Preparation method of FTY720-loaded platelet membrane biomimetic nanobubbles

Abstract

The application discloses a preparation method of FTY720-loaded platelet membrane biomimetic nanobubbles, and belongs to the technical field of biological medicines. The platelet membrane biomimetic nanobubbles are composed of a platelet membrane, a nanobubble core and an S1P receptor immunomodulatory drug FTY720. The biomimetic nanobubbles have good biocompatibility and targeting property, can be quickly and efficiently adhered to damaged blood vessels, thrombus and inflammation and other lesion sites of cardiovascular and cerebrovascular diseases after injection, can effectively induce the M2 anti-inflammatory phenotype conversion of immune cells such as macrophages and microglia in the lesions through the controllable release of FTY720, can reduce the adhesion of other immune cells such as T lymphocytes, can regulate the balance of pro-inflammatory / anti-inflammatory reactions, can reduce the lesion area, can improve the immune regulation treatment effect of cardiovascular and cerebrovascular diseases, and can reduce the side effects of FTY720.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the construction of a platelet membrane biomimetic nanobubble loaded with FTY720 and its application in immunomodulatory therapy for cardiovascular and cerebrovascular diseases. Background Technology

[0002] Currently, cardiovascular and cerebrovascular diseases are the leading cause of death worldwide. With socio-economic development, unhealthy lifestyles are becoming increasingly prominent, and the impact of cardiovascular and cerebrovascular diseases on residents' health is becoming more significant. In 2018, cardiovascular disease was the leading cause of death among urban and rural residents in my country, accounting for 46.66% in rural areas and 43.81% in urban areas, with rural areas accounting for a higher percentage than urban areas. Overall, the rate is rising rapidly, placing an increasingly heavy economic burden on residents and society, and has become a major public health problem.

[0003] In the development of cardiovascular and cerebrovascular diseases such as atherosclerosis, ischemic stroke, and acute myocardial infarction, inflammatory responses mediated by damaged endothelial cells, activated platelets, and various immune cells play a crucial role and are also important intervention targets for the treatment of these diseases. Currently, various therapeutic studies targeting the inflammatory response in cardiovascular and cerebrovascular diseases are underway, including the use of non-specific anti-inflammatory drugs such as corticosteroids, complement cascade inhibition therapy, and targeted therapies targeting neutrophil CD11 / CD18, endothelial cell P-selectin, and inflammatory cytokines such as IL-1β and IL-6. These studies have yielded some results in inhibiting atherosclerotic plaque growth and regulating inflammation after reperfusion injury in ischemic stroke and acute myocardial infarction in animal studies. However, in actual clinical applications, these anti-inflammatory treatment strategies have not achieved ideal results. Therefore, clarifying the mechanisms of inflammation occurrence and development, selecting effective therapeutic drugs for targeted delivery, and ultimately achieving effective regulation of the inflammatory process within the lesion are key issues and challenges in designing novel anti-inflammatory treatment strategies for cardiovascular and cerebrovascular diseases. FTY720 is an immunomodulatory drug for the treatment of multiple sclerosis and multiple organ transplantation. It is also the only S1P receptor agonist approved by the FDA for human clinical use. Regarding anti-inflammatory and immunomodulatory effects, it has been reported to be effective in the treatment of cardiovascular and cerebrovascular diseases such as ischemic stroke, atherosclerosis, and cell protection in acute myocardial infarction. Therefore, FTY720 is a potential clinical therapeutic drug for anti-inflammatory and immunomodulatory purposes in cardiovascular and cerebrovascular diseases.

[0004] Achieving efficient drug delivery while minimizing side effects is crucial for the development of novel drug delivery systems. With the advancement of nanotechnology and nanomaterials, nano-drug delivery systems constructed using polymers, phospholipids, and other materials have significantly improved drug delivery efficiency while reducing side effects, showing promising application prospects. Among these, nanobubbles possess excellent biocompatibility and ultrasound imaging enhancement capabilities, enabling safe, targeted delivery of drugs and therapeutic gases, as well as controllable, barrier-crossing release mediated by ultrasound fields, and are widely used in the diagnosis and treatment of cardiovascular diseases. In recent years, biomimetic nanomaterials based on autologous cell membranes have gradually become a research hotspot. Their key advantage lies in the fact that biomimetic nanomaterials retain the natural components and functions of cell membranes.

[0005] As the pathogenesis of cardiovascular and cerebrovascular diseases reveals, platelets not only participate in the recognition of damaged blood vessels and thrombus formation, but also play a crucial role in mediating the recruitment and adhesion of immune cells. Therefore, platelets are an important early target in the diagnosis and treatment of cardiovascular and cerebrovascular diseases. Utilizing the natural targeting function of platelet biomembranes, constructing biomimetic nanobubbles loaded with FTY720 platelet membranes can provide a safe and effective solution for the targeted delivery of FTY720 to lesions in cardiovascular and cerebrovascular diseases and for anti-inflammatory immune regulation. Summary of the Invention

[0006] To address the problems in the treatment of cardiovascular and cerebrovascular diseases, such as low drug delivery efficiency, unclear immune regulation mechanisms, and unsatisfactory treatment intervention effects, this invention utilizes platelet membranes, which are closely related to the occurrence and development of cardiovascular and cerebrovascular diseases, as a material to design and construct a biomimetic nanobubble carrying FTY720. This FTY720-loaded platelet membrane biomimetic nanobubble has a nanoscale size and retains the natural properties of the platelet membrane, enabling targeted delivery of FTY720, reducing its side effects, and improving the therapeutic effect of immune regulation in cardiovascular and cerebrovascular diseases.

[0007] The technical solution of this invention is: a biomimetic nanobubble for platelet membranes loaded with FTY720, as described in this invention.

[0008] The platelet membrane is prepared by separating the platelet contents through a platelet membrane extraction method to obtain purified platelet membrane vesicles, i.e., platelet membranes.

[0009] The platelet membrane extraction method is one of mechanical lysis extraction, hypotonic lysis extraction, and repeated freeze-thaw extraction.

[0010] Furthermore, the biomimetic nanobubble includes a bubble membrane shell and a nanobubble core;

[0011] The preparation method is as follows: purified platelet membrane and FTY720 molecules are adsorbed onto the gas-liquid interface of nanobubbles and reassembled by compression shear self-assembly.

[0012] Furthermore, the bubble membrane shell is prepared by assembling platelet membranes and FTY720 molecules.

[0013] Furthermore, the gas in the nanobubble core is one or two of air, oxygen, nitrogen, hydrogen, nitric oxide, helium, or sulfur hexafluoride.

[0014] Furthermore, the size of the platelet membrane biomimetic nanobubbles is 50-100 nm.

[0015] Furthermore, a method for preparing platelet membrane biomimetic nanobubbles loaded with FTY720 is characterized by the following specific operational steps:

[0016] Step (1): Resuspend platelet membranes in FTY720 saline solutions of different concentrations, and use water bath sonication to bind FTY720 saline solutions to platelet membranes to obtain a suspension.

[0017] Step (2): The prepared suspension is transferred to a pressure-induced shear bubble generator and different extrusion speeds are applied. By repeatedly injecting gas into the suspension, the gas-liquid interface is disturbed to generate nanobubbles. Under the action of water flow shear force, the platelet membrane and FTY720 physiological saline solution are adsorbed, fused and reassembled at the gas-liquid interface of the nanobubbles. Finally, biomimetic nanobubbles of platelet membrane loaded with FTY720 are obtained.

[0018] Furthermore, in step (1), the concentration of the FTY720 saline solution is 0.05-0.5 mg / mL.

[0019] Furthermore, in step (2), the different extrusion speeds applied by the pressure shear bubble generator are 10-50 mm / s, and the number of repeated injections is 100-500 times.

[0020] Furthermore, the prepared FTY720-loaded platelet membrane biomimetic nanobubbles are used for targeted drug delivery and immunomodulatory therapy for cardiovascular and cerebrovascular diseases.

[0021] Furthermore, the cardiovascular and cerebrovascular diseases include, but are not limited to, acute ischemic stroke, acute myocardial infarction, atherosclerosis, and microcirculatory disorders.

[0022] The beneficial effects of this invention are as follows: This invention provides a biomimetic nanobubble containing FTY720 from a platelet membrane. By constructing the FTY720-loaded biomimetic nanobubble using autologous platelet membranes, compared to traditional drug delivery nanogroups based on polymers, synthetic phospholipids, and other synthetic materials, its composition and preparation method are simpler, resulting in higher manufacturability and consistency. Furthermore, since nanobubbles are flexible structures, the FTY720-loaded platelet membrane biomimetic nanobubbles can more closely resemble natural platelets in shape and structure, which is more conducive to their targeting function. The biomimetic nanobubbles constructed by this invention can target and deliver the FDA-approved immunomodulatory drug FTY720 to lesions in cardiovascular and cerebrovascular diseases, effectively and timely regulating various immune cells such as macrophages and microglia within the lesions, providing a new strategy for the clinical treatment of cardiovascular and cerebrovascular diseases. Attached Figure Description

[0023] Figure 1 A schematic diagram illustrating the construction process of the platelet membrane biomimetic nanobubbles loaded with FTY720 provided by the present invention;

[0024] Figure 2 This is a transmission electron microscope image of the platelet membrane biomimetic nanobubbles loaded with FTY720 provided in Example 1;

[0025] Figure 3 Near-infrared fluorescence imaging and quantitative analysis of platelet membrane biomimetic nanobubbles loaded with FTY720 used for ischemic stroke targeting in Experiment Example 1.

[0026] Figure 4 M-mode ultrasound image of cardiac function evaluation after reperfusion of acute myocardial infarction and the use of FTY720-loaded platelet membrane biomimetic nanobubbles for anti-inflammatory regulation in Experiment Example 2.

[0027] Figure 5 This is an M-mode ultrasound evaluation image of normal cardiac function in Comparative Example 1. Detailed Implementation

[0028] To more clearly illustrate the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to the accompanying drawings:

[0029] like Figure 1 The present invention describes a platelet membrane biomimetic nanobubble loaded with FTY720. The structure of the biomimetic nanobubble includes a bubble membrane shell and a nanobubble core. It is prepared by adsorbing purified platelet membrane and FTY720 molecules onto the gas-liquid interface of the nanobubble and then reassembling them through a compressive shear self-assembly method.

[0030] Furthermore, the platelet membrane is purified into platelet membrane vesicles after the platelet contents are separated by a platelet membrane extraction method.

[0031] The platelet membrane extraction methods include, but are not limited to, mechanical lysis extraction, hypotonic lysis extraction, and repeated freeze-thaw extraction. Preferably, repeated freeze-thaw extraction is used to extract the platelet membrane.

[0032] Furthermore, the bubble membrane shell is prepared by assembling platelet membranes and FTY720 molecules.

[0033] Furthermore, the gas type of the nanobubble core includes, but is not limited to, one or two of air, oxygen, nitrogen, hydrogen, nitric oxide, helium, and sulfur hexafluoride, preferably sulfur hexafluoride gas.

[0034] Furthermore, the size of the platelet membrane self-assembled nanobubbles is 50-100 nm.

[0035] Furthermore, a method for preparing a platelet membrane biomimetic nanobubble loaded with FTY720 includes the following steps:

[0036] (1) Separate the platelet cell membrane and organelles to obtain a purified platelet membrane;

[0037] (2) Platelet membranes were resuspended in FTY720 saline solutions of different concentrations and the FTY720 saline solution was used to initially assemble and combine with the platelet membrane by water bath ultrasound to obtain a suspension.

[0038] The concentration of the FTY720 saline solution is 0.05-0.5 mg / mL; preferably, the concentration of the FTY720 saline solution is 0.1 mg / mL.

[0039] (3) The suspension obtained in step (2) is transferred to the pressure shear bubble generator and different extrusion speeds are applied. By repeatedly injecting gas into the suspension, the gas-liquid interface is disturbed to generate nanobubbles. Under the action of water flow shear force, the platelet membrane and FTY720 physiological saline solution are adsorbed, fused and reassembled at the gas-liquid interface of the nanobubbles. Finally, the platelet membrane biomimetic nanobubbles loaded with FTY720 are obtained.

[0040] The compression speed applied by the pressure shear bubble generator is 10-50 mm / s, preferably 30 mm / s;

[0041] The number of injections is 100-500; 200 is preferred.

[0042] (4) The obtained platelet membrane biomimetic nanobubbles loaded with FTY720 were used in a mouse model of cardiovascular and cerebrovascular diseases to evaluate the immune regulation effect.

[0043] Furthermore, the platelet membrane biomimetic nanobubbles carrying FTY720 are used for targeted drug delivery and immunomodulatory therapy in cardiovascular and cerebrovascular diseases (mouse model of cardiovascular and cerebrovascular diseases).

[0044] The cardiovascular and cerebrovascular diseases (mouse models of cardiovascular and cerebrovascular diseases) include, but are not limited to, acute ischemic stroke, acute myocardial infarction, atherosclerosis, and microcirculatory disorders.

[0045] Example 1:

[0046] The following steps were performed to prepare platelet membrane biomimetic nanobubbles loaded with FTY720:

[0047] (1) Platelet membranes were extracted using a repeated freeze-thaw method, with a concentration of 1×10⁻⁶. 9 The fresh platelet suspension was frozen and thawed three times at -80℃, then centrifuged at 4000g to separate the platelet cell membrane and organelles, and then resuspended and washed three times with physiological saline to obtain purified platelet membrane.

[0048] (2) Prepare a 0.1 mg / mL FTY720 saline solution and resuspend the platelet membrane vesicle precipitate in step (1). The resulting mixture is initially mixed by ultrasonic treatment in a 100W, 42KHz water bath for 5 minutes, so that the FTY720 saline solution is initially assembled and combined with the platelet membrane.

[0049] (3) Transfer the suspension obtained in step (2) into a pressure-induced shear bubble generator. Inject sulfur hexafluoride gas into the suspension at a speed of 30 mm / s to disturb the gas-liquid interface and generate nanobubbles. Repeat the injection 200 times. Under the influence of water flow shear force, the platelet membrane and FTY720 saline solution are adsorbed, fused, and reassembled at the nanobubble gas-liquid interface to prepare a biomimetic nanobubble structure of platelet membrane loaded with FTY720. The adsorption and recombination process of platelet membrane and FTY720 saline solution at the nanobubble gas-liquid interface is as follows: Figure 1 As shown;

[0050] The microstructure of the FTY720-loaded platelet membrane biomimetic nanobubbles prepared in Example 1 was characterized using transmission electron microscopy, such as... Figure 2 As shown, it exhibits a good hollow bubble structure, uniform particle size of 50-100 nm, and obvious high-contrast membrane shell structure and low-contrast bubble core structure.

[0051] Example 2:

[0052] The execution steps are the same as in Example 1, except that the concentration of FTY720 saline solution used for resuspending platelet membrane vesicle precipitation is 0.25 mg / mL.

[0053] Example 3:

[0054] The execution steps are the same as in Example 1, except that the injection rate of sulfur hexafluoride gas in the device during the compressive shearing preparation process is 50 mm / s.

[0055] Experimental Example 1:

[0056] Platelet membrane nanobubbles loaded with FTY720, prepared in Example 1, were used to construct a mouse model of ischemic stroke. The mice were randomly divided into a control group and a treatment group. 200 μL of FTY720-loaded platelet membrane nanobubbles labeled with near-infrared fluorescent dye were injected into the mice via the tail vein. Near-infrared fluorescence imaging was used to collect near-infrared fluorescence signals from the mouse heads before injection (Pre) and at 2, 4, 8, 12, and 24 hours after injection to evaluate the targeting effect on ischemic stroke lesions. The results are as follows: Figure 3 As shown, the intensity of infrared fluorescence signal in the head of mice in the blank group did not change significantly during the collection period; while the intensity of near-infrared fluorescence signal in the head of mice injected with platelet membrane biomimetic nanobubbles loaded with FTY720 increased rapidly and was significantly stronger than that in the control group during the subsequent observation period. Furthermore, the fluorescence signal in the right lesion hemisphere was stronger than that in the left normal hemisphere, indicating that the platelet membrane biomimetic nanobubbles loaded with FTY720 constructed in this invention have good targeting of ischemic stroke lesions.

[0057] Experimental Example 2:

[0058] Platelet membrane nanobubbles loaded with FTY720 prepared in Example 1 were selected, and a mouse model of myocardial infarction-reperfusion injury was constructed. 200 μL of FTY720-loaded platelet membrane nanobubbles were injected into the mice via the tail vein twice, on days 0 and 3 after model establishment. After 28 days of treatment, left ventricular ejection fraction (EF) and left ventricular fractional shortening (FS) were analyzed using M-mode ultrasound. The results are as follows: Figure 4 As shown, the immunomodulatory treatment with platelet membrane nanobubbles loaded with FTY720 resulted in a cardiac EF of 45.44±3.52% and a FS of 22.06±2.00% in mice, while the cardiac EF of untreated mice was 33.03±3.80% and the FS was 15.34±1.97%. The results indicate that the cardiac function of mice was significantly improved after treatment (P<0.001), demonstrating that the platelet membrane nanobubbles loaded with FTY720 provided by this invention have a good immunomodulatory therapeutic effect on myocardial infarction-reperfusion injury.

[0059] Comparative Example 1:

[0060] This comparative example is the M-mode ultrasound analysis and quantitative EF and FS of cardiac function in mice in the sham-operated group in Experiment Example 2. The results showed that the EF of the non-infarcted mouse heart was 66.55±4.35%, and the FS was 36.25±3.56%.

[0061] Finally, it should be understood that the embodiments described in this invention are only used to illustrate the principles of the embodiments of this invention; other variations may also fall within the scope of this invention; therefore, as examples rather than limitations, alternative configurations of the embodiments of this invention can be regarded as consistent with the teachings of this invention; correspondingly, the embodiments of this invention are not limited to the embodiments explicitly introduced and described in this invention.

Claims

1. A method for preparing FTY720-loaded platelet membrane biomimetic nanobubbles, characterized by, The following steps are performed to prepare the FTY720-loaded platelet membrane biomimetic nanobubbles: (1), the platelet membrane is extracted by repeated freeze-thaw method, and the fresh platelet suspension with a concentration of 1 x 10 9 / mL is frozen and thawed for 3 times at-80℃, then centrifuged at 4000 g to separate the platelet cell membrane and organelles, and washed with normal saline for 3 times to obtain the purified platelet membrane; (2) A FTY720 normal saline solution with a concentration of 0.1-0.25 mg / mL is prepared, and the platelet membrane vesicle precipitate in step (1) is resuspended. The resulting mixture is preliminarily mixed by water bath ultrasonic treatment at 100 W and 42 KHz for 5 min, so that the FTY720 normal saline solution is preliminarily assembled and combined with the platelet membrane; (3) The suspension obtained in step (2) is transferred into a pressure-induced shear bubble generating device, and sulfur hexafluoride gas in the device is injected into the suspension at a speed of 30-50 mm / s to disturb the gas-liquid interface and generate nanobubbles. The injection is repeated 200 times, and under the action of water flow shear force, the platelet membrane and the FTY720 normal saline solution are adsorbed, fused and reassembled on the gas-liquid interface of the nanobubbles to prepare FTY720-loaded platelet membrane biomimetic nanobubble structures.

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