Tetrahedral framework nucleic acid-polydatin compound as well as preparation method and application thereof in inhibition of kidney stone

Abstract

The invention provides a tetrahedral framework nucleic acid-polydatin compound as well as a preparation method and application thereof in inhibiting kidney stone, and belongs to the field of biomedical materials. The compound disclosed by the invention is a compound formed by mixing tetrahedral framework nucleic acid and polydatin; the tetrahedral framework nucleic acid is synthesized by self-assembly of four single-stranded DNA (deoxyribonucleic acid) with nucleotide sequences as shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4. The compound disclosed by the invention is targeted to renal tubules in vivo, and the tetrahedral framework nucleic acid and polydatin in the compound play a synergistic effect to inhibit the formation of calcium oxalate, so that the formation of kidney stones and renal tubule injury induced by calcium oxalate crystals are inhibited. The compound provided by the invention is a promising treatment method for preventing kidney stone, treating kidney stone and preventing kidney stone relapse, provides a new treatment way for kidney stone diseases, and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials, and in particular relates to a tetrahedral framework nucleic acid-polydatin complex and a preparation method thereof and use thereof in inhibiting kidney stones. Background Art

[0002] Kidney stone disease is a condition characterized by the formation of solid mineral lumps within the urinary system. These stones vary in size and composition, and commonly include calcium oxalate, calcium phosphate, uric acid, and struvite. The development of kidney stones is usually due to the crystallization of minerals and salts in the urine, which leads to the aggregation and formation of solid structures within the urinary tract. Kidney stones can present with a range of consequences, from the immediate and severe pain that accompanies their passage through the urinary tract to more chronic and systemic effects. The obstruction caused by large stones can disrupt normal urine flow and increase pressure within the kidney, causing additional pain and potential tissue damage. It can also promote urinary tract infections, leading to complications and worsening symptoms. Over time, persistent or recurrent stones pose a risk of kidney damage, potentially leading to chronic kidney disease. Hematuria, or blood in the urine, may occur during the passage of the stone, indicating possible damage to the urinary tract. Most importantly, recurrent stones can perpetuate a cycle of discomfort for the patient, affecting the patient's quality of life and requiring ongoing treatment. Current treatments for kidney stones mainly include medication (mainly to help small stones pass), extracorporeal shock wave lithotripsy (ESWL), and surgical intervention (ureteroscopy, percutaneous nephrolithotomy, etc.). Despite advances in treatment modalities, the management of kidney stone disease remains challenging. The recurrence rate remains high, highlighting the need for more targeted preventive measures. In addition, current treatment options are not always universally effective, and the choice of intervention depends on various factors, including stone composition and patient characteristics. The complexity of the etiological factors that lead to stone formation requires a comprehensive understanding to develop more personalized and effective treatment strategies. In this context, exploring new approaches is critical to addressing existing challenges and enhancing the overall landscape of kidney stone disease management.

[0003] The pathophysiological process of kidney stone formation involves the interaction of multiple factors. Supersaturation of the urine with substances such as calcium, oxalate, and phosphate provides the conditions for nucleation, crystal formation and subsequent enlargement. Crystal aggregation, influenced by urine flow and inhibitors or promoters, leads to the development of kidney stones. These crystals may be embedded in an organic matrix and adhere to the surface of the kidney, promoting stone growth. Inflammatory reactions in the tissues surrounding the kidney can lead to tissue damage, and larger stones can obstruct the urinary tract, causing increased pressure and reflux, which may cause pain, infection, and kidney damage. Genetic and metabolic factors, including diseases such as hypercalciuria and hyperoxaluria, further exacerbate an individual's susceptibility to kidney stone formation.

[0004] Tetrahedral framework nucleic acid (tFNA) is a new type of nanomaterial derived from DNA. A large number of studies have demonstrated its efficacy in immunomodulation, anti-oxidative stress and anti-inflammatory responses in various diseases. Notably, it has low cytotoxicity and low clearance compared with other nanomaterials, and has good biocompatibility and biodegradability. Studies have reported that it can be used not only as a nanomedicine, but also as a nanocarrier to carry other small molecule drugs to penetrate biological barriers with large loading sites. Polydatin (PD), a crystalline drug compound extracted from Polygonum cuspidatum, has been used to treat diseases such as sepsis, burns and ischemia-reperfusion injury. Studies have shown that it has an effect on the cytoplasmic / mitochondrial reactive oxygen species nlrp3 inflammasome pathway.

[0005] However, there is no research on using tetrahedral framework nucleic acids to carry polydatin for the treatment of kidney stones. Summary of the invention

[0006] The purpose of the present invention is to provide a tetrahedral framework nucleic acid-polydatin complex and a preparation method thereof and use thereof in inhibiting kidney stones.

[0007] The present invention provides a tetrahedral framework nucleic acid-polydatin complex, which is a complex formed by mixing a tetrahedral framework nucleic acid and polydatin; the tetrahedral framework nucleic acid is self-assembled and synthesized by four single-stranded DNAs with nucleotide sequences such as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4.

[0008] Furthermore, when the tetrahedral framework nucleic acid and polydatin are mixed, the molar ratio of the tetrahedral framework nucleic acid to polydatin is 1:100 to 1:400.

[0009] Furthermore, the method for synthesizing the tetrahedral framework nucleic acid comprises the following steps: adding four single-stranded DNAs into TM buffer, maintaining at 90-99° C. for 10-20 min, and rapidly cooling to 0-4° C. and maintaining for more than 20 min.

[0010] Furthermore, the method for synthesizing the tetrahedral framework nucleic acid comprises the following steps: adding four single-stranded DNAs into TM buffer, maintaining at 95° C. for 10 minutes, and rapidly cooling to 4° C. and maintaining for more than 20 minutes.

[0011] Furthermore, the four single-stranded DNAs are four single-stranded DNAs in equal molar ratios.

[0012] The present invention also provides a method for preparing the aforementioned tetrahedral framework nucleic acid-polydatin complex, which comprises the following steps:

[0013] The tetrahedral framework nucleic acid and polydatin are added into a solvent, mixed and reacted, and then ultrafiltered to obtain the product.

[0014] Further,

[0015] The solvent is one or more of PBS and DMSO;

[0016] And / or, the reaction temperature is 20-30°C;

[0017] And / or, the reaction time is 4 to 6 hours;

[0018] And / or, the ultrafiltration uses a 30 KDa molecular weight membrane.

[0019] The present invention also provides the use of the aforementioned tetrahedral framework nucleic acid-polydatin complex in the preparation of a drug for preventing kidney stones, treating kidney stones and / or inhibiting the recurrence of kidney stones.

[0020] Furthermore, the drug is a drug that inhibits the formation of calcium oxalate.

[0021] Furthermore, the drug is a drug that inhibits oxidative stress, inhibits inflammation and / or inhibits ferroptosis.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] In summary, the present invention provides a tetrahedral framework nucleic acid-polydatin complex, in which polydatin is mutually bound to the tetrahedral framework nucleic acid through an embedded binding mode, and the tetrahedral framework nucleic acid is used as a carrier to improve the stability and release ability of polydatin, and maintains excellent ability to enter cells. At the same time, the complex of the present invention can inhibit oxidative stress and inflammation to inhibit ferroptosis. The complex of the present invention targets the renal tubules in vivo, and the tetrahedral framework nucleic acid and polydatin in the complex play a synergistic role in inhibiting the formation of calcium oxalate, thereby inhibiting kidney stone formation and renal tubular damage induced by calcium oxalate crystals. The complex of the present invention is a promising treatment method for preventing kidney stones, treating kidney stones, and preventing the recurrence of kidney stones, and provides a new treatment approach for kidney stone disease, with good application prospects.

[0024] 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.

[0025] 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

[0026] Figure 1 Schematic diagram of the preparation and characterization results of the tFNA-PD complex: A is a schematic diagram of the synthesis process of the tFNA-PD complex; B is the PAGE characterization results of the tFNA and tFNA-PD complex, lane 1 is S1, lane 2 is S2, lane 3 is S3, lane 4 is S4, lane 5 is tFNA, lane 6 is tFNA-PD (1:100), lane 7 is tFNA-PD (1:200), and lane 8 is tFNA-PD (1:400); C is the spectrum of PD (Polydatin), tFNA and tFNA-PD (tFNA-P, 1:100) between 200 and 500 nm; D is the spectrum of tFNA Figure 3 is the ratio of the GelRed (λex = 312 nm) fluorescence to the fluorescence of the tFNA-PD complexes with different PD ratios (tFNA:PD = 1:100-400); E is the TEM image of the tFNA-PD complex (1:100) (scale bar: 50 nm, 20 nm); F is the AFM image of the tFNA-PD complex (1:100) (scale bar: 20 nm, 10 nm); G is the particle size result of tFNA and tFNA-PD complex (1:100) measured by DLS; H is the Zeta potential result of tFNA and tFNA-PD complex (1:100) measured by DLS.

[0027] Figure 2The results of the tFNA-PD complex's anti-oxidative stress and anti-ferroptosis effects in vitro: A is the uptake of Cy5-labeled tFNA and tFNA-PD complex (tFNA-P) by HK2 cells after 2 hours of co-incubation, red is Cy5-labeled tFNA or tFNA-PD complex, green is cytoskeleton, blue is cell nucleus, scale bar is 20μm; B is the flow cytometry result showing ... Immunofluorescence intensity of tFNA and tFNA-PD complex (tFNA-P) in HK2 cells; C and D are the flow cytometry results and statistical analysis results of HK2 cell apoptosis in different treatment groups in the calcium oxalate-induced cell damage model; E is the effect of different concentrations of tFNA on HK2 cell viability; F and G are the ROS flow cytometry detection results and statistical analysis results of different treatment groups in the calcium oxalate-induced cell damage model, CTL in F is the Control group, CTL+Fer1 is the Control+Fer1 group, and CTL+tFNA-P is the Control+tFNA-P group; H is the LDH detection result of cells in different treatment groups; I is the malondialdehyde (MDA) detection result of cells in different treatment groups; J is the superoxide dismutase (SOD) detection result of cells in different treatment groups; K is the glutathione (GSH) detection result of cells in different treatment groups; L is the glutathione (GSH) / glutathione disulfide (GSSG) detection result of cells in different treatment groups; M is the Fe 2+ Detection results; N is the catalase (CAT) detection results of cells in different treatment groups; Data are expressed as mean ± SD; *P < 0.05, **P < 0.01 and ***P < 0.001 represent statistically significant differences between the marker groups.

[0028] Figure 3 The results of tFNA-PD complex inhibiting the formation of kidney stones: A is a schematic diagram of the animal model; B is a representative IVIS image of the ex vivo organ after tail vein injection of Cy5-labeled tFNA-PD complex; C is a representative immunostaining of the kidney after tail vein injection of Cy5-labeled tFNA-PD complex; green is lotus tetrakehner lectin (LTL); red is Cy5-labeled tFNA-PD complex; blue is cell nucleus; D is the body weight of mice in each group, KS+tFNA-600P in D is KS+tFNA-P; E is the creatinine result of each group; F is the blood urea nitrogen (BUN) result of each group; G is a three-dimensional image of the kidney of each group of mice reconstructed based on microCT; H is the statistical result of calcium oxalate crystals in Figure G; I is the HE staining of kidney sections, Von Kossa staining and polarization microscope staining results; J is the HE staining tubular injury score in Figure I; data are expressed as mean ± standard deviation; *P<0.05, **P<0.01 and ***P<0.001 represent statistically significant differences between the labeled groups.

[0029] Figure 4 The results of tFNA-PD complex inhibiting oxidative stress and ferroptosis in vivo: A is the dihydroethidium (DHE) staining analysis of ROS levels in kidney tissue sections (red is reactive oxygen species, blue is cell nuclei, scale bar is 50μm) and heme oxygenase 1 (HO-1) levels in kidney tissue sections (red is HO-1, blue is cell nuclei, scale bar is 50μm); B is the statistical analysis of HO-1 in Figure A; C is the TEM image of sections of the five groups of kidney stones; the scale bars are 2μm and 500nm; D is the statistical graph of ROS in Figure A; E is the GSH expression level of the five groups of tissues; F is the GSH / GSSG expression level of the five groups of tissues; G is the MDA expression level of the five groups of tissues; H is the Fe 2+ Expression level; I is the SOD expression level of five groups of tissues; J is the WB detection results of glutathione peroxidase 4 (GPX4), ferroptosis inhibitory protein 1 (FSP1), osteopontin (OPN), SMAD4, interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α); K is the statistical analysis result of glutathione peroxidase 4 (GPX4) and ferroptosis inhibitory protein 1 (FSP1); L is the statistical analysis result of osteopontin (OPN) and SMAD4; M is the statistical analysis result of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α); Data are expressed as mean ± SD; *P < 0.05, **P < 0.01 and ***P < 0.001 represent statistically significant differences between the marker groups. DETAILED DESCRIPTION

[0030] The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0031] Example 1: Synthesis of tetrahedral framework nucleic acid-polydatin complex (tFNA-PD) of the present invention

[0032] 1. Synthesis of tetrahedral framework nucleic acid (tFNA)

[0033] Tetrahedral framework nucleic acid (tFNA) is synthesized by self-assembly of four sequence-specific single-stranded DNA (ssDNA) (Sangon, Shanghai, China) through a PCR program (maintained at 95°C for 10 min, and rapidly cooled to 4°C for more than 20 min). The specific preparation method is as follows:

[0034] Four single-stranded DNAs S1, S2, S3, and S4 (specific sequences are shown in Table 1) were dissolved in TM buffer (10 mM Tris-HCl, 50 mM MgCl 2 , pH 8.0) so that the final concentrations of the four single-stranded DNAs were 1000 nM respectively. After being fully mixed, they were quickly heated to 95°C and maintained for 10 min, and then quickly cooled to 4°C and maintained for more than 20 min, thereby self-assembly synthesizing tetrahedral framework nucleic acid (tFNA).

[0035] Table 1. Specific sequences of the four single-stranded DNAs of the present invention

[0036]

[0037] 2. Synthesis of tetrahedral framework nucleic acid-polydatin complex (tFNA-PD)

[0038] Polydatin (PD) was added to the tFNA prepared above and mixed evenly, the concentration of PD was 25 μM, 50 μM, 75 μM, 100 μM, the concentration of tFNA was 250 nM, and the molar ratio of tFNA to PD was 1:100, 1:200, 1:300, 1:400. The mixed solution was shaken at 25°C for 6 hours, and then the residual ssDNA and PD were removed by ultrafiltration (30 kDa molecular weight membrane, Millipore, USA) at 25°C for 10 minutes at a speed of 4000 rpm to obtain a tFNA-PD complex.

[0039] 3. Characterization of tetrahedral framework nucleic acid-polydatin complex (tFNA-PD)

[0040] The successful synthesis of tFNA-PD complex was verified by polyacrylamide gel electrophoresis (PAGE), transmission electron microscopy (TEM), and atomic force microscopy (AFM), and its size and shape were observed. Dynamic light scattering DLS (Nano ZS, Malvern, England) was used to measure the particle size and Zeta potential of tFNA and tFNA-PD complex. The absorbance curves of tFNA, PD, and tFNA-PD complex were depicted by ultramicrospectrophotometer. The fluorescence of GelRed was detected by Varioskan LUX microplate reader (ThermoScientific, USA).

[0041] result:

[0042] like Figure 1 As shown in A: The synthesis of tFNA-PD complex is divided into two steps. First, tFNA is self-assembled from four equimolar amounts of ssDNA. Subsequently, tFNA is complexed with PD by stirring for 6 h.

[0043] like Figure 1 As shown in B: PAGE results proved that the tFNA-PD complex was successfully synthesized, in which the position of the tFNA-PD complex moved up as the PD ratio increased.

[0044] like Figure 1 As shown in C: Figure 1 The C spectrum showed that the characteristic absorption peaks of the tFNA-PD complex were close to tFNA (260 nm) and PD (306 nm), indicating that PD was successfully carried by tFNA without any by-products.

[0045] The binding mode of tFNA and PD was determined by GelRed dye competition. The results are shown in Figure 1 As shown in D: When tFNA was incubated with 1xGelRed for 20 minutes, fluorescence was excited, while when tFNA-PD was incubated with GelRed, the fluorescence intensity of GelRed decreased. This means that the position where PD interacts with tFNA is the same double-stranded DNA double helix groove as GelRed.

[0046] Figure 1 E and 1F are TEM and AFM images of the tFNA-PD complex, and the AFM image shows that the tFNA-PD complex is about 20 nm. The same results were obtained from the TEM image, and a triangular structure was observed by TEM.

[0047] Figure 1 G and 1H are the particle size and Zeta potential results of tFNA and tFNA-PD complex measured by DLS. Figure 1 G shows that the particle size of tFNA is 14.21 nm, while the particle size of tFNA-PD complex is 18.17 nm. Figure 1 The zeta potential of tFNA is -4.54 mV, while the zeta potential of the tFNA-PD complex is -11.6 mV in H. These differences indicate that the tFNA-PD complex was successfully synthesized and is relatively stable compared with tFNA.

[0048] Therefore, the present invention successfully prepares a nano material - tFNA-PD complex.

[0049] The beneficial effects of the present invention are demonstrated below through specific test examples.

[0050] Experimental Example 1: Antioxidative stress and ferroptosis inhibition effects of tFNA-PD complex in vitro

[0051] 1. Experimental methods

[0052] (1) Cellular uptake of Cy5-labeled tFNA and tFNA-PD complex

[0053] In Table 1, the 5' end of S1 was connected to Cy5 to obtain Cy5-labeled S1 (Cy5-S1). Cy5-S1 was used to prepare Cy5-labeled tFNA and Cy5-labeled tFNA-PD complex (the molar ratio of tFNA to PD was 1:100) according to the method of Example 1 for the following experiment.

[0054] HK2 cells (human renal cortical proximal tubule epithelial cells) were grown in 6-well plates. Subsequently, the cells were incubated with Cy5-loaded tFNA (Cy5-tFNA, tFNA concentration was 250nM) or Cy5-loaded tFNA-PD (Cy5-tFNA-PD, tFNA concentration was 250nM) for 4 hours. After the cells were incubated for 2 hours, they were washed with PBS, fixed with 4% paraformaldehyde, and stained with phalloidin labeled with 4',6-diamino-2-phenylindole (DAPI) and fluorescein isothiocyanate (FITC). In order to evaluate the cellular uptake of tFNA and tFNA-PD at 1-4h, the results were obtained by confocal microscopy (FV3000, Olympus) and flow cytometry (tune NxT, Thermo Fisher).

[0055] (2) Cytotoxicity assay

[0056] HK2 cells were seeded in 96-well plates one day before administration. The control group was cultured with regular culture medium, while different concentrations of tFNA (62.5 nM, 125 nM, 250 nM, and 500 nM) were added to the HK-2 cells in the treatment group, and the cell viability was determined using CCK-8 after incubation for 24 hours.

[0057] (3) Calcium oxalate-induced cell damage model

[0058] HK2 cells (human renal cortical proximal tubule epithelial cells) were selected as target cells because the formation of kidney stones mainly affects the renal tubules. One day before the start of treatment, HK2 cells were seeded in 6-well plates and divided into 6 groups: control group (Control), control + Fer1 group (Control + Fer1), control + tFNA-PD complex group (Control + tFNA-P), model group (COM), model + Fer1 group (COM + Fer1), model + tFNA-PD complex group (COM + tFNA-P).

[0059] The control group was cultured in conventional culture medium, and 100 μg / ml calcium oxalate (C 2 CaO 4) were cultured for 24 hours to simulate calcium oxalate crystal-induced cell damage. Ferrostatin-1 (Fer1) is a compound that has been studied for inhibiting cell ferroptosis and is widely used as a gold standard in previous studies. The modeling + Fer1 group and the modeling + tFNA-PD complex group were added with drugs (Fer1 or tFNA-PD complex) and cultured for 24 hours after modeling to detect drug efficacy. The control + Fer1 group and the control + tFNA-PD complex group were added with drugs (Fer1 or tFNA-PD complex) and cultured for 24 hours to detect drug toxicity. The dosage of Fer1 was 2 μM. The tFNA-PD complex used in the experiment was the tFNA-PD complex prepared according to the method of Example 1 (the molar ratio of tFNA to PD was 1:100), and the tFNA concentration was 250 nM.

[0060] (4) Cell apoptosis detection

[0061] Annexin V-FITC / 7-AAD apoptosis kit (Elabscience, catalog number E-CK-A212) was used to evaluate the protective effect of calcium oxalate on cell apoptosis. After treating the cells in each group according to (3), the cells in each group were incubated with Annexin V-FITC and 7-AAD. Then, a flow cytometer (Symphony A5, BD, USA) was used to analyze the apoptosis results.

[0062] (5) ROS detection

[0063] After the cells were treated according to the method in (3), the ROS levels in six groups of cells (control group, control + Fer1 group, control + tFNA-PD complex group, model group, model + Fer1 group, model + tFNA-PD complex group) were detected by kit and flow cytometer.

[0064] (6) Oxidative stress system detection

[0065] After the cells were treated according to the method in (3), the expression of lactate dehydrogenase (LDH), lipid oxidation (MDA), and iron ion (Fe) in the six groups of cells (control group, control + Fer1 group, control + tFNA-PD complex group, model group, model + Fer1 group, and model + tFNA-PD complex group) was detected by kit. 2+ ), superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and GSH / GSSG levels.

[0066] 2. Experimental results

[0067] The present invention selected HK2 cells as target cells to evaluate their uptake of tFNA-PD complexes. Figure 2As shown in A, 2 hours after administration, the confocal image of HK2 cells confirmed the presence of tFNA and tFNA-PD complex in HK2 cells, indicating that the complex of the present invention can be well taken up by cells. Figure 2 The flow cytometry results of B showed that the immunofluorescence intensity of tFNA and tFNA-PD complexes in HK2 cells continued to increase from 0h to 4h, and the absorption of tFNA and tFNA-PD complexes in HK2 cells was significant. The above results indicate that tFNA and tFNA-PD complexes can be delivered to the kidneys, especially renal tubular cells, and have the potential to innovate targeted treatment of kidney stone disease.

[0068] CCK8 results showed ( Figure 2 E) tFNA had no significant toxic effect on HK2 cells (P>0.05). Among them, tFNA (250nM) had the strongest effect on promoting cell viability, and the cell growth was significantly faster than that of the control group (P<0.05).

[0069] Ferroptosis can cause significant cell death, so the present invention performed apoptosis flow cytometry analysis. Ferroptosis-1 (Fer1) is a compound that has been studied for its potential role in inhibiting a type of cell death called ferroptosis and has been studied in various iron poisoning-related diseases. 100μg / mL calcium oxalate was added to HK2 cells (COM group) to simulate the damage induced by calcium oxalate crystals. The apoptosis results showed that Fer1 and tFNA-P had no obvious toxic effects on HK2, but the cells died significantly after calcium oxalate administration (P<0.0001)( Figure 2 C and 2D). Fer1 can successfully protect HK2 cells from calcium oxalate-induced damage (P<0.01). The more exciting finding is that compared with Fer1, the tFNA-PD complex has a better protective effect on calcium oxalate-induced HK2 cell damage, with a cell death rate of only about 10% (P<0.0001, compared with COM, P<0.001, compared with Fer1). It can be seen that the tFNA-PD complex of the present invention can effectively protect HK2 cells from calcium oxalate-induced damage.

[0070] Lactate dehydrogenase (LDH) is an enzyme present in almost all body tissues. When cells die, intracellular LDH will be released outside the cell. Therefore, the present invention also tests the release of extracellular LDH. The LDH release results show ( Figure 2H), there was no significant change among the control group, control + Fer1 group, and control + tFNA-PD complex group, but the LDH release in the COM group increased significantly (P<0.0001). On the other hand, Fer1 (modeling + Fer1 group) and tFNA-PD complex (modeling + tFNA-PD complex group) successfully inhibited the release of LDH, indicating that Fer1 and tFNA-PD complex have good cell protection effects (P<0.001, P<0.0001).

[0071] One of the most notable features of ferroptosis is the upregulation and uncontrolled intracellular oxidative stress. Cellular reactive oxygen species (ROS) assay kit was used to assess cellular oxidative stress. Cellular ROS flow cytometry assay showed ( Figure 2 F and 2G), Fer1 and tFNA-PD complex had no effect on the cellular ROS level in the control + Fer1 group and the control + tFNA-PD complex group (P>0.05), the modeling group (COM) showed that calcium oxalate could significantly increase the cellular ROS level (P<0.0001), the modeling + Fer1 group and the modeling + tFNA-PD complex group showed that Fer1 and tFNA-PD complex could significantly reduce the increased cellular ROS level caused by calcium oxalate (P<0.05, P<0.01), indicating the effectiveness of its anti-oxidative stress ability.

[0072] In addition, the present invention also evaluated the level of malondialdehyde (MDA). The results showed that ( Figure 2 I), the oxidative stress level increased significantly after calcium oxalate administration, but was inhibited by Fer1 and tFNA-PD complex, and the inhibitory effect of tFNA-PD complex was significantly better than that of Fer1, and there was also a significant difference between the two.

[0073] On the other hand, the present invention evaluates superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and GSH / GSSG, which are indicators of the anti-oxidative stress system. Figure 2 As shown in Figures 2J, 2K, 2L, and 2N, these anti-oxidative stress system indicators showed severe depletion after calcium oxalate injury (P<0.0001), and with the addition of Fer1 and tFNA-PD complex, the anti-oxidative stress system retained its function (P<0.05, P<0.0001, P<0.05, P<0.01), further indicating that tFNA-PD complex can effectively resist oxidative stress.

[0074] Another characteristic of ferroptosis is Fe 2+ By extracting the cell mixture, the present invention found that after calcium oxalate induction, Fe 2+The level was significantly increased (P<0.0001), but it could be inhibited by Fer1 and tFNA-PD complex (P<0.001, P<0.01)( Figure 2 M).

[0075] In vitro experiments showed that ferroptosis was involved in calcium oxalate-induced cell damage, and Fer1 and tFNA-PD complexes could inhibit ferroptosis, thereby protecting renal tubular cells from damage. Among them, the protective effect of tFNA-PD complex was better than that of Fer1.

[0076] Experimental Example 2: tFNA-PD complex inhibits the formation of kidney stones

[0077] The following experiments were carried out using tFNA and tFNA-PD complexes (the molar ratio of tFNA to PD was 1:100) prepared by the method of Example 1.

[0078] 1. Experimental methods

[0079] (1) Establishment of animal model

[0080] C57BL / 6J mice (Chengdu Jicui Experimental Animal Co., Ltd.) were individually housed in ventilated cages with unrestricted access to sterilized food and water and were adaptively fed for 7 days. The experimental protocol was approved by the Animal Research Ethics Committee of West China Hospital, Sichuan University, and all procedures were performed in accordance with established animal protocols. To establish a kidney stone disease model (modeling group), 8-week-old male mice were intraperitoneally injected with a single dose of glyoxylic acid (80 mg / kg body weight; Sigma-Aldrich, Shanghai, China) daily for 7 consecutive days to induce a mouse renal calcium oxalate stone model. Mice in the sham operation group were injected with an equal amount of PBS for 7 days. The tFNA group, PD group and tFNA-PD complex (1:100) group were given 100 μL of drugs every day starting from the third day of modeling (the concentration of tFNA was 250 nM, the concentration of PD was 25 μM, and the concentration of tFNA in the tFNA-PD complex was 250 nM). On the seventh day of modeling, the blood, kidneys and major organs (heart, liver, spleen and lung) of mice in each group were collected for detection. The experiment was divided into 5 groups: sham operation group (Sham), modeling group (KS), modeling + tFNA group (KS+tFNA), modeling + PD group (KS+PD), modeling + tFNA-PD complex group (KS+tFNA-PD).

[0081] (2) In vivo imaging

[0082] After the animal model was established as described above, in order to evaluate the biodistribution of the tFNA-PD complex in vivo, the present invention also intravenously injected the Cy5-labeled tFNA-PD complex into mice 3 days after modeling. Fluorescence signal monitoring uses IVIS spectrum to perform in vitro organ fluorescence imaging at 1h, 2h, 4h and 8h respectively. Fix with OCT embedding solution and freeze. Tissue sections are stained with DAPI and lotus lectin (LTL). Fluorescence images are captured using a sliding scanner (VS200, Olympus).

[0083] (3) Weight measurement

[0084] After the mice in each group were treated according to (1), the body weight of the mice in each group was measured every day.

[0085] (4) Creatinine and blood urea nitrogen testing

[0086] After each group of mice was treated according to (1), fresh blood from each mouse was collected and centrifuged at 12,000 rpm for 10 minutes to separate the supernatant. These supernatants were then processed by an automatic biochemical analyzer using a commercially available kit (Rochester) for biochemical analysis.

[0087] (5) Micro CT

[0088] After the mice in each group were treated according to (1), the collected kidney specimens were fixed between the radiation source and the camera, rotated 0.9° to 180° along the vertical long axis, and scanned using Micro CT to determine whether there were high-density stone-like substances, and photographed and three-dimensionally reconstructed. Quantitative analysis was performed using Image-Pro Plus 6 software to calculate the number of crystals represented by the "area" index. The higher the score or value, the more kidney crystals there are.

[0089] (6) HE / VK staining of renal tissue sections

[0090] After treating each group of mice according to (1), kidney tissues were fixed with paraformaldehyde or OCT gel and then embedded in paraffin. The paraffin-embedded tissues were then sliced ​​into 5 μm thin sections for further analysis. Hematoxylin and eosin (H&E) staining, as well as von Kossa staining, were performed according to the manufacturer's protocol (Solarbio, China). Stone crystals were observed and photographed using a polarized light microscope. In HE-stained sections, tubular injury was scored semi-quantitatively on a scale of 0 to 5: 0 = normal histology; 1 = degeneration only, no necrosis; 2 to 5, <25, <50, <75, >75% of epithelial cells, showing necrosis, degeneration, regeneration, tubular dilation, protein casts, and interstitial lymphocyte infiltration, respectively.

[0091] 2. Experimental results

[0092] Kidney stone recurrence is a major problem in treatment. From a clinical perspective, surgical treatment of kidney stones can only remove visible kidney stones, but cannot treat calcium oxalate crystals formed in the renal parenchyma. In order to evaluate the inhibitory effect of tFNA and tFNA-PD complexes on kidney stone recurrence, it is necessary to start treatment after calcium oxalate formation. Using glyoxylic acid induction, male C57BL / 6 mice formed calcium oxalate crystals on the third day. The present invention starts treatment on the third day ( Figure 3 A). First, in order to evaluate the biodistribution of tFNA-PD complexes in vivo, tFNA-PD complexes were injected into the mouse tail vein, and the mice were killed at different time points (1h, 2h, 4h, 8h). The results showed that tFNA-PD complexes mainly accumulated in the liver, followed by kidneys, spleen, lungs and heart ( Figure 3 B) It is worth noting that the tFNA-PD complex has a rapid distribution behavior and can be quickly distributed to various organs. The concentration of the tFNA-PD complex reaches a peak 1 hour after injection and then gradually decreases, indicating that it requires repeated administration.

[0093] Since the most affected microstructure during calcium oxalate-induced injury is the renal tubule, the present invention further studied whether the tFNA-PD complex can be distributed to the tubular cells. The frozen sections were stained with the renal tubule markers lotus tetrakehelium lectin (LTL) 37 and 4', 6-diamino-2-phenylindole (DAPI). Immunofluorescence results showed that there was obvious accumulation of tFNA-PD complexes in the renal cortex where the proximal tubules are located ( Figure 3 C) Magnified image analysis further showed that proximal tubular cells were the main cells that accumulated tFNA-PD complexes.

[0094] Body weight represents the overall condition of the mouse. Figure 3 D. After injection of glyoxylic acid, the body weight of mice decreased significantly, while that of the sham operation group remained stable. Under the action of polydatin (KS+PD), tFNA (KS+tFNA), and tFNA-PD complex (KS+tFNA-P), the overall body weight of mice began to reverse on the 3rd day, and finally showed significant differences on the 7th day. This shows the protective effect of polydatin, tFNA, and tFNA-PD complex in alleviating calcium oxalate-induced organ damage.

[0095] In order to further determine the renal function, the present invention extracted mouse serum and measured its two key renal function parameters: creatinine and blood urea nitrogen (BUN). The results showed that ( Figure 3E and 3F), tFNA, polydatin (PD), and tFNA-PD complex can significantly protect renal function (creatinine: P<0.0001, P<0.0001, P<0.0001; BUN: P<0.0001, P<0.0001, P<0.0001), among which tFNA-PD complex has the most significant effect in protecting renal function, which is significantly different from the use of tFNA or PD alone (P<0.0001). This shows that tFNA-PD complex plays a synergistic role in protecting renal function.

[0096] The present invention further uses MicroCT to detect the formation of calcium oxalate crystals. Human kidney stones usually form a large stone in the pelvis, while the mouse kidney stone model presents small calcium oxalate spots / crystals in the parenchyma. The three-dimensional image of the mouse kidney reconstructed based on microCT shows that the crystal formation in the kidney increases significantly after glyoxylic acid injection ( Figure 3 G and 3H). PD alone could not reduce the formation of calcium oxalate crystals, while tFNA and tFNA-PD complex could significantly reduce the formation of calcium oxalate crystals.

[0097] In order to further evaluate the damage of calcium oxalate crystals to the kidneys, the present invention took kidneys from each group and stained the kidney sections with H&E staining, such as Figure 3 I. The renal tubules in the kidney stone group were significantly dilated, which may be the result of calcium oxalate blocking the renal tubules. Polydatin (PD) and tFNA-PD complex can significantly reduce renal tubular damage (P<0.001, P<0.0001)( Figure 3 I and 3J).

[0098] To further verify the microCT results, the present invention also sliced ​​the kidneys and observed them using Von Kossa staining and polarization microscopy. The Von Kossa staining kit can stain calcium to dark brown areas, making it easier to visualize. Consistent with the microCT results, polydatin (PD) has no inhibitory effect on calcium oxalate crystals, and the use of tFNA and tFNA-PD complexes can significantly reduce the formation of calcium oxalate crystals. Further polarization microscopy images are also consistent with these results. It shows that polydatin (PD) and tFNA work synergistically.

[0099] Experimental Example 3: tFNA-PD complex inhibits ferroptosis by inhibiting oxidative stress, thereby reducing renal tubular damage

[0100] The following experiment was carried out using the tFNA-PD complex prepared by the method of Example 1 (the molar ratio of tFNA to PD was 1:100).

[0101] 1. Experimental methods

[0102] (1) Establishment of animal model

[0103] C57BL / 6J mice (Chengdu Jicui Experimental Animal Co., Ltd.) were individually housed in ventilated cages with unrestricted access to sterilized food and water and were adaptively fed for 7 days. The experimental protocol was approved by the Animal Research Ethics Committee of West China Hospital, Sichuan University, and all procedures were performed in accordance with the established animal protocol. The experiment was divided into five groups: sham group (Sham), sham group + Fer1 group (Sham + Fer1 group), modeling group (KS), modeling + Fer1 group (KS + Fer1), and modeling + tFNA-PD complex group (KS + tFNA-PD). The modeling group was intraperitoneally injected with a single dose of glyoxylic acid (80 mg / kg body weight; Sigma-Aldrich, Shanghai, China) every day for 7 consecutive days to induce a mouse renal calcium oxalate stone model; the sham operation group was injected with an equal amount of PBS for 7 days; the sham+Fer1 group, KS+Fer1 group, and KS+tFNA-PD group were given 100 μL of drug (Fer1 concentration was 600 μM, tFNA concentration in tFNA-PD complex was 250 nM) every day starting on the third day of modeling. On the seventh day of modeling, the kidneys of mice in each group were taken for detection.

[0104] (2) Immunofluorescence staining

[0105] To further observe protein expression, immunofluorescence staining was performed. After treatment using the method described in (1), the kidney sections of each group of mice were fixed in cold 4% paraformaldehyde for 20 minutes and then treated with 0.5% Triton X-100 for 10 minutes. Then, the samples were blocked in 5% goat serum for 1 hour and incubated with the target primary antibody (ROS or HO-1) at 4°C overnight. After rinsing three times on the second day, the samples were incubated with the secondary antibody (1:500; Invitrogen, Carlsbad, USA) for 1 hour. Then, the cell nuclei were stained with DAPI and the cytoskeleton was stained with phalloidin. Finally, all samples were observed using a confocal laser microscope (A1R MP+, Nikon, Tokyo, Japan).

[0106] (3) Transmission electron microscopy.

[0107] After treatment with the method described in (1), the renal tissues of mice in each group were fixed with 2.5% glutaraldehyde and then with 1% osmium tetroxide. Subsequent steps included dehydration in a series of alcohols and embedding in epoxy resin. Ultrathin sections were prepared for transmission electron microscopy (TEM) examination and stained with uranyl acetate and lead citrate. TEM imaging was performed using a JEM-1400plus instrument.

[0108] (4) Oxidative stress system detection

[0109] After treatment by the method described in (1), the lipid oxidation (MDA) and iron ion (Fe 2+ ), superoxide dismutase (SOD), glutathione (GSH), and glutathione (GSH) / GSSG levels.

[0110] (5) Western blot analysis

[0111] After treatment by the method described in (1), the kidney tissue samples were rinsed with PBS and treated with protein extraction reagent (KeyGenBiotech, Nanjing, China) to extract proteins. The purified samples were then mixed with loading buffer (Beyotime, Shanghai, China) and heated at 100°C for 10 minutes. The target proteins were separated by SDS-PAGE gel and transferred to polyvinylidene fluoride (PVDF) membranes. Afterwards, the PVDF membranes were blocked with blocking solution (Beyotime, Shanghai, China) at room temperature for 20 minutes and incubated with proteins including GPX4, FSP1, OPN, SMAD4, IL-1β, IL-6, and TNF-α at 4°C overnight. After washing with TBST, the membranes were incubated with secondary antibodies (1:5000; Beyotime, Shanghai, China) for 1 hour. Finally, the protein bands on the membranes were detected by a chemiluminescence detection system (Bio-Rad, Hercules, USA).

[0112] 2. Experimental results

[0113] In order to further verify that the effect of tFNA-PD complex in treating kidney stones is to regulate ferroptosis, the present invention further examined the dihydroethidium (DHE) staining of kidney tissue sections. Compared with the kidney stone group (KS group), the tFNA-PD complex group had a significant decrease in cellular ROS ( Figure 4 A and 4D). Heme oxygenase 1 (HO-1) represents the lipid peroxidation status in tissues. Therefore, the present invention also uses kidney sections to stain HO-1. After treatment with tFNA-PD complex, the HO-1 level is significantly reduced ( Figure 4 A and 4B).

[0114] The TEM images of the modeling group of the kidney stone group showed obvious characteristics of ferroptosis, including overall mitochondrial atrophy, increased membrane density, and reduced or disappeared cristae. These morphological characteristics were significantly changed by inhibiting ferroptosis, especially in the tFNA-PD complex group ( Figure 4 C).

[0115] Tissue iron levels were also evaluated and the results showed that the tissue iron levels were increased in the kidney stone group, while they were significantly decreased in the tFNA-PD complex-treated group ( Figure 4H). Kidney stones lead to a significant increase in tissue MDA levels, while tFNA-PD complex can reduce oxidative stress ( Figure 4 G). Antioxidant stress parameters showed that kidney stones significantly damaged the antioxidant system. The SOD, GSH, and GSH / GSSG in the modeling group were significantly lower than those in the sham operation group. The tFNA-PD complex could restore the antioxidant system in the kidney and increase SOD, GSH, and GSH / GSSG ( Figure 4 E, 4F, and 4I).

[0116] Key parameters of ferroptosis inhibition markers were also evaluated, among which glutathione peroxidase 4 (GPX4) and ferroptosis inhibitory protein 1 (FSP1) decreased in the KS group, indicating that the anti-ferroptosis system was damaged, but was repaired by the tFNA-PD complex ( Figure 4 J and 4K).

[0117] The activation of the renal osteogenic pathway is believed to be related to the formation of stones. Therefore, the present invention also found that osteopontin (OPN) and SMAD4 were significantly activated in the kidney stone group, while the tFNA-PD complex group was significantly inhibited (OPN: P < 0.05, P < 0.01) (SMAD4: P < 0.05, P < 0.05) ( Figure 4 J and 4L).

[0118] Inflammatory factor markers such as interleukin-1β, interleukin-6, and tumor necrosis factor-α were significantly activated by kidney stones, but significantly inhibited by tFNA-PD (IL-1β: P<0.05, P<0.05)(IL-6: P<0.001, P<0.001)(TNF-α: P<0.05, P<0.05)( Figure 4 J and 4M).

[0119] In summary, the results of in vitro and in vivo experiments indicate that ferroptosis is an important mechanism of kidney stone disease and tFNA-PD can inhibit the formation of kidney stones by inhibiting ferroptosis.

[0120] In summary, the present invention provides a tetrahedral framework nucleic acid-polydatin complex, in which polydatin is mutually bound to the tetrahedral framework nucleic acid through an embedded binding mode, and the tetrahedral framework nucleic acid is used as a carrier to improve the stability and release ability of polydatin, and maintains excellent ability to enter cells. At the same time, the complex of the present invention can inhibit oxidative stress and inflammation to inhibit ferroptosis. The complex of the present invention targets the renal tubules in vivo, and the tetrahedral framework nucleic acid and polydatin in the complex play a synergistic role in inhibiting the formation of calcium oxalate, thereby inhibiting kidney stone formation and renal tubular damage induced by calcium oxalate crystals. The complex of the present invention is a promising treatment method for preventing kidney stones, treating kidney stones, and preventing the recurrence of kidney stones, and provides a new treatment approach for kidney stone disease, with good application prospects.

Claims

1. A tetrahedral framework nucleic acid-polydatin complex, characterized in that: It is a complex formed by mixing tetrahedral framework nucleic acid and polydatin; the tetrahedral framework nucleic acid is self-assembled and synthesized by four single-stranded DNAs with nucleotide sequences such as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.

4.

2. The tetrahedral framework nucleic acid-polydatin complex according to claim 1, characterized in that: When the tetrahedral framework nucleic acid and polydatin are mixed, the molar ratio of the tetrahedral framework nucleic acid to polydatin is 1:100 to 1:

400.

3. The tetrahedral framework nucleic acid-polydatin complex according to claim 1 or 2, characterized in that: The method for synthesizing tetrahedral framework nucleic acid comprises the following steps: adding four single-stranded DNAs into TM buffer, maintaining at 90-99° C. for 10-20 min, and rapidly cooling to 0-4° C. and maintaining for more than 20 min to obtain the tetrahedral framework nucleic acid.

4. The tetrahedral framework nucleic acid-polydatin complex according to claim 3, characterized in that: The method for synthesizing tetrahedral framework nucleic acid comprises the following steps: adding four single-stranded DNAs into TM buffer, maintaining at 95° C. for 10 minutes, and rapidly cooling to 4° C. and maintaining for more than 20 minutes to obtain the tetrahedral framework nucleic acid.

5. The tetrahedral framework nucleic acid-polydatin complex according to claim 4, characterized in that: The four single-stranded DNAs are four single-stranded DNAs in equal molar ratios.

6. The method for preparing the tetrahedral framework nucleic acid-polydatin complex according to any one of claims 1 to 5, characterized in that: It includes the following steps: The tetrahedral framework nucleic acid and polydatin are added into a solvent, mixed and reacted, and then ultrafiltered to obtain the product.

7. The preparation method according to claim 6, characterized in that: The solvent is one or more of PBS and DMSO; And / or, the reaction temperature is 20-30°C; And / or, the reaction time is 4 to 6 hours; And / or, the ultrafiltration uses a 30 KDa molecular weight membrane.

8. Use of the tetrahedral framework nucleic acid-polydatin complex according to any one of claims 1 to 5 in the preparation of a drug for preventing kidney stones, treating kidney stones and / or inhibiting the recurrence of kidney stones.

9. The use according to claim 8, characterized in that: The drug is a drug that inhibits the formation of calcium oxalate.

10. The use according to claim 8, characterized in that: The drug is a drug that inhibits oxidative stress, inhibits inflammation and / or inhibits ferroptosis.

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