Extraction method and application of tomato exosome-like nanovesicle

Abstract

The application discloses a kind of extraction method and application of tomato exosome-like nanovesicle, which comprises the following steps: first, tomato is treated under frozen condition, after thawing, peeling, juice processing, tomato juice is obtained;Then, differential gradient centrifugation is carried out in the way of increasing centrifugal force, the first supernatant is obtained, then ultracentrifugation, resuspension and filtration are carried out, exosome crude extract is obtained, is added into centrifuge tube preloaded with concentration gradient sucrose solution, and after sucrose density gradient centrifugation and purification, it is obtained.The extraction method can prepare tomato exosome-like nanovesicle with typical morphology, high yield and high purity.The obtained tomato exosome-like nanovesicle is rich in natural active substances, has low immunogenicity, good tissue penetration and delivery potential, can be stably taken up by cells and targeted to deliver to inflammation-related cells, further play the role of inhibiting inflammation, and can provide a new intervention approach and strategy for inflammatory reaction and inflammation-related diseases.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for extracting and applying tomato exosome-like nanovesicles. Background Technology

[0002] Plant exosome-like nanovesicles refer to membrane-bound vesicles with a diameter of approximately 30-300 nanometers isolated from plant tissues or organs. These nanovesicles are rich in bioactive components such as proteins, lipids, nucleic acids, and phytochemicals, possessing intrinsic bioactivity, low immunogenicity, good biocompatibility, and excellent tissue penetration capabilities, making them a highly attractive tool for the prevention and treatment of chronic diseases. They can serve as novel therapeutic agents to intervene in disease processes through their naturally loaded active molecules, and also as efficient and safe drug delivery systems to load therapeutic substances. Related studies have shown that various plant exosome-like nanovesicles derived from edible fruits and vegetables have significant bioactivity, naturally possessing multiple therapeutic properties such as anti-tumor, anti-inflammatory, anti-aging, antioxidant, and lipid-lowering effects. Furthermore, their raw material sources are widely available, production costs are low, and they are easy to scale up, demonstrating broad application prospects in the biomedical field.

[0003] Among numerous plant sources, tomato fruit has attracted much attention due to its rich content of lycopene, ascorbic acid, α-tocopherol, and various polyphenols. Lycopene, as the most potent antioxidant carotenoid, has been proven to possess multiple physiological functions, including protecting endothelial cells, inhibiting smooth muscle cell proliferation, regulating inflammatory pathways, reducing the risk of atherosclerosis, and alleviating liver oxidative stress and inflammatory damage. Exosome-like nanovesicles have been isolated from tomato fruit, encapsulating these natural bioactive molecules within their lipid bilayer structure. Compared to tomatoes, tomato exosome-like nanovesicles are rich in proteins, miRNAs, and high concentrations of lycopene and other phytochemicals. They transcend the dispersed structure of the plant itself, concentrating bioactivity through specific biogenetic pathways such as multivesicles, exhibiting excellent biocompatibility and stability. They can be stably internalized in the human body and regulate physiological processes. Furthermore, tomato exosome-like nanovesicles also possess the advantages of high bioavailability, good targeting, and strong tissue penetration of nanocarriers. Compared to directly consuming tomatoes, tomato exosome-like nanovesicles and their contents exhibit significantly improved bioavailability and targeting, with no immunogenicity risk, supporting large-scale sustainable production and representing a safer and more efficient biomedical tool. However, isolating plant exosome-like nanovesicles from complex plant matrices is extremely challenging. The main problem lies in effectively separating the target vesicles from co-precipitated cell debris, extracellular matrix insoluble polymers, and cell wall components. These impurities not only affect the reproducibility of experiments, as well as the purity and yield of the extracted plant exosome-like nanovesicles, but also interfere with downstream analysis and subsequent biotechnological applications.

[0004] Among related technologies, the most commonly used method for extracting tomato exosome-like nanovesicles is differential ultracentrifugation combined with sucrose density gradient centrifugation. This method involves multiple steps, is complex, and produces excessive impurities in the crude extract, resulting in low purity, low yield, and unstable efficacy of the tomato exosome-like nanovesicles. Furthermore, lycopene, a representative carotenoid active ingredient in tomatoes, possesses clear antioxidant and anti-inflammatory potential. However, as a lipid-soluble molecule, lycopene suffers from limited stability and bioavailability in aqueous environments, affecting its targeted delivery and localized action efficiency in in vitro and in vivo intervention systems. Macrophages play a crucial role in the occurrence, development, and resolution of inflammation. Under different microenvironmental stimuli, macrophages undergo polarization, mainly manifested as pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages. Inducing the transformation of M1 macrophages into M2 macrophages can promote inflammation resolution, while over-activated M1 macrophages can lead to uncontrolled inflammatory responses and tissue damage. Discovering active ingredients that can effectively inhibit the transformation of macrophages into pro-inflammatory M1 macrophages will provide new treatment ideas and intervention methods for these inflammatory-related diseases.

[0005] Therefore, there is an urgent need to develop a method for the extraction and preparation of tomato exosome-like nanovesicles that is reproducible, yields high purity, and has good targeted delivery effects. Furthermore, current interventions targeting macrophage phenotypic regulation still lack safe and sustainable natural source solutions, especially in effectively inhibiting M1-like macrophage inflammatory activation and rebalancing phenotypes within the inflammatory microenvironment. An active substance with high targeted delivery efficiency and stable action is urgently needed. Summary of the Invention

[0006] The first objective of this invention is to provide a method for extracting tomato exosome-like nanovesicles.

[0007] A second aspect of the present invention is to provide a tomato exosome-like nanovesicle.

[0008] The third aspect of this invention aims to provide a method for extracting the above-mentioned tomato exosome-like nanovesicles or the application of tomato exosome-like nanovesicles in the preparation of drugs or drug delivery carriers, specifically, the drugs include anti-inflammatory drugs or drugs that inhibit the transformation of macrophages into pro-inflammatory macrophage M1 type.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the present invention provides a method for extracting tomato exosome-like nanovesicles, comprising the following steps: S1. Place the tomatoes under freezing conditions, and after thawing, peel and juice them to obtain tomato juice; S2. The tomato juice is subjected to differential gradient centrifugation with increasing centrifugal force to obtain the first supernatant. Then, it is subjected to ultra-high speed centrifugation at 100,000~150,000 g, the precipitate is collected, resuspended in the first buffer solution, and filtered to obtain crude exosome extract. S3. Add the crude exosome extract to a centrifuge tube pre-filled with a sucrose solution of a concentration gradient, perform sucrose density gradient centrifugation, and collect the mixture containing tomato exosome-like nanovesicles. S4. The mixture containing tomato exosome-like nanovesicles is centrifuged at 100,000~150,000 g, the precipitate is collected, and the precipitate is resuspended in a second buffer solution to obtain the final product.

[0010] The method for extracting tomato exosome-like nanovesicles according to embodiments of the present invention has at least the following beneficial effects: (1) The present invention found that freezing treatment before juicing tomatoes, that is, freezing fresh tomatoes overnight at around -80°C and then thawing and juicing can effectively improve the extraction efficiency of vesicles. It is speculated that this is because low temperature freezing can mechanically damage the cell wall and membrane structure of tomatoes through the formation of ice crystals, which promotes the full release of intracellular vesicles in the subsequent homogenization, thereby improving the initial extraction efficiency.

[0011] (2) The present invention uses a filter to effectively filter the crude extract, and found that it can improve the uneven particle size distribution of tomato exosome-like nanovesicles, which helps to increase the yield of tomato exosome-like nanovesicles, reduce impurities, and make the obtained exosome-like nanovesicle products have higher purity.

[0012] In some embodiments of the present invention, in step S1, the freezing temperature is -75 to -85°C.

[0013] Freezing at -75 to -85°C causes water in cells to crystallize into smaller, denser ice crystals, powerfully and uniformly disrupting the cell walls from within. This facilitates the more thorough release of intracellular vesicles into the extracellular matrix, providing a higher initial vesicle concentration for subsequent juicing and centrifugation. Furthermore, tomato tissue is rich in various hydrolytic enzymes (such as pectinase and protease). During slow freezing at room temperature or -20°C, these enzymes may be activated due to cell damage, degrading the target vesicles and their carried bioactive molecules (such as proteins and RNA). Freezing at the temperature specified in this invention quickly halts almost all enzyme activity and biochemical reactions, maximizing the preservation of vesicle structural integrity and the bioactivity of their contents.

[0014] In some embodiments of the present invention, in step S1, the treatment time under freezing conditions is 6 to 12 hours.

[0015] In some embodiments of the present invention, step S2 includes differential gradient centrifugation, comprising first gradient centrifugation and second gradient centrifugation, wherein: The centrifugal force for the first gradient centrifugation is 300~500 g, and the centrifugation time is 8~20 min; The centrifugal force for the second gradient centrifugation is 2000~3000 g, and the centrifugation time is 20~45 min.

[0016] This invention helps to improve vesicle purity by setting two different gradient centrifugation (i.e., low speed + high speed centrifugation) to remove cell or tissue fragments and smaller organelles from tomato juice in sequence.

[0017] In some embodiments of the present invention, in step S2, the ultra-high speed centrifugation time is 60-90 minutes.

[0018] In some embodiments of the present invention, in step S2, the first buffer solution is a phosphate buffer solution.

[0019] In some embodiments of the present invention, in step S2, the pH value of the phosphate buffer solution is 7.2 to 7.4.

[0020] In some embodiments of the present invention, in step S2, the mass-to-volume ratio of the precipitate to the first buffer solution is 1:205~350, preferably 1:300.

[0021] In some embodiments of the present invention, in step S2, the filtration is performed using a filter with a pore size of 0.2~0.5μm.

[0022] In some embodiments of the present invention, in step S3, the centrifuge tube pre-filled with sucrose solutions of concentration gradients is sequentially filled with sucrose solutions of mass concentrations of 45-55 wt%, 25-35 wt%, 15-25 wt%, and 6-10 wt%.

[0023] Based on the density characteristics of tomato exosome-like nanovesicles, this invention optimizes the sucrose solution concentration gradient. It was found that in this extraction system, a sucrose solution concentration of approximately 50 wt% at the bottom layer is sufficient to capture all high-density impurities in the crude exosome extract. Furthermore, this invention introduces a 20 wt% sucrose solution gradient as a transition layer between 30 wt% and 8 wt% sucrose solutions. This helps avoid impurity retention and facilitates a gentler and more precise migration of vesicles to their equilibrium positions, thereby capturing vesicles with more typical morphology, higher particle count (higher yield), and fewer impurities (higher purity).

[0024] In some embodiments of the present invention, in step S3, the mass ratio of the sucrose solution with a mass concentration of 45-55 wt%, the sucrose solution with a mass concentration of 25-35 wt%, the sucrose solution with a mass concentration of 15-25 wt%, the sucrose solution with a mass concentration of 6-10 wt% to the crude exosome extract is (2-3):(2-3):(1-2):(1-2):1.

[0025] In some embodiments of the present invention, in step S3, the centrifugal force of the sucrose density gradient centrifugation is 100,000~180,000 g, and the centrifugation time is 1~3 h. Preferably, the centrifugal force of the sucrose density gradient centrifugation is 120,000~180,000 g.

[0026] In some embodiments of the present invention, in step S4, the centrifugation time is 60-90 min.

[0027] In some embodiments of the present invention, in step S4, the second buffer solution is a phosphate buffer solution.

[0028] In some embodiments of the present invention, in step S4, the pH value of the phosphate buffer solution is 7.2 to 7.4.

[0029] In some embodiments of the present invention, the mass-to-volume ratio of the precipitate to the second buffer solution is 1:80~120, preferably 1:100.

[0030] In a second aspect, the present invention provides a tomato exosome-like nanovesicle obtained by an extraction method as described in any of the first aspects.

[0031] In some embodiments of the present invention, the tomato exosome-like nanovesicles express CD81 protein and / or HSP70 protein.

[0032] A third aspect of the invention provides the use of the extraction method as described in any of the first aspects or the tomato exosome-like nanovesicles as described in the second aspect in the preparation of a drug or a drug delivery carrier.

[0033] Preferably, the drug includes an anti-inflammatory drug or a drug that inhibits the transformation of macrophages into pro-inflammatory macrophage M1 type.

[0034] The tomato exosome-like nanovesicles of the present invention have typical saucer-like or one-sided concave hemispherical morphological characteristics, which can be taken up by RAW264.7 macrophages and enter the cells. They can also inhibit the transformation of lipopolysaccharide (LPS)-stimulated macrophages into pro-inflammatory macrophages M1 type, thereby inhibiting the inflammatory response. Therefore, they can be used to relieve or improve inflammatory responses and inflammation-related diseases.

[0035] Furthermore, the tomato exosome-like nanovesicles extracted by the method of this invention can target and deliver hydrophobic active ingredients such as lycopene to inflammation-related cells in a form more suitable for cellular uptake, thereby increasing the local effective concentration and enhancing its functional stability.

[0036] Other features and advantages of the present invention will be set forth in the following description. Attached Figure Description

[0037] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 Transmission electron microscopy images of tomato exosome-like nanovesicles extracted by the methods of Examples 1 and 2 of this invention, where the scale bars of A to D are 100 nm, 200 nm, 500 nm and 200 nm, respectively. Figure 2 This is a transmission electron microscope image of tomato exosome-like nanovesicles extracted by the method of Comparative Example 1 of this invention. The scale bar is 100 nm. Figure 3 This is a transmission electron microscope image of tomato exosome-like nanovesicles extracted by the method of Comparative Example 2 of this invention. The scale bar is 100 nm. Figure 4 This is a transmission electron microscope image of tomato exosome-like nanovesicles extracted by the method of Comparative Example 3 of this invention. The scale bar is 500 nm. Figure 5 This is a transmission electron microscope image of tomato exosome-like nanovesicles extracted by the method in Comparative Example 4 of this invention. The scale bar is 100 nm. Figure 6 The NTA detection results are for tomato exosome-like nanovesicles extracted in Example 1 of this invention; Figure 7 The NTA detection results are shown for the tomato exosome-like nanovesicles extracted in Comparative Example 1 of this invention. Figure 8 The results of Western blot analysis of the characteristic proteins CD81 and HSP70 extracted from tomato exosome-like nanovesicles in Example 1 of this invention are shown.

[0038] Figure 9 This is a confocal image showing the uptake of tomato exosome-like nanovesicles prepared in Example 1 of this invention by RAW264.7 macrophages.

[0039] Figure 10 The image shows the RT-qPCR results of the inflammation-related gene IL-6 after RAW264.7 macrophages were treated with tomato exosome-like nanovesicles prepared in Example 1 of this invention.

[0040] Figure 11The image shows the RT-qPCR results of the inflammation-related gene iNOS after RAW264.7 macrophages were treated with tomato exosome-like nanovesicles prepared in Example 1 of this invention.

[0041] Figure 12 The Coomassie Brilliant Blue staining results show the total protein expression in tomato exosome-like nanovesicles in Example 2 of this invention. Detailed Implementation The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0042] The terms "preferred," "more preferably," etc., used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0043] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0044] In the description of this invention, the reference term "and / or" includes all and any combination of one or more of the associated listed items.

[0045] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0046] Example 1 This embodiment provides a method for extracting tomato exosome-like nanovesicles, including the following steps: 1. Obtain tomato juice Take 500g of fresh tomatoes, wash them, freeze them overnight at -80℃, thaw them at room temperature, peel and remove the stems, rinse them twice with pure water, and then put them into a juicer to extract the tomato juice.

[0047] 2. Obtain crude exosome extract The tomato juice obtained above was dispensed into 50 mL centrifuge tubes and subjected to gradient centrifugation at 4 °C. First, centrifuge at 300 g for 10 min, collect the supernatant, and then centrifuge at 2000 g for 30 min to obtain the first supernatant.

[0048] The first supernatant was then aliquoted into ultracentrifuge tubes and centrifuged at 135,000 g for 70 min. The precipitate was collected and then resuspended in PBS at pH 7.4 at a mass-to-volume ratio of 1:300. The precipitate was vortexed thoroughly and filtered through a 0.22 μm PES (polyethersulfone) membrane needle filter to obtain crude exosome extract.

[0049] 3. Sucrose density gradient centrifugation Sucrose solutions with mass concentrations of 50%, 30%, 20%, and 8% were prepared and added to ultracentrifuge tubes in descending order of concentration, with volumes of 2.5 mL, 2.5 mL, 1.5 mL, and 1.5 mL respectively. Finally, 1 mL of the crude exosome extract obtained above was added, and a total of 4 tubes were prepared. After balancing the ultracentrifuge tubes with PBS, they were centrifuged at 150,000 g for 2 h. The band containing tomato exosome-like nanovesicles was collected at the density boundary between the 50% and 30% sucrose solutions.

[0050] 4. Purification treatment The obtained bands containing tomato exosome-like nanovesicles were collected and placed into a new ultracentrifuge tube. They were centrifuged at 135,000 g for 70 min to remove residual sucrose solution from the bands. The supernatant was discarded, the precipitate was collected, and finally, PBS was added for resuspending to obtain tomato exosome-like nanovesicles, which were stored at -80℃ for later use.

[0051] Example 2 This embodiment provides a method for extracting tomato exosome-like nanovesicles, which differs from Example 1 in that the centrifugal force is different. Specifically, it includes the following steps: 1. Obtain tomato juice Take 500g of fresh tomatoes, wash them, freeze them overnight at -80℃, thaw them, peel and remove the stems, rinse them twice with pure water, and then put them into a juicer to extract the tomato juice.

[0052] 2. Obtain crude exosome extract The tomato juice obtained above was dispensed into 50 mL centrifuge tubes and subjected to gradient centrifugation at 4 °C. First, centrifuge at 500 g for 10 min, collect the supernatant, and then centrifuge at 3000 g for 20 min to obtain the first supernatant.

[0053] The first supernatant was then aliquoted into ultracentrifuge tubes and centrifuged at 135,000 g for 70 min. The precipitate was collected and then resuspended in PBS (pH 7.4) at a mass-to-volume ratio of 1:300. The precipitate was vortexed thoroughly and filtered through a 0.22 μm PES (polyethersulfone) membrane syringe filter to obtain crude exosome extract.

[0054] 3. Sucrose density gradient centrifugation Sucrose solutions with mass concentrations of 50%, 30%, 20%, and 8% were prepared and added to ultracentrifuge tubes in descending order of concentration, with volumes of 2.5 mL, 2.5 mL, 1.5 mL, and 1.5 mL respectively. Finally, 1 mL of the crude exosome extract obtained above was added, and a total of 4 tubes were prepared. After balancing the ultracentrifuge tubes with PBS, they were centrifuged at 135,000 g for 2.5 h. The band containing tomato exosome-like nanovesicles was collected at the density boundary between the 50% and 30% sucrose solutions.

[0055] 4. Purification treatment The obtained bands containing tomato exosome-like nanovesicles were collected and placed into a new ultracentrifuge tube. PBS solution was added to the entire tube, and the tube was centrifuged at 135,000 g for 70 min to remove residual sucrose solution from the bands. The supernatant was discarded, the precipitate was collected, and finally, PBS was added for resuspending to obtain tomato exosome-like nanovesicles, which were stored at -80℃ for later use.

[0056] Comparative Example 1 This comparative example provides a method for extracting tomato exosome-like nanovesicles, which differs from Example 1 in that the tomatoes were not subjected to -80℃ freezing treatment, and the sucrose density gradient and centrifugation parameters also differ. The specific steps include: 1. Obtain tomato juice Take 500g of fresh tomatoes, wash them, peel and remove the stems, rinse them twice with pure water, and then put them into a juicer to extract the tomato juice.

[0057] 2. Obtain crude exosome extract The tomato juice obtained above was dispensed into 50 mL centrifuge tubes and subjected to gradient centrifugation at 4 °C. First, centrifuge at 500 g for 10 min and collect the supernatant. Then, centrifuge at 2000 g for 15 min and finally centrifuge at 12000 g for 1 h to obtain the first supernatant.

[0058] The first supernatant was then aliquoted into ultracentrifuge tubes and centrifuged at 100,000 g for 70 min. The precipitate was collected and then resuspended in PBS at pH 7.4 at a mass-to-volume ratio of 1:300. The precipitate was vortexed thoroughly and filtered through a 0.22 μm PES (polyethersulfone) membrane needle filter to obtain crude exosome extract.

[0059] 3. Sucrose density gradient centrifugation Sucrose solutions with mass concentrations of 60%, 45%, 30%, and 8% were prepared and added to ultracentrifuge tubes in descending order of concentration, with volumes of 2.5 mL, 2.5 mL, 1.5 mL, and 1.5 mL respectively. Finally, 1 mL of the crude exosome extract obtained above was added, and a total of 4 tubes were prepared. The ultracentrifuge tubes were then balanced with PBS and centrifuged at 100,000 g for 2 h. The band containing tomato exosome-like nanovesicles was collected at the density boundary between the 50% and 30% sucrose solutions.

[0060] Then, the obtained bands containing tomato exosome-like nanovesicles were collected and placed into a new ultracentrifuge tube. PBS solution was added to the entire tube, and the tube was centrifuged at 100,000 g for 70 min to remove residual sucrose solution from the bands. The supernatant was discarded, the precipitate was collected, and finally, PBS was added for resuspending to obtain tomato exosome-like nanovesicles, which were stored at -80℃ for later use.

[0061] Comparative Example 2 This comparative example provides a method for extracting tomato exosome-like nanovesicles, which differs from Example 1 in that the tomatoes were not subjected to -80℃ freezing treatment, and the sucrose density gradient and centrifugation parameters also differ. The specific steps include: 1. Obtain tomato juice Take 500g of fresh tomatoes, wash them, peel and remove the stems, rinse them twice with pure water, and then put them into a juicer to extract the tomato juice.

[0062] 2. Obtain crude exosome extract The tomato juice obtained above was dispensed into 50 mL centrifuge tubes and subjected to gradient centrifugation at 4 °C. First, centrifuged at 300 g for 10 min and the supernatant was collected. Then, it was centrifuged at 2000 g for 30 min and finally centrifuged at 12000 g for 1 h to obtain the first supernatant.

[0063] The first supernatant was then aliquoted into ultracentrifuge tubes and centrifuged at 100,000 g for 1 h. The precipitate was collected and then resuspended in PBS at pH 7.4 at a mass-to-volume ratio of 1:300. After thorough vortexing, crude exosome extract was obtained.

[0064] 3. Sucrose density gradient centrifugation Sucrose solutions with mass concentrations of 60%, 45%, 30%, and 8% were prepared and added to ultracentrifuge tubes in descending order of concentration, with volumes of 2.5 mL, 2.5 mL, 1.5 mL, and 1.5 mL respectively. Finally, 1 mL of the crude exosome extract obtained above was added, and a total of 4 tubes were prepared. After balancing the ultracentrifuge tubes with PBS, they were centrifuged at 100,000 g for 2 h. The band containing tomato exosome-like nanovesicles was collected at the density boundary between the 45% and 30% sucrose solutions.

[0065] 4. Purification treatment The obtained bands containing tomato exosome-like nanovesicles were collected and placed into a new ultracentrifuge tube. PBS solution was added to the entire tube, and the tube was centrifuged at 100,000 g for 70 min to remove residual sucrose solution from the bands. The supernatant was discarded, the precipitate was collected, and finally, PBS was added for resuspending to obtain tomato exosome-like nanovesicles, which were stored at -80℃ for later use.

[0066] Comparative Example 3 This comparative example provides a method for extracting tomato exosome-like nanovesicles, which differs from Example 1 in that the tomatoes were not subjected to -80℃ freezing treatment, and the sucrose density gradient and centrifugation parameters also differ. The specific steps include: 1. Obtain tomato juice Take 500g of fresh tomatoes, wash them, peel and remove the stems, rinse them twice with pure water, and then put them into a juicer to extract the tomato juice.

[0067] 2. Obtain crude exosome extract The tomato juice obtained above was dispensed into 50 mL centrifuge tubes and subjected to gradient centrifugation at 4 °C. First, centrifuged at 400 g for 30 min, then at 800 g for 30 min, then at 2000 g for 30 min, and finally at 15000 g for 30 min to obtain the first supernatant.

[0068] The first supernatant was then aliquoted into ultracentrifuge tubes and centrifuged at 100,000 g for 2 h. The precipitate was collected and then resuspended in PBS at pH 7.4 at a mass-to-volume ratio of 1:300. After thorough vortexing, crude exosome extract was obtained.

[0069] 3. Sucrose density gradient centrifugation Sucrose solutions with mass concentrations of 60%, 45%, 30%, and 8% were prepared and added to ultracentrifuge tubes in descending order of concentration, with volumes of 2.5 mL, 2.5 mL, 1.5 mL, and 1.5 mL respectively. Finally, 1 mL of the crude exosome extract obtained above was added, and a total of 4 tubes were prepared. After balancing the ultracentrifuge tubes with PBS, they were centrifuged at 100,000 g for 2 h. The band containing tomato exosome-like nanovesicles was collected at the density boundary between the 45% and 30% sucrose solutions.

[0070] 4. Purification treatment The obtained bands containing tomato exosome-like nanovesicles were collected and placed into a new ultracentrifuge tube. PBS solution was added to the entire tube, and the tube was centrifuged at 100,000 g for 70 min to remove residual sucrose solution from the bands. The supernatant was discarded, the precipitate was collected, and finally, PBS was added for resuspending to obtain tomato exosome-like nanovesicles, which were stored at -80℃ for later use.

[0071] Comparative Example 4 This comparative example provides a method for extracting tomato exosome-like nanovesicles, which differs from Example 1 only in that the tomatoes were not subjected to -80°C freezing treatment; all other aspects are the same. Specifically, it includes the following steps: 1. Obtain tomato juice Take 500g of fresh tomatoes, wash them, peel and remove the stems, rinse them twice with pure water, and then put them into a juicer to extract the tomato juice.

[0072] 2. Obtain crude exosome extract The tomato juice obtained above was dispensed into 50 mL centrifuge tubes and subjected to gradient centrifugation at 4 °C. First, centrifuge at 300 g for 10 min, collect the supernatant, and then centrifuge at 2000 g for 30 min to obtain the first supernatant.

[0073] The first supernatant was then aliquoted into ultracentrifuge tubes and centrifuged at 135,000 g for 70 min. The precipitate was collected and then resuspended in PBS with a pH of 7.2-7.4 at a mass-to-volume ratio of 1:300. The precipitate was vortexed thoroughly and filtered through a 0.22 μm PES (polyethersulfone) membrane needle filter to obtain crude exosome extract.

[0074] 3. Sucrose density gradient centrifugation Sucrose solutions with mass concentrations of 50%, 30%, 20%, and 8% were prepared and added to ultracentrifuge tubes in descending order of concentration, with volumes of 2.5 mL, 2.5 mL, 1.5 mL, and 1.5 mL respectively. Finally, 1 mL of the crude exosome extract obtained above was added, and a total of 4 tubes were prepared. After balancing the ultracentrifuge tubes with PBS, they were centrifuged at 150,000 g for 2 h. The band containing tomato exosome-like nanovesicles was collected at the density boundary between the 50% and 30% sucrose solutions.

[0075] 4. Purification treatment The obtained bands containing tomato exosome-like nanovesicles were collected and placed into a new ultracentrifuge tube. They were centrifuged at 135,000 g for 70 min to remove residual sucrose solution from the bands. The supernatant was discarded, the precipitate was collected, and finally, PBS was added for resuspending to obtain tomato exosome-like nanovesicles, which were stored at -80℃ for later use.

[0076] Example 1: Observation using transmission electron microscopy This test example uses transmission electron microscopy (TEM) to observe the tomato exosome-like nanovesicles extracted in Examples 1-2 and Comparative Examples 1-4. The specific methods are as follows: Using a pipette, 10 μL of tomato exosome-like nanovesicle solutions extracted in Examples 1-2 and Comparative Examples 1-4 were respectively diluted with the same volume of PBS buffer at a 1:1 ratio. After thorough mixing, the solutions were dropped onto a copper grid (200 mesh), allowed to stand at room temperature for 3 min, and excess liquid was removed from the edge of the copper grid using clean filter paper and then air-dried. Negative staining was performed using 2% sodium phosphotungstenate solution, and the solutions were dried at room temperature for 5 minutes before imaging.

[0077] Figure 1 Transmission electron microscopy (TEM) images of tomato exosome-like nanovesicles extracted using the methods of Examples 1 and 2, wherein... Figure 1 Figures A, B, and C are images obtained in Example 1, while Figure D is an image obtained in Example 2. It can be seen that the tomato exosome-like nanovesicles extracted using the method of this invention contain a greater number of particles with regular morphology, exhibiting a typical saucer-like or one-sided concave hemispherical shape, and possessing high purity.

[0078] Figure 2 The image shows a transmission electron microscope image of tomato exosome-like nanovesicles extracted using the method in Comparative Example 1. The image shows that although the extracted tomato exosome-like nanovesicles have a high number of exosome particles, their morphology does not meet the general standard.

[0079] Figure 3 The image shows a transmission electron microscope (TEM) image of tomato exosome-like nanovesicles extracted using the method in Comparative Example 2. Tomato exosome-like nanovesicles can be observed, but they contain many impurities and have low purity.

[0080] Figure 4 The image shows a transmission electron microscope image of tomato exosome-like nanovesicles extracted using the method in Comparative Example 3. The image shows that the extracted tomato exosome-like nanovesicles have a high number of particles, but their morphology is also atypical and the content of impurities is high.

[0081] Figure 5 The image shows a transmission electron microscope (TEM) image of tomato exosome-like nanovesicles extracted using the method in Comparative Example 4. The morphology of these nanovesicles is blurry and unclear, and they contain a high amount of impurities.

[0082] Example 2: Nanovesicle tracking analysis This test example uses nanovesicle tracking analysis (NTA) to detect tomato exosome-like nanovesicles extracted in Examples 1-2 and Comparative Examples 1-4. The specific method is as follows: This example uses a laser light source to irradiate a nanovesicle suspension and detects the scattered light from the nanovesicles. The concentration of the nanovesicles is calculated by counting the number of scattered particles. Specifically, this includes using a ZetaView PMX 110 particle matrix to determine the particle count of the tomato exosome-like nanovesicles under 405 nm emission light, and then diluting the tomato exosome-like nanovesicles with PBS buffer to 1×10⁻⁶. 7 Particles / mL ~1×10 9 The particle size / mL was measured, and their size and mass were determined. Simultaneously, the particle trajectories of tomato exosome-like nanovesicles were analyzed.

[0083] in Figure 6 The NTA detection results of tomato exosome-like nanovesicles extracted in Example 1 are shown. Figure 7 The table shows the NTA detection results of tomato exosome-like nanovesicles extracted in Comparative Example 1. Table 1 shows the NTA detection results of tomato exosome-like nanovesicles extracted in each example and comparative example.

[0084] Table 1:

[0085] From Table 1, Figure 6 and Figure 7 The results showed that the NTA of tomato exosome-like nanovesicles obtained by the extraction method of this invention had a particle size of 3.3 × 10⁻⁶ before dilution. 10 The particle count was [number of particles / mL], with particle sizes mainly distributed at 165 nm and 210.1 nm. In contrast, in Comparative Example 1, the tomato exosome nanovesicles extracted using differential ultracentrifugation were not pre-treated with freezing; the NTA results showed a particle count of 2.00 × 10⁻⁶. 9 The particle count was approximately 1.11 × 10⁻⁶ particles / mL, with the particle size mainly around 93 nm. The particle counts for Comparative Examples 2, 3, and 4, after conversion, were 1.11 × 10⁻⁶ particles / mL. 10Particles / mL, 1.47 × 10 10 Particles / mL, 1.96 × 10 10 Particles / mL.

[0086] Based on the above experimental results, it can be clearly shown that the tomato exosome-like nanovesicle solution extracted using the method of the present invention contains more exosome-like nanovesicles and has a higher yield.

[0087] Example 3: Detection of characteristic protein expression in tomato exosome-like nanovesicles In this detection example, proteins were separated by Bis-Tris polyacrylamide gel electrophoresis, and then the expression of the tomato exosome-like nanovesicle characteristic proteins obtained in Example 1 was detected by Western blot. The specific experimental steps are as follows: (1) Electrophoresis: Tomato exosome-like nanovesicle samples were mixed with loading buffer and placed in a 95 ℃ metal bath for 10 min to allow the proteins to denature completely. Then, electrophoresis was performed. The electrophoresis conditions were: 80 V initially, 30 min until the sample entered the separating gel, then the voltage was increased to 120 V and electrophoresis was continued for about 70 min until the bromophenol blue was at the bottom of the separating gel, at which point the electrophoresis was terminated.

[0088] (2) Transfer: Cut a PVDF membrane of appropriate size, soak it in methanol for 10 minutes, and then place it in transfer buffer for later use. After prying open the glass plate, gently scrape off the stacking gel, and then cut out the gel block containing the target protein according to the protein marker indicator band. Carefully remove the gel from the glass plate, make a mark at the corresponding corner of the PVDF membrane, and stack it in a sandwich method of "filter paper-PVDF membrane-gel-filter paper", aligning the edges, and roll it back and forth several times with a glass rod to prevent small air bubbles from remaining. Clamp the electrophoresis clamp, with the membrane side facing the positive electrode of the transfer tank and the gel side facing the negative electrode. Place the ice box in the electrophoresis tank, fill it with pre-cooled 1× transfer buffer, close the lid, and connect the electrodes. Place the assembled electrophoresis tank on an ice bath, and fill the surrounding area with crushed ice to ensure low temperature. Then, turn on the power, set the constant voltage transfer to 100 V, the current to about 300 mA, and the time to 60 min.

[0089] (3) Blocking and incubation treatment: After the transfer, the PVDF membrane was washed once with 1×TBST buffer, and then added to a 5% skim milk blocking solution (prepared with TBST). The membrane was shaken at room temperature for 1 h, then the blocking solution was removed, 1×TBST buffer was added, and the membrane was shaken for 10 min. This process was repeated three times. After washing, the target band was excised according to the protein marker. The primary antibody was diluted to the required concentration with 5% skim milk or 5% BSA solution according to the antibody instructions. The PVDF membrane was placed in the diluted solution, placed on a shaker, and incubated overnight at 4 ℃.

[0090] (4) Color development: After incubation overnight, the primary antibody was recovered into a centrifuge tube and stored at -20 °C. The target band was placed on a shaker and washed three times with 1×TBST buffer for 10 min each time. The secondary antibody was diluted with 5% skim milk to the concentration required by the instructions. The target band was placed in the solution and incubated at room temperature for 1 h. Then, the membrane was placed on a shaker at room temperature and washed three times with 1×TBST buffer for 10 min each time. After washing, color development was performed. The luminescence solution was prepared according to the instructions of the Millipore Immobilon Western Chemilum HRP substrate ECL luminescence kit and was prepared fresh for each use.

[0091] (5) Data Acquisition and Analysis: Turn on the power to preheat the machine, turn on the computer, and open the software for later use. Remove the sample plate, and carefully place the membrane in the center of the sample plate of the gel imaging instrument with tweezers. Then, drop an appropriate amount of luminescent liquid to evenly cover the surface of the membrane. After adjusting the membrane to the optimal exposure area, close the sample plate. Adjust the instrument parameters and exposure time, acquire and save the image, and analyze it using ImageJ software.

[0092] Test results as follows Figure 8 As shown, the characteristic proteins CD81 and HSP70 were enriched in tomato exosome-like nanovesicles extracted using the method of this invention.

[0093] Example 4: Confocal verification of tomato exosome-like nanovesicles being taken up by RAW264.7 macrophages This test example uses the tomato exosome-like nanovesicles prepared in Example 1 as samples to verify macrophage uptake. The specific steps are as follows: First, tomato exosome-like nanovesicles were labeled with a lipid membrane fluorescent dye (PKH26). After removing the free dye, fluorescently labeled tomato exosome-like nanovesicles were obtained. Then, logarithmically growing RAW264.7 macrophages were seeded in confocal culture dishes / slides and cultured until confluence reached approximately 50%–70%. Finally, 5 μL of fluorescently labeled tomato exosome-like nanovesicles (1 × 10⁻⁶ particles) were added. 10 Cells were incubated with the particles for 1 h, 3 h, and 12 h, respectively. After incubation, the cells were thoroughly washed with PBS to remove any untaken particles. Cells were fixed with 4% paraformaldehyde, stained with DAPI, mounted, and observed and imaged using a confocal microscope.

[0094] The results are as follows Figure 9As shown, tomato exosome-like nanovesicles were fluorescently labeled and co-incubated with RAW264.7 macrophages, and intracellular fluorescence signals were observed using confocal microscopy. The results showed that green punctate fluorescence signals corresponding to the tomato exosome-like nanovesicles were observed in the cells at different incubation time points (1 h, 3 h, and 12 h). The signal intensity and the number of punctate particles increased with prolonged incubation time, indicating that the prepared tomato exosome-like nanovesicles can be effectively taken up by RAW264.7 macrophages and accumulate intracellularly, providing cellular evidence for their inflammatory and immunomodulatory effects.

[0095] Example 5: Effects of tomato exosome-like nanovesicles on LPS-induced expression of inflammation-related genes IL-6 and iNOS in RAW264.7 macrophages This test example uses the tomato exosome-like nanovesicles prepared in Example 1 as samples to study the effect of tomato exosome-like nanovesicles on the expression of macrophage inflammation-related genes. The specific steps are as follows: 1. Take RAW264.7 macrophages in the logarithmic growth phase, discard the culture medium, wash with PBS, scrape the cells or trypsin digest them and then stop digestion. Centrifuge at 900 rpm for 3 min, discard the supernatant, and prepare a single-cell suspension; adjust the cell density to 2×10⁶ cells / cells. 5 Cells were seeded into 6-well plates (2 mL of culture medium per well). Cells were cultured in a 5% CO2, 37 °C cell culture incubator for 24 h. When cell confluence reached approximately 60%–70%, the culture medium in the wells was discarded, the cells were washed once with PBS, and fresh culture medium was added.

[0096] 2. Set the groups as follows (3 replicates per group, or set the number of replicates according to the experimental design): (1) Control group (NC): only culture medium was added; (2) LPS group: Add LPS to the culture medium to a final concentration of 100 ng / mL (can be replaced according to actual needs); (3) Tomato exosome-like nanovesicle group: 5 μL of tomato exosome-like nanovesicles (particle number 3.30 × 10⁻⁶) were added to the culture medium. 10 ); (4) LPS + tomato exosome-like nanovesicle group: LPS (final concentration as above) and tomato exosome-like nanovesicles (dosage as above) were added to the culture medium at the same time.

[0097] Each group was incubated at 5% CO2 and 37℃ for 24 h (or replaced as needed).

[0098] 3. After incubation, total RNA was extracted from cells using a Total RNA Extraction Kit (such as Omega Total RNA Kit I or equivalent). cDNA was synthesized using a reverse transcription kit and amplified by RT-qPCR using the SYBR qPCR system. The qPCR program was set as follows: 95℃ for 3 min; 95℃ for 5 s, 60℃ for 30 s, for 40 cycles. Using the internal control gene β-actin as an internal control, relative quantification of IL-6 and iNOS was performed, using a 2... -ΔΔCt The method calculates the relative expression level.

[0099] Experimental results are as follows Figure 10 and Figure 11 As shown, compared with the control group, the mRNA expression levels of IL-6 and iNOS in macrophages of the LPS group were significantly increased; there was no significant difference between the tomato exosome-like nanovesicle treatment group and the control group; compared with the LPS group, the mRNA expression levels of IL-6 and iNOS in the LPS + tomato exosome-like nanovesicle group were significantly decreased, indicating that tomato exosome-like nanovesicles can inhibit the expression of LPS-induced macrophage inflammation-related genes, suggesting that they have biological activities to improve the inflammatory microenvironment and inhibit macrophage pro-inflammatory phenotypic transformation and inflammatory response.

[0100] Example 6: Coomassie Brilliant Blue Staining Detection of Total Protein Expression in Tomato Exosome-like Nanovesicles In this detection example, Bis-Tris polyacrylamide gel electrophoresis was used to separate proteins, and then Coomassie brilliant blue staining was used to detect the total protein distribution of the tomato exosome-like nanovesicles obtained in Example 2 above. The specific experimental steps are as follows: (1) Electrophoresis: Take tomato exosome-like nanovesicle samples and mix them with sample loading buffer. Place them in a 95℃ metal bath for 10 min to allow the proteins to denature completely. Then perform electrophoresis. Electrophoresis conditions: first 80 V voltage, 30 min until the sample enters the separating gel, then increase the voltage to 120 V and continue electrophoresis for about 70 min until the bromophenol blue is at the bottom of the separating gel, then stop electrophoresis.

[0101] (2) Fixation: After electrophoresis, prepare a staining container of suitable size and add sufficient fixative (usually 50% methanol + 10% acetic acid aqueous solution) for later use. After prying open the glass plate, gently scrape off the stacking gel, leaving the separating gel portion. Carefully remove the gel from the glass plate and gently slide it into the container containing the fixative, ensuring that the liquid completely submerges the gel. Place it on a shaker and fix it at room temperature for 1 hour to remove buffer components that interfere with staining and fix the proteins. Subsequently, discard the fixative, add deionized water, and wash three times on a shaker for 5 minutes each time.

[0102] (3) Staining treatment: After washing, add Coomassie Brilliant Blue staining solution (such as R-250 or G-250), ensuring the solution completely covers the gel. Place the container on a shaker and incubate at room temperature. According to the staining solution sensitivity instructions, stain for 1 hour to ensure that the dye molecules fully bind to the protein bands.

[0103] (4) Destaining: After staining, return the staining solution to the reagent bottle, place the gel on a shaker, and rinse it once with deionized water. Add the prepared destaining solution (an aqueous solution of methanol and acetic acid), place the gel in it, and destain on a shaker at room temperature. Observe the destaining situation every 30 minutes and replace with fresh destaining solution. Repeat several times until the blue background completely disappears and the protein bands are clearly visible.

[0104] (5) Data Acquisition and Analysis: Preheat the machine by turning on the power and turn on the computer, then open the software. Remove the sample plate, select the White Light Conversion Screen mode, and carefully place the gel in the center of the sample plate with tweezers, keeping the gel moist and flat with deionized water. After adjusting the gel to the optimal area of ​​the lens, turn off the sample plate. Adjust the instrument aperture, focus, and exposure time, acquire and save the image, and analyze the purity and relative content of the protein bands using ImageJ software.

[0105] Test results: such as Figure 12 As shown, the tomato exosome-like nanovesicles extracted using the method of this invention exhibit clear protein band distribution under Coomassie Brilliant Blue staining, with no obvious contaminating protein tails, indicating good sample purity. Furthermore, the rich protein expression profile displayed by Coomassie Brilliant Blue staining is consistent with… Figure 8 The consistent expression profiles of the characteristic proteins indicate that the extraction method is stable.

[0106] In summary, this invention provides a method and application for extracting tomato exosome-like nanovesicles. First, this invention involves freezing the tomatoes before juicing. It was found that freezing fresh tomatoes overnight at -80°C and then thawing before juicing effectively improves the extraction efficiency of the vesicles. This is presumably because low-temperature freezing can mechanically damage the tomato cell walls and membrane structures through ice crystal formation, promoting more complete release of intracellular vesicles during subsequent homogenization, thereby improving the initial extraction efficiency. Second, this invention also introduces a 0.22 μm PES (polyethersulfone) membrane needle filter to effectively filter the crude exosome extract. Results show that this filter can improve the uneven particle size distribution of tomato exosome-like nanovesicles, helping to increase the yield of tomato exosome-like nanovesicles, reduce impurities, and obtain a higher purity tomato exosome-like nanovesicle product. Furthermore, this invention optimized the parameters of differential ultracentrifugation and the sucrose density gradient, finding that using sucrose solutions with mass fractions of 8%, 20%, 30%, and 50%, and mixing them with crude exosome extract at specific volume ratios, helps increase the content of tomato exosome-like nanovesicles in the precipitate bands. Through improved and optimized extraction methods for tomato exosome-like nanovesicles, this invention significantly outperforms existing technologies in terms of yield, purity, morphological integrity, and process stability, demonstrating promising industrialization prospects. Moreover, the tomato exosome-like nanovesicles prepared by this invention can be efficiently taken up by macrophages, inhibiting the transformation of lipopolysaccharide-stimulated macrophages into pro-inflammatory M1 macrophages, thereby suppressing inflammatory responses and possessing potential for treating inflammatory-related diseases.

[0107] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A method for extracting tomato exosome-like nanovesicles, characterized in that, Includes the following steps: S1. Place the tomatoes under freezing conditions, and after thawing, peel and juice them to obtain tomato juice; S2. The tomato juice is subjected to differential gradient centrifugation with increasing centrifugal force to obtain the first supernatant. Then, it is subjected to ultra-high speed centrifugation at 100,000~150,000 g, the precipitate is collected, resuspended in the first buffer solution, and filtered to obtain crude exosome extract. S3. Add the crude exosome extract to a centrifuge tube pre-filled with a sucrose solution of a concentration gradient, perform sucrose density gradient centrifugation, and collect the mixture containing tomato exosome-like nanovesicles. S4. The mixture containing tomato exosome-like nanovesicles is centrifuged at 100,000~150,000 g, the precipitate is collected, and the precipitate is resuspended in a second buffer solution to obtain the final product.

2. The extraction method according to claim 1, characterized in that, In step S1, the freezing temperature is -75~-85℃; And / or, the treatment time under the freezing conditions is 6 to 12 hours.

3. The extraction method according to claim 1, characterized in that, In step S2, the differential gradient centrifugation includes a first gradient centrifugation and a second gradient centrifugation, wherein: The centrifugal force for the first gradient centrifugation is 300~500 g, and the centrifugation time is 8~20 min; The centrifugal force for the second gradient centrifugation is 2000~3000 g, and the centrifugation time is 20~45 min.

4. The extraction method according to claim 3, characterized in that, In step S2, the ultra-high speed centrifugation process takes 60-90 minutes. And / or, the first buffer is a phosphate buffer; And / or, the mass-to-volume ratio of the precipitate to the first buffer solution is 1:250~350; And / or, the filtration is performed using a filter with a pore size of 0.2~0.5μm.

5. The extraction method according to claim 1, characterized in that, In step S3, the centrifuge tubes pre-filled with sucrose solutions of concentration gradients are sequentially filled with sucrose solutions of mass concentrations of 45-55 wt%, 25-35 wt%, 15-25 wt%, and 6-10 wt%.

6. The extraction method according to claim 5, characterized in that, In step S3, the mass ratio of the sucrose solution with a mass concentration of 45-55 wt%, the sucrose solution with a mass concentration of 25-35 wt%, the sucrose solution with a mass concentration of 15-25 wt%, the sucrose solution with a mass concentration of 6-10 wt% to the crude exosome extract is (2-3):(2-3):(1-2):(1-2):

1.

7. The extraction method according to claim 5, characterized in that, In step S3, the centrifugal force of the sucrose density gradient centrifugation is 100,000~180,000 g, and the centrifugation time is 1~3 h.

8. The extraction method according to any one of claims 1 to 7, characterized in that, In step S4, the centrifugation time is 60-90 min; And / or, the second buffer is a phosphate buffer; And / or, the mass-to-volume ratio of the precipitate to the second buffer solution is 1:80~120.

9. A tomato exosome-like nanovesicle, characterized in that, Obtained by the extraction method as described in any one of claims 1 to 8; Preferably, the tomato exosome-like nanovesicles express CD81 protein and / or HSP70 protein.

10. The extraction method according to any one of claims 1 to 8 or the application of the tomato exosome-like nanovesicles according to claim 9 in the preparation of drugs or drug delivery carriers; Preferably, the drug includes an anti-inflammatory drug or a drug that inhibits the transformation of macrophages into pro-inflammatory macrophage M1 type.

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