Platelet membrane bionic nano-drug delivery system as well as preparation method and application thereof

Abstract

The invention provides a platelet membrane bionic nano-drug delivery system as well as a preparation method and application thereof, and relates to the technical field of medicines. According to the invention, through membrane engineering modification, a wrapping material obtained through hybridization of a platelet membrane and an NK cell exosome is used for loading ginsenoside Rg3 for the first time, a ginsenoside Rg3 delivery system with high encapsulation efficiency and high drug loading capacity is obtained, and the system has excellent stability and can be stored for a long time in normal-temperature and high-temperature environments; the ginsenoside Rg3-loaded platelet membrane bionic nano-drug delivery system is simple in preparation method and suitable for wide popularization and application; according to the ginsenoside Rg3-loaded platelet membrane bionic nano-drug delivery system, the lung cancer treatment effect of ginsenoside Rg3 can be effectively improved, a novel drug is provided for lung cancer treatment, and the drug is high in safety, high in biocompatibility, free of adverse reaction in the treatment process and obvious in anti-inflammatory effect.

Description

Technical Field

[0001] The present application relates to the field of medical technology, and in particular to a platelet membrane biomimetic nano-drug delivery system and a preparation method and application thereof. Background Art

[0002] Currently, the efficacy of many traditional drugs is significantly limited due to poor pharmacokinetics, easy elimination from the body, and high cytotoxicity. In recent years, nanoparticle technology, particularly nanodrug delivery systems, has garnered widespread attention and is increasingly being applied in medicine. Thanks to their small size, high targeting efficiency, and long circulation time, nanoparticles are ideal drug carriers, capable of improving in vivo targeting efficiency and reducing nonspecific adverse reactions. However, despite the significant attention and progress made in the medical field, several key challenges remain. These include the stability and drug resistance of nanodrug delivery systems, as well as suboptimal efficacy resulting from low target organ accumulation. Furthermore, the in vivo biocompatibility of nanodrug delivery systems needs to be improved to meet the requirements of precise disease treatment.

[0003] Platelets are small pieces of cytoplasm shed from the cytoplasm of mature megakaryocytes in the mammalian bone marrow. Although they comprise only 0.5%-1% of total blood cells, they play a vital role in the body. Recent studies have shown that platelet membranes can serve as biomimetic coatings, enabling nanoparticles to evade macrophage recognition, capture circulating tumor cells, and target sites of inflammation, making them ideal drug carriers. However, research on platelet-based drug delivery is limited. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides a platelet membrane biomimetic nano drug delivery system and its preparation method and application.

[0005] In one aspect, the present application provides a method for preparing a platelet membrane biomimetic nano-drug delivery system, the method comprising the following steps: Step 1: mixing and extruding the platelet membrane and the cell exosomes to obtain a hybrid material; Step 2: Mix ginsenosides with the hybrid material, stimulate the fusion of ginsenosides and the hybrid material by electroporation, and incubate.

[0006] Furthermore, the cell exosomes are selected from one or more of MSC cell exosomes, NK cell exosomes, and 293T cell exosomes.

[0007] Preferably, the cell exosomes are NK cell exosomes.

[0008] Furthermore, the volume ratio of the platelet membrane to the extracellular vesicles is 1:(0.5-2).

[0009] The volume ratio of the platelet membrane to the extracellular vesicles may be any value of 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, or 1:2.

[0010] Preferably, the volume ratio of the platelet membrane to the extracellular vesicles is 1:1.

[0011] Preferably, the extrusion conditions are 100-200 nm filter membrane extrusion 10-30 times More preferably, the extrusion condition is 20 times of extrusion through a 100 nm filter membrane.

[0012] Furthermore, the ginsenoside is selected from one or more of ginsenoside Rg3, ginsenoside Rb1, ginsenoside Rb2, and ginsenoside Rb3.

[0013] Preferably, the ginsenoside is ginsenoside Rg3.

[0014] Furthermore, the concentration of the ginsenoside is 0.1 mg / mL-0.3 mg / mL.

[0015] The concentration of the ginsenosides may be any value among 0.1 mg / mL, 0.2 mg / mL, and 0.3 mg / mL.

[0016] Preferably, the concentration of the ginsenoside is 0.1 mg / mL.

[0017] In a preferred embodiment, the solvent of the ginsenoside is water.

[0018] Furthermore, the volume ratio of the ginsenoside to the hybrid material is 1:(1-3).

[0019] The volume ratio of the ginsenoside to the hybrid material can be any value among 1:1, 1:2 and 1:3.

[0020] Preferably, the volume ratio of the ginsenoside to the hybrid material is 1:2.

[0021] Furthermore, the conditions for the electroporation method to stimulate the fusion of ginsenosides and the hybrid material include: a voltage of 300-400 V / cm and 2-4 electric shocks.

[0022] The voltage may be any value among 300 V / cm, 310 V / cm, 320 V / cm, 330 V / cm, 340 V / cm, 350 V / cm, 360 V / cm, 370 V / cm, 380 V / cm, 390 V / cm, and 400 V / cm.

[0023] The number of electric shocks can be any value among 2, 3, and 4.

[0024] Preferably, the voltage is 350 V / cm and the electric shock is performed 3 times.

[0025] Preferably, each electric shock lasts 10-30 ms, with an interval of 1-2 s.

[0026] More preferably, each shock is 10 ms long with a 1 s interval between shocks.

[0027] Preferably, the incubation conditions include shaking incubation at 36°C-38°C for 1-3 h.

[0028] More preferably, incubate at 37°C with shaking for 1 h.

[0029] In a preferred embodiment, the method comprises the following steps: Step 1: Platelet membranes and NK cell exosomes are mixed at a volume ratio of 1:(0.5-2), and extruded 10-30 times through a 100-200 nm filter membrane using a micro-extruder to obtain a hybrid material; Step 2: Dissolve ginsenoside Rg3 in water, and mix 0.1 mg / mL-0.3 mg / mL of ginsenoside Rg3 with the hybrid material in a volume ratio of 1:(1-3). After mixing, apply a voltage of 300-400 V / cm, and shock 2-4 times, each time for 10-30 ms, with an interval of 1-2 s. Use electroporation to stimulate its fusion, and incubate with shaking at 36°C-38°C for 1-3 h to obtain a bionic nanodrug delivery system of platelet membrane-wrapped ginsenoside Rg3.

[0030] Preferably, the method further comprises a purification step.

[0031] Those skilled in the art can use general methods for purification.

[0032] In a preferred embodiment, the purification comprises: after the incubation, adding anticoagulant buffer solution, centrifuging at 0.5-1 g for 10-30 min at room temperature, discarding the supernatant, and repeating 1-3 times to remove free ginsenoside Rg3 and solvent.

[0033] On the other hand, the present application also provides a platelet membrane biomimetic nano drug delivery system prepared by the method described.

[0034] Preferably, the platelet membrane biomimetic nano drug delivery system can achieve an encapsulation rate of ginsenosides of more than 80%; more preferably, it can reach 82.04%.

[0035] Furthermore, the ginsenoside is selected from one or more of ginsenoside Rg3, ginsenoside Rb1, ginsenoside Rb2, and ginsenoside Rb3.

[0036] Preferably, the ginsenoside is ginsenoside Rg3.

[0037] Preferably, the platelet membrane biomimetic nano drug delivery system has good stability and can be stored at 60° C. for more than 30 days.

[0038] On the other hand, the present application also provides a composition comprising the platelet membrane biomimetic nano drug delivery system.

[0039] The composition described in the present application may also be added with excipients, which may be appropriate solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, etc.

[0040] Preferably, the composition can be prepared as an injection.

[0041] The compositions described herein can be prepared by conventional methods, wherein one or more diluents or carriers may be added.

[0042] On the other hand, the present application also provides the use of the platelet membrane biomimetic nano drug delivery system or the composition in the preparation of lung cancer drugs.

[0043] Preferably, the lung cancer tumor inhibition rate can reach over 68%.

[0044] Preferably, the platelet membrane biomimetic nano-drug delivery system has high safety, high biocompatibility, and low adverse reaction rate during treatment.

[0045] Preferably, the defective rate is less than 10%, more preferably, 6.89%.

[0046] Preferably, the platelet membrane biomimetic nano drug delivery system or the composition also has anti-inflammatory effects.

[0047] The present invention has the following beneficial effects: 1. In this application, for the first time, platelet membranes and NK cell exosomes were hybridized to obtain a packaging material loaded with ginsenoside Rg3 through membrane engineering, resulting in a ginsenoside Rg3 delivery system with high encapsulation efficiency and high drug loading. The system also has excellent stability and can be stored for long periods of time in both room and high temperature environments. 2. The preparation method of the platelet membrane biomimetic nanodrug delivery system loaded with ginsenoside Rg3 is simple and suitable for wide promotion and application; 3. The platelet membrane biomimetic nanodrug delivery system loaded with ginsenoside Rg3 can effectively improve the therapeutic effect of ginsenoside Rg3 on lung cancer, providing a new drug for the treatment of lung cancer. The drug has high safety and biocompatibility, has no adverse reactions during the treatment process, and has obvious anti-inflammatory effects. DETAILED DESCRIPTION

[0048] In order to more clearly illustrate the overall concept of the application, the following is described in detail in the form of embodiments. In the following description, a large amount of specific details are provided to provide a more thorough understanding of the present invention. However, it will be apparent to those skilled in the art that the present invention can be implemented without the need for one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0049] Before further describing the specific embodiments of the present invention, it should be understood that the scope of the present invention is not limited to the specific embodiments described below. It should also be understood that the terminology used in the examples is intended to describe specific embodiments and is not intended to limit the scope of the present invention. The experimental procedures in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or according to the conditions recommended by the respective manufacturers.

[0050] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof. When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0051] Unless otherwise specified, in the following embodiments, the reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased from the market.

[0052] Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in microbiology, biochemistry, analytical chemistry, pharmacy, zoology, medicine, cell culture, and related fields conventional in the art.

[0053] In addition, the "water" mentioned in the present invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion exchange water, double distilled water, high-purity water, and purified water.

[0054] In the following examples, unless otherwise specified, % represents wt%, i.e., percentage by weight.

[0055] Those skilled in the art can obtain platelet membranes using common methods. In a preferred embodiment, the platelet membrane preparation method comprises: collecting blood from the abdominal aorta of adult SD rats, centrifuging the collected whole blood at 200 g for 20 minutes, removing the supernatant, centrifuging at 1800 g for 20 minutes at room temperature, washing the precipitate three times, and then centrifuging at 1800 g for 5 minutes to obtain platelets. The platelets are then placed in a 43°C water bath for 30 minutes, frozen and thawed three times, centrifuged at 8000 g for 15 minutes at room temperature, and the supernatant removed. The platelets are washed, resuspended, and sonicated at 20-25 kHz for 3 minutes in a water bath sonicator to obtain the platelet membrane.

[0056] MSC Cell Exosomes (Human) (Cat. No. 41220ES60), NK Cell Exosomes (Human) (Cat. No. 41221ES60), and 293T Cell Exosomes (Cat. No. 41219ES60) were purchased from Yisheng Biotechnology.

[0057] Ginsenoside Rg3 (CAS No.: 14197-60-5), ginsenoside Rb1 (CAS No.: 41753-43-9), ginsenoside Rb2 (CAS No.: 11021-13-9), and ginsenoside Rb3 (CAS No.: 68406-26-8) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0058] Example 1 Preparation of platelet membrane-encapsulated ginsenoside biomimetic nano drug delivery system Platelet membranes can act as biomimetic coatings, enabling nanoparticles to evade macrophage recognition, capture circulating tumor cells, and localize to sites of inflammation. However, due to the large molecular weight of ginsenosides, platelet membranes alone have limited encapsulation efficiency. In this example, platelet membrane-encapsulated ginsenoside preparation methods were optimized and screened through membrane engineering to enhance drug loading capacity and efficiency. The process is as follows.

[0059] Step 1: Platelet membranes and cell exosomes (MSC cell exosomes, NK cell exosomes, and 293T cell exosomes) were mixed at a volume ratio of 1:1 and extruded 20 times through a 100 nm filter membrane using a micro-extruder to obtain a hybrid material; Step 2: Dissolve ginsenosides (ginsenoside Rg3, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rb3) in water, and mix 0.1 mg / mL of ginsenosides with the hybrid material at a volume ratio of 1:1. After mixing, apply 300 V / cm voltage and shock twice, each time for 10 ms, with an interval of 1 s. Use electroporation to stimulate its fusion, and incubate with shaking at 37°C for 1 h. After the incubation, add anticoagulant buffer solution, centrifuge at 0.8 g for 20 min at room temperature, discard the supernatant and repeat 3 times to remove free ginsenosides and solvent, and obtain a bionic nanodrug delivery system of ginsenosides wrapped in platelet membrane.

[0060] The encapsulation efficiency (EE) of the platelet membrane-encapsulated ginsenoside biomimetic nanodrug delivery system was determined using an enzyme-linked immunosorbent assay (ELISA). The method involved measuring absorbance at a maximum absorption wavelength of 540 nm. A linear regression was performed using absorbance as the ordinate and concentration as the abscissa to construct a standard curve equation. The unbound drug concentration in the supernatant was then measured. The EE was calculated based on the initial concentration of the drug solution and the unbound drug concentration in the supernatant using the following formula. The results are shown in Table 1.

[0061] EE (%) = (initial concentration of drug solution - concentration of supernatant) / initial concentration of drug solution × 100%.

[0062] Table 1

[0063] As shown in Table 1, the optimal encapsulation effect for ginsenoside Rg3 was achieved when the hybrid material of NK cell exosomes and platelet membranes was used, with an encapsulation efficiency exceeding 70%. This example further screened and adjusted parameters such as the volume ratio of platelet membranes to exosomes, the volume ratio of ginsenosides to hybrid material, the voltage, and the number of shocks. The results are shown in Table 2.

[0064] Table 2

[0065] As shown in Table 2, the optimized platelet membrane-encapsulated biomimetic nano-drug delivery system for ginsenosides can achieve an encapsulation efficiency of over 80%. This method also effectively increases the drug loading capacity of ginsenoside Rg3 and its solubility in the product.

[0066] In summary, the preparation method of the optimized platelet membrane-encapsulated ginsenoside biomimetic nanodrug delivery system includes the following steps: Step 1: Platelet membranes and NK cell exosomes were mixed at a volume ratio of 1:1 and extruded 20 times through a 100 nm filter membrane using a micro-extruder to obtain a hybrid material; Step 2: Dissolve ginsenoside Rg3 in water, mix 0.1 mg / mL ginsenoside Rg3 with the hybrid material in a volume ratio of 1:2, apply 350 V / cm voltage and electric shock 3 times, each time for 10 ms, with an interval of 1 s, and stimulate its fusion by electroporation. Incubate at 37°C with shaking for 1 h. After the incubation, add anticoagulant buffer solution, centrifuge at 0.8 g for 20 min at room temperature, discard the supernatant and repeat 3 times to remove free ginsenoside Rg3 and solvent, and obtain a bionic nanodrug delivery system of ginsenoside Rg3 wrapped in platelet membrane.

[0067] The following experiment was carried out using the optimized platelet membrane-wrapped biomimetic nanodrug delivery system of ginsenoside Rg3 as a sample.

[0068] Example 2 Stability Evaluation In this example, the stability test was performed on the sample obtained by optimizing Example 1.

[0069] 1. Encapsulation efficiency stability test: In this example, a biomimetic nanodrug delivery system sample containing ginsenoside Rg3 encapsulated in platelet membranes, as obtained in Example 1, was placed in physiological saline and the dissolution of ginsenoside Rg3 was observed at different time points (0 h, 12 h, and 24 h). The concentration of ginsenoside Rg3 in the supernatant was measured using a microplate reader, and the concentration of ginsenoside Rg3 stably encapsulated in the platelet delivery system was calculated. The in vitro encapsulation stability was then observed, and the rate of change in encapsulation efficiency was calculated. The encapsulation efficiency was calculated using the same method as in Example 1, and the rate of change was calculated using the following formula. The results are shown in Table 3.

[0070] Change rate (%) = (initial encapsulation rate - final encapsulation rate) / initial encapsulation rate * 100% Table 3 Stability results

[0071] As can be seen from Table 3, the sample encapsulation efficiency did not change significantly, proving that the sample system was stable.

[0072] 2. Storage stability test: In this example, the platelet membrane-encapsulated ginsenoside Rg3 biomimetic nanodrug delivery system sample obtained in Example 1 was prepared as a lyophilized powder and dispensed into sealed bottles (0.5 g / bottle). The sample was stored at 60°C for 30 days. After 30 days, its morphology was observed and compared before and after the experiment. The results showed that its morphology did not change before and after storage, demonstrating the sample's good stability.

[0073] Example 3 Lung cancer treatment effect experiment In this example, the biomimetic nano-drug delivery system containing ginsenoside Rg3 encapsulated in platelet membranes obtained in Example 1 was used as a sample for lung cancer treatment testing. The method includes the following steps: 5×10 6 LLC (mouse lung cancer cell) cells were inoculated subcutaneously on the right inner thigh of BALB / c mice. The model was successfully established when a tumor appeared. Tumor size was assessed daily using a vernier caliper and tumor volume was calculated. When the tumor volume reached 90-100 mm 2 The treatment was started at 14:00. The mice were randomly divided into groups, and the dose of ginsenoside Rg3 in the sample was 25 mg / kg, and the drug was administered once every 2 days for a total of 4 times. An equal amount of commercially available ginsenoside Rg3 injection was used as a positive control, and a negative control group with an equal amount of normal saline injection was set up. After the experiment was completed, the mice were killed by cervical dislocation. After the mice were killed, they were quickly dissected and the tumor tissues of the mice were collected. The tumor volume and adverse reactions of the mice were calculated. Adverse reactions included but were not limited to weight loss, death, severe diarrhea, etc. The formulas for calculating the tumor inhibition rate and adverse reaction rate are as follows, and the results are shown in Table 4.

[0074] Tumor inhibition rate (TGI)% = (1-T / C) × 100, where T represents the tumor volume of the experimental group and C represents the tumor volume of the negative control group; Adverse reaction rate (%) = (number of mice with adverse reactions ÷ total number of mice treated with the drug) × 100%.

[0075] Table 4

[0076] As shown in Table 4, the platelet membrane-encapsulated biomimetic nano-drug delivery system of ginsenoside Rg3 obtained in Example 1 has a significant tumor inhibition effect, providing a new drug for the treatment of lung cancer. It is also highly safe and has a significantly reduced adverse reaction rate.

[0077] Example 4 Anti-inflammatory effect experiment In this example, IL-6 levels in each group of mice in Example 3 were detected using an ELISA kit. The test results are shown in Table 5.

[0078] Table 5

[0079] From the results in Table 5, it can be seen that the biomimetic nano drug delivery system of platelet membrane-encapsulated ginsenoside Rg3 obtained by optimization in Example 1 has an anti-inflammatory effect.

[0080] Example 5 In vivo biocompatibility evaluation In this example, in order to explore whether the bionic nano-drug delivery system of platelet membrane-wrapped ginsenoside Rg3 obtained by optimization in Example 1 would damage organs, the heart, liver, spleen, lung, and kidney of the experimental mice in Example 3 were collected and H&E sections were observed. The results of the H&E sections showed that the physiological structures of the above organs were normal, indicating that the delivery system has good safety and biocompatibility.

[0081] The foregoing is merely an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application should all be included within the scope of the claims of the present application.

Claims

1. A method for preparing a platelet membrane biomimetic nano drug delivery system, characterized in that: The method comprises the following steps: Step 1: mixing and extruding the platelet membrane and the cell exosomes to obtain a hybrid material; Step 2: Mix ginsenosides with the hybrid material, stimulate the fusion of ginsenosides and the hybrid material by electroporation, and incubate.

2. The preparation method according to claim 1, characterized in that The cell exosomes are selected from one or more of MSC cell exosomes, NK cell exosomes, and 293T cell exosomes.

3. The preparation method according to claim 1, characterized in that The volume ratio of the platelet membrane to the cell exosomes is 1:(0.5-2).

4. The preparation method according to claim 1, characterized in that The ginsenoside is selected from one or more of ginsenoside Rg3, ginsenoside Rb1, ginsenoside Rb2 and ginsenoside Rb3.

5. The preparation method according to claim 1, characterized in that The concentration of the ginsenoside is 0.1 mg / mL-0.3 mg / mL.

6. The preparation method according to claim 5, characterized in that The volume ratio of the ginsenoside to the hybrid material is 1:(1-3).

7. The preparation method according to claim 1, characterized in that The conditions for stimulating the fusion of ginsenosides and hybrid materials by electroporation include: voltage of 300-400 V / cm and electric shock times of 2-4 times.

8. The platelet membrane biomimetic nano drug delivery system prepared by the method according to any one of claims 1 to 7.

9. A composition, characterized in that The composition comprises the platelet membrane biomimetic nano drug delivery system as claimed in claim 8.

10. Use of the platelet membrane biomimetic nano drug delivery system according to claim 8 or the composition according to claim 9 in the preparation of lung cancer drugs.

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