Preparation and anti-tumor application of Ilisimu / copper-NLG919 bionic nanoparticles wrapped by tumor cell membranes

Abstract

The invention discloses preparation and anti-tumor application of tumor cell membrane coated Ilisimus / copper-NLG919 bionic nanoparticles, and particularly relates to a bionic nano-drug which is characterized in that NLG919, ES and Cu < 2 + > are used as active ingredients to construct a self-assembled drug system (ECN), and meanwhile, a tumor cell membrane is used for coating the ECN to form ECNM; compared with a traditional EC, the built ECNM has the advantages that the systemic circulation stability, tumor targeting and tumor accumulation of the EC are remarkably improved, copper death and chemical kinetic therapy (CDT) can be efficiently triggered, a tumor immunosuppression microenvironment (TIM) can be synergistically reversed, and then a higher tumor immunotherapy effect is achieved. The nano-drug disclosed by the invention has good biological safety, cycling stability and homologous tumor targeting property, the preparation method is simple and convenient, and materials and equipment are easy to obtain.

Description

Technical Field

[0001] The invention belongs to the field of biopharmaceuticals, and specifically discloses the preparation of ilisimol / copper-NLG919 bionic nanoparticles wrapped by tumor cell membranes and the anti-tumor application thereof. Background Art

[0002] In recent years, reversing the tumor immunosuppressive microenvironment (TIM) to activate autoimmune responses to fight tumors and their metastasis and recurrence has been widely studied. However, in the complex in vivo environment, plasma proteins can bind to drugs, causing them to be phagocytosed and cleared by the reticuloendothelial system, making it difficult to distribute to tumor tissues, which in turn leads to intravenous anti-tumor drug delivery strategies that are usually difficult to achieve therapeutic endpoints. Therefore, it is crucial to develop a formulation that is highly stable, concealed, and targeted. In recent years, cell membrane-coated nanoparticles have been used to "evade" the surveillance of immune cells in the body. For example, red blood cell membranes can significantly prolong the circulation time of nanoparticles in the body, but red blood cell membrane coatings lack tumor targeting specificity. In order to solve the above problems,

[0003] Previous studies have shown that the immunogenic death (ICD) of tumor cells can activate the tumor immune system. Immunogenically dead cells highly express tumor-associated antigens and damage-associated molecular patterns (DAMPs), such as calreticulin (CRT), adenosine triphosphate (ATP), and high-mobility group protein B1 (HMGB1). These substances recruit and activate dendritic cells to present antigens, promote T lymphocyte proliferation and differentiation, and then activate tumor immune responses. However, tumor immune responses are usually inhibited by TIMs. Copper ions are common ICD inducers. Cuproptosis induced by copper overload and chemodynamic therapy (CDT) are both believed to trigger ICD. Cuproptosis is a new cell death pathway discovered in recent years. It is a copper-dependent death caused by intracellular copper overload. It regulates ferredoxin 1 (FDX1) and induces fatty acylation aggregation of dihydrosulfamide S-acetyltransferase (DLAT) and downregulates iron-sulfur cluster proteins, ultimately causing protein toxic stress and leading to cell death. In addition, excess intracellular copper ions are reduced to cuprous ions by glutathione (GSH), and cuprous ions reduce hydrogen peroxide (H2O2) to generate toxic hydroxyl radicals (·OH) to trigger CDT. However, the copper content in tumor cells is low and is not enough to trigger copper-dependent copper death and CDT. In summary, increasing the copper ion content in tumor cells and regulating the tumor microenvironment to improve the efficiency of immune response is an attractive combined treatment strategy.

[0004] Elisimol (ES) is a highly efficient copper ion carrier with good biosafety and mitochondrial targeting. It can efficiently deliver copper ions into cells and induce copper death and CDT. However, the short half-life, poor stability and low tumor targeting efficiency of ES limit its clinical application. Summary of the invention

[0005] To solve the above problems, we disclose the preparation of tumor cell membrane-wrapped ilisimol / copper-NLG919 biomimetic nanoparticles and their anti-tumor application. The nanoparticles of the present invention activate ICD through copper death and CDT pathways, and reverse TIM to amplify tumor immune response, which can effectively inhibit tumor metastasis and recurrence while killing in situ tumors. The nanomedicine of the present invention has good biosafety, circulation stability and homologous tumor targeting, the preparation method is simple and convenient, and the materials and equipment are easy to obtain.

[0006] The present invention includes the following specific technical solutions:

[0007] A method for preparing tumor cell membrane-wrapped ilisimol / copper-NLG919 biomimetic nanoparticles, characterized in that it comprises the following steps:

[0008] S1. Extraction of tumor cell membranes: digestion and collection of tumor cells with pancreatic enzyme digestion solution, and suspension of the tumor cells in membrane protein extraction buffer. Ultrasonication with a probe is used to destroy the integrity of tumor cells, and ultracentrifugation is used to collect cell membranes.

[0009] S2. Preparation of core nanoparticles: dissolving elisemol ES and NLG919 in dimethyl sulfoxide (DMSO) and mixing; dropping copper chloride solution into the mixed solution and stirring at room temperature for several hours; centrifuging to remove unreacted copper chloride, dropping the supernatant into distilled water and stirring continuously for several decimal points; dialyzing to remove the organic solvent to obtain nanoparticles, storing them at low temperature, and naming them ECN;

[0010] S3. Preparation of coated nanomedicine:

[0011] The ECN obtained in step S2 and the tumor cell membrane obtained in step S1 are ultrasonically mixed under ice bath conditions; and the tumor cell membrane-wrapped ilisimol / copper-NLG919 biomimetic nanoparticles are obtained by centrifugation, which are named ECNM.

[0012] Furthermore, the above-mentioned method for preparing ilisimol / copper-NLG919 biomimetic nanoparticles wrapped by tumor cell membrane, wherein the tumor cells include but are not limited to breast cancer cells (such as 4T1 cells), lung cancer cells (such as A549 cells), gastric cancer cells (such as MGC-803 cells), liver cancer cells (such as HepG2 cells), colon cancer cells (such as HT-29 cells), pancreatic cancer cells (such as PANC-1 cells), prostate cancer cells (such as PC-3 cells), ovarian cancer cells (such as SKOV3 cells), melanoma cells (such as B16 cells) and leukemia cells (such as K562 cells).

[0013] Furthermore, in the method for preparing the above-mentioned tumor cell membrane-wrapped ilisimol / copper-NLG919 bionic nanoparticles, in step S1, the ultrasonic parameters are: 30%, On:Off=2:3, 30 min.

[0014] Furthermore, in the preparation method of the above-mentioned tumor cell membrane-wrapped ilisimol / copper-NLG919 bionic nanoparticles, in step S1, the ultracentrifugation step includes: centrifuging at 20,000 rpm at 4°C for 30 min, collecting the supernatant, and then centrifuging the supernatant at 100,000 rpm at 4°C for 30 min, collecting the precipitate, and obtaining the cell membrane.

[0015] Furthermore, in the above-mentioned method for preparing tumor cell membrane-wrapped elisemol / copper-NLG919 biomimetic nanoparticles, in step S2, the mass ratio of elisemol ES and NLG919 is 1:1; the mass ratio of elisemol ES and copper chloride is 1:2; and the mass-volume ratio of elisemol ES and DMSO is 5:1 mg / mL.

[0016] Furthermore, in the method for preparing the above-mentioned tumor cell membrane-wrapped ilisimol / copper-NLG919 biomimetic nanoparticles, in step S3, the tumor cell membrane and ECN are mixed in a mass ratio of 6:1.

[0017] Furthermore, the preparation method of the above tumor cell membrane-wrapped ilisimol / copper-NLG919 biomimetic nanoparticles,

[0018] The invention also discloses ilisimol / copper-NLG919 bionic nanoparticles wrapped with tumor cell membranes, which are prepared by the above method.

[0019] Furthermore, the present invention discloses the use of the bionic nanoparticles in preparing drugs for treating cancer.

[0020] Furthermore, in the above application, the cancer includes but is not limited to breast cancer, lung cancer, gastric cancer, liver cancer, colon cancer, pancreatic cancer, prostate cancer, ovarian cancer, melanoma, acute myeloid leukemia and lymphoma.

[0021] The invention also discloses a medicine for treating cancer, characterized in that the medicine contains the above-mentioned bionic nanoparticles.

[0022] Compared with the prior art, the present invention has the following outstanding beneficial effects:

[0023] The present invention discloses the preparation of tumor cell membrane-wrapped ilisimol / copper-NLG919 bionic nanoparticles and their anti-tumor application, and the principle is as follows:

[0024] In the present invention, NLG919 (an IDO1 inhibitor) which also has an unsaturated aromatic ring is combined with ES and Cu 2+ Blending, using ES and Cu 2+ Self-assembled nanoparticles (ECN) are constructed by the coordination bonds between NLG919 and ES molecules, as well as the π-π conjugation and hydrogen bonding between NLG919 and ES molecules; the addition of NLG919 can effectively prolong the half-life of ES, increase the stability of nanoparticles, and reduce the release of drugs in non-target sites; at the same time, NLG919, as a highly efficient indoleamine 2,3-dioxygenase 1 (IDO1) inhibitor, can reverse the tumor immunosuppressive microenvironment, thereby increasing the specific killing of tumor cells by T cells; in order to further improve the circulation stability and tumor targeting of self-assembled nanoparticles (ECN), tumor cell membranes are used to coat ECN to form tumor cell membrane-coated self-assembled nanoparticles (ECNM); tumor cell membrane-coated nanoparticles have high circulation stability and low immunogenicity, and can fuse with homologous tumor cells to enhance targeted uptake, which can significantly improve the tumor treatment effect of nanoparticles.

[0025] Several embodiments of the present invention can prove that the present invention has the following specific beneficial effects:

[0026] 1) The nanomedicine prepared by the present invention synergistically activates tumor immunity by inducing cell copper death / CDT and reversing TIM. Ilismox efficiently loads copper ions, which can effectively increase the copper ion content in cells, trigger cell copper death and CDT to activate tumor immunogenicity. NLG919 can reduce the inhibitory effect of the tumor microenvironment on cellular immunity, synergistically activate tumor immune response through multiple pathways, effectively kill tumor cells and inhibit metastasis and recurrence.

[0027] 2) The nanomedicine prepared by the present invention has good stability and tumor targeting. Ilismox efficiently binds to copper ions through coordination bonds, interacts with NLG919 through π-π conjugation and hydrogen bonds, promotes the self-assembly of nanoparticles and improves the stability of the system. The addition of NLG919 not only improves the shortcomings of Ilismox's short half-life and lack of tumor targeting, but also improves the stability of nanoparticles and reduces the release of drugs in non-target sites. In addition, the wrapping of tumor cell membranes can not only avoid the adsorption of intravascular substances and reduce the clearance of nanomedicines, but also achieve targeted uptake through membrane fusion with homologous tumor cells, and release drugs in response to high intracellular GSH.

[0028] 3) The method for preparing nanomedicine of the present invention is simple, the materials are easy to obtain, and no expensive reagents and equipment are involved. The nanomedicine has good storage stability and efficacy, and is suitable for clinical application and promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 (A) is the SDS-PAGE image of the outer cell membrane of the nanodrug ECNM, in which from left to right are marker, ECN, 4T1 cell membrane, a mixture of 4T1 cell membrane and ECN, and ECNM. It can be seen that ECNM retains most of the proteins on the 4T1 cell membrane; (B) is the immunoglobulin image of the marker detection on the 4T1 cell membrane, in which from left to right are 4T1 cells, 4T1 cell membrane, and ECNM;

[0030] Figure 2 The particle size distribution and electron microscope images of nanomedicine (A) EC, (B) ECN, (C) ECNM, (D) is the particle size;

[0031] Figure 3 Conditional release of GSH from ECNM at 37°C: (A) ES, (B)

[0032] NLG919, (CE) Storage stability of EC, ECN, and ECNM in different media;

[0033] Figure 4 (A) The cytotoxicity of conditioned medium with different concentrations of CuCl2 on 4T1 cells. The cytotoxicity of nanomedicines on 4T1 cells was detected by co-action of CuCl2 with different concentrations (B) 0, (C) 100 nM, and (D) 1 μM.

[0034] Figure 5 After the nanomedicine was co-incubated with 4T1 cells, the relative expression levels of (AB) FDX1 and (CD) IDO1 in the cells were determined by Western blotting;

[0035] Figure 6After the nanodrug was co-incubated with 4T1 cells, (A) DCFH-DA probe was used to perform fluorescence imaging of intracellular ROS, and the intracellular distribution of ROS was collected by laser confocal microscopy. Related kits were used to quantitatively measure the intracellular (B) ROS and (C) GSH contents.

[0036] Figure 7 After the nanomedicine was co-incubated with 4T1 cells, (A) CRT antibody-labeled fluorescence imaging and the kit detected the cell release of (B) ATP and (C) HMGB1;

[0037] Figure 8 Nanomedicine was injected into tumor-bearing mice through the tail vein. (A) In vivo imaging of fluorescently labeled nanomedicine in mice, as well as (B) fluorescence imaging and (C) quantitative fluorescence analysis of nanomedicine in major tissues and organs, (D) tumor volume changes and (E) final tumor weight. DETAILED DESCRIPTION

[0038] The embodiments of the present invention include the following main contents:

[0039] (1) The present invention prepares coated nanomedicines by first extracting tumor cell membranes, culturing and amplifying 4T1 cell lines, and using a membrane protein separation kit to obtain cell membranes: the steps include placing cells in a membrane protein extract, disrupting the cells with an ultrasonic probe, and then centrifuging to collect the cell membranes.

[0040] (2) The present invention is to prepare coated nanomedicines, and core nanoparticles are prepared by drug self-assembly. ES and NLG919 are mixed and dissolved in DMSO, and then CuCl2 solution is added dropwise to the mixed solution, and stirred for 6 hours at room temperature. The supernatant is then added dropwise to deionized water under continuous stirring and dialyzed to remove the organic solvent. The prepared nanoparticles are stored at 4°C and named ECN; the nanoparticles without NLG919 added during the preparation process are named EC; the obtained ECN is ultrasonically mixed with the tumor cell membrane under ice bath conditions, and the coated nanomedicine is obtained by centrifugation, which is named ECNM.

[0041] (3) The present invention characterizes the particle size and appearance of the nanomedicine, and identifies the cell membrane protein content of the outer layer of ECNM. The storage stability of the nanomedicine in PBS, DMEM and DMEM containing 10% FBS at 4°C, as well as the drug release behavior of the nanomedicine under GSH conditions, are measured.

[0042] (4) The present invention uses in vitro cell experiments to verify that ECNM activates cellular immunity.

[0043] Cytotoxicity analysis: 4T1 cells were co-incubated with CuCl2 conditioned medium and nanodrugs were co-incubated with 4T1 cells in CuCl2 conditioned medium. Cell activity was detected by MTT assay after 24 h.

[0044] Protein expression analysis: 4T1 cells were pre-incubated with INF-γ to stimulate IDO1 expression, and then nanomedicine was added for incubation. The expression of tumor immune-related enzyme IDO1 was detected by WB. The detection of copper death-related protein FDX1 was consistent with the above operation, but no pre-treatment with INF-γ was required.

[0045] Analysis of cell immunogenic death: After the nanodrug was co-incubated with 4T1 cells for 12 hours, the cells after administration were incubated with DCFH-DA, and the intracellular ROS distribution map was photographed by laser confocal microscopy. The intracellular ROS and GSH contents were detected by relevant kits. The cells after administration were immunofluorescently labeled with CRT, and the CRT fluorescence images were photographed by laser confocal microscopy. The contents of ATP and HMGB1 in the cell culture medium were detected by relevant kits.

[0046] (5) The present invention uses in vivo animal experiments to verify that ECNM reverses tumor immunosuppression.

[0047] In vivo and intratumoral distribution of drugs: Balb / c mice were inoculated with 4T1 cells to construct a tumor-bearing mouse model. The tumor-bearing mice were randomly divided into groups, and the nanodrugs were loaded with fluorescent dye DiR and intravenously injected. The mice were anesthetized at the set time points to take the fluorescence distribution map of the drug in the body. After the experiment, the mice were euthanized and the main organs were removed for fluorescence imaging.

[0048] Antitumor effect and immune activation of drugs: tumor volume was about 100mm 3 The tumor-bearing mice were randomly divided into groups and administered intravenously once every other day for a total of 5 times. The body weight and tumor volume of the mice were measured. The mice were euthanized 14 days later and the tumors were removed and weighed.

[0049] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0050] Example 1

[0051] 4T1 tumor cell membrane extraction.

[0052] 4T1 cells (Wuhan Sevier Biotechnology Co., Ltd., STCC20022P) were digested and collected using pancreatic enzyme digestion solution and suspended in membrane protein extraction buffer. Probe ultrasound (Nanjing Xianou Instrument, XO-650D) treatment was used to destroy the integrity of tumor cells, and ultracentrifugation was used to collect cell membranes. Membrane protein extraction buffer: Biyuntian, P0033. Probe ultrasound: Ultrasound: 30%, On:Off=2:3, 30min. Ultracentrifugation (BECKMAN COULTER, OptimaXPN-80): Centrifuge at 20000rpm at 4℃ for 30min, collect the supernatant, centrifuge at 100000rpm at 4℃ for 30min, collect the precipitate. Membrane protein detection see Figure 1 , (A) SDS-PAGE image of the outer cell membrane of the nanodrug ECNM, from left to right in the figure are marker, ECN, 4T1 cell membrane, a mixture of 4T1 cell membrane and ECN, and ECNM. It can be seen that ECNM retains most of the proteins on the 4T1 cell membrane; (B) is an immunoblot image of marker detection on the 4T1 cell membrane. From left to right in the figure are 4T1 cells, 4T1 cell membrane and ECNM. It can be seen that CD47, CD44 and Na+ in the outer cell membrane of ECNM + / K + -ATPases are expressed, and the absence of GAPDH in bands II and III indicates that the cell membrane has been successfully separated without affecting the protein diversity on the membrane.

[0053] Example 2

[0054] Specific operating methods and parameter conditions for the preparation of EC, ECN, and ECNM.

[0055] EC preparation: ES (Aladdin, STA-4783) (5 mg) was co-dissolved in 1 mL DMSO, and copper chloride (CuCl2, 10 mg) was added to the system. The system was stirred at room temperature for 2 hours, and unreacted CuCl2 was removed by centrifugation. Subsequently, the supernatant was dropped into 10 mL distilled water, and stirred for 2 hours, and dialyzed overnight to remove the organic solvent. The prepared EC was concentrated and stored at 4 ° C.

[0056] Preparation of ECN: ES (5 mg), NLG919 (Aladdin, N129887) (5 mg) were co-dissolved in 1 mL DMSO, and copper chloride (CuCl2, 10 mg) was added to the system. The system was stirred at room temperature for 2 hours, and unreacted CuCl2 was removed by centrifugation. Subsequently, the supernatant was dropped into 10 mL distilled water, and stirred for 2 hours, and dialyzed overnight to remove the organic solvent. The prepared ECN was concentrated and stored at 4 ° C.

[0057] ECNM preparation: 4T1 cell membrane fragments were mixed with ECN at a weight ratio of 6: 1. The mixture was sonicated in an ice bath for 5 minutes, and then the resulting nanoparticle ECNM was collected by centrifugation.

[0058] For the particle size, potential and morphology of EC, ECN and ECNM, see Figure 2 , the particle size distribution and electron microscope images of nano drugs (A) EC, (B) ECN, (C) ECNM, (D) is the particle size. It can be seen that the particle sizes of EC, ECN, and ECNM are slightly increased, and they have good dispersibility and solid spherical structure, among which ECNM has an obvious outer membrane structure.

[0059] Example 3

[0060] Drug release behavior assay.

[0061] The ECNM (calculated based on ES weight: 10 mg) was transferred into a dialysis bag (MW: 1000 kDa) (Shanghai Yuanye, SP131486) and placed in a flask filled with different buffer solutions (100 mL PBS, containing 0, 5 and 10 mM GSH). The flask was placed in a constant temperature oscillator (Changzhou Zhiborui Instrument, ZD-85) (100 rpm, 37 ° C), 1 mL of release medium was collected at intervals, and an equal volume of fresh buffer solution was added. The concentrations of NLG919 and ES were measured using high performance liquid chromatography (Shimadzu, LC-20AT) (HPLC). As Figure 3 As shown, ECNM releases (A) ES and (B) NLG919 in response to GSH at 37°C. It can be seen that in the absence of GSH, the cumulative release of ES and NLG919 within 48h is about 20%, while the cumulative release of 5mM GSH is about 55%, and 10mM GSH is about 65%, indicating that ECNM can respond to GSH to release drugs and increase the drug release of nanoparticles at the target site.

[0062] Example 4

[0063] Evaluation of drug storage stability.

[0064] The particle size was used to study the stability of different nanoparticles. Briefly, EC, ECN and ECNM were dispersed in PBS, DMEM and 10% FBS and stored at 4°C. At predetermined time points, the particle size changes of different samples were measured using a Malvern Zetasizer (NanoZS, UK). Figure 3 As shown in Figure 3, the storage stability of (CE)EC, ECN, and ECNM in different media. The experimental results show that the particle size of ECNM has almost no significant change within 10 days, and has good storage stability, while EC tends to aggregate more quickly than ECN.

[0065] Example 5

[0066] Cytotoxicity assay.

[0067] The cytotoxicity of the preparations in CuCl2-conditioned medium was determined by MTT assay. Briefly, 4T1 tumor cells were cultured at 1×10 3 The cells were seeded at a density of 100 cells per well into a 96-well plate. After the cells adhered, the culture medium was removed, and culture medium containing different concentrations of CuCl2, culture medium containing different concentrations of CuCl2 and preparations (including ES, EC, ECN and ECNM) were added to the 96-well plate. After incubation for 24 hours, 20 μL of MTT (5 mg / mL) (Wuhan Sevier Biotechnology Co., Ltd., G4104-100T) was added and further incubated for 4 hours. Thereafter, the culture medium was removed, 100 μL of DMSO was added to each well to dissolve the purple crystals, and the absorbance of each sample was measured at 450 nm. In addition, in order to study the copper transport capacity of different nanoparticles, the cytotoxicity of the nanoparticles at different copper concentrations was also measured (CuCl2 was added to the culture medium for the above-mentioned MTT experiment, CuCl2 content: 100 nM, 1 μM). Figure 4 As shown, (A) the cytotoxicity of conditioned medium with different concentrations of CuCl2 on 4T1 cells, and the toxicity test of nanomedicine on 4T1 cells with different concentrations of CuCl2 (B) 0, (C) 100nM, (D) 1μM. It can be seen that CuCl2 and ES themselves have no obvious toxicity to cells, but can bind to Cu 2+ and induce copper-dependent cell death. The initial Cu 2+ The higher the loading amount, the more obvious the cytotoxicity. ECNM showed the strongest cytotoxicity due to the promotion of cellular uptake by homologous cell membrane fusion.

[0068] Example 6

[0069] Western blot.

[0070] Western blot was used to determine the expression of IDO1 and FDX1 in cells after co-incubation with nanoparticles. For IDO1 expression, 4T1 tumor cells were plated at 5 × 10 4The cells were inoculated into 6-well plates at a density of 10 cells. After the cells adhered to the wall, the cells were pretreated with IFN-γ (50 ng / mL) (Nanjing Jiancheng Bioengineering Research Institute, H025-1-2) for 12 hours to stimulate cell IDO1 expression. Subsequently, nanoparticles (ES, EC, ECN and ECNM) were added to each well and incubated for 24 hours. Then, the cells were washed with PBS, digested with trypsin digestion solution and collected. The cells were treated with RIPA cell lysis buffer (Wuhan Sevier Biotechnology Co., Ltd., G2033-30ML) to collect proteins, and the protein concentration was measured using a BCA kit (Wuhan Sevier Biotechnology Co., Ltd., G2026-200T). The loading buffer was added to the protein sample, heated to 100°C and incubated for 15 minutes to denature the protein. The IDO1 protein was separated using SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with non-fat emulsion, incubated with IDO1 primary antibody (Wuhan Aimejie Technology Co., Ltd., HPA023149), HRP-labeled secondary antibody (Wuhan Aimejie Technology Co., Ltd., PMB-P50958) and ECL chemiluminescent reagent, and finally observed with an imaging system. The same method was used to measure the expression of FDX1 (Wuhan Aimejie Technology Co., Ltd., aviva-OAAN03998-50UL), and no IFN-γ pretreatment procedure was required when detecting FDX1 expression. Figure 5 As shown, after the nanomedicine was co-incubated with 4T1 cells, the relative expression levels of (AB) FDX1 and (CD) IDO1 in the cells were determined by immunoblotting. It can be seen that ECNM significantly reduced the expression of cellular FDX1 and IDO1, indicating that ECNM effectively induced copper cell death and reversed the immunosuppressive microenvironment of the tumor.

[0071] Detailed parameters: https: / / www.servicebio.cn / data-detail?id=2984&code=WBSYBG

[0072] Example 7

[0073] Evaluation of oxidative stress at the cellular level.

[0074] Intracellular ROS and GSH were measured using a ROS fluorescence assay kit and a reduced glutathione (GSH) colorimetric assay kit, respectively. 4 The cells were seeded into 6-well plates at a density of 10 cells / well. After the cells adhered, nanoparticles (ES, EC, ECN and ECNM) were added to each well and incubated for 12 hours. Thereafter, the cells were washed with PBS, and the ROS and GSH levels were measured according to the method described in the kit (E004-1-1, A006-1-1, Nanjing Jiancheng Bioengineering Institute).

[0075] In addition, DCFH-DA (Wuhan Saiweier Biotechnology Co., Ltd., G1706-100T) was also used as a fluorescent probe to detect ROS levels. Briefly, 4T1 tumor cells were plated at 5 × 10 4 The cells were seeded at a density of 100 cells per well in a 6-well plate with a slide. After the cells adhered, different nanoparticles (ES, EC, ECN and ECNM) were added to each well. After incubation for 6 hours, the cells were washed with PBS and incubated with DCFH-DA (10 μM) for another 2 hours in the dark. After fixation with 4% paraformaldehyde, the cells were washed with PBS and incubated with DAPI working solution (Wuhan Sevier Biotechnology Co., Ltd., GDP1024) for 5 minutes in the dark, and the cells were washed. Finally, a confocal laser scanning microscope (Olympus, FV3000) (CLSM) was used to observe and photograph. Figure 6 As shown, after the nanodrug was co-incubated with 4T1 cells, (A) DCFH-DA probe was used for fluorescence imaging of intracellular ROS, the intracellular distribution of ROS was acquired by laser confocal microscopy, and the related kits were used to quantitatively measure the intracellular (B) ROS and (C) GSH contents. It can be seen that ECNM significantly increased the intracellular ROS content while reducing GSH, disrupting the intracellular redox balance and inducing cell CDT death.

[0076] Example 8

[0077] ICD evaluation at the cellular level.

[0078] CRT expression was measured using CLSM. Briefly, 4T1 tumor cells were plated at 5 × 10 4 The density of each cell was inoculated into a 6-well plate with a crawling sheet. After the cells adhered to the wall, different nanoparticles (ES, EC, ECN and ECNM) were added to each well. After incubation for 24 hours, the culture medium was taken out, the cells were washed with PBS and fixed with 4% paraformaldehyde (Wuhan Saiweier Biotechnology Co., Ltd., G1101-25ML). Finally, the cells were incubated with an anti-CRT primary antibody (Wuhan Saiweier Biotechnology Co., Ltd., GB11587-100) and an Alexa488-coupled secondary antibody (Wuhan Saiweier Biotechnology Co., Ltd., GB25303), and CLSM was used to observe and take pictures.

[0079] For HMGB1 (Nanjing Jiancheng Bioengineering Institute, H257-1-2) and ATP (Nanjing Jiancheng Bioengineering Institute, A095-1-1) detection, cells were incubated with different nanoparticles (ES, EC, ECN, and ECNM) for 24 h. Subsequently, the culture medium was collected and the HMGB1 and ATP levels in the culture medium were measured using the corresponding kits. Figure 7As shown, after the nanodrug was co-incubated with 4T1 cells, (A) CRT antibody-labeled fluorescence imaging and the kit detected the cell release of (B) ATP and (C) HMGB1. It can be seen that ECNM increased the expression of CRT and the extracellular content of ATP and HMGB1, indicating that ECNM effectively activated the immunogenic death of cells.

[0080] Example 9

[0081] Organization distribution.

[0082] 4T1 tumor-bearing Balb / c mice were randomly divided into EC, ECN and ECNM groups. DiR (Shanghai Yuanye, Y18318-5mg) was used as a fluorescent probe to label different nanoparticles. 0.2mL of different DiR-labeled nanoparticles were intravenously injected into the blood system through the tail vein. At the predetermined time points, the mice were anesthetized for in vivo fluorescence imaging. At the end of the study, the main organs of the mice (including tumors, heart, liver, spleen, lungs, and kidneys) were collected for ex vivo fluorescence imaging. Figure 8 As shown, (A) in vivo imaging of fluorescently labeled nanomedicines in mice, as well as (B) fluorescence imaging and (C) quantitative fluorescence analysis of nanomedicines in major tissues and organs.

[0083] Detailed parameters: https: / / www.servicebio.cn / data-detail?id=4745&code=ZLLSYBG

[0084] Example 10

[0085] In vivo pharmacodynamic evaluation.

[0086] 4T1 tumor-bearing Balb / c mice were randomly divided into several groups, including (control group, ES group, EC group, ECN group and ECNM group, n=6). When the tumor volume reached about 100 mm 3 At 4 pm, 0.2 mL of different nanoparticles (ES concentration: 5 mg / kg) were injected intravenously through the tail vein every other day for a total of 5 injections. Tumor volume and body weight were measured every other day. The tumor volume was calculated as V = (a × b 2 ) / 2. (a represents the length of the tumor, b represents the width of the tumor). On day 14, the mice were sacrificed and the tumors were collected and measured for tumor weight. Figure 8 As shown, (D) tumor volume change and (E) final tumor weight, it can be seen that ECNM with high stability and homologous cell targeting significantly increased the accumulation of drugs in the tumor site, homologous cell membrane fusion promoted the uptake of ECNM, and high intracellular GSH promoted drug release. The results showed that ECNM had the strongest tumor growth inhibitory effect.

[0087] From the above examples, it can be seen that the present invention constructs a bionic nanomedicine, using NLG919, ES and Cu 2+ As the active ingredient, a self-assembled drug system (ECN) was constructed, and tumor cell membranes were used to coat ECN to form ECNM. Compared with the traditional ilisimol-copper nanocomplex (EC), the constructed ECNM significantly improved the systemic circulation stability, tumor targeting and tumor accumulation of EC, and can efficiently trigger copper death and CDT, synergistically reverse TIM, and thus achieve a higher tumor immunotherapy effect.

[0088] The core innovation of the present invention is the structure and preparation method of bionic nanomedicine. Elisimol and NLG919 are mixed and dissolved in DMSO, and then CuCl2 solution is added and stirred. Elisimol carries Cu through coordination bonds. 2+ , through hydrogen bonding and π-π conjugation, it interacts with NLG919 to self-assemble into a spherical nanostructure. The 4T1 cell membrane is extracted using a membrane protein separation kit, the cells are placed in a membrane protein extract, the cell membrane is collected by centrifugation after ultrasonic disruption, and the cell membrane is mixed with the spherical nanoparticles by ultrasonication to obtain the bionic nanomedicine ECNM with the cell membrane wrapped in the outer layer.

[0089] As well as therapeutic strategies: NLG919 improves the circulation stability and tumor enrichment of ES, and improves TIM. ECNM activates ICD by triggering copper death and CDT, and amplifies tumor immune response by improving TIM.

[0090] The above are only a few preferred embodiments of the present invention, which are described in a more specific and detailed manner, but they cannot be understood as limiting the scope of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for preparing tumor cell membrane-wrapped ilisimol / copper-NLG919 biomimetic nanoparticles, characterized in that: The following steps are involved: S1. Extraction of tumor cell membranes: digestion and collection of tumor cells with pancreatic enzyme digestion solution, and suspension of the tumor cells in membrane protein extraction buffer. Ultrasonication with a probe is used to destroy the integrity of tumor cells, and ultracentrifugation is used to collect cell membranes. S2. Preparation of core nanoparticles: dissolving ilisimol ES and NLG919 in dimethyl sulfoxide (DMSO) and mixing; Add copper chloride solution to the mixed solution and stir at room temperature for several hours; remove unreacted copper chloride by centrifugation, drop the supernatant into distilled water and continue stirring for several decimal points; perform dialysis to remove the organic solvent to obtain nanoparticles, store them at low temperature, and name them ECN; S3. Preparation of coated nanomedicine: The ECN obtained in step S2 and the tumor cell membrane obtained in step S1 are ultrasonically mixed under ice bath conditions; and the tumor cell membrane-wrapped ilisimol / copper-NLG919 biomimetic nanoparticles are obtained by centrifugation, which are named ECNM.

2. The method for preparing tumor cell membrane-encapsulated ilisimol / copper-NLG919 biomimetic nanoparticles according to claim 1, characterized in that: The tumor cells include, but are not limited to, breast cancer cells, lung cancer cells, gastric cancer cells, liver cancer cells, colon cancer cells, pancreatic cancer cells, prostate cancer cells, ovarian cancer cells, melanoma cells and leukemia cells.

3. The method for preparing tumor cell membrane-encapsulated ilisimol / copper-NLG919 biomimetic nanoparticles according to claim 1, characterized in that: In the step S1, the ultrasonic parameters are: 30%, On:Off = 2:3, 30 min.

4. The method for preparing tumor cell membrane-encapsulated ilisimol / copper-NLG919 biomimetic nanoparticles according to claim 1, characterized in that: In step S1, the ultracentrifugation step includes: centrifuging at 20,000 rpm at 4°C for 30 min, collecting the supernatant, and then centrifuging the supernatant at 100,000 rpm at 4°C for 30 min, collecting the precipitate, and obtaining the cell membrane.

5. The method for preparing tumor cell membrane-encapsulated ilisimol / copper-NLG919 biomimetic nanoparticles according to claim 1, characterized in that: In the step S2, the mass ratio of elisemomol ES to NLG919 is 1:1; the mass ratio of elisemomol ES to copper chloride is 1:2; and the mass volume ratio of elisemomol ES to DMSO is 5:1 mg / mL.

6. The method for preparing tumor cell membrane-encapsulated ilisimol / copper-NLG919 biomimetic nanoparticles according to claim 1, characterized in that: In step S3, the tumor cell membrane and ECN are mixed at a mass ratio of 6:

1.

7. Tumor cell membrane-wrapped ilisimol / copper-NLG919 biomimetic nanoparticles, characterized in that: Prepared by the method described in any one of claims 1 to 6.

8. Use of the bionic nanoparticles as claimed in claim 7 in the preparation of drugs for treating cancer.

9. The use according to claim 8, characterized in that: The cancers include, but are not limited to, breast cancer, lung cancer, stomach cancer, liver cancer, colon cancer, pancreatic cancer, prostate cancer, ovarian cancer, melanoma, acute myeloid leukemia, and lymphoma.

10. A drug for treating cancer, characterized in that: The medicine contains the biomimetic nanoparticles as claimed in claim 7.

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