Controlled and Tunable Loading and Release of Vesicles by Using Gigahertz Acoustics

Abstract

Controllable exchange of molecules between the interior and the external environment of vesicles is critical in drug delivery and micro/nano‐reactors. While many approaches exist to trigger release from vesicles, controlled loading remains a challenge. Herein, we show that gigahertz acoustic streaming generated by a nanoelectromechanical resonator can control the loading and release of cargo into and from vesicles. Polymer‐shelled vesicles showed loading and release of molecules both in solution and on a solid substrate. We observed deformation of individual giant unilamellar vesicles and propose that the shear stress generated by gigahertz acoustic streaming induces the formation of transient nanopores, with diameters on the order of 100 nm, in the vesicle membranes. This provides a non‐invasive method to control material exchange across membranes of different types of vesicles, which could allow site‐specific release of therapeutics and controlled loading into cells, as well as tunable microreactors.

Full text

German Edition:DOI:10.1002/ange.201810181
Controlled Release International Edition:DOI:10.1002/anie.201810181
Controlled and Tunable Loading and Release of Vesicles by Using
Gigahertz Acoustics
YaoLu
+,Wilke C. de Vries+,Nico J. Overeem+,Xuexin Duan,* Hongxiang Zhang,Hao Zhang,
WeiPang,Bart JanRavoo,* and Jurriaan Huskens*
Abstract: Controllable exchange of molecules between the
interior and the external environment of vesicles is critical in
drug delivery and micro/nano-reactors.While many
approaches exist to trigger release from vesicles,controlled
loading remains achallenge.Herein, we show that gigahertz
acoustic streaming generated by ananoelectromechanical
resonator can control the loading and release of cargo into
and from vesicles.Polymer-shelled vesicles showed loading
and release of molecules both in solution and on asolid
substrate.Weobserved deformation of individual giant uni-
lamellar vesicles and propose that the shear stress generated by
gigahertz acoustic streaming induces the formation of transient
nanopores,with diameters on the order of 100 nm, in the
vesicle membranes.This provides anon-invasive method to
control material exchange across membranes of different types
of vesicles,which could allow site-specific release of therapeu-
tics and controlled loading into cells,aswell as tunable
microreactors.
Current challenges in biomedical therapies lie mostly at its
interface with physics,chemistry,and engineering.Control
over matter at various length and time scales is targeted, by
drawing on concepts such as compartmentalization,[1] smart
materials,[2] and process triggers,[3] with the aim to design, for
example,artificial cells.[4,5] With biomimetic bilayer struc-
tures,artificial vesicles have been applied as ageneral tool to
achieve compartmentalization for various biomedical appli-
cations,such as drug carriers and micro/nanoscale reac-
tors.[6–11]
One of the key challenges in the development of vesicles
as reservoirs for localized storage and nano-vessels for
reactions is the controllable exchange of compounds between
the interior and the exterior of the vesicles,that is,the loading
or release of cargo into or from the vesicles.Numerous studies
have used triggers to quantitatively control the release of
molecular cargo from vesicles,which were internal such as
pH[12–14] and redox state[15–17] or external such as light,[2,18,19]
temperature,[20–22] ultrasound,[23–25] and magnetic field.[26–28] In
these triggered systems,the release rates have important
implications,for example,for the therapeutic activities of
various types of drug delivery systems.[29–31]
In contrast with the advancements in controlled release,it
is still highly challenging to achieve controlled loading. In
most cases,substances are preloaded into vesicles when they
are prepared by extrusion or sonication methods,[32,33] so that
the primary loading concentration is predetermined and
cannot be modified. However,inapplications such as micro/
nanoscale reactors or sustained-release carriers,control over
loading—preferentially remotely—is necessary to change the
amount or dosage of areagent at will.[34] Examples exist in
which pH-sensitive materials have been included into poly-
mer capsules to control the loading efficiency by tuning
proton gradients or temperature.[35–37] However,these meth-
ods are either limited by specific types of chemistry or are
restricted to cargos with specific properties.[38] Thus,con-
trolled loading and unloading of vesicles require general tools
that depend on the chemistry of neither the vesicles nor the
cargo.
So far, ultrahigh frequency acoustofluidics has rarely been
studied owing to the lack of such high-frequencyacoustic
devices.Inthis work, we report amethod for controlling both
loading and release of materials into and from vesicles
without damaging their structures using the gigahertz (GHz)
acoustic streaming generated by athin film-based nano-
electromechanical (NEMS) resonator.Such resonators have
recently been reported by us to generate high-speed (>ms@1)
acoustic streaming with strong forces (>nn), which has been
applied to enhance the solution mixing in microfluidic chips[39]
and to remove nonspecific binding at solid–liquid interfa-
ces.[40] Since acoustic streaming can exert mechanical forces
on cells that are immobilized at the solid–liquid interface,[41]
we envisaged that vesicles,which are soft and hollow
structures,would also be affected and could experience
[*] Dr.Y.Lu,[+] Prof. Dr.X.Duan, H. Zhang, Prof. Dr.H.Zhang,
Prof. Dr.W.Pang
State Key Laboratory of Precision Measuring Technology &Instru-
ments, Tianjin University
Tianjin 300072 (China)
E-mail:xduan@tju.edu.cn
Dr.Y.Lu,[+] N. J. Overeem,[+] Prof. Dr.J.Huskens
Molecular Nanofabrication group
MESA+Institute for Nanotechnology,University of Twente
7500 AE, Enschede (The Netherlands)
E-mail:j.huskens@utwente.nl
W. C. de Vries,[+] Prof. Dr.B.J.Ravoo
Organic Chemistry Institute and Center for Soft Nanoscience (SoN),
Westf-lische Wilhelms-Universit-tMfnster
Correnstr.40, 48149 Mfnster (Germany)
E-mail:b.j.ravoo@uni-muenster.de
[++]These authors contributed equally to this work.
Supportinginformation and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.201810181.
T2018 The Authors. Published by Wiley-VCH Verlag GmbH &Co.
KGaA. This is an open access article under the terms of the Creative
Commons AttributionNon-CommercialNoDerivs License, which
permitsuse and distribution in any medium, provided the original
work is properly cited, the use is non-commercial, and no
modifications or adaptations are made.
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mechanical deformation under such acoustic stimulation. We
hypothesized that owing to the fluidic nature of the lipid
membranes,mechanical deformation of the vesicles might
induce transient pores in the membrane,which would change
the membrane permeability and facilitate materials exchange
between the interior and exterior of the vesicles (Scheme 1).
Polymer-shelled vesicles (PSVs)[42,43] were used to dem-
onstrate the controlled loading and release of cargo both in
solution and on asolid substrate.The loading and unloading
rates were determined by fluorescence measurements.Real-
time deformation of giant unilamellar vesicles (GUVs) was
investigated to obtain insights into the mechanism of the
acoustic-controlled materials exchange,which was further
evaluated by finite element modeling (FEM) simulations.
Polystyrene nanoparticles (PS NPs) of different sizes were
loaded into the GUVs to test the uptake limits,thus to
estimate the pore size generated by the GHz acoustic
streaming. Since our approach is purely physical, using
hydrodynamic forces induced by acoustic streaming,it
provides anon-invasive way to control the loading and
release of various substances into or from vesicles with
different sizes and compositions.
To demonstrate the tunable loading and release of cargo,
we employed PSVs as amodel system because of their
reported use as ahighly stable nanocontainer for intracellular
delivery.[42, 44] As shown in Figure 1a,PSVs were prepared
from cyclodextrin vesicles,onto which adamantyl-functional-
ized poly(acrylic acid) (Ad-PAA) was attached by host–guest
recognition, followed by the cross-linking of the carboxylic
acid groups and conjugation with biotin to the PSV surface to
allow the specific immobilization of the vesicles on astrepta-
vidin (SAv)-coated glass substrate through biotin-SAvrecog-
nition. To facilitate the fluorescence characterization of the
loading and release,multiple PSVs were patterned on aglass
substrate using micromolding in capillaries (MIMIC).[45]
We first tested the controlled loading process.The
carboxyfluorescein (CF) dye was used as the cargo to be
loaded into PSVs to facilitate fluorescence imaging. Time-
dependent fluorescence images of the PSVs without and with
the acoustic stimulation are shown in Figure 1b.The surface-
patterned and empty PSVs were incubated in a5mmCF
solution. Without stimulation, the empty PSVs did not show
any fluorescence changes even after prolonged incubation
(15 min). However,the empty PSVs stimulated by acoustic
streaming at 100 mW exhibited an increased green fluores-
cence.Bygradually extending the duration from 5to15min,
ahigher fluorescence intensity was observed in the patterned
areas,which indicates that the CF dye was successfully loaded
into the vesicles and the loaded amount is dependent on the
stimulation time.The loading kinetics extracted from the
fluorescence measurements at different power levels (Fig-
ure S1) show that more CF dye is loaded into the vesicles at
higher power (Figure 1dand Figure S1 e) and that the loading
rate is approximately linear to the applied power (Fig-
ure S1 f). Controls in the absence of hypersound (Figure S1a–
d) indicate no fluorescence intensity increase,supporting the
conclusion that the intensities observed in the presence of
hypersound are caused by uptake into the vesicles.The data
shown in Figure S1 eindicates that at the applied CF
concentration and power level, the loading is not saturating
within 15 min. Theexperiments suggest that control over the
Scheme 1. Schematic of the acoustically controlled loading (top) and
release (bottom) of empty and pre-filled vesicles,respectively, through
the use of aNEMS-based acoustic resonator of 2.5 GHz.
Figure 1. Controlled loading into and release from immobilizedPSVs
under acoustic streaming. a) Schematic of the biotin–SAvbinding
motif used for the immobilization of the biotinylatedPSVs. The
chemical structure of the PSVsisshown in the zoomed-in cartoon.
Fluorescenceimaging of b) the loading of empty line-patterned PSVs
in asolution of 5mmCF and c) release from CF-encapsulated
(0.1 mm)PSVsinto water,without or with the acoustic stimulation at
100 mW for different durations. Scale bars=10 mm. Time-dependent
changes of fluorescence intensity with d) the loading (from experi-
ments shown in (b) and Figure S1) and e) the release of the CF dye
(from experiments shown in (c) and Figure S2) stimulated at different
power levels (100, 300, and 500 mW).
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loaded amount of cargo is feasible by variation of the power
and loading time.
To test the controlled release,immobilized PSVs that
were pre-loaded with CF were subjected to acoustic stream-
ing at 100 mW (Figure 1c). Compared to the sample without
stimulation, the acoustic streaming induced an obvious
decrease of fluorescence intensity in the patterned areas,
indicating asuccessful release of the CF dye.Similarly,by
increasing the power from 100 to 500 mW (Figure S2), the
green fluorescence of the patterned PSVs decreased faster
(Figure 1e)whereas the fluorescence intensity of the CF
released into the sealed chamber increased accordingly
(Figure S3a–d). Therelease kinetics derived from the fluo-
rescence changes in the sealed chamber show that the release
rate is approximately linearly dependent on the power of the
acoustic streaming (Figure S3e). As expected, the intensity
curves shown in Figure 1e follow an exponential decay trend
(as fitted in Figure S2e), which is also linearly dependent on
power (Figure S2 f), and complete release is achieved within
10–30 min.
We note that the relative fluorescence changes occurring
within the initial 5min of the release process (Figure 1e)are
larger than those observed during loading (Figure 1d), which
can be related to the different kinetics induced by the highly
different CF concentrations.
To evaluate possible damage to the vesicle structure
induced by the acoustic streaming, the cyclodextrin amphi-
philes in the PSVs were covalently labeled with rhodamine B.
Thered fluorescence intensity of rhodamine B-labeled PSVs
did not change after 20 min of acoustic stimulation at 500 mW
(Figure S4), confirming that the immobilized PSVs remained
intact and did not detach from the surface along the
acoustically generated vortex. Instead, the encapsulated dye
was released under the acoustic streaming.
These results demonstrate that the acoustic streaming-
triggered exchange of cargo from PSVs is abi-directional
process and that the (un)loading rates can be controlled by
the input power of the acoustic streaming.
In many applications,control over release is required
when the vesicles are suspended in asolution. Forthis
purpose,weinvestigated the dye release from PSVs in
solution by using ahome-built dialysis system (Figure 2a).
Theresonator was placed at the bottom of the system to
generate the acoustic streaming. CF-encapsulated PSVs were
preloaded in the bottom chamber and separated by adialysis
membrane from the top chamber.Pure buffer was placed in
the top chamber before any stimulation was applied.
Thefluorescence intensity of the released CF was
measured in the top chamber upon arelease from the PSVs
stimulated at 100, 300, and 500 mW (Figure S5) and plotted as
afunction of time.The release curves show acontinuously
increasing trend that gradually levels off to aplateau, the
height of which does not depend on the applied power
(Figure 2b). Theobserved lag time,most apparent at high
power, is attributed to the diffusion time needed for the
released dye to reach the upper chamber. Forassessing the
initial rates,this effect is ignored, and the initial sections of the
curves were fitted linearly.These initial slopes are regarded as
the release rates,which show alinear dependence on power
(Figure 2c). These results suggest that the acoustic streaming
induces the release of dye to alevel at which the concen-
tration is balanced between the two chambers.The final
concentration is not dependent on power;however, afaster
release of the dye is achieved at higher power.
Therelease in solution is more complex than that on
asurface.The high-velocity acoustic steaming accelerates the
motion of the suspended PSVs,which may enhance the
frequencies of vesicle–vesicle and vesicle–interface collisions.
Moreover,the high-speed mixing owing to the strong vortex
may facilitate the diffusion of the dye through the filter
membrane.Such combined effects will enhance the translo-
cation of the dye across the dialysis film.
To evaluate whether the acoustic streaming caused any
structural changes of the suspended PSVs,transmission
electron microscopy (TEM) was performed. Before the
acoustic stimulation, PSVs appeared as circular objects as
typically observed for vesicles (Figure 2d), and no apparent
difference was observed after the acoustic treatment at
500 mW for 20 min (Figure 2e). This suggests that the high-
speed collisions and mixing do not damage the vesicle
structures and that the release of cargo can be turned on
and off,which can be attributed to the formation of transient
nanopores in the membrane of the vesicle.The intact shape
and minimally affected size of the PSVs,which was also
confirmed by dynamic light scattering (DLS) measurements
(Figure S7), could indicate arelatively stable re-loading/
release capacity of PSVs under this switchable acoustic
trigger,but repeated loading/release was not attempted in
this study.
Figure 2. Controlled release from PSVsinsolution under acoustic
streaming. a) Chamber-based release system.Afilter membrane
(molecular weight cut-off=12–15 kDa) was sandwiched between two
PDMS chambers to keep the PSV-encapsulated CF dye in the lower
chamber,while the liberated CF dye can pass through the filter
membrane.Thus, the amount of the releaseddye can be determined
by measuring the fluorescence intensity in the top chamber.b)The
fluorescence intensities of the released CF dye as afunctionoftime,
and linear fits of the initial parts of these curves. The fluorescence
intensity at 0min was obtained by measuring the buffer solution in
the upper chamber without stimulation after waiting for 20 min (Fig-
ure S6). c) Initial release rates as afunction of power.TEM images of
the PSVsd)before and e) after the acoustic stimulation (500 mW for
20 min). Scale bars =100 nm.
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To study mechanical deformations of vesicles under
acoustic streaming, GUVs were immobilized on asupported
lipid bilayer (SLB) by the specific biotin–SAvinteraction
pairs (Figures S9). GUVs showed aclear deformation under
acoustic streaming but recovered their initial shape instantly
once the power was turned off.This dependence on the input
power (Figure S10 and Videos S1–S4) and the vertical
distance (Figure S11 and Videos S5–S8) were attributed,
respectively,tothe velocity and the spatial distribution of
the acoustic streaming.
To test the hypothesis that the mechanical deformation of
vesicles creates pores in the membrane and estimate the size
of these pores,wetried to load polystyrene nanoparticles (PS
NPs) of various sizes into GUVs under acoustic streaming.
Upon application of continuous acoustic streaming at
300 mW from afixed vertical distance of 100 mm(Figure S8 a)
to the GUVs for 10 min, we observed (Figure 3b)the blue
fluorescence from 50 nm PS NPs inside the vesicles.As
acontrol (Figure 3a), vesicles were incubated with PS NPs for
10 min without stimulation and blue fluorescence was not
observed inside the GUVs.This indicates that the acoustic
streaming is necessary to allow the transport of 50 nm PS NPs
into the vesicles.The power dependence and reproducibility
of the loading are shown in Figures S14 and S15, respectively.
To evaluate the size of the pores formed by acoustic
streaming, we performed similar experiments with PS NPs of
100 and 200 nm (Figure 3c,d and Figure S14), which indicated
that larger particles were progressively more difficult to
incorporate.Whenthe power of the acoustic streaming was
increased to 500 mW,both the blue (50 nm PS NPs) and the
orange fluorescence (100 nm PS NPs) intensities increased,
while the 200 nm PS NPs still stayed outside the vesicle
(Figure S14). Therefore,the loading of nanoparticles is power
dependent, which we assume is related to the dynamic pore
formation process generated during the deformation of
vesicles.Asaresult, the combination of vesicles with the
NEMS resonator can be used as asize-based filter to
exchange particles or other materials of specific sizes.
To understand the mechanism of vesicle deformation
induced by the acoustic streaming, we used a3Dfinite-
element model (FEM) simulation (details are provided in the
Supporting Information). Theresults show that the displace-
ment across the surface of the vesicle is non-uniformly
distributed under the acoustic streaming, which indicates the
deformation of the vesicle (Figure 4).
We have demonstrated that GHz acoustic streaming can
be used as ageneral tool to control the transfer of cargo into
and out of vesicles.PSVs,either immobilized on asurface or
suspended in solution, were used to study the acoustically
triggered loading and release processes.The kinetics of
loading and release can be tuned through the applied power
and potentially by other parameters such as cargo concen-
tration and vesicle type.Anincreased frequencyofthe device
can also possibly enhance the (un)loading process,byaccel-
erating the velocity of the acoustic streaming.For quantitative
prediction of the obtained cargo concentration after aloading
or release experiment, calibration may be needed, as done in
Figures 1and 2. Themechanical deformation of individual
GUVs was analyzed by simulation and experiment to under-
stand the materials exchange across the vesicle membrane.
We have proposed that transient nanopores are generated in
the membrane when vesicles are deformed by the acoustic
streaming. Theformation of nanopores increases the mem-
brane permeability and allows the transport of materials both
into and out of the vesicles.The size of these pores is such that
it allows transport of 100 nm, but not of 200 nm, PS NPs into
the GUVs.Both GUVs and PSVs stay intact during the
acoustic streaming,which confirms the non-invasive nature of
Figure 3. Loading of PS NPs into GUVsunder acoustic streaming.
CLSM images of TopFluor-labeled GUVs, immobilized on aSLB,
loaded with a,b) PS NPs of 50 (blue), c) 100 (orange) and d) 200 nm
(red fluorescence) without (control, (a)) and with (b,c,d) acoustic
streaming (300 mW,10min) applied from afixed distance of 100 mm.
Before imaging, the sampleswere rinsed three times to remove the
remaining PS NPs from the surrounding solution. Scale bars=10 mm.
Figure 4. FEM simulationsofvesicle deformation under acoustic
streaming. a) Simulated patterns of the acoustic streaming distributed
around the resonator.The vesicle was represented by an elastic and
hollow sphere (inside medium, water) surroundedbywater.The model
resonator was located above the vesicle. The 10-mmvesicle was
located at coordinates of (45 mm, 0 mm, @100 mm) relative to the
resonator center (Figure S16). The frequency of the resonatorwas
2.5 GHz. Simulated patterns of the displacement of the vesicle are
shown in the zoomed-in image (right). The right color bar indicates
the magnitude of the streaming velocity outside the vesicle from min
(blue) to max (red), while the left color bar indicates the magnitude of
the displacement of the vesicle from min (red) to max (pink). 3D plots
of b) the total shear stress in the xdirection and c) the mechanical
deformation in terms of aspect ratio of the vesicle at different power
levels (100, 300, and 500 mW) and different vertical distances (50,
100, 200, and 300 mm). The total shear stress in the xdirection was
obtained by the integration of the x-partial shear stressesacross the
surface of the vesicle.
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this acoustic approach. We showed that the GHz acoustic
poration effect can be applied to vesicles of different sizes and
compositions.Thus,itcan be used as aversatile tool to control
the release and uptake of different materials for different
types of vesicles.
Acknowledgements
X.D.acknowledges financial support from the Natural
Science Foundation of China (NSFC No.61674114,
91743110, 21861132001), National Key R&D Program of
China (2017YFF0204600), and the 111 Project (B07014).
N.J. O. and J.H. acknowledge financial support from the
Volkswagen Foundation (FlapChips project, 91-056). W.C.V.
acknowledges afellowship of the Fonds der Chemischen
Industrie.W.C.V.and B.J. R. sincerely thank the Deutsche
Forschungsgemeinschaft (DFG SFB858) for funding. Mat-
thias Tesch is acknowledged for providing assistance with the
synthesis of Ad-PAA, and Nadja Mçller for assistance with
TEM imaging.M.A. Abolghassemi Fakhree (Nanobiophysics
group,University of Tw ente) is thanked for assistance with
the GUV synthesis.Pieter H. Hamming is thanked for the
MatLab script for image analysis.
Conflict of interest
Theauthors declare no conflict of interest.
Keywords: controlled loading ·controlled release ·
gigahertz acoustic streaming ·transient nanopores ·vesicles
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Manuscript received:September 6, 2018
Acceptedmanuscript online: November 12, 2018
Version of record online: November 30, 2018
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