Drug-loaded hybrid nano-vesicle for targeted blocking of cross talk between sympathetic nerves and tumors and preparation method of drug-loaded hybrid nano-vesicle

Abstract

The invention belongs to the technical field of preparation of antitumor drugs, and particularly relates to a drug-loaded hybrid nano-vesicle for targeted blocking of cross talk between sympathetic nerves and tumors and a preparation method of the drug-loaded hybrid nano-vesicle. The drug-loaded hybrid nano-vesicle provided by the invention comprises a hybrid nano-vesicle formed by hydrating an M1 type macrophage source nano-vesicle and an acid-sensitive liposome, and a drug which is wrapped by the hybrid nano-vesicle and can inhibit cross talk between sympathetic nerves and tumors. The drug-loaded hybrid nano-vesicle can effectively target tumor tissue, degrade a compact extracellular matrix of the tumor, and can respond to a slightly acidic environment of the tumor to release the drug, so that efficient enrichment and deep penetration of the drug at a tumor part are realized, interaction between sympathetic nerves and tumors under a chronic stress condition is effectively blocked, and the drug-loaded hybrid nano-vesicle has a good application prospect. The nerve infiltration and neurotransmitter secretion in the tumor are inhibited, and the tumor immune microenvironment is improved, so that the tumor growth is effectively inhibited. In addition, the drug-loaded hybrid nano-vesicle is high in biological safety and has a good clinical application prospect.

Description

Technical Field

[0001] The present application belongs to the technical field of anti-tumor drug preparation, and more specifically, relates to a drug-loaded hybrid nanovesicle that targets and blocks crosstalk between sympathetic nerves and tumors, and a preparation method thereof. Background Art

[0002] The impact of nerves in the tumor microenvironment, particularly sympathetic nerves, on tumor development, progression, and drug resistance has attracted significant attention from international researchers. Norepinephrine, secreted by sympathetic nerves under chronic stress, can interact with β-adrenergic receptors on tumor cells and other cells in the tumor microenvironment, activating β-adrenergic receptors and influencing sympathetic nerve-tumor crosstalk through multiple pathways, promoting tumor development and drug resistance. Furthermore, recent studies have shown that chemotherapy drugs can increase β-adrenergic receptor expression in tumor cells, further exacerbating chemotherapy resistance. Targeted delivery of β-adrenergic receptor blockers may be an effective way to disrupt sympathetic nerve-tumor crosstalk and improve the efficacy of tumor chemotherapy. However, sympathetic nerve activity within tumors exacerbates collagen deposition in tumor tissue. Existing drugs are unable to overcome the dense matrix barrier within the tumor, making it difficult to achieve targeted delivery of β-adrenergic receptor blockers to tumor tissue and penetrate deep into the tumor.

[0003] Therefore, research and development of drugs that are tumor-targeted and can effectively degrade tumor extracellular matrix, achieve effective drug release in the micro-acidic environment of the tumor and efficient enrichment and deep penetration in the tumor site, block sympathetic nerve-tumor crosstalk, inhibit nerve development and neurotransmitter secretion in the tumor, and improve the tumor immune microenvironment are of great significance for effectively inhibiting tumor growth and improving chemotherapy effects. Summary of the Invention

[0004] In response to the defects of the existing technology, the purpose of this application is to provide a drug-loaded hybrid nanovesicle and its preparation method for targeted blocking of sympathetic nerve and tumor crosstalk, aiming to solve the problems that existing drugs cannot effectively degrade the tumor extracellular matrix, are difficult to achieve targeted delivery of drugs to tumor tissue and penetrate deep into the tumor, cannot effectively block sympathetic nerve-tumor crosstalk and lead to nerve development and neurotransmitter secretion in the tumor.

[0005] To achieve the above objectives, in the first aspect, the present application provides a drug-loaded hybrid nanovesicle that targets and blocks the crosstalk between sympathetic nerves and tumors, which includes hybrid nanovesicles formed by hydrating M1 macrophage-derived nanovesicles and acid-sensitive liposomes, and drugs encapsulated in the hybrid nanovesicles that can inhibit the crosstalk between sympathetic nerves and tumors.

[0006] Preferably, the acid-sensitive liposome is assembled from backbone lipids, acid-sensitive lipids and auxiliary lipids, and the liposome membrane structure is destroyed under the acidic tumor microenvironment.

[0007] Preferably, the above-mentioned backbone lipids include one or more of natural phospholipids, semi-synthetic phospholipids, fully synthetic phospholipids and their derivatives; the above-mentioned acid-sensitive lipids include one or more of acid-sensitive lipids containing protonated groups and acid-sensitive lipids containing acid-sensitive chemical bonds; the above-mentioned auxiliary lipids include one or more of polyethylene glycol-modified lipids and cationic lipids.

[0008] Preferably, the drug capable of inhibiting the crosstalk between sympathetic nerves and tumors is a β-adrenergic receptor blocker.

[0009] Preferably, the beta-adrenergic receptor blocker is selected from one or more of propranolol, metoprolol, bisoprolol, atenolol, arotinolol, labetalol and carvedilol.

[0010] Preferably, the M1 macrophages are obtained by subjecting any one of human peripheral blood monocytes, human monocyte cell lines, mouse bone marrow-derived macrophages or mouse monocyte-macrophage cell lines to M1 polarization treatment.

[0011] Preferably, the mass ratio of the M1 macrophage-derived nanovesicles, the drug, and the acid-sensitive liposomes is 1:(0.5-20):(1-50).

[0012] In a second aspect, the present application provides a method for preparing the above-mentioned drug-loaded hybrid nanovesicles, comprising the following steps: S1, assembling backbone lipids, acid-sensitive lipids and auxiliary lipids to form acid-sensitive liposomes; S2. Prepare M1 macrophage-derived vesicles by physical or chemical methods; S3. Hydrate the drug capable of inhibiting the crosstalk between sympathetic nerves and tumors, the M1 macrophage-derived vesicles, and the acid-sensitive liposomes, and then extrude and fuse them to obtain the drug-loaded hybrid nanovesicles.

[0013] Preferably, in step S1, the method for assembling acid-sensitive liposomes comprises any one of a thin film hydration method, a microfluidics method, a reverse phase evaporation method, a solvent injection method, an ultrasonic dispersion method or a freeze-thaw-extrusion method.

[0014] Preferably, in step S1, the molar ratio of the backbone lipid, acid-sensitive lipid and helper lipid is (5-8):(2-5):(0.2-3).

[0015] Preferably, the specific operation of step S3 is: placing the above-mentioned acid-sensitive liposomes in a buffer solution containing the above-mentioned M1 macrophage-derived vesicles to form acid-sensitive lipid vesicles, and then fusing the acid-sensitive lipid vesicles and the M1 macrophage-derived vesicles through multiple extrusions to form hybrid nanovesicles, while encapsulating the above-mentioned drug in the hybrid nanovesicles.

[0016] In a third aspect, the present application provides an anti-tumor drug, which comprises the drug-loaded hybrid nanovesicle.

[0017] Preferably, the anti-tumor drug further comprises a chemotherapeutic drug, which is a chemotherapeutic drug capable of promoting expression of β-adrenergic receptors in tumor cells.

[0018] Preferably, the chemotherapeutic drug is selected from one or more of gemcitabine or an anthracycline chemotherapeutic drug.

[0019] Preferably, the anthracycline chemotherapeutic drug is selected from one or more of doxorubicin, epirubicin, daunorubicin, epirubicin and pirarubicin.

[0020] Overall, compared with the prior art, the above technical solutions conceived by the present application mainly have the following technical advantages: (1) The drug-loaded hybrid nanovesicle provided by the present application for targeting and blocking sympathetic nerve-tumor crosstalk comprises a hybrid nanovesicle formed by hydration of M1 type macrophage-derived nanovesicles and acid-sensitive liposomes, and a drug capable of inhibiting sympathetic nerve-tumor crosstalk wrapped by the hybrid nanovesicle. The drug-loaded hybrid nanovesicle not only can degrade tumor extracellular dense matrix to reach deep tumor tissue to play a role, but also can effectively release the drug in response to the micro-acidic environment of the tumor, ensuring the multi-target effect of the drug in the tumor tissue. Further, the drug penetrating into the deep tumor tissue can inhibit tumor innervation and reduce neurotransmitter secretion by blocking the interaction between the sympathetic nerve and the tumor under chronic stress conditions, while the ability of the drug-loaded hybrid nanovesicle to reverse M2 type macrophages to M1 type, effectively blocks the sympathetic nerve-tumor crosstalk, inhibits the promoting effect of the sympathetic nerve on the tumor, improves the tumor immune microenvironment and tumor nerve microenvironment, and inhibits tumor growth.

[0021] (2) Compared with the administration of acid-sensitive liposomes (LIPs), hybrid nanovesicles (hNVs), drugs (Pro), drug-loaded acid-sensitive liposomes (Pro@LIPs) alone, the drug-loaded hybrid nanovesicle (Pro@hNVs) provided by the present application exhibits a synergistic effect in anti-tumor therapy, can effectively degrade tumor extracellular dense matrix, realize effective release of the drug in the micro-acidic environment of the tumor, and high-efficiency enrichment and deep penetration of the drug in the tumor site, block the sympathetic nerve-tumor crosstalk, inhibit tumor innervation and neurotransmitter secretion, improve the tumor immune microenvironment and tumor nerve microenvironment, and significantly inhibit tumor growth.

[0022] (3) The drug-loaded hybrid nanovesicles provided in this application can be combined with other chemotherapy drugs to significantly inhibit the neurogenesis and neurotransmitter secretion in tumors promoted by chemotherapy drugs, break the sympathetic nerve-tumor crosstalk induced by chemotherapy drugs, improve and enhance the effect of tumor chemotherapy, and achieve a synergistic anti-tumor effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 These are the test results of the drug-loaded hybrid nanovesicles prepared in the examples of the present application; content a is the FRET analysis of the drug-loaded hybrid nanovesicles prepared using different mass ratios of M1-NVs and phospholipids, and content b and content c are the protein profiles and the expression of the M1 macrophage marker CD86 of the drug-loaded hybrid nanovesicles prepared in the examples of the present application, respectively; Figure 2 Basic characterization of the drug-loaded hybrid nanovesicles (Pro@hNVs) prepared in the examples of this application; content a, content b, content c, and content d are TEM images, particle size, zeta potential, and acid-responsive drug release characteristics, respectively; Figure 3 The stability of the drug-loaded hybrid nanovesicles prepared in the examples of the present application; wherein content a, content b, and content c are the changes in particle size, zeta potential, and PDI after storage in PBS with or without 10% FBS for 14 days, respectively; Figure 4 The acid-responsive drug release characteristics of the drug-loaded hybrid nanovesicles (Car@hNVs) prepared in the examples of this application; Figure 5 The effect of the drug-loaded hybrid nanovesicles prepared in the examples of this application on ISO-stimulated Panc02 tumor cells; wherein content a and content b are respectively the viability and migration ability of Panc02 cells; Figure 6 The effect of Panc02 tumor cell conditioned medium pretreated with drug-loaded hybrid nanovesicles prepared in the examples of the present application on PC12 neural cells; wherein content a and content b are respectively the viability and axon length of PC12 neural cells; Figure 7 The ability of the drug-loaded hybrid nanovesicles prepared in the present embodiment to reverse polarize macrophages to M1 phenotype; wherein content a, content b, and content c are the relative expression levels of M1-related genes Tnf, CD80, + The proportion of macrophages and the content of pro-inflammatory factor TNF; Figure 8 The effect of drug-loaded hybrid nanovesicles prepared in the examples of the present application on nerve cells after reverse polarization of macrophages to M1 phenotype; wherein content a and content b are the effects of the supernatant of drug-loaded hybrid nanovesicles on the viability and axon length of PC12 cells after reverse polarization of macrophages to M1 phenotype; Figure 9 The ability of the drug-loaded hybrid nanovesicles prepared in the examples of this application to degrade the tumor extracellular matrix; wherein content a is the expression of MMP9 and MMP14 on the drug-loaded hybrid nanovesicles, content b is the MMPs enzyme activity, and content c is the type I collagen content in the tumor tissue of mice bearing orthotopic pancreatic cancer after treatment with the drug-loaded hybrid nanovesicles; Figure 10 The hybrid nanovesicles prepared in the examples of this application have the ability to significantly target tumor tissues; wherein content a is the in vivo imaging results of mice bearing orthotopic pancreatic cancer at different time points after intravenous injection of IR780-labeled LIPs and hNVs, respectively; content b is the quantitative analysis of the fluorescence intensity of the mouse tumor site; content c is the in vitro fluorescence imaging of pancreatic tumors and major organs; content d is the quantitative analysis of the fluorescence intensity of pancreatic tumors and major organs; Figure 11 The tumor penetration depth of the drug-loaded hybrid nanovesicles prepared in the examples of this application is shown in Figure 1. Content a shows the extravasation of drugs into the blood vessels of mice bearing orthotopic pancreatic cancer after intravenous injection of IR780-labeled LIPs and hNVs, and content b shows the relevant content of drugs extravasated from the blood vessels. Figure 12 is the weight of the tumor in mice bearing orthotopic pancreatic cancer after treatment with the drug-loaded hybrid nanovesicles prepared in the examples of the present application; Figure 13 is the number of tumor metastatic nodules after treating mice bearing orthotopic pancreatic cancer with the drug-loaded hybrid nanovesicles prepared in the examples of the present application; Figure 14 The effect of the drug-loaded hybrid nanovesicles prepared in the present application on the tumor microenvironment of mice bearing orthotopic pancreatic cancer; content a and content b are the content of M1 macrophages and M2 macrophages in pancreatic cancer tumor tissue, respectively, and content c is the content of CD8 + T cell content; Figure 15 The effect of drug-loaded hybrid nanovesicles prepared in this application example on the neural microenvironment of mice bearing in situ pancreatic cancer; wherein content a is TH in pancreatic cancer tumor tissue + The area ratio of sympathetic nerves, content b is the content of norepinephrine in pancreatic tumor tissue; Figure 16 The safety of the drug-loaded hybrid nanovesicles prepared in the examples of the present application; wherein content a is the weight of the mice after treatment with the drug-loaded hybrid nanovesicles, content b, content c, and content d are the levels of alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase in the serum of the mice after treatment with the drug-loaded hybrid nanovesicles, respectively; Figure 17The effect of the drug-loaded hybrid nanovesicles (Pro@hNVs) prepared in the examples of this application combined with chemotherapy drugs in the treatment of mice bearing orthotopic pancreatic cancer; wherein content a is the tumor weight, content b is the number of tumor metastatic nodules; Figure 18 The effect of drug-loaded hybrid nanovesicles (Pro@hNVs) prepared in this application example combined with chemotherapy drugs on the tumor microenvironment of mice bearing orthotopic pancreatic cancer; content a and content b are the content of M1 macrophages and M2 macrophages in pancreatic cancer tumor tissue, respectively, and content c is the content of CD8 + T cell content; Figure 19 The drug-loaded hybrid nanovesicles (Pro@hNVs) prepared in this embodiment can inhibit chemotherapy-induced neurogenesis and improve the tumor neural microenvironment after combining with chemotherapy drugs; wherein content a is TH in the tumor + The area ratio of sympathetic nerves, content b is the content of norepinephrine in pancreatic tumor tissue; Figure 20 The changes in tumor weight and neural microenvironment after the drug-loaded hybrid nanovesicles (Car@hNVs) prepared in the present application example combined with chemotherapy drugs were treated with mice bearing orthotopic breast cancer; content a is the tumor weight, content b is TH + The area ratio of sympathetic nerves; Figure 21 The effect of the drug-loaded hybrid nanovesicles (Aro@hNVs) prepared in the examples of this application combined with chemotherapy drugs in the treatment of mice bearing orthotopic pancreatic cancer; content a is the tumor weight, and content b is the number of tumor metastatic nodules. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solutions and advantages of this application more clear, the following further describes this application in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit this application.

[0025] In the description of the embodiments of this application, words such as "exemplary" or "for example" are used to indicate examples, illustrations, or descriptions. Any embodiment or design described as "exemplary" or "for example" in the embodiments of this application should not be construed as being preferred or advantageous over other embodiments or designs. Rather, the use of words such as "exemplary" or "for example" is intended to present the relevant concepts in a concrete manner.

[0026] In the description of the embodiments of the present application, unless otherwise specified, “plurality” means two or more.

[0027] The term "M1 macrophage-derived nanovesicles" refers to vesicles secreted by M1 macrophages. The term "hybrid nanovesicles" refers to vesicles formed by hydration of M1 macrophage-derived nanovesicles and acid-sensitive liposomes. The term "drug-loaded hybrid nanovesicles" refers to hybrid nanovesicles encapsulated with drugs that can inhibit sympathetic nerve-tumor crosstalk.

[0028] The present application provides a drug-loaded hybrid nanovesicle that targets and blocks crosstalk between sympathetic nerves and tumors, including hybrid nanovesicles formed by hydrating M1 macrophage-derived nanovesicles and acid-sensitive liposomes, and drugs encapsulated in the hybrid nanovesicles that can inhibit crosstalk between sympathetic nerves and tumors.

[0029] In this application, the acid-sensitive liposomes exhibit acid-responsive drug release properties. Their membrane structure changes in the acidic tumor microenvironment, rapidly releasing drugs encapsulated in the hybrid nanovesicles that can inhibit sympathetic nerve-tumor crosstalk. Any acid-sensitive liposomes reported in the prior art that exhibit acid-responsive drug release properties in an acidic tumor microenvironment are suitable for use in this application.

[0030] In some embodiments, the acid-sensitive liposomes are assembled from backbone lipids, acid-sensitive lipids, and helper lipids.

[0031] In the present application, the above-mentioned backbone lipids participate in forming the bilayer membrane structure of the acid-sensitive liposome, including one or more of natural phospholipids, semi-synthetic phospholipids, fully synthetic phospholipids and their derivatives.

[0032] In some embodiments, the backbone lipid is a fully synthetic phospholipid, preferably a C3-C30 fully synthetic phospholipid, and may be, but is not limited to, one or more of dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dilauroylphosphatidylcholine (DLPC), distearoylphosphatidylethanolamine (DSPE), and dioleoylphosphatidylethanolamine (DOPE). In a weakly acidic environment, dioleoylphosphatidylethanolamine (DOPE) undergoes a molecular conformational change from a lamellar phase to an inverted hexagonal phase (HII phase), disrupting the bilayer stability of the liposome membrane structure and acting like an acid-sensitive lipid. Therefore, DOPE can be used as both a backbone lipid and an acid-sensitive lipid. For example, when preparing acid-sensitive liposomes, DOPE can be selected as the backbone lipid to cooperate with other acid-sensitive lipids to achieve acid-responsive drug release. Alternatively, DOPE can be selected as the acid-sensitive lipid and cationic lipids (such as but not limited to DOTAP, DOTMA, DOEPC) can be used in collaboration to destroy the stability of the membrane structure and thus achieve acid-responsive drug release.

[0033] The above-mentioned acid-sensitive lipids in the present application are pH-sensitive and can change the liposome structure in an acidic tumor environment to release the encapsulated drugs, including one or more of acid-sensitive lipids containing protonated groups and acid-sensitive lipids containing acid-sensitive chemical bonds.

[0034] In some embodiments, the acid-sensitive lipid containing a protonatable group comprises one or more of an amino group (-NH2), a carboxyl group (-COOH), a phosphate group (-PO4H), or an imidazole group (-N=C(NH)NH). In some embodiments, the acid-sensitive lipid containing a protonatable group may be selected from, but not limited to, one or more of dioleoylphosphatidylethanolamine (DOPE), cholesterol hemisuccinate (CHEMS), oleic acid (OA), and palmitoylhomocysteine ​​(PHC).

[0035] In some embodiments, the acid-sensitive lipids containing acid-sensitive chemical bonds comprise one or more of amide bonds, hydrazone bonds, and imine bonds. In some embodiments, the acid-sensitive lipids containing acid-sensitive chemical bonds are selected from, but not limited to, amide bond lipids (e.g., mPEG-DMA-DSPE), hydrazone bond lipids (e.g., mPEG-Hz-CHEMS, mPEG-Hz-PE, DSPE-HYD-mPEG), and imine bond lipids (e.g., mPEG-Imine-PEI).

[0036] In the process of preparing acid-sensitive liposomes, those skilled in the art can select different helper lipids according to actual needs to impart or enhance the properties of acid-sensitive liposomes, such as stability, long circulation properties, acid sensitivity, etc. In some embodiments, the above-mentioned helper lipids include, but are not limited to, one or more of polyethylene glycol-modified lipids and cationic lipids. Exemplarily, polyethylene glycol-modified lipids are selected to enhance the stability and long circulation properties of acid-sensitive liposomes. The above-mentioned polyethylene glycol-modified lipids include, but are not limited to, one or more of mPEG-DSPE and mPEG-HZ-DSPE, wherein the molecular weight of the polyethylene glycol is 1000-6000, for example, 1000, 2000, 3000, 4000, 5000, 6000, etc. Exemplarily, cationic lipids are selected to cooperate with acid-sensitive lipids to enhance the instability of the membrane structure of acid-sensitive liposomes in the acidic tumor microenvironment, further enhance the acid sensitivity of acid-sensitive liposomes, and thus promote the release of drugs. The above-mentioned cationic lipids include but are not limited to one or more of 1,2-dioleoyl-3-trimethylamine propane (DOTAP), N-1-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMA), and 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC).

[0037] In some embodiments, the drug capable of inhibiting sympathetic nerve-tumor crosstalk is a beta-adrenergic receptor blocker. In some embodiments, the beta-adrenergic receptor blocker is selected from one or more of propranolol (Pro), metoprolol (Met), bisoprolol (Bis), atenolol (Ate), arotinolol (Aro), labetalol (Lab), and carvedilol (Car).

[0038] In some embodiments, the M1 macrophages are obtained by subjecting any one of human peripheral blood mononuclear cells, human mononuclear cell lines, mouse bone marrow-derived macrophages, or mouse mononuclear macrophage cell lines to M1 polarization. It is understood that the present application does not limit the method of M1 polarization treatment; any method that can produce M1 macrophages is suitable for the present application, such as, but not limited to, stimulation with one or more of interferon-γ (IFN-γ), lipopolysaccharide (LPS), TNF-α, IL-1β, or a Toll-like receptor (TLR) agonist.

[0039] In some embodiments, the mass ratio of the M1 macrophage-derived nanovesicles, the drug, and the acid-sensitive liposomes is 1:(0.5-20):(1-50), preferably 1:(0.5-20):(2-40), and more preferably 1:(0.5-20):(5-20).

[0040] In some embodiments, the particle size of the drug-loaded bacterial extracellular vesicles is 100 nm to 200 nm.

[0041] On the other hand, the present application also provides a method for preparing the above-mentioned drug-loaded hybrid nanovesicles, which comprises the following steps: S1, assembling backbone lipids, acid-sensitive lipids and auxiliary lipids to form acid-sensitive liposomes; S2. Prepare M1 macrophage-derived vesicles by physical or chemical methods; S3. Hydrate the drug capable of inhibiting sympathetic nerve-tumor crosstalk, the M1 macrophage-derived vesicles, and the acid-sensitive liposomes, and then extrude and fuse them to obtain the drug-loaded hybrid nanovesicles.

[0042] In some embodiments, in step S1, the method for assembling the acid-sensitive liposomes comprises any one of thin film hydration, microfluidics, reverse phase evaporation, solvent infusion, ultrasonic dispersion, or freeze-thaw-extrusion. In a preferred embodiment, the backbone lipid, acid-sensitive lipid, and helper lipid are dissolved in an organic solvent using the thin film hydration method, and the solvent is removed by evaporation under reduced pressure to form a uniform acid-sensitive liposome film. In some embodiments, the organic solvent comprises any one of chloroform, methanol, dichloromethane, or ethanol, or a combination of at least two thereof.

[0043] In some embodiments, in step S1, the molar ratio of the backbone lipid, the acid-sensitive lipid, and the helper lipid is (5-8):(2-5):(0.2-3).

[0044] In some embodiments, in step S2, M1 macrophages are subjected to ultrasound, extrusion, or microfluidics treatment to prepare M1 macrophage-derived vesicles.

[0045] In some embodiments, the specific operation of step S3 is: placing the above-mentioned acid-sensitive liposomes in a buffer solution containing the above-mentioned M1 macrophage-derived vesicles to form acid-sensitive lipid vesicles, and then fusing the acid-sensitive lipid vesicles and the M1 macrophage-derived vesicles through multiple extrusions to form hybrid nanovesicles, and at the same time encapsulating the above-mentioned drug capable of inhibiting sympathetic nerve and tumor crosstalk in the hybrid nanovesicles.

[0046] In some embodiments, in step S3, multiple extrusions are performed using a polycarbonate membrane, wherein the pore size of the polycarbonate membrane may be 0.1 μm to 0.5 μm, and the number of repeated extrusions is 10 to 50 times.

[0047] The present application also provides an anti-tumor drug, which includes the above-mentioned drug-loaded hybrid nanovesicles.

[0048] In some embodiments, the anti-tumor drug further comprises a chemotherapy drug, which is a chemotherapy drug capable of promoting the expression of β-adrenergic receptors in tumor cells.

[0049] In some embodiments, the chemotherapy drug is selected from one or more of gemcitabine or doxorubicin-based chemotherapy drugs. In some embodiments, the doxorubicin-based chemotherapy drug is selected from one or more of doxorubicin, epirubicin, daunorubicin, epirubicin, and pirarubicin.

[0050] In some embodiments, the drug-loaded hybrid nanovesicles and the chemotherapeutic drugs can be co-administered simultaneously, sequentially or separately. In addition, the simultaneous, sequential or separate administration methods are not limited to one time, and these administration methods can be repeated or combined.

[0051] In some embodiments, the drug-loaded hybrid nanovesicles and the chemotherapy drugs can be contained in an anti-tumor drug together with a pharmaceutically acceptable carrier, excipient and / or diluent.

[0052] In some embodiments, the anti-tumor drug can be administered by any method, such as, but not limited to, oral or parenteral administration. Parenteral administration methods can include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardial, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, rectal administration, and the like.

[0053] It will be appreciated that the total effective dose of the anti-tumor drug described herein can be administered to a patient as a single dose, or as multiple doses over a long period of time according to a fractionated treatment regimen. The content of the active ingredient in the anti-tumor drug can vary depending on the severity of the disease. A person skilled in the art can determine the appropriate effective dose based on the patient's age, weight, health status, and gender, the severity of the disease, diet, and excretion rate, as well as the formulation method, route of administration, and number of treatments.

[0054] In some embodiments, the tumor may be pancreatic cancer, breast cancer, prostate cancer, central nervous system (CNS) tumor, or primary CNS lymphoma, but is not limited thereto.

[0055] It should be understood that materials of the same or similar type, model, quality, properties, or functions as the reagents and instruments used in the following examples can be used to implement this application. The experimental methods used in the following examples are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0056] The following are examples: Example 1 Preparation and hybrid characterization of drug-loaded hybrid nanovesicles 1. Experimental steps 1) After LPS was used to induce macrophages for 48 h, M1 phenotype macrophages were obtained. The cells were cultured at a final concentration of 1×10 6 Cells were resuspended in ice-cold PBS at a concentration of 10 cells / mL. The cell suspension was sonicated on ice for 3 minutes at 50 W intermittently (1.5 seconds on, 2 seconds off) and then extruded 11 times using a microextruder (Avanti Polar Lipids, Alabama, USA) through 0.8 and 0.4 μm polycarbonate membrane filters (Whatman, Buckinghamshire, UK). The extruded suspension was centrifuged at 1500 g for 10 minutes at 4°C to remove cell debris, and the resulting supernatant was centrifuged at 14,000 g for 1 hour to collect M1 macrophage-derived vesicles (M1-NVs).

[0057] 2) DOPE, CHEMS, and DSPE-mPEG2000 were dissolved in chloroform at a molar ratio of 6:4:0.5 and placed in an eggplant-shaped flask. The solvent was evaporated under reduced pressure at 37°C, forming a uniform thin film (i.e., acid-sensitive liposomes) in the flask. The film was then hydrated in PBS containing M1-NVs at a mass ratio of M1-NVs to phospholipid of 1:10 at 37°C for 1 h. The film was stirred and vortexed, and then extruded 11 times through a 0.2 μm polycarbonate membrane filter using a microextruder to obtain uniformly dispersed hybrid nanovesicles (hNVs). Propranolol (Pro) was added during the hydration process to obtain drug-loaded hybrid nanovesicles (Pro@hNVs) at a mass ratio of Pro to phospholipid of 1:10. The resulting Pro@hNVs were then ultrafiltered using a 100 kDa ultrafiltration tube (Sigma-Aldrich) and washed three times. The resulting Pro@hNVs were stored at 4°C until further use.

[0058] 3) Verification of the binding of acid-sensitive liposomes (LIPs) to M1-NVs: In the experimental group, M1-NVs were labeled with DiI (excitation / emission = 549 / 565 nm) and DiD (excitation / emission = 644 / 663 nm). The labeled M1-NVs were then hydrated with LIPs at a mass ratio of 1:5, 1:10, and 1:20, respectively, and extruded onto 0.2 μm polycarbonate membranes using the same method for preparing hNVs. Fluorescence spectra were recorded using a fluorescence spectrophotometer with an excitation wavelength of 525 nm and emission wavelengths between 530 and 800 nm.

[0059] 4) To evaluate the successful construction of Pro@hNVs: Proteins were extracted using RIPA lysis buffer containing protease inhibitors, denatured at 95°C for 5 minutes, and loaded onto a 10% SDS-PAGE gel. Proteins were visualized using a fast silver staining kit. Western blot analysis of proteins from M1-like macrophages (control group 1), M1-NVs (control group 2), hNVs (control group 3), and Prp@hNVs (experimental group) was performed using rabbit anti-CD86 (Proteintech, cat No. 13395-1-AP, 1 / 1000 dilution) as the primary antibody and HRP-conjugated goat anti-rabbit IgG as the secondary antibody (Proteintech, cat No. SA00001-2, 1 / 5000 dilution). Protein detection was performed using an ECL chemiluminescence detection kit.

[0060] 2. Experimental results FRET analysis showed that when DiI- and DiD-labeled M1-NVs bound to LIPs, the fluorescence intensity of DiI at 565 nm increased, while the fluorescence intensity of DiD at 670 nm decreased ( Figure 1 Content a), indicating that LIPs fused with M1-NVs and were successfully inserted into the vesicles. SDS-PAGE analysis showed that the protein profiles of Pro@hNVs and hNVs were consistent with those of M1 macrophages and M1-NVs ( Figure 1 Content b), the presence of M1 macrophage marker CD86 in Pro@hNVs and hNVs further demonstrated the successful fusion of M1-NVs and LIPs ( Figure 1 Content c).

[0061] Example 2 Basic Characterization and Stability of Drug-Loaded Hybrid Nanovesicles 1. Experimental steps 1) The collection method of Pro@hNVs is the same as that in Example 1.

[0062] 2) Transmission electron microscopy (TEM) was used to observe the morphology of LIPs, hNVs, Pro@LIPs, and Pro@hNVs (control group 1-3 and experimental group, respectively), and dynamic light scattering (DLS) was used to evaluate their hydrodynamic diameter, zeta potential, and polydispersity index (PDI).

[0063] 3) Pro@LIPs (control group) and Pro@hNVs (experimental group) were dialyzed against PBS at pH 7.4 and pH 6.5, respectively, at 37°C and 150 rpm to evaluate the pH-responsive release of Pro. The amount of Pro released at different time points was determined using HPLC.

[0064] 4) DLS was used to monitor the time-dependent changes in the hydrodynamic diameter, zeta potential, and PDI of Pro@LIPs (control group) and Pro@hNVs (experimental group) in PBS with or without 10% FBS to evaluate their stability.

[0065] 2. Experimental results TEM images ( Figure 2 Content a) shows that the drug-loaded hybrid nanovesicles have a spherical vesicle structure. DLS analysis shows that compared with the control group, the average particle size and zeta potential of the drug-loaded hybrid nanovesicles remain basically unchanged, with an average particle size of 140-160 nm and a zeta potential of approximately -11 mV ( Figure 2 Content b and Figure 2Content c). The drug-loaded hybrid nanovesicles retained the pH-sensitive release properties of acid-sensitive liposomes, releasing approximately 72% of Pro at pH 6.5 and approximately 43% of Pro at pH 7.4 ( Figure 2 Content d).

[0066] The drug-loaded hybrid nanovesicles have excellent colloidal stability. After being placed in PBS containing 10% fetal bovine serum (FBS) or not for 14 days, the particle size ( Figure 3 Content a), Zeta potential ( Figure 3 Content b) and PDI ( Figure 3 Content c) showed no significant changes, indicating that the macrophage-derived nanovesicles still had excellent stability after hybridization and drug loading.

[0067] Example 3 Acid-responsive drug release performance of drug-loaded hybrid nanovesicles 1. Experimental steps 1) The method for collecting M1 macrophage-derived vesicles is the same as in Example 1.

[0068] 2) DPPC, mPEG-Hz-PE, and DSPE-mPEG2000 were dissolved in chloroform at a molar ratio of 7:5:0.5 and placed in an eggplant-shaped flask. The solvent was evaporated under reduced pressure at 37°C to form a uniform thin film (i.e., acid-sensitive liposomes) in the flask. The film was hydrated in PBS containing M1-NVs at a mass ratio of M1-NVs to phospholipid of 1:10 at 45°C for 1 h. The mixture was stirred and vortexed, and then extruded 11 times through a 0.2 μm polycarbonate membrane filter using a microextruder to obtain uniformly dispersed hybrid nanovesicles (hNVs). Carvedilol (Car) was added during the hydration process to obtain drug-loaded hybrid nanovesicles (Car@hNVs) at a mass ratio of 1:5. The mixture was then ultrafiltered using a 100 kDa ultrafiltration tube (Sigma-Aldrich) and washed three times. The prepared Car@hNVs were stored at 4°C until further use.

[0069] 3) Car@hNVs were dialyzed against PBS at pH 7.4 and pH 6.5, respectively, to evaluate the pH-responsive release of Car from Car@hNVs. The dialysis temperature was 37°C and the oscillation frequency was 150 rpm. The amount of Car released after 48 hours was determined by HPLC.

[0070] 2. Experimental results Drug-loaded hybrid nanovesicles (Car@hNVs) also have the pH-sensitive release characteristics of acid-sensitive liposomes, releasing about 69% of Car at pH 6.5 and 36% of Car at pH 7.4. Figure 4 ).

[0071] Example 4 Drug-loaded hybrid nanovesicles inhibit chronic stress-induced tumor cell proliferation and migration 1. Experimental procedures 1) The collection method of Pro@hNVs was the same as that of Example 1.

[0072] 2) Panc02 cells were incubated with PBS, LIPs, hNVs, Pro, Pro@LIPs, and Pro@hNVs (liposome concentration of 25 μg / mL and Pro concentration of 5 μM) in the presence of 5 μM isoproterenol (ISO) for 24 h. The cell viability was determined using a CCK-8 kit (Shanghai Yessen Biotech Co., Ltd.).

[0073] 3) After the Panc02 cells were pretreated with the above preparations for 24 h, the cells were collected, and 1.2 x 10 5 Panc02 cells were seeded into the upper chamber of a transwell insert with DMEM medium, and 10% FBS was added to the lower chamber. After 12 h, the insert was fixed with 4% paraformaldehyde (PFA) and stained with 5% crystal violet. The migrated cells were observed using a Ti-2 microscope, and the number of migrated cells was quantified using ImageJ.

[0074] 2. Experimental results Under the condition of pH 7.4, the drug-loaded hybrid nanovesicles had little effect on ISO-stimulated Panc02 cells; under the condition of pH 6.5, the drug-loaded hybrid nanovesicles significantly reduced the proliferation activity (content a) and migration ability (content b) of ISO-stimulated Panc02 cells. Figure 5 Figure 5

[0075] Example 5 Drug-loaded hybrid nanovesicles inhibit the promotion of tumor cells to nerve function under chronic stress 1. Experimental procedures 1) The collection method of Pro@hNVs was the same as that of Example 1.

[0076] ​​2) In the presence of 5 µM isoproterenol (ISO), Panc02 cells were treated with equal volumes of PBS, LIPs, hNVs, Pro, Pro@LIPs, and Pro@hNVs (liposome concentration was 25 µg / mL, Pro concentration was 5 µM), respectively, as control groups 1-5 and experimental groups, respectively. After 24 h, the culture medium was replaced with 1640 basal medium. After incubation with the cells for 24 h, the treated Panc02 cell-conditioned medium was collected and filtered with a 0.22 µm syringe filter to remove debris for subsequent experiments.

[0077] 3) In the neuronal cell proliferation assay, PC12 neurons were co-incubated with the above-collected Panc02 cell conditioned medium in a 96-well plate for 48 h, and the viability of PC12 neurons was detected using a CCK8 kit.

[0078] 4) In the neuronal axon growth experiment, PC12 neurons were co-incubated with the above-collected Panc02 cell-conditioned medium in a 12-well plate for 72 h. Neuronal growth was observed using a Ti-2 inverted fluorescence microscope and quantified using the Simple Neuron Tracer (SNT) plug-in of ImageJ.

[0079] 2. Experimental results ISO pre-treated Panc02 tumor cell culture medium significantly promoted nerve proliferation and axonal elongation. At pH 7.4, the conditioned medium of Panc02 cells pre-treated with drug-loaded hybrid nanovesicles could not inhibit this effect. At pH 6.5, drug-loaded hybrid nanovesicles significantly reduced the viability of PC12 nerve cells ( Figure 6 Content a) and axon length ( Figure 6 Content b).

[0080] Example 6 Drug-loaded hybrid nanovesicles reverse polarization of macrophages to M1 phenotype 1. Experimental steps 1) The collection method of Pro@hNVs is the same as that in Example 1.

[0081] 2) In the presence of 5 µM isoproterenol (ISO), Raw264.7 macrophages were treated with equal volumes of PBS, LIPs, hNVs, Pro, Pro@LIPs, and Pro@hNVs (liposome concentration was 25 µg / mL and Pro concentration was 5 µM), respectively, as control groups 1 to 5 and experimental groups.

[0082] 3) After 12 h of treatment, RNA was extracted from the treated macrophages, and the expression of the M1 phenotype-related gene Tnf was analyzed by real-time RT-PCR.

[0083] 4) After 24 h of treatment, cells were collected and stained with CD80 antibody. CD80 was analyzed by flow cytometry. + The proportion of macrophages.

[0084] 5) After 24 h of treatment, the cells were cultured in fresh RPMI 1640 medium for another 24 h. The supernatant was collected and filtered through a 0.22 µm syringe filter to remove debris. The content of the proinflammatory cytokine TNF was analyzed using an ELISA kit (Shenzhen Dakoway Biotechnology Co., Ltd.).

[0085] 2. Experimental results Macrophages were induced to M2 phenotype in ISO environment, but after being treated with drug-loaded hybrid nanovesicles, the expression of M1 phenotype-related gene Tnf was upregulated ( Figure 7 Content a), add CD80 + The proportion of macrophages ( Figure 7 Content b) and increase the content of pro-inflammatory factor TNF ( Figure 7 Content c) showed that drug-loaded hybrid nanovesicles could reverse the polarization of chronic stress-induced M2 macrophages to M1 phenotype.

[0086] Example 7 Drug-loaded hybrid nanovesicles reverse polarize macrophages to M1 and then kill neural cells 1. Experimental steps 1) The collection method of Pro@hNVs is the same as that in Example 1.

[0087] 2) In the presence of 5 µM isoproterenol (ISO), Raw264.7 macrophages were treated with equal volumes of PBS, LIPs, hNVs, Pro, Pro@LIPs, Pro@hNVs, PBS+Etan (TNF inhibitor), and Pro@hNVs+Etan (liposome concentration: 25 µg / mL, Pro concentration: 5 µM, and Etan concentration: 0.5 μg / mL), respectively. These cells were designated as control groups 1-5, experimental groups, and control groups 6-7, respectively. After 24 h, the culture medium was replaced with fresh RPMI 1640 medium. The cells were cultured for 24 h, and the conditioned medium was collected and filtered with a 0.22 µm syringe filter to remove debris for subsequent experiments.

[0088] 3) PC12 neurons were co-incubated with the filtered macrophage-conditioned medium in a 96-well plate for 48 h, and the viability of PC12 neurons was detected using a CCK8 kit.

[0089] 4) PC12 neurons were co-incubated with the filtered macrophage-conditioned medium in a 12-well plate for 72 h. Neuronal growth was observed using a Ti-2 inverted fluorescence microscope and quantified using the Simple Neuron Tracer (SNT) plugin in ImageJ.

[0090] 2. Experimental results Macrophages reversely polarized by drug-loaded hybrid nanovesicles can significantly kill PC12 neurons and reduce the proliferation of neurons ( Figure 8 Content a) and weakening of axon extension ( Figure 8 Content b).

[0091] Example 8 Drug-loaded hybrid nanovesicles effectively degrade tumor extracellular matrix 1. Experimental steps 1) The collection method of Pro@hNVs is the same as that in Example 1.

[0092] 2) Proteins were extracted using RIPA lysis buffer containing protease inhibitors and denatured at 95°C for 5 min. Western blotting was used to determine the expression of matrix metalloproteinase-9 (MMP9) and matrix metalloproteinase-14 (MMP14) in M1 macrophages, M1-NVs, hNVs, and Pro@hNVs (control groups 1–3 and experimental groups, respectively).

[0093] 3) The MMP activity assay kit (AAT Bioquest, Pleasanton, USA) was used to analyze the MMPs enzyme activity of M1-NVs, LIPs, hNVs, Pro@LIPs, and Pro@hNVs (control groups 1 to 4 and experimental groups, respectively) to evaluate their ability to degrade collagen in vitro.

[0094] 4) Construct a stress model in mice bearing orthotopic pancreatic cancer. 6 Panc02 pancreatic tumor cells were orthotopically injected into the pancreas of C57BL6 / J male mice to establish an orthotopic pancreatic cancer tumor model. The mice were placed in a 50 mL centrifuge tube every other day and subjected to restraint stress for 6 hours to receive chronic pressure stimulation. Pressure stimulation continued until the end of treatment.

[0095] 5) Seven days after Panc02 cell inoculation, the tumor-bearing mice from step 4) were randomly divided into six groups, with three mice in each group, and injected intravenously with equal volumes of PBS, LIPs, hNVs, Pro, Pro@LIPs, and Pro@hNVs (liposome concentration of 100 mg / kg, Pro concentration of 7 mg / kg), respectively. These groups were designated as control groups 1 to 5 and experimental groups, respectively. After treatment, the mice were killed, and tumor tissues were collected, fixed with 4% PFA, and cryosectioned. Immunofluorescence was used to detect the type I collagen content in the tumors to assess the ability of the mice to degrade the matrix in vivo.

[0096] 2. Experimental results Drug-loaded hybrid nanovesicles highly expressed MMP9 and MMP14 ( Figure 9Content a), and has higher MMP enzyme activity ( Figure 9 Content b) shows that drug-loaded hybrid nanovesicles have a strong ability to degrade extracellular matrix. In vivo experiments show that drug-loaded hybrid nanovesicles can significantly reduce the content of type I collagen in tumor tissue ( Figure 9 Content c) further demonstrated its ability to effectively degrade tumor extracellular matrix.

[0097] Example 9 Drug-loaded hybrid nanovesicles significantly target tumor tissues 1. Experimental steps 1) The collection method of Pro@hNVs is the same as that in Example 1.

[0098] 2) Constructing an orthotopic pancreatic cancer-bearing mouse stress model in the same manner as in step (4) of Example 8.

[0099] Four weeks after inoculation with Panc02 tumor cells, tumor-bearing mice were randomly divided into two groups of three. LIPs (control group) and hNVs (experimental group) labeled with IR780 (1 mg / kg) were injected intravenously for fluorescence imaging analysis. IR780 fluorescence was acquired using the Caliper IVIS Lumina II in vivo imaging system at various time points after injection. Mice were sacrificed 72 hours later, and major organs and tumors were harvested for IR780 fluorescence imaging.

[0100] 2. Experimental results In vivo imaging results showed that the fluorescence intensity of drug-loaded hybrid nanovesicles gradually increased with the increase of treatment time, which was significantly higher than that of the control group ( Figure 10 Content a and Figure 10 Content b) indicates that drug-loaded hybrid nanovesicles can exert stronger tumor targeting ability. Liver and lung tissues are rich in reticuloendothelial system and have a natural high accumulation tendency for drug-loaded nanovesicles, but in vitro imaging results show ( Figure 10 Content c and Figure 10 Content d), compared with the control group, the fluorescence accumulation of drug-loaded hybrid nanovesicles in tumor tissue was significantly enhanced, indicating that they can effectively evade recognition by the mononuclear phagocytic system and actively target and enrich tumor tissue.

[0101] Example 10 Drug-loaded hybrid nanovesicles can achieve deep drug penetration 1. Experimental steps 1) The collection method of Pro@hNVs is the same as that in Example 1.

[0102] 2) Constructing an orthotopic pancreatic cancer-bearing mouse stress model in the same manner as in step (4) of Example 8.

[0103] Four weeks after inoculation with Panc02 tumor cells, tumor-bearing mice were randomly divided into two groups of three and intravenously injected with IR780 (1 mg / kg) labeled LIPs (control group) or hNVs (experimental group) for fluorescence imaging analysis. Mice were sacrificed 72 hours later, and tumor tissues were collected, fixed with 4% PFA, embedded in paraffin, and sectioned. The tissues were labeled with a Cy3-conjugated CD31 antibody, and the vascular distribution of IR780 was observed using an FV3000 confocal microscope to assess its penetrating ability.

[0104] 2. Experimental results The experiment found that compared with the control group, the fluorescence intensity in the blood vessels treated with hybrid nanovesicles was the highest, indicating that the content of the hybrid nanovesicles that leaked out of the blood vessels was higher, indicating that the hybrid nanovesicles had a stronger ability to penetrate deep into tumors ( Figure 11 Content a and Figure 11 Content b).

[0105] Example 11 Drug-loaded hybrid nanovesicles inhibit pancreatic cancer tumor growth and metastasis 1. Experimental steps 1) The collection method of Pro@hNVs is the same as that in Example 1.

[0106] 2) A stress model of mice bearing orthotopic pancreatic cancer was constructed according to step (4) of Example 8. The tumor-bearing mice were randomly divided into 6 groups, with 6 mice in each group. They were intravenously injected with equal volumes of PBS, LIPs, hNVs, Pro, Pro@LIPs, and Pro@hNVs (liposome concentration was 100 mg / kg and Pro concentration was 7 mg / kg), respectively. The mice were divided into control groups 1 to 5 and experimental groups, respectively, and the injections were performed once every other day for a total of 7 injections. During the treatment period, the mice were continuously in a state of chronic stress. After the treatment, the mice were sacrificed, the tumors were collected for weighing and analysis, and the number of tumor metastasis nodules in the Panc02 model was counted.

[0107] 2. Experimental results Pancreatic cancer tumor growth was significantly inhibited after treatment with drug-loaded hybrid nanovesicles ( Figure 12 ) and the number of tumor metastatic nodules in the tumor decreased significantly ( Figure 13 ), and even better than the simple sum of the inhibitory effects of control group 3 (hNVs) and control group 4 (Pro), confirming the synergistic effect of the three components in the drug-loaded hybrid nanovesicles (Pro@hNVs).

[0108] Example 12 Drug-loaded hybrid nanovesicles improve the tumor immune microenvironment of pancreatic cancer 1. Experimental steps The tumor tissues of each treatment group collected in step (2) of Example 11 were ground and filtered to obtain a single cell suspension of tumor tissue. Antibodies were used to stain the cells, and flow cytometry was used to analyze the proportions of different immune cells.

[0109] 2. Experimental results Compared with other control groups, the content of M1 macrophages in pancreatic tumor tissue increased significantly after treatment with drug-loaded hybrid nanovesicles ( Figure 14 Content a), the content of M2 macrophages decreased significantly ( Figure 14 Content b), CD8 is closely related to anti-tumor immunity + T cell content increased significantly ( Figure 14 Content c) indicates that drug-loaded hybrid nanovesicles can effectively improve the tumor immune microenvironment in vivo.

[0110] Example 13 Drug-loaded hybrid nanovesicles improve the neural microenvironment of pancreatic cancer tumors 1. Experimental steps The tumor tissue collected in step (2) of Example 11 was subjected to immunofluorescence analysis to evaluate the intratumoral TH + The sympathetic nerve area was measured, and the tumor tissue was homogenized and the norepinephrine (NE) content in the tumor tissue was analyzed using an ELISA kit.

[0111] 2. Experimental results Compared with the control group, the sympathetic nerve area in pancreatic cancer tumor tissue was significantly decreased after treatment with drug-loaded hybrid nanovesicles ( Figure 15 Content a), the content of norepinephrine, which is closely related to sympathetic nerve activity, was also significantly downregulated ( Figure 15 Content b), which shows that drug-loaded hybrid nanovesicles can inhibit sympathetic nerve growth and axon growth in pancreatic cancer tumors, and effectively improve the neural microenvironment of pancreatic cancer.

[0112] Example 14 Safety of Drug-Loaded Hybrid Nanovesicles 1. Experimental steps The treatment was carried out according to step (2) of Example 11. The body weight of the mice was measured daily during the treatment. After the treatment, blood was collected from the mice's orbits, and the serum was collected to analyze the levels of alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase.

[0113] 2. Experimental results The drug-loaded hybrid nanovesicles treatment had no significant effect on the body weight of mice, indicating that the drug-loaded hybrid nanovesicles have good safety ( Figure 16 Content a). In addition, after treatment with drug-loaded hybrid nanovesicles, the serum levels of alanine aminotransferase ( Figure 16 Content b), aspartate aminotransferase ( Figure 16 Content c), lactate dehydrogenase ( Figure 16 The content of d) did not change significantly, further indicating that the drug-loaded hybrid nanovesicles have good safety.

[0114] Example 15 Drug-loaded hybrid nanovesicles (Pro@hNVs) improve the chemotherapy effect of pancreatic cancer 1. Experimental steps 1) The collection method of Pro@hNVs is the same as that in Example 1.

[0115] 2) According to step (4) of Example 8, a stress model of mice bearing orthotopic pancreatic cancer was constructed. The mice were randomly divided into 4 groups, each with 6 mice. The mice were intravenously injected with equal volumes of PBS, gemcitabine, Pro@hNVs, and gemcitabine + Pro@hNVs (control groups 1 to 3 and experimental groups, respectively). The dose of Pro in the treatment group was 7 mg / kg, injected once every other day, for a total of 7 injections; the dose of gemcitabine in the treatment group was 20 mg / kg, injected once every two days, for a total of 5 injections. During the treatment period, the mice were continuously in a state of chronic stress. After the treatment, the mice were sacrificed, the tumors were collected for weighing and analysis, and the number of tumor metastatic nodules in the Panc02 model was counted.

[0116] 2. Experimental results The drug-loaded hybrid vesicles combined with the chemotherapy drug gemcitabine exerted the strongest anti-tumor effect, with the smallest tumor weight and the least number of tumor metastases ( Figure 17 Content a and Figure 17 Content b), its tumor inhibition effect was significantly better than gemcitabine and Pro@hNVs, confirming the synergistic effect between the drugs.

[0117] Example 16 Drug-loaded hybrid nanovesicles combined with chemotherapy to improve tumor immune microenvironment 1. Experimental steps The tumor collected in step (2) of Example 15 was ground and filtered to obtain a single cell suspension of tumor tissue, and the cells were stained with antibodies, and the proportions of different immune cells were analyzed using flow cytometry.

[0118] 2. Experimental results After the drug-loaded hybrid nanovesicles were combined with the chemotherapy drug gemcitabine to treat tumors, the content of M1 macrophages in pancreatic tumor tissues increased significantly ( Figure 18 Content a), the content of M2 macrophages decreased significantly ( Figure 18 Content b), CD8 related to anti-tumor immunity + T cells ( Figure 18 The content of content c) also increased significantly, indicating that the drug-loaded hybrid nanovesicles can significantly improve the tumor immune microenvironment in combination with chemotherapy drugs.

[0119] Example 17 Drug-loaded hybrid nanovesicles inhibit chemotherapy-induced neurogenesis 1. Experimental steps The tumor tissue collected in step (2) of Example 15 was subjected to immunofluorescence analysis to evaluate the intratumoral TH + The sympathetic nerve area was measured, and the tumor tissue was homogenized and the norepinephrine (NE) content in the tumor tissue was analyzed using an ELISA kit.

[0120] 2. Experimental results The chemotherapy drug gemcitabine can promote the sympathetic nerve area and NE secretion in the tumor, while the drug-loaded hybrid nanovesicles can inhibit chemotherapy-induced neurogenesis. When combined with chemotherapy drugs, it can significantly improve the tumor neural microenvironment ( Figure 19 Content a and Figure 19 Content b) shows that drug-loaded hybrid nanovesicles combined with chemotherapy can effectively improve the therapeutic effect of the chemotherapy drug gemcitabine, providing a new treatment option for tumor treatment.

[0121] Example 18 Drug-loaded hybrid nanovesicles (Car@hNVs) improve the chemotherapy effect of breast cancer 1. Experimental steps 1) The collection method of Car@hNVs is the same as that in Example 3.

[0122] 2) Construct a stress model of mice bearing orthotopic breast cancer by placing 5×10 5 4T1 tumor cells were orthotopically injected into the mammary glands of BALB / C female mice to establish an orthotopic breast cancer model. The mice were placed in a 50 mL centrifuge tube every other day and subjected to restraint stress for 6 hours to receive chronic pressure stimulation. The pressure stimulation lasted until the end of treatment.

[0123] Seven days after inoculation with 4T1 tumor cells, tumor-bearing mice were randomly divided into four groups of six each and received equal volumes of PBS, doxorubicin, Car@hNVs, or doxorubicin plus Car@hNVs (control groups 1–3 and experimental groups, respectively). The Car@hNVs dose in the treatment group was 0.1 mg / kg, administered every other day for seven injections; the doxorubicin dose in the treatment group was 4 mg / kg, administered every two days for six injections. During treatment, the mice were maintained in a state of chronic stress. Following treatment, the mice were sacrificed, and tumors were harvested for weight and analysis. Changes in nerve density in the 4T1 model were also analyzed.

[0124] 2. Experimental results The drug-loaded hybrid nanovesicles (Car@hNVs) also exerted the strongest anti-tumor effect when combined with the chemotherapy drug doxorubicin, with the smallest tumor weight ( Figure 20Content a) confirmed the synergistic effect between drugs. The chemotherapy drug doxorubicin alone can promote the area of ​​sympathetic nerves in tumors, while the drug-loaded hybrid nanovesicles combined with chemotherapy drugs can inhibit chemotherapy-induced neurogenesis ( Figure 20 Content b) significantly improved the tumor neural microenvironment, indicating that drug-loaded hybrid nanovesicles combined with chemotherapy can effectively improve the therapeutic effect of chemotherapy drugs, providing an innovative idea for tumor treatment.

[0125] Example 19 Drug-loaded hybrid nanovesicles (Aro@hNVs) improve the chemotherapy effect of breast cancer 1. Experimental steps 1) The method for collecting M1 macrophage-derived vesicles is the same as in Example 1.

[0126] DSPC, DOPE, DOTAP, and DSPE-mPEG2000 were dissolved in a chloroform / ethanol mixture (2:1 by volume) at a molar ratio of 5:3:2:1. The lipid solution was then slowly injected into PBS preheated to 37°C containing M1-NVs using a 1 mL syringe. The mass ratio of M1-NVs to phospholipids was 1:5. Stirring was maintained during the injection process and continued for 30 minutes. The mixture was then extruded 11 times through a 0.2 μm polycarbonate membrane filter using a microextruder to obtain uniformly dispersed hybrid nanovesicles (hNVs). Arotinolol (Aro) was added during the injection process of the liposome preparation to obtain drug-loaded hybrid nanovesicles (Aro@hNVs). The mass ratio of Aro to phospholipids was 1:5. The mixture was then ultrafiltered using a 100 kDa ultrafiltration tube (Sigma-Aldrich) and washed three times. The prepared Aro@hNVs were stored at 4°C until further use.

[0127] 3) Constructing an orthotopic pancreatic cancer mouse stress model, 2.5×10 5 KPC pancreatic tumor cells were orthotopically injected into the pancreas of C57BL6 / J male mice to establish an orthotopic pancreatic cancer model. Mice were randomly subjected to any two of the following stressors daily (including: 4°C cold water swimming for 3 min, tail pinching for 5 min, food deprivation for 12 h, water deprivation for 12 h, cage tilt of 30° for 12 h, wet bedding for 3-4 h, removal of all bedding for 3-4 h, regular room lighting at night, physical restraint in a 50 mL centrifuge tube for 2 h, and bright light exposure for 30 min) until the end of treatment.

[0128] Tumor-bearing mice were randomly divided into four groups of four and received intravenous injections of equal volumes of PBS, gemcitabine, Aro@hNVs, or gemcitabine + Aro@hNVs (control groups 1–3 and experimental groups, respectively). The Aro@hNVs dose in the treatment group was 10 mg / kg, administered every other day for seven injections; the gemcitabine dose in the treatment group was 20 mg / kg, administered every two days for five injections. During treatment, the mice were continuously placed in a state of chronic stress. After treatment, the mice were sacrificed, and tumors were harvested for weight and analysis. The number of metastatic nodules in the KPC model was also counted.

[0129] 2. Experimental results The drug-loaded hybrid nanovesicles (Aro@hNVs) combined with the chemotherapy drug gemcitabine also exerted the strongest anti-tumor effect with the smallest tumor weight ( Figure 21 Content a) and the number of metastatic nodules is minimal ( Figure 21 Content b) further illustrates that the drug-loaded hybrid nanovesicles can effectively block sympathetic nerve-tumor crosstalk and enhance the chemotherapy effect of pancreatic cancer.

[0130] It is easy for those skilled in the art to understand that the above is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the scope of protection of the present application.

Claims

1. A drug-loaded hybrid nanovesicle that blocks crosstalk between sympathetic nerves and tumors, characterized in that: The invention comprises hybrid nanovesicles formed by hydrating M1 macrophage-derived nanovesicles and acid-sensitive liposomes, and drugs encapsulated in the hybrid nanovesicles that can inhibit crosstalk between sympathetic nerves and tumors.

2. The drug-loaded hybrid nanovesicle according to claim 1, characterized in that The acid-sensitive liposome is assembled from backbone lipids, acid-sensitive lipids and auxiliary lipids, and its liposome membrane structure changes under the acidic tumor microenvironment; The backbone lipids include one or more of natural phospholipids, semi-synthetic phospholipids, fully synthetic phospholipids and their derivatives; the acid-sensitive lipids include one or more of acid-sensitive lipids containing protonated groups and acid-sensitive lipids containing acid-sensitive chemical bonds; the auxiliary lipids include one or more of polyethylene glycol-modified lipids and cationic lipids.

3. The drug-loaded hybrid nanovesicle according to claim 1, characterized in that The drug capable of inhibiting crosstalk between sympathetic nerves and tumors is a beta-adrenergic receptor blocker; The beta-adrenergic receptor blocker is selected from one or more of propranolol, metoprolol, bisoprolol, atenolol, arotinolol, labetalol and carvedilol.

4. The drug-loaded hybrid nanovesicle according to claim 1, characterized in that The M1 macrophages are obtained by subjecting any one of human peripheral blood mononuclear cells, human mononuclear cell line, mouse bone marrow-derived macrophages or mouse mononuclear macrophage cell line to M1 polarization treatment.

5. The drug-loaded hybrid nanovesicle according to any one of claims 1 to 4, characterized in that: The mass ratio of the M1 macrophage-derived nanovesicles, the drug, and the acid-sensitive liposomes is 1:(0.5-20):(1-50).

6. A method for preparing drug-loaded hybrid nanovesicles according to any one of claims 1 to 5, characterized in that: The steps include: S1, assembling backbone lipids, acid-sensitive lipids and auxiliary lipids to form acid-sensitive liposomes; S2. Prepare M1 macrophage-derived vesicles by physical or chemical methods; S3. Hydrate the drug capable of inhibiting the crosstalk between sympathetic nerves and tumors, the M1 macrophage-derived vesicles, and the acid-sensitive liposomes, and then extrude and fuse them to obtain the drug-loaded hybrid nanovesicles.

7. The preparation method according to claim 6, characterized in that In step S1, the method for assembling and forming acid-sensitive liposomes includes any one of a thin film hydration method, a microfluidics method, a reverse phase evaporation method, a solvent injection method, an ultrasonic dispersion method, or a freeze-thaw-extrusion method; The molar ratio of the backbone lipid, the acid-sensitive lipid and the auxiliary lipid is (5-8):(2-5):(0.2-3).

8. The preparation method according to claim 6, characterized in that The specific operation of step S3 is: placing the acid-sensitive liposomes in a buffer solution containing the M1 macrophage-derived vesicles to form acid-sensitive lipid vesicles, and then fusing the acid-sensitive lipid vesicles and the M1 macrophage-derived vesicles through multiple extrusions to form hybrid nanovesicles, while encapsulating the drug in the hybrid nanovesicles.

9. An anti-tumor drug, characterized in that: It comprises the drug-loaded hybrid nanovesicles according to any one of claims 1 to 5.

10. The antitumor drug according to claim 9, characterized in that Also included are chemotherapy drugs that can promote the expression of β-adrenergic receptors in tumor cells; Preferably, the chemotherapy drug is selected from one or more of gemcitabine or doxorubicin chemotherapy drugs; The doxorubicin chemotherapy drug is selected from one or more of doxorubicin, epirubicin, daunorubicin, epirubicin and pirarubicin.

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