Acid-responsive bubble pump composite microneedle and its preparation method and application

Abstract

The present invention provides an acid-responsive bubble pump composite microneedle, and a preparation method and application thereof. The present invention uses carbonate and hyaluronic acid as raw materials, and then successfully prepares an acid-responsive composite microneedle by a template method. Experimental studies have confirmed that the bubble pump composite microneedle of the present invention is in the shape of a sharp quadrangular pyramid, has acid-responsive release characteristics, and the released CO2 bubbles can promote the penetration of drugs into the deep layer of the skin; and has good biocompatibility and mechanical properties, and can effectively break through the stratum corneum barrier. The bubble pump composite microneedle is loaded with the anti-tumor drug doxorubicin and copper sulfide nanoparticles, and achieves a better transdermal anti-melanoma treatment effect by combining photothermal therapy, chemotherapy and chemodynamic therapy. The bubble pump composite microneedle of the present invention significantly improves the transdermal efficiency of drugs, and has obvious advantages and broad prospects in the application of transdermal tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an acid-responsive bubble pump composite microneedle and a preparation method and application thereof. Background Art

[0002] Superficial malignant skin tumors pose a serious threat to human health due to their high incidence worldwide. Melanoma, in particular, is the most dangerous skin tumor, resulting in mortality in up to 90% of patients. Current treatments for melanoma typically involve surgical excision, cryotherapy, and chemotherapy. However, these traditional treatments all carry potential side effects, such as damage to normal tissue, scarring, pain, and infection. To overcome these limitations, transdermal drug delivery systems have been developed, offering the advantages of painlessness, sustained drug release, and convenient administration. However, due to the dense stratum corneum and lack of blood vessels in the dermis, transdermal drug delivery systems have low drug penetration efficiency, and single-agent treatments are less than ideal. These issues pose significant challenges to the effective treatment of superficial malignant skin tumors.

[0003] Microneedles, with tip heights ranging from 100 to 1000 microns, can directly penetrate the stratum corneum and deliver drugs to the dermis, making them a noninvasive, painless, and highly effective transdermal drug delivery system. In recent years, stimuli-responsive microneedles have become a research hotspot due to their potential to improve drug delivery targeting and reduce side effects. Doxorubicin inhibits cancer cell proliferation and transcription by interfering with DNA replication and RNA synthesis, exhibiting broad-spectrum anti-tumor effects. However, doxorubicin has significant systemic toxicity when used as a monotherapy or at high doses. Photothermal therapy, with its noninvasive, spatiotemporal controllable, and low-risk properties, is a novel alternative to traditional chemotherapy for improving cancer treatment. However, the efficacy of photothermal therapy can be compromised by photothermal tolerance. To address this challenge, chemodynamic therapy has emerged as another promising approach for cancer treatment. Its principle is to utilize the Fenton / Fenton-like reaction to convert intracellular hydrogen peroxide into toxic hydroxyl radicals. Inorganic nanoparticles, such as copper sulfide nanoparticles, possess both excellent photothermal and Fenton-like catalytic properties. Through a Fenton-like catalytic reaction, copper sulfide nanoparticles can deplete intracellular hydrogen peroxide and glutathione, leading to cancer cell apoptosis and exhibiting significant anti-tumor effects. Fucoidan, a natural polysaccharide extracted from marine brown algae, exhibits excellent biocompatibility and immunomodulatory activity, making it an ideal biomaterial for nanoparticle preparation. Therefore, the development of a novel microneedle that can combine photothermal therapy, chemotherapy, and chemodynamic therapy has the potential to address the aforementioned challenges in the current treatment of superficial skin malignancies. Summary of the Invention

[0004] To address the problems of low transdermal cancer drug penetration efficiency and unsatisfactory single-treatment efficacy, the present invention provides an acid-responsive bubble pump composite microneedle, its preparation method, and application. The composite microneedle not only penetrates the stratum corneum barrier but also responds to the acidic tumor microenvironment, generating carbon dioxide bubbles that, through the airflow, deliver copper sulfide nanoparticles and doxorubicin hydrochloride to deep lesions. Furthermore, the bubble pump composite microneedle can fully combine with photothermal therapy, chemotherapy, and chemodynamic therapy to achieve a synergistic anti-tumor effect.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides an acid-responsive bubble pump composite microneedle, which comprises a matrix material and an active ingredient simultaneously loaded on the matrix material; the matrix material is sodium hyaluronate and carbonate; the active ingredient is at least one of copper sulfide nanoparticles and doxorubicin hydrochloride.

[0007] Furthermore, the bubble pump composite microneedle comprises, by mass percentage, 75% to 90% sodium hyaluronate, 8% to 13% carbonate, 0% to 2% copper sulfide nanoparticles, 0% to 7% doxorubicin hydrochloride, and the remainder is water.

[0008] Furthermore, the carbonate is any one of sodium carbonate, sodium bicarbonate, calcium carbonate, and calcium bicarbonate.

[0009] Furthermore, the molecular weight of the sodium hyaluronate is 20 kDa-80 kDa; the copper sulfide nanoparticles use fucoidan as a stabilizer; and the concentration of doxorubicin hydrochloride is 40 mg / mL-50 mg / mL.

[0010] Furthermore, the density of the composite microneedles is 300 / cm 2 -400 pieces / cm 2 , the tip height is 500μm-700μm, and the tip distance is 300μm-400μm.

[0011] Furthermore, the backing layer of the bubble pump composite microneedle is sodium hyaluronate.

[0012] Furthermore, the bubble pump composite microneedle has rapid solubility and acid-responsive release properties, and the release of CO2 bubbles can promote deep penetration of drugs.

[0013] Furthermore, the copper sulfide nanoparticles and doxorubicin hydrochloride loaded in the bubble pump composite microneedle have good mechanical properties and biocompatibility.

[0014] The present invention also provides a method for preparing the bubble pump composite microneedle, comprising the following steps:

[0015] S1: Weigh the following mass percentages: 3%-5% anhydrous copper chloride, 20%-25% fucoidan, and 70%-80% sodium sulfide nonahydrate; dissolve the anhydrous copper chloride and fucoidan in 100 mL of ultrapure water and stir at room temperature to form a solution; dissolve the sodium sulfide in 1 mL of ultrapure water and add it dropwise to the copper chloride-fucoidan solution while stirring evenly; heat the solution at 90°C-110°C for 20-40 minutes, dialyze against ultrapure water, and freeze-dry to obtain copper sulfide nanoparticles;

[0016] S2: Prepare a 25%-35% aqueous solution of doxorubicin hydrochloride, copper sulfide nanoparticles, sodium hyaluronate, and carbonate with ultrapure water, heat to dissolve, cool to room temperature, and centrifuge for degassing to obtain a needle tip matrix solution;

[0017] S3: Prepare a 25%-35% sodium hyaluronate solution using ultrapure water, heat to dissolve, and centrifuge for degassing to obtain a backing matrix solution.

[0018] S4: Pour the needle tip matrix solution into the mold, centrifuge, remove the excess matrix solution, and repeat 1-3 times; add the backing matrix solution, centrifuge 1-3 times, dehydrate, and peel off to obtain the composite microneedle.

[0019] Furthermore, in step S1, the stirring rate is 200-600 rpm, and the dialysis time is 12-24 hours.

[0020] Furthermore, in steps S2 and S3, the heating temperature is 60° C.-90° C., the heating time is 5 minutes-10 minutes, the centrifugal speed is 4000-8000 rpm, and the centrifugal time is 3 minutes-8 minutes.

[0021] Furthermore, in step S4, the centrifugal speed is 3000-5000 rpm, the centrifugal time is 3-8 minutes, the dehydration temperature is 20-30° C., the dehydration time is 20-30 hours, and the moisture content after dehydration is less than 1%.

[0022] The present invention also provides the use of the bubble pump composite microneedle in preparing a preparation for resisting superficial skin tumors.

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

[0024] Furthermore, the bubble pump composite microneedle can respond and release in the acidic tumor microenvironment.

[0025] Furthermore, the bubble pump composite microneedle can effectively treat melanoma or breast cancer through multiple therapies.

[0026] Furthermore, the bubble pump composite microneedle can be used alone or in combination with photothermal therapy, chemotherapy, or chemodynamic therapy.

[0027] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0028] The bubble pump composite microneedles prepared by the present invention not only have rapid solubility and acid-responsive release properties, but also release CO2 bubbles to promote drug penetration deep into the skin. The bubble pump composite microneedles possess excellent mechanical properties and biocompatibility, and have shown significant tumor growth inhibition in melanoma-bearing mice. The addition of carbonates effectively enhances the dissolution rate of the composite microneedles in the skin and increases the depth of drug penetration, improving the effectiveness and patient compliance of transdermal cancer treatment. The combination of copper sulfide nanoparticles and doxorubicin hydrochloride effectively combines photothermal therapy, chemodynamic therapy, and chemotherapy. Therefore, the bubble pump composite microneedles of the present invention offer significant therapeutic advantages in transdermal cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a three-dimensional scanning image of the bubble pump composite microneedle of Example 2 of the present invention under a laser confocal microscope.

[0030] Figure 2 These are the delayed dissolution fluorescence images of the bubble pump composite microneedles of Examples 1 and 2 of the present invention.

[0031] Figure 3 The drug diffusion condition after the bubble pump composite microneedle of Example 1 and Example 2 of the present invention is inserted into the skin tissue; wherein Figure 3 a is the time-lapse fluorescence image at 0 and 30 minutes after inserting 2 mm thick poloxamer (F-127) gel simulating skin tissue; Figure 3 b is a cross-sectional fluorescence microscopy image after insertion into 1.5 mm thick pig skin.

[0032] Figure 4 The lateral diffusion of the drug after the bubble pump composite microneedle of Example 1, Example 2, and Example 3 of the present invention is inserted into the skin tissue; Figure 4 a is a top view under a fluorescence microscope after insertion into 1.5 mm thick pig skin; Figure 4 b is the quantitative graph of diffusion area.

[0033] Figure 5 is the transdermal release rate of the bubble pump composite microneedle of Example 8 of the present invention.

[0034] Figure 6 1 is a scanning electron microscope image and element distribution diagram of the bubble pump composite microneedle of Example 4 of the present invention.

[0035] Figure 7 Graphs showing the mechanical properties of the bubble pump composite microneedles of Examples 4, 5, 6, and 7 of the present invention.

[0036] Figure 8 is the transdermal measurement result of the bubble pump composite microneedle of the present invention; wherein, Figure 8 a is a diagram showing the effect of the bubble pump composite microneedle inserted into the skin in Example 4, Figure 8 b is a picture of the skin recovery after puncture by the bubble pump composite microneedle in Example 7.

[0037] Figure 9 The results are the evaluation results of the bubble pump composite microneedle of Example 4, Example 5, and Example 6 of the present invention in treating melanoma in vivo; wherein, Figure 9 a is the record of tumor volume at different time points after treatment with each preparation; Figure 9 b is a comparison of tumor photos of each group of preparations after the end of treatment on the 12th day; Figure 9 c is the statistical graph of tumor weight on day 12; Figure 9 d is the apoptosis indicator (TUNEL) staining and hematoxylin-eosin (H&E) staining images of the tumors of mice in each group. DETAILED DESCRIPTION

[0038] 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.

[0039] The preparation steps of copper sulfide nanoparticles in the present invention are:

[0040] The sample was weighed according to the following mass percentages: 3%-5% anhydrous copper chloride, 20%-25% fucoidan, and 70%-80% sodium sulfide nonahydrate; the anhydrous copper chloride and fucoidan were first dissolved in 100 mL of ultrapure water and stirred at room temperature to form a solution; the sodium sulfide was dissolved in 1 mL of ultrapure water and added dropwise to the copper chloride-fucoidan solution and stirred evenly; the solution was heated at 90°C-110°C for 20-40 minutes, dialyzed with ultrapure water, and freeze-dried to obtain copper sulfide nanoparticles.

[0041] Example 1

[0042] A method for preparing fluorescein isothiocyanate (FITC)-composite microneedles is as follows: 300 mg of sodium hyaluronate (molecular weight 50 kDa) is weighed, 1 mL of FITC aqueous solution (20 μg / mL) is added, and the mixture is heated to 80°C for 5 minutes to promote dissolution. The mixture is thoroughly mixed and centrifuged for degassing to obtain the needle tip matrix material. Simultaneously, 300 mg of sodium hyaluronate (molecular weight 50 kDa) is weighed, dissolved in 1 mL of ultrapure water, heated to 80°C for 5 minutes to promote dissolution, and centrifuged for degassing to obtain the backing matrix material.

[0043] The needle tip matrix material was poured into the mold, centrifuged, and the excess matrix material was removed, and the process was repeated twice. The backing matrix material was added, centrifuged twice, dehydrated to 1%, and peeled off to obtain the FITC-bubble pump composite microneedle.

[0044] Example 2

[0045] A method for preparing a FITC-bubble pump composite microneedle is as follows: 225 mg of sodium hyaluronate (molecular weight 50 kDa) and 75 mg of sodium bicarbonate are weighed, mixed, and then 1 mL of FITC aqueous solution (20 μg / mL) is added. The mixture is heated to 80°C for 5 minutes to promote dissolution. The mixture is thoroughly mixed and centrifuged for degassing to obtain the needle tip matrix material. Simultaneously, 300 mg of sodium hyaluronate (molecular weight 50 kDa) is weighed, dissolved in 1 mL of ultrapure water, heated to 80°C for 5 minutes to promote dissolution, and centrifuged for degassing to obtain the backing matrix material.

[0046] The needle tip matrix material was poured into the mold, centrifuged, and the excess matrix material was removed, and the process was repeated twice. The backing matrix material was added, centrifuged twice, dehydrated to 1%, and peeled off to obtain the FITC-bubble pump composite microneedle.

[0047] Example 3

[0048] A method for preparing a FITC-bubble pump composite microneedle is as follows: 270 mg of sodium hyaluronate (molecular weight 50 kDa) and 30 mg of sodium bicarbonate are weighed, mixed, and then 1 mL of FITC aqueous solution (20 μg / mL) is added. The mixture is heated to 80°C for 5 minutes to promote dissolution. The mixture is thoroughly mixed and centrifuged for degassing to obtain the needle tip matrix material. Simultaneously, 300 mg of sodium hyaluronate (molecular weight 50 kDa) is weighed, dissolved in 1 mL of ultrapure water, heated to 80°C for 5 minutes to promote dissolution, and centrifuged for degassing to obtain the backing matrix material.

[0049] The needle tip matrix material was poured into the mold, centrifuged, and the excess matrix material was removed, and the process was repeated twice. The backing matrix material was added, centrifuged twice, dehydrated to 1%, and peeled off to obtain the FITC-bubble pump composite microneedle.

[0050] Example 4

[0051] A copper sulfide nanoparticle-doxorubicin hydrochloride-hyaluronic acid-sodium bicarbonate bubble pump composite microneedle is prepared by weighing 225 mg of sodium hyaluronate (molecular weight 50 kDa), 75 mg of sodium bicarbonate, and 10 mg of copper sulfide nanoparticles, mixing them thoroughly, adding 1 mL of a 40 mg / mL doxorubicin hydrochloride aqueous solution, and heating to 80°C for 5 minutes to promote dissolution. The mixture is thoroughly mixed and centrifuged for degassing to obtain the needle tip matrix material. Simultaneously, 300 mg of sodium hyaluronate (molecular weight 50 kDa) is weighed and dissolved in 1 mL of ultrapure water, heated to 80°C for 5 minutes to promote dissolution, and centrifuged for degassing to obtain the backing matrix material.

[0052] The needle tip matrix material was poured into a mold, centrifuged, and excess matrix material was removed, and this process was repeated twice. The backing matrix material was added, centrifuged twice, dehydrated to 1%, and peeled off to obtain a copper sulfide nanoparticle-doxorubicin hydrochloride-hyaluronic acid-sodium bicarbonate bubble pump composite microneedle.

[0053] Example 5

[0054] A method for preparing a doxorubicin hydrochloride-hyaluronic acid-sodium bicarbonate bubble pump composite microneedle comprises: weighing 225 mg of sodium hyaluronate (molecular weight 50 kDa), 75 mg of sodium bicarbonate, and 40 mg of doxorubicin hydrochloride, mixing them thoroughly, adding them to 1 mL of ultrapure water, and heating them to 80°C for 5 minutes to promote dissolution. After thorough mixing, the mixture was centrifuged and degassed to obtain the needle tip matrix material. Simultaneously, 300 mg of sodium hyaluronate (molecular weight 50 kDa) was weighed and dissolved in 1 mL of ultrapure water, heated to 80°C for 5 minutes to promote dissolution, and centrifuged and degassed to obtain the backing matrix material.

[0055] The needle tip matrix material was poured into the mold, centrifuged, and the excess matrix material was removed, and the process was repeated twice. The backing matrix material was added, centrifuged twice, dehydrated to 1%, and peeled to obtain the doxorubicin hydrochloride-hyaluronic acid-sodium bicarbonate bubble pump composite microneedle.

[0056] Example 6

[0057] A method for preparing a copper sulfide nanoparticle-hyaluronic acid-sodium bicarbonate bubble pump composite microneedle comprises: weighing 225 mg of sodium hyaluronate (molecular weight 50 kDa), 75 mg of sodium bicarbonate, and 10 mg of copper sulfide nanoparticles, mixing them thoroughly, adding 1 mL of ultrapure water, and heating to 80°C for 5 minutes to promote dissolution. Thorough mixing and centrifugation for degassing yield the needle tip matrix material. Simultaneously, weighing 300 mg of sodium hyaluronate (molecular weight 50 kDa), dissolving it in 1 mL of ultrapure water, heating to 80°C for 5 minutes to promote dissolution, and centrifuging for degassing yields the backing matrix material.

[0058] The needle tip matrix material was poured into the mold, centrifuged, and the excess matrix material was removed, and repeated twice. The backing matrix material was added, centrifuged twice, dehydrated to 1%, and peeled to obtain the copper sulfide nanoparticle-hyaluronic acid-sodium bicarbonate bubble pump composite microneedle.

[0059] Example 7

[0060] A method for preparing a hyaluronic acid-sodium bicarbonate bubble pump composite microneedle is as follows: 225 mg of sodium hyaluronate (molecular weight 50 kDa) and 75 mg of sodium bicarbonate are weighed, mixed, and then 1 mL of ultrapure water is added. The mixture is heated to 80°C for 5 minutes to promote dissolution. The mixture is thoroughly mixed and centrifuged for degassing to obtain the needle tip matrix material. Simultaneously, 300 mg of sodium hyaluronate (molecular weight 50 kDa) is weighed, dissolved in 1 mL of ultrapure water, heated to 80°C for 5 minutes to promote dissolution, and centrifuged for degassing to obtain the backing matrix material.

[0061] The needle tip matrix material was poured into the mold, centrifuged, and the excess matrix material was removed, and the process was repeated twice. The backing matrix material was added, centrifuged twice, dehydrated to 1%, and peeled to obtain the hyaluronic acid-sodium bicarbonate bubble pump composite microneedle.

[0062] Example 8

[0063] A method for preparing a rhodamine B (RhB)-bubble pump composite microneedle is as follows: 225 mg of sodium hyaluronate (molecular weight 50 kDa) and 75 mg of sodium bicarbonate are weighed, mixed, and then 1 mL of a 10 mg / mL RhB aqueous solution is added. The mixture is heated to 80°C for 5 minutes to promote dissolution. The mixture is thoroughly mixed and centrifuged for degassing to obtain the needle tip matrix material. Simultaneously, 300 mg of sodium hyaluronate (molecular weight 50 kDa) is weighed, dissolved in 1 mL of ultrapure water, heated to 80°C for 5 minutes to promote dissolution, and centrifuged for degassing to obtain the backing matrix material.

[0064] The needle tip matrix material was poured into the mold, centrifuged, and the excess matrix material was removed, and the process was repeated twice. The backing matrix material was added, centrifuged twice, dehydrated to 1%, and peeled off to obtain the RhB-bubble pump composite microneedle.

[0065] Example 9

[0066] (1) Characterization of microneedle morphology

[0067] The FITC-bubble pump composite microneedle prepared in Example 2 was placed in a confocal dish and observed using a laser confocal microscope. Figure 1 As shown: It can be observed that the composite microneedles are composed of a 5×5 pointed quadrangular pyramid needle array, with a needle height of 600 μm, a base width of 330 μm, and a center distance of 600 μm.

[0068] (2) Dissolution performance test of bubble pump composite microneedle

[0069] The bubble pump composite microneedle was dissolved in PBS with a pH of 5.5, and the dissolution of the microneedle at different time points was recorded under a fluorescence microscope. Figure 2 As shown in Figure 2, upon contact with the PBS solution, the bubble pump composite microneedles of Examples 1 and 2 rapidly dissolved within 400 ms, with clearly visible bubbles, which also promoted the diffusion of FITC. The composite microneedles of Example 1, without the addition of NaHCO3, only swelled without generating bubbles, resulting in relatively slow FITC diffusion over a shorter distance.

[0070] (3) Diffusion performance test of bubble pump composite microneedle

[0071] F-127 hydrogel was used to simulate skin tissue to study whether it could increase the longitudinal penetration depth. The bubble pump composite microneedles of Example 1 and Example 2 were inserted into F-127 gel (30wt%) and the green fluorescence signal was recorded under a microscope. Figure 3 a, Cross-sectional fluorescence microscopy images collected every 30 minutes, showing that the penetration depth of the bubble pump composite microneedle in Example 2 increased from 350.24±3.49 μm (0 minute) to 469.83±1.07 μm (30 minutes), indicating that the CO2 bubbles generated by NaHCO3 significantly increased the diffusion distance of FITC.

[0072] In addition, the longitudinal penetration depth of the bubble pump composite microneedles of Examples 1 and 2 was also studied using pig skin. The penetration depth was tested on 2×2 cm pig skin with a thickness of 1.5 mm using the composite microneedles of Examples 1 and 2, respectively. The bubble pump composite microneedles of Examples 1 and 2 were inserted into the pig skin for 5 minutes, 10 minutes, and 20 minutes, respectively, and then cross-sectional analysis was performed at room temperature. 20 μm sections were prepared using a freezing microtome and observed using a laser confocal microscope. The results are shown in FIG. Figure 3 b shows that compared with the bubble pump composite microneedle treatment in Example 1, the tissue penetration depth of the bubble pump composite microneedle treatment in Example 2 was significantly increased 20 minutes after treatment.

[0073] In order to study the effect of adding NaHCO3 to microneedles on lateral diffusion, microneedles containing different amounts of NaHCO3 were inserted into ex vivo skin tissue. The lateral diffusion performance of the bubble pump composite microneedles of Examples 1, 2, and 3 was evaluated by measuring the diffusion area ratio of fluorescent molecules on a 1.5 mm thick pig skin surface under a fluorescence microscope. The results are shown in Figure 2. Figure 4 As shown, the top view taken within 1 minute confirmed that the diffusion area ratio increased, with the largest diffusion area ratio in Example 2. Therefore, the bubble pump composite microneedle with a NaHCO3 content of 25% was used for subsequent experiments.

[0074] (4) Transdermal release rate of bubble pump composite microneedle

[0075] The transdermal release rate of the microneedle can reflect its drug delivery efficiency. The composite microneedle prepared in Example 8 was used to measure its transdermal efficiency within 24 hours using a transdermal instrument. Fresh mouse abdominal skin was taken and placed on the transdermal instrument. Phosphate buffer (pH 5.5 and pH 7.4) was used as the release medium. After the microneedle was inserted into the skin, 1 mL of the release medium was taken out at different preset time points and the same volume of fresh medium was added. After the experiment, the RhB concentration in the release medium was measured using an ultraviolet spectrophotometer, and the cumulative release amount was calculated. The cumulative release curve is shown in Figure 2. Figure 5As shown, the percentage release at each time point at pH 5.5 was significantly greater than that at pH 7.4. The cumulative release rate reached 77% after 24 hours in a weakly acidic environment (pH 5.5), while the cumulative release rate in a neutral environment (pH 7.4) was only 21%, demonstrating its excellent acid-responsive transdermal release ability.

[0076] (5) Determination of the morphology and element distribution of bubble pump composite microneedles

[0077] The composite microneedle prepared in Example 4 was sprayed with gold for 90 seconds, and its surface morphology was observed using a scanning electron microscope at an accelerating voltage of 20.0 kV. Figure 6 As shown: It can be observed that the bubble pump composite microneedle needle is 600 μm high, and the energy spectrum analysis spectrum confirms the presence of Cu, S, Na, C and N, indicating that sodium bicarbonate, doxorubicin hydrochloride and copper sulfide nanoparticles are uniformly dispersed in the bubble pump composite microneedle prepared in Example 4.

[0078] (6) Mechanical properties test of bubble pump composite microneedle

[0079] The mechanical properties of microneedles determine their ability to penetrate the skin. The composite microneedles prepared in Examples 4, 5, 6, and 7 were placed on the lower plate of a texture analyzer. The compression rate was 1 mm / min, the deformation was 80%, and the average pressure change per needle during the compression process was recorded as follows: Figure 7 As shown in the figure, when the compression displacement was 400 μm, the mechanical strengths of the composite microneedles in each group were 0.36 N, 0.38 N, 0.37 N, and 0.39 N, respectively. These values far exceeded the minimum force of 0.058 N required to penetrate human skin, demonstrating that the composite microneedles can effectively penetrate the skin.

[0080] (7) Testing of the transdermal effect and skin recovery ability of bubble pump combined with microneedles

[0081] Take fresh pig skin, insert the composite microneedle prepared in Example 4 into the pig skin, press for 1 minute and then remove the backing layer. Figure 8 As shown in a: The microneedle left a clear red array on the pig skin, proving that the microneedle has good transdermal ability.

[0082] The bubble pump composite microneedle prepared in Example 7 was inserted into the back skin of the mouse. After pressing for 2 minutes, the microneedle was removed and the recovery of the mouse skin was observed in real time. Figure 8 As shown in Figure b: 10 minutes after microneedle treatment, the mouse back skin can be completely restored to normal without abnormal phenomena such as redness and swelling, which proves that the composite microneedle of the present invention has good in vivo biocompatibility.

[0083] (8) Evaluation of the efficacy of in vivo treatment of melanoma

[0084] C57BL / 6J mice (male, 5-6 weeks old) were anesthetized and depilated, and 1×10 6 melanoma cells. When the tumor volume reaches 100mm 3 Around 24 hours after the onset of leukemia, mice were randomly divided into five groups. On days 0, 2, 4, 6, 8, and 10, they were treated with the bubble pump composite microneedles of Example 4 plus 808 nm infrared light irradiation (Example 4+L), the bubble pump composite microneedles of Example 4, the bubble pump composite microneedles of Example 5, and the bubble pump composite microneedles of Example 6 plus 808 nm infrared light irradiation (Example 6+L), respectively. An untreated group was designated a blank control group. Tumor volume was recorded using a vernier caliper during this period. On day 12, mice were sacrificed and tumor tissues were collected in 4% paraformaldehyde. Tumor tissues were embedded in a mixture of polyethylene glycol and polyvinyl alcohol, sectioned, and stained with 4',6-diamidino-2-phenylindole (DAPI) and a cell apoptosis indicator (TUNEL). Tumor tissues were also embedded in paraffin and sectioned with hematoxylin-eosin (H&E) staining.

[0085] Figure 9 The results of the in vivo treatment of melanoma using the bubble pump composite microneedle + L of Example 4, the bubble pump composite microneedle of Example 4, the bubble pump composite microneedle of Example 5, and the bubble pump composite microneedle + L of Example 6 are shown. Figure 9 a. Compared with other groups, the tumor growth of the Example 4+L treatment group was significantly inhibited. The tumor inhibition effect of the Example 4+L treatment group was better than that of the Example 6+L treatment group and the Example 5 treatment group. Its mechanism may be related to the combined effects of photothermal therapy, chemodynamic therapy and chemotherapy. Similarly, the tumor photos and weights of mice in each group are shown in Figure 9 b and Figure 9 c, The tumor in the Example 4+L treatment group was the smallest. These results indicate that the Example 4 medication plus light irradiation group had a stronger therapeutic effect.

[0086] TUNEL staining and H&E staining results are as follows Figure 9 As shown in Figure d, compared with the untreated group, tumor cell damage and apoptosis were more pronounced in the Example 6 + L treatment group and the Example 5 treatment group. Tumor cell damage and necrosis in the Example 4 + L treatment group were more severe than those in the Example 4 treatment group. These data provide pathological evidence for the multiple therapeutic effects of the Example 4 + L treatment group.

[0087] The present invention uses carbonate and hyaluronic acid as raw materials and successfully prepares an acid-responsive bubble pump composite microneedle through a template method. The bubble pump composite microneedle is in the shape of a sharp quadrangular pyramid and has acid-responsive release characteristics. The release of CO2 bubbles can promote the penetration of drugs into the deep layers of the skin. At the same time, it has good biocompatibility and mechanical properties and can effectively break through the stratum corneum barrier. The acid-responsive bubble pump microneedle is loaded with the anti-tumor drug doxorubicin and copper sulfide nanoparticles to achieve transdermal anti-melanoma treatment. The bubble pump composite microneedle significantly improves the efficiency of drug transdermal transdermal therapy and has obvious advantages and broad application prospects in the application of multiple therapies for transdermal tumor treatment.

[0088] 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. An acid-responsive bubble pump composite microneedle, characterized in that: The bubble pump composite microneedle includes a matrix material and an active ingredient simultaneously loaded on the matrix material; the matrix material is sodium hyaluronate and carbonate; the active ingredients are copper sulfide nanoparticles and doxorubicin hydrochloride; the carbonate is sodium bicarbonate; the molecular weight of the sodium hyaluronate is 20 kDa-80 kDa; the copper sulfide nanoparticles are stabilized by fucoidan; and the concentration of the doxorubicin hydrochloride is 40 mg / mL-50 mg / mL.

2. The method for preparing the bubble pump composite microneedle according to claim 1, characterized in that: The following steps are involved: S1: Copper sulfide nanoparticles were prepared by hydrothermal method using anhydrous copper chloride, fucoidan and sodium sulfide as raw materials; S2: Sodium hyaluronate, carbonate, copper sulfide nanoparticles and doxorubicin hydrochloride are heated and mixed uniformly as raw materials, and then degassed to prepare a needle tip matrix solution; S3: Sodium hyaluronate is heated and dissolved, and then ultrasonically degassed to prepare a backing matrix solution; S4: pouring the needle tip matrix solution into a mold, adding the backing matrix solution after centrifugation, dehydrating by centrifugation, and peeling to obtain the bubble pump composite microneedle.

3. The preparation method according to claim 2, characterized in that The heating temperature of the hydrothermal method in step S1 is 80° C.-120° C., and the heating time is 10 minutes-40 minutes.

4. The preparation method according to claim 2, characterized in that The heating temperature in steps S2 and S3 is 60° C.-90° C., and the heating time is 5 minutes-10 minutes. The dehydration temperature in step S4 is 20° C.-30° C., and the dehydration time is 20 hours-30 hours. The moisture content after dehydration is less than 1%.

5. Use of the bubble pump composite microneedle according to claim 1 in the preparation of an anti-tumor preparation, characterized in that: The tumor is melanoma or breast cancer.

6. The use according to claim 5, characterized in that The bubble pump composite microneedle can be used alone or in combination with photothermal therapy, chemotherapy, or chemodynamic therapy.

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