Extracellular vesicles derived from Citrus plants: innovative applications in therapeutics and drug delivery

Abstract

Citrus-derived extracellular vesicles (Citrus-EVs) have attracted increasing interest as naturally occurring nanovesicles that possess intrinsic bioactivity and hold promise for drug delivery applications. Sourced from edible Citrus plants, these vesicles exhibit a lipid bilayer structure enriched with plant-specific proteins, nucleic acids, lipids, and secondary metabolites, which confer high biocompatibility, low immunogenicity, and favorable stability in physiological environments. This review summarizes recent progress in the isolation, purification, and characterization of Citrus-EVs, highlighting methodological approaches and current technical limitations. We also outline their demonstrated biological functions, including antitumor, anti-inflammatory, immunomodulatory, antioxidant, and gut microbiota-modulating activities, and discuss their emerging utility as versatile drug carrier platforms. In addition, we evaluate major strategies for cargo loading and delivery, along with representative applications in the therapeutic administration of small-molecule drugs, nucleic acids, and proteins. Finally, key challenges related to standardization, mechanistic insight, and clinical translation are presented, together with prospects for future research. Overall, this review aims to clarify the current landscape and translational potential of Citrus-EVs as natural nanodelivery systems for therapeutic applications.

Graphical abstract

Introduction

Extracellular vesicles (EVs) are lipid bilayer-enclosed nanostructures secreted by most eukaryotic cells and play a central role in intercellular communication by mediating the transfer of nucleic acids, proteins and lipids [1]. Among EV subtypes, exosomes have been most extensively investigated owing to their involvement in diverse physiological and pathological processes, including immune regulation, inflammation and cancer progression [2, 3]. Their capacity to traverse biological barriers and deliver functional cargos has positioned EVs as promising platforms for biomarker discovery and therapeutic delivery [4]. Plant-derived extracellular vesicles (PDEVs) have recently emerged as a distinct and rapidly expanding subclass of EVs. Structurally and functionally analogous to mammalian EVs, PDEVs exhibit several inherent advantages, including broad availability, low immunogenicity, high biocompatibility, and the ability to traverse biological barriers [5, 6]. Similar to animal-derived EVs, PDEVs possess a phospholipid bilayer architecture encapsulating nucleic acids, proteins, lipids, and plant-specific secondary metabolites, notably polyphenols, flavonoids and polysaccharides [7]. Accumulating evidence demonstrates that PDEVs exert diverse pharmacological activities, encompassing anti-inflammatory, antitumor as well as immunomodulatory effects, and can function as natural nanocarriers for targeted drug delivery in the treatment of tumors, inflammatory disorders, and infectious diseases [8–10].

With increasing recognition of PDEV-mediated cross-kingdom communication, several reviews have summarized their biological activities and translational potential, often focusing on representative sources such as ginger- or grape-derived vesicles, or providing a broad overview of PDEVs as a whole [11–13]. While these studies have been instrumental in establishing PDEVs as a novel class of bioactive nanomaterials, they predominantly emphasize shared characteristics across plant species and pay comparatively limited attention to source-dependent heterogeneity, particularly at the genus level [14]. As a result, the distinct compositional features, biological behaviors, and translational advantages of specific plant-derived EV subtypes may be underappreciated. To date, approximately 80 types of PDEVs have been reported, of which nearly 58% are derived from edible plants (e.g., tomato, ginger, grape, and citrus), while approximately 28% originate from medicinal plants (e.g., Panax, Ganoderma, Platycodon, and Citrus) [15]. Among these sources, Citrus plants represent one of the most extensively studied genera. As a classic example of medicinal-edible homologous plants, Citrus species have long been incorporated into both dietary practices and traditional medicine. As stated in the Yellow Emperor’s Inner Canon, “When consumed on an empty stomach, it serves as food; when taken by the ill, it acts as medicine,” underscoring the longstanding therapeutic value of Citrus plants [16].

Recently, Citrus-derived extracellular vesicles (Citrus-EVs) have attracted particular attention due to their abundant availability, high extraction yield, and consistent bioactivity. Notably, Citrus-EVs constitute a substantial proportion of reported edible plant-derived EVs explored as drug carriers, reflecting their prominent translational potential [17]. This prominence is likely attributable not only to the biochemical richness of Citrus tissues but also to several physicochemical and biological features that distinguish Citrus-EVs from other well-studied PDEVs, such as those derived from ginger or grape. First, Citrus fruits are globally cultivated agricultural products with stable supply chains, well-defined safety profiles, and suitability for large-scale processing, conferring practical advantages for the scalable production and quality control of Citrus-EVs [18]. Second, lipidomic analyses indicate that Citrus-EVs are enriched in plant-specific membrane lipids, particularly phosphatidylethanolamine and phosphatidic acid, which contribute to enhanced vesicle stability under gastrointestinal conditions and favor oral delivery applications [19, 20]. In contrast, such features are less consistently reported for ginger- or grape-derived vesicles. More importantly, Citrus-EVs intrinsically encapsulate diverse bioactive cargos, including flavonoids, limonoids, organic acids, and plant small RNAs, endowing them with endogenous antioxidant, anti-inflammatory, immunomodulatory, and metabolic regulatory activities [21–24]. This intrinsic bioactivity distinguishes Citrus-EVs from many other PDEVs that are primarily discussed as passive nanocarriers.

Despite rapidly accumulating experimental evidence, current studies on Citrus-EVs remain fragmented, with substantial heterogeneity in isolation protocols, characterization standards, and mechanistic interpretation [21, 25, 26]. Moreover, it is often unclear whether observed biological effects arise predominantly from the vesicular nanostructure, the endogenous plant-derived cargos, or their combined action [27]. These unresolved questions highlight the need for a focused and critical synthesis that goes beyond descriptive summaries and explicitly addresses source-specific characteristics, mechanistic plausibility, and translational constraints.

Against this background, the present review aims to provide a systematic and critical overview of Citrus-EVs as a distinct subclass of PDEVs, with a particular emphasis on their biogenesis, isolation and purification strategies, molecular composition, biological functions, and emerging applications in drug delivery. By explicitly comparing Citrus-EVs with other well-characterized PDEVs and animal-derived EVs, this review seeks to clarify what makes Citrus-EVs uniquely advantageous, as well as where their limitations lie. In addition, key challenges hindering their clinical translation, including standardization, mechanistic elucidation, pharmacokinetics, and safety evaluation, are discussed. Through this focused analysis, we aim to establish a conceptual framework that supports the rational development of Citrus-EVs as functional natural nanocarriers, rather than presenting a repetitive overview of plant-derived EVs.

Biogenesis, release and uptake of Citrus-EVs

Citrus-EVs represent a specific subset of PDEVs. While their overall biogenesis, secretion, and uptake exhibit conceptual similarities to mammalian EVs, Citrus-EVs also display unique characteristics linked to plant cellular architecture and physiology. Current evidence indicates that the formation of Citrus-EVs primarily occurs via two main pathways: an endosomal pathway that generates exosome-like vesicles and a plasma membrane-associated pathway responsible for microvesicle formation (Fig. 1). In the endosomal pathway, inward budding of the endosomal membrane results in the creation of intraluminal vesicles (ILVs) within multivesicular bodies (MVBs). The subsequent fusion of MVBs with the plasma membrane facilitates the extracellular release of ILVs as exosome-like vesicles. This process is generally understood to be regulated by the endosomal sorting complex required for transport (ESCRT), although ESCRT-independent mechanisms, such as ceramide- or CD63-related pathways, have also been proposed. The specific molecular details within plant systems remain incompletely elucidated [28]. In contrast, microvesicles are formed through outward budding directly from the plasma membrane, during which cargo is incorporated via membrane-associated sorting processes. However, the mechanistic basis for cargo selection and membrane remodeling in this pathway is still not well-defined for Citrus-EVs. A unique constraint in plants is the rigid cell wall, which presents an additional physical barrier to vesicle secretion compared to mammalian cells. In Citrus fruit cells, Citrus-EVs are believed to predominantly originate from MVBs. The fusion of the MVB outer membrane with the plasma membrane allows for the exocytic release of intraluminal vesicles into the extracellular space or the apoplastic compartment. Following their release, Citrus-EVs can be internalized by recipient cells through various uptake pathways. Reported mechanisms include clathrin-mediated endocytosis, caveolin-dependent endocytosis, macropinocytosis, phagocytosis, and lipid raft-mediated endocytosis. Alternatively, direct fusion with the recipient plasma membrane may occur, contingent upon the specific surface lipids and proteins of the vesicles, as well as the type of recipient cell [29].

Fig. 1.

Secretion and uptake mechanisms of EVs

According to the guidelines mentioned in MISEV 2023 [30], it is important to exercise caution in the use of terms related to presumed biogenesis pathways and to ensure that there is sufficient evidence to support them. The term “exosome” specifically refers to EVs released from internal compartments of cells via multivesicular bodies (MVBs). Unfortunately, most exosome isolation techniques fail to enrich for EVs produced by different mechanisms, and there are also difficulties in identifying subtype characteristics based on biogenetic mechanisms. Most of the existing literature regarding “exosomes” and “microvesicles” refers to a broad population of EVs rather than those released through specific biosynthetic pathways. Therefore, in this manuscript, we accept and adhere to the ISEV’s recommendation to use the term “EV”. A summary of the three main types of EVs (exosomes, microvesicles, and apoptotic bodies) is provided in Table 1.

Table 1.

The three main types of extracellular vesicles (exosomes, microvesicles, apoptotic bodies)

Category	Exosomes	Microvesicles	Apoptotic bodies
Origin	Released extracellularly from intraluminal vesicles after the fusion of intracellular multivesicular bodies with the plasma membrane	Formed by budding from the plasma membrane	Formed by plasma membrane invagination and cytoplasmic partitioning during apoptosis
Diameter range	30–150 nm	100 nm–1 μm	1–5 μm
Contents	mRNA, miRNA, non-coding RNA, proteins, MHC	mRNA, miRNA, non-coding RNA, proteins	Nuclear components, DNA, organelles
Protein markers	Alix, TSG101, HSC70, CD63, CD81, CD9	Selectin, integrins, CD40, MMP	Histones
Physiological functions	Intracellular communication, intercellular material exchange	Intracellular communication, intercellular transport of genetic material	Regulation of pathological and physiological processes

Compared with PDEVs, the biogenesis of animal-derived EVs is relatively conserved and mechanistically well defined, with the ESCRT machinery and its associated proteins (Vps4 and ALIX) serving as central regulators of MVB formation and EV production in mammalian systems [31]. In contrast, the biogenesis of PDEVs is more heterogeneous, frequently implicating the trans-Golgi network (TGN) as a major vesicle source and exhibiting stronger coupling to plant-specific stress-responsive signaling and lipid metabolic pathways [32]. These differences reinforce fundamental distinctions in vesicle origin and regulation between plant and animal kingdoms. Table 2 summarizes the comparison of biogenesis mechanisms between plant-derived and mammalian-derived EVs. Regarding cellular uptake mechanisms, the two also follow distinct delivery logics. Animal-derived vesicles typically achieve targeted delivery through specific recognition via ligand-receptor interactions; in contrast, the uptake of PDEVs such as those from Citrus relies more on non-specific interactions governed by the physicochemical properties of the membrane. This low-specificity uptake mode may serve as an important basis for their ability to achieve cross-kingdom delivery and overcome interspecies barriers. In summary, the fundamental differences in biogenesis and uptake mechanisms between plant- and animal-derived EVs render them complementary in functional characteristics and application potential, providing parallel and differentiated platforms for diverse applications in biomedicine and drug delivery.

Table 2.

Comparison of biogenesis mechanisms between plant-derived and mammalian-derived EVs

Comparative aspect	Plant-derived EVs	Mammalian-derived EVs
Primary site of origin	Released via endocytosis to form MVBs, which subsequently fuse with the plasma membrane and are secreted extracellularly [33].	Involves endocytosis and the formation of multivesicular bodies (MVBs); may also involve trafficking through the Golgi apparatus and trans-Golgi network, as well as direct budding from the plasma membrane [34, 35] (mainstream hypothesis).
Core regulatory mechanism	Highly dependent on the ESCRT complex and its associated proteins [36].	The ESCRT machinery may be involved; more closely associated with plant-specific lipid metabolism, GPI-anchored proteins, and stress signaling pathways[37] (partially confirmed/hypothetical).
Biogenesis status	Constitutive secretion and associated with specific cellular activation states.	Their biogenesis is closely linked to plant defense responses, developmental signals, and abiotic stresses [38] (hypothetical).
Markers	Tetraspanins (CD9, CD63, CD81), heat shock proteins (HSP70, HSP90), TSG101, ALIX, etc. [39]	PEN1, PATL1, TET8, as well as specific sphingolipids and phosphatidic acid [35].
Release process	No cell wall barrier; typically regulated by Rab GTPases (Rab27a/b, Rab11), which govern the trafficking of MVBs and their fusion with the plasma membrane [36].	Subject to a cell wall barrier; released through cell wall pores near plasmodesmata, or via enzymatic degradation of the cell wall to form channels for release.

Isolation and characterization of Citrus-EVs

Extraction, separation and purification of Citrus-EVs

PDEVs can be isolated in large quantities from diverse tissues, including roots, leaves, seeds and fruits. Nevertheless, the isolation of high-purity Citrus-EVs remains technically demanding because Citrus matrices are enriched in polysaccharides, pectins, pigments, and secondary metabolites that complicate downstream purification. To address these challenges, multiple isolation strategies have been developed, such as differential ultracentrifugation, sucrose density gradient centrifugation, ultrafiltration, size-exclusion chromatography (SEC), immunoaffinity-based capture, polymer-based precipitation, and electrophoretic dialysis [40–42]. Among these methods, ultracentrifugation-based protocols are most widely used for Citrus-EV preparation due to their relatively high yield and practical accessibility [43].

Ultracentrifugation (UC)

Differential ultracentrifugation separates vesicles based on differences in particle size and buoyant density and is considered a classical method for EV isolation (Fig. 2A). By stepwise increases in centrifugal force, large debris and cellular remnants are removed, followed by enrichment of vesicles within the exosomal size range (approximately 30–150 nm; density 1.13–1.19 g/mL) [44]. By virtue of its scalability and its ability to largely preserve vesicle integrity and bioactivity, this approach remains one of the most widely applied methods for Citrus-EV isolation. However, this approach has several inherent limitations, including poor suitability for highly viscous samples, prolonged processing times, and stringent equipment requirements due to the need for ultracentrifugation. Moreover, co-precipitation of soluble proteins and polysaccharide-rich components, such as pectin-like substances abundant in Citrus tissues, can compromise vesicle purity and increase separation complexity [45, 46]. Despite these drawbacks, ultracentrifugation remains a foundational technique for Citrus-EV studies. Representative protocols have been described for Citrus-EV isolation using this method. For example, Dolma et al. [17] isolated lemon-derived extracellular vesicles (LDEVs) by sequential low- and high-speed centrifugation at 4 ℃, followed by filtration and repeated ultracentrifugation at 110,000 × g to obtain exosome-like nanovesicles from Citrus limon L. juice (EXO-CLs). In a similar manner, Kilasoniya et al. [47] applied differential ultracentrifugation to isolate PDEVs from grapefruit (Citrus×paradisi) and tomato (Solanum lycopersicum), and subsequently demonstrated their feasibility as functional ingredients and targeted drug-delivery carriers.

Fig. 2.

Isolation and purification methods for Citrus-EVs extraction

Sucrose density gradient centrifugation (SDGC)

Sucrose density gradient centrifugation is considered a more refined approach for EV isolation, typically employing continuous or discontinuous sucrose gradients (commonly 8%, 15%, 30%, 45%, and 60% w/v). Purified EVs are generally enriched at the 30%–45% sucrose interface (Fig. 2B), allowing clear visualization and effective separation from coexisting impurities such as pigments and polysaccharides that are abundant in Citrus tissues. Although density gradient centrifugation yields vesicles with higher purity, it is labor-intensive and less amenable to large-scale production [48]. Using discontinuous density gradients, Stanly et al. [49] isolated membrane-bound vesicles from clementine juice by sequential low-speed centrifugation followed by purification on 1 M and 2 M sucrose/D₂O cushions, collecting vesicles floating above the 1 M layer. Similarly, Zhan et al. [50] combined differential centrifugation with sucrose density gradient ultracentrifugation to successfully enrich Citrus exosome-like nanoparticles (CELNs) at the 30%-45% sucrose interface. These studies highlight the utility of density gradients for mechanistic and compositional analyses requiring high-purity vesicles.

Size-exclusion chromatography (SEC)

SEC, also referred to as gel filtration chromatography (GFC), separates particles according to hydrodynamic radius using porous gel matrices such as Sepharose or Sephadex [51]. In this system, large particles elute rapidly, Citrus-EVs elute at intermediate volumes, and small molecules (e.g., free proteins, organic acids, and salts) elute last (Fig. 2C) [52]. SEC is regarded as a gentle purification technique that preserves vesicle integrity and biological activity, making it particularly suitable for downstream functional assays. SEC is effective in removing low-molecular-weight impurities and free flavonoids from Citrus extracts. However, its limited loading capacity and relatively high cost restrict its application for large-volume processing. Notably, Steć et al. [53] developed a hybrid approach combining ultrafiltration with SEC to isolate LDEVs, achieving improved purity and reproducibility.

Polymer precipitation (PP)

Polymer precipitation methods utilize water-soluble polymers, most commonly polyethylene glycol (PEG, typically PEG6000), to reduce vesicle solubility and induce precipitation via volume exclusion effects. This strategy enables EV enrichment using low-speed centrifugation and is operationally simple, making it suitable for large-scale extraction (Fig. 2D). However, PEG-based methods often yield vesicles with lower purity due to non-specific co-precipitation of proteins and flavonoids, which may interfere with subsequent bioactivity analyses [54]. Using this strategy, Tuo et al. [55] isolated Tangerine Peel exosome-like nanoparticles (TPELNs) from Yongquan honey orange peel by applying 8% PEG6000 to the supernatant obtained after sequential centrifugation at 6,000 × g and 10,000 × g, followed by purification through 0.22 μm filtration. Similarly, Li et al. [56] processed “Dahongpao” Citrus juice via stepwise low-speed centrifugation (2,000 ×g and 10,000 ×g) to remove cellular debris and lipids, filtered the supernatant through a 0.45 μm membrane, and precipitated vesicles using PEG6000, with subsequent washing and 0.22 μm filtration to obtain Citrus-derived EVs (CDEVs).

Electrophoretic Dialysis (ED)

The electrophoresis-dialysis approach integrates electrophoretic separation with dialysis-based purification. Differences in electrophoretic mobility enable the separation of EVs from coexisting proteins, while the semipermeable dialysis membrane concurrently removes salts and low-molecular weight impurities from the running buffer, yielding purified vesicles (Fig. 2E). Using a self-designed electrophoresis-dialysis system based on a standard laboratory electrophoresis apparatus and a 300 kDa dialysis membrane, Yang et al. [57] achieved the rapid and efficient isolation of LDEVs from lemon juice. Although cost-effective, this method requires careful operation and remains less widely adopted.

Emerging and combined strategies

Recent advances have introduced additional separation techniques, such as tangential flow filtration and ultrafiltration-based approaches, which enable continuous processing and improved preservation of vesicle structure [18, 23]. Owing to the chemical complexity of Citrus tissues and the heterogeneity of their constituents, reliance on a single isolation technique often fails to achieve optimal yield and purity. Consequently, current studies increasingly employ combinatorial strategies, such as coupling ultracentrifugation with sucrose density gradient centrifugation or integrating polymer-based precipitation with ultracentrifugation, to exploit methodological complementarity. These integrated workflows facilitate more efficient Citrus-EV isolation while substantially improving product purity. Table 3 summarizes the key features and application scenarios of the major Citrus-EV separation techniques.

Table 3.

Characteristics of main isolation methods for Citrus-EVs

Extraction method	Principle	Advantages	Disadvantages	Application	Morphology (TEM)	Size (NTA/DLS)	Zetapotential	Concentration	References
Ultracentrifugation (UC)	Differences in density and particle size	Simple operation; applicable to the extraction and isolation of most EVs	Not suitable for separating viscous liquids; Low yield and time-consuming	Lemon	nearly spherical	93.77 ± 12.31 nm	-3.46 ± 1.45mV	/	[17]
				Grapefruit(Citrus×paradisi)	sphericaloroval shapes	86–125 nm	-10mV	/	[47]
				Tangerine	cup or nearly round shapes	255 ± 18 nm	-16.3mV	0.5–0.75 µg/ml	[58]
				Grapefruit	/	105.7–396.1 nm	-49.2-1.52mV	/	[27]
				Orange	/	50–150 nm	-18.23 ± 2.7mV	/	[59]
				Grapefruit	rounded	147 ± 11 nm	/	9.09 × 1010±1.18 × 1010particles/mL	[60]
				Citrus. sinensis	round	167 ± 10 nm	/	/	[61]
Density Gradient Centrifugation (DGC)	Density difference	Easy observation of separation results; achievable for more purified EVs	More precise requirements for centrifugal speed and time; may damage the structure of EVs	Citrus clementina	/	103.3 ± 50 nm	/	1.16 × 109particles/mL	[49]
				Citrus	hemispherical	175.2 ± 21.8 nm	-7.153 ± 0.8563mV	1.66 × 108±9.52 × 106particles/mL	[50]
				Lemon	membrane-enclosed vesicle-like structure	/	-22.4mV	/	[62]
				orange	round and oval-shaped	154.5 ± 1.9 nm	/	/	[63]
Size Exclusion Chromatography (SEC)	Particle size	High sensitivity; wide applicability; relatively high purity	High cost; limited loading capacity, which restricts large-scale application	Lemon	spherical	40–600 nm			[53]
				Grapefruit	cup-shaped morphology	120–170 nm	/	About3.3 × 107particles/mL	[64]
Polymer Precipitation (PP)	Solubility	Simple operation; applicable to large-scale EVs extraction	High cost; low purity	Yongquan honey orange	spherical or spheroidal	166.1 nm	-15.85 ± 0.23mV	2.7 × 10¹⁰particles/mL	[55]
				Red mandarin (Citrus reticulata Blanco cv. ‘Dahongpao’)	spherical	190 nm	-5mV	/	[56]
Electrophoretic Dialysis (ED)	Charge-driven migration	Cost-effective; short time-consuming; high purity	Relatively cumbersome operation	Lemon	intact vesicles	50–150 nm	/	/	[57]
Tangential Flow Filtration (TFF)	Pressure-driven dynamic filtration technology	Effectively maintains structural integrity and biological activity	High cost; not suitable for large-volume samples	Citrus sinensis	spherical morphology	26.9 ± 0.11 nm	/	/	[18]
Ultrafiltration (UF)	Particle size	Simple operation; high extraction efficiency; no change to the biological activity of EVs	May cause deformation or damage to EVs; low purity	Citrus sinensis	vesicular structures	62 ± 12 nm	/	/	[23]

Impact of isolation strategies on biological activity, cargo integrity and reproducibility

While numerous methods have been developed for the isolation of Citrus-EVs, it is increasingly recognized that the choice of isolation strategy is not merely a technical consideration, but a critical determinant of downstream biological activity, cargo integrity, and experimental reproducibility. Different separation principles impose distinct physical and chemical stresses on vesicles, which may alter membrane integrity, surface composition, and functional outcomes.

Ultracentrifugation-based methods, although widely adopted due to their scalability and accessibility, subject vesicles to high centrifugal forces and prolonged processing times. These conditions may induce vesicle aggregation, partial membrane deformation, or loss of loosely associated surface proteins, potentially affecting cellular uptake behavior and immunomodulatory activity [65, 66]. In plant-derived systems rich in polysaccharides and secondary metabolites, ultracentrifugation is also prone to co-isolating non-vesicular contaminants, complicating functional attribution [67, 68]. Density gradient centrifugation improves purity by separating vesicles based on buoyant density and is therefore advantageous for compositional and mechanistic studies. However, repeated centrifugation steps and osmotic stress within sucrose gradients may compromise vesicle integrity and limit yield, restricting its applicability for large-scale or translational use [69, 70]. Size-exclusion chromatography is regarded as one of the gentlest isolation approaches, preserving vesicle morphology and bioactivity by avoiding high shear forces. Several recent studies have shown that SEC-isolated plant-derived EVs exhibit more consistent biological effects and improved reproducibility in functional assays [71, 72]. Nevertheless, its limited loading capacity and higher operational cost constrain its scalability. Polymer-based precipitation methods offer operational simplicity and high yield but suffer from low specificity [73, 74]. The co-precipitation of free proteins, flavonoids, and other Citrus-derived metabolites may artificially enhance or mask biological effects, thereby confounding interpretation of EV-specific activity. This issue is particularly relevant for Citrus-EVs, which are intrinsically enriched in bioactive small molecules.

Emerging filtration-based strategies, such as tangential flow filtration and ultrafiltration, enable continuous processing and improved batch-to-batch consistency [75, 76]. However, membrane adsorption and pressure-induced deformation remain unresolved challenges, necessitating further optimization.

Collectively, these observations indicate that isolation strategy selection directly influences the physicochemical properties, cargo composition, and functional readouts of Citrus-EVs. Variability in isolation protocols represents a major source of inconsistency across studies and limits cross-study comparability [77]. Therefore, future investigations should explicitly report isolation parameters, adopt orthogonal characterization methods, and consider method-dependent functional biases when interpreting biological outcomes. Establishing standardized, function-oriented isolation frameworks will be essential for advancing Citrus-EVs toward reproducible and translational applications.

Characterization of Citrus-EVs

Comprehensive characterization of Citrus-EVs is essential for elucidating their biological functions and evaluating their suitability as drug delivery systems. Current characterization strategies can be broadly classified into physical characterization and compositional analysis.

Physical characterization

Morphological characterization constitutes a fundamental component of Citrus-EV analysis and is commonly performed using scanning electron microscopy (SEM), transmission electron microscopy (TEM), cryo-electron microscopy (cryo-EM), and atomic force microscopy (AFM). These techniques provide complementary structural information: SEM primarily reveals surface topography, whereas TEM enables visualization of internal structure and overall morphology [78, 79]. TEM analyses indicate that Citrus-EVs typically exhibit spherical or cup-shaped morphologies comparable to those of animal-derived EVs, although subtle varietal differences exist. For instance, orange-derived EVs (ODEVs) often display more regular cup-shaped structures with relatively uniform diameters, whereas EVs from lemon and Citrus reticulata Blanco cv. “Dahongpao” may present more heterogeneous morphologies, likely reflecting tissue-specific influences [17, 56].

Particle size distribution and vesicle concentration are key quantitative parameters of EV characterization and are most frequently assessed using dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). DLS estimates particle size based on fluctuations in scattered light intensity and is rapid and easy to operate, but it is best suited for samples with narrow size distributions and is susceptible to interference from larger particles. In contrast, NTA provides both size distribution and absolute particle concentration, thereby offering a more reliable basis for dose normalization in downstream functional assays, particularly in cell-based studies [80, 81]. In addition, surface charge, typically expressed as zeta potential and measured by laser Doppler electrophoresis, critically influences EV colloidal stability and interactions with recipient cells. Available data indicate that Citrus-EVs generally range from 50 to 300 nm in diameter and exhibit zeta potentials from near neutrality to approximately − 50 mV, with variations depending on the Citrus source [15].

Component analysis

Lipids constitute the fundamental structural basis of EVs and are essential for both vesicle biogenesis and biological activity. The lipid bilayer of EVs is primarily composed of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidic acid (PA), with marked compositional heterogeneity across different EV sources [82]. Lipidomic analyses have shown that grapefruit-derived EVs (GDEVs) are enriched in PE (45.52%) and PC (28.53%), lipids associated with antioxidant, anti-inflammatory, and anti-colitis activities [27]. Similarly, orange juice-derived nanovesicles (ONVs) contain high levels of PE (40%) and PC (25%), along with PI (12%) and PA (5%) [59]. Comparative analyses indicate that Citrus-EVs exhibit a distinctive lipid signature, particularly a PE/PA ratio that contrasts with that observed in EVs from other plant sources such as grape or ginger.

Citrus-EVs also harbor a diverse proteome, predominantly comprising proteins involved in cytoskeletal organization, metabolic regulation, signal transduction, and vesicular transport and secretion. Proteomic characterization is commonly performed using SDS-PAGE, LC-MS/MS, and Western blotting [26]. For example, Tuo et al. [55] identified 1,984 proteins in TPELNs, which were enriched in pathways related to glutathione metabolism and α-linolenic acid metabolism, both closely linked to inflammation and immune regulation. In addition, Pocsfalvi et al. [83]. conducted comprehensive qualitative and quantitative proteomic analyses of EVs from four Citrus species, identifying approximately 600–800 proteins per sample and quantifying ~ 1,700 proteins using label-free approaches, thereby providing a valuable reference for comparative Citrus-EV proteomics.

EVs further encapsulate multiple classes of nucleic acids, including DNA, mRNA, miRNA, siRNA, and lncRNA, which participate in plant growth, development, environmental adaptation, and cross-kingdom biological regulation. Techniques such as Northern blotting, quantitative PCR, agarose gel electrophoresis, and next-generation sequencing are commonly employed for their detection. Functionally, miRNAs within Citrus-EVs have been shown to suppress the growth of Penicillium italicum hyphae on Citrus fruits, conferring resistance to fungal infection [84]. Moreover, grapefruit-derived nanovectors carrying miR-18a can modulate the IRF2 pathway to promote M1 macrophage polarization, thereby inhibiting liver metastasis of colon cancer [85].

Beyond macromolecules, Citrus-EVs retain bioactive small-molecule metabolites intrinsic to their plant origin, including polysaccharides, amino acids, flavonoids, saponins, alkaloids, and organic acids. Metabolomic profiling of tangerine juice-derived nanovesicles identified 35 metabolites spanning flavonoids, limonoids, cinnamic acid derivatives, carbohydrates, and lysophospholipids, with distinct enrichment patterns compared to the parent juice [57]. Compounds such as naringin, naringenin, vitamin C, citric acid, and citronellol have also been detected in GDEVs and LDEVs [22, 27, 62, 86, 87]. Collectively, these findings indicate that Citrus-EVs function as natural nanocarriers for small molecules, enhancing their aqueous solubility and transmembrane transport, which has important implications for elucidating the in vivo pharmacological mechanisms of Citrus-derived bioactives. The compositional features of Citrus-EVs are summarized in Table 4.

Table 4.

The essential active ingredients and key markers of Citrus-EVs

Biochemical profiles	Method	Plant source	Components or markers	Reference
Unique metabolite of certain Citrus-EVs	Bruker NMR spectrometer	Orange	carbohydrates (glucose, fructose, sucrose) and amino acids (alanine, asparagine isoleucine, threonine, leucine)	[59]
	ESI	Tangerine	organic acids, flavonoids, limonoids, cinnamic acid derivatives, lysophospholipids, carbohydrates, and a salicylic acid derivative.	[58]
	RP HPLC–ESI-Q-TOF-MS	Lemon	Polysaccharides, citric acid.	[22]
	HPLC	Orange	Vitamin C, Vitamin E and flavonoid components (naringin)	[88]
	GC-MS	Grapefruit	fructose, citric acid, glucose, sucrose, myo-inositol	[89]
Lipids	High-resolution mass spectrometry	Citrus	phosphatidylethanolamine, phosphatidylcholine, triglyceride	[50]
	triple quadrupole tandem mass spectrometer	Grapefruit	phosphatidylethanolamine, and phosphatidylcholine	[27]
	HPLC	Orange	phosphatidylethanolamine (PE) ( 40%), phosphatidylcholine (PC) ( 25%), phosphatidylinositol (PI), ( 12%) and phosphatidic acid (PA) (5%),	[59]
		Lemon	phosphatidylcholine (42.12%), diacylglycerol (17.05%), sphingomyelin (10.63%)	[62]
	LC-MS	Grapefruit	glycerophospholipids (50.05%), glycerophospholipids	[90]
		Lemon	glycerolipids(53.77%),glycerophospholipids(23.24%)
Proteins	MS	Citrus clementina	top-ranking proteins are clathrin, patellin-3 like, heat shock, actins, ATPase and CDC48 proteins.	[49]
	LC-MS	Tangerine	overall number of proteins in the three sets of parallel samples was 1984.	[55]
	LC-ESI-MS	Citrus fruit	altogether 1700 proteins.	[26]
	HPLC	Lemon	approximately 243 proteins	[62]
Nucleic acids	Illumina GAIIx	Orange	a total of 100 miRNAs were identified in EXO-CRs, including 96 known miRNAs and four novel miRNAs	[83]
	HPLC	Lemon	Micro RNA	[62]
	High-throughput RNA sequencing technology	Lemon	short RNA sequences (20–30 bp) with unknown functions	[91]
	Illumina Hiseq2500	Grapefruit	miRNA	[92]

Notably, a critical unresolved issue in Citrus-EVs research is the difficulty in discriminating vesicle-mediated bioactivity from the effects of free or co-isolated phytochemicals. Citrus tissues are intrinsically rich in flavonoids, organic acids, polysaccharides and other secondary metabolites, many of which possess intrinsic anti-inflammatory or antioxidant properties. During EV isolation, particularly when using ultracentrifugation or polymer precipitation, a fraction of these small molecules may remain co-isolated or loosely associated with vesicle membranes. As a consequence, biological effects observed in vitro or in vivo cannot be unequivocally attributed to vesicle-mediated delivery unless appropriate controls are implemented.

Emerging studies in the broader EV field emphasize the importance of including metabolite-depleted EVs, cargo-free EVs, and equivalent-dose free metabolite controls to disentangle carrier-mediated effects from intrinsic chemical bioactivity. However, such controls remain underutilized in current Citrus-EVs studies, representing a major limitation for mechanistic interpretation and cross-study comparability.

Despite the enormous potential demonstrated by research on Citrus and other PDEVs, significant gaps remain in the completeness and standardization of the characterization of their isolated products. These gaps limit the comparability between studies and the reliability of their conclusions. An analysis of currently published literature on Citrus-EVs reveals that most studies have not systematically followed the latest guidelines “MISEV 2023”, issued by the International Society for Extracellular Vesicles (ISEV) [30]. These deficiencies are mainly reflected in the following aspects: Firstly, there is a failure to adopt orthogonal approaches (i.e., multiple techniques with complementary principles) for cross-validation of particle concentration, size distribution, and biochemical composition. Secondly, the evaluation of non-vesicular co-isolated impurities is insufficient. Plant sap is rich in non-vesicular extracellular particles such as lipoproteins, free nucleic acids, and glycoprotein complexes. Few existing studies can fully demonstrate that the observed biological activities originate from EVs themselves rather than these co-isolated contaminants. In addition, there is limited coverage of key content emphasized in the MISEV guidelines, including the identification of surface-specific markers (e.g., characteristic lipid and protein markers of PDEVs), verification of EV purity (to exclude interference from impurities such as protein aggregates and fragments), and characterization of functional relevance.

To enhance the rigor and reproducibility of PDEV research, future studies must strive to establish a standardized system. We recommend facilitating domain consensus and calling on the PDEV research community to jointly develop supplementary guidelines that specify PDEV isolation protocols and functional characterization methods. This will thereby promote the translational application of Citrus-EVs in the food and biomedicine sectors.

Stability, toxicity and immunogenicity of Citrus-EVs

The stability of EVs is closely linked to structural integrity and is critical for preserving exosomal bioactivity and function. The physicochemical stability of Citrus-EVs is temperature dependent, allowing short-term storage at 4℃ and long-term preservation at -80℃; however, storage at 4℃ is associated with a gradual increase in particle size [88]. Stability is also pH sensitive, with Citrus-EVs exhibiting a more uniform size distribution under acidic conditions than in alkaline environments [56]. In vitro studies using simulated plasma further demonstrate that Citrus-EVs maintain colloidal stability for up to one month of storage [27]. Notably, Citrus-EVs display robust gastrointestinal stability, effectively protecting encapsulated cargos from pH fluctuations and digestive enzymes, whereas high-temperature boiling and ultrasonic treatment compromise vesicular structure and markedly reduce stability.

Exposure of biological systems to exogenous materials can elicit immune responses, raising concerns regarding the safety of extracellular vesicle-based delivery platforms. Similar to animal-derived EVs, Citrus-EVs display intrinsic compositional heterogeneity, which may theoretically confer cytotoxic or immunogenic risks. However, accumulating evidence indicates that Citrus-EVs exhibit low cytotoxicity and favorable biocompatibility, with no significant adverse effects observed across multiple in vitro cell models or in vivo animal studies. For example, no pathological abnormalities were detected in major organs, including the heart, liver, spleen, lung and kidney, within 24 h following oral administration of lipid-derived EVs, and grapefruit-derived therapeutic nanoparticles remained detectable in peripheral circulation for several days after intravenous injection without inducing inflammatory responses [8]. On the basis of their plant origin and phospholipid bilayer architecture, Citrus-EVs are thought to evade robust immune recognition and thus display low immunogenicity, minimizing the likelihood of excessive immune activation. Collectively, these attributes support their potential as safe and effective natural nanocarriers. Nevertheless, preparation strategies and artificial modifications may influence their biological behavior, underscoring the need for systematic toxicological assessments and comprehensive studies of immune interactions under diverse physiological and pathological conditions.

Differential characteristics between Citrus-EVs and animal-derived EVs

As an emerging bioactive delivery platform, Citrus-EVs offer distinct safety and functional advantages. Unlike animal-derived EVs, they are free from ethical concerns and pose a minimal risk of pathogen transmission; in contrast to synthetic liposomes, they intrinsically encapsulate diverse bioactive cargos, conferring broader and more versatile biological functions [93]. Consequently, elucidating the differences between PDEVs and animal-derived EVs with respect to isolation strategies, physicochemical properties, molecular composition, and delivery behavior has become a central focus in the field. Regarding isolation and purification, protocols for animal-derived EVs are relatively mature and optimized for high-purity recovery from complex biological fluids such as serum and cell culture supernatants. By comparison, Citrus-EVs are typically isolated from fruit juice or tissues rich in pectin, cellulose, and polysaccharides, necessitating rigorous removal of host-derived proteins and nucleic acids while preserving vesicle integrity. Morphologically, both PDEVs and animal-derived EVs display characteristic bilayered, cup-shaped or spherical structures under transmission electron microscopy. However, Citrus-EVs exhibit greater variability in size distribution, influenced by Citrus species, tissue origin, and extraction methodology. At the compositional level, Citrus-EVs differ markedly from animal-derived EVs in membrane lipid profiles, being enriched in plant-specific lipids such as phosphatidic acid, galactolipids and phytosterols, while containing little to no cholesterol. This distinctive lipid composition is thought to enhance vesicle stability and resistance to gastrointestinal conditions, an advantage for oral delivery applications. Molecular cargo constitutes the most fundamental distinction: animal-derived EVs predominantly transport regulatory nucleic acids, whereas Citrus-EVs are enriched in plant-specific small RNAs and secondary metabolites, including flavonoids and limonoids. These components underpin their cross-kingdom bioactivity and multifunctional delivery potential. In-depth investigation of these profound differences is the key to advancing Citrus-EVs from phenotypic observation to mechanism elucidation and engineered applications. To provide a clearer comparison between Citrus-EVs and animal-derived EVs, Table 5 summarizes their advantages and limitations.

Table 7.

Different administration approaches of Citrus-EVs

Administration approach	Plant source	Biodistribution	Effects	Reference
Oral	Grapefruit	Accumulated in the mid and distal small intestine, cecum and colon	Ameliorated DSS-induced colitis	[27]
		Stomach and small intestine	Enhanced targeted delivery efficiency and inhibited tumor growth	[121]
	Orange	Intestine	Regulated lipid metabolism	[59]
		Intestine	Reduced proteinuria and alleviated pathological damage in patients with IgA nephropathy	[118]
		Intestine	Enhanced cellular immune response	[61]
	Lemon	Kidney	Inhibited kidney stone formation	[62]
Intranasal	Grapefruit	Brain	Inhibited brain tumor growth in mice	[123]
Intraperitoneal	Grapefruit	Liver, lung, kidney and spleen tissues	Exerted targeting effects and inhibited tumor growth	[121]
	Lemon	Liver, spleen, kidney and tumor sites	Exhibited tumor-targeting capacity	[115]
Intravenous	Grapefruit	Liver, lung, kidney and spleen tissues	Exerted targeting effects and inhibited tumor growth	[121]
Intramuscular Injection	Grapefruit	Muscle tissue	/	[121]
Transdermal	Lemon	Skin tissue	Promoted wound healing	[99]
Plating	Citrus	SW480、HCT-116 and HS5 cells	Downregulating the intracellular phospholipase DDHD1 protein level exerts an anti-colorectal cancer effect.	[58]
	Lemon	Human CRC cell lines HCT-15, SW480, and NCI-60	Inhibits the growth of colorectal cancer cells with p53 inactivation and exerts an anti-colorectal cancer effect.	[86]
		SW480、LAMA84、MM1 and HS5 cells	Downregulating the protein level of acetyl-CoA carboxylase 1 exerts an anti-colorectal cancer effect.	[87]

Table 5.

Comparison of the advantages and limitations between Citrus-EVs and animal-derived EVs

Feature	Citrus-EVs	Animal-derived EVs
Source and accessibility	Widely available, low cost, and amenable to large-scale extraction	High cost; large-scale production requires stringent quality control
Safety	Free from animal pathogen risks, low immunogenicity, and suitable for oral administration	Pathogen contamination requires prevention and control; potential immunogenicity exists
Core components	Plant bioactive substances (e.g., flavonoids) with high stability	Animal signaling proteins and nucleic acids with strong bionic communication capacity
Main application directions	Food, health products, and oral delivery carriers	Disease diagnosis, targeted therapy, and regenerative medicine
Extraction and purification	Relatively simple methods but require removal of plant impurities; low standardization	Mature technologies but cumbersome processes; yield is constrained by cell status
Stability	High stability, suitable for the development of oral preparations; long-term storage stability requires further research	Stability is dependent on storage conditions; in vivo circulation half-life can be extended via engineering modification
Ethics and regulation	No animal ethics concerns; applicable to the food and cosmetics sectors	Subject to strict ethical oversight and medical product regulations

Application of Citrus-EVs in disease treatment

Citrus-EVs contain a diverse array of bioactive cargos, including proteins, nucleic acids, lipids and small-molecule metabolites, which collectively underpin their broad pharmacological potential. Among PDEVs, those originating from Citrus species are the most extensively investigated. Accumulating evidence indicates that Citrus-EVs exhibit multifaceted biological activities, such as antitumor, antioxidant, anti-inflammatory, antiviral, lipid-regulatory, microbiota-modulating, and wound-healing effects. Here, we have compiled current advances in the application of Citrus-EVs in disease therapy, with a particular focus on cancer and inflammatory disorders.

Anti-tumor activity

Among the reported biomedical applications, antitumor effects represent the most intensively investigated function of Citrus-EVs. Vesicles derived from multiple Citrus species, including lemon, grapefruit, sweet orange and lime, have demonstrated inhibitory effects against a range of cancer cell types, such as lung, skin, breast, gastric, and hematological malignancies. These effects are primarily mediated through the induction of cell-cycle arrest, activation of apoptotic pathways, modulation of oxidative stress, and regulation of the tumor immune microenvironment [89].

Anti-colorectal cancer

Citrus-EVs have shown particular promise in colorectal cancer models. Citrus limon L.-derived nanovesicles were shown to inhibit the proliferation of both wild-type and p53-deficient colorectal cancer cells following cellular internalization, suggesting a p53-independent antitumor mechanism [86]. In parallel, TPELNs loaded with DDHD1-siRNA effectively suppressed DDHD1 expression in SW480 human colorectal cancer cells by approximately 60%, highlighting the feasibility of Citrus-EVs as nucleic acid delivery vehicles [58]. Moreover, grapefruit-derived nanovectors delivering miR-18a were shown to prevent liver metastasis of colorectal cancer by inducing M1 macrophage polarization via modulation of the interferon regulatory factor 2 (IRF2) pathway [85]. Collectively, these studies illustrate both the intrinsic antitumor activity of Citrus-EVs and their capacity to function as targeted genetic delivery platforms.

Anti-breast cancer

Leveraging their superior safety and production scalability, Citrus-EVs have been incorporated into advanced drug-delivery platforms. A dual-delivery system combining Citrus limon L.-derived EVs with doxorubicin via three-dimensional bioprinting technology effectively suppressed cancer cell proliferation and migration, offering a novel therapeutic strategy for triple-negative breast cancer (TNBC) [94]. This approach underscores the potential of Citrus-EVs to synergize with conventional chemotherapeutics while reducing systemic toxicity.

Anti-skin cancer

Grapefruit-derived EVs (GDEVs) have demonstrated notable inhibitory activity against A375 melanoma cells. Mechanistic investigations revealed that these vesicles induced G2/M cell-cycle arrest, accompanied by downregulation of cyclin B1 and B2 and upregulation of the cell-cycle inhibitor p21 [89]. These findings suggest that Citrus-EVs can modulate key regulators of cell-cycle progression in malignant cells.

Anti-stomach cancer

LDEVs have also been investigated in gastric cancer models. Yang et al. [57] reported that LDEVs were efficiently internalized by gastric cancer cells, where they induced reactive oxygen species (ROS) accumulation, upregulated GADD45A expression, and triggered S-phase arrest and apoptosis. Importantly, antitumor efficacy was observed in both in-vitro and in-vivo models, indicating translational relevance.

Anti-leukemia

EVs derived from grapefruit and lemon selectively inhibit leukemia cell proliferation without cytotoxic effects on normal cells. GDEVs exert time-dependent antiproliferative effects comparable to high-dose (2 mM) ascorbic acid in U937 and K562 cell lines [60]. Lemon juice–derived nanovesicles, LDEVs, further demonstrated tumor-homing capacity and suppressed tumor growth in chronic myeloid leukemia xenograft models by activating TRAIL-mediated apoptosis and inhibiting angiogenesis [95].

Overall, Citrus-EVs exert antitumor effects through multiple mechanisms, including apoptosis induction, immune microenvironment modulation (such as M1 macrophage polarization), proliferation inhibition, and attenuation of metastasis and drug resistance. They function either as direct therapeutic agents carrying endogenous bioactives, like flavonoids, or as low-immunogenic nanocarriers facilitating drug delivery across physiological barriers such as the blood-brain barrier. However, the pronounced efficacy observed in vitro is often attenuated in vivo due to complex pharmacokinetics, tumor microenvironment heterogeneity, and immune-mediated clearance. Current research is constrained by three major challenges: incomplete elucidation of active cargos and signaling pathways, lack of standardized isolation and characterization protocols, and reliance on simplified preclinical models that limit translational predictability.

Anti-inflammatory and Immunomodulatory effects

Citrus-derived bioactive components exhibit well-documented anti-inflammatory properties, primarily through modulation of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB inflammatory-related signaling pathways [96]. Consistent with this, EVs derived from lemon, orange, grapefruit, and sweet orange display robust anti-inflammatory and immunoregulatory activities. LDEVs suppress ERK/NF-κB signaling and downregulate pro-inflammatory cytokines, including IL-6, IL-1β, and TNF-α [22]. Moreover, Citrus-EVs protect intestinal barrier integrity by regulating genes involved in inflammation (e.g., ICAM-1, HMOX-1) and tight-junction maintenance (e.g., OCLN, CLDN1, MLCK), thereby mitigating intestinal inflammatory injury [97]. Notably, GDEVs can be selectively internalized by intestinal macrophages, where they alleviate dextran sulfate sodium (DSS)-induced colitis in mice by upregulating heme oxygenase-1 (HO-1) expression and suppressing IL-1β and TNF-α production [27]. These observations accentuate the capacity of Citrus-EVs to modulate innate immune responses and inflammatory microenvironments.

Regulation of gut microbiota

Emerging evidence suggests that Citrus-EVs exert a modulatory effect on gut microbial homeostasis. Zhan et al. [50] demonstrated that CELNs markedly reduced the abundance of pathogenic taxa (containing Prevotellamassilia timonensis, Prevotellamassilia timonensis) in high-fat-diet-fed mice while enriching beneficial commensals (including Phocaeicola sartorii, Flintibacter hominis, Acetatifactor muris) and increasing short-chain fatty acid production. Medium and high doses restored microbial diversity in a dose-dependent manner, yielding a community structure closer to that of healthy controls. Similarly, TPELNs significantly alleviated DSS-induced intestinal inflammation by suppressing pro-inflammatory mediators, enhancing anti-inflammatory cytokine expression, reshaping the gut microbiota (such as Escherichia-Shigella species) toward beneficial genera (such as Lactobacillus species, Bifidobacterium species), and promoting anti-inflammatory metabolites (like indole carboxylic acid sulfate, sulfated bile acid) [55]. In addition, industrially produced lemon nanovesicles (iLNVs) conferred protection against 2,4-dinitrobenzenesulfonic acid (DNBS)-induced colitis in rats by attenuating inflammation and oxidative stress, partially reversing dysbiosis through enrichment of beneficial genera and reduction of harmful taxa [98]. Together, these studies imply that Citrus-EVs act as modulators of host-microbiota interactions, contributing to intestinal homeostasis.

Antioxidant stress

Oxidative stress refers to a pathophysiological state where the balance between the oxidative and antioxidant systems in the organism or cells is disrupted, leading to the accumulation of oxidizing substances such as ROS and subsequent damage to cellular components. It is recognized as one of the critical factors contributing to organismal aging and the pathogenesis of various diseases. Urzì et al. [21] showed that LDEVs suppressed ROS generation induced by hydrogen peroxide and ultraviolet exposure in human dermal fibroblasts, concomitant with activation of the AhR/Nrf2 signaling pathway. Baldini et al. [91] further provided evidence that exosome-like vesicles isolated from Citrus limon juice contained bioactive metabolites such as citrate and vitamin C and protected mesenchymal stromal cells from oxidative injury in vitro. Consistently, grapefruit-derived vesicles reduced inflammatory marker expression while upregulating antioxidant genes, reinforcing their dual antioxidative and anti-inflammatory functions [47].

Metabolic disease modulation

Hyperlipidemia and hyperglycemia are closely linked to disturbances in lipid and glucose metabolism. Edible Citrus-EVs have emerged as potential modulators of diet-related metabolic dysfunction. ONVs were shown to ameliorate obesity-associated intestinal alterations, enhance villus architecture, lower triglyceride levels, as well as regulate gene expression related to immune responses, barrier integrity, lipid absorption, and chylomicron secretion [59]. These effects collectively attenuated gastrointestinal inflammation induced by high-fat and high-sugar diets, suggesting that Citrus-EVs may help prevent or alleviate metabolic disorders through coordinated intestinal and systemic mechanisms.

Skin repair and wound healing

Citrus-EVs have also displayed considerable therapeutic promise in accelerating wound repair and angiogenic processes, alongside the preservation of skin integrity and youthful characteristics. Jin et al. [99] developed a semi-synthetic GelMA/DAS hydrogel incorporating LDEVs, which promoted M2 macrophage polarization, reduced pro-inflammatory cytokine release, enhanced angiogenesis and fibroblast proliferation, and accelerated wound healing in diabetic rat models. Additional study showed that grapefruit-derived vesicles improved keratinocyte viability and migration by reducing intracellular ROS and enhanced endothelial tube formation, underscoring their potential as plant-derived biotherapeutics for wound healing and anti-aging applications [100]. The compositional characteristics and multidimensional therapeutic potential of Citrus-EVs are Summarized in Fig. 3.

Fig. 3.

Summary of the compositional characteristics and multidimensional therapeutic potential of Citrus-EVs, which demonstrating a wide range of biological activities, indicating their significant effectiveness in preventing and treating various diseases

Although a growing body of evidence infers that Citrus-EVs exhibit broad pharmacological activities, their clinical translation remains constrained by several unresolved scientific issues. First, the underlying mechanisms of action are poorly defined, as it is often unclear whether the reported effects derive from the vesicular nanostructure itself or from the bioactive plant-derived cargos, posing a fundamental barrier to mechanistic interpretation. Second, current evidence relies predominantly on in vitro systems or immunodeficient animal models, which fail to capture the complexity of human physiology, intact immune responses, and disease pathophysiology, thereby limiting clinical relevance. In addition, systematic pharmacokinetic data are largely absent; critical parameters such as oral bioavailability, biodistribution, and metabolic fate remain insufficiently characterized, raising concerns regarding the translatability and safety of doses effective in vitro. Finally, the diverse biological functions attributed to Citrus-EVs may be context-dependent or even incompatible within human physiological systems, yet supporting evidence for such specificity is scarce. Addressing these challenges through rigorous mechanistic studies, clinically relevant disease models, and standardized preclinical evaluations will be essential before Citrus-EVs can be advanced toward credible clinical applications.

Drug loading and delivery strategies of Citrus-EVs

Citrus-EVs inherit the intrinsic bioactivity of their parent plants while possessing a lipid bilayer structure capable of encapsulating and transporting exogenous therapeutic cargos. Owing to their favorable biocompatibility, low immunogenicity, and tolerance to physiological environments, Citrus-EVs have emerged as attractive natural nanocarriers for drug delivery applications [5, 101, 102]. However, their delivery performance is highly dependent on loading strategy, cargo physicochemical properties, and administration route, necessitating a critical evaluation of their engineering approaches and translational limitations.

Benchmarking Citrus-EVs against traditional nanocarriers

Synthetic nanocarriers such as liposomes and polymeric nanoparticles have been widely developed and clinically applied for drug delivery. Liposomes provide high encapsulation efficiency and tunable composition but often suffer from limited in vivo stability, rapid clearance, and batch-to-batch variability. Polymeric nanoparticles offer structural robustness and controlled release but raise concerns related to biodegradability, long-term toxicity, and manufacturing complexity [45, 103].

In contrast, Citrus-EVs are naturally assembled vesicles derived from edible plant tissues. Their lipid composition and surface chemistry facilitate cellular uptake via endocytosis-related pathways, and multiple studies have reported preferential accumulation in inflamed or tumor-associated tissues following systemic or oral administration [27, 58]. Importantly, such “homing” behavior should not be overgeneralized. Current evidence indicates that tissue selectivity depends on administration route, disease microenvironment, and vesicle surface composition rather than representing an intrinsic universal property of Citrus-EVs [104, 105]. From a translational perspective, Citrus-EVs offer advantages in safety profile and oral administration feasibility but face challenges including heterogeneous composition, limited control over cargo loading, and insufficient standardization compared with synthetic systems. Therefore, Citrus-EVs should be regarded as complementary, rather than superior, alternatives to established nanocarriers.

Drug loading strategies for Citrus-EVs

Drug-loading strategies for Citrus-EVs can be broadly classified into exogenous and endogenous approaches, each associated with distinct advantages and limitations. Exogenous loading remains the most widely used strategy and involves introducing therapeutic cargos into isolated EVs through physical or chemical methods such as co-incubation, sonication, electroporation, membrane fusion, and freeze-thaw cycling (Fig. 4). While flexible and broadly applicable, exogenous loading is characterized by a fundamental trade-off between encapsulation efficiency and membrane integrity. Mild approaches such as passive incubation preserve vesicle structure but generally yield low loading efficiency, particularly for hydrophilic cargos [106]. In contrast, disruptive techniques such as sonication or electroporation can enhance cargo loading but may damage the lipid bilayer, alter surface proteins, and compromise the intrinsic bioactivity that distinguishes Citrus-EVs from synthetic carriers [107, 108].

Fig. 4.

Summary of common drug loading methods for Citrus-EVs. Drugs including nucleic acids, proteins, chemotherapy drugs, and small molecule drugs can be loaded into Citrus-EVs through these methods

Endogenous loading strategies aim to incorporate therapeutic molecules during vesicle biogenesis by manipulating plant metabolism or cultivation conditions. This approach better preserves vesicle integrity and cargo stability and may be advantageous for large-scale production. Conversely, it requires sophisticated genetic or agronomic interventions and remains at an early exploratory stage [109, 110]. As a whole, quantitative comparisons of loading efficiency, membrane preservation, and retained bioactivity across different strategies are still limited, highlighting the need for standardized evaluation frameworks.

Drug-delivery applications of Citrus-EVs

Citrus-EVs have been explored as carriers for small-molecule drugs, nucleic acids, and proteins, with small-molecule therapeutics representing the most extensively investigated cargo class. Accumulating evidence suggests that delivery performance depends on vesicle origin, loading strategy, surface engineering, and the physicochemical compatibility between cargos and endogenous vesicle components.

Grapefruit-derived extracellular vesicles (GDEVs)

Among Citrus species, GDEVs are the most intensively studied delivery system and are often regarded as a benchmark model for plant-derived nanocarriers [47, 111, 112]. Early mechanistic studies showcased that orally administered GDEVs preferentially accumulate in intestinal macrophages, a behavior attributed to their phosphatidic acid-enriched membranes and scavenger receptor-mediated uptake [29]. Leveraging this intrinsic tropism, Wang et al. [27]. encapsulated methotrexate (MTX) into GDEVs and demonstrated that the resulting formulation markedly attenuated colonic inflammation in a DSS-induced colitis mouse model while reducing systemic toxicity relative to free MTX. This work provided early in vivo evidence that plant-derived vesicles can improve therapeutic index through cell-selective delivery rather than passive biodistribution.

Beyond inflammatory disorders, GDEVs have been widely engineered for oncological applications. Using sonication, electroporation, or co-incubation strategies, GDEVs have been loaded with chemotherapeutic agents (e.g., doxorubicin and paclitaxel), anti-inflammatory drugs, and nucleic acids, achieving tumor growth suppression in glioma, colorectal, and breast cancer models [113]. Nevertheless, most efficacy data are derived from murine subcutaneous or orthotopic tumor models, and their translational relevance to human pharmacokinetics and tumor penetration remains uncertain.

To further enhance targeting specificity, surface functionalization approaches have been explored. Moon et al. [111] conjugated targeting aptamers to GDEVs via thiol-maleimide click chemistry, resulting in approximately two-fold increased uptake by brain endothelial cells. While these results highlight the modularity of Citrus-EVs, they also raise unresolved concerns regarding reproducibility, ligand density control, and the long-term safety of covalent surface modifications.

More sophisticated biohybrid designs have also been reported. Huang et al. [114] generated fused vesicles by combining CX5461-loaded GDEVs with CCR6-expressing nanovesicles derived from genetically engineered gingiva-derived mesenchymal stem cells. By exploiting the CCL20-CCR6 chemokine axis, these hybrid vesicles exhibited enhanced homing to inflamed skin and improved therapeutic efficacy in psoriasis and atopic dermatitis models. Despite their promising performance, such complex systems face substantial translational challenges, including scalability, regulatory classification, and quality control.

Lemon-derived extracellular vesicles (LDEVs)

LDEVs exhibit intrinsic anticancer activity and have been shown to inhibit colorectal cancer cell proliferation even in the absence of exogenous drug loading [86]. This inherent bioactivity makes LDEVs particularly attractive for combination therapy and resistance modulation. Xiao et al. [115]. developed an LDEV-based nanomedicine (HRED) by surface-modifying vesicles with heparin–cRGD and loading doxorubicin (DOX). Mechanistic investigations revealed that HRED entered DOX-resistant tumor cells through caveolin-mediated endocytosis, dissipated intracellular ATP levels, and consequently reduced ATP-dependent drug efflux, effectively reversing multidrug resistance in vivo.

Further enhancing tumor specificity, Yang et al. [116] developed hybrid vesicles (LEVBD) constructed by fusing LDEVs with homotypic breast cancer cell membrane fragments. This strategy enabled homologous tumor targeting and pH-responsive drug release within the acidic tumor microenvironment. While these studies underscore the versatility of LDEVs as engineering scaffolds, they also highlight a broader limitation: most enhanced targeting effects rely on additional membrane fusion or chemical modification steps, complicating standardization and large-scale manufacturing.

Sweet orange and other Citrus-derived EV

Extracellular vesicles derived from sweet orange (Citrus sinensis) have attracted growing attention for mucosal and oral delivery applications. Pomatto et al. [61, 117]. demonstrated that orange-derived EVs can successfully encapsulate mRNA encoding the SARS-CoV-2 S1 antigen and induce antigen-specific immune responses following oral or intranasal administration in animal models. Subsequent studies confirmed that these vesicles exhibit partial resistance to gastrointestinal degradation and retain bioactivity at room temperature, offering practical advantages over lipid nanoparticles for vaccine distribution. In addition to nucleic acids, orange-derived EVs loaded with dexamethasone sodium phosphate via electroporation significantly reduced proteinuria and renal pathology in IgA nephropathy mouse models [118]. Nonetheless, electroporation-induced membrane disruption and drug leakage remain unresolved issues, underscoring the trade-off between loading efficiency and vesicle integrity. Moreover, CDEVs loaded with tangeretin have demonstrated stronger antioxidant and anti-inflammatory activities compared to free tangeretin [56]. Detailed comparisons are summarized in Table 6.

Table 6.

Application of Citrus-EVs as carriers

Plant source	Loaded substance	Loading method	Experimental result	Reference
Grapefruit	HSP70	Incubation and sonication	Exhibited high drug loading efficiency and targeted delivery capability to glioma cells.	[45]
	Methotrexate	Co-incubation	Significantly reduced the toxic and side effects of methotrexate and enhanced the therapeutic effect on dextran sulfate sodium (DSS)-induced colitis in mice.	[27]
	Immunosuppressant CX5461	Electroporation	Significantly targeted and repaired tissue lesions by inhibiting inflammatory immune cells, and could be used for the treatment of autoimmune skin diseases.	[114]
	Aptamer	Hydrophobic insertion; Click chemistry	Extracellular vesicles (EVs) carrying aptamers successfully targeted hCMEC/D3 cells.	[111]
	BSA and HSP70 Proteins	Sonication	Efficiently delivered BSA and HSP70 to human peripheral blood mononuclear cells and colon cancer cells.	[119]
	siRNAs	Incubation	Successfully delivered to HaCaT cells and achieved gene inhibition.	[120]
	JSI-124; Folic Acid; Paclitaxel	Sonication	Successfully inhibited the growth of various types of tumors at different locations; not only enhanced the therapeutic effect of paclitaxel by targeting tumor tissues and inhibiting tumor growth, but also improved the delivery of siRNA to tumors.	[121]
	Leukocyte Membrane of Receptors Related to Inflammatory Response	Sonication	Could not only accurately home to inflamed tissues, but also target and deliver therapeutic agents to inflammatory tumor sites.	[122]
	Folic Acid and Polyethylenimine	Incubation and sonication	Could more effectively deliver miR17 to GL-26 tumor cells in mice via nasal administration, thereby effectively inhibiting brain tumors.	[123]
	Doxorubicin	Surface conjugation	Possessed high loading capacity and glioma targeting ability, which greatly promoted the cellular internalization of this delivery system and its anti - tumor proliferation capability.	[124]
	Salidroside	Sonication	Achieved synergistic effect, improving the blood - brain barrier penetration ability of the preparation and its neuroprotective effect.	[125]
	Trifunctional Platinum (IV)	Surface modification	Exhibited excellent anti - tumor activity, anti - metastatic activity and immunomodulatory activity.	[126]
Lemon	Gelatin Methacryloyl (GelMA)-Dialdehyde Starch (DAS) Hydrogel	Mixed loading	Participated in immune regulation, promoted the regeneration of blood vessels and fibrous tissues on macrophages, and had good biocompatibility and hemostatic performance. Moreover, it could promote the healing of diabetic wounds.	[99]
	Heparin-cRGD; Doxorubicin	EDC and NHS Catalysis; Incubation	Effectively overcame the multidrug resistance of doxorubicin (DOX) - resistant ovarian cancer.	[115]
	Doxorubicin	Sonication	Could effectively target homologous tumors in vivo, promote the sustained release of drugs, significantly inhibit tumor growth, and had no observable toxic and side effects.	[116]
Orange	SARS-CoV-2Vaccine	Cation-Mediated Interaction and Osmotic Shock	Precisely delivered to immune cells and stimulated immune response to fight against the new coronavirus.	[61]
	Dexamethasone Sodium Phosphate	Electroporation	Inhibited lymphocyte stimulation in vitro and alleviated renal pathological damage in IgA nephropathy (IgAN) mouse models.	[118]
	Curcumin	pH-driven method	Significantly improved the antioxidant activity.	[127]
Citrus	DDHD1-siRNA	Electroporation	Could effectively deliver the siRNA to human colorectal cancer cells, resulting in approximately 60% inhibition of target gene expression.	[27]
	Tangeretin	Passive diffusion method	After loading tangeretin, the antioxidant and anti - inflammatory activities were enhanced.	[56]

Engineering challenges and optimization strategies: stability, loading efficiency, and bioactivity preservation

Despite growing interest in Citrus-EVs as drug-delivery platforms, their engineering optimization remains constrained by several interrelated challenges encompassing formulation stability, inefficient encapsulation of hydrophilic cargos, loading-induced structural perturbation, and interference from endogenous Citrus metabolites. These issues collectively define the current performance ceiling of Citrus-EVs-based delivery systems and directly impact their translational feasibility.

Formulation stability and storage constraints

Long-term stability remains one of the most critical bottlenecks for Citrus-EVs-based formulations. During storage, EVs are prone to aggregation, cargo leakage, and alterations in surface charge and membrane organization, all of which can compromise delivery performance and reproducibility. Experimental studies have shown that repeated freeze-thaw cycles and prolonged storage at 4 ℃ can induce vesicle fusion and drug leakage, particularly for formulations loaded via disruptive methods such as sonication or electroporation [128, 129].

Cryopreservation and lyophilization with cryoprotectants (e.g., trehalose or sucrose) have been explored to improve storage stability, but their effectiveness is highly formulation-dependent [130]. Importantly, preservation of vesicle morphology does not necessarily correlate with retained biological activity or delivery efficiency, and stability claims should be supported by both physicochemical characterization (size, zeta potential, membrane integrity) and functional assays (cellular uptake, bioactivity retention) over clinically relevant storage durations [131].

Limited encapsulation efficiency of hydrophilic drugs

The lipid bilayer architecture of Citrus-EVs inherently favors the incorporation of lipophilic or amphiphilic molecules, resulting in consistently lower encapsulation efficiency (EE) for hydrophilic therapeutics compared with synthetic liposomes or polymeric nanoparticles [132, 133]. This limitation has been widely reported for nucleic acids, peptides, and small hydrophilic drugs [63].

Strategies such as cargo pre-complexation with cationic polymers or peptides, transient membrane fusion, and hybrid systems combining EV membranes with synthetic cores have been explored to enhance hydrophilic drug loading [134–136]. While these approaches can enhance EE, they also increase formulation complexity and introduce additional variables affecting reproducibility and regulatory acceptability. Notably, gains in loading efficiency are often accompanied by increased membrane perturbation or batch-to-batch variability, underscoring the absence of a universally optimal solution.

Loading-induced membrane disruption and bioactivity attenuation

High-efficiency loading methods, including sonication, electroporation, and freeze-thaw cycling, impose substantial mechanical or electrical stress on EV membranes. While these methods increase cargo encapsulation, they frequently disrupt lipid packing, alter surface protein orientation, and compromise the intrinsic bioactivity of Citrus-EVs.

This issue highlights a fundamental engineering trade-off: maximizing loading capacity often conflicts with preserving the biological features that motivate the use of EVs as biomimetic carriers. Emerging optimization strategies include parameter-controlled electroporation [137], microfluidic-assisted loading [138], and mild membrane fusion approaches [139], which aim to balance encapsulation efficiency with membrane preservation. However, quantitative comparisons of structural integrity and retained biological function across loading methods remain limited, reinforcing the need for standardized evaluation metrics.

Influence of endogenous Citrus metabolites on loading, stability and therapeutic interpretation

A defining characteristic of Citrus-EVs is their enrichment in endogenous flavonoids, organic acids and other secondary metabolites. These components can confer intrinsic anti-inflammatory or antioxidant activity and may synergize with loaded drugs. However, they can also compete with exogenous cargos for membrane association or vesicle lumen space, thereby influencing encapsulation efficiency, stability, and pharmacodynamic interpretation. For instance, enhanced antioxidant and anti-inflammatory effects observed in tangeretin-loaded Citrus-EVs may partly reflect additive or synergistic actions of endogenous flavonoids rather than improved delivery alone [78]. Without appropriate controls, it becomes difficult to distinguish carrier-mediated delivery effects from intrinsic vesicle bioactivity. Therefore, it is advocated to conduct comprehensive metabolomics analysis and cargo-free EVs controls to disentangle these effects and enable accurate interpretation of therapeutic outcomes [140]. Therefore, without rigorous control strategies to separate vesicle-mediated delivery effects from the pharmacological actions of endogenous Citrus metabolites, current therapeutic claims should be interpreted with caution. Future studies should prioritize mechanistic deconvolution rather than solely reporting enhanced efficacy.

Implications for therapeutic delivery and translational optimization

Collectively, these engineering challenges emphasize that Citrus-EVs are not plug-and-play delivery vehicles but context-sensitive systems requiring careful formulation optimization. Effective therapeutic translation will likely depend on balancing moderate loading efficiency with maximal structural and biological preservation, rather than pursuing aggressive loading strategies. Standardized evaluation frameworks integrating loading efficiency, membrane integrity, endogenous metabolite profiling, and functional outcomes are essential for meaningful cross-study comparison.

From a translational standpoint, addressing these challenges will require coordinated advances in vesicle engineering, quality control standardization, and comparative benchmarking against clinically approved nanocarriers. Without such efforts, the therapeutic potential of Citrus-EVs will remain constrained to proof-of-concept studies rather than progressing toward clinical implementation.

In vivo mechanisms of action of Citrus-EVs

Comprehensive understanding of the in vivo behavior of Citrus-EVs is a prerequisite for their development as therapeutic agents or drug-delivery platforms. As non-mammalian biomaterials, Citrus-EVs may pose a potential, albeit minimal, risk of immunogenicity, and their ability to traverse interspecies biological barriers represents a critical issue for clinical translation. Of note, the route of administration plays a decisive role in shaping their safety profile and in vivo behavior. While oral administration is generally favored for EV delivery due to its safety profile, mechanistic investigations typically adopt alternative routes, such as intravenous or local administration, to disentangle biodistribution patterns and functional outcomes. By virtue of their native phospholipid bilayer, Citrus-EVs display superior stability within the gastrointestinal environment relative to mammalian cell-derived EVs, positioning them as attractive candidates for oral delivery. Nonetheless, exposure to gastric acidity, bile salts, and digestive enzymes, coupled with intrinsically limited oral bioavailability, markedly constrains their functional integrity and delivery efficiency in vivo. Accordingly, rational engineering approaches, such as surface functionalization with targeting ligands or composite encapsulation strategies, are required to improve their in vivo performance and therapeutic efficacy.

Biodistribution and metabolism

In vivo biodistribution and metabolic fate constitute the foundation for understanding the biological functions of extracellular vesicles. At present, pharmacokinetic (PK) studies of PDEVs remain largely confined to early-stage preclinical investigations. Absorption represents the initial step in systemic entry, and PDEVs have been shown to undergo cellular uptake via multiple mechanisms, encompassing clathrin-dependent endocytosis, protein-mediated internalization, macropinocytosis, phagocytosis and lipid raft-associated pathways [141, 142]. Cui et al. [94] demonstrated that Citrus-EVs are internalized through endocytic processes in 4T1 and HCC-1806 tumor cells, as confirmed by colocalization analyses following in vitro co-incubation. Beyond oral and intravenous administration, alternative delivery routes such as transdermal and intranasal administration have been explored, offering the potential to bypass the blood-brain barrier. Zhuang et al. [123] revealed that intranasally administered PDEVs effectively suppressed malignant brain tumor growth, with fluorescence imaging revealing rapid accumulation in the olfactory bulb, hippocampus, thalamus and cerebellum. These observations underscore the pivotal role of administration route in dictating organ-specific distribution and, consequently, therapeutic efficacy.

Administration routes significantly influence tissue distribution patterns. Orally administered PDEVs predominantly localize to the gastrointestinal tract, whereas intravenously or intraperitoneally injected vesicles preferentially accumulate in highly perfused organs, including the liver and spleen [121] (Table 7). In addition, surface composition and targeting modifications critically affect organ-specific accumulation. Teng et al. [143] demonstrated that lipid composition governs the in vivo targeting behavior of PDEVs, with phosphatidylcholine-rich vesicles preferentially internalized by Ruminococcaceae and exhibiting reduced intestinal retention but enhanced hepatic accumulation. Under pathological conditions such as colitis and liver injury, Citrus-EVs display selective enrichment at inflammatory sites, likely attributable to increased vascular permeability and the upregulation of adhesion molecules [23].

In vivo metabolism of Citrus-EVs primarily occurs via lysosomal enzymatic degradation in macrophages within the liver and spleen. Metabolic byproducts are excreted through renal clearance, while a fraction of intact vesicles may be secreted into the intestine via biliary excretion, thereby participating in enterohepatic circulation. Using DiR-labeled vesicles, researchers tracked the in vivo fate of Citrus-EVs and observed substantial uptake by immune cell populations, including F4/80⁺ macrophages and DX5⁺ natural killer (NK) cells in the liver and spleen 72 h post-injection [121]. Notably, fluorescence signals persisted in the liver and spleen for up to 20 days, attesting to their in vivo stability and potential applicability in long-term management of chronic diseases. Nonetheless, most pharmacokinetic and biodistribution data are derived from small-animal studies. Whether these distribution patterns and clearance mechanisms translate to humans remains unresolved, pointing to the need for comprehensive pharmacokinetic assessment in clinically relevant models.

How to cross species barriers?

One of the most distinctive features of Citrus-EVs is their ability to traverse species barriers and mediate cross-kingdom communication. This phenomenon is thought to arise from a combination of physicochemical properties and biomimetic structural features. Citrus-EVs typically exhibit nanoscale dimensions (tens to hundreds of nanometers), enabling them to penetrate biological barriers such as intestinal epithelium, vascular endothelium, and cellular membranes via diffusion-assisted endocytosis. The phospholipid bilayer of Citrus-EVs confers robust protection to encapsulated cargos, including nucleic acids, proteins and small molecules, against enzymatic degradation during passage through harsh physiological environments such as the gastrointestinal tract. In addition, membrane lipid composition and surface biomolecules facilitate interactions with intestinal epithelial cells and immune cells, promoting uptake and translocation into systemic circulation or lymphatic pathways. Multiple studies have demonstrated that Citrus-EVs can be detected in organs such as the blood, liver, and spleen after oral administration, which directly confirms their intact absorption from the intestine and subsequent entry into the systemic circulation [121, 123, 143]. Furthermore, their liposoluble membrane structure also facilitates the penetration of barriers such as the blood-brain barrier and skin barrier.

It should be emphasized, however, that most proposed mechanisms of cross-species transport remain inferential. While experimental observations support the feasibility of cross-kingdom delivery, definitive molecular pathways, particularly receptor involvement and intracellular trafficking routes, have yet to be fully elucidated. Consequently, current models of Citrus-EV–mediated interspecies communication should be regarded as working hypotheses rather than established mechanisms.

Future directions and translational perspectives of Citrus-EVs

Despite rapid advances in the characterization and preclinical exploration of Citrus-EVs, their development remains at an early stage of translation. Progress toward clinical or practical application will require a transition from descriptive proof-of-concept studies to mechanism-driven validation, standardized manufacturing, and regulatory-aware engineering strategies. This section highlights emerging conceptual and technical directions that may support the rational development of Citrus-EVs as therapeutic agents and drug-delivery platforms.

TCM-inspired frameworks as hypothesis-generating tools for therapeutic exploration

Traditional Chinese Medicine (TCM) enhances human potential by regulating the balance of various bodily functions. Unlike Western medicine, which has traditionally focused more on specific molecular targets, the holistic approach of TCM offers additional therapeutic opportunities for metabolic diseases, chronic conditions, and other currently incurable disorders [144]. Traditional Chinese medicine (TCM) encompasses a long history of empirical use of Citrus-derived medicinal materials, particularly for the management of gastrointestinal dysfunction, inflammation, and metabolic imbalance. Citrus plants, which function as both edible and medicinal resources, are traditionally characterized by specific meridian tropisms that are thought to confer inherent targeting tendencies. Within classical TCM theory, Citrus-derived plants are prescribed for regulating qi flow and resolving dampness-heat, effects that show partial correspondence with contemporary understandings of immune modulation, inflammatory control, and gut-organ axis homeostasis.

In contemporary biomedical research, TCM principles can be used for guiding the selection of disease indications, administration routes, and biological endpoints for systematic investigation of Citrus-EVs, rather than serving as direct evidence of efficacy or targeting capability. For instance, the preferential use of Citrus-based formulations in gastrointestinal disorders within TCM practice aligns with emerging evidence that orally administered Citrus-EVs exhibit relative stability in the gastrointestinal tract and preferential interactions with intestinal immune cells. This convergence supports the rational design of focused pharmacological studies in inflammatory bowel disease, metabolic inflammation, and gut-associated immune regulation. Nevertheless, rigorous validation must rely on modern experimental models, quantitative pharmacokinetic analyses, and mechanistic investigations at the molecular and cellular levels.

Standardized production and mechanistic elucidation through multi-omics and single-cell technologies

A major bottleneck in the advancement of Citrus-EVs is the lack of standardized protocols for their production, isolation, and characterization. Substantial interspecies and intraspecies variability among Citrus sources results in pronounced differences in lipid composition, metabolite content, and vesicle-associated biomolecules. Future efforts should therefore establish comparative reference datasets encompassing particle size distribution, morphology, lipidomics, proteomics, and nucleic acid profiles across representative Citrus species and cultivars. Equally important, the mechanistic basis of Citrus-EV bioactivity and delivery behavior remains insufficiently defined. The integration of multi-omics approaches, including lipidomics, proteomics, transcriptomics, and metabolomics, with single-cell sequencing and spatially resolved analyses offers a powerful strategy to elucidate cell type-specific uptake pathways, intracellular trafficking routes, and downstream signaling events. Such approaches are critical for distinguishing carrier-mediated delivery effects from intrinsic vesicle bioactivity and for identifying molecular determinants of tissue selectivity.

From a translational perspective, a critical priority for future Citrus-EVs research lies in the systematic establishment of control frameworks that enable quantitative attribution of therapeutic efficacy. While multi-omics and single-cell technologies provide powerful tools for comprehensive cargo profiling and cell-specific uptake analysis, they must be complemented by rigorously designed functional controls to disentangle vesicle-mediated delivery effects from the intrinsic bioactivity of co-isolated plant metabolites. In this context, the integration of metabolomics-guided purification, vesicle integrity assessment, and side-by-side functional comparison between intact EVs, metabolite-depleted EVs, and equivalent-dose free metabolites will be essential to establish causality. Such methodological paradigms, which are already widely adopted in engineered mammalian EV studies, should be carefully adapted to accommodate the unique biochemical complexity and heterogeneity of plant-derived vesicles, thereby facilitating more reliable mechanistic interpretation and translational evaluation.

Development of next-generation targeted and hybrid delivery systems

Advances in nanotechnology provide multiple avenues for enhancing the delivery performance of Citrus-EVs through rational engineering. Surface modification strategies, ranging from covalent conjugation and bioorthogonal click chemistry to ligand insertion and membrane anchoring, allow the integration of targeting moieties, stealth-imparting coatings (notably polyethylene glycol and zwitterionic polymers), as well as cell-penetrating or receptor-binding peptides. While such modifications can improve circulation time and targeting efficiency, they also introduce challenges related to ligand density control, batch-to-batch reproducibility, and long-term safety.

Structural engineering approaches, such as lipid extraction and reassembly, membrane fusion, and hybrid systems combining EV membranes with synthetic cores, may further address limitations in drug-loading capacity and vesicle stability. However, increasing structural complexity inevitably raises concerns regarding manufacturability, scalability, and regulatory classification. Therefore, future development should prioritize a balanced evaluation of functional gain relative to added complexity, rather than pursuing maximal engineering sophistication.

In parallel, the exploration of alternative administration routes, spanning oral, intranasal, and mucosal delivery, represents a promising strategy to leverage the natural origin and favorable biocompatibility of Citrus-EVs. The incorporation of imaging labels and tracking methodologies may further facilitate quantitative assessment of biodistribution and delivery efficiency in vivo.

Translational barriers and regulatory considerations

The translation of Citrus-EVs from experimental systems to clinical applications faces substantial regulatory and manufacturing challenges. These include scalable isolation and purification methods, establishment of robust quality control parameters, assessment of batch consistency, and evaluation of potential allergenicity associated with Citrus-derived components.

At present, there are no unified regulatory guidelines specifically addressing plant-derived extracellular vesicles. This regulatory ambiguity complicates product classification, quality assurance, and clinical pathway definition. Alignment with existing extracellular vesicle standards, such as MISEV guidelines, combined with early engagement with regulatory agencies, will be critical for advancing Citrus-EVs toward clinical development.

Ultimately, the successful translation of Citrus-EVs will depend on realistic positioning: identifying therapeutic niches where their unique properties-oral compatibility, intrinsic bioactivity, and favorable safety profile-offer clear advantages over established nanocarriers. Without rigorous benchmarking and regulatory-aware design, Citrus-EVs are likely to remain confined to exploratory research rather than progressing to clinical implementation.

Conclusion

This article provides a comprehensive overview of recent advances in the extraction, preparation, physicochemical characterization, compositional analysis, and drug-loading strategies of Citrus-EVs. Particular emphasis is placed on emerging concepts and innovative application paradigms that position Citrus-EVs as promising platforms for therapeutic intervention and drug delivery. Collectively, the accumulated evidence suggests that Citrus-EVs possess considerable potential as a new generation of natural, efficient, and biocompatible nanocarriers, with prospective value in advancing both precision medicine and the modernization of TCM. Despite these encouraging developments, the translational advancement of Citrus-EVs remains constrained by several unresolved challenges. Future progress will require deeper mechanistic investigations to establish standardized production workflows, comprehensive molecular profiling, and robust, multidimensional quality control systems. In parallel, elucidation of cross-species transport mechanisms, rational optimization of targeting properties through surface engineering, and systematic evaluation of synergistic interactions between endogenous vesicle components and exogenously loaded therapeutics will be essential. From a translational perspective, priority should be given to validating the precision delivery capabilities of Citrus-EVs in clinically relevant disease contexts, particularly inflammatory disorders and malignancies. Achieving this goal will depend on the development of scalable manufacturing processes compliant with Good Manufacturing Practice (GMP) standards, as well as the establishment of innovative compatibility and formulation frameworks informed by TCM theory yet validated by modern pharmacological principles. Finally, the formulation of dedicated toxicological evaluation criteria and regulatory guidelines for plant-derived nanocarriers, coupled with rigorous assessment of dose-response relationships and long-term safety, will be critical for clinical progression. Through interdisciplinary integration spanning nanotechnology, systems biology, pharmacology, and regulatory science, Citrus-EVs may ultimately transition from experimental nanomaterials to clinically actionable delivery systems with broad therapeutic relevance.

Abbreviations

Citrus-EVs

Citrus-Derived Extracellular Vesicles

EVs

Extracellular Vesicles

PDEVs

Plant-Derived Extracellular Vesicles

LDEVs

Lemon-Derived Extracellular Vesicles

EXO-CLs

Exosome-Like Nanovesicles from Citrus Limon L

CELNs

Citrus Exosome-Like Nanoparticles

UC

Ultracentrifugation

SDGC

Sucrose Density Gradient Centrifugation

SEC

Size-Exclusion Chromatography

GFC

Gel Filtration Chromatography

PP

Polymer Precipitation

TPELNs

Tangerine Peel Exosome-Like Nanoparticles

CDEVs

Citrus-Derived Extracellular Vesicles

ED

Electrophoretic Dialysis

TFF

Tangential Flow Filtration

UF

Ultrafiltration

SEM

Scanning Electron Microscopy

TEM

Transmission Electron Microscopy

Cyro-EM

Cryo-Electron Microscopy

AFM

Atomic Force Microscopy

NTA

Nanoparticle Tracking Analysis

DLS

Dynamic Light Scattering

LDE

Laser Doppler Electrophoresis

PC

Phosphatidylcholine

PE

Phosphatidylethanolamine

PI

Phosphatidylinositol

PA

Phosphatidic Acid

GDEVs

Grapefruit-Derived Extracellular Vesicles

LCMS/MS

Liquid Chromatography-Mass Spectrometry

DOX

Doxorubicin

TNBC

Triple-Negative Breast Cancer

ROS

Reactive Oxygen Species

CML

Chronic Myeloid Leukemia

BBB

Blood-Brain Barrier

MAPKs

Mitogen-Activated Protein Kinases

ATP

Adenosine Triphosphate

ODEVs

Orange-Derived Extracellular Vesicles

DSS

Dextran Sulfate Sodium

EE

Encapsulation Efficiency

PK

Pharmacokinetics

FACS

Flow Cytometry

TCM

Traditional Chinese Medicine

Author contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Funding

This work was supported by the Key R&D program of Jiangxi province (No.20252BCG330031); the institutional-level research project of Jiangxi provincial institute of Traditional Chinese Medicine (ZYY2025B10); Supported by the earmarked fund for CARS-21; Jiangxi provincial agricultural machinery equipment R&D, manufacturing, promotion and application integration pilot project of Jiangxi provincial department of agriculture and rural affairs (YCTY202409).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

This research is a review paper and does not involve research in humans or animals.

Consent for publication

All the authors agree on the final submission.

Competing interests

The authors declare no competing interests.