Lock-release switching type triptolide nano-micelle as well as preparation method and application thereof

Abstract

The invention relates to the technical field of nano-drugs, and discloses a lock-release switching type triptolide nano-micelle as well as a preparation method and application thereof. The preparation method of the triptolide nano-micelle comprises the following steps: S1, carrying out esterification modification on a polyamide-amine dendritic polymer by using folic acid to obtain FA-PAMAM; s2: S2-1, dissolving the FA-PAMAM, the triptolide and the calycosin into a solvent, and rotating to form a film, so as to obtain a film A; adding a phosphoric acid buffer solution, hydrating, incubating, and filtering to obtain a hydrated solution; s2-2, dissolving functionalized polyethylene glycol in a solvent, and rotating to form a film to obtain a film B; and hydrating the membrane B by using a hydration solution, and filtering to obtain the triptolide nano-micelle. The triptolide nano-micelle can be used for drug preparation through carrier structure innovation, drug synergy and targeted intelligent drug release, and realizes high-efficiency and low-toxicity treatment of rheumatoid arthritis.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine technology, and discloses a lock-and-release switching type triptolide nanomicelle, its preparation method, and its application. Background Technology

[0002] Rheumatoid arthritis (RA) is a disabling chronic systemic autoimmune disease characterized by symmetrical, erosive synovitis. It can lead to cartilage destruction, bone damage, and disability, accompanied by multi-system complications. It not only severely impairs patients' physical function and quality of life, but also imposes a heavy economic burden.

[0003] Persistent chronic inflammation caused by macrophage polarization imbalance is a core driver of joint damage and high disability rates in rheumatoid arthritis (RA). During the pathological progression of RA, M1 macrophages infiltrating the synovial tissue secrete large amounts of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6, while simultaneously releasing matrix metalloproteinases (MMPs), creating a pro-inflammatory and tissue-degrading joint microenvironment. Meanwhile, the anti-inflammatory and repair functions of M2 macrophages are significantly inhibited, further exacerbating the vicious cycle of inflammation and structural joint damage. Therefore, targeting macrophages in the RA inflammatory microenvironment, by regulating their polarization balance and inhibiting the secretion of pro-inflammatory factors and the destructive activity of MMPs, has become a key direction for developing safe and effective RA treatment strategies.

[0004] Tripterygium wilfordii (TP) is an epoxy diterpenoid lactone compound extracted from the traditional Chinese medicine Tripterygium wilfordii, possessing both anti-inflammatory and immunosuppressive effects. While high doses of TP can exert a potent immunosuppressive effect, its severe hepatotoxicity and reproductive toxicity limit its application. Verbena isoflavones (CAL) are isoflavone components derived from Astragalus membranaceus, exhibiting multiple pharmacological effects including antioxidant, anti-inflammatory, and bone metabolism regulation. The combined use of TP and CAL can broaden the safe therapeutic window for TP-mediated macrophage polarization and achieve synergistic effects in anti-inflammation, immunomodulation, and bone protection; however, both have poor water solubility and other drug-like drawbacks.

[0005] Constructing targeted drug delivery systems based on nanotechnology and designing carriers that combine dose adaptability, low toxicity, and intelligent drug release characteristics specific to the microenvironment are key to achieving precise treatment of rheumatoid arthritis (RA) and reducing toxic side effects. Polyamide-amine dendritic macromolecules (PAMAMs), as hyperbranched polymer carriers, offer significant advantages over traditional carriers such as liposomes, nanoparticles, and exosomes, including superior drug loading capacity, ease of surface functionalization, and biocompatibility. Furthermore, due to their small particle size, PAMAMs possess the ability to effectively penetrate dense microenvironmental barriers. However, PAMAMs have a non-closed structure, which can easily lead to drug leakage and uncontrollable release, thus reducing targeted delivery efficiency. Therefore, overcoming the structural defects of PAMAMs and achieving controlled drug release through the combined delivery of TP and CAL is a critical issue that urgently needs to be addressed. Summary of the Invention

[0006] The purpose of this invention is to provide a lock-and-release switching type of triptolide nanomicelles, its preparation method, and its application, so as to solve the problems mentioned in the background art.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for preparing a lock-and-release switching type triptolide nanomicelles, comprising the following steps: S1: Esterification modification of polyamide-amine dendritic polymer with folic acid yields FA-PAMAM; S2: S2-1: Dissolve FA-PAMAM, triptolide, and verbascoside in a solvent, rotate to form a membrane, and obtain membrane A; add phosphate buffer solution, hydrate and incubate, filter, and obtain hydrated solution; S2-2: Functionalized polyethylene glycol is dissolved in a solvent and rotated to form a film, resulting in membrane B; membrane B is hydrated using an hydration solution and filtered to obtain triptolide nanomicelles.

[0008] Preferably, the preparation method of FA-PAMAM is as follows: S1-1: Folic acid is dissolved in dimethyl sulfoxide, dicyclohexylcarbodiimide and N-hydroxysuccinimide are added, the mixture is reacted, filtered, and freeze-dried to obtain folic acid activated ester; S1-2: Polyamide-amine dendritic polymer is dissolved in dimethyl sulfoxide, folic acid activated ester is added, the mixture is stirred and reacted, dialyzed, and dried to obtain FA-PAMAM.

[0009] Preferably, the FA-PAMAM comprises the following raw materials, in parts by mass: 1-2 parts folic acid, 0.5-1 parts dicyclohexylcarbodiimide, 0.3-0.8 parts N-hydroxysuccinimide, and 10-15 parts polyamide-amine dendrimer.

[0010] Preferably, the process parameters for the hydration incubation are: hydration incubation at 50~60℃ for 10~20 min.

[0011] Preferably, the triptolide nanomicelles comprise the following raw materials, by mass parts: 25-30 parts FA-PAMAM, 0.01-0.015 parts triptolide, 25-28 parts vernix isoflavone, and 20-25 parts functionalized polyethylene glycol dissolved in methanol.

[0012] Preferably, the functionalized polyethylene glycol is an MMP enzyme-sensitive PEG-removable type, including EEEPVGLIG-PEG2000-GILGVPEEE.

[0013] Preferably, the FA-PAMAM is further modified with methoxy polyethylene glycol succinimide to obtain modified FA-PAMAM, which is then used to prepare triptolide nanomicelles. The preparation process of the modified FA-PAMAM is as follows: FA-PAMAM is added to phosphate buffer, methoxy polyethylene glycol succinimide is added, and under nitrogen protection, the mixture is stirred, dialyzed, and lyophilized to obtain modified FA-PAMAM.

[0014] Preferably, the modified FA-PAMAM comprises the following raw materials, in parts by mass: 0.1 to 0.12 parts FA-PAMAM, 10 to 15 parts phosphate buffer, and 0.3 to 0.5 parts methoxy polyethylene glycol succinimide ester.

[0015] Preferably, the triptolide nanomicelles are targeted and can achieve lock-out / release switching.

[0016] Preferably, the triptolide nanomicelles can be used in the preparation of drugs for treating rheumatoid arthritis.

[0017] Compared with the prior art, the beneficial effects achieved by the present invention are: (1) Tripterygium wilfordii can exert a strong immunosuppressive effect, but it is accompanied by severe hepatotoxicity and reproductive toxicity, so its application is limited. In this scheme, it is used in combination with isoflavones to broaden its safe treatment window and improve the efficiency of macrophage polarization regulation, so as to achieve safe and efficient treatment.

[0018] (2) In order to further improve the poor water solubility of triptolide and verbascoside and improve their delivery accuracy, polyamide-amine dendritic macromolecules were selected as hyperbranched polymer carriers. Polyamide-amine dendritic macromolecules have the advantages of outstanding drug loading capacity, good biocompatibility and strong penetration ability. However, they are non-closed structures, which can easily lead to leakage of the encapsulated drug and lack of controllability of release, thereby reducing the targeted delivery efficiency. Therefore, this approach targets the core pathological features of rheumatoid arthritis, namely, the overexpression of matrix metalloproteinases (MMPs) in the synovial microenvironment and the high expression of folate receptors (FR) in activated macrophages. Folate (FA) is used to modify polyamide-amine dendritic macromolecules, enabling macrophages to actively target the drug through the specific binding of FA to FR, thereby regulating macrophage polarization. Furthermore, the outer layer is electrostatically linked to an MMP-sensitive removable PEG (EEEPVGLIG-PEG2000-GILGVPEEE). Upon entering the inflammatory microenvironment, MMP enzymes specifically degrade the PEG linkages, exposing the FA-modified core and further enhancing the active targeting binding to macrophages. Ultimately, this achieves precise and rapid drug release within the target cells. The amount of MMP-sensitive removable PEG added needs to be controlled; excessive addition will lead to an increase in the size of the nanomicelles.

[0019] (3) FA-PAMAM was further modified with methoxy polyethylene glycol succinimide to obtain modified FA-PAMAM, which was then used to prepare triptolide nanomicelles: polyamide-amine dendrimers encapsulate hydrophobic drugs through hydrophobic and electrostatic interactions. However, unmodified polyamide-amine dendrimers are prone to aggregation due to intermolecular hydrogen bonds, which leads to blockage of internal cavities and a reduction in encapsulation sites. After modification with polyethylene glycol, its strongly hydrophilic segments help to increase the encapsulation capacity and enhance the stability of the carrier in systemic circulation, thus preventing drug molecule leakage. At the same time, since the MMP enzyme-sensitive type connected to the outer layer through electrostatic forces can remove polyethylene glycol with similar segments, the affinity is greatly improved, and the overall stability is enhanced. However, when modifying polyamide-amine dendrimers with polyethylene glycol, the amount added should not be too much, otherwise it will affect the release rate.

[0020] In summary, through innovative carrier structure, drug synergy, and targeted intelligent drug release, triptolide nanomicelles with excellent therapeutic effects, high encapsulation rate, and lock-release switching capability were prepared, achieving highly efficient and low-toxicity treatment of RA. Attached Figure Description

[0021] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 Here is a structural characterization diagram: Figure 1 A is the infrared spectrum of FA-PAMAM; Figure 1 B is the 1H NMR spectrum of FA-PAMAM; Figure 1 C is the infrared spectrum of the nanomicelles prepared in Example 1; Figure 1 D is the transmission electron microscopy characterization of LD-PAMAM-TP&CAL; Figure 1 E represents the particle size distribution curve of LD-PAMAM-TP&CAL. Figure 1 F is the Zeta potential distribution curve of LD-PAMAM-TP&CAL; Figure 1 G is a quantitative statistical graph of the average particle size of each formulation; Figure 1 Quantitative statistical graph of the zeta potential of each H formulation; Figure 1 I is a quantitative statistical chart of the polydispersity index of each formulation; Figure 1 The figure shows the encapsulation efficiency of TP and CAL by LD-PAMAM-TP & CAL. Figure 1 K represents the in vitro cumulative release curve of TP; Figure 1 L represents the in vitro cumulative release curve of CAL; Figure 2 The diagram shows the effects of each formulation on RAW264.7 macrophages; among which... Figure 2A represents the growth curves of RAW264.7 cells after incubation with drug-loaded micelles at different concentrations of free TP. Figure 2 B represents the IC of each formulation. 50 Value statistical analysis chart; Figure 2 C represents the growth curves of RAW264.7 cells after incubation with different concentrations of CAL and TP. Figure 2 Figure D shows the flow cytometry results of RAW264.7 cell apoptosis after incubation with each formulation. Figure 2 E is a quantitative analysis graph of the results of cell apoptosis detected by flow cytometry; Figure 2 F represents the results of calcein AM / PI live / dead cell staining in RAW264.7 cells after incubation with different formulations; Figure 2 G represents a quantitative statistical graph of the results of calcein AM / PI live / dead cell staining; Figure 3 This image shows the LPS-induced uptake and intracellular distribution of different agents in RAW264.7 cells; among which... Figure 3 A is a fluorescence micrograph of LPS-induced RAW264.7 cells after different micelle treatments; Figure 3 Figure B shows the intracellular fluorescence intensity analysis of LPS-induced RAW264.7 cells after different micelle treatments. Figure 3 Figure C represents the flow cytometry results of the relative fluorescence intensity of LPS-induced RAW264.7 cells after different micelle treatments. Figure 3 D is a graph showing the relative fluorescence intensity analysis detected by flow cytometry. Figure 4 This diagram illustrates the regulatory effects of various formulations on the M1 / M2 phenotype of RAW264.7 macrophages; among which... Figure 4 Figure A shows the flow cytometry results of the effects of various formulations on the expression of CD86 (green fluorescent marker) phenotypic marker of macrophage M1; Figure 4 Figure B shows the flow cytometry results of the effects of each formulation on the expression of CD206 (red fluorescent label) phenotypic marker of macrophage M2; Figure 4 C and D are flow cytometry quantitative analysis diagrams of CD86 and CD206 expression levels after treatment with each formulation, respectively. Figure 4 E represents the immunofluorescence staining results of CD86 in macrophages after treatment with various agents; Figure 4 F represents the immunofluorescence staining results of CD206 in macrophages after treatment with various agents; Figure 4 G and H are quantitative analysis results of CD86 and CD206 immunofluorescence staining, respectively; Figure 5 The diagram shows the protective effects of different formulations on chondrocytes mediated by macrophages; among them Figure 5A represents the effect of different concentrations of M1 conditioned medium on chondrocyte proliferation in LPS-induced activated RAW264.7 cells; Figure 5 B represents the effect of conditioned medium on chondrocyte activity after treatment of RAW264.7 cells with various formulations; Figure 5 C represents the quantitative statistical results of the effect of different RAW264.7 cell conditioned media on chondrocyte apoptosis detected by flow cytometry. Figure 5 D represents the quantitative statistical results of fluorescence intensity on chondrocyte proliferation under different RAW264.7 cell conditioned media. Figure 5 E represents flow cytometry analysis of chondrocyte apoptosis after treatment with different RAW264.7 cell conditioned media. Figure 5 F represents the fluorescence staining results of chondrocyte proliferation after treatment with different RAW264.7 cell conditioned media.

[0022] Figure 6 Real-time imaging observations of CIA rats after injection of different formulations; Figure 7 This is a graph evaluating the efficacy of LD-PAMAM-TP&CAL in CIA model mice; where... Figure 7 A is a timeline diagram illustrating the establishment of the CIA rat model and the drug administration regimen. Figure 7 B represents the body weight change curves of CIA rats treated with different formulations; Figure 7 C represents the curves showing the changes in the arthritis index score of CIA rats treated with different formulations; Figure 7 D represents representative photographs of the hind paw lesion phenotypes in each formulation group of CIA rats at the experimental endpoint. Figure 7 E represents the experimental endpoint. X-ray images of the hind paws of CIA rats in each formulation group. Figure 7 F represents the HE staining results of ankle joint pathological sections of CIA rats after treatment with different preparations (scale bar = 100 μm). Figure 7 G represents the Safranin-Fix Green staining results of ankle joint pathological sections of CIA rats after treatment with different formulations (scale bar = 100 μm). Figure 8 Figure 1 shows the efficacy evaluation of different formulations after treating CIA rats; among them, Figure 8 A, B, and C represent the quantitative statistical results of ELISA detection of inflammatory factors IL-1β, TNF-α, and IL-6 in the blood. Figure 8 D, E, and F represent the quantitative statistical results of ELISA detection of bone injury-related factors TRAP 5b, RANKL, and OPG in the blood. Figure 8G and H represent the quantitative statistical analysis results of immunofluorescence staining of CD86 (marker of M1 macrophage) and CD206 (marker of M2 macrophage) in synovial tissue; (I) Immunofluorescence staining results of M1 macrophages (CD86-labeled, red fluorescence) and M2 macrophages (CD206-labeled, red fluorescence) in synovial tissue (scale bar = 50 μm). Figure 9 This is a diagram showing the safety evaluation results; in which... Figure 9 A represents the experimental endpoint. HE staining results of pathological sections of major organs of CIA rats after treatment with different preparations (red arrows indicate inflammatory cell infiltration, scale bar = 50 μm). Figure 9 B and C represent the quantitative statistical results of alanine aminotransferase and aspartate aminotransferase levels in the blood of CIA rats after treatment with different formulations. Detailed Implementation

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0024] It should be noted that there are no special restrictions on the suppliers of any of the raw materials involved in this invention. Exemplary examples include: TP and CAL provided by Chengdu Pufeed Biotechnology Co., Ltd. (purity 99.18%, Chengdu, China); the polyamide-amine dendritic polymer used is PAMAM-G5, purchased from Sigma-Aldrich; EEEPVGLIG-PEG2000-GILGVPEEE (polyethylene glycol modified at both ends by peptides (glutamic acid-glutamic acid-glutamic acid-proline-valine-glycine-leucine-isoleucine-glycine)) synthesized by Ruixi Biotechnology Co., Ltd. (Xi'an, China); the methoxy polyethylene glycol succinimide ester has the catalog number A0525947; and biphenylamine H33342 trichlorohydrochloride (Hoechst 33342) is provided by Aladdin Biotechnology Co., Ltd. (Shanghai, China). Folic acid (FA), penicillin / streptomycin solution (MA0110), phosphate-buffered saline (PBS), lipopolysaccharide (LPS), and the Annexin V-FITC / PI apoptosis detection kit were provided by Meilun Biotechnology Co., Ltd. (Dalian, China). CCK-8, IL-1β, IL-6, TNF-α ELISA kits and MMP2 enzyme were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Coumarin (Courule) and 1,1-octyldecyl-3,3,3-tetramethylindole tricarboxycyanine iodide (DiR) were provided by Nanjing Kaiji Biotechnology Development Co., Ltd. (Nanjing, China). FITC anti-mouse CD86 and PE anti-mouse CD206 were purchased from BioLegend Inc. (USA). Nitric oxide assay kits were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China); DMEM high-glucose medium was purchased from Gibco (USA); fetal bovine serum (SA211.02) was purchased from Cellmax; disposable cell culture equipment such as culture dishes and centrifuge tubes were purchased from NEST Ltd. Chicken type II collagen (CII), complete Freund's adjuvant (CFA), and incomplete Freund's adjuvant (IFA) were provided by Chondrex (USA). All other reagents used were of chromatographic grade.

[0025] RAW264.7 cells were obtained from Pucolife Biotechnology Co., Ltd. (Wuhan, China). Chondrocytes were purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd. (Jiangsu, China).

[0026] Female SD rats were purchased from Liaoning Changsheng Biotechnology Co., Ltd., with laboratory animal production license number SCXK(Liaoning) 2020-0001. The research protocol was approved by the Animal Research Ethics Committee of Shanxi University of Traditional Chinese Medicine (approval number 202503108). Furthermore, the care and use of all animals followed the principles of the "Guidelines for Ethical Review of Laboratory Animal Welfare" (GB / T35892-2018).

[0027] Unless otherwise specified, all figures below are parts by weight or mass ratios. Example 1: S1: 1 part folic acid was dissolved in 1500 parts dimethyl sulfoxide, 0.75 parts dicyclohexylcarbodiimide and 0.42 parts N-hydroxysuccinimide were added, and the reaction was carried out at room temperature for 2 hours. After filtration and freeze-drying, folic acid activated ester was obtained. 13.1 parts polyamide-amine dendrimer was added to 1500 parts dimethyl sulfoxide, and folic acid activated ester was added while stirring. The mixture was stirred at room temperature for 24 hours, and impurities were removed by dialyzing with distilled water for 48 hours. After freeze-drying, FA-PAMAM was obtained. S2: Dissolve 29 parts of FA-PAMAM, 0.01 parts of triptolide, and 25.69 parts of verbascoside in methanol and spin-coat a thin film at 40°C to obtain membrane A; add 1387 parts of phosphate buffer (pH 7.4), hydrate and incubate at 55°C for 15 min, and filter through a 0.22 μm microporous membrane as the hydration solution; dissolve 23 parts of functionalized polyethylene glycol in methanol and spin-coat a thin film to obtain membrane B; hydrate with the hydration solution and filter through a 0.22 μm microporous membrane to obtain triptolide nanomicelles, denoted as LD-PAMAM-TP&CAL.

[0028] Comparative Example 1: The difference from Example 1 is that TP and CAL were not introduced, but the rest is the same as Example 1, denoted as LD-PAMAM.

[0029] Comparative Example 2: The difference from Example 1 is that CAL was not introduced, but the rest is the same as Example 1, denoted as LD-PAMAM-TP.

[0030] Comparative Example 3: The difference from the Example is that no functionalized PEG was introduced, and the rest is the same as Example 1; it is denoted as FA-PAMAM-TP&CAL.

[0031] Performance Test 1: The FA-PAMAM, LD-PAMAM-TP&CAL, LD-PAMAM, LD-PAMAM-TP, and FA-PAMAM-TP&CAL samples from Examples 1 and Comparative Examples 1-3 were subjected to relevant performance tests. Encapsulation efficiency (EE) was determined by high-performance liquid chromatography (Agilent HPLC system); the chromatographic column was Agela-C18 (4.6 mm × 250 mm, 5 μm); the flow rate was 1 mL / min; the mobile phase for TP determination was methanol:water (40:6). The detection wavelength was 218 nm, the temperature was 25 °C, and the injection volume was 10 μL. The mobile phase for CAL determination was acetonitrile:water (35:65), the detection wavelength was 240 nm, the temperature was 30 °C, and the injection volume was 10 μL. Dialysis was performed using PBS buffer containing 10% FBS: First, 1 mL of FA-PAMAM-TP&CAL and LD-PAMAM-TP&CAL micelles were added to a dialysis bag with a molecular weight of 3500 Da, and the drug was released by slow shaking at 37 °C and 100 rpm. To simulate the microenvironment of arthritis, 1.5 μg / mL of MMPs enzymes were added to the released PBS buffer. Then, appropriate amounts of buffer were taken, and the contents of TP and CAL in the sample were measured at 2, 4, 6, 8, 10, 12, 24, 48, and 72 h. The results are as follows. Figure 1 As shown.

[0032] like Figure 1 As shown in A: the characteristic peak of free FA is at 1700 cm⁻¹. -1 The C=O stretching vibration peak of the nearby carboxyl group, the characteristic peak of PAMAM is at 3300 cm⁻¹. -1 The NH stretching vibration peak near the synthesized product FA-PAMAM was observed at 1690 cm⁻¹. -1 The new peak appearing at 3300 cm⁻¹ is attributed to the new amide bond formed after the reaction of FA with the terminal amino group of PAMAM. Furthermore, the peak at 3300 cm⁻¹... -1 The NH stretching vibration peak in the region also shifted and deformed, and its intensity decreased, indicating that PAMAM successfully participated in the reaction and folic acid was successfully grafted onto PAMAM.

[0033] like Figure 1 As shown in Figure B, characteristic NMR peaks from free FA are observed at chemical shifts δ~6.7 ppm, δ~4.7 ppm, and near δ~2.3 ppm. PAMAM's characteristic NMR peaks appear at chemical shifts δ~2.3 ppm, δ~2.6 ppm, and δ~3.2–3.3 ppm. The synthesized product FA-PAMAM exhibits a clear proton multiplet of the folic acid aromatic ring in the δ~6.5–8.7 ppm range, while the peak from δ=2.2 ppm to δ=3.5 ppm corresponds to the proton peak of the methylene group in PAMAM, confirming the successful introduction of the folic acid structure.

[0034] like Figure 1 As shown in C, the characteristic peak of TP is at 1756.03 cm⁻¹. -1 The stretching vibration of the carbonyl peak (C=O) of the ester, 3447.17 cm⁻¹ -1 The stretching vibration of the hydroxyl group (OH) is 1678.17 cm⁻¹. -1 The vibration of the double bond within the unsaturated ring. The characteristic infrared peak of CAL is at 3418.75 cm⁻¹. -1 The broad, intense peak produced by the stretching vibration of the hydroxyl group (OH) is at 1642 cm⁻¹. -1 A strong absorption peak is generated at the carbonyl group (C=O) of flavonoids. This peak can be observed at 1763.03 cm⁻¹ in LD-PAMAM-TP&CAL. -1 The stretching vibration of the characteristic ester carbonyl peak (C=O) of TP, and at 1616 cm⁻¹ -1 The presence of characteristic peaks for CAL at 3400 cm⁻¹ proves that TP and CAL were successfully encapsulated. Furthermore, TP and CAL exhibit peaks at 3400 cm⁻¹. -1 The hydroxyl groups present at the site moved and broadened within the drug-loaded micelles, possibly due to the interaction of the two drugs via hydrogen bonds, indicating that TP and CAL were encapsulated within the carrier.

[0035] like Figure 1 As shown in D: LD-PAMAM-TP&CAL is a regular sphere with smooth and undamaged particle edges and good morphological uniformity. Figure 1 The E and F values ​​indicate that the average particle size of LD-PAMAM-TP&CAL is (22.47±2.02) nm and the Zeta potential is (2.56±0.47) mV.

[0036] The particle size and zeta potential of FA-PAMAM, LD-PAMAM, FA-PAMAM-TP&CAL, LD-PAMAM-TP, and LD-PAMAM-TP&CAL were compared: The results are as follows Figure 1 As shown in G, H, and I, the particle sizes of both FA-PAMAM and FA-PAMAM-TP&CAL formulations are relatively small, approximately 5 nm; however, after functionalization with PEG, the particle size of the LD-PAMAM series formulations significantly increased (P<0.05). Simultaneously, the zeta potential decreased significantly from the markedly positive (18.90±0.83) mV of the FA-PAMAM formulation to near-neutral levels (P<0.05), a change that may be related to the hydrophilic encapsulation effect of PEG molecules.

[0037] The results are as follows Figure 1As shown in Figure J, in the LD-PAMAM-TP&CAL formulation, the encapsulation rates of drugs TP and CAL reached (93.93±1.97)% and (94.46±1.39)%, respectively, indicating that the carrier system has excellent encapsulation capacity for both drugs.

[0038] Depend on Figure 1 As shown in K and L, without the addition of MMPs, the FA-PAMAM-TP&CAL formulation exhibited a faster drug release rate, with cumulative release rates of CAL and TP of (81.48±4.95)% and (71.78±2.33)% at 72 h, respectively. In contrast, the release behavior of LD-PAMAM-TP&CAL was significantly slowed under the same conditions, with cumulative release rates of CAL and TP of only (29.37±4.93)% and (24.03±2.88)% at 72 h. In a simulated disease microenvironment (with the addition of MMPs), LD-PAMAM-TP&CAL exhibited typical responsive release characteristics, with cumulative release rates of CAL and TP of (71.55±0.29)% and (64.78±4.66)% at 72 h, respectively. This result confirms that the functionalized PEG-modified nanomicelles are MMP enzyme sensitive—under the action of highly expressed MMPs in the disease microenvironment, the PEG layer is degraded, the micelle structure disintegrates, thereby accelerating drug release, fully demonstrating the enzyme-sensitive targeted release characteristics of this formulation.

[0039] Example 2: S1: Dissolve 1 part folic acid in 1500 parts dimethyl sulfoxide, add 0.75 parts dicyclohexylcarbodiimide and 0.42 parts N-hydroxysuccinimide, react at room temperature for 2 h, filter, freeze dry to obtain folic acid activated ester; add 13.1 parts polyamide-amine dendrimer to 1500 parts dimethyl sulfoxide, add folic acid activated ester while stirring, stir at room temperature for 24 h, dialyze with distilled water for 48 h to remove impurities, freeze dry to obtain FA-PAMAM; Add 0.1 part of FA-PAMAM to 10 parts of phosphate buffer, add 0.4 parts of methoxy polyethylene glycol succinimide, stir at room temperature in the dark for 48 hours under nitrogen protection, dialyze to remove unreacted substances, and freeze dry to obtain modified FA-PAMAM. S2: 29 parts of modified FA-PAMAM, 0.01 parts of triptolide, and 25.69 parts of velvet isoflavone were dissolved in methanol and spun into a thin film at 40°C to obtain membrane A; 1387 parts of phosphate buffer with pH=7.4 were added, and the mixture was hydrated and incubated at 55°C for 15 min, then filtered through a 0.22 μm microporous membrane as the hydration solution; 23 parts of functionalized polyethylene glycol were dissolved in methanol and spun into a thin film to obtain membrane B; the mixture was hydrated with the hydration solution and filtered through a 0.22 μm microporous membrane to obtain triptolide nanomicelles, denoted as LD-PAMAM-TP&CAL.

[0040] Comparative Example 4: S1: 1 part folic acid was dissolved in 1500 parts dimethyl sulfoxide, 0.75 parts dicyclohexylcarbodiimide and 0.42 parts N-hydroxysuccinimide were added, and the reaction was carried out at room temperature for 2 h. After filtration and freeze-drying, folic acid activated ester was obtained. 13.1 parts polyamide-amine dendrimer was added to 1500 parts dimethyl sulfoxide, and folic acid activated ester was added while stirring. The mixture was stirred at room temperature for 24 h, and impurities were removed by dialyzing with distilled water for 48 h. After freeze-drying, FA-PAMAM was obtained. Add 0.1 part of FA-PAMAM to 10 parts of phosphate buffer, add 0.8 parts of methoxy polyethylene glycol succinimide, stir at room temperature in the dark for 48 hours under nitrogen protection, dialyze to remove unreacted substances, and freeze dry to obtain modified FA-PAMAM; S2: 29 parts of modified FA-PAMAM, 0.01 parts of triptolide, and 25.69 parts of velvet isoflavone were dissolved in methanol and spun into a thin film at 40°C to obtain membrane A; 1387 parts of phosphate buffer with pH=7.4 were added, and the mixture was hydrated and incubated at 55°C for 15 min, then filtered through a 0.22 μm microporous membrane as the hydration solution; 23 parts of functionalized polyethylene glycol were dissolved in methanol and spun into a thin film to obtain membrane B; the mixture was hydrated with the hydration solution and filtered through a 0.22 μm microporous membrane to obtain triptolide nanomicelles, denoted as LD-PAMAM-TP&CAL.

[0041] The encapsulation efficiency of CAL and TP and the cumulative release rate over 72 hours of LD-PAMAM-TP&CAL prepared in Example 2 and Comparative Example 4 were tested using the same method. In Example 2, the encapsulation efficiency of TP and CAL reached (95.91±0.83)% and (97.06±0.65)%, respectively, and the cumulative release rates of CAL and TP over 72 hours were (71.08±0.15)% and (64.34±2.97)%, respectively; in Comparative Example 4, TP... The encapsulation rates of CAL and TP reached (93.25±0.96)% and (94.09±0.88)%, respectively, and the cumulative release rates of CAL and TP over 72 hours were (69.08±0.15)% and (61.71±3.58)%, respectively. This shows that introducing an appropriate amount of polyethylene glycol to modify FA-PAMAM can improve the encapsulation rate without affecting the release rate. However, excessive introduction of polyethylene glycol will lead to performance degradation, so the amount added must be strictly controlled.

[0042] Performance Test 2: In vitro cytotoxicity assessment: The CCK-8 assay was used to evaluate the effects of TP, LD-PAMAM-TP, LD-PAMAM-TP&CAL, FA-PAMAM-TP&CAL, and LD-PAMAM-TP&CAL+MMPs on LPS (100 ng / mL) and IFN-γ (20 ng / mL) induced RAW264.7 cell viability. 1×10⁻⁶ cells in logarithmic growth phase were used as an example.4 Cells were seeded into 96-well plates and stimulated with LPS and IFN-γ at 37°C for 12 h. They were then treated with the above-mentioned preparations for 24 h. After that, the original culture medium was aspirated, and 100 μL of 10% CCK-8 working solution was added to each well. After incubation at 37°C for 2 h, the absorbance of the sample at 450 nm was measured using a microplate reader (HBS-1096A, Detai, Nanjing, China), and the 50% inhibition concentration was calculated. The CCK-8 assay was used to detect the attenuation effect of CAL on TP: RAW264.7 cells in logarithmic growth phase were seeded into 96-well plates. After cell adhesion, the cells were treated with TP (38 nM, 20 nM) and different concentrations of CAL for 24 h. Then, 10 μL of 10% CCK-8 working solution was added to each well and incubated at 37 °C for 2 h. The absorbance of the sample at 450 nm was measured using a microplate reader to assess cell viability. Annexin V-FITC / PI double staining flow cytometry was used to detect apoptosis in RAW264.7 cells: The effects of different agents on apoptosis of activated RAW264.7 cells were determined using annexin V / FITC / propidium iodide (PI) double staining. First, RAW264.7 cells in the logarithmic growth phase were... 5 Cells were seeded at a density of 100 cells / well into 6-well cell culture plates. After cell adhesion, the cells were induced for 12 h with LPS (100 ng / mL) and IFN-γ (20 ng / mL). Then, medium containing TP, TP & CAL, LD-PAMAM-TP, LD-PAMAM-TP & CAL, FA-PAMAM-TP & CAL, and LD-PAMAM-TP & CAL + MMPs micelles (TP concentration 20 nM, CAL concentration 50 μM) was added to each well. After culturing for 12 h, cell samples were collected and stained. Subsequently, the apoptosis rate was quantitatively analyzed by flow cytometry (BD Biosciences, New Jersey, USA). Calcein-AM / PI staining method was used to detect the viability of RAW264.7 cells: RAW264.7 cells were injected at a rate of 1 × 10⁶ cells per well. 4Cells were seeded at a density of [number] cells per well in 96-well plates. After cell adhesion, cells were induced for 12 h with LPS (100 ng / mL) and IFN-γ (20 ng / mL). Then, culture medium containing equal volumes of PBS, TP, TP & CAL, LD-PAMAM-TP, LD-PAMAM-TP & CAL, FA-PAMAM-TP & CA, and LD-PAMAM-TP & CAL + MMPs (TP concentration 20 nM, CAL concentration 50 μM) was added. After 48 h of culture, the culture medium was removed, and 100 μL of PBS containing calcein-AM (0.2 μM) and PI (2.5 μM) was added. The cells were incubated in the dark at 37°C for 30 min. Unbound dye was removed by washing the samples with PBS (0.01 mM), and the images were then observed using a fluorescence microscope (Nikon Eclipse E800, Nikon, Tokyo). Conclusions are as follows: Figure 2 As shown.

[0043] Depend on Figure 2 As shown in A, the survival rate of macrophages in all experimental groups decreased in a dose-dependent manner with increasing TP concentration; the half-maximal inhibitory concentration (IC50) of the free TP group, LD-PAMAM-TP group, FA-PAMAM-TP&CAL group, LD-PAMAM-TP&CAL+MMPs group, and LD-PAMAM-TP&CAL group was also significantly lower. 50 The concentrations of free TP were (37.42±5.80) nM, (47.37±4.52) nM, (48.78±3.42) nM, (65.57±4.09) nM, and (70.1±6.10) nM, respectively. 50 The IC50 value was significantly lower than that of each formulation group (P < 0.01); while the IC50 value of the LD-PAMAM-TP&CAL group was significantly lower than that of each formulation group (P < 0.01); 50 The highest value suggests that this formulation can most significantly reduce the cytotoxicity of TP.

[0044] Depend on Figure 2 As shown in B, the viability of RAW264.7 cells increased in a gradient with increasing CAL concentration, confirming that the combination of CAL and TP can antagonize the inhibitory toxicity of TP on macrophage proliferation in a dose-dependent manner.

[0045] Depend on Figure 2 As shown in C and D, compared with the free TP group, the apoptosis rate of the TP&CAL combination group was significantly reduced to 35.00%±2.76% (P<0.0001); at the same time, the apoptosis rate of the LD-PAMAM-TP&CAL group was also significantly lower than that of the LD-PAMAM-TP group (P<0.01). This indicates that the combination of CAL and TP can effectively reduce the apoptosis-inducing toxicity of TP. Furthermore, the apoptosis rate of each formulation group was lower than that of the free drug group, with the LD-PAMAM-TP&CAL group showing the most significant apoptosis inhibition effect.

[0046] Depend on Figure 2 As shown in E, F, and G, the proportion of live cells in the combination group was significantly higher than that in the single-drug group, and the proportion of cell death was lowest in the LD-PAMAM-TP&CAL group. This further confirms that the CAL combination strategy can significantly reduce the cytotoxicity of TP (in this detection system, live cells emit green fluorescence, and dead cells emit red fluorescence).

[0047] Performance Test 3: RAW264.7 cells were packed at 3 × 10⁶ cells per well. 5 Cells were seeded at a density of 100 g / mL in 6-well plates. After cell adhesion, they were cultured for 12 h under stimulation with LPS (100 ng / mL) and IFN-γ (20 ng / mL). The cell culture medium was then replaced with fresh medium containing fluorescent probe Cou-labeled formulations (Cou-PAMAM, FA-PAMAM-Cou, LD-PAMAM-Cou, LD-PAMAM-Cou+MMPs) (Cou concentration 3 μM). After 2 h of incubation, the medium was aspirated and discarded. The cells were then washed twice with PBS, fixed with 4% paraformaldehyde, and the nuclei were stained with Hoechst 33342 for 15 min. The cells were then observed and imaged using a fluorescence microscope. Flow cytometry was used to quantitatively analyze the uptake of different formulations by RAW264.7 cells, following the same steps described above. After treatment with different formulations, cells were collected, washed, and resuspended in 500 μL of PBS for subsequent flow cytometry analysis. Results Figure 3 As shown.

[0048] No green fluorescence signal was detected in the cells of the blank control group, but after culturing in drug-containing medium for 2 hours, cells in all treatment groups showed obvious green fluorescence, confirming that the preparation could be taken up by macrophages. Furthermore, the fluorescence intensity of the cells in the FA-PAMAM-Cou group was significantly higher than that in the blank control group and the Cou-PAMAM group (P < 0.0001). This difference stems from the fact that the FA modified on the surface of the preparation can specifically bind to the folate receptor highly expressed on the surface of macrophages, significantly improving drug uptake efficiency through receptor-mediated endocytosis. In contrast, the cell uptake rate of the LD-PAMAM-Cou group was significantly reduced. The core reason is that the outer enzyme-sensitive release functionalized PEG coating not only masked the FA target molecule, preventing it from binding to the receptor, but also further weakened the cells' phagocytic ability to absorb the preparation due to the increased particle size. The cellular uptake in the LD-PAMAM-Cou+MMPs group was significantly increased (P < 0.0001), suggesting that under the action of MMPs simulating the disease microenvironment, the outer PEG coating of the formulation was specifically cleaved, and the previously masked FA targeting molecules were re-exposed, thereby restoring the macrophages' ability to target and uptake the formulation. Flow cytometry quantitative analysis results were completely consistent with the trends observed by fluorescence microscopy, further validating the reliability of the above conclusions.

[0049] Performance Test 4: Regulatory Effects of Different TP-Loading Agents on the Polarization of M1 and M2 Macrophages: Immunofluorescence and flow cytometry were used to detect the effects of different agents on the M1 macrophage marker CD86 and the M2 macrophage marker CD206. RAW264.7 cells were divided into groups of 3 × 10⁻⁶ cells. 5 Cells were seeded at a density of [number] cells / well in 6-well plates and treated for 24 h with LPS (100 ng / mL), IFN-γ (20 ng / mL), or IL-4 (20 ng / mL) to induce polarization into M1 and M2 macrophages, respectively. A blank control group (untreated), a model group, and four treatment groups (LD-PAMAM-TP, LD-PAMAM-TP&CAL, FA-PAMAM-TP&CA, and LD-PAMAM-TP&CAL+MMPs, with TP concentration of 20 nM and CAL concentration of 50 μM) were established for intervention. After adding the corresponding FITC-CD86 and PE-CD206 fluorescent antibodies, the M1 / M2 phenotypic distribution was observed under a fluorescence microscope (green / red fluorescence labeled CD86-positive / CD206-positive cells, respectively). Flow cytometry analysis was performed using the same grouping design and drug treatment procedure. After incubation, cells were collected and analyzed by flow cytometry.

[0050] Depend on Figure 4The results showed that after treatment with FA-PAMAM-TP&CAL and LD-PAMAM-TP&CAL+MMPs, the positive rate of the M1 marker CD86 in LPS-induced RAW264.7 cells was significantly reduced (P < 0.0001), while the positive rate of the M2 marker CD206 was significantly increased (P < 0.0001), preliminarily suggesting the regulatory effect of the agents on macrophage polarization phenotype. Compared with the model group, the CD86 fluorescence intensity of cells in each agent treatment group was significantly reduced (P < 0.01), while the green fluorescence intensity of CD206 was significantly increased (P < 0.01), confirming that the agents can effectively regulate macrophage polarization. Further inter-group comparisons revealed that the LD-PAMAM-TP&CAL+MMPs group and the FA-PAMAM-TP&CAL group showed significantly better reductions in CD86 fluorescence and enhancements in CD206 fluorescence than the LD-PAMAM-TP and LD-PAMAM-TP&CAL groups (P < 0.01). The core mechanism of this difference lies in the fact that the functionalized PEG coating on the outer layer of the LD-PAMAM-TP&CAL group was not degraded. This not only masked the binding site of the FA ligand to the FR-β receptor on the macrophage surface but also reduced the cell's uptake efficiency of the drug. Therefore, its polarization-regulating effect was weaker than that of the FA-PAMAM-TP&CAL group (without PEG masking) and the LD-PAMAM-TP&CAL+MMPs group (PEG was degraded by MMPs). In addition, although the regulatory effect of the LD-PAMAM-TP&CAL group was weaker than the first two groups, it was significantly better than that of the LD-PAMAM-TP group (P<0.05). This result confirms that the combined application of TP and CAL has a synergistic effect in regulating macrophage polarization.

[0051] Performance Test 5: Investigating the protective effect of various formulations on chondrocytes by regulating macrophages: RAW264.7 cells were cultured at 3×10⁻⁶ cells / year. 5 Cells were seeded at a density of 100 cells / well in 6-well plates. Complete culture medium (blank group) and complete culture medium containing LPS (100 ng / mL) + IFN-γ were added to induce M1 polarization. After incubation for 24 h, the supernatant was collected to obtain macrophage M0-type conditioned culture medium (M0-CM) and M1-type conditioned culture medium (M1-CM). These were diluted to different concentrations and co-incubated with chondrocytes for 24 h and 48 h to assess cell viability. Figure 5 As shown in A, cell viability decreased significantly with prolonged co-incubation time and increased CM concentration.

[0052] M1 macrophages were treated for 24 h with FA-PAMAM-TP&CAL, LD-PAMAM-TP&CAL+MMPs, LD-PAMAM-TP, and LD-PAMAM-TP&CAL groups, respectively. CM cells were then collected and co-incubated with chondrocytes at a 40% concentration for 48 h. The effect on chondrocyte viability was observed using CCK8 assay. The experimental results are as follows: Figure 5 Compared with the untreated M1-CM group, all drug-treated groups significantly improved the inhibition of cell viability by M1-CM (P < 0.01), with the FA-PAMAM-TP&CAL group and the LD-PAMAM-TP&CAL+MMPs group showing the most significant effects. Figure 5 The apoptosis rates of chondrocytes in groups C, E, and M1-CM were relatively high. After treatment with different formulations, the apoptosis rates decreased in all groups, with the most significant decrease observed in FA-PAMAM-TP&CAL and LD-PAMAM-TP&CAL+MMPs (P < 0.01), at 14.03% and 20.64%, respectively. This indicates that FA-PAMAM-TP&CAL and LD-PAMAM-TP&CAL+MMPs can effectively inhibit inflammation-induced chondrocyte apoptosis. Fluorescence microscopy results are shown below. Figure 5 As shown in Figures D and F, EDU staining reflects cell proliferation status. The M1-CM group had a lower EDU positivity rate, indicating inhibition of cell proliferation under inflammatory conditions. Chondrocytes treated with the FA-PAMAM-TP&CAL group and the LD-PAMAM-TP&CAL+MMPs group exhibited the strongest fluorescence intensity, significantly higher than the M1-CM group, with a significantly increased EDU positivity rate (P < 0.01), and the best cell viability, consistent with the results of cytotoxicity and flow cytometry apoptosis. This indicates that they alleviated the inhibition of chondrocyte proliferation by M1 macrophages (CM). In summary, these results demonstrate that the FA-PAMAM-TP&CAL group and the LD-PAMAM-TP&CAL+MMPs group can significantly improve the inhibition of chondrocyte viability by the inflammatory microenvironment by regulating macrophage phenotype, effectively inhibiting apoptosis and promoting proliferation, thereby exerting a chondrogenic protective effect.

[0053] Performance test 6: Application effect of nano micelles: (1) CII powder was prepared into a 2 mg / mL solution with 0.1 mol of glacial acetic acid at 4℃. Then, an equal volume of CII solution was mixed with CFA and IFA in an ice bath at 4℃ and emulsified evenly. On the first immunization and the 7th day after the first immunization, 0.2 mL of CII / CFA emulsion was injected into the subcutaneous tissue at the base of the rat tail to establish a CIA model. On day 14 after the initial immunization, CIA rats were injected intravenously with equal volumes of PBS, TP, LD-PAMAM-TP, FA-PAMAM-TP&CAL, and LD-PAMAM-TP&CAL (TP: 60 μg / kg, CAL: 10 mg / kg). The drugs were then administered every 3 days, and the rats' body weight, paw changes, and paw swelling were observed and measured simultaneously. A total of 9 administrations were performed. (2) Each formulation was labeled with DiR as a fluorescent probe, and the targeting effect of each formulation was evaluated by in vivo fluorescence imaging in animals. The preparation of DiR-labeled formulations also adopted the secondary thin film dispersion method, in which the fluorescent probe: lipid material = 500:1, w / w. The successfully modeled CIA rats were divided into a saline control group, a free DiR group, a PAMAM-DiR group, a FA-PAMAM-DiR group, and an LD-PAMAM-DiR group (DIR dose 0.5 mg / kg), and administered the drugs via tail vein injection. The small animal in vivo imaging system was used to image the rats at 1, 3, 6, 9, 12, 24, 48, and 72 h after administration.

[0054] (3) After dehydrating the synovial tissue of CIA rats in each group, the tissue was embedded in paraffin and the effects of hematoxylin-eosin (HE), safranin-fast green staining and X-ray on the synovial tissue were evaluated.

[0055] (4) At the end of the experiment, blood was collected from each group of mice and plasma was prepared. The levels of relevant factors were detected using rat-specific ELISA kits for IL-1β, TNF-α, IL-6, TRAR 5b, RANKL, and OPG.

[0056] (5) After fixing, dehydrating, embedding and sectioning the joint tissue of CIA rats, the expression levels of M1 macrophage marker CD86 and M2 macrophage marker CD206 in the synovial tissue were detected by immunofluorescence to evaluate the regulatory effect of nanomicelles on macrophage polarization.

[0057] Conclusion: From Figure 6It was found that all micelle formulations showed fluorescence signals of varying intensities at the arthritis lesion sites in CIA rats after injection, while the blank control group and the free DiR group showed almost no specific fluorescence enrichment at the joint sites. Among them, the fluorescence signal at the joint sites of the FA-PAMAM-DiR group was significantly stronger than that of the free DiR group and the unmodified FA PAMAM-DiR group, confirming that the active targeting and enrichment of micelles to the lesion site can be achieved through the specific binding of folic acid FA to the highly expressed folic acid receptor on the surface of activated macrophages at the inflamed joint. Meanwhile, the LD-PAMAM-DiR group exhibited the best arthritis targeting effect: 72 hours after injection, the fluorescence signal intensity at the joint sites was significantly higher than that of all other experimental groups, and this fluorescence signal continued to enhance and remain at the joint sites for a longer period. This confirms that the nanomicelles constructed in this study can efficiently deliver drugs, achieving excellent targeting performance through PEGylation modification to prolong the retention time in the blood, MMP responsiveness, and FA-mediated active targeting.

[0058] Establish such as Figure 7 The CIA rat model and dosing timeline shown in Figure A. Body weight and arthritis scores were measured every three days during treatment with different formulations. Results are as follows: Figure 7 As shown in B and C, body weight increased normally, and arthritis scores showed a decreasing trend, with the most significant decrease observed in the LD-PAMAM-TP&CAL group. (CIA rats after drug administration) Figure 7 As shown in Figure D, compared with the control group, the model group showed significantly more severe redness, swelling, and deformation of the joints. After treatment with different micelles, the swelling of the joints in each group was relieved, showing a certain therapeutic effect. After treatment with LD-PAMAM-TP&CAL, there was almost no redness or swelling, approaching the normal group. Figure 7 As shown in Figure E, LD-PAMAM-TP&CAL micelles significantly reduced joint bone erosion and provided good protection for cartilage. Figure 7 As shown in Figure F, LD-PAMAM-TP&CAL showed the most significant therapeutic effect, with only a small amount of scattered inflammatory cell infiltration, similar to the normal control group. Synovial hyperplasia and pannus formation were effectively inhibited. This indicates that LD-PAMAM-TP&CAL significantly improves joint inflammation and cartilage erosion.

[0059] To further investigate the therapeutic mechanism of the formulation in RA, immunofluorescence staining was performed, such as... Figure 8 As shown in Figure I, the expression of M1 macrophage markers (CD86) and M2 macrophage markers (CD206) in the synovial tissue of the rat knee joint was detected. The results are as follows: Figure 8As shown in G and H, compared with the model group, all micelles reduced the expression level of CD86 in cartilage and synovium (P<0.01) and increased the expression level of CD206 (P<0.01). The LD-PAMAM-TP&CAL group showed the most significant reduction in CD86 and increase in CD206 (P<0.01), indicating that LD-PAMAM-TP&CAL can regulate macrophage polarization, inhibit M1 polarization, and promote M2 polarization. Inflammatory factors in rat serum, such as..., were analyzed by ELISA. Figure 8 As shown in Figures A through F, compared with the TP+CAL and LD-PAMAM-TP groups, the LD-PAMAM-TP&CAL and FA-PAMAM-TP&CAL groups significantly reduced the levels of inflammatory factors TNF-α, IL-1β, IL-6, and bone injury factors TRAR 5b and RANKL, and increased the expression level of osteoprotegerin OPG (P < 0.01), thereby alleviating joint inflammation and protecting cartilage tissue.

[0060] Performance Test 7: Safety Evaluation: After the last administration, the rats in each group were euthanized, and their hearts, livers, spleens, lungs, and kidneys were collected. The tissue samples were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, sectioned, stained with H&E, and the in vivo safety of micelles was evaluated by detecting alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the blood using the rate method. The results are as follows Figure 9 As shown in Figure A, the liver in the free drug group showed focal inflammatory infiltration, while the major organs in the drug-loaded micelle group showed no obvious pathological damage; serum ALT and AST levels were as follows. Figure 9 Figures B and C show that, compared with the normal group, the free drug group had significantly higher ALT and AST levels (P<0.01). There were no significant differences in the indicators of each micelle group compared with the normal group, indicating that micelles can reduce drug accumulation in normal organs and reduce liver toxicity.

[0061] In summary, the nanomicelles prepared in this study achieve the goal of highly efficient and low-toxicity treatment of RA through innovative carrier structure (lock-release switching PAMAM), TP&CAL drug synergy, and targeted intelligent drug release (MMP enzyme response + dual targeting), demonstrating excellent practical value.

[0062] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a lock-and-release switching type of triptolide nanomicelles, characterized in that: Includes the following steps: S1: Esterification modification of polyamide-amine dendritic polymer with folic acid yields FA-PAMAM; S2: S2-1: Dissolve FA-PAMAM, triptolide, and verbascoside in a solvent, rotate to form a membrane, and obtain membrane A; add phosphate buffer solution, hydrate and incubate, filter, and obtain hydrated solution; S2-2: Dissolve functionalized polyethylene glycol in a solvent, spin-form a film, and obtain film B; Membrane B was hydrated with an aqueous solution and filtered to obtain triptolide nanomicelles.

2. The method for preparing a lock-and-release switching type triptolide nanomicelle according to claim 1, characterized in that: The preparation method of FA-PAMAM is as follows: S1-1: Folic acid is dissolved in dimethyl sulfoxide, dicyclohexylcarbodiimide and N-hydroxysuccinimide are added, the reaction is carried out, filtered and freeze-dried to obtain folic acid activated ester; S1-2: Polyamide-amine dendritic polymer is dissolved in dimethyl sulfoxide, folic acid activated ester is added, the reaction is stirred, dialyzed and dried to obtain FA-PAMAM.

3. The method for preparing a lock-and-release switching type triptolide nanomicelle according to claim 1, characterized in that: The FA-PAMAM comprises the following raw materials, by mass: 1-2 parts folic acid, 0.5-1 parts dicyclohexylcarbodiimide, 0.3-0.8 parts N-hydroxysuccinimide, and 10-15 parts polyamide-amine dendrimer.

4. The method for preparing a lock-and-release switching type triptolide nanomicelle according to claim 1, characterized in that: The process parameters for the hydration incubation are: hydration incubation at 50~60℃ for 10~20 min.

5. The method for preparing a lock-and-release switching type triptolide nanomicelle according to claim 1, characterized in that: The triptolide nanomicelles comprise the following raw materials, by mass: 25-30 parts FA-PAMAM, 0.01-0.015 parts triptolide, 25-28 parts vernix isoflavone, and 20-25 parts functionalized polyethylene glycol dissolved in methanol.

6. The method for preparing a lock-and-release switching type triptolide nanomicelle according to claim 1, characterized in that: The functionalized polyethylene glycol is an MMP enzyme-sensitive PEG-removable type, including EEEPVGLIG-PEG2000-GILGVPEEE.

7. The method for preparing a lock-and-release switching type triptolide nanomicelle according to claim 1, characterized in that: The FA-PAMAM was further modified with methoxy polyethylene glycol succinimide to obtain modified FA-PAMAM, which was then used to prepare triptolide nanomicelles. The preparation process of the modified FA-PAMAM is as follows: FA-PAMAM is added to phosphate buffer, methoxy polyethylene glycol succinimide is added, and under nitrogen protection, the mixture is stirred, dialyzed, and lyophilized to obtain modified FA-PAMAM.

8. The method for preparing a lock-and-release switching type triptolide nanomicelle according to claim 7, characterized in that: The modified FA-PAMAM comprises the following raw materials, by mass parts: 0.1~0.12 parts FA-PAMAM, 10~15 parts phosphate buffer, and 0.3~0.5 parts methoxy polyethylene glycol succinimide ester.

9. The triptolide nanomicelles prepared by the method for preparing lock-and-release switching type according to any one of claims 1 to 7; characterized in that: The triptolide nanomicelles are targeted and can achieve lock-out / release switching.

10. An application of a lock-and-release switching type of triptolide nanomicelles, characterized in that: The triptolide nanomicelles described in claim 9 can be used in the preparation of a medicament for treating rheumatoid arthritis.

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