Plant-Derived Extracellular Vesicles: Natural Nanocarriers for Biotechnological Drugs

Abstract

Plant-derived extracellular vesicles (PDEVs) are lipid bilayer nanoparticles, naturally produced by plant cells, with sizes ranging from 50 to 500 nm. Recent studies have highlighted their great potential in the biotechnological and medical fields, due to their natural origin, high biocompatibility and intrinsic therapeutic properties. PDEVs contain a complex biological cargo of proteins, lipids, nucleic acids and secondary metabolites, including antioxidants and anti-inflammatory molecules, making them ideal for biomedical applications such as drug delivery. These vesicles play a key role in intercellular communication and gene regulation, proving to be particularly promising in personalized medicine. Recent studies have highlighted their ability to improve drug stability and bioavailability, optimizing targeted release and minimizing side effects. Despite some challenges, such as compositional variability and the need for standardized protocols, PDEVs are at the gunsight of innovative research aimed at improving their loading capacity and therapeutic specificity. This review aims to provide a comprehensive overview of PDEVs, exploring their structure, isolation methods, functional characteristics, and applications, highlighting their advantages over synthetic nanoparticles and animal-derived extracellular vesicles, leading to an innovative and sustainable solution for the development of new therapeutic approaches.

1. Introduction

In recent years, considerable research has supported a new and exciting concept that plant-derived extracellular vesicles (PDEVs), which are lipid bilayer nanostructures actively secreted by a wide variety of plant cells, have important bio-functional properties. These vesicles are typically between 50 and 500 nm in diameter and are found in many plant species [1,2,3]. PDEVs have been investigated for several uses, including as nutritional supplements, carriers of genetic material, drugs, and other useful biomolecules. In this review, we provide an overview of PDEVs, including their classification, characterization in terms of size and shape, purification methods, and possible uses. Extracellular vesicles have attracted much attention across various fields for over a century since they were first reported, because they play essential roles in the regulation of intercellular communication networks in multicellular organisms [4,5,6]. The recognition of PDEVs as bio-functional entities is relatively recent, ignited by the discovery of their presence in edible plants and their potential applications in biotechnology, nutrition, and medicine.

Compared to Animal-Derived Extracellular Vesicles (ADEVs), PDEVs offer significant advantages, such as greater availability, lower production costs, and reduced immunogenicity [7,8,9]. Furthermore, PDEVs are environmentally sustainable, making them promising alternatives for innovative therapeutic strategies. Recent research has highlighted their potential for enhancing the bioavailability of therapeutic agents, reducing adverse side effects through targeted delivery, and improving patient outcomes [1,10]. PDEVs serve as molecular cargo transporters, mediating the transfer of bioactive molecules like proteins, lipids, nucleic acids, and secondary metabolites. Additionally, they have shown promise as carriers for nucleic acids, including small interfering RNAs (siRNAs) and microRNAs (miRNAs), which can modulate gene expression in target cells. As more studies focus on elucidating the molecular mechanisms underlying PDEV biogenesis, cargo selection, and functional properties, it is becoming increasingly evident that these vesicles are highly adaptable and can be engineered to meet specific therapeutic needs [11,12].

2. Plant-Derived Extracellular Vesicles (PDEVs)

Plant-Derived Extracellular Vesicles (PDEVs) are lipid bilayer nanoscale structures actively secreted by plant cells into the extracellular environment, typically ranging from 50 to 500 nm in diameter. These vesicles play a crucial role in regulating plant physiology, and mediating processes such as nutrient transport, stress response, cellular signaling, and growth modulation [2,3,13,14]. PDEVs also facilitate communication between plant cells and engage in interactions with other organisms, such as bacteria, fungi, and animal cells, thereby establishing intricate networks vital for plant survival, adaptation, and defense mechanisms [15]. Structurally, PDEVs are composed of a lipid bilayer encapsulating a complex cargo of bioactive molecules, including nucleic acids, proteins, lipids, and metabolites. This diversity is central to their functional versatility, enabling PDEVs to modulate gene expression, elicit immune responses in recipient cells, and contribute to stress mitigation. They also play a pivotal role in activating defense-related genes and regulating metabolic processes in both donor and recipient cells [16]. The unique composition and multifaceted functional properties of these vesicles make them highly promising candidates for a wide range of biomedical applications, including drug delivery [17,18]. PDEVs have been shown to enhance the stability of therapeutic agents, improve bioavailability, and enable targeted delivery, thereby optimizing therapeutic efficacy [2]. Recent advancements have focused on engineering PDEVs to increase their loading capacity, specificity, and bioavailability, extending their applicability within the realm of precision medicine [19]. Their biocompatibility and natural origin position PDEVs as ideal candidates for sustainable and eco-friendly biotechnological drugs, providing significant advantages over conventional synthetic nanocarriers [19,20]. Research on PDEVs has demonstrated their role as mediators of intercellular communication within and between different species, highlighting their significance in cross-kingdom interactions [6,15]. This characteristic is particularly relevant in the field of drug delivery, as PDEVs can effectively transport therapeutic agents to target tissues, optimizing treatment outcomes and minimizing side effects [21]. Furthermore, PDEVs exhibit high biocompatibility and low immunogenicity, making them advantageous for clinical use compared to synthetic nanoparticles and animal-derived extracellular vesicles [19,20,22]. The expanding field of PDEV research continues to uncover new possibilities for harnessing the potential of these versatile structures in various biomedical applications. Through ongoing investigations and advancements, PDEVs hold great promise as a groundbreaking tool in the field of biotechnology, paving the way for innovative therapies, diagnostics, and targeted applications.

3. Biogenesis and Characterization of Plant-Derived Extracellular Vesicles

3.1. Biogenesis of Plant-Derived Extracellular Vesicles

Even though the earliest research on membrane-associated structures was conducted in the 1950s, it was not until the development of sophisticated microscopy methods like transmission electron microscopy (TEM), cryo-electron microscopy, and atomic force microscopy (AFM) that it became feasible to examine the morphology of these vesicles in great detail, which helped to clarify their biogenesis and release. Extracellular vesicles (EVs) are spherical structures that can have different origins within plant cells. The main biogenesis pathways include multivesicular bodies (MVBs), the exocyst-positive organelle (EXPO) complex, vacuoles, and autophagosomes (Figure 1) [13,23]. Fusion of MVBs with the plasma membrane is considered the most studied and best characterized mechanism for the release of extracellular vesicles. These multivesicular compartments contain small intraluminal vesicles, and in plants they act as sorting compartments, processing proteins between the trans-Golgi network and vacuoles. Extracellular vesicle biogenesis is regulated by specific proteins that mediate the transport and fusion of multivesicular compartments with the plasma membrane. Among these, Rab GTPases (such as Rab5 and Rab7) and SNARE complexes are crucial. In Arabidopsis, Rab5 and Rab7 are mainly located in multivesicular compartments and appear to facilitate fusion with the plasma membrane, thus releasing small vesicles into the extracellular space. Rab5 includes conventional subtypes (ARA7 and RHA1) and a plant-specific one (ARA6), while Rab7 includes eight protein variants [24]. Under normal conditions, the MON1–CCZ1 complex facilitates the maturation of Rab5 to Rab7 in multivesicular compartments, leading them to fuse with vacuoles for the degradation and recycling of materials. However, in response to infection by fungi such as Botrytis cinerea, an accumulation of ARA6 is observed on multivesicular compartments, colocalized with the TET8 protein at infection sites on the plasma membrane. This colocalization suggests that ARA6 may promote the fusion of the multivesicular compartment with the plasma membrane to respond to pathogenic attacks [25]. In addition to multivesicular compartments, the exocyst-positive organelle (EXPO) represents another pathway for extracellular vesicle biogenesis in plants. This complex was identified through the protein EXO70E2, which is a double-membrane structure similar to autophagosomes, but does not colocalize with known organelle markers, suggesting that they are separate entities. EXPO was observed to fuse with the plasma membrane, releasing its contents into the extracellular space [16,25,26]. This process may be crucial for the release of proteins without leader signals, such as S-adenosylmethionine synthase 2 (SAMS2), which plays a role in salt tolerance [25,27]. PDEVs can also arise from vacuoles and autophagosomes, internal structures involved in the defense response of plants. Vacuoles contain hydrolytic enzymes and defense proteins, such as Vacuolar Processing Enzymes (VPEs), which trigger the collapse of the vacuolar membrane in the event of viral infections to release cytoplasmic contents to compete with the pathogen [13,25,28]. In response to bacterial infections, vacuoles can fuse with the plasma membrane, releasing extracellular defense proteins to limit bacterial proliferation. It has also been observed that small intraluminal vesicles can form in vacuoles through fusion with multivesicular compartments, suggesting that some extracellular vesicles may arise from the fusion of vacuoles and the plasma membrane [25]. Once secreted into the extracellular space, vesicles must cross the plant cell wall, a physical barrier composed of lignin, pectin, and hemicellulose, which can hinder their passage. Recent studies have revealed the association of PDEVs with cell wall hydrolases, such as β-glucosidase and pectinesterase, which could temporarily destabilize the cell wall, facilitating the transit of vesicles [29]. In addition, the fluidity of the lipid structure of vesicles could allow them to compress to pass through the pores of the cell wall. Although these vesicles are normally spherical, it is possible that they assume tubular or distorted shapes to facilitate passage through the wall, as already observed in bacteria. Despite the progress in understanding the biogenesis of PDEVs, many aspects, such as the precise regulation of their secretion and the specific role of autophagosomes, remain unclear. Marker proteins such as TET8, TET9 and PEN1 represent promising starting points for the study of in situ localization, quantification and identification of these EVs. Deepening our knowledge of the molecular processes of extracellular vesicle sorting and release in plants may open new perspectives in biotechnology and plant defense sciences, offering tools to improve resistance to pathogens and tolerance to environmental stresses.

According to some research, plant hormones including salicylic acid and jasmonic acid have a crucial role in controlling the production of PDEVs, especially in stressful situations. In addition to the influence of plant hormones, environmental factors, including nutrient availability, temperature fluctuations, and the presence of pathogens, significantly affect the biogenesis of plant-derived extracellular vesicles [4,30,31]. For example, during a pathogen invasion, the synthesis of PDEVs often increases to provide defensive molecules to infection sites, thereby strengthening the plant’s response to biotic stresses. These adaptive mechanisms highlight the versatility of plants and their dependence on PDEVs to survive under dynamic environmental conditions.

3.2. Plant-Derived Extracellular Vesicles Molecular Cargo

The molecular cargo of PDEVs includes a wide range of bioactive molecules, such as proteins, lipids, RNAs, and metabolites. The protein content of PDEVs includes enzymes involved in plant defense, such as glucanases and proteases, as well as signaling molecules that regulate many of the physiological processes [2,32]. Recent studies have also identified the presence of chitinase in PDEVs, an enzyme capable of degrading chitin in fungal cell walls, conferring antifungal properties to PDEVs with potential applications in agriculture and clinical settings [29]. Lipids are a crucial component of PDEV membranes, ensuring vesicle stability and facilitating interactions with target cells. Among the most abundant lipids in PDEVs are phospholipids and sphingolipids, which allow vesicles to fuse with the cell membranes of recipient cells [14,33]. The presence of phytosterols, characteristic of plant vesicles, also confers anti-inflammatory effects, as evidenced by studies that have analyzed their effect on the modulation of the immune response in animal models. Glycolipids present in PDEVs improve their targeting capacity, interacting with specific receptors on mammalian cell surfaces, and increasing the precision of drug delivery [29,34]. This characteristic makes PDEVs a promising tool for the development of targeted and effective therapies against diseases such as cancer and autoimmune disorders. In addition to proteins and lipids, PDEVs contain different types of RNA, including mRNA and small non-coding RNAs such as microRNAs (miRNAs). These RNAs play key roles in gene regulation and are involved in interspecies communication, as plant RNAs can influence gene expression in mammals by binding to target mRNAs and fragmenting them. In the case of pathogen infections, plants use EVs to release miRNAs into the host, thereby silencing genes that contribute to the virulence of the pathogen. Recent studies have shown that EVs from edible plants are also able to influence the expression of genes in the mammalian genome, thanks to interkingdom interactions, through the transfer of sRNA [15,34]. In agreement with these findings, plant miRNAs have been observed to be naturally modified at the 3′ end by methylation, a process that increases their stability and protects them from degradation [24]. Plant miRNAs ingested by animals are taken up by intestinal epithelial cells and then enclosed in extracellular vesicles [35]. These vesicles protect miRNAs from degradation, allowing them to reach more distant cells, which can interact with endogenous RNAs and modulate gene expression [34]. Numerous studies indicate that these molecules also possess protective properties against some human diseases; for example, miRNA-156a, abundant in green vegetables such as cabbage, spinach, and lettuce, can slow the progression of atherosclerosis [36]. Recently, the presence of long non-coding RNAs (lncRNAs) have also been identified in PDEVs, broadening the therapeutic possibilities of these plant vesicles and offering new perspectives for treatments acting through epigenetic regulation [3,37]. The presence of secondary metabolites, such as carotenoids, polyphenols, and flavonoids, further enriches the cargo of PDEVs, making them ideal candidates for therapeutic applications by virtue of their antioxidant, antitumor, and immunomodulatory effects. Most of these compounds are packed into PDEVs during their biogenesis, providing additional health benefits [5,34]. These active metabolites are more concentrated in EVs than in plant cells; for example, ginger-derived EVs contain much higher amounts of 6-gingerol, 8-gingerol, and 10-gingerol, which are efficiently absorbed from the gut of rats [38]. Similarly, higher vitamin C content in strawberry-derived EVs has been shown to exhibit a potent antioxidant effect [39], just as broccoli-derived EVs containing high concentrations of sulforaphane were found to be effective in a mouse model of colitis [40]. It has also been demonstrated that PDEVs derived from different Aloe species carry photoinducible secondary metabolites such as anthraquinones, which have shown a phototoxic effect on human melanoma cells [41].

Table 1. Principal lipids, proteins, nucleic acids and secondary metabolites observed in PDEVs.

PDEV-Associated Component	Type	Source	References
Lipids	Phosphatidic acid (PA)	Ginger, Citrus, Grapefruit, Grape, Garlic, Tomato, Aloe, Orange, Tea flowers	[33,42,43,44,45,46,47,48,49,50]
	Phosphatidylethanolamine (PE)	Citrus, Grapefruit, Ginger, Olive Vegetation Water, Garlic, Tomato, Orange, Tea flowers	[42,43,46,48,49,51,52]
	Phosphatidylcholine (PC)	Grapefruit, Ginger, Garlic, Tomato, Aloe, Curcuma, Orange, Tea flowers	[33,43,45,46,47,48,49,53]
	Digalactosyldiacylglycerol (DGDG)	Garlic, Aloe, Orange	[45,47,48]
	Cholesterol esters (CE)	Tomato	[46]
	Diacylglycerols (DAG)	Tomato	[46]
Proteins	Aquaporins	Broccoli, Grape, Citrus	[54,55,56]
	Heat shock proteins	Grape, Citrus, Tomato, Ginger, Mulberry, Garlic	[44,56,57,58,59,60]
	Annexins	Grape, Citrus, Ginger, Sorghum bicolor, Arabidopsis thaliana, Bitter melon, Tea flowers	[44,49,56,58,61,62]
	Peroxidases	Citrus, Sunflower, Grapefruit, Tomato	[43,56,63,64]
	Glyceraldehyde 3 phosphate dehydrogenase	Bitter melon, Citrus, Sunflower, Aloe	[47,56,62,63]
Nucleic acids	miR-156	Grape, Ginger, Bitter melon, Coconut	[33,58,62,65]
	miR-159	Coconut, Bitter melon, Ginger, Grape, Strawberry, Broccoli	[33,39,54,58,62]
	miR-166	Coconut, Ginger, Bitter melon, Strawberry, Blueberry, Broccoli	[39,58,62,65,66,67]
	miR-167	Grape, Broccoli, Bitter melon	[33,62,67]
	miR-169	Grape, Hami melon	[33,68]
	miR-172	Kiwi, Grape, Broccoli, Ginger, Bitter melon, Cabbage	[33,68]
	miR-394	Grape, Broccoli, Bitter melon,	[33,62,67]
	miR-396	Strawberry, Ginger, Broccoli, Blueberry, Bitter melon,	[39,58,62,66,67]
	miR-398	Orange, Broccoli	[48,67]
	miT-408	Grape, Broccoli	[33,67]
Metabolites	Vitamin C	Citrus, Strawberry, Orange,	[39,48,69]
	Naringin	Grapefruit, Citrus	[43,70]
	Gingerol	Ginger	[50]
	Cannabidiol	Cannabis	[71]
	Trans-δ-Viniferin	Grape	[44]
	Anthraquinones	Aloe sp.	[41]
	Curcuminoids	Curcuma	[29]
	Sulforaphane	Broccoli	[4]

reports the main metabolites characterized to date in PDEVs.

3.3. Differences Between Plant-Derived Extracellular Vesicles and Animal-Derived Extracellular Vesicles

PDEVs and ADEVs are nanoparticles of growing interest, as they play key roles in intercellular communication. However, their dimensions, molecular composition and bioavailability are significantly different. PDEVs extracted from roots, fruits and fresh plants, are composed of lipids, proteins, nucleic acids and secondary metabolites such as flavonoids, polyphenols and terpenes. Although both plant and animal types contain proteins, lipids, and RNA, there are significant differences between the two types of EVs (Figure 2). ADEVs range in size from 30 to 150 nm, while PDEVs are larger, ranging in size from 50 to 500 nm. Regarding proteins, ADEVs contain proteins such as those involved in targeted fusion and membrane transport (e.g., ALIX, TSG101, CD9, CD63), while PDEVs are characterized by the presence of proteins such as actin, proteolytic enzymes, aquaporin, and reticulin heavy chains [22]. In terms of lipids, ADEVs are mainly composed of cholesterol, sphingomyelin, glycolipids, sphingolipids, and ceramides, while PDEVs contain lipids such as digalactosyldiacylglycerol and phosphatidylglycolamine (PE) [72]. Finally, although both types of vesicles transport RNA, ADEVs contain mRNA, miRNA, and lncRNA, while PDEVs are mainly rich in miRNA [22]. This composition allows PDEVs to interact with the gut microbiome and the human immune system, showing potentially beneficial effects in modulating inflammatory and immune responses [4,33,73]. Furthermore, PDEVs are naturally predisposed to oral administration due to their high stability in gastrointestinal conditions, which allows them to deliver bioactive molecules directly into the human body. This property clearly distinguishes PDEVs from their animal counterparts, as the intrinsic stability allows a more direct and targeted absorption through the digestive system [74].

3.4. Comparison Advantages of PDEVs over Synthetic Nanoparticles

The effectiveness of many drugs is often hindered by their inability to target specific tissues, leading to toxic side effects and reduced bioavailability. To address this issue, drug delivery systems have been developed to selectively transport molecules to targeted tissues, thereby enhancing their pharmacokinetic and pharmacodynamic profiles. These systems mainly include synthetic nanoparticles such as liposomes, lipid-based, and polymeric nanoparticles [75]. Such particles encapsulate therapeutic molecules or attach them to their surface, protecting them from degradation while ensuring controlled release at the intended site [76]. However, synthetic carriers still face challenges such as toxicity, uneven distribution in the body, and high production costs. In recent years, extracellular vesicles (EVs) have gained increasing attention as potential natural drug carriers. Due to their lipid-bilayer structure, EVs can encapsulate hydrophilic drugs in their aqueous core and hydrophobic compounds within their membrane [2]. Additionally, EVs mediate intercellular communication by transferring bioactive molecules [77]. Compared to synthetic nanoparticles, EVs exhibit superior biocompatibility and reduced toxicity in preclinical studies [78]. However, EVs derived from mammals pose risks such as immunogenicity when used in heterologous species. Even autologous applications carry concerns, including the potential spread of oncogenic traits to healthy cells or the transfer of viral nucleic acids [79]. A promising alternative to animal-derived EVs is plant-derived extracellular vesicles (PDEVs). These structures share many properties of mammalian EVs, such as the ability to transport both hydrophobic and hydrophilic drugs, but without the risks of immunogenicity or transmission of animal-derived pathogens. PDEVs have demonstrated high cellular uptake efficiency, strong stability in the gastrointestinal tract, and precise tissue-targeting capabilities [22]. Furthermore, the production of PDEVs is more cost-effective and environmentally sustainable compared to synthetic or animal-based counterparts [80].

4. Isolation and Characterization Techniques for PDEVs

4.1. Isolation Methods

The isolation method of PDEVs plays a crucial role in determining the yield and purity of the obtained samples since these factors can significantly affect the quality of the isolated vesicles. Additionally, the presence of contaminants that can co-isolate with EVs—such as cellular debris, lipoproteins, protein complexes, and aggregates—increases the complexity of the isolation process [81]. It has been demonstrated that isolation techniques significantly affect the physicochemical characteristics and downstream studies of EV cargo [82]. Although there is currently little research on PDEVs, in most cases the isolation techniques for mammal EVs have been adapted for EVs of plant origin. The most common technique for the isolation of PDEVs from apoplastic fluid involves a simple protocol based on the infiltration followed by centrifugation of tissues such as seeds or leaves. A key element of this process is the composition of the infiltration buffer, which must be optimized to minimize tissue damage and prevent contamination by intracellular contents [83]. Another method instead involves the gentle disruption of the cell wall. Once the apoplastic fluids are collected, several strategies can be adopted to separate the PDEVs, but the most widespread methods are ultracentrifugation, density gradient ultracentrifugation and size exclusion chromatography (SEC).

4.1.1. Ultracentrifugation

The ultracentrifugation protocol is derived from protocols used for mammal EVs, with initial low-speed centrifugations (500× g and 10 K× g) to eliminate debris and large organelles (such as mitochondria and chloroplasts), followed by ultracentrifugation carried out at higher speeds (40 K× g or 50 K× g, and finally 100 K× g). The pellet obtained at 40–50 K is generally considered enriched in EVs with classic dimensions between 30 and 200 nm. However, the pellet resulting from centrifugation at 100 K has been described differently: for sunflower seeds, it is similar to the one at 40 K both for protein content and EV size [84]. On the contrary, in studies on the Arabidopsis leaf apoplast, the pellet at 100 K seems to contain smaller particles, ranging in size from 10 to 17 nm [85]. For this reason, some works have focused on vesicles recovered from intermediate pellets (40–50 K), while others have studied those in the final fractions (100 K). This variability, with a particular focus on intermediate pellets, is a peculiarity of the plant field, since in mammalian studies EV separation is mainly based on pellets obtained at 10–20 K and 100 K. Differential ultracentrifugation is the most used method for EV isolation, mainly due to its operational simplicity and low cost. However, this technique has significant limitations that affect its efficacy. Ultracentrifugation does not allow a selective separation of EVs, as it also sediments non-specific particles, including protein and nucleic acid aggregates. Since vegetable juices are highly heterogeneous systems, all their components can precipitate once a certain centrifugal force is exceeded. Consequently, PDEVs isolated by this methodology can be contaminated by protein aggregates and nucleic acids, compromising both their purity and quality, with a negative impact on the accuracy of functional assessments [83,86].

4.1.2. Density Gradient Ultracentrifugation

Density gradient ultracentrifugation represents an alternative method to isolate PDEVs, which relies on extracellular vesicles’ capacity to suspend in various gradient areas and separate from other molecules in biofluids. Density gradient is commonly achieved with sucrose solutions (8%, 15%, 30%, 45%, 60%) or iodixanol (20–50%) [83,87]. The latter is preferred to preserve the size of the vesicles due to its lower density and iso-osmotic properties. In the case of PDEVs, some studies have observed multiple bands in the gradient; for example, EVs derived from turmeric are positioned between 8 and 30% and 30 and 45%, with a structure similar to mammalian exosomes [88]. Other similar results have been found in plants such as grapefruit, ginger, carrot and tomato [57,58,89,90]. Lower-density EVs are also important: those from turmeric (8–30%) reduce TNF-α, IL-6, and IL-1β, increasing HO-1, suggesting anti-inflammatory and antioxidant activities [88]. EVs from ginger, also 8–30%, activate Nrf2, inhibiting hepatic ROS, with potential beneficial effects for liver diseases [50].

4.1.3. Size Exclusion Chromatography (SEC)

Size Exclusion Chromatography (SEC), or gel filtration, is a technique that separates molecules based on their size and is commonly used for protein purification. SEC is based on the principle that larger molecules cannot penetrate the pores of the filter material and pass through the column more quickly, while smaller molecules, penetrating the pores, migrate more slowly, thus allowing separation. Different polymers have been developed to be used to fill SEC columns, such as dextrans (Sephadex), agarose (Sepharose), and polyacrylamide (Biogel P or Sephacryl), and specific commercial kits have also been created for the isolation of Evs [83]. Although SEC is a widely used method for isolating EVs from biofluids, its use for the separation of PDEVs is rather limited. However, some studies have also demonstrated its applicability for EVs of plant origin; for example, SEC has been used to isolate EVs from cabbage, cucumber, tomato, and carrot [57,90,91,92].

4.2. Characterization Techniques

Currently, the most widely used characterization techniques provide information on the size, concentration, morphology, presence of protein markers, protein concentration, and biochemical composition of PDEVs, which are essential to understanding their roles in cellular processes, intercellular communication and potential therapeutic applications [93]. These methods help to determine not only the physical properties of PDEVs, but also their molecular cargo, including proteins, lipids and RNA, which are essential to understand their biological functions. Morphological analysis techniques include electron microscopy and Nanoparticle Tracking Analysis (NTA), which provide complementary data on the shape, diameter distribution and concentration of PDEVs. Flow cytometry is widely used with animal EVs as it allows the profiling of surface markers and the analysis of the heterogeneity of vesicular populations, useful to distinguish subpopulations and their biochemical differences. However, specific markers for PDEVs are still poorly known, limiting the depth of research in this field. Furthermore, biochemical techniques such as Western Blotting and mass spectrometry identify vesicular content and markers, while molecular analyses investigate the nucleic component. The combined use of these methodologies provides a detailed profile of PDEVs, favoring diagnostic and therapeutic biotechnological applications [94,95].

4.2.1. Electron Microscopy

Electron microscopy is currently used to assess the morphology of PDEVs. Scanning Electron Microscopy (SEM) is able to observe the shape, size and surface distribution of vesicles, but exhibits some limitations, such as sample preparation, that can alter the natural morphology or the inability to visualize the lipid bilayer, a distinctive feature of EVs [96]. For finer details, Transmission Electron Microscopy (TEM) or cryo-EM are preferred, with resolutions of 1–3 nm and 5 nm, respectively [2,97]. TEM, combined with gold immunolabeling, also allows for the identification of proteins present in EVs, facilitating functional studies. Cryo-EM preserves the spherical structures of EVs in a hydrated environment, reducing fixation-related artifacts and allowing the acquisition of 3D tomographic images for detailed visualization. Other advanced techniques include Atomic Force Microscopy (AFM), which uses a probe to obtain three-dimensional topographic images at high resolution (less than 1 nm), ideal for observing EVs and other nanostructures [2].

4.2.2. Nanoparticle Tracking Analysis (NTA)

Nanoparticle Tracking Analysis (NTA) is a widespread technique that provides data on particle size and concentration of EVs. NTA combines laser light scattering microscopy with a camera to visualize and track suspension nanoparticles in real-time. NTA allows one to determine the size and total concentration of particles between 30 nm and 1 μm by tracking the Brownian motion of EVs [86,98]. Specific systems, equipped with a laser and camera, also detect fluorescent particles, which is useful for analyzing subpopulations of EVs labeled with specific antibodies or fluorophores [99,100]. This capability has been exploited to study the phenotype of vesicles and to identify molecular targets, such as miR-21 in lung cancer-derived EVs, with greater sensitivity than cytometry for particles smaller than 100 nm, such as exosomes [101]. NTA has proven to be particularly useful for characterizing the size distribution and molecular content of EVs, opening new perspectives in research on biomarkers and therapeutic targets.

4.2.3. Flow Cytometry

Flow cytometry is a standard technique to characterize EV surface markers, providing information on their biological origin. This technique allows one to analyze single particles as they pass through a laser ray, detecting light scattering signals: forward scattering (FSC) indicates size, while lateral scattering (SLS) provides data on morphology and internal structure [102,103]. One of its advantages is the ability to identify vesicle subpopulations through antibody-bound fluorescent markers, facilitating the simultaneous analysis of multiple surface proteins [104]. However, the application to PDEVs has significant limitations. The small size of EVs makes it difficult to distinguish the signal from the background, and the emitted fluorescence is often weak due to the small number of antigens per particle. Furthermore, lacking knowledge of specific markers for PDEVs complicates the identification of subtypes [4].

5. Biological Functions of PDEVs

5.1. Role in Plant-Plant Communication and in Plant-Microbe Interactions

PDEVs rich in miRNAs, bioactive lipids, and proteins act as extracellular messengers, facilitating cell–cell communication in a manner similar to mammalian extracellular vesicles. In fact, PDEVs play a key role in plant cell–cell communication, enabling information exchange between cells of the same species and between different species [28]. PDEVs also participate in essential cellular processes such as proliferation and differentiation, responding to a variety of stimuli, which may include environmental signals, such as changes in light or temperature, or biological signals, such as pathogen attacks. Research conducted on EVs from coconut water has shown their contribution to the fruit ripening process, supporting this function through RNA analysis [65]. PDEVs are involved in the transportation of phytohormones such as auxins, cytokinins and gibberellins, key elements for regulating plant growth and development [105]. Auxins, for example, are essential for root elongation and the formation of lateral roots, processes that are essential for optimizing water and nutrient absorption. Cytokinins, on the other hand, promote cell division and shoot development, while gibberellins are responsible for stem elongation, seed germination and the flowering process [106]. In addition to communication, PDEVs are strongly involved in plant defense mechanisms. They help eliminate harmful compounds from cells and actively participate in immune surveillance. Bacterial or fungal infections have been shown to enhance the production of plant EVs containing defense-related proteins, sRNAs, and lipid signals, highlighting their crucial role in plant immune mechanisms [13,107,108]. For example, a study on Arabidopsis infected with Golovinomyces orontii revealed exosome-like extracellular vesicles carrying PEN1/SYP121 and the ABC transporter PEN3. These compounds accumulate around the haustoria, forming a protective barrier that hinders fungal entry [109]. PDEVs extracted from the extracellular fluids of sunflower (Helianthus annuus) seedlings showed a heterogeneous composition, including cell wall restructuring enzymes and defensive proteins identified by proteomic analyses. When spores of the phytopathogenic fungus Sclerotinia sclerotiorum were exposed to purified sunflower EVs, growth arrest, structural alterations, and cell death were observed [13,63]. Likewise, De Palma et al. showed that EVs made from tomato roots efficiently inhibited both spore germination and germ tube development in diseases such Alternaria alternata, B. cinerea, and Fusarium oxysporum [64]. Although EV-mediated plant-fungal interactions have been extensively investigated, bacterial-plant interactions remain poorly understood. It has been demonstrated that Arabidopsis plants infected with Pseudomonas syringae produced twice the amount of EVs compared to uninfected plants. This result suggests that plant EVs may play a significant role in resistance against bacterial pathogens. Furthermore, EVs isolated from P. syringae-infected plants contained numerous proteins involved in the biotic stress response, supporting the hypothesis that these vesicles may act as vectors of immune signals during bacterial infections [85].

5.2. Role in Human Health and Therapeutic Applications

PDEVs have emerged as a promising resource for therapeutic applications in human health, due to their bioactive components that exhibit anti-inflammatory, antioxidant, and anticancer properties. Numerous studies have highlighted their ability to interact with mammalian cells, contributing to positive physiological effects. Plant-based treatments have been used for centuries, exploiting their natural forms or extracts of active compounds to prevent diseases and repair damage. However, only recently, attention has also focused on the use of PDEVs which have shown interesting biological properties, including anti-inflammatory, anticancer, antibacterial, antifungal and antioxidant effects [7,14,29,110]. These effects are mediated through various mechanisms, such as gene regulation, interaction with gut microbiota, macrophage activation, gene silencing and the presence of specific active compounds [111,112]. These natural therapeutic properties offer new perspectives for innovative treatments, both alone and in combination with other drugs and nanomedicines.

5.2.1. Anti-Inflammatory Effects of PDEVs

The immunomodulating and anti-inflammatory properties of PDEVs have attracted increasing attention. These vesicles can be used in a variety of biological scenarios since they not only have positive effects on the immune system but also remain stable under harsh conditions. Because of their capacity to regulate inflammatory responses in a variety of therapeutic settings, both in vitro and in vivo, as evidenced by multiple studies, PDEVs have shown promise in the treatment of chronic inflammatory illnesses such as colitis [88]. For example, ginger-derived EVs have been able to suppress NLRP3 inflammasome activation, a key protein involved in inflammatory diseases such as inflammatory bowel disease (IBD), reducing levels of pro-inflammatory cytokines such as IL-1β and IL-18 and preventing the assembly of the inflammasome itself. These anti-inflammatory effects are mediated by mechanisms such as the induction of autophagy and the prevention of focused cell death, as demonstrated in a clinical trial examining the effectiveness of ginger-derived EVs in patients with IBD [16,113]. Mulberry bark-derived EVs have been able to prevent colitis in animal models, possibly through AhR receptor activation, mediated by HSPA8 protein [59]. Furthermore, turmeric-derived EVs have shown potent anti-inflammatory effects in murine colitis models, modulating pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β and promoting the expression of the antioxidant gene HO-1, which contributes to the resolution of intestinal inflammation. Turmeric-derived EVs have also been associated with the inhibition of the NF-κB pathway, further increasing protection against intestinal inflammation [88]. On the other hand, orange-derived EVs have been found to be effective in reducing intestinal inflammation and restoring intestinal barrier integrity by modulating the expression of key genes such as HMOX-1, ICAM1, OCLN, CLDN1, and MLCK [114].

5.2.2. Antioxidant Properties of PDEVs

Reactive Oxygen Species (ROS) are generated during physiological processes such as aerobic respiration, as well as in response to external stimuli, such as xenobiotics, cytokines and bacterial invasion. ROS play an essential role in cell survival and proliferation, but when their production exceeds the organism’s defensive capacity, oxidative stress occurs, which damages crucial biomolecules such as DNA, proteins and lipids. This oxidative damage is implicated in the onset of several common diseases, including neurodegenerative disorders, cardiovascular diseases, diabetes and cancer. Natural antioxidants, such as polyphenols, carotenoids and vitamins, play a protective role against oxidative stress and have demonstrated anti-aging and anti-inflammatory effects. In this context, PDEVs emerge as promising therapeutic agents to counteract oxidative stress and associated pathological conditions, thanks to their antioxidant and anti-inflammatory properties. PDEVs act through the upregulation of antioxidant molecules such as nuclear factor erythroid 2 (Nrf2)-related enzyme, hemazole 1 (HO-1), and NAD(P)H quinone dehydrogenase 1 (NQO1), thus strengthening cellular defense mechanisms against oxidative damage [16,90]. For example, PDEVs extracted from Aloe vera peel showed significant antioxidant activity, dose-dependently increasing the expression of key genes such as Nrf2, CAT, HO-1, and SOD [7]. Similarly, PDEVs derived from carrots were able to prevent the downregulation of Nrf2 expression, protecting H9C2 cardiomyoblastic cells and SH-SY5Y human neuroblastoma cells from oxidative stress [90]. Strawberry-derived PDEVs may contribute to protection against oxidative damage and improve cell viability, due to their high concentration of vitamin C, anthocyanins, folic acid and flavonoids. Adipose-derived mesenchymal stem cells internalize strawberry vesicles without compromising their viability and, when pretreated with these vesicles, show protection against oxidative damage in a dose-dependent manner [39]. Blueberry-derived PDEVs have also been shown to reduce oxidative stress in cellular and animal models, by affecting the functionality of the mitochondrial protein Bcl-2 and preventing apoptosis in HepG2 cells. Furthermore, administration of these PDEVs in vivo improved insulin resistance and the expression of Nrf2-regulated detoxification/antioxidant genes, showing potential in the treatment of non-alcoholic fatty liver disease (NAFLD) [115]. Additionally, other plant sources such as grape juice, rich in polyphenols, have been shown to mitigate oxidative stress by modulating ROS levels in various cell models [44]. Vesicles derived from broccoli are known to enhance antioxidant defense systems, potentially through the activation of Nrf2 and other related pathways [116,117]. Citrus-derived PDEVs, rich in vitamin C and flavonoids, contribute to reducing oxidative stress [118]. Furthermore, apple-derived EVs have been shown to act as novel anti-inflammatory compounds that modulate the extracellular matrix production in dermal fibroblasts, reducing skin aging by inhibiting the TLR4/NF-κB inflammatory pathway, enhancing collagen synthesis, and decreasing extracellular matrix degradation [119].

5.2.3. Anticancer Properties of PDEVs

In recent years, PDEVs have emerged as a promising alternative in oncology research. Numerous studies have highlighted the ability of PDEVs to inhibit tumor cell proliferation, modulate the cell cycle, induce apoptosis and influence cancer metabolism, thus offering a multifactorial approach to oncology treatment. For example, PDEVs derived from Panax ginseng were able to induce macrophage polarization toward the M1 phenotype via Toll-like receptor (TLR)-4 and myeloid differentiation antigen 88 (MyD88) signaling pathways, with direct effects on melanoma cell apoptosis. Ceramides and proteins associated with PDEVs play a crucial role in macrophage activation, increasing their antitumor potential [120]. Similarly, PDEVs derived from Moringa oleifera seeds demonstrated a targeted action against tumor cells, inhibiting their viability through the regulation of the antiapoptotic protein Bcl-2 and the impairment of mitochondrial membrane potential [121]. Bitter melon nanovesicles have been shown to be effective against breast cancer, both in vitro and in vivo, by blocking tumor cell proliferation and migration. This effect has been attributed to their ability to induce the production of ROS and to impair mitochondrial function, thus triggering oxidative stress-mediated cell death pathways [122]. Similarly, tea flower vesicles have shown significant cytotoxic activity against breast cancer cells, with the particularity of also modulating the intestinal microbiota, suggesting potential systemic effects that could counteract metastasis [49]. Lemon PDEVs have been shown to inhibit the growth of chronic myeloid leukemia tumor cells in vivo, targeting the tumor and activating the TRAIL-mediated apoptotic process, with a significant reduction in the risk of cancer progression [123]. Some PDEVs, such as those derived from ginger, have also been shown to be effective against colorectal cancer, reducing the levels of cell cycle protein D1 RNA and inhibiting the proliferation of colon cancer cells [58]. Studies in lung cancer mouse models have highlighted the potential of Artemisia PDEVs, which improved the efficacy of PD-1 blockade through the remodulation of the tumor microenvironment and the reprogramming of tumor-associated macrophages. The mechanism seems to be linked to the activation of the cGAS-STING pathway induced by mitochondrial DNA, which explains their immunomodulatory effects [124]. PDEVs derived from Aloe arborescens, Aloe barbadensis, and Aloe chinensis have been shown to inhibit melanoma cell proliferation through the photoactivation of their anthraquinone content, leading to the production of ROS [41]. PDEVs derived from grapefruit mediated the inhibition of liver metastasis that is dependent on the induction of M1 macrophages (F4/80+IFNγ+IL-12+) [125]. PDEVs represent an extremely innovative therapeutic tool as they overcome the limitations related to low bioavailability, instability and safety issues of synthetic or animal vesicles, offering new opportunities for the treatment of cancer, both through direct action and by enhancing immune responses against tumors.

Table 2. PDEVs anti-inflammatory, antioxidant and anticancer effects.

PDEVs Source	Biological Activity	In Vitro/In Vivo Effects	References
Ginger	Anti-inflammatory	Suppression of NLRP3 inflammasome activation, a key protein involved in inflammatory diseases such as inflammatory bowel disease (IBD) and reduction in pro-inflammatory cytokines levels.	[113]
Mulberry bark	Anti-inflammatory	Stimulation of heat shock protein family A (Hsp70) member 8 (HSPA8) that mediated the activation of the AhR signaling pathway.	[59]
Turmeric	Anti-inflammatory	Regulation of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β and promotion of the expression of the antioxidant gene HO-1 in murine colitis models.	[53]
Orange	Anti-inflammatory	Reduction in intestinal inflammation and restoration of intestinal barrier integrity by modulating the expression of key genes such as HMOX-1, ICAM1, OCLN, CLDN1, and MLCK.	[114]
Garlic	Anti-inflammatory	Reduction in the expression of pro-inflammatory factors IFN-γ and IL-6 in LPS-stimulated HepG2 cells.	[126]
Carrot	Anti-inflammatory	Increased expression of the anti-inflammatory cytokine IL-10 in Raw 264.7 macrophages.	[72]
Cabbage	Anti-inflammatory	Decreased levels of pro-inflammatory cytokines IL-6 and IL-1β, along with COX-2, in LPS-stimulated Raw 264.7 macrophages, and suppression of NLRP3 inflammasome assembly and activation in primary macrophages from C57BL/6J mice, mediated by ginger’s anti-inflammatory properties.	[91]
Broccoli	Anti-inflammatory	Reduced expression of pro-inflammatory cytokines TNF-α, IL-17A, and IFN-γ, coupled with increased expression of the anti-inflammatory cytokine IL-10, in a DSS-induced colitis model using C57BL/6 (B6) mice.	[40]
Aloe vera	Antioxidant	Increase expression of key genes such as Nrf2, CAT, HO-1, and SOD.	[47]
Carrot	Antioxidant	Prevention of downregulation of Nrf2 expression, protecting H9C2 cardiomyoblastic cells and SH-SY5Y human neuroblastoma cells from oxidative stress.	[90]
Strawberry	Antioxidant	Protection against oxidative damage	[39]
Blueberry	Antioxidant	Reduction in oxidative stress by affecting the functionality of the mitochondrial protein Bcl-2 and preventing apoptosis in HepG2 cells.	[115]
Ginseng	Anticancer	Induction of melanoma cell apoptosis by macrophage polarization toward the M1 phenotype via Toll-like receptor (TLR)-4 and myeloid differentiation antigen 88 (MyD88) signaling pathways.	[120]
Moringa oleifera	Anticancer	Upregulation of the antiapoptotic protein Bcl-2 and impairment of mitochondrial membrane potential.	[121]
Bitter melon	Anticancer	Inhibition of breast cancer cells proliferation by induction of ROS production.	[122]
Tea flower	Anticancer	Cytotoxic activity against breast cancer cells via ROS generation.	[49]
Lemon	Anticancer	Growth inhibition of chronic myeloid leukemia tumor cells by activation of the TRAIL-mediated apoptotic process.	[123]
Ginger	Anticancer	Inhibition of the proliferation of colon cancer cells by reducing the levels of cell-cycle protein D1 RNA.	[58]
Aloe arborescens, Aloe barbadensis, Aloe chinensis	Anticancer	Inhibition of melanoma cell proliferation through anthraquinones photoactivation leading to ROS production.	[41]
Grapefruit	Anticancer	Inhibition of liver metastasis dependent on the induction of M1 macrophages (F4/80+IFNγ+IL-12+).	[125]
Grape	Anticancer	Apoptosis induction in colon-26 tumor and HT-29 adenocarcinoma cells.	[33]

reports the most widespread effects of PDEVs.

6. Enhancing PDEVs for Drug Delivery Applications

PDEVs can be used as drug delivery platforms by loading small molecules or nucleic acid-based drugs without undergoing degradation, making them an effective method to prevent drug damage [31]. PDEVs have high bioavailability, can be absorbed in the intestine, and penetrate deep into the skin, and, if administered intranasally, they are able to cross the blood–brain barrier and reach the brain [92]. Furthermore, PDEVs are considered safe due to their low toxicity and fewer side effects compared to synthetic lipid nanoparticles and animal-derived EVs. Most of the molecules used in PDEVs as cargo are nucleic acid-based drugs and small-molecule chemical drugs (Figure 3) [127].

6.1. Engineered PDEVs: Nucleic Acid Delivery

Several techniques using physical, chemical, or mechanical approaches are available to enable the introduction of genetic material into PDEVs. These methodologies allow PDEVs to be used as vehicles for the targeted delivery of nucleic acids, expanding their potential therapeutic applications. One of the most widely used techniques is electroporation, which applies an electric field to PDEVs, creating temporary pores in their lipid membrane. This process promotes the transfer of nucleic acids across the membrane and their subsequent incorporation into the vesicles. Another common method is sonication, which uses high-frequency sound waves to temporarily perturb the vesicle membrane, allowing the penetration of nucleic acids. A gentler approach is incubation, in which PDEVs are mixed with nucleic acids in a buffer solution. By varying parameters such as temperature, pH, and salt concentration, it is possible to favor the passive uptake of genetic material by the vesicles. Finally, chemical transfection involves the use of specific chemicals that increase the permeability of the PDEV membrane, making it more receptive to nucleic acids [12,127,128]. Each technique has advantages and limitations, and the choice depends on the type of nucleic acid, the nature of the PDEV, and the experimental objectives. These approaches represent versatile tools to exploit PDEVs as delivery platforms for innovative nucleic acid-based therapies. PDEVs are promising tools for the delivery of nucleic acids, such as siRNA and miRNA. A noteworthy example is grapefruit-derived PDEVs loaded with miR17, which have been shown to delay the growth of brain tumors in mice. This effect is possible due to the ability of PDEVs to reach the brain via nasal administration, allowing the transported miR17 to exert an inhibitory action. Furthermore, the addition of folic acid to PDEVs improves the targeting of folate receptor-positive brain tumors, providing a non-invasive treatment for brain diseases [129]. Different types of PDEVs lend themselves to different routes of administration. For example, acerola-derived PDEVs can deliver small RNAs to the digestive tract, with a peak of suppressive activity on target genes in the small intestine and liver within 24 h of administration, representing a valid option for the oral delivery of nucleic acids [130]. Similarly, ginger-derived PDEVs loaded with siRNA-CD98 showed effective action on colon tissue by reducing CD98 gene expression upon oral administration [131]. Broccoli-derived EVs were loaded with exogenous miRNAs with biologically active properties and then co-incubated with Caco-2 cells causing cytotoxicity. These results suggest that broccoli-derived EVs may increase the stability of therapeutic RNA, protecting it from degradation by RNases and digestive enzymes [67].

6.2. Surface Functionalization of PDEVs

In addition to carrying small therapeutic molecules, PDEVs have also been shown to improve the properties of inorganic nanoparticles by modifying their surfaces. For example, ginger-derived extracellular vesicles were used to coat nanocarriers, increasing their biocompatibility and persistence in the blood, and reducing their immunogenicity thanks to the integration of natural plant membranes [132]. Further research involved grapefruit-derived lipid nanovesicles modified with leukocyte-associated inflammatory protein receptors. This system was used for the targeted delivery of doxorubicin (DOX) to inflamed areas. The stability and detectability of the drug, assessed by spectrophotometry at 497 nm, were enhanced thanks to the coating of the vesicles [89]. In addition to carrying anticancer drugs, grapefruit-derived EVs can carry anti-inflammatory molecules such as curcumin. In mouse models of inflammation, vesicles modified with specific receptors showed a higher targeting efficiency than natural ones, optimizing drug release in inflamed areas [133]. Grapefruit-derived extracellular vesicles were functionalized with a maleimide-modified lipid moiety and conjugated with aptamers via click chemistry, enabling targeted drug delivery through enhanced selective cellular uptake in brain cell models (hCMEC/D3 and U87MG) [51]. Qiao et al. demonstrated that integrating electrodynamic platinum-palladium (Pt-Pd) nanosheets with ginger-derived extracellular vesicles improved blood circulation, promoted targeted accumulation at infection sites, and penetrated bacterial membranes. Additionally, this combination generated reactive oxygen species, enhancing synergistic electrodynamic and photothermal therapy with promising biocompatibility and in vivo applicability [134].

6.3. Oral Administration of PDEVs for Drug Delivery

PDEVs obtained from edible plant species have a high safety of use. They can efficiently encapsulate drugs and gene therapy agents, offering protection against gastrointestinal degradation and promoting absorption by the organism. Preclinical studies have shown that PDEVs do not significantly alter the physiological and biochemical parameters of laboratory animals, confirming their excellent biocompatibility [17]. In fact, oral administration is one of the preferred methods for drug intake, although it involves significant difficulties. The unfavorable environment of the gastrointestinal tract can compromise the efficacy of the drug, causing losses of active substances and hindering its absorption through the intestinal wall. In addition, orally administered drugs often show low bioavailability and may generate unwanted side effects [135]. On the other hand, the oral use of PDEVs is considered safe and promising, since these vesicles demonstrate high stability in the gastrointestinal tract. The reason why PDEVs are able to overcome the hostile environment of the gastrointestinal tract and reach target cells remains largely unknown. One possible explanation is that the lipid composition could be a determining factor. PDEVs are distinguished by their richness in specific lipids, such as PA, DGDG, MGDG, PC, PE and PI, in contrast to mammalian EVs, which are predominantly composed of CHOL, SM, PC and PE [136,137]. Previous studies suggest that lipid composition affects the formation, distribution and function of EVs in vivo, and could therefore explain their resistance to gastrointestinal digestion. Ginger-derived EVs were found to be highly stable in simulated gastrointestinal fluids, with effective uptake by intestinal epithelial cells and macrophages after oral administration [58]. Similarly, the study conducted by Wang et al. demonstrated that grapefruit-derived EVs maintained their stability both in acidic solutions and in simulated gastrointestinal fluids, showing remarkable resistance to enzymatic degradation [89]. In the same study, grapefruit PDEVs were used to encapsulate methotrexate (MTX). The authors compared the antiproliferative effect of encapsulated MTX versus the free drug, highlighting that most of the encapsulated MTX was selectively delivered to macrophages of the intestinal lamina propria. This approach significantly improved the anti-inflammatory properties of MTX while reducing its side effects, suggesting that PDEVs represent a promising system to increase the bioavailability of orally administered drugs. Similar evidence has emerged from studies on the use of PDEVs for the delivery of siRNA. Ginger-derived vesicles have been used to transport and protect siRNA from enzymatic degradation. Ginger-derived EVs have demonstrated the ability to deliver siRNA-CD98 to the intestine, effectively reducing the expression of the CD98 gene in colon cells [131].

7. Conclusions and Future Perspectives

Plant-derived extracellular vesicles represent a promising solution as biotechnological drugs. In fact, thanks to their natural origin, PDEVs are intrinsically safe, non-toxic and do not trigger immune responses. Furthermore, the production of plant material can offer a scalable alternative to the production limitations of synthetic or animal-derived EVs. Another important characteristic that distinguishes PDEVs is the presence of natural bioactive molecules cargo, such as proteins, lipids and miRNA, which play crucial functions in gene regulation and intercellular communication, giving PDEVs an intrinsic therapeutic potential. Other beneficial molecules contained in PDEVs include secondary metabolites with antioxidants and anti-inflammatory functions, which can be integrated with exogenous drugs to obtain synergistic effects. PDEVs are used in multiple contexts, ranging from the natural administration of nutrients and supplements to therapeutic support for pathologies such as cancer and inflammatory diseases. However, further studies are needed to better understand the pharmacological properties and delivery mechanism of PDEVs to fully exploit their therapeutic potential. Advances in bioengineering could further optimize their therapeutic potential, enabling more efficient and precise drug delivery. Surface modification strategies, for example, could be developed to improve targeting accuracy, allowing for personalized drug delivery systems tailored to individual patients and specific diseases. This would enable more effective treatments with fewer side effects, particularly for complex conditions such as cancer, neurodegenerative disorders, and genetic diseases. Moreover, the integration of PDEVs with cutting-edge technologies like gene editing or RNA-based therapies could open up new frontiers in precision medicine. By utilizing PDEVs as delivery vehicles for gene-editing tools or RNA molecules, it may be possible to directly correct genetic mutations or modulate gene expression in a highly targeted manner. This would enhance the therapeutic options available, offering more effective treatments for a wide range of previously untreatable diseases.