Journal of Controlled Release 266 (2017) 100–108

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Journal of Controlled Release

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

Cellular uptake of extracellular vesicles is mediated by clathrin-independent T

endocytosis and macropinocytosis
Helena Costa Verdera1, Jerney J. Gitz-Francois1, Raymond M. Schiffelers, Pieter Vader⁎
Laboratory of Clinical Chemistry and Hematology, University Medical Center Utrecht, Utrecht 3584 CX, The Netherlands

A R T I C L E I N F O A B S T R A C T

Keywords: Recent evidence has established that extracellular vesicles (EVs), including exosomes and microvesicles, form an
Extracellular vesicles endogenous transport system through which biomolecules, including proteins and RNA, are exchanged between
Exosomes cells. This endows EVs with immense potential for drug delivery and regenerative medicine applications.
Uptake Understanding the biology underlying EV-based intercellular transfer of cargo is of great importance for the
Endocytosis
development of EV-based therapeutics. Here, we sought to characterize the cellular mechanisms involved in EV
Spheroids
Macropinocytosis
uptake.
Internalization of fluorescently-labeled EVs was evaluated in HeLa cells, in 2D (monolayer) cell culture as
well as 3D spheroids. Uptake was assessed using flow cytometry and confocal microscopy, using chemical as well
as RNA interference-based inhibition of key proteins involved in individual endocytic pathways. Experiments
with chemical inhibitors revealed that EV uptake depends on cholesterol and tyrosine kinase activity, which are
implicated in clathrin-independent endocytosis, and on Na+/H+ exchange and phosphoinositide 3-kinase ac-
tivity, which are important for macropinocytosis. Furthermore, EV internalization was inhibited by siRNA-
mediated knockdown of caveolin-1, flotillin-1, RhoA, Rac1 and PAK1, but not clathrin heavy chain. Together,
these results suggest that EVs enter cells predominantly via clathrin-independent endocytosis and macro-
pinocytosis. Identification of EV components that promote their uptake via pathways that lead to functional
cargo transfer might allow development of more efficient therapeutics through EV-inspired engineering.

1. Introduction
Extracellular vesicles (EVs) are cell-derived membranous nano-
particles that enclose and transport complex molecules, including pro-
teins, nucleic acids, lipids and sugars, derived from the parent cell [1].
EVs have been found to function as natural carrier systems that effi-
ciently deliver their molecular cargo to recipient cells. EVs are released
by organisms ranging from prokaryotic cells to higher eukaryotes, and
in mammals these vesicles have been reported to be released from al-
most all cell types. EVs are generally classified into three subtypes,
apoptotic bodies, microvesicles and exosomes, according to their in-
tracellular origin [2]. However, no uniform EV nomenclature exists as
of yet, due to overlap in vesicle sizes and an absence of subtype-specific
markers [3]. Therefore, vesicle subtypes are collectively referred to as
EVs.
EVs have recently been demonstrated to act as a cell-to-cell com-
munication system involved in the natural transfer of macromolecules
between producer and recipient cells. As such, EVs can affect recipient
cell behavior, and modify physiological as well as pathological
processes [4]. For this reason, the potential usage of EVs for therapeutic
applications, including cell-free approaches for tissue engineering and
drug delivery, is increasingly being explored [5–7]. Very encouraging
proof-of-concept studies suggest that EVs may bear intrinsic properties
for anti-tumor immunotherapy [8], for treatment of immunological
disorders (e.g. graft-versus-host disease [9]) or for protecting or re-
generating tissue after e.g. kidney injury [10] or myocardial infarction
[11]. Furthermore, EVs may be modified for targeted drug delivery,
which has already been successfully demonstrated in small animal
studies for therapeutic delivery of RNA [12], protein [13], and small
molecules [14].
As EVs seem naturally capable of crossing biological barriers leading
to functional delivery of their cargo, it may be hypothesized that they
utilize native mechanisms for internalization and intracellular traf-
ficking [15]. Endocytosis has been reported to be the major route
through which EVs are engulfed by cells [16–19]. However, detailed
mechanisms remain unknown. For successful utilization of EVs as
therapeutic carrier systems, understanding the cellular entry routes that
these endogenous carriers follow to deliver their cargo into recipient

⁎
Corresponding author at: Laboratory of Clinical Chemistry and Haematology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands.
E-mail address: pvader@umcutrecht.nl (P. Vader).
1
Authors contributed equally

http://dx.doi.org/10.1016/j.jconrel.2017.09.019
Received 24 July 2017; Received in revised form 12 September 2017; Accepted 13 September 2017
Available online 14 September 2017
0168-3659/ © 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
H. Costa Verdera et al.
cells is of critical importance.
There are numerous mechanisms through which cells may inter-
nalize endocytic cargo. Generally, endocytosis is divided into two main
subgroups: phagocytosis and pinocytosis. Phagocytosis is a type of en-
docytosis that involves internalization of relatively large (> 1 μm)
particles and is typically restricted to specialized professional phago-
cytes. Pinocytosis, in contrast, is exhibited by all cells, and is commonly
classified into clathrin-dependent endocytosis (CDE), clathrin-in-
dependent endocytosis (CIE) and macropinocytosis (MP). CIE can be
further categorized into caveolae-, Arf-6, flotillin-1-, CDC42- and RhoA-
dependent endocytosis [20,21].
The ability of nanoparticles to deliver their cargo and elicit a bio-
logical response is strongly determined by the mechanism through
which they are taken up [22–24]. Here, we set out to identify the en-
docytic routes involved in internalization of EVs.
2. Materials and methods
2.1. Cell culture
A431 (human epidermoid carcinoma) cells (ATCC) and HeLa
(human cervical carcinoma) cells (ATCC) were cultured in Dulbecco's
Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine
serum (FBS) and 100 U/mL penicillin and 100 U/mL streptomycin.
Cells were cultivated at 37 °C and 5% CO2 under humidified conditions.
Cells were routinely checked for Mycoplasma contamination.
2.2. EV isolation
A431 cells were seeded in T175 flasks and cultured for 24 h, after
which medium was replaced by serum-free Opti-MEM (Thermo Fisher
Scientific) supplemented with 100 U/mL penicillin, 100 U/mL strepto-
mycin. After 48 h, EVs were isolated using a recently described size-
exclusion chromatography method [25]. Conditioned medium (CM)
was centrifuged for 10 min at 2000 ×g at 4 °C. Subsequently, CM was
vacuum filtered using 0.45 μm bottle top filters (Thermo Fisher Scien-
tific) and concentrated to ≤ 5 mL using 100 kD MWCO Amicon Ultra-
15 Centrifugal Filter Units (Millipore) at 4 °C. Concentrated CM was
loaded onto a HiPrep 16/60 Sephacryl S-400 HR gel filtration column
(GE Healthcare Life Sciences), equilibrated with phosphate-buffered
saline (PBS) and connected to an ÄKTA Start chromatography system
(GE Healthcare), both maintained at 4 °C. EVs were separated from
non-vesicular material using PBS as eluent. EV-containing fractions (as
determined by UV absorbance at 280 nm) were pooled, filtered through
a 0.45 μm filter and concentrated using 100 kD MWCO Amicon Ultra-
15 Centrifugal Filter Units at 4 °C.
2.3. Western blot analysis
Cells or EVs were lysed in RIPA buffer (Alfa Aesar) supplemented
with Protease Inhibitor Cocktail (Sigma-Aldrich). Lysates were cen-
trifuged at 12000 ×g for 15 min at 4 °C to remove insoluble material,
after which protein concentrations were determined using a MicroBCA
Protein Assay using bovine serum albumin (BSA) as a standard ac-
cording to the manufacturer's instructions. Lysates were mixed with
sample buffer containing dithiothreitol (or without for CD63 analysis),
heated to 95 °C for 10 min and subjected to electrophoresis on 4–12%
Bis-Tris polyacrylamide gels (Thermo Scientific). Proteins were blotted
on Immobilon-FL polyvinylidene difluoride membranes (Millipore),
after which membranes were blocked with 50% v/v Odyssey Blocking
Buffer (LI-COR Biosciences) in Tris-buffered saline (TBS). Subsequently,
membranes were probed with mouse anti-ALIX (1:1000, clone 3A9,
Abcam), mouse anti-CD63 (1:600, clone MEM-259, Abcam), mouse
anti-actin (1:1000, clone JLA20, Millipore), rabbit anti-calnexin
(1:1000, clone N3C2, Genetex), rabbit anti-CLTC (1:1000, polyclonal,
Abcam), rabbit anti-CAV1 (1:6000, clone EPR15554, Abcam), rabbit
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Journal of Controlled Release 266 (2017) 100–108
anti-CDC42 (1:6000, EPR15620, Abcam), rabbit anti-flotillin-1 (1:6000,
EPR6041, Millipore), rabbit anti-ARF6 (1:1000, EPR8357, Abcam),
mouse anti-RhoA (1:1000, 1B12, Abnova), mouse anti-Rac1 (1:1000,
23A8, Millipore), rabbit anti-PAK1 (1:1000, polyclonal, Abcam) or
rabbit anti-ANKFY1 (1:1000, polyclonal, Abgent) antibodies in 50% v/v
Odyssey Blocking Buffer in TBS with 0.1% Tween20 (TBS-T). Secondary
antibodies included Alexa Fluor 680 goat anti-mouse, Alexa Fluor 680
goat anti-rabbit (Thermo Fisher Scientific) and IRDye 800 donkey anti-
mouse (LI-COR Biosciences) and were applied at a 1:10000 dilution.
Protein bands were visualized on an Odyssey Infrared Imager (LI-COR
Biosciences) at 700 and 800 nm.
2.4. Nanoparticle tracking analysis
EV size distribution was determined with a Nanosight NS500 na-
noparticle analyser (Malvern Instruments) equipped with a 405 nm
laser. A camera level of 15–16 and automatic functions for all post-
acquisition settings except for the detection threshold were used. This
was fixed at 6. Using the script control function, three 30 s videos for
each sample were recorded. Analysis was performed with NTA 3.1
software.
2.5. Transmission electron microscopy
EVs were adsorbed to carbon-coated formvar grids for 15 min at RT.
Unbound EVs were removed by washing with PBS. Grids were fixated in
2% paraformaldehyde, 0.2% glutaraldehyde in PBS for 30 min at RT,
counterstained with uranyl-oxalate and embedded in a mixture of 1.8%
methyl cellulose and 0.4% uranyl acetate at 4 °C. Imaging was done on
a Jeol 1010 microscope (Jeol).
2.6. EV labelling and purification
EVs were labeled with 5 × 10−6 M PKH67 (Sigma-Aldrich) ac-
cording to the manufacturer's instructions. For removal of unin-
corporated label, XK-16/20 column (GE Healthcare) was packed with
sepharose CL-4B (Sigma-Aldrich) according to the manufacturer's in-
structions. Column was connected to a refrigerated ÄKTA Start chro-
matography system, equilibrated with PBS, and EV/dye mixtures were
injected. Pooled EV fractions were concentrated using 100 kD MWCO
Amicon Ultra-15 Centrifugal Filter Units at 4 °C.
2.7. Drug treatments
Cells were preincubated with inhibitors before EV addition. For
heparin (Sigma-Aldrich), cytochalasin D (Sigma-Aldrich), chlorproma-
zine (Sigma-Aldrich), genistein (Sigma-Aldrich), EIPA (Santa Cruz
Biotechnology) and wortmannin (Sigma-Aldrich), cells were pretreated
for 30 min. For simvastatin (Sigma-Aldrich), cells were pretreated for
20 h. Inhibitors were present throughout experiments.
2.8. Transfections
HeLa cells were seeded at a density of 50000 cells/well in 6-well
plates. After 24 h, cells were transfected with siRNA (sequences are
listed in Table 1) using Lipofectamine RNAiMax (Life Technologies)
according to the manufacturer's instructions. Transfection was repeated
after an additional 24 h. At 6 h after the second transfection, medium
was replaced and cells were incubated for 24 h and reseeded for EV
internalization assays, or for 48 h for Western blot analysis.
2.9. EV internalization assays
For flow cytometric analysis, HeLa cells were seeded at a density of
15000 cells/well in 24-well plates. After 24 h, cells were pretreated
with inhibitors as described above and then incubated with PKH67-
H. Costa Verdera et al.
Table 1
siRNA sequences.
CAV1 hsc.rnai.n001172895.12.1
CDC42 hsc.rnai.n044472.12.2
RhoA hsc.rnai.n001664.12.1
ARF6 hsc.rnai.n001663.12.2
Flot1 hsc.rnai.n005803.12.8
CLTC hsc.rnai.n004859.12.7
PAK1 hsc.rnai.n002576.12.1
Rac1 hsc.rnai.n018890.12.1
ANKFY1 hsc.rnai.n016376.12.1
siRNAs had a single 2-base 3′-overhang on the antisense strand and a blunt end modified with DNA bases (shown in lower case).
labeled EVs (from ~5 mL CM) for 4 h in the presence of inhibitors.
Subsequently, cells were washed with PBS, washed with acid wash
buffer (0.5 M NaCl, 0.2 M acetic acid) to remove membrane-bound EVs
and once more with PBS. Cells were trypsinized, fixated in 0.2% par-
aformaldehyde in PBS and subjected to flow cytometric analysis on a
FACSCanto (BD Biosciences).
For confocal microscopy analysis, HeLa cells were seeded at a
density of 4000 cells/well in 16-well chamber slides (Lab-Tek). After
24 h, cells were pretreated with inhibitors as described above and then
incubated with PKH67-labeled EVs (from ~20 mL CM) for 4 h in the
presence of inhibitors. Subsequently, cells were washed with PBS, wa-
shed with acid wash buffer (0.5 M NaCl, 0.2 M acetic acid) to remove
membrane-bound EVs and once more with PBS. Cells were fixated with
4% paraformaldehyde in PBS at room temperature for 20 min. After
fixation, slides were washed with PBS and mounted using Fluorsave
(Calbiochem). Confocal fluorescence imaging was done on a LSM700
laser scanning confocal microscope (Zeiss). Images were processed
using LSM Image Browser. Contrast and brightness parameter adjust-
ments were applied across whole images and equally across comparison
groups when necessary.
2.10. Spheroid formation and EV internalization assays
Spheroids were grown in a 96 well Insphero Gravity TRAP™ ULA
plate (Perkin Elmer). Wells were prewet with 10 μL of media. HeLa cells
were seeded at a density of 1000 cells/well, after which plates were
centrifuged at 250 × g for 2 min and allowed to incubate for 48 h prior
to treatment. Spheroids were pretreated with inhibitors as described
above and then incubated with PKH67-labeled EVs (from ~ 20 mL CM)
for 4 h in the presence of inhibitors. Subsequently, cells were washed
with PBS, washed with acid wash buffer (0.5 M NaCl, 0.2 M acetic acid)
to remove membrane-bound EVs and twice more with PBS. For flow
cytometric analysis, cells were trypsinized, fixed in 0.2% paraf-
ormaldehyde in PBS and subjected to flow cytometric analysis on a
FACSCanto (BD Biosciences). For confocal microscopy analysis, spher-
oids were fixated with 4% paraformaldehyde in PBS at room tem-
perature for 20 min. After fixation, spheroids were washed with PBS
and transferred to μView clear-bottom 96 well plates (Greiner Bio-One).
Confocal fluorescence imaging was done on a Yokogawa Cell Voyager
7000 (Yokogawa). Images were processed using Fiji. Contrast and
brightness parameter adjustments were applied across whole images
and equally across comparison groups when necessary.
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Journal of Controlled Release 266 (2017) 100–108
Sense 5′-CCUUCACUGUGACGAAAUACUGGtt-3′
Antisense 5′-AACCAGUAUUUCGUCACAGUGAAGGUG-3′
Sense 5′-CCACAAACAGAUGUAUUUCUAGUct-3′
Antisense 5′-AGACUAGAAAUACAUCUGUUUGUGGAU-3′
Sense 5′-CCCAGAUACCGAUGUUAUACUGAtg-3′
Antisense 5′-CAUCAGUAUAACAUCGGUAUCUGGGUA-3′
Sense 5′-CCUCUAACUACAAAUCUUAAUGAgc-3′
Antisense 5′-GCUCAUUAAGAUUUGUAGUUAGAGGUU-3′
Sense 5′-GGUGAAUCACAAGCCUUUGAGAAca-3′
Antisense 5′-UGUUCUCAAAGGCUUGUGAUUCACCUG-3′
Sense 5′-GCCUUUACAAGGAUGCAAUGCAGta-3′
Antisense 5′-UACUGCAUUGCAUCCUUGUAAAGGCUG-3′
Sense 5′-GCGAUCCUAAGAAGAAAUAUACAcg-3′
Antisense 5′-CGUGUAUAUUUCUUCUUAGGAUCGCCC-3′
Sense 5′-GGAACUAAACUUGAUCUUAGGGAtg-3′
Antisense 5′-CAUCCCUAAGAUCAAGUUUAGUUCCCA-3′
Sense 5′-ACAGCGAUCUGAAGAUAAAGGUUgg-3′
Antisense 5′-CCAACCUUUAUCUUCAGAUCGCUGUAC-3′
2.11. Statistical analysis
Comparisons between two groups were performed using in-
dependent two sample t-tests. Multiple-group testing was performed
using one-way ANOVA with Dunnett's post-hoc tests.
3. Results
EVs were isolated from conditioned medium from A431 cells using a
recently described procedure based on ultrafiltration followed by size-
exclusion chromatography [25]. EVs were collected in serum-free
medium, thus avoiding potential contamination with FBS-derived EVs.
Western Blot characterization revealed that EV marker proteins Alix
and CD63 were strongly enriched in EVs compared to cell lysate, while
the opposite was observed for calnexin, an integral protein of the en-
doplasmic reticulum (Fig. 1A). Actin was found at similar levels in EVs
and cell lysate. NTA showed that the majority of EVs were in a size
range of 60–250 nm, peaking at 100 nm (Fig. 1B). Transmission elec-
tron microscopy analysis indicated that EVs were spherical, membrane-
bound particles with most vesicles having a diameter around 100 nm
(Fig. 1C). To study internalization of EVs by HeLa cells, purified EVs
were fluorescently labeled with the membrane dye PKH67. Shortly after
incubation with EVs, a punctuated pattern of fluorescence could be
observed intracellularly, with increasing signal over time. Uptake was
inhibited by an excess of unlabeled EVs, as shown by flow cytometry
(Fig. 1D) and confocal microscopy analysis (Fig. 1E). This indicates that
EV uptake is mediated by specific processes and also excludes a sig-
nificant contribution of transfer of free PKH dye to the observed
fluorescent signal. EV internalization was completely inhibited at 4 °C
(Fig. 1F,G), which indicates that it is mediated by active, energy-de-
pendent endocytic processes, as opposed to passive membrane fusion.
Furthermore, EV internalization was found to be time-dependent
(Fig. 1H), and significantly inhibited in the presence of heparin
(Fig. 1I), a soluble analogue of heparan sulfate proteoglycans, as de-
scribed previously [26,27]. Cytochalasin D, an inhibitor of actin poly-
merization, significantly inhibited EV uptake, suggesting that endocytic
mechanisms requiring cytoskeletal remodeling are involved.
To unravel the contribution of specific endocytic processes to EV
internalization, recipient cells were pretreated with pharmacological
inhibitors known to interfere with different pathways. Chlorpromazine,
which blocks CDE by interfering with the association between clathrin
and the plasma membrane [28], did not inhibit, but slightly enhanced,
EV uptake, as shown by flow cytometry (Fig. 2A) and confocal micro-
scopy (Fig. 2B). This may be the result of a change in the activity of
other endocytic mechanisms as compensation for alterations in CDE, a
H. Costa Verdera et al. Journal of Controlled Release 266 (2017) 100–108

Fig. 1. EVs are internalized by HeLa cells. (A) Western Blot analysis of Alix, CD63, Actin and Calnexin in A431 cell lysate (CL) and EVs. (B) Size distribution profile of A431 EVs as
determined by NTA. (C) Electron microscopy analysis of A431 EVs. Scale bar represents 100 nm. (D,E) EV uptake by HeLa cells in the absence or presence of a five-fold excess of unlabeled
EVs uptake as quantified by flow cytometry (D) or visualized by confocal microscopy (E). Scale bars represent 10 μm. (F,G) EV uptake by HeLa cells at 37 °C or 4 °C as quantified by flow
cytometry (F) or visualized by confocal microscopy (G). (H) Time dependent EV uptake by HeLa cells. (I) EV uptake by HeLa cells in the absence or presence of 10 μg/mL heparin. (J) EV
uptake by HeLa cells in the presence of increasing concentrations of cytochalasin D. Values represent mean + S.E.M. *p < 0.05.

phenomenon known as cross-regulation [20]. In contrast, chlorproma-
zine dose-dependently inhibited uptake of transferrin, an established
CDE marker (Fig. S1), showing that chlorpromazine has the expected
activity in these cells. Together, these results suggest that CDE does not
significantly contribute to EV internalization. Genistein is a tyrosine
kinase inhibitor that causes local disruption of the actin network and
can inhibit the recruitment of dynamin to plasma membranes, both of
which are important in CIE [29]. EV uptake was dose-dependently in-
hibited by genistein, suggesting a role for CIE in their internalization
(Fig. 2C,D). As CIE includes several pathways which share a de-
pendency on cholesterol, we next pretreated cells with simvastatin, an
inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, an en-
zyme involved in the synthesis of cholesterol [30]. Simvastatin treat-
ment dose-dependently inhibited EV uptake (Fig. 2E), indicating that
the presence of cholesterol is indispensable for EV uptake, again sug-
gesting the importance of CIE as uptake route. For the study of MP, cells
were pretreated with 5-(N-ethyl-N-isopropyl)amirolide (EIPA), an in-
hibitor of Na+/H+ exchange, known to inhibit MP by lowering sub-
membranous pH, which affects Rac1 activation and actin assembly
[31]. EIPA dose-dependently restricted EV internalization (Fig. 2F,G).
Similar results were obtained for the phosphoinositide 3-kinase (PI(3)K)
inhibitor wortmannin (Fig. 2H). PI(3)K participates in the signaling
cascade that stimulates membrane ruffling and closes the macropino-
somes during MP [32]. Together, these findings suggest the importance
of clathrin-independent endocytosis and macropinocytosis for EV up-
take.
Interestingly, none of the inhibitors of either CIE or MP resulted in
complete inhibition of EV uptake, indicating a contribution from both
pathways. To evaluate whether inhibition of both pathways had an
additive effect, cells were pretreated simultaneously with both EIPA
and genistein. While each of the individual inhibitors reduced EV up-
take by ~50%, the combination treatment almost completely blocked
internalization (Fig. 3A), suggesting that CIE and MP are processes that
independently contribute. As constitutive MP occurs at very low levels
in epithelial cells, we next evaluated whether EVs themselves could
induce MP. After incubation with EVs, uptake of the fluid phase marker
dextran (70 kDa) increased substantially (Fig. 3B), indicating that EVs
are capable of stimulating macropinocytosis, and thereby their own
internalization.
Due to their reported low specificity in some cell types, chemical
inhibitors alone may not be sufficient to fully elucidate the mechanisms
that contribute to uptake of EVs. For example, genistein may also

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H. Costa Verdera et al. Journal of Controlled Release 266 (2017) 100–108

Fig. 2. EVs are internalized through tyrosine kinase-, cholesterol-, Na+/H+ exchangers-, and PI3K-dependent pathways. EV uptake by HeLa cells in the presence of increasing con-
centrations of (A,B) chlorpromazine, (C,D) genistein, (E) simvastatin, (F,G) EIPA or (H) wortmannin, as quantified by flow cytometry (A,C,E,F,H) or visualized by confocal microscopy
(B,D,G). Scale bars represent 10 μm. Values represent mean + S.E.M. *p < 0.05.

inhibit uptake via CDE by hindering recruitment of F-actin to clathrin-
coated pits, while wortmannin may display pleiotropic effects on en-
docytosis [33]. Furthermore, cholesterol is not only important for CIE,
but also for MP [34]. Therefore, we next sought to confirm our ob-
servations and evaluate the key contributors involved in EV
internalization through RNAi-mediated knockdown of specific proteins.
These proteins included clathrin heavy chain (CLTC), a major subunit of
clathrin, caveolin-1 (CAV-1), CDC42, Flotillin-1 (Flot1), ARF6, and
RhoA, key endocytic proteins regulating different subclasses of CIE, and
Rac1, PAK1 and ANKFY1, proteins previously shown to regulate MP

Fig. 3. Clathrin-independent endocytosis and macro-

pinocytosis independently contribute to EV internalization.
(A) EV uptake by HeLa cells in the presence of EIPA
(100 μM), genistein (200 μM) or the combination, as
quantified by flow cytometry. (B) Dextran uptake by HeLa
cells in the presence of increasing concentrations of EVs, as
quantified by flow cytometry. Values represent mean
+ S.E.M. *p < 0.05.

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H. Costa Verdera et al.
Fig. 4. Knockdown of CAV1, RhoA, Flotillin, Rac1 or PAK1 inhibits EV internalization. (A) Western Blot analysis of HeLa cells transfected with siRNA against key endocytic regulators
(clathrin heavy chain (CLTC), caveolin-1 (CAV1), CDC42, RhoA, ARF6, Flotillin, Rac1, PAK1 and ANKFY1) or negative control siRNA (NS). (B,C) EV uptake by HeLa cells pretreated with
indicated siRNAs, as quantified by flow cytometry (B) or visualized by confocal microscopy (C). Scale bars represent 10 μm. Values represent mean + S.E.M. *p < 0.05.
[21]. After two rounds of transfection, significant downregulation of
target proteins was observed (Fig. 4A). Depletion of CLTC had no effect
on EV internalization, confirming that uptake of EVs is independent of
CDE. EV internalization was significantly inhibited after knockdown of
caveolin-1, flotillin-1, and RhoA, but not ARF6 and CDC42, suggesting a
contribution of the caveolae-, flotillin-1-, and RhoA-dependent sub-
classes of CIE (Fig. 4B). Lastly, we observed a significant inhibition of
EV uptake upon downregulation of MP regulators Rac1 and PAK1. The
effects of knockdown of CLTC, Flotillin and PAK1 as markers of CDE,
CIE and MP, respectively, on EV internalization were confirmed by
confocal microscopy analysis (Fig. 4C).
Compared with 2D monolayers, culturing cells in 3D spheroids may
be more representative of the in vivo environment, and has been shown
to affect cell metabolism, interaction of cells with each other and the
extracellular matrix, as well as cell polarity and gene profiles (including
receptor expression) [35]. Therefore, we next evaluated endocytic
mechanisms involved in EV uptake into 3D spheroids. In contrast to the
2D monolayer, in which all cells had taken up EVs within a 4 h time
frame, uptake into spheroids was mainly restricted to the outer few cell
layers. Similar to results obtained using monolayer cultures, genistein
and EIPA, but not chlorpromazine, substantially inhibited EV uptake
(Fig. 5A). EIPA also displayed a slight effect on spheroid compactness
(brightfield pictures). Flow cytometric analysis revealed that both the
fluorescent signal per cell (Fig. 5B) as well as the percentage of EV-
positive cells (Fig. 5C) were significantly reduced after treatment with
genistein or EIPA. Taken together, our results show that EVs are in-
ternalized through CIE and MP in 2D monolayers as well as 3D spher-
oids.
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Journal of Controlled Release 266 (2017) 100–108
4. Discussion
EVs are gaining increasing interest from both academia and industry
as endogenous nanoparticles with properties that makes them highly
suitable for therapeutic applications such as regenerative medicine and
drug delivery [6,36]. Naturally equipped to deliver their biological
cargo, EVs are capable of modulating or reprogramming recipient cells.
However, whether these phenotypic responses are elicited may cru-
cially depend on the mechanism of EV uptake, similar to what has been
described for viruses [37] and synthetic nanoparticles [22,38].
In this study, using both pharmacological and RNAi-mediated in-
hibition of endocytic pathways, we show that EV internalization occurs
via independent contributions of CIE and MP. Following the assessment
of EV internalization in 2D monolayer conditions, similar evaluations
were performed in 3D spheroid culture. 3D culture more closely mimics
in vivo cellular organization (e.g. cellular polarization, interaction with
extracellular matrix), potentially including essential features required
for correct EV internalization and processing [39]. Similar inhibitory
effects of genistein and EIPA, but not chlorpromazine, were observed on
3D spheroids versus 2D cell culture, suggesting that our results may, to
the best of our knowledge, also be representative for the in vivo si-
tuation.
To study the involvement of CIE in EV uptake, we employed the
small molecule inhibitors genistein and simvastatin. For both inhibitors,
a dose-dependent effect on EV uptake was observed. Although often
used for membrane cholesterol depletion, recent work indicates that
statins may also have other effects on cells, including an effect on Rab
prenylation and glycosphingolipid biosynthesis [40]. Classically, me-
thyl-β-cyclodextrin and filipin, inhibitors known to affect structure and
function of cholesterol-rich membrane domains, are being used to study
H. Costa Verdera et al.
Fig. 5. Inhibition of clathrin-independent endocytosis or macropinocytosis attenuates EV internalization in spheroids. EV uptake by HeLa spheroids in the presence of chlorpromazine
(10 μM), genistein (200 μM) or EIPA (100 μM) as visualized by confocal microscopy (A) or quantified by flow cytometry (B,C). Pictures represent planes at the indicated depths within the
spheroids. Scale bars represents 100 μm. Values represent mean + S.E.M. *p < 0.05.
CIE [33]. However, these drugs may also interact with cholesterol in EV
membranes, thereby affecting EV membrane integrity, and thereby in-
directly influencing EV uptake [41]. Therefore, we also used RNAi to
inhibit expression of proteins with known involvements in specific
subclasses of CIE, and discovered significant contributions of CAV-1,
flotillin-1 and RhoA to EV uptake. Caveolins and flotillins constitute a
group of proteins that are enriched in specific microdomains within the
plasma membrane, known as lipid rafts. These microdomains contain
high concentrations of cholesterol and glycosphingolipids and are
known sites for endocytosis. Thus, lipid rafts may be involved in EV
uptake, as also previously suggested by others [16,42]. Our observation
that EV internalization was significantly inhibited even after partial
knockdown of RhoA (Fig.4A) suggests that RhoA-dependent en-
docytosis also plays a strong role. However, caution should be taken, as
possible contributions of RhoA to other internalization mechanisms
have not been completely ruled out, and interfering with RhoA sig-
naling is likely to affect many other cellular processes [20].
Our experiments using chemical inhibitors EIPA and wortmannin
suggest an important contribution for MP in EV internalization. To
confirm these observations, we silenced Rac1, PAK1 and ANKFY1,
proteins previously implicated to play important roles in MP. While
Rac1 and PAK1 inhibition attenuated EV internalization, we found no
significant effect after ANKFY1 knockdown. The reason for the apparent
lack of importance for ANKFY1 in this process is unknown. Although
PAK1 serves as target for both small GTP binding proteins Rac1 and
CDC42, our data seem to suggest that CDC42 is not involved in EV
uptake. This may be explained by the fact that Rac1 is thought to be
required for growth factor-induced MP, whereas CDC42 has a role in
constitutive MP, which occurs only at very low levels in HeLa cells
[43,44]. Concordantly, it has previously been shown that induction of
MP (through stimulation of growth factor receptors or Ras transfor-
mation) enhances EV uptake [45]. Here, we demonstrate that EVs
106
Journal of Controlled Release 266 (2017) 100–108
themselves can also stimulate MP, thereby potentiating their own up-
take. The mechanisms underlying this observation are unclear, but may
involve EV cargo-mediated activation of signaling pathways (e.g.
phosphatidylinositol-3-kinase (PI3K) [46], ras [47], and src [48]) that
trigger MP induction.
Our data that indicate that EVs are taken up via CIE and MP, but not
CDE, contrast with some results obtained by others. For example, CDE
was shown to be involved in uptake of prostate cancer cell-derived EVs
by mesenchymal stromal cells [49]. In this study, results from CAV1
knockdown experiments also ruled out an involvement of caveolae-
mediated endocytosis in the uptake process. Conversely, our results
coincide with others who revealed a lack of involvement of CDE, but
support a role for CIE in EV uptake [16,50]. Nevertheless, there are still
discrepancies in some of the observations, as suppression of CAV1 ex-
pression increased EV uptake by embryonic fibroblast cells [16], an
opposite effect to that observed here (Fig. 4B). Comparison and inter-
pretation of these different reports is however complicated by the fact
that, at least in some cell types, alterations in CAV1 expression may also
affect fluid phase as well as CDC42-dependent uptake [51,52]. Simi-
larly, while several reports suggested that cells internalize EVs via MP
[18,49,53], others did not find a role for MP in EV uptake [54,55].
However, these results were based on experiments using small molecule
inhibitors only. We provide further evidence supporting the involve-
ment of MP through RNAi-mediated depletion of proteins that are es-
sential for this process. Collectively, these findings demonstrate that EV
uptake may rely on a variety of endocytic mechanisms, depending on
the EV donor cell type and recipient cell type.
The observation that multiple, independently contributing path-
ways are involved in EV internalization presents the intriguing possi-
bility that distinct EV subpopulations are differentially processed by
cells. While it is generally accepted that cells release at least three types
of EVs, i.e. apoptotic bodies, microvesicles and exosomes, increasing
H. Costa Verdera et al.
evidence suggests that even within these groups, distinct subpopula-
tions can be discriminated [56,57]. These subpopulations express un-
ique surface protein repertoires and may therefore differentially in-
teract with recipient cells. These specific protein-protein interactions
may be important factors in the pathway selection of EV entry into cells
[41]. Our EV preparation, obtained using size-exclusion chromato-
graphy, likely contains a mixture of different EV subpopulations and
this may have resulted in different endocytic mechanisms being in-
volved. In addition, it is well-known that subpopulations of EVs differ in
physical properties, including size. Although non-phagocytic cells are,
in principle, able to internalize particles < 1 μm in size, particle size
can affect the internalization pathway. For example, the size limit for
particles to enter cells via clathrin-mediated endocytosis has been re-
ported to be ~200 nm [58,59]. In addition, multiple reports suggest
that size limitations of caveolae limit the internalization of nano-
particles larger than the size of individual caveolae (~ 50–100 nm)
[60,61]. There appears to be no upper size limit for cargo internalized
by macropinocytosis. In our experiments, according to NTA, the ma-
jority of EVs were between 50 and 150 nm in size, but also EVs larger
than 200 nm were observed. Whether EVs with different sizes are
preferentially internalized via specific pathways requires further re-
search.
Internalization through any of the endocytic pathways leads to de-
livery of endocytosed cargo (i.e. EVs and their cargo) into the en-
dosomal-lysosomal degradative pathway. Despite the multitude of
evidence that EVs are capable of delivering their cargo in a functional
manner (e.g. to the cytosol of target cells in the case of miRNA or
mRNA), the mechanisms underlying endosomal escape and/or active
transport of EV cargo out of endosomes remain completely unknown. In
this respect, the recent observation that EVs, internalized through MP,
displayed a higher cargo delivery efficiency to pancreatic cancer cells as
compared to liposomes, internalized through other mechanisms [53],
might indicate that MP is a productive entry pathway. Furthermore,
endosomes containing EVs have been shown to be targeted to scan the
endoplasmic reticulum as a possible site of cargo release, which would
allow directed delivery of EV-associated miRNA or mRNA into the
translation machinery [19]. These studies might provide new directions
for understanding the apparent efficacy of EV signaling.
In conclusion, we have shown that EVs enter HeLa cells via clathrin-
independent endocytosis and macropinocytosis, and not via clathrin-
dependent endocytosis. However, further research is warranted in order
to be able to define the contributions of the various uptake mechanisms
to EV function, and how this depends on the properties of EV producer
and recipient cells. Ultimately, a relationship between 1) pathways that
lead to functional delivery and 2) EV components that promote uptake
or ensure correct intracellular trafficking via those pathways may be
established. Identification of EV components that promote their uptake
via pathways that lead to functional cargo transfer will allow devel-
opment of more efficient delivery systems through EV-inspired en-
gineering. However, the apparent complexity of the uptake process and
the multiplicity of elements that seem to be involved highlight the
necessity of an interdisciplinary approach to such research.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.jconrel.2017.09.019.
Conflict of interest
None.
Acknowledgements
PV is supported by a VENI Fellowship from the Netherlands
Organisation for Scientific Research (NWO). The authors acknowledge
Cor Seinen (UMC Utrecht) for excellent assistance with electron mi-
croscopy and the Cell Microscopy Core, Department of Cell Biology,
Center for Molecular Medicine, UMC Utrecht for use of confocal
107
Journal of Controlled Release 266 (2017) 100–108
microscopy facilities.
References
[1] M. Yanez-Mo, P.R. Siljander, Z. Andreu, A.B. Zavec, F.E. Borras, E.I. Buzas, K. Buzas,
E. Casal, F. Cappello, J. Carvalho, E. Colas, A. Cordeiro-da Silva, S. Fais,
J.M. Falcon-Perez, I.M. Ghobrial, B. Giebel, M. Gimona, M. Graner, I. Gursel,
M. Gursel, N.H. Heegaard, A. Hendrix, P. Kierulf, K. Kokubun, M. Kosanovic,
V. Kralj-Iglic, E.M. Kramer-Albers, S. Laitinen, C. Lasser, T. Lener, E. Ligeti, A. Line,
G. Lipps, A. Llorente, J. Lotvall, M. Mancek-Keber, A. Marcilla, M. Mittelbrunn,
I. Nazarenko, E.N. Nolte-'t Hoen, T.A. Nyman, L. O'Driscoll, M. Olivan, C. Oliveira,
E. Pallinger, H.A. Del Portillo, J. Reventos, M. Rigau, E. Rohde, M. Sammar,
F. Sanchez-Madrid, N. Santarem, K. Schallmoser, M.S. Ostenfeld, W. Stoorvogel,
R. Stukelj, S.G. Van der Grein, M.H. Vasconcelos, M.H. Wauben, O. De Wever,
Biological properties of extracellular vesicles and their physiological functions,
Journal of extracellular vesicles 4 (2015) 27066.
[2] M. Colombo, G. Raposo, C. Thery, Biogenesis, secretion, and intercellular interac-
tions of exosomes and other extracellular vesicles, Annu. Rev. Cell Dev. Biol. 30
(2014) 255–289.
[3] S.J. Gould, G. Raposo, As we wait: coping with an imperfect nomenclature for ex-
tracellular vesicles, J. Extracell. Vesicles 2 (2013).
[4] E.L.A. S, I. Mager, X.O. Breakefield, M.J. Wood, Extracellular vesicles: biology and
emerging therapeutic opportunities, Nat. Rev. Drug Discov. 12 (2013) 347–357.
[5] T. Lener, M. Gimona, L. Aigner, V. Borger, E. Buzas, 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, Journal of extracellular vesicles 4 (2015)
30087.
[6] P. Vader, E.A. Mol, G. Pasterkamp, R.M. Schiffelers, Extracellular vesicles for drug
delivery, Adv. Drug Deliv. Rev. 106 (2016) 148–156.
[7] O.G. De Jong, B.W. Van Balkom, R.M. Schiffelers, C.V. Bouten, M.C. Verhaar,
Extracellular vesicles: potential roles in regenerative medicine, Front. Immunol. 5
(2014) 608.
[8] 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 thefirst
phase I clinical trial, J. Transl. Med. 3 (2005) 10.
[9] 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) 970–973.
[10] S. Bruno, C. Grange, M.C. Deregibus, R.A. Calogero, S. Saviozzi, F. Collino,
L. Morando, A. Busca, M. Falda, B. Bussolati, C. Tetta, G. Camussi, Mesenchymal
stem cell-derived microvesicles protect against acute tubular injury, J. Am. Soc.
Nephrol. 20 (2009) 1053–1067.
[11] R.C. Lai, F. Arslan, M.M. Lee, N.S. Sze, A. Choo, T.S. Chen, M. Salto-Tellez,
L. Timmers, C.N. Lee, R.M. El Oakley, G. Pasterkamp, D.P. de Kleijn, S.K. Lim,
Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury, Stem
Cell Res. 4 (2010) 214–222.
[12] 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).
[13] 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
vehicles for Parkinson's disease therapy, J. Control. Release 207 (2015) 18–30.
[14] 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) 2383–2390.
[15] M.E. Marcus, J.N. Leonard, FedExosomes: engineering therapeutic biological na-
noparticles that truly deliver, Pharmaceuticals (Basel) 6 (2013) 659–680.
[16] K.J. Svensson, H.C. Christianson, A. Wittrup, E. Bourseau-Guilmain, E. Lindqvist,
L.M. Svensson, M. Morgelin, M. Belting, Exosome uptake depends on ERK1/2-heat
shock protein 27 signaling and lipid raft-mediated endocytosis negatively regulated
by caveolin-1, J. Biol. Chem. 288 (2013) 17713–17724.
[17] C. Escrevente, S. Keller, P. Altevogt, J. Costa, Interaction and uptake of exosomes by
ovarian cancer cells, BMC Cancer 11 (2011) 108.
[18] D. Fitzner, M. Schnaars, D. van Rossum, G. Krishnamoorthy, P. Dibaj, M. Bakhti,
T. Regen, U.K. Hanisch, M. Simons, Selective transfer of exosomes from oligoden-
drocytes to microglia by macropinocytosis, J. Cell Sci. 124 (2011) 447–458.
[19] W. Heusermann, J. Hean, D. Trojer, E. Steib, S. von Bueren, A. Graff-Meyer,
C. Genoud, K. Martin, N. Pizzato, J. Voshol, D.V. Morrissey, S.E. Andaloussi,
M.J. Wood, N.C. Meisner-Kober, Exosomes surf on filopodia to enter cells at en-
docytic hot spots, traffic within endosomes, and are targeted to the ER, J. Cell Biol.
213 (2016) 173–184.
[20] S. Mayor, R.E. Pagano, Pathways of clathrin-independent endocytosis, Nat. Rev.
Mol. Cell Biol. 8 (2007) 603–612.
[21] G.J. Doherty, H.T. McMahon, Mechanisms of endocytosis, Annu. Rev. Biochem. 78
H. Costa Verdera et al.
(2009) 857–902.
[22] I.S. Zuhorn, R. Kalicharan, D. Hoekstra, Lipoplex-mediated transfection of mam-
malian cells occurs through the cholesterol-dependent clathrin-mediated pathway
of endocytosis, J. Biol. Chem. 277 (2002) 18021–18028.
[23] D. Vercauteren, M. Piest, L.J. van der Aa, M. Al Soraj, A.T. Jones, J.F. Engbersen,
S.C. De Smedt, K. Braeckmans, Flotillin-dependent endocytosis and a phagocytosis-
like mechanism for cellular internalization of disulfide-based poly(amido amine)/
DNA polyplexes, Biomaterials 32 (2011) 3072–3084.
[24] S. Uhrig, O. Coutelle, T. Wiehe, L. Perabo, M. Hallek, H. Buning, Successful target
cell transduction of capsid-engineered rAAV vectors requires clathrin-dependent
endocytosis, Gene Ther. 19 (2012) 210–218.
[25] 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.E. Andaloussi, Ultrafiltration with size-exclusion liquid chromatography for high
yield isolation of extracellular vesicles preserving intact biophysical and functional
properties, Nanomedicine (2015).
[26] H.C. Christianson, K.J. Svensson, T.H. van Kuppevelt, J.P. Li, M. Belting, Cancer cell
exosomes depend on cell-surface heparan sulfate proteoglycans for their inter-
nalization and functional activity, Proc. Natl. Acad. Sci. U. S. A. 110 (2013)
17380–17385.
[27] N.A. Atai, L. Balaj, H. van Veen, X.O. Breakefield, P.A. Jarzyna, C.J. Van Noorden,
J. Skog, C.A. Maguire, Heparin blocks transfer of extracellular vesicles between
donor and recipient cells, J. Neuro-Oncol. 115 (2013) 343–351.
[28] L.H. Wang, K.G. Rothberg, R.G. Anderson, Mis-assembly of clathrin lattices on
endosomes reveals a regulatory switch for coated pit formation, J. Cell Biol. 123
(1993) 1107–1117.
[29] L. Pelkmans, D. Puntener, A. Helenius, Local actin polymerization and dynamin
recruitment in SV40-induced internalization of caveolae, Science 296 (2002)
535–539.
[30] J.K. Liao, U. Laufs, Pleiotropic effects of statins, Annu. Rev. Pharmacol. Toxicol. 45
(2005) 89–118.
[31] M. Koivusalo, C. Welch, H. Hayashi, C.C. Scott, M. Kim, T. Alexander, N. Touret,
K.M. Hahn, S. Grinstein, Amiloride inhibits macropinocytosis by lowering sub-
membranous pH and preventing Rac1 and Cdc42 signaling, J. Cell Biol. 188 (2010)
547–563.
[32] J. Mercer, M. Schelhaas, A. Helenius, Virus entry by endocytosis, Annu. Rev.
Biochem. 79 (2010) 803–833.
[33] A.I. Ivanov, Pharmacological inhibition of endocytic pathways: is it specific enough
to be useful? Methods Mol. Biol. 440 (2008) 15–33.
[34] S. Grimmer, B. van Deurs, K. Sandvig, Membrane ruffling and macropinocytosis in
A431 cells require cholesterol, J. Cell Sci. 115 (2002) 2953–2962.
[35] S. Breslin, L. O'Driscoll, Three-dimensional cell culture: the missing link in drug
discovery, Drug Discov. Today 18 (2013) 240–249.
[36] J. Malda, J. Boere, C.H. van de Lest, P. van Weeren, M.H. Wauben, Extracellular
vesicles - new tool for joint repair and regeneration, Nat. Rev. Rheumatol. 12
(2016) 243–249.
[37] K.M. Hussain, K.L. Leong, M.M. Ng, J.J. Chu, The essential role of clathrin-mediated
endocytosis in the infectious entry of human enterovirus 71, J. Biol. Chem. 286
(2011) 309–321.
[38] X.X. Zhang, P.G. Allen, M. Grinstaff, Macropinocytosis is the major pathway re-
sponsible for DNA transfection in CHO cells by a charge-reversal amphiphile, Mol.
Pharm. 8 (2011) 758–766.
[39] B. Mateescu, E.J. Kowal, B.W. van Balkom, S. Bartel, S.N. Bhattacharyya, E.I. Buzas,
A.H. Buck, P. de Candia, F.W. Chow, S. Das, T.A. Driedonks, L. Fernandez-Messina,
F. Haderk, A.F. Hill, J.C. Jones, K.R. Van Keuren-Jensen, C.P. Lai, C. Lasser,
I.D. Liegro, T.R. Lunavat, M.J. Lorenowicz, S.L. Maas, I. Mager, M. Mittelbrunn,
S. Momma, K. Mukherjee, M. Nawaz, D.M. Pegtel, M.W. Pfaffl, R.M. Schiffelers,
H. Tahara, C. Thery, J.P. Tosar, M.H. Wauben, K.W. Witwer, E.N. Nolte-'t Hoen,
Obstacles and opportunities in the functional analysis of extracellular vesicle RNA -
an ISEV position paper, Journal of extracellular vesicles 6 (2017) (1286095).
[40] B. Binnington, L. Nguyen, M. Kamani, D. Hossain, D.L. Marks, M. Budani,
C.A. Lingwood, Inhibition of Rab prenylation by statins induces cellular glyco-
sphingolipid remodeling, Glycobiology 26 (2016) 166–180.
108
Journal of Controlled Release 266 (2017) 100–108
[41] L.A. Mulcahy, R.C. Pink, D.R. Carter, Routes and mechanisms of extracellular ve-
sicle uptake, Journal of extracellular vesicles 3 (2014).
[42] M.P. Plebanek, R.K. Mutharasan, O. Volpert, A. Matov, J.C. Gatlin, C.S. Thaxton,
Nanoparticle targeting and cholesterol flux through scavenger receptor type B-1
inhibits cellular exosome uptake, Sci Rep 5 (2015) (15724).
[43] A.J. Ridley, H.F. Paterson, C.L. Johnston, D. Diekmann, A. Hall, The small GTP-
binding protein rac regulates growth factor-induced membrane ruffling, Cell 70
(1992) 401–410.
[44] J. Mercer, A. Helenius, Virus entry by macropinocytosis, Nat. Cell Biol. 11 (2009)
510–520.
[45] I. Nakase, N.B. Kobayashi, T. Takatani-Nakase, T. Yoshida, Active macropinocytosis
induction by stimulation of epidermal growth factor receptor and oncogenic Ras
expression potentiates cellular uptake efficacy of exosomes, Sci Rep 5 (2015)
10300.
[46] J.L. Qu, X.J. Qu, M.F. Zhao, Y.E. Teng, Y. Zhang, K.Z. Hou, Y.H. Jiang, X.H. Yang,
Y.P. Liu, Gastric cancer exosomes promote tumour cell proliferation through PI3K/
Akt and MAPK/ERK activation, Dig. Liver Dis. 41 (2009) 875–880.
[47] K. Al-Nedawi, B. Meehan, R.S. Kerbel, A.C. Allison, J. Rak, Endothelial expression of
autocrine VEGF upon the uptake of tumor-derived microvesicles containing onco-
genic EGFR, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 3794–3799.
[48] M. Mineo, S.H. Garfield, S. Taverna, A. Flugy, G. De Leo, R. Alessandro, E.C. Kohn,
Exosomes released by K562 chronic myeloid leukemia cells promote angiogenesis in
a Src-dependent fashion, Angiogenesis 15 (2012) 33–45.
[49] T. Tian, Y.L. Zhu, Y.Y. Zhou, G.F. Liang, Y.Y. Wang, F.H. Hu, Z.D. Xiao, Exosome
uptake through clathrin-mediated endocytosis and macropinocytosis and mediating
miR-21 delivery, J. Biol. Chem. 289 (2014) 22258–22267.
[50] I. Hazan-Halevy, D. Rosenblum, S. Weinstein, O. Bairey, P. Raanani, D. Peer, Cell-
specific uptake of mantle cell lymphoma-derived exosomes by malignant and non-
malignant B-lymphocytes, Cancer Lett. 364 (2015) 59–69.
[51] Z.J. Cheng, R.D. Singh, E.L. Holicky, C.L. Wheatley, D.L. Marks, R.E. Pagano, Co-
regulation of caveolar and Cdc42-dependent fluid phase endocytosis by phospho-
caveolin-1, J. Biol. Chem. 285 (2010) 15119–15125.
[52] N. Chaudhary, G.A. Gomez, M.T. Howes, H.P. Lo, K.A. McMahon, J.A. Rae,
N.L. Schieber, M.M. Hill, K. Gaus, A.S. Yap, R.G. Parton, Endocytic crosstalk: cavins,
caveolins, and caveolae regulate clathrin-independent endocytosis, PLoS Biol. 12
(2014) e1001832.
[53] S. Kamerkar, V.S. LeBleu, H. Sugimoto, S. Yang, C.F. Ruivo, S.A. Melo, J.J. Lee,
R. Kalluri, Exosomes facilitate therapeutic targeting of oncogenic KRAS in pan-
creatic cancer, Nature (2017).
[54] D. Feng, W.L. Zhao, Y.Y. Ye, X.C. Bai, R.Q. Liu, L.F. Chang, Q. Zhou, S.F. Sui,
Cellular internalization of exosomes occurs through phagocytosis, Traffic 11 (2010)
675–687.
[55] A. Nanbo, E. Kawanishi, R. Yoshida, H. Yoshiyama, Exosomes derived from Epstein-
Barr virus-infected cells are internalized via caveola-dependent endocytosis and
promote phenotypic modulation in target cells, J. Virol. 87 (2013) 10334–10347.
[56] E. Willms, H.J. Johansson, I. Mager, Y. Lee, K.E. Blomberg, M. Sadik, A. Alaarg,
C.I. Smith, J. Lehtio, S. El Andaloussi, M.J. Wood, P. Vader, Cells release sub-
populations of exosomes with distinct molecular and biological properties, Sci Rep
6 (2016) 22519.
[57] J. Kowal, G. Arras, M. Colombo, M. Jouve, J.P. Morath, B. Primdal-Bengtson,
F. Dingli, D. Loew, M. Tkach, C. Thery, Proteomic comparison defines novel mar-
kers to characterize heterogeneous populations of extracellular vesicle subtypes,
Proc. Natl. Acad. Sci. U. S. A. 113 (2016) E968–977.
[58] J. Rejman, V. Oberle, I.S. Zuhorn, D. Hoekstra, Size-dependent internalization of
particles via the pathways of clathrin- and caveolae-mediated endocytosis,
Biochem. J. 377 (2004) 159–169.
[59] H.T. McMahon, E. Boucrot, Molecular mechanism and physiological functions of
clathrin-mediated endocytosis, Nat. Rev. Mol. Cell Biol. 12 (2011) 517–533.
[60] Z. Wang, C. Tiruppathi, J. Cho, R.D. Minshall, A.B. Malik, Delivery of nanoparticle:
complexed drugs across the vascular endothelial barrier via caveolae, IUBMB Life
63 (2011) 659–667.
[61] A. Akinc, G. Battaglia, Exploiting endocytosis for nanomedicines, Cold Spring Harb.
Perspect. Biol. 5 (2013) a016980.