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Accepted Manuscript
Functionalized extracellular vesicles as advanced therapeuticnanodelivery systems
Mei Lu, Haonan Xing, Zhe Xun, Tianzhi Yang, Xiaoyun Zhao,Cuifang Cai, Dongkai Wang, Pingtian Ding
PII: S0928-0987(18)30214-8DOI: doi:10.1016/j.ejps.2018.05.001Reference: PHASCI 4512
To appear in: European Journal of Pharmaceutical Sciences
Received date: 27 March 2018Revised date: 1 May 2018Accepted date: 3 May 2018
Please cite this article as: Mei Lu, Haonan Xing, Zhe Xun, Tianzhi Yang, Xiaoyun Zhao,Cuifang Cai, Dongkai Wang, Pingtian Ding , Functionalized extracellular vesicles asadvanced therapeutic nanodelivery systems. The address for the corresponding authorwas captured as affiliation for all authors. Please check if appropriate. Phasci(2017),doi:10.1016/j.ejps.2018.05.001
This is a PDF file of an unedited manuscript that has been accepted for publication. Asa service to our customers we are providing this early version of the manuscript. Themanuscript will undergo copyediting, typesetting, and review of the resulting proof beforeit is published in its final form. Please note that during the production process errors maybe discovered which could affect the content, and all legal disclaimers that apply to thejournal pertain.
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Functionalized extracellular vesicles as advanced therapeutic nanodelivery systems
Mei Lu a, Haonan Xing a, Zhe Xun b, Tianzhi Yang c, Xiaoyun Zhao d, Cuifang Cai a, Dongkai
Wang a,*, Pingtian Ding a,**
a School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, China
b China Institute of Metabolic Disease Research and Drug Development, China Medical
University, Shenyang, China
c Department of Basic Pharmaceutical Sciences, School of Pharmacy, Husson University,
Bangor, ME, USA
d School of life Science and Biopharmaceutics, Shenyang Pharmaceutical University,
Shenyang, China
* Address for correspondence:
Dongkai Wang, School of Pharmacy, Shenyang Pharmaceutical University, No.103, Wenhua
Road, Shenyang 110016, China. Tel: +86 24 23986310; +86 13998210961 (mobile). Fax: +86 24
23986310. E-mail: [email protected]. (D. Wang).
Pingtian Ding, School of Pharmacy, Shenyang Pharmaceutical University, No.103, Wenhua
Road, Shenyang 110016, China. Tel: +86 24 23986305; +86 13940375008 (mobile). Fax: +86 24
23986305. E-mail: [email protected]. (P. Ding).
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Abstract
Extracellular vesicles (EVs) are membrane enclosed vesicles that are shed by almost all cell
types, and play a fundamental role in cell-to-cell communication. The discovery that EVs are
capable of functionally transporting nucleic acid- and protein- based cargoes between cells,
rapidly promotes the idea of employing them as drug delivery systems. These endogenous vesicles
indeed hold tremendous promise for therapeutic delivery. However, issues associated with
exogenously administered EVs, including rapid clearance by the immune system, apparent lack of
targeting cell specificity, and insufficient cytoplasmic delivery efficiency, may limit their
therapeutic applicability. In this review, we discuss recent research avenues in EV-based
therapeutic nanodelivery systems. Furthermore, we narrow our focus on the development of
modification strategies to enhance the delivery properties of EVs, and elaborate on how to
rationally harness these functionalized vesicles for therapeutic delivery.
Keywords: Functionalization; Extracellular vesicles; Therapeutic nanodelivery systems; Targeting
capacity; Cytoplasmic delivery
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Abbreviations: AA-PEG, aminoethylanisamide-polyethylene glycol; AD, Alzheimer's disease;
BBB, blood-brain barrier; BDNF, brain derived neurotrophic factor; CML, Chronic Myeloid
Leukemia; CPPs, cell penetrating peptides; c(RGDyK, cyclo(Arg-Gly-Asp-D-Tyr-Lys) peptide;
DSPE, 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine; EGFR, endothelial growth factor
receptor; EVs, Extracellular vesicles; FA, folate; GALA, GALA peptide; GPI,
glycosylphosphatidylinositol; ICAM-1, intercellular adhesion molecule 1; imDCs, immature
dendritic cells; IL3, Interleukin 3; iRGD, internalizing arginine-glycine-asparagine; ISEV,
International Society for Extracellular Vesicles; Lamp2b, lysosome-associated membrane
glycoprotein 2b; LFA-1, lymphocyte function-associated antigen 1; MPS, mononuclear phagocyte
system; MSCs, mesenchymal stromal stem cells; MVBs, multivesicular bodies; PDGFR,
platelet-derived growth factor receptor; PEG, polyethylene glycol; PPI, proton pump inhibitors;
RVG, rabies viral glycoprotein; SIRPα, signal regulatory protein α; SMCNCs, superparamagnetic
magnetite colloidal nanocrystal clusters; VEGF, vascular endothelial growth factor; VSV-G, G
protein of vascular stomatitis virus; WFA, Withaferin A.
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1. Introduction
Extracellular vesicles (EVs), also termed as natural liposomes, are small membrane-enclosed
vesicles shed in an evolutionally conserved manner by cells ranging from prokaryotes to higher
eukaryotes (Colombo et al., 2014). With a size ranging from 40 to 1000 nm, EVs are composed of
a lipid bilayer decorated with various transmembrane proteins (Raposo and Stoorvogel, 2013;
Tkach and Thery, 2016). As defined primarily by subcellular origin, cells can secrete a multiple
subset of EVs. Among which, the most interest is focused on two major groups of EVs: exosomes
and microvesicles. Exosomes are originated from intraluminal budding of multivesicular bodies
(MVBs), while microvesicles are formed by directly budding from the plasma membrane (Thery
et al., 2009; Thery et al., 2002). EVs are capable of transferring biologically active molecules
between cells locally and at distance, thereby regulating gene expression and cellular function in
recipient cells (Valadi et al., 2007; van Dommelen et al., 2012) As such, these vesicles are
endorsed with a vast array of functions in a multitude of physiological processes and pathologies
(Baj-Krzyworzeka et al., 2007; Yanez-Mo et al., 2015), which has triggered new developments in
the field of diagnostics. Actually, the use of EVs as disease biomarkers has become an area of
intense investigation, given that healthy subjects and patients may secrete EVs with different
contents (Andreu et al., 2017; Barile and Vassalli, 2017). Moreover, giving the increased release of
EVs by diseased cells (cancer cells), recent clinical reports proposed to use the level of EVs
released into human body fluids as a reliable biomarker of diseases, specifically of cancers
(Cappello et al., 2017; Fais et al., 2016). For example, melanoma patients and prostate cancer
patients have remarkably higher levels of EVs in the plasma than healthy subjects, it is promising
to quantify EVs in the plasma of these patients and harness these endpoints in early diagnosis
(Logozzi et al., 2017; Logozzi et al., 2009). In addition to serving as disease biomarkers, EVs are
also emerging as a very valuable class of drug nanocarriers. In the last few years, excitement was
created to harness EVs as therapeutic nanodelivery systems because of their natural role in
transporting biological molecules amongst cells (Fuhrmann et al., 2015; Jiang et al., 2017). EVs
have been extensively studied as biological nanocarriers for a wide array of therapeutics, ranging
from small chemotherapeutic molecule to macromolecular miRNA, siRNA, DNA, and proteins in
various preclinical studies (Srivastava et al., 2016; Sun et al., 2010).
The therapeutic use of EVs as nanocarriers is boosted by their low immunogenicity and
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toxicity but high biological permeability to cross the blood-brain barrier (BBB) and
biocompatibility (El Andaloussi et al., 2013). Despite of these remarkable features, major
challenges still remain to be addressed before EVs can be used as competitive therapeutic
nanocarriers. In addition to issues concerning lack of efficient methods for isolation, loading,
scalable production, and quality regulation of EVs (Fais et al., 2016; Lener et al., 2015; Lu et al.,
2017), the intracellular delivery efficiency of EVs still remains unsatisfactory, especially after
systemic administration (Mulcahy et al., 2014; Nakase and Futaki, 2015; Smyth et al., 2015). For
efficient systemic drug delivery, therapeutic nanocarriers should possess three crucial features:
sufficiently long circulation time for effective tissue/organ accumulation, satisfactory penetration
and homing capacity to reach target tissues, and high cellular uptake and efficient endosomal
escape to achieve efficient cytoplasmic delivery (Haussecker, 2014). However, the unfavorable
pharmacokinetic profile, lack of targeting cell selectivity, and insufficient cytosolic delivery
efficiency of unmodified EVs may prohibit them from becoming clinically acceptable therapeutics.
For instance, naturally secreted EVs with no modifications to their composition may not readily
avoid the mononuclear phagocyte system (MPS) sequestration as assumed from their “self” nature
(van der Meel et al., 2014). After systemic administration, unmodified EVs have been described to
undergo swift removal from the circulation mediated by the MPS, which substantially limit their
accumulation in tissues of interest (Takahashi et al., 2013). In addition, EVs released by most cells
exhibit limited tropism to a specific cell type due to their highly complex and variable
composition (Barile and Vassalli, 2017). This may result in off-target effects when EVs are applied
for therapeutic delivery. Furthermore, the efficiency of these vesicles to overcome membrane
barriers, such as crossing the plasma membrane, and endosomal escape for cytoplasmic release of
their payloads, is still considered insufficient for therapeutic application . There is a competition
for the cellular uptake of these vesicles, as a considerable number of endogenous EVs are secreted
into the bodily fluids. On the other hand, the negative charge of EV membrane may also prevent
them from binding to negatively charged plasma membranes. Additionally, after internalization by
the recipient cell, EVs require endosomal escape from degradation by lysosomes, however, their
cytoplasmic release efficiency remains insufficient (Nakase and Futaki, 2015; Nakase et al., 2017).
To this end, strategies for enhancing the delivery properties of systemically delivered EVs are
greatly needed.
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Taking advantage of the possibility to modulate EV composition, the potential of EVs as drug
delivery vehicles can be further improved by incorporation of functional moieties. Recently,
strategies to modify the surface of EVs or encapsulate them with exogenous materials for better
delivery properties have been widely reported (Koh et al., 2017; Nakase and Futaki, 2015; Silva et
al., 2015; Tian et al., 2014). For example, it is plausible to decorate EVs with “stealth” polymer
coatings, such as polyethylene glycol (PEG) and dextran sulfate, typically applied in synthetic
nanoparticles, to provide them with greater disguise from MPS and extend their circulation
half-life (Kooijmans et al., 2016a; Watson et al., 2016). Additionally, engineering EVs to express
peptides, such as rabies viral glycoprotein (RVG) that binds to acetylcholine receptor expressed on
neuronal cells, enables EVs to efficiently pass through the blood-brain barrier and target siRNA to
the brain (Alvarez-Erviti et al., 2011). As another example, conjugating EVs with arginine-rich
cell penetrating peptides (CPPs) could activate the macropinocytosis pathway for effective cellular
uptake, thus enhancing the cytosolic delivery efficiency (Nakase et al., 2016). Generally,
incorporation of functional moieties on EV surface can be accomplished by using “cell
engineering” techniques, wherein donor cells are transfected with recombinant plasmids encoding
EVs’ membrane proteins (e.g. Lamp2b) fused to specific functional molecules. Alternatively,
functionalized EVs may be generated by employing “EV engineering”, wherein EVs are directly
modified through mechanisms of biochemical conjugation, post insertion, or electronic interaction.
In this review, we provide an update overview of EV-based therapeutic nanodelivery platforms
with focus on EV modification strategies, in order to efficiently harness functionalized EVs for
therapeutic delivery.
2. Unmodified EVs for therapeutic delivery
The advent of nanotechnology has brought a rapidly growing body of new nanoscaled drug
delivery vehicles, such as the most investigated lipid- and polymer-based nanocarriers (Duncan,
2003; Puri et al., 2009). Both of these nanoparticles have been effectively used to deliver a wide
range of therapeutics. However, they often fail to merge drug delivery efficiency with
biocompatibility, thus confronting issues such as potential toxicity and immunogenicity (Kumari
et al., 2010; Raemdonck et al., 2014). In this circumstance, EVs are increasingly emerged as a
fascinating drug delivery vehicle that outperforms synthetic nanoparticles. EVs have been shown
with strong protection for their contents and high permeability to traverse biological barriers, such
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as the BBB (Yang et al., 2017b). More importantly, These vesicles have been proposed to be less
immunogenic and toxic but with higher biodegradability than artificial delivery vehicles, possibly
due to their biological origin (Johnsen et al., 2014; Lai et al., 2013). These properties facilitate the
development of EVs as suitable nanocarriers for therapeutics and potential avenues for the
treatment of a number of diseases.
Many delivery approaches that take advantage of the innate delivery properties of EVs have
shown increased therapeutic efficacy (Martins-Marques et al., 2016; Momen-Heravi et al., 2014;
Sterzenbach et al., 2017). For instance, doxorubicin was successfully loaded into breast cancer
cell-derived EVs, which was more efficient than free drug in the treatment of breast cancer and
ovarian cancer mouse model. EVs loaded with doxorubicin displayed increased stability and
accumulation in tumor tissue, leading to a significant suppression of tumor growth and reduced
cardiotoxicity in mouse, a main side-effect of doxorubicin (Hadla et al., 2016). In a recent study,
Yang et al. demonstrated EVs from brain endothelial cells can ferry doxorubicin and paclitaxel
across the BBB in a zebra fish model, leading to the inhibition of tumor progression (Yang et al.,
2015). Whereas, both free drugs remained localized in the vasculature and could not penetrate the
BBB. Interestingly, bovine milk-derived EVs can also function as efficient carriers for
chemotherapeutics. Munagala et al. showed that EVs loaded with Withaferin A (WFA), not only
exhibited significantly higher efficacy in in vitro cell culture, but also possessed greater tumor
inhibitory effect in in vivo tumor models compared with free WFA (46% vs 23%) (Munagala et al.,
2016). Additionally, EVs possess the ability to deliver photodynamic molecules, thus holding
great potential in photodynamic therapy for cancer treatment (Kusuzaki et al., 2017). For instance,
Lessi et al. recently showed that EVs loaded with acridine orange exhibited increased
effectiveness and decreased toxicity in human melanoma cells, as compared to free acridine
orange. This was a completely new prototype for harnessing EVs in the treatment of cancers (Iessi
et al., 2017). These results confirmed EV formulations could be effectively used to enhance the
therapeutic efficacy of small chemotherapeutic molecules. However, it is noteworthy that
extracellular acidity probably represents the most important phenotype of malignant tumors (Fais
et al., 2014; Spugnini and Fais, 2017). This is a key point to tailor exosomes for anticancer therapy,
because it has been widely shown that the acidic milieu of cancer may serve as a selective
pressure to trigger the release of highly level of EVs by cancer cells, leading to cancer progression
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and metastasis (Logozzi et al., 2017; Parolini et al., 2009). To this end, it appears that antiacidic
molecules such as proton pump inhibitors (PPI) and buffers such as sodium bicarbonate and citrate,
may be utilized to improve the efficacy of anticancer therapy (Fais et al., 2014; Spugnini and Fais,
2017). For example, Federici and coworkers showed that PPI pretreatment can increase the
cellular uptake of cisplatin compared with untreated cells, leading to a significant inhibition of EV
release and a clear suppression of tumor growth both in vitro and in vivo (Federici et al., 2014).
Other than chemotherapeutics, unmodified EVs have also been extensively exploited to
shuttle small RNAs that have poor stability in the circulation. Yang’s group further employed brain
endothelial EVs to deliver vascular endothelial growth factor (VEGF) siRNA across the BBB to
the brain of zebra fish. Both in vitro and in vivo results showed siRNA was efficiently transported
to the brain and inhibited the tumor growth (Yang et al., 2017b). Similarly, marrow MSC-derived
EVs were therapeutically used to deliver miRNA (miR-146) to induce regression of glioma
growth (Katakowski et al., 2013). Despite of these encouraging results, Stremersch and coworkers
demonstrated that EVs appeared to be less effective than conventional liposome formulations in
the functional delivery of small RNAs. They reported evidence that EVs were unable to
functionally deliver cholesterol-conjugated siRNA and endogenous miRNA to mediate target gene
knockdown despite robust cellular uptake. In contrast, conventional anionic fusogenic liposomes
were capable of inducing a marked gene knockdown under equal experimental conditions. The
authors proposed that endolysosomal entrapment might be a main barrier that resulted in the
incapacity of unmodified EVs to guide cytosolic delivery of small RNAs (Stremersch et al., 2016).
While EVs have been widely explored to deliver small molecular drugs and RNAs, there are some
evidences that EV-mediated transfer of macromolecular DNA and protein is also possible. For
instance, Lamichhane and coworkers showed that DNA loading into EVs by electroporation
seemed to be size-dependent. Linear DNA above 750bp and plasmid DNA above 4.5kb was
observed to have very low loading efficiency (Lamichhane et al., 2015). Similarly, successful
loading of DNA and feasible cellular uptake of drug loaded EVs did not lead to transcription and
functional gene expression, underscoring other factors may limit the cytosolic delivery of EVs’
cargoes. Intracellular delivery of bioactive proteins is an attractive tool to replace missing or
poorly expressed proteins. In recent years, there is a growing passionate investigation bustle to
enrich proteins in EVs for improved delivery efficiency. For instance, Yuan et al. demonstrated
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that EVs derived from macrophages expressed the integrin lymphocyte function-associated
antigen 1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1), could deliver a cargo protein,
the brain derived neurotrophic factor (BDNF), traversing the BBB of mice after intravenous
administration. Moreover, an observable enhancement of delivery occurred during brain
inflammation, a common condition associated with many brain diseases (Yuan et al., 2017).
Collectively, EVs are characterized by several favorable features that highlight eloquently in
most studies where EVs may serve as powerful therapeutic nanocarriers. However, in some
circumstances they exhibited low capability of delivering their cargoes. To date, it still remains
elusive to explain why under some conditions EVs were very efficient for drug delivery, while in
other circumstance they exhibited dysfunctional. To this end, future research should be dedicated
towards elucidating the cellular mechanism behind successful EV-mediated delivery. Yet some
obstacles may lay behind these divergent results that need to be overcome for translation of EVs
into clinical therapies. Currently, no standard techniques have been established for the clinical
grade production, storage, and quality control of EV-based therapeutics (Fais et al., 2016; Lener et
al., 2015). In addition, safety issues, including general toxicity, potential immunogenicity,
immunotoxicity, and tumourigenicity, must be highlighted for therapeutic application of EVs in a
clinical setting (Lener et al., 2015). However, clinical translation of EVs is a long-standing issue,
which requires cooperation between researchers, clinicians, and competent authorities in EV
research field. Luckily, as a consequence of the substantial advance in this field, a level of
consensus on these challenges have been achieved by the International Society for Extracellular
Vesicles (ISEV), although it has not yet been fully implemented in clinical studies (Fais et al.,
2016). In addition to these considerable challenges that must be highlighted, strategies to
overcome several preclinical challenges of EVs, including short circulation time, apparent lack of
specific cell tropism, and insufficient cytoplasmic delivery efficiency are also essential to facilitate
the clinical translation of EVs (Alvarez-Erviti et al., 2011; Nakase et al., 2016; Smyth et al., 2015).
To this end, improving the delivery properties of EVs via surface modification or encapsulation
with exogenous materials is awaited with great interest to deal with these sticky issues, as they are
warranted to increase the circulation stability of intravenously injected EVs, improve their
targeting capability for better reaching tissues of interest, and facilitate EV payload delivery to the
cytosol of target cells. Rather encouragingly, the possibility to modify these vesicles and
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functionalize them with various functional moieties offers exiting opportunities to extend the
delivery potential of EVs beyond their native capability.
3. Modification techniques to enhance EVs’ delivery properties
For therapeutic applications, EVs need to be functionalized with specific moieties to improve
their delivery properties. In the case of synthetic nanocarriers, this is generally accomplished
through direct introduction of functional ligands on the particle surface. However,
functionalization appears to be more complex for EVs as their composition is dictated by the
donor cells. Nevertheless, multiple modification strategies have been successfully applied to
enhance the intracellular delivery efficiency of EVs (Kooijmans et al., 2016a; Tamura et al., 2017;
Tian et al., 2014). Generally, EV modification can be classified into “cell engineering” and “EV
engineering”. Provided that molecules delivered to cell membrane are naturally incorporated in
the budding microvesicles, while materials internalized inside the cells can be packaged into the
secreted exosomes, it can take advantage of such a biosynthesis process for EV modification by
genetic manipulation of their parent cells. Such a modification approach is called “cell
engineering”. While in “EV engineering”, surface functionalization is accomplished by direct
manipulation of EVs (Fig. 1). In this section, we will discuss these two EV modification
techniques.
3.1. Genetic engineering of producing cells for EV modification
“Cell engineering” hijacks a cellular biosynthesis process to generate modified EVs. By
using the cell’s own machinery for protein production, this approach allows for the expression of
proteins or peptides on EV surface with preserved direction and function (Zhao et al., 2016). A
general strategy of “cell engineering” is to fuse ligands with specific function to EV-enriched
membrane proteins. For example, in an early proof-of-concept study, neuron-targeting EVs were
generated by fusing RVG peptide with lysosome-associated membrane glycoprotein 2b (Lamp2b),
an EVs-associated membrane protein (Alvarez-Erviti et al., 2011). Likewise, engineering
immature dendritic cells (imDCs) to express a fusion construct of Lamp2b and
αν-integrin-specific iRGD peptide has been exploited to produce iRGD modified EVs (Tian et al.,
2014). However, lamp2b display of a muscle-specific peptide was shown to be minimally
effective at targeting muscle cells, illustrating a variable performance of this approach
(Alvarez-Erviti et al., 2011). Furthermore, concerns have also been raised about the long stability
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of Lamp2b-fused ligands, as the fused peptides tend to be proteolytic degradation in the endosome
before being sorted into EVs (Hung and Leonard, 2015). To suppress peptide loss, Hung and
coworkers pointed out that the introduction of a glycosylation motif on Lamp2b can protect
targeting construct from degradation, hence improving EV delivery to neuroblastoma cells in vivo
(Hung and Leonard, 2015).
In addition to Lamp2b, other EV-associated membrane proteins, such as platelet-derived
growth factor receptor (PDGFR), lactadherin, and Cytolysin A, have also been used as anchors
and fusion partners for protein domains/peptides of interest. Fusing functional ligands to the
transmembrane domain of PDGFR is a fascinating display system for EV modification (Ohno et
al., 2013). Through this method, EVs expressing endothelial growth factor receptor
(EGFR)-targeting peptide anchored by the transmembrane receptor of PDGF showed significantly
improved cell association to EGFR-expressing tumor cells (Ohno et al., 2013). In addition, the
C1C2 domain of lactadherin is another commonly used anchor for EV display of functional ligand
(e.g. antibodies against tumor biomarkers) (Delcayre et al., 2005). Recently, a similar approach
has been used to equip EVs with enhanced targeting properties. In this method, Escherichia coli
was engineered to express fusions between anti-HER2 affibody and the C-terminus of Cytolysin A,
which led to display of anti-HER2 affibody on the secreted EVs (Gujrati et al., 2014). However,
fusion of recombinant proteins or functional peptides to EV-specific membrane proteins may
impair the structural and functional integrity of these membrane proteins (Kooijmans et al., 2016c).
In this circumstance, a new EV marker glycosylphosphatidylinositol (GPI) can function as an
alternative anchor for the display of functional ligands. With such an approach, functional ligands
can be localized on GPI-rich EV surface through engineering EV-producing cells to express EGFR
nanobodies fused to GPI-anchor, to target cancer cells overexpressing higher level of EGFR
(Kooijmans et al., 2016a). Given that EVs are characterized to contain tightly packed membrane
domains (lipid rafts) which are enriched in GPI, GPI-fused nanobodies were strongly enriched in
EVs compared with their parent cells (Haraszti et al., 2016). Moreover, GPI anchoring may
potentially function as a versatile tool to incorporate a wide range of functional ligands, such as
antibodies, reporter proteins, and immune stimulatory molecules (Kooijmans et al., 2016a).
To address the limitation of conventional “cell engineering” methods, new EV
functionalization strategies have been explored. A novel approach is to produce fusogenic
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liposomes containing desired ligands, deliver them to cells through membrane fusion, and then
EVs equipped with functional ligands could be harvested (Lee et al., 2016). This method enables
an efficient and controlled manner to arm EVs with various functional ligands, such as peptides
and bio-orthogonal chemicals, without modification of EVs’ membrane proteins. In addition, such
a liposome-based cellular engineering method allows simultaneous modification of EVs with
multiple ligands with disparate functions (Lee et al., 2016). Additionally, surface modification can
also be achieved by simple incubating cells with, mainly hydrophobic, ligand of interest, and then
ligand may interact with the cell phospholipid bilayers driving spontaneously membrane insertion.
For example, lipidated AS1411 aptamer could spontaneously insert into cellular lipid bilayer,
resulting in efficient immobilization of targeting ligands onto cell membrane by simple incubation
(Wan et al., 2018). Afterwards, aptamer-functionalized EVs could be generated by repeated
mechanical extrusion of cells through multiple filters with different pore size. Overall, mechanical
extrusion approach may represent a rapid, easy, and economic technique to produce functionalized
EVs (Wan et al., 2018). Moreover, a completely different approach to surface engineering is to
encapsulate exogenous materials into cells, which can then be packaged into the secreted EVs. For
example, magnetically and optically targeting responsive EVs can be generated by incubating
macrophages with iron oxide nanoparticles and small molecule photosensitizers (Silva et al., 2013;
Silva et al., 2015). Then modified EVs could target specific areas of the body to achieve magnetic
and photodynamic targeting by applying an external magnetic field and optical stimulation.
However, challenges of this approach include difficulties in targeting deep tissues in the body, and
possible toxicity concerns associated with the administration of magnetic nanoparticles and
photosensitizers (Clavijo-Jordan et al., 2012).
3.2. Direct engineering of EVs
Conventional cell-based EV functionalization strategies are frequently criticized for
inefficiency, as only a low fraction of functional materials can be displayed on EV surface (Kim
and Eberwine, 2010). In addition, their production protocols are reported to be complex,
time-consuming, and not be readily applicable to all ligands, such as toxic proteins (Armstrong et
al., 2017). In contrast, direct EV engineering offers a more controllable and efficient tool to
incorporate high densities of functional moieties regardless of ligand type (Armstrong et al., 2017).
In addition, EVs are nonliving entities, hence, it is possible to use some chemical reagents and
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reaction conditions that could not be used in “cell engineering”. Nevertheless, there are still some
constraints, for example, the reaction conditions must be well controlled to avoid exposing EVs to
excessive solvent, temperature or pressure, as which may impair membrane integrity and denature
the functionality of membrane proteins (Wang et al., 2014).
Direct “EV engineering” is typically achieved by using covalent bioconjugation or
noncovalent modification, such as post insertion and electronic interaction. In covalent
conjugation strategies, chemical reactions are directly performed at EV surface under
biocompatible reaction conditions (Smyth et al., 2014). By employing chemical linkers, functional
ligands can be linked to amines, a reactive functional group expressed on EV membrane. This
reaction mechanism has been used to graft alkyne moieties onto EV surface. After conjugation
with membrane amines, the introduction of reactive alkyne bases allows a second click chemistry
reaction with model azide compounds (e.g. Azide-Fluor 545). As a consequence, functional
ligands can be stably conjugated onto the surface of isolated EVs (Smyth et al., 2014). As a
proof-of-principle, the result demonstrated chemical reactions were well suited for EV
modification, without significant effects on the structure integrity of EVs or their interaction with
recipient cells. Similarly, EVs could also be conjugated with cyclo(Arg-Gly-Asp-D-Tyr-Lys)
peptides [c(RGDyK)], a ligand with high affinity to integrin αvβ3 by using click chemistry
(bioorthogonal copper-free azide alkyne cyclo-addition). This technique was demonstrated to be
useful for rapid and large-scale production of functionalized EVs (Tian et al., 2018). In contrast to
covalent modification strategies, noncovalent functionalization approaches involve relatively mild
reaction conditions. As for post insertion, hydrophobic or lipids-conjugated ligands could
spontaneously insert into EV lipid bilayer through hydrophobic interaction. Through this approach,
nanobodies specific for EGFR were conjugated to the distal ends of lipid-PEG derivatives, which
formed nanobody-PEG-micelles in aqueous solution. Then, the resulted micelles were mixed with
EVs and incubated at elevated temperatures to facilitate micelle disintegration and spontaneous
integration of nanobody-PEG-lipids onto EV membrane. As a consequence, a
temperature-dependent transfer of nanobody-PEG-lipids to EV membrane was observed,
suggesting a post-insertion mechanism (Kooijmans et al., 2016b). It is noteworthy that post
insertion possesses general applicability, given that EVs from Neuro2A cells and platelets shared
similar transfer pattern of nanobody-PEG-lipids. In addition, the ease of ligand conjugation to
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PEGylated phospholipids makes it applicable to a wide range of functional ligands, such as
antibodies and peptides (Kooijmans et al., 2016b). Another noncovalent modification method is
based on electrostatic interaction. EVs are negatively charged with a zeta potential of
approximately -11.90mV (Saari et al., 2015), which can be exploited to equip EVs with cationic
moieties. For example, EV modification was achieved by simple mixing purified EVs with
cationized pullulan through an electrostatic interaction between both substances. In this approach,
pullulan was cationized with spermine to enhance its electrostatic interaction with the negatively
charged surface of EVs (Tamura et al., 2017). The advantages and disadvantages of “cell
engineering” and “EV engineering” are shown in Table 1.
4. Modified EVs for therapeutic delivery
Provided that unmodified EVs’ unfavorable characteristics may limit their widespread
application as drug delivery vehicles, EV modification could open up exciting opportunities for
improving their delivery capability and extending the repertoire of their payloads. The
introduction of function-specific moieties to EV surface might be an attractive strategy to endow
these vesicles with enhanced circulation time, increased targeting capacity, and improved
cytoplasmic delivery efficiency. Different strategies for EV functionalization are shown in Fig. 2.
In this section, we will elaborate on how to harness these modified EVs for efficient therapeutic
delivery.
4. 1. Modified EVs for extended circulation time
As EVs are abundantly present in our biological fluids, it seems logical to postulate that they
may be quite stable in the blood circulation when being used as therapeutic nanocarriers. However,
recent studies demonstrated that unmodified EVs from many cell lines all showed rapid clearance,
preferential accumulation in MPS associated organs, such as liver, spleen, and lungs, and followed
by swift elimination by bile excretion, renal filtration or phagocytosis by the immune system (Imai
et al., 2015; Saunderson et al., 2014; Smyth et al., 2015; Takahashi et al., 2013; Tian et al., 2014;
Wiklander et al., 2015). In the past decade, the fate of intravenously-injected EVs has been
extensively studied through different tools, such as luciferase labeling and fluorescence-mediated
imaging (Lai et al., 2014; Takahashi et al., 2013). Pharmacokinetic studies reported that EVs
abundance was decreased by more than a half from 30 to 60 minutes after systemic administration
(Lai et al., 2014). Recently, EVs were described to disappear very quickly from the circulation
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with a half-life of approximately two minutes, and completely disappear from the blood stream at
four hours upon intravenous injection (Saunderson et al., 2014; Takahashi et al., 2013).
Additionally, biodistribution of unmodified EVs showed a rapid distribution phase in the spleen,
liver, lung, and kidney at approximately 30 minutes after systemic injection, and followed by a
longer distribution phase reflecting elimination through the liver and kidney in 1 to 6 hours upon
intravenous administration (Lai et al., 2015; Lai et al., 2014). Of note, EV clearance was observed
to be much slower in macrophage-depleted mice than that in groups not subjected to macrophage
depletion treatment, illustrating a macrophage-dependent clearance of intravenous-injected EVs
from the blood circulation (Imai et al., 2015). In addition, it is proposed that the immune
recognition of exogenously-administered EVs is partly mediated by the exposure of externalized
phosphatidylserine on EVs to macrophages (Deschout et al., 2014; Feng et al., 2010). Together,
these results indicate that EVs display an unfavorable pharmacokinetic profile resembles that of
synthetic nanoparticles. Systemically administered EVs may not readily avoid MPS sequestration
as speculated from their wide existence in the blood circulation and “self” nature.
Considering that the rapid clearance feature along with minimal tissue/organ accumulation of
unmodified EVs may limit their application as drug delivery vehicles. Strategies to protract the
circulation time of EVs are urgently required. A common used approach to prevent swift removal
of nanoparticles from circulation by opsonization is to decorate these particles in a PEG corona
(Caracciolo, 2015). PEGylation has been shown to be a powerful way to prolong the circulation
half life of drug-loaded liposomes (Perche and Torchilin, 2013). Recently, PEGylation has been
explored to increase the circulation time of EVs (Table 2). Kooijmans and coworkers showed that
insertion of PEG-lipids to EV membrane endowed them with stealth properties, and significantly
extended their circulation time in mice after intravenous injection (Fig. 3). To be specific,
nanobody-PEG EVs could be detectable in blood for longer than 60 minutes after intravenous
injection. Whereas, unmodified EVs were rapidly cleared from the circulation within 10 minutes.
Noteworthy, incorporation of PEG chain in EV membrane reduced their interactions with
non-targeted cells, while at the same time, display of nanobodies on vesicle surface enhanced EV
association with tumor cells expressing EGFR (Kooijmans et al., 2016b). In another study by Kim
et al., modification of EVs with aminoethylanisamide-polyethylene glycol vector moiety
(AA-PEG) was demonstrated to significantly increase EVs’ circulation time in the blood
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circulation after systemic injection in mice, which allowed for robust accumulation in tumor and
greater in vivo therapeutic efficacy than non-modified EVs and free paclitaxel (Kim et al., 2018).
Based on these findings, modification of EVs with hydrophilic polymers though surface
engineering may prove crucial for prolonging EV circulation time, and represent a useful strategy
to the development of next generation of EV-based therapeutic nanodelivery systems. In addition,
concomitant functionalization of EVs with stealth properties and targeting capacity could enhance
EVs association with biomarkers of tumor cells, while at the same time reduce off-target effects.
However, it is of noted that PEG dilemma as observed in PEGylated liposomes, including reduced
cellular uptake/endosomal escape, and accelerated blood clearance after repeated administration,
could also occur to PEGylated EVs (Amoozgar and Yeo, 2012; Ishida et al., 2006). Therefore,
despite efficient shielding EVs from immune recognition through post insertion of PEG, it is
important to optimize several decoration parameters, such as grafting density and length of the
PEG chains. Another major consideration is that PEGylation greatly alters the surface composition
and behavior of EVs in vivo, which may lead to a question to what extent PEGylated EVs are
advantageous over synthetic nanocarriers. In this regard, future research should focus more on the
development of alternative functional ligands. For example, introduction of “self” proteins, such
as CD47, CD55, and CD59 to EV surface might help to protect them from phagocytotic clearance
and prolong circulation time (Clayton et al., 2003; Long and Beatty, 2013). In a recent study, EVs
were engineered to overexpress CD47, which interacted with SIRPα to produce a “don’t eat me”
signal in phagocytes, thus permitting EVs with a superior escape from phagocytosis by the MPS.
As a result, KRAS siRNA loaded EVs functionalized with CD47 suppressed multiple mouse
models of pancreatic cancer and significantly enhanced overall survival (Kamerkar et al., 2017).
4. 2. Modified EVs for improved targeting capability
Targeting delivery of drugs has been much sought to minimize off-target toxicity, reduce dose,
and enhance therapeutic efficacy. However, recent biodistribution studies revealed that after
systemic administration, naturally secreted EVs with no modifications accumulated predominantly
in MPS associated organs, such as the liver, spleen, and lung, and very few of EVs could be
delivered to target issues (Morishita et al., 2015; Ohno et al., 2013; Tian et al., 2014; Wiklander et
al., 2015). Such an unfavorable distribution characteristic may induce severe off-target effects and
limit the therapeutic application of unmodified EVs. Actually, EVs derived from most cells lack
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efficient targeting of specific cell types, especially after systemic administration, and this may
have become one of the most challenging concern for EV-mediate drug delivery (Sullivan et al.,
2017). Therefore, the targeting properties of EVs need to be improved before generic adoption of
EVs as a competitive therapeutic delivery vehicle can be considered.
EVs may be endowed with improved targeting properties by displaying cell-specific targeting
ligands on their surface or packaging exogenous targeting materials. Some frequently used
homing ligands include peptides, such as RVG targeting acetylcholine receptors on neurons (Yang
et al., 2017a), αν-integrin-specific internalizing arginine-glycine-asparagine (iRGD) peptide
(Sugahara et al., 2009), GE11 peptide binding specifically to EGFR (Klutz et al., 2011), and
fragment of Interleukin 3 (IL3) targeting IL3 receptor (Nievergall et al., 2014), proteins like
nanobodies (Kooijmans et al., 2018), specific targeting molecules such as folate (FA) (Zhang et al.,
2017), and iron oxide (Qi et al., 2016) (Table 3). For peptide targeting, a widely used strategy is to
insert the coding sequence of a targeting peptide in-frame in-between the coding sequences of
signal peptides and EV associated membrane proteins (Alvarez-Erviti et al., 2011; Hartman et al.,
2011). An early demonstration was reported by Alvarez-Erviti and colleagues, who equipped EVs
with Lamp2b-RVG peptide through genetic engineering of the donor cells with plasmid construct.
RVG-modified EVs efficiently delivered siRNA across the BBB to target acetylcholine receptor in
the brain, and silenced a gene of relevance to Alzheimer's disease (AD) (Alvarez-Erviti et al.,
2011). Upon the first description of engineered EVs for enhanced targeting capacity, many studies
have assessed the potential of targeting delivery via peptide-modified EVs. RVG equipped EVs
were also applied to shuttle opioid receptor mu siRNA across the BBB targeting neurons in mouse
brain (Liu et al., 2015). Particularly, the targeting capacity of RVG-modified EVs was quantified
to be two-fold greater in regard to the accumulation in the brain than non-targeted EVs (Liu et al.,
2015). Through the same approach, targeting peptide iRGD was successfully introduced to EVs
derived from immature denderitic cells to target breast cancer (Tian et al., 2014). Ohno et al.
provided one of the earliest proof-of-concept studies, where EV producing cells were engineered
to express the transmembrane domain of PDGFR fused to the GE11 peptide, a synthetic peptide
that binds specifically to EGFR. GE11 modified EVs could efficiently deliver let-7a miRNA to
EGFR-expressing xenograft beast cancer tissue in mice, leading to a marked inhibition in tumor
growth (Ohno et al., 2013). Recently, EV producing cells were engineered to express Lamp2b
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fused to a fragment of IL3. EVs equipped with IL3-Lamp2B loaded with Imatinib or with
BCR-ABL siRNA, were able to target Chronic Myeloid Leukemia (CML) cells and suppressed
cancer cell growth both in vitro and in vivo (Bellavia et al., 2017). More recently, Tian et al.
conjugated an integrin αvβ3-specific c(RGDyK) peptide onto EV surface via click chemistry. After
intravenous injection, c(RGDyK)-conjugated EVs could efficiently target the lesion region of
ischemic brain in mice, resulting in a significant suppression of the inflammatory response and
cellular apoptosis in the lesion region (Tian et al., 2018). Taken together, there are high
expectations that further development of peptide-modified EVs may help to usher in efficient
EV-based targeting delivery paradigms.
Apart from peptides, incorporating targeting proteins, such as nanobodies and SIRPα, to EV
surface has also been demonstrated as an attractive targeting strategy (Bryniarski et al., 2013; Koh
et al., 2017; Kooijmans et al., 2016a). Recently, Kooijmans et al. transfected EV producing cells
with vectors encoding EGFR nanobodies fused to GPI anchor signal peptides to target
EGFR-overexpressing tumor cells. Display of GPI-linked EGFR nanobodies on vesicle surface
significantly improved EV binding to tumor cells, which was dependent on EGFR density
(Kooijmans et al., 2016a). Alternatively, EGFR nanobody conjugated to PEG was introduced to
EV membrane via post-insertion mechanism, which greatly enhanced EVs binding to EGFR-
overexpressing tumor cells (Kooijmans et al., 2016b). More recently, Koh et al. generated a rather
sophisticated fusion protein containing SIRPα on top of the PDGFR. By applying PDGFR as a
pedestal, SIRPα was expressed on the surface of resulting EVs. SIRPα functionalized EVs
interfered CD47-SIRPa interaction between CD47-overexpressing cancer cells and bone marrow
derived macrophages, leading to enhanced tumor phagocytosis and significant inhibition of tumor
growth. The result manifested that modified EVs targeting to CD47-overexpressing cancer cells
may represent a versatile tool for cancer immunotherapy (Koh et al., 2017).
Additionally, EV surface can be modified with aptamers for increased targeting capacity (Pi
et al., 2018; Wan et al., 2018). Aptamers are oligonucleotide or peptide molecules that have high
affinity and specificity for their targets, and are commonly viewed as alternatives to antibodies for
targeting delivery (Chu et al., 2006). EVs functionalized with necleolin-specific AS1411 aptamer,
produced by simple extrusion of targeting ligands conjugated cells, exhibited enhanced targeting
capacity, improved therapeutic efficacy, and low systemic toxicity in vivo. In a follow-up study,
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EVs were effectively equipped with prostate-specific membrane antigen aptamer and EGFR
aptamer by simply incubating cholesterol-conjugated RNA aptamers with EVs. Both
reprogrammed-EVs displayed enhanced cancer-cell-specific targeting and efficient suppression of
tumor growth in prostate cancer xenograft and orthotopic breast cancer models, respectively (Pi et
al., 2018) .
An alternative decoration approach to enhance EVs’ tissue targetability is to modify them
with specific chemical compounds. For example, FA and other vitamin receptors are
overexpressed in many cancers of epithelial origin (e.g. colorectal cancer), and have been widely
employed to achieve tumor targeting (Munagala et al., 2016; Zhang et al., 2017). Recently,
Munagala et al. confirmed the feasibility of using drug loaded milk EVs functionalized with FA
for enhanced anti-tumor activity. FA-tagged EV formulations loaded with WFA exhibited
significant higher growth inhibition (74%) than unmodified EV formulations (50%) in mice
bearing human lung cancer (A549) xenografts. Furthermore, such a higher anti-tumor efficacy was
achieved by oral administration, suggesting the potential of effective oral anti-tumor therapy via
functionalized EV-based nanodelivery systems (Munagala et al., 2016). In another study, EVs
were modified with cationized pullulan, a polysaccharide with the ability to target hepatocyte
asialoglycoprotein receptors, which demonstrated not only higher targeting capacity, but also
enhanced therapeutic effect on liver injury (Tamura et al., 2017). Additionally, modification of
Paclitaxel-loaded EVs with AA-PEG moiety, a ligand with high affinity for sigma receptor
(Banerjee et al., 2004), led to greater accumulation of paclitaxel to lung cancer cells that
overexpressed with sigma receptor (Kim et al., 2018). Taken together, surface modification of EVs
may serve as a promising approach to enhance the targeting ability of these vesicles to desired
tissues and improve therapeutic efficacy.
Other than ligand modification, tissue-specific delivery may also be achieved by loading EVs
with magnetic nanoparticles. In a proof-of-concept study by Silva et al., macrophages were
encapsulated with therapeutic agents together with iron oxide nanoparticles. Consequently, EVs
loaded with therapeutics and magnetic nanoparticles were harvested. Magnetic EVs showed
enhanced targeting capacity and greater antitumor effects (Silva et al., 2015). More recently, Qi et
al. suggested superparamagnetic magnetite colloidal nanocrystal clusters (SMCNCs) was also
promising to endow EVs with magnetic-targeting properties. SMCN modified EVs were obtained
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by incubating mouse serum with transferring-conjugated SMCNCs and harvesting EVs with
superparamagnetic characteristics via magnetic separation. By applying external magnetic field,
SMCN-positive EVs loaded doxorubicin displayed greater accumulation at tumor sites, and
thereby resulting in greater suppression of tumor growth, compared with doxorubicin alone (Qi et
al., 2016). In another study, EV producing cells were first incubated with biotin-functionalized 1,
2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE)-PEG and FA-functionalized DSPE-PEG.
Then the magnetic and FA-functionalized EVs were obtained by conjugating FA/biotin-EVs with
SA-IONPs and recovered by magnetic separation. Magnetic and FA-functionalized EVs were
endowed with targeting properties and showed significantly enhanced antitumor efficacy both in
vitro and in vivo (Zhang et al., 2017).
4. 3. Modified EVs for enhanced cytoplasmic delivery efficiency
For efficient cytoplasmic delivery of EVs, a deep insight into their uptake mechanisms is
required. Generally, EVs gain entry into cells by either membrane fusion or endocytosis pathways
(Mulcahy et al., 2014). To be specific, there are a variety of endocytic pathways, including
clathrin-dependent endocytosis, and clathrin-independent pathways, such as caveolin-mediated
internalization, macropinocytosis, and lipid raft-mediated uptake (Mulcahy et al., 2014). In
addition, EVs uptake is likely to occur via more than one route, given that failures to completely
abrogate EV internalization upon the treatment with numerous inhibitors were frequently observed
(Barrã¨S et al., 2010; Escrevente et al., 2011; Morelli et al., 2004). For functional delivery of EV
contents into recipient cells, vesicles require in some way to fuse with cell membrane, either direct
at the plasma membrane or fusion with the limited endosomal membranes after cellular uptake
(Stranford and Leonard, 2017). EVs derived from specific cell types are capable of fusing with
target cells due to the expression of specific membrane proteins. For instance, it was reported that
EVs from DCs could bypass the endosomal-lysosomal pathway by directly fusing with the plasma
membrane through their tetraspanin CD9 interacting with surface glycoproteins on the target cells,
thus offering an efficient pathway for cytoplasmic delivery (Montecalvo et al., 2012; Van den
Boorn et al., 2011). However, the cellular uptake of EVs secreted by most cells is accomplished by
endocytosis (Morelli et al., 2004; Thery et al., 2009; Tian et al., 2010), which is considered
unsatisfactory for therapeutic use (Nakase and Futaki, 2015). First, there is a fierce competition
between the exogenously administered EVs and large number of endogenous EVs presented in the
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body fluids (e.g. approximately 3,000,000 EVs per microliter in the blood) (Vlassov et al., 2012).
Besides, the low cellular uptake efficiency of EVs may be partly due to the charge repulsion
existing between EVs with negatively charged cell membranes (Nakase et al., 2015). Furthermore,
when EVs are taken up by recipient cells and trafficked in the endosomal/lysosomal compartments,
EV cargo may suffer from acidification and digestion during the process of endosomal maturation
(Mindell, 2012). As a result, this may reduce the biological activity of EV’s contents before their
release to the cytosol. In fact, inefficient escape from endosomes into the cytosol represents one of
the major obstacles for intracellular targeting of EVs, which is similar to synthetic nanomaterials
(Morishita et al., 2017; Nakase and Futaki, 2015). Therefore, an important consideration for
EV-based delivery vehicles is to endow them with a functionality to breach the endosomal
membrane for the cytoplasmic release of their cargoes.
Although, a number of strategies have been reported to arm EVs with targeting properties,
approaches to enhance the cellular uptake of EVs and promote cytosolic release of their payloads
are relatively limited (Table 4). Nevertheless, several encouraging results have been recently
reported. Owing to the similarities in composition, function, and biogenesis process between EVs
and viruses (Nolte-'t Hoen et al., 2016), it has been suggested that incorporation of viral
components to EV membrane may be a useful tool to improve the intracellular delivery efficiency
of EVs (Koppers-Lalic et al., 2013). For example, by taking advantages of the pH-sensitive
properties of G protein of vascular stomatitis virus (VSV-G), Yang et al. engineered EVs to
express VSV-G on the surface to promote direct membrane fusion with recipient cells under acidic
conditions (e.g. in muscle tissues). As a result, VSV-G modified EVs facilitated direct membrane
fusion with target cells and delivery of biologically active membrane protein glucose transporter-4
directly into the cell membrane of recipient cell (Fig. 3) (Yang et al., 2017c). Of note, in addition
to transfer of membrane protein, VSV-G functionalized EVs may be readily applicable to deliver
encapsulated payloads, such as small chemotherapeutics, RNAs, and proteins, to the cytosol of
target cells, bypassing endocytic trafficking. An attractive strategy to promote endosomal escape
via membrane fusion at low pH is to use a pH-sensitive fusogenic GALA peptide. In a recent study,
Nakase et al. developed an efficient method to enhance the cellular uptake and cytosolic release of
EVs by combining cationic lipids with GALA peptide. Cationic lipids that acted as a “glue” to
facilitate cellular uptake by mitigating charge repulsion between EVs and recipient cells, while
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GALA peptide mediated membrane fusion of endosome and EVs at low pH, and promoted
cytosolic release of encapsulated ribosome-inactivating protein saporin (Fig. 3) (Nakase and
Futaki, 2015). Similarly, display of GALA peptide on the surface of EVs was also employed to
enhance tumor antigen presentation capacity for improved antitumor therapy (Morishita et al.,
2017).
An alternative strategy to improve EVs’ cytoplasmic delivery efficiency is to activate
macropinocytosis. Induction of macropinocytosis is accompanied by actin reorganization, ruffling
of plasma membrane, and engulfment of large volumes of extracellular fluid, which could lead to
the enhancement in cellular EV uptake efficiency (Nakase et al., 2015). With the intrinsic ability
to translocate into most cells both in vitro and in vivo, CPPs have been frequently used to enhance
the cellular uptake of nanoparticles (Tiwari et al., 2014; van Asbeck et al., 2013). Recently,
El-Andaloussi has reported an approach to incorporate CPPs on EV surface via fusion with
Lamp2b protein to enhance cellular translocation of siRNA (Elandaloussi et al., 2012). More
recently, Nakase and coworkers suggested that displaying arginine-rich CPPs on EV surface was
able to induce macropinocytosis and boost effective cellular uptake of EVs (Fig. 3) (Nakase et al.,
2016). Furthermore, the number of arginine residues in the peptide sequences could affect the
cellular EV uptake efficiency. Hexadeca-arginine peptide (R16) modified EVs loaded with saporin
showed effective anticancer activity, possibly due to the higher endosomal membrane perturbation
ability of R16 than that of shorter oligoarginines (Nakase et al., 2017). Still, as the authors
presented, further research is needed to elucidate the scaffold effects on the functionality of CPPs
other than the number of arginine residues (Nakase et al., 2017). Recently, Akishiba developed a
novel endosomolytic peptide (L17E) by introducing one glutamic acid residues into the
hydrophobic face. L17E possessed two distinct characteristics, including preferential disruption of
negatively charged membranes (endosomal membranes) over slightly less negatively charged
plasma membranes, and promotion of cellular uptake by activating macropinocytosis (Akishiba et
al., 2017). The author further demonstrated that L17E functionalized EVs showed effectiveness in
cytoplasmic delivery of EV encapsulated dextran (Akishiba et al., 2017).
5. Conclusion
The discovery that EVs are capable of transferring biomolecules between cells and altering
the recipient cells' phenotype enables them to be attractive candidates for drug delivery vehicles.
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EVs may possess several advantages over conventional drug delivery systems, such as high
biocompatibility and safety, low immunogenicity, and ability to traverse biological barriers.
However, the rapid clearance of unmodified exogenously administered EVs highlights the need to
protect them from the recognition by MPS and protract their circulation half-life. Besides, in order
to achieve targeted delivery, their intrinsic targeting capacity should be further tuned. Furthermore,
the efficiency of EVs to overcome biological barriers for reaching intracellular targets, still
remains unsatisfactory. In this context, for therapeutic applications, EVs need to be engineered to
prolong their circulation time, improve targeting properties, and to enhance cytoplasmic delivery
efficiency. Considering that multiple EVs’ biological interaction occurs through surface interaction,
surface engineering is fundamental for the development of advanced EV-based nanodelivery
systems. “Cloacking” EVs with hydrophilic moieties, such as PEG derivatives, has been proven to
confer these vesicles with stealth properties, allowing them to evade clearance by the immune
system. The incorporation of targeting ligands to EVs imparts them with enhanced targeting
properties, enabling a greater accumulation at target site and reducing off-targets effects on
healthy tissues. Display of VSV-G, GALA or other functional ligands on EV surface, has been
demonstrated to be attractive tools to boost the cellular uptake and cytosolic delivery of their
payloads. Together, functionalized EVs, offering the tantalizing prospect of extending the delivery
capability of native vesicles, may represent the next generation of therapeutic nanodelivery
vehicles that combines the biological features of EVs, such as high biocompatibility and low
immunogenicity, with the enhanced delivery properties of functional moieties. For EV
modification, “cell engineering” enables display of ligands with predictable orientation on EV
surface, however, may possess complexity, inefficiency, and long stability issues, (Richards et al.,
2016). While direct “EV engineering” allows for a controllable and efficient incorporation of
various ligands, but pose possible risk of damaging EVs’ structural and biological integrity. As
each modification techniques has certain benefits and drawbacks, when selecting a modification
approach, it is essential to consider the complexity of the modification systems, the properties of
functional ligands, and downstream applications (Armstrong et al., 2017). To this end, there is a
continuous need to develop novel modification strategies to efficiently maximize the potential of
modified EVs in therapeutic delivery. As an example, liposome-based cellular engineering has
been proven to be capable of introducing multiple ligands of divergent functionality (Lee et al.,
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2016). This is quite fascinating as EVs could be simultaneously functionalized with
immune-evading, targeting, and uptake-enhancing ligands to prolong their circulation half-life,
targeting capacity, and cytoplasmic delivery efficiency.
However, several challenges remain to reach maximum potential of modified EVs in clinic.
First, it warrants thorough investigation dedicated towards assessing the potential effects of
modification techniques on the biological functionality of EVs. This is because manipulation of
EV surface may alter the proper orientation of EVs’ membrane proteins, and expose certain
antigens that could be recognize by the immune system (Mout et al., 2012). This in turn can be a
potential source of immunogenic reactions that may cause immune responses on potential patients,
especially during chronic treatments. Modification process also bares the risk of damaging the
functionality of ligands. In this circumstance, a crucial concern is to distinguish between cellular
uptake and functional delivery. As when the functional ligands on vesicle surface are inactivated,
EVs can still be internalized by recipient cells via nonspecific endocytosis. However, inactivated
ligands would be unable to trigger specific signal cascades. For example, EVs may be unable to
fuse with target cells and release their payloads to the desired compartment of recipient cell when
the ligands on their surface are inactivated (Hung and Leonard, 2016). Therefore, the first step for
modified EVs to reach clinical use is the assessment of their functionality and potential
immunogenicity. Additionally, effective modification of EVs and harnessing them for therapeutic
delivery highlights the need for gaining deeper insight into EV biology. EVs are nonliving entities,
which may benefit modifications under harsher modification conditions compared with cells.
However, the significant smaller particle size and more rigid membrane of EVs as compared with
cells would possibly make it more complex for surface engineering (Parolini et al., 2009). Hence,
addressing our knowledge gaps is critical for empowering continued innovation for developing
novel modification techniques. Lastly, exploring more functional moieties may considerably assist
in improving the delivery properties and broadening the therapeutic applications of EVs. Ideal
candidates may possess several features, such as competent functionality, small molecular weight,
high chemical and thermal stability, and facile selection and production protocols (Revets et al.,
2005). With the use of these moieties, it would, therefore, be possible to achieve higher circulation
stability, greater tissue specificity, and improved delivery efficiency of EVs following systemic
administration, which may enhance the bioavailability and therapeutic efficacy of EV payloads.
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Together, resolution of these challenges through energetic collaboration of scientists from different
disciplines could minimize the future bottlenecks in the development of novel functionalization
strategies and boost these functionalized nanovesicles towards efficient and safe drug delivery in
clinic.
Conflict of interest:
The authors declare no conflict of interests relevant to this work.
Acknowledgements:
The authors are grateful for the financial support from the National Natural Science
Foundation of China (Grant No. 81373335).
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Figure and Table captions:
Fig. 1. Schematic illustration of EV modification techniques. Functionalized EVs can be
generated by using “Cell engineering”, wherein EV producing cells are genetically manipulated.
Alternatively, EVs can be directly functionalized through “EV engineering”.
Fig. 2. Schematic representation of functional ligands with divergent functions that can be
introduced to EVs for improved delivery properties. EVs can be endowed with prolonged
circulation time, increased targeting capacity, and enhanced cytoplasmic delivery efficiency by
incorporation of PEG moieties (e.g., VEGF nanobody-PEG and AA-PEG), targeting ligands (e.g.,
targeting peptides, nanobodies, and SIRPα), and functional proteins or peptides (e.g., VSV-G,
GALA, and CPPs).
Fig. 3. Schematic depiction of functionalized EVs to achieve efficient cytoplasmic delivery of
their payloads. Decoration of EVs in PEG corona (e.g., EGFR-nanobody-PEG) enables them to
escape from rapid phagocytosis by the MPS and permit long circulation half-life. Modification of
EVs with targeting ligands (e.g., GE11) allows them to bind target cells via receptor-ligand
interaction. Furthermore, EVs equipped with viral proteins (e.g., VSV-G) allow for direct
cytoplasmic delivery of their payloads via membrane fusion. Arginine-rich CPP-modifed EVs can
actively induce macropinocytosis for efficient cellular uptake. After cellular uptake via
receptor-mediated endocytosis or macropinocytosis, EVs containing fusogenic ligands (e.g.
GALA) can efficiently escape from endosome for cargo release to the cytoplasm of recipient cell
via backfusion with endosome membrane.
Table 1. Overview of advantages and disadvantages of EV modification techniques.
Table 2. Ligands used to endow EVs with prolonged circulation time.
Table 3. Ligands used to equip EVs with increased targeting capacity.
Table 4. Ligands used to arm EVs with enhanced cytoplasmic delivery efficiency.
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Table 1
Overview of advantages and disadvantages of EV modification techniques.
Modification
strategies
Advantages Disadvantages Ref.
Cell engineering
Fusion with EV
membrane
proteins
Incorporation of EVs with
preserved direction and
function
Inefficient, complex, and
time-consuming;
Variable performance;
Possible degradation of
fused ligands during EV
biogenesis;
Possibly compromise the
functionality of EVs’
membrane proteins;
May not be readily
applicable to all ligands
(Zhao et al.,
2016)
(Alvarez-Erviti
et al., 2011)
(Hung and
Leonard, 2015)
(Armstrong et
al., 2017)
Fusion with GPI No impairment of
structural and functional
integrity of EVs’
membrane proteins;
Enrichment of functional
ligands compared with
parent cells;
Applicable to multiple
ligands
Inefficient, complex, and
time-consuming protocol;
Possible degradation of
fused ligands in the
endosome during EV
biogenesis;
May not be readily
applicable to all ligands
(Kooijmans et
al., 2016a)
(Kim and
Eberwine,
2010)
Liposome-based
cellular
engineering
Efficient and controlled
protocols;
Without modification of
EVs membrane proteins;
Allows for simultaneous
incorporation of EVs with
multiple functional
ligands;
Possibly applicable to
multiple ligands
Possibly complex and
time-consuming
(Lee et al.,
2016)
Mechanical
extrusion
Rapid and easy to
perform;
Economic production
process
Likely to alter the structure
and orientation of ligands
and EVs’ membrane proteins
during the extrusion process
Unknown immunogenicity
(Wan et al.,
2018)
Packaging
exogenous
No modification of EVs’
membrane proteins;
Difficult to target deep
tissues in vivo;
(Silva et al.,
2013)
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materials Applicable to a wide array
of EVs and ligands
Potential toxic issues of
exogenous materials
(Silva et al.,
2015)
EV engineering
Chemical
conjugation
Rapid and easy to
perform;
Controllable and scalable
protocols;
Compatible with some
chemical reagents and
reaction conditions that
can not be used in “Cell
engineering”;
Have general applicability
to a wide range of ligands;
Allow for incorporation of
high ligand density
Excessive conditions may
impair membrane integrity
and denature the
functionality of EVs’
membrane proteins;
Induce potential
immunogenicity and toxicity;
Requirement of complex
purification steps
(Smyth et al.,
2014)
(Tian et al.,
2018)
Post insertion Fast and simple
manipulation;
Mild modification
condition;
Have general applicability
to multiple EVs and ligand
types
Likely low efficiency of ligand
incorporation;
Unknown ligand
incorporation stability
(Kooijmans et
al., 2016b)
(Kim et al.,
2018)
Electrostatic
interaction
Fast and simple
manipulation;
Mild modification
condition;
Have general applicability
to multiple EVs and ligand
types
Possibly low efficiency of
ligand incorporation;
Unknown ligand
incorporation stability
(Tamura et al.,
2017)
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Table 2
Ligands used to endow EVs with prolonged circulation time.
Ligands Modification
approach
Origin of EVs Outcome Ref.
EGFR-nanobody-PEG Post insertion Human
epidermoid
carcinoma cells
Prolonged
circulation
time and
improved cell
specificity
(Kooijmans et
al., 2016b)
AA-PEG Post insertion Macrophages Prolonged
circulation
time and
improved cell
specificity
(Kim et al.,
2018)
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Table 3
Ligands used to equip EVs with increased targeting capacity.
Targeting ligands Modificati
on
approach
Origin of
EVs
Target Outcome Ref.
RVG peptide Fusion
with
Lamp2b
imDCs Acetylcholine
receptor
Greater
accumulation
in the brain
and specific
gene
knockdown
(Alvarez-Er
viti et al.,
2011)
(Liu et al.,
2015)
iRGD peptide Fusion
with
Lamp2b
imDCs αν-integrin Significant
inhibition of
breast cancer
without overt
toxicity
(Tian et al.,
2014)
GE11 peptide Fusion
with
PDGFR
HEK293
cells
EGFR Marked
suppression of
breast cancer
in mice
(Ohno et
al., 2013)
IL3 peptide Fusion
with
Lamp2b
HEK293T
cells
IL3 receptors
overexpressed
on CML
Significant
regression of
cancer cell
growth
(Bellavia
et al.,
2017)
c(RGDyK) Click
chemistry
MSCs integrin αvβ3 Efficiently
suppression of
inflammarory
response in
lesion region
(Tian et al.,
2018)
EGFR nanobody Fusion
with GPI
Neuro2A
cells
EGFR
overexpressed
tumor cells
Increased
tumor
targeting
capacity
(Kooijmans
et al.,
2016a)
EGFR-nanobody-PE
G
Post
insertion
Neuro2A
cells
EGFR
overexpressed
tumor cells
Improved
targeting
capacity and
extended
circulation
time
(Kooijmans
et al.,
2016b)
SIRPα Fusion
with
PDGFR
HEK293T
cells
CD47 Remarkably
augmented
tumor
phagocytosis
(Koh et al.,
2017)
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and anti-tumor
T cell
responses
Lipid-conjugated
AS1411 aptamer
Hydropho
bic
insertion
DCs Necleolin Significantly
improved
targeting
capacity and
therapeutic
efficacy
(Wan et al.,
2018)
Cholesterol-conjug
ated EGFR aptamer
Post
insertion
HEK293T
cells
EGFR Significant
inhibition of
orthotopic
breast cancer
(Pi et al.,
2018)
FA Cell
engineerin
g
Bovine
milk;
Macrophag
es
FA receptor Improved
antitumor
efficacy
(Munagala
et al.,
2016)
(Zhang et
al., 2017)
Cationized pullulan Electrostat
ic
interaction
MSCs Hepatocyte
asialoglycoprot
ein receptors
Enhanced
anti-inflammat
ory effect in
liver injury
(Tamura et
al., 2017)
AA-PEG Post
insertion
Macrophag
es
Sigma receptor Greater
targeting
capacity and
extended
circulation
time
(Kim et al.,
2018)
Iron oxide
nanoparticles
Cell
engineerin
g
Macrophag
es
- Enhanced
anti-tumor
effect
(Silva et al.,
2015 )
SMCNCs Cell
engineerin
g
Blood - Increased
anti-tumor
effect
(Qi et al.,
2016)
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Table 4
Ligands used to arm EVs with enhanced cytoplasmic delivery efficiency.
Ligands Modification
approach
Origin of EVs Outcome Ref.
VSV-G protein Cell engineering HEK293T cells Mediating membrane
fusion for cargo delivery
directing recipient cell
membrane
(Yang et al.,
2017c )
GALA peptide
and cationic
lipids
EV engineering;
electrostatic
interaction
Hela cells Efficient cellular uptake
and cytoplasmic release
(Nakase et
al., 2015)
GALA peptide Cell
engineering;
chemical
conjugation
Murine
melanoma
B16BL6 tumor
cells
Efficient cytosolic delivery
and enhanced tumor
antigen presentation
(Morishita
et al., 2017)
Arginine-rich
CPPs (R8)
Chemical
conjugation
Hela cells Activation of
macropinocytosis and
increased cellular uptake
(Nakase et
al., 2016)
Arginine-rich
CPPs (R4, R8,
R12, R16)
Chemical
conjugation
Hela cells R16 CPPs achieved
maximum cellular uptake
and showed effective
anticancer activity
(Nakase et
al., 2017)
L17E peptide Post insertion Hela cells Perturbation of endosomal
membrane and efficient
cytoplasmic delivery
(Akishiba et
al., 2017)
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Graphics Abstract
Figure 1
Figure 2
Figure 3
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