CN112675149B - Preparation method and application of brain-targeted drug delivery system loaded with cyclosporin A - Google Patents

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 cyclosporin A

technical field

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

Background technique

Ischemic stroke is one of the leading causes of disability and death. At present, the main treatment for ischemic stroke is to restore cerebral blood perfusion in the ischemic area as soon as possible, but when the ischemic brain tissue is reperfused with blood, it will cause a series of re-injury. The mechanism of cerebral ischemia/reperfusion injury is complex, including many pathophysiological changes, such as energy metabolism disorders, oxidative stress, excitatory amino acid toxicity and inflammatory damage. Recent studies have shown that mitochondria play an important role in cerebral ischemia/reperfusion injury. During cerebral ischemia, the supply of oxygen and glucose in cerebral ischemic tissue is insufficient, and the synthesis of adenosine triphosphate in cells is blocked, which leads to the disorder of cellular energy metabolism, and further causes the accumulation of intracellular lactic acid and ion disturbance. During reperfusion, the pH of the ischemic brain tissue rises rapidly, and the voltage-gated channels on the inner mitochondrial membrane are opened, resulting in the abnormal opening of the mitochondrial permeability transition pore (mPTP). The abnormal opening of mPTP can cause osmotic swelling and rupture of the outer membrane of mitochondria, and free radicals are produced and released into the cytoplasm. Excessive free radicals in the cytoplasm can directly cause damage to biological macromolecules such as proteins, nucleic acids and lipids. , which can destroy the calcium ion pump on the mitochondrial membrane, reduce the mitochondrial membrane potential and further aggravate the opening of mPTP, forming a vicious circle. At the same time, a large number of free radicals in the ischemic area can also activate the metalloproteinase proproteins, degrade the neurovascular matrix, lead to the breakdown of the blood-brain barrier, and cause more serious damage to the cells in the ischemic area.

Cyclosporine A (cyclosporine A, CsA) is a classic mPTP inhibitor. CsA can combine with mitochondrial cyclosporine, thereby inhibiting the opening of mitochondrial mPTP, reducing the release of ROS and cytochrome c in the cytoplasm, reducing cell apoptosis, and thus exerting neuroprotective effects. However, after CsA enters the blood circulation, it is mainly distributed in tissues with high fat content, such as fat, liver, adrenal gland and pancreas, etc., but it rarely enters the central nervous system.

The ferritin particle is a hollow spheroid with a hollow lumen that can be used to encapsulate the drug. Moreover, the ferritin drug carrier can recognize and bind to the transferrin receptor 1 (TfR1) on the surface of brain capillary endothelial cells, mediated by TfR1, cross the blood-brain barrier, enrich in the ischemic area of the brain and release its own carry of chemotherapy drugs. Therefore, we constructed CsA-loaded ferritin (Fn) nanoparticles (CsA@Fn) to increase the distribution of CsA in the brain ischemic area, inhibit the opening of mPTP in nerve cells, and inhibit the release of ROS in the cytoplasm. Thereby reducing the damage of nerve cells; at the same time, the reduction of ROS in ischemic tissue can also avoid the excessive activation of metalloproteinase proproteins, reduce the degradation of neurovascular matrix, protect the blood-brain barrier, and improve the treatment of cerebral ischemia through dual approaches. /Effect of reperfusion injury.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a preparation method and application of a brain-targeted drug delivery system loaded with cyclosporin A, adopt a new method to load CsA into the hollow structure of ferritin, and use it for the treatment of cerebral ischemia/ reperfusion injury. CsA@Fn can cross the blood-brain barrier mediated by TfR1 on the surface of brain capillary endothelial cells and accumulate in the brain ischemic area, releasing CsA, inhibiting the opening of mPTP in nerve cells, and inhibiting the release of ROS in the cytoplasm, thereby reducing the Nerve cell damage; at the same time, the reduction of ROS in ischemic tissue can also avoid the excessive activation of metalloproteinase proproteins, reduce the degradation of neurovascular matrix, protect the blood-brain barrier, and improve the treatment of cerebral ischemia/regeneration through dual approaches Effects of perfusion injury.

The technical scheme of the present invention is: a preparation method of a brain-targeted drug delivery system loaded with cyclosporin A, which is characterized in that the ferritin is denatured in acetone and renatured in distilled water by using the characteristics of CsA was dispersed in 55% acetone solution ((the volume ratio of acetone to water was 55:45), and after 30 min of airtight stirring, the acetone was removed by volatilization, the ferritin was renatured, and the solubility of CsA decreased to form nanoparticles CsA@Fn; CsA @Fn can cross the blood-brain barrier, deliver CsA into the cerebral ischemic area, effectively accumulate in the cerebral ischemic tissue, significantly increase the density of nerve cells in the cerebral ischemic area, and be used for the treatment of cerebral ischemia/reperfusion injury ; where the particle size of CsA@Fn is 25 nm, the potential is mV, and the drug loading of CsA is 10.4%.

Features of the technical solution of the present invention:

①The present invention utilizes the characteristics of denaturation of ferritin in acetone and renaturation in distilled water to disperse ferritin and CsA in 55% acetone (acetone:water=55:45), and after airtight stirring for 30min, volatilize and remove acetone, Ferritin renatures, while the solubility of CsA decreases, loading CsA into the hollow structure of ferritin.

②The present invention utilizes the feature that ferritin can combine with TfR1 on the surface of cerebral capillary endothelial cells to deliver CsA into the cerebral ischemia area for the treatment of cerebral ischemia/reperfusion injury.

Innovation of the present invention: ① A new drug loading method of ferritin is developed, which utilizes the characteristics of denaturation of ferritin in acetone and renaturation in distilled water to load the drug into the hollow structure of ferritin. ②Constructed a drug delivery system that can cross the blood-brain barrier while reducing nerve cell damage and protecting the blood-brain barrier to treat cerebral ischemia/reperfusion injury.

Description of drawings

Fig. 1 Particle size of CsA@Fn. (A) Particle size distributions of CsA@Fn and Fn; (B) TEM images of CsA@Fn and Fn.

Fig. 2 Stability of CsA@Fn in different media. where n=5,

Figure 3. Kinetic curves of cumulative release of CsA from CsA@Fn in PBS buffers with different pH. where n=3,

**P<0.01，与游离CsA相比，^##P<0.01，与CsA@BSA相比。Fig. 4 Efficiency of CsA@Fn penetrating the blood-brain barrier in vitro. n=3,

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

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

**P<0.01，与生理盐水组相比，^##P<0.01，与CsA@Fn相比。Fig. 6 The effect of CsA@Fn on the infarct size of ischemic stroke. (A) Typical pictures of cerebral infarction in mice in each group after drug treatment. (B) The relative infarct volume of brain tissue in each group of mice after drug treatment. n=3,

**P<0.01 vs. saline group, ^## P<0.01 vs. CsA@Fn.

Fig. 7 Effects of drug treatment on ROS in ischemic area of ischemic stroke mice.

Figure 8. Effect of drug treatment on apoptosis of ischemic cerebral ischemic area in mice with ischemic stroke.

Figure 9 Effect of drug treatment on Evanslan leakage in ischemic stroke mice.

**P<0.01，与生理盐水组相比，^##P<0.01，与CsA@Fn相比。Figure 10. Neurological function scores of mice in each group after drug treatment. n=3,

**P<0.01 vs. saline group, ^## P<0.01 vs. CsA@Fn.

Figure 11 Nissl staining to examine the effects of drug treatment on nerve cells in mice with ischemic stroke.

Figure 12 Silver-plated staining to investigate the effect of drug treatment on the nerve myelin of ischemic stroke mice.

Detailed ways

1 Research methods

1.1 Preparation and characterization of CsA@Fn

(1) Preparation of CsA@Fn by solvent-based denaturation/renaturation self-assembly method: CsA@Fn was prepared by solvent evaporation method by taking advantage of the characteristics of ferritin denaturation in acetone and renaturation in water. Weigh 50 mg of ferritin, add it to 50 ml of mixed solvent (acetone: water = 55:45), seal and stir at room temperature for 30 min to denature ferritin; weigh 150 mg of CsA, dissolve it in 0.5 ml of acetone and add it to the above ferritin solution , sealed and stirred at room temperature for 10 minutes to fully mix ferritin and CsA; the above solution was added to an evaporating dish, and stirred at room temperature to promote the volatilization of acetone. CsA in , namely CsA@Fn.

(2) Characterization of particle size and potential of CsA@Fn: Take 100 μl of the above CsA@Fn solution, add 1.5 ml of distilled water to dilute, and use zeta potential and laser particle size analyzer to measure the particle size, zeta potential and polydispersity coefficient of CsA@Fn (PDI). At the same time, a small amount of CsA@Fn solution was taken, dropped on a glass slide, dried in a vacuum drying oven at 35 °C for 2 h, and plated with gold. The morphology of CsA@Fn was observed by field emission scanning electron microscopy.

(3) Investigation of the stability of CsA@Fn: A certain amount of CsA@Fn was measured and dispersed in distilled water, PBS (pH 7.4), 10% FBS and DMEM solutions, respectively. Stability in the above media.

(4) Determination of CsA@Fn drug loading: the chromatographic column is Dikma ODS C18 column (250mm×4.6mm, 5μm), the detection wavelength is 210nm, and the mobile phase composition is acetonitrile/water=90/10(v/v), Flow rate: 1.0 ml/min, injection volume: 20 μl, column temperature: 55°C. Weigh 10 mg of CsA@Fn lyophilized powder, add 1 ml of methanol, sonicate for 2 min, filter with 0.22 μm microporous membrane, inject 20 μl of sample according to the above chromatographic conditions, record the peak area, calculate the concentration of CsA according to the working curve, and calculate the CsA Drug loading of CsA in @Fn.

(5) Drug release characteristics of CsA@Fn in different media: Weigh a certain amount of CsA@Fn, disperse it in PBS buffer solutions of different pH (5.0, 7.4), and move it to a dialysis bag with a molecular weight cut-off of 5000 Da. Placed in 60 ml of PBS buffer solution of corresponding pH, and dialyzed at 37°C. At 1, 2, 4, 8, 12, 24, 48, and 96 h, take 200 μl of CsA@Fn in the dialysis bag and add 200 μl of blank release medium. The concentration of CsA was determined by HPLC, and the concentration of ferritin was determined by BCA method, and the accumulation was calculated. The amount of drug released, and the drug release curve was drawn.

1.2 Efficiency of CsA@Fn penetrating the blood-brain barrier in vitro

Obtain bEnd3 cells, add DMEM medium containing 10% fetal bovine serum, the cell concentration is 1×10 ⁵ cells/ml, take 300 μl into the transwell donor pool, add DMEM medium containing 10% fetal bovine serum to the receiving pool, each Change the culture medium every 2 days. After 7 days, the resistance value between the transwell receiving pool and the donor pool was detected by a resistance meter. When the resistance value was greater than 200Ω/cm ² , it was considered that the in vitro blood-brain barrier model was successfully constructed. Aspirate and discard the culture medium in the donor pool and the recipient pool, add CsA@Fn (the concentration of CsA is 50 μg/ml) in the donor pool, and add serum-free DMEM medium in the recipient pool to keep the liquid level of the donor pool and the recipient pool flush. , after 2, 4, and 8 hours of incubation, the culture medium in the receiving pool was collected, lyophilized, added with 200 μl methanol, sonicated for 2 min, filtered with a 0.22 μm microporous membrane, and the concentration of CsA in the receiving pool was detected by HPLC. Transport efficiency at the blood-brain barrier.

1.3 Distribution of CsA@Fn in the brain of stroke mice

(1) The blood flow of the right middle cerebral artery in mice was blocked for 1 h by suture method, and the mouse MCAO model was established. Mice were anesthetized with isoflurane and fixed on an incubator. An incision was made in the middle of the mouse neck, the common carotid artery, internal carotid artery and external carotid artery were separated, the right common carotid artery and internal carotid artery were pulled up, the proximal end of the right external carotid artery was ligated, and the external carotid artery was cut off. An incision was made at the stump of the external carotid artery, and a suture was inserted. After the wounds were sutured, the mice were placed in an incubator to keep warm. After 1 h of hypoxia, the suture incision was cut, the suture was pulled out, and the incision was sutured. 100 μl of Cy7.5-labeled CsA@Fn (with a CsA concentration of 500 μg/ml) was injected into the tail vein within 5 min, and 24 h later, the brain tissue was obtained, and after TTC staining, the distribution of CsA@Fn in the infarcted area was observed by a live imager. The heart, liver, spleen, lung, kidney and other organs and tissues were obtained, and the distribution of CsA@Fn in normal tissues was observed by in vivo imager.

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

1.4 The therapeutic effect and mechanism of CsA@Fn on ischemic stroke

(1) Animal groups: sham operation group (sham), operation group (MACO), free CsA group (2.5mg/kg), CsA@BAS group (2.5mg/kg), CsA@Fn group (1.25, 2.5mg/kg) kg).

(2) Evaluation of the effect of CsA@Fn in the treatment of ischemic stroke: After the model was successfully established, the mice in the experimental group were administered by tail vein injection. The neuroprotective effect of CsA@Fn on MACO mice; the mouse brain tissue was obtained, cut into 5 slices, stained with TTC, and the effect of CsA@Fn on the cerebral infarction area of MACO mice was observed by stereo microscope; the mouse brain tissue was obtained, Paraffin sections, H&E staining and TUNEL staining were used to observe the effects of CsA@Fn on the morphology and apoptosis of mouse brain tissue in MACO mice.

(3) Evaluation of the protective effect of CsA@Fn on the blood-brain barrier: 24 hours after the administration of the tail vein of MACO mice, Evans blue solution was injected into the tail vein. The distribution of brain tissue; paraffin section, the distribution of Evans blue in the brain tissue was observed by fluorescence microscope, and the protective effect of CsA@Fn on the blood-brain barrier was investigated.

(4) Evaluation of the protective effect of CsA@Fn on cerebral neurons: After 24 hours of administration to the tail vein of MACO mice, brain tissue was obtained, paraffin sectioned, Nissl staining and silver-plated staining, and the effect of CsA@Fn on cerebral neurons was observed. protective effect.

2 Experimental results

2.1 Characterization of CsA@Fn

The particle size and potential of CsA@Fn were measured by nanoparticle size and zeta potential analyzer, and the results are shown in Figure 1. The particle size of CsA@Fn is 25 nm, which is basically the same as that of Fn, the potential is mV, and the drug loading of CsA is 10.4%. The stability test results of CsA@Fn are shown in Figure 2. In distilled water, PBS buffer, 10% FBS solution and DMEM solution, the particle size of CsA@Fn did not increase significantly within 5 days, suggesting that in the above media, CsA @Fn stable for 5 days.

The in vitro drug release properties of CsA@Fn were investigated by HPLC, and the results are shown in Figure 3. In the PBS buffer solution of pH5.0, the release of CsA@Fn increased significantly, and within 24h, more than 75% of CsA could be released.

2.2 The penetration effect of CsA@Fn on the blood-brain barrier

First, we constructed an in vitro blood-brain barrier model to investigate the efficiency of CsA@Fn across the blood-brain barrier in vitro. The results showed that CsA@Fn could cross the blood-brain barrier in a time-dependent manner, and the transport efficiency was significantly higher than that of free CsA and CsA@BSA. When the incubation time was 8 h, 38.5% of CsA@Fn could cross the blood-brain barrier ( Figure 4).

Next, we established a mouse cerebral ischemia perfusion model to investigate the distribution of CsA@Fn in ischemic brain tissue. The results showed that the distribution of CsA@Fn in the brain tissue of the sham operation group was 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 that of CsA@BSA, and CsA@Fn The distribution in ischemic brain tissue was significantly higher than that in normal brain tissue (Figure 5).

2.3 Therapeutic effect of CsA@Fn on cerebral ischemia-reperfusion injury

The results of TTC staining showed that CsA@Fn could dose-dependently reduce the size of cerebral infarction in mice, and the effect was significantly better than the CsA@BSA group and the free CsA group (Figure 6). The results of H&E staining showed that in the cerebral ischemia area of the MCAO model mice, obvious infarcts could be found, accompanied by a large number of inflammatory cells infiltration, and at the same time, the nuclei of neurons were pyknotic and chromatin was condensed. However, after CsA@Fn treatment, no obvious infarction was found in the cerebral ischemic area, and the infiltration of inflammatory cells was reduced. The results of ROS staining showed that after cerebral ischemia was perfused, the ROS in the cerebral ischemic area was significantly enhanced, and CsA@Fn could dose-dependently reduce ROS in the cerebral ischemic area, and the effect was better than the same dose of CsA and CsA@BSA ( Figure 7). The results of TUNEL staining showed that CsA@Fn could dose-dependently reduce the number of apoptotic cells, and the effect was significantly better than the CsA@BSA group and the free CsA group (Figure 8).

2.4 Neuroprotective effect of CsA@Fn

After 30 min of tail vein injection of Evanslan, the accumulation of Evanslan in cerebral ischemic tissue could be clearly observed in the brain tissue of the MCAO model mice, while almost no Evanslan could be observed in the brain tissue of the CsA@Fn administration group. The distribution of Evanslan, suggesting that CsA@Fn can protect the integrity of the blood-brain barrier and reduce the penetration of Evanslan in brain tissue (Figure 9).

The neurological function of the mice in each group was scored by a 5-point scale. The results showed that the neurological function score of the MCAO model mice was the highest, reaching 3.4 points, while the neurological function score of the CsA@Fn treatment group was the lowest, which was 1.1 points. It was suggested that CsA@Fn could reduce the damage to the nervous system caused by ischemic stroke (Figure 10). Nissl staining and silver-plating staining showed that CsA@Fn could reduce the nerve damage caused by cerebral ischemia-reperfusion and significantly increase the density of nerve cells in the ischemic area of the brain (Figure 11-12).

3 Conclusions:

The solvent-based denaturation/renaturation self-assembly method can load CsA into ferritin (Fn) nanocages and improve the solubility of CsA. CsA@Fn can cross the blood-brain barrier and effectively accumulate in cerebral ischemic tissue. Compared with CsA and CsA@BSA, CsA@Fn can not only improve the integrity of the blood-brain barrier in MCAO mice, but also reduce the cerebral infarct size and neuron damage, and significantly improve the protection of CsA against cerebral ischemia-reperfusion injury. It has certain application prospects.

Claims (2)

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.

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