阅读说明：本技术 一种jo蛋白锚定的载药免疫细胞及其制备方法和应用 (JO protein anchored drug-loaded immune cell and preparation method and application thereof ) 是由 黄来强 吴佳敏 陈华清 冯春燕 代小勇 李婧 于 2021-09-30 设计创作，主要内容包括：本发明涉及一种JO蛋白锚定的载药免疫细胞及其制备方法和应用,具体公开了一种JO蛋白锚定的免疫细胞,所述免疫细胞表面上通过化学键偶联JO蛋白,和或者在免疫细胞表面引入偶联JO蛋白的连接剂,将JO蛋白插嵌在免疫细胞上；其中所述的连接剂为具有磷脂或磷脂类似物、聚乙二醇和活性基团的缀合物；所述免疫细胞中包含活性药物,所述的免疫细胞选自T细胞、B细胞或巨噬细胞。本发明证明JO蛋白对免疫细胞的促渗效果,将JO蛋白连接到免疫细胞,在实现JO促渗效果的同时,减少化药与JO蛋白连用时的给药次数。(The invention relates to a JO protein anchored drug-loaded immune cell and a preparation method and application thereof, and particularly discloses a JO protein anchored immune cell, wherein JO protein is coupled on the surface of the immune cell through a chemical bond, and/or a JO protein coupling connecting agent is introduced on the surface of the immune cell, and the JO protein is inserted into the immune cell; wherein the linking agent is a conjugate having a phospholipid or phospholipid analog, polyethylene glycol, and an active group; the immune cell contains active medicine and is selected from T cell, B cell or macrophage. The invention proves the penetration promoting effect of the JO protein on immune cells, and the JO protein is connected to the immune cells, so that the JO penetration promoting effect is realized, and the administration times of the chemical drugs and the JO protein when used together are reduced.)

1. The JO protein anchored drug-loaded immune cell is characterized in that JO protein is coupled on the surface of the immune cell through a chemical bond, and/or a connecting agent for coupling JO protein is introduced on the surface of the immune cell, so that JO protein is inserted into the immune cell;

wherein the linking agent is a conjugate comprising a phospholipid or a phospholipid analogue and an active group; the immune cell comprises an active drug;

preferably, the immune cell is selected from a T cell, a B cell or a macrophage;

preferably, the linker is selected from the group consisting of DMPE-PEG-NHS, DSPE-PEG-NHS, DPPE-PEG-NHS, DLPE-PEG-NHS, DMPE-PEG-OH, DSPE-PEG-OH, DPPE-PEG-OH, DLPE-PEG-OH, DMPE-PEG-COOH, DSPE-PEG-COOH, DPPE-PEG-COOH, DLPE-PEG-COOH, DMPE-NHS, DSPE-NHS, DPPE-NHS, DLPE-NHS, DMPE-OH, DSPE-OH, DPPE-OH, DLPE-OH, DMPE-COOH, DSPE-COOH, DPPE-COOH, DLPE-COOH and similar lipid intermediates;

preferably, the active drug is a chemical drug, protein, polypeptide, cytokine or radiotherapeutic agent with an anti-tumor effect.

2. The JO protein-anchored drug-loaded immune cell of claim 1, wherein the chemical bond is coupled with the JO protein by activating carboxyl on the surface of the JO protein and then coupling with amino or hydroxyl on the surface of the immune cell;

the method for introducing the JO protein coupling agent comprises the steps of coupling the JO protein coupling agent with the JO protein, and inserting the conjugate on the cell membrane of the immune cell through similar compatibility;

preferably, the activating agent used for the carboxyl group activation is at least one selected from 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).

3. A JO protein anchored liposome or nanoparticle, wherein JO protein is coupled to the surface of the liposome or nanoparticle through a chemical bond, or a JO protein-coupled linker is introduced to the surface of the liposome or nanoparticle, so that JO protein is inserted into the liposome or nanoparticle;

wherein the linking agent is a conjugate comprising a phospholipid or a phospholipid analogue and an active group;

preferably, the linker is selected from the group consisting of DMPE-PEG-NHS, DSPE-PEG-NHS, DPPE-PEG-NHS, DLPE-PEG-NHS, DMPE-PEG-OH, DSPE-PEG-OH, DPPE-PEG-OH, DLPE-PEG-OH, DMPE-PEG-COOH, DSPE-PEG-COOH, DPPE-PEG-COOH, DLPE-PEG-COOH, DMPE-NHS, DSPE-NHS, DPPE-NHS, DLPE-NHS, DMPE-OH, DSPE-OH, DPPE-OH, DLPE-OH, DMPE-COOH, DSPE-COOH, DPPE-COOH, DLPE-COOH and similar lipid intermediates.

4. The liposome or nanoparticle of claim 3, wherein the liposome or nanoparticle is further loaded with an active ingredient;

preferably, the active ingredient is a chemical drug, protein, polypeptide, cytokine or radiotherapeutic agent with an anti-tumor effect.

5. The liposome or nanoparticle of claim 3 or 4, wherein the JO protein-conjugated linker is introduced by inserting the conjugate into the liposome or nanoparticle surface or bilayer with similar compatibility after the linker is linked to the JO protein;

the method for coupling the JO protein by the chemical bond comprises the steps of activating carboxyl on the surface of the JO protein and then coupling the activated carboxyl with amino or hydroxyl on the surface of a liposome or a nanoparticle;

preferably, the activating agent used for the carboxyl group activation is at least one selected from 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).

6. The method of preparing a JO protein anchored drug-loaded immune cell of claim 1 or 2, wherein the method of preparation comprises the steps of:

1) activating the JO protein by an activator; or the JO protein is connected with the JO protein through a connecting agent, and then the JO protein is stored at room temperature for 72-168 hours for stabilization;

2) reacting the activated JO protein with immune cells to couple the JO protein on the surfaces of the immune cells; or reacting the JO protein of the coupling connecting agent with immune cells to embed the JO protein of the coupling connecting agent into the surface of an immune cell membrane;

3) and after separation and purification, incubating the obtained immune cells and active drugs together to obtain the JO protein anchored drug-loaded immune cells.

7. The method according to claim 6, wherein the molar ratio of the activator to the JO protein in the step 1) is 50-200: 1; the mol ratio of the connecting agent to the JO protein in the step 2) is 1-500: 1.

8. The method for preparing JO protein-anchored liposomes or nanoparticles according to any one of claims 3 to 5, comprising the steps of mixing a linker to which JO protein is coupled with a phospholipid as a bilayer or a polymer for preparing nanoparticles, and preparing the liposomes or nanoparticles.

9. The JO protein anchored drug-loaded immune cell of any one of claims 1-2, the liposome or nanoparticle of any one of claims 3-5 for use in the preparation of an anti-tumor drug.

10. The use of claim 9, wherein the tumor is a solid tumor, a hematological tumor, or a lymphoma.

Technical Field

The invention belongs to the field of biology, and particularly relates to a preparation method and application of JO protein anchored to lipid membrane component particles such as T cells, macrophages and other cells and liposomes.

Background

The existence of intercellular junctions, such as tight junctions, intermediate junctions and desmosomes, limits the intercellular permeation of drugs with molecular weights greater than 400 daltons, which is an important reason for the poor efficacy of solid tumor chemotherapy. Meanwhile, the killing effect of NK cells on solid tumors is further limited due to the connection between cells, so that CAR-T cell therapy cannot always make a breakthrough in the treatment of solid tumors. JO protein (junction initiator) is found and optimized in the mechanism of adenovirus infection of epithelial cells, and can be combined with desmosomal DSG2, so that tight connection among cells is remodeled, and a channel is opened among the cells, thereby facilitating further penetration of drugs and immune cells. The gold nano-particles can be used as a penetration enhancer, the aggregation of the gold nano-particles in A549 solid tumors can be obviously improved, the treatment effect of chemotherapeutic drugs such as cisplatin and DOxil can be improved, and the drug resistance of tumor cells can be overcome. Meanwhile, Ines et al prove that JO protein can improve the treatment effect of polyclonal antibody drugs on tumors. In the above applications, JO protein is used as an adjuvant and needs to be injected intravenously before the formal start of chemotherapy, and these studies have focused on blood-related tumors or lymphomas, and there has been no report on its application to infiltration of solid tumors.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a simple permeation-promoting strategy, which utilizes the characteristics of phospholipid bimolecules and polyamino of cell membranes, modifies JO protein on the surfaces of T cells and other types of cells by an EDC method or a one-step method, realizes the increase of the permeability of immune cells, and simultaneously reduces the administration frequency of chemotherapeutic drugs.

One aspect of the invention provides a JO protein anchored drug-loaded immune cell, wherein JO protein is coupled on the surface of the immune cell through a chemical bond, or a coupling agent of the JO protein is introduced on the surface of the immune cell, and the JO protein is inserted into the immune cell;

wherein the linking agent is a conjugate comprising a phospholipid or a phospholipid analogue and an active group, and the immune cell comprises an active drug.

Further, the active agent is endocytosed into the immune cells by co-incubation with the immune cells prior to use by the immune cells.

Further, the immune cell is selected from a T cell, a B cell or a macrophage.

Further, the T cell is a human T cell.

Further, the chemical bond coupling JO protein method is that carboxyl on the surface of JO protein is activated and then coupled with amino or hydroxyl on the surface of immune cells.

Further, the method of introducing the linker is to intercalate the conjugate into the cell membrane of the immune cell by similar compatibility after the linker is conjugated to the JO protein.

Further, the activating agent used for activating the carboxyl group is at least one selected from 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).

Further, the linker is selected from the group consisting of DMPE-PEG-NHS, DSPE-PEG-NHS, DPPE-PEG-NHS, DLPE-PEG-NHS, DMPE-PEG-OH, DSPE-PEG-OH, DPPE-PEG-OH, DLPE-PEG-OH, DMPE-PEG-COOH, DSPE-PEG-COOH, DPPE-PEG-COOH, DLPE-PEG-COOH, DMPE-NHS, DSPE-NHS, DPPE-NHS, DLPE-NHS, DMPE-OH, DSPE-OH, DPPE-OH, DLPE-OH, DMPE-COOH, DSPE-COOH, DPPE-COOH, DLPE-COOH and similar lipid intermediates.

Further, the active drug is a chemical drug, protein, polypeptide, cytokine or radiotherapeutic agent having an antitumor effect, such as paclitaxel, docetaxel, doxorubicin, cisplatin, camptothecin, daunorubicin, methotrexate, tumor necrosis factor, docetaxel, mitomycin, fluorouracil, gemcitabine, cyclophosphamide, vinblastine, interleukin 18.

In another aspect, the invention provides a JO protein anchored drug-loaded liposome or nanoparticle, wherein the surface of the liposome or nanoparticle is coupled with JO protein through a chemical bond, or a linker coupled with JO protein is introduced on the surface of the liposome or nanoparticle, so that the JO protein is inserted into the liposome or nanoparticle;

wherein the linking agent is a conjugate comprising a phospholipid or a phospholipid analogue and an active group.

Further, the liposomes or nanoparticles are loaded with an active ingredient.

Furthermore, the active ingredients are chemical drugs, proteins, polypeptides, cytokines or radiotherapeutic preparations with anti-tumor effects.

Further, the chemical bond coupling JO protein method is that carboxyl on the surface of JO protein is activated and then coupled with amino or hydroxyl on the surface of liposome or nano-particle.

Further, the method for introducing the linker is to couple the linker to the JO protein, and then insert the conjugate on the surface of the liposome or nanoparticle or in the bilayer through similar compatibility.

Further, the activating agent used for activating the carboxyl group is at least one selected from 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).

Further, the linker is selected from the group consisting of DMPE-PEG-NHS, DSPE-PEG-NHS, DPPE-PEG-NHS, DLPE-PEG-NHS, DMPE-PEG-OH, DSPE-PEG-OH, DPPE-PEG-OH, DLPE-PEG-OH, DMPE-PEG-COOH, DSPE-PEG-COOH, DPPE-PEG-COOH, DLPE-PEG-COOH, DMPE-NHS, DSPE-NHS, DPPE-NHS, DLPE-NHS, DMPE-OH, DSPE-OH, DPPE-OH, DLPE-OH, DMPE-COOH, DSPE-COOH, DPPE-COOH, DLPE-COOH and similar lipid intermediates.

In yet another aspect, the present invention provides a method for preparing JO protein anchored drug-loaded immune cells, comprising the steps of:

1) activating the JO protein by an activator; or the JO protein is connected with the JO protein through a connecting agent, and then the JO protein is stored at room temperature for 72-168 hours for stabilization;

2) reacting the activated JO protein with immune cells to couple the JO protein on the surfaces of the immune cells; or reacting the JO protein of the coupling connecting agent with immune cells to embed the JO protein of the coupling connecting agent into the surface of an immune cell membrane;

3) and after separation and purification, incubating the obtained immune cells and active drugs together to obtain the JO protein anchored drug-loaded immune cells.

Further, the activator is at least one of EDC and NHS.

Further, the linker is selected from the group consisting of DMPE-PEG-NHS, DSPE-PEG-NHS, DPPE-PEG-NHS, DLPE-PEG-NHS, DMPE-PEG-OH, DSPE-PEG-OH, DPPE-PEG-OH, DLPE-PEG-OH, DMPE-PEG-COOH, DSPE-PEG-COOH, DPPE-PEG-COOH, DLPE-PEG-COOH, DMPE-NHS, DSPE-NHS, DPPE-NHS, DLPE-NHS, DMPE-OH, DSPE-OH, DPPE-OH, DLPE-OH, DMPE-COOH, DSPE-COOH, DPPE-COOH, DLPE-COOH and similar lipid intermediates.

Further, the separation and purification method is centrifugation and washing at least 3 times with PBS of 50 times or more volume of the cell suspension.

Further, the molar ratio of the activator to the JO protein in step 1) is 50-200: 1. E.g., 60:1, 70:1, 80:1, 90:1, 100:1, 110:1, 120:1, 130:1, 140:1, 150:1, 160:1, 170:1, 180:1, 190: 1.

Further, the molar ratio of the linking agent to the JO protein in the step 2) is 1-500: 1. E.g., 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 110:1, 120:1, 130:1, 140:1, 150:1, 160:1, 170:1, 180:1, 190:1, 200:1, 250:1, 300:1, 350:1, 400:1, 450:1, 500: 1.

Further, the reaction time in the step 2) is 24 to 108 hours. For example, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, and 96 hours.

Further, the incubation method in step 3) is to disperse the cells in a solution containing the active drug for incubation.

Further, the concentration of the active drug in the solution is 1-150 μ g/mL, and the incubation time is 1-6 hours.

In still another aspect, the present invention provides a method for preparing the JO protein-anchored drug-loaded liposome or nanoparticle, which comprises the steps of mixing a linker conjugated to the JO protein with a phospholipid as a bilayer or a polymer for preparing a nanoparticle, and preparing the drug-loaded liposome or nanoparticle.

The invention further provides application of the JO protein anchored drug-loaded immune cell, the drug-loaded liposome or the drug-loaded nanoparticle in preparation of antitumor drugs.

Further, the tumor is a solid tumor, a hematologic tumor or a lymphoma. Examples of the cancer include lung adenocarcinoma, liver cancer, lung cancer, bile duct cancer, head and neck tumor, breast cancer, cervical cancer, ovarian cancer, prostate cancer, stomach cancer, intestinal cancer, leukemia, and pancreatic cancer.

Advantageous effects

1. The cells are immune cells derived from human bodies, so that the safety is high; and due to the tumor homing effect, the tumor can be actively gathered to the tumor part, the gathering speed to the tumor part is very quick, and the gathering can be finished 15 minutes after the tumor enters blood. Meanwhile, the cells lose activity within 24 hours after drug loading, which reduces side effects such as immune storm and the like possibly caused by T cell input.

2. The bio-safety of the JO protein and lipid intermediates such as EDC or DMPE-PEG-NHS, DSPE-PEG-NHS and the like is high.

3. The permeation promoting strategy provided by the invention can improve the difficult problem of permeation of nano-drugs or cell preparations in solid tumors. The penetration promoting function of the JO protein is directly connected with the therapeutic cells for the first time, and the therapeutic effect is far higher than that of free drugs and drug-loaded cells of unmodified JO protein.

4. The preparation method is simple and easy to implement, and convenient to operate and popularize.

5. The invention proves the penetration promoting effect of the JO protein on immune cells for the first time, the JO protein is connected to the immune cells Jurkat cells through a one-step method, and the administration times of the chemical drugs and the JO protein when being used together are reduced while the JO penetration promoting effect is realized.

6. The invention utilizes the lipid component of the cell membrane, and according to the similar intermiscibility principle and the characteristics of polyamino, the JO protein is quickly and efficiently modified on the surface of the cell membrane by a connecting agent through an EDC method or a one-step method, and can not be endocytosed by cells within 24 hours.

Drawings

FIG. 1 confocal microscopy of modification of Cy 5-labeled JO protein into Jurkat cells (blue is Cy 5-labeled JO protein)

FIG. 2 confocal laser microscopy was performed to observe the penetration of Jurkat cells and Jurkat cells modified with JO protein into A549 spherical tissues. The first row represents the experimental results of the control group (unmodified JO protein) and the second group represents the experimental results of JO protein-modified Jurkat cells. (green for A549 cells and red for DII dye, marker for Jurkat cells).

FIG. 3 is a confocal laser scanning microscope for observing the penetration of JO protein modified Jurkat cells in A549 spherical tissues. (green for A549 cells, blue for Cy5, labeling for JO protein).

FIG. 4 confocal laser microscopy of JO protein-modified and unmodified Jurkat cells in an animal model of A549 carcinoma in situ. Wherein A shows the result of modifying JO with DID-labeled Jurkat cells to give Jurkat-JO-DID, and B shows the result of Jurkat-DID with DID-labeled Jurkat cells.

FIG. 5 shows the results of killing tumor cells by Jurkat-JO-DOX.

FIG. 6 shows the result of Jurkat-JO-DOX induced apoptosis.

FIG. 7 shows the results of tumor cytotoxicity studies with different concentrations of JO protein.

FIG. 8 shows the results of the tumor chemotactic assay of Jurkat cells.

FIG. 9 is the tumor size micro-CT results of lung tumors at the mid-stage of cancer progression after dosing.

FIG. 10 is the tumor size micro-CT results of lung tumors at advanced stages of cancer progression after dosing.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below, but the present invention is not to be construed as being limited to the implementable range thereof.

Example 1 preparation of JO protein anchored T cells

Dissolving JO protein in water, wherein the concentration range is 1-200 mg/mL;

JO protein, EDC in a ratio of 1: 100 is dissolved in PBS and reacted for 72 hours at room temperature;

③ adding the carboxyl activated JO protein obtained in the step two into Jurkat cells, and incubating for 4 hours at 37 ℃ by a shaking table. 1000 rpm, centrifuge for 3 minutes, and wash three times with PBS. Thus obtaining the JO protein anchored T cell.

EXAMPLE 2 Permeability test

1) Cy 5-labeled JO protein-modified Jurkat cell permeation assay

The experimental method comprises the following steps: cy 5-labeled JO protein (JO-Cy5) was modified on the Jurkat cell surface as described above and observed 24 hours later with a confocal microscope.

The experimental results are shown in FIG. 1, and the more blue color on the Jurkat cell surface is evident as the concentration of JO-Cy5 is increased, indicating that JO-Cy5 is successfully modified to the Jurkat cell surface and has a positive correlation with the concentration of JO-Cy 5.

2) Experiment of penetration depth of JO protein modified and unmodified Jurkat cells in A549-GFP tumor organoid

The experimental method comprises the following steps: constructing an A549-GFP tumor organoid by using the GFP-expressing A549; JO was anchored to Jurkat cells by the methods described above, and Jurkat cells were labeled with the red dye DII, as specified in the DII specification. The JO concentration was quantified and added to the A549-GFP organoids at a final JO concentration of 5. mu.g/mL. After 24 hours, the observation was carried out by a confocal laser microscope. As a control group, dio-labeled Jurkat cells were used without modification of JO protein (Jurkat alkone).

The results of the experiment are shown in FIG. 2, and from the results of the multi-layer scan, as organoids were gradually advanced into the nucleus, DII distribution was clearly seen at 20 μm and 30 μm for the Jurkat-JO group, while DII distribution for the control group (Jurkat cells not linked to JO protein) was significantly weaker than that of the Jurkat-JO group. With the further progress of the scan at 40 μm and 50 μm, the red color distribution of Jurkat-JO group was somewhat weaker than that of the control group, indicating that JO-modified Jurkat cells reached deeper sites of organoids and had better penetration effect than the control group.

3) Penetration experiment of JO protein modified Jurkat cells in A549-GFP tumor organoid

The experimental method comprises the following steps: constructing an A549-GFP tumor organoid by using the GFP-expressing A549; JO was anchored to Jurkat cells by the method of example 1 and JO protein was labeled with Cy 5. The JO concentration was quantified and added to the A549-GFP organoids at a final JO concentration of 5. mu.g/mL. After 2 hours, the sample was observed by a confocal laser microscope.

The results of the experiment are shown in FIG. 3, and the JO-modified Jurkat cells were distributed to 50 μm of the A549 organoid within 2 hours, indicating that the JO-modified Jurkat cells were able to penetrate to the center of the organoid.

4) DID-labeled nude mouse experiments

The experimental method comprises the following steps: establishing an A549 in-situ cancer model, wherein specific references are as follows: doi:10.1016/j. biomaterials.2018.08.035.

Jurkat cells were labeled with DID (blue), see DID instructions (Thermo). Two groups of experiments are set, A, JO is modified by DID marked Jurkat cells to obtain Jurkat-JO-DID; b, DID-labeled Jurkat cells as a control group (Jurkat-DID). Jurkat-JO-DID and Jurkat-DID were injected into the tail vein, respectively, and 24 hours later, nude mice were sacrificed, and the tumors were frozen and sectioned, and stained with CD31 for tumor blood vessels and DAPI for cell nuclei. Observed with a confocal laser microscope.

The results of the experiment are shown in FIG. 4. As can be seen from A, Jurkat-JO-DID is widely distributed in tumors and still distributed in less vascularly distributed sites, while for the Jurkat-DID group it is distributed centrally around the vessels. It is shown that Jurkat-JO-DID, after reaching the tumor vessels, can further penetrate into the surrounding tumor tissue with a deeper tumor distribution effect.

Example 3 preparation of JO protein anchored drug-loaded T cells

Dissolving cytarabine (DOX) in DMSO at a concentration of 1-20 mg/mL;

adding the JO protein anchored T cells obtained in the step (1) into the solution obtained in the step (1), controlling the concentration of DOX to be 1-150 mu g/mL, and incubating for 1-6 hours at 37 ℃;

③ 1000 turns, centrifugates for 3 minutes, and washes with Phosphate Buffer Solution (PBS) for three times. Obtaining the Jurkat-JO-DOX of the Jurkat cell anchored by the JO protein loaded with the adriamycin.

Example 4 antitumor test experiment of Jurkat-JO-DOX

The CCK-8 method uses Jurkat and Jurkat-JO as carriers of drug delivery respectively, and discusses the anti-tumor capability after loading DOX. Experiments compared the killing ability of Jurkat-DOX modified and drug-loaded cells to tumor cells without JO modification (free DOX), and Jurkat-JO-DOX modified and drug-loaded cells at different concentrations (in DOX concentration) on the cellular level.

The experimental results are shown in FIG. 5, which proves that the Jurkat cells are loaded with DOX, and the lung adenocarcinoma cell line A549 Jurkat loaded with DOX (Jurkat-DOX) and the modified JO protein loaded with DOX (Jurkat-JO-DOX) both show certain anti-tumor effect. Under the same DOX concentration (0.5 mu M), especially under low concentration (below 2 mu M), the tumor killing capability of Jurkat-JO-DOX is obviously superior to that of Jurkat-DOX, and the anti-tumor effect is obviously higher than that of free DOX.

Example 5 Effect of Jurkat-JO-DOX on tumor cell apoptosis

The cell apoptosis detection kit Annexin V-647/PI is used for investigating the anti-tumor capability of Jurkat and Jurkat-JO as carriers for drug delivery after loading DOX.

The results of the experiments are shown in FIG. 6, the results of apoptosis are consistent with those of FIG. 5, the antitumor effects of Jurkat-JO-DOX and Jurkat-DOX are superior to those of free DOX, and the antitumor effect of Jurkat-JO-DOX is better, and the antitumor mechanism is derived from apoptosis of cells. Jurkat cells alone cannot kill the tumor, and the effect of the T cells is eliminated.

Example 6 examination of whether JO protein is toxic to A549

The JO protein and the cells are adopted for co-incubation, the experimental result is shown in figure 7, the experimental result shows that JO protein with different concentrations (5-40 mug/mL) can not cause cytotoxicity, and meanwhile, the anti-tumor effect of the Jurkat cells loaded with DOX is obviously superior to that of Free DOX.

Example 7 examination of Jurkat's tumor tropism

The transfwell experiment verifies the chemotactic capacity of Jurkat cells for 48 hours of culture medium in which A549 cells are cultured. Fresh serum-free medium was used as a negative control, and medium containing 20% serum was used as a positive control.

The A549 culture medium of 48 hours simulates the in-vivo tumor environment, the chemotactic capacity of Jurkat cells to tumors is studied by a transwell experiment, the experimental result is shown in figure 8, and the result shows that the Jurkat culture medium to the A549 is 1.2 times of that of a control group. That is, Jurkat cells have a certain tumor chemotactic capacity.

Example 8 antitumor Effect of Jurkat-JO-DOX and Jurka-DOX in vivo

After dosing with Jurkat-JO-DOX and Jurka-DOX, respectively, in a pulmonary tumor animal model, the tumor size of the pulmonary tumor at the mid and late stages of cancer progression after dosing was observed with micro-CT. The results of the experiment are shown in FIGS. 9-10, where FIG. 9 is the tumor size profile of the lung tumor at the mid-stage of cancer progression after dosing; FIG. 10 is a plot of tumor size of lung tumors at an advanced stage of cancer progression after dosing.

From experimental results, the penetration capability of the drug carrier in the solid tumor can be enhanced by taking the Jurkat cells as the drug delivery carrier, and in order to further optimize a drug delivery system, a JO protein (Jurkat-JO) is introduced on the Jurkat cells, and the penetration capability of the Jurkat cells in the solid tumor is better than that of unmodified Jurkat cells.

Jurkat and Jurkat-JO are respectively used as drug carriers to load DOX to obtain Jurkat-DOX and Jurkat-JO-DOX, and under the same DOX concentration, the in vivo and in vitro anti-tumor (A549) effects of the Jurkat-DOX and the Jurkat-JO-DOX are obviously superior to those of a free DOX group.