A nano delivery system based on exosome membrane fusion and a preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a nano delivery system based on exosome membrane fusion and a preparation method and application thereof, which comprises: (1) a prodrug complex formed by paclitaxel (PTXL) and IR780; (2) a liposome core loaded with the prodrug complex, and a membrane material of the liposome core comprises: DSPE-PEG-MAP, DSPE-PEI, phospholipid and cholesterol; and (3) a homologous tumor cell-derived exosome membrane fused to the surface of the liposome core. The application realizes the design of multi-level delivery in the same nanoparticle by means of the optimization of the properties of the liposome membrane composition, the fusion of the exosome and the liposome membrane, the mixed biomimetic nanoparticle particle size, the preparation of the PTXL targeted prodrug and the like, so as to improve the treatment effect at different levels.

Description

Technical Field

[0001] The present invention relates to a nano-delivery system based on exosome membrane fusion and a preparation method and application thereof, belonging to the field of biomedicine technology. Background Art

[0002] The research and application of nanocarriers and delivery technologies in oncology are rapidly developing. The use of nanoscale carriers not only improves the delivery of anti-tumor drugs but also addresses many practical challenges in cancer treatment, enabling efficient collaboration between multiple therapeutic approaches and expanding the potential for effective cancer cures. Consequently, the research and development of novel anti-tumor nanodrug carriers and nanoformulations has become a cutting-edge and hot topic across multiple disciplines, including medicine and nanomaterials, with a continuous stream of relevant research findings.

[0003] Nanocarriers offer advantages such as small size, high surface-to-volume ratio, and excellent physicochemical properties. Drug loading within nanocarriers can modulate drug pharmacokinetic and pharmacodynamic properties, such as improving in vivo stability, prolonging blood circulation time, and enabling controlled drug release, thereby enhancing the therapeutic index. The main goals of developing nanodrug formulations are to achieve both nonspecific and specific targeting and delivery, enhance safety and biocompatibility, and improve pharmacokinetic properties. The composition of nanocarriers varies depending on the materials used, including phospholipids, lipids, dextran, chitosan, various synthetic polymers, as well as carbon, silica, or metals. Despite significant advances in nanoscience and the introduction of some nanoformulations into clinical practice, their true advantages and value have yet to be fully realized in medicine. Currently, the following existing technical approaches are primarily used to optimize nanocarrier materials: 1. Encapsulating drugs within hydrophilic nanocarriers improves the solubility and chemical stability of anticancer drugs. Second, anticancer drugs are loaded into nanocarriers or biodegradable compounds are coupled to synthetic polymers to protect anticancer drugs from biodegradation or excretion. Third, functionalized nanoformulations are constructed to improve their penetration ability and selectively deliver chemotherapy targets or targeted compounds to tumor cells or stromal cells to improve the biodistribution of anticancer drugs and enhance targeting. Fourth, nanocarriers can be designed to release their payloads after triggering, thereby producing stimulus-sensitive nanomedicines. Fifth, multifunctional nanocarriers are constructed. Multifunctional nanocarriers have the ability to perform multiple functions simultaneously, such as co-delivering multiple drugs for combination therapy, mediating multiple targeted delivery, or synergistic diagnosis and treatment.

[0004] Despite significant progress in nanocarrier technology, existing technologies still have problems such as insufficient targeting, low drug loading, lack of flexibility, and chemical resistance, which restrict their effectiveness in clinical applications. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the present invention provides a nano-delivery system based on exosome membrane fusion, and a preparation method and application thereof.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] One of the objectives of the present invention is to provide a nano-delivery system based on exosome membrane fusion, comprising:

[0008] (1) A prodrug complex formed by paclitaxel (PTXL) and 2-(2-(2-chloro-3-((1,3-dihydro-3,3-dimethyl-1-propyl-2H-indol-2-ylidene)ethylidene)-1-cyclohexen-1-yl)vinyl)-3,3-dimethyl-1-propylindolium iodide (IR780), the prodrug complex being named PTXL-SS-IR780 and having the following structural formula:

[0009] ;

[0010] (2) a liposome core encapsulating the prodrug complex, wherein the membrane material of the liposome core comprises: DSPE-PEG-MAP, DSPE-PEI, phospholipid and cholesterol;

[0011] (3) Exosome membranes derived from homologous tumor cells fused to the surface of the liposome core.

[0012] IR780 iodide is a near-infrared, lipophilic, cationic heptamethylamine fluorescent dye. It exhibits excellent fluorescence intensity and is used as a photothermal therapy agent. Its absorption spectrum at 780 nm has led to its use as a sonodynamic therapy agent in medical therapeutic applications.

[0013] The above-mentioned DSPE-PEG-MAP is a compound composed of DSPE-PEG (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol) connected to activatable cell-penetrating peptides (ACPPs). ACPPs that can be activated by matrix metalloproteinase-2 / 9 (MMP-2 / 9) are introduced. In lung cancer cell tissues with high MMP-2 / 9 expression, the cell-penetrating peptide can be activated, allowing it to carry the nanocarrier across the membrane efficiently.

[0014] The above-mentioned DSPE-PEI is a lipid-polymer material formed by covalently linking phospholipids (DSPE) and polyethyleneimine (PEI). The lipid material with a "proton sponge effect" synthesized using PEI can assist liposomes in "lysosomal escape" and improve drug delivery efficiency.

[0015] Furthermore, the mass fractions of the components in the liposome core are: DSPE-PEG-MAP: 6%-8%; DSPE-PEI: 6%-8%; phospholipids: 62%-70%; cholesterol: 16%-20%.

[0016] Furthermore, the exosome membranes are derived from A549 lung cancer cells.

[0017] Furthermore, the particle size of the system is 140nm-160nm.

[0018] Furthermore, the mass ratio of the prodrug complex to the liposome core is 1:9; the mass ratio of the drug-loaded liposome to the exosome is 5:1.

[0019] A second object of the present invention is to provide a method for preparing the nano-delivery system based on exosome membrane fusion as described above, comprising the following steps:

[0020] S1, synthesis of 2'-Succinyl-PTXL (2'-succinyl-paclitaxel) and IR780-SS-NH2 (IR780-disulfide-amino), respectively;

[0021] S2, 2'-Succinyl-PTXL synthesized in step S1 and IR780-SS-NH2 are subjected to an amidation reaction to generate a prodrug complex PTXL-SS-IR780;

[0022] S3, using DSPE-PEG-MAP, DSPE-PEI, phospholipids and cholesterol as membrane materials, the thin film hydration ultrasonic method was used to encapsulate the PTXL-SS-IR780 from step S2 to form drug-loaded liposomes;

[0023] S4. Perform membrane fusion of the drug-loaded liposomes obtained in step S3 with the exosomes derived from tumor cells.

[0024] Further, in step S1:

[0025] The specific steps of synthesizing the 2'-Succinyl-PTXL are as follows: dissolving paclitaxel in N,N-dimethylformamide (DMF), adding succinic anhydride, stirring, washing, and drying to obtain;

[0026] The specific steps for synthesizing the IR780-SS-NH2 are as follows: cystamine dihydrochloride and TEA are dissolved in N,N-dimethylformamide, IR780 is added, and the mixture is stirred in nitrogen, followed by dialysis with deionized water and freeze-drying.

[0027] Furthermore, the amidation reaction in step S2 specifically comprises the following steps:

[0028] Dissolve 2'-Succinyl-PTXL in DMSO:H2O, then add N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC). The reaction time is 1.5h-2.5h. Then, slowly add IR780-SS-NH2 under nitrogen. The reaction temperature is 20℃-30℃ and stirred for 20h-30h.

[0029] The reaction equation of the above reaction process is as follows:

[0030] .

[0031] Furthermore, in step S3, the specific parameters of the thin film hydration ultrasonic method are: hydration temperature is 20°C-30°C, hydration time is 25min-35min, ultrasonic power is 150W-250W, and intermittent ultrasonication is 3min-6min.

[0032] Furthermore, in step S4, during the membrane fusion process, the drug-loaded liposomes and exosomes are sonicated for 10 min to 20 min respectively, and then mixed and sonicated for 12 min to 16 min.

[0033] The third object of the present invention is to provide an application of the nano-delivery system based on exosome membrane fusion as described above, which is mainly used to prepare drugs for phototherapy, chemotherapy or combined phototherapy / chemotherapy.

[0034] The beneficial effects of the present invention are as follows: by cleverly adjusting the membrane composition of liposomes, the fusion of exosomes and liposome membranes, optimizing the properties of hybrid biomimetic nanoparticles such as particle size, and preparing PTXL-targeted prodrugs, the present invention rationally designs the hierarchical structure and functional units of nanoparticles, integrates multi-level delivery at the systemic circulation, cellular level, and organelle level into the same nanoparticle, and realizes the design of multi-level delivery in the same nanoparticle, thereby improving the therapeutic effect at different levels. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the preparation process of the biomimetic nano-delivery system according to an embodiment of the present invention;

[0036] Figure 2 Transmission electron micrographs of exosomes, liposomes, and biomimetic nano-delivery systems according to embodiments of the present invention;

[0037] Figure 3 Flow cytometry results and mean fluorescence intensity graphs of the embodiment of the present invention and control group 1;

[0038] Figure 4 These are confocal fluorescence images of the embodiment of the present invention and control group 1;

[0039] Figure 5 Figure for the mitochondria targeting ability result of the embodiment of the present application;

[0040] Figure 6 Figure for the lung cancer cell targeting result of the embodiment of the present application;

[0041] Figure 7 Figure for the cell uptake result of the embodiment of the present application and control group 1;

[0042] Figure 8 Figure for the photothermal ability result of the embodiment of the present application;

[0043] Figure 9 Figure for the light stability result of the embodiment of the present application;

[0044] Figure 10 Figure for the calnexin expression result of the embodiment of the present application;

[0045] Figure 11 Figure for the release result of high mobility group protein 1 of the embodiment of the present application. DETAILED DESCRIPTION

[0046] The principles and features of the present application are described below in conjunction with the accompanying drawings, and the examples are only used to explain the present application and are not used to limit the scope of the present application.

[0047] The specific embodiments of the present application mainly include the following three aspects:

[0048] The first part is the preparation of the mixed membrane biomimetic nanodelivery system (MAP-PEI-Clip@PTXL-IR780 NPs): 2'-Succinyl-PTXL and IR780-S-S-NH2 are synthesized respectively, and PTXL-S-S-IR780 is generated by amidation reaction of 2'-Succinyl-PTXL and IR780-S-S-NH2. Subsequently, PTXL-S-S-IR780 is loaded by using thin film hydration ultrasonic method with DSPE-PEG-MAP, DSPE-PEI, phospholipid and cholesterol as membrane materials, and then membrane fusion is performed with A549 lung cancer cells (human non-small cell lung cancer cells) homologous exosomes to construct MAP-PEI-Clip@PTXL-IR780 NPs and characterize them. A549 homologous exosomes are mainly obtained by ultrahigh speed centrifugation.

[0049] The second part is the investigation of multi-stage delivery properties, which is investigated from the aspects of cell membrane penetration property, "lysosome escape" property, lung cancer cell targeting property, mitochondria targeting ability, etc.

[0050] The third part is the anti-cancer effect under the guidance of imaging, mainly including the investigation of near-infrared fluorescence imaging guiding ability, the investigation of light stability and photothermal heating, the investigation of reversing drug resistance, the investigation of the ability of ICD induced by combination of phototherapy and chemotherapy, the investigation of drug metabolism in vivo, the investigation of lung cancer treatment efficiency, the investigation of in vivo biological safety evaluation, etc.

[0051] Examples

[0052] I. Synthesis of 2'-Succinyl-PTXL

[0053] PTXL 100 mg was dissolved in DMF, and succinic anhydride 150 mg was added and stirred at room temperature for 3 hours. TLC was monitored with chloroform: acetone (15:1, v / v) as eluent (Rf=0.4), and vacuum dried. Then 10 mL of distilled water was added, stirred and filtered. The precipitate was dissolved in acetone, water was added, and vacuum dried to white solid. The deuterated chloroform was used as a solvent, and the 1 H-NMR spectrum was recorded on an AM-400 spectrometer.

[0054] II. Synthesis of IR780-S-S-NH2

[0055] Cystamine dihydrochloride 405 mg (1.8 mmol) and TEA 1.25 mL were dissolved in DMF, and then IR780 300 mg was added and stirred under nitrogen for 4 hours. Subsequently, it was dialyzed with deionized water and freeze-dried to obtain IR780-S-S-NH2.

[0056] III. Synthesis of PTXL-S-S-IR780

[0057] 80 mg of 2'-succinyl-PTXL was dissolved in DMSO:H2O (1:1, v / v), and then NHS 23 mg and EDC 31 mg were added and reacted for 2 hours. Then 117.5 mg of IR780-S-S-NH2 was slowly added under nitrogen, and stirred for 24 hours. The obtained PTXL-S-S-IR780 was dialyzed with methanol (MWCO=1000 Da) for one day, and then dialyzed with deionized water for one day and freeze-dried. The 1 H-NMR spectrum was recorded.

[0058] IV. Preparation of drug-loaded liposomes

[0059] Dissolve 1 mg of DSPE-PEG-MAP, 1 mg of DSPE-PEI, 9.025 mg of phospholipids, 2.375 mg of cholesterol, and 1.125 mg of PTXL-SS-IR780 in anhydrous ethanol, sonicate, and remove the ethanol by rotary evaporation at 45°C. Rehydrate for 30 minutes and sonicate for 4 minutes, repeating the sonication cycle for 3 seconds and 3 seconds. The drug-to-lipid mass ratio of the drug-loaded liposomes was 1:9.

[0060] 5. Exosome Extraction

[0061] A549 lung cancer cells at 70%-80% growth were incubated in serum-free RPMI-1640 medium. After 24-48 hours, the supernatant was collected and centrifuged at 300 × g for 10 minutes at 4°C to remove dead cells and cell debris. The supernatant was then removed by ultracentrifugation at 120,000 × g for 70 minutes at 4°C. The precipitated exosomes were resuspended in PBS and stored at -80°C until ready for use.

[0062] 6. Membrane Fusion

[0063] The drug-loaded liposomes and exosomes were sonicated for 15 min separately and then mixed and sonicated for 15 min.

[0064] The mass ratio of the drug-loaded liposomes to the exosomes is 5:1.

[0065] The particle size of the nanocarrier MAP-PEI-Clip@PTXL-IR780NPs prepared in this example is 140nm-160nm (see Figure 2 ).

[0066] Control group 1

[0067] According to the method of the above example, PEI-Clip@PTXL-IR780 NPs without membrane-penetrating peptide (DSPE-PEG-MAP) modification, MAP-Clip@PTXL-IR780 NPs without DSPE-PEI modification, and MAP-PEI-lip@PTXL-IR780 NPs without exosome modification were prepared simultaneously.

[0068] PEI-Clip@PTXL-IR780 NPs and MAP-Clip@PTXL-IR780 NPs and the nanocarriers of the present invention were tested for their membrane penetration ability and lysosomal escape function. The results showed that due to the modification of DSPE-PEG-MAP and DSPE-PEI, the present invention exhibited excellent membrane penetration ability and lysosomal escape function (see Figure 3 、 Figure 4 ).

[0069] Investigation of cell membrane penetration properties

[0070] The nanoparticles modified with or without the membrane-penetrating peptide (DSPE-PEG-MAP) were co-incubated with A549 cells, and the membrane-penetrating properties of the MAP-PEI-Clip@PTXL-IR780 NPs of the present invention on lung cancer cells were investigated by flow cytometry (FCM). Figure 3 (a) in the middle shows the flow cytometry results of different nanoparticles after being labeled with Dio (cell membrane green fluorescence) and incubated with A549 cells at different time points. Figure 3 (b) shows the mean fluorescence intensity graph, showing that the mean fluorescence intensity of the nanoparticles modified with the cell-penetrating peptide (DSPE-PEG-MAP) (MAP-PEI-Clip@PTXL-IR780NPs) was significantly higher than that of the nanoparticles without cell-penetrating peptide modification (PEI-Clip@PTXL-IR780NPs) at 2, 4, and 8 hours of treatment, and the intensity increased over time. This demonstrates that the cell-penetrating peptide can efficiently carry nanocarriers across the membrane.

[0071] Investigation of lysosomal escape performance

[0072] After reaching the acidic lysosomes, MAP-PEI-Clip@PTXL-IR780 NPs must first escape the lysosomes and then be transported to the mitochondria. To study the lysosomal escape ability of MAP-PEI-Clip@PTXL-IR780 NPs, green fluorescent markers were used, red fluorescent markers were used to label lysosomes, FITC (green fluorescence) was used to label MAP-PEI-Clip@PTXL-IR780 NPs, and lysosome kits were used to label A549 cell lysosomes. CLSM (confocal fluorescence microscopy) was used to detect the "lysosomal escape" effect of PEI-modified nanoparticles. The results are shown in Figure 2. Figure 4 .

[0073] Figure 4 These are confocal fluorescence images of different nanoparticles labeled with FITC (green fluorescence) and incubated with A549 cells at different time points. Figure 4The results showed that MAP-PEI-Clip@PTXL-IR780 NPs began to be engulfed by acidic lysosomes at 2 hours; most of them were localized in lysosomes at 4 hours, and the Pearson coefficient was less than 0.5 at 6 hours, indicating that the nanoparticles did not colocalize with lysosomes and escaped from lysosomes, avoiding lysosomal degradation. However, the Pearson coefficient of MAP-Clip@PTXL-IR780 NPs was still greater than 0.5 at 6 hours, indicating that MAP-Clip@PTXL-IR780 NPs were localized in lysosomes at 6 hours, which greatly increased the degradation of the nanomaterial. This is because the present invention adds DSPE-PEI to the lipid layer. The PEI (polyetherimide) molecules in it are rich in various amine groups, which have buffering capacity in a wide pH range and have a proton sponge effect, causing the lysosomes to become unstable. It can assist the liposomes in "lysosomal escape", thereby facilitating the escape from the MAP-PEI-Clip@PTXL-IR780 NPs lysosome and improving the drug delivery efficiency.

[0074] Mitochondrial targeting ability assessment

[0075] The PTXL-SS-IR780 and MAP-PEI-Clip@PTXL-IR780 NPs were taken to test their mitochondrial targeting ability. Specifically, A549 cells were cultured with PTXL-SS-IR780 and MAP-PEI-Clip@PTXL-IR780 NPs to verify their mitochondrial targeting and subcellular localization abilities. The results showed that the present invention has excellent mitochondrial targeting ability (see Figure 5 ). Figure 5 The fluorescence images of different nanoparticles after being labeled with Dio (cell membrane green fluorescence) and incubated with A549 cells for 8 hours show that the red fluorescence of the PTXL-SS-IR780 and MAP-PEI-Clip@PTXL-IR780 NPs groups is consistent with the green fluorescence, which confirms that the nanocarrier of the present invention is localized in the mitochondria and achieves effective mitochondrial targeting.

[0076] Mitochondria are key organelles in cells that regulate energy metabolism and maintain production. Introducing mitochondrial targeting function can increase the delivery resolution of nanomaterials to the organelle level, enabling them to be specifically targeted to the mitochondria of A549 cells, thereby improving the sensitivity to chemotherapy and phototherapy.

[0077] Lung cancer cell targeting investigation

[0078] A549, MDA-MB-231 (human breast cancer cells), HCT-116 (human colon cancer cells), and SW-620 (human colon cancer cells) cells were co-cultured with nanoparticles, and the targeting ability of MAP-PEI-Clip@PTXL-IR780NPs to homologous and heterologous tumor and normal cells was evaluated by FCM (flow cytometry). Figure 6 (a) is the flow result diagram. Figure 6 (b) is Figure 6 (a) Quantitative graph of mean fluorescence intensity. Figure 6 It can be seen that the fluorescence intensity in A549 cells was the highest after treatment with MAP-PEI-Clip@PTXL-IR780NP, indicating that MAP-PEI-Clip@PTXL-IR780NPs nanoparticles have the strongest targeting ability to homologous A549 cells, which is significantly higher than other cancer cell lines.

[0079] MAP-PEI-lip@PTXL-IR780 NPs and MAP-PEI-Clip@PTXL-IR780 NPs labeled with green fluorescent dye were co-incubated with A549 cells for 2, 4, and 6 hours. Figure 7 It can be seen that the fluorescence intensity of A549 cells treated with MAP-PEI-Clip@PTXL-IR780 NPs was significantly higher than that of the MAP-PEI-lip@PTXL-IR780 NPs-treated group, and had a time-dependent uptake behavior.

[0080] This is because the MAP-PEI-Clip@PTXL-IR780 NPs of the present invention are fused into liposomes using exosomes from the A549 cell line, thus giving the nanocarrier particles the ability to target the A549 cell line.

[0081] Exosome biomimetic technology is currently attracting significant attention. By integrating exosome features onto the surface of nanoparticles, they can impart external "markers" similar to those of actual cancer cells. Exosome-inspired nanoparticles can leverage the biological properties of cancer cells to enhance their targeting and recognition capabilities within the body. Liposomes share a similar membrane structure to exosomes and are more easily modified. Fusion of cancer cell membranes with liposome membranes can create nanosystems with homologous targeting and functionality, potentially offering innovative strategies for cancer treatment.

[0082] In vitro photothermal performance

[0083] IR780 is a near-infrared fluorescent dye with excellent biocompatibility and biomarker capabilities. It is widely used in nanocarriers in photothermal therapy, tumor targeted imaging, and photodynamic therapy.

[0084] To compare the photothermal effects of MAP-PEI-Clip@PTXL-IR780 NPs and PTXL-SS-IR780, the samples were exposed to NIR laser (808 nm, 1 W / cm 2 The photothermal stability of MAP-PEI-Clip@PTXL-IR780 NPs and PTXL-SS-IR780 was studied. The samples were irradiated for 15 minutes, then naturally cooled to the initial temperature after the laser was turned off. This process was repeated three times, and the temperature changes were recorded.

[0085] Figure 8 As can be seen, under near-infrared light (808 nm), the temperature of PBS (phosphate buffered saline) did not change significantly, while the temperature of the other groups rose rapidly, indicating that the IR780-loaded nanovesicles can convert near-infrared light into heat energy. The warming effect of MAP-PEI-Clip@PTXL-IR780 NPs increased with increasing concentration. At the same concentration, the warming effect of MAP-PEI-Clip@PTXL-IR780 NPs was significantly higher than that of PTXL-SS-IR780, demonstrating that the MAP-PEI-Clip@PTXL-IR780 NPs of this embodiment have excellent photothermal capacity.

[0086] Figure 9 It can be seen that after three laser on / off cycles, the temperature of the MAP-PEI-Clip@PTXL-IR780 NPs solution exceeded 45°C, while the temperature of PTXL-SS-IR780 alone dropped to below 35°C, indicating that the MAP-PEI-Clip@PTXL-IR780 NPs of the present invention have better photothermal stability than PTXL-SS-IR780. This is because the encapsulation of the lipid layer reduces the degradation of PTXL-SS-IR780. Therefore, MAP-PEI-Clip@PTXL-IR780 NPs can achieve effective photothermal conversion and have good photothermal stability.

[0087] Investigation of the ability of combined phototherapy and chemotherapy to induce ICD

[0088] Immunogenic cell death (ICD) is a form of cell death that activates the immune system to fight tumors. It releases damage-associated molecular patterns (DAMPs) to attract and activate immune cells, thereby enhancing anti-tumor immune responses. Damage-associated molecular patterns primarily include cell-surface-exposed calreticulin (CRT), extracellularly released adenosine triphosphate (ATP), and high-mobility group box 1 protein (HMBG1). Combining phototherapy and chemotherapy to induce ICD can produce a more sustained anti-cancer effect.

[0089] A549 cells were treated with PBS (control), PTXL-S-S-IR780, PTXL-S-S-IR780 plus laser, MAP-PEI-lip@PTXL-IR780 NPs plus laser, and MAP-PEI-Clip@PTXL-IR780 NPs plus laser, and after 24 h of incubation, the surface localization of CRT (calreticulin) was visualized using immunofluorescence, and the extracellular release of HMGB1 and ATP was determined using an HMGB1 ELISA kit (Bi Yun Tian, Hangzhou, China) and an ATP assay kit (Bi Yun Tian, Hangzhou, China).

[0090] Results are shown in Figure 10 and Figure 11 , Figure 10 Red fluorescence in the figure indicates CRT, Figure 11 is the release of HMGB1, and it can be seen that the red fluorescence of A549 cells treated with MAP-PEI-Clip@PTXL-IR780 NPs after incubation and laser irradiation is the strongest, and the release of HMGB1 is the most. Thus, the results show that the MAP-PEI-Clip@PTXL-IR780 NPs of the application effectively induce ICD under laser irradiation, enhance the anti-tumor immune response, and further promote cytotoxicity and apoptosis through the synergistic effect of phototherapy and chemotherapy.

[0091] Lung cancer is a highly complex disease, and its treatment has always been one of the focuses of the medical field. In view of the limitations of traditional treatment methods (including drug resistance, lack of precision treatment methods, etc.) and the problem of insufficient targeting of traditional nanometer drug delivery materials in treatment, the application is based on the synergistic effect of phototherapy and chemotherapy, and a mixed biomimetic membrane nanosystem is constructed by fusing natural lung cancer exosomes with liposome membranes. Through a carefully designed multi-stage delivery method, precise treatment of lung cancer is achieved.

[0092] The application designs a multi-stage precise treatment plan for lung cancer at the levels of systemic circulation, cell level, and organelle level, so that it can recognize the biological microenvironment it is in, dynamically adjust the interaction, continuously cross the delivery barrier, and maximize the delivery effect of the chemotherapy and phototherapy agents. The application effectively avoids drug resistance of chemotherapy drugs, significantly enhances the treatment effect, and simultaneously induces ICD, opening up a new way for cancer immunotherapy.

[0093] The beneficial effects of the application are achieved through the following three points:

[0094] i) Sequential and synergistic delivery of chemotherapy agents and phototherapy agents to ensure that the two treatment agents can reach the lung cancer cells at the same time and exert the maximum therapeutic effect;

[0095] ii) Combining lung cancer homology targeting with mitochondrial targeting. This strategy not only enhances the efficacy of phototherapy but also precisely localizes chemotherapy drugs to lung cancer tissue, avoiding chemotherapy resistance mechanisms and significantly improving chemotherapy efficacy.

[0096] iii) Combined induction of ICD generates a powerful anti-tumor immune response. This effect can promote immune system activity, further enhance the therapeutic effect on lung cancer, and maximize the synergistic effect of phototherapy and chemotherapy.

[0097] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A nano-delivery system based on exosome membrane fusion, characterized in that: include: (1) A prodrug complex formed by paclitaxel and IR780, the prodrug complex is named PTXL-SS-IR780, and has the following structural formula: ； (2) a liposome core encapsulating the prodrug complex, wherein the membrane material of the liposome core comprises: DSPE-PEG-MAP, DSPE-PEI, phospholipid and cholesterol; (3) Exosome membranes derived from homologous tumor cells fused to the surface of the liposome core.

2. The nano-delivery system based on exosome membrane fusion according to claim 1, characterized in that The mass fraction of each component in the liposome core is: DSPE-PEG-MAP:6%-8%; DSPE-PEI:6%-8%; Phospholipids: 62%-70%; Cholesterol: 16%-20%.

3. The nano-delivery system based on exosome membrane fusion according to claim 1, characterized in that The exosome membranes were derived from A549 lung cancer cells.

4. The nano-delivery system based on exosome membrane fusion according to claim 1, characterized in that The particle size of the system is 140nm-160nm.

5. The nano-delivery system based on exosome membrane fusion according to claim 1, characterized in that The mass ratio of the prodrug complex to the liposome core is 1:8-10; the mass ratio of the drug-loaded liposome to the exosome is 3-5:1-2.

6. A method for preparing a nano-delivery system based on exosome membrane fusion according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1, synthesis of 2'-Succinyl-PTXL and IR780-SS-NH2; S2, 2'-Succinyl-PTXL synthesized in step S1 and IR780-SS-NH2 are subjected to an amidation reaction to generate a prodrug complex PTXL-SS-IR780; S3, using DSPE-PEG-MAP, DSPE-PEI, phospholipids and cholesterol as membrane materials, the thin film hydration ultrasonic method was used to encapsulate the PTXL-SS-IR780 from step S2 to form drug-loaded liposomes; S4. Perform membrane fusion of the drug-loaded liposomes obtained in step S3 with the exosomes derived from tumor cells.

7. The method for preparing the nano-delivery system based on exosome membrane fusion according to claim 6, characterized in that: In step S1: The specific steps of synthesizing the 2'-Succinyl-PTXL are as follows: dissolving paclitaxel in N,N-dimethylformamide, adding succinic anhydride, stirring, washing, and drying to obtain; The specific steps for synthesizing the IR780-SS-NH2 are as follows: cystamine dihydrochloride and triethylamine are dissolved in N,N-dimethylformamide, IR780 is added, and the mixture is stirred in nitrogen, followed by dialysis with deionized water and freeze-drying.

8. The method for preparing the nano-delivery system based on exosome membrane fusion according to claim 6, characterized in that: The specific steps of the amidation reaction in step S2 are: Dissolve 2'-Succinyl-PTXL in DMSO:H2O, then add N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. The reaction time is 1.5h-2.5h. Then, slowly add IR780-SS-NH2 under nitrogen. The reaction temperature is 20℃-30℃ and stirred for 20h-30h.

9. The method for preparing the nano-delivery system based on exosome membrane fusion according to claim 6, characterized in that: In step S3, the specific parameters of the thin film hydration ultrasound method are: hydration temperature of 20°C-30°C, hydration time of 25min-35min, ultrasound power of 150W-250W, and intermittent ultrasound of 3min-6min; in step S4, during the membrane fusion process, the drug-loaded liposomes and exosomes are ultrasonically subjected to 10min-20min respectively, and then mixed and ultrasonicated for 12min-16min.

10. A use of the nano-delivery system based on exosome membrane fusion according to any one of claims 1 to 5, characterized in that: It is mainly used to prepare drugs for phototherapy, chemotherapy or photo / chemotherapy combined therapy.

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