Lenalidomide-loaded nanohydrogel microneedle patch and its preparation method

Abstract

This invention relates to the field of microneedle patch technology, specifically to a lenalidomide-loaded nanohydrogel microneedle patch and its preparation method. A dual-network hydrogel matrix is ​​constructed using methacrylamide gelatin and methacrylamide hyaluronic acid, and PLGA-PEG nanoparticles loaded with lenalidomide are encapsulated as microneedle patches. The resulting microneedle patch exhibits high mechanical strength and can effectively penetrate the skin; its hydrogel matrix can rapidly dissolve and release the drug, significantly improving local drug concentration and cumulative release rate.

Description

Technical Field

[0001] This invention relates to the field of microneedle patch technology, specifically to a lenalidomide-loaded nanohydrogel microneedle patch and its preparation method. Background Technology

[0002] Rheumatoid arthritis (RA) is a multisystemic, inflammatory, autoimmune disease that primarily affects peripheral joints. The autoimmune response leads to vasculitis, tissue edema, fibrinoid degeneration, and inflammatory cell infiltration in the skin and connective tissues, ultimately resulting in ulcer formation. Currently, there are no unified clinical guidelines for the diagnosis and treatment of RA-related ulcers. Common treatment methods are similar to those for other chronic ulcers, including topical treatments and oral medications, but their effectiveness is limited.

[0003] Addressing the limitations of current treatments, the use of microneedle patches—a safe, efficient, and painless drug delivery mechanism—offers a potential treatment for improving skin ulcers in RA patients. Summary of the Invention

[0004] The main objective of this invention is to provide a lenalidomide-loaded nanohydrogel microneedle patch and its preparation method, so as to provide a better way to administer lenalidomide (Len).

[0005] To achieve the above objectives, the present invention provides a method for preparing a lenalidomide-loaded nanohydrogel microneedle patch, comprising the following steps: S1, Preparation of PLGA-PEG / Len nanoparticle suspension: Weigh lactide-glycolic acid copolymer PLGA-PEG and lenalidomide Len according to the preset drug concentration, dissolve them in acetonitrile and then add them dropwise to pure water. After the solvent evaporates, ultrafiltration and centrifugation are performed to obtain PLGA-PEG / Len nanoparticle suspension. S2, Preparation of methacrylamide gelatin: Weigh gelatin at a ratio of 4-6g:40-60ml and add it to phosphate buffered saline (PBS). Heat to 55-65℃ and stir until completely dissolved. Add methacrylic anhydride at a ratio of 4-6g:3.5-4.5g. Stir and react for 3-5 hours. Transfer the filtrate to a dialysis bag, dialyze and purify at 45-55℃, freeze at -70-90℃, and freeze-dry to obtain methacrylamide gelatin. S3, Microneedle mold preparation; S4, Needle casting solution preparation: Prepare a suspension of methacrylamide gelatin, methacrylamide hyaluronic acid, and PLGA-PEG / Len nanoparticles according to the ratio of 2-3% hydrogel concentration of methacrylamide hyaluronic acid and 1-1.5% hydrogel concentration of methacrylamide gelatin in the final casting solution; after mixing, centrifuge the casting solution to remove air bubbles generated during the dissolution process to obtain the needle casting solution; S5, Preparation of backing layer material casting liquid: Weigh Prussian polysaccharide and add it to pure water to dissolve and obtain a 240-260 mg / mL backing layer casting liquid. Centrifuge the backing layer casting liquid to remove the air bubbles generated during the dissolution process to obtain the backing layer material. Step S6, casting microneedles: The casting liquid is added dropwise to the surface of the microneedle mold. The microneedle mold is centrifuged to allow the casting liquid to fully enter the tip of the microneedle mold. After centrifugation, the microneedle mold is placed in a sealed vacuum tank and heated in a water bath. During the process, the vacuum is evacuated to -0.8 kPa to remove bubbles. After removing bubbles, the surface of the microneedle mold is scraped off. After repeated heating and concentration at 30-35℃, the excess liquid on the surface of the microneedle mold is scraped off. The microneedles are then cured by ultraviolet light. Step S7, casting the backing layer and demolding: Place the microneedle mold that has been photocured flat with the micropores facing upward, drip the prepared backing layer casting liquid into the microneedle mold, dry it at 30-40℃ and then take it out. After demolding, the microneedle patch is obtained.

[0006] Preferably, in step S1, the mass ratio of lactide-glycolic acid copolymer (PLGA-PEG) to lenalidomide (Len) is 5:1, and the concentration of the PLGA-PEG / Len nanoparticle suspension is 500 μg / mL.

[0007] Preferably, step S1 includes: dissolving 5 mg of lactide-glycolic acid copolymer PLGA-PEG and 1 mg of nalidomide Len in acetonitrile and slowly adding the solution dropwise to pure water, stirring magnetically at 450 r / min for 12 h, removing impurities from the solution using a 0.22 μm filter membrane, and centrifuging at 3000 r for 3 min using an ultrafiltration centrifuge tube to remove free Len from the solution, thereby obtaining a PLGA-PEG / Len nanoparticle suspension.

[0008] Preferably, step S2 includes: weighing gelatin at a ratio of 5g:50ml and adding it to PBS, heating to 60℃ and stirring until completely dissolved, slowly adding methacrylic anhydride at a ratio of 5g:4 gelatin over 1 hour, stirring for about 2 hours, filtering the resulting solution after the reaction, transferring the filtrate to a dialysis bag and dialysis at 50℃ for 5 days, transferring the dialysis sample to -80℃ for freezing, and after 1 day, transferring it to a freeze dryer for 72 hours to obtain methacrylated gelatin.

[0009] Preferably, step S3 includes: placing the microneedle mold into a beaker, adding an appropriate amount of ultrapure water and detergent, ultrasonically cleaning it in an ultrasonic cleaner for 10 minutes, rinsing it clean with ultrapure water, and then placing the microneedles in a vacuum drying oven to dry them thoroughly for 1 hour.

[0010] Preferably, the centrifugation speed for removing bubbles generated during the dissolution process in steps S4 and S5 is 3000 rpm, and the centrifugation time is 3-8 min.

[0011] Preferably, step S6 includes: using a pipette to drop needle casting liquid onto the surface of the microneedle mold; placing the microneedle mold in a centrifuge tube and centrifuging at 3000 rpm for 3 minutes to ensure the needle casting liquid fully enters the tip of the microneedle mold; placing the centrifuged microneedle mold in a sealed vacuum chamber and heating it in a 50°C water bath; during this period, evacuating to -0.8 kPa for 2 minutes to remove bubbles; then gently scraping off air bubbles from the surface of the microneedle mold with a pipette tip until no air bubbles are generated on the surface of the microneedle mold base pores; placing the microneedle mold in an oven and heating it at 30-35°C; heating and concentrating the mixture multiple times; after concentrating for about 5 hours, scraping off the excess liquid from the surface of the microneedle mold; turning on the UV curing lamp and adjusting the distance between the light source and the microneedles; adjusting the irradiation area of ​​the UV lamp so that the spot size is consistent with the microneedle area; irradiating each microneedle for 2 minutes to allow it to photocur and solidify.

[0012] Preferably, step S7 includes: placing the photocured microneedle mold flat with the micropores facing upwards, uniformly dripping the prepared backing layer casting liquid into the microneedle mold, drying it in a 35°C oven until the liquid is formed, removing it and placing it at room temperature, and after the microneedles are demolded, using tweezers to remove the microneedles from the mold to obtain the microneedles.

[0013] The present invention also provides a lenalidomide-loaded nanohydrogel microneedle patch, which is prepared by the preparation method described above.

[0014] Compared with the prior art, the lenalidomide-loaded nanohydrogel microneedle patch and its preparation method in this invention have at least the following technical effects and advantages: 1. By administering medication locally through microneedle patches, a high local drug concentration can be formed at the site of skin ulcers, avoiding the limitations of systemic medication, while exerting an etiological treatment effect, effectively promoting the healing of RA-related skin ulcers and improving treatment outcomes.

[0015] 2. Impurities and free drug were removed through ultrafiltration, centrifugation, and dialysis purification, improving the purity of PLGA-PEG / Len nanoparticles. Ultrasonic cleaning and drying of the microneedle mold, along with centrifugation to remove bubbles from the casting solution, reduced the impact of impurities and bubbles on microneedle formation, ensuring stable quality of the prepared microneedle patches and effective drug delivery. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0017] Figure 1 The UV absorption spectra of Len, PLGA-PEG, and PLGA-PEG / Len are shown. Figure 2 a is the absorbance graph of different concentrations of Len at 306 nm. Figure 2 b represents the Len standard curve; Figure 3 a represents the particle size distribution curve of NPs. Figure 3 b is the potential diagram. Figure 3 c and Figure 3 d is a transmission electron microscope image of PLGA-PEG / Len; Figure 4 The graph shows the stability test results for PLGA-PEG / Len NPs. Figure 5 a is the experimental diagram of CCK8. Figure 5 b is a diagram of the hemolysis experiment; Figure 6 This is a drug release rate curve; Figure 7 GelMA and Gel NMR 1H NMR spectra; Figure 8 a is a schematic diagram of the hydrogel before photocuring. Figure 8 b is a schematic diagram of the hydrogel after photocuring; Figure 9 a and Figure 9 b is a schematic diagram of the microneedles in macroscopic view. Figure 9 c is an image under a microneedle optical microscope. Figure 9 d is the image under a microneedle scanning electron microscope; Figure 10 This is a force-displacement curve of the microneedle. Figure 11 This is a schematic diagram of microneedle dissolution. Figure 12 This is a diagram illustrating the skin's recovery process.

[0018] Figure 13 a represents the change in mouse body weight. Figure 13 b represents the number of swollen joints in the mouse. Figure 13 c represents the change in the thickness of the mouse's hind paw. Figure 13 d represents the change in the mouse's arthritis index score. Figure 13 The image above shows a comparison of swelling in the left and right hind paws of a mouse.

[0019] Figure 14 The graph shows the efficacy of skin ulcer healing in each group of mice.

[0020] Figure 15 The curves showing the skin healing rate of mice in each group.

[0021] Figure 16Image of mouse skin stained with H&E and Masson staining.

[0022] Figure 17 Image a shows the staining of IL-6 and TNF-α in skin tissue. Figure 17 bc is a quantitative graph showing the number of IL-6 and TNF-α positive cells. Detailed Implementation

[0023] The technical solutions in this embodiment will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0025] It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0026] Mechanism Analysis: TNF-α, IL-1, and IL-6 play important roles in rheumatoid arthritis (RA). They exert their effects through pro-inflammatory mechanisms, activation of infiltrating inflammatory cells, and destruction of cartilage and bone tissue, and are widely expressed in skin tissue. In normal skin healing, appropriate inflammatory factors can promote the inflammatory response, accelerate granulation tissue formation, and promote tissue repair. However, in RA patients, excessive or continuous secretion of these factors by cells accelerates the synthesis and secretion of large amounts of inflammatory factors, disrupting the balance of the body's cytokine network and triggering an inflammatory response. Simultaneously, inflammatory factors also enhance the phagocytic capacity of macrophages and monocytes, accelerate the synthesis and release of various chemokines such as oxygen free radicals, further damaging wound tissue, aggravating wound injury, and leading to poor wound healing or even infection. Therefore, targeting the pathogenesis of RA skin ulcers, the use of anti-TNF-α drugs holds promise for better therapeutic effects. Len, a derivative of thalidomide, belongs to the second-generation immunomodulatory imide class of drugs and has immunomodulatory, anti-angiogenic, and anti-tumor effects. Len's anti-TNF-α activity is 50,000 times that of Thalidomide, with significantly reduced neurotoxicity and reproductive toxicity. In addition to significantly downregulating TNF-α, Len can also inhibit the production of IL-1 and IL-6, which helps heal skin ulcers in patients with rheumatoid arthritis (RA). Len is currently widely used clinically for various connective tissue disease-related skin lesions and immune-related small vessel vasculitis. Numerous clinical cases have shown that after various immunosuppressants and glucocorticoids failed to treat RA-related skin ulcers, treatment with anti-TNF-α agents such as Len resulted in good wound healing and a reduced recurrence rate. However, oral administration has limited efficacy due to low local concentrations. Addressing the limitations of current treatment methods, delivering Len via microneedle patches—a safe, efficient, and painless delivery method—offers a potential therapeutic approach for improving skin ulcers in RA patients.

[0027] Please combine them together Figures 1-17 The method for preparing a lenalidomide-loaded nanohydrogel microneedle patch according to one embodiment of the present invention includes the following steps: Step S1: Weigh 5 mg of lactide-glycolic acid copolymer (hereinafter referred to as PLGA-PEG) and 1 mg of lenalidomide (hereinafter referred to as Len), dissolve them in 1 ml of acetonitrile, and slowly add them dropwise to 10 mL of pure water. Stir magnetically at 450 r / min for 12 h. Remove impurities from the solution using a 0.22 μm filter membrane. Centrifuge at 3000 r for 3 min using a 50 mL ultrafiltration centrifuge tube to remove free Len from the solution, and obtain PLGA-PEG / Len nanoparticles (NPs) suspension. Step S2: Weigh 5.0g of gelatin and add it to 50.0mL of PBS. Heat to 60℃ and stir until completely dissolved. Slowly add 4mL of methacrylic anhydride over 1 hour and stir for about 2 hours. After the reaction is complete, filter the resulting solution. Transfer the filtrate to a dialysis bag and dialyze at 50℃ for 5 days. After dialysis, transfer the sample to -80℃ and freeze. After 1 day, transfer it to a freeze dryer for 72 hours to obtain methacrylated gelatin. Step S3: Place the microneedle mold into a beaker, add an appropriate amount of ultrapure water and a little detergent, ultrasonically clean it in an ultrasonic cleaner for 10 minutes, then rinse it with ultrapure water, and put the microneedles into a vacuum drying oven to dry them thoroughly for 1 hour. Step S4: Weigh 125 mg of methacrylamide gelatin (hereinafter referred to as GelMA) and methacrylamide hyaluronic acid (hereinafter referred to as HAMA), and dissolve them completely in PLGA-PEG / Len nanoparticle suspension so that the concentrations of the two hydrogels in the final casting liquid are 2.5% (w / v) for HAMA and 1.25% (w / v) for GelMA. Then centrifuge the casting liquid at 3000 rpm for 5 min to remove the air bubbles generated during the dissolution process. Step S5: Using Prussian polysaccharide as the microneedle backing material, weigh an appropriate amount of Prussian polysaccharide and add it to ultrapure water to fully dissolve it to obtain a 250 mg / mL backing casting solution. Place the casting solution in a centrifuge and centrifuge at 3000 rpm for 5 min to remove the air bubbles generated during the dissolution process. Step S6: Use a pipette to drop the needle casting liquid onto the surface of the microneedle mold. Place the mold in a 50mL centrifuge tube and centrifuge at 3000rpm for 3min to ensure the casting liquid fully enters the needle tip. After centrifugation, place the mold in a 500mL sealed vacuum chamber and heat it in a 50℃ water bath. During this process, evacuate to -0.8kPa for 2min to remove bubbles. Then, use a pipette tip to gently scrape off the air bubbles on the mold surface (repeat twice) until no air bubbles are generated on the surface of the mold base pores. Place the mold in an oven and heat it at 30℃. Repeat the heating and concentration process. After about 5 hours of concentration, scrape off the excess liquid from the mold surface. Turn on the UV curing lamp (wavelength: 504nm) and adjust the distance between the light source and the microneedles. Adjust the irradiation area of ​​the UV lamp so that the spot size is consistent with the microneedle area. Irradiate each microneedle for 2min to allow it to photocur and solidify. Step S7: Place the photocured microneedle mold flat with the micropores facing upwards, and evenly drip the prepared backing layer casting liquid into the mold. Dry it in a 35°C oven until the liquid is formed, then remove it and place it at room temperature. After the microneedles are demolded, use tweezers to remove the microneedles from the mold to obtain the microneedles.

[0028] The following is a description of the source of the reagent materials used in this embodiment: 1. Len, Solarb China; 2. PLGA-PEG, Unisco China; 3. Acetonitrile, Abclonal (China); 4. Fetal bovine serum, Solarbio China; 5. Penicillin-streptomycin bispecific antibiotic solution, Solarbio China; 6. Gel, Solarb China; 7. MA, Chondrex (USA); 8. HAMA, Chondrex (USA); 9. Prussian polysaccharides, Chondrex (USA); 10. CCK-8 solution, Solarbio China.

[0029] In this embodiment, 10 mg of Len was weighed and placed in a 10 mL volumetric flask. Pure water was added to dilute to the mark to obtain a 1 mg / mL Len stock solution. An appropriate amount of the stock solution was weighed and placed in separate 10 mL volumetric flasks. Water was added to the mark to prepare reference solutions of 1.5625, 3.125, 6.25, 12.5, 25, 50, 100, and 200 μg / mL. A wavelength scan at 200 nm was performed using a UV spectrophotometer to confirm the detection wavelength. The vertical axis represents absorbance, and the horizontal axis represents mass concentration. A standard curve was plotted by selecting the appropriate detection wavelength.

[0030] Take 500 μL of the PLGA-PEG / Len NPs suspension prepared above, add 500 μL of acetonitrile to disrupt the nanoparticle structure and release the drug. Measure the absorbance of the nanoparticle suspension using UV-Vis spectrophotometry. Calculate the Len drug concentration using the standard curve formula obtained above, and calculate the nanoparticle encapsulation efficiency and drug loading rate. The formulas for encapsulation efficiency and drug loading rate are as follows: Encapsulation efficiency (%) = C × V (after membrane) / W (input Len) × 100% Drug loading rate (%) = C × V (after membrane) / W (Len added) + W (PLGA-PEG added) × 100%.

[0031] Len solution and PLGA-PEG / LenNPs suspension were measured repeatedly 5 times under the same chromatographic conditions. The full wavelength scan results and the encapsulation efficiency and drug loading rate of PLGA-PEG / LenNPs suspension were recorded each time. Prepare 2 mL of PLGA-PEG / LenNPs suspension of known concentration. Weigh an appropriate amount of Len and dissolve it in 2 mL of acetonitrile. Add the solutions to pre-treated and cleaned dialysis bags, tying both ends tightly. Gently squeeze the tied dialysis bags to ensure no drug leakage, then place them in 50 mL centrifuge tubes with 30 mL of dissolution medium. Perform three parallel measurements, shaking at 70 rpm for 60 h. Take 500 μL samples at 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 48, and 60 h, and calculate the drug concentration at each time point. Immediately after sampling, add 500 μL of the same medium and calculate the drug release rate at each time point. The calculation formula is as follows: Release rate (%) = C × 30 / W (input amount) × 100%.

[0032] The cell compatibility of PLGA-PEG / LenNP was tested using human umbilical vein endothelial cells (HUVECs). HUVECs were placed in endothelial cell culture medium containing 10% fetal bovine serum, 1% penicillin-streptomycin solution, and 1% endothelial cell growth supplement, and cultured in a cell culture incubator at 37°C and 5% CO2. HUVECs were then seeded at a density of 1×10⁴ cells / well in 96-well plates and returned to the incubator for overnight culture to allow complete adhesion. Subsequently, an appropriate amount of NP suspension was added to each well to achieve a final NP concentration of (0, 25, 50, 100, 150, 200 µg / mL) in the culture medium, and the cells were co-incubated. Then, 10 μL of CCK-8 solution was added to each well, and after 3 hours of incubation, the optical density at 450 nm was measured using a microplate reader. Cell viability was calculated using the following formula: Cell viability (%) = [(As-Ab) / (Ac-Ab)] × 100% As: Absorbance of experimental wells (including cells, culture medium, CCK-8 solution and PLGA-PEG / LenNPs) Ac: Absorbance of control wells (containing cells, culture medium, and CCK-8 solution, but excluding PLGA-PEG / LenNPs). Ab: Absorbance of blank wells (including culture medium and CCK-8 solution, excluding cells and PLGA-PEG / LenNPs).

[0033] One mL of fresh ocular blood from DBA / 1J mice was collected in an EDTA anticoagulant tube and centrifuged at 750 g for 5 min. Red blood cells were obtained after washing five times with PBS buffer. 100 μL of red blood cells were added to EP tubes containing 0.9 mL of deionized water, PBS buffer, and different concentrations of NP suspension (0, 25, 50, 100, 200 µg / mL). The deionized water group served as the positive control, the PBS group as the negative control, and the other groups as the experimental groups. After mixing, the EP tubes were placed in an air bath-thermal shaker and incubated at 37 °C for 4 h. Then, the tubes were centrifuged at 750 g for 5 min to obtain the supernatant. Finally, the optical density of the supernatant at 540 nm was measured using a microplate reader, and the hemolysis rate of the sample was calculated using the following formula: Hemolysis rate (%) = (NPs - negative control) / (positive control - negative control) × 100%.

[0034] Microneedle molds were obtained by precisely drilling conical holes in a flexible polydimethylsiloxane (PDMS) substrate using laser etching technology. Each mold contains 225 holes (array of 15×15), with a hole depth of 600μm, a diameter of 250μm, and a center-to-center distance of 550μm between adjacent holes.

[0035] The microstructure of PLGA-PEG / LenNPs was observed using a transmission electron microscope. An appropriate amount of PLGA-PEG / LenNPs suspension was dropped onto a copper mesh covered with a carbon film, ultrasonically dispersed, and then the copper mesh was rapidly dried.

[0036] The particle size and zeta potential of PLGA and PLGA-PEG / LenNPs were determined using a dynamic light scattering particle size analyzer. The prepared NPs suspension was stored at 4℃, and its particle size and PDI were measured at 0, 1, 2, 3, 4, 5, 6 and 7 days. The data were recorded and the trend was plotted.

[0037] Take an appropriate amount of GelMA and gelatin (hereinafter referred to as Gel) and dissolve them in 600 μL. Detect Gel and GelMA using a nuclear magnetic resonance spectrometer.

[0038] The morphology of the microneedles (MNs) was observed using a digital camera, optical microscope, and scanning electron microscope. This included the substrate diameter of the prepared MN patch, the tip height, and the distance between the tips of adjacent MNs and the substrate.

[0039] The mechanical strength of MN depends on the strength of the needle body. The strength of the microneedle body is tested using a universal tensile and compression tester. The force sensor speed is kept constant at 2 mm / min, and the maximum compression force is set to 100 N.

[0040] The prepared MN patch was fixed onto the skin of 5 SD rats using an MN ejector syringe, with the needle tip fully inserted. The microneedle patch was removed at 1, 2, 3, 4 and 5 minutes after insertion, and photographed under an optical microscope to observe the dissolution of the microneedles.

[0041] SD rats were anesthetized and their limbs were immobilized. Microneedles were inserted into the skin on the back of the rats using an MN ejector syringe and secured with medical tape. After 5 minutes, the tape was removed, the microneedles were withdrawn, and the skin recovery was observed. The skin sites where the microneedles were inserted were photographed every 0.5 hours to observe the skin recovery.

[0042] The following describes the characterization of lenalidomide-loaded nanohydrogel microneedle patches.

[0043] (1) Characterization of PLGA-PEG / Len nanoparticles and microneedles.

[0044] Len, in a full-wavelength ultraviolet scan of 200–800 nm, shows absorption peaks at 219 nm, 245 nm, and 306 nm, respectively. Figure 1 The ultraviolet absorption at 306 nm was found to be largely unaffected and relatively large, leading to the determination of 306 nm as the detection wavelength for Len. The relative standard deviation (RSD) of the five repeatability tests was 0.17 (Table 1), indicating good repeatability of the method.

[0045] Table 1 Repeatability of Len Peak Value The absorbance of different concentrations of Len at 306 nm was measured. Figure 2 a) Linear regression yielded the regression equation A = 55291C + 15579 (R² = 0.999), indicating that the Len solution exhibits good linearity within the concentration range of 1.5625–200 μg / mL, meeting the requirements. Figure 2 b).

[0046] PLGA-PEGNPs can be observed to be spherical nanoparticles under transmission electron microscopy. Figure 3 cd), the hydrated particle size is around 58nm ( Figure 3 a) The measured PDI was below 0.2. The potential of PLGE-PEG / LenNPs was -20.1 ± 1.253 (a). Figure 3 b). The encapsulation efficiency and drug loading rate were 91.2±2.37% and 17.5±1.53%, respectively (Table 2).

[0047] Table 2 PLGA-PEG, PLGA-PEG / Len particle size / zeta potential / encapsulation efficiency / drug loading The changes in particle size and polydispersity index (PDI) of PLGA-PEG / LenNP after storage at 4℃ for 7 days were investigated. The particle size of NP remained stable at around 58.0 nm, and the PDI was below 0.2. Figure 4 The particle size of PLGA-PEG / LenNP did not change significantly and remained relatively stable.

[0048] PLGA-PEG / LenNP, as a product released after microneedle administration, comes into direct contact with skin tissue; therefore, evaluating the biosafety of PLGA-PEG / LenNP is crucial. With increasing NP suspension concentration, HUVECs cell viability showed no significant change, remaining above 95%. Figure 5 a) indicates that the NP suspension has low cytotoxicity. The hemolysis experiment showed strong hemolysis in the positive control group, while all NP suspensions in each group exhibited good blood compatibility and no hemolysis was observed. Figure 5 b). All the above experiments demonstrate that PLGA-PEG / LenNP has excellent biocompatibility.

[0049] Len is completely released in approximately 24 hours. PLGA-PEG / LenNPs exhibit a burst release, with peak release reaching approximately 61% in about 4 hours. Figure 6 ).

[0050] Table 3. Drug release rate (h) at each time point Compared to the spectrum of unmodified Gel, GelMA showed new characteristic peaks at 5.497 and 5.258 ppm (red arrows), which are characteristic C=C peaks on MA. The decrease in the proton peak of the lysine methylene group at 2.548-3.041 ppm (red box) indicates that MA reacted with Gel, MA was successfully grafted, and the synthesis of GeIMA was successful. Figure 7 ).

[0051] The cross-linking mechanism of hydrogels plays an important role in structure formation and degradation. The photocurable hydrogel prepared in this experiment is a slightly yellowish liquid after curing, with a uniform and delicate texture. It can rapidly gel after ultraviolet irradiation, and the photocuring effect is good. Figure 8 ).

[0052] The microneedles consist of a 15×15 microneedle array. Figure 9 ab). The microneedles are conical in shape, with a height of approximately 600 μm, a tip-to-tip distance of approximately 550 μm, and a base diameter of approximately 250 μm. Figure 9 (cd). The mechanical properties of the microneedles were tested using a universal testing machine. The stress of the microneedle patch can reach 30N, or 0.14N / needle.

[0053] Based on their different functional mechanisms, hydrogel microneedles can be divided into dissolution microneedles and phase change microneedles. The microneedles used in this study are dissolution microneedles. These microneedles dissolve or degrade in the skin and do not need to be removed from the patient's skin during use. After insertion into the skin, the drug carried by the dissolution microneedle is released into the body along with the dissolution of the microneedle, and the drug delivery efficiency is higher. The microneedles prepared in this experiment completely dissolved within approximately 30 minutes of skin insertion, and the PLGA-PEG / LenNPs were completely released. Figure 11 ).

[0054] Although microneedles can enter skin tissue painlessly, non-invasively, and without infection, they still create numerous "micropores" on the skin surface. In this experiment, the skin surface basically returned to normal 120 minutes after the microneedles were inserted. Figure 12 This indicates that the damage caused by microbes to the tissue is negligible.

[0055] By removing the full thickness of the skin on the back of CIA mice, skin ulcers were artificially created to mimic the skin ulcers of RA patients. The prepared GelMA-HAMA@PLGA-PEG / Len microneedles were used for treatment, and the efficacy was evaluated.

[0056] (2) CIA mouse model DBA mice were subcutaneously injected at multiple points on their backs and tail bases with a collagen emulsion (a 1:1 mixture of bovine type II collagen and Freund's complete adjuvant), 1.0 ml per rat. Ten days after the initial immunization, a booster immunization was administered, with another 1.0 ml emulsion (a 1:1 mixture of bovine type II collagen and Freund's incomplete adjuvant) injected subcutaneously into the back and tail base. Seven days prior to the second immunization, the mice were fed a high-fat diet. Approximately two days after the second immunization, the mice developed arthritis symptoms with joint redness and swelling. We regularly observed and recorded the redness and swelling of the toes and ankles of the mice in each group, recording the paw swelling score every 2-3 days.

[0057] CIA mouse data measurement: Each mouse was ear-tagged, and its weight was recorded weekly from the start of rearing. After successful CIA model establishment, the number of swollen joints and the thickness of the hind paws were measured every 3 days, and an arthritis index score was calculated. The arthritis index scoring criteria are shown in the table below.

[0058] Table 4. CIA Mouse Arthritis Index Scoring Criteria (3) Mouse grouping and skin ulcer modeling Twenty male DBA / 1J mice were randomly divided into five experimental groups, as detailed in the table below. Except for the DBA group, the other four groups underwent CIA modeling using the method described below. Skin ulceration was induced in all mice simultaneously after modeling was completed.

[0059] Skin ulcer modeling method: CIA mice under general anesthesia were fixed face down on a board, their limbs restrained. Hair was shaved and skin prepared on the side of the mouse's back, slightly off-center to prevent scratching and interference with the experiment. Filter paper soaked in gentian violet solution was placed on the shaved area using tweezers, and gently pressed to imprint a marking area of ​​the same size and shape as the paper on the rat's back. The epidermis of the marked area was then cut away along the edge of the marking. The skin ulcer model in the DBA control group followed the same steps as above, except that the mice used DBA.

[0060] Mice with skin ulcers were housed individually after the ulceration model was established. The interventions for each group are shown in Table 8. Bedding was changed regularly to maintain a dry and clean environment. Wound diameter was measured daily, wound area was calculated, and data were recorded to plot the healing rate curve. After 14 days, mice were sacrificed, and skin samples from the wound area were collected to assess local skin pathology. The healing rate was calculated using the following formula: Healing rate (%) = (W - Wn) / W × 100% W represents the wound area on day 1, and Wn represents the wound area on day n.

[0061] Table 5. Mouse grouping and treatment (4) Mouse skin histopathology HE staining 1) Dewax in environmentally friendly dewaxing agents 1, 2 and 3 in sequence, then rinse with anhydrous ethanol, 95% ethanol, 85% ethanol and 75% ethanol for 5 minutes each, and rinse with running water for 1 minute.

[0062] 2) Stain with hematoxylin staining agent and rinse with running water.

[0063] 3) Dissolve the hydrochloric acid in alcohol for 2 seconds, then rinse with running water.

[0064] 4) Stain with eosin solution for 20 seconds, add 95% ethanol to adjust the color for 5 seconds, and add anhydrous ethanol 1 and 2 for dehydration for 2 minutes.

[0065] 5) Clearing agent is used to make the product transparent, seal it, and then examine it under a microscope.

[0066] Masson staining 1) After dewaxing and dehydration, place in fixative overnight and rinse with running water.

[0067] 2) Stain with hematoxylin for 5-10 minutes, then rinse with running water.

[0068] 3) Hydrochloric acid is used for alcohol differentiation, then rinsed with running water; lithium carbonate is used for blueing, then rinsed with running water.

[0069] 4) Dye with magenta solution for 5-10 minutes, then rinse with running water.

[0070] 5) Counterstain with aniline blue solution for 5 minutes.

[0071] 6) Treat with 1% glacial acetic acid for 1 minute, then dehydrate with 95% alcohol multiple times.

[0072] 7) Dehydrate with anhydrous alcohol, clear with xylene, and seal with resin.

[0073] Immunohistochemistry (IL-6, TNF-α) 1) Dewax in environmentally friendly dewaxing agents 1, 2, and 3 in sequence, then rinse with anhydrous ethanol, 95% ethanol, and 75% ethanol for 5 minutes each. Wash three times with distilled water, then soak.

[0074] 2) Add citrate antigen retrieval solution to the pressure cooker and immerse it in boiling buffer for 2 minutes. Rinse the pressure cooker with cold water and allow the retrieval solution to cool naturally to room temperature. Remove the slides from the retrieval solution and rinse them three times with distilled water for 5 minutes each time.

[0075] 3) The repaired sections were soaked in 3% H2O2 at room temperature in the dark for 30 minutes, and then rinsed with distilled water.

[0076] 4) After grouping the stroke circles, place them in TBST.

[0077] 5) Add 10% serum of the same source as the secondary antibody and incubate at room temperature for 30 minutes.

[0078] 6) Remove the serum, add 50-100 μL of primary antibody working solution to each slide, and incubate overnight.

[0079] 7) Rinse 3 times with TBST, add 50-100 μL of secondary antibody working solution to each slice, incubate at 37°C for 45 minutes, rinse 3 times with TBST, and then soak 3 times, each time for 3 minutes.

[0080] 8) Discard the TBST and add 50 μL of freshly prepared DAB colorimetric solution.

[0081] 9) After staining the nucleus with hematoxylin, wash it clean, then after differentiation with hydrochloric acid and alcohol, wash it clean again after a few seconds of blueing with the blueing solution.

[0082] 10) Soak the stained slides in two separate tanks of anhydrous alcohol and let them air dry at room temperature.

[0083] 11) Environmentally friendly sealing tablets.

[0084] All data from this experiment were statistically processed using SPSS 26.0 software. One-way ANOVA was used for comparisons between groups, LSD test was used for pairwise comparisons of homogeneous variances, and other tests were used for pairwise comparisons of unequal variances. t test, P >0.05 indicates no significant difference; P The difference <0.05 is statistically significant.

[0085] The body weight of mice in all groups increased, but the body weight increase in the CIA mouse group was slower than that in the normal control group. Figure 13 a). On days 2-3 after the second immunization, except for the normal control group, paw swelling was observed in mice in other groups. The number of swollen joints and the thickness of the hind paws reached their peak on day 44. Figure 13 bc). Following a second immunization, CIA mice experienced a rapid and significant increase in their arthritis index. In the late stages, they exhibited joint stiffness, inability to place their feet on the ground while walking, and other mobility impairments. Figure 13 d) CIA mouse model was successfully established.

[0086] In vivo animal experiments were conducted using a skin ulcer model. On day 14, all mouse wounds closed, but the degree of skin recovery was superior in the DBA, hydrogel, and MN groups compared to the CIA and NP groups (Figure 14). The degree of wound healing varied significantly among the groups in the first 7 days. Comparing the wound closure rates in each group, the CIA mouse group had the lowest closure rate on day 7. Mice treated with the MN group showed a significantly higher healing rate than the CIA group (P<0.0001), indicating that MN intervention significantly promoted the healing of skin ulcers in CIA mice. Conversely, the healing rate in the CIA group was significantly lower than that in the DBA group (P<0.0001).

[0087] Figure 14 In the diagram, blue, yellow, and green represent the wound area on days 0, 3, and 7, respectively. NP group: PLGA-PEG / Len NP intervention group; hydrogel group: GelMA-HAMA@PLGA-PEG / Len hydrogel intervention group; MN group: GelMA-HAMA@PLGA-PEG / Len microneedle intervention group.

[0088] Figure 15 In the middle, the NP group: PLGA-PEG / Len NP intervention group; hydrogel group: GelMA-HAMA@PLGA-PEG / Len hydrogel intervention group; MN group: GelMA-HAMA@PLGA-PEG / Len microneedle intervention group.

[0089] Table 6. Skin healing rate (%) of mice in each group during the first 7 days. Note: Compared with the DBA group #### P <0.0001; compared with the CIA group, P <0.0001.

[0090] NP group: PLGA-PEG / Len NP intervention group; hydrogel group: GelMA-HAMA@PLGA-PEG / Len hydrogel intervention group; MN group: GelMA-HAMA@PLGA-PEG / Len microneedle intervention group.

[0091] (5) Pathological analysis of mouse skin H&E staining The epidermis and skin appendages of mice in all groups showed repair and regeneration. In the DBA group, subcutaneous histology showed clear stratification with no increase in cellular components. In the CIA group, the subcutaneous tissue showed focal and widespread thickening, with increased collagen and cellular components, manifested as abundant fibroblast proliferation and collagen hyperplasia. Numerous lymphocytes were observed randomly interspersed among the proliferating collagen fibers or clustered independently, infiltrating the subcutaneous tissue. Compared to the CIA group, the severity of subcutaneous tissue lesions decreased sequentially in the NP, hydrogel, and MN groups, with progressively decreasing subcutaneous tissue thickness and cellular components. The NP group still showed significant collagen hyperplasia and lymphocyte infiltration in the subcutaneous tissue, the hydrogel group mainly showed a small amount of collagen hyperplasia and lymphocyte infiltration, and the MN group showed only a very small amount of collagen hyperplasia and lymphocyte infiltration in its skin pathology.

[0092] Masson staining Except for the DBA group, all other groups showed positive areas of collagen fibers of varying sizes in the subcutaneous tissue, appearing blue. In the CIA group, the positive areas of subcutaneous collagen fibers were clustered and regionally distributed, with a relatively dense blue color. The NP group showed fewer positive areas of subcutaneous collagen fibers than the CIA group, mainly appearing in an interspersed pattern, approximately uniformly distributed throughout the thickened subcutaneous tissue. The hydrogel group showed a further reduction in positive areas of collagen fibers compared to the NP group, with a distribution pattern similar to the NP group. The MN group had the fewest positive areas of collagen fibers, mainly concentrated in the deep layers of the subcutaneous tissue, exhibiting a linear distribution.

[0093] Figure 16 middle: NP group: PLGA-PEG / Len NP intervention group; hydrogel group: GelMA-HAMA@PLGA-PEG / Len hydrogel intervention group; MN group: GelMA-HAMA@PLGA-PEG / Len microneedle intervention group.

[0094] TNF-α Immunohistochemical staining No increased cellular components were observed in the subcutaneous tissue of the DBA group, and no TNF-α positive cells were observed, with only a small number of false-positive cells. In the CIA mouse group, the number of TNF-α positive cells in the subcutaneous tissue was relatively large, appearing in clusters and concentrated at the edges of lymphocyte aggregation areas or interspersed between lymphocyte aggregation areas (black arrows). These TNF-α positive cells were large, with abundant, dark brown cytoplasm and blue nuclei. Compared to the CIA group, the NP group still showed a large number of TNF-α positive cells in the subcutaneous tissue, appearing in clusters or scattered, mainly in the proliferating connective tissue or at the edges of lymphocyte aggregation areas (black arrows). In the hydrogel group, TNF-α positive cells were large, individually distributed in the dermis and surrounding proliferating connective tissue, with strongly granular cytoplasm (black arrows). The MN group showed almost no obvious TNF-α positive cell populations in the subcutaneous tissue.

[0095] IL-6 immunohistochemical staining No increase in cellular components was observed in the subcutaneous tissue of the DBA group, therefore no IL-6 positive cells were observed. In the CIA group, the number of positive cells in the subcutaneous tissue was relatively large, scattered, and singly distributed in areas of lymphocyte aggregation (black arrows). The cytoplasm of IL-6 positive cells showed moderate brown staining, while the nuclei showed blue staining. Compared to the CIA group, a small number of IL-6 positive cells were observed in the subcutaneous tissue of the NP group, randomly and singly distributed in areas of lymphocyte aggregation (black arrows), with no obvious IL-6 positive staining observed in the surrounding proliferating connective tissue. Almost no obvious IL-6 positive cell populations were observed in the subcutaneous tissue of the hydrogel and MN groups.

[0096] Figure 17 middle, P<0.0001, P<0.001, P<0.01, P<0.05.

[0097] NP group: PLGA-PEG / Len NP intervention group; hydrogel group: GelMA-HAMA@PLGA-PEG / Len hydrogel intervention group; MN group: GelMA-HAMA@PLGA-PEG / Len microneedle intervention group.

[0098] The above are merely preferred embodiments of the present invention and do not limit the scope of the patent. Any equivalent structural or procedural transformations made based on the description and drawings of the present invention, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A method of preparing a nanohydrogel microneedle patch loaded with lenalidomide, characterized by, The method comprises the steps of: S1, preparing a PLGA-PEG / Len nanoparticle suspension: according to a predetermined drug concentration, lactide-glycolide copolymer PLGA-PEG and lenalidomide Len are weighed, dissolved in acetonitrile, and then dropped into pure water. After the solvent is volatilized, the PLGA-PEG / Len nanoparticle suspension is obtained by ultrafiltration centrifugation; S2, preparing methacrylated gelatin: according to the ratio of 4-6 g:40-60 ml, gelatin is weighed and added to a phosphate buffer solution PBS, heated to 55-65 DEG C and stirred until completely dissolved. According to the ratio of gelatin: methacrylic anhydride 4-6 g:3.5-4.5, methacrylic anhydride is added, and the filtrate is transferred to a dialysis bag after stirring for 3-5 hours. After dialysis purification at 45-55 DEG C, freeze at-70-90 DEG C, and freeze-dry to obtain methacrylated gelatin; S3, microneedle mold preparation; S4, needle body casting liquid preparation: according to the ratio of 2-3% of the hydrogel concentration of methacrylated hyaluronic acid and 1-1.5% of the hydrogel concentration of methacrylated gelatin in the final casting liquid, methacrylated gelatin, methacrylated hyaluronic acid and PLGA-PEG / Len nanoparticle suspension are prepared; After mixing, the casting liquid is centrifuged to remove the bubbles generated during the dissolution process to obtain the needle body casting liquid; S5, backing layer material casting liquid preparation: Prussian polysaccharide is weighed and added to pure water to obtain a 240-260 mg / mL backing layer casting liquid. The backing layer casting liquid is centrifuged to remove the bubbles generated during the dissolution process to obtain the backing layer material; Step S6, casting microneedle: the needle body casting liquid is added to the surface of the microneedle mold, and the needle body casting liquid is fully introduced into the microneedle mold tip by centrifuging the microneedle mold. The microneedle mold is placed in a sealed vacuum tank and heated in a water bath. During the process, the vacuum is extracted to-0.8 kPa to remove bubbles. The surface bubbles of the microneedle mold are scraped off, and the excess liquid on the surface of the microneedle mold is scraped off after multiple heating and concentration at 30-35 DEG C. The microneedle is cured by ultraviolet light; Step S7, casting backing layer and demolding: the microneedle mold that has been photo-cured and formed is placed flat with the micropores facing up. The prepared backing layer casting liquid is dropped into the microneedle mold, and the microneedle patch is obtained after drying at 30-40 DEG C and demolding.

2. The process for the preparation of the nano-hydrogel microneedle patch loaded with lenalidomide as claimed in claim 1 wherein, The mass ratio of lactide-glycolide copolymer PLGA-PEG and lenalidomide in step S1 is 5:1, and the concentration of the PLGA-PEG / Len nanoparticle suspension is 500 μg / mL.

3. The method of claim 2, wherein the nanohydrogel microneedle patch loaded with lenalidomide is prepared by, In step S1, 5 mg of lactide-glycolide copolymer PLGA-PEG and 1 mg of lenalidomide Len are dissolved in acetonitrile and slowly added to pure water. The solution is stirred at 450 r / min for 12 h. The solution is filtered with a 0.22 μm filter to remove impurities. The solution is centrifuged at 3000 r for 3 min using an ultrafiltration centrifuge tube to remove free Len, and a PLGA-PEG / Len nanoparticle suspension is obtained.

4. The process for preparing the nano-hydrogel microneedle patch loaded with lenalidomide as claimed in claim 1, wherein, The step S2 includes: weighing gelatin according to the ratio of 5g:50ml, adding it into PBS, heating to 60℃ and stirring until completely dissolved, slowly adding methacrylic anhydride according to the ratio of 5g:4 within 1h, stirring for about 2h, after the reaction is completed, the obtained solution is suction filtered, the obtained filtrate is transferred to a dialysis bag and dialyzed and purified at 50℃ for 5 days, the sample after dialysis is transferred to-80℃ for freezing, after 1 day, it is transferred to a freeze dryer for 72h, and methacrylated gelatin is obtained.

5. The process for preparing the nano-hydrogel microneedle patch loaded with lenalidomide as claimed in claim 1, wherein, The step S3 includes: placing the microneedle mold into a beaker, adding appropriate amount of ultrapure water and detergent, ultrasonic cleaning in an ultrasonic cleaner for 10min, and then rinsing with ultrapure water, and placing the microneedle into a vacuum drying oven for sufficient drying for 1h.

6. The process for preparing the nano-hydrogel microneedle patch loaded with lenalidomide as claimed in claim 1, wherein, The centrifugal speed for removing the bubbles generated in the dissolving process in the steps S4 and S5 is 3000 rpm, and the centrifugal time is 3-8min.

7. The process for preparing the nano-hydrogel microneedle patch loaded with lenalidomide as claimed in claim 1, wherein, The step S6 includes: using a pipette gun to drop the needle body casting liquid onto the surface of the microneedle mold, placing the microneedle mold in a centrifuge tube and centrifuging at 3000 rpm for 3min, so that the needle body casting liquid fully enters the tip of the microneedle mold, placing the microneedle mold in a sealed vacuum tank after centrifugation, heating in a water bath at 50℃, during which the vacuum is extracted to-0.8kPa to remove bubbles for 2min, then using a pipette gun head to gently scrape off the bubbles on the surface of the microneedle mold until no bubbles are generated on the surface of the microneedle mold, placing the microneedle mold into an oven and heating at 30-35℃, and concentrating multiple times, after about 5h of concentration, scraping off the excess liquid on the surface of the microneedle mold, turning on the ultraviolet curing lamp and adjusting the distance between the light source and the microneedle, adjusting the irradiation area of the ultraviolet lamp so that the spot size is consistent with the area of the microneedle, and irradiating each microneedle for 2min to make it photo-cured and formed.

8. The process for preparing the nano-hydrogel microneedle patch loaded with lenalidomide as claimed in claim 1, wherein, The step S7 includes: placing the microneedle mold that has been photo-cured and formed flat with the micropores upward, uniformly dropping the prepared backing layer casting liquid onto the microneedle mold, drying in a 35℃ oven until the liquid is formed, then taking it out and placing it at room temperature, after the microneedle is demolded, using tweezers to take out the microneedle from the mold, and obtaining the microneedle.

9. A nanohydrogel microneedle patch loaded with lenalidomide, characterized by: The preparation method is prepared by using any one of claims 1-8. The preparation method is prepared by using any one of claims 1-8.

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