CN108543074A - The nano medicament carrying system and its preparation that a kind of excretion body for oncotherapy wraps up - Google Patents

Abstract

The invention discloses an exosome-wrapped nano-drug-loading system for tumor treatment and its preparation. The exosome-wrapped nano-drug-loaded system utilizes intracellular drug-loaded nanomaterials and then excretes them through the cells. After obtaining, the drug-loaded nanomaterial is loaded with anti-tumor drugs, including at least one of chemotherapy drugs, drugs for immunotherapy, and drugs for modifying the tumor microenvironment. Compared with the prior art, the present invention provides a new approach for biofilm-based bioprocessing nanoparticles by improving the composition and structure of the key external components of the nano drug-carrying system, the biofilm. The present invention uses exosome-wrapped nano-drug delivery system, which can greatly retain the composition and structure of exosomes, and the obtained exosome-wrapped nano-drug delivery system has good stability and tumor targeting in the process of blood circulation .

Description

An exosome-wrapped nano-drug delivery system for tumor treatment and its preparation

technical field

The invention belongs to the field of nanomaterials and oncology, and more specifically relates to a nano drug-carrying system wrapped in exosomes for tumor treatment and its preparation, especially a method for improving the tumor accumulation and deep penetration of anti-tumor drugs 1. Preparation and application of nano-drug delivery system for targeting and killing tumor stem cells.

Background technique

Tumor recurrence and metastasis are the most important factors that cause the low survival rate of cancer patients. Studies have shown that tumor recurrence and metastasis are closely related to cancer stem cells (CSCs). The drug resistance of CSCs is one of the main reasons for the failure of tumor therapy, mainly including the following aspects: (1) The high expression of ABC transporter in CSCs is the primary factor for their drug resistance; (2) CSCs are in a quiescent state in G0 phase It is very important to maintain drug resistance; (3) CSCs have more efficient DNA repair ability; (4) CSCs are located in the deep part of tumor tissue with hypoxia, and it is difficult for anti-tumor drugs to enter these cells. At present, the treatment of CSCs mainly focuses on their biological characteristics, including quiescent state, poorly differentiated state, and abnormal signaling pathways. Although a variety of inhibitors or promoters can significantly inhibit the characteristics of CSCs in vitro, once in vivo, the targeting and specificity of the treatment plan is particularly important, and small molecule drugs will also be used in the treatment of CSCs. Normal tissue cell uptake, with potential toxic side effects. Therefore, drugs for the treatment of CSCs need to have certain tumor targeting ability.

Due to its unique advantages, such as the EPR effect, the ability to couple targeting molecules on the surface, and the ability to achieve drug co-delivery, the nano-drug delivery system is very beneficial to the targeted therapy of CSCs. At present, a variety of nano-drug delivery systems have achieved a high degree of drug accumulation in CSCs by co-delivering CSCs drug resistance-related regulators and specific killer drugs, as well as surface-modified CSCs targeting ligands. However, this treatment strategy is still difficult to achieve the ideal therapeutic effect of CSCs, mainly because: (1) The ideal nano-drug delivery system targeting CSCs needs to be able to highly accumulate in tumor tissue, penetrate deep into tumor tissue, and be taken up by CSCs in large quantities , but the above methods are difficult to meet these characteristics at the same time; (2) CSCs do not have a common marker protein, the marker proteins of different CSCs are different from each other, and some marker proteins are also highly expressed in ordinary stem cells, so by coupling targeting ligand It is a potential threat to improve the targeting of CSCs in vivo; (3) The above-mentioned nano-drug delivery system is often complex in synthesis process, and has potential toxicity and side effects due to its "non-self" characteristics. Therefore, it is necessary to develop a therapeutic strategy that efficiently targets and kills CSCs with extremely low toxicity.

Currently, biofilm-based bioprocessed nanoparticles are widely used in tumor therapy. Based on this method, many specific properties have been endowed with nano-drug delivery systems, for example, nanoparticles wrapped in red blood cell membranes have longer blood circulation capabilities, nanoparticles wrapped in tumor cell membranes have efficient homologous targeting capabilities, stem cell-derived nanoghosts have good tumor targeting ability and so on. This natural modification method can avoid potential toxic side effects such as PEG dilemma caused by chemical modification. However, in this bioprocessing process, the partial destruction of the cell membrane structure causes the loss of some membrane proteins that play important functions, resulting in a decrease in the stability of the membrane-encapsulated nano-drug delivery system in blood circulation and tumor targeting. Exosomes are extracellular vesicles secreted by cells with a particle size between 30-100 nm. Exosomes have good biological stability and biocompatibility, low immunogenicity and low toxicity in the body. Studies have found that exosomes exhibit excellent cell uptake and tumor targeting capabilities due to the special proteins on their surface. At the same time, some exosomal proteins, such as CD81, CD9, etc., can degrade the extracellular matrix, thereby improving tumor penetration and CSCs targeting. Although these existing exosomes have good effects, the existing exosome technology has the problems of low yield, low drug loading, and long preparation process, which affect the practical application.

Contents of the invention

In response to the above defects or improvement needs of the prior art, the purpose of the present invention is to provide an exosome-encapsulated nano-drug delivery system and its preparation for tumor treatment, wherein the key external components of the nano-drug delivery system The composition and structure of the biofilm, the overall process design of the corresponding preparation method, and the parameters and conditions of each step (such as the incubation step of endocytosis and efflux) are improved. Compared with the existing technology, it is based on biofilm. Bioprocessing nanoparticles offers a new avenue. The present invention uses exosome-wrapped nano-drug delivery system, which can greatly retain the composition and structure of exosomes, and the obtained exosome-wrapped nano-drug delivery system has good stability and tumor targeting in the process of blood circulation . The present invention can especially solve the problem of poor targeting and killing effects of CSCs. After the drug-carrying system enters the blood through intravenous administration, it can efficiently accumulate in tumor tissue, penetrate deeply into the deep part of the tumor, and significantly improve the uptake behavior of CSCs; significantly Inhibit the growth of liver cancer and melanoma lung metastases, and significantly reduce the proportion of CSCs.

In order to achieve the above purpose, according to one aspect of the present invention, there is provided an exosome-wrapped nano-drug delivery system for tumor treatment, which is characterized in that the exosome-wrapped nano-drug delivery system utilizes endocytosis The drug-loaded nanomaterial is then obtained after the cell efflux, and the drug-loaded nanomaterial is loaded with anti-tumor drugs, and the anti-tumor drugs include at least one of chemotherapy drugs, immunotherapy drugs, and drugs that reconstruct the tumor microenvironment A sort of.

As a further preference of the present invention, the cells include at least one of tumor cells, tumor stem cells, immune cells, tumor-associated fibroblasts, mesenchymal stem cells, myeloid-derived suppressor cells, and regulatory T cells; Tumors corresponding to tumor cells and the tumor stem cells include acute leukemia, lymphoma, prostate cancer, thyroid cancer, esophageal cancer, bone cancer, gastric cancer, breast cancer, lung cancer, ovarian cancer, choriocarcinoma, cervical cancer, uterine body Cancer, liver cancer, bladder cancer, skin cancer, colon cancer or rectal cancer; the immune cells include T lymphocytes, B lymphocytes, K lymphocytes, NK lymphocytes, mast cells, or mononuclear phagocyte system.

As a further preference of the present invention, the nanomaterials in the drug-loaded nanomaterials include nanoliposomes, carbon-based nanomaterials, silicon-based nanomaterials, metal nanomaterials, semiconductor quantum dots, up-conversion materials, and polymer nanomaterials at least one of; the carbon-based nanomaterials preferably include at least one of graphene oxide, carbon nanotubes, and nanodiamonds; the silicon-based nanomaterials preferably include at least one of porous silicon, mesoporous silicon, and silicon dots One; the metal nanomaterial preferably includes at least one of gold nanoparticles, silver nanoparticles, and transition metal nanoparticles.

As a further preference of the present invention, the nanomaterial in the drug-loaded nanomaterial is a nanoparticle material, and the particle size of the nanoparticle material is 1-1000 nm.

As a further preference of the present invention, the antitumor drug loaded by the drug-loaded nanomaterial preferably includes treatment of acute leukemia, lymphoma, prostate cancer, thyroid cancer, esophageal cancer, bone cancer, gastric cancer, breast cancer, lung cancer, ovarian cancer, villi One or more of chemotherapy drugs for membranous epithelial cancer, cervical cancer, uterine body cancer, liver cancer, bladder cancer, skin cancer, colon cancer, rectal cancer, drugs for immunotherapy or drugs for modifying the 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 treatment, which is characterized in that it includes the following steps:

S1: co-incubating drug-loaded nanomaterials with cells;

S2: centrifuging to remove the drug-loaded nanomaterials not taken up by the cells;

S3: adding fresh medium without drug-loaded nanomaterials to continue incubation;

S4: Collect the drug-loaded nanomaterials effluxed by the cells by centrifugation, that is, obtain the nano-drug-loaded system wrapped in exosomes for tumor treatment.

As a further preference of the present invention, in the step S1, the co-incubation time is 1-96h.

As a further preference of the present invention, the step S2 specifically collects the cells at a low temperature of 0-10°C with a centrifugal force of 100-1000g, and washes the cells with pre-cooled phosphate buffer until no Free nano drug loading system;

The step S4 specifically includes centrifuging at a low temperature of 0-10°C 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 at a centrifugal force of 10000-20000g for 30- After 120 minutes, the nano-drug-loaded system effluxed out of the cells was obtained; after washing with pre-cooled phosphate buffer, the nano-drug-loaded system encapsulated by exosomes was obtained.

As a further preference of the present invention, in the step S3, the incubation is placed in a cell culture incubator at 37°C and 5% CO ₂ under cell culture conditions, or is first irradiated with ultraviolet light for 5-240min Then place it in a cell culture incubator and incubate for 12-96h.

According to another aspect of the present invention, the present invention provides the application of the above-mentioned exosome-encapsulated nano-drug delivery system for tumor treatment in the preparation of anti-tumor drugs.

Through the above technical scheme conceived by the present invention, compared with the prior art, since the exosome-wrapped nano-drug delivery system is used to form the exosome-wrapped nano-drug delivery system for tumor treatment, specifically, the anti- The nano drug-loaded material of the tumor drug is co-incubated with the cells, so that the cells endocytose the drug-loaded nano-material, and then utilizes the cell efflux to obtain an effluxed nano drug-loaded system wrapped in exosomes. The exosome-wrapped nano-drug delivery system used for tumor treatment in the present invention is obtained by expelling and endocytosed drug-loaded nanomaterials, and has the following characteristics: the nano-drug delivery system consists of an exosome structure on the surface and internal drug-loaded nanomaterials; the uptake behavior and killing effect of the nano-drug-loaded system in tumor cells and tumor stem cells are significantly better than unwrapped drug-loaded nanomaterials, and can accumulate more in tumor tissues and penetrate Enter the deep part of the tumor tissue; the nano-drug delivery system can produce significant inhibitory effects on various tumors with a very low amount of anti-tumor drugs.

The present invention proposes for the first time the idea of using the cell efflux nano-drug loading system to wrap exosomes on the surface of the nano-drug loading system. The existing nano-drug loading system membrane encapsulation technology is mainly obtained by crushing the biomembrane and co-extruding the nano-drug loading system through the filter membrane. This method is likely to cause damage to the cell membrane structure, which in turn leads to the loss of functional proteins on the membrane surface. , which is detrimental to the stability, targeting performance, etc. of the nano-drug delivery system. However, the preparation method in the present invention is milder and can preserve the complete structure of the exosome membrane most completely, thereby exerting better functions. For the first time, the present invention constructs a membrane-wrapped nano-drug delivery system by expelling nanoparticles from cells. After stimulating cells with the nano-drug delivery system, the yield of exosomes can be significantly increased by more than 34 times, and the drug-loading capacity can be increased by more than 2 times, and it is time-consuming Can be shortened by half. The anti-tumor drug loaded by the drug-loaded nanomaterial in the present invention can be a chemotherapeutic drug, an immunotherapy drug, a photothermal therapy drug or a small-sized nanoparticle with a photothermal effect that directly inhibits tumor growth, or an immune co-stimulatory molecule that adjuvantly treats tumors , monoclonal antibody, or at least one of the drugs that can affect the tumor microenvironment such as tumor tissue extracellular matrix and tumor stromal cells, for example, it can treat acute leukemia, lymphoma, prostate cancer, thyroid cancer, esophageal cancer, bone cancer, One or more chemotherapy drugs for gastric cancer, breast cancer, lung cancer, ovarian cancer, choriocarcinoma, cervical cancer, uterine body cancer, liver cancer, bladder cancer, skin cancer, colon cancer, and rectal cancer.

The present invention also preferably controls the incubation process of endocytosis and efflux, by controlling the type of cells and the parameter conditions (such as incubation time, etc.) The exosomes formed by the exosomes have a high content of exosomes, which makes the exosome-wrapped nano-drug delivery system have good stability and tumor targeting as a whole.

The technical solution of the present invention has the following beneficial effects:

(1) Covering the exosome structure on the surface of drug-loaded nanomaterials to improve the stability of drug-loaded nanomaterials and drug sustained-release effect;

(2) After the drug-loaded nanomaterials were incubated with the cells, the production of exosomes was significantly increased;

(3) The uptake behavior of exosome-encapsulated nano-drug delivery system in tumor cells and CSCs was significantly improved;

(4) The exosome-encapsulated nano-drug delivery system has a significantly enhanced killing effect on tumor cells and CSCs;

(5) The exosome-wrapped nano-drug delivery system can highly accumulate in tumor tissue and penetrate deeply into the deep part of tumor tissue.

Description of drawings

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

Figure 1B is the transmission electron microscope observation result of E-PSiNPs of the present invention, wherein, the left picture is the control group, and the right picture is the experimental group (the scale in the figure is 20nm);

Fig. 2 is the energy spectrum analysis result of E-PSiNPs of the present invention under FTEM;

Fig. 3 is the colocalization observation result of CD63 protein on the surface of E-PSiNPs of the present invention and internal PSiNPs;

Fig. 4 is the Western blot result of the exosome-associated protein on the surface of E-PSiNPs of the present invention;

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

Figure 6 shows the uptake behavior of DOX@E-PSiNPs of the present invention in H22CSCs;

Figure 7 shows the uptake behavior of DOX@E-PSiNPs of the present invention in B16CSCs;

Figure 8 shows the cytotoxicity of DOX@E-PSiNPs of the present invention to H22 tumor cells;

Figure 9A shows the effect of DOX@E-PSiNPs of the present invention on the number of H22 tumor clone spheres cultured in 3D soft fibrin glue;

Figure 9B shows the effect of DOX@E-PSiNPs of the present invention on the size of H22 tumor clone spheres cultured in 3D soft fibrin glue;

Figure 10 shows the cytotoxicity of DOX@E-PSiNPs of the present invention to B16 tumor cells;

Figure 11A is the effect of DOX@E-PSiNPs of the present invention on the number of B16 tumor clone spheres cultured in 3D soft fibrin glue;

Figure 11B is the effect of DOX@E-PSiNPs of the present invention on the size of B16 tumor clone spheres cultured in 3D soft fibrin glue;

Figure 12 shows the accumulation behavior of DOX@E-PSiNPs of the present invention in tumor tissues of tumor-bearing mice;

Figure 13 shows the accumulation behavior of DOX@E-PSiNPs of the present invention in CSCs in tumor tissues of tumor-bearing mice;

Figure 14 shows the deep penetration behavior of DOX@E-PSiNPs of the present invention in tumor cloning spheres in vitro;

Figure 15 shows the deep penetration behavior of DOX@E-PSiNPs of the present invention in tumor tissue in vivo;

Fig. 16A is the tumor growth curve of mice after DOX@E-PSiNPs of the present invention were injected intravenously into liver cancer subcutaneous tumor model mice;

Fig. 16B is the weight of the mouse tumor tissue after intravenous injection of DOX@E-PSiNPs of the present invention into liver cancer subcutaneous tumor model mice;

Fig. 16C is the experimental result of the survival period of the mouse after DOX@E-PSiNPs of the present invention was injected intravenously into the subcutaneous tumor model mouse of liver cancer;

Figure 17A shows the proportion of side population cells in the mouse tumor tissue after DOX@E-PSiNPs of the present invention was injected intravenously into the liver cancer subcutaneous tumor model mice;

Figure 17B shows the number of tumor clone spheres formed by monodisperse cells obtained from dispersed mouse tumor tissue in 3D soft fibrin glue after DOX@E-PSiNPs of the present invention was injected intravenously into subcutaneous tumor model mice of liver cancer;

Figure 17C shows the size of tumor clone spheres formed by monodisperse cells obtained from dispersed mouse tumor tissue in 3D soft fibrin glue after DOX@E-PSiNPs of the present invention was injected intravenously into subcutaneous tumor model mice of liver cancer;

Figure 18A shows the number of lung tumor nodules in mice with DOX@E-PSiNPs injected intravenously into mice with melanoma lung metastases;

Figure 18B is the result of H&E staining of lung tissue after DOX@E-PSiNPs of the present invention was injected intravenously into mice with melanoma lung metastases;

Figure 18C shows the experimental results of survival of mice with DOX@E-PSiNPs of the present invention injected into mice with melanoma lung metastases through intravenous injection;

Figure 19A shows the number of tumor clone spheres formed by monodisperse cells obtained from dispersing mouse lung tumor nodules in 3D soft fibrin glue after DOX@E-PSiNPs of the present invention was injected intravenously into mice with melanoma lung metastases;

Figure 19B shows the size of tumor clone spheres formed by monodisperse cells obtained from dispersed mouse lung tumor nodules in 3D soft fibrin glue after DOX@E-PSiNPs of the present invention were injected intravenously into mice with melanoma lung metastases.

Detailed ways

In order to make the object, technical solution and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not constitute a conflict with each other.

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

H22 mouse liver cancer cells, human liver cancer cell line Bel7402 cells, and mouse skin cancer cell line B16 can all be purchased from ATCC Corporation of the United States or CCTCC, the Chinese Type Collection Center.

BALB/C mice and C57 mice were purchased from the Medical Experimental Animal Center of Wuhan University, weighing 18-20 grams;

Adriamycin was purchased from Sigma.

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

1. Experimental materials and reagents

H22 mouse liver cancer cells, doxorubicin (with red fluorescence), boron-doped p<100> silicon wafer (resistance 0.00055-0.0015Ω-cm) were purchased from Virginia Company, USA

2. Experimental steps

(1) Place the silicon chip in a mixed solution of concentrated H ₂ SO ₄ and H ₂ O ₂ (volume ratio v:v=3:1) for 15 minutes, wash it with ultrapure water for 3 times, and then dry it for later use. Install the silicon wafer with the polished side up in the electrochemical etching device, add HF/ethanol solution with a volume ratio of 4:1, and after continuous corrosion for 300s at a current intensity of 165mA/ ^cm2 , a dark red film appears on the surface of the silicon wafer. After washing for 3 times, continue to add 3.3% HF/ethanol solution (mass ratio), and act for 90s under the current intensity of 4.5mA/cm ² , to peel off the porous silicon film from the silicon substrate. Aspirate the HF solution, wash gently with absolute ethanol for 3 times, collect the porous silicon film, and transfer it to the aqueous solution for ultrasonication for 16 hours, centrifuge at 8000g for 20 minutes, collect the supernatant, and place it in a water bath at 60°C for 3-6 hours to stimulate PSiNPs Fluorescence (PSiNPs, i.e. porous silicon nanoparticles).

Dissolve 10 mg of PSiNPs in 5 mL of ultrapure water, add 1 mL of 10 mg/mL DOX aqueous solution and continue stirring overnight, centrifuge at 10,000 g for 10 min, and discard the supernatant. Wash the precipitate with ultrapure water, centrifuge at 10,000 g for 10 min, and repeat 3 times. Drug-loaded PSiNPs (DOX@PSiNPs) were stored in ultrapure water at 4°C until use.

(2) 10 ⁷ H22 tumor cells were cultured in a sterile petri dish with a diameter of 10 cm. After overnight culture, the medium was discarded, and after washing with phosphate buffered saline (PBS), 6 mL of serum-free RPMI 1640 medium containing 200 μg/mL PSiNPs was added to continue incubation. After 1-96h (such as 1h, 6h, 12h, 24h, 48, 96h), collect the cells in the culture dish by centrifugation; after washing the collected cells with PBS, add 6mL of fresh medium to it and continue to incubate for 12-96h (such as 12h, 24h , 48h, 96h). Take the medium, centrifuge at 5000g to collect the supernatant, and centrifuge at 10000g or 20000g for 30min or 120min to obtain a precipitate, which is the effluxed porous silicon nanoparticles (E-PSiNPs).

(3) E-PSiNPs and PSiNPs were dissolved in PBS, and the particle size and zeta potential distribution were detected using a nanoparticle size and zeta potential meter with a 633nm He-Ne laser. The specific setting conditions are: the temperature is 25°C, and the equilibration time is 120s. At the same time, place the copper mesh with the carbon film side up on the disposable PE gloves, drop a certain concentration of E-PSiNPs or PSiNPs on the surface of the copper mesh, and let it balance for 5 minutes. After drying the liquid on the surface of the copper mesh with filter paper, put The copper grid was placed on the filter paper and stood overnight, and the morphology of the sample was observed by TEM; the energy spectrum scanning of the sample was performed using the scanning mode of FTEM, and the elemental composition of the sample was analyzed.

3. Experimental results

The experimental results found that the incubation time of the nano-drug delivery system with the cells (1h, 6h, 12h, 24h, 48, 96h), the time for the cells to continue to incubate in fresh medium (12h, 24h, 48h, 96h) and the final centrifugation to obtain Centrifugal force (10000g, 20000g) and time (30min, 120min) of exosome-wrapped porous silicon nanoparticles. Under these conditions, exosome-wrapped porous silicon nanoparticles can be obtained. Incubate the nano-drug delivery system with the cells for a longer time, greater centrifugal force, and shorter centrifugation time while ensuring the yield, so as to ensure a higher yield Reduce time consumption and special requirements for instruments and equipment. In the present invention, the preferred nano drug-carrying system is incubated with the cells for 12 hours, the cells are incubated in fresh medium for 24 hours, the centrifugal force is 20000g, and the centrifugation time is 30min (the final yield difference between centrifugation for 30min and 120min is not large), in the shortest possible time get a higher yield. The final yield of exosomes in the present invention is that ¹⁰⁷ cells can produce about 60 μg of exosomes, while the conventional ultra-high-speed centrifugation method can only obtain about 1.8 μg of exosomes, that is, after stimulating the cells with the nano drug delivery system It can significantly increase the yield of exosomes by more than 34 times.

It was found by DLS detection that the average particle size distribution of E-PSiNPs and PSiNPs was about 280nm and 120nm (Figure 1A), and the zeta potential was -11mV. Transmission electron microscope images showed that E-PSiNPs had irregular morphology and a film-like structure attached to the surface (Fig. 1B). The presence of Si element in the energy spectrum analysis results proves that the structure seen in the figure is indeed a porous silicon nanoparticle (Fig. 2). We used the same method (the size of the process parameters used in each step, the type and concentration of the reagents used, etc. were kept constant) to prepare the effluxed DOX-loaded porous silicon nanoparticles (DOX@E-PSiNPs) and the efflux The gold nanoparticles of rows (the preparation process of gold nanoparticles can refer to the prior art), all obtained similar results.

Example 2: Identification of surface membrane structure of exosome-encapsulated nano-drug delivery system

1. Experimental materials and reagents

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

2. Experimental steps

(1) Take a certain amount of E-PSiNPs, add 500 μL 5% BSA solution and incubate for 30 min, continue to add 10 μL FITC-CD63 antibody, incubate overnight at 4°C with shaking, centrifuge at 20,000g for 30 min, discard the supernatant, and wash 3 times with PBS. Take 20 μL of the E-PSiNPs solution incubated with FITC-CD63 and drop it on the confocal dish. After fully spreading, use the FV1000 confocal microscope to observe the co-localization of the green fluorescence of the FITC-CD63 antibody and the red fluorescence of PSiNPs. The specific parameters are: PSiNPs: Ex=488nm, Em=680nm; FITC: Ex=488nm, Em=520nm.

(2) Take E-PSiNPs, exosomes and corresponding cells with the same protein amount, and detect the expression of exosome marker proteins CD63 and TSG101 in cell-derived E-PSiNPs by Western blotting, and use Calnexin protein derived from endoplasmic reticulum as Negative control, β-action as internal control. 3. Experimental results

Immunofluorescence staining results showed that the green fluorescence of the FITC-CD63 antibody was completely colocalized with the red fluorescence of PSiNPs, indicating that there was CD63 protein on the surface of E-PSiNPs (Figure 3). Through western blot analysis, we further proved that there are a large number of exosomal marker proteins CD63 and TSG101 on the surface of H22-derived E-PSiNPs, and used Calnexin protein as a positive control to exclude the possibility that E-PSiNPs originated from the cytoplasm (Figure 4, control group 1 It is the cell lysate of H22 cells, the control group 2 is the exosomes of H22 cells collected by conventional ultra-high speed centrifugation, and the experimental group is E-PSiNPs). Similarly, E-PSiNPs derived from Bel7402 cells were prepared by the same preparation method as in Example 1, which also proved that a large number of exosome marker proteins CD63 and TSG101 also existed on the surface of E-PSiNPs derived from Bel7402 cells.

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

1. Experimental materials and reagents

H22 mouse liver cancer cells, doxorubicin, 3D soft fibrin glue, 3D soft fibrin glue (1) In vitro tumor cell uptake behavior

2×10 ⁵ H22 were suspended and cultured in the cell culture plate. After 24 hours, the medium was removed, and 1 mL of free DOX, DOX@PSiNPs or DOX@E- The serum-free medium of PSiNPs was incubated for 4 hours under normal cell culture conditions of 37°C and 5% CO ₂ , the cells were collected, washed 3 times with PBS, and centrifuged at 250g for 10 minutes. Add 500 μL of pre-cooled PBS to the cell pellet to resuspend the cells, pass through a 200-mesh sieve, and detect intracellular DOX fluorescence with a FC500 flow cytometer. Specific detection parameters: Ex=488nm, the emission light is detected as FL2 fluorescence channel.

(2) In vitro CSCs uptake

H22 cells were seeded in 3D soft fibrin glue and cultured to obtain H22CSCs. Add free DOX, DOX@PSiNPs and DOX@E-PSiNPs with final DOX concentrations of 0.5, 1, and 2 μg/mL to 2×10 ⁵ H22CSCs, incubate at 37°C and 5% CO ₂ for 4 h, collect the cells, PBS Wash 3 times, resuspend cells in 500 μL pre-cooled PBS, pass through a 200-mesh sieve, and detect intracellular DOX fluorescence with FC500 flow cytometer. Specific detection parameters: Ex=488nm, the emission light is detected as FL2 fluorescence channel.

3. Experimental results

H22 cells were co-incubated with DOX@E-PSiNPs derived from H22 cells. The results of flow cytometry analysis showed that compared with DOX@PSiNPs and free DOX, the fluorescence intensity of intracellular DOX in the DOX@E-PSiNPs treatment group increased by 5%. (Figure 5, control group 1 is free DOX; control group 2 is DOX@PSiNPs, that is, porous silicon nanoparticles loaded with anti-tumor drug doxorubicin; experimental group is DOX@E-PSiNPs). In H22CSCs, as the concentration of DOX increased, the fluorescence intensity of DOX in the DOX@E-PSiNPs treatment group gradually increased, indicating that the endocytosis of DOX@E-PSiNPs by CSCs was concentration-dependent. When the DOX concentration was 2 μg/mL, the incorporation of DOX@E-PSiNPs treatment group was 2.1 and 1.7 times that of free DOX and DOX@PSiNPs, respectively, indicating that DOX@E-PSiNPs had better CSCs targeting ability (Fig. 6. The control group 1 is free DOX, the control group 2 is DOX@PSiNPs, and the experimental group is DOX@E-PSiNPs). Similarly, we used DOX@E-PSiNPs derived from Bel7402 cells and co-incubated them with Bel7402 cells and their CSCs, which also proved that DOX@E-PSiNPs exhibited significant tumor cell and CSCs uptake behavior.

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

In order to study whether DOX@E-PSiNPs targeting CSCs is universal, we seeded a certain number of B16 cells in 3D soft fibrin glue, and collected a large number of B16F10 tumor stem cells after 5-7 days of culture, and further studied the effect of B16CSCs on Uptake behavior of DOX@E-PSiNPs derived from H22 cells.

1. Experimental materials and reagents

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

2. Experimental steps

B16CSCs were obtained by inoculating mouse skin cancer cell B16 cells in 3D soft fibrin glue. Add free DOX, DOX@PSiNPs and DOX@E-PSiNPs with final DOX concentrations of 0.5, 1, and 2 μg/mL to 2× ¹⁰⁵ B16CSCs, incubate at 37°C, 5% CO ₂ for 4 hours, collect cells, PBS Wash 3 times, resuspend cells in 500 μL pre-cooled PBS, pass through a 200-mesh sieve, and detect intracellular DOX fluorescence with FC500 flow cytometer. Specific detection parameters: Ex=488nm, the emission light is detected as FL2 fluorescence channel.

3. Experimental results

After co-incubating B16CSCs with DOX@E-PSiNPs derived from H22 cells, the results of flow cytometry analysis showed that at different drug concentrations, the intracellular DOX content of B16CSCs in the DOX@E-PSiNPs treatment group was significantly higher than that of free DOX and DOX@PSiNPs group (Figure 7, control group 1 is free DOX, control group 2 is DOX@PSiNPs, and experimental group is DOX@E-PSiNPs), indicating that DOX@E-PSiNPs derived from H22 can be taken up by B16CSCs in large quantities, indicating that DOX Targeting CSCs by @E-PSiNPs is universal.

Example 5: Cytotoxicity of exosome-encapsulated nano-drug delivery system on tumor cells and CSCs in vitro

1. Experimental materials and reagents

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

2. Experimental steps

(1) Tumor cytotoxicity in vitro

H22 cells were seeded in a 96-well plate at a cell density of 8×10 ³ . After overnight incubation, the medium was removed, and 100 μL of free DOX, DOX@PSiNPs or DOX@E-PSiNPs containing DOX concentrations of 0.25, 0.5, and 1 μg/mL were added. After incubating in serum-free medium at 37°C and 5% CO ₂ for 24 h, 10 μL of CCK-8 solution was added to each well and incubated for 4 h, and the absorbance at 450 nm of each well was detected using a 318C microplate reader.

(2) In vitro CSCs toxicity

400/well H22 cells were seeded in 96-well plates covered with 1 mg/mL 3D soft fibrin glue, and cultured at 37°C and 5% CO ₂ . After 5 days, discard the medium, add 100 μL of free DOX, DOX@PSiNPs or H22 cell-derived DOX@E-PSiNPs with a final DOX concentration of 2 μg/mL, and incubate for 24 h at 37 °C and 5% CO ₂ . Discard the supernatant, add 0.1% trypan blue solution to each well, stain for 1 min, discard the dye solution, wash 5 times with PBS, observe and take pictures under a microscope, and count the number and size of live tumor cell spheres.

3. Experimental results

The results of in vitro tumor cytotoxicity experiments showed that compared with free DOX and DOX@PSiNPs, H22 cell-derived DOX@E-PSiNPs showed stronger killing effect on H22 cells (Figure 8, control group 1 was free DOX, control group 1 Group 2 is DOX@PSiNPs, and the experimental group is DOX@E-PSiNPs). In the in vitro CSCs toxicity experiment, after co-incubation of DOX@E-PSiNPs with H22 tumor clonal spheres grown in 3D soft fibrin glue, the number of surviving tumor clonal spheres (Figure 9A, control group 1 is free DOX, control group 2 DOX@PSiNPs, DOX@E-PSiNPs in the experimental group) and size (Fig. 9B, free DOX in control 1, DOX@PSiNPs in control 2, DOX@E-PSiNPs in the experimental group) were significantly reduced, indicating that DOX @E-PSiNPs have a significant killing effect on CSCs. In summary, DOX@E-PSiNPs has a significant killing effect on tumor cells and can kill CSCs efficiently.

Example 6: The universality of exosome-encapsulated nano-drug delivery system to the cytotoxicity of tumor cells and CSCs

To investigate whether the killing effect of DOX@E-PSiNPs on CSCs is universal, we tested the killing effect of DOX@E-PSiNPs derived from H22 cells on CSCs of mouse skin cancer cell B16.

1. Experimental materials and reagents

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

2. Experimental steps

(1) Tumor cytotoxicity test in vitro

B16 cells were seeded in a 96-well plate at a cell density of 8×10 ³ . After overnight incubation, the medium was removed, and 100 μL of free DOX, DOX@PSiNPs or DOX@E-PSiNPs containing DOX concentrations of 0.25, 0.5, and 1 μg/mL were added. After incubating in serum-free medium at 37°C and 5% CO ₂ for 24 h, 10 μL of CCK-8 solution was added to each well and incubated for 4 h, and the absorbance at 450 nm of each well was detected using a 318C microplate reader.

(2) In vitro CSCs toxicity test

400 mouse skin cancer cell B16 cells/well were inoculated in a 96-well plate covered with 1 mg/mL 3D soft fibrin glue, and cultured at 37°C and 5% CO ₂ . After 5 days, discard the medium, add 100 μL of free DOX, DOX@PSiNPs or H22 cell-derived DOX@E-PSiNPs with a final DOX concentration of 2 μg/mL, and incubate for 24 h at 37 °C and 5% CO ₂ . Discard the supernatant, add 0.1% trypan blue solution to each well, stain for 1 min, discard the dye solution, wash 5 times with PBS, observe and take pictures under a microscope, and count the number and size of live tumor cell spheres.

3. Experimental results

The results of in vitro tumor cytotoxicity experiments showed that compared with free DOX and DOX@PSiNPs, different concentrations of DOX@E-PSiNPs derived from H22 cells could significantly kill B16 cells, indicating that DOX@E-PSiNPs has universality in killing tumor cells (Figure 10, control group 1 is free DOX, control group 2 is DOX@PSiNPs, and experimental group is DOX@E-PSiNPs). The results of in vitro CSCs toxicity experiments showed that after co-incubation of H22-derived DOX@E-PSiNPs with B16 tumor clonal spheres in 3D soft fibrin glue, the number of surviving tumor clonal spheres (Fig. 11A, control group 1 is free DOX, control group 1 Group 2 was DOX@PSiNPs, experimental group was DOX@E-PSiNPs) and size (Fig. 11B, control 1 was free DOX, control 2 was DOX@PSiNPs, experimental group was DOX@E-PSiNPs) were significantly decreased, It shows that DOX@E-PSiNPs is universal in killing CSCs.

Example 7: Accumulation behavior of exosome-encapsulated nano-drug delivery system in tumor tissue

1. Experimental materials and reagents

H22 mouse liver cancer cells, BALB/c mice

2. Experimental steps

(1) Tumor tissue accumulation experiment diagram;

2×10 ⁶ H22 tumor cells were subcutaneously injected into the outer thigh of BALB/c mice (18-20 g) to construct a mouse H22 subcutaneous tumor model. When the tumor size reached 250 mm ³ , 0.5 mg/kg free DOX, DOX@PSiNPs or DOX@E-PSiNPs were injected into the tail vein, and the mice were treated with cervical dislocation 24 hours later. The mouse tumors, heart, liver, spleen, lung, Kidneys were washed 3 times with PBS, the tissue surface was blotted dry, each tissue was weighed and 100 μL RIPA lysate was added, and the tissue was ground using a tissue grinder. After grinding, absorb the grinding solution, centrifuge at 10,000 g for 10 min, take out the supernatant solution and store it in the dark at 4°C until testing. DOX standard solutions of different concentrations were prepared using the supernatant solutions of each tissue blank control group after grinding and centrifugation. The fluorescence intensity of DOX in the grinding supernatant solution of each tissue was detected by high performance liquid chromatography at 488nm.

3. Experimental results

DOX@E-PSiNPs were injected into tumor-bearing mice through the tail vein. After 24 hours, each tissue was ground, and the DOX content in the grinding liquid of each tissue was quantitatively analyzed. The results showed that the DOX content of DOX@E-PSiNPs in the tumor site was respectively 3.9 times that of DOX and 1.7 times that of DOX@PSiNPs, indicating that DOX@E-PSiNPs has significant tumor tissue targeting ability (Figure 12, control group 1 is free DOX, control group 2 is DOX@PSiNPs, and experimental group is DOX@ E-PSiNPs).

Example 8: Uptake behavior of exosome-encapsulated nano-drug delivery system in CSCs of tumor tissue

1. Experimental materials and reagents

H22 mouse liver cancer cells, BALB/c mice

2. Experimental steps

2×10 ⁶ H22 tumor cells were subcutaneously injected into the outer thigh of BALB/c mice (18-20 g) to construct a mouse H22 subcutaneous tumor model. When the tumor volume reached 250 mm ³ , 0.5 mg/kg free DOX, DOX@PSiNPs, and DOX@E-PSiNPs were injected into the tail vein. After 24 hours, the mice were killed by cervical dislocation, and the tumor tissues were removed and washed with PBS. Place the tumor tissue in the stem cell medium and cut it into mung bean size, remove the medium, add 1mg/mL Collagenase, digest at 37°C for 2h, and shake once every 15min to ensure full digestion. Squeeze the digested tumor tissue with a rough ground glass sheet until there is no blocky structure, absorb the digested cell suspension, centrifuge at 1200g for 5min, discard the supernatant, wash the cell pellet with PBS for 3 times, and pass the solution through a 200-mesh sieve to obtain a single tumor tissue. cell suspension. The single-cell suspension was divided into two parts: one part was used to detect the fluorescence of DOX in all tumor cells under the condition of 488nm/FL2 using FC500 flow cytometer; the other part of the cell suspension was mixed with Hoechst 33342 dye (final concentration 5 μg/mL) , and incubate at 37°C in the dark for 90 minutes. At the same time, cells co-incubated with 50 μM verapamil and 5 μg/mL Hoechst 33342 were used as the control group. Immediately after the incubation, the cells were placed on ice, centrifuged at 250 g for 5 minutes at 4°C, removed the supernatant, and pre-cooled Wash 3 times with PBS, resuspend the cells in pre-cooled PBS, and store on ice. DOX fluorescence in side population cells was analyzed using a CytoFlex flow cytometer. The specific analysis method is as follows:

1) Circle live cells in the SSC-A and FSC-A scatter plots;

2) Construct a scatter diagram with horizontal and vertical coordinates of Ex=405nm, Em=450nm and Ex=405nm, Em=660nm, compare the verapamil treatment group with the untreated group, and circle a part of the cells that disappeared, which is the side group cell;

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

3. Experimental results

After injecting DOX@E-PSiNPs into tumor-bearing mice through the tail vein according to the method of Example 7, the tumor tissue was taken out and dispersed into single cells. It was found by flow cytometry that compared with free DOX and DOX@PSiNPs treatment group, DOX@E-PSiNPs treatment group intracellular DOX fluorescence intensity of side population cells increased by 64% and 71%, respectively. The above results further proved that DOX@E-PSiNPs had significant CSCs targeting ability in vivo experiments (Figure 13, control group 1 was free DOX, control group 2 was DOX@PSiNPs, and experimental group was DOX@E-PSiNPs).

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

1. Experimental materials and reagents

H22 mouse liver cancer cells, BALB/c mice, 3D soft fibrin glue, FITC-CD31 antibody

2. Experimental steps

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

4×10 ² /well H22 cells were seeded in 3D soft fibrin glue according to the method described above. When the tumor cell spheres grew to the 5th day, discard the medium, wash with PBS three times, add free DOX, DOX@PSiNPs or DOX@E-PSiNPs serum-free medium with a final DOX concentration of 10 μg/mL, and incubate for 24 h. Remove the medium, wash with PBS for 3 times, transfer the soft fibrin glue in the well to the confocal dish, and use the FV1000 confocal microscope in Z-axis scanning mode to observe the DOX fluorescence at different depths from the surface of the tumor cell sphere. The specific detection parameters are set as follows: Ex=559nm, Em=590nm.

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

10 ⁶ H22 liver cancer cells were subcutaneously injected into the root of the right thigh of BALB/c mice (about 18 g) to construct a mouse H22 subcutaneous tumor model. When the tumor volume grew to about 250 mm ³ , free DOX, DOX@PSiNPs or DOX@E-PSiNPs with a DOX concentration of 0.5 mg/kg were injected into the tail vein. After 24 hours, the mice were sacrificed by cervical dislocation, and the tumor tissues were taken out and processed by frozen sections. . FITC-CD31 antibody was incubated at 37°C for 30 min to label blood vessels in tumor slices. Use FV1000 confocal microscope to detect DOX red fluorescence and FITC green fluorescence on the slices at 559/580nm and 488/520nm, respectively. The DOX fluorescence distribution on the straight line from the tumor vessels to the inside of the tumor was counted using Image J software.

3. Experimental results

After co-incubating DOX@E-PSiNPs with tumor cloning spheres, the Z-axis scanning images of the confocal microscope showed that the DOX fluorescence of the tumor cell spheres treated with DOX@E-PSiNPs was significantly stronger than that of free DOX and DOX at the same penetration depth. @PSiNPs treatment group (Fig. 14). Further, DOX@E-PSiNPs were injected into the tumor-bearing mice through the tail vein, and the tumor tissues were taken out, sectioned, and stained with FITC-CD31 antibody to mark the tumor blood vessels. The results of confocal microscopy showed that DOX The @E-PSiNPs group can still detect obvious DOX fluorescence signals, while in the DOX and DOX@PSiNPs treatment, obvious fluorescence signals can only be detected at depths of 25 μm and 120 μm, and no obvious DOX fluorescence signals can be detected further deeper (Fig. 15). The above results verified that DOX@E-PSiNPs have obvious deep tumor penetration ability.

Example 10: Inhibitory effect of exosome-encapsulated nano-drug delivery system on mouse liver cancer subcutaneous tumor model 1. Experimental reagents and materials

H22 mouse liver cancer cells, BALB/c mice

2. Experimental steps

2×10 ⁶ H22 tumor cells were subcutaneously injected into the outer thigh of BALB/c mice (18-20 g) to construct a mouse subcutaneous tumor model. ^When the tumor size reached 50 mm, the mice were equally divided into 6 groups, 14 in each group, and PBS, E-PSiNPs, DOX, DOX@PSiNPs, DOX@E-PSiNPs were injected into the tail vein (the final concentration of DOX was 0.5 mg/ kg) or 4 mg/kg of high-dose free DOX, administered once every 2 days, a total of 5 times, the tumor size was measured at a fixed time every day. On the 17th day after the first administration, 8 animals were randomly selected from each group to continue the survival experiment. The mice were sacrificed by cervical dislocation, the tumor tissues were taken out, washed and dried, weighed and photographed.

(2) Killing effect on tumor stem cells

3. Experimental results

The results of tumor growth curves showed that DOX@E-PSiNPs significantly inhibited tumor growth, and its tumor inhibitory effect was better than that of high-dose free DOX administration group (Fig. 16A, 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). After the administration was completed, the weighing results of the removed tumor tissues also showed that DOX@E-PSiNPs significantly inhibited tumor growth (Figure 16B, control group 1 was normal saline group, control group 2 was E-PSiNPs, control group 3 was Free DOX, control group 4 is DOX@PSiNPs, control group 5 is high-dose free DOX group, experimental group is DOX@E-PSiNPs). The results of survival period also found that after administration of DOX@E-PSiNPs, the survival period of tumor-bearing mice was prolonged by 30 days, which was significantly better than that of other experimental groups, including the high-dose free DOX group (Fig. 16C, control group 1 is the 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: The 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 liver cancer cells, BALB/c mice

2. Experimental steps

According to the method of Example 10, the mouse liver cancer subcutaneous tumor model was administered and treated. After the administration was completed, the mice were killed by cervical dislocation, and the tumor tissues were taken out, washed and dispersed into single tumor cells, and analyzed by CytoFlex flow cytometry. Side group analysis, detecting the proportion of side group cells in each tumor tissue after different drug treatments. Take the monodispersed tumor cells and count them, inoculate them in a 96-well plate containing 1mg/mL 3D soft fibrin glue at a cell density of 800/well, and culture them for 5 days at 37°C and 5% CO2, then observe and count the tumor cells Number and size of balls.

3. Experimental results

The above results show that the proportion of side population cells in the tumor tissue after DOX@E-PSiNPs treatment is significantly reduced, indicating that DOX@E-PSiNPs can significantly kill side population 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 dispersed single cells were seeded in 3D soft fibrin glue and cultured for a period of time, and the results showed that the number of tumor clone spheres formed by tumor cells in the DOX@E-PSiNPs group in 3D soft fibrin glue (Figure 17B, control group 1 was physiological 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, experimental group was DOX@E-PSiNPs) and size (Fig. 17C , 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 ) was significantly reduced, indicating that DOX@E-PSiNPs can greatly inhibit the stemness of tumor cells.

Example 12: Inhibitory effect of exosome-encapsulated nano-drug delivery system on mouse melanoma lung metastases

1. Experimental reagents and materials

Mouse skin cancer cell line B16, C57 mice

2. Experimental steps

5×10 ⁵ B16F10 cells were injected into C57BL/6 mice by tail vein injection. After 2 days, the mice were divided into 6 groups on average, 14 in each group, and PBS, E-PSiNPs, DOX, DOX@PSiNPs, DOX@E-PSiNPs (final concentration of DOX was 0.5 mg/kg) or 4 mg/kg were injected into the tail vein. kg of high-dose free DOX, administered every 2 days for a total of 5 doses. On the 17th day after the first administration, 8 animals were randomly selected from each group to continue the survival experiment. The remaining 6 mice were killed by cervical dislocation, and the lung tissues were taken out, and the number of black tumor nodules in the lungs was observed and counted. Three lung tissues from each group were placed in 4% PFA, treated at 4°C for 24 hours, sectioned and processed by H&E staining.

3. Experimental results

DOX@E-PSiNPs were injected into mice with melanoma lung metastases through the tail vein. After the administration, we found that the number of lung tumor nodules in the DOX@E-PSiNPs administration group was significantly lower than that of the rest of the control group (Fig. 18A, 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). The lung tissue was sectioned and stained with H&E. We observed the number of lung tumor nodules under a microscope and obtained the same result (Fig. 18B, control group 1 is normal saline group, control group 2 is E-PSiNPs, control group 3 is free DOX , control group 4 was DOX@PSiNPs, control group 5 was high-dose free DOX group, and experimental group was DOX@E-PSiNPs). Through the mouse survival experiment, we further proved that DOX@E-PSiNPs has a significant inhibitory effect on metastases, and the survival period of mice administered with DOX@E-PSiNPs was significantly improved (Figure 18C, control group 1 is 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 13: Killing effect of exosome-encapsulated nano-drug delivery system on CSCs in mouse melanoma lung metastases

1. Experimental reagents and materials

Mouse skin cancer cell line B16, C57 mice

2. Experimental steps

According to the method in Example 12, the mouse melanoma lung metastases were administered. After the administration was completed, the black tumor nodules in the lung tissue of each administration treatment group were taken out in the stem cell culture medium, dispersed into single tumor cells and Count, seed at a cell density of 800/well in a 96-well plate containing 1mg/mL 3D soft fibrin glue, and observe the size and number of tumor cell spheres after culturing at 37°C and CO ₂ for 5 days.

3. Experimental results

The scattered tumor cells were inoculated in 3D soft fibrin glue and cultured. After a period of time, the size and number of tumor clone spheres formed were counted. The results showed that the number of tumor clone spheres formed by tumor cells in the DOX@E-PSiNPs treatment group (Fig. 19A, 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, experimental group is DOX@E- PSiNPs) and size (Fig. 19B, 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, experiment group was DOX@E-PSiNPs) was significantly lower than the rest of the administration groups, indicating that DOX@E-PSiNPs administration treatment can significantly inhibit the stemness of CSCs.

In addition to the specific cell types and antitumor drug types used in the above examples, the drug-loaded nanomaterial cells suitable for endocytosis through efflux in the present invention can also include tumor cells (acute leukemia, lymphoma, breast cancer, Lung cancer, ovarian cancer, choriocarcinoma, 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 cells, mast cells, mononuclear phagocyte system), tumor-associated fibroblasts, mesenchymal stem cells (Mesenchymal stem cells, MSCs), bone marrow-derived suppressor cells (myeloid-derived suppressor cells, MDSCs), regulatory T cells ( Treg cells), etc., these cells all meet the conventional definition in this field; the anti-tumor drugs pre-loaded by drug-loaded nanomaterials in the present invention can include the treatment of acute leukemia, lymphoma, breast cancer, lung cancer, ovarian cancer, choriocarcinoma, One or more chemotherapy drugs for cervical cancer, liver cancer, bladder cancer, skin cancer, colon cancer, rectal cancer and various other solid tumors.

Those skilled in the art can easily understand that the above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention, All should be included within the protection scope of the present invention.

Claims (10)

1. the nano medicament carrying system that a kind of excretion body for oncotherapy wraps up, which is characterized in that excretion body package is received Rice drug-loading system is obtained after the extracellular row again using cell endocytic medicament-carried nano material, and the medicament-carried nano material is negative It is loaded with antitumor drug, the antitumor drug includes in the drug of chemotherapeutics, immunotherapy medicaments, reconstruct tumor microenvironment At least one.

2. the nano medicament carrying system wrapped up as described in claim 1 for the excretion body of oncotherapy, which is characterized in that described thin Born of the same parents include tumour cell, tumor stem cell, immunocyte, tumour associated fibroblast cell, mescenchymal stem cell, derived from bone marrow Inhibit at least one of cell and regulatory T cells；The tumour cell and the corresponding tumour packet of the tumor stem cell Include acute leukemia, lymthoma, prostate cancer, thyroid cancer, cancer of the esophagus, osteocarcinoma, gastric cancer, breast cancer, lung cancer, oophoroma, suede Trichilemma epithelioma, cervix cancer, carcinoma of uterine body, liver cancer, carcinoma of urinary bladder, cutaneum carcinoma, colon cancer or the carcinoma of the rectum；The immunocyte packet Include T lymphocytes, bone-marrow-derived lymphocyte, K lymphocytes, NK lymphocytes, mast cell or mononuclear phagocyte system.

3. the nano medicament carrying system wrapped up as described in claim 1 for the excretion body of oncotherapy, which is characterized in that the load Nano material in medicine nano material include nano liposomes, c-based nanomaterial, Si-based nanometer material, metal nano material, At least one of semiconductor-quantum-point, up-conversion and polymer nano material；The c-based nanomaterial is preferably wrapped Include at least one of graphene oxide, carbon nanotube, Nano diamond；The Si-based nanometer material preferably include porous silicon, At least one of mesoporous silicon, silicon point；The metal nano material preferably includes Jenner's grain of rice, nano grain of silver, transition metal and receives At least one of grain of rice.

4. the nano medicament carrying system wrapped up as described in claim 1 for the excretion body of oncotherapy, which is characterized in that the load Nano material in medicine nano material is nano-particle material, and the grain size of the nano-particle material is in 1-1000nm.

5. the nano medicament carrying system wrapped up as described in claim 1 for the excretion body of oncotherapy, which is characterized in that the load The antitumor drug of medicine nanomaterial loadings preferably includes treatment acute leukemia, lymthoma, prostate cancer, thyroid cancer, food Road cancer, osteocarcinoma, gastric cancer, breast cancer, lung cancer, oophoroma, chorioepithelioma, cervix cancer, carcinoma of uterine body, liver cancer, carcinoma of urinary bladder, Cutaneum carcinoma, the chemotherapeutics of colon and rectum carcinoma, for immunotherapy medicaments or for being transformed in the drug of tumor microenvironment It is one or more.

6. a kind of preparation method for the nano medicament carrying system that excretion body for oncotherapy wraps up, which is characterized in that including with Lower step：

S1：Medicament-carried nano material is incubated altogether with cell；

S2：Centrifugation removes the medicament-carried nano material that the cell does not absorb；

S3：The fresh culture medium without medicament-carried nano material is added to continue to be incubated；

S4：The medicament-carried nano material extracellularly arranged through this is collected by centrifugation to get receiving to the excretion body package for oncotherapy Rice drug-loading system.

7. the preparation method of the nano medicament carrying system wrapped up as claimed in claim 6 for the excretion body of oncotherapy, feature It is, in the step S1, the time being incubated altogether is 1-96h.

8. the preparation method of the nano medicament carrying system wrapped up as claimed in claim 6 for the excretion body of oncotherapy, feature It is, the step S2 is specifically the cell to be collected with the centrifugal force of 100-1000g, using pre- in a low temperature of 0-10 DEG C Cold phosphate buffer cleans the cell, until there is no free nano medicament carrying systems in solution；

The step S4 is specifically to remove the cell in culture solution under 0-10 DEG C of low temperature with the centrifugal force of 100-1000g, The cell fragment in removal culture solution is centrifuged with 3000-5000g, is obtained with the centrifugal force 30-120min of 10000-20000g To the nano medicament carrying system extracellularly arranged；To get the nanometer wrapped up to excretion body after being cleaned using the phosphate buffer of precooling Drug-loading system.

9. the preparation method of the nano medicament carrying system wrapped up as claimed in claim 6 for the excretion body of oncotherapy, feature It is, in the step S3, the incubation is placed in cell incubator, in 37 DEG C, 5%CO₂Cell culture condition under incubate It educates, or makes first to be incubated 12-96h with being positioned in cell incubator again after ultraviolet light 5-240min.

10. prepared by the nano medicament carrying system wrapped up for the excretion body of oncotherapy as described in claim 1-5 any one Application in antitumor drug.