Flow Cytometry Role in Unlocking New Frontiers for Nanomedicine Applications of Plant‐Derived Vesicles

ABSTRACT

Extracellular vesicles (EVs) are nanoscale membrane‐bound vesicles released by any cell type under both physiological and pathological conditions. They carry a wide array of bioactive molecules, including microRNAs (miRNAs), lipids, proteins and other small biomolecules, and therefore play a key role in intercellular communication by transferring functional cargo between cells. Plant‐derived EVs (PDVs) are secreted by plant cells and, because of their natural abundance, PDVs are a compelling alternative to synthetic nanoparticles, particularly for drug delivery applications, due to their biocompatibility, low immunogenicity and intrinsic ability to encapsulate and transport therapeutic molecules. PDVs can enhance drug delivery by improving efficacy and safety, reducing dosage and toxicity, and enabling environmentally sustainable approaches. They also hold promises in diagnostics and nutraceuticals, where their immunomodulatory effects, disease‐specific molecular signatures, and incorporation into functional foods may lead to significant health benefits. Despite these advances, challenges remain in standardizing PDVs isolation and characterization techniques and in ensuring reproducibility for clinical translation. Moreover, the transformative potential of PDVs to drive pharmaceutical and nutraceutical innovation and to promote sustainable healthcare solutions has also been underlined. This review provides a comprehensive analysis of PDVs, integrating research findings from 2014 to 2025 to summarize their sources, biochemical composition, isolation, characterization and numerous uses in the fields of biomedical and biotechnological research. Emphasis has been placed on the use of flow cytometry as an emerging, robust, and rapid technique for PDVs identification and characterization, underlining its relevance in clinical and nutraceutical applications.

Abbreviations

ACNVs

Asparagus cochinchinensis derived vesicles

AFM

atomic force microscopy

BBB

blood‐brain barrier

BDVS

broccoli derived vesicles

CCD

charge‐coupled device

circRNAs

circular RNAs

Cryo‐EM

cryo‐electron microscopy

DLS

dynamic light scattering

DSS

dextran sulfate sodium

dSTORM

direct stochastic optical reconstruction microscopy

ET

tetraspanins

EVs

extracellular vesicles

FC

flow cytometry

FCM

flow cytometry

FicoVes

opuntia ficus‐indica vesicles

GDNs

ginger‐derived nanoparticles

GDVs

Grape‐derived vesicles;

GELNs

ginger‐derived EVs

HSP

heat shock proteins

HSP70

heat shock protein 70

LAMP1

lysosomal marker

LCD

lipophilic dyes

LDEVs

lemon‐derived EVs

LDVs

lemon derived vesicles

lncRNAs

long non‐coding RNAs

MDEVs

mammalian derived extracellular vesicles

MSCs

mesenchymal stem cells

MVBs

multivesicular bodies

MVs

microvesicles

NTA

nanoparticle tracking analysis

OSCC

oral squamous cell carcinoma

PA

phosphatidic acid

PDVs

plant derived vesicles

PEG

polyethylene glycol

PEN1

penetration1

PN

panax notoginseng

SEC

size‐exclusion chromatography

SEM

scanning electron microscopy

sRNAs

small non‐coding RNAs

TDI

time delay integration

TEM

transmission electron microscopy

TFF

tangential flow filtration

TSG101

tumour susceptibility gene 101

UC

ultracentrifugation

UF

ultrafiltration

WB

Western blotting

1. Introduction

EVs are small, non‐replicating, heterogeneous membranous structures, naturally released by all cell types (Welsh et al. 2024; Simeone, Bologna, et al. 2020; Lanuti et al. 2025). They carry surface and intraluminal bioactive compounds, including miRNAs, lipids, proteins and small biomolecules (van Niel et al. 2018; Subha et al. 2023; Catitti, De Bellis, et al. 2022; Piro et al. 2024; Brocco, Simeone, et al. 2022; Brocco, de Bellis, et al. 2022; Brocco et al. 2025). When first described, mammalian‐derived EVs (MDEVs) were believed to be cellular debris and mainly served to clear cellular waste (Shkryl et al. 2022; O'brien et al. 2022). However, research over the past decade has unveiled their critical role in intercellular communication and the transport of bioactive molecules that act as cellular signals. By mediating the transfer of these molecules, EVs facilitate cellular messaging and influence both physiological and pathological processes in multicellular organisms (Shkryl et al. 2022; O'brien et al. 2022). Animal‐derived EVs are typically classified by their size and biogenesis into three main categories: exosomes (50–150 nm), microvesicles (MVs) (100–1000 nm) and apoptotic bodies (100–5000 nm) (Subha et al. 2023). More recently, additional EV subtypes have been described, including large endosomes (1000–10,000 nm) and migrasomes, released by migrating cells (Subha et al. 2023). Exosomes, spherical membrane‐bound structures, originate from multivesicular bodies (MVBs) and are released into the extracellular space upon fusion with the plasma membrane (Mathivanan et al. 2010). Exosomes play a key role in intercellular communication by transporting a variety of molecules, including mRNAs, miRNAs, bioactive lipids and proteins (Rayner and Hennessy 2013). They are characterized by the expression of specific proteins, such as members of the tetraspanin family (CD81, CD82, CD9 and CD63), as well as flotillin, tumour susceptibility gene 101(TSG101), Alix and several heat‐shock proteins, including HSP90, HSP70, HSPA5, Chaperonin Containing TCP1 Subunit 2 (CCT2) and HSP60 (Colombo et al. 2014; Yokoi et al. 2015). The lipid composition of membranes typically includes sphingomyelin, cholesterol, phosphatidylinositol, ceramide, phosphatidylethanolamine and phosphatidylserine (Todorova et al. 2017). MVs, also known as microparticles (MPs), are formed through the outward budding of the plasma membrane and range in size from approximately 50 to over 1000 nm (Gould and Raposo 2013). MVs carry key proteins such as integrins, selectins and CD40, and their membranes are particularly rich in cholesterol, diacylglycerol and phosphatidylserine at concentrations higher than those found in exosomes (He, Rodrigues, et al. 2021). Their biogenesis involves alterations in membrane phospholipid asymmetry and cytoskeletal reorganization, allowing portions of the cytoplasm to be encapsulated during their release. MVs reflect the activation status of their parent cells (Raizada et al. 2024). Apoptotic bodies are released by apoptotic cells and, like other EVs subtypes, play a role in intercellular communication (Pagotto et al. 2023). Apoptotic bodies are characterized by the expression of the typical exosomal marker CD63, along with the lysosomal marker (LAMP1) and the stress‐related protein heat‐shock protein 70 (HSP70), which is upregulated during apoptosis (Kakarla et al. 2020; Akers et al. 2013).

EVs have been identified in many biological fluids, such as blood (plasma or serum), urine, saliva, breast milk, amniotic fluid, cerebrospinal fluid, pleural effusion and bronchoalveolar fluid (Ciferri et al. 2021). Over the past decade, the potential of EVs as biomarkers and therapeutic agents has expanded significantly (Catitti, Cufaro, et al. 2022; Catitti, De Bellis, et al. 2022; Ghelardini et al. 1979). Numerous studies have shown that some EV‐associated nucleic acids and proteins play critical roles in the development and progression of cancer, as well as in neurodegenerative, infectious and autoimmune diseases (Pieragostino et al. 2018; Brocco et al. 2020). Mammalian‐derived EVs stemming from some specific sources, such as mesenchymal stem cells, have displayed therapeutic potential in various conditions (Efthymakis et al. 2022; Whittier et al. 1987). Additionally, they offer significant advantages as drug delivery systems by addressing challenges such as low bioavailability and unintended side effects (Elsharkasy et al. 2020).

Plant‐derived vesicles (PDVs) are gaining attention in the nutraceutical field for their natural origin, excellent biocompatibility and capacity to transport bioactive compounds, including proteins, lipids and RNAs across biological barriers, enabling efficient delivery to target cells and positioning them as strong candidates for oral or topical therapeutic applications (Lo et al. 2024).

Interestingly, given that EVs represent an evolutionarily conserved mode of intercellular communication, all kingdoms of life, including plants, produce and use EVs as signalling mediators (Simeone, Bologna, et al. 2020). Therefore, PDVs have recently garnered considerable interest due to their low cytotoxicity, low immunogenicity, high yield and potential efficacy (Qiang et al. 2024). This review outlines recent advancements in isolation and characterizations, focusing on the innovative use of flow cytometry for PDVs assessment and innovative applications of PDVs within nanomedicine, with an emphasis on supporting sustainable healthcare solutions. It aims to drive transformative advancements in biomedicine, with a focus on developing efficient and targeted drug delivery systems for clinical applications. Furthermore, current literature employs a confusing array of terms for plant‐origin extracellular vesicles (e.g., plant‐derived exosome‐like nanoparticles, plant‐derived extracellular vesicles, plant‐derived nanovesicles, plant‐derived nanocarriers, plant exosome‐like nanovesicles, plant‐derived nanovesicles and edible plant‐derived nanovesicles) to describe nano‐ and micro‐sized vesicles (50–1000 nm) isolated from plants (Li et al. 2025). To ensure clarity, we adopt plant‐derived extracellular vesicle (PDVs) as the standard nomenclature for this review.

2. General Features of PDVs

PDVs typically range from 30 to 500 nm, although EVs isolated from carrots can reach sizes of 1500 nm PDVs have been isolated from a wide array of fruits and vegetables (Kim, Li, et al. 2022). These include ginger, tomato, cabbage, Momordica charantia, garlic, carrot, Solanum nigrum, ginseng and broccoli (Nueraihemaiti et al. 2025). Similar to MDEVs, PDVs have shown inherent anticancer and anti‐inflammatory (Shkryl et al. 2022). Proteomic analysis has revealed that PDVs are rich in enzymes vital for cell (de la Canal and Pinedo 2018; Rutter and Innes 2018). Furthermore, PDEVs contain sRNAs, suggesting a role in gene regulation that may influence cell proliferation and differentiation (Urzì et al. 2021). These intrinsic therapeutic characteristics are readily translatable to human health, as such effects have proven beneficial with no reported adverse effects observed to date (Alzahrani et al. 2023). PDEVs demonstrate excellent biocompatibility and remain stable in the human body, even when administered orally PDEVs possess the ability to function as innovative, biologically derived drug‐delivery systems, capable of encapsulating and transporting both hydrophilic and hydrophobic compounds to target sites. Despite their considerable promise, PDVs face major challenges, including issues related to scalability, efficiency and lack of standardized isolation and preparation methods leading to inconsistent results (Cui et al. 2020). Tackling these issues will require advances in isolation techniques, the formulation of comprehensive regulatory standards, and the establishment of scalable manufacturing processes.

3. Biochemical Composition of PDVs

PDVs could also be rearranged to emphasize their role as carriers of a diverse array of biomolecules that influence their pharmacokinetics and pharmacological functions. PDVs have been found to contain a broad spectrum of cargo, including proteins, small RNAs, lipids and nucleic acids. Among the primary lipid components of PDVs are phosphatidic acid, phosphatidylcholine, digalactosyl diacylglycerol, monogalactosyldiacylglycerol and phytosterols (Kilasoniya et al. 2023). Notably, PDVs have been shown to transport specific small RNAs capable of modulating various biological processes, including inter‐kingdom communication. For example, Arabidopsis thaliana‐derived vesicles contain small RNAs such as tiny RNAs (tyRNAs, 10–17 nucleotides in length), long non‐coding RNAs (lncRNAs), circular RNAs (circRNAs), and other regulatory sRNAs involved in cross‐species signalling (Kilasoniya et al. 2023). Furthermore, PDVs carry lipids that are particularly relevant in preserving their integrity and shielding their cargo from degradation during gastric digestion (Kilasoniya et al. 2023). For instance, phosphatidic acid (PA) is vital for membrane fission and fusion processes likely influenced by cytoskeletal rearrangements (Rome 2019) and contributes to the retention of EVs within the intestinal tract (Suharta et al. 2021). PA plays a key role in the formation of EVs by shaping the membrane and recruiting specific proteins from the cell. Phosphatidylcholine (PC), by contrast, mediates the transport of PDVs from the intestine to the liver (Nemati et al. 2022). Polyunsaturated lipids, such as phosphatidylethanolamine (PE) and PC enhance membrane flexibility, which is crucial for endocytosis, because this process relies on membrane deformability (Nemati et al. 2022). Cholesterol, a stabilizing component found in mammalian exosomes and liposomes, is notably absent in PDVs (Yang et al. 2018). Glycolipids, including digalactosyldiacylglycerol and monogalactosyldiacylglycerol, are essential in maintaining membrane stability, particularly during lyophilization (Wang et al. 2025). However, the lipid composition varies across plant sources; for example, ginger‐derived EVs (GELNs) are rich in PA and digalactosyldiacylglycerol, whereas grapefruit EVs (GDEVs) predominantly contain PE, PC and phosphatidylinositol (Fan et al. 2022). Additionally, the lipids of PDVs allow interaction with the intestinal microbiota, influencing bacterial composition and distribution (Teng et al. 2018). GELNs rich in PA are more readily taken up by Lactobacillus rhamnosus, promoting its growth in the gut (Yang et al. 2018). Proteins carried by PDVs, primarily of cytosolic origin, vary among species. Proteins commonly shared across multiple species include channel proteins, such as aquaporins and chloride channels, as well as cytosolic proteins like proteolytic enzymes and actin (Yáñez‐Mó et al. 2015). Overall, PDVs exhibit a lower protein content than MDEVs, except for lemon‐derived EVs (LDEVs), which display a higher protein concentration and share 56.7% similarity with MDEVs (Yang et al. 2018). The function of these proteins may be modulated by environmental factors, including pathogen exposure or plant stress. Surface proteins, such as tetraspanins (TET) and integrins, play a crucial role in cellular adhesion and cargo transfer, as evidenced by the reduced uptake in liver cancer cells after the depletion of these surface proteins from EVs (Kumar et al. 2024). Plants are also rich in secondary metabolites with therapeutic potential. For example, GELNs contain 6‐gingerol and 6‐shogaol, which are renowned for their antioxidant, anti‐inflammatory and antitumour properties (Zhao et al. 2024). PDVs derived from lemon and strawberry contain vitamin C, whereas those from grapefruit are abundant in naringenin, both of which exhibit antioxidant activity (Fawzy et al. 2025). Secondary plant metabolites may enhance the efficacy of PDVs role as a drug delivery platform. A meta‐analysis on the application of PDVs in cancer therapy has highlighted their non‐toxic and safe nature when administered orally, further supporting their potential as drug delivery systems (Fawzy et al. 2025) (Figure 1).

FIGURE 1.

Overview structure and generalized composition of PDVs, which include both lipid‐bilayer–enclosed vesicles and non‐vesicular particles such as lipid–protein complexes and nanostructured biomolecular aggregates. Surface‐associated proteins support adhesion and molecular transfer, while the lipid membrane and matrix components contribute to structural integrity and stability. PDVs carry proteins, non‐coding RNAs, lipids and secondary metabolites acting as mediators of intercellular communication and as natural carriers for therapeutic molecules, underlying their antioxidant, anti‐inflammatory and anticancer activities.

4. Comparative Features of Plant‐ and Mammalian‐Derived Vesicles

MDEVs and PDVs display distinct proteomic profiles, lipid profiles and structural characteristics. PDVs frequently contain plant‐specific proteins, including those associated with chloroplasts and cell walls, whereas MDEVs are typically enriched in proteins involved in cellular signalling and membrane trafficking (Kim et al. 2023). Unique PDVs molecules have been identified and are summarized in Table 1. Among them, Penetration 1 (PEN1), a membrane‐associated protein critical for plant immune responses that regulates callose deposition during pathogen attack, has been identified as a biomarker of PDVs (de la Canal and Pinedo 2018). Interestingly, PEN1 does not co‐localize with the Rab5‐type GTPase plant‐unique Rab GTPase (ARA6) marker, suggesting that PEN1‐positive and TET8‐positive PDVs follow distinct biogenetic pathways. Specific plant TET, TET8 and TET9 from Arabidopsis thaliana (AtTET8 and AtTET9), have been implicated in plant responses to fungal infections by Botrytis cinerea (Table 1). These TET have been observed to co‐localize with the multivesicular body (MVB) marker ARA6 both intracellularly and within EVs at the sites of fungal invasion (Ding et al. 2014). Plants send small RNAs in EVs to fungal pathogens to silence virulence genes (Cai et al. 2018). TET8 and TET9 are structurally similar to mammalian tetraspanin CD63, with which they share less than 30% homology (Cai et al. 2018).

TABLE 1.

Markers used for PDVs identification (Rome 2019; Pinedo et al. 2021; Rodríguez de Lope et al. 2024).

Category	Marker protein	Species/Source	Functions
Transmembrane proteins	PEN1 (SYP121)	Arabidopsis, Nicotiana benthamiana	Syntaxin is involved in vesicle trafficking; used as a marker for microvesicles
	TET8 (Tetraspanin‐8)	Arabidopsis, Citrus	Orthologue of mammalian CD63; enriched in exosomes; associated with multivesicular bodies (MVBs)
Cytosolic proteins	GAPDH	Arabidopsis, Nicotiana benthamiana, Sunflower	Enzyme involved in glycolysis; detected in EV proteomes; potential marker for cytosolic content
	HSP70, HSP90	Citrus, Sunflower, Tomato	Heat shock proteins; involved in protein folding; commonly found in EVs; potential markers for stress‐related proteins
	Actin	Arabidopsis, Ginger, Citrus	Cytoskeletal protein; involved in cell structure and motility; detected in EVs; potential marker for cytoskeletal content
Endosomal proteins	Rab GTPases (e.g., RabA2a, Rab7)	Arabidopsis, Citrus	Small GTPases involved in vesicle trafficking; detected in EVs; potential markers for endosomal origin
	Clathrin Heavy Chain	Arabidopsis, Citrus, Sunflower	Protein involved in clathrin‐mediated endocytosis; detected in EVs; potential marker for endocytic vesicles
Cell wall remodeling	Pectin Methylesterase	Pollen	Enzyme involved in cell wall modification; detected in EVs; potential marker for cell wall‐related proteins
	Patellins (PATL‐1, PATL‐2)	Arabidopsis, Citrus	Membrane‐trafficking proteins; involved in cytokinesis; detected in EVs; potential markers for membrane trafficking.
Aquaporins	Aquaporins (e.g., PIP1, PIP2)	Citrus, Broccoli, Grape	Water channel proteins; involved in water transport; detected in EVs; potential markers for membrane stability
Exocytosis complex	EXO70E2	Arabidopsis	Component of the exocytosis complex; detected in EVs; potential marker for exocytotic vesicles
ESCRT complex proteins	VPS4	Arabidopsis, Nicotiana benthamiana	Protein involved in vesicle scission; not detected in natural plant EVs; not a marker for plant EVs

Moreover, PDVs generally exhibit a different lipid composition, reflecting their unique cellular environments and physiological functions when compared to MDEVs (Sall et al. 2023). These differences not only indicate divergent biogenesis pathways but also suggest that the lipid makeup of PDVs contributes to their stability in extracellular environments, their selective uptake by recipient cells, and their involvement in plant‐specific signalling mechanisms. Additionally, the unique lipid profile of PDVs may play a role in facilitating their ability to cross species barriers, further highlighting their potential in therapeutic and agricultural applications (Yáñez‐Mó et al. 2015).

5. PDVs Isolation Methods

As previously emphasized, PDVs play a vital role in the cross‐kingdom transport of bioactive molecules, and their potential as novel therapeutic agents, as well as their involvement in plant defence mechanisms, has been well established. However, isolating pure and intact EVs from plants remains a considerable challenge. Various physical methods can be employed as sample pre‐treatment to disrupt the plant tissues and extract the juice from different plant parts before formal isolation. So far, studies have concentrated on purifying EVs from extracellular apoplastic fluids (e.g., after buffering infiltration‐centrifugation of leaves to recover the apoplastic content). The apoplast denotes the region outside the plasma membrane and includes the cell wall and the intercellular spaces (Lian et al. 2022). To obtain apoplastic washing fluid, prior to isolation, an infiltration solution is usually introduced into plant tissues (typically leaves or roots) by creating pressure differentials. Rutter and co‐author improved a method developed for isolating EVs from the apoplastic wash from Arabidopsis leaf tissue by using vacuum infiltration and ultra‐speed centrifugation (Rutter and Innes 2017). However, most published reports used other methods that involve the extraction of fruit juice, naturally contained within vacuoles in several cell layers in the pericarp, by either gently pressing the fruits/plants or harshly grinding them in a mixer/blender (Cui et al. 2020). Those methods are likely to result in tissue/cell rupture, that however, could increase intracellular contamination (Cui et al. 2020; Pérez‐Bermúdez et al. 2017; Zeng, Deng, et al. 2024). While the first, gentler technique might involve only slight contamination from cellular disruption due to mild operation conditions, the second probably will result in the recovery of artificial nanoparticles/microsomes as a result of disrupted cellular membranes, as well as native intracellular vesicles that are released when the cell is broken open (György et al. 2011).

It has been demonstrated that pre‐treating the plant juice before the isolation process significantly improves both the yield and purity of the resulting PDVs (Lian et al. 2022). Tris‐HCl is frequently added to juice from citrus plants to remove co‐purifying pectin. Once the plant juice has been obtained, PDVs isolation can be carried out (Lian et al. 2022).

After obtaining the juice, to recover PDVs, different isolation methods are currently available, including ultracentrifugation (UC), size‐exclusion chromatography (SEC), ultrafiltration (UF), polyethylene glycol (PEG) precipitation, density gradient centrifugation and tangential flow filtration (TFF). This plethora of employed methods (Table 2), better discussed in the next sections, underscores the relevance of reaching a consensus on standardized protocols in plant‐based EV research (Visan et al. 2022).

TABLE 2.

Different EV isolation methods: Advantages and disadvantages (Konoshenko et al. 2018; Zhao et al. 2021).

Isolation techniques	Advantages	Disadvantages	Yield and purity	Isolation time
Ultracentrifugation	Easy; Affordable; Excellent quality	The process is susceptible to contamination by protein aggregates, is time‐intensive, demands large sample volumes, depends on costly equipment, yields low recovery rates, and poses a high risk of structural damage to the vesicles	Limited yield and purity	More than 4 h
Density gradient ultracentrifugation	Superior purity; Exosome subpopulations are isolatable	Involves significant manual effort, results in higher material loss, and entails a complicated protocol	Limited yield and high purity	More than 16 h
Immunoaffinity technique	High specificity; Produces highly pure preparative samples	Expensive; Limited binding capacity; May miss vesicles lacking the selected surface antigen; Difficult to scale up large sample volumes	Low yield and high purity	4–8 h per sample
Tangential flow filtration	Enables simultaneous concentration and diafiltration with consistent parameters; High scale‐up	Operational complexity; Slower process and complex calculations	High yield and High purity	More than 1 h

5.1. Ultracentrifugation

Ultracentrifugation (UC) or differential ultracentrifugation is a widely adopted technique and defined as a ‘gold standard’ method for isolating EVs from cell culture supernatants and biological fluids (Williams et al. 2018). The procedure for isolating PDVs largely mirrors that are used for mammalian EVs. Differential centrifugation steps have been employed to isolate PDVs from Arabidopsis leaves apoplastic washing fluid (He, Cai, et al. 2021). After obtaining the apoplastic fluid, different centrifugation steps are adopted; it is usually centrifuged at 2000 × g for 30 min at 4°C to remove large cellular debris. The resulting supernatant is then passed through a 0.45 µm filter to eliminate larger vesicles, transferred to ultracentrifuge tubes, and further centrifuged at around 10,000 × g for 30 min at 4°C, usually carried out to remove remaining large vesicles. The PDVs fraction is subsequently isolated and pelleted by UC step at 100,000 × g for more than 1 h. To eliminate any potential protein aggregates, the pellet is generally washed with PBS and subjected to a second round of UC step at 100,000 × g for 1 h at refrigerated temperature (Huang et al. 2021).

Despite the UC is a widely used method for isolating EVs it has several drawbacks (Table 2), among the lack of standardization protocol and repeatability, the results of UC isolation are often highly dependent on the operator's skill, particularly during the critical steps of pellet resuspension and supernatant aspiration, further, the high risk of structural damage (loss, fusion and distortion) to the vesicles due to the high shear forces generated during high‐speed centrifugation, potentially affecting their biological activity and integrity (Konoshenko et al. 2018). The lack of standardization and poor scalability of UC are significant limitations for clinical and industrial applications, driving the need for more robust and reproducible isolation methods.

5.2. Density Gradient Ultracentrifugation

Although centrifugation is widely used for isolating PDVs with acceptable levels of purity, certain applications may require additional purification steps because of EVs heterogeneity. This heterogeneity arises because different EV types exhibit overlapping sedimentation characteristics, often leading to mixed populations in the UC pellets (He et al. 2023). A commonly employed method for further resolving this heterogeneity is density gradient fractionation, which separates vesicles based on their flotation rates and equilibrium densities. This technique is typically performed using sucrose or iodixanol (OptiPrep) gradients applied to EV pellets obtained from differential centrifugation (Pinedo et al. 2021). Sucrose gradient centrifugation has been used to purify PDVs from the P100 fraction, the pellet obtained after high‐speed UC (100,000 × g) and containing vesicles of 50–200 nm in diameter. In contrast, iodixanol gradients have proven effective in resolving EV subtypes from the P40 fraction (pellet obtained after centrifugation at 40,000 × g) containing vesicles ranging in size from approximately 30 nm to 5 µm in diameter (He et al. 2023). Since centrifugation at 100,000 × g (P100) results in a higher enrichment of PDVs compared to 40,000 × g (P40), iodixanol density gradient centrifugation has been specifically employed for further purification and density profiling of P100‐derived EVs using a top‐loading approach. Using a TET8 antibody in combination with transmission electron microscopy (TEM), it was observed that the majority of TET8‐positive EVs were concentrated in the third fraction of the gradient, exhibiting an average density of 1.08 g/mL in iodixanol (Huang et al. 2021). This density is notably comparable to that of small EVs in animal systems (Yang et al. 2023).

Despite the interesting purification approach of the density gradient fractionation some drawback need to be highlighted such as protocol variations due to differences in the type of gradient medium (e.g., iodixanol, sucrose), gradient concentration, sample loading method (top vs. bottom loading), contamination with non‐EV particles such as protein aggregates, lipoproteins and other nanoparticles which can have buoyant densities that overlap with EVs or co‐precipitate during high‐speed spins.

5.3. Immunoaffinity Technique

Immunoaffinity capture‐based isolation is regarded as the most advanced technique for purifying specific EV subtypes (Kowal et al. 2016). This method employs antibodies that selectively bind to EVs expressing surface protein markers (Huang et al. 2021). Tetraspanins (TETs), such as CD81 and CD63, are frequently used for immunocapture because of their high abundance on EV membranes (Yáñez‐Mó et al. 2015). A similar approach has been developed for plants, using antibody‐coated beads targeting the PDVs marker TET8 (He, Cai, et al. 2021). For immunoaffinity capture to succeed, the antibody must recognize a region of the protein marker that is exposed on the surface of the EV. A TET8 antibody was specifically designed to bind the large extracellular loop of the transmembrane domains (EC2) of the protein, enabling efficient isolation of TET8‐positive EVs from the P100 fraction (He, Cai, et al. 2021). This method provides a straightforward and effective strategy for isolating specific EV subtypes in plants. For example, TET8‐positive EVs isolated via this technique have been shown to carry EV‐enriched small RNAs and RNA‐binding proteins, including Argonaute 1, RNA helicases RH11 and RH37, and annexins ANN1 and ANN2 (Liu and Chen 2018).

The immunoaffinity capture represents an ideal method for the targeted analysis of cargo content in defined EV subpopulations, the technique has a low yield and introduces a selection bias, isolating only EVs with the target marker, and thus providing an incomplete picture of the entire population. Furthermore, the costs are high due to the need for high‐quality specific antibodies, limiting scalability and making the method suitable for small research volumes but not for large‐scale production.

5.4. Tangential Flow Filtration

Tangential Flow Filtration (TFF) is a membrane‐based technique used to concentrate and purify EVs by allowing continuous flow across the filter surface, reducing clogging and preserving vesicle integrity. TFF offers a scalable, reproducible and low‐contamination alternative to conventional ultracentrifugation for EV isolation. Unlike UC, TFF efficiently concentrates vesicles with higher yield, reduced co‐isolation of macromolecules or aggregates, and greater batch‐to‐batch consistency, thereby addressing key issues of reproducibility, purity and standardization in EV preparation (Busatto et al. 2018; Kawai‐Harada et al. 2024).

The initial step involves filtration through sterile hollow fibre polyether sulfone membranes with a pore size of 0.65 µm, effectively removing cellular debris (Chen et al. 2024). After this pre‐filtration, the samples undergo a second filtration step using membranes with a 500 kDa molecular weight cut‐off, which serves to eliminate free biomolecules. To ensure the purity of the filtration process, all membranes are pre‐rinsed with sterile phosphate‐buffered saline (PBS, pH 7.4) at a volume three times greater than that of the sample (Sukreet et al. 2021). To protect the integrity of the vesicles and minimize shear stress, the input flow rate is carefully controlled at 80 mL/min, maintaining a shear force below 2000 s. After filtration, EVs are concentrated to approximately 50 mL and can be subjected to six rounds of diafiltration using a sucrose buffer composed of 5% sucrose, 50 mM Tris, and 2 mM MgCl₂. This process ensures the removal of contaminants and buffer exchange. Finally, the purified EVs in the sucrose buffer are further concentrated to a final volume of 6–9 mL, making them suitable for downstream analyses (Visan et al. 2022).

Besides the advantages of TFF, the system presents several drawbacks. Although the protocols specify controlled flow rates to minimize shear stress, if flow rates are inadequately controlled, they can induce damaging forces or aggregation. Membrane‐related issues, such as fouling (clogging), remain a challenge affecting efficiency, and non‐specific adsorption to membrane materials (e.g., polyether sulfone—PES) can lead to EV loss. The method also requires optimization in terms of pore size, flow rate and pressure for a specific sample type, as a ‘one‐size‐fits‐all’ protocol is difficult to achieve universally. Furthermore, TFF necessitates specialized equipment and technical knowledge for proper operation and maintenance.

6. Characterization of PDVs

The characterization of PDVs is essential for understanding their structure, composition and biological functionality. Despite the characterization of mammalian‐EVs being well standardized to date, there is a lack of standardization protocols in this scientific area (Welsh et al. 2024). A rigorous characterization complying with recent MISEV guidelines has not been yet implemented due to the lack of bona fide reference markers because their composition and properties can vary significantly depending on the plant source, extraction method and growth conditions (Rodríguez de Lope et al. 2025)of their diverse origins and complex composition, numerous characterization methods have been developed to deepen our understanding of PDVs, their biological roles and to explore their potential in therapeutic, agricultural, food and nutraceutical applications (Liu et al. 2024).

6.1. Particle Size Measurement

The particle size and surface potential of EVs are generally assessed by Dynamic Light Scattering (DLS) and Nanoparticle Tracking Analysis (NTA) techniques (Chen et al. 2024). Vesicles derived from different sources display a wide range of particle sizes and are typically characterized by a negative zeta potential such as GELNs (100–500 nm, −10 to −30 mV (Man et al. 2021), LDVS (50–150 nm, −15 to −30 mV (Kunhi Mahin 2021), GDVS (50–200 nm, −20 to −30 mV (Rome 2019) and broccoli derived vesicles (BDVS) (∼100 nm, −10 to −25 mV) (Hossain et al. 2022). DLS determines the average particle size by monitoring fluctuations in light intensity caused by EV Brownian motion (Kruse et al. 2022). In contrast, NTA enables the visualization and tracking of individual EVs, providing detailed data on both particle concentration and size, thereby offering an accurate representation of their physical state. This technique is particularly well‐suited for analyzing particle size and concentration in complex samples (Bachurski et al. 2019), although precise measurement requires the sample to fall within a defined concentration range. Furthermore, NTA fluorescence mode facilitates the identification of specific EVs phenotypes by reducing background interference (Comfort et al. 2021).

6.2. PDVs Morphology

The morphology of EVs can be assessed using electron microscopy techniques, including scanning electron microscopy (SEM), transmission electron microscopy (TEM), and cryo‐electron microscopy (cryo‐EM) (Chuo et al. 2018). SEM provides detailed information on the surface structure of EVs, whereas TEM offers insight into their internal architecture and morphology (Chuo et al. 2018). However, the dehydration processes required during TEM sample preparation could lead to structural shrinkage and morphological artefacts. Despite this limitation, TEM remains widely used because of its capacity to deliver high‐resolution images at the nanoscale. In contrast, cryo‐EM allows visualization of EVs morphology under cryogenic conditions, effectively preserving structural integrity by avoiding dehydration‐induced distortions (Doerr 2022). Atomic Force Microscopy (AFM) is a powerful technique used to characterize PDVs size, three‐dimensional shape and surface properties (Farina and Hwang 2020). A newer AFM mode, called frequency‐modulation AFM, measures how the tip interacts with the sample surface, helping detect local stiffness, friction and adhesion (Skliar and Chernyshev 2019).

6.3. Marker Detection

Surface proteins act as markers and play a crucial role in the identification of both EVs and PDVs. Despite a constant and exponential growth of PDVs studies, to date, there is a lack of standardization, mainly related to rigorous characterization complying with MISEV guidelines due to the lack of bona fide reference markers, which instead are fully identified for EVs from mammalian cell origin (Rodríguez de Lope et al. 2025). Commonly used markers for mammalian‐EVs include CD9, CD63, CD81 and TSG101 (Welsh et al. 2024). However, such markers are quite difficult to use for PDVs for which plant‐unique homologues, beside to TET8, PEN1, germin‐like proteins and calreticulins, also aquaporins, vacuolar‐typeATPase complex subunits, fasciclin‐like arabinogalactan proteins FLA 10 and FLA13, syntaxins can be used for the characterization and monitoring (Rodríguez de Lope et al. 2025). Variables such as growth conditions and environmental factors influence the specific protein composition of PDVs. Proteomic analyses have highlighted the relevance of these protein profiles, particularly in medicinal plants (Barile et al. 2017). Several protein families have been identified in PDVs, some of which overlap with those found in MDEVs. These include proteins involved in hydrolysis, biosynthesis, membrane binding, network‐structure formation and small GTPase activity. In addition, plant‐specific proteins, such as those associated with chloroplasts and cell walls, have been detected. Despite the diversity of proteins identified in PDVs, as previously observed, the characterization of distinct protein markers remains an active area of research (Zhou et al. 2020; Rodríguez de Lope et al. 2025).

Different techniques can be used to analyze the PDVs phenotype, among them, TEM coupled with immunogold labelling, allows the detection and localization of proteins on the surface of EVs (Corona et al. 2023), while Western blotting (WB) enables the detection of specific proteins within PDVs preparations, providing insight into their expression profiles (Kowal et al. 2017). Interestingly, super‐resolution techniques such as direct Stochastic Optical Reconstruction Microscopy (dSTORM) have been recently implemented in EV studies. These systems allow profiling of individual EVs based on surface marker expression, most notably TET, such as CD9 and CD81. However, detection sensitivity can be affected by antibody aggregation and background fluorescence, necessitating careful sample preparation (Shao et al. 2018). Notably, these techniques present certain limitations: while TEM and dSTORM do not allow for statistically robust analyses, WB is not suitable for investigating complex samples. Flow cytometry is emerging as a powerful and robust single‐vesicle methodology, theoretically allowing in‐depth characterization of complex samples in a short time.

7. Flow Cytometry as a New Technique for EV Identification, Subtyping, Size Measurement and Separation

Flow cytometry (FCM) is a robust and powerful technique allowing multiplex analyses at a single event level (Théry et al. 2018; Welsh et al. 2024; Lee et al. 2008; Welsh et al. 2023). Flow cytometry is now extensively employed for the characterization of MDEVs, yet its application to PDVs remains limited. Nonetheless, the fundamental principles useful for the detection and quantification of animal EVs by FCM are equally applicable to PDVs. It must be underlined that the main FCM challenges in EV characterization include their small size. However, recent advances in nanotechnology have improved the sensitivity and resolution of FCM, enabling more accurate analysis of small particles and providing deeper insights into EV heterogeneity across sources such as animal, plant and microbial systems. FCM is a widely used method for high‐throughput, multiparametric analysis and quantification of EVs (Lanuti et al. 2025). It works by detecting light scatter and fluorescence signals as individual particles pass through a laser beam, providing information on morphological EVs features (side and forward scatter measurements) (Welsh et al. 2024). A key advantage of this technique is its ability to simultaneously assess multiple markers on single EVs(de Sousa et al. 2023). FCM provides several key advantages for PDVs characterization being a high‐throughput method, FCM enables rapid analysis of thousands of particles per second, producing large datasets that may reveal the size and heterogeneity of PDVs populations, and allows accurate quantification of their concentration by using dedicated volumetric counting devices or beads of known concentration as an internal standard. Furthermore, flow cytometry allows simultaneous multiparametric detection of different surface/intraluminal proteins, lipids and nucleic acids, for phenotypical and functional subtyping of PDVs (Ward et al. 2023)

7.1. Conventional Flow Cytometry Platforms: Strengths and Limitations

Conventional flow cytometry offers a highly versatile platform for the study of PDVs, providing rapid, quantitative and high‐throughput single‐particle analysis. However, accurate and reproducible analysis of PDVs by FCM requires careful optimization and standardization of measurement parameters, including instrument calibration, threshold settings and sample preparation protocols. Quantitative sizing and concentration measurements of EVs by FCM rely heavily on calibration strategies. Traditionally, calibration beads made of polystyrene or silica with well‐defined diameters and refractive indices have been employed to correlate light scatter with particle size. However, this approach is fraught with complications, as EVs differ substantially from these synthetic particles in optical properties and internal composition. The refractive index of EVs, which typically lies between 1.37 and 1.42, is significantly lower than that of polystyrene (1.59) or silica (1.43–1.47), resulting in markedly reduced light scattering for vesicles of comparable diameter (Welsh et al. 2023). Consequently, the direct use of bead‐based calibration often leads to an underestimation of EV size and poor comparability between instruments. A well‐documented example demonstrates that a gate defined by 200‐ and 400‐nm polystyrene beads encompassed EVs between 300 and 800 nm on one cytometer, whereas the same gate corresponded to EVs between 800 and 1900 nm on another platform, thus revealing how the same calibration reference may yield incompatible results across instruments (Welsh et al. 2023). To address these discrepancies, the use of model‐based calibration has been proposed. By applying Mie theory, light scatter signals can be converted into diameter estimates that consider the refractive index of the particle, the wavelength of the laser, and the optical configuration of the instrument. In a multi‐institutional study involving 46 FCM, van der Pol and colleagues demonstrated that the implementation of scatter‐based Mie modelling improved inter‐laboratory reproducibility of platelet‐derived EV measurements, reducing the coefficient of variation from 139% (when using bead‐based gates) to 81%. The same study revealed that approximately one quarter of instruments were unable to detect 400‐nm polystyrene beads, underscoring the heterogeneity of detection limits among commercial cytometers (van der Pol, van Gemert, et al. 2018). Fluorescence calibration, often performed using beads standardized in molecules of equivalent soluble fluorochrome (MESF), provides an alternative means of quantifying molecular abundance per vesicle. Nevertheless, conventional MESF beads were designed for cellular fluorescence ranges and are usually several orders of magnitude brighter than labelled EVs, which imposes the need for extrapolation into low‐intensity regions and increases uncertainty (Welsh et al. 2023). Recent developments, including low‐MESF calibrators and the use of Rosetta calibration software, have improved reproducibility and linearity, yielding coefficients of variation below 20% for mesenchymal stromal cell‐derived EVs samples (Lucchetti et al. 2023). The combined evidence indicates that accurate sizing and quantification of EVs require calibration procedures that reflect the optical and physical properties of biological vesicles, rather than relying solely on synthetic bead standards (Lucchetti et al. 2023). Importantly, according to Welsh et al., critical assay controls for PDVs FCM must include buffer‑only controls to assess background noise and instrument cleanliness, buffer‑plus‑reagent controls to quantify signal from unbound dyes or antibodies, unstained PDV samples to define autofluorescence, and detergent‐treated controls to verify that detected events are membrane‑enclosed vesicles rather than detergent‐resistant particles (Welsh et al. 2024). Fluorescence minus one (FMO) control should also be included whenever acquiring a multicolour panel to accurately set gating boundaries (Marchisio et al. 2020). Finally, it must also be considered that the height signal should be used in the area, since it more reliably reflects transient signals from extracellular vesicles than area measurements and helps mitigate issues related to background noise and coincidence (Welsh et al. 2023).

Importantly, one of the main limitations regarding EV analysis is the lower and upper Limit of Detection (LoD) of the instrument used, representing the largest and smallest signal that can be detected in the case of PDV analysis by FCM, the main issue concerns the lower LoD. It is determined by calibrating the instrument either in terms of molecules of MESF units, using fluorescence reference beads, or in terms of particle size, using scatter‐based sizing standards (such as the Rosetta Calibration system). The LoD does not represent a uniquely defined lower threshold, but rather a complex function influenced by background signal and detector efficiency (Welsh et al. 2023). In conventional FCM, the LoD generally falls within a broad range; as discussed in the Compendium of Single Extracellular Vesicle Flow Cytometry by Welsh et al. (2023), theoretical LoD values reported for different instruments span approximately 110–300 nm in diameter (Neves et al. 2024; Marchisio et al. 2020; van der Pol, van Gemert, et al. 2018). Another critical point is the choice of triggering strategies that significantly influence data reliability. Because most EVs scatter light below the detection threshold of conventional FCM, fluorescence‐based triggering, typically through membrane dyes or labelled antibodies, offers greater specificity and improves discrimination of EVs from background noise and non‐vesicular debris (Lucchetti et al. 2020; Libregts et al. 2018). Recently, it has been demonstrated that the use of a fluorescence‐based trigger such as the PE channel detecting CD90, a surface antigen highly expressed on mesenchymal stem cells (MSCs) and their derived EVs significantly enhances conventional flow cytometry sensitivity, especially for small EVs (100–200 nm) that are otherwise lost when triggering on SSC (Simeone, Celia, et al. 2020). This technical adjustment has been extended to the entire EV compartment analysis by using pan‐EV dyes that improve the resolution of conventional flow cytometers (Nolan and Duggan 2018).

To overcome scatter sensitivity limitations, FCM microspheres can also be used to detect EV surface proteins. Large microspheres capture EVs regardless of their surface composition, or antibody‐conjugated microspheres can capture EVs expressing a specific antigen (Welsh et al. 2024). However, this method also has limitations. It precludes the analysis at the single‐vesicle level, and, for PDVs, it is particularly challenging to identify specific markers for their selective capture (Bokun et al. 2025; van der Pol et al. 2012). Another important limitation when conventional FCM is used to study PDVs and EVs in general is the occurrence of the swarming phenomenon. Swarming, a specific form of coincidence detection, occurs when multiple small particles, such as EVs, are simultaneously present within the laser interrogation point and are therefore detected as a single event. Typically, swarming manifests as a deviation from linearity between sample dilution and event rate, along with an increase in median fluorescence or scatter intensity (van der Pol et al. 2012; Welsh et al. 2017b; Kuiper et al. 2021). It is, therefore, recognized as a major source of artefacts in EV FCM. Extending flow cytometry into this small‐particle domain, therefore, requires substantial optimization and rigorous validation of measurement and calibration strategies (Shahrokhian and Salimian 2018). To minimize swarm detection, the literature consistently recommends reducing particle concentration through appropriate dilution, using low flow rates, generally between 3 and 12 µL per minute, selecting smaller flow core sizes, preventing particle aggregation through filtration or dispersing agents, and routinely verifying linearity between event rate and concentration to ensure accurate single‐particle detection (van der Pol et al. 2012).

7.2. Alternatives to Conventional Flow Cytometry for the Study of PDVs

To overcome the limitations of conventional FCM, dedicated instruments specifically designed for EVs analysis, so‐called nano‐flow cytometers, have been recently developed. Imaging flow cytometry has also been successfully employed to improve single‐particle detection and characterization.

7.2.1. Nano‐Flow Cytometers

To overcome conventional FCM limitations for EV analyses, new nano‐ FCM platforms have been developed, being able to enhance resolution and sensitivity for high‐precision, single‐EV profiling, although they often require more stringent sample preparation. Together, these complementary technologies form a powerful analytical framework that advances our ability to explore PDV biology and translate their potential into diagnostic and therapeutic innovations (Botha et al. 2021). A range of cytometers, optimized for small‐particle detection, offers complementary capabilities in terms of sensitivity, detection range and optical configurations (López de las Hazas et al. 2023). Among them, the CytoFlex Nano (Beckman Coulter) allows users to analyze EVs from 40 nm to 1 µm, simultaneously analyzing five side‐scatter channels, theoretically improving the sensitivity and resolution of the instrument (Kim et al. 2024). The Delaware Flow Nano Cytometer (Kinetic River) is a five‐laser instrument, equipped with high‐sensitivity collection lenses. It offers up to three scatters and six fluorescence channels. The Shasta fluidics system ensures stable flow, while the Cavour monitor allows for real‐time laser alignment. The FlowNanoAnalyzer (nanofcm) can be used for the multiparameter characterization of natural and synthetic nanoparticles in the range 7–1000 nm at the single‐particle level (Choi et al. 2019). The FACSymphony A1 Cell Analyzer (Becton Dickinson) can be equipped with a Small Particle Detector, an optional side scatter (SP SSC) module designed to detect particles as small as 90 nm. It enhances sensitivity and reduces background noise through low‐noise electronics, high‐power lasers and a tight beam spot (Kim et al. 2024).

7.2.2. Imaging Flow Cytometry

Imaging FCM, by means ImagestreamX MKII (ISX), from EMD Millipore, FACSDiscovery S8 and FACSDiscovery S6 from BD Biosciences, combines all the benefits of conventional flow cytometry, such as high‐throughput analysis of scatter and multiple fluorescence parameters, with the added capability of capturing images of each measured particle or cell (Stoner et al. 2016). Thanks to its highly sensitivity, imaging flow cytometry combines the quantitative capabilities of conventional flow cytometry with high‐resolution imaging, making it particularly valuable for the study of PDVs by enabling the precise detection and visualization of individual PDVs as small as 100 nm, distinguishing them from non‐vesicular particles or debris, and allowing detailed phenotypic profiling through fluorescent labeling of membrane lipids and plant‐specific surface markers such as tetraspanins or ankyrin‐repeat proteins. Imaging flow cytometry also facilitates the real‐time tracking of vesicle cargo uptake (e.g., proteins, RNA) into recipient cells to investigate their roles in intercellular signalling and cross‐kingdom communication. While imaging flow cytometry has been extensively applied in MDEVs research, its use for PDVs studies remains limited, presenting a promising opportunity for future applications aimed at exploring PDVs heterogeneity, function, and therapeutic applications through standardized, image‐verified analysis (Woud et al. 2022). In more detail, in place of photomultiplier tubes (PMTs), the ISX employs charge‐coupled device (CCD) cameras for signal detection. CCDs offer a wider dynamic range, lower electronic noise, and greater quantum efficiency, making them ideal for detecting low‐intensity signals. The ISX also utilizes Time Delay Integration (TDI), where pixel intensities on the CCD are accumulated over longer integration times (milliseconds rather than microseconds). This prolonged exposure, together with slower flow rates, greatly improves sensitivity without introducing additional readout noise (Camacho et al. 2017). Additionally, TDI data collection provides a continuous 100% duty cycle, ensuring that every particle passing through the imaging region is captured without relying on a separate trigger. This design eliminates much of the noise and inefficiency common in traditional FCM and enhances the system's overall sensitivity for detecting small and dimly fluorescent particles (Zhu et al. 2014).

On the other hand, FACSDiscover A8 Cell Analyzer is a powerful, all‐in‐one system that combines cutting‐edge spectral flow cytometry and real‐time imaging through the integration of BD SpectralFX Technology and BD CellView Image Technology, offering up to 78 fluorescence detectors and eight scatter/imaging detectors, and a streamlined workflow designed to reduce setup complexity and produce reliable, reproducible single‐cell data previously unattainable with conventional systems (Shi et al. 2024). FACSDiscover S8 Cell Sorter, equipped with BD CellView Image Technology and BD SpectralFX Technology, is the first‐of‐its‐kind spectral flow cytometer with sort‐capable image analysis, combining high‐resolution real‐time imaging with advanced spectral detection to provide simultaneous spatial, morphological and phenotypic insights during sorting, allowing the isolation of rare or previously unresolvable cell types by correlating imaging data with conventional flow cytometry and downstream analytical results (Paulsen 2024).

7.3. Fluorescent‐Activated Cell Sorting for EV Isolation

Some advanced cytometers, called fluorescence‐activated cell sorters, allow the physical sorting of PDVs into defined subpopulations for further molecular analyses (van der Pol, Sturk, et al. 2018).

It has been demonstrated that by using lipophilic dyes (e.g., LCD), viability markers such as phalloidin, and panels of fluorescent labelled antibodies or plant‐specific probes targeting EV‐associated proteins (such as TET or membrane ankyrin‐repeat proteins), precise discrimination of EVs from background particles and non‐vesicular noise can be achieved by conventional flow cytometers. Furthermore, coupling this method with fluorescence‐activated cell sorting enables the selective enrichment and isolation of distinct EVs subpopulations, thereby facilitating downstream investigations into their functional roles in plant‐to‐plant communication, host–microbe interactions, and cross‐kingdom molecular signalling (Xu et al. 2024; Welsh et al. 2023). Sorting techniques, such as flow cytometry and immunoaffinity‐based methods, offer substantial advantages over conventional EVs isolation approaches particularly when applied to PDVs by enabling the highly specific and pure isolation of distinct EVs subpopulations based on surface markers such asTET, phosphatidylserine or unique plant membrane proteins (e.g., ankyrin repeats), which is not possible with traditional methods like ultracentrifugation or polymer‐based precipitation that often co‐isolate proteins, lipoproteins or cell debris (Zhao et al. 2021). These sorting methods preserve the structural integrity and biological functionality of PDVs by minimizing shear stress and avoiding harsh processing conditions, which is critical for maintaining their therapeutic potential, such as the anti‐inflammatory properties observed in ginger‐derived or grapefruit‐derived EVs. Moreover, sorting allows for high‐throughput and scalable processing, especially with advancements in microfluidic flow cytometry enabling efficient screening, quality control and batch analysis in both research and clinical contexts (Clos‐Sansalvador et al. 2022). This precision also facilitates the development of targeted therapies, as sorted PDVs can be engineered or selected to carry specific RNA, protein or metabolite cargo for delivery to inflamed or diseased tissues, while also supporting biomarker discovery by isolating EVs associated with specific physiological conditions. Finally, the compatibility of sorting methods with downstream assays such as proteomics, transcriptomics and functional cell‐based studies makes them highly versatile, although their application requires careful marker selection, technical expertise and comes with higher costs and complexity compared to traditional EV isolation techniques (Chiang and Chen 2019).

7.4. Technical Limitations and Platform Comparison in FCM

Technical limitations and platform variability remain major challenges in flow cytometry, influencing the accuracy, sensitivity and reproducibility of EVs and PDV measurements.

7.4.1. Inter‐Platform Variability and the Need for Standardization

Instrumental variability remains a major obstacle to the comparability of EV flow cytometry data. Even cytometers of identical models and manufacturers may differ significantly in light scatter sensitivity, fluorescence detection efficiency, and flow rate calibration. Studies coordinated within the METVES and METVES II initiatives have demonstrated that nominally identical instruments operating under the same flow rate setting may differ up to sixfold in their actual sample throughput (Kuiper et al. 2021). Such discrepancies critically affect the estimation of particle concentration and preclude reliable inter‐laboratory comparisons. Efforts toward standardization have therefore focused on establishing traceable calibration protocols and developing EV‐relevant reference materials. Flow rate calibration using volumetric methods or fluorescent counting beads is necessary for accurate concentration determination. Scatter calibration based on theoretical models, rather than bead diameter alone, allows for instrument‐specific correction of optical geometry and refractive index effects (van der Pol, van Gemert, et al. 2018). Fluorescence calibration, expressed in MESF units, must be reported together with information on calibration bead characteristics and intensity range to ensure interpretability across studies. Equally important is the comprehensive reporting of all acquisition parameters, including cytometer model, laser wavelength, optical configuration, triggering channel, threshold value and gating strategy, as well as verification of single‐particle detection through dilution linearity tests (Welsh et al. 2017a). The absence of such methodological transparency remains a major limitation in the current EV cytometry literature (Bohacova et al. 2024).

7.4.2. Methodological Considerations and Best Practices

The goal remains in the establishment of standardized, traceable and reproducible measurement frameworks that ensure cross‐platform and cross‐laboratory comparability. As emphasized in recent methodological studies (van der Pol, van Gemert, et al. 2018; Welsh et al. 2023), rigorous calibration, transparent reporting and interlaboratory validation will be essential prerequisites for transforming EV flow cytometry from a semi‐quantitative analytical approach into a reproducible and quantitative discipline capable of supporting clinical translation.

8. Cargo Characterization

PDVs play a critical role in cross‐kingdom sRNA transport, as demonstrated in the interaction between Arabidopsis and the fungal pathogen Botrytis cinerea. Arabidopsis releases vesicles that deliver host‐derived sRNAs into fungal cells, where these sRNAs suppress the expression of genes associated with virulence (Cai et al. 2018). Studying how sRNAs from host organisms affect pathogens can help identify important genes linked to disease. It has also been found that sRNAs generated from inserted genes (transgenes) can be delivered into fungi via EVs, showing promise for agricultural applications. A better understanding of the mechanism of actions of PDVs in this cross‐kingdom RNA transfer could lead to new methods of using artificial RNAs to protect crops during growth and post‐harvest (Cai et al. 2018). The P100 pellet, obtained during EV isolation, is especially useful for studying small RNAs inside EVs. To confirm that these sRNAs are inside the vesicles (and not just stuck to the outside), the P100 fraction can be treated with enzymes like nucleases or a mix of proteinase and nuclease. If the sRNAs are still present after this treatment but disappear when exposed to Triton X‐100, a detergent that disrupts EV membranes, it shows they were inside the vesicles. For example, sRNAs such as TAS1c‐siR483, TAS2‐siR453 and miRNA166 have been found inside EVs using this method (Huang et al. 2021). In addition to small RNAs, proteins and lipids, various metabolites have also been found in some PDVs. Plants are known to produce many types of metabolites that influence biological processes and human health. Recent research has identified several classes of these compounds, including carotenoids, flavonoids, saponins and glucosinolates. For example, GELNs contain active compounds like 6‐gingerol and 6‐shogaol, which are believed to contribute to their anti‐inflammatory effects (Edo et al. 2025). One study showed that 6‐shogaol in GELNs could activate the Nrf2 pathway via TLR4/TRIF signalling, helping to improve alcohol‐related liver damage in mice. Interestingly, orange juice‐derived EVs did not contain vitamin C or naringenin, two major bioactive compounds in orange juice. Instead, they were rich in sugars, amino acids, alcohols and other small molecules (Tinnirello 2025). Similarly, EVs from Catharanthus roseus (CLDENs) contained vinpocetine and other basic compounds but lacked key anti‐cancer agents like vincristine or vinblastine (Hao et al. 2024).

9. PDVs in Nanomedicine Applications

PDVs isolated from multiple edible species offer significant advantages due to their high biocompatibility, biodegradability and abundance, making them promising candidates for cell‐free therapeutic applications. Plants release different kinds of EVs that serve various functions, such as supporting cell structure, promoting growth, interacting with fungi, strengthening the plant's immune system and protecting against harmful microbes. These EVs can be extracted from many edible plants and are considered more natural, safe and widely available, making them a good option for cell‐free therapies. Studies in vitro and in vivo suggest that PDVs might offer advantages over traditional synthetic drug carriers, opening new directions for drug delivery. Researchers are currently working on using PDVs to carry drugs more effectively, but some obstacles still need to be overcome before they can be used in medical treatments (Xu et al. 2023). The structure and transport capacity of PDVs enhance their potential for drug delivery (Table 4), and the advantages of using PDVs over synthetic nanoparticles and mammalian‐derived EVs for drug delivery have been fully demonstrated (Dad et al. 2021). PDVs outperform artificially synthesized nanoparticles (e.g., liposomes) in terms of safety, low immunogenicity, improved cellular uptake, greater stability in the gastrointestinal tract and targeted delivery capabilities (Shinge et al. 2022). Moreover, PDVs offer the benefit of a simpler preparation method, whereas synthetic nanoparticles require more complex manufacturing processes, such as membrane extrusion or micro‐emulsification. Unlike synthetic nanoparticles, which are designed solely for drug delivery and lack intrinsic therapeutic properties (Witwer and Wolfram 2021), PDVs possess innate therapeutic, antitumour, regenerative and anti‐inflammatory capabilities (Figure 3). Furthermore, compared with EVs derived from mammalian cells or bacteria, PDVs exhibit advantages such as small size for deep‐tissue penetration, plasma‐membrane similarity and remarkable physicochemical stability under varying pH and temperature conditions (Lin et al. 2022). PDVs minimize the risks of unwanted gene or protein transfer and harmful immunogenic responses before clinical application. Additionally, PDVs can diffuse through the blood‐brain barrier (BBB) without triggering inflammatory responses or necrosis (Wang et al. 2022). PDVs are widely regarded as highly safe. As naturally occurring nanoparticles secreted by plants and already present in many foods consumed by humans, PDVs are well‐tolerated by the immune system and exhibit excellent biocompatibility (Anusha et al. 2022). Their non‐toxic nature has been confirmed in studies conducted on both animal and plant models (Umezu et al. 2021), with no reported impact on organ pathology, haemolytic activity or levels of cellular inflammatory markers (Sarvarian et al. 2022). Moreover, the encapsulation of therapeutic agents such as doxorubicin (DOX) within PDVs has been shown to reduce adverse effects while enhancing therapeutic outcomes (Cui et al. 2020).

TABLE 4.

Route of administration of PDVs, advantages, and disadvantages.

Administration route	The initial site	Advantages	Disadvantages
Intravenous injection	Mainly in the liver and spleen	Superior bioavailability compared to all delivery methods; No absorption or first‐pass metabolism barriers.	Repeated injections over a prolonged period should be avoided.
Oral administration	Mainly in the gastrointestinal tract	The first‐pass effect results in only 20%–30% bioavailability; improves patient compliance.	This leads to fluctuations and heightens side effects.
Intraperitoneal injection	Gastrointestinal tract and lymph nodes	Bioavailability of up to 80%, with a large absorption area and high capacity.	Extremely irritating; challenging to penetrate the abdominal wall.
Subcutaneous injection	Mainly in injection sites or lymph nodes	40% bioavailability, primarily used for localized delivery.	Limited to small doses of drugs with Strong pharmacological effects.

FIGURE 3.

The potential of vesicles derived from mammalian and plant cells. Their main aesthetic functions include anti‐scarring, anti‐ageing and anti‐pigmentation effects. Thanks to these properties, EVs are gaining interest in the field of skincare and cosmetic applications.

PDVs are obtained from widely accessible plant sources, offering a cost‐effective and scalable supply of raw materials. Compared to EVs derived from animal cells, PDVs substantially reduce the expenses associated with cell culture reagents, while also requiring less time and labour for large‐scale production (Alzahrani et al. 2023). The cost‐to‐yield ratio for PDVs is estimated to be approximately 300 times more favourable than that of EVs derived from animal cells (Cui et al. 2020; Wang, Xiong, et al. 2025). The lipid membranes of PDVs serve as a protective barrier for bioactive compounds, shielding them from external factors such as fluctuations in pH, heat and light, thereby contributing to their remarkable stability (Torres et al. 2021). PDVs exhibit an extended circulation time within the body, facilitating sustained drug activity and the gradual accumulation of therapeutic effects (Ly et al. 2023). Furthermore, PDVs demonstrate considerable resistance within the gastrointestinal environment, remaining stable and well‐tolerated. For instance, EVs derived from grapes have shown resistance to degradation by saliva, gastric acid and proteolytic enzymes, enabling them to cross the intestinal tract, penetrate the mucus layer, and be absorbed by intestinal stem cells in mice (Ju et al. 2013). Similarly, EVs derived from ginger have been shown to maintain high stability in simulated gastrointestinal fluids (Zeng, Liu, et al. 2024). Furthermore, EVs from grapefruit display exceptional stability under both acidic and simulated gastrointestinal conditions, affirming their strong resistance to digestive processes (Woith et al. 2019).

As natural delivery systems, PDVs inherently contain bioactive compounds from their source plants, conferring beneficial biological properties such as antioxidant, anti‐inflammatory and anticancer effects (Alzahrani et al. 2023). For instance, EVs derived from lemons have been shown to carry micronutrients such as vitamin C and citrate, offering antioxidant protection to human cells (Cui et al. 2020). BDVS, which contain sulforaphane, a bioactive compound present in certain cruciferous vegetables, have been found to prevent colitis in mice (Rutter and Innes 2017). Similarly, vesicles derived from ginger have been shown to transport 6‐gingerol and 6‐shogaol, two compounds known for their anti‐cancer, anti‐inflammatory and antioxidant activities, and have demonstrated notable hepatoprotective effects (Cui et al. 2020). In brain cancer therapy, grapefruit‐derived EVs have been engineered to deliver miR‐17, a microRNA with anti‐tumour properties. By coating EVs with folic acid and polyethylenimine and administering them intranasally, miR‐17 was efficiently delivered to the brain and selectively taken up by GL‐26 tumour cells. This delivery system suppressed MHC I expression in the cancer cells, leading to natural killer (NK) cell activation and enhanced tumour cell elimination (Zhuang et al. 2016). Lemon‐derived vesicles have also shown promise in overcoming drug resistance in ovarian cancer. Functionalization of these vesicles with heparin‐cRGD (HR) allowed for targeted delivery of doxorubicin. The treatment inhibited P‐glycoprotein (P‐gp) expression, reduced ATP production, and limited the energy available for drug efflux, thereby enhancing intracellular drug accumulation and reversing chemotherapy resistance (Xiao et al. 2022). Despite the promise of PDVs as natural carriers capable of crossing biological barriers, their clinical application is hindered by safety concerns. Key issues include the quality instability of plant sources and the complex composition of extracts, which can contain impurities linked to potential health risks. Indeed, the main limitations are linked not to bioactivity but to variability, scalability and quality control, challenges that arise from their biological origin and that require advanced standardization frameworks (Ferroni and Zavan 2025)

9.1. Nanotechnological Enhancements and Intrinsic Potential of PDVs

All these remarkable features, combined with the discovery of their intrinsic therapeutic properties, make PDVs ideal candidates for drug delivery applications (Shinge et al. 2022). As noted above, the use of PDVs represents a promising strategy in biomedicine for targeted delivery of drugs and bioactive molecules. One of the most studied techniques for enhancing the stability of EVs is encapsulation at both microscopic and nanoscopic levels. This involves enclosing the vesicles within protective matrices to improve their chemical stability. For example, micro‐ and nano‐encapsulation use biocompatible polymers or liposomes to create a ‘barrier’ around the vesicles, shielding them from harmful environmental conditions and thereby increasing their resistance and durability. In recent years, PDVs have been employed as carriers for transporting therapeutic molecules, including chemotherapeutic agents, siRNAs, DNA expression vectors and proteins (Dad et al. 2021). Many engineered EVs serve as carriers to transport siRNA and miRNA into the body, and stability and specificity are key requirements (Wei et al. 2023). Studies demonstrated that the stability of exosomes of mammalian origin loaded with siRNA will be reduced, because the modification of targeted substances will destroy the integrity of the exosome biofilm, resulting in extremely fast degradation of exosomes in the systemic circulation (El Andaloussi et al. 2013). This aspect has great significance to the purification technology of PDVs and the loading of target substances (Wei et al. 2023, Doerr 2022). These strategies include co‐incubation, electroporation, sonication and osmotic shock. Additionally, these techniques enable the controlled release of encapsulated bioactive compounds. Controlled and targeted release optimizes the bioavailability of active molecules, enhancing therapeutic efficacy (Klojdová et al. 2023). Some studies have shown that nanotechnology‐based controlled‐release systems can bypass physiological barriers, such as the gastrointestinal tract, ensuring that a larger quantity of EVs reaches the target tissues. The application of these nanotechnological techniques represents a significant advancement in the delivery of drugs and other functional molecules, paving the way for new forms of personalized and targeted therapy (Malik et al. 2023). Although research in this field is still in its early stages, preliminary results are promising, suggesting that these approaches could revolutionize the clinical use of EVs (García‐Alonso et al. 2004).

The instability of PDVs is the key reason for the limitation and questioning of the repeatability and expansion of many studies on PDVs and a critical but often overlooked factor in creating commercial EV‐based products. Due to their natural, nano‐scale nature, EVs are fragile and easily damaged by environmental stressors. If stability is not properly controlled, the final product can lose effectiveness, perform inconsistently or even cause adverse reactions. PDVs are particularly vulnerable to the harsh conditions of manufacturing, shipping, storage and formulation (Ferroni and Zavan 2025). PDVs are susceptible to a range of destabilizing conditions, including elevated temperature (causing vesicle deformation and cargo degradation), repeated freeze‐thaw cycles (leading to membrane rupture and aggregation), UV exposure (which oxidizes lipids and degrades nucleic acids) and exposure to common formulation agents like preservatives or surfactants that can disrupt membrane integrity (Kim, Park, et al. 2022; Rawat et al. 2025)

10. Biological Properties and Therapeutic Potential of PDVs

PDVs show significant potential as therapeutic agents due to their natural availability, ease of extraction and unique biological characteristics (Table 3). Evidence from both in vitro and in vivo studies indicates that PDVs possess intrinsic therapeutic effects that may aid in disease management and support overall human health. In addition, PDVs serve as an effective, biocompatible delivery system for therapeutic agents (Zhu et al. 2022). Indeed, PDVs are considered emerging natural tool for treating various diseases due to their ability to deliver drugs and therapeutic molecules directly to target cells demonstrating anti‐inflammatory, antioxidant and anticancer effects (Figure 2) in preclinical studies, while remaining safe, biocompatible, biodegradable and capable of crossing biological barriers, thus making them suitable for applications such as cancer therapy, inflammation treatment, skin repair and other future clinical application (Li et al. 2025). Plants represent a valuable natural resource for medicine, offering considerable potential in disease treatment due to their widespread availability and accessibility (Yue et al. 2023). Numerous plant species exhibit properties such as anti‐inflammatory, antimicrobial and tumour‐suppressing effects, which continue to drive progress in plant‐based therapeutic research (Cao et al. 2023). Fruits, such as grapefruit, lemon, orange, strawberry, blueberry and pomegranate have been identified as rich sources of EVs with antitumour, antioxidant and anti‐inflammatory properties, targeting conditions such as melanoma, gastric and ovarian cancers and chronic inflammation. Additionally, tea leaves have been explored as a source of EVs, demonstrating promising results in cancer therapy, particularly for breast cancer (Kilasoniya et al. 2023).

TABLE 3.

Overview of various PDVs, highlighting their origins and therapeutic properties.

Plant source	Origin	Size and cargo	Therapeutic properties	Study type	References
Ginger (Zingiber officinale)	Root	220–290 nm, contains 6‐gingerol and 6‐shogaol	Antioxidant, anti‐inflammatory, and anticancer activities; Enhances Nrf2 nuclear translocation in macrophages; Effective against colorectal cancer.	In vivo study	(Man et al. 2021)
Lemon (Citrus limon)	Fruit	50–119 nm, rich in vitamin C and citric acid	Antioxidant properties; Induces apoptosis in gastric cancer cells; Activates TRAIL‐mediated apoptotic pathways; Inhibits chronic myeloid leukaemia.	In vitro study	(Yang et al. 2025).
Grapefruit (Citrus paradisi)	Fruit	119–220 nm, contains naringin	Antioxidant and anti‐inflammatory effects; Regulates Wnt signalling; Inhibits liver metastasis; Enhances M1 macrophage activation.	In vivo study	(Zhang et al. 2024)
Strawberry (Fragaria × ananassa)	Fruit	50–119 nm, High in anthocyanins and vitamin C	Antioxidant properties; Protects mesenchymal stem cells from oxidative stress; Enhances Nrf2 activity.	In vitro study	(Wang, Xiong, et al. 2025)
Tea Flower (Camellia sinensis)	Flower	50–200 nm	Accumulates breast tumours and lung metastatic sites; Inhibits tumour growth; Modulates gut microbiome; Induces apoptosis in breast cancer cells.	In vitro and in vivo studies	(Gong et al. 2022; Yang et al. 2025)
Asparagus cochinchinensis	Root	119–220 nm	Exhibits anti‐proliferative activity against hepatocellular carcinoma cells; Induces apoptosis.	In vitro and in vivo studies	(Zhang et al. 2021)
Ginseng (Panax ginseng)	Root	50–220 nm	Induces macrophage polarization toward M1 phenotype; Activates TLR4 and MyD88 signalling pathways; Enhances antitumour effects.	In vitro and in vivo studies	(Cho et al. 2021)
Mulberry (Morus alba)	Root bark	119–220 nm	Exhibits anti‐inflammatory effects; Activates COPS8 intestinal epithelial cells; Potential agent for intestinal‐associated inflammatory diseases.	In vitro study	(Garrett et al. 2024)
Coconut (Cocos nucifera)	Water	119–220 nm	Supports growth of probiotics; Inhibits growth of harmful bacteria; Contains miRNAs; Potential antimicrobial properties.	In vitro study	(Yu et al. 2019)
Arabidopsis thaliana	Plant tissue	50–119 nm	Transfers small RNA to fungal pathogens; Suppresses fungal gene expression; Potential antifungal properties.	In vitro study	(Jokhio et al. 2024)
Apple (Malus domestica)	Fruit	200–220 nm	Modulates TLR4‐induced signalling; Downregulates NF‐κB pathway; Enhances collagen synthesis; Potential for skin regeneration.	In vitro study	(Trentini et al. 2022)
Aloe vera	Leaf	119–220 nm	Upregulates Nrf2; Promotes migration of HaCaT cells and fibroblasts; Enhances skin wound healing; Induces angiogenesis.	In vitro study	(Kim and Park 2022)
Cabbage (Brassica oleracea)	Leaf	201–220 nm	Suppresses apoptosis; Promotes epithelial cell repair; Enhances proliferation and migration.	In vitro study	(You et al. 2021)
Pomegranate (Punica granatum)	Fruit	119–220 nm, Contains phenolic acids, flavonoids, amino acids, terpenoids and tannins	Exhibits antioxidant and anti‐inflammatory effects.	In vitro study	(Zhao et al. 2024)
Grape (Vitis vinifera)	Fruit	119–220 nm, Contains trans‐δ‐viniferin	Antioxidant properties; Potential anticancer effects.	In vitro study	(Shkryl et al. 2024)
Cannabis (Cannabis sativa)	Plant tissue	200–220 nm, Contains cannabidiol	Antioxidant and anti‐inflammatory effects.	In vitro study	(Tajik et al. 2022)
Turmeric (Curcuma longa)	Root	198–220 nm, Contains curcumin	Inhibits pro‐inflammatory cytokines; Enhances antioxidant gene expression.	In vitro and in vivo study	(Gao et al. 2022)

FIGURE 2.

Therapeutic applications and potential modifications of PDVs. PDVs can be used in the treatment of various diseases, including cancer, immune disorders and metabolic diseases. They also serve as carriers for therapeutic agents. PDVs can be engineered or modified to deliver drugs like doxorubicin and methotrexate, or to transport specific miRNAs, enhancing their medical efficacy and targeting capabilities.

Nevertheless, traditional methods for developing plant‐based medicines often encounter challenges arising from the inherent complexity of natural compounds. This complexity can sometimes lead to toxicity or adverse effects, even when therapeutic efficacy is observed (Yuan et al. 2016). Furthermore, the precise mechanisms underlying the therapeutic actions of many plant‐derived medicines remain poorly understood, complicating efforts to determine their interactions with disease pathways. To overcome these limitations, innovative strategies are needed to isolate and purify the active constituents of plant‐based treatments (Zhang et al. 2024). Such advancements would not only clarify their mechanisms of action but also expand their potential applications in clinical disease management.

The route of administration plays a crucial role in determining the therapeutic efficacy of PDVs. Common delivery routes include oral, intravenous, intranasal and topical applications, each offering unique benefits and limitations (Table 4). Understanding the pharmacokinetics and biodistribution associated with each route is essential for optimizing the clinical potential of PDVs (Jain 2019).

While numerous studies have reported therapeutic effects of PDVs, comprehensive studies, particularly those focused on potential negative effects, are lacking. This gap of research on potential adverse effects is the primary reason their safety profile remains under question (Wei et al. 2023).

10.1. Anti‐Inflammatory Properties

Inflammation is a natural defence mechanism triggered by abnormal stimuli such as pathogenic microorganisms or cellular injury. This essential biological response serves to eliminate harmful agents and supports tissue repair and regeneration. However, when inflammation becomes excessive or persists in the absence of effective anti‐inflammatory regulation, it can result in irreversible tissue damage and contribute to the onset of complex diseases, including cancer, cardiovascular conditions and neurological disorders (Nolan and Duggan 2018). At present, nonsteroidal anti‐inflammatory drugs (e.g., aspirin) and glucocorticoids (e.g., dexamethasone) are routinely employed to manage inflammatory responses. Nonetheless, these conventional therapies are not without limitations, including the risk of developing drug resistance. Consequently, there is growing interest in identifying and developing plant‐based anti‐inflammatory agents as promising alternative treatments (Lou et al. 2024).

In vitro, studies demonstrate that PDVs can attenuate inflammatory responses in immune and epithelial cell lines. They have been shown to reduce the secretion of pro‐inflammatory cytokines such as TNF‐α, IL‐6 and IL‐1β, while promoting anti‐inflammatory mediators. Mechanistically, PDVs appear to interfere with NF‐κB signalling and oxidative stress pathways, thereby decreasing inflammatory gene expression (Nemati et al. 2022). Furthermore, another in vitro study showed that EVs isolated from Pueraria mirifica through gradient filtration combined with high‐speed centrifugation significantly suppressed the expression of pro‐inflammatory factors in M1‐like macrophages (Wu, Li, et al. 2024). Lemon‐derived vesicles (LDVs) have been shown to reduce the expression of pro‐inflammatory cytokines like IFN‐γ and TNF‐α, while simultaneously increasing levels of anti‐inflammatory cytokines such as IL‐10 and IL‐9 on murine and primary human macrophages (Raimondo et al. 2022). Nanovesicles were isolated from various plants, such as grapefruit, ginger, turmeric, garlic, cilantro, aloe vera, dandelion, lavender and cactus and were tested for their effects on NLRP3 inflammasome activity in bone marrow‐derived macrophages (BMDMs). Most of the vesicles had only mild effects, either slightly increasing or decreasing inflammation, based on Caspase‐1 activation and IL‐1β release. However, GDVs, especially at higher concentrations (up to 3 × 10^{1 0}/mL), strongly reduced both Caspase‐1 activity and IL‐1β levels after a 16‐h treatment followed by inflammasome activation. GDVs were easily taken up by the macrophages and lowered the release of IL‐18 and LDH, showing a reduction in inflammation and cell death. The anti‐inflammatory effect was mainly linked to the lipid content in GDVs rather than their proteins or RNAs, highlighting the promising natural inhibitory activity (Teng et al. 2022).

The anti‐inflammatory effects of GELNs were tested in vivo in 6–8‐week‐old C57BL/6 mice with colitis caused by Dextran Sulfate Sodium (DSS). EVs, obtained through ultra‐high‐speed centrifugation, were given orally and taken up by intestinal cells and immune cells in the colon. The results showed that these EVs significantly reduced inflammation and supported healing of the gut lining, highlighting their promise as new anti‐inflammatory treatments, especially when compared to EVs from Pueraria lobata (di Raimo et al. 2024). Murine models indicate that oral or systemic administration of PDVs can alleviate inflammation in conditions such as colitis, liver injury and arthritis. These vesicles not only dampen inflammatory cytokine production but also promote tissue repair and gut microbiota balance. Their natural origin and biocompatibility make them particularly attractive compared to synthetic nanocarriers (Huang et al. 2023). Early translational studies suggest that PDVs may exert anti‐inflammatory benefits in humans and large animals as well. For example, grape‐ and ginger‐derived vesicles have been reported to reduce intestinal inflammation and support mucosal healing in preclinical trials. Although direct evidence in clinical human studies remains limited, the available data support their potential as dietary supplements or therapeutic agents for inflammatory diseases (Wei et al. 2023) (Table 5).

TABLE 5.

Summary of the most recent drug loading attempts on EVs of plant origin.

Source	Drug	Particle size	Method	Loading efficacy	Application	Ref.
Acerola	hsa‐miR‐340	245–352 nm	Incubation at 30 min	60%	Gene‐suppressing in intestine	(Chen et al. 2024)
Aloe	Indocyanine Green	138.7–220 nm	ND	3:2 ratio loading	Skin cancer therapy	(Kim et al. 2023)
Bitter Melon	5‐Fluorouracil	100–200 nm	Sonication	n.d.	Oral squamous carcinoma	(Choi et al. 2015)
Broccoli	miRNAs	35–300 nm (mean 174.3 nm)	Lipofection	n.d.	Cell antiproliferation	(García‐Alonso et al. 2004)
Cabbage	miR‐184, Doxorubicin	100 nm	Incubation at 37°C	Doxorubicin or Cabex loading	Antitumour effects	(Nguyen et al. 2023)
Celery	Doxorubicin	111.8–113.7 nm	Incubation at 37°C	87%	Antitumour effects	(Kim et al. 2023)
Cucumber	DiI perchlorate	167 nm	Incubation & vortexing	n.d.	Skin therapy	(di Raimo et al. 2024)
Ginger	Survivin RNA	123.5–124.5 nm	Incubation at 37°C	80%	Antitumour effects	(Musi and Bongiovanni 2023)
Ginger	siRNA‐CD98	189.5 nm	Sonication & Extrusion	61 ± 8%	Ulcerative colitis treatment	(Zhang et al. 2017)
Grapefruit	DOX‐loaded nanoparticles	135 ± 5 nm	ND	1 µg EVs = 1.01‐1.9 µg DOX	Glioma therapy	(Garaeva et al. 2021)

Abbreviation: ND, not determined.

10.2. Antioxidant Properties

Oxidative stress arises from an imbalance in reactive oxygen species (ROS), often instigated by neutrophilic inflammatory infiltration, leading to cellular damage and death. This imbalance plays a crucial role in the pathogenesis of numerous diseases. Many plants naturally exhibit antioxidant properties, and widely used clinical antioxidants such as polyphenolic compounds, vitamin C and vitamin E are typically derived from plant sources (Kim et al. 2023). However, the purification efficiency of these natural antioxidants remains low; extraction techniques are often suboptimal, and the compounds are rapidly metabolized in the body. PDVs from plants contain a variety of plant‐derived antioxidants that may help address oxidative stress‐related diseases. Given the significant role of oxidative stress and ROS in the development of various diseases, there is an urgent need for more effective and standardized methods to isolate and enrich antioxidant components from PDVs. Such advancements could enhance their potential in reducing ROS levels, offering a valuable contribution to the prevention and management of ROS‐related pathological conditions (Lian et al. 2022).

In an in vivo study, high concentrations of EVs, isolated from carrots using SEC combined with UF, exhibited low cytotoxicity and a clear reduction of oxidative stress and apoptosis in H9C2 rat cardiomyoblast. These results underscore the therapeutic potential of carrot‐derived EVs in the treatment of conditions such as myocardial infarction and Parkinson's disease (Choi et al. 2015). Likewise, EVs extracted from blueberries via sequential low‐speed centrifugation in the presence of class I chitinases demonstrated antioxidant activity by lowering pro‐inflammatory factors and total glutathione levels (Zhu et al. 2024). Furthermore, EVs isolated from blueberries also showed high drug‐loading efficiency, successfully encapsulating up to 80% of curcumin, thereby highlighting their promise as effective carriers for therapeutic agents (Nguyen et al. 2023).

PDVs from fruits (e.g., grapefruit, lemon, blueberry) reduced ROS generation and lipid peroxidation in oxidative stress–induced cell models. They enhanced cell viability, mitochondrial function and expression of endogenous antioxidant enzymes such as superoxide dismutase (SOD) and catalase. GDVS, rich in polyphenols, showed protective effects against hydrogen peroxide–induced oxidative damage in epithelial and neuronal cells in vitro study (Ramírez‐Hernández et al. 2023).

Oral or systemic administration of PDVs demonstrated antioxidative effects in rodent models of liver injury, colitis and neuroinflammatory models, reduced malondialdehyde (MDA) levels, restored antioxidant enzyme activity, and lowered inflammatory cytokines. For example, GELNs alleviated oxidative stress–driven intestinal inflammation in mice by reducing ROS and enhancing gut epithelial defences (Huang et al. 2023).

Early reports suggest PDVs are biocompatible, non‐toxic and orally bioavailable, making them promise nutraceutical. In dietary supplementation studies (animal and limited human pilot data), PDVs from edible plants improved markers of oxidative balance, such as increasing glutathione levels and decreasing oxidative stress biomarkers. Although large‐scale human trials are still limited, the natural antioxidant content and safe oral delivery position PDVs as potential therapeutic or preventive tools for managing oxidative stress‐related conditions (Yang et al. 2025).

As previously mentioned, PDVs are promising natural biomaterials for the treatment of oxidative stress‐induced damage and oxidative stress‐related diseases, including chronic skin wounds, carcinogenesis and skin ageing (Kim et al. 2023). Dermal extracellular vesicles (DEVs) demonstrated the capacity to rebalance the mitochondrial membrane potential, which is a crucial indicator of mitochondrial integrity. Following exposure to oxidative stress, treatment with PDVs restored the membrane potential to near‐normal levels within the first 24 h. Consistently, PDVs also decreased mitochondrial superoxide production, further underscoring their beneficial effect on mitochondrial performance. Moreover, oxidative stress is known to diminish levels of essential ageing‐related molecules, such as sirtuin proteins. Di Raimo and co‐authors revealed that PDVs enhanced sirtuin 1 expression, reinforcing their prospective application in anti‐ageing skin therapies (di Raimo et al. 2024).

10.3. Antitumour Properties

Cancer treatment presents numerous challenges, including drug resistance, limited targeting capability and significant adverse effects. Existing therapeutic approaches, such as interventional procedures, immunotherapy, chemotherapy and radiotherapy, are restricted by factors including inter‐individual variability, differing levels of treatment tolerance, surgical limitations, drug‐induced toxicity and substantial financial costs (Musi and Bongiovanni 2023). These limitations highlight the ongoing difficulty in achieving effective cancer prevention and treatment.

PDVs offer several advantages, including low toxicity, broad availability and cost‐effectiveness, positioning them as promising candidates for the development of novel anti‐cancer therapies (Musi and Bongiovanni 2023; Qiang et al. 2024). Accordingly, the use of PDVs in oncology represents a critical area for research and innovation, and continued research is essential to unlock their full clinical potential.

PDVs have shown strong anticancer potential by modulating cell communication, improving the tumour microenvironment, inhibiting tumour vesicle release and enhancing drug sensitivity. They can reduce chemotherapy resistance and side effects by targeting various signalling pathways. Their adaptable nature allows them to deliver therapeutic molecules directly to cancer cells, with antitumour effects that vary by plant source, highlighting the potential for customized cancer therapies (Tang et al. 2024). PDVs isolated from fruits and vegetables (e.g., grapes, ginger, lemon and broccoli) have been shown to inhibit proliferation of various human cancer cell lines, including colon, liver and breast cancer cells, by triggering apoptosis, reducing metastatic potential, and suppressing tumour‐promoting signalling pathways (Ly et al. 2023).

Bitter melon‐derived EVs have been shown to enhance the effectiveness of the chemotherapeutic agent 5‐fluorouracil (5‐FU) in treating oral squamous cell carcinoma (OSCC). These vesicles not only increase the cytotoxicity but also reduce resistance to 5‐FU by downregulating the expression of the inflammasome component NLRP3. Additionally, bitter melon vesicles induce apoptosis in OSCC cells by triggering ROS‐mediated mitochondrial damage, a process potentially mediated by the MAP30 protein (Yang, Zhang, et al. 2021).

The therapeutic potential of EVs derived from artemisinin was explored by administering them intraperitoneally to adult C57 mice with lung cancer. The results indicated that mitochondrial DNA carried by these EVs activated the cGAS–STING signaling pathway, thereby remodelling the tumour microenvironment. This activation led to the reprogramming of tumour‐associated macrophages, converting them from a tumour‐promoting to an anti‐tumour phenotype, which enhanced immune responses and produced significant anti‐tumour (Wang, Meng, et al. 2025). Similarly, EVs isolated from Centella asiatica via high‐speed centrifugation were found to carry cancer‐targeting microRNAs and were shown to inhibit HepG2 cell proliferation by modulating metabolic pathways involved in amino acid and lipid synthesis, underscoring the potential of such PDVs as innovative tools in cancer immunotherapy and plant‐based nanomedicine (Chang et al. 2025). In vitro studies demonstrate the ability of PDVs to deliver bioactive molecules or siRNAs into cancer cells efficiently, suggesting a role as drug‐delivery nanocarriers (Yang et al. 2024).

In one study, researchers functionalized PDVs with the HA1 aptamer to load azithromycin, targeting HER2‐positive breast cancer cells (Shen et al. 2025). In another approach, grapefruit EVs coated with membranes from activated leukocytes enriched in LFA‐1, CXCR1 and CXCR2 exhibited improved targeting of cancer cells. These engineered vesicles successfully delivered doxorubicin to CT26 colon cancer cells, resulting in significant tumouricidal effects (Tan et al. 2022).

One notable example is Asparagus cochinchinensis (ACNVs), a medicinal plant known for its anticancer and antioxidant properties. An in vitro study by Zhang et al. (2021) demonstrated that EVs from ACNVs inhibited liver cancer cell growth and promoted apoptosis, primarily through phagocytosis. When modified with PEG to improve circulation and reduce immune clearance, these vesicles are more effectively accumulated at tumour sites. This PEGylation not only boosted their therapeutic impact but also minimized adverse reactions. GELNs inhibited tumour growth and promoted apoptosis in colorectal cancer xenograft models. PDVs are generally non‐toxic, show high stability in gastrointestinal conditions, and can cross biological barriers, making them attractive for oral cancer therapy in animals (Qiang et al. 2024). Preclinical animal studies highlight the safety profile of PDVs, with no significant adverse effects reported after repeated administration. According to Xu et al. (2023), early research indicates that PDVs show promise as natural, biocompatible vehicles for cancer drugs, which could enhance treatment effectiveness and minimize side effects, though clinical evidence remains limited.

10.4. Modulation of Gut Microbiota and Treatment of Inflammatory Diseases

The gut microbiota plays a crucial role in maintaining host health and can be influenced by dietary habits. However, disruptions to the intestinal mucosa, caused by unhealthy diets or certain diseases, can lead to imbalances in the microbiota, often necessitating medical intervention (Zhang 2022).

PDVs, particularly those from vegetables rich in nucleic acids such as RNA, found in beans, peas and lentils, have shown potential in modulating the gut microbiota and enhancing host physiological functions after intestinal absorption (Wu, Kan, et al. 2024). These EVs help strengthen the intestinal barrier, improve immune responses and alleviate metabolic disorders associated with microbiota imbalances (Hao et al. 2024). By regulating the composition and functions of the gut microbiota, PDVs positively influence gut health and overall well‐being, positioning them as a promising therapeutic option for gastrointestinal disorders.

PDVs, such as those from mulberry bark, carrots, ginseng and ginger, have shown significant anti‐inflammatory effects in animal models of colitis and other chronic inflammatory diseases. These EVs help reduce inflammation by inhibiting key signalling pathways, promoting antioxidant activity, modulating immune responses and improving gut health. Their bioactive contents, including proteins, lipids and RNAs, contribute to the suppression of pro‐inflammatory cytokines, enhancement of anti‐inflammatory factors like IL‐10, and restoration of intestinal barrier function and microbiota balance (Liu et al. 2024).

One in vivo study examined the effects of EVs derived from kidney beans on obesity induced by a high‐fat diet (Diez‐Sainz et al. 2022). Oral administration of kidney bean‐derived EVs resulted in significant reductions in body and liver weight while improving obesity markers in rats (Zhu et al. 2024). These EVs also enhanced gut microbiota diversity, reduced diet‐induced obesity and promoted the production of short‐chain fatty acids.

In a similar vein, constipation, a common gastrointestinal issue that affects quality of life, can be managed by modulating gut microbiota. A study on BDVs in a mouse model of loperamide‐induced constipation found that oral administration of PDVs significantly increased bowel movements and accelerated intestinal peristalsis (Duan et al. 2023). This effect was attributed to the modulation of gut microbiota and tryptophan metabolism, effectively alleviating constipation. These findings highlight the capacity of PDVs to stabilize within the gut, influence microbial communities, and provide innovative therapeutic solutions for conditions such as obesity and constipation by targeting gut microbiota and metabolic pathways (Wu, Duan, et al. 2022).

PDVs reduced pro‐inflammatory cytokine release (e.g., TNF‐α, IL‐6) in macrophage and epithelial cell lines stimulated with macrophages to recognize lipopolysaccharide (LPS) (Li et al. 2025). Oral delivery of GELNs in DSS‐induced colitis mice reduced colon inflammation, lowered immune infiltration and restored microbiota balance, liver inflammation (Zhang et al. 2016).

Furthermore, GDVs showed hepatoprotective effects by modulating gut–liver axis signaling (Wang et al. 2026) and broccoli‐derived EVs influenced gut microbiota metabolites linked to brain inflammation (Cui et al. 2024).

10.5. Wound Healing

PDVs play a promising role in wound healing by modulating various cellular processes involved in the healing cascade. They promote cell proliferation, migration, and differentiation, enhance collagen production, and reduce inflammation. Additionally, PDVs stimulate angiogenesis, the formation of new blood vessels, which is crucial for wound repair (Narauskaitė et al. 2021).

Tan et al. (2024) successfully isolated ginseng‐derived vesicles (GDVs) using a rapid separation method and demonstrated their potential in treating diabetic skin ulcers. Under high glucose conditions, GDVs enhance endothelial cell proliferation, migration and tissue remodelling by reducing oxidative stress and promoting anaerobic glycolysis. In diabetic mouse models (db/db), topical application of GDVs via sterile dressings significantly accelerated wound healing and angiogenesis, leading to complete re‐epithelialization. The treatment was found to be safe and biocompatible. Overall, GDVs show strong promise as plant‐based nanotherapeutics for promoting vascular regeneration and healing in diabetes‐related wounds (Zhu et al. 2024).

11. Aesthetics Agents

Anti‐scaring, anti‐ageing, and anti‐pigmentation are some of the most desired goals in facial skin care and beauty treatments. PEVs naturally carry a wide array of biomolecules, including proteins, lipids, DNA and various RNA species, all of which are essential for intercellular communication and regulating molecular activities within recipient cells. Furthermore, EVs are biocompatible, easily absorbed by cells, and can be directed towards specific tissues for targeted effects. In therapeutic applications, PDVs have shown promise in promoting skin health by accelerating wound healing, reducing skin pigmentation, minimizing wrinkles and preventing scarring (Kim et al. 2021; Foo et al. 2021).

Studies on PDVs in skin diseases have found that they promote skin regeneration just like mammalian EVs. These discoveries have raised growing interest in using PDVs in the cosmeceutical industry, especially as a natural, animal‐free alternative. Examples include EVs from various plant sources such as Codium fragile (C. fragile), Sargassum fusiform (S. fusiform), Dendropanax morbifera (D. morbifera) and Panax ginseng (P. ginseng). Specific types of plant EVs include those from aloe vera peels (A‐EVs), leaves (LEVs), stems (SEVs), ginseng roots (GrEVs) and ginseng cell culture supernatants (GcEVs) (Kee et al. 2022). Furthermore, PDVs have been introduced as a novel nanocarrier to enhance the positive effects in cosmeceuticals, offering several advantages that make them excellent alternatives to traditional nanocarriers in cosmeceutical applications aimed at improving skin health (Kee et al. 2022).

11.1. Reduce Scar Formation

There are various methods available to reduce or eliminate scarring, including surgery, laser therapy, chemical peels, dermabrasion, steroid or collagen injections and topical ointments.

Numerous studies have investigated the role of PDVs in scar reduction, with most findings suggesting that PDVs can accelerate wound healing and enable scarless repair. This process is linked to more organized collagen fibres, reduced cross‐linking and a smoother epidermal surface. Moreover, EVs have been shown to support skin cell proliferation, migration and angiogenesis, further enhancing their regenerative potential (Foo et al. 2021).

PDVs from Dendropanax morbifera leaves (LEVs) and stems (SEVs) showed no cytotoxicity and reduced melanin levels in B16BL6 melanoma cells, with LEVs exhibiting stronger anti‐melanogenic effects. LEVs also significantly suppressed melanin synthesis in a human epidermis model by regulating melanogenesis‐related genes (Kim et al. 2020).

11.2. Anti‐Ageing Properties

Over the years, a range of anti‐ageing techniques, including cosmetics, chemical peels, phototherapy and micro‐needling, have been developed to enhance the appearance of ageing skin. However, the effects of these methods are often temporary. Consequently, EVs have attracted considerable attention as a promising alternative, owing to their significant role in regulating numerous biological processes associated with skin ageing (Yang, Lee, et al. 2021).

EVs isolated from aloe vera peels (A‐EVs), known for their antioxidant‐rich composition, demonstrated excellent compatibility with human skin cells. A‐EVs reduced intracellular ROS levels and activated key antioxidant pathways (Nrf2, HO‐1, CAT and SOD), while promoting keratinocyte and fibroblast migration, indicating potential for skin rejuvenation and wound healing (Huang et al. 2023).

11.3. Anti‐Pigmentation Properties

Recent research has demonstrated that EV treatment can reduce UVB‐induced melanin production and promote the degradation of melanosomes, suggesting that EVs contain bioactive factors that may help prevent skin hyperpigmentation (Wu, Zhang, et al. 2022). PDEVs have shown substantial potential in the inhibition of melanogenesis. PDEVs isolated from yam bean (Pachyrhizus erosus) demonstrated their anti‐melanogenic activity in a zebrafish model, underscoring their possible application in skin‐lightening formulations (Kusnandar et al. 2025). The primary mechanism underlying the anti‐pigmentation effects of PDEVs is the regulation of microphthalmia‐associated transcription factor (MITF), leading to the downregulation of tyrosinase (TYR), tyrosinase‐related protein 1 (TYRP1) and tyrosinase‐related protein 2 (TYRP2), which collectively suppress melanin biosynthesis. For example, vesicles derived from Atractylodes lancea and Panax ginseng have been reported to decrease melanin content by reducing the expression of these melanogenic enzymes (Hsiao and Fisher 2014). In a related study, it was found that endothelial cell‐derived EVs attenuated melanogenesis and improved basement membrane integrity by inhibiting the TXNIP/NLRP3/IL‐18 signaling axis, a critical upstream regulator of MITF (Byun et al. 2024). Furthermore, EVs derived from Dendropanax morbifera suppressed melanin synthesis through modulation of the α‐MSH–MC1R pathway, resulting in reduced MITF expression and superior anti‐pigmentation efficacy compared to arbutin in human epidermal models (Nan et al. 2025).

12. Nutraceutical Application of PDVs

PDVs are emerging as a promising platform in the field of nutraceuticals due to their natural origin, biocompatibility and ability to carry bioactive molecules such as proteins, lipids and RNAs. They cross biological barriers and deliver their therapeutic cargo to target cells, thus making them ideal candidates for oral or topical administration.

In nutraceutical applications, PDVs have shown potential to modulate immune responses, reduce oxidative stress and support gut health, which are key determinants in preventing and managing chronic diseases (Tiwari et al. 2025). Their antioxidant and anti‐inflammatory properties contribute to the maintenance of overall health and the mitigation of conditions such as inflammatory bowel disease, metabolic syndrome and even skin disorders. As natural nanocarriers, they offer a safe and efficient platform for the delivery of plant‐based nutrients and therapeutic compounds, opening new avenues for functional foods and dietary supplements aimed at promoting wellness and disease prevention (Lo et al. 2024).

Another significant advantage of PDVs is their ability to cross biological barriers, such as the intestinal epithelium and, in some cases, the blood–brain barrier, thereby facilitating systemic distribution of their cargo without inducing toxicity or immune reactions (Azizi et al. 2024). This makes them particularly suitable for nutraceutical applications targeting brain health, immune modulation and systemic inflammation. Furthermore, their plant origin reduces the risk of zoonotic transmission or oncogenic DNA transfer, which are concerns with PDVs (Jiang et al. 2024). For instance, encapsulation of curcumin within turmeric‐derived EVs has been shown to improve its solubility and therapeutic efficacy against oxidative stress and inflammation (Li et al. 2021). Opuntia Ficus‐indica (FicoVes) vesicles possess a distinctive blend of antioxidant, anti‐inflammatory, and tissue‐regenerative properties, along with excellent biocompatibility and the ability to influence gene expression. These features position them as a promising candidate in nutrigenomics, especially for developing treatments for skin conditions like psoriasis, dermatitis and eczema. Incorporating FicoVes into gel or patch‐based formulations may help maintain their stability and enhance their effectiveness, with patches offering the added benefit of controlled, sustained release during topical application. However, further research, including in vivo studies, is needed to confirm these benefits and fully assess their therapeutic potential (Naselli et al. 2024).

13. Conclusion

This review provides a comprehensive analysis of PDVs, detailing their classification, biochemical composition, isolation and characterization methods, and comparing them with MDEVs. It also highlights their broad applications in nanomedicine, drug delivery and nutraceuticals. PDVs are naturally secreted nanoscale vesicles enriched with bioactive molecules such as proteins, lipids, RNAs and metabolites. Their inherent features, including biocompatibility, low immunogenicity, structural stability and the feasibility of large‐scale production, position them as a highly promising platform for future innovation across multiple sectors.

Recent advancements in isolation techniques, including differential centrifugation, density gradient fractionation, immunoaffinity capture and tangential flow filtration, have significantly improved the purity and specificity of PDVs preparations. However, their biological complexity, reflected in their diverse formation mechanisms, cargo compositions and release pathways, contributes to substantial heterogeneity. This presents major challenges in standardization and characterization, especially in the absence of bona fide reference markers, thereby offering an important avenue for future research to develop scalable and reproducible analytical frameworks.

Importantly, this review underscores how FCM represents a transformative analytical tool capable of addressing several current limitations in PDVs research. Its capacity for high‐throughput, multiparametric, single‐particle analysis allows rapid identification, quantification and phenotypic profiling of PDVs, even within highly heterogeneous samples. Advances in nanoscale flow cytometry, optimized triggering strategies, standardized calibration approaches and the emergence of imaging FCM have collectively enabled unprecedented sensitivity and accuracy in detecting small vesicles across different plant species.

Furthermore, this review highlights the therapeutic versatility of PDVs, which exhibit anti‐inflammatory, antioxidant, antitumour and microbiota‐regulating properties. Their ability to cross biological barriers and deliver functional cargo makes them excellent candidates for nano‐delivery systems in both pharmaceutical and cosmetic applications. Nevertheless, rigorous testing is needed to determine how isolation and characterization techniques influence the physicochemical and biological properties of PDVs. Only after addressing these variables combined with comprehensive in vivo, pharmacokinetic, and safety evaluations can PDVs be confidently advanced toward clinical or commercial integration.

Additionally, PDVs hold significant promise for the development of functional foods and nutraceuticals, offering novel strategies for health promotion and disease prevention. Their incorporation as carriers of bioactive compounds in oral supplements or topical formulations opens the door to innovative product development. Future research should aim to elucidate the molecular mechanisms underlying PDV activity, explore their potential as diagnostic biomarkers, and engineer PDVs with enhanced targeting capabilities. Collectively, these efforts will help unlock the full clinical and commercial value of PDVs.

PDVs also display cytocompatibility, anti‐ageing, whitening and regenerative properties, reinforcing their potential for cosmetic and therapeutic dermatological applications. However, current evidence is primarily limited in vitro studies, and further clinical validation is essential.

Although PDVs hold significant translational promise, several challenges hinder their progression toward clinical use. These include difficulties in large‐scale production, the need for efficient and reliable purification strategies, and the absence of harmonized regulatory guidelines. Addressing these obstacles will require continued advancements in isolation technologies, the establishment of standardized regulatory frameworks, and the development of scalable manufacturing processes. By consolidating recent progress in PDV isolation, characterization and application, this review emphasizes their potential to drive sustainable healthcare innovations. With continued research and technological refinement, PDVs may pave the way for transformative advances in biomedicine, particularly through the development of targeted and effective drug‐delivery platforms suitable for clinical implementation.

Author Contributions

Tamer Esmail: writing – review and editing, writing – original draft, conceptualization, data curation, methodology. Sabino Porro: writing – review and editing, writing – original draft, data curation. Francesca D'ascanio: writing – original draft. Domenico De Bellis: writing – original draft. Arianna Aquilini‐mummolo: writing – original draft. Giulia Colasante: writing – original draft. Pasquale Simeone: writing – review and editing. Ayesha Younas: writing – original draft. Marwa Balaha: writing – original draft, methodology, data curation. Mariagiulia Filoso: writing – review and editing. Luana D'onofrio: writing – review and editing. Melania Dovizio: writing – review and editing, conceptualization. Eleonora Aruffo: writing – review and editing, conceptualization. Piero Di Carlo: writing – review and editing, conceptualization. Lorenzo Secondi: writing – review and editing, conceptualization. Federica Flammini: writing – review and editing, supervision, conceptualization. Tiziana Pietrangelo: writing – review and editing, conceptualization. Chiara Porro: writing – review and editing, conceptualization. Patrizia Ballerini: writing – review and editing, conceptualization. Angelo Cichelli: writing – review and editing, conceptualization. Paola Lanuti: writing – review and editing, supervision, conceptualization.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

No new data were generated or analyzed in this study.