Ginsenoside Rg1 delivery nanometer material based on tetrahedral DNA and application of ginsenoside Rg1 delivery nanometer material in preparation of anti-myocardial ischemia drugs

Abstract

The invention discloses a ginsenoside Rg1 delivery nano-material based on tetrahedral DNA (deoxyribonucleic acid) and application of the ginsenoside Rg1 delivery nano-material in preparation of anti-myocardial ischemia drugs, and belongs to the field of nano-materials. Based on a programmable oligonucleotide self-assembly technology, a tetrahedral DNA nano-structure (TDN) is constructed and formed, accurate connection and spatial modification are realized by introducing an N-containing modified CREKA myocardial targeting peptide fragment, and ginsenoside Rg1 is further loaded to form a composite nano-drug system Rg1 (at) pTDN. The materials are all biocompatible components, and have good biological safety and tissue tolerance in vivo. The Rg1 (at) pTDN has good myocardial tissue targeting ability and cellular uptake efficiency, can realize stable, targeted and efficient delivery of drugs, is suitable for cardiovascular diseases such as MIRI, AMI and the like, and has clinical transformation and industrialization potential.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of nanomaterials, and particularly relates to a ginsenoside Rg1 delivery nanomaterial based on tetrahedral DNA and application thereof in preparation of an anti-myocardial ischemia drug. BACKGROUND

[0002] Myocardial ischemia-reperfusion injury (MIRI) is a major difficulty in the treatment of acute coronary heart disease, and its pathological mechanism involves oxidative stress, endoplasmic reticulum stress and activation of multiple cell death pathways. At present, there is still a lack of intervention strategies with strong targeting and small side effects.

[0003] Ginsenoside Rg1 is a natural active ingredient extracted from ginseng, which has good antioxidant and anti-apoptotic properties and can reduce myocardial cell damage caused by MIRI. Unfortunately, Rg1 has poor stability and low bioavailability in vivo, which limits its therapeutic potential.

[0004] DNA nanostructures, especially tetrahedral DNA nanostructures (TDNs), have shown great application prospects in the field of drug delivery in recent years due to their precise and controllable spatial conformation, excellent cell uptake capacity and programmable modification properties.

[0005] In order to improve the effect of Rg1 on anti-myocardial ischemia injury, the present application is proposed. SUMMARY

[0006] The present application aims to overcome the shortcomings of the prior art, and the first object is to provide a ginsenoside Rg1 delivery nanomaterial based on tetrahedral DNA, and the second object is to provide application of the ginsenoside Rg1 delivery nanomaterial based on tetrahedral DNA in preparation of an anti-myocardial ischemia drug.

[0007] The above objects of the present application are achieved by the following technical solutions: A ginsenoside Rg1 delivery nanomaterial based on tetrahedral DNA, and a preparation method thereof, comprising the following steps: (1) Construction of tetrahedral DNA nanomaterial TDN The single-stranded nucleic acids shown in SEQ ID NO. 1-4 are hybridized and combined according to an equivalent molar ratio to obtain TDN, and then concentrated; (2) Preparation of CREKA-oligo complex The azide-modified CREKA peptide is coupled with the DBCO-modified single-stranded DNA shown in the following sequence by click chemistry to obtain a CREKA-oligo complex, and then purified. DBCO modified single-stranded DNA: 5' DBCO-TTTCGTACGATCATAGATCAAT; (3) Preparation of pTDN The TDN was reacted with an excess of CREKA-oligo complex, and the CREKA-oligo complex that was not successfully loaded onto the TDF was removed to obtain pTDN. (4) Preparation of Rg1@pTDN nanomaterial The pTDN was incubated with an excess of ginsenoside Rg1, and the excess ginsenoside Rg1 was removed to obtain the product.

[0008] Preferably, the hybridization binding conditions in step (1) are as follows: TM buffer of 50 mM MgCl2 and 10 mM Tris-HCl, pH 8.0 as solvent, heating to 95°C for 10 min, and then cooling to 4°C for 20 min.

[0009] Preferably, the click chemistry reaction conditions in step (2) are as follows: the azide-modified CREKA peptide and the DBCO-modified single-stranded DNA are dissolved in PBS buffer at pH 7.4, and the reaction is stirred at room temperature.

[0010] Preferably, in step (2), 12% denaturing polyacrylamide gel electrophoresis is used for purification.

[0011] Preferably, the reaction conditions in step (3) are as follows: the CREKA-oligo and TDN are incubated at 95°C for 5 min, cooled to 25°C at a gradient of 1°C / min, and then incubated for 30 min for annealing.

[0012] Preferably, in step (3), Amicon Ultra-0.5ml 100kD centrifugal filter is used for ultrafiltration to remove excess CREKA-oligo during purification.

[0013] Preferably, the incubation conditions in step (4) are as follows: incubation at room temperature for 24 hours.

[0014] Preferably, in step (4), Amicon Ultra-0.5ml 30kD centrifugal filter is used to remove excess ginsenoside Rg1.

[0015] The above-mentioned any ginsenoside Rg1 delivery nanomaterial is used in the preparation of a drug for resisting myocardial ischemia.

[0016] The above-mentioned any ginsenoside Rg1 delivery nanomaterial is used in the preparation of a drug for preventing or treating myocardial ischemia-reperfusion injury.

[0017] Beneficial effects The application is based on programmable oligonucleotide self-assembly technology, constructs a tetrahedral DNA nanostructure (TDN), and realizes precise connection and spatial modification by introducing a CREKA myocardial targeting peptide segment containing N3 modification, and further loads ginsenoside Rg1 to form a composite nanomedicine system Rg1@pTDN. All materials are biocompatible components, and Rg1@pTDN has good biological safety and tissue tolerance in vivo. The CREKA modification significantly improves the drug enrichment degree in the myocardial ischemic area, and Rg1@pTDN has good myocardial tissue targeting ability and cell uptake efficiency, can realize stable, targeted and efficient drug delivery, is suitable for cardiovascular diseases such as MIRI and AMI, and has clinical transformation and industrialization potential. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 For the construction and physicochemical property characterization of Rg1@pTDN nanosystem, wherein A is the construction schematic of Rg1@pTDN, including the self-assembly of tetrahedral DNA nanostructure (TDN), the modification of CREKA peptide segment and the loading process of Rg1; B is the dynamic light scattering (DLS) detection of the hydration particle size of different components, which shows that the particle size of Rg1@pTDN is the largest, indicating that the structure assembly is successful; C is the Zeta potential determination result of each structure system, which is all negatively charged, and the surface charge changes slightly after loading Rg1, showing good colloidal stability; D is the atomic force microscope (AFM) imaging, which shows that Rg1@pTDN presents a typical uniform triangular shape with clear and complete structure edges; E is the agarose gel electrophoresis result, which verifies the gradual assembly process of the structure: with the gradual access of DNA chain and CREKA, the band mobility gradually slows down, indicating that the molecular weight increases; Lane 1 is marker, Lane 2-4 is different degrees of assembly, Lane 5 is pTDN structure containing CREKA; F is the HPLC-MS / MS quantification of the encapsulation efficiency of Rg1 at different initial concentrations (n=3), the result shows that there is a saturation trend in the loading of Rg1; G is the in vitro release experiment, Rg1@pTDN releases slowly in PBS, and releases significantly faster in the presence of 10 μM H2O2, showing ROS-responsive release characteristics (n = 3); Figure 2 For the in vivo biological safety evaluation of Rg1@pTDN; wherein A is the HE staining section of heart, lung, spleen, liver and kidney tissues; B, C and D are serum biochemical indicators of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and creatinine (CR); Figure 3Evaluation of myocardial targeting and cellular uptake ability of Rg1@pTDN, wherein A is the ex vivo fluorescence imaging result of mouse major organs (brain, heart, liver, spleen, lung, kidney); B is the laser confocal microscope observation of Cy5.5-labeled Rg1@pTDN uptake in H9C2 myocardial cells; Figure 4 Myocardial protection effect of Rg1@pTDN in a mouse MIRI model, wherein A is the survival rate analysis of mice within 72 hours after coronary ligation reperfusion; B is the serum myocardial enzyme CK-MB level detection result, Rg1@pTDN can effectively reduce the release of myocardial cell damage-related enzymes; C is a schematic diagram of M-mode echocardiogram, the left ventricular systolic function of the Rg1@pTDN group is significantly improved; D is the analysis result of cardiac function parameters, the left ventricular ejection fraction (LVEF) and the fractional shortening (FS) of the Rg1@pTDN treatment group are significantly improved, indicating that it has a significant protective effect in maintaining cardiac function; Figure 5 To investigate the antioxidant effect of Rg1@pTDN; wherein A is the H&E staining of the heart of each group; B is the content level of lipid peroxidation product malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the myocardial tissue of each group. DETAILED DESCRIPTION

[0019] The substantial content of the present application will be specifically introduced below in combination with examples, but the protection scope of the present application is not limited thereto.

[0020] Example 1: Construction of Rg1@pTDN nano system with myocardial targeting and ROS response ability I. Materials and reagents All oligonucleotide chains were synthesized by Shanghai Shengong Bioengineering Co., Ltd., and purified by high performance liquid chromatography, dissolved in ultrapure water, quantified to 100 μM, aliquoted and stored at−20 °C for experimental use; the specific sequence is shown in the following table. The Cys-Arg-Glu-Lys-Ala-N3 (N3-CREKA) peptide used in the experiment was purchased from Guoping Pharmaceutical Co., LTD, and the ginsenoside Rg1 was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Buffer and reagents include TM buffer (50 mM MgCl2, 10 mM Tris-HCl, pH 8.0), PBS buffer (pH 7.4), TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.3), 1×TAE / Mg 2+The buffer (pH 8.0) and nucleic acid dye GelRed; the solubilization of ginsenoside Rg1 was performed using 10% methanol solution. The consumables used in the experiment included Amicon Ultra centrifugal column filters (30 kDa, 0.5 ml, Millipore Corporation), dialysis membranes (30 kDa, Solarbio, Beijing, China) and disposable zeta potential cell; in addition, DNase I (6 U mL-1) was used as an enzymatic reagent for related experiments. The DNA sequences used in the experiment are shown in the following table.

[0021] ; II. Experimental methods 1. Construction and sequence design of tetrahedral DNA nanomaterials (TDNs) Four complementary single-stranded oligonucleotides (ssDNA, SEQ ID NO. 1-4) were designed in the present application. The DNA oligonucleotides were diluted in deionized water and quantified by ultraviolet-visible absorption spectrophotometry (Hitachi U-3010, Japan). The DNA oligonucleotides were added to the TM buffer (50 mM MgCl2and 10 mM Tris-HCl, pH 8.0) at an equivalent molar ratio (the molar ratio of the four DNA oligonucleotides was 1:1:1:1), and the mixture was heated to 95°C for 10 min and then cooled to 4°C for 20 min using a thermal cycler to complete the preparation of TDNs. Subsequently, the TDN complex was further concentrated by centrifugal column filters (Amicon Ultra-30 kDa, Millipore Corporation).

[0022] 2. Preparation of CREKA-oligo complex We will contain azide (N3) group CREKA peptide (Cys-Arg-Glu-Lys-Ala-N3) and DBCO modified single chain DNA (oligo) by click chemistry reaction coupling. The specific steps are as follows: first, the C-terminal of CREKA peptide is modified with azide (N3) group to form CREKA peptide containing azide (N3) group (Cys-Arg-Glu-Lys-Ala-N3); the 5' end of single chain DNA (oligo) is connected with DBCO (Dibenzocyclooctyne) group by chemical modification to form DBCO modified single chain DNA (oligo). Then dissolve the azide modified CREKA peptide and DBCO modified single chain DNA in phosphate buffer (PBS, pH 7.4), mix and stir gently at 25°C overnight (12h). At this time, the azide group and the DBCO group undergo click reaction to form a stable chemical bond, connecting the CREKA peptide and the single chain DNA together to form the CREKA-oligo complex. Finally, the obtained CREKA-oligo complex is purified by 12% denaturing polyacrylamide gel electrophoresis (PAGE), and stored at -20°C for standby.

[0023] 3. Preparation of pTDN CREKA-oligo (5 μM) and TDN (1 μM) were incubated at 95°C for 5 min, then cooled to 25°C at a gradient of 1°C / min, and incubated for 30 min to anneal to prepare pTDFs. The cargo molecules (i.e. CREKA-oligo) that failed to load onto TDFs were removed by ultrafiltration using Amicon Ultra-0.5ml 100kD centrifugal filters.

[0024] 4. Loading method of ginsenoside Rg1 This application uses electrostatic interaction combined with hydrogen bonding, and Rg1 is attached to the pTDN double helix structure to form Rg1@pTDN. The drug loading rate is determined by ultraviolet absorption method and high performance liquid chromatography (HPLC).

[0025] Different concentrations (20, 40, 80, 120, 160 μM) of ginsenoside Rg1 solution (10% methanol as solvent) were incubated with pTDN structure (1 μM) at room temperature for 24 hours, and the excess ginsenoside Rg1 was removed using Amicon Ultra-0.5ml 30kD centrifugal filters (Millipore Corporation) to obtain the tetrahedral structure Rg1@pTDN loaded with ginsenoside Rg1. The loading rate of ginsenoside Rg1 on the pTDN structure was determined by HPLC-MS / MS method. The calculation formula is as follows: Encapsulation efficiency (%) = (initial mass of Rg1 - remaining mass of Rg1) / initial mass of Rg1 x 100% 5. Physicochemical property characterization of Rg1@pTDN nanosystem Based on programmable oligonucleotide self-assembly technology, tetrahedral DNA nanostructure (TDN) was constructed, and by introducing N3-modified CREKA myocardial targeting peptide segment, precise connection and spatial modification were realized, and further loading of ginsenoside Rg1 formed a complex nanomedicine system Rg1@pTDN, and the process diagram is as follows Figure 1 The dynamic light scattering (DLS) data showed that the particle size increased during the structure assembly process, the average particle size of TDN was 5.04 nm, the pTDN formed after CREKA modification was 10.21 nm, and the Rg1@pTDN after loading Rg1 was 12.45 nm, which verified the success of the peptide segment modification and drug loading process Figure 1 Zeta potential determination showed that all structures were negatively charged, and the potential slightly increased during the loading process (TDN: -10.96 mV; pTDN: -7.396 mV; Rg1@pTDN: -8.674 mV), which indicated that the complex system had good colloidal stability Figure 1 Atomic force microscopy (AFM) imaging showed that it had regular shape, clear edge and triangular pyramid shape, which was consistent with the designed three-dimensional configuration characteristics Figure 1 The electrophoresis results further verified the integrity of the structure assembly process: with the access of DNA chain and CREKA, the migration rate gradually decreased, reflecting the gradual increase of molecular weight and the successful assembly Figure 1 Rg1 can be stably loaded in pTDN structure, showing concentration-dependent loading characteristics. When the initial Rg1 concentration increased from 20 μM to 160 μM, the encapsulation rate decreased from about 80% to about 41%, suggesting that as the drug concentration increased, the loading sites of pTDN tended to be saturated, showing a typical binding saturation behavior. This result shows that the number of effective binding sites on the surface of pTDN is limited, and there is an upper limit of drug loading capacity Figure 1 Considering the encapsulation efficiency, drug utilization rate and synthesis cost, etc., the final study determined to assemble 1 μM pTDN with 40 μM Rg1 molar ratio for preparing Rg1@pTDN complex. Under this condition, the encapsulation rate of Rg1 was 72.75% ± 4.40%, with good repeatability and loading stability. Further in vitro release experiment showed that Rg1@pTDN in neutral buffer (PBS, pH 7.4) showed sustained release, while in the presence of 10 μM H2O2, the release rate was more than 80% within 24 h, showing a typical ROS response characteristic, reflecting its adaptability to the lesion microenvironment Figure 1The application provides a stable and controllable platform for subsequent targeted delivery and controlled release intervention of Rg1 in the ischemic myocardial injury area.

[0026] Example 2: Rg1@pTDN has good biological safety and tissue tolerance in vivo In animal experiments, in order to simplify the calculation of the dose of administration, the dose of Rg1@pTDN is uniformly converted according to the initial input dose of Rg1 (mg / kg), that is, the dose is calibrated mainly according to the content of Rg1. In the system safety evaluation, different doses of Rg1@pTDN (0, 2.5, 10 and 40 mg / kg) were injected into C57BL / 6 mice through the tail vein, and no abnormal behavior or death was observed in the animals of each group within 72 hours, indicating that the risk of acute toxicity is low. HE staining was used to observe the tissue sections of the heart, liver, spleen, lung and kidney, and no obvious necrosis, edema or inflammatory cell infiltration was observed, and the tissue structure was complete, indicating that Rg1@pTDN did not cause pathological damage. Figure 2 In the middle A, it was found that the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and creatinine (CR) in each dose group had no significant difference with the blank control group, indicating that the system did not cause obvious adverse reactions at the level of liver and kidney function. Figure 2 In the middle B, according to the results of histology and biochemical indicators, Rg1@pTDN showed good biological safety in vivo within the effective dose range, which laid a safe foundation for its further in vivo treatment application.

[0027] Example 3: Rg1@pTDN has good myocardial tissue targeting ability and cell uptake efficiency To verify the targeted delivery characteristics of Rg1@pTDN, the in vivo tissue distribution of Cy5.5-labeled Rg1@pTDN was analyzed by imaging. After 6 hours of Cy5.5-Rg1@pTDN injection through the tail vein of mice, the main organs were detected ex vivo, and the results showed that the fluorescence intensity in the heart tissue was significantly higher than that in other organs (such as liver, spleen, lung and kidney), indicating that it showed obvious myocardial enrichment characteristics in vivo. Figure 3 In the middle A, this result confirmed the targeting effect of the CREKA peptide, which helped the localization and accumulation of Rg1@pTDN in the myocardial injury area. At the cellular level, the uptake of Cy5.5-labeled Rg1@pTDN in H9C2 myocardial cells was observed by laser confocal microscope. After 4 hours of incubation, uniform and significant red fluorescence signals were observed in the cytoplasmic region, indicating that the nano system could be efficiently endocytosed by myocardial cells and had good cell delivery efficiency. Figure 3 The above results fully prove that Rg1@pTDN has good targeted delivery ability from the aspects of tissue and cells, which provides support for its precise drug delivery in ischemic myocardial injury intervention.

[0028] Example 4: Rg1@pTDN significantly improves heart function and reduces acute myocardial injury in MIRI mouse model I. Experimental materials 1. Experimental animals Male C57BL / 6 mice, 8 weeks old, weighing 20-25 g, were provided by Guangdong Ziruabio. The mice were raised in a SPF level animal room with a temperature of 22±2 ℃, humidity of 55±5%, 12 h light / dark cycle, free access to food and water, and adaptive feeding for 7 days.

[0029] 2. Drugs and reagents Ginsenoside Rg1, phosphate buffered saline (PBS), physiological saline, 4% paraformaldehyde, 1.5% sodium pentobarbital solution (for injection), isoflurane. pTDN, Rg1@pTDN were prepared according to the method of Example 1.

[0030] 3. Consumables and instruments 8-0 silk, syringe (1 mL, 5 mL specifications), tracheal intubation kit, small animal respirator, thoracotomy surgical instruments (ophthalmic scissors, forceps, retractor), echocardiograph (Visualsonics Vevo2100), table top high speed centrifuge.

[0031] II. Experimental methods 1. Modeling, grouping and administration The myocardial ischemia-reperfusion model was established according to conventional methods in the art. Sodium pentobarbital (50 mg / kg, intraperitoneal injection) was used to induce anesthesia before surgery, and 1.0%-1.5% isoflurane was used to maintain anesthesia during surgery. The depth of anesthesia was judged by toe pinch reflex. After anesthesia, tracheal intubation was performed and connected to a small animal respirator. The chest was opened at the left 4th intercostal space, the heart was exposed, and the LAD was ligated 1-2 mm below the left atrial appendage with 7-0 silk. Myocardial pallor and ST segment elevation on electrocardiogram were observed to confirm ischemia. After 30 minutes of ischemia, the ligature was loosened and reperfusion was restored for 72 hours. The sham operation group was only threaded without ligation. After surgery, the mice were placed on a 37 ℃ constant temperature heating plate for warmth until spontaneous breathing stabilized and the tracheal tube was removed.

[0032] The mice were randomly divided into groups, with 11 mice in each group, and the administration was performed once at 24 hours before surgery and at the beginning of reperfusion.

[0033] (1) Sham operation group (Sham): thoracotomy without LAD ligation, and equal volume of physiological saline was injected into the tail vein; (2) Model group (MIRI): myocardial ischemia-reperfusion, and equal volume of physiological saline was injected into the tail vein; (3) Free Rg1 group (Rg1): After myocardial ischemia-reperfusion, free Rg1 was injected into the tail vein at a dose of 7.275 mg / kg (the dose is equivalent to the actual delivery dose of Rg1 in Rg1@pTDN, which is used to evaluate the efficacy difference between free form and nano delivery system at the same effective dose); (4) pTDN group: After myocardial ischemia-reperfusion, pTDN was injected into the tail vein at a dose of 15.88 mg / kg (equimolar to pTDN in Rg1@pTDN group, to exclude the influence of the carrier itself); (5) Rg1@pTDN group: After myocardial ischemia-reperfusion, Rg1@pTDN was injected into the tail vein at a dose of 10 mg / kg (the actual dose is about 7.275 mg / kg Rg1 + 15.88 mg / kg pTDN, see the description below); (3), (4), (5) group dose explanation: In this study, Rg1@pTDN is a complex nano-drug system with a clear initial ratio (pTDN:Rg1 = molar ratio 1:40, encapsulation rate 72.75% ± 4.40%). Therefore, the dose in animal experiments is calibrated according to the initial Rg1 added amount in the preparation process (i.e. 10 mg / kg), rather than the actual drug content after loading into the body. This expression method conforms to the conventional practice in the field of nano-drugs, and is conducive to maintaining the consistency between batches and the comparability of subsequent product development. Although the input dose of Rg1@pTDN is 10 mg / kg, due to the loading rate of 72.75%, the actual delivery dose of Rg1 is about 7.275 mg / kg. In order to scientifically compare the efficacy difference under different drug forms: the free Rg1 group uses a free Rg1 dose of 7.275 mg / kg to ensure consistency with the actual delivery dose of Rg1 in the Rg1@pTDN group, so as to exclude the bias caused by the difference in drug dose; the pTDN group is dosed according to the molar amount of pTDN required for the above delivery dose (corresponding to 15.88 mg / kg) to maintain consistency with the amount of pTDN used in the Rg1@pTDN group, in order to exclude the intervention effect of the carrier itself.

[0034] 2. Sample collection and processing The survival of mice within 72 h of reperfusion was observed, and the survival curve was drawn. Mice that survived to 72 h were collected under deep anesthesia by cardiac puncture, and about 0.8-1.0 mL of blood was collected. After anticoagulation, centrifugation at 1500 g for 15 min at 4°C, the supernatant plasma was aliquoted and stored at -80°C. The heart was taken and washed with cold physiological saline and then fixed with 4% paraformaldehyde for histological examination.

[0035] 4. Detection method (1) Echocardiography for cardiac function After 72 h of reperfusion, transthoracic echocardiography was performed using a high-resolution small-animal ultrasound imaging system (Vevo 2100, VisualSonics, Canada) equipped with a 30 MHz linear probe. M-mode images were obtained at the level of the papillary muscle, and left ventricular ejection fraction (LVEF) and fractional shortening (FS) were measured in the short-axis view, and the results were calculated by the system software.

[0036] (2) Serum myocardial enzyme detection Immediately after the mice were sacrificed, blood was collected and centrifuged at 1500 g for 15 min to separate the serum. The serum creatine kinase isoenzyme (CK-MB) activity was measured using a commercial detection kit (Nanjing Jiancheng Biological Engineering Institute, China) according to the instructions.

[0037] (3) Myocardial tissue oxidative stress level detection The ischemic area of the left ventricular tissue was washed with ice-cold physiological saline, weighed, and homogenized according to the kit instructions. The supernatant was centrifuged at 12000 rpm for 10 min and used for detection. The malondialdehyde (MDA) content was detected by the thiobarbituric acid (TBA) colorimetric method at a detection wavelength of 532 nm; the superoxide dismutase (SOD) activity was detected by the xanthine oxidase method at a detection wavelength of 550 nm; and the glutathione peroxidase (GSH-Px) activity was detected by the DTNB method at a detection wavelength of 412 nm. All kits were purchased from Nanjing Jiancheng Biological Engineering Institute (China).

[0038] (4) Histological detection The heart tissue was taken to make paraffin sections and perform HE staining.

[0039] III. Experimental results The 72-hour survival rates of the mice in each group were as shown in Table 1. Figure 4 Compared with the control group, the 72-hour survival rate of the mice in the model group was significantly reduced, indicating that the modeling was successful; compared with the model group, the 72-hour survival rate of the mice in the free Rg1 group and the pTDN group was not significantly improved, but the 72-hour survival rate of the mice in the Rg1@pTDN group was significantly improved, indicating that the Rg1@pTDN had a significantly better effect on the prognosis of the acute phase of ischemia-reperfusion than the same dosage of Rg1.

[0040] The myocardial injury enzyme indicators of the mice in each group were as shown in Table 1. Figure 4CK-MB levels in the Rg1@pTDN group were significantly lower than those in the model group, the free Rg1 group or the pTDN group, indicating that Rg1@pTDN can effectively reduce the rupture of cardiomyocytes and enzyme release induced by reperfusion, and the effect is significantly better than that of the same dosage of Rg1 (see

[0041] In terms of cardiac function evaluation, it can be seen from the M-mode echocardiogram that the left ventricular systolic activity of the Rg1@pTDN group was significantly enhanced Figure 4 C). Further quantitative analysis showed that the left ventricular ejection fraction (LVEF) and the fractional shortening (FS) of the Rg1@pTDN group were significantly higher than those of the model group, the free Rg1 group or the pTDN group, indicating that it has a positive effect on maintaining left ventricular pumping function and the effect is significantly better than that of the same dosage of Rg1 (see Figure 4 D, Table 3 and Table 4).

[0042] Table 1 Survival of mice in each group after 72 hours ; Table 2 CK-MB levels (mean ± standard deviation, U / L) of each group ; Table 3 Left ventricular ejection fraction (LVEF, mean ± standard deviation, %) of each group ; Table 4 Left ventricular fractional shortening (FS, mean ± standard deviation, %) of each group ; It can be seen that Rg1@pTDN can effectively improve the survival rate, protect myocardial tissue and improve heart function in the MIRI model in vivo, providing experimental basis for its prevention and treatment potential in myocardial ischemia-reperfusion injury.

[0043] Further investigate its antioxidant effect, after 72 hours of ischemia-reperfusion, histological and biochemical analysis of myocardial tissue. H&E staining showed that the myocardial fibers of the model group were arranged in disorder, edema and obvious inflammatory cell infiltration, while the above pathological damage of the Rg1@pTDN treatment group was significantly reduced, and the myocardial structure integrity was significantly improved (see Figure 5 A). Biochemical test results showed that the level of lipid peroxidation product malondialdehyde (MDA) was significantly reduced in the Rg1@pTDN group, while the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were significantly increased, indicating that the body's antioxidant defense ability was enhanced (seeFigure 5 The results showed that Rg1@pTDN had a significant antioxidant effect on myocardial tissue (Fig. 3, Table 5, Table 6 and Table 7). In the results, the antioxidant effect of Rg1@pTDN was significantly better than that of Rg1 with the same dosage. The results showed that Rg1@pTDN could effectively alleviate the oxidative stress caused by ischemia-reperfusion, thereby playing an important role in myocardial protection.

[0044] Table 5 MDA levels of myocardial tissue in each group (mean ± standard deviation, nmol / mg protein) ; Table 6 SOD activity of myocardial tissue in each group (mean ± standard deviation, U / mg protein) ; Table 7 GSH-Px activity of myocardial tissue in each group (mean ± standard deviation, U / mg protein) ; Based on programmable oligonucleotide self-assembly technology, a tetrahedral DNA nanostructure (TDN) was constructed, and by introducing a CREKA myocardial targeting peptide segment containing N3 modification, precise connection and spatial modification were realized, and further loading of ginsenoside Rg1 formed a composite nanomedicine system Rg1@pTDN. All materials are biocompatible components, and Rg1@pTDN has good biological safety and tissue tolerance in vivo. CREKA modification significantly improves the drug enrichment degree in myocardial ischemic areas, and Rg1@pTDN has good myocardial tissue targeting ability and cell uptake efficiency, can realize stable, targeted and efficient drug delivery, is suitable for MIRI, AMI and other cardiovascular diseases, and has clinical transformation and industrialization potential.

Claims

1. A tetrahedral DNA-based ginsenoside Rg1 delivery nanomaterial, characterized in that, The preparation method comprises the following steps: (1) Construction of tetrahedral DNA nanomaterial TDN The single-stranded nucleic acids shown in SEQ ID NO. 1-4 are combined by hybridization in an equivalent molar ratio to obtain TDN, which is concentrated; (2) Preparation of CREKA-oligo complex The azide-modified CREKA peptide is coupled with the DBCO-modified single-stranded DNA shown in the following sequence by click chemistry reaction to obtain the CREKA-oligo complex, which is purified; DBCO-modified single-stranded DNA: 5' DBCO-TTTCGTACGATCATAGATCAAT; (3) Preparation of pTDN TDN is reacted with excess CREKA-oligo complex, and the CREKA-oligo complex that is not successfully loaded on TDF is removed to obtain pTDN; (4) Preparation of Rg1@pTDN nanomaterial pTDN is incubated with excess ginsenoside Rg1, and the excess ginsenoside Rg1 is removed to obtain the Rg1@pTDN nanomaterial. 2.The ginsenoside Rg1 delivery nanomaterial of claim 1, wherein, In step (1), the hybridization combination condition is that TM buffer solution of 50 mM MgCl2 and 10 mM Tris-HCl, pH 8.0 is used as a solvent, heating to 95°C for 10 min, and then cooling to 4°C for 20 min. 3.The ginsenoside Rg1 delivery nanomaterial of claim 1, wherein, In step (2), the click chemistry reaction condition is that the azide-modified CREKA peptide and the DBCO-modified single-stranded DNA are dissolved in PBS buffer solution at pH 7.4, and the reaction is stirred at room temperature. 4.The ginsenoside Rg1 delivery nanomaterial of claim 1, wherein, In step (2), 12% denatured polyacrylamide gel electrophoresis is used for purification. 5.The ginsenoside Rg1 delivery nanomaterial of claim 1, wherein, In step (3), the reaction condition is that CREKA-oligo and TDN are incubated at 95°C for 5 min, cooled to 25°C at a gradient of 1°C / min, and then incubated for 30 min for annealing. 6.The ginsenoside Rg1 delivery nanomaterial of claim 1, wherein, In step (3), Amicon Ultra-0.5ml 100kD centrifugal filter is used for ultrafiltration to remove excess CREKA-oligo. 7.The ginsenoside Rg1 delivery nanomaterial of claim 1, wherein, In step (4), the incubation condition is incubation at room temperature for 24 hours. 8.The ginsenoside Rg1 delivery nanomaterial of claim 1, wherein, In step (4), Amicon Ultra-0.5ml 30kD centrifugal filter is used to remove excess ginsenoside Rg1.

9. Use of the ginsenoside Rg1 delivery nanomaterial according to any one of claims 1-8 in the preparation of a drug for resisting myocardial ischemia.

10. Use of the ginsenoside Rg1 delivery nanomaterial according to any one of claims 1-8 in the preparation of a drug for preventing or treating myocardial ischemia-reperfusion injury.

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