A triptolide-loaded photothermal-driven motor microneedle for treating rheumatoid arthritis and a preparation method thereof

Abstract

The present invention provides a triptolide-loaded photothermal-driven motor microneedle for treating rheumatoid arthritis, so as to improve the effectiveness and patient compliance of TG in clinical treatment of RA, and at the same time improve the efficiency of subcutaneous local tissue drug delivery by percutaneous administration; at the same time, a method for preparing the above-mentioned novel skin drug delivery preparation is provided; it comprises the following contents: a triptolide-loaded photothermal-driven motor microneedle for treating rheumatoid arthritis, comprising the following components: 300-500 parts of a silicon nanobottle modified with folic acid PEG, 15-20 parts of triptolide, 5-30 parts of perfluorohexane, 5-10 parts of indocyanine green, 100-200 parts of tetradecanol, and 1-10 parts of pure water.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical preparations and relates to a skin external preparation, and in particular to a triptolide-carrying photothermal-driven motor microneedle and a preparation method thereof. Background Art

[0002] Tripterygium wilfordii Hook.F. is a woody vine of the genus Tripterygium in the family Celastraceae. It is also known as water moss, yellow rattan, yellow rattan wood, yellow medicine, yellow wax rattan, vegetable insect medicine, and broken intestine grass. "Clinical Chinese Medicine" describes it as bitter, pungent, cold, and highly toxic. It enters the liver and kidney meridians. The main effects are to dispel rheumatism, promote blood circulation and dredge collaterals, and clear away heat and detoxify. Triptolide (TP) is an alkaloid extracted from the peeled root of Tripterygium wilfordii. It is an effective ingredient for treating diseases such as RA. It is the first anti-inflammatory and immunomodulatory Chinese herbal medicine studied and used in my country, and is known as the "Chinese herbal hormone". TP has anti-inflammatory, anti-tumor, and immunomodulatory effects. It is clinically used to treat RA, purpuric nephritis, nephrotic syndrome, diabetic nephropathy, and other autoimmune diseases and kidney diseases. TP is used to treat rheumatoid arthritis, mainly acting on T cells, B cells and dendritic cells, inhibiting the infiltration of proinflammatory factors and inflammatory cells, promoting cell apoptosis and activating anti-inflammatory pathways, and repairing damaged cartilage and bone tissue to a certain extent. However, the commercially available Tripterygium wilfordii polyglycosides are prone to cause adverse reactions and hepatotoxicity, reproductive toxicity, blood and hematopoietic system toxicity, cardiac toxicity, skin and mucosal toxicity, etc.

[0003] Microneedles (MNs) are minimally invasive devices consisting of needle arrays of about 50-900 μm in length. They are manufactured through microfabrication technology of different materials and geometric shapes. MNs can deliver drugs to the subepidermis and bypass the stratum corneum barrier. Therefore, compared with traditional injection and oral drug delivery strategies, microneedle drug delivery is safe, convenient, and painless, and can easily overcome the stratum corneum barrier to achieve drug delivery. Dissolvable microneedles (DMNs) do not leave needle fragments under the skin, minimizing the risk of inflammation caused by foreign bodies. Studies have found that triptolide soluble microneedle patches can avoid the first-pass metabolism of the drug in the liver and gastrointestinal toxicity, and can release the drug stably and for a long time; however, although the microneedle transdermal route can avoid the safety issues caused by joint cavity injection to a certain extent, increase the transdermal absorption of the drug and improve patient compliance, it does not have the advantages of joint-targeted drug delivery, and it also cannot avoid the rapid lymphatic clearance of the drug in the joint area. Therefore, it is necessary to further design drug delivery carriers to achieve targeted delivery of drugs and prolong their retention in the site of joint inflammation. Summary of the invention

[0004] The purpose of the present invention is to address the shortcomings of existing preparations and provide a photothermal-driven motor microneedle loaded with triptolide for the treatment of rheumatoid arthritis, so as to improve the effectiveness and patient compliance of TG in the clinical treatment of RA, while improving the efficiency of transdermal drug delivery to subcutaneous local tissues; another purpose of the present invention is to provide a method for preparing the above-mentioned new skin drug delivery preparation.

[0005] A triptolide-loaded photothermal-driven motor microneedle for treating rheumatoid arthritis, characterized in that it comprises the following components:

[0006] 300-500 parts of folic acid PEG-modified silicon nanobottle, 15-20 parts of triptolide, 5-30 parts of perfluorohexane, 5-10 parts of indocyanine green, 100-200 parts of tetradecanol, and 1-10 parts of pure water.

[0007] A triptolide-loaded photothermal-driven motor microneedle for treating rheumatoid arthritis, characterized in that the folic acid PEG-modified silicon nanobottle is obtained by reacting (3-chloropropyl)trimethoxysilane with butyl orthosilicate and then reacting with folic acid PEG-silane.

[0008] A method for preparing a triptolide-loaded photothermal-driven motor microneedle for treating rheumatoid arthritis, characterized in that it comprises the following steps:

[0009] Step 1: Prepare a silicon nanobottle by reacting (3-chloropropyl)trimethoxysilane and butyl orthosilicate;

[0010] Step 2, reacting folic acid PEG-silane with the nanobottle to generate a folic acid PEG-modified silicon nanobottle;

[0011] Step 3, loading triptolide, tetradecanol, perfluorohexane, and indocyanine green into a silicon nanobottle modified with folic acid PEG to obtain a triptolide photothermal drive motor;

[0012] Step 4: Mix the triptolide photothermal drive motor and the microneedle matrix evenly, pour them into the microneedle mold, and dry them to obtain the triptolide photothermal drive motor microneedle.

[0013] A method for preparing a triptolide-loaded photothermal-driven motor microneedle for treating rheumatoid arthritis, characterized in that the volume ratio of (3-chloropropyl)trimethoxysilane to butyl orthosilicate is 1:10, the molecular weight of the folic acid PEG-silane is 2000, the weight ratio of the triptolide, tetradecanol, perfluorohexane, indocyanine green and folic acid PEG-modified silicon nanobottle is 2:20:50:1:40, and the concentration of the hyaluronic acid matrix is ​​100 mg / mL.

[0014] A method for preparing a triptolide-loaded photothermal-driven motor microneedle for treating rheumatoid arthritis, characterized in that the steps of preparing a silicon nanobottle by reacting (3-chloropropyl)trimethoxysilane and butyl orthosilicate are specifically as follows:

[0015] (1) Weigh a certain amount of polyvinyl pyrrolidone and dissolve it in n-pentanol, add citric acid ammonium solution and anhydrous ethanol, and vortex;

[0016] (2) adding a certain amount of (3-chloropropyl)trimethoxysilane and butyl orthosilicate, vortexing, then ultrasonicating, and reacting under stirring at room temperature;

[0017] (3) After the reaction is completed, centrifuge, wash with anhydrous ethanol, and dry to obtain a silicon nanobottle.

[0018] A triptolide-loaded photothermal-driven motor microneedle for treating rheumatoid arthritis, characterized in that the citric acid-ammonia aqueous solution is a 0.2 M citric acid-ammonia aqueous solution prepared by dissolving 2.58 g of citric acid in ammonia water to a volume of 50 mL.

[0019] A preparation method of a triptolide-loaded photothermal-driven motor microneedle for treating rheumatoid arthritis, characterized in that the steps of reacting folic acid PEG-silane with a nanobottle to form a folic acid PEG-modified silicon nanobottle are specifically as follows:

[0020] (1) Weighing a certain amount of the above-mentioned silicon nanobottle and dispersing it in a mixed solution of anhydrous ethanol and ammonia water;

[0021] (2) Add folic acid PEG-silane solution dropwise in a 50°C water bath and stir for 24 h;

[0022] (3) After the reaction is completed, the silica nanobottle is washed with anhydrous ethanol and dried to obtain the folic acid PEG-modified silica nanobottle.

[0023] A method for preparing triptolide-loaded photothermal-driven motor microneedles for treating rheumatoid arthritis, characterized in that the folic acid PEG-silane solution is obtained by dissolving 2 mg of folic acid PEG-silane in 2 mL of DMSO.

[0024] A preparation method of a triptolide-loaded photothermal-driven motor microneedle for treating rheumatoid arthritis, characterized in that triptolide, tetradecanol, perfluorohexane, and indocyanine green are loaded into a folic acid PEG-modified silicon nanobottle to obtain a triptolide-loaded photothermal-driven motor. The specific steps are:

[0025] (1) Take perfluorohexane and load it into the above-mentioned folic acid PEG-modified silicon nanobottle by negative pressure.

[0026] (2) Add triptolide, indocyanine green, tetradecanol, and methanol and stir for 30 min;

[0027] (3) adding pure water and centrifuging for washing to obtain the triptolide photothermal drive motor.

[0028] A method for preparing triptolide-loaded photothermal-driven motor microneedles for treating rheumatoid arthritis, characterized in that perfluorohexane is pre-treated with oxygen enrichment.

[0029] A method for preparing a triptolide-loaded photothermal-driven motor microneedle for treating rheumatoid arthritis, characterized in that the triptolide-loaded photothermal-driven motor and the microneedle matrix are uniformly mixed and then poured into a microneedle mold, and dried to obtain the triptolide-loaded photothermal-driven motor microneedle in the following steps:

[0030] (1) Dissolve hyaluronic acid in an appropriate amount of ultrapure water and let stand to defoam;

[0031] (2) adding the above-mentioned triptolide photothermal drive motor to the hyaluronic acid solution and mixing them evenly to obtain a drug-containing microneedle matrix;

[0032] (3) The drug-containing microneedle matrix is ​​added to the PDMS mold, vacuumed, and dried after removing bubbles to obtain the triptolide-loaded photothermal-driven motor microneedle.

[0033] A method for preparing triptolide-loaded photothermal-driven motor microneedles for treating rheumatoid arthritis, characterized in that the concentration of the hyaluronic acid solution is 100 mg / mL, the microneedle array of the microneedle PDMS mold is 10×10, and the needle tip length is 600 μm.

[0034] The triptolide in the present invention is extracted from the effective part of the traditional Chinese medicine Tripterygium wilfordii, meets the national pharmacopoeia standards, and is a commercially available product.

[0035] The nanocarrier in the present invention is obtained by hydrolysis polymerization of (3-chloropropyl)trimethoxysilane and butyl orthosilicate. (3-chloropropyl)trimethoxysilane can be replaced by (3-mercaptopropyl)trimethoxysilane, propyltrimethoxysilane, 1,2-bis(triethoxysilyl)ethane, etc. Butyl orthosilicate can be replaced by tetraethyl orthosilicate, tetra(2-ethylbutyl)silicate, etc.

[0036] Compared with the traditional injection and oral administration strategies, microneedle administration is safe, convenient, painless, and can easily overcome the barrier of the stratum corneum to achieve drug delivery, but it does not have the advantages of joint-targeted drug delivery of intra-articular injection, and it is also impossible to avoid the rapid lymphatic clearance of drugs at the joint site. Nanoparticle-based systems have the potential to improve the positioning of drugs in cartilage, but the dense and highly charged extracellular matrix of cartilage cells constitutes an obstacle to drug administration. Polymer nanoparticles can achieve different degrees of cartilage positioning through passive and active targeting. Active targeting uses specific biochemical properties to selectively target the inflammatory site, further improving specificity, efficiency and residence time in inflammatory cells. Therefore, FA and PEG co-modified nanocarriers are designed in the present invention to target macrophage FA receptors and improve the retention capacity of drug carriers at inflammatory sites.

[0037] Photothermally driven nanomotors have attracted much attention in recent years. They can perform reversible and repeatable motion behaviors and achieve on / off functions through radiation. Such nanomotors show obvious advantages in in vivo applications, including effective propulsion in biological fluids, high drug loading, strong drug dragging ability, and enhanced tissue retention ability. However, due to random loading, nanomotors have poor asymmetry and low thermophoretic force. In the present invention, asymmetric nanoparticles are prepared in order to obtain stronger driving force.

[0038] The present invention designs a photothermal effect-induced perfluorohexane gasification-driven nanomotor drug delivery system, which is beneficial to achieve active targeted drug delivery in the RA joint cavity.

[0039] In the present invention, indocyanine green is used as a photothermal material, and the temperature rises under near-infrared light irradiation to cause perfluorohexane to gasify.

[0040] In the present invention, the perfluorohexane saturated with oxygen is vaporized by photothermal material under the action of near-infrared light to generate microbubbles to promote the release and distribution of the drug.

[0041] The tetradecanol in the present invention is insoluble in water and is used as a bottle stopper to prevent drug leakage.

[0042] Research has found that the present invention uses the photothermal effects of asymmetric nanobottles, folic acid PEG-silane, indocyanine green and perfluorohexane, and utilizes the transdermal drug delivery characteristics of microneedles to produce triptolide-loaded photothermal-driven motor microneedles with enhanced targeting and drug delivery. The microneedles have better transdermal properties and are safer and more compliant than traditional oral and intra-articular injections. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0044] Figure 1 Transmission electron microscopy images of nanobottles prepared under different conditions.

[0045] Figure 2 Cumulative release curves of FAPEG-SNM@TP and SNM@TP under illumination and no illumination conditions.

[0046] Figure 3 Effects of different preparations on inflammatory factors of cells in vitro (AC: effects of each preparation on inflammatory factors of cells in vitro; D: comparison of cell activity after treatment with each preparation group and original drug at different concentrations; E: mean fluorescence intensity).

[0047] Figure 4 Cell apoptosis after incubation with different preparations for 12 h (from left to right: TP, SNM@TP, SNM@TP NIR, FAPEG-SNM@TP, FAPEG-SNM@TP NIR).

[0048] Figure 5 Qn-t curve and Qe-t curve of TP in ex vivo skin.

[0049] Figure 6 Drug retention of TP in ex vivo skin. DETAILED DESCRIPTION

[0050] The present invention is further described below in conjunction with specific examples. The methods are conventional methods unless otherwise specified, and the raw materials can be obtained from public commercial channels unless otherwise specified.

[0051] The particle size and particle size distribution, morphology, encapsulation efficiency, spectral analysis, skin permeability, photothermal conversion and in vitro release of the triptolide-loaded photothermal motor described in the embodiments of the present invention, and the evaluation methods of the morphology, puncture ability, solubility, mechanical strength and other indicators of the triptolide-loaded photothermal motor (FAPEG-SNM@TP) microneedle are as follows:

[0052] 1. Particle size and its distribution evaluation method

[0053] The particle size distribution and zeta potential of FAPEG-SNM@TP were determined by dynamic light scattering particle size analyzer.

[0054] 2. Morphological observation

[0055] Take a small amount of FAPEG-SNM@TP and distilled water to dilute it to an appropriate mass concentration, place it in an ultrasonic oscillator to disperse it evenly, draw a small amount of the suspension and drop it onto the copper mesh, dry it naturally, let it stand for a while to allow the nanomotor to deposit on the copper mesh, and observe the morphology and electron microscopic particle size of the nanobottle and nanomotor under a transmission electron microscope.

[0056] 3. In vitro release

[0057] The in vitro drug release characteristics of FAPEG-SNM@TP were investigated by direct release method. The nanoparticles were dispersed in 4 mL of PBS solution containing 0.05% Tween 80, sealed in an Eppendroff tube, placed in a constant temperature water oscillator at 37±0.5℃, and oscillated at 135 rpm. Centrifuged (5000 rpm, 5 min) at 15, 30, 60, 120, 180, 240, 300, and 360 min, and 1 mL of supernatant was aspirated for later use. The remaining release medium in the tube was discarded, 4 mL of release medium was added, and oscillation in the water bath oscillator was continued. The concentration of triptolide in the release medium was determined by HPLC.

[0058] Each sample was tested in triplicate. The release curves of the illuminated group and the non-illuminated group were compared to investigate the effect of NIR illumination on the in vitro drug release of FAPEG-SNM@TP. The laser was irradiated for 10 min at 120 min and 240 min, respectively (power 3 W / cm 2 ) to determine the changes in drug release before and after NIR irradiation.

[0059] 4. FAPEG-SNM@TP cytotoxicity

[0060] The CCK-8 method was used to evaluate the cytotoxicity. Raw264.7 cells in the logarithmic growth phase were cultured at 1×10 4 Cells / well were inoculated in a 96-well plate, and blank wells were set as control wells, and cells in the blank culture medium of the scaffold were set as blank wells. Eight parallel experimental wells were set for each concentration (divided into light exposure and non-light exposure), and LPS was added for activation and cultured overnight. The culture medium was discarded, and preparations containing different concentrations were added and incubated in the incubator for 4 hours. The light group was irradiated with 808nm laser for 5 minutes (1W / cm 2 ).

[0061] After the illumination, 100 μL of diluted CCK-8 solution (CCK-8: blank culture medium = 1:9) was added to each well of all groups and incubated in the incubator for 1 h. The absorbance value of each well at 450 nm was measured by an ELISA instrument, and the cell survival rate was calculated by the formula.

[0062]

[0063] 5. Pharmacodynamic evaluation at the cell level

[0064] In vitro determination of proinflammatory cytokines. RAW 264.7 cells were seeded at 1.5×105 per well of a 6-well plate. After incubation at 37°C for 12 h, different dosage forms were added, with PBS as a blank control. After 6 h, LPS was added to the control group for activation. After 16 h, the cell supernatant was collected and its expression level was detected. The contents of TNF-α, IL-6 and IL-1β in the cell culture supernatant were detected, and ELISA tests were performed according to the protocol of the ELISA kit.

[0065] Example 1

[0066] Blank nanoparticles (SNM-1) consist of the following components in parts by weight:

[0067]

[0068] Specifications and sources of main components: n-pentanol (AR, 98%, Shanghai McLean Reagent Co., Ltd.), polyvinyl pyrrolidone (K30, Beijing Solebow Technology Co., Ltd.), anhydrous ethanol (pharmaceutical grade, 99.5%, Shanghai McLean Reagent Co., Ltd.), (3-chloropropyl)trimethoxysilane (98%, Shanghai Aladdin Biochemical Technology Co., Ltd.), butyl orthosilicate (98%, Shanghai McLean Reagent Co., Ltd.), sodium citrate (98%, Shanghai Aladdin Biochemical Technology Co., Ltd.), ammonia water (GR, 25-28%, 35.05MW, Shanghai Aladdin Biochemical Technology Co., Ltd.).

[0069] The preparation steps of blank nanoparticles (SNM-1) are as follows:

[0070] 1) Weigh the amount of polyvinyl pyrrolidone in the formula and dissolve it in n-pentanol, add citric acid ammonium solution and anhydrous ethanol, and vortex;

[0071] 2) adding the formulated amount of (3-chloropropyl)trimethoxysilane and butyl orthosilicate, vortexing, then ultrasonicating, and reacting at room temperature with stirring for 2.5 hours;

[0072] 3) After the reaction, centrifuge and wash with anhydrous ethanol.

[0073] Example 2

[0074] Blank nanoparticles (SNM-2) consist of the following components in parts by weight:

[0075]

[0076] Specifications and sources of main components: n-pentanol (AR, 98%, Shanghai McLean Reagent Co., Ltd.), polyvinyl pyrrolidone (K30, Beijing Solebow Technology Co., Ltd.), anhydrous ethanol (pharmaceutical grade, 99.5%, Shanghai McLean Reagent Co., Ltd.), (3-chloropropyl)trimethoxysilane (98%, Shanghai Aladdin Biochemical Technology Co., Ltd.), butyl orthosilicate (98%, Shanghai McLean Reagent Co., Ltd.), sodium citrate (98%, Shanghai Aladdin Biochemical Technology Co., Ltd.), ammonia water (GR, 25-28%, 35.05MW, Shanghai Aladdin Biochemical Technology Co., Ltd.).

[0077] The preparation steps of blank nanoparticles (SNM-2) are as follows:

[0078] 1) Weigh the amount of polyvinyl pyrrolidone in the formula and dissolve it in n-pentanol, add citric acid ammonium solution and anhydrous ethanol, and vortex;

[0079] 2) adding the formulated amount of (3-chloropropyl)trimethoxysilane and butyl orthosilicate, vortexing, then ultrasonicating, and reacting at room temperature with stirring for 2.5 hours;

[0080] 3) After the reaction, centrifuge and wash with anhydrous ethanol.

[0081] Example 3

[0082] Blank nanoparticles (SNM-3) consist of the following components in parts by weight:

[0083]

[0084]

[0085] Specifications and sources of main components: n-pentanol (AR, 98%, Shanghai McLean Reagent Co., Ltd.), polyvinyl pyrrolidone (K30, Beijing Solebow Technology Co., Ltd.), anhydrous ethanol (pharmaceutical grade, 99.5%, Shanghai McLean Reagent Co., Ltd.), (3-chloropropyl)trimethoxysilane (98%, Shanghai Aladdin Biochemical Technology Co., Ltd.), butyl orthosilicate (98%, Shanghai McLean Reagent Co., Ltd.), sodium citrate (98%, Shanghai Aladdin Biochemical Technology Co., Ltd.), ammonia water (GR, 25-28%, 35.05MW, Shanghai Aladdin Biochemical Technology Co., Ltd.).

[0086] The preparation steps of blank nanoparticles are as follows:

[0087] 1) Weigh the amount of polyvinyl pyrrolidone in the formula and dissolve it in n-pentanol, add citric acid ammonium solution and anhydrous ethanol, and vortex;

[0088] 2) adding the formulated amount of (3-chloropropyl)trimethoxysilane and butyl orthosilicate, vortexing, then ultrasonicating, and reacting at room temperature with stirring for 2.5 hours;

[0089] 3) After the reaction, centrifuge and wash with anhydrous ethanol.

[0090] Example 4

[0091] The composition and preparation method of the triptolide-loaded photothermal motor (FAPEG-SNM@TP) are as follows:

[0092] Blank nanoparticles (SNM-2) consist of the following components in parts by weight:

[0093]

[0094] Specifications and sources of main components: n-pentanol (AR, 98%, Shanghai McLean Reagent Co., Ltd.), polyvinyl pyrrolidone (K30, Beijing Solebow Technology Co., Ltd.), anhydrous ethanol (pharmaceutical grade, 99.5%, Shanghai McLean Reagent Co., Ltd.), (3-chloropropyl)trimethoxysilane (98%, Shanghai Aladdin Biochemical Technology Co., Ltd.), butyl orthosilicate (98%, Shanghai McLean Reagent Co., Ltd.), sodium citrate (98%, Shanghai Aladdin Biochemical Technology Co., Ltd.), ammonia water (GR, 25-28%, 35.05MW, Shanghai Aladdin Biochemical Technology Co., Ltd.).

[0095] The preparation steps of blank nanoparticles are as follows:

[0096] 1) Weigh the amount of polyvinyl pyrrolidone in the formula and dissolve it in n-pentanol, add citric acid ammonium solution and anhydrous ethanol, and vortex;

[0097] 2) adding the formulated amount of (3-chloropropyl)trimethoxysilane and butyl orthosilicate, vortexing, then ultrasonicating, and reacting at room temperature with stirring for 2.5 hours;

[0098] 3) After the reaction, centrifuge and wash with anhydrous ethanol.

[0099] The folic acid modified nanoparticles (FASNM-1) are composed of the following components in parts by weight:

[0100]

[0101] Specifications and sources of the main components: FAPEG-silane (2000 molecular weight, Beijing Huawei Ruike Chemical Co., Ltd.), ammonia water (GR, 25-28%, 35.05MW, Shanghai Aladdin Biochemical Technology Co., Ltd.), anhydrous ethanol (pharmaceutical grade, 99.5%, Shanghai McLean Reagent Co., Ltd.).

[0102] The preparation steps of folic acid modified nanoparticles are as follows:

[0103] 1) Weigh the amount of nanobottles in the formula and disperse them in a mixed solution of anhydrous ethanol and ammonia water;

[0104] 2) Add the formulated amount of folic acid PEG-silane solution dropwise in a 50°C water bath;

[0105] 3) After the reaction is completed, wash with anhydrous ethanol.

[0106] The triptolide-loaded photothermal drive motor (FAPEG-SNM@TP) is composed of the following components in parts by weight:

[0107]

[0108] Specifications and sources of the main components: triptolide (99%, reference substance, Sichuan Chengdu Zhibiao Huachun Biotechnology Co., Ltd.), perfluorohexane (AR, 98%, Shanghai Myrel Chemical Technology Co., Ltd.), indocyanine green (75%, Shanghai Aladdin Biochemical Technology Co., Ltd.), methanol (HPLC, 6 Anhui Tiandi High Purity Solvent Co., Ltd.), pure water was purified by PALL ultrapure water instrument.

[0109] The preparation steps of the triptolide-loaded photothermal drive motor are as follows:

[0110] 1) Take the formulated amount of perfluorohexane into the nanobottle through negative pressure;

[0111] 2) Add the formulated amount of triptolide, indocyanine green, tetradecanol, and methanol, and stir for 30 minutes;

[0112] 3) Add a certain amount of pure water and centrifuge to wash.

[0113] Example 5

[0114] The composition and preparation method of the folate-free and PEG-modified triptolide-loaded photothermal motor (SNM@TP) are as follows:

[0115] Blank nanoparticles (SNM-2) consist of the following components in parts by weight:

[0116]

[0117] Specifications and sources of main components: n-pentanol (AR, 98%, Shanghai McLean Reagent Co., Ltd.), polyvinyl pyrrolidone (K30, Beijing Solebow Technology Co., Ltd.), anhydrous ethanol (pharmaceutical grade, 99.5%, Shanghai McLean Reagent Co., Ltd.), (3-chloropropyl)trimethoxysilane (98%, Shanghai Aladdin Biochemical Technology Co., Ltd.), butyl orthosilicate (98%, Shanghai McLean Reagent Co., Ltd.), sodium citrate (98%, Shanghai Aladdin Biochemical Technology Co., Ltd.), ammonia water (GR, 25-28%, 35.05MW, Shanghai Aladdin Biochemical Technology Co., Ltd.).

[0118] The preparation steps of blank nanoparticles are as follows:

[0119] 1) Weigh the amount of polyvinyl pyrrolidone in the formula and dissolve it in n-pentanol, add citric acid ammonium solution and anhydrous ethanol, and vortex;

[0120] 2) adding the formulated amount of (3-chloropropyl)trimethoxysilane and butyl orthosilicate, vortexing, then ultrasonicating, and reacting at room temperature with stirring for 2.5 hours;

[0121] 3) After the reaction, centrifuge and wash with anhydrous ethanol.

[0122] The folate-free and PEG-modified triptolide-loaded photothermal motor (SNM@TP) is composed of the following components in parts by weight:

[0123]

[0124]

[0125] Specifications and sources of the main components: triptolide (99%, reference substance, Sichuan Chengdu Zhibiao Huachun Biotechnology Co., Ltd.), perfluorohexane (AR, 98%, Shanghai Myrel Chemical Technology Co., Ltd.), indocyanine green (75%, Shanghai Aladdin Biochemical Technology Co., Ltd.), methanol (HPLC, 6 Anhui Tiandi High Purity Solvent Co., Ltd.), pure water was purified by PALL ultrapure water instrument.

[0126] The preparation steps of the triptolide-loaded photothermal drive motor are as follows:

[0127] 1) Take the formulated amount of perfluorohexane into the nanobottle through negative pressure;

[0128] 2) Add the formulated amount of triptolide, indocyanine green, tetradecanol and methanol and stir for 30 minutes;

[0129] 3) Add a certain amount of pure water and centrifuge to wash.

[0130] The blank nanoparticles prepared in the above example were observed for particle size potential and morphology. Figure 1 , Table 1. The in vitro release, cell pharmacology and cytotoxicity of the triptolide-loaded photothermal motors, folic acid-free and PEG-modified triptolide-loaded photothermal motors prepared in the above examples were investigated. Figure 2 , Figure 3 , Figure 4 .

[0131] The results showed that as the reaction time progressed, at different concentrations, the opening of the nanoparticles showed a bottleneck-like structure, and the shape of the nanoparticles was slightly different. At a concentration of 0.18 mol / L, the bottle of SNM-1 nanoparticles was smaller, and the bottleneck connecting to the external environment was longer, forming a tadpole shape, which may affect drug loading and drug release. 1 At the concentration, the SNM-2 bottle body is round with a short bottleneck, similar to the shape of a flask. The shape of SNM-3 nanoparticles at 0.36 mol / L is similar to that at 0.2 mol / L, but some nanoparticles do not have a bottleneck structure.

[0132] Phase change materials can achieve phase change according to the change of ambient temperature, thereby achieving the purpose of controlled release of drugs. The FAPEG-SNM@TP and SNM@TP prepared in this study are excited by near-infrared light, and the temperature rises, causing the tetradecanol to achieve solid-liquid transformation, thereby releasing the drug. This experiment used the direct release method to investigate the release characteristics of FAPEG-SNM@TP and SNM@TP. The results are as follows Figure 2 As shown. Under light conditions, FAPEG-SNM@TP and SNM@TP released 50.99±0.04% and 54.40±3.38% of the total amount of drug, respectively, at 6 h. Under no light conditions, FAPEG-SNM@TP and SNM@TP released 21.13±2.89% and 16.87±2.87% of the total amount of drug, respectively, at 6 h. The results show that tetradecanol can control drug release and prevent drug leakage to a certain extent.

[0133] Proinflammatory cytokines, such as TNF-α, IL-6, and IL-1β, play an important role in the immune-related pathogenesis of RA. Therefore, in order to preliminarily verify the in vitro anti-inflammatory ability of FAPEG-SNM@TP and SNM@TP, the cytokine content in the supernatant of RAW264.7 cells treated with LPS was detected using an Elisa kit. Figure 3 Based on the CCK-8 experiment, the levels of TNF-α, IL-6 and IL-1β in the supernatant of each group after LPS treatment were measured. Each treatment group was able to inhibit their increase, but there was no statistical difference between the groups, proving that triptolide can also inhibit the production of inflammatory factors at a lower dosage concentration.

[0134] The in vitro cytotoxicity of free drugs and different preparations on RAW264.7 cells under the action of different concentrations of TP was studied, and the biocompatibility of FAPEG-SNM@TP and SNM@TP and the effect of photothermal effect on cells were evaluated. After incubation for 4 h, the viability of cells in each group was detected by CCK-8 method. Figure 4 At a concentration of 0.2 μmol / L, the cytotoxicity of SNM@TP and FAPEG-SNM@TP was significantly lower than that of free TP. The cell survival rate decreased after SNM@TP illumination, and there was a very significant difference between SNM@TP NIR and the original drug group. At a concentration of 0.4 μmol / L, there was a very significant difference between the FAPEG-SNM@TP group and the original drug group, and the survival rate of the original drug group was less than 80%. At a concentration of 0.6 μmol / L, the survival rate of the original drug group was less than 80% and the survival rate of SNM@TP NIR was less than 50%. At a concentration of 0.8 μmol / L, the survival rate of the original drug group was less than 60%, and the survival rate of the SNM@TP NIR group was less than 40%. At a concentration of 1 μmol / L, the survival rates of the FAPEG-SNM@TP, FAPEG-SNM@TP NIR and SNM@TP groups were 80%.

[0135] The IC50 values ​​were 0.6322 μmol / L for SNM@TP, 0.5737 μmol / L for SNM@TP NIR, 0.7970 μmol / L for FAPEG-SNM@TP, 0.6296 μmol / L for FAPEG-SNM@TP NIR, and 0.4993 μmol / L for free TP. The overall trend was that the higher the TP concentration of the preparation, the lower the cell survival rate. Illumination would cause certain damage to cells, and modification of FAPEG-silane could reduce the damage to cells caused by irradiation. These results showed that the cell viability of the preparation without modification of FAPEG-silane was significantly lower than that of the modified group under the stimulation of near-infrared light. On the other hand, it was found that the cell viability of the modified preparation under illumination was higher than that of the unmodified group. Therefore, it was speculated that FAPEG-silane could reduce the damage to cells caused by irradiation to a certain extent. In general, the cell activity of FAPEG-silane was significantly higher than that of the unmodified preparation, indicating that it had good biocompatibility.

[0136] Table 1 Particle size, potential and particle size distribution of nanobottles prepared under different conditions

[0137]

[0138] Delivery Mechanism Investigation

[0139] 1. Ex vivo skin penetration

[0140] Male SD rats were injected intraperitoneally with 3% sodium pentobarbital. After the rats were anesthetized, most of the mouse hair was shaved with a shaver, and then the remaining mouse hair was shaved with an electric shaver. The skin was removed, the fat tissue and fascia were peeled off, and after repeated washing with saline, it was dried with filter paper, and then wrapped with tin foil and placed in a -80℃ refrigerator for standby use. When used, the skin wrapped in tin foil was taken out from the -80℃ refrigerator, carefully opened, the skin was taken out, and it was soaked in saline for 30 minutes before use. 20% ethanol saline was injected into the receiving chamber of the Franz diffusion cell as the receiving liquid, and the excess air in the receiving chamber was discharged by ultrasound. The drug-loaded microneedles were placed on the treated ex vivo rat skin and a certain force was applied. After 1 minute, they were fixed with medical tape. The integrity of the skin must be ensured during the treatment process. After the microneedles were dissolved, near-infrared light was irradiated for 10 minutes. After the treatment was completed, the skin was placed on a 12mL receiving chamber, with the skin stratum corneum facing up and the skin tissue facing the receiving chamber, to ensure that the effective diffusion area of ​​the skin was in contact with the liquid surface of the receiving liquid. After the microneedle fixes the supply chamber and the receiving chamber, the diffusion cell is placed on a constant temperature magnetic stirrer, and the speed is 300r / min to simulate the blood and tissue fluid circulation under the human skin, and the temperature is kept constant at (37±5)℃. After 1, 2, 4, 6, 8, 10, 12, 24, and 36 hours of transdermal permeation, all the receiving liquid is collected and placed in an evaporating dish for water bath evaporation and 1mL of methanol is added. After dissolution, it is filtered through a 0.22μm microporous filter membrane and used as a sample for content determination. After sampling, an equal amount of fresh receiving liquid is added in time. Sample analysis is performed according to the content determination chromatographic conditions.

[0141] Calculation of steady-state permeation rate (Jss) and time lag (Tlag): Linear regression was performed on the steady-state permeation segment (straight line portion) in the Qn-t curve. The slope of the obtained straight line is Jss (μg·cm -2 ·h -1 ), the time corresponding to the intersection of the straight line and the horizontal axis is Tlag(h). The results are shown in Table 2.

[0142] Statistical methods: Graphpad prism software was used to perform statistical analysis on the experimental data, and One-way Anova was used. All data were expressed as mean ± standard deviation (x ± s). When P < 0.05, the difference was significant; when P < 0.01, the difference was extremely significant.

[0143] 2. Ex vivo skin retention

[0144] Blank skin sample processing: Take a blank skin sample, thaw it at room temperature, cut a 1cm 2, put it in a glass homogenizer, add 3mL of extraction solvent methanol, grind it thoroughly to get skin homogenate. Vortex it thoroughly for 1min, centrifuge it at 4℃ and 1500r / min for 20min, transfer the supernatant to a 5mL centrifuge tube, take 20L of supernatant after centrifugation, and inject it according to the content determination chromatographic conditions for analysis.

[0145] Treatment of drug-containing skin samples: After the in vitro skin permeation test (36 hours), remove the skin, rinse the inner and outer surfaces repeatedly with physiological saline for 5 times, and use filter paper to absorb excess water on the skin surface. Take the treated skin sample and cut it into a suitable size (same as the blank skin group, if it is less than 1cm2, use blank skin to make up). Extraction is the same as blank skin sample treatment, and the sampling and analysis process is the same as the in vitro skin permeation experiment.

[0146] Calculation of drug retention (Qm):

[0147] Qm is calculated as follows:

[0148] Qm=X / A

[0149] Where X is the drug retention amount (g) measured in each skin sample after 36 hours of drug application; A is the effective penetration area

[0150] Statistical methods: Same as the above-mentioned treatment method for ex vivo skin penetration.

[0151] 3. In vivo fluorescence imaging of rats

[0152] In vivo imaging of drug release: To detect the distribution of nanoparticles in arthritis model rats, SNM@TP and FAPEG-SNM@TP (1 ml of microneedle matrix contained 3.5 mg of the preparation, respectively) were used to pierce the stratum corneum with microneedles, and the preparations were released after the microneedles dissolved.

[0153] The IVIS small animal in vivo fluorescence imaging system was used to observe the distribution of nanoparticles in rats after different administration times (1, 2, 4, 8, 12, and 24 hours). Isoflurane anesthesia was used during the operation, with an initial dose of 3% and 1% during imaging. The spectral separation method was used to obtain fluorescence information in the experiment. The preparation contains ICG, which can be used as fluorescence information to reflect the distribution of nanoparticles. It can be excited by external light with a wavelength range of 750 to 810 nm and emit near-infrared light with a wavelength of about 808 nm.

[0154] 4. In vitro skin penetration results

[0155] From Table 2 and Figure 6It shows that at 36h, the skin permeation percentage of triptolide in FAPEG-SNM@TP NIR is about 3.31 times that of the TP original drug group. The cumulative skin percentage of triptolide in FAPEG-SNM@TP is about 1.71 times that of the original drug group. The skin permeation percentages of triptolide in SNM@TP NIR and SNM@TP are 1.98 and 2.14 times that of the original drug group, respectively. It can be seen that the permeation percentage of triptolide in the microneedle group is higher than that of the original drug group. Microneedles are a transdermal device that can overcome the barrier of the stratum corneum. Therefore, the cumulative release rate of the microneedle group is higher than that of the original drug group. Microneedles can effectively deliver drugs, improve the bioavailability of drugs, and can also effectively deliver nanoparticles to the subcutaneous tissue.

[0156] 5. Ex vivo skin retention results

[0157] Depend on Figure 5 It can be seen that after the 36-hour transdermal test, except for the FAPEG-SNM@TP group, the skin retention of the other groups was significantly higher than that of the original drug group. The drug retention in the skin of the FAPEG-SNM@TP light-irradiated group after 36 hours was 29.45 times that of the original drug group; the drug retention in the skin of the SNM@TP light-irradiated group was 58.69 times that of the original drug group. Among them, there was a significant difference in the skin retention of the two groups of light-irradiated preparations compared with the original drug group (P < 0.0001). Figure 5 and Figure 6 The results showed that there was no significant difference in the cumulative release of SNM@TP and SNM@TP in the illumination group after 36 hours, but the retention of the illumination group was 5.32 times that of SNM@TP; the retention of the FAPEG-SNM@TP illumination group in the whole skin after 36 hours was 5.67 times that of the FAPEG-SNM@TP group. Illumination can not only increase drug release, but also increase drug retention.

[0158] Table 2 In vitro skin permeation parameters of TP in drug-loaded microneedles and original drug groups

[0159]

[0160]

[0161] 6. Results of in vivo fluorescence imaging of rats

[0162] The real-time fluorescence imaging results of CIA rats at different time points within 24 hours after treatment with different microneedle preparations are shown in Figure 2. Figure 4-5After 8 hours, the fluorescence intensity of each group decreased. For FAPEG-SNM@TP microneedles, the fluorescence was strongly concentrated in the inflamed joint area. At 24 hours, the fluorescence intensity of the FAPEG-SNM@TP group was slightly higher than that of the other groups, indicating that the preparation could stay in the inflamed area to exert its effect. At 24 hours, the fluorescence intensity of the illumination group was lower, which may be because the ICG fluorescence was weakened after NIR laser irradiation.

[0163] 7. Experimental results of delivery mechanism investigation

[0164] The results of the in vitro skin experiment showed that the microneedle group could significantly increase the penetration and retention of the drug. Near-infrared light irradiation could significantly increase the retention of the drug in the skin. The results of the in vivo fluorescence imaging experiment showed that FAPEG-silane modification could make the preparation accumulate at the site of inflammation and prolong the retention of the preparation in the joint cavity.

[0165] In addition, it should be noted that the names of the specific reagents and raw materials described in this specification may be different. All equivalent or simple changes made according to the principles and embodiments of the present invention are included in the protection scope of the present invention. Those skilled in the art of the present invention may make various modifications or supplements to the specific embodiments described or replace them in a similar manner, as long as they do not deviate from the structure of the present invention or exceed the scope defined by the claims, they should all fall within the protection scope of the present invention.

Claims

1. A triptolide-loaded photothermal-driven motor microneedle for treating rheumatoid arthritis, characterized in that: The invention comprises the following components: 300-500 parts of silicon nanobottle modified with folic acid PEG, 15-20 parts of triptolide, 5-30 parts of perfluorohexane, 5-10 parts of indocyanine green, 100-200 parts of tetradecanol, and 1-10 parts of pure water; The method for preparing the triptolide-loaded photothermal-driven motor microneedle for treating rheumatoid arthritis comprises the following steps: Step 1: Prepare a silicon nanobottle by reacting (3-chloropropyl)trimethoxysilane and butyl orthosilicate; Step 2, reacting folic acid PEG-silane with silicon nanobottle to generate folic acid PEG-modified silicon nanobottle; Step 3, 15-20 parts of triptolide, 100-200 parts of tetradecanol, 5-30 parts of perfluorohexane, and 5-10 parts of indocyanine green are loaded into 300-500 parts of folic acid PEG-modified silicon nanobottles to obtain a triptolide photothermal drive motor; Step 4: Mix the triptolide photothermal drive motor and the microneedle matrix evenly, pour them into the microneedle mold, and dry them to obtain the triptolide photothermal drive motor microneedle.

2. The method for preparing a triptolide-loaded photothermal-driven motor microneedle for treating rheumatoid arthritis according to claim 1, characterized in that: The volume ratio of the (3-chloropropyl)trimethoxysilane to butyl orthosilicate is 1:10, and the molecular weight of the folic acid PEG-silane is 2000.

3. The method for preparing a triptolide-loaded photothermal-driven motor microneedle for treating rheumatoid arthritis according to claim 1, characterized in that: The steps of preparing a silicon nanobottle by reacting (3-chloropropyl)trimethoxysilane and butyl orthosilicate include: Step 1: Weigh a certain amount of polyvinyl pyrrolidone and dissolve it in n-pentanol, add citric acid ammonium solution and anhydrous ethanol, and vortex; Step 2: Add a certain amount of (3-chloropropyl)trimethoxysilane and butyl orthosilicate, vortex, then sonicate, and react under stirring at room temperature; Step 3: After the reaction is completed, centrifuge, wash with anhydrous ethanol, and dry to obtain a silicon nanobottle.

4. The method for preparing a triptolide-loaded photothermal-driven motor microneedle for treating rheumatoid arthritis according to claim 3, characterized in that: The citric acid ammonium solution is prepared by dissolving 2.58 g of citric acid in ammonia water and then making the volume to 50 mL of 0.2 M citric acid ammonium solution.

5. The method for preparing a triptolide-loaded photothermal-driven motor microneedle for treating rheumatoid arthritis according to claim 1, characterized in that: The specific steps of reacting folic acid PEG-silane with nanobottle to form folic acid PEG-modified silicon nanobottle are as follows: Step 1, weigh a certain amount of the above silicon nanobottle and disperse it in a mixed solution of anhydrous ethanol and ammonia water; Step 2: In a 50°C water bath, add a folic acid PEG-silane solution dropwise and stir to react for 24 hours. The folic acid PEG-silane solution is prepared by dissolving 2 mg of folic acid PEG-silane in 2 mL of DMSO. Step 3: After the reaction is completed, wash with anhydrous ethanol and dry to obtain a silicon nanobottle modified with folic acid PEG.

6. The method for preparing a triptolide-loaded photothermal-driven motor microneedle for treating rheumatoid arthritis according to claim 1, characterized in that: The specific steps of loading triptolide, tetradecanol, perfluorohexane, and indocyanine green into a silicon nanobottle modified with folic acid PEG to obtain a triptolide photothermal driven motor include: Step 1: taking perfluorohexane and loading it into the above-mentioned silicon nanobottle modified with folic acid PEG by negative pressure, wherein the perfluorohexane has been previously treated with oxygen enrichment; Step 2: Add triptolide, indocyanine green, tetradecanol and methanol, and stir for 30 minutes; Step 3, adding pure water and centrifuging for washing to obtain the triptolide photothermal drive motor.

7. The method for preparing a triptolide-loaded photothermal-driven motor microneedle for treating rheumatoid arthritis according to claim 1, characterized in that: The steps of mixing the triptolide photothermal drive motor and the microneedle matrix evenly and pouring them into the microneedle mold and drying them to obtain the triptolide photothermal drive motor microneedles are as follows: Step 1: Dissolve hyaluronic acid in appropriate amount of ultrapure water and let stand to defoam; Step 2: Add the above-mentioned triptolide photothermal drive motor to the hyaluronic acid solution and mix evenly to obtain a drug-containing microneedle matrix; Step 3: Add the drug-containing microneedle matrix to the PDMS mold, evacuate the mold, remove bubbles and dry it to obtain the triptolide-loaded photothermal-driven motor microneedle.

8. The method for preparing a triptolide-loaded photothermal-driven motor microneedle for treating rheumatoid arthritis according to claim 7, characterized in that: The concentration of the hyaluronic acid solution was 100 mg / mL, the microneedle array of the microneedle PDMS mold was 10×10, and the needle tip length was 600 μm.

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