An immunotherapy nano-delivery system with synergistic radiotherapy and its preparation method and application

Abstract

The present invention belongs to the field of medical technology, and specifically relates to an immunotherapy nano-delivery system with synergistic radiotherapy, and its preparation method and application. The nano-delivery system includes a manganese dioxide nanosheet, and a cancer cell membrane coated on the outside of the manganese dioxide nanosheet; the manganese dioxide nanosheet is loaded with an immune drug. Based on the hypoxic environment of the tumor, the present invention develops a manganese dioxide-loaded drug nano-delivery system that can generate oxygen, which achieves the activation of the STING pathway while overcoming the hypoxic conditions, and has a radiotherapy sensitization effect. The cancer cell membrane-coated manganese dioxide nano-delivery loaded with R848 constructed by the present invention has the dual functions of synergistic radiotherapy and immunotherapy, which can further increase the anti-tumor effect, and has a certain reference significance for constructing a nano-drug delivery system for synergistic tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to an immunotherapy nano-delivery system with synergistic radiotherapy, and a preparation method and application thereof. Background Art

[0002] In recent years, radiotherapy has been widely used in clinical practice as a mainstream cancer treatment method. In addition to directly killing cancer cells, ionizing radiation during radiotherapy can also lead to immunogenic cell death (ICD) and the release of tumor antigens, thereby enhancing a certain level of anti-tumor immune response. In addition, immunotherapy represented by the delivery of immunomodulators or checkpoint blockers and cancer vaccines also provides new methods for cancer treatment. At present, many literatures have also explored the combination of radiotherapy and immunotherapy to inhibit tumor growth and metastasis.

[0003] However, the immune response induced by radiotherapy is often suppressed, and the tumor microenvironment (TME) may greatly reduce the killing function of T cells. On the one hand, there are high levels of immunosuppressive cells in this site, such as regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and M2 macrophages, which can inhibit the anti-tumor immune response induced by radiotherapy; on the other hand, the hypoxia state in the tumor site can activate the hypoxia-inducible factor (HIF) signaling pathway, leading to increased VEGF levels, further stimulating tumor growth and increasing tumor invasiveness. Therefore, it is urgent to develop strategies that can reverse the immunosuppressive TME and induce strong anti-tumor immunity to achieve better therapeutic effects.

[0004] In addition, current drugs such as immunomodulators for immunotherapy have poor water solubility and low bioavailability, which means they are rapidly distributed and quickly cleared from the body before reaching the tumor site, which greatly limits their practical application. Therefore, there is an urgent need to develop better drug delivery systems to provide a more localized and longer-lasting immune response, thereby improving the safety and effectiveness of immunotherapy drugs. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide an immunotherapy nanodelivery system with synergistic radiotherapy, and its preparation method and application, which achieves good targeted drug delivery performance to provide a more localized and longer-lasting immune response, and overcomes the hypoxic environment and regulates the function of the microenvironment of the site, while achieving activation of the STING pathway, having a radiotherapy sensitization effect, and has important research value and practical significance for enhancing anti-tumor immune response.

[0006] To achieve the above objectives, the technical solutions adopted by the present invention include:

[0007] In a first aspect, the present invention provides an immunotherapy nano-delivery system with synergistic radiotherapy, wherein the nano-delivery system comprises a manganese dioxide nano-sheet and a cancer cell membrane coated on the outside of the manganese dioxide nano-sheet; the manganese dioxide nano-sheet is loaded with an immune drug.

[0008] Nanosheets are a type of two-dimensional nanomaterial with a suspended sheet structure and a large specific surface area. They can bind to drugs through electrostatic, hydrogen bonding and non-covalent interactions (such as van der Waals forces). Manganese dioxide is a type of transition metal material with redox activity that can consume excess H in tumor sites. 2 O 2 Oxygen is produced to relieve the hypoxia in the tumor site, further enhancing the immune response induced by radiotherapy. In addition, studies have shown that ionizing radiation can activate the interferon gene stimulator (STING) pathway, stimulate the production of type I interferon and the secretion of proinflammatory cytokines, thereby promoting the maturation of dendritic cells and triggering innate and adaptive immunity to fight cancer. 2+ The cGAS-STING pathway can be activated by increasing the overall catalytic activity of the cGAS enzyme and improving the affinity of cGAMP to STING, thereby further enhancing the anti-tumor immune response. Therefore, the development of a manganese dioxide-based immunodrug nanodelivery system for coordinated tumor radiotherapy will be a very meaningful research direction.

[0009] Cancer cell membrane coating can mimic natural cell membranes and reduce their clearance by reticuloendothelial cells. In addition, studies have also shown that because cancer cell membranes have negative charges, phospholipid bilayers and specific protein expression, nanoparticles coated with cancer cell membranes have strong targeting to homologous cancer cells, can activate immune responses to a certain extent, and give the nano-delivery system good stability and biocompatibility.

[0010] Through experimental research, it was found that the nano-delivery system of the present invention can be combined with radiotherapy to effectively overcome the radiation resistance of cancer cells, while inducing the production of reactive oxygen species to damage tumor cell DNA, further increasing the level of immunogenic cell death, releasing damage-related molecular patterns such as ATP, HMGB1 and CRT, increasing the uptake of tumor antigens by antigen-presenting cells-dendritic cells, and inducing an immune response; in addition, the nano-delivery system can consume the excessive H in the tumor site. 2 O 2 Oxygen is generated to overcome the hypoxic microenvironment at the tumor site and improve the immune tolerance at the tumor site, further enhancing the immune response induced by radiotherapy; in addition, the nano drug delivery system can achieve a more localized and sustained release of immune drugs and Mn 2+, thereby inducing stronger anti-tumor immunity by promoting the release of inflammatory factors such as TNF-α and the activation of the cGAS-STING signaling pathway, providing new possibilities for improving anti-tumor effects and having certain reference significance for constructing a nano-drug delivery system for synergistic tumor treatment.

[0011] Preferably, the immune drug includes an immunomodulator, a checkpoint blocker and a tumor vaccine. Preferably, the immune drug is R848.

[0012] Resiquimod (R848) is a small molecule immune adjuvant based on imidazoquinolinone, which can bind to TLR7 / 8 to trigger an immune response. R848 can convert MDSCs into antigen presenting cells (APCs) and polarize the macrophage phenotype from M2 to M1, thereby regulating the immunosuppressive microenvironment and ultimately inducing an anti-cancer response. The present invention effectively solves the problem of poor water solubility of R848 by loading the nano delivery system with R848 for tumor immunotherapy, significantly improves its bioavailability, and achieves a more stable and lasting immune response.

[0013] Preferably, the cancer cell membrane comprises a breast cancer cell membrane.

[0014] In the experiment of the present invention, a nano-delivery system was prepared by coating manganese dioxide nanosheets loaded with immune drugs with breast cancer cell (4T1) membranes, which achieved strong targeting to homologous breast cancer cells and could activate immune responses to a certain extent, providing new possibilities for improving the therapeutic effect of breast cancer.

[0015] Preferably, the method for preparing the manganese dioxide nanosheet loaded with immune drugs comprises the following steps:

[0016] (1) The manganese dioxide nanosheet aqueous solution was mixed with mPEG-NH 2 mixing, ultrasonicating, stirring, centrifuging, removing the supernatant to obtain a precipitate, and washing the precipitate to obtain a nanocomposite material;

[0017] (2) mixing the nanocomposite material of step (1) with the immune drug, ultrasonicating, stirring, centrifuging, removing the supernatant to obtain a precipitate, and washing the precipitate with an alcohol solvent to obtain drug-loaded manganese dioxide nanosheets.

[0018] Preferably, in step (1), the mass ratio of the manganese dioxide nanosheet aqueous solution to mPEG-NH2 is 2:(0.5-1.5).

[0019] mPEG-NH 2 As a surface modifier, mPEG-NH 2The addition of mPEG-NH 2 As a highly biocompatible polymer, it can improve the biocompatibility of drug-loaded nanosheets. The appropriate dosage ratio can ensure the biocompatibility and biosafety of the nanosheets in vivo. The experimental investigation found that when the manganese dioxide nanosheet aqueous solution and mPEG-NH 2 When the mass ratio of is 2:(0.5-1.5), the prepared nanodelivery system can achieve good drug loading efficiency and stability, and has a suitable drug sustained-release effect, thereby achieving a more stable and lasting drug immune response.

[0020] Preferably, in step (2), the mass ratio of the nanocomposite material to the immune drug is 1:(1-2).

[0021] Experimental research found that the dosage ratio of the nanocomposite material and the immunomodulator drug has an important influence on the release rate of the final nanodelivery system and the biological activity of the drug. When the mass ratio of the nanocomposite material and the immunomodulator is 1: (1-2), the nanodelivery system can ensure the stable and uniform distribution of the drug on the nanocarrier, avoid drug aggregation or excessive release, thereby improving the stability and durability of the immune drug, and can achieve effective release in the target tissues or cells, thereby improving the biological activity of the drug.

[0022] In a second aspect, the present invention provides a method for preparing the above-mentioned nano-delivery system, comprising the following steps:

[0023] (1) The manganese dioxide nanosheet aqueous solution was mixed with mPEG-NH 2 mixing, ultrasonicating, stirring, centrifuging, removing the supernatant to obtain a precipitate, and washing the precipitate to obtain a nanocomposite material;

[0024] (2) mixing the nanocomposite material of step (1) with the immune drug, ultrasonicating, stirring, centrifuging, removing the supernatant to obtain a precipitate, and washing the precipitate with an alcohol solvent to obtain drug-loaded manganese dioxide nanosheets;

[0025] (3) collecting the digested cancer cells, centrifuging, discarding the supernatant, resuspending the cell pellet with PBS, centrifuging, and discarding the supernatant to obtain a cell pellet;

[0026] (4) mixing physiological saline, water and PMSF to prepare a hypotonic buffer solution, wherein the mass ratio of the physiological saline, water and PMSF is 75:25:1;

[0027] (5) resuspending the cell pellet from step (3) in the hypotonic buffer from step (4) for 1-1.5 h, then sonicating with an ultrasonic disruptor, centrifuging at 1000-2000 rpm for 5-10 min, and collecting the supernatant;

[0028] (6) centrifuging the supernatant of step (5) at 15000-17000 rpm for 1-1.5 h, discarding the supernatant to collect the cell membrane fragments, washing with PBS, and freeze-drying to obtain cancer cell membranes;

[0029] (7) suspending and mixing the cancer cell membrane and the drug-loaded manganese dioxide nanosheets of step (2) in water, and performing ice bath sonication to obtain a mixed solution;

[0030] (8) Extruding the mixed solution of step (7) through a polycarbonate membrane to obtain a drug-loaded nanosheet coated with a cancer cell membrane, i.e., the nano-delivery system.

[0031] Preferably, in step (7), the mass ratio of cancer cell membrane to drug-loaded manganese dioxide nanosheets is 1:(0.5-1.5).

[0032] Experimental research found that when the mass ratio of cancer cell membrane and drug-loaded manganese dioxide nanosheets is 1: (0.5-1.5), it can ensure that the cancer cell membrane fully covers the surface of the drug-loaded nanomaterial, improve the stability of the drug delivery system, and to a certain extent avoid the loss or leakage of immune drugs, thereby improving the bioavailability of the drug.

[0033] In a third aspect, the present invention also provides the use of the above-mentioned nano-delivery system in the preparation of drugs for treating tumors.

[0034] Preferably, the tumor is breast cancer.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] (1) Based on the hypoxic environment of tumors, the present invention has developed a manganese dioxide-loaded drug nanodelivery system that can generate oxygen (i.e., the immunotherapy nanodelivery system of the present application), which can activate the STING pathway while overcoming the hypoxic conditions and has a radiotherapy sensitization effect.

[0037] (2) The present invention constructs a cancer cell membrane-coated manganese dioxide nano-delivery loaded with R848 (i.e., the immunotherapy nano-delivery system of the present application), which has the dual functions of synergizing radiotherapy and immunotherapy, can further increase the anti-tumor effect, and has certain reference significance for constructing a nano-drug delivery system for synergistic tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The prepared MnO 2 、MnO 2 -R848 and MnO 2 -Particle size distribution (A) and Zeta potential diagram (B) of R848@CM.

[0039] Figure 2 The prepared MnO 2 -R848@CM nano-delivery system in vitro radiotherapy effect evaluation diagram, where A shows different concentrations of MnO with (right) and without (left) X-ray irradiation 2 、MnO 2 -R848 and MnO 2 -R848@CM treated 4T1 cell viability; Figure B shows the cell viability of different treatment groups (PBS, H 2 O 2 、MnO 2 、MnO 2 -R848 and MnO 2 -R848@CM) ROS production; Figure C shows the ROS production in different treatment groups (PBS, H 2 O 2 、MnO 2 、MnO 2 -R848 and MnO 2 -R848@CM) cell apoptosis.

[0040] Figure 3 The different treatment groups (PBS, H 2 O 2 、MnO 2 、MnO 2 -R848 and MnO 2 -R848@CM) 2 AX (A), ATP (B), HMGB1 (C), and CRT (D) production diagrams, wherein Negative Control (+) in Figure A, Negative Control (+) in Figure B, Control (+) in Figure C, and Negative Control (+) in Figure D all represent the results obtained by X-ray irradiation alone.

[0041] Figure 4 The prepared MnO 2 -R848@CM nano-delivery system in vitro immune effect evaluation diagram, where A is different in vitro treatment groups (PBS, MnO 2 、R848、MnO 2 -R848, MnO 2 -R848@CM and LPS) BMDCs maturation; Figure B shows the experimental design of different treatment groups (PBS, MnO 2 、R848、MnO 2 -R848, MnO 2-R848@CM and LPS) BMDCs maturation flow cytometry; Figure C shows the maturation of BMDCs in different treatment groups (PBS, MnO 2 、R848、MnO 2 -R848, MnO 2 -R848@CM and LPS) TNF-α secretion; Figure D shows the secretion of TNF-α in different treatment groups (PBS, MnO 2 、R848、MnO 2 -R848, MnO 2 -R848@CM and LPS)4T1 and BMDCs co-incubation experimental design of BMDCs maturation; Figure E shows the in vitro irradiation and different treatment groups (PBS, MnO 2 、R848、MnO 2 -R848, MnO 2 Flow cytometry of BMDCs maturation after co-incubation of BMDCs with -R848@CM and LPS)4T1. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0043] Example 1. Manganese dioxide nanosheets (MnO 2 -R848) preparation method

[0044] The specific method is as follows:

[0045] (1) diluting a commercial manganese dioxide nanosheet dispersion (1 mg / ml) with milliq water to obtain a nanosheet solution with a concentration of 0.1 mg / ml;

[0046] (2) Mix the nanosheet solution and mPEG-NH 2 Mix at a mass ratio of 2:1, disperse by ultrasonic for 30 min, and stir at low temperature (4 °C) overnight;

[0047] (3) The mixed solution of step (2) was taken out, centrifuged at 14000 rpm for 20 min, and rinsed with milliq water to remove the unattached mPEG-NH 2 ;

[0048] (4) The nanocomposite obtained after centrifugation was mixed with R848 at a mass ratio of 1:1.5, ultrasonically dispersed for 30 min, and stirred at low temperature (4 °C) for 24 h;

[0049] (5) After the reaction, the mixed solution of step (4) was taken out, centrifuged at 14000 rpm for 20 min, and washed twice with methanol to remove free R848 to obtain drug-loaded manganese dioxide nanosheets (MnO 2 -R848).

[0050] Example 2. Nano-delivery system (MnO 2 -R848@CM) preparation method

[0051] The specific method is as follows:

[0052] (1) diluting a commercial manganese dioxide nanosheet dispersion (1 mg / ml) with milliq water to obtain a nanosheet solution with a concentration of 0.1 mg / ml;

[0053] (2) Mix the nanosheet solution and mPEG-NH 2 Mix at a mass ratio of 2:1, disperse by ultrasonic for 30 min, and stir at low temperature (4 °C) overnight;

[0054] (3) The mixed solution of step (2) was taken out, centrifuged at 14000 rpm for 20 min, and rinsed with milliq water to remove the unattached mPEG-NH 2 ;

[0055] (4) The nanocomposite obtained after centrifugation was mixed with R848 at a mass ratio of 1:1.5, ultrasonically dispersed for 30 min, and stirred at low temperature (4 °C) for 24 h;

[0056] (5) After the reaction, the mixed solution of step (4) was taken out, centrifuged at 14000 rpm for 20 min, and washed twice with methanol to remove free R848 to obtain drug-loaded nanosheets (MnO 2 -R848);

[0057] (6) Collect the digested 4T1 cells, centrifuge at 1500 rpm for 5 min, and discard the supernatant;

[0058] (7) Resuspend the cell pellet with PBS, centrifuge at 1500 rpm for 5 min, and discard the supernatant;

[0059] (8) preparing a hypotonic buffer solution (physiological saline: milliq water: PMSF = 75:25:1), resuspending the cell pellet obtained in step (7) with the hypotonic buffer solution and treating at 4°C for 1 h;

[0060] (9) Ultrasonicate the solution in (8) for 5 min using an ultrasonic disruptor (50% amplitude, 5 s on, 5 s off);

[0061] (10) The cell solution after ultrasound was centrifuged at 1500 rpm for 10 min and the precipitate was discarded to remove intracellular substances;

[0062] (11) The supernatant collected in step (10) was centrifuged at 17000 rpm for 1 h to collect the cell membrane fragments, which were then washed once with PBS and freeze-dried overnight to obtain 4T1 cancer cell membranes;

[0063] (12) The prepared 4T1 cancer cell membrane was mixed with the drug-loaded nanosheet (MnO 2 -R848) were mixed and suspended in milliq water at a mass ratio of 1:1 and ultrasonicated on ice for 0.5 h;

[0064] (13) The nanomaterial and cell membrane mixture of step (12) was extruded through 800 nm and 400 nm polycarbonate membranes 11 times each to obtain cancer cell membrane-coated drug-loaded nanosheets (R848-MnO 2 @CM), namely the nano delivery system.

[0065] The prepared MnO was characterized by dynamic light scattering (DLS) and Malvern particle analyzer. 2 、MnO 2 -R848 and MnO 2 -R848@CM particle size distribution and Zeta potential were measured. The results are as follows Figure 1 MnO 2 、MnO 2 -R848 and MnO 2 -R848@CM have average particle sizes of 126.82nm, 88.69nm, and 165.61nm, respectively; the Zeta potentials are -20.3mV, -15.67mV, and -21.5mV, respectively; MnO 2 The surface of the nanosheets is negatively charged, and the loading of R848 can mask its surface charge. 2 The increase in Zeta potential of the R848 group indicated the loading of R848; the surface of cancer cell membranes was negatively charged, compared with MnO 2 -R848 group, MnO 2 -R848@CM has a lower surface potential, which confirms the encapsulation of drug-loaded nanosheets by cancer cell membranes.

[0066] Example 3. MnO 2 -Evaluation of the in vitro radiotherapy effect of R848@CM nano-delivery system

[0067] 1. Experimental Methods

[0068] In order to evaluate the MnO 2-R848@CM nano-delivery system in vitro radiotherapy effect, this example conducted the following experiments. The specific method is as follows:

[0069] 1.1 In vitro cytotoxicity assay

[0070] In the cytotoxicity experiment, 4T1 cells were treated with different concentrations ([Mn = 0.1, 0.25, 0.5, 1, 2, 2.5, 5 and 10 μg / ml]) of MnO 2 、MnO 2 -R848 and MnO 2 -R848@CM were incubated for 24 hours, and the cell viability was determined by CCK8 method. 2 、MnO 2 -R848 and MnO 2 -R848@CM were incubated for 24 h and then irradiated with X-rays (6 Gy), and the cells were incubated for another 24 h, and then the cell viability was detected by the CCK8 method.

[0071] 1.2 Detection of reactive oxygen species (ROS) generation in vitro

[0072] 4T1 cells were first treated with PBS (Negative Control group), H 2 O 2 (Positive Control Group), MnO 2 、MnO 2 -R848 and MnO 2 -R848@CM ([Mn] = 5 μg / ml) was incubated for 12 h, and then H 2 The DCF-DA probe was incubated for 30 minutes. During in vitro radiotherapy, the cells were irradiated with 6 Gy of X-rays and incubated for 3 hours. The production of cellular ROS was measured using the Bio-Tech Reactive Oxygen Species Detection Kit (S0033S) with the aid of an inverted fluorescence microscope.

[0073] 1.3 In vitro cell apoptosis studies

[0074] 4T1 cells were first treated with PBS (Negative Control group), H 2 O 2 (Positive Control Group), MnO 2 、MnO 2 -R848 and MnO 2-R848@CM (

Mn

[0075] 1.4 In vitro DNA damage studies

[0076] 4T1 cells were first treated with PBS (Negative Control group), MnO 2 、MnO 2 -R848 and MnO 2 -R848@CM ([Mn] = 5 μg / ml) for 12 h. During in vitro radiotherapy, cells were irradiated with 6 Gy of X-rays and incubated for another 24 h. The cells were stained with 488-conjugated goat anti-rabbit IgG and DAPI and then imaged using a confocal microscope.

[0077] 1.5 In vitro immunogenic cell death study (ICD)

[0078] (1) In vitro detection of ATP production. 4T1 cells were first treated with PBS (Negative Control group), MnO 2 、MnO 2 -R848 and MnO 2 -R848@CM (

Mn

[0079] (2) In vitro detection of changes in intracellular HMGB1 content. 4T1 cells were first treated with PBS (Negative Control group), MnO 2 、MnO 2 -R848 and MnO 2 -R848@CM (

Mn

[0080] (3) In vitro detection of CRT expression. 4T1 cells were first treated with PBS (Negative Control group), MnO 2 、MnO 2 -R848 and MnO 2 -R848@CM ([Mn] = 5 μg / ml) for 12 h. During in vitro radiotherapy, cells were irradiated with 6 Gy of X-rays and incubated for another 24 h. The cells were stained with 488-conjugated goat anti-rabbit IgG and DAPI and then imaged using a confocal microscope.

[0081] 2. Experimental conclusion

[0082] Figure 2-3 The prepared MnO 2 -Evaluation results of in vitro radiotherapy effect of R848@CM nano-delivery system. Figure 2 As shown in A, mouse breast cancer cells (4T1) were used to test the MnO 2 -R848@CM was used for biocompatibility evaluation. 2 When the content was 10 μg / mL, the cell viability of 4T1 cells was still above 80%, indicating that it had good biocompatibility; however, after X-ray irradiation, the survival rate of 4T1 cells was significantly reduced. Figure 2 B DCFH-DA was used as a ROS probe to measure the intracellular ROS levels after different treatments. Compared with other groups, MnO 2 -R848@CM+X-ray treatment group can significantly promote the generation of ROS. Cell apoptosis experiment ( Figure 2 C) It was further confirmed that compared with the Control group (17.76%), MnO 2 -R848@CM+X-ray treatment group (34%) can increase the apoptosis rate of cancer cells and induce cancer cell death.

[0083] In addition, γ-H 2 AX was analyzed by immunofluorescence staining, and the results were as follows Figure 3 As shown in A, with MnO 2 Compared with R848@CM or X-ray irradiation alone, MnO 2 -R848@CM+X-ray treatment significantly increased the level of DNA damage in cancer cells, indicating that MnO 2 -R848@CM can increase the generation of ROS and induce significant DNA damage under X-ray irradiation, which is likely derived from the MnO 2The redox properties of MnO can alleviate the hypoxic microenvironment and provide the possibility for radiosensitization. In addition, studies have shown that ionizing radiation can cause immunogenic cell death (ICD) and trigger the release of damage-associated molecular patterns (DAMPs); when ICD occurs, ATP and HMGB1 are released from the nucleus to the extranucleus, and calreticulin (CRT) is translocated to the surface of cancer cells; therefore, MnO 2 -R848@CM nanoplatform and radiotherapy-induced ICD were evaluated, and the results were Figure 3 B, 3C and 3D show that with MnO 2 Compared with the R848@CM or X-ray treatment groups, MnO 2 -R848@CM+X-ray treatment enhanced the release of ATP and HMGB1; in addition, significant expression of CRT was observed on the extracellular surface of the combined treatment group. These results indicate that MnO 2 -R848@CM+X-ray irradiation treatment can significantly increase the level of immunogenic cell death and release damage-related molecular patterns such as ATP, HMGB1 and CRT.

[0084] Example 4. R848-MnO 2 Evaluation of the in vitro immune effect of @CM nano-delivery system

[0085] 1. Experimental Methods

[0086] In order to evaluate the MnO 2 -In vitro immune effect of R848@CM nano-delivery system, this example conducted the following experiments. The specific method is as follows

[0087] 1.1 In vitro determination of the maturation of bone marrow-derived dendritic cells (BMDCs)

[0088] First, BMDCs were obtained from the bone marrow of 8-week-old Balb / c mice and implanted into 12-well plates. Then, the BMDCs were mixed with PBS (Negative Control group), LPS (1 μg / mlL), MnO 2 、R848、MnO 2 -R848 and MnO 2 -R848@CM (

Mn

[0089] 1.2 Determination of BMDCs maturation under co-culture conditions

[0090] 4T1 cells were first treated with PBS (Negative Control group), MnO 2 、R848、MnO 2 -R848 and MnO 2 -R848@CM (

Mn

[0091] 2. Experimental results

[0092] The experimental results can be found in Figure 4 BMDCs were incubated with PBS, LPS (1 μg / mL), MnO 2 、R848、MnO 2 -R848 and MnO 2 -R848@CM were co-incubated to study the maturation of BMDCs in vitro. Figure 4 As shown in B, with MnO 2 Compared with the R848 (76.2%) and R848 (78%) treatment groups, the maturity of BMDCs after MnO2-R848@CM treatment was significantly improved to 84.3%, indicating that R848 and MnO 2 There is a certain synergistic effect in promoting the maturation of BMDCs; Elisa test further confirmed that MnO 2 -R848@CM also produced more inflammatory factor TNF-α in the corresponding culture supernatant, indicating that the STING pathway in BMDCs was effectively activated, resulting in a stronger anti-tumor immune response ( Figure 4 C) Since ionizing radiation-induced ICD can trigger the release of DAMPs, which in turn promotes the maturation of antigen-presenting cells-dendritic cells and increases the uptake of tumor antigens, activates T cells, and induces immune response, so Figure 4 D method to design the experiment, the results are as follows Figure 4 As shown in E, the BMDCs in the X-ray treated group were more mature (81.4%) compared with those in the group without X-ray irradiation (73.4%), while MnO 2 -R848@CM+X-ray synergistic treatment group showed a higher BMDCs maturation level (85.2%). The above conclusions indicate that MnO 2-R848@CM can not only act as a radiosensitizer but also has the potential to synergize radiotherapy immune activation.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. An immunotherapy nano-delivery system with synergistic radiotherapy, characterized in that: The nano delivery system comprises a manganese dioxide nanosheet and a cancer cell membrane coated outside the manganese dioxide nanosheet; the manganese dioxide nanosheet is loaded with an immune drug; The immune drug is R848; the cancer cell membrane is a breast cancer cell membrane; The preparation method of the nano delivery system comprises the following steps: (1) mixing the manganese dioxide nanosheet aqueous solution with mPEG-NH2, ultrasonicating, stirring, centrifuging, removing the supernatant to obtain a precipitate, and washing the precipitate to obtain a nanocomposite material; (2) mixing the nanocomposite material of step (1) with R848, ultrasonicating, stirring, centrifuging, removing the supernatant to obtain a precipitate, and washing the precipitate with an alcohol solvent to obtain drug-loaded manganese dioxide nanosheets; (3) Collect the digested breast cancer cells, centrifuge, discard the supernatant, resuspend the cell pellet with PBS, centrifuge, discard the supernatant, and obtain the cell pellet; (4) mixing physiological saline, water and PMSF to prepare a hypotonic buffer solution, wherein the mass ratio of the physiological saline, water and PMSF is 75:25:1; (5) resuspending the cell pellet from step (3) in the hypotonic buffer from step (4) for 1-1.5 h, then sonicating with an ultrasonic disruptor, centrifuging at 1000-2000 rpm for 5-10 min, and collecting the supernatant; (6) centrifuging the supernatant of step (5) at 15000-17000 rpm for 1-1.5 h, discarding the supernatant to collect the cell membrane fragments, washing with PBS, and freeze-drying to obtain breast cancer cell membranes; (7) suspending and mixing the breast cancer cell membrane and the drug-loaded manganese dioxide nanosheets of step (2) in water, and ultrasonicating in an ice bath to obtain a mixed solution; (8) extruding the mixed solution of step (7) through a polycarbonate membrane to obtain drug-loaded nanosheets coated with breast cancer cell membranes, i.e., the nano-delivery system; In the step (1), the mass ratio of the manganese dioxide nanosheet aqueous solution to mPEG-NH2 is 2:(0.5-1.5); In the step (2), the mass ratio of the nanocomposite material to R848 is 1:(1-2).

2. A method for preparing the nano delivery system according to claim 1, characterized in that: The following steps are involved: (1) mixing the manganese dioxide nanosheet aqueous solution with mPEG-NH2, ultrasonicating, stirring, centrifuging, removing the supernatant to obtain a precipitate, and washing the precipitate to obtain a nanocomposite material; (2) mixing the nanocomposite material of step (1) with R848, ultrasonicating, stirring, centrifuging, removing the supernatant to obtain a precipitate, and washing the precipitate with an alcohol solvent to obtain drug-loaded manganese dioxide nanosheets; (3) Collect the digested breast cancer cells, centrifuge, discard the supernatant, resuspend the cell pellet with PBS, centrifuge, discard the supernatant, and obtain the cell pellet; (4) mixing physiological saline, water and PMSF to prepare a hypotonic buffer solution, wherein the mass ratio of the physiological saline, water and PMSF is 75:25:1; (5) resuspending the cell pellet from step (3) in the hypotonic buffer from step (4) for 1-1.5 h, then sonicating with an ultrasonic disruptor, centrifuging at 1000-2000 rpm for 5-10 min, and collecting the supernatant; (6) centrifuging the supernatant of step (5) at 15000-17000 rpm for 1-1.5 h, discarding the supernatant to collect the cell membrane fragments, washing with PBS, and freeze-drying to obtain breast cancer cell membranes; (7) suspending and mixing the breast cancer cell membrane and the drug-loaded manganese dioxide nanosheets of step (2) in water, and ultrasonicating in an ice bath to obtain a mixed solution; (8) extruding the mixed solution of step (7) through a polycarbonate membrane to obtain drug-loaded nanosheets coated with breast cancer cell membranes, i.e., the nano-delivery system; In the step (1), the mass ratio of the manganese dioxide nanosheet aqueous solution to mPEG-NH2 is 2:(0.5-1.5); In the step (2), the mass ratio of the nanocomposite material to R848 is 1:(1-2).

3. The method for preparing the nano delivery system according to claim 2, characterized in that: In the step (7), the mass ratio of breast cancer cell membrane to drug-loaded manganese dioxide nanosheets is 1:(0.5-1.5).

4. Use of the nano delivery system as claimed in claim 1 in the preparation of drugs for treating tumors.

5. The use according to claim 4, characterized in that The tumor is breast cancer.

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