Temperature response controlled release system based on base-like pairing rule and application of temperature response controlled release system in preparation of rheumatoid arthritis treatment medicine

Abstract

The invention discloses a methotrexate temperature response controlled release system based on a base-like pairing rule as well as a construction method and application of the methotrexate temperature response controlled release system. According to the controlled release system, thymine-1-acetic acid modified polydopamine coated magnetic ferroferric oxide nano-particles are used as a nano-carrier, and hydrogen bond combination is formed by utilizing a 2, 4-diamino disc pyridine structure in MTX and TAA on the surface of FPT NPs through a base-like pairing rule, so that the nano-composite drug (FPT-MTX NPs) is constructed. According to the FPT-MTX NPs, the excellent paramagnetism of the Fe3O4NPs is utilized, and the MTX can be accurately delivered to a targeted joint part under the action of an external magnetic field. The MTX loading capacity of the FPT NPs reaches up to 0.42 mg mg <-1 >, and the photothermal conversion efficiency (eta) of the FPT-MTX NPs reaches 55.04%. Under the condition of 60 DEG C, the release rate of MTX can reach 77.7%.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and particularly relates to a temperature-responsive controlled release system based on a base pair-like pairing rule and its application in the preparation of drugs for treating rheumatoid arthritis. Background Art

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease, and its main clinical manifestations include joint pain, swelling, morning stiffness and fatigue, resulting in limited motor function of patients. The histopathological features of RA include macrophage and T cell infiltration, hyperplasia of the synovial lining layer, pannus formation, and cartilage and bone destruction. These pathological changes may ultimately lead to joint deformity and loss of motor function, severely reducing the quality of life of patients. Therefore, early detection, accurate diagnosis and timely treatment are crucial for RA patients.

[0003] At present, the treatment methods of RA mainly include physical therapy, drug therapy and surgical therapy, among which drug therapy plays a dominant role. Commonly used drugs include glucocorticoids, non-steroidal anti-inflammatory drugs (NSAIDs), disease-modifying antirheumatic drugs (DMARDs) and new biological agents, etc. These drugs mainly relieve symptoms by immunosuppression or targeted inhibition of specific inflammatory mediators. However, due to the high cost of new biological agents and the serious side effects of glucocorticoids, DMARDs have become the most widely used treatment regimen for RA. Among them, MTX is widely used for the single treatment of RA or combined treatment with biological agents because of its low price, wide treatment range and significant curative effect.

[0004] MTX inhibits the excessive immune response of RA patients through anti-inflammatory, anti-angiogenic and analgesic effects, and effectively delays joint destruction. However, MTX has the defects of hemolytic properties and insufficient targeting ability. Long-term administration may cause serious gastrointestinal reactions, liver and kidney damage and other toxic side effects, which have attracted much attention in the academic community and clinical applications. During the course of RA, the main characteristics of the microenvironment are enhanced angiogenesis and increased vascular permeability. Based on this, the MTX delivery system aims to utilize these unique pathological features to improve the therapeutic effect of MTX. Hydrophilic nanocarriers have a longer circulation time in the blood and can be enriched at the lesion site through the enhanced vascular permeability at the inflammatory site and the uptake of inflammatory cells, similar to the enhanced permeability and retention effect in tumor tissues. However, the efficacy of passive targeting in MTX delivery is still limited. Summary of the Invention

[0005] The present invention provides a temperature-responsive controlled release system based on base-pairing rules and its application in the treatment of RA, which can solve the problem of significant toxicity of MTX during the treatment of RA. We prepared a nanocarrier (FPT NPs) by coating Fe3O4 NPs with PDA modified with TAA. Through base-pairing rules, 2,4-DAPT in MTX binds to TAA on the surface of FPT NPs through hydrogen bonds to form a nano-composite drug (FPT-MTX NPs). Under the action of an external magnetic field, FPT-MTX NPs can be targeted and enriched in the joint cavity. Under near-infrared light irradiation, PDA and Fe3O4 NPs act synergistically to increase the local temperature, resulting in the breakage of the hydrogen bond between MTX and TAA and the release of MTX. At the same time, photothermal therapy is achieved by heating up, so as to achieve the synergistic effect of chemotherapy and photothermal therapy. The loading capacity of FPT NPs for MTX is as high as 0.42 mg mg-1, and the photothermal conversion efficiency (η) of FPT-MTX NPs reaches 55.04%. At 60 °C, the release rate of MTX can reach 77.7%. The present invention loads drugs through hydrogen bonds based on base-pairing rules, with simple assembly and controllable drug release.

[0006] To solve the technical problems of the present invention, the technical solution proposed is: an MTX temperature-responsive controlled release system based on base-pairing rules, the controlled release system comprising: a nanocarrier FPT NPs composed of magnetic iron oxide nanoparticles Fe3O4 NPs coated with polydopamine PDA modified with thymine-1-acetic acid TAA, and methotrexate MTX bound to TAA on the surface of FPT NPs through base complementary pairing principles; wherein, TAA on the surface of FPT NPs binds to 2,4-diaminopteridine 2,4-DAPT in MTX through hydrogen bonds to form a nano-composite drug FPT-MTX NPs.

[0007] Preferably, the controlled release system uses PDA modified with TAA coated on Fe3O4 NPs as the nanocarrier FPT NPs. Through base-pairing rules, hydrogen bonds are formed by using the 2,4-DAPT structure in MTX and TAA on the surface of FPT NPs, thereby constructing the nano-composite drug FPT-MTX NPs.

[0008] Preferably, the particle size range of the FPT-MTX NPs is 200-300 nm.

[0009] To solve the technical problems of the present invention, another technical solution proposed is: the preparation method of FPT-MTX NPs includes the following steps:

[0010] (1) Preparation of Fe3O4 NPs: FeCl3·6H2O, 1,6 - hexanediamine, and anhydrous sodium acetate were stirred and dissolved in ethylene glycol, then placed in a reaction kettle and heated for reaction for 6 h. The precipitate was collected by magnetic absorption, washed with distilled water and ethanol to obtain Fe3O4 NPs;

[0011] (2) Preparation of PDA - modified FP NPs: Fe3O4 NPs were dispersed in an alkaline aqueous solution and surface - modified with polydopamine PDA under mechanical stirring to obtain FP NPs;

[0012] (3) Preparation of FPT NPs: Thymine - 1 - acetic acid TAA and FP NPs were dispersed in an acetonitrile solution containing 1 - (3 - dimethylaminopropyl) - 3 - ethylcarbodiimide (N - (3 - dimethylaminopropyl) - N'-ethyl - carbodiimide, EDCI) and 1 - hydroxybenzotriazole (1 - Hydroxybenzotriazole, HOBT), stirred at room temperature, and the precipitate was collected by magnetic absorption. After washing with distilled water and ethanol, FPT NPs were obtained;

[0013] (4) Preparation of FPT - MTX NPs: FPT NPs were dispersed in an aqueous solution containing MTX, stirred at room temperature, and the FPT - MTX NPs were collected by magnetic absorption.

[0014] Preferably, in step (1), after the reactants were stirred and dissolved, the supernatant was taken and placed in the reaction kettle. First, it was heated to 160 °C and stabilized for 10 min, then heated to 200 °C for reaction for 6 h, and the obtained product was treated by magnetic absorption.

[0015] Preferably, in the process of preparing the carrier FP NPs in step (2), the mass concentration of Fe3O4 NPs in the aqueous solution is 0.3 mg / mL -1 . In the preparation method of the controlled - release system, in step (3), the mass ratio of FP NPs to TAA is 1:1.

[0016] Preferably, in step (4), the mass ratio of FPT NPs to MTX is 2:1.

[0017] Another technical solution proposed to solve the technical problem of the present invention is: the application of the temperature - responsive controlled - release system based on the base - pair - like pairing rule in the preparation of drugs for rheumatoid arthritis.

[0018] Preferably, the application of the temperature - responsive controlled - release system based on the base - pair - like pairing rule in the preparation of drugs for photothermal therapy.

[0019] Beneficial effects:

[0020] The present invention discloses a temperature-responsive controlled release system of methotrexate (MTX) based on base-pairing-like rules, and its construction method and application. The controlled release system uses magnetic iron oxide nanoparticles (Fe3O4 NPs) coated with polydopamine (PDA) modified with thymine-1-acetic acid (TAA) as a nanocarrier (Fe3O4@PDA@TAANPs, FPT NPs). Through base-pairing-like rules, the 2,4-diaminopteridine (2,4-DAPT) structure in MTX forms a hydrogen bond with TAA on the surface of FPT NPs, thereby constructing a nano-composite drug (FPT-MTX NPs). FPT-MTX NPs utilize the excellent paramagnetism of Fe3O4 NPs. Under the action of an external magnetic field, MTX can be accurately delivered to the targeted joint site. In addition, the abundant amino and hydroxyl groups on the surface of PDA can effectively scavenge reactive oxygen species (ROS) in the joint cavity, and cooperate with MTX to significantly improve the therapeutic effect. In the present invention, the loading capacity of MTX on FPT NPs is as high as 0.42 mg mg-1, and the photothermal conversion efficiency (η) of FPT-MTX NPs reaches 55.04%. Under the condition of 60 °C, the release rate of MTX can reach 77.7%. The present invention loads drugs through hydrogen bonds based on base-pairing-like rules, with simple assembly and controllable drug release.

[0021] Fe3O4 NPs is a magnetic material with good biocompatibility. It not only has the general advantages of magnetic nanoparticles, such as small volume, large specific surface area, low toxicity, low cost and high paramagnetism, but also can achieve precise enrichment and efficient penetration at the targeted site under the guidance of an external magnetic field, and is easy to assemble with other drug molecules. Fe3O4 NPs can achieve photothermal conversion under near-infrared light irradiation, thereby increasing the local temperature and realizing the synergistic treatment of hyperthermia and MTX, further improving the therapeutic effect of RA.

[0022] Based on the principle of base pairing, we designed a temperature-responsive MTX delivery system for the treatment of RA in the present invention.

[0023] Fe3O4 NPs have excellent paramagnetism and can achieve precise enrichment and efficient penetration of the targeted site under the action of an external magnetic field. By assembling Fe3O4 NPs with PDA and MTX, FPT-MTX NPs can deliver MTX to the joint site under the guidance of an external magnetic field. After reaching the targeted site, irradiating the joint area with excitation light of a specific wavelength, the photothermal conversion effect of PDA can increase the local temperature, resulting in the breaking of hydrogen bonds and the release of MTX. In addition, the abundant amino and hydroxyl groups on the surface of PDA can effectively scavenge reactive oxygen species (ROS) in the joint cavity and act synergistically with MTX to significantly improve the therapeutic effect.

[0024] The FPT-MTX NPs in the present invention have good photothermal conversion ability and can provide sufficient energy for hydrogen bond breaking.

[0025] The present invention has good magnetic targeting ability and can effectively reach the targeted site under the condition of an external magnetic field, and release MTX by heating under the irradiation of a near-infrared laser, reducing the toxic and side effects of MTX and enhancing its targeting specificity to the RA inflammatory site.

[0026] The temperature-responsive controlled release system based on the base-pairing rule of the present invention has a better targeting effect on the joint site than MTX used alone.

[0027] The temperature-responsive controlled release system based on the base-pairing rule of the present invention has a better therapeutic effect on RA than the single MTX drug treatment. Description of the Drawings

[0028] Figure 1 It is the temperature-responsive controlled release system based on the base-pairing rule of the present invention.

[0029] Figure 2 It is the transmission electron microscope image of FPT-MTX NPs.

[0030] Figure 3 It is the DLS column chart of Fe3O4 NPs and FPT-MTX NPs.

[0031] Figure 4 It is the ultraviolet absorption graph of FPT-MTX NPs.

[0032] Figure 5 It is the XRD detection graph of Fe3O4 NPs and FPT-MTX NPs.

[0033] Figure 6 It is the magnetic detection graph of FPT-MTX NPs, (A) before magnetic attraction; (B) after magnetic attraction.

[0034] Figure 7MTX release behavior of FPT-MTX NPs at different temperatures.

[0035] Figure 8 For the study of the photothermal conversion performance of FPT-MTX NPs, (A) The thermal heating of FPT-MTX NPs (0.5 mg mL-1) under different light intensities; (B) After removing the light source, the natural cooling curve of the FPT-MTX NPs aqueous solution was linearly fitted with the logarithmic cooling; (C) Five heating-cooling thermal cycle experiments.

[0036] Figure 9 For the photothermal effect imaging map of FPT-MTX NPs after tail vein injection under 808 nm excitation light irradiation targeting the ankle joint cavity of CIA model mice.

[0037] Figure 10 Quantification map of the expression levels of inflammatory factors in lipopolysaccharide (LPS)-stimulated RAW264.7 cells treated with different drugs (Control, LPS, MTX, FPT NPs, FPT-MTX NPs), (A) TNF-α; (B) IL-1β; (C) IL-6; (D) IL-10

[0038] Figure 11 For the cumulative and fluorescence intensity changes of Cy5-NHS-labeled FPT-MTX NPs in the ankle joints of CIA model mice over time after tail vein injection of FPT-MTX NPs under the conditions of presence and absence of an external magnetic field.

[0039] Figure 12 For the determination of the activity of RAW 264.7 cells using the CCK-8 kit for FPT-MTX NPs and other drug treatment groups (Control, MTX, FPT-MTX NPs, FPT-MTX NPs + 808 nm laser irradiation).

[0040] Figure 13 For the treatment effect diagrams of FPT-MTX NPs and other drug treatment groups (Control, CIA models, MTX, FPT-MTX NPs, FPT-MTX NPs + 808 nm laser irradiation) in mice, (A) Changes in the swelling degree and (B) Difference analysis of the swelling degree of the feet of CIA model mice; (C) Score and (D) Difference analysis of the score of the feet of CIA model mice.

[0041] Figure 14After treatment with different drugs, the physical picture (A), CT imaging picture (B) of the foot joints of CIA model mice after 45 days of treatment, and the staining of foot joint sections (C).

[0042] Figure 15 (A) Immunochemical staining map of the foot joints of CIA model mice and quantitative analysis of the expression levels of immune factors after treatment with different drugs; (B) TNF-α, (C) IL-1β, (D) IL-6, (E) IL-10. Detailed implementation mode

[0043] The following combines examples and specific situations to elaborate in detail on the specific implementation mode of the present invention, but it does not limit the protection scope of the present invention. Unless otherwise specified, all reagents and equipment are commercially available.

[0044] Example 1

[0045] MTX is the abbreviation of methotrexate

[0046] Tris is the abbreviation of tris(hydroxymethyl)aminomethane

[0047] DA·HCl is the abbreviation of dopamine hydrochloride

[0048] TAA is the abbreviation of thymine-1-acetic acid

[0049] EDCI is the abbreviation of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0050] HOBT is the abbreviation of 1-hydroxybenzotriazole

[0051] ACN is the abbreviation of acetonitrile

[0052] According to Figure 1 As shown, a preparation method of a temperature-responsive controlled release system based on base pairing rules includes the following steps:

[0053] The first step: Preparation of Fe3O4 NPs: Dissolve FeCl3·6H2O (1.0 g), 1,6-hexanediamine (2.1 g) and anhydrous sodium acetate (2.0 g) in 50 mL of ethylene glycol, and then transfer the supernatant to a reaction kettle. After stabilizing the reaction kettle at 160 °C for 10 min, raise the temperature to 200 °C and allow the mixture to react for 6 h. After the reaction kettle cools to room temperature, separate the crude Fe3O4 NPs from the obtained mixture by magnetic attraction. Then wash the Fe3O4 NPs twice with distilled water and anhydrous ethanol respectively.

[0054] Step 2: Preparation of FP NPs: Redisperse Fe3O4 NPs (15 mg) in distilled water (50 mL). Add Tris (30 mg) and stir until completely dissolved, then add isopropanol (3 mL). After the mixture is stabilized for 10 min, add DA·HCl (50 mg). Stir the resulting system mechanically at 900 rpm at room temperature for 2 h. After the reaction, separate the FP NPs from the resulting mixture by magnetic separation, and then wash the FP NPs twice with distilled water and absolute ethanol respectively.

[0055] Step 3: Preparation of FPT NPs: Dissolve TAA (10 mg) and EDCI (10 mg) in ACN (10 mL), and continuously stir the mixture for 30 min for activation. Subsequently, add HOBT (5 mg) and FP NPs (10 mg) in sequence, and allow the reaction to proceed at room temperature for 12 h. After the reaction, separate the FPT NPs from the resulting mixture by magnetic separation, and then wash the FPT NPs twice with distilled water and absolute ethanol respectively.

[0056] Step 4: Preparation of FPT-MTX NPs: Add MTX (4 mg mL-1, 250 μL) to the dispersed FPT NPs system (0.4 mg mL-1, 5 mL). Then stir the mixture at 11000 rpm at room temperature for 12 h. Separate the FPT-MTX NPs from the resulting mixture by magnetic separation.

[0057] Figure 2 After the FPT-MTX NPs are thoroughly washed with pure water and absolute ethanol, they are ultrasonically dispersed in ethanol, dropped on a copper grid, and observed by transmission electron microscopy (TEM, JEOL JEM 2100F, JPN). As Figure 2 shown, we observed that both the appearance morphologies of Fe3O4 NPs and FPT-MTX NPs are regular spherical and the sizes are uniform, and a good coating structure is formed on the outer layer. In addition, all the nanoparticles maintain a good dispersion state without obvious accumulation.

[0058] Figure 3 Take an appropriate amount of Fe3O4 NPs and ultrasonically disperse them sufficiently in pure water to form a suspension. Take 1 mL and place it in a cuvette (the height should not exceed 2 / 3 of the cuvette). After wiping the cuvette clean, place it in the measurement chamber. Face the side with a triangle on the cuvette towards yourself, cover the machine cover and detect the particle size distribution of Fe3O4 NPs. The method for detecting the particle size distribution of FPT-MTX NPs is the same as that of Fe3O4 NPs. According to DLS analysis, the average particle size of the synthesized Fe3O4 NPs is about 169.9 ± 25.0 nm, and the average particle size of the synthesized FPT-MTX NPs is about 197.6 ± 25.0 nm.

[0059] Figure 4 Prepare aqueous solutions of Fe3O4 NPs (0.095 mg / mL), PDA (0.120 mg / mL), TAA (0.135 mg / mL), FPT-MTX NPs (0.500 mg / mL) and a DMSO solution of MTX (0.150 mg / mL). Take 200 μL of each of the above and add it to 3 mL of pure water, and measure the ultraviolet absorbance at 250 - 500 nm using an ultraviolet spectrophotometer. As Figure 4 shown, FPT-MTX NPs have characteristic absorption peaks corresponding to each monomer.

[0060] Figure 5 After completely drying the Fe3O4 NPs and FPT-MTX samples at 60 °C, grind them into powder. Avoid sample contamination during the grinding process and try to make the particle size reach the micron level. Spread the ground sample evenly on the sample stage, and gently press it with another piece of glass to form a flat surface for the sample. Fix the sample so that the sample is perpendicular to the incident direction of the X-ray. The commonly used X-ray wavelength is Cu Kα (λ = 0.15418 nm). As Figure 5 shown, the XRD peaks of FPT-MTX NPs have a strong correspondence with the characteristic peaks of the inverse spinel structure (JCPDS 19 - 629), indicating that the crystal structure of Fe3O4 NPs is still retained after surface modification with PDA, TAA, and MTX.

[0061] Figure 6 Take an appropriate amount of FPT-MTX NPs and place them in absolute ethanol. After fully ultrasonic dispersion, place them on a flat table and let them stand under the condition of an external magnet. Take a photo record after the magnet completely adsorbs the material to observe the magnetic adsorption performance of the material. As Figure 6 shown, in the presence of an external magnetic field, FPT-MTX NPs can be completely adsorbed in about 30 s, and it still retains good magnetism.

[0062] Figure 7 First, uniformly disperse FPT-MTX NPs (0.4 mg mL-1, 0.5 mL) in a 1.5 mL centrifuge tube, and then place it in a water bath set at different temperatures (37 °C, 45 °C, 55 °C and 60 °C) for heat treatment. At different time points (0, 1, 2, 3, 5, 7 and 10 h), collect the samples, perform magnetic adsorption, extract the supernatant, and measure its ultraviolet absorbance at 354 nm. Then calculate the MTX content in the supernatant according to the standard UV-Vis absorption curve of MTX. As Figure 7 shown, with the increase in temperature, the release rate of MTX on FPT-MTX NPs increases significantly, and the release rate is as high as 77.7% at 60 °C.

[0063] Figure 8 FPT-MTX NPs at 0.5 mg mL-1 were evenly dispersed in 1.5 mL centrifuge tubes, and the heating conditions under different excitation light intensities were analyzed. Subsequently, the thermal stability of FPT-MTX NPs was evaluated by conducting five consecutive cycles of heating and cooling experiments under 808 nm, 1 W cm-2 light illumination, and the photothermal conversion rate was calculated to be 55.04% through the cooling curve.

[0064] Figure 9 CIA model mice were randomly divided into two groups, including the Control group and the FPT-MTX NPs plus magnetic field treatment group. Six hours after tail vein injection of FPT-MTX NPs (50 μg), the ankle joints of the mice were irradiated with a laser (808 nm, 0.5 W cm-2), and the corresponding thermal imaging maps were recorded with an infrared thermal imager. As Figure 9 shown, the average temperature of the FPT-MTX NPs plus magnetic field treatment group increased by 14.80 ± 0.96 °C, while that of the Control group was only 3.23 ± 0.45 °C. The results indicated that FPT-MTX NPs could be effectively enriched at the targeted site under the condition of an external magnetic field.

[0065] Figure 10 The results of detecting the mRNA expression levels of cytokines by reverse transcription quantitative PCR (RT-qPCR) are presented. The specific steps are as follows: First, Raw 264.7 cells were lysed with TRIzol reagent (Invitrogen) to extract total RNA, and the quality and concentration of the RNA were evaluated using a Nanodrop spectrophotometer. Subsequently, cDNA was synthesized according to the instructions of the HiScript III RT SuperMix kit (Vazyme). Real-time PCR reactions were performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme) and an ABI 7500 sequence detection system (Applied Biosystems). In the experiment, Raw 264.7 macrophages were seeded in 6-well plates (5 × 106 cells / well) and activated with LPS (100 ng mL-1). Subsequently, different drugs (MTX, 23.66 ng; FPT NPs, 56.34 ng; FPT-MTX NPs, 80.00 ng) were added respectively, and incubation was continued for 6 h. Finally, the relative mRNA expression levels of TNF-α, IL-6, IL-1β, and IL-10 were calculated by the 2-ΔΔCt method, with β-actin as the internal reference gene. As Figure 10As shown, the MTX and FPT-MTX NPs treatment groups significantly reduced the expression of IL-6, IL-1β, and TNF-α in RAW264.7 cells after LPS stimulation, indicating that MTX and FPT-MTX NPs effectively inhibited the inflammatory process. Notably, the increase in IL-10 in the FPT-MTX NPs group was much higher than that in the potent MTX group, suggesting that FPT-MTX NPs alleviate the inflammatory response of rheumatoid arthritis by enhancing the function of M2 macrophages while inhibiting the activation of M1 macrophages, thereby relieving symptoms and preventing joint damage.

[0066] Figure 11 The experimental results of CIA model mice are shown. The mice were randomly divided into three groups: control group (Control), FPT-MTX NP treatment group, and FPT-MTX NP plus magnetic field treatment group. After intravenous injection of Cy-5-labeled FPT-MTX NP (50 μg), magnetic strips were fixed at the ankle joints of the mice. In vivo fluorescence imaging was performed at different time points (0.5, 1, 2, 4, and 6 h). The results showed that under the action of an external magnetic field, the retention of FPT-MTX NPs at the ankle joint site was significantly enhanced, thus greatly improving the therapeutic effect while reducing the toxic side effects of MTX.

[0067] Figure 12 The results of four groups of experiments are shown: control group (Control), MTX group, FPT-MTX NPs group, and FPT-MTX NPs + 808 nm light irradiation treatment group. The specific experimental procedures are as follows: Raw 264.7 cells were seeded in 96-well plates (2×104 cells / well) and cultured in a CO2 incubator (5% CO2, 37 °C) for 24 h. After removing the medium, fresh medium containing the drug was added and incubation was continued for 12 h. After light treatment, incubation was continued for 24 h. Subsequently, CCK-8 reagent was added and the cell viability was measured using a microplate reader. As Figure 12 shown, compared with MTX, FPT-MTX NPs showed lower cytotoxicity when the concentration increased. After irradiation with 808 nm laser, the cell killing ability of FPT-MTX NPs was significantly enhanced, which was attributed to the photothermal effect promoting the release of MTX, thereby restoring its activity.

[0068] Figure 13The CIA model mice were divided into five groups, including a control group (Control), a CIA model group, an MTX treatment group (8.0 μg), an FPT-MTX NPs treatment group (26.7 μg), and an FPT-MTX NPs + 808 nm light irradiation treatment group (26.7 μg, light conditions: 3 min, 0.5 W cm-2). All mice were injected with the corresponding drugs via the tail vein. After administration, the ankle joints of the mice were wrapped with magnetic strips for 6 h. During the treatment period, the body weight of the mice was monitored every two days, and the joints were scored and the degree of swelling was measured. Figure 13 As shown, in the MTX treatment group and the FPT-MTX NPs + 808 nm light irradiation treatment group, the swelling of the hind paw joints of the mice was significantly improved.

[0069] Figure 14 Pictures of the ankle joints of the mice, CT imaging, and the staining results of ankle joint sections after 45 days of treatment are shown. In the MTX treatment group and the FPT-MTX NPs + 808 nm light irradiation treatment group, the swelling of the hind paw joints of the mice was significantly improved. In addition, the CT imaging results showed that the degree of bone erosion in these two groups was reduced. Compared with the group injected with the same dose of free MTX, the group of mice injected with FPT-MTX NPs and treated with light showed better protection against ankle joint bone erosion. Next, the analysis of ankle joint tissue sections by hematoxylin-eosin staining (H&E), safranin O-fast green staining (SO-FG), and TRAP kit staining showed that a large amount of proteoglycan loss, synovial hyperplasia, and severe bone destruction occurred in the ankle joints of the CIA model group and the FPT-MTX NPs group of mice. Although the cartilage damage in the mice of the free MTX treatment group was alleviated, the inflammatory cell infiltration and synovial hyperplasia were still relatively severe. The mice injected with FPT-MTX NPs and then treated with light showed the best treatment effect among all the treatment groups.

[0070] Figure 15Representative immunochemical staining results of mouse joints, including TNF-α, IL-1β, IL-6, and IL-10, as well as quantitative analysis of TNF-α, IL-1β, IL-6, and IL-10 in the joint tissues of arthritic mice after treatment, are shown. The results of the study indicate that MTX and FPT-MTX NPs inhibited the expression of TNF-α, IL-1β, and IL-6, respectively. Meanwhile, we observed a significant increase in the expression of IL-10 after treatment with FPT-MTX NPs, suggesting that the nanodrug could effectively inhibit the inflammatory process. In the RA microenvironment, macrophages exhibit different polarization states, including M1 and M2 types, which have opposite roles in the immune response. M1 macrophages are pro-inflammatory cells that secrete cytokines such as TNF-α, IL-1β, and IL-6, exacerbating the inflammatory response; while M2 macrophages have an anti-inflammatory effect and secrete cytokines such as IL-10 that promote tissue repair. Our study shows that FPT-MTX NPs can alleviate the inflammation of rheumatoid arthritis by inhibiting the activation of M1 macrophages and enhancing the function of M2 macrophages, thereby relieving symptoms and preventing joint damage.

Claims

1. A MTX temperature-responsive controlled release system based on a pseudo-base pairing rule, characterized in that, The controlled release system includes: a nanocarrier FPT NPs composed of magnetic iron oxide nanoparticles Fe3O4 NPs coated with polydopamine PDA modified by thymine-1-acetic acid TAA, and methotrexate MTX that binds to TAA on the surface of FPT NPs through the principle of base-like complementary pairing; wherein, TAA on the surface of FPT NPs binds to 2,4-diaminopteridine 2,4-DAPT in MTX through hydrogen bonds to form a nano-composite drug FPT-MTX NPs.

2. The temperature-responsive controlled release system based on the base-pairing-like rule according to claim 1, characterized in that: This controlled release system uses PDA-coated Fe3O4 NPs modified with TAA as the nanocarrier FPT NPs. Through the base pairing rule, hydrogen bonds are formed between the 2,4-DAPT structure in MTX and TAA on the surface of FPT NPs, thereby constructing the nano-composite drug FPT-MTX NPs.

3. The temperature-responsive controlled release system based on the base-pairing-like rule according to claim 1, characterized in that, The particle size range of the FPT-MTX NPs is 200 - 300 nm.

4. The preparation method of the temperature-responsive controlled release system based on the base-pairing-like rule according to claim 1, wherein The preparation method of FPT-MTX NPs includes the following steps: (1) Preparation of Fe3O4 NPs: FeCl3·6H2O, 1,6-hexanediamine, and anhydrous sodium acetate are stirred and dissolved in ethylene glycol, then placed in a reaction kettle and heated to react for 6 h. The precipitate is collected by magnetic absorption, washed with distilled water and ethanol to obtain Fe3O4 NPs; (2) Preparation of FP NPs modified with PDA: Fe3O4 NPs are dispersed in an alkaline aqueous solution and surface-modified with polydopamine PDA under mechanical stirring to obtain FP NPs; (3) Preparation of FPT NPs: Thymine-1-acetic acid TAA and FP NPs are dispersed in an acetonitrile solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (N-(3-dimethylaminopropyl)-N'-ethyl-carbodiimide, EDCI) and 1-hydroxybenzotriazole (1-Hydroxybenzotriazole, HOBT), stirred at room temperature, the precipitate is collected by magnetic absorption, washed with distilled water and ethanol to obtain FPT NPs; (4) Preparation of FPT-MTX NPs: FPT NPs are dispersed in an aqueous solution containing MTX, stirred at room temperature, and the FPT-MTX NPs are collected by magnetic absorption.

5. The preparation method of the controlled release system according to claim 4, characterized in that, In the step (1), after the reactants are stirred and dissolved, the supernatant is taken and placed in a reaction kettle. First, it is heated to 160 °C and stabilized for 10 min, then heated to 200 °C and reacted for 6 h. The obtained product is subjected to magnetic absorption treatment.

6. The preparation method of the controlled release system according to claim 4, characterized in that, During the preparation of the carrier FPNPs in step (2), the mass concentration of Fe3O4 NPs in the aqueous solution is 0.3 mg / mL -1 .

7. The preparation method of the controlled release system according to claim 4, characterized in that, In the step (3), the mass ratio of FP NPs to TAA is 1:

1.

8. The preparation method of the controlled release system according to claim 4, characterized in that, In the step (4), the mass ratio of FPT NPs to MTX is 2:

1.

9. Use of the temperature-responsive controlled release system based on the base-like pairing rule according to claim 1 in the preparation of drugs for rheumatoid arthritis.

10. Use of the temperature-responsive controlled release system based on the base-like pairing rule according to claim 1 in the preparation of drugs for photothermal therapy.

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