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Application of Fluorinated Polymers in the Intracellular Delivery of Proteins and Small Peptides

A polymer and protein technology, applied in other methods of inserting foreign genetic materials, recombinant DNA technology, etc., can solve problems affecting protein biological activity and safety, complex synthesis, etc., and achieve good biocompatibility and low cytotoxicity , low cost effect

Active Publication Date: 2021-12-24
EAST CHINA NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These chemical modifications are not only complex to synthesize, but also often affect the biological activity and safety of proteins

Method used

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  • Application of Fluorinated Polymers in the Intracellular Delivery of Proteins and Small Peptides
  • Application of Fluorinated Polymers in the Intracellular Delivery of Proteins and Small Peptides
  • Application of Fluorinated Polymers in the Intracellular Delivery of Proteins and Small Peptides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Synthesis and preparation of a protein transfection material library based on polyethyleneimine and fluorinated alkane functional groups

[0058] Three fluorinated alkanes (3-(perfluoro-n-propyl)-1,2-epoxypropane; 3-(perfluoro-n-butyl)-1,2-epoxypropane; 3-(perfluoro-n-propyl) n-hexyl)-1,2-propylene oxide) was grafted onto branched polyethyleneimine through epoxy ring-opening reaction according to different feeding ratios, and 12 kinds of cationic polymers modified by fluorinated alkanes functional groups were obtained for protein transfection Complex.

[0059] In a specific embodiment, the preparation method of the protein transfection material F3-3 shown in formula (2) is: 3-(perfluoro-n-butyl)-1,2-propylene oxide according to the feed ratio of 108: 1 Add 25,000 Dalton branched polyethyleneimine solution dissolved in methanol, stir and react at room temperature for 48 hours, separate and purify to obtain protein transfection material F3-3.

[0060] Protein...

Embodiment 2

[0063] Example 2: Screening of transfection efficiency of protein transfection material library

[0064] The protein transfection material prepared in Example 1 and BSA-FITC formed a complex at room temperature, and then transfected on HeLa cells, and the protein transfection efficiency of the material was evaluated by detecting the green fluorescence level in living cells. The specific method is as follows: culture HeLa cells in a 48-well plate and incubate for 24 hours, mix 4 microliters of BSA-FITC with different doses of materials respectively, add 50 microliters of serum-free medium and incubate for 30 minutes. After adding 150 microliters of serum-free medium, the cells were incubated with the cells for 4 hours before processing the cells. The efficiency of BSA-FITC transfection was evaluated by quantitatively analyzing the green fluorescence level in living cells by flow cytometry. The specific method is as follows: 4 hours after BSA-FITC protein transfection, the cell...

Embodiment 3

[0066] Embodiment 3: the comparison of transfection efficiency of protein transfection material F4-1, F3-3 and the method of protein modification penetrating peptide (TAT)

[0067] After the BSA protein surface was grafted with different proportions of penetrating peptides (TAT) (respectively 2, 4, and 6), compared with the protein transfection materials F4-1 and F3-3 in Example 1, BSA-FITC transfection Dyeing efficiency. The specific method is: the protein transfection materials F4-1, F3-3 prepared in Example 1 and BSA-FITC form a complex at room temperature, and then transfect on HeLa cells, by detecting the green fluorescence in living cells Evaluate the protein transfection efficiency of the material horizontally. Penetrating peptide modified BSA-FITC was used as a control. The specific method is as follows: culture HeLa cells in a 48-well plate and incubate for 24 hours, mix 4 micrograms of BSA-FITC with different doses of materials respectively, add 50 microliters of s...

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Abstract

The present invention provides an application of a fluorine-containing polymer represented by formula (1) in the intracellular delivery of proteins and small peptides, the fluorine-containing polymer includes branched polyethyleneimine and fluorine-containing alkane functional groups, The fluorine-containing alkane functional group is covalently connected to the branched polyethyleneimine; the fluorine-containing macromolecule can be used as a transfection carrier for proteins and / or small peptides. The method of using the fluorine-containing polymer as an intracellular delivery carrier of proteins and small peptides provided by the invention can achieve high transfection efficiency, has little toxicity to cells during the transfection process, and can maintain the biological activity of proteins and small peptides after transfection.

Description

technical field [0001] The invention relates to the fields of biotechnology, macromolecular chemistry, cell biology and the like, in particular to the application of fluorine-containing macromolecules in the intracellular delivery of proteins and small peptides. Background technique [0002] Protein transfection refers to the introduction of exogenous proteins, small peptides, etc. into cells, so as to achieve the purpose of disease treatment and biological research. The main challenge of current protein transfection lies in the lack of efficient and safe transfection techniques. Due to the large molecular weight and hydrophilic characteristics of proteins, small peptides and other molecules, more than 70% of the proteins expressed in the genome cannot pass through the cell membrane barrier, which requires the help of protein transfection technology. The existing protein transfection methods mainly include (1) modifying the penetrating peptide on the protein or small peptid...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/87
CPCC12N15/87
Inventor 程义云张振京
Owner EAST CHINA NORMAL UNIV
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