CN115120611A

CN115120611A - NO donor micelle composition and preparation method and application thereof

Info

Publication number: CN115120611A
Application number: CN202211059786.5A
Authority: CN
Inventors: 刘微; 房元英; 罗晓健; 饶小勇; 钟荣生; 陆浩伟; 张爱玲; 张尧; 杨婧; 何双凤
Original assignee: Jiangxi University of Traditional Chinese Medicine; Jiangxi Bencao Tiangong Technology Co Ltd
Current assignee: Jiangxi University of Traditional Chinese Medicine; Jiangxi Bencao Tiangong Technology Co Ltd
Priority date: 2022-09-01
Filing date: 2022-09-01
Publication date: 2022-09-30
Anticipated expiration: 2042-09-01
Also published as: CN115120611B

Abstract

The invention discloses an NO donor type micelle composition, a preparation method and application thereof, wherein the NO donor type micelle composition is composed of an NO donor type polymer and an insoluble drug with an anti-ischemia-reperfusion injury neuroprotection effect. The micelle composition treats and prevents ischemic stroke through dual-channel synergy of medicines and NO endothelial relaxation factors, and can achieve the effects of neuroprotection after cerebral ischemia reperfusion surgery and reduction of recurrence probability of stroke.

Description

NO donor micelle composition, and preparation method and application thereof

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to an NO donor micelle composition, and a preparation method and application thereof.

Background

Currently, drug therapy is the main means for protecting the cerebral ischemia reperfusion injury. Curcumin treats cerebral apoplexy through antioxidation, anti-inflammation and other effects. But curcumin has poor water solubility and low bioavailability, is difficult to transport to a brain injury part due to the existence of a blood brain barrier, and limits the application of curcumin in cerebral apoplexy diseases. How to overcome the problem of poor solubility of insoluble compounds including curcumin and break through the problem that blood brain barrier becomes the first bottleneck problem of the application of the research of medicinal preparations in the brain. With the development of nanotechnology, a nano drug delivery system becomes a potential targeted drug delivery system for optimizing the therapeutic effect of drugs, and the shape, size and surface design is carried out through a biological, physical or chemical modification strategy, so that the nano drug delivery system becomes an effective method and a research hotspot for improving the brain drug delivery efficiency.

The drug is encapsulated in a polymeric micelle core formed by the block copolymer, so that the solubility and the bioavailability of the insoluble drug can be improved, the in-vivo distribution behavior of the insoluble drug can be changed, the in-vivo circulation time can be prolonged, the effects of synergy and attenuation can be achieved, and the neuroprotective effect of the drug on cerebral ischemia reperfusion can be fully exerted.

In addition to high morbidity, cerebral apoplexy relapse is also a main cause of death and disability of patients, and cerebrovascular stenosis is one of the main factors influencing acute ischemic stroke relapse. Therefore, after the first onset of cerebral infarction, except neuroprotection, it is very important to dilate blood vessels, improve the function of vascular endothelium and prevent the reoccurrence of cerebral apoplexy. The clinical common means for preventing cerebral apoplexy comprise the application of a cerebral vessel stent and a vasodilator. The cerebrovascular stent operation easily causes poor adherence and incomplete dilatation of the stent, leads to secondary blockage of blood vessels, promotes the reformation of thrombus, and needs to take long-term anticoagulant drug treatment after the operation. Sodium nitroprusside and other clinical vasodilators have serious adverse reactions of digestive system, cardiac function and liver and kidney functions, while curcumin has obvious drug effect of antioxidant neuroprotection, but has poor capability of dilating blood vessels. In order to treat CIRI nerve injury and dilate blood vessels and prevent cerebral infarction recurrence, neuroprotective drugs, vasodilator drugs and anticoagulant drugs are required to be taken at the same time, so that the medication burden of patients is increased, and adverse drug reactions are increased. If the dilated blood vessels and the curcumin neuroprotective drugs are wrapped in the micelle drug delivery system together, the problems of low drug-loading rate, uncertain drug-loading proportion, compatibility change and the like exist.

The research shows that: the vascular wall cell is used as an action mechanism of a target spot, and the vascular wall cell can further play a role in protecting endothelium and preventing thrombosis through regulating the aspects of oxidative damage, permeability, cell factors, blood coagulation, fibrinolysis system and the like of vascular endothelium. NO as an endothelin-relaxing factor, one of the least molecular bioactive substances discovered to date, by entering cells with Fe in guanylate cyclase ²⁺ Combines and improves guanylate cyclase activity, increases cellular cGMP content, and cGMP is used as a second messenger, activates cell protein kinase PKG, regulates ion channel, and reduces intracellular Ca ²⁺ The concentration, which phosphorylates the myosin light chain, causes a vasodilatory effect on vascular smooth muscle, and is involved in the immediate regulation of blood pressure and the local regulation of blood flow. Under normal conditions, cerebral bloodEndothelial cells release NO continuously to maintain a constant state of vasodilation of the blood vessel while inhibiting leukocyte infiltration and platelet aggregation and adhesion on their surface. When vascular endothelial cells are damaged by oxygen radicals and the like, NO release is reduced, and thrombosis is easily caused.

Synthesizing NO donor material type polymer auxiliary materials, and constructing a delivery system with synergistic effect of innovative drug molecules and the polymer auxiliary materials and having industrial and clinical application prospects. Aiming at the microenvironment after CIRI operation and the etiology of cerebral apoplexy recurrence, the treatment effect of the drug delivery system is improved from two aspects of neuroprotection and blood vessel dilatation. In cerebral apoplexy ischemia period, NO is released from damaged parenchyma by using NO donor type micelle composition, microcirculation is improved, local blood flow is increased, vascular endothelial function is improved, and damage of hypoxia-reoxygenation to endothelial cells is inhibited, so that tissue protection is realized; the reperfusion period relies on curcumin to exert neuroprotective effects. The pharmaceutical composition with the NO dual-channel synergy on the drug molecules and the auxiliary material donor can simultaneously treat cerebral ischemia reperfusion neuroprotection and prevent ischemic stroke.

Disclosure of Invention

The invention aims to solve the problems and pathogenesis of diseases which need to be solved urgently in clinic, creatively proposes the dual-channel synergistic effect of intervention of active ingredients of medicaments and relaxation of NO endothelium on micelle polymers according to animal experiment results, treats cerebral ischemia-reperfusion injury and prevents relapse of cerebral apoplexy.

In order to achieve the purpose, the technical scheme provided by the invention is as follows:

the invention provides an NO donor type micelle composition, which comprises an NO donor type micelle material and an insoluble medicine with an anti-cerebral ischemia-reperfusion injury neuroprotection effect. The insoluble drug is curcumin.

Preferably, the NO donor micelle composition is prepared from the following raw materials in parts by weight: 1-10 parts of curcumin, 1-30 parts of NO donor micelle material, 1-30 parts of surfactant and 1-60 parts of freeze-drying protective agent.

Preferably, the NO donor-type micelle composition is composed of the following raw materials in parts by weight: 6 parts of curcumin, 15 parts of NO donor micelle material, 15 parts of surfactant and 30 parts of freeze-drying protective agent.

Preferably, the NO donor type micelle material is composed of one or more amphiphilic block polymer NO donors, and the amphiphilic block polymer NO donor is an amphiphilic diblock polymer NO donor, an amphiphilic triblock polymer NO donor or an amphiphilic multiblock polymer NO donor. Such as one or more of PLGA-NO, PLA-NO, mPEG-PLGA-NO, mPEG-PGA-NO, PGA-NO.

Wherein the PLGA is polylactic acid-glycolic acid, L/G =50/50 or 75/25, and the molecular weight is 5000-20000;

PLA is polylactic acid, and the molecular weight of PLA is 500-5000;

PGA is polyglutamic acid, and the molecular weight is 5000-30000;

the mPEG is polyethylene glycol monomethyl ether, and the molecular weight of the mPEG is 500-5000.

Preferably, the NO donor type micelle material is mPEG-PLA-NO, and the structural formula of the mPEG-PLA-NO is as follows:

；

wherein n1=6 ~ 100, n2=6 ~ 100.

Preferably, the surfactant is selected from one or more of tweens, polyoxyethylene fatty alcohol ethers, polyoxyethylene alkyl phenol ethers, polyoxyethylene fatty acid esters, polyoxyethylene polyoxypropylene copolymers, fatty acid sorbans and polyoxyethylene sorbitan fatty acid esters.

Preferably, the lyoprotectant is selected from one or more of trehalose, mannitol, sucrose, glucose, sodium chloride, lactose, sorbitol, dextran, glycerol, and glycine.

The invention also provides a preparation method of the NO donor micelle composition, which comprises the following specific preparation steps: mixing curcumin, an NO donor micelle material and a surfactant according to parts by weight, putting the mixture into an organic solvent for full dissolution, adding purified water, stirring uniformly, adding a freeze-drying protective agent, sterilizing, and freeze-drying to obtain a finished product of the NO donor micelle composition.

The sterilization method is one or more of a filtration sterilization method and a moist heat sterilization method.

The organic solvent is one or more selected from ethanol, methanol, diethyl ether, chloroform, dichloromethane, isopropanol, tert-butanol, n-butanol, and ethyl acetate.

Preferably, the preparation method of the NO donor-type micelle composition of the present invention specifically comprises: mixing 6 parts of curcumin, 15 parts of mPEG-PLA-NO and 15 parts of Tween surfactant, putting the mixture into 20 mL of ethanol for full dissolution, adding purified water, stirring uniformly, adding 30 parts of mannitol, sterilizing, and freeze-drying to obtain the finished product of the NO donor micelle composition.

The invention also provides application of the NO donor micelle composition in preparation of a medicament for preventing neuroprotection and relapse in cerebral ischemia-reperfusion injury. Preferably, the administration is intravenous.

Compared with the prior art, the invention has the beneficial effects that:

1) the synergistic treatment of cerebral ischemia reperfusion injury and prevention of cerebral apoplexy relapse, the improvement of long-term survival rate and the reduction of disability rate;

2) the effects of synergy and attenuation are achieved, and the administration dosage of the neuroprotective medicament is reduced, so that adverse reactions are reduced;

3) avoid the side effects of poor adherence, incomplete expansion and the like of the stent in the operation of the cerebrovascular stent, and reduce the adverse reaction caused by expanding blood vessels and anticoagulant drugs.

The invention proves the synergistic effect of the NO donor micelle and the medicine combination, and can improve microcirculation, increase local blood flow, improve the function of vascular endothelium and inhibit the injury of hypoxia-reoxygenation to endothelial cells on the premise of medicine intervention by utilizing NO, thereby reducing the cerebral infarction volume of mice, improving the neurological function and playing a role in synergistic interaction. The anti-cerebral ischemia reperfusion injury effect of the micelle is far better than that of a drug micelle without an NO donor.

In a word, the NO donor type micelle composition can simultaneously utilize double channels of medicine intervention and NO endothelial target effect to synergistically treat and prevent ischemic stroke and obviously relieve cerebral ischemia-reperfusion injury.

Drawings

FIG. 1 is a graph of the particle size of the NO donor type micelle composition Cur-PLA-NO prepared by the present invention;

FIG. 2 is a DSC measurement chart of the NO donor-type micelle composition Cur-PLA-NO prepared by the present invention;

FIG. 3 is an XRD measurement diagram of the NO donor type micelle composition Cur-PLA-NO prepared by the invention;

FIG. 4 is a graph showing the in vitro release behavior of curcumin and NO in the NO donor type micelle composition Cur-PLA-NO prepared by the present invention;

FIG. 5 is a graph of the in vitro blood brain barrier permeability versus different agent groups in an experimental study of the present invention;

FIG. 6 is a graph of the in vivo neurological function scores of different formulation groups in an experimental study of the invention;

FIG. 7 is a graph of TTC infarct volume for different groups of formulations in an experimental study of the invention;

FIG. 8 is a comparison of calculated cerebral infarct volumes for different groups of formulations in the experimental study of the present invention;

FIG. 9 is a graph of the water maze heat zones for different formulation groups in an experimental study of the present invention;

FIG. 10 is a graph comparing residence times in target quadrants of the water maze for different formulation groups in an experimental study of the present invention;

FIG. 11 shows the effect of different groups on the expression of eNOS, iNOS, nNOS in 6h and 24h cerebral tissues of MCAO mice (middle cerebral artery occlusion, MCAO) in experimental studies of the present invention (a) the concentration of nNOS in 6h MCAO mice after drug injection; (b) the nNOS concentration in the MCAO mouse brain changes 24 hours after the medicine is injected; (c) the iNOS concentration in the MCAO mouse brain changes 6h after the drug is injected; (d) the iNOS concentration in the MCAO mouse brain changes 24 hours after the drug is injected; (e) the eNOS concentration in the brain of the MCAO mice changed 6h after the injection of the drug; (f) change in eNOS concentration in the brain of MCAO mice 24h after drug injection ].

Detailed Description

In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

The embodiment provides a preparation method of an NO donor type micelle composition, which comprises the steps of mixing 6 parts of curcumin, 15 parts of mPEG-PLA-NO and 15 parts of surfactant (Tween) according to parts by weight, fully dissolving the mixture in 20 mL of ethanol, adding purified water, uniformly stirring, adding 30 parts of freeze-drying protective agent (mannitol), sterilizing, and freeze-drying to obtain a finished NO donor type curcumin micelle (Cur-PLA-NO).

Comparative example 1

The comparative example provides a preparation method of a non-NO donor type micelle composition, which comprises the steps of mixing 6 parts of curcumin, 15 parts of mPEG-PLA and 15 parts of surfactant (Tween) according to parts by weight, placing the mixture into 20 mL of ethanol for full dissolution, adding purified water, stirring uniformly, adding 30 parts of freeze-drying protective agent (mannitol), sterilizing, and freeze-drying to obtain a non-NO donor type curcumin micelle finished product (Cur-PLA).

Characteristics of NO-donor type micelle composition

1. Evaluation of Cur-PLA-NO in vitro morphology

The in vitro properties of the NO donor-type curcumin micelle (Cur-PLA-NO) prepared in example 1 and the non-NO donor-type curcumin micelle (Cur-PLA) prepared in comparative example 1 were measured, and the results are shown in table 1 below and fig. 1, 2 and 3.

TABLE 1 Cur-PLA-NO and Cur-PLA in vitro Properties

。

As can be seen from Table 1, the Cur-PLA-NO and Cur-PLA have controllable particle size (< 50 nm) and stable quality, and can be used for comparative study of later experiments.

In FIG. 1, the appearance of Cur-PLA-NO is observed by a transmission electron microscope and is spherical or spheroidal; in FIGS. 2 and 3, the melting points and crystal forms of different formulation groups (Cur-PLA-NO, curcumin, mPEG-PLA-NO micelle, physical mixture of curcumin and mPEG-PLA-NO micelle) are determined by Differential Scanning Calorimetry (DSC) and X-ray diffraction (XRD), and the results show that curcumin in Cur-PLA-NO is completely loaded in the micelle core and is in an amorphous state.

Cur-PLA-NO: Cur-PLA-NO micelles prepared in example 1;

mPEG-PLA-NO micelles: blank mPEG-PLA-NO micelles, i.e. micelles without curcumin added;

physical mixture of curcumin and mPEG-PLA-NO micelles: physical mixture of curcumin bulk drug and blank mPEG-PLA-NO micelle.

2. Evaluation of Cur-PLA-NO in vitro Release behavior

In order to evaluate the release behavior of Cur-PLA-NO in the in vivo circulation process, a PBS solution containing Tween 80 surfactant and L-cysteine is used as a dissolution medium, a dissolution experiment is carried out by a flow cell dissolution instrument, the curcumin content is measured by HPLC, the NO content is measured by an ultraviolet spectrophotometry, and the release behavior of curcumin and NO in Cur-PLA-NO is evaluated. The Origin Pro 2018C software is used for fitting the release rates of curcumin and NO (see Table 2), and the result shows that the release process of curcumin and NO conforms to a first-order kinetic equation and sudden release does not exist. The cumulative release amount of NO in the first 3h is 32.67 +/-1.73%, and the release tends to be rapid first and slow later and the slow release tends to be more and less first (see figure 4). Compared with NO, the cumulative release amount of curcumin in the first 3h is only 4.65 +/-0.19%, and the release amount reaches 55.43 +/-0.006% when the curcumin is in a sustained release trend until 60 h. The different release trends of curcumin and NO are in line with the demands of antioxidant neuroprotection in microenvironment and perfusion phase of NO deficiency in CIRI infarct phase.

TABLE 2 fitting equation of curcumin and NO in vitro release model in Cur-PLA-NO

。

3. Evaluation of Cur-PLA-NO in vitro blood brain Barrier Permeability

bEnd.3 cells (1X 10) ⁵ Cells/well) were inoculated on a transwell membrane (1.0 μm pore size, precoated with 2% w/v gelatin), cultured in a medium containing fetal bovine serum (10% v/v), an in vitro blood brain barrier model was established, and the blood brain barrier permeability of different formulations (curcumin solution, Cur-PLA and Cur-PLA-NO) was evaluated by comparing the curcumin content in the solution that permeates the transwell membrane. The results are shown in FIG. 5 (P < 0.05, P < 0.01, and differences were significant when P < 0.05). The results show that the curcumin solution, Cur-PLA and Cur-PLA-NO can penetrate the blood brain barrier to different degrees in vitro, which is related to that curcumin is a small molecule with strong lipid solubility. Compared with a curcumin solution and Cur-PLA, Cur-PLA-NO has higher permeability, because the furan ring compound is added into the mPEG-PLA block, the chemical composition, the length and the lipophilicity of the copolymer block are changed, and the copolymer block shows more positive affinity to mouse brain endothelial cells. Whether it can increase the amount of medicine entering brain in vivo is also related to the permeability of blood brain barrier and the binding ability of blood serum protein. In the MCAO model of mice, blood brain barrier permeability is increased, and the negative charge on the surface of Cur-PLA-NO can reject serum protein. These are all beneficial to Cur-PLA-NO entering the brain through the blood brain barrier to play a therapeutic role.

Secondly, research on anti-ischemia reperfusion injury neuroprotective effect of NO donor micelle composition

The study on the anti-cerebral ischemia reperfusion injury of C57BL/6 mice was carried out on the NO donor-type curcumin micelle (Cur-PLA-NO) prepared in example 1 and the non-NO donor-type curcumin micelle (Cur-PLA) prepared in comparative example 1, and the details are as follows:

Cur-PLA-NO neuro-protection research for resisting cerebral ischemia reperfusion injury

1. Establishment of animal model

A C57BL/6 mouse MCAO model is established by adopting a wire-bolt blocking method, which comprises the following steps: a mouse MCAO model is prepared under a body type microscope, and 3% sodium pentobarbital is used as a mouse injection sedative to expose common carotid artery, internal carotid artery and external carotid artery. The external carotid artery to the internal carotid artery were surgically ligated with 4-0 nylon thread, the right middle cerebral artery was occluded with a silicone-coated monofilament for 30 minutes and then the tap was removed, the internal carotid artery was ligated and the wound sutured, and blood perfusion was resumed. The body temperature of the mice is kept constant from molding to the time of mouse recovery.

2. Dosing regimens

Taking 126 male C57BL/6 mice 23-25 g, randomly dividing MCAO preoperatively into 6 groups, namely a normal saline group, a normal control group, a pseudo-operation group, a Cur-PLA-NO group, a comparative Cur-PLA group and a positive control edaravone group, wherein each group comprises 21 mice, 3 mice are used for nerve function scoring research, 15 mice are used for cerebral infarction volume research, 3 mice are used for water maze behaviourology research, administration is carried out on each group at different time points, zero time points are taken to represent administration before MCAO model building, other time points are taken after model building, and the single administration dose of each group is 0.72 mg/kg.

Normal control group: healthy mice without any treatment were injected with normal saline at the tail vein.

The sham operation group: the plug is inserted into the bifurcation of the middle cerebral artery and stopped, other follow-up work is consistent with the MCAO model, and the tail vein is injected with normal saline.

Physiological saline group: MCAO model mice were injected with normal saline tail vein.

Cur-PLA-NO group: MCAO model mice, tail vein injection NO donor curcumin micellar solution.

Comparative Cur-PLA group: MCAO model mice, tail vein injection of non-NO donor curcumin micellar solution.

Positive control edaravone group: the edaravone solution was injected into the tail vein of MCAO model mice (prepared by dissolving edaravone powder in DMSO and diluting the solution with sterile water for injection to a DMSO concentration of 0.1% or less).

3. Test items and results

(1) Neurological scoring study

Each group of mice was dosed prior to modelling and the animals were allowed to recover 24 hours before assessment of behaviour and the mice were scored according to the Zea Longa criteria.

The scoring criteria were: 0 point, no nerve damage symptoms; 1 minute, the contralateral forepaw can not be fully extended out; 2, the rear part turns to the opposite side; 3 min, and performing non-precision drilling towards the opposite side; 4 minutes, the walking can not be carried out independently; and 5 points, unconsciousness is lost. The result shows that the mice in the normal saline group have obvious nerve motor dysfunction, the administration group can obviously improve the symptom of nerve dysfunction, and the nerve function score of the Cur-PLA-NO group is lower than that of the comparative Cur-PLA group and is equivalent to that of the positive control edaravone group (see figure 6).

(2) Cerebral infarction volume research (TTC staining)

Each group of mice was administered with a single dose at 0h, 3h, 6h, 12h and 24h, respectively, and after reperfusion for 48h, the brain tissue of the mice was taken for TTC staining, and the cerebral infarction volume of each group was measured.

The cervical vertebra is removed, the mouse is killed, the brain is taken out, the olfactory bulb is taken out, the frozen state is 30min, 2mm brain tissue coronal sections are continuously collected, 1% TTC solution is used for staining for 30min at 37 ℃, the peduncle area is white, and the non-peduncle area is red. Sections were photographed and analyzed using ImageJ software 1.51j8 (see fig. 7), and all data were statistically analyzed using SPSS 20.0 software (see fig. 8, P < 0.05, P < 0.01, P < 0.001, with differences noted when P < 0.05, and NS indicating no significant differences).

The results of fig. 7 and 8 show that the Cur-PLA-NO group can significantly reduce the cerebral infarction volume of mice compared to the saline group. The effect of Cur-PLA-NO on reducing the cerebral infarction volume is equivalent to that of the positive control edaravone group in 0h, 3h and 6h, and is obviously superior to that of the comparative Cur-PLA group. Under the condition of 0h (before modeling) administration, the drug effect is optimal, so in the later stage endothelial cell protection effect test, a single administration mode before modeling is adopted for further research.

(3) Behavioral study of Water maze

The Morris Water Maze (MWM) is mainly used for testing the learning and memory ability of experimental animals on spatial position and orientation.

The 6 groups of mice were administered once in the tail vein on

days

0, 3, 6, 9, 12 and 15 in this order, and the total dose was 6 times per group. Each group of mice was trained for directional navigation starting from day 15 after dosing. The time and trajectory that the mouse found the platform in the first quadrant of the water maze was observed at the same time every day. After continuous training for 5 days, the residence time and the track of different groups of mice in a target quadrant are monitored by adopting a water maze track tracking system.

The results are shown in fig. 9 and fig. 10 (P is less than 0.05, P is less than 0.01, when P is less than 0.05, the difference is significant, and NS shows NO significant difference), which indicates that the residence time of the Cur-PLA-NO group mice in the target quadrant is significantly longer than that of the comparative Cur-PLA group, and is basically the same as that of the positive control edaravone group, and indicates that the continuous intravenous administration of Cur-PLA-NO group has a certain synergistic effect on the recovery of the neural memory function after cerebral ischemia reperfusion of the mice.

(II) research on endothelium relaxation effect of Cur-PLA-NO

NO can obviously improve the neural prognosis of ischemic stroke, and is mainly generated by three nitric oxide synthases, namely endothelial NO (eNOS), Induced NO (iNOS) and neural NO (nNOS), and plays different roles in the ischemic stroke. By measuring the generation of three nitric oxide synthases before and after the administration, the effects of the preparation on endothelium relaxation and blood circulation improvement are researched.

1. Dosing regimens

30 male C57BL/6 mice 23-25 g are taken, MCAO models are prepared according to the method shown in the item of animal model establishment under the 'one-Cur-PLA-NO anti-cerebral ischemia reperfusion injury neuroprotection research', the MCAO models are randomly divided into 5 groups before modeling, namely a normal saline group, a normal control group, a Cur-PLA-NO group, a comparative Cur-PLA group and a positive control edaravone group, and each group comprises 6 mice. Preferably, the single administration mode before the molding is carried out, and the administration dosage is 0.72 mg/kg.

2. Test items and results

After administration and modeling, each group was divided into 2 groups at random, 3 mice in each group were killed by cervical dislocation at the prescribed time points (6 h, 12 h) and brain tissues were taken out, olfactory bulbs and cerebellum were removed, left and right brains were weighed individually, 9mL of PBS solution was added to 1g of tissue, samples were homogenized thoroughly (30 Hz, 90 s) by a full-automatic sample rapid grinding apparatus, centrifuged for 20min (4 ℃, 3000 r/min), supernatants were carefully collected, and three nitric oxide synthases of eNOS, iNOS and nNOS were determined.

The results are shown in FIG. 11 (P < 0.05, P < 0.01, and P < 0.001, and when P < 0.05 the difference was significant, NS showed NO significant difference), and the groups administered (Cur-PLA-NO, Cur-PLA, and edaravone) were able to reduce nNOS expression to some extent at 6h and 24h after injection, relative to the saline group [ see (a) in FIG. 11 and (b) in FIG. 11 ], which is related to the use of early stage inflammatory drugs. Compared with the healthy mice of the normal control group, the iNOS level of the mice of the normal saline group shows a remarkable rising trend in two periods, and the phenomenon is particularly obvious in the damaged right brain, which is related to the gradual rise of the iNOS level of the central nervous system after the inflammatory stimulation. However, the expression of iNOS in the right brain of each group of mice was significantly reduced after the drug injection, and iNOS injury was alleviated [ see (c) in FIG. 11 and (d) in FIG. 11 ]. For the expression of eNOS, except the group of Cur-PLA-NO, other drugs did not increase the expression of eNOS which is beneficial to the endothelium relaxation effect [ see (e) in FIG. 11 and (f) in FIG. 11 ], which is caused by that NO released by Cur-PLA-NO stimulates brain endothelial cells to further regulate cerebral microvascular blood flow. The results show that Cur-PLA-NO can up-regulate eNOS through the NO donor material, inhibit iNOS and nNOS, expand microvasculature, improve cerebral microcirculation, and promote the recovery of ischemia reperfusion injury and vasodilatation of mice.

Finally, it should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and not intended to limit the present invention, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or some technical features thereof can be replaced. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An NO donor-type micelle composition, characterized in that: the NO donor-type micelle composition comprises an NO donor-type micelle material and an insoluble drug with an anti-cerebral ischemia-reperfusion injury neuroprotection effect, wherein the insoluble drug is curcumin.

2. The NO donor-type micelle composition of claim 1, wherein: the NO donor micelle composition is prepared from the following raw materials in parts by weight: 1-10 parts of curcumin, 1-30 parts of NO donor micelle material, 1-30 parts of surfactant and 1-60 parts of freeze-drying protective agent.

3. The NO donor-type micelle composition of claim 2, wherein: the NO donor micelle composition is prepared from the following raw materials in parts by weight: 6 parts of curcumin, 15 parts of NO donor micelle material, 15 parts of surfactant and 30 parts of freeze-drying protective agent.

4. The NO donor-type micelle composition of claim 2, wherein: the NO donor type micelle material is composed of one or more amphiphilic block polymer NO donors, and the amphiphilic block polymer NO donor is an amphiphilic diblock polymer NO donor, an amphiphilic triblock polymer NO donor or an amphiphilic multiblock polymer NO donor.

5. The NO donor-type micelle composition according to claim 2, wherein: the surfactant is selected from one or more of Tween, polyoxyethylene fatty alcohol ether, polyoxyethylene alkylphenol ether, polyoxyethylene fatty acid ester, polyoxyethylene polyoxypropylene copolymer, fatty acid sorbitan and polyoxyethylene sorbitan fatty acid ester.

6. The NO donor-type micelle composition of claim 2, wherein: the freeze-drying protective agent is selected from one or more of trehalose, mannitol, sucrose, glucose, sodium chloride, lactose, sorbitol, dextran, glycerol and glycine.

7. The NO donor-type micelle composition according to claim 4, wherein: the NO donor type micelle material is mPEG-PLA-NO, the mPEG is polyethylene glycol monomethyl ether with the molecular weight of 500-5000, the PLA is polylactic acid with the molecular weight of 500-5000, and the structural formula of the mPEG-PLA-NO is as follows:

；

wherein n1=6 ~ 100, n2=6 ~ 100.

8. A method for preparing the NO donor-type micelle composition according to any one of claims 2 to 7, wherein: the preparation method comprises the following specific steps: mixing curcumin, an NO donor micelle material and a surfactant according to parts by weight, putting the mixture into an organic solvent for full dissolution, adding purified water, stirring uniformly, adding a freeze-drying protective agent, sterilizing, and freeze-drying to obtain a finished product of the NO donor micelle composition.

9. The method for preparing an NO donor-type micelle composition according to claim 8, wherein: the preparation method comprises the following specific steps: mixing 6 parts of curcumin, 15 parts of mPEG-PLA-NO and 15 parts of Tween surfactant, putting the mixture into 20 mL of ethanol for full dissolution, adding purified water, stirring uniformly, adding 30 parts of mannitol, sterilizing, and freeze-drying to obtain the finished product of the NO donor micelle composition.

10. Use of the NO-donor-type micelle composition according to any one of claims 1 to 7 for preparing a medicament for neuroprotection and relapse prevention in cerebral ischemia-reperfusion injury.