CN111982999B - Detection method of small interference RNA loaded liposome in external preparation - Google Patents
Detection method of small interference RNA loaded liposome in external preparation Download PDFInfo
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- CN111982999B CN111982999B CN202010844109.9A CN202010844109A CN111982999B CN 111982999 B CN111982999 B CN 111982999B CN 202010844109 A CN202010844109 A CN 202010844109A CN 111982999 B CN111982999 B CN 111982999B
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- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
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Abstract
The invention relates to the field of biological agent detection, in particular to a detection method of a loaded small interfering RNA liposome in an external preparation. The method for detecting the small interfering RNA liposome loaded in the cosmetic or skin external preparation can simply, conveniently and low-cost detect whether the small interfering RNA liposome loaded in the cosmetic or skin external preparation is contained or not, can detect the binding state of the small interfering RNA in the cosmetic or skin external preparation and the liposome, has the characteristics of high sensitivity, simplicity and low cost, has good practical value, and provides powerful technical support for popularization of the cosmetic or skin external preparation containing the small interfering RNA liposome.
Description
Technical Field
The invention relates to the technical field of biological agent detection, in particular to a detection method of a loaded small interference RNA liposome in an external preparation.
Background
The small interfering RNA (Small interfering RNA, siRNA) is a small double-stranded RNA molecule which contains about 20-25 bases and can form an RNA-induced silencing complex (RNA-induced silencing complex, RISC) with endonuclease, exonuclease, helicase and the like, so as to trigger sequence-specific post-transcriptional gene silencing phenomenon started by double-stranded RNA. The small interfering RNA is mainly characterized in that the small interfering RNA can down regulate or shut down the expression of specific genes, has the characteristics of strong specificity and high efficiency, and can silence certain genes which can cause diseases. Has become a potential medicine for treating a plurality of diseases and is also applied to cosmetics or external preparations for skin in a small amount. The liposome nanoparticle drug delivery system loaded with the small interfering RNA is a positively charged nanoparticle formed by self-assembling a cationic liposome or chitosan modified cationic liposome with the chemically synthesized small interfering RNA in an aqueous solution. The small interfering RNA loaded liposome is added into a cosmetic or skin external preparation, which belongs to the brand new technical field, so the detection of the small interfering RNA loaded in the cosmetic or skin external preparation and the detection of the combined state of the small interfering RNA and the liposome in the cosmetic or skin external preparation become the difficult problems to be solved.
The current detection methods for siRNA mainly comprise gel electrophoresis, PCR quantitative method, intrusion detection and ELISA quantitative detection method. The gel electrophoresis method is generally used for detecting pure siRNA with known band size, and the siRNA is dyed by dye, but when small interfering RNA liposome is added into cosmetics or external skin dosage forms, the small interfering RNA is added in small quantity, and the small interfering RNA content is low, and the color of the cosmetics is interfered, so that the sensitivity of the conventional gel electrophoresis method is low, fluorescence is difficult to identify, and the binding state of the small interfering RNA in cosmetics and the liposome is difficult to distinguish.
The loaded small interfering RNA liposome is a positively charged nanoparticle formed by self-assembling a cationic liposome or chitosan modified cationic liposome with chemically synthesized small interfering RNA in an aqueous solution. The small interfering RNA loaded liposome is added into a cosmetic or skin external preparation, which belongs to the brand new technical field, so the detection of the small interfering RNA loaded in the cosmetic or skin external preparation and the detection of the combined state of the small interfering RNA and the liposome in the cosmetic or skin external preparation become the difficult problems to be solved.
The current detection methods for siRNA mainly comprise gel electrophoresis, PCR quantitative method, intrusion detection and ELISA quantitative detection method. The gel electrophoresis method is generally used for detecting pure siRNA with known band size, and the siRNA is dyed by dye, but when small interfering RNA liposome is added into cosmetics or external skin dosage forms, the small interfering RNA is added in small amount, the small interfering RNA content is low, and the color of the cosmetics is interfered, so that the sensitivity of the conventional gel electrophoresis method is low, fluorescence is difficult to identify, and the binding state of the small interfering RNA in the cosmetics and the liposome is difficult to distinguish.
Disclosure of Invention
The invention aims to solve the technical problems that: in order to solve the problems in the prior art, an improved detection method for small interfering RNA loaded liposomes in external preparations is provided, which solves the problems in the prior art.
The technical scheme adopted for solving the technical problems is as follows: a detection method of small interfering RNA loaded liposome in an external preparation comprises the following steps:
(1) Mixing the external preparation to be tested with DEPC sterile water, centrifuging, and taking out a supernatant;
(2) Dyeing the lower supernatant in step (1) with a dye R, NEB #b7024 violet gel loading dye, respectively;
(3) And (3) performing agarose gel electrophoresis after staining, and judging whether the small interfering RNA liposome loaded and the encapsulation state of the small interfering RNA are contained according to the banding condition by taking the small interfering RNA as a control group.
Further, for the purpose of compounding and mixing, the mixing ratio of the external preparation and the DEPC sterile water in the step (1) is 1:1 to 1:5.
further, in order to ensure the centrifugal efficiency, the rotational speed of the centrifugation in the step (1) is 3000-13000 rpm, and the centrifugation time is 1-30 min.
Further, for the purpose of co-dyeing, the dye R in the step (2) is 0.1-10 mM EDTANa2, 0.01-1 mM bromophenol blue, 0.1-5% glycerol and 0.1-5% Ficol400, and is dissolved in DEPC sterile water.
Further, for convenience of use, the type of the purple gel loading dye described in step (2) is neb#b7024.
NEB#B7024 purple gel-like dye has a phenomenon that small interfering RNA in a self-assembled state with liposome is separated from the assembled state, while dye R does not have the phenomenon, the loaded small interfering RNA liposome is taken as a positive control in the electrophoresis process, and the encapsulation state of the small interfering RNA in a cosmetic or skin external preparation containing the loaded small interfering RNA liposome is judged according to the condition of the strip.
The beneficial effects of the invention are as follows:
(1) The invention utilizes the influence difference of different dyes on the self-assembly state of the small interfering RNA, combines the centrifugation and dilution methods to reduce the color interference of cosmetics, and designs a simple and quick method for detecting the combination state of the small interfering RNA and the liposome in the cosmetics or skin external preparation;
(2) The invention further discovers that the assembly state of the small interfering RNA and the liposome cannot be influenced by the mixing of DEPC sterile water and the small interfering RNA liposome, and the DEPC sterile water and the small interfering RNA liposome can be detected by the method even if the added amount of the loaded small interfering RNA liposome is diluted to be very small, so that the accuracy is high and the sensitivity is good;
(3) The detection method of the loaded small interfering RNA liposome is simple, convenient and low in cost, can detect the combination state of small interfering RNA and the liposome in the cosmetic or skin external preparation, has good practical value, and provides powerful technical support for popularization of the cosmetic or skin external preparation containing the small interfering RNA liposome.
Drawings
The invention will be further described with reference to the drawings and examples.
FIG. 1 is a graph showing the result of electrophoresis of loaded small interfering RNA liposomes under the action of different dyes;
FIG. 2 is a graph of electrophoresis results of loaded small interfering RNA liposomes after centrifugation;
FIG. 3 is a graph of electrophoresis results of loaded small interfering RNA liposomes at different water dilutions;
FIG. 4 is an agarose gel electrophoresis chart after staining in example 4;
FIG. 5 is an agarose gel electrophoresis pattern after staining in example 5;
FIG. 6 is an agarose gel electrophoresis pattern after staining in example 6.
Detailed Description
The present invention will be described in further detail below with reference to specific implementation methods and application examples of the embodiments, and with reference to implementation data. It should be understood that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way. The terms used in the present invention generally have meanings and operation methods commonly understood by those of ordinary skill in the art unless otherwise indicated. Unless otherwise specified, all reagents involved in the examples of the present invention are commercially available products and are commercially available.
Reagent:
DEPC sterile water: adding 1ml of DEPC water into 1L of ultrapure water, stirring and mixing in a fume hood overnight, and sterilizing at 121 ℃ for 20min for standby;
the dye R is prepared according to the following proportion: 0.1-10 mM EDTANa2, 0.01-1 mM bromophenol blue and 0.1-5% glycerol, 0.05-5% Ficol400, dissolved in DEPC sterile water;
preparation of 1×tae buffer: 0.0372g of EDTA Na2 2H2O,0.242g Tris,0.0571ml glacial acetic acid is dissolved in 400ml of DEPC sterile water to 500ml of constant volume for preparation;
preparation of 1% agarose gel: 0.3g Agarose (Agarose) was added to 30ml 1 XTAE buffer, and after microwave dissolution, 3. Mu. lEB dye was added and mixed well to form a gel.
Example 1 as shown in fig. 1:
the embodiment verifies the assembled state of the loaded small interfering RNA liposome and the liposome under the action of different dyes.
Loaded small interfering RNA liposomes were stained with dye R (self-assembled, 1.2mM EDTANa2, 0.037mM bromophenol blue and 2.5% glycerol, 0.1% Ficol400, dye concentration 10-fold) and NEB#B7024 purple gel loading dye (available from NEB Co.) at 1-fold final concentrations, respectively. And gel electrophoresis is carried out on the stained loaded small interfering RNA liposome and a control group by using the small interfering RNA control group respectively. FIG. 1 is a graph showing the result of electrophoresis of loaded small interfering RNA liposomes under the action of different dyes; in the figure, 1 is a small interfering RNA control group, A is dye R staining, B is NEB#B7024 purple gel dye-loading staining. The dye R-dyed loaded small interfering RNA liposomes have obvious bright colors in the loading wells, no bands are in the agarose gel, and the loaded small interfering RNA liposomes dyed by the NEB#B7024 purple gel loading dye have no bright fluorescence in the loading wells, but instead have bands in the gel. Therefore, the dye R does not damage the assembly state of the small interfering RNA in the loaded small interfering RNA liposome and the liposome, and has no influence on the assembly state, while the NEB#B7024 purple gel loading dye can untangling the assembly state of the small interfering RNA in the loaded small interfering RNA liposome and the liposome, so that bright fluorescence does not exist in a loading hole of an electrophoresis result, and a band appears in the gel.
Example 2 as shown in fig. 2:
this example demonstrates the effect of different centrifugation speeds on the self-assembled state of small interfering RNAs in loaded small interfering RNA liposomes.
6 parts of 100. Mu.l of small interfering RNA liposomes were taken and separated into two groups, each group was centrifuged at 3000rpm, 8000rpm, and 13000rpm for 30min at 4℃with the addition of dye R (self-assembled, 1.2mM EDTANa2, 0.037mM bromophenol blue and 2.5% glycerol, 0.1% Ficol400, at a dye concentration of 10-fold) to one group and NEB#B7024 purple gel-loading dye (available from NEB Co.) to the other group at a final dye concentration of 1-fold. And the small interference RNA control group is used for carrying out electrophoresis on the loaded small interference RNA liposome after dye dyeing
FIG. 2 is a graph of electrophoresis results of loaded small interfering RNA liposomes after centrifugation; in the figure, 1 is a small interference RNA control, 2-4 are respectively 3000rpm, 8000rpm, 13000rpm after centrifugation of dye R dyeing group, 5-7 is 3000rpm, 8000rpm, 13000rpm after centrifugation of NEB#B7024 purple gel on the dye group.
The dye R has obvious bright color in each sample loading hole in the dye R staining group, but no band exists in agarose gel, which indicates that the small interfering RNA and the liposome are still in an assembled state, the small interfering RNA cannot be electrophoresed into the gel, and the small interfering RNA and the liposome cannot be in a disassembled state due to the addition of the dye R and the change of the centrifugal rate. Whereas each loaded small interfering RNA liposome stained with neb#b7024 purple gel loading dye, small interfering RNA and liposomes were disassembled, regardless of the centrifugation rate, and small interfering RNA bands appeared in each lane. Therefore, the dye R and centrifugation at a certain speed can not influence the assembly state of small interfering RNA in the loaded small interfering RNA liposome and the liposome, and the NEB#B7024 purple gel loading dye can release the assembly state of small interfering RNA in the loaded small interfering RNA liposome and the liposome.
Example 3 as shown in fig. 3:
this example verifies that water dilution affects loaded small interfering RNA liposomes on the assembled state of small interfering RNA and liposomes at different centrifugation speeds.
DEPC sterile water dilution load small interfering RNA liposomes (produced by Digitalis Gene technologies (Nanj) Inc.), dilution fold 1,2 and 3 times; the diluted loaded small interfering RNA liposomes were centrifuged at 3000rpm and 13000rpm for 20min, respectively, and then stained with the dye R and NEB#B7024 purple gels, the dye concentration was the same as in example 2, and each loaded small interfering RNA liposome stained was electrophoresed with a small interfering RNA control. FIG. 3 is a graph of electrophoresis results of loaded small interfering RNA liposomes at different water dilutions; in the figure, 1 is a small interference RNA control group, 2, 4, 6 is diluted 1 times, 2 times, 3 times of R staining group, 3, 5, 7 is diluted 1 times, 2 times, 3 times of NEB#B7024 purple gel sample dye staining group.
The agarose gel in the control group has obvious strips, and the sample loading hole has no bright color; the loaded small interfering RNA liposome added with dye R has obvious bright color in each sample loading hole under the dilution multiple of 1 to 3 times, and no band exists in agarose gel; the small interference RNA in the loaded small interference RNA liposome and the liposome are still in an assembled state, and the small interference RNA liposome is diluted by water, so that the assembled state of the small interference RNA in the loaded small interference RNA liposome and the liposome is not influenced. Whereas small interfering RNA loaded liposomes stained with NEB#B7024 purple gel loading dye, the small interfering RNA was assembled separately from the liposomes regardless of dilution.
As shown in fig. 4, example 4:
in the embodiment, different dyes are used for detecting the loaded small interfering RNA liposome in the cosmetics and detecting the assembly state of the loaded small interfering RNA and the liposome in the cosmetics.
400 μl of Miaojia nucleic acid myo-transmission essence emulsion (added with 5% small interfering RNA liposome, purchased from Meinai Biotechnology Co., ltd.) is mixed with 400 μl of DEPC sterile water at a ratio of 1:1, and left standing at normal temperature for 5min for use.
Centrifuging 1:1 mixed solution of cosmetics and DEPC sterile water at 3000rpm for 20min, removing cosmetic color interference, respectively using dye R and NEB#B7024 purple gel loading dye to dye the centrifuged supernatant, and performing agarose gel electrophoresis on the dye-dyed supernatant with a small interference RNA control group. FIG. 4 is a graph of agarose gel electrophoresis after staining; in the figure, 1 is a small interfering RNA control group, A is a cosmetic group stained with NEB#B7024 purple gel loading dye, and B is a dye R stain group. There was a distinct band in the agarose gel lanes in the control, no light in the loading wells, no band in lane a, and no band in lane B, light in the loading wells. Therefore, the loaded small interfering RNA liposome in the cosmetic to be tested is proved, and the small interfering RNA and the liposome are in an assembled state.
As shown in fig. 5, example 5:
in the embodiment, the detection of the small interfering RNA liposome with load and the assembly state of the small interfering RNA liposome in the external preparation form of the skin by using the external essence is verified by using different dyes.
Preparing external application essence of skin external preparation, adding hydroxyethyl cellulose 0.1ml, alkyl glucoside 0.5ml and glycerin 5ml into 83.15ml ultrapure water, uniformly mixing, swelling for 24 hours, adding 6ml butanediol, 3ml 1, 2-hexanediol and 2ml p-hydroxyacetophenone, uniformly mixing, sterilizing at 121 ℃ for 20min, taking out, cooling, adding 0.1ml phenoxyethanol and 0.05ml filtered sterilized sodium hyaluronate into an ultra-clean bench, and uniformly mixing to obtain the essence. The prepared external application essence is clear and transparent viscous liquid, does not contain impurities, and is added with 5% of small-load interfering RNA liposome.
Mixing the external application essence containing small interfering RNA liposome (5%) with DEPC sterile water at a ratio of 1:1, centrifuging at 3000rpm for 20min, removing color interference, respectively dyeing the lower clear liquid by using dye R and NEB#B7024 purple gel-like dye, and performing agarose gel electrophoresis on the lower clear liquid after dye dyeing by using a small interfering RNA control group. FIG. 5 is an agarose gel electrophoresis pattern after staining; in the figure, 1 is a small interfering RNA control group, A is a dye R staining group, and B is a NEB#B7024 purple gel-loading dye staining group. As can be seen from FIG. 5, there is a distinct band in the agarose gel lanes in the control, and no light color in the loading wells, and a band in lanes B, and no band in lanes A, and a light color in the loading wells. The external application essence of the skin external application dosage form to be tested contains small interfering RNA loaded liposome, and the small interfering RNA and the liposome are in an assembled state.
As shown in fig. 6, example 6:
the present example illustrates the essence milk raw material (without small interfering RNA liposome) provided by Nanjing Ruibo technology, and proves that the present invention can be used for detecting whether the external preparation contains the loaded small interfering RNA liposome.
Mixing the essence milk raw material with DEPC sterile water in a ratio of 1:1, centrifuging at 3000rpm for 20min, removing color interference, respectively using dye R and NEB#B7024 purple gel-loading dye to dye the lower clear liquid, and performing agarose gel electrophoresis on the lower clear liquid after dye dyeing by using a small interference RNA control group. FIG. 6 is an agarose gel electrophoresis pattern after staining; in the figure, 1 is a small interfering RNA control group, A is a dye R staining group, and B is a NEB#B7024 purple gel-loading dye staining group. As can be seen from FIG. 6, the A, B lane has no RNA band, which proves that the invention can be used for detecting whether the external preparation contains the loaded small interfering RNA liposome.
The above examples are provided for illustrating the technical concept and features of the present invention and are intended to enable those skilled in the art to understand the contents of the present invention and to implement the same, and are not intended to limit the scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention should be construed to be included in the scope of the present invention.
Claims (3)
1. A detection method of small interfering RNA loaded liposome in an external preparation is characterized by comprising the following steps: comprises the following steps:
(1) Mixing the external preparation to be tested with DEPC sterile water, centrifuging by a centrifugal device, and taking out a supernatant;
(2) Dyeing the lower clear liquid in the step (1) by using a dye R and a purple gel loading dye respectively;
(3) After dyeing, agarose gel electrophoresis is carried out, small interfering RNA is used as a control group, whether the small interfering RNA liposome loaded and the encapsulation state of the small interfering RNA are contained or not is judged according to the strip condition, and the model of the purple gel loading dye is NEB#B7024;
the dye R is 0.5-5 mM EDTA ANa2, 0.01-0.5 mM bromophenol blue and 0.5% -5% glycerol, and is dissolved in DEPC sterile water.
2. The method for detecting the small interfering RNA loaded liposome in the external preparation according to claim 1, which is characterized in that: the mixing proportion relation of the external preparation and DEPC sterile water is 1:1 to 1:5.
3. The method for detecting the small interfering RNA loaded liposome in the external preparation according to claim 1, which is characterized in that: the rotating speed of the centrifugal device is 3000-13000 rpm, and the centrifugal time of the centrifugal device is 1-30 min.
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| US8206904B2 (en) * | 2002-12-18 | 2012-06-26 | Third Wave Technologies, Inc. | Detection of nucleic acids |
| CN102113998B (en) * | 2011-02-24 | 2013-11-06 | 北京大学 | Separation and purification method for liposome containing encapsulated material |
| CN102226810A (en) * | 2011-03-28 | 2011-10-26 | 中国人民解放军第三军医大学第三附属医院 | Electrochemical immunoassay method based on Dopamine embedded liposome |
| CN106501525A (en) * | 2016-09-30 | 2017-03-15 | 江苏大学 | A kind of method of albumen or polypeptide drug envelop rate in measure lipid vesicle |
| CN108201533A (en) * | 2016-12-16 | 2018-06-26 | 南京理工大学 | A kind of N- succinyl-chitosan nanoparticle pharmaceutical delivery systems of load siRNA and its preparation method and application |
| CN110037988A (en) * | 2019-04-22 | 2019-07-23 | 广东和康众联健康产业管理有限公司 | Liposome nano granule drug delivery system of load siRNA and its preparation method and application |
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