CN115927345A - DNA nano material targeting HIF-1 alpha and preparation method and application thereof - Google Patents

Abstract

The invention discloses a DNA nano material of targeting HIF-1 alpha, a preparation method and application thereof, which consists of a regular tetrahedron DNA nano material and a complementary single-stranded DNA; the regular tetrahedron DNA nano material is formed by self-assembling four single-stranded DNAs (deoxyribonucleic acids) of TA, TB, TC and TD in an equimolar mode, the nucleotide sequence of the regular tetrahedron DNA nano material is sequentially shown as SEQ ID NO. 01-04, four end points of the regular tetrahedron DNA nano material are respectively provided with a cantilever single-stranded DNA, and each cantilever single-stranded DNA is provided with multiple nucleotide sequences specifically combined with HIF-1 alpha; the complementary single-stranded DNA is complementarily paired with the above-mentioned cantilever single-stranded DNA. The invention can realize the anti-tumor effect of inhibiting the growth and the metastasis of tumor cells by hijacking HIF-1 alpha.

Description

HIF-1 alpha-targeting DNA nano material and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a DNA nano material targeting HIF-1 alpha, and a preparation method and application thereof.

Background

Most organisms require cellular respiration through the use of oxygen to generate energy to sustain normal vital activities. Hypoxic condition of hypoxic superficial tissue cells. Higher animals rely primarily on the blood vessels of red blood cells to carry oxygen, which is transported to the tissue cells. The growth of cells in the tumor tissue is fast, the cells are compact, and the oxygen consumption is fast; tumor cells distant from blood vessels are often in a hypoxic state. Tumor cell hypoxia is closely related to malignant progression of tumors. Hypoxia promotes metabolic reprogramming, angiogenesis and tumor distant metastasis of tumors. Therefore, the hypoxic state of targeted tumor cells has long been regarded as a potential tumor treatment regimen by researchers.

The hypoxic condition can activate a hypoxic signal pathway. Hypoxia-induced factors (HIFs) are key factors of Hypoxia signaling pathways. The family of HIFs includes O ₂ The susceptive alpha subunits (HIF-1 alpha, HIF-2 alpha and HIF-3 alpha) and O ₂ The insensitive beta subunit (HIF-1 beta). When the cell is in a normal state, the alpha subunit is degraded by the VHL-mediated ubiquitin-proteasome pathway to be in an inactive state; when the cell is in a hypoxic state, the alpha subunit will no longer be degraded and transported into the nucleus to associate with the beta subunit to form a heterocomplex. At this time, the hypoxia inducible factor can be used as a transcription factor to be combined with DNA to regulate the transcription of downstream target genes and regulate a series of physiological processes such as hemoglobin generation, iron transport, sugar metabolism, angiogenesis, cell growth and differentiation and the like. The activity of HIFs is lower because normal tissues are often in a normal oxygen supply state compared to tumor tissues. The activation of the hypoxic signal pathway is closely related to the malignant progression of the tumor, so that the targeted HIFs are designed to inhibit the hypoxic signal pathway, can inhibit the malignant progression of the tumor without affecting normal tissues, and have important significance.

Currently, a series of advances have been made in the research to target hypoxic phenomena to treat tumors. Comprises designing the medicine directly with the HIFs target spot; gene therapy for HIFs; or to target important downstream factors regulated by hypoxic signaling, such as the mTOR or UPR signaling pathway. A series of small molecule inhibitors targeting HIFs were found to inhibit activation of tumor hypoxia signals by different mechanisms. Benzopyranyl 1,2,3-triazole, BIX01294, IDF-11774, etc. can inhibit activation of the hypoxic signaling pathway by promoting VHL degradation of HIF-1 α [6]. Cardenolides can inhibit activation of hypoxic signaling pathway by inhibiting the transcription factor activity of HIFs. However, these drugs have the problem of poor specificity, and can act on other targets besides the HIFs, such as HDACs. Therefore, the development of drugs that target more specifically to HIFs remains elusive.

In recent years, nanotechnology has been developed. The development of nano-drugs targeting HIFs has also attracted attention from related researchers [8]. The combination of different forms of nano material and tumor hypoxia proves its application in inhibiting tumor growth, raising sensitivity of radiotherapy and chemotherapy and treating tumor immunity. Researchers have developed a series of nanomaterials that inhibit the activation of HIFs by binding to hemoglobin, carrying oxygen to hypoxic target cells. Researchers also use different forms of nano materials as carriers for inhibiting the HIFs, and the nano materials are combined to be applied to anti-tumor treatment. For example, the lipid nanocapsule is used as a carrier of an HIFS inhibitor Acridine and applied to antitumor therapy. Lipid nanocapsules can protect Acridine from being rapidly degraded and prolong the onset time of the drug [14]. The nano material can also be used as an siRNA carrier of the HIFs, so that siRNA targeting the HIFs is conveyed to hypoxic tumor cells, and the expression of the HIFs is inhibited. However, the research of designing nano drugs directly targeting the HIFs by using nano materials is still insufficient at present.

The DNA nano material is a natural material with good self-assembly characteristics, and plays an indispensable role in the fields of biosensing, bioimaging, drug delivery, cell regulation and the like due to the advantages of strong encoding property, convenience in synthesis, easiness in modification, high stability, structural diversity and the like. The DNA nano material can be individually designed by changing a DNA base sequence, the size, the shape and the function of the nano material are accurately controlled, the synthesis cost is relatively low, and the biocompatibility is good. The DNA nano material is proved to be capable of being used as a biological carrier of an anti-tumor medicament to be applied to the anti-tumor medicament to play a role in cells. Because the DNA nano material has good biocompatibility, but whether the DNA nano material can be directly used as a nano medicament to target a corresponding anti-tumor target point or not has no related report at present. And no corresponding report exists in the research of applying the DNA nano material to the targeted HIFs to realize the anti-tumor effect.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a DNA nano material targeting HIF-1 alpha.

Another object of the present invention is to provide a method for preparing the DNA nanomaterial.

It is still another object of the present invention to provide the use of the above DNA nanomaterial.

The technical scheme of the invention is as follows:

a DNA nano material targeting HIF-1 alpha is composed of a regular tetrahedron DNA nano material and a complementary single-chain DNA,

the regular tetrahedron DNA nano material is formed by self-assembling four single-stranded DNAs (deoxyribonucleic acids) of TA, TB, TC and TD in an equimolar mode, the nucleotide sequence of the regular tetrahedron DNA nano material is sequentially shown as SEQ ID NO. 01-04, four end points of the regular tetrahedron DNA nano material are respectively provided with a cantilever single-stranded DNA, and each cantilever single-stranded DNA is provided with multiple nucleotide sequences specifically combined with HIF-1 alpha;

the complementary single-stranded DNA is complementarily paired with the above-mentioned cantilever single-stranded DNA.

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

Further preferably, the nucleotide sequence of the specific binding HIF-1 alpha is shown in SEQ ID NO. 06.

The preparation method of the DNA nano material comprises the following steps: fully mixing TA, TB, TC and TD with equimolar concentration and the complementary single-stranded DNA in a 1 XTE-Mg buffer solution with the pH value of 8.0, incubating for 5min at 95 ℃, and finally rapidly cooling to room temperature to synthesize the regular tetrahedron DNA nano material by one-step self-assembly.

In a preferred embodiment of the invention, the concentrations of said TA, TB, TC and TD in said 1 XTE-Mg buffer are all 10uM.

Further preferably, the concentration of the complementary single-stranded DNA in the 1 XTE-Mg buffer is 40uM.

In a preferred embodiment of the present invention, the formulation of the 1 XTE-Mg buffer is: tris.HCl 10mM final concentration, EDTA 1mM and 10mM final concentrationmM MgCl ₂ 。

The application of the DNA nano material in preparing anti-tumor drugs.

An antitumor pharmaceutical composition comprises the DNA nanomaterial as the effective component.

In a preferred embodiment of the present invention, the active ingredient is the above-mentioned DNA nanomaterial.

The beneficial effects of the invention are:

the invention designs a novel, easily synthesized and universal DNA nano material to target HIF-1 alpha and realize the function of anti-tumor treatment based on that the transcription factor HIF-1 alpha can be specifically combined with a specific DNA sequence NCGTGN. The specific action principle is shown in fig. 1, the DNA nanomaterial of the present invention can enter tumor cells by endocytosis, and four extended cantilever single-stranded DNAs thereof contain multiple HIF-1 α -specific binding DNA sequences, and HIF-1 α can be hijacked and trapped in cytoplasm by binding any one of the cantilever single-stranded DNAs of the DNA nanomaterial, thereby preventing the DNA nanomaterial from being transported to the nucleus to exert transcription factor activity to regulate the expression of downstream target genes and finally be degraded; because HIF-1 alpha regulates the expression of important target genes in tumor cells in an anaerobic state to maintain the growth and metastasis of the tumor cells, the DNA nano-material can realize the anti-tumor effect of inhibiting the growth and metastasis of the tumor cells by hijacking the HIF-1 alpha.

Drawings

Fig. 1 is a functional diagram of the present invention.

FIG. 2 is a structural view of DNA nanomaterials (TDNs) in example 1 of the present invention.

FIG. 3 is a diagram of gel electrophoresis in an example 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).

FIG. 4 is an AFM characterization of TDNs targeted to HIF-1 α in example 1 of the invention.

FIG. 5 is a graph showing the results of analysis of binding of TDNs to HIF-1. Alpha. In example 2 of the present invention, wherein (A) in U251 cells, the binding of TDNs to HIF-1. Alpha. In normal culture and hypoxic culture is analyzed; (B) Binding of TDNs to HIF-1 alpha in B16-F10 cells was analyzed in normal and hypoxic culture.

FIG. 6 is a graph showing the results of experiments in which TDNs inhibit the growth of tumors in example 2 of the present invention, in which (A) CCK-8 detected the effect of TDNs on the proliferation of tumor cells in normal culture and in hypoxic culture in U251 cells; (B) In B16-F10 cells, CCK-8 detects the effect of TDNs on the proliferation of tumor cells under normal culture and hypoxic culture; (C) Observing the effect of TDNs on the tumor proliferation in a C57/BL6 melanoma-bearing mouse by virtue of in-vivo imaging of a small animal; (D) analyzing the fluorescein intensity statistics of the tumor in (C); (E) survival curve analysis of mice in (C); * P <0.05, P <0.01.

FIG. 7 is a graph showing the results of experiments in which TDNs inhibit tumor metastasis in example 2 of the present invention, in which (A) in B16-F10 cells, cell scratch experiments were performed to examine the effect of TDNs on tumor cell migration in normal culture and in hypoxic culture; (B) In B16-F10 cells, detecting the effect of TDNs on tumor cell migration under normal culture and hypoxic culture by a collagen invasion experiment; (C) Statistically analyzing the ratio of the scratch distance of each group of B16-F10 cells after migrating for 24 hours to the scratch distance of 0 hour in the step (A); (D) Statistically analyzing the distance of each group of B16-F10 cells invading into the collagen tissue after 24h in the step (B); (E) Observing the effect of TDNs on the tumor metastasis in a C57/BL6 melanoma-bearing mouse by virtue of in-vivo imaging of a small animal; (F) statistical analysis of fluorescein intensity of the tumor in (E); (G) survival Curve analysis of mice in (E). * P <0.05,. P <0.01.

Detailed Description

The technical solution of the present invention is further illustrated and described by the following detailed description in conjunction with the accompanying drawings.

Example 1

The structure of the HIF-1 alpha-targeting DNA nanomaterial prepared in the embodiment is as follows:

the HIF-1 alpha-targeting DNA nanomaterial (TDNs, detailed structure shown in FIG. 2) prepared in this example is composed of regular tetrahedral DNA nanomaterial and a complementary single-stranded DNA, wherein,

the regular tetrahedron DNA nano material is formed by equimolar self-assembly of four single-stranded DNAs (TA, TB, TC and TD), the nucleotide sequences of the regular tetrahedron DNA nano material are sequentially shown as SEQ ID No. 01-04 (see Table 1 below), four end points of the regular tetrahedron DNA nano material are respectively provided with a cantilever single-stranded DNA, and each cantilever single-stranded DNA is provided with a multiple repetitive nucleotide sequence ncgtgn specifically combined with HIF-1 alpha (shown as SEQ ID No.06, used for combination with a hypoxia inducible factor and is a continuous tandem repetitive sequence of a specific DNA combination motif of the HIF-1 alpha so as to enhance the combination performance of the repetitive nucleotide sequence);

complementary single-stranded DNA, the nucleotide sequence of which is shown in SEQ ID NO.05 (see Table 1 below) is complementary-paired with the cantilever single-stranded DNA.

TABLE 1

The specific preparation method of the TDNs of this example: fully mixing TA, TB, TC, TD (10 uM) and 40uM complementary single-stranded DNA with equal molar concentration in a 1 XTE-Mg buffer solution, incubating at 95 ℃ for 5min, and finally rapidly cooling to room temperature to synthesize specific multi-antenna TDNs through one-step self-assembly and store at 4 ℃; wherein the 1 XTE-Mg buffer is Tris.HCl of 10mM, EDTA of 1mM and MgCl of 10mM of the final concentration ₂ The pH was 8.0.

Purity requirement or evaluation index: this example demonstrates the successful formation and purity evaluation of TDNs by Polyacrylamide (PAGE) gel electrophoresis. As shown in FIG. 3, TA is in the lowest molecular weight band (lane a), while TDNs composed of 5 strands are in the highest molecular weight band (lane e). The molecular weights of the products of each lane are arranged in increasing order as one step for lanes a (TA chain alone), lanes b (TA hybridized to TB chain), lanes c (TA, TB, and TC chain), lanes d (TA, TB, TC, and TD chain hybridized), and lanes e (TDNs targeted to HIF-1. Alpha.), corresponding to TDNs. Also, a single and bright band was obtained in lane e, indicating that the TDNs synthesized in this example were of high purity.

Other core technical indications: this example further confirmed the successful synthesis of TDNs using Atomic Force Microscopy (AFM) (as shown in figure 4). From the AFM images and x-y planes, typical DNA tetrahedral morphology and height can be observed, and the synthesized TDNs exhibit nano-structures with uniform size. From the 0.34nm pitch per base pair, the theoretical values of the tetrahedral side length and cantilever of the designed TDNs were calculated to be 5.44nm and 13.26nm, respectively, in this example, but actually, the height (7.72 nm) obtained at AFM was less than the sum of the side length and cantilever 18.7nm due to the structural collapse of three-dimensional TDNs into two-dimensional TDNs.

Example 2 antitumor Effect of TDNs prepared in example 1

(1) TDNs can bind HIF-1 alpha:

in this example, synthesized TDNs were labeled with biotin and co-cultured with glioblastoma cell U251 and melanoma cells B16-F10, and then introduced into the cells. And culturing the cells under normal culture conditions and hypoxic culture conditions for 24h. Cell lysates were harvested, TDNs were enriched using streptavidin magnetic beads, and HIF-1. Alpha. Bound to TDNs was detected using Western blot. This example found that TDNs bound to HIF-1 α under normal culture conditions, but that TDNs bound to HIF-1 α was less, and binding of TDNs to HIF-1 α was greatly increased under hypoxic conditions, as shown in FIGS. 5A-B, subject to low-level expression of HIF-1 α under normal culture conditions.

(2) TDNs inhibit proliferation of tumor cells and tumor growth in animals:

in this example, TDNs were co-cultured with U251 and B16-F10 cells, and cell proliferation was measured using CCK-8. In this example, it was found that U251 cells have good ability to resist the inhibition effect of hypoxic on cell proliferation, and TDNs can significantly inhibit the proliferation of U251 cells under the condition of hypoxic on the 5 th day of culture, as shown in fig. 6A. The B16-F10 cells were slightly different in the case that on day 2 of culture, the B16-F10 cells had good ability to resist the inhibition of cell proliferation by hypoxia, while TDNs were able to significantly inhibit the proliferation of B16-F10 cells under hypoxic conditions on day 2 of culture, but on day 4 of culture, hypoxia was able to significantly inhibit the proliferation of B16-F10 cells, but DNA tetrahedral nanopharmaceuticals were able to further inhibit the proliferation of B16-F10 cells, as shown in fig. 6B. The above results indicate that the DNA tetrahedral nano-drug can inhibit the proliferation of tumor cells under hypoxic condition.

This example establishes melanoma in situ neoplasia model by inoculating B16-F10 cells into C57/BL6 mice. In this example, it was found that the DNA tetrahedral nano-drug can significantly inhibit the growth of melanoma in mice, as shown in FIGS. 6C-D. And can significantly prolong the survival time of tumor-bearing mice, as shown in fig. 6E.

(3) TDNs inhibit migration of tumor cells and tumor metastasis in animals:

in this example, TDNs were co-cultured with B16-F10 cells and cell migration was examined using a cell scratch assay. This example shows that TDNs can inhibit migration of B16-F10 cells under hypoxic treatment. As shown in fig. 7A and 7C, under normal conditions, cell scratch was significantly reduced after 24h due to the migratory ability of B16-F10 cells, and the process was not affected by TDNs. However, under hypoxic conditions, after 24h, scratches of cells not affected by NA tetrahedral nano-drugs were significantly reduced, while scratches affected by TDNs were limited. Indicating that the TDNs can inhibit the migration of B16-F10 cells under hypoxic treatment. This example cultured B16-F10 cells on a collagen-formed matrix, as shown in FIGS. 7B and 7D, and found that under normal conditions, after 24 hours, the B16-F10 cells were able to invade collagen tissues for a certain distance due to the invasion capacity of the B16-F10 cells, and that the process was not affected by TDNs. However, under hypoxic conditions, after 24h, cells not affected by NA tetrahedral nano-drugs can have invasive capacity, while the distance of invasion of cells affected by TDNs to collagen tissues is significantly reduced. The TDNs are shown to be capable of inhibiting the invasion of B16-F10 cells under hypoxic treatment. The above results indicate that the DNA tetrahedral nano-drug can inhibit the migration and invasion of tumor cells under hypoxic condition.

This example establishes a melanoma metastasis model by injecting B16-F10 cells into C57/BL6 mice via tail vein. In this example, it was found that the DNA tetrahedral nano-drug can significantly inhibit the metastasis of melanoma in mice, as shown in FIGS. 7E-F. And can significantly prolong the survival time of tumor-bearing mice, as shown in fig. 7G.

The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims.

Claims (10)

1. A HIF-1 alpha targeting DNA nanomaterial, comprising: which consists of a regular tetrahedron DNA nano material and a complementary single-stranded DNA,

the regular tetrahedron DNA nano material is formed by self-assembling four single-stranded DNAs (deoxyribonucleic acids) of TA, TB, TC and TD in an equimolar mode, the nucleotide sequence of the regular tetrahedron DNA nano material is sequentially shown as SEQ ID NO. 01-04, four end points of the regular tetrahedron DNA nano material are respectively provided with a cantilever single-stranded DNA, and each cantilever single-stranded DNA is provided with multiple nucleotide sequences specifically combined with HIF-1 alpha;

the complementary single-stranded DNA is complementarily paired with the above-mentioned cantilever single-stranded DNA.

2. The DNA nanomaterial of claim 1, wherein: the nucleotide sequence of the complementary single-stranded DNA is shown in SEQ ID NO. 05.

3. The DNA nanomaterial of claim 2, wherein: the nucleotide sequence of the specific binding HIF-1 alpha is shown in SEQ ID NO. 06.

4. The method for producing a DNA nanomaterial according to any one of claims 1 to 3, wherein: the method comprises the following steps: fully mixing TA, TB, TC and TD with equimolar concentration and the complementary single-stranded DNA in a 1 XTE-Mg buffer solution with the pH value of 8.0, then placing the mixture at 95 ℃ for incubation for 5min, and finally rapidly cooling the mixture to room temperature to synthesize the regular tetrahedral DNA nano material by one-step self-assembly.

5. The method of claim 4, wherein: the concentrations of TA, TB, TC and TD in the 1 XTE-Mg buffer were all 10uM.

6. The method of claim 5, wherein: the concentration of the complementary single-stranded DNA in the 1 XTE-Mg buffer was 40uM.

7. The method according to any one of claims 4 to 6, wherein: the formula of the 1 XTE-Mg buffer solution is as follows: tris.HCl at a final concentration of 10mM, EDTA at 1mM and MgCl at 10mM ₂ 。

8. Use of the DNA nanomaterial of any one of claims 1 to 3 in the preparation of an anti-tumor drug.

9. An antitumor pharmaceutical composition characterized by: the effective components of the composition comprise: the DNA nanomaterial of any one of claims 1 to 3.

10. An antitumor pharmaceutical composition as claimed in claim 9 wherein: the DNA nanomaterial of any one of claims 1 to 3 as an active ingredient.

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