Separation microneedle of loaded lipidosome nanoreactor as well as preparation method and application of separation microneedle

Abstract

The invention provides a separation microneedle of a loaded lipidosome nanoreactor as well as a preparation method and application of the separation microneedle, and belongs to the technical field of biological medicines. The preparation method comprises the following steps: by taking phospholipid, cholesterol, tetramethoxysilane and 3-aminopropyltriethoxysilane as raw materials, entrapping cis-platinum, 5-aminolevulinic acid and catalase to prepare a liposome nano-reactor; polyvinylpyrrolidone, citric acid, sodium bicarbonate and regenerated silk cellulose are used as raw materials, a liposome nano reactor is dispersed in a microneedle body through a pouring method, and the microneedle capable of being rapidly separated is successfully prepared. Experimental research proves that the separation microneedle has good biocompatibility and mechanical properties, can effectively break through cuticle and realizes efficient transdermal delivery of a liposome nanoreactor. The liposome nanoreactor can be locally planted in deep tumor tissues, continuously supplies oxygen through enzymatic reaction, effectively improves the difficulty of insufficient oxygen supply in tumor photodynamic therapy, and has obvious advantages and wide prospects in the aspects of skin tumor photodynamic therapy application and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a separation microneedle loaded with a liposome nanoreactor, and a preparation method and application thereof. Background Art

[0002] Melanoma is a highly malignant and lethal skin cancer characterized by excessive tissue proliferation and high oxygen demand, leading to a hypoxic microenvironment within the tumor. Surgery, chemotherapy, radiotherapy, and immunotherapy, as existing traditional treatments, have greatly improved the survival of cancer patients. However, surgery is relatively risky, traumatic, and has many complications; chemotherapy is non-specific, poorly targeted, and has drug resistance; radiotherapy has low patient tolerance and compliance; immunotherapy is complex and requires long-term survival of reprogrammed cells, which hinders its further application. Photodynamic therapy has the characteristics of minimal trauma, spatiotemporal control, and convenient local administration, and has been clinically used in the treatment of melanoma. Although the emerging photodynamic therapy and its combination therapy have inhibited the malignant proliferation of tumors to a certain extent, the problem of insufficient oxygen supply to photodynamic therapy caused by the hypoxic microenvironment of the tumor remains a key constraint limiting its clinical efficacy.

[0003] To date, researchers have sought to alleviate the oxygen shortage associated with photodynamic therapy (PDT). These strategies include: 1) direct delivery of exogenous oxygen to the tumor; 2) in situ oxygen generation within the tumor; 3) reducing oxygen consumption by inhibiting tumor cell respiration; and 4) alleviating the hypoxic tumor microenvironment through inhibition of the hypoxia-inducible factor-1 (HIF) signaling pathway. However, these strategies can only partially mitigate the low efficiency of PDT caused by the hypoxic tumor microenvironment and fail to fundamentally provide a continuous supply of oxygen for PDT. Furthermore, due to the complexity and variability of the tumor microenvironment, single therapies are often insufficient to inhibit the rapid malignant growth of tumors. Modern PDT research focuses on nanoparticle delivery strategies for synergistic tumor treatment, primarily in combination with chemotherapy and immunotherapy. While these nanoparticles for PDT have achieved significant improvements in improving the tumor hypoxic microenvironment and enhancing efficacy, they still face the challenge of extremely low drug accumulation in tumor tissue.

[0004] For superficial malignant tumors of the skin, microneedles offer significant advantages in improving drug accumulation in superficial tumor lesions. They offer customizable length and shape, efficient transdermal delivery (directly penetrating the stratum corneum to deliver drugs to the dermis), and are non-invasive and painless. Therefore, developing a rapidly separating microneedle that can continuously provide oxygen for tumor photodynamic therapy, enabling multi-therapy combinations and increasing drug accumulation in tumor tissues, could potentially address the current issues with photodynamic therapy, which often result in poor tumor treatment efficacy due to hypoxia in the tumor microenvironment and the use of a single treatment modality. Summary of the Invention

[0005] To address the problems of low photodynamic therapy efficiency due to tumor hypoxia, suboptimal monotherapy, and suboptimal therapeutic effects caused by insufficient drug accumulation in tumor tissue, the present invention provides a separation microneedle loaded with a liposome nanoreactor, as well as its preparation method and application. The separation microneedle provided by the present invention not only provides continuous oxygen supply for tumor photodynamic therapy but also, through microneedle-assisted delivery, increases the accumulation of anti-tumor nanoparticles in deep tumor tissues. Furthermore, the rapid separation microneedle can fully combine photodynamic therapy, chemotherapy, and immunotherapy to exert a synergistic anti-tumor effect.

[0006] To achieve the above objectives, the present invention adopts the following technical solutions: The present invention provides a separation microneedle loaded with a liposome nanoreactor. The separation microneedle comprises 550 to 3000 parts of a microneedle body and 1 to 5 parts of a liposome nanoreactor loaded in the microneedle body, calculated in parts by weight; the microneedle body contains polyvinyl pyrrolidone, citric acid, sodium bicarbonate and regenerated silk cellulose; the liposome nanoreactor contains phospholipids, cholesterol, tetramethoxysilane, 3-aminopropyltriethoxysilane and a loaded active ingredient.

[0007] Furthermore, the microneedle body includes a functional backing layer and a rapidly detachable microneedle layer. In parts by weight, the functional backing layer contains 400 to 2000 parts of polyvinyl pyrrolidone, 55 to 270 parts of citric acid, and 70 to 350 parts of sodium bicarbonate; the rapidly detachable microneedle layer contains 50 to 300 parts of regenerated silk cellulose; the liposome nanoreactor is loaded in the rapidly detachable microneedle layer, and the content of the liposome nanoreactor is 1 to 5 parts.

[0008] Furthermore, the liposome nanoreactor includes liposomes and silica nanocapsules; by weight, the liposomes contain 0.3-1.5 parts of glycosylated phosphatidylcholine, 0.3-1.5 parts of phosphatidylcholine and 0.05-0.5 parts of cholesterol; the silica nanocapsules contain 0.1-0.5 parts of tetramethoxysilane and 0.03-0.3 parts of 3-aminopropyltriethoxysilane.

[0009] Furthermore, the loaded active ingredients are cisplatin, 5-aminolevulinic acid and catalase.

[0010] Furthermore, the amount of catalase used is 0.02-0.2 parts (the enzymatic activity of catalase is 500 U / mg-5000 U / mg), the amount of cisplatin used is 0.05-0.5 parts, and the amount of 5-aminolevulinic acid used is 0.1-1 parts, calculated in parts by weight.

[0011] Furthermore, the molecular weight of the polyvinyl pyrrolidone is 10-100 kDa, and the molecular weight of the regenerated silk cellulose is 100-150 kDa.

[0012] The present invention also provides a method for preparing the liposome nanoreactor for rapid separation of microneedles, comprising the following steps: S1: preparing catalase-loaded silica nanocapsules by using the reverse microemulsion in situ encapsulation method with the aforementioned amounts of tetramethoxysilane, 3-aminopropyltriethoxysilane, and catalase; S2: preparing a liposome nanoreactor by a thin film dispersion method using the amount of glycosylated phosphatidylcholine, phosphatidylcholine, cholesterol, the silica nanocapsules loaded with catalase, cisplatin and 5-aminolevulinic acid; S3: stirring and mixing the regenerated silk cellulose and the liposome nanoreactor in the required amount, and degassing to obtain a needle matrix solution; stirring and mixing the polyvinyl pyrrolidone, sodium bicarbonate, and citric acid in the required amount, and degassing to obtain a functional backing layer matrix solution; S4: pouring the needle body matrix solution into a mold, vacuum compressing and drying; adding the functional backing layer matrix solution, vacuum compressing and drying, and peeling and demoulding to obtain the separated microneedles.

[0013] Furthermore, in step S1, the ultrasonic power of the reverse microemulsion in situ encapsulation technology is 300 W to 600 W, the ultrasonic time is 3 minutes to 10 minutes, the stirring speed is 500 to 1000 rpm, and the volatilization time is 12 hours to 24 hours.

[0014] Furthermore, in step S2, the temperature of the rotary evaporator for the thin film dispersion method is 40° C. to 80° C., and the hydration dispersion time is 10 minutes to 30 minutes.

[0015] Furthermore, in step S3, the stirring speed is 500-1000 rpm, and the stirring time is 1 hour-3 hours.

[0016] Furthermore, in step S4, the drying temperature is 25° C. to 60° C., and the drying time is 8 hours to 24 hours.

[0017] The present invention also provides the use of the separation microneedle in preparing medicine for treating superficial skin tumors.

[0018] Furthermore, the superficial skin tumor is melanoma or breast cancer.

[0019] Furthermore, the rapid separation microneedle can be used alone or in combination with photothermal therapy, chemotherapy, or chemodynamic therapy.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. The separation microneedles prepared by the present invention not only have the characteristics of rapid needle dissociation and promotion of deep drug penetration in tumors, but can also continuously supply oxygen for tumor photodynamic therapy to improve the anti-tumor efficacy.

[0021] 2. The separation microneedle has good mechanical skin-breaking properties and biocompatibility, and shows obvious tumor growth inhibition effect on melanoma mice; the liposome nanoreactor can use the high concentration of hydrogen peroxide in the tumor microenvironment as a substrate to carry out enzymatic reaction to continuously produce oxygen, providing a continuous oxygen supply for photodynamic therapy while improving the immunosuppression caused by the tumor hypoxic microenvironment, thereby improving the efficacy of photodynamic combined with immunotherapy.

[0022] 3. The present invention effectively combines photodynamic therapy, chemotherapy, and immunotherapy by using a combination of cisplatin and 5-ALA. Experiments conducted in this invention have demonstrated that the rapid separation microneedles provided by this invention have significant therapeutic advantages in treating superficial skin tumors, and therefore have promising and broad market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 1 and 2 are side views, scanning electron microscope images, and three-dimensional scanning images under a laser confocal microscope of the separation microneedles in Examples 1 and 2 of the present invention; wherein, a is a side view and a partially enlarged view of the separation microneedle in Example 1; b is a scanning electron microscope image of the separation microneedle in Example 1; c is a locally enlarged scanning electron microscope image of a single needle of the separation microneedle in Example 1; d is a three-dimensional scanning image of the separation microneedle in Example 2 under a laser confocal microscope.

[0024] Figure 2 This is a scanning electron microscope side view and element distribution diagram of a single needle of the liposome nanoreactor rapid separation microneedle in Example 8 of the present invention.

[0025] Figure 3 This is a diagram showing the mechanical properties of the rapid separation microneedle in Example 1 of the present invention.

[0026] Figure 4 This is a graph showing the dissolution of the Rhodamine B-rapid separation microneedles in Example 2 of the present invention in a PBS solution over time.

[0027] Figure 5 This is a graph showing the oxygen production over time of the liposome nanoreactor rapid separation microneedle in Example 3 of the present invention.

[0028] Figure 6These are the pharmacodynamic evaluation results of the liposome nanoreactor rapid separation microneedle in the in vivo treatment of melanoma in Examples 3, 4, 5, 6, 7, and 8 of the present invention; wherein a is the tumor volume record at different time points after treatment for each group of preparations; b is the statistical graph of tumor weight on the 14th day; and c is a comparison of tumor photos of each group of preparations after the end of treatment on the 14th day.

[0029] Figure 7 These are staining images of pathological sections and apoptotic sections of melanoma treated in vivo using the liposome nanoreactor for rapid separation of microneedles according to Examples 3, 4, 5, 6, 7 and 8 of the present invention. DETAILED DESCRIPTION

[0030] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the technical solutions of the present invention are further described below with reference to the accompanying drawings and specific embodiments. In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods; the materials and reagents used can be purchased from biological or chemical reagent companies.

[0031] The preparation steps of the liposome nanoreactor in the present invention are: The following weight parts were weighed: 0.3-1.5 parts of glycosylated phosphatidylcholine, 0.3-1.5 parts of phosphatidylcholine, and 0.05-0.5 parts of cholesterol were used for the liposomes; 0.1-0.5 parts of tetramethoxysilane and 0.03-0.3 parts of 3-aminopropyltriethoxysilane were used for the silica nanocapsules. Silica nanocapsules were prepared by mixing 0.1-0.5 parts of tetramethoxysilane, 0.03-0.3 parts of 3-aminopropyltriethoxysilane, and 0.02-0.2 parts of catalase.

[0032] Glycosylated phosphatidylcholine, phosphatidylcholine, cholesterol, the silica nanocapsule, cisplatin and 5-aminolevulinic acid are mixed as raw materials to prepare a solution, the solvent is rotary evaporated at a temperature of 40°C to 80°C, 2 to 5 mL of ultrapure water is added and hydrated and dispersed for 10 to 30 minutes, and a liposome nanoreactor is prepared by a thin film dispersion method. Example 1

[0033] This embodiment provides a method for preparing a blank layered rapid separation microneedle, the specific steps of which are: 1. Weigh 150 mg of polyvinylpyrrolidone K30, 20 mg of citric acid, and 25 mg of sodium bicarbonate, mix with 0.5 mL of anhydrous ethanol, and stir at room temperature until completely dissolved to obtain a functional backing layer solution.

[0034] 2. Weigh 18 mg of regenerated silk cellulose and mix it with 0.3 mL of ultrapure water. Stir until completely dissolved to obtain a 6% (w / v) regenerated silk cellulose solution.

[0035] 3. Add 0.1 mL of a 6% (w / v) regenerated silk cellulose solution to the microneedle mold and vacuum evacuate for 30 minutes, repeat this cycle three times, and dry at 37°C for 8 hours to evaporate the solvent, anhydrous ethanol. Add 0.5 mL of the functional backing layer solution to the grooves of the previously dried microneedle mold. Vacuum evacuate for 30 minutes, repeat this cycle three times, and dry at 37°C for 8 hours to evaporate the solvent, ultrapure water. Demold to obtain blank, layered, rapid-detachment microneedles. Example 2

[0036] This embodiment provides a method for preparing Rhodamine B-rapid separation microneedles, the specific steps of which are: 1. Weigh 150 mg of polyvinylpyrrolidone K30, 20 mg of citric acid, and 25 mg of sodium bicarbonate, mix with 0.5 mL of anhydrous ethanol, and stir at room temperature until completely dissolved to obtain a functional backing layer solution.

[0037] 2. Weigh 18 mg of regenerated silk cellulose and mix it with 0.3 mL of ultrapure water. Stir until completely dissolved to obtain a 6% (w / v) regenerated silk cellulose solution.

[0038] 3. Weigh 0.6 mg of rhodamine B and dissolve it in 0.1 mL of 6% (w / v) regenerated silk cellulose solution. Add the regenerated silk cellulose to the microneedle mold and vacuum evacuate for 30 minutes, repeat this cycle three times, and dry at 37°C for 8 hours to evaporate the solvent, anhydrous ethanol. Add 0.5 mL of the functional backing layer solution to the grooves of the previously dried microneedle mold. Vacuum evacuate for 30 minutes, repeat this cycle three times, and dry at 37°C for 8 hours to evaporate the solvent, ultrapure water. Demold to obtain the rhodamine B-rapid separation microneedles. Example 3

[0039] This embodiment provides a method for preparing a liposome nanoreactor loaded with catalase for rapid separation of microneedles, the specific steps of which are as follows: 1. Weigh 150 mg of polyvinylpyrrolidone K30, 20 mg of citric acid, and 25 mg of sodium bicarbonate, mix with 0.5 mL of anhydrous ethanol, and stir at room temperature until completely dissolved to obtain a functional backing layer solution.

[0040] 2. Weigh 36 mg of regenerated silk cellulose and mix it with 0.3 mL of ultrapure water. Stir until completely dissolved to obtain a 12% (w / v) regenerated silk cellulose solution.

[0041] 3. After mixing 0.04 mg of tetramethoxysilane, 0.012 mg of 3-aminopropyltriethoxysilane and 0.0006 mg of catalase, silica nanocapsules loaded with catalase were prepared using the reverse microemulsion in situ encapsulation method.

[0042] 4. 0.1 mg of glycosylated phosphatidylcholine, 0.1 mg of phosphatidylcholine, 0.03 mg of cholesterol and 0.05 mg of silica nanocapsules loaded with catalase were prepared into liposome nanoreactors using a thin film dispersion method. The liposome nanoreactors were then mixed with 12% (w / v) regenerated silk cellulose solution in a volume ratio of 1:1 to prepare a 6% (w / v) regenerated silk cellulose solution loaded with catalase-lipid nanoreactor.

[0043] 5. Disperse the catalase-loaded lipid nanoreactor in 0.6 mL of 6% (w / v) regenerated silk cellulose solution as the first casting solution. Subsequently, use a pipette to drop 0.1 mL of the first casting solution directly onto the mold surface. Vacuum evacuate for 30 minutes, repeat three times to compress the regenerated silk cellulose into the microneedle cavity, and then dry in a 37°C oven for 8 hours to evaporate the solvent water.

[0044] 6. For the functional backing layer, add 0.5 mL of an ethanol-based premixed solution containing PVP K30 (30 wt%), citric acid (4 wt%), and sodium bicarbonate (5 wt%) as a second casting solution to the microneedle mold dried in the previous step to form the effervescent backing layer. Continue drying in a 37°C oven for another 8 h to evaporate the anhydrous ethanol solvent. Demold to obtain the catalase-lipid nanoreactor-loaded rapid-detachment microneedles. Example 4

[0045] This embodiment provides a method for preparing a rapid separation microneedle of a liposome nanoreactor loaded with 5-aminolevulinic acid, the specific steps being: 1. Weigh 150 mg of polyvinylpyrrolidone K30, 20 mg of citric acid, and 25 mg of sodium bicarbonate, mix with 0.5 mL of anhydrous ethanol, and stir at room temperature until completely dissolved to obtain a functional backing layer solution.

[0046] 2. Weigh 36 mg of regenerated silk cellulose and mix it with 0.3 mL of ultrapure water. Stir until completely dissolved to obtain a 12% (w / v) regenerated silk cellulose solution.

[0047] 3. 0.1 mg of glycosylated phosphatidylcholine, 0.1 mg of phosphatidylcholine, 0.03 mg of cholesterol and 0.06 mg of 5-aminolevulinic acid were prepared into liposomes by thin film dispersion method, and then mixed with 12% (w / v) regenerated silk cellulose solution in a volume ratio of 1:1 to prepare 6% (w / v) regenerated silk cellulose solution loaded with 5-aminolevulinic acid liposomes.

[0048] 4. Disperse the 5-aminolevulinic acid-loaded liposomes in 0.6 mL of 6% (w / v) regenerated silk cellulose solution as the first casting solution. Subsequently, use a pipette to drop 0.1 mL of the first casting solution directly onto the mold surface. Vacuum exhaust for 30 minutes, and repeat three times to compress the regenerated silk cellulose into the microneedle cavity. Then, dry in an oven at 37°C for 8 hours to evaporate the solvent water.

[0049] 5. For the backing layer, add 0.5 mL of an ethanol-based premix containing PVP K30 (30 wt%), citric acid (4 wt%), and sodium bicarbonate (5 wt%) as the second casting solution to the microneedle mold dried in the previous step to form the effervescent backing layer. Continue drying in a 37°C oven for another 8 h to evaporate the anhydrous ethanol solvent. Demold to obtain the 5-aminolevulinic acid liposome-loaded rapid-detachment microneedles. Example 5

[0050] This embodiment provides a method for preparing a cisplatin-loaded liposome nanoreactor for rapid separation of microneedles, the specific steps of which are as follows: 1. Weigh 150 mg of polyvinylpyrrolidone K30, 20 mg of citric acid, and 25 mg of sodium bicarbonate, mix with 0.5 mL of anhydrous ethanol, and stir at room temperature until completely dissolved to obtain a functional backing layer solution.

[0051] 2. Weigh 36 mg of regenerated silk cellulose and mix it with 0.3 mL of ultrapure water. Stir until completely dissolved to obtain a 12% (w / v) regenerated silk cellulose solution.

[0052] 3. 0.1 mg of glycosylated phosphatidylcholine, 0.1 mg of phosphatidylcholine, 0.03 mg of cholesterol and 0.02 mg of cisplatin were prepared into liposomes by thin film dispersion method, and then mixed with 12% (w / v) regenerated silk cellulose solution in a volume ratio of 1:1 to prepare 6% (w / v) regenerated silk cellulose solution loaded with cisplatin liposomes.

[0053] 4. Disperse the cisplatin-loaded liposomes in 0.6 mL of 6% (w / v) regenerated silk cellulose solution as the first casting solution. Subsequently, use a pipette to drop 0.1 mL of the first casting solution directly onto the mold surface. Vacuum exhaust for 30 minutes, and repeat three times to compress the regenerated silk cellulose into the microneedle cavity. Then, dry in an oven at 37°C for 8 hours to evaporate the solvent water.

[0054] 5. For the backing layer, add 500 µ A premixed ethanol solution containing PVP K30 (30 wt%), citric acid (4 wt%), and sodium bicarbonate (5 wt%) was added to the microneedle mold dried in the previous step as the second casting solution to form the effervescent backing layer. The mold was then dried in a 37°C oven for another 8 h to evaporate the anhydrous ethanol solvent. The mold was then removed to obtain the cisplatin-loaded liposome-loaded rapid-detachment microneedles. Example 6

[0055] This example provides a method for preparing catalase-loaded silica nanocapsules / / cisplatin liposome nanoreactor rapid separation microneedles, the specific steps of which are as follows: 1. Weigh 150 mg of polyvinylpyrrolidone K30, 20 mg of citric acid, and 25 mg of sodium bicarbonate, mix with 0.5 mL of anhydrous ethanol, and stir at room temperature until completely dissolved to obtain a functional backing layer solution.

[0056] 2. Weigh 36 mg of regenerated silk cellulose and mix it with 0.3 mL of ultrapure water. Stir until completely dissolved to obtain a 12% (w / v) regenerated silk cellulose solution.

[0057] 3. After mixing 0.04 mg of tetramethoxysilane, 0.012 mg of 3-aminopropyltriethoxysilane and 0.0006 mg of catalase, silica nanocapsules loaded with catalase were prepared using the reverse microemulsion in situ encapsulation method.

[0058] 4. 0.1 mg of glycosylated phosphatidylcholine, 0.1 mg of phosphatidylcholine, 0.03 mg of cholesterol, 0.05 mg of silica nanocapsules loaded with catalase, and 0.02 mg of cisplatin were prepared into a liposome nanoreactor using a thin film dispersion method. The mixture was then evenly mixed with a 12% (w / v) regenerated silk cellulose solution in a 1:1 volume ratio to prepare a 6% (w / v) regenerated silk cellulose solution of catalase-loaded silica nanocapsules / cisplatin liposome nanoreactor.

[0059] 5. Disperse the catalase / cisplatin-loaded liposome nanoreactor in 0.6 mL of 6% (w / v) regenerated silk cellulose solution as the first casting solution. Subsequently, use a pipette to drop 0.1 mL of the first casting solution directly onto the mold surface. Vacuum exhaust for 30 minutes, and repeat three times to compress the regenerated silk cellulose into the microneedle cavity. Then, dry it in an oven at 37°C for 8 hours to evaporate the solvent water.

[0060] 6. For the backing layer, add 0.5 mL of an ethanol-based premixed solution containing PVP K30 (30 wt%), citric acid (4 wt%), and sodium bicarbonate (5 wt%) as the second casting solution to the microneedle mold dried in the previous step to form the effervescent backing layer. Continue drying in a 37°C oven for another 8 h to evaporate the anhydrous ethanol solvent. Demold to obtain the catalase / cisplatin-loaded liposome nanoreactor rapid separation microneedles. Example 7

[0061] This example provides a method for preparing a catalase-loaded silica nanocapsule / 5-aminolevulinic acid liposome nanoreactor for rapid separation of microneedles, the specific steps of which are as follows: 1. Weigh 150 mg of polyvinylpyrrolidone K30, 20 mg of citric acid, and 25 mg of sodium bicarbonate, mix with 0.5 mL of anhydrous ethanol, and stir at room temperature until completely dissolved to obtain a functional backing layer solution.

[0062] 2. Weigh 36 mg of regenerated silk cellulose and mix it with 0.3 mL of ultrapure water. Stir until completely dissolved to obtain a 12% (w / v) regenerated silk cellulose solution.

[0063] 3. After mixing 0.04 mg of tetramethoxysilane, 0.012 mg of 3-aminopropyltriethoxysilane and 0.0006 mg of catalase, silica nanocapsules loaded with catalase were prepared using the reverse microemulsion in situ encapsulation method.

[0064] 4. 0.1 mg of glycosylated phosphatidylcholine, 0.1 mg of phosphatidylcholine, 0.03 mg of cholesterol, 0.05 mg of silica nanocapsules loaded with catalase and 0.06 mg of 5-aminolevulinic acid were prepared into liposome nanoreactors by thin film dispersion method, and then mixed with 12% (w / v) regenerated silk cellulose solution in a volume ratio of 1:1 to prepare 6% (w / v) regenerated silk cellulose solution of silica nanocapsules loaded with catalase / 5-aminolevulinic acid liposome nanoreactors.

[0065] 5. Disperse the catalase / 5-aminolevulinic acid liposome nanoreactor in 0.6 mL of 6% (w / v) regenerated silk cellulose solution as the first casting solution. Subsequently, use a pipette to drop 0.1 mL of the first casting solution directly onto the mold surface. Vacuum exhaust for 30 minutes, and repeat three times to compress the regenerated silk cellulose into the microneedle cavity. Then, dry it in an oven at 37°C for 8 hours to evaporate the solvent water.

[0066] 6. For the backing layer, add 0.5 mL of an ethanol-based premixed solution containing PVP K30 (30 wt%), citric acid (4 wt%), and sodium bicarbonate (5 wt%) as the second casting solution to the microneedle mold dried in the previous step to form the effervescent backing layer. Continue drying in a 37°C oven for another 8 h to evaporate the anhydrous ethanol solvent. Demold to obtain the catalase / 5-aminolevulinic acid liposome nanoreactor-loaded rapid-detachment microneedles. Example 8

[0067] This embodiment provides a method for preparing a liposome nanoreactor for rapid separation of microneedles loaded with catalase / 5-aminolevulinic acid / cisplatin, the specific steps of which are as follows: 1. Weigh 150 mg of polyvinylpyrrolidone K30, 20 mg of citric acid, and 25 mg of sodium bicarbonate, mix with 0.5 mL of anhydrous ethanol, and stir at room temperature until completely dissolved to obtain a functional backing layer solution.

[0068] 2. Weigh 36 mg of regenerated silk cellulose and mix it with 0.3 mL of ultrapure water. Stir until completely dissolved to obtain a 12% (w / v) regenerated silk cellulose solution.

[0069] 3. After mixing 0.04 mg of tetramethoxysilane, 0.012 mg of 3-aminopropyltriethoxysilane and 0.0006 mg of catalase, silica nanocapsules loaded with catalase were prepared using the reverse microemulsion in situ encapsulation method.

[0070] 4. 0.1 mg of glycosylated phosphatidylcholine, 0.1 mg of phosphatidylcholine, 0.03 mg of cholesterol, 0.05 mg of silica nanocapsules loaded with catalase, 0.06 mg of 5-aminolevulinic acid and 0.02 mg of cisplatin were prepared into liposome nanoreactors by thin film dispersion method, and then mixed with 12% (w / v) regenerated silk cellulose solution in a volume ratio of 1:1 to prepare 6% (w / v) regenerated silk cellulose solution loaded with catalase / 5-aminolevulinic acid / cisplatin liposome nanoreactors.

[0071] 5. Disperse the catalase / 5-aminolevulinic acid / cisplatin-loaded liposome nanoreactor in 0.6 mL of 6% (w / v) regenerated silk cellulose solution as the first casting solution. Subsequently, use a pipette to drop 0.1 mL of the first casting solution directly onto the mold surface. Vacuum exhaust for 30 minutes, and repeat three times to compress the regenerated silk cellulose into the microneedle cavity. Then, dry it in an oven at 37°C for 8 hours to evaporate the solvent water.

[0072] 6. For the backing layer, add 0.5 mL of an ethanol-based premixed solution containing PVP K30 (30 wt%), citric acid (4 wt%), and sodium bicarbonate (5 wt%) as the second casting solution to the microneedle mold dried in the previous step to form the effervescent backing layer. Continue drying in a 37°C oven for another 8 h to evaporate the anhydrous ethanol solvent. Demold to obtain the catalase / 5-aminolevulinic acid / cisplatin-loaded liposome nanoreactor rapid separation microneedles. Example 9

[0073] 1. Morphological characterization of rapid separation microneedles The blank rapid separation microneedle prepared in Example 1 was placed on the laboratory bench, and a bright field side view of the rapid separation microneedle was taken using a camera in macro mode; the Rhodamine B-rapid separation microneedle prepared in Example 2 was placed in a confocal dish and observed using a laser confocal microscope. Figure 1 As shown in a and d: It can be observed that the rapid separation microneedles are sharp conical prisms, composed of an array, and the needle height is 750 μ m, base width is 130 μ m.

[0074] 2. Rapid separation of microneedle micromorphology and element distribution The liposome nanoreactor prepared in Example 8 was rapidly separated from the microneedle by gold spraying for 90 s, and its surface morphology was observed by scanning electron microscopy at an accelerating voltage of 20.0 kV. Figure 1 As shown in b and c: It can be observed that the rapid separation of microneedles is 750 μ m; Energy spectrum analysis spectrum such as Figure 2 As shown, the presence of P, Si and Pt was confirmed, indicating that the liposome nanoreactor was uniformly dispersed in the liposome nanoreactor rapid separation microneedle prepared in Example 8.

[0075] 3. Mechanical performance test of bubble pump rapid separation microneedle The mechanical properties of the microneedles determine their ability to penetrate the skin. The rapid separation microneedles of Example 8 were placed on the lower plate of the texture analyzer. The compression rate was 1 mm / min and the deformation was 80%. The average pressure change per needle during the compression process was recorded as follows: Figure 3 As shown. When the compression displacement is 400 μ At 1.5 m, the mechanical strength of the rapid-detachment microneedles was 1.2 N / needle. These values ​​far exceed the minimum force of 0.058 N required to penetrate human skin, demonstrating that the rapid-detachment microneedles can effectively penetrate the skin.

[0076] 4. Dissolution performance test of rapid separation microneedles The bubble pump was used to quickly separate the microneedles and place them in PBS. A dissolved oxygen meter was used to detect the oxygen production. Figure 4 As shown, once the Rhodamine B-rapid detaching microneedles of Example 2 came into contact with the PBS solution, they began to dissolve rapidly within 1 second, with bubbles clearly visible. As the time extended to 16 seconds, the rapid detaching microneedles of Example 2 were almost completely disintegrated.

[0077] 5. Rapid separation microneedle oxygen production capacity test The liposome rapid separation microneedles of Example 2 were placed in PBS containing 1 mM H2O2, and the dissolution of the microneedles at different time points was recorded time-lapse under a fluorescence microscope. Figure 5 As shown, the liposome nanoreactor microneedles of Example 8 rapidly separated and gradually converted the H2O2 in the solution into oxygen as time went on, indicating that the liposome nanoreactor microneedles of Example 8 continuously produced oxygen using H2O2 as a substrate.

[0078] Example 10: Evaluation of the effect of in vivo treatment of melanoma C57BL / 6J mice (male, 5-6 weeks old) were anesthetized and depilated, and 5×10 5 melanoma cells. When the tumor volume reaches 50 mm 3 At about 40 days, the mice were randomly divided into 7 groups. Microneedle patch treatment was performed on days 0, 2, 4, 6, 8, 10, and 12 after grouping, and 635 nm laser irradiation (200 mW / cm 2 , 10 minutes). Tumor volume was recorded with a vernier caliper during this time. On day 14, mice were sacrificed and tumor tissue was collected in 4% paraformaldehyde. Tumor tissue was embedded in a mixture of polyethylene glycol and polyvinyl alcohol, sectioned, and stained with 4',6-diamidino-2-phenylindole (DAPI) and an apoptosis indicator (TUNEL). Tumor tissue was also embedded in paraffin and sectioned with hematoxylin and eosin (H&E).

[0079] Figure 6 The results of the rapid separation of microneedles from liposome nanoreactors for the treatment of melanoma in vivo are shown in Examples 3, 4, 5, 6, 7, and 8. Figure 6As shown in Figure a, the tumor growth of the group treated with Example 3 was significantly inhibited compared with the other groups. The tumor inhibition effect of the group treated with Example 3 was better than that of the reference groups in other examples. The mechanism may be related to the combined effects of photodynamic therapy, chemotherapy and immunotherapy. Similarly, the tumor weight of mice in each group is shown in the figure and photos. Figure 6 In Figures b and c, the tumor in the group treated with Example 8 was the smallest. These results indicate that the group treated with medication plus light according to Example 8 had a stronger therapeutic effect.

[0080] The results of pathological sections and apoptosis sections are as follows Figure 7 As shown in Figure 1, compared with the untreated group, the tumor cell damage and apoptosis in the group treated with Example 8 were the most obvious. These data provide pathological evidence for the multiple therapeutic effects of the group treated with Example 8.

[0081] The present invention uses phospholipids, cholesterol, tetramethoxysilane and 3-aminopropyltriethoxysilane as raw materials, and simultaneously encapsulates cisplatin, 5-aminolevulinic acid and catalase to prepare a liposome nanoreactor; using polyvinyl pyrrolidone and regenerated silk cellulose as raw materials, and simultaneously loading the liposome nanoreactor, a fast-separation microneedle that can be quickly separated is successfully prepared by a template method. The fast-separation microneedle has a sharp cone shape, has good biocompatibility and mechanical properties, can effectively break through the stratum corneum, and realizes efficient transdermal delivery and release of the liposome nanoreactor. The liposome nanoreactor fast-separation microneedle can continuously supply oxygen, effectively improving the dilemma of insufficient oxygen supply in tumor photodynamic therapy. It has obvious advantages and broad application prospects in the application of photodynamic therapy for skin tumors.

[0082] The above embodiments are intended to illustrate rather than limit the technical solutions described in the present invention. Although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the present invention, and all technical solutions and improvements thereof that do not depart from the spirit and scope of the present invention should be included within the scope of the claims of the present invention.

Claims

1. A separation microneedle loaded with a liposome nanoreactor, characterized in that: The separation microneedle includes 550 to 3000 parts of a microneedle body and 1 to 5 parts of a liposome nanoreactor loaded in the microneedle body, calculated in parts by weight; the microneedle body contains polyvinyl pyrrolidone, citric acid, sodium bicarbonate and regenerated silk cellulose; the liposome nanoreactor contains phospholipids, cholesterol, tetramethoxysilane, 3-aminopropyltriethoxysilane and loaded active ingredients.

2. The separation microneedle according to claim 1, characterized in that The microneedle body includes a functional backing layer and a rapidly detachable microneedle layer. In parts by weight, the functional backing layer contains 400-2000 parts of polyvinyl pyrrolidone, 55-270 parts of citric acid, and 70-350 parts of sodium bicarbonate; the rapidly detachable microneedle layer contains 50-300 parts of regenerated silk cellulose; the liposome nanoreactor is loaded in the rapidly detachable microneedle layer, and the content of the liposome nanoreactor is 1-5 parts.

3. The separation microneedle according to claim 1, characterized in that The liposome nanoreactor comprises liposomes and silica nanocapsules; in parts by weight, the liposomes contain 0.3-1.5 parts of glycosylated phosphatidylcholine, 0.3-1.5 parts of phosphatidylcholine and 0.05-0.5 parts of cholesterol; the silica nanocapsules contain 0.1-0.5 parts of tetramethoxysilane and 0.03-0.3 parts of 3-aminopropyltriethoxysilane.

4. The separation microneedle according to claim 1, characterized in that The loaded active ingredients are cisplatin, 5-aminolevulinic acid and catalase.

5. The separation microneedle according to claim 1, characterized in that The molecular weight of the polyvinyl pyrrolidone is 10-100 kDa, and the molecular weight of the regenerated silk cellulose is 100-150 kDa.

6. The method for preparing the separation microneedle according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: S1: Tetramethoxysilane, 3-aminopropyltriethoxysilane and catalase were encapsulated by reverse microemulsion in situ method to prepare catalase-loaded silica nanocapsules; S2: preparing a liposome nanoreactor by a thin film dispersion method using glycosylated phosphatidylcholine, phosphatidylcholine, cholesterol, the silica nanocapsules loaded with catalase, cisplatin and 5-aminolevulinic acid; S3: stirring and mixing the regenerated silk cellulose and the liposome nanoreactor, and degassing to obtain a needle matrix solution; then stirring and mixing polyvinyl pyrrolidone, sodium bicarbonate, citric acid and anhydrous ethanol, and degassing to obtain a functional backing layer matrix solution; S4: pouring the needle body matrix solution into a mold, vacuum compressing and drying; adding the functional backing layer matrix solution, vacuum compressing and drying, and peeling and demoulding to obtain the separated microneedles.

7. The preparation method according to claim 6, characterized in that In the reverse microemulsion in situ encapsulation method in step S1, the ultrasonic power is 300 W to 600 W, the ultrasonic time is 2 minutes to 10 minutes, the stirring speed is 500 to 1000 rpm, and the volatilization time is 12 hours to 24 hours.

8. The preparation method according to claim 6, characterized in that The rotary evaporation temperature of the thin film dispersion method in step S2 is 40°C to 80°C, and the hydration dispersion time is 10 minutes to 30 minutes; the drying temperature in step S4 is 25°C to 60°C, and the drying time is 8 hours to 24 hours.

9. Use of the separation microneedle according to any one of claims 1 to 5 in the preparation of a drug for treating superficial skin tumors.

10. The use according to claim 9, characterized in that The superficial skin tumor is melanoma or breast cancer; the separation microneedles can be used alone or in combination with photothermal therapy, chemotherapy and immunotherapy.

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