MiRNA nano-composite based on tetrahedral framework nucleic acid as well as preparation method and application of miRNA nano-composite

Abstract

The invention provides a miRNA nano-composite based on tetrahedral framework nucleic acid as well as a preparation method and application of the miRNA nano-composite. The specific miRNA is loaded on the tetrahedral framework nucleic acid to form the nanocomposite, and the nanocomposite has an excellent inhibition effect on human tongue squamous cell carcinoma cells, can be used for treating human tongue squamous cell carcinoma, provides a new treatment strategy for human tongue squamous cell carcinoma, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of nucleic acid nanomedicines, and in particular relates to a miRNA nanocomplex based on tetrahedral framework nucleic acid, and a preparation method and application thereof. Background Art

[0002] Tongue cancer is the most common oral cancer with the poorest prognosis. Human tongue squamous cell carcinoma is a common malignant tumor. It mainly occurs in the squamous epithelial cells of the oral mucosa and usually manifests as lumps or ulcers on the surface of the tongue. These lumps may be irregular and accompanied by pain and bleeding symptoms. According to existing data, tongue squamous cell carcinoma is highly invasive and has a tendency to metastasize, especially to cervical lymph node metastasis. Treatment methods usually include surgery, radiotherapy, chemotherapy, and photodynamic therapy. However, these treatments are often accompanied by serious side effects and limited therapeutic effects.

[0003] DNA nanomaterials (tetrahedral framework nucleic acids tFNAs) are nucleic acid molecules synthesized by base complementary pairing of several DNA single strands (usually 4). They have good stability in the body and can be used as carriers of certain drugs. They also have good biocompatibility, editability and biosafety. On the one hand, they have the potential to be used as drug carriers. On the other hand, there are also reports showing that DNA tetrahedrons themselves have certain pharmacological activities.

[0004] So far, there has been no report on the successful use of DNA tetrahedral nucleic acid structure combined with miRNA to inhibit tongue squamous cell carcinoma cells and achieve the treatment of human tongue squamous cell carcinoma. Summary of the invention

[0005] The purpose of the present invention is to provide a miRNA nanocomplex in tetrahedral framework nucleic acid to promote apoptosis of human tongue squamous cell carcinoma cells, thereby effectively treating human tongue squamous cell carcinoma.

[0006] The present invention provides a miRNA nanocomplex based on tetrahedral framework nucleic acid, which is characterized by: it is obtained by mixing DNA tetrahedral framework nucleic acid and miRNA and then incubating them;

[0007] The DNA tetrahedral framework nucleic acid is synthesized by self-assembly of four DNA single strands; the nucleotide sequences of the four DNA single strands are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 respectively;

[0008] The nucleotide sequence of the miRNA is shown as SEQ ID NO.5 or SEQ ID NO.6.

[0009] Furthermore, the method for synthesizing the DNA tetrahedral framework nucleic acid comprises the following steps: adding four DNA single strands into TM buffer, maintaining at 95° C. for 10 min, and maintaining at 4° C. for more than 20 min, to obtain.

[0010] Furthermore, the molar ratio of the DNA tetrahedral framework nucleic acid to the miRNA is 1:0.5-2.

[0011] Furthermore, the molar ratio of the DNA tetrahedral framework nucleic acid to the miRNA is 1:1.

[0012] The present invention also provides a method for preparing the aforementioned miRNA nanocomplex, which comprises the following steps:

[0013] The DNA tetrahedral framework nucleic acid and miRNA are mixed and incubated to obtain the product.

[0014] Furthermore, the incubation temperature is 25 to 35° C.; and / or the incubation time is 10 to 30 minutes.

[0015] The present invention also provides a use of the aforementioned miRNA nanocomplex in preparing a drug for treating human tongue squamous cell carcinoma and / or inhibiting metastasis of human tongue squamous cell carcinoma.

[0016] Furthermore, the drug is a drug that inhibits the activity of human tongue squamous cell carcinoma.

[0017] Finally, the present invention provides a drug for treating human tongue squamous cell carcinoma and / or inhibiting metastasis of human tongue squamous cell carcinoma, which comprises the aforementioned miRNA nanocomplex and a pharmaceutically acceptable carrier.

[0018] The miRNA nanocomplex prepared by the present invention by carrying specific miRNA on tetrahedral framework nucleic acid can significantly inhibit the activity of human tongue squamous cell carcinoma cells. In vitro cell experiments show that the miRNA in the nanocomplex of the present invention has a certain inhibitory effect on human tongue squamous cell carcinoma cells, and the tetrahedral framework nucleic acid not only has no inhibitory effect on human tongue squamous cell carcinoma cells, but has a promoting effect. However, the miRNA and tetrahedral framework nucleic acid that have opposite effects on the activity of human tongue squamous cell carcinoma cells are combined together, which unexpectedly improves the activity of miRNA in inhibiting human tongue squamous cell carcinoma cells. The excellent effect of the nanocomplex of the present invention in inhibiting the activity of human tongue squamous cell carcinoma cells makes it possible to treat human tongue squamous cell carcinoma, providing a new treatment strategy for patients with human tongue squamous cell carcinoma, and has good application prospects.

[0019] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0020] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the PAGE result of TR, which verifies that TR was prepared successfully.

[0022] Figure 2 This is the HPCE result of TR, which verifies that TR was successfully prepared.

[0023] Figure 3 Zeta potential diagram of tFNAs and TR.

[0024] Figure 4 Figure 2 shows the particle size of tFNAs and TR.

[0025] Figure 5 TEM image of TR, scale bar is 50 nm.

[0026] Figure 6 This is the AFM morphology result of TR.

[0027] Figure 7 The results of the CCK8 experiment to determine cell viability. a is the cell viability after incubating cells with different concentrations of TR for 24 hours; b is the cell viability after incubating cells with miRNA, PYM, TR and tFNAs for 24 hours; data analysis: mean ± standard deviation (SD) (n = 3). DETAILED DESCRIPTION

[0028] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0029] Example 1. Preparation of TR Nanocomplex Based on Tetrahedral Framework Nucleic Acid (TR)

[0030] 1. Experimental Methods

[0031] 1. Synthetic tFNAs

[0032] Four DNA single strands S1, S2, S3 and S4 (sequences as shown in Table 1) were added to 96 μL TM buffer (10 mM Tris-HCl, 50 mM MgCl2, pH = 8.0) in an equimolar ratio, and the final concentration of the four DNA single strands was 1 μM. After thorough mixing, the mixture was rapidly heated to 95°C and maintained for 10 minutes, and then rapidly cooled to 4°C and maintained for 20 minutes to obtain tetrahedral framework nucleic acids, referred to as tFNAs.

[0033] Table 1 Single-stranded DNA sequences for preparing tFNAs

[0034]

[0035] 2. Synthetic TR

[0036] miRNA sequences:

[0037]

[0038] The 5' end of the S3 chain is connected to a sticky end (SEQ ID NO.7:

[0039] TTGACCTGTGAATT), this sticky end does not participate in base complementary pairing during the self-assembly process of tFNAs, so the sticky end will hang on a vertex of the tetrahedral structure in the form of a single strand.

[0040] 1 μL of miRNA sense strand was mixed with 1 μL of tFNAs of the same concentration and then allowed to stand at room temperature for half an hour to prepare a tetrahedral framework nucleic acid-based miRNA nanocomplex named TR.

[0041] The beneficial effects of the TR prepared by the present invention are demonstrated by experimental examples below.

[0042] Experimental Example 1: Characterization of TR of the present invention

[0043] 1. Experimental Methods

[0044] (I) Verification of the synthesis of tFNAs and TR by polyacrylamide gel electrophoresis (PAGE)

[0045] The experiment was conducted using 8% PAGE gel. The four DNA single strands (S1, S2, S3, S4), tFNAs and TR were mixed with 6x loading buffer and loaded, and then electrophoresed in 1xTAE electrophoresis buffer for 80 minutes at a constant voltage of 80V. After removing the PAGE gel, it was stained with Gel-red for 20 minutes in a light-proof environment, and finally exposed to a chemiluminescent imager.

[0046] (II) Verification of the synthesis of tFNAs and TR by high-performance capillary electrophoresis

[0047] DNA single strands (S1, S2, S3, S4), tFNAs and TR were loaded according to the instructions of high performance capillary electrophoresis, and simulated gel images were obtained according to instrument analysis.

[0048] (III) Transmission electron microscopy (TEM) determination of the morphology of tFNAs and TR

[0049] Dilute the TR solution 3-5 times, take a small amount and drop it on the copper mesh. Dye it for 2 minutes and dry it. Then use an instrument to observe the shape and size of TR.

[0050] (IV) Atomic force microscopy (AFM) determination of the morphology of tFNAs and TR

[0051] The synthesized TR solution was diluted 3-5 times, and 1 mL of the sample was placed in the measuring cell. The morphology of tFNA-Que was measured by AFM using the tapping mode, and the particle size of the material was measured according to the instructions.

[0052] (V) Dynamic light scattering (DLS) determination of particle size and zeta potential of tFNAs and TR

[0053] The synthesized tFNAs and TR solutions were diluted 10-20 times. 1 mL of the sample was taken and added to the potential dish and the particle size dish respectively. Subsequently, the two containers were placed in the potential particle size analyzer for detection, and the obtained data were analyzed.

[0054] 2. Experimental Results

[0055] (I) PAGE verification of TR synthesis results

[0056] PAGE results are as follows Figure 1 As shown, it can be seen that the lanes from left to right are S1, S2, S3, S4, tFNAs, TR. It can be seen that tFNAs is located between 180-200bp, and there is no obvious miscellaneous band. In the second lane on the right, it can be seen that the molecular weight of TR is about 200bp. As the concentration of miRNA increases, the migration rate of TR decreases. Therefore, the molecular weight of TR in the figure is greater than that of tFNAs. It proves that TR was successfully synthesized.

[0057] (II) Synthesis of TR verified by efficient capillary

[0058] The position and banding of tFNAs in the lanes were observed by high-performance capillary electrophoresis. Figure 2 As shown, the tetrahedral framework nucleic acid tFNAs is located at about 180 bp in the second lane on the right, without obvious miscellaneous bands, which is consistent with the results of the PAGE run, once again proving the successful synthesis of TR.

[0059] (III) DLS detection of particle size and Zeta potential of tFNA-Que

[0060] Zeta potential of tFNAs and TR Figure 3 As shown in Figure 2, the particle size of tFNAs and TR is shown in Figure 2. Figure 4As shown, it can be seen that after tFNAs were loaded with miRNA to form a TR complex, the zeta potential changed from -6.71±0.74mV to -8±1.01mV, and the particle size changed from 12.91±0.79nm to 13.57±1.37nm.

[0061] (IV) TEM determination of TR morphology

[0062] TEM images of TR are shown in Figure 2. Figure 5 As shown, transmission electron microscopy was used to observe that TR exhibited a tetrahedral structure.

[0063] (V) AFM measurement of TR morphology

[0064] The AFM results of TR are shown in Figure 6 As shown in the figure, the miRNA can be observed to be clearly attached to the top of tFNAs by AFM, further proving the successful synthesis of TR. The right figure is an enlarged image, and the tetrahedral framework structure of TR is observed.

[0065] Experimental Example 2: Cell Experiment

[0066] 1. Experimental Methods

[0067] (I) Experimental cells

[0068] Human tongue squamous cell carcinoma cells (SCC9) were used for the experiments.

[0069] (II) CCK8 assay to determine cell viability

[0070] SCC9 cells were grown at 6×10 3 The cells were seeded in 96-well plates at a density of 100 and incubated overnight at 37°C in 5% CO2.

[0071] Subsequently, DMEM / HIGH complete medium containing different molar concentrations of TR (TR concentrations were 62.5nM, 125nM, and 250nM, respectively) was first added to the well plate, and after 24 hours of incubation, the cells were washed with phosphate buffered saline (PBS). Subsequently, 90μL of culture medium and 10μL of CCK8 solution were added to each well, and then cultured at 37°C for 1 hour. After incubation, the 96-well plate was placed in an ELISA reader, and the absorbance of each group was detected at a wavelength of 450nm.

[0072] To detect the effects of microRNA-149-3p mimics, PYM and TR intervention on the activity of SCC9 (human tongue squamous cell carcinoma cells), DMEM / F-12 culture medium containing microRNA-149-3p mimics, bleomycin (PYM), TR and tFNAs were added to the well plate (the final concentrations of microRNA-149-3p mimics, PYM, TR and tFNAs were all 250 nM). No drugs were added to the control group, and the culture was continued for 24 hours, and the OD value was finally detected.

[0073] (III) Data Statistics

[0074] All experiments were repeated at least three times to ensure reliability, and the experimental data were statistically analyzed using GraphPad Prism software version 9.0. For comparisons between groups, we used t-test or one-way analysis of variance (ANOVA) methods. The statistical significance level was set at *p<0.05, **p<0.01, ***p<0.001, and "ns" indicated no statistical significance.

[0075] 2. Experimental results - CCK8 assay to determine cell viability

[0076] The results of cell activity experiment (CCK8 experiment) are as follows Figure 7 As shown, from Figure 7 a It can be seen that at a certain concentration, TR has a significant inhibitory effect on squamous cell carcinoma cells.

[0077] Specifically, different concentrations of TR have different effects on cell activity. When the concentration is 62.5 nM, TR promotes the activity of SCC9 cells. When TR is 125 nM, SCC9 cells are higher than the control group. When the TR concentration is 250 nM, TR can significantly inhibit the activity of SCC9 cells.

[0078] Figure 7 b It can be seen that under the same concentration conditions, tFNAs have no inhibitory effect on the activity of squamous cell carcinoma cells, but have a promoting effect. Both RNA-149-3p mimics and TR have an inhibitory effect on the activity of squamous cell carcinoma cells, but the effect of TR is significantly improved compared to the inhibitory effect of miRNA.

[0079] In summary, the miRNA in the nanocomplex of the present invention has a certain inhibitory effect on human tongue squamous cell carcinoma cells, and the tetrahedral framework nucleic acid not only has no inhibitory effect on human tongue squamous cell carcinoma cells, but has a promoting effect. However, the miRNA and tetrahedral framework nucleic acid, which have opposite effects on the activity of human tongue squamous cell carcinoma cells, are combined together, which unexpectedly improves the activity of miRNA in inhibiting human tongue squamous cell carcinoma cells. The excellent effect of the nanocomplex of the present invention in inhibiting the activity of human tongue squamous cell carcinoma cells makes it possible to treat human tongue squamous cell carcinoma, providing a new treatment strategy for patients with human tongue squamous cell carcinoma, and has good application prospects.

Claims

1. A miRNA nanocomplex based on tetrahedral framework nucleic acid, characterized in that: It is obtained by mixing DNA tetrahedral framework nucleic acid and miRNA and then incubating them; The nucleotide sequence of the miRNA is shown in SEQ ID NO.5-6.

2. The miRNA nanocomplex according to claim 1, characterized in that: The DNA tetrahedral framework nucleic acid is synthesized by self-assembly of four DNA single strands; the nucleotide sequences of the four DNA single strands are shown in SEQ ID NOs. 1 to 4 respectively.

3. The miRNA nanocomplex according to claim 1, characterized in that: The method for synthesizing the DNA tetrahedral framework nucleic acid comprises the following steps: adding four DNA single strands into TM buffer, maintaining at 95° C. for 10 minutes and maintaining at 4° C. for more than 20 minutes to obtain the nucleic acid.

4. The miRNA nanocomplex according to any one of claims 1 to 3, characterized in that: The molar ratio of the DNA tetrahedral framework nucleic acid to the miRNA is 1:0.5-2.

5. The miRNA nanocomplex according to any one of claims 1 to 3, characterized in that: The molar ratio of the DNA tetrahedral framework nucleic acid to the miRNA is 1:

1.

6. A method for preparing the miRNA nanocomplex according to any one of claims 1 to 5, characterized in that: It includes the following steps: The DNA tetrahedral framework nucleic acid and miRNA are mixed and incubated to obtain the product.

7. The method according to claim 6, characterized in that: The incubation temperature is 25 to 35° C.; and / or the incubation time is 10 to 30 minutes.

8. Use of the miRNA nanocomplex according to any one of claims 1 to 5 in the preparation of a drug for treating human tongue squamous cell carcinoma and / or inhibiting metastasis of human tongue squamous cell carcinoma.

9. The use according to claim 8, characterized in that: The drug is a drug for inhibiting the activity of human tongue squamous cell carcinoma.

10. A drug for treating human tongue squamous cell carcinoma and / or inhibiting metastasis of human tongue squamous cell carcinoma, characterized in that: It comprises the miRNA nanocomplex according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

Images