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A self-assembled nucleic acid nanotube preparation, preparation method and application

A technology of nucleic acid nanotubes and nano-preparations, which is applied in drug combinations, pharmaceutical formulations, cardiovascular diseases, etc., to achieve the effects of reducing mRNA and protein expression levels, inhibiting abnormal proliferation, and enhancing autophagy

Inactive Publication Date: 2017-09-08
THE SECOND AFFILIATED HOSPITAL ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, how to design and develop a clinically applicable siRNA delivery system for the treatment of pulmonary arterial hypertension is still a huge challenge.

Method used

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  • A self-assembled nucleic acid nanotube preparation, preparation method and application
  • A self-assembled nucleic acid nanotube preparation, preparation method and application
  • A self-assembled nucleic acid nanotube preparation, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Synthesis of Tubular Nucleic Acid Nano-Preparations

[0055] Dry powder each sequence of mTOR siRNA (SEQ ID NO: 1), L (SEQ ID NO: 2), M (SEQ ID NO: 3), S (SEQ ID NO: 4) and S' (SEQ ID NO: 5) Samples were diluted with sterilized deionized water to 0.2 μg / μl; 2+ Compounded in buffer solution (pH 8.0), nucleic acid nanotubes (DNA nanotubes) were self-assembled and synthesized under the following conditions: 5 minutes at 95°C, 30 minutes at 65°C, 30 minutes at 50°C, 30 minutes at 37°C, and 30 minutes at 22°C . Subsequently, a determined dose of mTOR siRNA was added into the nanotube solution and left for another 30 min. 500 μl of tubular nanotube preparation solution was obtained. The molar ratio of mTOR siRNA, L, M, S and S' in the preparation is 6:1:3:3:3, and can be used as a medicine for treating pulmonary hypertension.

[0056] The atomic force microscope scanning image of the tubular nucleic acid nanopreparation structure is shown in figure 2 ;

[00...

Embodiment 2

[0059] Example 2: Effects of Different Doses of Transfection Reagents on the Transfection of Tubular Nucleic Acid Nano-Preparations

[0060] Digest the PASMC in the logarithmic growth phase with 0.25% trypsin, climb the cells, and seed the cells in a 24-well plate (built-in coverslip) at 37°C, 5% CO 2 After culturing in the incubator for 48 hours, it can be used for transfection when the cell density reaches 50% to 60%; the experimental grouping is: tubular nucleic acid nano preparation (50nM): the ratio of X-tremeGENE siRNA is 1:0, 1: 0.25, 1:0.5, 1:1, and 1:2, transfect the DNA nanotube nanopreparation carrying mTOR siRNA according to the X-tremeGENE siRNA transfection reagent instructions; after transfecting the nanopreparation, place it at 37°C, 5% CO 2 Incubate for 24 hours in the incubator. Afterwards, discard the culture medium of the 24-well plate, gently rinse with 37°C PBS for 3 min×3 times; add 1ml of 4% paraformaldehyde along the culture plate wall, fix at room te...

Embodiment 3

[0063] Example 3: Effects of Different Dosages of Tubular Nucleic Acid Nano-Preparations on Cell Transfection

[0064] The concentrations of tubular nucleic acid nano-preparations were designed to be divided into five concentrations: 0nM, 12.5nM, 25nM, 50nM and 100nM. During laser confocal observation, the concentration of Cy3-mTOR siRN was set at 12.5nM as the control group. Cy3-mTOR siRNA alone was used as a control for each concentration, and a blank control group was set up. The experimental operation of the confocal laser confocal observation of the cell transfection efficiency experiment and the flow cytometer detection of the average fluorescence density is the same as that of Example 2.

[0065] According to laser confocal Figure 5A and Figure 5B, the Cy3-mTOR siRNA carried by tubular nucleic acid nanocarriers showed red fluorescence distribution in cells, and with the increase of tubular nucleic acid self-assembled nano-preparations, the amount of DNA nanotubes enteri...

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Abstract

The present invention relates to a self-assembled nucleic acid nanotube preparation and its preparation method and application. The preparation comprises mTOR siRNA shown in sequence SEQ ID NO: 1, L shown in sequence SEQ ID NO: 2, sequence shown in SEQ ID M as shown in NO:3, S as shown in SEQ ID NO:4 and S' as shown in SEQ ID NO:5. Nucleic acid cross-links through complementary base pairing of single-stranded nucleic acid molecules into tubular nucleic acid nano-preparations. The preparation makes siRNA double-helical and self-assembles into the nanotube system, making siRNA not easily degraded by nucleases, forming a composite nanotube structure, giving full play to the advantages of nanoparticles being easily taken up by cells, and significantly enhancing the transfection of cells efficiency. Therefore, the siRNA enters the cell structure stably, maintains the original biological activity, regulates cell autophagy and proliferation, can effectively inhibit the abnormal growth of pulmonary artery smooth muscle cells, and can be used to prepare drugs for treating pulmonary hypertension.

Description

technical field [0001] The invention relates to a biological preparation, in particular to a tubular nucleic acid nano preparation containing siRNA, a preparation method and application thereof. Background technique [0002] The development of universal drug carriers is the most important research topic in nanomedicine. Many drug delivery systems have been reported in previous studies, such as: synthetic polymers, cationic liposomes, carbon nanotubes, cyclodextrin materials, nanoparticles, viral capsids, etc. The effectiveness of these delivery systems has been clearly demonstrated; however, most of them are unnatural or exogenous molecules that lack tissue specificity and are potentially toxic. Recent studies on the structure of DNA have allowed it to be further applied to the field of biology. DNA is a natural component in the human body. It is relatively stable, biodegradable, and non-immunogenic. Therefore, self-assembled DNA nanostructures have great potential in the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K9/14A61K48/00A61K31/7088A61P11/00A61P9/12
Inventor 王关嵩尤再春王应明王贝诺钱航杨俊俊
Owner THE SECOND AFFILIATED HOSPITAL ARMY MEDICAL UNIV
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