Ultrasound-controllable multifunctional microneedle patch as well as preparation method and application thereof

Abstract

The invention discloses an ultrasonic controllable multifunctional microneedle patch as well as a preparation method and application thereof, and belongs to the technical field of biological materials. The preparation method comprises the following steps: preparing crystalline CoW-LDH nanosheets from Co (NO3) 2.6 H2O, Na2WO4. 2H2O and NaOH, dispersing the crystalline CoW-LDH nanosheets in PBS (Phosphate Buffer Solution), centrifuging and collecting the crystalline CoW-LDH nanosheets, and re-dispersing the crystalline CoW-LDH nanosheets in ultrapure water to obtain amorphous CoW-LDH nanosheets; adding PEG (Polyethylene Glycol), ultrasonically dispersing, stirring at room temperature, centrifuging, collecting and centrifugally washing with ultrapure water to obtain a PEG modified amorphous CoW-LDH nanosheet; and adding microalgae and an amorphous CoW-LDH nanosheet modified by PEG (Polyethylene Glycol) to prepare the ultrasonic controllable multifunctional microneedle patch. The multifunctional microneedle patch disclosed by the invention is high in biological safety, can realize ultrasonic controllability, has a sonodynamic treatment effect and an oxygen supply effect, remarkably inhibits melanoma recurrence and remarkably promotes wound healing, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomaterials, and particularly relates to an ultrasonically controllable multifunctional microneedle patch as well as a preparation method and application thereof. BACKGROUND

[0002] Melanoma is the most deadly and metastatic skin cancer. Surgery has always been the standard treatment method, which can remove visible tumor tissue and adjacent skin. However, due to incomplete resection, residual invasive cancer cells often lead to recurrence or metastasis. In addition, patients with malignant tumors and full-thickness skin defects, extensive open wounds, are at risk of wound infection and long-term inflammation problems, and the recurrence of melanoma and wound healing disorders pose a major challenge to postoperative patients. Therefore, it is urgent to develop a more efficient and safe treatment method to cope with the recurrence of melanoma after surgery and promote wound healing.

[0003] As a non-invasive treatment method, sonodynamic therapy (SDT) refers to the use of ultrasound (US) to irradiate a sonosensitizer to generate reactive oxygen species (ROS) to eliminate residual tumor cells. Two-dimensional layered double hydroxides (2D-LDH) have diverse chemical compositions and structures, and have good biocompatibility, degradability and acid responsiveness, and can be used as an excellent sonosensitizer. Studies have shown that a simple crystalline-to-amorphous transformation of 2D-LDH nanosheets can greatly improve its sonodynamic effect. However, in the microenvironment of tumor tissue, hypoxia is caused by severe damage to microvessels and increased oxygen demand of rapidly proliferating tumor cells, and the hypoxic tumor microenvironment limits the generation of ROS, thereby reducing the efficacy of SDT.

[0004] Hypoxia is one of the hallmarks of the tumor microenvironment, and due to uncontrolled cell proliferation, it exceeds the ability of the original blood vessels to deliver oxygen, thus failing to meet the oxygen demand. More and more studies have shown that hypoxia plays an important role in the progression, metastasis and drug resistance of tumors to chemotherapy, radiotherapy and SDT. In addition, a severe hypoxic environment can hinder wound healing. In order to regenerate skin tissue, it is crucial to create a microenvironment that can release oxygen at the postoperative wound site. Therefore, hypoxia remains a major obstacle in the process of cancer treatment and wound healing. Oxygen supply is particularly important for improving the efficiency of tumor treatment by alleviating hypoxia, and wound healing is promoted by regulating cell proliferation, migration and neovascularization.

[0005] In nature, microalgae have a complex photosynthesis system that can achieve efficient photosynthesis and oxygen production. Chlorella (Chl) is a green microalgae known for its ability to generate oxygen through photosynthesis and has been applied in various wound healing. Chl has been certified by the US Food and Drug Administration as a safe food. Therefore, Chl can relieve tumor immunosuppression by continuously adjusting the tumor hypoxic microenvironment and enhance the efficacy of SDT. It has been reported that gaseous oxygen can only penetrate the skin to 300 microns, while dissolved oxygen can penetrate the skin more than 700 microns. Existing oxygen therapy, such as hyperbaric oxygen and gaseous oxygen therapy, mainly relies on gaseous oxygen, which has poor effect on skin penetration. Therefore, local delivery of dissolved oxygen is considered an effective method to improve the tumor hypoxic microenvironment, and it is important to provide oxygen locally rather than just deliver oxygen. Currently, the common use of microalgae is to coat them in hydrogels to promote their penetration to the wound site, but often cannot reach the deep layers of the wound, resulting in insufficient oxygen supply in the wound. Therefore, developing a multifunctional oxygen-generating carrier that ensures continuous oxygen supply to the deep tissues of the skin is key to promoting wound healing.

[0006] Microneedles (MNs) are micrometer-scale arrays with a height of 150 to 1500 microns, a width of 50 to 250 microns, and a needle tip thickness of 1 to 25 microns. As a transdermal drug delivery system, microneedles create microchannels by penetrating the stratum corneum without damaging blood vessels or nerves, allowing active molecules to passively diffuse. Microneedles have many advantages in transdermal drug delivery, including safety, convenience, minimally invasive, enhanced drug bioavailability, and delivery of effective drugs, and are used in many fields. Therefore, using the delivery capacity of microneedles, it is expected to deliver microorganisms and sonosensitizers to postoperative wound sites, thereby effectively eliminating residual tumors and promoting wound healing. SUMMARY

[0007] To solve the problem of easy recurrence of melanoma and wound healing disorder in the prior art, the main purpose of the present application is to provide an ultrasonically controllable multifunctional microneedle patch (Chl / LDH-MN) loaded with Chl and 2D-LDH, which has high biological safety and can be ultrasonically controllable. The rich sonodynamic therapy effect of 2D-LDH and the oxygen supply effect of Chl exhibit significant ability to inhibit melanoma recurrence and significantly promote wound healing.

[0008] Another object of the present application is to provide a preparation method of the ultrasonically controllable multifunctional microneedle patch, which is simple in process and easy to scale up.

[0009] Still another purpose of the present application is to provide the use of the ultrasonically controllable multifunctional microneedle patch in the preparation of a product for preventing the recurrence of melanoma after surgery and repairing skin lesions, which can effectively prevent the recurrence of melanoma after surgery and promote the regeneration of skin tissue and wound healing after surgery.

[0010] To achieve the above-mentioned purposes, the present application adopts the following technical solutions:

[0011] In a first aspect of the present application, a preparation method of an ultrasonically controllable multifunctional microneedle patch is provided, comprising the following steps:

[0012] Step 1: preparing crystalline CoW-LDH nanosheets, comprising: dissolving Co(NO3)2·6H2O in ultrapure water, magnetically stirring to obtain solution A; dispersing Na2WO4·2H2O in ultrapure water, adding nitric acid, and ultrasonically dispersing until completely dissolved to obtain solution B; dissolving NaOH in ultrapure water to obtain solution C; under room temperature and N2 protection conditions, slowly adding solution A to solution B, continuously stirring, and slowly adding solution C to the above-mentioned mixed solution until the pH value is 9-10, magnetically stirring, and transferring the obtained precipitate to a Teflon-lined autoclave for heating reaction, repeatedly centrifuging and washing the obtained reaction product with ultrapure water to obtain crystalline CoW-LDH nanosheets;

[0013] Step 2: preparing amorphous CoW-LDH nanosheets, comprising: dispersing the crystalline CoW-LDH nanosheets in PBS, centrifuging and collecting, and re-dispersing in ultrapure water to obtain amorphous CoW-LDH nanosheets;

[0014] Step 3: preparing PEG-modified amorphous CoW-LDH nanosheets, comprising: adding PEG to the suspension of the amorphous CoW-LDH nanosheets, ultrasonically dispersing, stirring at room temperature, centrifuging, collecting, and centrifuging and washing with ultrapure water to obtain PEG-modified amorphous CoW-LDH nanosheets;

[0015] Step 4: culturing Chl in F / 2 medium, and the culture conditions include: pH value of 6-8, temperature of 20-23℃, salinity of 38%-34%, shaking 2-3 times per week, and subculturing every 15-20 days;

[0016] Step 5: preparing the ultrasound-controllable multifunctional microneedle patch, comprising: adding LAP powder into a PBS solution to obtain a LAP solution, stirring in a 40-50 DEG C water bath in the dark; adding methacrylated hydrogel (GelMA) into the LAP solution to obtain a GelMA solution, oscillating in a 60-70 DEG C water bath in the dark; filtering the GelMA solution with a 0.22 mu m filter screen, and adding Chl and PEG-modified amorphous CoW-LDH nanosheets, heating a vacuum instrument to 50 DEG C, adding the GelMA solution into each microneedle, opening a vacuum valve, 3 minutes each time, repeating 10 times, sharpening the needle tip with a 405 nm LED lamp, and lightening for 1 minute; adding polyvinyl alcohol (PVA) powder into a PBS solution to obtain a PVA solution, and heating in a 60 DEG C water bath in the dark; after the needle tip of the microneedle mold is sharpened, adding the PVA solution, incubating at 35 DEG C for 12 hours, demolding, and obtaining the ultrasound-controllable multifunctional microneedle patch.

[0017] Preferably, in step 1, the molar ratio of Co(NO3)2·6H2O, Na2WO4·2H2O and NaOH is 1:1:100.

[0018] Preferably, in step 1, the heating reaction is carried out at a temperature of 80 DEG C for 24 hours.

[0019] Preferably, in step 5, the mass ratio of LAP powder, methacrylated hydrogel and PVA powder is 1:2:2.

[0020] Preferably, in step 5, the amount of Chl added is (1-9) x 10 9 cells.

[0021] Preferably, in step 5, the amount of PEG-modified amorphous CoW-LDH nanosheets added is 10 mg / kg of GelMA solution.

[0022] In a second aspect, the present application provides an ultrasound-controllable multifunctional microneedle patch prepared by the method.

[0023] In a third aspect, the present application provides use of the ultrasound-controllable multifunctional microneedle patch in the preparation of a product for preventing postoperative recurrence of melanoma and repairing skin lesions.

[0024] Compared with the prior art, the present application has at least the following beneficial effects:

[0025] 1. The present application develops an ultrasound-controllable multifunctional microneedle patch carrying PEG-modified amorphous CoW-LDH nanosheets, which has excellent ROS production capacity as a sonosensitizer under the action of ultrasound, can effectively induce cell apoptosis and eliminate tumors, and has antibacterial ability to promote wound healing.

[0026] 2、The active chlorella filled in the ultrasonic controllable multifunctional microneedle patch in the application helps to transport dissolved oxygen to the deep layer of the skin, and improves the tumor hypoxic microenvironment; on the one hand, the oxygen production system of chlorella plays a key role in cancer treatment based on SDT, antibacterial and wound healing; on the other hand, by effectively up-regulating the expression of vascular endothelial growth factor, the generation of new blood vessels is accelerated and the healing of postoperative wounds is promoted, which can inhibit tumor recurrence and promote wound healing without scar, and has wide application prospect.

[0027] 3、The ultrasonic controllable multifunctional microneedle patch loaded with microalgae and two-dimensional layered double hydroxide in the application has excellent effects of preventing melanoma recurrence after operation and repairing skin lesions. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 (a) Transmission electron microscopy image and corresponding element distribution map of the amorphous LDH in the example, (b) Pseudo-color scanning electron microscopy image of Chl and (c) Fluorescence microscopy image; (d) Scanning electron microscopy image of microneedle; (e) Fluorescence microscopy image of Chl / LDH-MN microneedle.

[0029] Figure 2 It is H&E staining image of Chl / LDH-MN microneedle after penetrating into mouse skin tissue in the example.

[0030] Figure 3 It is in vitro tumor cell killing and antibacterial effect in the example; wherein: (a) Cell viability of B16F10 cells treated by different groups; (b) Representative image of live / dead staining of B16F10 cells; (c) Bacterial colony plate image of Staphylococcus aureus and (d) Escherichia coli.

[0031] Figure 4 It is cell migration scratch experiment of HUVEC in the example.

[0032] Figure 5 It is the antitumor effect of Chl / LDH-MN microneedle in vivo in the example; wherein: (a) Schematic diagram of primary tumor treatment process in vivo; (b) Average growth curve of tumors treated by different groups (n=5); (c) Tumor growth curve treated by different groups (n=5).

[0033] Figure 6 It is the effect of Chl / LDH-MN microneedle in promoting skin lesion repair in vivo in the example; wherein: (a) Representative picture of skin wound size in different treatment groups within 14 days; (b) Statistical data of wound area in different treatment groups. DETAILED DESCRIPTION

[0034] For a more complete understanding of the technical solutions, objectives and advantages of the present application, the following will be further described in detail in conjunction with the drawings and specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present application, not all. It should be noted that for those skilled in the art, other embodiments obtained without departing from the concept of the present application are all within the scope of protection of the present application.

[0035] In the following examples, Chl was purchased from Shanghai Guangyu Biotechnology Co., Ltd.

[0036] Healthy C57BL / 6 mice (~20g) were purchased from Slaek Company and raised in the Experimental Animal Center of the Clinical Medical Science Park of the Tenth People's Hospital of Tongji University.

[0037] B16F10 tumor-bearing mouse models were constructed by Zhongshan Hospital, Fudan University.

[0038] B16F10 cells and HUVEC cells were purchased from Shanghai Yuanchuang Biotechnology Co., Ltd.

[0039] Example 1

[0040] This example prepares an ultrasonically controllable multifunctional microneedle patch (Chl / LDH-MN), the steps are as follows:

[0041] (1) Preparation of crystalline CoW-LDH nanosheets: First, Co(NO3)2·6H2O (0.5 mmol) was dissolved in 20 mL of ultrapure water, magnetically stirred (500 rpm, 10 min), and named solution A. Na2WO4·2H2O (0.5 mmol) was dispersed in 30 mL of ultrapure water, then nitric acid (0.4 mL, 5.6 mmol) was added, and Na2WO4·2H2O was completely dissolved by ultrasonic dispersion (40 kHz, 10 min), named solution B. NaOH (0.05 mol) was dissolved in 50 mL of ultrapure water and fully dissolved, named solution C. Under room temperature and N2 protection conditions, solution A was slowly added to solution B, and stirring (500 rpm, 10 min) was continued. Solution C was slowly added to the mixed solution of solution A and solution B until the pH value was 9.70, and magnetic stirring (500 rpm, 1.5 h) was carried out. The obtained precipitate was transferred to a Teflon-lined autoclave and reacted at 80°C for 24 h. Finally, repeated centrifugal washing with ultrapure water (6000 rpm, 5 min) for 4 times to obtain crystalline CoW-LDH nanosheets.

[0042] (2) Preparation of amorphous CoW-LDH nanosheets: Crystalline CoW-LDH nanosheets were dispersed in PBS (pH 4.0) for 6 h, and the obtained amorphous CoW-LDH nanosheets were collected by centrifugation (6000 rpm, 5 min) and then re-dispersed in ultrapure water.

[0043] (3) Preparation of PEG-modified amorphous CoW-LDH nanosheets: To improve its biocompatibility, PEG was used for modification. PEG (0.06 mol) was added to the suspension of amorphous CoW-LDH nanosheets, and ultrasonic dispersion (40 kHz, 30 min) was performed, followed by stirring (300 rpm, 12 h) at room temperature. The product was collected by centrifugation (6000 rpm, 5 min) and washed with ultrapure water for 4 times to obtain PEG-modified amorphous CoW-LDH nanosheets.

[0044] (4) Cultivation of Chlorella (Chl): Chl was cultivated in F / 2 medium, and the cultivation conditions were as follows: pH value was maintained at 6-8, temperature was 20-23 °C, and salinity was 38-34%. Chl was shaken 2-3 times per week, and was generally subcultured for 15-20 days.

[0045] (5) Preparation of Chl / LDH-MN microneedle patch: 50 mg of LAP powder was added to 20 mL of PBS solution to prepare a 0.25% LAP solution, which was stirred (300 rpm, 15 min) in a water bath at 40-50 °C in the dark. 100 mg of GelMA was added to 1 mL of the LAP solution to prepare a 10% GelMA solution, which was oscillated (200 rpm, 30 min) in a water bath at 60-70 °C in the dark.

[0046] To prevent low-temperature gelation, the GelMA solution was filtered through a 0.22 μm filter screen, and Chl and PEG-modified amorphous CoW-LDH nanosheets (Chl = 10 9 cells, PEG-modified amorphous CoW-LDH nanosheets = 10 mg / kg) were added. The vacuum instrument was heated to 50 °C, 400 μL of the 10% GelMA solution was added to each microneedle, the vacuum valve was opened for 3 minutes each time, and the process was repeated for 10 times. The needle tips were sharpened using a 405 nm LED lamp, and the light was irradiated for 1 minute. 100 mg of PVA powder was added to 500 μL of PBS solution to prepare a 20% PVA solution, which was heated in a water bath at 60 °C for 2 h in the dark. After the needle tips of the microneedle mold were sharpened, 300 mL of the 20% PVA solution was added, and the mold was kept in a 35 °C incubator for 12 h, followed by demolding to obtain the Chl / LDH-MN microneedle, which was characterized as Figure 1 shown.

[0047] Effect Example 1

[0048] Verification of solubility and skin penetration ability of Chl / LDH-MN microneedles

[0049] To evaluate the skin penetration ability of Chl / LDH-MN microneedles, the microneedles were used to penetrate the skin of anesthetized mice, and the hematoxylin and eosin (H&E) staining images of the skin tissue clearly showed the broken stratum corneum, as shown by the black arrows in Figure 2 The results showed that the Chl / LDH-MN microneedles could effectively penetrate the dermis layer of the mouse skin.

[0050] Example 2

[0051] Verification of tumor cell killing and antibacterial effects of Chl / LDH-MN microneedles

[0052] Firstly, B16F10 cells were treated with different groups (G1 = Control, G2 = US, G3 = Chl / LDH, G4 = LDH + US, G5 = Chl + US, G6 = Chl / LDH + US, 1.0 MHz, 1.0 W / cm 2 , 5 min) and cell viability was evaluated using the Biyun Tian CCK8 kit according to the instructions Figure 3 (a), and the results showed that the cell viability of B16F10 cells treated with Chl / LDH + US was significantly reduced, indicating the effectiveness of this treatment strategy for killing B16F10 tumor cells. In addition, cell viability was determined using the Biyun Tian Calcein / Iodine Propidium (PI) staining kit according to the instructions, and most of the tumor cells incubated with Chl / LDH showed red fluorescence staining after ultrasonic irradiation, indicating excellent tumor cell killing effect Figure 3 (b). Compared with other treatments, the number of S. aureus and E. coli colonies after LDH + US and Chl / LDH + US treatment was sharply reduced, indicating that Chl / LDH has excellent antibacterial ability through sonodynamic effect Figure 3 (c, 3d).

[0053] Example 3

[0054] Verification of the effect of Chl / LDH system in promoting skin lesion repair in vitro

[0055] Effective wound healing depends on the angiogenesis process within the wound tissue. To investigate the pro-angiogenic properties of Chl / LDH, the effect of Chl / LDH system on HUVEC cell migration and tube formation was evaluated by wound healing and in vitro angiogenesis experiments. To evaluate the migration ability of HUVEC cells under various group treatments, a cell scratch experiment was performed to simulate the ability to promote wound healing at the cellular level. First, a vertical scratch was made in the adherent HUVECs cells with a 10 μL pipette tip, and fresh medium containing different components was added and the ultrasound group was irradiated with ultrasound (G1 = Control, G2 = US, G3 = Chl / LDH, G4 = LDH + US, G5 = Chl + US, G6 = Chl / LDH + US, 1.0 MHz, 1.0 W / cm 2 , 5 min) for 24 h, and the cell migration ability was recorded using a fluorescence microscope. Before the assay, the HUVEC cells were stained with calcein for cell visualization under the fluorescence microscope Figure 4 . The cell migration ability was analyzed by Image-j software, and the results showed that Chl / LDH + US treated HUVECs could accelerate the migration of HUVECs compared to the control group.

[0056] Example 4

[0057] Evaluation of the effect of Chl / LDH-MN microneedles on in vivo anti-tumor treatment and promotion of skin lesion repair

[0058] A melanoma xenograft tumor mouse model was constructed, and B16F10 tumor cells (1 x 10 6 ) were injected subcutaneously into female C57BL / 6 mice (~ 20 g). On day 0, most of the tumor tissue was surgically removed, leaving the intact tumor tissue margin, which was then randomly divided into 6 groups and given the following different treatments (n = 5):

[0059] G1 = Control: control (no treatment);

[0060] G2 = MN + US: empty microneedles were applied to the postoperative wound site and ultrasound irradiation was performed;

[0061] G3 = Chl / LDH-MN: microneedles loaded with Chl and LDH were applied to the postoperative wound site and LED light irradiation was performed (LDH = 10 mg / kg, Chl = 10 9 cells);

[0062] G4 = LDH-MN + US: microneedles loaded with LDH were applied to the postoperative wound site (LDH = 10 mg / kg) and ultrasound irradiation was performed;

[0063] G5=Chl-MN+US: Chl-loaded microneedle patch was applied to the postoperative wound site (Chl=10 9 cells) and irradiated with LED lamp and ultrasound;

[0064] G6=Chl / LDH-MN+US: Chl and LDH-loaded microneedle patch was applied to the postoperative wound site (LDH=10 mg / kg, Chl=10 9 cells) and irradiated with LED lamp and ultrasound;

[0065] LED lamp parameters: 13.5 W, 20 min;

[0066] Ultrasound parameters: 1.0 MHz, 1.0 W / cm 2 , 5 min.

[0067] During the period of 0-12 days, the tumor volume of mice was measured every 2 days. The tumor volume was calculated according to the formula (tumor length) x (tumor width)2 / 2 and the tumor growth curve was plotted. The tumor growth curves of different treatment groups are shown in Figure 5 From the tumor average growth curve in Figure 5 and the tumor growth curve of each group, it can be seen that Chl / LDH-MN microneedle has an anti-tumor effect under the action of ultrasound, which significantly delays tumor growth.

[0068] The wound healing process was tracked within two weeks by measuring the change in wound size. First, a full-thickness skin defect wound was constructed on the back of the mice to simulate the skin damage after melanoma surgery, which was then randomly divided into 6 groups and given different treatments (n=3):

[0069] G1=Control: Control (no treatment);

[0070] G2=MN+US: Empty microneedle mold was applied to the postoperative wound site and irradiated with ultrasound;

[0071] G3=Chl / LDH-MN: Chl and LDH-loaded microneedle patch was applied to the postoperative wound site and irradiated with LED lamp (LDH=10 mg / kg, Chl=10 9 cells);

[0072] G4=LDH-MN+US: LDH-loaded microneedle patch was applied to the postoperative wound site (LDH=10 mg / kg) and irradiated with ultrasound;

[0073] G5=Chl-MN+US: Chl-loaded microneedle patch was applied to the postoperative wound site (Chl=10 9 cells) and irradiated with LED lamp and ultrasound;

[0074] G6 = Chl / LDH-MN+US: Microneedles loaded with Chl and LDH are applied to the postoperative wound site (LDH = 10 mg / kg, Chl = 10 mg / kg). 9 (cells), and subjected to LED light irradiation and ultrasonic irradiation;

[0075] LED light parameters: 13.5W, 20 minutes;

[0076] Ultrasound parameters: 1.0MHz, 1.0W / cm 2 5 minutes.

[0077] Record initial skin wound images within 14 days and analyze changes in wound size. Figure 6 a) The results showed that, compared with other groups, the Chl / LDH-MN microneedles stimulated by ultrasound exhibited a significantly higher wound healing rate. Figure 6 b) and showed better wound healing and smaller scars by day 14, indicating that ultrasound-controlled multifunctional microneedles have excellent skin wound healing capabilities.

[0078] The above are merely preferred embodiments of the present invention and are not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing an ultrasound-controlled multifunctional microneedle patch, characterized in that, Includes the following steps: Step 1: Preparation of crystalline CoW-LDH nanosheets, including: dissolving Co(NO3)2·6H2O in ultrapure water and magnetically stirring to obtain solution A; dispersing Na2WO4·2H2O in ultrapure water, adding nitric acid, and ultrasonically dispersing until completely dissolved to obtain solution B; fully dissolving NaOH in ultrapure water to obtain solution C; under room temperature and N2 protection conditions, slowly adding solution A to solution B while continuously stirring, and slowly adding solution C to the above mixed solution until the pH value is 9-10, magnetically stirring, transferring the obtained precipitate to a Teflon-lined autoclave, heating for reaction, and repeatedly centrifuging and washing the obtained reactants with ultrapure water to obtain crystalline CoW-LDH nanosheets; Step 2: Preparation of amorphous CoW-LDH nanosheets, comprising: dispersing the crystalline CoW-LDH nanosheets in PBS, centrifuging and collecting, and redispersing in ultrapure water to obtain amorphous CoW-LDH nanosheets; Step 3: Preparation of PEG-modified amorphous CoW-LDH nanosheets, including: adding PEG to a suspension of the amorphous CoW-LDH nanosheets, ultrasonically dispersing, stirring at room temperature, centrifuging, collecting and centrifuging and washing with ultrapure water to obtain PEG-modified amorphous CoW-LDH nanosheets; Step 4: Chlorella Chl is cultured in F / 2 medium under the following conditions: pH 6-8, temperature 20-23℃, salinity 38%-34%, shaken 2-3 times per week, and passaged every 15-20 days. Step 5: Preparation of ultrasonically controllable multifunctional microneedle patches, including: adding LAP powder to PBS solution to obtain LAP solution, stirring in a water bath at 40-50℃ in the dark; adding methacrylated hydrogel GelMA to LAP solution to obtain GelMA solution, oscillating in a water bath at 60-70℃ in the dark; filtering GelMA solution through a 0.22μm filter, adding PEG-modified amorphous CoW-LDH nanosheets obtained in step 3 and Chl cultured in step 4, heating the vacuum instrument to 50℃, adding GelMA solution to each microneedle, opening the vacuum valve, repeating for 3 minutes each time, 10 times, sharpening the needle tip with a 405nm LED lamp, and irradiating for 1 minute; adding polyvinyl alcohol (PVA) powder to PBS solution to obtain PVA solution, heating in a water bath at 60℃ in the dark; sharpening the needle tip of the microneedle mold, adding PVA solution, keeping at 35℃ for 12 hours, demolding, and obtaining the final product.

2. The method for preparing the ultrasound-controlled multifunctional microneedle patch according to claim 1, characterized in that, In step 1, the molar ratio of Co(NO3)2·6H2O, Na2WO4·2H2O and NaOH is 1:1:

100.

3. The method for preparing the ultrasound-controlled multifunctional microneedle patch according to claim 1, characterized in that, In step 1, the heating reaction temperature is 80°C and the heating reaction time is 24 hours.

4. The method for preparing the ultrasound-controlled multifunctional microneedle patch according to claim 1, characterized in that, In step 5, the mass ratio of LAP powder, GelMA, and PVA powder is 1:2:

2.

5. The method for preparing the ultrasound-controlled multifunctional microneedle patch according to claim 1, characterized in that, In step 5, the amount of Chl added is (1~9)×10 9 cells.

6. The method for preparing the ultrasound-controlled multifunctional microneedle patch according to claim 1, characterized in that, In step 5, the amount of PEG-modified amorphous CoW-LDH nanosheets added is 10 mg / kg GelMA solution.

7. An ultrasound-controlled multifunctional microneedle patch, characterized in that, The ultrasonically controllable multifunctional microneedle patch is obtained by the preparation method of any one of claims 1 to 6.

8. The use of the ultrasound-controlled multifunctional microneedle patch of claim 7 in the preparation of products for preventing postoperative recurrence of melanoma and repairing skin lesions.

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