DNA nanomaterial for tumor synergistic therapy and preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a DNA nanomaterial for tumor synergistic treatment and a preparation method and application thereof. The self-assembled long DNA nanocomposite is generated through RCA reaction, and then manganese oxide is generated on the surface through one-step biomimetic mineralization method. The method is simple, the obtained multifunctional DNA nanomaterial has good stability, dispersibility, biocompatibility and safety, compared with the self-assembled long DNA nanocomposite, the responsiveness degradation ability is enhanced, and the targeting of the nanomaterial is improved. Moreover, the multifunctional DNA nanomaterial can not only knock down the PD-L1 protein expression of tumor cells to avoid immune escape, but also can significantly activate the cGAS-STING signal to produce inflammatory cytokines, and can be used for synergistically enhancing the anti-tumor immune response.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine. More specifically, it relates to a DNA nanomaterial for tumor synergistic treatment and a preparation method and application thereof. BACKGROUND

[0002] In addition to traditional surgery, chemotherapy and radiotherapy, tumor immunotherapy has become a research hotspot in the past decade, and activating the innate immune system and regulating adaptive immunity has become a new strategy for tumor immunotherapy. However, current studies have shown that the tumor microenvironment (TME) that promotes tumors and immune suppression can promote tumor growth and immune escape, directly affecting the effectiveness of tumor immunotherapy.

[0003] Programmed Cell Death-Ligand 1 (PD-L1) is a type I transmembrane protein highly expressed in most tumors. PD-L1 on the membrane of tumor cells inhibits T cell proliferation and activation by binding to PD-1 on T cells, inhibits cytokine release, and thus leads to immune suppression. The RNA cleavage activity of DNAzyme can participate in gene silencing under the catalysis of metal cations, which indicates that DNAzyme can be used to cut PD-L1 mRNA to avoid immune escape. For example, Chinese Patent Application CN115261384A discloses a DNAzyme targeting PD-L1 gene, a targeted delivery carrier, and a tumor-targeting nanocomposite. DNAzymes Dz1-Dz10 with high target gene recognition and cleavage activity are screened and designed, and a DNAzyme delivery carrier APFP with tumor targeting is prepared. The APFP is electrostatically assembled with the DNAzyme to prepare an APFP / Dz1-Dz10 nanocomposite. The tumor cell-targeted delivery of the DNAzyme is achieved, the expression of PD-L1 in tumor cells is significantly inhibited, and CD8 + T cell tumor killing activity is successfully activated, thereby delaying the progression of metastatic melanoma in mice. However, this method requires a lot of effort and time to design and screen DNAzymes with high target gene recognition and cleavage activity, and the material preparation method is complex, making it difficult to produce industrially.

[0004] In addition, studies have shown that cyclic GMP-AMP synthase (cGAS) is a key cytoplasmic DNA sensing enzyme that can catalyze the conversion of GTP and ATP into the second messenger cyclic GMP-AMP (cGAMP), trigger the Stimulator of Interferon Genes (STING), induce the production of type I interferons and other cytokines, and play an important role in the innate immune signaling pathway; STING agonists can activate immune stimulating cells including dendritic cells, change the tumor microenvironment and induce the production of tumor-specific T cells, thereby achieving the effect of anti-tumor immunotherapy. DNA sensing mediated by cGAS-STING is the key to initiating anti-tumor immunity, but DNA-based cGAS-STING agonists have poor application effects due to low cell permeability and poor biological stability. SUMMARY

[0005] The technical problem to be solved by the present application is to overcome the defects and shortcomings of the existing deoxyribozyme carrier or drug screening difficulty, complex preparation method, low cell permeability of DNA-based cGAS-STING agonists, poor biological stability, and provide a preparation method of DNA nanomaterial for tumor synergistic therapy, which is simple in operation, and the obtained DNA nanomaterial can activate the PD-L1 pathway and the STING pathway at the same time, relieve the immunosuppressive state of tumor cells, and has a significant effect of inhibiting tumor cell growth.

[0006] The purpose of the present application is to provide a DNA nanomaterial for tumor synergistic therapy prepared by the preparation method.

[0007] Another purpose of the present application is to provide the use of the DNA nanomaterial in the preparation of an anti-tumor drug.

[0008] The above-mentioned purposes of the present application are achieved by the following technical solutions:

[0009] A preparation method of a DNA nanomaterial for tumor synergistic therapy, specifically comprising the following steps:

[0010] S1, annealing assembly of DNA primers and linear templates, adding rapid ligation reaction buffer and rapid T4 DNA ligase reaction to connect completely, forming a circular DNA template;

[0011] S2, adding phi29 DNA polymerase, sodium chloride, dNTP, BSA, complementary strands and phi29 DNA polymerase buffer to the circular DNA template obtained in step S1, RCA reaction is complete, inactivate phi29 DNA polymerase to stop RCA reaction, washing, obtaining self-assembled long DNA nanocomplexes;

[0012] S3, biomimetic mineralization reaction of the long DNA nanocomposite obtained in step S2 with manganese salt under alkaline conditions is completed, and the DNA nanomaterial is obtained by separation and purification;

[0013] The sequence of the DNA primer is shown in SEQ ID NO. 1, the sequence of the linear template is shown in SEQ ID NO. 2, and the sequence of the complementary strand is shown in SEQ ID NO. 3.

[0014] The present application provides a preparation method of a multifunctional DNA nanomaterial for tumor synergistic treatment. The long DNA nanocomposite generated by RCA is self-assembled as a core, and the surface is covered with biomimetic manganese oxide. On the basis of the existing RCA to generate DNA nanoclusters and the combination of metal oxides through electrostatic interaction, the present application directly generates metal oxides on the surface of the DNA nanocomposite through one-step biomimetic mineralization, greatly shortening the preparation time of the multifunctional DNA nanomaterial. The reduced particle size of the nanomaterial makes it more conducive to aggregation in tumor tissues during long circulation in blood. The one-step biomimetic mineralization method can avoid the aggregation problem caused by electrostatic interaction and better fully cover and protect the internal core structure. In addition, the complementary strand is added during the RCA process, so that the multifunctional DNA nanomaterial has a local double-stranded structure, which enhances the activation of the cGAS-STING pathway.

[0015] The method is simple to operate and is very suitable for large-scale industrial production.

[0016] Further, in step S1, the annealing assembly conditions are 90-95 DEG C for 5-10 min, cooling to 25 DEG C at a rate of 1-60 DEG C / min and retaining for 1-30 min, and then cooling to 4 DEG C.

[0017] Further, in step S1, the molar ratio of the DNA primer to the linear template is (1-2):1.

[0018] Preferably, in step S1, the annealing assembly is reacted in ultrapure water.

[0019] Further, in step S1, the rapid ligation reaction buffer includes 132 mM Tris-HCl, 20 mM MgCl2, 2 mM DTT, 2 mM ATP, 15% polyethylene glycol, and the pH is 7.6.

[0020] Further, in step S1, the reaction ligation complete conditions are 20-30 DEG C, and the reaction ligation is 30-40 min.

[0021] Further, in step S2, the RCA reaction conditions are 25-37 DEG C, and the reaction is 0.5-1.5 h.

[0022] Further, in step S2, the inactivation condition is 65-75℃ for 10 min or more.

[0023] Further, in step S2, the washing is washing with 1-5% dilute ammonia water for 2-3 times.

[0024] Further, in step S3, the manganese salt is selected from one or more of manganese chloride and manganese sulfate.

[0025] Further, in step S3, the alkaline condition is the addition of one or more of sodium hydroxide, potassium hydroxide and ammonia water.

[0026] Further, in step S3, the condition of the biomimetic mineralization reaction is 25-37℃ for 30-45 min.

[0027] Preferably, in step S3, the separation and purification are centrifugation, washing, resuspension in HEPES buffer and collection of the precipitate.

[0028] In addition, the present application also claims the DNA nanomaterial for tumor synergistic treatment prepared by the preparation method.

[0029] Preferably, the DNA nanomaterial is a long DNA nanocomposite self-assembled inside, in the form of a coil, covered with manganese oxide on the surface, and has a particle size of 90-110 nm.

[0030] Under the stimulation of the tumor microenvironment, the DNA nanomaterial of the present application can effectively induce the liquid-phase condensation of cGAS and activate the STING signal to produce inflammatory cytokines based on the synergistic effect of the release of manganese ions and the long and dense DNA structure. At the same time, due to the release of manganese ions, the DNAzyme in the long DNA chain also cuts PD-L1 mRNA, knocking down the expression of PD-L1 on the surface of tumor cells, so as to relieve the immunosuppressive state of tumor cells and enhance the effect of anti-tumor immune response. Moreover, the DNA nanomaterial has no obvious hemolytic behavior and cytotoxicity and has good biological safety, which is conducive to clinical use.

[0031] Therefore, the present application also claims the use of the DNA nanomaterial in the preparation of anti-tumor carriers and drugs.

[0032] The present application has the following beneficial effects:

[0033] The application generates self-assembled long DNA nanocomposites through RCA reaction, and then generates manganese oxide on the surface through one-step biomimetic mineralization method, the method is simple, the obtained multifunctional DNA nanomaterial has good stability, dispersibility, biocompatibility and safety, compared with the self-assembled DNA nanocomposites, the responsiveness degradation ability is enhanced, and the targeting of the nanomaterial is improved. And the multifunctional DNA nanomaterial of the application can not only knock down the expression of tumor cell PD-L1 protein to avoid immune escape, but also can significantly activate cGAS-STING signal to produce inflammatory cytokines, and can be used for synergistically enhancing the anti-tumor immune response. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 The synthesis flowchart of the DNA nanomaterial for tumor synergistic treatment of the application.

[0035] Figure 2 The transmission electron microscope image of the DNA nanomaterial obtained by the application.

[0036] Figure 3 The data statistical diagram of the hydration particle size and distribution coefficient experiment results of the DNA nanomaterial obtained by the application in PBS for 7 days.

[0037] Figure 4 The agarose gel electrophoresis diagram of the RCA reaction of the primer template of the application.

[0038] Figure 5 The DNAzyme shearing PD-L1 simulation substrate gel electrophoresis diagram of the application.

[0039] Figure 6 The data statistical diagram of the influence of the DNA nanomaterial of the application on the activity of human umbilical vein endothelial HUVEC cells.

[0040] Figure 7 The data statistical diagram of the influence of different concentrations of DNA nanomaterials of the application on the activity of mouse melanoma cells.

[0041] Figure 8 The live and dead staining diagram of the DNA nanomaterial and the long DNA nanocomposite on mouse melanoma cells of the application.

[0042] Figure 9 The flow cytogram of the apoptosis of the DNA nanomaterial on mouse melanoma cells of the application.

[0043] Figure 10 The data statistical diagram of the influence of the DNA nanomaterial of the application on the RNA level of mouse melanoma cells.

[0044] Figure 11Statistical chart of data of the effect of the DNA nanomaterial of the present application on the protein level of mouse melanoma cells.

[0045] Figure 12 Statistical chart of the hemolysis experimental results of the DNA nanomaterial of the present application at different concentrations. DETAILED DESCRIPTION

[0046] The present application will be further described in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and devices used in the present application are conventional reagents, methods and devices in the technical field.

[0047] In the present application, the DNA sequences involved are shown in Table 1, wherein the DNAzyme is a sequence known to have a shearing effect on mouse PD-L1 mRNA, and the remaining sequences are self-designed sequences.

[0048] Table 1 DNA sequences involved in the present application

[0049]

[0050] *Explanation of SEQ ID NO. 5 in the specification sequence listing: In the table, A and U in "rA" and "rU" are RNA, which is a shearing site; according to the editing rules of WIPO Sequence software, the nucleotide sequence must only contain the symbols listed in "WIPO ST.26 Annex I Part 1", and the base "t" in the RNA sequence is "U", so SEQ ID NO. 5 in Table 1 of the specification of the present application is substantially the same as SEQ ID NO. 5 in the sequence listing.

[0051] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0052] Example 1 Preparation method of a DNA nanomaterial for tumor synergistic therapy

[0053] The preparation method of the DNA nanomaterial for tumor synergistic therapy specifically comprises the following steps:

[0054] S1, 4 μL of 10 μM DNA primer and 2 μL of 10 μM linear template were mixed in 3 μL of ultrapure water, and an annealing program (95°C for 5 minutes, cooling to 25°C at a rate of 1°C per minute, and then quickly cooling to 4°C) was used in PCR assembly, and then 10 μL of rapid ligation reaction buffer (132 mM Tris-HCl, 20 mM MgCl2, 2 mM DTT, 2 mM ATP, 15% polyethylene glycol (PEG 6000), pH 7.6@25°C) and 1 μL of rapid T4 DNA ligase were added, and the reaction was connected at 25°C for 30 minutes in a PCR instrument to form a circular DNA template.

[0055] S2, 43 μL of ultrapure water, 2 μL of phi29 DNA polymerase, 10 μL of 800 mM sodium chloride, 10 μL of 10 mM dNTP (A\T\G\C base mixture), 4 μL of 5 mg / mL BSA, 1 μL of 100 μM complementary chain, and 10 μL of phi29 DNA polymerase buffer were added to the circular DNA template obtained in step S1, and RCA reaction was carried out at 37°C for 1 hour, and phi29 DNA polymerase was inactivated at 75°C for 10 minutes to terminate the RCA reaction; after the reaction was completed, the self-assembled long DNA nanocomposite (referred to as DNC for short) was obtained by centrifugal washing with 1% dilute ammonia water for 3 times.

[0056] S3, the long DNA nanocomposite obtained in step S2 was biomimetic mineralized with 4 μM of manganese chloride in a sodium hydroxide alkaline environment for 30 minutes, and after the reaction was completed, it was centrifuged, washed, and resuspended in HEPES buffer for standby, to obtain multifunctional DNA nanomaterials (referred to as DNCM for short).

[0057] The specific synthesis flowchart is shown in Figure 1 , and the transmission electron microscope image of the obtained DNA nanomaterial is shown in Figure 2 As can be seen from the figure, the DNA nanomaterial is uniformly distributed and in a thread-like shape, with a particle size of 90-110 nm; the content of manganese element in the DNA nanomaterial obtained by ICP-OES is greater than 200 μM.

[0058] The hydration particle size and distribution coefficient of the DNA nanomaterial in PBS for 7 days were determined, and the results are shown in Figure 3 As can be seen from the figure, the DNA nanomaterial has good stability in PBS within 7 days, and the distribution coefficient PDI is less than 0.25, which proves that it has good dispersibility.

[0059] Example 2 A preparation method of DNA nanomaterials for tumor synergistic treatment

[0060] The preparation method of the DNA nanomaterials for tumor synergistic treatment specifically comprises the following steps:

[0061] S1, 5 μL of 10 μM DNA primer and 5 μL of 10 μM linear template were mixed in a PCR instrument to assemble through an annealing program (95°C for 5 minutes, cooling to 25°C at a rate of 1°C per minute and remaining for 1 minute, and then rapidly cooling to 4°C), and then 10 μL of rapid ligation reaction buffer and 1 μL of rapid T4 DNA ligase were added to form a circular DNA template through ligation reaction at 20°C for 40 minutes in the PCR instrument.

[0062] S2, 42 μL of ultrapure water, 2 μL of phi29 DNA polymerase, 10 μL of 800 mM sodium chloride, 10 μL of 10 mM dNTP (A\T\G\C base mixture), 4 μL of 5 mg / mL BSA, 1 μL of 100 μM complementary chain, and 10 μL of phi29 DNA polymerase buffer were added to the circular DNA template obtained in step S1, and RCA reaction was carried out at 37°C for 1.5 hours, phi29 DNA polymerase was inactivated at 65°C for 10 minutes to terminate the RCA reaction; after the reaction was completed, 1% dilute ammonia water was used for centrifugal washing twice to obtain self-assembled long DNA nanocomposites (referred to as DNC).

[0063] S3, the long DNA nanocomposites obtained in step S2 were biomimetic mineralized with 8 μM manganese chloride in a sodium hydroxide alkaline environment for 40 minutes, and after the reaction was completed, centrifugal washing and resuspension in HEPES buffer were carried out for standby, to obtain multifunctional DNA nanomaterials (referred to as DNCM).

[0064] Example 3 A preparation method of DNA nanomaterials for tumor synergistic treatment

[0065] The preparation method of the DNA nanomaterials for tumor synergistic treatment specifically comprises the following steps:

[0066] S1, 5 μL of 10 μM DNA primer and 5 μL of 10 μM linear template were mixed in a PCR instrument to assemble through an annealing program (95°C for 5 minutes, cooling to 25°C at a rate of 1°C per minute and remaining for 1 minute, and then rapidly cooling to 4°C), and then 10 μL of rapid ligation reaction buffer and 1 μL of rapid T4 DNA ligase were added to form a circular DNA template through ligation reaction at 20°C for 40 minutes in the PCR instrument.

[0067] S2, 42 μL of ultrapure water, 2 μL of phi29 DNA polymerase, 10 μL of 800 mM sodium chloride, 10 μL of 10 mM dNTP (A\T\G\C base mixture), 4 μL of 5 mg / mL BSA, 1 μL of 100 μM complementary chain and 10 μL of phi29 DNA polymerase buffer were added to the circular DNA template obtained in step S1, and RCA reaction was carried out at 37°C for 1.5 hours, phi29 DNA polymerase was inactivated at 65°C for 10 minutes, and RCA reaction was terminated; after the reaction was completed, the self-assembled long DNA nanocomposite (referred to as DNC for short) was obtained by centrifugal washing twice with 1% dilute ammonia water.

[0068] S3, the long DNA nanocomposite obtained in step S2 was biomimetic mineralized with 6 μM manganese chloride in a sodium hydroxide alkaline environment for 40 minutes, and after the reaction was completed, it was centrifuged, washed, resuspended in HEPES buffer for standby, and multifunctional DNA nanomaterial (referred to as DNCM for short) was obtained.

[0069] Application Example 1 verifies the occurrence of RCA reaction

[0070] Taking the raw materials, intermediate products and final products of each process of Example 1 of the application as an example, the successful occurrence of RCA reaction is verified, and the specific method comprises the following steps:

[0071] S1, wash the glue making mold and comb with distilled water and dry, place on the glue making flat plate, put the comb in place and adjust to level with the level, standby;

[0072] S2, prepare the electrophoresis buffer: add 1×TBE buffer powder into 1 L of ultrapure water, stir and ultrasonically dissolve to prepare 1×TBE buffer, standby;

[0073] S3, prepare 3% agarose gel: weigh 3.0 g of agarose, add 100 mL of electrophoresis buffer, heat and dissolve, then add 10 μL of YeaRed nucleic acid dye (10000× diluted to 1×), continue to heat and gently shake to mix the dye and the gel evenly;

[0074] S4, glue making: pour the 3% agarose gel suspension into the prepared mold while hot, stand at room temperature until solidification, then gently pull out the comb vertically upward, and the electrophoresis lane is prepared, standby;

[0075] S5, sample loading, set the electrophoresis conditions to 100 V for 80 minutes, and the composition of each lane is as follows: ① DNA molecular weight standard Marker, ② DNA primer, ③ linear template, ④ circular DNA template formed after ligase ligation, ⑤ long DNA nanocomposite after adding polymerase.

[0076] Results are shown in Figure 4From the result figure, it can be seen that the DNA strand in the channel 5 is blocked in the hole, verifying the successful occurrence of the RCA reaction.

[0077] Application Example 2: Agarose gel electrophoresis experiment of DNAzyme cleavage substrate

[0078] With reference to the specific operation in Application Example 1, a 4% agarose gel is prepared, 1 μL of 100 μM DNAzyme (SEQ ID NO. 4) and 1 μL of 100 μM PD-L1 simulation substrate (SEQ ID NO. 5) are added to manganese chloride solutions with different concentrations (0, 50, 100, 200, 400, 800, 1000 μM), incubated at 37°C for 2h, and DNA molecular weight marker is loaded, and the electrophoresis condition is set to 100V for 100 minutes.

[0079] The results are shown in Figure 5 From the result figure, it can be seen that the DNAzyme can cleave the substrate at a concentration of 200 μM; the manganese element content of the obtained DNA nanomaterial in the embodiment is greater than 200 μM, indicating that the obtained multifunctional DNA nanomaterial can activate the cleavage function of the DNAzyme.

[0080] Application Example 3: Safety test of DNA nanomaterial

[0081] To prove that the obtained DNA nanomaterial (taking the DNA nanomaterial DNCM obtained in Example 1 as an example) has no killing effect on normal cells and has good biological safety, 100 μL (1×10 5 ) of human umbilical vein endothelial HUVEC cells are inoculated in each well of a 96-well plate, inoculated for 24h, and the cell adhesion is good, then the medium in each well is aspirated; the blank group is added with DNA nanomaterial-free medium DMEM, and the experimental group is added with medium containing different concentrations of DNA nanomaterial (4, 20, 40, 100, 200 μg / mL) prepared with DMEM, and is placed in an incubator for 24 hours; after incubation, the supernatant is aspirated, washed with PBS for three times, and serum-free medium DMEM containing CCK-8 is added, and CCK-8 accounts for 1 / 10 of the volume of the cell culture medium (10 μL CCK8+90 μL DMEM); placed in an incubator for 1 hour, and when the color turns orange, the 96-well plate is taken out, and the absorbance at 450 nm is detected by an enzyme marker, and the cell viability is calculated.

[0082] The results are shown in Figure 6 As can be seen from the figure, the DNA nanomaterial of the present application has no obvious effect on the cell viability of HUVEC cells at different concentrations, and has good biological safety.

[0083] Application Example 4: Effect of DNA nanomaterial on mouse melanoma cells

[0084] 1. To test the tumor cell killing effect of the obtained DNA nanomaterial (taking the DNA nanomaterial DNCM obtained in Example 1 as an example), 100 μL (1 × 10⁻⁶) was seeded into each well of a 96-well plate. 5 Melanoma B16F10 cells were seeded and cultured for 24 hours. After the cells adhered well, the culture medium in each well was aspirated. The blank group was treated with DMEM culture medium without DNA nanomaterials, while the experimental groups were treated with DMEM culture medium containing different concentrations of DNA nanomaterials (4, 20, 40, 100, and 200 μg / mL). The cells were incubated for 24 hours. After incubation, the supernatant was aspirated, washed three times with PBS, and serum-free DMEM culture medium containing CCK-8 was added. CCK-8 accounted for 1 / 10 of the cell culture medium volume (10 μL CCK8 + 90 μL DMEM). The cells were incubated for 1 hour. When the color turned orange, the 96-well plate was removed and the absorbance at 450 nm was measured using a microplate reader to calculate cell viability.

[0085] See results Figure 7 As can be seen from the figure, the DNA nanomaterial of the present invention has a significant tumor-killing effect, which is positively correlated with the concentration; the higher the concentration, the better the tumor-killing effect.

[0086] 2. To verify that the obtained DNA nanomaterial (taking the DNA nanomaterial DNCM obtained in Example 1 as an example) has the effect of killing tumor cells, 1 ml (1×10⁻⁶) was seeded into each well of a 12-well plate. 6 Melanoma B16F10 cells were seeded and cultured for 24 hours. After good cell adhesion, the culture medium in each well was aspirated. The control group was treated with DMEM culture medium without DNA nanomaterials, while the experimental groups were treated with DMEM culture medium at a concentration of 200 μg / mL containing different drugs (DNA nanomaterials DNCM and long DNA nanocomplexes DNC). Both groups were incubated for 24 hours. After incubation, the supernatant was aspirated, and the cells were washed twice with PBS. Calcein-AM / PI dye was prepared using DMEM. After aspirating the supernatant, 500 μL of dye was added to each well, and the cells were incubated at 37°C in the dark for 30 minutes. The cells were washed twice with DMEM, and then resuspended in 500 μL of DMEM for fluorescence imaging. The results are shown in [link to results]. Figure 8 The scale bar is 100μm.

[0087] As can be seen from the figure, the DNC group showed no obvious red fluorescence, while the DNCM group showed obvious red fluorescence, indicating that DNCM has a significant killing effect on B16F10 cells.

[0088] 3、After 24h of incubation in the above-mentioned experiment 2, the supernatant was aspirated, the adherent cells were washed once with PBS, an appropriate amount of trypsin cell digestion solution was added to digest the cells, and the cells were incubated at room temperature until the adherent cells could be blown down by gentle blowing. The trypsin cell digestion solution was then aspirated; the cells were gently blown down and transferred to a centrifuge tube, centrifuged at 1000g for 5 minutes, the supernatant was discarded, and the cells were gently resuspended with PBS and counted. 100,000 resuspended cells were taken, centrifuged at 1000g for 5 minutes, the supernatant was discarded, 195 μl of Annexin V-FITC binding solution was added to gently resuspend the cells, 5 μl of Annexin V-FITC was added, and the mixture was gently mixed. Then, 10 μl of iodinated propylthionine staining solution was added, the mixture was gently mixed, and the mixture was incubated at room temperature (20-25°C) for 20 minutes in the dark. Subsequently, the mixture was placed in an ice bath, and immediately detected by flow cytometry. The results are shown in Figure 9 .

[0089] As can be seen from the result graph, the cells in the DNCM group underwent significant apoptosis compared with the control group, indicating that DNCM has a significant effect on killing tumor cells.

[0090] 4、After 24h of incubation in the above-mentioned experiment 2, the supernatant was aspirated, the adherent cells were washed once with PBS, the well plate was placed on ice and pre-cooled Trizol lysis solution was added to lyse the cells. After the cells were completely lysed, the lysis solution and cell debris in the well plate were aspirated into a 1.5 mL EP tube, chloroform was added, the mixture was vortexed for 15 seconds, and the mixture was allowed to stand at room temperature for 3 minutes. The mixture was then centrifuged at 4°C at 12000 rpm for 15 minutes. After centrifugation, the liquid was divided into three layers: the upper layer was colorless liquid RNA, the middle layer was white DNA, and the bottom layer was pink protein. The upper aqueous RNA was carefully aspirated and transferred to a new 1.5 mL EP tube, and isopropanol was added. The mixture was gently inverted 10 times, a white flocculent precipitate appeared, and the mixture was allowed to stand at room temperature for 10 minutes. The mixture was then centrifuged at 4°C at 12000 rpm for 15 minutes, the supernatant was discarded, and the residual liquid in the 1.5 mL EP tube was aspirated with a pipette. 75% pre-cooled ethanol was added to float the RNA precipitate, and the precipitate was washed thoroughly (twice). The mixture was then centrifuged at 4°C at 12000 rpm for 10 minutes. The supernatant was removed, and the 75% ethanol was aspirated. The mixture was allowed to evaporate. DEPC water was added to each tube to dissolve and mix the precipitate, and the concentration and purity of the mixture were determined in a Nanodrop. Reverse transcription cDNA synthesis and quantitative PCR analysis were then performed. The results are shown in Figure 10 , wherein I is the PBS control group, II is the DNC group, and III is the DNCM group.

[0091] As can be seen from the figure, group III can significantly knock down the expression of PD-L1 mRNA, and at the same time increase the expression of IFN beta mRNA, a downstream signal molecule of the cGAS-STING pathway, indicating that the DNA nanomaterial obtained by the application can not only knock down the expression of PD-L1 mRNA of tumor cells, but also significantly activate the expression of the cGAS-STING signal molecule IFN beta mRNA, and can be used to synergistically enhance the anti-tumor immune response.

[0092] 5、After incubating for 24h in the above-mentioned experiment 2, the supernatant was aspirated, washed with pre-cooled PBS for 2 times, the PBS was aspirated and then RIPA protein lysate and protease and phosphatase inhibitors were added and dispersed in dots, and the lysate RIPA was dispersed as evenly as possible. The cells were collected into a 1.5mL EP tube (pre-cooled) on ice for 30min, and then centrifuged at 4 degrees and 12000rpm for 15min. The supernatant was placed in a new pre-cooled EP tube, labeled, and the concentration was measured by BCA. The protein concentration was adjusted with protein loading buffer, and then boiled at 100 degrees for 5min before loading for protein electrophoresis and development. The results are shown in Figure 11 , wherein I is the PBS control group, II is the DNC group, and III is the DNCM group.

[0093] As can be seen from the figure, the expression of PD-L1 protein in group III is significantly decreased, and the expression of downstream signal protein molecules of the cGAS-STING pathway is significantly increased, indicating that the DNA nanomaterial obtained can not only knock down the expression of PD-L1 protein of tumor cells to avoid immune escape, but also significantly activate the cGAS-STING signal to produce inflammatory cytokines, and can be used to synergistically enhance the anti-tumor immune response.

[0094] Application Example 5 Hemolysis experiment of DNA nanomaterial

[0095] The DNA nanomaterial DNCM (taking the DNA nanomaterial DNCM obtained in Example 1 as an example) was dispersed and diluted with water to 5μg / mL, 10μg / mL, 25μg / mL, 50μg / mL, 100μg / mL, and 200μg / mL. 0.2mL of the above-mentioned solutions with different concentrations were mixed with 0.2mL of treated blood (500μL of fresh blood was added to 4.5mL of physiological saline and centrifuged for 5-8 times at 3000rpm for 10min, until the supernatant was clear and transparent, and then the supernatant was discarded and the physiological saline was added to 5mL) and 0.6mL of physiological saline, and then incubated at 37℃ for 4h. After that, centrifugation was performed at 3000rpm for 10min, and the supernatant was aspirated to measure the absorbance at OD 541nm. The hemolysis rate was calculated, and the results are shown in Figure 12 .

[0096] As can be seen from the figure, the hemolysis rate of the obtained DNA nanomaterial is less than 5%, indicating that the DNA nanomaterial has good biocompatibility.

[0097] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.

Claims

1. A method for preparing DNA nanomaterials for tumor synergistic therapy, characterized in that, Specifically comprising the following steps: S1, annealing assembly of DNA primers and linear templates, adding rapid ligation reaction buffer and rapid T4 DNA ligase reaction ligation, complete formation of circular DNA template; S2, adding phi29 DNA polymerase, sodium chloride, dNTP, BSA, complementary strand and phi29 DNA polymerase buffer to the circular DNA template obtained in step S1, complete RCA reaction, inactivate phi29 DNA polymerase to terminate RCA reaction, wash, and obtain self-assembled long DNA nanocomplexes; S3, biomimetic mineralization reaction of the long DNA nanocomplexes obtained in step S2 with manganese salt under alkaline conditions, separation and purification, and obtain DNA nanomaterials; The sequence of the DNA primer is shown in SEQ ID NO. 1, the sequence of the linear template is shown in SEQ ID NO. 2, and the sequence of the complementary strand is shown in SEQ ID NO.

3.

2. The method of claim 1, wherein, In step S1, the molar ratio of the DNA primers to the DNA linear templates is (1-2):

1.

3. The preparation method according to claim 1, characterized in that, In step S1, the annealing assembly conditions are 90-95℃ for 5-10 min, cooling to 25℃ at a rate of 1-60℃ / min and retaining for 1-30 min, and then cooling to 4℃.

4. The preparation method according to claim 1, characterized in that, In step S1, the complete reaction ligation conditions are 20-30℃ for 30-40 min.

5. The method of claim 1, wherein the step of forming the first and second layers is performed by a process selected from the group consisting of: sputtering, evaporation, and chemical vapor deposition. In step S2, the RCA reaction conditions are 25-37℃ for 0.5-1.5 h.

6. The method of claim 1, wherein the step of forming the first and second layers is performed by a process selected from the group consisting of: sputtering, evaporation, and chemical vapor deposition. In step S3, the manganese salt is selected from one or more of manganese chloride and manganese sulfate.

7. The method of claim 1, wherein the step of forming the first and second layers is performed by a process selected from the group consisting of: sputtering, evaporation, and chemical vapor deposition. In step S3, the alkaline conditions are addition of one or more of sodium hydroxide, potassium hydroxide and ammonia.

8. The method of claim 1, wherein the step of forming the first and second layers is performed by a process selected from the group consisting of: sputtering, evaporation, and chemical vapor deposition. In step S3, the biomimetic mineralization reaction conditions are 25-37℃ for 30-45 min.

9. The DNA nanomaterials for tumor synergistic treatment prepared by the preparation method of any one of claims 1-8.

10. The use of the DNA nanomaterials of claim 9 in the preparation of anti-tumor carriers and drugs.

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