Co-assembled nanodrug for treating ischemic stroke and preparation method thereof

Abstract

The application discloses a kind of co-assembled nanomedicine and preparation method thereof in the field of medicine for treating ischemic stroke, which respectively dissolves oleanolic acid and tripterine in dichloromethane, then is added to PVA solution, ultrasonic treatment, stirring to remove organic solvent, collects nanoparticle and washes, freeze-drying to obtain co-assembled nanomedicine.The co-assembled nanomedicine prepared according to the above preparation method has certain protective effect on ischemic stroke, the dosage is significantly reduced, has smaller particle size, can inhibit or reduce the hepatotoxicity and nephrotoxicity of tripterine, so that it has smaller toxicity and stronger protective effect, and provides a new idea for the development of drugs for treating ischemic stroke.

Description

TECHNICAL FIELD

[0001] The present application relates to a pharmaceutical composition, in particular to a drug for treating ischemic stroke and a preparation method thereof. BACKGROUND

[0002] Stroke is the second leading cause of death worldwide, among which ischemic stroke accounts for 87%. Ischemic stroke has the characteristics of high incidence, high mortality and high recurrence rate. The main treatment methods in clinical practice are thrombolysis and thrombectomy, which have time window limitations and risks of bleeding. The main auxiliary drug is edaravone, and there is no more neuroprotective agent with definite efficacy. Studies have shown that natural pentacyclic triterpenoid extract tripterine (C 29 H 38 04) has obvious protective effect on ischemic stroke, but it has certain hepatotoxicity and nephrotoxicity. The deficiency is that the drugs derived from tripterine are mostly monomer drugs or nanometer monomer drugs extracted directly, which have large molecular weight, large dosage and large toxicity, and the use mode is intragastric administration or intraperitoneal injection. SUMMARY

[0003] The purpose of the present application is to provide a co-assembled nanodrug for treating ischemic stroke and a preparation method thereof. The co-assembled nanodrug can be used for treating ischemic stroke, and can inhibit or reduce the hepatotoxicity and nephrotoxicity of tripterine, has small dosage and small toxicity.

[0004] The purpose of the present application is achieved by a preparation method of a co-assembled nanodrug for treating ischemic stroke, comprising the following steps:

[0005] (1) Dissolve oleanolic acid and tripterine in dichloromethane respectively to prepare equal-concentration solutions with a concentration of 3 mg / ml to 5 mg / ml;

[0006] (2) Mix the above-mentioned tripterine solution and oleanolic acid solution according to the volume ratio (3:7) to (7:3), and then add to 2.5% PVA aqueous solution with a volume of 2.5 to 3.5 times of the mixture, and mix uniformly;

[0007] (3) Use an ultrasonic probe to ultrasonically treat the mixture to emulsify the solution, and form nanoscale small molecules through self-assembly characteristics;

[0008] (4) Add the formed emulsion drop by drop into 0.3% PVA aqueous solution with a volume of 5 to 10 times and a magnetic stirring speed of 400 rpm, and continuously stir at room temperature for 6 to 8 hours to remove the organic solvent;

[0009] (5) Centrifugal separation and collection of nanoparticles, followed by washing to remove free drugs and excess PVA; and freeze-drying to obtain a co-assembled nanodrug.

[0010] Further, in step (2), the tripterine solution and the oleanolic acid solution are mixed in a volume ratio of 6:4, and then added to a mixed volume of 3 times of 2.5% PVA aqueous solution. The optimal administration ratio of the co-assembled nanodrug for treating ischemic stroke according to the present application is 6:4 (weight ratio of the amount of tripterine to oleanolic acid), and the optimal concentration for use in mice is 4 mg / kg.

[0011] Further, in step (3), the mixture is subjected to ultrasonic treatment at a power of 40 W for 80-100 s using an ultrasonic probe.

[0012] Further, in step (5), centrifugal separation is performed at 4°C and a centrifuge speed of 12000 rpm, and the nanoparticles are collected after centrifugal separation for 30 min. Washing is performed twice using double-distilled water.

[0013] Compared with the prior art, the co-assembled nanodrug prepared according to the preparation method has the following beneficial effects: the drug raw material tripterine is a natural pentacyclic triterpenoid compound with multiple biological activities, and has a strong anti-inflammatory effect and a certain protective effect on ischemic stroke. After co-assembly with oleanolic acid, the administration amount is significantly reduced, the particle size is smaller, the liver and kidney toxicity of tripterine can be inhibited or reduced, the drug has smaller toxicity and stronger protective effect, and the developed drug composition can be directly injected into the animal body through the tail vein. A new idea is provided for the development of drugs for treating ischemic stroke. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 The morphology of the nanoparticles of the present application and the control group is shown in the figure, A1 is a tripterine nanoparticle, A2 is an oleanolic acid nanoparticle, and A3 is a tripterine-oleanolic acid co-assembled nanoparticle.

[0015] Figure 2 The infrared spectrum of the nanoparticles is shown in the figure.

[0016] In the infrared spectrum, the chemical bonds are shifted, which proves that the co-assembled drug is not a mixture of the two drugs, but a new nanoparticle that has interacted.

[0017] Figure 3 The morphology of the tripterine-oleanolic acid co-assembled nanoparticles with different proportions is shown in the figure.

[0018] Figure 4 The comparison chart of the protective effects of nanoparticles with different concentrations is shown in the figure.

[0019] Figure 5 The comparison chart of the treatment effects of nanoparticles and tripterine is shown in the figure.

[0020] Figure 6 Toxicity of nanoparticles to neurons; a, emodin-oleanolic acid co-assembly nanoparticles; b, emodin nanoparticles; c, oleanolic acid nanoparticles. DETAILED DESCRIPTION Example 1

[0021] Emodin-oleanolic acid co-assembly nanoparticles were prepared by emulsification solvent evaporation method. Emodin and oleanolic acid were dissolved in dichloromethane to prepare equal concentration solutions with a concentration of 3 mg / ml-5 mg / ml. Then 1 ml of emodin solution, 1 ml of oleanolic acid solution, and 1 ml of mixed solution of emodin and oleanolic acid at a volume ratio of 1:1 were added to 3.0 mL of 2.5% PVA aqueous solution (W / v). PVA was used as a medium to make the drugs uniformly dispersed in the aqueous solution. Then the mixture was subjected to ultrasonic treatment at a power of 40 W for 90 s (on for 10 s and off for 5 s, 6 cycles) to emulsify the solution and form nanoscale small molecules by self-assembly characteristics. The formed emulsion was added dropwise to 30 mL of 0.3% PVA aqueous solution (W / v) stirred at a magnetic stirring speed of 400 rpm, and stirring was continued at room temperature for 6-8 hours to remove the organic solvent. Finally, the nanoparticles were collected by centrifugation at 4°C and 12000 rpm for 30 minutes, then washed twice with double distilled water to remove free drugs and excess PVA, and freeze-dried for use.

[0022] The three groups of drugs obtained were emodin-oleanolic acid co-assembly nanoparticles, emodin nanoparticles, and oleanolic acid nanoparticles. The emodin-oleanolic acid co-assembly nanoparticles were the target product co-assembly nanomedicine of the present application, and the other two groups were control groups.

[0023] In Figure 1 In the scanning electron microscope, the size, particle size, uniformity, etc. of emodin nanoparticles and oleanolic acid nanoparticles were significantly different from those of emodin-oleanolic acid co-assembly nanoparticles, while in the Figure Two In the infrared spectrum, it can be seen that the chemical bonds of the co-assembly nanoparticles have undergone obvious shift, so we believe that the co-assembly nanomedicine is a new nanoparticle different from the monomer nanomedicine. Subsequently, as Figure 3 we optimized the conditions for the nanoparticles. Equal concentrations of emodin solution and oleanolic acid solution were mixed at a volume ratio of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1 to prepare 9 groups of solutions. One milliliter of each mixed solution was used to obtain nine co-assembly nanoparticles with different drug ratios by the above method. The drug release rate of each group was determined by the dialysis method, and the results are shown in Table 1.Figure Three The scanning electron microscope results, the preferred group with smaller particle size and uniform morphology is: the volume ratio of tripterine solution and oleanolic acid solution is: (3:7)~(7:3), and the most preferred is: 6:4.

[0024] After volume and weight conversion, the nanoparticles with smaller particle size and more uniform morphology of tripterine: oleanolic acid = 6:4 (weight ratio) are selected as the subsequent treatment drug. The nanoparticles are dissolved with normal saline to prepare tripterine-oleanolic acid co-assembled nanoparticle solutions of 3 mg / ml, 4 mg / ml, 5 mg / ml, and 6 mg / ml for the treatment of ischemic stroke.

[0025] Experimental object: ICR mice, provided by the Comparative Medicine Center of Yangzhou University.

[0026] Take 6-8 weeks old, male mice, weighing 25-30 g, and divide them into groups: normal group; MCAO model group; MCAO plus four dose groups (3 g / kg, 4 g / kg, 5 g / kg, 6 g / kg) of the application group. Drug administration during ischemia-reperfusion, 24 hours later, behavioral determination, and then take the tissue for staining.

[0027] As Figure 4 , TTC staining, dark color represents surviving tissue, and light color represents infarction. It can be seen that the light color area is smallest at 4 mg / kg, i.e., the infarction volume is smallest.

[0028] Grouping: control group, model group, tripterine-oleanolic acid co-assembled nanoparticle (different doses) treatment group.

[0029] Through TTC staining, it is found that when the drug concentration is 4 mg / kg, the mouse brain infarction volume is significantly reduced, proving that the drug has obvious protective effect on ischemic stroke.

[0030] Figure 5 The figure compares the treatment effects of co-assembled nanoparticles and tripterine.

[0031] In the figure, A. Statistical graph of TTC staining infarction area (the higher the value, the larger the infarction area); B. Neurological function score (the higher the score, the worse the neurological function); C. Rotarod test (the shorter the time, the worse the motor function); D. Corner test (the higher the value, the more severe the asymmetric sensory motor defect); E. Lateral arm placement test (the lower the score, the worse the sensory motor function); F. Open field test (the closer the walking distance, the worse the motor function).

[0032] Grouping: control group, model group, tripterine-oleanolic acid co-assembled nanoparticle treatment group, and tripterine nanoparticle treatment group.

[0033] The behavior of the mice was evaluated by the rotating rod experiment, the rotating angle experiment, the side arm placement experiment and the open field experiment, and it was found that the drug can obviously improve the feeling and movement function of the mice, and the protection of the co-assembled nano drug is obviously stronger than that of the equal quality of tripterine nanoparticles. Figure 5

[0034] Figure 6 The toxicity of the nanoparticles to neurons is compared in the graph.

[0035] The three graphs are, in turn, tripterine-oleanolic acid co-assembled nanoparticles, tripterine nanoparticles and oleanolic acid nanoparticles.

[0036] Through in vitro experiments, the drug toxicity was determined, the HT22 cells were inoculated in a 96-well plate at a density of 400 cells per well, 24 hours later, when the cell density reached 70-80%, the drug was added, and the cell viability was determined by using CCk8 reagent 24 hours after the drug was added, it can be seen that the toxicity of the co-assembled nanoparticles is less than that of the monomer nanoparticles, especially when the drug concentration is 0.5 μg / ml, 1 μg / ml.

[0037] Therefore, the optimal administration ratio of the nano drug for treating ischemic cerebral stroke is 6:4 (weight ratio of tripterine: oleanolic acid), and the optimal concentration for use in the tail vein of the mice is 4 mg / kg.

[0038] The present application is not limited to the above-mentioned embodiments, and on the basis of the technical solutions disclosed in the present application, those skilled in the art can make some substitutions and modifications to some technical features according to the disclosed technical content without creative labor, and these substitutions and modifications are all within the protection scope of the present application.​

Claims

1. A method for preparing a co-assembled nanodrug for treating ischemic stroke, characterized in that, It comprises the following steps: (1) dissolve oleanolic acid and tripterine in dichloromethane respectively to prepare equal-concentration solutions with a concentration of 3 mg / ml-5 mg / ml; (2) mix the tripterine solution and the oleanolic acid solution according to a volume ratio of (3:7)-(7:3), and then add them into 2.5% PVA aqueous solution with a volume ratio of 2.5-3.5, and mix them uniformly; (3) use an ultrasonic probe to perform ultrasonic treatment on the mixture to emulsify the solution and form nanoscale small molecules through self-assembly characteristics; (4) add the formed emulsion drop by drop into 0.3% PVA aqueous solution with a volume ratio of 5-10 and a magnetic stirring speed of 400 rpm, and continuously stir at room temperature for 6-8 hours to remove the organic solvent; (5) centrifugally separate and collect the nanoparticles, and then wash to remove free drugs and excessive PVA; and obtain the co-assembled nanodrugs after freeze-drying. 2.The preparation method of the co-assembled nanodrug for treating ischemic stroke according to claim 1, characterized in that, In step (2), the tripterine solution and the oleanolic acid solution are mixed according to a volume ratio of 6:4, and then added into 2.5% PVA aqueous solution with a volume ratio of 3. 3.The preparation method of the co-assembled nanodrug for treating ischemic stroke according to claim 1, characterized in that, In step (3), the mixture is ultrasonically treated for 80-100 s at a power of 40 W by using an ultrasonic probe. 4.The preparation method of the co-assembled nanodrug for treating ischemic stroke according to claim 1, characterized in that, In step (5), the centrifugal separation is performed at 4°C and a centrifuge speed of 12000 rpm, and the nanoparticles are collected after centrifugal separation for 30 minutes. 5.The preparation method of the co-assembled nanodrug for treating ischemic stroke according to claim 1, characterized in that, In step (5), the washing is performed twice with double-distilled water.

6. A co-assembled nanodrug for treating ischemic stroke, characterized in that, The co-assembled nanodrugs are obtained by the method according to any one of claims 1-5. The co-assembled nanodrugs are obtained by the method according to any one of claims 1-5.

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