Tumor cell exosome, anti-tumor immune drug delivery system and preparation method and application of anti-tumor immune drug delivery system

Abstract

The invention provides a tumor cell exosome, an anti-tumor immune drug delivery system and a preparation method and application of the anti-tumor immune drug delivery system, the tumor cell exosome is derived from starved and stimulated tumor cells, the tumor cell exosome is coated with nano drug particles for delivery, and the exosome has the effects of enhancing immune cell infiltration and remodeling an immune microenvironment; due to the homing effect of the exosome, the medicine and the exosome are more easily accumulated in the tumor, and the targeting property is improved; due to coating and camouflage of the biological membrane of the exosome membrane, the exosome membrane is prevented from being recognized, attacked and removed by an organism, and the biological safety is improved, so that the exosome and the nano-drug act on the tumor together to achieve a synergistic effect.

Description

Technical Field

[0001] The present application belongs to the technical field of tumor immunotherapy, and in particular, relates to a tumor cell exosome, an anti-tumor immune drug delivery system, and a preparation method and application thereof.

[0002] This research was funded by the China Postdoctoral Science Foundation (No. 2024M763792). Background Art

[0003] At present, the main means of tumor treatment are surgery, chemotherapy, radiotherapy, targeted therapy, immunotherapy, etc. Among them, immunotherapy aims to activate the human immune system and rely on its own immune function to kill cancer cells and tumor tissues, so it can prolong the patient's survival, improve the patient's quality of life, and reduce the side effects of traditional treatments. Existing immunotherapy includes the use of exosomes from various immune cells and various drug delivery nanoparticles that can be used for immunotherapy, which can improve the effect of anti-tumor immunity to a certain extent. For example, NK cell exosomes are rich in various anti-tumor factors including granzyme B, Fas ligand, etc., which have a killing effect on tumor cells (Frontiers in Immunology, 2021, 12, 771551); T cell-derived exosomes (Journal of Extracellular Vesicles, 2023, 12, 1-17).

[0004] However, immunotherapy still faces many technical challenges, including: 1) The tumor microenvironment itself is often immunosuppressive, and immune cell infiltration is insufficient, resulting in poor immunotherapy efficacy; 2) Immunotherapy drugs lack targeting and are prone to toxic side effects. 3) Although cutting-edge nanomedicines have improved targeting to a certain extent, their main components are organic polymer materials or inorganic materials, which lack biocompatibility. Therefore, efficacy, targeting, and biosafety need to be improved.

[0005] In particular, exosomes derived from tumor cells generally promote tumor growth and therefore generally have no therapeutic effect and may even have a counterproductive effect. Summary of the invention

[0006] The present application provides a tumor cell exosome, an anti-tumor immune drug delivery system, and a preparation method and application thereof to solve the problems existing in the related technologies. The technical solution is as follows:

[0007] In a first aspect, an embodiment of the present application provides a tumor cell exosome, wherein the exosome is induced to be produced by tumor cells through amino acid starvation stimulation.

[0008] In one embodiment, the exosomes are starved in vitro using a culture medium without amino acids to culture tumor cells, and the cell culture supernatant is collected and centrifuged to obtain a supernatant containing starvation-stimulated exosomes.

[0009] In one embodiment, the culture medium is EBSS medium, wherein the glucose content is 4.5 g / L.

[0010] In one embodiment, the duration of hunger stimulation is 12-36 hours.

[0011] In one embodiment, the centrifugation is performed using gradient centrifugation.

[0012] In one embodiment, the gradient centrifugation process is: 300g centrifugation for 10 min, 2000g centrifugation for 10 min, 10000g centrifugation for 30 min, and 110000g centrifugation for 70 min.

[0013] In a second aspect, an embodiment of the present application provides an anti-tumor immune drug delivery system, the components of which include nanoparticles and the above-mentioned tumor cell exosomes coating the nanoparticles; the nanoparticles are composed of components including polylactic acid polyethylene glycol amino groups and drugs.

[0014] In one embodiment, the drug is a compound of formula I:

[0015]

[0016] In one embodiment, the average molecular weight of the polylactic acid in the polylactic acid polyethylene glycol amino group is 1000-5000; the average molecular weight of the polyethylene glycol is 1000-5000.

[0017] In one embodiment, the mass ratio of the structural compound represented by Formula I to the polylactic acid polyethylene glycol amino group is (1-2):20.

[0018] In one embodiment, the nanoparticles are prepared by the following method: a DMSO solution of the structural compound represented by Formula I and polylactic acid polyethylene glycol amino is added dropwise into distilled water, and then dialyzed to obtain the nanoparticles.

[0019] In one embodiment, the volume ratio of DMSO to distilled water is (1-2.5):10.

[0020] In one embodiment, the addition of the compound of formula I and the DMSO solution of polylactic acid polyethylene glycol amino is performed under high-speed vortex state.

[0021] In one embodiment, dialysis uses a 20,000 KD dialysis bag.

[0022] In a third aspect, the present invention provides a method for preparing an anti-tumor immune drug delivery system, comprising the following steps:

[0023] The tumor cell exosomes are mixed with the nanoparticles and then ultrasonically treated in an ice bath to prepare the anti-tumor immune drug delivery system.

[0024] In one embodiment, the mass ratio of the tumor cell exosomes to the nanoparticles is (1-1.5):1.

[0025] In one embodiment, the ultrasonic treatment time is 4-8 min.

[0026] In one embodiment, separation is performed by centrifugation after ultrasonic treatment, and the centrifugation conditions are: centrifugation at a speed of 30,000 g for 15-45 min.

[0027] In a fourth aspect, the embodiments of the present application provide the use of the above-mentioned tumor cell exosomes or the above-mentioned anti-tumor immune drug delivery system in the preparation of anti-tumor drugs.

[0028] The advantages or beneficial effects of the above technical solution include at least:

[0029] A tumor cell exosome of the present application is derived from tumor cells stimulated by starvation, and has the function of enhancing immune cell infiltration and reshaping the immune microenvironment; and its homing effect makes it easier to accumulate in the tumor, thereby improving the targeting; in particular, the exosomes are rich in chemokine CXCL9, which has the function of recruiting CD8+T cells into tumor tissues, thereby achieving the effect of tumor killing.

[0030] In the anti-tumor immune drug delivery system of the present application, exosomes and nano-drugs act on tumors together to achieve a synergistic effect; at the same time, due to the homing effect of tumor-derived exosomes, nanoparticles coated with exosomes are more likely to accumulate in the tumor, thereby improving targeting; and due to the coating and camouflage of the exosome membrane, the nanoparticles are prevented from being recognized, attacked, and cleared by the body, thereby improving biosafety.

[0031] The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present application will be readily apparent by reference to the accompanying drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In the accompanying drawings, unless otherwise specified, the same reference numerals throughout the multiple drawings represent the same or similar parts or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings only depict some embodiments disclosed in the present application and should not be regarded as limiting the scope of the present application.

[0033] Figure 1 A flow chart for preparing the anti-tumor immune drug delivery system for this application;

[0034] Figure 2 The particle size distribution diagram of EEVs prepared in the example and DEVs in the control group;

[0035] Figure 3 The Western blot test results of EEVs prepared in the example and DEVs in the control group are shown in FIG.

[0036] Figure 4 The graphs are the changes in the volume and weight of the tumor in the mouse subcutaneous tumor model treated with PBS buffer, EEVs prepared in the example, and DEVs in the control group, as well as the H&E staining of the tumor tissue;

[0037] Figure 5 Figure 2 is the result of flow cytometry detection of tumor in mouse subcutaneous tumor model treated with PBS buffer, EEVs prepared in Example and DEVs in control group;

[0038] Figure 6 This is the H NMR spectrum of the compound of formula I synthesized in Example 3;

[0039] Figure 7 The mass spectrum of the compound of the structure shown in Formula I synthesized in Example 3;

[0040] Figure 8 The particle size distribution diagram of SPN@EEVs prepared in Example 4, EEVs prepared in Example 1, and DEVs of the control group; surface potential diagram;

[0041] Fig. 9 Transmission electron micrographs of SPN@EEVs prepared in Example 4, EEVs prepared in Example 1, and DEVs of the control group; and SDS-PAGE electrophoresis images;

[0042] Fig.10 This is a diagram showing the results of in vitro experiments in Example 5 to verify the regulation of SPN@EEVs on macrophage activity;

[0043] Fig.11 This is a diagram showing the results of in vitro experiments in Example 5 to verify the regulation of SPN@EEVs on T cell activity;

[0044] Fig.12 This is a diagram showing the tumor targeting experimental results of SPN@EEVs prepared in Example 4;

[0045] Fig.13The graphs are the volume and weight changes of the tumor in the mouse subcutaneous tumor model treated with PBS buffer, SPN prepared in Example 4, SPN@EEVs and EEVs prepared in Example 1, and the H&E staining of the tumor tissue;

[0046] Fig.14 This is a flow cytometry test result of the mouse subcutaneous tumor model treated with PBS buffer, SPN prepared in Example 4, SPN@EEVs and EEVs prepared in Example 1. DETAILED DESCRIPTION

[0047] In the following, only some exemplary embodiments are briefly described. As those skilled in the art will appreciate, the described embodiments may be modified in various ways without departing from the spirit or scope of the present application. Therefore, the drawings and descriptions are considered to be exemplary and non-restrictive in nature.

[0048] Tumor-derived exosomes have a homing effect and are more likely to accumulate in tumors, which improves targeting. In addition, due to the biological membrane of the exosome membrane, they can avoid being recognized, attacked, and cleared by the body, and have biological safety. However, tumor cell-derived exosomes themselves generally promote tumor growth and generally have no therapeutic effect, and may even have a counterproductive effect.

[0049] The present application discovered that the exosomes produced by tumor cells after starvation stimulation have anti-tumor immune effects, and that the exosomes themselves not only have immune functions, but can also be used as a drug delivery platform to encapsulate different nanoparticles to achieve synergistic immunotherapy and further enhance targeting.

[0050] Therefore, the present application provides a tumor cell exosome, wherein the exosome is induced to be produced by tumor cells through amino acid starvation stimulation.

[0051] In this application, amino acid starvation stimulates tumor cells, in which the expression of OGT enzyme increases, and O-GlcNAc can be added to the STING receptor to form O-GlcNAc glycosylated STING, further triggering the NF-kB signaling pathway to promote tumor cells to release a large number of exosomes EEVs. The exosomes are rich in chemokine CXCL9, which has the function of recruiting CD8+T cells into tumor tissues to achieve tumor killing effects. In addition, exosomes EEVs can be used to coat various nanodrugs, and play a synergistic role of "1+1>2" with nanodrugs, while improving the targeting and biocompatibility of drugs.

[0052] As one embodiment, the exosomes are starved to stimulate tumor cells in vitro using a culture medium without amino acids, and the cell culture supernatant is collected and centrifuged to obtain a supernatant containing starvation stimulated exosomes.

[0053] As one embodiment, the culture medium is an EBSS culture medium, wherein the glucose content is 4.5 g / L. Conventional EBSS culture medium contains 1 g / L of glucose, while normal DMEM culture medium contains 4.5 g / L of high glucose. All EBSS culture mediums used in this embodiment are supplemented with high glucose, i.e., 4.5 g / L, to ensure that the cells will not be starved for glucose, but only starved for amino acids.

[0054] As one embodiment, the duration of hunger stimulation is 12-36 hours.

[0055] As one embodiment, centrifugal separation adopts gradient centrifugation; the process of gradient centrifugation is: 300g centrifugation for 10 min, 2000g centrifugation for 10 min, 10000g centrifugation for 30 min, and 110000g centrifugation for 70 min.

[0056] The present application also provides an anti-tumor immune drug delivery system, the components of which include nanoparticles and the above-mentioned tumor cell exosomes coating the nanoparticles; the nanoparticles are composed of components including polylactic acid polyethylene glycol amino groups and drugs.

[0057] Exosome EEVs can be used to encapsulate various nanodrugs, because the exosomes of the present application themselves have a certain tumor killing effect, and thus they play a synergistic role with nanodrugs of "1+1>2". At the same time, due to the homing effect of tumor-derived exosomes, the nanoparticles encapsulated by the exosomes are more likely to accumulate in the tumor, thereby improving the targeting of delivery; and due to the coating and camouflage of the exosome membrane, the nanoparticles are prevented from being recognized, attacked, and cleared by the body, thereby improving biosafety. The nanoparticles of the present application are composed of components including polylactic acid polyethylene glycol amino groups and drugs. The polylactic acid polyethylene glycol amino groups have good biocompatibility and water solubility. The hydrophilic block polyethylene glycol (PEG) and hydrophobic block polylactic acid (PLA) they have can achieve self-assembly in aqueous solution, and the amino groups contained therein can interact with the carboxyl groups on the cell membrane, thereby achieving targeted delivery. The specific process is as follows: Figure 1 shown.

[0058] As one embodiment, the average molecular weight of the polylactic acid in the polylactic acid polyethylene glycol amino is 1000-5000; the average molecular weight of the polyethylene glycol is 1000-5000; the properties of the polymerization can be fine-tuned by adjusting the average molecular weight of the polymer.

[0059] As one embodiment, the drug is a compound having a structure shown in Formula I:

[0060]

[0061] In this embodiment, a drug compound having a structure shown in Formula I is designed and synthesized based on SHP-099 and the E3 ubiquitinase ligand Pomalidomide; the SHP-099 and the E3 ubiquitinase ligand Pomalidomide are connected via a linker.

[0062] In this embodiment, the linker is -COCH2CH2OCH2CH2OCH2CH2-, which connects the amino group on the piperidine group of SHP-099 and the amino group of Pomalidomide respectively.

[0063] In this embodiment, the compound of the structure shown in Formula I is prepared by the following preparation method:

[0064] Step S1, amino-diethylene glycol-propionic acid tert-butyl ester reacts with 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione in the presence of DIPEA in an organic solvent;

[0065] Step S2, deprotecting the reaction product of step S1;

[0066] Step S3, the product of step S2 is reacted with SHP-099 in the presence of DIPEA in an organic solvent, and the reaction is completed to obtain a compound of formula I.

[0067] In this embodiment, in step S1, the organic solvent is DMSO, and the reaction conditions are 80-100° C. for 6-36 hours.

[0068] In this embodiment, the deprotection in step S2 is carried out in a dichloromethane solution using trifluoroacetic acid at 30-45° C. for 1-3 h.

[0069] In this embodiment, the reaction in step S3 is carried out at 20-30° C. for 12-36 hours.

[0070] As one embodiment thereof, the nanoparticles are prepared by the following method:

[0071] The DMSO solution of the structural compound shown in Formula I and polylactic acid polyethylene glycol amino was added dropwise into distilled water, and then dialyzed to obtain the nanoparticles.

[0072] As one embodiment, the mass ratio of the compound of formula I to the polylactic acid polyethylene glycol amino group is (1-2): 20. At this mass ratio, the polylactic acid polyethylene glycol amino group self-assembles to encapsulate the compound of formula I to form nano micelles.

[0073] As one embodiment thereof, the volume ratio of DMSO to distilled water is (1-2.5):10;

[0074] As one embodiment, the compound of formula I and the DMSO solution of polylactic acid polyethylene glycol amino are added dropwise under high-speed vortex state.

[0075] As one embodiment, dialysis uses a 20000KD dialysis bag.

[0076] The present application also provides a method for preparing an anti-tumor immune drug delivery system, comprising the following steps:

[0077] The tumor cell exosomes and nanoparticles are mixed and then ultrasonically treated in an ice bath to prepare the anti-tumor immune drug delivery system.

[0078] In the present application, exosomes are mixed with nanoparticles by ultrasound, and the exosomes can encapsulate the nanoparticles to obtain an anti-tumor immune drug delivery system.

[0079] As one embodiment, the mass ratio of the tumor cell exosomes to the nanoparticles is (1-1.5):1.

[0080] As one embodiment, the ultrasonic treatment time is 4-8 minutes.

[0081] As one embodiment, separation is performed by centrifugation after ultrasonic treatment, and the centrifugation conditions are: centrifugation at a speed of 30,000 g for 15-45 minutes.

[0082] The following is a further description with reference to specific embodiments.

[0083] Example 1

[0084] In vitro, tumor cells were starved using EBSS (glucose content of 4.5 g / L) without amino acids, and cells were cultured with complete medium as control. After 24 h, the cell culture supernatant was collected and separated by gradient centrifugation at 300 g for 10 min, 2000 g for 10 min, 10000 g for 30 min, and 110000 g for 70 min to obtain starvation-stimulated exosomes (EEVs) and control group exosomes (DEVs).

[0085] The starvation-stimulated exosomes (EEVs) and the control exosomes (DEVs) obtained in Example 1 were characterized by a particle size meter, and the particle size distribution was as follows: Figure 2 shown.

[0086] from Figure 2 It can be seen that the particle sizes of exosomes in the two groups of DEVs and EEVs are similar and evenly distributed, which is the size of exosomes.

[0087] The exosome marker proteins of the starvation-stimulated exosomes (EEVs) and the control group exosomes (DEVs) obtained in Example 1 were analyzed by Western blot and compared with the cell lysate proteins. The results are as follows: Figure 3 As shown in A.

[0088] from Figure 3 A It can be found that DEVs and EEVs have obvious expression of exosome marker proteins, and both express exosome-related marker proteins, indicating that exosomes were successfully isolated.

[0089] The chemokines were further analyzed by Western blot. Figure 3 As shown in B.

[0090] from Figure 3 According to the results of B, chemokines CCL5, CCL20, and CXCL9 were significantly upregulated in the EEVs group after starvation stimulation.

[0091] Example 2

[0092] A subcutaneous tumor model of Balb / C mice was constructed using 4T1 breast cancer cells, and the mice were divided into three groups: PBS, normal exosomes DEVs (exosomes from normal cultured cells), and starvation-stimulated exosomes EEVs were injected through the tail vein. The tumor volume of the mice was monitored and recorded every other day. The results are shown in Figure 4 A and Figure 4 As shown in B, mice were killed at the end of the study (day 19) and the tumors were weighed. Figure 4 C; and the tumor tissue was sampled and stained with H&E. The results are shown in Figure 4 As shown in D.

[0093] Figure 4 The experimental results of AC suggested that the EEVs starvation exosome group significantly inhibited tumor growth. Figure 4 H&E staining in D showed that EEVs significantly induced tumor cell necrosis, indicating its anti-tumor killing activity.

[0094] Tumor tissue was collected and made into single cell suspension, and immune flow cytometry was used to explore the infiltration of various immune cells in the tumor. Figure 5 As shown in AD.

[0095] By detecting immune cells in the tumor by flow cytometry, it was found that the proportion of CD8+T cells in the EEVs-treated group was significantly increased, and the expression of Granzyme B was increased, indicating that the killing toxicity of CD8+T cells was significantly enhanced. In addition, the proportion of CD80+ / CD86+ cells in the tumor-infiltrating lymph nodes in the EEVs-treated group increased, and the expression of MHC-II in DC cells increased, indicating that EEVs promote the maturation of DC cells and facilitate their antigen presentation.

[0096] Example 3

[0097] Synthesis of SHP2-PROTAC: 86.19 mg of amino-diethylene glycol-tert-butyl propionate was dissolved in 4 mL of ultra-dry DMSO, and 126 μL of DIPEA was added and stirred for 5 min. After adding 102 mg of 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione, the mixture was refluxed at 90 ° C for 18 h. After cooling, the reaction solution was poured into 100 mL of saturated brine and extracted with 100 mL of ethyl acetate. The product in ethyl acetate was washed with 50 mL of saturated brine, dried over Na2SO4, filtered and concentrated to obtain an intermediate product. 11.0 mg of the intermediate product was dissolved in 2 mL of dichloromethane, 1 mL of trifluoroacetic acid was added dropwise, and the mixture was reacted in a water bath at 40 ° C for 2 h. The product was concentrated by rotary evaporation. 15.3 mg of the product was dissolved in 1 mL of DMF, followed by the addition of 16.2 μL of DIPEA, 9.72 mg of HATU and 8.19 mg of SHP-099, after stirring at room temperature for 24 h, the product was poured into 100 mL of saturated brine to precipitate, and extracted with 100 mL of ethyl acetate, the organic layer products were combined, washed with 50 mL of saturated brine, dried over Na2SO4, filtered and concentrated, and the resulting mixture was purified by preparative HPLC (10-100% acetonitrile / 0.1% TFA) to obtain the final product, a compound of the structure shown in Formula I, named SHP2-PROTAC.

[0098] 1 H-NMR(600MHz,DMSO)δ(ppm):11.13(s,1H),7.66(s,1H),7.61(s,1H),7.50(s,2H),7.44(s,1H),7.33(s,1H),7.13(s,1H),7.0 8(s,1H),5.04(s,1H),3.69–3.12(m,8H),2.97–2.61(m,2H),2.37(s,2H),2.17(s,3H),2.07(s,1H),1.48(s,2H),1.32(s,3H); such as Figure 6 shown.

[0099] LC-MS spectrum of the synthetic product SHP2-PROTAC: According to the molecular formula C 36 H 41 HRMS (ESI) calculated for Cl2N8O7 m / z [M+H] + 767.24, m / z [M+2H] 2+ is 384.12, and the actual measurements are 767.24 and 384.14 respectively; Figure 7shown.

[0100] Example 4

[0101] 8 μL of 100 mg / mL high molecular weight polymer PLA 2k -PEG 2k -NH2 DMSO solution was mixed evenly with 8 μL, 10 mM SHP2-PROTAC DMSO solution, and then added dropwise to 100 μL ddH2O under high-speed vortexing. Then, it was dialyzed in water for 24 h using a 20000 kD dialysis bag to remove DMSO and unloaded free drugs to obtain nanomicelles SPNs;

[0102] SPNs and exosomes EEVs were then mixed in a mass ratio of 1:1, and exosome-coated nanoparticles were formed after ice-bath ultrasonication for 5 min. The mixture was centrifuged at 30,000 g for 30 min to remove free exosomes not used for coating, thus obtaining the final anti-tumor immune drug delivery system, named SPN@EEVs.

[0103] SPNs, EEVs, and SPN@EEVs were subjected to dynamic light scattering (DLS) detection, and the particle size distribution was as follows Figure 8 As shown in A. The particle size distribution results indicate that SPNs, EEVs, and SPN@EEVs are nanoparticles with uniform particle size distribution, with diameters ranging from 80 nm to 120 nm.

[0104] The surface potential is Figure 8 As shown in B, after coating, the surface potential of SPN@EEVs turned negative compared with SPNs, which is consistent with EEVs, suggesting that EEVs may be coated on its outer surface.

[0105] SPNs, EEVs, and SPN@EEVs were observed by transmission electron microscopy. Fig. 9 As shown in A, transmission electron microscopy shows that each group of nanoparticles has a uniform morphology.

[0106] SPNs, EEVs, and SPN@EEVs were subjected to SDS-PAGE electrophoresis. Fig. 9 As shown in (B), in SDS-PAGE electrophoresis, SPN@EEVs and EEVs had consistent protein bands, indicating successful encapsulation of exosomes.

[0107] Example 5

[0108] (1) THP-1 macrophages were cultured in vitro and divided into four groups. The cells were treated with PBS, SPNs, EEVs, and SPN@EEVs for 24 h. The cells were collected and analyzed by flow cytometry. The results were as follows: Fig.10 As shown in AB.

[0109] from Fig.10 AB shows that SPNs and SPN@EEVs can significantly upregulate the expression of M1 macrophage cytokine HLA-DR and downregulate the expression of M2 macrophage cytokine CD206.

[0110] qPCR test was performed, and the results were as follows Fig.10 C, it was found that SPN@EEVs could significantly upregulate the expression of M1 macrophage factors iNOS, TNF-α, IL-1β, IL-12, CCL3 and IL-6, and downregulate the expression of M2 macrophage factors LYVE-1, TGF-β, CD206 and IL-10. Fig.10 The results suggest that SPN@EEVs can significantly induce macrophage polarization to the M1 subtype in vitro and have anti-tumor immune killing potential.

[0111] (2) CTLL-2T lymphocytes were cultured in vitro and divided into four groups. The cells were treated with PBS, SPNs, EEVs, and SPN@EEVs for 24 h. The cells were collected and analyzed by flow cytometry. The results were as follows: Fig.11 as shown.

[0112] Fig.11 It was found that Granzyme B expression was significantly upregulated in the SPN@EEVs group, suggesting its function in activating T cell killing.

[0113] Example 6

[0114] Similar to the steps of preparing SPN@EEVs in Example 4, nanoparticles Cy5.5-NPs loaded with fluorescent dye Cy5.5 were prepared and coated with exosomes by the above-mentioned ultrasonic method to obtain EEV-coated Cy5.5-NPs.

[0115] The subcutaneous tumor model was inoculated in Balb / C mice, and in vivo fluorescence imaging was performed after Cy5.5-NPs and EEV-coated Cy5.5-NPs were injected via the tail vein. The results are shown in Fig.12 As shown in A; organs were collected for in vitro fluorescence imaging, and the results were as follows Fig.12 B and Fig.12 As shown in C.

[0116] Fig.12 A shows that in the mouse living imaging experiment, EEV-coated Cy5.5-NPs can accumulate in the tumor tissues of living mice and stay there for a longer time.

[0117] Fig.12 B and Fig.12C shows that EEV-coatedCy5.5-NPs have stronger tumor targeting in the in vitro organ imaging experiment, indicating that exosome coating significantly enhances the accumulation of nanoparticles in tumors, suggesting that its targeting effect is better.

[0118] Example 7

[0119] The subcutaneous tumor model was inoculated in Balb / C mice and divided into four groups, which were injected with PBS, SPNs, EEVs and SPN@EEVs through the tail vein. The tumor volume of the mice was monitored and recorded every other day. The results are shown in Fig.13 A and Fig.13 As shown in B, mice were killed at the end of the study and the tumors were weighed. Fig.13 C; and the tumor tissue was sampled and stained with H&E. The results are shown in Fig.13 As shown in D.

[0120] from Fig.13 A shows that the volume of SPN@EEVs tumors is significantly smaller than that of tumors treated in other groups. Fig.13 B also shows that over time, the tumor volume of the SPN@EEVs group changed the least, and the tumor volume basically did not change after about 10 days. After the mice were killed, the tumor weight of the mice treated with the SPN@EEVs group was the lightest, as shown in Figure 2. Fig.13 As shown in C, the tumor was significantly inhibited after treatment with SPN@EEVs. Fig.13 H&E staining of D showed tumor tissue necrosis, indicating that SPN@EEVs can significantly induce tumor cell necrosis in the tumor. Therefore, the above results show that SPNs and EEVs have a synergistic effect in the SPN@EEVs formed by SPNs and EEVs.

[0121] At the end of the study (16 days), the mice were killed and the tumor tissues were collected and prepared into single cell suspensions. The results were detected by flow cytometry. Fig.14 As shown in AC.

[0122] from Fig.14 It can be found that in AC, SPN@EEVs treatment significantly upregulated the proportion of CD8+T cells, upregulated the proportion of CD80+F4 / 80+ macrophages, and downregulated the proportion of CD206+F4 / 80+ macrophages, indicating that SPN@EEVs can promote CD8+T cell infiltration and has the ability to promote macrophage polarization to M1 type, thereby reshaping the immune microenvironment.

[0123] In summary, this application discovered for the first time that exosomes released by tumor cells under starvation stimulation have the effect of enhancing immune cell infiltration and reshaping the immune microenvironment; and exosomes themselves can be used as a drug delivery platform. In this application, SHP2 PROTAC is used as a model drug, and PLA-PEG is used to assemble it into nanoparticles SPN, and then exosomes are coated on the outside as an anti-tumor immune drug delivery system, which has targeting and biocompatibility. The SPN and exosomes can synergistically inhibit and kill tumors, and have a good tumor treatment effect.

[0124] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification and the features of different embodiments or examples, unless they are contradictory.

[0125] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of this application, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0126] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any technician familiar with the technical field can easily think of various changes or substitutions within the technical scope disclosed in the present application, which should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. A tumor cell exosome, characterized in that: The exosomes are induced to be produced by tumor cells through amino acid starvation stimulation.

2. The tumor cell exosomes according to claim 1, characterized in that The exosomes are starved and cultured in vitro using a culture medium without amino acids to stimulate tumor cells, and the cell culture supernatant is collected and centrifuged to obtain a supernatant containing starvation-stimulated exosomes.

3. The tumor cell exosomes according to claim 1 or 2, characterized in that The culture medium is EBSS culture medium, wherein the glucose content is 4.5 g / L; the starvation stimulation time is 12-36 h; The centrifugal separation was performed by gradient centrifugation; the process of gradient centrifugation was as follows: 300 g centrifugation for 10 min, 2000 g centrifugation for 10 min, 10000 g centrifugation for 30 min, and 110000 g centrifugation for 70 min.

4. An anti-tumor immune drug delivery system, characterized in that: The components of the delivery system include nanoparticles and the tumor cell exosomes according to any one of claims 1 to 3 that encapsulate the nanoparticles; the nanoparticles are composed of components including polylactic acid polyethylene glycol amino groups and drugs.

5. An anti-tumor immune drug delivery system according to claim 4, characterized in that: The drug is a compound of formula I: The average molecular weight of polylactic acid in polylactic acid polyethylene glycol amino is 1000-5000; the average molecular weight of polyethylene glycol is 1000-5000; The mass ratio of the structural compound shown in formula I to the polylactic acid polyethylene glycol amino group is (1-2):

20.

6. An anti-tumor immune drug delivery system according to claim 4 or 5, characterized in that: The nanoparticles are prepared by the following method: The DMSO solution of the structural compound shown in Formula I and polylactic acid polyethylene glycol amino was added dropwise into distilled water, and then dialyzed to obtain the nanoparticles.

7. An anti-tumor immune drug delivery system according to claim 6, characterized in that: The volume ratio of DMSO to distilled water is (1-2.5):10; Add the compound of formula I and the DMSO solution of polylactic acid polyethylene glycol amino group dropwise under high-speed vortex state; The dialysis was performed using a 20000KD dialysis bag.

8. The method for preparing the anti-tumor immune drug delivery system according to any one of claims 4 to 7, characterized in that: The following steps are involved: The anti-tumor immune drug delivery system is prepared by mixing the tumor cell exosomes according to any one of claims 1 to 3 with nanoparticles and then ultrasonically treating the mixture in an ice bath.

9. The method for preparing the anti-tumor immune drug delivery system according to claim 7, characterized in that: The mass ratio of the tumor cell exosomes to the nanoparticles is (1-1.5):1; The ultrasonic treatment time is 4-8min; After ultrasonic treatment, separation is performed by centrifugation. The centrifugation conditions are: centrifugation at a speed of 30,000 g for 15-45 minutes.

10. Use of the tumor cell exosomes according to any one of claims 1 to 3 or the anti-tumor immune drug delivery system according to any one of claims 4 to 9 in the preparation of anti-tumor drugs.

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