Review
Plant Exosome-like Nanovesicles: Emerging
Therapeutics and Drug Delivery Nanoplatforms
Haseeb Anwar Dad,1 Ting-Wei Gu,1 Ao-Qing Zhu,1 Lu-Qi Huang,2 and Li-Hua Peng1
1College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, P.R. China; 2National Resource Centre for Chinese Materia Medica, China Academy of
Chinese Medical Sciences, Beijing 100700, P.R. China
Plant exosome-like nanovesicles, being innately replete with
bioactive lipids, proteins, RNA, and other pharmacologically
active molecules, offer unique morphological and composi-
tional characteristics as natural nanocarriers. Furthermore,
their compelling physicochemical traits underpin their modu-
lative role in physiological processes, all of which have fostered
the concept that these nanovesicles may be highly proficient in
the development of next-generation biotherapeutic and drug
delivery nanoplatforms to meet the ever-stringent demands
of current clinical challenges. This review systemically deals
with various facets of plant exosome-like nanovesicles ranging
from their origin and isolation to identification of morpholog-
ical composition, biological functions, and cargo-loading
mechanisms. Efforts are made to encompass their bio-
therapeutic roles by elucidating their immunological modu-
lating, anti-tumor, regenerative, and anti-inflammatory roles.
We also shed light on re-engineering these nanovesicles into
robust, innocuous, and non-immunogenic nanovectors for
drug delivery through multiple stringent biological hindrances
to various targeted organs such as intestine and brain. Finally,
recent advances centered around plant exosome-like nanove-
sicles along with new insights into transdermal, transmem-
brane and targeting mechanisms of these vesicles are also
elucidated. We expect that the continuing development of plant
exosome-like nanovesicle-based therapeutic and delivery nano-
platforms will promote their clinical applications.
Exosome-like nanovesicles (ELNVs), which are biological nanostruc-
tures of 40–150 nm, are secreted by most types of cells and relay in-
formation between cells and organisms across all three kingdoms of
life.1,2 Although earlier perceived to be cellular debris and hence
undervalued, ELNVs are now acknowledged as crucial entities to
regulate physiological functions of multicellular organisms in an
intercellular transmission manner.3 Despite a growing appreciation
of the importance of ELNVs in mammals, and the fact that plant
ELNVs (PELNVs) were observed first around 15 years earlier than
mammalian ELNVs,4,5 nothing much was known about PELNVs
beyond the old microscopy images suggesting their existence.6,7
When aiming to bloom new therapeutic approaches, scientists must
surmount numerous challenges, including issues with delivery, tissue
specificity, potential off-target effects, safety, toxicity, and large-scale
Molecular Therapy Vol. 29 No 1 January 2021 ª 2020 The American Society of Gene and Cell Therapy.
production costs. Addressing these challenges, PELNVs represent
themselves as avant-garde innocuous and eco-friendly robust and
feasible carriers for nanomedicine. In their comparison, synthesized
nanoparticles are linked with complications such as immunogenicity,
cytotoxicity, and complex fabrication process requirements.8
Contrarily, PELNVs, owing to their natural source and composition,
enjoy the perks of being undetected by the immune system, and hence
enhanced circulation periods and higher bioavailability. Moreover,
unlike mammals, plants do not harbor zoonotic or human pathogens.
Thus, PELNVs also take the edge over mammalian cell-derived
ELNVs with their non-immunogenic and innocuous traits. This pre-
cedence is also owing to their capability to showcase efficient uptake
at the target site, high output of delivery of therapeutic agents, and
cost-efficient production.9 PELNVs lodged in the paramural space
of plants are identical in morphology and functionality to their
mammalian analog and are recognized for their importance.10 They
not only contribute in plant cell crosstalk but also to inter-kingdom
communication.11 Although there are only limited numbers of
PELNV-related reviews, possibly because of only a handful of original
research articles in this field, some of which can be found else-
where,12–14 unfortunately, none of them has covered all aspects of
PELNVs. Herein, we have attempted to comprehensively review
and consolidate the position of PELNVs as entities poised to become
next-generation therapeutic and drug delivery nanoplatforms for
desired clinical outcomes. The present review is also meant to give
a quick glance to readers of all investigations directed toward PELNVs
and PELNV-derived nanovectors and their isolation techniques along
with identification of morphological composition. More importantly,
we elucidate the translation of their innate biological functions into
diversified therapeutic actions, and we also emphasize the immense
potential of manipulating them as a safe and sustainable drug delivery
nanoplatform, while highlighting the limitations and research gaps
for enhancing delivery objectives.
Biogenesis and Physiological Actions of PELNVs
Evidence of the existence of PELNVs has been being built since 1967.7
In earlier findings, the existence of multivesicular bodies (MVBs) in
https://doi.org/10.1016/j.ymthe.2020.11.030.
Correspondence: Li-Hua Peng, PhD, College of Pharmaceutical Sciences, Zhejiang
University, 866# Yuhangtang Road, Hangzhou 310058, P.R. China.
E-mail: lhpeng@zju.edu.cn
13
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plant cells and their fusion with plasmalemma led to jettison ELNVs
into the extracellular space in higher plants.6,7 In the ensuing years,
similar morphology vesicular structure was found to be involved in
the processes of cell differentiation causing secondary wall thick-
ness.15–17 The evolution of understanding the biogenesis and secre-
tion of PELNVs can be found in the literature.18–22 In short, PELNV
biogenesis is initiated with formation of the trans-Golgi network or
early endosome, which matures multivesicular endosomes (MVEs)
or MVBs (Figure 1). The characteristic intraluminal vesicles (ILVs),
which actively and selectively incorporate RNAs, including mRNAs,
microRNAs (miRNAs), and other non-coding RNAs along with
lipids and DNA, dwell inside MVBs, which are ultimately integrated
with plasmalemma to release PELNVs.23 PELNVs, being an integral
constituent of a plant’s physiological machinery, execute several
crucial roles within their own kingdom. For example, PELNVs are
14 Molecular Therapy Vol. 29 No 1 January 2021
Figure 1. Biogenesis of PELNVs and Their Functions
in Plant Physiology
PELNVs are formed by inward budding of MVBs with the
content in similar orientation as in the plasmalemma, and
fusion of MVBs with plasmalemma permits the release of
PELNVs.
extensively fraught with cell wall remodeling
enzymes and defense proteins (Figure 1).24
Mounting evidence highlights their role as being
the front line of plant defenses in plant infections
and in the developmental processes.25–27 In
recent years, in-depth understanding of PELNVs
gave scientists an impetus to direct their investi-
gations toward exploring the interspecies or
inter-kingdom communication role of PELNVs
and to look for developmental space in therapeu-
tics and drug delivery.
Preparation, Re-engineering, and Cargo
Loading of PELNVs
The farthermost objective of the isolation tech-
nique is to formulate PELNV-based nanostruc-
tures to accomplish the ultimate clinical outcome
for curing ailments.28 There are two types of
nanoplatforms that can be used for therapeutics
and drug delivery purposes, with one being
PELNVs obtained in their pristine form and the
other being PELNV-derived nanovectors. Here-
in, we elaborate the preparation methods of
both kinds along with cargo loading techniques
currently being investigated to equip them for
drug delivery.
Preparation of PELNVs
The overlapped size range and rather indistin-
guishable morphological similarities necessitate
the current efforts in designing a feasible isolation method of
ELNVs from the masses of the subpopulation of extracellular vesi-
cles (EVs). In this consideration, the differential ultracentrifugation
(UC) technique is commonly used and has acquired the benchmark
status in isolation and purification of ELNVs owing to its ease of use
and inexpensiveness.29–32 The technique mechanism operates on the
size and density variations of particles.28 PELNV source material or
plant juice is put through a series of centrifugations with gradually
enhancing speed. Each subsequent speedy centrifugation is sub-
jected to longer duration than the previous one to sediment the
higher density particles, and then pellets obtained in each centrifu-
gation are disposed. In the next step, previously acquired superna-
tant is subjected to a further higher speed of 100,000  g to retrieve
the pellet accommodating the PELNVs, which is subsequently re-
suspended and washed in a small amount of phosphate-buffered
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Figure 2. Isolation and Preparation of PELNVs
PELNVs could be isolated and prepared by series of centrifugations, including ultracentrifugation and sucrose gradient ultracentrifugation.
saline (PBS) (Figure 2).33 The PELNV yield obtained after the basic
procedure of differential UC is usually contaminated with nucleic
acids and protein agglomerates.34 Therefore, for further purification,
homogenized suspension is subjected to sucrose gradient ultracen-
trifugation (SGC) (8%, 15%, 30%, 45%, and 60%) at an elevated
speed greater than 150,000  g for 120 min. This step holds para-
mount importance in obtaining uncontaminated PELNVs. PELNVs
float in the sucrose bands depending on the elevated g-force
sedimentation and various speeds of sucrose gradients.35 Mostly,
sucrose gradients of the 30%/45% layer are harvested as the pre-
eminent source of PELNVs. To obtain the ultra-pure PELNV yield,
disposal of the pellets after 100,000  g UC and use of an additional
UC at 135,000  g manage to get rid of other extraneous EVs.
Although this benefits in achieving purity of PELNVs, it drops
the PELNV yield concentration.36,37
Repeated pelleting of PELNVs, under high centrifugal force of differ-
ential UCs, may result in compromising the PELNV structural integ-
rity and cause agglomeration.38 This complication may be averted by
incorporating a thin layer of high-density iso-osmotic material as a
cushion at the bottom of the centrifuge tube, and as a result PELNVs
will never spin out as pellets at the bottom and will give enhanced
yield and quality.28,39 In addition to differential centrifugation,
commercially available ELNV isolation kits are also utilized for
ELNV isolation by researchers. However, this technique has been
reported to co-precipitate other EVs alongside ELNVs as well as
RNA-protein complexes.40
Re-engineering of PELNVs
It is a grueling task to prepare uniform-sized PELNVs because they
vary in size from approximately 50 to 500 nm between and even
within species, which is major hindrance in their exploitation for
drug delivery. Moreover, efficient drug loading is one major concern
that is not readily achievable in purified PELNVs in pristine form.
Hence, it is indispensable to conceive a method to formulate uni-
form-sized nanoparticles repeatedly with competent drug loading.41
For the fortification and re-engineering of PELNVs as drug delivery
nanoplatforms, researchers have successfully adopted the Bligh and
Dyer technique (a well-reported liquid-liquid extraction method) to
extract nanolipids from PELNVs, and extracted nanolipids are
processed through a 200-nm liposome extruder, which ultimately
re-engineers them into a uniform-size nanoplatform termed
“PELNV-derived nanovectors.”42
Despite that PELNVs intrinsically express some lipids and cell adhe-
sion molecules such as phosphatidic acid (PA) that naturally promote
binding to particular recipient cells,43 most targeting achievements of
PELNVs are through its natural biodistribution. PELNV-derived
nanovectors also offer the possibility of tailoring their surface, which
can broaden the scope of desired target ability.44 In this regard, spe-
cific bioactive molecules can be immobilized onto the surface of
PELNV-based nanoplatforms to develop displays more compatible
to cellular surface receptors. For example, functionalizing the surface
of PELNV-based nanoplatforms more specifically to tumors can
assist in intensifying anti-tumor characteristics associated with the
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aforementioned nanoplatforms.45 By virtue of convenient surface
tailoring, ginger-derived ELNVs were functionalized with arrowtail
pRNA-3WJ and folic acid (FA) for ligand display and further used
to systematically deliver genes to the tumor site.46 The most common
strategy for the surface functionalization of PELNV-based nanoplat-
forms is the incubation method, which involves the mechanisms of
hydrophobic interaction, the diffusion process, and electrostatic
interaction.47 In addition to this, some ligands that are immobilized
on the surface of PELNVs can act as tags for bio-imaging applica-
tions.48 It was reported that grapefruit-derived nanovectors were
labeled with lipophilic carbocyanine (DiR) dye that enabled their
in vivo tracking by fluorescence.9 Furthermore, click chemistry opens
the door for bioconjugation of targeting macromolecules or small
molecules such as imaging agents to the surface of PELNVs via cova-
lent bonds.49
Careful and selective treatment of the surface of PELNVs can also
enhance the nucleic acid delivery capability of PELNVs. For instance,
the hybridization of polyethylenimine with grapefruit-derived nano-
vectors not only enhanced gene delivery to brain tumor but also
diminished the cytotoxicity characteristic of polyethylenimine
considerably.9 Similarly, introduction of cell-penetrating peptide to
the lipid bilayer structure of PELNVs has reportedly resulted in
boosting the cellular uptake and tissue penetration.50 Moreover, to
maximize the therapeutic functionalization of PELNV-based nano-
platforms, drug molecules can also be conjugated with their surface.46
Cargo Loading Techniques
To achieve superior effects against disease, besides the endogenous
components, PELNVs could be laden with exogenous therapeutic
molecular drugs such as protein, expression vectors, siRNA, and
DNA.51,52 Different strategies have been devised to lade ELNVs with
therapeutic drug molecules (Figure 3). These methods first entail prep-
aration of PELNVs from plant and later loaded with drug molecules
via direct manipulation of PELNVs. Mechanistically, for cargo loading
of ELNVs, passive and active cargo loading techniques are generally
used.53 The passive drug loading technique comprises an incubation
method, in which PELNVs are incubated with drug molecules at a
certain temperature. This drug encapsulation strategy is driven by
diffusion action and lipophilic interaction between the drug molecule
and lipid bilayer of PELNVs.54,55 In addition to passive loading, the
sonication technique, an active cargo loading method, is used, which
distorts the ELNV membrane structure briefly for successful diffusion
of cargo into ELNVs. The membrane structure of ENLVs reverts to its
intact integrity after the cargo loading. It also has been published that
this method takes precedence by boosting the cargo loading capability
up to 11-fold over passive cargo loading.56 In most conditions,
PELNVs retain a negatively charged surface that can attract oppositely
charged drugs such as doxorubicin and ultimately support the loading
of drug into their inner cavity by the sonication technique.57
Conversely, it also has been reported that negatively charged com-
pounds such as FA and neutrally surfaced drugs such as curcumin
can also be ensnared in PELNVs despite modifying their biological
activities. Reported data suggested that lipophilic properties of these
16 Molecular Therapy Vol. 29 No 1 January 2021
compounds can lead toward their encapsulation by surmounting the
surface-associated electrostatic forces of nanovesicles. Some findings
illustrated that siRNAs and DNAs, being strongly negatively charged
molecules, could be laden into PELNVs and exhibited their desired
activity. However, the cargo loading efficiencies were found to be sub-
par to those of positively charged molecules.58
Despite these substantial advantages, the peril of impairment of the
innate structure of PELNVs during brief distortion for cargo loading
demands solution. As PELNVs are innately laden with some biomol-
ecules, there might be the requirement of unloading some of the com-
ponents of PELNVs. However, there has not been any attempt related
to unloading ELNVs. This proposition may help in augmenting the
ELNV-oriented delivery, lest ELNV integrity should remain intact
during unloading procedure. Moreover, massive challenge of success-
ful RNA loading onto PELNVs still persists, particularly in elevated
levels for non-viral gene delivery. In this context, several studies
have demonstrated that physical electroporation can be exploited
for cargo loading, in particular RNA loading onto ELNVs, and its
loading efficiency can be elevated by optimization of ELNVs and
the siRNA ratio and voltage supplied during electroporation.59,60
However, electroporation fails to incorporate larger molecules such
as mRNA and DNA with the same efficiency as for small RNAs.61
Besides this, other RNA loading techniques harnessing the biological
process during ELNV formation can also be exploited such as passive
loading in which augmented intracellular RNA load can be acquired
by transfecting the source cells with desired cargo via viral or non-
viral nanovectors.62 These strategies have the potential to obviate
the current procedural limitations associated with RNA loading
onto PELNVs and will likely expand the horizons of gene delivery.
Characterization
Morphological Characterization
The flourishing advancements in ELNV-based nanotechnology de-
mand precision and variety of characterization techniques to acquire
a more elaborative view of the morphological and physiochemical
traits of prepared PELNV-based nanoplatforms. It is important to
devise and implement multiple characterization techniques to iden-
tify the diverse subpopulations of EVs and is vital to the design of
PELNV-based therapeutics or drug nanocarriers. Transmission
electron microscopy (TEM) is used for ultrastructure analysis of the
subcellular status of PELNVs.63,64 As an illustration, multifarious
ELNVs derived from grapefruit, ginger, carrots, and tomatoes have
been reported.65 In addition, atomic force microscopy (AFM) can
also be used to develop the structural and size-related understanding
of individual PELNVs. AFM asserts its superiority to typical optical
microscopy by ascertaining resolution of fractions of nanome-
ters.33,66,67 Size determination and zeta potential evaluation of
dispersed PELNVs can be done by dynamic light scattering (DLS),
also termed quasi-elastic light scattering, photon correlation spectros-
copy.65 Being non-intrusive and ultra-sensitive, DLS needs only little
sample volume to compute accurate and precise size, which makes
it the benchmark technique in evaluation of size distribution of sus-
pension particulates in multiple scientific disciplines.68–70 In addition
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Figure 3. Re-engineering Method for PELNVs and Drug Loading Procedure
The Bligh and Dyer extraction method is used for nanolipid extraction from PELNVs to re-engineer them into nanovectors. The drug loading procedure comprises the
sonication technique for drug loading onto re-engineered nanovectors and an incubation method for drug loading onto pristine PELNVs.
to this, nanoparticle tracking analysis (NTA) is also gaining popu-
larity among researchers to quantify the number of ELNVs in a sam-
ple volume and determine their size distribution.
Biochemical Characterization
The chemical composition profiles of PELNVs maintain significant
discrepancies from those of mammalian-derived ELNVs in terms
of their protein, lipid, and RNA content.65 Compositional analyses
of lipids, nucleic acids, and proteins of PELNVs are appreciated
as important characterization criteria for the quality control of
PELNVs.71,72 Plant and mammalian cell-derived ELNVs share
mutual common techniques used for chemical component charac-
terization. For confirmation of the origin of ELNVs, immunoblot-
ting of specific proteins is the most widely practised approach.
With the advancements in technology, ELISA, sodium dodecyl
sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE),73 liquid
chromatography-tandem massspectroscopy,74,75 a microfluidic elec-
trophoresis analyzer, and high-throughput small RNA sequencing
have been established for deep analysis of the components of
PELNVs.76,77 In particular, colorimetric assays such as bicincho-
ninic acid (BCA), fluorimetric assays, SDS-PAGE, and western blot-
ting are popular for protein analysis.78 Another highly sophisticated
molecular characterization technique is Raman spectroscopy, which
displays the chemical structure of PELNVs by developing a certain
spectrum of biomolecules such as peptides and nucleic acids
residing in them.79 Moreover, microarray analysis, digital droplet
PCR, and next-generation sequencing techniques have been estab-
lished for the RNA content analysis of PELNVs.80 For the lipidomic
analysis of PELNVs, the techniques most often used are a sulfo-
phospho-vanillin assay and total reflection Fourier transform
infrared spectroscopy.81,82 Additionally, fluorescence measurement
of lipophilic fluorescent dyes such as DiR can also correspond to
lipid quantification, as those dyes will only give fluorescence when
integrated into the lipid bilayer of PELNVs.83
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PELNVs generally have three major components: lipids, proteins, and
nucleic acids.84
Identification of Lipids
Lipids are essential components of lipid bilayer structures of
PELNVs.85 Lipid profiling of PELNVs has revealed two distinct group
of lipids, phospholipids and glycerol lipids, which have been found to
be vital for PELNV stability, uptake, and other biological functions.86
Many PELNVs harbor PA, which modulates membrane fission and
fusion, being the dominant lipid mediator. It can also influence
PELNV uptake, as it triggers cytoskeleton rearrangement and protein
regulation in vesicular endocytosis.43 Besides PA, PELNVs also amass
diverse lipid content such as phosphatidylethanolamine (PE), phos-
phatidylcholine (PC), phosphatidylinositol (PI), and diacylglycerol
(DG). It is noteworthy that, among the aforementioned lipids, PC
has been suggested to have the capability to vector PELNVs from
the intestine to the liver in an augmented manner. It is also note-
worthy that all PELNVs investigated so far were found to be devoid
of any cholesterol content.51,87,88 Since several studies suggest that
PELNV lipids play a biologically active role in recipient cells, a thor-
ough understanding of each lipid component that how it modulates
its actions will be beneficial in the development of effective
PELNV-based therapeutic strategies.
Identification of Proteins
According to the International Society for Extracellular Vesicles, sci-
entists are encouraged to analyze vesicles with respect to proteins,
which are found to be abundant or deficient in ELNVs, as proteins
are integral to their functionality.89 PELNV proteins can be mainly
categorized into transmembrane proteins and other plasmalemma-
associated proteins. Currently among transmembrane proteins,
CD63, CD81, and CD9 have been identified as possible markers of
mammalian cell-derived ELNVs.90–92 However, markers of PELNVs
have not been clearly reported yet and demand further investigations.
It was reported that protein contents of ginger-derived ELNVs
happen to be predominantly cytosolic, and fewer membrane trans-
porter proteins were identified, such as aquaporins and chloride
channels.87 In addition, some reports have also suggested that
grapes,86 lemons,30 and Arabidopsis93 have been found to harbor
heat shock proteins and aquaporin proteins. Other plasmalemma-
associated proteins mainly include metabolic enzymes, signal trans-
duction factors, adhesion factors, cytoskeleton proteins, and ubiqui-
tin.94,95 There is a need of considerable research for identification
of a broad range of PELNV proteins, which possibly will reveal their
roles in biological and pharmacological activities.
Identification of RNAs
The most fascinating trait of ELNVs is their ability to ferry various
RNA species among cells. RNAs in PELNVs mainly include
mRNA, miRNA, and small RNA (sRNA), among which mRNA
and non-coding miRNA can regulate the levels of RNA and proteins
in recipient cells, thus affecting cell morphology and function.96–98
Hitherto, most studies on ELNV-mediated communication have
been concentrated on notable RNA species such as miRNAs and
18 Molecular Therapy Vol. 29 No 1 January 2021
mRNAs. However, naked miRNAs are unstable and prone to subjec-
tion to immediate decomposition by ubiquitous RNases, and their ul-
timate packaging inside ELNVs circumvents their early obliteration
by RNases.63,99,100 Furthermore, a dietary plant’s characteristics along
with nutrigenomic influences may redesign the miRNA constitution
and other components of PELNVs.101 Arabidopsis thaliana-derived
ELNVs are found to be laden with miRNAs, sRNAs (carrying 18–
24 nt), and tiny RNAs (tyRNAs) (RNA with 10–17 nt). Co-existence
of the aforementioned RNAs and cargo in the apoplast indicates
specific sorting mechanisms for RNA loading and then use for
delivery to distant sites.102 In addition, PELNVs often contain un-
usual noncoding RNAs, although it is not completely understood
how ELNVs process them and what is their influence on uptake of
recipient cells and consequently in cell communication, which will
require further investigations to be elucidated.
PELNVs as Nano-Therapeutics for Diseases
Biological functions of the PELNVs, in their natural pristine struc-
tural integrity with intact bioactive cargoes after mere isolation
from plants, eventuate in mitigating pathological conditions in other
kingdom’s species and provide multiple therapeutic alternatives (Fig-
ure 4). The attributes that make PELNVs fascinating for therapy are
their plant provenance and enhanced safety profile along with their
versatile therapeutic potential rooted in their active source plants.65
Immunological Modulation Effects
Currently, PELNVs have revealed their salubrious immunological
regulating actions on the gastrointestinal (GI) haemostasis.52 It is
widely recognized that inflammation is a part of the innate immunity
mechanism. If left uncontrolled, it can evolve into acute or chronic in-
flammatory diseases and sometimes also turn into the deep-rooted
cause of most chronic ailments.103 PELNVs have been found to exert
their anti-inflammatory effect by regulating the immunological
actions of hosts. Moreover, miRNAs of PELNVs regulate the inter-
kingdom communication between gut microbiota and the host
immune system, which is translated into the homeostatic balance
between immunity and gut microbiota (Figure 5). In fact, after inter-
nalization into the host’s recipient cells, PELNVs trigger a plethora of
intracellular signaling cascades, which modulate cellular responses
and bring about tissue homeostasis.52 In the recurrent inflammatory
disorders of intestine such as colitis, intestinal macrophages get
deprived of their tolerogenic traits. Wang et al.104 demonstrated
that grapefruit-derived ELNVs have a propitious impact on intestinal
immune homeostasis by fortifying the anti-inflammatory capability
of intestinal macrophages, which ultimately alleviate dextran sulfate
sodium (DSS)-induced colitis in mice with no toxicity. After the
successful uptake of grapefruit-derived ELNVs by intestinal macro-
phages, these nanoplatforms upregulate the expression of heme-oxy-
genase-1 (HO-1) and interleukin (IL)-10 and suppress the production
of IL-6, IL-1b, and tumor necrosis factor (TNF)-a. Moreover,
miRNAs of ginger- and grapefruit-derived ELNVs were also reported
to target the genes panel in gut probiotic Lactobacillus rhamnosus
(LGG) alongside growing its number in mice gut and consequently
prompting anti-microbial immunity and wellbeing of the gut. Similar
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Figure 4. Overview of Biological Functions of PELNVs from a Variety of Plant Sources and Their Translation into Therapeutic Applications
(A) Summary of the biological functions of PELNVs derived from plants. (B) Elucidation of therapeutic mechanisms of PELNVs in immunological modulation, tissue repair, and
intestinal transporters modulation.

investigations on broccoli, grapes, and carrots have also been re- there has been no report up to now of any immunogenic response
ported, which gave credence to the impressive role of PELNVs induced by PELNVs that makes them safe and biocompatible. Besides
in immunological modulation therapeutics.52,65 Interestingly, that, transgenic plant-derived virus-like particles have been

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Figure 5. Mechanism of PELNV miRNAs Regulating the Inter-kingdom Communication between Gut Microbiota and the Host Immune System
PELNV mRNA-mediated targeting of the LGG monooxygenase ycnE increase the yields of indole-3-carboxaldehyde (I3A), which leads to inducing the production of IL-22.
IL-22 can improve the barrier function and ameliorate colitis in mice.
demonstrated as an effective nanoplatform to produce vaccines owing
to their ability to trigger a desired immunogenic response.105,106
Anti-tumor Activities
Cancerous tumors pose a grave danger to human health, and medic-
inal plants play important roles in the treatment of tumors to make up
for the shortcomings of modern medicine.107 The rise of miRNA
research has led people to gradually realize that miRNA is closely
related to tumor proliferation and apoptosis.108–110 With the further
study of ELNVs, more and more research has shown that plant-
derived miRNAs can span species and play a certain regulatory role
in the human body.111 For instance, ginger-derived ELNVs show
promising results in curtailing colorectal tumorigenesis by diminish-
ing pro-inflammatory cytokines and suppressing intestinal epithelial
cell proliferation and apoptosis, as overexpression of cyclin D1
mRNA suggests early cancer onset and tumor progression. It was
also found that ginger-derived ELNVs reduced cyclin D1 mRNA
levels in mouse colorectal cancer models, corroborating the anti-
tumor action of ginger-derived ELNVs.87 In addition, Citrus limon
L.-derived ELNVs were also explored for their role in the curtailment
of tumor growth. They were able to curtail the in vivo tumor develop-
ment in chronic myeloid leukemia (CML) by tumor targeting, oxida-
20 Molecular Therapy Vol. 29 No 1 January 2021
tive stress reduction, and most importantly by activating TNF-related
apoptosis-inducing ligand (TRAIL)-mediated apoptotic cell death.112
Regenerative Effects
Humans ingest PELNVs during the whole span of their life, but very
limited information has seen daylight regarding their functionality
and impact on the intestinal tract. Ju et al.86 explored the role of
grape-derived ELNVs on DSS-induced colitis in mouse models. It
was shown that they have the capability to diffuse through and enter
intestinal stem cells and thereupon induce the proliferation of Lgr5hi
intestinal stem cells and regenerate the intestinal epithelial tissue.
Moreover, these nanoplatforms can upregulate the expression of
pluripotent stem cell markers such as SOX2, Oct4, and Klf4, which
will prompt the homeostasis of intestinal epithelial tissue. In addition,
these findings also set the precedent for future implications of grape-
derived ELNVs for inducing the proliferation of Lgr5hi stem cells in
other ailments.113 Moreover, ginger-derived ELNVs exhibited the
role in curtailing the expression of the secreted protein hemopexin
and also influenced the expression of other mitochondrial and
cytoplasmic proteins such as heat shock protein, axin, and kinesin
to promote intestinal wound healing.87 In one remarkable investiga-
tion, wheat-derived ELNVs induced skin regeneration by triggering
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proliferative and migratory actions in a dose-dependent manner on
epithelial, endothelial, and dermal fibroblasts in in vitro studies.
Moreover, expression of the mRNA level of collagen type I was
elevated significantly. Aside from these proliferative and migratory
actions, wheat-derived ELNVs were reported to be proangiogenic in
nature by triggering tube-like structure formation in a human umbil-
ical vein endothelial cell (HUVEC) line, which suggested that
wheat-derived ELNVs have the capability to induce vascular forma-
tion during the wound healing process. These findings present exam-
ples for future exploration of wheat-derived ELNVs in in vivo studies
for wound healing development.114
Hepatoprotection
Researchers explored the use of ginger-derived ELNVs for amelioration
of alcohol-induced liver damage in mice. Alcohol-derived metabolites
can stimulate pro-inflammatory cytokines, transforming growth factor,
TNF-a, reactive oxygen species (ROS), and collagen, which lead to liver
deterioration. Ginger-derived ELNVs suppress ROS, which contributes
to hepatoprotection. Owing to shogaols and other bioactive con-
stituents, they can mediate the activation of nuclear factor erythroid
2-related factor 2 (Nrf2). Reportedly, Nrf2 stimulation leads to the
expression of liver detoxifying and antioxidant genes, which collectively
contributes to hepatoprotection. In conclusion, ginger-derived ELNVs
can induce hepatocyte metabolism and detoxifying and antioxidant de-
fenses, which are vital for hepatoprotection.115
PELNVs as Drug Delivery Nanoplatforms
ELNVs are committed to ferry a payload between cells in their near
vicinity to faraway sites, but they can also deliver payloads to other
species in inter-kingdom communication. Their unique design and
transport capability accentuate their potential for use in drug
delivery.116 The advantages of PELNV-based nanoplatforms over
synthesized nanoplatforms and mammalian-derived ELNVs for
drug delivery have been demonstrated. First, the selection of ELNVs
as a drug delivery nanoplatform demands strict scrutiny because their
innate biological actions and derivation source play pivotal roles in
their immunogenic acceptance. For example, ELNVs derived from
mammalian cancerous cells pose the risk of transmitting pro-
cancerous traits to recipient cells.117 PELNVs and artificially synthe-
sized nanoparticles such as liposomes and micelles share common
essential traits, including a comparable lipid bilayer structure and
the ability to deliver both hydrophilic and hydrophobic cargo.118,119
However, when set in comparison, there are stark differences that
show that PELNVs fare better against artificially synthesized nano-
particles in terms of being innocuous and having low immunogenic
effects, enhanced cellular uptake, higher stability in the GI tract
(GIT), and specific target ability.120 Moreover, PELNV-based nano-
platforms offer the benefit of a comparatively less complex prepara-
tion method, whereas artificially synthesized nanoparticles such as
liposomes require complex fabrication processes involving mem-
brane extrusion and microemulsification, among others.121 More-
over, additional rigorous modifications of synthetic nanoparticles
for cargo loading and coating refinements, e.g., polyethylene glycol
(PEG) coatings to develop satisfactory immune tolerance, are also
required.122 On the one hand, such PEG coatings extend the circula-
tion time and help with the immune tolerance to some extent; on the
other hand, these coatings can hamper the interaction between the
nanoparticle and target cells, thereby diminishing the drug bio-
distribution in target tissue.123 Moreover, it also has been reported
that repeated administration of PEG-coated liposome has contributed
to formation of anti-PEG antibodies, resulting in ineffective ther-
apy.124,125 Furthermore, synthetic nanoparticles only serve the
purpose of delivering drug molecules and do not contain any innate
therapeutic capability.
In comparison, PELNVs rose to new heights during the last decade
with the discovery of their innate therapeutic capabilities in cross-
kingdom communication such as anti-tumor, regenerative, and
anti-inflammatory activities. Moreover, PELNVs take the lead by of-
fering the merits of small size for deep tissue penetration, slightly
negative zeta potential for extended circulation,126 resemblance to
the plasma membrane, and pronounced physicochemical stability
under varying pH values and temperatures.79,127 When set in com-
parison with liposomes, their homing capability to tumor tissues is
10-fold higher, denoting a higher specificity for tumor targeting.128
Contrary to mammalian or bacterial cell-derived ELNVs, PELNVs
abate the potential perils of unwanted genetic or protein transfer
and a detrimental immunogenic response before clinical applica-
tion.129 Additionally, PELNV-derived nanovectors have reportedly
been successful in traversing mammalian hindrances such as
diffusing through the blood-brain barrier (BBB), which avoids any
excitation of inflammatory response or necrosis, unlike artificially
fabricated liposomes.51 Moreover, lipid bilayer structure of PELNVs
shrouds their cargo securely and evades enzymatic decomposition
by proteinases and nucleases.130 All of these stellar features coupled
with discovery of their intrinsic therapeutic actions make PELNVs
an ideal for their advent into the arena of drug delivery applications.
Here, we shed light on PELNV-based nanoplatforms for targeted
drug delivery.
Colon-Targeted Delivery
PELNVs proved themselves to be phenomenally biocompatible
through oral administration and successful in achieving targeted co-
lon drug delivery.86 This is because PELNVs are naturally capable of
enhancing the bioavailability of bioactive compounds via keeping
the load stable and secure.131,132 One study showed that ELNVs
with their liquid-ordered phase membrane resist detergent lysis to
a greater degree than do other subtypes of EVs.133 Moreover, it
has also come to light that PELNVs are resistant to digestive en-
zymes such as pepsin, intestinal pancreatin, and bile extract solution.
This stellar feature helps them in circumventing the decomposition
of their cargo by the gastric or intestinal environment and delivering
it to the desired colon site securely. However, it is still not well
understood how PELNVs resist the harsh GI environment and ac-
quire the safe entry into intestinal cells while keeping their payload
intact. In one attempt, grapefruit-derived ELNVs were loaded with
methotrexate to be aimed at intestinal macrophages. Upon oral de-
livery of methotrexate-loaded conjugate, it was revealed that these
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Figure 6. Schematic Illustration of Transdermal Delivery of PELNVs
PELNVs could penetrate the stratum corneum though three pathways: transcellular routes, intercellular routes, and appendageal routes.
conjugates effectively targeted F4/80+ macrophages localized in the
intestinal lamina propria through micropinocytosis and clathrin-
dependent cellular uptake pathways. After internalization these con-
jugates made significant decline in the DSS colitis-associated weight
loss and reversed the shortening of colon length along with its
obvious anti-inflammatory response by curbing the production of
proinflammatory cytokines TNF-a, IL-1b, and IL-6 in mice.
Furthermore, the side effects of methotrexate significantly subsided
when delivered by grapefruit-derived ELNVs as compared to free
methotrexate. Therefore, the compelling innate tendency of PELNVs
to augment anti-inflammatory actions of intestinal macrophages
coupled with cargo delivery ability put forth an interesting solution
for the prevention or treatment of autoimmune diseases and colon
cancer.104 Finally, yet importantly, PELNVs also show the potential
of being exploited as a personalized oral delivery tool because grape-
derived ELNVs were found to target intestinal cells, whereas grape-
fruit-derived ELNVs targeted macrophagess.86 Therefore, such re-
ports foreshadow the possible personalized modulation of the ac-
tions of recipient cells according to therapeutic goals.
Transdermal Delivery
There are generally two pathways for transdermal permeation of
exogenous agents. One is the physical channels of skin appendages
with adequate micrometer diameter to provide passage to nanove-
sicles.134 Mechanistically, it is speculated that once PELNVs are topi-
22 Molecular Therapy Vol. 29 No 1 January 2021
cally applied on skin, they will penetrate via a “lipid-rich channel”
coating on hair follicles.135,136 There are two ways that PELNVs could
reach the hair shafts.137 PELNVs could reach the hair shafts by either
entering hair matrix cells and moving farther by cell differentiation or
via direct entry into hair shafts from the hair tip or else the side of hair
shaft.138 In addition, PELNVs structurally resemble liposome (i.e., bi-
layered phospholipid structure and flexible diameters),139 the skin
penetration of which through trans-follicle routes has previously
been reported by our group and others.140–142 In addition to the
trans-follicle route, another route to enter dermis is through penetra-
tion of the stratum corneum (SC). This route follows two pathways,
i.e., transcellular and intercellular routes.135 Owing to the similarity
of the membrane surface between PELNVs and mammalian cells,
PELNVs are thought to be efficient in passing through the SC both
by the transcellular and intercellular routes through the lipid fusion
effect (Figure 6). In addition, the near-infrared-induced hyperthermia
effect can cause the configuration change of the membrane and hence
increase the permeability of the SC, which could also enhance the
penetration of ELNVs. Recently, a study surfaced that gave credence
to the use of PELNVs in transdermal drug delivery. It was reported
that broccoli-derived ELNVs, being highly lipophilic, represent a
high entrapment efficacy and can be deployed to penetrate deeply
into the skin tissue. Enhanced fusion of broccoli-derived ELNVs
with keratinocytes was corroborated by the transport of an encapsu-
lated fluorescent agent into the cells and also by the presence of
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Figure 7. Modification of PELNVs for Targeted Drug Delivery
(A–D) Targeted drug delivery applications of (A) pristine PELNVs and (B) PELNV re-engineered nanovectors, with further elucidation of surface functionalization of pristine
PELNVs, and (C and D) surface modification of re-engineered nanovectors for achieving cargo loading and targeted delivery.
broccoli proteins in the plasmalemma of keratinocytes. Hence,
PELNVs can also be envisaged as an efficient conduit to transdermal
drug delivery.143 Furthermore, considering enhanced skin perme-
ation and cargo entrapment efficacy, PELNVs can be exploited for
transdermal gene delivery for skin melanomas based on our past
experience in transdermal gene delivery.110
Tumor-Targeted Delivery
The present chemotherapeutic approaches in the fight against cancer
confront two interwoven impediments of low target specificity and
higher toxicity. To tackle both of these challenges, great efforts have
been made toward development of multiple new drug delivery sys-
tems such as cell membrane capsules144 and multiple generations of
liposomes, but such efforts are still faced with issues of toxicity and
target specificity.145 Conversely, PELNV-derived re-engineered
nanovectors have proven themselves to be innocuous and non-
immunogenic with target ability. As a case in point, scientists have
been successful in re-engineering nanovectors from ginger-derived
ELNVs with the aim to deliver doxorubicin to colon tumor cells.
These nanovectors efficiently internalized into Colon-26 tumor
model cells and inhibited their growth. It is noteworthy here that pro-
nounced anti-tumor action was not solely due to doxorubicin, but it
was also partly due to suppression of oxidative stress and pro-inflam-
matory cytokine levels induced by the inherent nature of ginger-
derived ELNVs. Additionally, it was suggested that ginger-derived
nanovectors opportunistically triggered doxorubicin release in acidic
pH, exploiting the acidic extracellular microenvironment of tumors
and consequently indicating diminished doxorubicin side effects.
Furthermore, it was reported that they were innocuous, biocompat-
ible, and able to be produced economically on a large scale. On a
comparative investigation with artificially synthesized liposome,
they happen to be superior to liposome in terms of biocompatibility,
safety, apoptosis, electric cell-substrate impedance, and pH-depen-
dent profile for drug release.57
In another investigation, grapefruit-derived nanovectors were
demonstrated for the co-delivery of FA and paclitaxel to target the
CT26 colon cancer model (Figure 7B) and the human SW620 colon
cancer severe combined immunodeficiency (SCID) mouse model
cells intravenously. Moreover, these nanovectors were reported to
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be innocuous with higher aiming efficacy, as they did not traverse the
placental barrier when injected intravenously in pregnant mice. This
paves the way for potential use of grapefruit-derived nanovectors in
pregnant women for delivering certain drugs in the future.51 In sub-
sequent research by the same group, they carried out the modification
of grapefruit-derived nanovectors by coating them with an inflamma-
tory chemokine receptor-enriched plasma membrane (Figure 7C). As
a result, these nanoplatforms were targeted to the inflamed CT26 co-
lon tumor and 4T1 breast tumor in mouse models to deliver the
chemotherapeutic agent doxorubicin and anti-inflammatory drug
curcumin. It was also reported that leukocyte function-associated an-
tigen-1 (LFA-1) and CXC chemokine receptor 2 (CXCR2) on the sur-
face of coated grapefruit-derived nanovectors effectuated the in-
flamed tumor target specificity.130 It cannot be ignored that not
only do PELNVs play the role of delivery vehicle for chemothera-
peutic drugs, but they also execute their inherent anti-tumor thera-
peutic action simultaneously. However, it is noteworthy that the ther-
apeutic role is dependent on selection of the plant source of PELNVs.
For instance, naringin, a key flavanone in grapefruit,146 can be hydro-
lyzed by intestinal microbiota into its active metabolite naringenin,
which displays anti-tumor action.146,147 Moreover, PELNVs have
the ability to disrupt the cascade of several protein expressions vital
for tumor development. For instance, PELNVs have been found to
promote anti-tumor action by inhibiting cyclin D1 mRNA expression
and by activating cyclic guanosine monophosphate (cGMP)-depen-
dent protein kinase, which ultimately assist in tumor suppression
by inhibiting T cell transcription signaling.87 Additionally, innate
characteristics of PELNVs to upregulate the expression of pro-
apoptotic molecules and suppression of angiogenic factors such as cy-
tokines exert anti-proliferative and apoptotic actions on cancerous
cells while sparing normal cells.112 Therefore, considering all of these
aspects, it can be proposed that integration of natural PELNVs and
chemotherapy could be harnessed as a viable strategy to eradicate
cancer in the future.
Gene Delivery
Previously, non-viral delivery vectors have been deployed to deliver
nucleic acid-based drugs to disrupt the gene expression of multiple
diseases. However, such vectors typically have the innate risks of
immunogenicity and toxicity.148,149 Although PELNVs are neophytes
in the arena of gene delivery, they provide a solution to aforemen-
tioned complications and also go a notch above by traversing strin-
gent biological barriers such as the BBB. For instance, grapefruit-
derived nanovectors were tasked to carry exogenous miRNA
(miR17) to mouse brain tumor via the intranasal route (Figure 7D),
as reports had suggested that major histocompatibility complex class
I (MHC class I) is among the prominent targeted genes of miR17. Af-
ter administration, grapefruit-derived nanovectors were able to
achieve quick intracerebral tumor internalization for miR17 delivery,
which downregulated the MHC class I expression of tumor cells by
the activation of natural killer cells, thus inhibiting the tumor
growth.9 Similarly, Li et al.46 successfully silenced the survivin gene
expression in subcutaneous tumor cells via intravenous delivery of
siRNA by ginger-derived ELNVs (Figure 7A). It is highlighted here
24 Molecular Therapy Vol. 29 No 1 January 2021
that ginger-derived ELNVs innately harbor 6-gingerol and 6-shogaol,
which are pharmacologically bioactive molecules with prominent
anti-oxidative and anti-tumor actions.112 Furthermore, researchers
successfully transferred siRNA-CD98 to intestinal epithelial tissue
by ginger-derived nanovectors, and they reported that these orally
administered nanoplatforms impressively were retained in the upper
colon and ileum and diminished CD98 gene expression and curbed
the inflammation caused by ulcerative colitis and also prevented co-
litis-associated cancer.58 Moreover, apart from efficient siRNA-
CD98 delivery and its high activity, anti-inflammatory action could
also be partially attributed to structural components of ginger-derived
nanovectors, which curb pro-inflammatory cytokines (TNF-a, IL-1b,
and IL-6) and promote the induction of anti-inflammatory cytokines
(IL-10 and IL-22).88,112 Hence, it can be deduced that intrinsic ther-
apeutic action coupled with gene delivery capability raise the thera-
peutic index of PELNV-based therapies.
Cellular Uptake and Biodistribution of PELNVs
The driving mechanisms behind the cellular internalization and cargo
delivery of ELNVs into recipient cell cytosol are still poorly identified.
The pivotal step of ELNV internalization is the entry step into the
recipient cell. ELNV cellular internalization into recipient cells,
occurring either via a non-specific process such as micropinocytosis
or macropinocytosis, or through a specific receptor-dependent route,
is still ambiguous. On the grounds that multifarious ELNVs share a
common surface proteome, it is speculated that one of them assumes
the role of general ligand for the cell receptor, facilitating ELNV inter-
nalization.150 Additionally, ELNV cellular internalization has been re-
ported to happen via endocytic pathways such as clathrin-dependent
and clathrin-independent pathways. Even so, this ELNV feature lacks
consensus among researchers. Currently, it is theorized that the deter-
minative factor behind the internalization route is recipient cell types
rather than ELNVs themselves.151
PELNV-based nanoplatforms laden with drug molecules have been
reported to be introduced into experimental subject animals via
oral,58 intravenous,46 intramuscular, and intranasal routes.51 There
are multiple reported pathways involved in the cellular internaliza-
tion of PELNV-based nanoplatforms such as phagocytosis, macro-
pinocytosis, clathrin-mediated endocytosis, and caveolae-mediated
endocytosis (Figure 8).57,104 Biodistribution and bioavailability of
these drug-laden nanoplatforms are influenced by multiple factors,
e.g., selection of administration route, source plant’s natural charac-
teristics, surface charge, and size dimension. In vivo biodistribution
of several DiR-labeled PELNVs and re-engineered nanovectors have
been evaluated by fluorescently enabled high-imaging systems.
Ginger-derived nanovectors were reported to be impressively re-
tained in stomach, colon, and upper ileum even after 12 h of oral
administration.58 Similarly, ginger-derived PELNVs prefer to
continue to dwell inside intestine upon oral administration.88 In
contrast, grapefruit-derived nanovectors were concentrated in mac-
rophages of the lamina propria of cecum and colon of small intes-
tine and later preferentially migrated to the liver as well.104 In addi-
tion, upon intranasal delivery of DiR-labeled grapefruit-derived
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Figure 8. Elucidation of Multiple Pathways of Cellular Internalization/Uptake of PELNVs after Introduction into the Subject
Facilitated diffusion, endocytosis, and direct fusion are shown.
nanovectors in mice, elevated intensity of fluorescence in lungs and
brain was detected, thus demonstrating brain tumor target ability.51
Intravenously administered DiR-labeled ginger-derived nanovec-
tors and FA-coupled ginger-derived nanovectors exhibited
augmented levels of fluorescence in the liver of mice, suggesting a
higher accumulation and probable contribution toward detoxifica-
tion. Meanwhile, relatively lower fluorescence was visible in other
organs such as heart, kidney, and spleen. Fluorescence intensity
of DiR-labeled, FA-coupled ginger-derived nanovectors was found
to be identifiable in blood circulation after 48 h. Moreover, it was
reported to be 2.8-fold higher than its counterpart, suggesting
enhanced tumor target ability, drug delivery, and diminished sys-
temic toxicity to normal organs.57 Based on these findings, it can
be deduced that PELNV-based nanoplatforms are equipped to be
retained at the target site for the desired duration with no toxicity.
PELNV Toxicity and Immunogenicity
An ideal drug delivery system must ensure its superlative non-toxicity
and non-immunogenicity with no side effects in in vitro and in vivo as
well.152,153 Even though there have been meagre attempts to find their
cytotoxic effects on living subjects, PELNVs show a fascinating
biocompatible nature so far because of their natural provenance.
For instance, in probes related to cytotoxic potential effects of grape-
fruit-derived ELNVs in mice, several markers such as pro-inflamma-
tory cytokines and serum levels of liver enzymes, e.g., alanine trans-
aminase (ALT) and aspartate transaminase (AST), were
quantitatively measured. Mice were pre-administered with grape-
fruit-derived ELNVs and commercially available DOTAP (1,2-dio-
leoyl-3-trimethylammonium-propane)-DOPE (dioleoylphosphati-
dylethanolamine) liposomes. ALT, AST, and pro-inflammatory
cytokine levels were reported to be notably elevated in liposomal-
treated mice and, surprisingly, no elevation was recorded in the
PELNV-treated mice group. In addition to this, no pathological
modification or necrosis at all was observed in any of the histological
samples of liver, kidneys, spleen, and lungs in the PELNV-treated
mice group.51 Moreover, in vitro MTT (3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide) assays also revealed compara-
ble result to earlier mentioned findings on PELNVs, where PELNVs
had far less impact on viability in both RAW 264.7 and colon-26
cell lines as compared to liposomes.58 These reports suggest that ar-
tificially synthesized nanoplatforms induce an immune response in
the host, meaning that they are sequestered by filtering through or-
gans, and, hence, clearance of these nanomaterials is enhanced as a
result. Conversely, during investigations, it has become explicit that
PELNVs are phenomenally non-immunogenic and in fact have
strong supportive immunomodulatory actions that bring about tissue
homeostasis and contribute to an organism’s wellbeing.
In conclusion, PELNV-based nanoplatforms display a wide range of
advantages with respect to biocompatibility, stability, biodistribution,
prolonged half-life, and cellular internalization. Additionally, one of
the key benefits of using PELNVs for drug delivery is their ability
to reduce the side effects of a loaded drug when administered into
experimental subjects.104 All of these characteristics make them
poised to provide a safe, robust, and feasible approach for targeted
drug delivery.
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Clinical Trials
There are currently three PELNV-based clinical trials registered by
the same sponsor, i.e., the University of Louisville. Grape-derived
ELNVs are currently in clinical trials and being tested on humans
for averting oral mucositis associated with chemoradiation therapy
for head and neck cancer (ClinicalTrials.gov: NCT01668849).
Furthermore, aloe- and ginger-derived ELNVs are also under human
clinical trials for the treatment and mitigation of symptoms (e.g., in-
sulin resistance and chronic inflammation) associated with polycystic
ovary syndrome (PCOS) (ClinicalTrials.gov: NCT03493984). In addi-
tion to their role as an active pharmaceutical ingredient, clinical trials
for harnessing these PELNVs as drug delivery nanoplatforms are
underway where they are being investigated for encapsulating the
hydrophobic drug curcumin for delivery to normal colon tissue and
colon tumor (ClinicalTrials.gov: NCT01294072).
The ELNV-mediated therapeutic approach is expected to be the
cornerstone of a paradigm shift toward advanced cancer therapies.
In particular, rising interest in anti-tumor action of PELNVs is due
to their dual functionality, i.e., an intrinsic therapeutic capability
and the ability to deliver chemotherapeutic drugs. The anti-tumor ac-
tion of PELNVs is highlighted by their ability to interfere in the
expression of multiple proteins implicated in tumor progression.
Additionally, TRAIL-mediated apoptotic cell death, anti-angiogenic
actions, and pharmacologically active components of PELNVs also
cast a spotlight on their anti-neoplastic actions.87,112 In addition,
owing to elevated permeability and the retention effect, PELNVs
tend to lodge inside tumor tissues containing ill-formed blood vessels
than they do in normal tissues.128 Furthermore, PELNVs also display
the possibility of re-engineering them with tumor-specific targeting
proteins, peptides, or antibodies for precise therapy. At the same
time, clinical translation of ELNVs is still thought to be limited by
the lack of appropriate, scalable, and cost- and time-effective nano-
technologies for the purification and loading of EVs. Moreover, as
part of daily nutrition, clinical utilization of PELNVs may pose
different concerns than human cell-based medicinal EV products.
Notably, some doubts pertaining to cell-to-cell transmission of the
plant toxin trichosanthin via ELNVs have been raised.154 Thus,
possible perils linked to the transfer of toxins and allergens via
PELNVs should be addressed before considering them for clinical
studies.
Challenges and Perspectives
PELNVs are important conveyers of information between cells
through the transmission of various proteins, bioactive lipids, and ge-
netic information to alter the phenotype and function of recipient
cells. Thus, PELNVs have now been implicated in numerous biolog-
ical and pathological processes not only between plant cells, but also
between cells of other species. However, there still lies ambiguity as to
whether communication to recipient cells is specific or stochastic.
Moreover, their exploitation as nanotherapeutics or to deliver pro-
teins, nucleic acids, and other drug cargoes across various biological
barriers has been garnering much attention recently. However, until
recently, our understanding of the cellular and molecular mecha-
26 Molecular Therapy Vol. 29 No 1 January 2021
nisms that govern PELNV biogenesis, release, and functions remains
limited. Applicable isolation techniques are still time-consuming in
acquiring pure PELNVs and demand to be rectified to achieve the
commercial and therapeutic translations of PELNVs. There is a dire
need for standardization of qualitative and quantitative analyses to
develop better comparative interpretations of results. In addition,
there are still unresolved concerns of regulatory affairs of PELNV
therapeutic and drug delivery applications as there has been no agree-
ment yet on how they should be classified for approval. Similarly, no
consensus has been reached thus far related to routes followed by
PELNVs for internalization or for cargo delivery to recipient cell, as
well as how PELNV uptake or delivery induces transformations in
recipient cells.
One major limitation pertaining to PELNVs is their inadequacy to
carry any high quantity of drug load. The membrane fusion tech-
nique145 could be harnessed to amalgamate artificially synthesized
liposomes with PELNVs to achieve augmented cargo loading
capability. Resultant hybrid nanoplatforms may offer tractable
physicochemical characteristics owing to feasible manipulation of
the integrated liposomal part. Additionally, these hybrid nanoplat-
forms could be explored for delivery of CRISPR-Cas9 expression vec-
tors for gene therapy. This technique might be helpful in obviating
immunogenicity, cytotoxicity, and long-term expression because of
the small size of PELNVs and their ability to traverse stringent biolog-
ical barriers and escape from the mononuclear phagocyte system
(MPS).
In addition, our current knowledge of PELNV physiology, diver-
sity, internalization, and cargo delivery is still limited to yield
clear mechanistic conclusions about precisely how PELNVs
interact with and modify acceptor cells. Determining whether
internalization occurs through macropinocytosis or micropinocy-
tosis and/or receptor-mediated pathways, and whether these pro-
cesses result in cargo delivery, will be essential to understand and
control PELNV uptake and delivery of cargoes for clinical pur-
poses. Intrinsic biochemical traits endow PELNVs with profound
potential of implementation into the drug delivery system.
PELNVs are already innately laden with bioactive compounds.
Further weaponizing these nanovesicles with drugs poses perils
of interactions between innate bioactive components and exoge-
nously loaded drugs. Hence, this demands extensive and in-depth
investigations for developing design strategies to load drug mole-
cules into these nanovesicles without any potential interactions.
Moreover, an additional process of unloading innate bioactive
compounds from nanovesicles prior to drug loading could be
used; however, the unloading step should not compromise surface
morphology, size, and biological functions. Most therapeutic ef-
fects achieved by PELNVs are through their natural bio-
distribution. However, PELNVs could be imparted with target
specificity by investigating surface tailoring of PELNVs to selec-
tively amass at far away and difficult-to-target tumors without
causing any immunogenicity. Second, PELNVs can be used as
an exemplary vehicle in the future for drugs that need to be
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transported across the BBB because they can traverse the BBB
and obviate toxicity and rapid drug clearance complexities by
MPS associated with other nanocarriers. Third, as PELNVs struc-
turally resemble liposomes along with their ability to penetrate
skin, they can be envisaged as potential delivery nanoplatforms
for transcutaneous vaccination as well.136 In order to acquire ver-
satile biomedical applications of PELNVs, the concept of amal-
gamation of natural organic PELNVs with inorganic nanomateri-
als such as magnetic nanoparticles endowed with photothermal
conversion traits is propounded. These envisioned hybrid nano-
platforms might provide the opportunity to explore the theranos-
tic multifunctionality with augmented targeted drug delivery
coupled with localized hyperthermia under the influence of exter-
nally manipulated factors such as magnetic field.
In conclusion, ELNVs have shown many advantages, especially in
biocompatibility, cellular uptake, and targeting capability. Current
synthetic drug delivery systems, including inorganic and polymeric
nanoparticles, are intrinsically foreign materials and have much
more potential toxicity and immunogenicity. In comparison,
PELNVs, which are natural and endogenic, are considered as much
more biocompatible with multiple types of biofunctions along with
the parent cells. We have made some great strides during the last
decade toward understanding the biomedical potential of PELNVs,
but in fact we have just started to scratch the surface of what they
can do. Certainly, we will encounter some challenges regarding the
future implementations of PELNVs in therapeutics and drug delivery
exploration. Nevertheless, these impediments will be resolved, as has
been witnessed in any newly burgeoning field. It is envisaged that the
multidisciplinary approach of integrating biological science, drug de-
livery techniques, nanotechnologies, and engineering will steer the
course for development of PELNV-based therapeutics and novel de-
livery nanoplatforms.
ACKNOWLEDGMENTS
The study was supported by the National Key Research and Develop-
ment Program of China (2018YFC1105404), the Key Project at
Central Government Level (2060302), and the National Natural Sci-
ence Foundation of China (81473145).
AUTHOR CONTRIBUTIONS
L.-H.P. and L.-Q.H. conceived the project. H.A.D., T.-W.G., and
A.-Q.Z. wrote the manuscript. All authors discussed the contents
and commented on the manuscript.
DECLARATION OF INTERESTS
The authors declare no competing interests.
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