Microneedle co-loading asiaticoside and near-infrared photothermal agent, and preparation method and application thereof

Abstract

The application discloses a microneedle co-loading asiaticoside and near-infrared photothermal agents, and a preparation method and application thereof. The microneedle adopts a layered structure, and hyaluronic acid and polyvinylpyrrolidone are used as a skeleton matrix. Asiaticoside, near-infrared photothermal agent IR1048 and octa-arginine are loaded on the needle tip, and asiaticoside is loaded on the base. The mechanical strength of a single needle is about 0.46 N, the needle can smoothly penetrate into the dermis layer and is basically dissolved within 10 min, and in-situ precise release of active ingredients is realized. In combination with 1064 nm laser irradiation, the application degrades amyloid in the dermis layer through the photothermal effect of IR1048, and cooperates with asiaticoside to inhibit abnormal cell proliferation and promote collagen regeneration, so that the lesion is effectively removed and the skin microenvironment is reshaped. The microneedle has no obvious skin irritation, has good blood compatibility, and has no systemic toxicity. The application provides a safe and efficient transdermal delivery new strategy with clinical conversion potential for skin amyloidosis.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation technology, specifically relating to a microneedle co-loaded with asiaticoside and a near-infrared photothermal agent, its preparation method, and its application. Background Technology

[0002] Cutaneous amyloidosis (CA) is a chronic metabolic skin disease characterized by the deposition of amyloid protein within the skin without affecting other organs. It is mainly classified into macular amyloidosis, lichenification, and nodular amyloidosis. Clinically, it primarily manifests as persistent itching, changes in appearance, and localized roughness and dryness, severely impacting patients' quality of life. Current treatments include drug therapy and physical therapy. Topical medications, such as corticosteroids, only relieve symptoms; long-term use can easily lead to skin atrophy and recurrence. Oral medications, such as retinoids and immunomodulators, have significant side effects. In physical therapy, photothermal therapy utilizes photothermal transducers to convert near-infrared light energy into heat energy to damage diseased cells, showing promising application potential. However, CA is often accompanied by thickened stratum corneum and dense amyloid protein deposition in the dermis, making it difficult for traditional topical medications to penetrate to the dermal target area. Microneedle drug delivery systems can painlessly and minimally invasively penetrate the thickened stratum corneum to achieve precise drug delivery into the dermis. Soluble hyaluronic acid microneedles, in particular, exhibit good biocompatibility and are biodegradable, showing promising potential in CA treatment. Asiaticoside (AS) has effects such as promoting wound healing, anti-inflammation, and inhibiting fibroblast proliferation. Previous experiments have confirmed that AS microneedle administration can improve skin lesions in rats with carcinoma (CA), but the specific mechanism remains unclear. Furthermore, monotherapy has drawbacks such as long treatment duration and slow onset of action; combining drug therapy with photothermal therapy can synergistically enhance efficacy. Therefore, developing a safe and effective drug delivery system that achieves precise drug delivery, synergistic effects of photothermal and drug action, is of great significance for the treatment of CA. Summary of the Invention

[0003] The purpose of this invention is to provide a microneedle co-loaded with asiaticoside and a near-infrared photothermal agent, its preparation method, and its application.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] A microneedle co-loaded with asiaticoside and a near-infrared photothermal agent, the microneedle comprising a tip and a substrate; the tip is based on hyaluronic acid and polyvinylpyrrolidone K90 as a matrix, and is simultaneously loaded with asiaticoside, a near-infrared photothermal agent and octameric arginine; the substrate is based on polyvinylpyrrolidone K90 as a matrix, and is loaded with asiaticoside.

[0006] Furthermore, the near-infrared photothermal agent is IR1048.

[0007] The preparation method of the microneedles co-loaded with asiaticoside and near-infrared photothermal agent includes the following steps:

[0008] (1) Hyaluronic acid and polyvinylpyrrolidone K90 were added to pure water to prepare a mixed aqueous solution. After swelling at room temperature, asiaticoside, near-infrared photothermal agent and octameric arginine were added and stirred until completely dissolved and mixed to obtain a needle tip matrix solution.

[0009] (2) Prepare an aqueous solution of polyvinylpyrrolidone K90 with pure water, add asiaticoside, stir until completely dissolved and mixed, and prepare a microneedle base solution.

[0010] (3) Add the tip matrix solution to the PDMS microneedle mold and centrifuge to allow the tip matrix solution to fully enter the mold cavity at the tip of the mold; scrape off the excess tip matrix solution on the surface of the mold and centrifuge to remove residual air bubbles in the mold cavity and make the tip matrix structure in the mold cavity more compact; then dry the mold to allow the tip matrix to initially solidify.

[0011] (4) Add microneedle base solution to the dried mold until it completely covers all the mold cavities, centrifuge to make the microneedle base solution uniformly fill the remaining mold cavities; add more microneedle base solution to cover the mold surface, then dry the mold, and after demolding, you will get microneedles co-loaded with asiaticoside and near-infrared photothermal agent.

[0012] Furthermore, in step (1), the mixed aqueous solution contains 20% hyaluronic acid and 10% polyvinylpyrrolidone K90; the needle tip matrix solution contains 0.5% asiaticoside, 1% near-infrared photothermal agent, and 0.5% octameric arginine.

[0013] Furthermore, in step (2), the mass fraction of polyvinylpyrrolidone K90 in the aqueous solution is 10%; and the mass fraction of asiaticoside in the microneedle base solution is 0.5%.

[0014] Furthermore, in step (3), the centrifugation conditions are a temperature of 4°C, a rotation speed of 4000 rpm, and a time of 5 min; the drying conditions are a temperature of 40°C and a time of 0.5 h.

[0015] Furthermore, in step (4), the centrifugation conditions are a temperature of 4°C, a rotation speed of 4000 rpm, and a time of 3 min; the drying conditions are a temperature of 40°C and a time of 6 h.

[0016] The above-mentioned microneedles co-loaded with asiaticoside and near-infrared photothermal agent are used in the preparation of drugs for treating cutaneous amyloidosis.

[0017] The above-mentioned microneedles co-loaded with asiaticoside and near-infrared photothermal agent are used in the preparation of drugs for inhibiting HSF cells and HaCaT cells.

[0018] The significant advantages of this invention are:

[0019] This invention successfully constructed a composite microneedle system co-loaded with asiaticoside and a near-infrared photothermal agent. This system combines photothermal therapy with the inhibitory effect of asiaticoside on abnormal proliferation and promotes regeneration. Octamericarginine is used as a skin penetration enhancer to improve drug penetration and retention in the deep skin layers. Through a "break-and-build" treatment approach, it safely and efficiently removes dermal amyloid deposits and reshapes the healthy skin microenvironment, providing a novel transdermal delivery strategy with significant clinical translational potential for localized cutaneous amyloidosis. Attached Figure Description

[0020] Figure 1 Microneedle morphology characterization results. A, Front view of BMN microneedle; B, Microscopic image of BMN microneedle; CD, SEM image of BMN microneedle; E, Front view of AI-MN@R8 microneedle; F, Microscopic image of AI-MN@R8 microneedle; GH, SEM image of AI-MN@R8 microneedle.

[0021] Figure 2 Microneedle mechanical strength results. A, Microneedle stress-displacement results; B, Mechanical strength results of a single microneedle (n=3).

[0022] Figure 3 Results of microneedle skin puncture depth. A, BMN microneedle skin puncture depth; B, AI-MN@R8 microneedle skin puncture depth.

[0023] Figure 4 : Thermal performance characterization results of microneedles. A, Thermal performance results of free IR1048; B, Thermal performance results of AI-MN@R8 microneedles; C, Photothermal imaging images of different groups.

[0024] Figure 5 Results of photothermal stability of AI-MN@R8 microneedles.

[0025] Figure 6 Results of photothermal conversion efficiency of microneedles. A, Photothermal conversion efficiency of free IR1048; B, Photothermal conversion efficiency of AI-MN@R8 microneedles.

[0026] Figure 7 Results of in vivo dissolution time of microneedles. A, Dissolution time of BMN microneedles; B, Dissolution time of AI-MN@R8 microneedles; C, Quantitative results of microneedle dissolution length.

[0027] Figure 8 Results of in vitro hemolysis experiments using microneedles. A, Results of in vitro hemolysis experiments using BMN microneedles; B, Results of in vitro hemolysis experiments using AI-MN@R8 microneedles.

[0028] Figure 9Results of BMN microneedle cytotoxicity assays. A, cytotoxicity results against HSF; B, cytotoxicity results against HaCaT.

[0029] Figure 10 Results of experiments on the cytotoxicity of different formulations to HSF cells.

[0030] Figure 11 Results of experiments on the cytotoxicity of different formulations to HaCaT cells.

[0031] Figure 12 Histological evaluation results of the skin barrier repair process after microneedle puncture.

[0032] Figure 13 TEM image of insulin-induced fibrinogen.

[0033] Figure 14 Macroscopic observation of an animal model of cutaneous amyloidosis.

[0034] Figure 15 Congo red staining results of histopathological sections from the modeling area of ​​an animal model of cutaneous amyloidosis.

[0035] Figure 16 H&E staining results of histopathological sections from the modeling area of ​​an animal model of cutaneous amyloidosis.

[0036] Figure 17 Results of weight changes in rats during treatment.

[0037] Figure 18 Results of skin recovery in rats during treatment.

[0038] Figure 19 Comparison of Congo red, H&E, and Masson staining results after treatment.

[0039] Figure 20 : Results of thiamine S staining after treatment.

[0040] Figure 21 Results of Ki67 and TUNEL staining after treatment.

[0041] Figure 22 H&E staining results for heart, liver, spleen, lungs, and kidneys. Detailed Implementation

[0042] To make the technical solution of the present invention easier to understand, the present invention will be further described below in conjunction with specific embodiments. It should be noted that the following embodiments are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0043] The specifications of the polydimethylsiloxane (PDMS) microneedle mold involved in the following examples are as follows: needle pitch 650μm, needle height 800μm, bottom size 320μm, microneedle patch size 17.5mm×17.5mm, and array number 385.

[0044] Example 1: Preparation of microneedles co-loaded with asiaticoside and near-infrared photothermal agent

[0045] S1: Hyaluronic acid (HA) and polyvinylpyrrolidone K90 (PVP K90) were mixed with pure water to prepare a mixed aqueous solution, wherein the mass fraction of HA was 20% and the mass fraction of PVP K90 was 10%. The mixed aqueous solution was allowed to swell at room temperature for 8 hours. Then, asiaticoside (AS), near-infrared photothermal agent IR1048 and octameric arginine (R8) were added sequentially and stirred until completely dissolved and mixed to obtain a needle tip matrix solution, wherein the mass fraction of AS was 0.5%, the mass fraction of IR1048 was 1%, and the mass fraction of R8 was 0.5%.

[0046] S2: Prepare a 10% aqueous solution of PVP K90 with pure water, add asiaticoside (AS), stir until completely dissolved and mixed to obtain a microneedle base solution, wherein the mass fraction of AS is 0.5%.

[0047] S3: Add the tip matrix solution to the PDMS microneedle mold and centrifuge at 4℃ and 4000rpm for 5 min to ensure the solution fully penetrates the mold cavity at the tip. Scrape off any excess solution from the mold surface and recycle it. To remove any residual air bubbles and make the tip matrix structure more compact, centrifuge again at 4℃ and 4000rpm for 5 min. Then, dry the mold at 40℃ for 0.5 h.

[0048] S4: Add the microneedle base solution to the dried mold until it completely covers the entire mold cavity. Centrifuge at 4℃ and 4000rpm for 3 minutes to ensure that the microneedle base solution uniformly fills the remaining mold cavity. Add more microneedle base solution to cover the mold surface. Then, dry the mold at 40℃ for 6 hours. After demolding, the microneedles co-loaded with asiaticoside and near-infrared photothermal agent are obtained, denoted as AI-MN@R8.

[0049] Dermoscopic characterization tests showed that the ratio of the tip height to the total height of the AI-MN@R8 microneedles was 0.24:1.

[0050] Comparative Example 1: Preparation of blank microneedles

[0051] S1: Hyaluronic acid (HA) and polyvinylpyrrolidone K90 (PVP K90) were mixed with pure water to prepare a mixed aqueous solution, wherein the mass fraction of HA was 20% and the mass fraction of PVP K90 was 10%. The mixed aqueous solution was swollen at room temperature for 8 hours to obtain the needle tip matrix solution.

[0052] S2: Prepare a 10% aqueous solution of PVP K90 with pure water, stir until completely dissolved and mixed to obtain a microneedle substrate solution.

[0053] S3: Add the tip matrix solution to the PDMS microneedle mold and centrifuge at 4℃ and 4000rpm for 5 min to ensure the solution fully penetrates the mold cavity at the tip. Scrape off any excess solution from the mold surface and recycle it. To remove any residual air bubbles and make the tip matrix structure more compact, centrifuge again at 4℃ and 4000rpm for 5 min. Then, dry the mold at 40℃ for 0.5 h.

[0054] S4: Add the microneedle base solution to the dried mold until it completely covers the entire mold cavity. Centrifuge at 4℃ and 4000rpm for 3 minutes to ensure that the microneedle base solution uniformly fills the remaining mold cavity. Add more microneedle base solution to cover the mold surface. Then dry the mold at 40℃ for 6 hours. After demolding, the blank microneedles are obtained and denoted as BMN.

[0055] Dermoscopic characterization tests showed that the ratio of the tip height to the total height of the BMN microneedles was 0.24:1.

[0056] Comparative Example 2: Preparation of microneedles loaded with asiaticoside

[0057] S1: Hyaluronic acid (HA) and polyvinylpyrrolidone K90 (PVP K90) were mixed with pure water to prepare a mixed aqueous solution, wherein the mass fraction of HA was 20% and the mass fraction of PVP K90 was 10%. The mixed aqueous solution was allowed to swell at room temperature for 8 hours, and then asiaticoside (AS) was added and stirred until completely dissolved and mixed to obtain a needle tip matrix solution, wherein the mass fraction of AS was 0.5%.

[0058] S2: Prepare a 10% aqueous solution of PVP K90 with pure water, add asiaticoside (AS), stir until completely dissolved and mixed to obtain a microneedle base solution, wherein the mass fraction of AS is 0.5%.

[0059] S3: Add the tip matrix solution to the PDMS microneedle mold and centrifuge at 4℃ and 4000rpm for 5 min to ensure the solution fully penetrates the mold cavity at the tip. Scrape off any excess solution from the mold surface and recycle it. To remove any residual air bubbles and make the tip matrix structure more compact, centrifuge again at 4℃ and 4000rpm for 5 min. Then, dry the mold at 40℃ for 0.5 h.

[0060] S4: Add the microneedle base solution to the dried mold until it completely covers the entire mold cavity. Centrifuge at 4℃ and 4000rpm for 3 minutes to ensure that the microneedle base solution uniformly fills the remaining mold cavity. Add more microneedle base solution to cover the mold surface. Then dry the mold at 40℃ for 6 hours. After demolding, the microneedles loaded with asiaticoside are obtained, denoted as AS-MN.

[0061] Dermoscopic characterization tests showed that the ratio of the tip height to the total height of the AS-MN microneedles was 0.24:1.

[0062] Comparative Example 3: Preparation of microneedles carrying a single near-infrared photothermal agent

[0063] S1: Hyaluronic acid (HA) and polyvinylpyrrolidone K90 (PVP K90) were mixed with pure water to prepare a mixed aqueous solution, wherein the mass fraction of HA was 20% and the mass fraction of PVP K90 was 10%. The mixed aqueous solution was allowed to swell at room temperature for 8 hours, and then near-infrared photothermal agent IR1048 was added and stirred until completely dissolved and mixed to obtain a needle tip matrix solution, wherein the mass fraction of IR1048 was 1%.

[0064] S2: Prepare a 10% aqueous solution of PVP K90 with pure water, stir until completely dissolved and mixed to obtain a microneedle substrate solution.

[0065] S3: Add the tip matrix solution to the PDMS microneedle mold and centrifuge at 4℃ and 4000rpm for 5 min to ensure the solution fully penetrates the mold cavity at the tip. Scrape off any excess solution from the mold surface and recycle it. To remove any residual air bubbles and make the tip matrix structure more compact, centrifuge again at 4℃ and 4000rpm for 5 min. Then, dry the mold at 40℃ for 0.5 h.

[0066] S4: Add the microneedle base solution to the dried mold until it completely covers the entire mold cavity. Centrifuge at 4℃ and 4000rpm for 3 minutes to ensure that the microneedle base solution uniformly fills the remaining mold cavity. Add more microneedle base solution to cover the mold surface. Then dry the mold at 40℃ for 6 hours. After demolding, the microneedles loaded with near-infrared photothermal agent are obtained, denoted as IR1048-MN.

[0067] Dermoscopic characterization tests showed that the ratio of the tip height to the total height of the IR1048-MN microneedles was 0.24:1.

[0068] Example 2: Physicochemical properties and in vitro performance characterization of microneedles

[0069] 2.1 Morphological Characterization

[0070] The surface morphology and microstructure of AI-MN@R8 and BMN microneedles were characterized using scanning electron microscopy.

[0071] The results are as follows Figure 1 As shown, both AI-MN@R8 and BMN microneedles have complete and undamaged arrays, with sharp and uniform needles, providing a good structural basis for efficient skin penetration. The sharp needle tips can penetrate the stratum corneum with less force, thus reducing skin damage and pain during the puncture process. The AI-MN@R8 microneedles have a smooth surface and dense internal structure, with no observed bubbles or drug precipitation; due to the color of the near-infrared photothermal agent IR1048, its needle tips appear black. Furthermore, the microstructure of AI-MN@R8 is highly similar to that of BMN, indicating that IR1048 and AS are uniformly dispersed in the microneedle matrix, which provides strong assurance for the mechanical strength of the microneedles.

[0072] 2.2 Mechanical strength test

[0073] The mechanical strength of individual AI-MN@R8 and BMN microneedles was tested using a texture analyzer.

[0074] The results are as follows Figure 2 As shown, the mechanical strength of a single BMN microneedle is 0.45±0.03N, and that of an AI-MN@R8 microneedle is 0.46±0.02N, with no significant difference between the two, indicating that the drug loading process did not significantly affect the mechanical strength of the microneedles. According to literature, the critical force required for microneedle penetration into the skin is approximately 0.058N / needle. The mechanical strength of both AI-MN@R8 and BMN microneedles far exceeds this threshold, fully meeting the requirements for transdermal drug delivery, ensuring structural integrity during puncture, and effectively penetrating the stratum corneum to reach the target site. Furthermore, the consistency in strength between AI-MN@R8 and BMN microneedles further confirms that IR1048 and AS are uniformly dispersed in the microneedle matrix, without compromising the structural integrity of the microneedles.

[0075] 2.3 Skin puncture performance test

[0076] SD rats were harvested, their back hair was shaved and removed, and the microneedling insertion area was demarcated. One BMN microneedle and one AI-MN@R8 microneedle were inserted into the rat's back skin and held for 10 minutes. Subsequently, the rats were euthanized by anesthesia with 10% urethane, and the back skin tissue was dissected. Samples were fixed in 4% paraformaldehyde, embedded, sectioned, and stained with hematoxylin and eosin (H&E) for histological comparative analysis.

[0077] The results are as follows Figure 3 As shown, both AI-MN@R8 and BMN microneedles successfully penetrated the dense stratum corneum and epidermis, leaving well-defined, smooth-edged puncture channels in the dermis. Measurements showed that the actual insertion depths of AI-MN@R8 and BMN microneedles were approximately 600 μm and 580 μm, respectively.

[0078] 2.4 Photothermal conversion performance test

[0079] 2.4.1 Characterization of photothermal properties

[0080] Free IR1048 and AI-MN@R8 microneedles were dissolved in pure water to prepare homogeneous solutions of 20, 30, 40, 50, and 60 µg / mL (based on IR1048), respectively, and placed in 1.5 mL EP tubes. 200 µL of each solution was added to a 96-well plate, with pure water as a blank control. A handheld near-infrared laser (1064 nm, 1 W / cm²) was used. 2 Irradiate for 10 minutes, and use an infrared thermal imager to record the solution temperature and thermal image in real time.

[0081] The results are as follows Figure 4 As shown, all solutions exhibited a heating trend upon laser irradiation, with a rapid temperature increase in the first 5 minutes followed by a gradual plateau. The highest temperature of the system showed a significant concentration dependence: after 10 minutes of irradiation, the temperature of the 60 µg / mL IR1048 solution rose from room temperature to approximately 64 °C; although pure water showed slight background endothermic reaction under the same laser conditions, its temperature rise was significantly lower than that of the experimental groups, fully verifying the excellent photothermal conversion capability of IR1048 itself. Further comparison revealed that after IR1048 was loaded onto microneedles, the temperature rise curve of the AI-MN@R8 microneedle solution at the same concentration was basically consistent with that of free IR1048, and the final temperature of the 60 µg / mL group was even slightly higher. These results indicate that loading IR1048 onto microneedles neither affected laser penetration nor weakened the photothermal conversion efficiency of IR1048.

[0082] 2.4.2 Photothermal stability investigation

[0083] AI-MN@R8 microneedles were dissolved in pure water to prepare a homogeneous solution of 30 µg / mL (based on IR1048) and placed in a 1.5 mL EP tube. 200 µL was added to a 96-well plate and analyzed using a handheld near-infrared laser (1064 nm, 1 W / cm²). 2 After irradiation for 10 minutes, the laser was turned off and allowed to cool for 10 minutes. This "laser on-off" cycle was repeated three times in the same hole. The temperature rise and fall curves and thermal images were recorded in real time using an infrared thermal imager.

[0084] The results are as follows Figure 5 As shown, each 1064nm laser irradiation lasting 10 minutes resulted in a rapid temperature increase; after the laser was turned off, the temperature gradually decreased to the initial baseline. After three complete heating and cooling cycles, the temperature change curves of each cycle highly overlapped, and no significant decrease in the system's maximum temperature was observed. This result fully demonstrates that IR1048 loaded in microneedles possesses good photothermal stability, and the microneedle matrix effectively protects the structural integrity of the photothermal agent, preventing its photodegradation under laser irradiation.

[0085] 2.4.3 Photothermal conversion efficiency

[0086] The photothermal conversion efficiency of free IR1048 and AI-MN@R8 microneedles was investigated using infrared thermal imaging. 30 µg / mL (based on IR1048) of IR1048 solution and 600 µL of AI-MN@R8 microneedle solution were respectively placed in 24-well plates, with an infrared thermal imager at 1064 nm and 1 W / cm². 2 Under continuous irradiation conditions, temperature changes were recorded in real time until the temperature reached a plateau. After the laser was turned off, the samples were allowed to cool naturally for 10 minutes, and the cooling curve was recorded again. Each group of samples was measured in triplicate. The photothermal conversion efficiency of each group was calculated based on the heating-cooling curves to evaluate the photothermal performance of IR1048 and AI-MN@R8 microneedles.

[0087] The results are as follows Figure 6 As shown. Both sets of cooling data show a good linear relationship, with the free IR1048 group showing R... 2 =0.99, AI-MN@R8 microneedle group R 2 =0.96, indicating that the data calculation is accurate and reliable. The calculation results show that the photothermal conversion efficiency of the free IR1048 solution is 29.53%; while after loading with AI-MN@R8, the photothermal conversion efficiency of IR1048 not only did not decrease, but actually increased to 33.58%. It is speculated that this is due to the HA / PVP K90 polymer network in the microneedles acting as an isolation agent, effectively inhibiting the aggregation of hydrophobic IR1048 molecules in the aqueous solution, thereby mitigating fluorescence quenching caused by aggregation. These results fully demonstrate that this microneedle system can effectively protect and further enhance the heat generation performance of photothermal materials.

[0088] 2.5 Investigation of In-Volume Dissolution

[0089] SD rats were used, and the hair on their backs was shaved. After the skin recovered for 24 hours, the microneedle insertion area was demarcated. AI-MN@R8 and BMN microneedles were inserted into the skin, and pressure was applied for 0, 1, 3, 5, 10, and 20 minutes before removal. The microneedles were then placed under an optical microscope to observe their dissolution state and changes in needle length.

[0090] The results are as follows Figure 7 As shown, after the microneedles come into contact with the subcutaneous tissue fluid, they exhibit extremely rapid water absorption and dissolution characteristics: within just 1 minute of insertion, the originally sharp needle tip begins to absorb water and become blunt; at 3-5 minutes, the needle height decreases significantly, and more than half of the needle structure has dissolved; by 10 minutes, the main body of the needle has basically disappeared, leaving only tiny protrusions on the base; at 20 minutes, the needle has completely dissolved and fused with the base.

[0091] Example 3: Biosafety Evaluation of Microneedles

[0092] 3.1 In vitro blood compatibility evaluation

[0093] Blood was collected from the marginal ear vein of New Zealand rabbits, and fresh whole blood was collected in centrifuge tubes with a heparin concentration of 10 IU / mL. The tubes were centrifuged at 4℃ and 4000 rpm for 10 min, and the supernatant was discarded. Ten volumes of physiological saline were added, and the mixture was thoroughly mixed and the supernatant was discarded. This process of resuscitation, centrifugation, and washing was repeated three times until the supernatant was clear and transparent. The supernatant was then discarded, yielding compacted red blood cells. A 2% (v / v) red blood cell suspension was prepared using PBS according to the red blood cell volume and stored at 4℃ for later use. Physiological saline was used as the negative control group, and Triton X-100 was used as the positive control group. AI-MN@R8 and BMN microneedles were prepared in PBS to create concentration gradients ranging from 1.25 to 20 mg / mL, and incubated at 37℃ for one day to serve as the solutions for each experimental group. After adding samples to each group, the mixture was thoroughly mixed and incubated at 37℃. After the reaction, each sample was centrifuged at 2500 rpm for 5 min. The absorbance of the supernatant was measured at a wavelength of 540 nm. The hemolysis rate was calculated based on the detection results, and a hemolysis rate greater than 5% was used as the criterion for determining the occurrence of hemolysis. The formula for calculating the hemolysis rate is as follows: Hemolysis rate (%) = (A experimental group - A negative control group) / (A positive control group - A negative control group) × 100%.

[0094] The results are as follows Figure 8As shown, combined with macroscopic observation and absorbance measurement results of centrifuge tubes, it can be seen that: in the positive control group, Triton X-100 completely ruptured the red blood cell membrane, releasing a large amount of hemoglobin, and the supernatant was bright red, with a hemolysis rate of 100%; in the negative control group, the red blood cells in the saline were intact and deposited at the bottom of the tube, and the supernatant was clear and transparent. Within the investigated concentration range of 1.25~20 mg / mL, the BMN group ( Figure 8 A) and AI-MN@R8 group ( Figure 8 B) The appearance of the test tubes at each concentration gradient was consistent with the negative control group. The supernatant was clear and colorless, and firm red blood cell sediment was visible at the bottom of the tubes. No macroscopic hemolysis was observed. Further quantitative calculations of absorbance at 540 nm showed that both BMN microneedles and AI-MN@R8 microneedles co-loaded with AS and IR1048 exhibited extremely low hemolysis rates at all concentrations, far below the internationally stipulated hemolysis safety threshold of 5%. This result confirms that the microneedle components do not cause osmotic pressure imbalance of the red blood cell membrane or damage to the lipid bilayer. The prepared co-loaded microneedles have excellent blood compatibility, providing reliable blood safety assurance for subsequent in vivo transdermal drug delivery.

[0095] 3.2 In vitro cytotoxicity evaluation

[0096] Human skin fibroblasts (HSF) and immortalized human keratinocytes (HaCaT) were cultured separately in DMEM complete medium and incubated at 37°C with 5% CO2. Cells in the logarithmic growth phase were harvested and cultured at a concentration of 1×10⁻⁶ cells / cells. 3 Cells were seeded at a density of 100 cells / well in 96-well plates and cultured until the cell density reached 80%. The original culture medium was then aspirated and replaced with diluted culture medium containing different test substances, and cultured for another 24 hours. 10 μL of CCK-8 solution was added to each well, and after incubation for 4 hours, the absorbance was measured at 450 nm. The laser irradiation group received a 1064 nm laser (1 W / cm²) 4 hours after drug administration. 2 Irradiate for 5 minutes. Cell viability is calculated using the following formula: Cell viability (%) = (A experimental group - A blank group) / (A control group - A blank group) × 100%.

[0097] The results are as follows Figure 9 As shown, under light-protected conditions, within a concentration range of 0.78–50 mg / mL, BMN microparticles effectively target HSF cells (…). Figure 9 A) and HaCaT cells ( Figure 9B) No significant inhibitory effect on cell proliferation was observed in either concentration group. Cell viability remained stable at approximately 100% in all concentration groups, with no statistically significant difference compared to the control group. Even at a high concentration of 50 mg / mL, the toxicity of BMN microparticles to both skin target cells was negligible. These results indicate that the HA / PVP K90 polymer network has good biosafety and does not produce toxic side effects on the proliferation and physiological metabolism of normal skin cells after dissolution, making it a safe and reliable transdermal delivery carrier.

[0098] like Figure 10 As shown in Figure A, under light-protected conditions, the survival rate of HSF cells remained above 80% within the tested concentration range for both free AS and AS-MN. Similarly, the AI-MN@R8 group did not show significant cell proliferation inhibition without laser intervention. However, after 1064nm laser irradiation, the cell viability of the AI-MN@R8+Laser group decreased in a concentration-dependent manner; when the AS concentration reached 200 μg / mL (corresponding to an IR1048 concentration of 30 μg / mL), the cell survival rate dropped to approximately 60%; at the highest administered concentration, the survival rate was only about 30%, demonstrating a significant cell-killing effect. Figure 10 B further verified the photothermal effect of IR1048: without laser irradiation, free IR1048 showed extremely low toxicity at all concentrations, with cell viability still approaching 100% at the highest concentration (39 μg / mL); however, cell viability significantly decreased in the IR1048+Laser group, confirming that free IR1048 can affect cell activity through efficient photothermal conversion. The photothermal effect of the IR1048-MN+Laser group was basically the same as that of the free group, indicating that the HA / PVP K90 polymer network did not weaken the photothermal performance of the photothermal agent.

[0099] like Figure 11 As shown in Figure A, under light-protected conditions, free AS, AS-MN, and AI-MN@R8 did not significantly inhibit HaCaT cell proliferation within the tested concentration range, and cell viability remained stable above 95%. After irradiation with a 1064nm laser, cell viability in the AI-MN@R8+Laser group showed a significant concentration-dependent effect: at lower concentrations (AS 50μg / mL, corresponding to IR1048 24μg / mL), cell viability dropped to approximately 75%; at the highest concentration, the viability was only about 20%, demonstrating a highly efficient in vitro photothermal effect. Figure 11 The results of B further clarified the contribution of the photothermal effect: free IR1048 and AI-MN@R8 had extremely low toxicity in the absence of laser irradiation; after the introduction of laser, the IR1048+Laser group, the IR1048-MN+Laser group, and the AI-MN@R8+Laser group all caused massive death of HaCaT cells.

[0100] Based on the combined in vitro experimental results of HSF and HaCaT, two core target cells of the skin, AI-MN@R8 exhibits extremely high biocompatibility against dermal and epidermal cells under normal physiological conditions. Under NIR-II region 1064nm laser excitation, the system can rapidly exert a photothermal effect, achieving highly efficient killing of local cells.

[0101] 3.3 Skin repair performance after puncture

[0102] SD rats were harvested, their back hair was shaved and removed, and microneedling areas were demarcated. BMN microneedles and AI-MN@R8 microneedles were inserted into the skin, and pressure was maintained for 10 minutes before removal. Rats were euthanized by anesthesia with 10% urethane at 0, 24, and 48 hours. Back skin tissue was dissected, and samples were fixed in 4% paraformaldehyde, embedded, sectioned, and stained with H&E for histological observation.

[0103] The results are as follows Figure 12 As shown in the figure, clear pinholes were visible in tissue sections from both the BMN and AI-MN@R8 treatment groups at time 0h. After microneedle removal, the skin initiated its own repair process: 24h after removal, the pinholes in both groups were largely closed, and no acute toxic reactions such as erythrocyte exudation, extensive inflammatory cell infiltration, or tissue necrosis were observed in the dermis; 48h after removal, the skin in the pinhole area had almost completely recovered, the epidermis was reconstructed into a complete layered structure, the stratum corneum re-covered the surface, and the collagen fibers in the dermis were tightly arranged. The overall tissue morphology was not significantly different from that of healthy skin without puncture, and no signs of scar hyperplasia were observed. These results indicate that the mechanical puncture of the skin by microneedles is transient and completely reversible.

[0104] Example 4: Evaluation of the in vivo therapeutic effect of micro-targeted therapy on cutaneous amyloidosis

[0105] 4.1 Establishment of experimental animal and cutaneous amyloidosis models

[0106] Male SD rats were used. Recombinant human insulin powder was prepared into a 1 mg / mL solution, and the pH was precisely adjusted to 2 with 0.1 mol / L NaOH. The solution was sealed and incubated in a 63°C water bath in the dark for 6 hours. The reaction was terminated at 4°C to obtain the insulin fibrillation inducer. 100 μL of the insulin fibrillation inducer was injected intradermally into the prepared skin area on the rat's back every other day, with the needle inserted at approximately a 15° angle. Successful injection was considered to result in the formation of a distinct, translucent wheal. The model was maintained for 45 consecutive days. During this period, the macroscopic morphology of the local skin was observed daily, and the system's safety was assessed by regular weighing.

[0107] 4.2 Experimental grouping and dosing regimen

[0108] After the model was established, the rats were randomly divided into 8 groups (n=5):

[0109] Normal control group: Healthy rats, raised in parallel;

[0110] Model control group: Model rats, with an equal volume of physiological saline injected into the skin lesion area;

[0111] Positive drug group: Model rats, with clobetasol propionate cream applied to the skin lesion area twice daily;

[0112] Blank microneedle group: Model rats were treated with BMN microneedles;

[0113] Single-drug microneedle group: Model rats were treated with AS-MN microneedles;

[0114] Photosensitive microneedle plus laser group: Model rats were given IR1048-MN microneedles and laser irradiation;

[0115] Drug-loaded microneedle group: Model rats were treated with AI-MN@R8 microneedles without laser irradiation;

[0116] Drug-loaded microneedles plus laser group: Model rats were given AI-MN@R8 microneedles and laser irradiation.

[0117] Each microneedling group received medication every two days. During the procedure, the affected area was cleaned with an alcohol swab. The microneedles were precisely aligned and pressed vertically with the thumb for 15 minutes to ensure penetration into the stratum corneum. Medical tape was then used to secure the microneedles, and the dressing was left on for 1 hour to allow the needle tip matrix to fully dissolve. For the photothermal therapy group, a 1064nm near-infrared laser (1W / cm²) was immediately used after the dressing was applied. 2 The treated area was vertically irradiated for 6 minutes. Throughout the treatment cycle, rat weight was measured every two days at fixed times, and a weight change curve was plotted. On days 0, 6, 12, 18, 24, 30, and 36 of treatment, the skin lesions were photographed and recorded. After treatment, rats were sacrificed, and tissue samples from the lesion area were fixed, embedded, sectioned, and stained with Congo red (CR), H&E, and Masson staining to observe histopathological changes. A highly specific Thioflavin S fluorescent probe was used for tissue staining and quantification. Heart, liver, spleen, lung, and kidney samples were collected for H&E staining to assess systemic toxicity. On day 7 of treatment, samples were collected for Ki67 immunohistochemical staining (to assess cell proliferation) and TUNEL staining (to detect apoptosis).

[0118] 4.3 Characterization of animal models of cutaneous amyloidosis

[0119] Figure 13 This is a TEM image of an insulin fibrillation inducer. The image shows dense, robust, and slender yet flexible mature amyloid fibers. Numerous individual fibers are intertwined and highly cross-linked, forming a three-dimensional network structure.

[0120] Figure 14Macroscopic observation of an animal model of cutaneous amyloidosis. The model presents typical chronic lesions. Acute generalized erythema is visible around the modeling area; well-defined, prominently raised localized plaques and solid nodules form on the surface of the area. The nodule surface is covered with brownish hyperkeratosis, accompanied by rough desquamation and pigmentation. The nodules are clustered, significantly raised, and the overall appearance is characteristic of lichenification.

[0121] Congo red staining combined with polarized light microscopy is the gold standard recognized in clinical pathology for identifying specific amyloid deposits. Under bright-field microscopy, amyloid plaques are specifically stained brick red or orange-red; while under polarized light excitation, due to their highly regular β-sheet spatial arrangement, the deposits exhibit the characteristic apple-green birefringent optical features. Figure 15 The results of Congo red staining of histopathological sections from the model area are shown. Numerous deeply stained, dense, homogeneous, amorphous brick-red clumps are visible within the papillary dermis of the model. Under polarized light microscopy, these red-stained areas exhibit strong apple-green birefringence, appearing as dense, coarse, and clustered fluorescent patches.

[0122] To further investigate the microscopic histological changes behind the macroscopic skin lesions, H&E staining was used to perform pathological morphological evaluation of skin sections from the rat model area. Figure 16 As shown, the skin tissue structure of healthy rats was intact, with loosely and orderly arranged collagen fiber bundles in the dermis, and no abnormal substance deposition or inflammatory cell infiltration was observed. In stark contrast, the model rats exhibited histopathological features of amyloidosis, with dense, brick-red homogeneous deposits in the dermis.

[0123] 4.4 Monitoring of rat body weight changes

[0124] The weight change curves of rats in each group during treatment are shown in the figure below. Figure 17 As shown, except for the positive control group where weight gain slowed in the mid-to-late stages, all other groups of rats maintained a steady natural growth trend. The delayed weight gain in the positive control group objectively reflects the systemic metabolic suppression that may be caused by long-term topical application of potent glucocorticoids. The steady weight gain in the other groups proves that repeated microneedle puncture and local photothermal therapy are mild and safe, and did not cause systemic toxic side effects.

[0125] 4.5 Observation of the macroscopic morphology of skin lesions

[0126] Macroscopic changes in skin lesions in rats of different groups during treatment are as follows: Figure 18As shown in the figure, the recovery of each group varied during the 36-day treatment period: In the model group, acute redness and swelling subsided and nodules softened after modeling was stopped, but rough scabs and pigmentation remained until the end of the observation period, which may be related to the strong tissue regeneration and heterologous protein metabolism capacity of rodents; the positive control group recovered relatively quickly in the early stage due to the anti-inflammatory effect of hormones, but tended to stagnate in the middle and late stages. In contrast, the AI-MN@R8+Laser group showed the fastest nodule and keratin exfoliation speed under the synergistic effect of drug delivery and photothermal action, and ultimately achieved a high degree of physiological recovery in skin color, smoothness, and hair follicle structure.

[0127] 4.6 Histopathological analysis of skin lesions

[0128] Multiple recombinant staining results of skin lesions in rats of each group after treatment are as follows: Figure 19 As shown in the figure, in the normal control group, the collagen fibers in the dermis were coarse and loosely arranged; in the model group, obvious brick-red amyloid lesions were visible under CR staining, and dense eosinophilic masses were observed in H&E staining. Masson staining showed that these masses crowded out the space of normal collagen, causing the blue collagen fibers to break and atrophy. In the single intervention groups (BMN, AS-MN, IR1048-MN+Laser), there were still varying degrees of residual eosinophilic masses in the dermis, and collagen regeneration was poor. However, AI-MN@R8+Laser showed a significant therapeutic effect: CR and H&E staining showed that the dense amyloid masses were significantly degraded, but the dermal stroma showed a loose structure in the repair phase, indicating that the tissue was in the late repair stage; Masson staining clearly showed a large number of newly formed blue collagen fibers, which were thinner and looser than normal collagen bundles, but this indicated that the dermal microenvironment was actively recovering towards physiological remodeling, which provided a solid histological basis for the complete cure of cutaneous amyloidosis.

[0129] 4.7 Evaluation of the effect of amyloid protein clearance in skin lesions

[0130] Highly sensitive Thioflavin S fluorescence staining further revealed the ability of each group to clear amyloid protein, as shown in the results. Figure 20 As shown, large areas of strong green fluorescent clumps remained in the dermis in both the model group and the BMN group, confirming that relying on autologous metabolism or simple physical puncture cannot fundamentally break down stubborn deep amyloid protein. The fluorescence signal in the positive control group was significantly weakened, confirming the strong inhibitory effect of the hormone; although the fluorescence in the AS-MN group and the IR1048-MN+Laser group showed convergence, significant fragments remained, suggesting that single treatment has limited efficacy. In contrast, the fluorescence signal in the AI-MN@R8+Laser group was almost completely quenched, and the quantitative fluorescence value showed no significant difference from the normal control group. This conclusively proves that, through targeted delivery and photothermal synergy, this system successfully decomposed the rigid spatial conformation of the amyloid β-sheet, fundamentally eliminating the pathogenic protein.

[0131] 4.8 Analysis of cell proliferation and apoptosis in skin lesion tissue

[0132] The Ki67 immunohistochemical staining results of the skin lesions of rats in each group on day 7 of treatment are as follows: Figure 21 As shown, the model group showed a large number of positive expressions in the epidermis and dermis, indicating significant abnormal cell proliferation; Ki67 expression remained at a high level in each single intervention group; while on day 7 of AI-MN@R8+Laser combined treatment, Ki67 positive cells were extremely rare, indicating that it can effectively block the abnormal proliferation process in the lesion site.

[0133] The corresponding TUNEL apoptosis staining results are as follows: Figure 21 As shown, the AI-MN@R8+Laser group had a clean background with no obvious apoptosis signal, while the model group showed diffuse strong positivity due to persistent inflammation, suggesting that the therapy cleared the lesions without causing a large number of apoptosis of surrounding healthy cells.

[0134] 4.9 Systemic Safety Evaluation

[0135] The H&E staining results of the major organs of rats in each group after treatment are as follows: Figure 22 As shown, the tissue morphology of the heart, liver, spleen, lungs, and kidneys in all intervention groups, including AI-MN@R8+Laser, was basically consistent with that of the normal control group, with no pathological damage such as cell degeneration, necrosis, congestion, or edema observed. This confirms that the microneedle combined therapy system exerts an effective local effect while possessing excellent in vivo biocompatibility and extremely high systemic safety.

[0136] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A microneedle co-loaded with asiaticoside and a near-infrared photothermal agent, characterized in that: The microneedle comprises a tip and a substrate; the tip uses hyaluronic acid and polyvinylpyrrolidone K90 as the backbone matrix and is simultaneously loaded with asiaticoside, a near-infrared photothermal agent and octameric arginine; the substrate uses polyvinylpyrrolidone K90 as the backbone matrix and is loaded with asiaticoside.

2. The microneedle according to claim 1, characterized in that: The near-infrared photothermal agent is IR1048.

3. A method for preparing microneedles as described in any one of claims 1 to 2, characterized in that: Includes the following steps: (1) Hyaluronic acid and polyvinylpyrrolidone K90 were added to pure water to prepare a mixed aqueous solution. After swelling at room temperature, asiaticoside, near-infrared photothermal agent and octameric arginine were added and stirred until completely dissolved and mixed to obtain a needle tip matrix solution. (2) Prepare an aqueous solution of polyvinylpyrrolidone K90 with pure water, add asiaticoside, stir until completely dissolved and mixed, and prepare a microneedle base solution. (3) Add the needle tip matrix solution into the PDMS microneedle mold and centrifuge to allow the needle tip matrix solution to fully enter the mold cavity at the tip of the mold; Scrape off excess needle-tip matrix solution from the mold surface, centrifuge to remove residual air bubbles in the mold cavity and make the needle-tip matrix structure in the mold cavity more compact; The mold is then dried to allow the needle tip matrix to initially solidify; (4) Add microneedle base solution to the dried mold until it completely covers all the mold cavities, centrifuge to make the microneedle base solution evenly fill the remaining mold cavities; add more microneedle base solution to cover the mold surface, then dry the mold, and after demolding, you will get microneedles co-loaded with asiaticoside and near-infrared photothermal agent.

4. The preparation method according to claim 3, characterized in that: In step (1), the mass fraction of hyaluronic acid in the mixed aqueous solution is 20%, and the mass fraction of polyvinylpyrrolidone K90 is 10%; the mass fraction of asiaticoside in the needle tip matrix solution is 0.5%, the mass fraction of near-infrared photothermal agent is 1%, and the mass fraction of octameric arginine is 0.5%.

5. The preparation method according to claim 3, characterized in that: In step (2), the mass fraction of polyvinylpyrrolidone K90 in the aqueous solution is 10%; the mass fraction of asiaticoside in the microneedle base solution is 0.5%.

6. The preparation method according to claim 3, characterized in that: In step (3), the centrifugation conditions are 4°C, 4000 rpm, and 5 min; the drying conditions are 40°C and 0.5 h.

7. The preparation method according to claim 3, characterized in that: In step (4), the centrifugation conditions are 4°C, 4000 rpm, and 3 min; the drying conditions are 40°C and 6 h.

8. The use of the microneedles as described in any one of claims 1 to 2 in the preparation of a medicament for treating cutaneous amyloidosis.

9. The use of the microneedles as described in any one of claims 1 to 2 in the preparation of drugs for inhibiting HSF cells and HaCaT cells.

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