CN108956540A - A kind of SPR method of quick screening charge reversal type cationic gene carriers - Google Patents
A kind of SPR method of quick screening charge reversal type cationic gene carriers Download PDFInfo
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- CN108956540A CN108956540A CN201810406862.2A CN201810406862A CN108956540A CN 108956540 A CN108956540 A CN 108956540A CN 201810406862 A CN201810406862 A CN 201810406862A CN 108956540 A CN108956540 A CN 108956540A
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Abstract
The invention discloses a kind of SPR methods of quickly screening charge reversal type cationic gene carriers, main innovation is can to monitor load and releasability of the technical characterstic of structure of matter variation come on-line evaluation charge reversal type cationic gene carriers to DNA online, in real time using SPR technique, a kind of method for establishing external quickly screening-gene carrier.Intracorporal reactive oxygen species are simulated with hydrogen peroxide or enzyme etc., online study carrier is under the reactive oxygen species effect of various concentration to the releasing effect of DNA.It is intended to help to evaluate the performance of genophore, shortens the genophore performance evaluation time, the method for establishing the excellent genophore of quick screenability, the research and development of accelerated gene drug.For this method without marking sample, experimental procedure is simple, and detection is fast, visual phenomenon, is a kind of ideal research method of the excellent genophore of external Fast Evaluation, screenability.
Description
Technical field
The present invention relates to the appraisal procedure technical fields of genophore performance, more particularly to a kind of quickly screening charge reversal
The SPR method of type cationic gene carriers.
Background technique
Gene therapy is the effective ways of current treatment genetic disease, but DNA volume is big and the property shadow of negative electrical charge
The intake of cell membrane is rung, and the dissociative DNA being injected intravenously can be by the serum nuclease fast degradation in blood, disadvantage mentioned above
Gene therapy is all limited in clinical application.Gene therapy there are aiming at the problem that, the positive searching of people is effective
Genophore carries out drug delivery.Non-viral gene vector is due to good biocompatibility, safety and can be large quantities of
The advantages that amount production, is concerned by people.Cation carrier is since it is with hypotoxicity and transfection effect in numerous genophores
The high feature of rate is compared to the research for being more broadly useful for gene therapy for other non-viral gene vectors.But it is cationic
Carrier/DNA composite nanoparticles be it is thermodynamically stable, be typically difficult to dissociate, this is greatly hindered in cell to DNA
Release, influence transfection efficiency.Then have researcher have devised can reactive oxygen species effect under occur from positive charge to
The cation carrier of negative electrical charge transformation, this kind of carrier occur fastly in the relatively high sick cell of tumour cell isoreactivity oxygen cluster
The charge reversal of speed, released dna, to solve the problems, such as that cation carrier transfection efficiency is low.However, charge reversal type cation
If carrier thermal stability and carried out in cell or tissue to the load of DNA and releasing research, higher reality is not only needed
Condition and complicated experimental procedure are tested, and cannot it is monitored and be observed online, in real time, increases experimental study
Time cannot evaluate the performance of carrier in shorter time again.Therefore, establish one kind can it is online in vitro, quickly,
Real-Time Evaluation carrier loads DNA and the research method of releasability is for shortening the genophore performance evaluation time, establishes fast
The method of the excellent carrier of fast screenability, the research and development of accelerated gene drug are significant.
Surface plasma body resonant vibration instrument (SPR) is due to, high sensitivity label-free with determinand, real-time monitoring reaction, back
The advantages that scape interference is small is widely used in the fields such as physics, chemistry, biology and life science.The present invention can using SPR technique
On online fast slowdown monitoring film the characteristics of structure of matter variation, a kind of external online, Fast Evaluation cationic gene carriers are established
The method for sensing of performance.
Summary of the invention
Technical problems based on background technology, the present invention provides a kind of label-free, sensitive, online research charges
The SPR method that counter-rotative type cationic gene carriers deliver DNA and discharge can be carried quickly in online detection with this method
The combination and dissociation of body and any DNA solve to influence DNA release and the thermodynamic stability of the cation carrier of transfection efficiency are asked
Topic.This method have the advantages that it is simple, quickly, can be with on-line checking.
Why the present invention can be realized above-mentioned purpose, it is characterised in that:
1. load and releasability of the on-line evaluation charge reversal type cationic gene carriers to DNA carry out just carrier
Step evaluation and screening.
2. on-line evaluation carrier/DNA composite nanoparticle is in the fixation on golden film surface and in various concentration active oxygen object
It is that application of the carrier in cell or living body provides safeguard to the releasability of DNA under matter effect.
3. the available the method for the performance of the charge reversal type cationic gene carriers based on different principle is evaluated, from
And carrier is screened.
Technical scheme is as follows:
A kind of SPR method of quick screening charge reversal type cationic gene carriers, comprising the following steps:
A, various concentration is injected in the golden film surface flow for having modified a certain concentration charge reversal type cationic gene carriers
DNA, the difference type surface plasma base member voluntarily built using laboratory resonated instrument, and on-line real time monitoring carrier is to DNA
Capture situation, the variation of lower spr signal, load capacity of the evaluation carrier to DNA are acted on according to various concentration DNA；
B, the carrier for synthesizing cation carrier and DNA under the conditions of certain N/P ratio/DNA complex nanometer granule；
C, a certain concentration carrier/DNA complex nanometer granule golden film surface flow injection various concentration is being modified
Hydrogen peroxide acts on the variation of lower spr signal, release conditions of the evaluation nanoparticle to DNA according to various concentration hydrogen peroxide.
Preferably, the hydrogen peroxide could alternatively be any reactive oxygen species.Such as peroxide, biological enzyme, artificial
Enzyme, oxygen radical etc..
Preferably, the available the method for the performance of the charge reversal type cationic gene carriers based on different principle is sieved
Choosing.
The SPR method of quick screening charge reversal type cationic gene carriers of the invention, on-line evaluation charge reversal type
Load and releasability of the cationic gene carriers to DNA carry out preliminary assessment and screening to carrier；On-line evaluation carrier/DNA
Composite nanoparticle the fixation on golden film surface and under the effect of various concentration reactive oxygen species to the releasability of DNA, be
Application of the carrier in cell or living body, which provides, to be ensured early period；Charge reversal type cationic gene carriers based on different principle
The available the method for performance evaluated, to be screened to carrier.
The invention has the beneficial effects that:
1) the SPR online study method that charge reversal type cationic gene carriers load DNA and discharge, without marking sample
Product, easy to operate, testing result quicklook.
2) by this method can online, quickly study charge reversal type cationic gene carriers to the load of DNA with
And the release action under reactive oxygen species effect to DNA, more intuitive evaluation is carried out to the performance of carrier.Use hydrogen peroxide
Or enzyme etc. simulates intracorporal reactive oxygen species, release of the online study carrier under the reactive oxygen species effect of various concentration to DNA
Effect.
3) this method is suitable for the research of the charge reversal type cationic gene carriers performance based on different principle, is a kind of
Simply, quickly online study charge reversal type cation carrier grinds the method for delivery and the release of drug for pharmaceutical carrier
Study carefully and provide a kind of new pharmaceutical carrier evaluation method, facilitates the quick screening of pharmaceutical carrier.
Present invention hydrogen peroxide or enzyme etc. simulate intracorporal reactive oxygen species, activity of the online study carrier in various concentration
To the releasing effect of DNA under oxygen species effect.It is intended to help to evaluate the performance of genophore, when shortening genophore performance evaluation
Between, the method for establishing the excellent carrier of quick screenability, the research and development of accelerated gene drug.This method is real without marking sample
It is simple to test step, detection is fast, and visual phenomenon is a kind of ideal research method of external Fast Evaluation genophore performance.
Detailed description of the invention
Fig. 1: after immobilization carrier, the spr signal figure of the DNA of flow injection various concentration；
Fig. 2: the carrier of synthesis/DNA composite nanoparticle SEM phenogram；
Fig. 3: to the spr signal figure of DNA release after immobilization carrier/DNA composite nanoparticle in golden film.
Specific embodiment
Embodiment 1:
In the golden film surface flow injection various concentration for having modified a certain concentration charge reversal type cationic gene carriers
Hydrogen peroxide shows plasma primitive resonance instrument, on-line real time monitoring carrier pair using the difference type that laboratory is voluntarily built
The capture situation of DNA.Variation with acting on lower spr signal according to various concentration DNA, load capacity of the evaluation carrier to DNA.Accordingly
Spr signal curve graph it is as shown in Figure 1.
Embodiment 2:
By carrier and DNA, synthetic vectors/DNA complex nanometer granule is sub under the conditions of certain N/P ratio first, is repairing later
The hydrogen peroxide of the golden film surface flow injection various concentration of a certain concentration nanoparticle, such as 10 are adornd-5M-10-3M, online
The spr signal variation under the effect of various concentration hydrogen peroxide is observed, studies nanoparticle to DNA by comparing the size of signal
Release and concentration of hydrogen peroxide relationship, thus online, intuitively evaluation pharmaceutical carrier different activities oxygen concentration cell
In to the releasability of genomic medicine.Corresponding nanoparticle phenogram is as shown in Figure 2；Spr signal curve is as shown in Figure 3.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (6)
1. a kind of SPR method of quickly screening charge reversal type cationic gene carriers, which comprises the following steps:
A, in the golden film surface flow injection various concentration for having modified a certain concentration charge reversal type cationic gene carriers
DNA, the difference type surface plasma base member resonance instrument voluntarily built using laboratory, on-line real time monitoring carrier is to DNA's
Situation is captured, the variation of lower spr signal, load capacity of the evaluation carrier to DNA are acted on according to various concentration DNA；
B, by cation carrier and DNA, synthetic vectors/DNA complex nanometer granule is sub under the conditions of certain N/P ratio；
C, in the peroxide for having modified a certain concentration carrier/DNA complex nanometer granule golden film surface flow injection various concentration
Change hydrogen, the variation of lower spr signal, release conditions of the evaluation nanoparticle to DNA are acted on according to various concentration hydrogen peroxide.
2. the SPR method of quickly screening charge reversal type cationic gene carriers as described in claim 1, which is characterized in that
The hydrogen peroxide could alternatively be any reactive oxygen species.
3. the SPR method of quickly screening charge reversal type cationic gene carriers as claimed in claim 2, which is characterized in that
The reactive oxygen species can be peroxide, biological enzyme, artificial enzyme, any one in oxygen radical.
4. the SPR method of quickly screening charge reversal type cationic gene carriers as described in claim 1, which is characterized in that
This method, load and releasability of the on-line evaluation charge reversal type cationic gene carriers to DNA, tentatively comments carrier
Valence and screening.
5. the SPR method of quickly screening charge reversal type cationic gene carriers as described in claim 1, which is characterized in that
This method on-line evaluation carrier/DNA composite nanoparticle is made in the fixation on golden film surface and in various concentration reactive oxygen species
It is that application of the carrier in cell or living body provides guarantee early period with the lower releasability to DNA.
6. the SPR method of quickly screening charge reversal type cationic gene carriers as described in claim 1, which is characterized in that
The available this method of the performance of charge reversal type cationic gene carriers based on different principle is evaluated, thus to carrier into
Row screening.
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| CN104306987A (en) * | 2014-09-24 | 2015-01-28 | 北京化工大学 | Method for constructing multifunctional gene treatment vectors based on gold nanoparticles |
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|---|---|---|---|---|
| US6218112B1 (en) * | 1996-12-23 | 2001-04-17 | Cobra Therapeutics Limited | Optimization of gene delivery and gene delivery system |
| CN1500149A (en) * | 2001-02-05 | 2004-05-26 | Methods and kits for identifing scavengers of reactive oxygen species (Ros) | |
| CN103197081A (en) * | 2013-04-17 | 2013-07-10 | 无锡优创生物科技有限公司 | Protein chip and preparation and detection method thereof |
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| CN104306987A (en) * | 2014-09-24 | 2015-01-28 | 北京化工大学 | Method for constructing multifunctional gene treatment vectors based on gold nanoparticles |
| CN106645038A (en) * | 2016-12-27 | 2017-05-10 | 中国科学院大学 | Quantitative detection method of O-GlcNAc |
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