Exosomes also called ectosomes, exovesicles, or exosome-like vesicles are uniformly round, membrane-covered structures (He et al., 2021) with a diameter of 30–150 nm (Figure 1) (Tauro et al., 2012). Exosomes are produced by the constitution of multivesicular bodies and secreted to the extracellular milieu after integration into the plasma membrane (Mathivanan et al., 2010; Żmigrodzka et al., 2016). Exosomes are known for their role in intercellular communication and can transport mRNA, bioactive lipids, and proteins (Rayner and Hennessy, 2013; Robbins and Morelli, 2014) and regulate gene/protein expression. Upon contact, exosomes deliver molecules that can confer new characteristics and/or reproduce recipient cells. They are composed of a certain number of specific proteins, notably the tetraspanin family (CD81, CD82, CD9 and CD63, flotillin, TSG101, and Alix) and heat choc proteins (HSP90, HSP70, HSPA5, CCT2, and HSP60) (Théry et al., 2009; AYokoi et al., 2015; Ciardiello et al., 2016; Ha et al., 2016). Exosomal membranes are composed of sphingomyelin, cholesterol, phosphatidylinositol, ceramide, phosphatidylethanolamine, phosphatidylserine, etc. (Tamkovich et al., 2016).

Microvesicles (MV) also called microparticles (MP) are vesicles that bud by burgeoning from the plasma membrane and has a size of 50–1,000 nm or even more (Figure 1) (Gould and Raposo, 2013). MV contains essential protein markers including integrins, selectins, and CD40. Their membranes possess cholesterol, diacylglycerol, and phosphatidylserine in greater quantities than exosomes (He et al., 2021). Their constitution is based on its principal mechanisms by a change of membrane phospholipids and the modification of the cytoskeleton of the cell. These MVs then implicate part of the cell’s cytoplasm. They also reflect the state of activation of their cell of origin or its apoptosis (Boireau and Elie-Caille, 2021).

Apoptotic bodies also called apoptosomes are vesicles with the dimension of 1–5 µm delivered by cells in apoptosis (Figure 1) (Zitvogel et al., 1998). They are essentially liberated during cellular apoptosis (Poon et al., 2014). Apoptotic bodies are cleared to assure a “clean” elimination of the cellular content following apoptosis. This content has a strong immunogenic potential, and its release in the extracellular milieu induces local inflammatory reactions. By embedding them in vesicles, the contents of the cell will be eliminated by macrophages that recognize the PS expressed on the apoptotic bodies, thus avoiding any inflammatory responses (Zitvogel et al., 1998). These apoptotic bodies’ function is not limited to the clearance of cellular components since they also participate in intercellular communication.

Extracellular vesicles are distinguished into three groups based on their formation mechanism: exosomes, microvesicles (MV), and apoptotic bodies (AB) (Table 1; Figure 1).

Plant-derived VEs are heterogeneous groups of vesicles containing different functions, mainly multivesicular bodies (MVBs), autophagosomes, vacuoles, and exocyst-positive organelles (EXPOs). The size of plant-derived nanovesicles is generally between 50 and 1,000 nm and varies according to plant origin and isolation technique (see Table 2). Plant-derived extracellular vesicles conduct heterogeneous cargos containing different biomolecules (proteins, small RNAs, lipids, and nucleic acids) and are primarily composed of phosphatidic acid, phosphatidylcholine, di galactosyl diacylglycerol, monogalactosyldiacylglycerol, and phytosterols (Liu et al., 2020a; Kocholata et al., 2022). The group of phospholipids occupies several functions including stability, vesicle liberation, and intercellular communication, and participates in the mechanism of membrane fusion. In addition, phosphatidylcholine and phosphatidylethanolamine are known for their important roles in strengthening therapeutic activities (antioxidant, antipolitic, and anti-inflammatory) (Wang et al., 2014). The presence of lipid composition in the vesicle membrane has an important role in intercellular interactions and in maintaining vesicle stability under physiological and pathological conditions (Xu et al., 2023). However, small RNAs are present in plant-derived nanovesicles that are capable of regulating biological functions, notably inter-kingdom communication to ensure communication between species as has been indicated in Arabidopsis EVs (Zhang et al., 2016b) in the presence of tiny RNAs (ty RNA) with a length of 10–17 nucleotides, long non-coding RNAs (lncRNA), circular RNAs (circRNA), and small RNAs (sRNA) (Cai et al., 2018). RNAs can be identified in the exterior of EVs, where they can be secured by RNA-binding proteins against enzymatic destruction. In addition, PENETRATION1 was identified between the Golgi complex and the plasma membrane and established as a plant-derived nanovesicle (PDNVs) biomarker (Wang et al., 2010). Syntaxins, which are localized in the membrane of the SNARE family were also established as integrated proteins that are implicated in the transport of vesicles inside the cells (Rutter and Innes, 2017). It has been demonstrated that PENETRATION1 does not localize with ARA6, so it indicates that the biogenic pathway of TET8-positive EVs is not the same as that of PEN1-positive EVs. Certain plant tetraspanins, notably Arabidopsis thaliana TETRASPANIN 8 and TETRASPANIN 9 (AtTET8 and AtTET9) are identified explicitly in infection by the fungal pathogen Botrytis cinerea and colocalize with the Arabidopsis MVB marker Rab5-type GTPase ARA6 inside the cell and EVs at fungal infection sites (Ding et al., 2014). In addition, the Exocyst-positive organelle (EXPO) was recently found to combine with the plasma membrane and release Exo70E2-positive cells in the intercellular spaces during live-cell imaging in plants using the immunostaining technique. The Exo70E2 secretion pathway is free of MVB pathways, and EXPO is unaffected by inhibitors of secretion and endocytosis in protoplasts.

Plant-derived nanovesicles generally present as a spherical structure when isolated which could promote cell wall passage. Plant-derived nanovesicle biological compounds, which include proteins, small RNAs, and metabolites, are unique in every case and depend on the original cell (Pegtel and Gould, 2019). These two biomarkers PENETRATION1 and TETRASPANIN 8 of EVs, which are not located in the same places are classified in two categories of plant EVs. Tetraspanins occupy a very important role in biogenesis, especially cargo selection, membrane fusion, and exosome absorption (Colombo et al., 2014). Plant-derived nanovesicles’ biogenesis pathway is similar to the exosome biogenesis pathway in that plant nanovesicles associated with TETRASPANIN 8/TETRASPANIN 9 (TET8/TET9) are produced by multivesicular bodies (MVBs). PENETRATION1 is a plant-derived nanovesicle biomarker found in the plasma membrane (Rutter and Innes, 2017). It has been established that trafficking between the Golgi and the plasma membrane is mediated by PEN1. There is no coincidence between PEN1 and ARA6, which determines that the biogenic pathway of TET8 EVs is not the same as that of PEN1 EVs (He et al., 2021). However, another pathway of EV biogenesis has been observed in the EXPO (EXocyst-Positive Organelles), presented generally as a spherical double-membrane structure, compared to the autophagosome, which has been identified in Arabidopsis. Although they present similarities with autophagosomes, which combine with the plasma membrane and liberate membrane vesicles in the cell wall, these vesicles are considered to be exosomes secreted by EXPO (Wang et al., 2010). Indeed, EXPO can combine with Exo70E2 passing the plasma membrane and thus releasing plant-derived nanovesicles (Figure 2). Plant-derived nanovesicles of RNAs are identified in encapsulated EVs, but they are outside the nanovesicles and can be defended by RNA-binding proteins from enzymatic degradation. As has been proved, ty-RNAs of Arabidopsis thaliana are abundant in EVs in proportion to cellular RNAs (Baldrich et al., 2019), and only siRNAs of the same RNA precursors can be discovered in EVs. RNA-binding proteins (RBPs) appear to have an important role in loading RNA into EV precursors and in the stability of exotic RNAs. Membrane polysomes could be a site for RNA loading into vesicles. Plant-derived nanovesicles are derived from the endocytic pathway when membranes invaginate during late endocytosis to form intraluminal vesicles (ILVs) inside multivesicular bodies (MVBs) (Zhang et al., 2017). ILVs are generated when premature endosomes integrate with the peripheral membrane layer. They encapsulate plant-derived nanovesicles in MVBs, which combine with the plasma membrane to release exosomes. Although the biogenesis of plant-derived nanovesicles has not been well studied, there is evidence for at least three distinct pathways (Figure 2) (Cai et al., 2019; Nemati et al., 2022).

The types of EVs are MVs and EXOs, and CA has various modes of biogenesis. MVs are derived from bourgeoning from the plasma membrane, while EXOs originate in the endosomal system in the form of intraluminal vesicles (ILVs), which are released when MVBs fuse with the plasma membrane, and apoptotic bodies are essentially liberated by bourgeoning from a cell undergoing apoptosis to apoptotic bodies (Poon et al., 2014) (Figure 3). However, even if the formation of MV and EXO occurs at different cell locations, intracellular mechanisms may be implicated in the formation of the two entities, depending on the cell type (van der Pol et al., 2014). According to the discovery in this study (van der Pol et al., 2014), the results indicated that T lymphocytes were capable of forming EVs at the level of the plasma membrane consisting of the characteristics of EXOs. Then, the intracellular mechanisms implied in the two formations simultaneously alter the possibility of establishing different sub-populations of EVs (Colombo et al., 2014).

Ultracentrifugation is the most frequently used method for isolating EVs. This method has been used for studying EVs derived from cell culture supernatants and biological fluids (Campoy et al., 2016; Coughlan et al., 2020). Some advantages of ultracentrifugation are that it can isolate EVs from large volumes of biological fluids, requires a relatively restricted set of reactants and consumables, and has no impact on EVs apart from gravitational force and pipetting (as no chemicals that are likely to interfere with downstream EV analysis are used). To reduce debris caused by co-sedimentation and contamination of preparations by cell lysis products, this phase also includes several sub-phases, firstly by centrifugation at 300–400 × g for 10 min, secondly sedimentation of a large proportion of the cells at 2000 × g to remove cell debris, thirdly at 10,000 × g to eliminate biopolymer aggregates and apoptotic bodies, and finally to obtain contiguous EVs in the supernatant, which are increased by ultracentrifugation at (100,000–200,000 × g) over 2 h.

Diverse techniques have been invented to filter EVs. These include cross-flow filtration (CFT) and centrifugal ultrafiltration (UF). In the last stage, high pressure is used to increase the density of smaller particles, permitting them to pass through the membrane without being blocked by clumps. In addition, UF provides a cut-off point for damage to its membranes due to the weight of the particles. This permits the removal of any contaminants found in extracellular vesicles using TFF. Sequential UF, consisting of a succession of filtration steps with a gradual reduction of the cut-off point, can permit EV isolation and enrichment from complex biological fluids (Boireau and Elie-Caille, 2021).

Immunocapture or magnetic sorting is a technique that permits the retention of EVs expressing a surface antigen (e.g., CD9, CD63, or CD81) using monoclonal antibodies (directed against this antigen) coupled to magnetic beads retained on a column by magnets. In the absence of a magnetic field, EVs positive with this antigen are then eluted. The isolation of sub-populations of EVs can also be performed by immuno-capture using antibodies specifically directed against antigens carried by EVs, such as tetraspanins (Blandin and Le Lay, 2020). This technique consists of isolating a particular sub-category of EVs from balls coated with an antibody that recognizes the specific protein marker exposed on EV membranes. It can also prevent contamination of isolated EVs by cytoplasmic proteins or RNA (Van Deun et al., 2014).

This is a method that uses polymer solutions with dextran to induce phase separation or extracts that will induce the precipitation (at low-speed centrifugation) of VEs by its surfactant properties, and polyethylene glycol (PEG) retains the macromolecules and other molecular components. The precipitation of EVs by polymer mixtures (Dextran, polyethylene glycol, or PEG) permitting phase separations at a low centrifugation speed and the precipitation of EVs is the basic principle of many commercial kits (e.g., Exo Quick TCTM). These last kits present the advantage of being rapid and easy to use but often lead to the co-precipitation of macromolecular protein complexes, lipoproteins, or immunoglobulins (Blandin and Le Lay, 2020). PEGs of diverse molecular weights have been used for many years for the precipitation of proteins, nucleic acids, viruses, and other small particles (Yamamoto et al., 1970). The technique uses a diminution of and the solvency of elements in super hydrophilic polymer solutions, PEG. The procedure is limited to the combination of polymer solution and sample, incubating and pelleting the EVs by low-speed centrifugation (1,500 × g).

In conclusion, there are other methods for isolating extracellular vesicles, such as microfluidic systems, immunological separation, and microfluidics. This number continues to grow in parallel with technological advances.

At this moment, various methods have been used to isolate mammalian-derived EVs, primarily ultracentrifugation, differential centrifugation, density gradient, and size exclusion chromatography. However, not all these methods are adapted to the evolution of extracellular vesicle production. These methods require several complicated and repetitive centrifugation steps to remove all debris. In addition, these steps are not yet well-detailed in the literature. This observation has permitted some researchers to develop new methods to purify EVs using simple procedures such as those mentioned in the following studies such as isolation of EVs derived from human milk by ultracentrifugation and filtration. For example, separate milk samples by different centrifugations (6,500 × g at 4°C for 30 min and 12,000 × g at 4°C for 1 h) were used to remove debris. Next, skimming was affected using 0.45 and 0.22 μm filters to remove any remaining debris. The filtered supernatant was centrifuged at 135,000× g for 90 min at 4°C to granulate the exosomes (Reif et al., 2020; You et al., 2021). In another study, EVs derived from milk were isolated by differential centrifugation; for density gradient, the milk was centrifuged twice at 3,000 × g, and then the milk supernatant was subjected to differential centrifugation at 5,000 × g and 10,000 × g in new, sterilized SW40 tubes. The supernatant at 10,000 × g was again added to a sucrose gradient (ranging from 2.0 to 0.4 M sucrose) and centrifuged at 192,000 × g for 15–18 h. At the final stage, the samples were collected, combined, and centrifuged at 100,000 × g for 65 min, the supernatant removed, and the EV pellets aliquoted and stored at 80°C (Van Herwijnen et al., 2018a). Bovine milk was isolated by the acetic acid/ultracentrifugation (AA/UC) method. The first skimmed milk was heated for 10 min at 37°C, then mixed with acetic acid, followed by centrifugation at 10,000 g for 10 min at 4°C. The supernatant was filtered through a 0.22 μm membrane and designated as lacto-serum. Lacto-serum was ultracentrifuged at 210,000 g for 70 min at 4°C. An EV pellet was resuspended with phosphate-buffered saline (PBS) for clean-up, and the remaining precipitates were removed by centrifugation at 10,000 g for 5 min at 4° (András and Toborek, 2015). In Table 3 we have summarized several studies on the Isolation of mammalian-derived extracellular vesicles.

There are several methods for the isolation of plant-derived nanovesicles (PDNVs), for example, differential ultracentrifugation (UC) combined with sucrose density gradient centrifugation, PEG precipitation, size exclusion chromatography (SEC), ultra-filtration membrane separation, etc., Nevertheless, in practical terms (extraction rate, extraction time, and purity) none of these methods are excellent, but the combination of several methods seems to be the best possible solution. The advantages and disadvantages of these techniques are established. Differential ultracentrifugation and the extraction method are the most used techniques for isolating nanovesicles of plant origin, then they are purified by density centrifugation on a sucrose gradient. Different types of nanovesicles can be selected based on centrifugation density and sucrose gradient (Yang et al., 2018a). The main stage of the UC step is to prepare the sample including mixing, pressing, or grinding, then plant-derived nanovesicles (by differential ultracentrifugation and sucrose gradient ultracentrifugation) are isolated and purified. In a centrifuge, the supernatant is initially centrifuged at a low speed to remove fiber debris in the plant tissue; in a second step, the centrifugation speed is gradually increased to remove finer particles while preserving plant-derived EVs in the supernatant. Evidently, as the number of centrifugations increases, the speed of centrifugation gradually increases and the duration of centrifugation becomes longer and longer. After three centrifugations at a relatively low speed, the supernatant is maintained. At this point, a centrifugal force of between 100,000 and 150,000 g is selected for the second centrifugation. (Li et al., 2023a). (See Figure 5). In Table 4 we have summarized several studies on the Isolation of Plant-derived extracellular vesicles.

The biological activities of exosome-type nanovesicles, in their intact natural structural integration with their bioactive cargoes after their simple isolation from plant EVs, diminish pathological situations in species of other reigns and provide multiple therapeutic alternatives (Mu et al., 2014; Dad et al., 2021).

To give an overview of the potential therapeutic application of plant-derived extracellular vesicles (P -EVs) based on plant types, plant comestibles have in recent years been the focus of numerous studies, which have revealed encouraging specificities that explain their availability, biocompatibility, and biodegradability. These EVs could well be necessary for the therapy of many diseases. (Kameli et al., 2021).

More importantly, they consist of several advantageous properties such as Ginger which has various beneficial properties including anti-oxidant (Hung et al., 2017), antibacterial (Dinicola et al., 2014), anti-inflammatory (Rome, 2019), anti-cancer (Vislocky and Fernandez, 2010), and regenerative potential for various diseases (Brahmbhatt et al., 2013). In addition, plant-derived nanovesicles are continents of biomolecules including proteins, drugs, DNA vectors, and siRNAs, and are delivered to target tissues (Nemati et al., 2022). It is for this cause that the biomolecules of plants have gotten much attention from researchers for their potential to improve health and defend against diverse infections.

In recent years, in the field of nanotechnology, nanovesicles derived from edible plants have attracted the attention of scientists for their drug delivery potential, as these particles can deliver hydrophobic and hydrophilic therapeutic agents to disease target sites. We will describe studies that have proven the therapeutic efficacy of nanovesicles derived from edible plants (grape, grapefruit, ginger, lemon, and carrot).

Ginger has intrinsic chemical compositions, such as shoal and gingerol, possessing several health benefits. In addition, multiple studies have indicated the therapeutic effects of ginger, including these studies (Zhang et al., 2016b; Zhu and He, 2023) where the authors have studied the effects of ginger in regeneration. Ginger-derived nanovesicles have demonstrated their role in reducing the expression of secreted hemotoxic protein and influencing the expression of other mitochondrial and cytoplasmic proteins such as heat shock protein, axing, and kinesin for intestinal wound recovery (Kim et al., 2022). Further studies have been interested in the effect of ginger in antitumor activities; the research found that plant-derived miRNAs are capable of crossing species and performing a veritable regulatory role in the human organism (Liu et al., 2017). Furthermore, ginger-derived nanovesicles have been demonstrated to reduce cyclin D1 RNA levels in mice with colorectal cancer (Zhang et al., 2016b). Studies indicate that ginger has hepatoprotective properties against ethanol- and acetaminophen-induced hepatotoxicity of carbon tetrachloride (Zhuang et al., 2015).

Grapefruit-derived nano-vectors (GNVs) presented varieties of therapeutic agents, including chemotherapeutics, DNA expression vectors, siRNAs, and proteins such as antibodies. It has been reported that particles purified from grapefruit indicated high stability and can carry several agents such as curcumin and Zymosan that were functionally active. Grapefruit-derived nanovesicles had similarities in size and structure to mammalian-derived exosomes. They consisted of proteins, lipids, and miRNA and have been assimilated by intestinal macrophages and stem cells. In another study, Wang et al. coated grapefruit-derived nanoparticles (GDNV) rich in active anti-inflammatory leukocyte receptors (IGNV). Using various animal models of inflammation-induced diseases, they have demonstrated that IGNVs target inflamed tumor tissue better than GDNVs. Moreover, this targeting of inflamed tissue was significantly inhibited by blocking LFA-1 or CXCR1/CXCR2 on IGNV membranes. Wang et al. reported that grapefruit-derived nanovesicles could release chemotherapeutic agents, siRNAs, DNA expression vectors, and proteins in different cell types. They also simultaneously administered grapefruit-derived nanovesicles and folic acid, and reported that this strategy significantly increased the efficiency of targeting cells expressing folic acid receptors. They then demonstrated that these nanovesicles improved chemotherapy-induced tumor growth inhibition in CT26 and SW620 cell-derived tumors in mice (Wang et al., 2015).

Nanovesicles derived from Citrus limon (lemon) contain many benefits, including antibacterial, antifungal, anti-inflammatory, anticancer, hepato-regenerative, and cardioprotective activities (Bhavsar et al., 2007; Kim et al., 2012; Riaz et al., 2014; Parhiz et al., 2015; Otang and Afolayan, 2016). The pharmacological potential of citrus lemon is determined by its rich chemical composition such as phenolic acids, coumarins, carboxylic acids, amino acids, and vitamins. In addition, some studies have demonstrated that citrus lemon nanovesicles inhibited the growth of chronic myeloid leukemia (CML) tumors in vivo by specifically reaching tumor sites and activating the TRAIL-mediated apoptosis process. Also, they were studied for their role in reducing tumor growth. They were capable of curbing in vivo tumor development in chronic myeloid leukemia (CML) by targeting tumors, reducing oxidation, and reducing cancer risk (Raimondo et al., 2015). In another study, citrus limon-derived nanovesicles were shown to possess cell growth inhibitory effects, primarily in p53-inactivated CRC cell lines, via the macropinocytosis pathway. The results of this study indicate that p53 inactivation activated macropinocytosis activity and that citrus lemon-derived nanovesicles had a cell growth inhibitory effect via the macropinocytosis pathway (Takakura et al., 2022).

Extracellular vesicles derived from carrot contains phytochemicals, namely, phenolics, carotenoids, polyacetylenes, and ascorbic acid. These chemical products help reduce the risk of cancer and cardiovascular disease because of their antioxidant, anti-inflammatory, plasma lipid-modifying, and anti-tumor properties. The role of polyphenols in the prevention of degenerative diseases, such as cancer, cardiovascular diseases, and neurodegenerative diseases has been reported. Carrot (Daucus carota) is also used in research for its medical effects.

Carrot juice also contains glutathione, an antioxidant that protects against free radicals. It presents potent anti-inflammatory properties that permit the relief of rheumatic and arthritic symptoms (Metzger et al., 2008). A study establishing that carrot-derived nanovesicles have anti-inflammatory and antioxidant effects that can restore glucose tolerance and cardiovascular and liver functions has been assessed in an in vivo model (Poudyal et al., 2010). In another study, EVs derived from carrots (Carex) were studied as a novel biomaterial with antioxidant functions in cardiomyoblastoma and neuroblastoma cells. The results indicated similar properties to EVs, and their antioxidant and apoptotic effects in cardio-myoblasts and neuroblastoma cells were further studied. Carex significantly inhibited ROS production and apoptosis induction; therefore, the antioxidant effect of Carex may be more effective in the early phase of diseases. Carex presented low cytotoxicity in H9C2 cardiomyocytes and SH-SY5Y neuroblastoma cells when high levels of Carex were delivered to cells. In addition, Carex inhibited the reduced expression of antioxidant molecules, including Nrf-2, HO-1, and NQO-1, in both models (Kim and Rhee, 2021).

Grape (Vitis vinifera) nanovesicles and their bioactive compounds have several pharmacological activities such as antioxidation and risk reduction and contain several active components including flavonoids, polyphenols, anthocyanins, proanthocyanidins, procyanidins, and resveratrol, a derivative of stilbene. It has a wide range of pharmacological and therapeutic effects, such as antioxidant, anti-inflammatory, and antimicrobial activities, as well as cardioprotective, hepatoprotective, and neuroprotective effects.

Wang et al. (2013); Zhuang et al., 2016 reported that grape-derived nanovesicles contain microRNA, proteins, and lipids. Although these nanovesicles do not resemble mammalian cell-derived exosomes, their structure and composition are similar to mammalian cell-derived exosomes (Ju et al., 2013). Grape-derived nanovesicles consist of proteins such as aquaporins and HSP70 proteins enriched in phosphatidylethanolamine. A study has indicated that grape-derived nanovesicles present unique transport characteristics and biological functions (Ju et al., 2013; Yu et al., 2020). These nanovesicles can traverse the intestinal mucosal barrier and be taken up by mouse intestinal stem cells, significantly inducing intestinal stem cells through the Wnt/β-catenin pathway (Yu et al., 2020). In addition, nanovesicle grape derivatives can reduce many of the risk factors associated with cancer, cardiovascular health, age-related cognitive diseases, and neurodegenerative diseases. These effects are generally attributed to the function of flavonoids in grapes, antioxidant activity, and increased nitric oxide production (Vislocky and Fernandez, 2010).

In terms of therapeutic applications, the extracellular vesicles released by plant cells possess several therapeutic agents, including drugs, proteins, DNA vectors, and siRNAs, and deliver them to target tissues, making them promising natural resources for modern drug discovery. In addition, they also reduce the risk of various pathologies as mentioned above, including cancer, chronic inflammatory diseases, and others. Because of their drug delivery potential, as these particles can deliver both hydrophobic and hydrophilic therapeutic agents to targeted disease sites, there has been a lot of attention from scientists which shows that extracellular vesicles are positioned as an ideal candidate for therapeutic applications. However, plant-derived nanovesicles can locate intrinsically in target tissues, which is one of the most important characteristics of a targeted delivery system. All aspects of plant-derived nanovesicles have not yet been fully identified and described, as they represent a new concept in the field of nanomedicine. The therapeutic potential of plant-derived nanovesicles has recently been demonstrated in several disease models and has been widely applied in the development of new drugs to treat particular diseases or maintain healthy body functions. A variety of plants have been used for the isolation of therapeutically effective exosomes presenting diverse functionalities. Table 5 lists important plants that have been used to extract plant-derived nanovesicles and their therapeutic applications.

In conclusion, to advance therapeutically, drug nanocarriers require a thorough evaluation of their physicochemical characteristics and communications in various biological environments (Herrmann et al., 2021). While liposomes have been largely evaluated, EVs have indicated unique properties that make them superior for drug delivery systems (Kooijmans et al., 2016). Plant-derived nanovesicles can be used as drug carriers as they present advantageous properties including low immunity, tissue-specific targeting, safety, large-scale production, preferred negative zeta potential values, and the ability to load many biomolecules. In Table 5, we present a summary of another plant-derived nanovesicle for therapeutic use.

We will discuss different studies that attribute various therapeutic functions to mammalian-derived extracellular vesicles, e.g., extracellular vesicles of an endosomal origin that are released by different cell types (neurons) serve to remove unwanted proteins in a drainage system and also serve to intercellularly transport their cargo: a specific set of proteins, RNAs, and lipids. Recently, extracellular vesicles derived from mouse neuroblastomas captured Aβ, resulting in reduced amounts of Aβ, decreased amyloid deposition, and reduced Aβ-induced synaptotoxicity in the hippocampus. This demonstrates the role of neuroblastoma cell extracellular vesicles in Aβ release (Yuyama et al., 2014). Results in another study were also similarly described on neuron-derived EVs (Yuyama et al., 2015). On the other hand, endothelial extracellular vesicles secreted by activated or apoptotic endothelial cells may be capable of participating in both toxic and positive effects of the vascular endothelial response. These effects may include anticoagulation, anti-inflammatory effects, angiogenesis, endothelial survivance, and endothelial regeneration (Dignat-George and Boulanger, 2011). In another study of miRNAs presented in human and porcine milk, EVs focused on a variety of genes that were involved in the regulation of the epithelial barrier and neonatal defense; these miRNAs associated with breast milk EV contributed to the subsequent guided development of the newborn (Van Herwijnen et al., 2018b). However, another study indicated that extracellular vesicles in pig milk also attenuated deoxynivalenol-induced changes in body weight and intestinal epithelial growth in mice. These extracellular vesicles inhibited cell proliferation and the creation of tight junction proteins and reduced deoxynivalenol-induced apoptosis. EVs also increased the expression of miR-181a, miR-30c, miR-365-5p, and miR-769-3p in IPEC-J2 cells, then reduced the expression of their target genes in the p53 pathway, and finally attenuated DON expression by promoting cell proliferation and tight junctions and inhibiting apoptosis to induce lesion (Xie et al., 2020). In addition, a study of cow’s milk-derived extracellular vesicles that reduced primary tumor growth and attenuated the progression of weight loss problems in cancer-related body weight loss was examined using the C-26 colorectal tumor model. Their discoveries highlighted the role of milk-derived extracellular vesicles in interspecies communication and their considerable context-dependent role in regulating cancer progression and metastasis. However, their results also suggested that milk-derived extracellular vesicles possess antiproliferative properties on cancer cells in vitro (Zhuang et al., 2016), as demonstrated in similar studies (Munagala et al., 2016). It has been indicated that peripheral endothelial cell-derived EVs may be involved in the modulation of innate immunity (Palette et al., 2013). In addition, human dermal microvascular endothelial cells have selectively sequestered cytoplasmic RNA degradation mechanisms in exosomes, which could also be involved in gene regulation. It has been established that dendritic cell-derived EVs can be focused to deliver siRNAs to neurons, microglia, and oligodendrocytes in the mouse brain (András and Toborek, 2015; Somiya et al., 2018). In another study, EVs derived from umbilical cord polyvalent mesenchymal stromal cells presented anti-apoptotic, pro-angiogenic, and antifibrotic activities, and immunomodulatory effects; effects similar to those of their source cells (Rohde et al., 2019).

In terms of therapeutic application, after confronting a series of challenges when developing a therapeutic approach including toxicity, safety, target specificity and large-scale production, leading to several targeting strategies for drug delivery in preclinical and clinical settings. Mammalian-derived nanovesicles are promising for clinical applications, both as biomarkers and as therapeutic vectors. As proven by scientists, EVs can be used as therapeutic agents or as drug cargo for reaching a specific target thanks to their membrane protein (Yang et al., 2018b). While the drug is encapsulated in extracellular vesicles, the advantage is that EVs act as a drug carrier, protecting the drug and transmitting it safely to the target site. Numerous studies have indicated that vesicles derived from mesenchymal stem cells (MSCs) appear especially useful for enhancing recovery after various injuries. As has been indicated in mice, injection of MSC-derived EVs suppressed hypoxia-induced inflammation and hypertension (Lee et al., 2012). MSC EVs can exert a neuroprotective effect after a brain lesion (Xin et al., 2012). Similarly, MSC EVs release miR-16 and other molecules to mouse breast cancer cells, exerting a decrease in vascular endothelial growth factor expression and a reduction in tumor growth (Lee et al., 2013). More recently, a study has demonstrated that VEs derived from macrophages or liver sinusoidal cells treated with interferon-α deliver antiviral RNA and proteins to hepatocytes, thereby reducing hepatitis B virus replication (Li et al., 2013). How different natural EVs promote these diverse responses remain to be elucidated. Drug loading into EVs can be achieved by these two strategies, either by directly loading the drug into exosomes or/and by loading the drug to target the mother cell during exosome biogenesis (Mittelbrunn and Sanchez-Madrid, 2010). Moreover, in the case of lipophilic drug switching, the mechanism is relatively simple due to the interaction between the lipid bilayer of EVs and the drug, which occurs via a hydrophobic interaction (Shtam et al., 2013). Such methods as electroporation, incubation, sonication, and thawing have been performed for the exogenous loading of EVs with drugs (Jo et al., 2014).

The use of EVs as drug carriers presents several advantages but must conform to good manufacturing practice (GMP) (Chen et al., 2020). There are several important aspects of nanovesicle GMP. The low large-scale productivity of nanovesicles is one of the major problems encountered in the implementation of nanovesicle-based therapeutics; the main issues to be known about the various isolation methods are the physicochemical and purity properties of exosomes for the high-quality, uniquely shaped nanovesicle community and the standardization of storage requirements. The evolution of the therapeutic potential of nanovesicles by enriching them with therapeutic biomolecules is a simple way of improving their therapeutic potential (Abou-El-Enein et al., 2013). To use nanovesicles as reliable therapeutic agents, scalable manufacturing processes are needed to produce exosomes rapidly, cost-effectively, and reproducibly. In Table 6, we present a summary of another mammalian nanovesicle for therapeutic use.