A biomimetic nanosheet composite preparation, a preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and relates to a kind of bionic nanosheet composite preparation and its preparation method and application, the preparation raw material of the bionic nanosheet composite preparation includes two-dimensional nanosheet, cell membrane and targeting antibody, the two-dimensional nanosheet is wrapped by the cell membrane, the targeting antibody is coupled to the cell membrane surface, and the two-dimensional nanosheet is arsenic nanosheet.The application constructs targeting antibody-bionic cell membrane-nanosheet three-layer composite preparation, integrates active targeting recognition, bionic interface modification and nanosheet carrier treatment function in one, and has good treatment effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a biomimetic nanosheet composite formulation, its preparation method, and its application. Background Technology

[0002] Small cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer with limited therapeutic efficacy. Nanomedicine delivery systems, represented by two-dimensional nanosheets (such as arsenene, black phosphorus, and graphene), offer new avenues for precision treatment of SCLC due to their ultra-high specific surface area and tunable surface chemistry. To enhance the accumulation of nanomedicines at tumor sites, actively targeting and modifying nanosheets with antibodies against tumor cell surface-specific antigens (such as DLL3, which is highly expressed in approximately 80% of SCLC patients) is one effective method to improve drug targeting. Furthermore, biomimetic nanotechnology, such as encapsulating the nanosheet core with cell membranes (macrophage membranes, erythrocyte membranes, etc.), can endow nanomedicines with "stealth" capabilities, prolong their in vivo circulation time, and may also endow them with additional biointerface functions.

[0003] Despite the progress made in improving the efficiency of nanomedicine delivery, the following drawbacks still exist when applying these technologies to the precision treatment of SCLC: single-targeting strategies have limitations. Relying solely on antibody-mediated active targeting, nanomedicines may still be rapidly cleared by the immune system in vivo, resulting in insufficient absolute amounts reaching the tumor site. While relying solely on cell membrane biomimetic camouflage can prolong circulation time, it lacks the ability to actively recognize tumor cells, resulting in limited tumor enrichment efficiency.

[0004] In summary, there is currently a lack of integrated solutions for efficient and universally applicable targeted delivery platforms. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a biomimetic nanosheet composite formulation, its preparation method, and its application.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a biomimetic nanosheet composite formulation, wherein the raw materials for preparing the biomimetic nanosheet composite formulation include two-dimensional nanosheets, cell membranes and targeting antibodies, wherein the cell membrane encapsulates the two-dimensional nanosheets, and the targeting antibody is coupled to the surface of the cell membrane, wherein the two-dimensional nanosheets are arsenene nanosheets.

[0008] Existing technologies lack an integrated targeted delivery scheme that organically combines active targeted recognition with biomimetic cell membrane modification, especially for the targeted modification application of SCLC nanosheets, which is rarely reported. Therefore, this invention provides a nanosheet composite formulation encapsulated by a biomimetic cell membrane modified with a targeting antibody, along with a method for preparing the composite formulation and its application as a targeted delivery system, particularly as a targeted delivery platform for small cell lung cancer.

[0009] This invention utilizes DLL3 antibody-mediated active targeting, achieving higher uptake efficiency in DLL3-positive SCLC cells than the untargeted control group. In a 3D tumor microsphere model, the targeted modification group penetrated to the core of the microspheres, while the control group was only distributed on the surface, indicating increased penetration depth. In a mouse model of pulmonary orthotopic tumors, the fluorescence enrichment at the tumor site was higher than that in the control group. No hemolysis was observed in the in vitro hemolysis experiment. No pathological abnormalities were found in the main organs of the mice after HE staining, and no abnormalities were found in the blood routine and blood biochemical indicators.

[0010] In some embodiments, the raw materials for preparing the biomimetic nanosheet composite formulation include, by mass parts, 1-2 parts of two-dimensional nanosheets, 1-2 parts of cell membrane, and 1-2 parts of targeting antibody.

[0011] The specific point values ​​for 1-2 parts can be selected as 1 part, 1.2 parts, 1.4 parts, 1.6 parts, 1.8 parts, 2 parts, etc. Other specific point values ​​within the above range can also be selected, which will not be elaborated here.

[0012] In some embodiments, the raw materials for preparing the biomimetic nanosheet composite formulation also include drugs.

[0013] In some embodiments, the arsenene nanosheets have a thickness of 1-20 nm and a particle size of 100-200 nm.

[0014] Thickness can be selected from 1 nm, 2 nm, 4 nm, 6 nm, 8 nm, 10 nm, 12 nm, 14 nm, 16 nm, 18 nm, 20 nm, etc., and particle size can be selected from 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0015] In some embodiments, the cell membrane is derived from any one or a combination of at least two of macrophages, erythrocytes, tumor cells, and stem cells.

[0016] In some embodiments, the surface of the cell membrane is labeled with azide.

[0017] In some embodiments, the targeting antibody includes any one or a combination of at least two of anti-DLL3, anti-HER2, anti-EGFR, and anti-PD-L1.

[0018] In some embodiments, the drug is loaded onto two-dimensional nanosheets.

[0019] In some embodiments, the loading method includes physical adsorption, π-π stacking, electrostatic adsorption, or covalent coupling.

[0020] In some embodiments, the mass ratio of the drug to the arsenene nanosheets is (0.1-0.5):1.

[0021] The specific point values ​​in (0.1-0.5) can be selected from 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, etc. Other specific point values ​​within the above range can also be selected, which will not be elaborated here.

[0022] In some embodiments, the drug includes BMN673.

[0023] The oxygen-containing vacancies on the surface of the two-dimensional nanosheets (arsenene) can catalyze the Fenton-like reaction in tumor cells to produce ROS, inducing DNA damage. Simultaneously, they serve as a carrier to load the PARP inhibitor BMN673, which inhibits DNA damage repair, thus achieving a synergistic anti-tumor effect. A biomimetic cell membrane (MH-S cell membrane) encapsulates the nanosheets, providing a biomimetic interface and reducing non-specific clearance. A targeting antibody (aDLL3) is coupled to the membrane surface, specifically recognizing the DLL3 antigen highly expressed in SCLC cells, achieving active targeted delivery.

[0024] In some embodiments, the arsenene nanosheets are prepared by a method comprising the following steps:

[0025] (1) Mix arsenic, N-methylpyrrolidone and ascorbic acid, use probe ultrasound, centrifuge and take the supernatant;

[0026] (2) Centrifuge the supernatant at high speed and collect the precipitate to obtain the product.

[0027] In some embodiments, the cell membrane is prepared by a method comprising the steps of incubating cells with tetraacetylated N-azidoacetaminomannose and then extracting the cell membrane.

[0028] In some embodiments, the power of the ultrasound is 200-600 W, and the duration is 20-24 h.

[0029] The power can be selected from 200 W, 250 W, 300 W, 350 W, 400 W, 450 W, 500 W, 550 W, 600 W, etc., and the time can be selected from 20 h, 21 h, 22 h, 23 h, 24 h, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0030] In some embodiments, the ratio of arsenic, N-methylpyrrolidone, and ascorbic acid is (500-550) mg:(25-30) mL:(100-150) mg.

[0031] The specific point values ​​in (500-550) can be selected from 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, etc.; the specific point values ​​in (25-30) can be selected from 25, 26, 27, 28, 29, 30, etc.; the specific point values ​​in (100-150) can be selected from 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0032] In some embodiments, the centrifugal force is 500-2000 xg and the time is 5-10 min.

[0033] The centrifugal force can be selected from 500 xg, 800 xg, 1000 xg, 1200 xg, 1500 xg, 1800 xg, 2000 xg, etc., and the time can be selected from 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0034] In some embodiments, the high-speed centrifugation is performed at a speed of 10,000-14,000 rpm for 5-10 minutes.

[0035] The engine speed can be selected from 10000 rpm, 10500 rpm, 11000 rpm, 11500 rpm, 12000 rpm, 12500 rpm, 13000 rpm, 13500 rpm, 14000 rpm, etc., and the time can be selected from 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, etc. Other specific values ​​within the above range can also be selected, which will not be described in detail here.

[0036] In some embodiments, the mixed incubation temperature is 36.5-37.5°C and the time is 48-72 h.

[0037] Temperatures can be selected from 36.5℃, 36.8℃, 37℃, 37.2℃, 37.5℃, etc., and time can be selected from 48 h, 52 h, 56 h, 60 h, 64 h, 68 h, 72 h, etc. Other specific point values ​​within the above range can also be selected, which will not be elaborated here.

[0038] In some embodiments, the final concentration of tetraacetylated N-azidoacetaminomannose in the incubation system is 45-50 μM, such as 45 μM, 46 μM, 47 μM, 48 μM, 49 μM, 50 μM, etc. Other specific values ​​within the above range can be selected, and will not be described in detail here.

[0039] In a second aspect, the present invention provides a method for preparing a biomimetic nanosheet composite formulation according to the first aspect, the preparation method comprising:

[0040] (1) Arsenylene nanosheets were mixed with distearate phosphatidylethanolamine-polyethylene glycol solution, and subjected to rotary evaporation under light-protected conditions using a probe for ultrasonic treatment to obtain PEGylated arsenylene nanosheets.

[0041] (2) Mix PEGylated arsenene nanosheets with cell membrane solution, sonicate in water bath and centrifuge to obtain membrane-encapsulated PEGylated arsenene nanosheets;

[0042] (3) Mix the membrane-coated PEGylated arsenene nanosheet solution with the antibody solution, centrifuge to collect the precipitate, and you will get the product.

[0043] In some embodiments, the mass ratio of arsenic in the arsenene nanosheets to distearate phosphatidylethanolamine-polyethylene glycol is (2-4):1.

[0044] The specific point values ​​in (2-4) can all be selected from 2, 2.2, 2.5, 2.8, 3, 3.2, 3, 3.2, 3.5, 3.8, 4, etc. Other specific point values ​​within the above range can also be selected, which will not be elaborated here.

[0045] In some embodiments, the solvent of the distearylphosphatidylethanolamine-polyethylene glycol solution is methanol, and the concentration of distearylphosphatidylethanolamine-polyethylene glycol in the solution is 2-3 mg / mL, such as 2 mg / mL, 2.2 mg / mL, 2.4 mg / mL, 2.6 mg / mL, 2.8 mg / mL, 3 mg / mL, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0046] In some embodiments, the probe uses ultrasound with a power of 200-600W for 10-15 minutes.

[0047] The power can be selected from 200 W, 250 W, 300 W, 350 W, 400 W, 450 W, 500 W, 550 W, 600 W, etc., and the time can be selected from 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0048] In some embodiments, the mass ratio of arsenic in the PEGylated arsenene nanosheets to the mass ratio of membrane proteins in the cell membrane solution is (1-2):(1-2).

[0049] The specific point values ​​in (1-2) can all be selected from 1, 1.2, 1.4, 1.6, 1.8, 2, etc. Other specific point values ​​within the above range can also be selected, which will not be elaborated here.

[0050] In some embodiments, the concentration of membrane proteins in the cell membrane solution is 1-2 mg / mL, such as 1 mg / mL, 1.2 mg / mL, 1.4 mg / mL, 1.6 mg / mL, 1.8 mg / mL, 2 mg / mL, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0051] In some embodiments, the power of the water bath ultrasound is 100-300W, the duration is 1-1.5 h, and the temperature is 0-15℃.

[0052] The power can be selected from 100 W, 120 W, 150 W, 180 W, 200 W, 220 W, 250 W, 280 W, 300 W, etc., the time can be selected from 1 h, 1.1 h, 1.2 h, 1.3 h, 1.4 h, 1.5 h, etc., and the temperature can be selected from 0℃, 1℃, 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃, 9℃, 10℃, 11℃, 12℃, 13℃, 14℃, 15℃, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0053] In some embodiments, the concentration of arsenic in the membrane-encapsulated PEGylated arsenene nanosheet solution is 0.5-1 mg / mL, such as 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0054] In some embodiments, the mass of arsenic to the mass of antibody is (1-2):1. The specific point values ​​in (1-2) can be selected from 1, 1.2, 1.4, 1.6, 1.8, 2, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0055] In some embodiments, the volume ratio of the membrane-encapsulated PEGylated arsenene nanosheet solution to the antibody solution is (1-2):1. The specific values ​​in (1-2) can be selected from 1, 1.2, 1.4, 1.6, 1.8, 2, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0056] In some embodiments, the mixing temperature is 35-40°C and the time is 2-3 hours.

[0057] Temperatures can be selected from 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, etc., and time can be selected from 2 h, 2.2 h, 2.4 h, 2.6 h, 2.8 h, 3 h, etc. Other specific values ​​within the above range can also be selected, which will not be elaborated here.

[0058] In some embodiments, the PEGylated arsenene nanosheets are further mixed with a drug before being mixed with the cell membrane solution.

[0059] In some embodiments, the mixing with the drug specifically includes: slowly adding the drug solution dropwise to the PEGylated arsenene nanosheet solution, sonicating and stirring in the dark, and centrifuging.

[0060] The term "slow addition" refers to adding 8-12 μL at a time, such as 8 μL, 9 μL, 10 μL, 11 μL, 12 μL, etc. Other specific values ​​within the above range can be selected, and will not be elaborated on here.

[0061] In some embodiments, the mass ratio of arsenic to drug in the PEGylated arsenene nanosheets is 1:(0.1-0.5). The specific values ​​in (0.1-0.5) can be selected from 0.1, 0.2, 0.3, 0.4, 0.5, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0062] In some embodiments, the ultrasound duration is 3-5 minutes and the power is 100-300W.

[0063] The time can be selected as 3 min, 3.2 min, 3.5 min, 3.8 min, 4 min, 4.2 min, 4.5 min, 4.8 min, 5 min, etc., and the power can be selected as 100 W, 120 W, 150 W, 180 W, 200 W, 220 W, 250 W, 280 W, 300 W, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0064] In some embodiments, the stirring time in the dark is 3-4 hours, such as 3 hours, 3.2 hours, 3.5 hours, 3.8 hours, 4 hours, etc. Other specific values ​​within the above range can be selected, and will not be described in detail here.

[0065] In some embodiments, the preparation method of the antibody solution includes: mixing the antibody solution with dibenzocyclooctene-succinimide ester, ultrafiltration, and resuspending the concentrate in PBS buffer.

[0066] In some embodiments, the molar ratio of the antibody to dibenzocyclooctene-succinimide ester is 1:(5-10). The specific point values ​​in (5-10) can be selected from 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, etc. Other specific point values ​​within the above range can be selected, and will not be described in detail here.

[0067] In some embodiments, the concentration of antibody in the antibody solution is 1-2 mg / mL, such as 1 mg / mL, 1.2 mg / mL, 1.5 mg / mL, 1.8 mg / mL, 2 mg / mL, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0068] Thirdly, the present invention provides the application of the biomimetic nanosheet composite formulation according to the first aspect in the preparation of tumor-targeting drugs.

[0069] In some embodiments, the tumor includes small cell lung cancer.

[0070] Compared with the prior art, the present invention has the following beneficial effects:

[0071] This invention is the first to construct a three-layer composite formulation of "targeted antibody-biomimetic cell membrane-nanosheet," integrating active targeting recognition, biomimetic interface modification, and nanosheet carrier / therapeutic function for targeted delivery and treatment of small cell lung cancer. Two-dimensional nanosheets (preferably arsenene) serve as both drug carriers and, by utilizing their surface oxygen vacancies to catalyze a Fenton-like reaction to generate ROS and induce DNA damage, possess therapeutic activity. Cell membranes are metabolically labeled with tetraacetylated N-azidoacetaminomannose (Ac4ManAz) to express azide groups on their surface, providing reaction sites for subsequent click chemical coupling. Cell membranes are extracted using repeated freeze-thaw cycles and differential centrifugation, extruded into membrane vesicles, and then co-extruded with nanosheets to complete encapsulation, preserving the intact membrane proteins. The targeted antibody is modified with DBCO and then coupled with the azide groups on the surface of the membrane-encapsulated nanosheets via a click chemical reaction, achieving directional and efficient antibody attachment to the membrane surface. The two-dimensional nanosheet material, the type of targeted antibody, the cell membrane source, and the loaded active drug are all substitutable, forming a universal targeted delivery platform. Attached Figure Description

[0072] Figure 1 This is a flowchart of drug preparation.

[0073] Figure 2 These are the results of atomic force microscopy (AFM) characterization of the three-dimensional morphology and thickness of the nanosheets (scale bar 100 nm). Figure A shows arsenene nanosheets, Figure B shows As NS@PEG-BMN673, and Figure C shows aDLL3-M@As NS@PEG-BMN673.

[0074] Figure 3 The morphology of aDLL3-M@As NS@PEG-BMN673 and the distribution of As, C, N, O and P elements on the nanosheet surface were characterized by scanning electron microscopy / energy dispersive spectroscopy (SEM-EDS) (scale bar = 100 nm).

[0075] Figure 4 The phagocytosis of As NS@PEG-BMN673-Cy5.5 (without DLL3-targeting antibody modification) and aDLL3-M@As NS@PEG-BMN673-Cy5.5 (DLL3-targeting antibody modification) in H82 cells (DLL3 positive) at different time points (2 h, 4 h and 8 h) is shown (scale bar = 20 μm).

[0076] Figure 5The phagocytosis of As NS@PEG-BMN673-Cy5.5 (without DLL3-targeting antibody modification) and aDLL3-M@As NS@PEG-BMN673-Cy5.5 (DLL3-targeting antibody modification) in H1046 cells (DLL3 negative) at different time points (2 h, 4 h and 8 h) is shown (scale bar = 20 μm).

[0077] Figure 6 The results are in vivo imaging at different time points after inhalation of As NS@PEG-BMN673-Cy7 (without DLL3-targeting antibody modification) and aDLL3-M@As NS@PEG-BMN673-Cy7 (DLL3-targeting antibody modification) in the Luci-H82 pulmonary orthotopic tumor mouse model.

[0078] Figure 7 The results show the cell viability after 48 h of incubation with BMN673 monotherapy at 10 nM, As NS@PEG monotherapy at 10 μg / mL (based on the As concentration), and combined administration at the above concentrations (As to BMN673 mass ratio of 1:0.38).

[0079] Figure 8 This is a cytotoxicity experiment of H82 cells treated with different drug groups for 48 hours.

[0080] Figure 9 This is an experiment on DNA damage in H82 cells treated with different drug groups for 48 hours. Detailed Implementation

[0081] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0082] Example 1

[0083] This embodiment provides a method for preparing a biomimetic nanosheet composite formulation, the preparation flowchart of which is shown below. Figure 1 The preparation method includes:

[0084] (1) Weigh 500 mg of arsenic powder and 100 mg of ascorbic acid, add 25 mL of N-methylpyrrolidone (NMP) to a 50 mL centrifuge tube, and seal the tube. Sonicate with a probe (334 W, 80%, 10 s / 5 s, ice bath, 20 h). Centrifuge at 500 rpm for 1 min and collect the supernatant. Aliquot the supernatant into 2 mL centrifuge tubes, centrifuge at 14000 rpm for 5 min, discard the supernatant, and resuspend the precipitate in 25 mL of NMP containing 100 mg of ascorbic acid to obtain the As NS (arsenene nanosheets) stock solution. Centrifuge the stock solution at 10000 rpm for 5 min, resuspend the precipitate in PBS, measure the 210 nm OD value, and calculate the concentration according to the standard curve Y = 0.0002x + 2.6883.

[0085] (2) Take the mother liquor containing 15 mg As, centrifuge at 10000 rpm for 5 min, and aspirate the supernatant. Wash the precipitate twice with methanol. Disperse the precipitate in 20 mL of 2 mg / mL DSPE-PEG5K methanol solution at a mass ratio of As:DSPE-PEG5K (number average molecular weight of 5000 Da) = 3:1, sonicate with a probe for 10 min (334 W, 80%, 10 s / 5 s, ice bath), evaporate to dryness in the dark, and resuspend in 10 mL of ultrapure water to obtain As NS@PEG.

[0086] (3) Prepare BMN-673 DMSO stock solution (25 mg / mL). Take 2 mg As NS@PEG, centrifuge and resuspend in 2 mL PBS, then transfer to a small brown glass bottle. Add 32 μL of BMN673 stock solution (As:BMN673=1:0.4) dropwise in 4 portions under magnetic stirring, sonicate in a water bath for 5 min (200W), stir in the dark for 4 h, centrifuge at 10000 rpm for 5 min, and resuspend the precipitate in PBS to an As concentration of 1 mg / mL to obtain As NS@PEG-BMN673.

[0087] (4) Mouse alveolar macrophage cell line (MH-S cells) was incubated with 50 μM Ac4ManAz for 72 h, cells were collected, washed with PBS, and membrane protein extraction reagent A (containing 1 mM PMSF) was added. Membrane protein extraction reagent A was purchased from Beyotime's cell membrane extraction kit. The cells were placed on ice for 10 min and subjected to three freeze-thaw cycles (frozen at -80℃ for 20 min and then treated at 37℃ for 20 min). The cells were centrifuged at 700g for 10 min to collect the supernatant, centrifuged at 14000g for 30 min to collect the membrane, resuspended in PBS, and squeezed repeatedly through a 400nm filter membrane 11 times to obtain cell membrane vesicles. The concentration was measured by BCA (standard curve y=0.5531x+0.1421), and azidized cell membrane vesicles were obtained.

[0088] (5) Take As NS@PEG-BMN673 (0.5 mg as As), centrifuge at 10000 rpm for 5 min, resuspend the pellet in 0.5 mL of azide-treated cell membrane solution (membrane protein concentration 1 mg / mL), and sonicate in a water bath (200W) for 1 h (with ice pack). Centrifuge at 10000 rpm for 5 min, and resuspend the pellet in PBS to an As concentration of 1 mg / mL to obtain M@As NS@PEG-BMN673.

[0089] (6) Ultrafiltration of aDLL3 antibody (1 mg) to 0.1 M NaHCO3 (pH 8.3), addition of 15 μL DBCO-NHS (20 mg / mL DMSO), and gentle shaking at room temperature for 2 h, followed by ultrafiltration to remove free DBCO-NHS, and resuspending in PBS to 1 mg / mL to obtain DBCO-aDLL3. Mix 0.5 mL M@As NS@PEG-BMN673 (As concentration 1 mg / mL) with 0.5 mL DBCO-aDLL3 (1 mg / mL), and gently shake at 37℃ for 2 h. Centrifuge at 10000 rpm for 5 min, and resuspend the precipitate in PBS to 1 mg / mL As concentration to obtain aDLL3-M@As NS@PEG-BMN673.

[0090] The results of atomic force microscopy (AFM) characterization of the three-dimensional morphology and thickness of nanosheets are as follows: Figure 2 As shown in the figure, Figure A represents arsenene nanosheets, Figure B represents As NS@PEG-BMN673, and Figure C represents aDLL3-M@As NS@PEG-BMN673.

[0091] The morphology and surface elemental distribution results of aDLL3-M@As NS@PEG-BMN673 are as follows: Figure 3 As shown.

[0092] This embodiment was supported by the following projects: 1. National Natural Science Foundation of China (NSFC) Youth Fund (32201137): Construction of nano-arsenic preparations based on a new synthetic lethal strategy and its research on the treatment of small cell lung cancer; 2. Provincial Medical and Health Program Youth Innovation Project (2023RC182): Construction of nano-tin preparations administered via the airway for sonodynamic therapy of lung cancer and its "vaccine-like" effect.

[0093] Example 2

[0094] In vitro targeting experiments:

[0095] Materials: SCLC cell lines H82 (DLL3 positive) and H1048 (DLL3 negative). Cultured in DMEM medium (10% fetal bovine serum, 1% penicillin-streptomycin) at 37°C with 5% CO2.

[0096] Samples: Cy5.5-labeled As NS@PEG-BMN673 (without DLL3-targeting antibody modification) and aDLL3-M@As NS@PEG-BMN673 (DLL3-targeting antibody modification), both prepared at an As concentration of 1 mg / mL.

[0097] Methods: H82 and H1048 cells were seeded into confocal culture dishes (5×10⁻⁶ cells / mL). 4 (samples / plate), incubated for 12 h. Add the above samples to a final concentration of As 50 μg / mL and incubate for 4 h. Wash twice with PBS, fix with 4% paraformaldehyde for 15 min, and stain with Hoechst 33342 for 10 min. Observe under a confocal microscope and quantify fluorescence intensity using ImageJ.

[0098] Result: As Figure 4 and Figure 5 As shown, in H82 cells, the intracellular fluorescence intensity of the DLL3-targeting antibody-modified group was significantly higher than that of the group without DLL3-targeting antibody modification. In H1048 cells, there was no statistically significant difference in fluorescence intensity between the two groups. This indicates that aDLL3 modification can specifically enhance the uptake by DLL3-positive cells.

[0099] This embodiment was supported by the following projects: 1. National Natural Science Foundation of China (NSFC) Youth Fund (32201137): Construction of nano-arsenic preparations based on a new synthetic lethal strategy and its research on the treatment of small cell lung cancer; 2. Provincial Medical and Health Program Youth Innovation Project (2023RC182): Construction of nano-tin preparations administered via the airway for sonodynamic therapy of lung cancer and its "vaccine-like" effect.

[0100] Example 3

[0101] In vivo tumor-targeted enrichment experiment:

[0102] Animal model: A Luci-H82 lung orthotopic tumor model was constructed using 6-8 week old BALB / c nude mice. Luci-H82 cells in logarithmic growth phase were harvested, digested with trypsin, and resuspended in PBS to a concentration of 2×10⁻⁶. 7 / mL. Mice were instilled with 50 μL of cell suspension (1×10⁻⁶) via airway. 6 (One per animal), tumor formation was confirmed by live imaging 2 weeks later.

[0103] Samples: Cy7-labeled As NS@PEG-BMN673 (without DLL3-targeting antibody modification) and aDLL3-M@As NS@PEG-BMN673 (DLL3-targeting antibody modification), both prepared at an As concentration of 1 mg / mL.

[0104] Methods: Tumor-bearing mice were randomly divided into two groups of three each. Each mouse received 50 μL (As 50 μg) via the airway. In vivo imaging was performed at 1 h, 6 h, 12 h, and 24 h post-administration, with excitation wavelength of 750 nm and emission wavelength of 770 nm. Mice were sacrificed 24 h later, and tumor tissue and major organs (heart, liver, spleen, lung, and kidney) were collected for fluorescence imaging and quantification.

[0105] Result: As Figure 6 As shown, the fluorescence intensity at the tumor site in the DLL3-targeting antibody modified group reached its peak 6 hours after drug administration, and strong fluorescence could still be detected 24 hours later; the fluorescence at the tumor site in the non-DLL3-targeting antibody modified group was weak and almost disappeared after 24 hours. Quantitative analysis showed that the tumor fluorescence intensity at 6 hours in the DLL3-targeting antibody modified group was significantly higher than that in the non-targeting control group. Ex vivo organ fluorescence imaging showed that the fluorescence in the DLL3-targeting antibody modified group was mainly concentrated in the lung (tumor site), with weak fluorescence in other organs; in the control group, the fluorescence in lung tissue was weak, with a small amount distributed in the liver and spleen.

[0106] Conclusion: Modification with DLL3-targeting antibodies can significantly improve the tumor targeting enrichment efficiency of nanosheets in a mouse model of SCLC lung orthotopic tumor.

[0108] Example 4

[0109] Determining the synergy ratio:

[0110] The SCLC cell line H82 was divided into a single-drug group (BMN673), a single-drug group (AsNS@PEG), and a combination drug group (BMN673+AsNS@PEG). The synergistic index of different drug ratios was compared.

[0111] Q = E (a+b) / (E a +E b -E a ×E b )

[0112] Where Q is the combination index (CI); Q less than 0.85 indicates antagonism; 0.85 ≤ Q < 1.15 indicates additive effects; Q ≥ 1.15 indicates synergistic effects.

[0113] E a and E b It is the cell proliferation inhibition rate of a single drug.

[0114] E (a+b) It is the cell proliferation inhibition rate of combined drug therapy.

[0115] BMN673 monotherapy at 10 nM, As NS@PEG monotherapy at 10 μg / mL (based on As concentration), and combined administration at the above concentrations (As to BMN673 mass ratio of 1:0.38), under incubation conditions of 48 h, CI = 1.26 > 1.15, as shown in the results. Figure 7 As shown, the two have a significant synergistic anti-tumor effect.

[0116] Experiments showed that the synergistic effect was only achieved at specific concentration ratios. When BMN673 was administered alone at 5 nM, As NS@PEG was administered alone at 10 μg / mL (based on the concentration of As), and when the above concentrations were combined (as to BMN673 mass ratio of 1:0.19), CI = 1.01 < 1.15 after 48 h of incubation. When BMN673 was administered alone at 15 nM, As NS@PEG was administered alone at 10 μg / mL (based on the concentration of As), and when the above concentrations were combined (as to BMN673 mass ratio of 1:0.57), CI = 1.12 < 1.15 after 48 h of incubation.

[0117] Drug loading efficiency:

[0118] The nanosheets were loaded with drugs in accordance with steps (1)-(3) in Example 1. The only difference between steps (1)-(3) in Example 1 and those in Example 1 is that the mass ratio of As to BMN673 is different.

[0119] In this embodiment, the drug was loaded with As at a mass ratio of 10:3, 10:4, and 10:5 to BMN673. After resuspending the precipitate to an As concentration of 1 mg / mL, the supernatant was collected by centrifugation at 4500 rpm for 10 min and the BMN673 content was determined.

[0120] Methods for determining and calculating loading rate: The content of BMN673 was determined by liquid chromatography-mass spectrometry (LCMS).

[0121] BMN673 drug loading rate (%) = (BMN673 feed amount - unloaded BMN673 content in the supernatant after centrifugation of the drug-loaded mixture) / (As NS@PEG-BMN673 total mass) × 100%

[0122] Table 1

[0123]

[0124] The results above show that the drug loading efficiency is high when the mass ratio of As to BMN673 is 10:4 and 10:5. Furthermore, the synergistic effect between the drugs at the As to BMN673 mass ratio of 10:4 (10:3.8 in the prepared As NS@PEG-BMN673) has been verified in the aforementioned cytotoxicity experiments. Therefore, the group with an As to BMN673 mass ratio of 10:4 exhibits high drug loading efficiency, synergistic effect, and complies with the principle of reducing drug dosage.

[0125] To further verify the therapeutic effect of drug loading with an As to BMN673 mass ratio of 10:4 and the effect of targeted modification on the therapeutic effect, we further verified the cytotoxic and DNA damage effects of the SCLC cell line H82.

[0126] Cytotoxicity assay groups: control group, BMN673, As NS@PEG, As NS@PEG-BMN673, aDLL3-M@AsNS@PEG-BMN673. When As was used in combination with BMN673, the mass ratio was 10:3.8, meaning 1 μg / mL As corresponds to 1 nM BMN673. The control group received no treatment. Figure 8 These are the results of a cytotoxicity experiment on H82 cells treated with different drug groups for 48 hours.

[0127] DNA damage experimental groups: control group, G1 (BMN673), G2 (As NS@PEG), G3 (As NS@PEG-BMN673), and G4 (aDLL3-M@As NS@PEG-BMN673). In group G1, the concentration of BMN673 was 10 nM. In group G2, the concentration of As was 10 μg / mL. When As and BMN673 were used together, the mass ratio was 10:3.8, meaning 10 μg / mL As corresponds to 10 nM BMN673. Figure 9 These are the results of DNA damage experiments on H82 cells treated with different drug groups for 48 hours.

[0128] The results above show that the As NS@PEG-BMN673 loading group has stronger anti-tumor activity and DNA damage induction effect than the single drug group. DLL3 targeted modification can further enhance the DNA damage effect and anti-tumor effect.

[0129] This embodiment was supported by the following projects: 1. National Natural Science Foundation of China (NSFC) Youth Fund (32201137): Construction of nano-arsenic preparations based on a new synthetic lethal strategy and its research on the treatment of small cell lung cancer; 2. Provincial Medical and Health Program Youth Innovation Project (2023RC182): Construction of nano-tin preparations administered via the airway for sonodynamic therapy of lung cancer and its "vaccine-like" effect.

[0130] The above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A biomimetic nanosheet composite formulation, characterized in that, The raw materials for preparing the biomimetic nanosheet composite formulation include two-dimensional nanosheets, cell membranes, and targeting antibodies. The cell membrane encapsulates the two-dimensional nanosheets, and the targeting antibody is coupled to the surface of the cell membrane. The two-dimensional nanosheets are arsenene nanosheets.

2. The biomimetic nanosheet composite formulation according to claim 1, characterized in that, The raw materials for preparing the biomimetic nanosheet composite formulation include, by mass parts, 1-2 parts of two-dimensional nanosheets, 1-2 parts of cell membrane, and 1-2 parts of targeting antibody; And / or, the raw materials for preparing the biomimetic nanosheet composite formulation also include drugs; And / or, the thickness of the arsenene nanosheets is 1-20 nm and the particle size is 100-200 nm; And / or, the cell membrane may be derived from any one or a combination of at least two of macrophages, erythrocytes, tumor cells, and stem cells; And / or, the surface of the cell membrane is labeled with azide; And / or, the targeting antibody includes any one or a combination of at least two of anti-DLL3, anti-HER2, anti-EGFR, and anti-PD-L1.

3. The biomimetic nanosheet composite formulation according to claim 2, characterized in that, The drug is loaded onto two-dimensional nanosheets; the loading method includes physical adsorption, π-π stacking, electrostatic adsorption, or covalent coupling. And / or, the mass ratio of the drug to the arsenene nanosheets is (0.1-0.5):1; And / or, the drug includes BMN673.

4. The biomimetic nanosheet composite formulation according to any one of claims 1-3, characterized in that, The arsenene nanosheets were prepared by a method comprising the following steps: (1) Mix arsenic, N-methylpyrrolidone and ascorbic acid, use probe ultrasound, centrifuge and take the supernatant; (2) Centrifuge the supernatant at high speed and collect the precipitate to obtain the product; And / or, the cell membrane is prepared by a method comprising the steps of: incubating cells with tetraacetylated N-azidoacetaminomannose, and then extracting the cell membrane.

5. The biomimetic nanosheet composite formulation according to claim 4, characterized in that, The power of the ultrasound is 200-600 W, and the duration is 20-24 h; And / or, the ratio of arsenic, N-methylpyrrolidone, and ascorbic acid is (500-550) mg:(25-30) mL:(100-150) mg; And / or, the centrifugal force is 500-2000 xg and the time is 5-10 min; And / or, the high-speed centrifugation is performed at a speed of 10,000-14,000 rpm for 5-10 min; And / or, the temperature for the mixed incubation is 36.5-37.5℃, and the time is 48-72 h; And / or, the final concentration of tetraacetylated N-azidoacetaminomannose in the incubation system is 45-50 μM.

6. The method for preparing the biomimetic nanosheet composite formulation according to any one of claims 1-5, characterized in that, The preparation method includes: (1) Arsenylene nanosheets were mixed with distearate phosphatidylethanolamine-polyethylene glycol solution, and subjected to rotary evaporation under light-protected conditions using probe ultrasound to obtain PEGylated arsenylene nanosheets. (2) Mix PEGylated arsenene nanosheets with cell membrane solution, sonicate in water bath and centrifuge to obtain membrane-encapsulated PEGylated arsenene nanosheets; (3) Mix the membrane-coated PEGylated arsenene nanosheet solution with the antibody solution, centrifuge to collect the precipitate, and you will get the product.

7. The preparation method according to claim 6, characterized in that, The mass ratio of arsenic in the arsenene nanosheets to the mass ratio of distearate phosphatidylethanolamine-polyethylene glycol is (2-4):

1. And / or, the solvent of the distearylphosphatidylethanolamine-polyethylene glycol solution is methanol, and the concentration of distearylphosphatidylethanolamine-polyethylene glycol in the solution is 2-3 mg / mL; And / or, the ultrasonic power of the probe is 200-600W, and the duration is 10-15 min; And / or, the mass ratio of arsenic in the PEGylated arsenene nanosheets to the mass ratio of membrane proteins in the cell membrane solution is (1-2):(1-2); And / or, the concentration of membrane proteins in the cell membrane solution is 1-2 mg / mL; And / or, the power of the water bath ultrasound is 100-300W, the time is 1-1.5 h, and the temperature is 0-15℃; And / or, the concentration of arsenic in the membrane-encapsulated PEGylated arsenene nanosheet solution is 0.5-1 mg / mL; And / or, the mass of the arsenic to the mass of the antibody is (1-2):1; And / or, the volume ratio of the membrane-encapsulated PEGylated arsenene nanosheet solution to the antibody solution is (1-2):1; And / or, the mixing temperature is 35-40°C, and the time is 2-3 h.

8. The preparation method according to claim 6 or 7, characterized in that, The PEGylated arsenene nanosheets are further mixed with drugs before being mixed with the cell membrane solution; And / or, the mixing with the drug specifically includes: adding the drug solution dropwise to the PEGylated arsenene nanosheet solution, sonicating and stirring in the dark, and centrifuging; And / or, the mass ratio of arsenic to drug in the PEGylated arsenene nanosheets is 1:(0.1-0.5); And / or, the ultrasound duration is 3-5 minutes, and the power is 100-300W; And / or, the stirring time in the dark is 3-4 hours; And / or, the method for preparing the antibody solution includes: mixing the antibody solution with dibenzocyclooctene-succinimide ester, ultrafiltration, and resuspending the concentrate in PBS buffer; And / or, the molar ratio of the antibody to dibenzocyclooctene-succinimide ester is 1:(5-10); And / or, the concentration of antibody in the antibody solution is 1-2 mg / mL.

9. The use of the biomimetic nanosheet composite formulation according to any one of claims 1-5 in the preparation of tumor-targeting drugs.

10. The application according to claim 9, characterized in that, The tumors include small cell lung cancer.

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