A tetrahedral framework nucleic acid carrying siRNA, a preparation method thereof and use thereof in preparing an anti-aging drug

Abstract

The application provides a tetrahedral framework nucleic acid carrying siRNA, a preparation method thereof and an application thereof in preparing anti-aging drugs, and belongs to the technical field of medicines.The siRNA for inhibiting the expression of rapamycin complex 1 (mTORC1) is carried on the tetrahedral framework nucleic acid to form a tetrahedral framework nucleic acid complex, the tetrahedral framework nucleic acid complex can exert a synergistic anti-aging effect, and the tetrahedral framework nucleic acid complex has a good application prospect in preparing anti-aging drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a tetrahedral framework nucleic acid carrying siRNA, its preparation method, and its use in the preparation of anti-aging drugs. Background Technology

[0002] In recent years, aging has gradually become a major global population trend. As people age, their physiological functions gradually decline. Therefore, the concept of healthy aging is receiving increasing attention. Maintaining the physical functions of the elderly and improving their quality of life is one of the important research directions in the field of anti-aging.

[0003] Rapamycin complex 1 (mTORC1) is central to cellular regulation of growth and metabolism in response to external stimuli. With age, mTORC1 signaling levels abnormally increase, leading to age-related diseases. Inhibition of mTORC1 has been shown to prolong the lifespan of various preclinical species, including yeast, nematodes, fruit flies, and mice. Knockout or mutation of the Raptor gene (a specific component of mTORC1) extends the lifespan of nematodes. Tissue-specific Raptor deficiency, by inhibiting mTORC1 signaling, can alleviate various age-related pathologies. Therefore, targeting mTORC1 provides an innovative strategy for anti-aging.

[0004] With the continuous development of nanodelivery systems, more and more therapeutic siRNA drugs are entering clinical trials, and some have even been approved for market launch. However, siRNA alone has problems such as difficulty in targeting cells, degradation by nucleases during delivery, and renal clearance, and its therapeutic efficacy needs further improvement. Summary of the Invention

[0005] In order to overcome the shortcomings of the existing technology, the purpose of this invention is to provide a tetrahedral framework nucleic acid carrying siRNA, a method for its preparation, and its use in the preparation of anti-aging drugs.

[0006] This invention provides a tetrahedral framework nucleic acid complex, which is a product prepared from siRNA and tetrahedral framework nucleic acid, wherein the molar ratio of siRNA to tetrahedral framework nucleic acid is 1:(0.5-10), and the siRNA is siRNA that inhibits the expression of rapamycin complex 1.

[0007] Furthermore, the molar ratio of the siRNA to the tetrahedral framework nucleic acid is 1:1.

[0008] Furthermore, the tetrahedral framework nucleic acid is self-assembled from four DNA single strands, the sequences of which are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, respectively;

[0009] The siRNA is self-assembled from two single-stranded RNA molecules, the sequences of which are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0010] The present invention also provides a method for preparing the above-mentioned tetrahedral framework nucleic acid complex, the method comprising the following steps: mixing siRNA and tetrahedral framework nucleic acid and reacting them to obtain the tetrahedral framework nucleic acid complex.

[0011] Furthermore, the reaction conditions are: first, the reaction is heated, and then the reaction is cooled.

[0012] The heating reaction is carried out at a temperature of 80-100℃ for 5-15 minutes.

[0013] The cooling reaction is carried out at a temperature of 0-10℃ for a time of 10-30 minutes.

[0014] Furthermore, the heating reaction is carried out at a temperature of 95°C for a duration of 10 minutes;

[0015] The cooling reaction was carried out at a temperature of 4°C for 20 minutes.

[0016] The present invention also provides the use of the above-mentioned tetrahedral framework nucleic acid complex in the preparation of anti-aging drugs.

[0017] Furthermore, the drug is a drug that inhibits the expression of Raptor mRNA and / or inhibits the expression of aging markers.

[0018] Furthermore, the drug is a drug that reduces the number of senescent cells.

[0019] Furthermore, the drug is a pharmaceutical preparation made with the tetrahedral framework nucleic acid complex as the active ingredient and pharmaceutically acceptable excipients.

[0020] Experimental results show that the siRNA that inhibits the expression of rapamycin complex 1 is loaded onto a tetrahedral framework nucleic acid, and the resulting tetrahedral framework nucleic acid complex can exert a synergistic anti-aging effect. This tetrahedral framework nucleic acid complex has good application prospects in the preparation of anti-aging drugs.

[0021] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0022] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0023] Figure 1 .siR-TDN box Structural characterization results: (a) Composite siR-TDN box (b) Schematic diagram of the structure; box (c) High performance liquid chromatogram; (d) 5% PAGE image showing successful loading of cy5 fluorescent siRNA into siR-TDN. box In the complex; (d)siR-TDN box Dynamic light scattering measurement of particle size; (e)siR-TDN box Zeta potential; (f)siR-TDN box Transmission electron microscopy image (scale bar: 100 nm); (g)siR-TDN box AFM images (scale bar: 50 nm); (h) 5% PAGE gel images showing incubation at 37°C for 24 h with different concentrations (0%, 1%, 2%, 4%, 6%, 8% and 10%) of fetal bovine serum (siR-TDN). box (Merger), TDN box (blue) and siRNA (red); (i) 5% PAGE gel images show the effects of different concentrations of RNase A (0, 12.5, 25, 50, 100, 200 and 400 μg / mL) on siR-TDN. box Serum stability of siR-TDN in 10% fetal bovine serum at 0, 1, 2, 4, 6, 12 and 24 h; (j) 5% PAGE gel images showing siR-TDN at 37 °C. box After 1 h of incubation with unloaded free siRNA, (k) 5% PAGE gel images showed that siRNA and siR-TDN were incubated at 37 °C for different times (0, 1, 2, 4, 6, 12, 24 h) after incubation with 25 μg / mL RNase A at 37 °C. box The amount of free radicals.

[0024] Figure 2 .siR-TDN boxDistribution results in cells and tissues: (a) siR-TDN box (a) Sample introduction diagram; (b) Flow cytometry analysis of siR-TDN exposure box (c) Cellular uptake of young and senescent fibroblasts after different time periods (3, 6, and 12 h); confocal fluorescence images showing siR-TDN in young and senescent fibroblasts after 3, 6, and 12 hours of incubation. box Cellular uptake (blue: DAPI, green: Cytoskeleton, red: siR-TDN) box (d) Statistical analysis of cell uptake rate by flow cytometry; (e) Statistical analysis of fluorescence intensity in confocal images; (f) Intraperitoneal injection of siR-TDN box In vivo fluorescence images of organs from C57BL / 6 mice at different time points (0.5, 1, 3, 6, 12, 24 h).

[0025] Figure 3 Results of anti-cellular senescence experiments: (a) Schematic diagram of anti-senescence cell treatment; (b) Detection of Raptor mRNA expression by qPCR (senescent cells versus siRNA, TDN). box siR-TDN box (c) Representative images of senescent cells and control cells in the presence of SA-β-Gal dye (green, scale bar: 100 μm); (d) Statistical analysis of the percentage of SA-β-Gal positive cells in mouse NIH / 3T3 cells after treatment; (e) Immunoblotting detection of changes in the protein levels of senescence markers (p21 and p16), mTORC1 pathway (Raptor, S6 and p-S6), and SASP factors (IL-6 and TNF-α) after doxorubicin-induced senescence; (f) Measurement of the expression levels of senescent NIH / 3T3 proteins p21, p16, Raptor, p-S6 / S6, IL-6, and TNF-α; Visualization of the percentage of p21-positive (g), p16-positive (h), IL-6-positive (i), and TNF-α-positive (j) cells after senescence induction (cytoskeleton: green; cell nucleus (DAPI): blue; target protein: red, scale bar: 20 μm).

[0026] Figure 4 Results of experiments on aging in mice exposed to doxorubicin: (a) Schematic diagram of the mouse experiment; (b) Survival curves of mice with accelerated aging; (Dox n=20, Dox+siR-TDN) box(n=20); (c) Quantification of grip strength test changes; turning time (d) and descent time (e) of the pole climbing test; (f) time spent on the swivel; (g) total distance traveled on the swivel; (h) representative images (left) and quantification (right) of H&E staining of liver sections, (scale bar, 50 μm); (i) visualization (left) and quantification (right) of the percentage of SA-β-gal positive senescent cells in the liver, (scale bar, 100 μm); (j) Left: representative visualization of p21 immunofluorescence in the liver of doxorubicin-exposed mice after indicated treatment (scale bar, 50 μm), right: quantification of the percentage of p21 positive senescent cells in the liver.

[0027] Figure 5 Results of anti-natural aging mouse aging bodily function experiments: (a) siR-TDN box Experimental design for treating aged mice; (b) Mouse appearance (representative images of young mice, aged mice, and treated aged mice); (c) Grip strength of mice in each group; (d) Turning time of mice in the pole climbing experiment; (e) Time to descend from the pole in the pole climbing experiment; (f) Time spent on the rotating bar in the rotundus experiment; (g) Total distance traveled by mice on the rotating bar in the rotundus experiment; (h) Open field experiment; (i) Time and distance of mice traversing the central region in the open field experiment, and total time and distance of mice moving in the new environment; (j) Serum biochemical tests (physiological saline or siR-TDN). box Changes in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA), and urea (U) levels in young and old mice after 2 months of treatment; (k) blood glucose during intraperitoneal glucose tolerance test.

[0028] Figure 6 Results of age-related changes in anti-natural aging mice: (a) Visualization of aging-associated galactosidase staining (SA-β-gal) in the liver, kidney, and lungs of 3-month-old and 19-month-old mice (scale bar, 50 μm); (b) Representative H&E plots of the effects on aged mice; (c) Representative immunofluorescence images of p21+ cells in the liver, kidney, and lungs of aged mice (scale bar, 50 μm); (d, e) Immunoblotting detection of aging markers (p21 and p16) and mTORC1 pathway (Raptor, S6, and p-S6) protein expression in the liver, kidney, and lungs of aged mice (scale bar, 50 μm). Detailed Implementation

[0029] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0030] Example 1: Preparation of tetrahedral framework nucleic acid carrying siRNA

[0031] One double-stranded siRNA (including the vector strand and the cargo strand, with a side length of 21 bp, see Table 1) and four single-stranded DNA strands (S1-S4, with a side length of 30 bp, see Table 1) were added to TM buffer (1×10⁻⁶). -4 M Tris-HCl, 5 × 10 -4 The mixture was prepared in a solution of MgCl2·6H2O (pH = 8.0), with all five strands having a final concentration of 1 μM. The entire reaction system was heated to 95 °C in a thermal circulator, then rapidly cooled to 4 °C and maintained for 20 min to obtain tetrahedral framework nucleic acid (siR-TDN) carrying siRNA. box Store at 4℃ until used.

[0032] Table 1. Specific sequences of double-stranded siRNA and 4 single-stranded DNA molecules.

[0033]

[0034] *In this context, lowercase letters represent RNA base sequences, and uppercase letters represent DNA base sequences.

[0035] The following describes the preparation of control samples using control examples.

[0036] Compare with Example 1, Preparation of Tetrahedral Framework Nucleic Acids

[0037] Four DNA single strands (S1-S4, side length 30bp, see Table 1) were added to TM buffer (1×10⁻⁶). -4 M Tris-HCl, 5 × 10 -4 The mixture was prepared in a solution of MgCl2·6H2O (pH = 8.0), with all four chains having a final concentration of 1 μM. The entire reaction system was heated to 95 °C in a thermal circulator for 10 min, then rapidly cooled to 4 °C and maintained for 20 min to obtain tetrahedral framework nucleic acids (tFNAs, abbreviated as TDN). box Store at 4°C until use. (Comparative Example 2: Preparation of NC-siRNA)

[0038] Add one double-stranded siRNA (including the vector strand and the cargo strand) to TM buffer (1×10⁻⁶). -4 M Tris-HCl, 5 × 10 -4 Mix in MgCl2·6H2O (pH=8.0) to obtain double-stranded siRNA at a final concentration of 1 μM. Heat the entire reaction system to 95°C in a thermal cycler for 10 min, then rapidly cool to 4°C and maintain for 20 min to obtain NC-siRNA, which can be stored at 4°C for later use.

[0039] The sequence of NC-siRNA is as follows:

[0040] sense:UUCUCCGAACGUGUCACGUTT(SEQ ID NO.7);

[0041] anti-sense: ACGUGACACGUUCGGAGAATT (SEQ ID NO. 8).

[0042] The following experimental examples demonstrate the beneficial effects of the present invention.

[0043] Experimental Example 1: Structural Characterization of the Product

[0044] 1. Experimental Methods

[0045] The siR-TDN assay was validated using polyacrylamide gel electrophoresis (PAGE), high-performance capillary electrophoresis (HPCE), transmission electron microscopy (TEM), and atomic force microscopy (AFM). box Whether the synthesis was successful and the size and shape of the tFNAs. Dynamic light scattering (DLS) was used to measure siR-TDNs. box The particle size and zeta potential.

[0046] 2. Experimental Results

[0047] Figure 1 (ag) shows siR-TDN box The structural characterization results. Figure 1 b. The siR-TDN was validated using high performance liquid chromatography (HPCE). box Synthesis and molecular weight Figure 1 c indicates that siRNA was successfully loaded into siR-TDN. box In the complex, Figure 1 (dg) results show siR-TDN box The molecular weight (approximately 413 bp) is slightly larger than that of tFNAs (approximately 337 bp). siR-TDN box It exhibits an average size of approximately 15.75 nm and a Zeta potential of approximately -6.66 mV. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) images show that siR-TDN... box It exhibits a unique tetrahedral shape.

[0048] Experiment Example 2, Stability Experiment

[0049] 1. Experimental Methods

[0050] Storage stability, serum stability, and enzyme stability were assessed by polyacrylamide gel electrophoresis (PAGE). The specific procedures are as follows:

[0051] (1) Storage stability: The samples prepared in Example 1 were placed in environments of 4°C and 25°C for 1 to 7 days to test their storage stability;

[0052] (2) Serum stability: The samples prepared in Example 1 were incubated with fetal bovine serum of different concentrations (0%, 1%, 2%, 4%, 6%, 8% and 10%) at 37°C for 24 h, or with 10% fetal bovine serum for different reaction times (0, 1, 2, 4, 6, 12 and 24 h) to test serum stability.

[0053] (3) Enzyme stability: The samples prepared in Example 1 were incubated with different concentrations (0, 12.5, 25, 50, 100, 200 and 400 μg / mL) of RNase A at 37°C for 1 h, or incubated with 25 μg / mL RNase A for different time gradients (0, 1, 2, 4, 6, 12 and 24 h) to test enzyme stability.

[0054] 2. Experimental Results

[0055] Serum stability results as follows Figure 1 As shown in h, with the gradual increase in the concentration of fetal bovine serum (FBS) during co-incubation (1-10%), the nanobox structure gradually disintegrated, and more and more free siRNAs were released. However, it was observed that after 24 hours of incubation with 10% FBS, 60% of the undegraded free siRNAs remained, and clear bands were identified. After co-incubation with 10% FBS for different times, some siRNAs were still encapsulated in the nanobox at 12 hours, and were not completely disintegrated until 24 hours. Then, siR-TDN was... box After incubation with different concentrations of RNase A for 1 hour, when the enzyme concentration was increased to 400 μg / mL, only 10.9% of the undegraded free siRNA remained, but the nanobox exoskeleton remained intact. Figure 1 Next, siR-TDN was further investigated. box The degradation curve of siRNA over time in 25 μg / mL RNase A showed that a band was still visible at 12 hours. Figure 1 This indicates that siR-TDN box This can give siRNA good storage stability, facilitate transportation, and protect siRNA from degradation by enzymes and serum under conditions that mimic the in vivo environment.

[0056] Experiment Example 3: Cell Experiment

[0057] 1. Experimental Methods

[0058] (1) Cell culture: In vitro study of siR-TDN using NIH / 3T3 mouse embryonic fibroblasts. boxAnti-aging effects were observed. Cells were cultured in DMEM medium containing 10% fetal bovine serum. Cell senescence was induced by doxorubicin, specifically by administering 0.1 μM doxorubicin twice daily, with subsequent experiments performed after 7 days. Cell senescence was induced by doxorubicin at a final concentration of 200 nM (i.e., the double-stranded siRNA from Example 1), NC-siRNA, and TDN, respectively. box siR-TDN box Alternatively, cells were treated with rapamycin (Rapa) at a final concentration of 50 μg / L for 24 h. The group of young cells that were not induced by doxorubicin was designated as the Control group.

[0059] (2) Cellular uptake: The Cy5 fluorescent group was linked to the sense strand of the siRNA to obtain Cy5-siR-TDN. box Experimental flow cytometry and confocal laser scanning microscopy analysis of siR-TDN box Cell entry into young (Control) and senescent (DOX-sen) cells.

[0060] (3) In vivo imaging: Six-week-old male C57BL / 6 mice were used to study Cy5-siR-TDN. box Distribution in the body. Hair was removed from the abdomen of mice with depilatory cream the day before the experiment. 1 nM Cy5-siR-TDN was injected intraperitoneally 24 / 12 / 6 / 3 / 1 / 0.5 h before imaging. box 100 μL. After capturing whole-body fluorescence images under isoflurane gas anesthesia, the mice were euthanized and dissected for further organ fluorescence imaging.

[0061] (4) Senescence-associated β-galactosidase (SA-β-Gal): The senescence-associated β-Gal activity in sample cells was determined according to the manufacturer's instructions (C0602; Beyotime, China). The well plates were sealed with a sealing membrane to prevent evaporation of the staining medium. After overnight incubation, the cells were washed with PBS and observed under a bright-field microscope. Western blotting and immunofluorescence staining were used to analyze the expression of senescence-associated proteins p16 and p21; the expression of senescence-associated secretory phenotypes (IL-6, TNF-α); and the expression of the mTORC1 signaling pathway (S6, p-S6).

[0062] 2. Experimental Results

[0063] Figure 2 The siR-TDN was displayed. boxDistribution in cells and tissues. Flow cytometry analysis showed that at 3 hours, young cells (Control) took up more siR-TDN than doxorubicin-induced senescent cells (DOX-sen). box However, there was no significant difference in cell uptake between the two types at 6h and 12h. Figure 2 At 12 hours, almost 90% of siR-TDN box It is taken up and enters the cell. Fluorescence microscopy reveals that young fibroblasts are spindle-shaped, while senescent cells become flattened, larger, and have enlarged nuclei. siR-TDN box After 12 hours of treatment, almost 100% of the cells showed red fluorescence, evenly distributed in the cytoplasm. Figure 2 ). with siR-TDN box After 24 hours of incubation, the fluorescence intensity of Cy5 in the young cell (Control) group and the senescent cell (DOX-sen) group remained at the same level, with no statistically significant difference. Figure 2 This suggests siR-TDN. box It has an excellent ability to enter senescent cells.

[0064] Regarding drug distribution in vivo, bioluminescence imaging analysis results showed that siR-TDN box Thirty minutes after intraperitoneal injection, the Cy5 fluorescence signal of siRNA was first transported via the circulatory system to tissues with high blood flow rates, such as the heart, liver, and lungs. Fluorescence began to appear in the kidneys one hour after administration, with a fluorescence peak not observed until three hours later, confirming the efficacy of siR-TDN. box The renal excretion of siRNA is relatively slow. 24 hours after administration, fluorescent signals remained in the liver and kidneys of mice, suggesting that the nanobox encapsulation may help protect siRNA from clearance. Figure 2 Therefore, siR-TDN box It can be considered a highly efficient siRNA delivery system that can rapidly penetrate senescent cells, which is key to improving the bioavailability of therapeutic siRNAs.

[0065] Figure 3 The siR-TDN was displayed. box It can alleviate doxorubicin-induced cell senescence. Figure 3 a is a schematic diagram of the anti-aging cell experimental treatment. Figure 3 b. qPCR was used to detect the interaction between senescent cells and siRNA and TDN. box siR-TDN box Raptor mRNA expression after 24 hours of incubation. It can be seen that siRNA treatment increased Raptor mRNA expression, and TDN...box siR-TDN box Raptor mRNA expression decreased after treatment. Further comparisons were made with siRNA and TDN in Table 1. box and siR-TDN box The difference in Raptor mRNA expression between the treated group and the aging control Dox group revealed that siR-TDN... box The expression reduction effect of Raptor mRNA after treatment was greater than that after siRNA treatment and TDN treatment. box The sum of the reductions in Raptor mRNA expression after treatment indicates that siR-TDN box It exhibited a synergistic effect in reducing the expression of Raptor mRNA.

[0066] Table 1. Raptor mRNA expression after treatment in different experimental groups

[0067]

[0068] Figure 3 c shows representative images of senescent cells and control cells in the presence of SA-β-Gal dye, and in Figure 3 Table 2 presents a statistical analysis of the percentage of SA-β-Gal positive cells in NIH / 3T3 cells of mice after treatment. It can be seen that siRNA and TDN... box siR-TDN box Both siRNA and Rapa treatments reduced the number of senescent cells. Further comparisons of siRNA and TDN in Table 2 are also shown. box siR-TDN box The percentage reduction in senescent cells relative to the Dox-sen group after Rapa treatment, siR-TDN box The effect of treatment on reducing senescent cells was greater than that of siRNA treatment and TDN treatment. box The sum of the effects of siR-TDN in reducing senescent cells after treatment indicates that... box It exhibits a synergistic effect in reducing the number of doxorubicin-induced senescent cells.

[0069] Table 2. Number of senescent cells in the presence of SA-β-Gal dye

[0070]

[0071] Figure 3 e used immunoblotting to detect changes in the protein levels of aging markers (p21 and p16), mTORC1 pathway (Raptor, S6, and p-S6), and SASP factors (IL-6 and TNF-α) after doxorubicin-induced aging, and in Figure 3(fg) presents the statistical analysis of the expression levels of p21 (see Table 3), p16, Raptor, p-S6 / S6, IL-6, and TNF-α. It can be seen that siRNA and TDN... box siR-TDN box Both siRNA and Rapa reduced p21 expression after treatment of doxorubicin-induced senescent cells. Further comparisons of siRNA and TDN in Table 3 are also shown. box siR-TDN box The percentage of p21 expression reduced by Rapa treatment compared to the Dox-sen group was found to be lower in siR-TDN. box The reduction in p21 expression was greater after treatment with siRNA than after treatment with TDN. box The sum of the p21 expression reduction effects after treatment indicates that siR-TDN box It exhibits a synergistic effect in reducing p21 expression in senescent cells.

[0072] Table 3. p21 expression after treatment in different experimental groups

[0073]

[0074] The above experimental results show that siR-TDN box It can effectively inhibit doxorubicin-induced cell senescence, and siR-TDN box The anti-aging effect is greater than that of siRNA and TDN. box The combined anti-aging effects have a synergistic effect.

[0075] Experiment Example 4: In vivo animal experiments

[0076] 1. Experimental Methods

[0077] To induce accelerated aging in male C57BL / 6 mice, doxorubicin was administered intraperitoneally twice, 10 days apart, at a dose of 10 mg / kg. In lifespan experiments, to detect lifespan extension in mice, they were treated with doxorubicin and siR-TDN. box (200 uL, every other day) until all mice died. In the healthy lifespan experiment, to test the physical function of mice, mice were intraperitoneally injected with doxorubicin (twice, 10 days apart, at a dose of 10 mg / kg) and drugs (the drugs in each group were siRNA (1 μM, 200 uL), TDN, etc.). box (1μM, 200uL), siR-TDN box(1 μM, 200 uL) or rapamycin (4 mg / Kg); every other day, for 4 weeks. Naturally aged C57BL / 6 mice (also known as aged mice, male, 17 months old) and young mice (4 weeks old at purchase) were used as controls. Mice in the control group and doxorubicin group were given 200 uL of physiological saline. At the end of the experiment, the mice were euthanized and samples were collected.

[0078] (2) Behavioral experiments

[0079] a) Grip strength: Mice were placed on top of a grid strength meter (YLS-13A) so that they gripped the grid with all four paws. The mice's tails were pulled until they were released, and grip strength was recorded over five trials. Grip strength (g) is averaged, excluding the maximum and minimum data points.

[0080] b) Pole climbing experiment: The apparatus used consisted of a wooden stick with a diameter of 1 cm, a length of 50 cm, and an incline of 45°, and a wooden ball with a diameter of 2.5 cm at the top of the stick. Researchers had the mice face up with their front paws on the ball, and then recorded the time it took them to turn around and climb down the stick.

[0081] c) Spinning bar test: To assess the mouse's motor coordination and balance. The device was set to accelerate from 4 revolutions to 44 revolutions over 300 seconds. The time and distance the mouse fell from the bar were recorded. Each mouse underwent two tests, with a 1-hour interval between each test.

[0082] d) Open field test: to evaluate the exploratory behavior of mice in an unfamiliar environment. Place the mice in a 50cm×50cm×50cm square open field and record the time and distance the mice travel in exploring the central area.

[0083] (3) Intraperitoneal glucose tolerance test

[0084] After a specified fasting period, mice were intraperitoneally injected with glucose (2 g / kg). Blood glucose levels were recorded using a glucometer at 0, 15, 30, 60, and 120 minutes.

[0085] (4) Biochemical index tests

[0086] Serum was collected and serum biochemical tests were performed. In aging mice, aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea, and creatinine (CRE) showed an increasing trend, but the differences were not statistically significant. This indicates that liver and kidney function tends to decline in aging mice, but without substantial damage.

[0087] 2. Experimental Results

[0088] Figure 4 The siR-TDN was displayed. box It slowed down the aging process in mice exposed to doxorubicin. Figure 4'a' is a schematic diagram of a mouse experiment. Figure 4 b shows that in siR-TDN box Treatment improved and prolonged the survival curve of aging mice. Figure 4 (cg) shows siR-TDN box Behavioral results of mice exposed to doxorubicin after treatment. Figure 4 c shows the quantitative results of grip strength in mice after treatment in each group. It can be seen that, compared with the Dox group, siRNA and TDN... box siR-TDN box Both siRNA and Rapa treatments enhanced grip strength in mice. Further comparisons of siRNA and TDN in Table 4 are also shown. box siR-TDN box The percentage increase in grip strength in mice treated with Rapa compared to the Dox group was found to be significant for siR-TDN. box The grip strength enhancement effect in mice treated with this method was greater than that after siRNA treatment and TDN treatment. box The sum of the grip strength enhancement effects after treatment indicates that siR-TDN box It exhibited a synergistic effect in enhancing grip strength in Dox-induced aging mice.

[0089] Table 4. Quantification of changes in grip strength in mice after treatment in each group

[0090]

[0091] Figure 4 h The results of inflammatory areas in mice after treatment were obtained by H&E staining liver sections; Figure 4 i shows the visualization (left) and quantitative (right) analysis results of the percentage of SA-β-gal positive senescent cells in the liver. Among them, Figure 4 The image shows a representative visualization (left) of p21 immunofluorescence in the liver of doxorubicin-exposed mice after indicated treatment, and quantitative results (right) of the percentage of p21-positive senescent cells in the liver. It can be seen that, compared with the Dox group, siRNA and TDN... box siR-TDN box The number of p21-positive senescent cells decreased after both siRNA and Rapa treatment. Further comparisons of siRNA and TDN in Table 5 are also shown. box siR-TDN box The percentage reduction in p21-positive senescent cells after Rapa treatment relative to the Dox group was found to be significant for siR-TDN. box The reduction effect of p21-positive senescent cells after treatment was greater than that after siRNA treatment and TDN treatment. box The sum of the reduction effects of siR-TDN on p21-positive senescent cells indicates that... boxIt exhibited a synergistic effect in reducing the number of senescent cells in Dox-induced aging mice.

[0092] Table 5. Percentage of p21-positive senescent cells in the liver

[0093]

[0094] Figure 5 The siR-TDN was displayed. box It can improve physical skills in aged mice (referring to naturally aging mice). Results showed that compared to young mice (left), aged mice (middle) had grayish back fur, less luster, and larger fur volume. siR-TDN box The fur on the backs of the mice in the treatment group (right) regained its dark and glossy color, but their body weight was not significantly different from that of the saline treatment group. Figure 5 b). The mice's physical function was then assessed through a series of functional tests, all of which showed a decline in older mice. Compared to the saline-treated group, siR-TDN... box Treatment significantly increased grip strength in aged mice. Figure 5 c) Maximum rotation time ( Figure 5 f) Total distance traveled Figure 5 g), and reduced turning time and climb-down time ( Figure 5 d,e). Open-field experiments showed that, compared to young control mice, older mice explored the central region for less time and distance, while siR-TDN... box The treated mice showed an increased tendency to explore central nervous system regions. Figure 5 The functional analysis results of these mice showed improvements in motor function, balance, exercise endurance, and spontaneous exploration ability. Serum biochemical tests were performed to determine possible changes in liver and kidney function in aged mice. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea, and creatinine (CRE) showed a trend of increase in aged mice, but the differences were not statistically significant. Figure 5 j). This indicates that liver and kidney function tends to decline in older mice, but without substantial damage. siR-TDN box The treatment did not damage liver or kidney function. Considering that rapamycin treatment can lead to glucose intolerance, the treatment of siR-TDN... box Mice treated for 8 weeks underwent a glucose tolerance test, and siR-TDN was found to be effective. box It does not affect the glucose tolerance of aged mice. Figure 5 k).

[0095] Figure 6 The siR-TDN was displayed. boxIt can alleviate age-related changes in aged mice. Results showed that senescent cells accumulated in different tissues of aged mice, consistent with the observed increase of SA-β-gal positive cells in the liver, kidney, and lungs of aged mice. siR-TDN box Reduced the number of SA-β-gal positive cells in the liver, kidney, and lung tissues of aged mice ( Figure 6 a) Aging is often accompanied by the accumulation of low-level chronic inflammation, which in turn accelerates aging, creating a vicious cycle. Extensive inflammatory cell infiltration can be observed in liver, kidney, and lung sections of aged mice, with siR-TDN... box It inhibited inflammation in the liver, kidney, and lung tissues of naturally aging mice. Figure 6 b).

[0096] Immunofluorescence staining results showed that p21 expression was upregulated in the liver, kidneys, and lungs of naturally aging mice, and siR-TDN was also observed. box After 8 weeks of treatment, the mice showed effective senescence clearance ability. Figure 6 c). Protein quantification analysis showed that, compared with the young control group and the saline treatment group, siR-TDN... box Treatment reduced the expression level of Raptor in aged mice, thereby inhibiting mTORC1 activity (as detected by pS6 / S6). Furthermore, the expression of aging markers, including the CDK inhibitors p16 and p21, was also reduced. Figure 6 d) Downward adjustment. In summary, siR-TDN box It demonstrates highly effective silencing of Raptor with no obvious side effects.

[0097] The above experimental results show that siR-TDN box It can effectively inhibit doxorubicin-induced aging and natural aging in vivo, and siR-TDN box The anti-aging effect is greater than that of siRNA and TDN. box The combined anti-aging effects have a synergistic effect.

[0098] In summary, this invention provides a tetrahedral framework nucleic acid loaded with siRNA, its preparation method, and its use in the preparation of anti-aging drugs. This invention loads siRNA that inhibits the expression of rapamycin complex 1 (mTORC1) onto a tetrahedral framework nucleic acid, forming a tetrahedral framework nucleic acid complex that exhibits a synergistic anti-aging effect. This tetrahedral framework nucleic acid complex shows promising application prospects in the preparation of anti-aging drugs.

Claims

1. The use of tetrahedral framework nucleic acid complexes in the preparation of anti-aging drugs, characterized in that, The tetrahedral framework nucleic acid complex is a product prepared from siRNA and tetrahedral framework nucleic acid, wherein the molar ratio of siRNA to tetrahedral framework nucleic acid is 1:1, and the siRNA is siRNA that inhibits the expression of rapamycin complex 1. The tetrahedral framework nucleic acid is self-assembled from four DNA single strands, the sequences of which are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, respectively. The siRNA is self-assembled from two single-stranded RNA molecules, the sequences of which are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

2. The use according to claim 1, characterized in that, The preparation method of the tetrahedral framework nucleic acid complex includes the following steps: mixing siRNA and tetrahedral framework nucleic acid and reacting them to obtain the tetrahedral framework nucleic acid complex.

3. The use according to claim 2, characterized in that, The reaction conditions are: first heat the reaction, then cool it down. The heating reaction is carried out at a temperature of 80-100℃ for 5-15 minutes. The cooling reaction is carried out at a temperature of 0-10℃ for a time of 10-30 minutes.

4. The use according to claim 3, characterized in that, The heating reaction was carried out at a temperature of 95°C for 10 minutes. The cooling reaction was carried out at a temperature of 4°C for 20 minutes.

5. The use according to claim 1, characterized in that, The drug is a drug that inhibits the expression of Raptor mRNA and / or inhibits the expression of aging markers.

6. The use according to claim 1, characterized in that, The drug is a drug that reduces the number of senescent cells.

7. The use according to any one of claims 1-6, characterized in that, The drug is a pharmaceutical preparation made with a tetrahedral framework nucleic acid complex as the active ingredient and pharmaceutically acceptable excipients.

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