A dual-aptamer modified membrane fusion liposome and a preparation method and application thereof

Abstract

The application discloses a kind of double aptamer modified membrane fusion liposome and its preparation method and application, belong to the field of biotechnology.The mesenchymal stem cell membrane is mixed with drug-loaded liposome, and is extruded after ultrasonic, and mesenchymal stem cell membrane fusion liposome is obtained;Mesenchymal stem cell membrane fusion liposome is incubated with double aptamer, and double aptamer modified mesenchymal stem cell membrane fusion liposome is obtained.The double aptamer modified membrane fusion liposome can specifically recognize chemotactic factor and adhesion molecule in bone marrow microenvironment, actively penetrates bone marrow sinusoidal barrier, and enriches drug in bone marrow lesion area;It also has the molecular system of target response type imaging function, realizes the accurate positioning and imaging of multiple myeloma cell.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and particularly relates to a dual-aptamer modified membrane fusion liposome, its preparation method, and its application. Background Technology

[0002] Multiple myeloma (MM) is a hematologic malignancy caused by the clonal proliferation of neoplastic plasma cells in the bone marrow, and is the second most common hematologic malignancy. MM remains an incurable cancer, and the ultimate challenge in its treatment is relapse and drug resistance. The tumor microenvironment in the bone marrow provides a natural protective shield for MM cells, promoting their proliferation, survival, and drug resistance through intercellular contact and cytokine secretion. Proteasome inhibitors, such as bortezomib, are core therapeutic agents, but due to their lack of targeting, while inhibiting tumor cells, they also damage normal tissues (especially nerve cells), leading to dose-limiting toxicities such as severe peripheral neuropathy. Furthermore, tumor cells can develop resistance to bortezomib by upregulating proteasome subtypes and activating alternative signaling pathways.

[0003] Liposomes are nanovesicles formed from a phospholipid bilayer, structurally similar to cell membranes. They are biocompatible, non-immunogenic, and can improve the water solubility of chemotherapeutic drugs. They were the first nanoscale drug delivery system (DDS) approved for clinical use. However, their application in hematologic diseases is limited due to insufficient targeting. Cell membrane biomimetic technology is a technique that mimics the structure and function of cell membranes. By combining the properties of cell membranes with artificial materials, it develops biomimetic materials or systems with specific functions. Most cell membrane biomimetic nanoparticles mainly utilize the recognition and binding capabilities of membrane proteins, but lack efficient membrane fusion capabilities. They still primarily enter cells via endocytosis, failing to address the problem of lysosomal degradation.

[0004] Current research primarily focuses on drug delivery for multiple myeloma using liposomes or membrane-mimetic nanoparticles alone. However, these methods are easily phagocytosed by lysosomes, resulting in poor drug delivery efficacy. There are currently no studies on the use of dual-aptamer combined with stem cell membrane fusion liposomes for the treatment of multiple myeloma. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a dual-aptamer modified membrane fusion liposome, its preparation method and application, wherein the prepared product targets tumor sites and has a good inhibitory effect on tumors.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a dual-aptamer modified membrane fusion liposome, comprising a mesenchymal stem cell membrane, a drug-loaded liposome, and a nucleic acid dual-aptamer; the mass ratio of the drug-loaded liposome to the mesenchymal stem cell membrane is 1:0.05-0.20; the mesenchymal stem cell membrane and the drug-loaded liposome are mixed and ultrasonically extruded to obtain the mesenchymal stem cell membrane fusion liposome; the molar ratio of the mesenchymal stem cell membrane fusion liposome to the nucleic acid dual-aptamer is 900-1100:1; the nucleic acid dual-aptamer is BCMA-Clivia, and the nucleotide sequence of BCMA-Clivia is shown in SEQ ID NO: 1.

[0007] Preferably, the drug-loaded liposome comprises liposomes and a drug; the mass ratio of the liposomes to the drug is 5-15:1.

[0008] The present invention provides a method for preparing the dual-aptamer modified membrane fusion liposome, comprising the following steps: mixing mesenchymal stem cell membrane with drug-loaded liposomes, sonicating and then extruding to obtain mesenchymal stem cell membrane fusion liposomes; incubating the mesenchymal stem cell membrane fusion liposomes with nucleic acid dual-aptamers to obtain dual-aptamer modified mesenchymal stem cell membrane fusion liposomes.

[0009] Preferably, the ultrasound conditions include: pulse mode, duty cycle of 35-45%, 0.5-1.5s on, 0.5-1.5s off, and a total time of 5-7 minutes.

[0010] Preferably, the pore size of the extrusion is 150-250 nm.

[0011] Preferably, the incubation temperature is 32-40℃ and the incubation time is 1-3h.

[0012] Preferably, the nucleic acid dual aptamer is modified with cholesterol.

[0013] Preferably, the cholesterol-modified nucleic acid dual aptamer is Chol-BCMA-Clivia, with the nucleotide sequence 5'-chol-SEQ ID NO:4-3'.

[0014] This invention provides the application of the dual-aptamer modified membrane fusion liposomes or the dual-aptamer modified membrane fusion liposomes obtained by the preparation method in the preparation of drugs for treating hematologic systemic diseases.

[0015] Preferably, the hematologic systemic diseases include multiple myeloma and B-cell lymphoma.

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) The mesenchymal stem cell membrane fusion liposome of the present invention, relying on the specific receptors naturally present on the surface of the mesenchymal stem cell membrane, can specifically recognize chemokines and adhesion molecules in the bone marrow microenvironment, actively penetrate the bone marrow sinusoidal barrier, and directionally enrich the drug in the bone marrow lesion area (such as multiple myeloma lesions). Compared with traditional non-targeting liposomes, the drug concentration in bone marrow tissue is significantly increased and the drug distribution in non-target organs (such as liver and kidney) is greatly reduced, effectively solving the core problem of poor bone marrow targeting of drugs in the clinical treatment of hematological diseases, and laying the foundation for precise drug action on the lesion site.

[0017] (2) On the one hand, this invention combines the characteristics of membrane-fusion liposomes to directly fuse with cells and deliver drugs directly to diseased bone marrow cells, avoiding the accumulation of drugs in normal tissues; on the other hand, it utilizes CD44 of bone marrow mesenchymal stem cells to prevent phagocytosis by immune cells. This characteristic significantly reduces the damage of traditional therapeutic drugs (such as chemotherapy drugs) to normal hematopoietic stem cells, immune cells, and peripheral tissue cells in the bone marrow, reduces the risk of adverse reactions such as bone marrow suppression and immune dysfunction, and significantly improves the safety of treatment.

[0018] (3) This invention utilizes the allosteric properties of aptamers to construct an imaging switch that activates upon the presence of BCMA (B cell maturation antigen). In the absence of BCMA, the aptamer exhibits a specific closed conformation, with its internal binding sites for fluorescent RNA and the substrate NBSI obscured by the spatial structure, resulting in a closed imaging signal. When the aptamer binds to BCMA on the surface of multiple myeloma cells, the binding force of BCMA induces conformational rearrangement (i.e., allosteric change), fully exposing the previously obscured binding sites for fluorescent RNA and NBSI. At this point, the fluorescent RNA can rapidly bind to the substrate NBSI to form a fluorescently active complex, triggering the activation of the fluorescence signal. This BCMA-dependent allosteric response mechanism ensures that the imaging signal is specifically generated only on the surface of multiple myeloma cells, completely eliminating background fluorescence interference from untargeted areas and significantly improving the signal-to-noise ratio and accuracy of the imaging. Attached Figure Description

[0019] Figure 1 This is a diagram of the nupack structure of BCMA-FAM.

[0020] Figure 2 This is a diagram of the nupack structure of the fluorescent RNA (Clivia).

[0021] Figure 3 This is a diagram of the nupack structure of BCMA-Clivia.

[0022] Figure 4The expression of CD44, CD73, and CD90 on the surface of membrane-fused liposomes of dual aptamers.

[0023] Figure 5 This is a TEM characterization image.

[0024] Figure 6 The image shows the full-wavelength UV scan of bortezomib (left) and the standard curve (right).

[0025] Figure 7 A diagram illustrating the formation of mesenchymal stem cell membrane fusion liposomes to validate latex microbeads.

[0026] Figure 8 The results determined the optimal membrane fusion ratio for mesenchymal stem cell membrane fusion with liposomes.

[0027] Figure 9 The results are from a time gradient experiment on the fusion of mesenchymal stem cells with liposomes.

[0028] Figure 10 The results of the connection between the BCMA aptamer and MM.1S were determined.

[0029] Figure 11 The results show the incubation time for the fusion of membrane-fused liposomes with Chol-BCMA-Clivia. The left image shows the electrophoresis results, and the right image shows the grayscale statistical results.

[0030] Figure 12 Imaging verification results of mesenchymal stem cell membrane fusion liposomes modified with dual aptamers.

[0031] Figure 13 In vitro cytotoxicity validation results of drug-loaded mesenchymal stem cell membrane fusion liposomes. Detailed Implementation

[0032] This invention provides a dual-aptamer modified membrane fusion liposome, comprising a mesenchymal stem cell membrane, a drug-loaded liposome, and a nucleic acid dual-aptamer; the mass ratio of the drug-loaded liposome to the mesenchymal stem cell membrane is 1:0.05-0.20, preferably 1:0.15; the mesenchymal stem cell membrane and the drug-loaded liposome are mixed and ultrasonically extruded to obtain the mesenchymal stem cell membrane fusion liposome; the molar ratio of the mesenchymal stem cell membrane fusion liposome to the nucleic acid dual-aptamer is 900-1100:1, preferably 1000:1; the nucleic acid dual-aptamer includes BCMA-Clivia, and the nucleotide sequence of BCMA-Clivia is as shown in SEQ ID NO:1.

[0033] In this invention, the drug-loaded liposome comprises liposomes and a drug; the mass ratio of the liposomes to the drug is 5-15:1, preferably 10:1. The drug in this invention includes bortezomib. The preparation of liposomes according to the present invention includes the following steps: Dimyristic phosphatidylcholine, DOTAP, and DSPE-PEG-NH2 are mixed in a molar ratio of 70-80:15-25:3-6, dissolved in chloroform, resulting in a final solution of 2.5-3.5 ml chloroform and 0.5-1.5 ml methanol solution; the water bath temperature is set at 37-39℃ and 80-110 rpm, and the mixture is rotary evaporated to form a film. 0.5-1.5 ml PBS is added for hydration, and the mixture is placed on a shaker at 43-47℃, 110-130 rpm, for 0.5-1.5 h until complete hydration. The mixture is then sonicated at 35-45%, with a 1-second on / off cycle for 1.5-2.5 min. The mixture is then extruded sequentially at 190-210 nm and 90-110 nm to obtain liposomes. The preparation of drug-loaded liposomes according to the present invention includes the following steps: hydrating the liposomes with PBS (pH=8.5) containing 190-210 mM mannitol and 45-55 mM meglumine, placing them on a shaker at 43-47℃, 110-130 r, for 0.5-1.5 h until complete hydration, sonicating in pulse mode with a duty cycle of 35-45%, 1 second on and 1 second off, for a total time of 1.5-2.5 min, and extruding them sequentially at 190-210 nm and 90-110 nm; dialyzing the above solution with a dialysis bag (8 kDa-10 kDa) for 22-26 h, replacing the in vitro liposome solution with PBS solution at pH=5.8-6.2; adding BTZ solution dissolved in DMSO, and incubating at room temperature for 23-25 ​​h at a liposome:BTZ mass ratio of 8-12:1 to obtain BTZ-loaded liposomes.

[0034] This invention also provides a method for preparing the dual-aptamer modified membrane fusion liposomes, comprising the following steps: mixing mesenchymal stem cell membranes with drug-loaded liposomes, sonicating, and then extruding to obtain mesenchymal stem cell membrane fusion liposomes; incubating the mesenchymal stem cell membrane fusion liposomes with nucleic acid dual-aptamers to obtain dual-aptamer modified membrane fusion liposomes. This invention uses a cell membrane protein extraction kit to extract mesenchymal stem cell membranes from mesenchymal stem cells. This invention uses a thin-film hydration method to prepare liposomes. This invention utilizes a pH gradient method to encapsulate bortezomib. This invention forms mesenchymal stem cell membrane fusion liposomes through ultrasonic co-extrusion.

[0035] In this invention, the conditions for ultrasound include: pulse mode, duty cycle of 35-45%, 0.5-1.5s on, 0.5-1.5s off, total time 5-7 min; preferably pulse mode, duty cycle of 38-42%, 0.8-1.3s on, 0.8-1.3s off, total time 5.5-6.5 min; further preferably pulse mode, duty cycle of 40%, 1s on, 1s off, total time 6 min.

[0036] In this invention, the pore size of the extruded material is 150-250 nm, preferably 180-230 nm, and more preferably 200 nm. After ultrasonic extrusion, the material is placed on ice for 15-25 min, preferably 18-22 min, and more preferably 20 min.

[0037] In this invention, the incubation temperature of the mesenchymal stem cell membrane fusion liposome and the nucleic acid dual aptamer is 32-40℃ and the time is 1-3h; preferably, the temperature is 36℃ and the time is 2h.

[0038] In this invention, the nucleic acid dual aptamer is modified with cholesterol. The cholesterol portion of the modified aptamer can directly insert into the hydrophobic core region of the liposome through hydrophobic interactions, forming a stable liposome-cholesterol-aptamer complex. Combining the allosteric properties of the aptamer with the biological characteristic of high BCMA expression on the surface of multiple myeloma cells, a molecular system with targeted-responsive imaging capabilities is constructed, enabling precise localization and imaging of multiple myeloma cells. The cholesterol-modified nucleic acid dual aptamer of this invention is Chol-BCMA-Clivia, with the nucleotide sequence 5'-chol-SEQ ID NO:4-3'.

[0039] This invention also provides the application of the dual-aptamer modified membrane fusion liposomes or the dual-aptamer modified membrane fusion liposomes obtained by the preparation method in the preparation of drugs for treating hematologic diseases. The hematologic diseases mentioned in this invention include multiple myeloma, B-cell lymphoma, etc.

[0040] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0041] Unless otherwise specified, the following embodiments are all conventional methods.

[0042] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0043] Example 1 1. Extraction of mesenchymal stem cell membrane (MSCM) Immortalized human bone marrow mesenchymal stem cells (Xinrun Biotechnology cells and their corresponding culture medium IH1003-5) in the logarithmic growth phase were collected. Cells were digested with 0.25% trypsin, and the cell pellet was collected after centrifugation at 600×g for 5 min. The pellet was washed with PBS. Cell membrane and cytoplasmic protein extraction were performed using a kit (Beyotime P0033): the cells were resuspended in reagent A containing PMSF (reagent A in the Beyotime P0033 kit) and incubated on ice for 15 min. The resulting stem cell suspension was rapidly frozen at -80℃, followed by rapid thawing in a 37℃ water bath. This freeze-thaw cycle was repeated three times. Homogenization was performed for 30-50 cycles. If significant cell clumps remained, further disruption was achieved using sonication. Microscopic observation ensured 70%-80% cell disruption. The cells were centrifuged at 700g for 10 min at 4℃ to remove nuclei and undisrupted cells. The supernatant was collected (30-50 μl of residue was avoided to prevent contamination). The collected supernatant was centrifuged at 14000g for 30 min at 4℃ to remove the supernatant, precipitate cell membrane fragments, resuspend in PBS, and a portion was used for BCA protein concentration determination to calculate the specific amount (by weight) required for subsequent binding to liposomes. The remainder was stored at -80℃ for later use. The BCA protein concentration determination procedure included: adding 200-300 μl of reagent B (reagent B from the Beyotime P0033 kit) to the extracted cell membrane precipitate, vortexing vigorously for 5 seconds, then incubating on ice for 5-10 min, repeating 1-2 times; centrifuging at 14000g for 5 min at 4℃, and collecting the supernatant, which is the cell membrane protein, for BCA protein concentration determination.

[0044] 2. Preparation of liposomes (Lipo) Accurately weigh DMPC (dimyristoyl phosphatidylcholine, Aladdin-D130420), DOTAP (dioleoyltrimethylammonium propane, Aladdin-D130438), and DSPE-PEG-NH2 (distearate-phosphatidylethanolamine-polyethylene glycol-amino) to a molar ratio of 75:20:5. Dissolve in chloroform and add to a round-bottom flask, resulting in a final solution of 3 ml chloroform and 1 ml methanol. Set the water bath temperature to 38℃ and 100 rpm, and use a rotary evaporator to form a film. Add 1 ml PBS for hydration, and place on a shaker at 45℃ and 120 rpm for 1 h until hydration is complete. Sonicate at 40%, 1 second on, 1 second off, for 2 min. Extrude using a 200 nm and 100 nm extruder sequentially to obtain liposomes.

[0045] 3. Preparation of bortezomib (BTZ)-loaded liposomes (Lipo@BTZ) The liposomes prepared above were hydrated in PBS (pH=8.5) containing 200 mM mannitol and 50 mM meglumine, and placed on a shaker at 45°C and 120 rpm for 1 h until hydration was complete. The mixture was then sonicated in pulse mode with a duty cycle of 40%, on and off for 1 second at a time, for a total time of 2 min. The mixture was extruded using 200 nm and 100 nm extruders sequentially. The solution was dialyzed for 24 h using a dialysis bag (8 kDa-10 kDa) (Yuanye SP131264) to replace the liposomes with PBS solution at pH=6. BTZ solution dissolved in DMSO was added, and the mixture was incubated at room temperature for 24 h at a liposome:BTZ mass ratio of 10:1 to obtain BTZ-encapsulated liposomes.

[0046] 4. Preparation and validation of drug-loaded mesenchymal stem cell membrane fusion liposomes (MLipo@BTZ) After mixing BTZ-loaded liposomes and mesenchymal stem cell membranes at a mass ratio of 1:0.15, the mixture was subjected to pulsed sonication with a duty cycle of 40%, 1 second on and 1 second off for a total time of 6 minutes. The mixture was then extruded using a 200nm extruder to obtain drug-loaded mesenchymal stem cell membrane fused liposomes, which were then placed on ice for 20 minutes for later use.

[0047] 5. Design of nucleic acid dual aptamer structures (1) The BCMA aptamer can bind to BCMA, which is highly expressed on the surface of MM.1S cells, thereby targeting MM.1S cells; The nucleotide sequence of BCMA-FAM is: 5'-AGUGCAAGACGUUCGCAGAUUAGCGAAAAGAGGGUCUCAUUGACUAGUAC-3' (5'-SEQ ID NO: 2-3'), and the nupack structure is as follows. Figure 1 As shown.

[0048] (2) Fluorescent RNA Clivia can bind to the substrate NBSI in the presence of the substrate and produce fluorescence.

[0049] The nucleotide sequence of the fluorescent RNA Clivia is: 5'-GAGAUUGUAAACAGCGCGACACUC-3' (5'-SEQ ID NO: 3-3'), and the nupack structure is as follows. Figure 2 As shown.

[0050] (3) Function of the dual aptamer: One part is used to target MM cells, and the other part is used to bind to NBSI to generate a reaction. The nucleotide sequence of the BCMA-Clivia dual aptamer is: 5'-AGUGCAAGACGUUCGCAGAUUAGCGAAAAGAGGGUCUCAUUGACUAGUACGAGAUUGUAAACAGCGCGACACUC-3' (5'-SEQ ID NO:1-3'), and the nupack structure is as follows. Figure 3 As shown.

[0051] (4) Cholesterol-modified nucleic acid dual aptamer Chol-BCMA-Clivia: 5'-Chol-AAAAAAGUGCAAGACGUUCGCAGAUUAGCGAAAAGAGGGUCUCAUUGACUAGUACGAGAUUGUAAACAGCGCGACACUC-3' (5'-Chol-SEQID NO:4-3'), synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0052] 6. Ligation of drug-loaded mesenchymal stem cell membrane fusion liposomes and cholesterol-modified nucleic acid dual aptamers The drug-loaded mesenchymal stem cell membrane fusion liposomes were incubated with cholesterol-modified nucleic acid dual aptamers at a molar ratio of 1000:1 at 36°C for 2 hours to obtain dual aptamer-modified drug-loaded mesenchymal stem cell membrane fusion liposomes (MLipo-BC@BTZ).

[0053] Experimental Example 1 1. Validation of mesenchymal stem cell membrane The expression of CD44, CD73, and CD90 was verified in the following liposomes: simple liposomes (Lipo, prepared in Example 1), drug-loaded liposomes (Lipo@BTZ, prepared in Example 1), mesenchymal stem cell membranes (MSCM, prepared in Example 1), mesenchymal stem cell membrane fusion liposomes (MLipo, prepared without BTZ in the MLipo@BTZ preparation in Example 1), drug-loaded mesenchymal stem cell membrane fusion liposomes (MLipo@BTZ, prepared in Example 1), dual-aptamer modified mesenchymal stem cell membrane fusion liposomes (MLipo-BC, prepared without BTZ in the MLipo-BC@BTZ preparation in Example 1), and dual-aptamer modified drug-loaded mesenchymal stem cell membrane fusion liposomes (MLipo-BC@BTZ, prepared in Example 1). The verification results of CD44, CD73, and CD90 expression are as follows: Figure 4 As shown, the extracted cell membrane is a mesenchymal stem cell membrane, and the liposomes, after being modified with drugs, membranes, and aptamers, did not affect the expression of proteins on the mesenchymal stem cell membrane.

[0054] 2. Liposome structure verification and preparation of fluorescent liposomes The Lipo, Lipo@BTZ, MLipo@BTZ, and MLipo-BC@BTZ prepared in Example 1 were characterized by TEM. The results are as follows: Figure 5 As shown, this indicates that liposomes form a bilayered vesicle-like structure.

[0055] Preparation of fluorescent liposomes (Lipo-FITC): Accurately weigh DMPC, DOTAP, DSPE-PEG-NH2, and DSPE-PEG-FITC (distearate phosphatidylethanolamine-polyethylene glycol-fluorescein isothiocyanate) to a molar ratio of 75:20:2.5:2.5. Dissolve in chloroform and add to a round-bottom flask. The final solution consists of 3 ml of chloroform and 1 ml of methanol. Set the water bath temperature to 38℃ and 100 rpm, and use a rotary evaporator to evaporate the solution into a film. Add 1 ml of PBS for hydration and place on a shaker at 45℃ and 120 rpm for 1 h until hydration is complete. Sonicate at 40% for 1 second on and 1 second off for 2 min. Extrude the film using a 200 nm and 100 nm extruder sequentially. Store at 4℃ for later use.

[0056] 3. Encapsulation of bortezomib (BTZ) Different concentrations of BTZ solutions were prepared using PBS solution. The absorbance values ​​of these solutions were measured using a UV spectrophotometer. A standard curve was constructed using the absorbance values ​​at 204 nm for different concentrations. The BTZ-encapsulated liposomes prepared in step 3 of Example 1 were subjected to dialysis to remove free BTZ. The external solution was replaced with PBS solution at pH 7.4. The corresponding absorbance values ​​were measured using a UV spectrophotometer, and the free BTZ concentration was obtained by substituting the values ​​into the standard curve. The calculated BTZ encapsulation efficiency was 70.65 ± 1.68%, and the drug loading rate was 6.599 ± 0.15%. After membrane and aptamer modification, the BTZ encapsulation efficiency was 57.85 ± 1.68%, and the drug loading rate was 4.789 ± 0.13%. The full-wavelength scan and standard curve are shown below. Figure 6 As shown. The formulas for encapsulation efficiency and drug loading rate are as follows: Encapsulation efficiency = (Amount of drug encapsulated in the nanoformulation / Total amount of drug initially added) × 100%; Drug loading rate = (Weight of drug contained in the nanoformulation / Total weight of the nanoformulation) × 100%.

[0057] 4. Validation of mesenchymal stem cell membrane fusion with liposomes The mesenchymal stem cell membrane prepared in step 1 of Example 1 was stained with Dil membrane dye and washed with PBS to remove unbound dye. Fluorescent liposomes prepared according to step 2 of this experimental example and the stained mesenchymal stem cell membrane were mixed at a mass ratio of 1:0.15, sonicated in pulse mode with a duty cycle of 40%, 1s on and 1s off, for a total time of 6 min, and extruded using a 200nm extruder to obtain fluorescent mesenchymal stem cell membrane fusion liposomes (MLipo-FITC). 10 μg of the fluorescent mesenchymal stem cell membrane fusion liposomes were incubated with 4 μL of aldehyde / sulfate latex beads (Thermo Fisher Scientific-A37304) at room temperature for 45 min, followed by the addition of 100 μL of blocking buffer (PBS solution containing 1% FBS) and incubation for 30 min. The membrane was washed twice by centrifugation with PBS (6000 rpm, 3 min), the supernatant was discarded, and the precipitate was resuspended in 50 μL of PBS (containing 1% FBS). Take 10 μl of PBS (containing 1% FBS), add 10 μl of the above solution, drop it onto a glass slide, and observe directly under a confocal microscope. The MLipo-FITC group consisted of fluorescent mesenchymal stem cell membrane fusion liposomes prepared in the above steps; the direct mixing group consisted of directly mixing the stained mesenchymal stem cell membrane with the fluorescent liposomes prepared in step 2 of this experimental example. The results are as follows: Figure 7 As shown, red fluorescence represents the mesenchymal stem cell membrane, and green fluorescence represents liposomes. The two co-localize after ultrasound, indicating that the mesenchymal stem cell membrane and liposomes form a membrane-fused liposome structure.

[0058] Experiment Example 2 1. Determination of the optimal ratio of mesenchymal stem cell membrane fusion liposomes Different groups of fluorescent mesenchymal stem cell membrane fusion liposomes were prepared according to different mass ratios of fluorescent liposomes to mesenchymal stem cell membranes. The preparation of the mesenchymal stem cell membrane was the same as step 1 of Example 1, the preparation of the fluorescent liposomes was the same as step 2 of Experimental Example 1, and the preparation of the fluorescent mesenchymal stem cell membrane fusion liposomes was the same as step 4 of Experimental Example 1.

[0059] MM.1S cells were seeded into 24-well plates. After reaching a suitable cell density, the cells were washed three times with PBS, and then stained with Dil staining solution for 7-10 min. Excess dye was removed, and the cells were washed 2-3 times with PBS. Mesenchymal stem cell membrane fusion liposomes prepared at different mass ratios were added to different wells and incubated at 37°C for 1 h, followed by three washes with PBS. The cells were then fixed with 4% PFA for 10 min, washed three times with PBS, and mounted using a DAPI-containing anti-fluorescence quenching mounting medium. The results were observed directly under a confocal microscope. Figure 8As shown, the mass ratio of fluorescent liposomes to mesenchymal stem cell membranes is 1:0.15. Red fluorescence represents the MM.1S cell membrane, and green fluorescence represents the mesenchymal stem cell membrane fusion liposomes. The two are co-located, and the green fluorescence does not enter the cytoplasm, indicating that the formed mesenchymal stem cell membrane fusion liposomes can fuse with MM.1S cells without endocytosis, thus avoiding lysosomal degradation.

[0060] 2. Validation of the time gradient of mesenchymal stem cell membrane fusion and liposome membrane fusion Fluorescent mesenchymal stem cell membrane fusion liposomes were prepared according to a mass ratio of fluorescent liposomes to mesenchymal stem cell membrane of 1:0.15. The preparation of the mesenchymal stem cell membrane was the same as step 1 of Example 1, the preparation of the fluorescent liposomes was the same as step 2 of Experimental Example 1, and the preparation of the fluorescent mesenchymal stem cell membrane fusion liposomes was the same as step 4 of Experimental Example 1.

[0061] MM.1S cells were seeded into 24-well plates. After reaching a suitable cell density, the cells were washed three times with PBS, and then stained with Dil staining solution for 7-10 minutes. Excess dye was removed, and the cells were washed 2-3 times with PBS. The prepared mesenchymal stem cell membranes were fused to liposomes and incubated at different time gradients (15 min, 30 min, 1 h, 2 h, 3 h, 4 h). After incubation, the cells were washed three times with PBS. The cells were fixed with 4% PFA for 5-10 minutes, washed three times with PBS, and mounted with a DAPI-containing anti-fluorescence quenching mounting medium. The cells were then observed directly under a confocal microscope. The results are as follows: Figure 9 As shown, when fluorescent mesenchymal stem cell membrane fusion liposomes were co-incubated with MM.1S cells for 4 hours, the green fluorescence remained on the cell membrane surface and did not enter the cytoplasm, indicating that the formed mesenchymal stem cell membrane fusion liposomes could fuse with MM.1S cells and did not undergo endocytosis for a long time.

[0062] 3. Binding of BCMA aptamers to MM.1S cells MM.1S cells and control K562 cells were seeded into 24-well plates. After reaching an appropriate cell density, the culture medium was removed, and the cells were washed three times with PBS. The plates were then blocked with 5% BSA solution at 37°C for 1 hour, followed by three washes with PBS. 1 μM BCMA-FAM solution (BCMA-FAM from step 5 of Example 1) was added, and the plates were incubated at 37°C for 1 hour, followed by three washes with PBS. The plates were then mounted with anti-fluorescence quenching mounting medium and observed directly under a confocal microscope. Results are as follows: Figure 10 As shown, only MM.1S cells exhibited strong green fluorescence, while control cells K562 showed almost no uptake of BCMA-FAM, indicating that BCMA-FAM can target MM.1S cells.

[0063] 4. Determination of incubation time for the fusion of mesenchymal stem cell membrane liposomes and nucleic acid dual aptamers. Referring to Example 1, mesenchymal stem cell membrane fusion liposomes were prepared at a mass ratio of unloaded liposomes (Lipo) to MSC membranes of 1:0.15. The liposomes were incubated with Chol-BCMA-Clivia at a molar ratio of 1000:1 at 36°C for 0 h, 0.5 h, 1 h, 1.5 h, and 2 h. The reaction solutions obtained from each group were then analyzed by electrophoresis. Electrophoretic analysis was also performed on the unloaded liposomes (Lipo group), mesenchymal stem cell membranes (MSCM group), mesenchymal stem cell membrane fusion liposomes (MLipo group), and Chol-BCMA-Clivia (Group A).

[0064] Specific steps: After mixing the above-mentioned samples with the corresponding loading buffer, prepare a 10% PAGE gel and separate the samples at a constant voltage of 100V; after electrophoresis, perform appropriate staining on the gel and record the bands, observe whether there are obvious bands at the loading wells, and measure the gray value of the bands in the loading wells to determine the optimal incubation time. Results are as follows: Figure 11 The results showed that incubation at 36℃ for 2 hours yielded the best results, and the gray values ​​of the loading wells indicated that the aptamer chain was successfully ligated to the mesenchymal stem cell membrane fusion liposomes.

[0065] Experimental Example 3 Imaging verification of the drug-loaded mesenchymal stem cell membrane fusion liposomes prepared in Example 1 Drug-loaded mesenchymal stem cell membrane fusion liposomes modified with dual aptamers were co-incubated with MM.1S and K562 at 37°C for 4 hours, containing 1 mM Mg. 2+ Wash three times with PBS to remove unlinked liposomes, add substrate NBSI, incubate at 37°C for 1 h, and observe directly using a confocal microscope (excitation 492 nm, emission 595 nm). Figure 12 The results showed that the merged image was formed by combining the bright field (left side of the channel map) and the fluorescence image (right side of the channel map). The results showed that strong fluorescence was only observed in cells with high BCMA expression (MM.1S), while no fluorescence signal was observed in cells with low or no BCMA expression (K562). This result suggests that cells with high BCMA expression may produce disease signals.

[0066] Experiment Example 4 The effects of free BTZ, drug-loaded liposomes (Lipo@BTZ, prepared in Example 1), drug-loaded mesenchymal stem cell membrane fusion liposomes (MLipo@BTZ, prepared in Example 1), and dual-aptamer modified drug-loaded mesenchymal stem cell membrane fusion liposomes (MLipo-BC@BTZ, prepared in Example 1) on MM.1S cell viability were verified.

[0067] After counting the MM.1S cells in the logarithmic growth phase, the cells were seeded at an adjusted density into 96-well plates, with 100 μL of cell suspension added to each well to ensure a cell count of approximately 1 × 10⁻⁶ cells per well. 5 According to the experimental design, sample solutions with different treatment conditions were added to each well. A blank control group (containing only culture medium), a negative control group (untreated cells), and experimental treatment groups were set up, with at least 6 replicates for each group. After 24 hours of treatment, 10 μL of CCK-8 reagent was added to each well, gently mixed, and incubated at 37℃ and 5% CO2 for another 3 hours. After incubation, the absorbance (OD450) of each well was measured at 450 nm using a microplate reader. Background correction was performed using the blank control wells. Cell viability was expressed and compared as the OD value of each treatment group relative to the control group. Cell viability = (experimental group absorbance - blank group absorbance) / (control group absorbance - blank group absorbance) × 100%. The data were normalized, and the results are shown below. Figure 13 As shown, under the same drug concentration, the MLipo-BC@BTZ group exhibited stronger cell-killing ability compared to the Free BTZ group. p <0.05, which is statistically significant.

[0068] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle 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 biaptamer-modified membrane fusion liposome, characterized in that, Including mesenchymal stem cell membranes, drug-loaded liposomes, and nucleic acid dual aptamers; The mass ratio of drug-loaded liposomes to mesenchymal stem cell membranes was 1:0.05-0.20; Mesenchymal stem cell membranes were mixed with drug-loaded liposomes and then extruded by ultrasonication to obtain mesenchymal stem cell membrane fusion liposomes; the molar ratio of mesenchymal stem cell membrane fusion liposomes to nucleic acid dual aptamers was 900-1100:1; The nucleic acid dual aptamer includes BCMA-Clivia, the nucleotide sequence of which is shown in SEQ ID NO:

1.

2. The membrane fusion liposome modified with dual aptamers as described in claim 1, characterized in that, The drug-loaded liposome comprises liposomes and a drug; the mass ratio of the liposomes to the drug is 5-15:

1.

3. The method for preparing the biaptamer-modified membrane fusion liposomes as described in claim 1 or 2, characterized in that, Includes the following steps: Mesenchymal stem cell membranes were mixed with drug-loaded liposomes, sonicated, and then extruded to obtain mesenchymal stem cell membrane fusion liposomes; the mesenchymal stem cell membrane fusion liposomes were incubated with nucleic acid dual aptamers to obtain dual aptamer-modified mesenchymal stem cell membrane fusion liposomes.

4. The preparation method according to claim 3, characterized in that, The ultrasound conditions include: pulse mode, duty cycle of 35-45%, 0.5-1.5s on, 0.5-1.5s off, and a total time of 5-7 minutes.

5. The preparation method according to claim 3, characterized in that, The pore size of the extruded material is 150-250 nm.

6. The preparation method according to claim 3, characterized in that, The incubation temperature is 32-40℃ and the time is 1-3 hours.

7. The preparation method according to claim 3, characterized in that, The nucleic acid dual aptamer is modified with cholesterol.

8. The preparation method according to claim 7, characterized in that, The cholesterol-modified nucleic acid dual aptamer is named Chol-BCMA-Clivia, with the nucleotide sequence 5'-chol-SEQ ID NO:4-3'.

9. The use of the aptamer-modified membrane fusion liposomes as described in claim 1 or 2, or the aptamer-modified membrane fusion liposomes obtained by the preparation method according to any one of claims 3-8, in the preparation of drugs for treating hematologic systemic diseases.

10. The application as described in claim 9, characterized in that, The hematologic disorders mentioned include multiple myeloma and B-cell lymphoma.

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