Amlodipine drug targeted delivery system, preparation method therefor, and use thereof

Abstract

Disclosed are an amlodipine drug targeted delivery system and a preparation method therefor. The targeted delivery system can improve the targeting property of amlodipine in preventing hepatic ischemia-reperfusion injury and is simple to prepare, highly feasible, and highly practical. The amlodipine drug targeted delivery system (Am@EXOs) prepared in the present invention enables a targeted distribution of amlodipine in the livers of hepatic ischemia-reperfusion injury model mice and precise inhibition of calcium ion overload within hepatocytes by means of a minimum dose of amlodipine, thereby reducing cell injury, improving the targeting property of amlodipine in preventing hepatic ischemia-reperfusion injury, and reducing the cost of using amlodipine.

Description

Amlodipine drug targeted delivery system and preparation method and application thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical technology, and particularly relates to amlodipine drug targeted delivery system and preparation method and application thereof. BACKGROUND

[0002] Previous studies have shown that extracellular vesicles (EXOs) are nanoparticles released by cells into the extracellular microenvironment, which can alleviate liver injury by repairing autophagy, immunosuppression and promoting tissue regeneration. Recently, delivery systems based on mesenchymal stem cell-derived extracellular vesicles (MSCs-EXO) have received extensive attention, so it is one of the widely prospects to prevent and treat liver ischemia-reperfusion injury by using the targeting and infiltration of MSCs-EXO to enhance the drug delivery performance and further improve the prevention and treatment efficiency of liver ischemia-reperfusion injury.

[0003] Mesenchymal stem cell-derived extracellular vesicles have similar biological characteristics to the mesenchymal stem cells from which they are derived. In recent years, extracellular vesicles have gradually attracted attention as non-cell products due to their small size, low immunogenicity, strong tissue penetration, long circulation half-life, high stability, and low risk of use. In addition to their own functions of maintaining tissue development and function, regulating immunity, resisting oxidative stress, and promoting regeneration, mesenchymal stem cells and their extracellular vesicles can also be used as biological carriers to deliver bioactive substances and treat diseases. Bone marrow mesenchymal stem cells (BMSCs) have immunomodulatory and autocrine functions, and their extracellular vesicles (EXOs) play an important role in these functions. EXOs can be directly applied or used as carriers for therapeutic drugs. BMSC EXOs can cross biological barriers and directly enter target cells, so it can be seen that EXOs based on BMSCs as a targeting system to deliver drugs to prevent and treat diseases has great significance for medical research.

[0004] Calcium channel blockers can prevent intracellular calcium overload caused by liver ischemia-reperfusion injury (IRI) and further play a liver protection function by preventing oxidative stress. Amlodipine is a L-type calcium channel blocker and is safe and stable in lowering blood pressure, and has become the most commonly used antihypertensive drug. Studies have found that amlodipine combined with gemcitabine can greatly reduce the drug resistance of pancreatic cancer patients to gemcitabine, thereby improving drug efficacy and reducing the incidence of distant metastasis and prolonging the survival rate of patients. Another study found that amlodipine treatment has important significance in the treatment of non-alcoholic fatty liver disease combined with hypertension. Therefore, amlodipine is an effective measure to prevent intracellular calcium overload caused by liver IRI. SUMMARY

[0005] Invention purposes: The purpose of the present application is to provide an amlodipine drug targeted delivery system and a preparation method thereof, which can improve the targeting of amlodipine in preventing liver ischemia-reperfusion injury, and the preparation method is simple, feasible, and practical.

[0006] Technical solutions: The present application provides an amlodipine drug targeted delivery system (Am@EXOs), which uses an extracellular vesicle as a carrier, the carrier is loaded with amlodipine drug, and the extracellular vesicle is derived from bone marrow mesenchymal stem cells.

[0007] Further, the particle size of the extracellular vesicle is 100-120 nm.

[0008] The present application also provides a preparation method of the amlodipine drug targeted delivery system.

[0009] 1) Bone marrow mesenchymal stem cells are cultured in an exosome-free complete culture medium;

[0010] 2) The cell culture medium obtained in step 1) is centrifuged for multiple times, and cells, dead cells and cell debris are removed in sequence, and finally the precipitate is collected;

[0011] 3) The precipitate obtained in step 2) is resuspended in a phosphate buffer solution, thereby obtaining an extracellular vesicle derived from bone marrow mesenchymal stem cells;

[0012] 4) Amlodipine is loaded into the extracellular vesicle by using an ice bath ultrasonic method;

[0013] 5) After the mixture obtained in step 4) is incubated in an incubator, free amlodipine is removed, thereby obtaining an amlodipine drug targeted delivery system.

[0014] Further, the step 1) is to culture the fifth generation of bone marrow mesenchymal stem cells in an exosome-free complete culture medium for 2 days.

[0015] Further, the pH of the phosphate buffer solution is 7-8.

[0016] Further, the weight ratio of amlodipine to extracellular vesicle in step 4) is 5-1:1-5, preferably 1:1.

[0017] Further, the incubation condition in step 5) is incubation at 37℃ for 1 h.

[0018] The present application also provides the use of the amlodipine drug targeted delivery system in the preparation of a drug for liver ischemia-reperfusion injury.

[0019] Advantages:

[0020] (1) The BMSC-derived cell extracellular vesicle (EXOs) provided by the application has simple extraction mode, and has good biocompatibility, low immunogenicity, targeting and other advantages.

[0021] (2) The amlodipine drug targeted delivery system (Am@EXOs) prepared by the application has simple and convenient preparation mode and certain feasibility.

[0022] (3) The amlodipine drug targeted delivery system (Am@EXOs) prepared by the application enables amlodipine to be targeted to the liver of a liver ischemia-reperfusion injury model mouse, realizes precise inhibition of calcium ion overload in liver cells with a minimum dose of amlodipine, thereby reducing cell damage, improves the targeting of amlodipine in preventing liver ischemia-reperfusion injury, and reduces the use cost of amlodipine. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is a schematic diagram of the preparation process of BMSC-derived EXOs and Am@EXOs.

[0024] Figure 2 is the characterization of Am@EXOs: Figure a is a transmission electron microscope (TEM) image of EXOs and Am@EXOs, the scale size is 100 nm; Figure b is the particle size distribution of EXOs and Am@EXOs analyzed by DLS; Figure c is the potential map of EXOs and Am@EXOs; Figure d is the Western blot analysis of EXOs and Am@EXOs markers CD63, CD9 and TSG101; Figure e is the encapsulation efficiency of EXOs and Am@EXOs in three different proportions, Figure f is the drug loading of EXOs and Am@EXOs in three different proportions; Figure g is the in vitro cumulative drug release of Am@EXOs in PBS.

[0025] Figure 3 is the biocompatibility of Am@EXOs: Amlodipine, EXOs and Am@EXOs cell live and dead map.

[0026] Figure 4 is the in vivo targeting ability of Am@EXOs: Figure a is the fluorescence signal of EXOs or Am@EXOs labeled with Cy5.5 intravenously injected into mice, and imaging after 6, 12 and 48 hours of treatment; Figure b is the ex vivo fluorescence signal of organs collected from mice sacrificed after 6 h of treatment.

[0027] Figure 5 is the in vivo therapeutic effect of Am@EXOs in IRI mouse model: Figure a is the determination of liver function ALT and AST levels of mice in the control group (Control), liver ischemia-reperfusion modeling group (IRI) and treatment group (EXOs, Am@EXOs); Figure b is the survival curve of mice in the control group (Control), liver ischemia-reperfusion modeling group (IRI) and treatment group (EXOs, Am@EXOs) after treatment. Detailed Implementation

[0028] To enhance understanding of the present invention, the present invention will be further described in detail below with reference to embodiments and accompanying drawings. These embodiments are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0029] In the following examples, the first-generation BMSCs (mouse bone marrow-derived) were purchased from the American Type Culture Collection (ATCC) in the United States.

[0030] Example 1: Preparation of Am@EXOs

[0031] Figure 1 is a schematic diagram of the preparation process of the drug-targeting carrier. First, extracellular vesicles (EXOs) derived from bone marrow mesenchymal stem cells are obtained by high-speed centrifugation. Then, the EXOs are loaded with the calcium blocker amlodipine using an ice bath sonication method to prepare the drug-targeting carrier Am@EXOs.

[0032] The specific steps are as follows:

[0033] (1) After the first generation of BMSCs (from mouse bone marrow) were revived, they were cultured and passaged continuously. The fifth generation of BMSCs with a density of 80-90% were cultured in exosome-free complete medium for 2 days.

[0034] (2) Collect the cell supernatant from (1) cultured for 2 days into a centrifuge tube and centrifuge at 300 xg speed and 4℃ temperature for 10 minutes to remove cells;

[0035] (3) Collect the cell supernatant after step (2) into a new centrifuge tube and centrifuge at 2000 xg speed and 4℃ temperature for 10 minutes to remove dead cells;

[0036] (4) Collect the cell supernatant after step (3) into a new centrifuge tube and centrifuge at 10000 xg speed and 4℃ temperature for 30 minutes to remove dead cell debris.

[0037] (5) Collect the cell supernatant after step (4) into an ultracentrifuge tube and ultracentrifuge at 100,000 xg speed and 4°C for 90 minutes to obtain precipitate (EXOs).

[0038] (6) After discarding the supernatant of the cells in the ultracentrifuge tube in step (5), 10 ml of phosphate buffer (PBS) was added to wash the precipitate (EXOs) at the bottom of the tube, and then the washed precipitate (EXOs) was obtained by ultracentrifugation at 100000 xg, 4°C for 90 minutes. After slowly discarding the phosphate buffer (PBS) in the centrifuge tube, 100 μL of phosphate buffer (PBS) was sucked up by a 1 ml syringe to blow the bottom of the tube and resuspend the EXOs, and the resuspension (EXOs) was collected and stored at -80°C;

[0039] (7) 500 μg of amlodipine was mixed with 500 μg of EXOs in 10 mL of PBS (pH = 7.4) for 30 minutes (r.t, 200 rpm), and then ice bath ultrasonic treatment was performed in an ultrasonic cell crusher (VCX130) with the following settings: 40% amplitude, 3 cycles of 15s, 90s of continuous, 2 minutes of cooling between each cycle. After ultrasonic treatment, it was placed in a 37°C incubator for 1 h, and further ultrafiltration through a 200KD ultrafiltration membrane was performed three times to remove excess free amlodipine, and finally Am@EXOs were obtained.

[0040] According to the transmission electron microscope observation of the morphology of the EXOs and Am@EXOs prepared in Example 1 (Figure 2a), it was found that both the pure EXOs and the Am@EXOs loaded with amlodipine had uniform size distribution, and the average diameters were 106.7 nm and 148.6 nm respectively (Figure 2b). By detecting the zeta potential of EXOs and Am@EXOs, due to the successful loading of amlodipine, the zeta potential of the two kinds of nanoparticles changed from about -20 mV to about -26 mV (Figure 2c). Next, Western blotting further verified the expression of exosome markers (CD63, TSG101, CD9) of EXOs and Am@EXOs, and the results showed that the loading did not change the surface proteins of EXOs (Figure 2d).

[0041] Example 2

[0042] The difference from Example 1 is that in step (7), the weight ratio of amlodipine to EXOs is 5:1, that is, 2500 μg of amlodipine is mixed with 500 μg of EXOs in 10 mL of PBS (pH = 7.4) for 30 minutes (r.t, 200 rpm).

[0043] Example 3

[0044] The difference from Example 1 is that in step (7), the weight ratio of amlodipine to EXOs is 1:5, that is, 500 μg of amlodipine is mixed with 2500 μg of EXOs in 10 mL of PBS (pH = 7.4) for 30 minutes (r.t, 200 rpm).

[0045] Example 4 Encapsulation efficiency and drug loading

[0046] Amlodipine and EXOs were loaded by ultrasound at the weight ratio of 1:1 (Example 1), 5:1 (Example 2), and 1:5 (Example 3), respectively. The encapsulation efficiency of EXOs was detected by a microplate reader. The results showed that the encapsulation efficiency was 64% when amlodipine and EXOs were mixed at a ratio of 5:1, 61% when mixed at a ratio of 1:5, and 92% when mixed at a ratio of 1:1 (Figure 2e). The amount of amlodipine in EXOs was also detected, and the drug loading rate of EXOs was calculated. The results showed that the drug loading rate was 6.6% when amlodipine and EXOs were mixed at a ratio of 1:5, and about 9% when mixed at a ratio of 5:1 and 1:1 (Figure 2f), indicating that a better encapsulation efficiency and drug loading rate can be achieved when amlodipine and EXOs are mixed at a ratio of 1:1.

[0047] Example 5 In vitro cumulative drug release detection

[0048] Am@EXOs were placed in dialysis bags to simulate in vivo drug release behavior. PBS (7~8) was used as the drug release medium, and the content of amlodipine outside the dialysis bag was detected at fixed time points to draw the drug release curve. The in vitro cumulative drug release of Am@EXOs (Example 1) in PBS was detected (Figure 2g), and it was found that amlodipine was uniformly released over time, and the release reached a stable state after 36 hours, with a release rate of about 73%.

[0049] Example 6 Biocompatibility

[0050] Amlodipine, EXOs, and Am@EXOs (Example 1) were co-cultured with LO2 cells for 24 hours, and the cells were subjected to live and dead staining. The live cells were green and the dead cells were red. Fluorescence microscopy showed (Figure 3) that the dead cells in the amlodipine, EXOs, and Am@EXOs groups were fewer, indicating good biocompatibility.

[0051] Example 7 Targeting ability of Am@EXOs

[0052] EXOs and Am@EXOs prepared in Example 1 were labeled with Cy5.5 and injected into the tail vein. The targeting ability of EXOs and Am@EXOs was detected by tracking at different time points using an in vivo imaging system. The imaging system clearly observed the signal related to Cy5.5-labeled EXOs, which remained at a high level for 24 hours (Figure 4a). During this period, Am@EXOs had similar performance, indicating that encapsulation did not affect the targeting ability of EXOs. Ex vivo imaging was performed 48 hours after tail vein administration, and fluorescence signals were detected in all collected organs, mainly in the liver (Figure 4b).

[0053] Example 8 In vivo therapeutic effect of Am@EXOs in IRI mouse model

[0054] Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are two most commonly used liver function analysis biomarkers, and the liver function is affected by detecting the levels of the two (Fig. 5a, b). Compared with the control group, the ischemia-reperfusion group showed high levels of ALT and AST. While free amlodipine has a certain liver function protection effect, EXOs group has almost no protective effect, while Am@EXOs (1:1) shows good liver protection effect. As shown in Fig. 5c, the survival rate of control animals was 100% (24 / 24), while the survival rate decreased to 8.3% (2 / 24) after ischemia-reperfusion, and the survival rate of amlodipine mice before ischemia-reperfusion was not significantly increased, and the EXOs group almost had no effect on the survival rate of mice. In mice treated with Am@EXOs (1:1), the survival rate increased to 67% (16 / 24). Hematoxylin and eosin tissue sections showed that there was no abnormality in the liver tissue of the control group, and obvious tissue structure disorder and severe cell necrosis were observed in the IRI group. Free amlodipine has a certain protective effect on liver structure and cell viability, and EXOs group has almost no protective effect. Am@EXOs treatment protects the structural integrity of liver tissue (Fig. 5d).

[0055] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An amlopidine drug targeting delivery system, characterized in that, The delivery system takes extracellular vesicles as carriers, the carriers are loaded with amlodipine drugs, and the extracellular vesicles are derived from bone marrow mesenchymal stem cells.

2. The amlodipine drug targeted delivery system according to claim 1, wherein, The particle size of the extracellular vesicles is 100-120 nm.

3. The amlodipine drug targeted delivery system according to claim 1 or 2, characterized in that, The preparation method is as follows: 1) Bone marrow mesenchymal stem cells are cultured in exosome-free complete medium; 2) The cell culture medium obtained in step 1) is centrifuged for multiple times, and cells, dead cells and cell debris are removed in sequence, and finally the precipitate is collected; 3) The precipitate obtained in step 2) is resuspended in a phosphate buffer solution, and bone marrow mesenchymal stem cell-derived extracellular vesicles are obtained; 4) Amlodipine is loaded into the extracellular vesicles by ice bath ultrasonic method; 5) After the mixture obtained in step 4) is incubated in an incubator, free amlodipine is removed, and an amlodipine drug targeted delivery system is obtained.

4. The amlodipine targeted delivery system according to claim 3, characterized in that, The step 1) is to culture the fifth generation of bone marrow mesenchymal stem cells in exosome-free complete medium for 2 days.

5. The amlodipine targeted delivery system according to claim 3, characterized in that, The pH of the phosphate buffer solution is 7-8.

6. The amlodipine drug targeted delivery system according to claim 3, characterized in that, The weight ratio of amlodipine to extracellular vesicles in step 4) is 5-1:1-5.

7. The amlodipine drug targeted delivery system according to claim 6, characterized in that, The weight ratio of amlodipine to extracellular vesicles in step 4) is 1:

1.

8. The amlodipine drug targeted delivery system according to claim 3, wherein the hydrophilic polymer is polyethylene glycol. The incubation condition in step 5) is incubation at 37℃ for 1 h.

9. Use of the amlodipine drug targeted delivery system according to any one of claims 1-8 in the preparation of a drug for liver ischemia-reperfusion injury.

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