A DNA nanomaterial targeting HIF-1 alpha and a preparation method and application thereof

Abstract

The application discloses a DNA nanomaterial targeting HIF-1 alpha and a preparation method and application thereof, which is composed of a tetrahedral DNA nanomaterial and a complementary single-stranded DNA; the tetrahedral DNA nanomaterial is self-assembled by TA, TB, TC and TD four single-stranded DNAs in equal molarities, nucleotide sequences of which are shown in SEQ ID NO. 01 to 04 in sequence, and each of four end points of the tetrahedral DNA nanomaterial has a cantilever single-stranded DNA, and each of the cantilever single-stranded DNAs has multiple nucleotide sequences specifically combined with HIF-1 alpha; the complementary single-stranded DNA is complementary to the cantilever single-stranded DNA. The application can realize the anti-tumor effect of inhibiting tumor cell growth and metastasis by hijacking HIF-1 alpha.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a DNA nanomaterial targeting HIF-1α, its preparation method, and its application. Background Technology

[0002] Most organisms require oxygen for cellular respiration to generate energy and maintain normal life activities. Hypoxia represents the state of oxygen deficiency in tissue cells. Higher animals mainly rely on red blood cells in blood vessels to carry oxygen to tissue cells. Tumor tissues, due to their rapid cell growth and dense cell structure, consume oxygen quickly; tumor cells, far from blood vessels, are often in a hypoxic state. Tumor cell hypoxia is closely related to the malignant progression of tumors. Hypoxia can promote metabolic reprogramming, angiogenesis, and distant metastasis in tumors. Therefore, targeting the hypoxic state of tumor cells has long been considered a potential cancer treatment and has attracted much attention from researchers.

[0003] Hypoxia can activate the hypoxia signaling pathway. Hypoxia-induced factors (HIFs) are key components of this pathway. The HIF family includes O2-sensitive α subunits (HIF-1α, HIF-2α, and HIF-3α) and O2-insensitive β subunits (HIF-1β). Under normal cellular conditions, the α subunits are degraded and inactivated by the VHL-mediated ubiquitin-proteasome pathway. Under hypoxic conditions, the α subunits are no longer degraded and are transported to the nucleus to bind with the β subunits, forming a heterologous complex. In this state, hypoxia-induced factors can act as transcription factors, binding to DNA to regulate the transcription of downstream target genes and modulating a series of physiological processes such as hemoglobin production, iron transport, glucose metabolism, angiogenesis, and cell growth and differentiation. Compared to tumor tissues, normal tissues are often under normal oxygen supply, resulting in lower HIF activity. The activation of the hypoxia signaling pathway is closely related to the malignant progression of tumors. Therefore, designing drugs that target HIFs to inhibit the hypoxia signaling pathway can inhibit the malignant progression of tumors without affecting normal tissues, which is of great significance.

[0004] Currently, research on targeting hypoxia to treat tumors has made a series of advances. These include drug design directly targeting HIFs; gene therapy targeting HIFs; and targeting important downstream factors regulating hypoxia signaling, such as the mTOR or UPR signaling pathways. A series of small molecule inhibitors targeting HIFs have been discovered, which can inhibit the activation of tumor hypoxia signaling through different mechanisms. Benzopyranyl 1,2,3-triazole, BIX01294, and IDF-11774 can inhibit the activation of hypoxia signaling pathways by promoting the degradation of HIF-1α by VHL. Cardenolides can inhibit the activation of the hypoxia signaling pathway by suppressing the transcription factor activity of HIFs. However, these drugs currently suffer from a lack of specificity, acting on other targets besides HIFs, such as HDACs. Therefore, developing more specific drugs targeting HIFs remains a long and arduous task.

[0005] In recent years, with the rise of nanotechnology, the development of nanomedicines targeting HIFs has also attracted the attention of researchers. Different forms of nanomaterials, combined with tumor hypoxia, have been shown to inhibit tumor growth, enhance radiotherapy and chemotherapy sensitivity, and aid in tumor immunotherapy. Researchers have developed a series of nanomaterials that bind to hemoglobin, carrying oxygen to hypoxic target cells to inhibit HIF activation. Other researchers have used different forms of nanomaterials as carriers for HIF inhibition, combining the two for anti-tumor therapy. For example, lipid nanocapsules have been used as carriers for the HIF inhibitor acridine in anti-tumor therapy. Lipid nanocapsules can protect acridine from rapid degradation, prolonging the drug's onset time. Nanomaterials can also serve as siRNA carriers for HIFs, delivering HIF-targeting siRNAs to hypoxic tumor cells and inhibiting HIF expression. However, research on designing nanomedicines that directly target HIFs using nanomaterials is currently insufficient.

[0006] DNA nanomaterials, as natural materials with excellent self-assembly properties, play an indispensable role in fields such as biosensing, bioimaging, drug delivery, and cell regulation due to their advantages including strong coding ability, convenient synthesis, ease of modification, high stability, and structural diversity. DNA nanomaterials can be personalized by altering the DNA base sequence, precisely controlling their size, shape, and function. Their synthesis cost is relatively low, and they exhibit good biocompatibility. DNA nanomaterials have been shown to serve as biological carriers for anti-tumor drugs, enabling these drugs to exert their effects within cells. However, despite their good biocompatibility, whether DNA nanomaterials can be directly used as nanomedicines to target specific anti-tumor targets is currently unreported. Furthermore, there are no reports on the application of DNA nanomaterials to target HIFs for anti-tumor effects. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a DNA nanomaterial that targets HIF-1α.

[0008] Another object of the present invention is to provide a method for preparing the above-mentioned DNA nanomaterials.

[0009] Another object of the present invention is to provide applications of the above-mentioned DNA nanomaterials.

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

[0011] A DNA nanomaterial targeting HIF-1α, consisting of a tetrahedral DNA nanomaterial and a complementary single-stranded DNA,

[0012] The tetrahedral DNA nanomaterial is self-assembled from four single-stranded DNA strands of TA, TB, TC and TD in equal molar amounts. Its nucleotide sequences are shown in SEQ ID NO. 01 to 04. Each of the four ends of the tetrahedral DNA nanomaterial has a cantilevered single-stranded DNA strand, and each cantilevered single-stranded DNA strand has multiple nucleotide sequences that specifically bind to HIF-1α.

[0013] The complementary single-stranded DNA pairs complementaryly with the aforementioned cantilevered single-stranded DNA.

[0014] In a preferred embodiment of the present invention, the nucleotide sequence of the complementary single-stranded DNA is shown in SEQ ID NO. 05.

[0015] The preparation method of the above-mentioned DNA nanomaterial includes the following steps: equimolar concentrations of TA, TB, TC and TD and the complementary single-stranded DNA are thoroughly mixed in a 1×TE-Mg buffer at pH 8.0, then incubated at 95°C for 5 min, and finally rapidly cooled to room temperature to achieve one-step self-assembly synthesis of the tetrahedral DNA nanomaterial.

[0016] In a preferred embodiment of the present invention, the concentrations of TA, TB, TC and TD in the 1×TE-Mg buffer are all 10 μM.

[0017] More preferably, the concentration of the complementary single-stranded DNA in 1×TE-Mg buffer is 40 uM.

[0018] In a preferred embodiment of the present invention, the formulation of the 1×TE-Mg buffer is: 10 mM Tris.HCl, 1 mM EDTA and 10 mM MgCl2.

[0019] The application of the above-mentioned DNA nanomaterials in the preparation of anti-tumor drugs.

[0020] An antitumor drug composition comprising the aforementioned DNA nanomaterial as its active ingredient.

[0021] In a preferred embodiment of the present invention, the effective component is the aforementioned DNA nanomaterial.

[0022] The beneficial effects of this invention are:

[0023] This invention utilizes the specific binding of transcription factor HIF-1α to the specific DNA sequence NCGTGN to design a novel, easily synthesized, and universal DNA nanomaterial to target HIF-1α and achieve anti-tumor therapeutic effects. Its specific mechanism of action is as follows: Figure 1 As shown, the DNA nanomaterial of the present invention can enter tumor cells via endocytosis. Its four outwardly extending single-stranded DNA arms contain multiple HIF-1α-specific binding DNA sequences. HIF-1α can be hijacked and trapped in the cytoplasm by binding to any one of the single-stranded DNA arms of the DNA nanomaterial, thereby preventing it from being transported to the cell nucleus to exert transcription factor activity to regulate the expression of downstream target genes, and ultimately being degraded. Since HIF-1α regulates the expression of important target genes in hypoxic tumor cells to maintain tumor cell growth and metastasis, this DNA nanomaterial can achieve an anti-tumor effect by hijacking HIF-1α to inhibit tumor cell growth and metastasis. Attached Figure Description

[0024] Figure 1 This is a schematic diagram illustrating the working principle of the present invention.

[0025] Figure 2 This is a structural diagram of the DNA nanomaterials (TDNs) in Example 1 of the present invention.

[0026] Figure 3 The image shown is a gel electrophoresis diagram in an embodiment of the present invention, wherein marker (lane M), TA (lane a), TA-TB (lane b), TA-TB-TC (lane c), TA-TB-TC-TD (lane d), and TDNs (lane e).

[0027] Figure 4 This is an AFM characterization diagram of TDNs targeting HIF-1α in Example 1 of the present invention.

[0028] Figure 5 The figure shows the analysis results of TDNs binding to HIF-1α in Example 2 of the present invention. (A) Analysis of the binding of TDNs and HIF-1α under normal culture and hypoxic conditions in U251 cells; (B) Analysis of the binding of TDNs and HIF-1α under normal culture and hypoxic conditions in B16-F10 cells.

[0029] Figure 6The figures show the experimental results of TDNs inhibiting tumor growth in Example 2 of this invention. (A) CCK-8 assay of U251 cells to detect the effect of TDNs on tumor cell proliferation under normal and hypoxic conditions; (B) CCK-8 assay of B16-F10 cells to detect the effect of TDNs on tumor cell proliferation under normal and hypoxic conditions; (C) In vivo imaging of small animals to observe the effect of TDNs on tumor proliferation in C57 / BL6 melanoma-bearing mice; (D) Statistical analysis of fluorescein intensity of tumors in (C); (E) Survival curve analysis of mice in (C); *P<0.05, **P<0.01.

[0030] Figure 7 Figure 2 shows the experimental results of TDNs inhibiting tumor metastasis in Example 2 of this invention. (A) Cell scratch assay in B16-F10 cells to detect the effect of TDNs on tumor cell migration under normal and hypoxic conditions; (B) Collagen invasion assay in B16-F10 cells to detect the effect of TDNs on tumor cell migration under normal and hypoxic conditions; (C) Statistical analysis of the ratio of scratch distance after 24 h to scratch distance at 0 h for each group of B16-F10 cells in (A); (D) Statistical analysis of the distance B16-F10 cells invaded collagen tissue after 24 h for each group in (B); (E) In vivo imaging observation of the effect of TDNs on tumor metastasis in C57 / BL6 melanoma-bearing mice; (F) Statistical analysis of fluorescein intensity in tumors in (E); (G) Survival curve analysis of mice in (E). *P<0.05, **P<0.01. Detailed Implementation

[0031] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0032] Example 1

[0033] The specific structure of the DNA nanomaterial targeting HIF-1α prepared in this embodiment is as follows:

[0034] The DNA nanomaterials (hereinafter referred to as TDNs) targeting HIF-1α prepared in this embodiment have detailed structures. Figure 2 It consists of a tetrahedral DNA nanomaterial and a complementary single-stranded DNA, wherein,

[0035] The tetrahedral DNA nanomaterial is self-assembled from four single-stranded DNA molecules of TA, TB, TC and TD in equal molar amounts. Its nucleotide sequences are shown in SEQ ID NO. 01 to 04 (see Table 1 below). Each of the four ends of the tetrahedral DNA nanomaterial has a cantilevered single-stranded DNA molecule. Each cantilevered single-stranded DNA molecule has multiple repeating nucleotide sequences ncgtgn that specifically bind to HIF-1α (used for binding to hypoxia-inducible factor, which is a continuous tandem repeating sequence of the HIF-1α specific DNA binding motif to enhance its binding performance).

[0036] The complementary single-stranded DNA, whose nucleotide sequence is shown in SEQ ID NO.05 (see Table 1 below), is complementary to the above-mentioned cantilevered single-stranded DNA.

[0037] Table 1

[0038] TA <![CDATA[ acacgctcacgaccacgaccacgaacacgc ttttttttttacattcctaagtctgaaacattacagcttgctacacgagaagagccgccatagta(SEQ ID NO.01)]]> TB <![CDATA[tatcaccaggcagttgacagtgtagcaagctgtaatagatgcgagggtccaatactttttttttt acacgctcacgaccacgaccacgaacacgc (SEQ ID NO.02)]]> TC <![CDATA[tcaactgcctggtgataaaacgacactacgtgggaatctactatggcggctcttctttttttttt acacgctcacgaccacgaccacgaacacgc (SEQ ID NO.03)]]> TD <![CDATA[ttcagacttaggaatgtgcttcccacgtagtgtcgtttgtattggaccctcgcattttttttttt acacgctcacgaccacgaccacgaacacgc (SEQ ID NO.04)]]> Complementary single-stranded DNA <![CDATA[ gcgtgttcgtggtcgtggtcgtgagcgtgt (SEQ ID NO.05)]]>

[0039] The specific preparation method of TDNs in this embodiment is as follows: Equimolar concentrations of TA, TB, TC, TD (10 uM) and 40 uM complementary single-stranded DNA are thoroughly mixed in 1×TE-Mg buffer, incubated at 95°C for 5 min, and then rapidly cooled to room temperature to synthesize specific multi-tentacle TDNs in one step, and stored at 4°C; wherein the 1×TE-Mg buffer is composed of 10 mM Tris.HCl, 1 mM EDTA and 10 mM MgCl2, and the pH is 8.0.

[0040] Purity requirements or evaluation indicators: This example uses polyacrylamide gel electrophoresis (PAGE) to verify the successful formation and purity evaluation of TDNs. For example... Figure 3 As shown, TA is in the lowest molecular weight band (lane a), while TDNs composed of 5 chains are in the highest molecular weight band (lane e). The molecular weights of the products in each lane are arranged in ascending order as follows: lane a (single TA chain), lane b (TA and TB chain hybridization), lane c (TA, TB, and TC chain hybridization), lane d (TA, TB, TC, and TD chain hybridization), and lane e (TDNs targeting HIF-1α), corresponding to the step-by-step composition of TDNs. Furthermore, a single and bright band was obtained in lane e, indicating that the TDNs synthesized in this embodiment have high purity.

[0041] Other key technical indicators: This embodiment utilizes atomic force microscopy (AFM) to further confirm the successful synthesis of TDNs (e.g., Figure 4(As shown). Based on the AFM images and the xy plane, the typical tetrahedral morphology and height of DNA can be observed, and the synthesized TDNs exhibit uniform nanostructures. Given a base pair spacing of 0.34 nm, the theoretical values ​​for the tetrahedral side length and cantilever of the designed TDNs were calculated to be 5.44 nm and 13.26 nm, respectively. However, in reality, due to the structural collapse of the three-dimensional TDNs into two-dimensional TDNs, the height obtained in the AFM (7.72 nm) is less than the sum of the side length and cantilever (18.7 nm).

[0042] Example 2: Antitumor effect of TDNs prepared in Example 1

[0043] (1) TDNs can bind to HIF-1α:

[0044] In this embodiment, synthesized TDNs were biotin-labeled and co-cultured with glioblastoma U251 cells and melanoma B16-F10 cells to allow them to enter the cells. The cells were cultured under normal and hypoxic conditions for 24 hours. Cell lysates were harvested, and TDNs were enriched with streptavidin magnetic beads. Western blot was used to detect HIF-1α bound to the TDNs. This embodiment found that under normal culture conditions, TDNs can bind to HIF-1α, but due to the low expression level of HIF-1α under normal culture conditions, the amount of HIF-1α bound by TDNs is relatively small. Under hypoxic conditions, the binding of TDNs to HIF-1α increases significantly. Figure 5 As shown in AB.

[0045] (2) TDNs inhibit the proliferation of tumor cells and tumor growth in animals:

[0046] In this embodiment, TDNs were co-cultured with U251 and B16-F10 cells, and cell proliferation was detected using CCK-8 assay. This embodiment found that U251 cells exhibited good resistance to the inhibitory effect of hypoxia on cell proliferation, while TDNs significantly inhibited the proliferation of U251 cells under hypoxic conditions on day 5 of culture. Figure 6 As shown in Figure A, the situation with B16-F10 cells is slightly different. On day 2 of culture, B16-F10 cells exhibit good resistance to the inhibitory effect of hypoxia on cell proliferation. TDNs significantly inhibited the proliferation of B16-F10 cells under hypoxic conditions on day 2. However, on day 4, hypoxia significantly inhibited the proliferation of B16-F10 cells, but DNA tetrahedral nanomedicines further inhibited the proliferation of B16-F10 cells, as shown in Figure A. Figure 6 As shown in Figure B. These results demonstrate that DNA tetrahedral nanomedicines can inhibit the proliferation of tumor cells under hypoxic conditions.

[0047] In this embodiment, B16-F10 cells were seeded into C57 / BL6 mice to establish an in situ melanoma tumorigenesis model. This embodiment demonstrated that DNA tetrahedral nanomedicine significantly inhibited melanoma growth in mice. Figure 6 As shown in CD. It can significantly prolong the survival time of tumor-bearing mice. Figure 6 As shown in E.

[0048] (3) TDNs inhibit tumor cell migration and tumor metastasis in animals:

[0049] In this embodiment, TDNs were co-cultured with B16-F10 cells, and cell migration was detected using a cell scratch assay. This embodiment found that TDNs could inhibit the migration of B16-F10 cells under hypoxic treatment. Figure 7 As shown in A and 7C, under normal conditions, due to the migration ability of B16-F10 cells, cell scratches significantly decreased after 24 hours, and this process was unaffected by TDNs. However, under hypoxic conditions, after 24 hours, cell scratches unaffected by NA tetrahedral nanomedicines significantly decreased, while scratches on cells affected by TDNs showed limited reduction. This indicates that TDNs can inhibit the migration of B16-F10 cells under hypoxic treatment. In this example, B16-F10 cells were cultured on a collagen-forming matrix, such as... Figure 7 As shown in B and 7D, this embodiment found that under normal conditions, due to the invasive ability of B16-F10 cells, after 24 hours, B16-F10 cells could invade collagen tissue a certain distance, and this process was unaffected by TDNs. However, under hypoxic conditions, after 24 hours, cells not treated with NA tetrahedral nanomedicines still possessed invasive ability, while the invasion distance of cells treated with TDNs into collagen tissue was significantly reduced. This indicates that TDNs can inhibit the invasion of B16-F10 cells under hypoxic treatment. These results demonstrate that DNA tetrahedral nanomedicines can inhibit the migration and invasion of tumor cells under hypoxic conditions.

[0050] In this embodiment, a melanoma metastasis model was established by injecting B16-F10 cells into C57 / BL6 mice via tail vein. This embodiment demonstrated that DNA tetrahedral nanomedicine significantly inhibited melanoma metastasis in mice. Figure 7 As shown in EF. It can significantly prolong the survival of tumor-bearing mice. Figure 7 As shown in G.

[0051] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A DNA nanomaterial targeting HIF-1α, characterized in that: It consists of tetrahedral DNA nanomaterials and a complementary single-stranded DNA. This tetrahedral DNA nanomaterial is self-assembled from four single-stranded DNA molecules of equal molarity: TA, TB, TC, and TD. Its nucleotide sequences are shown in SEQ ID NO. 01 to 04, respectively. Each of the four ends of the tetrahedral DNA nanomaterial has a cantilevered single-stranded DNA, and each cantilevered single-stranded DNA has multiple nucleotide sequences that specifically bind to HIF-1α. The complementary single-stranded DNA pairs complementaryly with the aforementioned cantilevered single-stranded DNA, and its nucleotide sequence is shown in SEQ ID NO.

05.

2. The method for preparing DNA nanomaterials according to claim 1, characterized in that: The process includes the following steps: equimolar concentrations of TA, TB, TC, and TD, along with the complementary single-stranded DNA, are thoroughly mixed in a 1×TE-Mg buffer at pH 8.0, incubated at 95°C for 5 min, and then rapidly cooled to room temperature to achieve one-step self-assembly synthesis of the tetrahedral DNA nanomaterial.

3. The preparation method according to claim 2, characterized in that: The concentrations of TA, TB, TC, and TD in the 1×TE-Mg buffer were all 10 μM.

4. The preparation method according to claim 3, characterized in that: The concentration of the complementary single-stranded DNA in 1×TE-Mg buffer was 40 uM.

5. The preparation method according to any one of claims 2 to 4, characterized in that: The formulation of the 1×TE-Mg buffer is: 10 mM Tris.HCl, 1 mM EDTA and 10 mM MgCl2.

6. The application of the DNA nanomaterial of claim 1 in the preparation of antitumor drugs, wherein the tumor is melanoma.

7. An antitumor drug composition, characterized in that: Its active ingredients include: the DNA nanomaterial as described in claim 1.

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