A liposome-loaded nanoreactor-loaded microneedle and a preparation method and application thereof

Abstract

The application provides a kind of liposome nano-reactor loaded separation microneedle and its preparation method and application, belong to the field of biological medicine.The application uses phospholipid, cholesterol, tetramethoxysilane and 3-aminopropyl triethoxysilane as raw material, encapsulates cisplatin, 5-aminoacetylpropionic acid and catalase, and prepares liposome nano-reactor;With polyvinylpyrrolidone, citric acid, sodium bicarbonate and regenerated silk fibroin as raw material, the liposome nano-reactor is dispersed in microneedle body by pouring method, and a kind of microneedle that can be quickly separated is successfully prepared.The experimental research of the application proves that the separation microneedle has good biocompatibility and mechanical property, can effectively break through the stratum corneum, realize the efficient transdermal delivery of liposome nano-reactor.The liposome nano-reactor can be locally planted in deep tissue of tumor, continuously supply oxygen through enzymatic reaction, effectively improve the oxygen supply shortage of tumor photodynamic therapy, and has obvious advantages and broad prospects in the application of skin tumor photodynamic therapy.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a separated microneedle loaded with a liposome nanoreactor and a preparation method and application thereof. BACKGROUND

[0002] Melanoma is a highly malignant and fatal skin cancer, with excessive tissue hyperplasia and high oxygen demand, resulting in the presence of hypoxic microenvironment in the tumor. Surgery, chemotherapy, radiotherapy and immunotherapy as the existing traditional treatment methods have greatly improved the survival rate of cancer patients. However, surgery has relatively high risk, large trauma, and many complications, chemotherapy has non-specificity, poor targeting, drug resistance, radiotherapy has low patient tolerance and compliance, and immunotherapy has treatment complexity and long-term survival of reprogrammed cells, which hinder its further application. Photodynamic therapy has the characteristics of small trauma, time and space controllability, and convenient local drug use, and has been clinically used for the treatment of melanoma. Although the emerging photodynamic therapy and its combination therapy can inhibit the malignant proliferation of tumors to some extent, the problem of insufficient oxygen supply for photodynamic therapy caused by the hypoxic microenvironment of the tumor is still a key restricting factor limiting its clinical efficacy.

[0003] So far, researchers have been committed to alleviating the oxygen supply shortage of photodynamic therapy, including: 1) directly delivering exogenous oxygen to the tumor; 2) generating oxygen in situ in the tumor; 3) reducing oxygen consumption by inhibiting tumor cell respiration; 4) inhibiting the hypoxia-inducible factor-1 (HIF) signaling pathway to alleviate the hypoxic microenvironment of the tumor. However, these strategies can only alleviate the problem of low photodynamic therapy efficiency caused by the hypoxic microenvironment of the tumor to some extent, and cannot fundamentally provide continuous oxygen for photodynamic therapy. Moreover, due to the complexity and variability of the tumor microenvironment, a single therapy is often insufficient to inhibit the rapid malignant proliferation of tumors. Modern photodynamic therapy mainly uses combination therapy (such as combination with chemotherapy and immunotherapy) as the main cooperative treatment of nanomedicine delivery strategies for tumors. Although these photodynamic combination therapy nanomedicines have been further improved in terms of improving the hypoxic microenvironment of the tumor and enhancing the efficacy, they still face the difficulty of extremely low drug accumulation in tumor tissue.

[0004] For skin superficial malignant tumors, microneedles have shown great advantages in improving drug accumulation in superficial tumor lesions, with the characteristics of designed length / shape, efficient transdermal delivery (which can directly penetrate the stratum corneum to deliver drugs to the dermis), non-invasive and painless, etc. Therefore, the preparation of a rapid separated microneedle that can continuously provide oxygen for photodynamic therapy of tumors, multi-therapy combination, and improve drug accumulation in tumor tissue, will be expected to solve the above problems of low tumor treatment effect caused by the hypoxic microenvironment of the tumor and single treatment method in photodynamic therapy. SUMMARY

[0005] In order to solve the problems of low efficiency of photodynamic therapy caused by tumor hypoxia, unsatisfactory single therapy and unsatisfactory treatment effect caused by insufficient drug accumulation in tumor tissue, the application aims to provide a separated microneedle loaded with liposome nanoreactor and a preparation method and application thereof. The separated microneedle provided by the application can not only continuously supply oxygen for tumor photodynamic therapy, but also increase the accumulation amount of anti-tumor nanomedicine in deep tumor tissue by means of microneedle assisted delivery. The fast separated microneedle can fully combine photodynamic therapy, chemotherapy and immunotherapy to play a synergistic anti-tumor effect.

[0006] In order to achieve the above-mentioned purpose, the application adopts the following technical solutions to achieve the above-mentioned purpose:

[0007] The application provides a separated microneedle loaded with liposome nanoreactor, which comprises 550-3000 parts of a microneedle body and 1-5 parts of a liposome nanoreactor loaded in the microneedle body by weight; the microneedle body comprises polyvinylpyrrolidone, citric acid, sodium bicarbonate and regenerated silk fibroin; and the liposome nanoreactor comprises phospholipid, cholesterol, tetramethoxysilane, 3-aminopropyl triethoxysilane and a loaded active ingredient.

[0008] Further, the microneedle body comprises a functional backing layer and a fast-separated microneedle layer, the functional backing layer contains 400-2000 parts of polyvinylpyrrolidone, 55-270 parts of citric acid and 70-350 parts of sodium bicarbonate by weight; the fast-separated microneedle layer contains 50-300 parts of regenerated silk fibroin; and the liposome nanoreactor is loaded in the fast-separated microneedle layer, and the content of the liposome nanoreactor is 1-5 parts.

[0009] Further, the liposome nanoreactor comprises a liposome and a silica nanocapsule; the liposome contains 0.3-1.5 parts of glycosylphosphatidylcholine, 0.3-1.5 parts of phosphatidylcholine and 0.05-0.5 parts of cholesterol by weight; and the silica nanocapsule contains 0.1-0.5 parts of tetramethoxysilane and 0.03-0.3 parts of 3-aminopropyl triethoxysilane.

[0010] Further, the loaded active ingredient is cisplatin, 5-aminoacetylpropionic acid and catalase.

[0011] Further, the amount of the catalase is 0.02-0.2 parts (the enzyme activity of the catalase is 500 U / mg-5000 U / mg), the amount of the cisplatin is 0.05-0.5 parts, and the amount of the 5-aminoacetylpropionic acid is 0.1-1 parts by weight.

[0012] Further, the polyvinylpyrrolidone has a molecular weight of 10-100 kDa, and the regenerated silk fibroin has a molecular weight of 100-150 kDa.

[0013] The application further provides a preparation method of the liposome nanoreactor rapid separation microneedle.

[0014] S1: the hydrogen peroxidase-loaded silicon dioxide nanocapsules are prepared by using reverse microemulsion in-situ encapsulation method with the dosages of tetramethoxysilane, 3-aminopropyl triethoxysilane and hydrogen peroxidase;

[0015] S2: the liposome nanoreactor is prepared by using film dispersion method with the dosages of glycosylated phosphatidylcholine, phosphatidylcholine, cholesterol, the hydrogen peroxidase-loaded silicon dioxide nanocapsules, cisplatin and 5-amino levulinic acid;

[0016] S3: the needle body matrix solution is prepared after the regenerated silk fibroin and the liposome nanoreactor are stirred and mixed, and then degassed; the functional backing layer matrix solution is prepared after the polyvinylpyrrolidone, sodium bicarbonate and citric acid are stirred and mixed, and then degassed;

[0017] S4: the needle body matrix solution is poured into a mold, vacuum compression dried, then the functional backing layer matrix solution is added, vacuum compression dried, and then the separation microneedle is obtained by stripping and demolding.

[0018] Further, the ultrasonic power of the reverse microemulsion in-situ encapsulation technology in step S1 is 300 W-600 W, the ultrasonic time is 3 minutes-10 minutes, the stirring speed is 500-1000 rpm, and the volatilization time is 12 hours-24 hours.

[0019] Further, the temperature of the rotary evaporator in the film dispersion method in step S2 is 40 ℃-80 ℃, and the hydration dispersion time is 10 minutes-30 minutes.

[0020] Further, the stirring speed in step S3 is 500-1000 rpm, and the stirring time is 1 hour-3 hours.

[0021] Further, the drying temperature in step S4 is 25 ℃-60 ℃, and the drying time is 8 hours-24 hours.

[0022] The application further provides application of the separation microneedle in preparation of a drug for treating superficial skin tumors.

[0023] Further, the superficial skin tumor is melanoma or breast cancer.

[0024] Further, the quick separation microneedle can be used alone or in combination with photothermal therapy, chemotherapy, and chemical kinetics therapy.

[0025] Compared with the prior art, the application has the following advantages and beneficial effects:

[0026] 1. The quick separation microneedle prepared by the application not only has the characteristics of quick disintegration of the needle body and promotion of drug penetration into deep tumor layers, but also can continuously supply oxygen for tumor photodynamic therapy to improve the anti-tumor effect.

[0027] 2. The quick separation microneedle has good mechanical skin breaking performance and biocompatibility, and shows obvious tumor growth inhibition effect on melanoma mice; the liposome nanoreactor can continuously produce oxygen by enzymatic reaction using high-concentration hydrogen peroxide in the tumor microenvironment as a substrate, thereby providing continuous oxygen supply for photodynamic therapy, improving the immunosuppression caused by the tumor hypoxic microenvironment, and improving the efficacy of photodynamic combined immunotherapy.

[0028] 3. The application of cisplatin and 5-ALA effectively combines photodynamic therapy, chemotherapy, and immunotherapy. The experiments prove that the quick separation microneedle provided by the application has obvious treatment advantages in the treatment of skin superficial tumors, and therefore has good and wide market application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 is a side view, a scanning electron microscope image, and a three-dimensional scanning image under a laser confocal microscope of the quick separation microneedle in Example 1 and Example 2 of the application; wherein a is a side view and a partial enlarged view of the quick separation microneedle in Example 1; b is a scanning electron microscope image of the quick separation microneedle in Example 1; c is a single-needle scanning electron microscope image of the quick separation microneedle in Example 1; d is a three-dimensional scanning image of the quick separation microneedle under a laser confocal microscope in Example 2.

[0030] Figure 2 is a single-needle scanning electron microscope side view and an element distribution image of the liposome nanoreactor quick separation microneedle in Example 8 of the application.

[0031] Figure 3 is a mechanical property diagram of the quick separation microneedle in Example 1 of the application.

[0032] Figure 4 is a dissolution diagram of rhodamine B-quick separation microneedle in Example 2 of the application in PBS solution over time.

[0033] Figure 5 is an oxygen production diagram of the liposome nanoreactor quick separation microneedle in Example 3 of the application over time.

[0034] Figure 6 These are the pharmacodynamic evaluation results of the rapid separation of microneedles using liposome nanoreactors in vivo for the treatment of melanoma according to Examples 3, 4, 5, 6, 7, and 8 of this invention; wherein, a is the record of tumor volume at different time points after treatment with each group of preparations; b is the tumor weight statistics on day 14; and c is a comparison of tumor photos after treatment with each group of preparations on day 14.

[0035] Figure 7 These are staining images of pathological and apoptotic sections of melanoma treated in vivo using liposome nanoreactors for rapid separation of microneedles in embodiments 3, 4, 5, 6, 7, and 8 of this invention. Detailed Implementation

[0036] To better illustrate the purpose, technical solution, and advantages of the present invention, the technical solution of the present invention will be further described below in conjunction with 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.

[0037] The preparation steps of the liposome nanoreactor in this invention are as follows:

[0038] The following parts by weight were weighed: liposomes consisted of 0.3-1.5 parts glycosylated phosphatidylcholine, 0.3-1.5 parts phosphatidylcholine, and 0.05-0.5 parts cholesterol; silica nanocapsules consisted of 0.1-0.5 parts tetramethoxysilane and 0.03-0.3 parts 3-aminopropyltriethoxysilane. Silica nanocapsules were prepared by mixing 0.1-0.5 parts tetramethoxysilane, 0.03-0.3 parts 3-aminopropyltriethoxysilane, and 0.02-0.2 parts catalase as raw materials and then using reverse microemulsion in-situ encapsulation technology.

[0039] Glycosylated phosphatidylcholine, phosphatidylcholine, cholesterol, the aforementioned silica nanocapsules, cisplatin, and 5-aminolevulinic acid were mixed as raw materials to prepare a solution. The solvent was then evaporated by rotary evaporation at a temperature of 40 ℃ to 80 ℃. 2 to 5 mL of ultrapure water was added to hydrate and disperse the solution for 10 to 30 minutes. The liposome nanoreactor was prepared by thin-film dispersion method. Example 1

[0040] This embodiment provides a method for preparing blank layered rapid separation microneedles, the specific steps of which are as follows:

[0041] 1. Weigh 150 mg of polyvinylpyrrolidone K30, 20 mg of citric acid and 25 mg of sodium bicarbonate and mix with 0.5 mL of anhydrous ethanol. Stir at room temperature until completely dissolved to obtain a functional backing layer solution.

[0042] 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.

[0043] 3. Take 0.1 mL of 6% (w / v) regenerated silk cellulose solution, add it to the microneedle mold, vacuum degas for 30 minutes, cycle three times, and dry at 37 ℃ for 8 hours to evaporate the solvent (anhydrous ethanol). Take 0.5 mL of the functional backing layer solution and add it to the groove of the microneedle mold after the previous drying step, vacuum degas for 30 minutes, cycle three times, and dry at 37 ℃ for 8 hours to evaporate the solvent (ultrapure water). Demold to obtain blank layered rapid separation microneedles. Example 2

[0044] This embodiment provides a method for preparing rhodamine B-rapid separation microneedles, the specific steps of which are as follows:

[0045] 1. Weigh 150 mg of polyvinylpyrrolidone K30, 20 mg of citric acid and 25 mg of sodium bicarbonate and mix with 0.5 mL of anhydrous ethanol. Stir at room temperature until completely dissolved to obtain a functional backing layer solution.

[0046] 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.

[0047] 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 solution to the microneedle mold, vacuum degas for 30 minutes, cycle three times, and dry at 37 ℃ for 8 hours. After drying, evaporate the solvent (anhydrous ethanol). Add 0.5 mL of the functional backing layer solution to the groove of the microneedle mold after the previous drying step, vacuum degas for 30 minutes, cycle three times, and dry at 37 ℃ for 8 hours. After drying, evaporate the solvent (ultrapure water), demold, and obtain the Rhodamine B-rapid separation microneedles. Example 3

[0048] This embodiment provides a method for rapidly separating microneedles using a liposome nanoreactor loaded with catalase. The specific steps are as follows:

[0049] 1. Weigh 150 mg of polyvinylpyrrolidone K30, 20 mg of citric acid and 25 mg of sodium bicarbonate and mix with 0.5 mL of anhydrous ethanol. Stir at room temperature until completely dissolved to obtain a functional backing layer solution.

[0050] 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.

[0051] 3. After mixing 0.04 mg tetramethoxysilane, 0.012 mg 3-aminopropyltriethoxysilane and 0.0006 mg catalase, silica nanocapsules loaded with catalase were prepared by in-situ encapsulation using a reverse microemulsion method.

[0052] 4. 0.1 mg of glycosylated phosphatidylcholine, 0.1 mg of phosphatidylcholine, 0.03 mg of cholesterol, and 0.05 mg of catalase-loaded silica nanocapsules were prepared by thin-film dispersion to obtain liposome nanoreactors. These liposomes were then mixed with 12% (w / v) regenerated silk cellulose solution at a volume ratio of 1:1 to prepare a 6% (w / v) regenerated silk cellulose solution for catalase-lipid nanoreactors.

[0053] 5. The lipid nanoreactor loaded with catalase was dispersed in 0.6 mL of 6% (w / v) regenerated silk cellulose solution as the first casting solution. Then, 0.1 mL of the first casting solution was directly dripped onto the mold surface using a pipette. The mold was evacuated under vacuum for 30 min and circulated three times to compress the regenerated silk cellulose into the microneedle cavity. The mold was then dried in an oven at 37 °C for 8 h to evaporate the solvent water.

[0054] 6. For the functional backing layer, 0.5 mL of a premixed ethanol solution containing PVP K30 (30 wt%), citric acid (4 wt%), and sodium bicarbonate (5 wt%) was added as a second casting solution to the dried microneedle mold from the previous step to form an effervescent backing layer. The mold was then dried again in an oven at 37 °C for 8 h to evaporate the anhydrous ethanol solvent. After demolding, the rapid separation microneedles loaded with catalase-lipid nanoreactors were obtained. Example 4

[0055] This embodiment provides a rapid separation microneedle preparation method for a liposome nanoreactor loaded with 5-aminolevulinic acid, the specific steps of which are as follows:

[0056] 1. Weigh 150 mg of polyvinylpyrrolidone K30, 20 mg of citric acid and 25 mg of sodium bicarbonate and mix with 0.5 mL of anhydrous ethanol. Stir at room temperature until completely dissolved to obtain a functional backing layer solution.

[0057] 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.

[0058] 3. Liposomes were prepared by thin-film dispersion of 0.1 mg glycosylated phosphatidylcholine, 0.1 mg phosphatidylcholine, 0.03 mg cholesterol and 0.06 mg 5-aminolevulinic acid. The liposomes were then mixed with 12% (w / v) regenerated silk cellulose solution at a volume ratio of 1:1 to prepare a 6% (w / v) regenerated silk cellulose solution loaded with 5-aminolevulinic acid liposomes.

[0059] 4. The liposomes loaded with 5-aminolevulinic acid were dispersed in 0.6 mL of 6% (w / v) regenerated silk cellulose solution as the first casting solution. Then, 0.1 mL of the first casting solution was directly dripped onto the mold surface using a pipette. The mold was then evacuated under vacuum for 30 min and circulated three times to compress the regenerated silk cellulose into the microneedle cavity. After that, the mold was dried in an oven at 37 °C for 8 h to evaporate the solvent water.

[0060] 5. For the backing layer, 0.5 mL of a premixed ethanol solution containing PVP K30 (30 wt%), citric acid (4 wt%), and sodium bicarbonate (5 wt%) was added as a second casting solution to the dried microneedle mold from the previous step to form an effervescent backing layer. The mold was then dried again in an oven at 37 °C for 8 h to evaporate the anhydrous ethanol solvent. After demolding, the rapid separation microneedles loaded with 5-aminolevulinic acid liposomes were obtained. Example 5

[0061] This embodiment provides a method for rapidly separating microneedles using a cisplatin-loaded liposome nanoreactor, the specific steps of which are as follows:

[0062] 1. Weigh 150 mg of polyvinylpyrrolidone K30, 20 mg of citric acid and 25 mg of sodium bicarbonate and mix with 0.5 mL of anhydrous ethanol. Stir at room temperature until completely dissolved to obtain a functional backing layer solution.

[0063] 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.

[0064] 3. Liposomes were prepared by thin-film dispersion of 0.1 mg glycosylated phosphatidylcholine, 0.1 mg phosphatidylcholine, 0.03 mg cholesterol and 0.02 mg cisplatin. The liposomes were then mixed with 12% (w / v) regenerated silk cellulose solution at a volume ratio of 1:1 to prepare a 6% (w / v) regenerated silk cellulose solution loaded with cisplatin liposomes.

[0065] 4. The cisplatin-loaded liposomes were dispersed in 0.6 mL of 6% (w / v) regenerated silk cellulose solution as the first casting solution. Then, 0.1 mL of the first casting solution was directly dripped onto the mold surface using a pipette. The mold was then evacuated under vacuum for 30 min and circulated three times to compress the regenerated silk cellulose into the microneedle cavity. After that, the mold was dried in an oven at 37 ℃ for 8 h to evaporate the solvent water.

[0066] 5. For the backing layer, use 500 µ A premixed ethanol solution containing PVP K30 (30 wt%), citric acid (4 wt%), and sodium bicarbonate (5 wt%) was added as a second casting solution to the microneedle mold dried in the previous step to form an effervescent backing layer. The mold was then dried again in an oven at 37 °C for 8 h to evaporate the anhydrous ethanol solvent. After demolding, the cisplatin-loaded liposome-based rapid separation microneedles were obtained. Example 6

[0067] This embodiment provides a method for rapidly separating microneedles using silica nanocapsules loaded with catalase / cisplatin liposome nanoreactors. The specific steps are as follows:

[0068] 1. Weigh 150 mg of polyvinylpyrrolidone K30, 20 mg of citric acid and 25 mg of sodium bicarbonate and mix with 0.5 mL of anhydrous ethanol. Stir at room temperature until completely dissolved to obtain a functional backing layer solution.

[0069] 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.

[0070] 3. After mixing 0.04 mg tetramethoxysilane, 0.012 mg 3-aminopropyltriethoxysilane and 0.0006 mg catalase, silica nanocapsules loaded with catalase were prepared by in-situ encapsulation using a reverse microemulsion method.

[0071] 4. Liposome nanoreactors were prepared by thin-film dispersion using 0.1 mg glycosylated phosphatidylcholine, 0.1 mg phosphatidylcholine, 0.03 mg cholesterol, 0.05 mg catalase-loaded silica nanocapsules, and 0.02 mg cisplatin. The liposome nanoreactors were then mixed with 12% (w / v) regenerated silk cellulose solution at a volume ratio of 1:1 to prepare a 6% (w / v) regenerated silk cellulose solution of catalase-loaded silica nanocapsules / cisplatin liposome nanoreactors.

[0072] 5. The catalase / cisplatin liposome nanoreactor was dispersed in 0.6 mL of 6% (w / v) regenerated silk cellulose solution as the first casting solution. Then, 0.1 mL of the first casting solution was directly dropped onto the mold surface using a pipette. The mold was evacuated under vacuum for 30 min and circulated three times to compress the regenerated silk cellulose into the microneedle cavity. The mold was then dried in an oven at 37 °C for 8 h to evaporate the solvent water.

[0073] 6. For the backing layer, 0.5 mL of a premixed ethanol solution containing PVP K30 (30 wt%), citric acid (4 wt%), and sodium bicarbonate (5 wt%) was added as a second casting solution to the dried microneedle mold from the previous step to form an effervescent backing layer. The mold was then dried again in an oven at 37 °C for 8 h to evaporate the anhydrous ethanol solvent. After demolding, the microneedles were obtained from the catalase / cisplatin liposome nanoreactor for rapid separation. Example 7

[0074] This embodiment provides a method for rapidly separating microneedles using silica nanocapsules loaded with catalase / liposome nanoreactors containing 5-aminolevulinic acid. The specific steps are as follows:

[0075] 1. Weigh 150 mg of polyvinylpyrrolidone K30, 20 mg of citric acid and 25 mg of sodium bicarbonate and mix with 0.5 mL of anhydrous ethanol. Stir at room temperature until completely dissolved to obtain a functional backing layer solution.

[0076] 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.

[0077] 3. After mixing 0.04 mg tetramethoxysilane, 0.012 mg 3-aminopropyltriethoxysilane and 0.0006 mg catalase, silica nanocapsules loaded with catalase were prepared by in-situ encapsulation using a reverse microemulsion method.

[0078] 4. Liposome nanoreactors were prepared by thin-film dispersion using 0.1 mg glycosylated phosphatidylcholine, 0.1 mg phosphatidylcholine, 0.03 mg cholesterol, 0.05 mg catalase-loaded silica nanocapsules, and 0.06 mg 5-aminolevulinic acid. The liposome nanoreactors were then mixed with 12% (w / v) regenerated silk cellulose solution at a volume ratio of 1:1 to prepare a 6% (w / v) regenerated silk cellulose solution of catalase-loaded silica nanocapsules / 5-aminolevulinic acid liposome nanoreactors.

[0079] 5. The catalase / 5-aminolevulinic acid liposome nanoreactor loaded with catalase was dispersed in 0.6 mL of 6% (w / v) regenerated silk cellulose solution as the first casting solution. Then, 0.1 mL of the first casting solution was directly dripped onto the mold surface using a pipette. The mold was evacuated under vacuum for 30 min and circulated three times to compress the regenerated silk cellulose into the microneedle cavity. The mold was then dried in an oven at 37 °C for 8 h to evaporate the solvent water.

[0080] 6. For the backing layer, 0.5 mL of a premixed ethanol solution containing PVP K30 (30 wt%), citric acid (4 wt%), and sodium bicarbonate (5 wt%) was added as a second casting solution to the dried microneedle mold from the previous step to form an effervescent backing layer. The mold was then dried again in an oven at 37 °C for 8 h to evaporate the anhydrous ethanol solvent. After demolding, the microneedles were obtained from the catalase / 5-aminolevulinic acid liposome nanoreactor for rapid separation. Example 8

[0081] This embodiment provides a method for rapidly separating microneedles using a liposome nanoreactor loaded with catalase / 5-aminolevulinic acid / cisplatin. The specific steps are as follows:

[0082] 1. Weigh 150 mg of polyvinylpyrrolidone K30, 20 mg of citric acid and 25 mg of sodium bicarbonate and mix with 0.5 mL of anhydrous ethanol. Stir at room temperature until completely dissolved to obtain a functional backing layer solution.

[0083] 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.

[0084] 3. After mixing 0.04 mg tetramethoxysilane, 0.012 mg 3-aminopropyltriethoxysilane and 0.0006 mg catalase, silica nanocapsules loaded with catalase were prepared by in-situ encapsulation using a reverse microemulsion method.

[0085] 4. Liposome nanoreactors were prepared by thin-film dispersion using 0.1 mg glycosylated phosphatidylcholine, 0.1 mg phosphatidylcholine, 0.03 mg cholesterol, 0.05 mg silica nanocapsules loaded with catalase, 0.06 mg 5-aminolevulinic acid, and 0.02 mg cisplatin. The liposome nanoreactors were then mixed with 12% (w / v) regenerated silk cellulose solution at a volume ratio of 1:1 to prepare a 6% (w / v) regenerated silk cellulose solution for the catalase / 5-aminolevulinic acid / cisplatin liposome nanoreactors.

[0086] 5. The catalase / 5-aminolevulinic acid / cisplatin liposome nanoreactor was dispersed in 0.6 mL of 6% (w / v) regenerated silk cellulose solution as the first casting solution. Then, 0.1 mL of the first casting solution was directly dripped onto the mold surface using a pipette. The mold was evacuated under vacuum for 30 min and circulated three times to compress the regenerated silk cellulose into the microneedle cavity. After that, the mold was dried in an oven at 37 °C for 8 h to evaporate the solvent water.

[0087] 6. For the backing layer, 0.5 mL of a premixed ethanol solution containing PVP K30 (30 wt%), citric acid (4 wt%), and sodium bicarbonate (5 wt%) was added as a second casting solution to the dried microneedle mold from the previous step to form an effervescent backing layer. The mold was then dried again in an oven at 37 °C for 8 h to evaporate the anhydrous ethanol solvent. After demolding, the microneedles loaded with catalase / 5-aminolevulinic acid / cisplatin liposome nanoreactor for rapid separation were obtained. Example 9

[0088] 1. Morphological characterization of rapidly separated microneedles

[0089] The blank rapid separation microneedle prepared in Example 1 was placed on the experimental stage, and a bright-field side view photograph 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. The side view of the microneedle is shown below. Figure 1 As shown in Figures a and d: It can be observed that the rapidly separating microneedles exhibit a sharp, conical, prismatic shape, arranged in an array, with a needle height of 750 mm. μ m, base width is 130 μ m.

[0090] 2. Rapid separation and determination of the microstructure and elemental distribution of microneedles

[0091] The liposome nanoreactor prepared in Example 8 was rapidly separated from the gold-sprayed microneedles for 90 s. The surface morphology was observed using a scanning electron microscope at an accelerating voltage of 20.0 kV. Figure 1 As shown in Figures b and c: the height of the rapidly separating microneedle is 750 mm. μ m; Energy spectral analysis spectrum as follows 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 microneedles prepared in Example 8.

[0092] 3. Mechanical performance test of rapid separation of microneedles using a bubble pump

[0093] The mechanical properties of microneedles determine their ability to penetrate the skin. In Example 8, the rapidly separated microneedles were placed on the lower plate of a texture analyzer, compressed at a rate of 1 mm / min, with a deformation of 80%. The average pressure change per needle during compression was recorded. Figure 3 As shown. When the compression displacement is 400... μ At m, the mechanical strength of the rapid separation microneedle is 1.2 N / needle. These values ​​far exceed the minimum force of 0.058 N required to penetrate human skin, proving that the rapid separation microneedle can effectively pierce the skin.

[0094] 4. Rapid separation of microneedles: dissolution performance test

[0095] The microneedle was rapidly separated by a bubble pump and placed in PBS. Oxygen production was then monitored using a dissolved oxygen meter. Figure 4 As shown. Once the Rhodamine B-rapid separation microneedles of Example 2 come into contact with the PBS solution, they begin to dissolve rapidly within 1 second, with clearly visible bubbles. As the time extends to 16 seconds, the rapid separation microneedles of Example 2 almost completely disintegrate.

[0096] 5. Rapid separation of microneedles and oxygen production capacity test

[0097] The liposome rapid separation microneedles from Example 2 were placed in PBS containing 1 mM H2O2. The dissolution of the microneedles at different time points was observed under a fluorescence microscope after a time delay. Figure 5 As shown, the liposome nanoreactor of Example 8 rapidly separates microneedles, gradually converting H2O2 in the solution into oxygen over time, indicating that the microneedles of the liposome nanoreactor of Example 8 continuously generate oxygen using H2O2 as a substrate.

[0098] Example 10: Evaluation of the efficacy of in vivo treatment for melanoma

[0099] Male C57BL / 6J mice (5-6 weeks old) were anesthetized and shaved. 5×10⁻⁶ mg of the solution was injected intradermally into the right back of the mice. 5 One melanoma cell. When the tumor volume reaches 50 mm. 3 At approximately 10:00 AM, mice were randomly divided into 7 groups. Microneedle patch therapy was administered on days 0, 2, 4, 6, 8, 10, and 12 after grouping, followed by 635 nm laser irradiation (200 mW / cm²) 6 hours after each administration. 2 (10 minutes). Tumor volume was recorded using calipers during this period. Mice were sacrificed on day 14, and tumor tissue was collected in 4% paraformaldehyde. The tumor tissue was then embedded in a mixture of polyethylene glycol and polyvinyl alcohol, sectioned, and stained with 4',6-diamidinyl-2-phenylindole (DAPI) and an apoptosis indicator (TUNEL). Simultaneously, the tumor tissue was embedded in paraffin and sectioned for hematoxylin and eosin (H&E) staining.

[0100] Figure 6 Examples 3, 4, 5, 6, 7, and 8 present the results of studies on the rapid separation of microneedles using liposome nanoreactors for in vivo treatment of melanoma. Figure 6 As shown in Figure a, tumor growth in the Example 3 treatment group was significantly inhibited compared to other groups. The tumor-suppressing effect of the Example 3 treatment group was superior to that of the other example control groups, and its mechanism may be related to the combined effects of photodynamic therapy, chemotherapy, and immunotherapy. Similarly, tumor weight diagrams and photographs of mice in each group are shown in Figure a. Figure 6 In groups b and c, the tumors in the treatment group of Example 8 were the smallest. These results indicate that the treatment group of Example 8 with administration plus light irradiation had a strong therapeutic effect.

[0101] The results of pathological sections and apoptosis sections are as follows: Figure 7 As shown, compared with the untreated group, the treatment group of Example 8 exhibited the most significant tumor cell damage and apoptosis. These data provide pathological evidence for the multiple therapeutic effects of the treatment group of Example 8.

[0102] This invention utilizes phospholipids, cholesterol, tetramethoxysilane, and 3-aminopropyltriethoxysilane as raw materials, simultaneously loading cisplatin, 5-aminolevulinic acid, and catalase to prepare liposome nanoreactors. Using polyvinylpyrrolidone and regenerated silk cellulose as raw materials, and simultaneously loading the liposome nanoreactors, a rapidly separable microneedle was successfully prepared via a template method. The rapidly separable microneedle exhibits a sharp, conical shape, good biocompatibility and mechanical properties, and can effectively penetrate the stratum corneum, achieving efficient transdermal delivery and release of the liposome nanoreactors. The rapidly separable microneedle of the liposome nanoreactor can continuously supply oxygen, effectively improving the predicament of insufficient oxygen supply in photodynamic therapy for tumors. It has significant advantages and broad application prospects in the application of photodynamic therapy for skin tumors.

[0103] The above embodiments are for illustrative purposes only and are not intended to limit the technical solutions described in this invention. Although this specification has described the invention in detail with reference to the above embodiments, those skilled in the art should understand that they can still make modifications or equivalent substitutions to the invention, and all technical solutions and improvements that do not depart from the spirit and scope of the invention should be covered within the scope of the claims of this invention.

Claims

1. An isolated microneedle loaded with a liposome nanoreactor, characterized in that, The separation microneedle comprises a microneedle body 550-3000 parts and a liposome nanoreactor 1-5 parts loaded in the microneedle body, the microneedle body comprises a functional backing layer and a fast separation microneedle layer, the functional backing layer contains polyvinylpyrrolidone 400-2000 parts, citric acid 55-270 parts and sodium bicarbonate 70-350 parts by weight; the fast separation microneedle layer contains regenerated silk cellulose 50-300 parts; the liposome nanoreactor is loaded in the fast separation microneedle layer, the liposome nanoreactor comprises a liposome, a silica nanocapsule and a loaded active ingredient, the liposome contains glycosylated phosphatidylcholine 0.3-1.5 parts, phosphatidylcholine 0.3-1.5 parts and cholesterol 0.05-0.5 parts by weight; the silica nanocapsule contains tetramethoxysilane 0.1-0.5 parts and 3-aminopropyl triethoxysilane 0.03-0.3 parts; the loaded active ingredient is cisplatin, 5-aminoacetic propionic acid and catalase; The preparation method of the separation microneedle comprises the following steps: S1: tetramethoxysilane, 3-aminopropyl triethoxysilane and catalase are used to prepare silica nanocapsules loaded with catalase by a reverse microemulsion in-situ encapsulation method; S2: glycosylated phosphatidylcholine, phosphatidylcholine, cholesterol, the silica nanocapsules loaded with catalase, cisplatin and 5-aminoacetic propionic acid are used to prepare a liposome nanoreactor by a thin film dispersion method; S3: after regenerated silk cellulose and the liposome nanoreactor are stirred and mixed, a needle body matrix solution is prepared after degassing; after polyvinylpyrrolidone, sodium bicarbonate, citric acid and anhydrous ethanol are stirred and mixed, a functional backing layer matrix solution is prepared after degassing; S4: the needle body matrix solution is poured into a mold, vacuum compression drying is performed, then the functional backing layer matrix solution is added, vacuum compression drying is performed, and then the separation microneedle is obtained by peeling off the mold.

2. The separating microneedle of claim 1, wherein, The molecular weight of the polyvinylpyrrolidone is 10-100 kDa, and the molecular weight of the regenerated silk cellulose is 100-150 kDa.

3. The separating microneedle of claim 1, wherein, In step S1, the ultrasonic power of the reverse microemulsion in-situ encapsulation method is 300 W-600 W, the ultrasonic time is 2 minutes-10 minutes, the stirring speed is 500-1000 revolutions / minute, and the volatilization time is 12 hours-24 hours.

4. The separating microneedle of claim 1, wherein, In step S2, the rotary evaporation temperature of the thin film dispersion method is 40℃-80℃, and the hydration dispersion time is 10 minutes-30 minutes; in step S4, the drying temperature is 25℃-60℃, and the drying time is 8 hours-24 hours.

5. Use of the isolated microneedle of claim 1 or 2 for the manufacture of a medicament for the treatment of a superficial skin tumor, characterized in that, The superficial skin tumor is melanoma; the separation microneedle can be used alone or in combination with photothermal therapy, chemotherapy and immunotherapy.

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