Preparation method and application of membrane fusion liposome

Abstract

The invention belongs to the technical field of biomedicine, and relates to a preparation method and application of a membrane fusion liposome. A drug-loaded cationic liposome and a CCR2 overexpression macrophage membrane are mixed and extruded, and the macrophage membrane is promoted to be integrated into double layers of the liposome, so that the membrane fusion liposome is obtained. According to the membrane fusion liposome, on one hand, PSMP is eliminated, recruitment of inflammatory macrophages is destroyed, and inflammation is relieved from the outside; on the other hand, the delivered STING inhibitor blocks the STING pathway in the injured renal tubular epithelial cells from the inside, the inflammation level is doubly relieved, and the synergistic anti-inflammatory and kidney protection effects are achieved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a method for preparing membrane-fused liposomes and their applications. Background Technology

[0002] The novel chemokine PSMP (PC3-secreted microprotein) plays a crucial initiation role in the pathogenesis of IBD (such as ulcerative colitis). Produced by colonic epithelial cells in the early stages of inflammation, PSMP acts as a "pioneer" in inflammatory initiation. By specifically binding to and activating its receptor CCR2, it drives Ly6C⁺CCR2⁺ monocytes / macrophages to migrate and infiltrate the intestinal inflammatory site, thereby amplifying the inflammatory response. Inhibiting PSMP function through neutralizing antibodies and other methods can significantly reduce the severity of colitis, decrease macrophage aggregation, and reduce the production of pro-inflammatory factors (such as IL-6, TNF-α, and CCL2). Therefore, targeting the PSMP / CCR2 interaction provides a novel strategy for developing drugs to treat IBD.

[0003] The chemokine PSMP is specifically highly expressed in human and mouse liver fibrosis tissues induced by various etiologies. Hepatocytes become the main cellular source of PSMP under the stimulation of injury signals (such as HMGB-1 and IL-33). PSMP drives the liver fibrosis process in two ways by binding to its receptor CCR2: on the one hand, it chemotactically polarizes monocytes / macrophages, triggering an inflammatory storm; on the other hand, it directly activates hepatic stellate cells, leading to excessive extracellular matrix deposition. Simultaneously, PSMP gene knockout or the use of the PSMP-specific neutralizing antibody 3D5 can effectively inhibit liver inflammation and fibrosis, and reverse existing fibrosis in therapeutic models. Therefore, inhibiting PSMP provides a starting point for the development of anti-liver fibrosis drugs.

[0004] The chemokine PSMP is specifically highly expressed in the renal tissue, urine, and plasma of patients with acute kidney injury (AKI). Produced by damaged renal tubular epithelial cells, PSMP attracts inflammatory macrophages to infiltrate the kidneys and polarize them to the M1 phenotype by specifically binding to its receptor CCR2, thereby driving renal inflammation and damage. Treatment with a PSMP-specific neutralizing antibody (3D5) significantly reduced mortality in AKI model animals and effectively improved renal function, alleviating renal tubular damage and inflammation; this effect was CCR2-dependent. Therefore, targeting PSMP provides a therapeutic strategy for AKI, for which effective drugs are currently lacking.

[0005] In summary, PSMP (PC3-secreted microprotein), a novel chemokine secreted by epithelial cells of damaged tissue, plays a central role in various diseases, including colitis, liver fibrosis, and acute kidney injury. PSMP specifically binds to and activates its receptor CCR2, driving inflammatory monocytes / macrophages to infiltrate the lesion site, thereby initiating and amplifying tissue inflammation, damage, and fibrosis. Currently, inhibiting PSMP function using neutralizing antibodies has significantly alleviated disease progression and improved tissue function in various animal models; however, using neutralizing antibodies (such as 3D5) to bind to and inactivate PSMP is merely a "blocking" strategy.

[0006] Furthermore, the "mitochondrial damage – mtDNA leakage – cGAS-STING activation – kidney inflammation" signaling axis is the core pathological mechanism of fibrosis in acute kidney injury (AKI) and chronic kidney disease (CKD); however, specifically delivering drugs, especially small molecule inhibitors, to the main diseased cells (renal tubular epithelial cells) is extremely difficult. For example, PLGA, a carrier now widely used in drug delivery system (DDS) research, is not only unable to be effectively retained due to the abundance of negatively charged heparan sulfate proteoglycans on the surface of the glomerular basement membrane and renal tubular epithelial cells, but may also be rejected by the negatively charged glomerular filtration barrier.

[0007] Existing kidney disease intervention technologies have two major pain points: On the one hand, interventions targeting the PSMP-CCR2 pathway are limited to "downstream blocking" and lack active intervention strategies that can capture and clear PSMP at the upstream of inflammation initiation, making it difficult to curb the initiation of inflammation and fibrosis at the root; on the other hand, small molecule inhibitors targeting the cGAS-STING pathway lack a precise delivery system that targets renal tubular epithelial cells, making it difficult to effectively reach the target site and limiting their clinical application.

[0008] Therefore, developing an upstream intervention strategy that can efficiently capture and clear inflammatory cells before PSMP recruits them, and simultaneously constructing a targeted delivery system that can overcome the renal electrostatic repulsion barrier and precisely deliver small molecule inhibitors to the cytoplasm of damaged renal tubular epithelial cells, to achieve synergistic or precise intervention on the two core pathological pathways, is of great significance for improving the treatment effect of kidney diseases (AKI and CKD) and promoting the clinical translation of related technologies. It is also a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0009] The purpose of this invention is to provide a method for preparing membrane-fused liposomes and their applications, overcoming the shortcomings of existing technologies. This invention employs the following technical solutions to achieve its objective: One aspect of the present invention provides a method for preparing membrane-fused liposomes, comprising the following steps: Drug-loaded cationic liposomes were mixed with CCR2-overexpressing macrophage membranes, sonicated, and then extruded 5 to 21 times through an extrusion membrane with a pore size of 0.1 to 0.4 μm to promote the integration of macrophage membranes into the bilayer of liposomes, thus obtaining membrane-fused liposomes.

[0010] Drug-loaded cationic liposomes were prepared by the following method: Step 1-1: The carboxyl group of 4-carboxyl-2,2,6,6-tetramethylpiperidine-1-oxy radical (4-carboxyl-TEMPO) was activated and reacted with DPSE-PEG-NH2 at pH 7.2±0.4. After dialysis purification, DPSE-PEG-TEMPO was obtained. Steps 1-2: Neutral phospholipids, cationic lipids, and DPSE-PEG-TEMPO were dissolved in a solvent, and then a STING inhibitor was added. The solvent was removed by rotary evaporation to obtain a lipid film. Phosphate buffer was added to the lipid film for hydration treatment, followed by sonication. The liposome solution was then continuously extruded through an extrusion membrane with a pore size of 0.1-0.4 μm for 5-21 times to obtain drug-loaded cationic liposomes.

[0011] In step 1-1: Preferably, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are used together for carboxyl activation.

[0012] Preferably, the carboxyl activation time is 10~60 min.

[0013] Preferably, the molar ratio of EDC, NHS and 4-carboxy-TEMPO is 1~2:1~2:1.

[0014] Preferably, the molar ratio of 4-carboxy-TEMPO to DPSE-PEG-NH2 is 5~20:1.

[0015] Preferably, the reaction temperature is 10~40℃ and the reaction time is 15~48 h.

[0016] Preferably, the molecular weight cutoff of the dialysis bag is 200~1000 Da.

[0017] In steps 1-2: Preferably, the cationic lipid is one or more of (2,3-dioleoyl-propyl)-trimethylamine (DOTAP), 1,2-dioleoyloxy-3-(dimethylamino)propane (DODAP), and dimethyltriphenylmethylammonium chloride (DMTAP). (2,3-dioleoyl-propyl)-trimethylamine (DOTAP) is preferred.

[0018] Preferably, the neutral phospholipid is one or more of 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), 1,2-distearatel-sn-glycerol-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), and 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine (DPPC). Preferably, it is 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC).

[0019] Preferably, the mass ratio of neutral phospholipids, cationic lipids, and DPSE-PEG-TEMPO is 30~45:7~15:5.

[0020] Preferably, the amount of STING inhibitor added accounts for 3 to 10 wt% of the total mass. Here, the total mass refers to neutral phospholipids, cationic lipids, DPSE-PEG-TEMPO, and STING inhibitor.

[0021] Preferably, the solvent is one or more of dichloromethane, methanol, chloroform, acetone, and ethanol.

[0022] Preferably, the rotary evaporation parameters include: temperature of 20~40℃, time of 10~50 min, rotation speed of 50~300 rpm, and pressure of 100~800 bar.

[0023] Preferably, the hydration treatment parameters include: temperature of 30~50℃, time of 10~50min, rotation speed of 50~300rpm, and pressure of 100~400 bar.

[0024] CCR2-overexpressing macrophage membranes were extracted from macrophages with stable CCR2 overexpression. The extraction method included the following steps: CCR2-overexpressing macrophages (RAW 264.7-CCR2) were established by plasmid electroporation. The CCR2-overexpressing macrophages were suspended in hypotonic buffer containing protease inhibitors and the cells were disrupted by squeezing. The supernatant was collected by low-speed centrifugation and the precipitate was collected by high-speed centrifugation. The precipitate was resuspended in phosphate buffer and sonicated. The precipitate was then extruded 5 to 21 times through an extrusion membrane with a pore size of 0.1 to 0.4 μm to obtain a cell membrane suspension.

[0025] Preferably, the hypotonic buffer comprises: 5-15 mM Tris-HCl, 0.5-2 mM MgCl2, and 0.5-2 mM protease inhibitor; the pH of the hypotonic buffer is 7.2±0.3.

[0026] Preferably, the protease inhibitor is one or more of benzyl sulfonyl fluoride (PMSF), 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), and diisopropyl fluorophosphate (DFP).

[0027] The parameters for low-speed centrifugation include: a centrifugation speed of 1000~5000 g, a centrifugation temperature of 1~6℃, and a centrifugation time of 20~40 min; the supernatant is collected by low-speed centrifugation to remove the precipitate, and this process can remove cell nuclei, mitochondria, etc.

[0028] The parameters for high-speed centrifugation include: a centrifugation speed of 15,000~30,000 g, a centrifugation temperature of 1~6℃, and a centrifugation time of 45~90 min; the precipitate is collected by high-speed centrifugation, and the supernatant is removed. This step can obtain the cell membrane.

[0029] In this paper, the pH of the phosphate buffer solution is 7.2 ± 0.3. The preferred ultrasonic treatment parameters include: ultrasonic power of 50–200 W, time of 1–8 min, and temperature of 10–40 °C. The extruded membrane can be one or more of the following: polycarbonate (PC) membrane, polyethersulfone membrane, polyamide membrane, cellulose acetate membrane, cellulose nitrocellulose membrane, and polytetrafluoroethylene membrane.

[0030] A second aspect of the present invention provides a membrane-fused liposome, which is prepared by the above preparation method.

[0031] A third aspect of this invention provides the application of membrane-fused liposomes in the preparation of drugs for clearing PSMP. The membrane-fused liposomes of this invention exhibit excellent PSMP clearance performance and can be directly used as the core active ingredient to prepare drugs for in vitro and in vivo PSMP clearance.

[0032] A fourth aspect of the present invention provides the use of membrane-fused liposomes in the preparation of medicaments for treating colitis, liver fibrosis, or acute kidney injury.

[0033] PSMP plays a central role in colitis, liver fibrosis, and acute kidney injury. The membrane-fused liposomes of this invention exhibit excellent PSMP clearance performance, efficiently capturing and eliminating inflammatory cells before they are recruited by PSMP, thus blocking the "PSMP-CCR2-inflammation / fibrosis" pathway at its source and inhibiting the progression of these diseases. Furthermore, the membrane-fused liposomes of this invention are loaded with a STING inhibitor, allowing them to precisely target renal tubular epithelial cells and release the STING inhibitor. This ensures the STING inhibitor effectively reaches its target site, blocking the cGAS-STING pathway and potentially offering therapeutic benefits for liver fibrosis and acute kidney injury.

[0034] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention utilizes the high affinity binding properties of CCR2-overexpressing cell membranes to actively respond to PSMP highly expressed in the target lesion area, achieving the highest degree of targeting specificity and enrichment efficiency. Furthermore, through the internalization of liposome nanoparticles, PSMP can be captured and cleared before it recruits large numbers of monocytes / macrophages, intervening from the very upstream of inflammation and potentially providing a more potent and durable therapeutic effect than simple blockade. In addition, the cell membrane used is derived from macrophages, which themselves express receptors for other inflammatory factors (such as TNF-α and IL-6). Therefore, the membrane-fused liposome nanomaterials of this invention can also simultaneously clear other inflammatory factors, producing excellent anti-inflammatory effects.

[0035] 2. This invention creatively utilizes the positively charged property of cationic liposomes, which can interact with the negatively charged glomerular basement membrane and renal tubular epithelial cells through electrostatic adsorption and possible endocytosis, thus solving the rejection problem of negatively charged carriers such as PLGA and achieving active renal targeting and efficient retention. Secondly, cationic liposomes can more effectively deliver poorly water-soluble STING inhibitors (such as C-176) to the cytoplasm of renal tubular epithelial cells, which is the site where cGAS senses mtDNA, and is crucial for acting on the intracellular target STING. Finally, for the treatment of acute kidney injury (AKI), rapid drug onset is required to block the cytokine storm, and the rapid release characteristics of cationic liposomes are perfectly matched to this.

[0036] 3. The introduction of the TEMPO group enables the invention to have real-time MRI localization function. The enrichment of liposomes in the kidney can be directly observed through MRI signals, realizing non-invasive diagnosis and drug distribution tracking. Moreover, the nitric oxide free radical TEMPO is an all-organic, metal-free material, which eliminates all risks caused by metal toxicity from the source (such as the NSF caused by gadolinium contrast agents widely used in clinical practice), making it safer for patients with renal insufficiency.

[0037] 4. This invention creates a synergistic therapeutic modality of PSMP clearance and STING antagonist delivery. On the one hand, clearing PSMP disrupts the recruitment of inflammatory macrophages, alleviating inflammation from the "external"; on the other hand, the delivered C-176 blocks the STING pathway within damaged renal tubular epithelial cells from the "internal," thus providing a dual relief of inflammation levels.

[0038] This invention develops a novel membrane-fused liposome that combines the advantages of both cell membranes and liposomes. Due to CCR2 overexpression, the membrane-fused liposome of this invention exhibits superior active targeting ability and enrichment efficiency against inflammatory lesions in acute kidney injury. This not only increases the concentration of drug C-176 at the lesion site, but also allows the membrane-fused liposome to directly remove the key PSMP chemokine from the source (providing a more potent and longer-lasting therapeutic effect than "blocking"). This synergistic effect with the mechanism of C-176 blocking the STING pathway achieves an anti-inflammatory and renal protective effect greater than the sum of its parts. Attached Figure Description

[0039] Figure 1 DSPE-PEG of Example 1 2000 -Infrared spectrum of TEMPO.

[0040] Figure 2 DSPE-PEG of Example 1 2000 -TEMPO, CCR2 Electron paramagnetic resonance image of MM / TP-SALP.

[0041] Figure 3 DSPE-PEG of Example 1 2000 - TEMPO's proton NMR spectrum.

[0042] Figure 4 TP-SALP of Example 1 CCR2 MM, CCR2 Transmission electron microscope image of MM / TP-SALP.

[0043] Figure 5 TP-SALP of Example 1 CCR2 MM, CCR2 Dynamic light scattering characterization diagram of MM / TP-SALP.

[0044] Figure 6 TP-SALP of Example 1 CCR2 Particle size change of MM / TP-SALP after 7 days of storage in PBS buffer.

[0045] Figure 7 Real-time quantitative PCR images of RAW 264.7 and RAW 264.7-CCR2.

[0046] Figure 8 Immunofluorescence images of RAW 264.7 and RAW 264.7-CCR2.

[0047] Figure 9 TP-SALP of Example 1 CCR2 MM / TP-SALP, CCR2 Western blot images of MM and MM / TP-SALP and MM as shown in Comparative Example 1.

[0048] Figure 10 TP-SALP of Example 1 CCR2 MM / TP-SALP, CCR2 Polyacrylamide gel electrophoresis images of MM and comparative example 1, MM / TP-SALP and MM.

[0049] Figure 11 TP-SALP of Example 1 CCR2 MM, CCR2 Fluorescence resonance energy diagram of MM / TP-SALP.

[0050] Figure 12 TP-SALP of Example 1 CCR2 MM, CCR2 Fluorescence colocalization pattern under a confocal microscope for MM / TP-SALP.

[0051] Figure 13 For different concentrations CCR2 MRI performance characterization of MM / TP-SALP at 1.5 T.

[0052] Figure 14 As in Example 1 CCR2 In vitro targeting data of MM / TP-SALP.

[0053] Figure 15 As in Example 1 CCR2 MM, TP-SALP, CCR2 In vitro PSMP clearance efficiency graphs of MM / TP-SALP and Comparative Example 1.

[0054] Figure 16 As in Example 1 CCR2 Graph showing the cytotoxicity results of MM / TP-SALP. Detailed Implementation

[0055] In the description of this invention, unless otherwise stated, the numerical range "a~b" represents a shortened representation of any combination of real numbers between a and b, and includes both a and b. "Multiple" includes two, three, four, five, or more.

[0056] The technical solution of the present invention will be further described and illustrated below with reference to specific embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are only for the purpose of helping to understand the present invention and are not intended to limit the specific scope of the present invention. Furthermore, the accompanying drawings used herein are merely for better illustrating the content disclosed in the present invention and do not limit the scope of protection. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commonly used in the art, and the methods used in the embodiments are all conventional methods in the art.

[0057] Example 1 The membrane-fused liposomes of this embodiment were prepared by the following method: 1. Synthesis of drug-loaded cationic liposomes (TP-SALP) 1-1. Under anhydrous and oxygen-free conditions, 4-carboxy-TEMPO (10 equivalents) was first mixed with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 12 equivalents) and N-hydroxysuccinimide (NHS, 12 equivalents) in DMSO solvent and magnetically stirred at 700 rpm / min for 30 min at room temperature to activate the carboxyl groups. Then, DPSE-PEG was slowly added dropwise using a constant pressure dropping funnel. 2000 -NH2 solution (1 equivalent), and the pH of the final solution system was adjusted to 7.5 with triethylamine (TEA), and the reaction was carried out at room temperature for 24 h. After the reaction was completed, the product was purified by dialyzing with a 300 Da dialysis bag for 72 h, and then freeze-dried and stored at -20 °C for later use.

[0058] 1-2. Mix DMPC, DOTAP, and DPSE-PEG 2000 TEMPO was dissolved in dichloromethane and methanol (volume ratio 4:1) at a mass ratio of 36:9:5, and then the STING inhibitor (C-176) was added. C-176 accounted for a certain percentage of the total mass of the components (i.e., DMPC, DOTAP, DPSE-PEG). 2000 The total amount of TEMPO and C-176 was 5 wt%. Organic solvents were removed using a rotary evaporator (300 bar, 100 rpm / min, 30℃, 30 min) to form a lipid film on a round-bottom flask. The bottom of the flask was purged with nitrogen for 10 min to remove as much unevaporated organic solvent as possible. A suitable amount of PBS buffer solution (pH 7.4) was added for hydration treatment (200 bar, 150 rpm / min, 40℃, 30 min). Then, the solution was sonicated at room temperature (80 W, 3 min, 3 s on and 3 s off cycle repeated (3 s-3 s)). The liposome solution was then extruded 15 times through a 0.2 μm polycarbonate membrane fitted with a liposome extruder to obtain liposomes with uniform particle size.

[0059] 2. Overexpression of macrophage membrane ( CCR2 Extraction of MM) After ensuring the cells were free of contamination and constructing a plasmid containing the target gene CCR2, Neon was used. TM Plasmids were electrotransfected into RAW 264.7 macrophages using a transfection instrument, and transfection efficiency was monitored using an EGFP control group. Forty-eight hours after transfection, successfully transduced cells were screened using a drug to obtain a macrophage cell line stably overexpressing CCR2 (RAW264.7-CCR2). RAW 264.7-CCR2 cells were then cultured in DMEM medium with 10% FBS and maintained at 37°C under a humid atmosphere of 5% CO2 until 80% confluence. Cells were then collected by centrifugation (1000 rpm / min, 5 min) (approximately 2 × 10⁶ cells / min). 8 Cell pellet was resuspended in ice-cold hypotonic buffer (pH 7.4) containing 10 mM Tris-HCl, 1 mM MgCl2, and 1 mM benzosulfonyl fluoride (PMSF) for 8 min to lyse cells. The cells were then extruded 7 times using a microextruder without a polycarbonate membrane to further disrupt the cell structure. The supernatant was collected by centrifugation at 4°C and 4000 g for 30 min to remove the nucleus and mitochondria. The supernatant was then centrifuged at 4°C and 20000 g for 1 h to collect the cell membrane pellet. The supernatant was discarded, and the pellet was resuspended in 1 mM PBS buffer (pH 7.4). The pellet was sonicated at room temperature (80 W, 3 min, 3 s–3 s). The cell membrane suspension was then extruded 15 times through a 0.2 μm polycarbonate membrane and stored at -80°C for later use. A portion of the cell membrane suspension was also diluted appropriately, and the protein concentration was determined using a BCA protein quantification kit.

[0060] 3. Membrane-fused liposomes ( CCR2 Preparation of MM / TP-SALP The mass concentration of the liposome solution was calculated based on the amount of feed used in the previous liposome synthesis. The cell membrane suspension and drug-loaded liposomes were mixed at a protein-lipid ratio of 1:100, ultrasonicated (80 W, 3 min, 3 s-3 s), and then continuously extruded through a 0.2 μm polycarbonate membrane 15 times to promote the integration of macrophage cell membranes into the bilayer of liposomes.

[0061] Figure 1 As can be seen from this, DSPE-PEG 2000 -TEMPO relative to DSE-PEG 2000 -NH2 at 1740 cm -1 The change in the absorption peak of the carbonyl stretching vibration indicates the formation of a new amide bond.

[0062] Figure 2 In the middle, the intermediate product DSPE-PEG 2000 -TEMPO and final materials CCR2 Both MM / TP-SALP exhibit the characteristic triplet of TEMPO; Figure 3 In the figure, 0.8-1.2 ppm represents methyl and methylene groups from TEMPO, indicating that TEMPO was successfully modified onto the material. From... Figure 2 and Figure 3 It can be seen that the chemical coupling of TEMPO was successful.

[0063] The particle size and potential of nanoparticles are characterized by nanoparticle size analyzers, such as... Figure 5 As shown: the average particle size of TP-SALP is 146 nm, and its zeta potential is 24.01 mV; CCR2 The average particle size of MM is 194 nm, and its zeta potential is -22.14 mV; CCR2 The average particle size of MM / TP-SALP was 155 nm, and its zeta potential was 16.42 mV. The liposome particle size increased after membrane fusion (results consistent with...). Figure 4 (The results show similar particle size), and the potential is lowered due to the integration of positively charged cationic liposomes into the negatively charged cell membrane.

[0064] TP-SALP, CCR2 MM / TP-SALP were dispersed separately in phosphate buffer at pH 7.4, and the particle size change of the materials was monitored over one week in PBS. Figure 6 As shown, the particle size variation of liposomes and membrane-fused liposomes is very small, indicating that the material has good in vitro stability.

[0065] Comparative Example 1 The membrane fusion liposome (MM / TP-SALP) of Comparative Example 1 was prepared by the following method: 1. The synthesis of drug-loaded cationic liposomes (TP-SALP) is the same as in Example 1.

[0066] 2. Extraction of macrophage membranes (MM) RAW 264.7 macrophages were cultured in DMEM medium containing 10% FBS and maintained at 37°C under a humid atmosphere of 5% CO2 until 80% confluence. Cells were then collected by centrifugation (1000 rpm / min, 5 min) (approximately 2 × 10⁶ cells / min). 8Cell pellet was resuspended in ice-cold hypotonic buffer (pH 7.4) containing 10 mM Tris-HCl, 1 mM MgCl2, and 1 mM benzosulfonyl fluoride (PMSF) for 8 min to lyse cells. The cells were then extruded 7 times using a microextruder without a polycarbonate membrane to further disrupt the cell structure. The supernatant was collected by centrifugation at 4°C and 4000 g for 30 min to remove the nucleus and mitochondria. The supernatant was then centrifuged at 4°C and 20000 g for 1 h to collect the cell membrane pellet. The supernatant was discarded, and the pellet was resuspended in 1 mM PBS buffer (pH 7.4). The pellet was sonicated at room temperature (80 W, 3 min, 3 s–3 s). The cell membrane suspension was then extruded 15 times through a 0.2 μm polycarbonate membrane and stored at -80°C for later use. A portion of the cell membrane suspension was also diluted appropriately, and the protein concentration was determined using a BCA protein quantification kit.

[0067] 3. Preparation of membrane fusion liposomes (MM / TP-SALP) The mass concentration of the liposome solution was calculated based on the amount of feed used in the previous liposome synthesis. The cell membrane suspension and drug-loaded liposomes were mixed at a protein-lipid ratio of 1:100, ultrasonicated (80 W, 3 min, 3 s-3 s), and then continuously extruded through a 0.2 μm polycarbonate membrane 15 times to promote the integration of macrophage cell membranes into the bilayer of liposomes.

[0068] Figure 7 The real-time quantitative PCR results show the overexpression of CCR2 in RAW 264.7 cells transfected with the plasmid: CCR2 mRNA was overexpressed by more than 8000-fold. Figure 8 Immunofluorescence images of RAW 264.7 cells and plasmid-transfected RAW 264.7 cells are shown: the fluorescence intensity reflects the effective overexpression of CCR2 in RAW 264.7 cells. This indicates that plasmid transfection successfully overexpressed CCR2 in RAW cells.

[0069] Protein imprinting characterizes the CCR2 target protein in each sample, such as Figure 9 As shown, only the two groups of CCR2 protein overexpression bands had significantly higher gray values; and after the preparation of membrane fusion liposomes, the overexpressed CCR2 protein was still well preserved.

[0070] The total protein of each sample was characterized by SDS-PAGE, such as... Figure 10 As shown, after membrane fusion, various membrane proteins are still well preserved; the membrane fusion process does not denature membrane proteins.

[0071] Liposomes, cell membranes, and membrane-fused liposomes were labeled with a pair of FRET dyes, respectively. Membrane fusion was characterized by fluorescence resonance energy transfer (FRET) experiments. Figure 11As shown, a non-radiative resonance transfer occurs at 565 nm, indicating that the cell membrane and liposomes have fully fused.

[0072] Liposomes, cell membranes, and membrane-fused liposomes were labeled with two different dyes, and fluorescence co-localization was performed using confocal microscopy. Figure 12 As shown, the two can be co-located under a confocal microscope; the cell membrane and liposomes have undergone effective fusion.

[0073] Configure a series of concentration gradients CCR2 MM / TP-SALP solution was used to characterize the MRI properties of the material at 1.5 T, such as... Figure 13 As shown: CCR2 MM / TP-SALP exhibits good T1 imaging signal at a certain concentration, indicating that... CCR2 MM / TP-SALP has good MRI performance.

[0074] Transwell experiments were performed using a cell-embedded version to simulate the material's in vitro targeting of the inflamed renal tubules via blood vessels; the fluorescence intensity of the nanoparticles was measured by dye labeling in the lower chamber (e.g., Figure 14 As shown in the figure, CCR2 overexpression in the cell membrane endows the material with stronger targeting efficiency, proving that the CCR2 overexpression material has enhanced inflammation targeting ability.

[0075] HK-2 cells were stimulated to express PSMP with LPS, and the activated culture medium was collected. Samples were then... CCR2 MM, TP-SALP, CCR2 MM / TP-SALP and MM / TP-SALP were co-incubated with the drug, and the PSMP concentration was detected using an ELISA kit. The results are as follows: Figure 15 As shown: Observed CCR2 MM (CM is) CCR2 The PSMP concentration decreased less in MM samples, TP-SALP samples, and MM / TP-SALP samples, while CCR2 overexpression was observed in... CCR2 The MM / TP-SALP material showed the greatest reduction in PSMP concentration, indicating that the membrane-fused liposomes obtained by fusing CCR2-overexpressing macrophage membranes into liposomes have excellent PSMP clearance capabilities.

[0076] Different concentrations of TP-SALP, CCR2 MM / TP-SALP was co-incubated with HK-2 cells, and cell viability was detected using a CCK8 assay kit. The results are as follows: Figure 16 As shown, cell viability remained good under different concentrations of material treatment, indicating that the material has good cell compatibility.

[0077] All aspects, embodiments, and features of this invention should be considered illustrative in all respects and not limiting of the invention; the scope of the invention is defined only by the claims. Other embodiments, modifications, and uses will become apparent to those skilled in the art without departing from the spirit and scope of the invention as claimed.

[0078] In the preparation method of this invention, the order of the steps is not limited to the listed order. For those skilled in the art, variations in the order of the steps without creative effort are also within the scope of protection of this invention. Furthermore, two or more steps or actions can be performed simultaneously.

[0079] Finally, it should be noted that the specific embodiments described herein are merely illustrative examples of the invention and are not intended to limit the implementation of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them; it is neither necessary nor possible to exemplify all embodiments here. However, these obvious variations or modifications derived from the essential spirit of the invention still fall within the scope of protection of the invention, and interpreting them as any additional limitation would contradict the spirit of the invention.

Claims

1. A method for preparing a membrane-fused liposome, characterized by, The method comprises the following steps: The drug-loaded cationic liposome and the CCR2 overexpression macrophage membrane are mixed, and after ultrasonic treatment, the mixture is continuously extruded 5-21 times through an extrusion membrane with a pore size of 0.1-0.4 μm to promote the integration of the macrophage membrane into the bilayer of the liposome, so as to obtain the membrane fusion liposome; The drug-loaded cationic liposome is prepared by the following method: Step 1-1: After the carboxyl group of 4-carboxyl-2,2,6,6-tetramethylpiperidine-1-oxyl radical is activated, the activated carboxyl group is reacted with DPSE-PEG-NH2 at pH 7.2±0.4, and then the product is purified by dialysis to obtain DPSE-PEG-TEMPO; Step 1-2: The neutral phospholipid, the cationic lipid and the DPSE-PEG-TEMPO are dissolved in a solvent, and then the STING inhibitor is added, and the solvent is removed by rotary evaporation to obtain a lipid film; the phosphate buffer is added to the lipid film, and the lipid film is hydrated and treated, and then the liposome solution is ultrasonically treated, and then the liposome solution is continuously extruded 5-21 times through an extrusion membrane with a pore size of 0.1-0.4 μm to obtain the drug-loaded cationic liposome.

2. The production method according to claim 1, characterized by, In step 1-1: The carboxyl group is activated by using 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide in combination; The carboxyl group activation time is 10-60 min; The molar ratio of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide and 4-carboxyl-TEMPO is 1-2:1-2:1; The molar ratio of 4-carboxyl-2,2,6,6-tetramethylpiperidine-1-oxyl radical to DPSE-PEG-NH2 is 5-20:1; The reaction temperature is 10-40℃, and the reaction time is 15-48 h; The molecular weight cut-off of the dialysis bag is 200-1000 Da.

3. The preparation method according to claim 1, characterized in that, In step 1-2: The cationic lipid is one or more of (2,3-dioleoyl-propyl)-trimethylamine, 1,2-dioleoyloxy-3-(dimethylamino)propane, and dimethyl triphenylphosphonium chloride; And / or, the neutral phospholipid is one or more of 1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine; And / or, the mass ratio of the neutral phospholipid, the cationic lipid and DPSE-PEG-TEMPO is 30-45:7-15:

5.

4. The preparation method according to claim 1, characterized in that, The rotary evaporation parameters include: temperature 20-40℃, time 10-50 min, rotation speed 50-300 rpm, and pressure 100-800 bar; And / or, the hydration treatment parameters include: temperature 30-50℃, time 10-50 min, rotation speed 50-300 rpm, and pressure 100-400 bar.

5. The method of claim 1, wherein, The CCR2 overexpression macrophage membrane is prepared by the following method: The CCR2 stable overexpression macrophage is established by plasmid electrotransformation, the CCR2 stable overexpression macrophage is suspended in a hypotonic buffer containing a protease inhibitor, and the cell is broken by extrusion, the supernatant is obtained by low-speed centrifugation, the precipitate is obtained by high-speed centrifugation, the precipitate is resuspended in a phosphate buffer, and the cell membrane suspension is obtained by ultrasonic treatment and continuous extrusion for 5-21 times through an extrusion membrane with a pore size of 0.1-0.4 μm.

6. The production method according to claim 5, characterized by, The hypotonic buffer comprises 5-15 mM Tris-HCl, 0.5-2 mM MgCl2 and 0.5-2 mM protease inhibitor, and the pH of the hypotonic buffer is 7.2±0.

3. The protease inhibitor is one or more of benzylsulfonyl fluoride, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride and diisopropyl fluorophosphate.

7. The preparation method according to claim 5, characterized in that, The low-speed centrifugation step parameters comprise that the rotation speed of low-speed centrifugation is 1000-5000 g, the centrifugation temperature is 1-6℃, and the centrifugation time is 20-40 min. And / or, the high-speed centrifugation step parameters comprise that the rotation speed of high-speed centrifugation is 15000-30000 g, the centrifugation temperature is 1-6℃, and the centrifugation time is 45-90 min.

8. A membrane-fused liposome, characterized by, The membrane fusion liposome is prepared by the preparation method of any one of claims 1-7.

9. Use of the membrane fusion liposome of claim 8 in the preparation of a PSMP drug.

10. Use of the membrane fusion liposome of claim 8 in the preparation of a drug for treating colitis, liver fibrosis or acute kidney injury.

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