DNA Hydrogel with Cascade Reaction Function, Preparation Method and Application Thereof

Abstract

The present invention discloses a DNA hydrogel with cascade reaction function, its preparation method and application. The carrier is a pure DNA system, which is formed by means of rolling circle replication. During the rolling circle replication process, an AS1411 nucleic acid aptamer sequence with targeting function and an antisense DNA sequence of hypoxia-inducible factor HIF-1α are generated. A nucleic acid aptamer capable of targeting tumor cells is designed on the linear template, and cholesterol is designed on the nucleic acid aptamer sequence. After high-speed centrifugation and standing, self-assembly forms a DNA hydrogel. This DNA hydrogel has been successfully applied to the drug sustained-release system, has good biocompatibility, and simultaneously loads chlorin e6 (Ce6), heme and glucose oxidase (GOx), showing broad prospects in the fields of inhibiting the growth of malignant tumor cells and biomedicine, etc.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical nanomaterials, and particularly relates to a DNA hydrogel with cascade reaction function, a preparation method thereof, and an application thereof. Background Art

[0002] Solid tumors are characterized by severe hypoxia, elevated levels of hydrogen peroxide (H2O2), and overexpression of glutathione (GSH). The special tumor microenvironment promotes the spread and metastasis of cancer, making it difficult to completely cure cancer through a single therapy. It has been found that many nanoparticles for cancer treatment have good therapeutic effects and low toxicity and side effects, such as DNA nanoparticles, silicon nanoparticles, etc. Although a large number of experimental studies have been carried out, the tumor diagnosis and treatment methods based on nanomaterials still face huge challenges.

[0003] Due to its precise Watson-Crick pairing and simple synthesis process, DNA has been applied to many research fields, among which DNA nanotechnology is an important application. G-quadruplex is a higher-order structure formed by folding DNA or RNA rich in tandem repeats of guanine (G). When G-quadruplex forms a complex with heme (G4 / H), it will have catalase activity and catalyze the reaction mediated by H2O2.

[0004] Starvation therapy is an emerging treatment method that inhibits tumor growth by blocking nutrient supply. Since blocking the supply of nutrients and energy to tumor cells only delays the process of tumor deterioration, it is still difficult to achieve an ideal therapeutic effect. Therefore, the development of a synergistic therapy combined with starvation therapy has far-reaching significance in the treatment of cancer. Photodynamic therapy (PDT) has developed rapidly due to its high selectivity, low side effects, and good adaptability. However, hypoxia-inducible factor-1α (HIF-1α), as one of the most important transcription factors mediating the cellular hypoxia response, can enable tumors to adapt to the hypoxic environment by promoting tumor angiogenesis, increasing the glycolysis level of cells, etc. A large number of experimental results show that in the hypoxic microenvironment of tumors, the PDT treatment effect is significantly reduced.

[0005] In view of the above existing problems and the advantages of DNA hydrogels, it is urgent to study a novel DNA nanohydrogel for treating malignant tumors and a preparation method thereof. Summary of the Invention

[0006] The technical problem to be solved by the present invention is: to overcome the defects of a single treatment method and provide a preparation of a hydrogel for highly efficient treatment of malignant tumors formed by self-assembly of DNA molecules, which has the advantages of high efficiency, low toxicity and side effects, and good biocompatibility.

[0007] The technical solution of the present invention is:

[0008] Preparation method of DNA hydrogel with cascade reaction function. First, a linear DNA replication template is designed, and a hydrogel of a pure DNA system is formed by rolling circle replication. The linear DNA is designed with a complementary sequence of AS1411 nucleic acid aptamer and a complementary sequence of antisense DNA of hypoxia-inducible factor HIF-1α. During the replication process, a large number of antisense DNA of HIF-1α and AS1411 nucleic acid aptamer sequences are generated. Then, the cholesterol-modified nucleic acid aptamer AS1411 sequence is hybridized through the Watson-Crick base complementary pairing principle, and after centrifugation, a DNA hydrogel nanostructure is self-assembled.

[0009] Furthermore, the nucleotide sequence of the linear DNA replication template is as shown in SEQ ID No.1, and the 5'-end is phosphorylated.

[0010] Furthermore, the nucleic acid aptamer is FAM-AS1411-Chol, which is a nucleotide sequence shown in SEQ ID No.3 and has a FAM group modified at the 5'-end and cholesterol modified at the 3'-end.

[0011] Preferably, the specific steps are as follows:

[0012] (1) Anneal the linear DNA template shown in SEQ ID No.1 with the 5'-end phosphorylated and the SEQ ID No.2 primer at the same concentration.

[0013] (2) Add T4 ligase and T4 ligase buffer, and maintain at room temperature for 6 h to form a DNA transcription template.

[0014] (3) Add phi29 DNA polymerase, dNTP, phi29 DNA polymerase buffer, and BSA, and maintain at 30 °C for 8 h to form a high-polymer DNA hydrogel carrier.

[0015] (4) Mix the DNA hydrogel carrier obtained in step (3) with the AS1411 nucleic acid aptamer targeting tumor cells in a buffer solution containing K + , maintain at 95 °C for 5 min, slowly cool to 25 °C, place in a 4 °C refrigerator for 2 h, and centrifuge at 8000 rpm to form the final DNA hydrogel carrier.

[0016] (5) Mix the DNA hydrogel heme and chlorin e6 obtained in step (4), maintain at 25 °C for 2 h, slowly cool to 25 °C, pass through a 10 kDa ultrafiltration tube, centrifuge at 8000 rpm for 5 min and wash 3 times, and then replace the solvent with sterile water to form a DNA hydrogel loaded with Ce6 and Hemin.

[0017] (6) Mix the DNA hydrogel obtained in step (4) with hemin and chlorin e6, maintain at 25 °C for 2 h, slowly cool to 25 °C, pass through a 10 kDa ultrafiltration tube, centrifuge at 8000 rpm for 5 min and wash 3 times, then replace the solvent with sterile water to form a DNA hydrogel loaded with Ce6 and Hemin.

[0018] (7) Dissolve glucose oxidase in 5 mM bicarbonate buffer (pH 8.3) to 1 mg / mL, add 200 mg / mL acrylamide (AAm) to 1 mL of glucose oxidase solution, stir at 4 °C for 10 min, add N-(3-aminopropyl) methacrylamide (APMAAm), glycerol dimethacrylate (GDA), 30 μL of ammonium persulfate and 3 μL of N,N,N',N'-tetramethylethylenediamine under stirring, continue the reaction for 4 h, and then replace the buffer with phosphate buffered saline (pH 7.4) in an ultracentrifugation device (MWCO 30 kDa, Millipore) to remove excess monomers and initiators. That is, glucose oxidase nanoparticles N(GOx) wrapped with a positively charged polyelectrolyte are obtained.

[0019] (8) Mix the DNA hydrogel obtained in step (6) with N(GOx) obtained in step (7) in a certain proportion, mix on a shaker at 25 °C for 30 min, and ultrafilter with a 100 kDa ultrafiltration tube to form a final DNA hydrogel with cascade reaction function.

[0020] Further, the annealing treatment method is: heat to 95 °C in a buffer containing K + and maintain for 5 min, then cool to 25 °C at 1 °C / min to form a G-quadruplex of the AS1411 nucleic acid aptamer.

[0021] Further, the reagent ratio for the preparation of N(GOx) is: the molar ratio of AAm / APMAAm / GDA is 10 / 1 / 0.15.

[0022] In the present invention, the G-quadruplex formed by the hemin and AS1411 (AH) has peroxidase catalytic activity and can catalyze the generation of oxygen from endogenous hydrogen peroxide in tumors.

[0023] The GOx consumes oxygen and glucose to generate gluconic acid and hydrogen peroxide.

[0024] The Ce6 converts oxygen into cytotoxic singlet oxygen to achieve photodynamic therapy of malignant tumors.

[0025] The complementary sequence of the HIF-1α antisense DNA can be complementary paired with the HIF-1α antisense DNA sequence, thereby down-regulating HIF-1α and further promoting the photodynamic therapy of tumors.

[0026] Application of the DNA hydrogel with cascade reaction function in targeted therapeutic drugs for malignant tumors.

[0027] The nucleic acid aptamer used is the aptamer selected by the research group of Liu Zhuang from Soochow University through screening. It specifically binds to nucleolin on the surface of tumor cells, thus achieving targeted drug delivery. The DNA gene nanoprobe constructed as a control group in the subsequent experimental work also has a certain targeted therapeutic effect.

[0028] Cholesterol (Chol) has hydrophobicity, which can promote the formation of nanostructures of DNA transcription copies and also enhance the stability of the structure.

[0029] Hydrogel is a hydrophilic polymer with a three-dimensional network structure. The supramolecular hydrogel used in the present invention is directly constructed with DNA, and the formation of the hydrogel is realized by DNA self-assembly technology and high-speed centrifugation. This hydrogel is precisely assembled through the Watson-Crick and Hoogsteen base complementary pairing principles, with short time consumption and good biocompatibility, and has broad prospects in the fields of inhibiting the growth of targeted tumor cells and biomedicine.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The DNA nucleic acid hydrogel of the present invention has been successfully applied to the drug slow-release system, with good biocompatibility. The addition of the G4 quadruplex structure enables this DNA hydrogel to simultaneously deliver multiple therapeutic genes and therapeutic drugs, and also has broad prospects in the fields of inhibiting the growth of targeted tumor cells and biomedicine. Brief Description of the Drawings

[0032] Figure 1 It is the schematic diagram of the treatment of tumor cells by the DNA hydrogel with cascade reaction function of the present invention;

[0033] Figure 2 It is the verification of the formation of DNA hydrogel nano-hydrogel by 2% agarose gel electrophoresis in Example;

[0034] Figure 3 It is the transmission electron microscope images of DNA nano-hydrogels of different samples in Example (hydrogel N without FAM-AS1411-Chol, hydrogel NF with FAM-AS1411-Chol, DNA hydrogel loaded with Ce6, Hemin, N(GOx));

[0035] Figure 4 It is the Zeta potential diagram of the hydrogel at different stages in Example;

[0036] Figure 5 The seven-day stable size diagram of the DNA hydrogel with cascade reaction function for the example;

[0037] Figure 6 The seven-day stable potential diagram of the DNA hydrogel with cascade reaction function for the example;

[0038] Figure 7 The circular dichroism spectrum of the G4 aptamer of AS1411 with loading capacity for the example;

[0039] Figure 8 Verification of the consumption of oxygen to produce gluconic acid by the final DNA hydrogel with cascade reaction function for the example;

[0040] Figure 9 Verification of the consumption of H2O2 to produce O2 by the final DNA hydrogel with cascade reaction function for the example;

[0041] Figure 10 For the example, the final DNA hydrogel with cascade reaction function consumes O2 to produce 1 Verification of O2;

[0042] Figure 11 For the example, the final DNA hydrogel with cascade reaction function consumes O2 inside cells to produce 1 Verification of O2;

[0043] Figure 12 CLSM detection diagram of the uptake of nano-hydrogel particles by targeted 4T1 cells, A549 cells and non-targeted L02 cells for the example;

[0044] Figure 13 Flow cytometry diagram of the uptake of nano-hydrogel particles by targeted 4T1 cells, A549 cells and non-targeted L02 cells for the example;

[0045] Figure 14 Detection diagram of the cytotoxicity of 4T1 cells with different therapeutic drugs for the example;

[0046] Figure 15 Detection diagram of the cytotoxicity of L02 cells with different therapeutic drugs for the example;

[0047] Figure 16 Uptake of the final DNA hydrogel with cascade reaction function in mice for the example;

[0048] Figure 17 One of the comparisons of the therapeutic effects of different therapeutic drugs on mice for the example;

[0049] Figure 18 Comparison diagram of the therapeutic effects of different therapeutic drugs on mice for the example;

[0050] Figure 19 To monitor the change in the body weight of mice during the treatment process in the examples;

[0051] Figure 20 For HE staining analysis of the main organs of the mice in the examples. Detailed implementation manners

[0052] A preparation method of a DNA hydrogel with a cascade reaction function for targeted therapy of malignant tumors. First, a linear DNA replication template is designed, and a hydrogel of a pure DNA system is formed by rolling circle replication. The linear DNA is designed with a complementary sequence of the AS1411 nucleic acid aptamer and a complementary sequence of the antisense DNA of hypoxia-inducible factor HIF-1α. During the replication process, a large amount of antisense DNA of HIF-1α and AS1411 nucleic acid aptamer sequences are generated. Then, the cholesterol-modified nucleic acid aptamer AS1411 sequence is hybridized through the Watson-Crick base complementary pairing principle, and after centrifugation, a DNA hydrogel nanostructure is self-assembled.

[0053] The nucleotide sequence of the linear DNA replication template is shown in SEQ ID No.1, and the 5'-end is phosphorylated.

[0054] The nucleic acid aptamer targeting tumor cells is FAM-AS1411-Chol, which is the nucleotide sequence shown in SEQ ID No.3 and is modified with a FAM group at the 5'-end and cholesterol at the 3'-end.

[0055] The self-assembled DNA hydrogel is used as a biological carrier for drug delivery, and chlorin e6 (Ce6), heme, and GOx are loaded.

[0056] The DNA nano-hydrogel with a cascade reaction function can load Ce6, heme, and GOx, and all can be used as a control group for subsequent work.

[0057] The DNA nano-hydrogel and the cultured 4T1 cells are incubated in an incubator. The culture environmental conditions are 37°C and a 5% CO2 concentration. After culturing and incubating for a certain time, relevant detections are carried out.

[0058] The DNA hydrogel is applied to mice, and drugs of different preparations are used as a control group for experiments. The treatment effect is observed by monitoring the changes in tumor volume and tissue changes in the mice.

[0059] The DNA nano-hydrogel was simply characterized by transmission electron microscopy (TEM), and then its treatment effect was tested by laser confocal imaging (CLSM), CCK-8 cytotoxicity method, and flow cytometry, etc., to verify the inhibitory growth effect of the drug on the targeted 4T1 tumor cells.

[0060] The DNA hydrogel carrier with cascade reaction function for targeted therapy of malignant tumors provided by the present invention, its preparation method and application use DNA nanohydrogel with gene regulation function as the carrier, which has good biocompatibility. The inhibitory growth effect of this nanoprobe on targeted cells, 4T1 tumor cells, has great application potential in the development of tumor-related research and treatment fields.

[0061] The oligonucleotide sequences used in this experiment are shown in Table 1.

[0062] Table 1 Oligonucleotide sequences used

[0063]

[0064] 1. The preparation process of the DNA nanohydrogel is as follows:

[0065] (1) Take the linear DNA template shown in SEQ ID No.1 with 5'-end phosphorylation and anneal it with the primer of SEQ ID No.2 at the same concentration;

[0066] (2) Add T4 ligase and T4 ligase buffer, and maintain at room temperature for 6 h to form a DNA transcription template;

[0067] (3) Add phi29 DNA polymerase, dNTP, phi29 DNA polymerase buffer and BSA, and maintain at 30 °C for 8 h to form a polymer DNA hydrogel carrier;

[0068] (4) Mix the DNA hydrogel carrier obtained in step (3) with the AS1411 nucleic acid aptamer targeting tumor cells in a buffer solution containing K + and maintain at 95 °C for 5 min, slowly cool to 25 °C, place in a 4 °C refrigerator for 2 h, and centrifuge at 8000 rpm to form the final DNA hydrogel carrier.

[0069] (5) Mix the DNA hydrogel heme and chlorin e6 obtained in step (4), maintain at 25 °C for 2 h, slowly cool to 25 °C, pass through a 10 kDa ultrafiltration tube, centrifuge at 8000 rpm for 5 min and wash 3 times, and then replace the solvent with sterile water to form a DNA hydrogel loaded with Ce6 and Hemin;

[0070] (6) Mix the DNA hydrogel obtained in step (4) with heme and chlorin e6, maintain at 25 °C for 2 h, slowly cool to 25 °C, pass through a 10 kDa ultrafiltration tube, centrifuge at 8000 rpm for 5 min and wash 3 times, and then replace the solvent with sterile water to form a DNA hydrogel loaded with Ce6 and Hemin;

[0071] (7) Dissolve glucose oxidase in 5 mM bicarbonate buffer (pH 8.3) to 1 mg / mL. Add 200 mg / mL acrylamide (AAm) to 1 mL of the glucose oxidase solution, stir at 4 °C for 10 min. While stirring, add N-(3-aminopropyl) methacrylamide (APMAAm), glycerol dimethacrylate (GDA), 30 μL of ammonium persulfate, and 3 μL of N,N,N',N'-tetramethylethylenediamine, and continue the reaction for 4 h. Then, change the buffer with phosphate buffered saline (pH 7.4) in an ultrafiltration device (MWCO 30 kDa, Millipore) to remove excess monomers and initiators. Thus, glucose oxidase nanoparticles N(GOx) encapsulated with a positively charged polyelectrolyte are obtained.

[0072] (8) Mix the DNA hydrogel obtained in step (6) and N(GOx) obtained in step (7) in a certain proportion, mix on a shaker at 25 °C for 30 min, and ultrafilter with a 100 kDa ultrafiltration tube to form the final DNA hydrogel with cascade reaction function.

[0073] To ensure the successful loading of N(GOx), the reagent ratio used in the preparation of N(GOx) is as follows: the molar ratio of AAm / APMAAm / GDA is 10 / 1 / 0.15.

[0074] According to the principle Figure 1 shown in the steps, first, a high molecular weight DNA nano-hydrogel was successfully prepared based on processes such as DNA self-assembly and RCA reaction, as Figure 2 . As Figure 3 , transmission electron microscopy (TEM) was used to analyze the size and morphology of the unmodified cholesterol DNA nanoflowers N prepared by the RCA reaction and the DNA hydrogel NF after cholesterol compression, indicating that the nanoflowers have a spherical porous sponge-like nanostructure, which has certain advantages for drug loading. The size of the uncompressed cholesterol DNA nanoflowers is about 1 μm, and the size of NF after cholesterol compression is about 459 nm. Finally, the size of NF-CHG is about 615 nm. Since the hydrophobicity of cholesterol can compress the hydrogel in a certain space, cholesterol plays an important role in the self-assembly process of the hydrogel.

[0075] At Figure 4Among them, according to the results, it can be seen that the Zeta potential of the DNA nanoflower is around -28.3 mV. When the FAM-AS1411-Chol auxiliary strand DNA is added, due to cholesterol relieving the negative charge, the charge rises to -23.9 mV. Since both GOx and NF are negatively charged, and at the same time, to ensure the enzyme activity of GOx, it is impossible to load NF during the RCA process. Therefore, a positively charged polyelectrolyte that can be acid-degraded is coated on the surface of negatively charged GOx to form N(GOx), thereby realizing the loading of GOx.

[0076] Figure 5 、 Figure 6 , the results show that the hydrodynamic size distribution and Zeta potential of NF-CHG remain stable within 7 days, which confirms its good stability in solution. To prove the key role of G-quadruplex in the loading of photosensitizer and heme, circular dichroism analysis was performed on the samples, as Figure 7 , the results are consistent with the expectations. Each sample shows a positive peak at 262 nm and a negative peak at 238 nm, confirming the stability and drug-loading ability of G-quadruplex in the samples.

[0077] 2. For the characterization of the cascade reaction function of the DNA nanohydrogel with cascade reaction function, the specific operation process is as follows:

[0078] As Figure 8 shown, the pH values of NF-CHG solutions with different pH values without glucose treatment remain unchanged under the original acidic conditions. When glucose is added, the pH of the NF-CHG (pH 5.4) solution gradually decreases with the extension of time within 30 min (ΔpH = 1.03666), while the change in the pH of the NF-CHG (pH 7.4) solution is only 0.13, which can be ignored. The results can show that under acidic conditions, due to the presence of N(GOx), NF-CHG has the ability to oxidize and decompose glucose and consume oxygen.

[0079] As Figure 9 shown, in the presence of hydrogen peroxide, since the hydrogen peroxide in NF-CHG is decomposed by AH, a certain amount of O2 will be generated in the solution, and the oxygen concentration increases from 7.87 mg / L to 10.38 mg / L. These results indicate that NF-CHG has the catalytic ability to decompose glucose and generate oxygen.

[0080] As Figure 10 , after treatment and irradiation with NF-CHG, the DCF fluorescence intensity increases sharply. However, in the absence of NF-CHG or light irradiation, the DCF fluorescence remains unchanged at a low level. The results show that NF-CHG can effectively generate 1 O2 under light.

[0081] As Figure 11, using DCFH-DA as a marker, the ROS production ability of NF-CHG in living cells was measured. When taking laser confocal fluorescence imaging, since the FAM fluorescence channel conflicts with the ROS detection fluorescence channel, therefore, samples without DCFH-DA were detected using the same laser intensity as a control. The results showed that NF-CHG has a high ROS production ability.

[0082] 3. Regarding the targeted cell uptake of DNA nano-hydrogels with cascade reaction functions and their cytotoxicity to targeted cells:

[0083] 4T1 cells (A549 cells, L02 cells) were cultured in a laser confocal special culture dish with DMEM culture medium. After incubation for 12 h, NF-CHG (0.12 μM) was added and co-incubated with the cells for a certain time. After washing 3 times with PBS and staining with Hochest33342 for 20 min, it was observed by CLSM. In addition, the cell uptake of NF-CHG was also measured by flow cytometry.

[0084] The results are as Figure 12 , NF-CHG preferentially accumulates in 4T1 and A549 cells rather than L02 cells. As Figure 13 , consistent with the confocal results, the fluorescence analysis measured by flow cytometry also shows that NF-CHG preferentially accumulates in 4T1 and A549 cells rather than L02 cells.

[0085] The cytotoxicity was determined by the CCK-8 method. 4T1 cells and L02 cells were cultured in 96-well plates. After 12 h, different drug analyses were performed in each well. Subsequently, 10 μL of CCK-8 was added to each well. After co-culturing for 1 h, the absorbance at 450 nm was measured with an enzyme-labeled instrument. As Figure 14 , [[ID=ID=19]] 15 shown, the cytotoxicity of the drug to 4T1 is significantly stronger than that to L02 cells. It is proved that NF-CHG with a synergistic therapeutic effect has great superiority in cancer treatment compared with single starvation therapy or photodynamic therapy.

[0086] 4. In vivo detection experiment

[0087] First, a subcutaneous tumor-bearing mouse model of 4T1 cells was established. After adapting and feeding the spare nude mice, an appropriate amount of 4T1 cells was inoculated, and continuous tracking and observation were carried out. The tumor grew to about 100 mm 3After that, the mice in which the model was successful were randomly divided into six groups, with five mice in each group. Treatments with PBS, Ce6+light, NF-C+light, NF-CH+light, NF-CHG, and NF-CHG+light were performed via the tail vein respectively, and the injection doses and frequencies were the same for each group. After the injection operation, continuous tracking and observation were carried out, the tumor volumes of the mice were tracked and recorded. After the experiment was terminated, the mice were sacrificed, the tumors were dissected, the tissues were anatomized, and the tumors and tissues were fixed with tissue fixative.

[0088] Figures 16 to 20 It is a live data analysis chart of 4T1 tumor-bearing mice after administration of different drugs. Through Figure 16 It can be seen that after 4 h of tail vein injection, NF-CHG began to accumulate near the tumor. As time extended, the fluorescence of NF-CHG increased steadily until it reached the peak at 10 h. Subsequently, the fluorescence at the tumor site gradually decreased from 24 h. This indicates that NF-CHG can accumulate in tumor tissues.

[0089] Figure 17 、 18 It can be seen the therapeutic effects of different drugs. Figure 19 As shown, the injection of drugs had little effect on the body weight of the mice, indicating that the drugs had good biocompatibility. The tissues fixed with 4% paraformaldehyde were paraffin-embedded, cryosectioned, and stained with H@E to analyze the morphological differences of tumor cells and cell proliferation. From Figure 20 It can be seen that there was no obvious metastasis in all organs. It was initially considered that the tumor-bearing system of malignant tumor cells was constructed in the experiment, and there might be some rejection with the mouse source itself.

[0090] In summary, the DNA nano-hydrogel therapeutic platform with cascade reaction function designed in this study achieved targeted combined therapy for tumor cells. The functions of this platform in this experiment were mainly manifested in the following aspects: (Ⅰ) GOx consumed oxygen and glucose to generate gluconic acid and hydrogen peroxide. (Ⅱ) The G-quadruplex formed by heme and AS1411 (AH) had peroxidase catalytic activity and could catalyze the generation of oxygen from endogenous hydrogen peroxide in tumors. (Ⅲ) Ce6 converted oxygen into cytotoxic singlet oxygen to achieve PDT. At the same time, the complementary sequence of HIF-1α antisense DNA could down-regulate HIF-1α by complementary pairing with the HIF-1α antisense DNA sequence, further promoting PDT. Laser confocal imaging and flow cytometry were used to detect and analyze the uptake of the hydrogel by breast cancer cells in vitro, and CCK8 was used to verify the cytotoxicity. A nude mouse model was established, and good therapeutic effects were presented by tracking the body weight, tumor size of the mice and subsequent pathological analysis.

[0091] The above-described embodiments merely represent specific implementation manners of the present application. The description is relatively specific and detailed, but it should not be construed as a limitation on the protection scope of the present application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the technical solution of the present application, several modifications and improvements can still be made, and these all fall within the protection scope of the present application.

Claims

1. Preparation method of DNA hydrogel with cascade reaction function, characterized in that, It includes the following steps: Firstly, design a linear DNA replication template, and form a hydrogel of a pure DNA system through rolling circle replication. In the linear DNA, design a complementary sequence of the AS1411 nucleic acid aptamer and a complementary sequence of the antisense DNA of hypoxia-inducible factor HIF-1α. During the replication process, a large number of antisense DNA of HIF-1α and AS1411 nucleic acid aptamer sequences are generated; Then, hybridize the cholesterol-modified nucleic acid aptamer AS1411 sequence through the Watson-Crick base complementary pairing principle, and self-assemble to form a DNA hydrogel nanostructure after centrifugation; The nucleotide sequence of the linear DNA replication template is shown in SEQ ID No.1, and the 5'-end is phosphorylated; The AS1411 nucleic acid aptamer is FAM-AS1411-Chol, which is a nucleotide sequence shown in SEQ ID No.3, with a FAM group modified at the 5'-end and cholesterol modified at the 3'-end; The DNA nano-hydrogel with cascade reaction function is loaded with Ce6, heme and GOx.

2. The preparation method of the DNA hydrogel with cascade reaction function according to claim 1, characterized in that: The specific steps are as follows: (1) Anneal the linear DNA template shown in SEQ ID No.1 with a phosphorylated 5'-end and the primer at the same concentration; (2) Add T4 ligase and T4 ligase buffer, and maintain at room temperature for 6 h to form a DNA transcription template; (3) Add phi29 DNA polymerase, dNTP, phi29 DNA polymerase buffer and BSA, and maintain at 30 °C for 8 h to form a polymer DNA hydrogel carrier; (4) The DNA hydrogel carrier obtained in step (3) and the AS1411 nucleic acid aptamer targeting tumor cells are maintained at 95 °C for 5 min in a buffer solution containing K + and slowly cooled to 25 °C, placed in a refrigerator at 4 °C for 2 h, and centrifuged at 8000 rpm to form the final DNA hydrogel carrier; (5) Mix the DNA hydrogel heme and chlorin e6 obtained in step (4), maintain at 25 °C for 2 h, slowly cool to 25 °C, pass through a 10 kDa ultrafiltration tube, centrifuge at 8000 rpm for 5 min and wash 3 times, then replace the solvent with sterile water to form a DNA hydrogel loaded with Ce6 and heme; (6) Dissolve glucose oxidase in 5 mM pH 8.3 bicarbonate buffer to 1 mg / mL, add 200 mg / mL acrylamide AAM to 1 mL glucose oxidase solution, stir at 4 °C for 10 min, add N-(3-aminopropyl) methacrylamide APMAAm, glycerol dimethacrylate GDA, 30 μL ammonium persulfate and 3 μL N,N,N',N'-tetramethylethylenediamine under stirring, continue the reaction for 4 h, and then replace the buffer with phosphate buffered saline at pH 7.4 in an ultracentrifugation device to remove excess monomers and initiators; namely, obtain glucose oxidase nanoparticles N(GOx) wrapped with a positively charged polyelectrolyte; (7) Mix the DNA hydrogel obtained in step (5) and N(GOx) obtained in step (6) in a certain proportion, mix on a shaker at 25 °C for 30 min, and ultrafilter with a 100 kDa ultrafiltration tube to form the final DNA hydrogel with cascade reaction function.

3. The preparation method of the DNA hydrogel with cascade reaction function according to claim 2, characterized in that: The annealing treatment method in step (1) is as follows: heating in a buffer solution containing K + to 95 °C and maintaining for 5 min, then cooling at 1 °C / min to 25 °C to enable the AS1411 nucleic acid aptamer to form a G-quadruplex.

4. The preparation method of the DNA hydrogel with cascade reaction function according to claim 2, characterized in that: The reagent ratio used for the preparation of N(GOx) in step (7) is: the molar ratio of AAm / APMAAm / GDA is 10 / 1 / 0.

15. A DNA hydrogel with a cascade reaction function obtained by the preparation method according to any one of claims 1 to 4.

6. Use of the DNA hydrogel with a cascade reaction function according to claim 5 in the preparation of a breast cancer targeted therapeutic agent.

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