Multi-mechanism nano-drug based on tetrahedral framework nucleic acid and application of multi-mechanism nano-drug in acute kidney injury treatment

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses a multi-mechanism nano-drug based on tetrahedral framework nucleic acid and application of the multi-mechanism nano-drug in acute kidney injury treatment, the nano-drug is a tFNA / GA (at) s i RNOX4 nano-composite system and is composed of the tetrahedral framework nucleic acid (tFNA), glycyrrhetinic acid (GA) and small interfering RNA (s iRNOX4) targeting NADPH oxidase 4; by adopting a single-carrier double-load strategy, effective co-delivery of the GA and the siRNOX4 is realized, the problems of poor solubility of the GA and low delivery efficiency of the si RNA are solved, and the pharmaceutical composition has definite kidney targeting property, can be massively accumulated in the kidney, especially in a renal tubule area, improves the concentration of a drug at a diseased region, and enhances the treatment effect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a multi-mechanism nano-drug based on tetrahedral framework nucleic acid and application thereof in treatment of acute kidney injury. BACKGROUND

[0002] Acute kidney injury is a critical illness with a very high clinical mortality rate, and the hospital mortality rate is as high as 50%. The core pathological mechanism is the vicious cycle of oxidative stress and inflammation. At present, there is a lack of specific treatment in clinical practice, and the main treatment is supportive treatment.

[0003] In the existing intervention strategies, anti-oxidant single-drug treatment, such as vitamin E and N-acetylcysteine, can alleviate the injury by eliminating active oxygen, but clinical trials have proved that high-dose NAC cannot significantly reduce the risk of AKI after cardiac surgery. Anti-inflammatory drugs such as glucocorticoids can inhibit the NF-κB pathway to reduce the release of inflammatory factors, but systemic application can easily cause side effects such as infection and osteoporosis. Natural product treatment such as glycyrrhetinic acid has been proved to have multi-target effect-activating Nrf2 / HO-1 antioxidant pathway and inhibiting NF-κB. Gene therapy such as siRNA targeting NOX4 can silence the key enzyme of ROS generation in the kidney, but naked siRNA is easily degraded by nucleases and lacks targeting.

[0004] In the above intervention strategies, single-drug treatment cannot break the pathological vicious cycle; GA has poor water solubility, and the oral bioavailability is less than 1%, and it lacks kidney targeting, and systemic administration can easily cause gastrointestinal side effects; naked siRNA is easily degraded by serum nucleases, and the kidney accumulation efficiency is low; the particle size of traditional liposome / polymer carriers is too large to pass through the glomerular filtration membrane to achieve passive targeting; cationic carriers can easily cause non-specific adsorption, increasing the liver and kidney toxicity.

[0005] Therefore, the tFNA / GA@siRNOX4 nano-composite system is constructed, the solubility of GA and the delivery efficiency of siRNA are solved by the “single-carrier double-loading” strategy, and the effective treatment of AKI is achieved. SUMMARY

[0006] The purpose of the present application is to provide a multi-mechanism nano-drug based on tetrahedral framework nucleic acid and its application in the treatment of acute kidney injury, so as to solve the problems raised in the background.

[0007] To achieve the above purpose, the present application provides the following technical scheme:

[0008] A multi-mechanism nano-drug based on tetrahedral framework nucleic acid, the nano-drug is a tFNA / GA@siRNOX4 nano-composite system, which is composed of tetrahedral framework nucleic acid (tFNA), glycyrrhetinic acid (GA) and small interfering RNA (siRNOX4) targeting NADPH oxidase 4.

[0009] The tFNA is formed into a tetrahedral structure by self-assembly of four single-stranded DNAs;

[0010] The siRNOX4 is loaded on the surface of the tFNA;

[0011] The GA is embedded in the double-stranded groove of the tFNA.

[0012] Preferably, the siRNOX4 is one of human siRNOX4 and mouse siRNOX4, the sequence of the human siRNOX4 is forward 5'-3' GGGACAAGAUUUGAAUACATT, and the sequence of the mouse siRNOX4 is forward 5'-3' GAAGUAUCAGACAAAUGUATT.

[0013] Preferably, the assembly molar ratio of the tFNA, the GA and the siRNOX4 is tFNA:GA:siRNOX4 = 1:300:4.

[0014] A preparation method of a multi-mechanism nanodrug based on a tetrahedral framework nucleic acid, comprising the following steps:

[0015] (1) Four equal molar single-stranded DNAs (S1, S2, S3, S4) are dissolved in a TM buffer containing 50 mM MgCl2 and 10 mM Tris-HCl, incubated at 95℃ for 10 minutes, and then incubated at 4℃ for 30 minutes to obtain tFNA, which is stored at 4℃ for standby;

[0016] (2) siRNOX4 is added to the tFNA solution obtained in step (1) according to a molar ratio of tFNA to siRNOX4 of 1:4, and incubated at 37℃ for 2 hours to obtain tFNA@siRNOX4;

[0017] (3) The GA is dissolved in DMSO, centrifuged after ultrasonic treatment to obtain a concentrated GA solution, and the tFNA@siRNOX4 is mixed with the GA solution according to a molar ratio of tFNA@siRNOX4 to GA of 1:300, and incubated at 4℃ with gentle stirring for 6 hours to obtain a tFNA / GA@siRNOX4 nanocomposite system.

[0018] A pharmaceutical composition comprising the multi-mechanism nanodrug of any one of the above and a pharmaceutically acceptable carrier.

[0019] An application of the multi-mechanism nanodrug in the preparation of a drug for treating acute kidney injury, including cisplatin-induced, glycerol-induced or renal ischemia-reperfusion injury.

[0020] Compared with the prior art, the present application has the following beneficial effects:

[0021] (1) The “single carrier dual-load” strategy was adopted to achieve effective co-delivery of GA and siRNOX4, solving the problems of poor GA solubility and low siRNA delivery efficiency.

[0022] (2) It has clear kidney targeting and can accumulate in large quantities in the kidney, especially in the renal tubules, thereby increasing the concentration of the drug in the lesion site and enhancing the therapeutic effect.

[0023] (3) Silencing the NOX4 gene by siRNOX4 reduces ROS generation from the source, while GA activates the Nrf2 / HO-1 pathway and inhibits NF-κB, forming a bidirectional therapeutic network that synergistically reduces oxidative stress and inflammation. The therapeutic effect is significantly better than single-drug treatment.

[0024] (4) It can significantly improve renal function and increase survival rate in both cisplatin- and glycerol-induced AKI models without obvious toxic side effects and is highly safe. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the preparation and characterization of tFNA / GA@siRNOX4 of the present invention; (A) Synthesis schematic; (B) PAGE verification of tFNA assembly; (C) PAGE verification of tFNA / GA@siRNOX4 assembly; (D) Complex particle size distribution; (E) and (F) TEM / AFM morphology analysis; (G) Zeta potential; (H) GA loading efficiency; (I) Hemolysis test;

[0026] Figure 2 Schematic diagram of the efficacy of the animal model of the present invention; (A) Cisplatin AKI model design; (BF) Body weight change; (G) Survival rate; (HJ) Kidney appearance and renal function indicators (BUN / CRE); (KO) Improvement of renal tissue pathology; (PS) Downregulation of inflammatory factors;

[0027] Figure 3 Schematic diagram of the efficacy of the animal model of the present invention; (A) Design of glycerol-induced AKI model; (B) H&E staining of renal tissue; (CD) Renal function index (BUN / CRE); (EF) Improvement of renal tissue pathology;

[0028] Figure 4 Schematic diagram of the safety evaluation of the present invention; (A) comparison of limb edema; (B) H&E staining of major organs; (CG) dynamic monitoring of liver function (AST / ALT) and renal function (BUN / CRE). DETAILED DESCRIPTION

[0029] With reference to the accompanying drawings on the basis of the embodiments of the present application, the technical solutions in the embodiments of the present application will be described clearly and completely. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0030] Embodiment one:

[0031] Please refer to Figure 1 The preparation method of a multi-mechanism nanodrug based on a tetrahedral framework nucleic acid shown in the figure comprises the following steps:

[0032] (1) Synthesis of tFNA: Dissolve four equal molar amounts of single-stranded DNA (S1, S2, S3, S4) in a TM buffer solution containing 50 mM MgCl2 and 10 mM Tris-HCl (pH 8.0), so that the concentration of each single-stranded DNA is 10 μM, incubate at 95°C for 10 minutes, and then incubate at 4°C for 30 minutes to obtain tFNA, which is stored at 4°C for standby use.

[0033] (2) Preparation of tFNA@siRNOX4: siRNOX4 is added to the tFNA solution obtained in step 1 at a molar ratio of tFNA to siRNOX4 (sequence is forward 5'-3' GAAGUAUCAGACAAAUGUATT) of 1:4, and incubated at 37°C for 2 hours to obtain tFNA@siRNOX4.

[0034] (3) Preparation of tFNA / GA@siRNOX4: Dissolve GA in DMSO, ultrasonically treat for 10 minutes, and then centrifuge at 3000 rpm for 5 minutes to obtain a concentrated GA solution; mix tFNA@siRNOX4 with the GA solution at a molar ratio of tFNA@siRNOX4 to GA of 1:300, and incubate at 4°C with gentle stirring for 6 hours to obtain a tFNA / GA@siRNOX4 nanocomposite system.

[0035] Embodiment two:

[0036] Please refer to Figure 1 The characterization detection of the tFNA / GA@siRNOX4 nanocomposite system is as follows:

[0037] Polyacrylamide gel electrophoresis (PAGE) verification: 12% polyacrylamide gel electrophoresis is used to verify the assembly of tFNA and the assembly of tFNA with siRNOX4 and GA;

[0038] As Figure 1 B, Figure 1 C and Figure 1As shown in FIG. H, the tFNA band lags significantly behind the single-stranded S1-S4, indicating that the tFNA is successfully assembled; after binding with siRNOX4, the migration rate of the tFNA complex is reduced, and the original siRNOX4 band completely disappears, confirming that the tFNA is successfully assembled with siRNOX4; the GA loading does not significantly change the structure and molecular weight of tFNA or tFNA@siRNOX4;

[0039] Particle size and zeta potential determination: The particle size and zeta potential of tFNA, tFNA@siRNOX4, tFNA / GA and tFNA / GA@siRNOX4 were determined using a dynamic light scattering instrument;

[0040] As shown in FIG. H, the tFNA band lags significantly behind the single-stranded S1-S4, indicating that the tFNA is successfully assembled; after binding with siRNOX4, the migration rate of the tFNA complex is reduced, and the original siRNOX4 band completely disappears, confirming that the tFNA is successfully assembled with siRNOX4; the GA loading does not significantly change the structure and molecular weight of tFNA or tFNA@siRNOX4; Figure 1 D and Figure 1 As shown in FIG. H, the tFNA band lags significantly behind the single-stranded S1-S4, indicating that the tFNA is successfully assembled; after binding with siRNOX4, the migration rate of the tFNA complex is reduced, and the original siRNOX4 band completely disappears, confirming that the tFNA is successfully assembled with siRNOX4; the GA loading does not significantly change the structure and molecular weight of tFNA or tFNA@siRNOX4;

[0041] Morphology observation: The morphology of tFNA / GA@siRNOX4 was observed by transmission electron microscopy (TEM) and atomic force microscopy (AFM), as shown in FIG. Figure 1 E-F, showing that it is uniformly dispersed, has a uniform particle size, and has a clear triangular geometric structure;

[0042] Stability test: tFNA / GA@siRNOX4 was incubated with PBS and 10% fetal bovine serum (FBS) at 37°C for different time points, and its stability was evaluated by gel electrophoresis;

[0043] The results show that tFNA / GA@siRNOX4 retains about 65% and 40% integrity, respectively, after 24 hours, indicating that it has good physiological stability;

[0044] Hemolysis experiment: tFNA / GA@siRNOX4 of different concentrations (0.5 μM, 1 μM, 2 μM, 4 μM) was incubated with red blood cells, as shown in FIG. Figure 1 I, showing that even at a concentration of 4 μM, its hemolytic activity is still very low, indicating high safety.

[0045] Example Three:

[0046] Please refer to Figure 2 As shown in FIG. H, the tFNA band lags significantly behind the single-stranded S1-S4, indicating that the tFNA is successfully assembled; after binding with siRNOX4, the migration rate of the tFNA complex is reduced, and the original siRNOX4 band completely disappears, confirming that the tFNA is successfully assembled with siRNOX4; the GA loading does not significantly change the structure and molecular weight of tFNA or tFNA@siRNOX4;

[0047] (1) Model establishment: 6-8 weeks old male C57BL / 6 mice were randomly divided into 6 groups: control group, cisplatin (Cis) group, Cis + tFNA group, Cis + tFNA / GA group, Cis + tFNA@siRNOX4 group and Cis + tFNA / GA@siRNOX4 group, except the control group, the rest of the mice were injected with 20 mg / kg cisplatin to induce AKI, and the control group was injected with the same amount of normal saline.

[0048] (2) Drug treatment: 24 hours after cisplatin injection, each group was injected with 100 μL of corresponding treatment material with a concentration of 1 μM through the tail vein, and the control group was injected with the same amount of normal saline.

[0049] (3) Index detection:

[0050] Body weight and survival rate: record the body weight change and 7-day survival rate of mice, as shown in Figure 2 B-G, the body weight of Cis group mice decreased significantly, and the 7-day survival rate was 0; while the body weight of tFNA / GA@siRNOX4 group decreased significantly, and the 7-day survival rate reached 80%.

[0051] Renal function index: detect the levels of blood urea nitrogen (BUN) and creatinine (CRE) in serum, the results show that the levels of BUN and CRE in tFNA / GA@siRNOX4 group are significantly reduced, close to normal levels.

[0052] Pathological histological analysis: H&E and PAS staining of kidney, the results show that the renal tubular structure of tFNA / GA@siRNOX4 group is basically normal, and the pathological damage is the lightest.

[0053] Immunohistochemical analysis: detection of KIM-1, TNF-α and IL-6 expression in kidney, as shown in Figure 2 K-O, the expression of the three in tFNA / GA@siRNOX4 group is significantly reduced.

[0054] Western blot analysis: detection of NOX4, HO-1 and other proteins in kidney, as shown in Figure 2 P-S, the expression of NOX4 in tFNA / GA@siRNOX4 group is significantly reduced, and the expression of HO-1 is significantly increased.

[0055] Example four:

[0056] Please refer to Figure 3 The treatment effect experiment of tFNA / GA@siRNOX4 nanocomposite system in glycerol-induced AKI model is as follows:

[0057] (1) Model establishment: 6-8 weeks old male C57BL / 6 mice were fasted for 16 hours, and then induced AKI by intraperitoneal injection of 50% glycerol, and the control group was injected with the same amount of normal saline.

[0058] (2) Drug treatment: After glycerol injection, 100 μL of corresponding treatment material with a concentration of 1 μM was injected into the tail vein of each group, and the control group was injected with the same amount of normal saline.

[0059] (3) Index detection:

[0060] Kidney function index: detection of BUN and CRE levels in serum, such as Figure 3 C-D shows that the BUN and CRE levels of the tFNA / GA@siRNOX4 group are significantly reduced.

[0061] Pathological histological analysis: H&E and PAS staining of the kidney, such as Figure 3 B shows that the tubular injury of the tFNA / GA@siRNOX4 group is significantly reduced.

[0062] TUNEL apoptosis detection: such as Figure 3 E-F shows that the tFNA / GA@siRNOX4 group has the least number of apoptotic cells and the most significant anti-apoptotic effect.

[0063] Example Five:

[0064] Please refer to Figure 4 As shown in the figure, the safety evaluation of the tFNA / GA@siRNOX4 nanocomposite system is as follows:

[0065] General state observation: observation of the overall morphology and appearance of the limbs of the mice, such as Figure 4 A shows that there is no significant difference between the control group and the tFNA / GA@siRNOX4 treatment group, and there is no abnormality such as limb edema.

[0066] Organ pathological examination: H&E and PAS staining of the heart, liver, spleen, lung and kidney of the mice, such as Figure 4 B shows that the results show that the organ morphology is normal at each time point, and there is no obvious pathological damage.

[0067] Serum biochemical index detection: detection of AST, ALT, BUN and CRE levels in serum within 30 days, such as Figure 4 C-G shows that the results are within the normal range and have no significant difference with the control group.

[0068] Although embodiments of the present application have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A multi-mechanism nanomedicine based on tetrahedral framework nucleic acid, characterized in that: The nanomedicine is a tFNA / GA@siRNOX4 nanocomposite system, which is composed of tetrahedral framework nucleic acid (tFNA), glycyrrhetinic acid (GA) and small interfering RNA (siRNOX4) targeting NADPH oxidase 4; The tFNA is formed by self-assembly of four single-stranded DNAs into a tetrahedral structure; The siRNOX4 is loaded on the surface of tFNA; The GA fits into the double-stranded groove of tFNA.

2. A multi-mechanism nanomedicine based on tetrahedral framework nucleic acid according to claim 1, characterized in that: The siRNOX4 is one of human siRNOX4 and mouse siRNOX4, the sequence of the human siRNOX4 is forward 5′–3′GGGACAAGAUUUGAAUACATT; the sequence of the mouse siRNOX4 is forward 5′–3′GAAGUAUCAGACAAAUGUATT.

3. The multi-mechanism nanomedicine based on tetrahedral framework nucleic acid according to claim 1, characterized in that: The assembly molar ratio of tFNA, GA and siRNOX4 is tFNA:GA:siRNOX4=1:300:

4.

4. The method for preparing a multi-mechanism nanomedicine based on tetrahedral framework nucleic acid according to claim 1, characterized in that: The following steps are involved: (1) Four equimolar single-stranded DNAs (S1, S2, S3, and S4) were dissolved in TM buffer containing 50 mM MgCl2 and 10 mM Tris-HCl, incubated at 95°C for 10 minutes, and then incubated at 4°C for 30 minutes to obtain tFNA, which was then stored at 4°C until use; (2) adding siRNOX4 to the tFNA solution obtained in step (1) at a molar ratio of tFNA to siRNOX4 of 1:4, and incubating at 37°C for 2 hours to obtain tFNA@siRNOX4; (3) GA was dissolved in DMSO, ultrasonicated, and then centrifuged to obtain a concentrated GA solution. tFNA@siRNOX4 was mixed with the GA solution at a molar ratio of tFNA@siRNOX4 to GA of 1:300, and the mixture was gently stirred and incubated at 4°C for 6 h to obtain a tFNA / GA@siRNOX4 nanocomposite system.

5. A pharmaceutical composition comprising the multi-mechanism nanodrug according to any one of claims 1-4 and a pharmaceutically acceptable carrier.

6. Use of the multi-mechanism nanomedicine according to any one of claims 1 to 4 in the preparation of a drug for treating acute kidney injury, characterized in that: The acute kidney injury includes cisplatin-induced acute kidney injury, glycerol-induced acute kidney injury or renal ischemia-reperfusion injury acute kidney injury.

Images