Preparation and application of self-assembled nano preparation for starting cancer immune cycle

Abstract

The invention belongs to the technical field of immunotherapy preparations, and particularly discloses a self-assembled nano preparation for starting cancer immune cycle, the main body of the self-assembled nano preparation is spherical particles jointly formed by catalase, lactate oxidase, sorafenib and chlorin e6, and the self-assembled nano preparation is prepared by adopting a'one-pot method '. The nano preparation disclosed by the invention has the capabilities of consuming lactic acid and generating oxygen and active oxygen. In a tumor microenvironment, the nano preparation is combined with a photodynamic therapy to effectively induce tumor cells to generate immunogenic death, promote activation and presentation of antigen presenting cells, collect a large number of immune effector cells in a tumor and reduce accumulation of immunosuppressive cells. And the nano preparation can be enriched to a tumor part in vivo within 2 hours, and has an obvious effect on inhibiting the growth of breast tumor cells in cooperation with a photodynamic therapy.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunotherapy preparations, and specifically relates to the preparation and application of a self-assembled nano preparation for initiating cancer immune circulation. Background Art

[0002] Effective activation of each event in the cancer immunity cycle (CIC) is a prerequisite for significant results in cancer immunotherapy. Insufficient presentation of tumor-associated antigens, limited tumor infiltration, and immune escape, influenced by the immunosuppressive tumor microenvironment, are the reasons for the failure of CIC. In clinical practice, immune checkpoint blockade therapy has shown exciting clinical value and strong therapeutic potential, but its response rate is relatively low due to the stagnation of CIC. Therefore, it is urgent to develop strategies targeting the suppressive tumor microenvironment to restart the activation of CIC to improve the anti-tumor efficacy.

[0003] Insufficient presentation of tumor-associated antigens is a typical feature of the tumor microenvironment. Immunogenic cell death is a special form of cell death that promotes the massive release of tumor-associated antigens from dying cells. Photodynamic therapy, as a common method of inducing immunogenic death, has received extensive attention in cancer treatment. However, due to the immunosuppressive effects of the tumor microenvironment, photodynamic therapy alone often has a low tumor clearance rate. Abnormal blood vessels and hypoxia in tumors severely hinder the activation and infiltration of T cells, indirectly leading to the failure of CIC. It has been reported that vascular endothelial growth factor inhibitors can induce tumor vascular normalization and significantly enhance the infiltration of immune cells in tumors, thereby improving the efficacy of immunotherapy. In addition, it has been observed clinically that the combination of vascular endothelial growth inhibitors and immune checkpoint blockade therapy can improve the clinical outcomes of a variety of cancers. In addition, lactate is a ubiquitous immunosuppressive molecule in the tumor microenvironment, which significantly impairs anti-tumor immunity by inhibiting effector T cells, promoting immune escape, and maintaining an acidic environment. Downregulating lactate production, interfering with lactate transporters, or directly consuming lactate can reprogram the immunosuppressive tumor microenvironment to enhance anti-tumor immunity. It has been reported that lactate transporter inhibitors significantly enhanced the therapeutic effect of immune checkpoint blockade therapy in a mouse breast cancer model. Lactate oxidase-based nanomedicines have been reported to reverse tumor immunosuppression and improve immunotherapy efficacy by reducing lactate concentrations in tumors. Although these strategies can alleviate immunosuppression to a certain extent, the anti-tumor immune response is a cyclic process, and limitations at each step may limit the generation of optimal immunotherapy effects. Therefore, targeting multiple key steps of CIC is needed to provide more powerful strategies that can reverse the immunosuppressive tumor microenvironment and trigger effective anti-tumor immunity. Summary of the invention

[0004] In order to solve the above technical problems, the present invention proposes a self-assembled nanopreparation for initiating a cancer immune cycle, the main body of which is spherical particles composed of catalase, lactate oxidase, sorafenib and dihydrochlorin e6, and is prepared by a "one-pot method". The prepared nanopreparation has a significant anti-tumor effect.

[0005] In order to achieve the above object, the present invention is achieved through the following technical solutions:

[0006] On the one hand, the present invention provides a self-assembled nanoformulation for initiating a cancer immune cycle, wherein the nanoformulation is a spherical particle composed of catalase, lactate oxidase, sorafenib and dihydrochlorin e6.

[0007] In a second aspect, the present invention provides a method for preparing the self-assembled nanoformulation for initiating cancer immune cycle, comprising the following steps:

[0008] The catalase solution was added to a sterile tube and placed in an ice bath. The lactate oxidase solution, the dihydrochlorin e6 solution, and the sorafenib solution were added under ultrasound. After thorough mixing, the nanoformulation was obtained by centrifugation, washing, and drying.

[0009] Preferably, the mass ratio of the catalase solution, the lactate oxidase solution, the sorafenib solution and the dihydrochlorin e6 solution is 6:1:0.43:0.33.

[0010] Preferably, the catalase solution, lactate oxidase solution, sorafenib solution and dihydrochlorin solution are mixed and reacted under ultrasonic conditions for 15 minutes, and the mixed solution is then centrifuged at 4° C. and a centrifugal force of 15,000*g for 10 minutes, washed twice with ultrapure water and then freeze-dried.

[0011] In a third aspect, the present invention provides the use of the nanoformulation prepared by the above method in anti-tumor treatment, wherein the nanoformulation promotes the infiltration of effector cells into the tumor, kills tumor cells, and reduces the number of immunosuppressive cells in the tumor.

[0012] The beneficial effects of the present invention are:

[0013] Compared with the prior art, the nanoformulation of the present invention is a spherical particle composed of catalase, lactate oxidase, sorafenib and dihydrochlorin e6, which has the ability to consume lactic acid and produce oxygen and reactive oxygen. In the tumor microenvironment, the nanoformulation combined with photodynamic therapy effectively induces immunogenic death of tumor cells, promotes activation and presentation of antigen-presenting cells, recruits a large number of immune effector cells in the tumor, and reduces the accumulation of immunosuppressive cells. And the nanoformulation of the present invention can be enriched in large quantities at the tumor site in vivo within 2 hours, and has a significant effect in synergizing with photodynamic therapy in inhibiting the growth of breast tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0015] Figure 1 The morphology characterization of the nanoformulation CLSC, where a) is the TEM morphology of CLSC, scale: 100 nm; b) is the particle size distribution diagram of CLSC.

[0016] Figure 2 The general performance characterization results of nano-preparation CLSC, where a) is the distribution diagram of the main elements of CLSC, scale: 20nm; b) is the UV-visible absorption spectrum of Ce6, SF and CLSC; c) is the consumption of lactic acid by CLSC and LOD; d) is the catalytic production of O2 by CLSC and CAT in the presence of H2O2; e) is the production of O2 by CLSC and Ce6 under 660nm laser irradiation 1 O2.

[0017] Figure 3 Schematic diagram of the biodistribution of CLSCs in vivo, where a) is the real-time fluorescence image of 4T1 tumor-bearing mice at different time points after intravenous injection of CLSCs; b) is the quantitative analysis of the fluorescence signal of CLSCs at the tumor site at different time points; c) is the in vitro fluorescence image of the main organs and tumors of 4T1 subcutaneous tumor-bearing mice 12 hours after administration.

[0018] Figure 4 Schematic diagram of the effect of CLSC-assisted photodynamic therapy on breast cancer, where a) is the change in mouse tumor volume (n=5); b) is the tumor weight on day 18 (n=5); c) is a picture of the tumor on day 18; d) is the survival curve of tumor-bearing mice in different treatment groups (n=8); the results are expressed as mean ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0019] Figure 5 Schematic diagram of CLSC-photodynamic therapy-induced anti-tumor immune response, where a) is a representative flow cytometry plot and quantitative analysis of immunoregulatory T cells; b) is a representative flow cytometry plot and quantitative analysis of CD8+T cells; the results are expressed as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. DETAILED DESCRIPTION

[0020] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0021] Catalase (CAT), lactate oxidase (LOD), sorafenib (SF), and chlorin (Ce6).

[0022] Example 1

[0023] Preparation and characterization of nanoformulated CLSC

[0024] 1) CLSCs were prepared by the one-pot method: 0.3 ml of CAT solution (7 mg / mL) was added to a 15 mL sterile tube and placed on ice. Then, 70 μL of LOD solution (5 mg / mL), 5.04 μL of Ce6 solution (38.8 mM) and 16.80 μL of SF solution (19.4 mM) were slowly added under ultrasound. The reaction mixture was ultrasounded for 15 min. After 15 min, the mixed solution was centrifuged (4 °C, 10 min, 15000 × g), and the CLSC precipitate was washed twice with ultrapure water. The CLSC precipitate was freeze-dried and weighed for subsequent experiments.

[0025] 2) Particle size determination: A 1.0 mg / mL CLSC solution was prepared and the particle size of the nanoformulation CLSC was determined using a Zetasizer Pro (Malvern).

[0026] 3) Morphology measurement: 10 μL of 10.0 mg / mL CLSC solution was dripped onto a 200-mesh carbon-coated copper grid. After standing for 1 min, the sample was aspirated along the edge of the copper grid with filter paper. Then 10 μL of ultrapure water was dripped. After standing for 1 min, the sample was aspirated along the edge of the copper grid with filter paper. The morphology was observed using a 120 kV transmission electron microscope (FEI Tecnai G2F30, America).

[0027] The experimental results are as follows Figure 1 shown. Figure 1 a shows that the nanoformulation CLSC is uniformly dispersed, with a particle size of approximately 223 nm; Figure 1 b shows that the morphology of the nanoformulated CLSC is a spherical structure with a diameter of about 150 nm. In summary, CLSC was successfully prepared by the one-pot method; under ultrasonic conditions, CLSC can be assembled into a stable and uniform spherical structure.

[0028] Example 2

[0029] Determination of encapsulation efficiency and drug loading of CLSC

[0030] 1) During the preparation of CLSCs, all centrifugation supernatants were saved and used to determine the encapsulation efficiency and drug loading of SF and Ce6 in CLSCs.

[0031] a. The content of free SF in the supernatant was determined by high performance liquid chromatography (HPLC, Waters e2695, USA). HPLC elution conditions: XBridge C18 column (5 μm, 4.6×250 mm); 60% B isocratic elution (Phase A: 100% water + 0.1% trifluoroacetic acid; Phase B: 20% water / 80% acetonitrile + 0.1% trifluoroacetic acid).

[0032] b. Measure the free Ce6 content in the supernatant at a wavelength of 400 nm using an ELISA reader

[0033] d. Calculation formula:

[0034] LE(%)=We / Wm×100%

[0035] Where LE represents the percentage of drug loading in the preparation; We represents the amount of drug encapsulated in the nanoformulation; and Wm represents the total weight of the drug-loaded nanoformulation.

[0036] EN%=(1-Cf / Ct)×100%

[0037] Where EN represents the encapsulation efficiency of the drug in the preparation. Its expression is: Where Cf is the amount of free drug; Ct is the total amount of nano preparation.

[0038] The experimental results are shown in Table 1. In CLSC, the encapsulation efficiency of SF was 92.64%, and the drug loading rate was 5.23%; the encapsulation efficiency of Ce6 was 84.82%, and the drug loading rate was 3.69%.

[0039] Table 1. Encapsulation efficiency and drug loading of sorafenib and chlorin in CLSCs

[0040]

[0041] Example 3

[0042] General performance characterization of nanoformulated CLSC

[0043] 1) Mapping analysis and UV absorption spectroscopy were used to determine that SF and Ce6 were successfully encapsulated in CLSC; 2) The ability of CLSC to consume lactic acid was examined using a lactic acid content determination kit; 3) The ability of CLSC to catalyze the production of oxygen was examined using a dissolved oxygen meter to monitor the oxygen content; 4) The ability of CLSC to promote the production of reactive oxygen species under 660nm light source was examined using a fluorescence spectrophotometer.

[0044] 1) Mapping analysis

[0045] 10 μL of 10.0 mg / mL CLSC solution was dripped on a 200-mesh carbon-coated copper grid. After standing for 1 min, the sample was aspirated along the edge of the copper grid with filter paper. Then 10 μL of ultrapure water was added. After standing for 1 min, the sample was aspirated along the edge of the copper grid with filter paper. The elemental morphologies of C, N, O, S, and F were collected using a transmission electron microscope.

[0046] 2) UV absorption spectrum

[0047] Ce6, SF and CLSC solutions were prepared at 9.23, 13.08 and 250 μg / mL, respectively, and the absorption spectra of Ce6, SF and CLSC were detected by UV spectrophotometer (Youke T-U7S). The best detection wavelengths of Ce6 and SF were about 400 nm and 260 nm, respectively.

[0048] 3) Lactic acid consumption

[0049] 26 μg of free LOD and CLSC containing an equal amount of LOD were dissolved in 2 mL of PBS solution and placed in a dialysis bag sealed at both ends. The dialysis bag was placed in a 15 mL centrifuge tube containing sodium lactate solution (5 mg / mL, 6 mL) and incubated in a shaker at 37 °C for 2 h. Subsequently, 20 μL of sodium lactate solution was collected from the centrifuge tube at 0, 10, 20, 30, 40, 60, and 90 min, and the lactate content was detected using a lactate assay kit.

[0050] 4) Production of oxygen

[0051] 182 μg of free CAT and CLSC containing an equal amount of CAT were dispersed in a hypoxic H2O2 solution (250 μM, 10 mL) and stirred at room temperature for 15 min. The concentration of dissolved oxygen in the solution containing CAT and CLSC was monitored by using a dissolved oxygen meter (Orion StarA213) with readings every 5 seconds.

[0052] 5) Production of singlet oxygen

[0053] The fluorescence intensity change of SOSG fluorescent probe at the detection wavelength of 530nm was used as the determination index of singlet oxygen generation. The operation was as follows: first, 2mL 100μg / mL CLSC solution and 2mL 3.69μg / mL Ce6 solution were prepared, and 5μL SOSG fluorescent probe (500μM) and 5μL H2O2 solution (100mM) were added to the solution respectively, and then the singlet oxygen generation was measured under near-infrared laser irradiation (660nm, 20mW / cm 2 ), and the fluorescence intensity of SOSG in CLSC and Ce6 solutions was measured using a fluorescence spectrophotometer (PerkinElmer LS55) every 30 seconds.

[0054] The experimental results are as follows Figure 2 shown. Figure 2 a shows the overlapping distribution of elements C, N, O, F, and S in CLSC, indicating that CAT, LOD, SF, and Ce6 are encapsulated in CLSC; Figure 2 b shows that CLSC has characteristic absorption peaks of SF and Ce6 at wavelengths of 285 nm and 400 nm, respectively, indicating that SF and Ce6 are encapsulated in CLSC; Figure 2 c shows that CLSCs continuously consumed lactate, and the concentration decreased from 57.2 mM to 28 mM within 90 min, which was similar to the free LOD; Figure 2 d shows that after adding CLSC, O2 in the H2O2 solution was slowly released, and the maximum O2 release amount was equal to that of free CAT, indicating that CLSC retained the catalytic performance of CAT; Figure 2 e shows that CLSC catalyzes the conversion of oxygen into 1 O2, indicating that CLSCs retain the photosensitizer properties of Ce6.

[0055] Example 4

[0056] In vivo biodistribution of CLSC

[0057] Small animal in vivo imaging was used to detect the enrichment of CLSCs in tumor sites and other organs after CLSCs were administered to mice via the tail vein.

[0058] The specific experimental methods are as follows:

[0059] 1) Three 5-6 week old female Balb / c mice were selected and injected subcutaneously on the left side of the back of each mouse with 2x 10 5 After the inoculation of 4T1 breast cancer cells, the mouse status and tumor growth were observed every 2 days.

[0060] 2) When the left tumor volume reaches 250mm 3 Around 24 hours after the operation, mice were injected with CLSC (25 mg / kg) via tail vein. Then, at 0, 2, 4, 6, 8, and 12 hours, the images were taken using a small animal in vivo imaging system ( Lumina III, PerkinElmer) to collect Ce6 fluorescence signals (excitation light: 670 nm; emission light: 720 nm). At the same time, after 12 hours, the mice were euthanized, and the liver, kidney, spleen, lung, and tumor tissues of the mice were collected, and the Ce6 fluorescence signals (excitation light: 670 nm; emission light: 720 nm) were collected using a small animal living imager.

[0061] The experimental results are as follows Figure 3 shown. Figure 3a and 3b show that CLSCs accumulate in mouse tumors after administration. From the results of quantitative analysis of fluorescence signals, it was found that the fluorescence signal of CLSCs first increased and then decreased over time. The fluorescence intensity of Ce6 was the highest at 2h, indicating that 2h after administration was the best time for 660nm light treatment. Figure 3 c shows that in the ex vivo tissue 12 hours after administration, CLSCs were clearly observed to accumulate in the tumor. This result suggests that CLSCs are significantly retained in the tumor, which is conducive to the anti-tumor effect of CLSCs.

[0062] Example 5

[0063] Effect of CLSC-assisted photodynamic therapy on breast cancer

[0064] The breast cancer model was used to evaluate the effect of CLSC treatment on tumor growth. First, a breast cancer mouse tumor model was established, and then the tumor-bearing mice were treated with CLSC. The tumor volume and survival cycle of the mice were monitored to evaluate the effect of CLSC in inhibiting breast cancer growth.

[0065] The specific experimental methods are as follows:

[0066] 1) Twenty 5-6 week old Balb / c female mice were selected and routinely raised in a barrier environment.

[0067] 2) 2x 10 5 4T1 breast cancer cells were used to observe the tumor growth of mice every 2 days.

[0068] 3) Wait until the tumor volume grows to 100mm 3 The mice were randomly divided into 4 groups: PBS, SF, CLSC, and CLSC(+). On days 0, 2, and 4 of the experimental period, each group of mice was injected with SF (1.30 mg / kg) and CLSC (25 mg / kg) via tail vein injection. The mice in the CLSC(+) group were illuminated by a 660 nm laser (0.5 W / cm 2 ) irradiated the tumor site for 5 min. Starting from day 0 of the experiment, the survival status of mice in each group was recorded every 2 days, and the tumor volume was calculated (tumor volume = 1 / 2 x the longest side of the tumor x the shortest side of the tumor x the shortest side of the tumor). At the same time, the tumor volume exceeded 1500mm 3 The end point of the experiment.

[0069] 4) After the experiment, the tumor tissues of mice in each group were collected, photographed and weighed

[0070] The experimental results are as follows Figure 4 shown. Figure 4a shows that compared with the PBS group, SF treatment had no significant inhibitory effect on mouse tumor growth, CLSC treatment had a slight inhibitory effect on tumor growth, and CLSC(+) treatment significantly inhibited the growth of breast cancer. Figure 4 bc showed that 18 days after administration, the average tumor weight of the CLSC(+) group was only 246.4 mg, which was 6.78, 5.70 and 4.34 times less than that of the PBS, SF and CLSC-treated groups, respectively. Figure 4 d shows that CLSC(+) significantly prolonged the survival period of tumor-bearing mice. The median survival time after CLSC(+) treatment was 25.5 days. In contrast, mice in the PBS, SF and CLSC groups all reached the humane endpoint within 20 days. In summary, it was found that CLSC synergistic photodynamic therapy effectively inhibited the growth of breast cancer and the effect was significant.

[0071] Example 6

[0072] CLSCs synergistically induce anti-tumor immune response through photodynamic therapy

[0073] Based on the obvious inhibitory effect of CLSC-synergistic photodynamic therapy on breast cancer growth, the anti-tumor growth mechanism of CLSC-synergistic photodynamic therapy was further studied.

[0074] The specific experimental methods are as follows:

[0075] 1) Twelve 5-6 week old Balb / c female mice were selected and routinely raised in a barrier environment.

[0076] 2) 2x 10 5 4T1 breast cancer cells were used to observe the tumor growth of mice every 2 days.

[0077] 3) Wait until the tumor volume grows to 100mm 3 The mice were randomly divided into 4 groups: PBS, SF, CLSC, and CLSC(+). On days 0, 2, and 4 of the experimental period, each group of mice was injected with SF (1.30 mg / kg) and CLSC (25 mg / kg) via tail vein injection. The mice in the CLSC(+) group were illuminated by a 660 nm laser (0.5 W / cm 2 )Irradiate the tumor site for 5 minutes.

[0078] 4) On the fifth day of the experimental cycle, all mice were killed by dislocation, and the mouse tumors were collected and cut into pieces of 2-4 mm in size to obtain tumor single cell suspensions as follows:

[0079] a) Tumors were dissociated into single cell suspension using a tumor dissociation kit (RWD DHTE-5001) and a single cell suspension preparation instrument (RWDDSC-400).

[0080] b) Filter the cell suspension using a 70 μm cell sieve and centrifuge (450 g, 5 min).

[0081] c) The cells were resuspended in red blood cell lysis buffer, placed at room temperature for 5 min, and centrifuged (450 g, 5 min).

[0082] d) Use the cell viability dye FVS 510 (1x PBS dilution, dilution ratio 1:1000) for staining at room temperature in the dark for 15 minutes. Gently wash the cells 3 times with 1% FBS / PBS and centrifuge (450g, 5min).

[0083] e) The cells were resuspended with rabbit anti-mouse CD16 / 32 antibody (1% FBS / PBS dilution, dilution ratio 1:100), incubated on ice for 10 min in the dark, and centrifuged (450 g, 5 min).

[0084] f) The cells were resuspended with rabbit anti-mouse CD45 (diluted with 1% FBS / PBS, dilution ratio 1:200), rabbit anti-mouse CD3 (diluted with 1% FBS / PBS, dilution ratio 1:200), rabbit anti-mouse CD4 (diluted with 1% FBS / PBS, dilution ratio 1:200), rabbit anti-mouse CD8 (diluted with 1% FBS / PBS, dilution ratio 1:200) and rabbit anti-mouse CD25 (diluted with 1% FBS / PBS, dilution ratio 1:200) antibodies in sequence, incubated on ice in the dark for 10 min, and centrifuged (450g, 5 min).

[0085] g) The cells were gently washed three times with 1% FBS / PBS, resuspended in 1% FBS / PBS, and analyzed by flow cytometry (BD FACS Aria II). Suppressive regulatory T cells (Treg): CD4 + CD25 + ; Effector cells (CD8 + T): CD3 + CD8 + .

[0086] The experimental results are as follows Figure 5 shown. Figure 5 a shows that the number of Treg cells in the tumor of the CLSC(+) group was significantly reduced, and the number of Treg cells decreased by 49.3% compared with that of the PBS group. Figure 5b shows that the proportion of CD8+T cells in CLSC(+) tumors increased significantly, which was 10.6, 5.51, and 3.79 times that of PBS, SF, and CLSC tumors, respectively. In summary, it was found that CLSC-synergistic photodynamic therapy can reduce the infiltration of immunosuppressive cells and increase the infiltration of immune effector cells. These findings support the view that CLSC-synergistic photodynamic therapy can reverse the tumor immunosuppressive microenvironment to activate antitumor immunity.

[0087] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all details in detail, nor do they limit the invention to only the specific embodiments described. Obviously, many modifications and changes can be made according to the content of this specification.

Claims

1. A self-assembled nanoformulation for initiating cancer immunity cycle, characterized in that: The nano preparation is spherical particles composed of catalase, lactate oxidase, sorafenib and dihydrochlorin e6.

2. The method for preparing the self-assembled nanoformulation for initiating cancer immune cycle according to claim 1, characterized in that: The method comprises the following steps: adding a catalase solution into a sterile tube and placing it in an ice bath, adding a lactate oxidase solution, a dihydrochlorin e6 solution and a sorafenib solution under ultrasound, mixing thoroughly, centrifuging, washing and drying to obtain a nano preparation.

3. The method for preparing the self-assembled nanoformulation for initiating cancer immune cycle according to claim 2, characterized in that: The mass ratio of the catalase solution, the lactate oxidase solution, the sorafenib solution and the dihydrochlorin e6 solution is 6:1:0.43:0.

33.

4. The method for preparing the self-assembled nanoformulation for initiating cancer immune cycle according to claim 3, characterized in that: The catalase solution, lactate oxidase solution, sorafenib solution and dihydrochlorin e6 solution were mixed and reacted for 15 minutes under ultrasonic conditions, and then the mixed solution was centrifuged at 4° C. and a centrifugal force of 15000*g for 10 minutes, washed twice with ultrapure water and then freeze-dried.

5. Use of the self-assembled nanoformulation for initiating cancer immune cycle prepared according to any one of claims 2 to 4 in anti-tumor treatment, wherein the nanoformulation promotes the infiltration of effector cells into the tumor, kills tumor cells, and reduces the number of immunosuppressive cells in the tumor.

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