A photocrosslinked HA-Tyr shell-core structured hydrogel microneedle, its preparation method and application

Abstract

This invention discloses a photocrosslinked HA-Tyr (hyaluronic acid-tyramine) shell-core structured hydrogel microneedles, their preparation method, and applications, belonging to the field of biomedical technology. This invention involves adding an HA-Tyr solution to a microneedle template to form a hydrogel shell, and then adding photosensitizer nanoparticles into the hydrogel shell to prepare the photocrosslinked HA-Tyr shell-core structured hydrogel microneedles; the hydrogel comprises hyaluronic acid-tyramine, a photocrosslinking agent, and an oxidant. This invention combines photocrosslinked hydrogel with microneedles to form a shell-core structured microneedle, which allows for reasonable control of drug release and precise controllable drug delivery. The photocrosslinked HA-Tyr shell-core structured hydrogel microneedles with different mechanical strengths in this invention can reasonably control drug release and are effectively used for the treatment of melanoma.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a photocrosslinked HA-Tyr shell-core structure hydrogel microneedle, its preparation method, and its application. Background Technology

[0002] Hyaluronic acid (HA) is a non-sulfated glycosaminoglycan found in the natural extracellular matrix. It is a biodegradable, non-toxic, biocompatible, low-immunogenic, and non-inflammatory linear polysaccharide that can interact with CD44 receptors on cell surfaces, directly affecting tissue homeostasis and promoting cell proliferation and migration. HA hydrogels have been widely used in osteoarthritis treatment, surgical wound healing, embryo implantation, drug delivery, and tissue engineering. However, natural HA has poor mechanical strength and is easily degraded by hyaluronidase in vivo. Physical or chemical cross-linking can be introduced to improve the mechanical strength of HA and overcome the limitations of in vivo application. Introducing tyramine (Tyr) into the HA backbone to form hyaluronic acid-tyramine (HA-Tyr) can induce enzymatic or photocrosslinking reactions, forming hydrogels with a three-dimensional cross-linked structure. This effectively improves mechanical strength and controls drug release rates, and has been widely used in the biomedical field.

[0003] Photocrosslinking offers advantages such as low curing heat, controllable crosslinking degree, precise spatiotemporal control, and ease of operation. Depending on the light source used, it can be divided into ultraviolet (UV) crosslinking and visible light crosslinking. UV light has poor biocompatibility and does not easily penetrate tissues, resulting in a significantly lower treatment depth compared to visible light crosslinking. Commonly used crosslinking agents for visible light crosslinking include Ru(II) polypyridine complexes, riboflavin (RF), and camphorquinone. The covalent crosslinking mediated by Ru(II) polypyridine complexes and ammonium persulfate (APS) mainly generates tyrosine free radicals and forms di-Tyr bridges between the two aromatic rings, resulting in HA-Tyr photocrosslinked hydrogels. By changing the concentration and ratio of Ru and APS, as well as the irradiation time, the crosslinking rate and mechanical properties of HA-Tyr can be controlled, yielding HA-Tyr hydrogels with different mechanical strengths and drug release rates to meet the requirements of different drug delivery methods. Ru(II) polypyridine complexes and ammonium persulfate (APS) have certain biotoxicity, but they are relatively safe at cross-linking concentrations, and the toxicity of APS can be reduced as it is consumed by the cross-linking reaction.

[0004] Microneedling, as a novel form of transdermal drug delivery, not only effectively avoids the first-pass effect in the liver and gastrointestinal digestion problems associated with oral medications, but also offers better patient compliance and maintains skin integrity compared to injections. The biggest challenge facing traditional transdermal drug delivery is the protective function of the stratum corneum. The skin is the body's natural barrier, and the transdermal rate, penetration amount, and retention rate of most drugs are difficult to achieve the desired therapeutic effect. Microneedling can effectively break through the physical and lipid barriers of the stratum corneum, allowing for controlled and precise delivery of drugs to specific skin layers and other tissues in a minimally invasive manner. Furthermore, combining microneedles with nanoparticles can better promote transdermal drug absorption, further enhancing the transdermal effect.

[0005] Currently, there is no observed application of photocrosslinked HA-Tyr hydrogels in microneedles. Summary of the Invention

[0006] Purpose of the Invention: The purpose of this invention is to address the shortcomings of existing technologies by providing a photocrosslinked HA-Tyr shell-core structured hydrogel microneedle. Based on the controllable mechanical strength and crosslinking speed of photocrosslinked HA-Tyr hydrogel, this invention synthesizes photocrosslinked HA-Tyr shell-core structured hydrogel microneedles encapsulating photosensitizer nanoparticles. This improves the drug transdermal rate, further controls the release of photosensitizers, enhances the antitumor effect of drugs, and provides more theoretical support for the application of hydrogel microneedles in the biomedical field.

[0007] Technical solution: The objective of this invention is achieved through the following technical solution:

[0008] This invention provides a photocrosslinked HA-Tyr shell-core structured hydrogel microneedle. An HA-Tyr solution is added to a microneedle template to form a hydrogel shell, and then photosensitizer nanoparticles are added to the hydrogel shell to prepare the photocrosslinked HA-Tyr shell-core structured hydrogel microneedle. The hydrogel comprises hyaluronic acid-tyramine, a photocrosslinking agent, and an oxidizing agent.

[0009] Preferably, the photocrosslinking agent is a Ru(II) polypyridine complex, and the oxidant is ammonium persulfate (APS).

[0010] Preferably, the molar ratio of the photocrosslinking agent to the oxidant is 1:1 to 30.

[0011] More preferably, the molar ratio of the photocrosslinking agent to the oxidant is 1:10.

[0012] This invention also provides a method for preparing the above-mentioned photocrosslinked HA-Tyr shell-core structured hydrogel microneedles, comprising the following steps:

[0013] (1) Adjust the pH of the HA-Tyr solution to alkaline, add the photocrosslinking agent solution and the oxidant solution to obtain a mixed solution A;

[0014] (2) Add mixed solution A to the surface of microneedle template, and react with light to obtain photocrosslinked HA-Tyr hydrogel, and dry to obtain microneedle A;

[0015] (3) Mix the HA aqueous solution and photosensitizer nanoparticles in equal proportions to obtain mixed solution B;

[0016] (4) Add mixed solution B to the surface of microneedle A, add microneedle base, and dry to obtain photocrosslinked HA-Tyr shell-core structured hydrogel microneedles.

[0017] This invention mainly uses a template method to prepare hydrogel microneedles, combining nanocarriers (i.e., photosensitizer nanoparticles) with microneedles to achieve precise and controllable drug release and improve drug delivery efficiency.

[0018] Preferably, in step (1), the concentration of the HA-Tyr solution is 0.5-2%; the concentration of the photocrosslinking agent solution is 50-600 μM; and the concentration of the oxidant solution is 0.5-6 mM.

[0019] Furthermore, the concentration of the HA-Tyr solution is 2%, the concentration of the photocrosslinking agent solution is 50–300 μM, and the concentration of the oxidant solution is 0.5–3 mM.

[0020] Furthermore, the concentration of the photocrosslinking agent solution is 300 μM, and the concentration of the oxidant solution is 3 mM.

[0021] This invention controls the mechanical strength of hydrogels by changing the concentration of oxidant and photocrosslinking agent, thereby forming a hydrogel microneedle shell with adjustable mechanical properties and crosslinking rate, and thus achieving precise and controllable drug release.

[0022] Preferably, in step (2), the microneedle template is a polydimethylsiloxane (PDMS) mold; the illumination time is 0 to 20 min; more preferably 1 to 5 min; and even more preferably 3 min.

[0023] Furthermore, the PDMS mold has a pyramidal array of holes, the mold is a 10×10 array, the microneedle height is 1000μm, the microneedle base diameter is 300μm, and the microneedle spacing is 750μm.

[0024] Preferably, in step (3), the concentration of the HA aqueous solution is 1.25-5%, the photosensitizer nanoparticles are Ce6 nanoparticles, and the concentration of the Ce6 nanoparticles is 5-15 mg / mL.

[0025] Furthermore, the concentration of the HA aqueous solution is 5%; the concentration of the Ce6 nanoparticles is 15 mg / mL.

[0026] Furthermore, the Ce6 nanoparticles are SCLMs / Ce6. Ce6 drugs have poor solubility, and SCLMs / Ce6 can significantly improve their solubility. In addition, the nanoparticles improve transdermal efficiency and enhance anti-tumor effects.

[0027] Preferably, in step (4), the microneedle base is a PMMA base, which can effectively prevent the drug from diffusing on the base and can concentrate it at the needle tip.

[0028] A preferred embodiment of the preparation method of the present invention is as follows:

[0029] (1) Dissolve HA-Tyr in PBS buffer, add 0.1M NaOH to adjust the pH to 6-8 to obtain HA-Tyr solution; dissolve Ru(II) polypyridine complex in PBS buffer to obtain Ru(II) polypyridine complex solution; dissolve APS in PBS buffer to obtain APS solution; add Ru(II) polypyridine complex solution and APS solution to HA-Tyr solution to obtain mixed solution A;

[0030] (2) Add mixed solution A to the surface of PDMS microneedle template, centrifuge at 4000 r / min and remove the supernatant, place under blue light for 3 min to react and obtain photocrosslinked HA-Tyr hydrogel, dry at room temperature overnight to obtain microneedle A;

[0031] (3) Dissolve HA in water to obtain an aqueous solution of HA; mix the HA solution and Ce6 nanoparticle solution in equal proportions to obtain mixed solution B;

[0032] (4) Add the mixed solution B to the surface of microneedle A, centrifuge at 4000 r / min and remove the surface solution, add the PMMA base, and dry at room temperature to obtain photocrosslinked HA-Tyr shell-core structured hydrogel microneedles.

[0033] This invention also provides the application of the above-mentioned photocrosslinked HA-Tyr shell-core structured hydrogel microneedles in photodynamic therapy for tumors. The photocrosslinked hydrogel shell of this invention has good biocompatibility. Microneedles prepared with such hydrogel as the shell can achieve controllable swelling and drug release under the skin, which is of great significance for enhancing the sustained and controlled release of microneedle transdermal drug delivery systems.

[0034] Beneficial effects:

[0035] (1) The photocrosslinked HA-Tyr shell-core structure hydrogel microneedles prepared by the present invention can effectively improve the disadvantage of uncontrollable mechanical strength in HA microneedles. By using photocrosslinked hydrogel as the shell of the shell-core structure microneedles, the mechanical strength can be effectively improved, and the mechanical strength and drug release rate can be precisely controlled, thereby enhancing the transdermal effect of the drug, prolonging the drug retention time in the body, greatly enhancing the transdermal rate of Ce6 nanoparticles, and achieving sustained drug release.

[0036] (2) The photocrosslinked hydrogel used in this invention can effectively control the crosslinking strength of the hydrogel by changing the concentration of the oxidant and the concentration of the photocrosslinking agent, thereby effectively changing the mechanical strength of the hydrogel and controlling the drug release rate. At the same time, Ru(II) polypyridine complex and APS have good biocompatibility at the crosslinking concentration and have good application prospects in the biomedical field. Attached Figure Description

[0037] Figure 1 The UV absorption spectrum of HA-Tyr is shown below.

[0038] Figure 2 Images of HA-Tyr hydrogels with different crosslinking agent concentrations;

[0039] Figure 3 The G' and G” values ​​are for HA-Tyr hydrogels with different crosslinking agent concentrations;

[0040] Figure 4 ROS generation for Ce6 and Ce6 / SCLMs;

[0041] Figure 5 The images show the morphological characteristics of HAT@SCLMs / Ce6-MN with different crosslinking agent concentrations under an optical microscope.

[0042] Figure 6 SEM images of HAT@SCLMs / Ce6-MN with different crosslinking agent concentrations;

[0043] Figure 7 Mechanical properties of HAT@SCLMs / Ce6-MN with different crosslinking agent concentrations;

[0044] Figure 8 The in vitro skin penetration ability of HAT@SCLMs / Ce6-MN with different cross-linking agent concentrations;

[0045] Figure 9 In vitro release curves of HAT@SCLMs / Ce6-MN from the needle tip for different crosslinking agent concentrations;

[0046] Figure 10 The survival rate of HA-Tyr hydrogels with different cross-linking agent concentrations after co-incubation in A375 cells for 48 h is shown in the figure.

[0047] Figure 11 The survival rate of A375 cells after co-incubation for 24 hours with different concentrations of HA and SCLMs / Ce6 mixed solutions. Detailed Implementation

[0048] The technical solution of the present invention will be described in detail below through specific embodiments, but the scope of protection of the present invention is not limited to the embodiments described.

[0049] In the following examples, the unit M represents mol / L.

[0050] In the following examples, Ru(II) polypyridine complex is abbreviated as Ru; Ce6 nanoparticles are abbreviated as SCLMs / Ce6; photocrosslinked HA-Tyr shell-core structure hydrogel microneedles are abbreviated as HAT@SCLMs / Ce6-MN; microneedles without hydrogel shell are abbreviated as SCLMs / Ce6-MN; photocrosslinking agent and oxidant are collectively referred to as crosslinking agent.

[0051] Example 1: Preparation of HA-Tyr photocrosslinked hydrogel

[0052] 1. Synthesis of HA-Tyr (Compound 1)

[0053] (1) Dissolve 55 kDa sodium hyaluronate (1 g, 2.6 mmol) in 100 mL of distilled water, add tyramine hydrochloride (451 mg, 2.6 mmol) to the solution; then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDC.HCl (548 mg, 2.86 mmol) and N-hydroxysuccinimide NHS (332 mg, 2.88 mmol) to the solution at the same time to induce a conjugation reaction;

[0054] (2) As the reaction proceeded, the pH of the solution was maintained at 4.7 with 0.1M HCl; the solution was stirred overnight at room temperature, and the pH was adjusted to 7.0 with 0.1M NaOH.

[0055] (3) Transfer the solution to a 3000 Da dialysis bag, dialyze with 0.1 M NaCl solution for 2 days, then dialyze with a mixture of distilled water and ethanol (3:1, v / v) for 1 day, and finally dialyze with distilled water for 1 day; freeze-dry the purified solution to obtain HA-Tyr;

[0056] (4) UV measurement of HA-Tyr grafting rate

[0057] Figure 1 The UV absorption spectrum of HA-Tyr is shown. The UV results indicate that HA-Tyr has UV absorption at 274 nm, confirming the correct synthesis of compound 1. The grafting rate of compound 1 is 6.61%.

[0058] 2. Preparation of HA-Tyr photocrosslinked hydrogels

[0059] (1) Preparation of APS solution: Dissolve 34.23 mg of APS solid in 500 μL of PBS to obtain 300 mM APS solution;

[0060] (2) Preparation of Ru solution: Dissolve 11.23 mg Ru(II) polypyridine complex (CAS No.: 50525-27-4, purchased from Maclean) in 500 μL PBS to obtain 30 mM Ru solution;

[0061] (3) Weigh 6 mg of HA-Tyr flocculent and dissolve it in 300 μL of PBS with pH 6 to 8 to obtain HA-Tyr solution. Add 50, 100, 300, 600 μM Ru and 0.5, 1, 3, 6 mM APS solutions to the solution and vortex to mix. Place the solution under a blue light lamp for 3 min to form a gel.

[0062] Figure 2 For HA-Tyr hydrogels with different crosslinking agent concentrations, Figure 2 Image A shows a hydrogel with a Ru concentration of 50 μM and an APS concentration of 0.5 mM. Figure 2 Image B shows a hydrogel with a Ru concentration of 100 μM and an APS concentration of 1 mM. Figure 2 C is an image of a hydrogel with a Ru concentration of 300 μM and an APS concentration of 3 mM; Figure 2 Image D shows a hydrogel with a Ru concentration of 600 μM and an APS concentration of 6 mM. Crosslinking agents of various concentrations and ratios can stably form a gel. The hydrogel becomes more stable with increasing crosslinking agent concentration, reaching its peak stability at a Ru concentration of 300 μM and an APS concentration of 3 mM. However, further increases in crosslinking agent concentration lead to decreased stability. Excessive crosslinking agent concentration may cause over-crosslinking of HA-Tyr, damaging the hydrogel.

[0063] 3. Rheological property determination of HA-Tyr photocrosslinked hydrogel

[0064] 20 mg of HA-Tyr flocculent was dissolved in 1000 μL of PBS (pH 6–8). 50, 100, 300, and 600 μM Ru and 0.5, 1, 3, and 6 mM APS solutions were added and vortexed. The mixture was then irradiated under blue light for 3 min to form a gel, and allowed to stand in the dark for 2 h to prepare hydrogels with Ru concentrations of 50 μM, 0.5 mM APS, 100 μM, 1 mM, 300 μM, 3 mM, and 600 μM and 6 mM APS. The rheometer (Discovery HR-2, TA) had an electrically heated parallel plate diameter of 40 mm, a clamping distance of 500 μm, and a testing temperature of 25 °C. The dynamic time-scan frequency was set to 1 Hz, and the stress was fixed at 1%, to monitor the changes in the mechanical properties of different colloids.

[0065] Figure 3 The values ​​represent the storage modulus (G') and loss modulus (G”) of HA-Tyr hydrogels with different crosslinking agent concentrations.

[0066] like Figure 3 As shown, the mechanical strength of the hydrogel increases with increasing crosslinking agent concentration, reaching its maximum at a Ru concentration of 300 μM and an APS concentration of 3 mM. Further increasing the crosslinking agent concentration decreases the mechanical strength of the hydrogel, possibly due to excessive crosslinking between HA and Tyr, which can damage the hydrogel. The rheological property results are consistent with the gelation results, further demonstrating that changing the crosslinking agent concentration can control the mechanical strength and crosslinking rate of the hydrogel. Example 2: Effect of HA on the ROS generation capacity of SCLMs / Ce6

[0067] Using ABDA (Maclean) as a reactive oxygen species indicator, the ROS generation capacity of HA+SCLMs / Ce6 and SCLMs / Ce6 in aqueous solution under NIR light irradiation was investigated. In aqueous solution, ABDA effectively captured and reacted with ROS, reducing its maximum UV absorbance at 401 nm. Aqueous solutions of HA+SCLMs / Ce6 and SCLMs / Ce6 were mixed with ABDA solution to achieve a final ABDA concentration of 50 μg / mL and a final Ce6 concentration of 10 μg / mL. The mixed solutions were then exposed to laser irradiation (650 nm, 0.2 W / cm²). 2 At 0, 2, 4, 6, 8 and 10 min, the absorbance of the mixed solution at 401 nm was measured using a UV-Vis spectrophotometer to determine the generation of ROS.

[0068] Figure 4 ROS generation for Ce6 and Ce6 / SCLMs. For example... Figure 4As shown, the UV absorbance of ABDA decreased significantly with increasing illumination time. The absorbance ratio of SCLMs / Ce6 decreased to 0.66 at 10 min, while the absorbance ratio of the group with added HA solution decreased to 0.37 after 10 min of laser irradiation. This indicates that the addition of HA solution can significantly improve the ROS generation ability of Ce6. SCLMs / Ce6 nanoparticles tend to aggregate in aqueous solution, affecting their ROS generation ability. HA solution disperses the SCLMs / Ce6 nanoparticles, effectively improving their ROS generation ability and thus enhancing their antitumor effect.

[0069] Example 3: Preparation of photocrosslinked HA-Tyr shell-core structured hydrogel microneedles HAT@SCLMs / Ce6-MN

[0070] (1) Preparation of the HAT@SCLMs / Ce6-MN hydrogel shell:

[0071] Dissolve 20 mg of HA-Tyr in 1 mL of PBS buffer solution, add 10 μL of Ru solution and 10 μL of APS solution to obtain HA-Tyr (300 μM Ru, 3 mM APS) solution;

[0072] The PDMS template was plasma-cleaned for 10 seconds, then 60 μL of HA-Tyr (300 μM Ru, 3 mM APS) solution was added to the surface of the PDMS microneedle template. After centrifugation at 4000 rpm for 10 min to remove excess solution, the template was irradiated under blue light for 3 min. After drying at room temperature overnight, the HA-Tyr photocrosslinked hydrogel microneedle shell (high crosslinking agent concentration) was obtained.

[0073] The same method was used to prepare HA-Tyr photocrosslinked hydrogel microneedle shells containing 50 μM Ru and 0.5 mM APS (low crosslinking agent concentration) and HA-Tyr photocrosslinked hydrogel microneedle shells containing 100 μM Ru and 1 mM APS (medium crosslinking agent concentration).

[0074] The PDMS template used has a pyramidal array of holes, which is a 10×10 array, with a microneedle height of 1000μm, a microneedle base diameter of 300μm, and a microneedle spacing of 750μm.

[0075] (2) Preparation of HAT@SCLMs / Ce6-MN: 80 μM of 5% HA aqueous solution containing 15 mg / mL SCLMs / Ce6 was added to the template surface of the HA-Tyr photocrosslinked hydrogel microneedle shell. The mixture was centrifuged at 4000 rpm for 10 min, and excess solution was removed. 250 mg of 40% PMMA solution was added as the substrate for the microneedles, and the mixture was dried at room temperature for two days. The resulting microneedle array was carefully separated from the mold to obtain HAT@SCLMs / Ce6-MN.

[0076] (3) Morphological and dimensional characterization of HAT@SCLMs / Ce6-MN: Optical images of HAT@SCLMs / Ce6-MN and SCLMs / Ce6-MN with different crosslinking agent concentrations were taken using a digital camera equipped with a macro lens. The length, base width, and tip spacing of the microneedles were observed and characterized. The microneedles were attached to the surface of a copper block with conductive adhesive, sputtered with gold, and then observed and characterized using a SEM (Hitachi Regulus 8100) at an accelerating voltage of 5.0 kV. The overall structure and structural integrity of the microneedles were then observed and characterized.

[0077] like Figure 5 , Figure 6 As shown, A, B, C, and D are optical and SEM images of SCLMs / Ce6-MN and HAT@SCLMs / Ce6-MN with low, medium, and high crosslinking agent concentrations, respectively. It can be seen that both HAT@SCLMs / Ce6-MN and SCLMs / Ce6-MN with different crosslinking agent concentrations exhibit good morphology, displaying a pyramid-like structure, with a measured base height and side length of ~420 μm and ~140 μm, respectively. Furthermore, the mechanical strength of the photocrosslinked hydrogel does not affect the macroscopic structure of the MN array.

[0078] (4) Mechanical properties of HAT@SCLMs / Ce6-MN

[0079] Compression tests were performed using a universal testing machine (TH-8203S, Suzhou Tuobo Machinery Equipment Co., Ltd., China) equipped with a load cell (80N). A force perpendicular to the stainless steel disc was applied to the microneedle at a speed of 0.5 mm / min, stopping when the compression distance reached 700 μm. Force-distance curves were then plotted, and the force at a compression distance of 500 μm was compared among various HAT@SCLMs / Ce6-MN.

[0080] Figure 7 Mechanical properties of HAT@SCLMs / Ce6-MN with different crosslinking agent concentrations.

[0081] like Figure 7As shown in Figure A, the mechanical properties of SCLMs / Ce6-MN and HAT@SCLMs / Ce6-MN with low crosslinking agent concentration are similar. With the increase of crosslinking agent concentration, the mechanical properties of the hydrogel also increase, and the mechanical properties of the microneedles are also improved.

[0082] like Figure 7 As shown in Figure B, at a displacement of 0.5 mm, the compressive forces of each microneedle array (100 needles) for SCLMs / Ce6-MN and HAT@SCLMs / Ce6-MN with low, medium, and high crosslinking agent concentrations are 24.811 N, 26.345 N, 33.581 N, and 39.3545 N, respectively. Furthermore, there are significant differences in the mechanical strength between HAT@SCLMs / Ce6-MN with high crosslinking agent concentration and SCLMs / Ce6-MN, and between HAT@SCLMs / Ce6-MN with low crosslinking agent concentration. Therefore, a high crosslinking agent concentration yields HAT@SCLMs / Ce6-MN with the highest mechanical strength.

[0083] Example 4: In vitro skin penetration and drug release of HAT@SCLMs / Ce6-MN

[0084] 1. HAT@SCLMs / Ce6-MN's in vitro skin penetration ability

[0085] Three different concentrations of crosslinking agent HAT@SCLMs / Ce6-MN and SCLMs / Ce6-MN were placed tip-down on pigskin, and thumb pressure was applied for 5 minutes. After pressure, excess Ce6 was removed with a cotton swab. The pigskin was then placed in a chemiluminescence imaging analyzer (Tanon 4600SF, China) for fluorescence imaging. The excitation wavelength was 400 nm, the emission wavelength was 670 nm, and the exposure time was fixed at 50 ms. Finally, the average fluorescence intensity of the skin was analyzed using ImageJ software.

[0086] Figure 8 The in vitro skin penetration ability of HAT@SCLMs / Ce6-MN with different cross-linking agent concentrations was evaluated. Figure 8 As shown in Figure A, there was no significant difference in skin fluorescence intensity between skin treated with SCLMs / Ce6-MN and HAT@SCLMs / Ce6-MN at low crosslinking agent concentrations. Furthermore, the fluorescence intensity of skin treated with HAT@SCLMs / Ce6-MN increased with increasing crosslinking agent concentration. Figure 8Further quantitative analysis, shown in Figure B, revealed that the mean fluorescence intensity of skin treated with high cross-linking agent concentrations of HAT@SCLMs / Ce6-MN was 1.7 times and 1.45 times that of skin treated with low cross-linking agent concentrations of HAT@SCLMs / Ce6-MN and SCLMs / Ce6-MN, respectively. Therefore, high cross-linking agent concentrations of HAT@SCLMs / Ce6-MN exhibited the highest Ce6 transdermal delivery efficiency in the porcine skin model.

[0087] 2. In vitro drug release of HAT@SCLMs / Ce6-MN

[0088] The tip of the microneedle was scraped off with a knife and placed in an EP tube, with 400 μL of water added as the release medium. The EP tube was placed in a constant temperature shaking incubator at 37°C and a rotation speed of 60 rpm. At 5 min, 10 min, 20 min, 40 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h, 200 μL of the supernatant release solution was collected, and the same volume of release medium was added. The concentration of Ce6 in the collected solution was determined by fluorescence analysis, and the cumulative release amount of Ce6 was calculated according to the Ce6 standard curve.

[0089] Figure 9 In vitro release curves of HAT@SCLMs / Ce6-MN from the needle tip at different cross-linking agent concentrations. Figure 9 As shown, the mechanical strength of the hydrogel shell increases with increasing cross-linking agent concentration, resulting in a slower release rate and a lower release amount of Ce6. The SCLMs / Ce6-MN tip exhibits a burst release in water, releasing 60.1% of Ce6 in just 10 minutes. In contrast, the high-concentration HAT@SCLMs / Ce6-MN only cumulatively releases 19.8% in 10 minutes, with a cumulative release rate of 76.3% after 24 hours, showing a significant difference from SCLMs / Ce6-MN. Furthermore, the release rate of Ce6 gradually increases with decreasing cross-linking strength. Therefore, HAT@SCLMs / Ce6-MN with a photocrosslinked HA-Tyr hydrogel shell can achieve slow drug release, avoiding burst release. Simultaneously, the drug release rate can be effectively controlled by adjusting the cross-linking strength of the HAT@SCLMs / Ce6-MN hydrogel shell.

[0090] Example 5: Investigation of the biocompatibility of HA-Tyr photocrosslinked hydrogel

[0091] The MTT assay was used to detect the in vitro cytotoxicity of the HA-Tyr photocrosslinked hydrogel prepared in Example 1 of this invention. Human malignant melanoma cells A375 (ATCC, USA) were seeded at a density of 3000 cells per well in 96-well plates and incubated at 37°C with 5% CO2 for 24 h. Afterward, the culture medium was discarded, and 200 μL of HA-Tyr photocrosslinked hydrogel solutions with different crosslinking agent concentrations (0-400 μM) were added, and the cells were cultured for another 48 h, with five replicates for each concentration. After 48 h, 20 μL of MTT reagent (Beyotime Biotechnology Co., Ltd.) (5 mg / mL) was added, and the cells were incubated for another 4 h. After removing the culture medium, 200 μL of dimethyl sulfoxide (DMSO) was added to dissolve the formed formazan crystals. The absorbance at 490 nm for each well was measured using a microplate reader (Sunrise™, Tecan, Austria). The corresponding cell viability was calculated using Origin software to assess the biocompatibility of the samples.

[0092] Figure 10 The survival rate of HA-Tyr hydrogels with different cross-linking agent concentrations after co-incubation in A375 cells for 48 hours is shown in the figure. Figure 10 As shown, after incubation for 48 hours with HA-Tyr hydrogel solutions of high, medium, and low crosslinking agent concentrations (0-400 μM), cell viability remained above 80%, indicating that the HA-Tyr photocrosslinked hydrogels exhibit low cytotoxicity and good biocompatibility. Furthermore, within the crosslinking agent concentration range used in this invention, the photocrosslinked HA-Tyr hydrogels all exhibited good biocompatibility.

[0093] Example 6: In vivo antitumor efficacy study of SCLMs / Ce6 and HA mixed solution (core).

[0094] Human malignant melanoma cells A375 were seeded at a density of 3000 cells per well in 96-well plates and incubated at 37°C with 5% CO2 for 24 h. Afterward, the culture medium was discarded, and 200 μL of a mixed solution of SCLMs / Ce6 and HA at different concentration gradients (SCLMs / Ce6 concentration range of 0-50 μg / mL, HA aqueous solution concentration of 0-333 μg / mL) was added, and the cells were cultured for another 24 h, with 5 replicates for each concentration. At 4 h of incubation, each well was treated with a 650 nm laser at 1 W / cm². 2 The cells were irradiated with an average light intensity for 1 min, with a dark group (without light) serving as a control. After 24 h, 20 μL of MTT reagent (5 mg / mL) was added, and incubation continued for another 24 h. After removing the culture medium, 200 μL of dimethyl sulfoxide (DMSO) was added to dissolve the formed formazan crystals. The absorbance of each well at 490 nm was measured using a microplate reader. The corresponding cell viability was calculated using Origin software to investigate the toxicity of the samples to A375 cells.

[0095] Figure 11 The survival rate of A375 cells after co-incubation for 24 hours with different concentrations of HA and SCLMs / Ce6 mixed solutions is shown in the figure. Figure 11 As shown, SCLMs / Ce6 exhibited low cytotoxicity under untreated conditions, with cytotoxicity increasing with increasing concentration. At a concentration of 50 μg / mL, cell viability decreased to 40.5%. Cells were then subjected to a 650 nm laser at 1 W / cm². 2 After 1 min of irradiation with average light intensity, the cytotoxicity of SCLMs / Ce6 significantly increased, with cell viability decreasing to 45.5% at a concentration of 12.5 μg / mL and to 3.2% at a concentration of 50 μg / mL. The IC50 values ​​for the light-irradiated and non-illuminated groups were [not specified in the original text]. 50 The concentrations were 11.4 μg / mL and 34.5 μg / mL, respectively, with the non-illuminated group showing significantly lower toxicity than the illuminated group. Therefore, under illumination, SCLMs / Ce6 can effectively kill tumor cells and has a good inhibitory effect on melanoma proliferation.

[0096] As described above, although the invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A method for preparing photocrosslinked HA-Tyr shell-core structure hydrogel microneedle, characterized in that, The HA-Tyr solution is added to the microneedle template to form a hydrogel shell, and photosensitizer nanoparticles are added to the hydrogel shell to prepare the photo-crosslinked HA-Tyr shell-core structure hydrogel microneedle; the hydrogel comprises hyaluronic acid-tyramine, a photo-crosslinking agent and an oxidizing agent; the photo-crosslinking agent is a Ru (II) polypyridyl complex, and the oxidizing agent is ammonium persulfate APS; the photosensitizer nanoparticles are Ce6 nanoparticles; the Ce6 nanoparticles are SCLMs / Ce6; the preparation method comprises the following steps: (1) adjusting the pH of the HA-Tyr solution to be alkaline, adding a photo-crosslinking agent solution and an oxidizing agent solution to obtain a mixed solution A; (2) adding the mixed solution A to the surface of the microneedle template, and performing light irradiation to obtain a photo-crosslinked HA-Tyr hydrogel, and drying to obtain microneedle A; (3) mixing the HA aqueous solution and the photosensitizer nanoparticles in equal proportions to obtain a mixed solution B; (4) adding the mixed solution B to the surface of the microneedle A, adding a microneedle base, and drying to obtain the photo-crosslinked HA-Tyr shell-core structure hydrogel microneedle; In step (1), the concentration of the HA-Tyr solution is 0.5% to 2%; the concentration of the photo-crosslinking agent solution is 50 to 600 μM; and the concentration of the oxidizing agent solution is 0.5 to 6 mM.

2. The production method according to claim 1, characterized by, The concentration of the HA-Tyr solution is 2%.

3. The preparation method according to claim 1, characterized in that, The concentration of the photo-crosslinking agent solution is 300 μM, and the concentration of the oxidizing agent solution is 3 mM.

4. The method of claim 1, wherein, In step (2), the microneedle template is a polydimethylsiloxane (PDMS) mold; and the light irradiation time is 1 to 20 min.

5. The preparation method according to claim 4, characterized in that, The light irradiation time is 3 min.

6. The preparation method according to claim 4, characterized in that, The PDMS mold has a pyramidal hole array, the mold is a 10×10 array, the microneedle height is 1000 μm, the microneedle base diameter is 300 μm, and the microneedle spacing is 750 μm.

7. The preparation method according to claim 1, characterized in that, In step (3), the concentration of the HA aqueous solution is 1.25 to 5%, and the concentration of the Ce6 nanoparticles is 5 to 15 mg / mL.

8. The preparation method according to claim 7, characterized in that, The concentration of the HA aqueous solution is 5%, and the concentration of the Ce6 nanoparticles is 15 mg / mL.

9. The photo-crosslinked HA-Tyr shell-core structure hydrogel microneedle prepared by the preparation method of any one of claims 1 to 8.

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