Advanced Drug Delivery Reviews 106 (2016) 148156

Contents lists available at ScienceDirect

Advanced Drug Delivery Reviews

journal homepage: www.elsevier.com/locate/addr

Extracellular vesicles for drug delivery

Pieter Vader a,, Emma A. Mol b, Gerard Pasterkamp a,b, Raymond M. Schiffelers a,
a
Department of Clinical Chemistry and Hematology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands
b
Department of Experimental Cardiology, University Medical Center Utrecht, the Netherlands, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands

a r t i c l e i n f o a b s t r a c t

Article history: Extracellular vesicles (EVs) are cell-derived membrane vesicles, and represent an endogenous mechanism for in-
Received 7 January 2016 tercellular communication. Since the discovery that EVs are capable of functionally transferring biological infor-
Received in revised form 11 February 2016 mation, the potential use of EVs as drug delivery vehicles has gained considerable scientic interest. EVs may
Accepted 17 February 2016
have multiple advantages over currently available drug delivery vehicles, such as their ability to overcome natu-
Available online 27 February 2016
ral barriers, their intrinsic cell targeting properties, and stability in the circulation. However, therapeutic applica-
Keywords:
tions of EVs as drug delivery systems have been limited due to a lack of methods for scalable EV isolation and
Extracellular vesicles efcient drug loading. Furthermore, in order to achieve targeted drug delivery, their intrinsic cell targeting prop-
exosomes erties should be tuned through EV engineering. Here, we review and discuss recent progress and remaining chal-
microvesicles lenges in the development of EVs as drug delivery vehicles.
drug delivery 2016 Elsevier B.V. All rights reserved.
isolation
biodistribution
targeting
nanomedicine

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
2. Isolation of extracellular vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
3. Loading of extracellular vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
3.1. Loading of cells before extracellular vesicle isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
3.2. Loading of extracellular vesicles after isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
4. Biodistribution and targeting of extracellular vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
4.1. Circulation time and biodistribution of extracellular vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
4.2. Interactions of extracellular vesicles with the immune system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
4.3. Specic targeting of extracellular vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
5. Therapeutic effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
6. Extracellular vesicle-mimetic nanovesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
Conict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

This review is part of the Advanced Drug Delivery Reviews theme issue on Biologically-
inspired drug delivery systems.
Corresponding authors at: Department of Clinical Chemistry and Hematology ,
University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands.
E-mail addresses: pvader@umcutrecht.nl (P. Vader), r.schiffelers@umcutrecht.nl
(R.M. Schiffelers).
1. Introduction
Extracellular vesicles (EVs) are nano-sized membrane vesicles,
released by many, if not all, cell types. EV release has been found to
occur in many unicellular- as well as multicellular organisms, suggest-
ing that it represents an evolutionary-conserved process. Mammalian

http://dx.doi.org/10.1016/j.addr.2016.02.006
0169-409X/ 2016 Elsevier B.V. All rights reserved.
 P. Vader et al. / Advanced Drug Delivery Reviews 106 (2016) 148156
cells can release distinct types of EVs, including exosomes, microvesicles,
and apoptotic bodies. This classication is based on their intracellular or-
igin. Exosomes are thought to be the smallest vesicle type, with sizes
ranging from 40 to 100 nm. They originate from intraluminal budding
of multivesicular endosomes (MVE) and are released upon fusion of
MVEs with the plasma membrane. In contrast, microvesicles (also
referred to as ectosomes) are more heterogeneous in size and can
be larger (50 nm-1 m), and are formed through direct budding
from the plasma membrane. When cells are compelled to undergo
apoptosis, a heterogeneous population of vesicles (50 nm-5 m) is
released, which are named apoptotic bodies [1]. Despite these differ-
ences in origin, no uniform EV nomenclature exists as of yet, due to
the overlap in vesicle sizes and the absence of subtype-specic
markers. As a result, it remains difcult (if not impossible) to purify
and thereby distinguish between vesicle types [2]. In this review,
we therefore refer to all vesicle subtypes as extracellular vesicles
(EVs).
The cargo of EVs comprises small and long, coding and non-coding
RNAs (e.g. mRNA, miRNA, lncRNA), lipids and proteins [3,4]. Initially,
EVs were thought to act as garbage bags, with a main function in
discard of cellular waste. Over the last decade, however, scientic
interest in EVs has rapidly increased, after it was shown that biolog-
ical information packaged in EVs could be transferred between cells,
and alter the recipient cells phenotype. Cells can package a distinct
set of biomolecules into EVs via endogenous sorting mechanisms,
and release EVs constitutively or after stimulation [5,6]. EVs may
subsequently be internalized by target recipient cells, resulting in
transfer of mRNAs and miRNAs, which can result in production or
silencing of target proteins, respectively [79], and of proteins, in-
cluding membrane proteins [10,11]. EVs can be isolated from bodily
uids, including blood, urine, cerebrospinal uid and saliva [1214].
As their content reects the status of the donor cell, EVs may be ap-
plied in diagnostics, either as pathological biomarkers or to follow
treatment efcacy (reviewed in [15,16]). In addition, it has become
clear that, through their important role in intercellular communica-
tion, EVs affect various processes involved in health and disease. The
discovery that EVs make up a natural mechanism for information
transfer between cells has stimulated interest into their potential
use as a new drug delivery platform.
Despite considerable research in the last 50 years, the clinical
translation of conventional drug delivery platforms has been limited.
The efciency of these platforms to overcome barriers in macromol-
ecule drug transport, such as reaching the target tissue and engaging
intracellular targets, is still unsatisfactory [17]. In addition, concerns
related to immunogenicity and toxicity of non-natural delivery
systems remain. EVs on the other hand seem to have many features
of an ideal carrier system. The EV cargo is naturally protected from
degradation in the circulation [18]. EVs seem to possess intrinsic
cell targeting properties, and are able to overcome natural barriers
such as the blood-brain barrier [19,20]. Furthermore, it is likely
that EVs utilize endogenous mechanisms for uptake, intracellular
trafcking and subsequent delivery of their content in recipient
cells [21]. Importantly, EVs may be nearly non-immunogenic when
used autologously. Moreover, several clinical trials using EVs for im-
munotherapy have already demonstrated the safety of EV adminis-
tration in humans [2224].
Although EVs hold immense promise for therapeutic drug delivery,
clinical applications may critically depend on the development of
scalable EV isolation techniques and approaches for efcient drug
loading. Furthermore, improved methods to modify their in vivo
biodistribution, which is an important determinant of their thera-
peutic effect as it enables more specic drug delivery to target tis-
sues, are required. In this review, we discuss new ndings and
recent improvements on these issues, and summarize recent suc-
cesses in the use of EVs as drug delivery vehicles in animal disease
models.
149
2. Isolation of extracellular vesicles
One of the prerequisites for clinical application of EVs is standardiza-
tion of the isolation process with regards to yield, reproducibility, and
purity [25]. Furthermore, such an isolation method should be scalable
and robust. For large scale EV production, the manufacturing process se-
quentially involves expansion of the donor cell line, collection of the
conditioned medium, and EV purication. Thus far, several methods
for the isolation of EVs have been described. The most commonly used
method is differential ultracentrifugation (UC). EVs are isolated based
on sedimentation at high g-forces. Generally, this method comprises
low speed spins to remove cell debris, followed by high speed spins to
pellet EVs. Sucrose density gradients may subsequently be utilized to
separate vesicle types based on density, and to further purify vesicles
from protein aggregates [26,27]. Disadvantages of UC and sucrose gradi-
ents include the time-consuming protocol, operator-dependent yield,
and possible aggregation and rupture of EVs due to high shear forces
[28]. Isolation of EVs using UC may therefore not be useful for clinical
practice, and novel isolation techniques are topic of intensive investiga-
tion. Two distinct approaches for isolation can be discriminated.
The rst approach, immunoafnity isolation, is based on selective
capture of EVs that bear specic marker proteins on their surface. This
could be important when separation of EV subtypes is required, al-
though it is currently unknown whether specic subtypes are more or
less feasible for drug delivery purposes. Clayton et al. developed an iso-
lation method to capture EVs derived from antigen-presenting cells
(APCs) using antibody-coated magnetic beads. Using antibodies specic
for major histocompatibility complex (MHC) class II, a specic EV sub-
type (i.e. exosomes) could be isolated [29]. A different antibody-based
method to isolate EVs was described by Ashcroft et al., who used a
microuidic circuit to isolate CD41-positive platelet-derived EVs in plas-
ma. EVs were captured with an anti-CD41 antibody-coated mica surface
[30]. This standardized and quick method requires a very low amount of
plasma (10 l) and could be adjusted for other sources of EVs in the fu-
ture. However, the absence of well-dened EV markers may thus lead to
isolation of only specic EV subsets or EVs derived from specic cell
types, and successful elution of intact EVs from the beads might prove
challenging. Furthermore, immunoafnity isolation protocols are not
very attractive for clinical applications, since EVs are isolated at a very
small scale.
The second approach comprises methods that isolate EVs based on
their size. With these methods, considerable efforts have already been
made to improve scalability of EV isolation. Lamparski et al. showed in-
creased recovery of MHC class II-expressing EVs using a combination of
ultraltration and ultracentrifugation of EVs in a 30% sucrose/deuterium
oxide cushion, for the rst time showing that it is possible to isolate EVs
for clinical application [31]. However, a drawback of this method is the
low EV recovery, hindering application in large clinical studies. In an at-
tempt to isolate EVs using ltration techniques only, Heinemann et al.
developed an easy three-step protocol [32]. First, a 0.1 m pore size pol-
yethersulfone (PES) membrane was used to remove dead cells and cell
debris. The sample was then passed through a 500 kDa molecular
weight cut-off modied PES lter to remove free proteins and reduce
large volumes, followed by isolation of EVs using a 0.1 um Track Etch l-
ter. A comparison of sequential ltration versus UC showed that al-
though ltration resulted in a slight reduction of EV yield compared to
UC, it resulted in isolation of a more specic subset of EVs. The major ad-
vantage of this method is the fast and fully automatable protocol, al-
though non-specic EV protein binding to the membranes leading to
lower recovery may present a limitation.
The use of size-exclusion chromatography (SEC) for EV isolation
from plasma was rst described by Boing et al. [33]. Fractionation of
plasma using a sepharose CL-2B column resulted in fast and specic
separation of proteins, HDL, and EVs. SEC isolation also resulted in a
higher recovery of EVs compared to UC. Increased standardization of
EV isolation was reported by Welton et al., who employed a
150 P. Vader et al. / Advanced Drug Delivery Reviews 106 (2016) 148156
commercially available column [34]. Furthermore, collection of only the
EV-containing eluate fractions reduced the isolation time to 10-15 mi-
nutes. Recently, Nordin et al. reported the isolation of EVs from large
volumes of cell culture media by ultraltration combined with size-
exclusion chromatography (SEC) [35]. The sample volume was rst re-
duced by ultraltration using 100 kDa-cutoff spin lters, followed by EV
isolation using SEC. They demonstrated that this method resulted in a
higher EV yield when compared to UC. Furthermore, electron microsco-
py data showed more biophysically intact EVs after SEC isolation, which
had impact on their subsequent biodistribution in vivo. These novel
methods based on ltration and/or SEC could very well be adjusted
and optimized for large-scale clinical application, including for drug de-
livery purposes.
Taken together, these studies also underline that medical application
of EVs still poses signicant challenges. From a pharmaceutical perspec-
tive, the reproducibility of composition and purity is particularly de-
manding. Especially since we know that out of the different classes of
biomolecules associated with EVs, such as nucleic acids, proteins and
lipids, there are hundreds of species of each class that may contribute
to the overall effects. In many instances, the therapeutic molecules
have only partly been identied. This is important as identication of
the active agents in the composition dictates quality control. It is con-
ceivable that, similar as for stem cell therapy, a framework of Good Prac-
tices will need to be established in which also the particular
pharmacokinetic and pharmacodynamic qualities of the EV product
are dened on a case-by-case basis [25]. At the same time, the rst clin-
ical studies have already been conducted demonstrating that clinical ap-
plication of EVs is safe and feasible.
3. Loading of extracellular vesicles
The lipid bilayer membrane serves as a natural barrier to protect EV
cargo from degradation in the bloodstream. However, the presence of
this membrane as well as the EV endogenous content make loading of
exogenous cargo (i.e. therapeutics) into EVs challenging. Nevertheless,
several methods to load EVs have already been described. Here, we pro-
vide an overview of two different loading approaches. One approach is
based on loading of therapeutics into cells from which the EVs are de-
rived, which, using the endogenous loading machinery of the cells,
may result in subsequent EV loading with the drug of interest. The sec-
ond approach involves loading of EVs after their isolation. An overview
of the described loading techniques is schematically depicted in Fig. 1.
3.1. Loading of cells before extracellular vesicle isolation
EVs are natural carriers of small RNAs, including miRNAs. Binding of
miRNAs (or siRNAs) to a target mRNA can cause either mRNA degrada-
tion or repression of protein translation, resulting in target gene silenc-
ing. This process is known as RNA interference [36]. Small RNAs may
also be used therapeutically, for the treatment of diseases in which spe-
cic genes are overactive, such as cancer. However, RNAs typically re-
quire delivery to their site-of-action, which is the cytosol of target
cells. To achieve loading of small RNAs into EVs, transfection-based ap-
proaches have been proposed. Kosaka et al. showed that transfection of
HEK293 and COS-7 cells with a vector for expression of miR-16, -21,
-143, -146a, or -155 resulted in overexpression of these miRNAs in
cells and, as a result of endogenous RNA secretory mechanisms, in
their subsequent active release in EVs [37]. Subsequently, these
miRNA-containing EVs were able to induce gene silencing in recipient
COS-7 cells. Similarly, other reports have shown that using vector-
induced expression of small RNAs in cells, small RNA loading into EVs
can be achieved [38,39]. Alternatively, EV donor cells may be transfected
with small RNAs directly [40,41]. The main disadvantage of these ap-
proaches however is that the level of RNA that is incorporated into
EVs may depend on the RNA species and/or sequence, although the
mechanisms that are involved in RNA sorting into EVs largely remain
to be elucidated [5,6,42].
Although most studies to date have investigated small RNAs as ther-
apeutic cargo to be loaded in EVs, other types of therapeutics may also
be incorporated, including mRNAs, proteins, and small molecules.
Pascucci et al. recently showed successful loading of Paclitaxel (PTX)
into EVs, by incubating mesenchymal stromal cells (MSCs) with a high
dosage of PTX. Subsequently, MSCs produced EVs loaded with PTX,
which were shown to be able to inhibit in vitro tumor growth [43]. In ad-
dition, Tang et al. showed that tumor cells incubated with chemothera-
peutic drugs package these drug into EVs. To stimulate formation of
drug-loaded EVs, cells were irradiated with ultraviolet light to induce
apoptosis [44]. Lee et al. recently demonstrated for a variety of com-
pounds that delivery into cells using fusogenic liposomes also leads to
their loading into EVs released from these cells [45]. Although shown
to be feasible for loading of hydrophobic as well as hydrophilic com-
pounds, it is unclear whether this approach also leads to incorporation
of synthetic lipids from the liposomes into EVs.
3.2. Loading of extracellular vesicles after isolation
The second approach involves loading of EVs after their isolation. For
some hydrophobic drugs, EV loading may be achieved through direct
mixing. This approach was employed by Sun et al., who loaded EVs
with curcumin, an anti-inammatory drug, by simple incubation at
22C for 5 minutes. Retention in EVs increased curcumin in vitro solubil-
ity and stability, and in vivo bioavailability. Intraperitoneal injection of
curcumin-loaded EVs resulted in protection against lipopolysaccharide
(LPS)-induced septic shock in mice, suggesting that this loading ap-
proach may also be feasible for clinical use [46]. Mixing has also been
successfully employed to load PTX into EVs [47]. Others have shown
that mild sonication may further improve PTX incorporation [48].
For hydrophilic compounds, including RNA, a major obstacle is
formed by the lipid bilayer membrane, which restricts passive loading
into EVs. One suggested method to achieve small RNA loading after EV
isolation is electroporation. This approach is based on spontaneous
pore formation in membranes to compensate for changes in voltage
after stimulation with an electrical signal. Alvarez-Erviti et al. were the
rst to report apparent successful siRNA loading into EVs by electropo-
ration. Importantly, subsequent systemic administration of siRNA-
loaded EVs in mice resulted in inhibition of Beta-Site APP-Cleaving En-
zyme 1 mRNA and protein expression in the brain [19]. The use of elec-
troporation for siRNA loading was also investigated by Walhgren et al.
Encapsulation was conrmed by uorescent microscopy, northern blot-
ting, and ow cytometry. EV-mediated delivery of siRNA to monocytes
and lymphocytes was observed, which resulted in inhibition of
mitogen-activated protein kinase 1 in vitro [49]. These studies suggested
that electroporation could lead to encapsulation of small RNAs in EVs,
without affecting EV integrity and function. In contrast, when
Kooijmans et al. investigated the efciency of electroporation for
siRNA loading into EVs, no signicant differences in siRNA retention be-
tween samples electroporated in the presence or absence of EVs could
be measured [50]. Instead, they found that electroporation of siRNA re-
sults in formation of extensive siRNA aggregates even in the absence of
EVs, which could lead to possible overestimation of the siRNA encapsu-
lation efciency. Therefore, caution should be taken with the interpreta-
tion of studies using electroporation for loading in the absence of proper
control experiments. In addition, electroporation might cause aggrega-
tion or fusion of EVs themselves, as has been shown for liposomes
[51]. In an attempt to minimize EV aggregation after electroporation,
Hood et al. investigated the use of membrane stabilizers. The use of tre-
halose pulse media (TPM) was shown to maximize EV colloidal stability,
most likely due to the biologic stabilization properties of trehalose [52].
In an attempt to load EVs with the antioxidant enzyme catalase,
several loading strategies were investigated by Haney et al., includ-
ing simple incubation at room temperature, saponin-mediated
 P. Vader et al. / Advanced Drug Delivery Reviews 106 (2016) 148156 151

Fig. 1. Animated overview of extracellular vesicle loading strategies. Left panel: Loading of EVs via transfection of the donor cell with a vector encoding small RNA or via incubation of
the donor cell with small molecules. Using the endogenous sorting machinery of donor cell, EVs are subsequently loaded with the therapeutics. Right panels: Loading of EVs after their
isolation. Electroporation is based on spontaneous pore formation after stimulation with an electrical signal. Other approaches include incubation at RT, repeated freeze-thaw cycles,
application of ultrasonic frequencies, treatment with a detergent-like molecule (e.g. saponin), or extrusion. Abbreviations: EV = extracellular vesicle, RT = room temperature.

permeabilization, sonication, freeze-thaw cycles, and extrusion [53].
The highest loading efciencies were obtained with sonication, extru-
sion or saponin treatment, although sonication and extrusion of EVs re-
sulted in signicant size increases as observed by dynamic light
scattering, nanoparticle tracking analysis and atomic force microscopy.
EVs loaded by sonication or by saponin treatment protected neurons
against reactive oxygen species production in vitro and, after intranasal
administration, decreased microglial activity compared to injection of
free catalase or PBS in an acute brain inammation mouse model. Al-
though this study demonstrates the feasibility of these strategies for
loading EV with large proteins, it remains to be determined how
possible disruption of the EV integrity during sonication or extrusion
procedures or saponin treatment affects their immunogenicity. A simi-
lar comparison of strategies for loading of hydrophilic porphyrins was
performed by Fuhrmann et al [54]. They observed an eleven-fold in-
crease in loading efcacy after EV loading by saponin treatment com-
pared to loading by passive incubation or extrusion. They also
compared EV loading with hydrophobic and hydrophilic porphyrins
by passive incubation at room temperature or electroporation. EV load-
ing efciency was higher for hydrophobic porphyrins compared to the
hydrophilic compounds. Interestingly, in both cases electroporation in-
creased the encapsulation efciency compared to passive incubation.
152 P. Vader et al. / Advanced Drug Delivery Reviews 106 (2016) 148156
Hydrophobic porphyrin-loaded EVs showed improved cellular uptake
compared to free drug or porphyrins-loaded liposomes. Reduced cell vi-
ability was observed in cancer cells after treatment with porphyrin-
loaded EVs followed by irradiation. Together, these studies demonstrate
that various approaches for EV loading are starting to become available.
Still, selection of the most suitable method for clinical application re-
mains to be determined.
4. Biodistribution and targeting of extracellular vesicles
4.1. Circulation time and biodistribution of extracellular vesicles
In vivo biodistribution and targeting of specic tissues by ther-
apeutics are important factors for their effectiveness. Issues associ-
ated with administration of free drugs, including unfavorable
pharmacokinetics and lack of selectivity, may be overcome by the
use of delivery vehicles. For this reason, Wiklander et al. investi-
gated the tissue distribution of 1,1'-dioctadecyl-3,3,3',3'-
tetramethylindotricarbocyanine iodide (DiR)-labeled EVs from
various cell sources [55]. Twenty-four hours after intravenous
(i.v.) injection in mice, they observed the highest uorescent sig-
nal in the liver, followed by spleen, gastrointestinal tract, and
lungs. Furthermore, they showed that cell source, EV dose, and
route of administration affect EV distribution, which may have im-
plications for the design and feasibility of therapeutic studies using
EVs. Injection of higher EV doses resulted in relatively lower liver
accumulation compared to lower doses, possibly caused by satura-
tion of the mononuclear phagocyte system. Systematic comparison
of the biodistribution of intraperitoneally (i.p), subcutaneously (s.c),
and i.v. injected EVs revealed that i.p. and s.c. injections resulted in re-
duced EV accumulation in liver and spleen and enhanced pancreas
and GI tract accumulation compared to i.v. injections.
With a similar aim of visualizing EV biodistribution and clearance
in vivo, Lai et al. developed an elegant EV imaging reporter system
[56]. EVs were engineered to express a fusion of membrane-bound
Gaussia luciferase (Gluc) reporter and a biotin domain, allowing multi-
modal imaging. In a time frame of 30 minutes to 6 hours after i.v. injec-
tion of Gluc-EVs in athymic nude mice, highest bioluminescent signals
were observed in spleen and liver. Surprisingly, after subsequent
transcardial perfusion with PBS, highest Gluc-EV signals were observed
in kidneys, and perfused spleens showed only minimal signals across all
time points. This difference suggests that the majority of splenic accu-
mulation is caused by EV storage in the spleen rather than uptake by
the tissue itself. Comparable Gluc-EV signals before and after perfusion
were observed for liver and lungs, thus indicating active uptake of EVs in
these organs. Most EVs were cleared after 6 hours, as a result of active
uptake of EVs by cells or hepatic clearance. Imai et al. employed a similar
approach to assess the importance of macrophages in EV clearance.
After systemic administration of Gluc-lactadherin (Gluc-LA)-labeled
EVs derived from melanoma cells, macrophage-depleted mice
displayed higher Gluc-LA EV accumulation in liver, spleen, and lungs,
and delayed EV clearance from blood compared to untreated mice
[57]. This suggests an important role for macrophages in EV clearance.
Instead of following EV biodistribution using labeled EVs, Bala et al.
loaded miRNA-155, an inammatory mediator, into EVs and deter-
mined organ distribution of miRNA-155-loaded EVs after i.v. adminis-
tration in miRNA-155-/- mice. After perfusion, highest miRNA-155
signals were again observed in the liver, followed by adipose tissue
and lungs, whereas the lowest signals were observed in muscle and kid-
neys [58].
Together, these reports demonstrate that systemically administered
EVs (1) have a short half-life and (2) are rapidly taken up by the mono-
nuclear phagocyte system (MPS), particularly in the liver and spleen.
Seemingly, this mechanism of clearance resembles that described for
synthetic nanoparticles, such as liposomes [59]. Moreover, it was re-
cently shown that the EV clearance rate and biodistribution prole is
similar to that of phosphatidylcholine : cholesterol liposomes and lipo-
somes made from the lipid extract of EVs [60]. Further studies should re-
veal whether EV clearance by the MPS is in fact due to their intrinsic
properties, or whether it is a result of the exogenous characteristics of
EVs obtained from in vitro cultures, presence of non-self-labels that
are present on the EV surface, or the quantity of vesicles that are
injected [59]. Interestingly, Kooijmans et al. recently showed that pro-
viding stealth properties to EVs via introduction of polyethylene glycol
(PEGylation) onto the EV surface signicantly increased their circula-
tion time in mice. PEGylation of EVs may therefore be employed to in-
crease accumulation in tumors or at sites of inammation, which are
characterized by localized increased vascular permeability [61].
Besides reports describing biodistribution of EVs after systemic in-
jection, the possibility of EV administration via other routes has also
been investigated. For some therapeutic applications, local drug admin-
istration may actually be preferred, e.g. when targeting the central ner-
vous system. Zhuang et al. showed that intranasal administration of
mouse lymphoma EVs loaded with the anti-inammatory drug
curcumin resulted in their localization to the brain. Curcumin levels
peaked at 1 hour after administration, and a signicant amount could
still be detected after 12 hours. No toxicity or abnormal behavior was
observed in mice upon EV treatment [20]. In addition, intranasal admin-
istration of catalase-loaded EVs resulted in higher EV accumulation in
brain tissue after 4 hours in a mouse model of Parkinsons disease, com-
pared to intravenous injection [53]. Establishing the most efcient EV
administration route for each application may therefore help to improve
therapeutic efcacy.
4.2. Interactions of extracellular vesicles with the immune system
EVs are predominantly submicron particles, which gives them a
large surface area to volume ratio. In addition, because of their particu-
late nature, they can also display repeating molecular patterns on their
surface which can be important in their interactions with the immune
system. Together, these EV qualities constitute a reactive surface for
molecules and cells, especially after injection into the blood stream.
Within plasma, several protein cascades might interact with the ve-
sicular surface. Classically, phosphatidylserine present on the extracel-
lular vesicle surface has been described to recruit several
phosphatidylserine-binding proteins of the coagulation cascade. The
vesicles provide a catalytic surface by bringing the proteins in close
proximity to each other, thereby increasing reaction speed. As a result,
EVs are generally described to promote coagulation and thrombosis.
Another important protein cascade in plasma is the complement
system. Complement has been described to be activated by liposomes
and contribute to their clearance [62]. For EVs, the situation seems to
be more complex. For human antigen presenting cells, for example,
EVs contain CD55 and CD59 membrane regulators of complement.
Both regulators were shown to be important in limiting complement
deposition on the vesicles as well as EV lysis [63]. These results indicate
that EVs carry complement inhibitors that may promote their survival
in the blood stream. At the same time, another study on tumor cell de-
rived EVs showed that these activated both C5a and soluble S protein
bound C5b-9, in a concentration dependent manner [64]. Activation
was dependent on the presence of calcium indicating that activation oc-
curred through the classical or lectin pathway. Interestingly, EVs from
malignant cell lines were more potent in complement activation than
EVs from a non-malignant cell line. Taken together these results indicate
that the source and composition of EVs is important in their net effect on
the complement cascade.
The intrinsic tissue distribution prole of isolated EVs underlines
that a large fraction of EVs interacts with the RES. In particular macro-
phages in liver and spleen are responsible for EV uptake, which could
impact the cellular phenotype. This intrinsic uptake by antigen-
presenting cells has been regarded as a possible route for vaccination.
Because of the resemblance of EV composition to the composition of
 P. Vader et al. / Advanced Drug Delivery Reviews 106 (2016) 148156
the parental cell, EVs can contain antigens, for example oncogenes. As
such, the EVs could represent a platform for tumor vaccination. Al-
though some studies support dendritic cell- and tumor-derived EVs as
anticancer vaccines (reviewed in [65]), recent literature suggests that
EVs are predominantly immunosuppressive [6668]. It is likely that
the condition of the immune system and the state and composition of
the parental cell dictates the outcome of the encounter of immune sys-
tem and EVs. An important pathophysiological role of this interaction is
illustrated in a recent paper by Costa-Silva et al. [69]. Here they showed
that uptake of pancreatic carcinoma-derived exosomes by Kupffer cells
in the liver caused these cells to secrete transforming growth factor-
beta and hepatic stellate cells to produce bronectin. This environment
promoted inux of bone marrow-derived macrophages that secreted
macrophage migration inhibitory factor that was shown to be involved
in liver metastasis of the pancreatic cancer cells. This study underlines
the important consequences of interaction of EVs with components of
the immune system.
4.3. Specic targeting of extracellular vesicles
As described above, administration of exogenous EVs seems to result
mainly in accumulation in the spleen and liver. However, several studies
have shown that it is possible to engineer EVs in such a way, that in-
creased targeting to other tissues or specic cell types is achieved. A
well-studied strategy to equip EVs with targeting properties is via trans-
fection of EV-producing cells to drive expression of targeting moieties
fused with EV membrane proteins. To accomplish increased targeting
to the brain, Alvarez-Erviti et al. engineered dendritic cells (DCs) to ex-
press a fusion protein of an EV membrane anchor, lysosome-associated
membrane glycoprotein 2b (Lamp2b), and brain-specic rabies viral
glycoprotein (RVG), a peptide that binds the acetylcholine receptor.
EVs isolated from these cells were shown to express RVG on their sur-
face. After tail vein injection of siRNA-loaded EVs, knockdown of
BACE1 mRNA and protein was demonstrated in the brains of mice, for
the rst time showing the potential of EVs as targeted drug delivery sys-
tems [19]. Wiklander et al. indeed found increased accumulation of
these RVG-targeted EVs in the brain after systemic administration com-
pared to unmodied EVs [55]. Similarly, Tian et al. showed that expres-
sion of a fusion of Lamp2b and v integrin-specic iRGD peptide
(internalizing RGD peptide CRGDKGPDC) in immature DCs resulted in
expression of these peptides on the surface of EVs. Injection of modied
EVs loaded with the chemotherapeutic drug doxorubicin in tumor-
bearing mice resulted in delivery of doxorubicin to the tumor site [70].
However, Hung & Leonard found that peptides fused to the N-
terminus of Lamp2b are not detected in cells and on EVs, presumably
as a result of degradation by endosomal proteases [71]. Glycosylation
of these peptides protected against proteolytic degradation, which en-
hanced expression on EVs and increased target binding. Subsequent to
the study of Alvarez-Erviti et al., they also investigated the inuence of
engineered glycosylation on the uptake of RVG-displaying EVs by neu-
roblastoma cells. EVs expressing glycosylated RVG peptides fused to
Lamp2b were more efciently internalized in vitro compared to EVs ex-
pressing non-glycosylated peptides. Therefore, glycosylation of
targeting peptides could possibly enhance tissue targeting.
To achieve targeted delivery to epidermal growth factor receptor
(EGFR)-expressing tumors, Ohno et al. displayed EGFR-specic GE11-
peptides fused to transmembrane domains of platelet-derived growth
factor receptor on EVs. They showed that miRNA-loaded EVs derived
from the human embryonic kidney cell line HEK293 modied to ex-
press the GE11-peptide displayed increased accumulation in EGFR-
expressing breast tumors in mice compared to untargeted EVs [72].
Together, these data highlight the possibilities of engineering of EVs to
enhance specic tissue targeting, which could contribute to making
EVs applicable as drug delivery systems.
Besides ligand-mediated targeting, magnetic drug targeting, i.e. en-
hanced drug delivery to a chosen tissue by application of a magnetic
153
eld gradient, represents a non-invasive alternative strategy to enhance
therapeutic efcacy. Delivery of drugs to tissues using magnetic iron
oxide nanoparticles bound to a therapeutic agent was already intro-
duced in the 1970s [73,74]. Recently, Silva et al. provided proof-of-
concept of using the combination of EVs and magnetic targeting for
tissue-specic delivery. Therapeutics and iron oxide nanoparticles
were together incubated with macrophages, which resulted in produc-
tion of EVs loaded with both the therapeutic and magnetic nanoparti-
cles. Magnetic targeting resulted in enhanced as well as spatially
modulated EV and drug uptake by cancer cells in vitro [75]. However,
disadvantages of this method when applied in vivo may include difcul-
ties to target deep tissues in the body, and toxicity issues associated
with the use of iron nanoparticles.
5. Therapeutic effects
Despite the challenges in the isolation, loading and targeting of EVs,
several reports have already demonstrated their potential for thera-
peutic drug delivery in various animal models of disease. The major-
ity of these studies have focussed on cancer therapy. Due to their
nanosize, EVs may achieve passive targeting to tumors via the en-
hanced permeation and retention (EPR) effect [76]. One of the rst
studies that described the use of EVs for drug delivery to tumors
was performed by Mizrak et al. EVs derived from the cell line HEK-
293 were loaded with the suicide gene cytosine deaminase fused to
uracil phosphoribosyltransferase to induce cell death in nerve sheath
tumor cells upon addition of the prodrug 5-uorocytosine. Weekly
intratumoral injections of engineered EVs led to signicant tumor re-
gression in mice after 2 months [77]. Later, Ohno et al. showed that
EGFR- targeted EVs loaded with let-7a miRNA delivered this miRNA
to EGFR-expressing breast tumors upon systemic delivery in
RAG2-/- mice, which signicantly inhibited tumor development
[72]. However, downregulation of let-7a target genes could not be
demonstrated. In addition, Zhang et al. utilized EVs to deliver
siRNA against transforming growth factor 1 (TGF1), a well-
studied target for cancer treatment, to sarcoma cells [78]. Using a
subcutaneous tumor model in mice, they showed that i.v. injection
of TGF-1 siRNA-loaded EVs strongly suppressed TGF-1 expression
and downstream signaling in the tumors, and inhibited primary
tumor growth as well as formation of lung metastases. Interestingly,
siRNA doses used in this study were surprisingly low (0.4 pmol per
injection). This may indicate that EVs were highly efcient in deliv-
ering their cargo to the target cells, or that other EV-mediated effects
contributed to the observed inhibition of tumor growth.
Besides for delivery of small RNA molecules, which critically depend
on intracellular delivery to conduct their silencing function, EVs have
also been employed to deliver chemotherapeutic agents, with the aim
to enhance their efcacy and reduce side effects. For example, Tian
et al. observed signicantly improved suppression of breast tumor
growth after i.v. injection of integrin-targeted, dendritic cell-derived
EVs loaded with the chemotherapeutic drug doxorubicin in mice, com-
pared to free drug. Moreover, doxorubicin was shown to cause less car-
diac damage, the most important dose-limiting side effect of the drug,
when packaged in EVs [70]. Furthermore, PTX-loaded EVs were shown
to be more effective for inhibiting growth of Lewis lung carcinoma me-
tastases than Taxol, a commercially available formulation of PTX [48].
Tang et al. used two different tumor models to show benecial effects
of packaging chemotherapeutic drugs into EVs [44]. Repeated i.p. injec-
tions of cisplatin-loaded EVs improved long-term survival of ovarian
cancer-bearing mice, as compared to cisplatin only. Furthermore, i.v. in-
jection of doxorubicin-loaded EVs delayed growth of established subcu-
taneous hepatocarcinoma. Importantly, these EV treatments did not
adversely affect liver or kidney functions, which are typical side effects
observed after administration of the free drugs. Based on results from
this study (Fig. 2), a phase II clinical trial has been initiated which
aims to evaluate the effect of chemotherapeutic drugs encapsulated in
154 P. Vader et al. / Advanced Drug Delivery Reviews 106 (2016) 148156

Fig. 2. Schematic overview of preclinical studies that formed the basis for the rst clinical trials using extracellular vesicles for drug delivery. Left panel: Loading of the anti-
inammatory agent curcumin into EVs was achieved via simple incubation at 22C. Curcumin-loaded EVs were shown to attenuate autoimmune encephalitis and LPS-induced septic
shock in mice. Right panel: EVs loaded with chemotherapeutic drugs inhibited hepatocarcinoma in mice after tail vein injection. EV loading was achieved by rst incubating tumor
cells with chemotherapeutic drugs. Then, to stimulate formation of drug-loaded EVs, cells were irradiated with ultraviolet light to induce apoptosis. Abbreviations: EV = extracellular
vesicle, LPS = lipopolysaccharide, i.p. = intraperitoneal, UV = ultraviolet.

EVs on malignant ascites and pleural effusion in advanced cancer
patients. Patients are locally injected four times a week, and both
therapeutic as well as side effects are recorded (clinicaltrials.gov,
NCT01854866).
A second ongoing clinical trial (phase I) in which EVs are being eval-
uated as drug delivery systems is based on two studies that showed
benecial effects of encapsulating anti-inammatory compounds into
EVs (Fig. 2). Firstly, Sun et al. showed that EVs as vehicles for curcumin
increase its solubility, stability, and bioavailability. Furthermore,
curcumin-loaded EVs protected mice from lipopolysaccharide (LPS)-
induced septic shock [46]. In a follow-up study, daily intranasal admin-
istrations of curcumin packaged in EVs delayed and attenuated experi-
mental autoimmune encephalomyelitis, an effect not observed after
administration of curcumin alone. Mechanistically, treatment success
was likely caused by increased induction of apoptosis in microglial
cells [20]. The clinical trial aims to study the ability of plant EVs to
deliver curcumin to colon tumors. Outcome measures include
curcumin concentrations in normal and cancerous tissue after oral
administration and safety as well as tolerability (compared to
curcumin alone) as determined by adverse events (clinicaltrials.
gov, NCT01294072).
Besides for cancer, EVs have been proposed as therapeutic delivery
vehicle for the treatment of Parkinsons disease (PD). PD is associated
with reduced levels of several brain enzymes such as catalase, super-
oxide dismutase, and other antioxidants. To increase brain levels of
catalase, Haney et al. intranasally administered catalase-loaded EVs
in a mouse model of PD [53]. Catalase-loaded EVs were shown to re-
duce microglial activation and protect neurons against reactive oxy-
gen species more efciently compared to free catalase. Altogether,
these results, although preliminary, have demonstrated that EVs
are promising candidate drug carriers for the treatment of a variety
of diseases.
6. Extracellular vesicle-mimetic nanovesicles
Although substantial progress has been made in the development of
natural EVs as drug delivery vehicles, the relatively low recovery of EVs
produced by mammalian cells remains an obstacle for large-scale EV
production. For this reason, the possibility of generating EV-mimetic
vesicles with a substantially greater yield has attracted recent attention.
Methods for preparing EV-mimetics are based on obtaining articially
generated nanovesicles from broken cells, which resemble the structur-
al and physical features of EVs. For example, Jang et al. produced drug-
loaded nanovesicles by serial extrusion of monocytes through lters in
the presence of chemotherapeutics, and isolated these vesicles from
free drugs using an OptiPrep density gradient [79]. This production
method resulted in a 100-fold increased vesicle yield compared to isola-
tion of naturally produced EVs. Nanovesicles were shown to express
lymphocyte function-associated antigen-1 (LFA-1) that can bind endo-
thelial cell adhesion molecules (CAMs), overexpressed on activated en-
dothelial cells, such as found in tumors. Importantly, i.v. injection of
these drug-loaded nanovesicles in mice resulted in their accumulation
in tumor tissue and subsequent reduction of tumor growth, without
the toxic side effects present after injection of the free drug. Interesting-
ly, EV-mimetic nanovesicles were found to be similarly effective as EVs
harvested from the same cells. An alternative method to generate EV-
mimetic nanovesicles was proposed by Yoon et al. Living cells were
sliced with microfabricated silicon nitride blades of 500 nm while
owing through a standardized microuidic system. After slicing,
nanovesicles were spontaneously formed through self-assembly of
membrane fragments. Vesicles were shown to be composed of a lipid
bilayer membrane and carry various membrane proteins, intracellular
proteins and RNAs. The addition of polystyrene latex beads during slic-
ing resulted in encapsulation of beads in the nanovesicles, with efcien-
cies up to 30% [80]. These successful examples demonstrate the
 P. Vader et al. / Advanced Drug Delivery Reviews 106 (2016) 148156
potential of these methods to generate nanovesicles for drug delivery
on a large scale. However, whether these cell-derived EV-mimetic vesi-
cles also possess the envisioned advantages of natural EVs, including
low immunogenicity and intrinsic capacity to utilize endogenous mech-
anisms for uptake and intracellular trafcking in recipient cells, is cur-
rently unknown.
7. Conclusions
EVs are natural membrane vesicles involved in intercellular commu-
nication. The ability of EVs to transfer their content to recipient cells via
endogenous uptake mechanisms makes them attractive candidates for
application in drug delivery. However, current obstacles to overcome
before deployment of EVs in large clinical trials are the need for both
scalable EV isolation methods, and more efcient drug loading ap-
proaches for different therapeutics. Especially ultraltration- and SEC-
based EV isolation approaches seem promising for large-scale clinical
application, although further optimizations remain to be implemented.
An additional challenge remains shifting in vivo biodistribution of EVs
from non-specic organ accumulation to accumulation in desired tis-
sues. Although considerable efforts have been made in targeting specic
cell types by engineering EVs to express cell-type specic ligands, one of
the major obstacles remains inefcient drug delivery to target tissues.
Gaining more insight into fundamental EV biology, in particular into
how EVs target specic cell types, and whether different EV subtypes
serve different physiological roles, could therefore contribute to further
improvements in the development of EVs for drug delivery.
Conict of interest statement
RMS is the CSO of Excytex, and on the scientic advisory board of JSR
Micro NV.
Acknowledgements
PV is supported by a VENI Fellowship (# 13667) from the
Netherlands Organisation for Scientic Research (NWO). The work of
EAM is part of the Project SMARTCARE-II of the BioMedical Materials in-
stitute, co-funded by the Dutch Ministry of Economic Affairs, Agricul-
ture and Innovation and the Netherlands CardioVascular Research
Initiative (CVON): the Dutch Heart Foundation, Dutch Federation of
University Medical Centers, the Netherlands Organization for Health
Research and Development, and the Royal Netherlands Academy of
Sciences. The work of RMS on cell-derived membrane vesicles is sup-
ported by a European Research Council starting grant (# 260627)
MINDS in the FP7 ideas program of the European Union.
References
[1] M. Colombo, G. Raposo, C. Thery, Biogenesis, secretion, and intercellular interactions
of exosomes and other extracellular vesicles, Annu. Rev. Cell Dev. Biol. 30 (2014)
255289.
[2] S.J. Gould, G. Raposo, As we wait: coping with an imperfect nomenclature for extra-
cellular vesicles, J. Extracellular Vesicles 2 (2013).
[3] G. Raposo, W. Stoorvogel, Extracellular vesicles: Exosomes, microvesicles, and
friends, J. Cell Biol. 200 (2013) 373383.
[4] S.M. van Dommelen, P. Vader, S. Lakhal, S.A.A. Kooijmans, W.W. van Solinge, M.J.A.
Wood, R.M. Schiffelers, Microvesicles and exosomes: Opportunities for cell-
derived membrane vesicles in drug delivery, J. Control. Release 161 (2012) 635644.
[5] D. Koppers-Lalic, M. Hackenberg, I.V. Bijnsdorp, M.A.J. van Eijndhoven, P. Sadek, D.
Sie, N. Zini, J.M. Middeldorp, B. Ylstra, R.X. de Menezes, T. Wurdinger, G.A. Meijer,
D.M. Pegtel, Nontemplated Nucleotide Additions Distinguish the Small RNA Compo-
sition in Cells from Exosomes, Cell Rep. 8 (2014) 16491658.
[6] M.F. Bolukbasi, A. Mizrak, G.B. Ozdener, S. Madlener, T. Strobel, E.P. Erkan, J.B. Fan,
X.O. Breakeeld, O. Saydam, miR-1289 and "Zipcode"-like Sequence Enrich
mRNAs in Microvesicles, Mol. Ther.Nucleic Acids 1 (2012).
[7] H. Valadi, K. Ekstrom, A. Bossios, M. Sjostrand, J.J. Lee, J.O. Lotvall, Exosome-
mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic ex-
change between cells, Nat. Cell Biol. 9 (2007) 654659.
[8] J. Ratajczak, K. Miekus, M. Kucia, J. Zhang, R. Reca, P. Dvorak, M.Z. Ratajczak, Embry-
onic stem cell-derived microvesicles reprogram hematopoietic progenitors:
155
evidence for horizontal transfer of mRNA and protein delivery, Leukemia 20
(2006) 847856.
[9] D.M. Pegtel, K. Cosmopoulos, D.A. Thorley-Lawson, M.A. van Eijndhoven, E.S.
Hopmans, J.L. Lindenberg, T.D. de Gruijl, T. Wurdinger, J.M. Middeldorp, Functional
delivery of viral miRNAs via exosomes, Proc. Natl. Acad. Sci. U. S. A. 107 (2010)
63286333.
[10] J. Skog, T. Wurdinger, S. van Rijn, D.H. Meijer, L. Gainche, M. Sena-Esteves, W.T.
Curry Jr., B.S. Carter, A.M. Krichevsky, X.O. Breakeeld, Glioblastoma microvesicles
transport RNA and proteins that promote tumour growth and provide diagnostic
biomarkers, Nat. Cell Biol. 10 (2008) 14701476.
[11] K. Al-Nedawi, B. Meehan, J. Micallef, V. Lhotak, L. May, A. Guha, J. Rak, Intercellular
transfer of the oncogenic receptor EGFRvIII by microvesicles derived from tumour
cells, Nat. Cell Biol. 10 (2008) 619624.
[12] M.P. Caby, D. Lankar, C. Vincendeau-Scherrer, G. Raposo, C. Bonnerot, Exosomal-like
vesicles are present in human blood plasma, Int. Immunol. 17 (2005) 879887.
[13] T. Pisitkun, R.F. Shen, M.A. Knepper, Identication and proteomic proling of
exosomes in human urine, Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 1336813373.
[14] Y. Ogawa, Y. Miura, A. Harazono, M. Kanai-Azuma, Y. Akimoto, H. Kawakami, T.
Yamaguchi, T. Toda, T. Endo, M. Tsubuki, R. Yanoshita, Proteomic Analysis of Two
Types of Exosomes in Human Whole Saliva, Biol. Pharm. Bull. 34 (2011) 1323.
[15] L. Urbanelli, S. Buratta, K. Sagini, G. Ferrara, M. Lanni, C. Emiliani, Exosome-based
strategies for Diagnosis and Therapy, Recent patents on CNS drug discovery,
102015 1027.
[16] C. Lasser, Exosomes in diagnostic and therapeutic applications: biomarker, vaccine
and RNA interference delivery vehicle, Expert. Opin. Biol. Ther. 15 (2015) 103117.
[17] Time to deliver, Nat. Biotechnol. 32 (2014) 961.
[18] N.B.Y. Tsui, E.K.O. Ng, Y.M.D. Lo, Stability of endogenous and added RNA in blood
specimens, serum, and plasma, Clin. Chem. 48 (2002) 16471653.
[19] L. Alvarez-Erviti, Y.Q. Seow, H.F. Yin, C. Betts, S. Lakhal, M.J.A. Wood, Delivery of
siRNA to the mouse brain by systemic injection of targeted exosomes, Nat.
Biotechnol. 29 (2011) (341-U179).
[20] X.Y. Zhuang, X.Y. Xiang, W. Grizzle, D.M. Sun, S.Q. Zhang, R.C. Axtell, S.W. Ju, J.Y. Mu,
L.F. Zhang, L. Steinman, D. Miller, H.G. Zhang, Treatment of Brain Inammatory Dis-
eases by Delivering Exosome Encapsulated Anti-inammatory Drugs From the
Nasal Region to the Brain, Mol. Ther. 19 (2011) 17691779.
[21] M.E. Marcus, J.N. Leonard, FedExosomes: Engineering Therapeutic Biological Nano-
particles that Truly Deliver, Pharmaceuticals (Basel) 6 (2013) 659680.
[22] B. Escudier, T. Dorval, N. Chaput, F. Andre, M.P. Caby, S. Novault, C. Flament, C.
Leboulaire, C. Borg, S. Amigorena, C. Boccaccio, C. Bonnerot, O. Dhellin, M.
Movassagh, S. Piperno, C. Robert, V. Serra, N. Valente, J.B. Le Pecq, A. Spatz, O.
Lantz, T. Tursz, E. Angevin, L. Zitvogel, Vaccination of metastatic melanoma patients
with autologous dendritic cell (DC) derived-exosomes: results of therst phase I
clinical trial, J. Transl. Med. 3 (2005) 10.
[23] M.A. Morse, J. Garst, T. Osada, S. Khan, A. Hobeika, T.M. Clay, N. Valente, R.
Shreeniwas, M.A. Sutton, A. Delcayre, D.H. Hsu, J.B. Le Pecq, H.K. Lyerly, A phase I
study of dexosome immunotherapy in patients with advanced non-small cell lung
cancer, J. Transl. Med. 3 (2005) 9.
[24] S. Dai, D. Wei, Z. Wu, X. Zhou, X. Wei, H. Huang, G. Li, Phase I clinical trial of autol-
ogous ascites-derived exosomes combined with GM-CSF for colorectal cancer, Mol.
Ther. 16 (2008) 782790.
[25] T. Lener, M. Gimona, L. Aigner, V. Borger, E. Buza s, G. Camussi, N. Chaput, D.
Chatterjee, F.A. Court, H.A. Del Portillo, L. O'Driscoll, S. Fais, J.M. Falcon-Perez, U.
Felderhoff-Mueser, L. Fraile, Y.S. Gho, A. Gorgens, R.C. Gupta, A. Hendrix, D.M.
Hermann, A.F. Hill, F. Hochberg, P.A. Horn, D. de Kleijn, L. Kordelas, B.W. Kramer,
E.M. Kramer-Albers, S. Laner-Plamberger, S. Laitinen, T. Leonardi, M.J. Lorenowicz,
S.K. Lim, J. Lotvall, C.A. Maguire, A. Marcilla, I. Nazarenko, T. Ochiya, T. Patel, S.
Pedersen, G. Pocsfalvi, S. Pluchino, P. Quesenberry, I.G. Reischl, F.J. Rivera, R.
Sanzenbacher, K. Schallmoser, I. Slaper-Cortenbach, D. Strunk, T. Tonn, P. Vader,
B.W. van Balkom, M. Wauben, S.E. Andaloussi, C. Thery, E. Rohde, B. Giebel, Applying
extracellular vesicles based therapeutics in clinical trials - an ISEV position paper, J.
Extracellular Vesicles 4 (2015) 30087.
[26] C. Thery, S. Amigorena, G. Raposo, A. Clayton, Isolation and characterization of
exosomes from cell culture supernatants and biological uids, Curr. Protoc. Cell
Biol. (2006) 22 (Chapter 3, Unit 3).
[27] M. Aalberts, F.M. van Dissel-Emiliani, N.P. van Adrichem, M. van Wijnen, M.H.
Wauben, T.A. Stout, W. Stoorvogel, Identication of distinct populations of
prostasomes that differentially express prostate stem cell antigen, annexin A1,
and GLIPR2 in humans, Biol. Reprod. 86 (2012) 82.
[28] D.D. Taylor, S. Shah, Methods of isolating extracellular vesicles impact down-stream
analyses of their cargoes, Methods 87 (2015) 310.
[29] A. Clayton, J. Court, H. Navabi, M. Adams, M.D. Mason, J.A. Hobot, G.R. Newman, B.
Jasani, Analysis of antigen presenting cell derived exosomes, based on immuno-
magnetic isolation and ow cytometry, J. Immunol. Methods 247 (2001) 163174.
[30] B.A. Ashcroft, J. de Sonneville, Y. Yuana, S. Osanto, R. Bertina, M.E. Kuil, T.H.
Oosterkamp, Determination of the size distribution of blood microparticles directly
in plasma using atomic force microscopy and microuidics, Biomed. Microdevices
14 (2012) 641649.
[31] H.G. Lamparski, A. Metha-Damani, J.Y. Yao, S. Patel, D.H. Hsu, C. Ruegg, J.B. Le Pecq,
Production and characterization of clinical grade exosomes derived from dendritic
cells, J. Immunol. Methods 270 (2002) 211226.
[32] M.L. Heinemann, M. Ilmer, L.P. Silva, D.H. Hawke, A. Recio, M.A. Vorontsova, E. Alt, J.
Vykoukal, Benchtop isolation and characterization of functional exosomes by se-
quential ltration, J. Chromatogr. A 1371 (2014) 125135.
[33] A.N. Boing, E. van der Pol, A.E. Grootemaat, F.A. Coumans, A. Sturk, R. Nieuwland,
Single-step isolation of extracellular vesicles by size-exclusion chromatography, J.
Extracellular Vesicles 3 (2014).
156 P. Vader et al. / Advanced Drug Delivery Reviews 106 (2016) 148156
[34] J.L. Welton, J.P. Webber, L.A. Botos, M. Jones, A. Clayton, Ready-made chromatogra-
phy columns for extracellular vesicle isolation from plasma, J. Extracellular Vesicles
4 (2015) 27269.
[35] J.Z. Nordin, Y. Lee, P. Vader, I. Mager, H.J. Johansson, W. Heusermann, O.P. Wiklander,
M. Hallbrink, Y. Seow, J.J. Bultema, J. Gilthorpe, T. Davies, P.J. Fairchild, S. Gabrielsson,
N.C. Meisner-Kober, J. Lehtio, C.I. Smith, M.J. Wood, S. El Andaloussi, Ultraltration
with size-exclusion liquid chromatography for high yield isolation of extracellular
vesicles preserving intact biophysical and functional properties, Nanomed.:
Nanotechnol., Biol. Med. 11 (4) (May 2015) 879883.
[36] S.M. Elbashir, J. Harborth, W. Lendeckel, A. Yalcin, K. Weber, T. Tuschl, Duplexes of
21-nucleotide RNAs mediate RNA interference in cultured mammalian cells, Nature
411 (2001) 494498.
[37] N. Kosaka, H. Iguchi, Y. Yoshioka, F. Takeshita, Y. Matsuki, T. Ochiya, Secretory mech-
anisms and intercellular transfer of microRNAs in living cells, J. Biol. Chem. 285
(2010) 1744217452.
[38] N. Kosaka, H. Iguchi, Y. Yoshioka, K. Hagiwara, F. Takeshita, T. Ochiya, Competitive
interactions of cancer cells and normal cells via secretory microRNAs, J. Biol.
Chem. 287 (2012) 13971405.
[39] Q. Pan, V. Ramakrishnaiah, S. Henry, S. Fouraschen, P.E. de Ruiter, J. Kwekkeboom,
H.W. Tilanus, H.L. Janssen, L.J. van der Laan, Hepatic cell-to-cell transmission of
small silencing RNA can extend the therapeutic reach of RNA interference (RNAi),
Gut 61 (2012) 13301339.
[40] Y. Zhang, D. Liu, X. Chen, J. Li, L. Li, Z. Bian, F. Sun, J. Lu, Y. Yin, X. Cai, Q. Sun, K. Wang,
Y. Ba, Q. Wang, D. Wang, J. Yang, P. Liu, T. Xu, Q. Yan, J. Zhang, K. Zen, C.Y. Zhang, Se-
creted monocytic miR-150 enhances targeted endothelial cell migration, Mol. Cell
39 (2010) 133144.
[41] Y. Akao, A. Iio, T. Itoh, S. Noguchi, Y. Itoh, Y. Ohtsuki, T. Naoe, Microvesicle-mediated
RNA molecule delivery system using monocytes/macrophages, Mol. Ther. 19 (2011)
395399.
[42] A.O. Batagov, V.A. Kuznetsov, I.V. Kurochkin, Identication of nucleotide patterns
enriched in secreted RNAs as putative cis-acting elements targeting them to
exosome nano-vesicles, BMC Genomics 12 (Suppl. 3) (2011) S18.
[43] L. Pascucci, V. Cocce, A. Bonomi, D. Ami, P. Ceccarelli, E. Ciusani, L. Vigano, A.
Locatelli, F. Sisto, S.M. Doglia, E. Parati, M.E. Bernardo, M. Muraca, G. Alessandri, G.
Bondiolotti, A. Pessina, Paclitaxel is incorporated by mesenchymal stromal cells
and released in exosomes that inhibit in vitro tumor growth: A new approach for
drug delivery, J. Control. Release 192 (2014) 262270.
[44] K. Tang, Y. Zhang, H.F. Zhang, P.W. Xu, J. Liu, J.W. Ma, M. Lv, D.P. Li, F. Katirai, G.X.
Shen, G.M. Zhang, Z.H. Feng, D.Y. Ye, B. Huang, Delivery of chemotherapeutic
drugs in tumour cell-derived microparticles, Nat. Commun. 3 (2012).
[45] J. Lee, J. Kim, M. Jeong, H. Lee, U. Goh, H. Kim, B. Kim, J.H. Park, Liposome-Based En-
gineering of Cells To Package Hydrophobic Compounds in Membrane Vesicles for
Tumor Penetration, Nano Lett. 15 (2015) 29382944.
[46] D. Sun, X. Zhuang, X. Xiang, Y. Liu, S. Zhang, C. Liu, S. Barnes, W. Grizzle, D. Miller,
H.G. Zhang, A novel nanoparticle drug delivery system: the anti-inammatory activ-
ity of curcumin is enhanced when encapsulated in exosomes, Mol. Ther. 18 (2010)
16061614.
[47] H. Saari, E. Lazaro-Ibanez, T. Viitala, E. Vuorimaa-Laukkanen, P. Siljander, M.
Yliperttula, Microvesicle- and exosome-mediated drug delivery enhances the cyto-
toxicity of Paclitaxel in autologous prostate cancer cells, J. Control. Release 220
(2015) 727737.
[48] M.S. Kim, M.J. Haney, Y. Zhao, V. Mahajan, I. Deygen, N.L. Klyachko, E. Inskoe, A.
Piroyan, M. Sokolsky, O. Okolie, S.D. Hingtgen, A.V. Kabanov, E.V. Batrakova, Devel-
opment of exosome-encapsulated paclitaxel to overcome MDR in cancer cells,
Nanomed.: Nanotechnol., Biol. Med. (Nov. 14 2015), http://dx.doi.org/10.1016/j.
nano.2015.10.012 [Epub ahead of print].
[49] J. Wahlgren, L.K.T. De, M. Brisslert, F. Vaziri Sani, E. Telemo, P. Sunnerhagen, H.
Valadi, Plasma exosomes can deliver exogenous short interfering RNA to monocytes
and lymphocytes, Nucleic Acids Res. 40 (2012) e130.
[50] S.A. Kooijmans, S. Stremersch, K. Braeckmans, S.C. de Smedt, A. Hendrix, M.J. Wood,
R.M. Schiffelers, K. Raemdonck, P. Vader, Electroporation-induced siRNA precipita-
tion obscures the efciency of siRNA loading into extracellular vesicles, J. Control.
Release 172 (2013) 229238.
[51] N.G. Stoicheva, S.W. Hui, Electrofusion of cell-size liposomes, Biochim. Biophys. Acta
1195 (1994) 3138.
[52] J.L. Hood, M.J. Scott, S.A. Wickline, Maximizing exosome colloidal stability following
electroporation, Anal. Biochem. 448 (2014) 4149.
[53] M.J. Haney, N.L. Klyachko, Y. Zhao, R. Gupta, E.G. Plotnikova, Z. He, T. Patel, A.
Piroyan, M. Sokolsky, A.V. Kabanov, E.V. Batrakova, Exosomes as drug delivery vehi-
cles for Parkinson's disease therapy, J. Control. Release 207 (2015) 1830.
[54] G. Fuhrmann, A. Serio, M. Mazo, R. Nair, M.M. Stevens, Active loading into extracel-
lular vesicles signicantly improves the cellular uptake and photodynamic effect of
porphyrins, J. Control. Release 205 (2015) 3544.
[55] O.P. Wiklander, J.Z. Nordin, A. O'Loughlin, Y. Gustafsson, G. Corso, I. Mager, P. Vader,
Y. Lee, H. Sork, Y. Seow, N. Heldring, L. Alvarez-Erviti, C.E. Smith, K. Le Blanc, P.
Macchiarini, P. Jungebluth, M.J. Wood, S.E. Andaloussi, Extracellular vesicle in vivo
biodistribution is determined by cell source, route of administration and targeting,
J. Extracellular Vesicles 4 (2015) 26316.
[56] C.P. Lai, O. Mardini, M. Ericsson, S. Prabhakar, C.A. Maguire, J.W. Chen, B.A. Tannous,
X.O. Breakeeld, Dynamic biodistribution of extracellular vesicles in vivo using a
multimodal imaging reporter, ACS Nano 8 (2014) 483494.
[57] T. Imai, Y. Takahashi, M. Nishikawa, K. Kato, M. Morishita, T. Yamashita, A.
Matsumoto, C. Charoenviriyakul, Y. Takakura, Macrophage-dependent clearance of
systemically administered B16BL6-derived exosomes from the blood circulation in
mice, J. Extracellular Vesicles 4 (2015) 26238.
[58] S. Bala, T. Csak, F. Momen-Heravi, D. Lippai, K. Kodys, D. Catalano, A. Satishchandran,
V. Ambros, G. Szabo, Biodistribution and function of extracellular miRNA-155 in
mice, Sci. Rep. 5 (2015) 10721.
[59] R. van der Meel, M.H.A.M. Fens, P. Vader, W.W. van Solinge, O. Eniola-Adefeso, R.M.
Schiffelers, Extracellular vesicles as drug delivery systems: Lessons from the lipo-
some eld, J. Control. Release 195 (2014) 7285.
[60] T. Smyth, M. Kullberg, N. Malik, P. Smith-Jones, M.W. Graner, T.J. Anchordoquy,
Biodistribution and delivery efciency of unmodied tumor-derived exosomes, J.
Control. Release 199 (2015) 145155.
[61] S.A. Kooijmans, L.A. Fliervoet, R. van der Meel, M.H. Fens, H.F. Heijnen, P.M. van
Bergen En Henegouwen, P. Vader, R.M. Schiffelers, PEGylated and targeted extracel-
lular vesicles display enhanced cell specicity and circulation time, J. Control. Re-
lease 224 (2016) 7785.
[62] S.M. Moghimi, I. Hamad, Liposome-mediated triggering of complement cascade, J.
Liposome Res. 18 (2008) 195209.
[63] A. Clayton, C.L. Harris, J. Court, M.D. Mason, B.P. Morgan, Antigen-presenting cell
exosomes are protected from complement-mediated lysis by expression of CD55
and CD59, Eur. J. Immunol. 33 (2003) 522531.
[64] B. Whitehead, L. Wu, M.L. Hvam, H. Aslan, M. Dong, L. Dyrskjot, M.S. Ostenfeld, S.M.
Moghimi, K.A. Howard, Tumour exosomes display differential mechanical and com-
plement activation properties dependent on malignant state: implications in endo-
thelial leakiness, J. Extracellular Vesicles 4 (2015) 29685.
[65] N. Chaput, J. Taieb, F. Andre, L. Zitvogel, The potential of exosomes in immunother-
apy, Expert. Opin. Biol. Ther. 5 (2005) 737747.
[66] J.E. Hellwinkel, J.S. Redzic, T.A. Harland, D. Gunaydin, T.J. Anchordoquy, M.W. Graner,
Glioma-derived extracellular vesicles selectively suppress immune responses,
Neuro Oncol. (Sep. 18 2015) [Epub ahead of print].
[67] M. Monguio-Tortajada, R. Lauzurica-Valdemoros, F.E. Borras, Tolerance in organ
transplantation: from conventional immunosuppression to extracellular vesicles,
Front. Immunol. 5 (2014) 416.
[68] L. Kordelas, V. Rebmann, A.K. Ludwig, S. Radtke, J. Ruesing, T.R. Doeppner, M. Epple,
P.A. Horn, D.W. Beelen, B. Giebel, MSC-derived exosomes: a novel tool to treat
therapy-refractory graft-versus-host disease, Leukemia 28 (2014) 970973.
[69] B. Costa-Silva, N.M. Aiello, A.J. Ocean, S. Singh, H. Zhang, B.K. Thakur, A. Becker, A.
Hoshino, M.T. Mark, H. Molina, J. Xiang, T. Zhang, T.M. Theilen, G. Garcia-Santos, C.
Williams, Y. Ararso, Y. Huang, G. Rodrigues, T.L. Shen, K.J. Labori, I.M. Lothe, E.H.
Kure, J. Hernandez, A. Doussot, S.H. Ebbesen, P.M. Grandgenett, M.A.
Hollingsworth, M. Jain, K. Mallya, S.K. Batra, W.R. Jarnagin, R.E. Schwartz, I. Matei,
H. Peinado, B.Z. Stanger, J. Bromberg, D. Lyden, Pancreatic cancer exosomes initiate
pre-metastatic niche formation in the liver, Nat. Cell Biol. 17 (2015) 816826.
[70] Y. Tian, S. Li, J. Song, T. Ji, M. Zhu, G.J. Anderson, J. Wei, G. Nie, A doxorubicin delivery
platform using engineered natural membrane vesicle exosomes for targeted tumor
therapy, Biomaterials 35 (2014) 23832390.
[71] M.E. Hung, J.N. Leonard, Stabilization of exosome-targeting peptides via engineered
glycosylation, J. Biol. Chem. 290 (2015) 81668172.
[72] S. Ohno, M. Takanashi, K. Sudo, S. Ueda, A. Ishikawa, N. Matsuyama, K. Fujita, T.
Mizutani, T. Ohgi, T. Ochiya, N. Gotoh, M. Kuroda, Systemically injected exosomes
targeted to EGFR deliver antitumor microRNA to breast cancer cells, Mol. Ther. 21
(2013) 185191.
[73] K.J. Widder, A.E. Senyei, D.F. Ranney, Magnetically responsive microspheres and
other carriers for the biophysical targeting of antitumor agents, Adv. Pharmacol.
Chemother. 16 (1979) 213271.
[74] A.E. Senyei, S.D. Reich, C. Gonczy, K.J. Widder, In vivo kinetics of magnetically
targeted low-dose doxorubicin, J. Pharm. Sci. 70 (1981) 389391.
[75] A.K. Silva, N. Luciani, F. Gazeau, K. Aubertin, S. Bonneau, C. Chauvierre, D. Letourneur,
C. Wilhelm, Combining magnetic nanoparticles with cell derived microvesicles for
drug loading and targeting, Nanomed.: Nanotechnol., Biol. Med. 11 (2015) 645655.
[76] H. Maeda, Macromolecular therapeutics in cancer treatment: the EPR effect and be-
yond, J. Control. Release 164 (2012) 138144.
[77] A. Mizrak, M.F. Bolukbasi, G.B. Ozdener, G.J. Brenner, S. Madlener, E.P. Erkan, T. Strobel,
X.O. Breakeeld, O. Saydam, Genetically engineered microvesicles carrying suicide
mRNA/protein inhibit schwannoma tumor growth, Mol. Ther. 21 (2013) 101108.
[78] Y. Zhang, L. Li, J. Yu, D. Zhu, X. Li, H. Gu, C.Y. Zhang, K. Zen, Microvesicle-mediated
delivery of transforming growth factor beta1 siRNA for the suppression of tumor
growth in mice, Biomaterials 35 (2014) 43904400.
[79] S.C. Jang, O.Y. Kim, C.M. Yoon, D.S. Choi, T.Y. Roh, J. Park, J. Nilsson, J. Lotvall, Y.K. Kim,
Y.S. Gho, Bioinspired exosome-mimetic nanovesicles for targeted delivery of che-
motherapeutics to malignant tumors, ACS Nano 7 (2013) 76987710.
[80] J. Yoon, W. Jo, D. Jeong, J. Kim, H. Jeong, J. Park, Generation of nanovesicles with
sliced cellular membrane fragments for exogenous material delivery, Biomaterials
59 (2015) 1220.