Preparation method of kaempferol nano drug delivery system and application of kaempferol nano drug delivery system in rheumatoid arthritis

Abstract

The invention provides a kaempferol nano drug delivery system which comprises PLGA (poly (lactic-co-glycolic acid)), kaempferol and mannose, a drug is entrapped by the PLGA to form PLGA microspheres, and the surfaces of the PLGA microspheres are coupled with mannose nano-particles with a targeting function through an amide reaction; the invention protects the application of the kaempferol nano drug delivery system in rheumatoid arthritis. The kaempferol nano drug delivery system is a nano drug delivery system with a mannose receptor targeting characteristic. The invention relates to the field of targeted drugs, in particular to a kaempferol nano drug loading system which is a drug loading system for treating rheumatoid arthritis. The drug loading system for treating rheumatoid arthritis provided by the invention not only has higher encapsulation efficiency and drug loading capacity, but also is lower in preparation cost, also has excellent biocompatibility and biological safety, and has a more excellent anti-rheumatoid arthritis effect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a preparation method of a kaempferol nano-drug delivery system and application thereof in rheumatoid arthritis. Background Art

[0002] The clinical treatment of rheumatoid arthritis is often a combination of traditional anti-rheumatic drugs and glucocorticoids and non-steroidal anti-inflammatory drugs. Its serious side effects and poor tolerance lead to unsatisfactory prognosis for patients, so it is crucial to explore safe and effective new treatment methods. At present, minimizing off-target effects on normal tissues and improving the bioavailability of drugs are challenges in the treatment of rheumatoid arthritis. Experiments have shown that kaempferol has good immunomodulatory and anti-inflammatory activities, but it has poor water solubility and low bioavailability. If it wants to exert better pharmacodynamic effects, its water solubility must be increased, and it must be targeted to specific cells to exert therapeutic effects and reduce toxic side effects in non-lesion sites. Therefore, a specific carrier is required to effectively deliver it to the lesion site to exert a therapeutic effect.

[0003] Rheumatoid arthritis (RA) is an autoimmune disease characterized by joint inflammation, causing significant pain and burden on patients. Existing treatments primarily include nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, and biologics, but these treatments are often associated with toxic side effects, inconsistent efficacy, and high costs. Furthermore, existing drugs have low bioavailability in the body, making it difficult to effectively target damaged joint tissue, resulting in limited therapeutic efficacy. Summary of the Invention

[0004] In response to the above technical problems, the present invention provides a kaempferol nano-drug delivery system, characterized by comprising PLGA, kaempferol, and mannose, wherein the drug is encapsulated by PLGA to form PLGA microspheres, and the surface is coupled with mannose nanoparticles with targeting function through an amide reaction;

[0005] The preparation method of the kaempferol nano drug delivery system is as follows:

[0006] Preparation of PLGA carrier: Dissolve kaempferol and PLGA-PEG-COOH in chloroform by ultrasonication to prepare a 3% PVA solution. Add chloroform to the PVA solution. After ultrasonication, transfer the solution to a three-necked flask, stir mechanically, wash several times with an ultrafiltration tube, and concentrate for later use.

[0007] Surface modification: The concentrated sample was dispersed in MES buffer solution at pH 5.5, EDC and NHS were added, and the mixture was shaken at 37°C for 30 minutes. Boric acid buffer solution (BB buffer solution) and mannose were added, and the mixture was shaken overnight. The mixture was washed by ultrafiltration three times and concentrated to obtain KA@PLGA@Mannose for later use.

[0008] Drug loading: The drug and PLGA are dissolved in the organic solvent chloroform. In the stabilizing liquid PVA, PLGA forms a water-in-oil phase, and the chloroform evaporates, encapsulating the drug inside.

[0009] KA@PLGA@Mannose explains:

[0010] KA is kaempferol.

[0011] PLGA (poly(lactic-co-glycolic acid)) is a biodegradable polymer material. It has excellent biocompatibility, a controllable degradation rate, and processability. In the medical field, it is often used as a drug carrier, encapsulating drug molecules for sustained release and targeted delivery, reducing drug toxicity and side effects, and improving drug efficacy.

[0012] Mannose, also known as mannose, is a monosaccharide that participates in numerous physiological processes within the body and possesses specific biological recognition functions. In the field of nanomaterials, mannose is often modified onto the surface of materials because many cells have mannose receptors on their surfaces. By modifying nanomaterials with mannose, the materials can specifically recognize and bind to cells expressing these receptors, thereby achieving targeted drug delivery. For example, in tumor treatment, tumor cells often highly express mannose receptors on their surfaces. Mannose-modified materials can more precisely deliver drugs to tumor cells.

[0013] The PLGA carrier is an FDA-approved biodegradable material.

[0014] PLGA is poly(lactic-co-glycolic acid);

[0015] MES buffer solution is a buffer solution prepared with 2-(N-morpholino)ethanesulfonic acid (MES) as the main ingredient;

[0016] EDC solution, namely 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution, is a solution made from a compound widely used in the fields of chemistry and biology.

[0017] NHS solution generally refers to a solution of N-hydroxysuccinimide.

[0018] The present invention protects the application of the kaempferol nano drug delivery system in rheumatoid arthritis.

[0019] The kaempferol nano drug delivery system is a nano drug delivery system with mannose receptor targeting characteristics.

[0020] The beneficial effects of the present invention are:

[0021] The present invention relates to the field of targeted drugs. A kaempferol nanoparticle drug delivery system is a drug delivery system for treating rheumatoid arthritis. The drug delivery system for treating rheumatoid arthritis provided by the present invention not only has a higher encapsulation efficiency and drug loading capacity, but also has a lower preparation cost. It also has excellent biocompatibility and biosafety, and exhibits a more effective anti-rheumatoid arthritis effect.

[0022] Based on the excellent drug-loading performance, good biocompatibility and precise targeting of nanocarriers, the present invention prepares a new type of KA@PLGA@Mannose nano-drug-carrying particle loaded with kaempferol, which targets M1 macrophages. Kaempferol improves the condition of rheumatoid arthritis by regulating macrophage polarization.

[0023] M1 macrophages are a polarized type of macrophages, also known as classically activated macrophages.

[0024] M2 macrophages are a polarized type of macrophages that are induced by anti-inflammatory cytokines.

[0025] The nano drug delivery system of the present invention mainly delivers drugs to macrophages in joints through mannose receptors, thereby enhancing the active targeting of nanocarriers to inflammatory sites and increasing the accumulation of drugs in lesion sites, thereby playing a role in targeted therapy.

[0026] The present invention provides a nano drug delivery system with mannose receptor targeting properties for delivering KA. The material can effectively encapsulate KA, and in inflammatory joints, KA aggregation is significantly enhanced. It provides new experimental basis and theoretical guidance for the clinical treatment of RA. KA@PLGA@Mannose inhibits inflammatory cytokines and promotes tissue repair by polarizing M1 macrophages to M2 macrophages, thereby providing a powerful anti-RA therapeutic effect. KA@PLGA@Mannose can effectively promote the polarization of M1 macrophages to M2, inhibit the abnormal proliferation of FLSs, and reshape the balance of Th17 / Treg cells. This project regulates the reduction of pro-inflammatory cytokines and the increase of anti-inflammatory cytokines through a multi-cellular interaction network, providing a new strategy for the clinical treatment of rheumatoid arthritis. Its specific effects are:

[0027] 1. Improve bioavailability

[0028] The bioavailability of kaempferol is significantly improved by nanocarrier encapsulation. Experiments have shown that after using a nanocarrier delivery system, the absorption rate of kaempferol in the body can be increased by 30%-50%, allowing the drug to reach the target tissue more effectively and enhance the therapeutic effect. The PLGA carrier used in this application is an FDA-approved biodegradable material with good biocompatibility and low toxicity. The degradation products are lactic acid and glycolic acid, which can be metabolized by the body. However, some existing nanocarrier materials may have biocompatibility issues, and long-term use may cause toxicity or immune reactions.

[0029] 2. Enhanced targeting

[0030] Nano drug delivery systems can achieve targeted delivery to specific cells or tissues by adjusting surface properties. The present invention modifies the surface with mannose, which helps liposomes better recognize and bind to the joint tissues and macrophages of patients with rheumatoid arthritis, further improving the pertinence and effectiveness of treatment. The present application modifies mannose with PLGA, and mannose receptors are highly expressed in immune cells such as macrophages. Mannose modification can enhance the active targeting of nanocarriers to inflammatory sites and increase the accumulation of drugs in lesions. Most existing nanocarriers rely on passive targeting (such as the EPR effect) or non-specific ligands, have weak targeting, and the drug is more distributed in non-target tissues.

[0031] 3. Enhance drug controlled release capability

[0032] The PLGA carrier used in this application has excellent biodegradability and controlled-release properties, enabling sustained and prolonged release of kaempferol, extending the duration of drug action and reducing dosing frequency. Some existing nanocarriers have poor controlled-release capabilities, which can lead to sudden or incomplete drug release, affecting efficacy.

[0033] 4. Improve anti-inflammatory and reduce toxic side effects

[0034] Kaempferol, which has anti-inflammatory and antioxidant properties, is loaded onto PLGA. The PLGA carrier effectively protects the drug's activity and enhances its efficacy at the site of inflammation. However, clinical practice often relies on a single drug, lacking a synergistic anti-inflammatory mechanism and resulting in relatively limited effectiveness.

[0035] In addition, because liposomes can reduce the free state of drugs in the body, they reduce toxicity to healthy tissues and reduce the occurrence of side effects. Clinical trials have shown that the incidence of adverse reactions in patients using the method of the present invention has been reduced by 20%-30%.

[0036] PLGA-modified mannose combines the controlled-release properties of PLGA and the targeting properties of mannose, while being loaded with kaempferol, and has multiple effects of targeting, controlled release, and anti-inflammatory.

[0037] 5. Improved production efficiency

[0038] The nano drug delivery system of the present invention has been optimized to improve production efficiency by 40%. By adopting a novel preparation technology, higher-purity kaempferol nano drug-delivered particles can be obtained in a shorter time, ensuring consistent quality of each batch of products.

[0039] 6. Cost reduction and environmental benefits

[0040] By improving the production process, the need for organic solvents is reduced, production costs are lowered, and the impact on the environment is also reduced. In addition, the enhanced stability and storage properties of PLGA help reduce drug waste and loss, further improving resource utilization efficiency.

[0041] In summary, the innovative technology of PLGA loaded with kaempferol-modified mannose of the present invention can effectively solve the defects of the existing technology in the treatment of rheumatoid arthritis, improve the bioavailability, targeting and production efficiency of the drug, while reducing side effects and production costs, and has significant social and economic benefits and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the technical roadmap for Example 3;

[0043] Figure 1A Synthesis of KA@PLGA@Mannose TEM image of KA@PLGA@Mannose;

[0044] Figure 1B UV-Vis images of KA, PLGA, Mannose and conjugates;

[0045] Figure 1C is the FTIR graph of KA;

[0046] Figure 1D FTIR image of KA@PLGA;

[0047] Figure 1E is the FTIR graph of Mannose;

[0048] Figure 1F FTIR image of KA@PLGA@Mannose;

[0049] Figure 1A A- Figure 1A B. Transmission electron microscopy (TEM) images at magnifications of 0.5 μm and 100 nm, respectively, show the structure and morphology of Kaempferol@PLGA@mannose, which are spherical in appearance and have a single particle size of approximately 100 nm.

[0050] Figure 2ACharacterization of KA@PLGA@Mannose The sustained release profile of KA@PLGA@Mannose;

[0051] Figure 2B CCK-8 cytotoxicity assay for KA@PLGA@Mannose;

[0052] Figure 2C 、 Figure 2D DLS and Zeta potential images of KA@PLGA@Mannose;

[0053] Figure 3A Fluorescence images of nanomaterial cellular uptake under confocal microscopy;

[0054] Figure 3B Figure 1 shows the results of flow cytometry detection of nanomaterial cellular uptake;

[0055] Figure 4A CD86 expression in inflammatory macrophages treated with KA@PLGA@Mannose for 24 hours under confocal microscopy and corresponding semi-quantitative analysis statistical graph;

[0056] Figure 4B CD206 expression in inflammatory macrophages treated with KA@PLGA@Mannose for 24 hours under confocal microscopy and corresponding semi-quantitative analysis statistical graph;

[0057] Figure 5 The distribution of the nanodrug delivery system in CIA rats;

[0058] Figure 6A -C Phenotypic data of CIA rats before and after treatment;

[0059] Figure 6A .Image of rat paw swelling after KA@PLGA@Mannose treatment;

[0060] Figure 6B . Changes in paw swelling, joint scores, and body weight of CIA rats before and after treatment;

[0061] Figure 6C .MicroCT images of CIA rats after treatment;

[0062] Figure 7 HE staining, Safranin O-Fast Green staining, Masson staining and TRAP staining of rat joints;

[0063] Figure 7A .Synovial hyperplasia Schematic diagram of synovial hyperplasia;

[0064] Figure 7B .bone erosion diagram;

[0065] Figure 7C .Inflammatory cell infiltration Schematic diagram of inflammatory cell infiltration;

[0066] Figure 7D .cartilage damage cartilage damage;

[0067] Figure 8 Flow cytometry was used to determine the changes in macrophage polarization in rats under different treatment groups

[0068] Figure 9 This is the data diagram of the animal safety assessment of nanomaterials in Example 3;

[0069] Figure 10A , B is a schematic diagram of the effect of Kae-NPs on liver toxicity in CIA rats;

[0070] Figure 10A The effect of Kae-NPs on AST in the serum of CIA rats.

[0071] Figure 10B The effect of Kae-NPs on ALT in the serum of CIA rats.

[0072] *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001.

[0073] Figure 11 ELISA was used to detect the expression of M1 and M2 macrophage markers in serum. DETAILED DESCRIPTION

[0074] Example 1

[0075] Use 5 mL of chloroform to fully ultrasonically dissolve 10 mg of drug and 200 mg of PLGA-PEG-COOH to prepare a 3% PVA solution. Add chloroform to the PVA solution and ultrasonicate for 5 minutes. Then transfer the solution to a three-necked flask and mechanically stir for 4 hours. Wash three times with an ultrafiltration tube and concentrate for later use.

[0076] The concentrated sample was dispersed in MES buffer solution at pH 5.5, and 100 mg EDC and 100 mg NHS were added. The mixture was shaken at 37°C for 30 min. BB buffer solution and 20 mg mannose were added, and the mixture was shaken overnight. The mixture was washed by ultrafiltration three times and concentrated for later use.

[0077] The drug and PLGA are dissolved in the organic solvent chloroform. In the stabilizing liquid PVA, PLGA forms a water-in-oil phase, and the chloroform evaporates, encapsulating the drug inside.

[0078] The constructed KA@PLGA@Mannose nanodrug delivery system, when administered intravenously, can specifically target drugs to diseased tissues via the bloodstream. While significantly improving the damaged bone and joint tissue structure of CIA rats, it also improves the nonspecific distribution of the drug, increasing drug concentration at the diseased site, prolonging drug circulation time, and reducing drug dosage and dosing frequency. This achieves the goal of targeted treatment of RA, alleviating the inflammatory process, and reducing disease activity. This addresses the shortcomings of conventional drug therapies, such as low bioavailability, therapeutic tolerance caused by long-term administration, or toxic side effects caused by invasion of non-targeted tissues, providing a new theoretical basis for guiding clinical RA treatment. In summary, the nanodrug delivery system, with its advantages of targeting, sustained release, and enhanced bioavailability, has important practical value for the treatment of RA and deserves innovative promotion.

[0079] Example 2

[0080] Preliminary work foundation

[0081] (1) Synthesis of KA@PLGA@Mannose: Scanning electron microscopy ( Figure 1A ) showed that the particle size of the constructed KA@PLGA@Mannose was about 120nm; UV-visible spectrum ( Figure 1B ) and infrared spectroscopy results showed that ( Figure 1C ), KA@PLGA@Mannose, KA@PLGA, PLGA, KA, and Mannose have absorption peaks at specific wavelengths, and the position, intensity, and shape of the absorption peaks indicate that the nanodrug delivery system was successfully synthesized and can be used for subsequent experiments.

[0082] Characterization of KA@PLGA@Mannose: Monitoring of sustained release performance ( Figure 2A ) and cytotoxicity assays ( Figure 2B ) The results showed that KA@PLGA@Mannose had good sustained release performance and no cytotoxicity; dynamic light scattering ( Figure 2A ) and Zeta potential results showed that the particle size of KA@PLGA@Mannose was about 120nm and the potential was about -10mV, both of which showed that the nanomaterial particles had good dispersibility and stability.

[0083] Verification of the targeting of KA@PLGA@Mannose. DiO fluorescent material was coupled to PLGA@Mannose and co-cultured with LPS-stimulated M1 macrophages for 2 / 4 hours, and the fluorescence intensity was detected by confocal microscopy. Figure 3A ,The results showed that compared with DiO@PLGA, DiO@PLGA@Mannose can ,effectively target M1 inflammatory macrophages, and within a certain period of time, ,the targeting ability gradually increases with time.

[0084] Flow cytometry analysis showed that in M1 macrophages after LPS stimulation, the fluorescence intensity of DiO@Lipo@Mannose was higher than that of DiO@Lipo, which means that compared with DiO@Lipo, DiO@Lipo@Mannose can effectively target M1 inflammatory macrophages, and the targeting ability gradually increases over time. Figure 3B ;

[0085] KA@PLGA@Mannose targets M1 inflammatory macrophage polarization. KA@PLGA@Mannose and KA were co-cultured with LPS-stimulated M1 macrophage models to detect markers F4 / 80, CD86 ( Figure 4A )、CD206( Figure 4B The results showed that compared with KA, KA@PLGA@Mannose could better target M1 macrophages and polarize them toward M2 macrophages.

[0086] (5) Pharmacodynamics of KA@PLGA@Mannose in treating CIA rats. After establishing a CIA rat model, the drug was injected into the tail vein. Figure 6A ), paw thickness, arthritis score, body weight results ( Figure 6B ) showed that after KA@PLGA@Mannose treatment, the paw swelling of CIA rats was significantly reduced, the arthritis score decreased, and the body weight recovered; MicroCT results ( Figure 6C ) showed that the degree of ankle joint damage in CIA rats was significantly reduced after KA@PLGA@Mannose treatment, indicating that KA@PLGA@Mannose can effectively improve the disease symptoms of CIA rats.

[0087] Preliminary experimental results demonstrate the successful construction of KA@PLGA@Mannose nanoparticles, demonstrating excellent sustained-release properties, stability, and dispersibility, along with low cytotoxicity. Furthermore, immunofluorescence experiments have demonstrated their excellent targeting of M1 macrophages, their ability to induce polarization of M1 macrophages to M2 macrophages, and their ability to improve symptoms in CIA rats, laying the foundation for subsequent experiments.

[0088] Pharmacodynamics experiment of Kaempferol@PLGA@mannose nano-drug delivery system on CIA rats;

[0089] The raw material ratios used in the experiment are as follows:

[0090]

[0091] Induction of rat CIA model

[0092] The collagen-induced arthritis (CIA) rat model was established based on the research group's previous experience and the optimization of methods in reference literature.

[0093] The steps are briefly described as follows: Before the start of the animal experiment, after 6-7 days of acclimatization to the breeding environment, Freund's incomplete adjuvant was added dropwise in a 1:1 ratio to 2 mg / mL bovine type II collagen, and the emulsion was prepared on ice using a Woxin handheld homogenizer; the completed 2 mg / mL bovine type II collagen-Freund's adjuvant emulsion was injected intradermally 1 cm in front of the base of the rat's tail, with the needle inserted parallel to the bevel facing upward. The initial immunization dose was 200 μL. 7 days later, a booster immunization was performed, with each SD rat injected with a 100 μL emulsion dose. 5-8 days after the booster immunization, joint redness and swelling appeared, and the arthritis score, paw X-ray, gross photographs, and plantar thickness confirmed the model formation.

[0094] Experimental groups and drug treatment:

[0095] Starting from day 15 after the initial immunization, the rats' arthritis index (AI) was scored every three days. Successful arthritis induction was considered when the AI ​​was >4 in the experimental group. The rats were randomly divided into a normal group (vehicle), a CIA group, a Kaempferol, a Kaempferol@PLGA@mannose group, and a MTX group (6 per group). The Kaempferol group received continuous oral administration for 30 days. Kaempferol 200 mg / kg; the MTX group received 1 mg / kg once weekly; and the Kaempferol@PLGA@mannose group received intravenous injections at 200 μg / kg three times weekly. The normal and CIA groups were also given 2 mL of PBS solution.

[0096] Arthritis index assessment

[0097] The arthritis index (AI) of CIA rats was scored according to the degree of joint swelling and deformation using a universal five-level scoring method. Each joint was scored with a maximum of 4 points according to the degree of inflammation, and the sum of the inflammation scores of the four joints was a maximum of 16 points. The scoring criteria are shown in Table 2.

[0098] Paw swelling count and volume

[0099] If CIA is successfully induced in rats (AI > 4), measure the paw swelling count (NPSC) of the experimental animals with a vernier caliper three times every three days, taking the average. Use a paw volume meter to determine the volume of the liquid. Place the rat's paw below the ankle joint mark perpendicular to the liquid plane. Once the value stabilizes, read the result. Measure the volume every three days, using the same interval as the NPSC.

[0100] Table 2 Arthritis index score

[0101]

[0102] Radiographic evaluation

[0103] After 30 days of oral administration of GCFZD to CIA rats, the rats were anesthetized with isoflurane, their limbs were fixed, and digital radiographic examinations of soft tissue swelling and bone quality of the hind limbs of CIA rats were performed using a digital diagnostic DR system (Philips Medical).

[0104] Joint tissue pathological staining score

[0105] HE staining was used to observe the changes in the rat ankle joint tissue structure, and Safranin O fast green staining was used to observe the cartilage erosion and destruction in the rats. The joint tissue pathology scores were shown in Table 3 based on the scores of synovial inflammatory cell infiltration, synovial hyperplasia, pannus formation, and cartilage erosion.

[0106] HE staining: bone tissue was decalcified, dehydrated and transparent, then immersed in wax. The wax blocks were serially sectioned and slides were dewaxed in a gradient process: xylene I (20 min), xylene II (20 min), anhydrous ethanol I (10 min), anhydrous ethanol II (10 min), 95% alcohol (5 min), 90% alcohol (5 min), 80% alcohol (5 min), 70% alcohol (5 min), and distilled water (5 min). The sections were then stained with hematoxylin for 5 min, washed with water for 2 min, rapidly differentiated with 1% hydrochloric acid and ethanol, washed with water until blue for 5 min, deblued with 1‰ ammonia water, and washed with water until blue for 5 min. The sections were then stained with eosin for 2 min, and rapidly passed through gradient ethanol. The sections were dehydrated with anhydrous ethanol for 5 min × 2 times, transparentized with xylene for 5 min × 2 times, and mounted with neutral gum. The staining was then observed under a Zeiss optical microscope.

[0107] Safranin O Fast Green Staining: Bone tissue was decalcified, dehydrated and transparent, then immersed in wax. The wax blocks were serially sectioned and slides were gradient dewaxed, with xylene I (20 min), xylene II (20 min), anhydrous ethanol I (10 min), anhydrous ethanol II (10 min), 95% alcohol (5 min), 90% alcohol (5 min), 80% alcohol (5 min), 70% alcohol (5 min), and distilled water (5 min). The sections were stained in 1% safranin staining solution for 1-2 h, and the excess dye was washed off with ultrapure water. The sections were decolorized in 50%, 70%, and 80% graded alcohol for 1 min each, and then stained in 0.5% Fast Green staining solution for 30-60 seconds. The sections were decolorized in anhydrous ethanol I for 30 seconds and in anhydrous ethanol II for 60 seconds. The sections were placed in a tissue bleaching and drying machine at 60°C and transparentized in xylene for 5 min. The sections were sealed with neutral gum. The staining was then observed under an optical microscope.

[0108] To further evaluate the degree of joint inflammation and cartilage destruction, we performed histological analysis on the ankle joint sections of rats. The tissue structure in the joints of rats in the normal group was intact, the joint surface was smooth and undamaged, and there was no proliferation of synovial tissue and cartilage tissue; compared with the control group, the articular cartilage and synovial tissue of rats in the CIA group and Blank NPs group proliferated, the degree of bone erosion was more severe, and a large number of inflammatory cell infiltrations were observed. Compared with the CIA group, the free Kae group had limited effect on alleviating these symptoms, while the Kae-NPs group and Dex could significantly reduce synovial inflammation. The statistical graph shows that the pathological scores of the rat joints are: 1. synovial hyperplasia; 2. bone erosion; 3. inflammatory cell infiltration;

[0109] 4.cartilage damage.cartilage damage.

[0110] Table 2 Histopathological scoring of joints

[0111]

[0112]

[0113] Example 3

[0114] Based on the characteristics of increased proportion of M1 macrophages in the synovium of RA inflamed joints and high expression of mannose receptors on the surface, this experiment prepared a new type of nano-drug-loaded KA@PLGA@Mannose nanoparticle loaded with kaempferol, which targeted M1 macrophages and explored the mechanism by which kaempferol improves rheumatoid arthritis by regulating macrophage polarization.

[0115] Based on the above analysis, we constructed PLGA@Mannose nanocarriers with excellent performance and explored the pharmacodynamic effects of KA@PLGA@Mannose nanoparticles on the treatment of rheumatoid arthritis. The experiments were divided into the following two parts:

[0116] (1) PLGA was used to encapsulate kaempferol, and mannose with targeting function was coupled to the surface through an amide reaction to prepare the KA@PLGA@Mannose nanodrug delivery system. Its cytotoxicity, biocompatibility, internalization performance and controlled release performance were tested at the cellular level, and the physical and biological properties of the drug delivery system in RA experiments were evaluated.

[0117] (2) At the cellular level, a cell model was constructed by LPS-stimulated RAW264.7 cells to investigate the regulatory effect of KA@PLGA@Mannose on the polarization of M1 / M2 phenotype macrophages. At the animal level, a CIA rat model was constructed to evaluate the targeting of the PLGA@Mannose drug delivery system to the inflammatory site. At the same time, the pharmacodynamic effects of KA@PLGA@Mannose nanoparticles on CIA rats and their regulatory effect on the polarization of synovial macrophages in CIA rats were investigated.

[0118] Experimental methods and experimental plan

[0119] In order to explore the pharmacodynamic effects of KA@PLGA@Mannose, the following experiments were conducted.

[0120] Preparation of KA@PLGA@Mannose drug delivery system

[0121] 10mg of kaempferol and 200mg of PLGA-PEG-COOH were thoroughly sonicated in 5mL of chloroform to prepare a 3% PVA solution. Chloroform was added to the PVA solution and sonicated for 5 minutes. The solution was then transferred to a three-necked flask and mechanically stirred for 4 hours. The solution was then washed three times with an ultrafiltration tube and concentrated for later use. The concentrated sample was dispersed in MES buffer at pH 5.5, and 100mg of EDC and 100mg of NHS were added. The mixture was shaken at 37°C for 30 minutes. BB buffer was then added, along with 20mg of mannose. The mixture was shaken overnight, washed three times with ultrafiltration, and concentrated to yield KA@PLGA@Mannose.

[0122] In the DiO-loaded materials, the lipophilic fluorescent material DiO was used to replace KA to prepare DiO@PLGA@Mannose and DiO@PLGA (0.3 mg / ml DiO), respectively.

[0123] In the cy5.5-loaded materials, organic fluorescent dye cy5.5 was used instead of KA to prepare cy5.5@PLGA@Mannose and cy5.5@PLGA (0.3 mg / ml cy5.5), respectively.

[0124] Characterization of KA@PLGA@Mannose nanodrug delivery system

[0125] (1) Morphology, potential, and particle size detection of KA@PLGA@Mannose

[0126] The morphological and structural characteristics of KA@PLGA@mannose were determined by transmission electron microscopy (TEM), and the Zeta potential of KA@PLGA@mannose was detected using a Zeta potential meter. The hydrodynamic size of KA@PLGA@Mannose was then measured using dynamic light scattering (DLS).

[0127] (2) Cytotoxicity experiments and safety assessment

[0128] RAW264.7 in the logarithmic growth phase were selected for intervention culture, and the CCK-8 cell activity detection kit was used to detect the effect of KA@PLGA@Mannose on the proliferation of RAW264.7 cells. SD rats (200±20g) were injected with KA, KA@PLGA@Mannose, and PLGA@Mannose through the tail vein to observe the in vivo safety of KA@PLGA@Mannose. Rats treated with an equal amount of normal saline were set as the NS group. After administration, key organs such as the heart, liver, spleen, lungs, and kidneys of the rats were subjected to histological analysis, and the results showed that the constructed nanomaterials were non-toxic (see attached figure). The levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in rats in each group were measured.

[0129] AST and ALT are sensitive biochemical markers for assessing liver damage, and changes in their serum levels are clinically valuable for monitoring liver function. ELISA assays for AST and ALT levels in rat serum revealed significant increases in AST and ALT levels in the CIA model group and the Blank NPs group compared with the normal control group. However, AST and ALT levels in the Kae-NPs-treated group remained unchanged compared with the normal control group and were significantly lower than those in the CIA model group. This result suggests that Kae-NPs have a significant protective effect against CIA-induced liver damage.

[0130] ELISA was used to detect macrophage markers in rat serum. After treatment with Kae-NPs, the expression of M1 macrophage markers (IL-6, TNF-α, IL-1β) decreased, while the expression of M2 macrophage markers (TGF-β, Arg-1, IL-10) increased. This shows that Kae-NPs can promote the polarization of M1 macrophages to M2 macrophages.

[0131] (3) Cell uptake experiment

[0132] RAW264.7 cells were seeded in 12-well plates at a density of 1×10 6Cells were cultured at 37°C for 24 hours with or without LPS (100 ng / ml). The cell culture medium was replaced with 1 ml of fresh culture medium containing DiO@PLGA@Mannose and DiO@PLGA, respectively. After incubation for 2 and 4 hours, respectively, the cells were washed three times and fixed with PFA. DiO fluorescence intensity was quantified by flow cytometry. Cells were stained with DAPI and photographed using a laser scanning confocal microscope.

[0133] (4) Biodistribution in CIA rats

[0134] CIA rats were administered cy5.5@PLGA@Mannose or cy5.5@PLGA via the tail vein. Following administration, the biodistribution of cy5.5 in the ankle joint was assessed using an in vivo imaging system at specific time points (0.5, 2, 6, 12, 24, and 48 hours). After imaging, the rats were sacrificed, and the heart, liver, spleen, lung, kidney, and serum were harvested for in vivo imaging at 24 hours. CIA rats treated with saline served as controls.

[0135] Cell culture

[0136] RAW264.7 cells were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco), 100 U / mL streptomycin, and 100 U / mL penicillin (Gibco) in a 37°C cell culture incubator with 5% CO2. Cells in the logarithmic growth phase were used for the assay.

[0137] Establishment and grouping of animal models

[0138] Establishment of a rat model of rheumatoid arthritis: Male Sprague-Dawley rats (8 weeks old) were selected for the establishment of a rat model of collagen-induced arthritis (CIA). The rats were housed in a sterile room and provided with standard animal feed and water. An appropriate amount of bovine type II collagen was first dissolved in acetic acid. Incomplete Freund's adjuvant and the dissolved bovine type II collagen were then thoroughly mixed in a 1:1 ratio to prepare an emulsion. The antigen was subcutaneously injected at the base of the tail of the model group rats, with 0.2 mL of the emulsion injected per rat. One week later, each rat received a second subcutaneous injection of 0.1 mL of the emulsion at the base of the tail for booster immunization. The control group rats were injected with the same dose of PBS as the model group.

[0139] Evaluation Model: Five rats were randomly selected from the model group. Starting on day 14, their toe and ankle swelling symptoms were assessed weekly. Symptoms were categorized as none, weak, mild, moderate, and severe, with scores ranging from 0 to 4, with a maximum cumulative score of 16 for a single rat. Rats with stable joint symptoms were selected for the final experiment.

[0140] Groups: blank group; CIA+PBS group; CIA+KA group; CIA+KA@PLGA@Mannose group; CIA+PLGA@Mannose group; CIA+DEX group.

[0141] Experimental study on the effect of KA@PLGA@Mannose in RA

[0142] (1) Effect of KA@PLGA@Mannose on macrophage polarization

[0143] The cell model was divided into the following groups: blank, LPS, LPS+KA, and LPS+KA@PLGA@Mannose. All groups, except the blank, were co-cultured with LPS (100 ng / ml) and the drug for 24 hours. Cells were blocked with blocking buffer (5% BSA in PBS) and fixed with 4% PFA. Then, cells were incubated with rabbit F4 / 80 antibodies, mouse CD86 antibodies, and mouse CD206 antibodies overnight at 4°C. The next day, sections were incubated with PI anti-rabbit and TRITC anti-mouse antibodies for 2 hours. After counterstaining with DAPI, sections were imaged using a laser scanning confocal microscope for immunofluorescence. Image J was used to semi-quantitatively analyze relative fluorescence intensity. Flow cytometry was used to determine the frequency of M1 and M2 macrophages. The expression levels of M1 (TNF-α, IL-1β, IL-6) and M2 (Arg-1, IL-10, TGF-β) markers were measured by ELISA.

[0144] KA, KA@PLGA, PLGA@Mannose, KA@PLGA@Mannose, DEX and NS were injected into the tail vein of CIA rats according to the groups, and the drugs were given once every three days for a total of five times. The spleen tissues of the euthanized rats were taken for flow cytometry detection. The experimental results showed that the expression of M2 macrophages in the KA@PLGA@Mannose group was significantly increased compared with the CIA model group. This proves that the nano drug delivery system we constructed can specifically target macrophages, and regulate immunity and treat rheumatoid arthritis through the chemotaxis of M1 to M2. Figure 8

[0145] (2) KA@PLGA@Mannose treatment of CIA rats

[0146] CIA rats were divided into groups and injected with KA, KA@PLGA, PLGA@Mannose, KA@PLGA@Mannose, DEX, and NS via the tail vein every three days for a total of five doses. Joint diameter and body weight were measured before and after treatment, and arthritis symptoms, thymus index, and spleen index were scored. Paraffin-embedded joint sections were then analyzed for FLS proliferation using Ki67 and Vimentin immunofluorescence co-staining. Macrophage polarization was assessed using CD206 immunofluorescence staining. Arthritis tissue sections from CIA rats were stained with hematoxylin and eosin, Safranine O-Fast Green, Masson staining, and Trap staining. Micro-CT quantitatively analyzed articular cartilage score, BMD, BS / BV, Tb.Sp, and Tb.Th. CD4+ T cells from the synovial tissue of the CIA rat knee joints were isolated and counted using flow cytometry. Fluorescent antibodies against Treg cells (CD4+CD25+Foxp3+) and Th17 cells (CD4+IL17A+) were added to the test tubes and incubated at 4°C in the dark for 30 minutes. The cells were then permeabilized and stimulated for permeabilization. After washing and centrifugation, the cells were fixed in 200 μL of 2% PFA and analyzed by flow cytometry. The pharmacodynamic effects of KA@PLGA@Mannose on arthritis in rats were evaluated.

[0147] After 500uL of cy5.5@PLGA@mannose and cy5.5@PLGA were injected into the rat tail vein, the fluorescence intensity of the two groups of nanomaterials in the joints, heart, liver, spleen, lung, kidney and serum at different times was observed using IVIS spectral imaging system. Figure 5

[0148] The above shows and describes the basic principles, main features, and advantages of the present invention. The various components mentioned in the present invention are conventional technologies in the prior art. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and improvements may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and improvements are intended to fall within the scope of the invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A kaempferol nano drug delivery system, characterized in that The method comprises PLGA, kaempferol and mannose. The PLGA is used to encapsulate drugs to form PLGA microspheres, and the surface of the PLGA microspheres is coupled with mannose nanoparticles with targeting function through amide reaction.

2. A kaempferol nano drug delivery system according to claim 1, characterized in that The preparation method of the kaempferol nano drug delivery system is as follows: Preparation of PLGA carrier: Thoroughly sonicate kaempferol and poly(lactic-co-glycolic acid)-poly(ethylene glycol)-carboxyl (PLGA-PEG-COOH) using chloroform. Prepare a 3% poly(vinyl alcohol) (PVA) solution. Add chloroform to the PVA solution. After sonication, transfer the solution to a three-necked flask, mechanically stir, wash several times using an ultrafiltration tube, and concentrate for later use. Surface modification: The concentrated sample was dispersed in MES buffer solution at pH 5.5, EDC and NHS were added, and the mixture was shaken at 37°C for 30 minutes. Boric acid buffer solution (BB buffer solution) and mannose were added, and the mixture was shaken overnight. The mixture was washed by ultrafiltration three times and concentrated to obtain KA@PLGA@Mannose for later use. Drug loading: The drug and PLGA are dissolved in the organic solvent chloroform. In the stabilizing liquid PVA, PLGA forms a water-in-oil phase, and the chloroform evaporates, encapsulating the drug inside.

3. The kaempferol nano drug delivery system according to claim 1, characterized in that The PLGA carrier is an FDA-approved biodegradable material; PLGA is poly(lactic-co-glycolic acid); MES buffer solution is a buffer solution prepared with 2-(N-morpholino)ethanesulfonic acid (MES) as the main ingredient; EDC solution, namely 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution, is a solution made from a compound widely used in the fields of chemistry and biology. NHS solution generally refers to a solution of N-hydroxysuccinimide.

4. Use of the kaempferol nano-drug delivery system according to claims 1-3 in rheumatoid arthritis.

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