CN105732981A - Modified polyethyleneimine, a gene vector composition, and a preparing method and applications of the gene vector composition - Google Patents
Modified polyethyleneimine, a gene vector composition, and a preparing method and applications of the gene vector composition Download PDFInfo
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Abstract
The invention provides modified polyethyleneimine, and a preparing method and applications of the modified polyethyleneimine. The modified polyethyleneimine is prepared through modifying polyethyleneimine with different molecular weights with phenylboronic acid compounds. The modified polyethyleneimine has an active targeting property for tumor cell membranes, and can carry genes to form a gene vector composition used for gene therapy. The gene vector composition is uniform in particle size, proper in charge, stable in properties, low in cell toxicity, high in cell transfection efficiency, high in target power, and good in biocompatibility and biodegradability. The modified polyethyleneimine and the gene vector composition can be used for the gene therapy. Preparing processes of the modified polyethyleneimine and the gene vector composition are simple, high in reaction degree controllability and capable of industrial production.
Description
Technical field
The present invention relates to bio-carrier technical field, particularly to a kind of modified polyethyleneimine, genophore complex and its preparation method and application.
Background technology
Gene therapy is by importing to exogenous gene to repair the deleterious gene causing that the dcc gene of disease or suppression cause disease in target cell core, thus reaching the purpose for the treatment of disease.Successful gene therapy depends on efficient gene carrier, and common carrier is divided into viral vector and non-viral vector.And viral vector limits its development because there is great potential safety hazard；Non-virus carrier mostly is cationic polymer, has easy preparation, a transport, and safely and effectively, the advantage such as non-immunogenicity, it has also become one of study hotspot of genophore.
It is the class non-viral vector that application is wide at present that polymine (is called for short PEI), and it, rich in cation, has bigger buffer capacity, and DNA, adherent cell are had stronger adhesion.But, the transfection efficiency of PEI is relevant with its molecular weight, though molecular weight has stronger transfection protocol at the PEI of more than 20KDa, but also has stronger cytotoxicity simultaneously, and the PEI of small-molecular-weight (2000) is though cytotoxicity is low, but its transfection efficiency is low.And in prior art, when PEI is used as genophore by the derivant that chemical modification obtains, tumor cell is not still had active targeting, stability and biodegradability poor.
Therefore, it is necessary to improved the active targeting of PEI by the means of chemical modification, reduce its cytotoxicity simultaneously, improve inside and outside transfection efficiency, so that extensive use can be able to as a kind of gene vector material.
Summary of the invention
Based on above-mentioned technical problem, the invention provides a kind of modified polyethyleneimine and preparation method thereof, additionally provide a kind of genophore complex and preparation method thereof.Modified polyethyleneimine toxicity prepared by the present invention is low, targeting is strong, transfection efficiency is high, can be applicable to gene vector system.
First aspect, the invention provides a kind of modified polyethyleneimine, shown in the following formula I of chemical structural formula of described modified polyethyleneimine:
In formula I, R is H or the substituent group as shown in formula II, and Re is-CH3、NH2Or the substituent group as shown in formula III；The number of the substituent group as shown in formula II is the natural number of 1～40, and m and n is the degree of polymerization, and m is the natural number of 1～300, and n is the natural number of 1～200, and n+m is the natural number of 20～450；R in formula II or formula III1、R2、R3、R4、R5Substituent group has at least one
Preferably, the R in formula II or formula III1、R2、R3、R4、R5Substituent group is separately selected from-H ,-COOH ,-NO2,-Br ,-Cl ,-F andIn any one.
Preferably, the weight average molecular weight of the modified polyethyleneimine shown in formula I is 1000-25000.
Second aspect, the preparation method that the invention provides a kind of modified polyethyleneimine, comprise the following steps:
(1) take the phenylboronic acid compound shown in following formula IV and be dissolved in the first solvent, and add catalyst, dehydrant, polymine, amidated graft reaction 12-48h is carried out at 20-50 DEG C, obtain reactant liquor, wherein, the mass ratio of polymine and phenylboronic acid compound is 0.5-10
In formula IV, R1、R2、R3、R4、R5Having at least one is
(2) in reactant liquor, add the second solvent crystallization, collect precipitation, obtain described modified polyethyleneimine, wherein, shown in the following formula I of chemical structural formula of described modified polyethyleneimine:
In formula I, R is H or the substituent group as shown in formula II, and Re is-CH3、-NH2Or the substituent group as shown in formula III；The number of the substituent group as shown in formula II is the natural number of 1～40, and m and n is the degree of polymerization, and m is the natural number of 1～300, and n is the natural number of 1～200, and n+m is the natural number of 20～450；R in formula II or formula III1、R2、R3、R4、R5Substituent group has at least one
Preferably, in described step (2), collected precipitation is carried out following purification process: described crude product is dissolved in distilled water, in the bag filter that molecular cut off is 600-7000, dialyse 24-72 hour with deionized water, freeze-dried obtain the modified polyethyleneimine after purification.
As described in the present invention, described distilled water, refer to through redistillation deionized water once.
Preferably, in described formula II, formula III or formula IV, R1、R2、R3、R4、R5Substituent group is separately selected from-H ,-COOH ,-NO2,-Br ,-Cl ,-F andIn any one.
More preferably, in step (1), described phenylboronic acid compound is any one in 2-Carboxybenzeneboronic acid, 3-Carboxybenzeneboronic acid, 4-Carboxybenzeneboronic acid, 3-carboxyl-5-nitrobenzene boronic acid, the bromo-3-Carboxybenzeneboronic acid of 5-, 4-carboxyl-3-chlorophenylboronic acid, 4-carboxyl-3-fluorobenzoic boric acid or 3-carboxyl-5-flurophenyl boronic acid.
Preferably, in step (1), described polymine is molecular weight is the branched polyethylene imine of 800-25000, wherein, shown in the structural formula of described polymine such as following formula (V):
In formula (V), m and n is the degree of polymerization, and m is the natural number of 1～300, and n is the natural number of 1～200, and n+m is the natural number of 20～450, and Re substituent group is-CH3Or-NH2。
As described in the present invention, by the polymine of the compound-modified different molecular weight of phenylboronic acid, the polymine that a series of phenylboronic acid is compound modified is obtained.
Preferably, the weight average molecular weight of the modified polyethyleneimine as shown in formula I is 1000-25000.
Preferably, in described step (1), the percent grafting of phenylboronic acid compound is 5%-40%, and described percent grafting is the ratio of phenylboric acid unit on each macromolecular chain.
Preferably, step (1) described condensing agent includes N, any one in N-dicyclohexylcarbodiimide (be called for short DCC), N, N-DIC (being called for short DIC) and 1-ethyl-(3-dimethylaminopropyl) carbon imidodicarbonic diamide hydrochlorate (abbreviation EDC).
Preferably, in described step (1), the mole of the condensing agent of addition is 1-5 times of phenylboronic acid compound mole.
Preferably, step (1) described catalyst includes any one in N-hydroxy-succinamide, N-hydroxy thiosuccinimide and 1-hydroxy benzo triazole.
Preferably, in described step (1), the mole of the catalyst of addition is 1-2 times of phenylboronic acid compound mole.
As described in the present invention, described catalyst can be described as again activator, often with described condensing agent coupling, for the synthesis of amide.Carboxylic acid compound is also unstable with the addition intermediate radical of carbodiimide class condensing agent, if be converted into corresponding Acibenzolar or active amide without acylation catalyst, then can form by-product by self-rearrangement.
Preferably, in step (1), described first solvent is ethanol, methanol, distilled water, dimethyl sulfoxide and N, at least one in N '-dimethyl Methanamide.
Preferably, in step (2), described second solvent is at least one in ether, methyl ether and n-butyl ether.
Add the second solvent, be to make the product in step (2) described reactant liquor precipitate out as much as possible.
The third aspect, the invention provides a kind of genophore complex, described genophore complex includes gene and genophore, gene described in described genophore load, wherein, the mass ratio of genophore and gene is 1-50:1, and described genophore is the modified polyethyleneimine that the method described in first aspect present invention or second aspect present invention prepares, shown in the following formula I of structural formula:
In formula I, R is H or the substituent group as shown in formula II, and Re is-CH3、-NH2Or the substituent group as shown in formula III；The number of the substituent group as shown in formula II is the natural number of 1～40, and m and n is the degree of polymerization, and m is the natural number of 1～300, and n is the natural number of 1～200, and n+m is the natural number of 20～450；R in formula II or formula III1、R2、R3、R4、R5Substituent group has at least one
Preferably, in described formula II, formula III, R1、R2、R3、R4、R5Substituent group is separately selected from-H ,-COOH ,-NO2,-Br ,-Cl ,-F andIn any one.
Preferably, described gene includes one or more the combination in any in DNA, RNA, siRNA (siRNA), micro rna (microRNA), plasmid, but is not limited to this.
Preferably, described genophore complex can be processed into aqueous solution, lyophilized powder genophore injection, for gene therapy.
Fourth aspect, the preparation method that the invention provides a kind of genophore complex, comprise the following steps:
(1) 1-100 part genophore is taken, it is dissolved in 1-100 part distilled water, obtain genophore solution, described genophore is the modified polyethyleneimine that the modified polyethyleneimine described in first aspect present invention or the method described in second aspect present invention prepare, shown in the following formula I of its structural formula:
In formula I, R is H or the substituent group as shown in formula II, and Re is-CH3、-NH2Or the substituent group as shown in formula III；The number of the substituent group as shown in formula II is the natural number of 1～40, and m and n is the degree of polymerization, and m is the natural number of 1～300, and n is the natural number of 1～200, and n+m is the natural number of 20～450；R in formula II or formula III1、R2、R3、R4、R5Substituent group has at least one
(2) in the described genophore solution of step (1), add 1-10 part gene, after vortex mixed, hatch, obtain genophore complex.
Preferably, in described formula II, formula III, R1、R2、R3、R4、R5Substituent group is separately selected from-H ,-COOH ,-NO2,-Br ,-Cl ,-F andIn any one.
Preferably, in step (2), described gene includes at least one in DNA, RNA, siRNA, microRNA, plasmid, but is not limited to this.
Preferably, the mass ratio of genophore and gene is 1-50:1.
As described in the present invention, described in hatch and directly at room temperature react, need not heat or lower the temperature, described incubation temperature is 20-37 DEG C.
Preferably, the time hatched described in is 10-30min.
Preferably, described genophore complex can pass through dilution, aqueous solution injection is made in concentration, for gene therapy.
Preferably, described genophore complex, also by techniques such as lyophilizing, makes lyophilized injectable powder, for gene therapy.
Preferably, the particle diameter of described genophore complex is 20-200nm.
Preferably, the Zeta potential of described genophore complex is 30-50mv.
5th aspect, the modified polyethyleneimine described in first aspect present invention or the genophore complex as described in third aspect present invention, can be used for preparing gene therapy medicament.
As described in the present invention, the polysaccharide on cell membrane (such as glycoprotein, glycolipid etc.) plays a very important role in being mutually distinguishable of cell, for instance in the pathological process such as generation and transfer of cancer, the glycosyl on cell membrane plays pivotal role.And phenylboric acid is a kind of many saccharide acceptors of important cell membrane, boric acid base group in phenylboric acid molecule can with the cis-1 in carbohydrate molecule, and 2 or 1,3-dihydroxy forms reversible cycliborate, therefore, carbohydrate molecule is had combination and identification ability preferably by phenylboronic acid compound.On different cells, the distribution of glycoprotein is different, and the metabolism of cancerous cell is very fast, and the glycoprotein Content of cell surface is higher.Based on this, genophore complex is different with degree with the quantity that normal cell is taken in by cancerous cell.
Modified polyethyleneimine of the present invention, can pass through to wrap up genetic stew and form the genophore complex that Stability Analysis of Structures, uniform particle diameter, electric charge are moderate, and due to the modification of phenylboric acid part, tumor cell is had stronger active targeting by described genophore complex；Described modified PE I is cationic polymer simultaneously, and glycoprotein that can be electronegative with cell surface is attracted by electrostatic interaction, hence into cell.Active targeting, passive target dual function under, tumor cell is strong to the picked-up ability of described genophore complex, and transfection is good.
Beneficial effects of the present invention includes the following aspects:
1) adopting the compound-modified low-molecular-weight polymine of phenylboronic acid, obtain modified polyethyleneimine, and be used as genophore, tumor cell membrane has active targeting, cell transfecting efficiency is high, and cytotoxicity is little；
2) the genophore complex that phenylboric acid modified polyethyleneimine is formed through load genetic stew, uniform particle diameter, electric charge are moderate, and biocompatibility and biodegradability are good, have good stability；
3) modified polyethyleneimine of the present invention, genophore complex preparation method simple and easy to do, the extent of reaction is controlled, can industrialized production.
Accompanying drawing explanation
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, the accompanying drawing used required in embodiment or description of the prior art will be briefly described below.Specific embodiment described herein only in order to explain the present invention, is not intended to limit the present invention.
Fig. 1 is the modified polyethyleneimine PEI-PBA that the embodiment of the present invention 1 prepares1H-NMR nuclear-magnetism is composed；
Fig. 2 is the grain size distribution of the genophore complex that the embodiment of the present invention 2 prepares；
Fig. 3 is cytotoxicity test result figure in effect example 1 of the present invention；
Fig. 4 is in-vitro transfection experimental result picture in effect example 2 of the present invention；
Fig. 5 is cell endocytic experimental result picture in effect example 3 of the present invention.
Detailed description of the invention
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described, it is clear that described embodiment is only a part of embodiment of the present invention, rather than whole embodiments.Based on the embodiment in the present invention, the every other embodiment that those of ordinary skill in the art obtain under not making creative work premise, broadly fall into the scope of protection of the invention.
The preparation of the polymine (PEI-PBA) that embodiment 14-Carboxybenzeneboronic acid is modified
(1) the 4-Carboxybenzeneboronic acid (being abbreviated as PBA) of 100mg is weighed, it is dissolved in 10mL methanol solvate, add the N-hydroxy-succinamide (being abbreviated as NHS) of 100mg, under room temperature, magnetic agitation is uniform, add 1-(3-the dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (being abbreviated as EDC) of 280mg, room temperature activates 4 hours, the 4-Carboxybenzeneboronic acid after being activated；
(2) taking 100mL reaction bulb, (Mw=1800 is abbreviated as PEI to the polymine of addition 300mg1.8K) and 10mL methanol, then the 4-Carboxybenzeneboronic acid after activation is added in reaction bulb, react 24 hours at 25 DEG C；
(3) after reaction terminates, add 200mL ether in step (2) described reaction bulb, so 2 times repeatedly, it is precipitated thing, collect precipitation, by precipitate and separate, vacuum drying, obtain the polyethylenimine derivates (being abbreviated as PEI-PBA) that crude product 4-Carboxybenzeneboronic acid is modified；
(4) described crude product is dissolved in appropriate distilled water, in the bag filter that molecular cut off is 600, dialyse 24 hours with deionized water, within every 8 hours, change water-dialyzing once, use freeze dryer lyophilizing afterwards again, freeze-dried obtain the polyethylenimine derivates PEI-PBA after purification.
The reaction scheme of the present embodiment 1 is as follows:
Taking the PEI-PBA after appropriate purification, a part is dissolved in D2O, carries out1H-NMR analyzes, it is determined that structure.
Fig. 1 is the NMR spectra of the PEI-PBA polymer that embodiment 1 prepares, from figure 2 it can be seen that the peak of 2.0 3.0ppm is CH2CH2The proton peak of NH unit, the peak of 7.2 7.6ppm is the proton peak of phenyl unit on phenylboric acid, thus illustrating that phenylboric acid successfully modifies polymine.
The preparation of embodiment 2PEI-PBA genophore complex and sign
(1) weighing 10mg genophore, be dissolved in 10mL distilled water, obtain genophore solution (concentration is 1mg/mL), described genophore is the polyethylenimine derivates PEI-PBA that embodiment 1 prepares；
(2) take the described genophore solution 6.4mL of step (1), add 1 μ gRNA, after vortex mixed 5min, carry out under room temperature hatching 20min, obtain PEI-PBA genophore complex.
Fig. 2 is the grain-size graph of the genophore complex formed after implementing the PEI-PBA load gene that the 2 4-Carboxybenzeneboronic acid prepared are modified, described particle diameter is to be obtained by dynamic light scattering method measurement on Malvern nano particle size instrument, as can be seen from Figure 2, the particle diameter of PEI-PBA genophore complex is more uniform, and mean diameter is 156nm.
The numerical value of Zeta potential is relevant to the stability of colloidal dispersion system, and Zeta potential more high (absolute value), system stability is more good.By Malvern ParticleSizer record PEI-PBA genophore complex Zeta potential be 40.7mV, electric charge is moderate, illustrates that its character is more stable, for its be used as genophore lay a good foundation.
The preparation of embodiment 3PEI-PBA genophore complex
PEI-PBA genophore complex is prepared according to the technical scheme in the embodiment of the present invention 2, PEI-PBA used prepares in the embodiment of the present invention 1, the difference is that, in the present embodiment, the genetic stew of PEI-PBA load is green fluorescent protein plasmid (pEGFP-N1), specifically refer to table 1.
The preparation of embodiment 4PEI-PBA genophore complex
PEI-PBA genophore complex is prepared according to the technical scheme in the embodiment of the present invention 2, PEI-PBA used prepares in the embodiment of the present invention 1, the difference is that, in the present embodiment, the genetic stew of PEI-PBA load is fluorescently-labeled siRNA (FAM-siRNA), specifically refer to table 1.
The preparation of the polymine that embodiment 53-carboxyl-5 nitrobenzene boronic acid is modified
(1) 3-carboxyl-5 nitrobenzene boronic acid of 40mg is weighed, it is dissolved in 10mL dimethyl sulfoxide solvent, add the N-hydroxy-succinamide (being abbreviated as NHS) of 50mg, under room temperature, magnetic agitation is uniform, add the dicyclohexylcarbodiimide (being abbreviated as DCC) of 120mg, room temperature activates 4 hours, 3-carboxyl-5 nitrobenzene boronic acid after being activated；
(2) taking 100mL reaction bulb, (Mw=10000 is abbreviated as PEI to the polymine of addition 100mg10K) and 20mL dimethyl sulfoxide, then 3-carboxyl-5 nitrobenzene boronic acid after activation is added in reaction bulb, react 48 hours at 20 DEG C；
(3), after reaction terminates, 300mL methyl ether is added in step (2) described reaction bulb, 2 times so repeatedly, it is precipitated thing, collects precipitation, by precipitate and separate, vacuum drying, obtains the polyethylenimine derivates that crude product 3-carboxyl-5 nitrobenzene boronic acid is modified；
(4) described crude product is dissolved in appropriate distilled water, in the bag filter that molecular cut off is 3500, dialyse 48 hours with deionized water, within every 8 hours, change water-dialyzing once, use freeze dryer lyophilizing afterwards again, freeze-dried obtain the polyethylenimine derivates after purification.
The reaction scheme of the present embodiment is as follows:
The preparation of the polymine that embodiment 64-carboxyl-3-chlorophenylboronic acid is modified
(1) the 4-carboxyl-3-chlorophenylboronic acid of 50mg is weighed, it is dissolved in 10mL methanol solvate, add the N-hydroxy-succinamide (being abbreviated as NHS) of 50mg, under room temperature, magnetic agitation is uniform, add 1-(3-the dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (being abbreviated as EDC) of 150mg, room temperature activates 4 hours, the 4-carboxyl-3-chlorophenylboronic acid after being activated；
(2) taking 100mL reaction bulb, (Mw=3000 is abbreviated as PEI to the polymine of addition 120mg3K) and 15mL methanol, then the 4-carboxyl-3-chlorophenylboronic acid after activation is added in reaction bulb, react 24 hours at 50 DEG C；
(3), after reaction terminates, 250mL ether is added in step (2) described reaction bulb, 2 times so repeatedly, it is precipitated thing, collects precipitation, by precipitate and separate, vacuum drying, obtains the polyethylenimine derivates that crude product 4-carboxyl-3-chlorophenylboronic acid is modified；
(4) described crude product is dissolved in appropriate distilled water, in the bag filter that molecular cut off is 1000, dialyse 72 hours with deionized water, within every 8 hours, change water-dialyzing once, use freeze dryer lyophilizing afterwards again, freeze-dried obtain the polyethylenimine derivates after purification.
The reaction scheme of the present embodiment is as follows:
The preparation of the polymine that embodiment 74-Carboxybenzeneboronic acid is modified
(1) the 4-Carboxybenzeneboronic acid (being abbreviated as PBA) of 100mg is weighed, it is dissolved in 10mL methanol solvate, add the N-hydroxy-succinamide (being abbreviated as NHS) of 80mg, under room temperature, magnetic agitation is uniform, add 1-(3-the dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (being abbreviated as EDC) of 250mg, room temperature activates 4 hours, the 4-Carboxybenzeneboronic acid after being activated；
(2) taking 100mL reaction bulb, (Mw=10000 is abbreviated as PEI to the polymine of addition 50mg10K) and 10mL methanol, then the 4-Carboxybenzeneboronic acid after activation is added in reaction bulb, react 48 hours at 25 DEG C；
(3), after reaction terminates, 200mL ether is added in step (2) described reaction bulb, 2 times so repeatedly, it is precipitated thing, collects precipitation, by precipitate and separate, vacuum drying, obtains the polyethylenimine derivates that crude product 4-Carboxybenzeneboronic acid is modified；
(4) described crude product is dissolved in appropriate distilled water, in the bag filter that molecular cut off is 3500, dialyse 72 hours with deionized water, within every 12 hours, change water-dialyzing once, use freeze dryer lyophilizing afterwards again, freeze-dried obtain the polyethylenimine derivates after purification.
The preparation of the polymine that embodiment 84-carboxyl-3-fluorobenzoic boric acid is modified
(1) the 4-carboxyl-3-fluorobenzoic boric acid of 20mg is weighed, it is dissolved in 10mLN, in dinethylformamide solvent, add the N-hydroxy-succinamide (being abbreviated as NHS) of 30mg, under room temperature, magnetic agitation is uniform, adding 1-(3-the dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (being abbreviated as EDC) of 50mg, room temperature activates 4 hours, the 4-carboxyl-3-fluorobenzoic boric acid after being activated；
(2) taking 100mL reaction bulb, (Mw=1800 is abbreviated as PEI to the polymine of addition 200mg1.8K) and 20mLN, dinethylformamide, then the 4-carboxyl-3-fluorobenzoic boric acid after activation is added in reaction bulb, react 24 hours at 25 DEG C；
(3), after reaction terminates, 300mL ether is added in step (2) described reaction bulb, 2 times so repeatedly, it is precipitated thing, collects precipitation, by precipitate and separate, vacuum drying, obtains the polyethylenimine derivates that crude product 4-carboxyl-3-fluorobenzoic boric acid is modified；
(4) described crude product is dissolved in appropriate distilled water, in the bag filter that molecular cut off is 600, dialyse 24 hours with deionized water, within every 6 hours, change water-dialyzing once, use freeze dryer lyophilizing afterwards again, freeze-dried obtain the polyethylenimine derivates after purification.
Comparative example 1 plasmid pEGFP-N1 and PEI1.8KThe preparation of the genophore complex formed
Plasmid pEGFP-N1 and carrier PEI is prepared according to the technical scheme in the embodiment of the present invention 21.8KThe genophore complex formed, the difference is that, in the present embodiment, used carrier is commercial PEI1.8K, genetic stew is plasmid pEGFP-N1, specifically refer to table 1.
(1) 5mgPEI is weighed1.8K, be dissolved in 5mL distilled water, obtain genophore solution (concentration is 1mg/mL), described genophore is reaction raw materials molecular weight be 1800 polymine (PEI1.8K)；
(2) take the described genophore solution 26.4 μ l of step (1), add 0.66 μ gpEGFP-N1, after vortex mixed 5min, carry out under room temperature hatching 20min, obtain PEI1.8KWith pEGFP-N1 complex.
Comparative example 2 plasmid pEGFP-N1 and PEI25KThe preparation of the genophore complex formed
Plasmid pEGFP-N1 and carrier PEI is prepared according to the technical scheme in the embodiment of the present invention 225KThe genophore complex formed, the difference is that, in the present embodiment, used carrier is commercial PEI25K, genetic stew is plasmid pEGFP-N1, specifically refer to table 1.
Comparative example 3 gene FAM-siRNA and PEI1.8KThe preparation of the genophore complex formed
PEI is prepared according to the technical scheme in the embodiment of the present invention 21.8KWith genophore complex, the difference is that, in the present embodiment, used carrier is commercial PEI1.8K, the genetic stew of load is FAM-siRNA, and carrier is for specifically refer to table 1.
The preparation of table 1PEI-PBA genophore complex
The Cytotoxic evaluation experiment of effect example 1 modified polyethyleneimine (PEI-PBA)
Description above it have been mentioned that, the transfection efficiency of PEI and its molecular weight have much relations, when molecular weight is less than 2000, it is generally acknowledged there is no transfection abilities, only when higher molecular weight (12-70KDa), polymine just shows high transfection efficiency, but along with the increase of polymine molecular weight, its cytotoxicity is consequently increased.Select the cladodification PEI of 25KDa as comparison in great majority are studied.
The present invention adopts MTT (tetrazolium bromide) colorimetric analysis to evaluate modified polyethyleneimine (PEI-PBA, embodiment 1 obtains, the molecular weight of raw material PEI is 1800) toxicity to MCF-7 cell, and with different molecular weight and not adorned PEI (PEI1.8K、PEI25K) compare, step is as follows:
(1) take in the culture medium that MCF-7 cell is placed in the hyclone that percentage by volume is 10%, and in CO2 gas incubator, at 37 DEG C, 5%CO2When cultivate 24h continuously；
(2) MCF-7 cell is inoculated on 96 orifice plates, 4000, every hole cell, after cultivating 24 hours, it is separately added into PEI-PBA, PEI of a series of concentration (0,20,40,60,80,100 μ g/mL)1.8K、PEI25K, with cell co-culture 24 hours；
(3) add the phosphate buffered solution (PBS, 5mg/mL) of 10mLMTT to every hole, mixture continues to cultivate 4h at 37 DEG C；
(4) drawing the upper strata nutrient solution of each orifice plate carefully, every hole adds 150mL dmso solution bluish violet crystallization first a ceremonial jade-ladle, used in libation 10min, utilizes microplate reader to test each hole light absorption value at 490nm.
As can be seen from Figure 3, under same concentrations, polymine PEI-PBA and the PEI that phenylboronic acid is compound modified25KCompare, all demonstrate stronger cytoactive, especially in higher concentrations；Illustrate that its cytotoxicity is far smaller than PEI25KCytotoxicity；The cytotoxicity of PEI-PBA is slightly slightly above the PEI of small-molecular-weight, unmodified modification1.8KCytotoxicity.Generally, the cytotoxicity of PEI-PBA is little, it is possible to carry out load genetic stew as excellent genophore.
The outer-gene transfection experiment of effect example 2 modified polyethyleneimine (PEI-PBA)
Polymine (the PEI-PBA that phenylboronic acid is compound modified, embodiment 1 obtains, the molecular weight of raw material PEI is 1800) Ex vivo cell transfection experiment, adopt expressing green fluorescent protein plasmid (pEGFP-N1) as reporter gene, PEI1.8K、PEI25KThe genophore complex that load pEGFP-N1 is formed compares group, is evaluated in 293T cell, and operating procedure is as follows:
(1) take in the culture medium that 293T cell is placed in the hyclone that percentage by volume is 10%, 37 DEG C, percentage by volume be 5% CO2 gas incubator in cultivate 24h continuously；
(2) 293T cell is inoculated on 24 orifice plates, every hole 3 × 104Individual cell, every hole is containing culture medium 500mL；
(3) after cultivating 24h, sop up the hyclone culture medium in orifice plate, wash 2 times with PBS, add the serum-free medium of 500mL in the orifice plate often organized；
(4) in each hole of 3 group of 24 orifice plate, it is separately added into plasmid pEGFP-N1 and carrier PEI1.8K、 PEI25K, (in Table 1, every hole, containing plasmid 0.66 μ g), mix homogeneously, changes complete medium into after cultivating 6-12 hour to the mass ratio of carrier and plasmid to the genophore complex that formed of PEI-PBA；
(5) after cultivating 36h, the fluorescence of the green fluorescent protein expressed in observation of cell, and transfect photo with fluorescence microscope shooting.
From fig. 4, it can be seen that the genophore complex of modified polyethyleneimine (PEI-PBA, embodiment 1 prepares) is all than the polymine (PEI of unmodified1.8K) demonstrate better transfection efficiency；And along with the increase of PEI-PBA Yu gene mass ratio, the positive charge of carrier surface is increasingly stronger, and transfection efficiency also increases therewith, and after certain limit, the transfection efficiency of PEI-PBA is more than PEI25K.This is just illustrating the introducing due to phenylboric acid, adds the polymine active targeting ability to tumor cell membrane surface, improves the transfection efficiency of cell.
The cell endocytic experiment of effect example 3 modified polyethyleneimine (PEI-PBA)
The cell endocytic experiment of the polymine (PEI-PBA, embodiment 1 obtains, and the molecular weight of raw material PEI is 1800) that phenylboronic acid is compound modified, adopts fluorescein-labeled siRNA (FAM-siRNA) as genetic stew, PEI1.8K、PEI25KThe genophore complex that load FAM-siRNA is formed compares group, and independent FAM-siRNA, as negative control, carries out endocytosis evaluation in MCF-7 cell, and operating procedure is as follows:
(1) take in the culture medium that MCF-7 cell is placed in the hyclone that percentage by volume is 10%, 37 DEG C, percentage by volume be 5% CO2 gas incubator in cultivate continuously；
(2) MCF-7 cell is inoculated on burnt 8 orifice plates of 4 groups of copolymerization (being placed in advance in sterile cover slips), every hole 1 × 104Individual small cell, every hole culture medium 300mL；
(3), after cultivating 24 hours, experimental group 1 adds FAM-siRNA；Experimental group 2,3 adds FAM-siRNA and PEI1.8K, the genophore complex (being followed successively by contrast enforcement 3, embodiment 4 to prepare) that formed of PEI-PBA；Experimental group 4 is initially charged micromolecular 4-Carboxybenzeneboronic acid (PBA), adds FAM-siRNA and the PEI-PBA genophore complex (embodiment 4 prepares) formed, and continues to cultivate 2 hours by the cell of each experimental group；
(4) contaminate lysosome with lysosome label Lyso-tracker, fix with 4% paraformaldehyde；Contaminate nucleus with Hochest33342 dyestuff, finally at confocal laser scanning microscope, and shoot photo.
The present embodiment, when shooting photo with Laser Scanning Confocal Microscope, respectively Hochest dyestuff, lysosome label Lyso-tracker, the exciting of fluorescein-labeled FAM-siRNA, launch and shoot under wavelength, and each group of photo is carried out overlapping combinations process.
Wherein, Lyso-Tracker takes on a red color fluorescence, and maximum excitation wavelength during detection is 577nm, and maximum emission wavelength is 590nm；The maximum excitation wavelength of Hoechst dyestuff is 346nm, and maximum emission wavelength is 460nm；Fluorescein FAM is a kind of green fluorescence material, and excitation wavelength 480nm launches wavelength 520nm.
Cell endocytic result is as it is shown in figure 5, owing to Hochest dyestuff, lysosome label contaminate nucleus, lysosome respectively, and therefore in they excite, emission wavelength ranges, the cell of four experimental grouies is all it is observed that characteristic fluorescence blue, red.
And unused carrier loaded genetic stew siRNA (experimental group 1), easily by nuclease hydrolysis and be with a large amount of negative charge and cell can not be entered by endocytosis, therefore after cultivating with cell, MCF-7 cell can only be dyeed by nucleus, lysosomal label, does not observe the green characteristic fluorescence of FAM-siRNA；PEI1.8KWith the FAM-siRNA genophore complex (experimental group 2) formed, also the green fluorescence of FAM-siRNA it is barely perceivable, verify that low-molecular-weight, not adorned PEI are relatively low to the transfection efficiency of tumor cell further, it is impossible to entered cell by endocytosis well.
And in experimental group 3, the genophore complex that PEI-PBA and FAM-siRNA is formed has very strong cellular uptake ability, in cell within a cell core and Cytoplasm, lysosome is dyeed by Hochest dyestuff, lysosome label Lyso-tracker, and fluorescently-labeled FAM-siRNA is under its excitation wavelength, the green fluorescence of emitting bright, and the cell overlap figure the 3 of Fig. 5 (row the 4th row) under combination can observe that siRNA endocytosis enters the position of cell well.This illustrates that PEI-PBA genophore complex has very excellent cell endocytic ability, just can be successfully delivered siRNA and enter cell, be primarily due to the introducing of phenylboric acid, enhance its active targeting ability after transfecting 2 hours.
Experimental group 4 is one and suppresses comparison diagram, it is initially charged micromolecular PBA, occupy the glycoprotein recognition site of cell surface, after adding PEI-PBA and the FAM-siRNA genophore complex formed, owing to remaining glycoprotein recognition site is few, it may be observed that the green fluorescence of more weak FAM-siRNA.
Above effect example shows, phenylboric acid modified polyethyleneimine is as genophore, and preparation technology is simple, and uniform particle diameter, electric charge are moderate, and cytotoxicity is little, and transfection is good, and targeting ability is strong, and cell endocytic ability is strong, is a kind of very excellent genophore.
Claims (10)
1. a modified polyethyleneimine, it is characterised in that the chemical structural formula of described modified polyethyleneimine is such as shown in formula I:
In formula I, R is H or the substituent group as shown in formula II, and Re is-CH3、-NH2Or the substituent group as shown in formula III；The number of the substituent group as shown in formula II is the natural number of 1～40, and m and n is the degree of polymerization, and m is the natural number of 1～300, and n is the natural number of 1～200, and n+m is the natural number of 20～450；R in formula II or formula III1、R2、R3、R4、R5Having at least one is
2. modified polyethyleneimine as claimed in claim 1, it is characterised in that in described formula II or formula III, R1、R2、R3、R4、R5Substituent group is separately selected from-H ,-COOH ,-NO2,-Br ,-Cl ,-F andIn any one.
3. the preparation method of a modified polyethyleneimine, it is characterised in that comprise the following steps:
(1) take the phenylboronic acid compound as shown in formula IV and be dissolved in the first solvent, and add catalyst, dehydrant, polymine, amidated graft reaction 12-48h is carried out at 20-50 DEG C, obtain reactant liquor, wherein, the mass ratio of polymine and phenylboronic acid compound is 0.5-10
In formula IV, R1、R2、R3、R4、R5Having at least one is
(2) in reactant liquor, add the second solvent crystallization, collect precipitation, obtain modified polyethyleneimine, shown in the following formula I of chemical structural formula of described modified polyethyleneimine:
In formula I, R is H or the substituent group as shown in formula II, and Re is-CH3、-NH2Or the substituent group as shown in formula III；The number of the substituent group as shown in formula II is the natural number of 1～40, and m and n is the degree of polymerization, and m is the natural number of 1～300, and n is the natural number of 1～200, and n+m is the natural number of 20～450；R in formula II or formula III1、R2、R3、R4、R5Having at least one is
4. the preparation method of modified polyethyleneimine as claimed in claim 3, it is characterised in that in described formula II, formula III or formula IV, R1、R2、R3、R4、R5Substituent group is separately selected from-H ,-COOH ,-NO2,-Br ,-Cl ,-F andIn any one.
5. the preparation method of the modified polyethyleneimine as described in claim 3 or 4, it is characterized in that, in described step (1), described catalyst includes any one in N-hydroxy-succinamide, N-hydroxy thiosuccinimide and 1-hydroxy benzo triazole；Described condensing agent includes N, N-dicyclohexylcarbodiimide, N, N ' any one in-DIC and 1-ethyl-(3-dimethylaminopropyl) carbon imidodicarbonic diamide hydrochlorate.
6. the preparation method of the modified polyethyleneimine as described in claim 3 or 4, it is characterized in that, described first solvent is ethanol, methanol, deionized water, dimethyl sulfoxide and N, at least one in N '-dimethyl Methanamide, described second solvent is at least one in ether, methyl ether and n-butyl ether.
7. a genophore complex, it is characterised in that include gene and genophore, gene described in described genophore load, wherein, the mass ratio of genophore and gene is 1-50:1, described genophore is modified polyethyleneimine, and its structural formula is such as shown in formula I:
In formula I, R is H or the substituted radical as shown in formula II, and Re is-CH3、-NH2Or the substituent group as shown in formula III；The number of the substituent group as shown in formula II is the natural number of 1～40, and m and n is the degree of polymerization, and m is the natural number of 1～300, and n is the natural number of 1～200, and n+m is the natural number of 20～450；R in formula II or formula III1、R2、R3、R4、R5Having at least one is
8. genophore complex as claimed in claim 7, it is characterised in that described gene includes one or more the combination in any in DNA, RNA, siRNA, microRNA, plasmid.
9. the preparation method of a genophore complex, it is characterised in that comprise the following steps:
(1) weigh 1-100 part genophore, be dissolved in 1-100 part distilled water, obtain genophore solution, shown in the following formula I of structural formula of described genophore:
In formula I, R is H or the substituted radical as shown in formula II, and Re is-CH3、-NH2Or the substituent group as shown in formula III；The number of the substituent group as shown in formula II is the natural number of 1～40, and m and n is the degree of polymerization, and m is the natural number of 1～300, and n is the natural number of 1～200, and n+m is the natural number of 20～450；R in formula II or formula III1、R2、R3、R4、R5Having at least one is
(2) in the genophore solution described in step (1), add 1-10 part gene, after vortex mixed, hatch, obtain genophore complex.
10. the modified polyethyleneimine as described in any one of claim 1-2 or the genophore complex as described in any one of the claim 7-8 application in preparing gene therapy medicament.
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