CN112675149B

CN112675149B - Preparation method and application of brain-targeted drug delivery system loaded with cyclosporine A

Info

Publication number: CN112675149B
Application number: CN202110001630.0A
Authority: CN
Inventors: 周四元; 刘道洲; 纪奇峰; 成颖; 刘苗; 宦梦蕾; 叶威良; 张邦乐
Original assignee: Air Force Medical University of PLA
Current assignee: Air Force Medical University of PLA
Priority date: 2021-01-04
Filing date: 2021-01-04
Publication date: 2022-09-16
Anticipated expiration: 2041-01-04
Also published as: CN112675149A

Abstract

The invention relates to the field of biological pharmacy, and discloses a preparation method and application of a cyclosporine A-loaded efficient brain-targeted drug delivery system. The drug delivery system comprises CsA and Fn, and CsA is wrapped in a cage-shaped structure of Fn by a solvent volatilization method by utilizing the characteristics that ferritin is denatured in acetone and can be renatured in water. The CsA @ Fn can pass through a blood brain barrier and accumulate in an ischemic area through a transferrin receptor on the surface of a brain capillary endothelial cell, and on one hand, the CsA @ Fn can protect the integrity of the blood brain barrier and reduce the accumulation of inflammatory factors in the cerebral ischemic area; on the other hand, CsA @ Fn taken by neuron cells can inhibit opening of mitochondrial permeability transition pore of neuron cells, reduce release of mitochondrial ROS and cytochrome C, inhibit apoptosis of neuron cells and play a role in neuroprotection.

Description

Preparation method and application of brain-targeted drug delivery system loaded with cyclosporine A

Technical Field

The invention relates to the field of biological pharmacy, in particular to a novel targeted drug delivery system for treating cerebral ischemia/reperfusion injury.

Background

Ischemic stroke is one of the leading causes of disability and death. At present, the main treatment means of ischemic stroke is to recover cerebral blood perfusion in an ischemic area as soon as possible, but when ischemic brain tissues are perfused again by blood, a series of secondary injuries can be caused. The mechanism of cerebral ischemia/reperfusion injury is complex and comprises a plurality of pathophysiological changes, such as energy metabolism disorder, oxidative stress, excitatory amino acid toxicity, inflammatory injury and the like. Recent studies have shown that mitochondria play an important role in cerebral ischemia/reperfusion injury. In cerebral ischemia, the supply of oxygen and glucose in cerebral ischemia tissues is insufficient, the synthesis of adenosine triphosphate in cells is blocked, the energy metabolism of the cells is disturbed, and the accumulation of intracellular lactic acid, ion disorder and the like are further caused. During reperfusion, the pH of ischemic brain tissue rapidly rises, and a voltage-gated channel on the inner mitochondrial membrane opens, resulting in abnormal opening of a mitochondrial permeability transition pore (mPTP). Abnormal opening of mPTP can cause osmotic swelling and mitochondrial outer membrane rupture, free radicals are generated in large quantity and released into cytoplasm, and excessive free radicals in the cytoplasm can directly cause damage to biomacromolecular substances such as protein, nucleic acid, lipid and the like, can damage a calcium ion pump on a mitochondrial membrane, reduce the mitochondrial membrane potential and further aggravate opening of m PTP, and form malignant cycle. Meanwhile, a large number of free radicals in an ischemic area can also activate metalloprotease proprotein, degrade a neurovascular matrix, cause blood brain barrier damage and cause more serious damage to cells in the ischemic area.

Cyclosporin a (CsA) is a classical mPTP inhibitor. CsA can be combined with mitochondrion cyclophilin, so as to inhibit opening of mitochondrion mPTP, reduce release of ROS, cytochrome c and the like in cytoplasm, reduce apoptosis and play a role in neuroprotection. However, after CsA enters blood circulation, it is mainly distributed in tissues with high fat content, such as fat, liver, adrenal gland and pancreas, but rarely enters the central nervous system.

The ferritin particles are hollow spheroids and the hollow cavity can be used to encapsulate the drug. Moreover, the ferritin drug carrier can recognize and combine with transferrin receptor 1(TfR1) on the surface of brain capillary endothelial cells, and the ferritin drug carrier can penetrate through a blood brain barrier under the mediation of TfR1, and enrich and release self-carried chemotherapeutic drugs in a cerebral ischemic area. Therefore, a CsA-loaded Ferritin (Fn) nanoparticle (CsA @ Fn) is constructed, distribution of CsA in a cerebral ischemia area is increased, opening of nerve cells mPTP is inhibited, release of ROS in cytoplasm is inhibited, and accordingly damage of nerve cells is reduced; meanwhile, the reduction of ROS in ischemic tissues can also avoid the over-activation of metalloprotease proprotein, reduce the degradation of neurovascular matrix, play a role in protecting blood brain barrier, and improve the effect of treating cerebral ischemia/reperfusion injury by dual ways.

Disclosure of Invention

The invention aims to provide a preparation method and application of a brain targeting drug delivery system loaded with cyclosporine A, and the novel method is adopted to load CsA into a ferritin hollow structure and is used for treating cerebral ischemia/reperfusion injury. The CsA @ Fn can pass through a blood brain barrier and be enriched in a cerebral ischemia area through the mediation of TfR1 on the surface of a brain capillary endothelial cell, so that CsA is released, the opening of a nerve cell mPTP is inhibited, and the release of ROS in cytoplasm is inhibited, thereby reducing the damage of the nerve cell; meanwhile, the reduction of ROS in ischemic tissues can also avoid the over-activation of metalloproteinase proprotein, reduce the degradation of neurovascular matrix, play a role in protecting blood brain barrier, and improve the effect of treating cerebral ischemia/reperfusion injury by dual ways.

The technical scheme of the invention is as follows: a preparation method of a brain targeting drug delivery system loaded with cyclosporine A is characterized in that ferritin and CsA are dispersed in 55% acetone solution (the volume ratio of acetone to water is 55:45) by utilizing the characteristics of denaturation of ferritin in acetone and renaturation of ferritin distilled water, the acetone is volatilized and removed after the mixture is stirred for 30min in a closed manner, ferritin renaturation is carried out, the solubility of CsA is reduced, nanoparticles CsA @ Fn are formed, the CsA @ Fn can penetrate through a blood brain barrier, the CsA is delivered to a cerebral ischemia area and effectively accumulated in the cerebral ischemia tissue, the density of nerve cells in the cerebral ischemia area is remarkably improved, and the brain ischemia/reperfusion injury is treated, wherein the particle size of the CsA @ Fn is 25nm, the potential is mV, and the drug loading capacity of the CsA is 10.4%.

The technical scheme of the invention has the characteristics that:

the method utilizes the characteristics of denaturation of ferritin in acetone and renaturation of ferritin distilled water, and comprises the steps of dispersing ferritin and CsA in 55% acetone (acetone: water: 55:45), stirring for 30min in a sealed manner, volatilizing to remove acetone, renaturing ferritin, reducing the solubility of CsA, and loading CsA into a ferritin hollow structure.

Secondly, the invention utilizes the characteristic that ferritin can be combined with TfR1 on the surface of the brain capillary endothelial cell to deliver CsA to the cerebral ischemia area for treating cerebral ischemia/reperfusion injury.

The invention has the innovativeness that: firstly, a novel ferritin drug loading method is developed, and the characteristics of denaturation of ferritin in acetone and renaturation in distilled water are utilized to load drugs into a hollow structure of ferritin. Secondly, a drug delivery system which can penetrate through a blood brain barrier, reduce nerve cell injury, protect the blood brain barrier and treat cerebral ischemia/reperfusion injury is constructed.

Drawings

FIG. 1 CsA @ Fn particle size. (A) The particle size distribution maps of CsA @ Fn and Fn; (B) transmission electron micrographs of CsA @ Fn and Fn.

Fig. 2 stability of CsA @ Fn in different media. Wherein n is 5, the total weight of the compound,

FIG. 3 kinetics of the cumulative release of CsA by CsA @ Fn in PBS buffer at different pH values. Wherein n is 3, and n is 3,

fig. 4 efficiency of CsA @ Fn penetration of the blood brain barrier in vitro. n is 3, and n is 3,

**P<0.01, compared to free CsA, ^## P<0.01, compared to CsA @ BSA.

Fig. 5 distribution of CsA @ Fn in the brains of mice model ischemic stroke.

Fig. 6 influence of CsA @ Fn on ischemic stroke infarct size. (A) Typical picture of cerebral infarction of each group of mice after drug treatment. (B) Relative infarct volume of brain tissue in each group of mice after drug treatment. n is 3, and n is 3,

**P<0.01, compared with the normal saline group, ^## P<0.01, compared to CsA @ Fn.

FIG. 7 effect of ROS in ischemic stroke mice after drug treatment.

FIG. 8 shows the effect of drug treatment on apoptosis in ischemic brain region of mouse in cerebral arterial thrombosis.

Figure 9 effect of drug treatment on evans leakage in mice with ischemic stroke.

Figure 10 neurological scores of groups of mice after drug treatment. n is equal to 3, and the total content of the N,

FIG. 11 Neisseria staining examines the effect of drug treatment on nerve cells of mice with ischemic stroke.

FIG. 12 silver plating staining to examine the effect of drug treatment on the neural myelin sheath of ischemic stroke mice.

Detailed Description

1 research methods

1.1 preparation and characterization of CsA @ Fn

(1) Preparing CsA @ Fn by a solvent-based denaturation/renaturation self-assembly method: the CsA @ Fn is prepared by a solvent volatilization method by utilizing the characteristics of denaturation of ferritin in acetone and renaturation in water. Weighing 50mg of ferritin, adding into 50ml of mixed solvent (acetone: water: 55:45), and stirring at room temperature under sealed condition for 30min to denature ferritin; weighing 150mg CsA, dissolving in 0.5ml acetone, adding into the ferritin solution, sealing and stirring at room temperature for 10min to mix ferritin and CsA; and adding the solution into an evaporation dish, stirring at room temperature to promote acetone volatilization, filtering for 10h by using a microporous filter membrane with the pore diameter of 0.22 mu m, and removing CsA which is not wrapped in ferritin to obtain CsA @ Fn.

(2) Particle size and potential characterization of CsA @ Fn: after diluting 100. mu.l of the CsA @ Fn solution with 1.5ml of distilled water, the particle size, zeta potential and polydispersity index (PDI) of CsA @ Fn were measured using a zeta potential and laser particle size meter. Meanwhile, a small amount of CsA @ Fn solution is dropwise added on a glass sheet, the glass sheet is placed in a vacuum drying oven at the temperature of 35 ℃ for drying for 2 hours, gold is plated, and the form of CsA @ Fn is observed by adopting a field emission scanning electron microscope.

(3) CsA @ Fn stability study: a certain amount of CsA @ Fn was measured, and dispersed in distilled water, PBS (pH 7.4), 10% FBS and DMEM solutions, and the stability of CsA @ Fn in the above medium was measured by a zeta potential and laser particle size analyzer.

(4) Determination of CsA @ Fn drug loading: the column was a Dikma ODS C18 column (250mm × 4.6mm, 5 μm), the detection wavelength was 210nm, the mobile phase composition was acetonitrile/water 90/10(v/v), the flow rate: 1.0ml/min, sample size: 20 μ l, column temperature: at 55 ℃. Weighing 10mg of CsA @ Fn freeze-dried powder, adding 1ml of methanol, performing ultrasonic treatment for 2min, filtering with a 0.22 mu m microporous membrane, injecting 20 mu l of sample according to the chromatographic conditions, recording the peak area, calculating the concentration of CsA according to the working curve, and calculating the drug loading of CsA in CsA @ Fn.

(5) Drug release profile of CsA @ Fn in different media: weighing a certain amount of CsA @ Fn, dispersing in PBS buffer solutions with different pH values (5.0 and 7.4), transferring into a dialysis bag with cut-off molecular weight of 5000Da, placing in 60ml of PBS buffer solution with corresponding pH value, and dialyzing at constant temperature of 37 ℃. At 1, 2, 4, 8, 12, 24, 48 and 96h, 200 mul of CsA @ Fn in the dialysis bag is taken and added with 200 mul of blank release medium, the concentration of CsA is measured by HPLC, the concentration of ferritin is measured by BCA method, the cumulative drug release amount is calculated, and a drug release curve is drawn.

1.2 the efficiency of CsA @ Fn penetration of the in vitro blood brain barrier

Obtaining bEnd3 cells, adding DMEM culture solution containing 10% fetal calf serum, and the cell concentration is 1x10 ⁵ At each ml, 300. mu.l was added to the transwell feeding cell, and DMEM medium containing 10% fetal bovine serum was added to the receiving cell, and the medium was changed every 2 days. After 7 days, detecting the resistance value between the transwell receiving pool and the transwell supplying pool by using a resistance meter, and when the resistance value is more than 200 omega/cm ² In time, the in vitro blood brain barrier model is successfully constructed. And (2) absorbing and removing the culture solution in the supply pool and the receiving pool, adding CsA @ Fn (the concentration of CsA is 50 mu g/ml) into the supply pool, adding serum-free DMEM culture solution into the receiving pool, keeping the liquid level of the supply pool and the liquid level of the receiving pool flush, incubating for 2, 4 and 8 hours, collecting the culture solution in the receiving pool, adding 200 mu l of methanol after freeze-drying, performing ultrasonic treatment for 2min, filtering by using a 0.22 mu m microporous filter membrane, detecting the concentration of CsA in the receiving pool by using HPLC (high performance liquid chromatography), and calculating the transport efficiency of CsA @ Fn across the blood brain barrier.

1.3 distribution of CsA @ Fn in mouse brain in stroke

(1) And blocking blood flow of the right middle cerebral artery of the mouse for 1h by adopting a wire-tying method to prepare a mouse MCAO model. Mice were anesthetized with isoflurane and fixed on a thermostatic table. An incision was cut in the middle of the mouse neck, the common carotid artery, the internal carotid artery and the external carotid artery were separated, the right common carotid artery and the internal carotid artery were pulled up, the right external carotid artery was ligated to the proximal brain end, and the external carotid artery was cut off. An incision is cut at the stump of the external carotid artery and a wire plug is inserted. After the wound was sutured, the mice were placed in an incubator for warming and insulation. After the oxygen deficiency lasts for 1h, the suture incision is cut, the thread plug is pulled out, and the incision is sutured. 100 mul of Cy7.5-labeled CsA @ Fn (the CsA concentration is 500 mug/ml) is injected into the tail vein within 5min, brain tissue is obtained after 24h, and after TTC staining, the distribution of the CsA @ Fn in the area of the peduncle is observed by using a living body imaging instrument. Organ tissues such as heart, liver, spleen, lung, kidney and the like are obtained, and the distribution of CsA @ Fn in normal tissues is observed by a living body imaging instrument.

(2) The brain tissue is fixed in 4% paraformaldehyde, paraffin is sliced, and the distribution of CsA @ Fn in the cerebral infarction area is observed by adopting a laser confocal microscope.

1.4 therapeutic action and mechanism of CsA @ Fn on cerebral arterial thrombosis

(1) Animal grouping: sham (sham), surgery (MACO), free CsA (2.5mg/kg), CsA @ BAS (2.5mg/kg), CsA @ Fn (1.25, 2.5 mg/kg).

(2) Evaluation of CsA @ Fn efficacy in treating ischemic stroke: after the model is successfully established, the drug is administered according to tail vein injection of the experimental group, after 24 hours, the nerve function of each group of mice is scored by adopting a 5-point method, and the nerve protection effect of CsA @ Fn on MACO mice is inspected; obtaining mouse brain tissue, cutting into 5 pieces, staining TTC, and observing the influence of CsA @ Fn on the cerebral infarction area of the MACO mouse by using a stereoscopic microscope; obtaining mouse brain tissue, paraffin sectioning, H & E staining and TUNEL staining, and observing the influence of CsA @ Fn on MACO mouse on the shape and apoptosis of mouse brain tissue.

(3) Evaluation of the protective effect of CsA @ Fn on the blood brain barrier: after the MACO mice are administrated in the tail vein for 24 hours, the tail vein is injected with the ivalas solution, after 30min, the heart is perfused with normal saline to obtain brain tissue, and the distribution of the ivalas in the brain tissue is observed; and (3) slicing paraffin, observing the distribution of Evans blue in brain tissues by adopting a fluorescence microscope, and inspecting the protection effect of CsA @ Fn on blood brain barrier.

(4) Evaluation of the protective effect of CsA @ Fn on cerebral neurons: after 24h of tail vein administration of MACO mice, brain tissues are obtained, paraffin sections are cut, Nie's staining and silver plating staining are carried out, and the protection effect of CsA @ Fn on cerebral neurons is observed.

2 results of the experiment

2.1 characterization of CsA @ Fn

The particle size and potential of CsA @ Fn were measured by a nanometer particle size and zeta potential analyzer, and the results are shown in FIG. 1. The particle size of CsA @ Fn is 25nm, the particle size is basically the same as that of Fn, the potential is mV, and the drug loading of CsA is 10.4%. The results of the stability experiment for CsA @ Fn are shown in fig. 2, and the particle size of CsA @ Fn in distilled water, PBS buffer, 10% FBS solution, and DMEM solution did not increase significantly within 5 days, indicating that CsA @ Fn remains stable within 5 days in the above medium.

In vitro drug release characteristics of CsA @ Fn were examined by HPLC, and the results are shown in FIG. 3. CsA @ Fn in PBS buffer at pH5.0, the release of CsA was significantly increased, and over 75% of CsA could be released within 24 h.

2.2 CsA @ Fn Effect on penetrating the blood brain Barrier

First, we constructed an in vitro blood brain barrier model and investigated the efficiency of CsA @ Fn in crossing the blood brain barrier in vitro. The results show that CsA @ Fn is able to cross the blood-brain barrier time-dependently and transport efficiency is significantly higher than free CsA and CsA @ BSA, with 38.5% of CsA @ Fn being able to cross the blood-brain barrier when the incubation time was 8h (fig. 4).

Next, we established a perfusion model of mouse cerebral ischemia, and examined the distribution of CsA @ Fn ischemic brain tissue. The results show that the distribution of CsA @ Fn in the brain tissue of the sham-operated group is significantly lower than that in the brain tissue of the model mice; the distribution of CsA @ Fn in the brain tissue of the model group was significantly higher than CsA @ BSA, and the distribution of CsA @ Fn in the ischemic brain tissue was significantly higher than that in the normal brain tissue (fig. 5).

2.3 therapeutic Effect of CsA @ Fn on cerebral ischemia-reperfusion injury

TTC staining results showed that CsA @ Fn was able to dose-dependently reduce cerebral infarct size in mice, and the effect was significantly better than that of the CsA @ BSA group and the free CsA group (fig. 6). H & E staining results show that in an ischemic area of an MCAO model mouse, an obvious infarction focus can be found, a large amount of inflammatory cell infiltration is accompanied, and meanwhile, the nucleus of a neuron is condensed and chromatin is condensed. After CsA @ Fn treatment, no obvious infarction focus is found in the cerebral ischemia area, and inflammatory cell infiltration is reduced. ROS staining results show that ROS in the cerebral ischemic region is remarkably enhanced after cerebral ischemia is perfused, CsA @ Fn can reduce ROS in the cerebral ischemic region in a dose-dependent manner, and the effect is better than that of CsA and CsA @ BSA at the same dose (figure 7). TUNEL staining results showed that CsA @ Fn was able to dose-dependently reduce the number of apoptotic cells and the effect was significantly better than the CsA @ BSA and free CsA groups (fig. 8).

2.4 neuroprotective Effect of CsA @ Fn

After 30min of tail vein injection of Evans blue, the accumulation of Evans blue in the cerebral ischemic tissue can be obviously observed in the cerebral tissue of MCAO model mice, while the distribution of Evans blue can hardly be observed in the cerebral tissue of CsA @ Fn administration group mice, which suggests that CsA @ Fn can protect the integrity of the blood brain barrier and reduce the penetration of Evans blue in the cerebral tissue (FIG. 9).

The nerve functions of the mice in each group are scored by a 5-point method, and the results show that the nerve function score of the MCAO model mouse is the highest and reaches 3.4 points, and the nerve function score of the CsA @ Fn treatment group is the lowest and is 1.1 points, which indicates that the CsA @ Fn can reduce the damage of cerebral arterial thrombosis to the nervous system (figure 10). Nie's staining and silver plating staining showed that CsA @ Fn was able to reduce nerve damage caused by cerebral ischemia-reperfusion, significantly increasing the density of nerve cells in cerebral ischemic areas (FIGS. 11-12).

And 3, conclusion:

the denaturation/renaturation self-assembly method based on the solvent can load the CsA into a ferritin (Fn) nano cage, and the solubility of the CsA is improved. CsA @ Fn is able to cross the blood brain barrier and accumulate efficiently in ischemic brain tissue. Compared with the CsA and the CsA @ BSA, the CsA @ Fn can improve the integrity of the blood brain barrier of the MCAO mouse, reduce the cerebral infarction area and the damage of neurons, obviously improve the protection effect of the CsA on cerebral ischemia-reperfusion injury, and has a certain application prospect.

Claims

1. A preparation method of a brain targeting drug delivery system loaded with cyclosporine A is characterized in that a solvent volatilization method is adopted to prepare CsA @ Fn by utilizing the characteristics of denaturation of ferritin in acetone and renaturation in water; weighing 50mg of ferritin, adding into 50ml of acetone/water mixed solvent, wherein the volume ratio of acetone to water is 55:45, and stirring at room temperature for 30min under sealed condition to denature ferritin; weighing 150mg CsA, dissolving in 0.5ml acetone, adding into the ferritin solution, sealing and stirring at room temperature for 10min to mix ferritin and CsA; and adding the solution into an evaporation dish, stirring at room temperature to promote acetone volatilization, filtering for 10h by using a microporous filter membrane with the pore diameter of 0.22 mu m, and removing CsA which is not wrapped in ferritin to obtain CsA @ Fn.

2. Use of the cyclosporin a-loaded brain-targeted delivery system of claim 1 in the preparation of a medicament for the treatment of cerebral ischemia and reperfusion injury.