Edible plant-derived extracellular vesicles serve as promising therapeutic systems

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Nano TransMed 2 (2023) 100004
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Nano TransMed
journal homepage: www.keaipublishing.com/en/journals/nano-transmed/
Edible plant-derived extracellular vesicles serve as promising therapeutic 
systems
Chun Yanga,1, Wenjing Zhangb,1, Muran Baia,1, Qiyuan Luoc, Qing Zhenga, Yao Xuc, Xiaoya Lic, 
Cheng Jiangb,⁎, William C. Chod,⁎, Zhijin Fane,⁎
a College of Basic Medicin, Beihua University, Jilin 132033, China 
b School of Medicine, The Chinese University of Hong Kong, Shenzhen 518172, China 
c Guangzhou University of Chinese Medicine, Guangzhou, China 
d Department of Clinical Oncology, Queen Elizabeth Hospital, 30 Gascoigne Road, Kowloon, Hong Kong, China 
e School of Medicine, South China University of Technology, Guangzhou 510006, China 
A R T I C L E I N F O
Keywords: 
Plant-derived extracellular vesicles 
Nanomedicine 
Therapeutic system 
Drug delivery 
Anti-inflammatory 
Cancer therapy
A B S T R A C T
Extracellular vesicles (EVs), which are natural nanocarriers characterized by a phospholipid bilayer structure, 
are released by living cells. They play a crucial role in the intercellular transport of proteins, nucleic acids, lipids, 
and metabolites, facilitating substance delivery and information exchange between cells. In light of recent nu-
merous studies, EVs has been found to transcend their basic role as mere delivery vehicle. Instead, they de-
monstrate an impressive array of biological activities, displaying preventive and therapeutic potential in miti-
gating various pathological processes encompassing cancer, neoplastic proliferation, infectious diseases, and 
oxidative trauma. Particularly, EVs derived from edible plants (EPDEVs) have been emphasized for their ex-
tensive range of physiological regulatory functions in animals and humans, with the potential for targeted drug 
delivery through oral administration. Leveraging these advantages, EPDEVs are expected to have excellent 
competitiveness in clinical applications or preventive healthcare products. This review provides a brief overview 
of the biogenesis, structure, and composition of EPDEVs, and summarizes their biological functions and me-
chanisms. It also analyzes the methods for isolating and purifying plant-EVs, assessing their advantages and 
disadvantages; discusses the latest advancements in biomedical applications, and concludes with a prospective 
insight into the research and development directions of EPDEVs.
1. Introduction
Extracellular vesicles (EVs) are small membrane-bound particles 
secreted by cells, which can carry a range of biological marcomolecules 
including proteins, nucleic acids, and metabolites [1–6]. Due to their 
low immunogenicity, good biocompatibility, and capacity for en-
gineered cargo loading with functional modules, EVs have emerged as a 
new generation of natural drug delivery systems [7–15]. However, their 
practical application is curtailed by the high production cost and low 
yield of animal cell-derived EVs. In contrast, edible plant-derived EVs 
(EPDEVs)，which can be produced in large quantities from plant sap, 
have been attracting mounting attention [16,17]. Moreover, recent 
studies have unveiled the therapeutic potential of plant-derived EVs 
(PDEVs) in tissue repair, [18] anti-inflammation, [19] and anti-tumor 
effects, [20,21] thereby enhancing the potential of EPDEVs as a pio-
neering nanotherapeutic system [22]. In this review, we give an over-
view of the biogenesis, structural composition, and biological functions 
of EPDEVs. We also highlight the latest advances in their biomedical 
applications with a special emphasis on their potential clinical appli-
cations in tissue repair, chronic inflammation, and anti-tumor 
Production and Hosting by Elsevier on behalf of KeAi
https://doi.org/10.1016/j.ntm.2023.100004 
Received 29 June 2023; Received in revised form 3 August 2023; Accepted 7 August 2023 
Available online 16 October 2023
2790-6760/© 2023 The Author(s). Publishing services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd. This is an open access article under the CC BY 
license (http://creativecommons.org/licenses/by/4.0/). 
]]]] 
]]]]]]
⁎ Corresponding authors.
E-mail addresses: jiangcheng@cuhk.edu.cn (C. Jiang), chocs@ha.org.hk (W.C. Cho), fanzhj5@mail.sysu.edu.cn (Z. Fan).
1 These authors contributed equally.
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treatments. Our aim is to explore the translational applications of EP-
DEVs in realm of nanomedicine (Fig. 1).
2. Genesis, structure and composition of plant-derived EVs
2.1. Biogenesis of plant-derived EVs
EVs are a diverse group of membranous vesicles that originate from 
endosomes and cell membranes and are released into the extracellular 
environment [23–26]. They can be categorized in mammals based on their 
mode of biosynthesis, including exosomes, microvesicles, apoptotic bodies, 
ectosomes, and migrasomes [24]. Exosomes, a prominent type of EVs, are 
primarily observed in immune and tumor cells and are surrounded by a 
phospholipid bilayer that bulges inwards through the endocytosome to 
form a multivesicular body (MVB) [27,28]. Subsequently, these exosomes 
are discharged via the fusion of MVBs with the cell membrane.
In recent years, a groundbreaking study by Rutter and Innes suc-
cessfully isolated and purified EVs from Arabidopsis leaf exoplastids 
[29]. These vesicles are known to contain a high concentration of stress- 
and defense-related proteins, including PEN1 (a plant synthetic protein). 
Notably, the secretion of these vesicles is increased during infection with 
Pseudomonas syringae strains and in response to salicylic acid, sug-
gesting a crucial role in the plant's immune response. Since then, nu-
merous researchers have isolated EVs from plant sap, although the me-
chanism of EV formation in plants remains unclear [30–32]. One theory 
suggests that unconventional protein secretion (UPS) in plants may lead 
to the formation of EVs [33]. Another study found that a significant 
proportion of the proteins contained in EVs extracted from citrus lemons 
overlap with proteins contained in mammalian exosomes, [30] implying 
the presence of exosomes in plants. Evidence of MVBs in plant cells dates 
back to 1967, [34] and higher plants have shown that fusion of MVBs 
with the plasma membrane can lead to the release of exosomes.
Transmission electron microscopy studies of barley leaf cells at-
tacked by fungi clearly revealed and confirmed the origin of plant MVBs 
in the cells' endosomes [35,36]. Polyvesicular bodies in plants have also 
been identified as prevesicular cavities, considered to be late 
endosomes of plants, and are involved in the plant's response to pa-
thogen invasion [37–41]. It has been reported that plants can block 
fungal invasion by forming papillae, which is an extracellular space 
deposited between the plasma membrane (PM) and cell wall [42,43]. 
The presence of MVBs in the invading subpapillary cytoplasm around 
the fungal haustrum is a manifestation of the fusion of MVBs and PM 
[35,43]. Some studies have observed the presence of exosome-like ve-
sicles in the papillary matrix, leading scholars to propose a possibility 
that PM-derived vesicles could fuse with endocytosis pathway orga-
nelles, and then MVB fuses with PM, releasing intracavitary vesicles 
(ILVs) as exosomes outside of the cell. These exosomes could eventually 
integrate into the defense structure, delivering substances to the papilla 
via unconventional secretion, similarM. Colella, P. Del Gaudio, M. Moros, 
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	Edible plant-derived extracellular vesicles serve as promising therapeutic systems
	1 Introduction
	2 Genesis, structure and composition of plant-derived EVs
	2.1 Biogenesis of plant-derived EVs
	2.2 Structure and composition of plant-derived EVs
	2.2.1 Lipids
	2.2.2 Proteins
	2.2.3 Nucleic acids
	2.2.4 Other active components
	3 Biological functions and mechanisms of Plant-derived EVs
	3.1 Biological functions of plant-derived EVs in Plants
	3.2 Regulation of animal physiological processes
	3.2.1 Anti-inflammatory properties
	3.2.2 Anti-tumor properties
	3.2.3 Tissue repair properties
	4 Engineering strategies for plant-derived EVs
	4.1 Separation strategy of Plant-derived EVs
	4.1.1 Differential centrifugation
	4.1.2 Density gradient centrifugation
	4.1.3 Ultrafiltration/filtration
	4.1.4 Immunomagnetic bead method
	4.1.5 Polymer precipitation
	4.1.6 Other separation methods
	4.2 Engineering strategy of plant-derived EVs
	5 Plant-derived EVs in the application of biomedical therapy
	5.1 Application of plant-derived EVs in Anti-inflammation
	5.2 Application of plant-derived EVs in tissue repair
	5.3 Application of plant-derived EVs in anti-tumour
	5.4 Application of plant-derived EVs in the regulation of gut microbiota
	6 Prospectives and challenges
	7 Conclusion and outlook
	Ethics approval and consent to participate
	Authors' contributions
	Consent for publication
	Funding
	mk:H1_35
	Declaration of Competing Interest
	Acknowledgements
	Referencesto exosomes in mammalian cells, 
contributing to innate immunity in plants as shown in previous studies 
[44–46].
In contrast to mammals and yeasts, plant MVBs are believed to 
be transformed by maturation of clathrin-coated tubular networks 
(TGN) in the Golgi stack matrix, which is similar to the function of 
early endosomes (EE) in mammalian cells. Some studies support the 
idea that EVs are derived from TGN/MVB [47,48]. It has been re-
ported that approximately 59% of EV proteins overlap with pro-
teins contained in TGN/EE/MVB.29 Additionally, the functional 
analysis of proteins contained in EVs extracted from grapefruit 
showed that most of these proteins correspond to plasma membrane 
proteins (45%) and vacuolar proteins (23.5%), supporting the 
presence of EV-derived intracavital vesicles in plants, similar to 
mammalian exosomes. Plant EV proteins are transported to exo-
somes through unconventional secretion pathways [29]. However, 
it has also been reported that EV proteins extracted from ginger 
mainly overlap with cytoplasmic proteins and rarely overlap with 
membrane proteins [32].
2.2. Structure and composition of plant-derived EVs
PDEVs are also feature as membranous vesicles with lipid bilayer for 
the basic skeleton and contain various active substances such as 
Fig. 1. Edible plant-derived EVs serve as promising therapeutic systems for nanomedicine. Edible plants including fruits, vegetables and medicinal plants are broken 
to isolate EVs from their juices and engineered for anti-inflammatory, anti-tumor, tissue repair and gut microbiota regulation. PDEVs: plant-derived extracellular 
vesicles.
C. Yang, W. Zhang, M. Bai et al. Nano TransMed 2 (2023) 100004
2
proteins and nucleic acids. The structure and composition of EVs are 
similar to those of animal origin, including lipid, protein, nucleic acid 
and other cytoplasmic components.
2.2.1. Lipids
Lipids are the important component of the PDEVs lipid bilayer, 
consisting of two distinct groups of lipids: phospholipids and glycerin 
lipid, which have been found to be critical for the stability, uptake, and 
other biological functions of PDEVs [49]. Some special lipids also give 
vesicles specific functions: Phosphatidylic acid (PA) is an important 
lipid mediator that controls membrane division and fusion, and can also 
influence PDEVs uptake by triggering cytoskeletal rearrangement and 
protein regulation during endocytosis [50–52]. In addition, PA con-
tained in PDEVs may explain the effect of EVs on the growth and 
proliferation of mammalian cells through its effect on the mTOR 
pathway [53]. The results that PA depletes ginger EV suggest that PA 
also plays a role in maintaining the timing and amount of PDEVs that 
accumulates in the gut. In addition to PA, PDEVs also accumulate a 
variety of lipids such as phosphatidylethanolamine (PE), phosphati-
dylcholine (PC), phosphatidylinositol (PI) and diacylglycerol (DG) [54]. 
Among them, PC has been found to promote the spread of EV from the 
gut to the liver [55]. What's more, the PDEVs lipids were also found to 
regulate the gut microbiota, leading to changes in the distribution of 
gut microbiota. It is also worth noting that none of the PDEVs in-
vestigated so far have any cholesterol. Several studies have shown that 
PDEVs lipids play bioactive roles in recipient cells, [56,57] so a thor-
ough understanding of the composition and role of each lipid will help 
develop PDEVs-based therapeutic strategies.
2.2.2. Proteins
Proteins form the material foundation for the functionality of 
PDEVs, hence the composition and content vary among different types 
of plant-EVs [58]. PDEVs proteins can be broadly categorized into two 
types: transmembrane proteins and other plasma membrane-related 
proteins. As for now, among transmembrane proteins, CD63, CD81 and 
CD9 have been identified as potential markers for exosomes derived 
from mammalian cells [59]. However, no studies have conclusively 
reported a marker protein for PDEVs. It has been reported that the 
protein content of ginger-derived plant-EVs predominantly consists of 
cytoplasmic substances, and it was identified that these vesicles contain 
fewer membrane transporters, such as water channel proteins and 
chloride channels [55]. Moreover, several reports suggest that plant- 
EVs derived from grapes, [50] lemons, [60] and Arabidopsis [29]
contain heat shock proteins and aquaporins. Other plasma membrane- 
related proteins predominantly encompass metabolic enzymes, signal 
transduction factors, adhesion factors, cytoskeletal proteins and ubi-
quitin [29,61]. Despite these discoveries, extensive research is still re-
quired to identify the broad spectrum of proteins in PDEVs [62]. Such 
an endeavor may unveil their roles in biological and pharmacological 
activity.
2.2.3. Nucleic acids
EVs can transport various RNA materials between cells. RNA in 
PDEVs mainly includes mRNA, miRNA and small RNA (sRNA), in which 
mRNA and non-coding miRNA can regulate the levels of RNA and 
protein in recipient cells, thereby affecting cell morphology and func-
tion [63,64]. Additionally, sRNA might serve a role in intercellular 
communication within plant EVs, providing a vital link in cellular in-
teractions. Interestingly, in mammalian cell-derived EVs, some specific 
sRNA was found to be preferentially loaded into PDEVs, further em-
phasizing the complexity and potential significance of these cellular 
interactions [36]. miRNA is small non-coding RNA in plants and ani-
mals that regulate protein levels after transcription. In a recent study, 
418 conserved miRNAs (ranging from 32 in ginger EV to 127 in soy-
bean) extracted from 11 edible fruits and vegetables were used to 
predict functional relationships between plant-derived miRNA and 
potential target genes in mammalian genomes through bioinformatics 
analysis, and these mammalian genes were found to be involved in 
immune response and cancer [31].
In-vitro functional validation showed that miR-168c regulates the 
glucocorticoid-induced leucine zipper protein (TSC22D3), [36] which 
plays a key role in the anti-inflammatory and immunosuppressant ef-
fects of glucocorticoids. A recent study on the regulatory effects of 
PDEVs on human microbiota showed that ath-mir167a derived from 
ginger EV may directly bind to Lactobacillus rhamnosus protein SpaC 
mRNA and regulate SpaC expression, [51] which can significantly re-
duce the translocation of Lactobacillus rhamnosus in peripheral blood 
and remain on the mucosal surface. In the same study, the author de-
monstrated that ginger EV-RNA can regulate the production of indole- 
3-acetaldehyde in lactic acid bacteria and affect the production of IL-22 
in host cells, thereby improving colitis. These findings suggest that 
PDEVs have broad cross-species regulatory potential. PDEVs have po-
tential in the field of infectious disease treatment and flora regulation.
To date, most studies of EV-mediated intercellular communication 
have focused on the more commonly recognized types of RNA, such as 
miRNA and mRNA [65,66]. Naked miRNA are unstable and easily 
broken down by ubiquitous RNA enzymes, but their encapsulation 
within EVs avoids this outcome. Arabidopsis derived PDEVs contain 
miRNA, sRNA (18–24 nt), and tiny RNA (tyRNA) (10–17 nt). The co-
existence of these RNA in apoptotic bodies suggests a specific sorting 
mechanism on RNA [67]. In addition, PDEVs often contain unusual 
non-coding RNA, however, it is not fully understood how PDEVs pro-
cesses them and their uptake into recipient cells as well as their impact 
on cell communication, therefore, further research is required to elu-
cidate these aspects [62].
2.2.4. Other active components
Apart from lipids, proteins, and nucleic acids, research has also in-
dicated that plant metabolites possess their own unique biologicalac-
tivities. [68,69] Specifically, secondary metabolites, while not crucial 
for cellular function or survival, nevertheless play a significant role in 
supporting the plant's resilience and adaptability within its environ-
ment. Secondary metabolites mainly include: (a) nitrogenous metabo-
lites, synthesized mainly from amino acids (e.g., alkaloids and gluco-
sides), (b) non-nitrogenous metabolites, such as phenolic compounds, 
which contain aromatic rings with hydroxyl groups (e.g., phenolic 
acids, coumarins, indole, flavonoid tannins and lignin), (c) terpenoids, 
Hydrocarbons derived from isoprene (e.g. plant volatiles, cardiac gly-
cosides, carotenoids, and sterols) [70]. These are of great use in the 
pharmaceutical and health products industry to cure many human and 
livestock diseases [71]. For example, dietary saponins have been found 
to reduce cholesterol in the blood, inhibit the growth of cancer cells, 
and stimulate the immune system [71]. Additionally, ginseng saponin 
has been proven to exhibit significant physiological activity. It provides 
protective effects for the liver, nervous system, and cardiovascular 
system, enhances immunity, and contributes to fatigue resistance. 
Moreover, its antioxidant and anti-tumor effects are particularly no-
table [72]. During their formation process, PDEVs also encapsulate 
these active ingredients, thereby conferring therapeutic functions.
3. Biological functions and mechanisms of Plant-derived EVs
PDEVs serve as carriers for intercellular transport and contain var-
ious biological active substances such as DNA, small RNA (sRNA), 
microRNA (miRNA), and proteins. They act as a conduit for both ma-
terial and information exchange between cells [33]. Of these compo-
nents, miRNA stands out as a potent regulator of gene expression. 
Within the source organism, miRNA performs a myriad of biological 
functions. Beyond its domestic roles, miRNA also has the capacity to 
regulate gene expression in neighboring organisms, demonstrating its 
significant influence within the larger biological community. Mean-
while, sRNA transfers between hosts and organisms through 
C. Yang, W. Zhang, M. Bai et al. Nano TransMed 2 (2023) 100004
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intercellular connections and induces gene silencing. This complex in-
terplay of bioactive substances transported via PDEVs results in various 
biological effects, such as anti-inflammatory, anti-viral, anti-fibrotic, 
anti-tumor effects by exerting their contents and participate in the de-
fense mechanism against pathogen invasion. The majority of PDEVs are 
edible, and they can serve as carriers for delivering targeted drugs with 
non-toxicity and low side effects, making them a hot research topic.
3.1. Biological functions of plant-derived EVs in Plants
PDEVs mainly play a role in plant intercellular communication. This 
includes intercellular and interspecies communication. PDEVs con-
taining miRNA, bioactive lipids, and proteins act as extracellular mes-
sengers, mediating intercellular communication in a manner similar to 
mammalian-EVs [73,74]. Proteomic analysis of sunflower seed EVs 
revealed that PDEVs are associated with the secretion of enzymes in-
volved in cell wall modification. Further, PDEVs are engaged in nu-
merous cellular activities. These include cell proliferation, differentia-
tion, and the orchestration of responses to various signals or triggers - 
which could be environmental cues, like light or temperature changes, 
or biological signals, such as the presence of pathogens. Zhao et al. 
founded that EVs isolated from mature and immature coconut water are 
involved in the maturation process of coconuts through RNA sequen-
cing [75].
Furthermore, various studies have shown that PDEVs play a sig-
nificant role in self-defense responses, particularly in inducible defense 
mechanisms, including the clearance of harmful substances from cells 
and participation in immune surveillance processes. It has been re-
ported that Arabidopsis secretes PDEVs at the site of Staphylococcus 
aureus infection and delivers sRNA into the fungus to silence critical 
pathogenic genes, playing a role in cross-species defense regula-
tion [76]. Adding to this understanding, research by Rutter et al. de-
monstrated an upsurge in PDEVs secretion following an infection by 
Pseudomonas syringae in Arabidopsis [29]. A similar increase was ob-
served after treatment with salicylic acid, suggesting PDEVs' active in-
volvement in plant immune responses. Hansen et al. found that vesicles 
can stop the growth of fungi in plants when they are invaded by fungi 
[77]. These data suggest that the secretion of EVs in plants contributes 
to the formation of early defense structures against pathogens. In 
summary, EVs are released in plant cells during pathogen invasion, 
helping to prevent the pathogen from entering the cell.
3.2. Regulation of animal physiological processes
3.2.1. Anti-inflammatory properties
Numerous studies have demonstrated the important role of ingested 
EPDEVs in alleviating intestinal inflammation [78,79]. Through con-
structing a model of intestinal epithelial cells and a mouse model of 
intestinal inflammation, researchers evaluated the protective effect of 
edible PDEVs on the intestinal barrier [50, 80, 81]. The structural basis 
of mechanical barriers is intact intestinal mucosal epithelial cells and 
tight connections between epithelial cells. Citrus-derived EVs can reg-
ulate the gene expression of tight junction proteins to restore intestinal 
barrier function, while grape EVs accelerate the recovery of the in-
testinal mucosal epithelium by inducing the proliferation of intestinal 
stem cells [82,83]. The intestinal microbiota is an important component 
of the intestinal mucosal barrier, and an imbalance in the intestinal 
microbiota can accelerate intestinal inflammation. Oral administration 
of PDEVs derived from tea leaves enhances the diversity and overall 
abundance of the gut microbiota, reducing the ratio of Firmicutes to 
Bacteroidetes and alleviating DSS-induced colitis [84]. PDEVs rich in 
lipids are preferentially absorbed by intestinal lactobacilli, targeting the 
regulation of ycnE production in Bifidobacterium animalis and inducing 
interleukin-22, and improving intestinal barrier function, thereby al-
leviating colitis. PDEVs can also target intestinal macrophages, upre-
gulating heme oxygenase-1 expression, suppressing the production of 
pro-inflammatory cytokines, and upregulating interleukin-10 (IL-10) 
secretion, thereby maintaining intestinal homeostasis [85,86]. PDEVs 
can also participate in regulating immune responses [51,55,87,88]. For 
example, PDEVs derived from fish mint can promote the conversion of 
M1-type activation induced by lipopolysaccharide to M2 phenotype and 
inhibit the activation of the nuclear factor kappa-B (NF-κB) and mi-
togen-activated protein kinase (MAPK) signaling pathways. In addition, 
PDEVs derived from chives can activate the NOD-like receptor protein 3 
(NLRP3) inflammasome and its downstream signaling pathways, in-
cluding caspase-1 cleavage, cytokine release, and pyroptosis of primary 
macrophages. Different PDEVs have slightly different mechanisms of 
action for anti-inflammatory molecules, which may be related to the 
types of nucleic acids carried by PDEVs. Xiao et al. used gene sequen-
cing technology to predict the miRNA gene sequences targeting cyto-
kines in PDEVs from five sources, including soybeans, cantaloupes, 
oranges, ginger, and tomatoes. The miRNA in PDEVs can regulate both 
intestinal barrier function and the function of dendritic cells, T cells, 
macrophages, and NF-κB-related signaling pathways to enhance im-
mune function in the body [31]. These findings reveal that PDEVs can 
protect the intestinal barrier, regulate inflammatory pathways, main-
tain intestinal homeostasis,and thus alleviate inflammation in the 
body.
3.2.2. Anti-tumor properties
Tumor is a complex systemic disease, which seriously threatens 
human life and health [89–92]. However, there is a lack of effective 
treatment in clinical practice [93]. Raimondo et al. demonstrated that 
EVs in citrus lemon juice could inhibit tumor cell growth by inducing 
the expression of TRAIL (TNF-related apoptosis-inducing ligand), in-
creasing the expression of pro-apoptotic genes Bad and Bax, and de-
creasing the expression of anti-apoptotic genes Survivin and Bcl-xl, 
without affecting normal cells [30]. Furthermore, it could also down- 
regulate the expression of vascular endothelial growth factor A (VEGF- 
A), interleukin-6 (IL-6), and interleukin-8 (IL-8) to inhibit angiogenesis. 
These findings suggest that lemon ELNs can inhibit cancer cell pro-
liferation by promoting TRAIL-mediated apoptosis and inhibiting the 
secretion of VEGF-A, IL-6, and IL-8.
It has been reported that ginseng-derived vesicles were quickly re-
cognized and internalized by macrophages, inducing M1 polarization 
and promoting the generation of reactive oxygen species (ROS), leading 
to apoptosis of melanoma cells in mice and inhibiting tumor growth 
[94]. Nanocarriers of ginger-derived EVs (grapefruit-derived lipid na-
noparticles, GDNV) can be used as carriers for 5-fluorouracil treatment 
of colon cancer [32]. The results have shown that colon cancer cells can 
effectively absorb GDNV, which can efficiently load 5-fluorouracil. 
Modified GDNVs are combined with targeting ligand folic acid, med-
iating the targeted delivery of 5-fluorouracil to the colon-26 tumors in 
vivo. Compared with free drugs, it enhances the inhibitory effect of 
chemotherapy on tumor growth. In another study, Shao et al. found 
that miRNA in licorice has a significant modulating effect on the gene 
expression of human immune cells, significantly inhibiting the expres-
sion of genes related to T cell differentiation, inflammation, and 
apoptosis [95]. In summary, PDEVs demonstrate multidimensional 
anti-tumor potential, including affecting tumor cells and the tumor 
microenvironment.
3.2.3. Tissue repair properties
Tissue repair plays a pivotal role in addressing the intricate re-
lationship between tissue senescence, damage, and disease. Skillful and 
timely tissue regeneration techniques not only counteract the effects of 
aging and injury but also hold the potential to preempt or alleviate the 
onset and advancement of various medical ailments. Recent studies 
have shown that PDEVs have potential tissue repair properties. Zhuang 
et al. orally administered ginger-derived vesicles to normal mice and 
found that they primarily accumulated in the liver and mesenteric 
lymph nodes, and could inhibit the generation of ROS in liver cells [96]. 
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Vesicle-treated mice with alcohol-induced liver injury showed a de-
crease in lipid droplets and triglyceride levels in the liver, as well as a 
reduction in liver weight, which were significantly better than those in 
the untreated model group, indicating the potential of ginger vesicles 
for preventing and treating alcohol-induced liver injury. Another study 
found that the traditional Chinese medicine Rhodiola contains sRNA 
(HJT-sRNA-m7) that can target proteins related to pulmonary fibrosis, 
reduce fibrosis factor expression, and improve pulmonary fibrosis 
symptoms [97]. Most of the lipid components in Rhodiola decoction are 
also present in Houttuynia, Dandelion, and Honeysuckle, and vesicles 
formed by lipids can facilitate the absorption of sRNA into the blood-
stream [98,99]. Oral sRNA as a therapeutic drug provides an innovative 
treatment strategy. Although these studies did not emphasize the re-
lationship between vesicle structure and RNA, the phenomenon of 
forming nanoparticles from medicinal plants during decoction is very 
common, and the separation and characterization methods are similar 
to those for juice-derived PDEVs. In essence, both are nanostructures 
containing multiple active components obtained from differently pro-
cessed plant samples.
Recently, Sahin et al. explored the potential role of wheat-derived 
EVs in treating skin wound healing by establishing an in vitro wound 
healing model [100]. The results showed that wheat EVs had a re-
markable pro-proliferation and migration effect on endothelial cells, 
epithelial cells, and dermal fibroblasts, promoting the formation of 
tube-like structures by endothelial cells, enhancing the expression of 
wound healing-related genes, modulating and coordinating the forma-
tion of blood vessels, and promoting wound healing. Furthermore, it 
was found that quercetin encapsulated in poly[lactic-co-glycolic acid] 
(PLGA) nanoparticles can be safely and controllably released, allowing 
cells to recruit, adhere, expand, and express cardiogenic proteins in the 
local myocardium, which is of great help for the treatment of cardio-
vascular indications. Zhang et al. found that miR168a, which is abun-
dant in rice, can bind to human/mouse low-density lipoprotein receptor 
adapter protein 1 (LDLRAP 1) mRNA, reducing the removal rate of low- 
density lipoprotein from mouse plasma and reducing the risk factors for 
cardiovascular disease [101].
4. Engineering strategies for plant-derived EVs
4.1. Separation strategy of Plant-derived EVs
4.1.1. Differential centrifugation
The separation of PDEVs from other substances by centrifugation is 
the most common separation method for PDEVs at present. Initially, a 
mild centrifugal force of 300 g is applied to remove cells from the cell 
culture medium. This is followed by stronger centrifugation, ranging 
from 1000 to 20,000 g, to eliminate large cell fragments and damaged 
organelles from the supernatant. Exosomes are then collected from the 
supernatant by means of high-speed centrifugation, typically between 
100,000 and 150,000 g. All of these centrifugal operations are carried 
out at 4 °C. A significant advantage of this approach is that the exo-
somes obtained are free from contamination by separation reagents, 
and the method accommodates a large number of separations while 
requiring small treatment samples. However, although ultra-fast cen-
trifugation is considered the "gold standard" for exosome extraction, it 
has numerous drawbacks. These include the relatively high cost of 
ultra-high-speed centrifugation equipment, the need for large sample 
sizes, and a long time for completion. Nevertheless, protein con-
tamination can still be present when observing exosomes under an 
electron microscope. Additionally, this method's separation efficiency is 
relatively low for plant samples with a high viscosity or more impurity 
particles, such as pueraria and yam [88].
4.1.2. Density gradient centrifugation
Density gradient centrifugation is a method that capitalizes on the 
different sedimentation coefficients of various components. Each 
component settles at its own rate under centrifugal force, forming bands 
within distinct density gradient regions. Common gradient materials 
include sucrose and ioxanol. Current findings suggest that exosomes 
can float at a sucrose gradient density of 1.15–1.19 g/mL. Based on this 
characteristic, samples can be ultra-centrifugally using a sucrose gra-
dient solution, enabling the identification of exosomes as they settle 
into different density regions. The process involved in the sucrose 
density gradient centrifugation method is intricate. It necessitates the 
advanced preparation of a continuous gradient concentration sucrose 
solution, which is placed at the bottom of the centrifuge tube, while the 
sample is positioned at the top. Centrifugation is then performed at 
100,000 xg at 4 ℃. While this method yields exosomes of high purity, itis not without its drawbacks. The process is complicated and time- 
consuming, and it fails to fully separate exosomes from proteins. 
Additionally, some researchers have indicated that the biological 
function of cell vesicles may be compromised when isolated via sucrose 
density gradient centrifugation. One can obtain PDEVs of high purity 
through density gradient centrifugation following differential cen-
trifugation, but this method has its limitations, especially in terms of 
scalability.
4.1.3. Ultrafiltration/filtration
Ultrafiltration is a method to separate samples of different scales 
under certain pressure by using the microporous structure of ultra-
filtration membrane. According to the size of exosomes, the filter 
membrane with corresponding pore size is used to filter the small and 
medium molecules of the sample to the other side of the membrane, 
while the large molecules are retained on the membrane to achieve the 
purpose of separation. Since the ultrafiltration membrane has been 
commercialized and the specifications are convenient, this method can 
be considered as an alternative to ultra-high speed centrifugation. Xu 
et al. improved the simple ultrafiltration method by connecting mem-
branes with different pore sizes (200, 100, 80, 50, 30 nm) in series to 
achieve rapid separation of exosomes of different sizes, and the capture 
efficiency was significantly higher than that of the ultrafiltration 
method [102].
The filtration method is a method to separate PDEVs based on 
molecular mass and size. The filtration method usually adopts fiber 
bundle filter and is often combined with ultrafiltration. Compared with 
differential centrifugal method, filtration method has lower pressure 
and better purification effect. However, due to the extrusion effect, the 
ultrafiltration/filtration process may change the structure of the PDEVs, 
and the filter membrane may have problems such as blockage and 
contamination. In addition, the filter is easily blocked by vesicles and 
other large molecules, which can easily cause the membrane to break 
under too much pressure.
4.1.4. Immunomagnetic bead method
The immunomagnetic bead method uses the functional groups on 
the surface of the magnetic bead to identify the surface protein of 
PDEVs, and realizes the separation of PDEVs under the action of mag-
netic field. Under the condition of specific identification and effective 
dissociation of PDEVs surface protein, the method can realize the se-
paration of specific PDEVs and its subtypes, the external magnetic field 
is easy to adjust, and the method has a good enrichment effect.
4.1.5. Polymer precipitation
Polymer precipitation method uses polymer competitive binding of 
water molecules to precipitate PDEVs from solution to achieve the 
purpose of separation. PDEVs is usually obtained by adding poly-
ethylene glycol (PEG) to the liquid sample containing PDEVs and then 
centrifuging at low speed. Polyethylene glycol is a water-soluble non- 
ionic compound with strong hydrophilicity, which can bind to the hy-
drophobic lipid bilayer, thus changing the solubility of exosomes and 
causing exosomes to precipitate. It has been reported that PEG level can 
affect exosome production, and the total proteins and RNA obtained 
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from exosomes are sufficient in quantity and quality for proteomics and 
sequencing analysis. Precipitation method is simple to operate, does not 
require special equipment, is more economical, and has high exosome 
production, but it will precipitate some non-exosome hydrophobic 
substances, resulting in insufficient exosome purity [103]. Recently, 
polymer-based co-precipitation kits have been developed, such as 
ExoQuick, TEI, etc., which can be used to extract exosomes from a 
variety of body fluids. Exosome precipitation could be obtained after 
the polymer precipitator ExoQuick was incubated with the sample at 
4 ℃ for 30 min, and then centrifuged at 1500 g at room temperature for 
30 min. Compared with the ultrafast centrifugation method, the kit 
method is simpler and shorter, and can achieve higher exosome pro-
duction. Exosome precipitation obtained by kit method contains more 
impurities, and samples from different sources need to use different kits 
for extraction, and the kits are more expensive. The method is simple to 
operate, but the purity of the extracted sample is not high. Residual PEG 
can increase the viscosity of the sample, and may affect the subsequent 
experiment [104].
4.1.6. Other separation methods
In addition to the above separation methods, there are dimensional 
exclusion chromatography, field flow separation, ultrasonic purifica-
tion technology, and separation methods based on surface component 
affinity. These methods are mainly based on the differences in the 
physical and biological properties of the impurities in the vesicles. For 
example, the exclusion method and the field flow separation method 
mainly use the size of the vesicle particle size for separation, and the 
affinity separation rule uses the affinity of the vesicle surface molecules 
for adsorption and elution, so as to achieve the purpose of separation 
and purification. In recent years, with the development of new isolation 
technologies, some new EVs isolation methods have been developed, 
such as nanoflow cytometry developed by Morales-Kastresana et al., to 
realize the isolation of EVs from immune cell lines and tumor cell lines 
[105]. The development of related separation technology can provide 
reference for the research of PDEVs, but different methods have their 
own advantages, and should be selected and optimized according to the 
research object and purpose. (See Table 1 for the advantages and dis-
advantages of each separation method.).
4.2. Engineering strategy of plant-derived EVs
Nanomedicine has provided new ideas for the diagnosis and treat-
ment of many diseases [118–122]. As natural nanocarriers, extra-
cellular vesicles have a wide range of biomedical applications. Natural 
PDEVs have limitations in terms of targeting, drug loading and func-
tional diversification. The engineered PDEVs are expected to overcome 
these problems and become a tool for targeted therapy. EV engineering 
methods can be divided into three categories: biological engineering, 
chemical engineering and physical engineering. Depending on the 
specific mode of operation, they can also be divided into drug loading, 
surface modification and hybridization.
Drug loading is the first problem to be faced by PDEVs as a drug 
delivery carrier. Depending on the nature of the drug, the loading 
method chosen is also different. Small molecules of fat-soluble drugs 
can be loaded into PDEVs through co-incubation. Small non-fat soluble 
molecules can be introduced into the vesicle by means of ultrasonic 
oscillation. The loading of macromolecules is difficult to be assisted by 
free diffusion and ultrasonic diffusion, and usually needs to be achieved 
by freezing and electroporation. The essence of these methods is to 
build a channel to meet the drug entry, so as to improve the loading 
efficiency of drugs.
During drug delivery, natural vesicles are achieved through their 
natural biological distribution, which includes targeting by means of 
enhanced permeability and retention (EPR) effects at tumor and in-
flammatory sites, as well as by transcellular operation of the endothelial 
system. However, there is still much room for improving the efficiency 
and specificity of these passive targeting actions. Surface modification 
can assemble specific targeting molecules to the surface of PDEVs to 
achieve affinity targeting with cell surface receptors. For example, 
ginger-derived PDEVs are functionalized by folic acid (FA) to achieve 
efficient affinity to targetcells and further deliver genes to tumor sites 
at the living level [123]. The most common strategy for surface func-
tionalization based on PDEVs is the incubation method, which involves 
mechanisms of hydrophobic interactions, diffusion processes, and 
electrostatic interactions. In addition, chemical coupling provides more 
possibilities for surface modification of PDEVs. The amino group, car-
boxyl group and sulfhydryl group on the surface of PDEVs are all 
common groups that are chemically coupled and can achieve firm 
modification under relatively mild conditions. The modification mode 
of chemical combination with biological metabolic reaction further 
improves the specificity of chemical modification and provides the 
possibility for in vivo modification. In conclusion, the surface mod-
ification of PDEVs can change the interaction mode of PDEVs with 
biological systems, achieve specific targeting affinity or change its 
original biological distribution, and ultimately is expected to achieve in 
vivo distribution and targeting according to human needs.
In order to explore the potential application of PDEVs, the hy-
bridization of PDEVs with other nanosystems has received extensive 
attention. For example, using the porous structure of metal-organic 
frameworks (MOFs) to improve the efficiency of drug loading, the 
packaging of PDEVs can greatly improve the biocompatibility of the 
hybrid system. At the same time, the bioimaging, stimulus response and 
Table1 
Methods for plant-derived EVs extraction. 
Methods Advantages Disadvantages Ref.
Ultrafast centrifugation Simple operation, no external pollution Time-consuming, require specific instruments [106,107]
Sucrose density gradient centrifugation 
method
High product purity Complex operation, cells may lose activity [108]
Size-exclusion chromatography High product purity, complete ultrastructure The number of purified products is small, limited by 
the instrument
[109]
Ultrafiltration Simple operation, low cost, high capture 
efficiency
Ultrafiltration membrane is easy to be blocked by 
macromolecular substances
[110]
Immunomagnetic bead method High product purity, good shape, high recovery Physiological salt concentration will affect biological 
activity
[111]
PEG precipitation method Simple operation, can achieve sequencing 
analysis
Low purity, making downstream analysis difficult to 
carry out
[112]
Microfluidic chip method Short time consuming, suitable for biological 
research
High requirements on equipment [113,114]
Asymmetric field flow separation method The separation range can be from a few 
nanometers to a few microns
Small amount of sample separation [115,116]
Ultrasonic nanofiltration technology Simple operation, no external pollution Expensive [117]
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catalytic activity of MOFs further enhance the application potential of 
hybridization systems. There have also been studies of hybridizing 
PDEVs with multifunctional liposomes, allowing new systems to in-
tegrate the advantages of both.
5. Plant-derived EVs in the application of biomedical therapy
PDEVs exhibit various fascinating biological functions and have 
wide-ranging applications in biomedical therapy. This review sum-
marizes the progress in their application in the areas of anti-in-
flammation, tissue repair, anti-tumor, and gut microbiota regulation, 
aiming to provide readers with the latest research insights (Fig. 2).
5.1. Application of plant-derived EVs in Anti-inflammation
PDEVs have great research value in anti-inflammatory aspects. 
Currently, the latest research has found that PDEVs have a promoting 
effect on the treatment of inflammatory diseases such as colitis and 
steatosis. They can reach the gastrointestinal tract in intact structures, 
participate in the renewal and regulation of the gastrointestinal tract 
microbiota. Currently, the most widely used are grapefruit-derived EVs, 
which can inhibit inflammation-mediated by inducing the production 
of heme oxygenase-1 and IL-10 for the treatment of DSS. Additionally, 
grapefruit EVs can also inhibit the production of pro-inflammatory 
cytokines, such as IL-1β, TNF-α, and IL-6, by intestinal macrophages, 
and reduce the levels of related chemokines, such as MCP-1, CXCL9, 
and CXCL10. Therefore, grapefruit-derived EVs have enormous poten-
tial for regulating intestinal immune responses and treating colitis in 
the future [80]. For the anti-inflammatory effect on colitis, broccoli- 
derived exosomes can regulate immune responses by targeting DCs to 
play an anti-inflammatory role [87]. In addition, the anti-inflammatory 
effect has also been found to some extent in liver disease. Ginger-de-
rived nanoparticles (GDN) loaded with gingerol can be targeted to liver 
cells and protect mice from alcohol-induced liver injury. Therefore, the 
anti-inflammatory effect of ginger still has great potential, playing an 
important role in protecting the intestine and digestive organs [81]. 
Furthermore, carrots have also been found to have strong anti-in-
flammatory effects in current research. Experiments have found that 
exosomes derived from carrots can induce the production of IL-10, 
promote the nuclear translocation of Nrf2, and inhibit the occurrence of 
inflammation, showing great potential for the inhibition of intestinal 
inflammation. Notably, curcumin, the bioactive constituent of turmeric, 
was evidenced in turmeric-derived EVs (Fig. 2A) [124]. In lipopoly-
saccharide (LPS)-induced acute infammation, turmeric-derived EVs 
showed excellent antiinfammatory and antioxidant properties. In mice 
colitis models, we demonstrated that orally administrated of turmeric- 
Fig. 2. Plant-derived EVs in the Application of Biomedical Therapy. A. Application of PDEVs in Anti-inflammation. B. Application of PDEVs in tissue repair. C. 
Application of PDEVs in anti-tumour. D. Application of PDEVs in the regulation of gut microbiota. 
(a) Reproduced with permission [124]. Copyright 2022, The Author(s). (b) Reproduced with permission [125]. Copyright 2023, Elsevier. (c) Reproduced with 
permission [126]. Copyright 2021, American Chemical Society. (d) Reproduced with permission [51]. Copyright 2018, Elsevier.
C. Yang, W. Zhang, M. Bai et al. Nano TransMed 2 (2023) 100004
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derived EVs could ameliorate mice colitis and accelerate colitis re-
solution via regulating the expression of the pro-infammatory cyto-
kines, including TNF-α, IL-6, and IL-1β, and antioxidant gene, HO-1. 
Results obtained from transgenic mice with NF-κB-RE-Luc indicated 
that turmeric-derived EVs-mediated inactivation of the NF-κB pathway 
might partially contribute to the protective effect of these particles 
against colitis.
Therefore, the anti-inflammatory effects of PDEVs are mainly con-
centrated in plants such as grapefruit, turmeric, and ginger, mainly 
used for inflammatory diseases in the digestive system such as colitis. 
For the potential anti-inflammatory effects, if applied to clinical treat-
ment in the future, it will mainly be used for the treatment of in-
flammatory diseases in the digestive system such as colitis. However, it 
is believed that PDEVs will discover the potential for other systemic 
diseases in the future, and drug development will also have greater 
prospects and breakthroughs.
5.2. Application of plant-derived EVs in tissue repair
In addition to their anti-inflammatory properties, PDEVs have en-
ormous potential for tissue repair. Wound healing is a physiological 
process that involves steady state, inflammation, proliferation, migra-
tion, and tissue remodeling. PDEVs can mediate wound healing by 
enhancing gene expression,inhibiting gene translation, or activating 
signaling pathways that are important for the wound healing process. 
Recent studies have found that wheat-derived exosomes play an im-
portant role in in vitro wound healing. The wound healing process re-
quires the replacement of the previously formed fibrin clot with type I 
collagen matrix, and collagen produced by fibroblasts controls cell 
adhesion and migration in skin healing. Wheat exosomes were found to 
enhance mRNA levels of type I collagen, leading to collagen production 
and increased fibroblast proliferation and migration during wound 
healing. Angiogenesis is a key stage in wound healing. When angio-
genesis occurs and endothelial cells are activated, they can transport 
nutrients to the wound site. Wheat-derived EVs can promote the for-
mation of tube-like structures by enhancing angiogenesis. Therefore, 
wheat-derived exosomes have great potential for future skin wound 
healing, and combining drugs with wheat-derived exosomes can more 
accurately accelerate skin wound healing [100]. For wound healing, 
grapefruit-derived vesicles can upregulate the expression of prolifera-
tion- and migration-related genes at a certain dose, increase the tube- 
forming ability of HUVEC cells, and promote wound healing, which is 
also a promising therapeutic tool for the future. In terms of tissue re-
pair, other cell-derived exosomes have made great breakthroughs and 
discoveries. In a recent study, Chen et al. encapsulated plant exosomes 
in hydrogels for spinal cord injury treatment (Fig. 2B). [125]. They 
reported that the hydrogel loaded with Flos Sophorae Immaturus-de-
rived exosomes allowed rapid improvement of the impaired motor 
function and alleviation of urination dysfunction by modulating the 
spinal inflammatory and oxidative conditions. This study provides an 
efficient and rapid acute-phase treatment plan for improving oxidative 
stress microenvironments after spinal cord injury and provides a the-
oretical basis for later clinical applications [127]. Therefore, there is 
still much to be explored and vast application prospects for the tissue 
repair function of PDEVs. It is believed that breakthroughs can be made 
in the treatment of cardiovascular, nervous, digestive, and other dis-
eases and the development of related drugs in the future.
5.3. Application of plant-derived EVs in anti-tumour
In recent years, an increasing number of studies have indicated the 
significant application prospects of PDEVs in anti-tumor therapy. 
Firstly, PDEVs can directly target tumor cells by carrying bioactive 
substances such as miRNA and proteins, exerting anti-tumor effects. 
Secondly, PDEVs can serve as effective drug carriers. These nanove-
sicles can be redesigned as harmless, non-immunogenic nano-carriers 
that can navigate through various biological barriers in the body to 
deliver drugs to various target organs, such as the intestines and the 
brain [62]. By encapsulating anti-tumor drugs internally, PDEVs en-
hance drug stability and bioavailability. Additionally, PDEVs can exert 
anti-tumor effects through various pathways, such as activating the 
immune system and modulating the tumor microenvironment. There-
fore, PDEVs have broad prospects for application in anti-tumor therapy 
and deserve further in-depth research and development.
Many PDEVs have demonstrated anti-cancer activity to a certain 
degree. Raimondo et al. investigated the application of lemon juice- 
derived nanovesicles in the treatment of chronic myeloid leukemia 
(CML). Lemon-derived nanovesicles exhibited the expected targeting 
effect in a CML model, significantly inhibiting tumor growth, suppres-
sing the secretion of various cytokines associated with angiogenesis, 
and inducing cell death mediated by TNF-α-related apoptosis-inducing 
ligand (TRAIL). In vivo biodistribution studies demonstrated that na-
novesicles targeted tumor tissue within 15 min and exhibited sustained 
action for over 24 h. Nanovesicles mediated cross-talk and eradicated 
cancer cells through TRAIL signaling, supporting their feasibility as 
chemotherapy drug carriers [128].
Grapefruit-derived nanocarriers (GNVs) carrying miR17 can be used 
for the treatment of brain tumors in mice. Folate-coated GNVs (FA- 
GNVs) enhance the targeting effect on folate receptor-positive GL-26 
brain tumors. Furthermore, FA-GNV-coated polyethyleneimine (FA- 
pGNV) not only enhances RNA-carrying capacity but also eliminates the 
toxicity of polyethyleneimine. Nasal administration of FA-pGNV car-
rying miR17 rapidly delivers the cargo to the brain and is selectively 
taken up by GL-26 tumor cells. The results demonstrate that mice 
treated with FA-pGNV/miR17 via intranasal delivery experience de-
layed brain tumor growth, offering a non-invasive therapeutic approach 
for treating brain-related diseases [129]. Nanovesicles can be isolated 
from Aloe vera gel and Aloe vera peel (gADNVs and rADNVs). gADNVs 
exhibit good structural stability, storage stability, and antioxidant ca-
pacity. They can be effectively absorbed by melanoma cells without 
toxicity in vitro and in vivo. Indocyanine green (ICG) encapsulated in 
gADNVs (ICG/gADNVs) demonstrates excellent stability under heating 
conditions and in serum, with a retention rate of over 90% after 30 days 
in gADNVs. It also effectively damages melanoma cells, inhibits mela-
noma growth, and outperforms free ICG and ICG liposomes. In addition 
to its ideal stability, gADNVs show remarkable skin penetration in mice, 
which may be advantageous for non-invasive transdermal drug delivery 
[130]. 5-fluorouracil (5-FU) is widely used in cancer treatment; how-
ever, 5-FU-mediated activation of the NOD-like receptor family pyrin 
domain-containing 3 (NLRP3) inflammasome induces resistance of oral 
squamous cell carcinoma (OSCC) cells to 5-FU. It has been shown that 
bitter melon-derived EVs (BMEVs) have intrinsic anti-inflammatory 
functions that can combat OSCC. BMEVs have the potential to enhance 
the therapeutic efficacy of OSCC treatment; they can significantly exert 
a synergistic effect with 5-FU on OSCC both in vitro and in vivo, in-
ducing S-phase cell cycle arrest and apoptosis. In addition, BMEVs have 
the potential to reduce 5-FU resistance: BMEVs significantly down-
regulate the expression of NLRP3, which reduces OSCC's resistance to 5- 
FU; the RNA of BMEVs can at least partially mediate anti-inflammatory 
bioactivity [131].
A recent study found that modified naturally derived grapefruit ev 
can improve glioblastoma systemic chemotherapy drug delivery diffi-
culties due to the blood-brain barrier/blood-brain tumor barrier (BBB/ 
BBTB) and limited tumor penetration (Fig. 2C) [126]. Heparin-based 
nano-particle-loaded doxorubicin (DNs) is attached to the surface of 
grapefruit EVs to create biomimetic EV-DNs. EV-DNs can enter glio-
blastoma tissue by receptor-mediated endocytosis and membrane fu-
sion, bypassing the blood-brain barrier and significantly promoting 
intracellular uptake and anti-proliferative ability, prolonging circula-
tion time, and achieving effective drug delivery. Thus, EV-DNs can 
significantly improve the efficacy of anti-glioma treatment. This 
strategy exhibits four times the drug-loading capacity, which is 
C. Yang, W. Zhang, M. Bai et al. Nano TransMed 2 (2023) 100004
8
unprecedented. Experimental evidence shows high accumulation of EV- 
DNs in glioblastoma tissue, maximizing their absorption in brain tu-
mors and exhibiting strong anti-glioblastoma efficacy in vivo.
5.4. Application of plant-derived EVs in the regulation of gut microbiota
With the advancement of scientific technology, increasing evidence 
suggests the immense potential of medicinal plants themselves or their 
major phytochemicals in the treatment of gastrointestinal diseases
[132].
PDEVs are a type of nano-sizedmembrane vesicle. With the progress 
of research, researchers have discovered and reported increasing 
numbers of EV-like nanoparticles in plants, fruits, and mushrooms. 
They possess excellent biochemical characteristics and inherent biolo-
gical functions, including: (1) good biocompatibility, (2) non-toxicity 
and low immunogenicity, (3) specific targeting abilities, (4) prolonged 
circulation and drug half-life, (5) high production capacity, (6) ability 
to cross the blood-brain barrier (BBB) besides the placenta. Compared 
to conventional methods, the delivery of drugs using PDEVs has the 
advantages of improving drug absorption, delivering drugs to the site of 
intestinal inflammation, and having high biocompatibility and no toxic 
side effects.[133].
Teng et al. investigated the effect of RNA from PDEVs on gut mi-
crobiome (Fig. 2D). [51] They identified small RNA and miRNA from 
ginger EVs that can regulate gut microbial composition and its meta-
bolites, and eventually inhibit mouse colitis. Mu et al. isolated and 
identified EVs from four edible plants, including grapes, grapefruits, 
ginger, and carrots, and confirmed that the size and structure of these 
EVs are similar to those of mammalian EVs. These PDEVs are absorbed 
by gut macrophages and stem cells and have biological effects on re-
cipient cells. For instance, ginger EVs induce heme oxygenase-1 (HO-1) 
and IL-10 expression in macrophages, while fruit EVs, including grapes 
and grapefruits, induce Wnt/TCF4 activation, which is crucial for 
maintaining intestinal homeostasis. Research has shown that plants 
communicate with mammalian cells (especially intestinal macrophages 
and stem cells) through EV nanoparticles, and the EVs derived from 
different types of plants have different biological effects on recipient 
mammalian cells [85].
Besides, PDEVs can be absorbed by gut microbiota and contain RNA 
that can alter microbial composition and host physiology. ELNs from 
ginger (GELN) are preferentially taken up by Lactobacillaceae in a li-
posome-dependent manner and contain microRNA that target various 
genes in Lactobacillus rhamnosus GG (LGG). One of these microRNA, 
GELN mdo-miR7267–3p, mediates the upregulation of LGG mono-
oxygenase ycnE, which leads to increased production of indole-3-al-
dehyde (I3A). ELN-RNA or I3A (a ligand of the aryl hydrocarbon re-
ceptor) are sufficient to induce IL-22 production, which is associated 
with the improvement of barrier function. These ELN-RNA's functions 
can be improved through an IL-22-dependent mechanism to alleviate 
mouse colitis. These findings reveal how plant products and their ef-
fects on the microbiota can be used to target specific host processes to 
alleviate diseases.
Therefore, the intake of probiotics, beneficial molecules, or micro-
organisms aimed at increasing the number of beneficial microbiota or 
their products in the gut can be beneficial to the host's health. From a 
therapeutic point of view, using liposomes derived from ginger ELNs, 
we successfully delivered miRNA orally to target gut bacteria to treat 
mouse colitis. This strategy provides an alternative approach for gene 
therapy of gut microbial dysbiosis-related diseases and provides a 
fundamental principle for oral delivery of therapeutic miRNA based on 
ELNs to treat diseases caused by dysbiosis. It can be imagined that the 
regulation of gut bacterial activity by miRNA that interacts with bac-
terial mRNA in a gene-specific manner will have many advantages over 
other methods, such as chemotherapy drugs that induce gut ecological 
imbalance and antibiotic treatment that promotes the rapid develop-
ment of antibiotic resistance. However, the concept of food-derived 
ELNs selectively taken up by gut microbiota and host cells is relatively 
new in gut physiology and health, and more exploration and research is 
needed [134].
6. Prospectives and challenges
The difficulty of large-scale preparation has been an important ob-
stacle to clinical transformation of animal vesicles. Although there are 
currently new cell culture models such as cell factories to obtain media 
containing EVs, thereby increasing the production of EVs. However, its 
operating conditions are strict and the training cost is high, which is not 
conducive to large-scale promotion. Unlike the preparation methods of 
animal derived-EVs, PDEVs can be separated directly from the sap 
without cell culture. Plants grow fast, can be massively expanded by 
asexual reproduction, and have easy access to their sap, which greatly 
simplifies the preparation of EVs. Also, humans can establish Good 
Agricultural Practices (GAP) to grow the plant, the quality control is 
easier compare to animal basis. The widespread use of PDEVs is ex-
pected to shift the preparation site of EVs from the laboratory to the 
farm or orchard, which is a major advantage of PDEVs compared with 
animal-derived EVs.
For PDEVs, oral administration, in addition to intravenous admin-
istration, still has great effects and research potential. Considering the 
origin of plant cells, making PDEVs edible could play a role in inter-
specific communication and serve as natural therapies for a variety of 
diseases. Grapeline-derived EVs (GDEVs), most of the oral GDEVs can 
be absorbed by intestinal macrophages. Studies have found that oral 
GDNS can carry methotrexate (MTX) and selectively target intestinal 
macrophages, which can effectively treat DSS induced colitis in mice. 
At the same time, oral GDEVs does not cause liver toxicity. Overall, oral 
delivery transports small molecules to intestinal macrophages in a non- 
cytotoxic manner [80]. Similar to grapefruit, grape-derived exosomes 
(GDEXO) can also be administered orally to treat intestinal in-
flammatory diseases. The latest study found that GDEXO, when ad-
ministered orally, protected mice from DSS-induced colitis and was 
administered in a manner that led to activation of beta-catenin and 
upregulation of intestinal stem cells [50]. In addition, carrot-derived 
nanoparticles also have the effect of anti-inflammatory [135]. Oral 
administration can induce the expression of IL-10, increase the nuclear 
translocation of Nrf, and target absorption by intestinal macrophages 
and stem cells, so as to treat intestinal diseases. Overall, there have 
been great breakthroughs in oral administration of EVs derived from 
plant cells, most of which focus on intestinal inflammatory diseases in 
the treatment of diseases. We believe that oral administration can have 
greater breakthroughs in the near future and can be effective in other 
systemic diseases.
In recent years, PDEVs has attracted the interest of many experts in 
microbiology, immunology and other fields, and research in this field 
has grown exponentially. These nano-sized particles have provided 
researchers with many interesting discoveries, making them promising 
for applications in human health and disease [136]. Despite the positive 
prospects of PDEVs in drug delivery, there are still many limiting fac-
tors that hinder its application. At first, it is urgent for us to standardize 
the research on PDEVs to clarify the regulatory mechanism of PDEVs 
function. Secondly, the immunogenicity and other safety risks of PDEVs 
as foreign substances in humans need to be carefully evaluated. Finally, 
the isolation of a single subtype of PDEVs is still a difficult problem, 
making the standardization of vesicles production an insurmountable 
obstacle, which will greatly hinder clinical applications. To solve these 
problems, more advanced technologies are needed to explore the 
characteristics of EVs.
7. Conclusion and outlook
PDEVs have shown satisfactory bioactivity against inflammation, 
cancer, microbial infections, and oxidative damage, particularly in the 
C. Yang, W. Zhang, M. Bai et al. Nano TransMed 2(2023) 100004
9
prevention or treatment of gastrointestinal diseases, cancer, cardio-
vascular and cerebrovascular disorders, among others. Additionally, 
PDEVs have the potential to target deliver small molecule drugs and 
nucleic acids through oral, transdermal, or injectable routes as natural 
drug carriers. Given the advantages mentioned above, PDEVs are ex-
pected to have excellent competitiveness in clinical applications or 
preventive healthcare products. This article provided a systematic re-
view of the research progress on PDEVs in biomedicine. By analyzing 
several representative cases, the logical design of engineered PDEVs in 
disease diagnosis and treatment was discussed. Engineered PDEVs have 
overcome the limitations of natural vesicles and animal vesicles, of-
fering greater hope for clinical translation. However, the research on 
using PDEVs for disease treatment is still in its early stages, and there 
are a series of issues that need to be addressed. Based on the research 
progress and the challenges in PDEVs research, we described what we 
believe could be the focus of future developments. 
1. Research on the interaction mechanisms between PDEVs and the 
human body, including immunogenicity, toxicology, and the study 
of PDEVs crossing biological barriers such as the intestinal mucosa 
and the blood-brain barrier. This research also includes in-
vestigating how PDEVs regulate human physiological processes. 
These studies will enrich our knowledge of the biomedical appli-
cations of PDEVs.
2. The development of plant resources, especially the development of 
traditional herbal medicines. Plants have been used as medicinal 
materials for thousands of years, but their mechanisms of action are 
still not well understood. PDEVs, as proven biopharmaceutical 
agents, could be a potential way for herbal medicines to exert their 
functions. This will drive the development of more herbal for-
mulations and functions.
3. With the introduction of synthetic biology and genetic engineering, 
there is potential to achieve engineered edible plants. Gene en-
gineering methods can design plants, mass-produce them, and pre-
pare active ingredients such as PDEVs. This may enable therapeutic 
agents to be converted into food supplements, achieving the concept 
of "food as medicine."
Ethics approval and consent to participate
Not applicable.
Authors' contributions
ZF, CJ and WC are responsible for conceptualization and manuscript 
revision. CY, MB, QL, YX, XL and QZ are contributors in writing the 
manuscript. MB and ZF are responsible for the visualization. MB, QL, 
YX, XL and QZ are responsible for literature collection and collation. All 
authors read and approved the final manuscript.
Consent for publication
Not applicable.
Funding
This work was supported by the Natural Science Foundation of Jilin 
Province (20230101368JC), Chinese University of Hong Kong 
(Shenzhen) startup funding (K10120220253) and GuangDong Basic and 
Applied Basic Research Foundation (2022A1515110206), National 
Natural Science Foundation of China (82002253).
Data Availability
Not applicable.
Declaration of Competing Interest
The authors declare that they have no competing interests.
Acknowledgements
Acknowledgments are extended to Dr. Robert Hein for his invalu-
able contribution in refining our manuscript.
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