A tetrahedral framework nucleic acid-triptertium wilfordii complex and its preparation method and application

Abstract

The present invention discloses a tetrahedral framework nucleic acid-triptertium complex and its preparation method and application, and belongs to the field of medical technology. The complex is a complex formed by mixing tetrahedral framework nucleic acid and triptertium, and the molar ratio of the tetrahedral framework nucleic acid and triptertium is 1: (20-25). The tetrahedral framework nucleic acid-triptertium complex of the present invention realizes drug delivery to a variety of target tissues / organs (especially the key target organ-hypothalamus) at the same time, and it itself hardly produces side effects. The complex can significantly reduce the inflammatory level of adipose tissue in obese mice, significantly improve glucose tolerance and insulin resistance in obese mice, significantly increase the energy consumption and heat production level of obese mice, and significantly reduce the blood leptin level of obese mice, and has broad application prospects in the preparation of weight loss drugs and drugs for the prevention of diabetes.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a tetrahedral framework nucleic acid-triptertium wilfordii complex and a preparation method and application thereof. Background Art

[0002] Obesity is a chronic metabolic disease caused by fat accumulation. It is the result of the interaction between genetic factors and lifestyle factors (such as high-fat diet). In addition to abnormal lipid metabolism, obesity can increase the risk of diseases such as hypertension, diabetes, and arthritis. In particular, systemic inflammation caused by obesity can induce insulin resistance, which is an important risk factor for diabetes. Currently, the number of obese people in the world has exceeded 1 billion, which has become a thorny issue in the field of global public health.

[0003] Tripterygium wilfordii is an active extract of Tripterygium wilfordii from traditional Chinese medicine, with a molecular formula of C 29 H 38 O4, the structure is as follows.

[0004]

[0005] Studies have found that celastrol can increase leptin sensitivity by upregulating the lepr-stat3 signaling pathway in the hypothalamus, restore the normal leptin content in obese bodies, and exert its weight loss effect (J. Liu, J. Lee, MA Salazar Hernandez, et al. Treatment of obesity with celastrol [J]. Cell, 2015, 161 (5): 999-1011.). Subsequent studies have also found that celastrol has targets for multiple tissues and organs in obese bodies, such as promoting energy consumption of adipose / muscle tissues through the HSF1-Pgc1α pathway, inhibiting obesity-induced liver metabolic damage through the Sirt1 pathway, and inhibiting adipose stem cell differentiation through CEBPA. However, on the one hand, as a small molecule monomer drug, tripterygium wilfordii has low bioavailability, which limits its biological effectiveness; on the other hand, high concentrations of tripterygium wilfordii can significantly inhibit the proliferation of vascular endothelial cells and produce toxicity to the body; in addition, although some tripterygium wilfordii drug delivery systems have been reported, these delivery systems cannot promote the distribution of tripterygium wilfordii in the key target organ, the hypothalamus. Therefore, it is urgent to develop new preparations that can not only improve the activity of tripterygium wilfordii, but also reduce the toxicity of tripterygium wilfordii and promote its distribution in the hypothalamus. Summary of the invention

[0006] In order to solve the above problems, the purpose of the present invention is to provide a tripterygium wilfordii-DNA tetrahedral complex and a preparation method and application thereof.

[0007] Specifically, the present invention provides a tetrahedral framework nucleic acid-triptertium wilfordii complex, wherein the tetrahedral framework nucleic acid-triptertium wilfordii complex is a complex formed by mixing tetrahedral framework nucleic acid and triptertium wilfordii, and the molar ratio of the tetrahedral framework nucleic acid to triptertium wilfordii is 1:(20-25).

[0008] Furthermore, the molar ratio of the tetrahedral framework nucleic acid to tripterygium wilfordii is 1:22.

[0009] Furthermore, the tetrahedral framework nucleic acid is self-assembled from four DNA single strands, and the sequences of the four DNA single strands are shown in SEQ ID NO.1-4 respectively.

[0010] Furthermore, the method for preparing the tetrahedral framework nucleic acid comprises the following steps: adding four DNA single strands into a solution, maintaining the solution at 95° C. for 10 minutes, cooling the solution to 4° C. and maintaining the solution for more than 20 minutes.

[0011] The present invention also provides a method for preparing the tetrahedral framework nucleic acid-triptertium wilfordii complex, which comprises the following steps: adding the tetrahedral framework nucleic acid and triptertium wilfordii into a solvent for mixed reaction to obtain the complex.

[0012] Furthermore, the reaction temperature is 2-6°C and the reaction time is more than 10 hours.

[0013] The present invention also provides a pharmaceutical composition, which is a preparation prepared by taking the above tetrahedral framework nucleic acid-triptertium wilfordii complex as an active ingredient and adding pharmaceutically acceptable excipients.

[0014] The present invention also provides the use of the tetrahedral framework nucleic acid-triptertium wilfordii complex in the preparation of weight loss drugs.

[0015] The present invention also provides the use of the tetrahedral framework nucleic acid-triptertium wilfordii complex in preparing a drug for preventing diabetes.

[0016] Furthermore, the drug is a drug that reduces the inflammatory level of adipose tissue, improves glucose tolerance, improves insulin resistance, increases energy expenditure and heat production levels, and / or reduces blood leptin levels.

[0017] The present invention verifies the drug binding mechanism and efficiency of the tetrahedral framework nucleic acid-triptertium wilfordii complex, the therapeutic effect on obesity and related complications, and the biological mechanism of action (including the mechanism of action on two key target organs / tissues (fat, hypothalamus)).

[0018] The tetrahedral framework nucleic acid-tripterin complex of the present invention can not only significantly reduce the toxicity of tripterin to vascular endothelial cells and improve its biocompatibility, but also can travel throughout the body as a "nano-patrol" and simultaneously achieve drug delivery to a variety of target tissues / organs (especially the key target organ - the hypothalamus).

[0019] The tetrahedral framework nucleic acid-triptertium wilfordii complex of the present invention can significantly reduce the inflammatory level of adipose tissue in obese mice, significantly improve glucose tolerance and insulin resistance in obese mice, significantly increase energy consumption and heat production levels in obese mice, and significantly reduce blood leptin levels in obese mice, and has broad application prospects in the preparation of weight loss drugs and drugs for preventing diabetes.

[0020] 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.

[0021] 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

[0022] Figure 1 :Synthesis and characterization of tFNA and t-Cel. (a) Schematic diagram of the synthesis process of tFNA and t-Cel. (b) Transmission electron microscopy observation of tFNA. (c) Atomic force microscopy observation of tFNA. (d) Concentration-absorbance standard curve of tripterygium wilfordii and loading efficiency of tripterygium wilfordii on tFNA. (e) Particle size of tFNA and t-Cel. (f) ζ potential of tFNA and t-Cel. (g) PAGE analysis of tFNA and t-Cel. (h) Competitive binding of Gel-Red / Hoechst33342 with t-Cel. (i) Stability under different conditions evaluated by PAGE (red: t-Cel, blue: tFNA).

[0023] Figure 2 : Cytotoxicity of different concentrations of Cel, tFNA and t-Cel (tFNA:Cel molar ratio is 1:22) on HUVEC.

[0024] Figure 3:Biodistribution results. (a) Biodistribution of tFNA and t-Cel (tFNA:Cel molar ratio of 1:22) at 1 min, 30 min, 60 min, and 90 min after intraperitoneal injection; (bd) Biodistribution of t-Cel (tFNA:Cel molar ratio of 1:50, 1:100, and 1:200) at 60 min after intraperitoneal injection.

[0025] Figure 4 .Anti-obesity effect analysis. (a) Schematic diagram of the establishment of the obesity model and the treatment regimen. (b) Body weight and body weight change ratio recorded during the 20-day treatment period (n=5). (c) Representative images of mice in different treatment groups on day 20. (d) Epididymal fat mass recorded on day 20 (n=5). (e) H&E staining and Oil Red staining were used to observe the cell diameter of epididymal adipocytes. (f) Oil Red staining and Sudan III staining were used to observe fat accumulation in the liver. Compared with the HFD group, ***, p<0.0001; compared with the t-Cel group, &&&, p<0.001, &&&&, p<0.0001. Figure 5 .Anti-inflammatory effect and glucose / insulin tolerance analysis. (a) Confocal observation of M1 / M2 polarization of macrophages in epididymal adipose tissue. (b) Expression levels of pro-inflammatory and anti-inflammatory genes detected by RT-PCR (n=3). (c) H&E staining observation of heart, lung and kidney. (d) IPGTT and corresponding area under the curve analysis (n=5). (e) IPITT and corresponding area under the curve analysis (n=5). Compared with the HFD group, *, p<0.05, ***, p<0.001, ***, p<0.01; compared with the t-Cel group, &, p<0.05, &&, p<0.01, &&&, p<0.01. Figure 6Energy expenditure and leptin sensitivity assessment. (a) Schematic diagram of the role of t-Cel-mediated energy expenditure in anti-obesity. (b) Measurement of oxygen consumption levels at 24°C (n=5). (c) Determination of 4-h cold tolerance at 4°C (n=5). (d) RT-PCR detection of the expression levels of energy expenditure-related genes (n=3). (e) Schematic diagram of the role of t-Cel regulation of appetite and leptin sensitivity in anti-obesity. (f) Average food intake recorded during the 20-day treatment period (n=5). (g) Plasma leptin concentrations during the 20-day treatment period were measured using ELISA (n=5). (h) Western blot images of ATF6, STAT3, and p-STAT3. (i) Semi-quantitative analysis of the relative levels of ATF6, p-STAT3, and the ratio of p-STAT3 / STAT3 (n=3). Compared with the HFD group, *, p<0.05, **, p<0.01, ***, p<0.001, ***, p<0.01; compared with the t-Cel group, &, p<0.05, &&, p<0.01, &&&, p<0.001, &&&. DETAILED DESCRIPTION

[0026] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0027] Example 1: Preparation of tetrahedral framework nucleic acid-tripterygium wilfordii complex

[0028] 1. Preparation of Tetrahedral Framework Nucleic Acids

[0029] Four sets of DNA single strands (S1, S2, S3, S4; sequences are shown in Table 1) with equal molar concentrations were dissolved simultaneously in TM buffer containing 10 mmol / L Tris-HCl and 50 mmol / L MgCl2·6H2O (pH ≈ 8.0). The mixture was heated at 95°C for 10 minutes and then kept at 4°C for 20 minutes to form tetrahedral framework nucleic acids (tFNA).

[0030] 2. Preparation of Tetrahedral Framework Nucleic Acid-Tripterine Complex

[0031] Subsequently, the TM buffer of tFNA was mixed with the DMSO solution of Cel, and the tFNA concentration in the mixed liquid was controlled to be 1 μM and the Cel concentration to be 22 μM (tFNA:Cel molar ratio was 1:22), and incubated at 4°C for 12 hours to form a tetrahedral framework nucleic acid-Celanoside complex (t-Cel).

[0032] Schematic diagram of the synthesis process of tFNA and t-Cel Figure 1 As shown in a.

[0033] Table 1. Sequences of the four DNA strands

[0034]

[0035] Example 2: Preparation of tetrahedral framework nucleic acid-tripterygium wilfordii complex

[0036] 1. Preparation of Tetrahedral Framework Nucleic Acids

[0037] Same as Example 1.

[0038] 2. Preparation of Tetrahedral Framework Nucleic Acid-Tripterine Complex

[0039] Subsequently, the TM buffer of tFNA was mixed with the DMSO solution of Cel, and the tFNA concentration in the mixed liquid was controlled to be 1 μM and the Cel concentration to be 20 μM (tFNA:Cel molar ratio was 1:20), and incubated at 4°C for 12 hours to form a tetrahedral framework nucleic acid-Celanoside complex (t-Cel).

[0040] Example 3: Preparation of tetrahedral framework nucleic acid-tripterygium wilfordii complex

[0041] 1. Preparation of Tetrahedral Framework Nucleic Acids

[0042] Same as Example 1.

[0043] 2. Preparation of Tetrahedral Framework Nucleic Acid-Tripterine Complex

[0044] Subsequently, the TM buffer of tFNA was mixed with the DMSO solution of Cel, and the tFNA concentration in the mixed liquid was controlled to be 1 μM and the Cel concentration was 25 μM (tFNA:Cel molar ratio was 1:25), and incubated at 4°C for 12 hours to form a tetrahedral framework nucleic acid-Celanoside complex (t-Cel).

[0045] The following experimental examples demonstrate the beneficial effects of the present invention. In the following experimental examples, all experiments were performed three times or more, and all quantitative data were expressed as mean ± standard deviation (mean ± SD). Statistical description and analysis were based on GraphPad Prism version 8.0.2. All comparisons between different groups were statistically evaluated using one-way analysis of variance (ANOVA) and Tukey's post hoc test. P values ​​less than 0.05 (p < 0.05) were considered statistically significant.

[0046] Experimental Example 1: Synthesis and Characterization

[0047] 1. Experimental Methods

[0048] To verify the successful synthesis of tFNA, the nanomorphology of the product (100 nM) was observed using transmission electron microscopy.

[0049] The loading efficiency of t-Cel was analyzed using an ELISA instrument. First, the absorbance of the Cel solution (gradient concentration: 20, 40, 60, 80, 100, 120, 140 and 160 μM) at a wavelength of 432 nm was recorded to obtain a concentration-absorbance standard curve. Then, a t-Cel solution containing 1 μM tFNA was centrifuged at 4000 rpm in a 3kD ultrafiltration membrane for 10 minutes (tFNA: Cel molar ratio was 1:100, 1:200, 1:300, 1:400, 1:500). The concentration of unloaded Cel was obtained according to the standard curve. The loading efficiency of Cel on tFNA was calculated according to the equation Δ = "C1-C2" / "C1" × 100% (Cl and C2 represent the total concentration and empty concentration of Cel, respectively).

[0050] In addition, the particle size and zeta potential of tRNA and t-Cel (both containing 100 nM tFNA) were measured using a laser particle size analyzer based on dynamic light scattering and electrophoretic light scattering, respectively. 8% PAGE using Gel-Red as a dye was used to verify the structural integrity of tFNA (1 μM) after binding to Cel.

[0051] The loading mechanism of Cel on tFNA was analyzed using a competitive binding assay. Gel Red was incubated with t-Cel solution (1 μM tFNA, tFNA:Cel molar ratio of 1:0, 1:100, 1:200, 1:300, 1:400, 1:500) at 4°C for 15 min and then analyzed using a microplate reader at a wavelength of 600 nm. Hoechst 33342 was incubated in t-Cel solution in the same manner and then analyzed using a microplate reader at a wavelength of 461 nm.

[0052] 2. Experimental results

[0053] tFNA is roughly triangular in shape under transmission electron microscopy ( Figure 1 b), the three-dimensional framework structure can be seen under atomic force microscopy ( Figure 1 c). Subsequently, the tetrahedral framework nucleic acid and tripterygium wilfordii were incubated and combined at different ratios (1:100-1:500) (4°C, 12 hours). The concentration-absorbance curve of tripterygium wilfordii was determined by spectrophotometry ( Figure 1 d), on this basis, the binding efficiency of tripterygium wilfordii and tetrahedral framework nucleic acid was determined by ultrafiltration method, which was greater than 80% at the selected loading ratio (1:100-1:500), which is better than the binding ability of tetrahedral framework nucleic acid with other types of small molecules (such as curcumin and temozolomide). In addition, the laser particle size analyzer determined that with the increase of tripterygium wilfordii loading, the particle size of the complex increased from about 10nm to about 60nm ( Figure 1e), and since celastrol is charge neutral, the electrical conductivity is still between -5mV and -10mV ( Figure 1 f), confirming the successful synthesis of the complex.

[0054] Furthermore, PAGE electrophoresis and Gel-Red staining showed ( Figure 1 g), after the tetrahedral framework nucleic acid was loaded with tripterygium wilfordii (1:100-1:500), its band position and brightness did not change significantly, which suggests that the loading of tripterygium wilfordii did not interfere with the original framework structure of the tetrahedral framework nucleic acid. More importantly, this suggests that tripterygium wilfordii and Gel-Red do not have the same DNA binding site. Consistently, the binding efficiency of Gel-Red and the complex (1:100-1:500) detected by the microplate reader was close to 100% ( Figure 1 h). In contrast, the binding efficiency of Hoechst 33342 to the complex (1:0-1:500) gradually decreased from 100% to about 10% ( Figure 1 h), indicating that tripterygium wilfordii and Hoechst 33342 have competitive DNA binding sites, namely, binding to the minor groove of the DNA double helix.

[0055] Experimental Example 2: Stability Evaluation

[0056] 1. Experimental Methods

[0057] The tFNA solution (1 μM) and t-Cel solution (1 μM tFNA, tFNA:Cel molar ratio of 1:22) were incubated at 4°C for different days (0 days, 3 days, 6 days, 9 days) and then analyzed by 8% PAGE to evaluate storage stability. In addition, the tFNA solution (1 μM) and t-Cel solution (1 μM, tFNA:Cel molar ratio of 1:22) were incubated with (1) different concentrations (0%, 2%, 4%, 6%) of fetal bovine serum at 37°C for 6 h and then analyzed by 8% PAGE to evaluate plasma stability; (2) different concentrations (0‰, 0.5‰, 1.5‰, 2.5‰) of DNase I were incubated at 37.4°C for 1 hour and then analyzed by 8% PAGE to evaluate enzyme stability.

[0058] 2. Experimental results

[0059] This experiment used PAGE to verify the stability of the product under different environments ( Figure 1i). After storage at 4°C for 0, 3, 6, and 9 days, the band positions and brightness of the tetrahedral framework nucleic acid and tetrahedral framework nucleic acid-tripterygium wilfordii complex remained almost unchanged, confirming good storage stability. After incubation with serum at 37°C for 6 hours, with the increase of serum concentration (0%, 2%, 4%, 6%), the band brightness of the tetrahedral framework nucleic acid and tetrahedral framework nucleic acid-tripterygium wilfordii complex decreased but did not completely disappear, suggesting that the two have certain serum stability and a more suitable degradation rate, which is conducive to drug delivery and release via blood administration. Similarly, after incubation with DNase I enzyme at 37°C for 6 hours, with the increase of enzyme concentration (0‰, 0.5‰, 1.5‰, 2.5‰), the band brightness of the tetrahedral framework nucleic acid and tetrahedral framework nucleic acid-tripterygium wilfordii complex decreased but did not completely disappear, suggesting that the two have certain enzyme stability, which is conducive to drug uptake under physiological conditions.

[0060] Experimental Example 3: Biocompatibility Experiment

[0061] 1. Experimental Methods

[0062] Human umbilical vein endothelial cells (HUVEC) were cultured in DMEM / F12 medium (Gibco, Thermo Fisher Scientific, USA) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. 3 / well) were cultured overnight in a 96-well plate and then incubated with tFNA, t-Cel (tFNA:Cel molar ratio of 1:22) and Cel (consistent with t-Cel concentration). The concentrations of tFNA in tFNA and t-Cel were set to 5, 10, 20 and 40 nM, and 1% fetal bovine serum was added. After 24 hours, the culture medium was discarded and treated with CCK-8 reaction solution (Biofive, Guangzhou, China) at 37°C for 30-40 minutes. Finally, the OD450 value was measured using a microplate reader.

[0063] 2. Experimental results

[0064] The cytotoxicity results of different concentrations of Cel, tFNA and t-Cel (tFNA:Cel molar ratio of 1:22) on human umbilical vein endothelial cells (HUVEC) are shown in Figure 2. Figure 2 As shown. It can be seen that Cel exhibits obvious HUVEC cytotoxicity, and high concentrations of Cel can significantly inhibit the proliferation of HUVEC cells and produce toxicity to the body. Compared with Cel, the cytotoxicity of tFNA is reduced, and t-Cel has basically no cytotoxicity and will not inhibit the proliferation of HUVEC cells.

[0065] The above experimental results show that the tetrahedral framework nucleic acid-triptertium wilfordii complex of the present invention can significantly reduce the toxicity of triptertium wilfordii to vascular endothelial cells and improve its biocompatibility.

[0066] Experimental Example 4: Animal Experiment

[0067] 1. Experimental Methods

[0068] A t-Cel solution (1 μM tFNA, tFNA:Cel molar ratio of 1:22) was prepared according to the method of Example 1. Referring to the preparation method of Example 1, the concentration of Cel was changed to prepare t-Cel solutions (1 μM tFNA, tFNA:Cel molar ratios of 1:50, 1:100, and 1:200, respectively) as control samples. tFNA was labeled with Cy-5 for fluorescence observation.

[0069] 1.1 Biodistribution in obese mice

[0070] C57BL / 6 mice (male, body weight 20±2g, n=25) were fed a high-fat diet (60% calories from fat, 20% calories from protein, 20% calories from carbohydrates) for 8 weeks to become obese mice. Obese C57BL / 6 mice were shaved for optimal in vivo imaging and anesthetized immediately before drug injection. Obese mice were randomly divided into five groups (n = 5 in each group), and the mice were intraperitoneally injected with: (1) tFNA solution (1 μM, 100 μL), (2) t-Cel solution (1 μM tFNA, tFNA:Cel molar ratio of 1:22), (3) t-Cel solution (1 μM tFNA, tFNA:Cel molar ratio of 1:50), (4) t-Cel solution (1 μM tFNA, tFNA:Cel molar ratio of 1:100), (5) t-Cel solution (1 μM tFNA, tFNA:Cel molar ratio of 1:200). tFNA was labeled with Cy-5 for fluorescence observation. The mice were observed at 1 minute, 30 minutes, 60 minutes and 90 minutes using an in vivo imaging system.

[0071] 1.2 Establishment of obesity model and treatment plan

[0072] C57BL / 6 mice (male, body weight 20±2g, n=20) were fed with a high-fat diet (60% calories from fat, 20% calories from protein, 20% calories from carbohydrates) for 8 weeks to become obese mice. Another group of C57BL / 6 mice (male, body weight 20±2g, n=5) were fed with a normal diet for 8 weeks as a negative control (Con group).

[0073] Obese mice were randomly divided into four groups (n = 5 per group) and injected intraperitoneally once every two days with (1) normal saline (HFD group), (2) tFNA (1 μM, 200 μL), (3) Cel (22 μM, 200 μL), and (4) t-Cel (1 μM tFNA, tFNA:Cel molar ratio of 1:22, 200 μL). After 20 days of treatment, all mice were sacrificed for further analysis. The in vivo experimental protocol was approved by the local ethics committee (20230302092).

[0074] 1.3 Analysis of anti-obesity effects

[0075] During the 20-day treatment period, the body weight (g) of all experimental mice was recorded every two days (n=5). Representative images of the degree of obesity of mice were recorded on the 20th day. After the mice were sacrificed, the epididymal adipose tissue was collected and weighed (n=5), fixed with 4% paraformaldehyde, and then H&E staining and oil red staining were performed to observe the diameter of adipocytes. At the same time, the liver was collected and stained with oil red and Sudan III to observe fat accumulation. The heart, lungs and kidneys were collected and H&E staining was performed for biosafety observation.

[0076] 1.4 Analysis of anti-inflammatory effects

[0077] Epididymal adipose tissue collected after mouse sacrifice was stained with DAPI, CD68 and iNOS to show M1 polarization of macrophages, or with DAPI, CD68 and CD206 to show M2 polarization of macrophages (n=3). In detail, fixed samples were treated with 0.5% Triton X-100 for 10 minutes, blocked with 5% goat serum for 1 hour, and incubated with primary antibodies (anti-CD68, anti-iNOS or anti-CD206) at 4°C overnight. The next day, the samples were rinsed three times with PBS and then incubated with the corresponding secondary antibodies (1:500) for 1 hour. The secondary antibody for CD68 was labeled with Cy-3, and the secondary antibodies for iNOS and CD206 were both labeled with FITC. At the same time, the nuclei were stained with DAPI for 15 minutes.

[0078] For real-time polymerase chain reaction, total RNA was dissociated by TRIzol and purified by RNA purification kit. After obtaining cDNA, target mRNAs were detected in RT-PCR analysis system, including TNF-α and IL-6 for M1 polarization, and TGF-β and Arg-1 for M2 polarization. GAPDH was used as a control.

[0079] 1.5 Glucose / insulin tolerance test

[0080] Before the mice were sacrificed on the 20th day, intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) (n=5) were performed. IPGTT was performed on mice injected with glucose (2g / kg-1) intraperitoneally after fasting for 12 hours. IPITT was performed on mice injected with insulin (0.75μ / kg-1) intraperitoneally after fasting for 4 hours. Blood glucose levels were detected from tail vein blood at 0 minutes, 30 minutes, 60 minutes, 90 minutes and 120 minutes after injection. The area under the curve (AUC) was determined by GraphPad Prism version 8.0.2.

[0081] 1.6 Energy consumption assessment

[0082] Energy expenditure levels were assessed on day 20 before mice were sacrificed.

[0083] For the measurement of oxygen consumption, all mice were housed in cages at 24°C with access to fresh food and water (n=5). After 48 hours of acclimatization, oxygen consumption levels were recorded using a 12-chamber CLMS in a standard light-dark cycle.

[0084] To test cold tolerance, all mice were individually housed in cages at 4°C with access to fresh water (n=5). Core body temperature was monitored using a thermometer after 4 hours of cold exposure.

[0085] To detect the expression levels of related genes, RT-PCR was used as described above (n=3). Target mRNAs included Pgc1α, CytC and Cox4β (mitochondrial function markers), Ucp1 and Prdm16 (fat browning markers), and Mcad and Cpt1α (β-oxidation markers). GAPDH was used as a control.

[0086] 1.7 Analysis of appetite and leptin resistance

[0087] For analysis of appetite, the average food intake (g / mouse / day) over the past three days was recorded on days 5, 10, 15, and 20 (n=5); plasma leptin concentrations were measured from tail vein blood on days 0, 5, 10, 15, and 20 (n=5) based on enzyme-linked immunosorbent assay.

[0088] 1.8 Western blotting

[0089] Western blotting was used to determine the expression levels of ATF6 and p-STAT3 (n=3), which reflect the levels of ER stress and leptin resistance in the hypothalamus collected on the 20th day after the mice were sacrificed. All protein samples were purified using cell protein extraction reagent, added to 5× loading buffer, mixed appropriately, placed in boiling water for 5 minutes, and stored at 20°C. The protein samples were transferred to polyvinylidene difluoride membranes blocked with 5% skim milk for 1 hour. The membranes were then incubated with primary antibodies against ATF6, anti-STAT3, or anti-p-STAT3 (1:1000) at 4°C overnight. The next day, the protein samples were incubated with the corresponding secondary antibodies for 60 minutes. Finally, the protein bands on the membrane were analyzed using an enhanced chemiluminescence detection system. β-actin was used as a control.

[0090] 2. Experimental results

[0091] 2.1 Biodistribution in obese mice

[0092] The biodistribution of tripterygium wilfordii in multiple target tissues / organs is the key to the simultaneous activation of different obesity-related pathways by tripterygium wilfordii, and the hypothalamus is a key target organ for the activation of obesity pathways. In vivo imaging shows ( Figure 3 a), after intraperitoneal injection of Cy5 fluorescently labeled tetrahedral framework nucleic acid into mice, it gradually distributed to the head, body and limbs within 90 minutes; the biological distribution of Cy5 fluorescently labeled tetrahedral framework nucleic acid-tripterygium wilfordii complex (tFNA:Cel molar ratio of 1:22) was basically consistent with it. Figure 3 a and Figure 3 bd It can be seen that the tetrahedral framework nucleic acid-triptertium wilfordii complex labeled with Cy5 fluorescence (tFNA:Cel molar ratio of 1:22) was widely distributed in the head, body and limbs of mice 60 minutes after intraperitoneal injection; however, the tetrahedral framework nucleic acid-triptertium wilfordii complex labeled with Cy5 fluorescence (tFNA:Cel molar ratio of 1:50, 1:100, 1:200) was almost not distributed in the brain of mice 60 minutes after intraperitoneal injection. This indicates that the tetrahedral framework nucleic acid-triptertium wilfordii complex prepared by controlling the tFNA:Cel molar ratio of 1:22 is more conducive to systemic distribution in obese mice, and is more conducive to the simultaneous activation of different obesity-related pathways (especially the key target organ-hypothalamus), exerting a significantly improved weight loss effect.

[0093] 2.2 Evaluation of weight loss effect

[0094] Obesity models were established in mice by inducing a high-fat diet, and the weight loss effect was evaluated after programmed drug administration (schematic diagram as shown in Figure 4a). The dosage and frequency of administration in this experiment are lower than those in the literature (J. Liu, J. Lee, MA Salazar Hernandez, et al. Treatment of obesity with celastrol [J]. Cell, 2015, 161 (5): 999-1011.), in order to reduce the potential side effects of celastrol and highlight the improvement of the compound on weight loss. Figure 4 As shown in b and c, after 20 days of different drug treatments, the weight of mice in the Con group, HFD group, and tFNA group continued to increase, while the weight of the Cel group began to decrease, and the weight of the t-Cel group decreased significantly by more than 10%. Figure 4 d), the epididymal fat mass of HFD group mice was significantly increased compared with that of Con group mice, which was not reduced by tFNA treatment, reduced by Cel treatment to some extent, and significantly reduced by t-Cel treatment, suggesting that the weight loss is closely related to the reduction of adipose tissue, and is less likely to be caused by drug toxicity.

[0095] Subsequently, epididymal adipose tissue and liver were histologically observed. HE staining and oil red staining showed (e.g. Figure 4 e), the diameter of epididymal adipocytes in the HFD and tFNA groups was significantly increased compared with the Con group, the diameter of epididymal adipocytes in the Cel group was reduced to a certain extent, and the diameter of epididymal adipocytes in the t-Cel group was significantly reduced, that is, the mass of stored lipids was significantly reduced. Oil red and Sudan III staining showed (as shown in Figure 4 f), the amount of fat deposits in the liver of the HFD and tFNA groups increased significantly, the amount of fat deposits in the liver of the Cel group decreased to a certain extent, and the amount of fat deposits in the liver of the t-Cel group decreased significantly, which is beneficial to maintaining liver health and inhibiting systemic obesity and complications.

[0096] 2.3 Analysis of anti-inflammatory effects

[0097] Inflammation level is an important marker of obesity progression. Adipose tissue inflammation further triggers systemic inflammation and glucose / insulin resistance, increasing the risk of systemic diseases such as diabetes. This experiment focused on the polarization of macrophages M1 / M2 in adipose tissue to reflect the level of inflammation. Confocal observation showed (such as Figure 5a), compared with the Con group, the expression of M1 marker iNOS in the HFD group and tFNA group increased, while the expression of M2 marker CD206 decreased. The M1 ratio of the Cel group decreased to a certain extent, and the M2 ratio increased to a certain extent; while the M1 ratio of the t-Cel group decreased significantly, and the M2 ratio increased significantly, indicating that the inflammatory level of adipose tissue was significantly reduced. q-PCR was further used to detect the expression levels of pro-inflammatory factor-related genes (TNF-α and IL-6) and anti-inflammatory factor-related genes (TGF-β and Arg-1) (e.g. Figure 5 b), the trend is consistent with the confocal results. In addition, HE staining showed that no obvious pathological changes were found in the heart, lung, and kidney of each treatment group (such as Figure 5 c), indicating that the drug has good biosafety.

[0098] 2.4 Glucose / insulin tolerance test

[0099] Furthermore, IPGTT was used to investigate the glucose tolerance level of each group of mice (e.g. Figure 5 d). Compared with the Con group, the HFD group and the tFNA group had a greater increase in blood glucose concentration after glucose injection (larger AUC area), which indicates a decrease in glucose tolerance. The increase in blood glucose concentration in the Cel group after glucose injection was reduced to a certain extent; while the increase in blood glucose concentration in the t-Cel group after glucose injection was significantly reduced and close to that of the Con group, indicating a significant improvement in glucose tolerance. Similarly, the insulin tolerance level of each group of mice was explored by IPITT (such as Figure 5 e). Compared with the Con group, the HFD group and the tFNA group showed a smaller decrease in blood glucose concentration after insulin injection (smaller AUC area), which indicates the occurrence of insulin resistance. The blood glucose concentration of the Cel group decreased to a certain extent after insulin injection; while the blood glucose concentration of the t-Cel group decreased significantly after insulin injection and was close to that of the Con group, which means that insulin resistance was significantly improved.

[0100] 2.5 Regulation of energy expenditure and leptin sensitivity

[0101] As mentioned above, t-Cel has significant weight loss and anti-inflammatory effects, and has a wide biodistribution in obese bodies. As a "nano-patrolman", t-Cel acts on multiple target tissues / organs in the body.

[0102] Obese bodies have energy metabolism disorders, such as mitochondrial function and fatty acid metabolism. Increasing energy consumption is a necessary mechanism for weight loss therapy. Oxygen consumption experiments reveal (e.g. Figure 6 b), compared with other groups, the respiratory oxygen consumption of the Cel group increased to a certain extent, while the respiratory oxygen consumption of the t-Cel group increased significantly. Figure 6c), compared with the Con group, the body temperature maintenance ability of the HFD group and the tFNA group decreased, the body temperature maintenance ability of the Cel group increased to a certain extent, and the body temperature maintenance ability of the t-Cel group increased significantly, approaching the Con group. The above suggests that the t-Cel group significantly increased the energy consumption and heat production level of obese mice (see the schematic diagram Figure 6 a).

[0103] Furthermore, the expression of energy expenditure-related genes in epididymal fat was detected by q-PCR (e.g. Figure 6 d), including pgc1α, mitochondrial function markers CytC and Cox4β, browning markers Ucp1 and Prdm16, and β-oxidation markers Mcad and Cpt1α. Compared with the Con group, the expression of energy expenditure-related genes in the HFD group and tFNA group generally decreased, the expression level of related genes in the Cel group increased to a certain extent, and the expression level of related genes in the t-Cel group increased significantly, approaching that in the Con group, which verified that t-Cel had a significant pharmacological effect on epididymal adipose tissue.

[0104] The hypothalamus of obese bodies is another important target. It has leptin resistance, which makes it difficult for leptin to regulate appetite. Improving hypothalamic leptin sensitivity is the primary mechanism by which tripterygium wilfordii exerts its weight-loss effect. This experiment regularly recorded the changes in the food intake of mice after administration, such as Figure 6 f. The food intake of the Con group, HDF group, and tFNA group hardly changed over time, and the food intake of the HFD group and tFNA group was lower than that of the Con group. This was due to the compensatory reduction in food intake caused by the increased fat content of the food. In contrast, the food intake of the Cel group decreased to a certain extent over time, while the t-Cel group decreased significantly, decreasing to about half of the original food intake. In addition, ELISA was used to regularly detect the blood leptin level of mice, as shown in Figure 5. Figure 6 g. The blood leptin levels of the Con group, HDF group, and tFNA group hardly changed over time, and the blood leptin levels of the HFD group and tFNA group were significantly higher than those of the Con group, which was due to excessive accumulation of leptin caused by reduced leptin sensitivity. The blood leptin level of the Cel group decreased to a certain extent, while the blood leptin level of the t-Cel group decreased significantly, decreasing to about 1 / 5 of that of the HFD group.

[0105] Furthermore, the expression of endoplasmic reticulum stress and leptin sensitivity-related proteins in the hypothalamus was detected by WB, such as Figure 6h,i. Endoplasmic reticulum stress is considered to be one of the main reasons for reducing leptin sensitivity, and ATF6 is one of the main markers of endoplasmic reticulum stress. It binds to molecular chaperones when the endoplasmic reticulum is in steady state, and is released when the endoplasmic reticulum is under stress, and can be detected. The expression levels of ATF6 in the HFD group and tFNA group were significantly higher than those in the Con group. The expression levels of ATF6 in the Cel group decreased to a certain extent, while the expression levels of ATF6 in the t-Cel group decreased significantly, confirming that it can significantly alleviate endoplasmic reticulum stress. In addition, the LepR-STAT3 signaling pathway is necessary for leptin to exert its effects. The STAT3 phosphorylation levels in the HFD group and tFNA group were significantly lower than those in the Con group. The STAT3 phosphorylation levels in the Cel group increased to a certain extent, while the STAT3 phosphorylation levels in the t-Cel group increased significantly, confirming the activation of the LepR-STAT3 signaling pathway and the normalization of leptin function. In summary, this experiment verified that t-Cel produced significant pharmacological effects on the hypothalamus (see schematic diagram Figure 6 e).

[0106] In summary, the present invention provides a tetrahedral framework nucleic acid-tripterin complex and its preparation method and application. The present invention verifies the drug binding mechanism and efficiency of the tetrahedral framework nucleic acid-tripterin complex, the therapeutic effect on obesity and related complications, and the biological mechanism of action. The complex travels throughout the body as a "nano-patrolman" and simultaneously delivers drugs to a variety of target tissues / organs (especially the key target organ - the hypothalamus), and it itself has almost no side effects, and has broad application prospects in the preparation of weight loss drugs and drugs for preventing diabetes.

Claims

1. A tetrahedral framework nucleic acid-tripterygium wilfordii complex, characterized in that: The tetrahedral framework nucleic acid-triptertium wilfordii complex is a complex formed by mixing tetrahedral framework nucleic acid and triptertium wilfordii, and the molar ratio of the tetrahedral framework nucleic acid to triptertium wilfordii is 1:22; The tetrahedral framework nucleic acid is self-assembled from four DNA single strands, and the sequences of the four DNA single strands are shown in SEQ ID NOs. 1-4 respectively.

2. The tetrahedral framework nucleic acid-triptertium wilfordii complex according to claim 1, characterized in that: The method for preparing the tetrahedral framework nucleic acid comprises the following steps: adding four DNA single strands into a solution, maintaining the solution at 95° C. for 10 minutes, cooling the solution to 4° C. and maintaining the solution for more than 20 minutes.

3. A method for preparing the tetrahedral framework nucleic acid-triptertium wilfordii complex according to any one of claims 1 to 2, characterized in that: The method comprises the following steps: adding tetrahedral framework nucleic acid and tripterygium wilfordii into a solvent and mixing them for reaction to obtain the product.

4. The method according to claim 3, characterized in that The reaction temperature is 2-6°C and the reaction time is more than 10 hours.

5. A pharmaceutical composition, characterized in that The pharmaceutical composition is a preparation prepared by using the tetrahedral framework nucleic acid-triptertium wilfordii complex as an active ingredient and adding pharmaceutically acceptable excipients.

6. Use of the tetrahedral framework nucleic acid-triptertium wilfordii complex according to any one of claims 1 to 2 in the preparation of weight loss drugs.

7. Use of the tetrahedral framework nucleic acid-triptertium wilfordii complex according to any one of claims 1 to 2 in the preparation of a drug for preventing diabetes.

8. The use according to claim 6 or 7, characterized in that The drug is a drug that reduces the inflammatory level of adipose tissue, improves glucose tolerance, improves insulin resistance, increases energy expenditure and heat production levels and / or reduces blood leptin levels.

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