CN105153339B - It is a kind of to aoxidize cationic polymer, preparation method and application that positive charge is removed in response - Google Patents
It is a kind of to aoxidize cationic polymer, preparation method and application that positive charge is removed in response Download PDFInfo
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- CN105153339B CN105153339B CN201510656807.5A CN201510656807A CN105153339B CN 105153339 B CN105153339 B CN 105153339B CN 201510656807 A CN201510656807 A CN 201510656807A CN 105153339 B CN105153339 B CN 105153339B
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- 0 CC(C)C(OC(C)C(C)*(*)(*)Cc(cc1)ccc1N(*)*)=O Chemical compound CC(C)C(OC(C)C(C)*(*)(*)Cc(cc1)ccc1N(*)*)=O 0.000 description 2
Abstract
The cationic polymer for removing positive charge is responded as gene delivery carrier, preparation method and application the invention discloses a kind of aoxidize, described charge reversal type gene delivery carrier is combined to by using 4 bromomethyl benzene boric acids and N, N lignocaine ethyl acrylate quaternary amine.Compared with prior art, different from conventional quaternary ammoniated carrier, the a large amount of positive charges of charge reversal type gene delivery carrier band for the oxidation response that the present invention is synthesized, DNA can be wrapped up well, but oxidizing condition that can be in the cell after into cell removes positive charge, generation charge reversal, negative electricity is changed into from positive electricity, and quick release goes out DNA and transfected.The carrier have efficiently, low toxicity the characteristics of, have a good application prospect.
Description
Technical field
The invention belongs to macromolecule and biological technical field, and in particular to a kind of oxidation response goes the cation of positive charge to gather
Compound, preparation method and the application as gene delivery carrier.
Background technology
Nucleic acid drug is transported in target cell to reach therapeutic purposes by gene therapy, has proved to be a kind of effective
The treating cancer with low side effect method.Conventional delivery vehicles can be divided into viral vector and non-viral load at present
Body.Non-virus carrier includes liposome, polymer, dendritic macromole, polypeptide of cation etc., has compared with viral vector
Good security, low immunogenicity, the advantages of biocompatibility is with a large amount of productions are easy to.But than viral vector, it is relatively low
Transfection efficiency be always perplex its application bottleneck, it is necessary to improve the transfection efficiency of non-virus carrier by chemical modification.
In non-virus carrier, cationic polymer be commonly used to neutralize DNA negative electricity and compress it into nano particle from
And protect DNA not to be degraded, and help it to enter cell.But the cationic polymerization formed by positive/negative electrostatic interaction
Thing/nucleic acid drug composite nanoparticles are thermodynamically stable, and it is difficult that dissociation is discharged that the nano-complex, which enters after cell,
Nucleic acid drug, causes nucleic acid drug to be difficult to play drug effect.Accordingly, it would be desirable to be designed to respond intracellular microenvironment and can be quick
The carrier of nucleic acid drug is discharged, the drug effect of nucleic acid drug is improved.Therefore, the present invention provides a kind of preparation method of polymer,
The polymer positively charged when extracellular can be made once entering to react after cell with being closely complexed with DNA
Positive charge disappears or becomes negatively charged, so as to quickly quickly be dissociated with DNA, promotes DNA expression.
Due to the physilogical characteristics that tumour is abnormal so that it discharges substantial amounts of active oxygen radical (ROS), including peroxidating
Hydrogen (H2O2), superoxide anion (O2), hydroxyl free radical (OH), these active oxygen radicals can be by phenyl boric acid or ester
Phenolic group is oxidized to, and then triggers the reaction that phenolic group benzylalcohol comes off, release (Broaders, K.E. applied to medicine；Grandhe,
S.；Frechet,J.M.,A biocompatible oxidation-triggered carrier polymer with
potential in therapeutics.J Am Chem Soc 2011,133(4),756-8.).Boric acid can be with glycol shape
The boron ester circularized, thus can for improve DNA and RNA in being conveyed with tumour cell or gene nucleotides it is mutual
Act on (Piest, M.；Engbersen,J.F.,Role of boronic acid moieties in poly(amido
Amine) s for gene delivery.J Control Release 2011,155 (2), 331-40.), and boric acid and boron
Ester does not have toxic side effect to human body.
Polymer in the present invention is exactly boronic acid containing or boron ester substituted benzyl quaternary ammonium salt, utilizes intracellular ROS Oxybenzenes
Boric acid or ester, the reversion for triggering its electric charge.Currently without the report of this decationizing polymer, equally also it is not prepared
The report of methods and applications.
The content of the invention
The quaternary ammoniated polymerization of cationic polymer phenyl boric acid benzyl that positive charge is removed in response is aoxidized the invention provides a kind of
Thing carries a large amount of positive charges, stable nano-complex can be formed by electrostatic self-assembled with the nucleic acid drug such as electronegative DNA.
Into after cell, boric acid or boron ester go phenolic group benzylalcohol to react when being aoxidized by ROS, and quaternary ammonium salt can be changed into tertiary amine, then may be used
Electronegative polymer is generated by self-catalysis ester linkage hydrolyzing, thus with electronegative nucleic acid drug it is mutually exclusive and with quick solution
From discharging nucleic acid drug and effectively transfected.
The cationic polymer for removing positive charge the present invention also discloses above-mentioned oxidation response is conveying load as gene
Body and its application in gene conveying.Prepared by the present invention be efficiently, the non-viral gene delivery vehicles of low toxicity, itself and DNA
The nano-complex of formation has high transfection efficiency.
It is a kind of to aoxidize the cationic polymer that positive charge is removed in response, it is characterised in that including following fragment：
In above formula：
R1、R2The alkyl or aromatic radical for C1-C6 independently；
R3、R4The alkyl acyl for H, C1-C6 independently；
M is 1-4 positive integer；
Anion is bromide ion or chlorion.
Preferably, the cationic polymer by polyacrylate, polymethacrylates or other containing one-level, two grades
The polymer of amino containing boric acid or boric acid ester group benzyl-trialkyl quaternary amine based compound reaction with preparing.
As further preferred, other described polymer containing one-level, secondary amine base include such as polyethyleneimine, polyamine group
Acid amides dendritic macromole.
Preferably, above-mentioned R3、R4For H, acetyl group or formoxyl, corresponding is boric acid or methyl borate, borogen,
R1、R2For methyl, ethyl；M be 1-3 positive integer, more preferably 1 or 2, be still more preferably 2.
Preferably, the cationic polymer is at least one of following compounds (1)~(4)：
N is the number 5-500 of repeat unit in above formula；More preferably 50-300；R1、R2Independently for C1-C6
Alkyl chain or aromatic radical, more preferably ethyl；PAMAM is at least one of 2-5 generations.
Preferably, wherein compound (1) and compound (2) any method in following two methods is prepared into
Arrive：
Method one：By N, N- disubstituted amido ethyl propylene acid esters or N, N- disubstituted amido ethylmethyl acrylate warp
Cross polymerisation and prepare N, N- disubstituted amido ethyl polyacrylate or N, N- disubstituted amido ethyl polymethyl
Acid esters, is then obtained with the reaction of boronate Bian bromine, boric acid ester group Bian bromine, boronate Bian chlorine or boric acid ester group Bian chlorine again；
Method two：By N, N- disubstituted amido ethyl propylene acid esters or N, N- disubstituted amido ethylmethyl acrylate are first
Reacted with boronate Bian bromine, boric acid ester group Bian bromine, boronate Bian chlorine or boric acid ester group Bian chlorine, generate corresponding acrylate list
Body, then carries out polymerisation and obtains；
The acrylate monomer includes bromo or chloro (2- acryloyl-oxies) ethyl (p- boric acid benzyl) diethyl ammonium, bromo
Or one kind in chloro (2- acryloyl-oxies) ethyl (p- borates benzyl) diethyl ammonium；
Preferably, described compound (1) structure is as follows：
Described compound (2) structure is as follows：
By taking above-claimed cpd (1) as an example, its preparation method is shown below：
Preferably, wherein compound (3) and compound (4) is prepared by following methods：Acrylate monomer is with gathering
Aziridine or PAMAM are prepared by Michael addition reaction；The acrylate monomer is included by bromo or chloro (2-
Acryloyl-oxy) ethyl (p- boric acid benzyl) diethyl ammonium, bromo or chloro (2- acryloyl-oxies) ethyl (p- borates benzyl) diethyl
One kind in ammonium.
Preferably, the structure of the compound (3) is as follows：
Its preparation method is shown below：
Preferably, the structure of the compound (4) is as follows：
Its preparation method is shown below：
The oxidation response of the present invention goes the cationic polymer of positive charge to be aoxidized by hydrogen peroxide etc. and remove quaternary ammonium
The lower quaternary ammonium salt of salt positive charge effect can be changed into tertiary amine, then generate electronegative carboxylic acid group by self-catalysis ester linkage hydrolyzing, with
Exemplified by above-claimed cpd (1), the process of its charge reversal is shown below：
This law additionally provides a kind of above-mentioned oxidation response and removes the cationic polymer of positive charge in conveying DNA and nucleic acid short chain
In application.
This law additionally provides cationic polymer the answering in conveying taxol that positive charge is removed in a kind of above-mentioned oxidation response
With.
Compared with prior art, the invention has the advantages that：
(1) the oxidation response that prepared by the present invention goes that positive charge counter-rotative type gene delivery carrier is simple in construction, be readily synthesized；
(2) it is different from general quaternary ammoniated carrier (as used the quaternary ammoniated poly- N of iodomethane, N- disubstituted amido ethyl propylenes
Acid esters) nucleic acid drug is wrapped up into the shortcoming that nano-complex that is too tight, being formed excessively is stablized, can not dissociated in the cell with DNA,
The lower quaternary ammonium salt of the quaternary ammoniated cationic polymer of boric acid (ester) benzyl prepared by present invention ROS effects in the cell can be changed into three-level
Amine, then generates electronegative carboxylic acid group by self-catalysis ester linkage hydrolyzing, promotes the nucleic acid drug dissociation with DNA and makes its effective
Transfection.
(3) the quaternary ammoniated cationic polymer of boric acid (ester) benzyl prepared by the present invention shows higher turn in cell
Dye is active (compared with PEI 25KDa), while having relatively low cytotoxicity.
Brief description of the drawings
Fig. 1 is polymer (1) HMP release profiles under oxidative conditions in the embodiment of the present invention 2；
Fig. 2 is the change of polymer (1) Zeta potential under oxidative conditions in the embodiment of the present invention 3；
Fig. 3 is the gel retardation assasy electrophoretogram of polymer (1)/DNA nano-complexes in the embodiment of the present invention 4；
Fig. 4 is the particle diameter distribution and Zeta potential figure of polymer (1)/DNA nano-complexes in the embodiment of the present invention 4；
Fig. 5 is the transmitted electron that polymer (1)/DNA nano-complexes N/P ratio is under the conditions of 13 in the embodiment of the present invention 4
Microscope figure；
Fig. 6 is that polymer (1)/DNA nano-complexes N/P ratio is in different mistakes under the conditions of 13 in the embodiment of the present invention 4
Hydrogen peroxide concentration it is lower 37 DEG C be incubated 1 hour after gel retardation assasy electrophoretogram；
Fig. 7 is the cytotoxicity figure for removing positive charge polymer of several oxidation responses in the embodiment of the present invention 5；
Fig. 8, Fig. 9, Figure 10 go what positive charge polymer and DNA were formed for what several oxidations in the embodiment of the present invention 6 were responded
The cell transfecting design sketch of nano-complex；
Figure 11 presses down for polymer (1)/DNA nano-complexes in the embodiment of the present invention 7 in various concentrations active oxygen radical
Cell transfecting figure under preparation (DPI) effect；
Figure 12 is transfection figure in the intratumor injection body of polymer (1)/DNA nano-complexes in the embodiment of the present invention 8.
Embodiment
The present invention provides some specific embodiments, but the present invention is not restricted by the embodiments.
Embodiment 1：
The synthesis of polymer (1)
Two methods synthetic polymer (1) can be used；
Synthetic method 1：
Monomeric acrylic N, N- diethylamino ethyl ester (2- (N, N-diethylaminoethyl) acrylate, DEAEA)
Vacuum distillation purifying is first being carried out before, DEAEA 5g are weighed, AIBN (azodiisobutyronitrile) 0.05g is 100 in molar ratio:
1 ratio mixing, bubbling argon deoxygenation in 30 minutes is heated to 65 DEG C of stirrings and carries out polymerisation in bulk, reaction 24 under anaerobic
Hour, then isolated and purified, dissolve a polymer in dichloromethane, precipitated with ice n-hexane, repeatedly for three times, drying is obtained
Polymer P DEAEA is pale yellow viscous liquid 4.5g, and yield is 90%.
Molecular weight determination is carried out to PDEAEA obtained above, assay method is gel permeation chromatography, polyethylene glycol
(PEG) PDEAEA prepared by standard items and embodiment is sample, dissolves the solution for being made into 10mg/mL with tetrahydrofuran respectively,
Standing is shaken up, with 0.22 micron of filtering with microporous membrane, take the μ L of filtrate sample introduction 20, record chromatogram, and calculating goes out PDEAEA
Number-average molecular weight be 10100, weight average molecular weight is 16400, and molecular weight distribution is 1.62.
PDEAEA 0.3g, 4- bromomethyl benzene boric acid 0.56g are weighed, is in molar ratio 1:1.5 ratio mixing is dissolved in N, N-
In solvent dimethylformamide, stir, react 24 hours at room temperature, after product ultra-pure water is dissolved, be placed in cutting after activation
Stay in the bag filter that molecular weight is 3500Da and dialyse 12, after dialysis terminates, with the filtering with microporous membrane that aperture is 0.22 micron, most
Pre-freeze, is freezed afterwards, obtains resulting polymer (1) B-PDEAEA for white solid 0.63g, yield is 92%, and structural formula is following (to be saved
Bromide ion is omited, similarly hereinafter)：
B-PDEAEA nuclear-magnetism detection data are as follows：
1H-NMR,D2O：δ=7.2-8.0 (4H, ArH), δ=4.0-4.6 (4H, ArHCH2N(CH2CH3)2CH2CH2), OOCCH- δ=3.0-3.6 (6H, ArHCH2N(CH2CH3)2CH2CH2), OOCCH- δ=2.3-2.6 (1H ,-
CH2), CH- δ=0.8-1.6 (8H, ArHCH2N(CH2CH3)2CH2CH2OOCCH-,-CH2CH-)。
The above-mentioned quaternary ammoniated ratio for obtaining polymer (1) is 100%.N is 59.
Synthetic method 2：
DEAEA 5g, 4- bromomethyl benzene boric acid 6.25g are weighed, DMF is dissolved in, room temperature reaction is stayed overnight, ether is precipitated three times, is dried
It is dry, obtain quaternary ammoniated monomer bromo (2- acryloyl-oxies) ethyl (p- boric acid benzyl) diethyl ammonium ((2-acryloyloxy)
Ethyl (p-boronic acid benzyl) diethylammonium bromide, B-DEAEA) 9.56g, yield is 85%,
Structure is following (omission bromide ion)：
Weigh bromo (2- acryloyl-oxies) ethyl (p- boric acid benzyl) diethyl ammonium monomer 5g, initiator ammonium persulfate
0.015g, is dissolved in water, and bubbling argon deoxygenation in 30 minutes is heated to 65 DEG C of stirrings under anaerobic, and reaction is placed in work after 8 hours
Molecular cut off after change is dialyses 12 hours in 3500Da bag filter, dialysis terminates rear pre-freeze, freezes, and obtains product polymerization
Thing (1) B-PDEAEA is white solid 4.35g, and yield is 87%.By control inventory can make the molecular weight (n) of polymer with
It is consistent made from method 1.The synthesis of polymer (2)
Weigh monomer methacrylic acid lignocaine ethyl ester (2- (N, N-diethylaminoethyl) methacrylate,
DEAEMA) 10g, initiator 2 isobutyl ethyl bromide 0.04mL, methanol 2mL are added in polymerization bottle 1, and bubbling 30 minutes weighs bromine
Change cuprous 0.04g, bipyridyl 0.088g vacuumizes the polymerization for being added to after logical argon gas and eliminating oxygen into another polymerization bottle 2
In bottle 1,30 DEG C of stirrings carry out ATRP polymerization, and reaction is stayed overnight, then isolated and purified, and dissolves a polymer in tetrahydrofuran, mistake
The short column of alundum (Al2O3) is sub twice, and eluant, eluent is THF, and concentrated by rotary evaporation is precipitated with ice n-hexane, repeatedly for three times, and drying is obtained
Polymer P DEAEMA is pale yellow viscous liquid 8.9g, and yield is 89%, and number-average molecular weight is 9400, and weight average molecular weight is
12400, n be 51.
Reactions and processing method of the polymer P DEAEMA with 4- bromomethyl benzene boric acids are identical with PDEAEA (referring to polymer
(1) synthetic method 1), the quaternary ammoniated resulting polymer (2) for obtaining product 100% is white solid, and yield is 90%, and it is tied
Structure formula is as follows：
The nuclear-magnetism detection data of polymer (2) are as follows：
1H-NMR, D2O：δ=7.2-8.0 (4H, ArH), δ=4.0-4.6 (4H, ArHCH2N(CH2CH3)2CH2CH2), OOCC- δ=3.0-3.6 (6H, ArHCH2N(CH2CH3)2CH2CH2), OOCC- δ=0.8-1.6 (11H, ArHCH2N
(CH2CH3)2CH2CH2OOCCH-,CH3C-,-CH2C-).
N is 80.The synthesis of polymer (3)
Weighing the polyethyleneimine that molecular weight is 10K, (PEI, molecular weight 10000, n is 230) 1g, B-DEAEA 8.5g, is taken out
Deaeration in condenser gas, argon gas protection is lower to add dry methanol, and 50 DEG C of stirrings carry out Michael addition reaction, and reaction is placed in after 3 days
Molecular cut off after activation is dialyses 20 hours in 3500Da bag filter, dialysis terminates rear pre-freeze, freezes, and obtains product and gathers
Compound (3) is white solid 8.3g, and yield is 87%, and structure is shown below：
The nuclear-magnetism detection data of polymer (3) are as follows：
1H-NMR, D2O：δ=7.2-8.0 (4H, ArH), δ=4.0-4.6 (4H, ArHCH2N(CH2CH3)2CH2CH2OOCCH2CH2), N- δ=3.0-3.8 (8H, ArHCH2N(CH2CH3)2CH2CH2OOCCH2CH2), N- δ=2.3-2.6
(6H,ArHCH2N(CH2CH3)2CH2CH2OOCCH2CH2N-,-CH2CH2), N- δ=0.8-1.6 (6H, ArHCH2N(CH2CH3)2CH2CH2OOCCH2CH2N-).N is 230.
The synthesis of polymer (4)
Weigh the 3rd generation PAMAM (1g) and B-DEAEA 3.4g and synthesized by the method for polymer (3) and obtain polymer (4)
White solid, yield is 85%, and structural formula is：
The nuclear-magnetism of polymer (4) detects that data are：
1H-NMR, D2O：δ=7.2-8.0 (8H, ArH), δ=4.0-4.6 (4H, ArHCH2N(CH2CH3)2CH2CH2OOCCH2CH2), N- δ=3.0-3.8 (256H, ArHCH2N(CH2CH3)2CH2CH2OOCCH2CH2N-,-
CONHCH2CH2N- ,-NCH2CH2), NHCO- δ=2.3-2.6 (248H, ArHCH2N(CH2CH3)2CH2CH2OOCCH2CH2N-,-
CONHCH2CH2N- ,-NCH2CH2NHCO-,-NCH2CH2), N- δ=0.8-1.6 (6H, ArHCH2N(CH2CH3)2CH2CH2OOCCH2CH2N-)。
Embodiment 2：
The oxidation response of polymer (1)
Weigh a certain amount of polymer (1) and be dissolved in ultra-pure water, concentration is 0.3mg/mL, add aqueous hydrogen peroxide solution, mistake
The final concentration of 1mM (every liter of mM representatives mM) of hydrogen oxide.The quinone that polymer (1) is generated under oxidative conditions in water quickly
It is changed into p-Hydroxybenzylalcohol (HMP), using 10% methanol aqueous solution as mobile phase, flow velocity is 1mL/min, uses high performance liquid chromatography
Method (HPLC) analyzes HMP, and appearance time is 3.5 minutes, can be obtained by the amount for monitoring the HMP that different time is discharged
HMP release profiles.The experiment proves that polymer poly compound (1) was quickly oxidized in 30 minutes under 1mM Hydrogen Peroxides,
Almost it is oxidized completely in 2 hours, testing result is referring to Fig. 1.
Embodiment 3：
The charge reversal that polymer (1) occurs under oxidative conditions
First group：Weigh a certain amount of polymer poly compound (1) and be dissolved in HEPES cushioning liquid (pH value is 7.4,10mM),
Concentration is 3mg/mL, adds the final concentration of 80mM of hydrogenperoxide steam generator.All the time the pH value for maintaining solution is incubation at 7.4,37 DEG C,
Determine Zeta potential of the solution in different time.
Second group：Weigh a certain amount of polymer (1) and be dissolved in HEPES cushioning liquid (pH value is 7.4,10mM), concentration is
3mg/mL, adds the final concentration of 80mM of aqueous hydrogen peroxide solution, solution ph is adjusted to after being incubated 2 hours at 5.0,37 DEG C, will
Solution ph is adjusted to 7.4, and the pH value that solution is maintained all the time is incubation at 7.4,37 DEG C, determines Zeta electricity of the solution in different time
Position.
Testing result is as shown in Fig. 2 the experiment proves that the Zeta potential of polymer (1) drops to 17mV, table from 28mV quickly
Bright quaternary ammonium salt is changed into tertiary amine under oxidative conditions, then by ester bond self-catalysis hydrolysis produce polyacrylic acid, potential gradually by
The negative transformation of forward direction, 0mV was changed into when 10 hours, while it has been found that can promote oxidation in sour environment, in pH value
To be incubated 2 hours under conditions of 5.0, charge reversal is only needed to 6 hours, it means that if polymer is escaped again after entering lysosome
Effusion comes, and the speed of charge reversal can be accelerated.
Embodiment 4：
The preparation of polymer (1)/DNA nano-complexes
Weigh a certain amount of polymer (1) and be dissolved in HEPES cushioning liquid (pH value is 7.4,10mM), concentration is 1mg/mL
(being designated as polymer (1) solution), the molten of 40 μ g/mL is diluted to by DNA with HEPES cushioning liquid (pH value is 7.4,10mM)
Liquid (is designated as DNA solution), is diluted to polymer (1) solution accordingly according to a series of N/P ratios (N/P mol ratios) of setting
Concentration, by volume 1:1 is quickly adding into DNA solution, vortex oscillation 10 seconds, is stored at room temperature 30 minutes, obtain it is a series of not
With the nano-complex solution of N/P ratio.
Physicochemical property to nano-complex solution obtained above is characterized：
1st, gel retardation assasy
A series of nanometer of the different N/P ratios (choosing the sample that N/P mol ratios are 3,5,7,9 respectively) prepared is taken to answer
The μ L of polymer solution 20, while taking 10 μ L pure plasmids DNA to compare, five samples are splined on into 0.8% agarose respectively (contains
0.5mg/mL ethidium bromides) prepare gel pore in, the 100mV in 1 × TAE buffer solutions, electrophoresis 30min.After electrophoresis terminates, put
Shot in ultraviolet gel imaging system, obtain electrophoretogram as shown in Figure 3.From figure 3, it can be seen that polymer (1) can be fine
Ground compresses DNA, is that can block DNA migration well when N/P is more than 3.
2nd, particle diameter distribution and Zeta potential are determined
Take a series of different N/P ratios of prepare (choosing the sample that N/P mol ratios are 3,5,7,9,11,13 respectively)
Nano-complex solution is surveyed using dynamic light scattering under the conditions of 25 DEG C to its size, Zeta potential and aggregation performance
It is fixed.Acquired results are calculated by DTS softwares.Every group of experiment is repeated 3 times and averaged.As a result as shown in figure 4, particle diameter
40nm is may be compressed to, Zeta potential is distributed between 15~20mV.
3rd, the transmission electron microscope observation of polymer (1)/DNA nano-complexes
The nano-complex that the N/P mol ratios prepared are 13 is taken, drop one is dripped on 400 mesh copper mesh, absorbed with filter paper many
Extraction raffinate body, drips phosphotungstic acid and carries out negative staining, surplus liquid is absorbed with filter paper, room temperature is dried naturally, uses transmission electron microscope
To observe the form of sample, transmission electron microscope picture is recorded.As a result as shown in figure 5, the nano-complex formed by electrostatic self-assembled,
Spherical nano particle is showed, form is regular, uniform, and particle diameter is in 40nm or so, the result surveyed with dynamic light scattering
It is basically identical.
4th, the gel retardation assasy under polymer (1)/DNA nano-complex oxidizing conditions
The nano-complex that the N/P mol ratios for taking 20 μ L to prepare are 13, under various concentrations concentration of hydrogen peroxide, 37 DEG C
It is incubated 1 hour, while with 10 μ L pure plasmids DNA and in 5mM H2O2DNA as reference substance, opposed within 1 hour in 37 DEG C of incubations
According in the gel pore for being then splined on 0.8% agarose (containing 0.5mg/mL ethidium bromides) preparation, in 1 × TAE buffer solutions
100mV, electrophoresis 30min.After electrophoresis terminates, it is placed in ultraviolet gel imaging system and shoots, obtain electrophoretogram as shown in Figure 6.From Fig. 6
In as can be seen that nano-complex in 0.5mM H2O2Concentration or more than 0.5mM H2O2Concentration is lower can to discharge DNA.
Embodiment 5：
The cytotoxicity experiment of polymer (1)~(4)
Detect that polymer exists using 3- (4,5- dimethylthiazoles -2) -2,5- diphenyltetrazolium bromide bromides (MTT) methods
The vitro cytotoxicity of A549 cells.By cell culture in 96 orifice plates, cell density is 5000/hole, and 100 are added in every hole
μ L culture mediums (10% (v/v) hyclone and 1% (v/v) green grass or young crops-Streptomycin Solution), PEI (25KDa) as a control group, cell
It is placed in 5%CO224h is cultivated in 37 DEG C of constant incubators of concentration and 95% humidity.After 24h, 100 μ L are added in each hole different
Concentration polymer culture medium solution (being respectively 5mg/ml, 10mg/ml, 20mg/ml, 40mg/ml, 80mg/ml), blank group is added
100 μ L culture medium solution, cell continues to cultivate 48h.After during culture 48h, 1100rpm centrifugations 6min is simultaneously abandoned in hole each to the greatest extent
Culture medium, adds 100 μ L MTT solution (being prepared using fresh culture, MTT concentration is 0.75mg/mL), cell continues to train
Support 3h.Thereafter the MTT nutrient solutions during 2800rpm centrifuges 7min and abandoned to the greatest extent per hole, add 100 μ L DMSO, and concussion makes in every hole
Crystallization be all dissolved in DMSO.The final absorbance with ELIASA test sample at 562nm and 620nm.Cell survival
Rate (percentage) is to be gone with the absorbance of experimental group divided by the absorbance of blank group is represented.Each group of data are same group
The average value in three holes of sample.As a result as shown in fig. 7, polymer (1) increases to the toxicity of A549 cells with the increase of concentration
Greatly, cell survival rate is higher than PEI (25KDa) under same concentrations.
Above-mentioned detection also is done to polymer (2)-(4) prepared in the same manner, testing result is equally shown in
Fig. 7, as shown in Figure 7 polymer (2)-(4) toxicity of A549 cells is increased with the increase of concentration, it is thin under same concentrations
Born of the same parents' survival rate is high more than PEI (25KDa).
Embodiment 6：
The cell transfection assays of polymer (1)-(4)/DNA nano-complexes
(1) luciferase gene is transfected
By A549 cell culture in 48 orifice plates, cell density is 25000/hole, contains 0.4mL culture mediums per hole, and
Cell is placed in 5%CO224h is cultivated in 37 DEG C of constant incubators of concentration and 95% humidity.It will be replaced afterwards per culture medium in hole
Be changed to 0.4mL culture mediums (serum FBS contents be 0% or 10%), and be separately added into prepare contain polymer (1)-(4)
A series of N/P ratios the μ L of nano-complex solution 50 (μ g of the DNAs of pGL4.13 containing luciferase 1), cultivate 4h.So
The culture medium containing nano-complex in most plate is abandoned afterwards, is replaced with fresh culture medium and is continued to cultivate to 48h.Discard culture medium,
100 μ 1 × cell pyrolysis liquids of L are added per hole, centrifuging and taking supernatant adds luciferase substrate, determined with chemiluminescence detector
Chemiluminescence intensity.Protein concentration is determined with Bradford protein detection kits.Chemiluminescence intensity is returned using protein concentration
One changes, and unit is every milligram of albumen luminous intensity (RLU/mg protein).Each group of data are three holes of companion specimens
Average value.As a result as shown in figure 8, the transfection of luciferase gene embody be cell integral level transfection, N/P for 13 it is poly-
The serum-free transfection value highest of compound (1)/DNA nano-complexes, PDEAEA more quaternary ammoniated than control group iodomethane (vehicle economy M, with
DNA is tightly combined, into cell after be difficult to escape from lysosome, it is impossible to DNA is discharged into progress and effectively transfected) transfection value is high
15400 times, it was demonstrated that DNA effectively can be discharged promotion really and turned by the charge reversal type gene delivery carrier of oxidation response
Dye.Its transfection value is also higher by 8 times than positive controls PEI/DNA simultaneously.What is more important, N/P ratio reach 60 when
Wait, polymer (1)/DNA nano-complexes show sero-fast characteristic, its transfection value in 10% serum can reach with
Serum-free is the same, and the transfection value than the PEI/DNA under equal conditions is higher by 100 times.
(2) Transfection of Enhanced Green Fluorescent
By A549 cell culture in 24 orifice plates, cell density is 80000/hole, per hole 0.8mL culture mediums, and will be thin
Born of the same parents are placed in 5%CO224h is cultivated in 37 DEG C of constant incubators of concentration and 95% humidity.It will be replaced with afterwards per culture medium in hole
0.8mL culture mediums (serum content be 0% or 10%), and add the nano-complex solution for preparing (polymer (1) is received
Rice compound, nitrogen phosphorus mol ratio is respectively 13,60) 100 μ L (μ g of DNA containing pEGFP 4), cultivates 4h.Then abandon in most plate
Culture medium containing nano-complex, replaces with fresh culture medium and continues to cultivate to 48h.After the completion of culture, 0.25% pancreas is used
Cell dissociation is collected into 15mL centrifuge tubes by enzyme/0.03%EDTA solution, and PBS solution is cleaned twice, and finally cell suspends
In 500 μ L PBS solutions, move into flow cytometer detection pipe and carry out flow cytomery.The excitation wavelength of green fluorescent protein is
488nm, launch wavelength is 510-540nm.In experiment, positive control be PEI/DNA nano-complexes, negative control be without appoint
The cell of where reason.Each group of data are the average value in three holes of companion specimens.As a result as shown in figure 9, under serum-free condition
The transfection value for polymer (1)/DNA nano-complexes that N/P is 13 is 78.4%, higher than positive controls PEI/DNA
(30.7%), under 10% serum condition, N/P is also influenceed the reduction of transfection value for 13 nano-complex by serum, but N/P is
60 nano-complex still shows very high transfection efficiency (90.0%), and now positive controls PEI/DNA transfection
Efficiency only has 5.0%.
(3) laser co-focusing observation egfp expression
A549 cells are incubated in radius 15mm culture vessel with glass bottom according to the cell concentration in 250000/ hole, per hole
1.5mL culture mediums, and cell is placed in 5%CO224h is cultivated in 37 DEG C of constant incubators of concentration and 95% humidity.Afterwards will
Culture medium replaces with 1.5mL culture mediums (serum content is 0% or 10%) in per hole, and adds the nano-complex prepared
(nano-complex of polymer (1), nitrogen phosphorus mol ratio is respectively 13,60) 300 μ L (μ g of DNA containing pEGFP 6) to solution, training
Support 4h.Then the culture medium containing nano-complex in most plate is abandoned, fresh culture is replaced with and continues to cultivate to 48h.Cultivate
Cheng Hou, is placed under laser confocal microscope and observes.The excitation wavelength of green fluorescent protein is 488nm, and launch wavelength is 510-
540nm, all photos are shot under 10 times of object lens, all the time using same light intensity.As a result as shown in Figure 10, PEI/DNA nanometers
Compound has only transfected a few cell, and simply the brightness of these cells is very high, and polymer (1)/DNA nano-complexes transfection
Cell is more uniformed, and this in terms of gene therapy to having great significance.For therapeutic gene, most cells can transfect production
Raw treatment albumen expresses a large amount of albumen far better than an only small part cell, because these albumen often only need to minimal amount just
The apoptosis of cell can be caused, therapeutic effect is reached.
Embodiment 7：
Cell transfection assays of polymer (the 1)/DNA nano-complexes under active oxygen radical inhibitor (DPI) effect
By A549 cell culture in 48 orifice plates, cell density is 25000/hole, contains 0.4mL culture mediums per hole, and
Cell is placed in 5%CO224h is cultivated in 37 DEG C of constant incubators of concentration and 95% humidity.It will be replaced afterwards per culture medium in hole
The serum free medium of 0.4mL DPI containing various concentrations (these concentration versus cell survival rates are without influence) is changed to, is incubated 0.5 hour
Afterwards, the μ L (pGL4.13 containing luciferase of polymer (1)/DNA nano-complexes solution 50 that the N/P prepared is 13 are added
μ g of DNA 1), cultivate 4h.Then abandon the culture medium containing nano-complex in most plate, replace with it is fresh contain with before
Culture medium containing comparable sodium DPI continues to cultivate to 10h.Culture medium is discarded, 100 μ 1 × cell pyrolysis liquids of L are added per hole,
Centrifuging and taking supernatant, adds luciferase substrate, and chemiluminescence intensity is determined with chemiluminescence detector.Protein concentration is used
Bradford protein detection kits are determined.Chemiluminescence intensity is normalized using protein concentration, and unit is every milligram of albumen hair
Luminous intensity (RLU/mg protein).Each group of data are the average value in three holes of companion specimens.As a result as shown in figure 11,
Cell transfecting efficiency treated DPI can decline two orders of magnitude, it was demonstrated that if cell does not have active oxygen radical, DNA is just
Effectively transfected it is difficult to discharge.
Embodiment 8：
Transfection experiment in the intratumor injection body of polymer (1)/DNA nano-complexes.
The BALB/c Female nude mices oxter inoculation 5 × 10 of 6~8 weeks6Individual A549 tumour cells, treat tumour length to about 200mm3
Two groups, every group six, difference intratumor injection polymer (1)/DNA and PEI/DNA nano-complex solution are randomly divided into afterwards
60 μ L (μ g of the DNAs of pGL4.13 containing luciferase 15), put to death nude mice after 48 hours, peel off tumour, add 4 times of volumes
1 × cell pyrolysis liquid, shred homogenate, 13500g is centrifuged 10 minutes, take supernatant, add luciferase substrate, use chemiluminescence
Detector determines chemiluminescence intensity.Protein concentration is determined with Bradford protein detection kits.Chemiluminescence intensity is utilized
Protein concentration is normalized, and unit is every milligram of albumen luminous intensity (RLU/mg protein).As a result as shown in figure 12, N/P is
The internal transfection of 13 polymer (1)/DNA nano-complexes is higher than PEI/DNA nano-complex 12 times, it was demonstrated that and polymer (1)/
DNA nano-complexes can be transfected effectively once tumor locus is reached.
In summary, the charge reversal type gene delivery carrier for the oxidation response that the present invention is built carries very strong positive electricity,
DNA can be wrapped up well, occur charge reversal under active oxygen radical effect that can be in the cell after entering cell, by
Positive electricity is changed into negative electricity, is escaped from lysosome, and released dna is transfected to nucleus.The carrier has higher transfection to live
Property and relatively low cytotoxicity, have a good application prospect.
Claims (10)
1. a kind of aoxidize the cationic polymer that positive charge is removed in response, it is characterised in that including following fragment：
In above formula：
R1、R2The alkyl or aromatic radical for C1-C6 independently；
R3、R4The alkyl acyl for H, C1-C6 independently；
M is 1-4 positive integer；
Anion is bromide ion or chlorion.
2. the cationic polymer of positive charge is removed in oxidation response according to claim 1, it is characterised in that the cation
Polymer is by polyacrylate, polymethacrylates or other are containing one-level, the polymer of secondary amine base and contain boric acid or boron
Perester radical benzyl-trialkyl quaternary amine based compound reaction is prepared.
3. the cationic polymer of positive charge is removed in oxidation response according to claim 2, it is characterised in that described other contain
One-level, the polymer of secondary amine base include polyethyleneimine, polyamine group acid amides dendritic macromole.
4. the cationic polymer of positive charge is removed in the oxidation response according to claim 1,2 or 3, it is characterised in that R3、R4
For H, acetyl group or formoxyl, R1、R2For methyl, ethyl.
5. the cationic polymer of positive charge is removed in oxidation response according to claim 1, it is characterised in that the cation
Polymer is at least one of following compounds (1)~(4)：
6. the cationic polymer of positive charge is removed in oxidation response according to claim 5, it is characterised in that wherein compound
(1) prepared with compound (2) any method in following two methods：
Method one：By N, N- disubstituted amido ethyl propylene acid esters or N, N- disubstituted amido ethylmethyl acrylate are by poly-
Close reaction and prepare N, N- disubstituted amido ethyl polyacrylate or N, N- disubstituted amido ethyl polymethacrylates,
Then obtained again with the reaction of boronate Bian bromine, boric acid ester group Bian bromine, boronate Bian chlorine or boric acid ester group Bian chlorine；
Method two：By N, N- disubstituted amido ethyl propylene acid esters or N, N- disubstituted amido ethylmethyl acrylate elder generation and boron
Acidic group Bian bromine, boric acid ester group Bian bromine, boronate Bian chlorine or the reaction of boric acid ester group Bian chlorine, generate corresponding acrylate monomer, so
Polymerisation is carried out afterwards to obtain；
The acrylate monomer includes bromo or chloro (2- acryloyl-oxies) ethyl (p- boric acid benzyl) diethyl ammonium, bromo or chlorine
One kind in generation (2- acryloyl-oxies) ethyl (p- borates benzyl) diethyl ammonium.
7. the cationic polymer of positive charge is removed in oxidation response according to claim 5, it is characterised in that wherein compound
(3) prepared with compound (4) by following methods：Acrylate monomer is added with polyethyleneimine or PAMAM by Michael
Prepared into reaction；The acrylate monomer is included by bromo or chloro (2- acryloyl-oxies) ethyl (p- boric acid benzyl) two
One kind in second ammonium, bromo or chloro (2- acryloyl-oxies) ethyl (p- borates benzyl) diethyl ammonium.
8. the cationic polymer of positive charge, its feature are removed in the oxidation response according to claim 5-7 any claims
It is, n is 5-500；PAMAM is at least one of second to the 5th generation.
9. the oxidation response described in claim 1-8 any claims go the cationic polymer of positive charge in conveying DNA and
Application in nucleic acid short chain.
10. the oxidation response described in claim 1-8 any claims removes the cationic polymer of positive charge in conveying Japanese yew
Application in alcohol.
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| CN109010838B (en) * | 2017-06-08 | 2020-11-17 | 浙江大学 | Application of compound containing tertiary amine N-oxide group as cell mitochondrion targeting carrier |
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