A DNA nanocage with TLR9 agonist activity and its application in the preparation of anti-tumor drugs

Abstract

The present invention belongs to the technical fields of biomedicine and molecular biology, and particularly relates to a DNA nanocage with TLR9 agonist activity and its application in the preparation of anti-tumor drugs. The DNA nanocage is formed by the precise base complementary pairing of oligonucleotides with CpG ODN activity, tumor-responsive nucleases and their substrates, and the obtained nanocage is an icosahedral cavity cage structure. The DNA nanocage can encapsulate anti-tumor drugs and / or drug carriers. Through experimental verification, the nanocage has a good activation effect on TLR9 of DC cells, can promote DC cells to secrete type I interferon, up-regulate the expression of co-stimulatory molecules, reverse the tumor microenvironment, and it can enhance the therapeutic effect on tumors, especially "cold" tumors in the immunological sense, and improve the survival rate of patients. Therefore, it has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical fields of biomedicine and molecular biology, and particularly relates to a DNA nanocage with TLR9 agonist activity and its application in the preparation of anti-tumor drugs. Background Art

[0002] The information disclosed in the background art of the present invention is only intended to increase the understanding of the overall background of the present invention, and is not necessarily regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Immunotherapy refers to the regulation of the body's immune function based on immunological principles and methods, aiming at the immune state of the body's immune system being too strong or too weak, and using artificial intervention measures to weaken or strengthen the body's immune response to achieve the purpose of treating diseases. Immunotherapy can enhance the ability of the immune system to target, recognize, and eliminate cancer cells in the body. In recent years, tumor immunotherapy has made great progress, providing hope for a variety of cancers. Immunotherapy mainly includes: immune checkpoint inhibitors (PD-1 / L1, CTLA-4), tumor vaccines, cell immunotherapy (CAR-T, CAR-NK), etc. Among them, immune checkpoint inhibitors block the binding of immune checkpoint molecules such as PD-1 and CTLA-4 expressed on the surface of T cells to their ligands on the surface of tumor cells, block the inhibitory effect of tumor cells on T cells, and enhance the anti-tumor ability of the body's immune system. Tumor vaccines include DNA vaccines, mRNA vaccine antigens / adjuvant vaccines, whole cell vaccines, etc., and produce a strong anti-tumor immune response by enhancing the presentation of tumor-associated antigens. Cell immunotherapy mainly involves transfusing chimeric antigen receptor T cells and NK cells into patients, and using the modified CAR-T / CAR-NK to recognize and kill tumor cells.

[0004] Tumor immunotherapy has developed rapidly in recent years and is a very promising tumor treatment method. Although it has great potential clinically, only a few patients respond to immunotherapy. The biggest obstacle to tumor immunotherapy is the tumor microenvironment. There is insufficient infiltration and impaired function of T cells in the tumor microenvironment. At the same time, tumor cells secrete a variety of cytokines and chemokines to promote the activation of surrounding stromal cells and immunosuppressive cells, resulting in a large infiltration of immunosuppressive cells such as regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment. Immunosuppressive cells further secrete immunosuppressive factors such as IL-6 and IL-10 to inhibit effector T cells and NK cells. Eventually, an immunologically "cold" tumor is formed.

[0005] TLR9 is a member of the Toll-like receptor family and belongs to a type of pattern recognition receptor. It can recognize corresponding pathogen-associated molecular patterns and damage-associated molecular pattern signals, thereby initiating innate immune responses and playing a role in regulating the body's homeostasis and host defense. TLR9 agonists can regulate the body's innate and adaptive immunity, enhance the immune response, thereby increasing the body's immune system's ability to recognize and kill tumor cells, showing significant efficacy in anti-tumor treatment and gradually becoming a hot topic in the field of tumor treatment. As a classic immune stimulant, TLR9 agonists exert immunological effects by activating TLR9. TLR9 is expressed in a variety of cells, especially highly expressed on the endosomal membrane of plasmacytoid dendritic cells (pDCs). TLR9 on the endosomal membrane of dendritic cells (DCs) can recognize CpG DNA of bacteria and viruses and effectively activate two major TLR9 signal transduction pathways: IRF7 and NF-κB. Activation of the IRF7 signaling pathway mediates the rapid production of IFN-α, which then activates NK cells, blocks immunosuppression, and promotes the homing of Th1 cells and CD8 + T cells to tumors. At the same time, activation of the NF-κB pathway can upregulate the expression of co-stimulatory molecules such as CD80, CD86, and CD40 on the surface of DCs, promoting the generation and activation of tumor-specific CD8 + T cells. Studies have shown that TLR9 agonists such as CpG ODN and lefitolimod can not only inhibit the occurrence and development of various cancers such as breast cancer, melanoma, and head and neck squamous cell carcinoma, but also inhibit the progression of tumors in distant sites. Currently, the FDA has approved a variety of TLR9 agonists for clinical trials. Such as SD-101 for melanoma, breast cancer, and head and neck squamous cell carcinoma, and Tilsotolimod in clinical phase II for the treatment of anti-PD-1 antibody-refractory melanoma and stage IIb-IV melanoma.

[0006] TLR9 agonists have the immunological properties of activating the body's innate immunity and reversing the tumor microenvironment. Existing studies have shown that treatment with TLR9 agonists can increase the secretion of chemokines and cytokines that recruit and activate anti-tumor immune cells, promote the tumor infiltration of T cells and DCs, while reducing the numbers of Tregs and MDSCs, enhancing the cytotoxic T lymphocyte response, and transforming the tumor microenvironment of murine pancreatic cancer into a lymphocyte-rich "hot" tumor phenotype. In addition, TLR9 agonists can affect tumor drug resistance mediated by pancreatic stellate cells by activating TLR9 on pancreatic stellate cells, changing the properties of pancreatic stellate cells, and reducing the expression of chemotherapy-resistant factors.

[0007] DNA nanocages are three-dimensional polyhedral nanostructures formed by the self-assembly of multiple synthetic DNA oligonucleotide strands through base pairing and hybridization. Common shapes include tetrahedrons, octahedrons, trigonal bipyramids, and dodecahedrons. DNA nanocages can easily combine with different chemical groups and biomolecules, endowing them with various functions.

[0008] DNA nanocages have many advantages as drug carriers: (1) They have a well-defined and predictable structure, with a porous structure, large internal space, and high loading capacity inside the cage; (2) They can respond to changes in the external environment and release the loaded drugs in an intelligent manner; (3) They have low cytotoxicity and do not cause severe immune stimulation reactions, being relatively safe; (4) They can protect the loaded drugs from the influence of the external environment; (5) They have good stability and are not easily degraded by ribozymes; (6) DNA nanocages do not require transfection reagents and can directly enter living cells, with good biocompatibility; (7) DNA nanocages have abundant structure modification sites, and through modification, targeted functions can be achieved. Therefore, DNA nanocages have good application prospects in drug carriers, biological detection, cell imaging, materials science, and other fields. Summary of the Invention

[0009] In view of the above-mentioned prior art, the purpose of the present invention is to provide a DNA nanocage with TLR9 agonist activity and its application in the preparation of anti-tumor drugs. The present invention particularly provides a DNA nanocage with TLR9 agonist-CpG ODN activity, which can enhance the therapeutic effect on tumors, especially "cold" tumors in the immunological sense, and improve the survival rate of patients. Based on the above research results, the present invention is completed.

[0010] Specifically, the technical solution of the present invention is as follows:

[0011] In the first aspect of the present invention, a DNA nanocage with TLR9 agonist activity is provided. The DNA nanocage is formed by the precise base complementary pairing of oligonucleotides with CpG ODN activity and tumor-responsive nucleases and their substrates. The resulting nanocage has an icosahedral cavity cage structure. Further, the DNA nanocage can encapsulate anti-tumor drugs and / or drug carriers.

[0012] The tumor-responsive nucleases include, but are not limited to, DNA enzymes and RNA enzymes; the tumor responsiveness can depend on factors such as H2O2, pH, and glutathione, etc., which are not specifically defined here. Correspondingly, based on specific tumor-responsive conditions, the tumor-responsive nucleases can be specifically designed.

[0013] Specifically, the DNA nanocage with TLR9 agonist activity (named Fe@DN) can be formed by oligonucleotides with murine TLR9 agonist CpG ODN sequences, H2O2-responsive DNAzyme 8-17 and its substrate 8-17D to construct an icosahedral DNA cage, while encapsulating and loading ferritin loaded with chloroquine phosphate.

[0014] In the second aspect of the present invention, there is provided a method for preparing the above DNA nanocage with TLR9 agonist activity, and the preparation method at least includes combining oligonucleotides with CpG ODN activity and a tumor-responsive nuclease and its substrate through DNA modular assembly technology.

[0015] In the third aspect of the present invention, there is provided the application of the above DNA nanocage with TLR9 agonist activity in the preparation of anti-tumor drugs.

[0016] In the fourth aspect of the present invention, there is provided an anti-tumor drug, which comprises the above DNA nanocage with TLR9 agonist activity, and the DNA nanocage with TLR9 agonist activity can be used as a drug active ingredient and / or a drug delivery system.

[0017] The anti-tumor drug may further include at least one drug active ingredient and / or at least one drug inactive ingredient.

[0018] In the fifth aspect of the present invention, there is provided a method for treating tumors, which includes administering a therapeutically effective dose of the above DNA nanocage with TLR9 agonist activity or anti-tumor drug to a subject.

[0019] The beneficial technical effects of the above one or more technical solutions:

[0020] 1. The above technical solution provides a method for preparing a DNA nanocage for synergistic cascade TLR9 activation and immunotherapy. A DNA cage is formed by oligonucleotides with CpG ODN activity and a tumor-responsive nuclease and its substrate, such as synthesized by murine CpG, human CpG, H2O2-responsive DNAzyme 8-17, etc., but not limited to a certain one.

[0021] 2. The above technical solution provides a method for preparing a DNA nanocage for synergistic cascade TLR9 activation and immunotherapy. The synthesized DNA cage can deliver drugs or drug carriers with appropriate particle sizes, including ferritin, platinum nanoparticles, gold nanoparticles, etc., but not limited to a certain one.

[0022] 3. The above technical solution provides a DNA nanocage Fe@DN for synergistic cascade TLR9 activation and immunotherapy and its preparation method. The drug particles prepared by this preparation method have a diameter of about 40 nm and have good passive targeting effect. It has been verified that the nanocage has a good activation effect on TLR9 of DC cells, can promote DC cells to secrete type I interferon, up-regulate the expression of co-stimulatory molecules, reverse the tumor microenvironment, and improve the survival rate of tumor-bearing mice. Therefore, it has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings forming a part of this invention are used to provide a further understanding of the invention. The schematic embodiments and descriptions thereof of the invention are used to explain the invention and do not constitute an improper limitation of the invention.

[0024] Figure 1 Synthesis schematic diagram of Fe@DN prepared in Example 1 of the present invention;

[0025] Figure 2 TEM image of Fe@DN prepared in Example 1 of the present invention;

[0026] Figure 3 Particle size distribution diagram of Fe@DN prepared in Example 1 of the present invention;

[0027] Figure 4 Zeta potential diagram of Fe@DN prepared in Example 1 of the present invention;

[0028] Figure 5 Experimental diagram of the effect of Fe@DN on the secretion level of type I interferon in DC in Example 2 of the present invention;

[0029] Figure 6 Experimental diagram of the effect of Fe@DN on the expression level of co-stimulatory molecules in DC in Example 2 of the present invention;

[0030] Figure 7 Tumor progression of mice after treatment with different preparations in Example 3 of the present invention;

[0031] Figure 8 Survival rate of mice after treatment with different preparations in Example 3 of the present invention;

[0032] Figure 9 HE staining of major organs of mice after treatment with different preparations in Example 3 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0033] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0034] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular forms are also intended to include the plural forms. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they specify the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0035] The present invention will be further described below in conjunction with specific examples. The following examples are only for explaining the present invention and do not limit its content. If the specific experimental conditions are not indicated in the examples, they are usually in accordance with conventional conditions or the conditions recommended by reagent companies; the reagents, consumables, etc. used in the following examples can be obtained from commercial sources without special instructions.

[0036] As mentioned above, tumor immunotherapy has developed rapidly in recent years and is a very promising tumor treatment method. However, the biggest obstacle to tumor immunotherapy is the "cold" tumor microenvironment. TLR9 agonists can regulate the innate and adaptive immunity of the body, enhance the immune response, thereby increasing the ability of the body's immune system to recognize and kill tumor cells, showing significant efficacy in anti-tumor treatment, and gradually becoming a hot spot in the field of tumor treatment.

[0037] In view of this, the present invention aims to provide a TLR9 agonist with better immune stimulation effect, reverse the "cold" tumor microenvironment and improve the effect of tumor immunotherapy, thereby providing a DNA nanocage that synergistically cascades TLR9 activation and immunotherapy.

[0038] Specifically, in a typical specific embodiment of the present invention, a DNA nanocage with TLR9 agonist activity is provided. The DNA nanocage is formed by the precise base complementary pairing of oligonucleotides with CpG ODN activity and tumor-responsive nucleases and their substrates. The resulting nanocage is an icosahedral cavity cage structure. Further, the DNA nanocage can encapsulate anti-tumor drugs and / or drug carriers.

[0039] Among them, the sources of the oligonucleotides with CpG ODN activity include, but are not limited to, human sources (such as human CpG) and non-human mammalian sources (such as murine CpG), and no specific limitations are made here.

[0040] The tumor-responsive nucleases include, but are not limited to, DNA nucleases and RNA nucleases; the tumor responsiveness can depend on, such as H2O2, pH, and glutathione, etc., and specific limitations are not made here. Correspondingly, based on specific tumor-responsive conditions, the tumor-responsive nucleases can be specifically designed. In a specific embodiment of the present invention, the tumor-responsive nuclease can be the H2O2-responsive DNAzyme 8-17; the responsiveness of the DNAzyme 8-17 to H2O2 can be achieved by modifying the phenylborate group on the DNAzyme 8-17. The phenylborate group is a group with high sensitivity and high selectivity to H2O2. Therefore, by modifying the phenylborate group on some nucleotides of the DNAzyme 8-17, the responsiveness to H2O2 is finally achieved.

[0041] The anti-tumor drugs include, but are not limited to, chemotherapeutic drugs, small molecule drugs, nucleic acid therapeutic drugs, steroid drugs, or combinations thereof; further, small molecule drugs (such as chloroquine phosphate) can be preferably used. The drug carriers include, but are not limited to, platinum nanoparticles, small molecule peptides, ferritin, gold nanoparticles, Fe nanoparticles, and Cu nanoparticles; further, ferritin can be preferably used.

[0042] Therefore, in a specific embodiment of the present invention, the DNA nanocage with TLR9 agonist activity (named Fe@DN) can be an icosahedral DNA cage formed by an oligonucleotide with a murine TLR9 agonist CpG ODN sequence, the H2O2-responsive DNAzyme 8-17, and its substrate 8-17D, and at the same time encapsulate and load ferritin loaded with chloroquine phosphate.

[0043] The design idea of the drug in the present invention is as follows: Fe@DN responds to the local H2O2 in the tumor, restores the activity of DNAzyme 8-17, cleaves the DNA cage of Fe@DN into two parts, and releases the ferritin drug carrier. After cleavage, the DNA nanocage is taken up by DC cells, activates TLR9 on the endosomal membrane, activates NK cells, blocks immunosuppression, promotes the homing of Th1 cells and CD8 + T cells to the tumor, and at the same time upregulates the expression of co-stimulatory molecules on the surface of DCs, promoting the generation and activation of tumor-specific CD8 + T cells, and reversing the "cold" tumor microenvironment.

[0044] Ferritin, as a drug carrier, targets tumor cells to release chloroquine phosphate, inhibits tumor cell autophagy, upregulates the expression of MHC class I molecules, and further improves immunogenicity, thereby generating an effective immune response. The DNA nanocage with TLR9 agonist activity provides a platform for the TLR9 activation / immunotherapy synergistic cascade, and it can be used as a flexible and powerful synergistic system to treat malignant tumors, especially "cold" tumors.

[0045] In one or more specific embodiments of the present invention, there is provided a method for preparing the above DNA nanocage having TLR9 agonist activity, and the preparation method at least includes combining an oligonucleotide with CpG ODN activity and a tumor-responsive nuclease and its substrate through DNA modular assembly technology.

[0046] Specifically, the DNA nanocage having TLR9 agonist activity is Fe@DN, and its preparation method includes:

[0047] S1. Formation of pentamers V, U, X, and L: Mix the phosphorylated oligonucleotide fragments of V1 to V5 in a buffer, heat and anneal; after incubation, equilibrate at low temperature to form pentamer V; similarly obtain pentamers U, X, and L;

[0048] S2. Preparation of VU5 and XL5: Thoroughly mix pentamer V and pentamer U, heat and anneal; after incubation, equilibrate at low temperature to form VU5 for subsequent chemical ligation; similarly obtain XL5;

[0049] S3. Chemical ligation: Non-enzymatic chemical ligation is used to link adjacent 3'-hydroxyl and 5'-phosphate groups; N-cyanoimidazole is used for the ligation reaction; the mixture is incubated at room temperature; after the ligation is completed, dialysis is carried out using a buffer;

[0050] S4. After chemical ligation, mix VU5, XL5, and DNAzyme 8-17-3BO-8-17D, add it to a solution containing ferritin loaded with chloroquine phosphate, heat and anneal; after incubation, equilibrate to obtain a solution;

[0051] S5. The solution obtained in step S4 is dialyzed to obtain Fe@DN.

[0052] In another specific embodiment of the present invention, in step S1,

[0053] The molar ratio of the phosphorylated oligonucleotide fragments of V1 to V5 is 1:0.5 - 2:0.5 - 2:0.5 - 2:0.5 - 2:0.5 - 2, preferably an equimolar ratio of 1:1:1:1:1. At this time, the molar concentration of each phosphorylated oligonucleotide fragment is 10 - 30 μM, preferably 20 μM;

[0054] Heating and annealing is heating at 85 - 95 °C for 10 - 30 min, preferably heating at 90 °C for 20 min, and then annealing to 20 °C at a rate of 0.1 - 1 °C / min (preferably 0.33 °C / min);

[0055] The incubation temperature can be 15 - 25 °C (preferably 20 °C), and the low temperature condition can be not higher than 5 °C, preferably 4 °C;

[0056] The nucleotide sequences of V1-V5 are shown in SEQ ID NO.1-5;

[0057] The nucleotide sequences of U1-U5 are shown in SEQ ID NO.6-10;

[0058] The nucleotide sequences of X1-X5 are shown in SEQ ID NO.11-15;

[0059] The nucleotide sequences of L1-L5 are shown in SEQ ID NO.16-20.

[0060] In another specific embodiment of the present invention, in the step S2,

[0061] The molar ratio of pentamer V to pentamer U is 1:1-10, preferably 1:5;

[0062] The heating and annealing are carried out at 45-55 °C for 3-6 h, preferably at 50 °C for 4 h, and then annealed to 20 °C at a rate of 0.1-1 °C / min (preferably 0.33 °C / min);

[0063] In another specific embodiment of the present invention, in the step S3,

[0064] The concentration of N-cyanoimidazole is 10-100 mM, preferably 50 mM;

[0065] In another specific embodiment of the present invention, NiCl2 is added in the step S3 to improve the ligation efficiency. Specifically, the concentration of NiCl2 is 10-100 mM, preferably 50 mM;

[0066] In another specific embodiment of the present invention, in the step S4,

[0067] The heating and annealing are carried out by heating to 45-55 °C for 3-6 h, preferably at 50 °C for 4 h, and then annealed to 20 °C at a rate of 0.1-1 °C / min (preferably 0.33 °C / min).

[0068] The molar ratio of VU5, XL5 and DNAzyme 8-17-3BO-8-17D is 1:1-5:5-15, preferably 1:1:10.

[0069] In another specific embodiment of the present invention, in the above preparation method, the buffer used can be sodium phosphate buffer.

[0070] In one or more specific embodiments of the present invention, there is provided the use of the above DNA nanocage having TLR9 agonist activity in the preparation of anti-tumor drugs.

[0071] The tumor is preferably a "cold" tumor with a high degree of immunosuppression, including but not limited to pancreatic cancer, breast cancer, osteosarcoma, testicular tumor, malignant melanoma, lung cancer, liver cancer, gastric cancer and esophageal cancer. In a specific embodiment of the present invention, taking pancreatic cancer as an example, the anti-tumor effect of the DNA nanocage Fe@DN with TLR9 agonist activity was verified, indicating that it has high safety, good biocompatibility, can significantly control tumor progression, and has a significant anti-tumor effect.

[0072] In one or more specific embodiments of the present invention, an anti-tumor drug is provided, and the anti-tumor drug contains the above-mentioned DNA nanocage with TLR9 agonist activity, and the DNA nanocage with TLR9 agonist activity can be used as a drug active ingredient and / or a drug delivery system.

[0073] In another specific embodiment of the present invention, the DNA nanocage with TLR9 agonist activity accounts for 1-99% of the total amount of the drug, and the drug is a unit dosage form suitable for single administration of an accurate dose.

[0074] In another specific embodiment of the present invention, the anti-tumor drug may further include at least one other drug active ingredient and / or at least one drug inactive ingredient.

[0075] Among them, the other drug active ingredients can be active substances with definite anti-tumor effects or active substances for tumor adjuvant therapy;

[0076] The anti-tumor active substances include but are not limited to cytotoxic drugs, nucleic acid synthesis inhibitors, nucleic acid transcription inhibitors, topoisomerase I inhibitors and tubulin inhibitors, which are not specifically limited herein.

[0077] The active substances for tumor adjuvant therapy include but are not limited to cytokines (such as G-CSF, GM-CSF, etc.), analgesics (such as aspirin, paracetamol, etc.), which are not specifically limited herein.

[0078] The above-mentioned at least one other drug active ingredient may or may not be loaded on the DNA nanocage, and those skilled in the art can design according to the actual situation.

[0079] The drug inactive ingredients can be carriers, excipients, diluents, etc. commonly used in pharmacy. Moreover, according to the usual methods, it can be made into oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, external preparations, suppositories and sterile injection solutions for use.

[0080] The carriers, excipients, diluents, etc. that can be included as non-drug active ingredients are well-known in the art, and those of ordinary skill in the art can determine that they meet clinical standards.

[0081] In yet another specific embodiment of the present invention, the carriers, excipients and diluents include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, arabic gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl paraben, propyl paraben, talc, magnesium stearate and mineral oil, etc.

[0082] In yet another specific embodiment of the present invention, the drugs of the present invention can be administered into the body by known methods. For example, they can be delivered systemically via intravenous injection or locally injected into the tissue of interest. Optionally, administration can be carried out via intravenous, transdermal, intranasal, mucosal or other delivery methods. Such administration can be carried out via a single dose or multiple doses. Those skilled in the art understand that the actual dose to be administered in the present invention can vary to a large extent depending on various factors, such as the target cells, the biological type or its tissue, the general condition of the subject to be treated, the route of administration, the mode of administration, and so on.

[0083] In one or more specific embodiments of the present invention, a method for treating tumors is provided, the method comprising administering to a subject a therapeutically effective dose of the above DNA nanocage having TLR9 agonist activity or the above anti-tumor drug.

[0084] In yet another specific embodiment of the present invention, the tumors are "cold" tumors with a high degree of immunosuppression, including but not limited to pancreatic cancer, breast cancer, osteosarcoma, testicular tumors, malignant melanoma, lung cancer, liver cancer, gastric cancer and esophageal cancer.

[0085] In yet another specific embodiment of the present invention, the subject can be a human and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, chimpanzees, etc., preferably human.

[0086] The subject refers to an animal that has already been the object of treatment, observation or experiment, preferably a mammal, and most preferably a human. The "therapeutically effective amount" refers to the amount of an active compound or agent, including the compounds of the present invention, that can cause a biological or medical response in a tissue system, animal or human that is sought by a researcher, veterinarian, doctor or other medical personnel, which includes alleviating or partially alleviating the symptoms of the disease, syndrome, disorder or condition being treated. It must be recognized that the optimal dosage and interval of administration of the active ingredient of the present invention are determined by its nature and external conditions such as the form, route and site of administration and the particular mammal being treated, and this optimal dosage can be determined by conventional techniques. It must also be recognized that the optimal course of treatment, i.e., the daily dosage of the compound within a specified time, can be determined by methods well known in the art.

[0087] The present invention will be further explained and illustrated by means of examples below, but it does not constitute a limitation to the present invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The test methods without specific conditions noted in the following examples are generally carried out under conventional conditions.

[0088] DNA material sequences used in the examples

[0089] As shown in Table 1 and Table 2 below, the oligonucleotide fragments with CpG ODN activity are shown in bold, and the lowercase letters are the sites of thiolation modification. BO is replaced by 4-(bromomethyl)phenylboronic acid pinacol ester (BO-br), and rA is adenosine ribonucleic acid.

[0090] Table 1

[0091]

[0092] Table 2

[0093]

[0094]

[0095] Example 1 Preparation and characterization of Fe@DN

[0096] (1) Formation of V, U, X, L pentamers: Taking V as an example, phosphorylated oligonucleotide fragments were mixed equimolarly at a concentration of 20 μM in sodium phosphate buffer, heated at 90 °C for 20 min, and then annealed to 20 °C at a rate of 0.33 °C / min. The sample was incubated at 20 °C and equilibrated at 4 °C to form pentamer V. The synthesis methods of other U, X, L pentamers are the same as that of pentamer V. 1～5 Phosphorylated oligonucleotide fragments were mixed equimolarly at a concentration of 20 μM in sodium phosphate buffer, heated at 90 °C for 20 min, and then annealed to 20 °C at a rate of 0.33 °C / min. The sample was incubated at 20 °C and equilibrated at 4 °C to form pentamer V. The synthesis methods of other U, X, L pentamers are the same as that of pentamer V.

[0097] (2) Preparation of VU5 and XL5: Taking VU5 as an example, 1:5 of V:U (20 μM) was fully mixed, heated to 50 °C for 4 h, and the sample was annealed to 20 °C at a rate of 0.33 °C / min. The sample was incubated at 20 °C and equilibrated at 4 °C to form VU5 for subsequent chemical ligation. The preparation method of XL5 is the same as that of VU5.

[0098] (3) Chemical ligation: Non-enzymatic chemical ligation was used to link adjacent 3'-hydroxyl and 5'-phosphate groups. N-cyanoimidazole (NCI, 50 mM) was used for the ligation reaction. 50 mM NiCl2 was added to improve the ligation efficiency. The mixture was incubated at 24 °C for 24 h. After the ligation was completed, dialysis was carried out with a dialysis bag with a pore size of 5.1 nm using sodium phosphate buffer.

[0099] (4) After chemical ligation, VU5, XL5, and DNAzyme 8-17-3BO-8-17D (prepared by incubating DNAzyme 8-17-3BO and 8-17D at a molar ratio of 1:1 with slow stirring at 25 °C for 8 h) were mixed at a molar ratio of 1:1:10 and added to the ferritin solution loaded with chloroquine phosphate. The mixture was heated to 50 °C for 4 h and annealed to 20 °C at a rate of 0.33 °C / min. The sample was incubated at 20 °C and equilibrated at 4 °C.

[0100] (5) The final solution in (4) was dialyzed using a dialysis bag with a pore size of 29 nm to obtain the Fe@DN solution.

[0101] The morphology of Fe@DN prepared in this example was characterized. The results of the TEM images are as Figure 2 shown, and the particle size distribution is as Figure 3 shown. The zeta potential measurement is as Figure 4 shown.

[0102] Example 2 Evaluation of DC Stimulation Experiment

[0103] In vitro, the tumor microenvironment was simulated. In an 80 μM H2O2 environment, Fe@DN (equivalent to 2 μg / mL of CpG ODN) was co-incubated with BMDCs. An equal volume of 1×PBS was used as a negative control, and 2 μg / mL of CpG ODN 1826 was used as a positive control. After co-incubation for 36 h, the culture supernatants of each group were taken into 2 mL EP tubes and centrifuged at 3000 g for 15 min. The supernatant was aspirated with a pipette into a new 2 mL EP tube for ELISA detection of the secretion of type I interferon. At the same time, the cells were treated, and the expression levels of co-stimulatory molecules such as CD80, CD86, and CD40 on the surface of BMDCs were detected by flow cytometry.

[0104] The detection results of the secretion level of type I interferon are as Figure 5 shown. From the measurement results, it can be seen that the ability of BMDCs to secrete IFN-α and IFN-β after treatment with Fe@DN was significantly increased. The detection results of the expression levels of co-stimulatory molecules are as Figure 6 shown. The expression levels of co-stimulatory molecules such as CD86, CD80, and CD40 on the surface of BMDCs were significantly increased after treatment with Fe@DN. It was proved that Fe@DN could effectively activate DCTLR9, promote the secretion of type I interferon by DCs, and up-regulate the expression levels of co-stimulatory molecules.

[0105] Example 3 In Vivo Antitumor Study of Fe@DN

[0106] In this example, pancreatic cancer was taken as an example to study the in vivo antitumor effect of Fe@DN.

[0107] Forty mice were used to establish an orthotopic model of pancreatic cancer. With the day of tumor-bearing defined as day 0, on day 7, the tumor-bearing mice were randomly divided into the above five treatment groups, with 8 mice in each group, namely the PBS group, the CQP group, the CpG group, the CQP+CpG group, and the Fe@DN group. Different treatment regimens were given, and the administration methods and doses were as follows: The PBS group was injected with 200 μL of 1×PBS via the tail vein; the CpG group was injected with 33 μg of CpG ODN 1826 into the tumor; the CQP group was injected with 1.2 mg of CQP intraperitoneally; the CQP+CpG group was injected with 1.2 mg of CQP intraperitoneally + 33 μg of CpG ODN 1826 into the tumor; the Fe@DN group was injected with an appropriate amount of Fe@DN (equivalent to 1.2 mg of CQP and 33 μg of CpG ODN) via the tail vein of each mouse. Among them, 3 mice in each group were anesthetized on days 7, 12, 17, and 22, and after intraperitoneal injection of the luciferase substrate solution (100 μL), the tumor progression of the mice was observed and photographed using a living imaging system. Another 5 mice in each group were continuously observed for 60 days, and the death of the mice was recorded, and the survival period of the mice in each treatment group was statistically analyzed. The main organs of the mice in each treatment group were taken, sectioned, and stained with H&E to observe tissue damage and pathological changes, which were used as systemic toxicity indicators.

[0108] The tumor progression of the mice in each treatment group was as Figure 7 shown, and the results showed that the Fe@DN treatment group could significantly control the tumor progression of the mice, showing a more significant therapeutic effect than other treatment groups. At the same time Figure 8 it was shown that compared with other treatment groups, Fe@DN could significantly prolong the survival period of the mice. It was proved that Fe@DN could exert a good anti-tumor effect in tumor-bearing mice.

[0109] As Figure 9 shown, compared with the control group of mice treated with PBS, the mice treated with Fe@DN had no visible tissue damage and pathological changes, indicating that the Fe@DN of the present invention has good biocompatibility.

[0110] Matters not covered in this invention are well-known techniques.

[0111] The above embodiments are only used to illustrate the technical concept and characteristics of the present invention, and their purpose is to enable those who are familiar with this technology to understand the content of the present invention and implement it accordingly, and it cannot be used to limit the protection scope of the present invention. All equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. A DNA nanocage with TLR9 agonist activity, characterized in that, The DNA nanocage is formed by the precise base complementary pairing of oligonucleotides with CpG ODN activity, a tumor-responsive nuclease, and its substrate, and the resulting nanocage is an icosahedral cavity cage structure; The DNA nanocage with TLR9 agonist activity is Fe@DN, and its preparation method includes: S1. Formation of pentamers V, U, X, and L: Mix the phosphorylated oligonucleotide fragments V1-V5 in a buffer, heat and anneal; after incubation, equilibrate at low temperature to form pentamer V; similarly obtain pentamers U, X, and L; S2. Preparation of VU5 and XL5: Thoroughly mix pentamer V and pentamer U, heat and anneal; after incubation, equilibrate at low temperature to form VU5 for subsequent chemical ligation; similarly obtain XL5; S3. Chemical ligation: Non-enzymatic chemical ligation is used to link adjacent 3'-hydroxyl and 5'-phosphate groups; N-cyanoimidazole is used for the ligation reaction; the mixture is incubated at room temperature; after ligation, dialysis is performed using a buffer; S4. After chemical ligation, mix VU5, XL5, and DNAzyme 8-17-3BO-8-17D, add it to a solution containing ferritin loaded with chloroquine phosphate, heat and anneal; after incubation, equilibrate to obtain a solution; S5. The solution obtained in step S4 is dialyzed to obtain Fe@DN; In the said step S1, the nucleotide sequences of V1-V5 are as shown in SEQ ID NO.1-5; The nucleotide sequences of U1-U5 are as shown in SEQ ID NO.6-10; The nucleotide sequences of X1-X5 are as shown in SEQ ID NO.11-15; The nucleotide sequences of L1-L5 are as shown in SEQ ID NO.16-20.

2. The DNA nanocage with TLR9 agonist activity according to claim 1, wherein The sources of the oligonucleotides with CpG ODN activity include human and non-human mammalian sources.

3. A method for preparing the DNA nanocage having TLR9 agonist activity according to any one of claims 1-2, characterized in that, The preparation method at least includes combining oligonucleotides with CpG ODN activity and a tumor-responsive nuclease and its substrate through DNA modular assembly technology.

4. The preparation method according to claim 3, characterized in that, The DNA nanocage with TLR9 agonist activity is Fe@DN, and its preparation method includes: S1. Formation of pentamers V, U, X, and L: Mix the phosphorylated oligonucleotide fragments V1-V5 in a buffer, heat and anneal; after incubation, equilibrate at low temperature to form pentamer V; similarly obtain pentamers U, X, and L; S2. Preparation of VU5 and XL5: Thoroughly mix pentamer V and pentamer U, heat and anneal; after incubation, equilibrate at low temperature to form VU5 for subsequent chemical ligation; similarly obtain XL5; S3. Chemical ligation: Non-enzymatic chemical ligation is used to link adjacent 3'-hydroxyl and 5'-phosphate groups; N-cyanoimidazole is used for the ligation reaction; the mixture is incubated at room temperature; after ligation, dialysis is performed using a buffer; S4. After chemical ligation, mix VU5, XL5, and DNAzyme 8-17-3BO-8-17D, add it to a solution containing ferritin loaded with chloroquine phosphate, heat and anneal; after incubation, equilibrate to obtain a solution; S5. The solution obtained in step S4 is dialyzed to obtain Fe@DN.

5. The preparation method according to claim 4, characterized in that, In the step S1, The molar ratio of the phosphorylated oligonucleotide fragments V1-V5 is 0.5-2:0.5-2:0.5-2:0.5-2:0.5-2, and at this time, the molar concentration of each phosphorylated oligonucleotide fragment is 10-30 μM; The heating and annealing are carried out at 85-95 °C for 10-30 min, and then annealed at a rate of 0.1-1 °C / min; The incubation temperature is 15-25 °C, and the low temperature condition is not higher than 5 °C; The nucleotide sequences of V1-V5 are shown in SEQ ID NO.1-5; The nucleotide sequences of U1-U5 are shown in SEQ ID NO.6-10; The nucleotide sequences of X1-X5 are shown in SEQ ID NO.11-15; The nucleotide sequences of L1-L5 are shown in SEQ ID NO.16-20; In the step S2, The molar ratio of the pentamer V and the pentamer U is 1:1-10; The heating and annealing are carried out at 45-55 °C for 3-6 h, and then annealed to 20 °C at a rate of 0.1-1 °C / min; In the step S3, The concentration of the N-cyanoimidazole is 10-100 mM; 10-100 mM NiCl2 is added in the step S3; In the step S4, The heating and annealing are carried out by heating to 45-55 °C for 3-6 h, and then annealed to 20 °C at a rate of 0.1-1 °C / min; The molar ratio of the VU5, XL5 and DNAzyme 8-17-3BO-8-17D is 1:1-5:5-15.

6. The application of the DNA nanocage with TLR9 agonist activity according to any one of claims 1-2 or the DNA nanocage with TLR9 agonist activity prepared by the preparation method according to any one of claims 3-5 in the preparation of an anti-tumor drug; The tumor is pancreatic cancer.

7. An anti-tumor drug, characterized in that, The anti-tumor drug comprises the DNA nanocage with TLR9 agonist activity according to any one of claims 1-2 or the DNA nanocage with TLR9 agonist activity prepared by the preparation method according to any one of claims 3-5.

8. The anti-tumor drug according to claim 7, wherein, The anti-tumor drug further comprises at least one drug active ingredient and / or at least one drug inactive ingredient.

Images