Tumor marker response starting type DNA nano system and application thereof in tumor cell imaging and inhibition

Abstract

The invention belongs to the technical field of tumor diagnosis and treatment products, and particularly relates to a tumor marker response starting type DNA nano system and application thereof in tumor cell imaging and inhibition. The tumor marker response starting type DNA nano system comprises an arch bridge DNA, an induction DNA, a hairpin A, a hairpin B and a hairpin C, wherein the sequence of the arch bridge DNA is as shown in SEQ ID NO: 2, the sequence of the induction DNA is as shown in SEQ ID NO: 3, the sequence of the hairpin A is as shown in SEQ ID NO: 4, the sequence of the hairpin B is as shown in SEQ ID NO: 5, and the sequence of the hairpin C is as shown in SEQ ID NO: 6. After the tumor marker response starting type DNA nano system and tumor cells are incubated together, the tumor cells can be detected and inhibited.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor diagnosis and treatment products, and specifically relates to a tumor marker response activation type DNA nanosystem and its application in tumor cell imaging and inhibition. Background Art

[0002] At present, the diagnosis of tumors in clinical practice mainly relies on pathological examination and imaging examination. Pathological examination is a pathological morphological method for examining pathological changes in cells, tissues or organs of the body. It is the gold standard for tumor diagnosis, but it will cause trauma to the human body. Imaging examination is the formation of images of the internal structures and organs of the human body, and plays an important role in the early screening of tumors. However, its diagnostic cost is high, the spatial resolution of the image is insufficient, and it is difficult to specifically and quantitatively detect tumor cells at the cellular level. Therefore, it is urgent to explore new tumor imaging methods to reduce costs while improving the accuracy of tumor imaging diagnosis.

[0003] At the same time, due to the large-scale use of anticancer drugs (such as doxorubicin, cisplatin, paclitaxel, etc.) in tumor treatment, multidrug resistance (MDR) cells have become one of the obstacles to chemotherapy drugs in clinical treatment. The emergence of MDR leads to a large amount of anticancer drugs in tumor cells. The accumulation of low-level drugs cannot effectively kill tumor cells, which eventually leads to chemotherapy failure. Therefore, it is urgent to develop a new and efficient chemotherapy drug or treatment method with a different inhibition mechanism from existing anticancer drugs to inhibit tumor cells. Summary of the invention

[0004] The present invention has developed a tumor marker responsive activation DNA nanosystem, in which in the presence of miR-18a, the DNA nanosystem assembles from a neck ring structure into a more stable Y-shaped structure, so that it can be prepared into a product for tumor diagnosis and / or treatment.

[0005] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0006] On the one hand, the present invention provides a tumor marker responsive activation type DNA nanosystem, which comprises: arch bridge DNA, induction DNA, hairpin A, hairpin B and hairpin C; wherein, the sequence of the arch bridge DNA is shown in SEQ ID NO:2, the sequence of the induction DNA is shown in SEQ ID NO:1, the sequence of the hairpin A is shown in SEQ ID NO:1, the sequence of the hairpin B is shown in SEQ ID NO:1, and the sequence of the hairpin C is shown in SEQ ID NO:1.

[0007] Preferably, the tumor marker response-activated DNA nanosystem further comprises ascorbic acid and CuSO4.

[0008] Preferably, the concentration of the arch bridge DNA is 3nM-5nM, the concentration of the inducing DNA is 3nM-4nM, and the concentrations of hairpin A, hairpin B and hairpin are 180μM-220μM, respectively.

[0009] Another aspect of the present invention provides a use of the tumor marker response activation type DNA nanosystem of the present invention in the preparation of a drug for treating a tumor, wherein the tumor contains microRNA-18a.

[0010] Preferably, the tumor in the above application is ovarian cancer.

[0011] In another aspect, the present invention further provides a use of the tumor marker response-activated DNA nanosystem of the present invention in the preparation of a product for diagnosing a tumor, wherein the tumor contains microRNA-18a.

[0012] Preferably, the tumor in the above application is ovarian cancer or breast cancer.

[0013] Preferably, the product in the above application includes a reagent, a kit or a microfluidic chip.

[0014] The beneficial effects of the present invention include at least:

[0015] (1) After the tumor marker response activation DNA nanosystem of the present invention is co-incubated with tumor cells containing the microRNA-18a marker, confocal image analysis is performed, and significant fluorescence is generated in the tumor cells, thereby achieving accurate diagnosis of the tumor.

[0016] (2) After the tumor marker response activation type DNA nanosystem in the present invention is co-incubated with tumor cells containing the microRNA-18a marker, the growth of tumor cells can be significantly inhibited, thereby achieving the treatment of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the construction method of the miR-18a response-activated DNA nano-diagnosis and treatment system provided in an embodiment of the present invention;

[0018] Figure 2 A fluorescence spectrum identification diagram of a DNA nano-diagnosis and treatment system provided by an embodiment of the present invention;

[0019] Figure 3 This is a tumor cell imaging and identification diagram based on DNA nanodiagnosis and treatment provided by an embodiment of the present invention;

[0020] Figure 4 The invention provides an embodiment of the invention for the identification of tumor cell inhibition based on the DNA nano-diagnosis and treatment system. DETAILED DESCRIPTION

[0021] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0022] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0023] An embodiment of the present invention provides a tumor marker responsive activation DNA nanosystem, which includes: arch bridge DNA, induction DNA, hairpin A, hairpin B and hairpin C; wherein the sequence of the arch bridge DNA is shown in SEQ ID NO:2, the sequence of the induction DNA is shown in SEQ ID NO:1, the sequence of the hairpin A is shown in SEQ ID NO:1, the sequence of the hairpin B is shown in SEQ ID NO:1, and the sequence of the hairpin C is shown in SEQ ID NO:1.

[0024] Specifically, the present invention selects the tumor marker miR-18a as the target, designs three different sequences of the neck ring structure DNA; uses the repeated freeze-thaw method to obtain the tumor cell lysate, adds it to the reaction system, and because it contains the highly expressed tumor marker miR-18a, the neck ring structure is induced to form a more stable "Y-type" structure through a chain displacement reaction, and replaces the target DNA so that the "Y-type" structure can be recycled. Copper ions are added, and under the action of sodium ascorbate, the "Y-type" structure is used as a template to form copper nanoclusters, and a tumor marker response activation DNA nanosystem is constructed; the details are as follows:

[0025] The arch bridge DNA (miR-18a complementary sequence) and the partially complementary inducing sequence are used as recognition probes for miR-18a; then hairpin A, hairpin B and hairpin C contain partial sequences of the CuNPs template respectively; among them, hairpin A, hairpin B and hairpin C are designed into a hairpin structure to lock part of the CuNPs synthesis template, and have a single-stranded protruding end at the 3′ end, and only when combined with a specific sequence can the strand displacement reaction be initiated to open the hairpin structure; when only miR-18a is present, it specifically binds to the miR-18a complementary sequence, releases the Trigger DNA, and specifically opens Hairpin A by binding to the protruding end of Hairpin A (single strand 1), triggering the strand displacement amplification reaction and forming an induced DNA-Hairpin A complex; at the same time, the double-stranded region of Hairpin A is opened, releasing the single-stranded region containing 2*; the 2* region of Hairpin A is base-complementarily paired with the protruding end of Hairpin B (i.e., single strand 2), further forming an induced DNA-Hairpin A-Hairpin B complex, releasing the single-stranded region containing 3*. Similarly, the 3* region of Hairpin B is complementary to the base pairing of the protruding end of Hairpin C (i.e., single-stranded 3), prompting the formation of the induced DNA-Hairpin A-Hairpin B-Hairpin C complex; Hairpin C mediates the chain displacement reaction again, and its 1* region is complementary to the base pairing of the protruding end of Hairpin A (single-stranded 1), but the generated DNA-Hairpin A-Hairpin B-Hairpin C complex is not very stable, which prompts the induced DNA to dissociate from the complex, leaving a stable Hairpin A-Hairpin B-Hairpin C complex, i.e., a nanostructure shaped like a Y-shaped structure; the dissociated induced DNA continues to trigger the chain displacement reaction, achieving the continuous release of the induced DNA and the generation of the nanostructure; when the induced DNA does not exist, the chain displacement reaction will not be triggered, and Hairpin A, Hairpin B and Hairpin C coexist in a metastable state; at this time, the formed "Y-shaped" DNA contains a complete CuNPs template.

[0026] In some specific examples, the tumor marker responsive activation DNA nanosystem further comprises ascorbic acid and CuSO4.

[0027] Specifically, sodium ascorbate (Ascorbate), Cu 2+ Reduced to Cu 0 The latter uses the formed "Y-shaped" DNA as a template to generate CuNPs, thereby realizing the construction of a miR-18a responsive activated DNA nano-diagnostic and therapeutic system.

[0028] In some specific examples, the concentration of the arch bridge DNA can be 3nM-5nM, such as 3.5nM, 4nM or 4.5nM, etc., the concentration of the induced DNA can be 3nM-4nM, 3.2nM, 3.5nM or 3.8nM, etc., and the concentrations of hairpin A, hairpin B and hairpin can be 180μM-220μM, such as 190μM, 200μM or 210μM, etc.

[0029] The embodiment of the present invention also provides a use of the tumor marker response activation type DNA nanosystem of the present invention in the preparation of a drug for treating a tumor, wherein the tumor contains microRNA-18a.

[0030] Specifically, the tumor marker responsive activation DNA nanosystem of the present invention can inhibit the growth of tumor cells having the microRNA-18a marker, thereby achieving the effect of treating tumors.

[0031] In some specific examples, the tumor in the above application is ovarian cancer.

[0032] Specifically, the tumor with microRNA-18a marker in the above application can be ovarian cancer. For example, after the tumor marker response activation DNA nanosystem in the present invention is co-incubated with A2780 cells, the survival rate of tumor cells in the experimental group is only 15.5%±4.1%, which is much lower than that of the control group (61.1%±6.8%).

[0033] The embodiment of the present invention also provides a use of the tumor marker response activation type DNA nanosystem of the present invention in the preparation of a product for diagnosing a tumor, wherein the tumor contains microRNA-18a.

[0034] Specifically, the tumor marker responsive activated DNA nanosystem of the present invention can perform cell imaging through fluorescence spectroscopy and can achieve tumor diagnosis through confocal fluorescence microscopy.

[0035] In some specific examples, the tumor in the above application is ovarian cancer or breast cancer.

[0036] For example, after the tumor marker responsive activation DNA nanosystem of the present invention was co-incubated with A2780 cells for 1 hour, confocal image analysis was performed, and significant fluorescence was generated in the tumor cells.

[0037] In some specific examples, the products in the above applications include reagents, kits or microfluidic chips.

[0038] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0039] 1. Construction of DNA Nanosystem

[0040] Example 1

[0041] In the following implementation, the oligonucleotide sequences used are shown in Table 1.

[0042] Table 1 Oligonucleotide sequences used

[0043]

[0044] (1) Design of DNA nanodiagnosis and treatment system

[0045] The tumor marker microRNA-18a (miR-18a) was selected as the target. Through rational design and simulation with DNAMAN software, the construction principle of a DNA nanodiagnosis and treatment system was shown in Figure 1, specifically as follows: first, a capture probe complex is designed, which includes a miR-18a complementary sequence and a partially complementary inducing sequence used as a recognition probe for miR-18a; then hairpin A, hairpin B and hairpin C respectively contain partial sequences of the CuNPs template; wherein, hairpin A, hairpin B and hairpin C are designed into a hairpin structure to lock part of the CuNPs synthesis template, and have a single-stranded protruding end at the 3′ end, and only when combined with a specific sequence can the strand displacement reaction be initiated to open the hairpin structure; when only miR-18a is present, it specifically binds to the miR-18a complementary sequence, releases Trigger DNA, and specifically opens Hairpin A by binding to the protruding end of Hairpin A (single strand 1), triggering the strand displacement amplification reaction, and forming an induced DNA-Hairpin A complex; at the same time, the double-stranded region of Hairpin A is opened, releasing a single-stranded region containing 2*; the 2* region of Hairpin A is complementary to the protruding end of Hairpin B (i.e., single strand 2) to further form an induced DNA-Hairpin A-Hairpin B complex, releases the single-stranded region containing 3*. Similarly, the 3* region of Hairpin B is complementary to the base of the protruding end (i.e., single-stranded 3) of Hairpin C, which promotes the formation of the induced DNA-Hairpin A-Hairpin B-Hairpin C complex; Hairpin C mediates the chain displacement reaction again, and its 1* region is complementary to the base of the protruding end (single-stranded 1) of Hairpin A, but the generated DNA-Hairpin A-Hairpin B-Hairpin C complex is not very stable, which prompts the induced DNA to dissociate from the complex, leaving behind a stable HairpinA-Hairpin B-Hairpin C complex, i.e., a Y-shaped nanostructure; the dissociated induced DNA (SEQ ID NO: 3) continues to trigger the chain displacement reaction, achieving the continuous release of the induced DNA and the generation of the nanostructure; when the induced DNA is not present, the chain displacement reaction will not be triggered, and Hairpin A, Hairpin B and Hairpin C coexist in a metastable state; at this time, the formed "Y-shaped" DNA contains a complete CuNPs template; sodium ascorbate (Ascorbate) is added, Cu 2+ Reduced to Cu 0 The latter uses the formed "Y-shaped" DNA as a template to generate CuNPs, thereby realizing the construction of a miR-18a responsive activated DNA nano-diagnostic and therapeutic system.

[0046] (2) Preparation of reaction solution

[0047] The arch bridge DNA (SEQ ID NO: 2) and the induction DNA (SEQ ID NO: 3) solutions were mixed, and the Hairpin A solution, Hairpin B solution and Hairpin C solution were mixed, and they were placed in a PCR instrument, pre-denatured at 95°C for 5 minutes, and then slowly cooled to room temperature at 1°C / min;

[0048] (3) Construction of Nanosystems

[0049] The complementary arch bridge DNA and induced DNA mixed solution were added to the cell lysate of MCF-7 and 293T cells respectively, and incubated at room temperature for 90 minutes; the complementary Hairpin A solution, Hairpin B solution and Hairpin C solution were added, and the incubation continued at room temperature for 90 minutes; the final concentration of the arch bridge DNA solution was 4nM, the final concentration of the induced DNA solution was 3.33nM, and the final concentration of the hairpin structure was 200μM; after the samples were prepared according to the above operation, they were placed at 4°C for subsequent use;

[0050] (4) Construction of nano-diagnosis and treatment system

[0051] To the prepared DNA nanosystem (the mixed solution), add an ascorbic acid solution with a final concentration of 3.84 mM, and incubate at room temperature for 30 minutes; then, add a CuSO4 solution with a final concentration of 66.67 mM;

[0052] (5) Fluorescence spectroscopy analysis of DNA nanodiagnosis and treatment systems

[0053] At room temperature, the fluorescence intensity of the experimental samples was recorded in the range of 300-400 nm using a fluorescence spectrophotometer F-7100 (Hitachi) to find the maximum excitation wavelength (λex); the fluorescence intensity of the above experimental samples was recorded in the range of 500-700 nm to find the maximum emission wavelength (λem); each group of samples was measured 3 times; the results are shown in Figure 2 As shown, the maximum excitation wavelength of the constructed DNA nano-diagnosis and treatment system is 345nm, and the maximum emission wavelength is 605nm.

[0054] 2. DNA Nanosystem Testing

[0055] (I) Tumor cell imaging test

[0056] A2780 cells were cultured at 10 5The cells were inoculated at a density of 10 cells / dish in a confocal culture dish, 90% 1640 culture medium was added, and the cells were cultured in a CO2 incubator for 24 hours. The prepared DNA nanodiagnosis and treatment system was added to the culture dish and incubated in a CO2 incubator, keeping the cells away from light. After 1 hour, the cells were removed from the incubator, the culture medium was removed, the cells were washed 3 times with 1×PBS, and the cells were suspended in 1×PBS for detection, keeping the cells away from light.

[0057] The above cell samples were observed using a confocal fluorescence microscope to observe the fluorescence imaging capability of the nano-diagnosis and treatment system. Figure 3 As shown, A2780 showed significant fluorescence enhancement, indicating that the constructed DNA nanodiagnostic and therapeutic system has imaging function for tumor cells.

[0058] (II) Tumor cell inhibition test

[0059] A2780 cells were cultured at 10 5 The cells were inoculated at a density of 10 cells / dish in a confocal culture dish, 90% 1640 culture medium was added, and the dish was cultured in a CO2 incubator for 24 hours. The prepared DNA nano-diagnosis and treatment system was incubated with human ovarian cancer cells A2780 at 37°C. After the target time was reached, fresh culture medium containing CCK-8 reagent was added to observe the tumor cell inhibition ability of the nano-diagnosis and treatment system.

[0060] The results are as follows Figure 4 As shown in the figure, with the extension of incubation time, the survival rate of A2780 decreased significantly, indicating that the constructed DNA nano-diagnosis and treatment system has an inhibitory function on tumor cells; while in the control group, that is, without the formation of the DNA nano-diagnosis and treatment system (fluorescent nanorobot), the survival rate of Cu 2+ The effect of ascorbic acid on cell survival was weak after co-incubation with cells.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. A tumor marker responsive activated DNA nanosystem, characterized in that: include: Arch bridge DNA, inducing DNA, hairpin A, hairpin B and hairpin C; wherein the sequence of the arch bridge DNA is shown in SEQ ID NO: 2, the sequence of the inducing DNA is shown in SEQ ID NO: 3, the sequence of the hairpin A is shown in SEQ ID NO: 4, the sequence of the hairpin B is shown in SEQ ID NO: 5, and the sequence of the hairpin C is shown in SEQ ID NO:

6.

2. The tumor marker response-activated DNA nanosystem according to claim 1, characterized in that: Also included are ascorbic acid and CuSO4.

3. The tumor marker response activated DNA nanosystem according to claim 1, characterized in that: The concentration of arch bridge DNA is 3nM-5nM, the concentration of inducing DNA is 3nM-4nM, and the concentrations of hairpin A, hairpin B and hairpin are 180μM-220μM, respectively.

4. Use of the tumor marker responsive activated DNA nanosystem according to any one of claims 1 to 3 in the preparation of a product for treating tumors, wherein the tumor marker is microRNA-18a.

5. The use according to claim 4, characterized in that: The tumor is ovarian cancer cells.

6. Use of the tumor marker responsive activated DNA nanosystem according to any one of claims 1 to 3 in the preparation of a product for diagnosing tumors, wherein the tumor marker is microRNA-18a.

7. The use according to claim 6, characterized in that: The tumor is ovarian or breast cancer cells.

8. The use according to claim 6 or 7, characterized in that: Products include reagents, kits or microfluidic chips.

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