Tumor microenvironment acid-responsive multifunctional DNA ditetrahedron as well as preparation method and application thereof

Abstract

The invention relates to a tumor microenvironment acid-responsive multifunctional DNA ditetrahedron as well as a preparation method and application thereof, and belongs to the technical field of nucleic acid drugs. The invention provides a double-DNA tetrahedral structure connected by an i-motif structure, wherein a first tetrahedron is loaded with an aptamer targeting IGFIIR and PD-L1, and a second tetrahedron is connected with CpG oligonucleotide. The DNA ditetrahedron is dissociated in a tumor subacid environment (pH is less than or equal to 6.8), and released difunctional units respectively target tumor cells and immune cells: aptIGFIIR / aptPD-L1 degrades PD-L1 through a lysosome way to relieve immunosuppression; the CpG activates the dendritic cells to enhance antigen presentation. The invention has the beneficial effects of accurate targeting, synergistic interaction and high stability, and has wide application prospects in the preparation of tumor drugs.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid drug technology, and relates to multifunctional DNA bitetrahedrons that are acid-responsive to the tumor microenvironment, their preparation methods, and applications. Background Technology

[0002] Radiotherapy inhibits tumor growth by inducing DNA damage in tumor cells. However, clinical practice and research have shown that radiotherapy alone can cause immunosuppression, leading to resistance in triple-negative breast cancer and resulting in poor treatment outcomes. With the exploration and development of immunotherapy in recent years, it has become a promising treatment for TNBC. Immune checkpoint inhibitors, such as PD-1 and PD-L1 inhibitors, can enhance the immune system's ability to target and destroy cancer cells. Nucleic acid drugs have advantages such as simple design, short development cycle, and strong targeting specificity, and are currently widely used in the treatment of tumors, genetic diseases, and viral infections. With their continued development and application in tumor treatment, the application of nucleic acid drugs in the treatment of TNBC is becoming possible.

[0003] A DNA tetrahedron is a three-dimensional framework nucleic acid structure formed by the self-assembly of four single-stranded DNA molecules with specific sequences through base pairing. It consists of four single-stranded DNA molecules forming six double-stranded edges through precise base pairing, creating a highly symmetrical tetrahedral spatial configuration. DNA tetrahedra possess several unique physicochemical properties: first, natural biocompatibility, as their constituent components are natural nucleotides, which do not trigger significant immune responses or cytotoxicity; second, structural rigidity, as the tetrahedral structure has high mechanical strength and can maintain a stable spatial configuration; third, programmability, allowing for the precise arrangement of functional molecules by designing extension or modification sites in the DNA sequence; and fourth, cell permeability, as their nanoscale size and unique spatial configuration enable efficient penetration of cell membranes via a caveolin-dependent pathway. These properties make DNA tetrahedra ideal nanocarriers and detection platforms, showing broad application prospects in the biomedical field.

[0004] Off-target effects caused by passive targeting strategies are a major challenge in drug delivery. Conventional DNA tetrahedra reach only small doses of the target tumor site in vivo. This is primarily because the surface of DNA tetrahedra lacks active targeting molecules, making them unable to specifically recognize tumor cells; furthermore, non-specific phagocytosis by the reticuloendothelial system leads to the clearance of large quantities of the carrier by the liver and spleen. In addition, the dense matrix and high interstitial pressure of tumor tissue also hinder the deep penetration of DNA tetrahedra, causing them to be mainly distributed around tumor blood vessels and unable to reach the core region.

[0005] Achieving precise spatiotemporal drug release is a key challenge for DNA tetrahedrons. Existing systems largely rely on environmental changes to trigger drug release, but these suffer from response lag and incomplete release. In enzyme-responsive DNA tetrahedrons, variations in the expression levels of target enzymes within tumor cells lead to fluctuations in drug release efficiency ranging from 20% to 80%. Drug burst release is also prevalent, with some vectors releasing more than 30% of their loaded drug before reaching the target site. Summary of the Invention

[0006] In view of this, the present invention aims to provide a multifunctional DNA double tetrahedron that is acid-responsive to the tumor microenvironment, a method for preparing a multifunctional DNA double tetrahedron that is acid-responsive to the tumor microenvironment, and an application of a multifunctional DNA double tetrahedron that is acid-responsive to the tumor microenvironment in the preparation of tumor drugs.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention provides a multifunctional DNA ditetrahedron that is acid-responsive to the tumor microenvironment. The multifunctional DNA ditetrahedron is formed by tetrahedron TDN1 and tetrahedron TDN2 linked by an i-motif structure. AptIGFIIR and AptPD-L1 are connected to the top of tetrahedron TDN1, and CpG is connected to the top of tetrahedron TDN2. An i-motif structure is formed by complementary pairing of one top of tetrahedron TDN1 and one top of tetrahedron TDN2. The i-motif structure is formed by complementary pairing of single nucleotides S1-1-C2 in tetrahedron TDN1 and S2-1-C2 in tetrahedron TDN2. The nucleic acid sequence of S1-1-C2 is shown in SEQ ID NO:10, and the sequence of S2-1-C2 is shown in SEQ ID NO:11.

[0009] Preferably, the four mononucleotides of the tetrahedral TDN1 are composed of S1-1-C2, S2, S3, and S1-4, and the four mononucleotides of the tetrahedral TDN2 are composed of S2-1-C2, S2, S3, and S2-4. The nucleotide sequence of S2 is shown in SEQ ID NO:4, the nucleotide sequence of S3 is shown in SEQ ID NO:5, the nucleotide sequence of S1-4 is shown in SEQ ID NO:6, and the nucleotide sequence of S2-4 is shown in SEQ ID NO:7.

[0010] Preferably, the nucleic acid sequence of AptIGFIIR is shown in SEQ ID NO:1, the nucleic acid sequence of AptPD-L1 is shown in SEQ ID NO:2, and the nucleic acid sequence of CpG is shown in SEQ ID NO:3;

[0011] Furthermore, the method for preparing the acid-responsive multifunctional DNA bitetrahedron of the tumor microenvironment comprises the following steps:

[0012] (a) Synthesis of tetrahedral TDN1: The single chains of S1-1-C2, S2, S3 and S1-4 were added to a reaction buffer containing 10 mM Tris-HCl and 50 mM MgCl2 in proportion, heated at 95 °C for 10 minutes and annealed at 4 °C for 20 minutes to obtain tetrahedral TDN1.

[0013] (b) Connecting aptamers: TDN1 was mixed with AptIGFIIR and AptPD-L1 and incubated at room temperature for 2 hours to obtain AptIGFIIR / AptPD-L1-TDN1;

[0014] (c) Synthesis of tetrahedral TDN2: The single chains of S2-1-C2, S2, S2, and S2-4 are synthesized into tetrahedral TDN2 in the same proportion as in step (a);

[0015] (d) CpG ligation: TDN2 was mixed with CpG sequence and incubated at room temperature for 2 hours to obtain CpG-TDN2;

[0016] (e) Assembly of bitetrahedrons: AptIGFIIR / AptPD-L1-TDN1 and CpG-TDN2 were mixed in proportion and ligated at room temperature for 8 hours to obtain the DNA bitetrahedrons;

[0017] Preferably, the molar ratio of the S1-1-C2, S2, S3, and S1-4 single chains is 1:1:1:1, and the molar ratio of the S2-1-C2, S2, S3, and S2-4 single chains is 1:1:1:1.

[0018] Preferably, the molar ratio of AptIGFIIR to tetrahedral TDN1 is 1:1, the molar ratio of AptPD-L1 to tetrahedral TDN1 is 2:1, and the molar ratio of CpG to tetrahedral TDN2 is 3:1.

[0019] Preferably, the molar ratio of AptIGFIIR / AptPD-L1-TDN1 to CpG-TDN2 is 1:1;

[0020] Furthermore, the application of the aforementioned acid-responsive multifunctional DNA bitetrahedron in the preparation of tumor drugs;

[0021] Furthermore, the application of the aforementioned tumor microenvironment acid-responsive multifunctional DNA bitetrahedron in the preparation of drugs for use in combination with radiotherapy;

[0022] Preferably, the tumor is triple-negative breast cancer.

[0023] The beneficial effects of this invention are as follows:

[0024] 1. Precise release in response to acid

[0025] By linking two DNA tetrahedra with an i-motif structure, the device efficiently unfolds (dissociation efficiency ≥90%) in the slightly acidic environment of tumors (pH ≤ 6.8), achieving precise spatiotemporal controlled release of dual-functional units (targeting tumor cells + activating immune cells) and avoiding accidental release from normal tissues. Optimizing the i-motif link length ensures structural stability under physiological conditions (pH 7.4), overcoming the technical bottleneck of low acid response efficiency in traditional nanocarriers.

[0026] 2. Dual-function collaborative design

[0027] aptIGFIIR / aptPD-L1-TDN1: Simultaneously targets insulin-like growth factor II receptor (IGFIIR) and programmed death ligand 1 (PD-L1), degrades PD-L1 in tumor cells via the lysosomal pathway, and relieves immunosuppression.

[0028] CpG-TDN2: Carries CpG oligonucleotides to activate dendritic cells (DCs), enhance antigen presentation capabilities, and form a synergistic "anti-inhibition + immune activation" dual pathway with PD-L1 degradation.

[0029] 3. Breakthrough in treatment efficacy

[0030] After combined radiotherapy, the tumor volume decreased from 1.27 cm in the control group. 3 Reduced to 0.10cm 3 Tumor weight decreased to 0.36g (compared to >1.0g in the control group), and survival was significantly prolonged. Radiotherapy combined with bitetrahedral induction of tumor cell apoptosis and immune cell infiltration was effective.

[0031] The aptamer released from the bitetrahedral is firmly linked to the CpG sequence through complementary base pairing, ensuring the efficient accumulation of functional molecules at the tumor site.

[0032] 4. Stability and security

[0033] Its stable structure solves the problem of easy degradation of nucleic acid drugs. No significant weight loss or organ damage was observed after intravenous injection, demonstrating excellent biocompatibility.

[0034] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0035] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein:

[0036] Figure 1 A schematic diagram illustrating the assembly process of tumor acid-responsive DNA bitetrahedrons;

[0037] Figure 2 Nucleic acid gel image showing the assembly process and results of tumor acid-responsive DNA bitetrahedrons;

[0038] (a) Agarose gel electrophoresis characterization of AptIGFIIR / AptPD-L1-TDN1 and (b) CpG-TDN2 nanoparticles; (c) Connection status of C1, (d) C2, and (e) C3 AptIGFIIR / AptPD-L1-TDN1-CpG-TDN2 nanoparticles;

[0039] Figure 3 Morphological and physicochemical characterization of the acid-responsive DNA ditetrahedrons of tumors; Detection results of C1-TDN1+C1-TDN2(a), C2-TDN1+C2-TDN2(b), and C3-TDN1+C3-TDN2(c) using a UV spectrophotometer;

[0040] (d) Particle size analysis of the two tetrahedra before and after synthesis at pH 7.4;

[0041] (e) Changes in the Zeta potential of AptIGFIIR / AptPD-L1-TDN1+CpG-TDN2 during tetrahedral synthesis with different i-motif connection lengths;

[0042] (f) Transmission electron microscope image of tetrahedral structure AptIGFIIR / AptPD-L1-TDN1+CpG-TDN2;

[0043] (g) Agarose gel electrophoresis results of DNA tetrahedral structures with different i-motif ligation lengths in an acidic environment;

[0044] (h) Agarose gel electrophoresis results of AptIGFIIR / AptPD-L1-TDN1 in PBS at different times;

[0045] (i) Agarose gel electrophoresis results of AptIGFIIR / AptPD-L1-TDN1 in 10% FBS solution at different times;

[0046] (j) Degradation rate of tetrahedral structure AptIGFIIR / AptPD-L1-TDN1+CpG-TDN2 over time (n=3);

[0047] (k) Agarose gel electrophoresis results of CpG-TDN2 in PBS at different times;

[0048] (l) Agarose gel electrophoresis results of CpG-TDN2 in 10% FBS at different times;

[0049] Figure 4 A schematic diagram illustrating the mechanism of action of the acid-responsive DNA ditetrahedron.

[0050] Figure 5 The acid-responsive DNA ditetrahedron can induce an effective immune response in solid tumors to inhibit tumor growth.

[0051] (a) Schematic diagram of the treatment regimen for 4T1 tumor-bearing mice;

[0052] (b) Comparison of antitumor effects among different treatment groups, scale bar = 2 cm;

[0053] (c) Monitoring of mouse weight at different time points (n=5);

[0054] (d) Tumor weight analysis at the end of the treatment period (n=5);

[0055] (e) Survival analysis of mice after different treatments (n=5);

[0056] (f) Tumor volume changes after different treatments (n=5), I: control, II: TDN1+TDN2, III: AptIGFIIR / AptPD-L1-TDN1+TDN2, IV: TDN1+CpG-TDN2, V: AptIGFIIR / AptPD-L1-TDN1+CpG-TDN2, VI: control + IR, VII: TDN1+TDN2+IR, VIII: AptIGFIIR / AptPD-L1-TDN1+TDN2+IR, IX: TDN1+CpG-TDN2+IR, X: AptIGFIIR / AptPD-L1-TDN2+IR;

[0057] (g) Bioluminescence detection of 4T1-Luc tumor progression in different treatment groups;

[0058] (hi) TUNEL (h) and H&E staining (i) analysis of tumor samples after different treatment regimens;

[0059] (jk) Serum TNF-α(j) and IL-2(k) levels in mice after different treatments: I: control, II: TDN1+TDN2, III: AptIGFIIR / AptPD-L1-TDN1+TDN2, IV: TDN1+CpG-TDN2, V: AptIGFIIR / AptPD-L1-TDN1+CpG-TDN2. Data are expressed as mean ± SEM (n=5). Detailed Implementation

[0060] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0061] The accompanying drawings are for illustrative purposes only and are schematic diagrams, not actual pictures. They should not be construed as limiting the invention. To better illustrate the embodiments of the invention, some parts in the drawings may be omitted, enlarged, or reduced, and do not represent the actual product dimensions. It is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings.

[0062] In the accompanying drawings of the embodiments of the present invention, the same or similar reference numerals correspond to the same or similar components. In the description of the present invention, it should be understood that if terms such as "upper," "lower," "left," "right," "front," and "rear" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, they are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, the terms used to describe positional relationships in the drawings are only for illustrative purposes and should not be construed as limiting the present invention. For those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.

[0063] Example 1

[0064] Synthesis of DNA tetrahedrons:

[0065] Four ssDNAs (S1, S2, S3, and S4) of the DNA tetrahedron were added to the reaction buffer (10 mM Tris-HCl, 50 mM MgCl2·6H2O) in a ratio of 1:1:1:1. The mixture was heated at 95°C for 10 minutes, and then immediately cooled to 4°C and placed for 20 minutes using a PCR thermal cycler (ABI, PCR System 9700) to prepare the synthesized DNA tetrahedron.

[0066] In the TDN1 tetrahedron, S1 is any one of the compounds S1-1-C1, S1-1-C2, and S1-1-C3, and S4 is S1-4. The nucleic acid sequences are shown in Table 1 below.

[0067] In the TDN2 tetrahedron, S1 is any one of the compounds S2-1-C1, S2-1-C2, and S2-1-C3, and S4 is S2-4. The nucleic acid sequences are shown in Table 1 below.

[0068] Synthesis of AptIGFIIR / AptPD-L1-TDN1:

[0069] To prepare the DNA tetrahedral synthesis solution, add 16 μL of PBS (1 L of deionized water containing 8.0 g NaCl, 0.2 g KCl, 1.44 g Na₂HPO₄, and 0.24 g KH₂PO₄) and 1 μL each of the four 100 mM tetrahedral single-stranded solutions to a 200 μL centrifuge tube and mix thoroughly. Add 19 μL of PBS and 1 μL of 100 mM ptIGFIIR solution and mix thoroughly. Add 18 μL of PBS and 2 μL of 100 mM ptPD-L1 solution and mix thoroughly. Set the PCR instrument program as follows: Step 1: Preheat at 95°C for 1 min; Step 2: Heat at 95°C for 15 min; Step 3: Anneal at 4°C for 2 h. After incubating the prepared solution in a PCR thermal cycler, mix the aptamer solution with the corresponding tetrahedral solution and incubate at room temperature for 2 h.

[0070] Synthesis of CpG-TDN2:

[0071] To prepare a DNA tetrahedral synthesis solution, add 16 μL of PBS (1 L of deionized water containing 8.0 g NaCl, 0.2 g KCl, 1.44 g Na₂HPO₄, and 0.24 g KH₂PO₄) and 1 μL each of four 100 mM tetrahedral single-stranded solutions to a 200 μL centrifuge tube and mix thoroughly. Then add 27 μL of PBS and 3 μL of 100 mM CpG solution and mix thoroughly. Set the PCR instrument program as follows: Step 1: Preheat at 95 °C for 1 min; Step 2: Heat at 95 °C for 15 min; Step 3: Anneal at 4 °C for 2 h. After incubating the prepared solution in a PCR thermal cycler, mix the aptamer solution with the corresponding tetrahedral solution and incubate at room temperature for 2 h.

[0072] Synthesis of AptIGFIIR / AptPD-L1-TDN1+CpG-TDN2:

[0073] The synthesized AptIGFIIR / AptPD-L1-TDN1 solution and CpG-TDN2 solution were mixed in a 1:1 ratio and left to stand at room temperature for 8 hours to obtain the AptIGFIIR / AptPD-L1-TDN1+CpG-TDN2 solution.

[0074] The bitetrahedral structure consists of two aptamers—tetrahedral structures—with different functions. First, images were obtained using agarose gel electrophoresis (...). Figure 2 (ab) shows that the migration speed of DNA tetrahedral lanes is slower than that of single-stranded DNA or other combinations of single-stranded DNA, proving the successful assembly of DNA tetrahedra. Simultaneously, the migration speed of DNA tetrahedra combined with different aptamers is slower than that of DNA tetrahedra, proving that different aptamers can be loaded onto DNA tetrahedra.

[0075] Table 1: Sequence List

[0076]

[0077]

[0078] Example 2

[0079] Screening of DNA Bi-Tetrahedral Linkage Length: To ensure the stable binding of the two tetrahedra of this nanoassembly under normal in vivo conditions, preventing self-separation, and to effectively release both tetrahedra in the acidic tumor microenvironment, the length of the linker sequence is crucial. We designed three i-motif linker sequences (C1, C2, and C3) of different lengths and synthesized DNA tetrahedral materials accordingly. The optimal linker length was then screened experimentally. Studies showed that excessively long or short linker sequences affect the stability and pH responsiveness of the assembly under normal conditions, highlighting the importance of exploring suitable linker lengths. First, to evaluate the linker performance under normal conditions, we synthesized tetrahedra of three different lengths and mixed them in specific proportions. After incubation at room temperature for 8 hours, agarose gel electrophoresis was performed for verification. The results were analyzed using... Figure 2 Based on the relative position changes of the bands, we can observe that the bitetrahedrons of the C1 connection length are basically not connected to each other, while the DNA tetrahedra of C2 and C3 lengths are connected to each other to form a whole.

[0080] To further evaluate the bonding performance of three different bonding lengths under normal conditions, we used a UV spectrophotometer to analyze the solutions before and after tetrahedral bonding of C1, C2, and C3. Figure 3The results showed that most of the two tetrahedra in the C1 design were not interconnected, while the connections between C2 and C3 were better. We then examined the changes in zeta potential before and after the ligation of two DNA tetrahedra with different connection lengths, obtaining similar conclusions. Figure 3 e) The AptIGFIIR / AptPD-L1-TDN1+CpG-TDN2 solution was dropped onto the carbon film, allowed to air dry, and its morphology was observed using a transmission electron microscope. Figure 3 We can observe the obvious double tetrahedral structure quite intuitively.

[0081] Example 3

[0082] Responsive Analysis of Tumor Acid-Responsive DNA Bitetrahedrons in Acidic Environment: To observe and analyze the interconnection of tetrahedral structures with different i-motif lengths and the separation of two tetrahedra in an acidic environment, we first synthesized bitetrahedral structures by mixing AptIGFIIR / AptPD-L1-TDN1 and CpG-TDN2 in a 1:1 ratio. Then, the pH of the solution was adjusted to 6.8. By observing the changes in band positions in agarose gel before and after pH adjustment, the response of the material in an acidic environment was analyzed. Figure 3 Analysis of the band positions in g reveals that C1-TDN1 and C1-TDN2 cannot connect to each other and therefore cannot respond; C2-TDN1 and C2-TDN2 can be fully connected at normal pH, but after the ambient pH changes to 6.8, the two tetrahedra separate, and the solution becomes a mixture of the two tetrahedra; C3-TDN1 and C3-TDN2 can be fully connected at normal pH, but after the ambient pH changes, only some tetrahedra separate, while most tetrahedra remain connected, indicating that the pH responsiveness of the C2 connection structure is better than that of C3.

[0083] To further verify the responsiveness of AptIGFIIR / AptPD-L1-TDN1+CpG-TDN2 under acidic conditions, we measured the particle size of the two tetrahedra before and after synthesis under normal pH conditions. Figure 3 d) The results further validated the bonding stability of our material under normal pH conditions, and that it could be completely separated under acidic pH conditions.

[0084] Stability assessment of tumor acid-responsive DNA bitetrahedrons: To verify the stability of our materials in PBS and 10% FBS, we synthesized AptIGFIIR / AptPD-L1-TDN1 and CpG-TDN2, respectively, and incubated them in PBS and 10% FBS solutions for different times. Their structures were then characterized by agarose gel electrophoresis. Figure 3 h and Figure 3k indicates that AptIGFIIR / AptPD-L1-TDN1 and CpG-TDN2 maintained their structural integrity and did not degrade even after being placed in PBS for 48 hours, while in a 10% FBS solution ( Figure 3 i and Figure 3 After 48 hours, the tetrahedral structure showed slight degradation, but the overall structure remained stable. Figure 3 The results also show that the degradation rate of the material structure has remained at a low level.

[0085] Example 4

[0086] In vivo evaluation of tumor acid-responsive DNA bitetrahedral combined with radiotherapy: The bitetrahedral assembly was used to study the in vivo antitumor effect in a tumor-bearing mouse model carrying subcutaneous 4T1 tumors. Nucleic acid materials were injected into the model mice via tail vein injection, with separate radiotherapy and non-radiotherapy groups. Treatment regimens for different samples via intravenous injection and radiotherapy are shown in Fig. 5a. Untreated model mice exhibited rapid tumor growth, with an average tumor volume of approximately 1.27 cubic centimeters after 21 days. Model mice treated with radiotherapy alone showed a slight tumor suppression effect, with a final tumor volume of approximately 1.10 cubic centimeters. Model mice treated with AptIGFIIR / AptPD-L1-TDN1 and CpG-TDN2 in combination with radiotherapy showed enhanced tumor suppression compared to model mice treated with radiotherapy alone. This may be due to the enhanced degradation of PD-L1 in tumor cells by AptIGFIIR / AptPD-L1-TDN1, which in turn enhances the immune system's inhibitory effect on tumor cells; and the activation of DCs by CpG-TDN2. The combination of AptIGFIIR / AptPD-L1-TDN1+CpG-TDN2 and radiotherapy demonstrated the most efficient 4T1 tumor suppression, with a final tumor volume of only about 0.10 cubic centimeters. This trend in tumor volume was also supported by changes in tumor weight, with the AptIGFIIR / AptPD-L1-TDN1+CpG-TDN2+IR group showing the lowest average tumor weight of only about 0.36 grams. Figure 5 d). The treatment regimen combining AptIGFIIR / AptPD-L1-TDN1+CpG-TDN2 with radiotherapy alleviated tumor development, and its model mice had the longest survival time. Figure 5 e). Histological evaluation of extracted tumor tissue samples by H&E staining and TUNEL assay showed that the model mice treated with AptIGFIIR / AptPD-L1-TDN1+CpG-TDN2 in combination with radiotherapy had the highest proportion of dead cells in their tumors, indicating that it can induce efficient 4T1 cell death and thus effectively inhibit tumor growth. Figure 5(h,i). Meanwhile, to evaluate the safety of the treatment regimen combining AptIGFIIR / AptPD-L1-TDN1+CpG-TDN2 with radiotherapy, we observed that mice treated with this regimen did not experience significant weight loss or major histological damage to key organs. These results demonstrate that the combination of AptIGFIIR / AptPD-L1-TDN1+CpG-TDN2 and radiotherapy can effectively inhibit tumor growth in vivo with a high safety profile. Subsequently, to further investigate the anti-tumor immune mechanism following treatment, tumor tissue was extracted for analysis of its immune cells and related cytokines. First, the total number of immune cells in the tumor was detected. The results showed a significant increase in the total number of immune cells (25.62%). Among them, the proportion of MDSCs after combined treatment with AptIGFIIR / AptPD-L1-TDN1+CpG-TDN2 and radiotherapy decreased by 11.87%, and the proportion of IFN-γ+CD8a+ T cells increased significantly, 23.31% higher than that of the control group. Figure 5 Further ELISA analysis of tumor tissue revealed that the secretion levels of pro-inflammatory cytokines IFN-γ, TNF-α, IL-2, and CXCL10 were significantly upregulated compared to the control group. Figure 5 These results indicate that the combination of AptIGFIIR / AptPD-L1-TDN1+CpG-TDN2 and radiotherapy demonstrates significant anti-tumor therapeutic effects both in vivo and in vitro by promoting anti-tumor immune responses and enhancing immune cell infiltration.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A multifunctional DNA bitetrahedron that is acid-responsive to the tumor microenvironment, characterized in that: The multifunctional DNA ditetrahedron is formed by connecting tetrahedron TDN1 and tetrahedron TDN2 through an i-motif structure. The top of tetrahedron TDN1 is connected to AptIGFIIR and AptPD-L1, and the top of tetrahedron TDN2 is connected to CpG. One top of tetrahedron TDN1 and tetrahedron TDN2 forms an i-motif structure through complementary pairing. The i-motif structure is formed by complementary pairing of single nucleotides S1-1-C2 in tetrahedron TDN1 and S2-1-C2 in tetrahedron TDN2. The nucleic acid sequence of S1-1-C2 is shown in SEQ ID NO:10, and the sequence of S2-1-C2 is shown in SEQ ID NO:

11.

2. The tumor microenvironment acid-responsive multifunctional DNA bitetrahedron according to claim 1, characterized in that: The four mononucleotides of the tetrahedral TDN1 are composed of S1-1-C2, S2, S3, and S1-4, and the four mononucleotides of the tetrahedral TDN2 are composed of S2-1-C2, S2, S3, and S2-4. The nucleotide sequence of S2 is shown in SEQ ID NO:4, the nucleotide sequence of S3 is shown in SEQ ID NO:5, the nucleotide sequence of S1-4 is shown in SEQ ID NO:6, and the nucleotide sequence of S2-4 is shown in SEQ ID NO:

7.

3. The multifunctional DNA bitetrahedron with acid-responsive tumor microenvironment according to claim 2, characterized in that: The nucleic acid sequence of AptIGFIIR is shown in SEQ ID NO:1, the nucleic acid sequence of AptPD-L1 is shown in SEQ ID NO:2, and the nucleic acid sequence of CpG is shown in SEQ ID NO:

3.

4. The method for preparing acid-responsive, multifunctional DNA bitetrahedrons in the tumor microenvironment according to any one of claims 1-3, characterized in that, The steps are as follows: (a) Synthesis of tetrahedral TDN1: The single chains of S1-1-C2, S2, S3 and S1-4 were added to a reaction buffer containing 10 mM Tris-HCl and 50 mM MgCl2 in proportion, heated at 95 °C for 10 minutes and annealed at 4 °C for 20 minutes to obtain tetrahedral TDN1. (b) Connecting aptamers: TDN1 was mixed with AptIGFIIR and AptPD-L1 and incubated at room temperature for 2 hours to obtain AptIGFIIR / AptPD-L1-TDN1; (c) Synthesis of tetrahedral TDN2: The single chains of S2-1-C2, S2, S2, and S2-4 are synthesized into tetrahedral TDN2 in the same proportion as in step (a); (d) CpG ligation: TDN2 was mixed with CpG sequence and incubated at room temperature for 2 hours to obtain CpG-TDN2; (e) Assembly of bitetrahedrons: AptIGFIIR / AptPD-L1-TDN1 and CpG-TDN2 were mixed in proportion and ligated at room temperature for 8 hours to obtain the DNA bitetrahedrons.

5. The preparation method according to claim 4, characterized in that: The molar ratio of the S1-1-C2, S2, S3, and S1-4 single chains is 1:1:1:1, and the molar ratio of the S2-1-C2, S2, S3, and S2-4 single chains is 1:1:1:

1.

6. The preparation method according to claim 5, characterized in that: The molar ratio of AptIGFIIR to tetrahedral TDN1 is 1:1, the molar ratio of AptPD-L1 to tetrahedral TDN1 is 2:1, and the molar ratio of CpG to tetrahedral TDN2 is 3:

1.

7. The preparation method according to claim 6, characterized in that: The molar ratio of AptIGFIIR / AptPD-L1-TDN1 to CpG-TDN2 is 1:

1.

8. The application of the tumor microenvironment acid-responsive multifunctional DNA bitetrahedron according to any one of claims 1-3 in the preparation of tumor drugs.

9. The use of the tumor microenvironment acid-responsive multifunctional DNA bitetrahedron according to any one of claims 1-3 in the preparation of a drug for use in combination with radiotherapy.

10. The application according to claim 7 or 8, characterized in that: The tumor is a triple-negative breast cancer.

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