CN108543074B - Exosome-encapsulated nano drug delivery system for tumor treatment and preparation thereof - Google Patents

Abstract

Translated fromChinese

本发明公开了一种用于肿瘤治疗的外泌体包裹的纳米载药系统及其制备，该外泌体包裹的纳米载药系统是利用细胞内吞载药纳米材料然后再经该细胞外排后得到的，所述载药纳米材料负载有抗肿瘤药物，包括化疗药物、用于免疫治疗的药物、用于改造肿瘤微环境的药物中的至少一种。本发明通过对关键的包裹纳米载药系统的外部组分生物膜的组成及结构等进行改进，与现有技术相比为基于生物膜的生物加工纳米粒提供了一种新的途径。本发明利用外泌体包裹纳米载药系统，能够大大保留外泌体的组成及结构，所得到的外泌体包裹的纳米载药系统在血液循环过程中具有良好的稳定性以及肿瘤靶向性。

The invention discloses an exosome-wrapped nano-drug-carrying system for tumor treatment and its preparation. The exosome-wrapped nano-drug-carrying system utilizes intracellular drug-carrying nanomaterials and then effluxes through the cell. Afterwards, the drug-loaded nanomaterial is loaded with anti-tumor drugs, including at least one of chemotherapeutic drugs, drugs for immunotherapy, and drugs for modifying tumor microenvironment. Compared with the prior art, the present invention provides a new way for biofilm-based bioprocessing nanoparticles by improving the composition and structure of the key external component biofilm encapsulating the nano-drug-carrying system. The present invention utilizes the exosome-wrapped nano-drug-carrying system, which can greatly preserve the composition and structure of exosomes, and the obtained exosome-wrapped nano-drug-carrying system has good stability and tumor targeting in the process of blood circulation .

Description

Exosome-encapsulated nano drug delivery system for tumor treatment and preparation thereof

Technical Field

The invention belongs to the field of nano materials and oncology, and particularly relates to an exosome-coated nano drug delivery system for tumor treatment and preparation thereof, in particular to preparation and application of a nano drug delivery system for improving tumor accumulation and deep penetration of an anti-tumor drug and targeting and killing tumor stem cells.

Background

The recurrence and metastasis of tumor are the most important factors causing the low survival rate of tumor patients. Studies have shown that tumor recurrence and metastasis are closely linked to tumor stem cells (CSCs). The drug resistance of CSCs is one of the main causes of failure of tumor therapy, and mainly includes the following aspects: (1) the ABC transporter with high expression of CSCs is the first factor for expressing drug resistance; (2) the CSCs are in a G0 static state and are important for maintaining drug resistance; (3) CSCs have a more efficient DNA repair capacity; (4) CSCs are located deep in hypoxic tumor tissue and are inaccessible to antineoplastic drugs. At present, CSCs are treated mainly for their biological characteristics, including quiescence, poorly differentiated states, and abnormal signaling pathways. Although in vitro experiments, various inhibitors or promoters can obviously inhibit the characteristics of the CSCs, once the CSCs enter the body, the targeting and specificity of a treatment scheme are particularly important, and small molecular drugs can be taken by normal tissue cells when the CSCs are treated, so that the CSCs have potential toxic and side effects. Therefore, drugs for CSCs therapy need to have a certain tumor targeting ability.

The nano drug-loaded system is very beneficial to the targeted therapy of CSCs due to the unique advantages of the nano drug-loaded system, such as EPR effect, surface couplable targeting molecules, capability of realizing drug co-delivery and the like. At present, a plurality of nano drug delivery systems realize high accumulation of drugs in CSCs by co-delivering a regulator related to drug resistance of CSCs, a specific killing drug, a surface modified CSCs targeting ligand and the like. However, this therapeutic strategy still has difficulty achieving the desired therapeutic effect on CSCs, mainly in: (1) the ideal target CSCs nano drug-carrying system needs to be capable of accumulating in tumor tissues highly, penetrating into the deep part of the tumor tissues deeply and being taken by CSCs in large quantity, but the method is difficult to meet the characteristics at the same time; (2) CSCs do not have a universal marker protein, the marker proteins of different CSCs are different from each other, and part of the marker proteins are highly expressed in common stem cells, so that the improvement of the targeting of the CSCs by coupling a targeting ligand has a potential threat; (3) the nano drug delivery system is complex in synthesis process and has potential toxicity and side effects due to the characteristics of non-self. Therefore, it is necessary to develop a therapeutic strategy that targets and kills CSCs with high efficacy and has very low toxic side effects.

Currently, bioprocessed nanoparticles based on biofilms are widely used in tumor therapy. Based on the method, a plurality of specific properties are endowed to a nano drug delivery system, for example, the nanoparticle wrapped by the erythrocyte membrane has longer blood long circulation capability, the nanoparticle wrapped by the tumor cell membrane has efficient homologous targeting capability, the nanoghosts derived from stem cells have good tumor targeting capability and the like. The natural modification mode can avoid potential toxic and side effects such as PEG predicament caused by chemical modification. However, in the biological processing process, partial destruction of the cell membrane structure causes loss of some membrane proteins which play important functions, resulting in reduction of stability and tumor targeting of the membrane-coated nano drug delivery system in the blood circulation process. Exosomes are extracellular vesicles secreted by cells with a particle size between 30-100 nm. The exosome has good biological stability and biocompatibility, low immunogenicity and low toxicity in the body. It was found that exosomes exhibit excellent cellular uptake and tumor targeting ability due to the specific proteins on the surface. Meanwhile, some exosome proteins, such as CD81, CD9, etc., are capable of degrading extracellular matrix, thereby enhancing tumor penetration and CSCs targeting. Although the existing exosomes have good effects, the existing exosome technology has the problems of low yield, small drug loading and long time consumption in the preparation process, and the practical application is influenced.

Disclosure of Invention

In view of the above drawbacks or needs for improvement in the prior art, an object of the present invention is to provide an exosome-encapsulated nano drug-loading system for tumor therapy and a preparation method thereof, wherein the composition and structure of an external component biofilm encapsulating the nano drug-loading system, the overall process design of a corresponding preparation method, and parameters, conditions, etc. of each step (such as an endocytosis and efflux incubation step) are improved, thereby providing a new approach for bio-processing nanoparticles based on a biofilm compared with the prior art. The invention utilizes the exosome to wrap the nano drug-carrying system, can greatly reserve the composition and the structure of the exosome, and the obtained exosome-wrapped nano drug-carrying system has good stability and tumor targeting property in the blood circulation process. The drug delivery system can particularly solve the problem of poor targeting and killing effects of CSCs, and after entering blood through intravenous administration, the drug delivery system can be efficiently accumulated in tumor tissues, deeply penetrates into deep parts of tumors and remarkably improves the uptake behavior of CSCs; obviously inhibit the growth of liver cancer and melanoma lung metastasis tumor, and obviously reduce the proportion of CSCs.

In order to achieve the above object, according to one aspect of the present invention, there is provided an exosome-encapsulated nano drug delivery system for tumor therapy, which is characterized in that the exosome-encapsulated nano drug delivery system is obtained by using a drug-loaded nanomaterial that is endocytosed by a cell and then discharged outside the cell, the drug-loaded nanomaterial being loaded with an anti-tumor drug, the anti-tumor drug comprising at least one of a chemotherapeutic drug, an immunotherapeutic drug, and a drug that reconstructs a tumor microenvironment.

As a further preferred of the present invention, the cell comprises at least one of a tumor cell, a tumor stem cell, an immune cell, a tumor-associated fibroblast, a mesenchymal stem cell, a bone marrow-derived suppressor cell, and a regulatory T cell; the tumor corresponding to the tumor cell and the tumor stem cell comprises acute leukemia, lymphoma, prostatic cancer, thyroid cancer, esophagus cancer, bone cancer, gastric cancer, breast cancer, lung cancer, ovarian cancer, chorioepithelioma, cervical cancer, uterine corpus cancer, liver cancer, bladder cancer, skin cancer, colon cancer or rectal cancer; the immune cell comprises a T lymphocyte, a B lymphocyte, a K lymphocyte, an NK lymphocyte, a mast cell, or a mononuclear phagocyte system.

As a further preferable aspect of the present invention, the nanomaterial in the drug-loaded nanomaterial comprises at least one of a nanoliposome, a carbon-based nanomaterial, a silicon-based nanomaterial, a metal nanomaterial, a semiconductor quantum dot, an upconversion material, and a polymer nanomaterial; the carbon-based nano material preferably comprises at least one of graphene oxide, carbon nano tubes and nano diamonds; the silicon-based nanomaterial preferably comprises at least one of porous silicon, mesoporous silicon and silicon dots; the metal nano-material preferably comprises at least one of gold nanoparticles, silver nanoparticles and transition metal nanoparticles.

As a further preferable mode of the invention, the nano material in the drug-loaded nano material is a nano particle material, and the particle diameter of the nano particle material is 1-1000 nm.

As a further preferred of the present invention, the drug-loaded nanomaterial-loaded antitumor drug preferably comprises one or more of a chemotherapeutic drug for treating acute leukemia, lymphoma, prostate cancer, thyroid cancer, esophageal cancer, bone cancer, gastric cancer, breast cancer, lung cancer, ovarian cancer, chorioepithelial cancer, cervical cancer, uterine corpus cancer, liver cancer, bladder cancer, skin cancer, colon cancer, and rectal cancer, a drug for immunotherapy, or a drug for modifying tumor microenvironment.

According to another aspect of the present invention, the present invention provides a method for preparing an exosome-encapsulated nano drug delivery system for tumor therapy, which is characterized by comprising the following steps:

s1: co-incubating the drug-loaded nanomaterial with cells;

s2: centrifuging to remove the drug-loaded nano material which is not taken up by the cells;

s3: adding a fresh culture medium without the drug-loaded nano material for continuous incubation;

s4: centrifugally collecting the drug-loaded nano material discharged by the cells to obtain the exosome-coated nano drug-loaded system for tumor treatment.

In a further preferred embodiment of the present invention, in the step S1, the co-incubation time is 1 to 96 hours.

As a further preferred embodiment of the present invention, the step S2 is specifically to collect the cells at a low temperature of 0-10 ℃ and a centrifugal force of 100-1000g, and wash the cells with pre-cooled phosphate buffer solution until no free nano drug-loaded system exists in the solution;

the step S4 is specifically to remove cells in the culture solution by centrifugation with a centrifugal force of 100-; and (3) cleaning the mixture by using a pre-cooled phosphate buffer solution to obtain the exosome-encapsulated nano drug delivery system.

In a further preferred embodiment of the present invention, in the step S3, the incubation is performed in a cell culture box at 37 ℃ and 5% CO₂Or the culture medium is placed in a cell culture box for incubation for 12-96h after being irradiated by ultraviolet light for 5-240 min.

According to another aspect of the invention, the invention provides the application of the exosome-encapsulated nano drug delivery system for tumor treatment in preparing an anti-tumor drug.

Compared with the prior art, the technical scheme provided by the invention has the advantages that the exosome-encapsulated nano drug-carrying system is adopted to form the exosome-encapsulated nano drug-carrying system for tumor treatment, and specifically, the nano drug-carrying material pre-loaded with antitumor drugs is co-incubated with cells, so that the drug-carrying nano material is endocytosed by the cells, and then the nano drug-carrying system which is discharged and encapsulated by exosomes is obtained by utilizing the discharge of the cells. The exosome-encapsulated nano drug-carrying system for tumor treatment is obtained by a drug-carrying nano material for cell efflux endocytosis, and has the following characteristics: the nano drug-carrying system consists of an exosome structure on the surface and a drug-carrying nano material in the nano drug-carrying system; the uptake behavior and the killing effect of the nano drug-loaded system in tumor cells and tumor stem cells are obviously superior to those of an uncoated drug-loaded nano material, and the nano drug-loaded system can be more accumulated in tumor tissues and penetrate into the deep parts of the tumor tissues; the nano drug-carrying system can generate obvious inhibition effect on various tumors with extremely low amount of antitumor drugs.

The invention firstly provides a preparation idea of utilizing a cell efflux nano drug-carrying system to wrap exosomes on the surface of the nano drug-carrying system. The existing nano medicine carrying system film wrapping technology is mainly obtained by a method of crushing a biological film and then extruding the biological film and the nano medicine carrying system together to penetrate through a filter membrane, the method easily causes damage to a cell membrane structure, further causes loss of functional proteins and the like on the surface of the membrane, and is unfavorable for the stability, targeting performance and the like of the nano medicine carrying system. The preparation method is milder, and can completely retain the complete structure of the exosome membrane, thereby playing a better role. According to the invention, the membrane-wrapped nano drug-carrying system is constructed by discharging nano particles outside cells for the first time, the yield of exosomes can be remarkably improved by more than 34 times after the nano drug-carrying system is used for stimulating cells, the drug-carrying capacity is improved by more than 2 times, and the consumption time can be shortened by half. The anti-tumor drug loaded by the drug-loaded nanomaterial can be a chemotherapeutic drug, an immunotherapy drug, a photothermal therapy drug or small-sized nanoparticles with photothermal effect for directly inhibiting tumor growth, or an immune co-stimulatory molecule and a monoclonal antibody for assisting in treating tumors, or at least one of drugs capable of influencing tumor microenvironments such as tumor tissue extracellular matrix and tumor stromal cells, for example, one or more chemotherapeutic drugs capable of treating acute leukemia, lymphoma, prostate cancer, thyroid cancer, esophageal cancer, bone cancer, gastric cancer, breast cancer, lung cancer, ovarian cancer, chorionic epithelial cancer, cervical cancer, endometrial cancer, liver cancer, bladder cancer, skin cancer, colon cancer and rectal cancer.

The invention also preferably controls the incubation processes of endocytosis and efflux, and by controlling the types of cells and the parameter conditions (such as incubation time and the like) of the related incubation processes, exosomes formed by endocytosis and efflux of the cells are formed outside the drug-loaded nanomaterial, the content of the exosomes is high, and the nano drug-loaded system wrapped by the exosomes has good stability and tumor targeting property as a whole.

The technical scheme of the invention has the following beneficial effects:

(1) the exosome structure is covered on the surface of the drug-loaded nano material, so that the stability and the drug slow-release effect of the drug-loaded nano material are improved;

(2) after the drug-loaded nano material is incubated with cells, the yield of exosomes is remarkably improved;

(3) the uptake behavior of the exosome-encapsulated nano drug delivery system in tumor cells and CSCs is remarkably improved;

(4) the killing effect of the exosome-encapsulated nano drug delivery system on tumor cells and CSCs is obviously enhanced;

(5) the exosome-coated nano drug delivery system can be highly accumulated in tumor tissues, and can penetrate into the deep parts of the tumor tissues in the deep parts.

Drawings

FIG. 1A is a particle size characterization of E-PSiNPs of the present invention;

FIG. 1B shows the observation results of E-PSiNPs of the present invention in a transmission electron microscope, wherein the left image is a control group and the right image is an experimental group (20 nm on the scale in the figure);

FIG. 2 shows the result of FTEM power spectrum analysis of E-PSiNPs of the present invention;

FIG. 3 shows the co-localization observation of the surface CD63 protein and the internal PSiNPs of the E-PSiNPs of the present invention;

FIG. 4 shows the Western blot results of the E-PSiNPs surface exosome-associated proteins of the present invention;

FIG. 5 shows the uptake behavior of DOX @ E-PSiNPs of the present invention in H22 tumor cells;

FIG. 6 is a graph showing the uptake behavior of DOX @ E-PSiNPs in H22CSCs according to the present invention;

FIG. 7 is a graph showing the uptake behavior of DOX @ E-PSiNPs in B16CSCs according to the present invention;

FIG. 8 is a graph showing the cytotoxicity of DOX @ E-PSiNPs of the present invention against H22 tumor cells;

FIG. 9A is a graph showing the effect of DOX @ E-PSiNPs of the present invention on the number of H22 tumor clonal balls cultured in 3D soft fibrin glue;

FIG. 9B is a graph showing the effect of DOX @ E-PSiNPs of the present invention on the size of H22 tumor clonal balls cultured in 3D soft fibrin glue;

FIG. 10 is a graph showing the cytotoxicity of DOX @ E-PSiNPs of the present invention against B16 tumor cells;

FIG. 11A is a graph showing the effect of DOX @ E-PSiNPs of the present invention on the number of B16 tumor clonal balls cultured in 3D soft fibrin glue;

FIG. 11B is a graph showing the effect of DOX @ E-PSiNPs of the present invention on the size of B16 tumor clonotypes cultured in 3D soft fibrin glue;

FIG. 12 is a graph showing the accumulation behavior of DOX @ E-PSiNPs of the present invention in tumor tissues of tumor-bearing mice;

FIG. 13 is a graph of the accumulation behavior of DOX @ E-PSiNPs of the present invention in CSCs in tumor tissue of tumor-bearing mice;

FIG. 14 is the deep penetration behavior of DOX @ E-PSiNPs of the present invention in vitro tumor clonal spheres;

FIG. 15 is a graph of the deep penetration behavior of DOX @ E-PSiNPs of the present invention in tumor tissue in vivo;

FIG. 16A is a graph showing the tumor growth curves of mice injected intravenously with DOX @ E-PSiNPs of the present invention into a hepatoma subcutaneous tumor model mouse;

FIG. 16B is a graph showing the weight of tumor tissue in mice, which are models of hepatoma subcutaneous tumors, after intravenous injection of DOX @ E-PSiNPs of the present invention;

FIG. 16C is the experimental results of the lifetime of mice after the DOX @ E-PSiNPs of the present invention enter the hepatoma subcutaneous tumor model mice by intravenous injection;

FIG. 17A is a graph showing the ratio of lateral population cells in tumor tissue of a mouse after intravenous injection of DOX @ E-PSiNPs of the present invention into a hepatoma subcutaneous tumor model mouse;

FIG. 17B shows the number of tumor clone balls formed in 3D fibrin glue by monodisperse cells obtained by dispersing tumor tissues of mice after DOX @ E-PSiNPs of the present invention enter the model mouse of liver cancer subcutaneous tumor by intravenous injection;

FIG. 17C shows the size of the tumor clone balls formed in 3D fibrin glue by the monodisperse cells obtained by dispersing the tumor tissue of the mouse after the DOX @ E-PSiNPs of the present invention enter the liver cancer subcutaneous tumor model mouse through intravenous injection;

FIG. 18A is a graph showing the number of tumor nodules in the lung of a mouse after intravenous injection of DOX @ E-PSiNPs of the present invention into a mouse with a melanoma lung metastasis;

FIG. 18B shows the H & E staining of lung tissue after intravenous injection of DOX @ E-PSiNPs of the present invention into mice with melanoma lung metastases;

FIG. 18C is a graph showing the results of a lifetime experiment of mice after intravenous injection of DOX @ E-PSiNPs of the present invention into a mouse with a melanoma lung metastasis;

FIG. 19A is the number of tumor clonal balls formed in 3D fibrin glue by monodisperse cells obtained by dispersing tumor nodules in the lung of a mouse after intravenous injection of DOX @ E-PSiNPs of the present invention into a melanoma lung metastatic tumor mouse;

FIG. 19B shows the size of the tumor clonal sphere formed by the monodisperse cells obtained by dispersing the tumor nodules in the lung of a mouse in 3D soft fibrin glue after the DOX @ E-PSiNPs of the present invention are injected intravenously into a melanoma lung metastatic tumor mouse.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

Various tumor cells, drugs and experimental animals were used in the following examples:

h22 mouse liver cancer cell, human liver cancer cell line Bel7402 cell, and mouse skin cancer cell line B16, all of which are available from American ATCC company or Chinese typical Collection CCTCC.

BALB/C mice, C57 mice, were purchased from Wuhan university medical laboratory animal center, weighing 18-20 grams;

doxorubicin was purchased from Sigma.

Example 1: preparation and characterization of exosome-encapsulated nano drug delivery system

1. Test materials and reagents

H22 mouse hepatoma cells, Doxorubicin (with red fluorescence), boron-doped p <100> silicon wafers (resistance 0.00055-0.0015. omega. -cm) were purchased from Virginia, Inc. of USA

2. Experimental procedure

(1) Putting the silicon chip in concentrated H₂SO₄And H₂O₂The mixed solution (v: v ═ 3:1) is washed with ultrapure water for 3 times for 15min, and then dried for standby. The polished surface of the silicon chip is upwards arranged in an electrochemical corrosion device, and HF/ethanol solution and 165mA/cm in volume ratio of 4:1 are added²After the silicon wafer is continuously corroded for 300s under the current intensity, a dark red film appears on the surface of the silicon wafer, the silicon wafer is washed for 3 times by absolute ethyl alcohol, and 3.3 percent of HF/ethanol solution (mass ratio) is continuously added, wherein the mass ratio of the HF/ethanol solution is 4.5mA/cm²The current intensity of (2) was applied for 90 seconds to peel the porous silicon film from the silicon substrate. Absorbing HF solution, gently cleaning with anhydrous ethanol for 3 times, and collecting porous silicon filmAnd the mixture is transferred into an aqueous solution for ultrasonic treatment for 16h, centrifuged at 8000g for 20min, the supernatant is collected and placed in a water bath at 60 ℃ for 3-6h to excite the fluorescence of the PSiNPs (PSiNPs, namely porous silicon nanoparticles).

10mg of PSiNPs were dissolved in 5mL of ultrapure water, and 1mL of a 10mg/mLDOX aqueous solution was added thereto and the mixture was stirred overnight, centrifuged at 10,000g for 10min, and the supernatant was discarded. The precipitate was washed with ultrapure water, centrifuged at 10,000g for 10min and repeated 3 times. The drug-loaded PSiNPs (DOX @ PSiNPs) were stored in ultrapure water and stored at 4 ℃ for further use.

(2) Get 10⁷A single H22 tumor cell was cultured in a 10cm diameter sterile petri dish. After overnight incubation, the medium was discarded, and after Phosphate Buffered Saline (PBS) washing, 6mL of serum-free RPMI 1640 medium containing 200. mu.g/mL of PSiNPs was added to continue incubation. After 1-96h (such as 1h, 6h, 12h, 24h, 48h and 96h), centrifugally collecting cells in a culture dish; after washing the collected cells with PBS, 6mL of fresh medium is added thereto for further incubation for 12-96h (e.g., 12h, 24h, 48h, 96 h). Collecting culture medium, centrifuging at 5000g to collect supernatant, centrifuging at 10000g or 20000g for 30min or 120min to obtain precipitate, which is discharged porous silicon nanoparticles (E-PSiNPs).

(3) E-PSiNPs and PSiNPs were dissolved in PBS, and the particle size and zeta potential distribution were measured using a nano-particle size and zeta potential meter with a 633nm He-Ne laser. The specific setting conditions are as follows: the temperature was 25 ℃ and the equilibration time 120 s. Meanwhile, placing the copper net with the carbon film side facing upwards on a disposable PE glove, dropwise adding E-PSiNPs or PSiNPs with a certain concentration on the surface of the copper net, balancing for 5min, sucking the liquid on the surface of the copper net with filter paper, placing the copper net on the filter paper, standing overnight, and observing the appearance of a sample by using a TEM (transmission electron microscope); and performing energy spectrum scanning on the sample by using the scanning mode of FTEM, and analyzing the elemental composition of the sample.

3. Results of the experiment

Experimental results show that the porous silicon nano-particles wrapped by exosomes can be obtained under the conditions of the incubation time of the nano-drug delivery system and the cells (1h, 6h, 12h, 24h, 48h and 96h), the continuous incubation time of the cells in a fresh culture medium (12h, 24h, 48h and 96h) and the centrifugal force (10000g, 20000g) and time (30min and 120min) for finally obtaining the porous silicon nano-particles wrapped by exosomes through centrifugationAnd (4) granulating. The nano drug delivery system and the cells are incubated for a longer time under the condition of ensuring the survival rate of the cells, the centrifugal force is higher, and the shorter centrifugal time is used under the condition of ensuring the yield, so that the consumption time can be reduced under the higher yield, and the special requirements on instruments and equipment can be ensured. According to the invention, the nano drug delivery system is preferably incubated with cells for 12h, the cells are incubated in a fresh culture medium for 24h, the centrifugal force is 20000g, the centrifugation time is 30min (the final yield difference between the centrifugation time of 30min and 120min is not great), and the higher yield can be obtained in the shortest possible time. The final yield of exosomes of the invention was 10⁷Each cell can generate about 60 mug of exosome, and only about 1.8 mug of exosome can be obtained by conventional ultra-high speed centrifugation method collection, namely, the yield of exosome can be remarkably improved by more than 34 times after the nano drug delivery system is used for stimulating the cells.

The average particle size distribution of E-PSiNPs and PSiNPs was found by DLS detection to be around 280nm and 120nm (FIG. 1A), and the zeta potential was-11 mV. The transmission electron microscope pictures show that the E-PSiNPs have irregular shapes and a layer of membranous structure is attached to the surface (figure 1B). The existence of Si element in the energy spectrum analysis result proves that the structure seen in the figure is the porous silicon nano-particle (figure 2). The same method is adopted (the size of the process parameters used in each step, the types and concentrations of the adopted reagents and the like are kept unchanged) to prepare the externally-discharged DOX-loaded porous silicon nanoparticles (DOX @ E-PSiNPs) and the externally-discharged gold nanoparticles (the preparation process of the gold nanoparticles can refer to the prior art), and similar results are obtained.

Example 2: exosome-encapsulated nano drug-loading system surface membrane structure identification

1. Test materials and reagents

FITC-CD63 antibody, CD63 antibody, TSG101 antibody, Calnexin antibody

2. Experimental procedure

(1) A certain amount of E-PSiNPs is added with 500 mu L of 5% BSA solution for incubation for 30min, 10 mu L of FITC-CD63 antibody is added continuously, the mixture is incubated overnight at 4 ℃ with shaking, 20,000g of the mixture is centrifuged for 30min, the supernatant is discarded, and the mixture is washed 3 times with PBS. 20 mu L of FITC-CD63 incubated E-PSiNPs solution is dripped on a confocal dish, after the solution is fully paved, the co-localization condition of the green fluorescence of FITC-CD63 antibody and the red fluorescence of PSiNPs is observed by using an FV1000 confocal microscope, and the specific parameters are as follows: PSiNPs: ex 488nm, Em 680 nm; FITC: ex 488nm and Em 520 nm.

(2) E-PSiNPs, exosomes and corresponding cells with the same protein amount are taken, an immunoblotting method is adopted to detect the expression conditions of exosome marker proteins CD63 and TSG101 in the E-PSiNPs from the cells, the Calnexin protein from the endoplasmic reticulum is used as a negative control, and the beta-action is used as an internal reference. 3. Results of the experiment

The immunofluorescent staining results showed that the green fluorescence of FITC-CD63 antibody completely colocalizes with the red fluorescence of PSiNPs, indicating the presence of CD63 protein on the surface of E-PSiNPs (FIG. 3). Through western blot analysis, we further demonstrated that a large number of exosome marker proteins CD63 and TSG101 exist on the surface of H22-derived E-PSiNPs, and that the potential that E-PSiNPs are derived from cytoplasm was excluded by using Calnexin protein as a positive control (fig. 4,control group 1 is a cell lysate of H22 cells,control group 2 is exosomes of H22 cells collected by a conventional ultracentrifugation method, and experimental group is E-PSiNPs). Similarly, the Bel7402 cell-derived E-PSiNPs prepared by the substantially same preparation method as in example 1 also demonstrated that there are a large number of exosome-marker proteins CD63 and TSG101 on the surface of Bel7402 cell-derived E-PSiNPs.

Example 3: uptake behavior of exosome-encapsulated nano drug delivery system in vitro tumor cells and CSCs thereof

1. Test materials and reagents

H22 mouse liver cancer cell, adriamycin, 3D soft fibrin glue (1) in vitro tumor cell uptake behavior

2×10⁵H22 was cultured in suspension in a cell culture plate, after 24 hours the medium was removed, 1mL of a serum-free medium containing free DOX, DOX @ PSiNPs or DOX @ E-PSiNPs at a DOX concentration of 0.5, 1, 2. mu.g/mL, respectively, was added to the cell culture plate, and the culture was incubated at 37 ℃ in 5% CO₂This normal cell culture was incubated for 4h, cells were harvested, washed 3 times with PBS, and centrifuged at 250g for 10 min. And adding 500 mu L of precooled PBS into the cell sediment to resuspend the cells, sieving the cells by a 200-mesh sieve, and detecting the intracellular DOX fluorescence by an FC500 flow cytometer. Specific detection parameters are as follows: ex ═488nm, emission light was detected as FL2 fluorescence channel.

(2) In vitro CSCs uptake

H22 cells were seeded into 3D soft fibrin glue and cultured to obtain H22 CSCs. To2X 10⁵Adding free DOX, DOX @ PSiNPs and DOX @ E-PSiNPs with DOX final concentration of 0.5, 1 and 2 mu g/mL into H22CSCs, and adding 5% CO at 37 DEG C₂Incubating for 4h under the condition, collecting cells, washing for 3 times by PBS, resuspending the cells by 500 mu L of precooled PBS, sieving by a 200-mesh sieve, and detecting the intracellular DOX fluorescence by an FC500 flow cytometer. Specific detection parameters are as follows: ex 488nm, emission light detected as FL2 fluorescence channel.

3. Results of the experiment

H22 cells and DOX @ E-PSiNPs from H22 cells are incubated together, and flow cytometry analysis results show that compared with the DOX @ PSiNPs and free DOX, the fluorescence intensity of intracellular DOX in DOX @ E-PSiNPs treatment groups is improved by about 5 times (fig. 5, acontrol group 1 is free DOX, acontrol group 2 is DOX @ PSiNPs, namely, anti-tumor drug doxorubicin-loaded porous silicon nanoparticles, and an experimental group is DOX @ E-PSiNPs). In H22CSCs, the DOX @ E-PSiNPs treatment group DOX fluorescence intensity gradually increased with the increase of DOX concentration, indicating that the endocytosis of DOX @ E-PSiNPs by the CSCs is concentration-dependent. At a DOX concentration of 2. mu.g/mL, the cell-entrance amount of the DOX @ E-PSiNPs-treated group was 2.1 and 1.7 times that of the free DOX and DOX @ PSiNPs, respectively, indicating that the DOX @ E-PSiNPs had better CSCs targeting ability (FIG. 6,control 1 was free DOX,control 2 was DOX @ PSiNPs, and the experimental group was DOX @ E-PSiNPs). Similarly, we used Bel7402 cell-derived DOX @ E-PSiNPs, and incubated with Bel7402 cells and CSCs thereof, and also demonstrated that DOX @ E-PSiNPs exhibit significant uptake behavior by tumor cells and CSCs.

Example 4: universality of uptake behavior of exosome-encapsulated nano drug delivery system in CSCs

To investigate whether DOX @ E-PSiNPs have universality for targeting CSCs, we further investigated the uptake behavior of H22 cell-derived DOX @ E-PSiNPs by B16CSCs by inoculating a certain number of B16 cells into 3D soft fibrin glue and collecting a large number of B16F10 tumor stem cells after culturing for 5-7 days.

1. Test materials and reagents

Mouse skin cancer cell line B16, adriamycin and 3D soft fibrin glue

2. Experimental procedure

Mouse skin cancer cells B16 cells were inoculated into 3D soft fibrin glue and cultured to obtain B16 CSCs. To2X 10⁵Adding free DOX, DOX @ PSiNPs and DOX @ E-PSiNPs with DOX final concentration of 0.5, 1 and 2 mu g/mL into B16CSCs, and adding 5% CO at 37 DEG C₂Incubating for 4h under the condition, collecting cells, washing for 3 times by PBS, resuspending the cells by 500 mu L of precooled PBS, sieving by a 200-mesh sieve, and detecting the intracellular DOX fluorescence by an FC500 flow cytometer. Specific detection parameters are as follows: ex 488nm, emission light detected as FL2 fluorescence channel.

3. Results of the experiment

After the B16CSCs and the H22 cell-derived DOX @ E-PSiNPs are co-incubated, flow cytometry analysis results show that the intracellular DOX content of the B16CSCs in the DOX @ E-PSiNPs treatment group is remarkably superior to that of free DOX and DOX @ PSiNPs groups under different drug concentrations (figure 7, acontrol group 1 is free DOX, acontrol group 2 is DOX @ PSiNPs, and an experimental group is DOX @ E-PSiNPs), which shows that the H22-derived DOX @ E-PSiNPs can be greatly absorbed by the B16CSCs, and shows that the DOX E-PSiNPs target CSCs have universality.

Example 5: exosome-encapsulated nano-drug delivery system is toxic to cells in tumor cells and CSCs in vitro

1. Test materials and reagents

H22 mouse liver cancer cell, human liver cancer cell line Bel7402 cell, 3D soft fibrin glue

2. Experimental procedure

(1) In vitro tumor cell toxicity

H22 cells at8X 10³The cells of (a) were seeded in a 96-well plate, the medium was removed after overnight incubation, 100. mu.L of a serum-free medium containing free DOX, DOX @ PSiNPs or DOX @ E-PSiNPs at a DOX concentration of 0.25, 0.5, 1. mu.g/mL, 5% CO at 37 ℃ was added₂After 24 hours of incubation under the conditions, 10 mu L of CCK-8 solution is added into each well for incubation for 4 hours, and the absorbance of each well at 450nm is detected by using a 318C microplate reader.

(2) Toxicity of CSCs in vitro

400 cells/well H22 were seeded on 3D plates plated at 1mg/mLFibrin glue in 96-well plates, 37 ℃ and 5% CO₂Culturing under the condition. After 5 days, the medium was discarded, 100. mu.L DOX was added to a final concentration of 2. mu.g/mL free DOX, DOX @ PSiNPs or H22 cell-derived DOX @ E-PSiNPs at 37 ℃ with 5% CO₂Incubate for 24h under conditions. Discarding the supernatant, adding 0.1% trypan blue solution into each well, staining for 1min, discarding the staining solution, washing with PBS for 5 times, observing and photographing under a microscope, and counting the number and size of the viable tumor cell balls.

3. Results of the experiment

In vitro tumor cytotoxicity test results show that H22 cell-derived DOX @ E-PSiNPs exhibit stronger killing effects on H22 cells than free DOX and DOX @ PSiNPs (FIG. 8,control group 1 is free DOX,control group 2 is DOX @ PSiNPs, and test group is DOX @ E-PSiNPs). In an in vitro CSCs toxicity test, after the DOX @ E-PSiNPs are incubated with H22 tumor clonosomes growing in 3D soft fibrin glue, the number (FIG. 9A,control 1 is free DOX,control 2 is DOX @ PSiNPs, and experimental group is DOX @ E-PSiNPs) and size (FIG. 9B,control 1 is free DOX,control 2 is DOX @ PSiNPs, and experimental group is DOX @ E-PSiNPs) of the surviving tumor clonosomes are both significantly reduced, which indicates that the DOX @ E-PSiNPs have significant CSCs killing effect. In conclusion, the DOX @ E-PSiNPs have obvious tumor cell killing effect and can kill CSCs efficiently.

Example 6: universality of exosome-encapsulated nano drug delivery system on cytotoxicity of tumor cells and CSCs (tumor suppressor cells)

To investigate whether the killing effect of DOX @ E-PSiNPs on CSCs has universality, we examined the killing effect of H22 cell-derived DOX @ E-PSiNPs on CSCs of mouse skin cancer cells B16.

1. Test materials and reagents

H22 mouse liver cancer cell, mouse skin cancer cell line B16, 3D soft fibrin glue

2. Experimental procedure

(1) In vitro tumor cell toxicity test

B16 cells at8X 10³The cells were seeded in 96-well plates at a density, after overnight incubation the medium was removed and 100. mu.L of the medium containing DOX at concentrations of 0.25, 0.5, 1. mu.g was addedmL free DOX, DOX @ PSiNPs or DOX @ E-PSiNPs in serum-free medium, 5% CO at 37 ℃₂After 24 hours of incubation under the conditions, 10 mu L of CCK-8 solution is added into each well for incubation for 4 hours, and the absorbance of each well at 450nm is detected by using a 318C microplate reader.

(2) In vitro CSCs toxicity test

400/well mouse skin cancer cells B16 were seeded in 96-well plates with 1mg/mL 3D soft fibrin glue at 37 deg.C with 5% CO₂Culturing under the condition. After 5 days, the medium was discarded, 100. mu.L DOX was added to a final concentration of 2. mu.g/mL free DOX, DOX @ PSiNPs or H22 cell-derived DOX @ E-PSiNPs at 37 ℃ with 5% CO₂Incubate for 24h under conditions. Discarding the supernatant, adding 0.1% trypan blue solution into each well, staining for 1min, discarding the staining solution, washing with PBS for 5 times, observing and photographing under a microscope, and counting the number and size of the viable tumor cell balls.

3. Results of the experiment

In vitro tumor cytotoxicity experiment results show that compared with free DOX and DOX @ PSiNPs, the DOX @ E-PSiNPs from H22 cells with different concentrations can obviously kill B16 cells, and the DOX @ E-PSiNPs have universality in killing tumor cells (fig. 10, acontrol group 1 is free DOX, acontrol group 2 is DOX @ PSiNPs, and an experimental group is DOX @ E-PSiNPs). In vitro CSCs toxicity test results show that after H22-derived DOX @ E-PSiNPs are incubated with B16 tumor clone spheres in 3D soft fibrin glue, the number (FIG. 11A,control group 1 is free DOX,control group 2 is DOX @ PSiNPs, and experimental group is DOX @ E-PSiNPs) and the size (FIG. 11B,control group 1 is free DOX,control group 2 is DOX @ PSiNPs, and experimental group is DOX @ E-PSiNPs) of the surviving tumor clone spheres are remarkably reduced, which indicates that the DOX @ E-PSiNPs kill CSCs with universality.

Example 7: accumulation behavior of exosome-encapsulated nano drug delivery system in tumor tissues

1. Test materials and reagents

H22 mouse hepatoma cell, BALB/c mouse

2. Experimental procedure

(1) Experimental graphs of tumor tissue accumulation;

2×10⁶a single H22 tumor cell was injected subcutaneously into BALB/c mice (18-20g) in sizeOutside the legs to construct a mouse H22 subcutaneous tumor model. When the tumor size reaches 250mm³In the preparation process, 0.5mg/kg free DOX, DOX @ PSiNPs or DOX @ E-PSiNPs is injected into tail vein, mice are treated by cervical dislocation method after 24h, tumors of the mice, heart, liver, spleen, lung and kidney are taken out, PBS is used for cleaning for 3 times, moisture on the surface of tissues is sucked, each tissue is weighed, 100 mu L RIPA lysate is added, and tissues are ground by using a tissue grinder. After grinding, the grinding fluid is sucked up, 10,000g of the grinding fluid is centrifuged for 10min, and the supernatant solution is taken out and placed in a dark place at 4 ℃ for storage and is to be tested. Different concentrations of DOX standard solutions were prepared using the supernatant solution after grinding and centrifugation of each tissue blank. Fluorescence intensity of DOX in each tissue triturated supernatant solution was measured using high performance liquid chromatography at 488 nm.

3. Results of the experiment

DOX @ E-PSiNPs enter tumor-bearing mice through tail vein injection, each tissue is ground after 24h, and the content of DOX in grinding fluid of each tissue is quantitatively analyzed, so that the result shows that the content of DOX in tumor parts of the DOX @ E-PSiNPs is respectively 3.9 times of free DOX and 1.7 times of the DOX @ PSiNPs, and the DOX @ E-PSiNPs have obvious tumor tissue targeting capacity (figure 12, acontrol group 1 is free DOX, acontrol group 2 is DOX @ PSiNPs, and an experimental group is DOX @ E-PSiNPs).

Example 8: uptake behavior of exosome-encapsulated nano drug delivery system in CSCs of tumor tissues

1. Test materials and reagents

H22 mouse hepatoma cell, BALB/c mouse

2. Experimental procedure

2×10⁶A mouse H22 subcutaneous tumor model was constructed by injecting H22 tumor cells subcutaneously into the outer thigh of BALB/c mice (18-20 g). When the tumor volume reaches 250mm³In the same time, the tail vein is injected with 0.5mg/kg free DOX, DOX @ PSiNPs, DOX @ E-PSiNPs, and the mice are killed by cervical dislocation after 24h, the tumor tissues are taken out, and PBS is cleaned. Placing the tumor tissue in a stem cell culture medium, cutting into mung bean size, removing the culture medium, adding 1mg/mL Collagenase, digesting at 37 ℃ for 2h, and shaking for 1 time every 15min to ensure full digestion. Squeezing digested tumor tissue with rough ground glass sheet until there is no block structure, sucking and removingCentrifuging 1200g of the cell suspension after the homogenization for 5min, discarding the supernatant, washing the cell precipitate for 3 times by PBS, and screening the solution by a 200-mesh screen to obtain the single cell suspension. The single cell suspension is divided into two parts: one part detects the fluorescence of DOX in all tumor cells under the condition of 488nm/FL2 by using an FC500 flow cytometer; mixing the other part of the cell suspension with Hoechst 33342 dye (the final concentration is 5 mug/mL), incubating for 90min at 37 ℃ in the dark, taking cells incubated by 50 mug M verapamil and 5 mug/mL Hoechst 33342 as a control group, immediately placing the cells on ice after the incubation is finished, centrifuging for 5min at 4 ℃ at 250g, removing supernatant, washing with precooled PBS for 3 times, and suspending the cells in precooled PBS for storage on ice. The side population cells were analyzed for DOX fluorescence using CytoFlex flow cytometry. The specific analysis method is as follows:

1) live cells were circled in SSC-A and FSC-A scattergrams;

2) constructing a scatter diagram with the horizontal and vertical coordinates of Ex-405 nm, Em-450 nm, Ex-405 nm and Em-660 nm, comparing the Verapamil-treated group with the untreated group, and circling a part of disappeared cells, wherein the part of disappeared cells are the lateral group cells;

3) the excitation and emission channels at 488nm/585nm were used to detect the intensity of DOX fluorescence in the side population of cells.

3. Results of the experiment

After injecting DOX @ E-PSiNPs into tumor-bearing mice through tail vein according to the method of example 7, tumor tissues are taken out and dispersed into single cells, and flow cytometry analysis shows that compared with free DOX and DOX @ PSiNPs treatment groups, the fluorescence intensity of intracellular DOX of side group cells in the tumor tissues of the DOX @ E-PSiNPs treatment group is respectively improved by 64% and 71%. The results further demonstrate that DOX @ E-PSiNPs have significant CSCs targeting ability in vivo experiments (FIG. 13,control 1 is free DOX,control 2 is DOX @ PSiNPs, and experimental is DOX @ E-PSiNPs).

Example 9: research on tumor deep penetration behavior of exosome-encapsulated nano drug delivery system

1. Test materials and reagents

H22 mouse hepatoma cell, BALB/c mouse, 3D soft fibrin glue, FITC-CD31 antibody

2. Experimental procedure

(1) Deep penetration behavior in 3D tumor cell spheres in vitro

4×10²Cells/well H22 were seeded in 3D soft fibrin glue as described previously. When the tumor cell balls grow to the 5 th day, the culture medium is discarded, PBS is washed for 3 times, serum-free culture mediums of free DOX, DOX @ PSiNPs or DOX @ E-PSiNPs with the DOX final concentration of 10 mu g/mL are respectively added for incubation for 24h, the culture medium is removed, PBS is washed for 3 times, the soft fibrin glue in the holes is transferred into a confocal dish, and the DOX fluorescence conditions at different depths from the surface of the tumor cell balls are observed in a Z-axis scanning mode of an FV1000 confocal microscope. The specific detection parameters are set as follows: ex 559nm and Em 590 nm.

(2) Study of tumor deep penetration behavior in tumor-bearing mice

10⁶A mouse H22 subcutaneous tumor model was constructed by injecting H22 hepatoma cells subcutaneously into the root of the right thigh of BALB/c mice (around 18 g). When the tumor volume grows to 250mm³At the left and right, free DOX, DOX @ PSiNPs or DOX @ E-PSiNPs with DOX concentration of 0.5mg/kg are injected into tail vein, and the mice are killed by cervical dislocation method after 24h, and tumor tissues are taken out and processed by frozen sections. FITC-CD31 antibody was incubated at 37 ℃ for 30min to label tumor section vessels. On-sections DOX red fluorescence and FITC green fluorescence were detected at 559/580nm and 488/520nm, respectively, using FV1000 confocal microscope. The distribution of DOX fluorescence on a straight line from the tumor vessel to the interior of the tumor was counted using Image J software.

3. Results of the experiment

After the DOX @ E-PSiNPs are incubated with the tumor clone spheres, the Z-axis scanning picture of a confocal microscope shows that the DOX fluorescence of the tumor cell spheres of the DOX @ E-PSiNPs treatment group is obviously stronger than that of the free DOX and the DOX @ PSiNPs treatment group under the same penetration depth (figure 14). Further, DOX @ E-PSiNPs were injected via tail vein into tumor-bearing mice, tumor tissues were removed, sectioned, and labeled with FITC-CD31 antibody for staining tumor vessels, and confocal microscopy showed that significant DOX fluorescence signals were still detected in the DOX @ E-PSiNPs group at a distance of 400 μm from the vessels, but significant fluorescence signals were only detected at depths of 25 μm and 120 μm at the farthest in the treatment of DOX and DOX @ PSiNPs, and no significant DOX fluorescence signals were detected when the treatment was continued (FIG. 15). The results prove that DOX @ E-PSiNPs have obvious tumor deep penetration capacity.

Example 10: inhibition effect of exosome-encapsulated nano drug delivery system on mouse liver cancersubcutaneous tumor model 1, and experimental reagent and material

H22 mouse hepatoma cell, BALB/c mouse

2. Experimental procedure

2×10⁶A mouse subcutaneous tumor model was constructed by injecting H22 tumor cells subcutaneously into the outer thigh of BALB/c mice (18-20 g). When the tumor size reaches 50mm³In this case, the mice were divided into 6 groups on average, 14 mice per group, and treated with PBS, E-PSiNPs, DOX @ PSiNPs, DOX @ E-PSiNPs (DOX final concentration of 0.5mg/kg) or high dose free DOX of 4mg/kg by tail vein injection, once every 2 days, for a total of 5 times, and the tumor size was measured at a fixed time per day. On day 17 after the first dose, 8 randomized doses per group were continued for life cycle experiments. Mice were sacrificed by cervical dislocation, tumor tissues were taken out, washed, dried, weighed and photographed.

(2) Killing effect on tumor stem cells

3. Results of the experiment

The tumor growth curve results show that DOX @ E-PSiNPs significantly inhibit tumor growth, and the tumor inhibition effect is superior to that of the high-dose free DOX administration group (FIG. 16A, acontrol group 1 is a normal saline group, acontrol group 2 is E-PSiNPs, acontrol group 3 is free DOX, acontrol group 4 is DOX @ PSiNPs, acontrol group 5 is a high-dose free DOX group, and an experimental group is DOX @ E-PSiNPs). The weighing of the removed tumor tissue after dosing was also shown to significantly inhibit tumor growth by DOX @ E-PSiNPs (fig. 16B,control 1 was saline,control 2 was E-PSiNPs,control 3 was free DOX,control 4 was DOX @ PSiNPs,control 5 was high dose free DOX, experimental was DOX @ E-PSiNPs). Survival results also show that after administration of DOX @ E-PSiNPs, the survival of tumor-bearing mice was prolonged by 30 days, which was significantly better than that of the remaining experimental groups, including the high dose free DOX group (fig. 16C,control group 1 was normal saline group,control group 2 was E-PSiNPs,control group 3 was free DOX,control group 4 was DOX @ PSiNPs,control group 5 was high dose free DOX group, and experimental group was DOX @ E-PSiNPs).

Example 11: killing effect of exosome-encapsulated nano drug delivery system on CSCs in mouse liver cancer subcutaneous tumor model

1. Experimental reagents and materials

H22 mouse hepatoma cell, BALB/c mouse

2. Experimental procedure

The mouse liver cancer subcutaneous tumor model is treated by administration according to the method of theembodiment 10, the mouse is killed by cervical dislocation after administration, tumor tissues are taken out, cleaned and dispersed into single tumor cells, lateral group analysis is carried out by using a CytoFlex flow cytometer, and the cell proportion of each tumor tissue lateral group after different administration treatments is detected. The number and size of tumor cell balls were counted, seeded at a cell density of 800 cells/well in a 96-well plate containing 1mg/mL 3D soft fibrin glue, and observed and counted after 5 days of culture at 37 ℃ under 5% CO 2.

3. Results of the experiment

The above results indicate that the ratio of the side group cells in the tumor tissue after the administration treatment of DOX @ E-PSiNPs is significantly reduced, which indicates that DOX @ E-PSiNPs can significantly kill the side group cells (FIG. 17A,control group 1 is normal saline group,control group 2 is E-PSiNPs,control group 3 is free DOX,control group 4 is DOX @ PSiNPs,control group 5 is high-dose free DOX group, and experimental group is DOX @ E-PSiNPs). The number of tumor clone balls formed by the tumor cells of the DOX @ E-PSiNPs group in the 3D soft fibrin glue (FIG. 17B,control group 1 is a physiological saline group,control group 2 is E-PSiNPs,control group 3 is free DOX,control group 4 is DOX @ PSiNPs,control group 5 is a high-dose free DOX group, and experimental group is DOX @ E-PSiNPs) and the size (FIG. 17C,control group 1 is a physiological saline group,control group 2 is E-PSiNPs,control group 3 is free DOX,control group 4 is DOX @ PSiNPs,control group 5 is a high-dose free DOX group, and experimental group is DOX @ E-PSiNPs) are remarkably reduced, which shows that the DOX @ E-PSiNPs can greatly inhibit the dry state of the tumor cells.

Example 12: inhibitory effect of exosome-encapsulated nano drug delivery system on mouse melanin lung metastasis tumor

1. Experimental reagents and materials

Mouse skin cancer cell lines B16, C57 mouse

2. Experimental procedure

5×10⁵A single B16F10 cell was injected tail vein into C57BL/6 mice. After 2 days, the mice were divided into 6 groups of 14 mice each, and treated with PBS, E-PSiNPs, DOX @ PSiNPs, DOX @ E-PSiNPs (DOX final concentration of 0.5mg/kg) or 4mg/kg of high dose free DOX administered every 2 days for a total of 5 times. On day 17 after the first dose, 8 randomized doses per group were continued for life cycle experiments. The remaining 6 mice were sacrificed by cervical dislocation, lung tissue was taken out, and the number of nodules of black tumor in lung was observed and counted. Each group was prepared by placing 3 lung tissues in 4% PFA, treating at 4 deg.C for 24H, slicing and H&And E, dyeing treatment.

3. Results of the experiment

DOX @ E-PSiNPs are injected into a mouse body with melanin lung metastasis tumor through tail vein, and after administration, the pulmonary tumor nodules of a DOX @ E-PSiNPs administration group are found to be significantly lower than those of other control groups (FIG. 18A, acontrol group 1 is a normal saline group, acontrol group 2 is E-PSiNPs, acontrol group 3 is free DOX, acontrol group 4 is DOX @ PSiNPs, acontrol group 5 is a high-dose free DOX group, and an experimental group is DOX @ E-PSiNPs). The same results were obtained by sectioning lung tissue and H & E staining, and observing the number of nodules in the lung by microscopy (FIG. 18B,control 1 is normal saline,control 2 is E-PSiNPs,control 3 is free DOX,control 4 is DOX @ PSiNPs,control 5 is high dose free DOX, and experimental is DOX @ E-PSiNPs). Through a mouse survival period experiment, the mice further prove that the DOX @ E-PSiNPs have a remarkable metastatic tumor inhibition effect, and the survival period of the mice of the DOX @ E-PSiNPs administration group is remarkably improved (FIG. 18C, acontrol group 1 is a physiological saline group, acontrol group 2 is E-PSiNPs, acontrol group 3 is free DOX, acontrol group 4 is DOX @ PSiNPs, acontrol group 5 is a high-dose free DOX group, and an experimental group is DOX @ E-PSiNPs).

Example 13: killing effect of exosome-encapsulated nano drug delivery system on CSCs in mouse melanoma lung metastasis tumor

1. Experimental reagents and materials

Mouse skin cancer cell lines B16, C57 mouse

2. Experimental procedure

After administration of the mouse melanoma lung metastases as in example 12, the black tumor nodules in lung tissue from each administration treatment group were removed from the stem cell culture medium, dispersed into individual tumor cells and counted, seeded at a cell density of 800/well in 96-well plates containing 1mg/mL 3D fibrin glue at 37 ℃ and CO₂Tumor cell pellet size and number were observed after 5 days of culture under the conditions.

3. Results of the experiment

The number of tumor clones formed by the tumor cells of the DOX @ E-PSiNPs treatment group (FIG. 19A,control group 1 is a normal saline group,control group 2 is E-PSiNPs,control group 3 is free DOX,control group 4 is DOX @ PSiNPs,control group 5 is a high dose free DOX group, experimental group is DOX @ E-PSiNPs) and the size (FIG. 19B,control group 1 is a normal saline group,control group 2 is E-PSiNPs,control group 3 is free DOX,control group 4 is DOX @ PSiNPs,control group 5 is a high dose free DOX group, experimental group is DOX @ E-PSiNPs) are significantly lower than the other administration groups, which indicates that the DOX-PSiNPs administration treatment can significantly inhibit the dry @ CSCs.

In addition to the specific cell types and anti-tumor drug species used in the above embodiments, the drug-loaded nanomaterial cells suitable for endocytosis by efflux in the present invention may also include tumor cells (acute leukemia, lymphoma, breast cancer, lung cancer, ovarian cancer, chorioepithelial cancer, cervical cancer, liver cancer, bladder cancer, skin cancer, colon cancer or rectal cancer), tumor stem cells, immune cells (including T lymphocytes, B lymphocytes, K lymphocytes, NK lymphocytes, mast cells, mononuclear phagocyte systems), tumor-associated fibroblasts, Mesenchymal Stem Cells (MSCs), myeloid-derived suppressor cells (MDSCs), regulatory T cells (Treg cells), and the like, which all satisfy the conventional definition in the art; the anti-tumor drug pre-loaded by the drug-loaded nanomaterial can comprise one or more of chemotherapeutic drugs for treating acute leukemia, lymphoma, breast cancer, lung cancer, ovarian cancer, chorioepithelioma, cervical cancer, liver cancer, bladder cancer, skin cancer, colon cancer, rectal cancer and various other solid tumors.

It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (8)

Translated fromChinese

1.一种用于肿瘤治疗的外泌体包裹的纳米载药系统，其特征在于，该外泌体包裹的纳米载药系统是利用细胞内吞载药纳米材料再经该细胞外排并经离心处理收集后得到的，所述载药纳米材料负载有抗肿瘤药物，所述抗肿瘤药物包括化疗药物、免疫治疗药物、重构肿瘤微环境的药物中的至少一种；1. a nano-drug-loading system wrapped by exosomes for tumor treatment, it is characterized in that, the nano-drug-carrying system of this exosome wraps is to utilize endocytosis drug-loaded nanomaterials to be effluxed through this cell again through the After being collected by centrifugation, the drug-loaded nanomaterials are loaded with anti-tumor drugs, and the anti-tumor drugs include at least one of chemotherapeutic drugs, immunotherapy drugs, and drugs for reconstructing tumor microenvironment;该用于肿瘤治疗的外泌体包裹的纳米载药系统是采用包括以下步骤的制备方法制备得到的：The exosome-encapsulated nano-drug delivery system for tumor treatment is prepared by a preparation method comprising the following steps:S1：将载药纳米材料与细胞共孵育；S1: co-incubating the drug-loaded nanomaterials with cells;S2：离心去除该细胞未摄取的载药纳米材料；S2: Centrifuge to remove the drug-loaded nanomaterials not taken up by the cells;S3：加入新鲜的不含载药纳米材料的培养基继续孵育；S3: Add fresh medium without drug-loaded nanomaterials to continue incubation;S4：离心收集经该细胞外排的载药纳米材料，即得到用于肿瘤治疗的外泌体包裹的纳米载药系统；S4: centrifugally collect the drug-loaded nanomaterials effluxed from the cells, that is, to obtain an exosome-coated nano-drug-loading system for tumor treatment;其中，所述步骤S2具体是在0-10℃的低温下，以100-1000g的离心力收集所述细胞，使用预冷的磷酸盐缓冲液清洗该细胞，直至溶液中不存在游离的纳米载药系统；Wherein, the step S2 is to collect the cells with a centrifugal force of 100-1000g at a low temperature of 0-10°C, and wash the cells with a pre-cooled phosphate buffer until there is no free nano-drug loading in the solution. system;所述步骤S4具体是在0-10℃低温下，以100-1000g的离心力离心去除培养液中的细胞，以3000-5000g离心去除培养液中的细胞碎片，以10000-20000g的离心力离心30-120min得到细胞外排的纳米载药系统；使用预冷的磷酸盐缓冲液清洗后，即得到外泌体包裹的纳米载药系统；The step S4 is specifically at a low temperature of 0-10° C., centrifuging with a centrifugal force of 100-1000g to remove cells in the culture medium, centrifuging at 3000-5000g to remove cell debris in the culture medium, and centrifuging with a centrifugal force of 10000-20000g for 30- 120min to obtain the nano-drug-carrying system for cell efflux; after washing with pre-cooled phosphate buffer, the nano-drug-carrying system encapsulated by exosomes is obtained;并且，所述细胞为肿瘤细胞、肿瘤干细胞中的至少一种；所述肿瘤细胞和所述肿瘤干细胞对应的肿瘤包括急性白血病、淋巴瘤、前列腺癌、甲状腺癌、食道癌、骨癌、胃癌、乳腺癌、肺癌、卵巢癌、绒毛膜上皮癌、子宫颈癌、子宫体癌、肝癌、膀胱癌、皮肤癌、结肠癌或直肠癌；In addition, the cells are at least one of tumor cells and tumor stem cells; the tumors corresponding to the tumor cells and the tumor stem cells include acute leukemia, lymphoma, prostate cancer, thyroid cancer, esophageal cancer, bone cancer, gastric cancer, Breast, lung, ovarian, chorioepithelial, cervical, uterine, liver, bladder, skin, colon or rectal cancer;所述载药纳米材料中的纳米材料为硅基纳米材料。The nanomaterials in the drug-loaded nanomaterials are silicon-based nanomaterials.2.如权利要求1所述用于肿瘤治疗的外泌体包裹的纳米载药系统，其特征在于，所述硅基纳米材料包括多孔硅、介孔硅、硅点中的至少一种。2. The exosome-encapsulated nano-drug delivery system for tumor treatment according to claim 1, wherein the silicon-based nanomaterial comprises at least one of porous silicon, mesoporous silicon, and silicon dots.3.如权利要求1所述用于肿瘤治疗的外泌体包裹的纳米载药系统，其特征在于，所述载药纳米材料中的纳米材料为纳米颗粒材料，所述纳米颗粒材料的粒径在1-1000nm。3. The exosome-wrapped nano-drug-loading system for tumor treatment according to claim 1, wherein the nano-material in the drug-loaded nano-material is a nano-particle material, and the particle size of the nano-particle material is at 1-1000nm.4.如权利要求1所述用于肿瘤治疗的外泌体包裹的纳米载药系统，其特征在于，所述载药纳米材料负载的抗肿瘤药物包括治疗急性白血病、淋巴瘤、前列腺癌、甲状腺癌、食道癌、骨癌、胃癌、乳腺癌、肺癌、卵巢癌、绒毛膜上皮癌、子宫颈癌、子宫体癌、肝癌、膀胱癌、皮肤癌、结肠癌、直肠癌的化疗药物、用于免疫治疗药物或用于改造肿瘤微环境的药物中的一种或多种。4. The exosome-wrapped nano-drug-loading system for tumor treatment according to claim 1, wherein the anti-tumor drugs loaded by the drug-loaded nanomaterials include the treatment of acute leukemia, lymphoma, prostate cancer, thyroid Cancer, esophagus cancer, bone cancer, stomach cancer, breast cancer, lung cancer, ovarian cancer, chorioepithelial cancer, cervical cancer, endometrial cancer, liver cancer, bladder cancer, skin cancer, colon cancer, rectal cancer chemotherapy drugs, used for One or more of immunotherapy drugs or drugs used to modify the tumor microenvironment.5.一种用于肿瘤治疗的外泌体包裹的纳米载药系统的制备方法，其特征在于，包括以下步骤：5. the preparation method of the nanometer drug-carrying system that is used for the exosome wrapping of tumor treatment, is characterized in that, comprises the following steps:S1：将载药纳米材料与细胞共孵育；S1: co-incubating the drug-loaded nanomaterials with cells;S2：离心去除该细胞未摄取的载药纳米材料；S2: Centrifuge to remove the drug-loaded nanomaterials not taken up by the cells;S3：加入新鲜的不含载药纳米材料的培养基继续孵育；S3: Add fresh medium without drug-loaded nanomaterials to continue incubation;S4：离心收集经该细胞外排的载药纳米材料，即得到用于肿瘤治疗的外泌体包裹的纳米载药系统；S4: centrifugally collect the drug-loaded nanomaterials effluxed from the cells, that is, to obtain an exosome-coated nano-drug-loading system for tumor treatment;其中，所述步骤S2具体是在0-10℃的低温下，以100-1000g的离心力收集所述细胞，使用预冷的磷酸盐缓冲液清洗该细胞，直至溶液中不存在游离的纳米载药系统；Wherein, the step S2 is to collect the cells with a centrifugal force of 100-1000g at a low temperature of 0-10°C, and wash the cells with a pre-cooled phosphate buffer until there is no free nano-drug loading in the solution. system;所述步骤S4具体是在0-10℃低温下，以100-1000g的离心力离心去除培养液中的细胞，以3000-5000g离心去除培养液中的细胞碎片，以10000-20000g的离心力离心30-120min得到细胞外排的纳米载药系统；使用预冷的磷酸盐缓冲液清洗后，即得到外泌体包裹的纳米载药系统；The step S4 is specifically at a low temperature of 0-10° C., centrifuging with a centrifugal force of 100-1000g to remove cells in the culture medium, centrifuging at 3000-5000g to remove cell debris in the culture medium, and centrifuging with a centrifugal force of 10000-20000g for 30- 120min to obtain the nano-drug-carrying system for cell efflux; after washing with pre-cooled phosphate buffer, the nano-drug-carrying system encapsulated by exosomes is obtained;并且，所述细胞包括肿瘤细胞、肿瘤干细胞中的至少一种；所述肿瘤细胞和所述肿瘤干细胞对应的肿瘤包括急性白血病、淋巴瘤、前列腺癌、甲状腺癌、食道癌、骨癌、胃癌、乳腺癌、肺癌、卵巢癌、绒毛膜上皮癌、子宫颈癌、子宫体癌、肝癌、膀胱癌、皮肤癌、结肠癌或直肠癌；In addition, the cells include at least one of tumor cells and tumor stem cells; the tumors corresponding to the tumor cells and the tumor stem cells include acute leukemia, lymphoma, prostate cancer, thyroid cancer, esophageal cancer, bone cancer, gastric cancer, Breast, lung, ovarian, chorioepithelial, cervical, uterine, liver, bladder, skin, colon or rectal cancer;所述载药纳米材料中的纳米材料为硅基纳米材料。The nanomaterials in the drug-loaded nanomaterials are silicon-based nanomaterials.6.如权利要求5所述用于肿瘤治疗的外泌体包裹的纳米载药系统的制备方法，其特征在于，所述步骤S1中，所述共孵育的时间为1-96h。6 . The method for preparing an exosome-coated nano-drug-loading system for tumor treatment according to claim 5 , wherein, in the step S1 , the co-incubation time is 1-96 h. 7 .7.如权利要求5所述用于肿瘤治疗的外泌体包裹的纳米载药系统的制备方法，其特征在于，所述步骤S3中，所述孵育是置于细胞培养箱中，在37℃、5％CO₂的细胞培养条件下孵育，或者是使先用紫外光照射5-240min后再放置于细胞培养箱中孵育12-96h。7. The method for preparing an exosome-wrapped nano-drug-loading system for tumor treatment according to claim 5, wherein in the step S3, the incubation is placed in a cell incubator at 37° C. , 5% CO₂ cell culture conditions, or first irradiated with UV light for 5-240min and then placed in a cell incubator for 12-96h incubation.8.如权利要求1－4任意一项所述用于肿瘤治疗的外泌体包裹的纳米载药系统在制备抗肿瘤药物中的应用。8. The application of the exosome-encapsulated nano-drug-loading system for tumor treatment according to any one of claims 1-4 in the preparation of anti-tumor drugs.

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