阅读说明：本技术 基于Toe-hold链置换的微小RNA-21细胞内成像及阿霉素药物递送方法 (Method for micro RNA-21 intracellular imaging and adriamycin drug delivery based on Toe-hold strand displacement ) 是由 常津 秦怡 宫晓群 王涛 彭伟盼 于 2019-09-10 设计创作，主要内容包括：本发明公开一种基于Toe-hold链置换的微小RNA-21细胞内成像及阿霉素药物递送方法,包括:基于金纳米粒子的探针制备；体外微小RNA-21的检测；阿霉素的加载与体外释放：将DNA-A、DNA-B、DNA-C互补配对后,加入阿霉素孵育,使阿霉素充分被嵌入DNA双链,之后利用不同浓度的RNA置换DNA-B、DNA-C进而将阿霉素从DNA双链中释放出来,通过酶标仪测定置换前后溶液中阿霉素的荧光值。本发明首次将Toe-hold链置换反应与肿瘤成像、诊断及治疗相结合,可检测出单个癌细胞中微小RNA-21的含量。(The invention discloses a method for micro RNA-21 intracellular imaging and adriamycin drug delivery based on Toe-hold strand displacement, which comprises the steps of preparing a probe based on gold nanoparticles; detecting in vitro micro RNA-21; loading and in vitro Release of Adriamycin: complementary pairing of DNA-A, DNA-B, DNA-C, adding adriamycin for incubation to enable adriamycin to be fully embedded into DNA double strands, replacing the DNA-B, DNA-C with RNA with different concentrations to further release the adriamycin from the DNA double strands, and measuring the fluorescence value of the adriamycin in the solution before and after replacement by a microplate reader. The invention combines the Toe-hold strand displacement reaction with tumor imaging, diagnosis and treatment for the first time, and can detect the content of micro RNA-21 in a single cancer cell.)

1. A method for micro RNA-21 intracellular imaging and adriamycin drug delivery based on Toe-hold strand displacement is characterized in that:

1) preparing a probe based on gold nanoparticles: complementary pairing is carried out on DNA-A marked with sulfydryl, DNA-B marked with Cy5 fluorescence and DNA-C marked with FAM fluorescence, which are designed, and then the paired three DNA chains are modified on the surface of the gold nano-particle through the sulfydryl DNA-A;

2) detection of in vitro microRNA-21: mixing micro RNA-21 with different concentrations with a probe solution, and after full reaction, detecting the change of FAM fluorescence intensity to realize the detection of the micro RNA;

3) loading and in vitro Release of Adriamycin: complementary pairing of DNA-A, DNA-B, DNA-C, adding adriamycin for incubation to enable adriamycin to be fully embedded into DNA double strands, replacing the DNA-B, DNA-C with RNA with different concentrations to further release the adriamycin from the DNA double strands, and measuring the fluorescence value of the adriamycin in the solution before and after replacement by a microplate reader.

2. The method for intracellular imaging of microRNA-21 and delivery of an adriamycin drug based on Toe-hold strand displacement as claimed in claim 1, wherein: the preparation of the probe based on the gold nanoparticles is as follows:

(1) weighing 24.5mg trisodium citrate into a flask, adding 37.5mL double distilled water, placing the flask on a heat collection magnetic stirrer, stirring and heating, adding 0.3mL chloroauric acid trihydrate when the liquid is about to boil, stirring and reacting for 10 minutes, stopping heating, naturally cooling to room temperature, centrifuging at 12000r for 20 minutes, concentrating and resuspending by using 10 times of double distilled water, and storing at 4 ℃;

(2) DNA-A, DNA-B and DNA-C were mixed in a molar ratio of 1: 2: 2-1: 4: 4 in PBS, reacting at 37 ℃ for 2 hours to make three DNA chains fully complementary and paired;

(3) mixing tris (2-carboxyethyl) phosphine and paired DNA according to the molar ratio of the mixture to the DNA-A of 100: 1, and rotating on a vortex instrument at low speed for 1 hour at room temperature;

(4) after the reaction is finished, 10 times of concentrated AuNP is added into the reaction solution according to the volume ratio of the AuNP to the current solution being 1: 1, and rotating on a vortex instrument at a low speed for 16-24 hours;

(5) adding 1M NaCl solution for multiple times to make its final concentration reach 0.3M;

(6) and (3) purifying by centrifugal separation, washing for multiple times by using PBS buffer solution to remove the DNA which is not modified on the surface of the AuNP particles, and finally resuspending by using PBS with the same volume as the added gold nanoparticles to obtain the DNA modified AuNP probe.

3. The method for intracellular imaging of microRNA-21 and delivery of an adriamycin drug based on Toe-hold strand displacement as claimed in claim 1, wherein: the detection of the in vitro micro RNA-21 is concretely as follows:

(1) mixing the DNA modified AuNP probe with microRNA-21 with different concentrations in equal volume (0-1000 nM);

(2) putting the mixed solution into a metal bath, and reacting for 1-2h at 37 ℃;

(3) after the reaction was completed, the FAM fluorescence intensity of the solution was measured.

4. The method for intracellular imaging of microRNA-21 and delivery of an adriamycin drug based on Toe-hold strand displacement as claimed in claim 1, wherein: the loading and in vitro release of doxorubicin are as follows: DNA-A, DNA-B, DNA-C is added according to a molar ratio of 1: 2: 2-1: 4: 4, reacting in PBS for 1-2h at 37 ℃ to ensure that the three are fully complementary and paired, and after the reaction is finished, enabling the molar ratio of the three to DNA-A to be 2: 1-10: 1, mixing the doxorubicin hydrochloride with the DNA solution, rotating the mixture on a vortex instrument at low speed for 1h at room temperature to embed the doxorubicin in a DNA double chain, and detecting the fluorescence values of the solution before and after loading the DOX (excitation wavelength: 475-;

the micro RNA-21 and mRNA were added to the solution to a final concentration of 1. mu.M, and the mixture was placed in a metal bath and reacted at 37 ℃ for 1 to 2 hours. After the reaction, the fluorescence intensity of doxorubicin solution before and after the reaction was measured.

5. The method for intracellular imaging of microRNA-21 and delivery of an adriamycin drug based on Toe-hold strand displacement as claimed in claim 1, wherein: detecting and imaging intracellular microRNA-21 and releasing adriamycin by adopting a probe and cell co-incubation mode:

1) adding the gold nanoparticle probe diluted by 20-30 times of complete culture medium into Hela cells of a cell culture dish which is full of 80-90% and 6cm, and culturing for 4-7h in a 37 ℃ incubator to ensure that the probe is fully endocytosed by the Hela cells;

2) washing with PBS for 2-3 times, and washing off probes which are not swallowed into cells;

3) and finally, adding a complete culture medium, observing under a fluorescence inverted microscope, performing cell imaging by using FAM, and qualitatively analyzing whether the adriamycin is released into cells from the probe DNA double strand according to whether the fluorescence of the adriamycin can be observed.

6. The method for intracellular imaging of microRNA-21 and delivery of an adriamycin drug based on Toe-hold strand displacement as claimed in claim 1, wherein: the quantification of the intracellular micro RNA-21 is to digest cells endocytosed by the probe by pancreatin, collect the FAM fluorescence value of 10000 cells by using a flow cytometer, and realize the quantitative detection of the micro RNA-21 according to a corresponding correlation formula of the FAM fluorescence value and the micro RNA-21 concentration established in vitro.

Technical Field

The invention relates to the field of gene diagnosis and drug delivery, in particular to a method for micro RNA-21 intracellular imaging and adriamycin drug delivery based on Toe-hold strand displacement.

Background

Micro RNA (mirna) is an endogenous, non-coding small molecule with a short length (about 22 nucleotides) and binds to messenger RNA to participate in the regulation of gene expression at the post-transcriptional level. Numerous studies have shown that the abnormal expression of microRNAs is closely related to various diseases, especially human tumors and cancers, and thus has important clinical significance in tumor diagnosis, treatment and prognosis. The detection methods of the micro-RNA are numerous, however, in situ imaging detection becomes a great challenge due to the problems that the probe is difficult to deliver, the cell signal path is easy to influence, the cleaning and separation cannot be carried out, and the like. In order to realize real-time monitoring and analysis of micro RNA in living cells, among a plurality of imaging technologies, the fluorescent probe-based fluorescent imaging technology has the advantages of simplicity, intuition, good selectivity, high sensitivity, rich information content and the like, and is widely used for detecting tumor markers in cells.

The gold nano-carrier has the advantages of passive targeting, enhanced drug toxicity and the like, and is widely applied to the fields of tumor diagnosis, imaging and the like. Secondly, the DNA modified nano-particles can realize the effective delivery and the controllable release of the drugs through the programmable sequence and the function of carrying anticancer drugs. The diagnosis and drug delivery integration strategy integrates the selective imaging characteristics of the fluorescent probe and the drug release effect, thereby realizing the integration effect of imaging of a specific target point and accurate drug release.

Based on the background, the invention provides a method for micro RNA-21 intracellular imaging and adriamycin drug delivery based on Toe-hold strand displacement, which realizes in-situ imaging detection of micro RNA-21 and simultaneously delivers an anticancer drug adriamycin to tumor cells to kill the tumor cells. In situ imaging detection of microRNA-21 was achieved by binding microRNA-21 to the Toe-hold1 and displacement of DNA sequence labeled with FAM fluorescence, doxorubicin was delivered by pre-embedding it into the probe DNA duplex, and release of doxorubicin was achieved by binding intracellular mRNA to the Toe-hold2 and displacement of one DNA strand of the doxorubicin-loaded site. Through the design of the probe, the release of the adriamycin can be started only under the condition that the microRNA exists in a large quantity, namely the probe enters a cancer cell. Finally, imaging of tumor cells and monitoring of drug release were performed by visual analysis of FAM and doxorubicin fluorescence.

Disclosure of Invention

The invention provides a method for micro RNA-21 intracellular imaging and adriamycin drug delivery based on Toe-hold strand displacement, aiming at overcoming the defects of the prior art.

The technical scheme of the invention is as follows: a method for micro RNA-21 intracellular imaging and adriamycin drug delivery based on Toe-hold strand displacement is described as follows:

1) the preparation method of the probe based on the gold nanoparticles comprises the following steps: complementary pairing is carried out on DNA-A marked with sulfydryl, DNA-B marked with Cy5 fluorescence and DNA-C marked with FAM fluorescence, and then the paired three DNA chains are modified on the surface of the gold nano-particle through the sulfydryl DNA-A.

2) Detection of in vitro microRNA-21: mixing micro RNA-21 with different concentrations with a probe solution, and after full reaction, detecting the change of FAM fluorescence intensity to realize the detection of the micro RNA;

3) doxorubicin loading and in vitro release assay: complementary pairing of DNA-A, DNA-B, DNA-C, adding adriamycin for incubation to enable adriamycin to be fully embedded into DNA double strands, replacing the DNA-B, DNA-C with RNA with different concentrations to further release the adriamycin from the DNA double strands, and measuring the fluorescence value of the adriamycin in the solution before and after replacement by a microplate reader.

The preparation of the probe for intracellular detection imaging and adriamycin release of the micro RNA-21 comprises the following steps:

the preparation of the probe for intracellular detection imaging and adriamycin release of the micro RNA-21 is as follows:

(1) weighing 24.5mg trisodium citrate into a flask, adding 37.5mL double distilled water, placing the flask on a heat collection magnetic stirrer, stirring and heating, adding 0.3mL chloroauric acid trihydrate when the liquid is about to boil, stirring and reacting for 10 minutes, stopping heating, naturally cooling to room temperature, centrifuging at 12000r for 20 minutes, concentrating and resuspending by using 10 times of double distilled water, and storing at 4 ℃;

(2) DNA-A, DNA-B and DNA-C were mixed in a molar ratio of 1: 2: 2-1: 4: 4 in PBS, reacting at 37 ℃ for 2 hours to make three DNA chains fully complementary and paired;

(3) mixing tris (2-carboxyethyl) phosphine and paired DNA according to the molar ratio of the mixture to the DNA-A of 100: 1, and rotating on a vortex instrument at low speed for 1 hour at room temperature;

(4) after the reaction is finished, 10 times of concentrated AuNP is added into the reaction solution according to the volume ratio of the AuNP to the current solution being 1: 1, and rotating on a vortex instrument at a low speed for 16-24 hours;

(5) adding 1M NaCl solution for multiple times to make its final concentration reach 0.3M;

(6) and (3) purifying by centrifugal separation, washing for multiple times by using PBS buffer solution to remove the DNA which is not modified on the surface of the AuNP particles, and finally resuspending by using PBS with the same volume as the added gold nanoparticles to obtain the DNA modified AuNP probe.

The invention relates to a micro RNA-21 intracellular detection imaging and adriamycin release in-vitro micro RNA-21 detection experiment, which comprises the following specific operation steps: mixing the DNA modified AuNP probe with micro RNA-21 with different concentrations in equal volume (0-1000nM), placing the mixed solution in a metal bath, and reacting at 37 ℃ for 1-2 h. After the reaction was completed, the FAM fluorescence intensity of the solution was measured.

The concrete description is as follows:

after the DNA-A chain is complementarily paired with the DNA-B, DNA-C, 10 bases are extended from the 5' end of the DNA-A chain to serve as a Toe-hold of the micro RNA, the micro RNA can be firstly combined to the Toe-hold of the DNA-A, and gradually complementarily paired with the DNA-A, the DNA-C chain is replaced, and then released, and the fluorescence of FAM is recovered after being far away from AuNP, so that the micro RNA can be quantitatively monitored. The length of the Toe-hold mediated strand displacement reaction can be combined and strand displacement can be realized only by micro RNA with 4 bases, and the process can be carried out spontaneously without participation of substances such as enzyme and the like, and is suitable for reaction in a cell complex liquid environment.

The adriamycin loading and in-vitro release test for micro RNA-21 intracellular detection imaging and adriamycin release is as follows: DNA-A, DNA-B, DNA-C is added according to a molar ratio of 1: 2: 2-1: 4: 4, reacting in PBS for 1-2h at 37 ℃ to ensure that the three are fully complementary and paired, and after the reaction is finished, enabling the molar ratio of the three to DNA-A to be 2: 1-10: 1, mixing the doxorubicin hydrochloride with the DNA solution, rotating the mixture on a vortex instrument at low speed for 1h at room temperature to embed the doxorubicin in a DNA double strand, and detecting the fluorescence values of the solution before and after loading the DOX by using a multifunctional microplate reader after the reaction is finished (the excitation wavelength is 475-. The micro RNA-21 and mRNA were added to the solution to a final concentration of 1. mu.M, and the mixture was placed in a metal bath and reacted at 37 ℃ for 1 to 2 hours. After the reaction, the fluorescence intensity of doxorubicin solution before and after the reaction was measured.

The concrete description is as follows: the adriamycin is an anthracycline antibiotic, has a wide anti-tumor spectrum, can be widely applied to treatment of a plurality of cancers such as blood system tumor, breast cancer, lung cancer and the like, the chromophore part of the planar aromatic molecule can be preferentially embedded between GC base pairs of a DNA double strand, the fluorescence of the DNA can be weakened after the DNA is embedded, the adriamycin can be released from the DNA double strand when the DNA-B, DNA-C is replaced by the micro RNA and the mRNA, the fluorescence can be recovered, and the change of the fluorescence intensity can be detected by an enzyme-labeling instrument.

And (3) carrying out detection imaging of intracellular micro RNA-21 and release of adriamycin by adopting a probe and cell co-incubation mode.

1) Adding the gold nanoparticle probe diluted by 20-30 times of the complete culture medium into the Hela cells of a 6cm cell culture dish which is full of 80-90%, and culturing for 4-7h in a 37 ℃ incubator to ensure that the probe is fully endocytosed by the Hela cells.

2) The probe that had not been engulfed into the cells was washed off by washing 2 to 3 times with PBS.

3) And finally, adding a complete culture medium, observing under a fluorescence inverted microscope, performing cell imaging by using FAM, and qualitatively analyzing whether the adriamycin is released into cells from the probe DNA double strand according to whether the fluorescence of the adriamycin can be observed.

The quantification of the intracellular micro RNA-21 is to digest cells endocytosed by the probe by pancreatin, collect the FAM fluorescence value of 10000 cells by using a flow cytometer, and realize the quantitative detection of the micro RNA-21 according to a corresponding correlation formula of the FAM fluorescence value and the micro RNA-21 concentration established in vitro.

The result of the detection

(1) And (3) qualitative detection: under the condition that micro RNA-21 is over-expressed in the Hela cells, the micro RNA-21 can be combined to a probe endocytosed cells and can replace DNA-C marked with FAM fluorescence to recover the fluorescence, so that the change of the FAM fluorescence can be observed under a fluorescence inverted microscope, and the imaging of the micro RNA-21 in the cancer cells is realized.

(2) And (3) quantitative detection: collecting FAM fluorescence values of a certain number of cells by using a flow cytometer, and calculating the concentration of the micro RNA-21 in the Hela cells according to a corresponding correlation formula of the FAM fluorescence intensity and the concentrations of different micro RNA-21.

The method for detecting and imaging micro RNA-21 in cells and delivering the adriamycin medicine, which is prepared by the invention, has the advantages that:

1. the invention combines the Toe-hold strand displacement reaction with tumor imaging, diagnosis and treatment for the first time, can detect the content of the micro RNA-21 in a single cancer cell, has the same detection result with other methods, and utilizes the micro RNA-21 to image the tumor cell, thereby providing a new idea for the integration of cancer diagnosis and treatment.

2. The probe designed by the invention can effectively diagnose and distinguish normal cells and tumor cells, realizes the effective release of the adriamycin in the tumor cells, so that the adriamycin can not be released in the normal cells in large quantity, and greatly reduces the toxic and side effects of the adriamycin on the normal cells.

Drawings

FIG. 1 is a graph showing that the gold nanoparticle probe prepared in example 1 of the present invention can be used for the release of microRNA-21 and doxorubicin: panel a is the color change of the solution after addition of RNA; and the graph B is agarose gel electrophoresis which proves that the DNA-B and the DNA-C are successfully replaced by the RNA (wherein 1 is a gold nano-particle probe, and 2-3 are respectively added with mRNA, micro-RNA and mRNA in the probe).

FIG. 2 shows that the gold nanoparticle probe prepared in example 1 of the present invention is used for in vitro detection of micro RNA-21:

a-detection fluorescence maps of micro RNA-21 with different concentrations; b-a detection trend graph of micro RNA-21 with different concentrations;

c-detection standard curve of micro RNA-21 with different concentrations.

FIG. 3 shows fluorescence intensities of DNA duplexes loaded with different concentrations of doxorubicin according to example 3 of the present invention.

FIG. 4 shows the horseradish peroxidase-labeled probe prepared in example 1(1) of the present invention for detecting and imaging intracellular microRNA-21 and releasing adriamycin:

in the figure are images of brightfield, FAM, doxorubicin and fusion, respectively.

Detailed Description

The invention will be further illustrated in the following examples, without however being restricted thereto.