A gene nanoprobe for targeted lung cancer treatment and its preparation method and application

A nano-probe and gene technology, applied in the field of biochemical nano-materials, can solve problems such as easy complications, poor effect, long treatment cycle, etc., and achieve good therapeutic effect, good synergistic therapeutic effect, and less toxic and side effects Effect

Active Publication Date: 2020-07-28
LINYI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Surgical treatment has disadvantages such as large trauma, high risk, and prone to complications, while radiotherapy has relatively large damage to normal tissues of the body, and the treatment cycle is relatively long.
Simply relying on one treatment method, the effect is not good
Moreover, it is easy to cause other damage to the body and cause complications while treating cancer, and there are disadvantages that cannot be ignored in specific experimental research and clinical application.

Method used

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  • A gene nanoprobe for targeted lung cancer treatment and its preparation method and application
  • A gene nanoprobe for targeted lung cancer treatment and its preparation method and application
  • A gene nanoprobe for targeted lung cancer treatment and its preparation method and application

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preparation example Construction

[0046] The present invention also provides a method for preparing the gene nanoprobe described in the above technical solution, comprising the following steps:

[0047] 1) Mix DNA self-assembled single strands and circular DNA templates in Tris-HCl buffer for annealing treatment. The conditions of the annealing treatment are: 95°C, 5min, and then reduce to 16-16°C at a rate of 0.5°C / min. At 28°C, a three-fork self-assembled structure carrier was obtained;

[0048] 2) Mixing the three-fork self-assembly structure carrier obtained in step 1) with T4 ligase and T4 ligase buffer, and performing a ring-forming reaction at 16° C. to obtain a three-fork self-assembly structure carrier containing a circular template;

[0049] 3) Mix the three-fork self-assembly structure carrier containing the circular template obtained in step 2) with the nucleotide triphosphate mixture, RNase inhibitor, T7 polymerase and T7 polymerase buffer, and perform rolling circle transcription at 37°C React f...

Embodiment 1

[0063] Preparation of RNA nanohydrogels:

[0064]The three DNA single strands (ASM-DNA-1, ASM-DNA-2, ASM-DNA-3) used to synthesize the three-fork self-assembly structure carrier and the Three-let7a, Three-miR 34a, The three circular DNA templates of Three-miR 145 were mixed together, and the Tris-HCl buffer was added to make the volume to 20 μL, so that the concentration of the above substances was 1×10 -6 mol / L. After annealing the above mixed solution (95°C, 5min, then drop 0.5°C per minute to 25°C), the three-pronged self-assembled structure carrier;

[0065] Add T4 ligase and its buffer solution and place at 16°C to form a circle to obtain a three-fork self-assembly structure carrier containing a circular template.

[0066] Then add nucleotide triphosphate mixture (rNTP), RNase inhibitor, T7 polymerase and its buffer to carry out RCT reaction at 37°C for 1.5h to obtain a carrier bound to circular DNA;

[0067] Mix the circular DNA-binding carrier and the targeting aptam...

Embodiment 2

[0071] Carrying out and optimizing DOX and TMPyP4:

[0072] Take 25 μM, 5 μL TMPyP4 solution, detect its ultraviolet absorption at 421 nm, then add different volumes (0-20 μL) of RNA nanohydrogels synthesized according to the required ratio, mix well and react at 37 °C for 2 h . Then, the supernatant was collected by high-speed centrifugation for detection by ultraviolet light absorption spectrum.

[0073] Similarly, similar to the above operation, adding different volumes (0-80 μL) of RNANHs to 20 μM, 100 μL DOX, as the amount of RNANHs added increases, the amount of DOX loaded on it will increase, and the rest will not be loaded into the water. The DOX on the gel will be free in the supernatant, and the supernatant after high-speed centrifugation is used for fluorescence detection by using the fluorescence characteristics of DOX itself.

[0074] The optimal amount of DOX and TMPyP4 that can be carried on the RNANHs can be detected by the above method. The optimization res...

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Abstract

The invention relates to a gene nanoprobe for targeted lung cancer treatment as well as a preparation method and application thereof, and belongs to the technical field of biochemical nano materials.The gene nanoprobe for the targeted lung cancer treatment comprises an RNA nano hydrogel and DOX and / or TMPyP4 loaded on the RNA nano hydrogel; the RNA nano hydrogel comprises a DNA self-assembled single chain, a cyclization DNA template and a targeted aptamer. The gene nanoprobe has a strong specific recognition ability, precise targeting, small toxic and side effects, high killing and injuring intensity against lung cancer cells, good therapeutic effect, and great potential in the application of the lung cancer treatment.

Description

technical field [0001] The invention relates to the technical field of biochemical nanomaterials, in particular to a gene nanoprobe for targeted lung cancer treatment and its preparation method and application. Background technique [0002] Lung cancer is one of the most common malignant tumors, and its mortality rate is at the forefront of all cancer-related deaths. In recent years, the threat of lung cancer to human health cannot be ignored and has become more and more serious. Therefore, it is very necessary to study and formulate effective treatment strategies to improve the treatment of lung cancer. [0003] For the treatment of lung cancer, the existing treatment techniques mostly use surgery and radiation therapy. However, surgical treatment has disadvantages such as large trauma, high risk, and prone to complications, while radiotherapy has relatively large damage to normal tissues of the body, and the treatment cycle is relatively long. Simply relying on one trea...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11A61K31/7105A61P35/00
CPCA61K31/7105A61P35/00C12N15/11
Inventor 李雪梅袁丹丹张家瑀丁来荣崔冰洁张书圣
Owner LINYI UNIVERSITY
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