carrier are dissolved in an oil phase solvent to prepare an oil phase; the oil phase and aqueous phase solvent are stirred in step I to prepare an emulsion; and the emulsion is stirred in step II, centrifuged, washed, and dried to prepare a second microsphere.

[0025] Optionally, the mass-volume percentage of the carrier to the oil phase solvent is 3%-5%;

[0026] Optionally, the mass percentage of vitamin D3 to the carrier is 2.5%-3.5%;

[0027] Optionally, the drying includes sublimation drying and desorption drying. The temperature for sublimation drying is -40°C to -20°C, and the temperature for desorption drying is 10°C to 30°C. The total drying time is 22 h to 26 h.

[0028] (B3) The mixing time is 8 min to 12 min.

[0029] In some embodiments of this application, the preparation methods of the first microsphere or the second microsphere each independently satisfy one or more of the following conditions:

[0030] (1) The oil phase solvent includes dichloromethane;

[0031] (2) The aqueous solvent includes a polyvinyl alcohol aqueous solution with a mass fraction of 0.5% to 1.5%;

[0032] (3) The volume ratio of the oil phase to the water phase is 1:(10~12);

[0033] (4) The stirring speed of stirring I is 700 rpm to 900 rpm;

[0034] (5) The temperature of stirring II is 20℃~30℃, and the duration is 3.5 h~4.5 h;

[0035] (6) The centrifugation speed is 2500 rpm to 3500 rpm and the duration is 8 min to 12 min.

[0036] One or more embodiments of this application provide a composite microsphere formulation comprising the composite microspheres described herein or composite microspheres prepared by the method described herein; and an acceptable excipient.

[0037] Optionally, the acceptable excipients include one or more of sodium chloride and sodium carboxymethyl cellulose;

[0038] Optionally, the sodium chloride in the acceptable excipient has a mass fraction of 0.9% to 1.0%;

[0039] Optionally, the mass fraction of the sodium carboxymethyl cellulose in the acceptable excipients is 0.4% to 0.6%;

[0040] Optionally, the composite microspheres have a mass fraction of 45% to 60% in the composite microsphere formulation.

[0041] One or more embodiments of this application provide the application of the composite microspheres, the composite microspheres prepared by the method, or the composite microsphere formulation in the preparation of products for preventing and treating hair loss and / or promoting hair regeneration, satisfying one or more of the following conditions:

[0042] (1) The hair loss prevention and / or hair regeneration promotion products include one or more of the following: subcutaneous implants, injectable suspensions and microneedle patches;

[0043] (2) The subjects of the products for preventing and treating hair loss and / or promoting hair regeneration include mammals;

[0044] (3) The hair loss prevention and / or hair regeneration promotion products are used to promote the proliferation of hair follicle stem cells and the survival of hair papilla cells, or one or more of these.

[0045] Compared with traditional technologies, this application has the following advantages:

[0046] This application provides composite microspheres with specific components, comprising a first microsphere and a second microsphere; the first microsphere comprises polyracemic lactic acid; and the second microsphere comprises vitamin D3 and a carrier. Implanting these composite microspheres subcutaneously can effectively cover the entire time window of hair follicle regeneration, improving the length and quality of regenerated hair. Attached Figure Description

[0047] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0048] Figure 1 Scanning electron microscope (SEM) images of PDLLA microspheres and VD3 microspheres. Left: PDLLA microspheres; Right: VD3 microspheres.

[0049] Figure 2 The degradation curves of PDLLA microspheres and the release curves of VD3 microspheres are shown.

[0050] Figure 3 The bar chart shows the results of new hair length and quality for each group. A to E correspond to Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3, and the blank control group, respectively.

[0051] Figure 4 To detect Piezo1 transcription by RT-qPCR.

[0052] Figure 5To detect Wnt10b transcription by RT-qPCR.

[0053] Figure 6 To detect VDR transcription by RT-qPCR.

[0054] Figure 7 To detect HO-1 transcription by RT-qPCR.

[0055] Figure 8 The total ROS level in rat skin tissue. Detailed Implementation

[0056] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. For example, features described or illustrated as part of one embodiment can be combined in a suitable manner in another embodiment to produce new embodiments. Furthermore, numerous details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0058] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0059] The terms “and / or,” “or / and,” and “and / or” as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. “Any and all combinations” includes any two related listed items, any more related listed items, or a combination of all related listed items. For example, “A and / or B” includes three parallel options: A, B, and “a combination of A and B.”

[0060] In this application, the terms "multiple", "various", "multiple times", "several", "several", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more or more.

[0061] In this application, "optionally", "optional", and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without".

[0062] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0063] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0064] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0065] A first aspect of this application provides composite microspheres, the composite microspheres comprising a first microsphere and a second microsphere; the first microsphere comprises polyracemic lactic acid; the second microsphere comprises vitamin D3 and a carrier.

[0066] In some embodiments of this application, the carrier includes one or more of PLGA, PCL, and liposomes.

[0067] In some embodiments of this application, the mass fraction of the polyracemic lactic acid in the first microspheres is ≥99%;

[0068] The mass ratio of the first microsphere to the second microsphere is (5~20):1, for example, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1.

[0069] In some embodiments of this application, the polyracemic lactic acid has a weight-average molecular weight of 80,000 Da to 120,000 Da and a polydispersity index of 1.5 to 2.0. For example, the polyracemic lactic acid has a weight-average molecular weight of 100,000 Da and a polydispersity index of 1.8.

[0070] In some embodiments of this application, the first microsphere satisfies one or more of the following conditions (A1) to (A3):

[0071] (A1) The average particle size is 25μm~50μm, for example, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50μm;

[0072] (A2) Particle size distribution width Span < 1.5;

[0073] (A3) The glass transition temperature is 40℃~44℃, for example, 40, 41, 42, 43, 44℃; and / or,

[0074] The average particle size of the second microsphere is 15μm to 35μm, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35μm.

[0075] A second aspect of this application provides a method for preparing the composite microspheres, comprising the following steps:

[0076] The first microsphere and the second microsphere are mixed evenly to prepare composite microspheres.

[0077] The composite microspheres, through the respective degradation / release kinetics of two independent microspheres, create a sequential activation effect over time after implantation, effectively covering the entire time window of hair follicle regeneration. Specifically, the first microsphere activates the Piezo1-M2-Wnt initiation pathway through mechanical stress, while the second microsphere provides antioxidant protection through the VDR-Nrf2 pathway. The peak biological effects of the two phases have a time difference (approximately 4-8 weeks) and overlap during the intermediate window, forming a three-stage relay pattern of "initiation → proliferation + protection → sustained protection".

[0078] In some embodiments of this application, the preparation method satisfies one or more of the following conditions (B1) to (B3):

[0079] (B1) The first microsphere is prepared by the following method:

[0080] The polyracemic lactic acid is dissolved in an oil phase solvent to prepare an oil phase; the oil phase and aqueous phase solvent are stirred in step I to prepare an emulsion; and the emulsion is stirred in step II, centrifuged, washed, frozen and dried to prepare the first microspheres.

[0081] Optionally, the polyracemic lactic acid has a mass fraction of 3% to 7% in the oil phase solvent;

[0082] Optionally, the drying includes sublimation drying and desorption drying. The temperature for sublimation drying is -40°C to -20°C, and the temperature for desorption drying is 10°C to 30°C. The total drying time is 22 h to 26 h. For example, the temperature for sublimation drying is -20°C, the temperature for desorption drying is 25°C, and the total drying time is 24 h.

[0083] (B2) The second microsphere is prepared by the following method:

[0084] Vitamin D3 and a carrier are dissolved in an oil phase solvent to prepare an oil phase; the oil phase and aqueous phase solvent are stirred in step I to prepare an emulsion; and the emulsion is stirred in step II, centrifuged, washed, and dried to prepare a second microsphere.

[0085] Optionally, the mass-volume percentage of the carrier to the oil phase solvent is 3%-5%, for example, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5%.

[0086] Optionally, the mass percentage of vitamin D3 to the carrier is 2.5%-3.5%, for example, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, or 3.5%.

[0087] Optionally, the drying includes sublimation drying and desorption drying. The temperature for sublimation drying is -40°C to -20°C, and the temperature for desorption drying is 10°C to 30°C. The total drying time is 22 h to 26 h. For example, the temperature for sublimation drying is -20°C, the temperature for desorption drying is 25°C, and the total drying time is 24 h.

[0088] (B3) The mixing time is 8 min to 12 min, for example, 8, 9, 10, 11, or 12 min.

[0089] In some embodiments of this application, the preparation methods of the first microsphere or the second microsphere each independently satisfy one or more of the following conditions:

[0090] (1) The oil phase solvent includes dichloromethane;

[0091] (2) The aqueous solvent includes a polyvinyl alcohol aqueous solution with a mass fraction of 0.5% to 1.5%, for example, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, or 1.5%;

[0092] (3) The volume ratio of the oil phase to the water phase is 1:(10~12), for example, 1:10, 1:11, 1:12;

[0093] (4) The stirring speed of stirring I is 700 rpm to 900 rpm, for example, 700, 750, 800, 850, 900 rpm;

[0094] (5) The temperature of stirring II is 20℃~30℃, for example 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30℃; the duration is 3.5 h~4.5 h, for example 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5 h;

[0095] (6) The centrifugation speed is 2500 rpm to 3500 rpm and the duration is 8 min to 12 min. For example, the centrifugation speed is 3000 rpm and the duration is 10 min.

[0096] A third aspect of this application provides a composite microsphere formulation comprising the composite microspheres described above or the composite microspheres prepared by the method described above; and an acceptable excipient.

[0097] Optionally, the acceptable excipients include one or more of sodium chloride and sodium carboxymethyl cellulose;

[0098] Optionally, the sodium chloride in the acceptable excipient has a mass fraction of 0.9% to 1.0%, for example, 0.9% or 1.0%;

[0099] Optionally, the mass fraction of the sodium carboxymethyl cellulose in the acceptable excipients is 0.4% to 0.6%, for example, 0.4%, 0.5%, or 0.6%.

[0100] Optionally, the mass fraction of the composite microspheres in the composite microsphere formulation is 45% to 60%, for example, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%.

[0101] A fourth aspect of this application provides the use of the composite microspheres, the composite microspheres prepared by the method, or the composite microsphere formulation in the preparation of products for preventing and treating hair loss and / or promoting hair regeneration, satisfying one or more of the following conditions:

[0102] (1) The hair loss prevention and / or hair regeneration promotion products include one or more of the following: subcutaneous implants, injectable suspensions and microneedle patches;

[0103] (2) The subjects of the products for preventing and treating hair loss and / or promoting hair regeneration include mammals;

[0104] (3) The hair loss prevention and / or hair regeneration promotion products are used to promote the proliferation of hair follicle stem cells and the survival of hair papilla cells, or one or more of these.

[0105] The embodiments of this application will be described in detail below with reference to some examples. It should be understood that these embodiments are only for illustrating this application and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, please refer to the guidelines given in this application first, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0106] In the following examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0107] The materials and reagents involved in the following embodiments are shown in Table 1.

[0108] Table 1. Sources of Materials and Reagents

[0109]

[0110] Example 1

[0111] 1. Preparation of PDLLA microspheres (first microsphere)

[0112] (1) Materials: PDLLA (Mw 100,000, PDI 1.8), dichloromethane (DCM, analytical grade), 1% (w / v) polyvinyl alcohol (PVA, Mw 30,000-70,000) aqueous solution.

[0113] (2) Steps: Dissolve PDLLA in DCM to prepare a 5% (w / v) solution; slowly add the oil phase (PDLLA / DCM solution) dropwise to a pre-cooled 1% PVA aqueous solution (oil phase:water phase = 1:10, v / v) under magnetic stirring (800 rpm); stir at room temperature for 4 hours to allow DCM to fully evaporate; collect the microspheres by centrifugation (3000 rpm, 10 minutes), wash three times with deionized water; freeze dry, wherein the sublimation drying temperature is -20℃, the desorption drying temperature is 25℃, and the total drying time is 24 hours to prepare white powdered PDLLA microspheres.

[0114] (3) Characterization:

[0115] a. Particle size distribution: Malvern Mastersizer 3000 laser particle size analyzer was used for dry loading at a pressure of 1 Bar; the Span was calculated as follows: Span = (D90 + D10) / D50; D50 = 32 ± 5 μm, Span < 1.5;

[0116] b. Morphology: Scanning electron microscopy (SEM) revealed that the PDLLA microspheres were regularly spherical with smooth surfaces. Figure 1 ;

[0117] c. Glass transition temperature (Tg): Determined by differential scanning calorimetry (DSC), Tg = 42 ± 2℃.

[0118] d. The mass fraction of polyracemic lactic acid is ≥99%.

[0119] 2. Preparation of VD3 microspheres (second microspheres, PLGA carrier)

[0120] (1) Materials: PLGA (lactic acid: glycolic acid = 75:25, Mw 50,000, PDI 1.6), vitamin D3 (purity ≥ 98%), DCM, 1% PVA aqueous solution.

[0121] (2) Steps: Dissolve PLGA in DCM to prepare a 4% (w / v) solution; add VD3 (3% of the weight of PLGA) and sonicate (200W, 5 minutes) to make it uniformly dispersed; prepare VD3 microspheres by the same emulsification-solvent evaporation method as for preparing PDLLA microspheres, i.e., slowly drop the oil phase (PLGA / DCM / VD3 solution) into a pre-cooled 1% PVA aqueous solution (oil phase:water phase = 1:10, v / v) under magnetic stirring (800 rpm); stir at room temperature for 4 hours to allow DCM to fully evaporate; collect the microspheres by centrifugation (3000 rpm, 10 minutes) and wash three times with deionized water; freeze dry using a Boyikang pilot5-8M freeze dryer, where the sublimation drying temperature is -20℃, the desorption drying temperature is 25℃, and the total drying time is 24 hours, and store in the dark.

[0122] (3) Characterization:

[0123] a. Particle size: D50 = 25 ± 4 μm;

[0124] b. Morphology: Scanning electron microscopy (SEM) revealed that the VD3 microspheres were regularly spherical with porous surfaces. Figure 1 ;

[0125] c. Drug loading: The actual drug loading was determined by high performance liquid chromatography (HPLC), and the encapsulation efficiency was 89%.

[0126] 3. Preparation of composite microspheres

[0127] (1) Physically mix PDLLA microspheres and VD3 microspheres at a mass ratio of 20:1 (PDLLA microspheres:VD3 microspheres = 20:1, i.e., VD3 accounts for approximately 0.13% of the total weight of microspheres) in a three-dimensional mixer for 10 minutes to ensure uniform distribution of the two types of microspheres. Dispense into sterile vials, seal with nitrogen gas, and store at 4°C protected from light.

[0128] (2) Reconstitute before use: Take the composite microsphere powder and add it to a suspension containing 0.9% NaCl and 0.5% sodium carboxymethyl cellulose to prepare an injection suspension with a microsphere concentration of 50 mg / mL.

[0129] 4. In vitro release kinetics study

[0130] (1) Degradation curve of PDLLA microspheres: PDLLA microspheres (50 mg) were placed in PBS (pH 7.4, 37 °C), and samples were taken periodically to determine the mass loss; the results are shown in Table 2.

[0131] Table 2. Mass loss of PDLLA microspheres

[0132]

[0133] (2) VD3 microsphere release curve: VD3-PLGA microspheres (containing approximately 1.35 mg of VD3) were placed in PBS containing 0.5% Tween 80, and samples were taken periodically. The VD3 concentration was determined by HPLC. A C18 column (4.6 mm × 150 mm, 5 μm) was used, with methanol-water (90:10) as the mobile phase for isocratic elution. The flow rate was set at 1.2 mL / min, the column temperature was controlled at 30 °C, and the detection was performed at a wavelength of 265 nm. The results are shown in Table 3.

[0134] Table 3. Cumulative Release of VD3 Microspheres

[0135]

[0136] (3) The PDLLA degradation rate (representing mechanical stress intensity) and VD3 release rate were plotted on the same time axis. The results are shown in […]. Figure 2 The peak mechanical stress of PDLLA occurs between 4 and 8 weeks (accelerated degradation period); the peak release of VD3 occurs between 8 and 16 weeks; there is a time difference of about 4 to 8 weeks between the two curves, and an effective overlap zone is formed between 4 and 12 weeks.

[0137] Example 2

[0138] This embodiment is a variation of Example 1, the difference being that the mass ratio of PDLLA microspheres to VD3 microspheres in the composite microspheres is 10:1. Specific operation: PDLLA microspheres and VD3 microspheres are physically mixed at a mass ratio of 10:1, i.e., 50mg of PDLLA microspheres + 5mg of VD3 microspheres (VD3 accounts for approximately 9.1% of the total microsphere weight). Everything else is the same as in Example 1.

[0139] Example 3

[0140] This embodiment is a variation of Example 1, the difference being that the mass ratio of PDLLA microspheres to VD3 microspheres in the composite microspheres is 5:1. Specifically, PDLLA microspheres and VD3 microspheres are physically mixed at a mass ratio of 5:1, i.e., 50 mg of PDLLA microspheres + 10 mg of VD3 microspheres (VD3 accounts for approximately 16.7% of the total microsphere weight). Everything else is the same as in Example 1.

[0141] Comparative Example 1

[0142] This comparative example is a comparative example of Example 1. The difference between this example and Example 1 is that the PDLLA single microspheres are individually packaged in sterile vials, sealed with nitrogen gas, and stored at 4°C in the dark.

[0143] Comparative Example 2

[0144] This comparative example is a comparative example of Example 1. The difference between this example and Example 1 is that the VD3 single microspheres are individually packaged into sterile vials, sealed with nitrogen gas, and stored at 4°C in the dark.

[0145] Comparative Example 3

[0146] This comparative example is a comparative example of Example 1, and the difference between it and Example 1 is that:

[0147] (1) Preparation of encapsulated microspheres: VD3 was directly added to the DCM solution of PDLLA (the content of VD3 was 2% of the weight of PDLLA) and single microspheres of “PDLLA encapsulated VD3” were prepared according to the method of Example 1.

[0148] (2) Release of encapsulated microspheres: In the encapsulation scheme, VD3 was released more than half in the first 4 weeks, which could not form a time mismatch with the mechanical stress peak of PDLLA, indicating that the independent microsphere mixing scheme is a necessary means to achieve time synergy. The release results are shown in Table 4.

[0149] Table 4. Release results of encapsulated microspheres

[0150]

[0151] Animal experiment (rat hair loss model)

[0152] (1) Animals: SD rats (Guangzhou Huateng Biomedical Co., Ltd.), female, 8 weeks old, weighing 180-220g, a total of 40 rats, randomly divided into 5 groups (n=8 / group).

[0153] (2) Model establishment: The hair on the back of the rats was shaved with an electric hair removal shaver, and hair removal cream (containing calcium thioglycolate) was applied and left on for 8 minutes before being wiped clean to establish a resting phase model. The hair removal area was approximately 4cm × 5cm.

[0154] (3) Administration: 24 hours after hair removal, each group was injected subcutaneously into the back with the corresponding preparation (0.5 mL / point, 4 points in total, distributed in a diamond shape), with an injection depth of about 3-5 mm.

[0155] (4) Observation indicators:

[0156] a. Length of new hair: On day 28, a circular skin area with a diameter of 2cm was taken and the length of new hair was measured. The specific method is as follows: when the hair in the hairless area on the back of the mouse enters the growth phase and reaches a suitable length (for easy removal), 20 new hairs are collected from the hairless area on the back of each group of mice every 1 day. The 3 longest hairs are selected, and their lengths are measured with calipers and recorded. The average value is then used for statistical analysis.

[0157] b. Quality of newly grown hair: On day 28 of the experiment, mice in each group were sacrificed by cervical dislocation, and newly grown hair samples were collected from the bald areas on the backs of each group of mice. The collected hair was weighed using a precise analytical balance, and the mass data of each group's samples were recorded. The average value was then used for statistical analysis to evaluate the impact of the experimental intervention on hair growth.

[0158] See results Figure 3 See Table 5.

[0159] Table 5. Hair regeneration experiment in a rat hair loss model

[0160]

[0161] Mechanism validation (Piezo1 and VDR pathways)

[0162] (1) Animals: SD rats, female, 8 weeks old, weighing 180-220g, a total of 160 rats, randomly divided into 5 groups (n=32 / group).

[0163] (2) Grouping: Same as Table 5.

[0164] (3) Detection time points: Day 3 (early start), Day 14 (start-up period), Day 42 (proliferation period), and Day 126 (maintenance period). At each time point, 8 mice in each group were randomly sacrificed by cervical dislocation and skin samples were taken for detection.

[0165] (4) Detection indicators:

[0166] a. Piezo1 pathway: RT-qPCR was used to detect the mRNA level of Piezo1;

[0167] b. Wnt pathway: RT-qPCR was used to detect the mRNA level of Wnt10b;

[0168] c. VDR pathway: RT-qPCR was used to detect the mRNA level of VDR;

[0169] d. Nrf2 pathway: RT-qPCR detection of HO-1 mRNA levels;

[0170] e. ROS detection kits are used to measure the total ROS level in tissues.

[0171] The primer sequences used in the detection are shown in Table 6.

[0172] Table 6. RT-qPCR primer sequence information

[0173]

[0174] The results are as follows Figures 4-8 As shown: On day 14, Piezo1 expression was significantly upregulated in groups A, B, and D; no significant changes were observed in groups C and E. On day 14, Wnt target gene expression in group A reached its peak, comparable to group B. On day 42, HO-1 expression remained high in group A; HO-1 expression in group B remained unchanged throughout the experiment, although ROS levels in group B began to rise from day 14; ROS levels in group A remained low on day 42, while both Wnt activity (maintaining proliferation) and VDR activity (antioxidant) were present. As a control, although Wnt pathway activity in group B had not yet decreased, VDR was still not activated, resulting in a state of "proliferation without protection" in group B. On day 126, the VDR pathway in group A remained active, while groups B and E were in an inactive state.

[0175] The mechanism of action of the composite microspheres is shown in Table 7.

[0176] Table 7. Mechanism of Action of Composite Microspheres

[0177]

[0178] (1) Startup phase (weeks 0-4): PDLLA dominant

[0179] After PDLLA microspheres are implanted subcutaneously, they absorb water and begin to degrade, slowly shrinking in volume (approximately 10-20%). This slow mechanical deformation is sensed by the Piezo1 mechanosensitive ion channels in the surrounding tissue, triggering calcium ion influx and activating the downstream YAP / TAZ signaling pathway. After YAP / TAZ enters the nucleus, it promotes macrophage polarization towards the M2 type (alternating activated type), and M2 macrophages secrete growth factors such as HGF, IGF-1, and TGF-β. These growth factors act on hair follicle stem cells (located in the hair follicle bulge and around the dermal papilla), upregulating the expression of genes such as Wnt10b and β-catenin, driving resting hair follicles into the anagen phase. During this stage, the VD3 microspheres have not yet entered their main release phase (cumulative release << 20%), and the VD3 pathway is only in a pre-activated state, not interfering with the PDLLA initiation signal.

[0180] (2) Transition period (4-12 weeks): Dual pathway overlap

[0181] PDLLA microspheres continue to degrade, maintaining a high level of mechanical stress; simultaneously, VD3 microspheres enter an accelerated release phase, with local VD3 concentrations gradually increasing. During this stage: the mechanical stress effect of PDLLA microspheres has not yet decreased to below 50% of baseline, and the VDR-Nrf2 pathway activation effect of VD3 microspheres has reached above 50% of baseline. The Wnt pathway maintains hair follicle stem cell proliferation; the VDR pathway begins to activate, promoting dermal papilla cell survival; the Nrf2 pathway gradually initiates, upregulating antioxidant enzymes such as HO-1 and NQO1, clearing ROS. The overlap of these two pathways forms a parallel support of "proliferation + protection." Without the protection of VD3, rapidly proliferating hair follicle cells produce large amounts of ROS under a high metabolic state, thus accelerating their entry into the regression phase.

[0182] (3) Maintenance phase (12-24 weeks and beyond): VD3 dominant

[0183] As PDLLA microspheres degrade in their later stages, mechanical stress gradually decreases, and the initiation signal weakens. At this point, VD3 release enters a plateau or slow-release phase, and the VDR-Nrf2 pathway becomes dominant: maintaining the stability of the dermal papilla vascular network; continuously inhibiting oxidative stress to prevent hair follicles from prematurely entering the regression phase; and regulating hair follicle cycle clock genes to prolong the duration of the anagen phase. Without the protection of this stage, hair follicles that enter the anagen phase after PDLLA initiation may prematurely enter the regression phase around 12-16 weeks, resulting in short-lived hair regeneration.

[0184] In summary, the polyracemic lactic acid-vitamin D3 composite microspheres provided in this application can promote the activation of hair follicle stem cells and hair growth, and can be used to prepare drugs or medical devices for the prevention and treatment of androgenetic alopecia, alopecia areata, hair loss or thinning hair after chemotherapy.

[0185] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0186] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. Composite microspheres, characterized in that, The composite microspheres comprise a first microsphere and a second microsphere; the first microsphere comprises polyracemic lactic acid; and the second microsphere comprises vitamin D3 and a carrier.

2. The composite microspheres according to claim 1, characterized in that, The carrier includes one or more of PLGA, PCL, and liposomes.

3. The composite microspheres according to claim 2, characterized in that, In the first microsphere, the mass fraction of the polyracemic lactic acid is ≥99%; in the second microsphere, the mass fraction of vitamin D3 is 2.5%~3%. The mass ratio of the first microsphere to the second microsphere is (5~20):

1.

4. The composite microspheres according to claim 1, characterized in that, The polyracemic lactic acid has a weight-average molecular weight of 80,000 Da to 120,000 Da and a polydispersity index of 1.5 to 2.

0.

5. The composite microspheres according to any one of claims 1 to 4, characterized in that, The first microsphere satisfies one or more of the following conditions (A1) to (A3): (A1) The average particle size is 25 μm to 50 μm; (A2) Particle size distribution width Span < 1.5; (A3) The glass transition temperature is 40℃~44℃; and / or, The average particle size of the second microsphere is 15 μm to 35 μm.

6. The method for preparing composite microspheres according to any one of claims 1 to 5, characterized in that, Includes the following steps: The first microsphere and the second microsphere are mixed evenly to prepare composite microspheres.

7. The method for preparing composite microspheres according to claim 6, characterized in that, The preparation method satisfies one or more of the following conditions (B1) to (B3): (B1) The first microsphere is prepared by the following method: The polyracemic lactic acid is dissolved in an oil phase solvent to prepare an oil phase; the oil phase and aqueous phase solvent are stirred in step I to prepare an emulsion; and the emulsion is stirred in step II, centrifuged, washed, frozen and dried to prepare the first microspheres. Optionally, the polyracemic lactic acid has a mass fraction of 3% to 7% in the oil phase solvent; optionally, the drying includes sublimation drying and desorption drying, the temperature of sublimation drying is -40℃ to -20℃, the temperature of desorption drying is 10℃ to 30℃, and the total drying time is 22 h to 26 h. (B2) The second microsphere is prepared by the following method: Vitamin D3 and a carrier are dissolved in an oil phase solvent to prepare an oil phase; the oil phase and aqueous phase solvent are stirred to prepare an emulsion. Furthermore, the emulsion is subjected to stirring II, centrifugation, washing, and drying to prepare the second microspheres; Optionally, the mass-volume percentage of the carrier to the oil phase solvent is 3% to 5%; Optionally, the mass percentage of vitamin D3 to the carrier is 2.5% to 3.5%; Optionally, the drying includes sublimation drying and desorption drying. The temperature for sublimation drying is -40°C to -20°C, and the temperature for desorption drying is 10°C to 30°C. The total drying time is 22 h to 26 h. (B3) The mixing time is 8 min to 12 min.

8. The method for preparing composite microspheres according to claim 7, characterized in that, The preparation methods of the first microsphere or the second microsphere each independently satisfy one or more of the following conditions: (1) The oil phase solvent includes dichloromethane; (2) The aqueous solvent includes a polyvinyl alcohol aqueous solution with a mass fraction of 0.5% to 1.5%; (3) The volume ratio of the oil phase to the water phase is 1:(10~12); (4) The stirring speed of stirring I is 700 rpm to 900 rpm; (5) The temperature of stirring II is 20℃~30℃, and the duration is 3.5 h~4.5 h; (6) The centrifugation speed is 2500 rpm to 3500 rpm and the duration is 8 min to 12 min.

9. A composite microsphere formulation, characterized in that, The composite microspheres comprising any one of claims 1 to 5 or the composite microspheres prepared by the method of any one of claims 6 to 8; and, acceptable excipients; Optionally, the acceptable excipients include one or more of sodium chloride and sodium carboxymethyl cellulose; Optionally, the sodium chloride in the acceptable excipient has a mass fraction of 0.9% to 1.0%; Optionally, the mass fraction of the sodium carboxymethyl cellulose in the acceptable excipients is 0.4% to 0.6%; Optionally, the composite microspheres have a mass fraction of 45% to 60% in the composite microsphere formulation.

10. The use of the composite microspheres according to any one of claims 1 to 5, the composite microspheres prepared by the method according to any one of claims 6 to 8, or the composite microsphere formulation according to claim 9 in the preparation of products for preventing and treating hair loss and / or promoting hair regeneration, shall meet one or more of the following conditions: (1) The hair loss prevention and / or hair regeneration promotion products include one or more of the following: subcutaneous implants, injectable suspensions and microneedle patches; (2) The subjects of the products for preventing and treating hair loss and / or promoting hair regeneration include mammals; (3) The hair loss prevention and / or hair regeneration promotion products are used to promote the proliferation of hair follicle stem cells and the survival of hair papilla cells, or one or more of these.

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