A natural cyclic peptide RA-V & DNA tetrahedron immune adjuvant and its preparation method and application

Abstract

The present invention discloses a natural cyclic peptide RA-V & DNA tetrahedron immune adjuvant and its preparation method and application, which belong to the field of biomedicine. The immune adjuvant includes a Rubiaceae type cyclic peptide compound RA-V and a DNA tetrahedron, and the DNA tetrahedron is a DNA tetrahedron with an arm length of 15-30bp formed by a DNA single strand. The present invention constructs a nanostructure assembled from 4 DNA single strands and RA-V, which specifically activates the sGAS-STING pathway through the recognition of double-stranded DNA by cGAS protein, thereby enhancing the body's immune function and preventing diseases such as viral infections and tumors. At the same time, by utilizing the good biological properties of DNA nanostructures, RA-V & DNA tetrahedron can effectively target disease lesions and exert the immunotherapy effect of the disease, which has great application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a natural cyclic peptide RA-V & DNA tetrahedron immune adjuvant, and a preparation method and application thereof. Background Art

[0002] Currently, the main clinical treatments for cancer are surgery, chemotherapy, and radiotherapy. While these treatments can improve patient survival, they are also associated with low patient tolerance and poor prognosis. Improving cancer treatment options and enhancing efficacy are pressing challenges. The advent of tumor immunotherapy has brought breakthrough progress in cancer treatment. Cyclic GMP-AMP synthase (cGAS) is an immune system accelerator. cGAS recognizes and binds to free double-stranded DNA in the cytoplasm, producing cGAMP and activating the downstream STING protein, which induces the production of type I interferon (IFN-β), thereby stimulating immune activity and combating disease development and progression. In the past few years, compounds that intervene in tumor development through the immunogenic cGAS-STING signaling pathway have emerged and have the potential for clinical development. However, these compounds suffer from limitations such as poor bioavailability, stability, and poor cancer selectivity, making them difficult to use in treatment. Therefore, the development of new drugs that stimulate the cGAS-STING pathway is of great research significance.

[0003] CN111420025A discloses a naturally derived cyclic peptide compound, RA-V, that can simultaneously activate both human and mouse cGAS-STING pathways. The compound, available in tablets and capsules, can be administered orally, by nasal inhalation, or rectally, and has demonstrated promising therapeutic effects against a variety of tumors, including colon cancer, melanoma, and gastric cancer. However, the cyclic peptide RA-V suffers from issues such as poor water solubility and insufficient targeting, limiting its further development.

[0004] DNA nanotechnology is a self-assembly technology that has been developed in recent years. With the good programmability of DNA sequences and the high specificity of base complementary pairing, DNA nanostructures with specified height, size and shape can be formed, which can meet the needs of drug design in a targeted manner and serve as a good carrier for delivering therapeutic drugs. Double-stranded DNA (dsDNA) is a natural substrate of cGAS protein, which can specifically activate the activity of cGAS protein and enhance the body's immune function. However, DNA nanostructures may be limited by many factors such as base sequence length and structure, which directly affects their ability to act as drug carriers and other functional effects. Therefore, rationally designing DNA nanostructures to enable them to play multiple functions such as drug carriers and immune activation activities is of great significance for the immunotherapy of tumor diseases. Summary of the Invention

[0005] The purpose of the present invention is to provide a natural cyclic peptide RA-V & DNA tetrahedron immune adjuvant and its preparation method and application to solve the problems existing in the above-mentioned prior art. The DNA tetrahedron and the natural cyclic peptide RA-V in the immune adjuvant can play a synergistic immune activation effect.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a natural cyclic peptide RA-V & DNA tetrahedron immune adjuvant, comprising a Rubiaceae type cyclic peptide compound RA-V and a DNA tetrahedron, wherein the DNA tetrahedron comprises a tetrahedron with an arm length of 15-30 bp formed by a DNA single chain.

[0008] Preferably, the ratio of the amount of the DNA tetrahedron to the Rubiaceae type cyclic peptide compound RA-V substance is (1:1)-(1:32).

[0009] Preferably, the tetrahedron comprises four DNA single strands, and the nucleotide sequences of the four DNA single strands are shown in SEQ ID NOs: 1-20.

[0010] The present invention also provides a method for preparing an immune adjuvant, comprising the following steps:

[0011] DNA tetrahedron and Rubiaceae type cyclic peptide compound RA-V are mixed in a substance amount of (1:1)-(1:32), incubated, centrifuged and supernatant discarded, and the precipitate is resuspended to prepare the natural cyclic peptide RA-V & DNA tetrahedron immune adjuvant.

[0012] Preferably, the concentration of the DNA tetrahedron solution in the mixed solution is 45-55 μM, the concentration of the RA-V solution is 400-16000 μM, and the solvent used for mixing is TEM buffer.

[0013] Preferably, the incubation conditions are: incubation at 4°C with shaking at 150-200 rpm for 2-3 hours;

[0014] The centrifugation conditions are: centrifugation at a speed of 8000-10000 rpm for 15-20 minutes.

[0015] Preferably, the method for preparing DNA tetrahedrons comprises the following steps: dissolving four DNA single strands separately, mixing them at equal concentrations and volumes, and self-assembling them into DNA tetrahedrons by programmed cooling through PCR.

[0016] Preferably, the programmed cooling of PCR is as follows: after stabilizing at 95°C for 5 minutes, the temperature is rapidly cooled to 4°C within 1 minute.

[0017] The present invention also provides the use of the immune adjuvant in the preparation of drugs for preventing and treating tumors, viral infections and bacterial infections. The tumors include, but are not limited to, rectal cancer.

[0018] The present invention also provides the use of the immune adjuvant in preparing medicines for improving immunity.

[0019] The present invention discloses the following technical effects:

[0020] (1) The present invention constructs a nanostructure composed of four single-stranded DNA and RA-V. This is the first time that the Rubiaceae-type cyclic peptide compound RA-V is found to form a whole after mixing with DNA tetrahedron. The operation is simple and expands the application of natural cyclic peptide RA-V as a drug.

[0021] (2) The preparation method of the immune adjuvant disclosed in the present invention mentions that DNA tetrahedron can not only be used as a drug carrier to improve the water solubility and targeting of the natural cyclic peptide RA-V, but also can be used as a natural reaction substrate of the cGAS protein, thereby maximizing the role of DNA tetrahedron in the natural cyclic peptide RA-V & DNA tetrahedron immune adjuvant.

[0022] (3) The present invention precisely controls the drug loading ratio of DNA tetrahedron and natural cyclic peptide RA-V, achieving efficient activation of the cGAS-STING signaling pathway. DNA tetrahedron and natural cyclic peptide RA-V can play a synergistic role in activating immunity.

[0023] (4) The natural cyclic peptide RA-V & DNA tetrahedron immune adjuvant prepared by the present invention has good in vivo anti-tumor activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1 Agarose gel electrophoresis images of DNA tetrahedrons with arm lengths of 15 bp, 20 bp, and 30 bp (A) and self-assembly schematic diagram (B);

[0026] Figure 2 Transmission electron microscopy characterization of a DNA tetrahedron with an arm length of 15 bp;

[0027] Figure 3 The figure shows the effect of DNA tetrahedrons with arm lengths of 15 bp, 20 bp, and 30 bp mixed with RA-V at the same concentration on stimulating cells to produce type I interferon (IFN-β);

[0028] Figure 4 The fluorescence spectra of DNA tetrahedron and RA-V mixed in different ratios;

[0029] Figure 5 The PAGE gel electrophoresis diagram of DNA tetrahedron and RA-V mixed in different ratios;

[0030] Figure 6 This is a graph showing the inhibitory effect of natural cyclic peptide RA-V & DNA tetrahedron immune adjuvant on the growth of mouse colorectal tumor (MC38);

[0031] Figure 7 This is a diagram showing the effect of natural cyclic peptide RA-V & DNA tetrahedron immune adjuvant in producing IgG antibodies after infection with specific antigen (OVA protein). DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] Example 1 Preparation of DNA tetrahedrons with arm lengths of 15 bp, 20 bp, and 30 bp

[0038] The four DNA single strands were dissolved in TEM buffer (10mM Tris, 1mM EDTA, 20mM MgCl2, pH=8.0) to a final concentration of 100μM for each strand, and the volume of each strand was 20μL. The four dissolved DNA single strands were mixed, and the volume of the mixed solution was 100μL. The mixed solution was subjected to gradient annealing using a gradient PCR instrument. The annealing procedure was as follows: 95°C for 5 minutes, then rapidly cooled to 4°C over one minute. The entire process took approximately 6 minutes, resulting in a final concentration of 25μM DNA tetrahedrons in a final volume of 100μL.

[0039] The sequences of the four single-stranded DNA are shown in SEQ ID NOs: 1-20:

[0040] 15bp DNA tetrahedron 1 sequence composition:

[0041] SEQ ID NO: 1:

[0042] 5'-AGGCAGACGAACATTCCTAAGTATTTATCACCCGCCATACGTATCACC-3';

[0043] SEQ ID NO: 2:

[0044] 5'-CTTGCTACACGAACTTAGGAATGTTCGAGAGGGTCCAATACCGAACAG-3';

[0045] SEQ ID NO: 3:

[0046] 5'-GGTGATAAAACGTGTAGCAAGCTGTAAGAGCATGCCCATCCAATGGCG-3';

[0047] SEQ ID NO: 4:

[0048] 5'-CCTCACTGCCTGGTGATACGAGGATGGGCATGCTCTACGGTATTGGAC-3'.

[0049] 15bp DNA tetrahedron 2 sequence composition:

[0050] SEQ ID NO: 5:

[0051] 5’-AGGCAGACGAACACTCCTGAGTATGTATCACCCGCCATACGTATCACC-3’;

[0052] SEQ ID NO: 6:

[0053] 5’-CTTGCTACACGAACTCAGGAGTGTTCGAGAGGGTCCACTACCGAACAG-3’;

[0054] SEQ ID NO: 7:

[0055] 5’-GGTGATACAACGTGTAGCAAGCTGTAAGAGCATGCCCATCCAATGGCG-3’;

[0056] SEQ ID NO: 8:

[0057] 5’-CCTCACTGCCTGGTGATACGAGGATGGGCATGCTCTACGGTAGTGGAC-3’.

[0058] Composition of the 15bp DNA tetrahedron 3 sequence:

[0059] SEQ ID NO: 9:

[0060] 5’-CTAGTAACGAACATTCCTAAGTATTTATCACCCGCCATACATAGATCA-3’;

[0061] SEQ ID NO: 10:

[0062] 5’-CTTGCTACACGAACTTAGGAATGTTCGAGAGGGTCCAATACCGAACAG-3’;

[0063] SEQ ID NO: 11:

[0064] 5’-GGTGATAAAACGTGTAGCAAGCTGTAAGAGCATGCCCATCCAATGGCG-3’;

[0065] SEQ ID NO: 12:

[0066] 5’-CCTCATACTAGTGATCTATGAGGATGGGCATGCTCTACGGTATTGGAC-3’.

[0067] Composition of the 20bp DNA tetrahedron sequence:

[0068] SEQ ID NO: 13:

[0069] 5’-AGGCAGTTGAGACGAACATTCCTAAGTCTGAAATTTATCACCCGCCATAGTAGACGTATCACC-3’;

[0070] SEQ ID NO: 14:

[0071] 5’-CTTGCTACACGATTCAGACTTAGGAATGTTCGACATGCGAGGGTCCAATACCGACGATTACAG-3’;

[0072] SEQ ID NO: 15:

[0073] 5’-GGTGATAAAACGTGTAGCAAGCTGTAATCGACGGGAAGAGCATGCCCATCCACTACTATGGCG-3’;

[0074] SEQ ID NO: 16:

[0075] 5’-CCTCGCATGACTCAACTGCCTGGTGATACGAGGATGGGCATGCTCTTCCCGACGGTATTGGAC-3’.

[0076] Composition of 30bp DNA tetrahedron sequences:

[0077] SEQ ID NO: 17:

[0078] 5’-AGATCTCGAACATTCCTAAGTCTGAAGATCCATTTATCACCAGCTGCTGCACGCCATAGTAGACGTATCACCTGTCCGTCTGAGGCAGTTGAG-3’;

[0079] SEQ ID NO: 18:

[0080] 5’-ACGATTACAGATCAAAGCTACTTGCTACACGAGGATCTTCAGACTTAGGAATGTTCGAGATCACATGCGAGGACTCGGTCCAATACCGTACTA-3’;

[0081] SEQ ID NO: 19:

[0082] 5'-ACGTGTAGCAAGTAGCTTTGATCTGTAATCGACTCTACGGGAAGAGCATGCCCATCCGGCTCACTACTATGGCGTGCAGCAGCTGGTGATAAA-3';

[0083] SEQ ID NO: 20:

[0084] 5'-AGAGCCGGATGGGCATGCTCTTCCCGTAGAGATAGTACGGTATTGGACCGAGTCCTCGCATGACTCAACTGCCTCAGACGGACAGGTGATACG-3'.

[0085] The synthesis of DNA tetrahedron was analyzed by 3% agarose gel electrophoresis. Figure 1 As shown. The self-assembly efficiency of DNA tetrahedron synthesis is high and the product purity is good. The DNA tetrahedron construction results were characterized by transmission electron microscopy. The results are shown in Figure 2 shown.

[0086] Example 2 Screening of Natural Cyclic Peptide RA-V & DNA Tetrahedron Immune Adjuvant System

[0087] (1) Cell culture: RAW264.7 cells were cultured in DMEM medium containing 10% fetal bovine serum (Gibco).

[0088] (2) Three DNA tetrahedrons with different arm lengths and natural cyclic peptides were mixed evenly at a molar ratio of 1:8;

[0089] (3) Different samples were added to RAW264.7 cells and grouped as follows: blank, RA-V alone, 15 bp DNA tetrahedron alone, 15 bp DNA tetrahedron + RA-V, 20 bp DNA tetrahedron alone, 20 bp DNA tetrahedron + RA-V, 30 bp DNA tetrahedron alone, and 30 bp DNA tetrahedron + RA-V. The incubation time was 6 h.

[0090] (4) PCR was used to measure the expression of IFN-β mRNA in RAW264.7 cells after different treatments, using GAPDH as the internal reference gene.

[0091] The upstream and downstream primers used in the PCR experiment are:

[0092] IFN-β upstream primer: 5′-CTATGAGATGCTCCAGAAC-3′;

[0093] IFN-β downstream primer: 5′-GATGATAGACATTAGCCAGGA-3′;

[0094] GAPDH upstream primer: 5′-TTGCGTCGCCAGGTGAAGA-3′;

[0095] GAPDH downstream primer: 5′-GCAGCGGCGCGAACACA-3′;

[0096] The PCR reaction system is as follows:

[0097] Table 1 PCR reaction system for IFN-β mRNA expression in RAW264.7 cells

[0098]

[0099] To ensure the reliability of the experimental data, three replicate wells were set up for each primer.

[0100] The PCR reaction conditions are as follows:

[0101] Table 2 PCR experimental conditions

[0102]

[0103] The preparation method of the Rubiaceae-type cyclopeptide compound RA-V can be referred to Hu, YY, et al. Rubipodanin B, a new cytotoxic cyclopeptide from Rubia podantha. Chemistry & Biodiversity, 2019, 16, e1800438; Chen, XQ, et al. Rubicordins AC, new cyclopeptides from Rubiacordifolia with cytotoxicity and inhibiting NF-κB signaling pathway. Tetrahedron, 2015, 71, 9673-967; Wang, Z., et al. Rubipodanin A, the first natural N-desmonomethyl Rubiaceae-type cyclopeptide from Rubia podantha, indicating an important role of the N9-methyl group in the conformation and bioactivity. PLoS ONE, 2015, 10, e0144950.

[0104] The experimental results are as follows Figure 3 As shown, after treatment with 15bp DNA tetrahedron + RA-V group, RAW264.7 cells expressed the most IFN-β mRNA, indicating that the combined immune stimulation effect of 15bp DNA tetrahedron + RA-V group was the best. In the present invention, 15bp DNA tetrahedron was finally selected as the drug delivery system and the cGAS protein reaction substrate.

[0105] Example 3 Preparation of Natural Cyclic Peptide RA-V & DNA Tetrahedron Immune Adjuvant

[0106] (1) Determination of system composition:

[0107] The natural cyclic peptide RA-V was dissolved in dimethyl sulfoxide to prepare a stock solution with a concentration of 18-20 μM for later use.

[0108] The DNA tetrahedron prepared in Example 1 was mixed with the natural cyclic peptide RA-V at different mass ratios to determine the maximum drug loading ratio. The mixing ratio of DNA tetrahedron and natural cyclic peptide RA-V was:

[0109] The ratios of DNA tetrahedron to RA-V were 1:1, 1:2, 1:4, 1:8, 1:16, and 1:32, respectively. The mixed solution was placed in a fluorescence spectrophotometer for measurement, using the fluorescence recovery of RA-V as a standard.

[0110] The experimental results are as follows Figure 4 and Figure 5 As shown. When the amount of DNA tetrahedron: amount of RA-V was 1:1, 1:2, 1:4, and 1:8, the fluorescence of RA-V at 310 nm was blocked. When the amount of DNA tetrahedron: amount of RA-V was 1:16, the fluorescence of RA-V at 310 nm was restored. This indicates that the maximum drug loading ratio of DNA tetrahedron to natural cyclic peptide RA-V in the natural cyclic peptide RA-V & DNA tetrahedron immune adjuvant is: amount of DNA tetrahedron: amount of RA-V = 1:16.

[0111] (2) System preparation

[0112] Mix the DNA tetrahedron and the natural cyclic peptide RA-V at a ratio of 1:16 (DNA tetrahedron:RA-V). Incubate at 4°C with shaking at 150-200 rpm for 2-3 hours (in this experiment, shaking at 150 rpm was used for 2 hours). Centrifuge the mixture at 8000-10000 rpm for 15-20 minutes (in this experiment, centrifugation at 10000 rpm was used for 20 minutes) using a 10kD ultrafiltration centrifuge tube. Resuspend the resulting precipitate in TEM solution (10mM Tris, 1mM EDTA, 20mM MgCl2, pH=8.0) to obtain the natural cyclic peptide RA-V & DNA tetrahedron immune adjuvant. The prepared natural cyclic peptide RA-V & DNA tetrahedron immune adjuvant should be used immediately.

[0113] Example 4 Evaluation of the anti-tumor effect of natural cyclic peptide RA-V & DNA tetrahedron immune adjuvant

[0114] Mouse colorectal cancer cells MC38 were diluted with normal saline to 2×10 7 100 μL of this cell suspension was inoculated into the right thigh of C57 mice and allowed to grow for 7 days to establish a tumor-bearing mouse model. Mice bearing well-grown tumors were randomly divided into the following groups: control, low-dose RA-V, high-dose RA-V, DNA tetrahedron, and RA-V & DNA tetrahedron. Administration was via the tail vein every other day, and the volume was measured. Tumor tissue was removed 12 days after administration.

[0115] The experimental results are as follows Figure 6 As shown, the experimental results showed that the tumor growth inhibition in the RA-V&DNA tetrahedron group was the most obvious after treatment, indicating that the RA-V&DNA tetrahedron immune adjuvant has good in vivo anti-tumor activity.

[0116] Example 5 Evaluation of the Antigen Response Effect of Natural Cyclic Peptide RA-V & DNA Tetrahedron Immune Adjuvant

[0117] C57 mice were used as a research model, and chicken ovalbumin (OVA) was used as a model antigen. Different samples were injected intramuscularly into the right thigh muscle of C57 mice. Samples were grouped as follows: blank, free OVA, OVA + RA-V, OVA + DNA tetrahedron, and OVA + RA-V + DNA tetrahedron. Peripheral blood was collected 7 days after intramuscular injection for OVA-IgG assay.

[0118] The experimental results are as follows Figure 7 As shown, the experimental results show that OVA+RA-V+DNA tetrahedron can better stimulate the activation of the immune system and produce specific antibodies in the body, indicating that RA-V&DNA tetrahedron immune adjuvant has good in vivo immune activation activity.

[0119] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A natural cyclic peptide RA-V & DNA tetrahedral immune adjuvant, characterized in that: It includes a Rubiaceae type cyclic peptide compound RA-V and a DNA tetrahedron, wherein the DNA tetrahedron includes a tetrahedron with an arm length of 15-30 bp formed by a DNA single strand; The ratio of the amount of the DNA tetrahedron to the Rubiaceae type cyclic peptide compound RA-V is (1:1)-(1:16); The DNA tetrahedron includes four DNA single strands. When the DNA tetrahedron is a tetrahedron with an arm length of 15bp formed by four DNA single strands, the nucleotide sequences of the four DNA single strands are shown as SEQ ID NOs: 1-4 or SEQ ID NOs: 5-8 or SEQ ID NOs: 9-12; when the DNA tetrahedron is a tetrahedron with an arm length of 20bp formed by DNA single strands, the nucleotide sequences of the four DNA single strands are shown as SEQ ID NOs: 13-16; when the DNA tetrahedron is a tetrahedron with an arm length of 30bp formed by DNA single strands, the nucleotide sequences of the four DNA single strands are shown as SEQ ID NOs: 17-20.

2. The method for preparing the immune adjuvant according to claim 1, wherein The following steps are involved: DNA tetrahedron and Rubiaceae type cyclic peptide compound RA-V were mixed in a substance amount of (1:1)-(1:16), incubated, centrifuged and the supernatant discarded, and the precipitate was resuspended to prepare the natural cyclic peptide RA-V & DNA tetrahedron immune adjuvant.

3. The preparation method according to claim 2, wherein The concentration of the DNA tetrahedron solution in the mixed solution is 45-55 μM, the concentration of the RA-V solution is 400-16000 μM, and the solvent used for the mixture is TEM buffer.

4. The preparation method according to claim 2, wherein Incubation conditions: incubate at 4°C with shaking at 150-200 rpm for 2-3 hours; The centrifugation conditions are: centrifugation at a speed of 8000-10000 rpm for 15-20 minutes.

5. The preparation method according to claim 2, wherein The preparation method of the DNA tetrahedron comprises the following steps: dissolving four DNA single strands respectively, mixing them in equal concentration and volume, and forming the DNA tetrahedron by self-assembly through programmed cooling through PCR.

6. The preparation method according to claim 2, wherein The programmed cooling of PCR was as follows: after stabilizing at 95° C. for 5 minutes, the temperature was rapidly cooled to 4° C. within 1 minute.

7. Use of the immune adjuvant according to claim 1 in the preparation of drugs for preventing and treating tumors, viral infections and bacterial infections.

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