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ROS-responsive gene delivery vector and its preparation method and application

A gene delivery and vector technology, applied in the field of biomedical materials, can solve the problems of low transfection efficiency and limited application of non-viral vectors

Active Publication Date: 2021-09-28
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the transfection efficiency of non-viral vectors is low, which limits its clinical application. Therefore, the development of safe, effective and controllable non-viral vectors has become a hot research field of gene therapy.

Method used

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  • ROS-responsive gene delivery vector and its preparation method and application
  • ROS-responsive gene delivery vector and its preparation method and application
  • ROS-responsive gene delivery vector and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1: gene delivery vector (POSS-G3-TK x ) preparation method

[0048] POSS-G3-TK x The synthetic route of figure 1 shown, including the following steps:

[0049] Step 1: Synthesis of ROS-sensitive novel thioketal linker (TK)

[0050] 3-mercaptopropionic acid (11.44g, 108.02mmol) and p-hydroxybenzaldehyde (6.0g, 49.1mmol) were dissolved in ethyl acetate, and the mixed solution was catalyzed with an appropriate amount of trifluoroacetic acid (TFA), and continuously React until p-Hydroxybenzaldehyde is consumed. The solvent was removed by rotary evaporation, and the obtained crude product was washed three times with dichloromethane and ice water, respectively. Finally, dry in a vacuum oven to obtain a white powder, which is a thioketal linker (TK) containing a carboxyl terminal residue.

[0051] Step 2: Preparation of active intermediate (TK-NHS active ester)

[0052] Thioketal linker (0.44g, 1.4mmol) and NHS (0.39g, 3.4mmol) were dissolved in the reaction ...

Embodiment 2

[0060] Embodiment 2: the preparation method of gene delivery carrier / DNA complex

[0061] Different qualities of POSS-G3-TK x Dissolve in HBG buffer (20mmol / L 4-hydroxyethylpiperazineethanesulfonic acid aqueous solution, adjust the pH value to 7.4 with NaOH, add 5% (w / v) glucose), mix gently with the same dose of plasmid DNA, and prepare POSS-G3-TK with different N / P ratio x / DNA complex, and then incubated at room temperature (25°C) for 30 min for subsequent experiments. Unless otherwise specified or agreed, the following POSS-G3-TK used in the present invention x The N / P ratio of the / DNA(OTD) complex was 40.

Embodiment 3

[0062] Example 3: Complex POSS-G3-TK x / DNA characterization

[0063] (1) Agarose gel electrophoresis to detect the compression ability of the vector to the gene

[0064] Confirmation of the complex POSS-G3-TK by agarose gel electrophoresis experiments x / DNA formation (agarose gel electrophoresis see figure 2 A), gel electrophoresis shows that when N / P≥10, POSS-G3-TK x can effectively compress the plasmid pEGFP-C1 and form a stable gene complex, indicating that POSS-G3-TK x It has good gene compression ability.

[0065] (2) Particle size and Zeta potential of the complex

[0066] Complex POSS-G3-TK x The particle size and Zeta potential of DNA / DNA are detected by Nano-ZS 90Nanosizer (MalvernInstruments Ltd, Worcestershire, UK) (the particle size and zeta potential of the complex are shown in figure 2 B). Polymer POSS-G3-TK x The particle size and potential of complexes formed with plasmid pEGFP-C1 at different N / P are shown in the figure. When N / P is 20 to 250, PO...

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Abstract

The invention discloses a ROS-responsive gene delivery carrier and its preparation method and application. The gene delivery carrier of the present invention is composed of a ROS-responsive novel thioketal linker (TK) and a POSS-based three-generation lysine star cationic polymer (POSS‑ G3) cross-linked. The cationic polymer gene delivery carrier provided by the invention has low cost, low cytotoxicity and high cell transfection efficiency, and is a very promising cationic polymer gene delivery carrier.

Description

technical field [0001] The invention belongs to the field of biomedical materials, and in particular relates to a ROS-responsive gene delivery carrier and its preparation method and application. [0002] technical background [0003] Gene therapy can not only be used to treat genetic diseases, but also a good strategy in the prevention and treatment of viral infections, diabetes, AIDS and cancer. However, due to the high molecular weight and negatively charged characteristics, free nucleic acid molecules cannot be effectively taken up by cells, and are easily degraded by intracellular and extracellular nucleases, and their bioavailability is extremely low. Therefore, suitable carriers are needed to transport them safely and effectively. Delivered to the target site and exert its effect. Compared with viral vectors, non-viral vectors do not have the disadvantages of cytotoxicity, immunogenicity, and size limitations for loading foreign genes, and also have the advantages of e...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/66C08J3/24C08L77/04
CPCC08J3/24C08J2377/04C12N15/66C12N15/85
Inventor 顾忠伟张瑜芯何一燕周洁马胜男
Owner NANJING TECH UNIV
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