A drug delivery system based on porous carbon nanospheres coated with macrophage membranes, drugs and preparation method and application thereof

Abstract

The present application relates to the technical field of biological medicine, in particular to a drug delivery system based on macrophage membrane-coated porous carbon nanospheres, a drug and a preparation method and application thereof. The drug delivery system provided by the present application is a high-efficiency and low-toxicity RA treatment scheme, which has a good clinical application prospect. Through the characteristics of precise targeted delivery and controlled release, the present application can reduce the amount of drug used in the treatment process, 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 application is expected to improve the quality of life of RA patients, reduce the side effects of drugs and harm to health, promote the progress of RA treatment, and has wide social needs and market potential.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a drug delivery system based on macrophage membrane-coated porous carbon nanospheres, the drug, its preparation method, and its application. Background Technology

[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 DMARDs (such as methotrexate (MTX), biologics, and Janus kinase (JAK) inhibitors) have been used to treat RA, the clinical remission rate (i.e., the target of preventing joint dysfunction) is only around 50%. This highlights the need to develop more effective treatment strategies. Against this backdrop, iguratimod (IGU), as a cost-effective DMARD, has shown promising potential in reducing bone destruction and inflammation in RA. However, due to its poor hydrophobicity, IGU's 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 inherent therapeutic potential, promising to enhance the treatment efficacy of RA. Therefore, optimizing the interaction between IGU and nanocarriers, improving its bioavailability, enhancing targeting to inflammatory sites, and achieving controlled release within the inflammatory environment of joints are key challenges. Exploring these synergistic effects is crucial for improving treatment efficacy and maintaining long-term inflammatory remission in RA.

[0003] The synovial microenvironment of inflammatory joints in rheumatoid arthritis (RA) is characterized by hypoxia, nutrient deficiency, acidic pH, elevated levels of reactive oxygen species (ROS), and excessive amounts of various pro-inflammatory factors. This pathological environment not only exacerbates synovial tissue proliferation and invasion but also presents challenges for the design of drug delivery systems, while simultaneously providing potential targets and response conditions. For example, hypoxic and acidic environments can be used to develop pH- or hypoxia-responsive nanocarriers, while high levels of ROS can trigger the release of nanomedicines. However, the dysregulation of synovial microenvironment homeostasis plays a central role in the progression of RA, making traditional anti-inflammatory therapies ineffective. Excessive ROS accumulation leads to immune regulatory imbalance, promoting the polarization of pro-inflammatory M1 macrophages and inhibiting the activation of anti-inflammatory M2 macrophages, resulting in persistent inflammation. Furthermore, the complex interactions between activated immune cells and pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) form a highly interactive inflammatory network, making 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 relapse after treatment. Restoring the immune homeostasis of the synovium is key to achieving sustained remission of RA. Therefore, in addition to targeted delivery and environmental responsiveness, nanocarriers that can eliminate pro-inflammatory factors could help enhance the therapeutic effect of IGU and improve clinical outcomes for RA patients.

[0004] Nanozymes are a class of nanomaterials with enzyme-like activity, attracting widespread attention in the biomedical field due to their high stability, multifunctional catalytic activity, and cost-effectiveness. However, metal-based nanozymes suffer from problems such as poor biodegradability, potential toxicity from metal ion release, and long-term accumulation in vivo. Therefore, it is necessary to find a method that can effectively control the level of inflammation in rheumatoid arthritis (RA). Summary of the Invention

[0005] The purpose of this invention is to provide a drug delivery system, drug, preparation method, and application based on macrophage membrane-coated porous carbon nanospheres, to address the problems existing in the prior art. This invention provides a drug delivery system based on macrophage membrane-coated porous carbon nanospheres (MM@PCNSs), specifically for targeted therapy of rheumatoid arthritis (RA). This drug delivery system combines macrophage membrane (MM) and porous carbon nanosphere (PCNSs) technologies, achieving precise targeting, dual-response controlled release, and effective modulation of the inflammatory microenvironment, demonstrating significant therapeutic potential.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This 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 with macrophage membranes.

[0008] Disruption of synovial microenvironment homeostasis in rheumatoid arthritis (RA) is a key factor affecting the therapeutic efficacy of DMARDs and post-treatment relapse in RA patients. This invention aims to address the low remission efficiency of existing DMARDs in clinical patients and maintain the inflammatory resolution period in patients receiving effective treatment. It develops a drug delivery system based on porous carbon nanospheres coated with macrophage membranes, achieving precise drug delivery and synergistic DMARD treatment with pH / photothermal dual response and pro-inflammatory microenvironment regulation. This maintains synovial microenvironment homeostasis, thereby achieving long-term control of RA inflammation levels.

[0009] Porous carbon nanospheres (PCNSs) can mimic the activities of glutathione peroxidase, catalase, and superoxide dismutase, while exhibiting excellent biocompatibility and stability. By regulating ROS levels in the synovial microenvironment of renal inflammatory disease (RA), they restore redox balance and promote the transformation of M1 macrophages into M2 macrophages. Nanozymes can effectively alleviate inflammation and joint damage. Furthermore, the unique electronic properties and broad absorption spectrum of PCNSs give them photoresponsive characteristics, which can be used to control drug release. To further enhance their therapeutic effect, this invention combines macrophage membranes (MMs) with porous carbon nanospheres (PCNSs). PCNSs can encapsulate macrophage membranes (MMs), a modification that not only improves biocompatibility and targeting of inflamed sites but also endows them with cytokine neutralizing capabilities. The receptors abundant on the surface of MMs can bind to and scavenge pro-inflammatory cytokines (such as IL-1β, IL-6, and TNF-α), effectively inhibiting inflammatory signal transduction and reducing excessive immune activation. This invention aims to provide a multifunctional treatment strategy by integrating targeted delivery, ROS clearance, and inflammation regulation technologies, utilizing MM-encapsulated PCNSs, which is expected to restore the homeostasis of the synovial microenvironment.

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

[0011] This invention provides a method for preparing the above-mentioned drug delivery system, the method comprising the following steps:

[0012] Aqueous polyacrylic acid, zinc oxide, and isopropanol were mixed evenly to obtain PAA-ZnNPs;

[0013] The PAA-ZnNPs were carbonized 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 isopropanol 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 conditions are: heating to 900°C at a heating rate of 5°C / min and calcining for 3 hours.

[0017] This invention provides the application of the above-described drug delivery system in the preparation of a medicament for treating rheumatoid arthritis.

[0018] More 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 medicament for treating rheumatoid arthritis, the medicament comprising the above-described drug delivery system and antirheumatic drug.

[0020] Preferably, the antirheumatic drug includes ellamod.

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

[0022] This invention provides a method for preparing the above-mentioned drug, the method comprising the following steps:

[0023] The drug is obtained by mixing the solution containing the drug delivery system and the antirheumatic drug solution.

[0024] Preferably, the mass ratio of the solution containing the drug delivery system to the antirheumatic 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; and the concentration of the antirheumatic drug in the antirheumatic 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 this 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 target and deliver drugs to the site of inflammation

[0029] The MM@PCNSs drug delivery system provided by this invention can specifically identify and target macrophages at the lesion site of rheumatoid arthritis by utilizing the targeting properties of the macrophage membrane. In vivo experiments in a CIA mouse model demonstrated that this drug system can effectively deliver drugs to the lesion site of arthritis, significantly increasing the local drug concentration compared to traditional drug delivery systems and avoiding side effects on healthy tissues.

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

[0031] The MM@PCNSs drug delivery system provided by this invention can achieve precise drug release under specific conditions in response to local acidic environments and NIR light irradiation. Experiments in a CIA mouse model showed that the local drug concentration at the site of inflammation significantly increased, while the drug concentration in healthy tissue remained at a low level, effectively reducing the toxicity and side effects of the drug 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 therapeutic efficacy and reducing drug waste.

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

[0033] MM@PCNSs possess nanozyme-like catalytic activity, enabling them to scavenge ROS generated in rheumatoid arthritis and neutralize pro-inflammatory cytokines. This property was validated by immunohistochemical analysis in a CIA mouse model. The results showed that in the group treated with MM@PCNSs / IGU, the inflammation level, bone erosion, and cartilage damage in the CIA mouse model were significantly reduced. Compared with the control group, the expression of inflammatory factors was significantly decreased, further demonstrating the effectiveness of MM@PCNSs in regulating the inflammatory microenvironment.

[0034] (4) Significant therapeutic effect

[0035] In a CIA mouse model, the treatment group using MM@PCNSs to deliver IGU drugs showed significant clinical efficacy. Compared with the control group, the treatment group mice exhibited significant improvements in joint swelling, pain, and dysfunction. Micro-CT analysis revealed significant bone erosion and articular cartilage protection in the treatment group, indicating that MM@PCNSs can effectively slow the progression of RA. Statistical analysis showed that the treatment group mice maintained stable body weight, reduced inflammation scores, and faster recovery of joint function. These data fully demonstrate the significant efficacy of the drug delivery system of this invention in the treatment of RA.

[0036] In summary, the drug delivery system provided by this invention is a highly efficient and low-toxicity treatment option for RA, with promising clinical application prospects. Through precise targeted delivery and controlled release, this invention can reduce the amount of drug used during treatment, minimize unnecessary waste, and improve the cost-effectiveness of treatment. From a social perspective, RA is a common chronic disease worldwide, severely impacting patients' quality of life. This invention, by providing a safer and more effective treatment method, is expected to improve the quality of life for RA patients, reduce drug side effects and health hazards, and promote the advancement of RA treatment, possessing broad social demand and market potential. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1 Flowchart for MM@PCNSs / IGU preparation and treatment;

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

[0040] Figure 3 The controllable drug release of MM@PCNSs / IGU is shown below: a) UV-Vis-NIR absorption spectroscopy results; b) temperature changes of PCNSs solutions with different concentrations under NIR laser irradiation; c) infrared thermographic image; d) effect of laser power density on temperature; e) photothermal durability of PCNSs; f) photothermal conversion efficiency and thermal relaxation behavior of PCNSs as reflected by the ΔT curve during laser irradiation and cooling; g) photothermal conversion efficiency calculated by Ln(θ); h) UV-Vis absorption spectra of IGU and IGU-loaded PCNS solutions; i) IGU release rate under different conditions.

[0041] Figure 4The targeting of MM@PCNSs / IGU is shown in the following figures: a) Fluorescence microscopy images showing the uptake of MM@PCNSs / Cy5.5 by inactive and activated HUVECs; b) Fluorescence microscopy images showing the endocytosis of MM@PCNSs / Cy5.5 by inactive and activated RAW264.7 cells; c) In vivo fluorescence imaging of mice 24 hours after injection of Cy5.5 and MM@PCNSs / Cy5.5; d) Fluorescence distribution in heart, liver, spleen, lung, kidney, and joint tissues of each group; e) Quantitative analysis of mean fluorescence intensity (MFI) of each organ; f) Representative immunofluorescence images of MM@PCNSs / Cy5.5 localization in inflamed synovium; g) Statistical graph of quantitative analysis of mean fluorescence intensity (MFI) in inflamed synovium.

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

[0043] Figure 6 The therapeutic effects of MM@PCNSs / IGU on the CIA mouse model are shown below; where a represents the treatment regimen for the CIA mouse model; bd represents the results of arthritis scores and paw swelling thickness surveys; e represents the results of H&E, Masson, and Safranin-O staining; f represents the results of bone erosion evaluation; and gk represents the evaluation results of bone mineral density (BMD), bone volume fraction (BV / TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular spacing (Tb.Sp).

[0044] Figure 7 The study aimed to assess the inhibitory effect of MM@PCNSs / IGU on in vivo inflammation levels; where ac represents the levels of IL-1β, IL-6, and TNF-α in serum and joint tissues; df represents the mRNA expression levels of IL-1β, IL-6, and TNF-α in synovial tissues; and g represents the results of immunofluorescence staining.

[0045] Figure 8 The biosafety of MM@PCNSs / IGU is defined as follows: a represents the mouse body weight survey results; bg represents the results of blood routine indicators (RBC, WBC, PLT) and serum biochemical indicators (ALT, AST, BUN); and h represents the results of tissue section analysis. Detailed Implementation

[0046] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of 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 terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0048] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0049] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

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

[0051] Example 1

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

[0053] The preparation process and treatment flowchart of MM@PCNSs / IGU are as follows: Figure 1 As 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 an aqueous solution of polyacrylic acid (0.2 g / mL). After stirring for 15 min, 72 mg of zinc oxide (ZnO) was added, and the solution was continuously sonicated for 10 min while shaking until the solution became clear. 600 mL of isopropanol (IPA) was added dropwise to the mixture at a rate of 1 drop per second using a dropping funnel. The mixture was then 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 finely in a mortar and placed in a tube furnace. High-purity argon gas was first introduced for 30 min, and then the temperature inside the tube furnace was increased to 900 °C at a rate of 5 °C / min. The sample was then calcined at 900 °C for 3 h to finally obtain PCNSs. RAW 264.7 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and incubated in a 37°C, 5% CO2 incubator. When the cell count was approximately 4 × 10⁻⁶ cells / year... 7 At this time, after washing with PBS, cells were collected by cell scraper and centrifuged at 800g for 5 min. Macrophage membrane fragments (MMs) were collected according to the instructions of the cell membrane protein extraction kit. The protein content of MMs was measured using BCA. MMs were then resuspended in 1 mL PBS and stored in liquid nitrogen. The frozen MMs were removed from the liquid nitrogen and rapidly thawed in a 37°C water bath. MMs and PCNSs were mixed at a 1:1 mass ratio and sonicated in an ultrasonic water bath. The mixture of MMs and PCNSs was then passed through a 0.45 μm pore size filter membrane using a liposome extractor, with repeated pressing 15 times. The mixture was then centrifuged at 700g for 10 min at room temperature. After removing the supernatant, MM@PCNSs were obtained. After resuspending with PBS, the MMs were stored at 4°C.

[0055] 1.2) Preparation of MM@PCNSs / IGU: 1 mg of iramod (IGU) and 1 mg of PCNSs were mixed in 1 mL of ethanol and mixed vertically for 24 h to obtain PCNSs / IGU. MM and PCNSs were mixed at a 1:1 mass ratio and then sonicated in an ultrasonic water bath. The mixture was then passed through a 0.45 μm pore size filter membrane using a liposome extractor, with repeated pressing 15 times. The mixture was then centrifuged at 700 g for 10 min at room temperature. After removing the supernatant, MM@PCNSs / IGU was obtained.

[0056] 1.3) Characterization of MM@PCNSs: The preparation process of MM@PCNSs includes a unique PAA-Zn nanoparticle synthesis and subsequent carbonization step, thereby obtaining 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. These nanoparticles were characterized by SEM (Sequencing on Microscopy). Figure 2 a) and TEM ( Figure 2 (b) Observation and confirmation. After drying, the PAA-Zn nanoparticles were calcined at high temperature to obtain PCNSs with the same particle size of approximately 180±10 nm. Figure 2 (ab in the original text). During calcination, PAA is transformed into a carbon framework, while the evaporation of Zn forms a mesoporous structure within the carbon structure. Elemental mapping of PCNSs ( Figure 2 c) shows a uniform distribution of carbon (C), while zinc (Zn) is virtually invisible, further verifying the successful synthesis of PCNSs. To further verify the synthesis effect, Raman spectroscopy and X-ray diffraction (XRD) analyses were employed. Figure 2 As shown in d, the Raman spectra of PCNSs show typical D and G bands, indicating the presence of carbon structures. Figure 2 In the XRD pattern shown by 'e', ​​diffraction peaks appear at (100) and (002), corresponding to the crystal planes of elemental carbon. Nitrogen adsorption-desorption isotherm testing ( Figure 2 f) in the figure represents a Type IV isotherm with a BET specific surface area of ​​561.1 m². 2 / g, and Figure 2 The g in the figure shows that it has a microporous (approximately 1.9 nm) and mesoporous (approximately 11 nm) structure. For example... 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 PCNSs are prone to aggregation in solution due to insufficient electrostatic repulsion. MM was encapsulated on the surface of PCNSs using conventional mechanical methods (ultrasound and extrusion techniques), thus forming MM@PCNSs. Figure 2As shown in Figure 'ab', the PCNSs surface is coated with a light gray membrane-like structure, indicating the formation of a typical "core-shell" structure. The macrophage membrane coating significantly alters the surface properties of the nanoparticles. For example... Figure 2 As shown in h, the zeta potential of MM@PCNSs increases to approximately -27.3 mV, which is much higher than that of uncoated PCNSs.

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

[0058] 2.1) UV-Vis-NIR absorption spectroscopy was performed on PCNSs, and the results of the UV-Vis-NIR absorption spectroscopy were as follows ( Figure 3 a) indicates that PCNSs exhibit a wide range of absorption in the NIR region, confirming their suitability for photothermal applications.

[0059] 2.2) To investigate its photothermal effect, PCNSs solutions of different concentrations (50, 100, 150, and 200 μg / mL) were irradiated with NIR lasers, and temperature changes were monitored. The results are as follows: Figure 3 As shown in b, with increasing PCNS concentration, the heating rate and final temperature also increase accordingly, while deionized water, as a control group, shows almost no temperature change. Infrared thermal imaging further confirms this trend. Figure 3 (c)

[0060] 2.3) Furthermore, laser power density plays a crucial role in heating efficiency, with a 100 μg / mL PCNSs solution heated at 0.7 W / cm². 2 1W / cm 2 and 1.4W / cm 2 Irradiation was performed continuously at the power density for 5 minutes, 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, a concentration of 100 μg / mL of PCNSs was subjected to four cycles of NIR laser irradiation and natural cooling. The specific experimental procedure was as follows: First, 100 μg / mL of PCNSs was irradiated at 808 nm and 1 W / cm². 2 The laser was continuously irradiated for 10 minutes, then the laser power was turned off, and the device was allowed to cool naturally for 10 minutes. This heating and cooling process was repeated four times. The temperature was recorded every 30 seconds using an infrared thermal imager, and a cyclic graph was generated based on the recorded temperature data to assess the stability of the photothermal performance. The photothermal durability was then tested. The results are as follows: Figure 3As shown in equation e, the maximum temperature remained stable after multiple heating-cooling cycles, indicating that PCNSs possess excellent photothermal durability. Furthermore, the photothermal conversion efficiency (η) of PCNSs was calculated, showing a efficiency of approximately 26%. Figure 3 The fg in the figure indicates that it can efficiently convert NIR light energy into heat energy, which is a key performance characteristic for achieving effective photothermal response.

[0062] 2.5) Drug loading and release experiments were conducted on MM@PCNSs. The specific experimental procedure was as follows: the absorbance of different concentrations of iramod (IGU) at 257 nm was measured to plot a standard curve. This experiment verified the therapeutic potential of MM@PCNSs. The results are as follows: Figure 3 As shown in Figure h, curve b is the UV-Vis absorption spectrum of IGU, and curve a is the IGU-loaded PCNS solution (the IGU-loaded PCNS solution was prepared by mixing 1 mg IGU and 1 mg PCNSs in 1 mL of ethanol and mixing for 24 h on a vertical mixer. After centrifugation, the absorbance of the IGU-loaded PCNSs supernatant at 257 nm was measured), demonstrating effective drug loading capacity with an encapsulation efficiency as high as 45.5%.

[0063] 2.6) The drug release behavior of MM@PCNSs / IGU was studied under different conditions. The specific experimental procedure was as follows: First, three parallel aliquots of 1 mg IGU-loaded PCNSs were taken at 37℃ in a water bath. One aliquot was dispersed in 1 mL of PBS solution with pH = 7.4, and the other two aliquots were dispersed in PBS solution with pH = 5.6. At certain time intervals, one aliquot of the 5.6 nanoparticle solution was treated with an 808 nm laser (1 W / cm²). 2 Irradiation was performed for 5 minutes, followed by centrifugation of three nanoparticle solutions at specific time points. The absorbance of the supernatant was measured. After the test, the supernatant was returned to the centrifuge tube, and the absorbance was measured again after a certain time interval. The experimental results are as follows: Figure 3 As shown in i in the figure. Under physiological conditions (pH 7.4), IGU showed an initial rapid release, followed by a sustained release trend; however, under acidic conditions simulating an inflammatory microenvironment (pH 5.6), the release rate increased significantly, demonstrating good pH responsiveness. 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 the figure. Figure 3 As shown in i in the figure. The results showed that when MM@PCNSs / IGU was irradiated with an 808nm laser, its IGU release rate was significantly faster than that of the unirradiated control group, regardless of whether it was at pH 7.4 or pH 5.6. This confirms that the photothermal effect of PCNSs can be used to promote drug release on demand, 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), followed by confocal fluorescence microscopy. The results showed that MM@PCNSs accumulated extensively around the inflamed endothelial cells. Figure 4 In the control group (a), MM@PCNSs showed the ability to selectively adhere to inflammatory blood vessels, while almost no binding was observed in the non-activated control group. 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-incubated with RAW264.7 macrophages. 2 × 10⁶ cells were added. 5 RAW 264.7 cells were seeded in confocal culture dishes and, after cell adhesion, incubated for 24 hours with medium containing 1 μg / mL LPS. RAW264.7 cells incubated in normal medium served as a control group. Next, DID-labeled MM@PCNSs were added to each group, followed by incubation at 37°C for another 4 hours. One group was then irradiated with laser for 10 minutes after 4 hours of incubation. The uptake capacity of RAW264.7 macrophages of Cy5.5-labeled MM@PCNSs was then investigated, and the results are shown below. Figure 4 b. Results showed that LPS-activated RAW264.7 macrophages exhibited significantly enhanced uptake of Cy5.5-labeled MM@PCNSs, while non-activated cells showed significantly less uptake. Figure 4 Figure b) shows that the inflammatory state enhances cellular uptake. Considering that inflammation is usually accompanied by a decrease in pH, this pH-responsive behavior promotes targeted drug release at the disease site. Furthermore, under 808 nm near-infrared (NIR) laser irradiation, the fluorescence signal in activated macrophages was further enhanced, confirming that MM@PCNSs can achieve drug release in response to both pH and NIR stimulation.

[0067] 3.3) In vivo fluorescence imaging

[0068] A CIA (collagen-induced arthritis) mouse model was constructed using the following steps: Female DBA / 1J mice aged 6-8 weeks were selected. First, complete Freund's adjuvant (CFA) and bovine CII were mixed in a 1:1 volume ratio and homogenized at high speed until completely emulsified. Then, 100 μL of the emulsion was injected intradermally into each mouse at a site 1.5 cm from the base of the tail. On day 21 after the first injection, a booster immunization was administered by mixing incomplete Freund's adjuvant (IFA) and bovine CII in a 1:1 volume ratio and emulsifying completely, following the same procedure. Subsequently, redness and swelling appeared in the joints of the mice. The joints of each mouse were measured and scored according to the following criteria:

[0069] 0: Normal, no erythema or swelling;

[0070] 1: Erythema or mild swelling is limited to the tarsal bone or ankle joint;

[0071] 2: Erythema or mild swelling spreads from the ankle joint toward the tarsal bone;

[0072] 3: Erythema or moderate swelling spreads from the ankle joint to the metatarsophalangeal joint;

[0073] 4: Erythema or severe swelling around the ankle, foot and toes, or stiffness in the joints of the limbs.

[0074] Through the above steps and scoring, a mouse model of arthritis (RA) can be established for studying the pathogenesis of the disease and testing the efficacy of novel drugs. On day 60 post-immunization, when the average arthritis score of the CIA mice reached 14 points, Cy5.5 and MM@PCNSs / Cy5.5 were injected via the tail vein, respectively. In vivo fluorescence imaging was then performed to further verify the targeting efficiency of MM@PCNSs. Results are as follows... Figure 4 As shown in c in the figure. 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 was performed on the major organs and inflamed joints of the CIA mouse model, and the results are shown in the figure. Figure 4 As shown in the figure, this result is consistent with the in vivo fluorescence imaging results, indicating that MM@PCNSs preferentially accumulate in inflamed synovium, while their non-specific distribution is significantly reduced. Furthermore, immunofluorescence staining of the inflammatory macrophage marker CD86 in a CIA mouse model further confirmed the selective localization of MM@PCNSs in inflamed synovial tissue. Figure 3The Cy5.5 group (simulating free drug circulation in vivo) showed almost no Cy5.5 signal, indicating that free drug molecules have difficulty effectively reaching and accumulating in inflamed synovial tissue. In contrast, MM@PCNSs showed significant enrichment, highlighting the targeting advantage of MM encapsulation. Furthermore, the co-localization results of MM@PCNSs and CD86 suggest that these nanoparticles can specifically bind to inflammatory macrophages, potentially achieving their therapeutic effect by regulating macrophages in the RA synovial microenvironment.

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

[0076] 4.1) This invention evaluates the scavenging effect of MM@PCNSs / IGU on ROS (including superoxide anions O2·). - The ability of hydroxyl radicals (·OH) and hydrogen peroxide (H2O2) to be removed was investigated. The specific experimental steps were as follows: ·OH removal: 1.8 mM ferrous sulfate heptahydrate (FeSO4·7H2O) and 5 mM hydrogen peroxide (H2O2) were added to 20 mL of deionized water and reacted at room temperature for 10 min under magnetic stirring, yielding a pale yellow solution. This is because the mixed solution underwent the Fenton reaction during this process. 2+ It reacts with H₂O₂ to produce highly toxic hydroxyl radicals (·OH). Then, 4.97 mg of salicylic acid (SA) was mixed into the above mixed solution, and stirred at room temperature for 30 min. At this point, the solution changed from light yellow to purplish-black, because ·OH reacts with SA to form 2,3-dihydroxybenzoic acid. The absorbance of the mixed solution at a wavelength of 510 nm was detected using a UV-Vis spectrophotometer. Next, a certain concentration of PCNSs was mixed into the solution and stirred for 1 h, 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 also needed to be prepared. 4.97 mg SA was dissolved in 20 mL of deionized water. Three groups of 2 mL SA solutions were taken, with 1 μL of H₂O₂ added to one group and 1 mg of FeSO₄·7H₂O added to the other. All six PCNSs solutions of different concentrations, along with the three control groups, were centrifuged. The absorbance of the supernatant at 510 nm was measured using a UV spectrophotometer. Finally, the scavenging ability of PCNSs for hydroxyl radicals was evaluated by comparing the absorbance values ​​of the supernatant. (O₂·7H₂O scavenging...) - PCNSs superoxide radicals (O2· -The scavenging effect was confirmed by measuring the photoreduction inhibition rate of nitrotetrazolam (NBT). In the experiment, riboflavin (20 μM), methionine (13 mM), and nitrotetrazolam (75 μM) were mixed in 20 mL of deionized water. This product exhibited a characteristic absorption peak at 560 nm in the UV-Vis absorption spectrum. Different concentrations of PCNSs (0 μg / mL, 3.125 μg / mL, 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, and 50 μg / mL) were then mixed with the above solution, and the test samples were continuously irradiated under a UV lamp for 15 min. Finally, the test samples and the control group (without PCNSs and without UV irradiation) were centrifuged, and the absorbance of the supernatant of different samples was measured (the test process required protection from light). The effect of PCNSs on O2· - The scavenging ability. Scavenging H2O2: Since hydrogen peroxide (H2O2) has a characteristic absorption peak at 240 nm in the UV-Vis absorption spectrum, different concentrations of PCNSs (0 μg / mL, 3.125 μg / mL, 6.25 μg / mL, 12.5 μg / mL, and 25 μg / mL) were added to 40 mM H2O2. The scavenging efficiency of PCNSs for H2O2 was detected by measuring the absorbance values ​​of the characteristic absorption peak of H2O2 in the test samples of different concentrations. The results are as follows... Figure 5 As shown in a, after adding PCNSs, O2· - The characteristic absorption peaks decreased significantly, indicating good scavenging ability; the scavenging rate reached 66.3% at a concentration of 50 μg / mL. Similarly, PCNSs also showed significant scavenging effects on ·OH and H2O2, with scavenging rates of 56.6% and 33.2%, respectively. Figure 5 (b and c in the text).

[0077] 4.2) This invention further evaluates the ·OH scavenging ability of PCNSs using electron paramagnetic resonance (ESR) spectroscopy combined with spin trapping technology. The specific experimental steps are as follows: Measurements were performed on a Brucker ESR5000 ESR spectrometer under the following conditions: X-band, microwave power 20mW, modulation frequency 100kHz, amplitude modulation 0.1mT, scan width 20mT, central magnetic field 324.5mT, and detection at room temperature. ·OH was generated using the Fenton reaction and captured with DMPO: 10μL H2O (23%), 10μL FeSO4·7H2O (0.4mmol / L), 10μL LDMPO (0.1mol / L), and 10μL sample or distilled water. The mixture was rapidly mixed and drawn into a quartz capillary. The ESR spectrum of ·OH was detected after 1 min under the above measurement parameters. Figure 5The figure d shows the ESR spectra of the solution containing spin trapping agent DMPO in the presence and absence of PCNSs: in the solution without PCNSs, a distinct 1:2:2:1 quartet signal can be observed, which is the characteristic signal of DMPO / ·OH adduct; while 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 scavenge ROS intracellularly, this invention measured the ROS level in activated macrophages. The specific experimental steps were as follows: 2 × 10 5 RAW264.7 cells were seeded in culture dishes and, after cell adhesion, incubated in medium containing LPS for 24 hours. RAW 264.7 cells incubated in normal medium served as a 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 dishes, and cultured for another 24 hours. Afterward, a ROS probe was added, and the cells were incubated at 37°C for 30 minutes. Cells were then collected, and ROS levels were detected by flow cytometry. Flow cytometry results ( Figure 5 As shown in e), MM@PCNSs can effectively scavenge ROS within LPS-activated macrophages. This scavenging ability is enhanced after loading IGU and further improved after laser irradiation, indicating that MM@PCNSs / IGU can enhance the antioxidant effect of chemotherapeutic drugs through photothermal effects.

[0079] 4.4) Western blot experiment: The specific experimental steps were as follows: MM and MM@PCNSs membrane proteins were centrifuged to remove the supernatant. Then, cell membrane proteins were extracted using a cell membrane protein extraction kit, and the supernatant was obtained. The concentration of the protein samples was measured using the BCA method. The membrane protein samples were mixed with RIPA and 5× loading buffer to calibrate the concentration of the membrane protein samples. The protein samples were heated in boiling water for 5 min to denature them. The membrane protein samples were separated by 10% SDS-PAGE at 120 V and transferred to a PVDF membrane at 300 mA. After blocking the membrane with 5% skim milk, the membrane was incubated overnight at 4 °C with primary antibodies against IL-1R1, IL-6R-α, and TNFR, and then incubated with secondary antibodies. Finally, the HRP signals of IL-1R1, IL-6R-α, and TNFR on the membrane were 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 encapsulation process with PCNSs. Figure 5(f) Results of detecting the concentration of pro-inflammatory factors in cell supernatant showed that MM@PCNSs could significantly neutralize environmental IL-1β, IL-6 and TNF-α. Figure 5 The presence of g in the formula indicates that it possesses the function of a "cytokine scavenger," effectively reducing the bioavailability of inflammatory factors.

[0080] 4.5) This invention evaluated the immunomodulatory effect of MM@PCNSs / IGU on LPS-activated macrophages. The specific experimental steps were as follows: RAW267.4 cells were injected at a rate of 1×10⁻⁶ cells / mL. 6 Cells were seeded at a density of [insert density here] on culture dishes. After cell adhesion, except for the blank control group, culture medium containing 1 μg / mL LPS was added for 24 h of induction treatment to establish an in vitro model of inflammation-activated macrophages. 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 dishes, and cultured at 37°C for another 24 h. Figure 5 As shown in h, the cell supernatant and cell volume were analyzed by ELISA and qPCR, respectively. ELISA results showed that MM@PCNSs significantly reduced the concentration of pro-inflammatory cytokines in the supernatant. Notably, the addition of IGU and controlled release via NIR laser further enhanced its anti-inflammatory effect and significantly inhibited cytokine secretion. Figure 5 (ik in the text). Furthermore, qPCR analysis provided information on changes in the transcriptional levels of inflammatory factors. Although MM@PCNSs themselves exhibit a relatively mild inhibitory effect on pro-inflammatory cytokines at the gene expression level, lower than the changes in secretion levels observed by ELISA, the addition of IGU and NIR-triggered release significantly reduced the gene expression levels of pro-inflammatory factors (ik). Figure 5 The ln in the figure highlights the synergistic advantages of targeted drug delivery and photothermal response controlled release.

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

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

[0083] A CIA mouse model was constructed, following the same procedure as in step "3.3" (in vivo fluorescence imaging). The CIA mouse model was then divided into six 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 for the CIA mouse model was as follows: Figure 6 As shown in a; healthy mice were used as the Healthy group.

[0084] PBS group: 0.2 mL of PBS was injected.

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

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

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

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

[0089] MM@PCNSs / IGU+NIR group: MM@PCNSs / IGU (IGU 5mg / kg) was injected 24 hours later and then irradiated with NIR for 10 minutes.

[0090] 5.2) Experimental methods and results

[0091] By day 45 of the experiment, the MM@PCNSs / IGU+NIR group showed the most significant improvement in arthritis severity, with significant reductions in both arthritis scores and foot swelling thickness. Figure 6 These results demonstrate that MM@PCNSs / IGU+NIR effectively inhibited the progression of arthritis in CIA mice. The significant reduction in joint inflammation and swelling indicates that the synergistic effect of IGU and NIR-responsive MM@PCNSs promoted 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] This invention performed histological analysis on the mouse ankle joint, using H&E, Masson staining, and Safranin-O staining. Figure 6(e). In the healthy group, the synovial tissue structure of the knee joint was normal, the articular cartilage surface was smooth, and the bone tissue was intact. In contrast, the PBS group showed a significant inflammatory response, including synovial tissue hyperplasia, inflammatory cell infiltration (indicated by black arrows in Masson staining), cartilage layer damage (indicated by black arrows in Safranin O staining), and invasion into the subchondral bone tissue. In contrast, the MM@PCNSs / IGU+NIR group showed minimal inflammatory cell infiltration, mild synovial tissue hyperplasia, no bone invasion, and a smooth cartilage surface. These findings indicate that MM@PCNSs / IGU+NIR can effectively inhibit the progression of RA-related joint damage, protect cartilage integrity, and reduce inflammatory cell infiltration. The inflammatory targeting of MM@PCNSs and its controlled release of IGU ensure local efficacy while reducing systemic side effects.

[0093] Bone erosion is an important indicator for assessing the severity of arthritis, and this invention uses micro-CT technology to evaluate it. Results showed that CIA mice treated with PBS exhibited severe bone erosion. Figure 6 In the study, bone mineral density (BMD), bone volume fraction (BV / TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) all decreased significantly, while intertrabecular spacing (Tb.Sp) increased significantly. Figure 6 (gk in the text). After treatment, all the above parameters improved. Especially in the MM@PCNSs / IGU+NIR treatment group, bone erosion was minimal, and all bone structure indicators almost returned to normal levels, indicating that its protective effect on bone structure was the most significant.

[0094] To evaluate the anti-inflammatory effects of different treatment regimens, this 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 Figure 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, indicating a significant inflammatory response in CIA mice. Notably, the levels of these cytokines were significantly reduced in the MM@PCNSs / IGU+NIR treatment group, which was superior to other treatment groups, indicating that this formulation is effective in suppressing RA inflammation. This synergistic anti-inflammatory effect is attributed to the presence of IGU and the ability of MM@PCNSs to neutralize pro-inflammatory cytokines. In addition, the photothermal properties of MM@PCNSs enhance tissue penetration and release of the drug, further improving the therapeutic effect. This invention also evaluated the mRNA expression levels of IL-1β, IL-6, and TNF-α in synovial tissue. Figure 7(df ​​in the text). Consistent with the protein level results, the gene expression levels of these three inflammatory factors in the MM@PCNSs / IGU+NIR group were significantly lower than those in other groups. Furthermore, the expression levels of IL-1β, IL-6, and TNF-α in joint tissue were detected by immunofluorescence staining. Figure 7 The results (g) also support the effectiveness of this treatment regimen in reducing inflammation. These findings indicate that MM@PCNSs / IGU+NIR can effectively alleviate RA-related joint inflammation by regulating cytokine production and inhibiting the inflammatory response.

[0095] Finally, the chronic toxicity of MM@PCNSs / IGU was evaluated at the end of the experiment. Mice treated with MM@PCNSs / IGU+NIR maintained stable body weight. Figure 8 (a) In addition, the mice's routine blood indicators (RBC, WBC, PLT) and serum biochemical indicators (ALT, AST, BUN) were similar to those of the healthy group. Figure 8 (bg in the text). Histological analysis showed no significant organ pathological abnormalities observed after treatment with MM@PCNSs / IGU or MM@PCNSs / IGU+NIR. Figure 8 The h in the figure indicates that MM@PCNSs / IGU has good biocompatibility and is suitable for long-term use in RA therapy.

[0096] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope 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 is a porous carbon nanosphere coated with a macrophage membrane; The method for preparing the drug delivery system comprises the following steps: Aqueous polyacrylic acid, zinc oxide, and isopropanol were mixed evenly to obtain PAA-Zn NPs; The PAA-Zn NPs were carbonized to obtain PCNSs; The PCNSs and macrophage membrane fragments are mixed evenly to obtain the drug delivery system.

2. The method for preparing the drug delivery system according to claim 1, characterized in that, The preparation method consists of the following steps: Aqueous polyacrylic acid, zinc oxide, and isopropanol were mixed evenly to obtain PAA-Zn NPs; The PAA-Zn NPs were carbonized 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 isopropanol 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℃ at a heating rate of 5℃ / min, and calcining for 3 hours.

5. The use of the drug delivery system of claim 1 in a delivery system for preparing a drug for treating rheumatoid arthritis.

6. A drug for treating rheumatoid arthritis, characterized in that, The drug includes the drug delivery system of claim 1 and the antirheumatic drug.

7. The drug according to claim 6, characterized in that, The antirheumatic drugs mentioned include ellamod.

8. The method for preparing the drug according to claim 6, characterized in that, The preparation method includes the following steps: The drug is obtained by mixing the solution containing the drug delivery system and the antirheumatic drug solution.

9. The preparation method according to claim 8, characterized in that, The mass ratio of the solution containing the drug delivery system to the antirheumatic 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 antirheumatic drug in the antirheumatic drug solution is 0.455 mg / mL.

Images