A drug delivery nanomaterial encapsulating a cell membrane and its preparation method

Abstract

This invention relates to the field of pancreatic cancer drug technology, and discloses a drug delivery nanomaterial encapsulating a cell membrane and its preparation method. The preparation method includes: dissolving PLGA, a sonosensitive agent IR780, and an immunoadjuvant R837 in a mixed organic solvent of dichloromethane and ethanol to form an oil phase; adding the oil phase to a PVA aqueous solution, ultrasonically emulsifying to form an emulsion, then adding an isopropanol aqueous solution, stirring and solidifying, and removing the organic solvent to obtain drug-loaded nanoparticles IR780 / R837@PLGA; finally, mixing the drug-loaded nanoparticles with the cell membrane of Panc02 pancreatic cancer cells in DEPC water and ultrasonically treating to obtain the drug delivery nanomaterial M-IR780 / R837@PLGA encapsulated by the cell membrane of Panc02 pancreatic cancer cells. This material can effectively target pancreatic tumors and prolong their in vivo circulation time, thereby enhancing the targeted therapy and immunotherapy effects of pancreatic cancer.

Description

Technical Field

[0001] This invention relates to the field of pancreatic cancer drug technology, and more particularly to a nanomaterial for drug delivery that encapsulates a cell membrane and a method for preparing the same. Background Technology

[0002] Pancreatic cancer is a highly malignant digestive system tumor with a five-year survival rate of less than 10%, posing a serious threat to human health. Currently, conventional treatments such as surgery, radiotherapy, and chemotherapy have limited effectiveness and poor patient prognosis, necessitating the development of novel and effective treatment methods. In recent years, immunotherapy, as an emerging treatment strategy that kills tumors by activating the body's anti-tumor immune response, has shown significant efficacy in various cancers. However, due to the abundance of immunosuppressive cells such as tumor-associated macrophages and tumor-associated fibroblasts in the pancreatic cancer tumor microenvironment, while lacking the infiltration of anti-tumor immune cells such as natural killer cells, the response rate of existing immunotherapies in pancreatic cancer treatment is low.

[0003] Sonodynamic therapy, as an emerging non-invasive tumor treatment strategy, utilizes ultrasound to activate sonosensitive agents accumulated at the tumor site, generating a large amount of reactive oxygen species, thereby effectively killing tumor cells. Despite its great potential in tumor treatment, the efficiency of its clinical application is still limited by issues such as poor tumor targeting, low bioavailability, and insufficient in vivo stability of sonosensitive agents and other drugs.

[0004] In recent years, nanocarrier technology has improved these shortcomings to some extent by encapsulating sonosensitizers and synergistic drugs. However, the characteristic of nanoparticles being rapidly cleared by the immune system means that the accumulation efficiency of drugs at tumor sites remains unsatisfactory. Improving the tumor-targeted accumulation of sonosensitizers and synergistic drugs is currently a key research area for enhancing the efficacy of sonodynamic therapy. Summary of the Invention

[0005] To address the clinical challenge of low response rates in immunotherapy for pancreatic cancer, and more specifically, to solve the problem of targeted accumulation of pancreatic cancer drugs on tumor cells, this invention provides a cell membrane-encapsulated drug delivery nanomaterial and its preparation method.

[0006] The specific technical solution of this invention is as follows:

[0007] This invention provides a method for preparing drug delivery nanomaterials that encapsulate cell membranes, comprising the following steps:

[0008] (1) Dissolve polylactic acid-glycolic acid copolymer (PLGA), sound sensitizer, and immune adjuvant in an organic solvent to obtain an oil phase;

[0009] (2) Using polyvinyl alcohol (PVA) solution as the aqueous phase, the oil phase is added to the aqueous phase, and after emulsification, solidification and removal of organic solvent, drug-loaded nanoparticles are obtained;

[0010] (3) The drug-loaded nanoparticles were resuspended in DEPC water, and then the cell membrane of Panc02 cells was added and sonicated to obtain drug delivery nanomaterials.

[0011] This invention, through the aforementioned steps, loads a sonosensitive agent and an immune adjuvant into a polylactic acid-glycolic acid copolymer to form a core, and then encapsulates the core surface with a cell membrane derived from Panc02 pancreatic cancer cells, achieving effective targeting and accumulation of the sonosensitive agent and immune adjuvant on pancreatic cancer tumor cells. Through the accumulation of the sonosensitive agent and immune adjuvant on the target tumor cells, the sonosensitive agent induces immunogenic cell death and releases endogenous antigens, while the immune adjuvant synergistically activates immune cells in the tumor microenvironment, reshaping the immunosuppressive microenvironment. Therefore, the drug delivery nanoparticles provided by this invention exhibit high therapeutic efficacy for pancreatic cancer.

[0012] Among them, the Panc02 cell membrane can increase the accumulation of PLGA at the target tumor site, reduce the clearance of the reticuloendothelial system and the non-specific uptake of healthy tissue.

[0013] Furthermore, by optimizing key process parameters such as oil phase composition, phase volume ratio, curing conditions, and membrane-to-core mass ratio, this invention successfully prepared drug delivery nanoparticles with uniform particle size and stable encapsulation efficiency. These drug delivery nanoparticles, passing through the cell membrane of Panc02 pancreatic cancer cells, can effectively target pancreatic tumors and prolong their in vivo circulation time. Based on the synergistic effect of the loaded sonosensitizer and immune adjuvant at the tumor site, they induce immunogenic cell death through ultrasound-activated sonodynamic therapy, and, combined with the adjuvant, activate the immune response, effectively enhancing the targeted and immunotherapeutic effects of pancreatic cancer.

[0014] Preferably, in step (1), the organic solvent is a mixture of dichloromethane and ethanol.

[0015] Further preferred, the volume ratio of dichloromethane to ethanol is 7~10:1.

[0016] Preferably, in step (1), the mass ratio of polylactic acid-glycolic acid copolymer to sound-sensitive agent is 100:3~10. The amount of immune adjuvant added can be adjusted as needed.

[0017] Preferably, in step (2), the mixing volume ratio of the oil phase and the water phase is 1~4:10.

[0018] Preferably, in step (2), the emulsification method is ultrasonic emulsification.

[0019] Further optimization involves using ultrasonic emulsification for 8-15 minutes, emulsifying until the system exhibits a uniform milkshake-like consistency.

[0020] Preferably, in step (2), the curing method is as follows: adding an isopropanol aqueous solution to the emulsion and stirring to cure; wherein the volume ratio of the emulsion to the isopropanol aqueous solution is 1~3:1, and the stirring time is 4~8 hours. Preferably, the volume percentage concentration of the isopropanol aqueous solution is 1~3%. The emulsion refers to the system obtained after ultrasonic emulsification.

[0021] Preferably, in step (3), the mass ratio of the cell membrane of Panc02 cells (quantified by membrane protein) to the drug-loaded nanoparticles is 1:45~60.

[0022] Preferably, in step (3), the nanoparticles are stirred after ultrasonication to stabilize their size and morphology. The stirring speed is 600-800 rpm, and the stirring time is 4-6 hours.

[0023] As a preferred option, the sound sensitizer is IR780 and the immune adjuvant is R837.

[0024] Based on the above preparation method, the present invention also provides a drug delivery nanomaterial, which is prepared by the above preparation method.

[0025] The above preparation method can produce drug delivery nanoparticles with uniform particle size and stable encapsulation efficiency, which can effectively target and accumulate the sonosensitive agent IR780 and the immunoadjuvant R837 on pancreatic cancer tumor cells. Based on this targeted accumulation, immunogenic death of tumor cells is induced by sonodynamic therapy, releasing endogenous antigens. Furthermore, the R837 adjuvant synergistically activates immune cells in the tumor microenvironment, reshaping the immunosuppressive microenvironment. Therefore, the drug delivery nanoparticles provided by this invention have a high therapeutic effect on pancreatic cancer.

[0026] Compared with the prior art, the present invention has the following technical effects:

[0027] The preparation method provided by this invention involves dissolving PLGA, the sonosensitive agent IR780, and the immunoadjuvant R837 in a mixed organic solvent of dichloromethane and ethanol to form an oil phase; adding the oil phase to an aqueous PVA solution, followed by ultrasonic emulsification to form an emulsion, then adding an aqueous isopropanol solution for stirring and solidification, and removing the organic solvent to obtain drug-loaded nanoparticles IR780 / R837@PLGA; finally, mixing the drug-loaded nanoparticles with the cell membrane of Panc02 pancreatic cancer cells in DEPC water and ultrasonically treating them to obtain drug delivery nanomaterials M-IR780 / R837@PLGA encapsulated by the cell membrane of Panc02 pancreatic cancer cells. This invention successfully prepared drug delivery nanoparticles with uniform particle size and stable encapsulation efficiency by optimizing key process parameters such as oil phase composition, phase volume ratio, solidification conditions, and membrane-to-core mass ratio. The drug delivery nanoparticles, passing through the cell membrane of Panc02 pancreatic cancer cells, can effectively target pancreatic tumors and prolong their circulation time in vivo. Furthermore, based on the synergistic effect of the sonosensitizer and immune adjuvant loaded on them at the tumor site, they can induce immunogenic cell death through ultrasound-activated sonodynamic therapy and activate the immune response in combination with adjuvants, effectively enhancing the targeted therapy and immunotherapy effects of pancreatic cancer. Attached Figure Description

[0028] Figure 1 Transmission electron microscope image of M-IR780 / R837@PLGA.

[0029] Figure 2 The hydration particle size and zeta potential of M-IR780 / R837@PLGA are given.

[0030] Figure 3 SDS-PAGE analysis of membrane protein modifications in M-IR780 / R837 / PLGA.

[0031] Figure 4 Subcellular localization map of M-IR780 / R837@PLGA.

[0032] Figure 5 Image showing the sonodynamic therapy effect of M-IR780 / R837@PLGA.

[0033] Figure 6 The results are for the detection of ATP release levels.

[0034] Figure 7 Immunofluorescence assay results for HMGB1 ectopic and CRT eversion.

[0035] Figure 8 The results are an evaluation of the peripheral immune effects of sonodynamic therapy combined with immunotherapy. Detailed Implementation

[0036] The present invention will be further described below with reference to embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0037] In the following examples, the polylactic acid-glycolic acid copolymer (CAS No. 34346-01-5) used had a molecular weight of 8000 Daltons, a copolymerization ratio of lactic acid to glycolic acid of 50:50, and carboxylic acid end groups. It was purchased from Xi'an Ruixi Biotechnology Co., Ltd. The cell membrane protein and plasma protein extraction kit was purchased from Beyotime Biotechnology Co., Ltd., product number P0033. The protein quantification kit BCA was purchased from Thermo Fisher Scientific.

[0038] In the following examples, ultrasonic emulsification is performed using an ultrasonic cell disruptor, model LS-1200B.

[0039] Example 1: Preparation of drug delivery nanomaterials encapsulating Panc02 cell membranes

[0040] The preparation of drug delivery nanoparticles M-IR780 / R837@PLGA includes the following steps:

[0041] (1) Preparation of IR780 / R837@PLGA aqueous solution: At room temperature, 40 mg PLGA and 1.6 mg IR780 were dissolved in 3.6 ml dichloromethane. 1 mg R837 was mixed with 0.4 ml anhydrous ethanol and then added to dichloromethane to obtain the oil phase. A PVA aqueous solution (2%, w / v) was prepared as the aqueous phase. The oil phase was then slowly added dropwise to 10 ml of the aqueous solution. After thorough mixing, the mixture was ultrasonically emulsified for 10 minutes in an ice bath using an ultrasonic cell disruptor until the system presented a uniform milkshake-like consistency, thus obtaining an O / W type nanoemulsion. 10 ml of 2% (volume ratio) isopropanol aqueous solution was added to the nanoemulsion for solidification. The mixture was stirred at 800 rpm for 5 hours in an ice bath using a magnetic stirrer to evaporate the organic solvents dichloromethane and ethanol. After stirring, the nanoparticles were collected using differential centrifugation: centrifugation at 4500 rpm for 10 min, collection of the supernatant, centrifugation at 12000 rpm for 20 min, collection of the precipitate, yielding IR780 / R837@PLGA. The nanoparticles were resuspended in 5 ml of DEPC water and stored at 4 °C.

[0042] (2) Extraction of Panc02 cell membrane: The cell membrane was extracted using a cell membrane protein and plasma protein extraction kit. The extraction reagent A containing 1 mM PMSF was mixed with mouse pancreatic cancer Panc02 cells at a ratio of 1 mL: 50 million cells. The mixture was placed on ice for 15 min and frozen and thawed 3 times. Then, it was centrifuged at 700 g for 10 min, and the supernatant was collected and centrifuged at 14000 rpm for 30 min. The precipitate was the Panc02 cell membrane.

[0043] (3) Preparation of M-IR780 / R837@PLGA aqueous solution: The mass of Panc02 cell membrane was quantified using a protein quantification kit. The mass ratio of cell membrane to IR780 / R837@PLGA was 1:50. Panc02 cell membrane and IR780 / R837@PLGA aqueous solution were mixed and placed in a bath ultrasonic cleaner for 5 minutes. Then, the mixture was stirred in an ice bath for 12 hours using a magnetic stirrer to obtain an aqueous solution containing M-IR780 / R837@PLGA nanoparticles.

[0044] Example 2: Preparation of drug delivery nanomaterials encapsulating Panc02 cell membranes

[0045] The preparation of drug delivery nanoparticles M-IR780 / R837@PLGA includes the following steps:

[0046] (1) Preparation of IR780 / R837@PLGA aqueous solution: At room temperature, 40 mg PLGA and 1.4 mg IR780 were dissolved in 3.5 ml dichloromethane. 1 mg R837 was mixed with 0.5 ml anhydrous ethanol and then added to dichloromethane to obtain the oil phase. A PVA aqueous solution (3%, w / v) was prepared as the aqueous phase. The oil phase was then slowly added dropwise to 10 ml of the aqueous solution. After thorough mixing, the mixture was ultrasonically emulsified for 15 minutes in an ice bath using an ultrasonic cell disruptor until the system presented a uniform milkshake-like consistency, thus obtaining an O / W type nanoemulsion. 10 ml of 2% (volume ratio) isopropanol aqueous solution was added to the nanoemulsion for solidification. The mixture was stirred at 800 rpm for 8 hours in an ice bath using a magnetic stirrer to allow the organic solvents dichloromethane and ethanol to evaporate. After stirring, the nanoparticles were collected using differential centrifugation: centrifugation at 4500 rpm for 10 min, collection of the supernatant, centrifugation at 12000 rpm for 20 min, collection of the precipitate, yielding IR780 / R837@PLGA. The nanoparticles were resuspended in 5 ml of DEPC water and stored at 4 °C.

[0047] (2) Extraction of Panc02 cell membrane: The cell membrane was extracted using a cell membrane protein and plasma protein extraction kit. The extraction reagent A containing 1 mM PMSF was mixed with mouse pancreatic cancer Panc02 cells at a ratio of 1 mL: 50 million cells. The mixture was placed on ice for 15 min and frozen and thawed 3 times. Then, it was centrifuged at 700 g for 10 min, and the supernatant was collected and centrifuged at 14000 rpm for 30 min. The precipitate was the Panc02 cell membrane.

[0048] (3) Preparation of M-IR780 / R837@PLGA aqueous solution: The mass of Panc02 cell membrane was quantified using a protein quantification kit. The mass ratio of cell membrane to IR780 / R837@PLGA was 1:45. The Panc02 cell membrane and IR780 / R837@PLGA aqueous solution were mixed and placed in a bath ultrasonic cleaner for 5 minutes. Then, the mixture was stirred in an ice bath for 12 hours using a magnetic stirrer to obtain an aqueous solution containing M-IR780 / R837@PLGA nanoparticles.

[0049] Example 3: Preparation of drug delivery nanomaterials encapsulating Panc02 cell membranes

[0050] The preparation of drug delivery nanoparticles M-IR780 / R837@PLGA includes the following steps:

[0051] (1) Preparation of IR780 / R837@PLGA aqueous solution: At room temperature, 40 mg PLGA and 4 mg IR780 were dissolved in 4.0 ml dichloromethane. 1 mg R837 was mixed with 0.4 ml anhydrous ethanol and then added to dichloromethane to obtain the oil phase. A PVA aqueous solution (2%, w / v) was prepared as the aqueous phase. The oil phase was then slowly added dropwise to 12 ml of the aqueous solution. After thorough mixing, the mixture was ultrasonically emulsified for 12 minutes in an ice bath using an ultrasonic cell disruptor until the system presented a uniform milkshake-like appearance, thus obtaining an O / W type nanoemulsion. 8 ml of 2% (volume ratio) isopropanol aqueous solution was added to the nanoemulsion for solidification. The mixture was stirred at 800 rpm for 6 hours in an ice bath using a magnetic stirrer to allow the organic solvents dichloromethane and ethanol to evaporate. After stirring, the nanoparticles were collected using differential centrifugation: centrifugation at 4500 rpm for 10 min, collection of the supernatant, centrifugation at 12000 rpm for 20 min, collection of the precipitate, yielding IR780 / R837@PLGA. The nanoparticles were resuspended in 5 ml of DEPC water and stored at 4 °C.

[0052] (2) Extraction of Panc02 cell membrane: The cell membrane was extracted using a cell membrane protein and plasma protein extraction kit. The extraction reagent A containing 1 mM PMSF was mixed with mouse pancreatic cancer Panc02 cells at a ratio of 1 mL: 50 million cells. The mixture was placed on ice for 15 min and frozen and thawed 3 times. Then, it was centrifuged at 700 g for 10 min, and the supernatant was collected and centrifuged at 14000 rpm for 30 min. The precipitate was the Panc02 cell membrane.

[0053] (3) Preparation of M-IR780 / R837@PLGA aqueous solution: The mass of Panc02 cell membrane was quantified using a protein quantification kit. The mass ratio of cell membrane to IR780 / R837@PLGA was 1:60. Panc02 cell membrane and IR780 / R837@PLGA aqueous solution were mixed and placed in a bath ultrasonic cleaner for 5 minutes. Then, the mixture was stirred in an ice bath for 16 hours with a magnetic stirrer to obtain an aqueous solution containing M-IR780 / R837@PLGA nanoparticles.

[0054] Example 4: Preparation of drug delivery nanomaterials encapsulating Panc02 cell membranes

[0055] The preparation of drug delivery nanoparticles M-IR780 / R837@PLGA includes the following steps:

[0056] (1) Preparation of IR780 / R837@PLGA aqueous solution: At room temperature, 40 mg PLGA, 1.6 mg IR780, and 1 mg R837 were dissolved in 4.0 ml dichloromethane to obtain the oil phase. A PVA aqueous solution (2%, w / v) was prepared as the aqueous phase, and then the oil phase was slowly added dropwise to 10 ml of the aqueous solution. After thorough mixing, the mixture was ultrasonically emulsified for 10 minutes in an ice bath using an ultrasonic cell disruptor until the system presented a uniform milkshake-like consistency, thus obtaining an O / W type nanoemulsion. 10 ml of 2% (v / v) isopropanol aqueous solution was added to the nanoemulsion for solidification. The mixture was stirred at 800 rpm for 5 hours in an ice bath using a magnetic stirrer to evaporate the organic solvent dichloromethane. After stirring, the nanoparticles were collected by differential centrifugation: centrifugation at 4500 rpm for 10 min, collection of the supernatant, and centrifugation at 12000 rpm for 20 min, collection of the precipitate, to obtain IR780 / R837@PLGA. Resuspend in 5 ml of DEPC water and store at 4°C.

[0057] (2) Extraction of Panc02 cell membrane: The cell membrane was extracted using a cell membrane protein and plasma protein extraction kit. The extraction reagent A containing 1 mM PMSF was mixed with mouse pancreatic cancer Panc02 cells at a ratio of 1 mL: 50 million cells. The mixture was placed on ice for 15 min and frozen and thawed 3 times. Then, it was centrifuged at 700 g for 10 min, and the supernatant was collected and centrifuged at 14000 rpm for 30 min. The precipitate was the Panc02 cell membrane.

[0058] (3) Preparation of M-IR780 / R837@PLGA aqueous solution: The mass of Panc02 cell membrane was quantified using a protein quantification kit. The mass ratio of cell membrane to IR780 / R837@PLGA was 1:50. Panc02 cell membrane and IR780 / R837@PLGA aqueous solution were mixed and then stirred in an ice bath for 12 hours with a magnetic stirrer to obtain an aqueous solution containing M-IR780 / R837@PLGA nanoparticles.

[0059] Example 5: Characterization of M-IR780 / R837@PLGA

[0060] (1) Morphology and particle size: The morphology of M-IR780 / R837@PLGA was observed using transmission electron microscopy. The product obtained in Example 1 was dropped onto a copper mesh, dried at room temperature, and then its morphology and structure were observed under a transmission electron microscope. The results are as follows: Figure 1 As shown. 2 mL of the product obtained in Example 1 was placed in a quartz cuvette and then placed in the sample cell for testing. The hydrated particle size distribution, zeta potential, and polydispersity index (PDI) were measured. The results are shown below. Figure 2 As shown.

[0061] Depend on Figure 1 As can be seen, the drug-loaded nanoparticles M-IR780 / R837@PLGA (polylactic acid-glycolic acid copolymer particles encapsulating sonosensitive agents and immunoadjuvants) are encapsulated by the cell membrane. Transmission electron microscopy revealed that the particle size of the cell membrane-inspired nanoparticles M-IR780 / R837@PLGA is approximately 150 nm, and the thickness of the cell membrane on one side is approximately 8 nm, consistent with existing literature reports on cell membrane thickness, demonstrating successful encapsulation by the Panc02 cell membrane. Figure 2As can be seen, the hydrated average particle size of the drug delivery nanoparticles obtained in Example 1 is 252.9 ± 66.57 nm, with a narrow particle size distribution and uniform particle size. The zeta potential is -7.55 mV, indicating electronegativity. Furthermore, the PDI is 0.171, demonstrating good dispersibility. The average particle size of the drug-loaded nanoparticles M-IR780 / R837@PLGA prepared in Examples 1 to 4 is shown in Table 1. Therefore, it can be seen that the average particle size of M-IR780 / R837@PLGA prepared in this invention is at the nanometer level.

[0062] Table 1 Average particle size (nm) Example 1 252.9±66.57 Example 2 270.3±70.25 Example 3 268.7±41.62 Example 4 432.1±73.16 .

[0063] (2) SDS-PAGE analysis of membrane protein modification of M-IR780 / R837@PLGA: Panc02 cell membrane (prepared in Example 1), Panc02 cell membrane proteins, M-IR780 / R837@PLGA sample (prepared in Example 1), and IR780 / R837@PLGA sample (prepared in Example 1) were added to reducing loading buffer and electrophoresed using a 10% SDS-PAGE gel. After electrophoresis, the gel was stained with Coomassie Brilliant Blue. The results are shown below. Figure 3 As shown.

[0064] By comparing the presence and location of the target bands, the successful modification of the membrane protein onto the surface of the nanoparticles was verified.

[0065] The preparation method of Panc02 cell membrane protein is as follows: Then, the cell membrane protein and cytoplasmic protein extraction kit is used for extraction. 200 μL of membrane protein extraction reagent B is added to the cell membrane precipitate and vortexed vigorously to mix. After incubation on ice for 10 min, the mixture is repeated twice. After centrifugation at 14000 g for 5 min at 4 ℃, the supernatant is collected as Panc02 cell membrane protein.

[0066] (3) Subcellular localization: Mouse pancreatic cancer Panc02 cells were localized at a rate of 2×10⁻⁶. 5 Cells were seeded at a density of 1 cell / well in 39 mm glass-bottom confocal culture dishes and cultured overnight. After cell adhesion, the M-IR780 / R837@PLGA nanoparticles obtained in Example 1 were co-incubated with the cells. Mitochondria were labeled with the mitochondrial fluorescent probe MitoTracker, and cell nuclei were labeled with Hoechst. The subcellular organelle localization of the biomimetic drug-loaded nanoparticles was performed using confocal fluorescence microscopy. The results are as follows: Figure 4 As shown.

[0067] Depend on Figure 4It is evident that M-IR780 / R837@PLGA can co-localize with MitoTraker, meaning that M-IR780 / R837@PLGA nanoparticles can target mitochondria.

[0068] Example 6: Anti-tumor effect at the cellular level

[0069] (1) Cell viability assessment: Cell viability was assessed using a CCK-8 assay kit to evaluate the cytotoxicity and sonodynamic killing effect of the drug on tumor cells. Panc02 cells were co-incubated with M-IR780 / R837@PLGA (obtained in Example 1) for 24 hours. The supernatant of each group was removed, and the cells were gently washed with PBS and then re-added to the culture medium for sonication. After culturing for another 24 hours, CCK8 reagent was added, and the cells were incubated in an incubator. The supernatant was then measured at 450 nm using a microplate reader. The results are shown in the figure below. Figure 5 .

[0070] Depend on Figure 5 It is evident that the M-IR780 / R837@PLGA nanoparticles exhibit concentration-dependent inhibition of the proliferation of mouse pancreatic cancer Panc02 cells, meaning that the viability of Panc02 cells decreases in a dose-dependent manner as the concentration of nanoparticles in the drug-containing culture medium increases.

[0071] (2) Immunogenic cell death assessment (ICD): Panc02 cells were co-incubated with M-IR780 / R837@PLGA (prepared in Example 1) for 24 hours and activated by sonication. The level of ATP release in the supernatant was detected by an ATP kit, and the results are shown in […]. Figure 6 .

[0072] Depend on Figure 6 As can be seen, compared with the blank control group, the M-IR780 / R837@PLGA treatment group alone, and the ultrasound treatment group alone, the extracellular ATP release in the M-IR780 / R837@PLGA combined with ultrasound treatment group was significantly increased. This result indicates that M-IR780 / R837@PLGA nanoparticles combined with sonodynamic therapy can significantly promote the release of extracellular ATP from mouse pancreatic cancer Panc02 cells.

[0073] Immunofluorescence was used to detect calreticulin (CRT) extravasated onto the cell membrane and HMGB1 ectopically displaced from the nucleus in Panc02 cells. The results are as follows: Figure 7 As shown, WGA represents the Panc02 cell membrane, exhibiting red fluorescence and clearly displaying the cell membrane outline of each group of cells. This allows for direct observation of cell adhesion, morphology, and density, clearly defining the cell membrane boundary. CRT exhibits green fluorescence and is used to observe the exposure of CRT on the cell membrane surface.

[0074] Depend on Figure 7 As can be seen, in the blank control group, the nanoparticle-only treatment group, and the ultrasound-only treatment group, the green fluorescence intensity of CRT was extremely weak, and there was almost no overlap of green fluorescence signals on the red cell membrane outline, indicating that in the normal and simple treatment groups of Panc02 cells, CRT did not cause significant cell membrane surface exposure, and ICD was not effectively induced. In the nanoparticle combined with ultrasound treatment group, the green fluorescence intensity of CRT was significantly enhanced, and most of the green fluorescence was concentrated on the red cell membrane outline, forming a clear overlap with the red cell membrane fluorescence of WGA-labeled cells, allowing for direct observation of the green fluorescence signal on the cell membrane surface, suggesting that SDT combined treatment can significantly promote the transfer and exposure of CRT to the cell membrane surface in Panc02 cells. DAPI showed blue fluorescence, clearly showing the cell nuclei of Panc02 cells in each group. The cell nuclei were intact, and the cell distribution and viability could be directly observed. HMGB1 showed green fluorescence, and the distribution of HMGB1 in the cell nucleus could be clearly observed, thus determining whether nuclear-cytoplasmic heterotopia had occurred. In the blank control group, the nanoparticle-only treatment group, and the ultrasound-only treatment group, green fluorescence (HMGB1) was mainly concentrated in the blue cell nuclei. In the combined channel image, green and blue fluorescence completely overlapped, with strong and concentrated fluorescence signals, indicating that HMGB1 was normally located in the nuclei of Panc02 cells in both the normal and simple treatment groups, without significant nuclear-cytoplasmic heterotopia. In the nanoparticle-ultrasound combined treatment group, the fluorescence intensity of HMGB1 changed significantly, and the green fluorescence signal in the blue cell nuclei was significantly weakened, suggesting that sonodynamic therapy can significantly promote the heterotopia of HMGB1 expression in Panc02 cells. The detection of three core ICD markers—ATP, CRT, and HMGB1—confirmed the ICD-inducing ability of M-IR780 / R837@PLGA-mediated sonodynamic therapy from different perspectives.

[0075] Example 7: Evaluation of the antitumor efficacy of sonodynamic therapy combined with PD-L1 therapy in animals

[0076] (1) Animal model construction: Orthotopic pancreatic cancer-bearing mice were constructed using Panc02-luci and divided into four groups: control group (PBS), M-IR780 / R837@PLGA + ultrasound (M-IRP + US), aPD-L1, and aPD-L1 + M-IR780 / R837@PLGA + ultrasound group (aPD-L1 + M-IRP + US). Tumor growth was monitored by in vivo fluorescence imaging. When the tumor grew to a suitable size, ultrasound irradiation was performed 24 hours after the nanomaterial was injected via the tail vein. A total of 5 treatments were performed. Two weeks after the start of treatment, the animals were sacrificed and the tumor and major organs were collected. M-IR780 / R837@PLGA is the M-IR780 / R837@PLGA nanoparticle obtained in Example 1.

[0077] (2) Evaluation of peripheral immune effects of sonodynamic therapy combined with immunotherapy: After euthanizing mice in each group, the spleens of the mice were collected, and flow cytometry analysis of immune cells in the spleens was performed to evaluate the activation of peripheral immunity. The results are shown in the figure. Figure 8 .

[0078] Depend on Figure 8 It is evident that, compared to the PBS control group, M-IRP+US, aPD-L1 monotherapy, and the combination of both significantly increased CD8 levels in the spleen. + The proportion of cytotoxic T cells to total T cells was increased, and the CD4 / CD8 ratio was reduced, indicating that the anti-tumor immune response was effectively activated; the combination therapy group showed the most significant effect, with its CD8 ratio being significantly lower. + The proportion of T cells reached its highest level, the CD4 / CD8 ratio dropped to its lowest level, and CD8... + The proportion of T cells in the total cell count was also significantly higher than in other groups, while CD4... + There was no statistically significant difference in the proportion of T cells among the total cells across the groups. This indicates that the combination therapy of M-IRP+US and aPD-L1 can more effectively remodel the spleen's immune microenvironment, preferentially enhance cytotoxic T cell-mediated anti-tumor immunity, and exhibit a significant synergistic immune activation effect.

[0079] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0080] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing a drug delivery nanomaterial that encapsulates a cell membrane, characterized in that: Includes the following steps: (1) Dissolve polylactic acid-glycolic acid copolymer, sound sensitizer, and immunoadjuvant in an organic solvent to obtain an oil phase; (2) Using polyvinyl alcohol solution as the aqueous phase, the oil phase is added to the aqueous phase, and after emulsification, solidification and removal of organic solvent, drug-loaded nanoparticles are obtained; (3) The drug-loaded nanoparticles were resuspended in DEPC water, and then the cell membrane of Panc02 cells was added and sonicated to obtain drug delivery nanomaterials.

2. The preparation method according to claim 1, characterized in that: In step (1), the organic solvent is a mixture of dichloromethane and ethanol.

3. The preparation method according to claim 2, characterized in that: The volume ratio of dichloromethane to ethanol is 7~10:

1.

4. The preparation method according to claim 1, characterized in that: In step (1), the mass ratio of polylactic acid-hydroxyacetic acid copolymer to sound-sensitive agent is 100:3~10.

5. The preparation method according to claim 1, characterized in that: In step (2), the mixing volume ratio of the oil phase and the water phase is 1~4:

10.

6. The preparation method according to claim 1, characterized in that: In step (2), the emulsification method is ultrasonic emulsification.

7. The preparation method according to claim 1, characterized in that: In step (2), the curing method is as follows: add an isopropanol aqueous solution to the emulsion and stir to cure; The volume ratio of the emulsion to the isopropanol aqueous solution is 1~3:1, and the stirring time is 4~8 hours.

8. The preparation method according to claim 1, characterized in that: In step (3), the mass ratio of the cell membrane of Panc02 cells to drug-loaded nanoparticles was 1:45~60, based on membrane protein quantification.

9. The preparation method according to claim 1, characterized in that: The sound sensitizer was IR780, and the immune adjuvant was R837.

10. The drug delivery nanomaterial prepared by the preparation method according to any one of claims 1 to 9.

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