Multifunctional DNA (deoxyribonucleic acid) nano assembly as well as preparation method and application thereof

Abstract

The invention discloses a multifunctional DNA nano assembly as well as a preparation method and application thereof. The multifunctional DNA nano assembly comprises DNA nano flowers, chemotherapeutic drugs are loaded on the DNA nano flowers, the surfaces of the DNA nano flowers loaded with the chemotherapeutic drugs are coated with metal polyphenol networks, and hyaluronic acid is connected to the surfaces of the metal polyphenol networks; the DNA nanoflower is obtained through a rolling circle amplification reaction, and a repetitive unit sequence of the DNA nanoflower is as shown in SEQ ID NO.3. The preparation method comprises the following steps: firstly, preparing DNA nanoflowers encoding deoxyribozyme sequences through a rolling circle amplification reaction, then loading a chemotherapeutic drug on the DNA nanoflowers, then coating the surface of the DNA nanoflowers with a metal polyphenol network, and finally carrying out hyaluronic acid functionalization, so that the prepared multifunctional DNA nano assembly can enter tumor cells in a targeting manner through hyaluronic acid, and the tumor cells can be targeted through the hyaluronic acid. The combined treatment of gene therapy, chemotherapy and photothermal therapy can be realized under the irradiation of 808 nm laser.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a multifunctional DNA nano-assembly for targeted delivery of DOX and deoxyribozyme and realizing multi-mode combined therapy, and a preparation method and application thereof. BACKGROUND

[0002] Adriamycin (DOX) is one of the effective chemotherapy drugs widely used in the treatment of various cancers, but DOX lacks selectivity for target tissues and cells, and can cause many side effects on various organs of the body, especially dose-dependent cardiotoxicity, and it is reported that 10-25% of patients receiving DOX treatment are prone to heart dysfunction.

[0003] Deoxyribozyme is a kind of catalytic DNA single strand, which usually performs enzyme function in the presence of cofactors. Among them, RNA cleavage type deoxyribozyme can inactivate target genes by cutting mRNA to regulate protein expression, and is considered to be a very promising tumor therapeutic agent. However, the lack of cofactors at the disease site, the presence of off-target effects, and the problem of easy degradation in the physiological environment limit the development of therapeutic deoxyribozymes. Moreover, the single-drug treatment based on deoxyribozyme is not ideal, and deoxyribozyme should be combined with other treatment methods to achieve synergistic and efficient therapeutic effect.

[0004] Photothermal therapy is a treatment method that uses photothermal conversion agents to convert light energy into local high temperature to achieve tumor cell ablation, which shows great clinical potential. However, most of the current photothermal nanomaterials have the disadvantages of complex preparation process, involvement of toxic additives and low drug loading capacity. Therefore, it is of great significance to find new photothermal nanomaterials.

[0005] Based on the above, it is of great significance to develop a multifunctional DNA nano-assembly for targeted delivery of DOX and deoxyribozyme and realizing multi-mode combined therapy. SUMMARY

[0006] In order to solve the above technical problems, the present application provides a multifunctional DNA nano-assembly and a preparation method and application thereof.

[0007] In a first aspect, the present application provides a multifunctional DNA nano-assembly, which is realized by the following technical scheme.

[0008] The multifunctional DNA nano-assembly comprises a DNA nanoflower, a chemotherapeutic drug is loaded on the DNA nanoflower, a metal polyphenol network is coated on the surface of the DNA nanoflower loaded with the chemotherapeutic drug, and hyaluronic acid is connected to the surface of the metal polyphenol network; the DNA nanoflower is obtained by rolling circle amplification reaction, and one repeat unit sequence of the DNA nanoflower is shown in SEQ ID NO. 3.

[0009] The DNA nanoflower of the application is hybridized from a DNA strand containing a deoxyribozyme repeat sequence and magnesium pyrophosphate, the deoxyribozyme is a 10-23 type deoxyribozyme which can specifically cut survivin mRNA, and the deoxyribozyme sequence is shown as SEQ ID NO. 4.

[0010] Further, the preparation method of the DNA nanoflower is as follows: mixing the phosphorylated linear template, the primer, the T4 DNA ligase buffer and ultrapure water, and after high-temperature denaturation treatment, stably treating at 4-25 ℃ for 1-4 hours, then adding the T4 DNA ligase, and after 16-37 ℃ reaction for 2-16 hours, a circular template-primer complex is obtained; mixing the circular template-primer complex, dNTP, DNA polymerase buffer, DNA polymerase and ultrapure water, and after 30 ℃ reaction for 2-10 hours, an amplification reaction is carried out, and the DNA nanoflower is obtained.

[0011] Further, the linear template sequence is shown as SEQ ID NO. 1; and the primer sequence is shown as SEQ ID NO. 2.

[0012] Further, the chemotherapy drug is selected from one of adriamycin, daunorubicin or nor daunorubicin.

[0013] Further, the method for loading the chemotherapy drug on the DNA nanoflower is as follows: dispersing the DNA nanoflower in ultrapure water, then adding the chemotherapy drug, and incubating at 25-37 ℃ in the dark for 0.5-24 hours, so as to obtain the DNA nanoflower loaded with the chemotherapy drug; wherein, 1-33 μmol of the chemotherapy drug is mixed with 1 g of the DNA nanoflower for incubation.

[0014] Further, the metal polyphenol network is one of tannic acid iron, gallic acid iron or gossypol copper.

[0015] Further, the method for coating the metal polyphenol network on the surface of the DNA nanoflower loaded with the chemotherapy drug is as follows: under vortex conditions, sequentially adding the polyphenol solution and the metal salt solution to the solution of the DNA nanoflower loaded with the chemotherapy drug, the mass ratio of the DNA nanoflower, the polyphenol and the metal salt is 1: (1.3-4): (0.3-1), after vortex, centrifugation is carried out, so as to obtain the DNA nanoflower coated with the metal polyphenol network.

[0016] Specifically, the method for coating the metal polyphenol network on the surface of the DNA nanoflower loaded with the chemotherapy drug is as follows: under vortex conditions, sequentially adding the tannic acid solution and the FeCl3·6H2O solution to the solution of the DNA nanoflower loaded with the chemotherapy drug, the mass ratio of the DNA nanoflower, the tannic acid and the FeCl3·6H2O is 1: (1.3-4): (0.3-1), after vortex, centrifugation is carried out, so as to obtain the DNA nanoflower coated with the metal polyphenol network.

[0017] Further, the method for coating the metal polyphenol network on the surface of the DNA nanoflower is as follows: the DNA nanoflower coated with the metal polyphenol network is dispersed in a hyaluronic acid solution, the mass ratio of the DNA nanoflower to the hyaluronic acid is 1: (5-20), and the solution is oscillated at 4-37 DEG C for 12-24 hours, and then centrifuged to obtain the hyaluronic acid functionalized nanoparticles.

[0018] In a second aspect, the present application provides a preparation method of a multifunctional DNA nanoassembler, which is realized by the following technical scheme.

[0019] The preparation method of the multifunctional DNA nanoassembler comprises the following steps:

[0020] S1. preparing a DNA nanoflower through a rolling circle amplification reaction;

[0021] S2. mixing the DNA nanoflower with a chemotherapeutic drug to load the chemotherapeutic drug;

[0022] S3. coating a metal polyphenol network on the surface of the DNA nanoflower loaded with the chemotherapeutic drug;

[0023] S4. performing hyaluronic acid functionalization on the surface of the metal polyphenol network.

[0024] Further, the preparation method of the DNA nanoflower is as follows: a phosphorylated linear template, a primer, a T4 DNA ligase buffer and ultrapure water are mixed, and after high-temperature denaturation treatment, the mixture is stabilized at 4-25 DEG C for 1-4 hours, then T4 DNA ligase is added, and the mixture is reacted at 16-37 DEG C for 2-16 hours to obtain a circular template-primer complex; the circular template-primer complex, dNTP, DNA polymerase buffer, DNA polymerase and ultrapure water are mixed, and the mixture is reacted at 30 DEG C for 2-10 hours to perform an amplification reaction, and then the DNA nanoflower is obtained.

[0025] Further, the method for loading the chemotherapeutic drug on the DNA nanoflower is as follows: the DNA nanoflower is dispersed in ultrapure water, and then the chemotherapeutic drug is added, and the mixture is incubated at 25-37 DEG C in the dark for 0.5-24 hours to obtain the DNA nanoflower loaded with the chemotherapeutic drug; wherein 1-33 micromoles of the chemotherapeutic drug is mixed with one gram of the DNA nanoflower for incubation.

[0026] Further, the method for coating the metal polyphenol network on the surface of the DNA nanoflower loaded with the chemotherapeutic drug is as follows: under vortex conditions, polyphenol solution and metal salt solution are sequentially added to the DNA nanoflower solution loaded with the chemotherapeutic drug, the mass ratio of the DNA nanoflower to the polyphenol to the metal salt is 1: (1.3-4): (0.3-1), and then the mixture is vortexed and centrifuged to obtain the DNA nanoflower coated with the metal polyphenol network.

[0027] Further, the method for functionalizing the metal polyphenol network surface with hyaluronic acid is as follows: dispersing the DNA nanoflower coated with the metal polyphenol network in a hyaluronic acid solution, the mass ratio of the DNA nanoflower to the hyaluronic acid being 1: (5-20), oscillating at 4-37 DEG C for 12-24 hours, and then centrifuging to obtain the hyaluronic acid functionalized nanoparticles.

[0028] In a third aspect, the application provides a use of the multifunctional DNA nanoassemblies, which is achieved by the following technical scheme.

[0029] The multifunctional DNA nanoassemblies are used for preparing a cancer treatment drug.

[0030] Further, the cancer includes breast cancer, liver cancer, and cervical cancer.

[0031] The application has the following beneficial effects.

[0032] (1) The magnesium pyrophosphate wrapped in the DNA nanoflower of the application is decomposed in the acidic environment of the lysosome in cells to produce Mg 2+ , which can realize self-sufficiency of metal cofactors and significantly improve the catalytic efficiency of deoxyribozymes;

[0033] (2) The metal polyphenol network coating of the application is pH-responsive and degradable in the acidic environment in cells, which leads to the exposure of the DNA nanoflower and subsequent collapse to form long single-stranded DNA and release DOX, and this cascade effect of the FDMH can effectively prevent the deoxyribozyme from being enzymolyzed during delivery;

[0034] (3) The combined treatment strategy of the application can achieve a treatment effect equivalent to that of high-concentration DOX at a lower DOX concentration, which greatly reduces the systemic toxicity caused by conventional chemotherapy. BRIEF DESCRIPTION OF DRAWINGS

[0035] In order to more clearly illustrate the specific embodiments of the application or the technical schemes in the prior art, the drawings needed in the specific embodiments or the prior art description will be briefly introduced as follows. Obviously, the drawings in the following description are some embodiments of the application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0036] Figure 1 is a schematic diagram of the preparation process and combined treatment mechanism of the multifunctional DNA nanoassemblies of the application;

[0037] Figure 2is an agarose gel electrophoresis diagram of a circular template-primer complex and DNA nanoflower prepared by the present application (wherein, lane M represents a DNA molecular weight marker, lane 1 represents a primer, lane 2 represents a phosphorylated linear template, lane 3 represents a circular template-primer complex, and lane 4 represents a DNA nanoflower);

[0038] Figure 3 is a SEM diagram (A) and a TEM diagram (B) of a DNA nanoflower prepared by the present application;

[0039] Figure 4 is a result diagram of a loading rate of DNA nanoflowers of different concentrations of the present application to DOX;

[0040] Figure 5 is a TEM diagram of FDM and FDMH prepared by the present application;

[0041] Figure 6 is a particle size distribution diagram (A) and a zeta potential diagram (B) of a DNA nanoflower, FD, FDM and FDMH prepared by the present application;

[0042] Figure 7 is a photothermal performance exploration diagram of FDMH of the present application (wherein, A: temperature change curve of FDMH of different concentrations with light irradiation time; B: temperature change curve of FDMH under laser irradiation of different powers; C: four photothermal cycle curves);

[0043] Figure 8 is a TEM diagram of DNA nanoflowers after treatment by PBS of pH 5.0 of the present application;

[0044] Figure 9 is a release rate diagram of DOX of FDMH of the present application under different pH;

[0045] Figure 10 is a polyacrylamide gel electrophoresis diagram of deoxyribozyme cutting ability of the present application (wherein, lane M represents a DNA molecular weight marker, lane 1 represents a deoxyribozyme, lane 2 represents a substrate, and lanes 3-9 represent mixtures of deoxyribozyme and substrate under different concentrations of Mg 2+ of the mixture after reaction for 5 hours);

[0046] Figure 11 is a polyacrylamide gel electrophoresis diagram of DNA nanoflower cutting ability of the present application (wherein, in diagram A, lane M represents a DNA molecular weight marker, lane 1 represents a substrate, and lane 2 represents a mixture of DNA nanoflower and substrate after treatment by PBS of pH 5.0 for 1 hour; in diagram B, lane M represents a DNA molecular weight marker, lane 1 represents a substrate, and lane 2 represents a mixture of DNA nanoflower and substrate after treatment by PBS of pH 7.4 for 1 hour);

[0047] Figure 12 Fig. 7 is a fluorescence microscope image (A) and a result graph (B) of detecting intracellular DOX fluorescence by flow cytometry of DOX released by the DNA nanoassemblies of the present application in cells;

[0048] Figure 13 Fig. 8 is a graph of cell survival rate of cells treated with different DNA nanoassemblies of the present application (where L represents 808 nm laser irradiation);

[0049] Figure 14 Fig. 9 is a graph of cell apoptosis rate of cells treated with different DNA nanoassemblies of the present application;

[0050] Figure 15 Fig. 10 is a graph of live and dead cell staining of cells treated with different DNA nanoassemblies of the present application;

[0051] Figure 16 Fig. 11 is a graph of morphological changes of 3D cell spheroids treated with different DNA nanoassemblies of the present application. DETAILED DESCRIPTION

[0052] Hereinafter, preferred embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. Before the description, it should be understood that the term used in the specification and the appended claims should not be construed as limited to general and dictionary meanings, but interpreted based on the meanings and concepts under the principles of the applicant's technology along with the scope of the present disclosure. Therefore, the description herein merely describes preferred examples of the application, but does not limit the scope of the application, and thus it should be understood that other equivalents and modifications could be made thereto without departing from the spirit and scope of the application.

[0053] In the present application, pyrophosphate ions are generated during the process of the rolling circle amplification reaction in which deoxyribonucleotide triphosphates participate in DNA elongation, and the pyrophosphate ions combine with Mg 2+ form insoluble magnesium pyrophosphate, which nucleates and grows in situ on the long-chain DNA produced by the rolling circle amplification reaction, and finally self-assembles with the long-chain DNA into a flower-shaped DNA-inorganic hybrid nanostructure (DNA nanoflower). Moreover, the DNA nanoflower has a large number of drug loading sites, which can load the chemotherapeutic drug DOX. The tannic acid and Fe 3+The tannic acid iron coating can be formed on the surface of the DNA nanoflower, which has excellent light-heat conversion capacity, and the tannic acid iron metal polyphenol network also has pH responsiveness. Under acidic conditions, the phenolic hydroxyl groups on the tannic acid are protonated, resulting in the disintegration of the tannic acid iron. Hyaluronic acid can specifically bind to the CD44 receptor overexpressed on the membrane of cancer cells, mediating the endocytosis of nanoparticles. Therefore, the multifunctional DNA nanoassemblies (FDMH) of the present application actively target cancer cells and enter cancer cells through the binding of hyaluronic acid to CD44 on the cell membrane. Under 808 nm laser irradiation, the tannic acid iron metal polyphenol network converts light energy into heat energy for photothermal therapy. As the incubation time is prolonged, the tannic acid iron coating on the FDMH is disintegrated in the acidic environment of the lysosome, resulting in the exposure of the DNA nanoflower, and the magnesium pyrophosphate wrapped on the DNA nanoflower is dissolved in the acidic environment to produce Mg 2+ which simultaneously causes the collapse of the DNA nanoflower to form long single-stranded DNA and releases DOX. The released Mg 2+ activates the deoxyribozyme on the long single-stranded DNA to cut the survivin mRNA in the cell, thereby inhibiting cell proliferation and achieving gene therapy, and the released DOX enters the nucleus for chemotherapy, ultimately realizing the combined therapy of gene therapy-chemotherapy-photothermal therapy.

[0054] The preparation method of the multifunctional DNA nanoassemblies of the present application is disclosed in detail below. The various reagents and instruments used in the following examples can be purchased on the market if not otherwise specified.

[0055] Example 1: Preparation of DNA nanoflower (DNF)

[0056] (1) Linear template phosphorylation: mix 5 μL of 10× T4 PNK enzyme buffer, 10 μL of 1 mM ATP, 5 μL of 100 μM linear template, 1 μL of 10 U / μL T4 PNK enzyme, and 29 μL of ultrapure water, and react at 37 °C for 30 minutes to transfer the phosphate group on the ATP to the 5' end of the linear template. Then heat at 90 °C for 5 minutes to inactivate the T4 PNK enzyme, and the 5' end phosphorylated linear template is obtained.

[0057] (2) Preparation of the circular template-primer complex: 5 μL of 100 μM primer, 10 μL of 10× T4 DNA ligase buffer, 33 μL of ultrapure water and 50 μL of the phosphorylated linear template obtained in step (1) of Example 1 were mixed, the mixed solution was heated at 95 ℃ for 5 minutes, then naturally cooled to 25 ℃, and was stably kept at 25 ℃ for 3 hours to allow the template strand and the primer strand to base complementarily pair. Then 2 μL of 350 U / μL T4 DNA ligase was added, and the reaction was carried out at 16 ℃ for 16 hours to circularize the template strand. Finally, the mixed solution was heated at 65 ℃ for 10 minutes to inactivate the T4 DNA ligase, and the circular template-primer complex was obtained.

[0058] Circular template / linear template:

[0059] 5'-AGCTAGCCTGGCCGAGGAAAACCGGAGCGGTCGTTGTAGCTAGCCTGGCCGAGGAAAACCGGAGCGGTCGTTGT-3' (SEQ ID NO. 1);

[0060] Primer:

[0061] 5'-CCAGGCTAGCTACAACGACCGC-3' (SEQ ID NO. 2).

[0062] (3) Preparation of the DNA nanoflower: 20 μL of the circular template-primer complex prepared in step (2) of Example 1, 8 μL of 5 mM dNTP, 5 μL of phi29 DNA polymerase buffer, 2 μL of phi29 DNA polymerase and 15 μL of ultrapure water were mixed, and the mixture was uniformly mixed and then reacted at 30 ℃ for 2 hours for amplification, and then was heated at 65 ℃ for 10 minutes to inactivate the phi29 DNA polymerase. Subsequently, 200 μL of ultrapure water was added to the mixed solution and ultrasonicated for 5 minutes to disperse the DNA nanoflower. Finally, the DNA nanoflower was obtained by centrifuging at 10000 rpm for 10 minutes, discarding the supernatant, and washing the obtained white precipitate twice with water.

[0063] The sequence of one repeat unit of the DNA nanoflower is:

[0064] 5'-CCTCGGCCAGGCTAGCTACAACGACCGCTCCGGTTTTCCTCGGCCAGGCTAGCTACAACGACCGCTCCGGTTTT-3' (SEQ ID NO. 3)

[0065] The results of agarose gel electrophoresis of the circular template-primer complex and the DNA nanoflower are as follows: Figure 2As shown, the migration speed of the circular template-primer complex is slower compared with the linear template, indicating that the circular template is successfully cyclized. Each circular template generates 67 tandem repeat sequences on average to form DNA nanoflowers hybridized with magnesium pyrophosphate. The DNA nanoflowers are retained in the gel pores due to their large molecular weight and diameter. Figure 3 The scanning electron microscope images (SEM images) and transmission electron microscope images (TEM images) of the synthesized DNA nanoflowers are shown in FIG. 2. The images show that the prepared DNA nanoflowers have a flower-like structure with a diameter of about 300 nm.

[0066] Example 2: Preparation of multifunctional DNA nanoassemblies (FDMH)

[0067] (1) Investigation of the loading capacity of DNA nanoflowers for DOX: 15 μg of the DNA nanoflowers prepared in Example 1 were dispersed in 100 μL of ultrapure water to obtain a DNA nanoflower solution with a concentration of 150 μg / mL. Then, 1 μL of 100 μM DOX was mixed with 0.2, 0.5, 1, 2, 5, 10, and 20 μL of the above DNA nanoflower solution with different concentrations (0.3, 0.75, 1.5, 3, 7.5, 15, and 30 μg / mL), respectively, and the volume was supplemented with ultrapure water to 100 μL to obtain mixed solutions of DOX and DNA nanoflowers with different concentrations. After incubation at 37 °C for 1 hour, the DOX-loaded DNA nanoflowers (DNF@DOX, FD) were obtained. The fluorescence intensity of DOX in each group was measured by a microplate reader, and the DOX loading rate was calculated. The excitation wavelength of DOX was 485 nm, and the emission wavelength was 595 nm.

[0068] (2) Preparation of FDMH: 15 μg of DNA nanoflowers prepared in Example 1 was dispersed in 97.5 μL of ultrapure water, then 2.5 μL of 10 μM DOX was added, and after mixing, it was incubated at 37 °C for 1 hour to obtain FD. Then 3 μL of 20 mg / mL tannic acid solution and 3 μL of 5 mg / mL FeCl3·6H2O solution were added to the FD solution under vortex conditions, and after vortexing for 10 seconds, centrifugation was performed at 10000 rpm for 10 minutes. The supernatant was discarded, and after washing once with water, a dark blue precipitate was obtained, which was DNA nanoflowers coated with metal polyphenol network (DNF@DOX@MPN, FDM). Subsequently, the FDM nanoparticles were dispersed in 150 μL of 2 mg / mL hyaluronic acid solution, and incubated at room temperature for 24 hours. After centrifugation at 10000 rpm for 10 minutes, the supernatant was discarded, and after washing twice with water, multifunctional DNA nanoassemblies (DNF@DOX@MNP@HA, FDMH) were obtained. The above preparation method obtained one FDMH. One FDMH was dispersed in 100 μL of ultrapure water to obtain FDMH with a concentration of 150 μg / mL (all subsequent experiments on the preparation of various DNA nanoassemblies were quantified by the mass of the initial DNA nanoflowers contained, so one FDMH obtained corresponds to 15 μg of DNA nanoflowers, and the concentration after dispersion in 100 μL of water is 150 μg / mL), and the concentration of DOX was 250 nM. Two FDMH were dispersed in 100 μL of ultrapure water to obtain FDMH with a concentration of 300 μg / mL, and the concentration of DOX contained was 500 nM, and so on.

[0069] As shown in Figure 4 , the concentration of DOX was kept constant (1 μM), and as the concentration of DNA nanoflowers gradually increased, the loading rate of DOX also gradually increased. When the concentration of DNA nanoflowers was 30 μg / mL, the loading rate of DOX exceeded 95%, proving that DNA nanoflowers can effectively load DOX, and calculation showed that 0.1 nmol of DOX can be loaded per 3 μg of DNA nanoflowers. Figure 5 The TEM images of FDM and FDMH are shown, and the image shows that the flower-like morphology of the DNA nanoflowers remains unchanged, and the coating on the outer layer of the nanoflowers can be observed, proving the successful coating of tannic acid iron and the successful functionalization of hyaluronic acid.

[0070] Figure 6 The particle size distribution and zeta potential of DNA nanoflowers, FD, FDM and FDMH are shown. The loading of DOX has little effect on the particle size and zeta potential of DNA nanoflowers. After coating with metal polyphenol network, the particle size of the nanoparticles increases, and the zeta potential also increases. After functionalization with hyaluronic acid, the particle size of the nanoparticles further increases, while the zeta potential decreases.

[0071] Example 3: Investigation of the photothermal performance of multifunctional DNA nanoassemblies

[0072] (1) 75, 150, 300 and 450 μg / mL solutions of FDMH prepared in Example 2 were prepared, respectively, with a total volume of 150 μL. The FDMH solution was irradiated under near-infrared laser with a wavelength of 808 nm and a power of 1.5 W / cm 2 for 10 minutes, and the solution temperature was recorded every 30 seconds.

[0073] (2) 150 μL of 150 μg / mL FDMH solution was irradiated with 808 nm near-infrared laser with a power of 0.5, 1, 1.5 and 2 W / cm 2 for 10 minutes, and the solution temperature was recorded every 30 seconds.

[0074] (3) 150 μL of 150 μg / mL FDMH solution was irradiated with 808 nm near-infrared laser with a power of 1.5 W / cm 2 for 10 minutes, then the laser was turned off, and when the solution temperature dropped to room temperature, the laser was turned on again. The laser was turned on and off repeatedly for 4 times, and the solution temperature was recorded every 30 seconds.

[0075] As shown in Figures Figure 7 A and 7B, the temperature of pure water and DNA nanoflowers increased slightly under near-infrared laser irradiation, while the temperature of FDMH solution increased with the extension of laser irradiation time and gradually reached equilibrium, and the highest temperature reached increased with the increase of concentration, and showed laser intensity dependence. Figure 7 C is a graph of 4 photothermal cycles, and the highest temperature of FDMH solution after 4 cycles decreased slightly but not much, indicating that FDMH has good photothermal stability.

[0076] Example 4: Feasibility of multifunctional DNA nanoassemblies for chemotherapy and gene therapy

[0077] (1) Verification of the pH responsiveness of DNA nanoflowers: 15 μg of DNA nanoflowers prepared in Example 1 were dispersed in 100 μL of PBS with pH 5.0, and TEM images were taken after 2 hours of reaction at room temperature.

[0078] (2) Verification of the pH responsiveness of metal polyphenol network: 30 μg of FDMH prepared in Example 2 was dispersed in 100 μL of PBS with pH 5.0 and 100 μL of PBS with pH 7.4, respectively. The fluorescence intensity of DOX in the solution was measured every half hour by a microplate reader, with an excitation wavelength of 485 nm and an emission wavelength of 595 nm.

[0079] (3) Deoxyribozyme cleavage ability investigation: 1.6 μL of 50 μM deoxyribozyme and 1.6 μL of 50 μM substrate were mixed in buffer (50 mM Tris-HCl, 150 mM NaCl) containing different Mg 2+ concentrations (0, 0.2, 0.5, 1, 2, 5 and 10 mM) with a total volume of 20 μL, and after incubation at 37 ℃ for 5 hours, polyacrylamide gel electrophoresis was performed.

[0080] The deoxyribozyme sequence is:

[0081] 5'-CCTCGGCCAGGCTAGCTACAACGACCGCTCCGG-3' (SEQ ID NO. 4);

[0082] The substrate sequence is:

[0083] 5'-CCGGAGCGGrArUGGCCGAGG-3' (SEQ ID NO. 5), which is a DNA / RNA chimeric sequence, wherein "rA" represents riboadenosine and "rU" represents ribouridine.

[0084] (4) DNA nanoflower cleavage ability investigation: 15 μg of DNA nanoflowers prepared in Example 1 were dispersed in 50 μL of PBS at pH 5.0 and 50 μL of PBS at pH 7.4, respectively, and after 2 hours of reaction, 5 μL of the solution was mixed with 1.6 μL of 50 μM substrate in buffer with a total volume of 20 μL, and after 5 hours of reaction at 37 ℃, polyacrylamide gel electrophoresis was performed.

[0085] Figure 8 The TEM image of the DNA nanoflowers after treatment with PBS at pH 5.0, due to the dissolution of magnesium pyrophosphate wrapped in the DNA nanoflowers in the acidic environment, the flower-like structure of the DNA nanoflowers disappeared, and long linear DNA chains appeared, which provided data support for the feasibility of subsequent gene therapy and DOX release.

[0086] Figure 9 The DOX release rate of FDMH at different pH, the phenolic hydroxyl groups on the polyphenol molecules are protonated under acidic conditions, leading to instability and even disintegration of the metal polyphenol network, and the exposed DNA nanoflowers also collapse in the acidic environment, releasing DOX, so the DOX release rate of FDMH at pH 5.0 is higher than that at pH 7.4.

[0087] Before exploring the cleavage ability of the DNA nanoflowers, the cleavage ability of the deoxyribozyme was first explored, as shown in Figure 10 the Mg 2+The band of substrate gradually weakened and the band of product gradually enhanced, indicating that the deoxyribozyme had excellent RNA cleavage ability, and the cleavage ability showed Mg 2+ concentration dependence. Subsequently, the DNA nanoflower and the acid-treated DNA nanoflower were mixed with the substrate and incubated, respectively, as shown in Figure 11 The acid-treated DNA nanoflower could cleave the substrate, while the DNA nanoflower without acid treatment could not cleave the substrate, because on the one hand the DNA nanoflower formed long single-stranded DNA in the acidic environment, and on the other hand the magnesium pyrophosphate dissolved to produce Mg 2+ Even if Mg 2+ is not added, the Mg 2+ dissolved can activate the deoxyribozyme on the long single-stranded DNA to cleave the substrate.

[0088] Example 5: Release of DOX in cells

[0089] (1) In order to observe more significant fluorescence of DOX in cells, FDM and FDMH loaded with high concentration of DOX were prepared, and the preparation steps were as follows: 15 μg of DNA nanoflower prepared in Example 1 was dispersed in 95 μL of ultrapure water, then 5 μL of 100 μM DOX was added, and after mixing, it was incubated at 37 ℃ for 1 hour, then 3 μL of 20 mg / mL tannic acid solution and 3 μL of 5 mg / mL FeCl3·6H2O solution were added to the above mixed solution under vortexing conditions, and vortexing was continued for 10 seconds, then centrifugation was performed at 10000 rpm for 10 minutes, and the supernatant was discarded, then water washing was performed once, and one portion of FDM was obtained. One portion of FDM was dispersed in 150 μL of 2 mg / mL hyaluronic acid solution, and incubated at room temperature for 24 hours under shaking, then centrifugation was performed at 10000 rpm for 10 minutes, and the supernatant was discarded, then water washing was performed twice, and one portion of FDMH was obtained. One portion of FDM or FDMH was dispersed in 500 μL of complete medium to obtain a solution of FDM or FDMH with a concentration of 30 μg / mL, and the concentration of DOX contained therein was 1 μM.

[0090] (2) Fluorescence microscopy of DOX in cells: HeLa cells were seeded at 1×10 5HeLa cells were seeded in confocal dishes at a density of 2 x 105cells per dish, incubated in an incubator for 24 hours, washed once with PBS, and then 500 μL of complete medium containing 1 μM free DOX or FDM or FDMH prepared in 500 μL of complete medium at a concentration of 30 μg / mL in Example 5 step (1) was added respectively for further incubation for 4, 8 or 12 hours. After washing the cells twice with PBS, Hoechst 33342 was added for staining for 10 minutes, and then the cells were washed twice with PBS before taking the microscope images. The excitation wavelength for DOX was 485 nm, and that for Hoechst 33342 was 350 nm.

[0091] (3) Flow cytometry for detecting the fluorescence intensity of DOX in cells: HeLa cells were seeded in 6-well plates at a density of 2 x 105cells per well, incubated in an incubator for 24 hours, washed once with PBS, and then 500 μL of complete medium containing 1 μM free DOX or FDM or FDMH prepared in 500 μL of complete medium at a concentration of 30 μg / mL in Example 5 step (1) was added respectively for further incubation for 4, 8 or 12 hours. After washing the cells once with PBS, the cells were trypsinized, and then 1 mL of complete medium was added to terminate the digestion. The cells were transferred into a centrifuge tube, centrifuged at 1000 rpm for 10 minutes, and then the supernatant was discarded. The cells were resuspended in PBS and then detected by flow cytometry. 5 (3) Flow cytometry for detecting the fluorescence intensity of DOX in cells: HeLa cells were seeded in 6-well plates at a density of 2 x 105cells per well, incubated in an incubator for 24 hours, washed once with PBS, and then 500 μL of complete medium containing 1 μM free DOX or FDM or FDMH prepared in 500 μL of complete medium at a concentration of 30 μg / mL in Example 5 step (1) was added respectively for further incubation for 4, 8 or 12 hours. After washing the cells once with PBS, the cells were trypsinized, and then 1 mL of complete medium was added to terminate the digestion. The cells were transferred into a centrifuge tube, centrifuged at 1000 rpm for 10 minutes, and then the supernatant was discarded. The cells were resuspended in PBS and then detected by flow cytometry.

[0092] Figure 12 A is the fluorescence microscope image of DOX in cells. Since DOX can quickly enter cells by diffusion, even if the incubation time is only 4 hours, the red fluorescence of DOX in cells treated with free DOX can be observed. Although FDMH can target into cells by interacting with CD44 on the cell membrane through hyaluronic acid, it takes a certain time for DOX to be released from FDMH. Therefore, the red fluorescence of cells treated with FDMH for 4 hours is weak, but as the incubation time is prolonged, DOX is gradually released, and the red fluorescence in cells gradually increases. The fluorescence intensity of DOX in cells treated with FDM is weak due to the lack of targeting to cancer cells. Figure 12 B is the result graph of the fluorescence intensity of DOX in cells detected by flow cytometry, which is consistent with the results of A. Figure 12 A is the fluorescence microscope image of DOX in cells. Since DOX can quickly enter cells by diffusion, even if the incubation time is only 4 hours, the red fluorescence of DOX in cells treated with free DOX can be observed. Although FDMH can target into cells by interacting with CD44 on the cell membrane through hyaluronic acid, it takes a certain time for DOX to be released from FDMH. Therefore, the red fluorescence of cells treated with FDMH for 4 hours is weak, but as the incubation time is prolonged, DOX is gradually released, and the red fluorescence in cells gradually increases. The fluorescence intensity of DOX in cells treated with FDM is weak due to the lack of targeting to cancer cells.

[0093] Example 6: Evaluation of the therapeutic effect of multifunctional DNA nanoassemblies

[0094] (1) In order to compare the treatment effects of different treatment methods, DNA nanoassemblies (DNF@MPN@HA, FMH) without DOX were prepared as a control. The preparation method of FMH is as follows: 15 μg of DNA nanoflowers prepared in Example 1 were dispersed in 100 μL of ultrapure water, and then 3 μL of 20 mg / mL tannic acid solution and 3 μL of 5 mg / mL FeCl3·6H2O solution were added under vortexing conditions. After vortexing for 10 seconds, centrifugation was performed at 10000 rpm for 10 minutes, and the supernatant was discarded. After washing once with water, DNF@MPN (FM) nanoparticles were obtained. Subsequently, the FM nanoparticles were dispersed in 150 μL of 2 mg / mL hyaluronic acid solution, and incubated at room temperature for 24 hours. Centrifugation was performed at 10000 rpm for 10 minutes, and the supernatant was discarded. After washing twice with water, FMH was obtained. The above preparation method prepared one FMH. Two FMHs were dispersed in 100 μL of ultrapure water to obtain a concentration of 300 μg / mL of FMH.

[0095] (2) CCK-8 experiment: HeLa cells were seeded in a 96-well plate at a density of 5000 cells per well, and cultured for 24 hours. The culture medium was discarded, and FMH (100 μL, 300 μg / mL) prepared in step (1) of Example 6 dispersed in complete culture medium or FDMH (100 μL, 300 μg / mL) prepared in Example 2 dispersed in complete culture medium was added. For groups that did not require near-infrared laser irradiation, the culture medium was discarded after 4 hours of incubation, and fresh culture medium was added for continued incubation for 20 hours. For groups that required near-infrared laser irradiation, the culture medium was discarded after 4 hours of incubation, and the cells were irradiated with a near-infrared laser with a wavelength of 808 nm and a power of 1.5 W / cm 2 for 5 minutes. Then the culture medium was discarded, and fresh culture medium was added for continued incubation for 20 hours. After incubation, the culture medium was discarded, and 110 μL of CCK-8-containing culture medium was added for incubation for 1 hour. Then the absorbance at 450 nm of each well was measured by an enzyme marker, and the cell survival rate was calculated.

[0096] (3) Annexin V-FITC / PI double staining method for detecting cell apoptosis: the cell treatment method was the same as step (2) of Example 6, after incubation, the culture medium was collected into a centrifuge tube (due to the small number of cells in the 96-well plate, 5 holes were set in each group, and one centrifuge tube was collected as one sample), the cells were washed once with PBS, and the PBS was also collected into the centrifuge tube, 30 μL of trypsin without EDTA was added to digest the cells, then 100 μL of complete culture medium was added to terminate the digestion, after blowing, the cells and culture medium were transferred into the centrifuge tube, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, the cells were washed once with PBS, 100 μL of 1x binding buffer, 5 μL of Annexin V-FITC and 10 μL of PI were added, incubated at room temperature for 15 minutes in the dark, and finally 400 μL of 1x binding buffer was added and mixed well before being detected on the machine.

[0097] (4) Calcein-AM / PI live and dead cell staining experiment: the cell treatment steps were the same as step (2) of Example 6, after incubation, the culture medium was discarded, 100 μL of Calcein-AM / PI working solution was added, and incubated in the incubator for 30 minutes, after incubation, the staining effect was observed under a fluorescence microscope, the excitation wavelength of Calcein was 494 nm, and the excitation wavelength of PI was 535 nm.

[0098] (5) 3D cell spheres: HeLa cells were seeded in a U-shaped 96-well ultra-low attachment culture plate at a density of 1500 cells per well, centrifuged at 1000 rpm for 5 minutes, and then cultured in the incubator for 2 days to form 3D cell spheres. FMH (100 μL, 600 μg / mL) dispersed in complete culture medium prepared in step (1) of Example 6 or FDMH (100 μL, 600 μg / mL) dispersed in complete culture medium prepared in Example 2 was added in the form of half medium replacement, for groups that needed near-infrared laser irradiation, after incubation for 4 hours, the cells were irradiated with a laser with a wavelength of 808 nm and a power of 1.5 W / cm 2 for 5 minutes. The morphology of the 3D cell spheres was photographed and recorded at the same time point every day. In addition, the culture medium was replaced once every two days in the form of half medium replacement during the entire experiment.

[0099] Figure 13The survival rate of cells after different treatments is shown in the graph. The FMH group shows a lower cell killing rate because it only undergoes gene therapy. The FDMH group and the FMH+L group undergo gene therapy combined with chemotherapy and gene therapy combined with photothermal therapy, respectively. Under the combined treatment of the two methods, the FDMH group and the FMH+L group show a higher cell killing rate compared with the FMH group. The FDMH+L group can achieve triple combination therapy of gene therapy, chemotherapy, and photothermal therapy, and thus shows the strongest cell killing rate under the same dose. Further, the apoptosis rate caused by different treatments was measured by flow cytometry. As shown in FIG. 5, the apoptosis rate of the FMH group was 9.05%, the apoptosis rates of the FMH+L group and the FDMH group were 25.82% and 17.51%, respectively, and the FDMH+L group caused the highest apoptosis rate of 52.51%. This result is consistent with the CCK-8 result. Subsequently, Calcein-AM / PI double staining provided visual evidence of combined treatment. As shown in FIG. 6, the FDMH+L group showed the most red fluorescent signals representing dead cells, which was consistent with the expectation. Since the 3D cell spheres can more accurately simulate the microenvironment in vivo in vitro, the present application also studied the changes in the morphology of the cell spheres after different treatments. As shown in FIG. 7, the FDMH+L group showed the smallest cell sphere volume after one week of treatment compared with the other groups. In general, these data all indicate that the FDMH shows good anti-tumor effect. Figure 14 Figure 15 Figure 16

[0100] The above merely illustrates the specific embodiments of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.​​​

Claims

1. A multifunctional DNA nanoassembler, characterized in that: The DNA nanoflower comprises a chemotherapy drug, a metal polyphenol network coated on the surface of the DNA nanoflower loaded with the chemotherapy drug, and hyaluronic acid connected to the surface of the metal polyphenol network. 2.The multifunctional DNA nanoassembly of claim 1, wherein: The preparation method of the DNA nanoflower is as follows: the phosphorylated linear template, the primer, the T4 DNA ligase buffer and ultrapure water are mixed, and after high-temperature denaturation treatment, the mixture is stabilized at 4-25 DEG C for 1-4 hours, then the T4 DNA ligase is added, and after 16-37 DEG C reaction for 2-16 hours, the circular template-primer complex is obtained; the circular template-primer complex, dNTP, DNA polymerase buffer, DNA polymerase and ultrapure water are mixed, and the mixture is reacted at 30 DEG C for 2-10 hours for amplification reaction, and the DNA nanoflower is obtained. 3.The multifunctional DNA nanoassembly of claim 2, wherein: The linear template sequence is shown as SEQ ID NO. 1; The primer sequence is shown as SEQ ID NO.

2. 4.The multifunctional DNA nanoassembly of claim 1, wherein: The chemotherapy drug is selected from one of adriamycin, daunorubicin or nor daunorubicin. 5.The multifunctional DNA nanoassembly of claim 1, wherein: The method for loading the chemotherapy drug on the DNA nanoflower is as follows: the DNA nanoflower is dispersed in ultrapure water, and then the chemotherapy drug is added, and the mixture is incubated at 25-37 DEG C in the dark for 0.5-24 hours to obtain the DNA nanoflower loaded with the chemotherapy drug; wherein 1-33 micromoles of the chemotherapy drug is mixed with 1 gram of the DNA nanoflower for incubation. 6.The multifunctional DNA nanoassembly of claim 1, wherein: The metal polyphenol network is one of tannic acid iron, iron gallate or copper gossypol. 7.The multifunctional DNA nanoassembly of claim 1, wherein: The method for coating the metal polyphenol network on the surface of the DNA nanoflower loaded with the chemotherapy drug is as follows: under vortex conditions, the polyphenol solution and the metal salt solution are sequentially added to the solution of the DNA nanoflower loaded with the chemotherapy drug, and the mass ratio of the DNA nanoflower, the polyphenol and the metal salt is 1: (1.3-4): (0.3-1), and after vortex, centrifugation is performed to obtain the DNA nanoflower coated with the metal polyphenol network. 8.The multifunctional DNA nanoassembly of claim 1, wherein: The method for connecting the hyaluronic acid to the surface of the metal polyphenol network is as follows: the DNA nanoflower coated with the metal polyphenol network is dispersed in the hyaluronic acid solution, and the mass ratio of the DNA nanoflower to the hyaluronic acid is 1: (5-20), and after oscillation reaction at 4-37 DEG C for 12-24 hours, centrifugation is performed to obtain the hyaluronic acid functionalized nanoparticles.

9. A method for preparing the multifunctional DNA nanoassemblies of any one of claims 1-8, characterized by: The method comprises the following steps: S1. preparing the DNA nanoflower by the rolling circle amplification reaction; S2. mixing the DNA nanoflower with the chemotherapy drug to load the chemotherapy drug; S3. coating the metal polyphenol network on the surface of the DNA nanoflower loaded with the chemotherapy drug; S4. performing hyaluronic acid functionalization on the surface of the metal polyphenol network.

10. The application of the multifunctional DNA nanometer assembly in the preparation of a drug for treating cancer according to any one of claims 1-8.

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