CN101492691A - Assembly method for core-shell structural gene support system and uses thereof - Google Patents
Assembly method for core-shell structural gene support system and uses thereof Download PDFInfo
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- CN101492691A CN101492691A CNA2008102076940A CN200810207694A CN101492691A CN 101492691 A CN101492691 A CN 101492691A CN A2008102076940 A CNA2008102076940 A CN A2008102076940A CN 200810207694 A CN200810207694 A CN 200810207694A CN 101492691 A CN101492691 A CN 101492691A
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Abstract
The invention discloses a method for assembling a gene vector system with a nuclear shell structure. The method has the following steps: complete ring-opening is carried out on small molecular weight polymine (PEI) to gather L-aspartame (PSI), alpha, beta-polyL-aspartame-g-polymine (AE) are obtained after purification, the AE is taken as a carrier to be assembled into a nano gene vector system with the nuclear shell structure with DNA; the invention is characterized in that the synthetic method of brush-shaped polycation is simple and the cost is low; as the used raw material is free from toxicum, the gene vector of the obtained brush-shaped polycation has low toxicity; the brush-shaped polycation and DNA can form nano-particle with the nuclear shell structure by charge interaction; the nano-particle has high gene transfection efficiency; base on a hydrophilic shell layer, the nano-particle can reduce absorption on protein, so that the nano-gene vector system with the nuclear shell structure has fine gene transfection efficiency with the existence of blood plasma and is expected to be used in treatment of in vivo genes.
Description
Technical field
The present invention relates to nano biological medicine technical field, specifically a kind of assemble method of core-shell structural gene support system and purposes.
Background technology
Gene therapy since can from gene level treatment or prevention ancestor genetic diseases or the day after tomorrow acquired disease, started the revolutionary new way of a treatment disease at biomedicine field.Although in the past more than 20 year, clinical trial not following thousand times, cause U.S. food and safety of medicine office not to approve any one gene therapy scheme so far yet because of its effect is undesirable, therefore, the transfection efficiency and the clinical safety thereof that improve therapeutic gene still are challenging problems.The virus type carrier is applied to most of clinical trials based on the high advantage of transfection efficiency, yet defectives such as its high immunogenicity, carinogenicity, non-guidance quality, repeatedly medication, preparation cost height make the non-viral gene type carrier that toxicity is low, immunogenicity is low, clinical safety is higher, be easy to prepare be subjected to more concern.
Polycation based on polymine (PEI) is easy to characteristics such as transformation because of its good gene transfection efficient, structure, become the object of people's broad research as genophore, but cause the transfection efficiency in blood plasma lower because of it is easy to the non-specific adsorption oxyphorase.Kissel etc. with polyethyleneglycol modified PEI (Bioconjugate Chem.2006,17,1209-1218), in the hope of reducing absorption, but reduced the endocytosis ability of cell to polymkeric substance/gene composite nano-granule to oxyphorase, transfection efficiency is undesirable.
Summary of the invention
The objective of the invention is to utilize linear low molecular weight polyethylene imines (PEI) open loop α, the polycation gene carrier of β-poly-(L-aspartic acid) synthetic brush and sac like, by lower molecular weight PEI to α, the high percentage of grafting of β-poly-(L-aspartic acid), improve lower molecular weight PEI " proton sponge effect ", promptly improve its gene transfection efficient and keep its hypotoxicity.To brush shape polycation α again, β-poly-(L-aspartic acid-g-polymine) (AE) can self-assembly forms the nanoparticle of nucleocapsid structure by coulombic interaction with DNA, this nanoparticle has high gene transfection efficient, cause is based on its hydrophilic shell, can reduce proteic absorption, thereby make the nano-gene carrier system of this nucleocapsid structure in the presence of blood plasma, have good gene transfection efficient, be expected to be used for the vivo gene treatment.
The object of the present invention is achieved like this:
(a) brush shape polycation is synthetic
With toxicity low, small molecular weight PEI is grafted on the low polymkeric substance of good water solubility, toxicity, obtains good water solubility, brush shape polycation that toxicity is low.(PSI), purifying gets α to be about to the complete open loop of small molecular weight PEI poly-(L-aspartimide), and β-poly-(L-aspartimide-g-polymine) (AE); Specifically:
(1) α, (PSI) synthetic of β-poly-(L-aspartimide)
The phosphoric acid that in the 1L round-bottomed flask, adds 25 gram (0.19mol) L-aspartic acid powder and 12.5 grams 85%, mix, in 185 ℃ of decompression reactions on the Rotary Evaporators 3 hours till no longer include water vapor and produce, add 150mL dimethyl formamide (DMF) solution stirring dissolved solids thing, under agitation the solution that obtains slowly is added drop-wise in the beaker that fills 1000mL water then, get white precipitate, leave standstill, supernatant liquor inclines, filter, solid is washed with water to neutrality, in 50 ℃ of vacuum-dryings 48 hours, get powdery solid-α, β-poly-(L-aspartimide) (PSI);
(2), polymine (PEI) open loop α, β-poly-(L-aspartimide)
Get the polymine (PEI) of lower molecular weight Mn:423 and the α that last step obtains, β-poly-(L-aspartimide) (PSI), it is dissolved in dimethyl formamide (DMF) solution respectively, 1: 3 in molar ratio (PSI/PEI) mixes again, stirs 22h under room temperature, after the filtration, solution is used
Dialysis tubing was at room temperature dialysed 6 days, and lyophilize must be brushed shape polycation { α, β-poly-(L-aspartimide-g-polymine) } (AE);
(b) assembling of core-shell structural gene support system
(a) step gained brush shape polycation (AE) and dna solution are pressed N: the P ratio is for (5,10,20,30 or 40 thorough mixing, room temperature left standstill 24 hours, got the AE/DNA composite nanometer particle and were core-shell structural gene support system.The stratum nucleare of nano particle is made up of by the mixture that coulombic interaction forms polymine and DNA, and the shell of nano particle is made of hydrophilic polymer.
The molecular weight of described brush shape polycation is not less than 10KDa.
Described core-shell structural gene support system is used for the gene transfection of HeLa, 293T or HepG2 cell, the especially gene transfection in the presence of serum.
The synthetic method that the present invention brushes the shape polycation is simple, and is with low cost; Because raw materials used nontoxic, the toxicity of gained brush shape polycation gene carrier is very low; This brush shape polycation and DNA can form the nanoparticle of nucleocapsid structure by coulombic interaction; This nanoparticle has high gene transfection efficient; Based on its hydrophilic shell, can reduce proteic absorption, thereby make the nano-gene carrier system of this nucleocapsid structure in the presence of blood plasma, have good gene transfection efficient, be expected to be used for the vivo gene treatment.
Description of drawings
Fig. 1 is brush shape polycation (AE) synthetic route chart
Fig. 2 is AE's
1H NMR spectrogram
Fig. 3 is an AE/DNA mixture gel electrophoresis figure
Fig. 4 is DNA protection and release experiment figure
Fig. 5 is N: the EF-TEM of AE/DNA mixture figure during P=10
Fig. 6 is the cytotoxicity of AE, (a) 239T; (b) HeLa; (c) HepG2
To be AE/DNA (pGL3) compare the transfection efficiency of different clones, (a) 239T at different N: P to Fig. 7; (b) HeLa; (c) HepG2
Fig. 8 is that AE/DNA is to the gene transfection efficient of HeLa cell in the presence of serum
Embodiment
Embodiment
α, β-poly-(L-aspartic acid-g-polymine) preparation (AE), the assembling of core-shell structural gene support system and structural characterization and performance evaluation
(1), α, (PSI) synthetic of β-poly-(L-aspartimide)
In the 1L round-bottomed flask, add the phosphoric acid of 25 gram (0.19mol) L-aspartic acid powder and 12.5 grams 85%, mix, in 185 ℃ of decompression reactions on the Rotary Evaporators 3 hours till no longer include water vapor and produce.Add 150mL DMF stirring and dissolving solids, under agitation the solution that obtains slowly is added drop-wise in the beaker that fills 1000mL water then, get white precipitate, leave standstill, the supernatant liquor that inclines filters, solid is washed with water to neutrality, in 50 ℃ of vacuum-dryings 48 hours, get powdery solid (PSI) 17 grams, productive rate 93%.
(2), small molecules PEI (Mn:423) opens α, β-poly-(L-aspartimide)
Earlier PEI423 and PSI are dissolved in DMF respectively, mix, under room temperature, stir 22h at 1: 3 in molar ratio (PSI/PEI).After the filtration, solution is used
Dialysis tubing was at room temperature dialysed 6 days, lyophilize.As shown in Figure 2, has very high percentage of grafting with PEI open loop PSI synthetic AE.
(3), gel electrophoresis
Prepared fresh different N: the AE/DNA mixture (nano particle) of P ratio, place under the room temperature and hatched 20 minutes, add 2 μ l dyestuffs, placed again 10 minutes, mixing solutions is loaded on the agar gel that contains ethidium bromide, in TAE electrophoresis buffered soln under the voltage of 100V electrophoresis after 40 minutes uv-exposure take pictures.As shown in Figure 3, PEI423 is little because of its molecular weight, even as N: when the P ratio is 30, and effective condensation DNA.Yet even AE/DNA mixture N: the P ratio is 3, and the migration of DNA is quite slow, illustrates that the AE/DNA mixture has very strong condensation ability to DNA.
(4), DNA protection and release experiment
N: P uses 1 μ lPBS or DNase-I at DNase/Mg respectively than the AE/DNA mixture and the DNA plasmid that are 5
2+Hatch in the digestion buffered soln.For making DNase inactivation and released dna, all samples was all handled 10 minutes with 4 μ l 250mM EDTA, handled with 8 μ l SDS again.Sample is at room temperature hatched 2h, adds 3 μ l dyestuffs then, further hatches 10 minutes, mixing solutions is loaded on the agar gel that contains ethidium bromide, in TAE electrophoresis buffered soln under the voltage of 50V electrophoresis after 1 hour uv-exposure take pictures.As shown in Figure 4, naked DNA is directly degraded by DNase-I in 30min, and N: P has effectively protected DNA in order to avoid it is by enzymic hydrolysis than the AE/DNA mixture that is 5.
(5), EF-TEM observes pattern
The concentration of DNA is that 20 μ g/ml are used for the EF-TEM test in the complex solution, get that 10 μ l N: P drip to clean copper mesh carefully than the AE/DNA mixture that is 10 and be that 7.4 massfractions are 1.5% phospho-wolframic acid dyeing 5 seconds, the preceding copper mesh drying at room temperature of imaging 5 minutes with pH.By Fig. 5 (a) as can be known, AE is at nanoscale condensation DNA.Fig. 5 (b) is N: the P ratio is 10 o'clock, the EF-TEM figure of AE/DNA mixture.
(6), cytotoxicity experiment
With initial density is 1 * 10
4Cells/well (HeLa and 293T), or initial density is 2 * 10
4Cells/well (HepG2) is inoculated in 96 orifice plates, hatches 18-20h in the substratum of 200 μ l, reaches 80% at treatment time inner cell degrees of fusion, substratum changes the polymkeric substance (1 that 100 μ l contain different concns into, 5,10,20 and 30 μ g/ml) fresh serum free medium.Continue to cultivate 2h with adding 20 μ lMTS behind the cell cultures 24h, measure absorbance, calculate the surviving rate of cell with ELISA
Cell survival rate (%)=(OD
Sample/ OD
Control) * 100
As shown in Figure 6, PEI423 is under survey concentration, and to 293T, HeLa and HepG2 clone does not almost have toxicity, and PEI25K or AE is along with the increase of concentration, and its toxicity also increases thereupon.Under same concentration, compare with the toxicity of PEI25K, AE almost is nontoxic, especially under high dosage.Even after polymer concentration is to cultivate 24h under the 30 μ g/ml, the cell of AE from survival rate still greater than 60%.By contrast, when cell is handled with PEI25K, the survival rate of cell descends about 40%.The major cause that AE toxicity is low is that its electric density is low, and the peptide main chain can be degraded.
(7), the outer-gene transfection efficiency is estimated
Initial density is 1 * 10
4Cells/well (HeLa and 293T), or initial density is 2 * 10
4Cells/well (HepG2), be seeded in 24 orifice plates, in the substratum of 1ml, cultivate and hatch 18-20h, when the cytogamy degree is 70-80% substratum being changed into is 500 μ l serum-frees or the substratum that contains 10% serum, and add N: P than being respectively (5,10,20,30 and 40) polymkeric substance/pGL3-control (1 μ g) mixture continues to cultivate 6 hours.At last substratum is changed into perfect medium and continue to cultivate 24h.Each transfection experiment repeats to do three times, transfection activity RLUs value representation.As shown in Figure 7, the gene transfection efficient of AE in 293T, HeLa and HepG2 clone compares from being monotonically increasing trend with N: P, work as N: the P ratio is controlled at 40, gene transfection efficient the best of AE, even be higher than PEI25K and liposome gene transfection efficient at 293T, HeLa and HepG2 cells.In addition, can also find out from Fig. 7 that the toxicity of AE is quite low.
As shown in Figure 8, observing the PEI423/DNA mixture under anti-phase fluorescent microscope is genetic expression blindly.Compare with the PEI25K/pEGFP-N2 mixture, can observe more GFP and express in the AE/pEGFP-N2 mixture, just explanation is as the efficient gene carrier for this, and AE has good prospect.
Claims (3)
1, a kind of assemble method of core-shell structural gene support system is characterized in that this method may further comprise the steps:
A), (AE) synthetic of brush shape polycation { α, β-poly-(L-aspartimide-g-polymine) }
(1) α, (PSI) synthetic of β-poly-(L-aspartimide)
The phosphoric acid that in the 1L round-bottomed flask, adds 25 gram (0.19mol) L-aspartic acid powder and 12.5 grams 85%, mix, in 185 ℃ of decompression reactions on the Rotary Evaporators 3 hours till no longer include water vapor and produce, add 150mL dimethyl formamide (DMF) solution stirring dissolved solids thing, under agitation the solution that obtains slowly is added drop-wise in the beaker that fills 1000mL water then, get white precipitate, leave standstill, supernatant liquor inclines, filter, solid is washed with water to neutrality, in 50 ℃ of vacuum-dryings 48 hours, get powdery solid-α, β-poly-(L-aspartimide) (PSI);
(2), polymine (PEI) open loop α, β-poly-(L-aspartimide)
Get the polymine (PEI) of lower molecular weight Mn:423 and the α that last step obtains, β-poly-(L-aspartimide) (PSI), be dissolved in dimethyl formamide (DMF) solution respectively with it, 1: 3 in molar ratio (PSI/PEI) mixes again, under room temperature, stir 22h, after the filtration, solution was at room temperature dialysed 6 days with the 3500A dialysis tubing, and lyophilize must be brushed shape polycation { α, β-poly-(L-aspartimide-g-polymine) } (AE);
B), the assembling of core-shell structural gene support system
With a step gained brush shape polycation (AE) and dna solution by N: the P ratio is 5,10,20,30 or 40 thorough mixing, leaves standstill under the room temperature 24 hours, must be core-shell structural gene support system by composite nanometer particle.
2, method according to claim 1 is characterized in that the molecular weight of described brush shape polycation is not less than 10KDa.
3, a kind of purposes of core-shell structural gene support system is characterized in that being used for the gene transfection of HeLa, 293T or HepG2 cell, especially the gene transfection in the presence of serum.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102174579A (en) * | 2011-01-19 | 2011-09-07 | 武汉大学 | Reducible and biodegradable comb type high polymer gene vector and preparation method of same |
| CN102516534A (en) * | 2011-11-14 | 2012-06-27 | 上海交通大学 | Polycation capable of being degraded into spermine, and synthesis method and nanoparticles thereof |
| CN109288815A (en) * | 2018-10-25 | 2019-02-01 | 南开大学 | A kind of preparation method and application of the multistage delivery nanoparticle of achievable cancer target delivery nucleic acid drug |
-
2008
- 2008-12-25 CN CNA2008102076940A patent/CN101492691A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102174579A (en) * | 2011-01-19 | 2011-09-07 | 武汉大学 | Reducible and biodegradable comb type high polymer gene vector and preparation method of same |
| CN102174579B (en) * | 2011-01-19 | 2013-03-06 | 武汉大学 | Reducible and biodegradable comb type high polymer gene vector and preparation method of same |
| CN102516534A (en) * | 2011-11-14 | 2012-06-27 | 上海交通大学 | Polycation capable of being degraded into spermine, and synthesis method and nanoparticles thereof |
| CN102516534B (en) * | 2011-11-14 | 2013-11-20 | 上海交通大学 | Polycation capable of being degraded into spermine, and synthesis method and nanoparticles thereof |
| CN109288815A (en) * | 2018-10-25 | 2019-02-01 | 南开大学 | A kind of preparation method and application of the multistage delivery nanoparticle of achievable cancer target delivery nucleic acid drug |
| CN109288815B (en) * | 2018-10-25 | 2020-12-22 | 南开大学 | Preparation method and application of multistage delivery nanoparticles capable of realizing targeted delivery of nucleic acid drugs to tumors |
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Open date: 20090729 |