Plant-derived extracellular vesicles: composition, function and clinical potential

Abstract

As membrane structural bodies secreted by cells, extracellular vesicles (EVs) have become a hot topic in the field of plant science in recent years. Plant-derived extracellular vesicles (PDEVs) play a key role in both plant physiological activities and the interaction between plants and the environment. These vesicles are involved in plant growth, development, and response to adversity by delivering signaling molecules, proteins, and other bioactive substances. Current studies have shown that PDEVs also exhibit promising potential for clinical applications, including as drug carriers, vaccine development, and biomarkers for disease diagnosis. In this paper, we review studies related to compositional characterization, isolation and purification methods, biological roles, and clinical applications of PDEVs. We aim to provide a systematic theoretical basis and practical guidance for future studies to promote their application in biomedical fields.

Introduction

EVs are lipid bilayer membrane-structured nanoscale vesicles released by cells into the external environment and are found in a wide variety of organisms, including mammals, plants, fungi, and bacteria [1, 2]. PDEVs have attracted much attention in the biomedical field. In recent years, numerous studies have highlighted the critical roles of PDEVs in facilitating cell–cell communication, mediating the exchange of bioinformation among different cells, and maintaining tissue homeostasis and organismal integrity [3]. Compared with animal-derived EVs, PDEVs are more efficient in production, have a shorter extraction cycle, are widely available, and are less immunogenic [4–6]. In addition, PDEVs are less cytotoxic, have superior biocompatibility [7–9], and can be targeted to specific tissues through endocytosis [10, 11]. The study on PDEVs not only provides new perspectives for understanding the physiological functions of plants but also offers a potential application platform for developing novel therapeutic strategies. As research on PDEVs continues, there is growing evidence that they have significant biological roles in the treatment of inflammation, tumors, and other diseases [12].

In 1967, researchers observed for the 1st time using electron microscopy that carrot cells were able to secrete vesicles [13]. In 2009, researchers have extracted vesicles similar to EVs from sunflower seeds. Some studies have shown that PDEVs and animal-derived EVs are similar in many ways [14]. Based on its natural source and unique physical and chemical properties, PDEVs show their potential as a new therapeutic method. These cheeses have excellent biocompatibility and can maintain structural stability even through oral routes [15–17]. The latest research data show that the biologically active substances carried by PDEVs have a regulatory effect on human health. These nanoscale carriers can be taken up by mammalian cells through endocytosis and membrane fusion. After internalization, PDEVs mainly have two fates: one is to form early endosomes and gradually mature into late endosomes, which will eventually be transported to lysosomes for degradation; The second is that it can destroy the endosomal membrane in an acidic environment, thereby releasing the active goods (proteins and nucleic acids) it carries into the cytoplasm and thus exerting biological functions [18, 19]. It is worth noting that the active ingredients contained in PDEVs are considered to be the material basis for their therapeutic effects. This natural delivery system not only maintains the activity of biomolecules but also achieves precise intracellular transmission [9, 20]. In addition to direct therapeutic effects, PDEVs have shown significant advantages as a novel drug carrier. Compared with traditional delivery systems, its unique membrane structure can load both hydrophilic and hydrophobic therapeutic substances [21]. This parental delivery characteristic allows PDEVs to effectively protect therapeutic components and achieve accurate delivery of targeted sites, providing innovative ideas for drug development [22].In addition, therapeutic drugs loaded in PDEVs can have synergistic effects with the inherent therapeutic activity of PDEVs themselves. This targeted delivery and enhanced efficacy significantly improve the overall therapeutic effect [23].Although the popularity of the research on PDEVs continues to rise, the field is still in its early stages of exploration. There are currently three core challenges: The structural analysis of PDEVs is incomplete, the separation and preparation lack unified standards, and the biological mechanism is still vague.

The aim of this review is to discuss the latest research progress on the compositional characteristics, isolation and purification techniques, biological functions, and clinical translational applications of PDEVs. In addition, the research strengths and challenges of PDEVs in the current field will be discussed, which will provide insight into the complexities associated with PDEVs research.

Biological composition of PDEVs

As the core link of quality control of PDEVs, component analysis is of importance due to component fluctuations caused by plant-derived differences. Similar to mammalian extracellular vesicles, the various biologically active molecules carried by PDEVs can regulate the physiological state and morphological changes of receptor cells [24]. Compared with animal cell vesicles, research on PDEVs has not been fully carried out. There are still obvious gaps in the systematic understanding of its biomolecular structure [25, 26]. The morphological and structural features of PDEVs are depicted in Fig. 1. This section will focus on the analysis of the biomolecular composition of PDEVs.

Fig. 1.

Biogenesis, morphological structure, and composition of PDEVs. Initially, the plasma membrane buds inward to form early endosomes which mature into late endosomes and communicate with the trans-Golgi network, leading to the formation of MVBs. Within MVBs, ILVs selectively accumulate and aggregate substances such as DNA, RNA, proteins, and lipids. These ILVs are subsequently released into the extracellular space upon fusion of MVBs with the plasma membrane, giving rise to PDEVs

Proteins

Of particular concern are members of the Annexin family, which are identified in citrus juice extracellular fluid, sunflower seeds and Arabidopsis leaf source EVs [27]. Studies have confirmed that such proteins play a regulatory role in plant biological/abiotic stress response [27]. In a notable study, garlic-derived PDEVs were subjected to trypsin treatment in order to remove surface proteins. The results demonstrated that trypsin-treated garlic-derived PDEVs exhibited reduced cellular uptake compared to the untreated counterparts, suggesting that membrane proteins may play a role in the internalization process of PDEVs [9]. Furthermore, an additional study revealed that aquaporins present on broccoli-derived PDEVs contribute to the maintenance of membrane stability [28].

Heat stress proteins (HSP60/70/80/90) are another critical component and are widely present in plant EVs, such as citrus, olives, grapes, and tomatoes. Several studies have shown that the enrichment characteristics of such molecular chaperone proteins are closely related to the molecular mechanisms in which plants cope with environmental stress [29, 30]. Studies have shown that members of the aquaporin family exist in PDEVs isolated from citrus, grapes, cauliflower, and Arabidopsis. This protein exerts a biological function by maintaining plasma membrane structural stability and regulating moisture permeability [31]. Ginger and PDEVs from Arabidopsis origin carry a variety of active ingredients. The existence of actin suggests that PDEVs may be involved in dynamic processes such as cell division, expansion, organelle movement and vesicle transportation [32, 33]. The 1/2/3 subtype of patellar protein was identified in EVs of Arabidopsis and citrus plants. According to annotations in the UniProt database, these carrier proteins may be involved in membrane transport events during the cell division phase, particularly in the cell plate formation process. By binding to hydrophobic molecules, they facilitate material transport between different cellular compartments, suggesting that the patella protein in PDEVs plays a critical regulatory role in plant physiological functions [30, 34]. PDEVs extracted from Arabidopsis thaliana and Citrus aurantium contain a variety of functional proteins, including synthetic protein clusters and isoforms such as RabA2a, RabB1c, and others [20]. It has been shown that these proteins are mainly involved in the regulation of vesicular transport, cytosolic, and secretory pathways, and that they play an important role in regulating the sorting and secretion of substances by PDEVs [29].

Lipids

PDEVs have significant differences with animal EVs in terms of lipid composition. The current research hotspots focus on the particularity of their lipid components. The main lipid components include phosphatidic acid (PA), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) [18, 20].These lipid characteristics not only affect the interaction ability of vesicles with animal cells but also determine the efficiency of vesicles being internalized and absorbed [3]. It is worth noting that the lipid composition of PDEVs is directly related to their biological function, and specific lipid components may regulate their physiological activities by changing the physical properties of the capsule or signaling pathways [35].

Studies have shown that PA ingredients can be extracted from various plants such as sunflower extraplasmic liquid, grapes, oranges, and ginger [20]. It is worth noting that PA not only plays a role in cell mitosis but also participates in regulating key biological processes such as membrane fusion and fission [20]. PE and PC have also been confirmed to exist in PDEVs of grapefruit, oranges and ginger [36]. The molecular structure and chemical properties of PA will significantly affect the changes in biofilm curvature [36]. As an important signal lipid, PA can effectively recruit specific cytoplasmic proteins to the cell membrane area [37], thereby regulating related physiological activities. Furthermore, the molecule participates in the vesicle formation process by promoting membrane curvature and recruiting specific proteins [38]. Studies have shown that the polyunsaturated fatty acids contained in PA, PE, and PC may reduce the rigidity of the membrane structure, and this characteristic plays an important role in membrane bending and morphological remodeling during endocytosis [38]. Sphingolipids isolated from Arabidopsis and tobacco may directly affect the dynamic properties of PDEVs membranes [39]. Glycosylated inositol phosphate ceramide has been proven to be the main sphingolipid component in plant tissues [39]. Although sphingomyelin has a clearer function in animal systems, such substances in plant systems also play a central role in key physiological processes such as pollen development, signal transduction, and biological/abiotic stress response [40].

In addition to their role in the uptake and internalization of PDEVs, lipids may also play a part in the intrinsic therapeutic activities of PDEVs. For example, PDEVs derived from grapefruit are enriched with PE and PC, which may underlie their antioxidant and anti-inflammatory effects [41, 42]. The current research field urgently needs to systematically analyze the spatial configuration and component characteristics of various lipid molecules in different PDEV samples [43].

Nucleic acid

The nucleic acid components in PDEVs include DNA and a variety of RNA types. MicroRNA, as a non-coding small RNA molecule composed of 22 nucleotides, realizes gene expression regulation by regulating the translation process of messenger RNA (mRNA), cleavage mRNA or inducing the expression of specific target genes [44, 45]. This type of functional molecule is commonly found in PDEVs from ginger, grapes, broccoli, and apples [46]. In the fields of cell therapy targeting and cross-border communication, microRNAs show unique regulatory value [47]. In vitro experiments have confirmed that the miRNA carried by PDEVs can effectively regulate the expression levels of inflammatory cytokines and cancer-related genes. Its action mechanism also includes targeting a variety of bacterial genes in the host microbiome, thereby regulating host microecology balance [18, 48]. Based on the silicone prediction model, the researchers identified 418 conserved microRNAs from 11 edible fruits and vegetables [49]. Bioinformatics analysis shows that the vast majority of microRNAs have the potential function of targeting and regulating key genes of human immune response and cancer signaling pathways [49]. Studies have demonstrated that host Arabidopsis cells secrete exosome-like extracellular vesicles to deliver siRNAs into fungal pathogen Botrytis cinerea [50]. This mechanism reveals that Arabidopsis enhances antifungal immunity through an EV-mediated cross-species RNA interference mechanism. However, identifying microRNAs in PDEVs faces technical challenges, mainly due to the lack of complete precursor microRNA sequence data for most plant species [51]. Combining existing evidence shows that microRNAs in PDEVs show important application prospects in regulating gene expression in specific pathological processes.

Metabolites

Plants produce large amounts of secondary metabolites that affect human health when consumed. We highlight currently reported metabolites in PDEVs from important plant sources. PDEVs extracted from broccoli have been shown to contain sulforaphane, with limited evidence suggesting it may have cancer-protective effects [52]. Grapefruit PDEVs include naringenin, fructose, citric acid, glucose, sucrose, myo-inositol, quinic acid, oxalic acid, glycolic acids, and aucubin, along with the amino acids leucine and isoleucine [48, 53, 54]. Notably, naringenin is recognized for its anti-tumor properties [55]. Strawberry PDEVs are particularly high in ascorbic acid [56]. EVs from apples contain flavonoids and furanocoumarins, which are known to be toxic to certain fungi and insects [57]. PDEVs from Javanese ginger and turmeric are rich in curcuminoids, celebrated for their antioxidant effects [53]. Tobacco-derived PDEVs include alkaloids and phenolics, while EVs from aconitituber contain small amounts of aconitine, hypaconitine, and mesoaconitine, which are toxic [54]. Overall, PDEVs are rich in diverse metabolites depending on their source, indicating significant potential for future therapeutic uses.

Isolation and purification of PDEVs

Before the formal separation process, plant soft tissues can be mechanically crushed to extract juice [58]. The apoplast refers to the space outside the plasma membrane of plant cells and includes the intercellular spaces and cell walls [27, 59]. Infiltration fluid is initially introduced into the plant tissue (typically leaves or roots) via a pressure gradient (e.g., manual pressurization). Subsequently, the infiltrated plant tissues are centrifuged at low speeds ranging from 700 to 5000 g to extract the apoplast washing fluid. Notably, it has been documented that root exudates from hydroponically grown plants can also be collected for the isolation of PDEVs [60]. To achieve this, the nutrient solution is replaced with ultrapure water to facilitate the collection of root exudates, which are then processed for PDEVs isolation. Common extraction methods include tissue grinding, pressing treatment, and multi-step purification processes such as ultracentrifugation, molecular exclusion chromatography, ultrafiltration technology, and immunoaffinity capture to achieve effective separation of PDEVs [61–63]. These methods leverage the physical characteristics of EVs (such as size, density, charge, solubility and surface protein distribution) to achieve efficient separation of vesicles from biological pollutants [64, 65]. Different methods for separating PDEVs have different yields and unique advantages and disadvantages. Researchers should make their choices based on experimental requirements (Fig. 2, Table 1).

Fig. 2.

Separation of PDEVs by ultrafiltration centrifugation, density gradient centrifugation, sedimentation, size exclusion chromatography, and immunoaffinity capture techniques Procedures and advantages and limitations of each method

Table 1.

The yield of PDEVs was isolated from different plants by different methods

Plant source	Isolation method	Yield	References
Strawberry	UC	18 ± 3 μg/250 ml juice	[56]
Ginger	UC	0.72 × 1010 particles/ml juice	[62]
Citrus limon	UC	3.38 × 1011 particles/mL juice	[65]
Ginseng	GUC	~ 500 mg/kg ginseng	[60]
ginger	GUC	6 × 107particles/mL juice	[72]
Ginger	UC/GUC	~ 50 mg/kg of ginger	[17]
Ginger	UC/GUC	4.2 × 109 particles/g ginger	[46]
Tomato	UC/GUC	3.8 × 1016 particles/kg tomato	[67]
Grape	UC/GUC	1.76 ± 0.15 g/kg Grape	[16]
Grapefruit	UC/GUC	2.21 ± 0.044 g/kg Grapefruit	[16]
Tomatoes	UC/GUC	0.44 ± 0.02 g/kg Tomatoes	[16]
Cabbage	SEC	1.504 × 1011 particles/ml juice	[62]
Red Cabbage	SEC	1.098 × 1011 particles/ml juice
Cucumber	SEC	0.263 × 1011 particles/ml juice
Pepper	SEC	0.135 × 1011 particles/ml juice
Tomato	SEC	0.119 × 1011 particles/ml juice
Ginger	Precipitation method	2–3.8 g/kg ginger	[74]
Garlic	Precipitation method	Not reported data	[75]
Arabidopsis	Immunoaffinity capture	Not reported data	[76]

Ultrafiltration centrifugation (UC)

Currently, ultracentrifugation is the most widely used separation method of EVs and is called the “gold standard” for the extraction and separation of EVs. Ultracentrifugation technology is based on the density and particle size of various components in the stock solution to obtain the required components, the technology is suitable for processing the separation of large-dose samples with significant differences in sedimentation coefficients. Today, ultracentrifugation separation technology is widely used to extract a wide range of plant EVs, including ginger, lemon, coconut, kiwi, cantaloupe, grapefruit, orange, tomato, blueberry, pea, and pear [66, 67]. With this method, labeling of EVs is not necessary, thus avoiding the risk of cross-contamination; in addition, it has advantages such as simple operation and low cost. However, ultracentrifugation requires multiple centrifugation steps (e.g., differential centrifugation + density gradient centrifugation) and the final recovery may be less than 30%. For low-concentration samples (e.g., serum, urine, etc.), a large amount of starting material may be required to obtain sufficient exosomes, limiting the application to small clinical samples. During high-speed centrifugation, other non-PDEV components (e.g., protein aggregates, lipoproteins, apoptotic vesicles, cellular debris, etc.) may be co-precipitated due to similar densities or sizes, which can affect downstream analysis (e.g., proteomics or RNA sequencing). To address this limitation, numerous researchers have integrated UC with density gradient centrifugation to achieve a higher purity in isolating PDEVs [68, 69]. In addition, the experiments are time-consuming, involve cumbersome steps, and impose high technical requirements on operators [70].

In summary, plant juice or extracts containing PDEVs undergo multiple rounds of centrifugation at progressively higher speeds to separate particles of different sizes and sedimentation rates. Initially, the juice is centrifuged at low speeds (500–10,000 g) to eliminate larger materials like fruit pulp and debris. The resulting supernatant is then subjected to higher speeds (40,000–100,000 g) for extended periods to remove smaller contaminants, allowing for the collection of PDEVs as a pure pellet [65]. Currently, there are no standardized UC protocols for isolating PDEVs, as various studies employ different centrifugation steps, speeds, and durations [71].

Density gradient centrifugation (DGC)

DGC is often used in conjunction with ultracentrifugation separation techniques to purify EVs by using the difference in sedimentation coefficients of different particles to form multiple bands. Gradient ultracentrifugation was conducted on samples enriched with nanometer-sized vesicles, which were isolated through differential ultracentrifugation (dUC) using a continuous sucrose gradient ranging from 8 to 45% (w/v) in a polypropylene ultracentrifugation tube. A sample containing 5 mg of protein was mixed with 500 µL of extraction buffer by stirring and pipetting, then centrifuged at 100,000 × g for 2 h at 4 °C. Six fractions were collected from the top to the bottom: Fr1 (5 mL), Fr2 (7 mL), Fr3 (Band 1, 7.5 mL), Fr4 (4.5 mL), Fr5 (Band 2, 3 mL), and Fr6 (11 mL). Each fraction was washed to eliminate sucrose by centrifuging at 100,000 × g for 1 h at 4 °C in extraction buffer. The resulting pellets were then resuspended in small amounts of extraction buffer [68].

Because of its high purity and economic advantages, sucrose d DGC technology has become a key separation method for plant-source EVs research. However, the high-viscosity sucrose solution may slow down the sedimentation rate of EVs, resulting in a longer precipitation time for EVs. Many plants, such as Asparagus, ginger, ginseng, and lemon, used this technique in the purification process of EVs [61, 72–74]. DGC is superior to differential ultracentrifugation in terms of purity but is still limited by low recoveries, operational complexity, and potential damage. This approach is suitable for studies requiring high purity and a sufficient sample size. However, in clinical translational or high-throughput settings, more efficient and standardized alternatives must be explored.

Precipitation method

The polymer precipitation technique has the advantages of easy operation and short time consumption, which is especially suitable for the determination of large-scale samples. However, the purity and recycling efficiency of EVs prepared by the current polymer precipitation technique are not high, which is prone to false positives, and the resulting polymer is not easy to completely remove, which is unfavorable to the subsequent functional assay. On this basis, polyethylene glycol (PEG) is suitable for the separation and enrichment of edible nanoparticles in ginger [75]. The researchers used an RNA purification and extraction kit to extract RNA from garlic-derived EVs [76]. This technique takes advantage of the reduced solubility of compounds in solutions containing superhydrophilic polymers, such as PEGs. The process involves combining the sample with the polymer solution, allowing for incubation, and then using low-speed centrifugation (1500 × g) to sediment the EVs. The resulting EV pellet is subsequently resuspended in PBS for additional analysis. It is worth noting that this technique can extract EVs from smaller sample sizes, making it more advantageous when extracting EVs from valuable or difficult-to-obtain plant samples. Precipitation methods are suitable for rapid crude extraction of PDEVs (e.g., initial screening or studies with large sample sizes), but their low purity and high impurity contamination limit the reliability of precision studies. For high-purity PDEVs (e.g., mechanism exploration or clinical diagnosis), it is recommended to combine with other purification techniques or choose alternative methods. Researchers have found that combining ultracentrifugation with the ExoQuick system based on polymer precipitation significantly improves the colloidal stability and separation purity of PDEVs [77].

Size exclusion chromatography

Size exclusion chromatography (SEC) is a method to separate EVs from other components in biological samples based on their size difference [78]. SEC is an effective technique for purifying EVs without the use of any chemical reagents. Microvesicles that were isolated using dUC and suspended in the correct volume of extraction buffer (500 µL for the 10 mL column and 250 µL for the 5 mL column) were introduced into the SEC column. A total of 30 fractions were collected, with each fraction measuring 500 µL for the 10 mL column and 250 µL for the 5 mL column, using extraction buffer for elution [68].

However, SEC is costly, requires specialized equipment, and imposes column usage frequency and spiking volume. Additionally, it is a more complex technique due to the co-separation of protein aggregates and lipoproteins [79, 80]. The main advantages of SEC include high efficiency, ease of operation, and shorter time consumption, which can improve the efficiency of EVs production and ensure the integrity of EVs. SEC is superior to ultracentrifugation and sedimentation in preserving the integrity of PDEVs and operational simplicity. SEC is one of the best methods for the separation performance of PDEVs and is combined with ultrafiltration or ultracentrifugation [81]. However, it remains limited by insufficient resolution, sample dilution, and low throughput. This method is suitable for studies with moderate purity requirements and small sample sizes (e.g., functional experiments). However, for ultra-high purity or large-scale clinical samples, it must be combined with other techniques or optimized separation strategies.

Immunoaffinity capture technology

Immunoaffinity capture technology, as a commonly used means of biocontainment and detection, is based on highly specific binding between antibodies and their specific antigens. The storage environment of EVs obtained by the immunoaffinity capture technique is stringent, so this technique is not suitable for isolating EVs on a large scale [82, 83]. Researchers used the immunoaffinity capture technique and sucrose density gradient centrifugation to isolate Arabidopsis-derived EVs and detected abundant nuclear small RNAs in the captured EVs, including TAS1c-small interfering RNA483, TAS2-small interfering RNA453, and tiny RNA166 [84]. Capture methods are suitable for high-specificity studies (e.g., specific PDEVs subpopulation analysis or diagnostic marker development). However, they are ill-suited for histologic studies or large-scale clinical applications due to high costs, incomplete target coverage, and potential sample damage. The choice should be weighed against the experimental objectives, or combined with non-marker-dependent methods to improve comprehensiveness.

Other methods

Several emerging techniques have facilitated the successful separation of PDEVs in experiments, providing convenience for future research and applications. Jackson et al. utilized a capillary channel polymer tip separation method based on hydrophobic interaction chromatography, achieving effective PDEVs separation in under 15 min and at a cost of less than $1, while ensuring purity and integrity [85]. Yang et al. integrated electrophoresis with a 300 kDa cut-off dialysis bag to effectively separate lemon-derived EVs, which were comparable in size and quantity to those obtained through traditional ultracentrifugation, resulting in considerable time and cost reductions [86]. Kırbaş et al. successfully isolated various biogenic PDEVs, including those from pomegranates, using a biphasic system that was both cost-effective and gentle, efficiently eliminating non-protein impurities [87]. It is important to note that the variability among plant species is much greater than that among animals, leading to notable differences in the size, structure, yield, purity, and dispersibility of EVs from different plants. Currently, there is no standardized method for isolating PDEVs, which poses a challenge for their clinical applications.

Therapeutic potential of PDEVs

The cross-species communication characteristics make PDEVs show significant clinical value, and its multi-component synergistic effect has stronger therapeutic potential than single-component drugs [54]. Compared with artificial synthetic delivery systems, PDEVs can effectively prevent epidemic elimination and maintain structural stability with their natural biocompatibility advantages, significantly improving drug bioavailability [88]. Studies have demonstrated that PDEVs can effectively penetrate biological barriers, such as the blood–brain barrier, without causing inflammation or necrosis. The latest scientific research progress has revealed diverse biological activities of PDEVs, including inhibiting tumor growth, regulating inflammatory response, maintaining intestinal homeostasis, and promoting anti-infective tissue regeneration [49, 89, 90] (Fig. 3). However, immune interactions and biodistribution of PDEVs during their application represent critical challenges that directly impact their safety and therapeutic efficacy. Current studies have shown that immunogenicity can be reduced by surface modifications. For example, polyethylene glycol modification reduces the recognition of EVs by the mononuclear phagocytic system and prolongs blood circulation time [91]. The biodistribution problem can be solved by targeting modification, e.g., RGD peptide-modified EVs can enhance tumor targeting [92].

Fig. 3.

Therapeutic potential and potential mechanisms of PVEDs in Anti-Tumor, Anti-Inflammation, Intestinal Homeostasis, and Regenerative Potential

Anti-tumor

PDEVs have been shown to play a pivotal role in suppressing tumor growth. Research on the anti-tumor mechanism of PDEVs shows that extracellular vesicles from different plant sources can not only regulate key biological behaviors such as proliferation, apoptosis, metabolic activities, and drug resistance of tumor cells but also reshape the tumor microenvironment. It is worth noting that this dual mechanism of action did not significantly affect normal cells [93, 94]. This multi-target action model significantly delays the development of malignant tumors and reduces the invasion and metastasis ability of tumor tissues [95, 96].

The particles of PDEVs extracted from lemon juice showed selective inhibition of tumor cell proliferation and had no significant effect on normal cell activity in vitreous [26]. Research has revealed that this mechanism of action is related to the activation of the TRAIL/Dr5 signaling pathway, and a specific pro-cancer apoptotic effect is achieved by inducing tumor necrosis factor-associated apoptotic ligand. TRAIL-mediated apoptosis pathway is characterized by precise targeting of cancer cells without damaging healthy cells [97, 98]. In a study conducted by Yan et al., Brucea javanica-derived EVs were demonstrated to deliver 10 functional microRNAs into 4T1 cells. These miRNAs effectively inhibited breast tumor growth by targeting the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway and promoting reactive oxygen species-and caspase-mediated apoptosis. Additionally, the PDEVs were found to modulate the physiological functions of endothelial cells, thereby suppressing vascular endothelial growth factor-mediated angiogenesis [99]. Experimental data show that PDEVs derived from lemon can effectively inhibit the growth of living tumors. PDEVs isolated in other citrus plants also exhibit this selective antitumor activity, indicating that this characteristic is universal in citrus plants [100]. This targeted anti-proliferative and pro-apoptotic mechanism can significantly mitigate the side effects arising from non-specific effects in traditional treatments. By ensuring therapeutic efficacy while enhancing treatment safety, it provides a novel research direction for cancer therapy.

It is worth noting that PDEVs show synergistic effects with current anti-cancer treatments. Chemotherapy may trigger inflammatory responses related to drug resistance and metastasis [101]. Taking oral squamous cell carcinoma (OSCC) as an example, the expression of protein No. 3 containing the nucleotide-binding domain of the NLR family may be unexpectedly upregulated due to chemotherapy drugs. This phenomenon also leads to drug resistance to 5‐fluorouracil (5-FU), which is often closely related to tumor progression and metastasis of OSCC [102, 103]. When bitter gourd source PDEVs and 5-FU are combined, this synergistic treatment plan significantly improves the efficacy of oral squamous cell carcinoma by enhancing the antiproliferative effect and cytotoxic effects. Compared to single-agent treatment with 5-FU or bitter gourd-derived PDEVs, the combined treatment group showed a more significant tumor suppressive effect, with increased programmed cell death observed in OSCC cells.

Anti-inflammation

PDEVs isolated from several sources are reported to modulate the expression of inflammation-related genes and ameliorate the inflammatory response [104, 105]. Inflammation is a defensive physiological response caused by the body to respond to external stimuli. However, an overactivated inflammatory response can cause acute and chronic diseases and cause tissue damage. Experimental data show that miR-396e-rich PDEVs significantly affect the regulation of macrophage metabolic pathways by targeting PFKFB3 protein, and achieve obesity prevention through the interaction mechanism between macrophages and adipocytes. Further studies have shown that this vesicle component can also regulate lipid metabolism processes through a macrophage-hepatocyte interaction network [106]. It is worth noting that grapefruit-derived EVs can specifically regulate the expression level of E-cadherin in intestinal epithelial cells. If the expression of this core protein that mediates intercellular adhesion junction is abnormal, it will directly lead to impaired intestinal barrier function and homeostasis imbalance [107]. Ginger-derived PDEVs, which also possess medicinal value, exhibit unique anti-inflammatory properties by bidirectionally regulating cytokine expression in both directions-inhibiting pro-inflammatory factors and activating pro-healing and anti-inflammatory factors. In addition, this type of nanovesicle can also effectively promote the survival and proliferation of intestinal epithelial cells [17].

PDEVs extracted from Panax notoginseng help reduce cerebral ischemia/reperfusion injury by promoting M2 polarization in microglia, a process that is partially influenced by lipid components [108]. PDEVs from Portulaca oleracea reduce pro-inflammatory cytokines, increase Interleukin-10 levels, and improve acute colitis in mice, showing potential for modulating gut microbiota in ulcerative colitis treatment [56]. In terms of mechanism, treatment with PDEVs modifies microbial metabolism and reprograms conventional CD4 + T cells into double-positive CD4 + CD8 + T cells. Additionally, PDEVs from ginger and lemon activate nuclear factor erythroid 2-related factor 2, which helps protect against oxidative damage [109].

Intestinal homeostasis

PDEVs contribute to intestinal homeostasis and have become a hot topic of research in the field of intestinal diseases. Due to the long-term exposure of the human digestive tract to PDEVs in edible plants, this naturally sourced therapeutic agent has significant clinical application advantages. The latest research reveals that PDEVs carrying microRNAs can be uptaken by intestinal microorganisms. This cross-border regulation can not only change the metabolic pattern of bacterial flora but also affect physiological functions through the microbial-host interaction mechanism [110, 111]. It is worth noting that turmeric extracts showed significant intestinal microbiota regulation in animal models. In the colitis model induced by dextran sodium sulfate, this substance can effectively restore the species abundance of intestinal microorganisms and improve community diversity, providing new ideas for the treatment of inflammatory bowel disease [112]. Research shows that PDEVs play multiple regulatory roles in intestinal health. PDEVs significantly alleviate colitis-related symptoms by repairing intestinal epithelial barrier function, inducing M2 macrophage polarization, and regulating the immune microenvironment [113]. Core gene analysis shows that PDEVs from buckwheat can regulate the key genes of the metabolic pathways of E. coli and Lactobacillus rhamnosus, effectively promoting the proliferation of intestinal microbial flora and enhancing microbial diversity [114]. Garlic-derived EVs can significantly inhibit the TLR4, MyD88 and NF-κB signaling pathway activities in DSS-induced colitis models while reducing the levels of proinflammatory factors. Notably, the characteristic component Peu-miR2916-p3 in EVs can specifically promote the proliferation of anti-inflammatory bacterial populations. These bacterial populations are closely associated with ameliorated colitis symptoms, revealing a synergistic mechanism among different components of PDEVs [115].

Regenerative potential

The regenerative potential of PDEVs has been widely investigated, encompassing wound healing, cell differentiation and tissue repair. In the field of tissue repair, ginseng PDEVs effectively promote skin cell proliferation, migration, and angiogenesis by activating the ERK and Akt/mTOR signaling pathways. Animal experiments demonstrate their dual effects of accelerating wound healing and alleviating inflammatory responses [116]. Studies have shown that nanovesicles derived from Astragalus can effectively enhance the proliferation ability of human bone marrow mesenchymal stem cells and significantly improve the expression level of estrogen receptor α. Experimental data show that this nanovesicle promotes the differentiation of human bone marrow mesenchymal stem cells into osteoblasts by upregulating the expression of bone morphogenetic protein 2 and the key osteogenesis transcription factor runt-related transcription factor 2. Zhao’s team confirmed in animal experiments that Gouqi-derived nanovesicles (GqDNVs) significantly improve the cross-sectional area and grip strength performance of the quadriceps femoris in the dexamethasone-induced muscular atrophy model by activating the AMPK pathway [117]. Metabolomic analysis revealed that GqDNVs exhibit excellent muscle regeneration therapeutic potential by enhancing the oxidative phosphorylation metabolic pathway [118].

In two distinct studies, EVs from wheat and grapefruit were found to promote skin regeneration by stimulating the proliferation and migration of human epidermal keratinocytes (HaCaT) in a dose-dependent manner [119, 120]. Both types of PDEVs also enhanced the formation of tube-like structures in human umbilical vein endothelial cells, indicating their proangiogenic properties and potential to facilitate vascular development during wound healing. Additionally, both studies noted that the wheat and grapefruit-derived EVs increased the mRNA levels of collagen type I in human dermal fibroblasts and HaCaT cells. Further research revealed that the grapefruit-derived EVs promoted the upregulation of several wound-healing factors, such as laminin, fibronectin, vimentin, and epidermal growth factor. Overall, these findings suggest that both wheat and grapefruit-derived EVs enhance skin regeneration by improving wound healing and closure [119, 120].

Potential applications as drug delivery systems

The lipid layer and internal aqueous phase of PDEVs endow them with the capability to encapsulate exogenous hydrophilic or hydrophobic molecules, such as chemotherapeutic drugs, mRNAs, miRNAs, siRNAs, proteins, while shielding them from degradation (Table 2) [21, 121–128]. It is worth noting that the latest research has proved that plant-derived vesicles have drug delivery capabilities, providing an important theoretical basis for their application as a new nanocarrier for clinical transformation [15, 129, 130]. Developing specific drug delivery mechanisms is crucial to improving treatment options for patients with various diseases [129–131]. Table 3 summarizes the safe dosages of PDEVs administered by different administration methods [8, 108, 132–136]. Dad et al. reported that PDEV can be used as a drug delivery system in two different ways [21]. Following isolation, these vesicles can be directly utilized for drug loading and delivery or modified into lipid-derived plant-derived nanovectors (PDNVs) (Fig. 4). PDEVs are becoming a new research hotspot to achieve this goal due to their low toxicity and easy penetration of mammalian barriers, and there is evidence that they may become an excellent nano delivery carrier.

Table 2.

Cargo encapsulation of PDEVs and enhanced therapeutic effects

Plant	Cargo	Disease model	Therapeutic effect or function	References
Grapefruit	HSP70	–	Dose-dependent cell protection from the etoposide-induced cytotoxicity	[121]
	Anti-luciferase siRNA	–	Suppress luciferase expression	[122]
Watermelon	miR146a-5p	Ovarian cancer	Regulation of IRAK1 and SERPINE1 expression to produce anti-angiogenic effect	[123]
Broccoli	miR159a miR159b-3p miR166b-3p miR403-3p	–	–	[124]
Kiwifruit	Sorafenib	HepG2 tumor xenograft model	Reduced dose-associated toxicity, hepatic targeting and uptake	[125]
Tomato	Curcumin	–	Inhibiting the expression of IL-1β and IL-6 on LPS-treated THP-1 Cell Line	[126]
Orange	mRNA	COVID-19	Delivering mRNAs into macrophages and expressing N, S1 and FS proteins, inducing a specific humoral and cell-mediated immune response in vivo	[127]
Ginger	siRNA-CD98	Ulcerative colitis	Specifically targeting colon and mediating CD98 gene inhibition	[128]
Celery	Doxorubicin	A549 subcutaneous tumor model	Lower toxicity and better anti-tumor efficacy	[21]

Table 3.

The safe dosage of PDEVs administered in different ways

Administration method	Safe dose range (for mice)	Advantage	Disadvantage	References
Oral	2–25 mg/kg	High safety, natural path, and good tolerance	Poor absorption and low utilization	[132, 133]
Intravenous injection	0.5–5 mg/kg	100% bioavailability	Immune response and embolism risk	[108, 134]
Intraperitoneal injection	~ 1–5 mg/kg	The technology is simple	There may be local irritation	–
Nasal/inhalation	5–20 μg per piece	Non-invasive	Local mucosal irritation	[135]
Smear	10–100 μg/mL	Extremely high security	Only for local treatment	[8]
Subcutaneous injection	3.75 mg/kg	Simple, local high concentration	The site of action is limited	[136]

Fig. 4.

PDEVs as drug delivery carriers. Drugs can either be loaded directly into unmodified PDEVs or into modified PDNVs

For instance, You et al. treated SW480 colon cancer cells with PDEVs derived from cabbage and red cabbage loaded with the chemotherapy drug doxorubicin (Dox) [63]. The isolated PDEVs were incubated with Dox for 4 h at 37 °C to obtain Dox-loaded PDEVs. After 72 h, it was reported that Dox-loaded cabbage-derived PDEVs could reduce cell viability of SW480 cells to 57.5%, comparable to the 61.0% with Dox only [63]. Grapefruit, as a plant source, has been proven to be used for nano drug delivery, and its derived nanoparticles are more efficient than liposomes in delivering the anti-inflammatory drug methotrexate, and show stronger mouse intestinal macrophage targeting capabilities [15]. Experiments have confirmed that the symptoms of colitis mice induced by dextran sulfate sodium were significantly relieved, which was manifested as a slowdown in weight and an improvement in colon shortening [15]. Coincidentally, scientific experiments have confirmed that exosomes extracted from grapefruit can accurately deliver DNA and proteins (including antibody components) to target cells without triggering a cytotoxic reaction. Experiments have also confirmed that when chemotherapy drugs such as Dox are targeted to tumor transport through plant EVs (such as grapefruit EVs), they can not only improve the drug delivery efficiency but also significantly enhance the therapeutic effect [129]. Beyond the direct use of PDEVs for drug delivery, scientists have also utilised lipids extracted from PDEVs to design nanovesicles known as PDNVs. Currently, researchers extract total lipids from the PDEVs by the Bligh and Dyer method. Briefly, the lipids of PDEVs are extracted using 2:1 (v/v) methanol: chloroform. Chloroform and water are then added, and the mixture is centrifuged to separate the organic phase. The organic phase is dried under nitrogen. The dried lipids are then resuspended in a buffer and sonicated before extrusion to form PDNVs. The PDNVs produced exhibit more uniform size distribution and display a spherical morphology under electron microscope [129]. These pioneering research results have established key theoretical support for the application of PDEVs in the field of nanomedicine.

Advantages and challenges

The interdisciplinary value shown by PDEVs research continues to stimulate scientific research enthusiasm, and its systematic development platform has made breakthrough progress in dimensions such as regenerative medicine, disease intervention, precise drug delivery, and diagnostic technology [137]. PDEVs are similar to animal-derived EVs, but they differ in physical properties, composition, distribution, and as therapeutic and drug delivery carriers (Table 4). Compared with conventional EVs, PDEVs show the following core advantages: (1) Their excellent biocompatibility, tissue penetration, and physical and chemical stability endow PDEVs with the ability to cross multiple physiological barriers, transmit biological information across species, and directly exert anti-inflammatory and anti-cancer effects in lesion sites as regulatory mediators; (2) PDEVs from natural sources have significant advantages over synthetic nanoparticles, including higher bioadaptation, structural stability, targeted delivery efficiency, circulating half-life and cellular uptake capacity. By carrying diverse therapeutic loads such as small molecule compounds, nucleic acid sequences, or recombinant proteins, the system can be developed into a cost-effective drug delivery platform; (3) Based on the abundant reserve of plant resources, PDEVs have the potential for large-scale industrial production [138]. Completed early-stage clinical studies involving PDEVs are now available. A preliminary clinical study (NCT04270023) evaluating apple-derived EVs for skin repair and anti-aging has been completed but has not yet been published. In addition, a Phase I trial (NCT04879810) is evaluating the safety of curcumin-derived EVs in patients with ulcerative colitis. However, currently, no Phase III or large-scale Phase II trials explicitly focusing on “PDEVs” are registered on ClinicalTrials.gov. The existing early-stage trials primarily focus on safety assessments. As the research on PDEVs becomes increasingly in-depth, the number of related patents is also on the rise. Table 5 summarizes the patents related to PDEVs in recent years.

Table 4.

Comparison between PDEVs and EVs of animal origin

Comparison type	PDEVs	Animal-derived EVs
Physical properties
Size	Overlaps with animal EVs, but some PDEVs may be larger	30–1000 nm
Membrane structure	Phospholipid bilayer, but containing plant-specific lipids (such as phosphatidic acid)	Phospholipid bilayer, containing cholesterol and sphingomyelin (a lipid specific to animals)
Stability	Acid and alkali resistant, high temperature (stronger adaptability to the plant’s living environment)	Sensitive to pH and temperature (easily degraded by protease)
Composition
Protein	Plant-specific proteins and metabolic enzymes	Transmembrane proteins, cell adhesion molecules, etc
Lipid	Phytosterols (such as β -sitosterol), phosphatidic acid	Cholesterol, sphingomyelin, phosphatidylserine
Nucleic acid	Plant miRNA (possibly cross-regulated), siRNA, mRNA	Animal miRNA, mRNA, lncRNA (stem cell EVs) have repair functions; Tumor EVs contain metastasis promoting factors
Others	Secondary metabolites (such as flavonoids and terpenoids)	Cytokines, growth factors
Distribution and source
Source	Secretions from fruits, leaves and roots	Stem cells, tumor cells (such as breast cancer, glioma)
Distribution	Plant body fluids (such as SAP, fruit juice), culture supernatants	Body fluids (blood, urine), cell culture supernatants, tissues
Release mechanism	Cell wall modification or secretion pathway release	Escrt-dependent/non-dependent pathways, plasma membrane germination
Drug carrier
Biocompatibility	Low immunogenicity (no risk of animal pathogens)	Autologous EVs have high immune compatibility (but allogeneic EVs may cause rejection)
Targeting	Natural tropism (such as intestinal targeting); It can be engineered and modified	Dependent on the characteristics of donor cells (such as natural targeting of tumor EVs to homologous tumors)
Production cost	Easy to scale up (low cost of plant cultivation)	The cost of stem cell culture is high. Tumor EVs need to be purified to remove carcinogenic components
Safety	No ethical disputes; No infectious pathogens	Strict screening is required (such as possible residual cancer-promoting substances in tumor EVs)

Table 5.

Patents related to PDEVs in recent years

Patent Number	Country	Year	Name
CN116687966	China	2023	Medicament for treating beriberi, separation method and containing device
CN117379565	China	2024	Plant-derived extracellular vesicles as well as content modification method and application of plant-derived extracellular vesicles
JP2025020285	Japan	2025	Plant-derived extracellular vesicle (Ev) composition and use of the same
KR1020190037162	Republic of Korea	2021	Composition including plant-derived extracellular vesicles
RU0002812553	Russian Federation	2024	compositions of extracellular vesicles (EVs) of plant origin and their use
CN115671146	China	2023	Plant-derived extracellular vesicles, uses thereof and products comprising same
KR102527607	Republic of Korea	2023	Plant-derived extracellular vesicles, composition containing same, and manufacturing method thereof
AU2022207638	Australia	2022	Composition comprising engineered plant-derived extracellular vesicles and use thereof as a vaccine
US20220142938	United States	2022	Plant-derived extracellular vesicle (EVs) compositions and uses thereof
WO2024143644	Republic of Korea	2022	Surface-modified plant-derived extracellular vesicles and targeted drug delivery system using same
WO2019066121	Republic of Korea	2017	Composition comprising plant-derived extracellular vesicles
US20250009827	United States	2025	Pharmaceutical, food, and cosmetic compositions and products

Although PDEVs have significant advantages, they still face multiple technical bottlenecks. First, while the current study confirms their multifunctional characteristics, numerous aspects of their mechanism of action remain poorly understood. For example, the interaction mechanism and specific modification principles of receptor cells are not yet clear, and the molecular mechanism of the endocytic pathway still needs to be further analyzed. Secondly, the complex biomolecules carried by PDEVs may induce potential adverse reactions, so systematic clinical trials must be completed before clinical application, focusing on verifying their pharmacokinetic characteristics, safety thresholds, formulation stability, and therapeutic efficacy. Short half-life, natural PDEVs are readily and rapidly cleared by the mononuclear phagocytosis system; non-specific enrichment: > 80% of the injected dose accumulates in the liver/spleen and only < 5% reaches the target tissue (e.g., tumor). Additionally, exosome separation technology lacks a standardized process, and different extraction methods lead to significant sample heterogeneity, which directly affects the repeatability of experimental data. Furthermore, there are significant challenges to regulatory compliance, as EVs are neither traditional drugs nor biosimilars, and the Food and Drug Administration (FDA)/European Medicines Agency (EMA) has yet to clarify their regulatory path. At the same time, PDEVs applications may face security issues. Source and preparation safety: If PDEVs are derived from heterologous cells, they may carry pathogens, carcinogens, or immunogenic molecules, leading to cross-infection or immune rejection. Cell fragments, apoptotic bodies, or endotoxins may be mixed in during the extraction process. Long-term retention of PDEVs may affect organ function (e.g., kidneys, liver), but there is currently a lack of long-term follow-up data. Finally, the research on plant exosome marker proteins is still in its early stages, and establishing a complete proteome analysis system will become a key breakthrough in cracking its biological functions [16, 139]. This limitation affects the accurate characterization of PDEVs.

Tackling the challenges mentioned above is crucial for research on PDEVs. Researchers must develop standardized methods for separation and purification, improve extraction techniques by combining differential centrifugation with density gradient centrifugation (like sucrose gradients) to increase purity, and create plant-specific affinity chromatography approaches (such as using lectin columns to capture PDEVs surface glycans). It is also important to address cross-species safety evaluations-for example, in gene-edited plant-derived cells-by removing immunogenic elements and building a plant miRNA host target database to predict potential off-target effects. Additionally, establishing quality control standards, identifying key biomarkers for PDEVs, forming international consensus guidelines, consulting FDA and EMA regulations for nanomedicines, and enhancing safety data for plant-derived substances are essential steps. Research in the field of PDEVs has significant research value, and these challenges are driving scientists toward more targeted and innovative solutions.

Conclusion

In summary, PDEVs represent a promising frontier in nanomedicine. This article summarizes the molecular composition, separation methods, biological functions, and therapeutic potential of PDEVs, and discusses the current research advantages and challenges. After decades of exploration and accumulation, PDEVs have developed into highly anticipated natural biologically active substances. This type of nanoparticle has excellent biological characteristics, including low immunoreactivity, strong stability, and excellent biological barrier penetration ability. As a therapeutic formulation, PDEVs exhibit significant efficacy in a variety of animal models; as a delivery vehicle, they can accurately transport anti-inflammatory and anti-tumor drugs to targeted sites. Although current research still faces technical bottlenecks, such as the need to optimize protocols for efficiently preparing high-purity PDEVs, the insufficient characterization of surface marker proteins, and the unclear mechanisms of action, the medical value of PDEVs has already begun to emerge. This biomaterial, which integrates multiple advantages, is emerging as a promising frontier in modern biomedicine and warrants continuous in-depth exploration by researchers.

Abbreviations

EVs

Extracellular vesicles

PDEVs

Plant-derived extracellular vesicles

HSP

Heat stress protein

PA

Phosphatidic acid

PE

Phosphatidylethanolamine

PC

Phosphatidylcholine

UC

Ultrafiltration centrifugation

DGC

Density gradient centrifugation

dUC

Differential ultracentrifugation

PEG

Polyethylene glycol

SEC

Size exclusion chromatography

PI3K

Phosphatidylinositol 3-kinase

mTOR

Mammalian target of rapamycin

ROS

Reactive oxygen species

OSCC

Oral squamous cell carcinoma

5-FU

5‐Fluorouracil

GqDNVs

Gouqi-derived nanovesicles

HaCaT

Human epidermal keratinocytes

PDNVs

Plant-derived nanovectors

Dox

Doxorubicin

FDA

Food and drug administration

EMA

European medicines agency

Author contributions

DFH, JC and MHZ searched for literature and wrote the first draft of this article. HBS, QJ, DWX, ZOP, TC and YLZ edited the manuscript. DYR and MJL reviewed the manuscript and polished the grammar. All authors contributed to the article and approved the submitted version.

Funding

This work was supported by the Ganzhou Science and Technology Plan Project (2023LNS37081 and GZ2024YLJ137), Jiangxi Provincial Health Commission Technology Plan Project (202510463) and Jiangxi Province Administration of Traditional Chinese Medicine Scienceand Technology Plan Project (2024B0303).

Data availability

Not applicable.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

All authors are aware and have agreed to publish.

Competing interests

The authors declare no conflict of interest. All authors agreed and reviewed the manuscript.