CN108948262B - Copolymer nucleic acid carrier containing metal coordination pyridine derivative ligand, preparation method and application - Google Patents
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Abstract
Copolymer nucleic acid carrier containing metal coordination pyridine derivative ligand, preparation method and application. The carrier consists of a ligand functional monomer, a hydrophilic functional monomer and a hydrophobic functional monomer, wherein the ligand functional monomer consists of a coordination metal, a coordination unit and a stimulus response unit. The non-viral vector material containing the metal coordination pyridine derivative ligand copolymer prepared by the controllable polymerization method has strong DNA binding capacity and acting force with cell membranes, and the obtained DNA compound has high endocytosis efficiency; after the complex enters cells, due to stimulation response, the metal ion ligand unit is disconnected from the copolymer main chain, DNA is released and expressed, and high transfection efficiency is shown.
Description
Technical Field
The invention belongs to the field of gene therapy and new materials, and particularly relates to a preparation method and application of a copolymer nucleic acid carrier containing a metal coordination pyridine derivative ligand.
Background
Gene therapy achieves the goal of treating disease by delivering therapeutically functional nucleic acid molecules to specific cells of a patient, and holds promise for the cure of many currently incurable major diseases in humans, such as cancer, hereditary diseases (e.g., hemophilia), infectious diseases (e.g., aids and rheumatoid diseases, etc.), and cardiovascular diseases. The key to successful gene therapy is how to construct a safe and efficient nucleic acid delivery system to deliver therapeutic genes to the site of treatment and to transfect them efficiently. Currently, viral vectors are mainly used clinically, and although viral vectors exhibit very considerable transfection efficiency, their potential immunogenicity limits clinical applications. On the contrary, non-viral vectors such as cationic polymers are the most important non-viral gene vectors, and have been rapidly developed in recent years due to the advantages of weak immunogenicity, strong nucleic acid loading capacity, easy structural modification and the like, however, nucleic acid coated by cationic polymers is negatively charged nucleic acid and excessive polycation to form nano nucleic acid complex particles with excessive positive charges, so that nanoparticles with excessive positive charges and negatively charged serum proteins or cell membrane components are easily aggregated in the in vivo circulation due to electrostatic interaction to form large particles which are rapidly eliminated by reticuloendothelial system (RES). In addition, the excessive positive charge of the nucleic acid complex is positively correlated with sustained cytotoxicity, inflammation, apoptosis, and the like. Therefore, based on the above-mentioned poor characteristics of the polycation-based nucleic acid complex nanoparticles, the development of low charge density or uncharged vectors is of great importance to promote the clinical application of non-viral vectors from the viewpoint of clinical application.
Disclosure of Invention
The invention aims to provide a preparation method and application of a multifunctional copolymer nucleic acid carrier containing a metal coordination pyridine derivative ligand, which has low charge density and aims at solving the problems that the existing polycation nucleic acid carrier has high positive charge density and has high cytotoxicity in vivo application.
The invention firstly synthesizes functional monomers containing pyridine derivative ligands with different compositions and structures, and synthesizes copolymer non-viral nucleic acid carrier materials containing metal coordination pyridine derivative ligands with fine structures by using a controllable free radical polymerization method (such as RAFT method), finally shows high transfection efficiency and is expected to be used for clinical treatment.
The technical scheme of the invention is as follows:
a copolymer nucleic acid carrier material containing metal coordination pyridine derivative ligand is prepared by copolymerizing a metal ligand functional monomer consisting of coordination metal, a coordination unit and a stimulus response unit with other hydrophilic functional monomers and hydrophobic functional monomers and performing metal coordination, wherein the copolymerization molar ratio of the metal ligand functional monomer to the hydrophilic functional monomer to the hydrophobic functional monomer is as follows: 1:(0.5-8):(0.5-10).
The coordination metal is zinc, calcium, magnesium, iron, aluminum, copper, europium or gadolinium; the coordination unit is a pyridine ring group or a polynitrogen ring group; the stimulus response unit is disulfide bond-containing monomer N, N' -bis (acryloyl) Cystamine (CBA), bis (2-methylpropene) ethoxy disulfide (DSDMA) or bis (2-propylene) ethoxy disulfide (DSDA); the hydrophilic functional monomer refers to methacrylate, acrylate, methacrylamide and acrylamide monomers which contain hydroxyl, polyethylene glycol chain segments or sugar-containing chain segments in side groups and have water solubility or strong hydrophilicity; the hydrophobic functional monomer is selected from methacrylate, acrylate, methacrylamide and acrylamide monomers with hydrophobic side groups.
A preparation method of the copolymer nucleic acid carrier material containing the metal coordination pyridine derivative ligand comprises the following steps:
uniformly mixing a metal ligand functional monomer, a hydrophilic functional monomer and a hydrophobic functional monomer in a molar ratio of 1 (0.5-8) to (0.5-10) in dimethylformamide, and then adding an initiator azobisisobutyronitrile AIBN or 4,4' -azobis (4-cyanovaleric acid) (ACVA) with a molar ratio of 0.06-0.08 and a chain transfer agent 4-cyanovaleric acid dithiobenzoic acid (CPADB) with a molar ratio of 0.1-0.4 according to the proportion of the metal ligand functional monomer as 1; transferring the mixture to a glass-sealed tube and treating 3 freeze-thaw cycles with liquid nitrogen to remove oxygen; the polymerization reaction is continued for 4 to 48 hours at the temperature of between 60 and 80 ℃, and then the mixture is cooled in an ice water bath; precipitating for three times in a solution of 10 times volume of reaction mixture with the volume ratio of diethyl ether to petroleum ether v/v of 1:2, and drying the product under vacuum to obtain a copolymer containing pyridine derivative ligand; obtaining the copolymer nucleic acid carrier containing the metal coordination pyridine derivative ligand after coordination with metal ion hydrated nitrate.
1) The prepared functional monomer containing the metal ligand and hydrophilic and hydrophobic functional monomers are integrated on the basis of giving consideration to the hydrophilic and hydrophobic water balance and coordination of materials, 4-cyanovaleric acid dithiobenzoic acid DB is taken as a chain transfer agent, azobisisobutyronitrile AIBN or 4,4' -azobis (4-cyanovaleric acid) ACVA is taken as an initiator, and a series of two-block, multi-block or random copolymers with the molecular weight of 3000-10000Da and different compositions and chain structures are synthesized through RAFT copolymerization.
2) And mixing and stirring the copolymer obtained above and equimolar compound containing the coordination metal at room temperature for 0.5-12h to form a metal ion ligand, thereby obtaining the copolymer nucleic acid carrier containing the metal coordination pyridine derivative ligand.
The application of the copolymer nucleic acid vector containing the metal coordination pyridine derivative ligand is mainly used for non-viral gene vectors, and the method comprises the following steps: mixing a copolymer nucleic acid carrier containing a metal coordination pyridine derivative ligand with deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in equal volume, and standing for 10-30 minutes at room temperature to prepare a DNA or RNA compound with the mass ratio of 5-60: 1; the complexes prepared were used to transfect different cell lines.
The invention has the advantages that:
the copolymer nucleic acid carrier material containing the metal coordination pyridine derivative ligand prepared by the controllable polymerization mode has strong DNA binding capacity and acting force with cell membranes, and the obtained DNA compound has high endocytosis efficiency; after the compound enters cells, due to stimulation response, the metal ion ligand unit is disconnected from the main chain of the copolymer, DNA is released and expressed, and high transfection efficiency is shown.
Drawings
FIG. 1 shows a 400M NMR spectrum of Zn-HDB.
FIG. 2 is a graph of green fluorescence transfection of 293T cells in vitro under different mass ratios of Zn-HDB/DNA. Commercial reagent Lipo2k and PEI25kTransfection was performed according to the respective transfection instructions.
Detailed Description
The invention will now be further illustrated by reference to specific examples which are provided for the purpose of illustration only and are not intended to limit the scope of the invention. It is to be noted that various modifications and changes may be made by those skilled in the art, and equivalents may fall within the scope of the invention defined by the claims appended hereto.
Example 1:
a copolymer nucleic acid carrier material containing metal coordination pyridine derivative ligands is prepared by polymerizing a pyridine derivative ligand-containing functional monomer consisting of a coordination unit dimethyl pyridine amine group and a stimulus response unit disulfide bond-containing monomer N, N' -bis (acryloyl) Cystamine (CBA) with a hydrophilic monomer hydroxyethyl methacrylate (HEMA) and a hydrophobic monomer N-butyl methacrylate by a controllable free radical, wherein the coordination metal is zinc (Zn) ions, and the structural formula of the copolymer nucleic acid carrier material of the zinc coordination pyridine derivative ligands is shown as follows:
the preparation method of the copolymer nucleic acid carrier material containing the metal coordination pyridine derivative ligand shown in the structural formula comprises the following steps:
1) preparation of metal ligand functional monomer DPASS
Dissolving 16mmol of 1, 4-p-dichlorobenzyl (Dx) in 20mL of dichloromethane, adding 8mmol of dimethylpyridine amine (DPA) and 40mmol of anhydrous potassium carbonate, stirring at room temperature for 48h, removing the solvent by rotary evaporation, and purifying the product by using a flash chromatography column to obtain DxDA; then, 2.4mmol DxDA is taken to be dissolved in ethanol, 10mmol 5-amino-1-pentanol (AP) and 12mmol anhydrous potassium carbonate are added to react at 60 ℃ overnight, the product is precipitated in distilled water and extracted by dichloromethane, an organic phase is dried by anhydrous sodium sulfate, and the organic phase is removed by rotary evaporation to obtain DxDA-AP; and dissolving 0.75mmol of DxDA-AP and 1.5mmol of N, N' -bis (acryloyl) Cystamine (CBA) in methanol, reacting for 48 hours at 40 ℃, and purifying the product by using a fast chromatography column to obtain the metal ligand functional monomer DPASS.
2) Preparation of copolymer Zn-HDB containing metal coordinated pyridine derivative ligand
Hydroxyethyl methacrylate (0.1mmol), DPASS (0.1mmol), n-butyl methacrylate (0.4mmol) were mixed in dimethylformamide, followed by the addition of initiator ACVA (0.0067mmol) and chain transfer agent CPADB (0.02 mmol). The mixture was transferred to a glass-sealed tube and treated with liquid nitrogen for 3 freeze-thaw cycles to remove oxygen. The polymerization was carried out at 70 ℃ for 48 hours, followed by cooling with ice water. Precipitating in ethyl ether-petroleum ether (v/v,1:2) solution for three times, and vacuum drying to obtain copolymer HDB. And (3) mixing and dissolving equal molar weight of HDB and zinc nitrate hexahydrate in methanol, stirring at 40 ℃ for 24 hours, and carrying out vacuum drying on the product to obtain Zn-HDB.
FIG. 1 shows the nuclear magnetic spectrum of Zn-HDB.
Example 2:
a copolymer nucleic acid carrier material containing metal coordination pyridine derivative ligand is composed of coordination metal, a pyridine derivative ligand functional monomer consisting of a coordination unit and a stimulus response unit, a hydrophilic monomer and a hydrophobic monomer, wherein the coordination metal is magnesium (Mg), the coordination unit is a dimethyl pyridylamine group, and the stimulus response unit is a disulfide bond-containing monomer bis (2-propylene) ethoxy disulfide (DSDA); the hydrophilic monomer is hydroxyethyl methacrylate; the hydrophobic monomer is isopropyl acrylamide.
A preparation method of the copolymer carrier Mg-HDN containing the metal coordination pyridine derivative ligand comprises the following steps:
1) preparation of functional monomer DDAD of metal ligand
Dissolving 20mmol of 1, 4-p-dichlorobenzyl (Dx) in 20mL of dichloromethane, adding 10mmol of dimethylpyridine amine (DPA) and 45mmol of anhydrous potassium carbonate, stirring at room temperature for 24h, removing the solvent by rotary evaporation, and purifying the product by using a flash chromatography column to obtain DxDA; then 3.0mmol of DxDA is dissolved in ethanol, 12mmol of 5-amino-1-pentanol (AP) and 12mmol of anhydrous potassium carbonate are added, the mixture reacts overnight at 65 ℃, a product is precipitated in distilled water and extracted by dichloromethane, an organic phase is dried by anhydrous sodium sulfate, and the organic phase is removed by rotary evaporation to obtain DxDA-AP; then 0.9mmol DxDA-AP and 1.8mmol bis (2-propylene) ethoxy disulfide (DSDA) are dissolved in methanol together, reaction is carried out for 24 hours at 40 ℃, and the product is purified by a fast chromatographic column to obtain a metal ligand functional monomer DDAD;
2) preparation of copolymer Mg-HDN containing metal coordination pyridine derivative ligand
Hydroxyethyl methacrylate (0.2mmol), DDAD (0.1mmol), N-isopropylacrylamide (0.6mmol) were mixed in dimethylformamide, and then initiator ACVA (0.0067mmol) and chain transfer agent CPADB (0.03mmol) were added. The mixture was transferred to a glass-sealed tube and treated with liquid nitrogen for 3 freeze-thaw cycles to remove oxygen. The polymerization was reacted at 70 ℃ for 48 hours, then cooled with ice water, and the reaction mixture was reacted in ether: precipitating for three times in petroleum ether (v/v,1:2) solution, and drying the product under vacuum to obtain HDN. Then HDN with Mg (NO)3)2·6H2Performing coordination on O to obtain Mg-HDN, Mg (NO)3)2·6H2The amount of O used was the amount of residual DDAD in each polymer.
Example 3:
the prepared copolymer containing metal coordination pyridine derivative ligand is applied to nucleic acid carriers for delivering deoxyribonucleic acid (DNA) into cells, and the method comprises the following steps: mixing the copolymer Zn-HDB containing the metal coordination pyridine derivative ligand prepared in the example 1 with DNA in the same volume, and standing for 15 minutes at room temperature to prepare DNA complexes with the mass ratios of 20, 40 and 60:1 respectively; the prepared complex is used for transfecting different cell lines, and the method comprises the following steps:
cell transfection procedure
1) Preparation of DNA complexes:
the DNA used was the green fluorescent protein plasmid pCMV-GFP and the luciferase plasmid pCMV-GLuc. The copolymer Zn-HDB containing the metal coordination pyridine derivative ligand prepared in example 1 and DNA were dissolved in acetate buffer solution of pH 5.2, respectively, mixed in equal volumes, and left to stand at room temperature for 15 minutes to obtain DNA complex nanoparticles.
2) In vitro cell transfection experiments:
human embryonic kidney cell line 293T at 5X 10 per well4Culturing the multiple cells in a 24-hole culture plate for 24 hours, adding the DNA compound nano particles, and continuously culturing for 48 hours, wherein the dosage of the DNA is 1 mu g/hole; the transfection was observed with an inverted fluorescent microscope when transfected with the green fluorescent protein plasmid pCMV-GFP and with a BioLux when transfected with the luciferase plasmid pCMV-GLucTMLuciferase assay kit to assess transfection efficiency.
FIG. 2 shows the copolymer Zn-HDB containing a metal-coordinated pyridine derivative ligand prepared in example 1, and a commercial transfection reagent Lipo2k and PEI25kPictures of GFP transfection in 293T cells. The figure shows that: transfection efficiency of copolymer Zn-HDB containing Metal coordinated pyridine derivative ligand prepared in example 1 with commercial transfection reagent Lipo2k and PEI25kAnd (4) the equivalent.
3) In vitro cytotoxicity experiments:
● human embryonic kidney cell line (293T), human colon cancer cell (HCT116), human cervical cancer cell (HeLa) and murine colon cancer cell (CT26) were plated at 1X 10 cells per well, respectively4Putting a plurality of cells into a 96-well culture plate, culturing for 24 hours, adding Zn-HDB/DNA compound nanoparticles with the dosage of 0.25 mu g/well, continuously culturing for 48 hours, adding 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazole bromide (MTT), and detecting cytotoxicity.
The copolymer nucleic acid vector Zn-HDB containing the metal coordination pyridine derivative ligand prepared by the invention has high transfection efficiency in a conventional cell line (293T) and various tumor cells, and in addition, the Zn-HDB nucleic acid vector shows excellent low cytotoxicity in the cells.
Example 4:
the prepared copolymer containing metal coordination pyridine derivative ligand is mainly used for non-viral gene vectors to transfer ribonucleic acid (RNA) into cells, and the method comprises the following steps: mixing the copolymer Mg-HDN containing the metal coordination pyridine derivative ligand prepared in the example 2 with RNA in the same volume, and standing for 20 minutes at room temperature to prepare DNA complexes with the mass ratios of 10, 20, 40 and 60:1 respectively; the prepared compound has high cell transfection efficiency and low cytotoxicity, and the method comprises the following steps:
cell transfection procedure
1) Preparation of RNA complexes:
the RNA is firefly luciferase silencing gene siLuc. The copolymer Mg-HDN containing metal coordination pyridine derivative ligand prepared in the above example 2 and RNA were dissolved in acetate buffer solution of pH 5.2, respectively, mixed in equal volumes, and left to stand at room temperature for 15 minutes to obtain RNA complex nanoparticles.
2) In vitro cell transfection experiments:
human colon cancer cells (HCT116) and human cervical cancer cells (HeLa) stably expressing firefly luciferase (firefly-luciferase) genes are respectively added at a rate of 1 × 10 per well4The cells are put into a 96-well culture plate, and are cultured for 24 hours, the copolymer Mg-HDN/RNA compound nano-particles containing the metal coordination pyridine derivative ligand are added, the dosage of the RNA is 5 pmol/well, the culture is continued for 24 hours, and the firefly luciferase detection kit is used for detecting the RNA interference efficiency.
3) In vitro cytotoxicity experiments:
human colon cancer cells (HCT116) and human cervical cancer cells (HeLa) stably expressing firefly luciferase (firefly-luciferase) genes are respectively added at a rate of 1 × 10 per well4The cells are put into a 96-well culture plate, cultured for 24 hours, added with copolymer Mg-HDN/RNA compound nano particles containing metal coordination pyridine derivative ligand, the dosage of RNA is 5 pmol/well, continuously cultured for 24 hours, added with 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazole bromide (MTT), and detected with cytotoxicity.
The copolymer Mg-HDN nucleic acid vector containing the metal coordination pyridine derivative ligand, prepared by the invention, shows high RNA interference efficiency in human colon cancer cells (HCT116) and human cervical cancer cells (HeLa), and has low cytotoxicity.
Claims (5)
1. A copolymer nucleic acid carrier containing metal coordination pyridine derivative ligand is prepared by copolymerizing a metal ligand functional monomer consisting of a coordination unit and a stimulus response unit with other hydrophilic functional monomers and hydrophobic functional monomers and performing metal coordination, wherein the copolymerization molar ratio of the metal ligand functional monomer to the hydrophilic functional monomer to the hydrophobic functional monomer is as follows: 1, (0.5-8) and (0.5-10);
the metal in the metal ligand is zinc, calcium, magnesium, iron, aluminum, copper, europium or gadolinium;
the coordination unit is a pyridine ring group or a polynitrogen ring group;
the stimulus response unit is disulfide bond-containing monomer N, N' -bis (acryloyl) Cystamine (CBA), bis (2-methylpropene) ethoxy disulfide (DSDMA) or bis (2-propylene) ethoxy disulfide (DSDA);
the hydrophilic functional monomer refers to methacrylate, acrylate, methacrylamide and acrylamide monomers which contain hydroxyl, polyethylene glycol chain segments or sugar-containing chain segments in side groups and have water solubility or hydrophilicity;
the hydrophobic functional monomer is selected from methacrylate, acrylate, methacrylamide and acrylamide monomers with hydrophobic side groups.
2. The metal complex pyridine derivative ligand-containing copolymer nucleic acid vector according to claim 1, wherein the hydrophilic functional monomer is hydroxyethyl methacrylate, hydroxyethyl acrylate, polyethylene glycol methacrylate, acrylamide, methacrylamide, or a mixture of two or more thereof.
3. The metal complex pyridine derivative ligand-containing copolymer nucleic acid vector according to claim 1, wherein the hydrophobic functional monomer is methyl methacrylate or ethyl methacrylate, isopropyl methacrylate, N-butyl acrylate, hexafluorobutyl methacrylate, trifluoroethyl methacrylate, dodecyl methacrylate, N-isopropylacrylamide or a mixture of a plurality of monomers.
4. A method for preparing a copolymer nucleic acid vector containing a ligand of a metal-coordinated pyridine derivative according to claim 1, comprising the steps of:
mixing a mixture of 1: 0.5-8: 0.5 to 10 portions of metal ligand functional monomer, hydrophilic functional monomer and hydrophobic functional monomer are evenly mixed in dimethylformamide, and then initiator azobisisobutyronitrile or 4,4' -azobis (4-cyanovaleric acid) (ACVA) with the molar ratio of 0.06 to 0.08 and chain transfer agent 4-cyanovaleric acid dithiobenzoic acid (CPADB) with the molar ratio of 0.1 to 0.4 are added according to the proportion of the metal ligand functional monomer of 1; transferring the mixture to a glass-sealed tube and treating 3 freeze-thaw cycles with liquid nitrogen to remove oxygen; the polymerization reaction is continued for 4 to 48 hours at the temperature of between 60 and 80 ℃, and then the mixture is cooled in an ice water bath; precipitating for three times in a solution with the volume ratio v/v of diethyl ether to petroleum ether being 1:2, and drying the product in vacuum to obtain a copolymer containing pyridine derivative ligand; obtaining the copolymer nucleic acid carrier containing the metal pyridine derivative ligand after coordination with metal ion hydrated nitrate.
5. Use of a copolymer nucleic acid vector comprising a ligand of a metal-coordinated pyridine derivative according to claim 1, wherein the nucleic acid vector is prepared by: mixing a copolymer nucleic acid carrier containing a metal coordination pyridine derivative ligand with deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in equal volume, and standing for 10-30 minutes at room temperature to prepare a DNA or RNA compound with the mass ratio of 5-60: 1; the resulting complexes were used to transfect a variety of different cell lines.
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| WO2005007810A2 (en) * | 2003-06-16 | 2005-01-27 | Grinstaff Mark W | Functional synthetic molecules and macromolecules for gene delivery |
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| CN107823653A (en) * | 2017-11-13 | 2018-03-23 | 南开大学 | The preparation and application for the compound that metal-ion ligand for non-viral gene carrier material is formed |
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| WO2005007810A2 (en) * | 2003-06-16 | 2005-01-27 | Grinstaff Mark W | Functional synthetic molecules and macromolecules for gene delivery |
| CN106496555A (en) * | 2016-11-23 | 2017-03-15 | 南开大学 | A kind of metal-complexing polycation gene carrier and its preparation method and application |
| CN107823653A (en) * | 2017-11-13 | 2018-03-23 | 南开大学 | The preparation and application for the compound that metal-ion ligand for non-viral gene carrier material is formed |
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| Amphiphilic Gold Nanoparticles Displaying Flexible Bifurcated Ligands as a Carrier for siRNA Delivery into the Cell Cytosol;Niikura K等;《APPLIED MATERIALS & INTERFACES》;20141224;第6卷(第24期);第22146-22154页 * |
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