Recent advances in micro/nanoscale intracellular delivery
Mengjie Sun, Xuexin Duan ⁎
State Key Laboratory of Precision Measuring Technology & Instruments, Tianjin University, Tianjin, 300072, China
Available online 28 December 2019
Cell membrane disruption
Intracellular delivery enables the efﬁcient drug deliveryinto various types of cells and has been a long-term stud-
ied topics in modern biotechnology. Targeted delivery with improved delivery efﬁcacy 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 ﬂexibility. 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 (http://creativecommons.org/licenses/by-nc-nd/4.0/).
The safe and efﬁcient 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.
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 difﬁcult.
The delivery normally requires
vectors, such as viruses or peptides, speciﬁc to target molecules.
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 efﬁciency
to achieve efﬁcient 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 efﬁciency and speciﬁcity, 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) 18–31
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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 efﬁciency
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
efﬁciency to solve these issues. In recent years, with the rapid develop-
ment of micro- and nanotechnologies, including microﬂuidic system
and lab-on-chip techniques, membrane disruption technologies at
Common target materials for intracellular delivery.
Cargo Category Challenge
All the small molecular structures
that have biological signiﬁcance.
Nanomaterials Quantum dots, nanoparticles (NPs),
and carbon nanotubes.
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
Difﬁcult to produce and to
May cause overexpression.
diversity and very sensitive
Fig. 1. Two approaches for intracellular delivery and their basic mechanisms.
19M. Sun, X. Duan / Nanotechnology and Precision Engineering 3 (2020) 18–31
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 microﬂuidics 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 ﬁeld 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
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, microﬂuidics, and lab-on-chip approaches and is
being developed to create more new opportunities.
Intracellular delivery dates back to the work of Barber et al.
used a very thin glass needle ﬁlled 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
and robotic systems,
has considerably im-
proved the t herapeutic efﬁciency 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 (50–500 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 speciﬁc 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.
ﬁrst 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 efﬁciently 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 modiﬁed or doped with the target
molecules by direct fabrication through micro/nanotechnology.
this case, biological macromolecules can enter cells without chemical
modiﬁcation 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 ﬂuorescently 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 efﬁciency 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 modiﬁcation. They cultured the cells for 24 h on
the nanostraw array (Fig. 2c) and then combined the nanostraw array
with the ﬂow channel; thus, the ﬂowing solution could ﬂow 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 difﬁcult to use in terms of
manufacturing process and cost, and their ﬁxed size is limited for spe-
ciﬁc applications. Given these conditions, breakthroughs and simpliﬁca-
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 microﬂuidic device or external technology.
This work advanced the use of nanostraws for a wide range of biomed-
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) 18–31
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 (ﬁxing 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
efﬁciencies of intracellular delivery of the two loading methods.
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 signiﬁcantly
improve the accuracy of drug delivery beyond traditional microneedle
transdermal delivery and risky microinjection. The NW/needle can be
ﬁxed 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 difﬁculty 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) microﬂuidic device integrated with nanostraw structures.
21M. Sun, X. Duan / Nanotechnology and Precision Engineering 3 (2020) 18–31
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 efﬁciency while reducing
cell damage. Therefore, noncontact stimulation of the external ﬁeld to
induce cell membrane permeability can bring more interesting beneﬁts
and more application prospects.
Electroporation is a relatively trivial physical method. This process
exposes the cells to anelectric ﬁeld, 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 ﬁelds 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 microﬂuidic 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 efﬁciently 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 ﬁeld, 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 efﬁciency.
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 microﬂuidic 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) 18–31
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,
microﬂuidic electroporation device has been developed for better trans-
fection efﬁciency and cell viability.
However, given the limited size
of microﬂuidic channels, the cell processing speed must be sacriﬁced to
achieve the balance between high ﬂux and low voltage, which is partic-
ularly important in the research of micro-nanoscale drug delivery.
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.
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 efﬁciency 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 efﬁciency
The ultrafast laser beam operates over a very small area
of action on cells, which limits its capability for full transfection. Dhakal
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 microﬂuidic technology has been popularly used
to increase the transfection efﬁciency and throughput. Uchugonova
built an ultrashort femtosecond (fs) laser-microﬂuidic cell trans-
fection platform to achieve optical reprogramming of large cell popula-
tions. In a microﬂuidic 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-microﬂuidic transfection platform
; (b) schematic of AuNP-mediated optoporation principle.
23M. Sun, X. Duan / Nanotechnology and Precision Engineering 3 (2020) 18–31
gene and cell therapies generally use several separate procedures for
gene transfer and cell separation or elimination. Lukianova-Hleb
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 speciﬁcity, PNBs of different sizes
were generated around cell-targeted gold nanoshells and nanospheres.
Simultaneously, high efﬁcacy, 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-speciﬁc 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 difﬁcult
scenarios such as in a living embryo.
The instrumentations required,
however, are generally expensive, complicated, and bulky.
Although light/electroporation can solve several intracellular delivery
challenges, their application in delivering some speciﬁc proteins and
nanomaterials is still limited. The damage caused by the energy ﬁeld
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. ﬁrst 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 ﬂowing
around to oscillate to generate sufﬁcient shear force to open up nearby
An ultrasound ﬁeld 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
ﬂuorescence protein–α-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, signiﬁcant 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 efﬁciency and safety of ultrasound therapy.
Horsley et al.
trasound to activate microbubbles to deliver high concentrations of
drugs into urothelial cells. As a common disease, urinary tract infection
still lacks efﬁcient 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 ﬁnding could affect
traditional oral antibiotic treatments. Although evidence supports the
therapeutic efﬁcacy 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 classiﬁcation 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.
speciﬁcally described the theory of
SAW and its interaction with particles and contact ﬂuids and divided
the SAW ﬂuids 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 ﬁnger 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 efﬁciency 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 efﬁciently 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 ﬂuorescence microscope. The transducer relied on a programmed
displacement platform to accurately adjust the position, and a ﬂuores-
cence microscope could detect changes in the ﬂuorescence intensity of
the treated cells. In general, the focused area of low-frequency ultrasound
at 1–5 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
Hypersound, deﬁned 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-ﬁlm 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 ﬂuid
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 device–liquid interface.
Lu et al.
GHz resonator with a ﬁeld 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.
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) 18–31
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 efﬁcient 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 ﬂuorescent 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) 18–31
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 ﬁeld 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 100–200 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 efﬁcient for
cell or tissue treatment, showing great potential for further delivery of
larger molecules or cargo.
2.5. Microﬂuidic system
Electroporation and sonoporation cause low cell viability or may
limit the delivery of materials due to electrical charges. Thus, recent
microﬂuidic delivery methods based on rapid cellular mechanical
deformation have become a prominent alternative in certain
In general, microﬂuidic 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-speciﬁc antigen uptake capacity.
This approach showed a limited success for electroporation, because
it relies on engineering and precisely controlled electric ﬁelds. Sharei
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 microﬂuidics 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 ﬁbroblasts, 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 modiﬁcation system
called cyto-PDMS. The design principle was similar to that of Sharei
. 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 ﬂow 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 ﬁ-
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 ﬁbroblast 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 efﬁciency. 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 ﬁeld was insufﬁcient to destroy the cell
membrane. Next, the deformed cells were exposed to a low-intensity
electric ﬁeld, 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 signiﬁcantly im-
proved transfection efﬁciency. It demonstrated the potential of ASP
platform in large-scale integration applications. This approach further
optimized the delivery efﬁciency of microﬂuidic 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 efﬁciency.
Different from the constriction function that disrupts the cell
membrane to increase permeability, Kizer et al.
designed a clog-
free and sheathless inertial microﬂuidic platform, named hydroporator,
based on a rapid mechanical cell deformation relying solely on hydrody-
namic cell shearing. This device utilized the inertia effect to ﬁrst 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 ﬂow, 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 ﬂuid 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 ﬁrst time showed the delivery capacity of different large
DNA nanostructures and their biostability in living cells. Given that de-
livery efﬁciency is related to ﬂow rate, a high ﬂow rate is required to
achieve efﬁcient delivery of larger molecules. However excessive ﬂow
rates may exacerbate cell death.
Li et al. designed a droplet microﬂuidic
platform for single-cell
transfection to achieve efﬁcient and consistent plasmid delivery of
lipoplex-mediated suspension cells.
delivery is extremely inefﬁcient (usually b5%) in transfection of
suspended cells (such as lymphocytes and hematopoietic cells for
The efﬁciency 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 ﬂuid compression
on cells increased when cells passed through the droplet pinch-off ori-
ﬁce, leading to increased membrane permeability and enhanced
lipoplex delivery into cells through endocytosis (Fig. 6e). The results
showed that the delivery efﬁciency 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) 18–31
Fig. 6. (a) Schematic representation of microﬂuidic 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 microﬂuidic 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) 18–31
work is expected to reprocess the transfected single cells to minimize
In addition, the combination of microﬂuidics with electropora-
tion has overcome a series of deﬁciencies, such as heat dissipation
of traditional electroporation. Extremely short electrode spacing
also could reduce the voltage requirements. Xu et al.
microchannel device containing both inlet and outlet electrodes to
generate an electric ﬁeld. High-speed ﬂuid 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.
manufacturing complexity of such type of devices often limits this
idea. Drug delivery with microﬂuidic system is often used at several
or single-cell level, allowing the accurate recording of electrical sig-
nals during delivery to reveal the speciﬁc 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 speciﬁcreagentsatspeciﬁclocations
in cell culture. This work ﬁlled the gap in the microﬂuidic 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 difﬁ-
cult. We aim to develop new methods that can meet the requirements
of high precision, large scale, and high ﬂexibility. 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 efﬁciently,
but the toxicity has been a barrier to clinical use.
A reliable control mechanism is essentially critical for next-
generation drug delivery system. Controlling drug delivery via signaling
is difﬁcult to achieve but is important for sophisticated delivery sys-
tems. The speciﬁcity 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.
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 difﬁcult-to-transfect cells. In particular, the devel-
opment of micronanotechnologyshows promise among emerging tech-
nologies in the ﬁelds 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 difﬁcult to perform in microarrays, microneedles
and microchannels have certain limitations in terms of the ﬂexibil-
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 diversiﬁed
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 ﬁnancial
interests or personal relationships that could have appeared to inﬂu-
ence the work reported in this paper.
Advantages and challenges for membrane disruption methods.
Methods Advantages Challenges
Nanoneedle Provides cellular nanoscale
Directly penetrates the
Can be used for many cell
Difﬁcult to implement on a large
Easily causes cell lysis.
Not suitable for high-throughput.
Electroporation High transfection
High cell death rate.
Slow microﬂow 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
Suitable for a wide range of
Loss of cytoplasmic content.
Cavitation could produce reactive
oxygen species that damage DNA,
requires additional chemicals in
Microﬂuidic Simple equipment and easy
Independent of physical
ﬁeld or the carrier.
Hydrodynamics does not
block the ﬂow passage.
The efﬁciency 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
The size of membrane poration
depends on precise shear forces.
28 M. Sun, X. Duan / Nanotechnology and Precision Engineering 3 (2020) 18–31
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).
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Mengjie Sun received the B.S.degree 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, microﬂuidics and
their interfaces with chemistry, biology, medicine, and envi-
31M. Sun, X. Duan / Nanotechnology and Precision Engineering 3 (2020) 18–31