ArticlePDF Available

Abstract and Figures

Intracellular delivery enables the efficient drug delivery into various types of cells and has been a long-term studied topics in modern biotechnology. Targeted delivery with improved delivery efficacy requires considerable requirements. This process is a critical step in many cellular-level studies, such as cellular drug therapy, gene editing delivery, and a series of biomedical research applications. The emergence of micro- and nanotechnology has enabled the more accurate and dedicated intracellular delivery, and it is expected to be the next generation of controlled delivery with unprecedented flexibility. This review focuses on several represented micro- and nanoscale physical approaches for cell membrane disruption-based intracellular delivery and discusses the mechanisms, advantages, and challenges of each approach. We believe that the deeper understanding of intracellular delivery at such low dimension would help the research community to develop more powerful delivery technologies for biomedical applications. Keywords: Drug delivery, Physical approaches, Cell membrane disruption, Low dimension
Content may be subject to copyright.
Recent advances in micro/nanoscale intracellular delivery
Mengjie Sun, Xuexin Duan
State Key Laboratory of Precision Measuring Technology & Instruments, Tianjin University, Tianjin, 300072, China
abstractarticle info
Available online 28 December 2019
Drug delivery
Physical approaches
Cell membrane disruption
Low dimension
Intracellular delivery enables the efcient drug deliveryinto various types of cells and has been a long-term stud-
ied topics in modern biotechnology. Targeted delivery with improved delivery efcacy requires considerable re-
quirements. This process is a critical step in many cellular-level studies, such as cellular drug therapy, gene
editing delivery, and a series of biomedical research applications. The emergence of micro- and nanotechnology
has enabledthe more accurate anddedicated intracellular delivery, andit is expected to be the next generation of
controlled delivery with unprecedented exibility. This review focuses on several represented micro- and nano-
scale physical approaches for cell membrane disruption-based intracellular delivery and discusses the mecha-
nisms, advantages, and challenges of each approach. We believe that the deeper understanding of intracellular
delivery at such low dimension wouldhelp the research community to develop more powerful delivery technol-
ogies for biomedical applications.
Copyright © 2020 Tianjin University. Publishing Service by Elsevier B.V. on behalf of KeAi Communications Co., Ltd.
This is an open access article under the CC BY-NC-ND license (
1. Introduction
The safe and efcient intracellular delivery of biologically active
macromolecules into living cells is a critical and challenging process in
For many different applications, such as cell analysis,
drug therapy, and gene transfection,
a range of materials, including
small molecules, nucleic acids, proteins, and nanomaterials, must be de-
livered to different kinds of cells. Given the strict regulation of the
plasma membrane, direct translocation of external materials is largely
For example, the cell membrane is an impassable barrier
for small hydrophilic molecules. Macromolecules (such as DNA, RNA,
and proteins) are hardly uptaken by cells without external help.
Table 1
summarizes the typical molecules and reagents that are cur-
rently important in cell biology and their challenges for intracellular de-
livery. Current transfection methods still feature many limitations. For
example, the delivery of large molecules into immune cells, stem cells,
and neurons remains difcult.
The delivery normally requires
vectors, such as viruses or peptides, specic to target molecules.
In ad-
dition, batch processing of cells often results in cell damage or drug res-
idue heterogeneity. Thus, the precise dose control and drug therapy
must be critically provided at the single-cell level. The development of
delivery methods that can improve safety, speed, cost, and efciency
to achieve efcient delivery of various molecules to various cells re-
mains a long-term challenge.
Current intracellular delivery can be generally divided into two ap-
proaches: carrier-based methods and membrane disruption techniques
(Fig. 1). In the delivery of materials with a carrier, the carrier can
completely pack the cargo and prevent its degradation. A carrier can
also utilize its own properties to enter thedesired intracellular compart-
ment and release the payload at appropriate time and conditions. As a
promising delivery method with a long history of research, viral
vectors are delivered into cells by means of viral infection, which re-
quires no external assistance.
Although the viral vectors possess
the advantages of high efciency and specicity, the nature of viruses
causes inevitable problems, such as in vivo immune response, vector
safety, and manufacturing complexity. Given these challenges,
biomimic lipid nanocarriers have become the most advanced non-
viral vectors in nucleic acid delivery, because they avoid the effects of
counterpart limitations. Nonetheless, the exact escaping mechanism re-
mains unclear. Most carriers require cellular uptake through endocytic
Thus, only limited combinations of cargo materials and
cell types are available. Certain carriers can fuse with the target mem-
brane through membrane fusion process assisted by membrane
Therefore, the direct delivery of cargo by the carrier with fu-
sion capability can avoid endocytosis.
Vesicles or microvesicles may
fuse with target cells and deliver macromolecules, such as DNA or pro-
teins, to avoid the delivery process and cytotoxicity of synthetic
The exact fusion mechanism still needs further study. Tar-
get cells and potential cargo materials may be inapplicable or escape
Nanotechnology and Precision Engineering 3 (2020) 1831
Corresponding author.
E-mail address: xduan@tju.ed (X. Duan).
2589-5540/Copyright © 2020 TianjinUniversity.Publishing Serviceby Elsevier B.V. on behalfof KeAi Communications Co., Ltd.This is an open access article under the CC BY-NC-ND l icense
Contents lists available at ScienceDirect
Nanotechnology and Precision Engineering
journal homepage:
By contrast, membrane disruption uses physical methods, including
mechanical, electrical, thermal, optical, chemical stimulation, etc. These
methods could generate discontinuous and transient nanopores in the
plasma membrane, resulting in the increased pore size of the membrane
to allow the diffusion of exogenous molecules or direct penetration to
cell membranes with solid conduits or carriers to release cargo in cells.
This approach is based on the rapid perturbation and healing of the
cell membrane, by which almost any type of submicron material can
be delivered regardless of carrier properties. Membrane disruption not
only facilitates nucleic acid delivery but also protein delivery, including
the delivery of antibodies,
transcription factors, and genome-editing
However, traditional membrane disruption methods
often pose challenges, such as (1) irreversible damages to cells due to
excessively strong physical disruption and reduced delivery efciency
due to inadequate stress; (2) limited throughput and scalability of sev-
eral methods; (3) physical disruptionto whole cell population, resulting
in passive operation of non-target cells.
Continuous efforts have been made to improve intracellular delivery
efciency to solve these issues. In recent years, with the rapid develop-
ment of micro- and nanotechnologies, including microuidic system
and lab-on-chip techniques, membrane disruption technologies at
Table 1
Common target materials for intracellular delivery.
Cargo Category Challenge
All the small molecular structures
that have biological signicance.
Structural diversity.
Chemical diversity.
Dependent transfer
Nanomaterials Quantum dots, nanoparticles (NPs),
and carbon nanotubes.
Structural diversity.
Chemical diversity.
Dependent transfer
The immune response.
Nucleic acids All oligonucleotides including DNA,
RNA, siRNA, mRNA, and miRNA.
DNA needs to be delivered
to the nucleus.
RNA is unstable.
Integration of DNA and
genome causes security
Proteins All amino acid combinations,
including antibodies, short peptides,
structural proteins, and
transcription factors.
Difcult to produce and to
May cause overexpression.
Features structural
diversity and very sensitive
tertiary structure.
Fig. 1. Two approaches for intracellular delivery and their basic mechanisms.
19M. Sun, X. Duan / Nanotechnology and Precision Engineering 3 (2020) 1831
micro/nanoscale are gradually emerging as alternative approaches for
intracellular delivery. Owing to their high-precision mechanical opera-
tion provided by modern micromachining technology, micro/nanoscale
membrane disruptions can now accurately manipulate membrane per-
turbations at the single-cell and subcellular levels. Micro-nano systems
can also provide accurate drug release rate and time control, avoiding
the unnecessary toxic reactions caused by excessive drug concentration
at the macro scale. Furthermore, given the size compatibility, these
technologies can be easily integrated with other microuidics or lab-
on-chips, largely facilitating the d ownstream cell analysis. In the follow-
ing sections, we will introduce the current methods of membrane dis-
ruption of interest in the eld of micro/nanotechnology and their
advantages and challenges.
2. Recent advances in micro/nanoscale intracellular delivery
The earliest membrane disruption-based intracellular delivery can
be traced back to the microinjection technique in 1911
tion transfection DNA technique was proven to be viable in 1982,
which led to the development of other membrane disruption tech-
niques, including optoporation
and sonoporation.
Many membrane
destruction techniques were later abandoned dueto drawbacks, such as
low throughput, high cost, and technical operation, except electropora-
tion, which was widely adapted. During the last decade, membrane dis-
ruption technology has been extensively combined with micro/
nanotechnology, microuidics, and lab-on-chip approaches and is
being developed to create more new opportunities.
2.1. Micro/nanoneedles
Intracellular delivery dates back to the work of Barber et al.
used a very thin glass needle lled with injection solutions to inoculate
living cells with substances such as bacteria. Traditional microinjection
techniques typically require cells to be immobilized on a substrate or
to be held in place by additional precision devices. The injection process
also requires the technician to possess precise and skilled operation,
which is slow and can only inject one cell at a time, thus limiting the
throughput. In recent years, the introduction of technologies, such as
automation equipment
and robotic systems,
has considerably im-
proved the t herapeutic efciency of microinjection technology, but con-
siderable throughput and scalability still need to be realized. Based on
microinjection research, studies have shown that one-dimensional mi-
croscale structures (microneedles) with sharp tips can penetrate skin,
cells, and other tissues, which enabled drug delivery and biological ther-
apy. Microneedles with a wide range of lengths (50500 μm) have been
applied in intradermal delivery applications.
Until the mid-1990s,
with the development of microelectronic industry, microneedles were
considered as an important research topic for drug delivery.
Microneedles can easily penetrate the human skin without irritate the
nerves, allowing drug penetration in a gentle and painless manner.
Micromachining technology can fabricate microneedles with different
types of materials, including semiconductors, metals, and polymers.
In general, polymer microneedles are widely used owing to advantages
of low toxicity, production cost, and risk of waste and good
Integrating microneedles onto traditional patches
has shown that a variety of drugs
, including proteins, antibodies,
and vaccines can be successfully achieved. Polymeric microneedle
patches are used in four different ways using to deliver drugs (Fig. 2a).
(1) Coated microneedles. The drug molecules are coated on the
microneedle indication; then, the microneedle could penetrate the
skin cuticle to release the drugs. (2) Dissolvable microneedles.
Microneedles are made by mixtures of soluble polymers and therapeu-
tic drugs. Microneedles require hours or days to fully dissolve and re-
lease the drug after being inserted into the skin. (3) Degradable
microneedles. Microneedles and patches are made from biodegradable
polymers. The drug will be released into the skin as the polymer
hydrolyzes. Thus, the sustained release of the therapeutic agent in a
constant dose can be achieved. (4) Bioresponsive microneedles.
Microneedle patches can respond to specic biosignals or environmen-
tal changes. Microneedles can deliver insulin and growth hormone
through animal skin. In recent years, studies have also shown that
microneedles can carry degradable NPs and release browning agents
for obesity treatment.
In addition, glycemic control of mouse
models is achieved using bioresponsive microneedle patches.
Most microneedles contain tips,which measure tens of microns, that
cannot be precisely located to a single cell, thus resulting in the non-
uniform delivery of plasmids or other macromolecules. Nanoneedles
with shorter lengths (b1μm) and sharp tips (b100 nm) were then de-
veloped to solve the issue. They provide better precision at the single-
cell level. In 2007, Kim et al.
rst demonstrated the penetration of
nanowire (NW) arrays into cells, which allowed the gene delivery to
mammalian stem cells. Onthis basis, different nanostructures, including
nanoneedles, nanopores, nanostraws, and other nanoscale structures,
have been developed. These works use needle-like (wire) structures
with high aspect ratio to directly penetrate the cells under the action
of gravity, the cells are inserted to form pores by nanoscale wires.
Kwak et al. described in detail the key role of NW arrays in cell
function-related applications. Penetrating NWs could efciently deliver
biomacromolecules and photoelectric stimulation, and high-density
non-penetrating NWs could be used to explore the interactions be-
tween nanostructured substrate and cell surface.
When the NW pen-
etrates the cell, the molecules could dissociate into the cytoplasm.
Therefore, the NW structure can be modied or doped with the target
molecules by direct fabrication through micro/nanotechnology.
this case, biological macromolecules can enter cells without chemical
modication or virus packaging. Shalek et al.
prepared vertical NWs
by chemical vapor deposition or standard semiconductor technology.
Previous studies have shown that cells could be spontaneously pene-
trated while being placed on the NW.
In Shalek's work, 1,1-
membrane HeLa cells were inoculated on green uorescently labeled
NW and observed this penetration process (Fig. 2b). This platform
could introduce siRNA, peptides, DNA, proteins, and impermeable in-
hibitors into challenging cell types, such as neurons and immune cells.
However, the NWs failed to penetrate cell immediately after contact.
They required the principal stress caused by cell diffusion or the tension
created by the adhesion of the cell membrane to the NWs.
nanostraw works similarly to microinjection. The high aspect ratio of
nanostraws allows their direct insertion into cells. Then, biomolecules
can diffusedirectly into the cell from the external environment through
the hollow structure, and the molecular delivery efciency can be accel-
erated with electrophoresis.
Xu et al.
created the rapid transmission
of second messenger Ca
by using a nanostraw device, without chem-
ical carrier and genetic modication. They cultured the cells for 24 h on
the nanostraw array (Fig. 2c) and then combined the nanostraw array
with the ow channel; thus, the owing solution could ow through
the bottom of the array, enabling Ca
from the solution to codiffuse
into hundreds of cells over several seconds. This method can achieve
the effective control of both intracellular delivery time and drug dosage.
However, nanoscale straws are relatively difcult to use in terms of
manufacturing process and cost, and their xed size is limited for spe-
cic applications. Given these conditions, breakthroughs and simplica-
tion are required in processing technology. In view of these problems,
He et al.
proposed a simpler preparation method by using O
etching to prepare nanostraws with controllable parameters of different
structures; they proved that DNA transfection can be achieved by com-
bining a nanopipette with a microuidic device or external technology.
This work advanced the use of nanostraws for a wide range of biomed-
ical devices.
Nanoneedles can directly penetrate the cell membrane to form
nanoscale holes. After the needle is withdrawn, exogenous molecules
could diffuse into the cytoplasm before the holes are healed. Park et al.
20 M. Sun, X. Duan / Nanotechnology and Precision Engineering 3 (2020) 1831
fabricated nanoneedle arrays
with 12 μm height and tapered to
b30 nm in diameter by etching of silicon.
The density of the
nanoneedle arrays ensures that each cell could be punctured by at
least one nanoneedle. The single-layer adherent cells were loaded
with a puncture (pressed into the cells by controlling the loading force
and puncture speed) or centrifugation (xing the nanoneedle array on
the centrifuge device, adjusting the different centrifugation speed and
time, and rotating the suspension cells in the solution) to compare the
efciencies of intracellular delivery of the two loading methods.
et al.
selected a diamond
with superior mechanical properties and
chemical inertness to prepare the nanoneedle arrays. They rinsed the
suspension cells onto a dense array of nanoneedles for a one-time,
high-throughput, cellular mechanical poration of the cells. Given that
the nanoneedles are short and condensed adequately, the cell mem-
branes were slightly disrupted to enhance molecular diffusion without
puncturing cells. This work had led to effective drugdelivery to resistant
cancer cells and is expected to be used to treat multiple resistant cancer
All three forms of one-dimensional nanostructures signicantly
improve the accuracy of drug delivery beyond traditional microneedle
transdermal delivery and risky microinjection. The NW/needle can be
xed on the support base, effectively avoiding the accumulation of tox-
icity of suspended nanomaterials (nanotubes, NPs, and suspended
NWs) in cells.
However, the area of the nanoneedle substrate limits
the number of cells cultured on the nanotip structure, and cell culture
and membrane permeation also require a long time. Collecting the re-
leased cells from nanoneedle substrates is a difculty that still requires
When delivering naked plasmid DNA that is not com-
plexed with lipofectamine,the transfection rate is extremely low, prob-
ably because the nanoneedle array could not simultaneously process
the cell and nuclear membranes. The diffusion of delivered DNA in the
Fig. 2. (a) Schematic of the mechanism by which a polymer mic roneedle patches deliver drugs; (b) Si NWs as nano needles for intr acellular delivery at the singl e-cell level
(c) microuidic device integrated with nanostraw structures.
21M. Sun, X. Duan / Nanotechnology and Precision Engineering 3 (2020) 1831
cytoplasm is restricted by structural proteins,
which might degrade
and prevent their entrance to the nucleus. Notably, the direct penetra-
tion of the cell membrane through the mechanical structure often
causes irreversible cell damages. This condition requires the continuous
innovation and optimization of preparation processes and materials of
micro/nanoneedles to provide high delivery efciency while reducing
cell damage. Therefore, noncontact stimulation of the external eld to
induce cell membrane permeability can bring more interesting benets
and more application prospects.
2.2. Electroporation
Electroporation is a relatively trivial physical method. This process
exposes the cells to anelectric eld, applying a voltage on the cell mem-
brane to form a transmembrane potential difference of about 0.5 V
induce transient pores in cell membrane, whereas exogenous sub-
stances in the surrounding environment are effectively delivered into
the cell by electrophoresis or promotion of the diffusion effect
(Fig. 3a). The earliest electroporation report was released in 1982. Neu-
mann et al.
used a pair of parallel plates to apply a certain voltage to
disperse suspended cells and proved that electroporation could be
used for mammalian DNA transfection. The advantages and disadvan-
tages of three electroporation scales (the earliest developed bulk elec-
troporation method and micro/nano-electroporation technology)
were summarized by Yang et al.
In contrast to macroscale methods,
electroporation combined with micro/nanotechnology can locate
electric elds at the single-cell level, considerably reduce the voltage
and heat effect, and avoid cell damages caused by high voltage.
Chang et al.
designed a microuidic system which contained
nanopore array and a series of interlaced U-shaped microcap structures
(Fig. 3b and c). After immersing and lifting the chip vertically into the
cell suspension, the U-shaped structure kept pointing upward. The
cells could be efciently captured in the U-shaped structure by gravity
and hydrodynamics and enabled good contact with the 400 nm
nanopores. The cells contacting with the nanopores can be
electroporated directly by the pulsed electric eld, and the surface-
charged macromolecule can quickly and directly enter the cells through
the nanopores by electrophoresis. This platform can effectively capture
and transfect primary mouse cardiomyocytes, a process that is almost
impossible to achieve by traditional electroporation because of the
large number of ion-transport proteins on sarcolemma. This condition
often leads to ion channel activation or abnormalities, resulting in
high cell death rate and low transfection efciency.
Given that the pore size of electroporation is extremely small to be
observed with an optical microscope, Guo et al.
adopted an electrical
measurement method to detect physiological behaviors inside and out-
side of the cell and comprehensively studied the cell electroporation
process. The novel microarray chip comprised four electrode units,
each of which had a pair of central electrodes at its center. The chip
was assembled on the printed circuit board, andthe polydimethylsilox-
ane (PDMS)cavity with inlet and outlet was attached to the chip to form
acellsamplechamber(Fig. 3d). After the cell suspension was inserted
Fig. 3. (a) Schematics of microuidic electroporation
; (b) rapidcapture of cells ontonanostructuresby dipping-trap method; (c) nanochannelelectroporationplatform
; (d) schematic
of the microarray chip combining (i) cell positioning, (ii) electroporation, and (iii) impedance measurement.
22 M. Sun, X. Duan / Nanotechnology and Precision Engineering 3 (2020) 1831
into the chip and stabilized, it was captured by the negative
dielectrophoresis force and positioned to the center electrode. Then,
the cells were electroporated in situ by the two center electrodes.
After electroporation, impedance analysis can be performed to detect
the cellular dynamic processes.
This setup is considerably less compli-
cated compared with a patch clamp
that could characterize single-
cell electroporation. Moreover, impedance analysis integrates multiple
functions, such as selective in situ electroporation, cell array localiza-
tion, and real-time electrical, measurement, and might have potential
applications in tumor therapy and pathological analysis. Recently,
microuidic electroporation device has been developed for better trans-
fection efciency and cell viability.
However, given the limited size
of microuidic channels, the cell processing speed must be sacriced to
achieve the balance between high ux and low voltage, which is partic-
ularly important in the research of micro-nanoscale drug delivery.
2.3. Optoporation
Using light in the form of a focused laser for cellular drug delivery,
optical transfection has also received research attention. As a noncon-
tact method, optics-based technology is important for the manipulation
of biological materials at the micron and submicron scale.
Small tran-
sient pores can be created by a variety of light forms to allow the deliv-
ery of plasmid DNA and other macromolecules. Light energy leads to
electron plasma, which causes the photochemically induced degrada-
tion of the membrane. Meanwhile, light energy can also generate cavi-
tation bubbles with an ultrashort lifetime on the membrane. Several
laser systems, including continuous-wave, pulsed picosecond, and
nanosecond lasers, have been used for optically mediated membrane.
These techniques afford a noncontact, fast, and sterile method to intro-
duce membrane impermeable molecules or uses a vector to deliver the
drug of interest. The efciency of delivery strongly depends on the cell
and/or drug types. Studies have used different forms of light to directly
introduce macromolecules into cultured cells. Using ultrafast pulsed
light for optical transfection offersselective targeting and high efciency
and viability.
The ultrafast laser beam operates over a very small area
of action on cells, which limits its capability for full transfection. Dhakal
et al.
used ultrafast near-infrared ray (NIR) laser microbeam to deliver
both single opsins and large multi-opsin constructs to target cells. The
optical delivery of multiple opsin-encoding genes leads to targeted ex-
pression and white-light activation.
The delivery of large fusion con-
structs of multiple spectrally separated opsins was achieved by
ultrafast NIR laser-mediated optoporation to obtain a high cell sensitiv-
ity to ambient broadband light, leading to visual restoration. This tech-
nology provides a novel idea for the functionalization of optical
transfection. Although various optical technologies are constantly evolv-
ing, many problems remain regarding the intracellular delivery of genes/
transcripts,including the low quality and reproducibility. Combining op-
tical transfection with microuidic technology has been popularly used
to increase the transfection efciency and throughput. Uchugonova
et al.
built an ultrashort femtosecond (fs) laser-microuidic cell trans-
fection platform to achieve optical reprogramming of large cell popula-
tions. In a microuidic tube with multiple genes, ultrashort laser pulses
induce transient membrane permeabilization, which enables the pro-
duction of high-quality and contamination-free induced pluripotent
stem cells (Fig. 4a).
Schomaker et al.
proposed another optical transfection method
combined photosensitive materials with laser treatment (Fig. 4b).
They incubated gold NPs (AuNP) with cells and exposed them to fs-
laser pulses. Photosensitive chemicals localized to the membranes of
endocytic vesicles could be activated by laser stimulation to induce lo-
calized membrane permeabilization of the cell.
Cell processing for
Fig. 4. (a) Illustration of fs-laser-microuidic transfection platform
; (b) schematic of AuNP-mediated optoporation principle.
23M. Sun, X. Duan / Nanotechnology and Precision Engineering 3 (2020) 1831
gene and cell therapies generally use several separate procedures for
gene transfer and cell separation or elimination. Lukianova-Hleb
et al.
developed a new approach of using plasmonic nanobubble
(PNB) for simultaneous transfection of target cells and elimination of
unwantedsubsets of other cells. Transient PNBs were generated around
a AuNP after a short-term laser irradiation. Laser energy was converted
into heat, causing a nanoscale explosion for transmembrane injection of
molecular cargo. Depending on cell specicity, PNBs of different sizes
were generated around cell-targeted gold nanoshells and nanospheres.
Simultaneously, high efcacy, selectivity, and no-damage delivery of
both molecular cargo and cell destruction were achieved using laser
pulse bulk treatment. Compared with other existing methods, this tech-
nology provides simultaneous and cell-specic multifunctionality. More
importantly, laser pulse bulk treatment has also been used in hard-to-
transfected cells, such as primary neurons and stem cells, and in difcult
scenarios such as in a living embryo.
The instrumentations required,
however, are generally expensive, complicated, and bulky.
2.4. Sonoporation
Although light/electroporation can solve several intracellular delivery
challenges, their application in delivering some specic proteins and
nanomaterials is still limited. The damage caused by the energy eld
and cytotoxicity are also unsolved. By contrast, ultrasound as a mild tech-
nology can promote the drug/gene delivery to cells (nuclei).
sonic stimulation can alter the cell membrane permeability, thereby
allowing the extracellular molecular absorption in a phenomenon called
sonoporation. In 1986, Fechheimer M et al. rst used ultrasound to load
exogenous macromolecules into suspension cells
and then transfect
DNA of mammalian cells.
Sonoporation can interfere with cell mem-
brane permeability in two ways: ultrasound microbubbles (cavitation)
and acoustic stimulation. In past studies, ultrasound was often used in
combination with microbubbles, because the addition of microbubbles
causes cavitation and enhances the effect of sonoporation. Cavitation
uses the pressure phase of ultrasonic waves to cause the microbubbles
to alternately contract and expand.
Cavitation bubbles generally refer
to sealed bubbles (10 μm in diameter) which were originally developed
for use in ultrasonic imaging.
The microbubbles vibrate steadily or col-
lapse violently when exposed to ultrasound, causing the liquid owing
around to oscillate to generate sufcient shear force to open up nearby
cell membranes.
An ultrasound eld can focus on local tissues and or-
gans in the microbubbles, thus improving targeted drug delivery.
This technology has been extensively studied and is expected to be an
effective tool for gene delivery.
Wang et al.
incorporated a green
uorescence proteinα-tubulin fusion protein to label the alpha-
tubulin cytoskeleton of HeLa cells and then stimulated these cells with
a single 1 MHz pulsed ultrasound and microbubbles (Fig. 5a and b).
When the acoustic pressure increased, or when the distance between
the microbubbles and the cells decreased, signicant cell deformation
could be observed, hence enhancing the membrane permeability and
disintegration of the cytoskeleton. Therefore, the proper control of
acoustic energy and microbubble-cell distance can effectively improve
the efciency and safety of ultrasound therapy.
Horsley et al.
used ul-
trasound to activate microbubbles to deliver high concentrations of
drugs into urothelial cells. As a common disease, urinary tract infection
still lacks efcient treatment. Oral antibiotics cannot penetrate the blad-
der wall well enough to accumulate to an effective concentration.
Therefore, in their work, the drugs and cultured cells were exposed to
the ultrasound chamber
with effective parameters, proving that the
amount of ultrasound-activated microbubbles released in the cells was
16 times higher than that without microbubbles. This nding could affect
traditional oral antibiotic treatments. Although evidence supports the
therapeutic efcacy of ultrasound-driven microbubbles, problems
with the transformation from basic research to clinical research
still need to be addressed. Roovers et al.
focused on acoustic set-
tings and microbubble-related parameters and envisioned new
technologies that provide additional control over treatment to pro-
vide better microbubble-assisted ultrasonic treatment program.
However, sonoporation often causes irreversible cell damage,
and many studies have discovered that the regeneration and colony
formation ability of cells after ultrasonic radiation are also relatively
The precise control of microbubbles and the exogenous
chemicals used to generate microbubbles remains urgent.
Without microbubbles, the acoustic formed by high-frequency sound
pressure (N10 MHz) only stimulates cell membrane permeability, which
has attracted people's attention. For example, Ding et al.
discussed the basic principle and classication of surface acoustic waves
(SAWs). Compared with cell exposure to low-frequency (b1MHz)bulk
ultrasonic wave, SAWs with high-frequency (N10 MHz) electromechani-
cal Rayleigh waves can effectively eliminate cavitation and excessive
shear damage to cells. Xie et al.
specically described the theory of
SAW and its interaction with particles and contact uids and divided
the SAW uids into two types: the traveling saw (TSAW) and the stand-
ing saw (SSAW). The SAW device is composed of a lithium niobate single-
crystal piezoelectric substrates and alternating nger pairs of straight in-
terdigitated transducer (IDT) placed on a piezoelectric base. For SAW-
promoted drug delivery, AC electrical signal is applied to the IDT, and
acoustic waves are generated and transmitted to the well plate where
cells are cultured. The cells are then exposed to the transient stimulation
of high-frequency acoustic pressure, which would effectively promote
membrane lipid reorganization. AuNPs and macromolecules have been
delivered into cells assisted with SAW.
The experimental results
showed that this method increased the delivery efciency of 20 kDa dex-
tran by N2-fold and that of 250 kDa dextran by 1.5-fold (Fig. 5c). Given
that cell membranes reseal almost instantly after acoustic stimulation
stops, this technique can efciently ensure cell viability. Yoon et al.
veloped an acoustic-transfection technique using a high-frequency ultra-
sonic transducer with a center frequency of N150 MHz in combination
with a uorescence microscope. The transducer relied on a programmed
displacement platform to accurately adjust the position, and a uores-
cence microscope could detect changes in the uorescence intensity of
the treated cells. In general, the focused area of low-frequency ultrasound
at 15 MHz usually reaches the millimeter level, which often affects a
large number of cells. However, this acoustic-transfection technique
can restrain the area with a 10 μm diameter, and the transfection
technology can be realized at the single-cell level through this focus-
ing capability (Fig. 5d). This technology enables the intracellular de-
livery of a variety of DNA plasmids, mRNA. and recombinant proteins
without microbubbles. Acoustic transfection can also provide a
CRISPR/Cas9 system to modify and reprogram the genome of a single
living cell.
Hypersound, dened as ultrasound with frequency N1 GHz and gen-
erated by bulk acoustic wave resonator, has been recently reported for
drug delivery applications.
In this type of device, thin-lm piezoelec-
tric material is sandwiched between two metal electrodes to achieve a
high resonate frequency. Such structure also guarantees the device sta-
bility at high power input (up to a few watts). The generated acoustic
waves propagate along the axial plane, further actuating the uid
When the GHz resonator contacts with the liquid, the acous-
tic wave energy could be attenuated rapidly, and a strong body force is
generated at the deviceliquid interface.
Lu et al.
fabricated the
GHz resonator with a eld effect transistor (FET) on the same chip.
Such composite device was applied to investigate the hypersound
poration effect on supported lipid bilayers (SLBs) in real time. Cyclic
voltammetry, atomic force microscopy, and laser scanning microscopy
were used together to characterize the nanopores.
The relationship
between membrane deformation and poration induced by hypersound
was carefully studied. As shown in Fig. 5e, SLB was covered on the
resonator, and the gold electrode connected with FET. The results
showed that the hypersound propagation deformed SLB and produced
nanopores. Thus, ions in the buffer solution could diffuse across the
membrane and induce potential changes across the membrane.
24 M. Sun, X. Duan / Nanotechnology and Precision Engineering 3 (2020) 1831
Zhang et al.
used the GHz resonator to develop a novel cell
poration method. Hypersound was used to stimulate cells and induce
the transient nanopores to achieve efcient delivery of exogenous
molecules. In this platform, cells were cultured at the bottom of the
chamber, and the target solution was added into the chamber. The
hypersound device was placed on the top of the chamber, and the
Fig. 5. (a) Schematic of cavitation
; (b) schematic of the acoustic exposure apparatus used to investigate the intracellular delivery of uorescent marker and cytoskeleton dynamics
induced by sonoporation
; (c) side (top) and perspective (bottom) view schematics of the experimental setup
; (d) acoustic transfection of adherent cells
; (e) schematic of the
integrated sensing system
; (f) hypersonic wave generated by a nanoelectromechanical resonator.
25M. Sun, X. Duan / Nanotechnology and Precision Engineering 3 (2020) 1831
position of the device was adjusted to optimize poration. DOX is a typ-
ical drug usually delivered into different cell lines, such as HeLa and
3T3 cells, at appropriate input power and treatment time (Fig. 5f). The
application of hypersound not only promotes the entry of DOX into
cells but also facilitates the uniform distribution of DOX in the nucleus.
The resulting strong acoustic streaming could exert large normal and
shear stress on cells and induce the transient nanopores in cell mem-
brane to improve the membrane permeability. Hyper-sonication
requires no micro bubble assistance. Thus, this process features advan-
tages compared with the conventional sonoporation. This work is a
breakthrough in the eld of cell uptake, especially in solving the nuclear
membrane barrier and cytoplasmic transport. Following this work,
polymer-wrapped mesoporous silica NPs encapsulated with DOX
were successfully delivered.
This research showed that hypersound
could promote drug carriers of 100200 nm to penetrate the cell mem-
brane directly, avoiding the slow release of endocytosis and the forma-
tion of endosomes.
This acoustic method is fast and efcient for
cell or tissue treatment, showing great potential for further delivery of
larger molecules or cargo.
2.5. Microuidic system
Electroporation and sonoporation cause low cell viability or may
limit the delivery of materials due to electrical charges. Thus, recent
microuidic delivery methods based on rapid cellular mechanical
deformation have become a prominent alternative in certain
In general, microuidic constriction results in
mechanical deformation of cells by passing them through a narrow
area 30%80% smaller than the cell diameter. The shear force and
pressure generated by this process can induce transient holes and
promote passive diffusion of macromolecules into cytoplasm. Mem-
brane pores can be rapidly sealed after intracellular delivery.
method is simple, controllable, fast, high-throughput, and suitable
for delivery of almost any macromolecule into almost any cell type.
This system shows potential in previously challenging cell types
(primary immune cells and stem cells) and materials. Szeto et al.
devised a simple approach that relied on microscale cell squeezing
and passive diffusion (Fig. 6a) to deliver the entire protein antigen
directly into B cells with low non-specic antigen uptake capacity.
This approach showed a limited success for electroporation, because
it relies on engineering and precisely controlled electric elds. Sharei
et al.
designed a cytoplasmic delivery method based on rapid me-
chanical deformation of cells. They believed that the size and fre-
quency of pores created by membrane rupture are related to the
shear and compression forces that the cells are subjected to. There-
fore, 45 parallel microuidics channels with different shrinkage
sizes and quantities were made by etching silicon and then sealed
by a Pyrex layer (Fig. 6b). The width and length of the constricted
area differed, resulting in varied degrees and duration of shear and
compression forces on the cell. This device was based on a parallel
channel design that could achieve a cell throughput as high as
20,000/s. This approach successfully delivered a range of cargos, in-
cluding carbon nanotubes, proteins and siRNA, into 11 types of
cells, including primary broblasts, embryonic stem cells, and a
range of immune cells. Lam et al.
developed a less expensive but
simple and rapid method using PDMS as a replacement device to
complete the construction of the cell squeezing platform. They de-
signed a cytoplasmic PDMS-based delivery and modication system
called cyto-PDMS. The design principle was similar to that of Sharei
et al's.
. PDMS was molded into microchannels and bonded to a
glass sheet. The cells and macromolecular materials entered from
the air inlet through the contraction area and exited from the air out-
let. The transparency of PDMS allowed direct and real-time charac-
terization of cells as they were passing through the constriction
area. This platform exhibited minimal buckling in the constriction
area but can withstand high shear forces, thus overcoming the
previous challenges of limiting ow rates due to the high pressure
sensitivity of PDMS.
The results indicated that this platform
could deliver cargos of different sizes into the cytoplasm of human -
broblasts with minimal effect on cell viability. In addition, the results
demonstrated that the recombinant enzyme-active Cre-protein
could be transferred into nucleus of the recipient broblast through
appropriate genome recombination. Squeezing as a platform eases
the use of a wide range of cargos without concerning complications,
such as the size and charge action. However, given the cell size differ-
ences and transmission inconsistency, cell clog ging of microchannels
and cargo delivery residual heterogeneity are still unsolved.
Meacham et al.
reported a method for coordinating mechanical
disruption of cell membranes and electrophoresis of DNA into cells to
increase transfection efciency. In this platform, the acoustic shear
force (ASP) technique was used to form a pressure gradient at the tip
of the nozzle by focusing the acoustic wave forcing the cell to pass
through the cell-scale hole and withstanding high shear force in a
short time, thereby forming a continuous transient poration
(Fig. 6c). In this process, only the high shear environment was used to
cause cell poration. The sound eld was insufcient to destroy the cell
membrane. Next, the deformed cells were exposed to a low-intensity
electric eld, which forced DNA into the cells by electrophoresis.
In this work, ASP technology achieved transfection of peripheral blood
mononuclear cells, and the ASP-ep coupling method signicantly im-
proved transfection efciency. It demonstrated the potential of ASP
platform in large-scale integration applications. This approach further
optimized the delivery efciency of microuidic squeezing and shearing
of cell membrane poration by both the diffusion of material molecules
into cells through poration and electrophoretic movement, which can
be actively driven to accelerate the delivery efciency.
Different from the constriction function that disrupts the cell
membrane to increase permeability, Kizer et al.
designed a clog-
free and sheathless inertial microuidic platform, named hydroporator,
based on a rapid mechanical cell deformation relying solely on hydrody-
namic cell shearing. This device utilized the inertia effect to rst mix the
cell suspensions with target materials and inserted them into the
microchannel. As shown in Fig. 6d, given their interactions with the sec-
ondary ow, cells could be aligned with the central channel and uni-
formly migrated to the stagnation point at the cross-junction where
they were stretched by uid dynamics. This process resulted in tran-
sient discontinuous porations in the cell membrane. Thus, targets in
the solution could enter the cytoplasm before the cell membrane was
This work has successfully delivered 2000 kDa dextran
and for the rst time showed the delivery capacity of different large
DNA nanostructures and their biostability in living cells. Given that de-
livery efciency is related to ow rate, a high ow rate is required to
achieve efcient delivery of larger molecules. However excessive ow
rates may exacerbate cell death.
Li et al. designed a droplet microuidic
platform for single-cell
transfection to achieve efcient and consistent plasmid delivery of
lipoplex-mediated suspension cells.
Lipoplex-mediated intracellular
delivery is extremely inefcient (usually b5%) in transfection of
suspended cells (such as lymphocytes and hematopoietic cells for
The efciency of lipoplex-mediated transfec-
tion depends on two factors: endocytic capability of target cells
and lipoplex size.
Through their devices, a single cellwas wrapped
in a dispersed droplet with negatively and positively charged lipids and
passed through a winding structure channel to experience chaotic
advection. Plasmids and liposomes are self-assembled into lipoplexes
in this process. Chaotic advection could aggravate the collision of
lipoplexes with cells. Thus, the shear force exerted by uid compression
on cells increased when cells passed through the droplet pinch-off ori-
ce, leading to increased membrane permeability and enhanced
lipoplex delivery into cells through endocytosis (Fig. 6e). The results
showed that the delivery efciency of three suspended cells increased
from 5% to 50%, solving the problem of intercellular variation. This
26 M. Sun, X. Duan / Nanotechnology and Precision Engineering 3 (2020) 1831
Fig. 6. (a) Schematic representation of microuidic squeezing for macromolecule delivery to cells
; (b) illustration of delivery mechanism showing the rapid deformation of a cell
generating transient membrane holes, as it passes through microuidic constriction. The illustration is an electron micrograph of current parallel channel design with blue cells
(c) schematic of combined mechanoporation/electrophoresis gene transfer method
; (d) schematic of the design and operating principles of the vector-free intracellular delivery
; (e) chip design and working mechanism. Scale bar: 100 μm.
27M. Sun, X. Duan / Nanotechnology and Precision Engineering 3 (2020) 1831
work is expected to reprocess the transfected single cells to minimize
immune rejection.
In addition, the combination of microuidics with electropora-
tion has overcome a series of deciencies, such as heat dissipation
of traditional electroporation. Extremely short electrode spacing
also could reduce the voltage requirements. Xu et al.
fabricated a
microchannel device containing both inlet and outlet electrodes to
generate an electric eld. High-speed uid shear stress and electro-
poration were used to induce cell membrane pores and promote
DNA delivery into cardiomyocytes. Hollow gold electrodes with
high aspect ratios could be combined with electroporation or
optoporation to induce transient nanopores on the cell membrane
with controlled timing and cellular localization.
cell electrical signals during intracellular delivery is critical to the
overall characterization of delivery effects.
However, the
manufacturing complexity of such type of devices often limits this
idea. Drug delivery with microuidic system is often used at several
or single-cell level, allowing the accurate recording of electrical sig-
nals during delivery to reveal the specic behaviors between the
such and the unaffected cells. In the work of Cerea et al.
time electrical recording was achieved in combination with single-
cell drug delivery. They designed a structure that could be used to re-
cord large cell populations. Hollow nanostructures and bottom
microchannels could deliver specicreagentsatspeciclocations
in cell culture. This work lled the gap in the microuidic process
of individual cells in the microelectrode array.
3. Features of an ideal intracellular delivery system
Over the years, we have developed several techniques to overcome
the barriers to intracellular delivery. Different methods feature their
own advantages (Table 2), and comparing their performances is dif-
cult. We aim to develop new methods that can meet the requirements
of high precision, large scale, and high exibility. Here, we present
guidelines that canbe used by researchers to develop new technologies
for intracellular delivery.
Minimal cell perturbation
Exogenous materials and physical forces can cause off-target effects
and toxicity to cells. The environmental factors, such as temperature,
that cause Brownian kicks may exert instability onto delivery systems.
Minimizing the exogenous decoration and manipulation is critical to
successful drug delivery systems.
A delivery system should be scalable given that the number of cells
requiring treatment could vary considerably. Not only the target but
also the production of delivery system should be scalable.
Suitability to cell types
An ideal delivery system should be compatible to any types of cells of
interest, including hard-to-transfect cells. This feature relies on a deliv-
ery mechanism independent of cell type or at least one appropriate
mechanism for each cell type.
Biocompatibility and safety
Compatibility is required for all drug delivery systems to avoid any
problem that might be caused by immune response. Safety is important
for clinical application. NPs, for example, can deliver drugs efciently,
but the toxicity has been a barrier to clinical use.
Control mechanism
A reliable control mechanism is essentially critical for next-
generation drug delivery system. Controlling drug delivery via signaling
is difcult to achieve but is important for sophisticated delivery sys-
tems. The specicity and dosage rely largely on control mechanism.
Furthermore, an ideal drug delivery system should control its behavior
by the environmental factors, such as the temperature, pH, or both.
Delivery systems should be economically reasonable and inexpensive.
4. Outlook
Compared with chemical methods, physics-based membrane dis-
ruption technology overcomes many challenges and avoids the side ef-
fects caused by viral vectors. This process holds potential to solve the
problems of treating difcult-to-transfect cells. In particular, the devel-
opment of micronanotechnologyshows promise among emerging tech-
nologies in the elds of biomedicine and clinical therapy. This discipline
also exhibits new scalability and controllability to treat a few cells andat
the single-cell level. Researchers may select different methods for vari-
ous applications. For example, electrical methods are more accessible
to single-channel independent control; acoustic cavitation and opti-
cal methods are difcult to perform in microarrays, microneedles
and microchannels have certain limitations in terms of the exibil-
ity of cell type, etc. The physical mechanisms of various methods ex-
hibit potential advantages and limitations. The combination two or
more of these techniques may provide more sustainable and inno-
vative options for intracellular delivery systems. A more thorough
understanding of the intracellular delivery mechanism will help
the biomedical research community to further develop more
powerful technologies in medical and industrial applications.
Micro-nanoscale manufacturing technology has become the key to
promote micro-systems to be more miniaturized and diversied
and integrated in large scale. If we could reasonably use their unique
advantages to cooperate, improvements in the intracellular delivery
system can be possibly achieved.
Declaration of Competing Interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inu-
ence the work reported in this paper.
Table 2
Advantages and challenges for membrane disruption methods.
Methods Advantages Challenges
Nanoneedle Provides cellular nanoscale
Directly penetrates the
Can be used for many cell
High-precision manufacturing
Difcult to implement on a large
Easily causes cell lysis.
Not suitable for high-throughput.
Electroporation High transfection
High cell death rate.
Slow microow processing.
Optoporation Local operation.
High accuracy at single-cell
Complex and costly equipment.
Damage to some molecules
(e.g., protein denaturation).
Sonoporation Transient exposure to
membrane disruption and
low cell damages.
Locate microbubbles and
target cavitation.
Suitable for a wide range of
cell types.
Loss of cytoplasmic content.
Cavitation could produce reactive
oxygen species that damage DNA,
requires additional chemicals in
conventional sonoporation.
Microuidic Simple equipment and easy
Independent of physical
eld or the carrier.
Hydrodynamics does not
block the ow passage.
The efciency of the squeeze cell
method correlates with cell size.
Works on suspended cells only and
considers the molecular residual
heterogeneity of materials.
Incompatible with cell
characterization methods.
The size of membrane poration
depends on precise shear forces.
28 M. Sun, X. Duan / Nanotechnology and Precision Engineering 3 (2020) 1831
This work was supported by National Natural Science Foundation of
China (NSFC No. 61674114, 91743110, 21861132001), National Key Re-
search and Development Program of China (2017YFF0204604), Tianjin
Applied Basic Research and Advanced Technology (17JCJQJC43600),
the Foundation for Talent Scientists of Nanchang Institute for Micro-
technology of Tianjin University, and the 111 Project (B07014).
1. Dixon JE, Osman G, Morris GE, et al. Highly efcient delivery of functional cargoes by
the synergistic effect of GAG binding motifs and cell-penetrating peptides. Proc Natl
Acad Sci U S A 2016;113(3):E291-9.
2. Bacolla A, Wang G,Vasquez KM. New perspectives on DNA and RNA triplexes as ef-
fectors of biological activity. PLoS Genet2015;11(12):1-12.
3. Sibbitts J, Sellens KA, Jia S, et al. Cellular analysis using microuidics. Anal Chem
4. Mantz A, Pannier AK. Biomaterial substrate modications that inuence cell-
material interactions to prime cellular responses to nonviral gene delivery. Exp
Biol Med 2019;244(2):100-13.
5. Adler AF, Leong KW. Emerging links between surface nanotechnology and endocy-
tosis: Impact on nonviral gene delivery. Nano Today 2010;5(6):553-69. https://doi.
6. Zhang R, Qin X, Kong F, et al. Improving cellular upta ke of therapeutic entities
through interaction with components of cell membrane. Drug Delivery 2019;26
7. Sharei A, Mao S,Langer R, et al. Intracellular delivery of biomoleculesby mechanical
deformation. Micro-Nanosystem. biotechnology 2016:143-76.
8. Hartman TE, Sar N, Genereux K, et al. Derivation and characterization of lines for
production of recombinant antibodies. Biotechnol Bioeng 2007;99(4):846-54.
9. Peer D. A daunting task: Manipulating leukocyte function with RNAi. Immunol Rev
10. Di Pisa M, ChassaingG, Swiecicki JM. When cationic cell-penetrating peptides meet
hydrocarbons to enhance in-cell cargo delivery. J Pept Sci 2015;21(5):356-69.
11. Stewart MP, ShareiA, Ding X, et al. In vitroand ex vivo strategiesfor intracellularde-
livery. Nature 2016;538(7624):183-92.
12. Thomas CE, Ehrhardt A, Kay MA. Progress and problems with the use of viral vectors
for gene therapy. Nat Rev Genet 2003;4(5):346-58.
13. Kay MA. State-of-the-art gene-based therapies: The road ahead. Nat Rev Genet
14. Khalil IA, Kogure K, Akita H, et al. Uptake pathways and subsequent intracellular
trafcking in nonviral gene delivery. Pharmacol Rev 2006;58(1):32-45. https://
15. Sahay G, Alakhova DY, Kabanov AV. Endocytosis of nanomedicines. J Control Re-
lease 2010;145(3):182-95.
16. Stewart MP, Lorenz A, Dahlman J, et al. Challenges in carrier-mediated intracellular
delivery: Moving beyond endosomal barriers. Wiley Interdiscip Rev Nanomedicine
Nanobiotechnology 2016;8(3):465-78.
17. Furusawa M, Nishimura T, Yamaizumi M, et al. Injection of foreign substances into
single cells by cell fusion. Nature 1974;249(5456):449-50.
18. Wood MJA. Extracellular vesicles: biology and emerging therapeutic opportunities.
Nat Publ Gr 2013;12(5):347-57.
19. Yang J, Tu J, Lamers GEM, et al. Membrane fusion mediated intracellular delivery of
lipid bilayer coated mesoporous silica nanoparti cles. Adv Healthc Mater 2017;6
20. Saari H, Lisitsyna E, Rautaniemi K, et al. FLIM reveals alternative EV-mediatedcellu-
lar up-take pathways of paclitaxel. J Control Release 2018;284:133-43. https://doi.
21. Marschall ALJ, Zhang C, Frenzel A, et al. Delivery of antibodies to the cytosol:
Debunking the myths. MAbs 2014;6(4):943-56.
22. Hendel A, Bak RO, Clark JT, et al. Chemically modied guide RNAs enhance CRISPR-
Cas genome editing in human primary cells. Nat Biotechnol 2015;33( 9):985-9.
23. Schumann K, Lin S, Boyer E, et al. Generation of knock-in primary human T cells
using Cas9 ribonucleoproteins. Proc Natl Acad Sci U S A 2015;112(33):10437-42.
24. Barber MA. A technic for the inoculation of bacteria and other substances into living
cells. J Infect Dis 1911.
25. Neumann E, Schaefer-Ridder M, WangY, et al. Gene transfer into mouse lyoma cells
by electroporation in high electric elds. EMBO J 1982;1(7):841-5.
10.1002/j.1460-2075. 1982.tb01257.x.
26. Tsukakoshi M, Kurata S, Nomiya Y, et al. A novel method of DNA transfection by
laser microbeam cell surgery. Appl Phys B Photophysics Laser Chem 1984;35(3):
27. FechheimerM, Boylan JF, Parker S, et al. Transfection of mammalian cellswith plas-
mid DNA by scrape loadingand sonication loading. Proc Natl Acad Sci U S A 1987;84
28. Chow YT, Chen S, Liu C, et al. A high-throughput automated microinjection system
for human cellswith small size. IEEE/ASME Trans Mechatronics 2016;21(2):838-50.
29. Shull G, Haffner C, Huttner WB, et al. Robotic platform for microinjection into single
cells in brain tissue. EMBO Rep 2019.
30. Ye Y, Yu J, Wen D, et al. Polymeric microneedles for transdermal protein delivery.
Adv Drug Deliv Rev 2018;127: 106-18.
31. Gerstel MS. Place VA. Drug Delivery Device US Patent 1976;3:964,482.
32. Madou MJ. Fundamentals of microfabrication and nanotechnology, three-volume set.
CRC Press. 2011.
33. Kim Y, Park J, Prausnitz MR. Microneedles for drug and vaccine delivery. Adv Drug
Deliv Rev 2012;64(14):1547-68.
34. Gossett DR, Tse HTK, Lee SA, et al. Hydrodynamic stretching of single cells for large
population mec hanical phenot yping. Proc Natl Acad Sci U S A 2012 ;109(20):
35. Jiang W, Tian Q, Vuong T, et al. Comparison study on four biodegradable polymer
coatings for controlling magnesium degradation and human endothelial cell adhe-
sion and spreading. ACS Biomater Sci Eng 2017;3(6):936-50.
36. Hye J, Shin JU, Hyeong S, et al. Biomaterials successful transdermal allergen de-
livery and allergen-specic immunotherapy using biodegradable microneedle
patches. Biomaterials 2018;150:38-48.
37. Yang J, Chen Z, Ye R, et al. Touch-actuated microneedle array patch for closed-loop
transdermal drug delivery. Drug Delivery 2018;25(1):1728-39.
38. Joyce JC, Carroll TD, Collins ML, et al. A microneedle patch for measles and rubella
vaccination is immunogenic and protective in infant rhesus macaques. J Infect Dis
39. Maurya A, Nanjappa SH, Honnavar S, et al. Rapidly dissolving microneedle patches
for transdermal iron replenish ment therapy. J Pharm Sci 2018; 107(6):1642 -7.
40. Zhang Y, Liu Q, Yu J, et al. Locally induced adipose tissue browning by microneedle
patch for obesity treatment. ACS Nano 2017;11(9):9223-30.
41. Kajimura S, Spiegelman BM, Seale P. Brown and beige fat: Physiological roles be-
yond heat generation. Cell Metab 2015;22(4):546-59.
42. Lu Y, AimettiAA, Langer R, et al. Bioresponsive materials. Nat Rev Mater 2017;2(1),
43. Kim W, Ng JK, Kunitake ME, et al. Inter facing silicon nanowires with mammalian cells. J
Am Chem Soc 2007;129(23):7228-9.
44. Kwak M, Han L, Chen JJ, et al. Interfacing inorganic nanowire arrays and living cells
for cellular function analysis. Small 2015;11(42):5600-10.
smll.201 501236.
45. Tian JH, Hu J, Zhang F, et al. Microelectronic engineering fabrication of high-density
metallic nanowires and nanotubes for cell culture studies,88 . 2011:1702-6. https://
46. Shalek AK, Robinson JT, Karp ES, et al. Vertical silicon nanowires as a universal plat-
form for delivering biomoleculesinto living cells. Proc Natl Acad Sci U S A 2010;107
47. Ha W, Montelius L, Samuelson L, et al. Gallium phosphide nanowires as a substrate
for cultured neu rons. Nano Lett 2007;7(10): 2960-5.
48. Jiang K, Fan D, Belabassi Y, et al. Medicinal surface modication of silicon nano-
wires: Impact on calcication and stromal cell proliferation. ACS Appl Mater Inter-
faces 2009;1(2):266-9.
49. Qi S, Yi C, Ji S, et al. Cell adhesion and spreading behavior on vertically aligned sili-
con nanowire arrays. ACS Appl Mater Interfaces 2009;1(1):30-4.
50. Turner AMP, Dowell N, Turner SWP, et al. Attachment of astroglial cells to
microfabricated pillar arrays of different geometries. J Biomed Mater Res 2000;51(3):
430-41. AID-JBM18N3.0.
51. Xu AM, Aalipour A, Leal-Ortiz S, et al. Quantication of nanowire penetration into
living cells. Nat Commun 2014;5, 3613.
52. Wu Y, Li L, Mao Y, et al. Static micromixer-coaxial electrospray synthesis of theranostic
lipoplexes. ACS Nano 2012;6(3):2245-52.
53. Xu AM, Kim SA, Wang DS, et al. Temporally resolved direct delivery ofsecond mes-
sengers into cells using nanostraws. Lab Chip 2016;16(13):2434-9.
54. He G, Chen HJ, Liu D, et al. Fabricationof various structures of nanostraw arrays and
their applications in gene delivery. Adv Mater Interfaces 2018;5(10):1-8. https://
55. Campbell SA. Fabrication engineering at the micro- and nanoscale New York,10 .
2008:356-436 doi:0199861226.
56. Paik SJ, Park S, Zarnitsyn V, et al. A highly dense nanoneedle array for intracellular
gene delivery. Tech Dig - Solid-State Sensors, Actuators, Microsystems Work. 2012:
149-52. hh2012.40.
57. Park S, Choi SO, Paik SJ, et al. Intracellular delivery of molecules using microfabricated
nanoneedle arrays. Biomed Microdevices 2016;18(1):1-13.
58. Zhu X, Kwok SY,Yuen MF, et al. Dense diamond nanoneedle arraysfor enhanced in-
tracellular delivery of drug molecules to cell lines. J Mater Sci 2015;50(23):7800-7.
29M. Sun, X. Duan / Nanotechnology and Precision Engineering 3 (2020) 1831
59. Yang Y, Yuen MF, Chen X, et al. Fabrication of arrays of high-aspect-ratio diamond
nanoneedles via maskless ecr-assisted microwave plasma etching. CrystEngComm
60. Zhang Y, AliSF, Dervishi E, et al.Cytotoxicity effects of graphene and single-wall car-
bon nanotubes in neural phaeochromocytoma-derived pc12 cells. ACS Nano 2010;4
61. Soenen SJ, Parak WJ, Rejman J, et al. (Intra)cellular stability of inorganic nanoparti-
cles: Effects on cytotoxicity, particle functionality, and biomedi cal applications.
Chem Rev 2015;115(5):2109-35.
62. Djuris AB, Leung YH, Ng AMC, et al. Toxicity of metal oxide nanoparticles: Mecha-
nisms, characterization, and avoiding experimental artefacts. Small 2015;11(1):
63. Low SP, Williams KA, Canham LT, et al. Evaluation of mammalian cell adhesion on
surface-modied porous silicon. Biomaterials 2006;27(26):4538-46. https://doi.
64. Peng J, Garcia MA, Choi JS, et al. Molecular recognition enables nanosubstrate-
mediated delivery of gene-encapsulated nanoparticles with high efciency. ACS
Nano 2014;8(5):4621-9.
65. Hou S, Choi JS, Chen KJ, et al. Supramolecular nanosubstrate-mediated delivery for
reprogramming and transdifferentiation of mammalian cells. Small 2015;11(21):
66. Shimizu N, Kamezaki F, Shigematsu S. Tracking of microinjected DNA in live
cells reveals the intracellular behavior and elimination of extrachromosomal
genetic material. Nucleic Acids Res 2005;33(19):6296-307.
67. Tsong TY. Electroporation of cell m embranes. Bio phys J 1991;60( 2):297-306.
68. Yang Z, Chang L, Chiang C, et al. Micro-/nano-electroporation for active gene
delivery. Curr Pharm Des 2015;21(42):6081-8.
69. Movahed S, Li D. Microuidics cell electroporation. Microuid Nanouid 2011;10
70. Chang L, Gallego-Perez D, Chiang CL, et al. Controllable large-scale transfection of
primary mammali an cardiomyocytes on a nanochannel array platform. Small
71. Guo X, Zhu R. Controllable in-situ cell electroporation with cell positioning and im-
pedance monitoring using micro electrode array. Sci Rep 2016;6(1), 31392. https://
72. Wiegert JS, Gee CE, Oertner TG. Single-cell electroporation of neurons. Cold Spring
Harb Protoc 2017;2017(2):135-8. prot094904.
73. Semenov I, Xiao S, Pakhomov AG. Electroporation by subnanosecond pulses. Bio-
chemistry Biophysics Reports 2016;6:253-9.
74. Ouyang M, Hill W, Lee JH, et al. Microscale symmetrical electroporator array as a
versatile molecular delivery sy stem. Sci Rep 20 17;7, 44757. https://doi.or g/10.
75. Hsi P, Christianson RJ, Dubay RA, et al. Acoustophoretic rapid media exchange and
continuous-ow electrotransfectionof primary human T cells for applicationsin au-
tomated cellular therapy manufacturing. Lab Chip 2019;19(18):2978-92. https://
76. Maragò OM, Jones PH, Gucciardi PG, et al. Optical trapping and manipulation of
nanostructures. Nat Nanotechnol 2 013;8(11):8 07-19. https://
77. Stevenson DJ, Gunn-Moore FJ, Campbell P, et al. Single cell optical transfection. J R
Soc Interface 2010;7(47):863-71.
78. Uchugonova A, König K, Bueckle R, et al. Targeted transfection of stem cells with
sub-20 femtosecond laser pulses. Opt Express 2008;16(13):9357.
79. Dhakal K, Batabyal S, Wright W, et al. Optical delivery of multiple opsin-encoding
genes leads to targeted expressi on and white-light activation. Light Sci Appl
80. Yang X, Xie H, Alonas E, et al. Mirror-enhanced super-resolution microscopy. Light:
Science & Applications 2016;5(6):e16134-8.
81. UchugonovaA, Breunig HG, BatistaA, et al. Optical reprogramming of human cellsin
an ultrashort femtosecond laser microuidic transfection platform. J Biophotonics
82. Schomaker M, Heinemann D, Kalies S, et al. Characterization of nanoparticle mediated
laser transfection by femtosecond laser pulses for applications in molecular medicine. J
Nanobiotechnology 2015;13(1):1-15.
83. Lukianova-Hleb EY, Mutonga MBG, Lapotko DO. Cell-specic multifunctional pro-
cessing of heterogeneous cell systems in a single laser pulse treatment. ACS Nano
84. Kohli V, Elezzabi AY. Lasersurgery of zebrash (Danio rerio) embryos using femto-
second laser pulses: Optimal parameters for exogenous material delivery, and the
lasers effect on short- and long-term development. BMC Biotechnol 2008;8:1-20.
85. Karki A, Giddings E, Carreras A, et al. Sonoporation as an Approach for siRNA deliv-
ery into T cells. Ultrasound Med Biol 2019;45( 12):3222-31.
86. Myers R, Grundy M, Rowe C, et al. Ultrasound-mediated cavitation does not de-
crease the activity of small molecule, antibody or viral-based medi cines. Int J
Nanomedicine 2018;13:337-49.
87. Sun S, Xu Y, Fu P, et al. Ultrasound-targeted photodynamic and gene dual therapy
for effectively inhibiting triple negative breast cancer by cationic porphyrin lipid
microbubbles loaded with HIF1α-siRNA. Nanoscale 2018;10:58-70. https://doi.
88. Rinaldi L, Folliero V, Palomba L, et al. Sonoporation by microbubblesas gene therapy
approach against liver cancer. Oncotarget 2018;9(63):32182-90.
89. Shapiro G, Wong AW, Bez M, et al. Multiparameter evaluation of in vivo gene deliv-
ery using ultrasound-guided, microbubble-enhanced sonoporation. J Control Re-
lease 2016;223:157-64.
90. Fechheimer M, Denny C, Murphy RF, et al. Measurement of cytopla smic pH in
Dictyostelium discoideum by using a new method for introducing macromolecules
into living cells. Eur J Cell Biol 1986;40(2):242-7.
91. Stride E. Physical principles of microbubbles for ultrasound imaging and therapy.
Cerebrovasc Dis 2009;27(SUPPL 2):1-13.
92. Shung KK. Diagnostic ultrasound: Past, present, and future. J Med Biol Eng 2011;31
93. Kooiman K, Vos HJ, Versluis M, et al. Acoustic behavior of microbubbles and impli-
cations for drug delivery. Adv Drug Deliv Rev 2014;72:28-48.
1016/j.add r.2014.0 3.003.
94. Yildirim A, Shi D, Roy S, et al. Nanoparticle-mediated acoustic cavitation enables
high intensity focused ultrasound ablation without tissue heating. ACS Appl Mater
Interfaces 2018;10:36786-95.
95. Lenta cker I, De Cock I, D eckers R, et al. Understanding u ltrasound induced
sonoporation: Denitions and u nderlying mechanisms. Adv Dru g Deliv Rev
96. Noble-Vranish ML, Song S, Morrison KP, et al.Ultrasound-mediated gene therapy in
swine livers using single-element, multi-lensed, high-intensity ultrasound trans-
ducers. Mol Ther - Methods Clin Dev 2018;10:179-88.
97. Wang M, Zhang Y, Cai C, et al. Son oporation-induced cell membrane perme-
abilization and cytoskeleton disassembly at varied acoustic and microbubble-cell
parameters. Sci Rep 2018;8(1):1-12.
98. Horsley H, Owen J, BrowningR, et al. Ultrasound-activated microbubbles as a novel
intracellular drug delivery system for urinary t ract infection. J Control Release
99. Defoor W, Ferguson D, Mashni S, et al. Safety of gentamicin bladder irrigations in
complex urolog ical cases. J Urol 2006;175(5 ):1861-4.
100. Carugo D, Owen J, Crake C, et al. Biologically and acoustically compatible chamber
for studying ultrasound-mediated delivery of therapeutic compounds. Ultrasound
Med Biol 2015;41(7):1927-37.
101. Roovers S, Segers T, Lajoinie G, et al. The role of ultrasound-driven microbubble
dynamics in drug delivery: From microbubble fundamentals to clinical transla-
tion. Langmuir 2019;35(31):10173-91.
102. Spurný P, Oberst J, Heinlein D. Photographicobservations of Neuschwanstein,a sec-
ond meteorite from the orbit of the Příbram chondrite. Nature 2003;423(6936):
103. Kaufman GE, Miller MW, Dan Grifths T, et al. Lysis and viability of cultured mam-
malian cells exposed to 1 MHz ultrasound. Ultrasound Med Biol 1977;3(1):21-5.
104. Ding X, Li P, Lin SCS, et al. Surface acoustic wave microuidics. Lab Chip 2013;13
105. Xie Y, Bachman H, Huang TJ. Acoustouidic methods in cell analysis. TrAC - Trends
Anal Chem 2019;117:280-90.
106. RamesanS, Rezk AR, Dekiwadia C, et al. Acoustically-mediatedintracellulardelivery.
Nanoscale 2018;10(27):13165-78.
107. Yoon S, Wang P, Peng Q, et al. Acoustic-transfection for genomic manipulation of
single-cells using high frequency ultrasound. Sci Rep 2017;7(1):1-11. https://doi.
108. Zhang Z, WangY, Zhang H, et al. Hypersonicporation: A new versatile cell poration
method to enhance cellular uptake using a piezoelectric nano-electromechanical
device. Small 2017;13(18), 1602962.
109. Cui W, Zhang H, ZhangH, et al. Localized ultrahigh frequencyacoustic elds induced
micro-vortices for submilliseconds microuidic mixing. Appl Phys Lett 2016;109
(25), 253503.
110. Cui W, Pang W, Yang Y, et al. Theoretical and experimental char acterizations of
gigahertz acoustic streaming in microscale uids. Nanotechnol Precis Eng 2019;2
111. Lu Y, Huskens J, Pang W, et al. Hyper sonic poration of supported lipid bilayers.
Mater Chem Front 2019;3(5):782-90.
112. Qu H, Yang Y, Chang Y, et al. On-chip integrated multiple microelectromechanical
resonators to enable the local heating, mixing and viscosity sensing for chemical re-
actions in a droplet. Sensors Actuators B Chem 2017;248:280-7.
113. Lu Y, Palanikumar L, Choi ES, et al. Hypersound-enhanced intracellular delivery
of drug-loaded mesoporous silica nanoparticles in a non-endosomal pathway.
ACS Appl Mater Interfaces 2019;11:19734-42.
114. Miyata K, Oba M, Nakanishi M, et al. Polyplexes from poly(aspartamide) bearing
1,2-diaminoethane side chains inducepH-selective, endosomal membrane destabi-
lization with amplied transfection and negligible cytotoxicity. J Am Che m Soc
115. Benjaminsen RV, Mattebjerg MA, Henriksen JR, et al. The possible "proton sponge "
effect of polyethylenimine (PEI) does not include change inlysosomal pH. Mol Ther
116. DiTommaso T, Cole JM, Cassereau L, etal. Cell engineeringwith microuidic squeez-
ing preserves functionality of primary immune cells in vivo. Proc Natl Acad Sci U S A
30 M. Sun, X. Duan / Nanotechnology and Precision Engineering 3 (2020) 1831
117. Klein A, Hank S, Raulf A, et al. Live-cell labeling of endogenous proteins with nano-
meter precision by transduced nanobodies. Chem Sci 2018;9(40):7835-42. https://
118. Ding X, Stewart MP, Sharei A, et al. High-throughput nuclear delivery and rapid ex-
pression of DNA via mecha nical and electrical cell-membrane disruption. Nat
Biomed Eng 2017;1(3):1-7.
119. Blazek AD, Paleo BJ, Weisleder N. Plasma membrane repair: A central process for
maintaining cellular homeostasis. Physiology 2015;30(6):438-48.
120. Szeto GL, Van Egeren D, Worku H, et al. Microuidic squeezing for intracellular an-
tigen loading in polyclonal B-cells as cellular vaccines. Sci Rep 2015;5:1-13. https://
121. Sharei A, Zoldan J, Adamo A, et al. A vector-free microuidic platform for intracellu-
lar delivery. Proc Natl Acad Sci U S A 2013;110(6):2082-7.
pnas.121870511 0.
122. Lam KH, Fernandez-Perez A, Schmidtke DW, et al. Functional cargo delivery into
mouse and human broblasts using a versati le microuidic device. Biomed
Microdevices 2018;20(3):1-14.
123. GervaisT, El-Ali J, Günther A, et al.Flow-induceddeformationof shallow microuidic
channels. Lab Chip 2006;6(4):500-7.
124. Saung MT, Sharei A, Adalsteinsson VA, et al. A size-selective intracellular delivery
platform. Small 2016;12(42):5873-81.
125. Meacham JM, Durvasula K, Degertekin FL, et al. Enhanced intracellular delivery via
coordinated acoustically driven shear mechanoporation and electrophoretic inser-
tion. Sci Rep 2018;8(1):1-10.
126. Zarnitsyn VG, Meacham JM, Varady MJ, et al. Electrosonic ejector microarray for
drug and gene delivery. Biomed Microdevices 2008;10(2):299-308. https://doi.
127. Sukharev SI, Klenchin VA, Serov SM, et al. Electroporation and electrophoretic DNA
transferinto cells-the effectof DNA interactionwith electropores.Biophys J 1992;63
128. Dimitrov DS, Sowers AE. Membrane electroporaton fast molecular exchange by
electroosmosis. Biochim Biophys Acta Biomembr 1990;1022(3):381-92. https://
129. Kizer ME, Deng Y, Kang G,et al. Hydroporator:A hydrodynamic cell membrane per-
forator for high-throughput vector-free nanomaterial intracellular deliver y and
DNA origami biostability evaluation. Lab Chip 2019;19(10):1747-54. https://doi.
130. Tsukakoshi M, Kurata S, Nomiya Y, et al. A novel method of DNA transfection by
laser microbeam cell surgery. Appl Phys B Photophysics Laser Chem 1984;35(3):
131. Cooper ST, McNeil PL. Membrane repair: Mechanisms and pathophysiology. Physiol
Rev 2015;95(4):1205-40.
132. Teh SY, Lin R, Hung LH, et al. Droplet microuidics. Lab Chip 2008;8(2):198-220.
133. Li X, A ghaamoo M, Li u S, et al . Lipoplex- medi ated single-cell tran sfec tion via
droplet microuidics. Small 2018;14(40):1-10.
134. Uchida E, Mizuguchi H, Ishii-WatabeA, et al. Comparison of the efciency andsafety
of non-viral vector-mediated gene transfer into a wide range of human cells. Biol
Pharm Bull 2002;25(7):891-7.
135. Maurisse R,De Semir D, Emamekhoo H, et al. Comparative transfection of DNA into
primary and transformed mammalian cells from different lineages. BMC Biotechnol
136. Palchetti S, Pozzi D, Marchini C, et al. Manipulation of lipoplex concentration at the
cell surface boosts transfec tion efciency in hard-to-transfect cells. Nanomed
Nanotechnol Biol Med 2017;13(2):681-91.
137. Maiti B, Kamra M, Karande AA, et al. Transfection efciencies of α-tocopherylated
cationic gemini lipids with hydroxyethyl bearing headgroups under high serum
conditions. Org Biomol Chem 2018; 16(11):1983- 93.
138. Digiacomo L, Palchetti S, Pozzi D, etal. Cationic lipid/DNA complexesmanufactured
by microuidics and bulk self-assembly exhibit different transfec tion behavior.
Biochem Biophys Res Commun 2018;503(2):508-12. https://
139. Mochizuki S, Nishina K, Fujii S, et al. The transfection efciency of calix[4]arene-
based lipids: The role of the alkyl chain length. Biomater Sci 2015;3(2):317-22.
140. Xu Z, Malhi M, Maynes J, et al. Microuidic delivery of genome-editting materials
into iPS-cardiomyocytes using sy nergistic electroporation and shear stress.
TRANSDUCERS 2017 - 19th Int Conf Solid-State Sensors. Actuators Microsystems
141. Messina GC, Dipalo M, La Rocca R, et al. Spatially, temporally, and quantitatively
controlled delivery of broad range of molecules into selected cells through plas-
monic nanotubes. Adv Mater 2015;27(44):7145-9.
142. Caprettini V, Cerea A, Melle G, et al. Soft electroporation for delivering molecules
into tightly adherent mammalian cells through 3D hollow nanoelectrodes. Sci Rep
143. Spira ME, HaiA. Multi-electrode array technologies for neuroscience and cardiology.
Nat Nanotechnol 2013;8(2):83-94.
144. Cerea A, Caprettini V, Bruno G, et al. Selective intracellulardelivery and intracellular
recordings combined in MEA biosensors. Lab Chip 2018;18(22):3492-500. https://
145. Gladkov A, Pigareva Y, Kutyina D, et al. Design of cultured neuron networks in vitro
with predened connectivity using asymmetric microuidic channels. Sci Rep
146. Grygoryev K, Herzog G, Jackson N, et al. Reversible integration of microuidic de-
vices with microelectrode arrays for neurobiological applications. Bionanoscience
Mengjie Sun received the from Tianjin University
in 2017, majoring in measurement and control technology
and instruments. She is currently pursuing a M.S. degree in
Tianjin University.Her research interests focus on intracellu-
lar delivery at the micro/nanoscale based on hypersound.
Xuexin Duan received his PhD degree at University of
Twente, Netherland (2010). After Postdoc stud ies at Yale
University, he moved to Tianjin University. Currently, he is
a full professor at State Key Laboratory of Precision Measur-
ing Technology & Instruments, Department of Precision In-
strument Engineering of Tianjin University. His research is
about MEMS/NEMS devices, microsystem, microuidics and
their interfaces with chemistry, biology, medicine, and envi-
ronmental science.
31M. Sun, X. Duan / Nanotechnology and Precision Engineering 3 (2020) 1831
... Alternative processing has been tested, and improved internalization rates have been achieved by modulating the cell permeability through biotechnological processes such as osmoporation (de Andrade et al. 2022b) and electroporation (Dimopoulos et al. 2021). Sonoporation, for example, is an emerging nonthermal processing technology able to improve the permeability of yeast envelopes by combining cavitation, heating, dynamic agitation, shear stresses, and turbulence (Sun and Duan 2020). However, so far, few studies have investigated its feasibility for enhancing the internalization rate of bioactive molecules into yeast biocapsules. ...
... Indeed, sonoporation is a nonthermal, mild, and straightforward permeabilization process which allows the release of inner biomolecules, such as fatty acids and polysaccharides (Wu et al. 2015), as well as the intracellular delivery of target biomaterials, such as genes (Ramesan et al. 2018). Through this technique, the conformational arrangement of the yeast envelope may be altered by newly formed pores/openings in the plasma membrane as a response to acoustic energy (Sun and Duan 2020). ...
Full-text available
The encapsulation of fisetin into S. cerevisiae cells through sonoporation coupled with drying is reported for the first time in the literature. To establish the best conditions to maximize the amount of internalized fisetin, the cell density (5–10% w/v), fisetin concentration (1–3 mg/mL), acoustic energy density (0–333.3 W/L), and drying method (freeze-drying and spray drying) were analyzed through a Box-Behnken experimental design (BBD) coupled with response surface methodology (RSM). Higher encapsulation efficiency (EE) was achieved with a cell density of 10% w/v, while fisetin concentration of 3 mg/mL favored the encapsulation yield (EY) and antioxidant activity (AA). Higher EE (67.7%), EY (25.7 mg/g), and AA (90%) were registered when an acoustic density of 333.3 W/L was used. Furthermore, both drying protocols promoted fisetin encapsulation, but through spray drying, the EE, EY, and AA were 11.5%, 11.1%, and 26.6% higher than via freeze-drying, respectively. This work proved that fully filled biocapsules were produced through sonoprocessing, and their morphology was influenced by the acoustic energy and drying process. Overall, these results open new perspectives for the application of sonoprocessing-assisted encapsulation, paving the way for developing innovative yeast-based delivery systems for lipophilic compounds such as fisetin. Key points • Sonoprocessing improves the encapsulation of fisetin into S. cerevisiae cells • Spray drying promotes fisetin loading into yeasts’ intracellular space and cavities • Fisetin binding with yeast extracellular agents are favored by freeze-drying
... In-cell NMR requires a long time, ≥3 h, to collect spectra during which cells die, lyse, and leak. Among the most commonly employed delivery techniques is electroporation, however, electroporated cells exhibit damage to membranes, mitochondria, protein, and DNA, decreases in ATP levels as well as increases in reactive oxygen species, ROS, and intracellular Ca 2+ concentrations, all of which can lead to cell death 27,28 . Thus effective use of electroporation requires optimization of a number of parameters including voltage, cuvette gap size, shape, length and number of pulses, cell size and concentration, buffer and temperature, to strike a balance between transfection efficiency and cell death. ...
... Unlike the case of VECT protein delivery, control experiments examining the supernatant of electroporated samples revealed sharp 1 H-15 N cross-peaks consistent with leakage of labeled target protein from the cells ( Supplementary Fig. 3). This likely reflects the loss of integrity of plasma and nuclear membranes and other organelles due to the electroporation process 19,27,28 . The combination of prolonged cell viability and the absence of cell leakage suggests that VECT is a simple and reliable method to deliver exogenous target proteins for longduration in-cell NMR studies. ...
Full-text available
High-resolution structural studies of proteins and protein complexes in a native eukaryotic environment present a challenge to structural biology. In-cell NMR can characterize atomic resolution structures but requires high concentrations of labeled proteins in intact cells. Most exogenous delivery techniques are limited to specific cell types or are too destructive to preserve cellular physiology. The feasibility of microfluidics transfection or volume exchange for convective transfer, VECT, as a means to deliver labeled target proteins to HeLa cells for in-cell NMR experiments is demonstrated. VECT delivery does not require optimization or impede cell viability; cells are immediately available for long-term eukaryotic in-cell NMR experiments. In-cell NMR-based drug screening using VECT was demonstrated by collecting spectra of the sensor molecule DARPP32, in response to exogenous administration of Forskolin. The microfluidic technique of cell volume exchange for convective transfer, VECT can be used to deliver the DARPP-32 protein into cells for in-cell NMR experiments.
... In addition to the general need for high cargo concentrations in diffusion-based delivery methods, this remains a major hurdle to be solved for effective siRNA transfection in an ACT context [129]. One potential strategy could be combining cell squeezing with electrophoresis to increase loading of siRNA to the cell cytoplasm [130,131]. While a high-shear environment can permeate the cell membrane, its combination with electrophoretic activity has been shown to enable active transport of DNA in Jurkat cells [131]. ...
T lymphocytes are the major drivers of antitumor immunity. The recent clinical success of adoptive T cell therapies and immune checkpoint inhibitors has demonstrated the strength of modulating T cell function in fighting cancer. Nonetheless, a significant fraction of patients remain unresponsive largely due to the immunosuppressive tumor environment that blunts T cell activity. Small interfering RNAs (siRNAs) offer the potential to sequence-specifically silence the expression of negative regulator genes in T cells in a transient manner, thereby releasing the block on anti-tumor responses. Despite the current focus on small molecule- and antibody-based immune checkpoint inhibitors as well as T cell-directed delivery of mRNA and genome editing machinery, the application of siRNA involves important clinical advantages. The recent surge of adoptive cell therapies and development of new and potent delivery approaches has enabled efficient siRNA delivery to T cells both ex vivo and in vivo. As such, siRNA molecules have a newfound potential to improve the proliferation, survival, tumor infiltration and potency of T cells in cancer immunotherapy. In this review, we briefly discuss the extracellular and intracellular delivery hurdles associated with siRNA therapy, in particular with regard to T cell targeting. We provide a timely and comprehensive overview of current and emerging delivery technologies used for siRNA transfection, discussing their strengths and weaknesses from a clinical as well as a manufacturing point-of-view. Finally, we critically review the current status and new potential avenues for modulating T cell function in cancer immunotherapy using siRNA.
... The reasons are manifold, some related to insufficient understanding of the molecular basis of many diseases [8], others to the difficulty of designing therapeutic molecules that effectively interfere with the functionality of fully folded proteins [9], or the presence of post-translational modifications of key proteins [10]. Additional difficulties are involved in delivering therapeutic molecules, particularly inside a cell [11]. Perhaps, most importantly, the continuous evolution of viruses and bacteria makes it difficult to design drugs and vaccines whose efficacy persists over time [12,13], which is a particular issue very relevant in the case of fast-mutating RNA viruses [14]. ...
Full-text available
In a recent paper, we proposed the folding interdiction target region (FITR) strategy for therapeutic drug design in SARS-CoV-2. This paper expands the application of the FITR strategy by proposing therapeutic drug design approaches against Ebola virus disease and influenza A. We predict target regions for folding interdicting drugs on correspondingly relevant structural proteins of both patho-genic viruses: VP40 of Ebola, and matrix protein M1 of influenza A. Identification of the protein targets employs the sequential collapse model (SCM) for protein folding. It is explained that the model predicts natural peptide candidates in each case from which to start the search for therapeutic drugs. The paper also discusses how these predictions could be tested, as well as some challenges likely to be found when designing effective therapeutic drugs from the proposed peptide candidates. The FITR strategy opens a potential new avenue for the design of therapeutic drugs that promises to be effective against infectious diseases.
... [58][59][60][61] Typical structures aiming to regularly porate cells may include 3D vertical nano needle/wires, [62,63] nano pillars, [64,65], hollow nanoelectrodes [66] and nano pyramids, [67,68] amongst others. [69][70][71][72][73] The integration of electrical/optical-based poration techniques with SERS is very interesting because it gives an insight into changes of the local microenvironment exploiting plasmonic nanostructures. These may be utilised not only for their ability to enhance Raman signals but also as multifunctional elements which enable other important functions such as monitoring of internal constituent changes [74] and drug delivery [75,76]. ...
Full-text available
The development of local plasmonic nano sensors which are sensitive, whilst remaining non-invasive with high cell viability is challenging but of great interest for the investigation of cellular processes. With this aim, we developed an effective SERS active biointerface to monitor cell activity and discriminate between undifferentiated and differentiated neurons through principal component analysis. We propose a plasmonic tipped Au nanopyramids (Au NPs) array, which benefits from high aspect ratios and sharp tips that are excellent SERS sensors. To realise the structure, we developed a large-scale inexpensive fabrication route based on the assembly of charged nanospheres used as a mask by means of a colloidal lithography technique, to better space and shape the NPs. Here we show that Au NPs with tip curvatures of 10 nm and localized plasmon resonance at 785 nm can non-destructively probe ND7/23 neurons. We prove that these tips allow us to track ND7/23 neurons on the SERS substrate, detecting both the membrane constituents, proteins and lipids, and even intracellular DNA/RNA fragments.
Cell poration technologies offer opportunities not only to understand the activities of biological molecules but also to investigate genetic manipulation possibilities. Unfortunately, transferring large molecules that can carry huge genomic information is challenging. Here, we demonstrate electromechanical poration using a core-shell-structured microbubble generator, consisting of a fine microelectrode covered with a dielectric material. By introducing a microcavity at its tip, we could concentrate the electrical field with the application of electric pulses and generate microbubbles for electromechanical stimulation of cells. Specifically, the technology enables transfection with molecules that are thousands of kDa even into osteoblasts and Chlamydomonas, which are generally considered to be difficult to inject. Notably, we found that the transfection efficiency can be enhanced by adjusting the viscosity of the cell suspension, which was presumably achieved by remodeling of the membrane cytoskeleton. The applicability of the approach to a variety of cell types opens up numerous emerging gene engineering applications.
The hydrodynamic method mimics the in vivo environment of the mechanical effect on cell stimulation, which not only modulates cell physiology but also shows excellent intracellular delivery ability. Herein, a hydrodynamic intracellular delivery system based on the gigahertz acoustic streaming (AS) effect is proposed, which presents powerful targeted delivery capabilities with high efficiency and universality. Results indicate that the range of cells with AuNR introduction is related to that of AS, enabling a tunable delivery range due to the adjustability of the AS radius. Moreover, with the assistance of AS, the organelle localization delivery of AuNRs with different modifications is enhanced. AuNRs@RGD is inclined to accumulate in the nucleus, while AuNRs@BSA tend to enter the mitochondria and AuNRs@PEGnK tend to accumulate in the lysosome. Finally, the photothermal effect is proved based on the large quantities of AuNRs introduced via AS. The abundant introduction of AuNRs under the action of AS can achieve rapid cell heating with the irradiation of a 785 nm laser, which has great potential in shortening the treatment cycle of photothermal therapy (PTT). Thereby, an efficient hydrodynamic technology in AuNR introduction based on AS has been demonstrated. The outstanding location delivery and organelle targeting of this method provides a new idea for precise medical treatment.
Some of the challenges of yeast encapsulation protocols are low phytochemical internalization rates and limited intracellular compartment of yeasts. This study uses an ultrasound‐assisted batch encapsulation (UABE) protocol to optimize the encapsulation of curcumin and fisetin by recovering non‐encapsulated biomaterial and further incorporating it into non‐loaded yeasts in three encapsulation stages (1ES, 2ES, and 3ES). The effect of selected acoustic energies (166.7 and 333.3 W L‐1) on the encapsulation efficiency (EE), yield (EY), and antioxidant activity retention were evaluated, and then, compared with a control process (without ultrasound treatment). Compared to the control, enhanced EEs were achieved for both curcumin (10.9% control to 58.5% UABE) and fisetin (18.6% control to 76.6% UABE) after 3ES and the use of 333.3 W L‐1. Similarly, the yeast maximum loading capacity was improved from 6.6 to 13.4 mg g‐1 for curcumin; and from 11.1 to 26.4 mg g‐1 for fisetin after UABE protocol. The antioxidant activity of produced biocapsules was positively correlated with the bioactive loaded content of yeasts when ultrasound treatment was applied. Overall, results from this study provide valuable information regarding UABE processes, and moreover, bring new and creative perspectives for the ultrasound technology in the food industry.
Intracellular delivery strategies are critical to the application of gene delivery, cell therapy, induced pluripotent stem cells, etc. Traditional methods such as viruses, liposomes, and electroporation always lead to high toxicity and cell damage, therefore, their applications can be limited. The mechanical method based on biological probes is less harmful to cells and keep the cell activities. Molecules can be delivered into the living cell quite accurately as well. So, the mechanical method is getting more and more attention. Biological probes methods include microinjection, nanotips and nanoneedle array, the following overview introduces the characteristics and applications of these three methods. In the end, we compared the advantages and disadvantages of these three methods and we make the prospects for subsequent development.
The use of synthetic nanomaterials as contrast agents, sensors, and drug delivery vehicles in biological research primarily requires effective approaches for intracellular delivery. Recently, the well-accepted microelectrophoresis technique has been reported to exhibit the ability to deliver nanomaterials, quantum dots (QDs) as an example, into live cells, but information about cell viability and intracellular fate of delivered nanomaterials is yet to be provided. Here we show that cell viability following microelectrophoresis of QDs is strongly correlated with the amount of delivered QDs, which can be finely controlled by tuning the ejection duration to maintain long-term cell survival. We reveal that microelectrophoretic delivered QDs distribute homogeneously and present pure Brownian diffusion inside the cytoplasm without endosomal entrapment, having great potential for the study of dynamic intracellular events. We validate that microelectrophoresis is a powerful technique for the effective intracellular delivery of QDs and potentially various functional nanomaterials in biological research.
Full-text available
Delivery of small interfering RNAs (siRNAs) into primary T cells is quite challenging because they are non-proliferating cells and are difficult to transfect with non-viral approaches. Because sonoporation is independent of the proliferation status of cells and siRNA acts in the cell cytoplasm, we investigated whether sonoporation could be used to deliver siRNA into mouse and human T cells. Cells mixed with Definity microbubbles and siRNA were sonicated with a non-focused transducer of center frequency 2.20 MHz producing ultrasound at a 10% duty cycle, pulse repetition frequency of 2.20 kHz and spatial average temporal average ultrasound intensity of 1.29 W/cm2 for 5 s and then examined for siRNA fluorescence by flow cytometry analysis. These sonoporation conditions resulted in high-efficiency transfection of siRNA in mouse and human T cells. Further, the efficacy of siRNA delivery by sonoporation was illustrated by the successful visualization of decreased methylation-controlled J protein expression in mouse and human CD8 T cells via Western blot analysis. The results provide the first evidence that sonoporation is a novel approach to delivery of siRNA into fresh isolated mouse and human T cells in vitro, and might be used for in vivo studies in the future.
Full-text available
Microinjection into single cells in brain tissue is a powerful technique to study and manipulate neural stem cells. However, such microinjection requires expertise and is a low-throughput process. We developed the "Autoinjector", a robot that utilizes images from a microscope to guide a microinjection needle into tissue to deliver femtoliter volumes of liquids into single cells. The Autoinjector enables microinjection of hundreds of cells within a single organotypic slice, resulting in an overall yield that is an order of magnitude greater than manual microinjection. The Autoinjector successfully targets both apical progenitors (APs) and newborn neurons in the embryonic mouse and human fetal telencephalon. We used the Autoinjector to systematically study gap-junctional communication between neural progenitors in the embryonic mouse telencephalon and found that apical contact is a characteristic feature of the cells that are part of a gap junction-coupled cluster. The throughput and versatility of the Autoinjector will render microinjection an accessible high-performance single-cell manipulation technique and will provide a powerful new platform for performing single-cell analyses in tissue for bioengineering and biophysics applications.
Full-text available
a r t i c l e i n f o Even as gigahertz (GHz) acoustic streaming has developed into a multi-functional platform technology for biochemical applications, including ultrafast microfluidic mixing, microparticle operations, and cellar or vesicle surgery , its theoretical principles have yet to be established. This is because few studies have been conducted on the use of such high frequency acoustics in microscale fluids. Another difficulty is the lack of velocimetry methods for microscale and nanoscale fluidic streaming. In this work, we focus on the basic aspects of GHz acoustic streaming, including its micro-vortex generation principles, theoretical model, and experimental characterization technologies. We present details of a weak-coupled finite simulation that represents our current understanding of the GHz-acoustic-streaming phenomenon. Both our simulation and experimental results show that the GHz-acoustic-induced interfacial body force plays a determinative role in vortex generation. We carefully studied changes in the formation of GHz acoustic streaming at different acoustic powers and flow rates. In particular, we developed a microfluidic-particle-image velocimetry method that enables the quantification of streaming at the microscale and even nanoscale. This work provides a full map of GHz acoustofluidics and highlights the way to further theoretical study of this topic.
Full-text available
Efficient cellular delivery of biologically active molecules is one of the key factors that affect the discovery and development of novel drugs. The plasma membrane is the first barrier that prevents direct translocation of chemic entities, and thus obstructs their efficient intracellular delivery. Generally, hydrophilic small molecule drugs are poor permeability that reduce bioavailability and thus limit the clinic application. The cellular uptake of macromolecules and drug carriers is very inefficient without external assistance. Therefore, it is desirable to develop potent delivery systems for achieving effective intracellular delivery of chemic entities. Apart from of the types of delivery strategies, the composition of the cell membrane is critical for delivery efficiency due to the fact that cellular uptake is affected by the interaction between the chemical entity and the plasma membrane. In this review, we aimed to develop a profound understanding of the interactions between delivery systems and components of the plasma membrane. For the purpose, we attempt to present a broad overview of what delivery systems can be used to enhance the intracellular delivery of poorly permeable chemic entities, and how various delivery strategies are applied according to the components of plasma membrane.
Full-text available
The development of new modalities for high-efficiency intracellular drug delivery is a priority for a number of disease areas. One such area is urinary tract infection (UTI), which is one of the most common infectious diseases globally and which imposes an immense economic and healthcare burden. Common uropathogenic bacteria have been shown to invade the urothelial wall during acute UTI, forming latent intracellular reservoirs that can evade antimicrobials and the immune response. This behaviour likely facilitates the high recurrence rates after oral antibiotic treatments, which are not able to penetrate the bladder wall and accumulate to an effective concentration. Meanwhile, oral antibiotics may also exacerbate antimicrobial resistance and cause systemic side effects. Using a human urothelial organoid model, we tested the ability of novel ultrasound-activated lipid microbubbles to deliver drugs into the cytoplasm of apical cells. The gas-filled lipid microbubbles were decorated with liposomes containing the non-cell-permeant antibiotic gentamicin and a fluorescent marker. The microbubble suspension was added to buffer at the apical surface of the bladder model before being exposed to ultrasound (1.1 MHz, 2.5 Mpa, 5500 cycles at 20 ms pulse duration) for 20 seconds. Our results show that ultrasound-activated intracellular delivery using microbubbles was over 16 times greater than the control group and twice that achieved by liposomes that were not associated with microbubbles. Moreover, no cell damage was detected. Together, our data show that ultrasound-activated microbubbles can safely deliver high concentrations of drugs into urothelial cells, and have the potential to be a more efficacious alternative to traditional oral antibiotic regimes for UTI. This modality of intracellular drug delivery may prove useful in other clinical indications, such as cancer and gene therapy, where such penetration would aid in treatment.
Full-text available
In the last couple of decades, ultrasound-driven microbubbles have proven excellent candidates for local drug delivery applications. Besides being useful drug carriers, microbubbles have demonstrated the ability to enhance cell and tissue permeability and as a consequence, drug uptake herein. Notwithstanding the large amount of evidence for their therapeutic efficacy, open issues remain. Due to the vast amount of ultrasound- and microbubble-related parameters that can be altered, and the variability in different models, the translation from basic research to (pre-)clinical studies has been hindered. This review aims at connecting the knowledge gained from fundamental microbubble studies to the therapeutic efficacy seen in in vitro and in vivo studies, with an emphasis on a better understanding of the response of a microbubble upon exposure to ultrasound and its interaction with cells and tissues. More specifically, we address the acoustic settings and microbubble-related parameters i.e. bubble size and physico-chemistry of the bubble shell that play a key role in microbubble-cell interactions and in the associated therapeutic outcome. Additionally, new techniques that may provide additional control over the treatment, such as monodisperse microbubble formulations, tunable ultrasound scanners and cavitation detection techniques, are discussed. An in-depth understanding of the aspects presented in this work could eventually lead the way to more efficient and tailored microbubble-assisted ultrasound therapy in the future.
Autologous cellular therapies based on modifying T cells to express chimeric antigen receptor genes have been highly successful in treating hematological cancers. Deployment of these therapies is limited by the complexity and costs associated with their manufacturing. Transitioning these processes from virus-based methods for gene delivery to a non-viral method, such as electroporation, has the potential to greatly reduce cost and manufacturing time while increasing safety and efficacy. Major challenges with electroporation are the negative impacts on cell health associated with exposure to high-magnitude electric fields, and that most commercial bulk electroporators are low-precision instruments designed for manually-operated, lower-throughput batch processing of cells. Negative effects on cell health can be mitigated by use of specialized electroporation medias, but this adds processing steps, and long-term exposure to these medias can reduce cell viability and efficacy. To enable automated, clinical-scale production of cellular therapies using electrotransfection in specialized medias, we developed a high-precision microfluidic platform that automatically and continuously transfers cells from culture media into electroporation media using acoustophoresis, and then immediately applies electric fields from integrated electrodes, limiting cell residence time in electroporation media to seconds, and enabling high transfection efficiency with minimum impact on cell viability. We tested our system by transferring primary human T cells from a standard cell media to electroporation media, and then transfecting them with mRNA encoding an mCherry fluorescent protein. We achieved a media exchange efficiency of 86% and transfection efficiency of up to 60%, with less than a 5% reduction in viability.
Cellular analysis is a central concept for both biology and medicine. Over the past two decades, acoustofluidic technologies, which marry acoustic waves with microfluidics, have significantly contributed to the development of innovative approaches for cellular analysis. Acoustofluidic technologies enable precise manipulations of cells and the fluids that confine them, and these capabilities have been utilized in many cell analysis applications. In this review article, we examine various applications where acoustofluidic methods have been implemented, including cell imaging, cell mechanotyping, circulating tumor cell phenotyping, sample preparation in clinics, and investigation of cell-cell interactions and cell-environment responses. We also provide our perspectives on the technological advantages, limitations, and potential future directions for this innovative field of methods.
The intracellular delivery efficiency of drug-loaded nanocarriers is often limited by biological barriers arising from the plasma membrane and the cell interior. In this work, the entering of doxorubicin (Dox)-loaded mesoporous silica nanoparticles (MSNs) into cytoplasm was acoustically enhanced through direct penetration with the assistance of hypersound of gigahertz (GHz) frequency. Both fluorescence and cell viability measurements revealed that the therapeutic efficacy of Dox-loaded MSNs were significantly improved by tuning the power and duration of hypersound on demand with a nanoelectromechanical (NEMS) resonator. Mechanism studies with inhibitors illustrated that the membrane defects induced by the hypersound-triggered GHz acoustic streaming facilitated the Dox-loaded MSNs of 100-200 nm to directly penetrate through the cell membrane instead of via the traditional endocytosis, which highly increased the delivery efficiency by avoiding the formation of endosomes. This acoustic method enables the drug carriers to overcome biological barriers of the cell membrane and the endosomes without the limitation of carrier materials, which provides a versatile way of enhanced drug delivery for biomedical applications.
The successful intracellular delivery of exogenous macromolecules is crucial for a variety of applications ranging from basic biology to the clinic. However, traditional intracellular delivery methods such as those relying on viral/non-viral nanocarriers or physical membrane disruptions suffer from low throughput, toxicity, and inconsistent delivery performance and are time-consuming and/or labor-intensive. In this study, we developed a single-step hydrodynamic cell deformation-induced intracellular delivery platform named "hydroporator" without the aid of vectors or a complicated/costly external apparatus. By utilizing only fluid inertia, the platform focuses, guides, and stretches cells robustly without clogging. This rapid hydrodynamic cell deformation leads to both convective and diffusive delivery of external (macro)molecules into the cell through transient plasma membrane discontinuities. Using this hydroporation approach, highly efficient (∼90%), high-throughput (>1 600 000 cells per min), and rapid delivery (∼1 min) of different (macro)molecules into a wide range of cell types was achieved while maintaining high cell viability. Taking advantage of the ability of this platform to rapidly deliver large molecules, we also systematically investigated the temporal biostability of vanilla DNA origami nanostructures in living cells for the first time. Experiments using two DNA origami (tube- and donut-shaped) nanostructures revealed that these nanostructures can maintain their structural integrity in living cells for approximately 1 h after delivery, providing new opportunities for the rapid characterization of intracellular DNA biostability.