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Method for preparing lipidosome/gel composite gene activation scaffold

A gene activation and liposome technology, applied in the field of biomedicine, can solve the problems of limitation, slow release rate, low transfection efficiency of naked DNA, etc., and achieve the effects of reasonable method design, simple preparation process, and good research and application prospects.

Active Publication Date: 2018-03-20
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Naked DNA has been successfully entrapped in gels prepared from materials such as collagen, pluronic / hyaluronic acid, polyethylene glycol, and sodium alginate. After implantation, in situ gene transfection and in vivo tissue repair have been achieved. , but the low transfection efficiency and slow release rate of naked DNA limit their application

Method used

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  • Method for preparing lipidosome/gel composite gene activation scaffold
  • Method for preparing lipidosome/gel composite gene activation scaffold
  • Method for preparing lipidosome/gel composite gene activation scaffold

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1: the preparation of TAT-LPD

[0034] First, blank cationic liposomes were prepared. Dissolve the two in chloroform according to the molar ratio of DOTAP:cholesterol=1:1, evaporate under reduced pressure on the rotary evaporator, remove the chloroform to obtain a lipid film, and hydrate with an appropriate amount of HEPES (10mM, pH 7.4) to make the final lipid concentration 10mM, ultrasonic for 3-5 minutes, use Extruder to push liposomes through 0.4μm membrane 11 times, and then push liposomes through 0.1μm membrane 11 times to prepare cationic liposomes with small particle size and uniform distribution, 4°C Save it for later use.

[0035] In the second step, the PRO / DNA complex is prepared. Accurately weigh a certain amount of protamine (PRO), dissolve it in HEPES (10mM, pH 7.4), and prepare a PRO solution with a concentration of 1mg / mL. Vortex and mix PRO and green fluorescent protein particle pEGFP-N1 according to the mass ratio of 0.6:1, and let it st...

Embodiment 2

[0038] Example 2: Preparation of 0% DSPE-PEG-TAT modified LPD

[0039] First, blank cationic liposomes were prepared. Dissolve the two in chloroform according to DOTAP:cholesterol=1:0.5 molar ratio, evaporate under reduced pressure on a rotary evaporator, remove chloroform, and obtain a lipid film, and hydrate with an appropriate amount of HEPES (10mM, pH 7.4) to make the final lipid concentration 10mM, ultrasonic for 3-5 minutes, use Extruder to push liposomes through 0.4μm membrane 11 times, and then push liposomes through 0.1μm membrane 11 times to prepare cationic liposomes with small particle size and uniform distribution, 4°C Save it for later use.

[0040] In the second step, the PRO / DNA complex is prepared. Accurately weigh a certain amount of protamine (PRO), dissolve it in HEPES (10mM, pH 7.4), and prepare a PRO solution with a concentration of 1mg / mL. Vortex and mix PRO and green fluorescent protein particle pEGFP-N1 according to the mass ratio of 0.8:1, and let ...

Embodiment 3

[0043] Example 3: Preparation of double-labeled TAT-LPD with fluorescein and rhodamine

[0044] First, fluorescein-labeled cationic liposomes are prepared. Dissolve the two in chloroform according to the molar ratio of DOTAP:cholesterol = 1:1, dip a small amount of fluorescein phospholipid Fluorescein DHPE (Molecular Probes, USA) into the above chloroform solution with a capillary tube, mix well, and reduce pressure on a rotary evaporator Evaporate and remove chloroform to obtain a lipid film, hydrate with an appropriate amount of HEPES (10mM, pH 7.4) to make the final lipid concentration 10mM, sonicate for 3 to 5 minutes, use an Extruder to push the liposome through the 0.4μm membrane 11 times, and then Push the liposomes through the 0.1 μm membrane 11 times to prepare fluorescein-labeled cationic liposomes with small particle size and uniform distribution, and store them in the dark at 4°C for later use.

[0045] The second step is to prepare the rhodamine-labeled PRO / DNA c...

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Abstract

The invention relates to a method for preparing a lipidosome / gel composite gene activation scaffold, belonging to the technical field of biomedicine. The method disclosed by the invention comprises the following steps: taking TAT modified liposome as a high-efficiency gene transfection vector, entrapping the vector into MMPs sensitive peptide cross-linked hyaluronic acid gel, and constructing theMMPs-sensitive gene activation scaffold in which the lipidosome is entrapped. The MMPs can be secreted by cells in the migration and proliferation process, so that the gel is degraded. On the one hand, a space is created for migration and proliferation of the cells, and a meshed structure of the gel is prevented from becoming a physical barrier of cell migration and proliferation; on the other hand, the TAT-liposome is released, and peripheral cells are subjected to high-efficiency in-situ transfection, so that growth factors are expressed and secreted, and tissue repair and regeneration can be promoted.

Description

technical field [0001] The invention relates to a preparation method of a tissue engineering scaffold, in particular to a preparation method of a matrix metalloproteinase-sensitive liposome / gel composite gene activation scaffold, belonging to the technical field of biomedicine. Background technique [0002] With the rapid development of medical technology, the use of tissue engineering technology to repair and regenerate defective tissues and organs has become a very potential therapeutic method. Growth factors are important cell nutrients used in tissue engineering. Cell proliferation, maintenance of cell survival and many other biological functions. However, most of the growth factors are protein drugs, and their half-life is relatively short. They tend to lose their biological activity in the body and can only produce effects in a short period of time. In addition, growth factors are expensive and costly to treat. [0003] In recent years, the development of gene therapy...

Claims

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Application Information

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IPC IPC(8): C12N15/88
CPCC12N15/88
Inventor 沈海俊王笑娜陶安军
Owner JIANGSU UNIV
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