Sinomenine hydrochloride loaded Jingui bean polysaccharide microneedle as well as preparation method and application of sinomenine hydrochloride loaded Jingui bean polysaccharide microneedle

Abstract

The invention relates to a sinomenine hydrochloride loaded chlorcosan polysaccharide microneedle as well as a preparation method and application thereof, and belongs to the technical field of sinomenine hydrochloride microneedles. The chlorpeas polysaccharide microneedle comprises a needle tip layer and a backing layer, the needle tip layer is prepared by performing primary drying on a mixed aqueous solution containing sinomenine hydrochloride and chlorpeas polysaccharide, and the backing layer is prepared by performing secondary drying on a chlorpeas polysaccharide aqueous solution. The sinomenine hydrochloride loaded Jingui bean polysaccharide microneedle has enough mechanical strength to penetrate through the stratum corneum, has biodegradability and good biocompatibility, is small in skin irritation, has good skin permeability and high drug loading capacity, and has flexibility and adhesion due to the fact that the microneedle is backed. When in use, the micro-needle can be tightly adhered to a movable joint part, so that dissolution is promoted, the insertion efficiency of the micro-needle is improved, and the stability of transdermal drug delivery is ensured.

Description

Technical Field

[0001] The invention belongs to the technical field of sinomenine hydrochloride microneedles, and particularly relates to a sinomenine hydrochloride-loaded sinomenine polysaccharide microneedle and a preparation method and application thereof. Background Art

[0002] Rheumatoid arthritis (RA) is a common chronic autoimmune inflammatory disease characterized by chronic inflammation of the synovial membrane, erosion of cartilage and bone tissue, and ultimately destruction of joint structure. Although effective treatments for RA have evolved from nonsteroidal anti-inflammatory drugs and glucocorticoids to modern biological agents and even gene therapy, long-term and frequent use of these drugs may lead to serious side effects such as cardiovascular disease and liver and kidney damage, often accompanied by impaired immune function, potential threats to life and health, and insufficient targeting of inflammatory joints. To date, there is still a lack of ideal therapeutic drugs and methods.

[0003] Microneedles (MNs) are an emerging transdermal drug delivery system that can avoid the first-pass effect, greatly reduce side effects, effectively improve the transdermal delivery efficiency of most drugs, and facilitate self-administration, thereby significantly improving patient compliance. Dissolvable microneedles are usually made of polymers. Polymer microneedles have the advantages of being painless, non-invasive, and having good biocompatibility, which effectively improves the transdermal delivery efficiency of most drugs. Traditional Chinese medicine polysaccharides have a wide range of pharmacological effects, including anti-inflammatory, antioxidant, anti-tumor, and immune regulation. At present, traditional Chinese medicine polysaccharides have been widely used in the manufacture of MNs. As a matrix material, they can not only reflect the intrinsic characteristics and biological functions of polysaccharides, but also provide good stability and high mechanical strength.

[0004] Sinomenine is an active ingredient extracted from the plant Sinomenispermacae Juss, which has anti-rheumatic, anti-inflammatory, immunomodulatory and anti-tumor pharmacological effects, and also has unique advantages in analgesia. Sinomenine hydrochloride (SIN) is a natural alkaloid found in Sinomenispermacae Juss, and was listed in the Chinese Pharmacopoeia (ChP) in 2005. Sinomenine hydrochloride (SIN-hcl) has been used in the clinical treatment of RA in the form of tablets or injections for many years, but Sinomenine hydrochloride (SIN) has a short half-life, low oral bioavailability, and rapid metabolism, resulting in high dosages that can easily cause side effects such as liver and kidney damage, thereby reducing its efficacy and hindering drug delivery to the target joint lesions. Therefore, it is very necessary to develop new routes of administration to improve the efficacy of Sinomenine hydrochloride and reduce side effects. Summary of the invention

[0005] In view of the above problems, the present invention provides a sinomenine hydrochloride-loaded sinomenine polysaccharide microneedle and a preparation method and application thereof.

[0006] The first object of the present invention is to provide a sinomenine hydrochloride-loaded sinomenine polysaccharide microneedle, comprising a needle tip layer and a backing layer, wherein the needle tip layer is made by drying a mixed aqueous solution containing sinomenine hydrochloride and sinomenine polysaccharide once, and the backing layer is made by drying the sinomenine hydrochloride polysaccharide aqueous solution twice.

[0007] Furthermore, the aqueous solution of Glechoma longituba polysaccharide is prepared by mixing Glechoma longituba polysaccharide and water.

[0008] Furthermore, in the aqueous solution of golden armor bean polysaccharide, the dosage ratio of golden armor bean polysaccharide to water is 100-120 mg:1 mL.

[0009] Preferably, the amount ratio of the golden armor bean polysaccharide to water is 100mg:1mL, 105mg:1mL, 110mg:1mL, 115mg:1mL, 120mg:1mL, 115mg:1mL, 120mg:1mL, 125mg:1mL, 130mg:1mL, 135mg:1mL, 140mg:1mL, 145mg:1mL or 150mg:1mL. Further preferably, the amount ratio of the golden armor bean polysaccharide to water is 150mg:1mL.

[0010] Furthermore, the golden bean polysaccharide (hereinafter referred to as PLP) (purity ≥ 95%) was purchased from Shanyang Lianfeng Biotechnology Co., Ltd. PLP is mainly composed of uronic acid and galactose, and the weight average molecular weight of PLP is 300 kDa.

[0011] The golden armor bean polysaccharide is prepared from white kidney beans through water extraction, filtration, concentration and spraying, wherein the processes of water extraction, filtration, concentration and spraying are well known in the technical field of polysaccharide extraction and are not specifically limited in the present invention.

[0012] Furthermore, the mixed aqueous solution containing sinomenine hydrochloride and sinomenine polysaccharide is prepared by mixing sinomenine hydrochloride, sinomenine polysaccharide, a water-soluble polymer material and water.

[0013] Furthermore, in the mixed aqueous solution containing sinomenine hydrochloride and sinomenine polysaccharide, the dosage ratio of sinomenine hydrochloride, sinomenine polysaccharide, water-soluble polymer material and water is 30-60mg:100-150mg:100-150mg:1mL.

[0014] Preferably, the usage ratio of sinomenine hydrochloride, golden bean polysaccharide, water-soluble polymer material and water is 50 mg: 100 mg: 100 mg: 1 mL.

[0015] Furthermore, the water-soluble polymer material is polyvinyl pyrrolidone.

[0016] Furthermore, the height of the needle tips in the needle tip layer is 600-700 μm, the width of the backing layer is 300-400 μm, and the distance between the needle tips in the needle tip layer is 650-750 μm.

[0017] Preferably, the needle tip height of the needle tip layer is 600 μm, 610 μm, 620 μm, 630 μm, 640 μm, 650 μm, 660 μm, 670 μm, 680 μm, 690 μm or 700 μm.

[0018] The width of the backing layer is 300 μm, 310 μm, 320 μm, 330 μm, 340 μm, 350 μm, 360 μm, 370 μm, 380 μm, 390 μm or 400 μm.

[0019] The distance between the needle tips in the needle tip layer is 650 μm, 660 μm, 670 μm, 680 μm, 690 μm, 700 μm, 710 μm, 720 μm, 730 μm, 740 μm or 750 μm.

[0020] The second object of the present invention is to provide a method for preparing sinomenine hydrochloride-loaded sinomenine polysaccharide microneedles, comprising:

[0021] Casting a mixed aqueous solution containing sinomenine hydrochloride and sinomenine polysaccharide into a mold, keeping it under vacuum for a predetermined time, scraping off excess solution on the mold, and drying it once to obtain a mold filled with a needle tip layer;

[0022] The aqueous solution of scutellaria baicalensis polysaccharide is poured onto the surface of the dry mold filled with the needle tip layer, dried twice, and peeled off to obtain scutellaria baicalensis polysaccharide microneedles loaded with sinomenine hydrochloride.

[0023] Furthermore, the predetermined time is 20-50 minutes.

[0024] Furthermore, the primary drying temperature is 15-30°C.

[0025] Furthermore, the secondary drying temperature is 30-40°C.

[0026] The third object of the present invention is to provide the use of the above-mentioned sinomenine hydrochloride-loaded golden bean polysaccharide microneedle in the preparation of drugs for treating rheumatoid arthritis.

[0027] Beneficial effects of the present invention:

[0028] The sinomenine hydrochloride-loaded sinomenine polysaccharide microneedles of the present invention have sufficient mechanical strength to penetrate the stratum corneum, are biodegradable and have good biocompatibility, are less irritating to the skin, and have good skin permeability and a higher drug loading capacity.

[0029] The microneedle backing of the present invention has flexibility and adhesion, and can be closely adhered to the active joint part during use, so as to promote dissolution and improve the insertion efficiency of the microneedle, and ensure the stability of transdermal drug delivery.

[0030] The microneedles in the present invention use scutellaria polysaccharide as the microneedle matrix, which takes advantage of scutellaria polysaccharide as a carrier for controlled release, and achieves the purpose of slow release of sinomenine hydrochloride. Moreover, scutellaria polysaccharide has anti-inflammatory and antioxidant capabilities, and is made into microneedles with sinomenine hydrochloride, which can minimize the damage of microneedles to the skin, and give full play to the advantages of scutellaria polysaccharide as a drug carrier and therapeutic material, reflecting the synergistic anti-inflammatory effect. In addition, when the microneedles are applied to the local joints to accurately target the synovial lesions of arthritis, joint swelling is reduced to the greatest extent, and synovial inflammation and hyperplasia are inhibited, thereby effectively improving the effect of sinomenine hydrochloride in treating RA.

[0031] Furthermore, through the experiment on the rheumatoid arthritis AIA rat model of the present invention, the experimental results show that the microneedle of the present invention has better therapeutic effect and safety than commercial methotrexate.

[0032] Other features and advantages of the present invention will be described in the following description, and partly become apparent from the description, or understood by practicing the present invention. The purpose and other advantages of the present invention can be realized and obtained by the structures pointed out in the description, claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] 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 some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0034] Figure 1 The infrared spectrum of golden armor bean polysaccharide according to an embodiment of the present invention is shown;

[0035] Figure 2 The optical microscope image and the scanning electron microscope image of the microneedle obtained according to Example 1 of the present invention are shown;

[0036] Figure 3 A fluorescence microscope image of the microneedle obtained according to Example 1 of the present invention is shown;

[0037] Figure 4 The mechanical properties of the microneedle obtained according to Example 1 of the present invention are shown;

[0038] Figure 5 The HE staining result of the microneedle insertion performance study obtained according to Example 1 of the present invention is shown;

[0039] Figure 6 The graph shows the recovery performance results of the microneedles obtained in Example 1 of the present invention after insertion into the rat skin;

[0040] Figure 7 The in vivo dissolution performance results of the microneedles obtained according to Example 1 of the present invention are shown;

[0041] Figure 8 The graph shows the penetration depth of the drug in the body after the microneedles obtained in Example 1 of the present invention are inserted into the skin;

[0042] Fig. 9 The cumulative release graph of sinomenine hydrochloride released in vitro from the microneedles obtained according to Example 1 of the present invention is shown;

[0043] Fig.10 The adhesion and rheology investigation graph of the microneedles obtained according to Example 1 of the present invention is shown;

[0044] Fig.11 The anti-inflammatory effect diagram and the synergistic anti-inflammatory effect diagram of the golden armor bean polysaccharide according to the embodiment of the present invention are shown;

[0045] Fig.12 A diagram showing the antioxidant effect of golden armor bean polysaccharide according to an embodiment of the present invention is shown;

[0046] Fig.13 The ankle joints of rats in each group after 21 days of drug administration according to the embodiment of the present invention are shown;

[0047] Fig.14 The figures show the H&E stained sections of the ankle joint tissues, the H&E stained sections of the synovial tissues and the Safranin Fast Green stained sections of the rats in each group 21 days after administration according to the examples of the present invention. DETAILED DESCRIPTION

[0048] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are 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.

[0049] The PLP (purity ≥ 95%) used in the embodiments of the present invention was purchased from Shanyang Lianfeng Biotechnology Co., Ltd.; sinomenine hydrochloride (purity ≥ 98%) was purchased from Xi'an Shennong Biotechnology Co., Ltd., and polyvinyl pyrrolidone (PVP-90) was purchased from Chengdu Haoyuanli Biological Company; the microneedle PDMS mold was customized by Henan Weinanopentium Biotechnology Co., Ltd.

[0050] Among them, the infrared spectrum of PLP is shown in Figure 1 ,from Figure 1 It can be seen that there are many characteristic peaks of polysaccharides in the infrared spectrum of PLP powder, including 3407.75 cm -1 The strong and broad sugar ring hydroxyl peak at 2928.63 cm -1 Weak asymmetric stretching vibration of C-Hs in methyl and methylene groups at 1735.81 cm -1 The characteristic peak of aldehyde acid is at 1641.20 cm -1 , the peak of glycosidic bond is at 1062.82cm -1 .

[0051] 1. Microneedle Preparation

[0052] Example 1

[0053] Preparation of Sinomenine hydrochloride-loaded Polysaccharide Microneedles (hereinafter referred to as SIN@PLP MNs):

[0054] The microneedle patch was prepared by vacuum casting in two steps using a polydimethylsiloxane (PDMS) mold. First, 100 mg of polyvinyl pyrrolidone (PVP) and 100 mg of golden bean polysaccharide were weighed and dissolved in 1 mL of pure water solution. Then, 50 mg of sinomenine hydrochloride was added to make it dispersed evenly as the needle tip layer solution, which was cast into the PDMS mold and kept in a vacuum of -0.08 MPa for 30 min to fully fill the tip of the mold. The excess needle tip solution was scraped off and dried naturally. Next, 150 mg of golden bean polysaccharide was dissolved in 1 mL of pure water solution, and the backing solution was obtained after full swelling. The backing solution was poured on the surface of the dry PDMS mold and placed in a 37°C oven until completely dry. Finally, it was peeled off from the PDMS mold to obtain golden bean polysaccharide microneedles loaded with sinomenine hydrochloride.

[0055] 2. Characterization of the physicochemical properties of sinomenine hydrochloride-loaded sinomenine polysaccharide microneedles

[0056] 2.1 Characterization of microneedle morphology

[0057] The morphology and size of the prepared microneedles were examined using an optical microscope. Heavy metal atoms were sprayed onto the surface of the prepared microneedles under vacuum conditions, and then the morphology of the microneedle patch was observed using a scanning electron microscope (SEM). The structure of the microneedles and the distribution of the drugs in the microneedles were imaged using an inverted fluorescence microscope. In order to prepare a double-layer MN patch with fluorescent labeling, the fluorescent dye fluorescein isothiocyanate (FITC) was used as a fluorescent probe instead of SIN, and rhodamine was used instead of golden bean polysaccharide to prepare the MN patch.

[0058] The results are as follows Figure 2 and Figure 3 As shown, Figure 2 (a) is an optical microscope image of the microneedle (scale bar is 100 μm). Figure 2 Middle b is a scanning electron microscope scan (scale bar is 100 μm), Figure 3 The morphology is shown under a fluorescence microscope.

[0059] from Figure 2 It can be seen that the microneedle prepared in Example 1 of the present invention has a height of about 650 μm, a base width of 360 μm, and a spacing between each microneedle of 700 μm.

[0060] To further examine the bilayer structure of SIN@PLP MNs, fluorescence microscopy was used. Figure 3 The drug-loaded needle tip labeled with FITC emits green fluorescence. Figure 3 As can be seen in the figure, at the bottom, the rhodamine-labeled polysaccharide backing and attached to the needle body showed red fluorescence, confirming the existence of the bilayer structure.

[0061] 2.2 Mechanical properties test

[0062] The mechanical strength of SIN@PLP MNs was measured using a texture analyzer (model: TMS-PRO, brand: FTC, USA). Each SIN@PLP MNs patch to be tested was fixed horizontally on a metal aluminum plate with the needle tip facing upwards. A cylindrical probe with a diameter of 10 mm was used for extrusion, with a trigger force of 0.05 N and a test speed of 30 mm / min until the extrusion deformation reached 40%. The force-displacement curve of the microneedle was recorded. Figure 4 .

[0063] According to existing research, a force of 0.1N is enough to penetrate the skin. Figure 4 It can be seen that the prepared microneedles can withstand a compression force of 0.64 N / needle with a displacement of 450 μm, indicating that the microneedle patch needle tip has sufficient mechanical strength to penetrate the stratum corneum.

[0064] 2.3 In vivo penetration depth study

[0065] To verify the puncture performance of the SIN@PLP MNs patch on living rats, the SD rats were anesthetized and the back hair was removed. The microneedles were removed after pressing with the SIN@PLP MNs patch for five minutes. The rats were then immediately killed and the skin after microneedle puncture was peeled off. The skin was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. HE staining was performed and the puncture depth was observed under a microscope. In order to enhance the visualization of the microneedle structure, a fluorescently labeled SIN@PLP MNs patch was prepared to evaluate the in vivo penetration depth of the microneedle. The MNs were vertically inserted into the ex vivo pig skin and the distribution of fluorescence in the skin was observed under a laser confocal microscope to characterize the puncture depth. The experimental results are shown in Figure 2. Figure 5 and Figure 8 shown.

[0066] Figure 5 The scale bar is 100 μm. The penetration experiment confirmed that SIN@PLP MNs can successfully penetrate the skin and reach a sufficient depth both in vivo and in vitro. In the in vivo experiment, H&E-stained rat skin tissue sections confirmed that MNs effectively penetrated the skin, and the insertion depth of SIN@PLP MNs was about 200 μm. In order to observe the distribution of drugs inserted into the ex vivo skin by SIN@PLP MNs, the pig skin with MNS inserted was scanned layer by layer using a laser confocal microscope. Figure 8 The results showed that the microneedle penetration depth in the vertical direction reached 500 μm, which can deliver SIN to the deeper layers of the skin to achieve drug delivery. These results show that SIN@PLP MNs exhibited superior penetration ability in the study, which is essential for effective transdermal drug delivery.

[0067] 2.4 Skin recovery test

[0068] To evaluate whether the SIN@PLP MNs patch would cause irreversible damage to the skin after application to the rat skin, the back skin of SD rats was pretreated, and then the SIN@PLP MNs were inserted for 30 seconds and then removed. The healing of the skin at different time points was observed and images were recorded with a digital camera. Figure 6 shown.

[0069] like Figure 6 As shown in the figure, just after the microneedle pierced the skin, it was observed that the SIN@PLP MNs patch left obvious pinhole-like marks after piercing the skin, indicating that the prepared microneedle can successfully penetrate the skin. After removing the microneedle, the pinhole gradually became blurred over time, and only redness appeared. After 15 minutes, the skin returned to a slightly red mark, and the pinhole gradually blurred. After 2 hours, the skin completely returned to its original state, indicating that the skin can recover in a relatively short period of time, thereby minimizing the damage to the skin caused by the microneedle.

[0070] 2.5 In vivo solubility test

[0071] The prepared SIN@PLP MNs patch was pressed onto the ex vivo rat skin to fully insert the needle tip. The morphology of the microneedles at different dissolution time points was observed under an optical microscope and photographed. Figure 7 shown.

[0072] like Figure 7 As shown in the figure, the solubility of the microneedle after piercing the skin directly affects the release and diffusion of the drug in the skin. After piercing the skin, the needle tip becomes obviously blunt after 5 minutes; the needle tip layer is completely dissolved after 10 minutes; and the backing layer slowly dissolves after 15 minutes; and completely dissolves after 20 minutes. This shows that the needle tip layer of the microneedle can dissolve quickly after piercing the skin, which is conducive to the rapid release of the SIN carried. The backing layer composed of PLP dissolves slowly, thereby increasing local retention and improving the efficiency of transdermal drug delivery.

[0073] 2.6 Determination of drug loading of sinomenine hydrochloride-loaded sinomenine polysaccharide microneedles The SIN content was determined by high performance liquid chromatography (HPLC), with a mobile phase of methanol-0.2% triethylamine (45:55), gradient elution, a flow rate of 1.0 ml / min, an injection volume of 10 uL, a column temperature of 30 °C, and a detection wavelength of 266 nm.

[0074] The microneedles were loaded with sinomenine hydrochloride (SIN). A piece of microneedle was dissolved in 1 mL of distilled water and ultrasonicated for 30 minutes. The obtained sample was filtered and the content of sinomenine hydrochloride was determined according to the established HPLC method. Each group of experiments was repeated 3 times. The drug loading of one piece of MNs was calculated to be about 2.893 mg. The measurement results show that the multifunctional microneedle patch we prepared has a high drug loading capacity and improves the effectiveness of transdermal drug delivery.

[0075] 2.7 In vitro release experiment

[0076] The in vitro transdermal delivery of SIN@PLP MNs was evaluated using a Franz diffusion cell at 300 rpm, 4 ml PBS as the dissolution medium, and the temperature was maintained at 32°C. At different time points, samples were collected and supplemented with an equal volume of fresh PBS. All collected samples were filtered and the supernatant was analyzed and the cumulative release rate of the drug was calculated according to the established HPLC method. Fig. 9 As shown;

[0077] Fig. 9The in vitro cumulative release curve of SIN from SIN@PLP MNs is shown. Due to the rapid dissolution of the needle tip, SIN was released rapidly within 1 hour of administration, and the cumulative release amount reached 32.0%±2.2%. The dissolved PLP then formed a viscous gel at the insertion site, and SIN was slowly released and diffused from PLP, slowing down from 1 hour to 4 hours and completely released at 12 hours. SIN@PLP MNs can significantly promote transdermal drug delivery, and the addition of PLP plays the advantage of slow degradation of the polymer carrier and release of drugs.

[0078] 2.8 Viscosity and rheological investigation of microneedle matrix solution

[0079] The fresh microneedle matrix solution in gel state was tested using a MCR101 rheometer (Anton Paar GmbH, Graz, Austria) at 25°C. The shear rate was gradually increased from 0.01 to 100 s -1 , and analyze the changes of shear viscosity and shear stress with shear rate. The results are as follows Fig.10 shown.

[0080] Fig.10 (a) is a schematic diagram of SIN@PLP MNs adhering to ex vivo skin. Fig.10 b is the graph showing the change of viscosity of PLP with shear rate, and the horizontal axis of b is the shear rate (s -1 ), the vertical axis is viscosity (mPa·s -1 ), Fig.10 c is the graph of the shear stress of PLP changing with shear rate, and the abscissa is the shear rate (s -1 ), the ordinate is shear stress (Pa).

[0081] from Fig.10 It can be seen from the data that the microneedle matrix solution prepared by PLP has a certain viscosity. The flexible backing made of golden bean can adhere to the drug delivery skin site when used, improving the stability and efficiency of microneedle drug delivery. The shear stress of PLP in the gel state increases with the increase of shear rate, while the shear viscosity decreases, showing the shear thinning of pseudoplastic non-Newtonian fluid. It is conducive to the uniform distribution of drugs in the microneedle matrix and is conducive to the preparation of microneedles by vacuum casting.

[0082] 2.9 Antioxidant effect of polysaccharides from golden bean

[0083] 2.9.1 Evaluation of antioxidant capacity

[0084] The antioxidant effect of different concentrations of PLP was quantitatively analyzed by ABTS experiment. After different groups of microneedles were dissolved in deionized water to obtain different concentrations, the ABTS clearance rate of each administration group was calculated using the ABTS method. The test results are shown in the figure below. Fig.12 As shown in ( Fig.12 (a) is the antioxidant effect of golden bean polysaccharide determined by ABTS experiment).

[0085] Quantitative analysis by ABTS experiment showed that ( Fig.12 As shown in a), the antioxidant capacity is positively correlated with the concentration. When PLP is 6 mg / mL, the ABTS clearance rate reaches about 58%, which shows that PLP has good antioxidant capacity.

[0086] 2.9.2 NO inhibition ability evaluation

[0087] NO production was quantified by Griess assay. RAW264.7 cells growing in logarithmic numbers were plated at 8 × 10 -4 The cells were inoculated at a density of 1 μg / mL in a 12-well plate and incubated for 24 h to allow the cells to fully adhere to the wall. Then, different concentrations of PLP were added for 2 h, and LPS (1 μg / mL) was added for another 12 h. Subsequently, the cell supernatant was collected and the absorbance at 540 nm was measured using an ELISA reader to calculate the NO content. The results are shown in Table 1. Fig.12 As shown in b ( Fig.12 (b) is the antioxidant effect of golden bean polysaccharide determined by Griess method).

[0088] from Fig.12 As can be seen in middle b, PLP has a significant inhibitory ability on NO concentration and is concentration-dependent.

[0089] 2.9.3 Anti-inflammatory effect of golden bean polysaccharide

[0090] RAW264.7 macrophages stimulated with lipopolysaccharide (LPS) were used as an inflammatory cell model to evaluate the anti-inflammatory effect and mechanism of SIN@PLP MNs in vitro. Fig.11 shown.

[0091] from Fig.11 As shown in ( Fig.11 (a) shows the anti-inflammatory effect of golden bean polysaccharide. The CCK8 method was used to evaluate the effect of different concentrations of PLP on the viability of LPS-induced RAW264.7 cells. PLP had a significant inhibitory effect on cell viability in a concentration-dependent manner.

[0092] Further synergistic anti-inflammatory experimental results such as Fig.11 As shown in b ( Fig.11 b is the result of the coordinated anti-inflammatory effect between golden bean polysaccharide (PLP) and sinomenine hydrochloride (SIN). In this figure, the "+" sign indicates the addition of drugs or LPS induction in the corresponding row. Fig.11 As can be seen in b, the simultaneous addition of SIN and PLP inhibited the activity of inflammatory cells more significantly. Therefore, it was inferred that PLP had a synergistic anti-inflammatory effect.

[0093] 3. Experimental efficacy test of sinomenine hydrochloride-loaded sinomenine polysaccharide microneedles

[0094] 3.1 Experimental methods

[0095] AIA has the same immunological and pathological characteristics as human RA. To study the pharmacodynamics of drug-loaded microneedles in adjuvant arthritis (AA) rats, an adjuvant arthritis (AIA) model was established in male SD rats. On day 0, 0.1 mL of complete Freund's adjuvant (CFA) containing 10 mg / ml Mycobacterium tuberculosis was injected into the right hind toe pad of the rats. From day 15, the rats were randomly divided into 6 groups (n=6 in each group): control group, model group, sinomenine hydrochloride subcutaneous injection group (SIN), golden bean polysaccharide microneedle group (MN), sinomenine hydrochloride loaded golden bean microneedle group (DMN), and methotrexate gavage group (MTX dose 1 mg / kg). The percutaneous administration group was fixed with medical tape and administered once every 3 days for a total of 6 times.

[0096] After the AIA model was established, the volume of the rats' hind feet was measured using a toe volume meter. The toe volume was measured every three days and the arthritis score was calculated. The weight of each group was weighed and the weight changes were recorded. Histological analysis: All rats were killed after the administration. The ankle joints were removed by dissection and fixed with 4% paraformaldehyde. The ankle joint tissues were decalcified, paraffin-embedded and sectioned. HE staining and safranin fast green SO staining were performed to observe inflammatory cell infiltration and cartilage damage. The experimental results are as follows Fig.13 Fig.14 shown.

[0097] 3.2 Test results

[0098] After 14 days of induction by injection of complete Freund's adjuvant (CFA) into the hind paws of rats in the model group, the hind paws of rats in the model group showed obvious swelling and redness compared with healthy rats, indicating that the adjuvant arthritis rat (AIA) model was successfully established. The MTX gavage group was used as a positive control. It is currently the most commonly used anti-rheumatic drug in clinical practice to improve RA diseases. However, long-term use of MTX has great adverse reactions and potential toxic effects. Compared with oral MTX, transdermal administration of MNs directly delivers SIN to the pathological area, avoiding gastrointestinal side effects and potential toxicity distributed in other organs. After 15 days of transdermal treatment, the thickness of the hind paws of rats in the MTX group of golden bean polysaccharide microneedles was significantly reduced, and the thickness of the SIN@PLP MNs group loaded with sinomenine hydrochloride was the lowest, which was basically consistent with the thickness of the hind paws of the control group, and the arthritis score was also significantly lower than that of the model group. These results show that the SIN@PLP MNs group can effectively inhibit the development of rheumatoid arthritis.

[0099] H&E staining, safranin fast green staining and immunostaining were used for histological analysis of the ankle joint. The rats in the AIA model group showed obvious narrowing or even disappearance of the joint cavity, synovial tissue hyperplasia accompanied by a large number of inflammatory cell infiltration and cartilage destruction. The results showed that in the MTX gavage group, cartilage erosion and synovial inflammation were alleviated. The SIN@PLP MNs group more effectively inhibited synovial hyperplasia, reduced inflammatory cell infiltration and restored the cartilage surface. H&E staining of the ankle joint and synovial tissue in the model group showed inflammatory cell infiltration, pan-membrane formation, and severe synovial hyperplasia. Safranin fast green staining directly reflects the structure of articular cartilage and subchondral bone tissue, red indicates cartilage and green indicates osteogenesis. The results showed that SO staining of the ankle joint in the model group was characterized by cartilage erosion, and the SIN@PLP MNs administration group had a significant therapeutic effect on the inhibition of cartilage erosion.

[0100] In summary, it is shown that the embodiment of the present invention successfully prepared soluble double-layer microneedle SIN@PLP MNs with golden bean polysaccharide as the main microneedle matrix, and studied their combined treatment of RA in rats. Its safety and effectiveness were verified in the AIA rat model, and the results showed that SIN@PLP MNs had better therapeutic effect and safety than commercial MTX, accurately targeted the synovial lesions of arthritis, reduced joint swelling, inhibited synovial inflammation and hyperplasia, and effectively avoided cartilage erosion and cartilage destruction. Therefore, the embodiment of the present invention has developed a safe and effective drug-loaded Chinese medicine polysaccharide microneedle patch, which enables anti-arthritis drugs to locally and selectively treat RA, reflecting the advantages and potential of composite materials with different properties for drug delivery.

[0101] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent substitutions for some of the technical features therein; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A sinomenine hydrochloride-loaded sinomenine polysaccharide microneedle, characterized in that: The invention comprises a needle tip layer and a backing layer. The needle tip layer is made by drying a mixed aqueous solution containing sinomenine hydrochloride and sinomenine polysaccharide once, and the backing layer is made by drying the sinomenine polysaccharide aqueous solution twice.

2. The sinomenine hydrochloride-loaded sinomenine polysaccharide microneedle according to claim 1, characterized in that: The aqueous solution of Glechoma longituba polysaccharide is prepared by mixing Glechoma longituba polysaccharide and water.

3. The sinomenine hydrochloride-loaded sinomenine polysaccharide microneedle according to claim 1, characterized in that: In the aqueous solution of golden armor bean polysaccharide, the dosage ratio of golden armor bean polysaccharide to water is 100-150 mg:1 mL.

4. The sinomenine hydrochloride-loaded sinomenine polysaccharide microneedle according to claim 1, characterized in that: The weight average molecular weight of the golden bean polysaccharide is 300 kDa.

5. The sinomenine hydrochloride-loaded sinomenine polysaccharide microneedle according to claim 1, characterized in that: The mixed aqueous solution containing sinomenine hydrochloride and sinomenine polysaccharide is prepared by mixing sinomenine hydrochloride, sinomenine polysaccharide, a water-soluble polymer material and water.

6. The sinomenine hydrochloride-loaded sinomenine polysaccharide microneedle according to claim 5, characterized in that: The mass ratio of sinomenine hydrochloride, sinomenine polysaccharide, water-soluble polymer material and water in the mixed aqueous solution containing sinomenine hydrochloride and sinomenine polysaccharide is 30-60mg:100-150mg:100-150mg:1mL.

7. The sinomenine hydrochloride-loaded sinomenine polysaccharide microneedle according to claim 5, characterized in that: The water-soluble polymer material is polyvinyl pyrrolidone.

8. The sinomenine hydrochloride-loaded sinomenine polysaccharide microneedles according to any one of claims 1 to 7, characterized in that: The height of the needle tips of the needle tip layer is 600-700 μm, the width of the backing layer is 300-400 μm, and the distance between the needle tips in the needle tip layer is 650-750 μm.

9. The method for preparing sinomenine hydrochloride-loaded sinomenine polysaccharide microneedles according to any one of claims 1 to 8, characterized in that: include: Casting a mixed aqueous solution containing sinomenine hydrochloride and sinomenine polysaccharide into a mold, keeping it under vacuum for a predetermined time, scraping off excess solution on the mold, and drying it once to obtain a mold filled with a needle tip layer; The aqueous solution of scutellaria baicalensis polysaccharide is poured onto the surface of the dry mold filled with the needle tip layer, dried twice, and peeled off to obtain scutellaria baicalensis polysaccharide microneedles loaded with sinomenine hydrochloride.

10. Use of the sinomenine hydrochloride-loaded sinomenine polysaccharide microneedles according to any one of claims 1 to 8 in the preparation of drugs for treating rheumatoid arthritis.

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