Tumor-Specific Enzyme-Responsive Nanomaterials for Photodynamic Therapy, Their Preparation and Applications

Abstract

The present invention provides a tumor-specific enzyme-responsive photodynamic therapy nanomaterial, which is composed of a metal-organic framework material, a single-stranded DNA named a-strand, and a single-stranded DNA named b-strand; the metal-organic framework material is a nanoparticle with abundant unsaturated metal coordination sites on its surface; the b-strand contains a reverse complementary region with respect to the a-strand, a quenching group is modified in the reverse complementary region and an apurinic / apyrimidinic site is contained, and after the apurinic / apyrimidinic site is excised, a short chain modified with the quenching group will be freed from the reverse complementary region; a phosphate group is modified at the 5'-end of the a-strand, the a-strand and the b-strand are reversely complementary to form a double-stranded DNA, and are fixed on the surface of the metal-organic framework material through the interaction between the phosphate group and the unsaturated metal coordination sites in the metal-organic framework material. The nanomaterial of the present invention realizes specific photodynamic therapy for tumor cells.

Description

Technical Field

[0001] The present invention relates to a tumor-specific enzyme-responsive photodynamic therapy nanomaterial and its preparation and application. Background Art

[0002] In recent years, metal-organic frameworks (MOFs), a class of emerging hybrid porous materials composed of metal ions or clusters bridged by organic ions, have attracted increasing attention. The superior properties of MOFs, such as well-defined pore sizes, tunable compositions and structures, tunable sizes, photothermal properties, photodynamic properties, high drug-loading capacities and good biocompatibility, have led to remarkable achievements in the field of nanomedicine applications of MOFs due to the facile nanoscale synthesis of MOFs and alternative functionalization through inclusion and surface chemistry. Among them, there are extensive applications in photodynamic therapy due to their superiority in terms of particle size and function. Photodynamic therapy (PDT) is a form of phototherapy involving light and photosensitive chemicals, which can cause cell death (phototoxicity) when used in combination with molecular oxygen. PDT has the ability to kill microbial cells, including bacteria, fungi and viruses. Photodynamic therapy has been proven to be a non-invasive treatment modality for treating local solid tumors. It can generate reactive oxygen species (ROS) under laser irradiation to induce high oxidative stress, thereby ablating tumor cells.

[0003] However, it is difficult to reduce the toxicity of nanoparticles to normal cells in the case of systemic administration in photodynamic therapy, resulting in relatively large side effects of photodynamic therapy on the body itself. Therefore, the development of tumor-specific stimulus-responsive photodynamic therapy is of great significance for precise targeted therapy. Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to develop a tumor cell-specific stimulus-responsive photodynamic therapy method and material. The object of the present invention is to provide a controllable photodynamic therapy nanomaterial to achieve the regulation of photodynamic effects during the treatment process.

[0005] To solve the above technical problems, the present invention provides a tumor-specific enzyme-responsive photodynamic therapy nanomaterial, which is composed of a metal-organic framework material (MOF), a single-stranded DNA named a-strand, and a single-stranded DNA named b-strand; the metal-organic framework material (MOF) is a nanoparticle with abundant unsaturated metal coordination sites on its surface; the b-strand contains a reverse complementary region with the a-strand, and a quenching group is modified in the reverse complementary region and contains an apurinic / apyrimidinic site (AP site). After the excision of the apurinic / apyrimidinic site (AP site), a short chain modified with a quenching group will be freed from the reverse complementary region; a phosphate group is modified at the 5'-end of the a-strand, and the a-strand and the b-strand are reversely complementary and combined into double-stranded DNA (dsDNA), and are fixed on the surface of the metal-organic framework material through the interaction between the phosphate group and the unsaturated metal coordination sites in the metal-organic framework material (MOF).

[0006] In the above nanomaterial, the metal-organic framework material can be a zirconium-based metal-organic framework material (Zr-MOF).

[0007] In the above nanomaterial, the zirconium-based metal-organic framework material (Zr-MOF) can adopt PCN-224. PCN-224 is a spherical nanoparticle produced by the reaction of ZrOCl2 and a porphyrin derivative (H2TCPP). It is assembled into a structure with ordered pores through the coordination bond between the ligand porphyrin molecule (H2TCPP) and the metal zirconium salt, and the Zr6 cluster appears repeatedly in the structure. The Zr6 cluster is an octahedral structure formed by connecting 6 Zr atoms with O atoms or OH groups. Due to the existence of the Zr6 cluster, PCN-224 has abundant unsaturated zircon (Zr) metal coordination sites on its surface and can form a Zr-P bond through coordination with the phosphate group.

[0008] In the above nanomaterial, the length of the short chain modified with a quenching group that is complementary to the a-strand is less than or equal to 9 nucleotides.

[0009] In the above nanomaterial, the a-strand and the b-strand are reversely complementary and combined into double-stranded DNA, and the distance between the phosphate group on the a-strand and the quenching group on the b-strand after combination is less than or equal to 20 nucleotides.

[0010] In the above nanomaterial, the quenching group can be modified at any position of the b-strand, preferably the 3'-end; the quenching group can be BHQ2 or BHQ3.

[0011] In the above nanomaterial, the nucleotide sequence of the a-strand can be as shown in Sequence 1 of the sequence listing, with a phosphate group modified at the 1st position.

[0012] Among the above-mentioned nanomaterials, the nucleotide sequence of the b strand can be as shown in Sequence Listing Sequence 2, where the base T at the 12th position is replaced with an AP site, and the 20th position is modified with a quenching group.

[0013] The present invention also provides a preparation method using the above-mentioned nanomaterials, including annealing and assembling the a strand and the b strand to form double-stranded DNA, and fixing the double-stranded DNA on the surface of the metal-organic framework material through the interaction between the phosphate group at the 5' end of the a strand and the unsaturated metal coordination sites in the metal-organic framework material, to obtain a tumor-specific enzyme-responsive photodynamic therapy nanomaterial.

[0014] The present invention also provides a product, the active ingredient of which is the above-mentioned nanomaterial.

[0015] In the above-mentioned product, the product can be a drug. The product can be used for treating cancer. The cancer can be lung cancer, such as non-small cell lung cancer.

[0016] To solve the above technical problems, the present invention also provides any one of the following applications of W1-W4:

[0017] W1. The application of the above-mentioned nanomaterials or preparation method in the preparation of products for enhancing the efficacy of cancer treatment drugs;

[0018] W2. The application of the above-mentioned nanomaterials or preparation method in the preparation of cancer treatment drugs;

[0019] W3. The application of the above-mentioned nanomaterials or preparation method in the preparation of products for inhibiting the growth of cancer cells;

[0020] W4. The application of the above-mentioned nanomaterials or preparation method in the preparation of products for reducing the cell viability of cancer cells.

[0021] In the above-mentioned applications, 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.

[0022] In the above-mentioned applications, the cancer can be lung cancer. The cancer cells can be lung cancer cells, such as A549 cells.

[0023] When the tumor-specific enzyme-responsive photodynamic therapy nanomaterial PCN-dsDNA(BHQ+APsite) constructed by the present invention enters tumor cells, the highly expressed APE1 in tumor cells can rapidly act on the AP site on the b strand of the nanoparticle, causing the quenching group modified on the b strand to dissociate from the nanoparticle, and the photodynamic effect of the nanoparticle is restored. A large amount of reactive oxygen species (ROS) can be generated by irradiating to excite the photodynamic effect to kill cells. At the same time, due to the increase in the intracellular redox level, the expression of APE1 will be further increased, and then more nanoparticles will restore the photodynamic effect, so as to achieve a positive feedback regulation. In normal cells, since the expression level of APE1 is relatively low and is not sufficient to restore the photodynamic effect of the nanomaterial, it has no killing effect on normal cells. The present invention combines metal-organic frameworks and nucleic acid nanostructures, comprehensively utilizes enzymatic reactions and photodynamic effects, and uses the mechanism of the cells themselves for amplification, realizing specific photodynamic therapy for tumor cells and causing high killing power to tumor cells on the premise of reducing the toxicity to normal cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the structural and schematic diagram of the tumor-specific enzyme-regulated photodynamic therapy material disclosed by the present invention.

[0025] Figure 2 It is the electron microscopy imaging diagram, electrophoresis characterization diagram and Zeta potential characterization diagram of the tumor-specific enzyme-responsive photodynamic therapy material disclosed by the present invention. Figure 2 Figure (A) is the electron microscopy imaging diagram of PCN-224; Figure 2 Figure (B) is the 2% agarose gel electrophoresis diagram, where lane 1 is dsDNA, lane 2 is free dsDNA, lane 3 is PCN-dsDNA(BHQ+APsite), and lane 4 is PCN-dsDNA(BHQ+APsite) after PCN degradation; Figure 2 Figure (C) is the Zeta potential diagram of PCN-224; Figure 2 Figure (D) is the Zeta potential diagram of PCN-dsDNA.

[0026] Figure 3 It is the photodynamic response characterization diagram of the tumor-specific enzyme-regulated photodynamic therapy material disclosed by the present invention in solution. Among them, PCN-dsDNA(BHQ+APsite) is the tumor-specific enzyme-regulated photodynamic therapy material disclosed by the present invention, PCN-dsDNA(BHQ) is the control without the APsite label, and PCN is PCN-224.

[0027] Figure 4ROS staining images of tumor-specific enzyme-regulated photodynamic therapy materials disclosed in the present invention in tumor cells A549. Among them, PCN-dsDNA(BHQ+APsite) is the tumor-specific enzyme-regulated photodynamic therapy material disclosed in the present invention, PCN-dsDNA(BHQ) is the control without the APsite label, PCN is PCN-224, CTRL is the blank control without material treatment, Hoechst is the nuclear dye, DCFH-DA is the ROS probe, and Merge is the combination of nuclear imaging and ROS imaging.

[0028] Figure 5 Cytotoxicity images of tumor-specific enzyme-regulated photodynamic therapy materials disclosed in the present invention against lung cancer cells A549. Among them, PCN-dsDNA(BHQ+APsite) is the tumor-specific enzyme-regulated photodynamic therapy material disclosed in the present invention, PCN-dsDNA(BHQ) is the control without the APsite label, PCN is PCN-224, and Contorl is the blank control without material treatment.

[0029] Figure 6 Western blot characterization images of the effect of tumor-specific enzyme-regulated photodynamic therapy materials disclosed in the present invention on the expression level of APE1 in lung cancer cells A549. Among them, PCN-dsDNA(BHQ+APsite) is the tumor-specific enzyme-regulated photodynamic therapy material disclosed in the present invention, and Control is the blank control without material treatment. Detailed implementation manners

[0030] The present invention will be further described in detail below in combination with the specific implementation manners. The provided examples are only for clarifying the present invention and not for limiting the scope of the present invention. The following examples can be used as a guide for those of ordinary skill in the art to make further improvements and do not constitute any limitation to the present invention in any way.

[0031] The experimental methods in the following examples are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all conventional biochemical reagents and can be obtained from commercial channels unless otherwise specified.

[0032] Example 1 Construction and effect verification of tumor-specific enzyme-responsive photodynamic therapy nanomaterials

[0033] 1. Construction of tumor-specific enzyme-responsive photodynamic therapy nanomaterials

[0034] It has been found that the photodynamic effect of photosensitizers can be reduced by the action of organic small molecule photoquenching groups (such as the black hole quencher series). Therefore, a switch that specifically responds to tumor markers can be assembled with a quenching group and a photosensitizer, enabling photodynamic therapy to be activated only within tumor cells, thereby reducing the toxicity of photodynamic therapy to normal tissues. Apurinic / apyrimidinic endonuclease 1 (APE1) is a multifunctional enzyme and is part of the base excision repair (BER) pathway. APE1 is responsible for processing almost all apurinic / apyrimidinic sites (AP sites) in mammalian cells and has been proven to be related to tumorigenesis, development, and metastasis. This enzyme is overexpressed in the cytoplasm of tumor cells and is considered a tumor-specific enzyme. Moreover, APE1 has a regulatory effect on the intracellular redox level, and its expression level will increase when the intracellular reactive oxygen species level rises. In summary, the inventors plan to use DNA molecules with quenching groups and AP sites to construct controllable photodynamic therapy nanomaterials with MOF to reduce the toxicity to normal tissues in photodynamic therapy.

[0035] The inventors constructed a tumor-specific enzyme-responsive photodynamic therapy nanomaterial named PCN-dsDNA(BHQ+APsite), and its structural diagram is shown in Figure 1 , which is composed of a metal-organic framework material (MOF), a single-stranded DNA named strand a, and a single-stranded DNA named strand b; the metal-organic framework material (MOF) is a nanoparticle with abundant unsaturated metal coordination sites on its surface; strand b contains a reverse complementary region to strand a, and a quenching group is modified in the reverse complementary region and contains an apurinic / apyrimidinic site (AP site). After the apurinic / apyrimidinic site (AP site) is excised, a short chain modified with a quenching group will be freed from the reverse complementary region; a phosphate group is modified at the 5' end of strand a, and strand a and strand b are reversely complementary and combined into double-stranded DNA (dsDNA), and are fixed on the surface of the metal-organic framework material through the interaction between the phosphate group and the unsaturated metal coordination sites in the metal-organic framework material (MOF).

[0036] In this embodiment, the metal-organic framework material is a zirconium-based metal-organic framework material (Zr-MOF), and specifically PCN-224 is used for the zirconium-based metal-organic framework material (Zr-MOF). PCN-224 is spherical nanoparticles produced by the reaction of ZrOCl2 and porphyrin derivative (H2TCPP). PCN-224 is assembled into a structure with ordered pores through the coordination bonds of ligand porphyrin molecules (H2TCPP) and metal zirconium salts, and the Zr6 cluster appears repeatedly in the structure. The Zr6 cluster is an octahedral structure formed by connecting 6 Zr atoms with O atoms or OH groups. Due to the existence of the Zr6 cluster, PCN-224 has abundant unsaturated metal ligand sites on its surface. In theory, it can form Zr-P bonds through coordination with the phosphate groups on DNA, enabling DNA to be connected to the surface of PCN-224, thereby preparing a functionalized metal-organic framework material. PCN-224 has good stability, biocompatibility, and optical properties. The average diameter of the spherical nanoparticles is 90 nm, and it can carry out a photodynamic effect under the action of visible light (670 nm), converting ground-state oxygen into reactive oxygen species (ROS), thereby killing cells and tissues.

[0037] The specific sequences of the a-chain and b-chain in this embodiment are shown in Table 1: The a-chain is 28 bases in full length, and the b-chain is 20 bases in full length; the phosphate group is labeled at the 5'-end of the a-chain, and the quenching group is labeled at the 3'-end of the b-chain; phosphate is modified at the 5'-end of the a-chain to ensure the interaction and connection between the nucleic acid and Zr on the surface of Zr-MOF; the 3'-end of the b-chain is modified with a quenching group (BHQ1), the 12th position is the AP site, and the 1st - 11th positions and the 13th - 20th positions are complementary to the a-chain; after complementary pairing, the distance between the phosphate group and the quenching group on the a-chain is 8 nucleotides, and the distance between the phosphate group and the AP site is 18 nucleotides; the AP site is 9 nucleotides away from the 3'-end of the quenching group to ensure that after the AP site is excised by the action of APE1, the length of the short chain modified with the quenching group complementary to the a-chain is short enough to free the nanoparticles, thereby restoring the photodynamic effect of the nanoparticles.

[0038] Table 1 Nucleic acid sequences

[0039]

[0040] Note: X is the AP site, and PO4 represents phosphorylation.

[0041] The working principle of PCN-dsDNA(BHQ+APsite) is as follows: in a laser environment, in normal cells, due to the low content of apurinic / apyrimidinic endonuclease 1 (APE1), the quenching group can block the progress of the photodynamic effect, so photodynamic therapy (PDT) cannot be carried out in normal cells. However, in a laser environment, the highly expressed APE1 in tumor cells can cleave the AP site on the material, resulting in the inability of the quenching group to continue blocking the photodynamic effect, thereby enabling the tumor-specific enzyme APE1-responsive photodynamic therapy nanomaterial PCN-dsDNA(BHQ+APsite) to carry out photodynamic killing in tumor cells and amplify through the cell's own mechanism (APE1 has a regulatory effect on the intracellular redox level, and its expression level will increase when the intracellular reactive oxygen species level rises).

[0042] The preparation method of the tumor-specific enzyme APE1-responsive photodynamic therapy nanomaterial PCN-dsDNA (BHQ+APsite) of the present invention includes annealing and assembling the a-chain and the b-chain into double-stranded DNA, and fixing the double-stranded DNA on the surface of the metal-organic framework material through the interaction between the phosphate group at the 5' end of the a-chain and the unsaturated metal coordination sites in the metal-organic framework material to obtain the tumor-specific enzyme-responsive photodynamic therapy nanomaterial. Specifically, it includes the following steps:

[0043] A. Synthesis and characterization of the photodynamic therapy nanomaterial PCN-224

[0044] Dissolve H2TCPP (50 mg), ZrOCl2·8H2O (150 mg) and benzoic acid (1400 mg) in DMF (50 mL), stir and mix, place at 90 °C for 5 hours, collect by centrifugation, wash 3 times with DMF to obtain PCN-224 nanoparticles, and suspend them in DMF for standby.

[0045] B. Preparation of labeled double-stranded DNA

[0046] Synthesize and modify the a-chain and the b-chain according to Table 1.

[0047] Prepare labeled double-stranded DNA (dsDNA): establish a 30 μL system, the reaction buffer environment is 1×HEPES buffer (pH 7.4), add the a-chain and the b-chain respectively to make their concentrations in the system both 10 μM, and the remaining volume is supplemented with ddH2O. Apply the annealing program to ensure the correct binding of the oligonucleotide chains of the double-stranded DNA (dsDNA) structure. The PCR annealing program is set as follows: 80 °C, 2 min; 70 °C, 2 min; 60 °C, 1 min; 50 °C, 1 min; 37 °C, 5 min; keep at 37 °C.

[0048] The a-chain and b-chain stably bind to obtain labeled double-stranded DNA. The distance between the phosphate group and the quenching group on the a-chain is 8 nucleotides, the distance between the phosphate group and the AP site is 18 nucleotides; the AP site is 9 nucleotides away from the 3'-end of the quenching group.

[0049] C. Synthesis of Tumor-Specific Enzyme-Responsive Photodynamic Therapy Nanomaterial PCN-dsDNA(BHQ+APsite)

[0050] React the PCN-224 synthesized in step A and the labeled double-stranded DNA prepared in step B overnight in HEPES buffer solution to obtain the tumor-specific enzyme-responsive photodynamic therapy nanomaterial PCN-dsDNA(BHQ+APsite), specifically as follows:

[0051] Take the prepared PCN-224, wash it three times with deionized water and then disperse it in water overnight to obtain an aqueous solution of PCN-224 with a concentration of 500 μg / mL; add 485 μL of deionized water to a 2 mL centrifuge tube, then add 30 μL of labeled double-stranded DNA (dsDNA, 10 μM), and slowly drip 300 μL of the aqueous solution of PCN-224 (500 μg / mL) into the centrifuge tube in a shaker. The reaction solution is shaken overnight on a rotary shaker, and then 100 μL of 1 M HEPES buffer (pH 7.4) is slowly added dropwise in a shaker. After brief sonication, continue shaking for 2 h. After the reaction is completed, centrifuge and wash twice with deionized water (12000 rpm, 10 min) to remove the free DNA from the system, and obtain the tumor-specific enzyme-responsive photodynamic therapy nanomaterial PCN-dsDNA(BHQ+APsite). Finally, redisperse the product PCN-dsDNA(BHQ+APsite) in deionized water, characterize its synthesis by electron microscopy imaging and 2% agarose gel electrophoresis.

[0052] The characterization results are shown in Figure 2 , such as Figure 2 Figure (A) shows that PCN-224 was successfully synthesized without aggregation and sedimentation, and Figure 2 the electrophoresis result in Figure (B) also shows that dsDNA was conjugated to the surface of the metal-organic framework PCN. Specifically, the band in lane 2 is dimmer than that in lane 1, and there is no band in lane 3. The reason is that the metal-organic framework affects the DNA dye and causes the band to disappear, while the band reappears in lane 4 after cleaving PCN-dsDNA with phosphoric acid, indicating that DNA was successfully conjugated to PCN. After DNA is conjugated to PCN, it will cause the potential on the surface of PCN nanoparticles to decrease, as shown in Figure 2 Figure (C) and Figure 2As shown in Figure (D), the potential on the surface of PCN-dsDNA nanoparticles is significantly lower than that on the surface of PCN, indicating the successful conjugation of PCN-dsDNA.

[0053] The quenching group on dsDNA is 8 bases away from the surface of the metal-organic framework PCN-224, and the AP site is 18 bases away from the surface of the metal-organic framework PCN-224.

[0054] A control was set up, that is, the c chain without labeled AP site in Table 1 (the base T at the 12th position was not replaced by the AP site, and other amino acid sequences and modifications were the same as the b chain) was used to replace the b chain, and other steps remained unchanged to obtain the control PCN-dsDNA(BHQ) without AP site label.

[0055] 2. Detection of the photodynamic effect of the tumor-specific enzyme-regulated photodynamic therapy material on the solution

[0056] Whether the photodynamic effect of the nanomaterial is turned on is determined by whether the AP site is labeled on the double-stranded DNA. The generation of reactive oxygen species (ROS) in the solution system is detected by DPBF. Reactive oxygen species can oxidize DPBF and reduce its fluorescence. The specific steps are as follows:

[0057] Disperse the nanomaterial PCN-dsDNA(BHQ+APsite) in the 1×TAE buffer system of a 96-well plate to form an aqueous solution of 40 μg / mL. Dilute APE1 1:20 with enzyme-free water, take 1 μL and add it to the system, react for 20 - 30 min, add diphenylisobenzofuran (abbreviation: DPBF, 100 mg / mL) dye, and laser treat for 10 min (670 nm, 100 mW / cm 2 ), and use a fluorescence spectrometer to detect its fluorescence intensity. Use PCN-dsDNA(BHQ) and PCN (using PCN-224) as controls.

[0058] The results are shown in Figure 3 , indicating that PCN-dsDNA(BHQ+APsite) turns on the photodynamic effect of the nanoparticles under the action of APE1, generates reactive oxygen species under laser treatment, oxidizes DPBF and causes its fluorescence to decrease. The PCN group also obtained similar results, further proving that APE1 turns on the photodynamic effect of PCN-dsDNA(BHQ+APsite). The PCN-dsDNA(BHQ) group lacks the switch AP site, so APE1 cannot turn on its photodynamic effect and thus cannot generate reactive oxygen species under light irradiation, so its fluorescence is higher.

[0059] 3. The tumor-specific enzyme-regulated photodynamic therapy material is used to kill lung cancer cells A549

[0060] Based on the above-synthesized tumor-specific enzyme-regulated photodynamic therapy material PCN-dsDNA(BHQ+APsite), the intracellular reactive oxygen species (ROS) level was detected using a ROS probe to determine the photodynamic effect of the nanomaterial. The tumor cells used were A549, with PCN-dsDNA(BHQ) and PCN-224 as controls, and a blank control was also set. The specific steps are as follows:

[0061] (1) Turn on the water bath and adjust the water temperature to 37 °C. Take out the A549 cells from liquid nitrogen, place them in water at 37 °C and quickly thaw, gently shaking during this period. After about 1 min, the cells are thawed. Wipe the cryopreservation tube clean with 75% alcohol, transfer it to the laminar flow hood. The laminar flow hood should be sterilized by ultraviolet light in advance and kept ventilated. Transfer the cells in the cryopreservation tube to a culture flask with a pipette on the laminar flow hood, and add 5 mL of prepared 1640 medium. Place the culture flask in an incubator at 37 °C and 5% carbon dioxide, and observe the cell growth under a microscope. If necessary, change the medium after 8 hours. Take out trypsin from the -20 °C refrigerator in advance, place it in a 37 °C water bath to melt, wipe the cryopreservation tube clean with 75% alcohol, and transfer it to the laminar flow hood. When the cells are cultured in an incubator at 37 °C and 5% carbon dioxide and grow to 80%-90%, they can be passaged. Aspirate the medium in the original culture flask with a pipette, pour in PBS to wash once, aspirate it with a pipette, add 1 mL of trypsin, covering the bottom of the flask, incubate at 37 °C for 1 min, add 3 mL of medium to stop digestion, observe under a microscope whether the cells change from adherent to free, gently pipette the cells with a pipette until the cells fall off the bottom of the flask, take 2 mL from the culture flask and transfer it to a new culture flask, add 3 mL of 1640 medium to the new culture flask, and place it in an incubator at 37 °C and 5% carbon dioxide for culture.

[0062] (2) Select a cell bottle with adherent cells covering about 80%, aspirate the medium, add PBS to wash, aspirate the PBS, add 1 mL of melted trypsin to digest for 1 min, add 1 mL of medium to stop digestion and pipette the cells until they are all suspended. Aspirate 1 mL of the medium and transfer it to a 1.5 mL centrifuge tube for centrifugation at 900 r / min for 10 min. After centrifugation, discard the supernatant, add 1 mL of 1640 medium and pipette to mix evenly. Aspirate 200 μL and place it in a confocal culture dish for overnight culture. Take 100 μL of nanoparticles (4 treatments: PCN-dsDNA(BHQ+APsite), PCN-dsDNA(BHQ), PCN-224, and blank) and add 100 μL of 1640 medium to mix evenly. Aspirate the medium in the overnight-cultured culture dish, add the above mixed medium, add it to the cells and incubate for 4 h, aspirate the medium, add the medium mixed with Hoechst 33342 dye, culture for 0.5 h, and irradiate with laser for 10 min (100 mW / cm 2 ).

[0063] (3) High Inclination Light Optics (HILO) microscopy detection

[0064] Take out the above-mentioned culture medium and aspirate it, wash it once with PBS to remove cell metabolites, and aspirate the PBS. Turn on the power of the total internal reflection microscope and the CCD, then open the corresponding program in the computer. Drop oil on the oil immersion lens, place the sample on the stage, turn on the laser and the mercury lamp, adjust the focus, find cells with better morphology, and photograph the cells under bright field, under the laser emission source, and under the mercury lamp emission source.

[0065] The results are shown in Figure 4 , indicating that the active oxygen species probe imaging of the PCN group and PCN-dsDNA (BHQ+APsite) has a relatively high fluorescence response, while the blank control and PCN-dsDNA (BHQ) have a relatively low fluorescence response. This shows that the photodynamic effect of PCN-dsDNA (BHQ) is relatively low and cannot generate a large amount of reactive oxygen species in tumor cells. However, due to the overexpression of APE1 in tumor cells, the photodynamic effect of PCN-dsDNA (BHQ+APsite) is activated and can generate a large amount of reactive oxygen species.

[0066] 4. CCK-8 assay for the cytotoxicity of tumor-specific enzyme-regulated photodynamic therapy materials against tumor cells

[0067] Based on the above-synthesized tumor-specific enzyme-regulated photodynamic therapy material PCN-dsDNA (BHQ+APsite), use the CCK-8 kit (Dojindo, PH632) to determine cell viability and evaluate the killing effect of PCN-dsDNA (BHQ+APsite) on tumor cells. The tumor cells used are A549, with PCN-dsDNA (BHQ) and PCN-224 as controls, and a blank control is also set. The specific implementation steps are as follows:

[0068] (1) Take out A549 cells from the CO2 incubator and observe them with an inverted microscope. If the cells have grown to cover the bottom of the culture flask and have good cell morphology, the cells can be seeded into a 96-well plate. Digest the cells from the culture flask according to the steps in Part 3 of this example. Aspirate 10 μL of the cell suspension onto a cell counting chamber and count the cells under the microscope to make the cell density 6.5×10 4 / mL; Add the cell suspension with a density of 6.5×10 4 / mL to the 96-well plate, add 100 μL to each well, and add 100 μL of PBS to each well around the 96-well plate (to prevent evaporation of the cell suspension). Place the seeded 96-well plate in the CO2 incubator and culture for 12 h.

[0069] (2) Take out the 96-well plate seeded with A549 cells from the CO2 incubator and place it on an inverted microscope for observation. If the cell density in the 96-well plate is uniform and the cell morphology is good, then sample addition can be carried out. Use a multi-channel pipette to aspirate the culture medium in the 96-well plate, and add 100 μL of PBS to each well for washing; add the prepared nanoparticles (4 treatments including PCN-dsDNA(BHQ+APsite), PCN-dsDNA(BHQ), PCN-224, and blank) to the 96-well plate seeded with A549 cells, 100 μL of sample to each well.

[0070] (3) After sample addition, place the 96-well plate in the CO2 incubator for incubation. After 12 h, take out the cells, irradiate with laser (670 nm) for 10 min, change the culture medium, and add 10 μL of CCK-8 to each cell sample well. Gently shake the 96-well plate to mix evenly. Subsequently, place the 96-well plate in the CO2 incubator for culture for 2 - 4 h until the cell mixture in the 96-well plate turns yellow. Use a multi-functional microplate reader to measure the OD450 value of each sample of A549 cells. Calculate the cell viability.

[0071] The results are shown in Figure 5 , indicating that since PCN-dsDNA(BHQ) has no switchable AP site, the overexpressed APE1 in lung cancer cells A549 cannot unlock its photodynamic effect, so its cell survival rate is relatively high (84.7%), while PCN-dsDNA(BHQ+APsite) depends on the overexpressed APE1 in A549 cells, and its photodynamic effect is unlocked, so its cell survival rate is relatively low (33.3%).

[0072] In summary, the tumor-specific enzyme-responsive photodynamic therapy nanomaterial PCN-dsDNA(BHQ+APsite) constructed in the present invention has low toxicity to normal cells and has significant killing effect specifically induced by APE1 on tumor cells. The present invention combines metal-organic frameworks and nucleic acid nanostructures, comprehensively utilizes enzymatic reactions and photodynamic effects, and amplifies using the mechanism of the cells themselves, realizing specific photodynamic therapy for tumor cells and causing high killing effect on tumor cells while reducing the toxicity to normal cells.

[0073] The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modification, use or improvement of the present invention, including those that depart from the scope disclosed in this application but are made by using conventional techniques known in the art. The application of some basic features can be carried out according to the scope of the appended claims below. Sequence Listing <110> Beijing University of Chemical Technology <120> Tumor-Specific Enzyme-Responsive Photodynamic Therapy Nanomaterials and Their Preparation and Applications <130> GNCSY212908 <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 28 <212> DNA <213> Artificial Sequence <400> 1 ggcgaggttt cgcgtcgcag cgtgcacg 28 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 cgtgcacgct gtgacgcgaa 20

Claims

1. A tumor-specific enzyme-responsive nanomaterial for photodynamic therapy, characterized in that: It is composed of a metal-organic framework material, a single-stranded DNA named a-strand, and a single-stranded DNA named b-strand; the metal-organic framework material is nanoparticles with abundant unsaturated metal coordination sites on the surface; the b-strand contains a reverse complementary region to the a-strand, a quenching group is modified in the reverse complementary region and contains an apurinic / apyrimidinic site, and after excision of the apurinic / apyrimidinic site, a short chain modified with a quenching group will be freed from the reverse complementary region; a phosphate group is modified at the 5'-end of the a-strand, the a-strand and the b-strand are reversely complementary and combined into double-stranded DNA, and are fixed on the surface of the metal-organic framework material through the interaction between the phosphate group and the unsaturated metal coordination sites in the metal-organic framework material; The metal-organic framework material is PCN-224; For the short chain modified with a quenching group, the length of its complementary pairing with the a-strand is less than or equal to 9 nucleotides; The a-strand and the b-strand are reversely complementary and combined into double-stranded DNA, and the distance between the phosphate group on the a-strand and the quenching group on the b-strand after combination is less than or equal to 20 nucleotides; The nucleotide sequence of the a-strand is as shown in Sequence 1 of the sequence listing, and a phosphate group is modified at the 1st position; The nucleotide sequence of the b-strand is as shown in Sequence 2 of the sequence listing, where the base T at the 12th position is replaced by an AP site, and a quenching group is modified at the 20th position.

2. The preparation method of the tumor-specific enzyme-responsive photodynamic therapy nanomaterial according to claim 1, characterized in that: The a-strand and the b-strand are annealed and assembled into double-stranded DNA, and the double-stranded DNA is fixed on the surface of the metal-organic framework material through the interaction between the phosphate group at the 5'-end of the a-strand and the unsaturated metal coordination sites in the metal-organic framework material, obtaining a tumor-specific enzyme-responsive photodynamic therapy nanomaterial.

3. Product, characterized in that: Its active ingredient is the tumor-specific enzyme-responsive photodynamic therapy nanomaterial described in Claim 1.

4. Application, characterized in that: For any of the following applications: W1. Application of the tumor-specific enzyme-responsive photodynamic therapy nanomaterial described in Claim 1 in the preparation of a product for enhancing the efficacy of cancer treatment drugs; W2. Application of the tumor-specific enzyme-responsive photodynamic therapy nanomaterial described in Claim 1 in the preparation of cancer treatment drugs; W3. Application of the tumor-specific enzyme-responsive photodynamic therapy nanomaterial described in Claim 1 in the preparation of a product for inhibiting the growth of cancer cells; W4. Application of the tumor-specific enzyme-responsive photodynamic therapy nanomaterial described in Claim 1 in the preparation of a product for reducing the cell viability of cancer cells.

5. The application according to claim 4, characterized in that: The cancer is lung cancer, and the cancer cells are lung cancer cells.

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