Metal coordination polycation gene vector and preparation method and application thereof
A polycation and gene carrier technology, applied in the field of metal-coordination polycation gene carrier and its preparation, can solve the problems of low transfection efficiency of stem cells, and achieve the effects of expanding applications, improving DNA binding ability, and improving endocytosis efficiency.
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Embodiment 1
[0025] A metal coordination polycation gene carrier, composed of a coordination metal, a coordination unit, a stimulus response unit and a polycation, the coordination metal is zinc (Zn), and the coordination unit is a lutidine amine group, The stimuli response unit is N,N'-bis(acryloyl)cystamine (CBA) containing disulfide bond monomer; the polycation is polyethyleneimine (PEI) with a weight average molecular weight of 1800, and the zinc coordination monomer modifies the polycation The Zn-PD structural formula obtained by PEI is as follows:
[0026]
[0027] In the structural formula: "M" represents the coordination metal Zn; "n" represents the number of metal ion ligands grafted on one polyethyleneimine (PEI) molecule, and n is 2 or 4.
[0028] A method for preparing the metal-coordinated polycation gene carrier Zn-PD, comprising the steps of:
[0029] 1) Preparation of stimuli-responsive ligand DDAC
[0030] Dissolve 16mmol of 1,4-p-dichlorobenzyl (Dx) in 20mL of dichlo...
Embodiment 2
[0035]A metal coordination polycation gene carrier, composed of a coordination metal, a coordination unit, a stimulus response unit and a polycation, the coordination metal is magnesium (Mg), and the coordination unit is a lutidine amine group, The stimuli response unit is bis(2-propylene) ethoxy disulfide (DSDA) containing disulfide bond monomer; the polycation is the first generation polyamide-amine (PAMAM), and the polycation PAMAM is obtained by modifying the polycation PAMAM with magnesium coordination monomer The structural formula of Mg-PAD is as follows:
[0036]
[0037] In the structural formula: "M" represents the coordination metal Mg; "n" represents the number of metal ion ligands grafted on one polyamide-amine (PAMAM) molecule, and n is 1 or 3.
[0038] A preparation method of the metal-coordinated polycation gene carrier Mg-PAD, comprising the steps of:
[0039] 1) Preparation of stimuli-responsive ligand DDAD
[0040] Dissolve 16mmol of 1,4-p-dichlorobenzy...
Embodiment 3
[0044] The application of the prepared metal-coordinating polycation gene carrier is used for non-viral gene carrier to deliver deoxyribonucleic acid (DNA) in cells. Let it stand for 10-30 minutes under high temperature to prepare a Zn-PD / DNA complex with a mass ratio of 5-60:1; the prepared complex is not only used to transfect conventional immortal cell lines, but also to transfect limited proliferation functional Cells - stem cells with low cytotoxicity, as follows:
[0045] Cell transfection steps of Zn-PD2 and Zn-PD4
[0046] 1) Preparation of Zn-PD / DNA complex:
[0047] The DNA used is the green fluorescent protein plasmid pCMV-GFP and the luciferase plasmid pCMV-GLuc. Dissolve Zn-PD2 or Zn-PD4 and DNA in acetate buffer at pH 5.2, mix equal volumes, and let stand at room temperature for 15 minutes to obtain a complex with a Zn-PD / DNA mass ratio of 5-20:1 ;
[0048] 2) In vitro cell transfection experiments:
[0049] The human embryonic kidney cell line 293T, rat bon...
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