Drug-loaded bacterial outer membrane vesicles for targeted intervention of tumor neural microenvironment and preparation method thereof

Abstract

The present invention belongs to the technical field of preparation of anti-tumor drugs, and more specifically, relates to a drug-loaded bacterial outer membrane vesicle that targets and intervenes in the tumor neural microenvironment and a preparation method thereof. The drug-loaded bacterial outer membrane vesicle includes a bacterial outer membrane vesicle, and a small molecule drug with a neuroinhibitory effect wrapped by the bacterial outer membrane vesicle, and the surface of the bacterial outer membrane vesicle is also modified with a nerve cell targeting molecule. The drug-loaded bacterial outer membrane vesicle provided by the present invention can effectively target nerves in the tumor microenvironment, while effectively blocking the interaction between nerves and tumors, inhibiting nerve infiltration and nerve axon growth in the tumor microenvironment, and thus inhibiting tumor growth. The preparation method of the present invention is simple and highly operable, and the prepared drug-loaded bacterial outer membrane vesicle has good biocompatibility and good clinical application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-tumor drug preparation, and more specifically, relates to a drug-loaded bacterial outer membrane vesicle for targeted intervention of the tumor neural microenvironment and a preparation method thereof. Background Art

[0002] Among the components of the tumor microenvironment (TME), intratumoral neural infiltration plays an important role in promoting tumor initiation and progression and is associated with poor prognosis. Similar to the critical role of the nervous system in tissues, intratumoral nerves promote the development and metastasis of solid tumors (such as pancreatic cancer, gastric cancer, breast cancer, prostate cancer, and glioma) and hematological tumors through paracrine signals such as neurotransmitters (such as glutamate, serotonin, and norepinephrine), direct electrochemical signals, and systemic nerve-tumor interactions. Because the crosstalk between nerves and tumors is a mutually beneficial process, nerves are also affected by tumor cells. Numerous studies have shown that tumor cell-derived neurotrophic factors, such as nerve growth factor (NGF), are the main drivers of tumor tissue recruitment of nerves, promotion of neural infiltration, and axonal growth. Among them, NGF appears to be the most critical regulator of tumor-induced neural infiltration. In addition to tumor cells, immune cells in the TME may also have an impact on intratumoral nerves, especially macrophages. This is because tumor cells can easily invade nerves, causing perineural invasion (PNI), which damages the nerves and then recruits M2 macrophages with tissue repair capabilities to promote nerve repair and growth.

[0003] Currently, interventions for tumor innervation include gene blockade (retrograde gene delivery to specific neurons using adeno-associated viral vectors), chemical blockade (using 6-hydroxydopamine, botulinum toxin, or capsaicin), and surgical denervation. In animal studies, denervation has shown significant inhibitory effects on tumor growth and metastasis. Furthermore, treatment with β-adrenergic blockers can reduce the progression of breast cancer, prostate cancer, and melanoma, and bupivacaine-loaded liposomes can significantly inhibit breast cancer growth and metastasis. These studies suggest that regulating nerves in the tumor microenvironment may provide new avenues for tumor treatment.

[0004] Bacterial outer membrane vesicles (OMVs) are naturally occurring spherical vesicles, primarily produced by Gram-negative bacteria through budding, with diameters ranging from 30 to 250 nm. OMVs contain bacterial antigens and a variety of pathogen-associated molecular patterns, such as lipopolysaccharide, flagellin, and peptidoglycan, and are promising immunoadjuvants. Furthermore, due to their ease of preparation, good biocompatibility, excellent stability, and unique tumor-targeting capabilities, OMVs have been widely used in anti-tumor drug delivery research.

[0005] Therefore, research and development of nano-drug delivery systems targeting tumor tissue nerves to block nerve-tumor crosstalk to inhibit tumor growth or synergistically enhance existing tumor therapies has important clinical application value. Summary of the Invention

[0006] In response to the deficiencies of the prior art, the object of the present invention is to provide a drug-loaded bacterial outer membrane vesicle for targeted intervention of the tumor neural microenvironment and a preparation method thereof, wherein bacterial outer membrane vesicles are used as drug carriers, and small molecule drugs with neuroinhibitory effects are encapsulated and the surface of the bacterial outer membrane vesicles is also modified with nerve cell targeting molecules. The corresponding drug-loaded bacterial outer membrane vesicles can effectively target nerves in the tumor microenvironment, which is beneficial for targeted intervention of tumors with high nerve density. At the same time, the drug-loaded bacterial outer membrane vesicles can effectively block the interaction between nerves and tumors, block nerve-tumor crosstalk, inhibit nerve infiltration and nerve axon growth in the tumor microenvironment, and thus inhibit tumor growth. In addition, the drug-loaded bacterial outer membrane vesicles can also effectively reverse polarize M2 tumor-associated macrophages, improve the tumor neural microenvironment, inhibit the growth of nerve cells and nerve axon generation, cause nerve damage, and affect nerve function. The three components in the drug-loaded bacterial outer membrane vesicles promote each other synergistically to inhibit tumor growth and improve the therapeutic efficacy. The preparation method of the present invention is simple and highly operable. The prepared drug-loaded bacterial outer membrane vesicles have good biocompatibility and have good clinical application prospects.

[0007] To achieve the above objectives, the present invention provides a drug-loaded bacterial outer membrane vesicle for targeted intervention of the tumor neural microenvironment, comprising bacterial outer membrane vesicles and small molecule drugs encapsulated by the bacterial outer membrane vesicles, wherein the small molecule drugs are small molecule drugs with neuroinhibitory effects; the surface of the bacterial outer membrane vesicles is also modified with nerve cell targeting molecules.

[0008] Preferably, the bacterial outer membrane vesicles include bacterial outer membrane vesicles derived from Escherichia coli, bacterial outer membrane vesicles derived from Akkermansia muciniphila or bacterial outer membrane vesicles derived from attenuated Salmonella.

[0009] Preferably, the mass ratio of the bacterial outer membrane vesicles, the nerve cell targeting molecule, and the drug is (1-20):1:(1-200).

[0010] Preferably, the small molecule drug is a Trk inhibitor, an anesthetic or other small molecule drug with neurosuppressive effects.

[0011] Preferably, the Trk inhibitor is one or more of larotrectinib, entrectinib, AZ23 and LOXO-195; the anesthetic is one or more of procaine, tetracaine and lidocaine; and the other small molecule drugs with neuroinhibitory effects are one or more of the neurotoxin botulinum toxin and 6-hydroxydopamine.

[0012] Preferably, the nerve cell targeting molecule comprises a nerve cell targeting peptide and a conjugate, the nerve cell targeting peptide is conjugated to the surface of the conjugate, the conjugate can be connected to the surface of the bacterial outer membrane vesicle by lipid intercalation, and the nerve cell targeting peptide is selected from one or more of NP41, RVG, HNP401, HNP402 and HNP403.

[0013] Preferably, the particle size of the drug-loaded bacterial outer membrane vesicles is 30 nm to 250 nm.

[0014] The present invention also provides a method for preparing the drug-loaded bacterial outer membrane vesicles, which is characterized by comprising the following steps:

[0015] S1. Incubating bacterial outer membrane vesicles with nerve cell targeting molecules, modifying the nerve cell targeting molecules onto the surface of the bacterial outer membrane vesicles by lipid intercalation, and collecting the bacterial outer membrane vesicles modified with the nerve cell targeting molecules;

[0016] S2. The bacterial outer membrane vesicles modified with the nerve cell targeting molecule are mixed and incubated with a small molecule drug, wherein the small molecule drug is a small molecule drug with a nerve inhibitory effect, and the drug-loaded bacterial outer membrane vesicles that target and intervene in the tumor neural microenvironment are collected.

[0017] Preferably, in step S1, the mass ratio of the bacterial outer membrane vesicles to the nerve cell targeting molecule is (1-20):1.

[0018] Preferably, in steps S1 and S2, the collection conditions are: collection at a centrifugal force of 2000 g to 150000 g at 4°C.

[0019] The present invention also provides a drug for treating cancer, which comprises the drug-loaded bacterial outer membrane vesicles and a chemotherapy drug.

[0020] Preferably, the chemotherapy drug is a chemotherapy drug that can promote tumor cells to highly express nerve growth factor, and the chemotherapy drug that can promote tumor cells to highly express nerve growth factor is selected from one or more of gemcitabine or doxorubicin chemotherapy drugs.

[0021] More preferably, the ammonia mycin chemotherapy drug is selected from one or more of doxorubicin, epirubicin, daunorubicin and pirarubicin.

[0022] In general, the above technical solutions conceived by the present invention have the following beneficial effects compared with the prior art:

[0023] (1) The present invention provides a drug-loaded bacterial outer membrane vesicle for targeted intervention in the tumor neural microenvironment, comprising a bacterial outer membrane vesicle and a small molecule drug encapsulated by the bacterial outer membrane vesicle, wherein the small molecule drug is a small molecule drug with a neurosuppressive effect; the surface of the bacterial outer membrane vesicle is also modified with a nerve cell targeting molecule. The drug-loaded bacterial outer membrane vesicle can effectively target nerves in the tumor microenvironment, block the interaction between nerves and tumors, and inhibit tumor growth while inhibiting neural infiltration and axon growth in the tumor microenvironment.

[0024] (2) The present invention modifies the bacterial outer membrane vesicles with nerve cell targeting molecules and uses them as carriers of the drug-loaded bacterial outer membrane vesicles of the present invention, which can cause small molecule drugs with neuroinhibitory effects to accumulate at the tumor site, effectively target the nerves in the tumor microenvironment, reduce the toxic side effects of small molecule drugs on the body, and effectively reverse polarize M2 tumor-associated macrophages to M1 type, improve the tumor neural microenvironment, and inhibit the growth of nerve cells and the generation of nerve axons. At the same time, the drug-loaded bacterial outer membrane vesicles are also wrapped with small molecule drugs with neuroinhibitory effects, which inhibit tumor nerve infiltration and nerve growth by blocking nerve-tumor crosstalk. The present invention proposes a new strategy for blocking nerve-tumor crosstalk to achieve the inhibition of tumor growth or synergistic enhancement of existing tumor therapies.

[0025] (3) The present invention has proved through experiments that the bacterial outer membrane vesicles in the drug-loaded bacterial outer membrane vesicles can effectively reverse the polarization of M2 tumor-associated macrophages to M1 type, improve the tumor neural microenvironment, and inhibit the growth of nerve cells and the generation of nerve axons. The bacterial outer membrane vesicles modified with nerve cell targeting molecules allow the encapsulated small molecule drugs to interact with nerve cells more, thereby making the effect of blocking nerve-tumor crosstalk more significant. The three promote each other synergistically, so that the drug-loaded bacterial outer membrane vesicles can significantly inhibit the proliferation of nerve cells and the generation of nerve axons of nerve cells, effectively inhibit the function of nerve cells, thereby effectively blocking the interaction between nerves and tumors, and can also significantly slow down the proliferation and migration of tumor cells promoted by nerve cells.

[0026] (4) Compared with the separate administration of OMV, NP-OMV, Lar, and Lar-OMV, the drug-loaded bacterial outer membrane vesicles (Lar@NP-OMV) provided by the present invention showed a synergistic effect in cancer treatment. Lar@NP-OMV can effectively target nerves in the tumor microenvironment, inhibit nerve growth, and then inhibit tumor growth, prolonging survival. Its therapeutic effect is significantly better than other control groups, and even better than the simple sum of the technical effects of single NP-OMV and single Lar.

[0027] (5) The drug-loaded bacterial outer membrane vesicles provided by the present invention can be combined with other chemotherapy drugs to achieve a synergistic anti-tumor effect, while having no obvious toxic side effects on the organism and having good biocompatibility. In a preferred embodiment, when the drug-loaded bacterial outer membrane vesicles provided by the present invention are combined with chemotherapy drugs that can promote high expression of NGF in tumor cells to treat cancer, tumor growth can be significantly inhibited. The synergistic effect between the drugs makes the effect of the combined treatment better than that of a single drug-loaded bacterial outer membrane vesicle or a single chemotherapy drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The results of the detection of drug-loaded bacterial outer membrane vesicles modified with neurobinding peptide NP41 in Example 1 are shown in FIG. Figure 1 A is the vesicle particle size, Figure 1 B is the vesicle zeta potential.

[0029] Figure 2 The results of the test of drug-loaded bacterial outer membrane vesicles modified with neurobinding peptide NP41 in Example 2 are shown in FIG. Figure 2 A is the vesicle particle size, Figure 2 B is the vesicle zeta potential.

[0030] Figure 3 This is the TEM image of the vesicles in Example 3.

[0031] Figure 4 is the uptake result of NP41 modified vesicles in Example 4; wherein Figure 4 A is the result of PC12 neurons taking up DiO-labeled vesicles. Figure 4 B shows the uptake of DiO-labeled vesicles by primary DRG neurons.

[0032] Figure 5 The distribution of vesicles in different tissues of tumor-bearing mice in Example 5; Figure 5 A is the in vivo imaging results of mouse orthotopic pancreatic tumors at different time points after tail vein injection of IR780-labeled OMVs and NP-OMVs. Figure 5 B is the quantitative fluorescence intensity of the tumor site, Figure 5 C is the in vitro fluorescence imaging of pancreatic tumors and major organs. Figure 5D is the quantitative analysis of the fluorescence intensity of pancreatic tumors and major organs. Figure 5 E is the co-localization analysis of NP-OMV and neural marker β3-tubulin, Figure 5 F is the semi-quantitative result of colocalization analysis.

[0033] Figure 6 The effect of the drug-loaded vesicles on the growth of PC12 neural cells in Example 6; Figure 6 A is the proliferation of PC12 neurons. Figure 6 B is the quantification of axon length of PC12 neurons.

[0034] Figure 7 The effect of the drug-loaded vesicles in Example 7 on tumor cells after acting on PC12 neural cells; Figure 7 A. Figure 7 B respectively shows the effects of drug-loaded vesicles on the proliferation and migration of Panc02 after acting on PC12 neural cells.

[0035] Figure 8 The effect of drug-loaded vesicles in Example 8 on reverse polarization of M2 macrophages; wherein Figure 8 A. Figure 8 B respectively shows the expression changes of M1-related surface characteristic molecules CD86 and TNFα after drug-loaded vesicles reverse polarized M2 macrophages, Figure 8 C. Figure 8 D shows the expression changes of M2-related surface characteristic molecules Mgl1 and Mrc1 after drug-loaded vesicles reverse polarized M2 macrophages.

[0036] Figure 9 The effect of drug-loaded vesicles on nerve cells after reverse polarization of M2 macrophages in Example 9; Figure 9 A. Figure 9 B respectively shows the effects of the supernatant after drug-loaded vesicles reverse polarized M2 macrophages on PC12 neuronal cell proliferation and PC12 neuronal cell axon generation.

[0037] Figure 10 The inhibitory effect of the drug-loaded vesicles in Example 10 on Panc02 in situ pancreatic cancer; wherein Figure 10 A is the weight of mouse tumor, Figure 10 B is the survival time of mice.

[0038] Figure 11 The effect of the drug-loaded vesicles in Example 11 on the neural microenvironment of Panc02 in situ pancreatic cancer; wherein Figure 11 A is the ratio of GAP43 positive area in mouse tumor tissue, Figure 11 B is the ratio of β3-tubulin positive area in mouse tumor tissue.

[0039] Figure 12 The safety of the drug-loaded vesicles in Example 12 on Panc02 orthotopic pancreatic cancer-bearing mice; wherein Figure 12 A is the weight of the mouse, Figure 12 B is the alanine aminotransferase content in mouse serum, Figure 12 C is the creatinine content in mouse serum.

[0040] Figure 13 This is the effect of gemcitabine on the NGF expression level of Panc02 cells in Example 13.

[0041] Figure 14 The inhibitory effect of the drug-loaded vesicles combined with gemcitabine on Panc02 in situ pancreatic cancer in Example 14; wherein Figure 14 A is the nerve density in mouse pancreatic tumor tissue, Figure 14 B is the norepinephrine content in mouse pancreatic tumor tissue, Figure 14 C is the weight of pancreatic tumor in mice, Figure 14 D is the survival time of mice.

[0042] Figure 15 The results show the uptake of RVG-modified vesicles by primary DRG neurons.

[0043] Figure 16 The inhibitory effect of the drug-loaded vesicles combined with doxorubicin on 4T1 in situ breast cancer in Example 16; wherein Figure 16 A is the nerve density in mouse breast tumor tissue, Figure 16 B is the weight of mouse mammary tumor. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are merely for the purpose of explaining the present invention and are not intended to limit the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0045] The present invention provides a drug-loaded bacterial outer membrane vesicle for targeted intervention of the tumor neural microenvironment, comprising a bacterial outer membrane vesicle and a small molecule drug encapsulated by the bacterial outer membrane vesicle, wherein the small molecule drug is a small molecule drug with a neuroinhibitory effect; the surface of the bacterial outer membrane vesicle is also modified with a nerve cell targeting molecule.

[0046] In some embodiments, the bacterial outer membrane vesicles can be bacterial outer membrane vesicles secreted by any Gram-negative bacteria. In some embodiments, the bacterial outer membrane vesicles include bacterial outer membrane vesicles derived from Escherichia coli, bacterial outer membrane vesicles derived from Akkermansia muciniphila, or bacterial outer membrane vesicles derived from attenuated Salmonella.

[0047] In some embodiments, the mass ratio of the bacterial outer membrane vesicles, the nerve cell targeting molecule, and the small molecule drug is (1-20):1:(1-200).

[0048] In some embodiments, the mass ratio of the bacterial outer membrane vesicles, the nerve cell targeting molecule, and the small molecule drug is (1-10):1:(1-100).

[0049] The small molecule drug described in the present invention is a small molecule drug with a neuroinhibitory effect, and the small molecule drug with a neuroinhibitory effect here can be a medically recognized small molecule drug with a neuroinhibitory effect. In some embodiments, the above-mentioned small molecule drug is a Trk inhibitor, an anesthetic or other small molecule drug with a neuroinhibitory effect. Among them, the above-mentioned Trk inhibitor can block the neurotrophic effect of NGF, and inhibit nerve infiltration and nerve growth by blocking the NGF-Trk signaling pathway; the above-mentioned anesthetic and other small molecule drugs with a neuroinhibitory effect can directly inhibit nerve growth. However, it is not known whether the neuroinhibitory effect of small molecule drugs is enhanced by combining with bacterial outer membrane vesicles modified with nerve cell targeting molecules. Therefore, the synergistic effect of cancer treatment by combining small molecule drugs with neuroinhibitory effects and bacterial outer membrane vesicles modified with nerve cell targeting molecules may be a technical feature of the present invention.

[0050] In some embodiments, the Trk inhibitor is one or more of larotrectinib, entrectinib, AZ23, and LOXO-195; the anesthetic is one or more of procaine, tetracaine, and lidocaine; and the other small molecule drugs with neuroinhibitory effects are one or more of the neurotoxin botulinum toxin and 6-hydroxydopamine.

[0051] In some embodiments, the neural cell targeting molecule comprises a neural cell targeting peptide and a conjugate, wherein the neural cell targeting peptide is conjugated to the conjugate, and the conjugate can be attached to the surface of bacterial outer membrane vesicles by lipid intercalation. In some embodiments, the conjugate includes but is not limited to DSPE PEG-Mal, NH2-PEG-DSPE, and N3-PEG-DSPE.

[0052] In some embodiments, the nerve cell targeting peptide is selected from one or more of NP41, RVG, HNP401, HNP402 and HNP403.

[0053] In some embodiments of the present invention, the neural cell targeting molecule is selected from one or more of DSPE PEG-NP41, DSPE PEG-RVG, NH2-PEG-RVG29, DSPE PEG-HNP401, DSPE PEG-HNP402 and DSPE PEG-HNP403.

[0054] In some embodiments, the particle size of the drug-loaded bacterial outer membrane vesicles is 30 nm to 250 nm.

[0055] In a preferred embodiment, the particle size of the drug-loaded bacterial outer membrane vesicles is 50 nm to 150 nm.

[0056] The present invention also provides a method for preparing the above-mentioned drug-loaded bacterial outer membrane vesicles, comprising the following steps:

[0057] S1. Incubating bacterial outer membrane vesicles with nerve cell targeting molecules, modifying the nerve cell targeting molecules onto the surface of the bacterial outer membrane vesicles by lipid intercalation, and collecting the bacterial outer membrane vesicles modified with the nerve cell targeting molecules;

[0058] S2. The bacterial outer membrane vesicles modified with the above-mentioned nerve cell targeting molecules are mixed and incubated with small molecule drugs, wherein the above-mentioned small molecule drugs are small molecule drugs with neuroinhibitory effects, and the drug-loaded bacterial outer membrane vesicles that target and intervene in the tumor neural microenvironment are collected.

[0059] As an optional manner, in the above-mentioned method for preparing drug-loaded bacterial outer membrane vesicles, step S1 specifically includes the following steps:

[0060] S1-1. Preparation of bacterial outer membrane vesicles: inoculating and culturing a single bacterial colony, collecting the bacterial solution by centrifugation, filtering and concentrating the bacterial solution, and collecting the bacterial outer membrane vesicles by centrifugation;

[0061] S1-2. Preparing a neuron targeting molecule: dissolving the conjugate in an organic solvent, mixing the neuron targeting peptide and the conjugate, stirring to carry out a coupling reaction, and then dialyzing and freeze-drying to obtain the neuron targeting peptide;

[0062] S1-3. The bacterial outer membrane vesicles are mixed and incubated with the nerve cell targeting molecules, and the nerve cell targeting molecules are modified onto the surface of the bacterial outer membrane vesicles by lipid intercalation, and the bacterial outer membrane vesicles modified with the nerve cell targeting molecules are collected.

[0063] The bacterial outer membrane vesicles of the present invention can be isolated from bacterial cultures according to various methods known in the art. The type of technique for isolating bacterial outer membrane vesicles from bacterial cultures is not particularly limited. For example, ultracentrifugation, density gradient ultracentrifugation, ultrafiltration, size exclusion chromatography, ion exchange chromatography, immunoaffinity capture, microfluidics-based separation, aqueous two-phase systems, or precipitation methods can be used.

[0064] In some embodiments, in step S1-1, the centrifugation condition is: 2000g-150000g for 10 minutes to 2 hours.

[0065] In some embodiments, in step S1-2, the coupling reaction is carried out under stirring at room temperature for 3 h to 5 h.

[0066] In some embodiments, in step S1-3, the mass ratio of the bacterial outer membrane vesicles to the nerve cell targeting molecule is (1-20):1; and the incubation time is 6 hours to 12 hours.

[0067] In some embodiments, in steps S1 and S2, the collection conditions are: collection at a centrifugal force of 2000 g to 150000 g at 4°C.

[0068] The present invention also provides a drug for treating cancer, which comprises the drug-loaded bacterial outer membrane vesicles and a chemotherapy drug.

[0069] In some embodiments, the above-mentioned chemotherapy drugs are chemotherapy drugs that can promote high expression of NGF in tumor cells. In practical applications, the above-mentioned chemotherapy drugs that can promote high expression of NGF in tumor cells are used for cancer treatment. Although they can kill tumor cells, they will also cause tumor cells to highly express NGF. Highly expressed NGF can promote nerve infiltration and neurite growth, which is not conducive to cancer treatment, resulting in the therapeutic effect of using chemotherapy drugs that can promote high expression of NGF in tumor cells alone being not ideal. However, it is not known whether the therapeutic effect of chemotherapy drugs that can promote high expression of NGF in tumor cells is enhanced by combining with outer membrane vesicles. Therefore, the synergistic effect of cancer treatment by combining chemotherapy drugs that can promote high expression of NGF in tumor cells and drug-loaded bacterial outer membrane vesicles may be a technical feature of the present invention.

[0070] In a preferred embodiment, the above-mentioned chemotherapy drugs capable of promoting high expression of NGF in tumor cells are selected from one or more of gemcitabine and doxorubicin chemotherapy drugs.

[0071] In a preferred embodiment, the above-mentioned adriamycin-based chemotherapy drug is selected from one or more of doxorubicin, epirubicin, daunorubicin and pirarubicin.

[0072] In one aspect of the present invention, the drug-loaded bacterial outer membrane vesicles and the chemotherapeutic drugs can be co-administered simultaneously, sequentially, or separately. In the present invention, "simultaneously" means that the two drugs are administered at the same time, while "sequentially" means that one drug is administered within 5 minutes, 10 minutes, or several hours after the administration of the other drug. However, the half-life of the drug administered first in the circulation is provided so that the two drugs are present at the same time at therapeutically effective doses. In addition, the methods of simultaneous, sequential, or separate administration are not limited to one time, and these methods of administration can be repeated or administered in combination.

[0073] In one aspect, the drug-loaded bacterial outer membrane vesicles and the chemotherapy drugs can be included in a drug for treating cancer together with a pharmaceutically acceptable carrier, excipient and / or diluent.

[0074] In another aspect, the drug-loaded bacterial outer membrane vesicles and the chemotherapy drugs can be provided in the form of separate drugs or in the form of a kit.

[0075] The cancer treatment drug of the present invention may comprise only the drug-loaded bacterial outer membrane vesicles and the chemotherapeutic drug, or may be formulated into a suitable form together with a pharmaceutically acceptable carrier and further including an excipient or diluent. Such carriers include all types of solvents, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads, and microsomes.

[0076] The cancer treatment drugs of the present invention can be administered to mammals, including humans, by any method. For example, the cancer treatment drugs can be administered orally or parenterally. Parenteral administration methods may include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardial, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal administration.

[0077] The cancer treating drug of the present invention can be formulated into a preparation for oral administration or parenteral administration according to the administration route described above.

[0078] The total effective dose of the medicine for treating cancer of the present invention can be administered to the patient in a single dose, or according to a fractionated treatment regimen, can be administered to the patient in long-term multiple doses. In the medicine for treating cancer of the present invention, the content of the active ingredient can vary according to the severity of the disease. The effective dose of the medicine for treating cancer depends on various factors, including the patient's age, weight, health status and gender, the severity of the disease, diet and excretion rate, as well as formulation, route of administration and number of treatments. Those skilled in the art can determine the appropriate effective dose of the medicine for treating cancer of the present invention by considering these factors, and therefore the total dose of the medicine for treating cancer is not limited. The medicine for treating cancer of the present invention as described above is not particularly limited to formulation, route of administration and method of administration, as long as the effect of the present invention is shown.

[0079] The present invention provides use of drug-loaded bacterial outer membrane vesicles and chemotherapy drugs in preparing cancer therapeutic agents.

[0080] The present invention provides a method for treating cancer, comprising administering an effective dose of a cancer treating drug to a subject in need thereof, wherein the cancer treating drug comprises bacterial outer membrane vesicles and a chemotherapy drug as active ingredients.

[0081] In one aspect of the present invention, the cancer may be pancreatic cancer, breast cancer, prostate cancer, central nervous system (CNS) tumor or primary CNS lymphoma, but is not limited thereto.

[0082] As used herein, the term "bacterial outer membrane vesicles" refers to vesicles secreted by Gram-negative bacteria.

[0083] The term "drug-loaded bacterial outer membrane vesicles" is the same as "drug-loaded vesicles", which refers to bacterial outer membrane vesicles whose surface is modified with nerve cell targeting molecules and encapsulates small molecule drugs with neuroinhibitory effects.

[0084] The term "effective dose" of the present invention refers to an amount that, when administered to a subject, shows an effect of improving, treating, detecting and diagnosing cancer, or inhibiting or slowing the progression of cancer. The "subject" can be an animal, preferably a mammal, particularly an animal including a human, or a cell, tissue, or organ derived from an animal. The subject can be a patient in need of these effects.

[0085] The above technical solution is described in detail below in conjunction with specific embodiments.

[0086] The main reagents and materials used in the following examples are from the following sources:

[0087] PC12 neural cells were purchased from the Cell Bank of the Chinese Academy of Sciences; primary DRG neural cells were extracted from the vertebrae of C57BL6 / J mice; C57BL6 / J male mice and SPF-grade BALB / C female mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.; Panc02 cells were purchased from Beijing Beina Chuanglian Biotechnology Research Institute; and mouse bone marrow-derived macrophages were purchased from the Shanghai Cell Bank.

[0088] Larotrectinib, entrectinib, and lidocaine were purchased from MedChemExpress; DSPE PEG-Mal was purchased from Shanghai Pengshuo Biotechnology Co., Ltd.; NP41 and RVG were purchased from Hefei Guopi Biotechnology Co., Ltd.; DSPE-PEG-NP41 and DSPE-PEG-RVG were constructed by conventional molecular biology methods; IR780 was purchased from Sigma-Aldrich; the cell membrane green fluorescent dye DiO was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.; IL-4 was purchased from Peprotech Biotechnology Co., Ltd.; TRIzol, reverse transcription kit, and SYBRGreen dye were purchased from TaKaRa; gemcitabine was purchased from Shandong Qilu Pharmaceutical Group Co., Ltd.; and doxorubicin was purchased from Sigma.

[0089] Example 1 Preparation of drug-loaded vesicles by incubating Escherichia coli outer membrane vesicles modified with neurobinding peptide NP41 with small molecule drugs

[0090] 1. Experimental Materials and Reagents

[0091] Escherichia coli Nissle 1917, larotrectinib, DSPE-PEG-NP41.

[0092] 2. Experimental Procedure

[0093] (1) Inoculate a single E. coli colony into LB medium and culture in a 37°C constant temperature shaker until OD 600 When the value is approximately 1.5, the culture can be stopped. At the end of the culture, the bacterial suspension was collected and centrifuged at 2000g for 10 minutes at 4°C. The supernatant was filtered through a 0.22 μm vacuum filter. The filtrate was concentrated and centrifuged at 150,000g for 2 hours. The precipitate was collected to obtain OMVs.

[0094] (2) Weigh 10 mg of NP41 and dissolve it in 0.5 mL of PBS (pH 8.0). Weigh 8.8 mg of DSPE PEG-Mal and dissolve it in 0.5 mL of N,N-dimethylformamide (DMF). Mix the two and add PBS to 12 mL. Stir and react at room temperature for 4 h. Dialyze the solution in PBS using a dialysis bag with a molecular weight cutoff of 3500 Da. After 24 h, lyophilize the liquid in the dialysis bag to obtain the neuronal cell-targeting peptide (DSPE-PEG-NP41).

[0095] (3) DSPE-PEG-NP41 and OMVs were evenly mixed at a mass ratio of 1:5, incubated at 37°C for 12 h, and centrifuged at 150,000 g for 1 h to obtain NP41-modified vesicles.

[0096] (4) The NP41-modified vesicles were dispersed in 0.5 mL of 0.9× PBS, mixed with 50 μg of larotrectinib, incubated at 37°C on a shaker for 4 h, centrifuged at 150,000 g for 1 h, washed with PBS, and centrifuged at 150,000 g for 1 h to obtain NP41-modified drug-loaded vesicles.

[0097] (5) The NP41-modified drug-loaded vesicles were dispersed in PBS, and their particle size and zeta potential were measured using a dynamic light scattering particle size analyzer. The NP41-modified drug-loaded vesicles served as the experimental group, the unmodified drug-loaded vesicles served as control group 1, the NP41-modified unmodified drug-loaded vesicles served as control group 2, and the unmodified drug-loaded vesicles served as control group 3.

[0098] 3. Experimental Results

[0099] like Figure 1 A. Figure 1 As shown in Figure B, the average particle size of the obtained vesicles is about 100-130 nm, and the Zeta potential is about -12 mV, indicating that the particle size and Zeta potential of the vesicles are basically unchanged after being modified with NP41 and loaded with larotrectinib.

[0100] Example 2 Preparation of drug-loaded vesicles by incubating Escherichia coli outer membrane vesicles modified with neurobinding peptide NP41 with entrectinib

[0101] 1. Experimental Materials and Reagents

[0102] Escherichia coli Nissle 1917, DSPE-PEG-NP41, entrectinib.

[0103] 2. Experimental Procedure

[0104] (1) The OMVs collection method was the same as in Example 1; the NP41-modified vesicles collection method was the same as in Example 1.

[0105] (2) The NP41-modified vesicles were dispersed in 0.5 mL of 0.9× PBS, mixed with 50 μg of entrectinib, incubated at 37°C on a shaker for 4 h, centrifuged at 150,000 g for 1 h, washed with PBS, and centrifuged at 150,000 g for 1 h to obtain NP41-modified drug-loaded vesicles.

[0106] (3) The NP41-modified drug-loaded vesicles were dispersed in PBS and placed at 4°C for different periods of time. The particle size and zeta potential were measured using a dynamic light scattering particle size analyzer. The NP41-modified drug-loaded vesicles served as the experimental group, the unmodified drug-loaded vesicles served as control group 1, the unmodified drug-loaded vesicles served as control group 2, and the unmodified drug-loaded vesicles served as control group 3.

[0107] 3. Experimental Results

[0108] The experimental results show that the average particle size of the obtained vesicles is about 130-150nm ( Figure 2 A), Zeta potential is about -10mV ( Figure 2 B), and after being placed at 4°C for a certain period of time, its average particle size and Zeta potential remained basically unchanged, indicating that the vesicles still had good stability after being modified with NP41 and loaded with entrectinib.

[0109] Example 3: Incubation of Attenuated Salmonella Outer Membrane Vesicles Modified with Neurotargeting Peptide NP41 with Small Molecule Drugs to Obtain Drug-Loaded Vesicles

[0110] 1. Experimental Materials and Reagents

[0111] Larotrectinib, DSPE-PEG-NP41, attenuated Salmonella (deficient in guanosine tetraphosphate synthesis).

[0112] 2. Experimental Procedure

[0113] (1) A single attenuated Salmonella colony was inoculated into LB medium and cultured in a 37°C constant temperature shaker until OD 600 When the value is approximately 1.5, the culture can be stopped. At the end of the culture, the bacterial suspension was collected and centrifuged at 2000g for 10 minutes at 4°C. The supernatant was filtered through a 0.22 μm vacuum filter. The filtrate was concentrated and centrifuged at 150,000g for 2 hours. The precipitate was collected to obtain OMVs.

[0114] (2) DSPE-PEG-NP41 and OMVs were evenly mixed at a mass ratio of 1:5, incubated at 37°C for 12 h, and centrifuged at 150,000 g for 1 h to obtain NP41-modified vesicles.

[0115] (3) The NP41-modified vesicles were dispersed in 0.5 mL of 0.9× PBS, mixed with 50 μg of larotrectinib, incubated at 37°C on a shaker for 4 h, centrifuged at 150,000 g for 1 h, washed with PBS, and centrifuged at 150,000 g for 1 h to obtain NP41-modified drug-loaded vesicles.

[0116] (4) The vesicles were fixed with 4% paraformaldehyde. After 10 minutes, the fixed vesicles were dropped onto a copper mesh placed on a sealing film. After standing for 1 hour, the vesicles were stained with 2% phosphotungstic acid solution for 5 minutes. The copper mesh was washed with ultrapure water three times to remove excess dye. The copper mesh was placed on filter paper and dried at room temperature. The morphology of the vesicles was observed using an HT7700 transmission electron microscope. NP41-modified drug-loaded vesicles served as the experimental group, non-NP41-modified drug-loaded vesicles served as control group 1, NP41-modified non-drug-loaded vesicles served as control group 2, and non-NP41-modified drug-loaded vesicles served as control group 3.

[0117] 3. Experimental Results

[0118] TEM results showed that the obtained vesicles had a regular spherical morphology and a particle size of about 50-150 nm ( Figure 3 ), indicating that the particle size of the attenuated Salmonella-derived vesicles did not change substantially after being modified with NP41 and loaded with drugs.

[0119] Example 4 Targeting of NP41-modified vesicles to neural cells in vitro

[0120] 1. Experimental Materials and Reagents

[0121] Escherichia coli Nissle 1917, DSPE-PEG-NP41, PC12 neural cells, and primary DRG neural cells.

[0122] 2. Experimental Procedure

[0123] (1) NP41-modified vesicles were prepared according to Example 1 and labeled with the cell membrane green fluorescent dye DiO.

[0124] (2) DRG primary neurons and PC12 neurons were cultured at 3×10 5 The cells were plated in 6-well plates at a density of 100 cells / well. After culturing for 12 h, the supernatant was discarded and fresh serum-free culture medium was added.

[0125] (3) DiO-labeled OMVs and DiO-labeled NP-OMVs were added to the above-mentioned DRG primary neuron culture plate, and co-cultured at 37°C and 5% CO2 for 6 h. After incubation, the supernatant was discarded, the cells were washed three times with PBS, trypsinized, centrifuged at 1200 rpm for 3 min, and resuspended in 300 μL PBS. The mean fluorescence intensity of DiO in the cells was measured using a CytoFlex S flow cytometer. The detection parameters were set as: Ex = 488 nm, and the detection filter was FITC. OMVs served as the control group, and NP-OMVs served as the experimental group. The method for PC12 neuronal cells to take up DiO-labeled vesicles was the same as that for DRG primary neuronal cells.

[0126] 3. Experimental Results

[0127] Flow cytometry results showed that compared with OMV, the fluorescence intensity of NP-OMV in PC12 neural cells was stronger ( Figure 4 A). Similarly, DRG primary neurons were able to take up more NP-OMV ( Figure 4 B).

[0128] Example 5 Tissue distribution of NP41-modified vesicles in vivo

[0129] 1. Experimental Materials and Reagents

[0130] Escherichia coli Nissle 1917, DSPE-PEG-NP41, C57BL6 / J mice.

[0131] 2. Experimental Procedure

[0132] (1) Construction of an orthotopic pancreatic cancer model in mice: A surgical incision was made 5-10 mm below the left costal arch of the mouse, and 25 μL of Panc02 cell suspension (containing 1×10 6 The Panc02 mouse pancreatic cancer orthotopic model was established by injecting 500 cells into the head of the mouse pancreas.

[0133] (2) Four weeks after tumor cell inoculation, the tumor-bearing mice were randomly divided into two groups, with three mice in each group. Three tumor-bearing mice were injected with the prepared IR780-labeled NP41-modified vesicles at a protein quantitative dose of 1.75 mg / kg via the tail vein as the experimental group, and three tumor-bearing mice were injected with the same dose of IR780-labeled vesicles without NP41 modification as the control group.

[0134] (3) At 0 h, 6 h, 12 h, 24 h, 48 h, and 72 h after intravenous injection, the IR780 fluorescence in mice was imaged and photographed using the IVIS Lumina II small animal imaging system, and the total fluorescence in the tumor area was quantitatively analyzed.

[0135] (4) 72 h after injection, the mice were killed by cervical dislocation, and the main organs (heart, liver, spleen, lung, kidney, brain) and pancreatic tumors were removed. After rinsing with saline, the surface moisture was wiped dry. IR780 fluorescence in vitro imaging was performed using IVIS Lumina II small animal imaging, and the total fluorescence was statistically analyzed.

[0136] (5) Mouse tumor tissues were fixed with 4% PFA, dehydrated with graded alcohol, made transparent, immersed in wax, and embedded in the tissue for tissue sectioning. Alexa Fluor 594-β3-tubulin antibody was used to label nerve fibers in the tumor tissues. The colocalization of IR780-labeled NP41-modified vesicles or non-NP41-modified vesicles and nerve fibers was observed using confocal microscopy and analyzed using Image J.

[0137] 3. Experimental Results

[0138] In vivo imaging results showed that the IR780 fluorescence signal gradually increased in tumor tissue over time and reached a peak at 24 hours after injection. The fluorescence intensity of NP41-modified vesicles labeled with IR780 in pancreatic tumor tissue was significantly higher than that of the vesicles without NP41 modification ( Figure 5 A, Figure 5B). In vitro imaging results also showed that NP41-modified vesicles were most abundant in pancreatic tumor tissue ( Figure 5 C, Figure 5 D). In addition, immunofluorescence staining results showed that NP41-modified vesicles could co-localize well with nerve fibers ( Figure 5 E, Figure 5 F). The above results indicate that NP41-modified vesicles can target nerves in pancreatic tumor tissues.

[0139] Example 6 Effects of drug-loaded vesicles on PC12 neural cells

[0140] 1. Experimental Materials and Reagents

[0141] Escherichia coli Nissle 1917, larotrectinib, DSPE-PEG-NP41, and PC12 neural cells.

[0142] 2. Experimental Procedure

[0143] (1) The collection of drug-loaded vesicles is the same as in Example 1.

[0144] (2) PC12 neural cells were cultured at 1×10 4 Cells were plated at a density of 100 μg / mL in a 96-well plate. After overnight culture, RPMI 1640 medium containing 50 ng / mL NGF (nerve growth factor) and 1% FBS (fetal bovine serum) and drug-loaded vesicles were added and incubated at 37°C, 5% CO2. After 24 hours, the supernatant was discarded, and fresh serum-free medium containing CCK8 reagent was added to each well and incubated for 4 hours. After the incubation period, the absorbance of the solution at a wavelength of 450 nm was measured using a microplate reader. PC12 neural cells to which NP41-modified drug-loaded vesicles (Lar@NP-OMV) were added at 5 μg / mL (protein amount) were used as the experimental group, PC12 neural cells without treatment were used as control group 1, PC12 neural cells to which vesicles (OMV) without NP41 modification were added at 5 μg / mL (protein amount) were used as control group 2, PC12 neural cells to which NP41-modified vesicles (NP-OMV) were added at 5 μg / mL (protein amount) were used as control group 3, PC12 neural cells to which larotrectinib (Lar) was added at 50 ng / mL were used as control group 4, and PC12 neural cells to which drug-loaded vesicles (Lar@OMV) without NP41 modification were added at 5 μg / mL (protein amount) were used as control group 5.

[0145] (3) PC12 neural cells were cultured at 1×10 4Cells were plated at a density of 1 / 4 in 12-well plates and cultured overnight. RPMI 1640 medium supplemented with 50 ng / mL NGF and 1% FBS and drug-loaded vesicles were then added and incubated at 37°C, 5% CO2. Axonal growth in each group was observed daily. 72 hours after drug addition, images were taken using a LEICA inverted fluorescence microscope. Neurite growth was quantitatively analyzed using the Simple Neurite Tracer plugin in Image J (Fiji), and average neurite length was calculated. PC12 neural cells to which NP41-modified drug-loaded vesicles (Lar@NP-OMV) were added at 5 μg / mL (protein amount) were used as the experimental group, PC12 neural cells without treatment were used as control group 1, PC12 neural cells to which vesicles (OMV) without NP41 modification were added at 5 μg / mL (protein amount) were used as control group 2, PC12 neural cells to which NP41-modified vesicles (NP-OMV) were added at 5 μg / mL (protein amount) were used as control group 3, PC12 neural cells to which larotrectinib (Lar) was added at 50 ng / mL were used as control group 4, and PC12 neural cells to which drug-loaded vesicles (Lar@OMV) without NP41 modification were added at 5 μg / mL (protein amount) were used as control group 5.

[0146] 3. Experimental Results

[0147] like Figure 6 As shown in A, compared with non-NP41-modified vesicles, NP41-modified vesicles, free larotrectinib and non-NP41-modified drug-loaded vesicles, NP41-modified drug-loaded vesicles (Lar@NP-OMV) can significantly inhibit NGF-induced PC12 neuronal cell proliferation and PC12 neuronal cell axonal generation ( Figure 6 B), even better than the simple sum of the technical effects of control group 3 (NP-OMV) and control group 4 (Lar). ​​These results demonstrate that NP41-modified larotrectinib-loaded vesicles can effectively inhibit the function of nerve cells and effectively block the NGF-Trk signaling pathway, thereby blocking the interaction between nerves and tumors, confirming the synergistic effect of the three components in the NP41-modified drug-loaded vesicles (Lar@NP-OMV).

[0148] Example 7 Effect of drug-loaded vesicles on tumor cell growth after acting on PC12 neural cells

[0149] 1. Experimental Materials and Reagents

[0150] Escherichia coli Nissle 1917, larotrectinib, DSPE-PEG-NP41, PC12 neural cells, and Panc02 cells.

[0151] 2. Experimental Procedure

[0152] (1) The collection of drug-loaded vesicles is the same as in Example 1.

[0153] (2) PC12 neural cells were cultured at 1×10 4 The cells were plated at a density of 100 cells / well in a 12-well plate and cultured overnight. 50 ng mL -1 The cells were cultured in RPMI 1640 medium supplemented with NGF and 1% FBS, along with the drug-loaded vesicles. After 72 hours of culture, the medium was replaced with fresh serum-free RPMI 1640 medium. After 24 hours, the supernatant was collected and filtered through a 0.22 μm sterile filter to remove debris and dead cells. This provided the conditioned medium from PC12 neurons treated with the different drugs and stored at -80°C until further use. PC12 neural cells to which NP41-modified drug-loaded vesicles (Lar@NP-OMV) were added at 5 μg / mL (protein amount) were used as the experimental group, PC12 neural cells without treatment were used as control group 1, PC12 neural cells to which vesicles (OMV) without NP41 modification were added at 5 μg / mL (protein amount) were used as control group 2, PC12 neural cells to which NP41-modified vesicles (NP-OMV) were added at 5 μg / mL (protein amount) were used as control group 3, PC12 neural cells to which larotrectinib (Lar) was added at 50 ng / mL were used as control group 4, and PC12 neural cells to which drug-loaded vesicles (Lar@OMV) without NP41 modification were added at 5 μg / mL (protein amount) were used as control group 5.

[0154] (3) Panc02 cells (mouse pancreatic cancer cells) were plated at 1×10 4 Cells were plated at a density of 100 cells / well in a 96-well plate. After overnight culture, the supernatant was discarded and conditioned medium from PC12 neurons treated with the above-mentioned different drugs was added. The cells were cultured at 37°C and 5% CO2. After 24 hours, the supernatant was discarded and fresh serum-free medium containing CCK8 reagent was added to each well for 4 hours. After the incubation period, the absorbance of the solution at a wavelength of 450 nm was measured using a microplate reader to evaluate the effect of NP41-modified drug-loaded vesicles on tumor cell proliferation after acting on neurons.

[0155] (4) Soak the scratch mold in a culture dish containing 75% alcohol and sterilize it by ultraviolet irradiation. Wash the residual alcohol on the mold with PBS and let it dry before use. Place the mold in a 12-well plate so that the bottom of the mold fits tightly with the surface of the well plate. Panc02 cells were cultured at 2×10 4Cells were plated at a density of 10 cells per well in the scratch mold. After overnight culture, the mold was gently removed, the supernatant discarded, and the cells were washed three times with PBS. The conditioned medium of PC12 neurons treated with the above-mentioned different drugs was added and cultured at 37°C and 5% CO2. The healing of the Panc02 cell scratches was observed and photographed using an inverted microscope at 0h and 12h of culture. The scratch area was statistically analyzed using Image J software, and the scratch healing ratio was calculated to evaluate the effect of NP41-modified drug-loaded vesicles on Panc02 cell migration after acting on neurons.

[0156] 3. Experimental Results

[0157] like Figure 7 As shown in A, NP41-modified drug-loaded vesicles (Lar@NP-OMV) significantly slowed down the proliferation of Panc02 cells promoted by neurons; in addition, the percentage of Panc02 cell scratch healing in the NP41-modified drug-loaded vesicle treatment group was the lowest ( Figure 7 B), even better than the simple sum of the technical effects of control group 3 (NP-OMV) and control group 4 (Lar), confirming the synergistic effect of the three components in the NP41-modified drug-loaded vesicles (Lar@NP-OMV). These results indicate that NP41-modified drug-loaded vesicles slowed the proliferation and migration of Panc02 cells promoted by neural cells.

[0158] Example 8 Effect of drug-loaded vesicles on reverse polarized M2 macrophages

[0159] 1. Experimental Materials and Reagents

[0160] Escherichia coli Nissle 1917, larotrectinib, DSPE-PEG-NP41, mouse bone marrow-derived macrophages, IL-4, TRIzol, reverse transcription kit, and SYBR Green dye.

[0161] 2. Experimental Procedure

[0162] (1) The collection of drug-loaded vesicles is the same as in Example 1.

[0163] (2) Drug-loaded vesicles were added to M2 bone marrow macrophages induced by IL-4 and co-cultured. After 24 hours, the cells were collected, total RNA was extracted, reverse transcribed into cDNA, and the expression of M1 and M2 related genes was detected by fluorescence quantitative PCR. PC12 neurons added with NP41-modified drug-loaded vesicles (Lar@NP-OMV) at 5 μg / mL (protein amount) were used as the experimental group, PC12 neurons without treatment were used as control group 1, PC12 neurons added with vesicles (OMV) without NP41 modification at 5 μg / mL (protein amount) were used as control group 2, PC12 neurons added with NP41-modified vesicles (NP-OMV) at 5 μg / mL (protein amount) were used as control group 3, PC12 neurons added with larotrectinib (Lar) at 50 ng / mL were used as control group 4, and PC12 neurons added with vesicles (Lar@OMV) without NP41 modification at 5 μg / mL (protein amount) were used as control group 5.

[0164] 3. Experimental Results

[0165] like Figure 8 A. Figure 8 B. Figure 8 C. Figure 8 As shown in Figure D, compared with free larotrectinib, vesicles without NP41 modification, vesicles modified with NP41, drug-loaded vesicles without NP41 modification, and drug-loaded vesicles modified with NP41 significantly increased the expression levels of M1-related genes CD86 and TNFα, and significantly downregulated the expression of M2-related genes Mgl1 and Mrc1, indicating that the vesicles provided by the present invention can effectively reverse polarize M2 macrophages.

[0166] Example 9 Effects of drug-loaded vesicles on neural cells after reverse polarization of M2 macrophages

[0167] 1. Experimental Materials and Reagents

[0168] Escherichia coli Nissle 1917, larotrectinib, DSPE-PEG-NP41, mouse bone marrow-derived macrophages, PC12 neural cells, IL-4, TRIzol, reverse transcription kit, and SYBR Green dye.

[0169] 2. Experimental Procedure

[0170] (1) The collection of drug-loaded vesicles is the same as in Example 1.

[0171] (2) Drug-loaded vesicles were added to the M2 bone marrow-derived macrophages induced by IL-4 for co-culture. After 24 hours, the supernatant was discarded, the cells were washed with PBS three times, and replaced with fresh serum-free RPMI 1640 medium. After further culture for 12 hours, the cell supernatant was collected to obtain the macrophage conditioned medium. PC12 neural cells to which NP41-modified drug-loaded vesicles (Lar@NP-OMV) were added at 5 μg / mL (protein amount) were used as the experimental group, PC12 neural cells without treatment were used as control group 1, PC12 neural cells to which vesicles (OMV) without NP41 modification were added at 5 μg / mL (protein amount) were used as control group 2, PC12 neural cells to which NP41-modified vesicles (NP-OMV) were added at 5 μg / mL (protein amount) were used as control group 3, PC12 neural cells to which larotrectinib (Lar) was added at 50 ng / mL were used as control group 4, and PC12 neural cells to which drug-loaded vesicles (Lar@OMV) without NP41 modification were added at 5 μg / mL (protein amount) were used as control group 5.

[0172] (3) PC12 neural cells were cultured at 1×10 4 Cells were plated in a 96-well plate at a density of 100 μL. After overnight culture, the supernatant was discarded and replaced with 100 μL of macrophage-conditioned medium and cultured for another 24 hours. After the culture was completed, 10 μL of CCK8 reagent was added to each well and incubated for 4 hours. The absorbance of the solution at a wavelength of 450 nm was measured using a microplate reader to calculate the cell survival rate.

[0173] (4) PC12 neural cells were cultured at 1×10 5 Cells were plated at a density of 100 cells / well in 12-well plates. After overnight culture, the supernatant was discarded and cultured in macrophage-conditioned medium at 37°C and 5% CO2. Axonal growth in each group was observed daily. After 72 hours of culture, images were taken using a LEICA inverted fluorescence microscope. Neurite growth was quantitatively analyzed using the Simple Neurite Tracing plugin in Image J (Fiji), and axon length was calculated to investigate the effect of NP41-modified drug-loaded vesicles (Lar@NP-OMV) on neuronal axonal formation after reverse polarization of M2 macrophages.

[0174] 3. Experimental Results

[0175] like Figure 9 As shown in A, compared with the PBS and free larotrectinib groups, the vesicles without NP41 modification, the vesicles modified with NP41, the drug-loaded vesicles without NP41 modification and the drug-loaded vesicles modified with NP41 could significantly inhibit the growth of PC12 neurons and the axonal formation of PC12 neurons ( Figure 9B) This indicates that the vesicles provided by the present invention can reverse polarize M2 macrophages, which is detrimental to the growth of PC12 neurons.

[0176] Example 10 Inhibitory Effect of Drug-Loaded Vesicles on Panc02 In Situ Pancreatic Cancer

[0177] 1. Experimental Materials and Reagents

[0178] Escherichia coli Nissle 1917, larotrectinib, DSPE-PEG-NP41, and male C57BL6 / J mice.

[0179] 2. Experimental Procedure

[0180] (1) The collection of drug-loaded vesicles is the same as in Example 1.

[0181] (2) The construction of the mouse Panc02 pancreatic cancer orthotopic model was the same as in Example 5.

[0182] (3) Seven days after tumor cell inoculation, the tumor-bearing mice were divided into six groups, each with 16 mice. PBS, OMV, NP-OMV, Lar, Lar@OMV, and Lar@NP-OMV were injected into the tail vein. The PBS-treated group was injected with 200 μL PBS. The non-drug-loaded vesicles (OMV-treated group and NP-OMV-treated group) were administered at a protein quantification of 1.75 mg / kg dispersed in 200 μL PBS. The Lar-treated group was administered at a dose of 0.25 mg / kg dispersed in 200 μL PBS. The drug-loaded vesicles (Lar@OMV-treated group and Lar@NP-OMV-treated group) were administered at a Lar dose of 0.25 mg / kg dispersed in 200 μL PBS. The drugs were administered once every three days for a total of six times. After the administration, eight mice were randomly selected from each group to continue the survival experiment. Lar@NP-OMV-treated C57BL6 / J mice served as the experimental group, PBS-treated C57BL6 / J mice served as control group 1, OMV-treated C57BL6 / J mice served as control group 2, NP-OMV-treated C57BL6 / J mice served as control group 3, free Lar-treated C57BL6 / J mice served as control group 4, and Lar@OMV-treated C57BL6 / J mice served as control group 5.

[0183] 3. Experimental Results

[0184] like Figure 10 As shown in A, after the administration, the weighing results of the tumor tissue removed showed that Lar@NP-OMV significantly inhibited the growth of pancreatic in situ tumors, and its tumor inhibition effect was significantly better than that of other control groups, confirming the synergistic effect of the three components in the NP41-modified drug-loaded vesicles (Lar@NP-OMV). The survival results also found that ( Figure 10B), Lar@NP-OMV treatment prolonged the survival of tumor-bearing mice, which was significantly better than other control groups.

[0185] Example 11 Effect of drug-loaded vesicles on the neural microenvironment of Panc02 in situ pancreatic cancer

[0186] 1. Experimental Materials and Reagents

[0187] Escherichia coli Nissle 1917, larotrectinib, DSPE-PEG-NP41, and male C57BL6 / J mice.

[0188] 2. Experimental Procedure

[0189] (1) The collection of drug-loaded vesicles is the same as in Example 1.

[0190] (2) The method of Example 10 was used to administer the drug to mice bearing pancreatic cancer orthotopic tumors.

[0191] (3) After the treatment, the mice were treated with drugs. The tumor tissues were removed and fixed with 4% PFA. After dehydration with different concentrations of alcohol gradient, paraffin embedding and sectioning were performed. Immunofluorescence staining was performed with β3-tubulin and GAP43 antibodies to study the changes in the tumor neural microenvironment after Lar@NP-OMV treatment. Lar@NP-OMV-treated C57BL6 / J mice served as the experimental group, PBS-treated C57BL6 / J mice served as control group 1, OMV-treated C57BL6 / J mice served as control group 2, NP-OMV-treated C57BL6 / J mice served as control group 3, free Lar-treated C57BL6 / J mice served as control group 4, and Lar@OMV-treated C57BL6 / J mice served as control group 5.

[0192] 3. Experimental Results

[0193] like Figure 11 A, Figure 11 As shown in Figure B, after drug treatment, the nerve density in the tumor tissue of tumor-bearing mice in the Lar@NP-OMV group was significantly reduced, even surpassing the simple sum of the technical effects of the NP-OMV and Lar groups, confirming the synergistic effect of the three components in the NP41-modified drug-loaded vesicles (Lar@NP-OMV). These results indicate that Lar@NP-OMV can effectively target nerves in the tumor microenvironment and inhibit nerve growth.

[0194] Example 12 Biosafety of Drug-Loaded Vesicles

[0195] 1. Experimental Materials and Reagents

[0196] Escherichia coli Nissle 1917, larotrectinib, DSPE-PEG-NP41, and male C57BL6 / J mice.

[0197] 2. Experimental Procedure

[0198] (1) The collection of drug-loaded vesicles is the same as in Example 1.

[0199] (2) The method of Example 10 was used to administer the drug to mice bearing pancreatic cancer orthotopic tumors.

[0200] (3) The weight of mice was weighed and recorded every day during the treatment. After the treatment, blood was collected from the mice's orbits and the supernatant was collected for blood biochemical index testing. The level of aspartate aminotransferase (AST) in the serum was tested to evaluate liver function; the level of creatinine (CREA) was tested to evaluate renal function. Lar@NP-OMV-treated C57BL6 / J mice were used as the experimental group, PBS-treated C57BL6 / J mice were used as control group 1, OMV-treated C57BL6 / J mice were used as control group 2, NP-OMV-treated C57BL6 / J mice were used as control group 3, free Lar-treated C57BL6 / J mice were used as control group 4, and Lar@OMV-treated C57BL6 / J mice were used as control group 5.

[0201] 3. Experimental Results

[0202] like Figure 12 As shown in A, after drug treatment, the body weight of the tumor-bearing mice in the Lar@NP-OMV group did not change significantly compared with the PBS group, and the serum aspartate aminotransferase and creatinine levels of the mice did not change significantly ( Figure 12 B, Figure 12 C) This indicates that the hepatotoxicity is very low and the drug-loaded vesicles modified with NP41 have basically no side effects on the body.

[0203] Example 13 Effect of Gemcitabine on NGF Expression Levels in Panc02 Cells

[0204] 1. Experimental Materials and Reagents

[0205] Gemcitabine, Panc02 cells, TRIzol, reverse transcription kit, and SYBR Green dye.

[0206] 2. Experimental Procedure

[0207] (1) Panc02 cells were plated at 2×10 5 The cells were plated in 6-well plates at a density of 10 cells / well and cultured for 12 h.

[0208] (2) Add 0.03 μg / mL gemcitabine to the above 6-well plate and treat the cells. After 24 hours, the cells were harvested, total RNA was extracted, reverse transcribed into cDNA, and the expression of NGF was detected by fluorescent quantitative PCR. Gemcitabine-treated Panc02 cells served as the experimental group, and untreated Panc02 cells served as the control group.

[0209] 3. Experimental Results

[0210] like Figure 13 As shown in the results, compared with the control group, gemcitabine can significantly increase the expression level of NGF, indicating that gemcitabine may promote neurogenesis in tumor tissue.

[0211] Example 14 Inhibitory effect of drug-loaded vesicles combined with gemcitabine on Panc02 in situ pancreatic cancer

[0212] 1. Experimental Materials and Reagents

[0213] Escherichia coli Nissle 1917, larotrectinib, DSPE-PEG-NP41, male C57BL6 / J mice, gemcitabine.

[0214] 2. Experimental Procedure

[0215] (1) The collection of drug-loaded vesicles is the same as in Example 1.

[0216] (2) The construction of the mouse Panc02 pancreatic cancer orthotopic model was the same as in Example 5.

[0217] (3) Seven days after tumor cell inoculation, the tumor-bearing mice were divided into four groups, each with 14 mice. PBS, gemcitabine (GEM), Lar@NP-OMV, and gemcitabine and Lar@NP-OMV were injected into the tail vein. The PBS dosage was 200 μL PBS; Lar@NP-OMV was administered at a Lar dosage of 0.25 mg / kg and dispersed in 200 μL PBS; GEM was administered at a dosage of 20 mg / kg and dispersed in 200 μL PBS; gemcitabine and Lar@NP-OMV were administered in a mixed manner, with the dosage being the sum of the dosages of the two drugs, dispersed in 200 μL PBS. The drugs were administered once every three days for a total of six times. After the administration, eight mice were randomly selected from each group to continue the survival experiment. C57BL6 / J mice treated with gemcitabine and Lar@NP-OMV were used as experimental groups, C57BL6 / J mice treated with PBS were used as control group 1, C57BL6 / J mice treated with gemcitabine were used as control group 2, and C57BL6 / J mice treated with Lar@NP-OMV were used as control group 3.

[0218] 3. Experimental Results

[0219] like Figure 14 As shown in A, after the administration, the tumor tissue was taken out and immunofluorescence staining was performed ( Figure 14 A) and ELISA ( Figure 14 B), the results showed that gemcitabine treatment increased the nerve density and norepinephrine content in pancreatic tumor tissue, indicating that gemcitabine can promote the growth of nerves in tumor tissue. By measuring tumor weight, it was found that gemcitabine combined with Lar@NP-OMV significantly inhibited the growth of pancreatic in situ tumors, and its tumor inhibition effect was significantly better than that of gemcitabine and Lar@NP-OMV ( Figure 14 C), confirming the synergistic effect between the drugs. Survival results ( Figure 14 D) also showed that gemcitabine combined with Lar@NP-OMV treatment prolonged the survival of tumor-bearing mice, significantly outperforming other control groups. In particular, while all mice in the control group receiving gemcitabine alone died by day 62, mice in the group receiving a combination of gemcitabine and Lar@NP-OMV survived until day 80, demonstrating that the combination of the two drugs can effectively enhance the pancreatic cancer treatment efficacy of gemcitabine.

[0220] Example 15 Targeting of RVG-modified vesicles to neural cells in vitro

[0221] 1. Experimental Materials and Reagents

[0222] Escherichia coli Nissle 1917, DSPE-PEG-RVG, and primary DRG neurons.

[0223] 2. Experimental Procedure

[0224] (1) RVG-modified vesicles were prepared according to Example 1, wherein the neuronal cell targeting molecule was DSPE-PEG-RVG, and then the vesicles were labeled with the cell membrane green fluorescent dye DiO.

[0225] (2) DRG primary neurons were treated according to the method of Example 4, and the mean fluorescence intensity of DiO in DRG primary neurons was measured by flow cytometry. OMV served as the control group, and RVG-OMV served as the experimental group.

[0226] 3. Experimental Results

[0227] Flow cytometry results showed that the fluorescence intensity of RVG-OMV in DRG primary neurons was stronger than that of OMV ( Figure 15 ), indicating that DRG primary neurons can absorb more RVG-OMV.

[0228] Example 16 Inhibitory effect of drug-loaded vesicles combined with doxorubicin on 4T1 in situ breast cancer

[0229] 1. Experimental Materials and Reagents

[0230] Escherichia coli Nissle 1917, lidocaine, DSPE-PEG-NP41, SPF-grade BALB / C female mice, and doxorubicin.

[0231] 2. Experimental Procedure

[0232] (1) The collection of drug-loaded vesicles is the same as in Example 1, wherein the small molecule drug is lidocaine.

[0233] (2) Construction of an in situ breast cancer model: 4T1 breast cancer cells in the logarithmic growth phase were digested with 0.05% trypsin for 1 min, centrifuged at 1000 rpm for 5 min, the supernatant was removed, the cells were resuspended and counted, washed three times with saline, and resuspended with saline to adjust the concentration to 1×10 7 50 μL of breast cancer cell suspension was injected into the right mammary fat pad of mice.

[0234] (3) When the breast tumor grows to 50-100 mm 3 Tumor-bearing mice were randomly divided into four groups, each consisting of six mice. PBS, doxorubicin (DOX), Lido@NP-OMV, or doxorubicin and Lido@NP-OMV were administered via the tail vein. PBS was administered in 200 μL of PBS; Lido@NP-OMV was administered at a dose of 1 mg / kg dispersed in 200 μL of PBS; DOX was administered at a dose of 4 mg / kg dispersed in 200 μL of PBS; and doxorubicin and Lido@NP-OMV were administered as a mixed dose, the sum of the doses of the two drugs alone, dispersed in 200 μL of PBS. Dosing was repeated every three days for a total of six doses. BALB / C mice treated with doxorubicin and Lido@NP-OMV served as the experimental group, while BALB / C mice treated with PBS served as control group 1, BALB / C mice treated with doxorubicin served as control group 2, and BALB / C mice treated with Lido@NP-OMV served as control group 3.

[0235] 3. Experimental Results

[0236] After the administration, the tumor tissue was taken out and immunofluorescence staining was performed. Figure 16 As shown in A, doxorubicin treatment increased nerve density in breast tumor tissue, indicating that doxorubicin can promote nerve growth in tumor tissue. By detecting tumor weight, it was found that doxorubicin combined with Lido@NP-OMV significantly inhibited the growth of breast in situ tumors, and its tumor inhibition effect was significantly better than that of doxorubicin and Lido@NP-OMV ( Figure 16 B), confirming the synergistic effect between the drugs.

[0237] It will be easily understood by those skilled in the art that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements 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 drug-loaded bacterial outer membrane vesicle for targeted intervention of the tumor neural microenvironment, characterized in that: The invention comprises bacterial outer membrane vesicles and small molecule drugs encapsulated by the bacterial outer membrane vesicles, wherein the small molecule drugs are small molecule drugs with neuroinhibitory effects; the surface of the bacterial outer membrane vesicles is also modified with nerve cell targeting molecules; The bacterial outer membrane vesicles are bacterial outer membrane vesicles secreted by any Gram-negative bacteria; The nerve cell targeting molecule includes a nerve cell targeting peptide and a conjugate. The nerve cell targeting peptide is conjugated to the surface of the conjugate. The conjugate can be connected to the surface of the bacterial outer membrane vesicle by lipid intercalation. The nerve cell targeting peptide is NP41 or RVG.

2. The drug-loaded bacterial outer membrane vesicles according to claim 1, characterized in that The bacterial outer membrane vesicles include bacterial outer membrane vesicles derived from Escherichia coli, bacterial outer membrane vesicles derived from Akkermansia muciniphila or bacterial outer membrane vesicles derived from attenuated Salmonella.

3. The drug-loaded bacterial outer membrane vesicles according to claim 1, characterized in that The mass ratio of the bacterial outer membrane vesicles, the nerve cell targeting molecule and the drug is (1-20):1:(1-200).

4. The drug-loaded bacterial outer membrane vesicles according to claim 1, characterized in that The small molecule drug is a Trk inhibitor, an anesthetic or other small molecule drug with a neuroinhibitory effect.

5. The drug-loaded bacterial outer membrane vesicles according to claim 4, characterized in that The Trk inhibitor is one or more of larotrectinib, entrectinib, AZ23 and LOXO-195; the anesthetic is one or more of procaine, tetracaine and lidocaine; and the other small molecule drugs with neuroinhibitory effects are one or more of the neurotoxin botulinum toxin and 6-hydroxydopamine.

6. A method for preparing drug-loaded bacterial outer membrane vesicles according to any one of claims 1 to 5, characterized in that: The steps include: S1. Incubating bacterial outer membrane vesicles with nerve cell targeting molecules, modifying the nerve cell targeting molecules onto the surface of the bacterial outer membrane vesicles by lipid intercalation, and collecting the bacterial outer membrane vesicles modified with the nerve cell targeting molecules; S2. The bacterial outer membrane vesicles modified with the nerve cell targeting molecule are mixed and incubated with a small molecule drug, wherein the small molecule drug is a small molecule drug with a nerve inhibitory effect, and the drug-loaded bacterial outer membrane vesicles that target and intervene in the tumor neural microenvironment are collected.

7. The preparation method according to claim 6, characterized in that In step S1, the mass ratio of the bacterial outer membrane vesicles to the nerve cell targeting molecule is (1-20):

1.

8. The preparation method according to claim 6, wherein In steps S1 and S2, the collection conditions are: collection at a centrifugal force of 2000 g to 150000 g at 4°C.

9. A drug for treating cancer, characterized in that: It comprises the drug-loaded bacterial outer membrane vesicles according to any one of claims 1 to 5, and further comprises a chemotherapy drug.

10. The drug for treating cancer according to claim 9, characterized in that The chemotherapy drug is a chemotherapy drug that can promote tumor cells to highly express nerve growth factor, and the chemotherapy drug that can promote tumor cells to highly express nerve growth factor 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 and pirarubicin.

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