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Programmable multi-DNA release from multilayered polyelectrolytes using gigahertz nano-electromechanical resonator

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Programmable multi-DNA release from multilayered polyelectrolytes using gigahertz nano-electromechanical resonator

Abstract and Figures

Background Controllable and multiple DNA release is critical in modern gene-based therapies. Current approaches require complex assistant molecules for combined release. To overcome the restrictions on the materials and environment, a novel and versatile DNA release method using a nano-electromechanical (NEMS) hypersonic resonator of gigahertz (GHz) frequency is developed. Results The micro-vortexes excited by ultra-high frequency acoustic wave can generate tunable shear stress at solid–liquid interface, thereby disrupting molecular interactions in immobilized multilayered polyelectrolyte thin films and releasing embedded DNA strands in a controlled fashion. Both finite element model analysis and experiment results verify the feasibility of this method. The release rate and released amount are confirmed to be well tuned. Owing to the different forces generated at different depth of the films, release of two types of DNA molecules with different velocities is achieved, which further explores its application in combined gene therapy. Conclusions Our research confirmed that this novel platform based on a nano-electromechanical hypersonic resonator works well for controllable single and multi-DNA release. In addition, the unique features of this resonator such as miniaturization and batch manufacturing open its possibility to be developed into a high-throughput, implantable and site targeting DNA release and delivery system.
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Guoetal. J Nanobiotechnol (2019) 17:86
https://doi.org/10.1186/s12951-019-0518-7
RESEARCH
Programmable multi-DNA release
frommultilayered polyelectrolytes using
gigahertz nano-electromechanical resonator
Xinyi Guo1, Hongxiang Zhang2, Yanyan Wang1, Wei Pang2 and Xuexin Duan1*
Abstract
Background: Controllable and multiple DNA release is critical in modern gene-based therapies. Current approaches
require complex assistant molecules for combined release. To overcome the restrictions on the materials and environ-
ment, a novel and versatile DNA release method using a nano-electromechanical (NEMS) hypersonic resonator of
gigahertz (GHz) frequency is developed.
Results: The micro-vortexes excited by ultra-high frequency acoustic wave can generate tunable shear stress at
solid–liquid interface, thereby disrupting molecular interactions in immobilized multilayered polyelectrolyte thin films
and releasing embedded DNA strands in a controlled fashion. Both finite element model analysis and experiment
results verify the feasibility of this method. The release rate and released amount are confirmed to be well tuned.
Owing to the different forces generated at different depth of the films, release of two types of DNA molecules with
different velocities is achieved, which further explores its application in combined gene therapy.
Conclusions: Our research confirmed that this novel platform based on a nano-electromechanical hypersonic reso-
nator works well for controllable single and multi-DNA release. In addition, the unique features of this resonator such
as miniaturization and batch manufacturing open its possibility to be developed into a high-throughput, implantable
and site targeting DNA release and delivery system.
Keywords: Controllable release, NEMS resonator, Gigahertz ultrasound, Acoustic streaming, Micro-vortexes,
Polyelectrolyte thin films
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and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/
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Background
Controlled release of drugs, especially macromolecular
therapeutic agents such as DNA, is commonly adopted
in a variety of fields, spanning from the basic researches
of biomedical materials to the application development
of gene-based therapies [13] due to their precise control
of the dosage, minimum side-effect and high treatment
efficacy [4]. To realize an effective controllable release,
numerous immobilization and encapsulation approaches
have been applied for the establishment of drug carri-
ers [58], among which one of the most extensively used
and most promising methods goes to the self-assembly
of polyelectrolytes through layer-by-layer (LbL) tech-
nique [9, 10]. e precise and nanometer-scaled control
over film thickness and drug capacity of this method
has been highlighted by numerous researchers [11, 12].
Besides, simply by adopting a certain condition that can
induce film disruption, release of DNA and other biologi-
cal molecules can be achieved. So far, approaches to pro-
moting LbL film disruption have been studied extensively
[1315], including (a) methods based on environment
changes, such as pH [16, 17], ionic strength [18] and
liquid temperature [19, 20], (b) methods using specific
materials that participate in certain kinds of chemical
reactions, such as reductively [21], enzymatically [22, 23]
and hydrolytically [24] degradable polyelectrolytes, and
(c) methods by applying external stimulus, such as light
Open Access
Journal of Nanobiotechnology
*Correspondence: xduan@tju.edu.cn
1 State Key Laboratory of Precision Measuring Technology & Instruments,
Tianjin University, Tianjin 300072, China
Full list of author information is available at the end of the article
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Page 2 of 11
Guoetal. J Nanobiotechnol (2019) 17:86
[25], electrochemical potentials [26, 27] and ultrasound
[28, 29]. Each of these outlined approaches keeps their
respective strengths, and many have been verified to be
suitable for controlled DNA release [3033]. However,
their disruptions rely either on critical environment fac-
tors or special chemical property of the polyelectrolytes,
which bring additional restrictions for practical use [11].
For example, intense environmental changes required in
certain release process may put forward a higher request
for the protection of molecular bioactivity and restrict
the application of the method in cell experiments and liv-
ing organisms, and the adoption of some special materi-
als in some cases to enhance the disruption may increase
the complexity and cost of DNA immobilization.
Another focus in the LbL-based release is the pro-
grammable release of multiple biological agents [3437].
Controlling the release rate of several targets in differ-
ent orders, such as sequential or parallel release, or even
separate and mutually exclusive release profiles, can
provide effective tools for combined drug therapy inves-
tigation and achieve a better efficacy. Till now, several
studies have achieved the multiple release of different
DNA constructs assembled by polyelectrolyte films, and
most of them adopts specific design of complex film
materials and structures to achieve the required release
behavior [32, 3840]. For example, Liu etal. [38] demon-
strated a film fabrication method using a set of specially
designed degradable cationic polymers performing dif-
ferent erosion speeds, which was applied to release two
different plasmids with distinct profiles; Jessel etal. [39]
reported the use of cationic cyclodextrins as an enhancer
for sequential and direct delivery of different DNA mole-
cules into cells in contact with the films. erefore, devel-
oping DNA release method that can realize controllable
and multiple release with simple and moderate disrup-
tion condition is in great demand.
Owning to the development of microsystem and nano-
technology, acoustic devices based on piezoelectric
materials have gained increasing attention in biochemi-
cal research field [4144] which is due to their low cost,
batch manufacturing, small volume and noninvasive to
biomolecules [4547]. Here, we demonstrated a novel
and versatile controlled release approach using gigahertz
ultrasound (hypersound) induced by a nano-electro-
mechanical acoustic resonator composed of ultra-thin
material layers (several tens to hundreds of nanometers
thick). e ultra-short attenuation distance of this high
frequency ultrasound wave provides a steep acoustic gra-
dient at concentrated active region, thus generates micro
vortexes which can offer a powerful shear stress on the
interfaces between the vortexes and the substrates and
effectively realize DNA release from polyelectrolyte films
deposited on surface. Results of our experiments verifies
that by tuning the power applied to the device and the
distance between device and LbL films, DNA release
rate and amount can be precisely controlled. We also
designed a multi-DNA release system by simply assem-
bling two kinds of DNA molecules with commonly used
cationic polymers into LbL films. e porous film prop-
erty making possible for flowing liquid to pass through
the nano-sized pores and interact with DNA molecules
seated on inner layers. us, concurrent release with dis-
tinct properties of two kinds of DNA molecules which are
located on different depth of the films was achieved due
to the gradually decreased fluid velocity and shear stress
from the outer layer to the inner layer. Other advantages
of this method such as mild and pure physical interac-
tions, simple operation and low power consumption
(several hundreds of milliwatts) open possibilities for it
to be further developed into a universal, high-throughput
and implantable invivo DNA release and delivery system.
Methods
Materials
Poly (allylamine hydrochloride) (PAH, MW = 120,000–
200,000) and linear poly (ethylene imine) (LPEI,
MW = 25,000) were obtained from Alfa Aesar (United
States). Poly (sodium 4-styrenesulfonate) (PSS,
MW = 70,000) was purchased from Sigma Aldrich Co.
(United States). Single-stranded DNA molecules of 75
base pairs (5 to 3: (T)15CTA ACT GC TGG GCG ATT
CTG GTG ACG CGG C A A CGA TGA TTG GGA ACG ATG
ATT GGG AACA) were synthesized by Sangon Biotech
Co., Ltd. (Shanghai, China), and were labeled by Alexa
Fluor 488 (DNA-Green) or CY3 (DNA-Red). All chemi-
cals were used as received without any further purifica-
tion. Deionized water (DI water, 18.25MΩ) was used for
the preparation of buffer, polymer and DNA solutions.
Preparation oftheLbL lms
Prior to film preparation, glass substrates and QCM
chips were cleaned by 5min rinsing in ethanol, 5 min
rinsing in DI water, nitrogen-blow drying, and 20 min
oxygen plasma treatment. 2 mg/ml PAH, 2 μM DNA,
28mg/ml PSS and 10mg/ml LPEI used for the fabrica-
tion of multilayered films were prepared in the presence
of 150mM NaCl (pH = 6.5). Solution of LPEI contains
10 mM HCl to facilitate polymer solubility. 6 bilayers
of PAH/DNA-Red were achieved using a layer-by-layer
(LbL) method: substrates were alternatively submerged
in PAH and DNA-Red solutions (15min each), and were
rinsed in a 150mM NaCl solution (pH = 6.5) for 5min
between the deposition of each two layers. Multilayered
films composed of [LPEI/PSS]3/PAH/DNA-Green/[PAH/
PSS]5/PAH/DNA-Red/PAH were applied in multiple
DNA release experiments, and its buildup procedures
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Guoetal. J Nanobiotechnol (2019) 17:86
were operated as described above. In QCM experiments,
films were assembled under a flowing condition (100μl/
min) assisted by a peristaltic pump (Ismatec, ISM596D)
and a flow module (Q-Sense, QFM 401), and the incuba-
tion time of each layer lay on QCM frequency variation
rate.
Device fabrication
Fabrication process of a nano-electromechanical hyper-
sonic resonator is illustrated in Additional file1: Figure
S1. Briefly, alternating layers of silicon dioxide (SiO2) and
aluminum nitride (AlN) were deposited on Si wafer using
plasma-enhanced chemical vapor deposition (PECVD)
and reactive sputtering respectively to form the Bragg
mirror for acoustic reflection. A 600 nm thick molyb-
denum (Mo) layer was further deposited by RF sputter-
ing and patterned by plasma etching to form the bottom
electrode. After that, 1000nm AlN was deposited as the
piezoelectric layer and patterned by reactive ion etching
(RIE) to expose bottom electrode for electrical connec-
tion. Finally, 60nm chromium (Cr) and 300nm gold (Au)
were evaporated and patterned by lift-off process, serving
as top electrodes and testing pads.
Controlled release system
Sinusoidal signal (1.56 GHz) applied to the resonator
was generated by a signal generator (Agilent, N5171B)
and pre-amplified by a power amplifier (Mini-Circuits,
ZHL-5W-422+), and resonators were wire-bonded to
evaluation boards (EVB boards) for signal transmission
and device performance characterization. Polydimethyl-
siloxane (PDMS) chambers with different heights were
sealed on resonator substrates to form liquid containers.
During DNA release experiments, PDMS chambers were
fulfilled with 150mM NaCl solution (pH = 6.5), and glass
substrates modified with multilayered films were covered
on the top of the chamber. By controlling power output
of the generator, hypersonic wave and micro-vortexes in
liquid can be stimulated and well-tuned. In QCM meas-
urements, 200 μl NaCl solution was added to an open
module (Q-Sense, QOM 401) loaded with a modified
QCM chip. A T-shaped EVB board with a resonator fac-
ing down was designed and inserted into the solution
during DNA release process, and the distance between
the resonator and QCM chip was controlled to 200μm.
Characterization
Fluorescence microscope (Olympus BX53) with a CCD
camera (Olympus DP73) was utilized to examine the
amount of DNA-Red remained on glass substrates dur-
ing LbL and DNA release process. Before characteriza-
tion, glass slides were rinsed with DI water and dried
in nitrogen. Fluorescent pictures of a fixed area on each
slide were taken at each time point using an exposure
time of 100ms, and the fluorescence intensity was cal-
culated using ImageJ. Fluorescent value in liquid was
detected using a Microplate spectrophotometer (ermo
Scientific, VARIOSKAN LUX, 548nm/580nm). Quartz
crystal microbalance (QCM, Q-Sense) was used to pro-
vide mass information, and all measurements were
performed at 35MHz resonant frequency. Surface topog-
raphy of multilayered films was characterized by scan-
ning electron microscope (SEM, FEI F50), and 2nm Au
was evaporated on glass substrates before SEM charac-
terization to enhance surface conductivity. Film thick-
ness was recorded by atomic force microscope (Bruker,
Dimension Icon, tapping mode). In multiple DNA release
experiments, DNA-Red and DNA-Green were measured
by a fiber optic spectrometer (NOVA-EX, Ideaoptics,
China). A scan range from 500 to 700nm was used, and
different optical filters were applied to ensure that only
one peak will be recorded in one measurement. e fluo-
rescent intensities of these two molecules were extracted
at 580nm and 537nm respectively. Gel electrophoresis
was used to characterize the integrity of DNA molecules
after the treatment of acoustic streaming. DNA sam-
ples (prepared in 150 mM NaCl solution) were loaded
into 1.0wt% agarose and ran at 120V for approximately
30min, and then the gel was photographed under UV
light using a fluorescent gel imaging and analysis system
(ChemiDoc XRS+, BIO-RAD).
Results anddiscussion
Release system andmechanism study
To realize the delicately controlled DNA release, a
delivery system integrated of nano-electromechanical
(NEMS) hypersonic resonator was established and shown
in Fig.1a. e NEMS resonator is composed of a piezo-
electric layer sandwiched between two metal electrodes
which is fabricated by CMOS compatible process. Opti-
cal image of the device is shown in Fig.1b, and electri-
cal property of the device is characterized which shows a
resonant frequency at 1.56GHz (Additional file1: Figure
S2). e golden pentagon on device surface indicates top
electrode and working area of the device, and the thick-
ness of each material layer in a resonator ranges from
several tens to several hundreds of nanometers. To form
a stable and adjustable test condition, PDMS chambers
with a diameter of 6.9mm were mounted and sealed on
the resonator. 150mM NaCl solution was filled in the
chamber during release experiments to imitate an envi-
ronment adaptable to human body fluid. Glass slides
modified with target molecules using LbL technique were
covered on the chamber and in contact with the liquid.
Our previous works have confirmed that under reso-
nator stimulation, the propagation and attenuation of
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Guoetal. J Nanobiotechnol (2019) 17:86
acoustic waves in liquid will form stable and powerful
micro-vortexes [48]. Generally, the energy leakage of
sound waves causes its pressure to decrease during its
propagation in liquid, and this part of assumed power is
converted to the momentum of flow motion. e ampli-
tude (A) of an acoustic wave can be described as
where
A0
is the initial wave amplitude,
β
is the attenua-
tion coefficient, and z is the distance between acoustic
source and measured point. e coefficient
β
describes
the attenuation rate, and is given by
Here,
µ
indicates fluid viscosity,
ω
denotes acoustic fre-
quency,
ρ
is liquid density and
c
is the sound velocity in
liquid. e equation clearly shows that acoustic attenu-
ation is frequency-squared dependent, and a higher fre-
quency leads to a much stronger energy decrease. For
example, when travel 200μm in fluid, sound waves of
1.5GHz have already dissipated to 3% of its initial ampli-
tude, but waves of 150 MHz still have 96% remained.
(1)
=A
e
(2)
β
=
2µω
2
3ρ
c3
Hence, traditional ultrasound (kHz–MHz) can hardly
generate powerful acoustic streaming, and controllable
release methods based on ultrasound usually rely on cav-
itation [28]. In our hypersonic resonator system, the ele-
vated acoustic frequency (1.56GHz) can provide a higher
fluid velocity, which certifies it to be a preferable tool for
the generation of localized high-speed micro-vortexes
which can be more moderate and easier controlled. e
three-dimensional finite element model (3D FEM) analy-
sis of the acoustic streaming is shown in Fig.1c. Liquid
above the pentagonal working area of the resonator is
accelerated by device resonation, moves upward from the
center, and returns through the edge. When the uplifted
fluid reaches the interface between liquid and solid,
which is glass substrates modified with DNA molecules
in our work, flow direction is forced to change. Fluid dis-
perses laterally, and the longitudinal component of fluid
velocity is attenuated to zero in a short distance. e ver-
tical (z direction) gradient of the lateral fluid velocity (
Vx
)
causes shear stress (
τ
) at the border, which can be defined
by the formula
(3)
τ
=µ
V
x
z
Fig. 1 Release system based on nano-electromechanical resonator and theoretical simulation. a Schematic of release system. Multilayered films
composed of [PAH/DNA]6 were deposited on glass surface through LbL technique. Micro-vortexes triggered by the nano-electromechanical
hypersonic resonator generate controlled shear forces on adsorbed molecules, disassemble the multilayered films, and thus release embedded
DNA strands. b Top view of the resonator under microscope. ce 3D FEM analysis of acoustic streaming and resulted shear force. c Micro-vortexes
formed under the stimulation of a hypersonic resonator at 1.56 GHz. Color bar indicates the distribution of fluid velocity and arrows describe flow
direction. d Shear stress distribution along x-direction on the plane 200 μm away from resonator surface. e Composited shear stress distribution on
x–y plane 200 μm away from resonator surface
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Guoetal. J Nanobiotechnol (2019) 17:86
is shear stress interacts with materials at the inter-
face, overcomes the electrostatic forces among poly-
electrolytes, and finally leads to the disassembly of the
multi-layered films including the embedded chemicals.
Figure 1d shows the one-dimensional distribution of
shear stress along x-direction on the top border 200μm
away from the resonator surface, and a two-dimensional
composited stress is described in Fig. 1e. Most intense
shear stress can be seen focused right above the fringe of
device working area, and gradually decreases outwards.
Although the value of shear stress outside the pentagon
region seems to be much smaller from the simulation,
our experimental results indicate that it’s enough for mul-
tilayer film disassembly, and the release efficiency is not
restricted by the small size of the hypersonic resonator.
Film disassembly triggered byanano‑electromechanical
hypersonic resonator
In this work, in order to attest to the universality of this
method, commercialized and commonly used polycation
PAH was used, and multi-layered films simply composed
of six bilayers of PAH/DNA-Red (single-stranded DNA
labeled by CY3) were applied as a release model. To con-
firm that the quality of our established film is adequate
for the following release experiments, deposition process
was monitored by fluorescence microscope (Additional
file1: Figure S3), and the steady increase of fluorescent
intensity indicates the successful built up of uniform
multi-layered polyelectrolyte thin films.
To trigger the film destruction and DNA release, signal
of 500mW power at 1.56GHz was applied to the reso-
nator, and the remaining fluorescent DNA on glass sub-
strate was recorded (Fig.2a). No obvious differences can
be observed from the control group (without resonator
stimuli) during 30min incubation, indicating a good sta-
bility of DNA-Red embedded in polyelectrolytes. Mean-
while, fluorescent intensities of the experiment group
present a sharp decrease. is clear distinction of the
release behavior proofs that the hypersonic resonator
can effectively disassemble the electrostatically adsorbed
DNA molecules. To confirm that this phenomenon is not
caused by the disruption of fluorescent molecules, we
further detected the variation of fluorescent intensities
in solution (Additional file1: Figure S4). e growth of
Fig. 2 Characterization of DNA release process. a Fluorescent images of LbL films immersed in buffer without (control group) and with (experiment
group, 500 mW) resonator stimuli, observed at 0, 10 and 30 min. b QCM frequency recovery during DNA release. Release effectiveness was
observed from baseline changing each time after 10 min treatment. The exponential fit has a correlation coefficient of 0.973. c SEM images of bare
glass (blank) and LbL films before (0 min) and after (60 min and 90 min) resonator stimuli
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Guoetal. J Nanobiotechnol (2019) 17:86
fluorescent value in liquid over treating time is consist-
ent with the result observed from glass surface, and the
quantitative analysis indicates a final DNA concentration
of 9.3nM.
To further prove the film release, mass and morphol-
ogy change of LbL films were also recorded in our study.
Real-time mass monitoring of the release process was
carried out using QCM (Additional file1: FiguresS5, S6;
Fig.2b). e mass change follows an exponential decay,
and 90 min are required to stop the frequency recov-
ery. Film morphology was further characterized by SEM
(Fig.2c). Glass substrate presents a smooth surface with
fine texture before film deposition. After LbL modifica-
tion, a much rougher structure with dense nano-sized
islands and particles was obtained. After 90min treat-
ment, hardly any sediment remains, and the glass sub-
strate almost returns to its original state. AFM detection
of film thickness (Additional file1: Figure S7) also reveals
that only 20.5% of the initial thickness remains after
90 min treatment. All these results indicate the effi-
cient removal of the materials adsorbed on glass surface.
Besides, the structural integrity of DNA molecules with
or without resonator stimuli was analyzed by agarose gel
electrophoresis (Additional file 1: Figure S8). All lanes
migrated to the same position, proving that this approach
can realize DNA release without producing any appreci-
able structural damages.
Controlled DNA release
According to the fluorescent and QCM measurement, we
conclude that film disassembly and DNA release induced
by the NEMS resonator is a progressive rather than an
immediate process. erefore, this technology is very
suitable for sustained drug release in a controlled man-
ner. Here, to further study the release kinetics and realize
controlled DNA release, two variables, power applied to
the resonator (hereinafter, power) and distance between
resonator and LbL films (hereinafter, height), are chosen
to be optimized. (Other factors influencing DNA release
are discussed in Additional file1: FiguresS9 and S10).
eoretically, power determines the energy absorbed
by liquid, thus influences the speed of the vortexes. With
higher power applied, vortexes can reach a higher veloc-
ity, attain a larger shear force within the same distance,
and thus realize a faster release rate. On the other hand,
under same power condition, height change produces
much more complex effects on the vortex formation
(Fig.3a). When the height is too small, the accelerating
distance of liquid above device surface is limited, thus
restricts the generation of high-speed vortex. When it
is too large, the large liquid volume actuated by a sin-
gle device will also constrain the maximum fluid speed.
However, the shear stress at solid–liquid interface shown
in Fig. 3b continues to decay exponentially within the
entire height range. is is due to the change of distance
between the vortex velocity center (where the maximum
velocity locates) and the interface. e maximum veloc-
ity in the vortex occurs about 60 μm away from reso-
nator. When the height is small, liquid–solid interface
locates very close to the velocity center, thus the resulted
shear stress can be large. On the contrary, a larger height
will lead to more severe energy attenuation during the
upward movement of fluid, and the actual influence
of fluid on interface will be weakened, thereby further
reduce its ability for film disassembly. In summary, by
altering power and height, shear stress changes mono-
tonically, and different release rates can also be achieved.
To confirm our deduction, experiments with three dif-
ferent powers (5mW, 100mW and 500mW) and heights
(200μm, 1500μm, and 3400μm) were carried out, and
the results demonstrate a power and height depend-
ent release character consistent with the above analysis
(Fig.3). Curve fitting in the figure shows an exponential
trend, and we can express this regularity by the following
equation
Here,
Y0
,
A
and
R0
are three parameters determined
by power and height. When treating time (t) is sufficient
enough, y can be represented by
Y0,
which indicates
ultimate DNA amount remained on surface.
AR0
refers to curve slope when
t=0
, and can be used to
describe the initial release rate. us, quantitative analy-
sis of release rate and released amount can be achieved
by calculating
Y0
and
AR0
. eir specific values were
extracted from fitted curves and were plotted in Fig.4 as
a function of power and height. We can conclude from
the figures that by altering height and power, the release
velocity and ultimate released quantity vary linearly. is
character provides us an opportunity to preset power and
height from a calibration curve according to required
release effectiveness.
Multiple DNA release andmechanism study
e results above have demonstrated that a hypersonic
resonator can realize a well-controlled release of a sin-
gle kind of DNA molecule. To meet the requirements of
multiple DNA release, two kinds of DNA strands were
embedded in multilayered films in our following stud-
ies, and the release character and mechanism were thor-
oughly studied.
Figure5a describes the modification procedure used in
this section. DNA-Red (labeled by red fluorescent mole-
cule, CY3) and DNA-Green (labeled by green fluorescent
molecule, AF488) were sequentially embedded at differ-
ent depth of the films. ese two molecules are identical
(4)
y
=Y
0
+Ae
R
0
t
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Guoetal. J Nanobiotechnol (2019) 17:86
except the fluorescent marker to guarantee that the
release effectiveness will not be influenced by their struc-
tures and properties. Substrates were pre-coated with 3
LPEI/PSS bilayers to improve interfacial property and
DNA adsorption. 5 bilayers of PAH/PSS were inserted
between two kinds of DNA molecules for better distinc-
tion. Fluorescent spectrums of these two DNA molecules
were separately recorded using a fiber optic spectrom-
eter system, and the results are displayed in Fig.5b. Fig-
ure5c shows the remained DNA percentages extracted
at 580nm and 537nm, and differential analysis of DNA
released during each period of observation is given in
Fig.5d.
A clear result obtained from Fig.5 is that instead of
a sequential and outside-in release character, deep-
seated DNA-Green and shallow-seated DNA-Red
are released concurrently, except that the release
rate of inner DNA-Green appears to be slower. is
phenomenon is attributed to the structure of poly-
electrolyte films established by LbL technique, which
has been reported to exhibit a porous and permeable
property [49]. During the release process, the up-flow-
ing liquid stands a chance to pass through the nano-
sized pores and reach the innermost films, thus creates
shear force on inner surfaces and further release part of
loosely bound DNA molecules. e inhibition of outer
materials makes the force descend with the increase
of depth, and a smaller shear force exerted on inner
molecules will evidently lead to a lower release rate.
To verify our analysis, 2D-FEM simulation is given in
Fig.6, in which staggered white stripes are applied to
represent molecule cross-sections for model simplifica-
tion, and other colored area indicates the space among
molecule strings. e intervals are amplified to provide
a clear observation of field distribution. e hyper-
sonic resonator is placed at the lower right corner of
Fig. 3 Tunable DNA release. Variation trends of maximum vortex speed (a) and interface shear stress (b) when changing the height were
theoretically analyzed using FEM simulation. Experimental release effectiveness under different power (a) and height (b) were recorded using
percentage of remained DNA as a function of resonator treating time, observed at 0, 5, 10, 20, 30, 50, 70 and 90 min
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Guoetal. J Nanobiotechnol (2019) 17:86
the simulation area (not shown here), and the distance
between the resonator and films is set at 200μm. Under
resonator stimuli, gradually varied color can be seen in
the graphic, indicating gradually decreased fluid veloc-
ity and shear stress from the outer layer to the inner
layer, thus confirms our explanations.
Here, a detailed description can be provided for the
entire multiple DNA release process. When hypersound
treatment started, both DNA-Red and DNA-Green
release according to the fluid velocity and shear stress
they received. After 10min, over 60% of outer DNA-Red
has been removed. Only small amounts of DNA-Red who
bond tightly to or even embedded in the internal mate-
rials are left on the surface, which lead to a much lower
release rate. After 30 min, the release of inner DNA-
Green also begins to slow down, indicating that most of
loosely bound DNA-Green molecules have been success-
fully released. 20min later, the release of outer DNA-Red
is almost accomplished, and its velocity approaches to
zero. Meanwhile, a slightly increased speed was observed
on inner DNA-Green (Fig.5d), which can be explained by
the larger shear force obtained due to the entire removal
of exterior materials and the exposure of DNA-Green to
the vortexes.
ese results clearly indicate that our NEMS resona-
tor is able to realize a differentiable multi-DNA release.
Different release rates can be obtained simply by using
different embedding levels. ere are no requirements
in the selection of polycation electrolytes and the struc-
tural difference between the target DNA molecules. Since
the device is CMOS compatible, the applied power can
be actually programmed, a tunable release of two or even
more kinds of target DNA molecules can be achieved,
which holds great significance in the area of medical
applications.
Conclusions
In summary, we developed a novel method to trigger
the disassembly of multilayered polyelectrolyte thin
films for controllable single and multi-DNA release
using nano-electromechanical hypersonic resonators.
Due to the ultra-short attenuation distance of hyper-
sonic waves in liquid, localized and high-speed micro-
vortexes are triggered, thus creating controlled shear
Fig. 4 Analysis of DNA release rate and amount. Release capacity (100%-
Y0
, indicating theoretical DNA amount which can be released from surface)
and initial release velocity (
A
R0
) were extracted from fitting curves in Fig. 3c, d and plotted here as a function of power (a, b) and height (c, d)
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Guoetal. J Nanobiotechnol (2019) 17:86
Fig. 5 Characterization of multi-DNA release. a Schematic of film composition and release process for multiple DNA release. b Spectrum result
of DNA remained on glass surface under resonator stimuli, observed at 0, 10, 30, 50, 70 and 90 min. Fluorescent intensities of DNA-Green and
DNA-Red were calculated at 537 nm and 580 nm, respectively. Power and height were set to 500 mW and 200 μm. c Percentage of remained DNA
as a function of resonator treating time. d Differential percentage of DNA released during each period of observation
Fig. 6 Simulated distributions of a fluid velocity and b shear stress within LbL films. White stripes indicate cross-sections of polymer and DNA
molecules, and are staggered and dispersed in colored fluid area to represent the interlaced and porous film structure
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Guoetal. J Nanobiotechnol (2019) 17:86
forces at liquid–solid interface for film disruption. Sim-
ply by tuning the power applied to the device and the
distance between device and embedded films, release
speed and amount can be precisely controlled, demon-
strating a good controllability of this approach. In addi-
tion, a unique feature that the vortexes can penetrate the
multilayered films and create shear forces on inner layers,
enables this approach to release multiple DNA molecules
at the same time with different speed, providing a sim-
ple method without using complex assistant molecules
for combined gene therapy. It is also noted that since
the hydrodynamic approach is a pure physical method
and the NEMS resonator is CMOS compatible, it can
be readily applied to different types of controlled release
applications.
Additional le
Additional le1. Additional figures and experimental details. Figure S1.
Fabrication process of a nano-electromechanical hypersonic resonator.
Figure S2. Electrical property of the resonator. Figure S3. Fluorescent
observation of film assembly. Figure S4. Fluorescence of released DNA in
liquid. Figure S5. Setup for QCM detection of film disassembly. Figure S6.
Real-time results of QCM detection of film disassembly. Figure S7. Film
thickness detection. Figure S8. DNA agarose gel electrophoresis. Figure
S9. Temperature control. Figure S10. Influence of different temperature.
Abbreviations
LbL: layer-by-layer; PAH: poly (allylamine hydrochloride); LPEI: linear poly (eth-
ylene imine); PSS: poly (sodium 4-styrenesulfonate); DNA-Red: DNA molecules
labeled by CY3; DNA-Green: DNA molecules labeled by Alexa Fluor 488; QCM:
quartz crystal microbalance; DI water: deionized water; SiO2: silicon dioxide;
AlN: aluminum nitride; Mo: molybdenum; Cr: chromium; PECVD: plasma-
enhanced chemical vapor deposition; RIE: reactive ion etching; EVB: evaluation
boards; PDMS: polydimethylsiloxane; SEM: scanning electron microscope; 3D
FEM: three-dimensional finite element model.
Acknowledgements
Not applicable.
Authors’ contributions
XG, XD and YW designed the experiments. XG performed the experiments
and data analysis and was a major contributor in writing the manuscript. XG
and HZ did the simulation. WP contributed the device design and fabrication.
XD supervised the experiments and edited the final version of the manuscript.
All authors read and approved the final manuscript.
Funding
The authors gratefully acknowledge financial support from the National
Natural Science Foundation of China (NSFC No. 61674114, 91743110,
21861132001), National Key R&D Program of China (2017YFF0204604), Tianjin
Applied Basic Research and Advanced Technology (17JCJQJC43600), the
Foundation for Talent Scientists of Nanchang Institute for Microtechnology of
Tianjin University, and the 111 Project (B07014).
Availability of data and materials
All data supporting this study are included in this published article and its
additional file.
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1 State Key Laboratory of Precision Measuring Technology & Instruments,
Tianjin University, Tianjin 300072, China. 2 College of Precision Instrument
and Opto-electronics Engineering, Tianjin University, Tianjin 300072, China.
Received: 31 December 2018 Accepted: 30 July 2019
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This study demonstrated the feasibility of using regenerable polyelectrolyte membranes to ultimately control the irreversible membrane fouling in a forward osmosis (FO) process. The regenerable membrane was fabricated by assembling multiple polyethyleneimine (PEI) and poly(acrylic acid) (PAA) bilayers on a polydopamine-functionalized polysulfone support. The resulting membrane exhibited higher water flux and lower solute flux in FO mode (with the active layer facing feed solution) than in PRO mode (with the active layer facing draw solution) using trisodium citrate as draw solution, most likely due to the unique swelling behavior of the polyelectrolyte membrane. Membrane regeneration was conducted by first dissembling the existing PEI-PAA bilayers using strong acid and then reassembling fresh PEI-PAA bilayers on the membrane support. It was found that, after the acid treatment, the first covalently bonded PEI layer and some realigned PAA remained on the membrane support, acting as a beneficial barrier that prevented the acid-foulant mixture from penetrating into the porous support during acid treatment. Water and solute flux of the regenerated membrane was very similar to that of the original membrane regardless of alginate fouling, suggesting an ultimate solution to eliminating the irreversible membrane fouling in an FO process. With a procedure similar to the typical membrane cleaning protocol, in-situ membrane regeneration is not expected to noticeably increase the membrane operational burden but can satisfactorily avoid the expensive replacement of the entire membrane module after irreversible fouling, thereby hopefully reducing the overall cost of the membrane-based water treatment system.
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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.
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Protein therapeutics are promising candidates for disease treatment due to their high specificity and minimal adverse side effects; however, targeted protein delivery to specific sites has proven challenging. Mesoporous silica nanoparticles (MSN) have demonstrated to be ideal candidates for this application, given their high loading capacity, biocompatibility, and ability to protect host molecules from degradation. These materials exhibit tunable pore sizes, shapes and volumes, and surfaces which can be easily functionalized. This serves to control the movement of molecules in and out of the pores, thus entrapping guest molecules until a specific stimulus triggers release. In this review, we will cover the benefits of using MSN as protein therapeutic carriers, demonstrating that there is great diversity in the ways MSN can be used to service proteins. Methods for controlling the physical dimensions of pores via synthetic conditions, applications of therapeutic protein loaded MSN materials in cancer therapies, delivering protein loaded MSN materials to plant cells using biolistic methods, and common stimuli-responsive functionalities will be discussed. New and exciting strategies for controlled release and manipulation of proteins are also covered in this review. While research in this area has advanced substantially, we conclude this review with future challenges to be tackled by the scientific community.
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Free-standing, stimuli-responsive polyelectrolyte multilayer films enabled by light-induced degradation of sacrificial compartments are introduced. Two examples- (i) a triple responsive film that uses light, redox, and pH for different functions, and (ii) different wavelengths of light for different functions-are described. This approach to multiresponsive materials offers simple design and chemical synthesis while enabling different stimuli to perform separate functions in the same material.