Tumor killing T cell extracellular vesicle release promoting method based on radio stimulation control

Abstract

The invention provides a tumor killing T extracellular vesicle release promoting method based on radio stimulation control, and belongs to the technical field of biological medicines.The yield of EVT secreted by T cells is remarkably increased by applying electrical stimulation to the T cells, an electrical stimulation system capable of being wirelessly controlled is established, efficient and controllable release of the T extracellular vesicles is achieved, and the tumor killing effect of the T extracellular vesicles is improved. The calcium ion dependency mechanism of the EVT is defined, and the function retention of the released EVT in tumor killing activity and tissue penetrability is verified.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically is a method for promoting the release of tumor-killing T cell extracellular vesicles based on radio stimulation control. Background Art

[0002] Recent studies have found that extracellular vesicles from T cells (EVs) T ) As a "cell-free substitute" for T cell function, EV has shown unique advantages in anti-tumor immunity. T It can carry a variety of T cell-derived immune effector molecules (such as perforin, granzyme, inflammatory factors, etc.), and exert anti-tumor effects through multiple mechanisms such as directly inducing tumor cell apoptosis and regulating the tumor microenvironment. T EVs have smaller size, higher tissue penetration, lower immunogenicity and stronger engineering potential. T Replacing T cells for tumor immunotherapy is theoretically highly feasible.

[0003] Although EV T Although EV has many advantages, its clinical application still faces significant challenges. T The therapeutic effect of T cells usually depends on high-dose administration, while EVs produced by T cells under natural conditions T The number is limited and it is difficult to meet the treatment needs. Secondly, traditional in vitro EV T The preparation process is complicated, and there are problems such as low separation and purification efficiency and attenuated biological activity, which restricts its large-scale industrial application. Summary of the Invention

[0004] The present invention provides a tumor-killing T cell extracellular vesicle (EV) based on radio stimulation control T ) release-promoting method, aimed at increasing T cell secretion of EVs T yield.

[0005] Technical solution: A method for promoting the release of tumor-killing T cell extracellular vesicles based on radio stimulation control, wherein the method promotes the secretion of T cell extracellular vesicles by applying electrical stimulation to T cells in vitro.

[0006] Furthermore, the conditions for applying electrical stimulation are: time of 5-30 minutes, voltage of 2-6 V. Preferably, time of 20 minutes, voltage of 4 V.

[0007] Furthermore, electrical stimulation is applied to T cells through a wireless micro-electrical stimulation system.

[0008] Furthermore, the wireless micro-electrical stimulation system includes a hydrogel encapsulation layer, a conductive electrode layer and a wireless functional module;

[0009] The hydrogel encapsulation layer is used to encapsulate T cells, maintain cell activity and allow the release of extracellular vesicles;

[0010] The conductive electrode layer is located in the hydrogel packaging layer and is connected to the wireless control module;

[0011] The wireless functional module is located in the hydrogel packaging layer and is used to implement non-invasive remote application of electrical stimulation signals.

[0012] During use, activated T cells are dispersed in PBS or DMEM solution and dripped onto the conductive electrode layer. The hydrogel mixture is then added to encapsulate the cells and device. After curing, the hydrogel has a porous structure that ensures the release of T cell exosomes.

[0013] Beneficial effect: This invention solves the problem of traditional EV T The present invention achieves efficient and controllable release of T cell extracellular vesicles by establishing a wirelessly controllable electrical stimulation system, clarifies its calcium ion dependence mechanism, and verifies the released EVs. T Functional retention in terms of tumor killing activity and tissue penetration is the key to EV T It provides innovative technical support for the large-scale preparation of and its application in tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 In this example, electrical stimulation promotes T cells to release EVs T Schematic diagram and result diagram; among them, a is a schematic diagram of electrically stimulated T cells; b is the relative quantification of EV secretion by cells before and after electrical stimulation (2V, 5min); d is a SEM image of the hydrogel, scale: 20 μm; e is a SEM image of T cells loaded on the device, scale: 10 μm; f is the effect of different voltages at the same time on the release of EVs by T cells; g is the effect of fixed voltage for different times on the release of EVTs by T cells; h is the change in the number of EVTs secreted by T cells before and after electrical stimulation under optimal conditions.

[0015] Figure 2 Figure 1 is a graph showing the experimental results of the effect of electrical stimulation on T cell activity in the examples; (a) shows the effect of different voltages at the same time on T cell activity; (b) shows the effect of a fixed voltage at different times on T cell activity; (c) confocal image of T cell live-dead staining under optimal electrical stimulation conditions, scale: 20 μm.

[0016] FIG3 is a graph showing the results of intracellular calcium ion content detection in the embodiment; wherein a is the Ca content in T cells after treatment under different conditions. 2+ Concentration levels; b is EV secretion by T cells under different conditions T c is the correlation analysis between the intracellular calcium ion concentration level and the amount of EV release.

[0017] Figure 4 shows EVs secreted by T cells in the example T It contains granzyme B test images; among them, a is the in vitro uptake of EVT by 4T1 tumor cells, scale: 20 μm; b is the in vitro uptake of EVT by CT26 tumor cells, scale: 20 μm.

[0018] Figure 5 shows the cytotoxicity test results of EVT in the example; wherein, a is the cytotoxicity of different concentrations of N-EV on 4T1 cells; b is the cytotoxicity of different concentrations of E-EV on 4T1 cells; c is the comparison of the killing ability of the same concentration of N-EV and E-EV on 4T1 cells.

[0019] FIG6 is a graph showing a comparison of the spheroid penetration ability of T cells and E-EVs in the examples, scale: 100 μm.

[0020] Figure 7 is a diagram showing the results of E-EV treatment inhibiting tumor progression in the 4T1 solid tumor model in the embodiment; wherein, a is a flowchart of the treatment of the 4T1 solid tumor model; b is the change in tumor volume of a single group of mice; c is a comparison of tumor volume changes in different groups of mice; d is the survival curve of different groups of mice; e is the change in body weight of different groups of mice; f is a physical picture of late-stage tumors in different groups of mice, scale: 1 cm; g is the weight of late-stage tumors in different groups of mice.

[0021] Figure 8 shows tumor tissue EVs in the example. T Representative images of immunofluorescence staining of granzyme B and fluorescence quantitative analysis results; a is the EV of tumor tissue in each group T a Representative images of immunofluorescence staining and fluorescence quantitative analysis, scale bar: 50 μm; b Representative images of immunofluorescence staining and fluorescence quantitative analysis of granzyme B in tumor tissues of each group, scale bar: 50 μm. DETAILED DESCRIPTION

[0022] The technical solutions of the present invention are described in detail below through examples, but the protection scope of the present invention is not limited to the examples.

[0023] The reagents and raw materials used in the following examples can all be purchased from the market.

[0024] Example 1: Construction of a wireless micro-electrical stimulation system device

[0025] like Figure 1 As shown in c, the wireless micro-electrical stimulation system device is mainly composed of a hydrogel encapsulation layer, a conductive electrode layer and a wireless functional module, and T cells are encapsulated in the hydrogel.

[0026] Hydrogel encapsulation layer: used to encapsulate T cells, maintain cell activity and allow sufficient release of extracellular vesicles;

[0027] Conductive electrode layer: located inside the hydrogel encapsulation layer and connected to the wireless control module;

[0028] Wireless functional module: located in the hydrogel encapsulation layer, used to achieve non-invasive remote application of electrical stimulation signals;

[0029] This wireless micro-electrical stimulation system can stably maintain the biological activity of encapsulated cells and has good biocompatibility and stimulation responsiveness.

[0030] Example 2: Using a wireless micro-electrical stimulation system device to promote T cell release of EVs T

[0031] In this example, the electrical stimulation conditions were optimized and standardized. T To find the optimal voltage, do the following:

[0032] Step 1: Using the device constructed in Example 1, T cells in a normal culture state are encapsulated in the hydrogel.

[0033] Activated T cells (CD8+ T cells) were dispersed in a PBS solution and dripped onto the electrode. A hydrogel solution was then added to encapsulate the cells and device. After curing, the hydrogel has a porous structure that allows for the release of exosomes from the T cells.

[0034] Electrical stimulation promotes T cell release of EVs T The schematic diagram is as follows Figure 1 As shown in a, the SEM image of the hydrogel without T cells is shown in Figure 1 As shown in d, the SEM image of the encapsulated hydrogel is Figure 1 As shown in e. Figure 1 Middle b is the relative quantitative graph of EV secretion by cells before and after electrical stimulation (2V, 5min).

[0035] Step 2: Under the condition of fixed electrical stimulation time of 10 min, test the effect of electrical stimulation voltage on EVs released by T cells. T impact.

[0036] 1) Specific operation: The electrical stimulation time was fixed at 10 min, and then different voltages (specifically 0, 2, 4, and 6 V) were applied to T cells in normal culture.

[0037] 2) Quantification of extracellular vesicles: First, 200 µL of the supernatant from the device after electrical stimulation was added to an ELISA plate. After 1 hour of adsorption, the supernatant was aspirated and a 1% bovine serum albumin solution in PBS was added. The plate was left to stand for 1 hour to fill the blank. The coating solution was then discarded (patted dry and blotted with lint-free paper). The plate was washed three times with 1× PBST (pH 7.2-7.4) (filling each well completely), for 3-5 minutes each wash. Next, 200 µL of cholesterol-modified HRP was added to each well. After incubation for 1 hour, the solution was removed and the plate was washed three times, following the same procedure as above. Next, 50 µL of substrate solution (TMB) was added to each well and color was developed at room temperature in the dark for 10 minutes. Finally, after color development, 50 µL of stop solution (2 mol / L H₂SO₄) was added to each well to terminate the reaction. The OD value was read at 450 nm using a microplate reader.

[0038] 3) Results: Figure 1 As shown in (f), electrical stimulation can promote the release of EVs by T cells. T , and the optimal voltage is 4V.

[0039] Step 3: Under the condition of applying voltage of 4 V, test the effect of electrical stimulation time on EV released by T cells. T impact.

[0040] 1) Specific operation: Under the condition of applying a voltage of 4 V, T cells in normal culture were subjected to different electrical stimulation times (specifically, the time was 0, 10, 20, and 30 minutes).

[0041] 2) Quantification of extracellular vesicles.

[0042] 3) Results: Figure 1 As shown in middle g, electrical stimulation can promote the release of EVs by T cells T Under the condition of applying voltage of 4V, the EVs released by T cells during continuous electrical stimulation for 20 min were T Relatively the most.

[0043] From the general steps 2 and 3, we can see that electrical stimulation promotes T cells to release EVs. T The optimal conditions are 4 V, 20 min. Figure 1 As shown in h, under this condition, T cell EVs TThe release amount was increased by about 13 times compared to the case without electrical stimulation.

[0044] NTA test results further showed that EV T The concentration is consistent with previous experimental results and is approximately 13 times that released under normal conditions.

[0045] Step 4: Detection of T cell activity after electrical stimulation.

[0046] 1) Specific operation: After the cells were electrically stimulated under different conditions, they were seeded into 96-well plates and cultured in a cell culture incubator for 24 h.

[0047] 2) Detection: To determine cell viability, CCK-8 assay kit or Calcein / PI cell viability and cytotoxicity assay kit were used to assess cell viability according to the manufacturer's instructions.

[0048] 3) Results: Effects of electrical stimulation on T cell activity Figure 2 As shown, Figure 2 Figure a shows the effect of different voltages of 0, 2, 4, and 6 V on T cell activity when the electrical stimulation time was fixed at 10 min. Figure 2 Middle b shows the effect of fixed electrical stimulation time of 4 V and different voltages of 0, 10, 20, and 30 min on T cell activity; Figure 2 Middle c is a confocal image of live-dead staining of T cells under optimal electrical stimulation conditions (Es: 4V, 20 min), scale bar: 20 μm.

[0049] By optimizing the electrical stimulation parameters, the optimal voltage was determined to be 4 V and the duration was 20 min. Under these conditions, the amount of T cell EV release was more than 13 times that of the natural state, and the cell activity was well maintained.

[0050] Step 5: Promote EV T Analysis of the release mechanism:

[0051] Calcium content colorimetric detection kit detection:

[0052] Procedure: Seed T cells into a six-well plate. Collect treated T cells and centrifuge at 600g for 5 minutes at 4°C to collect the cells. Discard the supernatant and flick the cells. Add 100 µL of cell lysis buffer to each well and gently flick the cells to fully lyse them. After complete lysis, centrifuge at 12,000g for 5 minutes at 4°C and collect the supernatant. For the assay, add 50 µL of supernatant and 150 µL of assay working solution to each well and mix thoroughly. Incubate at room temperature in the dark for 5-10 minutes. After incubation, measure the OD575 using a microplate reader and calculate the calcium content of the sample based on the standard curve.

[0053] Detection: Intracellular calcium concentration was measured using a calcium chromogenic assay kit according to the manufacturer's instructions.

[0054] result Figure 3 As shown, (a) is the Ca2+ level in T cells after treatment under different conditions. 2+ Concentration levels; (b) EV secretion by T cells after treatment with different conditions T (e) Correlation analysis between intracellular calcium ion concentration level and EV release amount under electrical stimulation conditions. Figure 3 In the table, Es means electrical stimulation treatment, 4V voltage electrical stimulation for 20 min; CaCl2 means treatment with calcium chloride solution; Es+EDTA means cells were treated with EDTA at the same time as electrical stimulation (4V voltage electrical stimulation for 20 min); Es+Cd 2+ Indicates that Cd was added under the regulation of electrical stimulation (4V voltage electrical stimulation for 20 min) 2+ Culture cells.

[0055] After 20 min of 4V electrical stimulation, intracellular calcium ions increased significantly Figure 3 As shown in a, the corresponding EV is significantly increased Figure 3 As shown in middle b; intracellular Ca 2+ The concentration is positively correlated with the amount of EV released. Figure 3 As shown in c.

[0056] Step 6: EV T Maintenance and verification of biological functions:

[0057] 1) Confocal analysis:

[0058] First, tumor cells were seeded onto a confocal dish, and then EV T After incubation with tumor cells for 4 hours, granzyme B staining was performed using the same staining procedures as flow cytometry. Finally, to facilitate the localization of granzyme B, the cell nuclei were also labeled. After staining, the staining was observed under a confocal microscope.

[0059] These EVs carrying granzyme B T Co-incubated with 4T1 cells, stained with granzyme B 4 hours later, and confocal imaging was performed after staining. The results showed that strong fluorescence signals were observed in tumor cells for both N-EV and E-EV ( Figure 4 A similar effect was observed in CT26 cells ( Figure 4 (b) demonstrates that EVs carrying granzyme B T It can be easily taken up by tumor cells and has no specificity.

[0060] 2) DiD labeling of T cells and EVs:

[0061] To obtain DiD-labeled T cells and EVs, appropriate amounts of T cells and EVs were incubated with 0.5 M DiD fluorescent dye at a 1:1 volume ratio at 25 °C for 2 h. Excess DiD dye was removed from T cells by centrifugation at 1000 g for 5 min, and excess DiD dye was removed from EVs by centrifugation at 150,000 g for 2 h.

[0062] Spheroid culture: Count the cultured tumor cells and seed them into a U-bottom 96-well plate at a density of 3,000-10,000 cells per well, 100 μL per well. Observe the cells after culturing in an incubator for 2-3 days. If clear spheroids have formed, the medium can be changed. When changing the medium, aspirate approximately half the volume of the culture medium and add an equal amount of fresh medium to prevent the spheroids from being disrupted. Once the spheroids are fully formed, carefully aspirate the medium, rinse with PBS, and then replenish with fresh medium. Add an appropriate amount of 100 μL of DiD-labeled T cells and EVs to the spheroids for co-incubation. After incubation, rinse the spheroids with PBS. After completion, transfer the spheroids to a confocal dish and observe the staining effect under a confocal microscope.

[0063] Because of the physical barriers at the border of solid tumors, T cells have difficulty in penetrating dense tumor tissues, resulting in insufficient infiltration, which is one of the bottlenecks faced by T cell therapy. T With a smaller volume, they can theoretically infiltrate into tumor tissue more easily than T cells. Although it has been proven that EVs released by electrically stimulated T cells can enter tumor cells as smoothly as EVs released by T cells under normal conditions, tumor cells cultured on a flat surface are different from solid tumors. In many aspects, the cells cultured on a flat surface have obvious differences in their actual morphology and structure from those under living conditions. In order to verify the EV T To test the penetrating ability of the spheroids, researchers cultured 3D tumor cell spheroids, which more closely resembled solid tumors, to simulate tumors. Once the spheroids were cultured, DiD-labeled T cells and E-EVs were co-incubated to simulate the actual penetrating properties of T cells and E-EVs. After incubation, confocal microscopy was used to image the spheroids in three dimensions at different depths. As expected, T cells clustered only at the edges of the spheroids and were unable to penetrate the interior, whereas E-EVs possessed strong penetrating ability ( Figure 6 ). Therefore, EV TIt overcomes the problem that T cells have difficulty passing through dense tumor tissue, resulting in insufficient infiltration.

[0064] 2) To validate EV in vitro T The cytotoxic effect on tumor cells was detected using a lactate dehydrogenase (LDH) cytotoxicity detection kit (Biyuntian).

[0065] LDH release is considered to be a safe and effective alternative to the previous use of radioactive 51Cr to label cells and then detect cell membrane integrity through 51Cr release. First, tumor cells were seeded into six-well plates, and different concentrations of EV were added to each well after one day of culture. T (12.5, 25, 50, 100, 200 μg / mL), and after incubation for 24 h, LDH release assay was performed according to the manufacturer's instructions.

[0066] Proven EV T Carrying the cytotoxic molecule granzyme B. To achieve this, EV T EVs need to be proven to replace T cells in anti-tumor T Cytotoxicity. By using different concentrations (12.5, 25, 50, 100, 200 μg / mL) of N-EV to co-incubate with 4T1 for 24 hours, the release of lactate dehydrogenase was detected to verify the cytotoxicity. The results showed that N-EV had obvious cytotoxicity and was concentration-dependent. As the concentration increased, the cytotoxicity of N-EV increased (Figure 5a). Then, the cytotoxicity of E-EV was explored using the same method. The results showed that E-EV not only had obvious cytotoxicity like N-EV (Figure 5b), but also at the same concentration, E-EV and N-EV had similar tumor cell killing effects (Figure 5c). The above results not only verified the EV T The cytotoxicity of EVs was not affected by electrical stimulation. T biological functions.

[0067] Functional verification has shown that the EVT released by electrical stimulation still carries cytotoxic molecules such as granzyme B, and has good tumor cell killing ability; it exhibits strong penetrability in the 3D tumor sphere model.

[0068] Step 7: EV T Verification of advantages in tumor tissue penetration:

[0069] Modeling and Therapeutic Evaluation of 4T1 Solid Tumor Models: Animal experiments were conducted according to protocols approved by the Animal Care and Use Committee of Nanjing University of Posts and Telecommunications. A 4T1 breast cancer model was established by subcutaneously injecting 2×106 murine 4T1 cells per Balb / c mouse. When tumor volume reached 50–100 mm, the 4T1 breast cancer model was established. 3 At around 400 days, the mice were randomly divided into 4 groups, with 5 mice in each group. They were treated with N-EV, T cells, and E-EV respectively, and PBS was used as a control. The body weight and tumor volume of the mice were monitored. At the end of the experiment, the tumor volume of the mice exceeded 1000 mm. 3 All mice were considered dead and euthanized, and their tumor tissues, blood, heart, liver, spleen, lung, and kidney were collected for further analysis.

[0070] After demonstrating in vitro that electrical stimulation can promote the release of EVs from T cells and that E-EVs and N-EVs have similar cytotoxicity, the specific therapeutic effect of E-EVs in inhibiting solid tumor growth in vivo was evaluated. 4T1 cells were inoculated subcutaneously into female Balb / c mice. Seven days later, the tumor-bearing mice were randomly divided into four groups of five mice each. Each group was treated with PBS, device-loaded T cells without electricity (N-EVs), T cells, and device-loaded T cells with electricity (E-EVs) every two days for a total of three treatments ( Figure 7 (a) The tumor volume and body weight of the mice were then monitored. Tumor volume data showed that the E-EV group significantly inhibited the growth of tumor volume in mice, showing a good therapeutic effect. Although the N-EV and T cell groups had a certain inhibitory effect on tumor volume growth, the therapeutic effect was far less than that of the E-EV group ( Figure 7 The tumor-bearing mice in the PBS group, N-EV group, and T cell group all died on the 36th, 45th, and 52th day, respectively. However, E-EV treatment significantly prolonged the survival of the mice, with two mice even surviving for more than 80 days ( Figure 7 During this period, the weight difference of mice in each group was not significant ( Figure 7 (e) This indicates that the treatment has good biosafety and does not cause significant systemic toxicity. After the monitoring, the tumors of all tumor-bearing mice were removed, photographed, and weighed. Compared with the PBS group, the N-EV and T cell treatment groups only inhibited tumor growth in the early stages, while E-EV treatment effectively inhibited tumor progression ( Figure 7 f and g).

[0071] In order to further explain why there is such a therapeutic effect, the tumor tissues of mice in each group were tested for EV T The results showed that the EVs infiltrating into the tumor in the E-EV treatment group TThe largest number and strongest immunofluorescence ( Figure 8 (a) (Because the T cells loaded into the device were membrane-labeled in advance, the EVs here T All of them were produced by T cells in the device, and the cells used in the T cell treatment group were not labeled.) The same is true for the immunofluorescence slice data of granzyme B. In addition, the number of tumor cell nuclei in the E-EV treatment group was significantly reduced, indicating that E-EV is more effective in killing tumor cells. Figure 8 Middle b).

[0072] In summary, the present invention can greatly improve EV by establishing a wirelessly controllable electrical stimulation system. T Release efficiency and yield increased by 13 times; EV T It can be released in situ and efficiently, significantly enhancing targeting and local drug concentration; maintaining EV T The biological function stability, strong cytotoxicity and tissue penetration ability; the electrical stimulation mechanism is clear, Ca 2+ The pathway is highly dependent and the mechanism is controllable.

[0073] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes may be made to the form and details without departing from the spirit and scope of the present invention.

Claims

1. A method for promoting the release of tumor-killing T cell extracellular vesicles based on radio stimulation control, characterized in that: Electrical stimulation of T cells in vitro promotes T cell extracellular vesicle secretion.

2. The method according to claim 1, characterized in that The conditions for applying electrical stimulation are: time 5-30 minutes, voltage 2-6V.

3. The method according to claim 1, characterized in that The conditions for applying electrical stimulation were: time 20 min, voltage 4V.

4. The method according to claim 1, wherein Electrical stimulation of T cells was applied via a wireless micro-electrical stimulation system.

5. The method according to claim 4, characterized in that The wireless micro-electrical stimulation system comprises a hydrogel encapsulation layer, a conductive electrode layer and a wireless functional module; The hydrogel encapsulation layer is used to encapsulate T cells, maintain cell activity and allow the release of extracellular vesicles; The conductive electrode layer is located in the hydrogel packaging layer and is connected to the wireless control module; The wireless functional module is located in the hydrogel packaging layer and is used to implement non-invasive remote application of electrical stimulation signals.

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