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P53 messenger RNA nanoparticles, preparation method thereof and application thereof in preparing drug for treating tumors

A technology of tumor drugs and nanoparticles, applied in DNA preparation, anti-tumor drugs, gene therapy, etc., can solve the problems of ineffective small molecule inhibitors, genome mutations, etc., and achieve the effect of enhancing chemotherapy sensitivity

Inactive Publication Date: 2020-04-10
HANGZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these attempts have achieved some success, both methods have significant application limitations. For example, small molecule inhibitors will have no effect on deletion mutations of the p53 tumor suppressor gene; p53-based DNA gene therapy has genomic Integration and potential risk of genome mutation

Method used

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  • P53 messenger RNA nanoparticles, preparation method thereof and application thereof in preparing drug for treating tumors
  • P53 messenger RNA nanoparticles, preparation method thereof and application thereof in preparing drug for treating tumors
  • P53 messenger RNA nanoparticles, preparation method thereof and application thereof in preparing drug for treating tumors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Preparation of p53 messenger RNA nanoparticles:

[0056] 1. In vitro synthesis of chemically modified p53mRNA:

[0057] Use in vitro transcription technology (IVT) to synthesize enhanced green fluorescent protein (EGFP) and p53 messenger RNA (mRNA). An untranslated region (UTR) is designed at the 5'end of RNA to enhance the translation initiation of mRNA. In order to improve its stability and translation efficiency, a reverse cap analog (ARCR) was further applied to the 5'end of the mRNA. In order to avoid immune stimulation caused by mRNA, 5-methyl-cytidine triphosphate (5'-Methyl-CTP) and pseudouridine-5'-triphosphate (Pseudo-UTP) were used to replace conventional cytidine triphosphate and uridine Triphosphoric acid. The specific process is:

[0058] The human p53 gene open reading frame (ORF) plasmid carrying the T7 promoter was purchased from Addgene. Digest with HindIII / ApaI endonuclease to form linearized DNA. Then, the open reading frame of p53 contain...

experiment example 1

[0076] Experimental example 1: Nanocarriers deliver p53mRNA to restore the tumor suppressor function of p53 in hepatocellular carcinoma (Hep3B) and non-small cell lung cancer cells (H1299):

[0077] Experimental methods and steps:

[0078] 1. Immunofluorescence (IF) staining: Hep3B cells were fixed with 4% paraformaldehyde (Electron MicroscopySciences) for 15 minutes at room temperature, and then in 0.2% polyethylene glycol octyl phenyl ether (Triton X-100)-phosphate Soak in the buffer for 10 minutes. The samples were further incubated with phosphate blocking buffer solution (containing 2% bovine serum albumin, 2% normal goat serum and 0.2% gel) at room temperature for 30 minutes. Subsequently, it was fixed with 4% paraformaldehyde (Electron Microscopy Sciences) for 15 minutes. Incubate the first antibody overnight at 4°C, wash with phosphate buffer, and then mix with Alexa Fluor 647-labeled goat anti-mouse IgG (Molecular Probe Company) in blocking buffer (1:1000 dilution) at ro...

experiment example 2

[0084] Experimental example 2: p53 angioplasty enhances the sensitivity of p53-deficient hepatocellular carcinoma and non-small cell lung cancer cells to the mTOR inhibitor everolimus:

[0085] Experimental methods and steps:

[0086] 1. Cell survival experiment: p53-inactivated Hep3B or H1299 cells were evenly spread in a 96-well plate at a density of 5000 cells / well. After 24 hours of cell attachment, everolimus of different drug concentrations was added. After culturing for 24 hours, add 0.1ml fresh complete medium to continue culturing for 24 hours, and use Alarma Blue to detect cell viability.

[0087] 2. Western blotting: in dissolution buffer (1mM ethylenediaminetetraacetic acid, 20mM Tris HCl PH 7.6, 140mM sodium chloride, 1% aprotinin, 1% ethyl phenyl polyethylene glycol, 1 mM methyl sulfonate Lyse cells or tumor tissues in acid fluoride solution and 1mM sodium vanadate), and add protease inhibitors (Cell Signaling Technology). The protein concentration was detected by B...

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Abstract

The p53 messenger RNA nanoparticles, a preparation method thereof and an application thereof in preparing a drug for treating tumors belong to the technical field of genetic engineering. The p53 messenger RNA nanoparticles are prepared by the following method: 1, chemically modified p53mRNA is synthesized in vitro; and 2, a nano-system is prepared by a self-assembly method to deliver the p53 messenger RNA in vivo. The p53 messenger RNA nanoparticles play an anti-tumor and chemosensitivity-enhancing therapeutic role by restoring the anti-tumor function of p53. The advantages of the invention are as follows: 1, the p53 messenger RNA in the invention is successfully delivered to the p53 inactivated liver cancer and lung cancer tumor cells through the nano-system to effectively and rapidly induce cell apoptosis and G1 phase cell cycle arrest in vivo and in vitro, thus significantly inhibiting the growth of tumor cells; and 2, the p53 messenger RNA delivered by the nanoparticles can enhancethe anti-tumor effect of a mTOR inhibitor everolimus by supplementing the tumor suppressor p53 deleted in the tumor.

Description

Technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to the technical field of p53 messenger RNA nanoparticle and its preparation method and application in the preparation of tumor drugs. Background technique [0002] In addition to the inactivation of tumor suppressor factors, the occurrence and development of cancer is also accompanied by abnormal activation of tumor growth-promoting functional pathways, such as the mammalian target of rapamycin (mTOR), a serine / threonine kinase that can promote cells Growth and proliferation. The mTOR signaling pathway is abnormally activated in 60% of tumors to increase the proliferation of tumor cells, including liver cancer and lung cancer. [0003] Everolimus (RAD001) is a typical mTOR inhibitor and has been clinically approved by the FDA for use in advanced kidney cancer, pancreatic neuroendocrine tumors and other types of cancer. However, everolimus cannot effectively improv...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K9/51A61K47/34A61K48/00A61K31/7105A61P35/00C12P19/34C12N15/10
CPCA61K9/5146A61K31/7105A61P35/00C12N15/10C12P19/34
Inventor 孔娜谢恬
Owner HANGZHOU NORMAL UNIVERSITY
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