Application of biomimetic targeted nano-controlled release carrier in preparation of osteoarthritis treating drugs

Abstract

This invention discloses the application of a biomimetic targeted nano-controlled release carrier in the preparation of drugs for treating osteoarthritis, belonging to the field of biomedical technology. The biomimetic targeted nano-controlled release carrier comprises a thermoresponsive nanocore and a functionalized cell membrane, with the functionalized cell membrane encapsulating the thermoresponsive nanocore. The thermoresponsive nanocore comprises magnetic iron oxide nanomaterials, an active drug, and a surface modifier. This invention utilizes genetic engineering technology to modify cells to overexpress Anti-Col1, extracting the cell membrane to encapsulate the nano-controlled release core and constructing a biomimetic targeted nano-controlled release system. This system precisely controls the heating temperature and time of the joint in the atrial fibrillary membrane (AMF) under magnetic field guidance after intravenous injection, achieving synergistic treatment of osteoarthritis (OA) drugs and magnetothermal therapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of biomimetic targeted nano-controlled release carriers in the preparation of drugs for the treatment of osteoarthritis. Background Technology

[0002] Osteoarthritis (OA) is a common chronic degenerative joint disease, predominantly affecting middle-aged and elderly individuals. It commonly impacts weight-bearing and highly active joints, severely threatening health and reducing quality of life. OA involves numerous pathological processes, characterized by progressive localized articular cartilage degeneration, joint inflammation, and pathological subchondral bone remodeling, leading to chronic pain, functional impairment, and even disability. However, due to limited understanding of its complex pathogenesis, effective medical methods for curing OA are still lacking in clinical practice.

[0003] Administering anti-inflammatory drugs is a conservative treatment method that protects articular cartilage, relieving symptoms and controlling disease progression. Osteoarthritis (OA) treatment involves both local intra-articular injection and systemic administration. Intra-articular injection has become the primary method of OA treatment, but it frequently causes adverse reactions such as poor medication adherence and tissue damage from repeated injections. Systemic administration requires high doses of medication orally or by injection to achieve effective local drug concentrations in bone tissue, which may lead to systemic toxicity in other organs or tissues. For example, long-term use of nonsteroidal anti-inflammatory drugs (NSAIDs) and opioid analgesics may cause gastrointestinal ulcers and addiction. To improve drug concentration at the joint site, developing drug delivery vehicles that target articular cartilage is one of the most effective strategies. Furthermore, drug delivery vehicles can protect the drug from degradation, improve drug solubility, and prolong systemic circulation and residence time, resulting in better OA treatment outcomes.

[0004] Furthermore, previous research indicates that localized hyperthermia of joints can not only promote blood circulation around the joint, reduce muscle spasms, and relieve pain, but also accelerate tissue regeneration and help reduce inflammation. Currently used hyperthermia methods include hot compresses and infrared irradiation. However, hot compresses are inefficient; near-infrared lasers have limited penetration depth within the body and can easily damage the skin at the application site. Therefore, developing drug delivery carriers that target articular cartilage in combination with hyperthermia is of great significance for the treatment of osteoarthritis (OA).

[0005] In recent years, nanotechnology, especially nanomedicine delivery systems, has received widespread attention. Benefiting from their small size (1–100 nm), high specific surface area, good drug loading efficiency, and targeted delivery capabilities, nanomaterials have, to some extent, improved the targeting of systemic drug delivery, prolonged the dosing cycle, and reduced nonspecific adverse reactions. Many nanoparticle-based drug delivery systems, such as polymers, liposomes, dendritic macromolecules, polymer nanoparticles, and inorganic nanoparticles, have been studied for intravenous injection in the treatment of osteoarthritis (OA). These nanotherapeutic strategies not only improve drug targeting and delivery efficiency but also enhance drug solubility and stability. However, the short half-life of nanoparticles due to immune clearance is the biggest challenge faced by traditional nanomedicine delivery systems. To address this issue, researchers have modified the surface of nanomaterials with polymers to form a hydration layer, reducing interactions with body fluids and enhancing systemic circulation time. However, studies have shown that repeated administration of polymer-coated nanoparticles can induce an immune response, producing antibodies and increasing the risk of clearance by the immune system. Furthermore, the uncontrollable drug release in vivo by traditional drug delivery systems severely reduces drug efficacy, and long-term use may lead to decreased patient compliance and systemic side effects. In addition, traditional surface chemical modification methods are inefficient and involve complex preparation processes. Most importantly, most inorganic materials cannot be degraded and excreted from the body, leading to their accumulation on the reticuloendothelial system and causing long-term toxicity. Therefore, the drawbacks of traditional nano-targeted delivery systems, such as easy immune clearance, lack of controlled release, and poor safety, severely limit their application in OA treatment.

[0006] Magnetic iron oxide nanomaterials (IONPs) are nanoparticles with unique magnetic properties that respond to external magnetic fields. Currently, IONPs have achieved great success in the field of tumor hyperthermia due to their unique magnetic targeting and magnetocaloric effects. Furthermore, due to their unique physical properties and ability to act at the cellular level, IONPs have been extensively studied as drug delivery agents for over thirty years. After being loaded with drug molecules, IONPs can be guided to diseased tissue via an external magnet, thereby improving drug efficacy and minimizing side effects.

[0007] Based on the aforementioned unique advantages, this invention designs IONPs for the synergistic treatment of OA drugs and magnetothermal therapy. Summary of the Invention

[0008] One objective of this invention is to provide a biomimetic targeted nano-release carrier, comprising a thermoresponsive nanocore and a functionalized cell membrane, wherein the functionalized cell membrane is coated on the outside of the thermoresponsive nanocore; The thermally responsive nanocore comprises magnetic iron oxide nanomaterials, an active drug, and a surface modifier. The magnetic iron oxide nanomaterials are magnetic Fe3O4 with sodium citrate as a surface ligand, the active drug is chondroitin sulfate, and the surface modifier is posalogum F108. The functionalized cell membrane is a macrophage membrane overexpressing the Anti-Col1 gene.

[0009] The second objective of this invention is to provide a method for preparing the above-mentioned biomimetic targeted nano-controlled release carrier, comprising the following steps: Step 1: Construct macrophages overexpressing the Anti-Col1 gene and extract the cell membrane; Step 2: Magnetic Fe3O4 with sodium citrate as the surface ligand, chondroitin sulfate and posalogum F108 are added to water to react and obtain thermally responsive nanocores. Step 3: Encapsulate the thermoresponsive nanocore with the cell membrane from Step 1 to obtain the biomimetic targeted nano-controlled release carrier.

[0010] Furthermore, in step 2, the concentrations of magnetic Fe3O4, chondroitin sulfate, and posalogne F108 with sodium citrate as the surface ligand were 50 mg / mL, 140 mg / mL, and 1%, respectively.

[0011] Furthermore, in step 3, the mass concentrations of the cell membrane and the thermally responsive nanonucleus are 1:1.

[0012] The third objective of this invention is to provide the application of the above-mentioned biomimetic targeted nano-controlled release carrier as an active ingredient in the preparation of osteoarthritis treatment drugs used in conjunction with navigation magnets and alternating magnetic fields.

[0013] Furthermore, the biomimetic targeted nano-controlled release carrier is the sole active ingredient.

[0014] Furthermore, the alternating magnetic field is set at 403 kHz.

[0015] This invention utilizes genetic engineering technology to modify cells to overexpress Anti-Col1, extracts the cell membrane to encapsulate a nano-controlled release core, and constructs a biomimetic targeted nano-controlled release system. This system precisely controls the heating temperature and time of the joint in the AMF (arterial magnetic field) after intravenous injection and magnetic field guidance, achieving synergistic treatment of OA (osteoarthritis) drugs and magnetothermal therapy. Attached Figure Description

[0016] Figure 1 This is a schematic diagram illustrating the synergistic drug and magnetothermal treatment of arthritis using the biomimetic targeted nano-controlled release carrier of this invention.

[0017] Figure 2 SEM particle size analysis of MNFC@CM. Wherein, (a) represents MNs, and (b) represents MNFC@CM.

[0018] Figure 3 The results show the biocompatibility of MNFC@CM. Among them, (a) shows the survival rate of chondrocytes, endothelial cells and macrophages after treatment with different concentrations of material (n = 5), and (b) shows the expression of inflammatory factors in different treatment groups after LPS-induced macrophage activation.

[0019] Figure 4 The effects of intra-articular injection of IONPs on the knee joint in the AMF. Among them, (a) shows the heating of the mouse knee joint in an alternating magnetic field, and (b) shows the temperature change curve of the mouse knee joint.

[0020] Figure 5 In the image, (a) shows the HE staining of a synovial section, and (b) shows the HE and Safranin-Fix-Green staining of an articular cartilage section.

[0021] Figure 6 This shows the heating effect on the mouse knee joint in an alternating magnetic field.

[0022] Figure 7 HE and Safranin-Fixed Green staining images of knee joint tissue sections. Detailed Implementation

[0023] Developing drug delivery carriers targeting articular cartilage in combination with hyperthermia is of great significance for the treatment of osteoarthritis (OA). However, traditional nano-targeted delivery systems suffer from drawbacks such as easy immune clearance, lack of controlled release, and poor safety. Magnetic iron oxide nanomaterials (IONPs) have been approved for cancer treatment in Europe due to their unique magnetocaloric effect, magnetic targeting, and biocompatibility. Preliminary experiments by the inventors showed that IONPs, after intra-articular injection, could precisely heat the knee joint of mice in an alternating magnetic field (AMF); they could also load drugs and reach the knee joint via intravenous injection under magnetic field guidance, but the enrichment content in the joint remained low. The reasons for this may be: ① Most IONPs are cleared by the immune system after being injected into mice via the tail vein, resulting in a very low concentration of material entering the bloodstream; ② Although IONPs themselves have some magnetic targeting properties, their targeting efficiency to the joint in vivo remains poor. Therefore, modifying IONPs to enhance joint targeting and reduce immunogenicity may enable synergistic treatment of OA drugs and magnetocaloric therapy.

[0024] Posalogum F108 (PF108) is a thermosensitive polymer that can be used to prepare temperature-sensitive controlled-release systems. It not only stably loads drugs at 37°C and physiological pH, but also expands and releases the drug at 41.8°C. Furthermore, it can act as a drug carrier, highly dispersing the drug and increasing its loading capacity. Therefore, PF108 can be used to modify IONPs to prepare magnetothermal-responsive nano-controlled-release systems. Applying AMF can induce hyperthermia, which not only enables long-range, on-demand drug release, enhancing targeted delivery, avoiding adverse exposures, and improving drug efficacy, but also provides therapeutic benefits.

[0025] Studies have shown that cell membrane-encapsulated nanoparticles not only exhibit good homing ability but also possess advantages such as low immunogenicity and long internal circulation time. Furthermore, modifying cell membranes using genetic engineering techniques can enhance their original functions or endow them with additional functional ligands, thereby strengthening targeting and further prolonging circulation time. Literature indicates that in early-stage osteoarthritis (OA), the surface of damaged cartilage highly expresses type I collagen (Col1). Encapsulating materials with Anti-Col1-overexpressing cell membranes can enhance joint targeting by binding to the specific recognition of receptors by antibodies. Therefore, this invention designs a biomimetic targeted nano-controlled-release system by modifying cells to overexpress Anti-Col1 and then extracting the cell membrane to encapsulate a nano-controlled-release core. This system precisely controls the heating temperature and time of the joint in the atrial fibrillary membrane (AMF) under magnetic field guidance after intravenous injection, achieving synergistic treatment of OA drugs and magnetothermal therapy.

[0026] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0028] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Example

[0029] I. Experimental Methods 1. Using genetically engineered cells to extract functionalized cell membranes, and constructing a biomimetic targeted nano-release system based on IONPs. (1) Construction and gene expression verification of Anti-Col 1 overexpression cells Anti-Col1 stable cell lines were established by overexpressing the Anti-Col1 gene in macrophages using slow-virus infection (Shanghai Heyuan Biotechnology Co., Ltd.).

[0030] (2) Extraction of Anti-Col 1 functionalized cell membrane Hypotonic lysis buffer containing 30 mM Tris-HCl (pH 7.5), 225 mM mannitol, 75 mM sucrose, 0.2 mM EGTA, and a mixture of protease and phosphatase inhibitors was added to the constructed Anti-Col1 functionalized macrophages. After incubation at room temperature for 15 minutes, the cells were homogenized multiple times using a Dunns homogenizer. The homogenized solution was centrifuged at 10,000 g for 25 min at 4 °C. The precipitate was discarded, and the supernatant was centrifuged again at 150,000 g for 35 min at 4 °C. After centrifugation, the membrane was resuspended once with EDTA and collected by centrifugation at 150,000 g for 35 min at 4 °C. Finally, the membrane was resuspended in 1 ml of 0.2 mM EDTA. Membrane protein concentrations were determined using a BCA kit. The membrane suspension was stored at -80 °C for subsequent use.

[0031] (3) Preparation and performance analysis of thermoresponsive nano-controlled release cores based on IONPs drug loading ① Preparation of CA@Fe3O4 (MNs) core stabilized by sodium citrate (CA) FeCl3·6H2O (6.48 g, 24.0 mmol) and trisodium citrate (1.2 g, 4.08 mmol) were dissolved in ethylene glycol (120 mL). NaAc (1.20 g) was added, and the mixture was stirred at 1000 rpm / min for 30 min. The resulting solution was sealed in PTFE-lined stainless steel autoclaves (25 mL × 8), heated at 200°C for 8 hours, and then cooled to room temperature. After removing the ethylene glycol, the black product was washed with ethanol and deionized water, and then dried to obtain the core CA@Fe3O4 (MNs).

[0032] ②Preparing thermoresponsive nano-controlled-release cores by modifying CA@Fe3O4 with PF108 to support drugs The prepared CA@Fe3O4 was surface-modified with posalogum F108 (PF108) and simultaneously loaded with chondroitin sulfate (Chs) to construct a thermoresponsive nano-controlled-release core. The specific experimental procedure is as follows: CA@Fe3O4 (50 mg / mL), Chs (140 mg / mL), and PF108 (1%) were mixed in 3 mL of water and stirred at room temperature and pH = 7 for 24 h. Excess Chs was removed by slow dialyzing using a dialysis bag. After dialysis, the brown solution (CA@Fe3O4@PF108-Chs) was collected and stored at 4 ℃ for later use.

[0033] (4) A biomimetic targeted nano-release system was constructed by encapsulating CM, and its characterization and performance analysis were performed. ① Construction of the biomimetic targeted nano-release system CA@Fe3O4@PF108-Chs@CM (MNFC@CM) The extracted functionalized cell membranes (based on protein concentration) were mixed with CA@Fe3O4@PF108-Chs at a mass concentration ratio of 1:1 and sonicated at room temperature for 10 min. The CA@Fe3O4@PF108-Chs@CM was collected using a magnet and resuspended in secondary water for later use.

[0034] ②Characteristics and performance analysis of biomimetic targeted nano-controlled release system Basic characterization analysis: The morphology, size, particle size distribution and zeta potential of the biomimetic targeted nano-release system were analyzed by scanning electron microscopy (SEM) and dynamic light scattering (DLS).

[0035] 2. Construct cell models to evaluate targeting and anti-inflammatory effects in vitro. (1) Evaluation of cell compatibility First, the cytotoxicity and IC50 of CA@Fe3O4@PF108-Chs@CM were investigated using the CCK-8 assay. 50 The values ​​were measured, and the specific operating steps are as follows: chondrocytes, macrophages, and endothelial cells were seeded in 96-well plates (1×10⁶ cells / well). 4 Cells / well were incubated at 37 °C for 24 h with 5% CO2. Cells were then co-incubated with dual-targeting IONPs (0.5, 2.0, 5.0, 10, 20, 50 μg / mL) containing different iron concentrations for 12 h, followed by incubation with CCK-8 for 2 h. Cell viability was measured using a microplate reader, and the IC50 was determined. 50 The value was used to analyze the cytotoxicity of the material in vitro.

[0036] (2) Evaluation of anti-inflammatory activity This invention aims to construct a cellular inflammation model by stimulating RAW 264.7 cells with LPS, and to verify the anti-inflammatory effect of a biomimetic targeted nano-controlled release system in vitro. The specific procedure is as follows: RAW 264.7 cells are seeded in 12-well plates at a density of 1 × 10⁻⁶ cells / well. 5 Cells were cultured at 37℃ for 24 h. After stimulating cells with 200 ng / mL LPS for 24 h, the culture medium was removed, and fresh DMEM medium containing Chs or CA@Fe3O4@PF108-Chs@CM was added to each well. The magnetothermal treatment group was placed in an alternating magnetic field at 403 kHz for 10 min, incubated for 24 h, washed twice with PBS, and treated with 500 μL Trizol reagent at 4℃ for 30 min. The mRNA expression levels of inflammatory factors TNF-α, IL-1β, and IL-6 were detected by quantitative RT-PCR.

[0037] 3. Establish an OA mouse model and evaluate in vivo efficacy through intra-articular and intravenous administration. (1) After administering medication via intra-articular injection and intravenous injection with magnetic field navigation, the heating temperature and time of the knee joint were precisely controlled. Taking into account the analysis results of magnetothermal efficiency, drug encapsulation, release efficiency and in vitro cellular anti-inflammatory efficacy, a high-performance biomimetic targeted nano-controlled release system was selected. By changing the material concentration, the targeting time of the navigation magnetic field and the power of the alternating magnetic field, the heating temperature of the mouse knee joint was precisely controlled.

[0038] Material concentration: The knee joints of mice injected with CA@Fe3O4@PF108-Chs@CM (1 time / day, 10 mg / mL / time) for different days were placed in an alternating magnetic field of 403 KHz, and the temperature changes of the knee joints were recorded using an infrared thermal imager.

[0039] Navigation magnetic field processing time: Mice were injected intravenously with CA@Fe3O4@PF108-Chs@CM (10 mg / mL) containing the same iron mass concentration (N=5). The magnetic targeting time of the magnet at the knee joint was varied, and the temperature change of the knee joint was recorded by infrared thermal imager in an alternating magnetic field of 403 kHz.

[0040] (2) Evaluation of therapeutic effect An OA mouse model was established, and the therapeutic effects of OA in mice were evaluated using behavioral, molecular biological, histopathological, immunohistochemical, and imaging methods.

[0041] Establishment of OA mouse model: Eight-week-old male ICR mice were selected, and after anesthesia, the knees were flexed at 90 degrees. 0 0.1 mg MIA (dissolved in 10 μl of sterile saline) was injected into the joint cavity of one knee through the patellar ligament using a 26-30G microsyringe. After injection, the knee joint was flexed and extended to distribute the drug solution evenly.

[0042] Histopathology: Mouse knee joints were fixed in 10% paraformaldehyde for 24 h and decalcified in 20% EDTA solution for 2 months. The specimens were then dehydrated and embedded in paraffin. Subsequently, the specimens were sectioned into 5.0 μm sections along the sagittal plane and stained with HE and Safranin O Fast Green. Histological changes were observed under a light microscope.

[0043] II. Experimental Results In this embodiment, CA@Fe3O4 (MNs) with a particle size of 70 nm was prepared, modified with PF108 and loaded with drugs, and then encapsulated with a cell membrane to obtain CA@Fe3O4@PF108-Chs@CM (MNFC@CM). The heating effect on the knee joint of mice and the therapeutic effect on OA were then investigated, and detailed descriptions are as follows: (1) Material synthesis and characterization The morphology and particle size of the prepared core-shell structured nanomaterials were characterized using scanning electron microscopy (SEM). Figure 2 As shown, the uncoated MNs core exhibits a relatively uniform (spherical / quasi-spherical) morphology, with particle sizes mainly distributed in the range of 60–80 nm, and an average particle size of approximately 70 nm. After coating with the outer layer material, the overall particle size of MNFC@CM significantly increases, with an average particle size of approximately 90 nm, and the particle size distribution is concentrated between 80–100 nm. The comparison before and after coating shows that the outer layer was successfully coated on the surface of the iron oxide core, and the coated particles still maintain good dispersion without obvious agglomeration. This result confirms the successful construction of core-shell structured nanomaterials.

[0044] (2) Validation of anti-inflammatory effects using in vitro cell models First, the cytotoxicity of MNFC@CM against chondrocytes, endothelial cells, and macrophages was determined using the CCK-8 assay. Figure 3 As shown in Figure a, MNFC@CM exhibits almost no toxicity to endothelial cells and macrophages at low concentrations, while demonstrating significant proliferative effects at high concentrations. For chondrocytes, compared to the control group, viability remained above ~90% even at the highest concentration of 50 μg / mL (Fe). These results indicate that MNFC@CM has good biocompatibility.

[0045] Based on the excellent in vitro cell compatibility of MNFC@CM, a cellular inflammation model was further constructed by stimulating RAW 264.7 cells with LPS, and the anti-inflammatory effect was verified in vitro. Figure 3 As shown in Figure b, compared with the control group, the expression levels of inflammatory factors (TNF-α, IL-1β, and IL-6) were higher in the MNFC@CM+AMF group due to LPS-induced macrophage activation; the expression of inflammatory factors was significantly reduced in the MNFC@CM+AMF group. These results indicate that MNFC@CM combined with magnetothermal therapy can inhibit the inflammatory microenvironment at the cellular level.

[0046] (3) After intra-articular administration, IONPs can precisely heat the knee joint and reduce OA. ① After intra-articular injection, the knee joint heating temperature can be precisely controlled by changing the concentration of AMF. MNFC@CM was injected intra-articularly into the joint cavity of OA mice to test its heating effect on the knee joint within the AMF. Figure 4As shown, the mouse knee joint can be rapidly heated to 41–43°C within 4 minutes; the heating temperature of the knee joint can be controlled by changing the number of times the material is administered. These results indicate that changing the concentration of IONPs in an alternating magnetic field can efficiently and precisely heat the mouse knee joint, providing an experimental basis for using IONPs to construct a nano-controlled release system to achieve drug and magnetothermal synergistic therapy for osteoarthritis (OA). ② After local magnetic thermotherapy to the joints, the inflammation of OA was significantly reduced. Based on precise control of the heating temperature of the MNFC@CM on the knee joint, the knee joints of OA mice were heated at 43℃ for 30 min, and this operation was repeated 5 times (once a day). Then, synovium and articular cartilage were collected, and the therapeutic effect of magnetothermal therapy on OA was investigated by pathological sectioning. Figure 5 As shown in Figure a, inflammatory cell infiltration, fibrous tissue hyperplasia, and synovial cell proliferation were observed in the synovial tissue of mice in the OA group, with disordered cell arrangement and a small amount of capillary proliferation in the surrounding tissue; while the inflammatory cell infiltration in the synovial tissue of mice treated with MNFC@CM+AMF was significantly reduced. Furthermore, HE and Safranin O Fast Green staining results of the articular cartilage showed that the surface of the articular cartilage in the OA group mice was significantly damaged, and the tangential fiber direction was shifted; although the tangential fiber direction was still shifted in the MNFC@CM+AMF group mice, the cartilage tissue was significantly intact. Figure 5 (b) The above results indicate that magnetothermal therapy based on IONPs can alleviate OA inflammation to some extent by moderately heating the knee joint.

[0047] (4) IONPs can be administered intravenously and guided by a magnetic field to reach the joint, achieving synergistic treatment of OA drugs and magnetothermal therapy. The above experimental results demonstrate that the prepared MNFC@CM can precisely heat the knee joint in an alternating magnetic field and alleviate OA symptoms to some extent. Subsequently, a preliminary study was conducted on the combined drug and magnetothermal therapy administered via tail vein injection to OA mice, as detailed below: ①IONPs, after intravenous administration, can reach the knee joint under magnetic field guidance, precisely heating the knee joint in the AMF. Building upon the aforementioned work, and leveraging the magnetic targeting properties of IONPs, this study investigated the heating effect of MNFC@CM injected via the tail vein and guided by a magnetic field on the mouse knee joint in an alternating magnetic field. After establishing an OA model using male ICR mice, they were randomly divided into four groups: Saline, Saline+AMF, MNFC@CM+AMF, and MNFC@CM+MT+AMF. The MNFC@CM+AMF and MNFC@CM+MT+AMF groups received a single tail vein injection of 20 mg / mL MNFC@CM every other day. In the MNFC@CM+MT+AMF group, a magnet (MT) was placed at the testis of mice for navigation for 4 hours per day. Then, the knee joints of all four groups of mice were placed in the AMF for 20 min, and the temperature changes of the mouse knee joints in the alternating magnetic field were recorded using infrared thermal imaging. Figure 6 As shown, the knee joint temperature of mice in the Saline group remained constant at around 34℃ in the AMF coil, while the knee joint temperature of mice in the Saline+AMF group increased to 38.5℃ due to the thermal radiation from the AMF coil. In the MNFC@CM+MT+AMF treatment group, after intravenous administration and 4 h of magnetic field navigation, the knee joint temperature of mice in the MNFC@CM+AMF group rose to 41.4℃ in the alternating magnetic field; while the knee joint temperature of the MNFC@CM+AMF group was only 39.0℃ without magnetic field navigation. These results indicate that IONPs, after intravenous administration, can reach the mouse knee joint under magnetic field navigation to achieve targeted and precise heating.

[0048] ②IONPs can achieve synergistic drug and magnetothermal therapy for OA after intravenous administration. After repeated treatment for 14 days in different groups of mice, knee joints were collected and analyzed using frozen sections and HE and Safranin-Fix Green staining to assess the treatment effect on osteoarthritis (OA). Figure 7 As shown, compared with the normal group (Ctrl) mice, OA mice treated with saline (Saline) showed severe damage to the articular cartilage layer. In the MNFC@CM+AMF group, without magnetic navigation, although the articular cartilage tissue defects were significantly restored compared with the Saline group, the tangential fiber direction was still offset, and the chondrocyte arrangement was disordered, indicating that the inflammation had not completely subsided. In contrast, the joint inflammation in the MNFC@CM+MT+AMF group mice was greatly improved, with no obvious cartilage tissue defects, continuous and intact tangential layers, and more regular chondrocyte arrangement. These results indicate that IONPs, administered intravenously with magnetic field navigation, can achieve synergistic drug and magnetothermal therapy for OA, providing experimental evidence for its potential clinical application.

Claims

1. A biomimetic targeted nanoreservoir carrier, characterized in that, It includes a thermoresponsive nanocore and a functionalized cell membrane, wherein the functionalized cell membrane is coated on the outside of the thermoresponsive nanocore; The thermally responsive nanocore comprises magnetic iron oxide nanomaterials, an active drug, and a surface modifier. The magnetic iron oxide nanomaterials are magnetic Fe3O4 with sodium citrate as a surface ligand, the active drug is chondroitin sulfate, and the surface modifier is posalogum F108. The functionalized cell membrane is a macrophage membrane overexpressing the Anti-Col1 gene.

2. The method of claim 1, wherein the preparation of the biomimetic targeted nanocontrolled release carrier is characterized by, Includes the following steps: Step 1: Construct macrophages overexpressing the Anti-Col1 gene and extract the cell membrane; Step 2: Magnetic Fe3O4 with sodium citrate as the surface ligand, chondroitin sulfate and posalogum F108 are added to water to react and obtain thermally responsive nanocores. Step 3: Encapsulate the thermoresponsive nanocore with the cell membrane from Step 1 to obtain the biomimetic targeted nano-controlled release carrier.

3. The production method according to claim 2, characterized by, In step 2, the concentrations of magnetic Fe3O4, chondroitin sulfate, and posalom F108 with sodium citrate as the surface ligand were 50 mg / mL, 140 mg / mL, and 1%, respectively.

4. The preparation method according to claim 2, characterized in that, In step 3, the mass concentrations of the cell membrane and the thermally responsive nanonucleus are 1:

1.

5. The use of the biomimetic targeted nano-controlled release carrier as an active ingredient in the preparation of an osteoarthritis treatment drug used in conjunction with a navigation magnet and an alternating magnetic field, as described in claim 1.

6. The application according to claim 5, characterized in that, The biomimetic targeted nano-controlled release carrier is the only active ingredient.

7. The application according to claim 5, characterized in that, The alternating magnetic field is 403 kHz.

Images