Cryomicroneedles loaded with TNF-α-siRNA and their applications

Abstract

The present invention discloses a frozen microneedle loaded with TNF-α-siRNA and its application, characterized in that the frozen microneedle is composed of TNF-α-siRNA and water. The present invention introduces a frozen microneedle patch encapsulating TNF-α-siRNA based on the characteristics of ice. The stability of siRNA can be ensured by the low-temperature production environment. It can easily release the drug continuously as the needle body melts after penetrating the skin to achieve transdermal drug delivery. The frozen microneedle has the advantages of convenience, no sharp waste residue, and no needle body breakage.

Description

Technical Field

[0001] The invention belongs to the technical field of medical devices, and specifically relates to a frozen microneedle loaded with TNF-α-siRNA and an application thereof. Background Art

[0002] Rheumatoid arthritis (RA) is a chronic, symmetrical, refractory, systemic autoimmune disease with inflammatory synovitis as the main symptom, which is mainly manifested by symmetrical joint swelling, morning stiffness, pain and dysfunction. At present, there are many treatment methods for RA, but there is no specific method to completely cure RA. Clinically, non-steroidal anti-inflammatory drugs, anti-rheumatic drugs, and glucocorticoids are widely used in the treatment of RA, but these drugs often have large side effects and are prone to gastrointestinal irritation. The toxic side effects of the central nervous system and cardiovascular damage caused by long-term use have become the main causes of life threats. Therefore, RA patients are in urgent need of a drug or treatment method with small side effects and good therapeutic effects. Acupoint therapy is an important part of the theory of traditional Chinese medicine therapy. It is based on the meridian theory. The drug is applied to specific acupoints through the meridian system. According to its stimulating effect on the acupoints, the local concentration of the drug in the affected area can be increased, the adverse reactions can be reduced, and it is more suitable for long-term medication. Compared with traditional Western medicine methods, it has more advantages in the treatment of RA.

[0003] Tumor necrosis factor-α (TNF-α) is the most important participant in the cascade of inflammatory responses and is a proinflammatory factor widely distributed in the serum and synovial fluid of RA patients. The immune network composed of many cytokines, including TNF-α, plays a key role in the development of RA. TNF-α can inhibit the expression of related proteins in the inflammatory signaling pathway and promote the development of inflammation, and is closely related to the severity of RA. Small interfering RNA (siRNA) is a technology that specifically knocks down target genes to reduce their expression and effectively treat related diseases. Therefore, the inflammatory progression of RA can be reduced by specifically inhibiting the expression of TNF-α, thereby alleviating the development of the disease. In 2001, Elbashir et al. first confirmed the existence of RNAi mechanism in mammalian cells. At present, since the in vivo siRNA drug delivery method is usually to dissolve siRNA and then inject it locally or systemically, there are disadvantages such as poor compliance due to strong skin irritation, which brings discomfort to patients, sharp waste residues and a significant risk of infection. At the same time, siRNA is highly hydrophilic, and the stratum corneum of the skin will prevent the transdermal effect of hydrophilic drugs, making it difficult for them to pass through the stratum corneum barrier of the skin and enter the blood to achieve therapeutic effects, which limits their clinical application. Therefore, it is very important to develop a safe and efficient transdermal delivery system for siRNA.

[0004] Microneedle technology is another new transdermal drug delivery method after traditional transdermal drug delivery. It can form micron-level channels in the skin to improve the permeability of drugs, and the channels can recover by themselves. It can accurately deliver drugs to specific skin layers or tissues without almost touching the dermis, greatly reducing the pain during use to simplify the drug delivery process. It can be used as a carrier for some drugs. Since siRNA usually needs to be dissolved in a nuclease-free (RNase) solution before use, and the stability of liquid siRNA is much lower than that of lyophilized powder siRNA, siRNA should be kept at a low temperature as much as possible after dissolution to avoid repeated freezing and thawing. Currently, the existing types of microneedles are made at room temperature or warm and dry conditions, and the stability of liquid siRNA cannot be guaranteed.

[0005] Cryo-microneedles are essentially a type of soluble microneedles. Since Henry first used microneedles for transdermal drug delivery in 1998, microneedle technology has received extensive attention and research from scholars. Similar to other types of microneedles, cryo-microneedles have the dual advantages of injection and transdermal drug delivery and are not limited to loading protein macromolecule drugs, nucleic acid drugs, vaccines and small molecule drugs. Currently, they can also provide in vivo treatment, such as therapeutic cells, predatory bacteria, etc. Compared with other types of microneedles, cryo-microneedles have good biocompatibility and can greatly retain the biological activity of the encapsulated drugs. They will not have the dimensional errors caused by dehydration during the drying process of ordinary soluble microneedles, and other problems such as allergies and rashes after other materials penetrate the skin. In addition, compared with injection, cryo-microneedles cause extremely small trauma to the skin. In addition, they can achieve precise positioning, high efficiency and safety during use, making them a very cutting-edge choice for transdermal drug delivery.

[0006] However, since TNF-α-siRNA is easily affected by the carrier, it is difficult to ensure its therapeutic activity using conventional microneedle carriers. Therefore, how to provide a frozen microneedle that can effectively ensure the therapeutic activity of TNF-α-siRNA and ensure the mechanical strength of the microneedle, while avoiding the frozen microneedle from melting quickly at room temperature, thereby making it difficult to use the frozen microneedle normally, is still a technical problem that needs to be urgently solved by technicians in this field. Summary of the invention

[0007] In view of the above shortcomings, the present invention provides a cryo-microneedle loaded with TNF-α-siRNA and its application. Specifically, in order to achieve the purpose of the present invention, the present invention intends to adopt the following technical solutions:

[0008] The present invention relates to a frozen microneedle loaded with TNF-α-siRNA, characterized in that the frozen microneedle is composed of TNF-α-siRNA and water. The frozen microneedle of the present invention has a simple formula, can effectively ensure its therapeutic activity, can ensure the mechanical strength of the frozen microneedle, and can also prevent the frozen microneedle from melting quickly at room temperature.

[0009] In a preferred embodiment of the present invention, the length of the cryo-microneedle body is 1000-2000 μm, and the length and width of the bottom are both between 400-800 μm.

[0010] In a preferred embodiment of the present invention, the cryo-microneedle contains 300-500 ng of TNF-α-siRNA per microneedle.

[0011] In a preferred embodiment of the present invention, the maximum load rupture force of the cryo-microneedle is 0.12-0.24 N / needle.

[0012] In a preferred embodiment of the present invention, the sequence of the TNF-α-siRNA is 5'-GCCACACCAAAGCAUUCAATT-3'.

[0013] In a preferred embodiment of the present invention, the water is ultrapure water, particularly preferably DEPC water.

[0014] Another aspect of the present invention relates to a method for preparing the above-mentioned cryo-microneedle, which is characterized by comprising the following steps:

[0015] Using a PDMS negative mold, the TNF-α-siRNA solution is added to the PDMS negative mold twice. After the first addition, the mold is vacuum treated and then centrifuged. Before the second centrifugation, the mold is rotated 180 degrees and then centrifuged again to allow the solution to fully enter the needle cavity; finally, a sufficient and appropriate amount of water is used as the mold base to fill the bottom of the mold, and the entire mold is cooled at 0-4°C, and then placed at -15 to -25°C for 2-4 hours, and frozen at -60°C to -100°C for 2-4 hours. Finally, the frozen microneedle is gently separated from the PDMS mold and frozen under liquid nitrogen conditions, and then stored at -60°C to -100°C. Compared with direct freezing at -80°C and gradient freezing, it is found that the needle tip cannot be completely peeled off from the mold when freezing at -80°C, while the frozen microneedle can be completely peeled off by using the gradient freezing of the present invention.

[0016] Another aspect of the present invention also relates to the use of the above-mentioned cryo-microneedles in the preparation of microneedles for treating rheumatoid arthritis.

[0017] In a preferred embodiment of the present invention, the microneedle for treating rheumatoid arthritis is used for Zusanli acupoint. TNF-α-siRNA cryo-microneedle administration via acupoints and non-acupoints was applied to the rabbit rheumatoid arthritis model induced by ovalbumin (OAV). TNF-α-siRNA cryo-microneedle administration via acupoints can further improve the efficacy of rheumatoid arthritis compared with transdermal administration at non-acupoints. Beneficial Effects

[0018] The present invention introduces a TNF-α-siRNA-encapsulated frozen microneedle patch based on the properties of ice. The stability of siRNA can be guaranteed by the low-temperature production environment. After penetrating the skin, the drug can be continuously released as the needle body melts, thereby achieving a transdermal drug delivery effect. The frozen microneedles have the advantages of being convenient, having no sharp waste residues, and not having needle body breakage. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 a) Preparation process of cryo-microneedle; b) 3D rendering of cryo-microneedle;

[0020] Figure 2 a) Array image of frozen microneedles; b) Needle-shaped image of frozen microneedles; c) Melting of frozen microneedles at room temperature; d) Melting of frozen microneedles on fingertips;

[0021] Figure 3 FAM-TNF-α-siRNA standard curve;

[0022] Figure 4 a) The results of rat skin puncture in vitro before staining; b) The results of rat skin puncture in vitro after staining; c) The penetration ability of cryo-microneedles at different exposure times;

[0023] Figure 5 Image of cryo-microneedle applied to the skin of a rabbit knee joint;

[0024] Figure 6 Investigation of the mechanical properties of cryo-microneedles and several commonly used polymer microneedles;

[0025] Figure 7 a Schematic diagram of the induction and drug intervention of RA rabbit model; b body weight change; c knee joint diameter change; Note: Compared with the control group, P<0.01; compared with the model group, #P<0.05, ##P<0.01; the same as the following charts.

[0026] Figure 8 Effects of TNF-α siRNA cryo-microneedling on serum TNF-α, IL-1β, IL-6, and IL-10 levels;

[0027] Fig. 9 Effects of TNF-α siRNA cryo-microneedling on microvascular blood flow in rabbits;

[0028] Fig.10 Effects of TNF-α siRNA cryo-microneedling on rabbit synovial tissue pathology;

[0029] Fig.11 RT-PCR was used to detect the expression of TNF-α, MMP-1, MMP-3, and TIMP-1 mRNA in rabbit synovial tissue;

[0030] Fig.12 Western-blot was used to detect the expression of related proteins in rabbit synovial tissue. DETAILED DESCRIPTION

[0031] In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Unless otherwise specified, the reagents involved in the embodiments of the present invention are all commercially available products and can be purchased through commercial channels.

[0033] Embodiment 1:

[0034] Experimental Materials

[0035] 1.1 Main Reagents

[0036] Ovalbumin (batch number C12654052, Shanghai Bio Biotechnology Co., Ltd.); Freund's complete adjuvant (batch number SLCF1289, SIGMA, USA); Rabbit tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10) ELISA detection kits (batch number 20211220, Nanjing Jiancheng Bioengineering Institute); TNF-α-siRNA specially modified with 2'OMe (5'—GCCACACCAAAGCAUUCAATT—3') was purchased from Suzhou Jima Gene Co., Ltd.; DEPC water (lot number R0021, Beyotime); 4% paraformaldehyde fixative (lot number AR1068, BOSTER); RIPA protein lysis buffer (lot number 20210826, Solarbio), BCA protein concentration assay kit (lot number 20210903, Solarbio); Trizol (lot number 10296028, Invitrogen); SuperScript III RT reverse transcription kit (lot number 11752050, ABI-invitrogen); Sybr qPCR mix (lot number 4472920, ABI-invitrogen); β-actin Antibodies (lot number F20040, Abways); MMP-1 antibody (lot number 20211010, Bioswamp), MMP-3 antibody (lot number 20211010, Bioswamp), TIMP-1 antibody (lot number 20211010, Bioswamp), fluorescent secondary antibody (lot number F300405, Abways).

[0037] 1.2 Main instruments

[0038] Low-temperature high-speed centrifuge TG-16M (Shanghai Luxiangyi Centrifuge Instrument Co., Ltd.); Constant temperature vacuum drying oven DZF-6090 (Shanghai Yiheng Technology Co., Ltd.); PDMS microneedle mold (Anhui Zhongding Yuxuan New Material Technology Co., Ltd.); Fully automatic embedding machine ES600 (Leica, Germany); Paraffin pathology slicer RM2235 (Leica, Germany); Handheld microscope 10085-1A (Xinxiang Optics); Texture analyzer CTX (Brookfield, USA); Optical microscope DM3000 (Leica, Germany); Enzyme-linked immunosorbent assay instrument SYNERGY H1 (Berteng Instrument Co., Ltd., USA); Vortex oscillator MS3 Basic (IKA, Germany); BI-2000A+ microcirculation detector (Chengdu Taimeng Software Co., Ltd.); Fluorescence quantitative PCR instrument StepOneSoftware (Applied Biosystems, USA); Electrophoresis instrument EPS 300 (Bio-Rad, USA); DYCZ-24DN Double vertical electrophoresis instrument (Beijing Liuyi Company); ChemiDocTMXRS+ gel imaging system (Bio-Rad Company, USA).

[0039] 1.3 Experimental animals

[0040] Male New Zealand rabbits, weighing 2.3-2.5 kg, were provided by the Experimental Animal Center of Heilongjiang University of Chinese Medicine. The experimental animal qualification certificate number is SYXK (black) 2021-004. The room temperature was controlled at (24.0 ± 2.0) °C, the relative humidity was maintained at about 50%, the instruments were disinfected regularly, the dark and light time were equally distributed for 12 hours, and the rabbits were adapted to feeding for 7 days. The animal experiments related to this experiment strictly followed the regulations of Heilongjiang University of Chinese Medicine on the management and use of experimental animals.

[0041] 2. Methods and Results

[0042] 2.1 Preparation of TNF-α-siRNA-loaded cryo-microneedles

[0043] During the preparation process, a PDMS negative mold with a needle body length of 1400 μm and a bottom width of 600 μm was used. A TNF-α-siRNA solution with a concentration of 20 μM prepared with an appropriate amount of DEPC water was added to the PDMS negative mold twice. After the first addition, the mold was vacuum treated for 5 min (-0.09-0.1Mpa) and then centrifuged at 3000 r / min for 5 min. Before the second centrifugation, the mold was rotated 180° and centrifuged to allow the solution to fully enter the needle cavity; finally, a sufficient and appropriate amount of DEPC water was used as the mold base to fill the bottom of the mold, and the entire mold was cooled at 4°C for 40 minutes, placed at -20°C for 3 h, and frozen in a -80°C refrigerator for 3 h. Finally, the frozen microneedle was gently detached from the PDMS mold and placed under liquid nitrogen at -196°C for 1 h. It was obtained and stored at -80°C to ensure its mechanical strength. The specific preparation process and 3D renderings are shown in the figure below. Figure 1 As shown in a and 1b.

[0044] 2.2 Morphology of cryo-microneedles

[0045] 2.2.1 Array morphology of cryo-microneedles

[0046] After the frozen mold is removed, use the naked eye ( Figure 2 a) and handheld microscope observation ( Figure 2 b) Its array. The entire frozen microneedle is a 15 mm × 15 mm square, colorless, uniform, transparent ice-like, with a flat basal layer, a complete needle body and the same spacing, forming a 10 × 10 array perpendicular to the backing and arranged regularly and completely. The stability of the frozen microneedles at room temperature and body temperature was also studied. The microneedle patch was taken out from -196°C liquid nitrogen conditions and placed at room temperature and body temperature respectively. A digital camera was used to observe its melting behavior. After being placed at room temperature for about 20 s, frost began to appear on the needle tip, and the needle tip began to melt after about 140 s. When placed at 37°C for about 50 s on the fingertips, the needle tip began to melt. There was no pain or discomfort on the fingertips during the continuous shooting process. The stability was evaluated based on the relationship between the needle tip length and the residence time. The results are as follows. Figure 2 As shown in c and 2d, the morphology of the frozen microneedles at RT is maintained slightly longer.

[0047] 2.2.2 TNF-α-siRNA in cryo-microneedles

[0048] Dissolve fluorescently labeled TNF-α-siRNA (FAM-TNF-α-siRNA) in an appropriate amount of 0.1% DEPC water to prepare FAM-siRNA stock solution. Take 0.1% DEPC water to dilute the stock solution to 0.05μg / μL, 0.10μg / μL, 0.15μg / μL, 0.20μg / μL, 0.25μg / μL, 0.30μg / μL standard solutions with different concentration gradients. Measure the OD value of each group by an enzyme marker at a wavelength of 492nm, and draw a standard curve based on the test results. The results are shown in Table 1. Figure 3 .

[0049] Table 1 Absorbance values ​​of standard solutions of different concentrations

[0050]

[0051] The standard curve of OD value (Y) against FAM-TNF-α-siRNA concentration (X) was obtained, and the standard curve equation was: y = 988.58x + 124.2 (R 2 = 0.9991), with high accuracy and good repeatability. According to the curve results, the OD value and FAM-TNF-α-siRNA concentration showed a good linear relationship in the range of 0.05-0.30μg / μL.

[0052] The prepared frozen microneedle patch was placed in 1 mL of DEPC water and dissolved. After the solution was completely dissolved, the absorbance value was measured at 492 nm. The concentration of FAM-TNF-α-siRNA was calculated according to the standard curve and the content was converted according to its molecular weight. Each frozen microneedle contained approximately (39.6±1.29) μg of TNF-α-siRNA (n=10).

[0053] 2.3 Mechanical strength of cryo-microneedles

[0054] Mechanical strength is one of the most important mechanical properties of soluble microneedles. The necessary condition for frozen microneedles to penetrate the stratum corneum and reach the dermis to successfully deliver drugs is that the needle body has sufficient mechanical strength. In this experiment, the mechanical strength of frozen microneedles was tested using a texture analyzer. The frozen microneedle patch was placed horizontally on a pre-cooled stainless steel plate, and then a 4 mm diameter cylindrical probe was used to measure the mechanical strength of the frozen microneedle at a speed of 0.05 mm min. -1 The probe moves vertically downward to the tip of the cryo-microneedle at a constant speed, and the force recording starts when the cylindrical probe contacts the tip. The changes in displacement and compression force are recorded to evaluate its mechanical strength. Through this method, the mechanical strength of cryo-microneedles and three other commonly used soluble microneedles (hyaluronic acid, polylactic acid and polyvinyl alcohol) are compared horizontally. Figure 4As shown, in this experiment, the compression force / displacement curves of the soluble microneedles made of polylactic acid (PLA) and polyvinyl alcohol (PVA) are similar, and the maximum compression force that each microneedle can withstand can reach an average of 0.2N; the maximum load rupture force of the frozen microneedle is about 0.18N, and the pressure at the tip of the microneedle reaches 0.03N to successfully penetrate the skin. The frozen microneedles prepared in this experiment can withstand 6 times the pressure, so it can ensure that they have sufficient mechanical strength to penetrate the skin.

[0055] 2.4 Ability of cryo-microneedles to penetrate ex vivo skin

[0056] Take the rat skin in vitro, remove the villi and excess tissue. After washing with saline and drying with filter paper, insert the frozen microneedle into the rat skin with a microneedle syringe. Observe the morphology of the needle hole in the skin and immediately stain it with 0.4% trypan blue solution. After 1 min, wash and observe the staining results ( Figure 4 a and 4b). Figure 4 a shows unstained skin with regular pinhole shapes; Figure 4 b shows the stained skin after adding trypan blue solution. The pinhole area has been stained, and the successful staining of the pinhole area indicates that cryoMNs have successfully penetrated the stratum corneum barrier. At the same time, we evaluated the penetration ability of cryomicroneedles into ex vivo rat skin at different exposure times at room temperature (RT). After removing the villi, subcutaneous fat and muscle tissue from the ex vivo rat skin, the cryomicroneedles at different RT exposure times (0s, 15s, 30s, 45s) were inserted into the ex vivo rat skin, and the skin was embedded and fixed with OCT embedding agent. The skin was frozen and cut into sections, stained, and the penetration was observed under a microscope, as shown in Figure 2. Figure 4 As shown in c.

[0057] 2.5 In vivo pharmacodynamics study of cryo-microneedles

[0058] 2.5.1 Establishment, grouping and treatment of rabbit RA model

[0059] Modeling: The ovalbumin solution was fully mixed and emulsified with an equal volume of Freund's complete adjuvant under ice bath conditions to prepare an ovalbumin-Freund's complete adjuvant (OVA) emulsion with a final concentration of 10 mg / mL. 1 mL of OVA emulsion was injected cumulatively at 4-6 points on the scapula and depilated areas of the back of the rabbit for sensitization, and the injection was continued every 7 days for three weeks. On the 21st day, 0.5 mL of ovalbumin solution (containing 5 mg of ovalbumin) was injected perpendicular to the skin on both sides of the patellar ligament at the midpoint of the line connecting the highest point of the tibial tubercle and the lower edge of the patella to strengthen the induction again. The control group was injected with an equal amount of normal saline.

[0060] Grouping: Thirty rabbits were randomly divided into 6 groups, control group (Control), model group (Model), TNF-α siRNA+non-acupoint group (NAP), NC-TNF-α siRNA+non-acupoint group (NC+NAP), TNF-α siRNA+acupoint group (AP), and NC-TNF-α siRNA+acupoint group (NC+AP).

[0061] Treatment: To study the effect of TNF-α siRNA cryo-microneedling on RA, the acupuncture points of Zusanli (located on the dorsolateral side of the hind limb, about 0.3 cm below the tibial tuberosity) on both sides of the knee joints of rabbits were used as the acupuncture points for drug administration, and the area above the knee was used as a non-acupuncture point for drug administration. Considering the feasibility of future experiments, the range was appropriately expanded (±5 mm), see Figure 5 a-5b. The AP group and the NAP group were given a cryo-microneedle patch loaded with TNF-α siRNA at the bilateral knee joint acupoints and non-acupoints, respectively. The NC+AP group and the NC+NAP group were given a cryo-microneedle patch loaded with NC-TNF-α siRNA at the bilateral knee joint acupoints and non-acupoints, respectively. Each group of rabbits started the corresponding treatment after the model was successfully established, and the treatment was given once every 2 days for a total of 4 weeks according to the body weight and the method of administration.

[0062] 2.5.2 Knee joint swelling and body weight changes in rabbits

[0063] The weight of the rabbits was measured every 7 days starting from the day of booster immunization, and the diameter of the knee joint of the rabbits was measured with a micrometer vernier caliper at the midpoint of the line between the highest point of the tibial tubercle and the lower edge of the patella. The changes in weight and knee joint diameter before and after the establishment of the model were recorded.

[0064] 2.5.3 Changes in immune organ index

[0065] The rabbits in each group were fasted for 4 hours, and the rabbits in each group were weighed and anesthetized. The spleen and thymus were separated, and the organ weight was measured 2 hours after the last administration. The immune organ index was calculated as follows: immune organ index = immune organ weight (mg) / rabbit body weight (g).

[0066] 2.5.4 Detection of serum inflammatory factor levels

[0067] All rabbits were fasted for 4 hours before the experiment. Rabbits in each group were anesthetized 1 hour after the last administration, and blood was collected from the ear vein. The blood was placed at room temperature for 2 hours, centrifuged at 3000 rpm for 10 minutes, and the supernatant was separated and stored at -20°C. The levels of TNF-α, IL-1β, IL-6, and IL-10 in serum were detected according to the instructions.

[0068] 2.5.5 Changes in microcirculation indicators

[0069] The magnification of the dissecting microscope of the microcirculation instrument was adjusted to 40×, and after calibration with a micrometer ruler, the microcirculation detector was used to detect the microcirculation arteriolar diameter (AD), venous diameter (VD), vascular resistance (resistance index, RI), comprehensive assessment (CA) and microvascular blood flow (BF) before and after treatment to evaluate the microcirculatory responsiveness.

[0070] 2.5.6 Pathological changes of synovial tissue

[0071] After treatment, rabbits were euthanized by ear vein air embolization, and the synovial tissue of the knee joint was fixed with 10% paraformaldehyde and then dehydrated with different concentrations of ethanol. After being transparentized with xylene, the tissue was embedded in paraffin and sliced. Finally, the slices were stained with hematoxylin-eosin (HE). The histopathological changes of the synovium were observed under a light microscope. The morphological changes of the synovial tissue in each group were compared.

[0072] 2.5.7 RT-PCR detection of MMP-1, MMP-3, and TIMP-1 mRNA expression in synovial tissue

[0073] We used RT-PCR to detect the expression levels of TNF-α, MMP-1, MMP-3, and TIMP-1 mRNA in the synovial tissue of each group of rabbits. The synovial tissue of each group of rabbits was taken, and the total RNA of the specimens was extracted using Trizol reagent. Then, the quality of the total RNA was detected using concentration and purity as indicators. The cDNA was synthesized using a reverse transcription kit. After the reaction was completed, the PCR product was scanned and imaged by 2% agarose gel electrophoresis and an automatic gel imaging analysis system. The relative expression of mRNA was expressed by the ratio of the density values ​​of the electrophoresis bands of the target gene and the internal reference gene. The primer sequences are shown in Table 2.

[0074] Table 2 Primer sequences

[0075]

[0076] 2.5.8 Western blot detection of the expression of MMP-1, MMP-3, and TIMP-1 related proteins in synovial tissue

[0077] After the treatment, the rabbits were euthanized, and the synovial tissue of the rabbit knee joint was obtained. After being quickly frozen under liquid nitrogen, it was fully ground. The RIPA lysis buffer was added and the tissue protein was shaken and lysed under ice bath conditions. After lysis, the supernatant was centrifuged and the total protein concentration of each group was determined by BCA method. The target protein was separated by SDS-PAGE buffer, and the protein was denatured by boiling water bath. The sample was loaded, electrophoresed, and wet transferred to PVDF membrane. The blocking solution was added and blocked at room temperature for 1 h. The primary antibody was added and incubated overnight at 4 °C, and the secondary antibody was incubated for 1 h. The gray value of related proteins was calculated by Image J software using β-action as the internal reference in the gel imaging system to detect the expression of related proteins.

[0078] 2.10 Statistical analysis

[0079] The obtained data were analyzed and processed using GraphPad 8.0.2 statistical software. The measured data were expressed as "mean ± standard deviation" (x ± s), and the inter-group comparison was performed using one-way analysis of variance. P < 0.05 indicated statistically significant differences.

[0080] 3 Results

[0081] 3.1 Morphology of cryo-microneedles

[0082] The prepared frozen microneedle patch is a 15mm×15mm square structure, colorless, uniform and transparent. The basal layer is flat, the needle body is intact, and the spacing is the same, forming a complete 10×10 array perpendicular to the backing. In addition, we also evaluated the stability of frozen microneedles at room temperature (25°C) and body temperature (37°C). The microneedle patch was taken out of liquid nitrogen and placed at room temperature and body temperature, and the melting behavior of the microneedles was observed with a digital camera. After about 20 seconds at 25°C, frost began to appear on the needle tip, and the needle tip began to melt after about 140 seconds. After about 50 seconds at 37°C, the needle tip began to melt, and no pain or discomfort occurred during continuous shooting. The frozen microneedle morphology at 25°C was retained slightly longer.

[0083] 3.2 Mechanical strength of cryo-microneedles

[0084] like Figure 6 As shown in the figure, when evaluating the mechanical stability of cryoMNs, the compression force / displacement curves of the soluble microneedles made of polylactic acid (PLA) and polyvinyl alcohol (PVA) are similar, and the maximum compression force that each microneedle can withstand can reach an average of 0.2N; the maximum load rupture force of the frozen microneedle is about 0.18N, and the pressure at the tip of the microneedle reaches 0.03N to successfully penetrate the skin. The frozen microneedles prepared in this experiment can withstand 6 times the pressure, so it can ensure that they have sufficient mechanical strength to penetrate the skin.

[0085] 3.4 Ability of cryo-microneedles to penetrate ex vivo skin

[0086] according to Figure 4 The results shown in c show that when the cryo-microneedles were kept at 24 ± 2.0 °C for 30 s, they could penetrate the dermis, but began to lose this ability at 45 s. The cryo-microneedles could penetrate the dermis (523 ± 63 μm) in <15 s at 24 ± 2.0 °C. The results show that the ability of cryo-microneedles to penetrate the skin decreases with the prolonged exposure time. Therefore, cryo-microneedles should be used within 30 s after being removed from storage conditions to ensure that they have sufficient penetration ability.

[0087] 3.5 Knee joint swelling and body weight changes in rabbits

[0088] Induction of RA rabbit model, changes in body weight and knee joint diameter Figure 7 As shown. After enhanced induction, the knee joint diameter of the model group increased significantly and the body weight decreased significantly compared with the control group (P<0.01). Compared with the model group, the knee joint swelling of the acupoint group (AP) and the non-acupoint group (NAP) was significantly reduced and the body weight was significantly increased (P<0.01). In addition, the changes in the AP group in alleviating knee joint swelling and weight gain were more obvious than those in the NAP group (P<0.01). Compared with the model group, the NC-TNF-α siRNA+acupoint group (NC+AP) and the NC-TNF-α siRNA+non-acupoint group (NC+NAP) showed almost no changes.

[0089] 3.6 Changes in immune organ index

[0090] The changes of spleen and thymus weights and immune organ indexes are shown in Table 3. The spleen and thymus weights and immune organ indexes of rabbits in the model group were significantly higher than those in the control group (P<0.01). Compared with the model group, the spleen and thymus weights and immune organ indexes of the acupoint group and non-acupoint group were significantly lower than those of the model group (P<0.05, P<0.01), while NC+AP and NC+NAP had almost no changes.

[0091] Table 3 Changes of immune organ index

[0092]

[0093] 3.7 Changes in serum inflammatory factor levels

[0094] Figure 8is the level of serum inflammatory factors in each group. The levels of TNF-α, IL-1β, and IL-6 in the serum of rabbits in the model group were significantly higher than those in the control group, while the level of IL-10 was significantly lower than that in the control group (P<0.01). Compared with the model group, the levels of TNF-α, IL-1β, and IL-6 in the serum of rabbits in the acupoint group and the non-acupoint group were significantly reduced (P<0.01), and IL-10 was significantly increased (P<0.05, P<0.01). The improvement of the indicators in the acupoint group was more obvious than that in the non-acupoint group. , while NAP and NC+NAP showed almost no changes compared with the model group.

[0095] 3.8 Changes in microcirculation indicators

[0096] Fig. 9 Table 4 shows the changes in microcirculation indexes and microvascular blood flow status of rabbits in each group. Compared with the normal group, the micromotion and venous vessel diameter of rabbits in the model group were significantly reduced, and the comprehensive score and vascular resistance were significantly increased (P<0.01); compared with the model group, the micromotion and venous vessel diameter of rabbits in the acupoint group and non-acupoint group were significantly increased, and the comprehensive score and vascular resistance were significantly reduced (P<0.05, P<0.01), and the changes in various microcirculation indexes in the acupoint group were more obvious than those in the non-acupoint group. However, NAP and NC+NAP had almost no changes. On the other hand, the loop morphology, blood flow velocity and loop state of rabbits in the control group were normal; the model group rabbits had microcirculation disorders such as significantly thinned microvessels, granular oscillatory blood flow, slow stasis, and red blood cell aggregation; compared with the model group, the microcirculation indexes of rabbits in the acupoint group and non-acupoint group were significantly improved. However, NC+AP and NC+NAP had almost no changes.

[0097] Table 4 Changes of microcirculation indexes

[0098]

[0099] 3.9 Pathological changes of synovial tissue

[0100] The synovial cells of the rabbit knee joints in the control group were orderly and complete, without pathological features such as proliferation and inflammatory cell infiltration ( Fig.10 ). Obvious pathological features were observed in the model group, such as massive proliferation of synovial tissue, infiltration of inflammatory cells, tissue congestion caused by tissue vascular dilation, and pannus formation; compared with the model group, although the synovial tissue pathological status of the acupoint group and the non-acupoint group was improved to a certain extent, the effect of the non-acupoint group was not ideal, while the acupoint group had neatly arranged tissue cells, a small amount of synovial cell proliferation, reduced inflammatory cell infiltration, and no pannus formation. NC+AP and NC+NAP showed almost no changes compared with the model group.

[0101] 3.10 Changes in the expression of MMP-1, MMP-3, and TIMP-1 mRNA in synovial tissue

[0102] The expressions of TNF-α mRNA, MMP-1 mRNA, MMP-3 mRNA and TIMP-1 mRNA in the synovial tissue of rabbits in each group were shown in Fig.11 Compared with the control group, the expression levels of TNF-α mRNA, MMP-1 mRNA, and MMP-3 mRNA in the model group were significantly increased (P<0.01), and the expression level of TIMP-1 mRNA was significantly decreased (P<0.01); compared with the model group, the expression levels of TNF-α mRNA, MMP-1 mRNA, and MMP-3 mRNA in the synovial tissue of the acupoint group and the non-acupoint group were significantly decreased (P<0.05, P<0.01), and the expression level of TIMP-1 mRNA was significantly increased (P<0.01); there was almost no change in NC+AP and NC+NAP.

[0103] 3.11 Changes in the expression of MMP-1, MMP-3, and TIMP-1 proteins in synovial tissue

[0104] The expression of related proteins in the synovial tissue of rabbits in each group is as follows Fig.12 Compared with the control group, the expression of MMP-1 and MMP-3 proteins in the synovial tissue of rabbits in the model group was significantly increased (P<0.01), and the expression of TIMP-1 protein was significantly decreased (P<0.01); compared with the model group, the expression of TNF-α, MMP-1, and MMP-3 proteins in the synovial tissue of the acupoint group and the non-acupoint group were significantly decreased (P<0.05, P<0.01), and the expression of TIMP-1 protein was significantly increased (P<0.01); NC+AP and NC+NAP had almost no changes.

[0105] The above describes the preferred embodiments of the present invention, but it is not intended to limit the present invention. Those skilled in the art may make improvements and changes to the embodiments disclosed herein without departing from the scope and spirit of the present invention.

Claims

1. A method for preparing frozen microneedles, characterized in that: The cryo-microneedle is composed of TNF-α-siRNA and water, and the preparation method comprises the following steps: A PDMS negative mold with a needle body length of 1400μm and a bottom width of 600μm was used. A TNF-α-siRNA solution with a concentration of 20μM prepared with an appropriate amount of DEPC water was added to the PDMS negative mold twice. After the first addition, the mold was vacuum treated at -0.09-0.1MPa for 5 minutes and then centrifuged at 3000r / min for 5 minutes. Before the second centrifugation, the mold was rotated 180° and then centrifuged again to allow the solution to fully enter the needle cavity. Finally, a sufficient and appropriate amount of DEPC water was used as the mold base to fill the bottom of the mold. The entire mold was cooled at 4℃ for 40 minutes, then placed at -20℃ for 3h, and frozen in a -80℃ refrigerator for 3h. Finally, the frozen microneedle was gently separated from the PDMS mold and frozen at -196℃ liquid nitrogen for 1h, and then stored at -80℃ to ensure its mechanical strength. The sequence of the TNF-α-siRNA is 5'-GCCACACCAAAGCAUUCAATT-3'.

2. The preparation method according to claim 1, wherein the maximum load rupture force of the frozen microneedle is 0.18N / needle.

3. The cryo-microneedle prepared by the preparation method according to claim 1.

4. Use of the cryomicroneedle according to claim 3 in preparing microneedles for treating rheumatoid arthritis.

5. The use according to claim 4, wherein the microneedle for treating rheumatoid arthritis is used at the Zusanli acupoint.

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