Soluble microneedle loaded with triptolide and salvianolic acid A as well as preparation method and application of soluble microneedle

Abstract

The invention provides a soluble microneedle loaded with triptolide and salvianolic acid A as well as a preparation method and application of the soluble microneedle, and relates to the technical field of pharmaceutical preparations. According to the invention, the soluble microneedle is utilized to load the triptolide nanocrystal and the salvianolic acid A, and the medicine which can directly act on the diseased joint of a patient with rheumatoid arthritis through local transdermal administration is prepared. By using the soluble microneedle loaded with the triptolide and the salvianolic acid A, the skin delivery capacity can be improved, accumulation of diseased regions is increased, meanwhile, side effects caused by systemic drug absorption are reduced, the curative effect is improved by using the two drugs together, and the soluble microneedle loaded with the triptolide and the salvianolic acid A has a better treatment effect on rheumatoid arthritis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical preparations, in particular to a soluble microneedle loaded with triptolide and salvianolic acid A, and a preparation method and application thereof. BACKGROUND

[0002] Rheumatoid arthritis (RA) is a common chronic inflammatory disease and systemic autoimmune disease, mainly involving the synovial joints, and with disease progression, it can also involve other parts of the body, including the heart, lungs, kidneys and other internal organs. Its characteristics are the destruction of immune regulation in the synovial membrane, the presence of systemic inflammation and autoantibodies, and eventually leading to severe damage and destruction of cartilage and bone. The main clinical manifestations of RA are chronic, peripheral, symmetrical and polyarthritic lesions. The main pathological features of RA patients are synovial intimal cell proliferation, synovial inflammation, many inflammatory cells in the interstitium, the formation of new microvessels and membranes, and the destruction of bone matrix and cartilage in the joint.

[0003] Triptolide (TP) is the main active ingredient extracted from Tripterygium wilfordii Hook F. It is known to have multi-target anti-RA activity, including inhibiting osteoclast differentiation and bone resorption, inducing fibroblast-like synoviocyte (FLS) apoptosis, inhibiting FLS proliferation, migration and invasion, regulating inflammatory response and inhibiting pathological angiogenesis. Its mechanism of action involves the regulation of TLR4 / NF-κB, JAK2 / STAT3 and MAPK signaling pathways.

[0004] Salvia miltiorrhiza is a commonly used medicinal material for treating RA in traditional Chinese medicine, and is often used in combination with Tripterygium. Its main active ingredient, salvianolic acid A (SAA), has been shown to have anti-RA effects, including inhibiting the production of inflammatory mediators, protecting chondrocytes, inhibiting extracellular matrix (ECM) degradation (such as down-regulating ADAMTS-5, MMP1, and MMP13 expression), and promoting ECM synthesis (such as increasing collagen II and aggrecan). The effects of SAA are related to the regulation of the NF-κB pathway.

[0005] Existing studies have shown that although TP has strong anti-RA activity, its clinical application is limited by potential adverse reactions such as liver toxicity. It is worth noting that studies have found that the combination of SAA and TP can enhance the anti-RA efficacy of TP and effectively reduce the toxic side effects of TP-induced liver damage. The liver-protective effect of SAA is related to the regulation of liver metabolic profiles and the expression of related genes.

[0006] Therefore, based on the well-defined anti-RA mechanisms of TP and SAA, and their synergistic (synergistic anti-RA) and toxicity-reducing (reduced TP hepatotoxicity) effects when used in combination, the development of a soluble microneedle loaded with triptolide and salvianolic acid A provides a basis for a more effective and safer RA treatment strategy. Summary of the Invention

[0007] In view of this, the present invention provides a soluble microneedle loaded with triptolide and salvianolic acid A, its preparation method, and its application. The microneedle acts directly on the affected skin of rheumatoid arthritis patients through local transdermal drug delivery, allowing for better penetration of the stratum corneum and avoiding injection pain. It accumulates at the lesion site, thereby successfully clearing the inflammatory response of rheumatoid arthritis. Simultaneously, compared with injection and oral administration, it reduces the toxicity and side effects associated with systemic drug absorption.

[0008] On one hand, the present invention provides a soluble microneedle loaded with triptolide and salvianolic acid A, comprising a tip matrix and a backing matrix loaded with triptolide nanocrystals, salvianolic acid A and blank microneedles.

[0009] Furthermore, the triptolide nanocrystals comprise triptolide raw material, stabilizer, aqueous phase, and organic phase.

[0010] Furthermore, the backing matrix comprises sodium carboxymethyl cellulose (CMC-Na) and water, wherein the amount of CMC-Na in the backing matrix is ​​0.3~0.6%.

[0011] Furthermore, the mixing ratio of the triptolide raw material to the stabilizer is 1:(0.5-3); and / or, the ratio of the organic phase to the aqueous phase is 1:(5-30).

[0012] Furthermore, the needle tip matrix is ​​chondroitin sulfate (CS) and polyvinylpyrrolidone-K30 (PVP-K30).

[0013] On the other hand, the present invention provides a method for preparing soluble microneedles loaded with triptolide and salvianolic acid A, comprising the following steps: Tripterygium wilfordii raw material and stabilizer were dissolved in anhydrous ethanol, vortexed and then sonicated to obtain an organic solution containing stabilizer as the organic phase. Use ultrapure water as the aqueous phase; At room temperature, the organic phase is injected into the aqueous phase while stirring, and the stirring is maintained during the process to obtain an initial suspension; After ultrasonic stabilization, the ethanol was removed by rotary evaporation to obtain triptolide (TP-NCs) nano-suspension. Tanshinone A was dissolved in the triptolide (TP-NCs) nano-suspension to obtain a first mixed solution. Then, chondroitin sulfate was weighed and mixed with PVP-K30 to obtain a mixed powder. The mixed powder was sprinkled on the surface of the first mixed solution and allowed to swell overnight to obtain a drug-containing needle matrix. CMC-Na was sprinkled on the water surface and allowed to swell overnight to obtain the backing matrix; The drug-containing needle matrix was placed in the cavity of the PDMS negative mold and vacuum injection was performed. After removal, excess drug-containing needle matrix was removed and dried to obtain the needle tip matrix. Then, the backing matrix was added and placed in the cavity of the PDMS negative mold and vacuum injection was performed. After removal, air bubbles were removed and the mixture was placed at room temperature and dried overnight. The next day, after demolding, the soluble microneedles loaded with triptolide and tanshinone A were obtained.

[0014] Furthermore, the ratio of the mixed powder to the first mixed solution is (4-6):4 (g / ml).

[0015] Furthermore, the ratio of the salvianolic acid A to the triptolide (TP-NCs) nano-suspension is (10-20):1 (mg / ml).

[0016] Furthermore, the vortex treatment time is 0.5 min to 3 min; The ultrasonic treatment time is 3-7 minutes, and the ultrasonic power is 180W. When the organic phase is injected into the aqueous phase, the temperature of the aqueous phase is 15℃-40℃; The rotary evaporation temperature is 35℃-50℃; The pressure value for vacuum injection is -0.07 MPa to -1 MPa; The drying temperature for the drug-containing needle matrix is ​​25℃-40℃, and the drying time is 30min-45min; The backing substrate is dried at a temperature of 20℃-40℃ for 10h-16h.

[0017] On the other hand, the present invention provides the application of soluble microneedles loaded with triptolide and salvianolic acid A in the preparation of drugs for treating rheumatoid arthritis.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The soluble microneedles of this invention deliver medication directly to the affected skin of rheumatoid arthritis patients via local transdermal administration. This allows for better penetration of the stratum corneum and avoids injection pain, accumulating at the lesion site and successfully clearing the inflammatory response of rheumatoid arthritis. Simultaneously, compared to injection and oral administration, it reduces the toxicity and side effects associated with systemic drug absorption.

[0019] 2. The preparation method provided by this invention utilizes antisolvent precipitation to prepare triptolide nanocrystals, and adds salvianolic acid A to the nanosuspension to enhance efficacy. The nanocrystals have small particle size and good water solubility, while Span 85 acts as a stabilizer to improve drug stability. Encapsulating them in soluble microneedles facilitates drug absorption and action. Furthermore, the preparation method provided by this invention is simple, convenient, and controllable. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments are briefly introduced below.

[0021] Figure 1 The structural formula of triptolide; Figure 2 The structural formula of salvianolic acid A is shown below. Figure 3 Laser irradiation image of the triptolide nanosuspension prepared in Example 1; Figure 4 Morphological image of the triptolide nanocrystals prepared in Example 1 under a scanning electron microscope; Figure 5 This is a differential scanning calorimetry (DSC) graph of the sample during performance testing. Figure 6 This is an X-ray diffraction analysis diagram of the sample during performance testing; Figure 7 The cumulative drug release percentage of the triptolide nano-suspension and triptolide raw material prepared in Example 1; Figure 8 External scanning electron microscope image of the soluble microneedles loaded with triptolide and salvianolic acid A prepared in Example 1; Figure 9 The test result curves are for the soluble microneedles loaded with triptolide and salvianolic acid A prepared in Example 1. Figure 10 The average body weight change of rats in each group over 21 days in the pharmacodynamic study; Figure 11 The severity of rheumatoid arthritis in rats of each group was scored over 21 days in a pharmacodynamic study.

[0022] Figure 12 The image shows a physical representation of the soluble microneedles loaded with triptolide and salvianolic acid A provided in an embodiment of the present invention. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0024] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] This invention provides a soluble microneedle loaded with triptolide and salvianolic acid A, comprising a tip matrix and a backing matrix loaded with triptolide nanocrystals, salvianolic acid A, and a blank microneedle.

[0029] It is understood that the soluble microneedles of this invention act directly on the affected skin of rheumatoid arthritis patients through local transdermal drug delivery. This allows for better penetration of the stratum corneum and avoids injection pain, accumulating at the lesion site and successfully clearing the inflammatory response of rheumatoid arthritis. Simultaneously, compared to injection and oral administration, it reduces the toxicity and side effects associated with systemic drug absorption.

[0030] In some embodiments, the triptolide nanocrystals comprise triptolide active pharmaceutical ingredient, stabilizer, aqueous phase, and organic phase.

[0031] Specifically, the stabilizer is a surfactant, preferably Span 85.

[0032] In some embodiments, the backing matrix comprises sodium carboxymethyl cellulose (CMC-Na) and water, and the amount of CMC-Na in the backing matrix is ​​0.3~0.6%, preferably 0.3~0.6%.

[0033] Specifically, the water is purified water.

[0034] In some embodiments, the mixing ratio of the triptolide raw material to the stabilizer is 1:(0.5-3); and / or, the ratio of the organic phase to the aqueous phase is 1:(5-30), preferably 1:10.

[0035] Specifically, the preferred mixing ratio of the triptolide raw material to the stabilizer is 1:2; In some embodiments, the needle tip matrix is ​​chondroitin sulfate (CS) and polyvinylpyrrolidone-K30 (PVP-K30).

[0036] Specifically, the mass ratio of chondroitin sulfate (CS) to PVP-K30 is 1:1.

[0037] This invention also provides a method for preparing soluble microneedles loaded with triptolide and salvianolic acid A, comprising the following steps: Tripterygium wilfordii raw material and stabilizer were dissolved in anhydrous ethanol, vortexed and then sonicated to obtain an organic solution containing stabilizer as the organic phase. Take distilled water as the aqueous phase; At room temperature, the organic phase is injected into the aqueous phase while stirring, and the stirring is maintained during the process to obtain an initial suspension; After ultrasonic stabilization, the ethanol was removed by rotary evaporation to obtain triptolide (TP-NCs) nano-suspension. Tanshinone A was dissolved in the triptolide (TP-NCs) nano-suspension to obtain a first mixed solution. Then, chondroitin sulfate was weighed and mixed with PVP-K30 to obtain a mixed powder. The mixed powder was sprinkled on the surface of the first mixed solution and allowed to swell overnight to obtain a drug-containing needle matrix. CMC-Na was sprinkled on the water surface and allowed to swell overnight to obtain the backing matrix; The drug-containing needle matrix was placed in the cavity of the PDMS negative mold and vacuum injection was performed. After removal, excess drug-containing needle matrix was removed and dried to obtain the needle tip matrix. Then, the backing matrix was added and placed in the cavity of the PDMS negative mold and vacuum injection was performed. After removal, air bubbles were removed and the mixture was placed at room temperature and dried overnight. The next day, after demolding, the soluble microneedles loaded with triptolide and tanshinone A were obtained.

[0038] Specifically, in the initial suspension, the concentration of the triptolide raw material is 0.3~0.7 mg / mL.

[0039] Specifically, the ratio of the mixed powder to the first mixed solution is 5:4 (g / ml).

[0040] Specifically, the ultrasonic power of the ultrasonic stabilization process is 180W.

[0041] Specifically, the mixing ratio of the triptolide raw material to the anhydrous ethanol is (3mg-7mg):1ml, preferably 5mg:1ml.

[0042] In some embodiments, the ratio of the mixed powder to the first mixed solution is (4-6):4 (g / ml), preferably 5:4 (g / ml).

[0043] In some embodiments, the ratio of the salvianolic acid A to the triptolide (TP-NCs) nano-suspension is (10-20):1 (mg / ml), preferably 15:1 (mg / ml).

[0044] In some embodiments, the vortex treatment time is 0.5 min to 3 min; The ultrasonic treatment time is 3-7 minutes, and the ultrasonic power is 180W. When the organic phase is injected into the aqueous phase, the temperature of the aqueous phase is 15℃-40℃; The rotary evaporation temperature is 35℃-50℃; The pressure value for vacuum injection is -0.07 MPa to -1 MPa; The drying temperature for the drug-containing needle matrix is ​​25℃-40℃, and the drying time is 30min-45min; The backing substrate is dried at a temperature of 20℃-40℃ for 10h-16h.

[0045] The preferred vortex treatment time is 0.5 min; the preferred ultrasonic treatment time is 5 min; when the organic phase is injected into the aqueous phase, the preferred aqueous phase temperature is 25℃; the preferred rotary evaporation temperature is 45℃; the preferred vacuum injection pressure is -0.08 MPa; the preferred drying temperature for the drug-containing needle matrix is ​​37℃, and the preferred drying time is 40 min; the preferred drying temperature for the backing matrix is ​​26℃, and the preferred drying time is 12 h.

[0046] Specifically, the triptolide (TP) and salvianolic acid A (SAA) used in the embodiments of the present invention were purchased from Shaanxi Cuikang Pharmaceutical Technology Co., Ltd.; the Span 85 used in the embodiments of the present invention were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; the polyvinylpyrrolidone-K30 (PVP-K30) and sodium carboxymethyl cellulose (CMC-Na) used in the embodiments of the present invention were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; the chondroitin sulfate (CS) used in the embodiments of the present invention were purchased from Xi'an Wanfang Biotechnology Co., Ltd.; and the anhydrous ethanol used in the embodiments of the present invention were purchased from Xilong Scientific Co., Ltd.

[0047] Example 1 Example 1 of this invention provides a method for preparing soluble microneedles (TP-NCs+SAA@DMN) loaded with triptolide and salvianolic acid A, comprising the following steps: S1. Accurately weigh 10 mg of stabilizer Span 85 and 5 mg of triptolide (TP) raw material, dissolve them in 1 mL of anhydrous ethanol, vortex for 0.5 min and sonicate for 5 min to obtain an organic solution containing stabilizer as the organic phase. S2. Take ultrapure water at a volume ratio of 1:10 between the organic phase and the aqueous phase, and use it as the aqueous phase. At 25°C, inject the organic phase into the aqueous phase while stirring, and keep stirring at a constant speed to obtain an initial suspension. S3. After stabilizing the prepared initial suspension with ultrasound at 180W, the ethanol was removed by rotary evaporation at 45℃ to obtain triptolide (TP-NCs) nano-suspension. S4. Dissolve 60 mg of salvianolic acid (SAA) in 4 mL of triptolide (TP-NCs) nano-suspension to obtain the first mixed solution. Then weigh 2.5 g of chondroitin sulfate (CS) and 2.5 g of PVP-K30 and mix them evenly to obtain a mixed powder. Sprinkle the mixed powder on the surface of the first mixture and let it swell overnight to obtain the drug-containing needle matrix. S5. Sprinkle 0.4g CMC-Na on the surface of 10mL purified water and allow it to swell overnight to obtain the backing matrix; S3. Place the drug-containing needle matrix into the cavity of the PDMS negative mold, inject the sample under vacuum at -0.08 MPa for 40 min, remove the sample, remove excess drug-containing needle matrix, and dry at 37°C for 40 min; then add the backing matrix into the cavity of the PDMS negative mold, inject the sample under vacuum at -0.08 MPa for 10 min, remove the sample, remove air bubbles, and dry at 26°C for 12 hours. The next day, demold the sample to obtain the drug-loaded microneedles.

[0048] Among them, the triptolide (TP-NCs) nanosuspension was irradiated with a laser pen as follows: Figure 3 As shown.

[0049] Example 2 Example 2 of this invention provides a method for preparing soluble microneedles (TP-NCs+SAA@DMN) loaded with triptolide and salvianolic acid A, comprising the following steps: S1. Accurately weigh 5 mg of stabilizer Span 85 and 10 mg of triptolide (TP) raw material, dissolve them in 1 mL of anhydrous ethanol, vortex for 0.5 min and sonicate for 3 min to obtain an organic solution containing stabilizer as the organic phase; S2. Take ultrapure water at a volume ratio of 1:5 between the organic phase and the aqueous phase, and use it as the aqueous phase. At 15°C, inject the organic phase into the aqueous phase while stirring, and keep stirring at a constant speed to obtain an initial suspension. S3. After stabilizing the prepared initial suspension with ultrasound at 180W, remove the ethanol by rotary evaporation at 35℃ to obtain triptolide (TP-NCs) nano-suspension. S4. Dissolve 40 mg of salvianolic acid (SAA) in 4 mL of triptolide (TP-NCs) nano-suspension to obtain the first mixed solution. Then weigh 2 g of chondroitin sulfate (CS) and 2 g of PVP-K30 and mix them evenly to obtain a mixed powder. Sprinkle the mixed powder on the surface of the first mixture and let it swell overnight to obtain the drug-containing needle matrix. S5. Sprinkle 0.3g CMC-Na on the surface of 10mL purified water and allow it to swell overnight to obtain the backing matrix; S3. Place the drug-containing needle matrix into the cavity of the PDMS negative mold, inject the sample under vacuum at -0.07 MPa for 40 min, remove the sample, remove excess drug-containing needle matrix, and dry at 25°C for 30 min; then add the backing matrix into the cavity of the PDMS negative mold, inject the sample under vacuum at -0.07 MPa for 10 min, remove the sample, remove air bubbles, and dry at 20°C for 10 hours. The next day, demold the sample to obtain the drug-loaded microneedles.

[0050] Example 3 Example 2 of this invention provides a method for preparing soluble microneedles (TP-NCs+SAA@DMN) loaded with triptolide and salvianolic acid A, comprising the following steps: S1. Accurately weigh 30 mg of stabilizer Span 85 and 10 mg of triptolide (TP) raw material, dissolve them in 1 mL of anhydrous ethanol, vortex for 3 min and sonicate for 7 min to obtain an organic solution containing stabilizer as the organic phase. S2. Take ultrapure water at a volume ratio of 1:30 for organic phase to aqueous phase and use it as the aqueous phase. At 40°C, inject the organic phase into the aqueous phase while stirring and maintain uniform stirring to obtain an initial suspension. S3. After stabilizing the prepared initial suspension with ultrasound at 180W, remove the ethanol by rotary evaporation at 50℃ to obtain triptolide (TP-NCs) nano-suspension. S4. Dissolve 80 mg of salvianolic acid (SAA) in 4 mL of triptolide (TP-NCs) nano-suspension to obtain the first mixed solution. Then weigh 3 g of chondroitin sulfate (CS) and 3 g of PVP-K30 and mix them evenly to obtain a mixed powder. Sprinkle the mixed powder on the surface of the first mixture and let it swell overnight to obtain the drug-containing needle matrix. S5. Sprinkle 0.6g CMC-Na on the surface of 10mL purified water and allow it to swell overnight to obtain the backing matrix; S3. Place the drug-containing needle matrix into the cavity of the PDMS negative mold, inject the sample under vacuum at -1 MPa for 40 min, remove it, remove excess drug-containing needle matrix, and dry at 40°C for 45 min; then add the backing matrix into the cavity of the PDMS negative mold, inject the sample under vacuum at -1 MPa for 10 min, remove it, remove air bubbles, and dry at 40°C for 16 hours. The next day, demold to obtain the drug-loaded microneedles.

[0051] Example 4 Example 2 of the present invention provides a method for preparing soluble microneedles (TP-NCs+SAA@DMN) loaded with triptolide and salvianolic acid A. The difference from Example 1 is that the amount of TP and Span 85 added in step S1 is 7 mg and 14 mg, respectively.

[0052] Example 5 Example 3 of the present invention provides a method for preparing soluble microneedles (TP-NCs+SAA@DMN) loaded with triptolide and salvianolic acid A. The difference from Example 1 is that the rotary evaporation temperature in step S2 is 50°C.

[0053] Example 6 Example 4 of this invention provides a method for preparing soluble microneedles (TP-NCs+SAA@DMN) loaded with triptolide and salvianolic acid A. The difference from Example 1 is that the vacuum injection pressure in step S3 is -0.07 MPa and the vacuum injection time is 50 min. Example 7 Example 5 of this invention provides a method for preparing soluble microneedles (TP-NCs+SAA@DMN) loaded with triptolide and salvianolic acid A. The difference from Example 1 is that the vacuum injection pressure in step S4 is -0.09 MPa and the vacuum injection time is 30 min. Example 8 Example 6 of the present invention provides a method for preparing soluble microneedles (TP-NCs+SAA@DMN) loaded with triptolide and salvianolic acid A. The difference from Example 1 is that the amount of CMC-Na added in step S4 is 0.5g.

[0054] Performance testing The performance of the triptolide (TP-NCs) nanosuspension and TP-NCs+SAA@DMN prepared in Example 1 was tested, including: (1) The average particle size, polydispersity index and zeta potential of TP-NCs were determined by dynamic laser scattering particle size analyzer. The measurements were performed in parallel three times and the results are shown in Table 1.

[0055] The results showed that the polydispersity index of TP-NCs was less than 0.3, indicating that the particle size was uniform and the particles were evenly dispersed.

[0056] Table 1. Average particle size, polydispersity index, and potential value of TP-NCs

[0057] (2) Morphological observation 10 μL of LTP-NCs was pipetted onto a silicon substrate, vacuum dried, and then attached to a conductive stage with conductive adhesive. Gold was then sputtered onto the substrate using an ion sputtering apparatus. The morphology was observed and photographed under a scanning electron microscope. The results are as follows: Figure 3 As shown, the prepared TP-NCs were found to be regular spherical with a relatively uniform distribution and a particle size of 150nm-200nm.

[0058] (3) Determination of drug loading Chromatographic conditions: The chromatography workstation was an Agilent 1260 Infinity liquid chromatography system, and the chromatographic column was a Supersil ODS2 (4.6 mm × 150 mm, 5 μm). The mobile phase was water:acetonitrile = 70:30 (v / v); the detection wavelength was 218 nm, the flow rate was 1 mL / min, the injection volume was 5 μL, and the column temperature was 30 °C.

[0059] Seven drug-loaded soluble microneedles were taken, and the tips of the microneedles were gently scraped off with a scalpel. The residue was placed in an EP tube and dissolved in 2 ml of ultrapure water. The tube was then shaken at 37°C and 100 rpm for 30 min on a shaker. Afterward, 1.5 ml of methanol was added, and the mixture was extracted by sonication for 20 min. The extract was then filtered through a 0.22 μm filter and the total drug concentration was determined by HPLC. The drug loading of each microneedle was calculated, and the average value was taken from three parallel determinations. The drug loading of TP in each microneedle was found to be 11.47 μg.

[0060] (4) Differential scanning calorimetry (DSC) Take appropriate amounts of TP active pharmaceutical ingredient, Span 85, TP-NCs lyophilized powder, mannitol, a physical mixture of TP active pharmaceutical ingredient and Span 85 (in the same proportion as the formulation), and another physical mixture of TP active pharmaceutical ingredient, mannitol, and Span 85 (in the same proportion as the formulation). Place each in a crucible, and place the sample into the annular area of ​​the sample cell for determination.

[0061] Scanning range: 40-260℃; Heating rate: 10℃ / min.

[0062] The results are as follows Figure 5 As shown.

[0063] The results showed that in the DSC spectra of TP and TP-NCs, a typical endothermic peak was observed at around 240℃, indicating their crystallinity, while TP-NCs did not have an endothermic peak between 40℃ and 260℃. It can be concluded that after TP was prepared into nanocrystals, its crystal form changed, transforming from a crystalline form to an amorphous structure. (5) X-ray diffraction analysis (XRD) Take appropriate amounts of TP active pharmaceutical ingredient, TP-NCs lyophilized powder, mannitol, Span 85 and mannitol lyophilized powder, and a physical mixture of TP active pharmaceutical ingredient, mannitol and Span 85 (in the same proportions as the formulation) for X-ray diffraction (XRD) analysis. The results are as follows: Figure 6 As shown.

[0064] The results showed that UPA and UPA-ES exhibited strong characteristic diffraction peaks at diffraction angles of 8.0°, 9.6°, 14.1°, 14.5°, 20.5°, 23.0°, and 24.8°, indicating that the drug exists in crystalline compound form. ES showed no peaks and exhibited a flat diffraction curve. Furthermore, the crystalline peaks at the above positions were found to have disappeared in UPA@ES-TPGS, possibly because UPA is uniformly dispersed amorphously within the liposome, indicating that the hydrophobic drug UPA was successfully encapsulated within the hydrophobic bilayer of the liposome.

[0065] The TP active pharmaceutical ingredient exhibits strong characteristic peaks at diffraction angles of 7.7°, 15.5°, 38.1°, and 46.1°. Mannitol shows distinct characteristic peaks at diffraction angles of 14.6°, 18.8°, and 23.4°. However, TP-NCs show no obvious characteristic peaks at these angles, exhibiting a relatively flat diffraction curve. Combined with DSC results, it is evident that most of the drug's crystalline form has transformed into an amorphous form. The appearance of new characteristic peaks is likely due to the lyophilization protectant mannitol.

[0066] (6) In vitro drug release behavior study The in vitro drug release behavior study of triptolide was conducted as follows: 2 mL of triptolide (TP-NCs) nanosuspension was placed into a pretreated dialysis bag (molecular weight cutoff 3500D). Simultaneously, 2 mL of TP methanol solution with the same drug content as the TP-NCs nanosuspension was placed into another pretreated dialysis bag. The bags were then sealed at both ends and placed into a container containing 50 mL of release medium. The mixture was stirred at a constant temperature of 37℃ and a constant speed of 100 rpm. At predetermined time intervals (10, 20, 40, 60, 90, 120, 180, and 210 min), 0.5 mL of release medium was aspirated, and 0.5 mL of isothermal release medium was added simultaneously. The released medium was filtered through a 0.22 μm microporous membrane, and the concentration of triptolide in the release medium was determined by HPLC. The cumulative release percentage (Q%) of triptolide was calculated using the following formula, and the results are shown below. Figure 7 As shown, the results indicate that the triptolide (TP-NCs) nanosuspension has a certain sustained-release effect compared to the UPA methanol solution, which can better control drug release and prolong the duration of action.

[0067] Q%=W t / W total ×100%(4-1) Among them: W t W represents the cumulative release of triptolide in the release medium at time point t. total The total amount of triptolide in the TP-NCs nano-suspension before the release experiment.

[0068] (7) Appearance and shape and scanning electron microscopy observation The TP-NCs+SAA@DMN were subjected to experimental morphology and scanning electron microscopy observation.

[0069] One TP-NCs+SAA@DMN wafer was laid flat on a planar conductive stage coated with double-layer conductive adhesive, or placed obliquely on an L-shaped conductive stage. Gold was sputtered using an ion sputtering system. The coated sample was then randomly imaged using a scanning electron microscope to observe its morphological characteristics and take photographs. The results are as follows: Figure 8As shown in Figure a, the microneedle tip shape is intact and unbroken. Figure b shows that the prepared microneedles have uniform shape and size, and the microneedle array is complete and aesthetically pleasing. This preparation method is convenient, simple, and highly reproducible.

[0070] (8) Determination of the mechanical strength of drug-loaded microneedles The mechanical properties of TP-NCs+SAA@DMN were characterized using a UniversalTA texture analyzer. A microneedle was fixed in the center of a stainless steel plate with its tip facing upwards, and the distance between the microneedle tip and the sensor was set to 0.8 mm. A 2 mm diameter cylindrical probe was compressed downwards at a constant speed of 0.1 mm / min. The probe displacement and corresponding resistance to the microneedle after contact with the microneedle tip were recorded. The results are as follows: Figure 9 As shown.

[0071] (9) Pharmacodynamic studies Newly purchased 6-8 week old male SD rats were acclimatized for one week. Hair was completely removed from the rats' hind limbs using a razor and depilatory cream, taking care not to damage the skin. The rats were allowed 24 hours to recover after hair removal to allow their skin to return to normal. Mice were randomly divided into four groups: Control group, Model group, Positive group (Diclofenac Diethylamine Emulgel), Blank Microneedle group (BlankDMN), Microneedle group loaded with Tanshinone A (SAADMN), Microneedle group loaded with Tripterygium wilfordii nanocrystals (TP-NCs@DMN), and Microneedle group loaded with Tripterygium wilfordii nanocrystals and Tanshinone A (TP-NCs+SAA@DMN), with 6 mice in each group.

[0072] Establishment of a rheumatoid arthritis model: Collagen solution was mixed with complete Freund's adjuvant (CFA) at a 1:1 ratio (v / v) and emulsified by shearing. Each SD rat was injected with a total of 200 μL, specifically 50 μL at the base of the tail, 50 μL subcutaneously in each of the left and right buttocks, and 25 μL in each of the left and right toes. This was the initial immunization, designated Day 0.

[0073] On Day 7, a booster immunization was performed by mixing the collagen solution with incomplete Freund's adjuvant (IFA) at a 1:1 ratio (v / v) and emulsifying it using a high-speed homogenizer. Each SD rat was injected with a total of 100 μL, specifically 20 μL into the tail base, 20 μL subcutaneously in each of the left and right buttocks, and 20 μL into each of the left and right toes.

[0074] After the model was established on Day 14, drug administration began. The blank microneedle group (BlankDMN), the salvianolic acid A-loaded microneedle group (SAADMN), the triptolide nanocrystal microneedle group (TP-NCs@DMN), and the triptolide nanocrystal and salvianolic acid A-loaded microneedle group (TP-NCs+SAA@DMN) were administered one microneedle to each side of the rat's hind limbs every two days. The normal group received no treatment, the positive control group received 90 mg / kg of salvianolic acid nanocrystal every two days, and the model group received the same volume of physiological saline.

[0075] The changes in body weight of mice in each group during the experiment are as follows: Figure 10 As shown in the figure. The results indicated that the body weight of rats in each group gradually increased and remained relatively stable overall, while the body weight of rats in the model group increased more slowly and ultimately fell below that of rats in other groups. The TP-NCs+SAA@DMN group showed a more pronounced trend in body weight gain, and its final body weight approached that of the normal group, indicating that the micro-targeted therapy has a certain therapeutic effect on RA rats.

[0076] The thickness of the paw pads of rats in each group was recorded every 4 days, and scores were calculated according to internationally accepted scoring standards. The specific scoring standards are shown in Table 2. The results are as follows: Figure 11 As shown, the higher the score, the more severe the joint damage.

[0077] Table 2 Scoring Criteria

[0078] Continuous observation revealed that the model group mice experienced persistent swelling and extremely reddish-purple toes during treatment. The positive drug group and the SAA+TP-NCs@DMN group showed the fastest reduction in redness and swelling, and their toes essentially returned to normal shape after treatment. The blank microneedle group showed minimal toe recovery, while the swelling in the other groups decreased, with the SAA+TP-NCs@DMN group showing the best results.

[0079] In summary, the soluble microneedles loaded with triptolide and tanshinone A provided by this invention can improve the efficacy of treating rheumatoid arthritis, and have a sustained-release effect and good biocompatibility, thus avoiding the side effects of systemic administration.

[0080] While embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations fall within the scope and spirit of the invention as set forth in the claims. Furthermore, the invention described herein may have other embodiments and can be implemented or carried out in various ways.

Claims

1. A soluble microneedle of triptolide and salvianolic acid A, characterized in that, The needle tip matrix and the backing matrix of the microneedle containing triptolide nanocrystals, salvianolic acid A and blank microneedle.

2. The tracheloside and salvianolic acid A soluble microneedle according to claim 1, characterized in that, The triptolide nanocrystals comprise triptolide raw material, stabilizer, water phase and organic phase. 3.The trachelogenin and salvianolic acid A-loaded dissolvable microneedle of claim 2, characterized in that, The backing matrix comprises sodium carboxymethyl cellulose (CMC-Na) and water, and the amount of CMC-Na in the backing matrix is 0.3-0.6%.

4. The tracheloside and salvianolic acid A soluble microneedle according to claim 3, characterized in that, The mixing ratio of the triptolide raw material to the stabilizer is 1:(0.5-3); and / or, the ratio of the organic phase to the water phase is 1:(5-30).

5. The trachelosant and salvianolic acid A soluble microneedle according to claim 4, characterized in that, The needle tip matrix is chondroitin sulfate (CS) and polyvinylpyrrolidone-K30 (PVP-K30).

6. A method for preparing the soluble microneedle of triptolide and salvianolic acid A according to any one of claims 1-5, characterized in that, The method comprises the following steps: The triptolide raw material and the stabilizer are dissolved in anhydrous ethanol, and then treated by vortex and ultrasonic to obtain a water phase containing ultrapure water; The organic phase is injected into the water phase under stirring at room temperature, and stirring is maintained during the injection to obtain a primary suspension; After the primary suspension is treated by ultrasonic stabilization, the ethanol is removed by rotary evaporation to obtain a triptolide (TP-NCs) nanosuspension; Salvianolic acid A is dissolved in the triptolide (TP-NCs) nanosuspension to obtain a first mixed solution, then chondroitin sulfate and PVP-K30 are mixed to obtain a mixed powder, and the mixed powder is scattered on the surface of the first mixed solution to swell overnight to obtain a drug-containing needle part matrix; CMC-Na is scattered on the water surface to swell overnight to obtain the backing matrix; The drug-containing needle part matrix is placed in the cavity of a PDMS negative mold, vacuum sampling is performed, the excess drug-containing needle part matrix is removed, and then the needle tip matrix is obtained after drying, and then the backing matrix is added to the cavity of the PDMS negative mold, vacuum sampling is performed, the bubbles are removed after taking out, and the backing matrix is placed at room temperature and dried overnight, and then demolding is performed the next day to obtain the triptolide and salvianolic acid A-containing soluble microneedle.

7. The preparation method according to claim 6, characterized in that, The solid-liquid ratio of the mixed powder to the first mixed solution is (4-6):4 (g / ml).

8. The preparation method according to claim 7, characterized in that, The solid-liquid ratio of the salvianolic acid A to the triptolide (TP-NCs) nanosuspension is (10-20):1 (mg / ml).

9. The preparation method of claim 8, wherein The vortex treatment time is 0.5 min-3 min; The ultrasonic treatment time is 3 min-7 min, and the ultrasonic power is 180 W; When the organic phase is injected into the water phase, the temperature of the water phase is 15℃-40℃; The rotary evaporation temperature is 35℃-50℃; The pressure value of the vacuum sampling is -0.07Mpa–-1Mpa; The temperature for drying the drug-containing needle part matrix is 25℃-40℃, and the drying time is 30 min-45 min; The temperature for drying the backing matrix is 20℃-40℃, and the drying time is 10h-16h.

10. The triptolide and salvianolic acid A-containing soluble microneedle of any one of claims 1 to 5 for use in the preparation of a drug for treating rheumatoid arthritis.

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