WO2018155880A1 - Liposome–nucleic acid nano-fusion for quantitative diagnosis of multiple ribonucleic acid markers, theoretical stability evaluation method therefor, application thereof, and production method therefor - Google Patents

Abstract

The present invention relates to: a liposome–nucleic acid nano-fusion for the quantitative diagnosis of multiple ribonucleic acid markers, the nano-fusion containing, in a liposome, a silica sphere having bound to the surface thereof a nucleic acid nano-structure having a first nucleic acid structure, which is branched and has a cyclic end, bonded with a second nucleic acid structure, which is branched and has a cyclic end; a theoretical stability evaluation method for the nano-fusion; an imaging system using the nano-fusion in disease diagnosis; and a production method for the nano-fusion. By using the liposome–nucleic acid nano-fusion according to the present invention, an advantage may be achieved whereby the difference in aspects of RNA expression among various cell lines may be quantitatively compared, and the difference among cells in a single cell line may be checked, and thus multiple intracellularly-expressed cancer-specific RNA markers acquired in an actual clinical setting may be diagnosed in real-time, and on the basis thereof, information required for diagnosis and treatment may be easily acquired.

Description

Liposome-Nucleic Acid Nanofusions for Quantitative Diagnosis of Multiple Ribonucleic Acid Markers, Methods for Evaluating Theoretical Stability, Applications thereof, and Methods for Preparing the Same

The present invention provides a liposome-nucleic acid nanofusion for quantitative diagnosis of multiple ribonucleic acid markers, an imaging system for confirming stability using the nanofusion molecular dynamics simulation technique, and utilizing the same for disease diagnosis, a disease diagnosis method, and a method for preparing the nanofusion. It is about.

Most cancer diagnosis currently uses materials harmful to the human body, such as metals and polymers, and it takes at least three days and a month or more for accurate diagnosis. And since most diagnostic agents do not generate light in the visible wavelength range, there is a limit in diagnosing cancer by eye only until the cancer progresses considerably and forms a tumor. For the same reason, there is a limit to the diagnosis being made in real time in vivo.

Accordingly, in recent years, a lot of research has been conducted to manufacture a diagnostic material using biocompatible materials in order to minimize human hazards. Nucleic acid is a material that attracts a lot of attention and is being produced in various forms through nanobiotechnology. The technology used to diagnose cancer by attaching phosphors to the nucleic acid nanostructures thus produced is drawing much attention.

However, since nucleic acid is a substance constituting the living body, when inserted into the living body, it is very likely to be broken down by enzymes and lose its original function. In addition, in order to minimize the non-specific binding of the diagnostic material to cancer cells as well as general cells, a process of binding to a target material is required. However, until now, studies that satisfy the above requirements at the same time have not been much progressed yet. In addition, cancer cells that do not express specific targets, such as triple negative breast cancer, have considerable difficulty in diagnosis.

On the other hand, according to the expression of the RNA of the target cell, it has been found that the diagnosis corresponding to the entire cancer cycle, and among them, the expression of a specific miRNA is known to change rapidly in early cancer. For this reason, the importance of miRNA as a diagnostic marker is increasing day by day. In addition, precise treatment is difficult due to tumor heterogeneity in cancer tissues occurring at one site, and it is important to obtain spatial-temporal information by checking the difference between cells for precise patient-specific treatment. It is considered to be.

Based on these needs, technologies such as single cell real time polymerase chain reaction and high-throughput sequencing have been developed, but they also have significant limitations in the time and cost required for analysis. Have. In addition, various diagnostic systems have been reported for real-time intracellular analysis using nanotechnology in order to overcome the above problems (Korea Patent No. 1464100), but because the intracellular delivery of the diagnostic system is largely dependent on intercellular characteristics, Quantitative comparisons by type are not possible, which causes problems in practical clinical applications.

Accordingly, there is a need for a high-performance cancer diagnostic agent capable of rapidly diagnosing cancer by comparing RNA expression patterns among various cell lines.

The present inventors completed the present invention by developing a liposome-nucleic acid nanofusion for quantitative diagnosis of multiple ribonucleic acid markers, which is capable of quantitative comparison between cells with high efficiency and low cost.

Accordingly, an object of the present invention is to provide a branched first nucleic acid structure having a cyclic terminal; And it is to provide a liposome-nucleic acid nano fusion for multiple ribonucleic acid marker quantitative diagnosis comprising a silica spherical body bonded to the surface of the nucleic acid nanostructures conjugated to the branched second nucleic acid structure having a cyclic terminal inside the liposome.

Another object of the present invention is to provide a biological diagnostic imaging system comprising the liposome-nucleic acid nanofusion for the multi-ribonucleic acid marker quantitative diagnosis.

Still another object of the present invention is to provide a method for preparing a liposome-nucleic acid nanofusion for multi-ribonucleic acid marker quantitative diagnosis.

Still another object of the present invention is to provide an evaluation method for theoretically evaluating the stability of the liposome-nucleic acid nanofusion for multi-ribonucleic acid marker quantitative diagnosis.

Still another object of the present invention is to provide a kit for diagnosing cancer comprising the liposome-nucleic acid nanofusion for multi-ribonucleic acid marker quantitative diagnosis.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention is a branched first nucleic acid structure having a cyclic terminal; And it provides a liposome-nucleic acid nano fusion for quantitative diagnosis of multiple ribonucleic acid markers comprising a silica spherical body bonded to the surface of the nucleic acid nanostructures conjugated to the branched second nucleic acid structure having a cyclic terminal inside the liposome.

In one embodiment of the present invention, the first nucleic acid structure may be a form in which linear nucleic acids selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 1 to 3 are combined in a Y-shaped branched form.

In another embodiment of the present invention, the second nucleic acid structure may be in a form in which linear nucleic acids selected from the group consisting of nucleotide sequences of SEQ ID NOs: 4 to 6 are combined in a Y-shaped branched form.

In another embodiment of the present invention, the linear nucleic acid may further include a phosphor at the 5 'end.

In another embodiment of the present invention, the first nucleic acid structure may comprise a nucleotide sequence of SEQ ID NO: 7 having a cyclic terminal complementary to the target RNA.

In another embodiment of the present invention, the second nucleic acid structure may comprise a nucleotide sequence of SEQ ID NO: 8 having a cyclic terminal complementary to the target RNA.

In another embodiment of the present invention, the first nucleic acid structure and the second nucleic acid structure may be further labeled with a phosphor and a quencher.

In another embodiment of the present invention, the nucleic acid structure may be one that is further labeled with a quencher at one end of the base sequence having a complementary sequence with the target RNA.

In another embodiment of the present invention, the silica sphere may be 50 to 90 nm.

In another embodiment of the present invention, the silica spherical surface is treated by sequentially treating aminopropyl trimethoxysilane and cyanuric chloride before the nucleic acid nanostructures are bonded to the surface. May be modified.

In another embodiment of the present invention, the liposome may be made of a cationic lipid.

In another embodiment of the present invention, the cationic lipid may be composed of a cationic lipid comprising a component of DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) and cholesterol.

The present invention also provides a biodiagnostic imaging system comprising the liposome-nucleic acid nanofusion.

In one embodiment of the present invention, the system may be to measure intracellular fluorescence.

The present invention also provides a method for diagnosing a disease comprising administering the liposome-nucleic acid nanofusion to a subject in need thereof and measuring fluorescence.

In one embodiment of the invention, the disease may be cancer.

In another embodiment of the present invention, the administration may be through oral administration, intravenous injection, intraperitoneal injection, intramuscular injection, arterial injection or subcutaneous injection.

In addition, the present invention provides a method for preparing liposome-nucleic acid nanofusions for quantitative diagnosis of multiple ribonucleic acid markers, comprising the following steps:

After preparing a branched first nucleic acid structure having a cyclic terminal and a branched second nucleic acid structure having a cyclic terminal, a nucleic acid nanostructure is prepared by conjugation with each other using a ligase enzyme (S1). ;

Adding a silicate solution to a solvent to synthesize silica nanoparticles (S2);

Sequentially treating aminopropyl trimethoxysilane and cyanuric chloride on the nanoparticles of step S2 (S3);

Preparing a nucleic acid nanostructure by reacting the nanoparticle body of step S3 with the nucleic acid nanostructure of step S1 (S4);

Adding a cationic lipid to the nucleic acid nanostructure of step S4, followed by ultrasonication to prepare a liposome-nucleic acid nanofusion (S5).

In one embodiment of the present invention, the solvent of the S2 step may be to include methanol, ethanol, distilled water, and ammonia water.

In another embodiment of the present invention, the methanol and ethanol may be mixed in a volume ratio of 6: 4 to 8: 2:

In addition, the present invention is an evaluation method for theoretically evaluating the stability of the liposome-nucleic acid nanofusion for multi-ribonucleic acid marker quantitative diagnosis,

The method provides an evaluation method comprising the step of measuring the hydrogen bonding energy generated inside the nanofusion.

In one embodiment of the present invention, the measurement may be performed at 35 to 40 ℃.

The present invention also provides a kit for diagnosing cancer comprising the liposome-nucleic acid nanofusion for multi-ribonucleic acid marker quantitative diagnosis.

Liposome-nucleic acid nanofusion for quantitative diagnosis of multiple ribonucleic acid markers according to the present invention has the effect of quantitative comparison of RNA expression patterns of various cell lines and the difference between cells in a single cell line. This enables multiple real-time diagnosis of cancer-specific RNA markers expressed in cells obtained in actual clinical practice, and has the advantage of easily and quickly obtaining information necessary for diagnosis and treatment.

In particular, high-sensitivity diagnosis is possible by targeting microribonucleic acid whose concentration is absolutely low in cells, and thus it is possible to predict the diagnosis and prognosis of early cancers in which relatively few target substances are expressed. Because it is possible to target a variety of target material, even difficult to diagnose carcinoma such as triple negative breast cancer can be easily diagnosed.

In addition, multi-colorimetric diagnostic evaluation according to various miRNA concentrations using various phosphors is possible, and heterogeneity between cells can be evaluated through diagnosis of various miRNAs in a single cell. In addition, since the liposome bilayer on the surface of the fusion slows down the degradation of the diagnostic body in vivo, there is an advantage that it is easy to apply in vivo.

1 is a diagram illustrating a method for preparing a liposome-nucleic acid nanofusion and a diagnostic method for diagnosing intracellular real-time multiple RNA markers of the present invention.

2 shows a method for producing a nucleic acid nanostructure (afc-DNA), and a lower part shows a result of confirming the synthesis of the nucleic acid nanostructure through electrophoresis.

3 is a diagram showing simulation conditions for confirming the three-dimensional structure of the nucleic acid nanostructures.

4 is a view showing the results of confirming the three-dimensional structure of the nucleic acid nanostructures through the oxDNA program in accordance with the conditions of FIG.

5 is a view showing a process and results of bonding the nucleic acid nanostructures to the surface of the silica nanoparticles, 5a is a schematic diagram of the bonding process, 5b is a result of confirming the binding stability (37 degrees) with temperature, 5c shows the particle size change according to the surface treatment, and 5d shows the result of visualizing the nucleic acid nanostructures attached to the silica surface through a fluorescence microscope.

6 is a view showing the results according to the size, the fluorescent signal amplification and the surface charge change according to the size of the liposome-nucleic acid nano fusion of the present invention.

FIG. 7 is a diagram showing the results obtained by treating liposome-nucleic acid nanofusions prepared in the present invention to two types of breast cancer cell lines (MCF-7 and SK-BR-3) and confirming that intracellular delivery is well performed.

8 is a result confirming that the nanofusion of the present invention has a multi-diagnostic ability, five breast cancer cell lines (HCC-1937, MCF-7, MDA-MB-453, MDA-MB-231 and SK-BR-3 After treating the liposome-nucleic acid nanofusion of the present invention, the result obtained by flow cytometry is compared with the results of RT-PCR which is currently used as a standard.

Figure 9a shows the result of measuring the target miRNA to the nucleic acid nanostructures, resulting in the increase in the resulting fluorescent signal, Figure 9b is mixed with the target miRNA in various concentrations, the fluorescent signal according to the concentration in the mixed solution Figure 9c shows the result of confirming the change of, Figure 9c is a view showing the result of converting the change of the fluorescent signal to color.

The present inventors have completed the present invention as a result of research to develop cancer cell diagnostics capable of real-time intracellular multiplex RNA marker diagnosis.

Accordingly, the present invention provides a branched first nucleic acid structure having a cyclic terminal; And a liposome-nucleic acid nano fusion for quantitative diagnosis of multiple ribonucleic acid markers, wherein the nucleic acid nanostructure to which the branched second nucleic acid structure having a cyclic terminal is conjugated is bound to the surface thereof in a liposome. It is characterized by providing a diagnostic imaging system.

In addition, the present invention provides a method for preparing a liposome-nucleic acid nanofusion for multi-ribonucleic acid marker quantitative diagnosis comprising the following steps:

After preparing a branched first nucleic acid structure having a cyclic terminal and a branched second nucleic acid structure having a cyclic terminal, a nucleic acid nanostructure is prepared by conjugation with each other using a ligase enzyme (S1). ;

Adding a silicate solution to a solvent to synthesize silica nanoparticles (S2);

Sequentially treating aminopropyl trimethoxysilane and cyanuric chloride on the nanoparticles of step S2 (S3);

Preparing a nucleic acid nanostructure by reacting the nanoparticle body of step S3 with the nucleic acid nanostructure of step S1 (S4);

Adding a cationic lipid to the nucleic acid nanostructure of step S4, followed by ultrasonication to prepare a liposome-nucleic acid nanofusion (S5).

In this case, if the preparation method can produce liposome-nucleic acid nanofusions according to the present invention, the order and / or configuration of the steps may be appropriately changed, but not limited to the above steps.

Liposome-nucleic acid nanofusions according to the present invention can be used for the diagnosis of a disease. For this purpose, in one embodiment of the present invention, two kinds of nucleic acid structures labeled with phosphors to be implemented in a cell are attached to silica spheres. The globular surface was coated with liposomes (see FIG. 1). At this time, in one embodiment of the present invention, but used as a spherical body for support, if the nano-spherical spheres that can be used in vivo can be used without limitation, the size of the prepared nano fusion can be injected into the body If the size and shape can be appropriately adjusted, but preferably can be prepared in a sphere having a diameter of 50 nm to 90 nm.

In the present invention, the first nucleic acid structure and the second nucleic acid structure may be prepared in the form of a branch by allowing linear nucleic acids having an adhesive end to be bonded in a Y-shaped branch form or repeatedly bonded in the Y-shaped branch form. Eggplant may be to label the phosphor at least three 5 'end, but is not limited thereto.

The nucleic acid construct has a cyclic terminus and is complementary to the target ribonucleic acid for complementary binding to intracellular target messenger ribonucleic acid (mRNA) or target microribonucleic acid (miRNA) to detect intracellular signals. The nucleic acid including the sequence and the short sequence which binds to the cyclic nucleic acid structure may be attached to one or more ends of the Y-branch. At this time, by further labeling a quencher at one end of the sequence complementary to the target ribonucleic acid, the cyclic nucleic acid structure binding to the target ribonucleic acid is a poster resonance energy transfer between the phosphor and the quencher (Forster Resonance Energy Transfer, FRET). That is, when the cyclic nucleic acid structure does not bind with the target ribonucleic acid, light does not occur due to the Foster resonance energy transfer effect, and when combined, light is generated from the phosphor.

Preferably, the phosphor may be fluorescein, Texas red, rhodamine, alexa, cyanine, BODIPY or coumarin, more preferably. Preferably 6-FAM, Texas 615, Alexa Fluor 488, Cy5, or Cy3, preferably the quencher is TAMRA, BHQ, Iowa Black RQ or MBNQ It may be (MGBNFQ, molecular grove binding non-fluorescence quencher), more preferably Iowa Black RQ, if the phosphor or quencher can be used in vivo is not limited to this can be appropriately used by those skilled in the art.

Liposomes of the present invention can vary in lipid composition to control interaction with cells according to the intended use. In one embodiment of the present invention, liposomes were prepared with a positively charged lipid so that the liposome-nucleic acid nanofusion according to the present invention could be introduced into the cytoplasm by non-specifically fusion with a cell membrane to a target cell, preferably the positively charged lipid. May be prepared using DOTAP (1,2-dioleoyl-3-trimethylammonium-propane), but is not limited thereto.

In addition, the liposome-nucleic acid fluorescence fusion of the present invention is a nucleic acid nano fusion (silica sphere having a nucleic acid nanostructure conjugated to a first nucleic acid structure and a second nucleic acid structure) and liposomes added to the same solution (solvent), respectively. It can be prepared by mixing, preferably the mixing weight ratio of the silica nanoparticles and liposomes surface-treated nucleic acid nanofusions is not limited, as in one embodiment of the present invention solution and liposomes containing nucleic acid nanofusions This solution may be mixed at a weight ratio of 8: 7, but is not limited thereto. In this case, the same solution (solvent) should be used to prevent rupture of liposomes due to osmotic pressure. Preferably, the solution may include distilled water or a phosphate solution such as PBS (Phosphate buffersaline), but is not limited thereto.

In addition, the solvent of the step S2 of the present invention may be one containing methanol, ethanol, distilled water, and ammonia water, the methanol and ethanol may be mixed in a volume ratio of 6: 4 to 8: 2 :.

The liposome-nucleic acid nanofusion body according to the present invention has a feature that enables quantitative diagnosis according to the expression of the color tone by labeling phosphors having different color tones at the ends of the first and second nucleic acid structures.

Therefore, the liposome-nucleic acid nanofusion of the present invention can be utilized as a bio-diagnostic imaging system, and the system captures multiple RNA markers to activate specific fluorescent substances, and according to fluorescence signal change and color conversion according to miRNA concentration, It has features that allow quantitative comparison between cells.

In addition, the present invention may provide a disease diagnosis method comprising administering the liposome-nucleic acid nanofusion to an individual in need of diagnosis of the disease and measuring intracellular fluorescence. The administration may be by oral administration, intravenous injection, intraperitoneal injection, intramuscular injection, arterial injection or subcutaneous injection method, the subject means a subject in need of diagnosis of the disease, more specifically human, or non -Means human mammals such as primates, mice, rats, dogs, cats, horses, or cattle. In addition, the diagnostic method is not limited to any one of the diseases because the nucleic acid sequence can be changed and liposome-nucleic acid fluorescent nanofusions can be prepared according to the type of disease requiring diagnosis. It may be to, and most preferably in accordance with an embodiment of the present invention may be for the purpose of diagnosing breast cancer.

Furthermore, the liposome-nucleic acid fluorescent nanofusion of the present invention may be prepared to include various materials because the interior thereof is empty. The material that can be included therein is not limited, but preferably includes a therapeutic agent to be used as a therapeutic system at the same time as the bio-diagnostic imaging.

In addition, the present invention is an evaluation method for theoretically evaluating the stability of the liposome-nucleic acid nanofusion for multi-ribonucleic acid marker quantitative diagnosis, the method comprising the steps of measuring the hydrogen bonding energy generated inside the nanofusions The evaluation method can be provided.

The evaluation method is carried out by oxDNA simulation, oxDNA is a simulation code developed to implement a large-particle DNA model, it is possible to theoretically evaluate the physical stability of nucleic acid nanofusions through molecular dynamics.

In the present invention, the measurement of the hydrogen bond energy is characterized in that it is carried out at 35 to 40 ℃, the evaluation method by measuring the hydrogen bond energy generated in the interior of the nano-fusion or nucleic acid nanostructures for a predetermined time (x10 ⁵ ) When it is confirmed that the hydrogen bonding energy is kept constant, it is determined that the prepared nanofusion or nucleic acid nanostructures have structural stability.

The present invention also provides a kit for diagnosing cancer comprising the liposome-nucleic acid nanofusion for multi-ribonucleic acid marker quantitative diagnosis.

In the present invention, the cancer may include breast cancer, bile duct cancer, bladder cancer, brain tumor, cervical cancer, chorionic cancer, colon cancer, endometrial cancer, esophageal cancer, gastric cancer, multiple myeloma, AIDS-related leukemia and adult T-cell lymphoma / leukemia, epithelial cancer, Liver cancer, lung cancer, lymphoma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer or renal cell cancer.

As used herein, the term "kit" may include one or more other component compositions, solutions or devices suitable for analytical methods.

In addition, the kit may include all biological or chemical reagents necessary for diagnosing cancer, differences between multiple cell lines of cancer, or between cells within a single cell line, essential elements necessary for performing PCR, and a guide. In addition to the above primer sets, PCR kits include test tubes or other suitable containers, reaction buffers (pH and magnesium concentrations vary), enzymes such as deoxynucleotides (dNTPs), Taq-polymerases and reverse transcriptases, DNases, RNAse inhibitors, DEPC-water, sterile water and the like. The guide is a printed document which explains how to use the kit, e.g. the reaction conditions presented. The instructions may include brochures in the form of pamphlets or leaflets, labels affixed to the kit, and instructions on the surface of the package containing the kit. In addition, the guide may include information disclosed or provided through an electronic medium such as the Internet. For the purposes of the present invention, liposome-nucleic acid nanofusions of the present invention may have been delivered intracellularly with a molecular beacon structure and nanostructures capable of specific binding to intercellular miRNAs within a single cell line or between different cell lines of a target cancer. At this time, since the nanostructure itself is composed of a labeled fluorescent factor capable of quantification, cancer can be diagnosed by confirming it through fluorescence signal increase and color conversion.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

EXAMPLE

In this example, for effective cancer diagnosis and clinical application, the target miRNA was selected and used as miR-21 and miR-22, miRNAs that are known to be closely related to cancer in the previous studies. Simple intracellular delivery of diagnostic probes has limitations that are difficult to quantitatively analyze due to cell-specific intracellular migration rates.For example, if the diagnostic probe is simply delivered in large quantities due to cellular characteristics, background noise of the diagnostic probe itself Because of this, even if there is no actual target miRNA, it is possible to cause positive false, and even if there is a target miRNA, it may cause negative false. Thus, the nanotechnology-based diagnostic probes developed to date have mostly the characteristics of relative quantification, and the present inventors attempt to overcome the above limitations, for the efficient diagnosis of intracellular miRNA, as shown in FIG. 1. Specific liposome-nucleic acid nanofusions have been developed.

Example 1 Preparation of Liposomal-Nucleic Acid Nanofusions

1-1. Nucleic Acid Nanostructure Synthesis and Molecular Dynamics Simulation

The sequence having the nucleotide sequence of the nucleic acid oligomer constituting the first nucleic acid structure (fc1-DNA) and the second nucleic acid structure (fc2-DNA) and the complementary sequence with the target RNA is shown in Table 1. After dissolving in TE buffer (10 mM Tris, pH 8.0, 0.1 mM EDTA), each nucleic acid oligomer was mixed in the same molar ratio (0.1 mM). Thereafter, the first nucleic acid structure and the second nucleic acid structure were synthesized by gradually lowering the temperature of the solution at 60 ° C. to 20 ° C. by 1 ° C. per minute.

The synthesis of the nucleic acid nanostructures (afc-DNA) was performed by conjugation through ligase enzymes between the first and second nucleic acid constructs as shown above in FIG. 2. The same mole number unit nucleic acid nanostructure mixture (0.1 mM) was added to 3 Weiss units of T4 ligase enzyme and buffer (4 ° C., 16 hours). As shown below in Figure 2, nucleic acid nanostructure synthesis was confirmed through electrophoresis.

The oxDNA program was used to confirm the three-dimensional structure of the synthesized nucleic acid nanostructure, specific simulation conditions are shown in FIG. 3, and the results are shown in FIG. 4. As can be seen in Figure 4, the nucleic acid structure and nucleic acid nanostructures synthesized in the present invention was confirmed that the hydrogen bond energy generated inside is kept constant, having stability.

TABLE 1

1-2. Synthesis of Silica Nanoparticles of Various Sizes and Attachment of Nucleic Acid Nanoparticles

For particle synthesis, methanol and ethanol were mixed in various volume ratios so that the final volume was 46 mL, and then 1 mL of distilled water and 3 mL of ammonia water (28-30 w / w%) were added and stirred for 10 minutes. Afterwards, 0.6 ml of Tetraethyl orthosilicate (TEOS) solution was slowly treated to prepare silica nanoparticles. The prepared silica nanoparticles were washed with ethanol three times or more through centrifugation (15,000 G, 30 minutes).

After rinsing with ethanol, 200 mg of silica nanoparticles were treated with 0.8 mL of APTMS (aminopropyltrimethoxysilane, (3-Aminopropyl) trimethoxysilane), 38 ml of ethanol, 2 ml of distilled water, and 0.25 ml of acetic acid. Added. After rinsing with ethanol and acetonitrile through three or more centrifugations, 115.2 mg of cyanuric chloride was treated and stirred for 2 hours. Washed three times or more with acetonitrile, ethanol, distilled water, borate buffer (Borate buffer, pH 8.5).

Finally, nucleic acid nanofusions were prepared by treating nucleic acid nanostructures in which 25 pmole amine groups per 4 mg of silica nanoparticles were substituted at the ends, and reacting for 16 hours or more. Unreacted nucleic acid nanostructures were removed by washing with distilled water through centrifugation.

1-3. Cationic Phospholipid Treatment

Silica nanoparticles to which the nucleic acid nanoparticles were bound were treated with cationic lipid DOTAP (1,2-dioleoyl-3-trimethylammonium-propane). Prior to the reaction, 3.5 mg of DOTAP phospholipid was dispersed by sonication in 1.4 ml of distilled water at 10% amplitude (amplitude, Qsonica Q500) (dispersed at 4 ° C. and stored at low temperature until reaction). The DOTAP solution and the nanofusion solution were mixed, reacted with stirring for 1 hour, and then washed with distilled water and physiological saline through centrifugation.

Example 2. Silica Nanoparticle Synthesis and Nucleic Acid Nanostructure Conjugation

In Example 2, various sizes of silica nanoparticle bodies were prepared for signal amplification and efficient intracellular delivery of nucleic acid nanostructures, thereby deriving an optimal size of silica nanoparticle bodies. Based on the synthesis method using a mixture of methanol and ethanol, silica nanoparticles having a size of 20 ~ 270 nm were synthesized.

Subsequently, aminopropyl trimethoxysilane and cyanuric chloride are sequentially treated on silica nanoparticles to bond the nucleic acid nanostructures to the surface, as shown in FIG. 5A. It was allowed to react with the amine group.

In addition, Figure 5b shows the result of checking how much the fluorescent factor attached to the surface of the silica drops over time at 37 ℃, the intensity of the fluorescence signal generated in the nucleic acid nanostructure is different depending on the size of the silica nanoparticles It confirmed that there was an optimum point.

Example 3 Cationic Phospholipid Treatment and Intracellular miRNA Detection

In the present invention, for efficient intracellular delivery and endosomal escape, silica nanoparticles coated with nucleic acid nanostructures (ie, nucleic acid nanofusions) are treated with DOTAP (Dioleoyl-trimethylammonium-propane), a cationic phospholipid. To prepare a liposome-nucleic acid nanofusion and use it for diagnosis. In this example, the size and surface charge change of the particle body according to the DOTAP treatment was confirmed.

As a result, as shown in Figure 5c, it can be seen that it is possible to attach the nucleic acid nanostructures to the surface of the silica nanoparticles of various sizes, and then to process the DOTAP to produce nano fusions of various sizes, methanol and ethanol When the final nano fusions were prepared using the silica nanoparticles produced at a mixing volume ratio of 8: 2, the size was 51.38 nm, the size was 80.56 nm at 6: 4, and the thickness was 134.21 nm. It can be seen that the size of 225.70 nm at 2: 8 and 264.40 nm at 0:10. In addition, as can be seen in Figure 5d, the nucleic acid nanostructure attached to the surface of the micro-sized silica beads through the same aminopropyl trimethoxysilane and cyanuric chloride chemical reaction fluorescence microscope (Zeiss Axiovert 200M, Carl Zeiss) Confirmed through to verify the adhesion of the nucleic acid nano fusion on the surface of the silica nanoparticles in the present invention.

6 shows the results of confirming the size according to the preparation step of the liposome-nucleic acid nanofusion of the present invention, the fluorescence signal amplification and the surface charge change according to the size. Fluorescence intensity was measured using a fluorescence photometer (SpectraMax M5, Molecular Devices), and the change of surface charge was measured by dynamic light scattering technique (Zetasizer Nano ZS, Malvern Instruments). Figure 6a schematically shows the manufacturing step of the present invention, attaching the nucleic acid nanostructures to the surface of the silica nanoparticles produced using various fractions of methanol / ethanol mixture, and finally treated with DOTAP to coat the surface It was. 6b and 6c show the images taken using the size of the silica spheres and the scanning electron microscope (JEM-3010, JEOL) according to the mixing volume ratio of methanol and ethanol, Figure 6d is bound according to the sphere size It shows the amount of the nucleic acid nanostructure and the fluorescent signal. According to the result, it can be seen that the amount of aggregates on the surface of the nucleic acid nanostructure is changed according to the size of the spherical body, thereby changing the size of the fluorescent signal. Figure 6e shows the surface charge change before and after the DOTAP treatment, it can be seen that the surface is coated with DOTAP (positive charge) through the result of the surface charge is changed from negative charge to positive charge when the DOTAP treatment.

The prepared liposome-nucleic acid nanofusions were treated with two types of breast cancer cell lines (MCF-7 and SK-BR-3) to confirm that intracellular delivery was well achieved. As shown in FIG. 7A, it was confirmed that the DOTAP was better delivered into the cells than the presence of DOTAP, and that the liposome-nucleic acid nanofusion having a final size of about 70 nm was best delivered into the cells.

As can be seen in Figure 7b, as confirmed by flow cytometry (MacsQuant VYB, Miltenyi Biotec), when the nucleic acid nanostructures of the present invention is delivered into the cell, the phosphors appear in the same proportion and the nucleic acid structures are delivered in the same ratio into the cell It can be confirmed.

To confirm real-time intracellular multiple diagnosis, liposomes of the present invention in five breast cancer cell lines (HCC-1937, MCF-7, MDA-MB-453, MDA-MB-231, and SK-BR-3) After treatment with the nucleic acid nanofusions, the results obtained through flow cytometry were compared with the results of RT-PCR, which is currently standard. All cancer cells were cultured in 24-well plates and treated with 0.5 mg of liposome-nucleic acid nanofusions on 1 x 10 ⁵ cells. After treatment, the cells were extracted after incubation for 2 hours at 37 ° C. and 5% CO ₂ concentration, and measured by flow cytometry. In the case of RT-PCR, it was measured using TaqMan Fast Universal PCR Master Mix (Applied Biosystems), and a miRNA specific primer kit was purchased from the company (miR-21: Hs04231424_s1, miR-22: Hs00993773_g1). . The expression level of the target miRNA was compared with the result obtained by the method proposed in the present invention and the result confirmed by RT-PCR in FIG. 8A. The primer sequences used are shown in Table 2 below. As a result, it was confirmed that the relative ratios of the results obtained in RT-PCR and the results obtained in the present invention are consistent with all five types of cancer cell tumors used.

As a result, it was confirmed that it is possible to quantitatively compare the expression patterns of miR-21 and miR-22 between cells, and to obtain data on the difference between cells in the same cell line.

Example 4. Development of nucleic acid nanostructures and confirmation of target miRNA detection ability

According to the previous embodiment, the liposome-nucleic acid nanofusion of the present invention is composed of a fluorescence beacon structure capable of specific binding to a target miRNA and a label fluorescent factor capable of quantifying the nanostructure itself when the nanostructure is delivered into a cell. It confirmed through. In addition, the structural stability of the synthesized liposome-nucleic acid nanofusions was verified by electrophoresis technique and molecular dynamic simulation.In the case of specific detection ability of the target miRNA, a comparison of the mixture of different concentrations of the miRNA with different concentrations of the miRNA was performed. Experiments confirmed that miRNA specificity and detectability were effective. The molecular dynamics simulation program used to confirm the structural stability was the freeware oxDNA. On the oxDNA simulation, the nucleic acid strand having the same nucleotide sequence used in the present invention was input, and it was confirmed in FIG. 4 that the internal energy was stably maintained over time. In addition, various concentrations of target miRNAs were treated to nucleic acid nanostructures in order to confirm the detectability, and the increase in fluorescence signal generated therefrom was measured using a fluorescence spectrometer (SpectraMax M5, Molecular Devices), and is shown in FIG. 9A. After assembling the nucleic acid nanostructures, it was confirmed that there was no change in the detection ability of the target miRNA, and mixed the target miR-21 and miR-22 at various concentrations, and the fluorescent signal was dependent only on the concentration of the target miRNA in the mixture The increase was confirmed in FIG. 9B. The fluorescence signal thus obtained is converted to color and shown in FIG. 9C.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above are to be understood in all respects as illustrative and not restrictive.

Claims (27)

1. Branched first nucleic acid structure having a cyclic terminal; And Liposome-nucleic acid nano fusion for quantitative diagnosis of multiple ribonucleic acid markers comprising a silica sphere in which the nucleic acid nanostructures conjugated to the branched second nucleic acid structure having a cyclic terminal bound to the surface inside the liposome.
2. The method of claim 1,

   The first nucleic acid structure is a liposome-nucleic acid nano fusion for quantitative diagnosis of multiple ribonucleic acid markers, characterized in that the linear nucleic acid selected from the group consisting of the base sequence of SEQ ID NO: 1 to 3 in the form of Y-shaped bound.
3. The method of claim 1,

   The second nucleic acid structure is a liposome-nucleic acid nano fusion for quantitative diagnosis of multiple ribonucleic acid markers, characterized in that the linear nucleic acid selected from the group consisting of the base sequence of SEQ ID NO: 4 to 6 in the form of a Y-shaped bound.
4. The method of claim 2 or 3,

   The linear nucleic acid is characterized in that it further comprises a phosphor at the 5 'terminal, liposome-nucleic acid nano fusion for multiple ribonucleic acid marker quantitative diagnosis.
5. The method of claim 2,

   Wherein the first nucleic acid structure is characterized in that it comprises a nucleotide sequence of SEQ ID NO: 7 having a cyclic terminal complementary to the target RNA, liposome-nucleic acid nano fusion for multiple ribonucleic acid marker quantitative diagnosis.
6. The method of claim 3, wherein

   The second nucleic acid structure is characterized in that it comprises a nucleotide sequence of SEQ ID NO: 8 having a cyclic terminal complementary to the target RNA, liposome-nucleic acid nano-fusion for multiple ribonucleic acid marker quantitative diagnosis.
7. The method of claim 1,

   The first nucleic acid structure and the second nucleic acid structure is characterized in that the phosphor and quencher is further labeled, liposome-nucleic acid nano fusion for quantitative diagnosis of multiple ribonucleic acid markers.
8. The method of claim 1,

   The silica sphere is characterized in that 50 nm to 80 nm, liposome-nucleic acid nano fusion for multi-ribonucleic acid marker quantitative diagnosis.
9. The method of claim 1,

   The silica spherical body is surface-modified by sequentially processing aminopropyl trimethoxysilane and cyanuric chloride before the nucleic acid nanostructures are bonded to the surface. Liposome-Nucleic Acid Nanofusion for Marker Quantitative Diagnosis.
10. The method of claim 1,

    The liposome is characterized by consisting of a cationic lipid, liposome-nucleic acid nano fusion for multi-ribonucleic acid marker quantitative diagnosis.
11. The method of claim 10,

    The cationic lipid is DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) and a cationic lipid comprising a cholesterol as a component, multi-ribonucleic acid marker quantitative diagnostic liposome-nucleic acid nano fusion.
12. Biological diagnostic imaging system comprising a liposome-nucleic acid nanofusion for multi-ribonucleic acid marker quantitative diagnosis of claim 1.
13. The method of claim 12,

    And the system measures intracellular fluorescence.
14. Method for preparing a liposome-nucleic acid nanofusion for multi-ribonucleic acid marker quantitative diagnosis comprising the following steps:

    After preparing a branched first nucleic acid structure having a cyclic terminal and a branched second nucleic acid structure having a cyclic terminal, a nucleic acid nanostructure is prepared by conjugation with each other using a ligase enzyme (S1). ;

    Adding a silicate solution to a solvent to synthesize silica nanoparticles (S2);

    Sequentially treating aminopropyl trimethoxysilane and cyanuric chloride on the nanoparticles of step S2 (S3);

    Preparing a nucleic acid nanostructure by reacting the nanoparticle body of step S3 with the nucleic acid nanostructure of step S1 (S4);

    Adding a cationic lipid to the nucleic acid nanostructure of step S4, followed by ultrasonication to prepare a liposome-nucleic acid nanofusion (S5).
15. The method of claim 14,

    The first nucleic acid structure is characterized in that the linear nucleic acid selected from the group consisting of the base sequence of SEQ ID NO: 1 to 3 in the form of a combination of Y-shaped branched, multi-ribonucleic acid marker quantitative diagnostic liposome-nucleic acid nano fusion method .
16. The method of claim 14,

    The second nucleic acid structure is characterized in that the linear nucleic acid selected from the group consisting of the nucleotide sequence of SEQ ID NO: 4 to 6 in the form of a combination of Y-shaped branched, multi-ribonucleic acid marker quantitative diagnostic liposome-nucleic acid nano fusion method .
17. The method according to claim 15 or 16,

    Wherein the linear nucleic acid is characterized in that it further comprises a phosphor at the 5 'terminal, method for producing a liposome-nucleic acid nano fusion for multiple ribonucleic acid marker quantitative diagnosis.
18. The method of claim 15,

    Wherein the first nucleic acid structure is characterized in that it comprises a nucleotide sequence of SEQ ID NO: 7 having a cyclic terminal complementary to the target RNA, liposome-nucleic acid nano fusion for multiple ribonucleic acid marker quantitative diagnosis.
19. The method of claim 16,

    The second nucleic acid structure is characterized in that the cyclic end comprises a nucleotide sequence of SEQ ID NO: 8 having a complementary sequence to the target RNA, multiple ribonucleic acid markers quantitative diagnostic liposome-nucleic acid nano fusion method for the preparation.
20. The method of claim 14,

    The first nucleic acid structure and the second nucleic acid structure is characterized in that the phosphor and quencher further labeled, liposome-nucleic acid nano fusion method for quantitative diagnosis of multiple ribonucleic acid markers.
21. The method of claim 14,

    The silica spherical body is characterized in that 50 nm to 80 nm, multi-ribonucleic acid marker quantitative diagnostic liposome-nucleic acid nano fusion method for diagnostics.
22. The method of claim 14,

    The cationic lipid is DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) and a cationic lipid comprising cholesterol as a component, a method for producing a liposome-nucleic acid nano fusion for quantitative diagnosis of multiple ribonucleic acid markers .
23. The method of claim 14,

    The solvent of the step S2 is methanol, ethanol, distilled water, and ammonia water, characterized in that, multi-ribonucleic acid marker quantitative diagnostic liposome-nucleic acid nano fusion method for manufacturing.
24. The method of claim 23, wherein

    Methanol and ethanol is 6: 4 to 8: characterized in that the volume ratio is mixed, the method for producing a liposome-nucleic acid nano fusion for multi-ribonucleic acid marker quantitative diagnosis.
25. An evaluation method for theoretically evaluating the stability of liposome-nucleic acid nanofusions for quantitative diagnosis of multiple ribonucleic acid markers according to claim 1,

    The method comprises the step of measuring the hydrogen bond energy generated inside the nanofusion, evaluation method.
26. The method of claim 25,

    The measurement method, characterized in that carried out at 35 to 40 ℃.
27. A cancer diagnostic kit comprising a liposome-nucleic acid nanofusion for quantitative diagnosis of multiple ribonucleic acid markers according to claim 1.

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