CN105295055A - Magnetic transfection reagent based on supramolecule self-assembly - Google Patents
Magnetic transfection reagent based on supramolecule self-assembly Download PDFInfo
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- CN105295055A CN105295055A CN201510830717.3A CN201510830717A CN105295055A CN 105295055 A CN105295055 A CN 105295055A CN 201510830717 A CN201510830717 A CN 201510830717A CN 105295055 A CN105295055 A CN 105295055A
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- cyclodextrin
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Abstract
The invention relates to a magnetic transfection reagent based on supramolecule self-assembly, wherein magnetic spheres prepared through a high temperature decomposition method and six-site fully-substituted alpha-cyclodextrin-PEI600 (CD-PEI) are assembled through self-assembly to form the magnetic transfection reagent. According to the present invention, the preparation method of the magnetic transfection reagent is provided, the structure of the magnetic transfection reagent is characterized, and the biological toxicity and the transfection performance of the magnetic transfection reagent at the cell level are characterized; the preparation method is mild, simple and green environmental protection; and the synthesized magnetic transfection reagent has characteristics of smaller particle size, surface having a lot of positive charges, good siRNA combination ability, low biological toxicity, high transfection efficiency, and good application prospects, and the performance of the magnetic transfection reagent as the siRNA vector is excellent.
Description
Technical field
The present invention relates to polymer chemistry, bio-medical material, magneticsubstance and nanotechnology research field, be specially a kind of magnetic transfection reagent based on Supramolecular self assembly.
Background technology
RNA interference (RNAi) has huge application potential treating targetedly in the diseases such as tumour, hereditary defect and diabetes.Although at present the mechanism of action of RNAi clearly, owing to lacking the carrier effectively sending siRNA, and cause its clinical application have received significantly limit.Although the RNAi based on virus vector can have targeting and higher transfection efficiency concurrently, the application of virus vector also may cause certain danger.Nano material is the non-virus carrier of a quasi-representative, generally has the advantages such as size is little, easy modification, and effectively can mediate siRNA and enter cell and induce RNAi effect.But from the result of study of nanotoxicology, most nano material all has cytotoxicity in various degree.The feature that Magnetofection reagent is easily modified with it, toxicity is low, transfection efficiency is high progresses into the visual field of people.Therefore, design a kind of can stablizing transduce siRNA and induce the research emphasis that the magnetic transfection reagent of RNAi effect becomes current.
Summary of the invention
The object of the invention is for above-mentioned existing problems, a kind of magnetic transfection reagent based on Supramolecular self assembly is provided, this transfection reagent utilizes this promising siRNA delivery system of magnetic transfection, surface is with a large amount of PEI, transfection reagent is made to present larger electropositivity, therefore can form the mixture of nano-scale with siRNA in aqueous, protection siRNA not by enzyme liberating, and improves by magnetic force the ability that it enters cell effectively; The present invention utilizes self-assembly to modify for magnetic nanoparticle, make its surface with a large amount of PEI, finally make magnetic transfection reagent, and pass through the sign of the aspect character such as particle diameter, current potential, transfection, prove that it has the character that mediation siRNA realizes RNAi effect, for further huge contribution has been made in research.
Technical scheme of the present invention:
A kind of magnetic transfection reagent based on Supramolecular self assembly, be that the magnetic ball made by high-temperature decomposition by self-assembly and 6 are entirely replaced α cyclodextrin-PEI600 (CD-PEI) and carry out assembling and formed, 6 structural formulas entirely replacing α cyclodextrin-PEI600 are as follows:
Based on a preparation method for the magnetic transfection reagent of Supramolecular self assembly, step is as follows:
1) assemble primitive 6 and entirely replace α cyclodextrin-PEI
600synthesis
With N, N-carbonyl dimidazoles (CDI) activates α cyclodextrin, dimethyl sulfoxide (DMSO) (DMSO) solution of α cyclodextrin is slowly added drop-wise to N, in the dimethyl sulphoxide solution of N-carbonyl dimidazoles, react 3 hours under nitrogen protection, obtained product is precipitated in ether, obtain intermediate product 6 and entirely replace α cyclodextrin acyl imidazoles, intermediate product is dissolved in dimethyl sulfoxide (DMSO), slowly be added drop-wise in the dimethyl sulphoxide solution of PEI600, react 5 hours under nitrogen protection, reaction solution is dialysed 72 hours, freeze-drying obtains solid product 6 and entirely replaces α cyclodextrin-PEI600, α cyclodextrin, N, N-carbonyl dimidazoles, the mol ratio of PEI600 is 1:1.2:10, α cyclodextrin-dimethyl sulphoxide solution and N, N-carbonyl dimidazoles-dimethyl sulphoxide solution concentration is 0.1mmol/mL, PEI
600-dimethyl sulphoxide solution concentration is 0.5mmol/mL, reaction formula is as follows:
2) self assembly magnetic transfection reagent
α cyclodextrin-PEI600 is entirely replaced water-soluble by above-mentioned 6, be made into the solution of 1mmol/mL, the magnetic ball prepared by high-temperature decomposition is dissolved in toluene and is made into 1mg/mL, isopyknic magnetic ball solution and 6 are replaced full the mixing of α cyclodextrin-PEI600 solution, vigorous stirring 8-10 hour, water phase separated, Magneto separate, wash three times, store in aqueous with the concentration of 1mg/mL, obtain target compound magnetic transfection reagent.
Advantage of the present invention is:
1) mainly have employed N in the chemosynthesis part of this research, N-carbonyl dimidazoles as activator, by cyclodextrin and PEI
600carry out coupling, reaction mechanism is clear and definite, condition probes into maturation, working method is simple, reproducible, is widely used; It is the self-assembly of mainly assembling primitive that the connection of magnetic ball between compound then have employed with cyclodextrin, assembling reaction has the features such as reaction conditions is simple, environmental protection compared to general chemical reaction, and reaction has certain reversibility, at present at high score chemical field, in the synthesis being widely used in functional polymer based on the self-assembly of cyclodextrin and finishing, and obtain good effect.
2) PEI as a kind of widespread use non-viral class transgenosis reagent because its higher bio-toxicity serious limit its development, but also there are some researches show that the PEI toxicity that molecular weight is lower is obviously lower, but also limit the efficiency of its gene transfection simultaneously, micromolecular PEI is enriched in the surface of magnetic ball by this gene transfection agent, transfection is strengthened, a kind of magnetic transfection reagent with low cytotoxicity and high transfection efficiency of final synthesis by the reactive force of externally-applied magnetic field.
3) the magnetic transfection reagent of final synthesis, particle diameter is less, and surface is with a large amount of positive electricity, has well in conjunction with the ability of siRNA, and bio-toxicity is low, transfection efficiency is high, as siRNA carrier superior performance, has a good application prospect.
Accompanying drawing explanation
The assembling primitive NMR of Fig. 1 synthesized by this patent schemes.
The FT-IR figure of the magnetic transfection reagent of Fig. 2 synthesized by this patent.
The thermogravimetric curve figure of the magnetic transfection reagent of Fig. 3 synthesized by this patent.
The agarose gel electrophoresis retardance figure of the magnetic transfection reagent of Fig. 4 synthesized by this patent.
The cytotoxicity figure of the hysteresis curve of the magnetic transfection reagent of Fig. 5 synthesized by this patent.
The transfection figure of the magnetic transfection reagent of Fig. 6 synthesized by this patent.
Embodiment
Below in conjunction with specific examples, the present invention is further illustrated, to understand the present invention better.
Embodiment:
A kind of magnetic transfection reagent based on Supramolecular self assembly, be that the magnetic ball made by high-temperature decomposition by self-assembly and 6 are entirely replaced α cyclodextrin-PEI600 (CD-PEI) and carry out assembling and formed, 6 structural formulas entirely replacing α cyclodextrin-PEI600 are as follows:
Based on a preparation method for the magnetic transfection reagent of Supramolecular self assembly, step is as follows:
1) synthesis that primitive 6 replaces α cyclodextrin-PEI600 is entirely assembled
With N; N-carbonyl dimidazoles (CDI) activates α cyclodextrin; 0.973g α cyclodextrin is dissolved in 10mL dimethyl sulfoxide (DMSO) (DMSO); 1.297gN, N-carbonyl dimidazoles is dissolved in dimethyl sulfoxide (DMSO), α cyclodextrin soln is slowly added drop-wise to N; in N-carbonyl dimidazoles solution; react 3 hours under nitrogen protection, obtained product is precipitated in ether, obtain intermediate product 6 and entirely replace α cyclodextrin acyl imidazoles.Intermediate product is dissolved in 10mL dimethyl sulfoxide (DMSO); slowly be added drop-wise to and be dissolved with in the 20mL dimethyl sulphoxide solution of 10gPEI600; react 5 hours under nitrogen protection, by reaction solution dialyse 72 hours, freeze-drying obtains solid product 6 and entirely replaces α cyclodextrin-PEI600.Weighing and obtaining product is 2.7g.Productive rate is 57.0%, and reaction formula is as follows:
The assembling primitive NMR of Fig. 1 synthesized by this patent schemes, and shows in figure: target compound 6 entirely replaces α cyclodextrin-PEI600 and is successfully synthesized.
2) self assembly magnetic transfection reagent
36mg6 position being replaced full α cyclodextrin-PEI600 is dissolved in 4mL water, the magnetic ball prepared by 4mg high-temperature decomposition is dissolved in 4mL toluene, magnetic ball solution and 6 are replaced α cyclodextrin-PEI600 solution entirely mix, vigorous stirring overnight, water phase separated, Magneto separate, washs three times, and the magnetic ball obtained is dissolved in 1mL water and obtains target magnetic transfection reagent.
The FT-IR figure of the magnetic transfection reagent of Fig. 2 synthesized by this patent, shows in figure: 6 entirely replace α cyclodextrin-PEI600 and have successfully been modified at magnetic ball surface.
The thermogravimetric curve figure of the magnetic transfection reagent of Fig. 3 synthesized by this patent, shows in figure: 6 modification ratios entirely replacing α cyclodextrin-PEI600 are about 16%.
The agarose gel electrophoresis retardance figure of the magnetic transfection reagent of Fig. 4 synthesized by this patent, shows in figure: when N/P ratio is 1:2, and transfection reagent can completely in conjunction with siRNA.
The sign of magnetic transfection reagent:
1) cytotoxic assay of magnetic transfection reagent
The toxicity of magnetic transfection reagent to 3T3 cell is measured by mtt assay.Be 5x10 by concentration
4the 3T3 cell suspension of/mL is inoculated in 96 well culture plates respectively, every hole 100 μ L cell suspension.At 5%CO
2in incubator, after cultivating 24 hours in 37 DEG C, in every hole, add the testing sample 10 μ L of concentration from 10 μ g/mL to 1000 μ g/mL.Control group adds 10 μ LPBS, and cell continued cultivation after 24 hours in 37 DEG C of incubators, adds the PBS solution (5mg/mL) of 10 μ LMTT and continue cultivation 4 hours in every hole.Sucking-off substratum, adds 150 μ L dissolve purple crystallization first a ceremonial jade-ladle, used in libations in every hole.The absorption value at 570nm place is measured by microplate reader.Each quality when control group does 6 Duplicate Samples.
Fig. 5 is the cytotoxicity figure of the hysteresis curve of the made magnetic transfection reagent of this patent.Represent in figure that material magnetic transfection reagent has very low cytotoxicity, when concentration reaches 100 μ g/mL, cell survival rate is still about 80%, and it is not obvious for Cytotoxic impact with or without externally-applied magnetic field, and its toxicity significantly lower than the transfection reagent PEI25KD of commercialization, has good application prospect
2) the transfection performance measurement of magnetic transfection reagent.
By GFP-Hela cell with 5x10
4the density in/hole is inoculated in 24 well culture plates, by complete 1640 culture medium culturing at 5%CO
2in incubator, cultivate 24 hours at 37 DEG C.Before transfection, remove substratum, with PBS (0.1M, pH7.4) buffer solution cell, every hole adds 1640 fresh serum free mediums of 400 μ L.Magnetic transfection reagent and antiGFP-siRNA are mixed with mass ratio 20:1, by mixed solution vortex mixing, compound system is left standstill half an hour, by fresh serum free medium and mixture mixing, make the ultimate density of siRNA be 60nM.Control group adds the PBS of equivalent.Join in 24 orifice plates respectively by 100 μ L complex solutions, cell cultivates the substratum removed after 5 hours containing mixture under the condition having magnetic field.After adding the fresh perfect medium of 500 μ L, cell is at 5%CO
2in incubator, continue cultivation at 37 DEG C 48 hours.By fluorescence photo, result is compared.
Fig. 6 is the transfection figure of the made magnetic transfection reagent of this patent, wherein: figure A is the cell fluorescence photo before transfection, figure B is the cell fluorescence photo after transfection, can find out that after carrying out transfection, most cells fluorescence disappears, explanation there occurs RNAi effect, and testimonial material has good siRNA transfection efficiency.
Claims (2)
1. the magnetic transfection reagent based on Supramolecular self assembly, it is characterized in that: be that the magnetic ball made by high-temperature decomposition by self-assembly and 6 are entirely replaced α cyclodextrin-PEI600 (CD-PEI) and carry out assembling and formed, 6 structural formulas entirely replacing α cyclodextrin-PEI600 are as follows:
2., as claimed in claim 1 based on a preparation method for the magnetic transfection reagent of Supramolecular self assembly, it is characterized in that step is as follows:
1) assemble primitive 6 and entirely replace α cyclodextrin-PEI
600synthesis
With N; N-carbonyl dimidazoles (CDI) activates α cyclodextrin; dimethyl sulfoxide (DMSO) (DMSO) solution of α cyclodextrin is slowly added drop-wise to N; in the dimethyl sulphoxide solution of N-carbonyl dimidazoles; react 3 hours under nitrogen protection, obtained product is precipitated in ether, obtain intermediate product 6 and entirely replace α cyclodextrin acyl imidazoles; intermediate product is dissolved in dimethyl sulfoxide (DMSO), is slowly added drop-wise to PEI
600dimethyl sulphoxide solution in, react 5 hours under nitrogen protection, dialysed by reaction solution 72 hours, freeze-drying obtains solid product 6 and entirely replaces α cyclodextrin-PEI
600the mol ratio of α cyclodextrin, N, N-carbonyl dimidazoles, PEI600 is 1:1.2:10, α cyclodextrin-dimethyl sulphoxide solution and N, N-carbonyl dimidazoles-dimethyl sulphoxide solution concentration is 0.1mmol/mL, and PEI600-dimethyl sulphoxide solution concentration is 0.5mmol/mL; Reaction formula is as follows:
2) self assembly magnetic transfection reagent
α cyclodextrin-PEI600 is entirely replaced water-soluble by above-mentioned 6, be made into the solution of 1mmol/mL, the magnetic ball prepared by high-temperature decomposition is dissolved in toluene and is made into 1mg/mL, isopyknic magnetic ball solution and 6 are replaced full the mixing of α cyclodextrin-PEI600 solution, vigorous stirring 8-10 hour, water phase separated, Magneto separate, wash three times, store in aqueous with the concentration of 1mg/mL, obtain target compound magnetic transfection reagent.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111187770A (en) * | 2020-01-08 | 2020-05-22 | 中国地质大学（武汉） | Homogeneous biomolecule nano particle and synthetic method thereof |
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| WO2003038103A1 (en) * | 2001-10-29 | 2003-05-08 | Atso Raasmaja | Method for gene transfection using synergistic combinations of cationic lipids and cationic polymers |
| CN102414116A (en) * | 2009-02-26 | 2012-04-11 | 加利福尼亚大学董事会 | A supramolecular approach for preparation of size controllable nanoparticles |
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| WO2003038103A1 (en) * | 2001-10-29 | 2003-05-08 | Atso Raasmaja | Method for gene transfection using synergistic combinations of cationic lipids and cationic polymers |
| CN102414116A (en) * | 2009-02-26 | 2012-04-11 | 加利福尼亚大学董事会 | A supramolecular approach for preparation of size controllable nanoparticles |
| CN103977772A (en) * | 2014-05-16 | 2014-08-13 | 大连理工大学 | Preparation method of cyclodextrin modified magnetic nano adsorbent and application thereof in hemodialysis adsorption system |
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| MEHDI KHOOBI，等: "Synthesis of polyethyleneimine (PEI) and β-cyclodextrin grafted PEI nanocomposites with magnetic cores for lipase immobilization and esterification", 《JOURNAL OF CHEMICAL TECHNOLOGY AND BIOTECHNOLOGY》 * |
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| CN111187770A (en) * | 2020-01-08 | 2020-05-22 | 中国地质大学（武汉） | Homogeneous biomolecule nano particle and synthetic method thereof |
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