A kind of amlodipine drug targeted delivery system and its preparation method and application

Abstract

The present invention discloses an amlodipine drug targeted delivery system and a preparation method thereof. The targeted delivery system can improve the targeting of amlodipine in preventing liver ischemia-reperfusion injury, and the preparation method is simple, with high feasibility and strong practicality. The amlodipine drug targeted delivery system (Am@EXOs) prepared by the present invention enables amlodipine to be distributed in the liver of a mouse model of liver ischemia-reperfusion injury, and achieves precise inhibition of calcium ion overload in liver cells with a minimum dose of amlodipine, thereby reducing cell damage, improving the targeting of amlodipine in preventing liver ischemia-reperfusion injury, and reducing the use cost of amlodipine.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to an amlodipine drug targeted delivery system and a preparation method and application thereof. Background Art

[0002] Previous studies have reported that extracellular vesicles (EXOs) are nanoparticles released by cells into the extracellular microenvironment, which can alleviate liver damage by repairing autophagy, suppressing immunosuppression, and promoting tissue regeneration. Recently, the delivery system based on mesenchymal stem cell-derived extracellular vesicles (MSCs-EXO) has received extensive attention. Therefore, utilizing its targeting and infiltration properties to enhance the delivery performance of drugs and further improve the prevention and treatment efficiency of liver ischemia-reperfusion injury is one of the broad prospects for preventing liver ischemia-reperfusion injury.

[0003] Extracellular vesicles derived from mesenchymal stem cells have similar biological properties to the mesenchymal stem cells from which they are derived. Due to their small size, low immunogenicity, strong tissue permeability, long circulation half-life, high stability, and low risk of use, extracellular vesicles have gradually attracted attention as non-cellular products in recent years. In addition to their own functions of tissue development and function maintenance, immune regulation, anti-oxidative stress and promotion of regeneration, mesenchymal stem cells and their extracellular vesicles can also be used as biological carriers to deliver bioactive substances to treat diseases. Bone marrow mesenchymal stem cells (BMSCs) have functions such as immunoregulation and autocrine secretion, and their extracellular vesicles (EXOs) play an important role in exerting these functions. EXOs can not only be used directly, but also as carriers of therapeutic drugs. BMSC EXOs can cross biological barriers and directly enter target cells. It can be seen that BMSC-based EXOs as a targeted system to deliver drugs to prevent and treat diseases is of great significance to medical research.

[0004] Calcium channel blockers can prevent intracellular calcium overload caused by hepatic ischemia-reperfusion injury (IRI), and further play a liver-protective function by preventing oxidative stress. Amlodipine is an L-type calcium channel blocker, and it is safe to use and has a stable blood pressure reduction effect. It has become the most commonly used antihypertensive drug. Studies have found that the combination of amlodipine and gemcitabine can greatly reduce the resistance of pancreatic cancer patients to gemcitabine, thereby improving the efficacy of the drug, reducing the incidence of distant metastasis and prolonging the survival rate of patients. Other studies have found that treatment with amlodipine is of great significance in the treatment of non-alcoholic fatty liver disease with hypertension. Therefore, using amlodipine to prevent intracellular calcium overload caused by liver IRI is an effective preventive measure for liver IRI. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention 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, highly feasible and practical.

[0006] Technical solution: The present invention provides an amlodipine drug targeted delivery system (Am@EXOs), wherein the delivery system uses extracellular vesicles as carriers, the carriers are loaded with amlodipine drugs, and the extracellular vesicles are derived from bone marrow mesenchymal stem cells.

[0007] Furthermore, the particle size of the extracellular vesicles is 100-120 nm.

[0008] The present invention also provides a method for preparing an amlodipine drug targeted delivery system:

[0009] 1) Bone marrow mesenchymal stem cells were cultured in complete medium without exosomes;

[0010] 2) centrifuging the cell culture medium obtained in step 1) multiple times to remove cells, dead cells and cell debris in turn, and finally collecting the precipitate;

[0011] 3) resuspending the precipitate obtained in step 2) in a phosphate buffer solution to obtain extracellular vesicles derived from bone marrow mesenchymal stem cells;

[0012] 4) Loading amlodipine into extracellular vesicles using ice bath sonication;

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

[0014] Furthermore, the step 1) is to culture the fifth generation bone marrow mesenchymal stem cells in a complete culture medium without exosomes for 2 days.

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

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

[0017] Furthermore, the incubation condition in step 5) is incubating at 37° C. for 1 h.

[0018] The present invention also provides application of the amlodipine drug targeted delivery system in preparing drugs for treating liver ischemia-reperfusion injury.

[0019] Beneficial effects:

[0020] (1) The BMSC-derived extracellular vesicles (EXOs) provided by the present invention are simple to extract and have the advantages of good biocompatibility, low immunogenicity, and targeting.

[0021] (2) The amlodipine drug targeted delivery system (Am@EXOs) prepared by the present invention is simple and convenient to prepare and has certain feasibility.

[0022] (3) The amlodipine drug targeted delivery system (Am@EXOs) prepared by the present invention enables the targeted distribution of amlodipine in the liver of mice with liver ischemia-reperfusion injury model, achieving precise inhibition of calcium ion overload in liver cells with a minimum dose of amlodipine, thereby reducing cell damage, improving the targeting of amlodipine in preventing liver ischemia-reperfusion injury, and reducing the cost of using amlodipine. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0025] Figure 3 Biocompatibility of Am@EXOs: Live-death plots of Amlodipine, EXOs and Am@EXOs cells.

[0026] Figure 4 The in vivo targeting ability of Am@EXOs: Figure a shows the in vivo fluorescence signal of mice intravenously injected with EXOs or Am@EXOs labeled with Cy5.5, and imaged 6, 12 and 48 hours after treatment; Figure b shows the in vitro fluorescence signal of organs collected from mice killed 6 hours after treatment.

[0027] Figure 5The diagram shows the in vivo therapeutic effect of the IRI mouse model of Am@EXOs: Figure a shows the determination of ALT and AST levels of liver function in each group of mice after the control group (Control), liver ischemia-reperfusion model group (IRI) and treatment group (EXOs, Am@EXOs); Figure b shows the survival curves of each group of mice after the control group (Control), liver ischemia-reperfusion model group (IRI) and treatment group (EXOs, Am@EXOs). DETAILED DESCRIPTION

[0028] In order to deepen the understanding of the present invention, the present invention will be further described in detail below in conjunction with embodiments and drawings. The embodiments are only used to explain the present invention and do not constitute a limitation on the protection scope of the present invention.

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

[0030] Example 1 Preparation of Am@EXOs

[0031] Figure 1 The figure is a schematic diagram of the preparation process of drug-targeted carriers. First, extracellular vesicles (EXOs) derived from bone marrow mesenchymal stem cells were obtained by high-speed centrifugation. Then, EXOs and calcium blocker amlodipine were loaded by ice bath ultrasound to prepare drug-targeted carriers Am@EXOs.

[0032] The specific steps are as follows:

[0033] (1) The first-generation BMSCs (derived from mouse bone marrow) were revived and cultured, and the fifth-generation BMSCs with a density of 80-90% were cultured in complete medium without exosomes for 2 days;

[0034] (2) Collect the cell supernatant cultured for 2 days in (1) into a centrifuge tube and centrifuge at 300 x g and 4°C for 10 minutes to remove the cells;

[0035] (3) Collect the cell supernatant after treatment in step (2) into a new centrifuge tube and centrifuge at 2000 x g and 4°C for 10 minutes to remove dead cells;

[0036] (4) Collect the cell supernatant after treatment in step (3) into a new centrifuge tube and centrifuge at 10,000 x g and 4°C for 30 minutes to remove dead cell debris;

[0037] (5) Collect the cell supernatant after treatment in step (4) into an ultracentrifuge tube and ultracentrifuge at 100,000 x g and 4°C for 90 minutes to obtain a precipitate (EXOs);

[0038] (6) After discarding the cell supernatant in the ultracentrifuge tube in step (5), add 10 ml of phosphate buffered saline (PBS) to wash the precipitate (EXOs) at the bottom of the tube, and then ultracentrifuge again at 100,000 x g, 4°C for 90 minutes to obtain the washed precipitate (EXOs). After slowly discarding the phosphate buffered saline (PBS) in the centrifuge tube, use a 1 ml syringe to draw 100 μL of phosphate buffered saline (PBS) to blow the bottom of the tube to resuspend the EXOs, and collect the resuspended solution (EXOs) and store it at -80°C;

[0039] (7) 500 μg of amlodipine and 500 μg of EXOs were mixed in 10 mL of PBS (PH = 7.4) for 30 minutes (rt, 200 rpm), and then subjected to ice bath sonication in an ultrasonic cell disruptor (VCX130) with the following settings: 40% amplitude, 3 cycles of 15 s, 90 s duration, and 2 minutes of cooling between each cycle. After sonication, the cells were placed in a 37 °C incubator for 1 h. After the incubation, the cells were further ultrafiltered three times using a 200 KD ultrafiltration membrane to remove excess free amlodipine, and finally Am@EXOs were obtained.

[0040] The morphology of EXOs and Am@EXOs prepared in Example 1 was observed by transmission electron microscopy ( Figure 2 a), it was found that pure EXOs and Am@EXOs loaded with amlodipine had uniform size distribution, with average diameters of 106.7 nm and 148.6 nm, respectively ( Figure 2 b). By detecting the zeta potential of EXOs and Am@EXOs, the zeta potential of the two nanoparticles changed from about -20 mV to about -26 mV due to the successful loading of amlodipine ( Figure 2 c). Next, immunoblotting was used to further verify the exosomal markers (CD63, TSG101, CD9) expressed by EXOs and Am@EXOs. The results showed that drug loading did not change the surface proteins of EXOs ( Figure 2 d).

[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 and 500 μg of EXOs are mixed in 10 mL PBS (PH=7.4) for 30 minutes (rt, 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 and 2500 μg of EXOs are mixed in 10 mL PBS (PH=7.4) for 30 minutes (rt, 200 rpm).

[0045] Example 4 Encapsulation efficiency and drug loading

[0046] Amlodipine and EXOs were ultrasonically loaded at a 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 an ELISA instrument. The results showed that the encapsulation efficiency of amlodipine and EXOs was 64% when 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 2 e). At the same time, the amount of amlodipine in EXOs was detected and the drug loading rate of EXOs was calculated. The results showed that the drug loading of amlodipine and EXOs was 6.6% when the ratio was 1:5, while the drug loading of 5:1 and 1:1 was about 9% ( Figure 2 f), indicating that the weight ratio of amlodipine to EXOs of 1:1 can achieve a good encapsulation efficiency and drug loading.

[0047] Example 5 In vitro cumulative drug release assay

[0048] Am@EXOs were placed in a dialysis bag to simulate the in vivo drug release behavior. PBS (7-8) was used as the drug release medium. The content of amlodipine outside the dialysis bag was detected at a fixed time point and the drug release curve was drawn. The in vitro cumulative drug release of Am@EXOs (Example 1) in PBS was detected ( Figure 2 g), it was found that amlodipine was released uniformly over time and reached a stable release rate 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 h, and the cells were stained for live and dead cells, with live cells in green and dead cells in red. Fluorescence microscopy showed ( Figure 3 ), the number of dead cells in the amlodipine, EXOs, and Am@EXOs groups was relatively small, indicating good biocompatibility.

[0051] Example 7 Targeting ability of Am@EXOs

[0052] The EXOs and Am@EXOs prepared in Example 1 were labeled with Cy5.5 and injected into the tail vein. The in vivo imaging system was used to track the targeting ability of EXOs and Am@EXOs at different time points. The signal associated with Cy5.5-labeled EXOs was clearly observed by the imaging system and remained at a high level for 24 hours ( Figure 4 a), 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 fluorescent signals were detected in all collected organs, mainly in the liver ( Figure 4 b).

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

[0054] Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are the two most commonly used biomarkers for liver function analysis. Figure 5 a, b). Compared with the control group, the ischemia-reperfusion group showed high ALT and AST levels. Free amlodipine had a certain protective effect on liver function, the EXOs group had almost no protective effect, and Am@EXOs (1:1) showed good liver protective effect. Figure 5 c Analysis showed that the survival rate of animals in the control group was 100% (24 / 24), while the survival rate dropped to 8.3% (2 / 24) after ischemia-reperfusion. The survival rate of mice gavaged with amlodipine before ischemia-reperfusion was not significant, and the EXOs group had almost no increase in 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 no abnormalities in the liver tissue of the control group, while obvious tissue structural disorder and severe cell necrosis were observed in the IRI group. Free amlodipine has a protective effect on liver structure and cell viability to a certain extent, while the EXOs group has almost no protective effect. Am@EXOs treatment protected the structural integrity of liver tissue ( Figure 5 d).

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An amlodipine drug targeted delivery system, characterized in that: The delivery system uses extracellular vesicles as carriers, the carriers are loaded with amlodipine drugs, the extracellular vesicles are derived from bone marrow mesenchymal stem cells, and the weight ratio of amlodipine to extracellular vesicles is 1:

1.

2. The amlodipine drug targeted delivery system according to claim 1, characterized in that: 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 steps are as follows: 1) Bone marrow mesenchymal stem cells were cultured in complete medium without exosomes; 2) centrifuging the cell culture medium obtained in step 1) multiple times to remove cells, dead cells and cell debris in turn, and finally collecting the precipitate; 3) resuspending the precipitate obtained in step 2) in a phosphate buffer solution to obtain extracellular vesicles derived from bone marrow mesenchymal stem cells; 4) Loading amlodipine into extracellular vesicles using ice bath sonication; 5) After incubating the mixture obtained in step 4) in an incubator, free amlodipine is removed to obtain an amlodipine drug targeted delivery system.

4. The amlodipine drug targeted delivery system according to claim 3, characterized in that: The step 1) is to culture the fifth generation bone marrow mesenchymal stem cells in a complete culture medium without exosomes for 2 days.

5. The amlodipine drug 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 incubation condition in step 5) is 1 h at 37°C.

7. Use of the amlodipine drug targeted delivery system as described in any one of claims 1 to 6 in the preparation of a drug for treating liver ischemia-reperfusion injury.

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