Drug delivery system based on macrophage membrane coating porous carbon nanospheres, drug and preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to a drug delivery system based on macrophage membrane coating porous carbon nanospheres, a drug and a preparation method and application of the drug delivery system. The drug delivery system provided by the invention is an efficient and low-toxicity RA treatment scheme, and has a good clinical application prospect. Through the characteristics of precise targeted delivery and controlled release, the dosage of the medicine in the treatment process can be reduced, unnecessary waste of the medicine is reduced, and the cost benefit of treatment is improved. From the perspective of social benefits, RA is a common chronic disease in the global range, and the living quality of a patient is seriously influenced. The invention provides a safer and more effective treatment method, which is expected to improve the life quality of RA patients, reduce the side effects of medicines and harm to health and promote the progress of RA treatment, and has wide social demands and market potential.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a drug delivery system based on macrophage membrane-coated porous carbon nanospheres, a drug, and a preparation method and application thereof. Background Art

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune disease that primarily affects the joints, leading to persistent inflammation of the synovial tissue and, ultimately, joint destruction. Although existing disease-modifying antirheumatic drugs (DMARDs) (such as methotrexate (MTX), biologics, and Janus kinase (JAK) inhibitors) have been used to treat RA, clinical remission rates (i.e., the goal of preventing joint dysfunction) are only approximately 50%. This highlights the need to develop more effective treatment strategies. In this context, iguratimod (IGU), a cost-effective DMARD, has shown promising potential in reducing bone destruction and inflammation in RA. However, due to the poor hydrophobicity of IGU, its drug absorption and bioavailability are limited, resulting in delayed therapeutic effects. Furthermore, long-term use of IGU may lead to infections, liver damage, and gastrointestinal adverse reactions, further limiting its clinical application. To overcome these limitations, nanoparticle-based drug delivery systems have emerged as a promising strategy. These nanocarriers not only enable targeted drug delivery and controlled release but also possess intrinsic therapeutic potential, potentially enhancing the therapeutic efficacy of RA. Therefore, optimizing the interaction between IGU and nanocarriers, improving its bioavailability, enhancing its targeting to inflammatory sites, and achieving controlled release in the inflammatory environment of joints are key challenges. Exploring these synergistic effects is crucial for improving therapeutic efficacy and maintaining long-term inflammatory remission in RA.

[0003] The synovial microenvironment of inflamed RA joints is characterized by hypoxia, nutrient deficiency, acidic pH, elevated reactive oxygen species (ROS) levels, and an excess of multiple proinflammatory cytokines. This pathological environment not only exacerbates synovial tissue proliferation and invasion but also presents challenges for the design of drug delivery systems. However, it also provides potential targets and response conditions. For example, hypoxia and acidic environments can be used to develop pH- or hypoxia-responsive nanocarriers, while high levels of ROS can trigger the release of nanodrugs. However, dysregulation of the synovial microenvironment plays a central role in the progression of RA, rendering traditional anti-inflammatory therapies ineffective. Excessive accumulation of ROS leads to an imbalance in immune regulation, promoting the polarization of proinflammatory M1 macrophages and inhibiting the activation of anti-inflammatory M2 macrophages, resulting in persistent inflammation. Furthermore, the complex interplay between activated immune cells and proinflammatory cytokines (IL-1β, IL-6, and TNF-α) creates a highly interactive inflammatory network that makes synovial proliferation and bone erosion difficult to control. This inflammatory imbalance is also a major reason for the limited efficacy of DMARDs, with many patients experiencing disease relapses despite treatment. Restoring synovial immune homeostasis is key to achieving sustained remission in RA. Therefore, in addition to targeted delivery and environmental responsiveness, nanocarriers that can eliminate proinflammatory cytokines will help enhance the therapeutic effect of IGU and improve the clinical outcomes of RA patients.

[0004] Nanozymes, a class of nanomaterials with enzyme-like activity, have attracted widespread attention in the biomedical field due to their high stability, multifunctional catalytic activity, and cost-effectiveness. However, metal-based nanozymes suffer from poor biodegradability, potential toxicity from metal ion release, and long-term accumulation in the body. Therefore, it is necessary to find a method that can effectively control inflammatory levels in RA over the long term. Summary of the Invention

[0005] The purpose of the present invention is to provide a drug delivery system based on macrophage membrane-coated porous carbon nanospheres, a drug, a preparation method thereof, and an application thereof, in order to solve the problems existing in the above-mentioned prior art. The present invention provides a drug delivery system based on macrophage membrane-coated porous carbon nanospheres (MM@PCNSs), particularly for the targeted treatment of rheumatoid arthritis (RA). The drug delivery system combines the two technologies of macrophage membrane (MM) and porous carbon nanospheres (PCNSs), achieving precise targeting, dual-response controlled release, and effective inflammatory microenvironment regulation, and has significant therapeutic potential.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a drug delivery system based on macrophage membrane-coated porous carbon nanospheres (MM@PCNSs drug delivery system), wherein the drug delivery system comprises porous carbon nanospheres coated by macrophage membranes.

[0008] Disturbances in the synovial microenvironment homeostasis of rheumatoid arthritis are a key factor affecting the therapeutic efficacy of DMARDs in RA patients and post-treatment relapse. This invention aims to address the low efficacy of existing DMARDs in clinical treatment and maintain effective inflammation resolution in patients. By developing a drug delivery system based on macrophage membrane-encapsulated porous carbon nanospheres, this system achieves precise drug delivery and synergistic DMARD therapy with dual pH / photothermal responsiveness and pro-inflammatory microenvironment regulation, maintaining synovial microenvironment homeostasis and ultimately achieving long-term control of RA inflammation.

[0009] Porous carbon nanospheres (PCNSs) can mimic the activities of glutathione oxidase, catalase, and superoxide dismutase, while also exhibiting excellent biocompatibility and stability. By regulating ROS levels in the synovial microenvironment of RA, they restore redox balance and promote the transformation of M1 macrophages to M2 macrophages. Nanozymes can effectively alleviate inflammation and joint damage. In addition, the unique electronic properties and broad absorption spectrum of PCNSs make them photoresponsive, making them useful for controlled drug release. To further enhance their therapeutic effects, the present invention combines macrophage membranes (MMs) with porous carbon nanospheres (PCNSs). PCNSs can coat macrophage membranes (MMs). This modification not only improves biocompatibility and targeting of inflammatory sites, but also imparts cytokine neutralization capabilities. The receptors rich in MM surfaces can bind to and clear proinflammatory cytokines (such as IL-1β, IL-6, and TNF-α), effectively inhibiting inflammatory signal transduction and alleviating immune overactivation. This invention intends to provide a multifunctional therapeutic strategy by integrating targeted delivery, ROS scavenging and inflammation regulation technologies using MM-coated PCNSs, which is expected to restore the homeostasis of the synovial microenvironment.

[0010] Further preferably, the drug delivery system can target the treatment of rheumatoid arthritis and has a synergistic regulatory effect on the homeostasis of the synovial microenvironment.

[0011] The present invention provides a method for preparing the above-mentioned drug delivery system, which comprises the following steps:

[0012] The polyacrylic acid aqueous solution, zinc oxide and isopropyl alcohol were mixed to obtain PAA-ZnNPs;

[0013] performing a carbonization treatment on the PAA-ZnNPs to obtain PCNSs;

[0014] The PCNSs and macrophage membrane fragments are mixed evenly to obtain the drug delivery system.

[0015] Preferably, the volume ratio of the polyacrylic acid aqueous solution to isopropyl alcohol is 1:2; and the mass ratio of polyacrylic acid to zinc oxide in the polyacrylic acid aqueous solution is 0.36 mg:72 mg.

[0016] Preferably, the carbonization treatment condition parameters are: heating to 900° C. at a heating rate of 5° C. / min and calcining for 3 hours.

[0017] The present invention provides use of the above-mentioned drug delivery system in preparing a drug for treating rheumatoid arthritis.

[0018] Further preferably, the drug can target and treat rheumatoid arthritis and has a synergistic regulatory effect on the homeostasis of the synovial microenvironment.

[0019] The present invention provides a medicine for treating rheumatoid arthritis, which comprises the above-mentioned drug delivery system and an anti-rheumatic drug.

[0020] Preferably, the anti-rheumatic drug includes iguratimod.

[0021] Further preferably, the drug can target and treat rheumatoid arthritis and has a synergistic regulatory effect on the homeostasis of the synovial microenvironment.

[0022] The present invention provides a method for preparing the above-mentioned medicine, which comprises the following steps:

[0023] The solution containing the drug delivery system and the anti-rheumatic drug solution are mixed to obtain the drug.

[0024] Preferably, the mass ratio of the solution containing the drug delivery system and the anti-rheumatic drug solution is 1:1.

[0025] Preferably, the concentration of the drug delivery system in the solution containing the drug delivery system is 1 mg / mL; the concentration of the anti-rheumatic drug in the anti-rheumatic drug solution is 0.455 mg / mL.

[0026] The present invention discloses the following technical effects:

[0027] The drug delivery system (MM@PCNSs) provided by the present invention combines macrophage membranes (MM) and porous carbon nanospheres (PCNSs), significantly improving the targeted therapeutic effect of rheumatoid arthritis (RA) and has the following technical effects:

[0028] (1) Precisely targeted drug delivery to the site of inflammation

[0029] The MM@PCNSs drug delivery system provided by this invention leverages the targeting properties of the macrophage membrane to specifically identify and target macrophages within rheumatoid arthritis lesions. In vivo experiments demonstrated that this drug delivery system effectively delivered drugs to arthritis lesions in a CIA mouse model. Compared to traditional drug delivery systems, the system significantly increased local drug concentration and avoided side effects on healthy tissues.

[0030] (2) Dual-response release characteristics of drugs

[0031] The MM@PCNSs drug delivery system provided by the present invention can respond to local acidic environments and NIR light irradiation, achieving precise drug release under specific conditions. Experiments in a CIA mouse model showed that the local concentration of the drug at the site of inflammation increased significantly, while the drug concentration in healthy tissue remained at a low level, effectively reducing the drug's toxicity and side effects on normal tissues. Through a dual response mechanism triggered by pH and NIR light, drug release can be precisely controlled, ensuring that the drug is released only in the required area (i.e., the site of inflammation), thereby improving the therapeutic effect and reducing drug waste.

[0032] (3) Environmental regulation and anti-inflammatory effects

[0033] MM@PCNSs possess nanozyme-like catalytic activity, which can scavenge ROS produced in rheumatoid arthritis and neutralize proinflammatory cytokines. This property was verified by immunohistochemical analysis in a CIA mouse model. The results showed that the MM@PCNSs / IGU-treated group showed a significant reduction in inflammation, bone erosion, and cartilage damage in the CIA mouse model of arthritis. Compared with the control group, the expression of inflammatory factors was significantly reduced, further demonstrating the effectiveness of MM@PCNSs in modulating the inflammatory microenvironment.

[0034] (4) Significant therapeutic effect

[0035] In a CIA mouse model, the treatment group using MM@PCNSs to deliver the IGU drug demonstrated significant clinical efficacy. Compared to the control group, the treated mice experienced significant improvements in joint swelling, pain, and dysfunction. Micro-CT analysis revealed significant bone erosion and cartilage preservation in the treated mice, demonstrating that MM@PCNSs effectively slowed the progression of RA. Statistical analysis revealed that the treated mice maintained stable body weight, had reduced inflammation scores, and experienced rapid recovery of joint function. These data fully demonstrate the significant efficacy of the drug delivery system in the treatment of RA.

[0036] In summary, the drug delivery system provided by the present invention is an efficient and low-toxic RA treatment solution with good clinical application prospects. Through the characteristics of precise targeted delivery and controlled release, the present invention can reduce the amount of drugs used during treatment, reduce unnecessary waste of drugs, and improve the cost-effectiveness of treatment. From the perspective of social benefits, RA is a common chronic disease worldwide, and the quality of life of patients is seriously affected. By providing a safer and more effective treatment method, the present invention is expected to improve the quality of life of RA patients, reduce the side effects of drugs and health hazards, promote the progress of RA treatment, and has broad social needs and market potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0038] Figure 1 Flow chart for the preparation and treatment of MM@PCNSs / IGU;

[0039] Figure 2 Preparation and characterization of MM@PCNSs / IGU; a is the SEM image; b is the TEM image; c is the elemental mapping of PCNSs; d is the Raman spectrum of PCNSs; e is the XRD pattern; f is the gas adsorption-desorption isotherm test results; g is the pore size statistics; h is the zeta potential statistics;

[0040] Figure 3 is the controlled drug release of MM@PCNSs / IGU; wherein, a is the UV-Vis-NIR absorption spectrum detection result; b is the temperature change of PCNSs solution with different concentrations under NIR laser irradiation; c is the infrared thermal imaging diagram; d is the effect of laser power density on temperature; e is the photothermal durability investigation result of PCNSs; f is the photothermal conversion efficiency and thermal relaxation behavior of PCNSs reflected by the ΔT curve during laser irradiation and cooling; g is the photothermal conversion efficiency calculated by Ln(θ); h is the UV-visible absorption spectrum of IGU and IGU-loaded PCNS solution; i is the IGU release rate under different conditions;

[0041] Figure 4is the targeting of MM@PCNSs / IGU; among them, a is a fluorescence microscopy image showing the uptake of MM@PCNSs / Cy5.5 by non-activated and activated HUVECs; b is a fluorescence microscopy image showing the endocytosis of MM@PCNSs / Cy5.5 by non-activated and activated RAW264.7 cells; c is the in vivo fluorescence imaging of mice after injection of Cy5.5 and MM@PCNSs / Cy5.5 (24 hours); d is the fluorescence distribution in the heart, liver, spleen, lung, kidney and joint tissues of each group; e is the quantitative analysis of the mean fluorescence intensity (MFI) of each organ; f is a representative immunofluorescence image of MM@PCNSs / Cy5.5 localization in the inflamed synovium; g is a statistical graph of the quantitative analysis of the mean fluorescence intensity (MFI) of the inflamed synovium;

[0042] Figure 5 is the scavenging effect of MM@PCNSs / IGU on ROS and pro-inflammatory cytokines; a is the scavenging effect of different concentrations of PCNSs on superoxide anions (O2· - b is the relative quantitative analysis of the residual hydrogen peroxide (H2O2) after incubation with different concentrations of PCNSs; c is the UV-visible spectra of H2O2 neutralized by different concentrations of PCNSs; d is the electron spin resonance (ESR) spectrum confirming the scavenging effect of PCNSs on hydroxyl radicals (·OH); e is the flow cytometry detection of the level of reactive oxygen species (ROS) in macrophages; f is the Western Blot analysis shows the protein expression levels of IL-1R1, IL-6R-α, and TNFR2 in MM and MM@PCNSs; g is ELISA detection of cytokine secretion levels after treatment with different concentrations of PCNSs and MM@PCNSs; h is a schematic diagram of the experimental flow (produced by www.biorender.com), showing the experimental steps for detecting cytokines in cell culture supernatants and macrophages by ELISA and qPCR; ik is the concentration detection results of IL-1β (i), IL-6 (j), and TNF-α (k) in cell culture supernatants; ln is the relative expression levels of proinflammatory cytokines IL-1β (l), IL-6 (m), and TNF-α (n) mRNA in macrophages;

[0043] Figure 6 Figure 3 is the therapeutic effect of MM@PCNSs / IGU on CIA mouse model; a is the treatment plan of CIA mouse model; bd are the arthritis score and foot swelling thickness investigation results; e are the H&E, Masson and Safranin-O staining results; f is the bone erosion evaluation result; gk are the bone mineral density (BMD), bone volume fraction (BV / TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and trabecular spacing (Tb.Sp) evaluation results;

[0044] Figure 7 is the inhibitory effect of MM@PCNSs / IGU on the level of inflammation in vivo; where ac is the detection results of the levels of IL-1β, IL-6 and TNF-α in serum and joint tissue; df is the detection results of the mRNA expression levels of IL-1β, IL-6 and TNF-α in synovial tissue; g is the result of immunofluorescence staining;

[0045] Figure 8 is the biosafety of MM@PCNSs / IGU; where a is the mouse weight survey result; bg are the test results of blood routine indicators (RBC, WBC, PLT) and serum biochemical indicators (ALT, AST, BUN); h is the tissue section analysis result. DETAILED DESCRIPTION

[0046] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0047] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0048] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0049] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0050] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0051] Example 1

[0052] (1) Preparation and characterization of MM@PCNSs / IGU

[0053] The preparation process and treatment flow chart of MM@PCNSs / IGU are shown in the figure. Figure 1 shown.

[0054] 1.1) Preparation of MM@PCNSs: 1.8 mL of polyacrylic acid (PAA) with a molecular weight of 1800 was added to 300 mL of deionized water to obtain a 0.2 g / mL PAA aqueous solution. After stirring for 15 minutes, 72 mg of zinc oxide (ZnO) was added. The solution was shaken and sonicated continuously for 10 minutes until the solution clarified. 600 mL of isopropyl alcohol (IPA) was added dropwise to the mixture at a rate of 1 drop per second using a dropping funnel. The mixture was stirred magnetically overnight to form PAA-ZnNPs. 900 mL of the PAA-ZnNPs solution was centrifuged, washed twice with ethanol, and dried in an oven. The dried sample was then ground in a mortar and placed in a tube furnace. High-purity argon was introduced for 30 minutes. The temperature in the tube furnace was then increased to 900°C at a rate of 5°C / min and calcined at 900°C for 3 hours to obtain PCNSs. RAW 264.7 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) in an incubator at 37°C and 5% CO2. When the cell number was approximately 4 × 10 7 After washing with PBS, cells were scraped and collected by centrifugation at 800 g for 5 min. Macrophage membrane fragments (MM) were collected according to the instructions of the cell membrane protein extraction kit. MM protein mass was measured using BCA assay. MM was then resuspended in 1 mL of PBS and stored in liquid nitrogen. The frozen MM was removed from liquid nitrogen and rapidly thawed in a 37°C water bath. MM was mixed with PCNSs in a 1:1 mass ratio and sonicated in an ultrasonic water bath. The MM and PCNSs mixture was then passed through a 0.45 μm pore size filter using a liposome extruder, repeatedly pushed 15 times, and centrifuged at 700 g for 10 min in a room temperature centrifuge. The supernatant was removed to obtain MM@PCNSs. After resuspending in PBS, the mixture was stored in a refrigerator at 4°C.

[0055] 1.2) Preparation of MM@PCNSs / IGU: 1 mg of iguratimod (IGU) and 1 mg of PCNSs were mixed in 1 mL of ethanol and mixed on a vertical mixer for 24 h to obtain PCNSs / IGU. MM and PCNSs were mixed in a 1:1 mass ratio and sonicated in an ultrasonic water bath. The MM and PCNSs mixture was then passed through a 0.45 μm pore size filter membrane using a liposome extruder, repeatedly pushed 15 times, and centrifuged in a centrifuge at room temperature at 700 g for 10 min. The supernatant was removed to obtain MM@PCNSs / IGU.

[0056] 1.3) Characterization of MM@PCNSs: The preparation process of MM@PCNSs includes a unique PAA-Zn nanoparticle synthesis and a subsequent carbonization step to produce carbon nanozymes (PCNSs). In this process, PAA coordinates with metal ions to form well-dispersed, network-like, and uniformly sized PAA-Zn nanoparticles with a diameter of approximately 180±10 nm. Figure 2 a) and TEM( Figure 2 b) was observed and confirmed. After the dried PAA-Zn nanoparticles were calcined at high temperature, PCNSs ( Figure 2 During the calcination process, PAA was converted into a carbon skeleton, while the evaporation of Zn formed a mesoporous structure in the carbon structure. Elemental mapping of PCNSs ( Figure 2 Figure c) shows that carbon (C) is evenly distributed, while zinc (Zn) is basically invisible, further verifying the successful synthesis of PCNSs. In order to further verify the synthesis effect, the present invention uses Raman spectroscopy and X-ray diffraction (XRD) analysis. Figure 2 As shown in (d), typical D bands and G bands appeared in the Raman spectrum of PCNSs, indicating the presence of carbon structures. Figure 2 The XRD pattern shown in Figure e shows diffraction peaks at (100) and (002), corresponding to the crystal plane of elemental carbon. Nitrogen adsorption-desorption isotherm test ( Figure 2 f) is a type IV isotherm with a BET surface area of ​​561.1 m 2 / g, and Figure 2 The g in the graph shows that it has microporous (about 1.9 nm) and mesoporous (about 11 nm) structures. Figure 2 As shown in h, the zeta potential of PAA-Zn is approximately -9.7 mV, while that of PCNSs is -9.1 mV, indicating that they are prone to aggregation in solution due to insufficient electrostatic repulsion. MM was coated on the surface of PCNSs using conventional mechanical methods (ultrasound and extrusion techniques), thus forming MM@PCNSs. Figure 2The ab in Figure 3 shows that the surface of PCNSs is covered with a light gray membrane structure, indicating the formation of a typical "core-shell" structure. The coating of macrophage membrane significantly changes the surface properties of nanoparticles. Figure 2 As shown in (h), the zeta potential of MM@PCNSs increased to about -27.3 mV, which is much higher than that of unencapsulated PCNSs.

[0057] (2) Controlled drug release of MM@PCNSs / IGU

[0058] 2.1) PCNSs were tested by UV-Vis-NIR absorption spectroscopy. The results of UV-Vis-NIR absorption spectroscopy were ( Figure 3 (a) shows that PCNSs exhibit broad absorption in the NIR region, confirming their suitability for photothermal applications.

[0059] 2.2) To study its photothermal effect, PCNSs solutions with different concentrations (50, 100, 150 and 200 μg / mL) were irradiated with NIR laser and the temperature changes were monitored. Figure 3 As shown in Figure b, as the PCNS concentration increases, the heating rate and final temperature also increase accordingly, while the deionized water control group has almost no temperature change. Infrared thermal imaging further verifies this trend ( Figure 3 c) in the above example.

[0060] 2.3) In addition, laser power density plays a key role in heating efficiency. When 100 μg / mL PCNSs solution was heated at 0.7 W / cm 2 , 1W / cm 2 and 1.4W / cm 2 The irradiation was continued for 5 min at a power density of 100 nm and the temperature change of the PCNSs solution was recorded. Figure 3 As shown in (d), the temperature of the PCNSs solution increases proportionally with the increase of power density.

[0061] 2.4) To evaluate the photothermal stability of PCNSs, four cycles of NIR laser irradiation and natural cooling were performed on PCNSs with a concentration of 100 μg / mL. The specific experimental steps were as follows: First, 100 μg / mL PCNSs were irradiated at 808 nm and 1 W / cm 2 The laser was irradiated continuously for 10 minutes, then the laser power was turned off and the temperature was allowed to cool naturally for 10 minutes. This heating and cooling operation was repeated four times. The temperature was recorded every 30 seconds using an infrared thermal imager, and a cycle diagram was made based on the recorded temperature data to evaluate whether the photothermal performance was stable. The photothermal durability was then tested. The results are as follows: Figure 3As shown in e, after multiple heating-cooling cycles, the maximum temperature remains stable, indicating that PCNSs have excellent photothermal durability. In addition, the photothermal conversion efficiency (η) of PCNSs was calculated, and the results showed that its photothermal conversion efficiency is about 26% ( Figure 3 fg in ), indicating that it can efficiently convert NIR light energy into thermal energy, which is a key performance for achieving effective photothermal response.

[0062] 2.5) Drug loading and release experiments were conducted on MM@PCNSs. The specific experimental steps were as follows: the absorbance of different concentrations of IGU at 257 nm was measured to draw a standard curve. This experiment verified the therapeutic potential of MM@PCNSs. The results are shown in Figure 2. Figure 3 As shown in h, curve b is the UV-visible absorption spectrum of IGU, and curve a is the IGU-loaded PCNS solution (the preparation method of the IGU-loaded PCNS solution is to mix 1 mg IGU and 1 mg PCNSs in 1 mL ethanol and mix them on a vertical mixer for 24 hours. After centrifugation, the absorbance of the IGU-loaded PCNSs supernatant at 257 nm is measured), showing effective drug loading capacity, and its encapsulation efficiency is as high as 45.5%.

[0063] 2.6) The drug release behavior of MM@PCNSs / IGU was studied under different conditions. The specific experimental steps were as follows: First, in a water bath at 37°C, three 1 mg portions of IGU-loaded PCNSs were taken in parallel. One portion was dispersed in 1 mL of PBS solution at pH = 7.4, and the other two portions were dispersed in PBS solution at pH = 5.6. At regular time intervals, one portion of the pH = 5.6 nanoparticle solution was irradiated with an 808 nm laser (1 W / cm 2 ) for 5 minutes, then at specific time points, three portions of the nanoparticle solution were centrifuged and the absorbance of the supernatant was measured. After the test, the supernatant was returned to the centrifuge tube and the absorbance of the supernatant was measured again after a certain interval. Figure 3 As shown in the figure i. Under physiological conditions (pH 7.4), IGU showed an initial rapid release followed by a sustained release trend; however, under acidic conditions (pH 5.6) that simulate the inflammatory microenvironment, the release rate increased significantly, showing good pH responsiveness. In order to further improve the efficiency of local drug release, the effect of NIR laser irradiation on the release behavior was also tested, and the results are shown in Figure 1. Figure 3 The results show that when MM@PCNSs / IGU was irradiated with an 808nm laser, the IGU release rate was significantly faster than that of the unirradiated control group, both at pH 7.4 and pH 5.6. This confirms that the photothermal effect of PCNSs can be used to promote on-demand drug release, thereby improving the precision of treatment.

[0064] (3) Targeting of MM@PCNSs / IGU

[0065] 3.1) Cy5.5-labeled MM@PCNSs were co-incubated with TNF-α-activated human umbilical vein endothelial cells (HUVECs) and then observed by confocal fluorescence microscopy. The results showed that MM@PCNSs accumulated in large quantities around the inflammatory endothelial cells ( Figure 4 In the non-activated control group, there was almost no binding, highlighting the selective adhesion ability of MM@PCNSs to inflamed blood vessels. In addition to adhesion, MM@PCNSs also exhibited selective uptake by macrophages in the inflammatory microenvironment.

[0066] 3.1) Cy5.5-labeled MM@PCNSs were co-cultured with RAW264.7 macrophages. 2×10 5 RAW 264.7 cells were plated in a confocal culture dish, and after the cells adhered, a culture medium containing 1 μg / mL LPS was added and incubated for 24 hours. RAW264.7 incubated with normal culture medium served as the control group. Next, DID-labeled MM@PCNSs were added to each group and then incubated at 37°C for another 4 hours. One group was incubated for 4 hours and then laser irradiated for 10 minutes. The uptake capacity of RAW264.7 macrophages for Cy5.5-labeled MM@PCNSs was then investigated, and the results are shown in Figure 2. Figure 4 The results showed that the uptake of Cy5.5-labeled MM@PCNSs by LPS-activated RAW264.7 macrophages was significantly enhanced, while that by non-activated cells was less ( Figure 4 (b) in the figure indicates that inflammation enhances cellular uptake. Given that inflammation is often accompanied by a decrease in pH, this pH-responsive behavior promotes targeted drug release at the disease site. Furthermore, upon 808nm near-infrared (NIR) laser irradiation, the fluorescence signal in activated macrophages was further enhanced, confirming that MM@PCNSs can achieve dual pH / NIR stimulus-responsive drug release.

[0067] 3.3) In vivo fluorescence imaging

[0068] The CIA (collagen-induced arthritis) mouse model was constructed. The specific steps are as follows: female DBA / 1J mice aged 6-8 weeks were selected. First, complete Freund's adjuvant (CFA) and bovine CⅡ were mixed at a volume of 1:1 and stirred at high speed under a homogenizer until completely emulsified. Then, each mouse was injected intradermally with 100 μL of emulsion, and the injection site was 1.5 cm away from the base of the tail. On the 21st day after the first injection, incomplete Freund's adjuvant (IFA) and bovine CⅡ were mixed at a volume of 1:1 and completely emulsified according to the same steps as before for booster immunization. Subsequently, the joints of the mice became red and swollen. Each foot of the mouse was measured and scored, and the scoring criteria were as follows:

[0069] 0: normal without erythema or swelling;

[0070] 1: erythema or mild swelling limited to the tarsal or ankle joints;

[0071] 2: Erythema or mild swelling extending from the ankle to the tarsus;

[0072] 3: erythema or moderate swelling extending from the ankle to the metatarsal joints;

[0073] 4: Erythema or severe swelling surrounding the ankles, feet, and toes, or stiffness in the joints of the limbs.

[0074] Through the above steps and scoring, an RA mouse model can be established to study the pathogenesis of the disease and test the efficacy of new drugs. On the 60th day after immunization, when the average arthritis score of CIA mice reached 14 points, Cy5.5 and MM@PCNSs / Cy5.5 were injected into the tail vein, respectively, and then in vivo fluorescence imaging was performed to further verify the targeting efficiency of MM@PCNSs. The results are shown in Figure 2. Figure 4 As shown in Figure c. In the CIA (collagen-induced arthritis) mouse model, the fluorescence signal of MM@PCNSs / Cy5.5 at the inflamed joints was significantly stronger than that of the Cy5.5 free dye control group, indicating that MM coating significantly enhanced the retention of nanoparticles at the disease site. Subsequently, in vitro fluorescence imaging of the major organs and inflamed joints of the CIA mouse model was performed, and the results are shown in Figure 4. Figure 4 As shown in the figure, the results are consistent with the in vivo fluorescence imaging results, showing that MM@PCNSs preferentially accumulate in the inflamed synovium, while the non-specific distribution is significantly reduced. In addition, immunofluorescence staining of the inflammatory macrophage marker CD86 in the CIA mouse model further confirmed the selective localization of MM@PCNSs in the inflamed synovial tissue ( Figure 3fg in Figure 1). The Cy5.5 group (simulating free drug circulation in vivo) showed virtually no Cy5.5 signal, indicating that free drug molecules struggle to effectively reach and accumulate in inflamed synovial tissue. In contrast, MM@PCNSs exhibited significant accumulation, highlighting the targeting advantage of MM coating. Furthermore, colocalization of MM@PCNSs with CD86 suggests that the nanoparticles specifically bind to inflammatory macrophages, potentially achieving their therapeutic effects by regulating macrophages in the synovial microenvironment of RA.

[0075] (4) Clearance effect of MM@PCNSs / IGU on ROS and pro-inflammatory cytokines

[0076] 4.1) The present invention evaluated the ability of MM@PCNSs / IGU to scavenge ROS (including superoxide anions O2 - The specific experimental steps are as follows: scavenging ·OH: 1.8mM ferrous sulfate heptahydrate (FeSO4·7H2O) and 5mM hydrogen peroxide (H2O2) were added to 20mL deionized water and reacted at room temperature for 10min under magnetic stirring to obtain a light yellow solution. This is because the mixed solution undergoes a Fenton reaction in this process, Fe 2+ Reacting with H2O2, it produces highly toxic hydroxyl radicals (·OH). Next, 4.97 mg of salicylic acid (SA) was added to the mixed solution and stirred at room temperature for 30 minutes. The solution then changed from light yellow to purple-black, due to the reaction of ·OH with SA to form 2,3-dihydroxybenzoic acid. The absorbance of the mixed solution at a wavelength of 510 nm was measured using a UV-visible spectrophotometer. Next, various concentrations of PCNSs were added to the solution and stirred for 1 hour, resulting in final concentrations of (0 μg / mL, 3.125 μg / mL, 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, and 50 μg / mL). Different control groups were also prepared. 4.97 mg of SA was dissolved in 20 mL of deionized water. Three groups of 2 mL SA solutions were taken, and 1 μL of H2O2 was added to one group, and 1 mg of FeSO4·7H2O was added to the other group. The six groups of PCNSs solutions with different concentrations and the three control groups were centrifuged together. The absorbance of the supernatant at 510 nm was measured using a UV spectrophotometer. Finally, the absorbance values ​​of the supernatant were compared to evaluate the scavenging ability of PCNSs on hydroxyl radicals. - :Superoxide radicals (O2· -) The scavenging effect was confirmed by measuring the photoreduction inhibition rate of nitro blue tetrazolium chloride (NBT). In the experiment, riboflavin (20μM), methionine (13mM) and nitro blue tetrazolium chloride (75μM) were mixed in 20mL of deionized water. The product has a characteristic absorption peak at 560nm in the UV-visible absorption spectrum. Then different concentrations of PCNSs (0μg / mL, 3.125μg / mL, 6.25μg / mL, 12.5μg / mL, 25μg / mL and 50μg / mL) were mixed with the above solution, and the test samples were placed under a UV lamp for continuous illumination for 15 minutes. Finally, the test samples and the control group (no PCNSs were added and no UV light was applied) were centrifuged, and the absorbance of the supernatant of different samples was detected (the test process needs to be kept away from light). The effect of PCNSs on O2· was evaluated by comparing the changes in absorbance values. - Scavenging ability of H2O2. Scavenging H2O2: Since hydrogen peroxide (H2O2) has a characteristic absorption peak at 240nm in the UV-visible absorption spectrum, different concentrations of PCNSs (0μg / mL, 3.125μg / mL, 6.25μg / mL, 12.5μg / mL and 25μg / mL) were added to 40mM H2O2. The absorbance values ​​of the characteristic absorption peaks of H2O2 in the test samples with different concentrations were measured to detect the scavenging efficiency of PCNSs on H2O2. The results are shown in Figure 2. Figure 5 As shown in a, after adding PCNSs, O2· - The characteristic absorption peak of PCNSs decreased significantly, indicating that it has good scavenging ability, with a clearance rate of 66.3% at a concentration of 50 μg / mL. Similarly, PCNSs also showed significant scavenging effects on ·OH and H2O2, with clearance rates of 56.6% and 33.2%, respectively. Figure 5 b and c).

[0077] 4.2) The present invention further evaluated the ·OH scavenging ability of PCNSs using electron paramagnetic resonance (ESR) spectroscopy combined with spin trapping technology. The specific experimental steps were as follows: Measurements were performed on a Brucker ESR5000 ESR spectrometer under the following conditions: X-band, microwave power 20 mW, modulation frequency 100 kHz, amplitude modulation 0.1 mT, sweep width 20 mT, central magnetic field 324.5 mT, and detection at room temperature. ·OH was generated by the Fenton reaction and trapped using DMPO: 10 μL H2O (23%), 10 μL FeSO4·7H2O (0.4 mmol / L), 10 μL DMPO (0.1 mol / L), and 10 μL sample or distilled water. The mixture was rapidly mixed and aspirated into a quartz capillary tube. The ESR spectrum of ·OH was measured for 1 minute under the above measurement parameters. Figure 5Figure d shows the ESR spectra of solutions containing the spin-trapping agent DMPO in the presence and absence of PCNSs: in the solution without PCNSs, an obvious 1:2:2:1 quartet signal can be observed, which is the characteristic signal of DMPO / ·OH adduct; after the addition of PCNSs, the signal is significantly weakened, indicating that it has good ·OH scavenging ability.

[0078] 4.3) To further evaluate its ability to clear ROS in cells, the present invention measured the ROS level in activated macrophages. The specific experimental steps were as follows: 2×10 5 RAW264.7 cells were plated in a culture dish, and after the cells adhered, a culture medium containing LPS was added and incubated for 24 hours. RAW 264.7 incubated with normal culture medium served as the control group. Subsequently, except for the positive control group, MM@PCNSs, MM@PCNSs+NIR, IGU, IGU / MM@PCNSs and IGU / MM@PCNSs+NIR were added to the culture dish and cultured for another 24 hours. After that, a ROS probe was added, and after incubation at 37°C for 30 minutes, the cells were collected and the ROS content was detected by flow cytometry. Flow cytometry results ( Figure 5 Figure (e) shows that MM@PCNSs can effectively scavenge ROS in LPS-activated macrophages. This scavenging ability is enhanced after loading with IGU and further improved after laser irradiation, indicating that MM@PCNSs / IGU can enhance the antioxidant effect of chemotherapy drugs through the photothermal effect.

[0079] 4.4) Western blot experiment. The specific experimental steps are as follows: MM and MM@PCNSs membrane proteins were centrifuged and the supernatant was removed. Then, the cell membrane proteins were extracted using a cell membrane protein extraction kit, and the supernatant was obtained. The concentration of the protein sample was measured using the BCA method. The membrane protein sample was mixed with RIPA and 5× loading buffer to calibrate the membrane protein sample concentration. The protein sample was heated in boiling water for 5 minutes to denature it. The membrane protein sample was separated by 10% SDS-PAGE at a voltage of 120V and transferred to a PVDF membrane at a current of 300mA. After blocking the membrane with 5% skim milk, the membrane was incubated with primary antibodies against IL-1R1, IL-6R-α, and TNFR overnight at 4°C, followed by incubation with secondary antibodies. Finally, the HRP signal of IL-1R1, IL-6R-α, and TNFR on the membrane was detected using Super ECL. Western blot analysis confirmed that the receptor membrane proteins of these cytokines were still retained on the surface of MM@PCNSs, although some membrane proteins were lost during the coating process with PCNSs ( Figure 5f). The results of detecting the concentration of pro-inflammatory factors in the cell supernatant showed that MM@PCNSs could significantly neutralize IL-1β, IL-6 and TNF-α in the environment ( Figure 5 g), indicating that it has the function of a "cytokine scavenger" and can effectively reduce the bioavailability of inflammatory factors.

[0080] 4.5) The present invention evaluated the immunomodulatory effect of MM@PCNSs / IGU on LPS-activated macrophages. The specific experimental steps were as follows: RAW267.4 cells were cultured at 1×10 6 The cells were plated on the culture dish at a density of 1 μg / mL. After the cells attached to the wall, a culture medium containing 1 μg / mL LPS was added to the culture dish, except for the blank control group, and the induction treatment was carried out for 24 hours to establish an in vitro inflammatory activated macrophage model. Subsequently, MM@PCNSs, MM@PCNSs+NIR, IGU, IGU / MM@PCNSs and IGU / MM@PCNSs+NIR were added to the culture dish, except for the positive control group, and cultured at 37°C for another 24 hours. Figure 5 As shown in h, the present invention performed ELISA and qPCR analysis on the cell supernatant and cell bodies, respectively. The ELISA results showed that MM@PCNSs significantly reduced the concentration of pro-inflammatory cytokines in the supernatant. It is worth noting that the addition of IGU and the controlled release achieved by NIR laser further enhanced its anti-inflammatory effect and significantly inhibited the secretion of cytokines ( Figure 5 In addition, qPCR analysis provides information on changes in the transcriptional levels of inflammatory factors. Although the inhibitory effect of MM@PCNSs on proinflammatory cytokines at the gene expression level is relatively mild, lower than the secretion level changes observed by ELISA, the addition of IGU and NIR-triggered release significantly reduced the gene expression levels of proinflammatory factors ( Figure 5 ln in), highlighting the synergistic advantages of targeted drug delivery and photothermal responsive controlled release.

[0081] (5) Therapeutic effect of MM@PCNSs / IGU on CIA mouse model

[0082] 5.1) Construction and grouping of CIA mouse models

[0083] The CIA mouse model was constructed in the same manner as in step 3.3, in vivo fluorescence imaging. The CIA mouse model was then divided into 6 groups: PBS group, MM@PCNSs group, MM@PCNSs+NIR group, IGU group, MM@PCNSs / IGU group, and MM@PCNSs / IGU+NIR group. The treatment regimen of the CIA mouse model was as follows: Figure 6 As shown in a; healthy mice were used as the Healthy group.

[0084] PBS group: PBS was injected with an injection volume of 0.2 mL.

[0085] MM@PCNSs group: MM@PCNSs were injected with an injection volume of 0.2 mL.

[0086] MM@PCNSs+NIR group: MM@PCNSs were injected at a volume of 0.2 mL, and NIR irradiation was given for 10 min 24 h after the injection of MM@PCNSs.

[0087] IGU group: injected with IGU 5 mg / kg.

[0088] MM@PCNSs / IGU group: injected with MM@PCNSs / IGU (IGU 5mg / kg).

[0089] MM@PCNSs / IGU+NIR group: NIR irradiation for 10 min was given 24 h after injection of MM@PCNSs / IGU (IGU 5 mg / kg).

[0090] 5.2) Experimental methods and results

[0091] On day 45 of the experiment, the MM@PCNSs / IGU+NIR group showed the most significant improvement in arthritis severity, with a significant decrease in arthritis score and foot swelling thickness ( Figure 6 bd in Figures 1 and 2). These results demonstrate that MM@PCNSs / IGU+NIR effectively inhibits arthritis progression in CIA mice. The significant reduction in joint inflammation and swelling suggests that the synergistic effect of IGU and NIR-responsive MM@PCNSs promotes drug accumulation and controlled release in inflamed joints. Compared with IGU monotherapy, the MM@PCNSs / IGU group exhibited superior anti-arthritis efficacy, highlighting the potential of nanoparticle-based drug delivery systems in the treatment of inflammatory arthritis.

[0092] The present invention conducted a histological analysis of the ankle joint of mice using H&E, Masson and Safranin-O staining ( Figure 6e). The synovial tissue structure of the knee joint in the healthy group was normal, with a smooth articular cartilage surface and intact bone tissue. In contrast, the PBS group showed a significant inflammatory response, including synovial tissue hyperplasia, inflammatory cell infiltration (indicated by the black arrow in Masson staining), cartilage layer damage (indicated by the black arrow in Safranin O staining), and invasion of the subchondral bone tissue. In contrast, the MM@PCNSs / IGU+NIR group showed minimal inflammatory cell infiltration, mild synovial tissue hyperplasia, no bone tissue invasion, and a smooth cartilage surface. These findings indicate that MM@PCNSs / IGU+NIR can effectively prevent the progression of RA-related joint damage, protect cartilage integrity, and reduce inflammatory cell infiltration. The inflammatory targeting of MM@PCNSs and its ability to control the release of IGU ensure localized drug efficacy while reducing systemic side effects.

[0093] Bone erosion is an important indicator for evaluating the severity of arthritis. The present invention evaluated it using micro-CT technology. The results showed that CIA mice treated with PBS had severe bone erosion ( Figure 6 f), bone mineral density (BMD), bone volume fraction (BV / TV), trabecular number (Tb.N) and trabecular thickness (Tb.Th) decreased significantly, while trabecular spacing (Tb.Sp) increased significantly ( Figure 6 After treatment, all of the above parameters improved. In particular, the MM@PCNSs / IGU+NIR-treated group showed minimal bone erosion, and all bone structural indicators returned to nearly normal levels, demonstrating its most significant protective effect on bone structure.

[0094] To evaluate the anti-inflammatory effects of different treatment regimens, the present invention detected the levels of pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α in serum and joint tissues. Figure 7 As shown in Figures ac, the levels of IL-1β, IL-6, and TNF-α in the PBS group mice were significantly higher than those in the healthy group, suggesting that there was a significant inflammatory response in the CIA mice. It is worth noting that the levels of the above cytokines in the MM@PCNSs / IGU+NIR treatment group decreased significantly, which was better than the other treatment groups, indicating that the preparation was effective in inhibiting RA inflammation. This synergistic anti-inflammatory effect is attributed to the presence of IGU and the ability of MM@PCNSs to neutralize proinflammatory cytokines. In addition, the photothermal properties of MM@PCNSs enhanced the tissue penetration and release of the drug, further improving the therapeutic effect. The present invention also evaluated the mRNA expression levels of IL-1β, IL-6, and TNF-α in synovial tissue ( Figure 7Consistent with the results at the protein level, the gene expression levels of these three inflammatory factors in the MM@PCNSs / IGU+NIR group were significantly lower than those in the other groups. Furthermore, the expression of IL-1β, IL-6, and TNF-α in joint tissues was detected by immunofluorescence staining ( Figure 7 These findings suggest that MM@PCNSs / IGU+NIR can effectively alleviate RA-related joint inflammation by regulating cytokine production and inhibiting inflammatory responses.

[0095] Finally, the present invention evaluated the chronic toxicity of MM@PCNSs / IGU at the end of the experiment. The body weight of mice treated with MM@PCNSs / IGU+NIR remained stable ( Figure 8 In addition, the blood routine indicators (RBC, WBC, PLT) and serum biochemical indicators (ALT, AST, BUN) of the mice were similar to those of the healthy group ( Figure 8 Tissue section analysis showed that no obvious organ pathological abnormalities were observed after treatment with MM@PCNSs / IGU or MM@PCNSs / IGU+NIR ( Figure 8 h), indicating that MM@PCNSs / IGU has good biosafety and is suitable for long-term use in RA therapy.

[0096] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A drug delivery system based on macrophage membrane-coated porous carbon nanospheres, characterized in that: The drug delivery system includes porous carbon nanospheres coated with macrophage membranes.

2. The method for preparing the drug delivery system according to claim 1, wherein The preparation method comprises the following steps: The polyacrylic acid aqueous solution, zinc oxide and isopropyl alcohol were mixed to obtain PAA-Zn NPs; performing a carbonization treatment on the PAA-Zn NPs to obtain PCNSs; The PCNSs and macrophage membrane fragments are mixed evenly to obtain the drug delivery system.

3. The preparation method according to claim 2, characterized in that The volume ratio of the polyacrylic acid aqueous solution to isopropyl alcohol is 1:2; the mass ratio of polyacrylic acid to zinc oxide in the polyacrylic acid aqueous solution is 0.36 mg:72 mg.

4. The preparation method according to claim 2, characterized in that The carbonization treatment conditions are as follows: heating to 900° C. at a heating rate of 5° C. / min and calcining for 3 h.

5. Use of the drug delivery system according to claim 1 in preparing a medicament for treating rheumatoid arthritis.

6. A drug for treating rheumatoid arthritis, characterized in that: The drug comprises the drug delivery system according to claim 1 and an anti-rheumatic drug.

7. The drug according to claim 6, characterized in that The anti-rheumatic drugs include iguratimod.

8. The method for preparing the medicine according to claim 6, characterized in that: The preparation method comprises the following steps: The solution containing the drug delivery system and the anti-rheumatic drug solution are mixed to obtain the drug.

9. The preparation method according to claim 8, characterized in that The mass ratio of the solution containing the drug delivery system and the anti-rheumatic drug solution is 1:

1.

10. The preparation method according to claim 8, characterized in that The concentration of the drug delivery system in the solution containing the drug delivery system is 1 mg / mL; the concentration of the anti-rheumatic drug in the anti-rheumatic drug solution is 0.455 mg / mL.

Images