Plant extracellular vesicle light loading method and device

Abstract

The invention belongs to the technical field of medicine preparation processes, and particularly relates to a plant extracellular vesicle light loading method and device. The method comprises the following steps: step 1, mixing a plant extracellular vesicle solution and an active ingredient solution, and incubating; step 2, treating the mixed solution by adopting illumination; wherein the light treatment conditions are as follows: red light, green light and yellow light are adopted for treatment, and the wavelength of the red light is selected from 615-625nm; the wavelength of the green light is selected from 510-520 nm; the wavelength of the yellow light is selected from 575-590 nm; the illumination time is selected from 5-30 minutes. The invention further provides a device for implementing the method. By optimizing the illumination condition, the device and the method can effectively improve the loading efficiency and the stability of the plant extracellular vesicles. Therefore, the invention has a good application prospect in the field of medicines.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparation processes, and particularly relates to a method and device for photo-loading of plant extracellular vesicles. Background Art

[0002] In the fields of biomedicine and materials science, the search for new materials with low toxicity, high biocompatibility, and multifunctional loading and delivery capabilities has always been a research hotspot and difficulty. In the prior art, although a variety of materials have been used in biomedical applications, most materials have problems such as high immunogenicity, limited loading capacity, and difficulty in functioning across species, making it difficult to meet the requirements of increasingly complex biomedical application scenarios.

[0003] Plant extracellular vesicles (EVs), which are nanoscale membranous vesicles secreted by plant cells, provide a new idea for solving the above problems. Plant EVs naturally possess the following characteristics: Firstly, they have good biocompatibility, low toxicity, and good immune tolerance, being suitable for various in vitro and in vivo application scenarios without triggering strong immune responses; Secondly, they have strong molecular loading capabilities and can carry various biomolecules such as proteins, nucleic acids, lipids, etc., as well as small molecule compounds, meeting the material loading requirements in different biomedical applications; Thirdly, they have the ability to transfer functions across species and have been proven to be able to play roles in signal transduction and functional regulation between different organisms, laying a foundation for their application in complex biological systems. For example, they have great potential in the research and application of biological interactions between plants and animals, animals and plants, and even different microorganisms.

[0004] Based on the above characteristics, plant EVs, as an ideal photosensitive material and functional molecule carrier, can be used in biomedical fields such as phototherapy, photodynamic therapy, drug delivery, gene editing vectors, etc., as well as in fields such as cosmetics and agricultural biotechnology, showing broad application prospects.

[0005] However, it has been found that under different process conditions, the loading efficiency of plant EVs for drugs varies significantly. Therefore, developing new drug preparation processes to improve the loading efficiency of plant EVs for drugs is an important research topic in this field. Summary of the Invention

[0006] Aiming at the problems of the prior art, the present invention provides a method and device for photo-loading of plant extracellular vesicles.

[0007] A method for photo-loading of plant extracellular vesicles includes the following steps:

[0008] Step 1: Mix a plant extracellular vesicle solution and an active ingredient solution and incubate.

[0009] Step 2: Treat the mixed solution with light.

[0010] Among them, the conditions for light treatment are as follows:

[0011] Treatment with red, green, and yellow light is adopted. The wavelength of the red light is selected from 615 - 625 nm; the wavelength of the green light is selected from 510 - 520 nm; the wavelength of the yellow light is selected from 575 - 590 nm; the light irradiation time is selected from 5 - 30 minutes.

[0012] Preferably, the light irradiation time is 10 minutes.

[0013] Preferably, in step 1, the mass concentration ratio of plant extracellular vesicles to the active ingredient in the mixed solution is 1:1 to 1:32.

[0014] Preferably, in step 1, the conditions for mixed incubation are incubation at 37 ± 0.5 °C for 30 - 60 minutes.

[0015] Preferably, in step 2, the conditions for the light treatment further include: the light intensity of the red light is selected from 1000 - 5000 mcd; the light intensity of the green light is selected from 2000 - 20000 mcd; the light intensity of the yellow light is selected from 1000 - 8000 mcd.

[0016] And / or, in step 2, the mixed solution is treated with rotating light, and the rotation speed of the light relative to the mixed solution is 20 - 25 revolutions per minute.

[0017] Preferably, it further includes step 3: quickly cooling the solution after the treatment in step 2 to room temperature.

[0018] Preferably, the plant extracellular vesicles are selected from kudzu root extracellular vesicles or hypericum perforatum extracellular vesicles;

[0019] And / or, the active ingredient is selected from at least one of proteins, small molecule drugs, nucleic acid drugs, polypeptides, or photosensitive active molecules.

[0020] The present invention also provides plant extracellular vesicles loaded with an active ingredient prepared by the above method for light loading of plant extracellular vesicles.

[0021] The present invention also provides a plant extracellular vesicle light loading bioreactor for implementing the above method for light loading of plant extracellular vesicles, including:

[0022] A fixing component for fixing a container, which is used to load a plant extracellular vesicle solution and an active ingredient solution;

[0023] A light source component, on which a red light source, a green light source, and a yellow light source for irradiating the container are provided.

[0024] Preferably, it further includes a rotating component, which is used to drive the fixed component or the light source component so that the fixed component and the light source component rotate relative to each other.

[0025] The present invention provides a new method for preparing plant extracellular vesicles loaded with active ingredients. By irradiating with three kinds of light, namely red light, green light and yellow light, it can promote the entry of active ingredients into plant extracellular vesicles, improve the effective loading rate and stability of plant extracellular vesicles, and significantly improve the quality of drugs. In a preferred embodiment, the present invention also optimizes process parameters (including wavelength, light intensity, illumination time, rotation speed, etc.), achieving an unexpected improvement in the effective loading rate. In addition, the method of the present invention is a pure physical method, only requiring light irradiation, which does not harm extracellular vesicles and does not introduce drugs with toxic and side effects. Therefore, the present invention has good application prospects.

[0026] Obviously, based on the above content of the present invention, according to the common general knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can be made.

[0027] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention. Description of the Drawings

[0028] Figure 1 is a schematic structural diagram of the outer cover in Embodiment 1 of the present invention;

[0029] Figure 2 is a schematic structural diagram of the LED lamp tube in Embodiment 1 of the present invention;

[0030] Figure 3 is the experimental result of the loading efficiency of the light loading of FITC-Dextran protein molecules by Pueraria lobata extracellular vesicles in Embodiment 2 of the present invention.

[0031] Among them, 1 - outer cover, 2 - LED lamp tube, 3 - liquid crystal display screen, 4 - fan, 5 - LED lamp. Detailed Description of the Embodiments

[0032] In the following examples and experimental examples, reagents and raw materials not specifically described are all commercially available products.

[0033] Example 1 Plant Extracellular Vesicle Light Loading Bioreactor

[0034] This example provides a bioreactor for promoting the light loading of plant extracellular vesicles. It includes:

[0035] A fixing component for fixing a container, which is used to hold a plant extracellular vesicle solution and an active ingredient solution;

[0036] A light source component, on which a red light source, a green light source and a yellow light source for irradiating the container are provided;

[0037] A rotating component for driving the fixing component or the light source component so that the fixing component and the light source component rotate relatively.

[0038] As a preferred embodiment, the structure of the bioreactor in this example is as Figure 1 、 2 shown. It includes a base and an outer cover 1. The outer cover 1 is 20 cm long, 20 cm wide and 16 cm high. The fixing component, the light source component and the rotating component are all arranged inside the outer cover 1. A liquid crystal display screen 3 is arranged on the surface of the outer cover 1, and a control board is arranged inside. The liquid crystal display screen 3 and the control board are electrically connected. A fan 4 can be arranged on the outer cover 1 to reduce the temperature inside the outer cover 1. The fixing component is fixedly connected to the outer cover 1 through a connecting structure.

[0039] The light source component includes an LED lamp tube 2, and there is a channel for accommodating the container inside the LED lamp tube 2. In this example, the container is preferably a transparent tube. LED lamps 5 for illuminating the container are arranged on the LED lamp tube 2. The LED lamps 5 are arranged in three parallel rows, and an LED red light source, an LED green light source and an LED yellow light source are respectively arranged. There are 12 LED lamps 5 in each row at equal intervals, and the row direction is parallel to the axis of the LED lamp tube 2. Preferably, the wavelength of the LED red light source is 615 - 625 nm, and the voltage is 1.9 - 2.5 V (Shenzhen Ouweitai Optoelectronics Co., Ltd., model: 503IRC - 85L / 12); the wavelength of the LED green light source is 510 - 520 nm, and the voltage is 3.0 - 3.2 V (Shenzhen Ouweitai Optoelectronics Co., Ltd., model: 504LGC); the wavelength of the LED yellow light source is 575 - 590 nm, and the voltage is 1.9 - 2.2 V (Shenzhen Ouweitai Optoelectronics Co., Ltd., model:

[0040] L503FYC - 24). Each LED lamp 5 is electrically connected to the control board. This light source component can provide the following intensities of light for the container: red light 1000 - 5000 mcd; green light 2000 - 20000 mcd; yellow light 1000 - 8000 mcd.

[0041] The rotating assembly includes a rolling bearing which is embedded in an opening of the outer cover 1. The outer ring of the rolling bearing is fitted with the outer cover 1, and the inner rings of the rolling bearing extend from both ends of the rolling bearing. There is a motor on the base, and the motor is connected to one end of the inner ring through a belt. The other end of the inner ring is provided with screw holes for fixing the LED lamp tube 2. In this embodiment, there are three screw holes, and there are screws in the screw holes. The end with the threaded hole is located outside the outer cover 1; the motor is electrically connected to the control board.

[0042] Through the bioreactor of this embodiment, a reaction solution that needs to be light-treated (such as a mixture of plant extracellular vesicle solution and active ingredient solution) can be loaded into the tube. Then, through the rotating LED lamp tube 2, the reaction solution is subjected to rotating light irradiation treatment. During the process of treating plant extracellular vesicles for light loading, the bioreactor of this embodiment contains specific stimulation wavelengths of a combination of multiple-band LED light sources, which can control the stimulation intensity and stimulation time to create the best conditions for vesicle loading; in addition, around the transparent tube, the light can rotate to ensure that the vesicles are fully and evenly irradiated by light and prevent the aggregation phenomenon generated during the vesicle treatment process, thereby improving the vesicle loading rate.

[0043] Example 2 Method for Light Loading of Plant Extracellular Vesicles

[0044] This embodiment uses the bioreactor of Example 1 for light loading of plant extracellular vesicles, including the following steps:

[0045] Step 1, Extract extracellular vesicles from Pueraria lobata: Grind 50 grams of Pueraria lobata leaves and stems with an extractor, pass the obtained juice through a filter paper, and then centrifuge at 10000g for 10 minutes for separation. Filter the supernatant with a 0.22μm membrane to remove large fragments, and then use an Amicon Ultra-4 PL 100kDa centrifugal filter (Merck Millipore, Darmstadt, Germany) to centrifuge the sample at a speed of 5000g at 4°C for 10 minutes to concentrate the extracellular vesicles. Take 10μl of the above extracellular vesicle suspension and dilute it to 1mL with PBS buffer without calcium and magnesium ions. Evaluate the particle size and zeta potential with a Nano Sight NS300. Suspend the extracellular vesicles in RIPA buffer and measure them by BCA analysis, with a concentration of 0.102 ± 0.003mg / mL.

[0046] Step 2, Mix the extracellular vesicles of Pueraria lobata obtained in Step 1 with the FITC-Dextran solution, and control the final concentration to be 25ug / mL for extracellular vesicles of Pueraria lobata and 25ug / mL for FITC-Dextran, and incubate at 37°C for 30 minutes;

[0047] Step 3, Load the mixed solution into a sterilized tube, load it into the bioreactor of Example 1, and perform light irradiation treatment on the mixed solution.

[0048] The light conditions are as follows:

[0049] The light exposure times are selected as 5, 10, 15, and 30 minutes (4 experimental groups).

[0050] The wavelength of the LED red light source is 615 - 625 nm, and the voltage is 1.9 - 2.5 V; the wavelength of the LED green light source is 510 - 520 nm, and the voltage is 3.0 - 3.2 V; the wavelength of the LED yellow light source is 575 - 590 nm, and the voltage is 1.9 - 2.2 V.

[0051] During light exposure, the red light intensity is selected as 6000 mcd; the green light intensity is selected as 28000 mcd; the yellow light intensity is selected as 20000 mcd.

[0052] The rotation speed of the lamp tube is: 20 revolutions per minute.

[0053] Step 4: After the light exposure, quickly cool the sample to room temperature to allow the outer exosome membrane to restore stability and prevent drug leakage.

[0054] Detect the loaded exosome sample by a fluorescence spectrometer. The loading efficiency can be determined by measuring the fluorescence intensity of FITC - Dextran. As Figure 3 shown, the results show that the highest light - loading efficiency is 78% at 10 - minute light treatment, showing significant statistical significance compared with other time points.

[0055] From the above embodiments, it can be seen that the present invention provides a photoreloading bioreactor for plant extracellular vesicles and a method for photoreloading plant extracellular vesicles. By optimizing the light conditions, the device and method of the present invention can effectively improve the loading efficiency and stability of plant extracellular vesicles. Therefore, the present invention has good application prospects in the medical field.

Claims

1. A method for light loading of plant extracellular vesicles, characterized in that, It includes the following steps: Step 1: Mix the plant extracellular vesicle solution and the active ingredient solution and incubate them; Step 2: Treat the mixed solution with light; Among them, the conditions for light treatment are: Treat with red, green, and yellow three-color light. The wavelength of red light is selected from 615 - 625 nm; the wavelength of green light is selected from 510 - 520 nm; the wavelength of yellow light is selected from 575 - 590 nm; the light exposure time is selected from 5 - 30 minutes.

2. The method for optical loading of plant extracellular vesicles according to claim 1, characterized in that: The light exposure time is 10 minutes.

3. The method for photo-loading of plant extracellular vesicles according to claim 1, wherein: In Step 1, the mass concentration ratio of plant extracellular vesicles to active ingredients in the mixed solution is 1:1 to 1:

32.

4. The method for optically loading extracellular vesicles of plants according to claim 1, wherein: In Step 1, the conditions for mixed incubation are to incubate at 37 ± 0.5 °C for 30 - 60 minutes.

5. The method for optically loading extracellular vesicles of plants according to claim 1, wherein: In Step 2, the conditions for light treatment further include: the light intensity of red light is selected from 1000 - 5000 mcd; the light intensity of green light is selected from 2000 - 20000 mcd; the light intensity of yellow light is selected from 1000 - 8000 mcd. And / or, in Step 2, the mixed solution is treated with rotating light, and the rotation speed of the light relative to the mixed solution is 20 - 25 revolutions per minute.

6. The method for light loading of plant extracellular vesicles according to claim 1, wherein: It further includes Step 3: Rapidly cool the solution treated in Step 2 to room temperature.

7. The method for optically loading extracellular vesicles of plants according to claim 1, characterized in that: The plant extracellular vesicles are selected from Pueraria lobata extracellular vesicles or Hypericum perforatum extracellular vesicles; And / or, the active ingredient is selected from at least one of proteins, small molecule drugs, nucleic acid drugs, polypeptides, or photosensitive active molecules.

8. The plant extracellular vesicle loaded with active ingredients prepared by the plant extracellular vesicle light loading method according to any one of claims 1 - 7.

9. A plant extracellular vesicle light-loading bioreactor for implementing the plant extracellular vesicle light-loading method according to any one of claims 1-7, characterized in that, It includes: A fixing component for fixing a container, and the container is used to hold the plant extracellular vesicle solution and the active ingredient solution; A light source component, on which a red light source, a green light source, and a yellow light source for irradiating the container are provided.

10. The extracellular vesicle light-loading bioreactor for plants according to claim 9, characterized in that: It further includes a rotating component for driving the fixing component or the light source component so that the fixing component and the light source component rotate relative to each other.

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