A method of isolating exosome extracts from plant sources to allow plant derived exosome-liposome hybrids with increased activity

Abstract

The invention belongs to a technique in which a substantial quantity of exosomes with increased activity is isolated from plants or similar sources. This is achieved by activating the plant derived fluids through physical, chemical, or biological means. Subsequently, the isolated and activated exosomes are merged with cationic liposomes to produce a hybrid molecule possessing elevated activity. This invention also aims to enhance the efficiency of acquiring hybrid nanovesicles derived from plants by eliminating cell wall remnants through the ultrafiltration process subsequent to the activation step. This resulting hybrid molecule finds applications in both pharmaceuticals and cosmetics.

Description

[0001] A METHOD OF ISOLATING EXOSOME EXTRACTS FROM PLANT SOURCES TO ALLOW PLANT DERIVED EXOSOME-LIPOSOME HYBRIDS WITH INCREASED ACTIVITY

[0002] Technical Field

[0003] The invention relates to a method for obtaining plant derived exosome-liposome hybrid molecules, comprising isolating exosomes from several plant sources by extracting through at least one activation step that activates plant cells, inducing them to release elevated levels of both quantity and activity, and combining the exosomes with increased activity with liposomes to produce the hybrid molecule exhibiting elevated activity levels.

[0004] The State of Art

[0005] Cell-to-cell communication is essential for maintaining tissue homeostasis and organismal integrity, with most communication mechanisms involving coordination between receptors and signaling molecules. Recently, nano-vesicle-mediated cell communication has gained prominence, particularly due to the ability of extracellular vesicles known as exosomes to transport various molecules from producing cells to target cells.

[0006] Studies in the prior art have indicated that plant-derived nanovesicles exhibit properties similar to mammalian exosomes, suggesting comparable functionalities. Additionally, it is believed that plant exosomes are primarily released through multi vesicular body-mediated extracellular release. Studies have demonstrated the isolation of exosomes from various plants, such as lemon, ginger, and carrot.

[0007] Exosomes, nano-sized vesicles typically ranging from 30 to 120 nm in diameter, are released by multiple cell types, including mesenchymal stem cells, immune cells (e.g., B and T cells, dendritic cells), epithelial cells, endothelial cells, and cancer cells. Exosomes are cell-specific, they possess the characteristics of the source which they isolated from. These lipid bilayer vesicles contain numerous proteins, lipids, and 3 ’-5’ exonucleases that function in a well-coordinated manner. Exosomes are found in both the nucleus and cytoplasm and possess the ability to shuttle between cells due to their small size and participate in RNA processing. Their capacity to access target cells makes them promising candidates for drug delivery. Studies have shown promising results for exosome-based treatments in various diseases, including cancer and neurodegenerative disorders. Exosome secretion occurs through a process called exocytosis, a vital cellular process, is essential for intracellular signaling and the growth of the plasma membrane. It involves the fusion of vesicles with the plasma membrane, releasing their contents outside the cell, a process known as intracellular trafficking. Exosome secretion is governed by the incorporation of multivesicular bodies (MVBs) fused with the plasma membrane, followed by the release of their cargo. MVBs, formed through inward budding into the endosomal lumen, mediate exosome secretion and transport endosome cargo molecules for degradation to lysosomes. They also facilitate the secretion of contents into the extracellular space through plasma membrane fusion, in addition to delivering cargo to lysosomes.

[0008] The mechanism underlying the development of MVBs, and intraluminal vesicles (ILVs) involves the endosomal sorting complex required for transport (ESCRT) mechanism, comprised of four protein- associated complexes (ESCRT-0, -I, -II, and -III), along with VPS4, VTA1, and ALIX proteins. This mechanism drives the formation of ILVs, which are essential for MVB formation, vesicle budding, and protein cargo sorting. The ESCRT process is initiated by the identification and sequestration of ubiquitinized proteins, followed by interactions between ESCRT-I and -II with ESCRT-III, facilitating vesicle budding. Eventually, the ESCRT-III complex isolates from the MVB membrane after splitting the buds to form ILVs, with energy provided by the Vps4 separation protein. Exosomes isolated from various cell types have been found to contain ESCRT components and ubiquitin proteins, suggesting the involvement of the ESCRT mechanism in exosomal biogenesis. Additionally, the exosomal protein Alix, associated with ESCRT proteins TSG101 and CHMP4, participates in cargo arrangement and membrane budding, further supporting the role of ESCRT components in exosome biogenesis.

[0009] Exosomes, released from MVBs into the extracellular space, are taken up by recipient cells through three mechanisms: fusion with the recipient cell's plasma membrane, receptor-ligand binding between exosome and recipient membranes, and endocytosis by phagocytosis. Exosomes have the ability to modulate molecular pathways upon entering recipient cells, affecting various biological functions.

[0010] Previous studies have reported that plant-derived nanovesicles share similar properties with mammalian exosomes. This suggests that plant-derived exosomes exhibit characteristics close to those of mammalian exosomes. Recent research indicates that exosomes derived from edible plants exhibit superior stability and biocompatibility in the body, with no observed toxicity or immunogenicity unlike exosomes sourced from mammalian cells. Notably, while exosomes from mammalian cells typically contain around 20% cholesterol, those from edible plants are devoid of cholesterol. Especially, when contrasted with animal tissue or cell culture solutions, the plant source offers the benefit of being capable of yielding a significant quantity. Moreover, the efficacy of plant-derived exosomes in wound healing and skin regeneration is attributed to their antioxidant or protein components derived from plants. According to the literature, the primary secretion mechanism of plant exosomes is thought to be accomplished by multivesicular body-mediated release into the extracellular space. Upon encountering external or internal stimuli such as stress, plant-derived nanovesicles are secreted via multivesicular bodies (MVBs), which share a similar morphology with mammalian exosomes. In addition to their morphological similarities, plant and mammalian exosomes exhibit resemblance in their molecular composition. Both types of exosomes contain nucleic acids, mRNA, and miRNA, albeit in varying amounts. For instance, mammalian exosomes are enriched with 100-300 miRNAs, while plant exosomes have fewer. While mammalian exosomes contain higher levels of cholesterol, plant exosomes contain a relatively higher proportion of phospholipids, with some also containing plant-specific lipids such as galactolipids. However, further research is needed to identify the lipid or protein content of plant exosomes more comprehensively.

[0011] Exosome-like nanovesicles isolated from edible plants have been shown to affect nutrition absorption and organ homeostasis. Interspecies communication occurs when exosome-like nanoparticles derived from various edible plants, such as grape, grapefruit, ginger, and carrot, exhibit anti-inflammatory properties in inflammatory bowel diseases. While the presence of exosome-like nanoparticles in plants is supported by evidence, their role in mediating molecular interactions between species requires further detailed studies. Previous studies have demonstrated the influence of plant exosomes on cancer progression and inflammation.

[0012] Novel techniques and kits have emerged for isolating exosomes and extracellular vesicles, supplementing the traditional ultracentrifuge technique. These advancements have been made possible by developments in science and technology, resulting in various isolation techniques with different effects on exosomes based on factors such as density, shape, size, and surface proteins.

[0013] Ultracentrifugation, the oldest and most commonly used method for exosome isolation, relies on segregating extracellular particles based on their size, density, and shape through sequential centrifugation. While ultracentrifugation offers the advantage of handling large sample quantities with high exosome yields, it also has drawbacks. The method's long centrifugation periods at high speeds can potentially damage extracellular vesicles, complicating downstream analysis.

[0014] Immunoaffinity capture, or "exosome fishing," isolates specific types of exosomes based on interactions between exosomal receptors and ligands anchored to purification columns. While advantageous for isolating specific exosome types, this method is costly, and yields are often low.

[0015] A recently developed method known as exosome precipitation utilizes an aqueous two-phase separation technique. In this method, a water-excluding polymer interface captures exosomes by altering their solubility or dispersibility. This approach offers several advantages, including low cost, ease of use, lack of requirement for expensive equipment, and the ability to handle large sample capacities with high yields.

[0016] Filtration is another technique that is a crucial process used to separate, clarify, modify, or concentrate liquid solutions, mixtures, or suspensions. In industries such as biotechnology, pharmaceuticals, and medicine, fdtration plays a vital role in the production, processing, and analysis of various products, including drugs, diagnostics, and chemicals. By removing or "filtering" the suspension medium, components in a suspension can be concentrated. Furthermore, selecting the appropriate fdter material, pore size, and other filter parameters has enabled the development of specialized filtration techniques for selectively isolating components from different sources, such as microbial cultures, blood, and various liquids like solutions, mixtures, or suspensions.

[0017] In the context of exosome isolation, filtration allows for the separation of small particles and soluble molecules from exosomes. During this process, the population of exosomes becomes concentrated by the filtration membrane. Another emerging technique, known as tangential flow filtration (TFF), combines permeable membrane filtration and flow to efficiently concentrate extracellular vesicles (EVs) from a colloidal matrix. TFF differs from traditional filtration methods as it operates tangentially along the liquid surface, preventing the formation of filter cake and enabling high efficiency in obtaining exosomes.

[0018] Any of these methods can be used for exosome isolation regardless of the source. However, with the plant- derived exosomes the cell wall of the plants is the main drawback. Thus, the presence of a cell wall in plant cells has long been perceived as a barrier to the production or perception of vesicles, leading to a lack of research on plant EVs in the subsequent decades. Plant EVs are rich in cell wall-modifying proteins, suggesting a potential mechanism wherein these vesicles degrade a portion of the cell wall to facilitate their passage. These enzymes which can be listed as 1,3-b-glucosidases, pectinesterases, polygalacturonases, b- galactosidases, and b-xylosidase / a-L-arabinofuranosidase-2, could actively perturb the cell wall, thereby facilitating their transmission. Furthermore, the identified proteins suggest that plant cells employ similar mechanisms as animal cells to release EVs, encompassing exosomes and microvesicles. Also, they have the capability to fuse with the plasma membrane of target cells, allowing for the release of their contents intracellularly. This fusion process might occur through clathrin-mediated endocytosis (CME) or via mechanisms independent of CME. Additionally, protein microdomains present on the plasma membrane, such as remorins, could facilitate the fusion of EVs and the release of their cargo.

[0019] Exosomes represent valuable biomaterials for the advancement of novel nano-carriers, particularly in the realm of functionally advanced drug delivery systems. One approach involves the creation of hybrid exosomes using the freezing -thawing method to exert control over and modify the performance of exosomal nano-carriers. Research has shown that by hybridizing exosomes containing specific membrane proteins isolated from genetically modified cells with various liposomes, new hybrid nano-carriers can alter cell interactions, lipid composition, or lipid properties.

[0020] Studies have primarily focused on engineering exosomes to enhance their efficiency through methods like surface modification and hybridization with synthetic nano-carriers such as liposomes. These hybrid exosomes, essentially novel biological nano-carriers, hold promise as a new avenue for transporting both hydrophilic and hydrophobic loads to recipient cells. The technology underlying hybrid exosomes involves fusing exosomes and liposomes through membrane fusion, resulting in a hybrid nanoparticle system.

[0021] Although various studies on exosomes and hybrid exosomes are present in the literature, there is currently no existing technology for the hybridization of plant-derived exosomes and their use as a delivery system.

[0022] In conclusion, while exosome-liposome hybridization is a known method today, ongoing developments aim to enhance the properties of the resulting hybrid molecule, making it a highly active molecule with superior properties.

[0023] The Purpose of the Invention

[0024] The main object of the invention is to isolate exosomes derived from plants with elevated levels of both quantity and activity, while also eliminating the plant cell wall, employing physical, chemical, or biological methodologies, as opposed to the mixed exosome populations typically obtained through current isolation and hybridization approaches. Furthermore, the goal is to create a hybrid molecule with enhanced activity using these isolated plant sourced exosomes. However, this outcome isn't the result of a novel hybridization process or a new method of hybridization. Instead, it is achieved through obtaining activated exosome molecules using a specific method. This inventive method enables the isolation of plant-derived exosome molecules with increased activity and quantity through biological, chemical, or physical means, which can then be hybridized with liposome molecules using established techniques, resulting in hybrid molecules with heightened activity.

[0025] In the invention, it is aimed to enhance the efficiency of acquiring hybrid nanovesicles derived from plants by eliminating cell wall remnants through the ultrafiltration process subsequent to the activation step. This resulting hybrid molecule finds applications in both pharmaceuticals and cosmetics.

[0026] The present invention introduces a method for isolating plant-derived exosomes, which combines centrifugation, ultrafiltration, and TFF systems with filters having a molecular weight cutoff (MWCO) of 100,000 Da to 500,000 Da. This method demonstrates high efficacy, ensuring thorough purification from large molecular impurities, ease of application, and seamless integration into large-scale production processes.

[0027] Another objective of the invention is to develop a method for isolating exosomes from the raw plant material, including one or more elements comprising pulp, shell, seed, stem, leaf, root, and flower.

[0028] Another objective of the invention is to provide a suitable extraction technique for the GMP production of plant-derived exosomes for medical and commercial products.

[0029] Another objective of the invention is to develop new combinations by taking advantage of the superior superior properties of different plant-derived exosomes and to develop new generation carrier systems with using liposomes.

[0030] Using exosomes derived from plants as drug delivery systems offers several advantages: They are sourced from natural origins, providing lower immunogenicity compared to synthetic materials and better compatibility with the body. Additionally, plant-derived exosomes contain components originating from cellular and molecular processes of plants, enhancing their compatibility with the human body and reducing the risk of tissue rejection. They can be easily modified through genetic engineering or other biotechnological methods, increasing the possibility of carrying different drugs or customizing carriers for specific treatment strategies. Production of plant-derived exosomes is generally more cost-effective than synthetic methods, and they are biodegradable, contributing to reduced environmental impact. Also, the size of plant-derived exosomes is bigger than the mammal derived exosomes. Thus, plant-derived exosomes are thought to be better carriers due to their larger size. These advantages make plant-derived exosomes appealing for drug delivery systems, offering further potential for research and development in this field.

[0031] With hybridization technology, plant-derived exosomes can be combined with liposomes loaded with precursors relevant to their intended application, becoming hybrid nanocarriers which we named as hybrid plantosome. The advantages of the hybrid plantosome include:

[0032] • Facilitating the release of the active product from the exosome and ensuring it falls within the cosmetic category.

[0033] • Enhancing the entry of the active product into cells.

[0034] • Preventing the degradation of the active product.

[0035] • Enabling the production of different active products for various purposes (hair, skin, wound care, cellulite).

[0036] • Allowing passage through the digestive system.

[0037] Naturally, hybrid exosomes with these membrane modifications make it easier for both the required genetic material for rejuvenation and the specific active substance for a particular area to reach the target. The method in the invention involves isolating exosomes from several plant sources by increasing their activity and number through physical, chemical, or biological activation. The aim is to obtain highly active hybrid molecules after hybridization due to the increased activity and number of exosome cells obtained through the method.

[0038] The primary objective is to enhance the number and activity of plant-derived exosomes through physical, chemical, or biological methods and obtain a greater number of exosomes capable of carrying more biological materials. This approach aims to produce more active hybrid plantosome with a higher number compared to non-activated exosome-liposome hybrid molecules.

[0039] Another key objective of the invention is to obtain a high number of exosomes with increased activity and to ensure that the new hybrid nano-carrier formed after hybridization with liposomes exhibits higher activity than molecules hybridized through normal processes.

[0040] The process, the subject of the invention, can be applied to a variety of plant types, allowing for the isolation of exosomes with increased activity from different botanical sources. This versatility enhances the potential applications of the method across various fields, including biotechnology, medicine, and cosmetics.

[0041] The novelty of the invention lies in the process of obtaining a higher number of exosomes with increased activity compared to conventional isolation methods. This is achieved by physically, chemically, or biologically activating plant cells and eliminating their cell walls before the hybridization process. By subjecting the plant cells to stress and inducing activation, they release not only the exosomes present outside the cell but also those contained within. In contrast, in conventional isolation processes without cell activation and cell wall elimination, only the exosomes outside the cell are typically isolated, while those within remain inaccessible.

[0042] Through the activation of plant cells before isolation, more active exosomes are released in higher quantities. Subsequently, these exosomes, which exhibit enhanced activity, are hybridized with cationic liposomes to form hybrid molecules with heightened activity.

[0043] In order to solve technical problem and fulfill the aims, the invention relates to a method where / in which high number and high-activity exosomes are isolated by activating plant or other sources with the physical, chemical or biological methods and then the isolated and activated exosomes are hybridized with cationic liposomes to obtain a hybrid molecule called as hybrid plantosome with high activity, wherein the method is characterized by comprising the steps of washing the selected plants to remove impurities, followed by gentle drying and shredding using a laboratory blender or any equivalent equipment. The resulting plant water undergoes gradual centrifugation at increasing speeds to collect the supernatant at each step. This supernatant is transferred to a large cylindrical chamber connected to another chamber fdled with glass beads. Emulsification occurs by transferring the plant juice supernatant between these chambers, facilitating the release of passive exosomes. Ultrafiltration then removes remaining plant cell wall residues, allowing smaller molecules like water, proteins, and exosomes to pass through while blocking larger ones. The purified liquid is collected for further exosome isolation using the tangential-flow filtration method. Finally, isolated exosome molecules are hybridized with liposome molecules using sonication to obtain hybrid plantosomes with high activity.

[0044] Inventors discloses a method of isolating a high number of exosome extracts with high-activity from animal-derived cord blood or fetal animal or animal milk or other animal-derived fluids to allow high- activity exosome-liposome hybridization is disclosed in the patent application numbered as WO2023121578. In addition to this, another document belonging to the inventors, PCT / TR2023 / 050037 relates to a portable and combined product that will enable obtaining activated exosome molecules, nanoparticles or protein molecules in certain sizes from various sources including cell culture, biological material, plant, blood plasma. Although various studies on exosomes and hybrid exosomes are present in the literature, there is currently no existing technology for the hybridization of plant-derived exosomes and their use as a delivery system.

[0045] For this invention, various plant sources were used to derive exosomes. First, the NTA analysis was conducted for exosome characterization from different plant sources shown in Figure 1A, IB and 1C. After proving that exosomes are successfully isolated, in Figure 2A, Lowry Assay was conducted to analyze the protein quantification. The highest plant source was found as olive leaves and berries. After that MTS Assay was conducted in order to observe the cell viability. The cells were treated with exosomes derived from different plant sources with different dosages to also find the active dosage in Figure 3A. According to the results, peach and grape with 200 pg / mL dosage, and melon with 100 pg / mL showed the highest cell viabilities. Then, combinations of these three plant sources were tested in Figure 3B. As a result, it was observed that the combinations with all three decreased the cell viability, while the combination of peach / grape and peach / melon have the highest. However, they showed no significant difference from each other. Then, Scratch Assay was performed on melon with lOOpg / mL, grape and peach with 200 pg / mL, and hybrid plantosome with 200pg / mL dosages. The images were taken at 0, 24, and 48 hours. It was observed that each one of them had better closure rate than the control group. However, there was no significant difference observed between the exosomes and hybrid plantosome. It was shown that hybrid plant sources and exosomes show similar characteristics and can be used as nanocarriers as well. The structural and characteristic features and all advantages of the invention outlined in the drawings below and in the detailed description made by referring these figures will be understood clearly, therefore the evaluation should be made by taking these figures and detailed explanation into consideration.

[0046] Description of the Figures

[0047] Figure 1A: Nanoparticle Tracking Analysis (NTA) applied to melon derived exosomes diluted 1: 100 with PBS.

[0048] Figure IB: Nanoparticle Tracking Analysis (NTA) applied to grape derived exosomes diluted 1: 100 with PBS.

[0049] Figure 1C: Nanoparticle Tracking Analysis (NTA) applied to peach derived exosomes diluted 1: 100 with PBS.

[0050] Figure 2A: Lowry Assay applied to chosen plant-derived exosomes.

[0051] Figure 3A: Cell viability analysis of the Human Dermal Fibroblast (HDF) cells at 24, 48 and 72 hours after the treatment with the different plant-derived exosomes applied with different dosages which are determined as 100 and 200pg / mL.

[0052] Figure 3B: Cell viability analysis of the Human Dermal Fibroblast (HDF) cells at 24, 48 and 72 hours after the treatment with the highest activities of hybrid plantosome combinations shown in Figure 3A with chosen active dosages applied as combined.

[0053] Figure 4A: Scratch Assay performed on single plant-derived exosomes and hybrid plantosome at 24, 48, and 72 hours.

[0054] Figure 5A: Optimization of emulsification cycles for enhanced extraction of intracellular exosomes using glass beads. Exosome concentrations were compared without any activation emulsification conditions.

[0055] Figure 5B: Optimization of emulsification cycles for enhanced extraction of intracellular exosomes using glass beads. Exosome concentrations were compared activation with glass beads for 10 cycles emulsification conditions. Figure 5C: Optimization of emulsification cycles for enhanced extraction of intracellular exosomes using glass beads. Exosome concentrations were compared with glass beads for 20 cycles emulsification conditions.

[0056] Figure 5D: Optimization of emulsification cycles for enhanced extraction of intracellular exosomes using glass beads. Exosome concentrations were compared with glass beads for 30 cycles emulsification conditions.

[0057] Detailed Explanation of the Invention

[0058] In this section, the preferred embodiment of the invention is clarified such that there is no limiting effect for the sake of better understanding the subject.

[0059] The invention relates to a method for obtaining plant derived exosome-liposome hybrid molecules. In this method, exosomes with elevated levels of quantity and activity are isolated from several plant sources. Subsequently, these active plant-derived exosomes are combined with liposomes to produce a novel hybrid molecule with enhanced activity. In this sense, the inventive method comprises steps of isolating exosomes from several plant sources by extracting through at least one activation step that activates plant cells, inducing them to release elevated levels of both quantity and activity, and combining the exosomes with increased activity with liposomes to produce the hybrid molecule exhibiting elevated activity levels. In the preffered embodiment, the method comprises further at least one ultrafiltration step that eliminates cell wall remnants after the activation step. Liposomes in the invention are cationic liposomes. Plant sources can be aloe vera, olive leave, melon, grape, peach, berry, mango and / or grapefruit.

[0060] The activation step employed in the method in the invention to enhance the activity of exosomes can take the form of physical, chemical, or biological means.

[0061] As is known in the literature, there are many exosome isolation methods. Some of these are known methods such as immunoprecipitation, nanofiltration and ultracentrifugation. In this invention, exosome isolation by tangential-flow filtration method is preferred.

[0062] In the physical activation step, first of all, for exosome isolation (extraction), selected plants are provided. These plants are washed with to remove unwanted elements. In this washing step, cold fresh water or PBS can be used. In the next step, the plants are dried gently and shredded, preferably using a laboratory blender or any equipment that is equivalent, with a blade rotating at a speed of 10 to 1,000 rpm. Instead of blender, mortar and pestle, and grinder can be used. The plant water obtained from the laboratory blender (or any any equivalent equipment) is gradually centrifuged to remove unwanted elements and debris. After removing the unwanted elements and debris, the centrifugation is applied to the plant juice. Preferably, in the first centrifugation, the plant juice is centrifuged at 1000 g for 10 minutes and the supernatant is collected without touching the pellet to new tubes. The same process is repeated as 2000 g for 20 min, 3000 g for 30 min, 10000 g for 60 min and 20000 g for 10 min, respectively, and the supernatant is collected to perform the activation process. Centrifugation steps were determined by considering various plant-derived extracellular vesicle isolation methods in the literature. The main purpose of following centrifugation steps successively at increasing speeds is to ensure the separation of components of different densities from the solution. Centrifugation helps separate particles of different sizes (e.g., cell debris, proteins) within the sample. This process increases the purity of the exosomes to be isolated later and helps remove unwanted residues. Therefore, these steps allows exosomes to be isolated more effectively.

[0063] After completing the centrifuge steps, the obtained supernatant is transferred into a cylindrical chamber. In this step, a Luer Lock tip can be used. The cylindrical chamber filled with the supernatant is connected to another air-tight chamber having 2 Luer Lock inlets and containing the medical glass beads (grade glass beads) where an empty cylindrical chamber with a Luer Lock inlet is connected to the other end of the chamber containing the glass beads. The plant juice supernatant is subjected to emulsification by transferring it back and forth between two cylindrical chambers, one filled with glass beads and the other empty, at a rate of at least once per second and with a minimum of 20 repetitions. This process causes the passive exosomes present in the plant juice supernatant to be physically emulsified as they pass through the glass beads, facilitating their active release into the external environment. For the elimination of the rest of the plant cell wall, the activated fragment is exposed to ultrafiltration, a specialized filtration technique. Activated supernatant fragment containing cell wall components is passed through the ultrafiltration system with pores typically ranging from 20 to 100 nanometers to filter out unwanted cell wall remnants or molecules that can be considered as waste within the obtained activated material. The membrane permits the passage of smaller molecules like water, proteins, and exosomes while selectively blocking larger ones like cell wall fragments. This process step effectively removes cell wall components, producing a clarified and purified liquid. Typically, the primary plant cell wall is thin, around 0.1 pm in thickness, whereas the secondary wall ranges from 2 to 10 pm thick. Consequently, the ultrafiltration membrane selected must effectively retain all such fragments of the cell wall. The choice of filtration membrane can differ depending on the sample being analyzed.

[0064] The activated and ultrafiltered fragment is collected in another chamber for the exosome isolation process, the activated and ultrafiltered fragment is collected in another chamber. The cylindrical chamber containing the supernatant and an empty cylindrical chamber are connected to a high-pressure tangential -flow filtration system. The filtration cycle is continued with the ratio of initial volume to final product volume varying from 10: 1 to 3: 1, depending on the concentration of the plant used, and the resulting exosome is collected in a new tube. According to various exosome isolation studies conducted with TFF, exosomes isolated using systems comprising membranes with cut-off values ranging from 100 to 500 kDa exhibit the preferred structural integrity and concentration. Therefore, in accordance with literature, concentration of the raw material between 10: 1 and 3: 1 will ensure the successful plant exosome isolation.

[0065] After the physical activation step, exosome molecules with increased activity are hybridized by liposome molecules. This hybridation is made by using the sonication method. In this wat, it is possible to obtain the hybrid nanoparticles with high activity, a great number of the exosome molecules with increased activity obtained as described above.

[0066] In the studies carried out within the scope of the invention, optimization of emulsification cycles for enhanced extraction of intracellular exosomes using glass beads was performed. Exosome concentrations were compared under different emulsification conditions: (Figure 5A) without any activation, (Figure 5B) activation with glass beads for 10 cycles, (Figure 5C) activation with glass beads for 20 cycles, and (Figure 5D) activation with glass beads for 30 cycles. The optimal number of emulsification cycles required for the efficient extraction of intracellular exosomes utilizing glass beads was investigated. Emulsification, a method known to facilitate the release of intracellular contents, including exosomes, under mechanical stress, was utilized. Experiments were conducted comparing the exosome concentrations obtained with varying numbers of emulsification cycles (10, 20, and 30 cycles) alongside a control group without activation. It was found that the concentration of exosomes was maximized when subjected to emulsification cycles. Conversely, lower concentrations were observed in samples without activation and those subjected to fewer (10) or more (30) cycles. Notably, structural deterioration was observed in exosomes emulsified 30 times, indicating the importance of optimizing the emulsification process.

[0067] Based on these findings, it is concluded that the optimal number of emulsification cycles for extracting intracellular exosomes using glass beads lies between 10 and 20. Emulsifying samples 20 times provides the most efficient extraction without causing significant structural damage. These findings have valuable implications for future experiments and protocols involving the extraction of intracellular exosomes, guiding researchers towards maximizing yield while minimizing structural damage.

Claims

CLAIMS1. A method for obtaining plant derived exosome-liposome hybrids, is characterized by comprising;• isolating exosomes from several plant sources by extracting through at least one activation step that activates plant cells, inducing them to release elevated levels of both quantity and activity,• combining the exosomes with increased activity with liposomes to produce the hybrid molecule exhibiting elevated activity levels.

2. A method according to claim 1, comprising at least one ultrafiltration step that eliminates cell wall remnants after the activation step.

3. A method according to claim 1 or 2, wherein the liposomes are cationic liposomes.

4. A method according to claim 1, wherein the activation step is in the form of physical, chemical, or biological means.

5. A method according to claim 1, wherein the plant sources is aloe vera, olive leave, melon, grape, peach, berry, mango and / or grapefruit.

6. A method according to claim 1, wherein the activation step comprises:• washing and drying the selected plants to remove the unwanted elements and debris,• obtaining plant water from plants,• centrifuging plant water obtained from the previous step, and collecting the supernatant, wherein in the first centrifugation, the supernatant is collected without touching the pellet to new tubes and in the second centrifugation, supernatant is collected to perform the activation step,• transferring obtained supernatant into a chamber with a Luer Lock tip, wherein the chamber filled with the supernatant is connected to another air-tight chamber having 2 Luer Lock inlets and containing the medical glass beads (grade glass beads) where an empty cylindrical chamber with a Luer Lock inlet is connected to the other end of the chamber containing the glass beads,• transferring the supernatant subjected to emulsification, and forth between two cylindrical chambers, one filled with glass beads and the other empty,• exposing the activated fragment to the ultrafiltration for the elimination of the rest of the plant cell wall,• passing the activated supernatant fragment containing cell wall components through the ultrafiltration system with pores typically ranging from 20 to 100 nanometers,• removing cell wall components, and producing a clarified and purified liquid,collecting the activated and ultrafiltered fragment in another chamber.• connecting the chamber containing the supernatant and empty chamber with high-pressure tangential-flow filtration system wherein the filtration cycle is continued with the ratio of initial volume to final product volume varying from 10: 1 to 3: 1, depending on the concentration of the plant used, and the resulting exosome is collected in a new tube.

7. A method according to claim 6, wherein exosome molecules with increased activity are hybridized by liposome molecules.

8. A method according to claim 6, wherein the second centrifugation is performed as 2000 g for 20 min, 3000 g for 30 min, 10000 g for 60 min and 20000 g for 10 min, respectively.

9. A method according to claim 6, comprising transferring the supernatant subj ected to emulsification, and forth between two cylindrical chambers, one filled with glass beads and the other empty, at a rate of at least once per second and with a minimum of 20 repetitions.

Images