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Controlled and Tunable Loading and Release of Vesicles Using Gigahertz Acoustics


Abstract and Figures

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. Here, we show that gigahertz acoustic streaming generated by a nanoelectromechanical resonator can control the loading and release of cargo into/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 in the vesicle membranes. The size of these pores was estimated to be on the order of 100 nm by loading nanoparticles of different sizes into the vesicles. Forming such pores with gigahertz acoustic streaming provides a non‐invasive method to control materials exchange across membranes of different types of vesicles. This method could allow site‐specific release of therapeutics and controlled loading into cells, as well as tunable microreactors.
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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
Yao Lu+, Wilke C. de Vries+, Nico J. Overeem+, Xuexin Duan,* Hongxiang Zhang, Hao Zhang,
Wei Pang, Bart Jan Ravoo,* 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 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 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 a non-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, as well as tunable
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 a general tool to
achieve compartmentalization for various biomedical appli-
cations, such as drug carriers and micro/nanoscale reac-
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, in applications such as micro/
nanoscale reactors or sustained-release carriers, control over
loading—preferentially remotely—is necessary to change the
amount or dosage of a reagent 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
So far, ultrahigh frequency acoustofluidics has rarely been
studied owing to the lack of such high-frequency acoustic
devices. In this work, we report a method for controlling both
loading and release of materials into and from vesicles
without damaging their structures using the gigahertz (GHz)
acoustic streaming generated by a thin film-based nano-
electromechanical (NEMS) resonator. Such resonators have
recently been reported by us to generate high-speed (>ms1)
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)
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)
W. C. de Vries,[+] Prof. Dr. B. J. Ravoo
Organic Chemistry Institute and Center for Soft Nanoscience (SoN),
Westflische Wilhelms-Universitt Mnster
Correnstr. 40, 48149 Mnster (Germany)
] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
 2018 The Authors. Published by Wiley-VCH Verlag GmbH &Co.
KGaA. This is an open access article under the terms of the Creative
Commons Attribution Non-Commercial NoDerivs License, which
permits use and distribution in any medium, provided the original
work is properly cited, the use is non-commercial, and no
modifications or adaptations are made.
1Angew. Chem. Int. Ed. 2018,57, 1 6  2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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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 a solid 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 a non-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 a model system because of their
reported use as a highly 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 a strepta-
vidin (SAv)-coated glass substrate through biotin-SAv recog-
nition. To facilitate the fluorescence characterization of the
loading and release, multiple PSVs were patterned on a glass
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 a 5 mmCF
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. By gradually extending the duration from 5 to 15 min,
a higher 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 1d and 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 S1 a–
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 S1e indicates that at the applied CF
concentration and power level, the loading is not saturating
within 15 min. The experiments 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 a NEMS-based acoustic resonator of 2.5 GHz.
Figure 1. Controlled loading into and release from immobilized PSVs
under acoustic streaming. a) Schematic of the biotin–SAv binding
motif used for the immobilization of the biotinylated PSVs. The
chemical structure of the PSVs is shown in the zoomed-in cartoon.
Fluorescence imaging of b) the loading of empty line-patterned PSVs
in a solution of 5 mmCF and c) release from CF-encapsulated
(0.1 mm) PSVs into 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).
Communications  2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2018,57,16
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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 a successful 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). The release 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 S3 e). As expected, the intensity
curves shown in Figure 1 e 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 5 min 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.
The red 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 a bi-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 a solution. For this
purpose, we investigated the dye release from PSVs in
solution by using a home-built dialysis system (Figure 2a).
The resonator 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 a dialysis
membrane from the top chamber. Pure buffer was placed in
the top chamber before any stimulation was applied.
The fluorescence intensity of the released CF was
measured in the top chamber upon a release from the PSVs
stimulated at 100, 300, and 500 mW (Figure S5) and plotted as
a function of time. The release curves show a continuously
increasing trend that gradually levels off to a plateau, the
height of which does not depend on the applied power
(Figure 2b). The observed lag time, most apparent at high
power, is attributed to the diffusion time needed for the
released dye to reach the upper chamber. For assessing 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 a linear dependence on power
(Figure 2c). These results suggest that the acoustic streaming
induces the release of dye to a level at which the concen-
tration is balanced between the two chambers. The final
concentration is not dependent on power; however, a faster
release of the dye is achieved at higher power.
The release in solution is more complex than that on
a surface. 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 a relatively 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 PSVs in solution under acoustic
streaming. a) Chamber-based release system. A filter 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 released dye can be determined
by measuring the fluorescence intensity in the top chamber. b) The
fluorescence intensities of the released CF dye as a function of time,
and linear fits of the initial parts of these curves. The fluorescence
intensity at 0 min 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 a function of power. TEM images of
the PSVs d) before and e) after the acoustic stimulation (500 mW for
20 min). Scale bars =100 nm.
3Angew. Chem. Int. Ed. 2018,57, 1 6  2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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To study mechanical deformations of vesicles under
acoustic streaming, GUVs were immobilized on a supported
lipid bilayer (SLB) by the specific biotin–SAv interaction
pairs (Figures S9). GUVs showed a clear 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, to the 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, we tried to load polystyrene nanoparticles (PS
NPs) of various sizes into GUVs under acoustic streaming.
Upon application of continuous acoustic streaming at
300 mW from a fixed 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
a control (Figure 3 a), 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 3 c,d and Figure S14), which indicated
that larger particles were progressively more difficult to
incorporate. When the 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. As a result, the combination of vesicles with the
NEMS resonator can be used as a size-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 a 3D finite-
element model (FEM) simulation (details are provided in the
Supporting Information). The results 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 a general tool to control the transfer of cargo into
and out of vesicles. PSVs, either immobilized on a surface 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. An increased frequency of the device
can also possibly enhance the (un)loading process, by accel-
erating the velocity of the acoustic streaming. For quantitative
prediction of the obtained cargo concentration after a loading
or release experiment, calibration may be needed, as done in
Figures 1 and 2. The mechanical 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. The formation 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 GUVs under acoustic streaming.
CLSM images of TopFluor-labeled GUVs, immobilized on a SLB,
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, 10 min) applied from a fixed distance of 100 mm.
Before imaging, the samples were rinsed three times to remove the
remaining PS NPs from the surrounding solution. Scale bars =10 mm.
Figure 4. FEM simulations of vesicle 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) surrounded by water. The model
resonator was located above the vesicle. The 10-mm vesicle was
located at coordinates of (45 mm, 0 mm, 100 mm) relative to the
resonator center (Figure S16). The frequency of the resonator was
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 stresses across the
surface of the vesicle.
Communications  2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2018,57,16
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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, it can be used as a versatile tool to control
the release and uptake of different materials for different
types of vesicles.
X.D. acknowledges financial support from the Natural
Science Foundation of China (NSFC No. 61176106,
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 a fellowship 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 Twente) is thanked for assistance with
the GUV synthesis. Pieter H. Hamming is thanked for the
MatLab script for image analysis.
Conflict of interest
The authors 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
Accepted manuscript online: November 12, 2018
Version of record online: && &&,&&&&
5Angew. Chem. Int. Ed. 2018,57, 1 6  2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
These are not the final page numbers!
Controlled Release
Y. Lu, W. C. de Vries, N. J. Overeem,
X. Duan,* H. Zhang, H. Zhang, W. Pang,
B. J. Ravoo,* J. Huskens*
&&&& &&&&
Controlled and Tunable Loading and
Release of Vesicles by Using Gigahertz
Sound control: Gigahertz acoustic
streaming generated by a nanoelectrome-
chanical resonator is used to control the
exchange of materials from vesicles by
inducing transient nanopores in the
vesicle membrane.
Communications  2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2018,57,1–6
These are not the final page numbers!
... Sound waves can reversibly alter the structure and morphology of nanovesicle membranes, allowing for the engineering and modification of vesicles at the molecular level [69][70][71]. For instance, sound waves such as ultrasound beams can improve drug loading [72], transportation [73], and release [74] from these vesicles for drug delivery applications. Moreover, sound waves such as low-intensity ultrasound (LIUS) can enhance the generation of EVs by regulating EV biosynthesis, shining light on the large-scale production of EVs, and making them a promising tool for translational medicine applications. ...
Full-text available
Extracellular vesicles are nano- to micro-scale, membrane-bound particles released by cells into extracellular space, and act as carriers of biomarkers and therapeutics, holding promising potential in translational medicine. However, the challenges remain in handling and detecting extracellular vesicles for disease diagnosis as well as exploring their therapeutic capability for disease treatment. Here, we review the recent engineering and technology advances by leveraging the power of sound waves to address the challenges in diagnostic and therapeutic applications of extracellular vesicles and biomimetic nanovesicles. We first introduce the fundamental principles of sound waves for understanding different acoustic-assisted extracellular vesicle technologies. We discuss the acoustic-assisted diagnostic methods including the purification, manipulation, biosensing, and bioimaging of extracellular vesicles. Then, we summarize the recent advances in acoustically enhanced therapeutics using extracellular vesicles and biomimetic nanovesicles. Finally, we provide perspectives into current challenges and future clinical applications of the promising extracellular vesicles and biomimetic nanovesicles powered by sound.
Artificial cells are constructed to imitate natural cells and allow researchers to explore biological process and the origin of life. The construction methods for artificial cells, through both top-down or bottom-up approaches, have achieved great progress over the past decades. Here we present a comprehensive overview on the development of artificial cells and their properties and applications. Artificial cells are derived from lipids, polymers, lipid/polymer hybrids, natural cell membranes, colloidosome, metal-organic frameworks and coacervates. They can be endowed with various functions through the incorporation of proteins and genes on the cell surface or encapsulated inside of the cells. These modulations determine the properties of artificial cells, including producing energy, cell growth, morphology change, division, transmembrane transport, environmental response, motility and chemotaxis. Multiple applications of these artificial cells are discussed here with a focus on therapeutic applications. Artificial cells are used as carriers for materials and information exchange and have been shown to function as targeted delivery systems of personalized drugs. Additionally, artificial cells can function to substitute for cells with impaired function. Enzyme therapy and immunotherapy using artificial cells have been an intense focus of research. Finally, prospects of future development of cell-mimic properties and broader applications are highlighted.
In aptamer-based assay schemes, aptamer probes not labeled with biomarkers have to be eliminated before testing, which may lead to a tremendous waste of precious probes. We herein propose a microfluidics system integrating an aptamer concentration gradient generator (Apt-CGG) and a dual single-cell culturing array (D-SCA), termed Mi-Apt-SCA. This facilitates the precise construction of a nanoscale-gradient microenvironment and the high-throughput profiling of single-cell growth/phenotypes in situ with the minimal consumption of Apt-probe. Unlike previous snakelike mixers, the choreographed winding-ravined aptamer dual-spiral micromixer (Apt-WD-mixer) in Apt-CGG could allow thorough blending to generate linear concentration gradients of aptamer (quasi-non-Newtonian fluid) under the action of continuous fluidic wiggles and bidirectional Dean flow. In contrast to other trap-like systems, the mild vortex allows single-cell growth in an ultra-tender fluidic microenvironment using triple-jarless single-cell culture capsules (TriJ-SCCs) in D-SCA (shear stress: 3.43 × 10-5 dynes per cm2). The minimum dosage of aptamer probe required for exploring PDL1 protein expression in two hepatoma cell lines is only one-900th of that required by conventional protocols. In addition, this approach facilitated the profiling of ITF-β/cisplatin-mediated single-cell/cell-cluster phenotypes.
Cell mechanical motion is a key physiological process that relies on the dynamics of actin filaments. Herein, a localized shear-force system based on gigahertz acoustic streaming (AS) is proposed, which can simultaneously realize intracellular delivery and cellular mechanical regulation. The results demonstrate that gold nanorods (AuNRs) can be delivered into the cytoplasm and even the nuclei of cancer and normal cells within a few minutes by AS stimulation. The delivery efficiency of AS stimulation is four times higher than that of endocytosis. Moreover, AS can effectively promote cytoskeleton assembly, regulate cell stiffness and change cell morphology. Since the inhibitory effect of AuNRs on cytoskeleton assembly, this AuNRs-AS system is able to inhibit or promote cell mechanical motion in a controlled manner by regulating the mechanical properties of cells. The bidirectional regulation of cell motion is further verified via scratch experiments, in which AuNRs-treated cells recover their motion ability through AS stimulation. In particular, the results of AuNRs-AS mechanical regulation on cell are related to the intrinsic properties of cell lines, revealing to more obvious effects on the cells with higher motor capacities. In summary, this acoustic technology has shown superiorities in controllable cell-motion manipulation, indicating its potential in building a multifunctional, integrated cytomechanics regulation platform.
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Polymeric porous capsules represent hugely promising systems that allow a size-selective through-shell material exchange with their surroundings. They have vast potential in applications ranging from drug delivery and chemical microreactors to artificial cell science and synthetic biology. Due to their porous core-shell structure, polymeric porous capsules possess an enhanced permeability that enables the exchange of small molecules while retaining larger compounds and macromolecules. The cross-capsule transfer of material is regulated by their pore size cut-off, which depends on the molecular composition and adopted fabrication method. This review outlines the main strategies for manufacturing polymeric porous capsules and provides some practical guidance for designing polymeric capsules with controlled pore size.
The dynamic functionalization of the nanoparticle surface with biocompatible coatings is a critical step towards the development of functional nano-sized systems. While covalent approaches have been broadly exploited in the stabilization of nanoparticle colloidal systems, these strategies hinder the dynamic nanosurface chemical reconfiguration. Supramolecular strategies based on specific host-guest interactions hold promise due to their intrinsic reversibility, self-healing capabilities and modularity. Host/guest couples have recently been implemented in nanoparticle platforms for the exchange and release of effector molecules. However, the direct exchange of biocompatible hydrophilic oligomers (e.g. peptides) for the modulation of the surface charge and chemical properties of nanoparticles still remains a challenge. Here, we show the intracellular reconfiguration of nanoparticles by a host/guest mechanism with biocompatible oligomeric competitors. The surface of gold nanoparticles was functionalized with cyclodextrin hosts and the guest exchange was studied with biocompatible mono and divalent adamantyl competitors. The systematic characterization of the size and surface potential of the host/guest nanoparticles allowed the optimization of the binding and the stabilization properties of these supramolecular systems. The in cellulo host/guest-mediated direct reconfiguration of the peptide layer at the surface of nanoparticles is achieved by controlling the valence of adamantane-equipped peptides. This work demonstrates that host/guest supramolecular systems can be exploited for the direct exchange of pendants at the surface of nanoparticles and the intracellular dynamic chemical reconfiguration of biocompatible colloidal systems.
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The construction of artificial cells with specific cell-mimicking functions helps to explore complex biological processes and cell functions in natural cell systems and provides an insight into the origins of life. Bottom-up methods are widely used for engineering artificial cells based on vesicles by the in vitro assembly of biomimetic materials. In this review, the design of artificial cells with a specific function is discussed, by considering the selection of synthetic materials and construction technologies. First, a range of biomimetic materials for artificial cells is reviewed, including lipid, polymeric and hybrid lipid/copolymer materials. Biomaterials extracted from natural cells are also covered in this part. Then, the formation of microscale, giant unilamellar vesicles (GUVs) is reviewed based on different technologies, including gentle hydration, electro-formation, phase transfer and microfluidic methods. Subsequently, applications of artificial cells based on single vesicles or vesicle networks are addressed for mimicking cell behaviors and signaling processes. Microreactors for synthetic biology and cell-cell communication are highlighted here as well. Finally, current challenges and future trends for the development and applications of artificial cells are described.
A surge of research in intracellular delivery technologies is underway with the increased innovations in cell‐based therapies and cell reprogramming. Particularly, physical cell membrane permeabilization techniques are highlighted as the leading technologies because of their unique features, including versatility, independence of cargo properties, and high‐throughput delivery that is critical for providing the desired cell quantity for cell‐based therapies. Amongst the physical permeabilization methods, sonoporation holds great promise and demonstrates to deliver a variety of functional cargos, such as biomolecular drugs, proteins, and plasmids, to various cells including cancer, immune, and stem cells. However, traditional bubble‐based sonoporation methods usually require special contrast agents. Bubble‐based sonoporation methods also have high chances of inducing irreversible damage to critical cell components, lowering the cell viability, and reducing the effectiveness of delivered cargos. To overcome these limitations, several novel non‐bubble‐based sonoporation mechanisms are under development. This review will cover both the bubble‐based and non‐bubble‐based sonoporation mechanisms being employed for intracellular delivery, the technologies being investigated to overcome the limitations of traditional platforms, as well as perspectives on the future sonoporation mechanisms, technologies, and applications. This review presents a comprehensive evaluation of the current state of sonoporation research and its advantages and limitations. Particularly, this review covers the current bubble‐based sonoporation mechanisms and the novel upcoming non‐bubble‐based sonoporation mechanisms and their respective technologies that are utilized to enhance intracellular delivery. This review concludes with a perspective on how the field of sonoporation can advance.
Rapid and personalized single-cell drug screening testing plays an essential role in acute myeloid leukemia drug combination chemotherapy. Conventional chemotherapeutic drug screening is a time-consuming process because of the natural resistance of cell membranes to drugs, and there are still great challenges related to using technologies that change membrane permeability such as sonoporation in high-throughput and precise single-cell drug screening with minimal damage. In this study, we proposed an acoustic streaming-based non-invasive single-cell drug screening acceleration method, using high-frequency acoustic waves (>10 MHz) in a concentration gradient microfluidic device. High-frequency acoustics leads to increased difficulties in inducing cavitation and generates acoustic streaming around each single cell. Therefore, single-cell membrane permeability is non-invasively increased by the acoustic pressure and acoustic streaming-induced shear force, which significantly improves the drug uptake process. In the experiment, single human myeloid leukemia mononuclear (THP-1) cells were trapped by triangle cell traps in concentration gradient chips with different cytarabine (Ara-C) drug concentrations. Due to this dual acoustic effect, the drugs affect cell viability in less than 30 min, which is faster than traditional methods (usually more than 24 h). This dual acoustic effect-based drug delivery strategy has the potential to save time and reduce the cost of drug screening, when combined with microfluidic technology for multi-concentration drug screening. This strategy offers enormous potential for use in multiple drug screening or efficient drug combination screening in individualized/personalized treatments, which can greatly improve efficiency and reduce costs.
Synthetic polymersomes have structure similarity to bio-vesicles and could disassemble in response to stimuli for "on-demand" release of encapsulated cargos. Though widely applied as a drug delivery carrier, the burst release mode with structure complete destruction is usually taken for most responsive polymersomes, which would shorten the effective drug reaction time and impair the therapeutic effect. Inspired by the cell organelles' communication mode via regulating membrane permeability for transportation control, we highlight here a biomimetic polymersome with sustained drug release over a specific period of time via near-infrared (NIR) pre-activation. The polymersome is prepared by the self-assembling amphiphilic diblock copolymer P(OEGMA-co-EoS)-b-PNBOC and encapsulates the hypoxia-activated prodrug AQ4N and upconversion nanoparticle (PEG-UCNP) in its hydrophilic centric cavity. Thirty minutes of NIR pre-activation triggers cross-linking of NBOC and converts the permeability of the polymersome with sustained AQ4N release until 24 h after the NIR pre-activation. The photosensitizer EoS is activated and aggravates environmental hypoxic conditions during a sustained drug release period to boost the AQ4N therapeutic effect. The combination of sustained drug release with concurrent hypoxia intensification results in a highly efficient tumor therapeutic effect both intracellularly and in vivo. This biomimetic polymersome will provide an effective and universal tumor therapeutic approach.
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Conspectus Cells are highly advanced microreactors that form the basis of all life. Their fascinating complexity has inspired scientists to create analogs from synthetic and natural components using a bottom-up approach. The ultimate goal here is to assemble a fully man-made cell that displays functionality and adaptivity as advanced as that found in nature, which will not only provide insight into the fundamental processes in natural cells but also pave the way for new applications of such artificial cells. In this Account, we highlight our recent work and that of others on the construction of artificial cells. First, we will introduce the key features that characterize a living system; next, we will discuss how these have been imitated in artificial cells. First, compartmentalization is crucial to separate the inner chemical milieu from the external environment. Current state-of-the-art artificial cells comprise subcompartments to mimic the hierarchical architecture of eukaryotic cells and tissue. Furthermore, synthetic gene circuits have been used to encode genetic information that creates complex behavior like pulses or feedback. Additionally, artificial cells have to reproduce to maintain a population. Controlled growth and fission of synthetic compartments have been demonstrated, but the extensive regulation of cell division in nature is still unmatched. Here, we also point out important challenges the field needs to overcome to realize its full potential. As artificial cells integrate increasing orders of functionality, maintaining a supporting metabolism that can regenerate key metabolites becomes crucial. Furthermore, life does not operate in isolation. Natural cells constantly sense their environment, exchange (chemical) signals, and can move toward a chemoattractant. Here, we specifically explore recent efforts to reproduce such adaptivity in artificial cells. For instance, synthetic compartments have been produced that can recruit proteins to the membrane upon an external stimulus or modulate their membrane composition and permeability to control their interaction with the environment. A next step would be the communication of artificial cells with either bacteria or another artificial cell. Indeed, examples of such primitive chemical signaling are presented. Finally, motility is important for many organisms and has, therefore, also been pursued in synthetic systems. Synthetic compartments that were designed to move in a directed, controlled manner have been assembled, and directed movement toward a chemical attractant is among one of the most life-like directions currently under research. Although the bottom-up construction of an artificial cell that can be truly considered “alive” is still an ambitious goal, the recent work discussed in this Account shows that this is an active field with contributions from diverse disciplines like materials chemistry and biochemistry. Notably, research during the past decade has already provided valuable insights into complex synthetic systems with life-like properties. In the future, artificial cells are thought to contribute to an increased understanding of processes in natural cells and provide opportunities to create smart, autonomous, cell-like materials.
We present the preparation of ligand conjugated redox-responsive polymer nanocontainers by the supramolecular decoration of cyclodextrin vesicles with a thin redox-cleavable polymer shell that displays molecular recognition units on its surface. Two widely different recognition motifs (mannose–Concanavalin A and biotin–streptavidin) are compared and the impact of ligand density is studied using aggregation assays, dynamic light scattering, and a fluorometric quantification of ligands. Microcontact printing is used to prepare streptavidin-patterned surfaces and the specific immobilization of biotin conjugated nanocontainers is demonstrated. As a prototype of a nanosensor, these tethered nanocontainers can sense a reductive environment and react by releasing a payload.
The development of smart drug delivery systems to realize controlled drug release for highly specific cancer treatment has attracted tremendous attention. Herein, nanoscale coordination polymers (NCPs) constructed from hafnium ions and bis-(alkylthio) alkene (BATA), a singlet-oxygen responsive linker, are fabricated and applied as nanocarriers to realize light-controlled drug release under a rather low optical power density. In this system, NCPs synthesized through a solvothermal method are sequentially loaded with chlorin e6 (Ce6), a photosensitizer, and doxorubicin (DOX), a chemotherapeutic drug, and then coated with lipid bilayer to allow modification with polyethylene glycol (PEG) to acquire excellent colloidal stability. The singlet oxygen produced by such NCP-Ce6-DOX-PEG nanocomposite can be used not only for photodynamic therapy, but also to induce the break of BATA linker and thus the destruction of nanoparticle structures under light exposure, thereby triggering effective drug release. Notably, with efficient tumor accumulation after intravenous injection as revealed by CT imaging, those NCP-Ce6-DOX-PEG nanoparticles could be utilized for combined chemo-photodynamic therapy with great antitumor efficacy. Thus, this work presents a unique type of NCP-based drug delivery system with biodegradability, sensitive responses to light, as well as highly efficient tumor retention for effective cancer combinational treatment.
Nonspecific binding (NSB) is a general issue for surface based biosensors. Various approaches have been developed to prevent or remove the NSBs. However, these approaches either increased the background signals of the sensors or limited to specific transducers interface. In this work, we developed a hydrodynamic approach to selectively remove the NSBs using a microfabricated hypersonic resonator with 2.5 gigahertz (GHz) resonant frequency. The high frequency device facilitates to generate multiple controlled micro-vortices which then create cleaning forces at the solid-liquid interfaces. The competitive adhesive and cleaning forces have been investigated using the finite element method (FEM) simulation, identifying the feasibility of the vortices induced NSB removal. NSB proteins have been selectively removed experimentally both on the surface of the resonator and on other substrates which contact the vortices. Thus, the developed hydrodynamic approach is believed to be a simple and versatile tool for NSB removal and compatible to many sensor systems. The unique feature of the hypersonic resonator is that it can be used as a gravimetric sensor as well, thus a combined NSB removal and protein detection dual functional biosensor system is developed.
Multi-compartmentalization is a key feature of eukaryotic cells, allowing separation and protection of species within the membrane walls. During the last years, several methods have been reported to afford synthetic multi-compartment lipidic or polymeric vesicles that mimic biological cells and that allow cascade chemical or enzymatic reactions within their lumen. We hereby report on the preparation and study of liposomes in polymersomes (LiPs) systems. We discuss on the loading and co-loading of lipidic nano-vesicles made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipentadecanoyl-sn-glycero-3-phosphocholine (diC15-PC), or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) inside the lumen of giant poly(butadiene)-b-poly(ethylene oxide) (PBut-b-PEO) polymersomes. These LiPs systems were characterized by confocal microscopy and UV-visible spectroscopy. We further demonstrate that we can achieve controlled sequential release of dyes from diC15-PC and DPPC liposomes at defined temperatures inside the giant PBut-b-PEO polymersomes. This controlled release could be used as a means to initiate cascade reactions on demand in confined micro-reactors.
We present self-assembly of redox-responsive polymer nanocontainers comprising a cyclodextrin vesicle core and a thin reductively cleavable polymer shell anchored via host-guest recognition on the vesicle surface. The nanocontainers are of uniform size, show high stability and selectively respond to a mild reductive trigger as revealed by dynamic light scattering, transmission electron microscopy, atomic force microscopy, a quantitative thiol assay and fluorescence spectroscopy. Live cell imaging experiments demonstrate a specific redox-responsive release and cytoplasmic delivery of encapsulated hydrophilic payloads such as the pH-probe pyranine, and the fungal toxin phalloidin. Our results show the high potential of these stimulus-responsive nanocontainers for cell biological applications requiring a controlled delivery.
We present self-assembly of redox-responsive polymer nanocontainers comprising a cyclodextrin vesicle core and a thin reductively cleavable polymer shell anchored via host-guest recognition on the vesicle surface. The nanocontainers are of uniform size, show high stability and selectively respond to a mild reductive trigger as revealed by dynamic light scattering, transmission electron microscopy, atomic force microscopy, a quantitative thiol assay and fluorescence spectroscopy. Live cell imaging experiments demonstrate a specific redox-responsive release and cytoplasmic delivery of encapsulated hydrophilic payloads such as the pH-probe pyranine, and the fungal toxin phalloidin. Our results show the high potential of these stimulus-responsive nanocontainers for cell biological applications requiring a controlled delivery.
On-chip integrating several functional components for developing integrated lab-on-a-chip microsystem remains as a challenge. In this work, by employing multiple microelectromechanical resonators both as actuators and sensors, on-chip heating, mixing and chemical reaction monitoring are successfully demonstrated. Mechanism studies using COMSOL simulations indicate that the local heating and mixing are induced by the acoustic wave attenuation during its transmission in liquid. On-line chemical reaction monitoring is realized by viscosity sensing using the same resonator through impedance analysis. Classic Diels-Alder reaction in a single droplet was performed to verify the feasibility of using such microsystem for mixing, heating and online reaction monitoring at microscale.
Efficient delivery of genes and therapeutic agents to the interior of the cell is critical for modern biotechnology. Herein, a new type of chemical-free cell poration method— hypersonic poration—is developed to improve the cellular uptake, especially the nucleus uptake. The hypersound (≈GHz) is generated by a designed piezoelectric nano-electromechanical resonator, which directly induces normal/shear stress and “molecular bombardment” effects on the bilayer membranes, and creates reversible temporal nanopores improving the membrane permeability. Both theory analysis and cellular uptake experiments of exogenous compounds prove the high delivery efficiency of hypersonic poration. Since target molecules in cells are accumulated with the treatment, the delivered amount can be controlled by tuning the treatment time. Furthermore, owing to the intrinsic miniature of the resonator, localized drug delivery at a confined spatial location and tunable arrays of the resonators that are compatible with multiwell plate can be achieved. The hypersonic poration method shows great delivery efficacy combined with advantage of scalability, tunable throughput, and simplification in operation and provides a potentially powerful strategy in the field of molecule delivery, cell transfection, and gene therapy.
This study reports the self-assembly of a novel polymer vesicles from an amphiphilic multiblock copolyamide, and the vesicles have shown a special structure of ultrathin wall thickness of about 4.5 nm and of a combined bilayer and monolayer packing model. Most interestingly, the vesicles are ultrasound-responsive and can release the encapsulated model drugs in response to ultrasonic irradiation.