Controllable inflammation-driven neutrophil-mediated tumor drug delivery system

Abstract

The invention provides a neutrophil-mediated tumor drug delivery system driven by controllable inflammation, and belongs to the technical field of medicines. The invention provides a medicine for treating tumors. The medicine comprises LPS (Lipopolysaccharide) and a tumor medicine delivery system, the tumor drug delivery system comprises neutrophile granulocyte and a therapeutic nano-material loaded into the neutrophile granulocyte. The LPS in the medicine provided by the invention can induce local acute inflammation at a tumor part so as to improve the targeting property of a tumor medicine delivery system on tumors and improve the treatment effect of the tumor medicine delivery system on the tumors. The result of the embodiment shows that the acute controllable inflammation model in the tumor is established by injecting the LPS into the triple-negative breast cancer tumor. The intra-tumor acute controllable inflammation model can amplify inflammatory signals of a tumor microenvironment, and can significantly improve the treatment effect of triple negative breast cancer while improving the drug targeting of a tumor drug delivery system.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a controllable inflammation-driven neutrophil-mediated tumor drug delivery system. Background Art

[0002] Cancer is the leading cause of death in the world, and the number of deaths continues to increase every year. Tumors are characterized by multiple mechanisms such as malignant proliferation, invasion and metastasis, and immune evasion to promote their own occurrence and development, and create a tumor microenvironment (TME) for themselves to further resist the body's immunity and maintain their own development, making surgery, radiotherapy and chemotherapy, the three major conventional treatments, still unable to effectively treat many cancers. The development of targeted drug delivery is one of the most important goals of drug research. In recent decades, the development of biocompatible nanomaterials has greatly improved the delivery efficiency of drugs in tumor sites, enhanced the tumor targeting of drugs, and reduced the toxic side effects of drugs, which has led to the rapid development of tumor targeted therapy, and some nanomedicines have been approved for routine clinical use. However, there are still many obstacles that hinder the widespread application of nanomedicine. For example, although the EPR effect and active targeting methods can regulate the biodistribution of nanomedicines to a certain extent, only a small part of nanomedicines can reach the tumor site, and most of them are cleared by the reticuloendothelial system (RES). In addition, most nanomaterials do not have a long enough blood half-life, and it is difficult to achieve deep penetration into tumor tissues. Therefore, it is very necessary to develop other new strategies for tumor-targeted drug therapy that are more efficient and less toxic to further improve the effect of tumor treatment. The research on cell carriers combined with nanomaterials provides a new idea for further realizing active targeted tumor treatment.

[0003] Nanomaterials with cells as carriers are used to prepare tumor-specific targeted anti-tumor drugs. They utilize the inherent properties of various cells, such as long circulation time in the blood, specific targeting of tumor microenvironment and tumor cells, ability to cross natural biological barriers in the body, rich surface ligands, deformation and migration ability, and unique cell signaling or metabolic mechanisms, etc., and combine them with nanomaterials with advantages such as safety, biodegradability and good biocompatibility. Nanomaterials are loaded into cells to prepare new anti-tumor drugs with tumor-specific targeting, and targeted nanotherapy for tumors is completed by cell delivery of nanomaterials. This is a cancer treatment strategy that maximizes the advantages of both while minimizing their inherent disadvantages, thereby enhancing the overall therapeutic effect.

[0004] Neutrophils are the most abundant type of granulocytes in the human body, accounting for 40% to 70% of all white blood cells in humans. As the first responders of the immune system, neutrophils have inherent migration and phagocytic abilities, which enable them to ingest nanoparticles and phagocytose dead red blood cells. Moreover, they can migrate across blood vessels to adjacent tissues and cross natural biological barriers. In addition, activated neutrophils can capture and kill pathogens by releasing extracellular traps (NETs) composed of depolymerized chromatin and intracellular granule proteins to the extracellular space. They have the ability to release the nanoparticles they have ingested, so neutrophils are natural candidates as cell carriers. However, the current use of neutrophils as drug carriers for the treatment of tumors generally has the problem of poor drug targeting and poor therapeutic effect on tumors. Summary of the invention

[0005] In view of the defects in the prior art, the purpose of the present invention is to provide a drug for treating tumors, which first increases the inflammatory response in the tumor, significantly improves the targeting of the tumor drug delivery system, and then significantly improves the therapeutic effect on the tumor.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] The invention provides a drug for treating tumors, comprising: LPS and a tumor drug delivery system; the tumor drug delivery system comprises neutrophils and therapeutic nanomaterials loaded into the neutrophils.

[0008] Preferably, the direct site of action of LPS is inside the tumor.

[0009] Preferably, the tumor drug delivery system is used after the LPS acts on the inside of the tumor for 6 to 12 hours.

[0010] Preferably, the neutrophils include animal bone marrow neutrophils and / or peripheral blood neutrophils.

[0011] Preferably, the therapeutic nanomaterials include nanocarriers and tumor therapeutic drugs;

[0012] The nanocarrier comprises any one or more of human serum albumin, liposome, mesoporous silica nanoparticles, graphene, micelle, nanocapsule and gold nanorod;

[0013] The tumor therapeutic drugs include any one or more of drugs that inhibit enzyme activity in tumor cells, drugs that inhibit tubulin formation, drugs that inhibit DNA formation, drugs that inhibit RNA formation, and drugs that cause tissue hypoxia.

[0014] Preferably, the tumor drug delivery system comprises BSA-DOX / NEs;

[0015] The preparation method of BSA-DOX / NEs comprises:

[0016] After mixing the BSA aqueous solution with β-mercaptoethanol, the disulfide bonds in the BSA are reversibly opened to obtain a protein solution;

[0017] The protein solution was mixed with a DOX·HCl aqueous solution and then ultrafiltered to obtain BSA-DOX nanomaterials;

[0018] Neutrophils were mixed with BSA-DOX nanomaterials and co-incubated to obtain BSA-DOX / NEs.

[0019] Preferably, the protein solution is adjusted to be alkaline and then mixed with the DOX·HCl aqueous solution.

[0020] The present invention provides the use of the drug described in the above technical solution in the preparation of drugs for treating tumors.

[0021] The present invention provides a model of acute controllable inflammation in a tumor, and the method for constructing the model of acute controllable inflammation in a tumor comprises:

[0022] An acute controllable intratumoral inflammation model was established by intratumoral injection of LPS.

[0023] Preferably, the tumor includes any one or more of breast cancer, acute leukemia, malignant lymphoma, bronchogenic carcinoma, ovarian cancer, soft tissue sarcoma, osteogenic sarcoma, rhabdomyosarcoma, Ewing sarcoma, blastoma, neuroblastoma, bladder cancer, thyroid cancer, prostate cancer, head and neck squamous cell carcinoma, testicular cancer, gastric cancer and liver cancer.

[0024] The present invention provides application of LPS in preparing a drug for improving the effect of a tumor drug delivery system in treating tumors; the tumor drug delivery system comprises neutrophils and therapeutic nanomaterials loaded into the neutrophils.

[0025] Beneficial effects of the present invention:

[0026] The present invention provides a drug for treating tumors, including: LPS and a tumor drug delivery system; the tumor drug delivery system includes neutrophils and therapeutic nanomaterials loaded into neutrophils. When the present invention uses the drug to treat tumors, LPS first acts directly on the inside of the tumor, induces controllable acute inflammation in the tumor tissue, amplifies the inflammatory signals in the tumor microenvironment, and then promotes the tumor drug delivery system with neutrophils as cell carriers to infiltrate a large number of tumor microenvironments, thereby achieving specific targeting of therapeutic nanomaterials loaded in the tumor drug delivery system. The drug of the present invention uses neutrophils as cell carriers to achieve more accurate tumor targeted treatment of anti-tumor drugs, and at the same time uses the characteristics of neutrophils to control the progress of systemic inflammatory reactions to ensure the safety of treatment. The drug provided by the present invention induces local acute inflammation at the tumor site to improve the targeting of the tumor drug delivery system to the tumor, and improves the therapeutic effect of anti-tumor drugs such as the tumor drug delivery system on the tumor. The present invention shows through the results of the embodiment that an acute controllable inflammation model in the tumor is established by injecting LPS into the triple-negative breast cancer tumor. The intratumor acute controllable inflammation model can amplify the inflammatory signals of the tumor microenvironment, and while improving the drug targeting of the tumor drug delivery system, it can significantly improve the therapeutic effect of the tumor drug delivery system on triple-negative breast cancer.

[0027] Further, the tumor drug delivery system described in the present invention may include neutrophils and therapeutic nanomaterials loaded into neutrophils. The present invention utilizes the inherent targeting characteristics of neutrophils, not only to achieve the specific accumulation of the tumor drug delivery system at the tumor site, but also to release therapeutic nanomaterials after the tumor drug delivery system is subjected to inflammatory stimulation in the tumor microenvironment, thereby achieving the precise release of anti-tumor drugs. The tumor drug delivery system of the present invention makes full use of the inherent properties of neutrophils and develops its potential as a natural carrier. The anti-tumor drug prepared by the tumor drug delivery system with neutrophils as carriers provided by the present invention, through the mediation of nanocarriers, ensures the drug loading of neutrophils, and avoids the influence of therapeutic drugs on the normal physiological activity of neutrophils. The present invention breaks through the obstacles encountered by nanomedicine in its development, solves the problems of short blood half-life of anti-tumor drugs in vivo in tumor treatment, low tumor targeting, and strong systemic toxicity and side effects, combines the advantages of cell carriers and nanomaterials, and further promotes the clinical application and development of nanomedicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0029] Figure 1 The immunohistochemical image of neutrophil infiltration in mouse tumor tissue after LPS was injected into mouse tumor in Example 1;

[0030] Figure 2 This is a graph showing the expression levels of inflammatory cytokines CXCL1 and CXCL2 in tumor tissues after LPS was injected into mouse tumors in Example 1;

[0031] Figure 3 This is a graph showing the expression levels of pro-inflammatory cytokines TNF-α and IL-6 in tumor tissues after LPS was injected into mouse tumors in Example 1;

[0032] Figure 4 This is a blood routine chart after LPS was injected into the mouse tumor in Example 1;

[0033] Figure 5 This is the blood biochemical profile after LPS was injected into the mouse tumor in Example 1;

[0034] Figure 6 This is a graph showing the expression levels of inflammatory cytokines in the serum of tumor-bearing mice after neutrophil infusion following intratumoral injection of LPS in mice in Example 2;

[0035] Figure 7 This is a dynamic scattering hydrated particle size (DLS) graph of the BSA-DOX nanomaterial prepared in Example 3;

[0036] Figure 8 This is a scanning electron microscope (SEM) image of the BSA-DOX nanomaterial prepared in Example 3;

[0037] Fig. 9 This is a diagram showing the cell morphology of BSA-DOX / NEs in Example 3 observed by Wright-Giemsa staining;

[0038] Fig.10 This is a laser scanning confocal microscopy image of BSA-DOX / NEs in Example 4;

[0039] Fig.11 The figure is a graph showing the in vitro killing effect of BSA-DOX / NEs with different concentrations of DOX on 4T1 cells without PMA treatment and after PMA treatment in Example 5;

[0040] Fig.12The in vitro killing effect of BSA-DOX / NEs with different concentrations of DOX on 4T1 cells at 24h and 48h after PMA treatment in Example 5;

[0041] Fig.13 This is the directional chemotaxis diagram of BSA-DOX / NEs for tumor tissues that induce controllable inflammation in Example 6;

[0042] Fig.14 This is a graph showing the inhibition of tumor volume in each group of mice in Example 7;

[0043] Fig.15 The photos of tumor tissues of mice in each group in Example 7;

[0044] Fig.16 The tumor weight of each group of mice in Example 7;

[0045] Fig.17 The figure shows the in vitro killing effect of BSA-DOX / NEs with different concentrations of DOX on PC3 cells without PMA treatment and after PMA treatment in Example 8;

[0046] Fig.18 The figures are the in vitro killing effects of BSA-DOX / NEs with different DOX concentrations on PC3 cells after PMA treatment in Example 8 at 24h and 48h. DETAILED DESCRIPTION

[0047] The invention provides a drug for treating tumors, comprising: LPS and a tumor drug delivery system; the tumor drug delivery system comprises neutrophils and therapeutic nanomaterials loaded into the neutrophils.

[0048] The medicine provided by the present invention uses LPS (lipopolysaccharide) to induce acute controllable inflammation in the tumor and a tumor drug delivery system for treating tumors. In the present invention, the direct site of action of the LPS is in the tumor. In an embodiment of the present invention, the present invention directly acts on the tumor by injecting LPS into the tumor. The present invention uses the body weight of the treated subject as the standard, and the injection amount of the lipopolysaccharide can be 1 mg / kg. As an optional embodiment of the present invention, taking a mouse tumor model as an example, the weight of the mouse model is 20 g. When performing the corresponding effect verification, the injection amount of LPS in the mouse tumor can be 0.02 mg.

[0049] The present invention can also inject the LPS into the tumor to construct an acute controllable inflammation model within the tumor.

[0050] The present invention uses LPS to act directly inside the tumor, which can induce controllable acute inflammation in the tumor tissue, amplify the inflammatory signals in the tumor microenvironment, and then promote the tumor drug delivery system with neutrophils as cell carriers to infiltrate a large number of them in the tumor microenvironment, thereby achieving specific targeting of the therapeutic nanomaterials loaded in the tumor drug delivery system, using neutrophils as cell carriers to achieve more accurate tumor targeted treatment, and at the same time using the characteristics of neutrophils to control the progression of systemic inflammatory response, thereby ensuring the safety of treatment.

[0051] The present invention preferably uses a tumor drug delivery system to treat tumors 6 to 12 hours after LPS acts on the inside of the tumor. As an optional embodiment of the present invention, the present invention uses a tumor drug delivery system to treat tumors by intravenous infusion. In the present invention, the injection volume of the tumor drug delivery system is preferably calculated based on the number of neutrophils. In the present invention, the number of neutrophils when the tumor drug delivery system is injected is preferably ≥1×10 6 It can also be 1×10 6 ~4×10 6 , or 3×10 6 indivual.

[0052] In the present invention, the tumor drug delivery system includes neutrophils and therapeutic nanomaterials loaded into the neutrophils. In the present invention, the neutrophils include animal bone marrow neutrophils and / or peripheral blood neutrophils. The present invention does not specifically limit the preparation method of the neutrophils, and any conventional preparation method in the art can be used. In the present invention, the therapeutic nanomaterials include nanocarriers and tumor therapeutic drugs; the therapeutic nanomaterials are composed of nanocarriers as tools, and the therapeutic drugs are loaded in the nanocarriers. As an optional embodiment of the present invention, the drug delivery system is constructed by neutrophils and nano drug carriers through endocytosis. The present invention does not specifically limit the construction method, and any conventional construction method in the art can be used.

[0053] As an optional embodiment of the present invention, the nanocarrier includes a positively charged, negatively charged or nearly neutral nanomaterial with a particle size of 1 to 500 nm. In the present invention, the nanocarrier includes any one or more of human serum albumin, liposomes, mesoporous silica nanoparticles, graphene, micelles, nanocapsules and gold nanorods; the tumor therapeutic drug includes any one or more of drugs that inhibit enzyme activity in tumor cells, drugs that inhibit tubulin formation, drugs that inhibit DNA formation, drugs that inhibit RNA formation and drugs that cause tissue hypoxia. In the present invention, the tumor therapeutic drug includes DOX·HCl. As an optional embodiment of the present invention, the tumor includes: any one or more of breast cancer, acute leukemia, malignant lymphoma, bronchial lung cancer, ovarian cancer, soft tissue sarcoma, osteosarcoma, rhabdomyosarcoma, Ewing's sarcoma, blastoma, neuroblastoma, bladder cancer, thyroid cancer, prostate cancer, head and neck squamous cell carcinoma, testicular cancer, gastric cancer and liver cancer. In the present invention, breast cancer includes triple-negative breast cancer.

[0054] The present invention has no special limitation on the preparation method of the tumor drug delivery system described in the above technical solution, and any conventional method in the art can be used for preparation. As an optional embodiment of the present invention, the preparation method of the tumor drug delivery system may include: first loading the therapeutic drug into a nanocarrier to obtain a therapeutic nanomaterial; and then loading the therapeutic nanomaterial into neutrophils to obtain a tumor drug delivery system.

[0055] The present invention first loads the therapeutic drug into a nanocarrier, and then loads the drug-loaded nanocarrier into neutrophils, and successfully prepares a drug delivery system with neutrophils as carriers. The prepared drug delivery system with neutrophils as carriers is intravenously injected into the human body. During the blood circulation process, neutrophils, as cell carriers of anti-tumor drugs, are stimulated by chemokines released from the acute inflammatory area of ​​the tumor site, pass through blood vessels, and actively target the tumor site along the chemokine concentration gradient. Neutrophils infiltrate the tumor site in large quantities, and the tumor site further releases inflammatory cytokines, further recruiting more neutrophils and other immune cells, thereby significantly enhancing the targeting efficiency of neutrophils carrying therapeutic nanomaterials, greatly improving the drug accumulation concentration at the tumor site, and prolonging the blood half-life of the drug in the human body, thereby providing precise targeted treatment for tumors.

[0056] The drug for treating tumors described in the present invention uses neutrophils loaded with therapeutic nanomaterials as a tumor drug delivery system, which induces controllable acute inflammation in tumor tissue, amplifies inflammatory signals in the tumor microenvironment, and promotes the large-scale infiltration of neutrophils in the tumor microenvironment, thereby achieving specific targeting of the nanomaterials loaded in the cells, using neutrophils as carriers to achieve more accurate tumor targeted treatment, and at the same time using neutrophils to control the progression of systemic inflammatory response to ensure the safety of treatment.

[0057] As an optional embodiment of the present invention, the tumor drug delivery system includes neutrophils and therapeutic nanomaterials loaded into neutrophils. The therapeutic nanomaterial can use bovine serum albumin as a nanocarrier. The therapeutic drug in the therapeutic nanomaterial can be doxorubicin hydrochloride (Doxorubicin, DOX·HCl). As an optional embodiment of the present invention, the therapeutic nanomaterial includes a spherical nanomaterial formed by coupling the nanocarrier bovine serum albumin with the therapeutic drug Doxorubicin. In the present invention, the spherical nanomaterial formed by coupling the nanocarrier bovine serum albumin with the therapeutic drug Doxorubicin can be referred to as BSA-DOX. The tumor drug delivery system of neutrophils carrying BSA-DOX in the present invention can be referred to as BSA-DOX / NEs.

[0058] The present invention provides a method for preparing BSA-DOX / NEs according to the above technical solution, comprising:

[0059] After mixing the BSA aqueous solution with β-mercaptoethanol, the disulfide bonds in the BSA are reversibly opened to obtain a protein solution;

[0060] The protein solution was mixed with a DOX·HCl aqueous solution and then ultrafiltered to obtain BSA-DOX nanomaterials;

[0061] Neutrophils were mixed with BSA-DOX nanomaterials and co-incubated to obtain BSA-DOX / NEs.

[0062] The present invention mixes the BSA aqueous solution with β-mercaptoethanol, and reversibly opens the disulfide bonds in BSA to obtain a protein solution. The present invention mixes the BSA aqueous solution with β-mercaptoethanol mainly to utilize β-mercaptoethanol to relax the compact structure of BSA, open the disulfide bonds, expose part of the hydrophobic domain, and serve as a hydrophobic drug binding site. As an optional embodiment of the present invention, the mass concentration of BSA in the BSA aqueous solution is 2 mg / mL. When the BSA aqueous solution is mixed with β-mercaptoethanol in the present invention, the volume of the β-mercaptoethanol can be 0.7% of the volume of the BSA aqueous solution. The present invention does not specifically limit the mixing method of the BSA aqueous solution and β-mercaptoethanol, and conventional mixing methods in the art can be used. After the BSA aqueous solution is mixed with β-mercaptoethanol, the present invention stirs for 3 minutes, reversibly opens the disulfide bonds in BSA, and obtains a protein solution. In the present invention, the stirring speed can be 500rpm.

[0063] After obtaining the protein solution, the present invention mixes the protein solution with the DOX·HCl aqueous solution and then ultrafilters to obtain BSA-DOX nanomaterials. The present invention preferably adjusts the protein solution to be alkaline; as an optional embodiment of the present invention, the adjustment can be adjusted to a pH of 10. After the present invention adjusts the protein solution to be alkaline, the DOX·HCl aqueous solution is slowly added, reacted for 0.5 to 1h, and ultrafiltered to obtain BSA-DOX nanomaterials. After the protein solution is mixed with the DOX·HCl aqueous solution, the reaction that occurs is mainly to bind DOX to the hydrophobic domain of the protein, thereby loading DOX. The present invention can use a NaOH solution with a molar concentration of 0.1M to adjust the pH. The present invention preferably slowly adds a NaOH solution when adjusting the pH. The present invention preferably adjusts the pH to 10. The main purpose of adjusting the pH to 10 in the present invention is to make the protein solution alkaline, and DOX·HCl becomes a hydrophobic drug under alkaline conditions, which is conducive to loading into BSA. After the pH is adjusted to 10, the present invention slowly adds a DOX·HCl aqueous solution to the protein solution. The actual effect of slowly adding the corresponding solution in the present invention is mainly to prevent liquid splashing and ensure that the liquid drips into the reaction bottle. In the present invention, the mass concentration of DOX·HCl in the DOX·HCl aqueous solution can be 3mg / mL. When the DOX·HCl aqueous solution is added in the present invention, the volume ratio of the DOX·HCl aqueous solution to the BSA aqueous solution can be 1:10. In the process of slowly adding the DOX·HCl aqueous solution, the present invention is preferably accompanied by stirring, and the stirring speed can be 500rpm. After the DOX·HCl aqueous solution and the protein solution are completely mixed, the stirring is carried out at 37°C for 1h; the stirring speed can be 500-800rpm. After the reaction is completed, the present invention preferably collects the supernatant of the reaction product and performs ultrafiltration to obtain BSA-DOX nanomaterials. The present invention preferably collects the supernatant of the reaction product by centrifugation; the centrifugal speed can be 10000rpm; the centrifugal time can be 10min. After obtaining the supernatant of the reaction product, the present invention ultrafilters the obtained supernatant. In the present invention, the ultrafiltration is preferably performed 3 times. In the present invention, each ultrafiltration can be performed using a 30kD ultrafiltration tube; the rotation speed of each ultrafiltration can be 5000rpm; and the time of each ultrafiltration can be 10min. After the ultrafiltration is completed, the present invention discards the filtrate, and the obtained retentate is the BSA-DOX nanomaterial. The average particle size of the BSA-DOX nanomaterial prepared by the method described in the above technical solution of the present invention is 160-180nm; the BSA-DOX nanomaterial is spherical.

[0064] After obtaining the BSA-DOX nanomaterial, the present invention mixes the neutrophils with the BSA-DOX nanomaterial and incubates them together for 1 hour to obtain BSA-DOX / NEs. The present invention preferably cultures the neutrophils in a culture medium and then mixes them with the BSA-DOX nanomaterial. In the present invention, the culture medium for neutrophil culture includes 1640 culture medium without fetal bovine serum; the culture temperature can be 37°C; the culture time can be 1 hour; the culture can be carried out under 5% CO 2 After the neutrophil culture is completed, the present invention adds the BSA-DOX nanomaterial to the culture medium of the neutrophils to mix the neutrophils with the BSA-DOX nanomaterial. In the present invention, when the BSA-DOX nanomaterial is added, it is preferred that the mass concentration of the BSA-DOX nanomaterial in the mixed system is 5 μg / mL. The temperature of the co-incubation in the present invention can be 37°C; the time of the co-incubation can be 1 hour; the co-incubation can be carried out at 5% CO 2 In the cell culture incubator. In the present invention, during the co-incubation process, the neutrophils incorporate the BSA-DOX nanomaterials into the cells through endocytosis, thereby obtaining a neutrophil drug delivery system. After the co-incubation is completed, the present invention preferably obtains BSA-DOX / NEs by centrifugation, washing and resuspension. The speed of the centrifugation in the present invention can be 1500rpm; the time of the centrifugation can be 3min. After the centrifugation is completed, the present invention discards the supernatant and washes it with PBS; the number of times of washing can be 3 times. The present invention mainly removes free nanomaterials by discarding the supernatant and washing. After the washing of the present invention is completed, the cell precipitate finally obtained is preferably resuspended with PBS. The preparation method of BSA-DOX / NEs provided by the present invention can significantly increase the uptake of BSA-DOX nanomaterials by neutrophils.

[0065] The present invention also provides the use of the drug described in the above technical solution in the preparation of a drug for treating tumors. In the present invention, the tumor includes: any one or more of breast cancer, acute leukemia, malignant lymphoma, bronchial lung cancer, ovarian cancer, soft tissue sarcoma, osteogenic sarcoma, rhabdomyosarcoma, Ewing sarcoma, blastoma, neuroblastoma, bladder cancer, thyroid cancer, prostate cancer, head and neck squamous cell carcinoma, testicular cancer, gastric cancer and liver cancer. In the present invention, the breast cancer includes triple-negative breast cancer.

[0066] The present invention provides a model of acute controllable inflammation in a tumor, and the method for constructing the model of acute controllable inflammation in a tumor comprises:

[0067] An acute controllable intratumoral inflammation model was established by intratumoral injection of LPS.

[0068] In the present invention, the tumor includes: any one or more of breast cancer, acute leukemia, malignant lymphoma, bronchogenic carcinoma, ovarian cancer, soft tissue sarcoma, osteogenic sarcoma, rhabdomyosarcoma, Ewing sarcoma, blastoma, neuroblastoma, bladder cancer, thyroid cancer, prostate cancer, head and neck squamous cell carcinoma, testicular cancer, gastric cancer and liver cancer. In the present invention, the breast cancer includes triple-negative breast cancer.

[0069] The present invention takes the body weight of the treated subject as the standard, and the injection amount of lipopolysaccharide can be 1 mg / kg. As an optional embodiment of the present invention, taking a mouse tumor model as an example, the weight of the mouse model is 20g, and when performing corresponding effect verification, the injection amount of LPS in the mouse tumor can be 0.02 mg.

[0070] The intratumor acute controllable inflammation model provided by the present invention uses the inflammatory tumor microenvironment for the first time to induce controllable acute inflammation in tumor tissue and amplify the inflammatory signals in the tumor microenvironment. The intratumor acute controllable inflammation model provided by the present invention can promote the infiltration of tumor drug delivery systems such as neutrophils in the tumor microenvironment, thereby realizing the specific targeting of nanomaterials loaded in the tumor drug delivery system, using neutrophils as carriers to achieve more accurate tumor targeted therapy, and using neutrophils to control the progress of systemic inflammatory response to ensure the safety of treatment. The present invention provides inducing local acute inflammation at the tumor site to improve the targeting of the tumor drug delivery system to the tumor and improve the therapeutic effect of anti-tumor drugs on the tumor. The present invention shows through the results of the examples that an intratumor acute controllable inflammation model is established by injecting LPS into triple-negative breast cancer tumors. The intratumor acute controllable inflammation model can amplify the inflammatory signals of the tumor microenvironment, and can significantly improve the therapeutic effect of triple-negative breast cancer while improving the drug targeting of the tumor drug delivery system.

[0071] The present invention provides the use of LPS in preparing a drug for improving the effect of a tumor drug delivery system in treating tumors; the tumor drug delivery system comprises neutrophils and therapeutic nanomaterials loaded into the neutrophils.

[0072] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0073] The raw materials used in the present invention, unless otherwise specified, are conventional commercially available products; unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art in the field to which the present invention relates.

[0074] The preparation method of the LPS solution used in the following examples is as follows: dissolve LPS solid powder in a sterile PBS solution, and vortex to mix thoroughly to prepare a 0.4 mg / mL LPS solution. The weight of the mice in the following experimental groups is 20 g.

[0075] Example 1

[0076] Establishment of controllable acute inflammation in the tumor of 4T1 tumor-bearing mouse model

[0077] (1) Take logarithmically growing 4T1 cells, digest them with trypsin, resuspend them in 1640 culture medium, and adjust the cell density with sterile phosphate buffered saline (PBS) for later use;

[0078] (2) BALB / c mice were used to establish a 4T1 tumor-bearing mouse model. Each BALB / c mouse was subcutaneously injected with (1) to obtain a concentration of 5×10 6 0.1 mL of 4T1 cell suspension of cells / L;

[0079] (3) When the subcutaneous tumor of the tumor-bearing mouse model established in (2) reaches 50 mm 3 Afterwards, 15 mice were randomly divided into 5 groups, each with 3 mice. One group was injected with 50 μL of PBS, and the other four groups were injected with 50 μL of LPS solution. The mass concentration of LPS in the LPS solution was 0.4 mg / mL. The four groups of tumor-bearing mice injected with LPS were euthanized after 6h, 9h, 12h, and 24h, respectively, and the tumor-bearing mice injected with PBS were euthanized after 24h. The tumor tissues of each group of mice were taken to prepare pathological sections, and immunohistochemical staining of neutrophil surface marker CD11b was performed.

[0080] (4) When the subcutaneous tumor of the tumor-bearing mouse model established in (2) reaches 50 mm 3 Afterwards, 18 mice were randomly divided into 6 groups, each with 3 mice. The control group was injected with 50 μL of PBS, and the other groups were injected with 50 μL of LPS solution. The mass concentration of LPS in the LPS solution was 0.4 mg / mL. Tumor tissues of mice were taken at 3h, 6h, 12h, 24h and 3d after LPS injection, and enzyme-linked immunosorbent assay (ELISA) was used to evaluate the expression level of inflammatory cytokines in tumor tissues after intratumoral injection of LPS.

[0081] (5) When the subcutaneous tumor of the tumor-bearing mouse model established in (2) reaches 50 mm 3Afterwards, 39 mice were taken and randomly divided into 11 groups, with 6 mice in the Control group and 3 mice in each of the other groups. The Control group was injected with PBS intratumorally, and the other groups were injected with 50 μL of LPS solution intratumorally, with the mass concentration of LPS in the LPS solution being 0.4 mg / mL. And after LPS injection, the blood of mice was taken for routine blood tests at 30 minutes, 3 hours, 6 hours, 9 hours, 12 hours, 24 hours, 3 days, 6 days, 9 days and 14 days of the experiment. The routine blood tests of the Control group (3 mice) were carried out immediately after the intratumoral injection of PBS. The blood biochemical tests of the Control group (3 mice) and the LPS group mice were carried out at 14 days.

[0082] Immunohistochemical staining results of neutrophil surface marker CD11b Figure 1 As shown, Figure 1 The scale bar in the figure is 100 μm. Figure 1 The immunohistochemical image shows neutrophil infiltration in tumor tissue of mice after intratumoral injection of LPS. The expression levels of inflammatory cytokines in tumor tissue after intratumoral injection of LPS are shown in Figure 2. Figures 2-3 shown. Figure 2 is the expression level of inflammatory cytokines CXCL1 and CXCL2 in tumor tissue after intratumoral injection of LPS; Figure 3 is the expression levels of proinflammatory cytokines TNF-α and IL-6 in tumor tissues after intratumoral injection of LPS. Figure 4 This is a blood routine test chart after intratumoral injection of LPS. Figure 5 shown. Figure 5 This is the blood biochemical diagram after LPS was injected into the mouse tumor. ** in the figure indicates significant difference, p<0.01, *** indicates extremely significant difference, p<0.001, and ns indicates no significant difference.

[0083] like Figure 1 As shown, the expression of CD11b in tumor tissue of tumor-bearing mice after LPS injection was significantly higher than that in the control group, and CD11b expression continued to increase from 6 hours to 12 hours, and began to decrease at 24 hours.

[0084] Depend on Figures 2-3 It can be seen that after intratumoral injection of LPS, the expression levels of neutrophil chemokines CXCL1 and CXCL2 in tumor tissue increased significantly. The expression levels of proinflammatory cytokines TNF-α and IL-6 increased significantly. Therefore, intratumoral injection of LPS successfully induced an inflammatory response in the tumor, promoted the accumulation of a large number of neutrophils, and was conducive to the precise targeting of neutrophil carriers at the tumor site after intravenous administration. The expression levels of these cytokines reached a peak 12 hours after intratumoral injection of LPS.

[0085] Depend on Figure 4 It can be seen that after intratumoral injection of LPS, the number of leukocytes and granulocytes in mice only increased briefly, and returned to normal levels 24 hours after LPS injection. Therefore, the optimal time for intravenous injection of neutrophil carriers is 12 hours after intratumoral injection of LPS.

[0086] Figure 5 The results showed that the liver and kidney function of tumor-bearing mice was not impaired when the liver and kidney function of the mice was evaluated 14 days after intratumoral injection of LPS. Although LPS can induce the desired acute inflammatory response in the tumor, it is crucial to prevent the systemic inflammatory response caused by LPS and achieve a controllable inflammatory response.

[0087] Example 2

[0088] (1) After BALB / c mice were killed by cervical dislocation, their femurs and tibias were stripped and soaked in 75% alcohol for 3 minutes. Then, they were taken to the cell room for operation. A 1 mL syringe was used to absorb tissue diluent (Beijing Solebold Technology Co., Ltd.), and gently inserted into the bone marrow cavity to flush out the bone marrow in the femur and tibia. This operation was repeated repeatedly until all the bone marrow was flushed out. The flushed bone marrow fluid was carefully spread into a centrifuge tube containing neutrophil separation solution (Beijing Solebold Technology Co., Ltd.), centrifuged at 1100g for 30 minutes, and the lower ring-shaped milky white cell layer was carefully aspirated into a 15 mL centrifuge tube. Cell washing solution (Beijing Solebold Technology Co., Ltd.) was added to 10 mL, and centrifuged at 250g for 10 minutes. The supernatant was discarded, and the cell precipitate was washed 3 times with cell washing solution to obtain pure neutrophils.

[0089] (2) Tumor-bearing mice were established according to the method of Example 1, and the subcutaneous tumor reached 50 mm 3 Afterwards, the mice were randomly divided into 6 groups, each with 3 mice. One group was injected with 50 μL of PBS, and the other five groups were injected with 50 μL of LPS solution. The mass concentration of LPS in the LPS solution was 0.4 mg / mL. Four of the five groups of tumor-bearing mice injected with LPS were given 5×10 5 , 1×10 6 , 2×10 6 , 4×10 6 Neutrophil infusion was performed and the expression of inflammatory cytokines in serum was measured to determine whether neutrophil infusion could alleviate the systemic inflammatory response induced by LPS.

[0090] The expression levels of inflammatory cytokines in the serum of tumor-bearing mice after neutrophil infusion after LPS injection into the tumor were as follows Figure 6 shown.

[0091] Neutrophil transfusion can effectively inhibit inflammation caused by bacterial infection in patients with neutropenia. Neutrophils can reduce the toxic effects of LPS. Neutrophil transfusion was performed on tumor-bearing mice 12 hours after intratumoral injection of LPS, and the expression of inflammatory cytokines in serum was measured to determine whether neutrophil transfusion can alleviate the systemic inflammatory response induced by LPS. Figure 6 The number of neutrophils transfused to each mouse reached 1×10 6 The expression levels of pro-inflammatory cytokines IL-6 and TNF-α in serum returned to normal levels within 2 hours after transfusion. The results showed that LPS can induce controllable transient acute inflammation in tumor tissues, and the development of systemic inflammation can be controlled by neutrophils.

[0092] Example 3

[0093] Preparation of nanomaterials with neutrophils as carriers as drugs for treating tumors:

[0094] (1) Preparation of BSA-DOX nanomaterials

[0095] 10 mg of bovine serum albumin BSA was dissolved in 5 mL of deionized water to obtain a BSA aqueous solution, which was placed in a round-bottom flask and stirred at 37 ° C and 500 rpm for several minutes, and then 35 μL of β-mercaptoethanol was added to the solution to reversibly open the disulfide bonds in BSA. After stirring for 3 minutes, a 0.1 M NaOH solution was slowly added to the flask to adjust the pH of the solution to 10, and then 1.5 mg of doxorubicin hydrochloride DOX·HCl was weighed and dissolved in 500 μL of deionized water to prepare a 3 mg / mL DOX·HCl aqueous solution, which was slowly added to the stirring solution and continued to stir in a 37 ° C water bath for 1 hour. Subsequently, the solution was centrifuged at 10000 rpm for 10 minutes, the supernatant was collected, and the filtrate was discarded after ultrafiltration three times with a 30 kD ultrafiltration tube at 5000 rpm. The final product was left on top, and the final product BSA-DOX nanomaterial was obtained.

[0096] like Figure 7 As shown in FIG. 1 , the dynamic scattering hydrated particle size (DLS) diagram of the prepared BSA-DOX nanomaterials is shown. The average particle size of the nanomaterials is 160 to 180 nm. Figure 8 As shown, it is a scanning electron microscope (SEM) image, and it can be seen that the BSA-DOX nanomaterial is spherical.

[0097] (2) Preparation of BSA-DOX / NEs

[0098] After BALB / c mice were killed by cervical dislocation, their femurs and tibias were stripped, soaked in 75% alcohol for 3 minutes, and then taken to the cell room for operation. A 1mL syringe was used to absorb tissue diluent (Beijing Solebow Technology Co., Ltd.), gently inserted into the bone marrow cavity, and the bone marrow in the femur and tibia was flushed out. This operation was repeated repeatedly until the bone marrow was completely flushed out. The flushed bone marrow fluid was carefully spread into a centrifuge tube containing neutrophil separation solution (Beijing Solebow Technology Co., Ltd.), centrifuged at 1100g for 30min, and the lower ring-shaped milky white cell layer was carefully drawn into a 15mL centrifuge tube, and cell washing solution (Beijing Solebow Technology Co., Ltd.) was added to 10mL, centrifuged at 250g for 10min, and the supernatant was discarded. The cell pellet was washed 3 times with cell washing solution to obtain pure neutrophils.

[0099] The neutrophils obtained above were placed in 1640 medium without fetal bovine serum (FBS) and incubated at 37°C and 5% CO 2 The prepared nanomaterials were cultured in a cell culture incubator for 1 h, and then added to the culture medium containing neutrophils (10 mL of 1640 culture medium containing 3×10 6 After fully shaking, the mixture was incubated in a cell culture incubator for 1 hour, taken out, centrifuged (1500 rpm, 3 min), the supernatant was discarded, free nanomaterials were removed, and the mixture was washed three times with PBS, the supernatant was discarded, and the cell pellet was finally resuspended with PBS to obtain BSA-DOX / NEs.

[0100] The cell morphology of the BSA-DOX / NEs obtained in this example was observed by Wright-Giemsa staining. Fig. 9 As shown, neutrophils maintained their complete cell morphology and lobed nuclear morphology before and after taking up the nanomaterials.

[0101] Example 4

[0102] Validation of neutrophil uptake of therapeutic nanomaterials following co-incubation with them:

[0103] (1) Take the BSA-DOX / NEs prepared in Example 3 and count 1×10 6 Neutrophils were centrifuged and the supernatant was discarded. They were resuspended in 1 mL of 4% tissue fixative (Beijing Solebow Technology Co., Ltd.) in a 2 mL centrifuge tube and the samples were placed in a 4°C refrigerator for fixation for 20 min.

[0104] (2) After fixation, the sample was taken out of the refrigerator, centrifuged (1500 rpm, 3 min), the supernatant was discarded, and the sample was washed three times with PBS. After resuspending in 1 mL of PBS, 2 μL of 4',6-diamidino-2-phenylindole Dapi and 2 μL of cell membrane green fluorescent probe Dio dye were added to the sample, and the sample was incubated and stained for 5 min in the dark;

[0105] (3) After staining is completed, the sample is taken out, centrifuged and the supernatant is discarded, washed three times with PBS, resuspended in 1 mL PBS, added to the confocal dish, and the sample is observed using a confocal laser scanning microscope (CLSM). The results are as follows: Fig.10 shown. Fig.10 Laser scanning confocal microscopy images of BSA-DOX / NEs, with a scale bar of 5 μm.

[0106] like Fig.10 As shown in the figure, the blue fluorescence is the neutrophil nucleus, the green fluorescence is the Dio-stained cell membrane, and the red fluorescence is DOX. It can be observed from the Merge diagram that DOX has entered the neutrophil membrane but not the nucleus. The results show that the neutrophils have successfully taken up the BSA-DOX nanomaterials, and also indicate the successful preparation of BSA-DOX / NEs. After centrifugation (1500rpm, 3min), the amount of BSA-DOX in the supernatant was detected, and the final amount of BSA-DOX taken up by the neutrophils was 7.921μg / 10 6 cell.

[0107] Example 5

[0108] In vitro killing experiment in which BSA-DOX / NEs produces neutrophil extracellular traps (NETs) under the stimulation of PMA and releases BSA-DOX to produce toxic effects on tumor cells:

[0109] First, triple-negative breast cancer cells 4T1 (about 3000 cells / well) were inoculated in a 96-well plate and incubated in 1640 cell culture medium for 24 hours. Then, according to the method for preparing BSA-DOX / NEs in Example 3, neutrophils were co-incubated with different concentrations (0μg / mL, 0.5μg / mL, 1μg / mL, 2μg / mL, 5μg / mL) of BSA-DOX to obtain BSA-DOX / NEs containing different concentrations of DOX. BSA-DOX / NEs with different concentrations of DOX were divided into two groups, and pretreated with and without PMA (100nM, phorbol ester, used to simulate inflammatory sites in the body) for 4 hours. Then, BSA-DOX / NEs without PMA treatment and PMA treatment were centrifuged at 2000rpm for 5 minutes. The supernatant was then incubated with 4T1 cells for different times (24h, 48h), and then the MTT solution was added, and the cell viability was detected according to the standard MTT method, as shown in FIG. Fig.11 and Fig.12 Each experimental group was subjected to three biological replicates. Fig.11 and Fig.12 The data are from three independent samples and are expressed as mean ± standard deviation. Fig.11 The in vitro killing effect of BSA-DOX / NEs with different concentrations of DOX on 4T1 cells without PMA treatment and after PMA treatment; Fig.12 The in vitro killing effects of BSA-DOX / NEs with different concentrations of DOX on 4T1 cells after PMA treatment at 24h and 48h.

[0110] Fig.11 The data were derived from three independent samples and expressed as mean ± standard deviation. Fig.11 It can be observed that after 48 hours of incubation, in both the groups without PMA treatment and the groups treated with PMA, the cell survival rate gradually decreased with the increase of BSA-DOX concentration, but the supernatant obtained by PMA treatment had a more significant killing effect on the cells; Fig.12 It can be observed that as the incubation time increases, the cell survival rate gradually decreases. The above experimental results prove that BSA-DOX / NEs will release more BSA-DOX at the inflammatory site, thereby killing tumor cells, and the longer the time, the stronger the killing effect.

[0111] Example 6

[0112] Targeted chemotaxis of BSA-DOX / NEs to tumor tissues inducing controlled inflammation

[0113] (1) Take logarithmically growing 4T1 cells, digest them with trypsin, resuspend them in 1640 culture medium, and adjust the cell density with sterile phosphate buffered saline (PBS) for later use;

[0114] (2) BALB / c mice were used to establish a 4T1 tumor-bearing mouse model. Each BALB / c mouse was subcutaneously injected with (1) to obtain a concentration of 5×10 6 0.1 mL of 4T1 cell suspension of cells / L;

[0115] (3) When the subcutaneous tumor of the tumor-bearing mouse model established in (2) reaches 50 mm 3 Afterwards, the mice were randomly divided into 2 groups: mice in group 1 were injected intratumorally with LPS (50 μL, 2 mg / mL), and mice in group 2 were injected intratumorally with PBS (50 μL);

[0116] (4) Take the BSA-DOX / NEs in Example 3 and incubate them with the cell membrane dye DiD (10 μM) for 10 min. Wash three times with ice-cold PBS. 12 h after intratumoral injection of LPS or PBS, both groups of mice were injected with DID-stained BSA-DOX / NEs (dosage of 3×10 per mouse) through the tail vein. 6 neutrophils), and major organs were collected for in vitro fluorescence imaging 12 hours after administration.

[0117] like Fig.13 As shown in the figure, in breast cancer mice injected with PBS intratumorally, BSA-DOX / NEs were mainly distributed in the liver and spleen. However, in breast cancer mice with controlled inflammation injected with LPS intratumorally, the accumulation of BSA-DOX / NEs in the liver was significantly reduced, while the accumulation in tumor tissue was significantly increased compared with the control group. This shows the importance of inducing controlled inflammation in tumors, which can effectively amplify inflammatory signals and significantly improve the tumor targeting ability of neutrophil vectors.

[0118] Example 7

[0119] BSA-DOX / NEs was applied to the 4T1 tumor-bearing mouse model to achieve tumor inhibitory effect.

[0120] (1) Take logarithmically growing 4T1 cells, digest them with trypsin, resuspend them in 1640 culture medium, and adjust the cell density with sterile phosphate buffered saline (PBS) for later use;

[0121] (2) BALB / c mice were used to establish a 4T1 tumor-bearing mouse model. Each BALB / c mouse was subcutaneously injected with (1) to obtain a concentration of 5×10 6 0.1 mL of 4T1 cell suspension of cells / L;

[0122] (3) When the subcutaneous tumor of the tumor-bearing mouse model established in (2) reaches 50 mm 3 Afterwards, the mice were randomly divided into 6 groups, with 3 mice in each group, and recorded as group 1, group 2, group 3, group 4, group 5 and group 6. Group 1, group 3 and group 4 were injected with PBS (50 μL) intratumorally, and group 2, group 5 and group 6 were injected with LPS (50 μL, 2 mg / mL) intratumorally. At 12 h and 3 days after intratumoral injection, group 1 and group 2 were injected with PBS via tail vein, group 3 and group 5 were injected with BSA-DOX nanomaterials (injection amount: 50 μg DOX per 20 g mouse), and group 4 and group 6 were injected with BSA-DOX / NEs cell vectors (injection amount: 3×10 6 cells).

[0123] (4) The tumor size was measured every 2 days, the mice were weighed, and the tumor volume was measured by measuring the length and width of the tumor with a vernier caliper. On the 14th day, the mice were euthanized. The BSA-DOX nanomaterial and the BSA-DOX / NEs cell vector were prepared according to the preparation method of Example 3 of the present invention. Three biological replicates were performed for each experimental group.

[0124] like Figures 14 to 16 As shown, Fig.14 The tumor-bearing mice were injected with PBS, PBS, BSA-DOX nanomaterials, BSA-DOX / NEs cell vectors, BSA-DOX nanomaterials, and BSA-DOX / NEs cell vectors through the tail vein. The 1st, 3rd, and 4th groups were injected with PBS, and the 2nd, 5th, and 6th groups were injected with LPS. The changes in tumor volume of the tumor-bearing mice within 14 days. Fig.15 The following are photos of tumors in each group of tumor-bearing mice after 14 days of the above treatment. Fig.16 The weight of tumors in each group of tumor-bearing mice after 14 days of treatment. Figures 14 to 16 It can be seen that the tumors of the tumor-bearing mice in group 1 injected with PBS in the tail vein grew rapidly within 14 days; the tumor-bearing mice in group 2 injected with LPS intratumorally and PBS in the tail vein had only a small inhibitory effect on tumor growth under the condition of inducing autoimmunity; the tumor-bearing mice in group 3 injected with PBS intratumorally and BSA-DOX nanomaterials in the tail vein had increased tumor growth inhibition compared with group 2 due to the passive targeting effect of BSA-DOX; the tumor-bearing mice in group 4 injected with PBS intratumorally and BSA-DOX / NEs cell vectors in the tail vein had no significant effect even after the addition of neutrophils with active targeting effect. The tumor area does not have enough inflammatory factors to make a large number of neutrophils migrate to the tumor site, so the growth of the tumor is only slightly inhibited; the 5 groups of tumor-bearing mice injected with LPS intratumorally and BSA-DOX nanomaterials into the tail vein, after stimulating autoimmunity and having the passive targeting effect of nanomaterials, the growth of the tumor is further inhibited; the 6 groups of tumor-bearing mice injected with LPS intratumorally and BSA-DOX / NEs cell carriers into the tail vein, acute inflammation causes a large number of neutrophil carriers to migrate to the tumor site, and the growth of the tumor is significantly inhibited. The above experimental results show that the anti-tumor drug prepared by the nanomaterial with neutrophils as carriers provided by the present invention can achieve tumor inhibition.

[0125] Example 8

[0126] In vitro killing experiment in which BSA-DOX / NEs produces neutrophil extracellular traps (NETs) under the stimulation of PMA and releases BSA-DOX to produce toxic effects on tumor cells:

[0127] First, prostate cancer cells PC3 (about 3000 cells / well) were inoculated in a 96-well plate and incubated in 1640 cell culture medium for 24 hours. Then, according to the method for preparing BSA-DOX / NEs in Example 3, neutrophils were co-incubated with different concentrations (0μg / mL, 0.5μg / mL, 1μg / mL, 2μg / mL, 5μg / mL) of BSA-DOX to obtain BSA-DOX / NEs containing different concentrations of DOX. BSA-DOX / NEs with different concentrations of DOX were divided into two groups, and pretreated with and without PMA (100nM, phorbol ester, used to simulate inflammatory sites in the body) for 4 hours. Then, BSA-DOX / NEs without PMA treatment and PMA treatment were centrifuged at 2000rpm for 5 minutes. The supernatant was then incubated with PC3 cells for different times (24h, 48h), and then the MTT solution was added to detect cell viability according to the standard MTT method, as shown in FIG. Fig.17 and Fig.18 Each experimental group was subjected to three biological replicates. Fig.17 and Fig.18 The data are from three independent samples and are expressed as mean ± standard deviation. Fig.11 The in vitro killing effects of BSA-DOX / NEs with different concentrations of DOX on PC3 cells without PMA treatment and after PMA treatment; Fig.12 The in vitro killing effects of BSA-DOX / NEs with different concentrations of DOX on PC3 cells after PMA treatment at 24h and 48h.

[0128] Fig.17 It can be observed that after 48 hours of incubation, in both the groups without PMA treatment and the groups treated with PMA, the cell survival rate gradually decreased with the increase of BSA-DOX concentration, but the supernatant obtained by PMA treatment had a more significant killing effect on the cells; Fig.18 It can be observed that as the incubation time increases, the cell survival rate gradually decreases. The above experimental results prove that BSA-DOX / NEs will release more BSA-DOX at the inflammatory site, thereby killing tumor cells, and the longer the time, the stronger the killing effect.

[0129] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A drug for treating tumors, characterized in that: include: LPS and tumor drug delivery system; the tumor drug delivery system includes neutrophils and therapeutic nanomaterials loaded into the neutrophils.

2. The drug according to claim 1, characterized in that The direct site of action of LPS is inside the tumor.

3. The drug according to claim 2, characterized in that: The tumor drug delivery system is used after the LPS acts inside the tumor for 6 to 12 hours.

4. The drug according to claim 1, characterized in that The neutrophils include animal bone marrow neutrophils and / or peripheral blood neutrophils.

5. The drug according to claim 1, characterized in that: The therapeutic nanomaterials include nanocarriers and tumor therapeutic drugs; The nanocarrier comprises any one or more of human serum albumin, liposome, mesoporous silica nanoparticles, graphene, micelle, nanocapsule and gold nanorod; The tumor therapeutic drugs include any one or more of drugs that inhibit enzyme activity in tumor cells, drugs that inhibit tubulin formation, drugs that inhibit DNA formation, drugs that inhibit RNA formation, and drugs that cause tissue hypoxia.

6. The drug according to claim 1, characterized in that: The tumor drug delivery system includes BSA-DOX / NEs; The preparation method of BSA-DOX / NEs comprises: After mixing the BSA aqueous solution with β-mercaptoethanol, the disulfide bonds in the BSA are reversibly opened to obtain a protein solution; The protein solution was mixed with a DOX·HCl aqueous solution and then ultrafiltered to obtain BSA-DOX nanomaterials; Neutrophils were mixed with BSA-DOX nanomaterials and co-incubated to obtain BSA-DOX / NEs.

7. The drug according to claim 6, characterized in that: The protein solution was adjusted to be alkaline and then mixed with a DOX·HCl aqueous solution.

8. Use of the drug according to any one of claims 1 to 7 in the preparation of a product for treating tumors.

9. An acute controllable inflammation model in a tumor, characterized in that: The method for constructing the intratumor acute controllable inflammation model comprises: An acute controllable intratumoral inflammation model was established by intratumoral injection of LPS.

10. The use of LPS in the preparation of a drug for improving the effect of a tumor drug delivery system in treating tumors; the tumor drug delivery system comprises neutrophils and therapeutic nanomaterials loaded into the neutrophils.

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