CpG-ODN modified DNA tetrahedron-alkannin compound as well as preparation method and application thereof

Abstract

The invention provides a CpG-ODN modified DNA tetrahedron-alkannin compound as well as a preparation method and application thereof, and belongs to the technical field of biological medicines, the compound overcomes the defects of poor water solubility and poor in-vitro stability of alkannin, and meanwhile, the compound can effectively induce an ICD effect, improve T cell infiltration in tumors and promote secretion of anti-tumor cell factors, so that the anti-tumor effect of the alkannin is improved. Good application prospects are realized in the treatment of the triple negative breast cancer.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a CpG-ODN modified DNA tetrahedron-shikonin complex and a preparation method and application thereof. Background Art

[0002] Female breast cancer is a highly harmful malignant tumor. It is a major public health problem in my country and even globally, seriously affecting women's health and life expectancy. Among them, triple negative breast cancer (TNBC) refers to breast cancer in which all three receptors are negative, namely 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 therapeutic targets, higher recurrence rate and greater metastatic potential, patients have a very poor survival prognosis. For many years, chemotherapy has been the standard treatment for TNBC, but the overall effect is poor, and drug-resistant recurrence and metastasis are prone to occur, with a median survival of 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 innovative treatment of TNBC. However, the "cold tumor" microenvironment of TNBC lacks immune cell infiltration and insufficient tumor immunogenicity, resulting in the fact that the cure of TNBC is still difficult to achieve. These key facts remind us that better innovative immunotherapies are urgently needed to prolong the survival of TNBC patients and achieve the cure of TNBC.

[0004] Shikonin (SK) is the main active ingredient isolated from the dried root of Lithospermum officinale, a traditional Chinese medicine. 16 H 16 O5, the structure is as follows:

[0005] In recent years, studies have found that shikonin has significant anti-tumor biological activity. 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). Ultimately, ICD can enhance the immune response and induce a specific immune response against tumor antigens. However, the non-selective toxicity and low water solubility of shikonin limit its use in anti-tumor therapy.

[0006] Unmethylated cytosine-phosphate-guanine oligonucleotide (CpG-ODN, abbreviated as CpG) is a very potential immune adjuvant with the sequence: 5'-TCCATGACGTTCCTGACGTT-3'. CpG can activate the host defense mechanism of mammals and stimulate a variety of immune cells to induce 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 proinflammatory 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 conducive to the immune enhancement effect of CpG.

[0007] The 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. These DNA nanostructures have been widely used in biomedical research. Among these DNA nanostructures, DNA tetrahedron (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. At present, there are no reports on the simultaneous delivery of shikonin and CpG using DNA tetrahedron. Whether DNA tetrahedron can be used to deliver shikonin and CpG simultaneously, and whether shikonin and CpG can exert a better synergistic therapeutic effect, requires further research. Summary of the invention

[0008] The present invention provides a CpG-ODN modified DNA tetrahedron-shikonin complex and a preparation method and application thereof. The complex solves the shortcomings of poor water solubility and poor in vitro stability of shikonin. At the same time, the complex can effectively induce 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 comprises a CpG-ODN modified DNA tetrahedron and shikonin.

[0011] Preferably, 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 shown in SEQ ID NOs. 1 to 4, respectively.

[0012] Further preferably, the CpG-ODN is modified by thiophosphorylation.

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

[0016] Preferably, the method for synthesizing 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.

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

[0018] The method for preparing 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: 25-30° C. and 200-400 rpm shaking for 4-6 hours.

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

[0022] Preferably, the ultrafiltration condition is: ultrafiltration is performed 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 CpG-ODN modified DNA tetrahedron-shikonin complex provided by the present invention has a simple preparation method and a controllable preparation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The figure is a schematic diagram of the synthesis of CpG-DT-SK and its characterization results. Among them: Figure A is a schematic diagram of the synthesis of CpG-DT-SK; Figure B is the native PAGE detection result of CpG-DT; Figure C is the result of fluorescence competition quenching experiment; Figure D is the UV-visible absorption spectrum of CpG-DT, SK and CpG-DT-SK; Figure E is the native PAGE detection result of CpG-DT and CpG-DT-SK; Figure F is the UV absorbance value of total SK (80μM) and free SK at 595nm; 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 The water solubility and in vitro stability test results of CpG-DT-SK. Among them: Figure A shows the pictures of CpG-DT, CpG-DT-SK and free SK dissolved in TM buffer respectively; Figure B shows the native PAGE test results of CpG-DT and CpG-DT-SK co-incubated with 10% FBS for 0h, 4h, 8h, 12h, and 20h; Figure C shows the semi-quantitative analysis results of the structural stability of CpG-DT and CpG-DT-SK by relative band intensity.

[0031] Figure 3The results of the cell uptake and cytotoxicity test of CpG-DT-SK are shown in Figure A. The laser scanning confocal microscope image of the cell uptake of CpG-DT and CpG-DT-SK by 4T1 cells; Figure B is the flow cytometry analysis of the cell uptake rate of CpG-DT and CpG-DT-SK by 4T1 cells; Figure C is the fluorescence quantitative analysis of the cell uptake rate of CpG-DT and CpG-DT-SK by 4T1 cells; Figure D is the cytotoxicity of CpG-DT, SK and CpG-DT-SK by 4T1 cells.

[0032] Figure 4 Figure 2 shows the results of in vitro ICD induction and immunostimulatory activity of CpG-DT-SK, wherein: Figure A shows the CRT immunofluorescence image of 4T1 cells in each treatment group; Figure B shows the quantitative determination results of ATP release from 4T1 cells in each treatment group; Figure C shows the quantitative determination results of HMGB1 release from 4T1 cells in each treatment group; Figure D shows the representative flow cytometry image of DC maturation in each treatment group; Figure E shows the results of ELISA kit determination of DC secretion of TNF-α; Figure F shows the results of ELISA kit determination of DC secretion of IFN-γ.

[0033] Figure 5 The results of biodistribution and antitumor activity determination in 4T1 tumor-bearing mice. Among them: Figure A is a schematic diagram of the timeline of the treatment experiment of 4T1 tumor-bearing mice; Figure B is the in vivo fluorescence images of 4T1 tumor-bearing mice at 1h and 6h after injection of Cy5-labeled CpG / SK, CpG-DT and CpG-DT-SK; Figure C is the tumor growth curve of mice in each treatment group; Figure D is the tumor tissue photos of mice in each treatment group; Figure E is the tumor weight of mice in each treatment group; F is the H&E and TUNEL staining images of tumor tissue sections of mice in each treatment group.

[0034] Figure 6 The in vivo anti-tumor immunostimulatory activity of CpG-DT-SK is determined. + and CD8 + Representative flow cytometry images of cells; Figure B shows CD4 + and CD8 + Representative immunofluorescence images of cells; Figure C shows the ELISA results of TNF-α in the serum of mice in each treatment group; Figure D shows the ELISA results of IFN-γ in the serum of mice in each treatment group.

[0035] Figure 7 The results of the biosafety evaluation of CpG-DT-SK are shown in Figure A. Figure A shows the weight curve of mice in each treatment group; Figure B shows the representative H&E staining images of the heart, liver, spleen, lung and kidney tissue sections of mice in each treatment group. DETAILED DESCRIPTION

[0036] The following is explained in conjunction with specific embodiments:

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

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

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

[0040] 1. Synthesis of CpG-ODN-modified DNA tetrahedron (CpG-DT)

[0041] CpG-DT was synthesized using the four DNA single strands S1, S2, S3, and S4 in Table 1 below. The specific steps were as follows: equimolar amounts of the four DNA single strands were mixed evenly in TM buffer (10 mM Tris, 5 mM MgCl2, pH 8.0) to make the final concentration of each DNA single strand 1 μM to obtain a mixed solution; the mixed solution was placed in a PCR instrument, and the following program was set for the reaction: 85°C for 5 min, 60°C for 5 min, and 4°C for 30 min; and a CpG-ODN-modified DNA tetrahedron (CpG-DT) solution was obtained.

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

[0043]

[0044]

[0045] 2. Preparation of CpG-ODN modified DNA tetrahedron-shikonin complex (CpG-DT-SK)

[0046] Shikonin (SK) dissolved in DMSO at a concentration of 10 mM was added to the CpG-DT solution prepared above and mixed evenly to obtain a final concentration of SK of 80 μM and a final concentration of CpG-DT of 1 μM to obtain a mixed solution; the mixed solution was incubated at 300 rpm and shaken at 25° C. for 6 h, and then centrifuged at 5000 rpm for 10 min using a 30K MWCO ultrafiltration tube (Millipore, USA) to remove residual SK and DNA single strands; TM buffer was added to the ultrafiltration tube after centrifugation to obtain a CpG-ODN-modified DNA tetrahedron-shikonin complex (CpG-DT-SK) solution.

[0047] 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 concentrate in the ultrafiltration tube after centrifugation, and the concentration of CpG-DT-SK was measured using a NanoDrop Lite (Thermo Fisher Scientific, USA) ultramicro spectrophotometer.

[0048] 3. Characterization of CpG-DT-SK

[0049] (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 strands S1, S1+S2 (consistent with the synthesis method of CpG-DT), S1+S2+S3 (consistent with the synthesis method of CpG-DT) and synthesized CpG-DT were electrophoresed by 8% native PAGE, and the size of the electrophoresis bands was observed. The results are as follows: Figure 1 As shown in Figure B, it can be seen from Figure B that the band size of CpG-DT is consistent with expectations, which shows that CpG-DT was successfully prepared.

[0050] (2) The binding of SK and CpG-DT was further demonstrated by fluorescence competitive quenching experiments. The specific method was as follows: Gelred fluorescent dye was added to 200 μL of 1 μM CpG-DT solution, mixed evenly and incubated for 1 hour; different amounts of SK were added to make the final concentrations of SK 0 μM, 40 μM, 80 μM, 120 μM, 160 μM, and 200 μM, respectively, mixed evenly and incubated for 6 hours; after the incubation was completed, the fluorescence spectrum between 450 and 800 nm was measured with an ELISA reader (Varioskan LUX, Thermo Fisher, USA) when the excitation light was 300 nm. The results are shown in Figure 2. Figure 1 As shown in Figure C, it can be seen from Figure C that the fluorescence intensity of GelRed (λex=300nm) decreases with the increase of SK concentration, which indicates that the binding of GelRed to CpG-DT is replaced by SK, and SK binds to CpG-DT by embedding.

[0051] (3) The UV-visible absorption spectrum was used to verify whether the above CpG-DT-SK was successfully prepared. The results were as follows: Figure 1 As shown in Figure D. As shown in Figure D, the characteristic absorption peaks of CpG-DT-SK have both CpG-DT (260nm) and SK (500-600nm), which indicates that SK is successfully loaded onto CpG-DT.

[0052] (4) The band sizes of CpG-DT and CpG-DT-SK in the gel were verified by native PAGE. The results are as follows: Figure 1As shown in Figure E. As shown in Figure E, the mobility of CpG-DT and CpG-DT-SK in the gel is very close, which indicates that the shape and size of CpG-DT and CpG-DT-SK are very similar.

[0053] (5) The loading rate of CpG-DT on SK was determined by 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, so that the final concentration of SK was 80 μM, to obtain a mixed solution; 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, and 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:

[0054]

[0055] Wherein, “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.

[0056] The results are as follows Figure 1 As shown in Figure F, the absorbance of total SK is 0.635, and the absorbance of free SK is 0.228. The above-mentioned loading rate calculation formula is used to calculate that the loading rate of CpG-DT to SK is about 64%. After conversion, the molar ratio between CpG-DT and SK in CpG-DT-SK is 1:51.

[0057] (6) The average particle size of CpG-DT and CpG-DT-SK was measured by a dynamic light scattering nanoparticle size analyzer (919s, Opptronix, China). Figure 1 As shown in Figure G. From Figure G, it can be seen that the hydrated particle sizes of CpG-DT and CpG-DT-SK are 11.9 nm and 12.5 nm, respectively.

[0058] (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 2. Figure 1 As shown in Figure H in FIG. It can be seen from Figure H that CpG-DT-SK is a monodisperse nanoparticle, and the size is consistent with expectations.

[0059] Experimental Example 1: Water solubility and in vitro stability of CpG-DT-SK

[0060] 1. Water solubility test

[0061] According to the method of Example 1, CpG-DT solution and CpG-DT-SK solution were prepared respectively, wherein the concentration of CpG-DT-SK solution was adjusted so that the final concentration of SK in CpG-DT-SK was 80 μM; at the same time, free SK with a final concentration of 80 μM was added to 100 μL TM buffer as a control group. After mixing evenly, the mixture was left for 24 hours to observe the homogeneity of each mixed solution.

[0062] The results are as follows Figure 2 As shown in Figure A in the figure, it can be seen from Figure A that precipitation appeared in the control group, while the CpG-DT-SK solution still maintained homogeneity, which indicates that CpG-DT combined with SK can increase the water solubility of SK.

[0063] 2. In vitro stability test

[0064] Equimolar amounts of CpG-DT and CpG-DT-SK were mixed with DMEM medium containing 10% FBS (fetal bovine serum) at a volume ratio of 1:4, and then incubated at 37°C for 0h, 4h, 8h, 12h, and 20h; finally, the serum stability was analyzed using 8% native PAGE.

[0065] The results are as follows Figure 2 As shown in Figure B, it can be seen from Figure B that when incubated for 0 to 12 hours, CpG-DT and CpG-DT-SK both showed a clear single band in the gel, which shows that CpG-DT and CpG-DT-SK have a certain tolerance to 10% fetal bovine serum. The semi-quantitative analysis results of relative band intensity are shown in Figure 2 As shown in Figure C, it can be seen from Figure C that after incubation for 20 hours, more than 50% of CpG-DT-SK remained intact, which shows that CpG-DT-SK has good in vitro stability, which provides a prerequisite for further in vivo research.

[0066] Experimental Example 2: Cellular Uptake and Cytotoxicity of CpG-DT-SK

[0067] 1. Cellular Uptake

[0068] Cy5 was coupled to the S1 chain, and the Cy5-labeled CpG-DT and CpG-DT-SK were synthesized using the same method as in Example 1. 54T1 cells (mouse triple-negative breast cancer cells) were inoculated into 35 mm confocal culture 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 culture dishes containing 4T1 cells, and incubated with the cells for 6 h, then the cells were rinsed with PBS buffer and fixed with 4% paraformaldehyde for 20 min, and then 100 nM Hoechst 33342 fluorescent dye was added to stain the cell nucleus for 10 min, and 50 nM LysoTracker green probe was added to stain the lysosome for 20 min; after staining, the cells were washed with PBS buffer to remove unreacted fluorescent dye; imaging was performed using a laser scanning confocal microscope (FV3000, Olympus, Japan).

[0069] The imaging results are as follows Figure 3 As shown in Figure A, it can be seen from Figure A that the fluorescence intensity of 4T1 cells treated with CpG-DT and CpG-DT-SK is similar; in addition, the LysoTracker green probe can co-localize with Cy5 fluorescence, which indicates that both CpG-DT and CpG-DT-SK can be taken up by 4T1 cells and accumulated in lysosomes, and after the DNA backbone is degraded by lysosomal enzymes, SK inserted in the DNA helix can be released from CpG-DT-SK.

[0070] Flow cytometry (FCM) was used to quantitatively evaluate cellular uptake. 4T1 cells were cultured in 6-well plates at 37°C for 12 h, and then cell culture medium without any drug (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, and incubated with cells for 6 h. Single-cell suspensions were prepared using trypsin digestion enzymes, and samples were examined using a flow cytometer (CytoFLEX, Thermo Fisher, USA).

[0071] Flow cytometry results Figure 3 As shown in Figure B, Figure 3 The results of the relevant quantitative analysis are consistent with those of Figure A. Figure 3 As shown in Figure C (****p<0.0001; ns, no statistical difference), it can be seen from Figure C that there is no statistical difference in the average fluorescence intensity of 4T1 cells after treatment with CpG-DT and CpG-DT-SK, which indicates that CpG-DT loaded with SK will not affect its cellular uptake.

[0072] 2. Cytotoxicity

[0073] 4T1 cells were cultured at a rate of 1×10 4 The cells were inoculated at a density of 100 / well in a 96-well plate and cultured overnight; CpG-DT, SK and CpG-DT-SK were added and incubated with the cells, where the concentrations of SK were 0.5μM, 1.0μM, 1.5μM, 2.0μM, 3.0μM and 4.0μM, respectively, and 3 parallel experiments were performed for each concentration; after incubation for 24h, CCK8 reagent was added and incubated for another 2h to obtain the cell culture fluid. The absorbance of the cell culture fluid at 450nm was measured using an ELISA reader (Varioskan LUX, Thermo Fisher, USA), and the cell survival rate was calculated according to the following formula:

[0074]

[0075] Wherein, "As" represents the absorbance of the experimental well (the absorbance of the well containing the test sample, cells, culture medium and CCK-8 reagent), "Ab" represents the absorbance of the blank well (the absorbance of the well containing the culture medium and CCK-8 reagent), and "Ac" represents the absorbance of the control well (the absorbance of the well containing cells, culture medium and CCK-8).

[0076] The results are as follows Figure 3 As shown in Figure D in the figure (*p<0.05, **p<0.01; ns, no statistical difference), it can be seen from Figure D that since CpG-DT delivered more SK into 4T1 cells, CpG-DT-SK exhibited enhanced cytotoxicity compared with SK; in addition, CpG-DT showed no cytotoxicity to 4T1 cells, indicating that it has excellent biocompatibility as a carrier.

[0077] Experimental Example 3: In vitro ICD induction by CpG-DT-SK and in vitro maturation detection of BMDC

[0078] 1. In vitro ICD induction study

[0079] The ability of CpG-DT-SK to induce the ICD cascade was determined, including observation of CRT exposure and determination of ATP and HMGB1 release, as follows:

[0080] (1) CRT exposure: 4T1 cells were cultured at a rate of 1×10 5Cells were seeded at a density of 100 cells / well into 12-well plates and cultured overnight at 37°C; cell culture medium without any drugs (Control), CpG-DT, SK, and CpG-DT-SK (the concentration of SK was 1.5 μM) were added and incubated for 24 hours; cells were harvested, fixed with 4% paraformaldehyde for 20 minutes, and incubated with 5% FBS for 60 minutes; then, cells were incubated with Alexa Fluor 488-labeled CRT antibody (Abcam, UK) at room temperature for 2 hours, and then the cell nuclei were stained with Hoechst 33342 fluorescent dye for 10 minutes; after staining, cells were washed with PBS buffer to remove unreacted fluorescent dye; imaging was performed using a laser scanning confocal microscope.

[0081] The imaging results are as follows Figure 4 As shown in Figure A, it can be seen from Figure A that CpG-DT-SK can enhance CRT exposure.

[0082] (2) Release of ATP and HMGB1: 4T1 cells were cultured at a rate of 1×10 5 Cells were seeded at a density of 100 cells / well into a 12-well plate and cultured at 37°C overnight; cell culture medium without any drugs (Control), CpG-DT, SK, and CpG-DT-SK (SK concentration was 1.5 μM) were added and incubated for 24 h; the culture medium was harvested and the concentrations of ATP and HMGB1 in the culture medium were determined using an ATP content detection kit (Beyotime, China) and an HMGB1 ELISA kit (Sango Biotech, China), respectively.

[0083] The results of ATP concentration determination are as follows Figure 4 As shown in Figure B, the results of the determination of HMGB1 concentration are as follows Figure 4 As shown in Figure C (*p<0.05; ***p<0.001; ns, no statistical difference); from Figures B to C, it can be seen that CpG-DT-SK can induce 4T1 cells to strongly excrete HMGB1 and ATP.

[0084] In summary, the exposure and release of CRT, HMGB1, and ATP can effectively induce the ICD effect, further promote the differentiation and maturation of DCs, and promote anti-tumor immune activity.

[0085] 2. In vitro maturation assay of BMDC

[0086] The effect of CpG-DT-SK on the maturation of BMDC (mouse bone marrow-derived dendritic cells) was determined in vitro using the Transwell system. The specific method is as follows:

[0087] 4T1 cells were seeded in the upper compartment of the 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 cultured for another 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) for 30 min at 4 °C; after staining, the maturation status of BMDCs was analyzed by flow cytometry; and the supernatant was collected for later use.

[0088] The results of flow cytometry analysis were as follows Figure 4 As shown in Figure D, it can be seen from Figure D that 4T1 cells treated with CpG-DT, SK, and CpG-DT-SK can increase the maturation rate of BMDC from 12.2% to 17.2%, 20.8%, and 30.1%, respectively, which indicates that CpG-DT-SK has a stronger promoting effect on BMDC maturation. This powerful effect may be attributed to the synergistic effect of ICD and CpG in promoting BMDC maturation.

[0089] The collected supernatants were tested for the expression levels of TNF-α and IFN-γ using ELISA kits (Solarbio, China). The results were as follows: Figure 4 As shown in Figures E to F (*p<0.05; **p<0.01; ***p<0.001; ns, no statistical difference). As shown in Figures E to F, 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.

[0090] Experimental Example 4: In vivo biodistribution and antitumor activity determination of CpG-DT-SK

[0091] The mice used in the experiment were female BALB / c mice (7-8 weeks old, 18-20 g, Jinan Pengyue Experimental Animal Breeding Co., Ltd.).

[0092] 4T1 cells were injected subcutaneously into the left upper abdomen of female BALB / c mice at a dose of 1×10 6 4T1 tumor-bearing (triple-negative breast cancer) models were constructed. The timeline diagram of the treatment experiment of 4T1 tumor-bearing mice is shown in the figure. Figure 5 As shown in Figure A, the specific steps are as follows:

[0093] First, 200 μL of Cy5-labeled CpG / SK (CpG+free SK), CpG-DT and CpG-DT-SK were injected into the tail vein of model mice, where 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 mice was measured at 1 h and 6 h using an imaging system (ABL-X5, Tanon, China).

[0094] The results are as follows Figure 5 As shown in Figure B, it can be seen from Figure B that 1h and 6h after tail vein injection, CpG-DT-SK showed stronger fluorescence signals in the tumor area than CpG-DT and CpG / SK, thus confirming the excellent in vivo tumor targeting effect of CpG-DT-SK.

[0095] When the tumor volume reaches about 80 mm 3 At the time of treatment, all mice were randomly divided into 4 groups for treatment. Each group was injected with PBS buffer (Control), SK (injection dose of 2 mg / kg), CpG-DT (injection dose of 13.2 mg / kg) and CpG-DT-SK (injection dose of 15.2 mg / kg) through the tail vein. The treatment was performed 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, the mice were killed, the tumors in the mice were removed, photographed and weighed, and the tumor volume was calculated according to the following formula:

[0096] Tumor volume = (length × width 2 ) / 2

[0097] The results are as follows Figure 5 As shown in Figures C to E in (****p<0.0001). As shown in Figure C, compared with the Control group, treatment with CpG-DT can slightly inhibit tumor growth, treatment with free SK can moderately inhibit tumor growth, and the tumor volume of mice treated with CpG-DT-SK is significantly smaller at the end of the entire treatment, indicating that CpG-DT-SK has a good tumor inhibition effect in vivo; As shown in Figures D to E, the size and weight measurement results of the tumor show the same trend as the change in tumor volume. Specifically, the tumor growth inhibition rate after treatment with CpG-DT-SK can reach 74.2%, which shows that the CpG-DT-SK prepared by the present invention has excellent therapeutic performance on primary tumors.

[0098] The tumor tissues were further analyzed by H&E and TUNEL staining. Figure 4As shown in Figure F. As shown in Figure F: In the H&E staining image, 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 integrated cell structure; in the TUNEL staining image, 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 show that compared with CpG-DT and free SK, CpG-DT-SK has the best anti-tumor efficiency.

[0099] Experimental Example 5: Determination of the in vivo anti-tumor immunostimulatory activity of CpG-DT-SK

[0100] Helper T cells (CD4) are crucial for regulating adaptive immunity, while CTLs (CD8) can directly kill cancer cells.

[0101] After the 4T1 tumor-bearing model mice were treated on the 18th day according to the method in Experimental Example 4, the mice were killed and their spleens, tumor tissues and serum were collected.

[0102] (1) The collected spleen was ground and lysed, and finally passed through a 200-mesh sieve to prepare a spleen single-cell suspension, and the cell concentration was adjusted to 1×10 7 Take 100 μL of the above single cell suspension and add 1 μL FITC-CD3e MonoclonalAntibody (11-0031-81, eBioscience, USA), 1.25 μL PerCP-Cyanine5.5-CD4 MonoclonalAntibody (45-0042-82, eBioscience, USA), 2.5 μL APC-eFluor TM 780-CD8a MonoclonalAntibody (47-0081-80, eBioscience, USA); incubate in the dark for 30 minutes, centrifuge at 4°C and 800g for 10 minutes; remove the supernatant, add 300 μL PBS buffer to resuspend, and detect using flow cytometer.

[0103] Test results such as Figure 6 As shown in 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.

[0104] (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, laser scanning confocal microscopy was used for imaging.

[0105] Test results such as Figure 6 As shown in Figure B, the immunofluorescence staining images of CD4 and CD8 in tumor tissue sections are similar to those in Figure 2. Figure 6 Panel A in Figure 2 shows consistent results.

[0106] (3) The collected serum was used to detect the expression levels of TNF-α and IFN-γ using ELISA kits.

[0107] Test results such as Figure 6 As shown in Figures C to D, Figure C shows the expression level of TNF-α in serum, and Figure D shows the expression level of TNF-γ in serum (**p<0.01; ***p<0.001; ns, no statistical difference). As shown in Figures C to D, after CpG-DT-SK treatment, both TNF-α and IFN-γ were significantly enhanced, 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, and thus enhance the anti-tumor immune response; at the same time, TNF-α combined with IFN-γ can significantly inhibit the growth of malignant tumor cells.

[0108] 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.

[0109] Test Example 6: Biosafety Evaluation of CpG-DT-SK

[0110] The 4T1 tumor-bearing model mice were treated for 18 days according to the method in Experimental Example 4. The weight of the mice was monitored and recorded every two days during the treatment until the 18th day. The weight monitoring results are as follows: Figure 7 As shown in Figure A in FIG, it can be seen from Figure A that no obvious abnormal changes in body weight were observed in each treatment group.

[0111] After treatment on day 18, the hearts, livers, spleens, lungs, and kidneys of the mice were collected for H&E staining for biosafety evaluation. Figure 7 As shown in Figure B, no obvious histological changes were found in the heart, liver, spleen, lung, and kidney of each treatment group by H&E staining.

[0112] 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.

2. The composite according to claim 1, characterized in that 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 shown in SEQ ID NOs. 1 to 4 respectively.

3. The composite according to claim 1, characterized in that The CpG-ODN is modified by phosphorothioate.

4. The composite according to claim 2, characterized in that The molar ratio between the four DNA single strands is 1:1:1:

1.

5. The composite according to claim 1, characterized in that The molar ratio between the CpG-ODN modified DNA tetrahedron and shikonin is 1:40-200.

6. The composite according to claim 1, characterized in that The molar ratio between the CpG-ODN modified DNA tetrahedron and shikonin is 1:

80.

7. The composite according to claim 2, 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; Preferably, the solvent is one or more of TM buffer, PBS buffer, and DMSO.

8. The method for preparing the CpG-ODN modified DNA tetrahedron-shikonin complex according to claim 1, characterized in that: The method comprises the following steps: adding CpG-ODN modified DNA tetrahedron and shikonin into a solvent to obtain a mixed solution, shaking the mixed solution at 200-400 rpm for 4-6 hours under the condition of 25-30°C, and ultrafiltration; Preferably, the solvent is one or more of TM buffer, PBS buffer, and DMSO; Preferably, the ultrafiltration condition is: ultrafiltration is performed using a 30 kDa molecular weight membrane.

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

10. 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; 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.

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