Near-infrared response type microneedle as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medical microneedles, and particularly relates to a near-infrared response type microneedle as well as a preparation method and application thereof. The construction method of the microneedle comprises the following steps: firstly, jointly loading a medicine and the carbon quantum dots on the ZIF-8 nanoparticles to form a composite material; then, the material is entrapped in hydrogel with methacrylic acid silk fibroin as a matrix, and the near-infrared response type microneedle is prepared. Under the irradiation of near-infrared light, the carbon quantum dots convert light energy into heat energy to promote drug release. Precise drug delivery under regulation and control of near-infrared light is realized, and the near-infrared light-controlled drug delivery system has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of medical microneedle technology, specifically relating to a near-infrared responsive microneedle, its preparation method, and its application. Background Technology

[0002] Osteoarthritis (OA) affects over 595 million people worldwide, primarily manifesting as chronic pain and degenerative joint disease. Current treatments range from nonsteroidal anti-inflammatory drugs (NSAIDs) to intra-articular injections, but limitations such as short duration of efficacy, numerous systemic adverse reactions, and significant invasiveness remain common. One of the key pathological features of OA is abnormal osteochondral interface and neurovascular invasion, accompanied by extracellular matrix (ECM) metabolic disorders, leading to structural damage and increased pain sensitivity.

[0003] Cryptochlorogenic acid (CCA), a natural polyphenol extracted from Japanese honeysuckle, possesses potent anti-inflammatory, antioxidant, and ferroptosis-inhibiting properties, making it a potential candidate drug for treating osteoarthritis (OA). However, its clinical application is limited by low bioavailability and insufficient targeted delivery technology, thus affecting its efficacy. Therefore, developing formulations capable of efficiently loading CCA and achieving precise delivery is of great significance for delaying OA progression and alleviating pain. Existing carriers, such as metal-organic frameworks (e.g., ZIF-8), while exhibiting good sustained-release properties, struggle to achieve on-demand, responsive drug release, remaining insufficient in terms of therapeutic controllability. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a near-infrared responsive microneedle, its preparation method, and its applications. It achieves near-infrared response, accelerates drug release on ZIF-8, and enables precise spatiotemporal control.

[0005] The first aspect of this invention provides a near-infrared responsive microneedle, which is prepared by mixing a drug and carbon quantum dots with ZIF-8 nanoparticles in an equal mass ratio and loading the mixture onto the ZIF-8 nanoparticles to obtain a composite material; the composite material is then embedded in a hydrogel based on methacrylated silk fibroin; the methacrylated silk fibroin has the characteristics of controllable sustained-release performance, excellent thermal stability, and high mechanical strength, and is suitable for long-term implantation and use as a sustained-release carrier; Under near-infrared irradiation, the carbon quantum dots of the near-infrared responsive microneedles absorb light and convert it into heat energy, causing the ZIF-8 nanoparticles to disintegrate and release the drug; the mass ratio of the carbon quantum dots to the ligands of the ZIF-8 nanoparticles is 10mg~11mg:330mg.

[0006] In another preferred embodiment, the drug is cryptochloronic acid.

[0007] In another preferred embodiment, the mass ratio of the composite material to the methacrylated silk fibroin hydrogel microneedles is 7.5~8:120.

[0008] A second aspect of the present invention provides a method for preparing the near-infrared responsive microneedles, characterized by comprising the following steps: Cryptochlorogenic acid, carbon quantum dots and ZIF-8 nanoparticles were mixed in an organic environment and reacted with zinc acetate solution to obtain a precipitate. After filtration and centrifugation, a composite material was obtained. The composite material, methacrylated silk fibroin, and photoinitiator were mixed in anhydrous sodium hydroxide, ultrasonically degassed, injected into a mold, and cured to obtain the near-infrared responsive microneedles.

[0009] In another preferred embodiment, the photoinitiator is (lithium phenyl(2,4,6-trimethylbenzoyl)phosphate).

[0010] In another preferred embodiment, the reagent used in the organic environment is methanol.

[0011] In another preferred embodiment, the curing refers to ultraviolet curing at a wavelength of 365 nm.

[0012] The third aspect of this invention provides the application of the near-infrared responsive microneedles in the treatment of osteoarthritis.

[0013] Compared with the prior art, the present invention has the following beneficial effects: This invention utilizes the efficient photothermal conversion capability of carbon quantum dots to promote drug release on ZIF-8, achieving precise spatiotemporal drug release; it constructs a three-in-one platform of "drug-photothermal-microneedle": organically combining the natural active ingredient cryptochlorogenic acid, the photothermal conversion agent carbon quantum dots, and minimally invasive microneedle technology, improving patient compliance while ensuring efficacy; focusing on osteoarthritis, it not only relieves pain but also intervenes in the disease process by inhibiting MMP3 / 9 / 13 expression, blocking abnormal neurovascular invasion, and protecting cartilage integrity; it has outstanding safety and translational potential: the components used, methacrylamide silk fibroin, carbon quantum dots, cryptochlorogenic acid, and ZIF-8, all have good biocompatibility, avoiding the systemic toxicity of traditional NSAIDs and the invasive risks of injection therapy; it achieves synergistic effects of chemotherapy and physical therapy: NIR irradiation not only triggers drug release, but its local thermal effect can also improve microcirculation, relieve muscle spasms, and enhance the overall therapeutic effect. Attached Figure Description

[0014] Figure 1 Figure 1 shows the microneedle preparation process and morphological characterization results.

[0015] Figure 2The images show the structural characterization of ZIF-8-CCA-CQDs, where a is a SEM image; b is the particle size distribution of ZIF-8; c is the particle size distribution of ZIF-8-CCA-CQDs; d is the infrared spectrum; and e is the X-ray diffraction pattern.

[0016] Figure 3 Figure 1 shows the performance analysis results of the hydrogel microparticles. Figure 2 shows the calibration curve of CCA absorbance at 365 nm relative to concentration; Figure 3 shows the curve of cumulative CCA release from ZIF-8-CCA-CQDs in vitro under physiological conditions over 72 hours; Figure 4 shows the thermal stability analysis of the hydrogel; Figure 5 shows the changes in the hydrogel under UV irradiation; Figure 6 shows the photothermal conversion effect of the hydrogel under NIR irradiation; and Figure 7 shows the results of different power densities (0.5-2.0 W·cm⁻¹). -2 The graph shows the temperature change over time at different ZIF-8-CCA-CQDs masses (2.5-10 mg); the graph shows the temperature change over time at different ZIF-8-CCA-CQDs masses (2.5-10 mg); the graph shows the time-dependent storage (G', black) and loss (G'', red) modulus analysis during gelation; and the graph shows the storage modulus (G′, black) and loss modulus (G″, red) analysis results over time during gelation.

[0017] Figure 4 Figure 1 shows the mechanical property analysis results of SFZICC MNs. Figure 2 shows the SEM image of the hydrogel microneedle patch. Figure 3 shows the SEM images of multiple regions (regions 1-8) on the surface, with corresponding energy-dispersive X-ray spectroscopy (EDS) elemental distribution maps showing the distribution of carbon (C), nitrogen (N), oxygen (O), and zinc (Zn). Figure 4 shows the complete array structure and conical tip morphology of the microneedles in SEM images, with multiple magnifications and scale bars of 500 μm (left), 100 μm (middle), and 200 μm (right). Figure 5 shows the morphological observation of microneedles loaded with Rhodamine B after penetrating mouse skin. Figure 6 shows the fluorescence imaging of condylar sections after applying microneedles under control and 808 nm near-infrared radiation conditions, with a scale bar of 30 μm.

[0018] Figure 5 The mechanical and biocompatibility analysis results of SFZICC MNs are shown in the following figures: a) a typical stress-strain curve of the hydrogel; b) infrared thermography of the hydrogel under laser irradiation; c) in vitro degradation curve of the hydrogel in phosphate-buffered saline (PBS) at 37°C; d) longitudinal in vivo fluorescence imaging after subcutaneous implantation of the hydrogel in mice; and e) hematoxylin-eosin (H&E) staining of tissue sections at the implantation site.

[0019] Figure 6Figure 1 shows the results of the biocompatibility assessment experiment; a) shows the results of the cell experiment, scale bar = 200 μm; b) shows the results of H&E staining of heart, liver, spleen, lung and kidney tissues of mice treated with SFZICC microneedles and control group, no significant histological abnormalities or inflammatory reactions were found in any organ, scale bar = 200 μm; c) shows the visual assessment results of hemoglobin release in the supernatant after incubation with red blood cells; d) shows the results of quantitative hemolysis rate, showing that hemolysis of ZIF-8, ZIF-8-CCA-CQDs, SilMA and SFZICC was negligible (<5%) (p>0.05 compared with control group), while Triton X-100 induced complete hemolysis (p<0.0001 compared with all groups).

[0020] Figure 7 The results of photothermal responsive microneedle patches in relieving pain and related negative emotions are shown in the following figures: a) Von Frey test results; b) Schematic diagram of mouse EEG recordings; c) Activity trajectory of mice in the elevated cross maze experiment; d) Activity trajectory of mice in the open field experiment; e) Comparison of EEG spectra after soft brush stimulation; f and g) Quantitative results of the elevated cross maze experiment, where f is the dwell time in the open arm and g is the number of times the open arm is entered; h and i) Quantitative results of the open field experiment, where h is the total movement distance and i is the movement time.

[0021] Figure 8 The images show the results of how photothermal-responsive microneedle patches improve the pathological structural changes in temporomandibular joint osteoarthritis (TMJ-OA). Images a-e show the results of H&E staining, Safranin O-Fix Green staining, scanning electron microscopy (SEM), silver glycine staining, and a representative stereomicroscopic image of the condylar tissue, respectively. The scale bar lengths are 100 μm for a-d and 200 μm for e. Image f shows the OARSI score. Image g shows the semi-quantitative analysis results of image a. Image h shows the semi-quantitative analysis results of image b.

[0022] Figure 9 The experimental results are shown in the target screening results. a is a representative image of immunofluorescence staining of condylar neurovascular markers (CD31 and VEGF) and (PGP9.5 and CGRP), with a scale bar of 30 micrometers. b to e are the semi-statistical analysis results of the images in a (n=3 per group; Kruskal-Wallis test was used, and Dunn test was used for post-hoc multiple comparisons).

[0023] Figure 10The results of the in vitro analysis of NIR-assisted SFZICC MNs therapy inhibiting neurovascularization are shown in the figures; a-c are representative images of EPC cells, a is a representative image of wound healing, b is a representative image of tube formation, and c is a representative image of crystal violet staining; d is the result of β3-tubulin immunofluorescence staining (green); e is the result of crystal violet staining after treatment with Control, Veh, SFZICC+NIR, CXB, NIR, or SFZICC; f is the result of cell migration distance; g is the result of the number of migrating cells; h is the result of the dendrite length. Each group has n=3. The Kruskal-Wallis test was used, and the Dunn test was used for post-hoc multiple comparisons.

[0024] Figure 11 The diagrams show the roles of CCA in the potential mechanisms of TMJ-OA. a) is a schematic diagram of the screening of CCA's mechanisms of action in TMJ-OA; b) is a network of OA disease targets and a screening network of core targets; c) shows the screening of CCA targets using four complementary databases: Swiss Target Prediction, SEA, Galaxy Dock, and COMET; d) is a diagram showing the analysis of potential CCA targets in TMJ-OA identified through reverse target screening; e) is a volcano plot of differentially expressed genes in TMJ-OA; f) is a heatmap of OA-related DEGs; g~i are GO analysis diagrams of nine core targets of the OA-targeting CCA pathway, where g represents biological processes (BP); h represents molecular functions (MF); and i represents cellular components (CC).

[0025] Figure 12 The diagrams show the results of molecular docking and molecular dynamics simulations of the binding interactions between CCA and MMPs. a) Molecular docking diagram of MMP3 and CCA; b) Molecular docking diagram of MMP9 and CCA; c) Molecular docking diagram of MMP13 and CCA; d) Root mean square deviation (RMSD) results of CCA with MMP3, MMP9, and MMP13 in a 100-nanosecond molecular dynamics simulation; e) Radius of gyration (Rg) results of MMP3, MMP9, and MMP13 in a 100-nanosecond molecular dynamics simulation; f) Number of hydrogen bonds formed between CCA atoms and MMP3, MMP9, and MMP13 in a 100-nanosecond molecular dynamics simulation; h) Free energy landscape (FELs) diagram of MMP3 obtained from principal component analysis (PCA) of molecular dynamics simulations; i) Free energy landscape diagram of MMP9 based on PCA; j) Free energy landscape diagram of MMP13 based on PCA.

[0026] Figure 13The diagrams show the binding interactions between CCA and MMPs, analyzed through molecular docking and molecular dynamics simulations. a) is the RMSF map, showing the local conformational flexibility of each protein in specific regions, with unique peak patterns corresponding to cyclic or hinge domains. b) is the residue-level decomposition results of the binding free energy of the MMP3-CCA, MMP9-CCA, and MMP13-CCA complexes. The decomposition analysis based on MM-PBSA reveals the contribution of individual residues to the overall binding affinity.

[0027] Figure 14 Figure (a) shows the results of MMPs expression inhibition in a photothermal microneedle patch model of osteoarthritis. Figure (a) shows a representative image of condyle immunofluorescence staining using MMP3, MMP9 and MMP13 markers, scale bar = 30 μm. Figures (b) to (d) show the semi-quantitative analysis of the immunofluorescence images in Figure (a) (n=3 per group; one-way ANOVA and Tukey post-hoc test). Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0029] The core pathological mechanism of pain and joint degeneration is abnormal neurovascular invasion at the osteochondral interface and dysregulation of extracellular matrix (ECM) metabolism. During the progression of osteoarthritis (OA), ECM degradation is accompanied by elevated levels of inflammatory cytokines, tissue hypertrophy, and angiogenesis. In subchondral bone, accelerated bone turnover is accompanied by vascular invasion, with vessels extending from the subchondral bone, crossing the tide line, and ultimately entering the articular cartilage. Cryptochloroic acid (CCA), a naturally occurring polyphenol found in honeysuckle, possesses significant anti-inflammatory, antioxidant, and ferroptosis-inhibiting properties, which have been well-established to be closely related to pain relief. Therefore, CCA-loaded drugs may hold significant potential for delaying OA progression and alleviating pain. However, the optimal delivery strategy for these drugs requires further investigation to improve targeting efficiency, prolong retention time, and achieve controlled release within the joint.

[0030] Microneedles (MNs) are a minimally invasive transdermal drug delivery technology with broad application prospects in the treatment of osteoarthritis (OA). Unlike traditional patches, microneedles can penetrate the stratum corneum to form micron-sized channels, enabling precise delivery of therapeutic drugs through the microcirculation system, achieving minimally invasive and painless pharmacological effects. This technology has been widely used in diabetes management, vaccine delivery, cancer treatment, alopecia, dermatology, and anesthesia. Among various microneedle technologies, hydrogel-based microneedles (HMNs) have become a focus of scientific research due to their high drug loading capacity, excellent biocompatibility, and simple preparation process. Therefore, hydrogel-based microneedles are considered an ideal carrier material for localized osteoarthritis drug delivery. However, their drug release behavior is relatively passive and lacks precise controlled release capabilities, requiring further optimization of material properties.

[0031] Carbon quantum dots (CQDs) and ZIF-8 are used to achieve precise controlled-release and long-acting sustained-release drug delivery, respectively. As zero-dimensional carbon-based nanomaterials, carbon quantum dots possess both excellent optical properties and good biocompatibility. These materials are particularly suitable for photothermal therapy due to their absorption characteristics in the near-infrared region. ZIF-8 is a biocompatible nanoporous metal-organic framework (MOF) that is widely used as a drug carrier due to its high drug loading capacity.

[0032] Currently, ZIF-8 nanoparticles loaded onto hydrogel microneedles are used for drug delivery. However, this relies solely on the sustained release of ZIF-8, representing a single drug delivery method. Furthermore, it cannot achieve on-demand release or response to NIR light. Additionally, the hydrogel substrate uses methacrylamide gelatin, which has poor stability and mechanical properties, making it unsuitable for osteoarthritis applications. Existing technologies also utilize ZIF-8-loaded ciprofloxacin, coated with polylactic-co-glycolic acid copolymer (PLGA), and doped with Fe3O4 nanoparticles to create an NIR-activated drug delivery system. However, this system relies on Fe3O4 to respond to NIR light, resulting in a low response rate.

[0033] Based on this, the present invention integrates ZIF-8 material embedded with carbon quantum dots (CQDs) into methacrylamide silk fibroin, enabling precise controlled release through photothermal triggering of ZIF-8 dissociation under NIR light irradiation. This precisely meets the need for stimulus-responsive therapy in osteoarthritis treatment, minimizing systemic side effects while significantly improving therapeutic efficacy.

[0034] This invention employs a temporomandibular joint (TMJ) osteoarthritis model induced by unilateral anterior crossed occlusion. Given that the TMJ is one of the most complex joints in the human body and prone to osteoarthritis, the TMJ-OA model has become a representative clinical model for osteoarthritis research. This invention develops a NIR photothermal responsive hydrogel microneedle, abbreviated as SFZICC microneedle, for targeted therapy of TMJ-OA. This system is constructed by loading CCA and CQDs separately onto ZIF-8 nanoparticles, which are then integrated into a methacrylated silk fibroin hydrogel matrix. Under NIR light irradiation, this minimally invasive microneedle patch not only achieves spatiotemporally controlled drug release through photothermal effects, effectively delivering active ingredients, but also provides physical therapy. This invention systematically evaluates the analgesic effect of the SFZICC microneedle patch in a validated osteoarthritis model, elucidating its mechanism of action and pathological neurovascularization process, thereby providing a new paradigm for osteoarthritis treatment.

[0035] The following is a detailed description of a near-infrared responsive microneedle, its preparation method, and its applications.

[0036] SilMA reagents were purchased from EFL Corporation, Suzhou, China. Quantum dots (CQDs) were provided by XFNANO Corporation, Nanjing, China. Cryptochlorophyllin (CCA) reagents were purchased from Targeted Molecules, Inc., USA. 2-Imidazolinone (2-MIN) and lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) reagents were purchased from Sigma-Aldrich, Inc., USA. Zinc acetate dihydrate was purchased from Sigma-Aldrich, Inc., St. Louis, Missouri, USA. Calcein / PI cell viability / cytotoxicity assay kits were purchased from Beyotime Biotechnology, Inc., Shanghai, China. Durbeco Modified Eagle Medium (DMEM) was purchased from Heklon, Inc., Logan, Utah, USA, and neural basal medium was purchased from Ingenium, Inc., Waltham, Massachusetts, USA.

[0037] 1. Synthesis of ZIF-8 and ZIF-8-CCA-CQDs ZIF-8 nanoparticles were prepared using a one-pot synthesis method. Specifically, 330 mg of 2-MIN was dissolved in 10 mL of methanol and sonicated for 5 min to obtain a 2-MIN solution. 150 mg of zinc acetate dihydrate was dissolved in 5 mL of deionized water and sonicated for 5 min to obtain a zinc acetate solution. Subsequently, the zinc acetate solution was added dropwise to the 2-MIN solution under magnetic stirring to obtain a mixture. The mixture was reacted at room temperature (37°C) for 15 min, and the white precipitate was collected by centrifugation at 10,000 rpm for 10 min. The precipitate was washed three times with 2 mL of methanol to remove impurities, yielding ZIF-8.

[0038] ZIF-8-CCA-CQDs were prepared using a one-pot synthesis method. The specific steps were as follows: 10 mg of CCA, 10 mg of CQDs, and 330 mg of 2-MIN were dissolved in 10 mL of methanol. After ultrasonic treatment for 5 min, a 2-MIN solution containing CCA was obtained. 150 mL of zinc acetate dihydrate was dissolved in 5 mL of deionized water. After ultrasonic treatment for 5 min, a zinc acetate solution was obtained. Subsequently, under magnetic stirring, the zinc acetate solution was added dropwise to the 2-MIN solution to react and obtain a mixture. The mixture was reacted at room temperature (23°C) for 15 min, and the white precipitate was collected by centrifugation at 10,000 rpm for 10 min. The precipitate was washed three times with 2 mL of methanol to remove impurities, yielding ZIF-8-CCA-CQDs. The specific preparation process is as follows: Figure 1 As shown.

[0039] The ZIF-8 and ZIF-8-CCA-CQDs prepared above were subjected to freeze-drying to obtain powders with good flowability, which were used for subsequent experiments.

[0040] 2. Fabrication of microneedle arrays The microneedle array was prepared using a polydimethylsiloxane (PDMS) mold. 7.5 mg of ZIF-8-CCA-CQDs, 120 mg of SilMA, and 0.25% LAP (by mass) were added to ddH₂O. After sonication for 10 min, the mixture was degassed under vacuum and pre-concentrated at 37°C for 12 hours. The resulting solution was then injected into the PDMS mold and cured using ultraviolet light at 365 nm for 10 min. Finally, the PDMS mold was carefully removed using sterile forceps to obtain the microneedle patch, denoted as MN.

[0041] 3. Performance Characterization 3.1 Morphological characterization of MN To evaluate the skin implantation effect and drug release performance of MN, MN loaded with Rhodamine B was implanted into excised porcine skin tissue, and morphological changes were monitored in real time using an optical microscope.

[0042] Figure 2 As shown in Figures a through c, the ZIF-8 particles exhibit a regular dodecahedral morphology with a relatively uniform particle size (approximately 160 nm). After loading CCA and CQDs, the particle size increases to approximately 205 nm, indicating successful loading. Figure 2 The middle d shows 1747cm -1 and 1694cm -1 The absorption peaks at 753 cm⁻¹ are attributed to the C=O stretching vibrations of the ester and carboxylic acid groups in the CCA molecule, respectively. -1 and 688cm -1 The peak at 2868 cm⁻¹ corresponds to the out-of-plane and in-plane bending vibrations of the imidazole ring in ZIF-8, while the peak at 2868 cm⁻¹ corresponds to the out-of-plane and in-plane bending vibrations of the imidazole ring in ZIF-8. -1With 2961cm -1 The peak at this point originates from the symmetric and asymmetric CH stretching vibrations of the methyl group (-CH3) in CQDs. This confirms that CCA and CQDs are successfully embedded in the ZIF-8 framework. Figure 2 As shown in e, X-ray diffraction confirmed the preservation of the ZIF-8 crystal framework.

[0043] Calibration curves for CCA were established using UV-Vis spectroscopy, revealing the sustained release of ZIF-8-CCA-CQDs, with over 85 μg·mL⁻¹ released within 72 hours. - ¹The CCA of ZIF-8-CCA-CQDs composite material demonstrates its effective sustained-release capability, such as Figure 3 As shown in a~b in the figures. Thermogravimetric analysis (TGA) revealed that ZIF-8 and ZIF-8-CCA-CQDs underwent significant thermal degradation upon heating. ZIF-8 remained stable up to 542 °C, followed by rapid mass loss (approximately 50% residue at 800 °C), while ZIF-8-CCA-CQDs began to degrade at 516 °C, with even less residue at 800 °C. The decreased stability in ZIF-8-CCA-CQDs is attributed to the decomposition and volatilization of CCA and CQDs. Their high stability between 200 and 500 °C confirms the preservation of the integrity of the ZIF-8 host framework, indicating that both materials possess suitable thermal stability. Figure 3 (c) Upon exposure to 405 nm UV light, the photoinitiator LAP is activated and generates free radicals. These free radicals subsequently initiate covalent cross-linking between the methacryloyl groups of SilMA, transforming the liquid precursor into a hydrogel with a three-dimensional network structure. Figure 3 (d) Under different 808 nm NIR irradiations, ZIF-8-CCA-CQDs exhibited a power-dependent photothermal conversion effect at 1.0 W·cm⁻¹. - At an irradiation power density of ², the temperature rapidly rises to over 40°C within 10 minutes. Figure 3 (e, f) in 1.0 W·cm - Under NIR irradiation, the sample containing 7.5 mg ZIF-8-CCA-CQDs exhibited a highly efficient photothermal response, reaching 40 °C within 10 min. Figure 3 (g in the sample). 7.5 mg ZIF-8-CCA-CQDs sample, administered at 1.0 W / cm² per cycle. - ²NIR irradiation for 10 min showed excellent stability during five on / off cycles, confirming its strong photothermal durability. Figure 3 In the figure, h), G' is black and G'' is red. Modulus analysis shows that a rapid sol-gel transition occurs at ~6 minutes. The cross-linking of G' and G'' confirms the effective in-situ cross-linking and formation of a stable hydrogel network. Figure 3 (i in the text).

[0044] A SilMA precursor solution loaded with uniformly dispersed ZIF-8-CCA-CQDs was crosslinked under 405 nm UV light to produce a robust SFZICC composite hydrogel. SEM confirmed the uniform dispersion of the nanocomposite within the hydrogel matrix. Rheological analysis showed a rapid transition from a fluid to a solid state upon stimulation. The storage modulus (G′) remained low at approximately 0.1 Pa for the first 5 minutes, characteristic of the sol-like state. Subsequently, a distinct crossover between the storage modulus (G′) and loss modulus (G″) was observed, a hallmark of gelation and the initiation of structural remodeling.

[0045] 3.2 Mechanical integrity and biocompatibility of SFZICC MNs (hydrogel microneedle patches) SFZICC MNs were manufactured by casting SFZICC composite material into a mold, followed by vacuum degassing, thermal concentration at 37°C for 12 hours, and UV curing. SEM and energy-dispersive X-ray spectroscopy (EDS) characterization showed that the MNs were smooth, conical, with uniform tip morphology, and uniformly distributed ZIF-8-CCA-CQDs. It is speculated that these nanoparticles concentrated at the tips contribute to the overall mechanical strength. Figure 4 (a~c in the original text).

[0046] Mechanical testing confirmed that MNs possess sufficient compressive strength and penetrating power to penetrate the stratum corneum without rupturing. An MN array with a tip diameter of 250 μm, a needle height of 400 μm, and a needle spacing of 580 μm maintained its structural integrity during application. Figure 4 (d in the text). Histological examination confirmed that rhodamine B-loaded MNs entered the subchondral bone at the osteochondral junction, and a significantly wider drug distribution area was observed after NIR irradiation. Figure 4 (e). Evaluation using a universal testing machine showed that the penetration power of MNs exceeded the recognized skin penetration threshold of 0.098 N per needle. Furthermore, the SFZICC composite material, i.e., SilMA containing ZIF-8-CCA-CQDs, exhibited superior mechanical strength compared to needles made entirely of SilMA, highlighting the reinforcing effect of the nanocomposite material. Figure 5 a). Under NIR irradiation (1.0 W·cm) -² Under 10 min, the temperature at the MNs region rapidly rose to 40.8℃. Figure 5 (b) indicates efficient photothermal performance. In the degradation study under in vitro conditions, the hydrogel samples were immersed in PBS (pH 7.4) at 37°C, the initial mass was recorded, and the samples were removed at predetermined time intervals, gently dried, and weighed to calculate the degradation rate, as shown in Figure b). Figure 5As shown in c, complete degradation was achieved within 40 days. In contrast, SilMA microneedles completely degraded in the in vivo environment within 21 days, a finding consistent with its in vitro biodegradable properties. Figure 5 (c, d) Histological analysis (H&E staining) showed that MN could effectively penetrate the skin without significant tissue damage. Figure 5 The "e" in the text supports the safety of this transdermal delivery system.

[0047] Comprehensive biocompatibility assessments confirmed the material's good biocompatibility. ATDC5 cells were seeded in 96-well plates (5 × 10³ cells per well) and cultured for 24 hours. Afterward, cells were treated with 7.5 mg / mL ZIF-8, 7.5 mg / mL ZIF-8-CCA-CQDs, 120 mg / mL SilMA, and 120 mg / mL SFZICC (a SilMA hydrogel matrix-based system with ZIF-8 nanoparticles loaded with CCA and CQDs) for 24 or 48 hours, respectively. Cell viability was assessed using the CCK-8 assay, and absorbance at 450 nm was measured. For live / dead cell staining, cells were stained with calcein-AM and propidium iodide for 15 minutes, and imaging was performed using a confocal laser scanning microscope. The control group, ZIF-8 group, ZIF-8-CCA-CQDs group, SilMA group, and SFZCC group all showed high cell viability (green, calcein-AM) and minimal cell death (red, propidium iodide); histological staining revealed no significant histological abnormalities or inflammatory responses in the heart, liver, spleen, lungs, or kidneys. It can be seen that at the cellular level, chondrocytes did not show signs of cytotoxicity in CCK-8 assays, while live / dead staining showed strong viability within 3 days. Figure 6 (a and b in the text).

[0048] The hemolysis assay was performed using mouse red blood cells (RBCs). Blood samples were centrifuged at 3000 rpm for 10 minutes, washed with PBS, and resuspended. The samples were then incubated with 2% red blood cell suspension (volume ratio) at 37°C for 1 hour. After centrifugation, the amount of hemoglobin released from the supernatant was measured at a wavelength of 540 nm. PBS and Triton X-100 were used as negative and positive controls, respectively. Figure 6 The c-value shows that, compared with the negative control, no significant color change was observed in any of the test materials, except for Triton X-100 (Beyotime, P0096-100ml), which served as a positive control. Figure 6The results showed that hemolysis was negligible (<5%) for ZIF-8, ZIF-8-CCA-CQDs, SilMA, and SFZICC (p>0.05 compared to the control group), while Triton X-100 induced complete hemolysis (p<0.0001 compared to all groups). These results demonstrate the excellent biocompatibility and low toxicity of the material.

[0049] 3.3 NIR-assisted SFZICC MNs alleviate pain in the TMJ-OA model A TMJ-OA model was induced in mice by fixing defective restorations to the left upper and lower anterior teeth after separation. Pain relief was assessed using behavioral and electrophysiological evaluations.

[0050] Mechanical hyperalgesia was assessed using von Frey fibers. Mice were allowed 60 minutes of acclimatization before the experiment, followed by vertical attachment of 0.008 g to 2.0 g of fibers to the TMJ region. A withdrawal threshold was defined as ≥3 positive reactions (head withdrawal or mouth wiping) in 5 tests. SFZICC MNs+NIR treatment significantly improved the mechanical pain threshold in the von Frey test, superior to the control group. Figure 7 The a) indicates that it has an effective analgesic effect.

[0051] Studies have shown that mice with chronic pain not only exhibit pain-related behaviors but also depression-like symptoms. Therefore, the elevated cross maze (EPM) and open field test (OFT) were further performed to assess anxiety and depression-related behaviors in these mice. In the open field test, mice were placed in a 50×50×40 cm experimental area, and their spontaneous activity was continuously recorded for 10 minutes. In the elevated cross maze test, the time mice spent in the open and closed arms was recorded separately, for a total duration of 5 minutes. In both the elevated cross maze and open field tests, treated mice exhibited reduced anxiety-like behaviors (…). Figure 7 c and d in the text are consistent with pain relief.

[0052] Electroencephalography (EEG) was performed after electrodes were implanted in the S1BF region. The facial area was stimulated 10 times with a soft brush (5 seconds each time, 30-second intervals), and the EEG responses were averaged. In the TMJ-OA mouse model, nociceptive information received by the spinal trigeminal tract nucleus is transmitted to the primary somatosensory barrel cortex of the cerebral cortex during pain transmission. Subsequently, changes in neuronal activity within the S1BF region of the mouse cerebral cortex were examined. EEG further revealed stable brainwave activity and inhibition of pain-related neural signals. Figure 7 (b, e in the text). These findings indicate that SFZICC+NIR effectively relieves TMJ-OA pain.

[0053] 3.4. NIR-assisted SFZICC MNs therapy improves TMJ-OA pathology. Histological and immunofluorescence analyses were performed by fixing condylar samples in 4% paraformaldehyde, embedding them, and then sectioning them. Routine staining with hematoxylin and eosin (H&E) was used. After decalcification, condylar sections were stained with safranin O-fast green and silver glycine, respectively. Immunofluorescence staining was performed using primary antibodies: PGP 9.5 (1:300, catalog number ab8189, Abcam), CGRP (1:400, catalog number 14959, CST), VEGF (1:300, catalog number sc-7269, Santa Cruz), and CD31 (1:300, catalog number sc-376764, Santa Cruz).

[0054] Histological analysis showed that NIR-assisted SFZICC MNs effectively alleviated the progression of TMJ-OA.

[0055] H&E and Safranin O-Fixed Green staining showed that the SFZICC+NIR group improved cartilage thinning and reduced proteoglycan deposition, with better results than the celecoxib (CXB) group. Figure 8 a and b in the text Figure 8 (g and h in the text). In the SFZICC+NIR group, the OARSI score was significantly reduced ( Figure 8 (f) SEM showed that the repair of microcracks at the condylar cartilage interface was better ( Figure 8 (c in the text)

[0056] Neurovascular invasion was assessed by glycine silver staining and immunofluorescence. NIR-assisted SFZICC MNs significantly reduced nerve fiber density and cartilage penetration. Figure 8 d in Figure 9 Stereoscopic microscopy and immunofluorescence confirmed a significant reduction in pathological angiogenesis. Figure 8 (e in the text).

[0057] In summary, these results indicate that NIR-assisted SFZICC MNs not only alleviate pain by inhibiting neurovascularization, but also improve the structural degeneration of TMJ-OA.

[0058] 3.5 NIR-assisted SFZICC MNs therapy inhibits neurovascularization in vitro Anti-angiogenic and anti-neurogenic effects were evaluated in vitro. Linear wounds were created using sterile 200 μL pipette tips. Endothelial progenitor cells (EPCs) were cultured in conditioned medium for 24 hours before imaging. In the tubular formation assay, Matrigel was coated onto 24-well plates and polymerized. EPCs (2 × 10⁶ cells per well) were seeded onto the gel surface and cultured for 48 hours before imaging. After fixation with 4% paraformaldehyde, staining was completed with crystal violet for 20 minutes. Trigeminal nerve (TG) tissue was isolated from rats aged 1–3 days after birth, aseptically processed, minced, and digested using a combination of collagenase and trypsin. After digestion, the cell suspension was collected by centrifugation, resuspended, and seeded into cell culture dishes for further culture. Crystal violet staining and immunofluorescence staining experiments were then performed. EPCs and TG neurons were divided into six groups: untreated group, VEGF (2.5 ng / mL), SFZICC+NIR, CXB (10 μmol / L), NIR, and SFZICC.

[0059] Experiments showed that NIR-assisted SFZICC MNs therapy extract strongly inhibited EPC migration, superior to CXB ( Figure 10 ab in Figure 10 fg in the middle). Tube formation experiments confirmed the effective inhibition of angiogenesis formation ( Figure 10 (c in the text)

[0060] In TG neurons, β3-tubulin and crystal violet staining showed significant inhibition of axonal growth. Figure 10 The de in Figure 10 The h in the figure indicates that NIR-assisted SFZICC MNs therapy has a dual inhibitory effect on neurogenesis.

[0061] 3.6 Bioinformatics Analysis of the Multi-Target Mechanism of Cryptochlorogenic Acid's Anti-TMJOA Pain Relief To elucidate the mechanism of CCA-mediated analgesia, a reverse target-finding method was employed. Figure 11 (a) From the Gene Cards database, 345 OA-related targets were identified, of which 87 (score > 30) were selected for analysis. A protein-protein interaction (PPI) network constructed using STRING and visualized using Cytoscape generated 86 nodes and 1366 edges; the first 21 nodes were defined as core TMJ-OA targets (a). Figure 11 (b) in the middle.

[0062] Using Swiss Target Prediction, Similarity Ensemble Approach (SEA), GalaxyDock, and COMET, 172 potential CCA targets were predicted, which were then filtered down to 118 human-related targets. An "CCA target network" (118 nodes, 505 edges) was constructed. Intersection analysis revealed 9 common targets: MMP1, MMP2, MMP3, MMP9, MMP13, ESR1, PTGS1, COMT, and F9. After excluding F9 (which has no network connectivity), an 8-node, 17-edge PPI network was generated. Figure 11 The text (cd) highlights MMPs as key participants.

[0063] Differentially expressed genes associated with osteoarthritis were obtained from the GEO database, and volcano plots and heatmaps were generated to visualize gene expression profiles. Figure 11 The results showed that key enriched biological processes (BPs) included collagen catabolism, extracellular matrix degradation, proteolysis, regulation of neuroinflammatory responses, positive regulation of vascular-associated smooth muscle cell proliferation, and receptor signaling pathways. Figure 11 In terms of molecular function (MF), the main enriched terms are endopeptidase activity, serine-type endopeptidase activity, metalloendopeptidase activity, metallopeptidase activity, peptidase activity, zinc ion binding, and collagen binding. Figure 11 The cellular components (CC) significantly affected are primarily the extracellular matrix, extracellular space, extracellular regions, collagen-containing extracellular matrix, and extracellular exosomes. Figure 11 (i). KEGG analysis involves pathways such as IL-17, relaxin signaling, and estrogen signaling. Figure 12 The term "e" suggests its role in tissue remodeling and inflammation.

[0064] 3.5. Verify that MMP3, MMP9, and MMP13 are key targets. Molecular docking revealed that CCA strongly binds to MMP3 (PDB: 4XCT), forms hydrogen bonds with R249, M247, and A189, and undergoes aromatic-H interactions with L188 (docking score: -8.32 kcal / mol). Figure 13 (a) CCA binds to R233, H224, and A217 via H bonds, and undergoes aromatic-H interactions with H224 and H201, binding to MMP9 (1B8Y) (-8.45 kcal / mol). Figure 13 (b) For MMP13 (5 UWL), H bonds with T245, I243 and A186 were observed (-8.05 kcal / mol) Figure 12 (ac in the text).

[0065] Molecular dynamics simulations showed that the complexes formed by MMP3, MMP9, and MMP13 with CCA were structurally stable: the root mean square deviation (RMSD) was stable at approximately 2.6 Å (MMP3), 2.7 Å (MMP9), and 3.3 Å (MMP13), respectively. Figure 12 The dynamic fluctuations in the radius of gyration (Rg) of the MMP3 / 9 / 13-CCA complex indicate a conformational change during the simulation, accompanied by a change in the overall structural compactness. Figure 12 (e). Solvent-accessible surface area (SASA) analysis showed slight fluctuations in the MMP3 / 9 / 13-CCA complex, indicating that small molecule binding induced conformational changes and altered the local binding microenvironment. Figure 12 (g in the text). Hydrogen bond analysis showed that there was a strong hydrogen bond interaction between MMP3 / 9 / 13 and CCA (average 4-6 hydrogen bonds). Figure 12 f in the text). Root mean square fluctuation (RMSF) analysis showed that the MMP3 / 9 / 13-CCA complex had low RMSF values ​​(mostly below 3 Å), indicating reduced residue flexibility and high structural stability. Figure 13 (a) The free energy landscape (FEL) is plotted on RMSD and Rg coordinates, showing the energy distribution during the molecular dynamics simulation of the MMP3 / 9 / 13-CCA complex. The color gradient from red (high energy) to blue (low energy) represents the decrease in free energy states. Figure 12 (hj in the text). Per-residue decomposition analysis based on free energy identified several key residues with high contribution scores in each complex: TYR248, MET247, LEU243, TYR245, ARG249, and HIS226 in MMP3; LEU218, GLU216, ALA217, LEU197, ARG233, and PHE232 in MMP9; and LEU218, THR247, LEU239, and PHE252 in MMP13. These residues are likely crucial for catalytic function and CCA binding. Figure 13 (b) in the middle.

[0066] The functional effects of the above targets were verified through in vivo experiments. A TMJ-OA model was used, and near-infrared assisted SFZICC microneedle therapy was applied. Immunofluorescence staining was performed using primary antibodies: MMP3 (1:300, catalog number GTX55709, GeneTex), MMP9 (1:300, catalog number GTX100665, GeneTex), and MMP13 (1:300, catalog number GTX100458, GeneTex). In vivo, UAC-induced upregulation of MMP3, MMP9, and MMP13 was significantly reversed by NIR-assisted SFZICC MNs therapy. Figure 14 This verified their role as functional targets.

[0067] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A near-infrared responsive microneedle, characterized by, The drug and the carbon quantum dots are mixed with the ZIF-8 nanoparticles at an equal mass ratio, and after being loaded on the ZIF-8 nanoparticles, a composite material is obtained; the composite material is embedded in a hydrogel based on methyl methacrylated silk fibroin to obtain the near-infrared responsive microneedle. The near-infrared responsive microneedle is under near-infrared irradiation, the carbon quantum dots absorb light and convert it into heat energy to make the ZIF-8 nanoparticles disintegrate, and the drug is released; the mass ratio of the carbon quantum dots to the ligand of the ZIF-8 nanoparticles is 10mg-11mg:330mg.

2. The near-infrared responsive microneedle of claim 1, wherein, The drug is phloroglucinolic acid.

3. The near-infrared responsive microneedle of claim 1, wherein, The mass ratio of the composite material to the methyl methacrylated silk fibroin hydrogel microneedle is 7.5-8:

120.

4. A method of preparing the near-infrared responsive microneedle according to any one of claims 1 to 3, characterized by, The method comprises the following steps: The phloroglucinolic acid, the carbon quantum dots and the ZIF-8 nanoparticles are mixed in an organic environment, and a zinc acetate solution is added for reaction to obtain a precipitate, which is filtered and centrifuged to obtain the composite material; The composite material, the methyl methacrylated silk fibroin and a photoinitiator are mixed in sodium hydroxide, ultrasonic degassing is performed, the mixture is injected into a mold, and solidification is performed to obtain the near-infrared responsive microneedle.

5. The method for preparing infrared-responsive microneedles according to claim 4, characterized in that, The photoinitiator is a lithium salt of phenyl(2,4,6-trimethylbenzoyl)phosphinic acid.

6. The method for preparing infrared-responsive microneedles according to claim 4, characterized in that, The reagent used in the organic environment is methanol.

7. The method for preparing infrared-responsive microneedles according to claim 4, characterized in that, The solidification refers to ultraviolet curing under a wavelength of 365nm.

8. Use of the near-infrared responsive microneedle according to any one of claims 1-3 in the treatment of osteoarthritis.

Images