Preparation method and application of pH / ROS dual-responsive functionalized biomimetic nanomaterial loaded with mannose

Abstract

The application provides a preparation method and application of a pH / ROS dual-responsive functionalized biomimetic nanomaterial loaded with mannose. The nanomaterial takes a pH / ROS dual-responsive material as a carrier and simultaneously loads a mannose drug. The prepared nanoparticles are wrapped with functionalized macrophage membranes on the surface of the nanoparticles by a lipid extruder to achieve targeted drug delivery. The functionalized biomimetic nanoparticles provided by the application can rapidly release mannose in an acidic and / or high active oxygen microenvironment, thereby effectively promoting the generation of cartilage cell extracellular matrix. The application provides a promising strategy for targeted treatment of intervertebral disc degeneration induced by cartilage injury.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials technology, specifically relating to a biomimetic nanomaterial, and more particularly to the preparation and application of a pH / ROS dual-responsive biomimetic nanomaterial loaded with mannose. Background Technology

[0002] Low back pain (LBP) is a major clinical problem and socioeconomic health burden worldwide. The reported prevalence of LBP is close to 30%, with a lifetime prevalence exceeding 80%. Studies have shown that lumbar intervertebral disc degeneration (IVDD) may be the main cause of nonspecific pain in LBP patients, also known as discogenic low back pain. Endplate injury is one of the key factors driving IVDD progression and mediating low back pain, forming a vicious cycle of "injury-degeneration-pain." The intervertebral disc consists of the nucleus pulposus, annulus fibrosus, and superior and inferior cartilaginous endplates. The cartilaginous endplates are the only pathway for nutrient supply to the nucleus pulposus, transporting nutrients such as glucose and oxygen through diffusion, while also providing mechanical cushioning between the vertebral body and the intervertebral disc. Endplate injury disrupts the mechanical balance of the intervertebral disc, accelerating degeneration and inducing pain. After endplate injury, the stress distribution between the vertebral body and the intervertebral disc becomes abnormal, significantly increasing the tension on the annulus fibrosus, easily leading to annulus fibrosus tearing and nucleus pulposus protrusion, which in turn compresses nerve roots or irritates the sinuvertebral nerve, directly inducing radicular pain or mechanical low back pain. Abnormal mechanical stimulation activates mechanosensitive pathways within the intervertebral disc, promoting the expression of related factors and further accelerating the IVDD process. Following endplate injury, the cartilage matrix is ​​destroyed, the nutrient supply to the nucleus pulposus is obstructed, nucleus pulposus cells gradually undergo apoptosis, and the synthesis and degradation of extracellular matrix (type II collagen, proteoglycans) decrease and increase, leading to dehydration, fibrosis, and loss of height in the intervertebral disc. This results in abnormally increased intervertebral mobility, stimulating aseptic inflammation in surrounding soft tissues, accompanied by elevated reactive oxygen species (ROS). Normal endplates and nucleus pulposus are not innervated, but after endplate injury, inflammatory factors induce nerve fibers to invade the damaged endplate and degenerated intervertebral disc from the vertebral bone tissue, significantly increasing the sensitivity of the intervertebral disc tissue to mechanical and chemical stimuli. Even slight vertebral movement can trigger significant low back pain.

[0003] Current treatments for intervertebral disc degeneration mainly include physical therapy, drug therapy, intradiscal injection therapy, and surgery. Drug therapy has side effects and poor long-term adherence; the efficacy of physical therapy and traditional Chinese medicine lacks standardized evidence-based medical support, and individual differences are significant; while hormone injections have a rapid onset of action, they can worsen cartilage degeneration; radiofrequency ablation and other neurodestructive treatments only block pain signals and cannot address the root causes of endplate damage and intervertebral disc degeneration, resulting in a high rate of pain recurrence. Surgery is highly invasive, carries risks of complications such as infection, bleeding, and nerve damage, and has a long postoperative recovery period. Therefore, endplate damage is the core link between intervertebral disc degeneration and low back pain, driving the "degeneration-pain" cycle through both biomechanical imbalance and biological inflammation. Current treatments all have the limitation of "treating the symptoms but not the root cause," failing to achieve physiological repair of the endplate and intervertebral disc.

[0004] In recent years, various nanomaterials have shown potential in alleviating discogenic pain. The MnO2@TMNP biomimetic nanoparticles developed by a team at Huazhong University of Science and Technology can scavenge reactive oxygen species (ROS) and capture pro-inflammatory factors, inhibiting macrophage M1 polarization, thereby reducing inflammation and blocking pain signaling pathways. A team at Wenzhou Medical University focused on mitochondrial function, designing PGA-Mn-TP04 and PGA-Cu@SS08 nanoparticles: the former targets mitochondria to scavenge ROS and repair mitochondrial function; the latter further combines with hydrogels to achieve sustained release and inhibit ferroptosis, thus effectively protecting nucleus pulposus cells and delaying degeneration. In addition, inorganic nanoenzymes based on microfluidic technology, such as nanospheres, cerium oxide, and iron tetrasulfide, also delay IVDD by scavenging ROS, regulating the immune microenvironment, or providing mechanical support. However, the in vivo stability and targeting efficiency of these nanomaterials still have some technical limitations. Therefore, how to effectively target and repair damaged endplates and regulate the inflammatory microenvironment to treat intervertebral disc degeneration is a pressing clinical problem to be solved. Summary of the Invention

[0005] The present invention aims to provide a pH / ROS dual-responsive functionalized biomimetic nanomaterial loaded with mannose, its preparation method and application, in order to solve the problem in the prior art that it is impossible to achieve physiological repair of the endplate and intervertebral disc, that is, to effectively target and repair damaged endplate and regulate the inflammatory microenvironment.

[0006] This invention provides a pH / ROS dual-responsive functionalized biomimetic nanomaterial loaded with mannose. The pH / ROS dual-responsive material is used as a carrier to load mannose drugs. The pH / ROS dual-responsive material is composed of components such as cyclodextrin (α-CD), eugenol (SA), and pinacol phenylboronic acid (PPTs).

[0007] This invention provides a method for preparing a pH / ROS dual-responsive biomimetic nanomaterial loaded with mannose, comprising: 1) SA-αCD, DMAP, and CDI-activated HPAP were sequentially dissolved in anhydrous DMSO, with a molar ratio of SA-αCD, DMAP, and CDI-activated HPAP of 1:30:6; the mixture was magnetically stirred for 72 h at room temperature and under argon protection. After the reaction was completed, an appropriate amount of deionized water was added to precipitate a white precipitate, which was then centrifuged to obtain SA-HPAP-αCD; preferably, SA-HPAP-αCD was purified by freeze-drying. 2) Dissolve mannose and SA-HPAP-αCD in a mixed solvent to obtain a drug-containing solution; wherein the mass ratio of mannose to SA-HPAP-αCD is 1:10; the mixed solvent is obtained by mixing methanol and dimethyl sulfoxide in a volume ratio of 3:1; 3) The drug-containing solution is added dropwise to the lipid dispersion solution, stirred rapidly for 3 minutes, then stirred at 200 rpm for 3 hours, and then placed at room temperature for self-assembly for 3 hours. Man NPs are obtained by centrifugation at 15000 rpm for 10 minutes. Preferably, the Man NPs are washed three times with 10.0 mL of double-distilled water and then resuspended in 0.2 mL of ultrapure water. 4) The Man NPs were mixed with the cell membrane at a mass ratio of 1:1, sonicated for 30 minutes, and then processed through a lipid extruder to obtain biomimetic nanomaterials.

[0008] In one embodiment of the invention, the hydrodynamic diameter of the biomimetic nanomaterial is 282 ± 1 nm.

[0009] In one embodiment of the invention, the cell membrane is the cell membrane of a macrophage that highly expresses CCR1.

[0010] In one embodiment of the present invention, the lipid dispersion is prepared by a method comprising the following steps: dispersing lecithin and DSPE-PEG2000, heating at 60-70°C for 20-45 min, and then cooling to room temperature; wherein the mass ratio of lecithin to DSPE-PEG2000 is 1:1-1.5.

[0011] The present invention further provides a pH / ROS dual-responsive biomimetic nanomaterial loaded with mannose prepared according to the above preparation method.

[0012] In one embodiment of the invention, the hydrodynamic diameter of the biomimetic nanomaterial is 282 ± 1 nm.

[0013] Another aspect of the present invention provides the use of the mannose-loaded pH / ROS dual-responsive biomimetic nanomaterial prepared according to the above-described preparation method, or the above-described biomimetic nanomaterial in the preparation of a medicament for treating intervertebral disc degeneration and / or related conditions.

[0014] In one embodiment of the invention, the use is for targeted repair of damaged endplates and / or regulation of the inflammatory microenvironment.

[0015] Another aspect of the present invention provides a pharmaceutical composition for treating intervertebral disc degeneration and / or related conditions, comprising the above-mentioned biomimetic nanomaterials.

[0016] The beneficial effects of this invention are as follows: 1. High safety, no acute toxicity was observed in in vivo experiments, good biocompatibility, and significantly reduced biotoxicity compared to free drugs.

[0017] 2. Strong targeting: By highly expressing specific receptors on the surface of macrophage membranes, and through the specific recognition and binding of CCL3 and CCR1, it achieves tissue enrichment and efficient cell internalization of degenerated tissues, reduces immune escape and improves drug utilization. 3. Intelligent response release: Mannose is rapidly released in the acidic (low pH) and high ROS microenvironment of intervertebral disc degeneration pathology (release rate of about 90% in 10h), and is stable under normal physiological conditions (release rate of only 45% in 48h), reducing off-target toxicity. Based on the above description of the present invention, and in accordance with common technical knowledge and conventional methods in the relevant fields, various other modifications, substitutions, or alterations can be made without departing from the basic technical concept of the present invention. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application, the following detailed description of non-limiting embodiments is provided using accompanying drawings, making their features, objectives, and advantages more apparent: Figure 1 These are transmission electron microscope images of Man NPs nanoparticles (a) and CR-Man NPs nanoparticles (b) in the embodiments of the present invention.

[0019] Figure 2 The particle size distribution and zeta potential diagrams of Man NPs nanoparticles (a) and CR-Man NPs nanoparticles (b) in the embodiments of the present invention are shown.

[0020] Figure 3 The figure shows the stability test results of Man NPs nanoparticles and CR-Man NPs nanoparticles in pure water and 10% (a) fetal bovine serum (b) in the embodiments of the present invention.

[0021] Figure 4 The images show the in vitro drug release curves of CR-Man NPs nanoparticles in different release media over 48 hours in embodiments of the present invention.

[0022] Figure 5 The results of cell viability and death staining and CCK-8 assays of CR-Man NPs nanoparticles in this embodiment of the invention are shown.

[0023] Figure 6 The results of the cellular phagocytosis experiment of CR-Man NPs nanoparticles in the embodiments of the present invention are shown.

[0024] Figure 7 The images show in vivo imaging results of mice at 2, 4, 8, 12, 24 and 48 hours after intravenous injection in this embodiment of the invention.

[0025] Figure 8 The above describes the CLSM results of frozen sections taken from mice 48 hours after intravenous injection in this embodiment of the invention. Figure 9 This is an MRI image of in vivo treatment with CR-Man NPs nanoparticles in an embodiment of the present invention. Detailed Implementation

[0026] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0027] Example 1: Preparation and Characterization of Materials 1. The preparation of biomimetic nanomaterials includes the preparation of carrier materials and the preparation of nanomaterials. The reagents for preparing the carrier materials were all purchased from Aladdin, and the synthesis method is as follows: 1) Synthesis of eugenol (SAA): In a 100 mL round-bottom flask, 7.41 g (40.68 mmol) of eugenol, 19.40 mL (182.81 mmol) of trimethyl orthoformate, and 2.04 g (8.24 mmol) of pyridinium 4-methylbenzenesulfonate (PPTs) were dissolved in 50 mL of methanol. The mixture was then heated under reflux at 75 °C for 12 h. The reaction progress was monitored by thin-layer chromatography using a 1:10 mixture of petroleum ether and ethyl acetate as the developer. After the reaction was complete, the system was cooled to room temperature, and 25 mL of saturated NaHCO3 solution was added and stirred for 15 min to quench the reaction. Subsequently, methanol was removed by rotary evaporation under reduced pressure. The reaction mixture was extracted three times with ethyl acetate, and the organic layer was collected and dried overnight in anhydrous magnesium sulfate (MgSO4). The next day, ethyl acetate was removed again by rotary evaporation under reduced pressure to obtain a golden-yellow, transparent, viscous liquid.

[0028] 2) Synthesis of SAA modified with cyclodextrin (α-CD) (CA-αCD): In a 250 mL round-bottom flask, dissolve 1.6 g (1.67 mmol) α-CD and 4.56 g (20 mmol) SAA in 14 mL anhydrous dimethyl sulfoxide (DMSO), then add 0.25 g (1 mmol) PPTs to the mixture. Stir the mixture at 60 °C for 72 h under argon protection. At the end of the reaction, add 140 mL of acetone to the mixture, causing a precipitate to form. Filter the precipitate under vacuum, wash with acetone, and dry under vacuum to obtain a white powder.

[0029] 3) Synthesis of CDI-activated HPAP: 2.77 g (11.8 mmol) of 4-(hydroxymethyl)phenylboronic acid pinacol ester (HPAP) was dissolved in 18 mL of anhydrous dichloromethane (DCM). Then, 3.83 g (23.6 mmol) of N,N'-carbonyldiimidazole (CDI) was added to the solution, and the mixture was reacted at room temperature for 30 min. After the reaction was complete, 20 mL of dichloromethane was added to the mixture, followed by washing three times with 30 mL of deionized water. The organic phase was further washed with saturated sodium chloride solution and dried overnight with anhydrous MgSO4. Finally, the solvent was removed by rotary evaporation under reduced pressure to obtain a white solid of CDI-activated HPAP.

[0030] 4) Synthesis of SA-HPAP-αCD: 1.02 g (0.6 mmol) SA-αCD was dissolved in 30 mL of anhydrous DMSO. 2.22 g (18 mmol) DMAP and 1.18 g (3.6 mmol) CDI-activated HPAP were added sequentially to the above solution. The mixture was magnetically stirred for 72 h at room temperature under argon protection. After the reaction was complete, 40 mL of deionized water was added to precipitate a white precipitate, which was obtained by centrifugation. The crude product was then purified by dialyzing in ultrapure water for 2 days (MWCO: 1000 Da). Finally, the white solid SA-HPAP-αCD was collected by freeze-drying.

[0031] 2. The synthesis methods of nanomaterials are as follows: 1) Preparation of drug-loaded nanomaterials: NPs were prepared via a nanoprecipitation / self-assembly method. 6.0 mg of lecithin (Aladdin) and 9.0 mg of DSPE-PEG2000 (Ruixi Biotechnology) were dispersed in 400 μL of anhydrous ethanol and 7.0 mL of deionized water by ultrasonication. The mixture was then slowly stirred at 65 °C (200 rpm) for 30 minutes to obtain a lipid dispersion.

[0032] Meanwhile, 5.0 mg of mannose (Aladdin) and 50.0 mg of SA-HPAP-αCD were dissolved in a mixed solvent containing 600 μL of methanol and 200 μL of dimethyl sulfoxide to obtain a drug-containing solution.

[0033] The drug-containing solution was added dropwise to a preheated lipid dispersion (1.0 mL / min), and then rapidly stirred for 3 minutes (600 rpm), followed by stirring at 200 rpm for 3 hours. After self-assembly at room temperature for 3 hours, Man NPs were collected by centrifugation at 15,000 rpm for 10 minutes. The collected NPs were washed three times with 10.0 mL of double-distilled water and then resuspended in 0.2 mL of ultrapure water for use.

[0034] 2) Extraction of cell membranes: Cell membranes were extracted using a cell membrane protein extraction kit (Beyotime). The specific steps are as follows: Culture approximately 20-50 million macrophages, wash once with PBS (Seville), and scrape the cells using a cell scraper. Centrifuge to collect the cells, aspirate the supernatant, and keep the cell pellet for later use. Gently resuspend the cell pellet in an appropriate amount of ice-cold PBS, centrifuge at 4°C, 600g for 5 minutes to precipitate the cells. Discard the supernatant, then centrifuge at 4°C, 600g for 1 minute to precipitate any residual liquid on the centrifuge tube wall and further precipitate the cells, aspirating as much residual liquid as possible. Add 1 ml of membrane protein extraction reagent A (pre-added with PMSF) to the 20-50 million cells, gently and thoroughly resuspend the cells, and incubate on ice for 10-15 minutes. Transfer the cell suspension to an appropriately sized ice-cold glass homogenizer (Lansberg), and homogenize for approximately 30-50 cycles. Centrifuge at 700g for 10 minutes at 4°C, and carefully collect the supernatant into a new centrifuge tube to remove cell nuclei and unbroken cells. Then centrifuge at 14000g for 30 minutes at 4°C to precipitate cell membrane debris.

[0035] 3) Cell membrane-coated drug-loaded nanomaterials: The cell membrane and nanoparticles were freeze-dried at a mass ratio of 1:1, diluted separately with pure water, mixed, and sonicated for more than 30 minutes. Then, the mixture was passed through a lipid extruder (Bohr Biotechnology) to coat the surface of the nanoparticles with the cell membrane, thus obtaining the biomimetic nanomaterial CR-Man NPs.

[0036] 3. Characterization methods: Two μL of freshly prepared nanoparticles were dispersed in 2 mL of pure water. The particle size and zeta potential of the nanoparticles were determined using dynamic light scattering (DLS) and laser Doppler velocimetry (Nano ZS, Malvern Instruments, UK). The morphology of the nanoparticles was characterized by transmission electron microscopy (TEM, JEM-1400, Tokyo, Japan).

[0037] 4. Results: This invention successfully prepared Man NPs and CR-Man NPs. Figure 1 Transmission electron microscopy (TEM) observations showed that the prepared Man NPs ( Figure 1 a) and CR-Man NPs ( Figure 1 b) All are spherical and monodisperse.

[0038] Figure 2 Dynamic light scattering (DLS) analysis showed that Man NPs and CR-Man NPs ( Figure 2 The hydrodynamic diameters of the nanoparticles were 275±5 nm and 282±1 nm, respectively. The zeta potentials of Man NPs and CR-Man NPs were -24.2±0.3 mV and -22.6±0.8 mV, respectively, indicating that cell membrane coating did not significantly affect the nanoparticle size, and the zeta potential decreased after cell membrane coating.

[0039] To test the stability of the nanoparticles, the freshly prepared nanoparticles were dispersed in 2 mL of ultrapure water or 10% fetal bovine serum (Semicin), and their particle size and polydispersity index were continuously measured at different time points. The test results are as follows: Figure 3 As shown, neither ManNPs nor CR-Man NPs exhibited significant particle size changes within 48 hours in either of the aforementioned liquids. This indicates that these nanoparticles remain stable in the bloodstream.

[0040] Experimental Example 2: In vitro drug release Test method: The in vitro mannose release behavior of CR-Man NPs was studied using dialysis.

[0041] 200 µL of freshly prepared nanoparticles were transferred into a dialysis bag (MWCO: 3500 Da), and then immersed in 40 mL of PBS (Seville), PBS containing 0.5 mM H2O2, PBS at pH 5.0, or PBS containing 0.5 mM H2O2 at pH 5.0. All release media were incubated at 37 °C with gentle shaking at 100 rpm. At predetermined time points, 4 mL of release media was removed and replaced with an equal volume of fresh media. The concentration of mannose was determined by high-performance liquid chromatography (HPLC, Shimadzu, Japan), and the cumulative drug release percentage was calculated.

[0042] like Figure 4 As shown, under normal physiological conditions (PBS pH=7.4), only about 48% of the mannose was released from the nanoparticles after 10 hours. Figure 4 (Purple lines), and then reached equilibrium. However, after incubation for 10 hours in an acidic medium with high ROS concentration, the drug release rate of CR-Man NPs reached approximately 90% ( Figure 4 (Blue line). Therefore, CR-Man NPs are able to efficiently and slowly release drugs in a simulated pathological microenvironment.

[0043] Experimental Example 3: In vitro cytotoxicity test Assay Methods: The in vitro cytotoxicity of free mannose and drug-loaded NPs on RAW264.7 cells (Provac) and cartilage endplate cells (Provac) was studied using the Cell Counting Kit-8 (Beyotime). Cells were counted at 1 × 10⁶ cells per well. 4 Cells were seeded at a density of [number] cells / well in 96-well plates and cultured for 24 h. Subsequently, cells were treated for 24 h with different concentrations of free mannose (Maclean) and drug-loaded NPs (0, 1.5, 3, 6.25, 12.5, 25, 50, or 100 µg / mL). Wells containing only cell culture medium served as controls. The absorbance of each culture was measured at 450 nm using a Thermo Multiskan Spectrum microplate reader (Thermo Fisher Scientific).

[0044] like Figure 5 As shown, in all treatment groups, the survival rates of RAW 264.7 cells and cartilage endplate cells decreased significantly with increasing drug concentration. CR-Man NPs exhibited superior inhibitory effects on cell growth compared to uncoated Man NP nanoparticles and free mannose. Compared to free drugs, nanoparticles have a stronger ability to be internalized by cells, resulting in a better cell growth effect for the nanotherapeutic agent. Therefore, the biotoxicity of CR-Man NPs was significantly lower than that of the unmodified nanoparticle group and significantly better than that of the free drug group, demonstrating that coating the nanoparticle surface with a cell membrane improves the biocompatibility of the material. Simultaneously, cell viability and mortality staining results also confirmed the good biocompatibility of the nanomaterial.

[0045] Experiment Example 4: Cellular Uptake of Nanoparticles RAW 264.7 cells and cartilage endplate cells were seeded in confocal culture dishes (Semic). After 24 h of cell adhesion, cells were treated with free Cy5 (source leaf), Cy5-labeled Man NPs (Cy5-Man NPs), or Cy5-labeled CR-Man NPs (Cy5-CR-Man NPs), with a final concentration of 4 µg / mL for 2 h, 4 h, and 8 h, respectively. At the specified time points, cells were washed three times with PBS and then fixed with 4% paraformaldehyde (Solepro) at room temperature. The nuclei were then stained with DAPI (Beyotime) for 10 min. Finally, the cellular uptake efficiency of the nanoparticles was observed using a confocal laser scanning microscope (CLSM; Carl Zeiss AG, Baden-Württemberg, Germany).

[0046] like Figure 6 As shown, compared with the free Cy5 group, the fluorescence intensity of the Cy5-Man NPs nanoparticle group was significantly enhanced in both RAW 264.7 cells and cartilaginous endplate cells, further confirming that the nanoparticles are more easily phagocytosed by cells than the free drug. Cellular uptake efficiency also increased with increasing incubation time. Compared with non-targeted nanoparticles and free Cy5, the membrane-modified nanoparticles exhibited the strongest fluorescence intensity in both RAW 264.7 cells and cartilaginous endplate cells. In conclusion, functionalized cell membrane-modified nanoparticles can be efficiently internalized by RAW 264.7 cells and cartilaginous endplate cells, thereby achieving targeted delivery to intervertebral disc degeneration lesions.

[0047] Experimental Example 5: Distribution of Nanoparticles in Vivo In vivo distribution experiments were conducted in male C57 mice (6-8 weeks old) with endplate injury-induced intervertebral disc degeneration. All mice were obtained from the AAALAC-certified Laboratory Animal Center of Army Medical University, Chongqing, China. Intervertebral disc degeneration was induced by microscopic endplate injury. Two weeks later, after successful model establishment using MRI, mice were intravenously injected with free Cy5, Cy5-labeled Man NPs (Cy5-ManNPs), or Cy5-labeled CR-Man NPs (Cy5-CR-Man NPs) at a dose of 1 mg / kg Cy5. Mice were anesthetized at predetermined time points after administration, and in vivo distribution images were acquired using an in vivo imaging system (Perkin Elmer, Waltham, USA). Forty-eight hours after injection, mice were sacrificed, and images of the modeling site and major organs were collected and imaged. Subsequently, fluorescence intensity within the modeling tissues was analyzed.

[0048] In addition, tissue samples were frozen and sectioned to 10 μm and fixed with 4% paraformaldehyde (Solepro), followed by staining of cell nuclei with DAPI (Beyotime). The distribution of nanoparticles in the intervertebral disc tissue was determined using confocal laser scanning microscopy (CLSM).

[0049] The results are as follows Figure 7 As shown, the fluorescence intensity in mice treated with free Cy5 decreased rapidly, indicating that free Cy5 was quickly cleared in vivo. However, a certain intensity of fluorescence was still observed in the intervertebral disc tissue 24 h or 48 h after administration of Cy5-Man NPs, indicating that the nanoparticles can prolong their circulation time in vivo. In mice treated with Cy5-CR-Man NPs, the fluorescence intensity at the model site remained stable 48 h after administration, suggesting that functionalized cell membrane modification can significantly prolong the retention time of nanoparticles in degenerated intervertebral disc tissue.

[0050] Furthermore, cryosection imaging further verified the penetrating ability of nanoparticles into degenerated tissues. Figure 8 The results showed that obvious red fluorescence signals were observed in the degenerated tissue after treatment with Cy5-CR-Man NPs, which further confirmed that functionalized cell membrane modification can improve the permeability of nanoparticles in degenerated tissues.

[0051] Note: Red: Cy5-labeled nanoparticles; Blue: DAPI-stained cell nuclei; Scale bar = 50 μm.

[0052] Experimental Example 6: Therapeutic Effect of In Vivo Intervertebral Disc Degeneration Intervertebral disc degeneration in mice was induced by damaging the endplate under a microscope. Two weeks later, after successful model establishment using MRI, mice were randomly divided into five groups. Every five days, mice were intravenously injected with PBS (I), free mannose (II), Man NPs (III), R-Man NPs (IV), or CR-Man NPs (V) at a dose of 2.5 mg / kg for four consecutive days. Twenty-five days later, MRI was used to observe the intervertebral disc lesions. Figure 9 ).

[0053] The results showed that after Cy5-CR-Man NPs treatment, the degenerated areas improved to some extent, and the intervertebral disc morphology was significantly improved compared with the control group, with the degeneration condition being alleviated. The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing biomimetic nanomaterials, comprising: 1) SA-αCD, DMAP, and CDI-activated HPAP were sequentially dissolved in anhydrous DMSO, with a molar ratio of SA-αCD, DMAP, and CDI-activated HPAP of 1:30:6; the mixture was magnetically stirred for 72 h at room temperature and under argon protection. After the reaction was completed, an appropriate amount of deionized water was added to precipitate a white precipitate, which was then centrifuged to obtain SA-HPAP-αCD; preferably, SA-HPAP-αCD was purified by freeze-drying. 2) Dissolve mannose and SA-HPAP-αCD in a mixed solvent to obtain a drug-containing solution; wherein the mass ratio of mannose to SA-HPAP-αCD is 1:10; the mixed solvent is obtained by mixing methanol and dimethyl sulfoxide in a volume ratio of 3:1; 3) The drug-containing solution is added dropwise to the lipid dispersion solution, stirred rapidly for 3 minutes, then stirred at 200 rpm for 3 hours, and then placed at room temperature for self-assembly for 3 hours. Man NPs are obtained by centrifugation at 15000 rpm for 10 minutes. Preferably, the Man NPs are washed three times with 10.0 mL of double-distilled water and then resuspended in 0.2 mL of ultrapure water. 4) The Man NPs were mixed with the cell membrane at a mass ratio of 1:1, sonicated for 30 minutes, and then processed through a lipid extruder to obtain biomimetic nanomaterials.

2. The preparation method according to claim 1, wherein, The hydrodynamic diameter of the biomimetic nanomaterial is 282±1 nm.

3. The preparation method according to claim 1 or 2, wherein, The cell membrane is the cell membrane of macrophages that highly express CCR1.

4. The preparation method according to any one of claims 1-3, wherein, The lipid dispersion is prepared by a method including the following steps: dispersing lecithin and DSPE-PEG2000, heating at 60~70℃ for 20~45 min and then cooling to room temperature; the mass ratio of lecithin to DSPE-PEG2000 is 1:1~1.

5.

5. The biomimetic nanomaterials prepared by the preparation method according to any one of claims 1-4.

6. The biomimetic nanomaterial as described in claim 5, wherein, The hydrodynamic diameter of the biomimetic nanomaterial is 282 ± 1 nm.

7. The use of the biomimetic nanomaterial prepared by the preparation method according to any one of claims 1-4, or the biomimetic nanomaterial according to claim 5 or 6, in the preparation of a medicament for treating intervertebral disc degeneration and / or related conditions.

8. The use as described in claim 7, wherein, The intended use is to target and repair damaged endplates and / or regulate the inflammatory microenvironment.

9. A pharmaceutical composition for treating intervertebral disc degeneration and / or related conditions, comprising the biomimetic nanomaterial as described in claim 5 or 6.

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