Nucleic acid nanocarriers and uses thereof

Abstract

The application discloses a nucleic acid nano drug carrier which can be used for small molecule anticancer drug platinum drug delivery and drug efficacy enhancement. The nucleic acid nano drug carrier has high sensitization property and can quickly enter eukaryotic cells. The carrier can accurately target and deliver small molecule drug platinum drugs into tumor cells, effectively kill tumor cells and regulate the expression of drug resistance genes in tumor cells. Compared with traditional drug carriers, the nano carrier is safe, non-toxic, high in drug loading capacity and can realize real-time monitoring, and has a wider application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to a nucleic acid nanocarrier and its application in the field of biotechnology. BACKGROUND

[0002] Cancer is one of the most important diseases threatening human health, and chemotherapy is one of the main means of tumor treatment. Currently, six platinum-based antitumor drugs have been approved for clinical antitumor treatment of various solid tumors. However, platinum drugs have obvious systemic toxicity, and clinical application is faced with major problems such as severe dose toxicity, drug inactivation, and drug resistance. Although the nanodelivery system constructed by liposomes, micelles, dendrimers, and polymers overcomes the defects of traditional platinum drug preparations, effectively controls drug delivery and release in vivo, thereby enhancing the therapeutic effect and reducing the toxic side effects, but it is limited by high toxicity, weak targeting, complex synthesis, and other factors, which hinders its wide application in clinical treatment. Compared with synthetic materials, endogenous material-derived carriers are very beneficial to the development of nanomedicine. With the development of DNA nanostructures, DNA is considered a good drug delivery carrier due to its high biocompatibility and unique advantages. People can control the synthesis of DNA nanostructures of different sizes and shapes as biosensors, drug targeted transport carriers, etc. DNA tetrahedron has the advantages of size controllability, structural stability, and easy modification, and is widely used for the delivery of small molecule drugs.

[0003] Various anticancer drugs, including platinum drugs, can cause DNA damage, and cancer cells repair DNA damage through the base excision repair pathway (BER), thereby developing drug resistance. In this pathway, apurinic / apyrimidinic endonuclease (APE1) plays an important role. Apurinic / apyrimidinic sites (AP sites) are easily formed at the damaged site, and APE1 can specifically recognize and cut the site to repair DNA. By inactivating the BER pathway of tumor cells and inhibiting the expression of APE1 enzyme, it is expected to enhance the therapeutic effect of platinum anticancer drugs. SUMMARY

[0004] The technical problem to be solved by the present application is how to target and efficiently deliver small molecule platinum drugs to tumor cells while sensitizing platinum drugs and enhancing drug efficacy.

[0005] To solve the above technical problems, the present application first provides a nucleic acid nanocarrier.

[0006] The nucleic acid nano-drug carrier provided by the application is composed of a DNA tetrahedron main structure with several elongated chains at the vertexes, and five short chains connected with the elongated chains through base complementary pairing; the five short chains are respectively: a DNA single strand containing an AS1411 aptamer, a siRNA single strand containing an inhibitor of deoxyribose nuclease APE1 enzyme, an RNA single strand containing a reverse complementary region of the siRNA single strand, and two nucleic acid probe DNA single strands for detecting the inhibition of deoxyribose nuclease APE1 enzyme; one of the two nucleic acid probe DNA single strands is modified with a fluorescent group at the 5' end, and the other is modified with a quenching group at the 3' end, and has an AP site at a position 8-10 bp adjacent to the quenching group; the two nucleic acid probe DNA single strands are complementary to the same elongated chain, and the fluorescent group and the quenching group are adjacent after complementary binding.

[0007] The working principle of the nucleic acid nano-drug carrier provided by the application is as follows: platinum drugs are delivered to cells through the DNA tetrahedron, and the DNA tetrahedron nanostructure is loaded with an AS1411 nucleic acid aptamer, an siRNA of APE1 enzyme, and a DNA probe for characterizing APE1 activity, respectively. The AS1411 aptamer is used to target the nucleolin protein on the surface of tumor cell membranes, so that the platinum drugs are accurately and precisely targeted and delivered to tumor cells; the siRNA containing the inhibitor of APE1 enzyme is used to inhibit the base excision repair pathway (BER) of tumor cells; and the DNA probe for characterizing the inhibition of APE1 enzyme is used to monitor the inhibition of APE1 enzyme in real time.

[0008] In the nucleic acid nano-drug carrier, the fluorescent group is J0E, HEX, VIC, R0X, CY3 or CY5, and the quenching group is BHQ2 or BHQ3.

[0009] In the nucleic acid nano-drug carrier, the DNA tetrahedron main structure with several elongated chains at the vertexes is composed of four DNA long single strands self-assembled, and the three vertexes of the tetrahedron have elongated chains. The nucleotide sequences of the four DNA long single strands can be specifically P1 (as shown in SEQ ID NO: 1), P2 (as shown in SEQ ID NO: 2), P3 (as shown in SEQ ID NO: 3), and P4 (as shown in SEQ ID NO: 4) in Table 1.

[0010] The two nucleic acid probe DNA single strands for detecting the inhibition of the deprotonated pyrimidine endonuclease APE1 enzyme can be specifically P5 (as shown in SEQ ID NO: 5) and P6 (as shown in SEQ ID NO: 6) in Table 1, both of which have sequences complementary to P1 (the sequence complementary to P1 of P5 is indicated by underlining, and the sequence complementary to P1 of P6 is indicated by a wave line): wherein the 5' end of P5 is modified with a fluorescent group; the 3' end of P6 is modified with a quenching group, and the position 8-10 bp adjacent to the quenching group has an AP site; and the fluorescent group and the quenching group of P5 and P6 are adjacent.

[0011] The DNA single strand containing the AS14111 aptamer in the nucleic acid nanocarrier can be specifically P7 (as shown in SEQ ID NO: 7, wherein the first to 23th positions are the sequence of the AS14111 aptamer) in Table 1, which has a sequence complementary to P3 (the 24th to 41th positions of SEQ ID NO: 7, which is complementary to the 62th to 84th positions of SEQ ID NO: 3).

[0012] The siRNA single strand for inhibiting the deprotonated pyrimidine endonuclease APE1 enzyme in the nucleic acid nanocarrier can be specifically the RNA single strand of P8 (as shown in SEQ ID NO: 8) in Table 1, which has a sequence complementary to P2 (the first to 20th positions of SEQ ID NO: 8, which is complementary to the first to 20th positions of SEQ ID NO: P2) and a sequence complementary to P9 (the 26th to 44th positions of SEQ ID NO: 8, which is complementary to the first to 19th positions of SEQ ID NO: 9). P8 and P9 are complementary to form APE1 siRNA.

[0013] The RNA single strand complementary to the reverse complementary region of the siRNA single strand in the nucleic acid nanocarrier is shown in SEQ ID NO: 9, which is complementary to the 26th to 44th positions of SEQ ID NO: 8.

[0014] The application also provides a preparation method of the nucleic acid nanocarrier, which comprises annealing and assembling each DNA single strand to obtain DTN, annealing and assembling each siRNA single strand to obtain siRNA, and annealing and assembling the DTN and the siRNA to obtain the nucleic acid nanocarrier.

[0015] The application also provides a use method of the nucleic acid nanocarrier, which comprises the step of loading the platinum drug into the nucleic acid nanocarrier.

[0016] The application also provides a product obtained by loading the drug into the nucleic acid nanocarrier.

[0017] In the product, the drug is a cancer treatment drug.

[0018] In the product, the cancer treatment drug can be a platinum drug. In an embodiment of the present application, the platinum drug is cisplatin or C8-cisplatin.

[0019] In the product, the product can be a drug. The product can be used for treating a disease, such as cancer. The cancer can be lung cancer, such as non-small cell lung cancer.

[0020] To solve the above technical problems, the present application also provides any one of the following W1-W5:

[0021] W1, application of the above nucleic acid nanocarrier in preparing a product for enhancing the efficacy of a cancer treatment drug;

[0022] W2, application of the above nucleic acid nanocarrier in preparing a cancer treatment drug;

[0023] W3, application of the above product in preparing a cancer treatment drug;

[0024] W4, application of the above product in preparing a product for inhibiting the growth of cancer cells;

[0025] W5, application of the above product in preparing a product for reducing the viability of cancer cells.

[0026] In the application, the cancer treatment drug can be a lung cancer treatment drug. The lung cancer treatment drug can be a non-small cell lung cancer treatment drug. Specifically, the cancer treatment drug can be cisplatin or C8-cisplatin.

[0027] In the application, the cancer can be lung cancer, such as non-small cell lung cancer. The cancer cells can be lung cancer cells, such as non-small cell lung cancer cells.

[0028] The nucleic acid nanocarrier for delivering small-molecule anticancer drug platinum drugs disclosed by the present application has the following characteristics: (1) the DNA tetrahedron skeleton main part carries cisplatin molecules into cells; (2) the expression of apurinic-apyrimidinic endonuclease APE1 in the gene repair process is inhibited by APE1 siRNA, the tumor cell damage repair pathway is inhibited, and the efficacy of platinum drugs is enhanced; (3) a nucleic acid probe is designed to detect and evaluate the inhibition effect of APE1 enzyme; (4) the loading of AS1411 aptamer can accurately target tumor cells and avoid damage to normal cells. It has been verified by experiments that the nucleic acid nanocarrier of the present application has high sensitization property and can quickly enter tumor cells. The carrier can accurately target the delivery of small-molecule drug platinum drugs to tumor cells, effectively kill tumor cells and regulate gene expression in tumor cells. Compared with traditional drug carriers, the nanocarrier is safe, non-toxic, has high drug loading capacity, and can be monitored in real time, and has a wider application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 Structure diagram of nucleic acid nanocarrier of embodiment 1 of the present application.

[0030] Figure 2 Electrophoresis characterization diagram of nucleic acid skeleton structure and nucleic acid nanocarrier of embodiment 1 of the present application. Wherein "+" represents adding the chain, and "-" represents not adding the chain. Figure 2 A diagram of FIG. 1 is an electrophoresis characterization diagram of step-by-step assembly of the nucleic acid skeleton structure, Figure 2 B diagram of FIG. 1 is an electrophoresis characterization diagram of assembly of the nucleic acid nanocarrier.

[0031] Figure 3 Atomic force microscope characterization diagram of the nucleic acid nanocarrier of embodiment 1 of the present application. Figure 3 A diagram of FIG. 2 is the nucleic acid nanocarrier, Figure 3 B diagram of FIG. 2 is the nucleic acid nanocarrier after loading drugs.

[0032] Figure 4 Cell uptake diagram of the nucleic acid nanocarrier of embodiment 1 of the present application to tumor cells A549. Wherein cy3 is the cell under the laser emission source with excitation wavelength of 520 nm, cy5 is the cell under the laser emission source with excitation wavelength of 620 nm, nucleus staining is Hoechst 33342, which is the cell under the mercury lamp emission source with excitation wavelength of 350 nm, and merge is the fluorescence overlap combination diagram. The dotted part is the content of APE1.

[0033] Figure 5 Cell survival rate diagram of the nucleic acid nanocarrier of embodiment 1 of the present application loaded with cisplatin and tetravalent platinum drug C8-cisplatin to tumor cells A549. The data shown are mean ± standard deviation, and the number of repetitions is 3.

[0034] Figure 6 Body weight change diagram of mice with treatment time in the tumor inhibition experiment of PTX model mice in embodiment 1 of the present application. The data shown are mean ± standard deviation, and the number of repetitions is 5.

[0035] Figure 7 Volume change diagram of tumor of mice with treatment time in the tumor inhibition experiment of PTX model mice in embodiment 1 of the present application. The data shown are mean ± standard deviation, and the number of repetitions is 5.

[0036] Figure 8 Picture of change of ex vivo tumor of mice in the tumor inhibition experiment of PTX model mice in embodiment 1 of the present application.

[0037] Figure 9 Bar chart of weight of ex vivo tumor of mice in the tumor inhibition experiment of PTX model mice in embodiment 1 of the present application. The data shown are mean ± standard deviation, and the number of repetitions is 5. DETAILED DESCRIPTION

[0038] The present application is further described in detail in connection with the preferred embodiments below, given solely by way of illustration and not by way of limitation. The following examples provided serve as a guide for further improvement by those skilled in the art, and do not in any way constitute a limitation on the present application.

[0039] In the quantitative test of the following examples, three repeated experiments were set up, and the results were averaged.

[0040] In the experimental methods of the following examples, unless otherwise specified, they are all conventional methods. In the following examples, unless otherwise specified, the materials, reagents, etc. can be obtained from commercial channels.

[0041] Experimental Example 1

[0042] I. Preparation of nucleic acid nano-drug carrier based on DNA nano-carrier loaded with platinum anticancer drugs

[0043] The inventors constructed a nucleic acid nano-drug carrier based on DNA nano-carrier loaded with platinum anticancer drugs, the structural diagram of which is shown in Figure 1 The 5 short chains are: DNA single strand containing AS1411 aptamer, siRNA single strand containing inhibition of apurinic endonuclease APE1 enzyme, RNA single strand containing reverse complementary region of the siRNA single strand, two nucleic acid probe DNA single strands containing detection of apurinic endonuclease APE1 enzyme inhibition.

[0044] In the above nucleic acid nano-drug carrier, the DNA tetrahedral main structure with several extended chains at the vertices is composed of 4 DNA long single strands, and the 3 vertices of the tetrahedron have extended chains. The nucleotide sequence of the 4 DNA long single strands can be specifically P1 (as shown in SEQ ID NO: 1), P2 (as shown in SEQ ID NO: 2), P3 (as shown in SEQ ID NO: 3), and P4 (as shown in SEQ ID NO: 4) in Table 1.

[0045] In the nucleic acid nanocarrier, the two nucleic acid probe DNA single strands for detecting the inhibition of apurinic / apyrimidinic endonuclease APE1 enzyme can be specifically as shown in P5 and P6 in Table 1, both of which have sequences complementary to P1 (the sequence of P5 complementary to P1 is indicated by underlining, and the sequence of P6 complementary to P1 is indicated by a wavy line). The 5' end of P5 is modified with a fluorescent group; the 3' end of P6 is modified with a quencher group, and the position 8-10 bp adjacent to the quencher group has an AP site; the fluorescent group and the quencher group of P5 and P6 are adjacent; the fluorescent group is selected from J0E, HEX, VIC, R0X, CY3 or CY5, and the quencher group is selected from BHQ2 or BHQ3; preferably, the fluorescent group is CY5, and the quencher group is BHQ3.

[0046] In the nucleic acid nanocarrier, the DNA single strand containing AS14111 aptamer can be specifically as shown in P7 in Table 1 (as shown in SEQ ID NO: 7, wherein positions 1-23 are the sequence of AS14111 aptamer), which has a sequence complementary to P3 (positions 24-41 of SEQ ID NO: 7, which is complementary to positions 62-84 of SEQ ID NO: 3).

[0047] In the nucleic acid nanocarrier, the siRNA single strand containing the apurinic / apyrimidinic endonuclease APE1 enzyme inhibitor can be specifically as shown in the RNA single strand of P8 in Table 1 (as shown in SEQ ID NO: 8), which has a sequence complementary to P2 (positions 1-20 of SEQ ID NO: 8, which is complementary to positions 1-20 of SEQ ID NO: P2) and a sequence complementary to P9 (positions 26-44 of SEQ ID NO: 8, which is complementary to positions 1-19 of SEQ ID NO: 9). P8 and P9 form APE1 siRNA.

[0048] In the nucleic acid nanocarrier, the RNA single strand containing the region complementary to the siRNA single strand is as shown in SEQ ID NO: 9, which is complementary to positions 26-44 of SEQ ID NO: 8.

[0049] Table 1 Sequences

[0050]

[0051]

[0052] The preparation method of the nucleic acid nano drug carrier comprises the following steps: 2.5 μM of P1-P7 is respectively added in a 50 μL system, 10×TM buffer is used as a buffer, and a preliminary nucleic acid nano drug carrier is formed by PCR annealing self-assembly. The preparation method of the 10×TM buffer is as follows: 968.8 mg of tris-hydroxymethyl aminomethane and 1.072 g of magnesium acetate are accurately weighed, poured into a 50 ml centrifuge tube, 40 ml of deionized water is added to dissolve, and ice acetic acid is used to adjust the pH value to 8.0. The PCR program is as follows: 80 ℃ for 10 min, 4 ℃ for 30 min. The prepared preliminary nucleic acid nano drug carrier is a DTN, which is stored in a 4 ℃ refrigerator.

[0053] Further, the siRNA double chain is prepared, the P8 and P9 RNA powders are taken out from the refrigerator, placed in a high-speed centrifuge, balanced, and the parameters are set as 4 ℃, 12,000 r / min and 2 min; the amount of DEPC water required is added on a sterile operation test bench according to the information on the siRNA tube outer package, and a siRNA solution with a concentration of 20 μM is prepared. The prepared DTN solution with a concentration of 1 μM is placed in an ultra-clean bench, a 200 μL enzyme-free PCR tube is taken, 17.5 μL of DEPC water, 5 μL of the prepared 10×TM buffer (prepared with DEPC water and sterilized), 25 μL of the DTN solution with a concentration of 1 μM, and 2.5 μL of the siRNA solution with a concentration of 20 μM are added in sequence, and the total system is 50 μL. Annealing. The PCR annealing program is as follows: 75 ℃ for 2 min, 65 ℃ for 1 min, 55 ℃ for 1 min, 45 ℃ for 1 min, and 37 ℃ for 1 min. The nucleic acid nano drug carrier is prepared and stored in a 4 ℃ refrigerator.

[0054] The nucleic acid nano drug carrier synthesized in the embodiment is characterized by using polyacrylamide gel electrophoresis, and the results are shown in Figure 2 , which shows that the synthesized nucleic acid skeleton structure and the nucleic acid nano drug carrier (DTN) are as shown in Figure 2 , and the experimental results are as shown in Figure 2 . As shown in A of Figure 2 , the gradual assembly process of the DNA tetrahedron can be clearly seen, the tetrahedron is composed of seven nucleic acid chains P1-P7, and since the larger the molecular weight of the nucleic acid in the Native-page is, the slower the migration speed in the lane is, with the continuous addition of the nucleic acid chain, the molecular weight increases, the electrophoresis band migration slows down, and finally a clear gradient appears, and no impurity band appears. Figure 2 . As shown in B of Figure 2 , the lane 3 in B of Figure 2Lane 2 of B figure of the siRNA double-stranded structure without adding siRNA, the increase of molecular weight indicates that the siRNA double-stranded structure is successfully assembled, and there is no degradation phenomenon, and the preparation is completed.

[0055] AFM characterization of nucleic acid nano drug carriers (DTN) and nucleic acid nano drug carriers carrying small molecule platinum drugs: dilute 1 μM nucleic acid nanostructure to 5 nM, drop 5 μL 5 nM DTN and 5 μL C8-cis-DTN on mica sheet, and dry. Use Bruker tapping mode probe OTESPA-R3 to scan and image. The results show that: Figure 3 The size of nucleic acid nano drug carriers in A figure is 15 nm, Figure 3 The size of nucleic acid nano drug carriers in B figure after loading small molecule platinum drugs is slightly increased, about 20 nm.

[0056] II. Uptake of nucleic acid nano drug carriers by A549 cells

[0057] A549 cells (Wuhan Punsai Life Science and Technology Co., Ltd., item number CL-0016) were selected. A549 cells with a confluence of 80% were taken out of the incubator, and 1 ml of trypsin was added for digestion. After centrifugation, RPMI-1640 medium was added to the cells, and they were blown evenly with a pipette gun. 200 ml was taken and added to a confocal culture dish, and the cells taken were about 100,000. They were cultured in a 37°C, 5% CO2 cell incubator for 12 h to allow the cells to adhere. The confocal culture dish was taken out, and the concentration of the two nucleic acid nano probes was 200 μL x 200 nM / dish. Each dish contained 180 μL of RPMI-1640 medium, 20 μL of 50 μL x 2 μM nucleic acid nanostructure, and 1 μL of Hoechst 33342 (2.5 μg / mL). After incubation in a 37°C, 5% CO2 incubator for several hours, the cells were photographed. Different nucleic acid nano drug carriers were added to the confocal culture dish, and the complete nucleic acid nano drug carrier treatment was used as a control without adding APE1 siRNA. The specific settings are as follows:

[0058] Complete nucleic acid nano drug carrier treatment: 200 μL of complete nucleic acid nano drug carrier was added to the culture system at a final concentration of 200 nM. The preparation method of the complete nucleic acid nano drug carrier is described in the first part of this example. The raw nucleotide chains used were P1, P2, P3, P4, P5, P6, P7, P8, and P9.

[0059] Nucleic acid nanocarrier treatment control without APE1 siRNA: 200 μL of the culture system was added to a final concentration of 200 nM nucleic acid nanocarrier without APE1 siRNA. The raw material nucleotide chains used were P1, P2, P3, P4, P5, P6, and P7 (the raw material nucleotides used were not connected to P8 and P9). Other parameters remained exactly the same as in the first part of this example.

[0060] After incubating in a 37°C incubator for 4 hours, the cells were imaged under a total internal reflection fluorescence microscope. The laser and mercury lamp were turned on, and the focus was adjusted to identify cells with good morphology. Cells were imaged under bright field, laser, and mercury lamp conditions. A 40× oil immersion lens was used. Cy5 excitation wavelength was 620 nm, and emission wavelengths were 640-720 nm. Cy3 excitation wavelength was 520 nm, and emission wavelengths were 540-640 nm. Hoechst 33342 excitation wavelength was 350 nm, and emission wavelength was 460 nm.

[0061] The results are as follows Figure 4 As shown, Figure 4 The middle (upper row) shows that -Cy5 in the nucleic acid nanoparticles without APE1 siRNA entered the cells, but the cell fluorescence was very strong, indicating that more APE1 protein was detected; Figure 4 middle( Figure 4 The lower row of pictures in the figure shows the fluorescence overlay of cy5 and cy3 after the complete nucleic acid nanocarrier enters the cell at the same time. It can be seen that the smart nucleic acid nanocarrier is successfully assembled. At the same time, the fluorescence of cy5 in the dotted part is observed to be weakened, indicating that the addition of APE1 siRNA has inhibited the expression of APE1 protein to a certain extent.

[0062] 3. Cytotoxicity of Nucleic Acid Nanocarriers to A549 Cells

[0063] 1: Cytotoxicity of cisplatin-loaded nucleic acid nanoparticles to A549 cells

[0064] 1.1 First, prepare a cisplatin solution. Cisplatin is a water-soluble platinum drug, and the cisplatin used is Aladdin, product number D109812. Use an analytical electronic balance to weigh 0.3 mg of cisplatin powder and dissolve it in 1 ml of sterile, mold-free water to prepare a 1 mM cisplatin solution. Dilute the solution to various concentrations using sterile, mold-free water. Store at 4°C, protected from light.

[0065] 1.2 Further connect the prepared nucleic acid nanocarrier with cisplatin molecules to obtain nucleic acid nanocarrier loaded with cisplatin molecules:

[0066] 1.2.1 The complete nucleic acid nanocarrier prepared according to the first part of the application was mixed with cisplatin solution in the dark at 4°C with constant shaking for 60 min. The cisplatin-nucleic acid nanocarrier was obtained by ultrafiltration three times with 0.5 mL 30K ultrafiltration centrifuge tube at 6000 rpm for 5 min.

[0067] 1.2.2 The nucleic acid nanocarrier prepared according to the first part of the application without APE1 siRNA was mixed with cisplatin solution in the dark at 4°C with constant shaking for 60 min. The cisplatin-nucleic acid nanocarrier (without siRNA) was obtained by ultrafiltration three times with 0.5 mL 30K ultrafiltration centrifuge tube at 6000 rpm for 5 min.

[0068] 1.3 A549 cells were taken out of the incubator for cell counting. A549 cells in good condition with a confluence of 80% were taken and cultured until the cell density counted under a microscope was 6.5 x 10 4 / mL. The cell mixture with a density of 6.5 x 10 4 / mL was added to a 96-well plate, 100 μL per well, and 100 μL of PBS was added to each well around the 96-well plate (to prevent evaporation of the cell mixture). The 96-well plate with the cells was placed in a CO2incubator for 12 h. The 96-well plate with the A549 cells was taken out of the CO2incubator and observed under an inverted microscope. If the cell density in the 96-well plate was uniform and the cell morphology was good, the sample was added, 10 μl per well. The final concentration of the nucleic acid nanocarrier was set to 200 nM; the final concentration of cisplatin, cisplatin-nucleic acid nanocarrier (without siRNA), and cisplatin-nucleic acid nanocarrier was set to 0.005, 0.05, 0.5, 5, 10, and 20 μM. After the sample was added, the 96-well plate was placed in a CO2incubator for incubation, and after 48 h, the cells were taken out and 10 μL of CCK-8 was added to each well, and the 96-well plate was gently shaken to mix the contents. Subsequently, the 96-well plate was placed in a CO2incubator for 2-4 h, and the cell mixture in the 96-well plate turned yellow. The OD 450 values of the A549 cells in each sample were measured using a multifunctional enzyme marker. The cell activity was calculated, and the results are shown in Figure 5 From the graph, it can be seen that the smart nanocarrier system can sensitize cisplatin, reduce cell viability, and enhance the drug efficacy, and the enhancement of the drug efficacy is related to the concentration.

[0069] 2: Toxicity of nucleic acid nanocarrier loaded with tetravalent platinum drug C8-cisplatin to A549 cells

[0070] 2.1 First, prepare the solution of the tetravalent platinum drug C8-cisplatin, C8-cisplatin is an oil-soluble platinum drug, and the C8-cisplatin used is prepared by the Institute of Chemistry, Chinese Academy of Sciences, and the synthesis literature is: Near-Infrared Light Irradiation Induced Mild Hyperthermia Enhances Glutathione Depletion and DNA Interstrand Cross-Link Formation for Efficient Chemotherapy. 0.7 mg of cisplatin powder is weighed using an analytical electronic balance, dissolved in 1 ml of DMSO, and prepared into an 8 mM solution of C8-cisplatin. The solution is diluted to different concentrations using sterile water, and stored at 4°C in the dark.

[0071] 2.2 Further, the prepared nucleic acid nano-drug carrier is connected with the C8-cisplatin molecule to obtain a C8-cisplatin-loaded nucleic acid nano-drug carrier:

[0072] 2.2.1 The complete nucleic acid nano-drug carrier prepared according to the first part of the application is mixed with the C8-cisplatin solution, and is uniformly shaken at 4°C in the dark for 60 min. The C8-cisplatin-nucleic acid nano-drug carrier is obtained by ultrafiltration three times with a 0.5 mL 30K ultrafiltration centrifuge tube at 6000 rpm for 5 min.

[0073] 2.2.2 The nucleic acid nano-drug carrier prepared according to the first part of the application without APE1 siRNA is mixed with the C8-cisplatin solution, and is uniformly shaken at 4°C in the dark for 60 min. The C8-cisplatin-nucleic acid nano-drug carrier (without siRNA) is obtained by ultrafiltration three times with a 0.5 mL 30K ultrafiltration centrifuge tube at 6000 rpm for 5 min.

[0074] 2.3 A549 cells are taken out of the thermostat incubator for detection, and A549 cells in good condition with a confluence of 80% are taken, and the cell density counted under a microscope is 6.5 x 10 4 / mL; the density of the cells is 6.5 x 10 4 / mL of cell mixture was added to a 96-well plate, 100 μL was added to each well, 100 μL of PBS was added to each well around the 96-well plate (to prevent evaporation of the cell mixture). The 96-well plate with the seeded cells was placed in a CO2incubator for 12 h. The 96-well plate with the seeded A549 cells was taken out of the CO2incubator and placed on an inverted microscope to observe. If the cell density in the 96-well plate was uniform and the cell morphology was good, the sample could be added, 10 μL of sample was added to each well. The final concentration of the nucleic acid nano-drug carrier was set to 200 nM; the final concentration of C8-cisplatin, C8-cisplatin-nucleic acid nano-drug carrier (without siRNA), C8-cisplatin-nucleic acid nano-drug carrier was set to 0.005 μM, 0.05 μM, 0.5 μM, 5 μM, 10 μM, and 20 μM. After the sample was added, the 96-well plate was placed in a CO2incubator for incubation, and after 48 h, the cells were taken out, 10 μL of CCK-8 was added to each well, and the 96-well plate was gently shaken to mix evenly. Subsequently, the 96-well plate was placed in a CO2incubator for 2-4 h, and the cell mixture in the 96-well plate turned yellow. The OD 450 values of the A549 cells in each sample were measured using a multifunctional enzyme marker. Figure 5 As can be seen from the figure, the nucleic acid nano-drug carrier can sensitize C8-cisplatin and enhance its efficacy.

[0075] Four, mouse tumor inhibition experiment

[0076] BALB / c-Nude mice (Nanjing Model Biological Product, item number NM-NSG-008) of 8-9 weeks old were selected, and after being fed to a body weight of about 20 g, a nude mouse PDX model was constructed, and all experiments met the ethical requirements. The specific process is as follows:

[0077] During the feeding process, the laboratory was required to maintain a constant temperature of 25°C, and the relative humidity of the air reached about 50%, which was monitored for 24 h. At the same time, the experimental mice were allowed to eat and drink freely, and the selected food was high-pressure sterilized SPF grade mouse feed, and the drinking water was ultrapure water. The method for constructing the nude mouse PDX model was the tumor block embedding method. After 7 days of adaptive feeding, the experimental mice were confirmed to have reached the expected modeling body weight (about 20 g), and daily animal observation confirmed that the physiological state was normal. The PDX model was constructed: the left axillary of the mouse was wiped with alcohol for disinfection, and the preserved tumor tissue was washed with serum-free RMIP 1640 medium. The tumor tissue was stored in a -80°C refrigerator, and the size was about 1 mm 3 around. The washed tumor tissue was injected subcutaneously with a syringe, and all the above operations were completed in a sterile clean bench. The growth state of the tumor and the physiological state of the mouse were observed at regular intervals every day. The growth state of the tumor of the constructed PDX model nude mouse was measured every day, and the tumor volume (mm 3) = 1 / 2 x long diameter, select the PDX model mice which tumor volume is more than 90mm 3 After that, select the mice with similar tumor size and good condition, and randomly divide them into 4 groups, each group with 5 mice. The 4 groups are treated as follows:

[0078] The negative control PBS buffer group: the drug used is PBS buffer.

[0079] The positive control cisplatin group: the drug used is cisplatin solution, 1 mM cisplatin solution (solvent is PBS buffer), the preparation method is referred to the method of 2.1 in step 2 of the third part of this embodiment.

[0080] The nucleic acid nano-drug carrier without inhibiting APE1 protein + C8-cisplatin group: the drug used is C8-cisplatin-nucleic acid nano-drug carrier (without siRNA) solution with a concentration of 3 μM (solvent is PBS buffer), the preparation method is referred to the method of 2.2.2 in step 2 of the third part of this embodiment.

[0081] The nucleic acid nano-drug carrier with inhibiting APE1 protein + C8-cisplatin group: the drug used is C8-cisplatin-nucleic acid nano-drug carrier solution with a concentration of 3 μM (solvent is PBS buffer), the preparation method is referred to the method of 2.2.1 in step 2 of the third part of this embodiment.

[0082] The administration method is tail vein injection, the administration dose is a fixed volume of 150 μl, to ensure the relative concentration of the drug is consistent (standardized by platinum content, 2 mg platinum / kg mouse), a total of 5 administrations.

[0083] Figure 6 The change of the body weight of the mice with the treatment time can be obtained from the curve trend, compared with the PBS injection group, the body weight of the mice using the nucleic acid nano-drug carrier (the nucleic acid nano-drug carrier with inhibiting APE1 protein + C8-cisplatin group and the nucleic acid nano-drug carrier without inhibiting APE1 protein + C8-cisplatin group) has no greater change, the body weight of the mice in the positive control cisplatin group decreases faster, and the body weight of the mice in the nucleic acid nano-drug carrier without inhibiting APE1 protein + C8-cisplatin group and the nucleic acid nano-drug carrier with inhibiting APE1 protein + C8-cisplatin group decreases slightly.

[0084] Figure 7 The change of the tumor volume of the mice with the treatment time is shown in the graph, from which it can be clearly seen that the tumor volume of the mice in the nucleic acid nano-drug carrier with inhibiting APE1 protein + C8-cisplatin group increases at the smallest rate, while the tumor volume of the mice in the other administration groups increases faster. At the same time, it can be seen from the graph that the tumor volume of the mice in the nucleic acid nano-drug carrier without inhibiting APE1 protein + C8-cisplatin group and the nucleic acid nano-drug carrier with inhibiting APE1 protein + C8-cisplatin group decreases slightly. Figure 8 and Figure 9The pictures of the tumors ex vivo (the mice were killed 24h after the last measurement of the tumor volume, and the tumor tissues were obtained) also reached the same conclusion. It was shown that the nucleic acid nanocarrier prepared in the application could quickly reach the tumor site and be enriched in the tumor site due to the passive targeting of the AS1411 aptamer; the nucleic acid nanocarrier loaded with the tetravalent platinum prodrug C8-Cispt was rapidly released to play a role under a high concentration of reducing agents; the siRNA of APE1 was released after entering the cell, inhibiting the expression of APE1 protein, inhibiting the self-repair of tumor cells, overcoming the platinum drug resistance problem to a certain extent, and achieving the most significant effect.

[0085] These results show that the nucleic acid nanocarrier for delivery of small-molecule anticancer platinum drugs disclosed in the application has high sensitization properties and can quickly enter eukaryotic cells. The carrier can accurately target the delivery of small-molecule drugs, platinum drugs, to tumor cells, effectively kill tumor cells and regulate gene expression in tumor cells. The nanocarrier is safe and non-toxic, has a high drug loading capacity, and can be monitored in real time, and has a wider application prospect compared with traditional drug carriers.

[0086] The above describes the present application in detail. For those skilled in the art, without departing from the spirit and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wider range under equivalent parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that the present application can be further improved. In summary, according to the principle of the present application, the present application intends to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which are outside the scope disclosed in the present application. Some basic features can be applied within the scope of the following attached claims. SEQUENCE LISTING <110> Beijing University of Chemical Technology <120> A nucleic acid nanocarrier and application thereof <130> GNCSY211258 <160> 9 <170> SIPOSequenceListing 1.0 <210> 1 <211> 103 <212> DNA <213> Artificial Sequence (Artificial Sequence) <400> 1 tttggacaaa atggcagcgt catatcaggg agggggtacc tgggtagtca gcgctaccac 60 acacagaact ccctacgaca ttggcatgag atactcggag acc 103 <210> 2 <211> 83 <212> DNA <213> Artificial Sequence (Artificial Sequence) <400> 2 cgcgatccgg atcgatcgat atttgtgcct agggtatcac gctttatctg tagctcgccc 60 ccgtctccga gtatctcatg ccc 83 <210> 3 <211> 84 <212> DNA <213> Artificial Sequence (Artificial Sequence) <400> 3 gtgataccct aggcacaaat gatgtcgtag ggagttctgt gcttggatca cgcgtcggtt 60 gttttttttt tttttttttt tttt 84 <210> 4 <211> 63 <212> DNA <213> Artificial Sequence (Artificial Sequence) <400> 4 gtggtagcgc tgactaccca cggggcgagc tacagataaa gaacaaccga cgcgtgatcc 60 aat 63 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence (Artificial Sequence) <400> 5 acgctgccat tttgtccaaa 20 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <400> 6 ggtaccccct cctgatatg 19 <210> 7 <211> 41 <212> DNA <213> Artificial Sequence <400> 7 ggtggtggtg gttgtggtgg tggaaaaaaa aaaaaaaaaa a 41 <210> 8 <211> 46 <212> RNA <213> Artificial Sequence <400> 8 aucgaucgau ccggaucgcg uuuccgucug guacgacugg aguacc 46 <210> 9 <211> 21 <212> RNA <213> Artificial Sequence <400> 9 uacuccaguc guaccagacc u 21

Claims

1. A nucleic acid nanoparticle drug carrier, characterized in that: The invention comprises a DNA tetrahedron main structure with several extended chains at its vertices, and five short chains connected to the extended chains through complementary base pairing; the five short chains are: a DNA single chain containing an AS1411 aptamer, a siRNA single chain containing an siRNA that inhibits the apurinic pyrimidine endonuclease APE1 enzyme, an RNA single chain containing a reverse complementary region to the siRNA single chain, and two nucleic acid probe DNA single chains containing a detection of the inhibition of the apurinic pyrimidine endonuclease APE1 enzyme; the two nucleic acid probe DNA single chains containing the detection of the inhibition of the apurinic pyrimidine endonuclease APE1 enzyme have a fluorescent group modified at the 5' end of one and a quenching group modified at the 3' end of the other, and an AP site is provided at a position 8-10 bp adjacent to the quenching group; the two nucleic acid probe DNA single chains complementarily bind to the same extended chain, and after complementary binding, the fluorescent group and the quenching group are adjacent to each other; The main structure of the DNA tetrahedron with several extended chains at the vertices is self-assembled by four long DNA single chains, and three vertices of the tetrahedron have extended chains; The sequences of the four long single-stranded DNAs are shown in sequence 1, sequence 2, sequence 3, and sequence 4 in the sequence listing, respectively; the sequences of the two single-stranded DNA nucleic acid probes for detecting the inhibition of the apurinic pyrimidine endonuclease APE1 enzyme are shown in sequence 5 and sequence 6 in the sequence listing, respectively; the sequence of the single-stranded DNA containing the AS14111 aptamer is shown in sequence 7 in the sequence listing; the sequence of the single-stranded siRNA containing the siRNA that inhibits the apurinic pyrimidine endonuclease APE1 enzyme is shown in sequence 8 in the sequence listing; the sequence of the single-stranded RNA containing the reverse complementary region to the single-stranded siRNA is shown in sequence 9 in the sequence listing; The nucleic acid nano drug carrier is used to load platinum drugs, and the platinum drug is C8-cisplatin.

2. The nucleic acid nanoparticle drug carrier according to claim 1, characterized in that: The fluorescent group is JOE, HEX, VIC, ROX, CY3 or CY5, and the quenching group is BHQ2 or BHQ3.

3. A method for preparing the nucleic acid nanoparticle drug carrier according to any one of claims 1-2, characterized in that: The method comprises annealing each DNA single strand to assemble DTN, annealing each siRNA single strand to assemble siRNA, and mixing DTN and siRNA and then annealing and assembling them to obtain a nucleic acid nano drug carrier.

4. A method for using the nucleic acid nanoparticle drug carrier according to any one of claims 1 to 2, characterized in that: The method comprises the steps of loading a platinum drug with the nucleic acid nano drug carrier; the drug is C8-cisplatin.

5. A product obtained by loading a drug onto the nucleic acid nanocarrier according to any one of claims 1-2; the drug is C8-cisplatin.

6. Any of the following W1-W5 applications: W1. Use of the nucleic acid nanoparticle drug carrier according to any one of claims 1 to 2 in the preparation of a product for enhancing the efficacy of cancer therapeutic drugs; W2. Use of the nucleic acid nanoparticle drug carrier according to any one of claims 1 to 2 in the preparation of cancer therapeutic drugs; W3. Use of the product according to claim 5 in the preparation of cancer therapeutic drugs; W4. Use of the product according to claim 5 in the preparation of a product for inhibiting the growth of cancer cells; W5. Use of the product according to claim 5 in the preparation of a product for reducing the viability of cancer cells.

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