Bio-switchable miRNA (micro Ribonucleic Acid) functionalized tetrahedral framework nucleic acid as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to biologically switchable miRNA (micro Ribonucleic Acid) functionalized tetrahedral framework nucleic acid as well as a preparation method and application thereof. According to the controllable miRNA delivery system based on tetrahedral framework nucleic acid, miRNA is encapsulated in a tetrahedron, and effective delivery and controllable release of miRNA are achieved in an RNA enzyme H response mode. The nucleic acid tetrahedron is composed of three single-stranded nucleic acids and miR-30a-5p, has high stability, is used as eye drops, realizes noninvasive and simple-operation treatment of uveal melanoma (UM), solves the problem of adverse reaction caused by an invasive treatment method in ophthalmic disease treatment, and improves patient compliance and life quality. According to the invention, the BiRDS carrying miR-30a-5p is applied to UM in the form of nano eye drops for the first time, the research blank of noninvasive inhibition of UM based on miRNA is filled, and the application prospect is very good.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a bio-switchable miRNA functionalized tetrahedral framework nucleic acid and a preparation method and use thereof. Background Art

[0002] Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. It originates from melanocytes, 90% of which are located in the choroid and the remaining 10% are located in the ciliary body and iris. Although UM is rare, it is highly invasive and has a poor prognosis. More than 50% of patients will develop systemic metastases after diagnosis, mainly liver metastases. Once metastasis occurs, the survival period is generally less than 1 year. Currently, the treatment strategies for UM are relatively limited, and no adjuvant therapy has been proven to be effective in preventing UM metastasis. Therefore, early treatment of uncertain lesions is particularly important, which helps prevent the occurrence of fatal UM. Local control of early UM can be achieved in a variety of ways, including radiotherapy, laser therapy, chemotherapy, and surgical intervention. However, these invasive treatments have significant adverse reactions, including retinal detachment, vision loss, and tumor recurrence. Therefore, in order to improve the prognosis of patients with early UM, it is imperative to develop a new, non-invasive, effective, and safe treatment strategy.

[0003] In recent years, microRNA (miRNA)-based gene therapy has become a new and effective method for treating human tumors and a hot topic of research. As a gene expression regulator, miRNA plays a vital role in the occurrence, development and metastasis of tumors. MiRNA can regulate the expression of target genes by binding to specific messenger RNA (mRNA) sequences, thereby affecting the proliferation, apoptosis and metastasis of tumor cells. Studies have shown that multiple miRNAs are abnormally expressed in UM cells. These miRNAs affect the growth and metastasis of UM by regulating key signaling pathways such as cell cycle, apoptosis, invasion and metastasis. Notably, miR-30a-5p has been found to be a tumor suppressor in many malignant tumors, including head and neck squamous cell carcinoma, non-small cell lung cancer, prostate cancer, renal cell carcinoma, breast cancer and hepatocellular carcinoma. In addition, a survival analysis of UM patients showed that the expression level of miR-30a-5p was positively correlated with overall survival. By improving these dysregulated miRNAs, it is expected to inhibit the growth and metastasis of UM. However, the stability, delivery efficiency, and ability to cross biological barriers of miRNA in gene therapy pose significant obstacles to its clinical application, especially in the eye with the blood-retinal barrier.

[0004] To address this problem, researchers have made every effort to develop innovative miRNA delivery systems. Among them, nanodelivery systems based on tetrahedral framework nucleic acids (tFNA) / tetrahedral DNA nanostructures (TDN) show considerable application potential due to their unique structure and properties. Studies have shown that tFNA can be used as a programmable carrier for transdermal administration of cutaneous malignant melanoma. In addition, the potential of tFNA-based miRNA delivery in ocular diseases includes corneal epithelial wound healing, retinal and choroidal pathological neovascularization, and retinal ischemia / reperfusion injury. However, this delivery method of integrating miRNA into the side chains or vertices of tFNA / TDN through sticky ends may affect the size of the nanomaterial and its penetration. In addition, exposed miRNA may become unstable and susceptible to enzymatic degradation.

[0005] Controlled release of miRNA in cells through cell environment, such as pH or intracellular enzymes, is one of the ways to solve the problem of unstable exposed miRNA and easy degradation by enzymes. RNase H is widely present in cells, and it can catalyze the degradation of the RNA part of DNA-RNA hybrids. Applying it to the tFNA miRNA delivery system may be a feasible way to achieve stable and efficient delivery of miRNA.

[0006] In summary, miR-30a-5p is a promising candidate molecule for improving the prognosis of early UM patients based on miRNA. However, due to the difficulty of miRNA in stably crossing the blood-retinal barrier and being easily degraded in the biological environment, its clinical application is greatly limited. Therefore, it is urgent to develop a delivery system that can stably deliver miR-30a-5p across the blood-retinal barrier and efficiently function in cells, so as to achieve non-invasive and effective treatment of uveal melanoma. Summary of the invention

[0007] In view of the problems of the prior art, the present invention provides a bio-switchable miRNA functionalized tetrahedral framework nucleic acid and a preparation method and use thereof, aiming to stably, efficiently and safely inhibit uveal melanoma.

[0008] The present invention provides a functionalized tetrahedral framework nucleic acid, wherein the tetrahedral framework nucleic acid is a BiRDS structure; the BiRDS structure comprises 1 to 3 RNA1s, and the nucleotide sequence of the RNA1 comprises SEQ ID NO.4.

[0009] Preferably, the functionalized tetrahedral framework nucleic acid is composed of three single-stranded nucleic acids S1, S2, S3 and RNA1 through base complementary pairing;

[0010] The molar ratio of S1 to S2, S3, and RNA1 is 1:1:1:1-3.

[0011] Preferably, the nucleotide sequence of S1 is SEQ ID NO.1.

[0012] Preferably, the nucleotide sequence of S2 is SEQ ID NO.2.

[0013] Preferably, the nucleotide sequence of S3 is SEQ ID NO.3.

[0014] The present invention provides a method for preparing the functionalized tetrahedral framework nucleic acid described in any one of the above items, which comprises the following steps:

[0015] The single-stranded nucleic acids S1, S2, S3 and RNA1 are mixed to obtain.

[0016] The present invention provides use of any of the above-mentioned functionalized tetrahedral framework nucleic acids in the preparation of drugs for treating post-stroke depression, cardiovascular disease, cancer, peripheral nerve injury, and knee osteoarthritis.

[0017] Preferably, the cancer is selected from the group consisting of head and neck squamous cell carcinoma, non-small cell lung cancer, prostate cancer, renal cell carcinoma, breast cancer, hepatocellular carcinoma, and uveal melanoma.

[0018] Preferably, the cancer is uveal melanoma.

[0019] The present invention provides a medicine for treating uveal melanoma, which is prepared by using any of the above-mentioned functionalized tetrahedral framework nucleic acids as active ingredients and adding pharmaceutically acceptable excipients.

[0020] The present invention provides a tFNA-based bio-switchable miRNA delivery system BiRDS, which can encapsulate miRNA in the nanomaterial of tFNA for protection, and realize the effective delivery and controllable release of miRNA in the form of RNase H response. BiRDS is a nucleic acid tetrahedron composed of three single-stranded nucleic acids S1-S3 and miR-30a-5p, wherein the middle part of the tetrahedron edge is a DNA-RNA hybridization region, and when RNase H is present, the RNA part of the DNA-RNA hybridization region is hydrolyzed to achieve the dissociation of the tetrahedral structure, thereby achieving the intracellular release of miR-30a-5p. Experiments have shown that BiRDS has high stability and good biocompatibility, can penetrate the blood-retinal barrier, and can be used as eye drops for the treatment of eye diseases. When used to treat UM, non-invasive and simple-to-operate treatment can be achieved, which is suitable for long-term and frequent administration, and solves the problem of adverse reactions caused by invasive treatment methods in the treatment of ophthalmic diseases, including pain, bleeding, inflammation, infection, and even choroidal / retinal detachment and visual impairment, etc., to improve patient compliance and quality of life. In summary, the present invention is the first to apply BiRDS carrying miR-30a-5p in the form of nano eye drops to UM, filling the research gap of non-invasive inhibition of UM based on miRNA and has good application prospects.

[0021] Definitions of terms used in the present invention: Unless otherwise stated, the initial definitions provided for groups or terms in this document apply to the groups or terms throughout the specification; for terms that are not specifically defined in this document, the meaning that a person skilled in the art can give them should be given based on the disclosure and context.

[0022] "BiRDS structure" refers to a tetrahedral framework structure composed of nucleic acids, which will dissociate under the action of RNase H. In the tetrahedral framework structure, one vertex contains 3 free deoxyribonucleotides, and at least one of the other three vertices contains 6 free ribonucleotides and 2 free deoxyribonucleotides; at least three edges contain 20 base pairs of double-stranded nucleic acids respectively; at least one edge has a 4 base pair DNA-RNA base complementary region.

[0023] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0024] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The experimental results and quantitative results of BiRDS synthesis verified by agarose electrophoresis;

[0026] Figure 2 The experimental results of BiRDS synthesis verified by high-efficiency capillary electrophoresis;

[0027] Figure 3 Transmission electron microscopy (TEM) and atomic force microscopy (AFM) images of BiRDS, scale bar is 100 nm;

[0028] Figure 4 The particle size distribution and zeta potential results of dynamic light scattering detection of TDN and BiRDS are shown in the figure;

[0029] Figure 5 The agarose electrophoresis results of BiRDS stability at room temperature and its quantitative results are shown;

[0030] Figure 6 The agarose electrophoresis results and quantitative results of BiRDS stability in a biological environment at 37°C are shown;

[0031] Figure 7 The fluorescence detection diagram of different nucleic acid drugs in MUM2B cells and their quantitative results;

[0032] Figure 8 The flow cytometry results and quantitative results of different nucleic acid drugs in MUM2B cells are shown;

[0033] Fig. 9 Figure 1 is a graph showing the viability of MUM2B cells measured by CCK8 reagent; Figure A is a graph showing the viability of MUM2B cells after treatment with different concentrations of BiRDS; Figure B is a graph showing the viability of MUM2B cells over time after treatment with different nucleic acid drugs;

[0034] Fig.10 This is a colony formation experiment result diagram of MUM2B cells after being treated with different nucleic acid drugs;

[0035] Fig.11 The results of the scratch experiment after MUM2B cells were treated with different nucleic acid drugs;

[0036] Fig.12 The quantitative results of colony formation experiment and scratch test after MUM2B cells were treated with different nucleic acid drugs;

[0037] Fig.13 This is a diagram showing the results of a transwell experiment after MUM2B cells were treated with different nucleic acid drugs;

[0038] Fig.14 This is a quantitative result diagram of the transwell experiment after MUM2B cells were treated with different nucleic acid drugs;

[0039] Fig.15 This is the flow cytometry result of apoptosis experiment after MUM2B cells were treated with different nucleic acid drugs;

[0040] Fig.16 It is a quantitative graph of the flow cytometry results of apoptosis experiments after MUM2B cells were treated with different nucleic acid drugs; UP is the result in the upper right quadrant of the flow cytometry graph, and LP is the result in the lower right quadrant of the flow cytometry graph;

[0041] Fig.17 Immunofluorescence staining of E2F7 after MUM2B cells were treated with different nucleic acid drugs;

[0042] Fig.18 Immunofluorescence staining of cleaved caspase-3 (C-Caspase-3) after MUM2B cells were treated with different nucleic acid drugs;

[0043] Fig.19 The quantitative results of immunofluorescence staining of E2F7 and C-Caspase-3 after MUM2B cells were treated with different nucleic acid drugs;

[0044] Fig. 20 This is the result of western blot assay for E2F7, caspase-3, and C-Caspase-3 in MUM2B cells;

[0045] Fig.21 This is a graph showing the expression of E2F7, caspase-3, and C-Caspase-3 in MUM2B cells as determined by western blot experiments;

[0046] Fig. 22 Fluorescence staining of different nucleic acid drugs penetrating into eye tissues;

[0047] Fig.23 This is a quantitative result of fluorescent staining of different nucleic acid drugs penetrating into eye tissues;

[0048] Fig.24 This is the hematoxylin and eosin (HE) staining image of the eye tissue of normal animals using BiRDS;

[0049] Fig.25 These are the tumor morphology diagrams and tumor weight and size statistics of UM animals treated with different nucleic acid drugs;

[0050] Fig.26The images are of the eyes of UM animals treated with different nucleic acid drugs and the HE staining images of eye tumors;

[0051] Fig. 27 Immunofluorescence staining of E2F7 after UM animals were treated with different nucleic acid drugs;

[0052] Fig.28 Immunofluorescence staining of C-Caspase-3 in UM animals after treatment with different nucleic acid drugs;

[0053] Fig.29 The quantitative results of immunofluorescence staining of E2F7 and C-Caspase-3 after UM animals were treated with different nucleic acid drugs;

[0054] Fig.30 This is the result of western blot experiment to determine E2F7, caspase-3, and C-Caspase-3 in UM animal tumor tissues;

[0055] Fig.31 This is the result of western blot experiment to determine the expression of E2F7, caspase-3, and C-Caspase-3 in UM animal tumor tissues;

[0056] Fig.32 This is the tdt-mediated dUTP nick end labeling (TUNEL) staining image;

[0057] Fig.33 This is the result of Ki-67 immunohistochemical staining;

[0058] Fig.34 These are the quantitative results of TUNEL staining, Ki-67 immunohistochemical staining, and IFN-γ assay by Elisa. DETAILED DESCRIPTION

[0059] In the following examples and experimental examples, reagents and materials not specifically described are all commercially available.

[0060] All oligonucleotide sequences and miRNAs were synthesized by Sangon Biotech Co., Ltd. (Shanghai).

[0061] Example 1 Functionalized tetrahedral framework nucleic acid and preparation method thereof

[0062] This embodiment provides a tetrahedral framework nucleic acid BiRDS for inhibiting uveal melanoma, which is prepared according to the following method:

[0063] BiRDS is composed of three single-stranded DNAs (S1-S3) and miR-30a-5p. S1-S3 and miR-30a-5p were added to TM buffer (10mM Tris-HCl, pH 7.5, 50mM magnesium chloride) at a molar ratio of 1:1:1:3, denatured at 95°C for 10min, and then rapidly cooled to 4°C for 20min on a thermal cycler. One-pot synthesis was performed based on the principle of base complementary pairing and self-assembly.

[0064] The nucleotide sequences of S1-S3 and miR-30a-5p are shown in the following table:

[0065]

[0066] Comparative Example 1

[0067] This comparative example provides the control samples used in the experiment:

[0068] 1. TDN

[0069] The sample was prepared by adding three single-stranded DNAs (S1-S3) in a molar ratio of 1:1:1 into TM buffer (10 mM Tris-HCl, pH 7.5, 50 mM magnesium chloride), denaturing at 95°C for 10 min, and then rapidly cooling to 4°C for 20 min on a thermal cycler.

[0070] The technical solution of the present invention is further illustrated by experiments below.

[0071] In order to facilitate experimental observation, the 5' end of miR-30a-5p was modified with fluorescent sulfo-cyanine 5 (Cy5) residue. In the comparative TDN sample, S1 was also modified with Cy5.

[0072] Experimental Example 1 Characterization of Functionalized Tetrahedral Framework Nucleic Acid BiRDS

[0073] In this experimental example, the BiRDS prepared in Example 1 was characterized.

[0074] 1. Experimental Methods

[0075] 1. Synthesis of BiRDS and characterization of its structure, size and other properties

[0076] The successful synthesis and structure of BiRDS were verified and observed by 2% agarose gel (105 V, 25 min), high-performance capillary electrophoresis, Zetasizer Nano ZS90 system (Malvern Panalytical, UK), atomic force microscopy (AFM, Shimadzu, Kyoto, Japan) and transmission electron microscopy (TEM, Hitachi, Ltd., Tokyo, Japan).

[0077] 2. Stability test of BiRDS in biological environment

[0078] BiRDS was stored in TM buffer at room temperature (25°C) for 1-7 days or incubated with 10% FBS (Corning Inc., NY, USA) at 37°C for 0.5-48 h. The activity and degradation of BiRDS were detected by agarose gel electrophoresis (105 V, 25 min), and the gel was exposed and analyzed using a gel imaging system (Bio-Rad, USA).

[0079] 2. Experimental Results

[0080] 1. Synthesis of BiRDS and its structure, size and other properties

[0081] The results of agarose gel electrophoresis experiments are shown in Figure 1 As shown in the figure: 3 single-stranded DNAs (S1-S3) and miR-30a-5p gradually synthesized BiRDS. There were three miR-30a-5p molecules in BiRDS, and its Cy5 fluorescence intensity was the strongest. Compared with the unprotected naked miR-30a-5p, the miR-30a-5p carried and delivered by BiRDS showed stronger stability and less degradation. The results of the capillary electrophoresis experiment are shown in the figure. Figure 2 As shown in Figure 2, the stable and efficient synthesis of BiRDS was further confirmed. Figure 3 ), BiRDS was observed to have a triangular nanocomposite structure, suggesting that it can promote its entry into tissues and cells through an angular attack mechanism. Dynamic light scattering (DLS) results are shown in Figure 4 As shown, the particle size of BiRDS is (10.94±1.31) nm and the zeta potential is (-7.52±2.78) mV; compared with the classic TDN, its size is smaller and its zeta potential is lower.

[0082] 2. Stability of BiRDS

[0083] The stability test results are as follows Figure 5 , 6 As shown in the figure, BiRDS can be stored at room temperature (25°C) for 7 days with an activity of 88.06% ± 3.61%. In addition, under the conditions of 10% fetal bovine serum (FBS) and 37°C, the activity of BiRDS can be maintained at 93.38% ± 2.52% for 24 hours, and it is only degraded at 48 hours, with an activity of 61.53% ± 13.01%. These experimental results provide a basis for the potential application of BiRDS in vivo.

[0084] The above results show that BiRDS was successfully prepared by the method of Example 1, and the triangular nanocomposite structure has the properties of small size, easy synthesis, and high stability, and can be used for an efficient and stable miRNA drug delivery system, and has great potential for clinical application.

[0085] Experimental Example 2 Effects of Functionalized Tetrahedral Framework Nucleic Acid BiRDS on Cells

[0086] The BiRDS and TDN samples used in this experimental example were prepared according to the method of Example 1 or Comparative Example 1. The sequence of miR-30a-5p is shown in Table 1.

[0087] 1. Experimental Methods

[0088] 1. Cell culture

[0089] MUM2B cells (Hunan Fenghui Biotechnology Co., Ltd., China), human invasive choroidal melanoma cells, were cultured at 37°C, 5% CO2, 90% RMPI 1640 medium plus 10% FBS and 100 U / mL penicillin / streptomycin (Hyclone, USA).

[0090] 2. Experimental Grouping

[0091] The experiment was divided into the following five groups: negative control (abbreviated as NC) group, miR-30a-5p (abbreviated as miR) group, miR combined with liposome (abbreviated as miR+Lipo) group, TDN group and BiRDS group. The nucleic acid drug in the NC group was single-stranded RNA synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) without transfection reagent; the nucleic acid drug in the miR group was naked miR-30a-5p without transfection reagent; the miR+Lipo group was transfected into MUM2B cells with the assistance of Lipofectamine 3000 transfection reagent according to the instructions (Thermo Fisher Scientific, USA). The nucleic acid drug in the TDN group was TDN; the nucleic acid drug in the BiRDS group was BiRDS. The content of nucleic acid drugs in each group was 100 nM. When the cells were treated with the above nucleic acid drugs, the concentration of FBS in the culture medium was 1% to reduce the formation of nanoparticle protein corona.

[0092] The nucleotide sequence of the single-stranded RNA of the NC group is SEQ ID NO.5:

[0093] uuguacuacacaaaaguacug.

[0094] 3. Cellular uptake

[0095] MUM2B cells were seeded in confocal culture dishes (Corning Inc., NY, USA) and cultured for 24 h. According to the grouping, the cells were treated with different nucleic acid drugs. After culturing in the dark for 24 h, the cells were fixed with 4% paraformaldehyde (Servicebio, China) for 60 min, stained with phalloidin (Servicebio, China) for 30 min, and stained with 4',6-diamidino-2-phenylindole (DAPI) (Servicebio, China) for 10 min. The cells were washed 3 times with PBS between each step, and finally blocked with 10% glycerol (Servicebio, China). The entry of nanomaterials into the cells was observed using a laser confocal microscope (FV3000, Olympus, Japan). The uptake of different nucleic acid nanomaterials by MUM2B cells was detected by flow cytometry (Beckman Coulter, USA).

[0096] 4. Cell Proliferation Assay

[0097] The effects of different nucleic acid drugs on UM cell proliferation were explored through CCK8 experiment and colony formation experiment.

[0098] In the CCK8 experiment, MUM2B cells were first evenly inoculated in a 96-well plate (Corning, USA), and after culturing for 24 hours, different nucleic acid drugs were added for treatment. Then, CCK8 reagent (HYK0301, MCE, USA) was added at 24 hours, 48 ​​hours, and 72 hours, and the OD value at 450 nm was measured with a microplate reader (Varioska LUX system, Thermo Scientific, USA). Each group was repeated 5 times, and the cell viability was finally calculated. In addition, 0, 50nM, 100nM, 150nM, 200nM, and 250nM BiRDS were used to treat the cells for CCK8 experiments.

[0099] In the colony formation experiment, MUM2B cells were first inoculated in 6-well plates, with 3 wells in each group. After 24 hours of culture, different nucleic acid drugs were added according to the grouping. The culture medium was changed every 2 to 3 days, and the cells were cultured for 7 to 14 days until visible clones appeared. The cells were fixed with 4% paraformaldehyde for 60 minutes, stained with crystal violet (Servicebio, China) for 15 minutes, and washed with PBS three times in each step. Finally, the number of clones was photographed and counted.

[0100] 5. Cell migration and invasion assay

[0101] Scratch assay and transwell assay were used to explore the effects of different nucleic acid drugs on the migration and invasion of UM cells.

[0102] In the scratch test, MUM2B cells were first evenly inoculated in a 6-well plate. After the cells were fully grown, a vertical line was drawn in the center of the well with a yellow tip. According to the grouping situation, different nucleic acid drugs were added, and cell migration was observed and recorded under an inverted optical microscope at 0h and 24h, and the migration distance was measured and the wound closure rate was calculated.

[0103] In the transwell experiment, Matrigel (Corning, 354234, USA) was first evenly spread on the bottom of a 24-well chamber (LABSELECT, 14341, China) to form a thin film, and then hydrated with serum-free medium. Cells were evenly inoculated in the upper chamber with serum-free medium, and different nucleic acid drugs were added according to the grouping situation. Complete medium containing 10% fetal bovine serum was added to the lower chamber to allow MUM2B cells to pass through the membrane from the upper chamber into the lower chamber. After 24 hours of culture, the cells at the bottom of the transwell insert were fixed with 4% paraformaldehyde for 15 minutes, stained with crystal violet for 10 minutes, and rinsed with PBS 3 times at each step. Finally, the number of invasive cells was observed under a microscope and recorded.

[0104] 6. Cell apoptosis experiment

[0105] MUM2B cells were evenly seeded in 6-well plates and cultured for 24 h before different nucleic acid drugs were added according to the grouping. Annexin V-FITC apoptosis detection kit (Beyotime, C1062L, China) was used for analysis using flow cytometry (Beckman Coulter, USA), and fluorescence compensation was adjusted using FlowJO v10 software (USA).

[0106] 7. Detection of the expression levels of E2F7, caspase-3 and cleaved caspase-3

[0107] In gene therapy for tumors, miRNA can bind to the mRNA of the target gene, thereby inhibiting the translation of the target gene into functional protein. Previous studies have shown that E2F7 is one of the direct downstream target genes of miR-30a-5p. Studies have shown that E2F7 can inhibit the apoptosis of prostate cancer cells, while E2F7 deficiency can induce apoptosis of skin cancer cells. As an apoptosis marker, caspase-3 is cleaved and activated under the stimulation of apoptotic signals, thereby initiating the apoptotic caspase cascade reaction.

[0108] To further verify the effects of different nucleic acid drugs on UM cell apoptosis, immunofluorescence and western blot methods were used to detect the expression levels of E2F7, caspase-3, and cleaved caspase-3 (C-Caspase-3), respectively.

[0109] Immunofluorescence method: First, MUM2B cells were inoculated in confocal culture dishes, and nucleic acid drugs were added the next day. After 24 hours of treatment, the cells were fixed with 4% paraformaldehyde for 30 minutes, the cell membrane was destroyed with 0.5% Triton X-100 (Servicebio, China), and blocked with 5% goat serum (Servicebio, China). Then, the primary antibody was used overnight at 4°C, where the dilution ratio of E2F7 antibody (24489-1-AP, proteintech, USA) was 1:200; the dilution ratio of cleaved caspase-3 antibody (AF-7022, Affinity, Jiangsu, China) was 1:100. The next day, the corresponding fluorescent secondary antibody (ZSGB-BIO, China) was incubated on a shaker for 60 minutes, where the secondary antibody was fluorescently labeled with Alexa Fluor 647 (red fluorescence). Then, the cytoskeleton and cell nucleus were stained with fluorescein isothiocyanate (FITC) and DAPI, respectively, and finally the confocal dish was sealed with 10% glycerol. The cells were washed three times with PBS in each step. The fluorescence intensity of the target protein was observed using a confocal microscope (FV3000, Olympus, Japan).

[0110] Western blot method: A whole protein extraction kit (KeyGEN, Jiangsu, China) was used to obtain the whole protein of the adherent MUM2B cells. According to the molecular weight of the protein, 10% separation gel and 5% stacking gel (Servicebio, China) were prepared. The voltage of the separation gel during electrophoresis was 130V, and the voltage of the stacking gel was 80V. The protein was transferred to a PVDF membrane (MILLIPORE, Germany) and carried out in an ice bath at 250mA for 1h. Blocking was performed with 5% skim milk powder for 1h. Primary antibody was used overnight. The antibodies used were as follows: E2F7 antibody (24489-1-AP, proteintech, USA) at a dilution ratio of 1:1000; caspase-3 antibody (ab184787, Abcam, USA) at a dilution ratio of 1:2000; cleaved caspase-3 antibody (AF-7022, Affinity, Jiangsu, China) at a dilution ratio of 1:2000; Actin antibody (380624, ZENBIO, USA) at a dilution ratio of 1:10000. The membrane was washed three times with Tris buffered saline (TBS) and Tween-20 (TBST) (Servicebio, China), incubated with the corresponding secondary antibody (ZSGB-BIO, China) for 1 h, washed three times with TBST, and then developed in an imaging system (Syngene, Bangalore, India), and the grayscale value of the bands was analyzed using ImageJ software.

[0111] 2. Experimental Results

[0112] 1. Cellular uptake of BiRDS

[0113] The results of confocal microscopy experiments are shown in Figure 7 As shown: MUM2B cells were observed to have the highest uptake of BiRDS and TDN, with no statistically significant difference between the two groups. Naked miR-30a-5p had limited ability to enter cells, while Lipofectamine showed a certain promoting effect on the entry of miR-30a-5p into cells. The results of flow cytometry assay ( Figure 8 ), the same phenomenon was also observed. The results show that BiRDS, like the classic TDN, has a high degree of cell permeability and can efficiently enter cells to exert its effects.

[0114] 2. BiRDS inhibits the proliferation, migration and invasion of UM cells

[0115] The results of CCK8 detection are as follows Fig. 9 As shown: BiRDS has the ability to inhibit cell proliferation, and between 0nM and 100nM, the inhibitory effect increases with increasing concentration, but with further increasing concentration, the inhibitory effect is no longer statistically significant. Therefore, 100nM is the optimal inhibitory concentration of BiRDS and is used in subsequent in vitro experiments. MUM2B cells were continuously treated with 100nM different nucleic acid drugs for 24h, 48h, and 72h. It was observed that the inhibitory effect of BiRDS on MUM2B cell proliferation increased with time and was significantly stronger than the miR group and miR+Lipo group. In the results of the colony formation experiment ( Fig.10 , 12 ), the number of cell colonies formed in the BiRDS group was the lowest compared with the miR group and miR+Lipo group, and the difference was statistically significant. This further confirmed that BiRDS can effectively inhibit the proliferation of UM cells.

[0116] The scratch test results are as follows Fig.11 , 12 As shown: BiRDS inhibited the healing of scratch wounds and showed the effect of inhibiting migration, and the inhibitory effect was stronger than that of the miR group and miR+Lipo group.

[0117] The results of the transwell experiment are as follows Fig.13 , 14 As shown: Compared with the NC group and TDN group, the number of cells penetrating the mold into the lower chamber in the miR group, miR+Lipo group and BiRDS group was significantly reduced, and the number of cells entering the lower chamber in the BiRDS group was significantly the lowest. BiRDS showed a significant ability to inhibit cell invasion.

[0118] The above experimental results show that BiRDS has a significant inhibitory effect on the proliferation, migration and invasion of MUM2B cells, and the known effect is stronger than that of the miR group and miR+Lipo group. BiRDS has the best inhibitory effect on proliferation at 100nM. The experimental results suggest that BiRDS carrying miR-30a-5p can effectively inhibit the malignant phenotype of UM cells in vitro, thereby potentially inhibiting the distant metastasis of UM and preventing the formation of lethal UM.

[0119] 3. BiRDS promotes UM cell apoptosis through the miR-30a-5p / E2F7 axis

[0120] The results of apoptosis experiments were as follows Fig.15 , 16 As shown: BiRDS significantly promoted the apoptosis of MUM2B cells compared with other experimental groups. There was no statistically significant difference between the miR group and the NC group. This may be due to the unstable structure, rapid degradation and poor cellular uptake of naked miR-30a-5p. With the help of Lipofectamine transfection reagent, the apoptosis rate of miR+Lipo group (6.26%±0.80%) was higher than that of miR group (3.40%±0.87%), but still much lower than that of BiRDS group (20.78%±0.97%). These results confirm the potential of BiRDS as a promising miRNA drug delivery system.

[0121] The results of immunofluorescence experiments of E2F7 are shown in Fig.17 , 19 As shown in Figure 2, compared with the NC group, the expression level of E2F7 in the miR group was downregulated and was significantly lowest in the BiRDS group. Fig.18 , 19 As shown: The expression of C-Caspase-3 in the BiRDS group was significantly enhanced, and while apoptosis had not yet occurred in the miR group and the NC group, the BiRDS group had triggered the strongest apoptotic caspase cascade.

[0122] Western blot results Fig. 20 , 21 As shown: Compared with naked miR-30a-5p and lipofectamine-assisted transfected miR-30a-5p, BiRDS-delivered miR-30a-5p can significantly downregulate the expression of E2F7 and caspase-3 in UM cells, while upregulating the expression of cleaved caspase-3, thereby leading to stronger cell apoptosis.

[0123] The results showed that BiRDS significantly promoted the apoptosis of MUM2B cells, and promoted the apoptosis of UM cells through the miR-30a-5p / E2F7 axis, which also showed that miR-30a-5p also played the role of a tumor suppressor in UM.

[0124] The above experimental results show that BiRDS is an efficient and stable miRNA delivery system, which can be efficiently taken up by MUM2B cells, inhibit the proliferation, migration and invasion of MUM2B cells, and promote the apoptosis of MUM2B cells, reflecting its potential for treating UM.

[0125] Experimental Example 3 Functionalized tetrahedral framework nucleic acid BiRDS corresponds to in vivo inhibition of UM

[0126] The BiRDS and TDN samples used in this experimental example were prepared according to the method of Example 1 or Comparative Example 1. The sequence of miR-30a-5p is shown in Table 1.

[0127] 1. Experimental Methods

[0128] 1. Establishment of UM nude mouse xenograft animal model

[0129] Animal research was approved by the Medical Ethics Committee of West China School of Stomatology, Sichuan University, with the approval number WCHSIRB-D-2024-507. Female Balb / c nude mice aged 3 weeks were purchased from Jiangsu Jianyao Technology Co., Ltd., China, and tumor models were established starting at 4 to 5 weeks of age. Mice were anesthetized with bromoethanol (Aibei, Nanjing, China) and 5х10 5 / 5μL of MUM2B cells in good logarithmic growth phase were inoculated into the choroid of mice through Hamilton microinjector under a stereo microscope (morcato, Germany), and then treated with erythromycin eye ointment. One day later, the model was successfully established.

[0130] 2. Animal grouping

[0131] The model mice were divided into 5 groups, 6 mice in each group, namely PBS group, TDN group, miR-30a-5p (referred to as miR) group, BiRDS group and programmed death ligand 1 (PD-L1) antibody group. The first four groups were injected with 1μL and 5μL eye drops twice a day; the PD-L1 antibody group was injected with 5mg / Kg anti-PD-L1 (HY-P99145, MCE, USA) through the tail vein once every three days as a positive control group. The mice were killed after 2 weeks and tumor tissue specimens were collected. The gross morphology of the tumor was photographed, and the tumor weight and diameter were measured.

[0132] The concentration of nucleic acid in the above eye drops is 1 μM, and the solvent is TM buffer.

[0133] 3. Biosafety Test

[0134] Normal mice (Balb / c nude mice) were divided into 3 groups, 6 in each group, namely PBS group, miR group, and BiRDS group. Each group was given 5 μL eye drops twice a day. Mice were killed after 2 weeks, and eye tissue specimens were collected. Hematoxylin and eosin (HE) staining experiments were performed, and HE staining experiments were determined using a HE staining kit (C0105S, Beyotime, China).

[0135] 4. Determination of penetration into eye tissues

[0136] The tumors removed from the eyes of nude mice were cut into tissue sections from the sagittal plane, fixed with 4% paraformaldehyde (Servicebio, China), stained with DAPI (Servicebio, China) for 10 min, washed three times with PBS, and finally blocked with 10% glycerol (Servicebio, China). The Cy5 signal was observed using a laser confocal microscope (FV3000, Olympus, Japan) to compare the penetration of different nucleic acid nanomaterials in eye tissues.

[0137] 5. Histological analysis

[0138] (1) Completely dissect the eyeballs and tumors of mice. The tumors removed from the eyes of nude mice were cut into tissue sections from the sagittal plane and subjected to immunofluorescence staining, HE staining, TUNEL detection, and immunohistochemistry experiments. E2F7 is a transcription factor that is highly expressed in cancer tissues and promotes cancer cell proliferation. Ki-67 immunohistochemistry staining was used to verify that miR-30a-5p targeted silencing of E2F7.

[0139] Immunofluorescence staining: The immunofluorescence staining was performed according to the steps of the immunofluorescence method described in Experimental Method 7 of Experimental Example 2. The difference was that the fluorescent label of the secondary antibody was Alexa Fluor 488 (green); and the cytoskeleton was not stained with FITC.

[0140] HE staining experiments were performed using a HE staining kit (C0105S, Beyotime, China).

[0141] Immunohistochemistry experiment: The paraffin sections were first subjected to antigen retrieval, incubated with Ki-67 antibody (ab16667, abcam, USA, 1:100) at room temperature for 1 h, then incubated with the corresponding secondary antibody for 1 h, stained with DAB colorimetric solution, and finally observed under an optical microscope (Olympus, Japan) for Ki-67 expression.

[0142] TUNEL detection was performed using a one-step TUNEL apoptosis detection kit (HY-K1078, MCE, USA).

[0143] (2) Mouse ocular tumor tissue was cut into pieces and prepared into a homogenous solution for western blot and ELISA detection.

[0144] The Western blot experiment was performed according to the steps of the western blot method described in Experimental Example 2, Experimental Method 7.

[0145] The IFN-γ concentration was detected using a BCA protein assay kit (P0012S, Beyotime, China) and an ELISA kit (BY-EM220140, BYabscience, China).

[0146] 4. Statistical analysis

[0147] The survival analysis data of miR-30a-5p for UM cancer patients were obtained from starBase online (starBase). The relevant images and bands were processed and analyzed using ImageJ software to obtain relevant quantitative data and three-dimensional thermal images. All experimental quantitative data were expressed as mean ± standard deviation (SD) and analyzed by GraphPad Prism 9 (GraphPad Software, USA) according to different groups and the corresponding statistical test methods. P value < 0.05 was considered statistically significant.

[0148] 2. Experimental Results

[0149] 1. Penetration of BiRDS in ocular tissues

[0150] The results of immunofluorescence staining experiments are shown in Fig. 22 , 23 As shown: In the fundus fluorescent sections, it was observed that the Cy5 fluorescence signal in the BiRDS group was significantly increased, which was about 16 times and 3 times that of the miR group and TDN group, respectively. This finding shows that BiRDS can effectively pass through the complex structure of the eye and reach the fundus tumor lesions. Therefore, BiRDS has the prerequisite for treating eye diseases in the form of eye drops. It promotes topical ocular medication and home treatment of a series of eye diseases, thereby improving patient compliance and efficacy.

[0151] 2. Biosafety of BiRDS

[0152] The results of HE staining experiments are as follows Fig.24 No obvious pathological changes were found under microscopic examination, and no significant differences were observed between the BiRDS group and the PBS group. The results showed that BiRDS has good biosafety and can be used for in vivo applications.

[0153] 3. BiRDS inhibits tumor growth

[0154] Tumor morphology, weight, and size Fig.25 As shown in the figure, the tumors in the BiRDS group had the smallest morphology. The tumor weight and diameter were (23.28±4.52) mg and (3.08±0.08) mm, respectively, which were not statistically significant compared with the PD-L1 antibody group. The eye conditions of the mice and the HE staining results of the tumor tissues are shown in the figure. Fig.26 As shown: In nude mice, the PBS group, TDN group, and miR group clearly showed significant proptosis and periorbital swelling. HE histological examination results showed that in the PBS group, UM grew significantly, broke through the base, invaded the peripheral nerves, glands, and muscle tissues, and caused complete destruction of the internal structure of the eye. In contrast, the BiRDS group and the pd-l1 antibody group showed the ability to retain the complete eye structure, with relatively limited tumor foci and an appearance similar to that of normal healthy eyes. The results showed that BiRDS nano eye drops can effectively inhibit the growth of UM in vivo.

[0155] 4. BiRDS nano eye drops inhibit UM in vivo via the miR-30a-5p / E2F7 axis

[0156] Immunofluorescence results of tumor tissues Figure 27-29 As shown in Figure 2, compared with the PBS group and the miR group, the expression level of E2F7 in the BiRDS group was significantly decreased, while the expression level of cleaved caspase-3 was significantly increased, leading to apoptosis of UM cells. Fig.30 , 31 As shown: In the BiRDS group, E2F7 and caspase-3 were significantly downregulated, while cleaved caspase-3 was significantly upregulated, and there was no statistically significant difference compared with the PD-L1 antibody group. This confirms that BiRDS nano eye drops can induce UM cell apoptosis through the miR-30a-5p / E2F7 axis.

[0157] In addition, the results of TdT-mediated dUTP nick end labeling (TUNEL) staining of tumor sections were as follows Fig.32 As shown: Tumor cell apoptosis occurred in the miR group, BiRDS group, and PD-L1 antibody group, and the apoptosis phenomenon in the latter two groups was particularly prominent. This is because the naked miR-30a-5p nano eye drops may penetrate the eyeball through the sclera to reach the tumor site at the fundus. In contrast, in addition to bypassing the sclera, BiRDS nano eye drops can also directly pass through the cornea. The results show that BiRDS has a good effect in inducing apoptosis.

[0158] The results of Ki-67 immunohistochemical staining are shown in Fig.33As shown: The percentage of ki-67 positive cells in the BiRDS group was the lowest (2.80% ± 0.62%), compared with the PBS group (14.33% ± 1.82%) and the miR group

[0159] There was a statistically significant decrease compared with the PD-L1 antibody group (10.79% ± 1.62%), but there was no statistically significant difference compared with the PD-L1 antibody group (5.22% ± 1.06%). Fig.34 ) also obtained a consistent phenomenon: compared with the PBS group and the miR group, a significant increase in IFN-γ was observed in the BiRDS group, and IFN-γ has the ability to activate the immune system and exert anti-tumor effects. It can be seen that BiRDS nano eye drops are an effective method to inhibit UM cell proliferation and promote cancer cell apoptosis through the miR-30a-5p / E2F7 axis in vivo.

[0160] The above results indicate that BiRDS has good biosafety and efficient ocular tissue penetration when applied to UM animals, and can be used as nano eye drops to effectively inhibit the growth of UM in vivo and induce apoptosis of cells in tumor tissues.

[0161] It can be seen from the above experimental examples that the present invention successfully prepared BiRDS, which has a triangular nanocomposite structure; BiRDS has the properties of small size, easy synthesis and high stability. BiRDS has a significant inhibitory effect on the proliferation, migration and invasion of MUM2B cells, and the inhibitory effect is stronger than that of miR-30a-5p transfected with commercial transfection reagents. BiRDS has the best inhibitory effect on proliferation at 100nM. When BiRDS is applied to UM animals, it has good biosafety and efficient eye tissue permeability, and can be used as nano eye drops; BiRDS effectively inhibits the growth of UM in vivo and induces apoptosis of cells in tumor tissues. The tumor inhibitory effect of BiRDS is significantly better than that of miR-30a-5p, and is comparable to the therapeutic effect of commercial tumor immunotherapy drugs.

[0162] Therefore, the present invention successfully prepared BiRDS in a buffer solution from three single-stranded nucleic acids S1-S3 and miR-30a-5p in a molar ratio of 1:1:1:3. It is a bio-switchable miRNA delivery system based on tFNA, which can encapsulate miRNA in tFNA nanomaterials for protection, and achieve effective delivery and controllable release of miRNA in an RNase H response manner. BiRDS has a tetrahedral structure, high stability, good biocompatibility, can penetrate the blood-retinal barrier, and can be used as eye drops for the treatment of eye diseases. When used to treat UM, miR-30a-5p is used to achieve non-invasive and simple-to-operate treatment, which is suitable for long-term and frequent administration, and solves the problem of adverse reactions caused by invasive treatment methods in the treatment of ophthalmic diseases, including pain, bleeding, inflammation, infection, and even choroidal / retinal detachment and visual impairment, thereby improving patient compliance and quality of life. In summary, the present invention is the first to apply BiRDS carrying miR-30a-5p in the form of nano eye drops to UM, filling the research gap of non-invasive inhibition of UM based on miRNA and has good application prospects.

Claims

1. A functionalized tetrahedral framework nucleic acid, characterized in that: The tetrahedral framework nucleic acid is a BiRDS structure; the BiRDS structure contains 1-3 RNA1, and the nucleotide sequence of RNA1 includes SEQ ID NO.

4.

2. The functionalized tetrahedral framework nucleic acid according to claim 1, characterized in that: The functionalized tetrahedral framework nucleic acid is composed of three single-stranded nucleic acids S1, S2, S3 and RNA1 through base complementary pairing; The molar ratio of S1 to S2, S3, and RNA1 is 1:1:1:1-3.

3. The functionalized tetrahedral framework nucleic acid according to claim 2, characterized in that: The nucleotide sequence of S1 is SEQ ID NO.

1.

4. The functionalized tetrahedral framework nucleic acid according to claim 2, characterized in that: The nucleotide sequence of S2 is SEQ ID NO.

2.

5. The functionalized tetrahedral framework nucleic acid according to claim 2, characterized in that: The nucleotide sequence of S3 is SEQ ID NO.

3.

6. The method for preparing the functionalized tetrahedral framework nucleic acid according to any one of claims 1 to 5, characterized in that: The steps include: The single-stranded nucleic acids S1, S2, S3 and RNA1 are mixed to obtain.

7. Use of the functionalized tetrahedral framework nucleic acid according to any one of claims 1 to 5 in the preparation of a drug for treating post-stroke depression, cardiovascular disease, cancer, peripheral nerve injury, and knee osteoarthritis.

8. The use according to claim 7, characterized in that: The cancer is selected from the group consisting of head and neck squamous cell carcinoma, non-small cell lung cancer, prostate cancer, renal cell carcinoma, breast cancer, hepatocellular carcinoma, and uveal melanoma.

9. The use according to claim 8, characterized in that: The cancer is uveal melanoma.

10. A drug for treating uveal melanoma, characterized in that: The invention is prepared by taking the functionalized tetrahedral framework nucleic acid as any one of claims 1 to 5 as an active ingredient and adding pharmaceutically acceptable auxiliary materials.

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