Nano composite material and micro-needle patch for treating acne and preparation method of nano composite material and micro-needle patch

Abstract

The invention discloses a nano composite material for treating acne, a microneedle patch and a preparation method of the nano composite material and the microneedle patch, and belongs to the technical field of biomedical materials. The nano composite material provided by the invention comprises nano particles of a core-shell structure taking silk fibroin nano particles and resveratrol as a core layer and ZIF-8 as a shell layer. The prepared nano composite material has excellent structural stability, the bioavailability of active ingredients can be improved, skin cell regeneration and collagen synthesis can be promoted while a good acne treatment effect is achieved, and therefore the skin state of an acne patient is improved; the propionibacterium acnes micro-needle patch prepared from the propionibacterium acnes micro-needle patch is easy to clinically transform and has a huge clinical application value.

Description

Technical Field

[0001] This application belongs to the technical field of biomedical materials, and particularly relates to a nanocomposite material for treating acne, a microneedle patch, and a preparation method thereof. Background Art

[0002] Acne is a chronic inflammatory skin disease of the pilosebaceous unit, which is prone to occur on the face and upper body, forming polymorphic skin lesions such as blackheads, papules, pustules, and nodules. Clinically, the treatment of acne mainly includes drug treatment, laser treatment, and chemical peeling: Although laser treatment and chemical peeling have good curative effects, these methods need to be carried out in professional institutions and have high costs, which are not suitable for all patients. Drug treatment includes taking oral antibiotics or using topical antibiotic creams, but due to the skin barrier effect, these drugs are often difficult to penetrate the skin to reach the affected area, thus limiting the treatment effect, and long-term use of oral antibiotics may lead to intestinal microbial imbalance and other side effects. In the prior art, Propionibacterium acnes can secrete extracellular polysaccharides to form biofilms, which are resistant to general antibacterial agents and host inflammatory cells, further increasing the treatment difficulty. Therefore, it is necessary to develop a product with excellent comprehensive performance to achieve effective and safe treatment of acne.

[0003] The prior art with the publication number of CN 116509789 A discloses a composition for treating acne, comprising: a biodegradable polymer and an acne treatment agent; wherein, the acne treatment agent includes a melanin inhibitor, a bactericidal and anti-inflammatory agent, a wound healing agent, and / or a pore shrinking agent. This composition can treat acne and make the microneedles have better hardness and dissolution rate.

[0004] However, the existing compositions for treating acne have the following problems: First, due to the existence of the skin barrier, the transdermal absorption rate of the drug is low, resulting in poor treatment effect; second, drug resistance is easily generated, increasing the treatment difficulty. Summary of the Invention

[0005] This application discloses a nanocomposite material for treating acne, a microneedle patch, and a preparation method thereof, aiming to solve the technical problems of poor stability of the nanocomposite material and poor treatment effect on acne.

[0006] To achieve the above object, the technical solution of this application is:

[0007] The first aspect of this application provides a nanocomposite material for treating acne, comprising: nanoparticles with a core-shell structure having silk fibroin nanoparticles and resveratrol as the core layer and ZIF-8 as the shell layer.

[0008] The second aspect of this application provides a preparation method of the nanocomposite material for treating acne described in the first aspect, and the preparation method includes:

[0009] Dissolve silk fibroin nanoparticles and resveratrol in ethanol, react them, and then freeze-dry to obtain RSL-NPs;

[0010] React the RSL-NPs, zinc nitrate hexahydrate, and 2-methylimidazole in water, separate and collect them to obtain the nanocomposite.

[0011] Preferably in combination with the second aspect, the mass ratio of the silk fibroin nanoparticles to resveratrol is 1:0.04 - 1 。

[0012] Preferably in combination with the second aspect, when dissolving the silk fibroin nanoparticles and resveratrol in ethanol for reaction, it is a continuous shaking reaction in the dark at room temperature for 24 h.

[0013] Preferably in combination with the second aspect, the molar ratio of zinc nitrate hexahydrate to 2-methylimidazole is 1:30 - 40.

[0014] Preferably in combination with the second aspect, the mass ratio of the RSL-NPs to the mixture of zinc nitrate hexahydrate and 2-methylimidazole is 1:20 - 40.

[0015] The third aspect of the present application provides a microneedle patch for treating acne, including the nanocomposite for treating acne described in the first aspect or the nanocomposite for treating acne prepared by the preparation method described in the second aspect.

[0016] Preferably in combination with the third aspect, it includes a backing layer and a needle tip layer;

[0017] The needle tip layer is composed of soluble microneedles arranged in an array on the backing layer, and the soluble microneedles are loaded with the nanocomposite for treating acne; the substrate of the needle tip layer further includes sodium hyaluronate;

[0018] The needle tip layer is a quadrangular pyramid, with a diameter of 300 - 500 μm, a height of 500 - 700 μm, and a needle tip spacing of 500 - 700 μm;

[0019] The backing layer is one or more of a PVA film, a PE film, a PVC film, a PET film, an EVA film, a PU film, and a PSA film.

[0020] The fourth aspect of the present application provides a preparation method of the microneedle patch for treating acne described in the third aspect, and the preparation method includes:

[0021] Add sodium hyaluronate to the aqueous solution of the nanocomposite for treating acne, mix it, and inject it into a microneedle array mold to make a microneedle array;

[0022] Inject the backing layer gel into the microneedle array mold for filling to make the backing, and demold after drying to obtain the microneedle patch.

[0023] The fifth aspect of the present application provides the application of the microneedle patch for treating acne prepared by the preparation method described in the fourth aspect in the preparation of anti-acne drugs.

[0024] Compared with the prior art, the advantages or beneficial effects of the embodiments of the present application at least include:

[0025] The nanocomposite provided by the present application is a core-shell structured nanoparticle with silk fibroin nanoparticles and resveratrol as the core layer and ZIF-8 as the shell layer. On the one hand, silk fibroin nanoparticles and resveratrol are made into the RSL-NPs core layer through electrostatic adsorption, and ZIF-8 is combined to form a core-shell structure, which has excellent structural stability; on the other hand, when treating acne, the shell layer ZIF-8 undergoes acid-responsive release in the acidic site caused by Propionibacterium acnes infection to produce free Zn 2+ , effectively killing bacteria; subsequently, the RSL-NPs in the core layer are released for inflammation regulation and skin repair, achieving a treatment sequence of first "eliminating" and then "healing", improving the bioavailability of active ingredients, promoting skin cell regeneration and collagen synthesis while achieving good acne treatment effects, thereby improving the skin condition of acne patients; thirdly, it can utilize the hyaluronidase released by Propionibacterium acnes to degrade the hyaluronic acid matrix, thereby releasing the core-shell structured nanocomposite, achieving the effect of precise drug delivery and effectively preventing the generation of drug resistance of Propionibacterium acnes. At the same time, its preparation raw materials all use bioactive polymers, which have excellent biocompatibility, and the microneedle patch made thereof is easy to be clinically transformed and has great clinical application value. Description of the Drawings

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are only some embodiments recorded in the present application. For those of ordinary skill in the art, other drawings can be obtained according to these drawings without creative efforts.

[0027] Figure 1 Scanning electron microscope images of SF-NPs, RSL-NPs, and RSL@ZIF-8 prepared in the embodiments of the present application;

[0028] Figure 2 Drug loading rate and encapsulation rate diagrams of RSL-NPs prepared in the embodiments of the present application;

[0029] Figure 3Live / dead staining images of L929 cells treated with SF-NPs, RSL-NPs, and RSL@ZIF-8 prepared in the embodiments of the present application;

[0030] Figure 4 CCK8 data graphs of different concentrations treated with SF-NPs prepared in the embodiments of the present application;

[0031] Figure 5 CCK8 data graphs of different concentrations treated with RSL-NPs prepared in the embodiments of the present application;

[0032] Figure 6 CCK8 data graphs of different concentrations treated with RSL@ZIF-8 prepared in the embodiments of the present application;

[0033] Figure 7 Hemolysis rate graphs of SF-NPs, RSL-NPs, and RSL@ZIF-8 prepared in the embodiments of the present application;

[0034] Figure 8 Physical images of the wound healing of L929 cells treated with SF-NPs, RSL-NPs, and RSL@ZIF-8 prepared in the embodiments of the present application;

[0035] Figure 9 Wound healing rate graphs of SF-NPs, RSL-NPs, and RSL@ZIF-8 prepared in the embodiments of the present application;

[0036] Figure 10 Scanning electron microscope images of Propionibacterium acnes treated with SF-NPs, RSL-NPs, and RSL@ZIF-8 prepared in the embodiments of the present application;

[0037] Figure 11 Colony images of Propionibacterium acnes treated with SF-NPs, RSL-NPs, and RSL@ZIF-8 prepared in the embodiments of the present application;

[0038] Figure 12 Bacterial viability graphs of Propionibacterium acnes treated with SF-NPs, RSL-NPs, and RSL@ZIF-8 prepared in the embodiments of the present application;

[0039] Figure 13 Physical image of the A1-microneedle patch prepared in the embodiments of the present application. Detailed implementation manners

[0040] Next, the technical solutions in the embodiments of the present application will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present application without creative efforts shall fall within the protection scope of the present application.

[0041] In the following description of this embodiment, the term "and / or" is used to describe the association relationship of associated objects, indicating that there can be three relationships. For example, A and / or B can represent: A exists alone, B exists alone, and the situation where A and B exist simultaneously. Wherein A and B can be singular or plural. The character " / " generally represents an "or" relationship between the associated objects before and after.

[0042] In the following description of this embodiment, the term "at least one" means one or more, and "a plurality" means two or more. "At least one of the following items (pieces)" or similar expressions refer to any combination of these items, including any combination of single item (piece) or plural items (pieces). For example, "at least one of a, b or c", or, "at least one of a, b and c" can both represent: a, b, c, a - b (that is, a and b), a - c, b - c, or a - b - c, where a, b, and c can be single or multiple respectively.

[0043] Those skilled in the art should understand that in the following description of the embodiments of the present application, the sequence numbers do not mean the order of execution, and some or all of the steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation to the implementation process of the embodiments of the present application.

[0044] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a" and "the" used in the embodiments of the present application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0045] It should be noted that all raw material reagents in the embodiments of the present application are purchased on the market or prepared by conventional methods well-known to those skilled in the art.

[0046] In a first aspect, an embodiment of the present application provides a nanocomposite for treating acne, including: nanoparticles with a core-shell structure having silk fibroin nanoparticles and resveratrol as the core layer and ZIF-8 as the shell layer.

[0047] Among them, on the one hand, silk fibroin nanoparticles and resveratrol are made into the RSL-NPs core layer through electrostatic adsorption, and ZIF-8 is combined to form a core-shell structure, which has excellent structural stability; on the other hand, when the prepared microneedles are used to treat acne, the shell layer ZIF-8 undergoes acid-responsive release in the acidic part caused by Propionibacterium acnes infection to produce free Zn 2+ , effectively killing bacteria; subsequently, the RSL-NPs in the core layer are released for inflammation regulation and skin repair, achieving a treatment sequence of first "eliminating" and then "healing", improving the bioavailability of active ingredients, and at the same time being able to greatly enhance the absorption efficiency; thirdly, it can utilize the hyaluronidase released by Propionibacterium acnes to degrade the hyaluronic acid matrix, thereby releasing the core-shell structured nanocomposite material, achieving the effect of precise drug delivery and effectively preventing the generation of drug resistance of Propionibacterium acnes. At the same time, its preparation raw materials all use bioactive polymers, which have excellent biocompatibility, and the prepared microneedle patch is easy to be clinically translated and has great clinical application value.

[0048] Secondly, the embodiment of the present application also provides a preparation method of the nanocomposite material for treating acne described in the first aspect, and the preparation method includes:

[0049] Dissolve silk fibroin nanoparticles and resveratrol in ethanol, react, and then freeze-dry to obtain RSL-NPs;

[0050] React the RSL-NPs, zinc nitrate hexahydrate and 2-methylimidazole in water, separate and collect to obtain the nanocomposite material.

[0051] It should be noted that the embodiment of the present application does not particularly limit the specific source of silk fibroin nanoparticles, which can be obtained through commercial purchase or synthesized by methods known in the art. For example, the preparation method adopted in the embodiment of the present application is:

[0052] Step 1: Use anhydrous Na 2 CO 3 to degum the silkworm cocoon shell: Cut the cocoon shell into pieces for later use; Prepare a 0.2M anhydrous Na 2 CO 3 solution and boil it (the mass-volume ratio of the silkworm cocoon to the anhydrous Na 2 CO 3 solution is 1:10); Put the cocoon shell pieces into the anhydrous Na 2 CO 3 solution and boil for 20 min, and add deionized water during this process to keep the system concentration stable; Take out the boiled silkworm cocoon and rinse it with deionized water multiple times; After rinsing, put it into the anhydrous Na 2 CO 3Solution, repeat the above steps; put the obtained silk into an oven to dry and obtain silk fibroin fibers;

[0053] Step 2: Use LiBr to dissolve silk fibroin fibers to obtain purified silk fibroin: Prepare a LiBr solution with a concentration of 9.3 M; Take the dried silk fibroin fibers and slowly add them to the prepared LiBr solution in multiple batches (the mass-volume ratio of silk fibroin to LiBr solution is 1:10 - 30); Stir and react for 4 h to ensure that the silk fibroin fibers are fully dissolved; After filtering the above regenerated silk fibroin solution with absorbent cotton, place it in a dialysis bag (cut-off molecular weight 8000 - 14000 Da) and dialyze for 72 - 96 h; Freeze-dry the dialyzed solution to obtain purified silk fibroin (SF);

[0054] Step 3: Prepare SF-NPs by the freeze-thaw method: Prepare an SF solution with a concentration of 2 mg / mL with deionized water and place it on a magnetic stirrer, and stir at a constant speed of 100 rpm; Subsequently, use isopropanol as a denaturant to induce the self-assembly of SF into SF-NPs, that is, slowly add isopropanol dropwise to the SF solution during stirring for reaction (isopropanol:SF = 1:10 (v / v)); Freeze the mixed solution overnight; Dissolve and centrifuge the frozen mixed solution at room temperature to obtain an SF-NPs precipitate, wash it with ethanol solution and water, centrifuge and freeze-dry it to obtain silk fibroin nanoparticles (SF-NPs).

[0055] It should be noted that in the preparation method adopted in this application, resveratrol is first bound to the surface of silk fibroin nanoparticles by electrostatic adsorption. Moreover, there are nucleation sites of ZIF-8 on the silk fibroin nanoparticles, which can bind ZIF-8 to the outer layer of the nanoparticles and form a core-shell structure.

[0056] In the examples of this application, the mass ratio of the silk fibroin nanoparticles to resveratrol is 1:0.04 - 1, and more preferably 1:1. Among them, silk fibroin is a natural protein with good biocompatibility and biodegradability, and can be used to prepare biomedical materials. Resveratrol scavenges free radicals through its antioxidant effect, blocks the positive feedback loop of oxidative stress and pro-inflammatory cytokines, and thus achieves the purpose of anti-inflammation. The concentration of resveratrol is 1 - 25 mg / mL. By controlling the mass ratio of the silk fibroin nanoparticles to resveratrol, the drug loading rate of the silk fibroin nanoparticles to resveratrol can be increased, so as to achieve the expected application effect.

[0057] In the examples of this application, when dissolving the silk fibroin nanoparticles and resveratrol in ethanol for reaction, it is preferably to react continuously with light avoidance and oscillation at room temperature for 24 h. Among them, through the oscillation reaction, resveratrol can be bound to the surface of the silk fibroin nanoparticles by electrostatic adsorption to form a structurally stable core layer material.

[0058] In the embodiments of the present application, the molar ratio of zinc nitrate hexahydrate to 2-methylimidazole is preferably 1:30 - 40. Among them, by controlling the molar ratio of zinc nitrate hexahydrate to 2-methylimidazole, the structure and morphology of the generated ZIF-8 can be effectively controlled.

[0059] In the embodiments of the present application, the mass ratio of the RSL-NPs to the mixture of zinc nitrate hexahydrate and 2-methylimidazole is preferably 1:20 - 40. Among them, by controlling the addition amount of each substance, ZIF-8 with a stable structure and morphology can be combined onto the nucleation sites of silk fibroin nanoparticles to form nanoparticles with a stable core-shell structure.

[0060] The third aspect of the present application provides a microneedle patch for treating acne, including the nanocomposite for treating acne described in the first aspect or the nanocomposite for treating acne prepared by the preparation method described in the second aspect.

[0061] In the embodiments of the present application, the microneedle patch for treating acne includes a backing layer and a needle tip layer; the needle tip layer is composed of soluble microneedles arranged in an array on the backing layer, and the soluble microneedles are loaded with the nanocomposite for treating acne; the substrate of the needle tip layer further includes sodium hyaluronate; the needle tip layer is a quadrangular pyramid with a diameter of 300 - 500 μm, a height of 500 - 700 μm, and a needle tip spacing of 500 - 700 μm; the backing layer is preferably one or several of PVA film, PE film, PVC film, PET film, EVA film, PU film, PSA film. Among them, the molecular weight of sodium hyaluronate is 10,000 - 100,000. Sodium hyaluronate within this molecular weight range has good solubility and good hardness of the prepared needle tips. By controlling the size, shape, and array spacing of the microneedles, the microneedles can achieve the best natural healing effect after insertion into the skin, with low irritation and no skin problems caused by the needle holes.

[0062] The fourth aspect of the present application provides a preparation method for the microneedle patch for treating acne described in the third aspect, and the preparation method includes: adding sodium hyaluronate to the aqueous solution of the nanocomposite for treating acne and mixing, injecting it into a microneedle array mold to make a microneedle array; injecting a backing layer gel into the microneedle array mold for filling to make a backing, and demolding after drying to obtain the microneedle patch. Among them, the process for preparing the microneedle patch in the present application is simple and easy to implement, with low cost and easy clinical transformation, and has broad application prospects.

[0063] The fifth aspect of the present application provides the use of the microneedle patch for treating acne prepared by the preparation method described in the fourth aspect in the preparation of anti-acne drugs. Among them, based on the above microneedle patch, the transdermal absorption rate of the drug is increased, the stability and penetration ability of the drug are enhanced, and the drug is released in a responsive manner in the acidic microenvironment of acne lesions, realizing precise drug delivery and achieving the treatment effect of antibacterial first and then anti-inflammatory.

[0064] The technical solutions of the present application will be further elaborated below in conjunction with specific embodiments.

[0065] Example 1

[0066] This example provides a preparation method of A1-nanocomposite, which specifically includes:

[0067] S101: Add 1 mL of an ethanol solution of 25.0 mg / mL SF-NPs to 1 mL of a resveratrol solution of 25 mg / mL, mix them, put them into a constant temperature oscillator, continuously oscillate in the dark at room temperature for 24 h, centrifuge at 11000 rpm for 15 min, and wash with deionized water multiple times to obtain silk fibroin nanoparticles and resveratrol composite material (denoted as RSL-NPs);

[0068] S102: Add 0.958 mL of zinc nitrate hexahydrate (0.037 g / mL) to 81.6 mL of deionized water containing 14.2 mg of RSL-NPs, and stir for 30 min; then add 17.39 mL of 2-methylimidazole to the above reaction system, and react at 25 °C for 1 h; after the reaction, centrifuge at 11000 rpm for 10 min, collect the precipitate, wash it 3 times with deionized water, and freeze-dry to obtain A1-nanocomposite (denoted as RSL@ZIF-8).

[0069] Example 2

[0070] The preparation method, component ratio, preparation operation and process parameters of the nanocomposite provided in this example are basically the same as those in Example 1, except that the mass concentration of the resveratrol solution in the first step of this example is 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, and correspondingly, A2-nanocomposite, A3-nanocomposite, A4-nanocomposite, A5-nanocomposite, A6-nanocomposite are obtained.

[0071] Example 3

[0072] This example provides a preparation method of A1-microneedle patch, which specifically includes:

[0073] S301: Preparation of needle-layer gel: Prepare a 10 mg / mL RSL@ZIF-8-NPs solution with deionized water. Weigh 0.1 g of sodium hyaluronate with a molecular weight of 100,000 and add it to 1 mL of the nano-drug solution successively. Each time, stir vigorously until the sodium hyaluronate is completely dissolved to obtain the needle-layer gel. Centrifuge the needle-layer gel at 3000 rpm for 3 min for degassing treatment;

[0074] S302: Preparation of backing-layer gel: Prepare a backing-layer gel with a 200 mg / mL PVA solution using deionized water. Centrifuge the backing-layer gel at 3000 rpm for 3 min for degassing treatment;

[0075] S303: Add the needle-layer gel to the mold groove with a medicine spoon (the base of the mold is a cube with a length of 10 mm, a width of 10 mm, and a height of 1.5 mm; the needle tip is a quadrangular pyramid, evenly distributed on the base, with a diameter of 300 μm, a height of 650 μm, and a needle tip spacing of 550 μm). Evacuate (0.09 - 0.1 MPa) and maintain for 2 min. Place the mold in a 50 mL sleeve, centrifuge at 4000 rpm for 3 min and then take it out, and scrape off the colloid with bubbles on the surface. Repeat this step 2 - 3 times until each needle hole is filled with gel;

[0076] S304: Add the backing-layer gel to the mold groove as the backing layer of the microneedle patch at a mass area concentration of 1.2 mg / mm 2+ ; Subsequently, place it at room temperature for static drying for 72 h, demold, and obtain the microneedle patch loaded with anti-acne drugs. The fabricated appearance is as Figure 13 shown.

[0077] Meanwhile, to verify the comprehensive performance of the nanocomposites prepared in the above examples, the present application provides the following comparative examples for detailed elaboration.

[0078] Comparative Example 1

[0079] In this comparative example, the preparation raw material SF-NPs in Example 1 is used as Comparative Example 1, denoted as B1-nanocomposite.

[0080] Comparative Example 2

[0081] In this comparative example, the preparation raw material RSL-NPs in Example 1 is used as Comparative Example 2, denoted as B2-nanocomposite.

[0082] To verify the appearance of the nanocomposites prepared in the examples of the present application, the intermediates fabricated in the examples are tested by scanning electron microscopy, and the results are as Figure 1 shown; among them, Figure 1 are the scanning electron micrographs of SF-NPs, RSL-NPs, and RSL@ZIF-8.

[0083] According to Figure 1 what is known, SF-NPs exhibit a spherical structure with a particle size of approximately 260 ± 18 nm; the particle size of RSL-NPs gradually increases with drug loading, approximately 540 ± 22 nm; the particle size of RSL@ZIF-8 is approximately 200 ± 46 nm, which may be due to the growth and nucleation of ZIF-8 squeezing, causing the nuclear layer sphere to shrink inward, thus resulting in a smaller particle size of the nanoparticles.

[0084] To verify the effect of the mass concentration of the resveratrol solution added on the prepared materials, the drug loading rate and encapsulation rate of the A1-nanocomposite, A2-nanocomposite, A3-nanocomposite, A4-nanocomposite, A5-nanocomposite, and A6-nanocomposite prepared in the examples were tested:

[0085] The nanocomposite was prepared by the co-culture method: 25 mg of freeze-dried SF-NPs was added to 1 ml of ethanol to prepare a 25 mg / mL silk fibroin nanoparticle solution, which was mixed with 1 mL of resveratrol ethanol solution and gently stirred in the dark for 24 hours. The loaded particles were centrifuged at 11000 rpm for 15 minutes at 4°C, washed with double-distilled water to remove the unloaded resveratrol, and used for further characterization and utilization by freeze-drying.

[0086] In the drug loading amount (DLC) and encapsulation rate (EE) experiments, the concentration of resveratrol in the mixed reaction system was 1 - 25 mg / mL. After extraction in ethanol, referring to the standard curve (0.5 - 9 μg / mL), the loading amount of resveratrol was measured using a UV-visible spectrophotometer at a wavelength of 320 nm. Briefly, 25 mg of dry RSL-NPs was extracted with 1 ml of fresh ethanol in a centrifuge tube. After gently shaking for 1 hour, the SF-NPs were removed by centrifugation at 11000 rpm, and then the resveratrol concentration was measured. The drug loading rate and encapsulation rate are respectively represented by Equation (1) and Equation (2):

[0087] Drug loading rate (%) = (mass of loaded drug / mass of nanoparticles) × 100 Equation (1)

[0088] Encapsulation rate (%) = (mass of loaded drug / mass of administered drug) × 100 Equation (2)

[0089] According to Figure 2 what is known, when the mass ratio of resveratrol to silk fibroin nanoparticles is 1:1, the drug loading rate is 11.92%. Considering the biocompatibility and the dosage of resveratrol to exert its efficacy, the mass ratio of resveratrol to silk fibroin nanoparticles of 1:1 is preferably used as the final concentration, that is, the A1-nanocomposite (denoted as RSL@ZIF-8).

[0090] To verify the comprehensive performance of the nanocomposite prepared in the embodiments of the present application, corresponding biocompatibility and antibacterial performance tests were carried out:

[0091] 1. Skin toxicity

[0092] To verify the skin toxicity of the nanocomposite, we usually use mouse fibroblast L929 cells as model cells. CCK-8 method and FDA / PI live-dead staining method were used for quantitative and qualitative detection. The specific method is as follows: L929 cells were cultured under the constant temperature culture conditions of 37 °C and 5% CO 2 concentration in DMEM complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution. When the cells grew to cover 80-90% of the culture flask area, the cells were digested with trypsin, and the cell concentration was adjusted to 104 cells / well with DMEM complete medium, and then inoculated into 96-well plates, with 3 parallels in each group. After culturing for 24 hours, different concentrations of nano-drug solutions were added to each group. Incubate for another 24 h, discard the cell culture medium, and slowly wash 2-3 times with PBS buffer. Subsequently, cell culture medium (serum-free) and CCK-8 mother liquor were mixed at a volume ratio of 10:1, and 100 μL of the mixture was added to each well. After incubating in the dark at 37 °C for 20-40 min, the absorbance value of each well (wavelength: 450 nm) was detected using a microplate reader.

[0093] Qualitative detection of cell viability by FDA / PI staining method: After co-incubating the nano-drug with the cells for 24 h, discard the cell culture medium, and slowly wash 2-3 times with PBS buffer. Subsequently, FDA and PI were diluted with PBS (dilution ratio: 1:1000) and added to each well, 100 μL per well. Then, the live-dead state of the cells was observed through a fluorescence microscope.

[0094] According to Figure 3 、 Figure 4 、 Figure 5 and Figure 6 it can be seen that the SF-NPs, RSL-NPs, and RSL@ZIF-8 nanoparticles prepared in the present application have no cytotoxic effect within a certain concentration range and have good biocompatibility at the cellular level.

[0095] 2. Cell hemolysis experiment

[0096] To determine the hemolytic activity of NPs and investigate their blood compatibility. First, the nanoparticles were prepared into a 1.8 mg / mL solution with PBS. Then, the stock solution was gradually diluted by half, and fresh mouse red blood cells (RBC, 5% v / v) were inoculated in an incubator at 37 °C for 1 hour. The RBC suspension served as the negative control, and the suspension treated with 2% Triton X-100 served as the positive control. Three replicate samples were provided for each group. After incubation, centrifuge for 10 min, and measure the OD value of the supernatant at 540 nm.

[0097] The calculation formula for the hemolysis rate is as follows: Hemolysis rate (%) = (Absorbance of sample group - Absorbance of negative control) / (Absorbance of positive control - Absorbance of negative control) × 100%.

[0098] According to Figure 7 it can be seen that the hemolysis rates of the three kinds of nanoparticles, namely B1-nanocomposite, B2-nanocomposite, and A1-nanocomposite, are all below 5% within the application concentration range, meeting the requirements of biocompatibility.

[0099] 3. Cell scratch assay

[0100] 1) Cell culture: Inoculate L929 cells into a 6-well plate, add 2 mL of DMEM medium containing 10% fetal bovine serum to each well, and adjust the cell density to 5×10 5 cells / well. Culture in an incubator at 37 °C and 5% CO 2 until the cell confluence reaches over 90%.

[0101] 2) Scratch treatment: Use a 200 μL sterile pipette tip to vertically scratch a straight line on the surface of the cell monolayer in each well, noting that the scratch width is consistent and the bottom cells are completely removed. Slowly wash the cells 3 times with PBS to remove cell debris.

[0102] 3) Nanoparticle treatment: Prepare a 200 μg / mL nanoparticle suspension, and add 2 mL of serum-free medium containing nanoparticles to each well (add an equal volume of serum-free medium without nanoparticles to the control group).

[0103] 4) Cell culture and observation: Place the 6-well plate in the incubator, use an inverted microscope to observe and photograph the cell migration at 0 h, 24 h, and 48 h, and record the scratch healing area.

[0104] Calculate the healing rate: Healing rate = (Initial scratch area - Residual scratch area) / Initial scratch area × 100%

[0105] According to Figure 8 and Figure 9 it can be seen that the RSL@ZIF-8 treatment group has the highest healing rate and cell migration rate, indicating that this group has the best repair effect.

[0106] 4. Propionibacterium acnes Test

[0107] To verify the effect on Propionibacterium acnes, the morphological changes of Propionibacterium acnes after treatment with different nanomaterials were observed by scanning electron microscopy. The pretreated bacterial cells were centrifuged at 4000 rpm for 10 min, washed twice with PBS, immediately fixed with 2.5% glutaraldehyde, and left overnight at 4 °C. Then, gradient treatment was carried out with 30, 50, 70, 80, 90, 95, and 100% ethanol, and dehydration was carried out for 15 min at each step. Finally, the morphology of the dried sample was observed by scanning electron microscopy.

[0108] According to Figure 10 it can be seen that after the bacterial suspension was incubated with RSL@ZIF-8, obvious morphological changes of damaged Propionibacterium acnes were visible, with irregular edges, wrinkled surfaces, and membrane perforations, indicating that the treatment from RSL@ZIF-8 could disrupt the integrity of the bacterial cell membrane, thereby causing the leakage of cell contents. For RSL-NPs and SF-NPs, their ability to kill bacteria was lower than that of RSL@ZIF-8, and the damage was smaller. The results showed that the main antibacterial mechanism of RSL@ZIF-8 might be related to membrane disruption and cytoplasmic leakage, showing great potential for the treatment of acne vulgaris with acne hyperplasia.

[0109] The agar plate colony counting method was used to visually evaluate the antibacterial activity of RSL@ZIF-8 nanoparticles. Before the experiment, Propionibacterium acnes (ATCC 6919) was cultured in BHI medium under anaerobic conditions at 37 °C for 48 h. Then, 100 μL of the Propionibacterium acnes suspension (2×10 6 CFU / mL) was incubated with PBS and different concentrations of RSL@ZIF-8 at 37 °C for 48 h. The bacterial suspension was diluted 10 3 times and spread on RCA plates, and incubated under anaerobic conditions for 48 h to count the colony-forming units (CFU).

[0110] According to Figure 11 it can be seen that in the concentration range of 0 - 300 μg / mL, as the concentration of RSL@ZIF-8 nanoparticles increased, the effect of inhibiting bacterial growth enhanced. The number of colonies decreased significantly at 300 μg / mL, and almost no colonies were visible, indicating that 300 μg / mL of RSL@ZIF-8 almost completely blocked the growth of bacteria. Combining the results of biocompatibility evaluation, 200 μg / mL of RSL@ZIF-8 was selected for subsequent experiments in this study.

[0111] A live / dead bacterial staining kit was used to qualitatively evaluate the viability of bacteria after different treatments. Briefly, a Propionibacterium acnes suspension (500 μL, 2×10 8CFU / mL) were incubated with different treatments for 24 h. Then the bacteria were collected, centrifuged at 8000 rpm for 5 min, and washed twice with PBS. The bacterial pellet was resuspended in 500 μL of the premixed dye solution and stained for 15 min in the dark at room temperature. After removing the excess dye, the bacterial samples were imaged using a confocal laser scanning microscope. The results are as Figure 12 shown.

[0112] According to Figure 12 it can be seen that the live / dead bacterial staining method was used to further study the integrity of the bacterial membrane in vitro. The membrane-permeable green fluorescent SYTO 9 probe can label both live and dead bacteria, while the red dye PI for nuclear staining can only label dead bacteria. After treatment with PBS alone, almost all the bacteria survived (green). For the SF-NPs treatment group, the bacteria were only labeled with very weak red fluorescence, indicating that the bactericidal effect was negligible. The green-labeled dead bacteria and the red-labeled dead bacteria were co-localized as yellow. In contrast, the percentage of dead bacteria (labeled red) in the RSL-NPs and RSL@ZIF-8 treatment groups was significantly increased, proving that the composite core-shell nanomaterials can significantly damage the integrity of the bacterial membrane.

[0113] Therefore, the nanocomposite provided in this application includes core-shell structured nanoparticles with silk fibroin nanoparticles and resveratrol as the core layer and ZIF-8 as the shell layer. The nanocomposite prepared in this application has excellent structural stability, can improve the bioavailability of active ingredients, promote skin cell regeneration and collagen synthesis while achieving good acne treatment effects, thereby improving the skin condition of acne patients; and can accurately deliver drugs, effectively prevent the generation of drug resistance of Propionibacterium acnes, and the prepared microneedle patch is easy to be clinically translated and has great clinical application value.

[0114] The various embodiments in this specification are described in a progressive manner. The same or similar parts between the various embodiments can be referred to each other, and the key points of each embodiment are the differences from other embodiments.

[0115] The above embodiments are only used to illustrate the technical solutions of this application, rather than to limit this application; although this application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of this application.

Claims

1. A nanocomposite material for treating acne, characterized in that: include: Nanoparticles with a core-shell structure, with silk fibroin nanoparticles and resveratrol as the core layer and ZIF-8 as the shell layer.

2. A method for preparing the nanocomposite material for treating acne according to claim 1, characterized in that: The preparation method comprises: The silk fibroin nanoparticles and resveratrol were dissolved in ethanol for reaction and then freeze-dried to obtain RSL-NPs; The RSL-NPs, zinc nitrate hexahydrate and 2-methylimidazole are reacted in water, separated and collected to obtain the nanocomposite material.

3. The method for preparing a nanocomposite material for treating acne according to claim 2, characterized in that: The mass ratio of the silk fibroin nanoparticles to resveratrol is 1:0.04-1 。 4. The method for preparing a nanocomposite material for treating acne according to claim 2, characterized in that: When the silk fibroin nanoparticles and resveratrol are dissolved in ethanol for reaction, the reaction is carried out under continuous oscillation at room temperature in the dark for 24 hours.

5. The method for preparing a nanocomposite material for treating acne according to claim 2, characterized in that: The molar ratio of the zinc nitrate hexahydrate to 2-methylimidazole is 1:30-40.

6. The method for preparing a nanocomposite material for treating acne according to claim 2, characterized in that: The mass ratio of the RSL-NPs to the mixture of zinc nitrate hexahydrate and 2-methylimidazole is 1:20-40.

7. A microneedle patch for treating acne, characterized in that: The invention comprises the nanocomposite material for treating acne according to claim 1 or the nanocomposite material for treating acne prepared by the preparation method according to any one of claims 2 to 6.

8. The microneedle patch for treating acne according to claim 7, characterized in that: comprising a backing layer and a needle tip layer; The needle tip layer is composed of soluble microneedles arranged in an array on a backing layer, and the soluble microneedles are loaded with a nanocomposite material for treating acne; The base material of the needle tip layer also includes sodium hyaluronate; The needle tip layer is a quadrangular pyramid with a diameter of 300-500 μm, a height of 500-700 μm, and a needle tip spacing of 500-700 μm; The backing layer is one or more of a PVA film, a PE film, a PVC film, a PET film, an EVA film, a PU film, and a PSA film.

9. A method for preparing a microneedle patch for treating acne according to any one of claims 7-8, characterized in that: The preparation method comprises: Adding sodium hyaluronate to the aqueous solution of the nanocomposite material for treating acne, mixing, and injecting into a microneedle array mold to prepare a microneedle array; The backing layer gel is injected into the microneedle array mold to fill it to form a backing, and then the backing is removed from the mold after drying to obtain the microneedle patch.

10. Use of the microneedle patch for treating acne prepared by the preparation method of claim 9 in the preparation of anti-acne drugs.

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