CN102516535B - Degradable imine polycation and synthetic method thereof, and nanoparticle - Google Patents
Degradable imine polycation and synthetic method thereof, and nanoparticle Download PDFInfo
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Abstract
The invention relates to degradable imine polycation which can be used for the delivery of nucleic acids, and a synthetic method thereof, and a nanoparticle According to the invention, PEI (polyethyleneimine) with low molecular weight and methylglyoxal are used for constructing the polycations which can be degraded to the initial state of the PEI with low molecular weight; and the synthetic method of the polycations comprises the following steps of: slowly and dropwise adding a methylglyoxal aqueous solution to an ethanol solution of the PEI, meanwhile, adding a dried molecular sieve; and using a magnetic stirrer for stirring under room temperature so as to carry out condensation reaction among primary amino, aldehyde group and ketocarbonyl. The polycations not only are degradable, but also release the low-molecular-weight PEI with unprotonated amino, and the degradation products of the polycations cannot produce acidic groups like that of other degradable polymers. The characteristics of the polycations can contribute to the escape of lysosome, and gene are released in cytoplasm with low surface charge. For the proton sponge effect, the lysosome is fractured by the osmotic pressure generated by the proton sponge effect. The amino bonds of the polycations exert the function of proton sponge.
Description
Technical field
The present invention relates to a kind of the degradable imine class polycation and its synthetic method, nano particle of the conveying available for nucleic acid (DNA and RNA).
Background technology
The key bottleneck that DNA and siRNA medicines move towards clinical practice from concept is the research and development of its internal induction system.In all kinds of genophores, Poly-cation compare with virus and liposome vectors supports that density and loading are big, prepare simply with gene, it is easy to the advantages such as chemical modification, there is also chemical toxicity is big, the body-internal-circulation cycle short (being unfavorable for targeting) shortcoming.Polyethyleneimine (the PEI of HMW, 25kDa) because its amino density is big, it is easy to wrap up negatively charged genetic stew and as higher nucleic acid substances (the DNA, RNA) delivery vehicles of the transfection activity being widely studied in vitro and in vivo earliest, but its high component alkyl skeleton can not be avoided can not to degrade brought cytotoxicity.Therefore it is to improve genetic stew transfection activity, reduce a Key Strategy of its cytotoxicity to design and build degradable polycation.Such as, it is an example known to the scientist of this area to build degradable polyethylene imines polycation gene carrier by the connecting key that can be broken in physiological conditions using the polyethyleneimine of small-molecular-weight.
Robert J.Lee of Ohio State University etc. are crosslinked polyethyleneimine class polycation [the Gosselin MA for obtaining high molecular weight degradable with low molecule amount PEI (PEI 800Da) respectively using the thiobis of crosslinking agent two (succinyl phosphorons amino propyl acid ester) (DSP) and double forminoethe of sulphur dipropyl two (DTBP) as bridging agent earliest, effective conveying genophore of Guo W, Lee RJ. low molecular weight polyethylenes reversible crosslink formation.Bioconjugate chemistry, [J] 2001；12：989-94.].The two contain disulfide bond derivative has the transfection activity suitable with commercially available HMW PEI (25KDa) in Chinese hamster (CHO) gonad cell, the introducing of disulfide bond allows polymer by internal go back original reagent glutathione reduction, so as to which disulfide bond disconnects, the small low molecule amount PEI of cytotoxicity is degraded into, but specific cell toxicity data is not provided in document.Then there is lot of documents to report polymer [the Kloeckner J for being crosslinked low molecule amount PEI (PEI 800Da) by the bridging agent that self can be broken containing disulfide bond, ester bond, amine ester bond etc., Wagner E, Ogris M. utilize the degradable poly cationic gene carriers that many amine oligomers are built.[J] Europe pharmacy .2006；29：The publications such as 414-25.].Although these researchs show notional feasibility, the PEI of small-molecular-weight and the low molecule amount PEI with various bridging agents fracture fragment are not still received in the application of human body.
In order that the low molecule amount PEI after degraded is without bridging agent fragment, Sung Wan Kim of University of Utah et al. are reported to be crosslinked using glutaraldehyde as bridging agent and low molecule amount PEI (1800Da), generate degradable polyethylene imines polycation [the Kim YH of imine linkage connection, Park JH, Kim SW, et al. build degradable polyethylene imines polycation gene carrier by acid-sensitive key.[J] control releases research 2005；103：209-19.].The characteristics of this structure is：After cellular uptake, the polycation of gene is condensed under the acid condition of endocytosis body and lysosome, is disconnected with the imine linkage that low molecule amount PEI is joined directly together, is generated the small component PEI of no cytotoxicity.But the paradigmatic structure of imine linkage is it cannot be guaranteed that this polycation carries sufficiently stable before nucleic acid enters cell, its transfection activity is more undesirable.
In order to improve the stability of imine structure, Jin and du be used as bridging agent using the glyoxal for the imines that can carry conjugatedπbond structure with primary amine formation, by human endogenous's property polyamino monomer --- spermine and spermidine aggregate into polycation, show preferable stability and gene transfection activity [Du Z, Jin T. using human endogenous's property polyamine monomers as basic building unit and it is degradable be human endogenous's property polyamine monomers polycation gene carrier, wrapped up for DNA (including DNA vaccination) with conveying .Jin T, 2009.].Therefore, we are by identical connecting key, using low molecule amount PEI be basic building unit and it is degradable be initial low molecular weight PEI polycation gene carrier.Early stage we by the use of glyoxal as bridging agent, the PEI of low molecule amount is polymerized to the degradable polycation gene carrier for initial low molecular weight PEI and glyoxal.Experiment in vitro shows good transfection and cytoactive.On this basis, we further synthesize the imines polycation for having no document report using pyroracemic aldehyde with the PEI of low molecule amount.
The content of the invention
It is an object of the invention to overcome deficiency of the prior art, there is provided a kind of degradable imine class polycation and its synthetic method, nano particle.The present invention returns to the polycation of the initial state of many amino molecules using the PEI of low molecule amount with pyroracemic aldehyde structure and degraded, wherein, the imines with conjugatedπbond is combined PEI primary amine with the aldehyde radical of pyroracemic aldehyde and ketone carbonyl generation；The chemical stability of polycation that the present invention is built preferably, can tight DNA and RNA, it is not degradable in cyclic process in vivo, take into account the stability up to before target cell and the biological responding entered after target cell.
The purpose of the present invention is achieved through the following technical solutions：
The present invention relates to a kind of degradable imine class polycation, structural formula is：
The invention further relates to a kind of synthetic method of above-mentioned degradable imine class polycation, comprise the following steps：
A, the pyroracemic aldehyde aqueous solution is slowly dropped in PEI ethanol solution at -10 DEG C~10 DEG C, while adding dry molecular sieve；Due to there is water generation in course of reaction, so adding molecular sieve water removal in reaction, be conducive to the progress of reaction and the growth of polymer molecular weight；
B, at 10 DEG C~30 DEG C using magnetic stirrer carry out condensation reaction, produce degradable imine class polycation.
It is preferred that, the pyroracemic aldehyde aqueous solution is slowly dropped in PEI ethanol solution at 0 DEG C in the step a.
It is preferred that, the PEI is low molecule amount PEI.
It is preferred that, the molecular weight of the low molecule amount PEI is 800Da.
It is preferred that, the obtained degradable imine class polycation is further isolated and purified, the purification procedures are as follows：The product is placed in low-temperature frozen dry vacuum after bag filter dialysis and removes water.
It is preferred that, the molecular weight of the bag filter is 10000；The time of the dialysis is 12~48 hours.
The invention further relates to a kind of nano particle, the nano particle is prepared by the method comprised the following steps：
A, add ultra-pure water or DEPC to be configured to said polycation solution the above-mentioned degradable imine class polycation, nucleic acid substances plus ultra-pure water or DEPC are configured to nucleic acid substances solution；
B, the said polycation solution is added rapidly in nucleic acid substances solution, mixed；
C, mixed solution is incubated at room temperature, produces the nano particle.
It is preferred that, the nucleic acid substances are DNA or RNA.
Compared with prior art, the beneficial effects of the present invention are：Instrument can not degrade the polycation gene carrier that the present invention is built in physiological conditions, and discharge in degradation process human endogenous's property polyamino monomer containing unprotonated amino, and their catabolite can buffer the acidity of endosome；This property can aid in escape lysosome, and gene is discharged in the cytoplasm of low surface charge.For proton sponge effect, lysosome is due to that the osmotic pressure that proton sponge effect (absorption proton) is produced ruptures；The amino linkage of polycation plays proton sponge effect.
Brief description of the drawings
Fig. 1 is Polyimine-MPEI synthetic route chart.
Fig. 2 is Polyimine-MPEI H-NMR spectrum.
Fig. 3 is Polyimine-MPEI IR spectrograms.
A is the gel electrophoresis figure of Polyimine-MPEI/DNA compounds in Fig. 4；B is the gel electrophoresis figure of Polyimine-MPEI/siRNA compounds.
Fig. 5 is the grain-size graph of Polyimine-MPEI/DNA compounds.
Fig. 6 is the Zeta potential figure of Polyimine-MPEI/DNA compounds.
Fig. 7 schemes for Polyimine-MPEI COS-7 cell transfectings activity.
Fig. 8 is Polyimine-MPEI COS-7 cell MTT toxicity figures.
Embodiment
Embodiments of the invention are elaborated below in conjunction with the accompanying drawings：The present embodiment is implemented lower premised on technical solution of the present invention, gives detailed embodiment and specific operating process.But protection scope of the present invention is not limited to following embodiments.
The present invention includes to the physico-chemical property characterizing method of polycation and genetic stew formation nano particle (being also polyplex)：Gel electrophoresis, dynamic light scattering and Zeta potential；Polycation is luciferase plasmids with the transfection activity of genetic stew formation nano particle (being also polyplex) and the DNA plasmid of toxicity test；Polycation is Cos-7 cells with the transfection activity of genetic stew formation nano particle (being also polyplex) and the cell of toxicity test.
The synthetic method of Polyimine-MPEI polycations
The synthetic route of polycation is as shown in Figure 1.Entirely react and carried out in the environment of anhydrous and oxygen-free, weigh a certain amount of PEI800Da, add the appropriate ethanol dissolving (while adding molecular sieve dry in right amount) newly steamed, then pyroracemic aldehyde (the 40w% aqueous solution) is weighed according to suitable stoichiometric proportion, under -10~10 DEG C (preferably 0 DEG C), pyroracemic aldehyde (the 40w% aqueous solution) is slowly dropped in said mixture.Then reaction is stayed overnight at room temperature.Filtering, filtrate is dialysed 12~48 hours in deionized water with bag filter, and low-temperature frozen dry vacuum obtains end-product after removing water.
Polyimine-MPEI H-NMR spectrum is as shown in Figure 2；Polyimine-MPEI IR spectrograms are as shown in Figure 3.From Fig. 2,3：According to PEI800 structure, the chemical shift δ of PEI800 main chain and the methylene of side chain is between 2.6-2.8.And the hydrogen atom chemical shift δ on the methylene on the polymer molecular chain synthesized is between 0.99-3.69, and the hydrogen atom chemical shift δ in carbon-to-nitrogen double bon on-CH=N- is in 7.2 or so and 7.5 or so position.Fig. 3 is the infrared spectrum of end-product obtained through dialysing, the spectrogram is analyzed knowable to, the middle strong absworption peak at 3421cm-1 is the stretching vibration of the N-H of primary amine symmetric and anti-symmetric.Absworption peak strong in being to have in the range of 1690-1630 (cm-1) in absorption frequency-C=N-, and on macromolecule Polyimine-MSP main chain, because carbon-to-nitrogen double bon is conjugated with other carbon-to-nitrogen double bon, so as to reduce the bond order of double bond, make the reduction of its double bond, that is, vibration frequency is reduced.So finding the middle strong absworption peak for having-C=N- at 1646 (cm-1) places.As can be seen here, Polyimine-MPEI target compounds have been synthesized.
Embodiment 2
Polyimine-MPEI and plasmid formation compound polyplex preparation
Detect that polycation (Polyimine-MPEI) is combined DNA or RNA in the case of the mass ratio different from DNA or RNA as genophore by electrostatic interaction.Weigh quantitative polycationic polymer, plus ultra-pure water or DEPC water are configured to 2mg/mL solution, then filtered with 0.22 μm of sterile filter, the concentration dilution of plasmid is into 1mg/mL, configure different quality than complex solution, need to keep the concentration of plasmid solution constant, then the concentration of said polycation solution is diluted according to different mass ratioes, note keeping the volume of the said polycation solution after diluting and plasmid solution equal, finally said polycation solution is added rapidly to mix in plasmid solution, 30min is incubated at room temperature, thus obtain a series of compound of mass ratioes.
Polyimine-MPEI and plasmid formation compound gel electrophoresis
Configuration quality is than 1.0% agarose solution, the heating for dissolving in micro-wave oven, 40mL solution is taken, in the beaker for pouring into special EB pollutions, about 40 μ L EB solution is added, poured into after stirring evenly in mould, plug after comb, about 30min, glue just solidifies, appropriate TAE buffer solutions are added in electrophoresis tank, Ago-Gel is put into electrophoresis tank waits loading.Then configuration different quality than complex solution, be incubated at room temperature 30min.The Marker of loading selects 1000-10000kb plasmid maker, and 1 μ L sample-loading buffer is first taken during loading, adds 5 μ L sample, after being well mixed, is added in gel pore.Plus 160 volts of voltage of voltage, blue bromophenol blue quickly can be moved to after the bottom of glue, and about 20min is taken pictures with ultraviolet gel imaging system.The Gel electrophoresis results of compound, as shown in figure 4, as shown in Figure 4：Band is from left to right naked DNA, polymer plasmid mass ratio 0.5,1,3,5,7, marker, 10,15,30,50,70,100 respectively.When Polyimine-MSP and DNA compound is before mass ratio 10, because carrier does not wrap DNA therefore can not block DNA migration, therefore bright band is shown.And because some plasmid is wrapped up by macromolecule, therefore band is in hangover form, and brightness is not so good as naked DNA.When in mass ratio >=10 due to carrier by DNA it is fully wrapped around live, therefore DNA is non-migratory in electrophoresis, does not have band in swimming lane, shows at mass ratio more than 10, and polymer has the ability of very strong complex gene material.
Embodiment 4
Polyimine-MPEI and plasmid formation compound particle diameter
The sample size that the measure of compound particle diameter needs is generally 1ml, plasmid solution and each 500 μ L of said polycation solution, and the concentration of plasmid is 20 μ g/mL, the dilution of said polycation solution is carried out according to mass ratio, the mass ratio of required measure is 0.5,1,3,5,7,10,15,20,30.At ambient temperature, the hydrated radius of compound is determined with Nano-S laser particle analyzers, refractive medium is set to water, and refractive index is 1.33, and viscosity is 0.8872cP, each sample is determined 3 times, mapping of averaging.The particle size determination result of compound, as shown in figure 5, as shown in Figure 5：Two kinds of carriers and the particle diameter of plasmid composite can be stablized in below 200nm.Macromolecule does not wrap DNA when mass ratio is 0.5, and DNA and macromolecule are in loose condition (of surface), causes particle diameter bigger than normal close, very unstable, data are bigger than normal.When ratio is between 0.5 to 20, polymer energy stable existence, size is between 100nm-200nm.
Polyimine-MPEI and plasmid formation compound Zeta potential
The dilution of said polycation solution is carried out according to mass ratio, the mass ratio of required measure is 0.5,1,3,5,7,10,15,20,30.Medium for dilution is preferably physiological saline or PBS.The surface charge of compound is determined using zetasizer 2000, sample minute is set to automatically, each sample is determined 3 times, mapping of averaging.The zeta potential measurement result of compound, as shown in fig. 6, as shown in Figure 6：With the increase of polymer and DNA plasmid mass ratio, Zeta potential is consequently increased, since mass ratio 15, and Zeta potential is just stablized in 10-15mV, illustrates that polymer and the nano particle that plasmid is formed are more stable.
Embodiment 6
Polyimine-MPEI and plasmid formation compound COS-7 cell transfection assays
First, in 48 porocyte culture plates, 0.5mL cell suspension is added, density is 5.0-10*104/ mL, overnight incubation.When 48 orifice plates are transfected, the amount that plasmid is added per hole is fixed, the plasmid per hole 500ng, the μ L of volume 25, polycation is configured to 2mg/mL solution, and be sterile filtered with 0.22 μm of filter membrane, according to the testing sample and the mass ratio of plasmid of setting, it is diluted to required ratio, the cumulative volume of said polycation solution is 25 μ L, then said polycation solution is added in the middle of the solution of plasmid, it is quick to mix, it is incubated 30min.The volume for so adding the compound per hole is 50 μ L, is 1/10th of cumulative volume (500 μ L), meets regulation.Each mass ratio does three multiple holes.Positive controls PEI25kDa, result during with its optimal plasmid than 2 (N/P=5) makees three control wells, in this period of incubation, cell is taken out from incubator, remove the culture medium for having serum, washed one time with 200 μ L PBS solution again, culture medium changes the culture medium of 250 μ L serum-free into, and then the compound being incubated is sequentially added into cell.After 4 hours, the culture medium of serum-free is removed, is added per hole and contains 10% hyclone and 1% dual anti-ideal culture medium, be further cultured for 48 hours, detect transfection results.
A series of transfection activity under the conditions of mass ratioes is investigated with reporter gene luciferase, as a result as shown in Figure 7.It is the PEI25kDa that mass ratio is 2 to set positive controls (N/P ratios are 10), it can be seen that, our the polycationic transfection efficiency of oneself design synthesis are suitable with the result of control group, with the increase of polycation and plasmid mass ratio, the transfection activity of the polycation of our oneself design synthesis is always maintained at constant, when mass ratio reaches 100: 1, transfection activity is not still reduced.Above-mentioned experiment shows that the Poly-cation that we build has good transfection activity and very low toxicity.
Embodiment 7
Polyimine-MPEI COS-7 cytotoxicity experiments
The toxicity of polycation is determined using mtt assay, using PEI25kDa as positive controls, the cell being left intact is negative control group.First, inoculating cell, by COS-7 cell dissociations, is diluted in the cell suspension that density is 1.0-5.0*104/mL, 96 orifice plates and 100 μ L, overnight incubation is added per hole.2mg/mL said polycation solution is diluted to different concentration gradients by second step, and final volume is 100 μ L, and positive controls PEI25kDa is diluted to the concentration gradient consistent with testing sample group.Cell is taken out, the culture medium for having serum is removed, is washed one time with 100 μ L PBS, add the culture medium without phenol red serum-free.Then the water that 100 μ L are added in said polycation solution and positive controls PEI25kDa, negative control group is added.After 4 hours, remove nutrient solution and said polycation solution, added per hole 100 μ L without phenol red serum free medium, 25 μ L MTT solution is added under the conditions of lucifuge, is placed in cell culture incubator and cultivates 6 hours.The crystallization situation of micro- Microscopic observation living cells, after being fully crystallized, then adds and is compareed at measurement result at 100 μ LDMSO placement 10min ELIASAs 570nm, 630nm.As a result it is as shown in Figure 8.Among COS-7 cells, the toxicity of polycation is much smaller than control group PEI25kDa.When 150 μ g/mL of concentration, cell survival rate still reaches 90%.
Claims (6)
1. a kind of degradable imine class polycation, it is characterised in that structural formula is：
2. a kind of synthetic method of degradable imine class polycation as claimed in claim 1, it is characterised in that comprise the following steps：
A, the pyroracemic aldehyde aqueous solution is slowly dropped in PEI ethanol solution at 0 DEG C, while adding dry molecular sieve；
B, the stirring at 10~30 DEG C, carry out condensation reaction, produce product；
The PEI is low molecule amount PEI；The molecular weight of the low molecule amount PEI is 800Da.
3. the synthetic method of degradable imine class polycation according to claim 2, it is characterised in that further isolated and purified to the obtained product, the purification procedures are as follows：The product is placed in low-temperature frozen dry vacuum after bag filter dialysis and removes water.
4. the synthetic method of degradable imine class polycation according to claim 3, it is characterised in that the molecular weight of the bag filter is 10000；The time of the dialysis is 12~48 hours.
5. a kind of nano particle, it is characterised in that the nano particle is prepared by the method comprised the following steps：
A, add ultra-pure water or DEPC to be configured to said polycation solution the degradable imine class polycation described in claim 1, nucleic acid substances plus ultra-pure water or DEPC are configured to nucleic acid substances solution；
B, the said polycation solution is added rapidly in nucleic acid substances solution, mixed；
C, mixed solution is incubated at room temperature, produces the nano particle.
6. nano particle according to claim 5, it is characterised in that the nucleic acid substances are DNA or RNA.
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| US20030022375A1 (en) * | 2001-07-05 | 2003-01-30 | Akira Itoh | Vector for reversible gene integration |
| CN102181053A (en) * | 2011-02-25 | 2011-09-14 | 苏州大学 | Hydrophobic-group-modified polyethyleneimine derivative and application thereof |
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| US20030022375A1 (en) * | 2001-07-05 | 2003-01-30 | Akira Itoh | Vector for reversible gene integration |
| CN102181053A (en) * | 2011-02-25 | 2011-09-14 | 苏州大学 | Hydrophobic-group-modified polyethyleneimine derivative and application thereof |
Non-Patent Citations (4)
| Title |
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| A Novel Non-Viral Vector for DNA Delivery Based on Low Molecular Weight, Branched Polyethylenimine: Effect of Molecular Weight on Transfection Efficiency and Cytotoxicity;Dagmar Fischer等;《Pharmaceutical Research》;19990512;第16卷(第8期);第1273-1279页 * |
| Biodegradable polymers as non-viral carriers for plasmid DNA delivery;Jordy Luten等;《Journal of Controlled Release》;20080303;第126卷(第2期);第97-110页 * |
| DagmarFischer等.ANovelNon-ViralVectorforDNADeliveryBasedonLowMolecularWeight Branched Polyethylenimine: Effect of Molecular Weight on Transfection Efficiency and Cytotoxicity.《Pharmaceutical Research》.1999 |
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