A CpG-ODN modified DNA tetrahedron-shikonin complex, its preparation method and application

Abstract

The present invention provides a CpG-ODN modified DNA tetrahedron-shikonin complex, its preparation method and application, belonging to the field of biomedical technology. This complex solves the disadvantages of poor water solubility and poor in vitro stability of shikonin. At the same time, this complex can effectively induce the ICD effect, increase the infiltration of T cells in tumors, and promote the secretion of anti-tumor cytokines, and has good application prospects in the treatment of triple-negative breast cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and specifically to a CpG-ODN modified DNA tetrahedron-shikonin complex, a preparation method thereof, and an application thereof. Background Art

[0002] Female breast cancer is a malignant tumor with extremely great harm, and is a major public health problem in China and even globally, seriously affecting the health and life expectancy of women. Among them, triple negative breast cancer (TNBC) refers to breast cancer in which all three receptors are negative. The three receptors are estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor-2 (HER-2). TNBC accounts for about 15% to 20% of all breast cancers. Due to the lack of effective treatment targets, higher recurrence rate, and greater metastasis potential, the survival prognosis of patients is extremely poor. For many years, chemotherapy has been the standard treatment method for TNBC, but the overall effect is not good, and it is easy to develop drug resistance, recurrence, and metastasis. The median survival period is only 12 to 18 months. Therefore, the treatment of TNBC is very difficult, and it is urgent to find more effective new strategies and new models for the treatment of TNBC.

[0003] The latest clinical application of cancer immunotherapy has brought hope for the innovative treatment of TNBC. However, the "cold tumor" microenvironment of TNBC lacks immune cell infiltration and insufficient tumor immunogenicity, resulting in the still difficult cure of TNBC. These key facts remind us that there is an urgent need for better innovative immunotherapies to extend the survival period of TNBC patients and achieve the cure of TNBC.

[0004] Shikonin (SK) is the main active ingredient isolated from the dried roots of the traditional Chinese medicine Lithospermum erythrorhizon, and its chemical formula is C 16 H 16 O5, and the structure is as follows:

[0005] ; In recent years, studies have found that shikonin has significant anti-tumor biological activities. More importantly, shikonin can trigger immunogenic cell death (ICD) and promote anti-tumor immune effects. The main characteristics of ICD are the exposure and release of a large number of damage-associated molecular patterns (DAMPs), including calreticulin (CRT), high-mobility group protein B1 (HMGB1), and adenosine triphosphate (ATP), etc. These molecules contribute to the differentiation and maturation of dendritic cells (DCs), and further promote the activation, proliferation, and tumor infiltration of cytotoxic T lymphocytes (CTLs). Finally, ICD can enhance the immune response and trigger a specific immune response against tumor antigens. However, due to the non-selective toxicity and low water solubility of shikonin, its use in anti-tumor treatment is limited.

[0006] Unmethylated cytosine-phosphate-guanine oligonucleotide (CpG-ODN, simply referred to as CpG) is a very promising immune adjuvant with the sequence: 5’-TCCATGACGTTCCTGACGTT-3’. CpG can activate the host defense mechanism of mammals, stimulate a variety of immune cells to trigger strong innate and adaptive immune responses. CpG can be specifically recognized by Toll-like receptor 9 (TLR9) expressed by dendritic cells, B cells, macrophages, etc., and stimulate these cells to produce a variety of pro-inflammatory cytokines, effectively improving the tumor immunosuppressive microenvironment and promoting T cell-mediated immune responses. CpG has been identified as a safe and effective immune adjuvant in basic research and clinical trials and is widely used in cancer immunotherapy. However, free CpG is difficult to be absorbed by cells and is easily degraded by nucleases. Loading CpG into target cells through a suitable drug delivery system will be beneficial to the exertion of the CpG immune enhancement effect.

[0007] Simultaneous delivery of different types of drugs requires a multifunctional drug delivery system to achieve synergistic therapy. In the past few decades, DNA nanotechnology has developed rapidly, and researchers have constructed 2D and 3D structures of various sizes or shapes, and these DNA nanostructures have been widely used in biomedical research. Among these DNA nanostructures, DNA tetrahedron (DNAtetrahedron, DT) has the advantages of excellent biocompatibility, negligible toxicity, programmability, good stability, and easy cellular uptake, providing an ideal platform for the targeted delivery of anticancer drugs. Currently, there is no relevant report on the simultaneous delivery of shikonin and CpG using DNA tetrahedron. Whether DNA tetrahedron can be used to simultaneously deliver shikonin and CpG and whether it can exert a better synergistic therapeutic effect of shikonin and CpG requires further research. Summary of the Invention

[0008] The present invention provides a CpG-ODN modified DNA tetrahedron-shikonin complex, its preparation method and application. This complex solves the disadvantages of poor water solubility and poor in vitro stability of shikonin. At the same time, this complex can effectively induce the ICD effect, increase T cell infiltration in tumors, and promote the secretion of anti-tumor cytokines, and has good application prospects in the treatment of triple-negative breast cancer.

[0009] The technical solution of the present invention is as follows:

[0010] A CpG-ODN modified DNA tetrahedron-shikonin complex, comprising a CpG-ODN modified DNA tetrahedron and shikonin.

[0011] Preferably, the CpG-ODN modified DNA tetrahedron is self-assembled and synthesized from four DNA single strands, and the nucleotide sequences of the four DNA single strands are shown in SEQ ID NOs. 1-4 respectively.

[0012] More preferably, the CpG-ODN is modified with phosphorothioate.

[0013] Preferably, the molar ratio between the four DNA single strands is 1:1:1:1.

[0014] Preferably, the molar ratio between the CpG-ODN modified DNA tetrahedron and shikonin is 1:40-200.

[0015] More preferably, the molar ratio between the CpG-ODN modified DNA tetrahedron and shikonin is 1:80.

[0016] Preferably, the synthesis method of the CpG-ODN modified DNA tetrahedron comprises the following steps: adding the four DNA single strands into a solvent, maintaining at 85 °C for 5 min, maintaining at 60 °C for 5 min, and maintaining at 4 °C for more than 30 min.

[0017] Preferably, the solvent is one or more of TM buffer, PBS buffer, and DMSO.

[0018] The preparation method of the CpG-ODN modified DNA tetrahedron-shikonin complex comprises the following steps: adding the CpG-ODN modified DNA tetrahedron and shikonin into a solvent to obtain a mixed solution, reacting the mixed solution, and ultrafiltering.

[0019] Preferably, the solvent is one or more of TM buffer, PBS buffer, and DMSO.

[0020] Preferably, the reaction conditions are: oscillating reaction at 200-400 rpm for 4-6 h at 25-30 °C.

[0021] Preferably, the concentration of the CpG-ODN modified DNA tetrahedron in the mixed solution is 1-20 μM.

[0022] Preferably, the ultrafiltration conditions are: ultrafiltration using a 30 kDa molecular weight membrane.

[0023] Application of the CpG-ODN modified DNA tetrahedron-shikonin complex in the preparation of a drug for treating triple-negative breast cancer.

[0024] Preferably, the CpG-ODN modified DNA tetrahedron-shikonin complex treats triple-negative breast cancer by inducing ICD effect, increasing T cell infiltration in tumors, and promoting the secretion of anti-tumor cytokines.

[0025] Beneficial effects:

[0026] (1)The CpG-ODN modified DNA tetrahedron-shikonin complex provided by the present invention improves the water solubility and in vitro stability of shikonin.

[0027] (2)The CpG-ODN modified DNA tetrahedron-shikonin complex provided by the present invention can effectively induce the ICD effect, increase T cell infiltration in tumors, and promote the secretion of anti-tumor cytokines, and has good application prospects in the treatment of triple-negative breast cancer.

[0028] (3)The preparation method of the CpG-ODN modified DNA tetrahedron-shikonin complex provided by the present invention is simple and the preparation process is controllable. Description of the drawings

[0029] Figure 1 Synthesis schematic diagram and characterization results of CpG-DT-SK. Among them: Figure A is the synthesis schematic diagram of CpG-DT-SK; Figure B is the native PAGE detection result of CpG-DT; Figure C is the result of fluorescence competitive quenching experiment; Figure D is the ultraviolet-visible absorption spectra of CpG-DT, SK and CpG-DT-SK; Figure E is the native PAGE detection results of CpG-DT and CpG-DT-SK; Figure F is the ultraviolet absorbance values of total SK (80 μM) and free SK at 595 nm; Figure G is the average particle size of CpG-DT and CpG-DT-SK; Figure H is the AFM image result of CpG-DT-SK.

[0030] Figure 2 Water solubility and in vitro stability test results of CpG-DT-SK. Among them: Figure A is the picture of CpG-DT, CpG-DT-SK and free SK dissolved in TM buffer respectively; Figure B is the native PAGE detection results of CpG-DT and CpG-DT-SK co-incubated with 10% FBS for 0 h, 4 h, 8 h, 12 h, 20 h; Figure C is the semi-quantitative analysis result of the structural stability of CpG-DT and CpG-DT-SK by relative band intensity.

[0031] Figure 3Results of cell uptake and cytotoxicity tests of CpG-DT-SK. Among them, Figure A shows the laser scanning confocal microscopy images of cell uptake of CpG-DT and CpG-DT-SK by 4T1 cells; Figure B shows the analysis of the cell uptake rate of CpG-DT and CpG-DT-SK by 4T1 cells by flow cytometry; Figure C shows the fluorescence quantitative analysis of the cell uptake rate of CpG-DT and CpG-DT-SK by 4T1 cells; Figure D shows the cytotoxicity of CpG-DT, SK, and CpG-DT-SK to 4T1 cells.

[0032] Figure 4 Results of in vitro ICD induction and immunostimulatory activities of CpG-DT-SK. Among them: Figure A shows the immunofluorescence images of CRT in 4T1 cells of each treatment group; Figure B shows the quantitative determination results of ATP release in 4T1 cells of each treatment group; Figure C shows the quantitative determination results of HMGB1 release in 4T1 cells of each treatment group; Figure D shows the representative flow cytometry images of DC maturation in each treatment group; Figure E shows the results of ELISA kit determination of TNF-α secreted by DC; Figure F shows the results of ELISA kit determination of IFN-γ secreted by DC.

[0033] Figure 5 Results of biodistribution and anti-tumor activity determination in 4T1 tumor-bearing mice. Among them: Figure A shows the schematic timeline of the treatment experiment of 4T1 tumor-bearing mice; Figure B shows the in vivo fluorescence images at 1 h and 6 h after injecting Cy5-labeled CpG / SK, CpG-DT, and CpG-DT-SK into 4T1 tumor-bearing mice; Figure C shows the tumor growth curves of mice in each treatment group; Figure D shows the tumor tissue photos of mice in each treatment group; Figure E shows the tumor weights of mice in each treatment group; Figure F shows the H&E and TUNEL staining images of tumor tissue sections of mice in each treatment group.

[0034] Figure 6 Determination of in vivo anti-tumor immunostimulatory activity of CpG-DT-SK. Among them: Figure A shows the representative flow cytometry images of CD4 + and CD8 + cells in the spleens of mice in each treatment group; Figure B shows the representative immunofluorescence images of CD4 + and CD8 + cells in tumor tissue sections of mice in each treatment group; Figure C shows the ELISA results of serum TNF-α of mice in each treatment group; Figure D shows the ELISA results of serum IFN-γ of mice in each treatment group.

[0035] Figure 7 Results of biosafety evaluation of CpG-DT-SK. Among them: Figure A shows the body weight curves of mice in each treatment group; Figure B shows the representative H&E staining images of tissue sections of the heart, liver, spleen, lung, and kidney of mice in each treatment group. Detailed implementation methods

[0036] The following is an illustration with specific embodiments:

[0037] The raw materials and equipment used in the following embodiments are all known products and are obtained by purchasing commercially available products.

[0038] Example 1: Preparation of CpG-ODN modified DNA tetrahedron-shikonin complex (CpG-DT-SK)

[0039] The synthesis schematic diagram of CpG-DT-SK is as shown in Figure A in Figure 1 and the specific method is as follows:

[0040] I. Synthesis of CpG-ODN modified DNA tetrahedron (CpG-DT)

[0041] Four DNA single strands S1, S2, S3, and S4 in Table 1 below are used to synthesize CpG-DT. The specific operation steps are as follows: Mix equimolar amounts of the four DNA single strands evenly in TM buffer (10 mM Tris, 5 mM MgCl2, pH 8.0) so that the final concentration of each DNA single strand is 1 μM to obtain a mixed solution; Place the mixed solution in a PCR instrument and set the following program for reaction: Maintain at 85°C for 5 min, 60°C for 5 min, and 4°C for 30 min; Obtain a CpG-ODN modified DNA tetrahedron (CpG-DT) solution.

[0042] Table 1. Nucleotide sequences of the four DNA single strands (“*” represents phosphorothioation, and the underlined part is the CpG sequence)

[0043]

[0044] II. Preparation of CpG-ODN modified DNA tetrahedron-shikonin complex (CpG-DT-SK)

[0045] Add shikonin (SK) with a concentration of 10 mM dissolved in DMSO to the above-prepared CpG-DT solution and mix evenly so that the final concentration of SK is 80 μM and the final concentration of CpG-DT is 1 μM to obtain a mixed solution; Incubate the mixed solution at 25°C with shaking at 300 rpm for 6 h, and then use a 30K MWCO ultrafiltration tube (Millipore, USA) to centrifuge at 5000 rpm for 10 min to remove residual SK and DNA single strands; Add TM buffer to the centrifuged ultrafiltration tube to obtain a CpG-ODN modified DNA tetrahedron-shikonin complex (CpG-DT-SK) solution.

[0046] The synthesized CpG-DT-SK solution was concentrated by centrifugation at 5000 rpm for 10 min using a 30K MWCO ultrafiltration tube. PBS buffer was added to the concentrated solution in the ultrafiltration tube after centrifugation, and the concentration of CpG-DT-SK was measured using a NanoDrop Lite (Thermo Fisher Scientific, USA) ultra-micro spectrophotometer.

[0047] III. Characterization of CpG-DT-SK

[0048] (1) The successful preparation of the above CpG-DT was verified by native PAGE (non-denaturing polyacrylamide gel). The specific method was as follows: DNA single strand S1, S1+S2 (the same synthesis method as CpG-DT), S1+S2+S3 (the same synthesis method as CpG-DT), and the synthesized CpG-DT were electrophoresed through 8% native PAGE respectively, and the sizes of the electrophoretic bands were observed. The results are as Figure 1 shown in Figure B of [reference]. It can be seen from Figure B that the band size of CpG-DT meets the expectation, indicating the successful preparation of CpG-DT.

[0049] (2) The binding of SK to CpG-DT was further demonstrated by a fluorescence competition quenching experiment. The specific method was as follows: Gelred fluorescent dye was added to 200 μL of a CpG-DT solution with a concentration of 1 μM, and after mixing evenly, it was incubated for 1 h; different amounts of SK were added continuously to make the final concentrations of SK 0 μM, 40 μM, 80 μM, 120 μM, 160 μM, and 200 μM respectively. After mixing evenly, it was incubated for another 6 h; after the incubation was completed, the fluorescence spectrum between 450 - 800 nm was measured using a microplate reader (Varioskan LUX, Thermo Fisher, USA) with an excitation light of 300 nm. The results are as Figure 1 shown in Figure C of [reference]. It can be seen from Figure C that the fluorescence intensity of GelRed (λex = 300 nm) decreases with the increase of SK concentration, indicating that the binding of GelRed to CpG-DT is replaced by SK, and SK binds to CpG-DT in an intercalating manner.

[0050] (3) The successful preparation of the above CpG-DT-SK was verified by ultraviolet-visible absorption spectroscopy. The results are as Figure 1 shown in Figure D of [reference]. It can be seen from Figure D that the characteristic absorption peaks of CpG-DT-SK have both CpG-DT (260 nm) and SK (500 - 600 nm), indicating that SK was successfully loaded onto CpG-DT.

[0051] (4) The band sizes of CpG-DT and CpG-DT-SK in the gel were verified by native PAGE. The results are asFigure 1 As shown in Figure E. It can be seen from Figure E that the migration rates of CpG-DT and CpG-DT-SK in the gel are very close, indicating that the shapes and sizes of CpG-DT and CpG-DT-SK are very similar.

[0052] (5) The loading rate of SK by CpG-DT was determined using a UV-visible spectrophotometer. The specific method was as follows: SK was added to a CpG-DT solution with a concentration of 1 μM such that the final concentration of SK was 80 μM to obtain a mixture. After mixing evenly, the mixture was incubated at 25 °C for 6 h. Free SK was removed by centrifugation using a 30K MWCO ultrafiltration tube. Finally, the absorbance at 595 nm was measured using a UV-visible spectrophotometer (Genesys 50, Thermo Fisher, USA), and the loading rate was calculated according to the following formula:

[0053]

[0054] In the formula, "total SK absorbance" represents the absorbance value of 80 μM SK at 595 nm, and "free SK absorbance" represents the absorbance value of SK not bound to CpG-DT at 595 nm.

[0055] The results are shown in Figure 1 Figure F. It can be seen from Figure F that the total SK absorbance value was 0.635 and the free SK absorbance value was 0.228. Calculated by the above loading rate calculation formula, the loading rate of CpG-DT for SK was approximately 64%. After conversion, the molar ratio between CpG-DT and SK in CpG-DT-SK was 1:51.

[0056] (6) The average particle sizes of CpG-DT and CpG-DT-SK were measured using a dynamic light scattering nanoparticle sizer (919s, Opptronix, China). The results are shown in Figure 1 Figure G. It can be seen from Figure G that the hydrated particle sizes of CpG-DT and CpG-DT-SK were 11.9 nm and 12.5 nm, respectively.

[0057] (7) The morphology and size of CpG-DT-SK were further confirmed by atomic force microscopy (AFM) (MultiMode 8, Bruker, Germany)). The AFM image results are shown in Figure 1 Figure H. It can be seen from Figure H that CpG-DT-SK was monodisperse nanoparticles and the size was as expected.

[0058] Test Example 1: Water solubility and in vitro stability of CpG-DT-SK

[0059] I. Water solubility test

[0060] Prepare CpG-DT solution and CpG-DT-SK solution respectively according to the method of Example 1. Among them, adjust the concentration of CpG-DT-SK solution so that the final concentration of SK in CpG-DT-SK is 80 μM; at the same time, add free SK with a final concentration of 80 μM to 100 μL of TM buffer as a control group. After mixing evenly, place for 24 h, and observe the uniformity of each mixed solution.

[0061] The results are as Figure 2 shown in Figure A in

[0062] II. In vitro stability test

[0063] Mix equimolar amounts of CpG-DT and CpG-DT-SK with DMEM medium containing 10% FBS (fetal bovine serum) at a volume ratio of 1:4, and then incubate at 37 °C for 0 h, 4 h, 8 h, 12 h, 20 h; finally, analyze serum stability by 8% native PAGE.

[0064] The results are as Figure 2 shown in Figure B in Figure 2 From Figure B, it can be obtained that when incubating for 0-12 h, both CpG-DT and CpG-DT-SK show clear single bands in the gel, indicating that both CpG-DT and CpG-DT-SK have certain tolerance to 10% fetal bovine serum. The semi-quantitative analysis results of the relative band intensity are as

[0065] shown in Figure C in

[0066] I. Cellular uptake

[0067] Couple Cy5 to the S1 strand, and synthesize Cy5-labeled CpG-DT and CpG-DT-SK by the same method as in Example 1. Add 5×10 54T1 cells (mouse triple-negative breast cancer cells) were seeded into 35-mm confocal dishes and cultured at 37 °C for 12 h. Cell culture medium without any drugs (Control), Cy5-labeled CpG-DT, and CpG-DT-SK (SK concentration: 1020 nM, CpG-DT concentration: 20 nM) were added to the confocal dishes containing 4T1 cells, respectively, and co-incubated with the cells for 6 h. Then the cells were rinsed with PBS buffer and fixed with 4% paraformaldehyde for 20 min. Subsequently, Hoechst 33342 fluorescent dye at a concentration of 100 nM was added to stain the cell nuclei for 10 min, and LysoTracker Green Probe at a concentration of 50 nM was added to stain the lysosomes for 20 min. After staining, the cells were washed with PBS buffer to remove the unreacted fluorescent dyes. Imaging was performed using a laser scanning confocal microscope (FV3000, Olympus, Japan).

[0068] The imaging results are shown in Figure 3 Figure A in

[0069] Flow cytometry (FCM) was used to quantitatively evaluate cell uptake. 4T1 cells were cultured in 6-well plates at 37 °C for 12 h. Then cell culture medium without any drugs (Control), Cy5-labeled CpG-DT, and CpG-DT-SK (SK concentration: 1020 nM, CpG-DT concentration: 20 nM) were added to the 6-well plates containing 4T1 cells, respectively. After co-incubation with the cells for 6 h, single-cell suspensions were prepared using trypsin digestive enzyme, and the samples were examined using a flow cytometer (CytoFLEX, Thermo Fisher, USA).

[0070] The flow cytometry results are shown in Figure 3 Figure B in Figure 3 which is consistent with the results in Figure A in Figure 3 Figure C in p (<0.0001; ns, no statistical difference). It can be seen from Figure C that there is no statistical difference in the mean fluorescence intensity of 4T1 cells treated with CpG-DT and CpG-DT-SK, indicating that loading SK with CpG-DT does not affect its cell uptake.

[0071] II. Cytotoxicity

[0072] The 4T1 cells were seeded in a 96-well plate at a density of 1×10 4 cells / well and cultured overnight. CpG-DT, SK, and CpG-DT-SK were added and co-incubated with the cells. The concentrations of SK were 0.5 μM, 1.0 μM, 1.5 μM, 2.0 μM, 3.0 μM, and 4.0 μM, respectively, and three parallel experiments were performed for each concentration. After incubation for 24 h, CCK8 reagent was added and incubation continued for 2 h to obtain the cell culture medium. The absorbance of the cell culture medium at 450 nm was measured using a microplate reader (Varioskan LUX, Thermo Fisher, USA), and the cell viability was calculated according to the following formula:

[0073]

[0074] In the formula, "As" represents the absorbance of the experimental well (the absorbance of the well containing the test sample, cells, medium, and CCK-8 reagent), "Ab" represents the absorbance of the blank well (the absorbance of the well containing the medium and CCK-8 reagent), and "Ac" represents the absorbance of the control well (the absorbance of the well containing cells, medium, and CCK-8).

[0075] The results are shown in Figure 3 Figure D (* p <0.05, ** p <0.01; ns, no statistical difference). As can be seen from Figure D, since CpG-DT delivers more SK into 4T1 cells, CpG-DT-SK exhibits enhanced cytotoxicity compared with SK. In addition, CpG-DT did not show cytotoxicity to 4T1 cells, indicating its excellent biocompatibility as a carrier.

[0076] Test Example 3: Detection of in vitro ICD induction by CpG-DT-SK and in vitro maturation of BMDC

[0077] I. In vitro ICD induction study

[0078] The ability of CpG-DT-SK to induce the ICD cascade was determined, including the observation of CRT exposure and the measurement of ATP and HMGB1 release. The specific methods are as follows:

[0079] (1) Exposure of CRT: The 4T1 cells were seeded in a 96-well plate at a density of 1×10 5Cells were seeded into a 12-well plate at a density of 1×10

[0080] per well and cultured overnight at 37°C. Cell culture medium without any drugs (Control), CpG-DT, SK, and CpG-DT-SK (SK concentration was 1.5 μM) were added and co-incubated for 24 h. Cells were harvested, fixed with 4% paraformaldehyde for 20 min, and incubated with 5% FBS for 60 min. Subsequently, the cells were incubated with an Alexa Fluor 488-labeled CRT antibody (Abcam, UK) for 2 h at room temperature, and then the cell nuclei were stained with Hoechst 33342 fluorescent dye for 10 min. After staining, the cells were washed with PBS buffer to remove unreacted fluorescent dye. Imaging was performed using a laser scanning confocal microscope. Figure 4 The imaging results are shown in Figure A of

[0081] . It can be seen from Figure A that CpG-DT-SK can enhance CRT exposure. 5 (2)Release of ATP and HMGB1: 4T1 cells were seeded into a 12-well plate at a density of 1×10

[0082] per well and cultured overnight at 37°C. Cell culture medium without any drugs (Control), CpG-DT, SK, and CpG-DT-SK (SK concentration was 1.5 μM) were added and co-incubated for 24 h. The culture medium was harvested, and the concentrations of ATP and HMGB1 in the culture medium were measured using an ATP content detection kit (Beyotime, China) and an HMGB1 ELISA kit (Sango Biotech, China), respectively. Figure 4 The measurement results of the ATP concentration are shown in Figure B of Figure 4 , and the measurement results of the HMGB1 concentration are shown in Figure C of p (* p <0.05; ***

[0083] <0.001; ns, no statistical difference). It can be seen from Figures B to C that CpG-DT-SK can induce strong excretion of HMGB1 and ATP by 4T1 cells.

[0084] II. In vitro maturation detection of BMDC

[0085] The effect of CpG-DT-SK on the maturation of BMDC (mouse bone marrow-derived dendritic cells) was measured in vitro using a Transwell system. The specific method is as follows:

[0086] 4T1 cells were seeded in the upper compartment of a Transwell system and co-cultured with cell culture medium without any drugs (Control), CpG-DT, SK, and CpG-DT-SK (SK concentration was 1.5 μM) for 24 h; then BMDCs were added to the lower compartment and the co-culture was continued for 24 h. BMDCs were harvested and stained with PE Anti-Mouse CD11c antibody (as a gate), FITC Anti-Mouse CD80 antibody, and APC Anti-Mouse CD86 antibody (Abcam, UK) at 4 °C for 30 min respectively; after staining, the maturation status of BMDCs was analyzed using a flow cytometer; meanwhile, the supernatant was collected for later use.

[0087] The results of flow cytometer analysis are shown in Figure 4 Figure D in. It can be seen from Figure D that CpG-DT, SK, and CpG-DT-SK-treated 4T1 cells can increase the maturation rate of BMDCs from 12.2% to 17.2%, 20.8%, and 30.1% respectively, indicating that CpG-DT-SK has a stronger promoting effect on BMDC maturation. This strong effect may be attributed to the synergistic effect of ICD and CpG in promoting BMDC maturation.

[0088] The collected supernatant was used to detect the expression levels of TNF-α and IFN-γ using an ELISA kit (Solarbio, China), and the results are shown in Figure 4 Figures E - F in (* p < 0.05; ** p < 0.01; *** p < 0.001; ns, no statistical difference). It can be seen from Figures E - F that after treatment with CpG-DT-SK, the secretion of TNF-α and IFN-γ can be significantly enhanced. The increased secretion of the above cytokines will inhibit the growth and metastasis of tumor cells and further promote the activation of T cells.

[0089] Experimental Example 4: Determination of in vivo biodistribution and anti-tumor activity of CpG-DT-SK

[0090] The experimental mice used were female BALB / c mice (7 - 8 weeks old, 18 - 20 g, Jinan Pengyue Laboratory Animal Breeding Co., Ltd.).

[0091] 4T1 cells were subcutaneously injected into the upper left abdomen of female BALB / c mice at a dose of 1×10 6 cells / mouse to establish a 4T1 tumor-bearing (triple-negative breast cancer tumor) model. The schematic diagram of the timeline for the treatment experiment of 4T1 tumor-bearing mice is shown in Figure 5 Figure A in, and the specific operation steps are as follows:

[0092] First, 200 μL of Cy5-labeled CpG / SK (CpG + free SK), CpG-DT, and CpG-DT-SK were respectively injected into the tail veins of the model mice. Among them, the concentration of SK was 51 μM, the concentration of CpG-DT was 1 μM, and the concentration of CpG was 4 μM. The fluorescence distribution of Cy5 in the mice was measured at the 1 h and 6 h time points using an imaging system (ABL-X5, Tanon, China).

[0093] The results are as Figure 5 shown in Figure B of

[0094] When the tumor volume reached approximately 80 mm 3 , all the mice were randomly divided into 4 groups for treatment. Each group was respectively injected with PBS buffer (Control), SK (injection dose: 2 mg / kg), CpG-DT (injection dose: 13.2 mg / kg), and CpG-DT-SK (injection dose: 15.2 mg / kg) via the tail vein. The treatment was carried out once every 2 days for a total of 5 times. The tumor volume was monitored and recorded every two days until the 18th day. After the treatment process was completed, the mice were sacrificed, and the tumors in the mice were excised, photographed, and weighed. The tumor volume was calculated according to the following formula:

[0095] Tumor volume = (length × width 2 ) / 2

[0096] The results are as Figure 5 shown in Figures C - E of p <0.0001). It can be seen from Figure C that compared with the Control group, the use of CpG-DT for treatment could slightly inhibit tumor growth, the use of free SK for treatment could moderately inhibit tumor growth, while the tumor volume of the mice treated with CpG-DT-SK was significantly smaller at the end of the entire treatment, indicating that CpG-DT-SK had a good tumor suppression effect in vivo; it can be seen from Figures D - E that the measurement results of the tumor size and weight showed the same trend as the change in tumor volume. Specifically, the tumor growth inhibition rate of the mice treated with CpG-DT-SK could reach 74.2%, indicating that the CpG-DT-SK prepared in the present invention had excellent therapeutic performance for primary tumors.

[0097] The tumor tissues were further analyzed by H&E and TUNEL staining. The analysis results are as Figure 5As shown in Figure F. It can be seen from Figure F that in the H&E stained images, compared with CpG-DT and free SK, the tumor tissue morphology after CpG-DT-SK treatment showed extensive nuclear atrophy and severe tumor cell death, without an integrated cell structure; in the TUNEL stained images, compared with CpG-DT and free SK, the tumor cells after CpG-DT-SK treatment were severely apoptotic (red dots represent apoptotic tumor cells). The above staining results indicate that the anti-tumor efficiency of CpG-DT-SK is the best compared with CpG-DT and free SK.

[0098] Experimental Example 5: Determination of in vivo anti-tumor immune stimulating activity of CpG-DT-SK

[0099] Helper T cells (CD4) are crucial for regulating adaptive immunity, while CTLs (CD8) can directly kill cancer cells.

[0100] On the 18th day after treating the 4T1 tumor-bearing model mice according to the method in Experimental Example 4, the mice were sacrificed, and the spleens, tumor tissues and sera of the mice were collected.

[0101] (1) The collected spleens were ground, lysed for red blood cells, and finally passed through a 200-mesh sieve to prepare single cell suspensions of the spleen, and the cell concentration was adjusted to 1×10 7 cells / mL. Take 100 μL of the above single cell suspension, and sequentially add 1 μL of FITC-CD3e Monoclonal Antibody (11-0031-81, eBioscience, USA), 1.25 μL of PerCP-Cyanine5.5-CD4 Monoclonal Antibody (45-0042-82, eBioscience, USA), 2.5 μL of APC-eFluor™ 780-CD8a Monoclonal Antibody (47-0081-80, eBioscience, USA); after incubating in the dark for 30 min, centrifuge at 4°C and 800 g for 10 min; aspirate the supernatant, add 300 μL of PBS buffer to resuspend, and use a flow cytometer for detection.

[0102] The detection results are as shown in Figure 6 Figure A. It can be seen from Figure A that after treatment with CpG-DT and SK, the active T cells in the spleen increased slightly, and after treatment with CpG-DT-SK, the abundance of active T cells in the spleen was the highest.

[0103] (2)The collected tumor tissues were prepared into paraffin-embedded sections, and then immunofluorescence staining of CD4 and CD8 was performed. The specific antibodies were as follows: CD8 alpha Monoclonal Antibody (MA1-10301, eBioscience, USA), Rhodamine (TRITC)–conjugated Goat Anti-Rat IgG (SA00007-7, Proteintech, USA), Anti-Mouse CD4 (65104-1-Ig, Proteintech, USA), Fluorescein (FITC)–conjugated Affinipure Goat Anti-Rat IgG (SA00003-11, Proteintech, USA); finally, imaging was performed using a laser scanning confocal microscope.

[0104] The detection results are as Figure 6 shown in Figure B of Figure 6 . It can be seen from Figure B that the immunofluorescence staining images of CD4 and CD8 in the tumor tissue sections showed consistent results with

[0105] Figure A of

[0106] . Figure 6 (3)The collected serum was used to detect the expression levels of TNF-α and IFN-γ using an ELISA kit. p <0.01; *** p <0.001; ns, no statistical difference). It can be seen from Figures C-D that after treatment with CpG-DT-SK, both TNF-α and IFN-γ were significantly enhanced, being 2.1 times and 2.3 times that of the Control group, respectively. The increase in IFN-γ secretion can improve the immunogenicity of tumor cells, promote the proliferation and differentiation of CTLs, thereby enhancing the anti-tumor immune response; at the same time, TNF-α can significantly inhibit the growth of malignant tumor cells when combined with IFN-γ.

[0107] The above results indicate that the CpG-DT-SK provided by the present invention can trigger a strong pro-inflammatory response, thereby inducing tumor regression.

[0108] Test Example 6: Biological safety evaluation of CpG-DT-SK

[0109] According to the method in Test Example 4, the 4T1 tumor-bearing model mice were treated for 18 days. During the treatment period, the body weights of the mice were monitored and recorded every two days until the 18th day. The body weight monitoring results are as Figure 7As shown in Figure A, it can be seen from Figure A that no obvious abnormal changes in body weight were observed in each treatment group.

[0110] After 18 days of treatment, the hearts, livers, spleens, lungs, and kidneys of the mice were collected for H&E staining to conduct biosafety evaluation. The results of H&E staining are as Figure 7 shown in Figure B. It can be seen from Figure B that no obvious histological changes were found in the H&E staining of the hearts, livers, spleens, lungs, and kidneys in each treatment group.

[0111] The above results indicate that the CpG-DT-SK provided by the present invention has good biocompatibility and biosafety in vivo.

Claims

1. A CpG-ODN modified DNA tetrahedron-shikonin complex, comprising a CpG-ODN modified DNA tetrahedron and shikonin, wherein the molar ratio between the CpG-ODN modified DNA tetrahedron and shikonin is 1:40 to 200; Among them, The CpG-ODN modified DNA tetrahedron is synthesized by self-assembly of four DNA single strands, and the nucleotide sequences of the four DNA single strands are respectively as shown in SEQ ID NOs. 1 to 4, and the molar ratio between the four DNA single strands is 1:1:1:1; Among them, the CpG-ODN is modified by phosphorothioation.

2. The composite according to claim 1, wherein, The molar ratio between the CpG-ODN modified DNA tetrahedron and shikonin is 1:

80.

3. The composite according to claim 1, characterized in that, The synthesis method of the CpG-ODN modified DNA tetrahedron comprises the following steps: adding four DNA single strands into a solvent, maintaining at 85 °C for 5 min, maintaining at 60 °C for 5 min, and maintaining at 4 °C for more than 30 min.

4. The composite according to claim 3, characterized in that, The solvent is one or more of TM buffer, PBS buffer, and DMSO.

5. The preparation method of the CpG-ODN modified DNA tetrahedron-shikonin complex according to claim 1, characterized in that, Comprising the following steps: adding the CpG-ODN modified DNA tetrahedron and shikonin into a solvent to obtain a mixed solution, oscillating and reacting the mixed solution at 200-400 rpm at 25-30 °C for 4-6 h, and ultrafiltrating.

6. The preparation method according to claim 5, characterized in that, The solvent is one or more of TM buffer, PBS buffer, and DMSO.

7. The preparation method according to claim 5, characterized in that, The conditions for ultrafiltration are: ultrafiltration using a 30 kDa molecular weight membrane.

8. The preparation method according to claim 5, characterized in that, The concentration of the CpG-ODN modified DNA tetrahedron in the mixed solution is 1-20 μM.

9. Use of the CpG-ODN modified DNA tetrahedron-shikonin complex according to claim 1 in the preparation of a drug for treating triple-negative breast cancer, wherein the CpG-ODN modified DNA tetrahedron-shikonin complex treats triple-negative breast cancer by inducing ICD effect, increasing T cell infiltration in tumors, and promoting the secretion of anti-tumor cytokines.

Images