CN106496555B - A kind of metal coordination polycation gene carrier and its preparation method and application - Google Patents

A kind of metal coordination polycation gene carrier and its preparation method and application Download PDF

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CN106496555B
CN106496555B CN201611034965.8A CN201611034965A CN106496555B CN 106496555 B CN106496555 B CN 106496555B CN 201611034965 A CN201611034965 A CN 201611034965A CN 106496555 B CN106496555 B CN 106496555B
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polycation
metal
coordination
stimuli responsive
ligand
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CN106496555A (en
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郭天瑛
刘帅
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Nankai University
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
    • C08G73/02—Polyamines
    • C08G73/0206—Polyalkylene(poly)amines
    • C08G73/0213—Preparatory process
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/48—Polymers modified by chemical after-treatment
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
    • C08G83/002—Dendritic macromolecules
    • C08G83/003—Dendrimers
    • C08G83/004—After treatment of dendrimers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Abstract

A kind of metal coordination polycation gene carrier, is made of coordinating metal, list of coordination units, stimuli responsive unit and polycation.The invention has the advantages that greatly improving polycation DNA binding ability and the active force with cell membrane by the metal-ion ligand for modifying the group containing stimuli responsive on polycation；After compound enters cell, the connection between metal-ion ligand and polycation is disconnected, and DNA is released, expresses, and shows high transfection efficiency, and metal coordination monomer of the invention has expanded application of the polycation in terms of non-viral gene vector significantly.

Description

A kind of metal coordination polycation gene carrier and its preparation method and application
Technical field
The invention belongs to gene therapy and field of new materials, and in particular to a kind of metal coordination polycation gene carrier and Preparation method and application.
Background technique
Gene therapy refers to that functional nucleic acid, which is delivered to specific tissue cell by certain means, carries out disease The process for the treatment of, it can be used for the treatment of many ancestor genetic diseases and the serious acquired disease day after tomorrow.However lack efficient Genophore have become the main obstacle for restricting gene therapy development, especially some functional cells (such as stem cell) Transfection efficiency is especially low, seriously hinders the development of gene regulation.Stem cell is a kind of with self-renewal capacity and differentiation The cell colony of ability is concerned in the fields such as organizational project and cell therapy.By gene regulation, stem cell can be divided into A variety of specific cells, or a series of angiogenesis factors of secretion or neurotrophic factor etc..Therefore seek efficient gene to carry The transfection that body is used as stem cell is still huge challenge.Nowadays, genophore is generally divided into viral vectors and non-virus carrier Two major classes.Although viral vectors shows considerable transfection efficiency, its potential immunogenicity limits clinical application. On the contrary, non-virus carrier has received widespread attention since its structure is controllable, is convenient for the advantages that modifying, is highly-safe, but its Efficiency need to be improved.
Cationic polymer is most important a kind of non-viral gene vector, such as polyethyleneimine (PEI), polylysine (PLL), polyamide-amide (PAMAM) etc..However the transfection efficiency of these polycations, it is especially non-to the transfection efficiency of stem cell It is often low.Therefore being chemically modified to it becomes a kind of effective ways for improving transfection efficiency.On the other hand, metal-ion ligand Its wide prospect in terms of bio-medical is imparted with the strong interaction of phosphoric acid components.Therefore, metal coordination monomer is used to Polycation is modified, can effectively improve its binding ability with DNA and enhances the phase interaction of compound Yu phosphorous cell membrane With promotion endocytosis finally obtains high transfection efficiency.
Summary of the invention
The purpose of the present invention is a kind of metal coordination polycation gene carrier and its system there are problem, are provided for above-mentioned Preparation Method and application synthesize a kind of metal-ion ligand first, modify polycation with it, finally show the transfection effect of superelevation Rate is expected to be used for clinical treatment；The preparation method of metal coordination Poly-cation is provided.
Technical solution of the present invention:
A kind of metal is coordinated polycation gene carrier, by coordinating metal, list of coordination units, stimuli responsive unit and poly- sun from Son composition, general structure are as follows:
Wherein: " M " represents coordinating metal；Lutidines amine component represents list of coordination units；Disulfide bond component represents stimulation Response unit；Ball represents polycation；" n " represents the quantity of a polycation molecules grafted metal ion ligand, n 1- 200；
The coordinating metal is zinc, calcium, magnesium, iron, aluminium, copper, europium or gadolinium；The list of coordination units be the group of class containing pyridine ring or More azo-cycle class groups；The stimuli responsive unit is the N of monomer containing disulfide bond, bis- (acryloyl) cystamines (CBA) of N'-, bis- (2- methyl Propylene) two sulphur (DSDA) of ethyoxyl two sulphur (DSDMA) or bis- (2- propylene) ethyoxyl；The polycation is weight average molecular weight Polyethyleneimine (PEI), the polylysine (PLL) of weight average molecular weight 500-300000 or the polyamides of algebra 1-5 of 600-25000 Amine-amine (PAMAM).
A kind of preparation method of the metal coordination polycation gene carrier, includes the following steps:
1) list of coordination units will be contained and stimuli responsive monomer is dissolved in solvent with molar ratio 1:1-10, be 25-90 in temperature 48h is reacted at DEG C, revolving removes solvent, and product flash chromatography column purification obtains stimuli responsive ligand；
2) 0.5- is mixed in above-mentioned stimuli responsive ligand and the equimolar compound containing coordinating metal at room temperature 12h is formed metal-ion ligand, then is mixed with this metal-ion ligand with polycation with molar ratio 1-200:1, in temperature 48h is reacted at a temperature of 25-90 DEG C, obtains metal coordination polycation gene carrier；Or by stimuli responsive ligand first with poly- sun from Son is reacted 48h at being 25-90 DEG C in temperature, is added and stimuli responsive ligand equimolar amounts with molar ratio 1-200:1 mixing Compound containing coordinating metal, is mixed 0.5-12h at room temperature, obtains metal coordination polycation gene carrier.
A kind of application of the metal coordination polycation gene carrier, is used for non-viral gene vector, the method is as follows: will Metal coordination polycation (M-PD) mixes in equal volume with DNA (DNA) or ribonucleic acid (RNA), stands at room temperature 10-30 minutes, M-PD/DNA the or M-PD/RNA compound of mass ratio 5-60:1 is made；Compound obtained is not only to transfect Conventional immortal cell line can also transfect limited proliferative functionality cell-stem cell.
The invention has the advantages that
The present invention by modifying the metal-ion ligand of the group containing stimuli responsive on polycation, greatly improve poly- sun from Sub DNA binding ability and the active force with cell membrane, improve the endocytosis efficiency of M-PD/DNA compound；On the other hand, multiple It closes after object enters cell, due to stimuli responsive, the connection between metal-ion ligand and polycation is disconnected, and DNA is released, table It reaches, shows high transfection efficiency, metal coordination monomer of the invention has been expanded polycation significantly and carried in non-viral gene Application in terms of body.
Detailed description of the invention
Fig. 1 is the 400M nuclear magnetic spectrogram of Zn-PD4.
Fig. 2 is external 293T cell green fluorescence of the Zn-PD2 and Zn-PD4 under the conditions of polymer/DNA mass ratio 10:1 Transfection figure.Commercialization reagent Xfect and PEI25kIt is transfected according to respective transfection specification.
Fig. 3 turns for external 293T cell green fluorescence of the Zn-PD2 and PD2 under the conditions of polymer/DNA mass ratio 10:1 Dye figure.
Fig. 4 is PEI1.8k, the external rBMSC cell of Zn-PD2 and Zn-PD4 under the conditions of polymer/DNA mass ratio 10:1 Green fluorescence transfection figure.Commercialization reagent Xfect and PEI25kIt is transfected according to respective transfection specification.
Fig. 5 is the PEI in rat bone marrow mesenchymal stem cells (rBMSC)1.8k, Zn-PD2 and Zn-PD4 are in polymer/DNA Transfection efficiency under the conditions of mass ratio 10:1.Commercialization reagent Xfect and PEI25kTurned according to respective transfection specification Dye.Compared with Xfect, P < 0.05 *.
Fig. 6 is the PEI in human marrow mesenchymal stem cell (hBMSC)1.8k, Zn-PD2 and Zn-PD4 are in polymer/DNA matter Amount is than the transfection efficiency under the conditions of 10:1.Commercialization reagent Xfect and PEI25kTurned according to respective transfection specification Dye.Compared with Xfect, P < 0.05 *.
Fig. 7 is the PEI in rat adipose-derived mesenchymal stem cells (rADSC)1.8k, Zn-PD2 and Zn-PD4 are in polymer/DNA Transfection efficiency under the conditions of mass ratio 10:1.Commercialization reagent Xfect and PEI25kTurned according to respective transfection specification Dye.Compared with Xfect, P < 0.05 *.
Specific embodiment
The present invention program is described further below in conjunction with specific embodiment but these embodiments are merely to illustrate this hair It is bright rather than limit the scope of the invention.It is necessary to note that those skilled in the art the present invention can be done various modifications or Change, these equivalent forms also fall within the scope of the appended claims of the present application.
Embodiment 1:
A kind of metal is coordinated polycation gene carrier, by coordinating metal, list of coordination units, stimuli responsive unit and poly- sun from Son composition, the coordinating metal are zinc (Zn), and list of coordination units is lutidines amine groups, and stimuli responsive unit is containing disulfide bond Bis- (acryloyl) cystamines (CBA) of monomer N, N'-；Polycation is the polyethyleneimine (PEI) of weight average molecular weight 1800, zinc coordination The Zn-PD structural formula that monomer modification polycation PEI is obtained is as follows:
In structural formula: " M " represents coordinating metal Zn；" n " represents polyethyleneimine (PEI) molecule grafted metal ion The quantity of ligand, n are 2 or 4.
A kind of preparation method of the metal coordination polycation gene carrier Zn-PD, includes the following steps:
1) preparation of stimuli responsive ligand DDAC
16mmol Isosorbide-5-Nitrae-is dissolved in 20mL methylene chloride benzyl dichloride (Dx), 8mmol lutidines amine is added (DPA) and 40mmol Anhydrous potassium carbonate 48h, is stirred at room temperature, revolving removes solvent, and product flash chromatography column purification obtains DxDA；Then, it takes 2.4mmolDxDA to be dissolved in ethyl alcohol, 10mmol 5- amino -1- amylalcohol (AP) and 12mmol Carbon Dioxide is added Potassium, overnight, product precipitates in distilled water for 60 DEG C of reactions, is extracted with methylene chloride, and organic phase is dry with anhydrous sodium sulfate, revolving Organic phase is removed, DxDA-AP is obtained；Again by bis- (acryloyl) cystamines (CBA) of 0.75mmol DxDA-AP and 1.5mmol N, N'- It is dissolved in methanol, 40 DEG C of reaction 48h jointly, product flash chromatography column purification obtains stimuli responsive ligand DDAC；
2) preparation of the genophore Zn-PD of metal coordination polycation
0.015mmol stimuli responsive ligand DDAC is dissolved in 1mL methanol, equimolar zinc nitrate hexahydrate, electromagnetism is added 3h is stirred, 0.0075mmol and 0.00375mmol PEI is separately added into1.8k, 60 DEG C of reaction 48h, product precipitates with ether, respectively Zn-PD2 and Zn-PD4 polymer is obtained, " 2 " and " 4 " respectively represent a polycation PEI1.8k2 and 4 have been modified on molecule A metal ligand molecule.
Fig. 1 is the nuclear magnetic spectrogram of Zn-PD4.
Embodiment 2:
A kind of metal is coordinated polycation gene carrier, by coordinating metal, list of coordination units, stimuli responsive unit and poly- sun from Son composition, the coordinating metal are magnesium (Mg), and list of coordination units is lutidines amine groups, and stimuli responsive unit is containing disulfide bond Bis- two sulphur (DSDA) of (2- propylene) ethyoxyl of monomer；Polycation is the polyamide-amide (PAMAM) of the first generation, and magnesium is coordinated monomer The Mg-PAD structural formula that modification polycation PAMAM is obtained is as follows:
In structural formula: " M " represents coordinating metal Mg；" n " represent polyamide-amide (PAMAM) molecule grafted metal from The quantity of sub- ligand, n are 1 or 3.
A kind of preparation method of the metal coordination polycation gene carrier Mg-PAD, includes the following steps:
1) preparation of stimuli responsive ligand DDAD
16mmol Isosorbide-5-Nitrae-is dissolved in 20mL methylene chloride benzyl dichloride (Dx), 8mmol lutidines amine is added (DPA) and 40mmol Anhydrous potassium carbonate 48h, is stirred at room temperature, revolving removes solvent, and product flash chromatography column purification obtains DxDA；Then, it takes 2.4mmolDxDA to be dissolved in ethyl alcohol, 10mmol 5- amino -1- amylalcohol (AP) and 12mmol Carbon Dioxide is added Potassium, overnight, product precipitates in distilled water for 60 DEG C of reactions, is extracted with methylene chloride, and organic phase is dry with anhydrous sodium sulfate, revolving Organic phase is removed, DxDA-AP is obtained；Again by bis- two sulphur of (2- propylene) ethyoxyl of 0.75mmol DxDA-AP and 1.5mmol (DSDA) it is dissolved in methanol, 40 DEG C of reaction 48h jointly, product flash chromatography column purification obtains stimuli responsive ligand DDAD；
2) preparation of the genophore Mg-PAD of metal coordination polycation
0.015mmol stimuli responsive ligand DDAD is dissolved in 1mL methanol, is separately added into 0.015mmol and 0.005mmol The polyamide-amide (PAMAM) of a generation, 60 DEG C of reaction 48h, then it is separately added into 0.015mmol magnesium nitrate, 3h, product is stirred at room temperature It is precipitated with ether, respectively obtains Mg-PAD1 and Mg-PAD3 polymer, " 1 " and " 3 " respectively represents a polycation PAMAM points 1 and 3 metal ligand molecules have been modified on son.
Embodiment 3:
The application of prepared metal coordination polycation gene carrier, is used for non-viral gene vector, passes into cell Pass DNA (DNA), the method is as follows: metal coordination polycation Zn-PD is mixed in equal volume with DNA, it is quiet at room temperature It sets 10-30 minutes, the Zn-PD/DNA compound of mass ratio 5-60:1 is made；Compound obtained is not only to transfect conventional nothing Proliferative cell system is limited, limited proliferative functionality cell-stem cell can be also transfected and cytotoxicity is very low, the method is as follows:
The cell transfecting step of Zn-PD2 and Zn-PD4
1) preparation of Zn-PD/DNA compound:
DNA used is green fluorescent protein plasmid pCMV-GFP and luciferase plasmids pCMV-GLuc.By Zn-PD2 or Zn-PD4 and DNA is dissolved in the acetate buffer of pH 5.2 respectively, isometric to mix, and is stored at room temperature 15 minutes, can obtain Zn-PD/ DNA mass ratio is the compound of 5-20:1；
2) Ex vivo cell transfection is tested:
By human embryonic kidney cell line 293T, rat bone marrow mesenchymal stem cells rBMSC, human marrow mesenchymal stem cell hBMSC With rat adipose-derived mesenchymal stem cells rADSC respectively with every hole 5 × 104Cell it is several enter 24 well culture plates, cultivate 24 hours, Above-mentioned Zn-PD/DNA complex nanometer granule is added, the dosage of DNA is 1 hole μ g/, continues to cultivate 48h；When with green fluorescence When albumen plasmid pCMV-GFP is transfected, transfected condition is observed with inverted fluorescence microscope, when with luciferase plasmids pCMV-GLuc When transfection, with BioLuxTMLuciferase Assay Reagent box assesses transfection efficiency.
Fig. 2 is Zn-PD2, Zn-PD4 and commercialization transfection reagent Xfect and PEI25kGFP transfection in 293T cell Picture.Show in figure: the transfection efficiency of Zn-PD2 and Zn-PD4 is superior to commercialization transfection reagent Xfect and PEI25k。
Fig. 3 is that the preceding GFP with after coordination of PD2 coordination transfects picture.Show in figure: PD2 transfection efficiency after zinc is coordinated It significantly improves.
Fig. 4 is PEI1.8k, Zn-PD2, Zn-PD4 and commercialization transfection reagent Xfect and PEI25kIn rBMSC cell GFP transfect picture.Show in figure: Zn-PD4, which is shown, is much better than commercialization transfection reagent Xfect and PEI25kStem cell turn Contaminate efficiency.
Fig. 5, Fig. 6 and Fig. 7 are PEI1.8k, Zn-PD2, Zn-PD4 and commercialization transfection reagent Xfect and PEI25k? Luciferase Transfection efficiency in tri- kinds of stem cells of rBMSC, hBMCS and rADSC.Three figures show Zn-PD4 all kinds of dry thin It is shown in born of the same parents and is much better than commercialization transfection reagent Xfect and PEI25kTransfection efficiency.
3) vitro cytotoxicity is tested:
By human embryonic kidney cell line 293T, rat bone marrow mesenchymal stem cells rBMSC, human marrow mesenchymal stem cell hBMSC With rat adipose-derived mesenchymal stem cells rADSC respectively with every hole 1 × 104Cell it is several enter 96 well culture plates, cultivate 24 hours, Zn-PD/DNA complex nanometer granule is added, the dosage of DNA is 0.25 hole μ g/, continues to cultivate 48h, 3- (4,5- diformazans are added Base thiazole -2) -2,5- diphenyltetrazolium bromide bromide (MTT), detects cytotoxicity.
Metal prepared by the present invention is coordinated polycation gene carrier Zn-PD, not only the tool in regular growth system (293T) There is high transfection efficiency, and also possess high transfection efficiency in various stem cells (rBMSC, hBMSC and rADSC), It is much better than commercialization transfection reagent PEI instantly25kAnd Xfect.In addition, Zn-PD genophore shown in above-mentioned cell it is excellent Low cytotoxicity more.
Embodiment 4:
The application of prepared metal coordination polycation gene carrier, is used for non-viral gene vector, passes into cell Pass ribonucleic acid (RNA), the method is as follows: metal coordination polycation (Mg-PAD) is mixed in equal volume with RNA, is stood at room temperature 10-30 minutes, the Mg-PAD/RNA compound of mass ratio 5-60:1 is made；Compound obtained has high in stem cell Transfection efficiency and low cytotoxicity, the method is as follows:
The cell transfecting step of Mg-PAD1 and Mg-PAD3
1) preparation of Mg-PAD/RNA compound:
RNA used is firefly luciferase cryptiogene siLuc.Mg-PAD1 or Mg-PAD3 and RNA is dissolved in respectively The acetate buffer of pH 5.2, it is isometric to mix, it is stored at room temperature 15 minutes, can obtain Mg-PAD/RNA mass ratio is 5-20:1's Compound；
2) Ex vivo cell transfection is tested:
The rat marrow mesenchyma of expressing luciferase stably (firefly-luciferase) gene is dry thin Born of the same parents rBMSC and human marrow mesenchymal stem cell hBMSC is respectively with every hole 1 × 104Cell it is several enter 96 well culture plates, cultivate 24 Hour, above-mentioned Mg-PAD/RNA complex nanometer granule is added, the dosage of RNA is the hole 5pmol/, continues to cultivate for 24 hours, with firefly Fireworm Luciferase Assay Reagent box detects RNA jamming effectiveness.
3) vitro cytotoxicity is tested:
The rat marrow mesenchyma of expressing luciferase stably (firefly-luciferase) gene is dry thin Born of the same parents (rBMSC-fluc) and human marrow mesenchymal stem cell (hBMSC-fluc) are respectively with every hole 1 × 104Cell it is several enter 96 Well culture plate is cultivated 24 hours, and Mg-PAD/RNA complex nanometer granule is added, and the dosage of RNA is the hole 5pmol/, continues to cultivate For 24 hours, 3- (4,5- dimethylthiazole -2) -2,5- diphenyltetrazolium bromide bromide (MTT) is added, detects cytotoxicity.
Metal prepared by the present invention is coordinated polycation gene carrier Mg-PAD, in stem cell rBMSC-fluc and hBMSC- High RNA jamming effectiveness is shown in fluc, and cytotoxicity is very low.

Claims (3)

1. a kind of metal is coordinated polycation gene carrier, it is characterised in that by coordinating metal, list of coordination units, stimuli responsive unit It is formed with polycation, general structure is as follows:
Wherein: " M " represents coordinating metal；Lutidines amine component represents list of coordination units；Disulfide bond component represents stimuli responsive Unit；Ball represents polycation；" n " represents the quantity of a polycation molecules grafted metal ion ligand, n 1-200；
The coordinating metal is zinc, calcium, magnesium, iron, aluminium, copper, europium or gadolinium；The list of coordination units is lutidines amine；The thorn Swashing response unit is the N of monomer containing disulfide bond, bis- (acryloyl) cystamines of N'-；The polycation is weight average molecular weight 600-25000 Polyethyleneimine, the polylysine of weight average molecular weight 500-300000 or the polyamide-amide of algebra 1-5.
2. metal as described in claim 1 is coordinated polycation gene carrier, which is characterized in that the stimuli responsive in general structure Unit can replace two sulphur of (2- metering system) ethyoxyl in pairs or bis- two sulphur of (2- propylene) ethyoxyl.
3. a kind of preparation method of metal coordination polycation gene carrier as claimed in claim 1 or 2, it is characterised in that including such as Lower step:
1) list of coordination units will be contained and stimuli responsive monomer is dissolved in solvent with molar ratio 1:1-10, in the case where temperature is 25-90 DEG C 48h is reacted, revolving removes solvent, and product flash chromatography column purification obtains stimuli responsive ligand；
2) 0.5-12h, shape is mixed in above-mentioned stimuli responsive ligand and the equimolar compound containing coordinating metal at room temperature It is mixed at metal-ion ligand, then with this metal-ion ligand with polycation with molar ratio 1-200:1, at 25-90 DEG C of temperature At a temperature of react 48h, obtain metal coordination polycation gene carrier；Or by stimuli responsive ligand elder generation and polycation with mole It is mixed than 1-200:1, reacts 48h at being 25-90 DEG C in temperature, add the gold containing coordination with stimuli responsive ligand equimolar amounts The compound of category, is mixed 0.5-12h at room temperature, obtains metal coordination polycation gene carrier.
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