A pathological microenvironment self-driven targeted nanorobot, a preparation method and application thereof

Abstract

The application discloses a pathological microenvironment self-driven precise targeted nanorobot and a preparation method and application thereof, and is prepared according to the following steps: (1) preparing a MOF carrier; (2) preparing a MOF@ISL&CORM-401 compound; (3) preparing an apoptotic cell membrane; (4) preparing an FH peptide modified apoptotic cell membrane: coupling FH peptide with a sequence of FHKHKSPALSPV and DSPE-PEG-NHS, mixing with apM under ultrasonic, centrifugal purification, and obtaining FM; (5) preparing a nanorobot: mixing the MOF@ISL&CORM-401 and the membrane protein of FM, asymmetrically wrapping, extruding through the membrane, and centrifugal to obtain the nanorobot. The self-driven nanorobot with the triple functions of self-driving, precise targeting and synergistic treatment is designed, the CORM-401-ROS reaction is utilized to provide active power to break through the synovial membrane barrier, the engineered cell membrane is utilized to realize the targeted enrichment of core pathological cells, the ISL and CO are utilized to repair the mitochondrial function, and the OA pathological cycle is blocked, so that a novel technical scheme is provided for the treatment of osteoarthritis from the symptomatic relief to the pathological reversal.

Description

Technical Field

[0001] This invention relates to the field of biomedical nanomaterials technology, specifically to a self-driven, precisely targeted nanorobot for pathological microenvironments, its preparation method, and its applications. Background Technology

[0002] Osteoarthritis (OA) is a globally prevalent degenerative joint disease, with a prevalence exceeding 50% in people over 65 years of age and over 80% in those over 75 years of age. Its core pathological features include cartilage loss, subchondral bone remodeling, osteophyte formation, and chronic synovitis. Patients often experience joint pain, functional impairment, and even deformities, severely reducing their quality of life. Current clinical treatment primarily focuses on symptomatic relief: late-stage patients rely on joint replacement surgery, while early and mid-stage patients use conservative treatments such as nonsteroidal anti-inflammatory drugs (NSAIDs) and opioids. However, these methods only alleviate pain and cannot break the pathological cycle of "synovial inflammation-cartilage damage-inflammatory amplification," and they also have side effects such as gastrointestinal bleeding and osteoporosis, limiting their clinical application.

[0003] The synovium, as the core regulatory tissue of the joint cavity, plays a crucial role in the pathological process of osteoarthritis (OA). Under normal conditions, the synovium secretes hyaluronic acid-rich synovial fluid to maintain joint lubrication and cartilage nutrition. Under pathological conditions, the synovium exhibits pathological thickening, accompanied by extracellular matrix remodeling and inflammatory cell infiltration, forming a dense physical barrier that hinders drug penetration. The applicant previously confirmed through single-cell sequencing that synovial fibroblasts and pro-inflammatory macrophages are the core driving cells for OA progression: synovial fibroblasts highly express tenascin-C (TNC) and collagen type XIV alpha1 chain (COL14A1), and proliferate abnormally, promoting synovial fibrosis; synovial macrophages accumulate and secrete large amounts of inflammatory factors such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), accelerating cartilage matrix degradation. Dysfunction of synovial fibroblasts and synovial macrophages is closely related to mitochondrial dysfunction, specifically manifested as reduced activity of mitochondrial respiratory chain complexes, excessive production of reactive oxygen species (ROS), and impaired mitochondrial autophagy. These mitochondrial abnormalities not only disrupt normal cellular physiological functions but also exacerbate pathological cellular activation, forming a vicious cycle and becoming a key factor in the disruption of the pathological microenvironment of osteoarthritis (OA).

[0004] Nanomedicine delivery systems offer new avenues for precision treatment of osteoarthritis (OA), with metal-organic frameworks (MOFs) attracting significant attention due to their high drug loading capacity, controllable degradation, and environmental responsiveness. Zeolitic imidazolate framework-8 (ZIF-8), a typical MOF, can achieve controlled drug release in the acidic microenvironment of the OA synovium and is easily surface-modified to enhance targeting, making it an ideal carrier for both targeting core cells and regulating mitochondrial function. However, in the pathological state of OA, the thickened synovial barrier (composed of multiple layers of proliferating cells, cross-linked fibers, and inflammatory edema fluid) significantly reduces permeability. Traditional nanocarriers rely solely on Brownian motion for passive diffusion, making it difficult to penetrate deep into the lesion areas of the synovium, thus limiting therapeutic efficacy. Summary of the Invention

[0005] To address the aforementioned technical problems, the first objective of this invention is to provide a method for preparing a self-driven, precisely targeted nanorobot for pathological microenvironments. The second objective is to provide a self-driven, precisely targeted nanorobot for pathological microenvironments, and the third objective is to provide its applications. This invention designs a self-driven nanorobot that integrates the triple functions of "self-driven, precisely targeted, and synergistic therapy." It utilizes the reaction of carbon monoxide-releasing molecule-401 (CORM-401) with reactive oxygen species (ROS) to provide active propulsion to overcome the synovial barrier. Through engineered cell membranes, it achieves targeted enrichment of core diseased cells. With the synergistic effect of isoliquiritin (ISL) and carbon monoxide (CO), it repairs mitochondrial function, blocks the pathological cycle of osteoarthritis (OA), and provides a novel technical solution for treating osteoarthritis from "symptomatic relief" to "pathological reversal."

[0006] To achieve the first objective mentioned above, the present invention provides the following technical solution: a method for preparing a self-driven, precisely targeted nanorobot for pathological microenvironments, characterized by preparation according to the following steps:

[0007] (1) Preparation of MOF support: 2-methylimidazolium methanol solution and zinc nitrate hexahydrate methanol solution were mixed and reacted, centrifuged, washed and dried to obtain MOF support;

[0008] (2) Preparation of MOF complex (MOF@ISL&CORM-401) loaded with isoliquiritin (ISL) and carbon monoxide-releasing molecule-401 (CORM-401): MOF was soaked in a mixed solution of ISL and CORM-401, loaded with sonication, centrifuged, and washed to obtain MOF@ISL&CORM-401;

[0009] (3) Preparation of apoptotic cell membrane (apM): RAW264.7 cells were induced to apoptosis by staurosporine (STS), and the cell membrane was extracted and purified to obtain apM;

[0010] (4) Preparation of FH peptide-modified apoptotic cell membrane (FM): The FH peptide with the synthetic sequence FHKHKSPALSPV was coupled with distearate-phosphatidylethanolamine-polyethylene glycol-N-hydroxysuccinimide (DSPE-PEG-NHS), then mixed with apM, sonicated, and centrifuged to obtain FM;

[0011] (5) Preparation of nanorobots: MOF@ISL&CORM-401 and FM were mixed at a protein mass ratio of 1:4, asymmetrically wrapped by electrostatic attraction, squeezed through the membrane, and centrifuged to obtain FM@MOF@ISL&CORM-401 nanorobots.

[0012] In the above scheme: in step (1), the concentration of 2-methylimidazole solution is 0.35M, the concentration of zinc nitrate hexahydrate solution is 0.05M, after mixing, stir at room temperature for 30-40min, centrifuge at 12000rpm, wash with ultrapure water, and dry at 65-70℃; the particle size of MOF carrier is 200-300nm.

[0013] In the above scheme: in step (2), the ISL concentration is 0.1-1 mg / mL, the CORM-401 concentration is 0.1-1 mg / mL, the ultrasonic power is 300-400 W, and the loading time is 24 h.

[0014] In the above scheme: in step (3), the cell apoptosis rate is ≥85%; cell membrane extraction is performed using a kit + glass homogenate - centrifugation method.

[0015] In the above scheme: in step (4), the molar ratio of FH peptide to DSPE-PEG-NHS is 1:2, and the coupling reaction is stirred at room temperature in the dark for 10-20h; the mass ratio of the coupling compound to apM is 1:5, and the ultrasonic power is 200-300W.

[0016] In the above scheme: In step (5), the electrostatic attraction method for asymmetric encapsulation is as follows: the well plate is subjected to plasma treatment to make its surface negatively charged, MOF@ISL&CORM-401 is resuspended in aqueous solution and dropped into the well plate, and then a negatively charged FM membrane is slowly added. Asymmetric encapsulation of MOF@ISL&CORM-401 is achieved by means of electrostatic interaction. The well plate is placed in a 4°C environment for static incubation to ensure that the encapsulation is sufficient and stable. The membrane is squeezed and centrifuged to obtain FM@MOF@ISL&CORM-401 nanorobots; wherein, the concentration of MOF@ISL&CORM-401 suspension is 1 mg / mL.

[0017] A method for preparing a self-driven, precisely targeted nanorobot for pathological microenvironments, as described above, yields a self-driven, precisely targeted nanorobot for pathological microenvironments. This nanorobot possesses a bilayer structure of "MOF core-FM coating," with the core loaded with ISL and CORM-401, and the coating containing FH targeting peptides. It exhibits a movement speed ≥20 μm / s in a ROS environment and significantly increases its targeting efficiency against fibroblasts and pro-inflammatory macrophages in the synovial mesophyll.

[0018] Specifically, this invention uses an MOF as the core carrier, carrying CORM-401 and ISL. Utilizing the energy or material gradient of the high ROS reaction between CORM-401 and the OA synovium, it provides active propulsion for the nanorobots, enabling them to overcome the thickened synovial physical barrier. Through an FH peptide-modified apoptotic cell membrane coating, the nanorobots acquire the ability to precisely target fibroblasts and pro-inflammatory macrophages in the synovial mesolayer, reducing drug distribution to non-target tissues. Through the synergistic effect of CO generated by CORM-401 and ISL, it dually repairs mitochondrial dysfunction in core diseased cells, inhibits abnormal fibroblast proliferation, downregulates pro-inflammatory macrophage activity, and blocks the OA pathological cycle.

[0019] The application of a self-driven, precisely targeted nanorobot in the pathological microenvironment in the preparation of drugs for treating osteoarthritis. This invention's nanorobot can actively move and overcome the synovial barrier using the energy or material gradient from the reaction of CORM-401 with ROS. It targets core diseased cells through engineered cell membranes, and ISL and CO can synergistically repair mitochondrial function, inhibit abnormal fibroblast proliferation and macrophage pro-inflammatory factor secretion, thus blocking the vicious cycle of "synovial inflammation-cartilage damage-inflammatory amplification," thereby treating osteoarthritis.

[0020] The osteoarthritis referred to is post-traumatic osteoarthritis, primary degenerative osteoarthritis, metabolic-related osteoarthritis, or infection-related osteoarthritis. The administration method is intra-articular injection, which can penetrate the thickened synovial barrier, synergistically regulate mitochondrial function, inhibit synovial inflammation and fibroblast proliferation, and promote cartilage repair.

[0021] Post-traumatic osteoarthritis (PTAI) is secondary osteoarthritis caused by previous joint trauma (such as intra-articular fractures, ligament injuries, meniscus injuries, etc.). Primary degenerative osteoarthritis (PDAI) is a primary degenerative disease of articular cartilage caused by age or long-term joint strain without a clear cause. Metabolic osteoarthritis includes gouty osteoarthritis induced by hyperuricemia (abnormal uric acid metabolism) and secondary osteoarthritis caused by obesity (overweight leading to joint biomechanical imbalance). Infection-related osteoarthritis specifically refers to suppurative osteoarthritis caused by pathogens such as bacteria and fungi infecting the joint cavity, leading to purulent inflammation and secondary cartilage and bone destruction.

[0022] The nanorobots of this invention offer a solution for overcoming biological barriers through their active locomotion capabilities. They can convert environmental energy (such as chemical and light energy) into mechanical motion, achieving targeted accumulation of substances in lesions. In the high-ROS microenvironment of OA synovial membrane, carbon monoxide-releasing molecules (CORM-401) can react with ROS to generate carbon monoxide (CO). The accompanying energy / mass gradient can drive the directional migration of the nanosystems, while CO can regulate mitochondrial metabolism. Meanwhile, isoglycyrrhizin (ISL), a natural active compound, can inhibit abnormal proliferation of fibroblasts, downregulate the secretion of pro-inflammatory factors from macrophages, and repair mitochondrial damage by improving mitochondrial respiratory chain function and promoting mitophagy. Furthermore, cell membrane nanocoating technology can endow carriers with "biocamouflage" properties. By genetically engineering targeted molecules on the cell membrane surface, precise identification of specific diseased cells can be achieved, solving the problems of poor targeting and low biocompatibility of traditional nanomaterials.

[0023] Beneficial effects

[0024] Compared with the prior art, the present invention has the following beneficial effects: (1) It breaks through the limitations of the traditional nanocarrier “passive diffusion + single targeting” and integrates the triple mechanism of “pathological microenvironment self-driving - engineered membrane precise targeting - dual drug synergistic regulation” for the first time. With the high ROS of OA synovium as the signal source, it realizes the pathological adaptation closed loop of “power generation - targeted enrichment - drug efficacy”, and solves the core problems of difficult penetration of synovial barrier and poor targeting of target cells. (2) The MOF carrier has both high drug loading rate and acid response degradation, ensuring the precise release of drugs in synovial lesions; the FM coating not only provides targeting but also improves biocompatibility; the CORM-401-ROS reaction provides active power to break through the thickened synovial barrier. (3) ISL and CO synergistically repair mitochondrial function and simultaneously inhibit the abnormal proliferation of synovial fibroblasts and the pro-inflammatory activity of macrophages, blocking the OA cycle from the core pathological link. Unlike the traditional single anti-inflammatory or analgesic regimen, it can achieve “pathological reversal”. (4) All steps use conventional experimental equipment (centrifugation, ultrasound, electrostatic adsorption), the reaction conditions are mild, the product particle size is uniform, the repeatability is good (batch-to-batch difference rate ≤5%), and it is easy to scale up production and clinical translation. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the structural design and fabrication process of nanorobots.

[0026] Figure 2 Characterization of nanorobots. A. Representative transmission electron microscopy (TEM) images of MOF; B, C. Representative TEM images and energy dispersive X-ray spectroscopy (EDS) of MOF@ISL&CORM-401; D, E. Flow cytometry detection and statistical analysis of asteroidin-induced apoptosis efficiency in RAW264.7 macrophages; F, G. TEM images and representative immunofluorescence images of FM@MOF@ISL&CORM-401; H. Visualization analysis of total protein content in different groups stained with Coomassie brilliant blue; I, J. Particle size and zeta potential of different groups. * indicates statistically significant differences between groups (P<0.05, P<0.01, P<0.001).

[0027] Figure 3 To assess the self-driving performance of nanorobots: A. Trajectories of different groups of nanomaterials in conditioned medium (ROS secreted by cells); B. Mean square displacement of different groups of nanomaterials; C. Average velocity of different groups of nanomaterials; D. Diffusion coefficient of different groups of nanomaterials. * Indicates statistically significant differences between groups (P<0.05, P<0.01, P<0.001).

[0028] Figure 4 To illustrate the in vitro targeting and cellular regulation effects of nanorobots. Representative immunofluorescence images and flow cytometry analysis of AF.RAW264.7 macrophages and FLSs cells after co-incubation with different nanomaterials; FLSs cells were stimulated with IL-1β, and RAW264.7 cells were stimulated with LPS to simulate an inflammatory state; cell nuclei were stained with DAPI (blue), and cell membranes were labeled with DiI (red); FITC-labeled nanoparticles exhibited green fluorescence. Scale bar: 20 μm; G. Real-time quantitative polymerase chain reaction was used to detect the gene expression levels of M1 polarization markers (such as inducible nitric oxide synthase and tumor necrosis factor-α) and M2 polarization markers (such as arginase-1, mannose receptor, interleukin-10, and interleukin-13) in RAW264.7 macrophages after co-incubation with different nanomaterials; H. Cell proliferation was detected in each group after different FLSs treatments using a cell proliferation assay kit; I. Cell migration in each group after different FLSs treatments.

[0029] Figure 5To repair mitochondrial function in cells using nanorobots. The following are bar charts showing the dynamic changes in oxygen consumption rate (OCR) and key indicators of mitochondrial respiratory function in A and B RAW.264.7 cells after different treatments. Experimental groups included: Group I (blank control), Group II (lipopolysaccharide (LPS) stimulation group), and Group VII (LPS combined with FM@MOF@ISL & CORM-401 co-treatment group). During the detection process, Oligomycin (oligomycin, an inhibitor of mitochondrial ATP synthase), FCCP (an uncoupling agent to induce maximal respiration), and Rotenone & antimycin A (rotenone & antimycin A, inhibitors of complexes I and III, blocking the mitochondrial respiratory chain) were added sequentially. OCR units were measured in pmol / min. Detection indicators included basal respiration, maximal respiration, ATP production, and proton leakage. The following are bar charts showing the dynamic changes in oxygen consumption rate (OCR) and key indicators of mitochondrial respiratory function in C and D FLSs cells after different treatments. The experimental groups were as follows: Group I was the blank control group; Group II was the interleukin-1β (IL-1β) stimulation group; and Group VII was the IL-1β combined with FM@MOF@ISL&CORM-401 co-treatment group. During the detection process, Oligomycin (oligomycin, an inhibitor of mitochondrial ATP synthase), FCCP (an uncoupling agent to induce maximal respiration), and Rotenone & antimycin A (rotenone & antimycin A, inhibitors of complexes I and III, blocking the mitochondrial respiratory chain) were added sequentially. The OCR unit was pmol / min. The detection indicators included basal respiration, maximal respiration, ATP production, and residual respiratory capacity; * indicates statistically significant differences between groups (P<0.05, P<0.01, P<0.001).

[0030] Figure 6 To investigate how nanorobots can suppress synovial inflammation and treat osteoarthritis by regulating mitochondrial function. A. Representative images of hematoxylin and eosin stained synovial tissue from different treatment groups in an osteoarthritis model; B. Representative images of immunohistochemical staining of interleukin-1β (IL-1β), interleukin-6 (IL-6), and cytochrome c oxidase subunit IV in synovial tissue from different treatment groups in an osteoarthritis model, respectively; E. Representative images of safranin and fast green stained articular cartilage from different treatment groups in an osteoarthritis model. Detailed Implementation

[0031] The present invention will now be further described with reference to the accompanying drawings and embodiments.

[0032] Example 1

[0033] Fabrication of self-driven, precisely targeted nanorobots for pathological microenvironments:

[0034] 1. MOF support preparation: 0.4 mL of methanol solution containing 2-methylimidazole (0.35 M) and 0.2 mL of methanol solution containing zinc nitrate hexahydrate (0.05 M) were mixed, stirred at room temperature for 30-40 min, centrifuged at 12000 rpm for 5 min, washed three times with ultrapure water, and dried at 65-70℃ to obtain white MOF powder. TEM showed that it had a rhombic dodecahedral shape with a particle size of about 200-300 nm. Figure 2 A).

[0035] 2. Preparation of MOF@ISL & CORM-401: MOF powder was immersed in 1 mL of a methanol mixture of 0.5 mg / mL ISL and 0.5 mg / mL CORM-401. Ultrasonic loading was performed for 24 h at a power of 300-400 W. The MOF@ISL & CORM-401 was collected by centrifugation. TEM showed that it was a rhombic dodecahedral shape with a particle size of approximately 200-300 nm. EDS showed the presence of sulfur, as did CORM-401, indicating no other sulfur content, thus confirming successful loading. Figure 2 (B, C).

[0036] 3. Preparation of apoptotic cell membranes

[0037] RAW264.7 cells were treated with 1 μM STS for 24 h, and the apoptosis rate was 89.2% as determined by flow cytometry. Figure 2 After extracting apoptotic cell membranes from D and E, apoptotic cell membranes were obtained as apoptotic membranes (apM). Cell membranes were extracted using a kit (cell membrane extraction) + glass homogenate-centrifugation method, which is a standard procedure. The first centrifugation conditions were: 4°C, 3000 rpm for 10 min, and the supernatant was discarded. The second centrifugation conditions were: 4°C, 14000 g for 30 min, and the supernatant was discarded, and the cell membranes were collected.

[0038] 4. Preparation of FH peptide-modified apoptotic cell membrane (FM): The FH peptide with the synthetic sequence FHKHKSPALSPV was conjugated with DSPE-PEG-NHS at a molar ratio of 1:2. The conjugation reaction was carried out at room temperature in the dark with stirring for 10-20 h. Then, it was mixed with apoM and sonicated at a mass ratio of 1:5. The sonication power was 200-300 W, and the mixture was centrifuged at 10000 g to obtain FM.

[0039] 5. Fabrication of nanorobots

[0040] The membrane protein mass ratio of MOF@ISL&CORM-401 (MOF@ISL&CORM-401 suspension concentration of 1 mg / mL) to FM was 1:4. The mixture was asymmetrically encapsulated by electrostatic attraction, squeezed through a membrane, and centrifuged to obtain FM@MOF@ISL&CORM-401 nanorobots.

[0041] The specific steps of the electrostatic attraction asymmetric encapsulation method are as follows: First, the 6-well plate is plasma-treated to make its surface negatively charged. Then, MOF@ISL&CORM-401 is resuspended in aqueous solution (MOF@ISL&CORM-401 suspension concentration is 1 mg / mL) and dropped into the well plate. The nanoparticles are uniformly dispersed and fixed on the surface of the well plate by centrifugation and electrostatic adsorption. Then, a negatively charged FM membrane is slowly added. Asymmetric encapsulation of MOF@ISL&CORM-401 is achieved by electrostatic interaction. Finally, the well plate is placed in a 4°C environment for static incubation overnight to ensure that the encapsulation is sufficient and stable. Then, the membrane is squeezed and centrifuged to obtain FM@MOF@ISL&CORM-401 nanorobots.

[0042] TEM and immunofluorescence results showed FM asymmetric encapsulation of MOF@ISL&CORM-401 ( Figure 2 SDS-PAGE showed that the FH peptide mainly co-migrated with cell membrane proteins, proving the modification was successful. Figure 2 The particle size is approximately 200-300 nm, and the zeta potential is positive. Figure 2 (I, J).

[0043] Example 2

[0044] FM@MOF@ISL&CORM-401's motion performance

[0045] FM@MOF@ISL & CORM-401 were dispersed in a culture medium containing M1 macrophages and inflammatory fibroblasts (containing H2O2) at a concentration of 0.5 mg / mL. The medium was placed in an NTA container for observation, and the movement trajectory was recorded using a CCD camera. The results showed that the nanorobots exhibited directional movement with an average speed of approximately 29.14 μm / s, while in the medium without CORM-401, the speed was approximately 19 μm / s. This demonstrates that the nanorobots can obtain active propulsion through the reaction of CORM-401 with ROS (simulated H2O2). Figure 3 (AD).

[0046] Example 3

[0047] In vitro targeting and cell regulation effects of FM@MOF@ISL&CORM-401

[0048] We used laser confocal microscopy to observe the uptake of different nanoparticles (MOF, MOF@FITC, MOF@CORM-401&FITC, FM@MOF@CORM-401&FITC) by RAW264.7 cells. FM@MOF@CORM-401&FITC was used because ISL lacks fluorescence; to directly visualize that the nanoparticles were indeed engulfed by cells, we replaced ISL with FITC, thus allowing for a more direct observation of the faster uptake of the nanorobots without affecting the experimental results. The conclusions are as follows: Figure 4 As shown in Figure A, under both LPS stimulation (inflammation model) and no LPS stimulation conditions, the intracellular fluorescence signal (FITC-labeled nanoparticles) in the FM@MOF@CORM-401&FITC group was significantly stronger than that in other groups. Furthermore, flow cytometry analysis revealed that in the inflammatory state, the proportion and fluorescence intensity of fluorescently positive cells in the FM@MOF@CORM-401&FITC group were significantly higher than those in other groups, further demonstrating its efficient uptake and targeting in inflammatory cells. Figure 4 (Group information: I: Blank, II: MOF, III: MOF@FITC, IV: MOF@CORM-401&FITC, V: LPS+MOF, VI: LPS+MOF@FITC, VII: LPS+MOF@CORM-401&FITC).

[0049] Similarly, under IL-1β stimulation (inflammation model) FLS conditions, the intracellular fluorescence signal (FITC-labeled nanoparticles) in the FM@MOF@CORM-401 & FITC group was significantly stronger than that in other groups. Figure 4 (Group information: I: Blank, II: MOF, III: MOF@FITC, IV: MOF@CORM-401&FITC, V: IL-1β+MOF, VI: IL-1β+MOF@FITC, VII: IL-1β+MOF@CORM-401&FITC).

[0050] Detection of mRNA expression of macrophage M1 / M2 type markers ( Figure 4 The results showed that, compared with the LPS group (which promoted M1 polarization), the FM@MOF@ISL&CORM-401 group significantly upregulated the expression of M2 markers (mannose receptor, interleukin-10, interleukin-13, arginase 1) and significantly downregulated the expression of M1 markers (inducible nitric oxide synthase 2, tumor necrosis factor α), indicating that it can effectively induce macrophage polarization from M1 to M2, exerting anti-inflammatory and tissue repair phenotypic regulatory effects. This was demonstrated by the CCK-8 assay. Figure 4 H) and scratch test ( Figure 4I) Analysis of FLS cell proliferation and migration: The FM@MOF@ISL&CORM-401 group significantly inhibited FLS cell proliferation and migration. (Group information: I: MOF, II: IL-1β, III: IL-1β+MOF, IV: IL-1β+MOF@ISL, V: IL-1β+MOF@CORM-401, VI: IL-1β+MOF@ISL&CORM-401, VII: IL-1β+FM@ISL&CORM-401).

[0051] Example 4

[0052] Mitochondrial repair function of FM@MOF@ISL&CORM-401

[0053] Using RAW264.7 cells and FLSs (fibroblast-like synovial cells) as models, mitochondrial respiratory function was detected by cellular oxygen consumption rate (OCR). The specific experiments were as follows: A cellular energy metabolism analysis system was used to detect changes in OCR. Key indicators such as basal respiration, maximum respiration, ATP production, and proton leak were analyzed by sequentially adding Oligomycin (an ATP synthase inhibitor), FCCP (an uncoupling agent), and Rotenone & antimycin A (respiratory chain complex inhibitors). Figure 5 (A). The results showed that compared with LPS (Group II), the LPS combined with FM@MOF@ISL&CORM-401 treatment group (Group VII) showed significantly improved basal respiration, maximal respiration, proton leakage, and ATP production capacity, with all indicators approaching normal levels. This indicates that FM@MOF@ISL&CORM-401 can effectively repair mitochondrial respiratory function in RAW264.7 cells and restore energy metabolism homeostasis. Figure 5 (B). Similarly, mitochondrial function in FLSs stimulated by IL-1β was analyzed using OCR, assessing basal respiration, maximal respiration, ATP production, and spare respiratory capacity. Figure 5 The results showed that mitochondrial functional indicators were significantly impaired in the IL-1β stimulation group (Group II), while basal respiration, maximal respiration, ATP production capacity, and reserve respiration capacity of the IL-1β combined with FM@MOF@ISL&CORM-401 treatment group (Group VII) were significantly restored, approaching normal levels. Figure 5(D). This indicates that FM@MOF@ISL&CORM-401 can effectively repair IL-1β-induced mitochondrial functional damage in FLSs and restore their energy metabolism potential. In summary, FM@MOF@ISL&CORM-401 can significantly repair mitochondrial function in RAW264.7 cell FLSs in vitro, restoring mitochondrial respiration capacity and energy metabolism levels.

[0054] Example 5

[0055] FM@MOF@ISL&CORM-401 In Vitro Treatment of Osteoarthritis

[0056] An osteoarthritis (OA) model of the knee joint was established in mice by transection of the anterior cruciate ligament (ACL). Four weeks post-surgery, nanomaterials were injected intra-articularly three times per week for four consecutive weeks. Mice that underwent ACL transection using MOF@ISL, MOF@CORM-401, MOF@ISL&CORM-401, or FM@MOF@ISL&CORM-401 were used as OA knee joints, while mice in the sham-operated group served as normal knee joints. All mice were treated with saline at the same time point. Four weeks after ACL transection, mice were sacrificed, and the right knee joint was harvested for examination. After decalcification, embedding, and sectioning, hematoxylin and eosin staining was used to observe synovial inflammation and hyperplasia, and safranin and fast green staining was used to observe cartilage degeneration. The staining results showed that the synovial tissue in the ACL transection group was significantly thickened, with significant inflammatory cell infiltration; the ACL transection + FM@MOF@ISL&CORM-401 group showed reduced synovial thickening and decreased inflammatory cell infiltration. Figure 6 Immunohistochemical results showed that, compared with the anterior cruciate ligament transection group, the expression of macrophage pro-inflammatory markers (interleukin-1β, interleukin-6) was significantly decreased and the expression of mitochondrial protein cytochrome c oxidase IV was significantly increased in the anterior cruciate ligament transection + FM@MOF@ISL&CORM-401 group. Figure 6 (BD). Safranin-Fix Green staining results showed that cytochrome c oxidase IV stained the cartilage matrix lightly, the cartilage surface was uneven, and the number of chondrocytes was reduced; the nanorobot group stained the cartilage matrix deeply, the cartilage surface was smoother, the number of chondrocytes increased, and the degree of cartilage degeneration was significantly reduced. Figure 6 (Group information: I: sham surgery group, II: anterior cruciate ligament transection group, III: anterior cruciate ligament transection + MOF@ISL group, IV: anterior cruciate ligament transection + MOF@CORM-401 group, V: anterior cruciate ligament transection + MOF@ISL&CORM-401 group, VI: anterior cruciate ligament transection + FM@MOF@ISL&CORM-401 group).

[0057] The self-driven, detachable biomimetic nanorobot (FM@MOF@ISL&CORM-401) prepared in this invention has good physicochemical properties and active movement capabilities. It can precisely target the core cells of OA synovial membrane, synergistically regulate mitochondrial function, effectively alleviate the pathological progression of OA, and provide a new and effective solution for OA treatment.

[0058] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a self-driven, targeted nanorobot for pathological microenvironments, characterized in that, Prepare according to the following steps: (1) Preparation of MOF support: 2-methylimidazolium methanol solution and zinc nitrate hexahydrate methanol solution were mixed and reacted, centrifuged, washed and dried to obtain MOF support; (2) Preparation of MOF@ISL&CORM-401 complex: MOF was soaked in a mixed solution of isoliquiritin ISL and CORM-401, ultrasonically assisted loading, centrifuged, and washed to obtain MOF@ISL&CORM-401; (3) Preparation of apoptotic cell membrane: RAW264.7 cells were induced to apoptosis by astrosporin STS, and the cell membrane was extracted and purified to obtain apM; (4) Preparation of FH peptide-modified apoptotic cell membrane: The FH peptide with the synthetic sequence FHKHKSPALSPV was coupled with DSPE-PEG-NHS, then mixed with apM, sonicated, and centrifuged to obtain FM; (5) Preparation of nanorobots: The membrane proteins of MOF@ISL&CORM-401 and FM were mixed at a ratio of 1:4, asymmetrically wrapped by electrostatic attraction, squeezed through the membrane, and centrifuged to obtain FM@MOF@ISL&CORM-401 nanorobots.

2. The method for preparing the self-driven, targeted nanorobot for pathological microenvironments according to claim 1, characterized in that: In step (1), the concentration of 2-methylimidazole solution is 0.35M, the concentration of zinc nitrate hexahydrate solution is 0.05M, after mixing, stir at room temperature for 30-40 min, centrifuge at 12000 rpm, wash with ultrapure water, and dry at 65-70℃; the particle size of MOF carrier is 200-300 nm.

3. The method for preparing the self-driven, targeted nanorobot for pathological microenvironments according to claim 2, characterized in that: In step (2), the ISL concentration is 0.1-1 mg / mL, the CORM-401 concentration is 0.1-1 mg / mL, the ultrasonic power is 300-400 W, and the loading time is 24 h.

4. The method for preparing the self-driven, targeted nanorobot for pathological microenvironments according to claim 3, characterized in that: In step (3), the apoptosis rate is ≥85%.

5. The method for preparing the self-driven, targeted nanorobot for pathological microenvironments according to claim 4, characterized in that: In step (4), the molar ratio of FH peptide to DSPE-PEG-NHS is 1:2, and the coupling reaction is stirred at room temperature in the dark for 10-20 hours; the mass ratio of the coupling compound to apM is 1:5, and the ultrasonic power is 200-300W.

6. The method for preparing the self-driven, targeted nanorobot for pathological microenvironments according to any one of claims 1-5, characterized in that, In step (5), the electrostatic attraction asymmetric encapsulation method is as follows: the well plate is subjected to plasma treatment to make its surface negatively charged, MOF@ISL&CORM-401 is resuspended in aqueous solution and dropped into the well plate, and then a negatively charged FM membrane is slowly added. Asymmetric encapsulation of MOF@ISL&CORM-401 is achieved by means of electrostatic interaction. The well plate is placed in a 4°C environment for static incubation to ensure that the encapsulation is sufficient and stable. The membrane is squeezed and centrifuged to obtain FM@MOF@ISL&CORM-401 nanorobots; the concentration of MOF@ISL&CORM-401 suspension is 1 mg / mL.

7. A method for preparing a pathological microenvironment self-driven targeted nanorobot according to any one of claims 1-6, resulting in a pathological microenvironment self-driven targeted nanorobot.

8. The use of the pathological microenvironment self-driven targeted nanorobot of claim 7 in the preparation of a drug for treating osteoarthritis.

9. The application according to claim 8, characterized in that: The osteoarthritis referred to is post-traumatic osteoarthritis, primary degenerative osteoarthritis, metabolic-related osteoarthritis, or infection-related osteoarthritis.

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