Drug carrier, DNA tetrahedron drug and preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a drug carrier, a DNA tetrahedron drug and a preparation method and application thereof. The application provides a drug carrier, which comprises single-stranded DNA and G-quadruplexes; the single-stranded DNA has a nucleotide sequence shown in Seq ID NO. 1, Seq ID NO. 2, Seq ID NO. 3, Seq ID NO. 4, Seq ID NO. 5 and Seq ID NO. 6; the G-quadruplex has a nucleotide sequence shown in Seq ID NO. 7; the single-stranded DNA is connected with each other to form a DNA tetrahedron structure; and the G-quadruplex is connected with the single-stranded DNA in a complementary manner. The application can effectively solve the technical problems of the existing drug carrier, such as complex synthesis, accumulation of toxic effects, difficulty in biological degradation, and easy degradation of a loaded nucleic acid drug in the body.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, and in particular relates to drug carriers, DNA tetrahedral drugs, their preparation methods and applications. Background Technology

[0002] Acute liver failure (ALF) is a critical clinical condition that can cause multiple organ dysfunction in a short period of time. It often leads to death due to complications such as cardiopulmonary failure, renal failure, gastrointestinal bleeding, and sepsis, resulting in an extremely high mortality rate. Currently, there is no effective comprehensive treatment. Because the pathogenesis of ALF is very complex and multiple factors can interact, the specific mechanisms are not yet fully understood. ALF is mainly characterized by a single-stage necrosis of hepatocytes, with the necrotic area ≥ 2 / 3 of the liver parenchyma, accompanied by severe degeneration of surviving hepatocytes, and the sinusoidal wall scaffold not collapsing or only partially collapsing. For patients with acute liver failure, orthotopic liver transplantation is usually considered the main treatment for ALF; however, unfortunately, a considerable number of patients die due to insufficient donors, high costs, or poor self-repair capabilities. In addition, stem cell-based therapies, such as embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs), and induced pluripotent stem cells (iPSCs), have become a hot topic in human liver disease research over the past decade. However, the exact mechanisms remain unclear and require further investigation. Their effective clinical application still has a long way to go. Despite the high heterogeneity and individual / regional differences in the clinical manifestations, biochemical changes, and pathological mechanisms of this disease, [further research is needed].

[0003] Reactive oxygen species (ROS) are known to be a significant factor in liver injury. Excessive ROS production can lead to inflammation and hepatocyte necrosis, exacerbating disease symptoms. Furthermore, ALF-induced activation of the innate immune system can also result in the production of large amounts of inflammatory mediators, further contributing to systemic disease and ALF damage. Especially in the early stages of liver injury, monocyte-derived macrophages have pro-inflammatory effects, exacerbating inflammation. Therefore, the development and application of interfering RNA, antioxidants, and anti-inflammatory drugs in recent years have generated considerable interest in ALF treatment. However, interfering RNA is easily degraded or off-target by various nucleases in plasma and kidneys, limiting its widespread clinical application. Antioxidants and anti-inflammatory drugs have short half-lives in the blood, severely hindering their effective utilization.

[0004] Currently, inorganic nanomaterials such as cerium oxide nanomaterials, quantum dots, magnetic nanoparticles, and carbon nanomaterials, and organic nanomaterials such as liposome nanoparticles and chemical polymer nanomaterials are used. These are typically delivered to specific tissue sites by specifically modifying nanomaterials with aptamers, peptides, or targeting antibodies. However, the synthesis of these delivery nanomaterials is complex; and most hard nanomaterials, such as gold, silver, copper, and manganese nanoparticles, have cumulative toxicity and are difficult to degrade, posing a potential risk of adverse reactions in clinical treatment. Furthermore, naked siRNA modified on the surface of nanomaterials is easily degraded in vivo, and the complex material synthesis process limits the large-scale application of these nanocarriers. Summary of the Invention

[0005] Based on this, this application provides drug carriers, DNA tetrahedral drugs, their preparation methods and applications, which can effectively solve the technical problems of existing drug carriers being complex to synthesize, having cumulative toxic effects and being difficult to biodegrade, and nucleic acid-loaded drugs being easily degraded in vivo.

[0006] The first aspect of this application provides a drug carrier, comprising:

[0007] Single-stranded DNA and G-quadruplex;

[0008] The single-stranded DNA has the nucleotide sequences shown in Seq ID NO.1, Seq ID NO.2, Seq ID NO.3, Seq ID NO.4, Seq ID NO.5 and Seq ID NO.6;

[0009] The G-quadruplex has the nucleotide sequence shown in Seq ID NO.7;

[0010] The ends of Seq ID NO.1, Seq ID NO.2, and Seq ID NO.3 are each connected to a first connector; the ends of Seq ID NO.4, Seq ID NO.5, and Seq ID NO.6 are each connected to a third connector; and the end of the G-quadruplex is connected to a second connector.

[0011] The sequence of the first connector is complementary to the sequence of the second connector;

[0012] The single-stranded DNA molecules are interconnected to form a DNA tetrahedral structure; the G-tetrahedral is complementary to the single-stranded DNA molecules through the second adapter.

[0013] The drug carrier of this application can be linked to nucleic acid drugs and chemical drugs, serving as a carrier capable of simultaneously delivering nucleic acid drugs and chemical drugs.

[0014] Specifically, the drug carrier of this application can connect a fourth adapter, which is complementary to the third adapter, to the end of the nucleic acid drug, so that the nucleic acid drug is complementary to the single-stranded DNA through the fourth adapter; the chemical drug can act as a G4 ligand and bind to the DNA tetrahedron through π-π stacking interaction, or the chemical drug can be planarly inserted between the GC-CG base pairs of the DNA through a chemical group (such as a quinone ring), or the chemical drug can bind to the DNA tetrahedron through electrostatic adsorption, or the chemical drug can be covalently linked to the DNA tetrahedron.

[0015] The second aspect of this application provides a DNA tetrahedral drug, including:

[0016] Single-stranded DNA, G-quadruplexes, nucleic acid drugs, and chemical drugs;

[0017] The single-stranded DNA has the nucleotide sequences shown in Seq ID NO.1, Seq ID NO.2, Seq ID NO.3, Seq ID NO.4, Seq ID NO.5 and Seq ID NO.6;

[0018] The G-quadruplex has the nucleotide sequence shown in Seq ID NO.7;

[0019] The ends of Seq ID NO.1, Seq ID NO.2, and Seq ID NO.3 are each connected to a first connector; the ends of Seq ID NO.4, Seq ID NO.5, and Seq ID NO.6 are each connected to a third connector; the end of the G-quadruplex is connected to a second connector; and the end of the nucleic acid drug sequence is connected to a fourth connector.

[0020] The sequence of the first connector is complementary to the sequence of the second connector; the sequence of the third connector is complementary to the sequence of the fourth connector.

[0021] The single-stranded DNA molecules are interconnected to form a tetrahedral structure; the G-quadruplex is complementary to the single-stranded DNA via the second adapter; the nucleic acid drug is complementary to the single-stranded DNA via the fourth adapter.

[0022] Specifically, the sequences of the first, second, third, and fourth adapters can be obtained through screening and software evaluation, and there is no fixed pattern. Their function is to construct DNA tetrahedrons from Seq ID NO.1 to Seq ID NO.6, and to load nucleic acid drugs onto these DNA tetrahedrons.

[0023] In another embodiment, the nucleic acid drug is selected from one or more of TNF-α siRNA, Bcl-2 siRNA, p53 siRNA, Rex1 siRNA, ApoB1 siRNA, NGF siRNA, MDM2 siRNA, Akt2 siRNA, raf antisense oligonucleotide, PLK1 antisense oligonucleotide, or CpG DNA.

[0024] Specifically, by designing the sequences of the first, second, third, and fourth linkers, DNA tetrahedrons can carry different nucleic acid drugs, including siRNA. Examples include TNF-α siRNA for anti-inflammatory treatment of liver failure, Bcl-2 siRNA targeting the anti-apoptotic protein Bcl2 for lung cancer treatment, p53 siRNA for acute renal failure, Rex1 siRNA regulating stem cell neural differentiation, ApoB1 siRNA for hypercholesterolemia, NGF siRNA for pancreatic cancer, MDM2 siRNA for non-small cell lung cancer, Akt2 siRNA for colorectal liver metastases, raf antisense oligonucleotides for malignant melanoma, PLK1 antisense oligonucleotides for primary gastric cancer, and CpG DNA regulating immunostimulatory activity. These technologies can simultaneously carry two or more different drugs to expand the range of diseases treated, and can also be designed to deliver different drugs to treat two different diseases simultaneously.

[0025] In another embodiment, the chemical drug is selected from one or more of antioxidants, antibiotics, antitumor drugs, or photosensitizers.

[0026] In another embodiment, the TNF-α-siRNA has the nucleotide sequence shown in Seq ID NO.8.

[0027] In another embodiment, the antioxidant is selected from one or more of the anionic compounds manganese porphyrin, iron porphyrin, vitamin C, and N-acetylcysteine; the antibiotic is selected from one or more of doxorubicin, mitoxantrone, daunorubicin, epirubicin, epirubicin, pirarubicin, and carrarubicin; the antitumor drug is selected from one or more of temozolomide, 5-fluorouracil (5-FU), paclitaxel, camptothecin, and platinum-based drugs; and the photosensitizer is selected from one or more of dihydroporphyrin e6 (Ce6), 5,10,15,20-tetra(1-methyl-4-pyridine)porphyrin tetra(p-toluenesulfonic acid) (TMPyP4), and protoporphyrin IX (PIX).

[0028] Specifically, DNA tetrahedra can carry not only specific anionic compounds like porphyrins, but also other drugs. For example, antioxidants like vitamin C and N-acetylcysteine ​​can effectively bind to DNA tetrahedra through covalent linkage. Antibiotics like doxorubicin can insert into the GC-CG base pairs of DNA via quinone ring planar insertion; mitoxantrone inserts into the DNA molecule from the minor groove and has a specific recognition effect on 5'-CG base pairs; daunorubicin, epirubicin, epirubicin, pirarubicin, and carrubicin can bind to DNA through intercalation. Antitumor drugs like temozolomide bind to DNA tetrahedra through electrostatic adsorption; platinum-based drugs can bind to DNA through intercalation; 5-fluorouracil, paclitaxel, and camptothecin can covalently link to DNA structures. Photosensitizers like dihydroporphyrin e6 can bind to DNA structures through covalent linkage, while TMPyP4 and PIX can directly bind to DNA through intercalation. Carrying different drugs can treat different diseases.

[0029] Specifically, the DNA tetrahedral nanodelivery system utilizes G-quadruplexes (G4-DNA) to carry the anionic compound manganese porphyrin (MnP4) through π-π stacking interactions. Six single-stranded DNA molecules self-assemble into a DNA tetrahedral structure through complementary pairing, and then carry nucleic acid drugs (such as the short interfering RNA functional nucleic acid sequence TNF-α-siRNA) through complementary pairing, enhancing the in vivo stability of nucleic acid drugs and achieving targeted delivery to specific organs.

[0030] In another embodiment, the DNA tetrahedral drug further includes a fluorescent dye that modifies the single-stranded DNA.

[0031] In another embodiment, the fluorescent dye is selected from one or more of FAM, Cy5, Cy7, Alexa Fluor 488, AMCA, Cy3, Cy5.5, Quasar 670, TAMRA, and Texas Red.

[0032] Specifically, the fluorescent dye can be modified on one or more single-stranded DNA strands.

[0033] A third aspect of this application provides a method for preparing the aforementioned DNA tetrahedral drug, characterized by comprising the following steps:

[0034] Step 1: Mix single-stranded DNA, G-quadruplex, and nucleic acid drugs in a buffer solution and heat to anneal, obtaining the reactants;

[0035] Step 2: Mix the reactants with chemical drugs to obtain DNA tetrahedral drugs.

[0036] In another embodiment, the molar ratio of the single-stranded DNA, the G-quadruplex, and the nucleic acid drug is 1:3:3; and the concentration of the chemical drug is 50 μM.

[0037] Specifically, the mass ratio of the single-stranded DNA, the G-quadruplex, and the nucleic acid drug is 1:3:3.

[0038] Specifically, the final concentration of the single-stranded DNA (the sum of the concentrations of Seq ID NO.1 to Seq ID NO.6) is 200 nM. Seq ID NO.1, Seq ID NO.2, Seq ID NO.3, Seq ID NO.4, Seq ID NO.5, and Seq ID NO.6 are of the same concentration and mixed in equal volumes.

[0039] Specifically, the buffer solution is Annealing Buffer™ Buffer (10mM Tris, 5mM MgCl2, pH 8.0).

[0040] Specifically, the chemical drug is the anionic compound manganese porphyrin, and the concentration of manganese porphyrin added is 20 μM.

[0041] In another embodiment, in step 1, the annealing temperature is 95°C and the annealing time is 5 min; in step 2, the mixing reaction temperature is 25°C and the mixing reaction time is 1 h.

[0042] Specifically, in step 1, the annealing temperature is 95°C and the annealing time is 5 min; in step 2, the mixing reaction temperature is 25°C and the mixing reaction time is 1 h.

[0043] The fourth aspect of this application discloses the application of the DNA tetrahedral drug in the preparation of liver disease treatment; the nucleic acid drug is TNF-α-siRNA, and the chemical drug is the anionic compound manganese porphyrin.

[0044] This application utilizes six single-stranded DNA to form a DNA tetrahedron, which can serve as a drug carrier. Nucleic acid drugs can be linked to this DNA tetrahedron through base complementary pairing. Simultaneously, chemical drugs can be loaded onto the DNA tetrahedron through positive and negative charge attraction, electrostatic adsorption, covalent linkage, and physical spatial intercalation, thus obtaining a DNA tetrahedron drug simultaneously loaded with both nucleic acid drugs and chemical drugs. This DNA tetrahedron drug directly targets and specifically accumulates in specific organs, which will help improve the precision treatment of various organ diseases, reduce damage to normal tissue cells, and improve treatment safety. The DNA tetrahedron acts as a drug carrier. Compared with other common metal nanomaterials and polymer nanomaterials, the DNA tetrahedron of this application has good biocompatibility and biodegradability, is non-toxic to the human body, and can be regarded as an endogenous biological nanoparticle. It is ultimately degraded into nucleotides in vivo and then reabsorbed and utilized in the nucleotide cycle. Furthermore, compared to traditional single-treatment methods, the DNA tetrahedron of this application can carry a variety of different nucleic acid drugs and chemical drugs, enabling simultaneous treatment of different diseases and promoting multimodal combination or synergistic therapy, thus overcoming the shortcomings of single-treatment methods. Moreover, the DNA tetrahedron drugs of this application possess characteristics such as simple and rapid synthesis, molecular characteristic recognition, and flexible programmability. Attached Figure Description

[0045] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0046] Figure 1 A schematic diagram of the structure of the DNA tetrahedral drug provided in the embodiments of this application and a schematic diagram of its effect on ALF mice;

[0047] Figure 2 Atomic force electron microscopy images and physicochemical characterizations of different drugs (Ⅰ.TDN, Ⅱ.MnP4, Ⅲ.TDN-G4, Ⅳ.TDN-siTNF-α, Ⅴ.TDN-siTNF-α / -G4, Ⅵ.TDN-siTNF-α / -G4-MnP4) provided for embodiments of this application.

[0048] Figure 3 Experiments were conducted to investigate the anti-inflammatory biological activity and M2 polarity conversion promotion activity of different drugs (Ⅰ.TDN, Ⅱ.MnP4, Ⅲ.TDN-G4, Ⅴ.TDN-siTNF-α / -G4, Ⅵ.TDN-siTNF-α / -G4-MnP4) provided in the embodiments of this application.

[0049] Figure 4Intracellular antioxidant bioactivity of different drugs (Ⅰ.TDN, Ⅱ.MnP4, Ⅲ.TDN-G4, Ⅴ.TDN-siTNF-α / -G4, Ⅵ.TDN-siTNF-α / -G4-MnP4) provided in the embodiments of this application.

[0050] Figure 5 The results of in vivo therapeutic effects of CCl4 pretreatment and different drugs (Ⅰ. TDN, Ⅱ. MnP4, Ⅲ. TDN-G4, Ⅳ. TDN-siTNF-α, Ⅴ. TDN-siTNF-α / -G4, Ⅵ. TDN-siTNF-α / -G4-MnP4) provided in the embodiments of this application are presented.

[0051] Figure 6 The results of mRNA sequencing analysis of liver tissue from ALF mice treated with the DNA tetrahedral drug TDN-siTNF-α / -G4-MnP4, as provided in the embodiments of this application, after 2 hours of treatment.

[0052] Figure 7 The stability results of the DNA tetrahedral drug TDN-siTNF-α / -G4 provided in the embodiments of this application.

[0053] Figure 8 The UV-Vis spectrum of the DNA tetrahedral drug TDN-siTNF-α / -G4 provided in the embodiments of this application, and the standard curve for the determination of MnP4 concentration in the loading efficiency of TDN-siTNF-α / -G4 loaded with MnP4.

[0054] Figure 9 The cytotoxicity of different concentrations of MnP4 on different cells is shown in the embodiments of this application.

[0055] Figure 10 The biocompatibility of different concentrations of TDN-siTNF-α / -G4 with different cells is shown in the embodiments of this application.

[0056] Figure 11 The laser confocal scanning microscopy (CLSM) imaging results of TDN-G4 / TNF-αsiRNA-FAM and lysosome colocalization in different cells provided in the embodiments of this application.

[0057] Figure 12 The results of different intracellular uptake of different drugs are provided for the embodiments of this application.

[0058] Figure 13The results of H&E staining scans of ALF mouse liver tissues treated with different drugs (Ⅰ.TDN, Ⅱ.MnP4, Ⅲ.TDN-G4, Ⅳ.TDN-siTNF-α, Ⅴ.TDN-siTNF-α / -G4, Ⅵ.TDN-siTNF-α / -G4-MnP4) provided in the embodiments of this application.

[0059] Figure 14 Neutrophil staining scan results of ALF mouse liver tissue treated with different drugs (Ⅰ.TDN, Ⅱ.MnP4, Ⅲ.TDN-G4, Ⅳ.TDN-siTNF-α, Ⅴ.TDN-siTNF-α / -G4, Ⅵ.TDN-siTNF-α / -G4-MnP4) provided in the embodiments of this application. Detailed Implementation

[0060] This application provides DNA tetrahedral drugs, their preparation methods, and applications, addressing the technical shortcomings of existing technologies such as complex synthesis processes for drug carriers, the cumulative toxicity of nanoparticles, their difficulty in biodegradation, and the potential for adverse reactions.

[0061] Existing manganese porphyrin (MnP4) has good ROS scavenging ability and is widely used in various biomedical applications. However, its short half-life in blood seriously hinders its effective utilization. Short interfering RNA (TNF-α-siRNA) is used to inhibit tumor necrosis factor α (TNF-α), which plays an important role in overcoming inflammation. However, because siRNA is easily degraded or off-target by various nucleases in plasma and kidneys, its widespread clinical application is limited.

[0062] This application discovers that G4-DNA can be used to deliver drugs and interfering RNA in vivo. G4-DNA has unique intrinsic biological properties and modulated cellular behavior functions, including high biocompatibility, low immunogenicity, good biological stability, and cellular behavior regulation.

[0063] This application utilizes the principle of DNA hybridization self-assembly to synthesize a six-armed TDN (DNA tetrahedron) of approximately 25 nm in length by self-assembling short DNA fragments (Seq ID NO.1, Seq ID NO.2, Seq ID NO.3, Seq ID NO.4, Seq ID NO.5, and Seq ID NO.6), a G-quadruplex (G4-DNA, i.e., Seq ID NO.7), and a therapeutic TNF-α-siRNA (a double-stranded RNA, including Seq ID NO.8 and Seq ID NO.9). Each of the eight DNA strands has a corresponding sticky end protrusion at its 3' end. Each DNA tetrahedron has a notch in the middle where the 5' and 3' ends of the oligonucleotide meet. Furthermore, each of the six arms of the DNA tetrahedron is 46 base pairs long. The protrusions at the notches are complementary to the protrusions of the siRNA chain (a therapeutic agent for inhibiting TNF-α gene expression) and the G4-DNA, and together they consist of 303 Watson-Crick base pairs. Figure 1 It is worth noting that anionic MnP4 can act as a G4-DNA ligand and bind to DNA tetrahedra through π-π stacking interactions, thereby enabling the simultaneous delivery of TNF-α-siRNA and MnP4 to exert anti-inflammatory and reactive oxygen species scavenging effects.

[0064] The technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0065] The reagents or drugs used in the following examples are either commercially available or self-made.

[0066] The following examples use TNF-α siRNA (abbreviated as siTNF-α).

[0067] Example 1

[0068] This application provides a DNA tetrahedral drug, and the specific preparation method is as follows:

[0069] like Figure 2 As shown in Figure A, six groups of materials were prepared according to the following method: I. TDN, II. MnP4, III. TDN-G4, IV. TDN-siTNF-α, V. TDN-siTNF-α / -G4, and VI. TDN-siTNF-α / -G4-MnP4.

[0070] Six single-stranded ssDNA molecules of equal concentration (Seq ID NO.1–Seq ID NO.6, with a molar ratio of 1:1:1:1:1:1) were mixed with a G4 strand (Seq ID NO.7) and a siTNF-α double strand (Seq ID NO.8 and Seq ID NO.9, with a molar ratio of 1:1) in Annealing Buffer™ Buffer (10 mM Tris, 5 mM MgCl2, pH 8.0) at a molar ratio of 1:3:3. The final concentration of each single-stranded ssDNA molecule was 200 nM. After thorough mixing, place the mixture in a metal bath and heat at 95°C for 5 minutes. Then, slowly cool it to room temperature to obtain mixture 1 (TDN-siTNF-α / -G4), labeled as TDN-siTNF-α / -G4.

[0071] Six single-stranded ssDNA molecules of equal concentration (Seq ID NO.1–Seq ID NO.6, with a molar ratio of 1:1:1:1:1:1:1) were mixed in Annealing Buffer™ (10 mM Tris, 5 mM MgCl2, pH 8.0), resulting in a final concentration of 200 nM for each ssDNA single strand. After thorough mixing, the mixture was heated at 95°C for 5 minutes in a metal bath and then slowly cooled to room temperature to obtain mixture 2, labeled TDN.

[0072] Six single-stranded ssDNA molecules of equal concentration (Seq ID NO.1–Seq ID NO.6, with a molar ratio of 1:1:1:1:1:1) were mixed with the G4 strand (Seq ID NO.7) at a 1:3 molar ratio in Annealing Buffer™ (10 mM Tris, 5 mM MgCl2, pH 8.0), resulting in a final concentration of 200 nM for each ssDNA single strand. After thorough mixing, the mixture was heated at 95°C for 5 minutes in a metal bath and then slowly cooled to room temperature to obtain mixture 3, labeled TDN-G4.

[0073] Six single-stranded ssDNA molecules (Seq ID NO.1–Seq ID NO.6, with a molar ratio of 1:1:1:1:1:1) and a double-stranded siTNF-α molecule (Seq ID NO.8 and Seq ID NO.9, with a molar ratio of 1:1) were mixed in an equal volume (1:3) with annealing buffer™ (10 mM Tris, 5 mM MgCl2, pH 8.0), resulting in a final concentration of 200 nM for each ssDNA single strand. After thorough mixing, the mixture was heated at 95°C for 5 minutes in a metal bath and then slowly cooled to room temperature to obtain mixture 4, labeled as TDN-siTNF-α.

[0074] (2) The above mixture 1 (TDN-siTNF-α / -G4) was incubated with MnP4 (final concentration 50 μM) at room temperature for 1 hour to obtain DNA tetrahedral drug, which was labeled as TDN-siTNF-α / -G4-MnP4.

[0075] Table 1

[0076]

[0077] Note: Regarding the modification pattern of the siTNF-α sequence, Seq ID NO.8 is siTNF-αsense. The uppercase letters represent ribonucleotides, and the lowercase letters (ct) represent deoxyribonucleic acid. Seq ID NO.9 is TNF-αsiRNA antisense. The uppercase letters with underscores in the Seq ID NO.9 sequence indicate 2′-O-methylribonucleotide modification.

[0078] (3) Atomic force microscopy (AFM) was used to detect the above-mentioned DNA tetrahedral drugs TDNs(Ⅰ), TDN-siTNF-α(Ⅳ), TDN-siTNF-α / -G4(V), and TDN-siTNF-α / -G4-MnP4(ⅤI). The solutions of these materials were diluted and dropped onto the surface of mica sheets, then freeze-dried overnight under vacuum before AFM detection. The results are as follows: Figure 2 As shown, Figure 2 (B) shows the results from atomic force microscopy. Figure 2 (C) The gel was run by non-denaturing polyacrylamide gel electrophoresis (PAGE) (8%, acrylamide / bis(acrylamide = 19:1)) at a constant voltage of 120V for 40 minutes. Then it was gently removed and placed in staining solution containing EB for about 15 minutes. The gel was then scanned and photographed under 312nm UV light in the dark. Figure 2 (D) Baseline calibration of the circular dichroism chromatograph was performed using distilled water. All spectra were obtained in a sample chamber purged with nitrogen. The scan range was 200–320 nm at room temperature, performed in a 1 mm wide quartz reaction vessel, with an instrument scan rate of 60 nm / min and an average of 5 consecutive scans. Figure 2 (E) The hydrodynamic diameters of TDNs (Ⅰ), TDN-siTNF-α (Ⅳ), TDN-siTNF-α / -G4 (V), and TDN-siTNF-α / -G4-MnP4 (ⅤI) were determined by dynamic light scattering (DLS). Figure 2 (F) The potentials of TDNs (Ⅰ), TDN-siTNF-α (Ⅳ), TDN-siTNF-α / -G4 (V), and TDN-siTNF-α / -G4-MnP4 (ⅤI) were measured using a particle size analyzer. GH) Cytotoxicity assay (1) Three cell lines, AML-12, BMDMs, and RAW264.7 (2×10⁻⁶), were subjected to cytotoxicity assay. 5 (1) Cells / wells were incubated overnight in 96-well plates containing 100 μl DMEM / F-12 and DMEM media, respectively. (2) Cells were then incubated for 24 hours with TDN-G4 / TNF-α-siRNA (25 nM, 50 nM, 125 nM, 200 nM, 250 nM), MnP4 (5 μM, 10 μM, 20 μM, 50 μM) and TDN-G4 / TNF-α-siRNA / MnP4 (MnP4 concentration: 20 μM). (3) Fresh medium containing CCK-8 reagent was used instead of the original medium for an additional 2 hours of incubation. The absorbance was measured at 450 nm using an ELISA reader. Untreated cells served as the control group. Each experiment was performed three times independently. Cell viability was calculated using the following formula: Cytotoxicity (%) = (A sample – A blank) / (A control – A blank). IJ) Intracellular TDN-G4 / TNF-α-siRNA uptake experiment. 1. Combine AML-12 and BMDMs, RAW264.7 (2×10) 5 Cells (cells / wells) were cultured in 6-well plates containing DMEM / F-12 and DMEM media, respectively, until the confluence reached approximately 80%-90% after 24 hours. 2. After washing three times with PBS buffer, cells were incubated with TDN-G4 / TNF-α-siRNA medium for 2 hours. 3. After cell culture, cells were washed 2-3 times with PBS buffer, then digested with pre-warmed trypsin for 1-2 min, and immediately collected with PBS buffer containing 2% FBS. 4. The collected cells were centrifuged at 1000 rpm for 5 min and washed twice with PBS buffer. 5. Finally, the fluorescence of TDN-G4 / TNF-α-siRNA in cells was quantified using flow cytometry. Fluorescence was measured by counting 20,000 events.

[0079] Figure 2 Results: Characterization of the TDN-G4 / TNF-α-siRNA / MnP4 co-delivery system: (B) Color bars indicate height in the image. Inset shows monodisperse TDNs, TDN-siTNF-α, TDN-siTNF-α / -G4, and TDN-siTNF-α / -G4-MnP4 on mica. (C) Non-denaturing gel electrophoresis shows the self-assembly process of TDN-siTNF-α / -G4 (M: Marker, lane 1: T1, lane 2: T1+T2, lane 3: T1+T2+T3, lane 4: T1+T2+T3+T4+T5+T6(TDN), lane 5: TDN-siTNF-α / -G4, T1 is Seq ID NO.1, T1+T2 is a mixture of Seq ID NO.1 and Seq ID NO.2 after annealing, T1+T2+T3 is a mixture of Seq ID NO.1, Seq ID NO.2 and Seq ID NO.3 after annealing, T1+T2+T3+T4+T5+T6 is Seq ID NO.1, Seq ID NO.2, Seq ID NO.3, Seq ID NO.4, Seq ID NO.5 and Seq ID NO.2, Seq ID NO.3, Seq ID NO.4, Seq ID NO.5 and Seq ID NO.3, Seq ID NO.4, Seq ID NO.5, and Seq ID NO.3, Seq ID NO.4, Seq ID NO.5, Seq ID NO.6 ... NO.6 The mixture after mixing and annealing. (D) CD spectrum analysis of G-quadruplex in TDN-G4. (E) Hydrodynamic diameters of TDNs (I), TDN-siTNF-α (IV), TDN-siTNF-α / -G4 (V), and TDN-siTNF-α / -G4-MnP4 (VI). (F) Zeta potentials of TDNs (I), TDN-siTNF-α (IV), TDN-siTNF-α / -G4 (V), and TDN-siTNF-α / -G4-MnP4 (VI). (G) Cytotoxicity of different concentrations of MnP4 to AML-12 cells over 24 hours. (h) Various concentrations of TDN-siTNF-α / -G4 over 24 hours. Scale bar, 50 μm. (J) FACS assessment to monitor the uptake of TDN-siTNF-α / -G4 and TDN-siTNF-α / -G4-MnP4 in AML-12 cells.

[0080] Example 2

[0081] This application provides embodiments of experiments on the regulation of inflammation by the DNA tetrahedral drugs TDN-siTNF-α / -G4 and TDN-siTNF-α / -G4-MnP4, specifically including:

[0082] Experimental flowchart as follows Figure 3 A and 3F.

[0083] Experimental results Figure 3Experimental steps for BE:

[0084] 1. After culturing RAW 264.7 and BMDMs immune cells for 24 hours, they were incubated for 4 hours in serum-free DMEM medium containing different materials. Positive control TNF-α-siRNA was transfected using Lipo3000 reagent.

[0085] 2. Then discard the culture medium and add DMEM medium containing serum and continue culturing for 20 hours.

[0086] 3. Next, lipopolysaccharide (LPS, 100ug / uL) was added and the cells were cultured for another 5 hours. Cells were collected, total RNA was extracted, and the expression of TNF-α and IL-6 gene mRNA in RAW 264.7 and BMDMs cells was detected by qRT-PCR.

[0087] II. Experimental Results Figure 3 Experimental steps for GH:

[0088] 1. After culturing RAW 264.7 and BMDMs immune cells for 24 hours, the medium was replaced with serum-free DMEM, and different material treatment groups were added and incubated for 4 hours. Positive control TNF-α-siRNA was transfected using Lipo3000 reagent.

[0089] 2. Then, replace the medium with serum-containing DMEM and continue culturing for 20 hours.

[0090] 3. Next, lipopolysaccharide (LPS, 100ug / uL) was added and cultured for another 5 hours. The supernatant was collected, and the contents of anti-inflammatory factors TNF-α and IL-6 secreted by RAW 264.7 and BMDMs cells were detected by enzyme-linked immunosorbent assay (ELISA).

[0091] Experimental results Figure 3 Experimental steps for EF):

[0092] 1. After culturing immune cells BMDMs for 24 hours, different material treatment groups were added and incubated for 4 hours. Positive control TNF-α-siRNA was transfected using Lipo3000 reagent.

[0093] 2. After washing twice with PBS, add DMEM medium containing LPS (0.2ug / uL) and continue culturing for 4 hours.

[0094] 3. Collect cells, extract total RNA and total protein, and use qRT-PCR to detect the mRNA expression of phenotypic markers M1 (TNF-α and iNOS) and M2 (ARG-1 and YM-1) in BMDMs macrophages.

[0095] IV. Experimental Results Figure 3 The experimental procedure of IJ) was as follows: Western blotting was used to detect the protein expression of phenotypic markers M1 (TNF-α and iNOS) and M2 (ARG-1 and YM-1) of BMDMs.

[0096] The results are as follows Figure 3 As shown, BC) showed the mRNA expression of TNF-α in RAW264.7 (B) and BMDMs (C) cells after 24 hours of treatment. D, E) showed the detection of TNF-β in the supernatant of RAW226.7 (D) and BMDMs (E) cells after 24 hours of different treatments. G, H) showed the mRNA expression of macrophage markers M1 (TNF-α and iNOS) and M2 (Arg-1 and Ym-1) in BMDMs under different conditions, which was evaluated by qRT-PCR analysis (n=3). IL) Western blotting was used to analyze the protein expression of macrophage markers M1 and M2 in BMDMs. β-tubulin was used as a control. Lipo represents siTNF-α transfected with Lipo3000 reagent. Statistical significance is expressed by bars and asterisks, where *P<0.05, **P<0.01, ***P<0.001, ***P<0.0001, and no significant difference was found in NS (n=3).

[0097] The results indicate that the DNA tetrahedral drugs TDN-siTNF-α / -G4(V) and TDN-siTNF-α / -G4-MnP4(VI) possess anti-inflammatory biological activity and alter the polarity of macrophages from the M1 pro-inflammatory phenotype to the M2 anti-inflammatory phenotype.

[0098] Example 3

[0099] The antioxidant bioactivity experiments of the DNA tetrahedral drugs TDN-siTNF-α / -G4 and TDN-siTNF-α / -G4-MnP4 provided in the embodiments of this application are as follows: Figure 4 As shown, it specifically includes:

[0100] Figure 4 A is a structural diagram of five materials: Ⅰ. TDN, Ⅱ. MnP4, Ⅲ. TDN-G4, Ⅴ. TDN-siTNF-α / -G4, Ⅵ. TDN-siTNF-α / -G4-MnP4.

[0101] Figure 4 BI methods include:

[0102] Figure 4The BC method was as follows: I.TDN, II.MnP4, III.TDN-G4, V.TDN-siTNF-α / -G4, and VI.TDN-siTNF-α / -G4-MnP4 were added to BMDMs and RAW264.7 cells induced with 10 μM H2O2 and cultured for 5 hours. Cell viability was detected by CCK-8 assay.

[0103] Figure 4 D) The method is as follows: SOD activity and superoxide radical inhibition rate in cells after different treatments are detected by SOD and superoxide radical kits.

[0104] Figure 4 The EF method was as follows: 1. RAW 264.7 and BMDMs immune cells were treated with different materials and cultured for 24 hours. 2. Then, the cells were replaced with fresh medium containing lipopolysaccharide (LPS, 1 mg / mL) and cultured for another 24 hours. 3. Then, diluted DCFH-DA (final concentration 10 μM) was added for staining for 30 min. 4. The cells were washed twice with serum-free medium, collected, and analyzed by flow cytometry.

[0105] Figure 4 Method G) is as follows: 1. Collect the treated cells (10 × 10⁻⁶). 5 1. Wash cells twice with PBS, resuspend cells in 500 μL staining buffer, add 5 μL Calcein-AM staining solution, and incubate in the dark for 20 minutes. 2. Then add 10 μL PI staining solution, mix gently, and incubate at 4°C in the dark for 10-20 minutes. 3. Wash cells with PBS, aspirate a drop of the mixture onto a glass slide, cover with a coverslip, and immediately perform fluorescence microscopy imaging. H)AML-12 cells were treated with CCL4 (20 mmol / mL) for 24 hours, the culture medium was removed, and a solution containing...

[0106] I. TDN, II. MnP4, III. TDN-G4, V. TDN-siTNF-α / -G4, and VI. TDN-siTNF-α / -G4-MnP4 were cultured in fresh medium for 24 hours, and cell viability was detected using CCK-8 assay.

[0107] Figure 4 The JK cell cycle detection method is as follows: 1. Collect cells (1-5) × 10 61. Centrifuge at 500-1000 rpm for 5 min and discard the culture medium. Wash twice with pre-cooled PBS and fix with 70% cold ethanol at 4°C for 24 hours. 2. Centrifuge for 5 min and discard the supernatant. Resuspend the cells in 100 μg RNase A solution and incubate at 37°C for 20 min. 3. Filter once through a 400-mesh sieve, centrifuge at 500-1000 rpm for 5 min, and discard the PBS. 4. Incubate with 1 mL of PI staining solution at 37°C for 20 min (PI concentration 50 μg / mL). 6. Detect by flow cytometry within 1 hour: PI fluorescence is excited by argon ions at an excitation wavelength of 488 nm and emitted at a wavelength greater than 630 nm, producing red fluorescence. The scatter plot of front-scattered light versus side-scattered light and the histogram of PI fluorescence can be analyzed.

[0108] Figure 4 BC) Effects of I. TDN, II. MnP4, III. TDN-G4, V. TDN-siTNF-α / -G4, and VI. TDN-siTNF-α / -G4-MnP4 on H2O2-induced cell death in BMDMs and RAW264.7 cells. D) Superoxide anion decomposition capacity after various treatments. EF) Reduction of ROS production in BMDMs and RAW264.7 cells by various treatments stimulated with LPS. BMDMs and RAW264.7 cells were stained with DCFH-DA and quantitatively analyzed 24 hours after different treatments. G) Live / dead staining of AML-12 cells pretreated with CCl4 using Calcein AM / PI, followed by incubation with different treatments. H) Percentage of proliferating AML-12 cells pretreated with CCl4 and then incubated with different treatments. Scale bar, 100 μm. I) Relative viability of AML-12, BMDMs, and RAW264.7 cells after 24 hours of different treatments. J, K) FACS cell cycle analysis and corresponding quantitative results of AML-12 cells treated for 24 hours with control (untreated), I. TDN, II. MnP4, III. TDN-G4, V. TDN-siTNF-α / -G4, and VI. TDN-siTNF-α / -G4-MnP4. Statistical significance is expressed by bars and asterisks, where *P<0.05, **P<0.01, ***P<0.001, and ***P<0.0001 (n=3).

[0109] As can be seen, the embodiments of this application demonstrate the antioxidant effect of TDN-G4 / TNF-α-siRNA / MnP4.

[0110] Example 4

[0111] This application provides in vivo therapeutic experiments of the DNA tetrahedral drug TDN-siTNF-α / -G4-MnP4, specifically including:

[0112] ALF animal model was established by intraperitoneal injection of 100 μL (4 μL / g) of 30% CCL4 into 6-week-old male C57 mice using male C57 BL / 6 animals. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and albumin (Alb) levels were measured, and histological changes were observed using light and electron microscopy. In addition, liver tissue was dissected from the mice and macroscopic changes were observed. Mice were randomly divided into 8 groups of 10 each, and injected intravenously via tail vein with 100 μL of physiological saline, 100 μL of TDN, 100 μL of MnP4, 100 μL of TNF-α-siRNA, 100 μL of TDN-G4, 100 μL of TDN / TNF-α-siRNA, 100 μL of TDN-G4 / TNF-α-siRNA, and 100 μL of TDN-G4 / TNF-α-siRNA-MnP4, respectively. Two hours and 24 hours after treatment, 200 μL of butorphanol was injected intraperitoneally, and 300 μL of blood was collected from the tail vein for testing liver function, kidney function, and inflammatory factors. The animals were then euthanized using CO2 inhalation, and the major organs (heart, liver, spleen, lung, and kidney) of each group of mice were collected and fixed in formalin solution. After 1-2 days, they were embedded in paraffin and sectioned. Hematoxylin-eosin staining was performed on the heart, liver, spleen, lung, and kidney sections, and Ki67 and Ly6g immunohistochemistry and UNEL assay were performed on the liver sections. Finally, the inhibition rates of superoxide radicals and hydroxyl radicals in the liver tissue were measured.

[0113] The results are as follows Figure 5 As shown, Figure 5A) Preparation and treatment plan for ALF mice. Intraperitoneal injection of CCl4 (IP); intravenous injection (IV). B) Digital photographs of major organs in ALF mice 2 hours after different treatments. C) Intravenous injection of control CCl4, Cy7 TDNsiTNF-α / -G4, and Cy7 TDN-siTNF-α. D) Hematological marker analysis of liver damage in ALF mice after different treatments. ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALB, albumin. Normal range (blue background). E) Detection of serum TNF-α and IL-6 in mice after different treatments. F) Hematological marker analysis of kidney damage in ALF mice 2 hours after different treatments, including urea (UN) and creatinine (CRE). Normal range (blue background). G) H&E staining (N, necrosis), neutrophil staining (SI, severe inflammation), TUNEL-positive cells (red nuclei), M1 (Cox-2, green) and M2 (CD206, red) macrophage markers, and Ki-67-positive cells (red nuclei) from ALF mouse liver tissue treated for 2 hours. Scale bar, 100 μm.

[0114] Example 5

[0115] This application provides the mRNA sequencing and transcriptomics analysis results of ALF mouse liver tissues treated with or without the DNA tetrahedral drug TDN-siTNF-α / -G4-MnP4, specifically including:

[0116] In the above-described embodiments, mRNA sequencing was performed on liver tissues of ALF mice treated with or without TDN-siTNF-α / -G4-MnP4. The results are as follows: Figure 6 As shown.

[0117] Figure 6A) Principal component analysis (PCA) score plot of differentially expressed genes in the liver of ALF mice with and without TDN-siTNF-α / -G4-MnP4 treatment. Each data point corresponds to the PCA analysis for each sample. B) Volcano plot showing significantly upregulated (Sig Up) and downregulated (Sig Down) genes in the ALF group and the TDN-siTNF-α / -G4-MnP4 treatment group. Yellow dots: upregulated genes (FC>2). Blue dots: downregulated genes (FC<0.5). Gray dots: no significant difference. C) Gene Ontology (GO) enrichment analysis of TDN-siTNF-α / -G4-MnP4 treatment-related pathways is highlighted in red (inflammatory response), green (negative regulation of apoptosis), blue (redox process), and purple (positive regulation of cell proliferation). D) Enrichment plot from GSEA analysis of the "TNF_SIGNALING_PATHWAY" and "apoptosis" gene sets. NES, normalized enrichment score. FDR (False Discovery Rate), |NES|>1, and FDR q-value <0.25 were considered different between the two groups. E) Heatmap of inflammatory response markers (red shading), negative regulation of apoptosis (green shading), antioxidants (blue shading), and positive regulation of cell proliferation (purple shading), selected from mRNA sequencing data after TDN-siTNF-α / -G4-MnP4 treatment (P<0.05). F) KEGG pathway analysis of differentially expressed genes using OmicShare tool. G) Protein-protein interaction (PPI) network of proteins encoded by differentially expressed genes. Circles represent genes, and the area of ​​the circle represents the degree of gene expression.

[0118] Example 6

[0119] This application provides stability experiments for the DNA tetrahedral drug TDN-siTNF-α / -G4, specifically including:

[0120] The DNA tetrahedral drug TDN-siTNF-α / -G4 was incubated with serum at 37°C, and then the samples were collected for PAGE analysis. The results are as follows: Figure 7 As shown. M, Marker; Lane 1, siTNF-α; Lane 2, siTNF-α in serum after 2 hours; Lane 3, TDN-siTNF-α / -G4 in TM buffer; Lane 4, TDN-siTNF-α / -G4 in serum after 2 hours.

[0121] Example 7

[0122] This application provides cytotoxicity and biocompatibility experiments of different drugs MnP4 and TDN-siTNF-α / -G4, specifically including:

[0123] Determination of the UV-Vis spectrum of MnP4 and the standard curve for concentration determination: 1. Incubate 200 nM TDN-G4 / TNF-α siRNA with 20 μM MnP4 at room temperature for 1 hour. 2. Then centrifuge the mixture at 1000 rpm for 10 minutes at room temperature. 3. Collect the supernatant and determine the amount of unloaded free MnP4 in the supernatant at 560 nm using a UV-Vis spectrophotometer. Calculation formulas for loading amount and loading efficiency: Final MnP4 loading amount = Initial MnP4 content - Unloaded free MnP4 content in the supernatant. MnP4 loading efficiency = (Final MnP4 loading amount / Initial MnP4 content) × 100%. Results are as follows: Figure 8 As shown, Figure 8 (A) shows the UV-Vis spectrum of MnP4. (B) shows the standard curve for MnP4 concentration determination.

[0124] After incubating cells with different concentrations of MnP4 for 24 hours, its cytotoxicity against (A) BMDM and (B) RAW264.7 cells was determined. Figure 9 The cytotoxicity of different concentrations of MnP4 on (A) BMDM and (B) RAW264.7 cells is shown.

[0125] The biocompatibility of different concentrations of TDN-siTNF-α / -G4 with (A) BMDMs and (B) RAW264.7 cells after incubation with cells for 24 hours was determined using the same method. Figure 10 The biocompatibility of different concentrations of TDN-siTNF-α / -G4 on (A) BMDMs and (B) RAW264.7 cells is shown.

[0126] Example 8

[0127] This application provides laser confocal scanning microscopy (CLSM) imaging experiments to detect the co-localization of TDN-G4 / TNF-α siRNA-FAM and lysosomes in different cells, specifically including:

[0128] 1. Under standard culture conditions, at 1×10 4 cells / cm 21. AML-12 cells, BMDMs cells, and Raw264.7 cells were seeded at a density of [insert density here] in 35 mm confocal culture dishes. 2. Cells were then washed twice with PBS buffer and co-cultured for 2 hours with FAM-labeled TDN-G4-TNF-α-siRNA. 3. Cells were then gently washed twice with pre-cooled PBS buffer. 10 μM of a lysosomal red fluorescent probe was added, and the cells were incubated for 30 minutes for specific staining of the lysosomes. 4. Cells were then stained with Hoechst 33342 for approximately 25 minutes. 5. Finally, cells were rinsed three times with PBS buffer. 6. The location of DN-G4-TNF-α-siRNA within the living cells was determined using a laser confocal scanning microscope. Fluorescence imaging of FAM-TDN-G4-TNF-α-siRNA and Hoechst 33342 was performed using a 60× oil immersion objective lens. The excitation wavelengths were 405 nm and 543 nm, respectively, and the corresponding emission wavelengths were in the range of 430 / 480 nm and 550 / 630 nm. Figure 11 CLSM images showing co-localization of TDN-G4 / TNF-α siRNA-FAM and lysosomes in AML-12 cells, BMDMs cells, and Raw 264.7 cells (Scale bars: 50 μm).

[0129] Example 9

[0130] This application provides embodiments of experiments on the uptake of different drugs in different cells, specifically including:

[0131] BMDMs and RAW264.7 cells were incubated with different drugs (II.MnP4, V.TDN-siTNF-α / -G4, VI.TDN-siTNF-α / -G4-MnP4) for 2 hours, respectively. Then, FACS (flow cytometry fluorescence sorting) was used to estimate the uptake of different drugs in (A) BMDMs and (B) RAW264.7 cells within 2 hours. The results are as follows: Figure 12 As shown.

[0132] Example 10

[0133] This application provides pathological experiments on liver tissues of ALF mice treated with different drugs, specifically including:

[0134] ALF mice treated with different drugs (Ⅰ. TDN, Ⅱ. MnP4, Ⅲ. TDN-G4, Ⅳ. TDN-siTNF-α, Ⅴ. TDN-siTNF-α / -G4, Ⅵ. TDN-siTNF-α / -G4-MnP4) for 24 hours had their liver tissues harvested for H&E staining and neutrophil staining. The results are as follows: Figure 13-14 As shown, Figure 13H&E staining (N, necrosis) of liver tissue from ALF mice treated 24 hours later. Scale bar, 100 μm; Neutrophil staining (SI, severe inflammation) of liver tissue from ALF mice treated 24 hours later. Scale bar, 100 μm.

[0135] The above examples demonstrate that the study of the molecular mechanisms related to the antioxidant, anti-inflammatory, and proliferation-promoting effects of the DNA tetrahedral drug TDN-siTNF-α / -G4-MnP4 elucidates the mechanism of multi-pathway synergistic treatment of ALF and verifies its rapid and significant efficacy (significant efficacy can be observed 2 hours after intravenous injection). Therefore, through experiments on the molecular mechanisms of TDN-siTNF-α / -G4-MnP4 in antioxidation, anti-inflammation, and proliferation, the intrinsic multi-mode mechanism of action of the TDN-siTNF-α / -G4-MnP4 complex against ALF has been elucidated. This will not only provide clinical reference for multi-mechanism combination therapy of ALF but also contribute to establishing a theoretical basis for the development of more effective, safe, and rapid DNA tetrahedral drug nanomaterials. Furthermore, animal experiments using a mouse model of ALF induced by the hepatotoxin carbon tetrachloride (CCl4) showed that TDN-siTNF-α / -G4-MnP4, as a therapeutic system, can not only alleviate ALF damage but also achieve passive and preferential accumulation in the liver. This will further improve the therapeutic effects of gene therapy and antioxidant drugs on ALF, while effectively avoiding damage to normal tissue cells and reducing side effects. This will open up new directions for the application and development of DNA tetrahedral drug materials and the treatment of various liver-related diseases.

[0136] In summary, the DNA tetrahedral drug carrier of this application possesses molecular recognition characteristics, controllable nanoscale size, and specific encoding capabilities. It directly targets or specifically accumulates in the liver, which will help improve the precision treatment of various liver diseases, reduce damage to normal tissue cells, and improve treatment safety. Furthermore, it exhibits excellent biocompatibility and biodegradability, is non-toxic to the human body, and can be considered as endogenous biological nanoparticles. In vivo, it is ultimately degraded into nucleotides, which then enter the nucleotide cycle for reabsorption and utilization. As can be seen from the above embodiments, the DNA tetrahedral drug multifunctional delivery system based on this application promotes multimodal combined or synergistic treatment of acute liver failure, overcoming many shortcomings of single treatment methods, and has no serious side effects.

[0137] The above description is only a preferred embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

[0138]

[0139]

[0140]

[0141]

[0142]

Claims

1. A DNA tetrahedral drug, characterized in that, include: Single-stranded DNA, G-quadruplexes, nucleic acid drugs, and chemical drugs; The single-stranded DNA has the nucleotide sequences shown in Seq ID NO.1, Seq ID NO.2, Seq ID NO.3, Seq ID NO.4, Seq ID NO.5 and Seq ID NO.6; The G-quadruplex has the nucleotide sequence shown in Seq ID NO.7; The ends of Seq ID NO.1, Seq ID NO.2, and Seq ID NO.3 are each connected to a first connector; the ends of Seq ID NO.4, Seq ID NO.5, and Seq ID NO.6 are each connected to a third connector; the end of the G-quadruplex is connected to a second connector; and the end of the nucleic acid drug sequence is connected to a fourth connector. The sequence of the first connector is complementary to the sequence of the second connector; the sequence of the third connector is complementary to the sequence of the fourth connector. The single-stranded DNA molecules are interconnected to form a tetrahedral structure; the G-quadruplex is complementary to the single-stranded DNA via the second adapter; the nucleic acid drug is complementary to the single-stranded DNA via the fourth adapter.

2. The DNA tetrahedral drug according to claim 1, characterized in that, The nucleic acid drug is selected from one or more of TNF-α siRNA, Bcl-2 siRNA, p53 siRNA, Rex1 siRNA, ApoB1 siRNA, NGF siRNA, MDM2 siRNA, Akt2 siRNA, raf antisense oligonucleotide, PLK1 antisense oligonucleotide, or CpG DNA.

3. The DNA tetrahedral drug according to claim 1, characterized in that, The chemical drug is selected from one or more of antioxidants, antitumor drugs, or photosensitizers.

4. The DNA tetrahedral drug according to claim 1, characterized in that, The chemical drug is selected from antibiotic drugs.

5. The DNA tetrahedral drug according to claim 2, characterized in that, The TNF-α-siRNA has the nucleotide sequence shown in Seq IDNO.

8.

6. The DNA tetrahedral drug according to claim 3, characterized in that, The antioxidant is selected from one or more of the anionic compounds manganese porphyrin, iron porphyrin, vitamin C, and N-acetylcysteine; the antitumor drug is selected from one or more of temozolomide, 5-fluorouracil, paclitaxel, camptothecin, and platinum-based drugs; the photosensitizer is selected from one or more of dihydroporphyrin e6, 5,10,15,20-tetra(1-methyl-4-pyridine)porphyrin tetra(p-toluenesulfonic acid), and protoporphyrin IX.

7. The DNA tetrahedral drug according to claim 4, characterized in that, The antibiotic is selected from one or more of doxorubicin, mitoxantrone, daunorubicin, epirubicin, epirubicin, pirarubicin, and carrubicin.

8. The method for preparing the DNA tetrahedral drug according to any one of claims 1 to 7, characterized in that, Includes the following steps: Step 1: Mix single-stranded DNA, G-quadruplex, and nucleic acid drugs in a buffer solution and heat to anneal, obtaining the reactants; Step 2: Mix the reactants with chemical drugs to obtain DNA tetrahedral drugs.

9. The preparation method according to claim 8, characterized in that, The volume ratio of the single-stranded DNA, the G-quadruplex, and the nucleic acid drug is 1:3:3; the concentration of the chemical drug added is 50 μM.

10. The preparation method according to claim 8, characterized in that, In step 1, the annealing temperature is 95 ℃ and the annealing time is 5 min; in step 2, the mixing reaction temperature is 25 ℃ and the mixing reaction time is 1 h.

11. The use of the DNA tetrahedral drug according to any one of claims 1 to 7 in the preparation of a drug for treating acute liver failure; wherein the nucleic acid drug is TNF-α-siRNA, and the chemical drug is the anionic compound manganese porphyrin.

Images