Framework nucleic acid machine targeting ALKBH5 and loaded with cisplatin and preparation method and application thereof

Abstract

The present invention discloses a framework nucleic acid machine targeting ALKBH5 and loaded with cisplatin, and a preparation method and application thereof, wherein the framework nucleic acid machine comprises a DNA tetrahedral framework nucleic acid and cisplatin loaded on the DNA tetrahedral framework nucleic acid; wherein the DNA tetrahedral framework nucleic acid is synthesized by hybridization of 4 DNA single strands, and at least one DNA single strand is connected with siRNA targeting ALKBH5. The framework nucleic acid machine provided by the present invention can play a synergistic role after cisplatin is combined with the DNA tetrahedral framework nucleic acid modified with siRNA targeting ALKBH5, significantly inhibiting the expression of ALKBH5 and inhibiting GBM cell proliferation and inducing GBM cell apoptosis, which can effectively improve the therapeutic effect of cisplatin on GBM, and can be used to prepare drugs for treating glioblastoma.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology and relates to a framework nucleic acid machine targeting ALKBH5 and loaded with cisplatin, and a preparation method and application thereof. Background Art

[0002] Glioblastoma (GBM) is the most common malignant tumor of the central nervous system, characterized by high mortality, high recurrence rate, low cure rate, and extremely poor prognosis. The first-line chemotherapy drug temozolomide (TMZ) can penetrate the blood-brain barrier (BBB) ​​to reach the tumor site, but its drug resistance is the main cause of postoperative treatment failure. In addition, no effective method has been found to overcome the drug resistance problem of temozolomide. There are many drugs that can be used clinically for tumor chemotherapy, but most commonly used tumor chemotherapy drugs except temozolomide have the problem of being unable to penetrate the blood-brain barrier to enter the brain tissue, and their efficacy is limited.

[0003] Cisplatin (CDDP) is a first-generation platinum chemotherapy drug with a simple structure and clear mechanism. It is a non-specific drug for the cell cycle. It usually binds to genomic DNA (gDNA) or mitochondrial DNA (mtDNA), causing DNA damage, blocking the production of DNA, mRNA and protein, preventing DNA replication, activating multiple transduction pathways, and ultimately leading to necrosis or apoptosis. However, due to the existence of the blood-brain barrier, cisplatin cannot penetrate the blood-brain barrier to enter the brain tissue to exert its effect, which severely limits its chemotherapy effect on glioblastoma, and is therefore rarely used to treat brain glioma.

[0004] The ALKB family is a nucleic acid oxidase (NAOX) that depends on iron and 2-oxoglutarate. The m6A demethylase ALKBH5 is one of the nine members of the ALKB family. It is highly expressed in glioblastoma and maintains the stemness of glioblastoma.

[0005] DNA tetrahedron (TDN) is a new type of nanobiomaterial that is widely used in the biomedical field due to its good biocompatibility, editability and biosafety. Due to its special spatial structure and appropriate size, TDN has great potential in cell uptake and tissue penetration, and TDN can function as a delivery carrier. Summary of the invention

[0006] The first object of the present invention is to provide a framework nucleic acid machine that targets ALKBH5 and loads cisplatin, so as to solve at least one of the above-mentioned technical problems.

[0007] The second object of the present invention is to provide a method for preparing the above-mentioned framework nucleic acid machine to solve at least one of the above-mentioned technical problems.

[0008] The third object of the present invention is to provide the use of the above-mentioned framework nucleic acid machine in the preparation of drugs for treating glioblastoma.

[0009] According to one aspect of the present invention, a framework nucleic acid machine targeting ALKBH5 and loaded with cisplatin is provided, which comprises a DNA tetrahedral framework nucleic acid and cisplatin, wherein cisplatin is bound to the DNA tetrahedral framework nucleic acid by electrostatic adsorption and covalent bond binding, thereby being loaded on the DNA tetrahedral framework nucleic acid.

[0010] In some embodiments, the DNA tetrahedral framework nucleic acid is synthesized by hybridization of four DNA single strands, and the nucleotide sequences of the four DNA single strands respectively include the nucleotide sequences shown in SEQ ID NOs: 1 to 4; wherein at least one DNA single strand is connected to an siRNA targeting ALKBH5.

[0011] The experimental results of in vivo / in vitro ALKBH5 silencing effect evaluation experiments, in vitro anti-GBM evaluation experiments, and in vivo anti-tumor experiments show that the framework nucleic acid machine provided by the present invention, cisplatin and DNA tetrahedral framework nucleic acid modified with siRNA targeting ALKBH5 can play a synergistic role after being combined by electrostatic adsorption and covalent bond binding, significantly inhibiting the expression of ALKBH5 and inhibiting GBM cell proliferation and inducing GBM cell apoptosis. The framework nucleic acid machine provided by the present invention can effectively improve the therapeutic effect of cisplatin on GBM and can be used to prepare drugs for treating glioblastoma.

[0012] In addition, the framework nucleic acid machine provided by the present invention has no in vitro cytotoxicity, has no effect on the size of multiple physiological indicators such as blood routine and blood biochemistry, has no adverse effects or toxic effects on organs, and is highly safe.

[0013] In some embodiments, the antisense nucleotide sequence of the siRNA targeting ALKBH5 includes a modified or unmodified nucleotide sequence as shown in SEQ ID NO:5.

[0014] In some embodiments, at least one DNA single strand is connected to the nucleic acid aptamer AS1411, wherein the nucleotide sequence of the nucleic acid aptamer AS1411 is shown in SEQ ID NO: 6. The nucleic acid aptamer AS1411 can specifically bind to the nucleolin on the cell membrane surface of the tumor cell, so that the drug delivery system containing the nucleic acid aptamer AS1411 is transported into the cell; or under the dual stimulation of the regulation of nucleolin and the nucleic acid aptamer AS1411, the tumor cell produces macropinocytosis to internalize the drug delivery system containing the nucleic acid aptamer AS1411. Therefore, the modification of the nucleic acid aptamer AS1411 can improve the uptake of the framework nucleic acid machine by tumor cells.

[0015] In some embodiments, at least one single strand of DNA is connected to a nucleic acid aptamer GS24, wherein the nucleotide sequence of the nucleic acid aptamer GS24 is shown in SEQ ID NO: 7. The nucleic acid aptamer GS24 can specifically bind to the transferrin receptor on the surface of vascular endothelial cells, and assist the drug delivery system containing the nucleic acid aptamer GS24 to pass through the cerebral vascular wall. Thus, the modification of the nucleic acid aptamer GS24 can assist the framework nucleic acid machine to pass through the blood-brain barrier and deliver cisplatin and siRNA targeting ALKBH5 to the tumor site.

[0016] In some embodiments, the nucleotide sequences of the four DNA single strands of the DNA tetrahedral framework nucleic acid are shown as SEQ ID NO: 1 and SEQ ID NO: 8 to 10, respectively. The DNA tetrahedral framework nucleic acid targets ALKBH5 and is modified with nucleic acid aptamer AS1411 and nucleic acid aptamer GS24.

[0017] According to another aspect of the present invention, a method for preparing a framework nucleic acid machine targeting ALKBH5 and loaded with cisplatin is provided, comprising the following steps:

[0018] The DNA single strands used for hybridization synthesis of DNA tetrahedral framework nucleic acid were added to TM buffer respectively, and after mixing, heated at 95°C for 10 min, and then cooled to 4°C and kept for 10 min to obtain DNA tetrahedral framework nucleic acid;

[0019] Mix the DNA tetrahedral framework nucleic acid and cisplatin, and incubate at 37°C for 3-4 h.

[0020] In some embodiments, the TM buffer has a pH of 8.0 and is composed of 10 mM Tris-HCl and 50 mM MgCl. 2 .

[0021] In some embodiments, the molar ratio of any two DNA single strands in the DNA single strands used for hybridization synthesis of DNA tetrahedral framework nucleic acid is 1:1.

[0022] In some embodiments, the molar ratio of DNA tetrahedral framework nucleic acid to cisplatin can be 1:(500-4000).

[0023] In some embodiments, the molar ratio of the DNA tetrahedral framework nucleic acid to cisplatin is 1: 2000. Thus, the drug loading efficiency of cisplatin is effectively improved.

[0024] The framework nucleic acid machine provided by the present invention has a simple preparation method. The prepared framework nucleic acid machine can deliver cisplatin while inhibiting ALKBH5 expression, thereby achieving the dual therapeutic effects of inhibiting tumor cell proliferation and promoting tumor cell apoptosis, increasing the sensitivity of glioblastoma to cisplatin chemotherapy, and improving the therapeutic effect. The framework nucleic acid machine can be used to prepare drugs for treating glioblastoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The schematic diagram of the synthesis route of the framework nucleic acid machine of the present invention;

[0026] Figure 2 This is the result of 8% polyacrylamide gel electrophoresis of TDN;

[0027] Figure 3 8% polyacrylamide gel electrophoresis results of TDN, TDN-AS1411, TDN-GS24 and TDN-AG-siRNA;

[0028] Figure 4 AFM image of TDN, scale bar 120 nm;

[0029] Figure 5 TEM image of TDN, scale bar 200 nm;

[0030] Figures 6 to 9 The results of DLS detection of particle size and Zeta potential of TDN, and particle size and Zeta potential of TDN-AG-siRNA@CDDP are shown in order;

[0031] Figures 10-12 The results of flow cytometry and quantitative analysis of the uptake of Cy5-labeled TDN-GS24 by bEnd.3, U87 and U251 cells at different time points are shown in sequence;

[0032] Fig.13 IVIS images of real-time fluorescence tracking of Cy5-labeled TDN (Cy5) and TDN-AS1411-GS24 (Cy5) in U87-luc tumor-bearing mice; representative bioluminescence and fluorescence images of the brain and other organs collected 60 min after injection and quantitative analysis of fluorescence values;

[0033] Fig.14The results of in vitro cytotoxicity evaluation of different DNA tetrahedrons are shown;

[0034] Fig.15 The hemolysis test and quantitative analysis results of different concentrations of TDN-AG;

[0035] Figures 16 and 17 These are representative confocal images of Cy5-labeled TDN-AS1411-GS24 uptake by U87 and U251 cells in the in vitro BBB model, where scale bar, 100 μm;

[0036] Fig.18 The figure shows the WB analysis results of the silencing efficiency of different drugs on ALKBH5 in vitro;

[0037] Fig.19 The figure shows the WB analysis results of different drug treatments on the silencing efficiency of ALKBH5 in tumor tissues of orthotopic GBM mouse models;

[0038] Figures 20-21 The cell activities of U87 and U251 GBM cells after treatment with different drugs at different concentrations are shown in sequence; the different color curves correspond to the same treatment groups. Fig. 27 ;

[0039] Fig. 22 This is the result of EdU proliferation experiment; cell nucleus: blue; EdU: red; scale bar, 100 μm;

[0040] Fig.23 The results of quantitative analysis of EdU staining;

[0041] Fig.24 The results of cell apoptosis were detected by flow cytometry;

[0042] Fig.25 WB analysis of the expression of γ-H2Ax, Bax, Bcl-2 and Caspase3 in GBM cells after treatment with different drugs;

[0043] Fig.26 Representative tumor bioluminescence images of mice on days 5, 10, 15, and 20 after tumor model establishment;

[0044] Fig. 27 Tumor growth curves to quantify bioluminescent signal intensity;

[0045] Fig.28 The Kaplan-Meier survival curve of treated mice; curves of different colors correspond to the same treatment groups. Fig. 27 ;

[0046] Fig.29The weight curve of mice from the beginning of tumor transplantation to the 20th day; the curves of different colors correspond to the same treatment groups. Fig. 27 ;

[0047] Fig.30 Schematic diagram of MRI and T2WI imaging results of representative mouse brain;

[0048] Fig.31 Representative tumor sections of IHC staining for γ-H2Ax, BAX, Bcl-2, and Caspase3 in different groups; scale bar, 100 μm;

[0049] Fig.32 H&E staining images of the main organs of normal mice treated with PBS or TDN-AG-siRNA@CDDP on the 1st and 7th days, scale bar, 100 μm;

[0050] Fig.33 The results of blood routine tests of normal mice on the 1st and 7th days after treatment with PBS or TDN-AG-siRNA@CDDP;

[0051] Fig.34 The results of blood biochemical indexes of normal mice treated with PBS or TDN-AG-siRNA@CDDP on the 1st and 7th days. DETAILED DESCRIPTION

[0052] The present invention is further described in detail below in conjunction with the embodiments. The examples are only for explanation and are not intended to limit the present invention in any way. Unless otherwise specified, the raw materials and reagents used in the examples are conventional products that can be obtained commercially; the experimental methods in the examples where specific conditions are not specified are usually carried out according to conventional conditions in the art or according to the conditions recommended by the manufacturer.

[0053] The DNA sequences involved in the present invention are shown in Table 1.

[0054] Table 1 DNA sequences involved in the present invention

[0055]

[0056] The schematic diagram of the synthetic route of the framework nucleic acid machine of the present invention is as follows Figure 1 shown.

[0057] The cell lines and animal sources used in the present invention are as follows:

[0058] U87MG, U251MG, and bEnd.3 cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and maintained in DMEM containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO 2 nourish.

[0059] DMEM medium, FBS and other cell culture supplies were purchased from Gibco (USA).

[0060] Female BALB / c mice (4 to 5 weeks old) were purchased from Guangdong Medical Laboratory Animal Center and maintained in the Laboratory Animal Center of Zhujiang Hospital of Southern Medical University. All animal experiments were performed in accordance with the procedures approved by the Laboratory Animal Ethics Committee (LAEC-2024-068).

[0061] The protein detection (Western Blot, WB) experiment involved in the present invention comprises the following steps:

[0062] Proteins from cells and mouse GBM tissues were extracted on ice using a membrane protein extraction kit (KeyGEN Biotechnology, China) and a whole cell lysis kit (KeyGEN Biotechnology, China), followed by quantification using a BCA assay kit (Beyotime, China). After quantification, protein lysates were added to 5× loading buffer (Fdbio science, China), separated by SDS-polyacrylamide gel electrophoresis, and then transferred to PVDF membranes (Millipore, USA). The membranes were incubated with the corresponding primary antibodies overnight and then with horseradish peroxidase (HRP)-labeled secondary antibodies (CST, USA) for 1 h. The bands were visualized using the ECL detection system of the ImageQuant LAS500 chemiluminescence technology (General Electric, USA). Protein expression was analyzed by ImageJ (National Institutes of Health, USA) software, with β-actin as a control.

[0063] In the present invention, the relevant experimental data were statistically analyzed using GraphPad Prism 9 (GraphPad Software, USA). The data were repeated ≥ 3 times, and the results were expressed as mean ± standard deviation (SD). t Test, one-way analysis of variance or two-way analysis of variance was used for comparison among multiple groups. Kaplan-Meier method was used to draw survival curves, and log-rank test was used to compare survival differences among groups. The symbol "ns" means not significant. * express P <0.05, ** express P <0.01, *** express P <0.001, **** express P <0.0001.

[0064] Example 1 Preparation of a framework nucleic acid machine targeting ALKBH5 and loaded with cisplatin

[0065] The steps include:

[0066] (1) DNA tetrahedral assembly

[0067] The DNA single strands were dissolved in DEPC water, purified by HPLC, and the concentration of the purified oligonucleotides was determined by measuring UV absorption at 260 nm.

[0068] In TM buffer (10 mM Tris-HCl and 50 mM MgCl 2 Equimolar amounts of purified single-stranded DNA S1, S2-AS1411, S3-GS24 and S4-siRNA were added to the culture medium (pH=8.0), wherein the final concentration of each single-stranded DNA was 1000 nM after dissolution. After dissolution, the DNA was thoroughly mixed and placed in a PCR instrument. After a heating program (heating at 95°C for 10 min, rapidly cooling to 4°C and maintaining for 20 min), the DNA tetrahedron modified with nucleic acid aptamers and connected to siRNA was self-assembled and synthesized, which was denoted as TDN-AG-siRNA (or TDN-AS1411-GS24-ALKBH5, TDN-AG-siALKBH5).

[0069] Similarly, different combinations of single-stranded DNA were used to prepare TDN, TDN-AS1411, TDN-GS24, TDN-AS1411-GS24 (or TDN-AG), TDN-siALKBH5 (or TDN-ALKBH5), TDN-AS1411-siALKBH5 (or TDN-A-siALKBH5), and TDN-GS24-siALKBH5 (or TDN-G-siALKBH5). Cy5-S1 was used instead of S1 to prepare TDN (Cy5), TDN-GS24 (Cy5), and TDN-AS1411-GS24 (Cy5) labeled with Cy5.

[0070] The synthesized TDN, TDN-AS1411, TDN-GS24, and TDN-AG-siRNA were detected by 8% polyacrylamide gel electrophoresis (PAGE). Figure 2~3 shown. Figure 2 In the figure, lanes 1 to 4 are DNA single strands S1 to S4, lane 5 is TDN, lane 6 is S1+S2+S3, lane 7 is S1+S2, and lane 8 is S1; Figure 3 In the figure, lanes 1 to 4 are TDN, TDN-AS1411, TDN-GS24 and TDN-AG-siRNA. PAGE results showed that TDN, TDN-AS1411, TDN-GS24 and TDN-AG-siRNA were successfully synthesized.

[0071] The synthesis, size, and shape of TDNs were examined and verified by atomic force microscopy (AFM) and transmission electron microscopy (TEM). Figures 4 and 5 shown. Figures 4 and 5 The successful synthesis of TDN was verified and its shape was shown.

[0072] (2) Synthesis of TDN-AG-siRNA@CDDP

[0073] CDDP was dissolved in DEPC water, and then TDN-AG-siRNA and CDDP were mixed at a molar ratio of 1:2000 and incubated at 37°C for about 3-4 hours. After incubation, the residual single-stranded DNA (ssDNA) and CDDP were removed by ultrafiltration (30 kDa molecular weight membrane, Millipore, USA) to obtain TDN-AG-siRNA loaded with CDDP (denoted as TDN-AG-siRNA@CDDP).

[0074] Similarly, TDN-AG@CDDP was prepared according to the above method for subsequent experiments.

[0075] The particle size and zeta potential of TDN and TDN-AG-siRNA@CDDP were measured by dynamic light scattering DLS (NanoZS, Malvern, UK). Figures 6 to 9 The results showed that the particle size of TDN was 6.82 nm and the Zeta potential was -4.00 mV, while the particle size of TDN-AG-siRNA@CDDP was 14.13 nm and the Zeta potential was -0.462 mV. These differences indicated that the nanoparticles TDN-AG-siRNA@CDDP were successfully synthesized and relatively stable.

[0076] Test Example 1 Evaluation of the BBB penetration ability of TDN-GS24

[0077] (1) Construction of in vitro BBB model

[0078] According to the method described in reference (DOI: 10.1002 / advs.202004929), an in vitro BBB model was constructed using bEnd.3 until the transendothelial electrical resistance (TEER) was between 200 and 300 Ω, where bEnd.3 was located in the upper chamber of the Transwell cell culture system and the lower chamber was U87MG or U251MG.

[0079] (2) Evaluation of TDN-GS24’s ability to penetrate the BBB

[0080] TDN-GS24 labeled with Cy5 (400 nM) was added to the upper chamber (bEnd.3) of the Transwell cell culture system and incubated for 0, 1, 2, 4, and 6 h. Cells in the upper and lower chambers were collected for flow cytometry (BD Biosciences, USA) to detect the fluorescence intensity of Cy5, and data were analyzed using FlowJo v10.8.1 (USA).

[0081] The results are as follows Figures 10-12 Flow cytometry results showed that the number of cells that took up Cy5 fluorescence gradually decreased in the upper chamber, while the number of cells that took up Cy5 fluorescence gradually increased in the lower chamber, indicating that TDN-GS24 could effectively cross the BBB and be internalized by GBM cells in vitro.

[0082] Experimental Example 2 Evaluation of the ability of TDN-GS24 to penetrate the BBB in vivo

[0083] (1) Establishment of GBM orthotopic mouse model (U87-luc tumor-bearing mice)

[0084] Under the guidance of a stereotaxic instrument (RWD, China), 2-5 μL of 5 × 10 6 PBS containing 100 U87-luc cells was injected into the brain of female BALB / c nude mice (4-5 weeks old). The injection site was the coordinate relative to the posterior lobe (2.0 mm posterior lobe, 2.0 mm lateral lobe, 3.0 mm ventral). After the orthotopic GBM model was established, bioluminescence imaging was performed on days 5, 10, 15, and 20 using the IVIS Spectrum imaging system to observe the growth of intracranial tumors. The body weight of mice was monitored regularly, and the survival curve was drawn using the Kaplan-Meier method.

[0085] (2) Evaluation of BBB penetration ability in vivo

[0086] 400 nM Cy5-labeled TDN-AS1411-GS24 and TDN were intravenously injected into U87-luc tumor-bearing mice, respectively, with an injection volume of 200 μL, and the position distribution of Cy5 fluorescence was tracked using the IVIS Spectrum imaging system (PerkinElmer, USA). Mice were killed 60 min after injection, and brain, heart, liver, spleen, lung, and kidney tissues were obtained and visualized using similar imaging parameters. LivingImage4.3.1 (PerkinElmer, USA) was used to quantitatively evaluate the fluorescence intensity.

[0087] The results are as follows Fig.13As shown. IVIS images show that fluorescence signals can be detected in the brain of U87-luc tumor-bearing mice after TDN (Cy5) and TDN-AS1411-GS24 (Cy5) injection. In the TDN-AS1411-GS24 (Cy5) group, the fluorescence mostly persisted in the brain over time due to the specific binding of GS24 and TfR, and the specific binding of AS1411 to nucleolin. In contrast, TDN (Cy5) showed peak fluorescence intensity at 10 minutes and was quickly eliminated from the brain due to the lack of two specific binding affinities.

[0088] In vitro fluorescence images showed that the fluorescence signals of TDN (Cy5) and TDN-AS1411-GS24 (Cy5) were significantly colocalized with the bioluminescent signals of GBM in the brain. The TDN-AS1411-GS24 (Cy5) group showed stronger fluorescence compared with the TDN (Cy5) group. In addition, TDN-AS1411-GS24 (Cy5) showed lower renal metabolism than TDN (Cy5) within the same time frame. These results indicate that TDN-AS1411-GS24 (Cy5) crosses the blood-brain barrier more effectively than TDN (Cy5), enhances GBM targeting, and minimizes renal metabolism.

[0089] Test Example 3 In vitro cytotoxicity evaluation

[0090] The CCK-8 detection kit was used to detect the cell activity after treatment with TDN, TDN-AS1411, TDN-GS24, TDN-siALKBH5, and TDN-AG-siRNA.

[0091] U87 cells were seeded in 96-well plates and pre-cultured in an incubator for 24 h. Subsequently, TDN, TDN-AS1411, TDN-GS24, TDN-siALKBH5, and TDN-AG-siRNA were added at concentrations of 0, 25, 50, 100, 200, and 400 nM for 48 h, the original culture medium was removed, CCK-8 solution was added, and the cells were incubated at 37°C for 2-4 h. The absorbance at 450 nm was measured with an ELISA reader to calculate the cell activity.

[0092] The results are as follows Fig.14 The results showed that after incubation with different DNA tetrahedrons at concentrations of 0, 25, 50, 100, 200, and 400 nM for 24 h, the cell activity of each group of cells did not change significantly, indicating that TDN, TDN-AS1411, TDN-GS24, TDN-siALKBH5, and TDN-AG-siRNA had no in vitro cytotoxicity.

[0093] Test Example 4 Hemolysis Test

[0094] 2 mL of blood was collected from the mouse orbit, added to an anticoagulant tube, and centrifuged (3000 rpm, 5 min) to obtain red blood cells. The resulting precipitate was collected after washing with PBS 5 times. It was then diluted to 10 times the initial volume with PBS. 200 μL of the diluted suspension was mixed with 800 μL of PBS, Triton-X 100 (0.025% PBS), and different concentrations of TDN-AG (25, 50, 100, 200, 400 nM) into 1 mL of the system. After incubation at 37°C for 2 h, the supernatant was centrifuged and the absorbance was measured at 541 nm. The hemolysis rate was calculated according to the formula: Hemolysis rate (%) = absorbance [(sample-negative control) / (positive control-negative control)] × 100%.

[0095] The results are as follows Fig.15 The results of the hemolysis experiment showed that TDN-AG did not induce hemolysis in the concentration gradient of 25, 50, 100, 200, and 400 nM.

[0096] Experimental Example 5 GBM cell targeting ability and uptake under in vitro BBB model

[0097] The in vitro BBB model was constructed using bEnd.3. The lower chamber was inoculated with U87 and U251 cell suspensions and pre-cultured in an incubator for 24 h. After the cells adhered to the wall, Cy5-labeled TDN-AS1411-GS24 (400 nM) was added to the upper chamber of the model. After culturing in the incubator for 0, 1, 2, 4, and 6 h, the culture medium was aspirated and the cells in the lower chamber were subjected to the following operations: washing three times with PBS, each for 5 min; then fixed with 4% paraformaldehyde for 30 min, the paraformaldehyde was aspirated, and the cells were washed three times with PBS, each for 5 min; then incubated with phalloidin (FITC-labeled), protected from light for 15 min, the phalloidin was aspirated, and the cells were washed three times with PBS, each for 5 min; then incubated with DAPI, protected from light for 5-10 min, the DAPI was aspirated, and the cells were washed three times with PBS, each for 5 min; after aspiration, protected from light, a confocal microscope (Nikon, Japan) was used to observe the distribution of Cy5 fluorescence signals in cells.

[0098] The results are as follows Figures 16 and 17 The results showed that TDN-AS1411-GS24 was gradually taken up by both U87 and U251 GBM cells within 0-6 h, and experienced the process of crossing the BBB, targeting the GBM cell membrane, and being taken up by GBM cells. The process of its uptake by GBM cells was "temporally and spatially ordered".

[0099] Experimental Example 6 In vitro gene silencing effect of TDN-AG-siRNA

[0100] The in vitro BBB model was constructed using bEnd.3. U87 cells were plated at 1.5×10 5 The density of cells was planted in the lower chamber. After 48 h of culture, the cell fusion rate was about 70%. PBS, siALKBH5, TDN-A-siALKBH5, TDN-G-siALKBH5, and TDN-AG-siRNA were added to the upper chamber, and the concentration of siALKBH5 in each group was 400 nM. Among them, Lipo3000 (ThermoFisher Scientific, USA) reagent was transfected with siALKBH5 strictly according to the instructions. After 48 h, the cells were collected for Western Blot (WB) analysis.

[0101] The results are as follows Fig.18 shown.

[0102] In the in vitro BBB model, protein collection and WB detection were performed on GBM cells in the lower chamber. WB results showed that TDN-AG-siRNA could significantly penetrate the BBB to target GBM cells and effectively silence the ALKBH5 gene.

[0103] Experimental Example 7 In vivo gene silencing effect of TDN-AG-siRNA

[0104] Using the orthotopic GBM mouse model, 5 days after the implantation of U87-luc cells in tumor-bearing nude mice, the mice were divided into 4 groups (n=6) by random number table method. PBS, FreeCDDP, TDN-AG-siRNA, TDN-AG-siRNA+CDDP, and TDN-AG-siRNA@CDDP were injected through the tail vein on days 10, 13, 16, and 19 after the implantation of U87-luc cells. The CDDP concentration in each group was maintained at 2.5 mg / mL, and the injection volume was 200 μL each time. On the 20th day from the date of tumor cell implantation, the mice were killed, and the tumor tissues were obtained. The expression of ALKBH5 protein in the tumor tissues was detected by WB.

[0105] The results are as follows Fig.19 The results showed that TDN-AG-siRNA@CDDP treatment could reduce the expression of ALKBH5 in intracranial transplanted tumors to a higher degree compared with other treatment groups.

[0106] Experimental Example 8 In vitro evaluation of TDN-AG-siRNA@CDDP against GBM

[0107] After the in vitro BBB model was successfully constructed, the GBM cells in the lower chamber of the Transwell cell culture system were incubated with PBS, Free CDDP, TDN-A-G+CDDP, TDN-AG-siRNA+CDDP, TDN-AG@CDDP, and TDN-AG-siRNA@CDDP in the upper chamber for 24 h. The CCK-8 method was used to detect the effect on cell viability, and the IC50 at a wavelength of 450 nm was determined. At the same time, the Click-iTEdU imaging kit (KeyGEN BioTECH, China) was used for detection and confocal microscopy was used for observation. All staining was performed according to the manufacturer's instructions. At the same time, U87 cells in the lower chamber were collected, and the degree of cell apoptosis was detected by flow cytometry using the AnnexinV-FITC / PI kit (KeyGENBioTECH, China). WB was used to detect the levels of γ-H2Ax, Bax, Bcl-2, and Caspase3 proteins in U87 cells in different treatment groups, and their grayscale values ​​were calculated by ImageJ.

[0108] The results are as follows Figures 20 to 25 shown.

[0109] Figures 20-21 The results showed that TDN-AG-siRNA@CDDP significantly increased the death of GBM cells. 50 The value is 9.224 μg / mL; IC 50 The value is 11.24 μg / mL.

[0110] Figure 22-23 The results of EdU cell proliferation experiments confirmed that TDN-AG-siRNA@CDDP treatment could significantly reduce the proliferation of GBM cells, and the differences were significant compared with TDN-AG-siRNA+CDDP and TDN-AG@CDDP groups ( P <0.05), indicating that after cisplatin and TDN-AG-siRNA are combined through electrostatic adsorption and covalent bond binding, the two can play a synergistic role and significantly improve the inhibitory effect on GBM cell proliferation.

[0111] Fig.24 The flow cytometry results shown further demonstrated that TDN-AG-siRNA@CDDP could induce GBM cell apoptosis to the highest extent.

[0112] Fig.25The WB experimental results shown in the figure indicate that TDN-AG-siRNA@CDDP can increase the expression of γ-H2Ax, Cleave-Caspase-3 and Bax, while decreasing the expression of Bcl-2, further confirming that it has a good function of inducing GBM cell apoptosis.

[0113] Experimental Example 9 In vivo anti-GBM activity of TDN-AG-siRNA@CDDP

[0114] Using the orthotopic GBM mouse model, 5 days after the implantation of U87-luc cells in tumor-bearing nude mice, the mice were divided into 4 groups (n=6) by random number table method. PBS, FreeCDDP, TAG+CDDP, TDN-AG-siRNA+CDDP, TDN-AG@CDDP, and TDN-AG-siRNA@CDDP were injected through the tail vein on days 10, 13, 16, and 19 after the implantation of U87-luc cells. The CDDP concentration in each group was maintained at 2.5 mg / mL, and the injection volume was 200 μL each time.

[0115] Body weight was monitored every 4 days from the day of tumor cell implantation. The IVIS Spectrum Imaging System was used for bioluminescence imaging to detect intracranial tumor growth on days 5, 10, 15, and 20. At the same time, the small animal magnetic resonance imaging system (BRUKER, USA) was used to observe the size of the intracranial tumor on day 20, and the images were analyzed using Paravision 6.0 (BRUKER, USA). The mice were then killed, and the brain tissue was obtained, fixed with 4% PFA, embedded in paraffin, and stained with immunohistochemistry (IHC). The paraffin-embedded tissue was sectioned at 4 μm.

[0116] IHC staining: Tissue sections were dewaxed, hydrated, and antigen recovered, blocked with 10% goat serum for 1 h at room temperature, and incubated with primary antibodies (Ki67, γ-H2Ax, BAX, Bcl-2) at 4°C overnight. After washing with PBS, the sections were reacted with HRP-labeled secondary antibodies. Observation was performed using diaminobenzidine (DAB) as a substrate. Digital images were acquired under a scanner (3DHISTECH, Hungary). Quantitative analysis was performed using ImageJ software.

[0117] The results are as follows Figures 26 to 31 shown.

[0118] The growth of U87-lucGBM tumors was monitored by luciferin bioluminescence. Fig.26 ), fluorescence values ​​were quantified by relative photon flux method ( Fig. 27 ). The results showed that the TDN-AG-siRNA@CDDP group could reduce the tumor fluorescence intensity to the greatest extent compared with other groups.

[0119] Fig.28 The Kaplan-Meier survival curves shown in the figure show that the median survival time of mice in the TDN-AG-siRNA@CDDP group was significantly longer than that in the other groups. Fig.29 The body weight curve shown shows that the body weight of mice in the TDN-AG-siRNA@CDDP group did not change much and had no acute toxicity.

[0120] Fig.30 The high-resolution MRI images shown show that the tumor volume in the TDN-AG-siRNA@CDDP group was the smallest, indicating that its therapeutic effect was the most significant.

[0121] Fig.31 The IHC results showed that among the treatment groups, the changes in apoptotic protein indicators in the TDN-AG-siRNA@CDDP group were the most obvious, which was consistent with Fig.25 The results are consistent with those of the in vitro apoptosis protein detection, indicating that the in vivo therapeutic effect of TDN-AG-siRNA@CDDP is very significant.

[0122] Experimental Example 10 Biosafety Evaluation of TDN-AG-siRNA@CDDP

[0123] To evaluate the biosafety of TDN-AG-siRNA@CDDP, mice were divided into two groups (n=6) using a random number table method and injected with PBS and TDN-AG-siRNA@CDDP via the tail vein, respectively. Compared with the PBS injection group, H&E staining of the brain, kidney, lung, spleen, liver, and heart was performed on the first and seventh days after TDN-AG-siRNA@CDDP (400nM, 200μL) injection. The biosafety of TDN-AG-siRNA@CDDP was further evaluated by blood routine and blood biochemistry.

[0124] The results are as follows Figures 32-34 shown.

[0125] like Fig.32 As shown in the figure, H&E staining was performed on the kidney, lung, spleen, liver and heart of mice on the 1st and 7th days after injection of TDN-AG-siRNA@CDDP. The staining results showed that all organs were normal without pathological changes, confirming that TDN-AG-siRNA@CDDP had no adverse effects or toxic effects on organs and had very good biosafety.

[0126] like Fig.33 and 34As shown, the mice were tested for routine blood tests on the 1st and 7th days after TDN-AG-siRNA@CDDP administration, including red blood cells (RBC), white blood cells (WBC), platelets (PLT), mean platelet volume (MPV), mean corpuscular volume (MCV), hematocrit (HCT), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin concentration (MCH), hemoglobin concentration (HGB); and blood biochemistry tests, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea nitrogen (BUN), alkaline phosphatase (ALP), creatinine (CR), direct bilirubin (D-BIL), total bilirubin (T-BIL), total bile acid (TBA), uric acid (UA). Compared with the PBS group (Ctrl) and the normal reference value, all the test indicators were within the normal range, indicating that TDN-AG-siRNA@CDDP was non-toxic and highly safe at the experimental dose.

[0127] Test Example 11 Drug loading efficiency test

[0128] TDN-AG-siRNA and CDDP were mixed in different ratios and incubated at 37°C for about 4 hours. The drug loading efficiency (DLE) of CDDP was detected by ICP-MS after ultrafiltration using a 30 kDa ultrafiltration tube. The results are shown in Table 2. The results showed that when the molar ratio of TDN-AG-siRNA to CDDP was 1:2000, the drug loading efficiency of CDDP could reach up to 71.15%.

[0129] Table 2 Drug loading efficiency of CDDP

[0130]

[0131] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the creative concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A framework nucleic acid machine targeting ALKBH5 and loaded with cisplatin, characterized in that: The framework nucleic acid machine comprises a DNA tetrahedral framework nucleic acid and cisplatin loaded on the DNA tetrahedral framework nucleic acid; the DNA tetrahedral framework nucleic acid is synthesized by hybridization of four DNA single strands, and the nucleotide sequences of the four DNA single strands are shown in SEQ ID NO: 1 and SEQ ID NO: 8-10, respectively.

2. The method for preparing a framework nucleic acid machine according to claim 1, characterized in that: The steps include: The DNA single strands used for hybridization synthesis of DNA tetrahedral framework nucleic acid were added to TM buffer respectively, and after mixing, heated at 95°C for 10 min, and then cooled to 4°C and kept for 10 min to obtain DNA tetrahedral framework nucleic acid; Mix the DNA tetrahedral framework nucleic acid and cisplatin, and incubate at 37°C for 3-4 h.

3. The method for preparing a framework nucleic acid machine according to claim 2, characterized in that: The pH of the TM buffer is 8.0, and its composition includes 10 mM Tris-HCl and 50 mM MgCl2.

4. The method for preparing a framework nucleic acid machine according to claim 3, characterized in that: In the DNA single strands used for hybridization synthesis of DNA tetrahedral framework nucleic acid, the molar ratio of any two DNA single strands is 1:

1.

5. The method for preparing a framework nucleic acid machine according to claim 4, characterized in that: The molar ratio of the DNA tetrahedral framework nucleic acid to cisplatin is 1:(500-4000).

6. The method for preparing a framework nucleic acid machine according to claim 5, characterized in that: The molar ratio of the DNA tetrahedral framework nucleic acid to cisplatin is 1:2000.

7. Use of the framework nucleic acid machine according to claim 1 in the preparation of drugs for treating glioblastoma.

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