CN102391509B - Polyethylenimine derivative, preparation method thereof and application thereof - Google Patents
Polyethylenimine derivative, preparation method thereof and application thereof Download PDFInfo
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- CN102391509B CN102391509B CN 201110386601 CN201110386601A CN102391509B CN 102391509 B CN102391509 B CN 102391509B CN 201110386601 CN201110386601 CN 201110386601 CN 201110386601 A CN201110386601 A CN 201110386601A CN 102391509 B CN102391509 B CN 102391509B
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Abstract
The invention discloses a polyethylenimine derivative. The polyethylenimine derivative is ploxamer modified polyethyleneimine. The invention also discloses a preparation method and an application of the polyethyleneimine derivative. The polyethyleneimine derivative of the invention, which has the advantages of small cytotoxicity and high transfection efficiency, can be used as a genetic vector.
Description
Technical field
The present invention relates to medical technical field, be specifically related to polyethylenimine derivates of a kind of poloxamer (Ploxamer, trade(brand)name Pluronic) modification and its production and application.
Background technology
Gene therapy is meant people's normal gene or medicative gene is imported defective or the performance therapeutic action of people's somatic target cell to correct gene by certain way.Because of exist in the body multiple can degradation of dna or the enzyme of RNA and dissimilar biological barriers, so therapeutic gene wants to arrive effectively target site, must be by means of certain carrier transportation.But plasmid DNA generally enters effective expression behind the nucleus, RNA (siRNA or miRNA) then is its regulating effect of performance in tenuigenin, therefore, how to make up dissimilar carriers, make it or can in tenuigenin, assemble, perhaps nuclear membrane can be penetrated and enrichment in nucleus is focus and the difficult point that present genophore is paid close attention to.Polymine (polyethylenimine, PEI) be a present widely used class non-viral gene vector, higher transfection efficiency is all arranged in vivo and in vitro, the transfection efficiency of PEI is relevant with its molecular weight, its molecular weight just can obtain satisfied transfection effect greater than 22000 o'clock, but also had higher cytotoxicity simultaneously, and the PEI of small molecular weight (Mw=2000) cytotoxicity is very little, but its transfection efficiency is low, can't reach the purpose of treatment.And the mixture that forms of PEI and DNA is very easily assembled in vivo and (as is deposited on the pulmonary capillary bed, then cause serious pulmonary infarction), the particle diameter of its carrier system and dispersion stabilization are all very responsive to buffer salt system and serum, and toxicity is big when causing being applied in the body, transfection efficiency is low, it is not good to distribute in pharmacokinetics and the body.In order to overcome its shortcoming, the general hydrophilic polymer of biocompatibility such as the polyoxyethylene glycol (PEG) of using modified PEI, to improve the stability of PEI/DNA mixture, reduce toxicity, but because the avidity that PEI is modified back and cell by hydrophilic polymer reduces, make goal gene can't enter cell, transfection efficiency descends.How improving the inside and outside transfection efficiency of PEI by the means of chemically modified, reduce cytotoxicity simultaneously, is the focus of research at present.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of polyethylenimine derivates, and this polyethylenimine derivates is the polymine of decorated by poloxamer, its good stability in vivo and in vitro, and cytotoxicity is little, and the transfection efficiency height.
In addition, also need to provide a kind of preparation method of polyethylenimine derivates and as the application of genophore.
In order to solve the problems of the technologies described above, the present invention is achieved through the following technical solutions:
In one aspect of the invention, provide a kind of polyethylenimine derivates, this polyethylenimine derivates is the polymine of decorated by poloxamer, and its chemical structural formula is as follows:
In the formula, PEI is a polymine, x=2-80, y=10-40.
Preferably, the molecular weight of described polymine is 2000.
Poloxamer is an amphipathic nature polyalcohol, is made up of hydrophilic polyoxyethylene chain (EO) and lipophilic polyoxypropylene chains (PO), and its chemical structural formula is as follows:
EO chain formation wetting ability shell can increase the colloidal stability of solid support material, reduces the cytotoxicity of PEI; And PO chain formation lipotropy kernel can increase the lipotropy of material, improves the avidity of pair cell, and then improves transfection efficiency.
Because the wetting ability of polyoxyethylene chain and the lipotropy of polyoxypropylene chains make this analog copolymer of poloxamer have surfactivity far from it, belong to nonionic surface active agent.According to hydrophile-lipophile balance value (HLB), poloxamer has the multiple product from the Ploxamer 401 (HLB=0.5) of extreme hydrophobicity to extremely hydrophilic Ploxamer 108 (HLB=30.5).
Preferably, poloxamer of the present invention is selected from: model is respectively the poloxamer of F68, P105, P123, L61 (Mw=8594,6554,5780,2022).Marque for poloxamer, its identify code is the letter (L: liquid of physical aspect under its room temperature of expression, P: pasty state, F: sheet (solid)) two of suffix or three bit digital, first figure place (and three bit digital situations under front two number) multiply by the approximate molecular mass of 300 expression hydrophobic materials polyoxypropylenes, and back one digit number x 10 is the shared per-cent of polyoxyethylene (for example L61 represents that molecular mass is the poloxamer that the polyoxypropylene of 1800g/mol has 10% polyoxyethylene formation).
Preferably, the reaction mol ratio of described poloxamer and polymine is 1: 2-10; Preferred, the reaction mol ratio of described poloxamer and polymine is 1: 5-10; Most preferred, the reaction mol ratio of described poloxamer and polymine is 1: 10.
In another aspect of this invention, provide a kind of preparation method of above-mentioned polyethylenimine derivates, may further comprise the steps:
With triphosgene and N-hydroxy-succinamide activation poloxamer two terminal hydroxy group;
With activatory poloxamer and polymine reaction, get the polymine of decorated by poloxamer.
In another aspect of this invention, also provide of the application of above-mentioned polyethylenimine derivates as genophore.
Polyethylenimine derivates of the present invention proves that through cytotoxicity experiment and in-vitro transfection cell experiment not only cytotoxicity is little, and the transfection efficiency height, is suitable for using as genophore.
Description of drawings
The present invention is further detailed explanation below in conjunction with the drawings and specific embodiments.
Fig. 1 is PEI 2000-g-poloxamer synthetic reaction scheme figure of the present invention;
Fig. 2 is the NMR figure spectrogram of the PEI 2000 after the embodiment of the invention 1 is modified;
Fig. 3 is PEI 2000 and the PEI 25kd cytotoxic assay figure as a result after the embodiment of the invention 2 is modified;
Fig. 4 is the PEI 2000 in-vitro transfection measurings figure as a result of PEI 2000 and unmodified after the embodiment of the invention 3 is modified;
Fig. 5 is that the embodiment of the invention 4 is utilized the PEI 2000 time dependent born of the same parents' of the going into situation maps after Laser Scanning Confocal Microscope is observed modification.
Embodiment
Polyethylenimine derivates of the present invention is the polymkeric substance that utilizes poloxamer to form as bridge chain molecule series connection low molecular weight polyethylene imines, and it is as genophore, and not only cytotoxicity is little, and the transfection efficiency height.
The following (see figure 1) of the preparation method of polyethylenimine derivates of the present invention:
(A) activation of poloxamer two terminal hydroxy group
Take by weighing the poloxamer after dewatering in right amount, add the triphosgene of twice molar weight, be dissolved in the mixing solutions of dry toluene and anhydrous methylene chloride, room temperature lower magnetic force stirring reaction spends the night.Vacuum is revolved to steam to remove and is desolvated, again with the dissolving of an amount of dry toluene and anhydrous methylene chloride, it is an amount of to add N-hydroxy-succinamide then, under the magnetic agitation, anhydrous triethylamine (being dissolved in the 4ml anhydrous methylene chloride) is dropwise added in the reaction solution, continue the about 4h of stirring reaction.Question response fully after, with reacting liquid filtering and once more vacuum revolve to steam to remove and desolvate residue obtained being dissolved in the 50ml ethyl acetate, get supernatant liquor behind the high speed centrifugation, rotary evaporation is flung to ethyl acetate, the cooling curing reactant is stored under-20 ℃ of drying conditionss, gets the product after two hydroxyls activate.
(B) the decorated by poloxamer polymine after the activation
Be dissolved in the 10ml anhydrous methylene chloride after PEI 2000 dewatered, two activatory poloxamers are dissolved in the 10ml anhydrous methylene chloride, simultaneously above-mentioned two liquid are splashed into slowly at the bottom of the 10ml anhydrous methylene chloride in the liquid, the saturated back of nitrogen room temperature magnetic agitation is spent the night, get supernatant behind the high speed centrifugation, rotary evaporation gets head product.The distilled water dissolving of head product after with deionization, the dialysis tubing that places Mwco=7000 is 4 ℃ of dialysis 4 days down, and every 24h changes the medium of once dialysing.With the solution lyophilize of dialysis back gained ,-20 ℃ of preservations.Get the polymkeric substance behind an amount of purifying, a part is water-soluble, utilizes infiltration gel chromatography (GPC) to measure its molecular weight; Another part is dissolved in D20, carries out 1H-NMR and analyzes, and determines structure.
The called after PEI 2000-ag-poloxamer (Pluronic) of the graftomer of gained, wherein, a represents the reaction feed ratio of PEI and poloxamer in this polymkeric substance, among the present invention, a can choose 2,5,10, and promptly the reaction feed ratio of PEI and poloxamer can be 2,5,10 in this polymkeric substance.The concrete name of gained graftomer can be respectively PEI 2000-2g-F68, PEI 2000-5g-F68, PEI 2000-10g-F68, PEI 2000-2g-P123, PEI 2000-5g-P123, PEI 2000-10g-P123, PEI 2000-2g-P105, PEI 2000-5g-P105, PEI 2000-10g-P105, PEI 2000-2g-L61, PEI 2000-5g-L61, PEI 2000-10g-L61.
The preparation of the polymine of embodiment 1 decorated by poloxamer
Take by weighing the poloxamer 1.2mmol (F68=10.31g after dewatering, P105=7.86g, P123=6.94g, L61=2.43g, BASF Aktiengesellschaft), be dissolved in the mixing solutions of dry toluene and anhydrous methylene chloride, add 0.712g triphosgene (2.4mmol), room temperature lower magnetic force stirring reaction spends the night.Vacuum is revolved to steam to remove and is desolvated, with an amount of dry toluene and anhydrous methylene chloride dissolving, add N-hydroxy-succinamide 0.274g (2.4mmol) then, under the magnetic agitation again, anhydrous triethylamine (0.4ml is dissolved in the 4ml anhydrous methylene chloride) is dropwise added in the reaction solution, continue the about 4h of stirring reaction.Question response fully after, with reacting liquid filtering and once more vacuum revolve to steam to remove and desolvate residue obtained being dissolved in the 50ml ethyl acetate, get supernatant liquor behind the high speed centrifugation, rotary evaporation is flung to ethyl acetate, the cooling curing reactant, the poloxamer after obtaining activating is stored under-20 ℃ of drying conditionss.
With PEI (0.10mmol, Mw=2000, sigma company) is dissolved in the 10ml anhydrous methylene chloride after dewatering, getting poloxamer after the dual-activeization (0.01,0.02,0.05mmol) is dissolved in respectively in the 10ml anhydrous methylene chloride, simultaneously above-mentioned two liquid are splashed into slowly at the bottom of the 10ml anhydrous methylene chloride in the liquid, the saturated back of nitrogen room temperature magnetic agitation is spent the night, and gets supernatant behind the high speed centrifugation, rotary evaporation gets head product.With the dissolving of the distilled water behind the deionization, place the dialysis tubing of Mwco=7000 to dialyse 4 days down at 4 ℃, every 24h changes the medium of once dialysing.With the solution lyophilize of dialysis back gained, obtain the reacting final product behind the purifying, preserve down for-20 ℃.Get the polymkeric substance behind an amount of purifying, a part is water-soluble, utilizes infiltration gel chromatography (GPC) to measure its molecular weight; Another part is dissolved in D2O, carries out
1H-NMR analyzes, and determines structure.Fig. 2 is the NMR collection of illustrative plates of a representative polymer, and as can be seen from Figure 2, the peak of 3.7-3.9ppm is-CH
2CH
2O-unit proton peak, 3.6-4.3ppm is-CH
2CH
2NH-unit proton peak, the peak of 1.1-1.4ppm is-CH
3The unit proton peak.Do not see other peak in the NMR spectrum, illustrate that this polymkeric substance has higher purity.
The Cytotoxic evaluation experiment of the polymine of embodiment 2 decorated by poloxamer
With the Hela cell inoculation on 96 orifice plates, about 10,000 cells in every hole, cultivate the PEI 25kd that adds a series of concentration (0,4,6,8,16,24,32 μ g/ml) after 24 hours respectively and modify after PEI 2000, continue to cultivate 24h, mtt assay is measured cytotoxicity.As can be seen from Figure 3 modified PEI 2000 compares with PEI 25kd, all demonstrated stronger cytoactive in each concentration, especially when high density, the Hela cell of handling with the PEI 2000 after modifying still shows very high biological activity, provide a valid approach for solving PEI as the excessive problem of genophore cytotoxicity, studying carefully its major cause is because the polymerisate of low-molecular-weight PEI and poloxamer can be degraded to micromolecular fragment in vivo, can and discharge by the somatocyte metabolism, reduce gathering.
The outer-gene transfection experiment of the polymine of embodiment 3 decorated by poloxamer
Transfection digests the Hela cell with 0.1% pancreatin+0.02%EDTA the day before yesterday, evenly be seeded on 24 orifice plates, make cell degree of converging reach 50%, every hole adds DMEM substratum (containing 10% foetal calf serum) 1ml, cultivate 24h, make Hela cell degree of converging reach 70%-80%.Before the transfection, change the DMEM substratum of serum-free, respectively the PEI 2000 of unmodified and modified PEI 2000 (PEI 2000-g-Pluronic) are formed mixture (w: w=10: 1 with plasmid pGL3, contain plasmid 3 μ g), join in 24 orifice plates, every kind of mixture repeats to do 3 holes, shakes 24 orifice plates gently and makes the mixture shop even, change the DMEM substratum that contains 10% foetal calf serum after hatching 4h, continue to cultivate.Measure the luciferin enzymic activity after 48 hours.
As can see from Figure 4, except F68, the PEI of various decorated by poloxamer demonstrates better transfection efficiency than the PEI of unmodified, and along with the increase of PEI feed ratio, the positive charge of surfaces of carrier materials is more and more stronger, and transfection efficiency also increases thereupon.F68 is water-soluble stronger poloxamer, and its ability aspect promotion carrier permeates cell membranes is relatively poor relatively, and therefore the solid support material transfection efficiency through its modification improves not obvious.
Embodiment 4 Laser Scanning Confocal Microscopes observe decorated by poloxamer polymine go into born of the same parents' situation
With the Hela cell inoculation in six orifice plates (putting into the sterility cover slide in advance), every hole 5 * 10
5Cell, cultivate 24h after, the PEI derivative (polymine of decorated by poloxamer) that the phycoerythrin mark is crossed mixes with DNA, hatches transfectional cell behind the 30min.Experiment divides four groups, is respectively PEI 2000-10g-F68 group, PEI 2000-10g-P123 group, PEI 2000-10g-P105 group and PEI 2000-10g-L61.By different time points, behind 1min, 5min, 10min, 20min, cover glass is picked up from six orifice plates, be placed on and drip in advance on the slide glass that 20ul glycerine is arranged, cell is towards slide glass, cover glass neutral gum mounting then, laser confocal microscope detects, the result as shown in Figure 5:
A (PEI2000-10g-F68, HLB=29): F68 is water-soluble stronger poloxamer, it can form stable micellar structure in water, be suitable for the solubilising of insoluble drug.But because of its lipotropism a little less than, so on saturating film certain difficulty is being arranged, it disperses more even in cell, therefore is fit to the sort of not needs and necessarily enters nucleus and express just effective medicine;
B (PEI2000-10g-P105, HLB=15): than F68, the PO/EO ratio of P105 diminishes, and hydrophilic composition diminishes in the molecule, and the HLB value is more little, therefore fat-soluble increase, easier permeate through cell membranes, as can be seen from Figure 5, when 30min, most of carrier has all entered cell, and part is gathered on the nuclear membrane;
(PEI2000-10g-P123, HLB=8): compare with P105 with F68, the PO/EO ratio of P123 is littler, and is fat-soluble bigger, easier permeate through cell membranes for C.When 30min, carrier enters cell fully, and has quite a few to pass nuclear membrane, enters in the nucleus.In the poloxamer of selected four kinds of models, the HLB value of B and C is more moderate, and the solubility property of carrier is better, and saturating film ability is stronger, and practicality is stronger;
D (PEI2000-10g-L61, HLB=3): L61 is fat-soluble the strongest in these four kinds of poloxamers, also just has the strongest saturating film ability, distribute at most in the cell and in the nuclear, when 30min, most of carriers have all entered nucleus, and therefore, this carrier is more suitable for entering nuclear medicine in needs.
Above experimental result shows that PEI through decorated by poloxamer is as genophore, toxicity is little, the transfection efficiency height, and obtain solid support material behind the decorated by poloxamer of different HLB values, when rotaring redyeing gene, show characteristic distributions in the different cells, can design its transport vehicle respectively according to it in the difference aspect solubility property and the cytolemma penetrativity as DNA or RNA.
The above embodiment has only expressed embodiments of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to claim of the present invention.Should be pointed out that for the person of ordinary skill of the art without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (8)
1. a polyethylenimine derivates is characterized in that, this polyethylenimine derivates is the polymine of decorated by poloxamer, and its chemical structural formula is as follows:
In the formula, PEI is a polymine, x=2-80, and y=10-40, the molecular weight of described polymine are 2000.
2. polyethylenimine derivates according to claim 1 is characterized in that, described poloxamer is selected from: model is the poloxamer of F68, P105, P123 or L61.
3. polyethylenimine derivates according to claim 1 is characterized in that, the reaction mol ratio of described poloxamer and polymine is 1: 2-10.
4. polyethylenimine derivates according to claim 3 is characterized in that, the reaction mol ratio of described poloxamer and polymine is 1: 5-10.
5. polyethylenimine derivates according to claim 4 is characterized in that, the reaction mol ratio of described poloxamer and polymine is 1: 10.
6. the preparation method of the described polyethylenimine derivates of claim 1 is characterized in that, may further comprise the steps:
With triphosgene and N-hydroxy-succinamide activation poloxamer two terminal hydroxy group;
With activatory poloxamer and polymine reaction, get the polymine of decorated by poloxamer.
7. the described polyethylenimine derivates of claim 1 is as the application of genophore.
8. application according to claim 7 is characterized in that, described genophore carrying DNA or RNA.
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| CN104098763B (en) * | 2014-07-23 | 2016-08-17 | 黄山学院 | A kind of sulfhydrylation poloxamer derivative carrier and its preparation method and application |
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