Advances in plant-derived extracellular vesicles: isolation, composition, and biological functions

Abstract

Plant-derived extracellular vesicles (PDEVs) are nanoscale vesicles released from plant cells into the extracellular space. While similar in structure and function to mammalian-derived EVs, PDEVs are unique due to their origin and the specific metabolites they carry. PDEVs have gained significant attention in recent years, with numerous reports isolating different PDEVs from various plants, each exhibiting diverse biological functions. However, the field is still in its early stages, and many issues need further exploration. To better develop and utilize PDEVs, it is essential to have a comprehensive understanding of their characteristics. This review provides an overview of recent advances in PDEV research. It focuses on the methods and techniques for isolating and purifying PDEVs, comparing their respective advantages, limitations, and application scenarios. Furthermore, we discuss the latest discoveries regarding the composition of PDEVs, including lipids, proteins, nucleic acids, and various plant metabolites. Additionally, we detail advanced studies on the multiple biological functions of PDEVs. Our goal is to advance our understanding of PDEVs and encourage further exploration in PDEV-based science and technology, offering insights into their potential applications for human health.

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1. Introduction

Effective cell communication is essential for the function of multicellular organisms, and extracellular vesicles (EVs) play a pivotal role in this process by transporting signaling molecules and facilitating intercellular communication.¹ EVs, which are lipid-bilayer-enclosed particles released by cells, lack the ability to replicate independently due to the absence of a functional nucleus.² EVs can be categorized into subtypes such as exosomes, microvesicles (MVs), and apoptotic bodies.³ However, most separation techniques do not differentiate EV subtypes based on their biogenesis, and distinguishing these subtypes remains a challenge due to the lack of universal molecular markers. Therefore, the International Society of Extracellular Vesicles (ISEV) advise against using biogenesis-based terminology unless EV subtypes are explicitly isolated and characterized.³ Although studies continue to uncover the mechanisms governing EV formation, especially via multivesicular bodies (MVB) or retrovirus budding, the ability to reliably modulate and control EV secretion is still in its early stages.

The first documented observation of mammalian-derived EVs (MDEVs) occurred in 1983 during studies on sheep reticulocytes.⁴ By 1987, Johnstone coined the term “exosome” to describe these EVs, following their identification in maturing reticulocytes.⁵ Since then, MDEVs have been extensively studied across various mammalian cell types, including cancer cells,^6,7 immune cells,⁸ and stem cells.⁹ MDEVs have been detected in multiple biological fluids such as urine, blood, and saliva, where they carry a cargo of proteins, nucleic acids, and lipids,^10,11 which are believed to facilitate intercellular communication.¹² As such, MDEVs have emerged as key regulators in physiological and pathological processes due to their pharmacological properties and biocompatibility. Despite the promise of MDEVs in therapeutic applications, challenges remain in their clinical translation, though some ongoing clinical trials are evaluating their efficacy.^13,14 Additionally, the use of MDEVs carries the risk of immune responses, which can result in adverse effects.¹⁵

In contrast, plant-derived extracellular vesicles (PDEVs) offer notable advantages, including their non-toxic, non-immunogenic nature, low-cost production from sustainable plant sources, and absence of human pathogenic contaminants.^16,17 These features make PDEVs highly promising candidates for use in drug delivery systems, medical therapeutics, or as health supplements. The literature uses various terms to describe EVs derived from plants, including plant-derived nanovesicles, plant exosome-like nanovesicles, and edible plant-derived EVs. For consistency, this review will refer to them collectively as PDEVs.

PDEVs have garnered significant attention for their unique ability to mediate interkingdom communication and their potential health benefits for humans.^18,19 However, despite the growing interest in PDEVs, research in this area is still in its infancy, and several obstacles hinder progress. For instance, standardized protocols for the isolation and characterization of PDEVs are lacking, and more research is needed to assess their stability and preservation. Furthermore, while models have been proposed to explain PDEV secretion,²⁰ a deeper understanding of their biogenesis mechanism is pivotal for understanding their precise biological functions.

This review provides a comprehensive analysis of recent findings concerning PDEVs, covering topics such as isolation and purification methods, physical properties, composition, stability, and preservation. We will also explore the potential biological functions of PDEVs and their application as drug carriers, suggesting directions for further research in biotherapeutics. Our goal is to inspire innovation in the field of PDEV-based science and technology, with an emphasis on the potential contributions of PDEVs to health and biomedical advancement.

2. Biogenesis of PDEVs

As shown in Fig. 1, the biogenesis of PDEVs is primarily governed by three major biological pathways: multivesicular bodies (MVB), exocyst-positive organelles (EXPO), and vacuoles. These pathways contribute to the heterogeneity of PDEV subtypes, with MVB and EXPO being considered the principal sources due to their ability to fuse with the plasma membrane and release vesicles into the extracellular space.²¹

Fig. 1 Biogenesis and secretion of PDEVs. (1) MVB pathway, PDEVs associated with tetraspanin 8/tetraspanin 9 (TET8/TET9), and penetration 1 (PEN1) are secreted from MVB. (2) EXPO pathway; (3) vacuoles pathway. ① Points to the TET8/TET9-dependent pathway; ② points to the PEN1-dependent pathway. ER: endoplasmic reticulum; EE: early endosome; MVB: multivesicular body; TET8, tetraspanin-like protein 8; TET9, tetraspanin-like protein 9; PEN1, peneration1. Note: the complete mechanisms of PDEV biogenesis are still not fully understood.

The first pathway, involving MVB, is well-characterized in plant cells. MVB are late endosomal compartments containing intraluminal vesicles, which typically range from 30 to 100 nm in diameter. The formation of these EVs is regulated by the endosomal sorting complex required for transport (ESCRT) machinery, which orchestrates the inward budding of the MVB membrane to generate intraluminal vesicles.²² Upon fusion of the MVB with plasma membrane, intraluminal vesicles are secreted into the extracellular space as PDEVs. In Arabidopsis, tetraspanin (Tets)-like genes TET8 and TET9 have been identified in EVs released from MVB, supporting their role in extracellular vesicle biogenesis.²³ These TET-containing vesicles are thought to mediate intercellular communication by delivering bioactive molecules. Additionally, PEN1-related EVs, which contain 10–17-nucleotide-long non-coding RNAs, have been found in Arabidopsis. The secretion of these vesicles can be inhibited by Brefeldin A, a known inhibitor of the conventional endoplasmic reticulum (ER)–Golgi vesicular trafficking pathway.²⁴

The second major biogenetic route for PDEVs is the EXPO pathway. EXPO are characterized by their unique double-membrane structure, which merges with the plasma membrane to release their internal vesicles into the extracellular environment.²⁵ Marker proteins such as Exo70H1 and Exo70B2 have been identified as essential components of EXPO-mediated vesicle trafficking. These proteins are implicated in plant innate immune responses, inducing pathogen-triggered immunity (PTI) and enhancing resistance to various pathogens in Arabidopsis thaliana.²⁶ This suggests that EXPO-derived vesicles may play a role in plant defense signaling, in addition to their potential involvement in other biological processes such as intercellular communication.

In addition to the MVB and EXPO pathways, vacuoles have also been proposed to contribute to the formation of PDEVs. Vacuoles in plant cells serve as multifunctional organelles involved in storage, degradation, and homeostasis. Small vacuoles (SVs) are believed to originate from the fusion of MVB, while central vacuoles are thought to form through the maturation of SVs and subsequent fusion events. Recent studies suggest a potential interplay between vacuole formation and MVB, wherein MVB transition into SVs, which later fuse to form the central vacuole.²⁷ This indicates that vacuolar vesicle trafficking pathways may be involved in PDEV biogenesis, though further research is required to fully elucidate the mechanisms underlying this process.

3. Tissue processing for PDEV extraction

The extraction of PDEVs requires approaches similar to those employed for MDEVs. The process involves two main stages: the preparation of plant tissues, and the isolation and purification of PDEVs. This section focuses on the initial step of tissue processing, while the subsequent section will explore the techniques used for PDEVs isolation and purification.

Tissue processing for PDEVs can be broadly categorized into two main approaches, as shown in Fig. 2. The first is the tissue-disruption method, which involves mechanical disruption to extract plant juice containing both vesicles and cell debris. This method often utilizes a wall breaker or blender, especially for plants with tougher texture.^28,29 The second approach is the tissue-infiltration method, which collects apoplastic wash fluid through negative pressure infiltration, aiming to maintain cellular integrity.²⁴

Fig. 2 Pre-processing methods for the isolation of PDEVs. (1) Tissue-disruption method: this method involves mechanical dissociation of plant tissue using a blender to extract plant juice, followed by centrifugation to remove cell debris and enrich for vesicles; (2) tissue-infiltration method: in this approach, a vesicle-isolation buffer (VIB) is infiltrated into the apoplastic space of plant cells using vacuum or pressure techniques. Apoplast washing fluid (AWF), containing vesicles from the apoplast, is then collected through subsequent centrifugation.

3.1. Tissue-disruption method

The tissue-disruption method relies on physical techniques to break down plant tissues, facilitating the extraction of vesicle-rich juice from components such as fruits, leaves, and stems. Common techniques include grinding, squeezing, and blending.^30–32 While these methods are effective at breaking down plant structures and can enhance the yield of PDEVs, they may also introduce cellular debris, including organelle membranes and plasma membrane fragments. For instance, a recent study found that PDEVs extracted from leaves contained proteins typically associated with intracellular organelles, suggesting contamination from disrupted cellular structures.³³

To improve the purity of PDEVs, modifications to tissue-disruption techniques have been implemented. For example, the addition of Tris-HCl to citrus-derived juice helps eliminate co-purifying pectin.³⁴ In the case of Catharanthus roseus, leaves are subjected to sequential cleaning, digestion with pectinase and cellulase, and centrifugation to remove protoplasts.³⁵ Despite the risk of contamination from intracellular material, the tissue-disruption method remains favored when higher quantities of PDEVs are required for therapeutic applications. Compared with the tissue-infiltration approach, this method yields a significantly higher concentration of PDEVs, although with a trade-off in purity.

3.2. Tissue-infiltration method

The tissue-infiltration method, primarily used for collecting apoplastic wash fluid, offers a gentler alternative to tissue disruption. The plant apoplast, located outside the plasma membrane but within the cell wall, is a key site for many biological processes, including nutrient transport and pathogen defense.³⁶ This method involves vacuum infiltration, wherein a buffer is introduced into plant tissues, such as leaves or roots, under vacuum pressure. After infiltration, excess surface liquid is carefully removed, and the tissues are subjected to low-speed centrifugation to collect apoplast washing fluid.^24,37

Compared with the tissue-disruption method, tissue-infiltration causes significantly less cell damage, resulting in PDEVs with higher purity.³³ However, this method has several limitations. First, the concentration of PDEVs obtained is typically low due to the dilution effect of the apoplast washing fluids. Second, vacuum application can still result in minor cell disruption, potentially leading to contamination from intracellular vesicles. Third, the introduction of external washing fluid may cause plant to secrete additional metabolites, altering the original metabolite profile of the apoplastic fluid.³⁶

4. Isolation and purification of PDEVs

The morphology of PDEVs closely resembles that of MDEVs, suggesting similarities in their characteristics. As a result, established methodologies for isolating MDEVs are also applicable to PDEVs, with modifications tailored to plant material. These isolation techniques exploit the unique physical and biochemical properties of PDEVs, as outlined in Fig. 3.

Fig. 3 Different separation methods for PDEV purification. (A) Differential ultracentrifugation (DUC); (B) gradient ultracentrifugation (GUC); (C) ultrafiltration (UF); (D) size-exclusion chromatography (SEC); (E) tangential-flow filtration (TFF); (F) asymmetric flow field-flow fractionation (AF4); (G) electrophoresis with dialysis (ELD); (H) polymer-based precipitation (PBP); (I) aqueous two-phase system (ATPS); (J) immunoaffinity (IA).

The methods are classified as follows: (I) density-based methods: these include differential ultracentrifugation (DUC) and gradient ultracentrifugation (GUC).³⁵ (II) Size-based methods: techniques such as ultrafiltration (UF),³⁸ size-exclusion chromatography (SEC),³⁹ electrophoresis coupled with dialysis (ELD),⁴⁰ tangential flow filtration (TFF),⁴¹ and asymmetric flow field-flow fractionation (AF4)⁴² are used. (III) Solubility- and partitioning-based methods: This category includes polymer-based precipitation (PBP)²⁹ and the aqueous two-phase system (ATPS).⁴³ (IV) Immunoaffinity (IA) capture-based methods.⁴⁴ Additionally, emerging technologies, such as microfluidic platforms, nano-flow cytometry, and nanoscale lateral displacement array technologies, are gaining traction for PDEV isolation. One innovative example is a capillary-channeled polymer (C-CP) fiber spin-down tip technique, which efficiently isolates PDEVs from various fruit and vegetable sources.⁴⁵

These isolation and purification methods are reviewed in detail, with a focus on their respective advantages and limitations. Table 1 provides a comprehensive comparison of these methods, aiding in the selection of appropriate protocols for specific research needs.

Table 1 The main advantages and limitations of purification methods for PDEVs

Methods	Principles	Advantages	Limitations	Ref.
Differential ultracentrifugation (DUC)	Differences in sedimentation rates	Simplicity, cost-effectiveness, low risk of contamination with additional isolation reagents	Risk of damaging, co-sediments contaminants, pellets tend to aggregate	49–52
Gradient ultracentrifugation (GUC)	Differences in the buoyant densities	High recovery and purity	Loss of sample, time-consuming, large centrifuge equipment, difficulty in scaling up production	19 and 53
Ultrafiltration (UF)	Difference in size	Allows for concurrent processing of different samples, easy to operate, preserves vesicle structure, high recovery	Membrane blockage, particle loss caused by adhesion to the membranes, longer operation times and inefficiencies	55 and 162
Size-exclusion chromatography (SEC)	Differences in size and molecular weight	Preserves vesicle structure, minimises co-isolation of contaminants	Requires specialized equipment, lengthy process, difficult to scale up	64 and 163
Tangential flow filtration (TFF)	Using a cross-flow filtration principle	Process large samples, more effective, scalable, and gentle, higher yield, reduced presence of single macromolecules and aggregates	The risk of clogging, high costs	41 and 59
Asymmetric flow field-flow fraction (AF4)	Hydrodynamic size-based	Highly reproducible, fast, label-free and gentle, efficient recovery, high resolution	The tedious sample-preparation procedure, the increased requirement for the EVs’ concentration, time-consuming	62 and 63
Electrophoresis coupled dialysis (ELD)	Differences in size across an applied electric field and membrane	Fast, time-saving, does not require specialized equipment	Low yields, manual process, may cause aggregation of vesicles	40 and 64
Polymer-based precipitation (PBP)	Altering solubility with polymers to increase sedimentation rate	User-friendly operation, low cost, process large samples	Contamination and retention of the polymer, difficulty in purification	67 and 163
Aqueous two-phase system (ATPS)	Differences in partition coefficient between two immiscible solvents	High efficiency and purity, less time, no additional equipment required	Contamination and retention of dextran	43, 68 and 69
Immunoaffinity (IA)	Antigen–antibody immune reaction occurs between exogenous antibodies and PDEV surface antigens, forming an immune complex	Able to isolate PDEVs of specific origin, high purity	Pending to establish better PDEV tags, different surface proteins cannot be identified, low recovery rates, small sample volume, high cost, possible damage to structure and activity	70–72

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4.1. Differential ultracentrifugation (DUC) and gradient ultracentrifugation (GUC)

DUC and GUC are widely utilized density-based techniques for PDEV isolation due to their simplicity and cost-effectiveness, earning them the designation of gold-standard methods.^46–48 DUC separates particles by size and density in a stepwise manner, progressively removing larger particles like cells, debris, apoptotic bodies, and microvesicles^49–51 (Fig. 3A). Despite its widespread use, a drawback of DUC is the potential co-sedimentation of contaminants such as protein aggregates during high-speed centrifugation.⁵² GUC, on the other hand, utilizes a gradient medium to separate particles based on density, forming distinct layers where vesicles settle at equilibrium position (Fig. 3B).⁵³ This method typically achieves higher purity but may result in lower PDEV yield compared with DUC.¹⁹

4.2. Ultrafiltration (UF)

UF is a widely employed technique for isolating PDEVs by using membrane filters with defined pore size, facilitating particle separation based on size (Fig. 3C). This process involves trapping larger particles, such as PDEVs, on the membrane surface, while allowing smaller impurities, such as proteins, to pass through. The result is a higher purity of PDEVs, contingent upon the pore size and molecular weight cut-off (MWCO) of the filtration membrane.⁵⁴

For instance, Lee et al. successfully employed UF to isolate PDEVs from the leaves and stems of Dendropanax morbifera, utilizing a 100 kDa MWCO centrifugal filter unit.³¹ Their study highlights UF's efficacy in maintaining the PDEVs’ structural integrity while selectively removing smaller contaminants. However, when comparing UF with other PDEV separation techniques, such as ultracentrifugation (UC) and polyethylene glycol (PEG)-based precipitation, UF demonstrated a higher level of protein contamination, despite its ability to preserve the PDEVs’ morphology more effectively.⁵⁵

The filtration mechanism of UF is typically characterized by dead-end filtration, where the sample media is applied perpendicularly to the membrane surface, forcing the entire sample volume through the filter at once. While this method is effective for batch processing, it is often associated with declining membrane permeability over time due to the accumulation of retained particles on the membrane surface, a phenomenon known as membrane fouling. This can result in extended filtration times, reduced throughput, and overall inefficiencies in the system.⁵⁶ To address these limitations, future improvements in UF systems could focus on optimizing membrane materials and configurations, potentially incorporating cross-flow filtration techniques to enhance efficiency and reduce fouling.

4.3. Size-exclusion chromatography (SEC)

SEC is a powerful and widely utilized technique for separating extracellular vesicles, including PDEVs, based on their size and hydrodynamic dimensions (Fig. 3D). The core principle of SEC involves a porous gel matrix, where larger particles are excluded from entering the pores and travel more rapidly through the column. In contrast, smaller molecules, such as free proteins and nucleic acids, penetrate the gel pores and are retained longer, resulting in slower flow rates and delayed elution.⁵⁷ SEC's unique mechanism makes it highly effective for isolating PDEVs while simultaneously removing smaller soluble contaminants. The porous stationary phase allows EVs to elute in the early fractions, while smaller impurities such as free proteins, nucleic acids, and other molecular contaminants remain temporarily trapped within the gel matrix.⁵⁸ This selective elution results in a purified PDEV fraction with minimal protein contamination, a critical factor for downstream functional studies.

In a study focusing on PDEVs from cabbage, SEC was employed in conjunction with UF to enhance isolation efficiency. The combination yielded a relatively homogeneous population of PDEVs, as confirmed by nanoparticle tracking analysis (NTA), demonstrating SEC's efficacy in providing a uniform size distribution of isolated vesicles.³⁰ Additionally, SEC has been successfully adapted for isolating PDEVs from various plant sources. For example, Elisa Garzo et al. described two distinct SEC-based methods for collecting PDEVs from the phloem sap of rice and melon, further showcasing the versatility of SEC in purifying vesicles from diverse plant tissues.³⁹

4.4. Tangential flow filtration (TFF)

TFF is a system that concentrates and filters out particles using a cross-flow filtration principle (Fig. 3E). Unlike traditional dead-end filtration methods, TFF directs the sample flow parallel to the membrane surface, reducing the risk of molecule buildup and membrane fouling. This continuous flow prevents clogging, ensuring that the entire volume is processed uniformly, making it particularly advantageous for large-scale applications.⁴¹ Compared with UC, TFF proves to be a more effective, scalable, and gentle technique. A comparative evaluation of TFF and UC on conditioned cell culture media demonstrated that TFF concentrates EVs with similar physicochemical properties as UC but with a higher yield, reduced presence of single macromolecules and aggregates (<15 nm in size), and enhanced batch-to-batch uniformity in half the processing time.⁵⁹ TFF has also been successfully employed for isolating PDEVs from various plant sources. For instance, PDEVs were efficiently obtained from Aster yomena callus and Aloe vera peels, demonstrating the versatility of this technique in processing diverse plant tissues.^60,61 These examples confirm TFF's scalability and its potential to facilitate the isolation of PDEVs for both research and therapeutic applications.

4.5. Asymmetric flow field-flow fractionation (AF4)

AF4 utilizes size-based separation similar to SEC, but offers greater precision and versatility. In this technique, the sample flows through a narrow channel while an external force field (e.g., gravitational or electric) is applied perpendicular to the flow direction (Fig. 3F). Larger particles experience stronger drag forces, causing them to migrate closer to the channel walls, while smaller particles remain suspended in the center due to Brownian motion. This differential movement based on size creates distinct layers, enabling efficient particle separation.⁶² The technique offers several advantages: AF4 is label-free, non-invasive, highly reproducible, and capable of separating particles within a broad size range, from a few nanometers to micrometers, making it ideal for isolating EV subpopulations.⁶³ Additionally, AF4 operates without physical obstructions or packing materials, minimizing potential contamination and ensuring gentle handing of PDEVs.

In a recent study, AF4 was employed to differentiate two distinct exosome subpopulations, large exosome vesicles (Exo-L, 90–120 nm) and small exosome vesicles (Exo-S, 60–80 nm), for human plasma.⁴² AF4 also identified a third, smaller particle population known as ‘exomes’ (∼35 nm), previously undetectable using conventional techniques.⁴²

4.6. Electrophoresis with dialysis (ELD)

ELD is a novel technique combining electrophoresis and dialysis to isolate PDEVs, introduced by Yang et al.⁶⁴ In this method, electrophoresis is used to drive particles through a semi-permeable membrane, effectively separating PDEVs from other macromolecules based on their charge and size (Fig. 3G). After separation, dialysis further purifies the vesicles by removing unwanted small molecules and contaminants. This method ensures that PDEVs remain intact, offering a size and yield comparable to UC-based approaches. In subsequent research, ELD was successfully employed to isolate PDEVs from bitter melon, showcasing its versatility in different plant sources.⁴⁰ ELD's potential lies in its ability to yield high-purity PDEVs with minimal loss, making it an appealing alternative to more conventional methods. However, the scalability of ELD remains a challenge, particularly for high-throughput applications.

4.7. Polymer-based precipitation (PBP)

PBP is an established method for isolating EVs using volume-excluding polymers like polyethylene glycol (PEG) to precipitate particles based on their solubility (Fig. 3H). This method creates a polymeric network that traps PDEVs, which are then collected through low-speed centrifugation.⁶⁵ Commercial kits often use this principle to isolate EVs. PBP is a rapid, easy-to-implement approach suitable for isolating PDEVs in laboratory settings. For instance, Kalarikkal et al. demonstrated the efficacy of PEG-based precipitation for isolating PDEVs from ginger.²⁹ The study reported that this method yielded a comparable amount of PDEVs to DUC, with minimal differences in size, surface charge, and biochemical composition between the two techniques. Additionally, further refinement of the PEG-based precipitation process, such as adjusting the pH, significantly improved yield without compromising PDEVs’ integrity.⁶⁶ However, one notable limitation of PBP is the potential for co-precipitating non-EV components, such as proteins, which may interfere with downstream analyses.⁶⁷ Therefore, while PBP is convenient, additional purification steps may be necessary depending on the application.

4.8. Aqueous two-phase system (ATPS)

ATPS involves the use of two polymers or polymer–salt mixtures to form a biphasic system that selectively partitions particles into distinct phases based on their affinity to each phase (Fig. 3I). This technique is especially useful for separating biomolecules with differing solubility properties. Recently, Savcı et al. employed ATPS to successfully isolate PDEVs from grapefruit, establishing its efficacy for PDEVs.⁶⁸ The process offers a low-cost, scalable alternative for separating PDEVs, especially in complex plant samples. ATPS allows for the efficient removal of contaminants such as proteins and nucleic acids while maintaining the structural integrity of PDEVs.⁶⁹ This method is also relatively gentle, minimizing the risk of damaging the vesicles. A modified version of ATPS incorporating an oil phase between aqueous phases was developed by Seo et al., which further enhanced the isolation of high-purity PDEVs.⁴³ Despite its advantages, ATPS can be more difficult to optimize than other methods due to the need to carefully select polymer systems and phase compositions for each specific sample type.

4.9. Immunoaffinity (IA)

IA capture-based methods isolate specific EV subpopulations by exploiting surface markers characteristic of these vesicles (Fig. 3J). By using antibodies that target proteins such as CD9, CD63, CD81, or plant-specific markers like TET8, IA provides a highly specific approach for PDEV isolation.⁷⁰ This technique offers unparalleled precision, allowing for the targeted isolation of EVs carrying specific markers, which is invaluable for studying subpopulations and analyzing their cargo.⁷¹ A notable application of this method was demonstrated by He et al., who used beads coated with anti-TET8 antibodies to isolate TET8-positive PDEVs from plant fractions.⁴⁴ While IA is highly specific, it often results in lower yields compared with physical separation methods, such as UC, due to the selective nature of antibody binding.⁷² Therefore, IA is generally employed in conjunction with other techniques when the objective is to isolate EV subtypes for detailed molecular analyses.

4.10. Combination strategies for enhanced PDEV isolation

The strategic combination of isolation methods is crucial for enhancing both the purity and yield of PDEVs. Frequently employed combinations include UC paired with GUC, as well as UF combined with SEC. These hybrid approaches maximize isolation efficiency by leveraging the complementary strengths of each method. For instance, UF combined with SEC minimizes protein contaminants in the PDEV fraction, producing vesicles with improved purity.³⁰ This combination is particularly effective when high throughput and reduced contamination are required, ensuring a more uniform population of vesicles.

Recent research in the field has also emphasized the combination of GUC with IA techniques to further isolate specific subclasses of EVs from complex vesicular mixtures.⁷¹ IA, through the use of antibodies targeting surface markers specific to certain EV subtypes, provides high specificity in isolating subpopulations of PDEVs. This is especially beneficial when isolating specific functional vesicles for targeted therapeutic or diagnostic applications. However, the increased specificity of IA can come at the expense of low yield, which must be taken into account for larger-scale applications.

While the combination of these methods enhances the quality of isolated PDEVs, it's important to acknowledge the associated trade-offs, including increased operational complexity and higher costs. As PDEV isolation techniques become more advanced, it is vital to balance efficiency, cost, and scalability. The development of cost-effective and reproducible combination strategies, alongside rigorous physicochemical characterization methods, will be key to differentiating various PDEV types and ensuring their suitability for specific downstream applications.

Additionally, the diverse range of plant sources from which PDEVs are derived presents unique challenges compared with isolating MDEVs. The structural and compositional variability among plant species affects the optimization of isolation techniques. For example, differences in plant fluid viscosity can significantly influence the effectiveness of UC, complicating the process.^73,74 This variation underscores the need for tailored isolation protocols that accommodate the distinct properties of different plant sources, ensuring consistent and high-quality outcomes. Future research should focus on customizing these protocols to address the unique challenges presented by each plant species. Table 2 summarizes the current technologies employed in preparing and detecting PDEVs across various tissue types, highlighting their comparative advantages and limitations.

Table 2 Technologies used to prepare and detect the physical characteristics of PDEVs from different tissues

Tissue	Sources	Pre-processing	Purification	Characterization	Ref.
NTA, nanoparticle tracking analysis; TEM, transmission electron microscopy; Nano FCM, nano flow cytometer; CM, confocal microscopy; DLS, dynamic light scattering; AFM, atomic force microscope; BCA, bicinchoninic acid.
Phloem	Oryza sativa	(1) Aphid stylectomy phloem sap-collection. (2) Centrifuged at 10 000g for 15 min at 4 °C	SEC	(1) NTA: 100–200 nm in size; Particle concentration: 3 × 10^8 particles per ml	39
	Cucumis melo	(1) Stem incision phloem sap-collection. (2) Centrifuged at 10 000 g for 15 min at 4 °C	SEC	(1) NTA: 70–150 nm in size; Particle concentration:4.7 × 10^8 particles per ml
Leaf	Catharanthus roseus	(1) The leaves were digested with cellulase (3 g per 100 ml) and pectinase (0.2 g per 100 ml) for 12 h. (2) Centrifuged at 3000 rpm for 20 min then 1000 rpm, 40 min at 4 °C. (3) Concentrated to about 100 ml by hollow fiber module	DUC and GUC	(1) TEM: rounded hollow vesicle shape. (2) Nano FCM: average diameter: 75.51 ± 10.19 nm. (3) Zetasizer Nano ZS: zeta potential: −21.8 mV	35
	Arabidopsis thaliana	(1) The leaves were placed in a 200 ml syringe and gently vacuumed with an infiltration buffer for 20 s. (2) Fixed on to a small plastic stick, then placed into a 50 ml conical tube. (3) Centrifuged for 10 min at 4 °C at 900g to collect the apoplastic washing fluid (AWF)	UC, GUC and IA	(1) TEM: rounded hollow vesicle shape; 30–150 nm in size. (2) CM: the TET8-positive EVs were pulled-down by TET8-specific antibody-linked beads	71
	Arabidopsis thaliana	(1) The leaves were placed inside a needleless syringe and infiltrated with vesicle infiltration buffer. (2) Fixed on to a small needleless syringe, then placed into a 50 ml conical tube. (3) Centrifuged for 30 min at 4 °C at 900g to collect the AWF	PBP	(1) TEM: typical cup-shaped. (2) DLS: average diameter: 266 nm; Zeta potential: −48 mV(12% PEG8000 fraction). (3) NTA: particle concentration: 6 × 10^6, 7 × 106, 1 × 10^7 particles per ml (5%, 8%, 12% PEG 8000fraction); yield: 4.5 × 10^9, 5.25 × 10^9, 7.5 × 10^9 particles per g (5%, 8%, 12% PEG 8000fraction)	106
	Portulaca oleracea	(1) The samples were thoroughly washed with running water and then placed into phosphate-buffered saline (PBS). (2) Homogenized using a blender	DUC and GUC	(1) TEM: round-shaped. (2) DLS: 30–400 nm in size (average diameter: 180 nm); zeta potential: −31.4 mV	134
Seed	Moringa oleifera	Prepared seed powder and then extracted by the water	UF	Flow cytometry analysis: 100–500 nm in size	38
Flower	Actinidia chinensis	Pollen grains were pelleted at 5000g for 15 min at 4 °C	DUC and UF	(1) NTA: 120–209 nm in size. (2) AFM: round-shaped	164
Peels	Aloe vera	(1) Pulverized using blender. (2) The mixture was centrifuged at 1000g, 2000g, 3000g, and 10 000g to remove impurities.	TFF and DUC	(1) TEM: round-shaped. (2) NTA: 50–200 nm in size (average diameter: 153.6 ± 51.4 nm); particle concentration: 5.35 ± 2.92 × 10^8 particles per ml. (3) BCA: protein concentration: 5.01 ± 0.27 μg mL^−1. protein ratio: 1.07 ± 0.42 × 10^9 particle per μg	61
Callus	Aster yomena	(1) Centrifuged at 1800g, 3500g, and 10 000g for 20 min at 4 °C. (2) Filtered through a 0.22 μm vacuum filter	TFF and DUC	(1) TEM: round-shaped. (2) DLS: average diameter: 225.2 ± 13 nm	60
Fruit	Momordica charantia	(1) Squeezed to obtain the juice. (2) The juice was sequentially centrifuged at 3000g for 10 min, and 10 000g for 20 min (3) filtered through a 0.22 μm pore filter	ELD	(1) TEM: cup-shaped. (2) NTA: 100–300 nm in size	40
	Lemon	(1) Squeezed to obtain the juice. (2) The juice was sequentially centrifuged at 3000g for 10 min, and 10 000g for 20 min (3) filtered through a 0.22 μm pore filter	ELD	(1) TEM: round-shaped. (2) NTA: 100–300 nm in size; particle concentration: 1.0 × 10^8 particles per ml	64
	Avocado	(1) Ground in sterile PBS using a glass homogenizer. (2) Filtration to remove any large fibers	PBS and UF	(1) TEM: round-shaped. (2) NTA: 50–120 nm in size; Particle concentration: 1.4 × 10^8particles per ml	165
Starchy tuber	Allium sativum	(1) Blender to obtain the juice. (2) Centrifuged for 10 min	ATPS	(1) TEM: irregularly-shape. (2) NTA: 50–100 nm in size; particle concentration: 1.3 × 10^8 particles per ml	107

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5. Physical characterization of PDEVs

Physical characterization is crucial not only for assessing the properties of PDEVs but also for distinguishing them from other co-isolated materials. It is widely accepted that PDEVs resemble vesicles of human or animal origin. Under electron microscopy, PDEVs have been confirmed to have a spherical, oval, or cup-shaped morphology.⁷⁵ They exhibit distinct membrane structures and come in subspherical or teratoid shapes of varying sizes. The key to their identification lies in whether the size of the isolated PDEVs conforms to the specified particle size and potential range. The particle size and potential of nanovesicles from different plant sources vary widely, with average particle sizes ranging from 30 nm to 200 nm and zeta potentials ranging from nearly neutral to about −50 mV.⁷⁶ This variability may be related to the different components contained in nanovesicles from different species, differences in biogenesis, and the various separation processes, all of which impact particle size.³³

Similar to MDEVs, the physical characteristics of PDEVs are typically evaluated by examining particle size distribution, concentration, morphology, and surface charge. Understanding these characteristics is vital for accurate analysis and differentiation of PDEVs. Various detection techniques, including transmission electron microscopy (TEM), scanning electron microscopy (SEM), cryo-EM, scanning-probe microscopy (SPM), and transmission electron atomic force microscopy (AFM), have been utilized to study PDEV morphology.^19,77

It's important to note that the morphology of PDEVs may vary depending on their source and the detection technique employed. For instance, PDEVs from ginseng often exhibit a spherical shape,³² while those from strawberry typically display a round or cup-shaped structure when observed using TEM.⁷⁸ Techniques like TEM and SEM, which involve sample fixation, dehydration, and staining, often result in a cup-shaped morphology due to vesicle dehydration and deformation.⁷⁹ In contrast, cryo-EM analyzes samples at extremely low temperatures, preserving their spherical morphology.⁷⁷

Quantification of PDEVs typically involves assessing particle numbers using methods such as dynamic light scattering (DLS), NTA, and laser transmission spectroscopy (LTS).^73,80,81 Differentiating specific subtypes of EVs is crucial for detailed analysis, and this can be achieved through multiparametric surface marker analysis using fluorescent dyes and antibodies on platforms like Nano FCM NanoAnalyzer (nFCM) and Particle Metrix Zeta View Fluorescence Nanoparticle Tracking Analyzer (F-NTA).⁸² Additionally, the technique of Localized Surface Plasmon Resonance (LSPR) has emerged as highly sensitive for exosome detection, enabling the analysis of protein marker expression profiles.⁸² These techniques hold promise for characterizing PDEVs, contingent on the identification of standard PDEV subtype markers and commercialized antibodies, due to the similarities between MDEVs and PDEVs.

6. Stability of PDEVs

As interest in PDEVs grows, understanding the factors influencing their stability is critical for advancing their application in biomedical research. Various factors, including temperature, chemical reagents, sonication, and pH, have been shown to affect PDEV stability. It is well documented that EVs from both mammalian and plant sources exhibit partial resistance to the harsh gastric environment of the gastrointestinal tract.^83,84 Fox example, PDEVs isolated from Catharanthus roseus (L.) Don leaves maintained their structural integrity under both acidic and alkaline conditions, demonstrating substantial resistance to degradation by DNase, RNase, and proteases. Notably, neither acid–base treatment nor a 30 min enzymatic digestion impaired their ability to stimulate nitric oxide release in RAW264.7 macrophage cells. However, exposure to surfactants and heat treatment was found to compromise the PDEVs’ stability,³⁵ highlighting potential vulnerabilities in certain conditions.

Despite the observed resistance of PDEVs to the gastrointestinal environment, the molecular mechanisms that confer this stability remain poorly understanding. Furthermore, while a few studies have evaluated the gastrointestinal digestion of PDEVs, most have focused on their cargo rather than the EVs themselves. These studies often report a dramatic reduction in cargo following digestion, yet the remaining components still exhibit biological activity.⁸⁵ This underscores the importance of further research into the mechanisms governing both PDEV stability and cargo retention in such environments.

The preservation method employed plays a pivotal role in maintaining PDEV stability. Researchers have explored various storage conditions, including the impact of pre- and post-separation procedures, temperature variations, and freeze–thaw cycles, on PDEV yield, composition, functionality, and particle integrity. Commonly, cryopreservation at −80 °C is used for storing isolated EVs, although temporary storage at 4 °C is also feasible. Lyophilization has emerged as a promising technique for longer-term preservation of EV products. However, prolonged storage at −80 °C can result in a decline in EV concentration and purity, increased particle size variability, and change in zeta potential, all of which can lead to shifts in the size–charge relationship of the EVs.⁸⁶

The inclusion of preservatives also significantly affects PDEVs stability. For instance, a study on PDEVs derived from Dendropanax morbifera leaves revealed that preservatives, particularly 1,3-butylene glycol and TMO, enhanced vesicle stability. Of the conditions tested, PDEVs stored with TMO at 4 °C demonstrated the highest stability.⁸⁷ In the context of MDEVs, lyophilization with cryopreservation agents (CPAs) such as trehalose, poly(vinylpyrrolidone) 40 (PVP40), dimethyl sulfoxide (DMSO), glycerin, propylene glycol, sucrose, and poly(ethylene glycol) has been shown to offer superior preservation compared with lyophilization alone.^88,89 These agents help mitigate the detrimental effects of lyophilization and improve the overall stability of the vesicles.

7. PDEV composition

To fully harness the therapeutic potential of PDEVs, it is crucial to achieve a comprehensive understanding of their biochemical composition. Current studies indicate that PDEVs are primarily composed of lipids, proteins, nucleic acid, and various plant metabolites, as depicted in Fig. 4. Advanced techniques such as flow cytometry-based methods, western blotting, enzyme-linked immunosorbent assay (ELISA), low-input RNA sequencing (RNA-Seq), quantitative PCR (qPCR), and liquid chromatography–tandem mass spectrometry (LC-MS/MS) have been employed to analyze these components.^90,91 Despite these technological advances, in-depth analyses of PDEV composition have been limited to only a few plant species, highlighting the need for broader research into the biochemical diversity of PDEVs.

Fig. 4 Composition of PDEVs. Lipids: phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylcholine (PC), digalactosyl diacylglycerol (DGDG), and monogalactosyl diacylglycerol (MGDG). Proteins: membrane proteins, heat shock proteins (HSP), tetraspanin proteins, endosomal sorting complex required for transport (ESCRT). Nucleic acids: DNA, mRNA, siRNA, and miRNA. Plant metabolites: curcumin, 6-gingerol, 6-shogaol, and β-glucan.

7.1. Lipids in PDEVs

The lipid bilayer of PDEVs, predominantly composed of various phospholipids, is integral to maintaining their structural integrity and stability. Key lipids in PDEVs include phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylcholine (PC), digalactosyl diacylglycerol (DGDG), and monogalactosyl diacylglycerol (MGDG), which are essential constituents found in both plant- and animal-derived vesicles.⁹² Notably, the lipid composition of PDEVs differs across plant species, which may affect their biological functions and interactions with recipient cells. For example, ginger- and grape-derived PDEVs are particularly rich in PA, while PE and PC are predominate in grapefruit- and garlic-derived PDEVs.^93,94

The lipid composition of PDEVs significantly influences their selective uptake by specific cell types. Grape PDEVs have shown a preferential affinity for intestinal stem cells,⁹⁵ whereas ginger PDEVs are selectively internalized by hepatocytes.⁹⁶ Intriguingly, microbial interactions also exhibit lipid-specific preferences: Lactobacillus rhamnosus favors PA-enriched ginger PDEVs, while members of the Ruminococcaceae family preferentially internalize PC-enriched grapefruit PDEVs.^95,96 These findings suggest that the lipid profiles of PDEVs act as molecular signals that guide their targeted uptake by specific cells, providing a promising mechanism for selective drug delivery.

In addition to the role they play in cellular uptake, PDEVs’ lipid components also contribute to their biological activity. For instance, lipids derived from ginger PDEVs have been shown to suppress activation of the nucleotide-binding domain and leucine-rich repeat-containing family pyrin domain containing 3 (NLRP3) inflammasome in bone marrow-derived macrophages.⁹⁷ Moreover, PA from ginger PDEVs has demonstrated inhibitory effects on the growth of the periodontal pathogen Porphyromonas gingivalis by interacting with its outer membrane proteins.⁹⁸ These findings suggest that PDEVs’ lipid content may play an active role in modulating immune responses and microbial interactions.

The structural flexibility of the phospholipid bilayer in PDEVs has also enabled the development of artificial nanovesicles for drug delivery.⁹⁹ Several research groups have utilized lipids from grapefruit-derived PDEVs to engineer grapefruit-derived nanovectors capable of delivering a range of therapeutic agents, including chemotherapeutics, small-interfering RNAs (siRNAs), DNA expression vectors, and proteins, to diverse cell types.^100–102 Compared with synthetic liposomes, PDEVs offer enhanced biocompatibility and lower immunogenicity due to their natural plant origin, making them ideal candidates for drug delivery applications.

Despite the critical roles that lipids play in the formation and functional properties of PDEVs, our current understanding of their lipidomics remains incomplete. More extensive lipidomic analyses of PDEVs across a broader range of plant species are needed to fully explore their potential as biocompatible carriers for targeted therapeutic interventions.

7.2. Proteins in PDEVs

Proteins play a vital role in the biological functions of PDEVs, contributing to various physiological and intercellular communication processes. For instance, studies on Solanum lycopersicum L. (tomato) root-secreted PDEVs have revealed the presence of cytosolic proteins involved in plant–microbe interactions and cell wall remodeling, suggesting a role in pathogen defense mechanisms.¹⁰³ Another study demonstrated that PDEVs derived from broccoli (Brassica oleracea) contain aquaporins, which help maintain membrane stability under stress conditions.¹⁰⁴ Additionally, membrane-bound proteins may facilitate the uptake and internalization of PDEVs into mammalian cells, similar to how lipids mediate cellular interactions.¹⁰⁵

Western blotting is commonly used to detect proteins within PDEV preparations. Several proteins, such as PEN1 and TET8, have been identified as pan-PDEV markers. These proteins are notably enriched in the lumen of Arabidopsis apoplast,^23,24,85 highlighting their potential as signature components of PDEVs. In recent studies, western blotting has been applied to analyze PDEVs purified from Arabidopsis thaliana using commercially available mammalian exosomal kits. These studies have identified the presence of tetraspanins (CD9, CD63, CD81) and ESCRT-related proteins such as TSG101 and ALIX, suggesting a shared molecular composition between PDEVs and MDEVs.¹⁰⁶ Moreover, PDEVs derived from garlic express several heat-shock proteins (HSPs), including HSP70, as well as tetraspanins CD9 and CD63.¹⁰⁷ Similarly, PDEVs from Cannabis sativa have been shown to contain elevated levels of tetraspanins (CD9, CD81) alongside HSPs such as HSP60, HSP70, and HSP90, further emphasizing the conservation of key protein families across plant species.¹⁰⁸ Recent investigations have proposed synthetic proteins such as PEN1, ABC transporter protein PEN3, and TET-8 as potential surface markers for PDEVs.¹⁰⁹ However, the consistent identification and validation of surface marker proteins specific to PDEVs remain a significant challenge for researchers, impeding the development of standardized isolation and characterization protocols.

7.3. Nucleic acids in PDEVs

Nucleic acids, recognized for their biomarker potential and functional roles, are essential components of PDEVs. Techniques such as low-input RNA sequencing (RNA-Seq) and quantitative PCR (qPCR) are commonly employed to identify specific nucleic acid sequences within PDEVs.¹¹⁰ The nucleic acid content, particularly RNAs, plays a crucial role in modulating biological functions in plants and may influence interspecies interactions at the molecular level.

The main classes of small RNAs (sRNAs) in plants, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), act as gene silencing signals. These sRNAs can move between cells, from roots to shoots, and even between host plants and pathogens.¹¹⁰ Notably, the ten most representative miRNA families have been identified in various food plants, and EVs are enriched in these miRNA families.⁸⁵ For instance, PDEVs from Arabidopsis thaliana contain several species of miRNAs and siRNAs, as well as an underexplored class of “tiny RNAs” (10 to 17 nucleotides) that are highly abundant in these vesicles.¹¹¹ Moreover, sequencing of sRNAs has revealed that miRNAs, typically 20–22 nucleotides in length, are capable of regulating human mRNA.¹¹² This suggests that sRNAs in PDEVs may mediate long-distance communication within plants and potentially function as cross-kingdom delivery agents. Target prediction analyses using tools like TargetScan indicate that miRNAs isolated from PDEVs have the potential to target and regulate mammalian genes involved in inflammatory and cancer-related pathways. Emerging evidence further supports the immunomodulatory and anticancer activities of plant-derived miRNAs.^113,114 While the focus has traditionally been on RNA within PDEVs, recent research has begun to explore the role played by DNA in these vesicles. Although DNA content in PDEVs has received comparatively less attention, there is growing evidence of its potential in disease biomarker discovery and intercellular communication.^115,116

7.4. Plant metabolites in PDEVs

Recent research has highlighted the presence of plant metabolites in PDEVs from various sources. For instance, polyphenols have been identified in SEC-purified PDEVs, including cyanidin 3-glucoside and cyanidin 3,5-diglucoside in PEDVs isolated from pomegranate, and hesperidin in PDEVs from orange.⁸⁵ Similarly, PDEVs isolated from turmeric have been found to contain curcumin, a hydrophobic polyphenol renowned for its potent anti-inflammatory properties.¹¹⁷ In another study, Zhang et al. demonstrated that PDEVs from ginger are enriched with bioactive compounds such as 6-gingerol and 6-shogaol, which possess comparable anti-inflammatory effects.⁹³ Additionally, high-performance liquid chromatography coupled with mass spectrometry (HPLC/MS) revealed that PDEVs from ginger, depending on their density, may contain specific metabolites.⁹⁶

In addition to polyphenols, other plant-derived bioactive compounds have been identified in PDEVs. For example, oat-derived PDEVs have been found to contain up to 20% β-glucan, significantly higher than the ≤4% β-glucan found in oat microparticles or oat flour.⁸¹ These findings underscore the enrichment of specific plant metabolites in PDEVs, with many of these compounds, such as curcumin, 6-gingerol, and 6-shogaol, being recognized for their therapeutic potential in treating various diseases.¹¹⁸

Interestingly, analysis of secondary metabolites in nanovesicles from different plant species has revealed that lipophilic molecules are often associated with vesicle membranes. This observation suggests that these metabolites are passively incorporated into vesicles due to their lipophilicity, rather than being actively packaged into the vesicles.¹¹⁹ The encapsulation of plant-derived metabolites into PDEVs raises important questions regarding whether this process enhances their bioactivity and bioavailability, a topic that warrants further investigation.

8. Biological functions of PDEVs

PDEVs, characterized by their diverse molecular cargo and distinct cystic structure, perform a wide range of biological activities. These vesicles, with their capacity to transfer lipids, proteins, nucleic acids, and metabolites, have been implicated in several physiological and therapeutic processes, which are illustrated in Fig. 5. This section explores the key biological functions of PDEVs, including their anticancer, anti-inflammatory, immunomodulatory, antioxidant, and drug delivery capabilities.

Fig. 5 Biological functions of PDEVs. PDEVs from diverse plant sources exhibit multiple biological effects by impacting cells or tissues. They can act as anticancer agents, immunomodulators, anti-inflammatory substances, antioxidants, regulators of gut microbiome and intestinal health, managers of metabolic syndrome, regenerative therapies, solutions for osteoporosis, treatments for COVID-19, and even serve as drug carriers.

8.1 Anticancer potential

In recent years, the therapeutic applications of EVs, especially MDEVs, in cancer treatment have garnered significant attention, with some advancing to clinical trials.¹²⁰ Parallel to this, PDEVs have emerged as promising anticancer agents, with an expanding body of research demonstrating their diverse and potent effects in oncology. These studies reveal that PDEVs can inhibit cancer cell proliferation, modulate the cell cycle, induce apoptosis, and influence cancer metabolism,¹⁹ suggesting a multifaceted approach for cancer treatment.

For example, PDEVs from Panax ginseng have shown the capacity to induce M1 macrophage polarization through Toll-like receptor (TLR)-4 and myeloid differentiation antigen 88 (MyD88)-mediated signaling pathways, promoting apoptosis in melanoma cells. The ceramide lipids and proteins contained within these vesicles appear to play a crucial role in this macrophage polarization,³² enhancing their anticancer potential. Similarly, PDEVs from Moringa oleifera seeds have demonstrated inhibitory effects on cancer cell viability and apoptosis induction. These effects are mediated through the regulation of B-cell lymphoma 2 (Bcl-2) protein expression and the disruption of mitochondrial membrane potential, implicating key mitochondrial pathways in their mechanism of action.³⁸ Moreover, PDEVs from bitter melon have exhibited significant anticancer activity against breast cancer in both in vitro and in vivo models. These vesicles were shown to suppress cancer cell proliferation and migration while inducing apoptosis by promoting reactive oxygen species (ROS) production and compromising mitochondrial function.¹²¹ The elevated ROS levels appear to trigger oxidative stress-mediated cell death pathways, providing a promising route for cancer treatment. In another study, PDEVs sourced from tea flower were found to exert cytotoxicity effects on breast cancer cells, inhibiting tumor growth and reducing migration capacity. Notably, these vesicles also modulated the gut microbiota, suggesting a potential systemic anticancer effect that could influence metastasis.³⁷ Further research on PDEVs from Atractylodes lancea rhizome demonstrated their ability to reduce melanin levels and tyrosinase activity in melanoma cells. These findings suggest that PDEVs may be effective in melanoma therapy, particularly in the modulation of melanogenesis pathways, which are critical in melanoma progression.¹²²

8.2. Immunomodulation and anti-inflammation

Imbalanced immune signaling triggers inflammation, which, if improperly controlled, can lead to chronic inflammatory conditions and various related diseases, posing significant health risks.^123,124 To address these risks, recent studies have explored the immunomodulatory and anti-inflammatory properties of PDEVs across various biological systems, both in vitro and in vivo.^60,125 A key discovery involves PDEVs from Pueraria lobata, an herb known for its medicinal and dietary uses, which effectively promoted M2 macrophage polarization, indicating their potential as therapeutic agents for resolving inflammation.¹²⁶ On the contrary, PDEVs sourced from ginseng suppress M2-like macrophage polarization, instead promoting the secretion of M1-macrophage-associated cytokines such as TNF-α, IL-12, and IL-6 in response to IL-4 and IL-13.³² This suggests that different PDEVs can have distinct, context-dependent immunomodulatory effects.

The NLRP3 inflammasome plays a critical role in regulating the immune response and is implicated in various inflammatory diseases, including inflammatory bowel disease (IBD).¹²⁷ Evidence suggests that PDEVs from ginger rhizome effectively suppress NLRP3 inflammasome activation, a key factor in modulating immune mechanisms. Specifically, ginger PDEVs inhibit inflammatory cascades, reduce levels of pro-inflammatory cytokines such as IL-1β and IL-18, and prevent the assembly of the NLRP3 inflammasome. These anti-inflammatory effects are mediated by mechanisms like the induction of autophagy and the prevention of focal cell death.⁹⁷ Supporting these findings, a clinical trial (ClinicalTrials.gov Identifier: NCT04879810) assessed the anti-inflammatory efficacy of ginger PDEVs in patients with IBD, demonstrating their potential as a novel therapeutic approach.

The interaction between PDEVs and target cells also plays a crucial role in mediating inflammatory responses. For instance, PDEVs from turmeric have exhibited potent anti-inflammatory effects in murine models of colitis. These vesicles modulate key pro-inflammatory cytokines, including TNF-α, IL-6, and IL-1β, while enhancing the expression of the antioxidant gene HO-1, thereby promoting the resolution of colitis.¹¹⁷ Additionally, turmeric PDEVs may inactivate the NF-κB pathway, further contributing to their protective effects against colitis. In another study, PDEVs from oranges (Citrus sinensis) were shown to mitigate intestinal inflammation and restore intestinal barrier integrity by regulating the expression of key genes, including HMOX-1, ICAM1, OCLN, CLDN1, and MLCK.¹²⁸ These findings highlight the therapeutic potential of PDEVs for maintaining gut homeostasis and treating intestinal disorders. Moreover, PDEVs have shown promise in addressing neuroinflammation. In a model of chronic brain inflammation induced by alcohol consumption, oat PDEVs were able to cross the blood–brain barrier and reduce the secretion of pro-inflammatory cytokines by microglial cells, thereby preventing ethanol-induced neurodegenration.⁸¹ Further illustrating the clinical relevance of PDEVs, a clinical trial (ClinicalTrials.gov Identifier: NCT01668849) evaluated the efficacy of grape exosomes in reducing the occurrence of oral mucositis during radiation and chemotherapy for head and neck cancers.

8.3. Antioxidant potential

Cells generate ROS during various physiological processes, such as aerobic respiration, or in response to external stimuli like xenobiotics, cytokines, and bacterial invasion.¹²⁹ Excessive ROS levels can result in oxidative stress,¹³⁰ which damages biomolecules and contributes to diseases such as neurodegenerative disorders, cardiovascular diseases, chronic wounds, carcinogenesis, and skin aging.¹³¹ PDEVs are recognized for their antioxidant properties, primarily through their anti-inflammatory actions. They enhance cellular defense mechanisms against oxidative damage by upregulating antioxidant molecules such as nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), and NAD(P)H quinone dehydrogenase 1 (NQO1).⁷⁵ Consequently, PDEVs hold potential as therapeutic agents in mitigating oxidative stress and its associated pathological conditions. For instance, research has demonstrated that PDEVs from Aloe vera peel exhibit significant antioxidant potential, showing dose-dependent upregulation of key antioxidant genes like Nrf2, CAT, HO-1, and SOD.⁶¹ Likewise, PDEVs from carrot prevent the downregulation of Nrf2 expression, protecting H9C2 cardiomyoblasts and human neuroblastoma SH-SY5Y cells from oxidative stress.⁷⁵

8.4. Regulation of the gut microbiome and intestinal health

The gut microbiome plays a vital role in maintaining intestinal health, influenced by factors such as genetics, early immunity development, diet, antibiotics use, and lifestyle choices.⁷⁵ Disruption of the microbiome can compromise the intestinal barrier, aggravating various diseases.¹³² Emerging research suggests that PDEVs have the potential to regulate the gut microbiome, thereby improving intestinal barrier function and strengthening immune response. For instance, PDEVs from turmeric, when orally administered, were shown to restore the integrity of a damaged intestinal barrier, modulate gut microbiota composition, and shift macrophage phenotypes toward an anti-inflammatory state.¹³³ Similarly, PDEVs from Portulaca oleracea L. were found to significantly alter intestinal flora diversity, ameliorating dextran sulfate sodium (DSS)-induced microbiota disturbances and promoting microbial structure recovery.¹³⁴ Additional studies involving PDEVs from carrot, grapefruit, and ginger indicate their potential therapeutic benefits in maintaining intestinal homeostasis through diverse cellular and intercellular mechanisms.⁹²

8.5. Management of metabolic syndrome

Metabolic syndrome refers to a cluster of risk factors that elevate the likelihood of developing cardiovascular disease. These factors include abdominal obesity, hypertension, impaired fasting glucose, elevated triglyceride levels, and low HDL cholesterol.¹³⁵ With societal changes and shifts in lifestyle, the global prevalence of metabolic syndrome is rising, currently affecting approximately 25% of the world's population.¹³⁶ Natural sources, particularly from dietary sources like vegetables and fruits, have gained widespread recognition for their potential in preventing, managing, and treating metabolic syndrome.¹³⁷ Consequently, researchers have begun investigating the regulatory effects of PDEVs on metabolic syndrome. For example, a study on PDEVs from orange juice demonstrated their ability to combat obesity by reducing triglyceride levels and modulating the gene expression of ANGPTL4.⁹¹ Similarly, PDEVs from dried nuts were found to contain conserved plant miRNAs, such as miR159a and miR156c, which effectively downregulated Tnfrsf1a protein levels, thereby attenuating the TNF-α signaling pathway in adipocytes and reducing inflammation in mice.¹³⁸ Moreover, a clinical trial (ClinicalTrials.gov Identifier: NCT03493984) is currently assessing the efficacy of PDEVs from Aloe vera for improving insulin resistance and chronic inflammation in patients with polycystic ovary syndrome.⁹⁹ These findings, along with ongoing clinical research, present promising avenues for using PDEVs in managing metabolic syndrome.

8.6. Regenerative potential

Regenerative medicine is an emerging and innovative field focused on repairing, engineering, or regenerating human cells, tissues, or organs to restore normal function.¹³⁹ Numerous biomaterials have been explored in regenerative medicine, and PDEVs have recently gained attention for their therapeutic potential, especially in skin tissue regeneration.¹⁴⁰ PDEVs sourced from wheat and grapefruit have shown promising results in skin regeneration, stimulating the proliferation and migration of HaCaT cells in a dose-dependent manner. These PDEVs also increased mRNA levels of collagen type I (COL1A1) in human dermal fibroblast (HDF) and HaCaT cells. Additionally, PDEVs from grapefruit were found to enhance the expression of key wound-healing factors such as laminin, fibronectin, vimentin, and epidermal growth factor, thereby improving wound closure and healing.^68,141 Moreover, PDEVs from Aloe vera demonstrated the ability to modulate inflammation while inhibiting myofibroblast differentiation and contraction, positioning them as a promising candidate for PDEV-based therapeutic approaches aimed at tissue regeneration.¹⁴²

8.7. Addressing osteoporosis

Osteoporosis is a systemic skeletal disorder characterized by an imbalance between bone resorption and formation, leading to decreased bone mass and mineral density.¹⁴³ Clinical treatment often involves bone resorption inhibitors such as estrogen, calcitonin, and bisphosphonates. However, long-term bisphosphonate use has been associated with severe side effects,¹⁴⁴ prompting the exploration of natural alternatives and supplements for managing and preventing osteoporosis. Notably, Hwang et al. developed PDEVs from yam, which demonstrated osteogenic activity by activating the BMP-2/p-p38-dependent Runx2 pathway.¹⁴⁵ These yam PDEVs promoted longitudinal bone growth and increased tibial mineral density in ovariectomized mice with induced osteoporosis, with corresponding improvements in osteoblast-related parameters. The PDEVs were also absorbed through the gastrointestinal tract, indicating effective oral bioavailability, while demonstrating systemic biosafety through histological and liver/kidney toxicity evaluations. This suggests yam PDEVs as a safe, orally effective therapeutic for osteoporosis. Furthermore, PDEVs from lemon significantly enhanced collagen synthesis, supporting bone matrix maintenance and overall bone health.¹⁴⁶ PDEVs from Cissus quadrangularis also exhibited potential in bone tissue engineering by promoting wound healing, reducing oxidative stress, and enhancing the function of human-derived mesenchymal stem cells (hMSCs) and the pre-osteoblast cell line MC3T3.¹⁴⁷

8.8. Treatment of COVID-19

Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is a global health crisis characterized by acute respiratory illness and systemic complications.¹⁴⁸ The virus spreads primarily through respiratory droplets, aerosols, and less frequently via fecal or urinary transmission, contributing to widespread person-to-person infection. Despite advancements in vaccination, there remains an ongoing demand for effective COVID-19 treatments. Several antiviral drugs, such as nirmatrelvir/ritonavir, remdesivir, and molnupiravir, have been authorized for COVID-19 management.¹⁴⁹ In addition, research is exploring complementary treatments, including traditional Chinese medicine (TCM) and medicinal plants.¹⁴⁹

PDEVs have gained attention due to their enrichment in bioavailable miRNAs, which have potential cross-kingdom regulatory functions. Recent studies have identified plant miRNAs within PDEVs from various sources, such as Hami melon, soybean, tomato, pear, grapefruit, pea, blueberry, and ginger, that target the SARS-CoV-2 transcriptome.^112,150 Preliminary findings indicate promising potential for these miRNAs in inhibiting SARS-CoV-2, offering a novel therapeutic avenue.¹⁵¹

8.9. Utilization of PDEVs as versatile drug carriers

Oral drug delivery is often preferred for its convenience; however, the degradation of drugs by gastric acids can significantly reduce their efficacy, making alternative delivery systems crucial. PDEVs, due to their acid resistance, ability to across physiological barriers, and storage stability, have emerged as promising candidates for drug delivery systems.¹⁵² These vesicles exhibit properties such as biocompatibility and biodegradability, akin to their mammalian counterparts, which make them suitable for delivering a wide range of therapeutic agents, including chemotherapeutic drugs, proteins, mRNAs, siRNAs, miRNAs, and DNA vectors.¹⁵³

Recent studies have explored PDEVs from edible plants such as grapefruit, broccoli, ginger, aloe, and lemon for their potential as drug carriers.¹⁵⁴ The loading of therapeutic agents into PDEVs is achieved through various methods, including co-incubation, electroporation, sonication, saponin treatment, freeze–thaw cycles, and osmotic shock,¹⁵⁵ each chosen based on the nature of the cargo. For instance, PDEVs from Catharanthus roseus (L.) Don leaves have demonstrated remarkable stability in gastrointestinal fluids, withstanding enzymatic digestion and extreme pH conditions, suggesting their potential as robust nanocarriers for oral drug delivery.³⁵ A clinical trial (ClinicalTrials.gov Identifier: NCT01294072) investigated the efficacy of PDEVs for enhancing the delivery of curcumin to normal colon tissue and colon cancers. Curcumin, known for its anti-inflammatory and anticancer properties, has limited bioavailability when administered orally. The trial aimed to address this limitation by utilizing PDEVs to deliver curcumin more effectively to colon cancers and surrounding normal tissues, offering a novel solution to curcumin's poor bioavailability.

Advances in PDEV-based drug delivery systems continue to evolve, further expanding their applications. For example, ginger PDEVs combined with Pd–Pt nanosheets have demonstrated efficacy for targeting infection sites, where the vesicle-mediated uptake by bacteria enhances antimicrobial effects.¹⁵⁶ Additionally, PDEVs from cucumber have shown potential in improving photodynamic therapy for the treatment of hypertrophic scars. A notable development is that of NDs@EV-RGD, which combines copper-based metal–organic framework nanodots (Cu-MOF NDs) with RGD-modified cucumber PDEVs. This innovative system has demonstrated efficient transdermal delivery, successfully overcoming the barrier posed by the dense stratum corneum, making it a promising approach for enhancing therapeutic outcomes in skin-related conditions.¹⁵⁷

9. Discussion

This review provides a comprehensive analysis of PDEVs, emphasizing their distinct advantages for biomedical applications. First, the abundance of plant materials offers a sustainable and cost-effective source for PDEV production, ensuring high yields and making them accessible for both research and commercialization. Additionally, using plant sources adheres to ethical standards, avoiding the reliance on animal-derived materials, which aligns with the growing demand for ethically sourced biomedical products. PDEVs possess diverse biological functions, including antioxidant, anti-inflammatory, and anticancer properties, positioning them as promising candidates for disease prevention and treatment. Moreover, their high bioavailability enhances absorption in the human body, amplifying their therapeutic potential. PDEVs also exhibit low toxicity and immunogenicity, making them safer for biomedical applications compared with synthetic or animal-derived vesicles.

Despite these advantages, several challenges must be addressed to fully unlock the potential of PDEVs. First, the lack of a unified nomenclature convention complicates the identification and classification of PDEVs across different studies, which hampers comparative research and the development of standardized methodologies. Second, the need for standardized biomarkers to consistently identify, isolate, and characterize PDEVs is critical. Without these markers, it becomes difficult to ensure reproducibility in research and clinical settings. Third, current preservation and transportation techniques are underdeveloped, limiting the clinical application of PDEVs. Understanding their stability during storage and transport is crucial for clinical and industrial applications. Moreover, the distribution and metabolism of PDEVs within the human body, particularly their fate in the gastrointestinal tract, remain poorly understood. Most studies to date have focused on the fate of the cargo carried by PDEVs, rather than the vesicles themselves, leaving significant gaps in our understanding of their therapeutic mechanisms and in vivo stability.

To fully exploit the potential of PDEVs, further research is required in several key areas. First, their stability in the gastrointestinal tract must be thoroughly investigated. The gastrointestinal environment poses challenges due to its varying pH levels and digestive enzymes. While some studies suggest that PDEVs may retain biological activity after digestion, the precise effects of these conditions on the vesicles themselves remain unclear. Future research should focus on both the stability of the PDEVs’ cargo and the integrity of the vesicles. Developing new techniques to track intact PDEVs within the gastrointestinal tract and assess their stability would provide valuable insights. For example, identifying biomarkers to track PDEVs in vivo and understanding their breakdown mechanisms are essential for predicting their efficacy and safety in various treatments. Advancements in imaging techniques could enable real-time monitoring of PDEVs’ movement within the body, offering insights into their biodistribution and potential off-target effects.⁹⁰ Additionally, studying the breakdown products of PDEVs may reveal how they are metabolized, and identifying the enzymes involved in this process could inform strategies to regulate PDEVs’ stability or enhance their therapeutic properties.⁹⁵

Second, a deeper understanding of the mechanisms underlying PDEV biogenesis and release is crucial for optimizing production and enhancing their therapeutic potential. Genetic manipulation techniques, such as knockout or knockdown models, should be employed to explore how specific genes influence PDEV biogenesis.¹⁵⁸ Additionally, transcriptomic and proteomic analyses could identify differentially regulated genes and proteins involved in vesicle formation. These data could then be used to construct regulatory networks and develop mathematical models simulating the PDEV biogenesis process.¹⁵⁹ Furthermore, the application of machine learning and artificial intelligence (AI) technologies, as potent tools for deciphering complex biological pathways, is well documented and could significantly enhance our understanding of PDEV formation and function.^160,161

Lastly, optimizing preservation and transportation techniques is vital for maintaining PDEV stability and functionality in clinical settings. Developing standardized methods for the isolation, purification, and characterization of PDEVs is essential to ensure reproducibility and reliability in both research and clinical applications. Addressing these challenges will pave the way for the effective utilization of PDEVs in biomedicine, potentially revolutionizing drug delivery, disease treatment, and therapeutic interventions.

10. Conclusion

In conclusion, while PDEVs present significant potential in disease prevention, treatment, and drug delivery, addressing key challenges, such as understanding their biogenesis, conducting compositional analysis, and identifying key marker proteins, is essential for achieving their standardized production and clinical application. Despite these hurdles, PDEVs hold great promise for future application in medicine and biotechnology. Their unique properties, including sustainability, bioavailability, and safety, position them as ideal candidates for various therapeutic uses. Overcoming the identified challenges will pave the way for the widespread clinical adoption of PDEVs, potentially revolutionizing the fields of medicine and drug delivery.

Author contributions

Conceptualization: Yao-Bo Zeng, Sibao Chen, Da-Jian Yang, Guo-Qing Chen; data curation and original draft preparation: Yao-Bo Zeng, Li-Sha Shen, Yong Yang, Xing Zhou, Guo-Qing Chen; figures and tables: Xun Deng, Lianbao Ye, Sibao Chen; review and editing: Sibao Chen, Da-Jian Yang, Guo-Qing Chen; all authors contributed to the article and approved the submitted version.

Data availability

The data presented in this study are available from the corresponding author upon reasonable request.

Conflicts of interest

All authors declared that there are no conflicts of interest.

Acknowledgements

This research was funded by the Shenzhen Science and Technology Innovation Commission (JCYJ20220531090802006), Innovation and Technology Fund, Mainland-Hong Kong Joint Funding Scheme (MHP/010/20), Chongqing Science and Technology Commission (CSTB2023NSCQ-MSX0155), Science and Technology Research Program of Chongqing Municipal Education Commission (KJQN202215127), and 2024 Guangdong Pharmaceutical University's “Discipline Training Excellence, Innovation and Quality Improvement” Engineering Team Project (2024ZZ05).

References

1. S. El Andaloussi, I. Mäger, X. O. Breakefield and M. J. Wood, Extracellular vesicles: biology and emerging therapeutic opportunities, Nat. Rev. Drug Discovery, 2013, 12, 347–357.
2. H. Kalra, G. P. Drummen and S. Mathivanan, Focus on extracellular vesicles: introducing the next small big thing, Int. J. Mol. Sci., 2016, 17, 170.
3. J. A. Welsh, D. C. Goberdhan, L. O'Driscoll, E. I. Buzas, C. Blenkiron, B. Bussolati, H. Cai, D. Di Vizio, T. A. Driedonks and U. Erdbrügger, Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches, J. Extracell. Vesicles, 2024, 13, e12404.
4. B.-T. Pan and R. M. Johnstone, Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: selective externalization of the receptor, Cell, 1983, 33, 967–978.
5. R. M. Johnstone, M. Adam, J. Hammond, L. Orr and C. Turbide, Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes), J. Biol. Chem., 1987, 262, 9412–9420.
6. H. Zhang, M. Yang, X. Wu, Q. Li, X. Li, Y. Zhao, F. Du, Y. Chen, Z. Wu and Z. Xiao, The distinct roles of exosomes in tumor-stroma crosstalk within gastric tumor microenvironment, Pharmacol. Res., 2021, 171, 105785.
7. C. Huang, Z. Liu, M. Chen, L. Du, C. Liu, S. Wang, Y. Zheng and W. Liu, Tumor-derived biomimetic nanozyme with immune evasion ability for synergistically enhanced low dose radiotherapy, J. Nanobiotechnol., 2021, 19, 1–10.
8. Z. Xu, S. Zeng, Z. Gong and Y. Yan, Exosome-based immunotherapy: a promising approach for cancer treatment, Mol. Cancer, 2020, 19, 1–16.
9. N. Wang, X. Li, Z. Zhong, Y. Qiu, S. Liu, H. Wu, X. Tang, C. Chen, Y. Fu and Q. Chen, 3D hESC exosomes enriched with miR-6766–3p ameliorates liver fibrosis by attenuating activated stellate cells through targeting the TGFβRII-SMADS pathway, J. Nanobiotechnol., 2021, 19, 1–26.
10. L. Li, L. Zhang, K. C. Montgomery, L. Jiang, C. J. Lyon and T. Y. Hu, Advanced technologies for molecular diagnosis of cancer: State of pre-clinical tumor-derived exosome liquid biopsies, Mater. Today Bio, 2023, 18, 100538.
11. K. Barreiro, A. C. Lay, G. Leparc, V. D. T. Tran, M. Rosler, L. Dayalan, F. Burdet, M. Ibberson, R. J. Coward and T. B. Huber, An in vitro approach to understand contribution of kidney cells to human urinary extracellular vesicles, J. Extracell. Vesicles, 2023, 12, 12304.
12. A. Thakur, A. Johnson, E. Jacobs, K. Zhang, J. Chen, Z. Wei, Q. Lian and H. J. Chen, Energy sources for exosome communication in a cancer microenvironment, Cancers, 2022, 14, 1698.
13. J. Kim, S. Li, S. Zhang and J. Wang, Plant-derived exosome-like nanoparticles and their therapeutic activities, Asian J. Pharm. Sci., 2022, 17, 53–69.
14. C. Fusco, G. De Rosa, I. Spatocco, E. Vitiello, C. Procaccini, C. Frigè, V. Pellegrini, R. La Grotta, R. Furlan and G. Matarese, Extracellular vesicles as human therapeutics: A scoping review of the literature, J. Extracell. Vesicles, 2024, 13, e12433.
15. Y.-J. Li, J.-Y. Wu, J. Liu, W. Xu, X. Qiu, S. Huang, X.-B. Hu and D.-X. Xiang, Artificial exosomes for translational nanomedicine, J. Nanobiotechnol., 2021, 19, 1–20.
16. N. Mu, J. Li, L. Zeng, J. You, R. Li, A. Qin, X. Liu, F. Yan and Z. Zhou, Plant-derived exosome-like nanovesicles: current progress and prospects, Int. J. Nanomed., 2023, 4987–5009.
17. J. Feng, Q. Xiu, Y. Huang, Z. Troyer, B. Li and L. Zheng, Plant–derived vesicle–like nanoparticles as promising biotherapeutic tools: Present and future, Adv. Mater., 2023, 35, 2207826.
18. K.-J. Lo, M.-H. Wang, C.-T. Ho and M.-H. Pan, Plant-Derived Extracellular Vesicles: A New Revolutionization of Modern Healthy Diets and Biomedical Applications, J. Agric. Food Chem., 2024, 72, 2853–2878.
19. M. Q. Lian, W. H. Chng, J. Liang, H. Q. Yeo, C. K. Lee, M. Belaid, M. Tollemeto, M. G. Wacker, B. Czarny and G. Pastorin, Plant–derived extracellular vesicles: recent advancements and current challenges on their use for biomedical applications, J. Extracell. Vesicles, 2022, 11, 12283.
20. N. Liu, L. Hou, X. Chen, J. Bao, F. Chen, W. Cai, H. Zhu, L. Wang and X. Chen, Arabidopsis TETRASPANIN8 mediates exosome secretion and glycosyl inositol phosphoceramide sorting and trafficking, Plant Cell, 2024, 36, 626–641.
21. Y. Cui, J. Gao, Y. He and L. Jiang, Plant extracellular vesicles, Protoplasma, 2020, 257, 3–12.
22. X. Li, H. Bao, Z. Wang, M. Wang, B. Fan, C. Zhu and Z. Chen, Biogenesis and function of multivesicular bodies in plant immunity, Front. Plant Sci., 2018, 9, 979.
23. Q. Cai, L. Qiao, M. Wang, B. He, F.-M. Lin, J. Palmquist, S.-D. Huang and H. Jin, Plants send small RNAs in extracellular vesicles to fungal pathogen to silence virulence genes, Science, 2018, 360, 1126–1129.
24. B. D. Rutter and R. W. Innes, Extracellular vesicles isolated from the leaf apoplast carry stress-response proteins, Plant Physiol., 2017, 173, 728–741.
25. J. Wang, Y. Ding, J. Wang, S. Hillmer, Y. Miao, S. W. Lo, X. Wang, D. G. Robinson and L. Jiang, EXPO, an exocyst-positive organelle distinct from multivesicular endosomes and autophagosomes, mediates cytosol to cell wall exocytosis in Arabidopsis and tobacco cells, Plant Cell, 2010, 22, 4009–4030.
26. T. Pečenková, M. Hála, I. Kulich, D. Kocourková, E. Drdová, M. Fendrych, H. Toupalová and V. Žárský, The role for the exocyst complex subunits Exo70B2 and Exo70H1 in the plant–pathogen interaction, J. Exp. Bot., 2011, 62, 2107–2116.
27. Y. Cui, W. Cao, Y. He, Q. Zhao, M. Wakazaki, X. Zhuang, J. Gao, Y. Zeng, C. Gao and Y. Ding, A whole-cell electron tomography model of vacuole biogenesis in Arabidopsis root cells, Nat. Plants, 2019, 5, 95–105.
28. M. Zhang, E. Viennois, C. Xu and D. Merlin, Plant derived edible nanoparticles as a new therapeutic approach against diseases, Tissue Barriers, 2016, 4, e1134415.
29. S. P. Kalarikkal, D. Prasad, R. Kasiappan, S. R. Chaudhari and G. M. Sundaram, A cost-effective polyethylene glycol-based method for the isolation of functional edible nanoparticles from ginger rhizomes, Sci. Rep., 2020, 10, 4456.
30. J. Y. You, S. J. Kang and W. J. Rhee, Isolation of cabbage exosome-like nanovesicles and investigation of their biological activities in human cells, Bioact. Mater, 2021, 6, 4321–4332.
31. R. Lee, H. J. Ko, K. Kim, Y. Sohn, S. Y. Min, J. A. Kim, D. Na and J. H. Yeon, Anti-melanogenic effects of extracellular vesicles derived from plant leaves and stems in mouse melanoma cells and human healthy skin, J. Extracell. Vesicles, 2020, 9, 1703480.
32. M. Cao, H. Yan, X. Han, L. Weng, Q. Wei, X. Sun, W. Lu, Q. Wei, J. Ye and X. Cai, Ginseng-derived nanoparticles alter macrophage polarization to inhibit melanoma growth, J. Immunother. Cancer, 2019, 7, 1–18.
33. Y. Liu, S. Wu, Y. Koo, A. Yang, Y. Dai, H. Khant, S. R. Osman, M. Chowdhury, H. Wei and Y. Li, Characterization of and isolation methods for plant leaf nanovesicles and small extracellular vesicles, Nanomedicine, 2020, 29, 102271.
34. G. Pocsfalvi, L. Turiák, A. Ambrosone, P. Del Gaudio, G. Puska, I. Fiume, T. Silvestre and K. Vékey, Protein biocargo of citrus fruit-derived vesicles reveals heterogeneous transport and extracellular vesicle populations, J. Plant Physiol., 2018, 229, 111–121.
35. X. Ou, H. Wang, H. Tie, J. Liao, Y. Luo, W. Huang, R. Yu, L. Song and J. Zhu, Novel plant-derived exosome-like nanovesicles from Catharanthus roseus: preparation, characterization, and immunostimulatory effect via TNF-α/NF-κB/PU. 1 axis, J. Nanobiotechnol., 2023, 21, 160.
36. B. M. O'Leary, A. Rico, S. McCraw, H. N. Fones and G. M. Preston, The infiltration-centrifugation technique for extraction of apoplastic fluid from plant leaves using Phaseolus vulgaris as an example, J. Visualized Exp., 2014, e52113.
37. Q. Chen, Q. Li, Y. Liang, M. Zu, N. Chen, B. S. Canup, L. Luo, C. Wang, L. Zeng and B. Xiao, Natural exosome-like nanovesicles from edible tea flowers suppress metastatic breast cancer via ROS generation and microbiota modulation, Acta Pharm. Sin. B, 2022, 12, 907–923.
38. M. Potestà, V. Roglia, M. Fanelli, E. Pietrobono, A. Gismondi, S. Vumbaca, R. G. Nguedia Tsangueu, A. Canini, V. Colizzi and S. Grelli, Effect of microvesicles from Moringa oleifera containing miRNA on proliferation and apoptosis in tumor cell lines, Cell death discovery, 2020, 6, 43.
39. E. Garzo, C. M. Sánchez-López, A. Fereres, C. Soler, A. Marcilla and P. Pérez-Bermúdez, Isolation of Extracellular Vesicles from Phloem Sap by Size Exclusion Chromatography, Curr. Protoc., 2023, 3, e903.
40. M. Yang, Q. Luo, X. Chen and F. Chen, Bitter melon derived extracellular vesicles enhance the therapeutic effects and reduce the drug resistance of 5-fluorouracil on oral squamous cell carcinoma, J. Nanobiotechnol., 2021, 19, 259.
41. K. S. Visan, R. J. Lobb, S. Ham, L. G. Lima, C. Palma, C. P. Z. Edna, L. Y. Wu, H. Gowda, K. K. Datta and G. Hartel, Comparative analysis of tangential flow filtration and ultracentrifugation, both combined with subsequent size exclusion chromatography, for the isolation of small extracellular vesicles, J. Extracell. Vesicles, 2022, 11, 12266.
42. H. Zhang, D. Freitas, H. S. Kim, K. Fabijanic, Z. Li, H. Chen, M. T. Mark, H. Molina, A. B. Martin and L. Bojmar, Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation, Nat. Cell Biol., 2018, 20, 332–343.
43. H. Seo, C. Nam, E. Kim, J. Son and H. Lee, Aqueous two-phase system (ATPS)-based polymersomes for particle isolation and separation, ACS Appl. Mater. Interfaces, 2020, 12, 55467–55475.
44. B. He, Q. Cai, L. Qiao, C.-Y. Huang, S. Wang, W. Miao, T. Ha, Y. Wang and H. Jin, RNA-binding proteins contribute to small RNA loading in plant extracellular vesicles, Nat. Plants, 2021, 7, 342–352.
45. K. K. Jackson, C. Mata and R. K. Marcus, A rapid capillary-channeled polymer (C-CP) fiber spin-down tip approach for the isolation of plant-derived extracellular vesicles (PDEVs) from 20 common fruit and vegetable sources, Talanta, 2023, 252, 123779.
46. A. Bosque, L. Dietz, A. Gallego-Lleyda, M. Sanclemente, M. Iturralde, J. Naval, M. A. Alava, L. Martínez-Lostao, H.-J. Thierse and A. Anel, Comparative proteomics of exosomes secreted by tumoral Jurkat T cells and normal human T cell blasts unravels a potential tumorigenic role for valosin-containing protein, Oncotarget, 2016, 7, 29287.
47. P. Li, M. Kaslan, S. H. Lee, J. Yao and Z. Gao, Progress in exosome isolation techniques, Theranostics, 2017, 7, 789.
48. N. Hadizadeh, D. Bagheri, M. Shamsara, M. R. Hamblin, A. Farmany, M. Xu, Z. Liang, F. Razi and E. Hashemi, Extracellular vesicles biogenesis, isolation, manipulation and genetic engineering for potential in vitro and in vivo therapeutics: An overview, Front. Bioeng. Biotechnol., 2022, 10, 1019821.
49. M. A. Livshits, E. Khomyakova, E. G. Evtushenko, V. N. Lazarev, N. A. Kulemin, S. E. Semina, E. V. Generozov and V. M. Govorun, Isolation of exosomes by differential centrifugation: Theoretical analysis of a commonly used protocol, Sci. Rep., 2015, 5, 1–14.
50. P. D. Subudhi, C. Bihari, S. K. Sarin and S. Baweja, Emerging role of edible exosomes-like nanoparticles (ELNs) as hepatoprotective agents, Nanotheranostics, 2022, 6, 365.
51. K. Sidhom, P. O. Obi and A. Saleem, A review of exosomal isolation methods: is size exclusion chromatography the best option?, Int. J. Mol. Sci., 2020, 21, 6466.
52. B. Talebjedi, N. Tasnim, M. Hoorfar, G. F. Mastromonaco and M. De Almeida Monteiro Melo Ferraz, Exploiting microfluidics for extracellular vesicle isolation and characterization: potential use for standardized embryo quality assessment, Front. Vet. Sci., 2021, 7, 620809.
53. L. M. Doyle and M. Z. Wang, Overview of extracellular vesicles, their origin, composition, purpose, and methods for exosome isolation and analysis, Cells, 2019, 8, 727.
54. E. J. Northrop-Albrecht, W. R. Taylor, B. Q. Huang, J. B. Kisiel and F. Lucien, Assessment of extracellular vesicle isolation methods from human stool supernatant, J. Extracell. Vesicles, 2022, 11, e12208.
55. N.-J. Liu, N. Wang, J.-J. Bao, H.-X. Zhu, L.-J. Wang and X.-Y. Chen, Lipidomic analysis reveals the importance of GIPCs in Arabidopsis leaf extracellular vesicles, Mol. Plant, 2020, 13, 1523–1532.
56. B. Van der Bruggen, in Fundamental modelling of membrane systems, Elsevier, 2018, pp. 25–70.
57. R. Stranska, L. Gysbrechts, J. Wouters, P. Vermeersch, K. Bloch, D. Dierickx, G. Andrei and R. Snoeck, Comparison of membrane affinity-based method with size-exclusion chromatography for isolation of exosome-like vesicles from human plasma, J. Transl. Med., 2018, 16, 1–9.
58. A. N. Böing, E. Van Der Pol, A. E. Grootemaat, F. A. Coumans, A. Sturk and R. Nieuwland, Single-step isolation of extracellular vesicles by size-exclusion chromatography, J. Extracell. Vesicles, 2014, 3, 23430.
59. S. Busatto, G. Vilanilam, T. Ticer, W.-L. Lin, D. W. Dickson, S. Shapiro, P. Bergese and J. Wolfram, Tangential flow filtration for highly efficient concentration of extracellular vesicles from large volumes of fluid, Cells, 2018, 7, 273.
60. W. S. Kim, J.-H. Ha, S.-H. Jeong, J.-I. Lee, B.-W. Lee, Y. J. Jeong, C. Y. Kim, J.-Y. Park, Y. B. Ryu and H.-J. Kwon, Immunological effects of aster yomena callus-derived extracellular vesicles as potential therapeutic agents against allergic asthma, Cells, 2022, 11, 2805.
61. M. K. Kim, Y. C. Choi, S. H. Cho, J. S. Choi and Y. W. Cho, The antioxidant effect of small extracellular vesicles derived from aloe vera peels for wound healing, Tissue Eng. Regener. Med., 2021, 18, 561–571.
62. S. Sitar, A. Kejžar, D. Pahovnik, K. Kogej, M. Tušek-Žnidarič, M. Lenassi and E. Žagar, Size characterization and quantification of exosomes by asymmetrical-flow field-flow fractionation, Anal. Chem., 2015, 87, 9225–9233.
63. H. Zhang and D. Lyden, Asymmetric-flow field-flow fractionation technology for exomere and small extracellular vesicle separation and characterization, Nat. Protoc., 2019, 14, 1027–1053.
64. M. Yang, X. Liu, Q. Luo, L. Xu and F. Chen, An efficient method to isolate lemon derived extracellular vesicles for gastric cancer therapy, J. Nanobiotechnol., 2020, 18, 1–12.
65. M. Y. Konoshenko, E. A. Lekchnov, A. V. Vlassov and P. P. Laktionov, Isolation of extracellular vesicles: general methodologies and latest trends, BioMed Res. Int., 2018, 2018, 8545347.
66. A. P. Suresh, S. P. Kalarikkal, B. Pullareddy and G. M. Sundaram, Low pH-based method to increase the yield of plant-derived nanoparticles from fresh ginger rhizomes, ACS Omega, 2021, 6, 17635–17641.
67. F. A. Coumans, A. R. Brisson, E. I. Buzas, F. Dignat-George, E. E. Drees, S. El-Andaloussi, C. Emanueli, A. Gasecka, A. Hendrix and A. F. Hill, Methodological guidelines to study extracellular vesicles, Circ. Res., 2017, 120, 1632–1648.
68. Y. Savcı, O. K. Kırbaş, B. T. Bozkurt, E. A. Abdik, P. N. Taşlı, F. Şahin and H. Abdik, Grapefruit-derived extracellular vesicles as a promising cell-free therapeutic tool for wound healing, Food Funct., 2021, 12, 5144–5156.
69. O. K. Kırbaş, B. T. Bozkurt, A. B. Asutay, B. Mat, B. Ozdemir, D. Öztürkoğlu, H. Ölmez, Z. İşlek, F. Şahin and P. N. Taşlı, Optimized isolation of extracellular vesicles from various organic sources using aqueous two-phase system, Sci. Rep., 2019, 9, 19159.
70. T. Shtam, R. Samsonov, A. Volnitskiy, R. Kamyshinsky, N. Verlov, M. Kniazeva, E. Korobkina, A. Orehov, A. Vasiliev and A. Konevega, Isolation of extracellular microvesicles from cell culture medium: Comparative evaluation of methods, Biochem. (Moscow), Suppl. Ser. B, Biomed. Chem., 2018, 12, 167–175.
71. Y. Huang, S. Wang, Q. Cai and H. Jin, Effective methods for isolation and purification of extracellular vesicles from plants, J. Integr. Plant Biol., 2021, 63, 2020–2030.
72. N. Zarovni, A. Corrado, P. Guazzi, D. Zocco, E. Lari, G. Radano, J. Muhhina, C. Fondelli, J. Gavrilova and A. Chiesi, Integrated isolation and quantitative analysis of exosome shuttled proteins and nucleic acids using immunocapture approaches, Methods, 2015, 87, 46–58.
73. B. D. Rutter and R. W. Innes, Growing pains: addressing the pitfalls of plant extracellular vesicle research, New Phytol., 2020, 228, 1505–1510.
74. A. Cvjetkovic, J. Lötvall and C. Lässer, The influence of rotor type and centrifugation time on the yield and purity of extracellular vesicles, J. Extracell. Vesicles, 2014, 3, 23111.
75. D. K. Kim and W. J. Rhee, Antioxidative effects of carrot-derived nanovesicles in cardiomyoblast and neuroblastoma cells, Pharmaceutics, 2021, 13, 1203.
76. M. Cao, N. Diao, X. Cai, X. Chen, Y. Xiao, C. Guo, D. Chen and X. Zhang, Plant exosome nanovesicles (PENs): green delivery platforms, Mater. Horiz., 2023, 10, 3879–3894.
77. Y. Yuana, R. I. Koning, M. E. Kuil, P. C. Rensen, A. J. Koster, R. M. Bertina and S. Osanto, Cryo-electron microscopy of extracellular vesicles in fresh plasma, J. Extracell. Vesicles, 2013, 2, 21494.
78. F. Perut, L. Roncuzzi, S. Avnet, A. Massa, N. Zini, S. Sabbadini, F. Giampieri, B. Mezzetti and N. Baldini, Strawberry-derived exosome-like nanoparticles prevent oxidative stress in human mesenchymal stromal cells, Biomolecules, 2021, 11, 87.
79. H. Shao, H. Im, C. M. Castro, X. Breakefield, R. Weissleder and H. Lee, New technologies for analysis of extracellular vesicles, Chem. Rev., 2018, 118, 1917–1950.
80. C. Y. Soo, Y. Song, Y. Zheng, E. C. Campbell, A. C. Riches, F. Gunn-Moore and S. J. Powis, Nanoparticle tracking analysis monitors microvesicle and exosome secretion from immune cells, Immunology, 2012, 136, 192–197.
81. F. Xu, J. Mu, Y. Teng, X. Zhang, K. Sundaram, M. K. Sriwastva, A. Kumar, C. Lei, L. Zhang and Q. M. Liu, Restoring Oat Nanoparticles Mediated Brain Memory Function of Mice Fed Alcohol by Sorting Inflammatory Dectin–1 Complex Into Microglial Exosomes, Small, 2022, 18, 2105385.
82. D. Fortunato, D. Mladenović, M. Criscuoli, F. Loria, K.-L. Veiman, D. Zocco, K. Koort and N. Zarovni, Opportunities and pitfalls of fluorescent labeling methodologies for extracellular vesicle profiling on high-resolution single-particle platforms, Int. J. Mol. Sci., 2021, 22, 10510.
83. M.-C. López de las Hazas, L. del Pozo-Acebo, M. S. Hansen, J. Gil-Zamorano, D. C. Mantilla-Escalante, D. Gómez-Coronado, F. Marín, A. Garcia-Ruiz, J. T. Rasmussen and A. Dávalos, Dietary bovine milk miRNAs transported in extracellular vesicles are partially stable during GI digestion, are bioavailable and reach target tissues but need a minimum dose to impact on gene expression, Eur. J. Nutr., 2022, 1–14.
84. L. del Pozo-Acebo, M.-C. L. de Las Hazas, J. Tomé-Carneiro, A. del Saz-Lara, J. Gil-Zamorano, L. Balaguer, L. A. Chapado, R. Busto, F. Visioli and A. Dávalos, Therapeutic potential of broccoli-derived extracellular vesicles as nanocarriers of exogenous miRNAs, Pharmacol. Res., 2022, 185, 106472.
85. M.-C. L. de Las Hazas, J. Tomé-Carneiro, L. del Pozo-Acebo, A. del Saz-Lara, L. A. Chapado, L. Balaguer, E. Rojo, J. C. Espín, C. Crespo and D. A. Moreno, Therapeutic potential of plant-derived extracellular vesicles as nanocarriers for exogenous miRNAs, Pharmacol. Res., 2023, 198, 106999.
86. S. Gelibter, G. Marostica, A. Mandelli, S. Siciliani, P. Podini, A. Finardi and R. Furlan, The impact of storage on extracellular vesicles: A systematic study, J. Extracell. Vesicles, 2022, 11, e12162.
87. K. Kim, J. Park, Y. Sohn, C.-E. Oh, J.-H. Park, J.-M. Yuk and J.-H. Yeon, Stability of plant leaf-derived extracellular vesicles according to preservative and storage temperature, Pharmaceutics, 2022, 14, 457.
88. K. B. Y. El Baradie, M. Nouh, F. O'Brien III, Y. Liu, S. Fulzele, A. Eroglu and M. W. Hamrick, Freeze-dried extracellular vesicles from adipose-derived stem cells prevent hypoxia-induced muscle cell injury, Front. Cell Dev. Biol., 2020, 8, 181.
89. M. M. Bahr, M. S. Amer, K. Abo-El-Sooud, A. N. Abdallah and O. S. El-Tookhy, Preservation techniques of stem cells extracellular vesicles: A gate for manufacturing of clinical grade therapeutic extracellular vesicles and long-term clinical trials, Int. J. Vet. Sci. Med., 2020, 8, 1–8.
90. X.-H. Xu, T.-J. Yuan, H. A. Dad, M.-Y. Shi, Y.-Y. Huang, Z.-H. Jiang and L.-H. Peng, Plant exosomes as novel nanoplatforms for microRNA transfer stimulate neural differentiation of stem cells in vitro and in vivo, Nano Lett., 2021, 21, 8151–8159.
91. E. Berger, P. Colosetti, A. Jalabert, E. Meugnier, O. P. Wiklander, J. Jouhet, E. Errazurig-Cerda, S. Chanon, D. Gupta and G. J. Rautureau, Use of nanovesicles from orange juice to reverse diet-induced gut modifications in diet-induced obese mice, Mol. Ther.–Methods Clin. Dev., 2020, 18, 880–892.
92. S. Rome, Biological properties of plant-derived extracellular vesicles, Food Funct., 2019, 10, 529–538.
93. M. Zhang, E. Viennois, M. Prasad, Y. Zhang, L. Wang, Z. Zhang, M. K. Han, B. Xiao, C. Xu and S. Srinivasan, Edible ginger-derived nanoparticles: A novel therapeutic approach for the prevention and treatment of inflammatory bowel disease and colitis-associated cancer, Biomaterials, 2016, 101, 321–340.
94. B. Wang, X. Zhuang, Z.-B. Deng, H. Jiang, J. Mu, Q. Wang, X. Xiang, H. Guo, L. Zhang and G. Dryden, Targeted drug delivery to intestinal macrophages by bioactive nanovesicles released from grapefruit, Mol. Ther., 2014, 22, 522–534.
95. S. Ju, J. Mu, T. Dokland, X. Zhuang, Q. Wang, H. Jiang, X. Xiang, Z.-B. Deng, B. Wang and L. Zhang, Grape exosome-like nanoparticles induce intestinal stem cells and protect mice from DSS-induced colitis, Mol. Ther., 2013, 21, 1345–1357.
96. X. Zhuang, Z.-B. Deng, J. Mu, L. Zhang, J. Yan, D. Miller, W. Feng, C. J. McClain and H.-G. Zhang, Ginger-derived nanoparticles protect against alcohol-induced liver damage, J. Extracell. Vesicles, 2015, 4, 28713.
97. X. Chen, Y. Zhou and J. Yu, Exosome-like nanoparticles from ginger rhizomes inhibited NLRP3 inflammasome activation, Mol. Pharm., 2019, 16, 2690–2699.
98. K. Sundaram, D. P. Miller, A. Kumar, Y. Teng, M. Sayed, J. Mu, C. Lei, M. K. Sriwastva, L. Zhang and J. Yan, Plant-derived exosomal nanoparticles inhibit pathogenicity of Porphyromonas gingivalis, Iscience, 2019, 21, 308–327.
99. T. Karamanidou and A. Tsouknidas, Plant-derived extracellular vesicles as therapeutic nanocarriers, Int. J. Mol. Sci., 2021, 23, 191.
100. X. Zhuang, Y. Teng, A. Samykutty, J. Mu, Z. Deng, L. Zhang, P. Cao, Y. Rong, J. Yan and D. Miller, Grapefruit-derived nanovectors delivering therapeutic miR17 through an intranasal route inhibit brain tumor progression, Mol. Ther., 2016, 24, 96–105.
101. Y. Teng, J. Mu, X. Hu, A. Samykutty, X. Zhuang, Z. Deng, L. Zhang, P. Cao, J. Yan and D. Miller, Grapefruit-derived nanovectors deliver miR-18a for treatment of liver metastasis of colon cancer by induction of M1 macrophages, Oncotarget, 2016, 7, 25683.
102. Q. Wang, X. Zhuang, J. Mu, Z.-B. Deng, H. Jiang, L. Zhang, X. Xiang, B. Wang, J. Yan and D. Miller, Delivery of therapeutic agents by nanoparticles made of grapefruit-derived lipids, Nat. Commun., 2013, 4, 1867.
103. M. De Palma, A. Ambrosone, A. Leone, P. Del Gaudio, M. Ruocco, L. Turiák, R. Bokka, I. Fiume, M. Tucci and G. Pocsfalvi, Plant roots release small extracellular vesicles with antifungal activity, Plants, 2020, 9, 1777.
104. M. d. C. Martínez-Ballesta, P. García-Gomez, L. Yepes-Molina, A. L. Guarnizo, J. A. Teruel and M. Carvajal, Plasma membrane aquaporins mediates vesicle stability in broccoli, PLoS One, 2018, 13, e0192422.
105. H. Song, B. S. Canup, V. L. Ngo, T. L. Denning, P. Garg and H. Laroui, Internalization of garlic-derived nanovesicles on liver cells is triggered by interaction with CD98, ACS Omega, 2020, 5, 23118–23128.
106. S. Jokhio, I. Peng and C.-A. Peng, Extracellular vesicles isolated from Arabidopsis thaliana leaves reveal characteristics of mammalian exosomes, Protoplasma, 2024, 1–9.
107. İ. Özkan, P. Koçak, M. Yıldırım, N. Ünsal, H. Yılmaz, D. Telci and F. Şahin, Garlic (Allium sativum)-derived SEVs inhibit cancer cell proliferation and induce caspase mediated apoptosis, Sci. Rep., 2021, 11, 14773.
108. A. O. Ipinmoroti, J. k. Turner, E. J. Bellenger, B. J. Crenshaw, J. Xu, C. Reeves, O. Ajayi, T. Li and Q. L. Matthews, Characterization of cannabis strain-plant-derived extracellular vesicles as potential biomarkers, Protoplasma, 2023, 260, 1603–1606.
109. M. Pinedo, L. de la Canal and C. de Marcos Lousa, A call for Rigor and standardization in plant extracellular vesicle research, J. Extracell. Vesicles, 2021, 10, e12048.
110. A. F. Samad, M. Sajad, N. Nazaruddin, I. A. Fauzi, A. M. Murad, Z. Zainal and I. Ismail, MicroRNA and transcription factor: key players in plant regulatory network, Front. Plant Sci., 2017, 8, 565.
111. P. Baldrich, B. D. Rutter, H. Z. Karimi, R. Podicheti, B. C. Meyers and R. W. Innes, Plant extracellular vesicles contain diverse small RNA species and are enriched in 10-to 17-nucleotide “tiny” RNAs, Plant Cell, 2019, 31, 315–324.
112. J. Xiao, S. Feng, X. Wang, K. Long, Y. Luo, Y. Wang, J. Ma, Q. Tang, L. Jin and X. Li, Identification of exosome-like nanoparticle-derived microRNAs from 11 edible fruits and vegetables, PeerJ, 2018, 6, e5186.
113. K. Kim, H. J. Yoo, J.-H. Jung, R. Lee, J.-K. Hyun, J.-H. Park, D. Na and J. H. Yeon, Cytotoxic effects of plant sap-derived extracellular vesicles on various tumor cell types, J. Funct. Biomater., 2020, 11, 22.
114. M. De Robertis, A. Sarra, V. D'Oria, F. Mura, F. Bordi, P. Postorino and D. Fratantonio, Blueberry-derived exosome-like nanoparticles counter the response to TNF-α-induced change on gene expression in EA. hy926 cells, Biomolecules, 2020, 10, 742.
115. L. Cambier, K. Stachelek, M. Triska, R. Jubran, M. Huang, W. Li, J. Zhang, J. Li and D. Cobrinik, Extracellular vesicle-associated repetitive element DNAs as candidate osteosarcoma biomarkers, Sci. Rep., 2021, 11, 94.
116. J. W. Clancy, C. S. Sheehan, A. C. Boomgarden and C. D’Souza-Schorey, Recruitment of DNA to tumor-derived microvesicles, Cell Rep., 2022, 38, 110443.
117. C. Liu, X. Yan, Y. Zhang, M. Yang, Y. Ma, Y. Zhang, Q. Xu, K. Tu and M. Zhang, Oral administration of turmeric-derived exosome-like nanovesicles with anti-inflammatory and pro-resolving bioactions for murine colitis therapy, J. Nanobiotechnol., 2022, 20, 206.
118. J.-W. Chen, S. Chen and G.-Q. Chen, Recent advances in natural compounds inducing non-apoptotic cell death for anticancer drug resistance, Cancer Drug Resist., 2023, 6, 729.
119. E. Woith, G. Guerriero, J.-F. Hausman, J. Renaut, C. C. Leclercq, C. Weise, S. Legay, A. Weng and M. F. Melzig, Plant extracellular vesicles and nanovesicles: focus on secondary metabolites, proteins and lipids with perspectives on their potential and sources, Int. J. Mol. Sci., 2021, 22, 3719.
120. F. Zhang, J. Guo, Z. Zhang, M. Duan, G. Wang, Y. Qian, H. Zhao, Z. Yang and X. Jiang, Application of engineered extracellular vesicles for targeted tumor therapy, J. Biomed. Sci., 2022, 29, 14.
121. T. Feng, Y. Wan, B. Dai and Y. Liu, Anticancer activity of bitter melon-derived vesicles extract against breast cancer, Cells, 2023, 12, 824.
122. T. Ishida, S. Morisawa, K. Jobu, K. Kawada, S. Yoshioka and M. Miyamura, Atractylodes lancea rhizome derived exosome-like nanoparticles prevent alpha-melanocyte stimulating hormone-induced melanogenesis in B16-F10 melanoma cells, Biochem. Biophys. Rep., 2023, 35, 101530.
123. J. Giraud and M. Saleh, Host–Microbiota Interactions in Liver Inflammation and Cancer, Cancers, 2021, 13, 4342.
124. S. Almasabi, A. U. Ahmed, R. Boyd and B. R. Williams, A potential role for integrin-linked kinase in colorectal cancer growth and progression via regulating senescence and immunity, Front. Genet., 2021, 12, 638558.
125. Z. Zhang, Y. Yu, G. Zhu, L. Zeng, S. Xu, H. Cheng, Z. Ouyang, J. Chen, J. L. Pathak and L. Wu, The emerging role of plant-derived exosomes-like nanoparticles in immune regulation and periodontitis treatment, Front. Immunol., 2022, 13, 896745.
126. J. Wu, X. Ma, Y. Lu, T. Zhang, Z. Du, J. Xu, J. You, N. Chen, X. Deng and J. Wu, Edible Pueraria lobata-derived exosomes promote M2 macrophage polarization, Molecules, 2022, 27, 8184.
127. Y. Zhen and H. Zhang, NLRP3 inflammasome and inflammatory bowel disease, Front. Immunol., 2019, 10, 411387.
128. S. P. Bruno, A. Paolini, V. D'Oria, A. Sarra, S. Sennato, F. Bordi and A. Masotti, Extracellular vesicles derived from citrus sinensis modulate inflammatory genes and tight junctions in a human model of intestinal epithelium, Front. Nutr., 2021, 8, 778998.
129. P. D. Ray, B.-W. Huang and Y. Tsuji, Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling, Cell. Signalling, 2012, 24, 981–990.
130. G. Storz and J. A. Imlayt, Oxidative stress, Curr. Opin. Microbiol., 1999, 2, 188–194.
131. H. J. Forman and H. Zhang, Targeting oxidative stress in disease: Promise and limitations of antioxidant therapy, Nat. Rev. Drug Discovery, 2021, 20, 689–709.
132. S. Jandhyala, R. Talukdar and C. Subramanyam, Role of the normal gut microbiota, World J. Gastroenterol., 2015, 21(29), 8787–8803.
133. C. Gao, Y. Zhou, Z. Chen, H. Li, Y. Xiao, W. Hao, Y. Zhu, C. T. Vong, M. A. Farag and Y. Wang, Turmeric-derived nanovesicles as novel nanobiologics for targeted therapy of ulcerative colitis, Theranostics, 2022, 12, 5596.
134. M.-z. Zhu, H.-m. Xu, Y.-j. Liang, J. Xu, N.-n. Yue, Y. Zhang, C.-m. Tian, J. Yao, L.-s. Wang and Y.-q. Nie, Edible exosome-like nanoparticles from portulaca oleracea L mitigate DSS-induced colitis via facilitating double-positive CD4+ CD8+ T cells expansion, J. Nanobiotechnol., 2023, 21, 309.
135. M.-A. Cornier, D. Dabelea, T. L. Hernandez, R. C. Lindstrom, A. J. Steig, N. R. Stob, R. E. Van Pelt, H. Wang and R. H. Eckel, The metabolic syndrome, Endocr. Rev., 2008, 29, 777–822.
136. M. G. Saklayen, The global epidemic of the metabolic syndrome, Curr. Hypertens. Rep., 2018, 20, 1–8.
137. S. Castro-Barquero, A. M. Ruiz-León, M. Sierra-Pérez, R. Estruch and R. Casas, Dietary strategies for metabolic syndrome: a comprehensive review, Nutrients, 2020, 12, 2983.
138. K. Aquilano, V. Ceci, A. Gismondi, S. De Stefano, F. Iacovelli, R. Faraonio, G. Di Marco, N. Poerio, A. Minutolo and G. Minopoli, Adipocyte metabolism is improved by TNF receptor-targeting small RNAs identified from dried nuts, Commun. Biol., 2019, 2, 317.
139. C. Mason and P. Dunnill, A brief definition of regenerative medicine, Regener. Med., 2007, 3(1), 1–5.
140. M. Rodrigues, N. Kosaric, C. A. Bonham and G. C. Gurtner, Wound healing: a cellular perspective, Physiol. Rev., 2019, 99, 665–706.
141. F. Şahin, P. Koçak, M. Y. Güneş, İ. Özkan, E. Yıldırım and E. Y. Kala, In vitro wound healing activity of wheat-derived nanovesicles, Appl. Biochem. Biotechnol., 2019, 188, 381–394.
142. O. Ramírez, F. Pomareda, B. Olivares, Y.-L. Huang, G. Zavala, J. Carrasco-Rojas, S. Álvarez, C. Leiva-Sabadini, V. Hidalgo and P. Romo, Aloe vera peel-derived nanovesicles display anti-inflammatory properties and prevent myofibroblast differentiation, Phytomedicine, 2024, 122, 155108.
143. T. D. Rachner, S. Khosla and L. C. Hofbauer, Osteoporosis: now and the future, Lancet, 2011, 377, 1276–1287.
144. T. Sözen, L. Özışık and N. Ç. Başaran, An overview and management of osteoporosis, Eur. J. Rheumatol., 2017, 4, 46.
145. J.-H. Hwang, Y.-S. Park, H.-S. Kim, D.-h. Kim, S.-H. Lee, C.-H. Lee, S.-H. Lee, J.-E. Kim, S. Lee and H. M. Kim, Yam-derived exosome-like nanovesicles stimulate osteoblast formation and prevent osteoporosis in mice, J. Controlled Release, 2023, 355, 184–198.
146. N. Baldini, E. Torreggiani, L. Roncuzzi, F. Perut, N. Zini and S. Avnet, Exosome-like nanovesicles isolated from Citrus limon L. exert anti-oxidative effect, Curr. Pharm. Biotechnol., 2018, 19, 877–885.
147. R. Gupta, S. Gupta, P. Gupta, A. K. Nüssler and A. Kumar, Establishing the Callus-Based Isolation of Extracellular Vesicles from Cissus quadrangularis and Elucidating Their Role in Osteogenic Differentiation, J. Funct. Biomater., 2023, 14, 540.
148. A. M. Carabelli, T. P. Peacock, L. G. Thorne, W. T. Harvey, J. Hughes, S. J. Peacock, W. S. Barclay, T. I. De Silva, G. J. Towers and D. L. Robertson, SARS-CoV-2 variant biology: immune escape, transmission and fitness, Nat. Rev. Microbiol., 2023, 21, 162–177.
149. N. I. o. Health, COVID-19 treatment guidelines panel: Coronavirus diseases 2019 (COVID-19) treatment guidelines, 2023.
150. S. P. Kalarikkal and G. M. Sundaram, Edible plant-derived exosomal microRNAs: Exploiting a cross-kingdom regulatory mechanism for targeting SARS-CoV-2, Toxicol. Appl. Pharmacol., 2021, 414, 115425.
151. S. Zhou, Y. Cao, F. Shan, P. Huang, Y. Yang and S. Liu, Analyses of chemical components and their functions in single species plant-derived exosome like vesicle, TrAC, Trends Anal. Chem., 2023, 117274.
152. P. Vader, E. A. Mol, G. Pasterkamp and R. M. Schiffelers, Extracellular vesicles for drug delivery, Adv. Drug Delivery Rev., 2016, 106, 148–156.
153. Y. Zhao, H. Tan, J. Zhang, B. Pan, N. Wang, T. Chen, Y. Shi and Z. Wang, Plant-Derived Vesicles: A New Era for Anti-Cancer Drug Delivery and Cancer Treatment, Int. J. Nanomed., 2023, 6847–6868.
154. Z. Fang and K. Liu, Plant-derived extracellular vesicles as oral drug delivery carriers, J. Controlled Release, 2022, 350, 389–400.
155. Z. Xu, Y. Xu, K. Zhang, Y. Liu, Q. Liang, A. Thakur, W. Liu and Y. Yan, Plant-derived extracellular vesicles (PDEVs) in nanomedicine for human disease and therapeutic modalities, J. Nanobiotechnol., 2023, 21, 114.
156. Z. Qiao, K. Zhang, J. Liu, D. Cheng, B. Yu, N. Zhao and F.-J. Xu, Biomimetic electrodynamic nanoparticles comprising ginger-derived extracellular vesicles for synergistic anti-infective therapy, Nat. Commun., 2022, 13, 7164.
157. T. Kong, K. Zhang, Y. Wang, Y. Ye, J. Hou, C. Xu, N. Zhao and F. J. Xu, Cucumber–Derived Extracellular Vesicle–Functionalized Metal–Organic Frameworks for Enhanced Photodynamic Therapy of Hypertrophic Scars, Adv. Funct. Mater., 2024, 2400379.
158. G.-N. Zhao, P. Zhang, J. Gong, X.-J. Zhang, P.-X. Wang, M. Yin, Z. Jiang, L.-J. Shen, Y.-X. Ji and J. Tong, Tmbim1 is a multivesicular body regulator that protects against non-alcoholic fatty liver disease in mice and monkeys by targeting the lysosomal degradation of Tlr4, Nat. Med., 2017, 23, 742–752.
159. A. C. Dixson, T. R. Dawson, D. Di Vizio and A. M. Weaver, Context-specific regulation of extracellular vesicle biogenesis and cargo selection, Nat. Rev. Mol. Cell Biol., 2023, 24, 454–476.
160. E. Serretiello, A. Smimmo, A. Ballini, D. Parmeggiani, M. Agresti, P. Bassi, G. Moccia, A. Sciarra, A. De Angelis and P. Della Monica, Extracellular Vesicles and Artificial Intelligence: Unique Weapons against Breast Cancer, Appl. Sci., 2024, 14, 1639.
161. H.-J. Park, J. M. Kelly, J. R. Hoffman, F. Takaesu, W. Schwartzman, A. Ulziibayar, T. Kitsuka, E. Heuer, A. Yimit and R. Malbrue, Computational analysis of serum-derived extracellular vesicle miRNAs in juvenile sheep model of single stage Fontan procedure, J. Extracell. Vesicles, 2022, 1, 100013.
162. C. Théry, K. W. Witwer, E. Aikawa, M. J. Alcaraz, J. D. Anderson, R. Andriantsitohaina, A. Antoniou, T. Arab, F. Archer and G. K. Atkin-Smith, Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines, J. Extracell. Vesicles, 2018, 7, 1535750.
163. M. Y. Konoshenko, E. A. Lekchnov, A. V. Vlassov and P. P. Laktionov, Isolation of extracellular vesicles: general methodologies and latest trends, BioMed Res. Int., 2018, 2018, 8545347.
164. C. Suanno, E. Tonoli, E. Fornari, M. P. Savoca, I. Aloisi, L. Parrotta, C. Faleri, G. Cai, C. Coveney and D. J. Boocock, Small extracellular vesicles released from germinated kiwi pollen (pollensomes) present characteristics similar to mammalian exosomes and carry a plant homolog of ALIX, Front. Plant Sci., 2023, 14, 1090026.
165. S. Sharma, M. Mahanty, S. G. Rahaman, P. Mukherjee, B. Dutta, M. I. Khan, K. R. Sankaran, X. He, L. Kesavalu and W. Li, Avocado–derived extracellular vesicles loaded with ginkgetin and berberine prevent inflammation and macrophage foam cell formation, J. Cell. Mol. Med., 2024, 28, e18177.

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