CN102268460A - Method for preparing photo-responsive PEGylation gene transfer system assembled on basis of subject and object - Google Patents
Method for preparing photo-responsive PEGylation gene transfer system assembled on basis of subject and object Download PDFInfo
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- 150000001875 compounds Chemical class 0.000 claims abstract description 6
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Abstract
The invention discloses a method for preparing a photo-responsive PEGylation gene transfer system assembled on the basis of a subject and an object. The method comprises the following steps of: (1) preparing a polyethyleneimine-grafted cyclodextrin solution at concentration of 0.1-1mg/mL and an azobenzene-modified polyethylene glycol solution at concentration of 0.4-2mg/mL from an N-2-hydroxyethyl piperazine-N-2-ethyl sulfonic acid buffer solution; (2) preparing a DNA (Deoxyribonucleic Acid) solution at the concentration of 50-500mug/mL from the buffer solution in the step (1); (3) mixing two solutions in the step (1), wherein the molar ratio of the cyclodextrin included in the polyethyleneimine-grafted cyclodextrin solution to the azobenzene-modified polyethylene glycol is 1:1-1:4, performing ultrasonic treatment for 15 minutes and standing for 1 hour to obtain a super molecular compound solution; and (4) adding the solution prepared by the step (3) into isovolumetric solution of the step (2), eddy-mixing and standing. According to the method disclosed by the invention, the stability of the system in physiological salt solution can be improved. Through photoisomerization of azobenzene, stripping of a PEG shell in an intracellular gene transfer system is realized, and the gene transfection efficiency can be effectively increased.
Description
Technical field
The present invention relates to the preparation method that a kind of photoresponse PEGization gene based on the Subjective and Objective assembling transmits system, it belongs to the preparation method and the technology of the new and effective non-virus gene delivery system of field of gene.
Background technology
Gene therapy is that people's normal gene or medicative gene are imported people's somatic target cell by certain way, with defective or the performance therapeutic action of correcting gene, thereby reaches the biomedical new technology for the treatment of the disease purpose.Because exposed nucleic acid molecule is difficult to stable existence in body fluid, can only realize the expression of low level in vivo.Though virus vector has higher transfection efficiency, problems such as potential toxicity, immunogenicity and target have limited its application in clinical treatment.Exploitation has highly effective and safe and tissue-specific high-biocompatibility non-virus gene delivery system has very important significance.
Cationic polymers becomes one of main developing direction of gene therapy vector owing to have characteristics such as preparation is simple, immunogenicity is low, year gene capacity is big.But this cationoid polymkeric substance assembly is assembled in vivo easily, and is cleared out of in the body by reticuloendothelial system, reduces the gene carrier and ties up to intravital cycling time and influence gene transfection efficient.The gene that preparation polyoxyethylene glycol (PEG) is changed transmits system and can effectively address the above problem, but the PEG shell has hindered dissociating of the interior assemblies of born of the same parents and crossed over the nuclear membrane ability simultaneously, thereby reduces the transfection efficiency of transmission system.The PEGization gene that structure has the environmental response characteristic transmits system, makes it keep stable in circulating in vivo, realizes effective disengaging of PEG shell when reaching target cell, has very important significance.Existing research mostly is that the PEG segment that the mode by chemical bonding will contain sensitive group is incorporated in the gene transmission system, but this method preparation complexity and can influence the ability of genophore association dna molecular.Along with development of fiber technology, light can be realized drug disposition is discharged regulation and control more accurately.The preparation method is simple for the means of supramolecule assembling simultaneously, therefore adopts supramolecule assembling means to prepare novel environmental response type non-virus gene delivery system and has wide application development prospect.
Summary of the invention
The purpose of this invention is to provide the preparation method that a kind of photoresponse PEGization gene based on the Subjective and Objective assembling transmits system.Its step is as follows:
1) adopt N-2-hydroxyethyl piperazine-N-2-ethyl sulfonic acid buffered soln, configuration concentration is that the polymine grafted cyclodextrin solution of 0.1～1 mg/mL and nitrogen benzide that concentration is 0.4～2 mg/mL are modified polyglycol solution;
2) adopt buffered soln in the step 1), configuration concentration is the dna solution of 50～500 μ g/mL;
3) two solution in the step 1) are mixed, the mol ratio that cyclodextrin that the polymine grafted cyclodextrin contains and nitrogen benzide are modified polyoxyethylene glycol is 1:1～1:4, leaves standstill 1 h behind the ultrasonic 15min, obtains super molecular compound solution;
4) solution with the step 3) preparation joins in the solution of isopyknic step 3) configuration, and whirlpool mixes and leaves standstill, and obtains to transmit system based on the photoresponse PEGization gene of Subjective and Objective assembling.
After described photoresponse PEGization gene transmission system based on the Subjective and Objective assembling adopted wavelength to be the long wave ultraviolet light irradiation 10-20 min of 365 nm, the PEG shell can effectively break away from.
The beneficial effect that the present invention is compared with prior art concrete:
1) present method preparation is simple.By the Subjective and Objective effect of nitrogen benzide and cyclodextrin, adopt the supramolecule package technique, the photoresponse PEGization gene that can prepare based on the Subjective and Objective assembling transmits system.
2) non-virus gene delivery system of present method preparation has responsiveness.By the photic isomery of nitrogen benzide, realize effective disengaging that gene transmits system PEG shell in the cell, can effectively improve gene transfection efficient.The responsiveness regulation and control are simple, use for 365 nm long wave uv irradiating for some time got final product.
Description of drawings
Fig. 1 is respectively under 10,20,30,40, the 80 and 100 μ g/mL DNA situation of dissociating for the PEI-CD/DNA assembly at the polyanion heparin concentration.
Fig. 2 is respectively under 10,20,30,40, the 80 and 100 μ g/mL DNA situation of dissociating for the photoresponse PEGization gene transmission system based on Subjective and Objective assembling is PEI-CD/Azo-PEG/DNA at the polyanion heparin concentration.
Fig. 3 is PEI-CD/DNA assembly and the Zeta-potential result of variations of PEI-CD/Azo-PEG/DNA assembly before and after photoresponse.
Fig. 4 is based on relative PEI with the PEI-CD/DNA assembly before and after the photoresponse PEGization gene transmission system response of Subjective and Objective assembling
25kHuman hepatoma HepG2 cell's transfection results of/DNA assembly.
Embodiment
The present invention adopts the supramolecule package technique, the Subjective and Objective by nitrogen benzide and cyclodextrin to and the light isomerism of nitrogen benzide, preparation photoresponse PEGization gene transmission system.Its step is as follows: 1) adopt N-2-hydroxyethyl piperazine-N-2-ethyl sulfonic acid buffered soln, configuration concentration is that the polymine grafted cyclodextrin solution of 0.1～1 mg/mL and nitrogen benzide that concentration is 0.4～2 mg/mL are modified polyglycol solution; 2) adopt buffered soln in the step 1), configuration concentration is the dna solution of 50～500 μ g/mL; 3) two solution in the step 1) are mixed, the mol ratio that cyclodextrin that the polymine grafted cyclodextrin contains and nitrogen benzide are modified polyoxyethylene glycol is 1:1～1:4, leaves standstill 1 h behind the ultrasonic 15min, obtains super molecular compound solution; 4) solution with the step 3) preparation joins in the solution of isopyknic step 3) configuration, and whirlpool mixes and leaves standstill, and obtains to transmit system based on the photoresponse PEGization gene of Subjective and Objective assembling.Have the Subjective and Objective effect between nitrogen benzide and the cyclodextrin, azobenzene molecule can be realized the transformation of cis-trans isomerism by the regulation and control of light simultaneously.There are greatest differences in the nitrogen benzide of cis-trans isomerism and the binding constant of cyclodextrin: trans nitrogen benzide is a hydrophobicity, can form inclusion compound with cyclodextrin; And cis-azobenzene is a wetting ability, can not form inclusion compound with nitrogen benzide.Therefore utilize this Subjective and Objective effect to the photic isomerism of nitrogen benzide, the polyoxyethylene glycol segment can be incorporated on the polyethyleneimine: amine molecule, obtain photoresponse PEGization gene transmission system based on the Subjective and Objective assembling.In target tissue, utilize the regulation and control of light to realize the trans cis that changes into of nitrogen benzide, cause nitrogen benzide and the effect of cyclodextrin Subjective and Objective to disappear, realize the assembling of separating of PEG, thereby realize assembly effectively dissociating and the high-level efficiency transfection in born of the same parents.
Embodiment 1:
Configuration NaCl concentration is that 20mM, pH are 7.4, concentration is N-2-hydroxyethyl piperazine-N-2-ethyl sulfonic acid (Hepes) buffered soln of 20mM.Adopt this buffered soln, configuration concentration is that polymine grafted cyclodextrin (PEI-CD, the PEI molecular weight 25000) solution of 1 mg/mL and nitrogen benzide that concentration is 2 mg/mL are modified polyoxyethylene glycol (Azo-PEG, PEG molecular weight 5000) solution.Select green fluorescent protein reporter gene pEGFP(4733bp for use) be goal gene, configuration concentration is the pEGFP solution of 500 μ g/mL.Get above-mentioned PEI-CD solution of 162 μ l and the above-mentioned Azo-PEG solution of 88 μ l, the mol ratio of CD and Azo-PEG is 1:1 among the PEI-CD, leaves standstill 1 h behind ultrasonic 15 min.Mix with 250 μ l dna solution equal-volume whirlpools then and leave standstill, prepare photoresponse PEGization gene transmission system based on the Subjective and Objective assembling.
Embodiment 2:
Adopting NaCl concentration is that 20 mM, pH=7.4, concentration are N-2-hydroxyethyl piperazine-N-2-ethyl sulfonic acid (Hepes) buffered soln of 20mM, configuration concentration is the polymine grafted cyclodextrin (PEI-CD of 0.1 mg/mL, PEI molecular weight 25000) solution and concentration are nitrogen benzide modification polyoxyethylene glycol (Azo-PEG, the PEG molecular weight 5000) solution of 1.5 mg/mL.Configuration concentration is the pEGFP solution of 50 μ g/mL.Get above-mentioned PEI-CD solution of 162 μ l and the above-mentioned Azo-PEG solution of 88 μ l, the mol ratio of CD and Azo-PEG is 1:4 among the PEI-CD, leaves standstill 1 h behind ultrasonic 15 min.Mix with 250 μ l dna solution equal-volume whirlpools then and leave standstill, prepare photoresponse PEGization gene transmission system based on the Subjective and Objective assembling.
Embodiment 3:
Adopting NaCl concentration is that 20 mM, pH=7.4, concentration are N-2-hydroxyethyl piperazine-N-2-ethyl sulfonic acid (Hepes) buffered soln of 20mM, configuration concentration is the polymine grafted cyclodextrin (PEI-CD of 1 mg/mL, PEI molecular weight 25000) solution and concentration are nitrogen benzide modification polyoxyethylene glycol (Azo-PEG, the PEG molecular weight 5000) solution of 0.4 mg/mL.Configuration concentration is the pEGFP solution of 50 μ g/mL.Get above-mentioned PEI-CD solution of 22 μ l and the above-mentioned Azo-PEG solution of 228 μ l, the mol ratio of CD and Azo-PEG is 1:2 among the PEI-CD, leaves standstill 1 h behind ultrasonic 15 min.Mix with 250 μ l dna solution equal-volume whirlpools then and leave standstill, prepare photoresponse PEGization gene transmission system based on the Subjective and Objective assembling.
Embodiment 4:
Adopting NaCl concentration is that 20 mM, pH=7.4, concentration are N-2-hydroxyethyl piperazine-N-2-ethyl sulfonic acid (Hepes) buffered soln of 20mM, configuration concentration is the polymine grafted cyclodextrin (PEI-CD of 0.5 mg/mL, PEI molecular weight 25000) solution and concentration are nitrogen benzide modification polyoxyethylene glycol (Azo-PEG, the PEG molecular weight 5000) solution of 1 mg/mL.Configuration concentration is the pEGFP solution of 50 μ g/mL.Get above-mentioned PEI-CD solution of 54 μ l and the above-mentioned Azo-PEG solution of 196 μ l, the mol ratio of CD and Azo-PEG is 1:4 among the PEI-CD, leaves standstill 1 h behind ultrasonic 15 min.Mix with 250 μ l dna solution equal-volume whirlpools then and leave standstill, prepare photoresponse PEGization gene transmission system based on the Subjective and Objective assembling.
Embodiment 5:
Adopting NaCl concentration is that 20 mM, pH=7.4, concentration are N-2-hydroxyethyl piperazine-N-2-ethyl sulfonic acid (Hepes) buffered soln of 20mM, configuration concentration is the polymine grafted cyclodextrin (PEI-CD of 1 mg/mL, the PEI molecular weight is 25000) solution and concentration is that the nitrogen benzide of 2 mg/mL is modified polyoxyethylene glycol (Azo-PEG, PEG molecular weight are 5000) solution.Configuration concentration is the pEGFP solution of 100 μ g/mL.Get above-mentioned PEI-CD solution of 54 μ l and the above-mentioned Azo-PEG solution of 196 μ l, the mol ratio of CD and Azo-PEG is 1:4 among the PEI-CD, leaves standstill 1 h behind ultrasonic 15 min.Mix with isopyknic dna solution whirlpool then and leave standstill 1 h, obtain photoresponse PEGization gene transmission system based on the Subjective and Objective assembling.
Embodiment 6:
The photoresponse PEGization gene based on the Subjective and Objective assembling of pressing embodiment 1 preparation transmits system, adopts dynamic light scattering and the stability of projection electron microscopy study assembly under physiology salt condition to find that its stability has raising to a great extent.Because the PEG segmental exists, its Zeta-potential obviously reduces.
Embodiment 7:
The photoresponse PEGization gene based on the Subjective and Objective assembling of pressing embodiment 2 preparations transmits system, a large amount of Anionic Protein matter environment in the analogue body, under the condition that the finite concentration polyanion exists, because the surface PEG segmental exists, the assembling physical efficiency is effectively protected dna molecular, avoids the destruction of polyanion; Stability in physiology salt condition obviously improves.Can prolong cycling time in vivo.
Embodiment 8:
The photoresponse PEGization gene based on the Subjective and Objective assembling of pressing embodiment 3 preparations transmits system, because shielding effect of PEG segmental and volume excluding effect, the Zeta-potential of assembly significantly reduces.Adopt gel electrophoresis to measure PEI-CD/DNA and PEI-CD/Azo-PEG/DNA assembly and be respectively under 10,20,30,40, the 80 and 100 μ g/mL DNA situation of dissociating at heparin concentration.Because the PEG segmental exists, tie up under the higher heparin concentration (40 μ g/mL) based on the photoresponse PEGization gene carrier of Subjective and Objective assembling and just to dissociate, and the PEI-CD/DNA assembly promptly dissociates under low concentration (10 μ g/mL), and the result as shown in Figure 1, 2.
Embodiment 9:
The photoresponse PEGization gene based on the Subjective and Objective assembling of pressing embodiment 4 preparations transmits system, adopts the Zeta-potential analyser to study its optical Response.At first the photoresponse PEGization gene carrier based on the Subjective and Objective assembling with the embodiment of the invention 4 preparations ties up to irradiation 10-20 min under the 365 nm long wave ultraviolet light sources.Because the PEG segmental exists, the photoresponse PEGization gene of assembling based on Subjective and Objective transmits the remarkable reduction of the Zeta-potential of system than PEI-CD/DNA assembly.After the illumination, the nitrogen benzide transconfiguration changes cis into, the Subjective and Objective effect of itself and cyclodextrin is eliminated, the PEG segment is deviate from, surface potential returns to the level near the PEI-CD/DNA assembly, illustrates that the photoresponse PEGization gene carrier cording based on the Subjective and Objective assembling of the embodiment of the invention 4 preparations has good photoresponse.The result as shown in Figure 3.
Embodiment 10:
The photoresponse PEGization gene transmission system based on the Subjective and Objective assembling by embodiment 5 preparations is carried out the gene transfection experiment.At first the photoresponse PEGization gene based on the Subjective and Objective assembling with the embodiment of the invention 5 preparations transmits system and PEI-CD/DNA assembly and PEI
25k/ DNA assembly in human hepatoma HepG2 cell's endocytosis 4.5 h, shines 10-20 min with the PEGization group then under distance ultraviolet source certain distance, behind transfection 48 h, adopt the quantitative analysis of cell streaming instrument.The PEG segmental exists, and being difficult in cell takes place effectively to dissociate arrives nucleus and make transfection efficiency reduce; PEI-CD/DNA assembly after the illumination response makes it dissociate easily and passes through nuclear membrane that transfection efficiency significantly improves because the PEG segmental is deviate from.The result as shown in Figure 4.
Claims (2)
1. preparation method based on the photoresponse PEGization gene transmission system of Subjective and Objective assembling is characterized in that its step is as follows:
1) adopt N-2-hydroxyethyl piperazine-N-2-ethyl sulfonic acid buffered soln, configuration concentration is that the polymine grafted cyclodextrin solution of 0.1～1 mg/mL and nitrogen benzide that concentration is 0.4～2 mg/mL are modified polyglycol solution;
2) adopt buffered soln in the step 1), configuration concentration is the dna solution of 50～500 μ g/mL;
3) two solution in the step 1) are mixed, the mol ratio that cyclodextrin that the polymine grafted cyclodextrin contains and nitrogen benzide are modified polyoxyethylene glycol is 1:1～1:4, leaves standstill 1 h behind the ultrasonic 15min, obtains super molecular compound solution;
4) solution with the step 3) preparation joins in the solution of isopyknic step 3) configuration, and whirlpool mixes and leaves standstill, and obtains to transmit system based on the photoresponse PEGization gene of Subjective and Objective assembling.
2. a kind of photoresponse PEGization gene based on the Subjective and Objective assembling according to claim 1 transmits the preparation method of system, after it is characterized in that described photoresponse PEGization gene transmission system based on the Subjective and Objective assembling adopts wavelength to be the long wave ultraviolet light irradiation 10-20 min of 365 nm, the PEG shell can effectively break away from.
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Cited By (3)
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| CN104109248A (en) * | 2014-06-20 | 2014-10-22 | 南开大学 | Aggregate based on bridged biscyclodextrin subject and object induced aggregation construction and application thereof |
| CN107434851A (en) * | 2017-09-06 | 2017-12-05 | 阿里生物新材料（常州）有限公司 | A kind of preparation method of Photosensitive hydrogel |
| CN114887604A (en) * | 2022-04-27 | 2022-08-12 | 吉林大学 | Preparation method and application of photoresponse type bifunctional magnetic material |
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2011
- 2011-07-20 CN CN 201110203677 patent/CN102268460A/en active Pending
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| 《 Chem. Commun.》 20100513 Ke Peng, et al. Light controlled protein release from a supramolecular hydrogel 4094-4096 2 第46卷, * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104109248A (en) * | 2014-06-20 | 2014-10-22 | 南开大学 | Aggregate based on bridged biscyclodextrin subject and object induced aggregation construction and application thereof |
| CN107434851A (en) * | 2017-09-06 | 2017-12-05 | 阿里生物新材料（常州）有限公司 | A kind of preparation method of Photosensitive hydrogel |
| CN114887604A (en) * | 2022-04-27 | 2022-08-12 | 吉林大学 | Preparation method and application of photoresponse type bifunctional magnetic material |
| CN114887604B (en) * | 2022-04-27 | 2023-01-31 | 吉林大学 | Preparation method and application of photoresponse type bifunctional magnetic material |
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Application publication date: 20111207 |