Mannosylated hybrid membrane packaged Cu / Zn-MOF drug delivery system and preparation method and application thereof

Abstract

The invention belongs to the technical field of drug delivery, and particularly relates to a mannosylation hybrid membrane encapsulated Cu / Zn-MOF drug delivery system and a preparation method and application thereof.The drug delivery system comprises mannosylation hybrid membrane encapsulated H-151-loaded Cu / Zn-MOF, the hybrid membrane is prepared by fusing lipidosome and a macrophage cell membrane, and the lipidosome and the macrophage cell membrane are combined to form the mannosylation hybrid membrane loaded H-151-loaded Cu / Zn-MOF drug delivery system. The preparation method comprises the following steps: dissolving H-151 in dimethyl sulfoxide, dropwise adding a Cu / Zn-MOF methanol suspension, stirring, centrifuging, and washing to obtain H-151-loaded Cu / Zn-MOF; mixing the macrophage membrane with lipidosome, performing ultrasonic treatment, and extruding through a polycarbonate membrane to obtain a hybrid membrane; preparing lipidosome by adopting DSPE-PEG-Man, and fusing the lipidosome with a macrophage membrane to obtain a mannosylation hybrid membrane; the H-151-loaded Cu / Zn-MOF is mixed with the mannosylation hybrid membrane, incubation and centrifugation are performed, and the H-151-loaded Cu / Zn-MOF encapsulated by the mannosylation hybrid membrane is obtained. The mannosylation hybrid membrane encapsulated Cu / Zn-MOF drug delivery system has the functions of accurate delivery, intelligent release, oxidation resistance and inflammation resistance, and accurate and efficient treatment of inflammatory diseases is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug delivery, and specifically relates to a mannosylated hybrid membrane encapsulated Cu / Zn-MOF drug delivery system, a preparation method and an application thereof. Background Art

[0002] Uveitis is an intraocular inflammatory disease that can seriously affect visual function. Its pathological process is usually accompanied by intraocular inflammatory cell infiltration, blood-retinal barrier destruction, abnormal accumulation of reactive oxygen species (ROS), and imbalance in the proportion of immune cell subsets. Commonly used clinical therapeutic drugs such as glucocorticoids have good anti-inflammatory effects, but their serious side effects (such as immunosuppression, osteoporosis, metabolic disorders, etc.) when used systemically and their lack of targeting limit their long-term and precise application. Therefore, there is an urgent need to develop a therapeutic strategy that can take into account both anti-inflammatory and ROS clearance.

[0003] In recent years, metal-organic frameworks (MOFs), as emerging nanocarrier materials, have been widely used in the field of drug delivery due to their high specific surface area, good drug encapsulation capacity and pH-responsive degradation properties. MOFs are loaded with anti-inflammatory drugs and used to treat inflammatory diseases. Although this metal-organic framework drug delivery system has the ability to sustain drug release and certain antioxidant properties, it has poor stability and targeting in the body and is easily cleared by the immune system. Summary of the Invention

[0004] In order to solve the problems in the prior art, the present invention provides a mannosylated hybrid membrane encapsulated Cu / Zn-MOF drug delivery system and its preparation method and application. By synergistically loading Cu / Zn-MOF with the STING antagonist H-151, combining hybrid membrane encapsulation and mannose surface functionalization, it achieves the purpose of precise delivery, intelligent release, antioxidant and anti-inflammatory functions, and realizes the purpose of precise and efficient treatment of inflammatory diseases.

[0005] Existing drug delivery systems generally have the following technical shortcomings: insufficient targeting: a single passive or active targeting strategy cannot take into account both enrichment at inflammatory sites and cell-specific delivery; uncontrollable drug release: some carriers lack response to the inflammatory microenvironment (such as pH), resulting in premature drug release or insufficient release rate; single function: existing solutions only focus on one function of anti-inflammatory or antioxidant, and lack synergistic effects.

[0006] The present invention solves the technical problem by adopting the following technical solutions:

[0007] The present invention aims to provide a mannosylated hybrid membrane encapsulated Cu / Zn-MOF drug delivery system, wherein the mannosylated hybrid membrane encapsulates H-151-loaded Cu / Zn-MOF, and the hybrid membrane is prepared by fusion of liposomes and macrophage membranes.

[0008] The copper-zinc bimetallic organic framework (Cu / Zn-MOF) combines antioxidant (mimicking the activity of multiple enzymes such as SOD and GPx) and drug delivery capabilities. Its porous structure can load drugs and responds to degradation in acidic microenvironments for controlled release. The Cu / Zn-MOF is loaded with the STING antagonist H-151, creating a synergistic anti-inflammatory and antioxidant effect. Furthermore, biomimetic membrane coating technology is incorporated into the nano-drug delivery system. By encapsulating cell membranes (such as macrophage membranes), the nanoparticles possess "self-recognition" capabilities, enabling passive targeting of inflamed tissues while prolonging their circulation time in the body. Further, combined with mannose (Man) modification, active targeting of inflammatory cells expressing mannose receptors is possible.

[0009] A method for preparing a mannosylated hybrid membrane encapsulated Cu / Zn-MOF drug delivery system comprises the following steps:

[0010] S1. Cu / Zn-MOF loaded with H-151: H-151 was dissolved in DMSO (dimethyl sulfoxide), and the Cu / Zn-MOF methanol suspension was added. The mixture was stirred, centrifuged, and then washed to obtain the H-151-loaded Cu / Zn-MOF.

[0011] S2. Fusion of liposomes and macrophage membranes to construct hybrid membranes: macrophage membranes and liposomes were mixed, sonicated, and then extruded through a polycarbonate membrane to obtain hybrid membranes;

[0012] S3. Preparation of mannosylated hybrid membrane: liposomes were prepared using DSPE-PEG-Man and fused with macrophage membranes to obtain mannosylated hybrid membranes;

[0013] S4. Mannosylated hybrid membrane encapsulating H-151-loaded Cu / Zn-MOF: H-151-loaded Cu / Zn-MOF was mixed with the mannosylated hybrid membrane, incubated, and centrifuged to obtain a mannosylated hybrid membrane encapsulating H-151-loaded Cu / Zn-MOF.

[0014] Furthermore, the method of loading H-151 on Cu / Zn-MOF includes: dissolving 10 mg of H-151 in 1 mL of DMSO, adding 20 mL of Cu / Zn-MOF methanol suspension (5 mg / mL) dropwise, stirring at room temperature in the dark for 12 h, centrifuging at 8000 rpm to remove free drugs, and washing with methanol three times.

[0015] Furthermore, the preparation method of Cu / Zn-MOF includes: weighing 446.25 mg of zinc nitrate hexahydrate (Zn(NO3)2·6H2O) and 241.56 mg of copper nitrate trihydrate (Cu(NO3)2·3H2O), dissolving them in 20 mL of anhydrous methanol to form solution A; weighing 3.284 g of 2-methylimidazole (2-MIM) and dissolving them in an equal volume of methanol to form solution B; adding solution B dropwise to solution A under magnetic stirring conditions, continuing to stir at room temperature for 4 hours, after the reaction is completed, centrifuging at 8000 rpm for 5 minutes, discarding the supernatant, washing the precipitate with methanol three times to remove impurities, and finally vacuum drying at 50°C to obtain Cu / Zn-MOF powder.

[0016] Furthermore, the method for constructing a hybrid membrane (LM) by fusing liposomes with macrophage membranes includes: mixing the macrophage membrane and liposomes in a mass ratio of 1:1, sonicating in an ice bath for 5 minutes, and then extruding through 0.4 μm and 0.2 μm polycarbonate membranes 15 times to promote membrane fusion, and finally obtaining a hybrid membrane.

[0017] Furthermore, the method for extracting the macrophage membrane includes: washing the RAW264.7 cells with PBS buffer, scraping the cells and collecting them in a 15 mL centrifuge tube, centrifuging at 4°C and 300g for 5 minutes to precipitate the cells, then adding pre-cooled hypotonic buffer containing protease antagonists and PMSF to resuspend them, and incubating them in an ice bath for 30 minutes, then transferring the cell suspension to a glass homogenizer and manually homogenizing them about 30 times under ice bath conditions to fully destroy the cell structure and release the cell membrane. The homogenate is then centrifuged at 4°C and 10,000g for 10 minutes, and the supernatant is collected. The supernatant is then ultracentrifuged at 4°C and 100,000g for 60 minutes, and the resulting precipitate is the macrophage membrane.

[0018] Furthermore, the preparation method of liposomes includes: dissolving soybean phospholipids (SPC) and DSPE-PEG2000 (phospholipid-methoxy polyethylene glycol) in chloroform at a mass ratio of 10:1, rotary evaporating at 40°C to form a uniform lipid film, and then hydrating the resulting film with PBS at 37°C for 15 minutes and ultrasonicating for 15 minutes to obtain small-sized liposomes.

[0019] Furthermore, the preparation method of the mannosylated hybrid membrane includes: dissolving soybean lecithin and DSPE-PEG-Man (phospholipid polyethylene glycol mannose) in chloroform at a mass ratio of 10:1, rotary evaporating at 40°C to form a uniform lipid film, hydrating the resulting film with PBS at 37°C for 15 minutes, ultrasonically breaking it to form liposomes, mixing the macrophage membrane and the liposomes at a mass ratio of 1:1, ultrasonicating in an ice bath for 5 minutes, and then extruding through 0.4μm and 0.2μm polycarbonate membranes 15 times to promote membrane fusion, and finally obtaining a mannosylated hybrid membrane.

[0020] Furthermore, the method of encapsulating H-151-loaded Cu / Zn-MOF with a mannosylated hybrid membrane includes: mixing the H-151-loaded Cu / Zn-MOF and the mannosylated hybrid membrane in a mass ratio of 1:5, incubating at 37°C for 2 hours, and centrifuging to remove unencapsulated particles to obtain a mannosylated hybrid membrane-encapsulated H-151-loaded Cu / Zn-MOF.

[0021] A mannosylated hybrid membrane encapsulated Cu / Zn-MOF drug delivery system or a preparation method of a mannosylated hybrid membrane encapsulated Cu / Zn-MOF drug delivery system and its application in the delivery of inflammatory drugs.

[0022] Compared with the prior art, the beneficial technical effects of the present invention are:

[0023] 1. The present invention synergistically loads Cu / Zn-MOF with the STING antagonist H-151, combines hybrid membrane encapsulation and mannose surface functionalization, and develops a novel nano-drug delivery system with precise delivery, intelligent release, antioxidant and anti-inflammatory functions, achieving precise and efficient treatment of experimental autoimmune uveitis (EAU) and even other inflammatory diseases.

[0024] 2. The present invention forms a "dual targeting" strategy by integrating liposomes and macrophage membranes (passive chemotaxis) with mannose modification (active targeting), solving the problem that single targeting technology cannot simultaneously achieve enrichment of inflammatory sites and cell-specific delivery, thereby better improving the therapeutic effect.

[0025] 3. The present invention utilizes the pH-responsive degradation characteristics of Cu / Zn-MOF and combines hybrid membrane modification to delay the drug release rate, thereby achieving precise controlled release in the inflammatory microenvironment (weakly acidic) and reducing drug leakage.

[0026] 4. The present invention constructs a multi-effect synergistic treatment system by utilizing the antioxidant capacity of Cu / Zn-MOF and the anti-inflammatory and immunomodulatory functions of the STING antagonist (H-151), thus breaking through the limitations of traditional single-function drug delivery systems.

[0027] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented in accordance with the contents of the specification. In order to make the above contents of the present invention and its objectives, features and advantages more obvious and easy to understand, the specific implementation methods of the present invention are specifically listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a transmission electron microscope (TEM) image of Cu / Zn-MOF in the present invention.

[0029] Figure 2Confocal fluorescence microscopy images of the hybrid membranes of the present invention (red = macrophage membrane, green = liposomes).

[0030] Figure 3 Western blot analysis of F4 / 80 and CD11b in the macrophage membrane, hybrid membrane encapsulated drug-loaded Cu / Zn-MOF and mannosylated hybrid membrane encapsulated drug-loaded Cu / Zn-MOF of the present invention.

[0031] Figure 4 This is a transmission electron microscopy (TEM) image of the mannosylated hybrid membrane encapsulated drug-loaded Cu / Zn-MOF of the present invention.

[0032] Figure 5 Graph showing the test results of potential measurement and particle size measurement of drug-loaded Cu / Zn-MOF, hybrid membrane-encapsulated drug-loaded Cu / Zn-MOF, and mannose-modified hybrid membrane-encapsulated drug-loaded Cu / Zn-MOF in the present invention.

[0033] Figure 6 This is a graph showing the cumulative release results of drug-loaded Cu / Zn-MOF and mannosylated hybrid membrane-encapsulated drug-loaded Cu / Zn-MOF at room temperature under different pH conditions (7.4 and 5.4) of the present invention.

[0034] Figure 7 The present invention is a mannosylated hybrid membrane encapsulating drug-loaded Cu / Zn-MOF for NO, O2 - and UV-visible absorption spectra of ·OH radical scavenging activity.

[0035] Figure 8 These are confocal fluorescence microscope images of the nanomaterial cell uptake in each group in Example 7 of the present invention.

[0036] Figure 9 These are the clinical scores and pathological scores of each group in Example 8 of the present invention. DETAILED DESCRIPTION

[0037] The technical solutions of the present invention are further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely exemplary illustrations and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0038] In addition, unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0039] Example 1

[0040] A method for preparing a mannosylated hybrid membrane encapsulated Cu / Zn-MOF drug delivery system comprises the following steps:

[0041] 1. Synthesis of Copper-Zinc Bimetallic Organic Framework (Cu / Zn-MOF)

[0042] A copper-zinc bimetallic organic framework (Cu / Zn-MOF) was synthesized using a solvothermal method. First, 446.25 mg of zinc nitrate hexahydrate (Zn(NO₃)₂·6H₂O) and 241.56 mg of copper nitrate trihydrate (Cu(NO₃)₂·3H₂O) were weighed and dissolved in 20 mL of anhydrous methanol to form solution A. Separately, 3.284 g of 2-methylimidazole (2-MIM) was dissolved in an equal volume of methanol to form solution B. Solution B was slowly added dropwise to solution A under magnetic stirring, and the reaction was continued with stirring at room temperature for 4 hours. After the reaction, the mixture was centrifuged at 8000 rpm for 5 minutes, the supernatant discarded, and the precipitate washed three times with methanol to remove impurities. Finally, the mixture was dried under vacuum at 50°C to obtain Cu / Zn-MOF powder.

[0043] 2. Loading of STING antagonist H-151

[0044] Dissolve 10 mg of H-151 in 1 mL of DMSO, and add 20 mL of a 5 mg / mL Cu / Zn-MOF methanol suspension dropwise. Stir at room temperature in the dark for 12 hours, centrifuge at 8000 rpm to remove free drug, and wash three times with methanol to obtain the H-151-loaded Cu / Zn-MOF.

[0045] 3. Construction of hybrid membrane (LM) by fusion of liposomes and macrophage membranes

[0046] (1) Extraction of macrophage membranes

[0047] First, RAW264.7 cells were washed with PBS buffer, scraped and collected in a 15 mL centrifuge tube, and centrifuged at 4°C and 300 g for 5 minutes to precipitate the cells. Pre-cooled hypotonic buffer containing protease antagonists and PMSF was then added to resuspend the cells and incubated in an ice bath for 30 minutes. The cell suspension was then transferred to a glass homogenizer and manually homogenized about 30 times in an ice bath to fully destroy the cell structure and release the cell membrane. The homogenate was then centrifuged at 4°C and 10,000 g for 10 minutes, and the supernatant was collected. The supernatant was then ultracentrifuged at 4°C and 100,000 g for 60 minutes. The resulting precipitate was the macrophage membrane.

[0048] (2) Preparation of liposomes

[0049] Liposomes were prepared by the thin film hydration method. Soybean lecithin and DSPE-PEG2000 were dissolved in chloroform at a mass ratio of 10:1 and rotary evaporated at 40°C to form a uniform lipid film. The resulting film was then hydrated with PBS at 37°C for 15 minutes and ultrasonically disrupted to form small-sized liposomes.

[0050] (3) Preparation of hybrid membrane

[0051] Hybrid membranes were obtained by an incubation-extrusion method. Macrophage membranes were mixed with liposomes at a mass ratio of 1:1, sonicated in an ice bath for 5 minutes, and then extruded 15 times through 0.4 μm and 0.2 μm polycarbonate membranes to promote membrane fusion.

[0052] 4. Mannose functionalized hybrid membrane (Man modification)

[0053] Soybean lecithin and DSPE-PEG-Man were dissolved in chloroform at a mass ratio of 10:1 and rotary evaporated at 40°C to form a uniform lipid film. The resulting film was then hydrated with PBS at 37°C for 15 minutes and ultrasonically broken to form liposomes. The macrophage membrane and liposomes were mixed at a mass ratio of 1:1, ultrasonicated in an ice bath for 5 minutes, and then extruded through 0.4μm and 0.2μm polycarbonate membranes 15 times to promote membrane fusion, finally obtaining a mannosylated hybrid membrane.

[0054] 5. Composite assembly to form the final drug delivery system

[0055] The H-151-loaded Cu / Zn-MOF was mixed with the mannosylated hybrid membrane at a mass ratio of 1:5 and incubated at 37°C for 2 hours. Unencapsulated particles were removed by centrifugation to obtain the drug-loaded Cu / Zn-MOF encapsulated by the mannosylated hybrid membrane.

[0056] like Figure 1 The transmission electron microscopy (TEM) images showed that Cu / Zn-MOF was successfully synthesized, and the synthesized Cu / Zn-MOF nanoparticles were uniformly dispersed and had a rhombic dodecahedral structure.

[0057] like Figure 4 The transmission electron microscope image of the Cu / Zn-MOF loaded with H-151 encapsulated in the mannosylated hybrid membrane of the present invention shows that the diameter of the Cu / Zn-MOF loaded with H-151 encapsulated in the mannosylated hybrid membrane is about 120 nm, and the membrane structure of the nanoparticles can be clearly observed.

[0058] The following verification experiments were conducted using the mannosylated hybrid membrane encapsulated Cu / Zn-MOF drug delivery system prepared in Example 1 of the present invention.

[0059] Example 2

[0060] To verify the fusion of the macrophage membrane and liposomes of the present invention, fluorescent dyes were used to label the two separately: the macrophage membrane was labeled with Cy5 red fluorescence, and the liposomes were labeled with FITC green fluorescence, so as to achieve co-localization observation under confocal microscopy (CLSM). The specific operation is as follows:

[0061] The extracted RAW264.7 cell membranes were resuspended in 1 mL of PBS and a Cy5-NHS (N-carboxysuccinimide modified with Cy5) solution was added. The cells were incubated with gentle shaking at room temperature for 30 minutes in the dark. The labeled cell membranes were then washed three times with PBS, centrifuged at 12,000 rpm for 10 minutes each time at 4°C to remove free, unbound Cy5 dye. To prepare the liposome phase mixture, FITC-DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-fluorescein labeling) was added to a chloroform solution of soybean lecithin and DSPE-PEG2000. The mixture was thoroughly mixed in the dark. Chloroform was then removed by rotary evaporation using conventional thin-film hydration techniques to form a lipid film, which was then hydrated with PBS to form the FITC-labeled liposomes. Cy5-labeled CM and FITC-labeled liposomes were mixed in an ice bath at a mass ratio of 1:1 and ultrasonicated for 5 minutes. The mixture was then extruded through 0.4 μm and 0.2 μm polycarbonate membranes 15 times each to obtain fluorescently labeled hybrid membranes. Confocal laser scanning microscopy (CLSM) imaging was performed. If significant red and green fluorescence overlap (i.e., yellow colocalization signal) was observed, the fusion was successful. Figure 2 As shown, the present invention successfully prepared the hybrid membrane.

[0062] Example 3

[0063] In order to verify whether the hybrid membrane encapsulated Cu / Zn-MOF loaded with H-151 and the mannose-modified hybrid membrane encapsulated Cu / Zn-MOF loaded with H-151 successfully retained the surface marker proteins of the macrophage membrane, Western blot (WB) analysis was further performed to detect the expression of two macrophage-specific membrane proteins, F4 / 80 and CD11b.

[0064] The experimental procedure is as follows: First, membrane proteins were extracted from Cu / Zn-MOF hybrid membranes encapsulating H-151 and Cu / Zn-MOF hybrid membranes encapsulating H-151. Both nanocomplexes were pelleted by ultracentrifugation at 4°C (12,000 rpm, 15 min). The supernatant was discarded and the cells were thoroughly lysed in RIPA lysis buffer (RIPA to PMSF, 100:1 volume ratio). The cells were incubated on ice for 30 min, and pulsed sonication (5 s × 3 times) was used to assist lysis. The supernatant was then collected by centrifugation (4°C, 10,000 rpm, 5 min) as the protein sample. Protein concentration was determined using the BCA assay. 20 μL of diluted protein sample and various concentrations of BCA standards were added to a 96-well plate. Subsequently, 200 μL of BCA working solution (reagent A:B = 50:1) was added. The cells were incubated at 37°C in the dark for 30 min. The OD value was measured at 562 nm using a microplate reader, and the protein concentration was calculated. After adding 1 / 4 volume of 5× Loading Buffer, denature at 100°C for 5 minutes. Adjust the sample volume to equal volume with 1× Loading Buffer, and then perform SDS-PAGE electrophoresis. Take a 4-20% gradient precast gel, remove the comb and check for leakage, and load the prestained marker and equal amount of protein sample respectively. First, pre-run at a constant voltage of 10V for 20 minutes, then run the upper gel at 85V and the lower gel at 130V. After electrophoresis, transfer the protein to a PVDF membrane. The membrane is first activated in methanol for 1 minute and assembled into a sandwich with the gel (gel side facing the black board and membrane facing the white board) to ensure that there are no bubbles. The transfer is completed using the semi-dry transfer method (400mA constant current, 33min). After the transfer is completed, wash the membrane with TBST three times at room temperature for 5 minutes each time, and then add 5% skim milk powder TBST solution to block at room temperature for 2 hours. After blocking, the membrane was washed three times with TBST and then incubated overnight at 4°C with primary antibody incubation solution (rabbit anti-F4 / 80 1:1000, rabbit anti-CD11b 1:1000, rabbit anti-β-actin 1:20,000). The primary antibody was removed the next day, and the membrane was washed three times with TBST before the secondary antibody (HRP-conjugated goat anti-rabbit IgG, 1:50,000) was added. The membrane was incubated on a slow rocker at room temperature for 2 hours, and the washing was repeated three times. Finally, a chemiluminescent developer (solution A:solution B = 1:1) was applied to the protein area of ​​the membrane. The reaction was protected from light for 10 minutes, and the protein band signal was acquired and imaged using a developer.

[0065] like Figure 3 The results showed that characteristic bands of F4 / 80 and CD11b could be detected in Cu / Zn-MOF loaded with H-151 and Cu / Zn-MOF loaded with H-151 encapsulated in hybrid membranes and mannose-modified hybrid membranes.

[0066] Example 4

[0067] In order to further verify the success of the hybrid membrane and mannose modification, the present invention measured the hydrodynamic particle size and Zeta potential of Cu / Zn-MOF loaded with H-151, Cu / Zn-MOF loaded with H-151 encapsulated by hybrid membrane, and Cu / Zn-MOF loaded with H-151 encapsulated by mannose-modified hybrid membrane, respectively.

[0068] The specific method is as follows: Take appropriate amounts of samples, ultracentrifuge at 4°C (12,000rpm, 10min) to precipitate, discard the supernatant, and resuspend thoroughly with 1mL of ultrapure water. Rinse the sample pool with ultrapure water before measurement, gently wipe the light path with dust-free paper, and dry it naturally to ensure that there are no water marks and particle residues. The sample is then slowly injected into the sample pool to avoid bubbles and ensure that the sample is evenly distributed. The particle size is determined by dynamic light scattering (DLS). The sample pool is placed in the particle size analyzer and the test is completed according to the standard operating procedure of the instrument. When performing Zeta potential detection, the sample needs to be injected into a special electrode container and tested according to the same process.

[0069] like Figure 5 As shown in the figure, the potential and particle size changes of drug-loaded Cu / Zn-MOF, hybrid membrane-encapsulated drug-loaded Cu / Zn-MOF, and mannose-modified hybrid membrane-encapsulated drug-loaded Cu / Zn-MOF further indirectly confirmed the successful loading of H-151 and the successful modification of the hybrid membrane and mannose.

[0070] Example 5

[0071] In order to evaluate the controlled release performance of the mannosylated hybrid membrane-encapsulated H-151-loaded Cu / Zn-MOF system constructed in the present invention under different physiological environments, an in vitro drug release experiment based on the dialysis method was designed.

[0072] The specific steps are as follows: 0.04 mL of each 10 mg / mL solution of H-151-loaded Cu / Zn-MOF and H-151-loaded Cu / Zn-MOF encapsulated with a mannosylated hybrid membrane was diluted 10-fold with sterile PBS to obtain 0.4 mL of working solution, which was then placed into a dialysis bag. The total H-151 content was controlled to be 0.4 mg. The dialysis bag containing the sample was then placed into a centrifuge tube containing 20 mL of release medium: PBS buffer at pH 7.4 and pH 5.4, respectively. Samples were taken at predetermined time points (0, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 24, 36, and 48 h). At each time point, 1 mL of release medium was removed from the dialysis bag, and an equal volume of fresh PBS was added to maintain a constant system volume. Three replicates were set for all samples. The H-151 absorption intensity of the sample solution was measured at 280 nm using a UV-visible spectrophotometer, and the drug concentration was calculated using the standard curve, and then the cumulative release percentage was calculated.

[0073] The experimental results show that Figure 6 As shown in the data, the cumulative release rate of drug-loaded Cu / Zn-MOF encapsulated by the mannosylated hybrid membrane at pH 5.4 reached 68.4% in 48 hours, which was significantly higher than 32.2% at pH 7.4; while the release rates of drug-loaded Cu / Zn-MOF at pH 5.4 and pH 7.4 were 53% and 20.3%, respectively, indicating that the mannosylated hybrid membrane encapsulated drug-loaded Cu / Zn-MOF has stronger controlled release ability and environmental response in the inflammatory micro-acidic environment, and can effectively reduce drug leakage.

[0074] Example 6

[0075] In order to evaluate the performance of the mannose-modified hybrid membrane-encapsulated H-151-loaded Cu / Zn-MOF system in scavenging reactive oxygen free radicals in vitro, the scavenging of nitric oxide (·NO), superoxide anion (·O2 - ) and hydroxyl radical (·OH) scavenging experiments.

[0076] The NO scavenging experiment used the Griess reagent method: 0.5 M sodium nitroprusside (SNP) solution was mixed with different concentrations of mannose-modified hybrid membrane-encapsulated H-151-loaded Cu / Zn-MOF (5, 10, 20, 40, and 80 μg / mL) and incubated for 90 minutes. Griess Reagent R1 and R2 were then added for 10 minutes. The absorbance was measured at a wavelength of 540 nm to evaluate the NO scavenging ability.

[0077] O2 - The scavenging experiment used the xanthine-xanthine oxidase system: a reaction solution containing 0.05 mM xanthine and 50 mU / mL xanthine oxidase was prepared in PBS (pH 7.4), and samples of different concentrations were added. After incubation at 37°C for 30 minutes, the absorbance was measured at a wavelength of 560 nm to evaluate the superoxide anion scavenging ability.

[0078] The OH scavenging experiment uses the Fenton reaction system: 0.2 mM FeSO4 reacts with 0.2 mM H2O2 to generate active hydroxyl radicals. After adding samples of different concentrations, the mixture is incubated at room temperature for 30 minutes. A specific probe dye is used to react to generate a colored product. The absorbance change at a wavelength of 510 nm is measured to evaluate the hydroxyl radical scavenging ability.

[0079] like Figure 7 The results showed that the mannose-modified hybrid membrane encapsulated drug-loaded Cu / Zn-MOF system can effectively eliminate nitric oxide (·NO), superoxide anions (·O2 - ) and hydroxyl radicals (·OH).

[0080] Example 7

[0081] In order to verify the targeted uptake ability of the Cu / Zn-MOF system loaded with H-151 encapsulated by the mannosylated hybrid membrane at the cellular level, the mouse-derived macrophage cell line RAW264.7 was used as a cell model, and the fluorescence distribution of the nanomaterials in the cells was observed by confocal laser scanning microscopy (CLSM).

[0082] In the experiment, each set of nanomaterials (H-151-loaded Cu / Zn-MOF, H-151-loaded Cu / Zn-MOF encapsulated in a hybrid membrane, and H-151-loaded Cu / Zn-MOF encapsulated in a mannosylated hybrid membrane) was uniformly modified with the red fluorescent probe Cy5. The labeling method involved adding Cy5-NHS active ester (10 μM) to a PBS dispersion of the materials (pH 7.4). After incubation at room temperature in the dark for 30 minutes, the materials were centrifuged to remove free dye and resuspended in serum-free DMEM medium for later use.

[0083] In the experiment, RAW264.7 cells were seeded in 24-well plates (about 5×10 4 After culturing to 70-80% confluency, the medium was replaced with serum-free medium, and each group of Cy5-labeled nanomaterials was added and incubated at 37°C. After incubation, the cells were washed three times with PBS and the cell nuclei were stained with Hoechst 33342.

[0084] The fluorescence distribution was observed using a confocal laser scanning microscope at excitation wavelengths of 405 nm (Hoechst) and 640 nm (Cy5). Figure 8 As shown in the figure, the red Cy5 fluorescence signal in the cells of the mannosylated hybrid membrane encapsulated drug-loaded Cu / Zn-MOF group was the strongest, which was much higher than that of the unencapsulated group and the hybrid membrane group without mannose modification, suggesting that it has a stronger intracellular uptake ability.

[0085] Example 8

[0086] An experimental autoimmune uveitis (EAU) model was established in female C57BL / 6J mice to evaluate the therapeutic effect of mannosylated hybrid membrane-encapsulated drug-loaded Cu / Zn-MOF system in alleviating ocular inflammation.

[0087] To establish the EAU model, 5 mg of IRBP651–670 was dissolved in 200 μL of DMSO and then added to 800 μL of PBS to prepare the antigen peptide stock solution. Separately, 50 mg of Mycobacterium tuberculosis was added to 10 mL of complete Freund's adjuvant (CFA) and mixed thoroughly for later use. The peptide and CFA were pipetted into two 5 mL syringes at a 1:1 volume ratio and then connected and mixed to a total volume of 2 mL. The mixed injection solution was sealed on ice for 1 hour. The solution was then poured into water and observed for the formation of an oily mass. On the day of modeling, mice were anesthetized with an intraperitoneal injection of 1.25% avertin. Subsequently, the emulsion was injected into the base of the tail (50 μL), the back of the neck (50 μL), the left and right groin (50 μL each), and the left and right axilla (30 μL each), totaling 260 μL. Immediately after awakening, 0.1 μg / μL PTX (10 μL) plus PBS (190 μL) was injected intraperitoneally. From day 7 to 12 after immunization, mice were treated with tail vein injections of PBS, H-151, H-151-loaded Cu / Zn-MOF, hybrid membrane-encapsulated H-151-loaded Cu / Zn-MOF, and mannosylated hybrid membrane-encapsulated H-151-loaded Cu / Zn-MOF. On day 13, the mice underwent clinical ocular assessment. Tropicamide was used to dilate the pupils, and slit-lamp observation was performed for signs of anterior segment inflammation. Anterior segment scoring criteria included corneal edema, conjunctival and ciliary congestion, anterior chamber inflammatory cell infiltration, and iris adhesions. On day 14, the mice were sacrificed by cervical dislocation. Eyeballs were immediately removed and fixed in fixative for 48 hours. Subsequently, the eyes were graded dehydrated, cleared, and paraffin-embedded. Paraffin sections were taken along the corneal-optic nerve axis and histologically observed and scored using H&E staining. Inflammatory cell infiltration, neovascularization, detachment, and wrinkling of the retina and choroid were also evaluated microscopically.

[0088] The experimental results show that Figure 9 As shown in the results, the drug-loaded Cu / Zn-MOF treatment group encapsulated by the mannosylated hybrid membrane showed the most significant therapeutic effect in both the clinical score and the histopathological score of ocular inflammation, which was superior to the other treatment groups, indicating that the drug-loaded Cu / Zn-MOF system encapsulated by the mannosylated hybrid membrane can most effectively improve EAU ocular inflammation.

[0089] The Cu / Zn-MOF of the present invention has both biomimetic enzyme activity and drug carrier function; Cu / Zn-MOF has superoxide dismutase (SOD) activity and exhibits multi-enzyme synergistic free radical scavenging ability; and exhibits pH-responsive degradation behavior, suitable for controlled release applications in inflammatory microenvironments. At the same time, the loading of the STING antagonist H-151 synergizes with the Cu / Zn-MOF to construct a multi-effect synergistic therapeutic system. The construction of a hybrid membrane (LM) of liposomes and macrophage membranes uses a 1:1 mass ratio to fuse liposomes and RAW264.7-derived macrophage membranes; a hybrid membrane structure with biomimetic functions is constructed to enhance immune clearance and tissue homing capabilities; and key membrane proteins (F4 / 80, CD11b) are retained. Mannose (Man) surface functionalization modification achieves active targeting: DSPE-PEG-Man is co-inserted into the membrane layer to achieve active recognition of cells with high expression of mannose receptors; and the synergistic effect of passive and active targeting is achieved at the same time, improving drug enrichment efficiency. The nanosystem of the present invention is constructed to form a nanomedicine system with anti-inflammatory and ROS elimination functions; it is suitable for the treatment of various inflammatory diseases, especially experimental autoimmune uveitis (EAU).

[0090] The serial numbers of the above embodiments of the present invention are for description only and do not represent the advantages or disadvantages of the embodiments.

[0091] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are protected by the present invention.

Claims

1. A mannosylated hybrid membrane encapsulated Cu / Zn-MOF drug delivery system, characterized in that: The mannosylated hybrid membrane encapsulated the H-151-loaded Cu / Zn-MOF, and the hybrid membrane was prepared by fusion of liposomes and macrophage membranes.

2. The method for preparing a mannosylated hybrid membrane-encapsulated Cu / Zn-MOF drug delivery system according to claim 1, characterized in that: The following steps are involved: S1. Cu / Zn-MOF loaded with H-151: H-151 was dissolved in DMSO, and the methanol suspension of Cu / Zn-MOF was added. The mixture was stirred, centrifuged, and then washed to obtain Cu / Zn-MOF loaded with H-151. S2. Fusion of liposomes and macrophage membranes to construct hybrid membranes: macrophage membranes and liposomes were mixed, sonicated, and then extruded through a polycarbonate membrane to obtain hybrid membranes; S3. Preparation of mannosylated hybrid membrane: liposomes were prepared using DSPE-PEG-Man and fused with macrophage membranes to obtain mannosylated hybrid membranes; S4. Mannosylated hybrid membrane encapsulating H-151-loaded Cu / Zn-MOF: H-151-loaded Cu / Zn-MOF was mixed with the mannosylated hybrid membrane, incubated, and centrifuged to obtain a mannosylated hybrid membrane encapsulating H-151-loaded Cu / Zn-MOF.

3. The method for preparing a mannosylated hybrid membrane-encapsulated Cu / Zn-MOF drug delivery system according to claim 2, characterized in that: The method for loading H-151 on Cu / Zn-MOF includes: dissolving 10 mg of H-151 in 1 mL of DMSO, adding 20 mL of Cu / Zn-MOF methanol suspension dropwise, stirring at room temperature in the dark for 12 hours, centrifuging at 8000 rpm to remove free drugs, and washing with methanol three times.

4. The method for preparing a mannosylated hybrid membrane-encapsulated Cu / Zn-MOF drug delivery system according to claim 3, characterized in that: The preparation method of Cu / Zn-MOF includes: weighing 446.25 mg of zinc nitrate hexahydrate and 241.56 mg of copper nitrate trihydrate, dissolving them in 20 mL of anhydrous methanol to form solution A; weighing 3.284 g of 2-methylimidazole, dissolving it in an equal volume of methanol to form solution B; adding solution B dropwise to solution A under magnetic stirring conditions, and continuing to stir at room temperature for 4 hours. After the reaction is completed, centrifugation is performed at 8000 rpm for 5 minutes, the supernatant is discarded, and the precipitate is washed three times with methanol to remove impurities, and finally vacuum drying is performed at 50°C to obtain Cu / Zn-MOF powder.

5. The method for preparing a mannosylated hybrid membrane-encapsulated Cu / Zn-MOF drug delivery system according to claim 2, characterized in that: The method for constructing a hybrid membrane by fusing liposomes with macrophage membranes includes: mixing the macrophage membrane and liposomes in a mass ratio of 1:1, ultrasonicating in an ice bath for 5 minutes, and then extruding through 0.4 μm and 0.2 μm polycarbonate membranes 15 times to promote membrane fusion, thereby finally obtaining a hybrid membrane.

6. The method for preparing a mannosylated hybrid membrane-encapsulated Cu / Zn-MOF drug delivery system according to claim 5, characterized in that: The method for extracting macrophage membranes includes: washing RAW264.7 cells with PBS buffer, scraping the cells and collecting them in a 15mL centrifuge tube, centrifuging at 4°C and 300g for 5 minutes to precipitate the cells, then adding pre-cooled hypotonic buffer containing protease antagonists and PMSF to resuspend them, and incubating them in an ice bath for 30 minutes. The cell suspension is then transferred to a glass homogenizer and manually homogenized about 30 times under ice bath conditions to fully destroy the cell structure and release the cell membrane. The homogenate is then centrifuged at 4°C and 10,000g for 10 minutes, and the supernatant is collected. The supernatant is then ultracentrifuged at 4°C and 100,000g for 60 minutes. The resulting precipitate is the macrophage membrane.

7. The method for preparing a mannosylated hybrid membrane-encapsulated Cu / Zn-MOF drug delivery system according to claim 6, characterized in that: The preparation method of liposomes includes: dissolving soybean lecithin and DSPE-PEG2000 in chloroform at a mass ratio of 10:1, rotary evaporating at 40°C to form a uniform lipid film, then hydrating the obtained film with PBS at 37°C for 15 minutes, and ultrasonically disrupting it to form liposomes.

8. The method for preparing a mannosylated hybrid membrane-encapsulated Cu / Zn-MOF drug delivery system according to claim 2, characterized in that: The preparation method for the mannosylated hybrid membrane involves dissolving soybean lecithin and DSPE-PEG-Man in chloroform at a mass ratio of 10:

1. Rotary evaporation is performed at 40°C to form a uniform lipid film, and the solvent is continuously evaporated to remove residual chloroform. The resulting film is then hydrated with PBS at 37°C for 15 minutes and sonicated for 15 minutes to obtain small-sized liposomes. Macrophage membranes and liposomes are mixed at a mass ratio of 1:1, sonicated in an ice bath for 5 minutes, and then extruded 15 times through 0.4 μm and 0.2 μm polycarbonate membranes to promote membrane fusion, resulting in the mannosylated hybrid membrane.

9. The method for preparing a mannosylated hybrid membrane-encapsulated Cu / Zn-MOF drug delivery system according to claim 2, characterized in that: The method for encapsulating H-151-loaded Cu / Zn-MOF with a mannosylated hybrid membrane includes: mixing the H-151-loaded Cu / Zn-MOF and the mannosylated hybrid membrane in a mass ratio of 1:5, incubating at 37° C. for 2 hours, and removing unencapsulated particles by centrifugation to obtain a mannosylated hybrid membrane-encapsulated H-151-loaded Cu / Zn-MOF.

10. Use of the mannosylated hybrid membrane-encapsulated Cu / Zn-MOF drug delivery system according to claim 1 or the preparation method of the mannosylated hybrid membrane-encapsulated Cu / Zn-MOF drug delivery system according to any one of claims 2 to 9 in the delivery of inflammatory drugs.

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