Self-assembled nanoparticles as well as preparation method and application thereof

Abstract

The invention relates to a self-assembled nanoparticle and a preparation method and application thereof.The self-assembled nanoparticle is obtained through self-assembly of cationic amino acid and flavonoid micromolecules, the molar ratio of the cationic amino acid to the flavonoid micromolecules is 30: 1-1: 1, the self-assembled nanoparticle is high in drug loading capacity, and the self-assembled nanoparticle can be used for preparing a drug carrier. The hydrogel can specifically target an inflammation part, eliminate oxidative stress and inflammation and inhibit osteoclast differentiation, has good biocompatibility and in-vivo safety, and is simple in preparation process, low in cost, easy to produce in batches, good in treatment effect and suitable for popularization and application. The compound has wide application prospects in preparation of drugs for preventing and / or treating inflammatory arthritis, preparation of arthritis targeted diagnosis and treatment agents, preparation of drug carriers and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of nanobiomaterials, in particular to a self-assembled nanoparticle and a preparation method and application thereof. BACKGROUND

[0002] Rheumatoid arthritis is an inflammatory disease characterized by chronic and systemic autoimmune response, and the pathological core is synovial inflammation and progressive tissue destruction caused by abnormal activation of the immune system. When the external pathogen or damage-associated molecular patterns are recognized by innate immune cells such as macrophages, T cells can be activated and B cells can produce autoantibodies, thereby triggering the release of a large amount of inflammatory factors, causing synovial hyperplasia, cartilage erosion and bone damage; patients often show joint swelling, limited mobility and joint deformity.

[0003] A large number of studies have shown that the pathological microenvironment of rheumatoid arthritis is characterized by oxidative stress and a large number of pro-inflammatory M1 macrophages and osteoclasts. Although some non-steroidal anti-inflammatory drugs can eliminate inflammation and relieve rheumatoid arthritis to some extent, they cannot eliminate the oxidative stress caused by immunogens and suppress joint destruction. In addition, there are also disease-modifying antirheumatic drugs (DMARDs), including methotrexate, leflunomide, hydroxychloroquine, etc. These drugs can control disease progression, reduce inflammatory response and delay joint structure damage by inhibiting immune cell proliferation and blocking inflammatory signal transduction. However, rheumatoid arthritis is characterized by multiple joint onset, and traditional drugs such as DMARDs and NSAIDs can relieve systemic inflammation to some extent, but lack tissue specificity, making it difficult to achieve effective enrichment at the inflammation site, leading to systemic exposure and common side effects such as liver and kidney dysfunction, gastrointestinal reactions and bone marrow suppression. Therefore, it is urgent to develop new targeted treatment methods that can specifically target the inflammation site of arthritis and have the functions of immune regulation, oxidative stress elimination and tissue repair.

[0004] Cationic amino acids (such as arginine and lysine) have shown unique application prospects in the treatment of inflammatory diseases, which is mainly due to their unique charge characteristics, metabolic participation ability and immune regulation function. This kind of molecules can target M1 macrophages at the inflammation site through the specific up-regulation of cationic amino acid transporters CAT-2 on the surface of pro-inflammatory macrophages, and effectively inhibit the differentiation process of osteoclasts by inhibiting the activation of key molecules (such as NF-κB and NFATc1) of the RANKL signaling pathway. In addition, cationic amino acids can also play an anti-inflammatory effect through multiple mechanisms such as regulating nitric oxide (NO) synthesis, affecting immune cell function and maintaining cellular redox homeostasis.

[0005] Nevertheless, the application of cationic amino acids still faces a series of key challenges. First, the pharmacokinetic behavior of cationic amino acids in their small molecule form is unsatisfactory, exhibiting problems such as short half-life, insufficient tissue retention, and difficulty in maintaining local concentrations, thus limiting the sustainability of therapeutic effects. Second, the safety and potential side effects of long-term use still require comprehensive evaluation, especially in humans, where high systemic doses of cationic amino acids may affect amino acid metabolic balance, gut microbiota composition, and T cell polarization, posing a risk of inducing immune imbalance or metabolic abnormalities.

[0006] Flavonoids (such as myricetin, luteolin, and kaempferol) are a class of polyphenolic secondary metabolites widely found in plants and diets. They exhibit significant antioxidant and anti-inflammatory potential in medical applications, particularly in the treatment of inflammatory diseases. Flavonoids exert their antioxidant effects through multiple mechanisms, including scavenging free radicals, inhibiting oxidative stress signaling pathways (such as NF-κB and MAPK), and regulating antioxidant enzyme activity. These functions endow them with powerful anti-inflammatory and cytoprotective capabilities.

[0007] The clinical application of small flavonoid molecules is still in its early stages. First, as a class of natural polyphenol small molecules, flavonoids have problems such as poor water solubility, low bioavailability, and easy rapid metabolism and elimination in the body, which often leads to insufficient effective concentration or non-specific distribution, thus limiting their therapeutic effects.

[0008] Currently, numerous studies have focused on using nanocarriers (such as mesoporous silica, polyvinylpyrrolidone encapsulation, and PLGA) or hydrogel systems (such as sodium alginate and chitosan) to deliver small flavonoids or bioactive molecules like arginine, aiming to enhance their stability, bioavailability, and targeted accumulation capabilities. However, these carrier systems still face challenges such as limited delivery efficiency, insufficient carrier biocompatibility, and the potential for degradation products to exacerbate the lesion microenvironment. For example, PLGA releases acidic products during degradation, leading to a decrease in local microenvironment pH, which may actually worsen the inflammatory response; other polymeric carriers also suffer from drawbacks such as low loading rates and lack of targeting.

[0009] There are currently no effective solutions to the problems of low delivery efficiency of carrier systems, low utilization of amino acids and flavonoids, insufficient biocompatibility of carriers, and the possibility that degradation products may exacerbate the deterioration of the lesion microenvironment. Summary of the Invention

[0010] The purpose of this invention is to address at least one deficiency in the prior art by providing a self-assembled nanoparticle, its preparation method, and its application. This approach bypasses traditional carriers and utilizes the drug molecules themselves for direct self-assembly, achieving higher drug utilization and lower systemic toxicity. This addresses problems in related technologies such as low carrier system delivery efficiency, low utilization of amino acids and flavonoids, insufficient carrier biocompatibility, and the potential for degradation products to exacerbate the deterioration of the lesion microenvironment.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of the present invention is to provide a self-assembled nanoparticle, wherein the nanoparticle is formed by the self-assembly of cationic amino acids and flavonoid small molecules, wherein the molar ratio of the cationic amino acids to the flavonoid small molecules in the nanoparticle is 30:1 to 1:1; preferably 20:1 to 10:1, more preferably 13 to 14:1.

[0012] Furthermore, the cationic amino acid includes at least one of arginine, histidine, and lysine; preferably, the cationic amino acid is arginine.

[0013] Further, the flavonoid small molecule includes at least one of flavonols and flavones; wherein the flavonols include at least one of myricetin, quercetin, and kaempferol, and the flavones include at least one of luteolin and apigenin; preferably, the flavonoid small molecule is myricetin.

[0014] Furthermore, the nanoparticles are spherical in shape with an average particle size of 180~240 nm; preferably, the average particle size is 200 nm.

[0015] A second aspect of the present invention is to provide a method for preparing nanoparticles as described in the first aspect, comprising the following steps: Flavonoid small molecule solution and cationic amino acid solution were prepared separately using flavonoid small molecules and cationic amino acids, respectively. Formaldehyde was then added to the cationic amino acid solution to obtain a mixed solution system. Flavonoid small molecule solution was then added to the mixed solution system, and the mixture was stirred, washed, and dried to obtain the self-assembled nanoparticles.

[0016] Furthermore, the molar ratio of the cationic amino acid to the flavonoid small molecule is 30:1 to 1:1; preferably 13 to 14:1.

[0017] Furthermore, the volume ratio of the flavonoid small molecule solution to the cationic amino acid solution is 1~3:2~6, the concentration of the flavonoid small molecule solution is 1~8 mg / mL, and the concentration of the cationic amino acid solution is 1~15 mg / mL.

[0018] Furthermore, the volume ratio of the flavonoid small molecule solution to the cationic amino acid solution is 1:4, the concentration of the flavonoid small molecule solution is 3-6 mg / mL (preferably 4 mg / mL), and the concentration of the cationic amino acid solution is 5-10 mg / mL (preferably 7.5 mg / mL).

[0019] Furthermore, the volume ratio of the cationic amino acid solution to formaldehyde is 100~140:1, preferably 120:1.

[0020] Furthermore, the preparation of the flavonoid small molecule solution is a mixed solution obtained by dissolving flavonoid small molecules in anhydrous ethanol.

[0021] Furthermore, the preparation of the cationic amino acid solution is a mixed solution obtained by dissolving cationic amino acids in deionized water.

[0022] Further, the stirring is performed by ultrasonic stirring at room temperature for 1-2 minutes, followed by continuous stirring at 600-1000 rpm at room temperature for 10-14 hours, wherein the ultrasonic parameters are 30-50 kHz; preferably, the ultrasonic parameters are 40 kHz for 1 minute; and then continuous stirring at 800 rpm at room temperature for 12 hours.

[0023] Furthermore, the cleaning process involves washing with pure water 2 to 3 times.

[0024] Further, after washing, the product is collected by centrifugation at 10,000 to 14,000 rpm for 12 to 18 minutes; preferably, it is collected by centrifugation at 12,000 rpm for 15 minutes.

[0025] Furthermore, the drying is vacuum freeze drying, which is carried out at a temperature of -90 to -70°C for 6 to 12 hours.

[0026] A third aspect of the present invention is to provide a self-assembled nano-targeting drug, wherein the active ingredient of the drug comprises self-assembled nanoparticles as described in the first aspect or self-assembled nanoparticles prepared by the preparation method described in the second aspect.

[0027] Furthermore, the drug is a self-assembled nano-targeted drug injection solution; the injection solution further includes a biocompatible solution; wherein the biocompatible solution includes at least one of physiological saline and phosphate buffer.

[0028] Furthermore, the physiological saline contains NaCl at a concentration of 0.9 wt%.

[0029] Further, the phosphate buffer is a 0.01 mmol / L phosphate buffer with pH = 7.4.

[0030] Furthermore, in the injection solution, the concentration of the self-assembled nanoparticles is 1 to 30 mg / mL, preferably 5 to 15 mg / mL, and more preferably 10 mg / mL.

[0031] By controlling the concentration of cationic amino acid-flavonoid small molecule self-assembled nano-targeted drugs in the injection solution within an appropriate range, the therapeutic effect and biocompatibility of the drug can be effectively balanced. If the concentration is too high, it may increase toxicity and make injection difficult; while if the concentration is too low, it may lead to insufficient efficacy or even treatment failure. Therefore, controlling the appropriate concentration range is crucial to ensuring both drug efficacy and safety.

[0032] Furthermore, using mice as subjects, the dosage of the self-assembled nano-targeted drug is 10-20 mg / kg; preferably 20 mg / kg.

[0033] Furthermore, the dosage of the self-assembled nano-targeted drug suitable for intravenous injection in humans is 0.5~2.5 mg / kg; preferably 1.6 mg / kg.

[0034] A fourth aspect of the present invention is to provide an application of a nano-targeted drug as described in the first aspect, or a preparation method as described in the second aspect, or a self-assembled nano-targeted drug as described in the third aspect, wherein the application is selected from one of the following applications: application in the preparation of a drug for treating inflammatory arthritis, application in the preparation of an arthritis-targeted diagnostic agent, or application in the preparation of a drug carrier.

[0035] The present invention adopts the above technical solution and has the following technical effects compared with the prior art: This invention utilizes the combination of cationic amino acids and flavonoid small molecules via the Mannich reaction, and the self-assembly of nanoparticles based on non-covalent interactions such as hydrophilic-hydrophobic interactions and π-π stacking, to achieve carrier-free self-assembly and nano-scale formation. This significantly improves drug loading and delivery efficiency, solving the problems of low utilization of amino acids and flavonoid small molecules and low delivery efficiency of carrier systems. Furthermore, the prepared nanoparticles exhibit excellent dispersion stability in physiological solutions and can be diluted with physiological saline or phosphate buffer for direct clinical use without the need for solubilizers. Simultaneously, these nanoparticles can specifically target joint inflammation sites. After intravenous injection, the nanoparticle drug can accumulate in pro-inflammatory M1 macrophages at the inflamed site via a cationic amino acid transporter 2 (CAT2)-mediated targeting mechanism, inducing their transformation to the anti-inflammatory M2 phenotype and effectively inhibiting osteoclast differentiation and function. In addition, the nanoparticles prepared in this application have a simple preparation process, low cost, are easy to mass-produce, and exhibit good therapeutic effects, showing broad application prospects in the preparation of drugs for the prevention and / or treatment of inflammatory arthritis, the preparation of targeted diagnostic agents for arthritis, and the preparation of drug carriers. Attached Figure Description

[0036] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are for illustrative purposes only, and do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a TEM image of arginine-myricetin self-assembled nanoparticles (MANPs) with a particle size of 200 nm in one embodiment of the present invention. Figure 2 This is a high-angle annular dark-field image of arginine-myricetin self-assembled nanoparticles (MANPs) and their corresponding elemental mapping diagram in one embodiment of the present invention. Figure 3 This is a Fourier transform infrared (FT-IR) spectrum of arginine-myricetin self-assembled nanoparticles (MANPs) in one embodiment of the present invention. Figure 4 This is the Raman spectrum of arginine-myricetin self-assembled nanoparticles (MANPs) in one embodiment of the present invention. Figure 5 This is a statistical graph showing the free radical scavenging efficiency of arginine-myricetin self-assembled nanoparticles (MANPs) at different concentrations in one embodiment of the present invention. Figure 6 This is a confocal laser scanning microscope (CLSM) image showing the results of co-incubating BMDM cells with MANPs-FITC for different times after different treatments in one embodiment of the present invention; Figure 7This is a CLSM result image of BMDM cells after different treatments (iNOS, CAT2) in one embodiment of the present invention; Figure 8 This is a confocal laser scanning microscope (CLSM) image showing the results of co-incubating BMDM cells with MANPs-FITC for different times after different treatments in one embodiment of the present invention; Figure 9 This is a confocal microscopy result of an experiment on reactive oxygen species scavenging LPS-induced BMDMs by arginine-myricetin self-assembled nanoparticles (MANPs) in one embodiment of the present invention. Figure 10 This is a flow cytometry image of the repolarization assay of LPS / IFN-γ induced BMDMs macrophages by arginine-myricetin self-assembled nanoparticles (MANPs) in one embodiment of the present invention; wherein, Q1 partition (CD86- CD206+) represents M2 macrophages; Q3 partition (CD86+ CD206-) represents M1 macrophages; Figure 11 This is an optical microscope image showing TRAP staining results of RAW264.7 cells after different treatments in one embodiment of the present invention. Figure 12 This is an in vivo fluorescence result at different time points after tail vein injection of arginine-myricetin self-assembled nanoparticles MANPs-Cy7 labeled with Cy7 into mice with rheumatoid arthritis in one embodiment of the present invention. Figure 13 This is a graph showing the changes in joint swelling and body weight over time in mice with rheumatoid arthritis treated with arginine-myricetin self-assembled nanoparticles (MANPs) in one embodiment of the present invention. Figure 14 This is an RT-qPCR result of arginine-myricetin self-assembled nanoparticles (MANPs) used to treat rheumatoid arthritis mice in one embodiment of the present invention. Figure 15 This is a Micro-CT image of the ankle joint of a mouse with rheumatoid arthritis treated with arginine-myricetin self-assembled nanoparticles (MANPs) for 21 days, according to one embodiment of the present invention. Figure 16 This is a graph showing the results of mineral density analysis of ankle bone in mice treated with arginine-myricetin self-assembled nanoparticles (MANPs) for 21 days, according to one embodiment of the present invention. Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental materials in the following embodiments that do not specify their source are all commercially available raw materials. The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to general international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise stated, all parts are parts by weight, and all percentages are percentages by mass. Unless otherwise defined or stated, all professional and scientific terms used in the present invention have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the methods of the present invention.

[0038] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the scope of the invention.

[0039] The flavonoid small molecules involved in this invention include at least one of flavonols and flavones; wherein, the flavonols include at least one of myricetin, quercetin, and kaempferol, and the flavones include at least one of luteolin and apigenin; their specific structural formulas are shown below: In one specific embodiment, the method for preparing self-assembled nanoparticles includes the following steps: (1) Take cationic amino acids and flavonoid small molecules with a molar ratio of 13~14:1. Dissolve the cationic amino acids in deionized water to obtain a cationic amino acid solution, and dissolve the flavonoid small molecules in ethanol to obtain a flavonoid small molecule solution.

[0040] (2) Take cationic amino acid solution and flavonoid small molecule solution at a volume ratio of 4:1 respectively. Add formaldehyde to cationic amino acid solution at a volume ratio of cationic amino acid solution: formaldehyde = 120:1. Then add flavonoid small molecule solution to cationic amino acid solution system through Pasteur pipette. Mix ultrasonically at room temperature for 40 kHz for 1 min to obtain mixed solution.

[0041] (3) The mixture was stirred continuously at 800 rpm for 12 h at room temperature. After washing with pure water 2 to 3 times, the product was collected by centrifugation (12000 rpm, 15 min) and then freeze-dried under vacuum to obtain self-assembled nanoparticles.

[0042] Other manufacturers not specifically labeled in the examples can be obtained through commercial purchases.

[0043] The above-mentioned cationic amino acid-flavonoid small molecule self-assembled nanoparticle targeted drugs have a simple, mild, pollution-free synthesis process, high yield, low cost, and are easy to mass-produce, showing excellent clinical application prospects in the biomedical field. Moreover, by optimizing material design (such as surface modification, size control, or composite structure construction), the biocompatibility and targeting of the drugs can be further improved, thereby promoting their application in biomedicine and other fields.

[0044] The present invention will be described by way of example below.

[0045] Example 1 - Preparation of self-assembled nanoparticles and their injection solutions This invention relates to a specific method for preparing self-assembled nanoparticles and their injection solution, specifically including the following steps: (1) Dissolve 0.9 g of arginine in 120 mL of deionized water to obtain an arginine solution (7.5 mg / mL); dissolve 0.12 g of myricetin in 30 mL of anhydrous ethanol to obtain a myricetin solution (4 mg / mL).

[0046] (2) Add 1 mL of formaldehyde to the arginine solution, and then gradually add the myricetin solution to the arginine solution through a Pasteur pipette. Stir the mixture at 40 kHz for 1 min at room temperature to obtain a mixture.

[0047] (3) Place a magnetic stir bar in the above mixture and stir overnight at 800 rpm and room temperature. Then wash with pure water 2-3 times, centrifuge (12000 rpm, 15 min) to collect the product, freeze dry under vacuum (dry at -90 to -70℃ for 6-12 h) to obtain arginine-myricetin self-assembled nanoparticles, hereinafter referred to as MANPs.

[0048] (4) Preparation of MANPs injection solutions: When performing in vivo injection, weigh a certain mass of MANPs powder according to the actual concentration and volume used, add the corresponding volume of biocompatible solution, such as physiological saline or phosphate buffer, to the sample vial according to the calculated ratio, and resuspend by sonication at 40 kHz for 5 min in a water bath to obtain MANPs injection solutions of different concentrations, with a concentration range of 5 ~ 50 mg / mL. In the above injection solutions, the preferred concentration of MANPs is 5 ~ 15 mg / mL, preferably 10 mg / mL.

[0049] Example 2 - Structural Analysis of Arginine-Myricetin Self-Assembled Nanoparticles This embodiment analyzes the arginine-myricetin self-assembled nanoparticles (MANPs) prepared in Example 1.

[0050] (1) Arginine-myricetin self-assembled nanoparticles (MANPs) were analyzed using transmission electron microscopy. TEM images and high-angle annular dark-field images and their corresponding elemental mapping maps were obtained. The results are as follows: Figure 1 , Figure 2 As shown.

[0051] from Figure 1 As can be seen from this, MANPs have a spherical structure with an average particle size of about 200 nm. The particle size is small and uniform, resulting in good dispersibility. Figure 2 As can be seen from this, MANPs are composed of C, N, and O elements, and the elements are evenly distributed.

[0052] (2) Fourier transform infrared (FTIR) and Raman spectroscopy analyses were performed on arginine-myricetin self-assembled nanoparticles (MANPs), and the results are as follows: Figure 3 or Figure 4 As shown.

[0053] from Figure 3 From this, we can know that MANPs are at 1675 cm. -1 A characteristic absorption peak similar to that in the arginine spectrum appears at [location missing], which can be attributed to the antisymmetric stretching vibration of the guanidine group, indicating that MANPs retain the guanidine residue of arginine; from [location missing] Figure 4 From this, we can see that the Raman spectra of MANPs are in the range of 1000–1750 cm⁻¹. -1 The interval features broad saddle peaks, 1367 and 1574 cm. -1 The peak values ​​are at 1330 cm⁻¹, corresponding to the concentration of myricetin at 1330 cm⁻¹. -1 OH bonds at 1617 cm -1 The C=C bond vibration at the location indicates that MANPs retain the aromatic ring structure of myricetin.

[0054] Example 3 - Free Radical Scavenging Experiment This embodiment is based on the arginine-myricetin self-assembled nanoparticles (MANPs) prepared in Example 1.

[0055] DPPH (1,1-Diphenyl-2-trinitrophenylhydrazine) is a stable free radical that appears deep purple in organic solvents and exhibits strong absorption at 517 nm. In the presence of antioxidants, these antioxidants can react with DPPH free radicals, reducing them and causing the solution to lighten in color and decrease in absorbance. By measuring the change in absorbance, the scavenging ability of a sample against DPPH free radicals can be calculated.

[0056] The prepared MANPs were dispersed in phosphate buffer (0.01 mmol / L, pH = 7.4) to prepare MANPs solutions of varying concentrations (5, 10, 25, 50, 75, 100, 125, 150 μg / mL). These solutions were then mixed with an equal volume of 0.1 mmol / L DPPH solution, and an equal volume of PBS was used as a blank control. After thorough mixing, the mixture was incubated in the dark for 30 min, and the absorbance was measured at 517 nm. The results are shown below. Figure 5 As shown.

[0057] from Figure 5 It is known that arginine-myricetin self-assembled nanoparticles (MANPs) exhibit concentration-dependent free radical scavenging, with 150 μg / mL MANPs able to scavenge 91.34% of DPPH free radicals. Example 4 - M1 BMDM Macrophage Targeting Experiment This embodiment is based on the arginine-myricetin self-assembled nanoparticles (MANPs) prepared in Example 1.

[0058] 1. To investigate whether MANPs can be specifically taken up by M1-polarized macrophages. Mouse bone marrow-derived macrophages (BMDMs) were prepared from wild-type C57BL / 6 mice (Suzhou Cyagen Biosciences Co., Ltd.). The specific steps were as follows: Bone marrow was isolated from the femur and tibia of 4-6 week old male mice; erythrocytes in the bone marrow were lysed using erythrocyte lysis buffer; to remove adhesive stromal cells, the collected bone marrow was cultured overnight in α-MEM containing 10% fetal bovine serum and 1% penicillin-streptomycin. Non-adhesive monocytes were collected and cultured for 7 days in complete medium containing M-CSF (20 ng / mL) to obtain BMDMs. After induced maturation, BMDMs were co-incubated with LPS / IFN-γ (100 ng / mL, 20 ng / mL) and IL-4 (20 ng / mL) for 12 hours to induce differentiation of BMDM macrophages into the M1 pro-inflammatory phenotype and the M2 anti-inflammatory phenotype, respectively. These were designated as the LPS / IFN-γ group (M1 positive control) and the IL-4 group (M2 positive control). The PBS group served as the M0 negative control. MANPs prepared using FITC fluorescent labeling were designated as MANPs-FITC. After co-incubation with MANPs-FITC (10 μg / mL, prepared using complete cell culture medium) for different times (10 min, 30 min, 1 h), BMDMs were imaged using laser confocal microscopy (CLSM). The results are shown below. Figure 6 As shown.

[0059] from Figure 6 The results showed that LPS / IFNγ-induced M1 macrophages exhibited significant green fluorescence signals after 10 min of incubation, with a significant increase in fluorescence signal at 30 min, and reached a peak fluorescence intensity after 1 h, which was significantly higher than that of the untreated group and the IL-4-induced M2 group at the same time point, indicating that MANPs can be specifically taken up by M1 polarized macrophages.

[0060] 2. Investigating the macrophage targeting mechanism of M1 type BMDMs Immunofluorescence staining (iNOS, CAT2) was performed on the established M1 positive control group, M2 positive control group, and M0 negative control group, followed by imaging using laser confocal microscopy (CLSM). The results are as follows: Figure 7 As shown.

[0061] from Figure 7 From this, we can know that: iNOS + The M1 type BMDMs showed significant red fluorescence signal of CAT-2 on their surface, while no obvious red fluorescence was observed in the M0 and M2 types.

[0062] To confirm whether CAT-2 is a key gene for MANPs targeting M1 macrophages, CAT-2 expression in M1 BMDM macrophages was inhibited by transfecting siRNA. MANPs-FITC (10 μg / mL, prepared in complete cell culture medium) were co-incubated with CAT-2-transfected and untransfected BMDMs for different times (10 min, 30 min, 1 h), followed by imaging using laser confocal microscopy (CLSM). The results are as follows: Figure 8 As shown.

[0063] from Figure 8 The results show that when CAT-2 expression is reduced, the specific uptake of MANPs by M1 macrophages is significantly reduced, indicating that cationic amino acid transporter 2 is a key protein for MANPs to target M1 macrophages.

[0064] Example 5 - Experiment on the scavenging of reactive oxygen species by BMDM macrophages This embodiment is based on the arginine-myricetin self-assembled nanoparticles (MANPs) prepared in Example 1.

[0065] Primary BMDM macrophages were obtained from the tibia and femur bone marrow of C57BL / 6 mice and matured for 7 days after induction with M-SCF 20 ng / mL. BMDM macrophages were first co-incubated with 100 ng / mL LPS and 20 ng / mL IFN-γ for 8 hours. Cells treated with LPS / IFN-γ were then further treated with L-arginine (20 μg / mL), myricetin (20 μg / mL), and MANPs (20 μg / mL, prepared using complete cell culture medium), respectively. PBS treatment served as a negative control. Intracellular ROS levels were detected using the DCFH-DA reactive oxygen species probe after 12 hours. The results are as follows: Figure 9 As shown.

[0066] from Figure 9 As can be seen, MANPs can effectively remove LPS / IFN-γ-induced intracellular reactive oxygen species at a concentration of 20 μg / mL, which has certain guiding significance for the actual dosage of the injection solution used for in vivo injection in animals.

[0067] Example 6 - Repolarization Experiment of BMDM Macrophages This embodiment is based on the arginine-myricetin self-assembled nanoparticles (MANPs) prepared in Example 1.

[0068] Primary BMDM macrophages were obtained from the tibia and femur bone marrow of C57BL / 6 mice. After maturation for 7 days with M-SCF 20 ng / mL, the BMDM macrophages were first co-incubated with LPS / IFN-γ (100 ng / mL, 20 ng / mL) and IL-4 (20 ng / mL) for 12 hours to induce differentiation into M1 pro-inflammatory phenotype and M2 anti-inflammatory phenotype, respectively, designated as LPS / IFN-γ group (M1 positive control group) and IL-4 group (M2 positive control group). LPS / IFN-γ-treated cells were then treated with IL-4 (20 ng / mL) and MANPs (20 μg / mL, prepared using complete cell culture medium) for 12 h, designated as treatment groups: LPS / IFN-γ + IL-4 group and LPS / IFN-γ + MANPs group. Flow cytometry was then used to analyze the expression of CD86 and CD206 in each group. The results are as follows: Figure 10 As shown.

[0069] from Figure 10 It can be seen that, compared with the LPS / IFN-γ group, the Q1 partition (CD86) of the treatment group LPS / IFN-γ + IL-4 group and LPS / IFN-γ + MANPs group is higher. - CD206 + The number of cells increased significantly, and the Q3 partition (CD86) showed a significant increase. + CD206 - The significant decrease in MAP cells indicates that MANPs can effectively induce pro-inflammatory M1 macrophages to differentiate into anti-inflammatory M2 macrophages.

[0070] Example 7 - Osteoclastization Induction Experiment of RAW264.7 Macrophages This embodiment is based on the arginine-myricetin self-assembled nanoparticles (MANPs) prepared in Example 1.

[0071] RAW264.7 cells were purchased from Cybio Biotechnology Co., Ltd. (Catalog No.: iCell-m047). RAW264.7 cells were induced to undergo osteoclast differentiation using DMEM complete medium containing RANKL (50 ng / mL) and M-CSF (20 ng / mL), and were simultaneously co-incubated with PBS, L-arginine (20 μg / mL), myricetin (20 μg / mL), and MANPs (20 μg / mL), with the corresponding fresh medium changed daily. DMEM complete medium was used as a negative control. On day 5, the cells were detected using a TRAP staining kit. Results are shown below. Figure 11 As shown, OC is DMEM complete medium (osteoclast induction medium) containing RANKL (50 ng / mL) and M-CSF (20 ng / mL).

[0072] from Figure 11 The results showed that the osteoclast-inducing medium significantly induced RAW264.7 cells to differentiate into osteoclasts. Compared with the PBS treatment group, the percentage of TRAP-positive area in each treatment group was significantly reduced. In addition, the MANPs treatment group showed the smallest percentage of TRAP-positive area, indicating that MANPs have a good potential to inhibit macrophage osteoclast differentiation.

[0073] Example 8 - Targeted Experiment in Rheumatoid Arthritis Mice This embodiment is based on the arginine-myricetin self-assembled nanoparticles (MANPs) prepared in Example 1.

[0074] 1. Preparation of injection solution: MANPs prepared using Cy7 fluorescent labeling were dispersed in phosphate buffer (0.01 mmol / L, pH = 7.4) to obtain MANPs-Cy7 injection solution with a concentration of 10 mg / mL.

[0075] 2. Construction of Rheumatoid Arthritis Mice: Female DBA / 1JGpt mice (6 weeks old) were obtained from Suzhou Cyagen Biotech Co., Ltd. Healthy DBA / 1JGpt mice (6 weeks old) were randomly divided into four groups: a control group (normal healthy group), a positive control group (adjuvant-induced rheumatoid arthritis RA+PBS), a low-dose MANPs treatment group (for adjuvant-induced rheumatoid arthritis mice), and a high-dose MANPs treatment group (for adjuvant-induced rheumatoid arthritis mice). All healthy 6-week-old mice were subcutaneously injected with a 1:1 mixture of complete Freund's adjuvant and bovine type II collagen (2 mg / mL) emulsion to induce primary immunization. Twenty-one days later, a secondary immunization was induced by subcutaneous injection of a 1:1 mixture of incomplete Freund's adjuvant and bovine type II collagen emulsion, thus constructing the rheumatoid arthritis mouse model.

[0076] 3. Detection: On the 7th day after the second immunization, three rheumatoid arthritis mice were injected with the above-mentioned injection solution at a dose of 10 mg / kg via the tail vein. The distribution and metabolism of MANPs-Cy7 in the mice were observed by in vivo fluorescence at 30 min, 2 h, 4 h and 8 h.

[0077] The above-mentioned injection solution was administered to rheumatoid arthritis mice via tail vein injection at a dose of 10 mg / kg. The distribution and metabolism of MANPs in the mice were observed using in vivo fluorescence at time points of 30 min, 2 h, 4 h, and 8 h. The results are as follows: Figure 12 As shown.

[0078] from Figure 12 The results show that MANPs are concentrated in the limbs and ankles (i.e., inflamed areas) of mice with rheumatoid arthritis, indicating that MANPs have the ability to target inflamed joints in mice with arthritis and can maintain a high drug concentration near the joint for a long time.

[0079] Example 9 - Treatment Experiment for Rheumatoid Arthritis in Mice This embodiment is based on the arginine-myricetin self-assembled nanoparticles (MANPs) prepared in Example 1.

[0080] 1. Preparation of injection solution: The prepared MANPs were dispersed in phosphate buffer (0.01 mmol / L, pH = 7.4) to obtain a MANPs injection solution with a concentration of 10 mg / mL.

[0081] 2. Experimental Treatment: Rheumatoid arthritis mice constructed in Example 8 were subjected to different treatments in different groups. The overall animal experiment lasted for 4 weeks (calculated from the second immunization). During this period, the body weight and ankle joint diameter of all mice were recorded every two days as one of the evaluation criteria for rheumatoid arthritis. The results are as follows: Figure 13 As shown; after the experimental evaluation period, synovial tissue was collected from all mice for RT-qPCR detection, and the results are as follows. Figure 14 As shown; the specific treatment is as follows: the above-mentioned injection solution was administered to the corresponding experimental groups of rheumatoid arthritis mice via tail vein injection at doses of 10 mg / kg and 20 mg / kg, respectively, as the MANPs 10 mg / kg treatment group and the MANPs 20 mg / kg treatment group; the control group (Normal) was not injected; the positive control group (RA+PBS) was injected with PBS, and the experimental observations were recorded.

[0082] from Figure 13 , 14 It can be seen that MANPs injection can restore the weight loss induced by rheumatoid arthritis in mice. Figure 13 Part B), to reduce joint swelling ( Figure 13 Part A); RT-qPCR results showed ( Figure 14 MANPs treatment can effectively reduce the expression of inflammatory factors CD86 and IL-1β in the synovial tissue of joints and increase the expression of anti-inflammatory factors CD206 and IL-10. A 10 mg / kg injection dose of MANPs can inhibit and reduce ankle swelling in mice with rheumatoid arthritis, and a 20 mg / kg dose has a better therapeutic effect.

[0083] On day 21 after MANPs injection treatment, mouse ankle joints were harvested for Micro-CT imaging analysis, and the results are as follows. Figure 15 , Figure 16 As shown.

[0084] from Figure 15 , 16 The results showed that MANPs (10 mg / kg, 20 mg / kg) significantly inhibited bone erosion in mice with rheumatoid arthritis and increased bone mineral density in the ankle joints of mice with rheumatoid arthritis.

[0085] Based on the difference between mouse experimental dose and human dose, according to the body surface area normalization formula (human equivalent dose (mg / kg) = mouse dose (mg / kg) × (mouse Km coefficient (3) / human Km coefficient (37)), the dose of arginine-myricetin nanomaterial in the injection solution suitable for human intravenous injection can be calculated to be around 1.6 mg / kg.

[0086] In summary, this invention utilizes the self-assembly of cationic amino acids and flavonoid small molecules to prepare self-assembled nanoparticles. The preparation process is simple, mild, pollution-free, high-yield, low-cost, and easily scalable. Furthermore, the prepared self-assembled nanoparticles have high drug loading capacity, can specifically target inflammatory sites, eliminate oxidative stress and inflammation, and inhibit osteoclast differentiation, while exhibiting good biocompatibility and in vivo safety. At a concentration of 150 μg / mL, it can scavenge 91.34% of DPPH free radicals, demonstrating not only that the self-assembled nanoparticles (MANPs) can be specifically taken up by M1-polarized macrophages, but also verifying that cationic amino acid transporter 2 is a key protein for MANPs targeting M1-type macrophages. The self-assembled nanoparticles (MANPs) prepared in this invention can effectively scavenge LPS / IFN-γ-induced intracellular reactive oxygen species at a concentration of 20 μg / mL, and have good potential to inhibit macrophage osteoclast differentiation. By dispersing the self-assembled nanoparticles in a biocompatible solution and preparing an injection for treatment in mice, it was verified that the nanoparticles not only have the ability to target inflamed joints in mice with arthritis, but also can maintain a high drug concentration near the joint for a long time, showing excellent clinical application prospects in the biomedical field.

[0087] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the description and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A self-assembled nanoparticle, characterized in that, The nanoparticles are formed by the self-assembly of cationic amino acids and flavonoid small molecules; wherein, in the nanoparticles, the molar ratio of the cationic amino acids to the flavonoid small molecules is 30:1 to 1:

1.

2. The nanoparticles according to claim 1, characterized in that, The cationic amino acid includes at least one of arginine, histidine, and lysine; and / or The flavonoid small molecules include at least one of flavonols and flavonoids; wherein the flavonols include at least one of myricetin, quercetin, and kaempferol, and the flavonoids include at least one of luteolin and apigenin.

3. The nanoparticles according to claim 1, characterized in that, The nanoparticles are spherical in shape and have an average particle size of 180~240 nm.

4. A method for preparing nanoparticles as described in any one of claims 1 to 3, characterized in that, Includes the following steps: Flavonoid small molecule solution and cationic amino acid solution were prepared separately using flavonoid small molecules and cationic amino acids, respectively. Formaldehyde was then added to the cationic amino acid solution to obtain a mixed solution system. Flavonoid small molecule solution was then added to the mixed solution system, and the mixture was stirred, washed, and dried to obtain the self-assembled nanoparticles.

5. The preparation method according to claim 4, characterized in that, The molar ratio of the cationic amino acids and flavonoid molecules is 30:1 to 1:1; and / or The volume ratio of the flavonoid small molecule solution to the cationic amino acid solution is 1-3:2-6, the concentration of the flavonoid small molecule solution is 1-20 mg / mL, and the concentration of the cationic amino acid solution is 1-15 mg / mL; and / or The volume ratio of the cationic amino acid solution to formaldehyde is 100~140:

1.

6. The preparation method according to claim 4, characterized in that, The preparation of the flavonoid small molecule solution is a mixed solution obtained by dissolving flavonoid small molecules in anhydrous ethanol; and / or The preparation of the cationic amino acid solution is a mixed solution obtained by dissolving cationic amino acids in deionized water; and / or The stirring process involves ultrasonic stirring at room temperature for 1-2 minutes, followed by continuous stirring at 600-1000 rpm at room temperature for 10-14 hours. The ultrasonic parameters are as follows: 30~50kHz; and / or The cleaning process involves washing with pure water 2-3 times; and / or After washing, the product was collected by centrifugation at 10,000 to 14,000 rpm for 12 to 18 minutes. The drying was performed by vacuum freeze drying at -90 to -70°C for 6 to 12 hours.

7. A self-assembled nano-targeting drug, characterized in that, The active ingredient of the drug includes self-assembled nanoparticles as described in any one of claims 1 to 3 or self-assembled nanoparticles prepared by any one of claims 4 to 6.

8. The self-assembled nano-targeting drug according to claim 7, characterized in that, The drug is a self-assembled nano-targeted drug injection solution; the injection solution also includes a biocompatible solution; wherein the biocompatible solution includes at least one of physiological saline and phosphate buffer.

9. The self-assembled nano-targeting drug according to claim 8, characterized in that, In the injection solution, the concentration of the self-assembled nanoparticles is 5 to 50 mg / mL.

10. The application of a nano-targeting particle as described in any one of claims 1-3, or a preparation method as described in any one of claims 4-6, or a self-assembled nano-targeting drug as described in any one of claims 7-9, characterized in that, The application is selected from one of the following: in the preparation of drugs for the prevention and / or treatment of inflammatory arthritis, in the preparation of targeted diagnostic agents for arthritis, and in the preparation of drug carriers.

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