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org Review

Surface-Mediated Intracellular Delivery by Physical Membrane

Disruption
Yangcui Qu,§ Yanxia Zhang,§ Qian Yu,* and Hong Chen

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ABSTRACT: Effective and nondestructive intracellular delivery of exogenous molecules and

other functional materials into living cells is of importance for diverse biological fundamental
research and therapeutic applications, such as gene editing and cell-based therapies. However,
for most exogenous molecules, the cell plasma membrane is effectively impermeable and thus
remains the greatest barrier to intracellular delivery. In recent years, methods based on surface-
mediated physical membrane disruption have attracted considerable attention. These methods
exploit the physical properties of the surface to transiently increase the membrane permeability
of cells come in contact thereto, thereby facilitating the efficient intracellular delivery of
molecules regardless of molecule or target cell type. In this Review, we focus on recent
progress, particularly over the past decade, on these surface-mediated membrane disruption-
based delivery systems. According to the membrane disruption mechanism, three categories
can be recognized: (i) mechanical penetration, (ii) electroporation, and (iii) photothermal
poration. Each of these is discussed in turn and a brief perspective on future developments in
this promising area is presented.
KEYWORDS: intracellular delivery, surface-mediated gene transfection, membrane disruption, mechanical penetration, electroporation,
photothermal poration

1. INTRODUCTION for recalcitrant cells. Moreover, an inherent shortcoming of all

Intracellular delivery of exogenous molecules and other
functional materials into living cells without compromising
cell viability plays an important role in various biological
fundamental research and therapeutic applications, ranging from
gene editing, such as clustered regularly interspaced short
palindromic repeats/associated protein 9 (CRISPR/Cas9)
technology for regulation of cell fate and behavior to ex vivo
cell-based therapies, such as the use of modified immune cells for
cancer treatment, and induced pluripotent stem cells for tissue
repair and regeneration.1−4 However, the 5−6 nm thick plasma
membrane that encloses the cell remains an impenetrable barrier
for most exogenous molecules;5 effective and nondestructive
delivery methods are, therefore, urgently needed.
Existing intracellular delivery methods can be divided into two
categories according to the delivery mechanism, that is, carrier-
based methods and membrane disruption-based methods.6−9 In
the former, the molecules to be delivered are packaged with a
carrier before entering the cell by endocytosis or by fusion with
the cell membrane.10,11 In recent years, various viral and
nonviral carriers have been developed. All of them, however,
have their own significant limitations. For example, viral carriers
raise concerns regarding cytotoxicity and immunogenicity, while
nonviral carriers, such as liposomes, cationic polymers, and
inorganic nanoparticles, usually require complex synthetic
procedures and suffer from low delivery efficiency, especially
carrier-based methods is lack of “universality”, that is, a given
carrier may not be suitable for a given molecule because of the
variability in molecular properties, such as size, charge, and
hydrophobicity.12−15
In contrast to carrier-based methods, membrane disruption-
based methods, and in particular, physical membrane disruption
(PMD)-based methods, deliver the molecules and materials into
cells by membrane penetration or permeabilization via optical,
electrical, mechanical, magnetic, thermal, and acoustic
means.16−18 PMD-based methods are also less dependent on
the physicochemical properties of the delivered molecule and
thus are able to deliver almost any exogenous molecule into the
cell. A series of PMD-based intracellular delivery systems
including direct penetration (e.g., microinjection),19 electro-
poration,20 magnetic perforation,21 acoustic perforation,22 and
optoporation23 have been developed; two categories can be
distinguished, that is, solution-mediated and surface-mediated.
Solution-mediated methods rely on physical “agents”, such as
Received: April 16, 2020
Accepted: June 19, 2020
Published: June 19, 2020

© 2020 American Chemical Society https://dx.doi.org/10.1021/acsami.0c06978

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Scheme 1. Schematic Illustration of Three Categories of Surface-Mediated Membrane Disruption-Based Intracellular Delivery
Systems
electric field and laser irradiation applied in solution to generate
transient holes in the cell membrane for high-efficiency delivery
of dispersed molecules.24−27 However, these methods suffer
generally from a lack of control of the process leading to
randomness, poor uniformity, and poor repeatability. In
addition, the required high voltage field or high energy laser
radiation may result in excessive membrane damage causing cell
death.
In contrast, surface-mediated PMD-based methods depend
on the physical properties of the surface to which the target cells
come in contact to increase membrane permeability tran-
siently.9,10 Efficient delivery of the molecule (loaded on the
surface or in solution) in an electric field or under laser
irradiation to virtually any cell type is thereby facilitated.
Compared with solution-mediated methods, surface-mediated
methods possess the additional merit that the surface can serve
as a reservoir for the molecules to be delivered, as well as an
agent for the localized delivery of molecules to surface-attached
cells.28 Also, because of the enhancing effect of surface-cell
interactions, the required voltage or laser energy is relatively low
thereby decreasing the loss of cell viability. Additionally, besides
surface-mediated PMD-based methods, there are also some
reports about surface-mediated chemical membrane disruption-
based methods that rely on some chemical promotors, such as
detergents to promote molecular delivery to cells by either
perturbing cell membrane or facilitating the internalization of
nanoneedles.29,30 These methods are covered in other
publications elsewhere.18,31
In this Review, we mainly focus on recent developments of
surface-mediated PMD-based methods for intracellular delivery
of exogenous molecules. These methods are discussed under
three headings: (i) mechanical penetration using nanoX arrays
(here X can be wire/needle/pillar/straw of uniform length that
is oriented perpendicular to the substrate) (see section 2); (ii)
electroporation using microfluidics, microelectrodes, micro/
nanopore membranes, or nanostraw arrays (see section 3); and
(iii) photothermal poration using surface-immobilized photo-
thermal agents such as gold nanomaterials and polydopamine
(see section 4) as illustrated in Scheme 1. A brief perspective is
then presented on approaches that show promise for the future.
It is noted that our Review does not want to cover all the aspects
of intracellular delivery by membrane disruption because of the
scope limit, so interested readers are suggested to read an
excellent review about the basic concepts, such as cell structures,
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cell membrane permeability intrinsic mechanisms, as well as
properties and functions of delivered molecules for the details.9
2. SURFACE-MEDIATED INTRACELLULAR DELIVERY
BY MECHANICAL PENETRATION
Mechanical penetration is one of the most direct approaches for
intracellular delivery by generating localized holes in the cell
membrane, through which the exogenous molecules enter the
cells. Surface-mediated mechanical penetration generally relies
on the nanoX arrays to penetrate the cell membrane.32−36 In the
past decade, two possible mechanisms of nanoX arrays for
intracellular delivery have been proposed. One mechanism is
that nanoX arrays with optimal density and aspect ratio can
penetrate cell membrane spontaneously, but the penetration is
limited for some specific cell types.37−42 In 2013, Melosh et al.
found that stiff cells could be penetrated by nanowires more
easily compared to regular and soft cells.32 Later, they
systematically studied the interaction between cells and hollow
nanowires and explored the influence of the geometry of
nanowires, the rigidity of the cell, and the adhesion of the cells
on the interaction between cell and nanowires. Their results
implied that spontaneous cell penetration by nanoX arrays is
possible, yet limited in scope, time scale, and cell types.42
Another mechanism is that the nanoX arrays themselves cannot
penetrate the cell membrane, but they are justly engulfed by cell
membrane.43−46 This claim was supported by the following
finding that only 7.1% hollow nanowires could spontaneously
penetrate cell membrane.47 However, the penetration efficiency
can be increased with the assistance of external force.48,49
Although the definite mechanism is still not clear, the nanoX
arrays have been widely used for intracellular delivery because of
the wide range of delivered molecules and target cell types. In
this section, we will introduce the development of surface-
mediated mechanical penetration approaches for intracellular
delivery that are divided into two categories based on the
structure of the nanoX arrays: (i) solid nanoX arrays and (ii)
hollow nanostraw arrays (as illustrated in Scheme 2 and
summarized in Table 1).
2.1. Solid NanoX Arrays. 2.1.1. Silicon Nanowire Arrays.
Silicon nanowire arrays (SiNWAs) refer to arrays of dense
silicon nanowires that are oriented perpendicular to the
substrate.83 Including the intrinsic biocompatibility of silicon,
the high surface-to-volume ratio and unique nanoporous
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Scheme 2. Schematic Illustration of Different Surface-
Mediated Mechanical Penetration Approaches Based on
NanoX Arraysa
a
(a) Intracellular delivery by mechanical penetration using solid
nanoX arrays without external force. (b, c) Intracellular delivery by
mechanical penetration using solid nanoX arrays with external force.
(b) The nanoX arrays were placed on the bottom and the target cells
adhered on the surface of nanoX arrays. (c) The nanoX arrays were
placed on the top to penetrate into cell monolayer. (d) Intracellular
delivery by mechanical penetration using hollow nanostraw arrays,
where the exogenous molecules are stored in bottom fluid storage
device.
structure and of SiNWAs provide them great potential for
biotechnology and biomedical applications.84 In general, there
are two main strategies for fabrication of SiNWAs including the
top-down strategy (e.g., metal-assisted chemical etching
(MACE) and reactive ion etching (RIE)) and the bottom-up
strategy (e.g., chemical vapor deposition (CVD) and laser
ablation).85−87 The SiNWAs discussed in this section are mainly
fabricated using CVD, MACE, and RIE methods.
In 2007, for the first time, Yang and Conklin et al.
demonstrated that SiNWAs containing nanowires with small
diameter and high aspect ratio could deliver plasmid DNA
(pDNA) to cells.37 The SiNWAs could directly penetrate mouse
embryonic stem cells (mESCs) without using external forces
(Figure 1a and b), and the penetrated cells survived and
proliferated for up to 3 days. Moreover, their preliminary results
indicated that the penetration of SiNWAs was beneficial for gene
transfection using HEK293T cells as model cell lines. However,
the transfection efficiency was very low (<1%).37 Later, Park et
al. applied SiNWAs for gene-specific manipulation of various
human and murine immune cells without compromising cell
viability.39 In particular, they delivered siRNA for gene silencing
to study the potential basis of clinical heterogeneity in chronic
lymphocytic leukemia.40 In addition, to deeply understand the
influence of physical parameters of SiNWAs on intracellular
delivery, a series of SiNWAs with different geometrical
structures were fabricated and the effects of physical parameters
of SiNWAs on intracellular delivery were examined. As shown in
Figure 1c−h, efficient delivery of molecules in smaller
suspension immune cells (e.g., native mouse B and T cells)
needed the use of SiNWAs in which nanowires were denser
(0.3−1 μm−2) and longer (2−3 μm). In contrast, larger and
adherent immune cells (e.g., DCs and MΦs) required the use of
SiNWAs in which nanowires were less dense (0.15−0.2 μm−2)
and slightly shorter (1−2 μm). The SiNWAs with fairly long
nanowires (>3 μm) might cause the nucleus rupture of adherent
immune cells, leading to severe cytotoxicity even cell death.40
The delivery efficiency of pristine SiNWAs is generally quite
low. To improve the efficiency, several groups modified SiNWAs
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with various chemicals (e.g., cationic polymers,50−53 poly-
dopomine,54 or supramolecular nanoparticles55,56) to increase
the loading amount of delivered molecules as well as the contact
between target cells and molecules. For example, we modified
SiNWAs with various cationic polymers, such as branched
polyethylenimine (PEI)50 and poly(N,N-dimethylamino)ethyl
methacrylate (PDMAEMA)51 and aminated poly(glycidyl
methacrylate) (PGMA)52 for loading pDNA and achieved
enhanced transfection of pDNA to HeLa cells. However, there
are still some limitations in these systems. Because these cationic
polymers were firmly immobilized on the surface of SiNWAs,
they cannot provide enough protection and quick release of the
pDNA into cytoplasm, restricting the transfection efficiency. In
addition, the high positive charge carried by these cationic
polymers caused the loss of viability of target cells. To further
improve the transfection efficiency and decrease the cytotox-
icity, we synthesized a branched PEI cross-linked by disulfide
bonds for modification of SiNWAs to load pDNA.53 When the
modified SiNWAs penetrated the membrane of target cells, the
disulfide bonds were broken by the high concentration of
glutathione in the cytoplasm, leading to the fast release of PEI
and preloaded pDNA. The “engineered” cells could be
efficiently harvested by trypsin digestion and the SiNWAs
surface could be regenerated by dithiothreitol treatment due to
the replaceable properties of disulfide bonds (Figure 2a).53 It is
noted that the process of modification of SiNWAs with polymers
was relatively complicated. Alternatively, Ito et al. developed a
polydopamine (PDA) coating on SiNWAs by a simple
deposition method (Figure 2b).54 The biocompatible PDA
coating could enhance the adhesion, spreading, and growth of
cells. Moreover, they provided a uniform porous layer on the
surface of nanowires to increase the area of surface with
functional groups for adsorption of siRNA. Compared with flat
silicon surface and unmodified SiNWAs, PDA-coated SiNWAs
exhibited more efficient delivery of siRNA to knock down the
expression of a specific gene via RNA interference.54
Codelivery or sequential delivery of diverse genes into one cell
plays a crucial role in cell reprogramming.1,88 One typical
example is to deliver four genes (Oct4, Sox2, KLF4, and c-MYC
(OSKM)) to fibroblasts for preparation of induced pluripotent
stem cells (iPSCs).89,90 However, it is still challenging to deliver
diverse genes together effectively because of the large volume of
complex of these genes. To solve this problem, Tseng et al.
developed a new gene delivery system based on adamantine
(Ad) modified SiNWAs (Ad-SiNWAs) and supramolecular
nanoparticle (SNP) vectors.56 The SNP vectors were synthe-
sized through a modular self-assembled approach from
polyamidoamine dendrimer anchored with Ad groups (Ad-
PAMAM), poly(ethylene glycol) anchored with an Ad group
(Ad-PEG), branched polyethylenimine grafted with β-cyclo-
dextrin (CD) grafted (CD-PEI), and delivered pDNA. Because
of the host−guest interactions between Ad and CD, as well as
electrostatic interactions between PEI and pDNA, the resulted
SNP vectors (DNA⊂SNPs) could encapsulate and compress
four kinds of pDNA to sharply reduce the volume of the complex
(Figure 3a).55 The DNA⊂SNPs were enriched on the surface of
Ad-SiNWAs and could be introduced through the cell
membranes to release pDNA inside cytoplasm after dynamic
dissociation. Using this system, direct reprogramming/trans-
differentiation of fibroblasts into a stem cell-like state or a
neuronal cell-like state were realized successfully (Figure 3b).56
They further extended the application range of this system,
achieving successful transfection for not only various adherent
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Table 1. Summary of Surface-Mediated Intracellular Delivery by Mechanical Penetrationa

materials delivered molecule cell line delivery efficiency cell viability ref
SiNWAs pDNA HEK293T <1% 37
SiNWAs pDNA/siRNA/protein/peptide HeLa/hFs/NPCs/HNs/DCs 95% 39
B/NK/MΦs/T cells 69−90% without 40
affecting
viability
SiNWAs pDNA HeLa 50
pDNA HeLa 51
siRNA/pDNA HeLa 80%(siRNA to HeLa) 90% 52
pDNA HeLa/SMMCs >60% >90% 53
SiNWAs siRNA A549-luc cells 70−80% 54
SiNWAs pDNA U87/HeLa/NIH3T3/MCF7/BC-1/Ramos/ 70−98% 55
Jurkat/BJ/hDFs/J1/mEFs/hNSCs
pDNA (OSKM/pNTFs) fibroblast cells 30−50% 56
SiNWAs pDNA hDPSCs 77% 57
SiNWAs pDNA HEK293/HeLa/hDPSCs/hFFs >85% (hDPSCs and >90% 58
HEK 293)
silicon pillar arrays pDNA hFFs/DPSCs 75% 59
silicon nanoneedle dextran DU145/HeLa 50% (calcein to 90% 60
arrays HeLa)
silicon nanoneedle pDNA NIH3T3 34% (pDNA) 42% 61
arrays (Cre recombinase)
silicon nanoneedle siRNA/pDNA/VEGF-165 gene HeLa 90% 41
arrays
silicon nanoneedle FITC-dextran/pYFP NIH3T3 45% (FITC-dextran, 62
arrays 70 kDa)
silicon nanoneedle quantum dots HeLa/skin/muscle 63
arrays
silicon microneedle PI HeLa 229 97.9% 97.7% 64
arrays
diamond luminescent iridium polypyridine A549 92% 65
nanoneedle arrays EthD1/Dextran/antibody/pDNA/ NIH3T3/neurons 45% (pGFP to 90% 48
quantum dots/polystyrene nanoparticles neurons)
carbon nanofiber pDNA CHO 66,
arrays 67
carbon nanofiber shRNA/pDNA CHO 53.1% (shRNA) 68
arrays
carbon nanofiber pDNA V97 69
arrays
carbon nanofiber pDNA V97 38
arrays
carbon nanosyringe quantum dots/pGFP NIH3T3/MSCs 34% (pGFP to 85% 70
arrays NIH3T3)
30% (pGFP to
MSCs)
carbon nanosyringe gene: VEGF-mRNA/pGFP HeLa/PC3/MDA-MB-231/MCF7/ 46% (pGFP to 71
arrays lymphocyte)
proteins: GFP/RFP/caspase-3 T47D/HepG2/primary lymphocyte 70.3% (GFP to
T47D)
79% (RFP to HepG2)
ZnO nanowire Hyg3/peptide/pDNA MCF7/Microalgal 6.52 × 10−3 49,
arrays 72
ZnO nanowire pGFP HEK293 60% 73
arrays 8% (pGFP)
CuO nanowires pDNA HeLa <1% without 74
affecting
viability
hollow nanoneedle dextran/pRFP HEK293 70−90% 29
arrays
hollow carbon PI/dextran/quantum dots/pYFP HEK293/L6 99% for dye delivery 97% 75
nanostraw arrays 84% for pDNA
transfection
hollow tapered quantum dots microalgae 9.8% 76
microtube arrays
Al2O3 nanostraw fluorescence dye/Co2+/EDTA/pGFP CHO 70% (Co2+) 77
arrays 75% (EDTA)
10% (pGFP)
Al2O3 nanostraw Ca2+ indicator CHO 90% 78
arrays

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Table 1. continued
materials delivered molecule cell line delivery efficiency cell viability ref
Al2O3 nanostraw Co2+/EDTA CHO 95% 47
arrays
Al2O3 nanostraw functional probes of glycolysylation CHO near 100% 79
arrays
Al2O3 nanostraw pRFP CHO 1.5% near 100% 80
arrays
hierarchical spiky staurosporine MCF7 84.6% 94.5% 81
microstraw arrays
Au nanostraw arrays calcein NIH3T3 85% 90% 82
a
Abbreviation: Silicon nanowire arrays (SiNWAs); Oct4, Sox2, KLF4, and c-MYC (OSKM, a pDNA encoding reprogramming factors, 13 kbp);
Ascl1, Brn2, Myt1l, and NeuroD1 (pNTFs, a pDNA encoding neuron specific transcription factors); pDNA encoding red/yellow/green
fluorescence protein (pRFP/pYFP/pGFP); green/red fluorescence protein (GFP, 26 kDa/RFP); ZnO-binding peptide (ZBP) conjugated with
FITC (ZBP-FITC); propidium iodide (PI, 0.66 kDa); vascular endothelial growth factor (VEGF); ethylenediamine-tetra-acetic acid (EDTA);
dextran (3 kDa, 70 kDa, 500 kDa); pDNA (5.2 kbp, 6 kbp); quantum dots (15−20 nm in diameter); polystyrene nanoparticles (200 nm in
diameter); human embryonic kidney cells (HEK293); human fibroblasts (hFs); rat neural progenitor cells (NPCs); hippocampal neurons (HNs);
dendritic cells (DCs); nature killer (NK) cells; microphages (MΦs); smooth muscle cells (SMMCs); breast cancer-1 cell (BC-1); human dermal
fibroblast (hDFs); mouse embryo fibroblast (mEFs); human neural stem cells (hNSCs); human dental pulp stem cells (hDPSCs); human foreskin
fibroblasts (hFFs); Chinese Hamster Ovary (CHO); Chinese hamster lung cells (V79); mesenchymal stem cells (MSCs).

Figure 1. Intracellular delivery by membrane penetration using SiNWAs fabricated via chemical vapor deposition. (a) Typical confocal microscopy
image and (b) a typical scanning electronic microscopy (SEM) image of mESCs penetrated with silicon nanowires. Adapted with permission from ref
37. Copyright 2007 American Chemical Society. (c−h) Representative SEM images of (c) mouse bone-marrow DCs, (d) B cells, (e) DCs, (f) MΦs,
(g) NK cells, and (h) T cells on top of SiNWAs with different geometric structures. Adapted with permission from ref 40. Copyright 2012 American
Chemical Society.

cells (HeLa, U87, MCF3, NIH3T3, and HEK293) but also
recalcitrant suspension cells (BC-1, Ramos, and Jurkat cells) and
primary cells (BJ, hDFs, J1, mEFs, and hNSCs). In particular,
sequential delivery of three pDNA encoding three different
proteins (pDNA encoding green fluorescence protein (pGFP),
mCherry (pmCherry), and E2-Crimson (pE2-Crimson)) to
U87 cells was achieved by adding three respective SNP vectors
with encapsulated pDNA 12, 24, and 36 h after the cell
settlement on Ad-SiNWAs. In addition, Ad-SiNWAs were
subcutaneously transplanted into one side of the mouse body,
followed by local injection of SNPs encapsulated with plasmid
encoding luciferase reporter (pGL3⊂SNPs). The expression of
luciferase in site with Ad-SiNWAs was significantly increased
compared to that detected in site without Ad-SiNWAs or in site
with smooth silicon surface modified with Ad, showing the
application potential of this system for in vivo gene delivery
(Figure 3c).55 However, it should be noted that this system has a
drawback regard to the hard harvest of “engineered” cell from
the surface of SiNWAs, which might limit its ex vivo
applications.
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2.1.2. Other Solid NanoX Arrays. In addition to chemical
modification to increase the loading amount of delivered
molecules, introduction of external force (e.g., centrifugal force
and squeeze force) to nanoX arrays can also enhance the delivery
efficiency by increasing the extent of membrane penetration. In
general, there are two classic models for applying external force
on nanoX arrays. The first one is to culture cells on the top of the
nanoX arrays and then to apply a top-down centrifugal force or
impact force on the adhered cells to promote deep penetration
of nanoX (as illustrated in Scheme 2b).49,65,71,72 For example,
with the assistance of centrifugal force, successful transfection of
pDNA was achieved not only for easy-to-transfect HepG2 cells
(more than 70% transfection efficiency) but also for hard-to-
transfect lymphocytes (about 46% transfection efficiency) using
carbon nanosyringe arrays (CNSAs), expanding the application
range of cell types and improving the transfection efficiency
(Figure 4).71 The second one is to place nanoX arrays on the top
of cells to squeeze the cells monolayer, promoting the
penetration of nanoX for the entry of exogenous molecules
into cells (as illustrated in Scheme 2c).48,61,62 For example, by
applying external squeeze force on diamond nanoneedle arrays,
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Figure 2. (a) Scheme of intracellular delivery of DNA and regeneration of SiNWAs modified with a branched PEI cross-linked by disulfide bonds.
PEICBA represents a branched PEI cross-linked by disulfide bonds that are sensitive to glutathione. Adapted with permission from ref 53. Copyright
2019 American Chemical Society. (b) Schematic illustration of preparation of PDA-coated SiNWAs for delivery of siRNA into cells. siRNA could be
easily bound to the surface of PDA-coated SiNWAs through the electrostatic interaction. Adapted with permission from ref 54. Copyright 2016
American Chemical Society.
various exogenous molecules including fluorescent dyes,
dextran, antibodies, quantum dots, nanoparticles, and pGFP
were delivered to NIH3T3 cells and hard-to-transfect primary
neuron cells effectively. The transfection efficiency of pDNA to
neurons was improved significantly from ∼1−5% by using
lipofection approach to ∼45% by using this system. Moreover,
after delivery process, the diamond nanoneedle arrays could be
removed from the cells to return back the normal cell culture
conditions.48
Carbon nanofiber arrays (CNFAs) are also a kind of nanoX
arrays for intracellular delivery. In 2003, McKnight et al.
demonstrated that penetration of CNFAs into living cells could
provide efficient delivery and expression of exogenous genes.
They prepared a tapered spiked CNFAs with a small tip
diameter of 20−50 nm for insertion directly into cells and a wide
base diameter of 1 μm for providing mechanical strength. The
pDNA loaded on the CNFAs by physical adsorption or covalent
interaction was delivered in CHO cells by penetration of CNFAs
alone or in combination with centrifugal force or pressing
force.66,67 In the following work, the CNFAs were coated with a
thin gold layer as an anchor for immobilization of pDNA
containing thiol or amino groups via covalent bonds. Compared
with unmodified CNFAs, the gold-coated CNFAs exhibited
increased loading capacity of pDNA, thus enhancing trans-
fection efficiency of pDNA encoding yellow fluorescent protein
(pYFP) to V97 cells.69 To better release the pDNA on the
CNFAs, they further optimized this platform by binding DNA
via disulfide bonds. When the nanofibers penetrated into the
cells, the pDNA was released effectively because of the breakage
of disulfide bonds by glutathione in cytoplasm, further
improving the transfection efficiency.38
Although the above-mentioned systems realize effective
intracellular delivery, one main drawback of most nanoX-
array-based systems is the insufficient cell harvest capability,
limiting their broad applications in ex vivo cell-based therapy.3,91
In some reports, the “engineered” cells were harvested by trypsin
digestion but the viability might be compromised. Aiming at this
limitation, Seo and Park et al. developed a microdevice
integrated with “intracellular delivery” and “cell harvest”
functions together. The microdevice was composed of three
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layers: a glass manifold layer associated with a pump (top layer),
a monolithic polydimethylsiloxane (PDMS) membrane (inter-
layer), and a microchannel layer with ZnO nanowire arrays
(bottom layer). Among them, the top layer and interlayer
together served as a pneumatic microvalve (Figure 5a). Once the
target cells were loaded in microchannel, the PDMS membrane
was properly bent down by supplying air through the pump, and
cells were squeezed onto ZnO nanowire arrays preloaded with
delivered molecules. After delivery, the PDMS membrane was
bent upward by supplying vacuum through the pump, thus the
“engineered” cells were harvested due to reverse-directional
pressure (Figure 5b).49
2.2. Hollow Nanostraw Arrays. Although solid nanoX
arrays are promising for intracellular delivery, they are still
restricted by some limitations. For example, as the delivery
process goes on, the molecules loaded on solid nanoX arrays
gradually taper off, causing a lack of continuous supply of
delivered molecules. In addition, it remains challenging to
control the dosage of delivered molecules precisely, and it is
cumbersome to codeliver or sequential deliver diverse molecules
into the same cell repeatedly. To this end, nanostraw arrays
consisting of a dense array of hollow nanotubes have been
developed.92−95 The bottom of nanostraw arrays is connected to
a fluid storage device or a microfluidic channel, thus the cells
adhered on the top of the nanostraw arrays can be continuously
and repeatedly delivered with the same or different molecules
through the bottom device/microchannel (as illustrated in
Scheme 2d).77,96,97 In addition, nanostraw arrays can achieve
precisely control of delivery dosage by modulating the
composition of external fluids. More importantly, codelivery
or sequential delivery of diverse molecules to the same cell can
be realized simply by adding or changing molecules in the fluid
storage/microchannel without repeated penetration of cell
membrane, those together solving the above-mentioned
problems related to the solid nanoX arrays.
In 2012, Sivan et al. developed silicon nanostraw arrays that
were established continuous fluidic access with a fluidic storage
device. Multiple delivery of fluorescently labeled dextran to the
same HEK293 cells was achieved. Especially, only the cells
connecting with nanostraws expressed red fluorescence when
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Figure 3. (a) Schematic illustration of preparation of DNA⊂SNPs and the interaction between Ad-SiNWAs and DNA⊂SNPs. The host−guest
interaction between the Ad motifs on Ad-SiNWAs and the β-cyclodextrin (CD) motifs of DNA⊂SNPs leads to dynamic assembly and local
enrichment of DNA⊂SNPs onto Ad-SiNWAs. Reproduced with permission from ref 55. Copyright 2014 American Chemical Society. (b) Schematic
illustration of intracellular delivery of OSKM encoding reprogramming factors and pNTFs encoding neuron specific transcription factors to fibroblasts
for cell reprogramming and cell transdifferentiation using Ad-SiNWAs, respectively. Reproduced with permission from ref 56. Copyright 2015 John
Wiley and Sons. (c) Schematic illustration of gene delivery in vivo using Ad-SiNWAs. Reproduced with permission from ref 55. Copyright 2014
American Chemical Society.

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Figure 4. Schematic illustration of intracellular delivery using CNSAs
combined with centrifugal force, where the CNSAs was placed on the
bottom and the target cells adhered on the surface of CNSAs. Adapted
with permission from ref 71. Copyright 2016 John Wiley and Sons.
Figure 5. Schematic illustration of (a) fabrication of the ZnO nanowire
array-incorporated microdevice. The microdevice was composed of
three layers: a glass manifold layer associated with a pump (top layer), a
monolithic polydimethylsiloxane (PDMS) membrane (interlayer), and
a microchannel layer with ZnO nanowire arrays (bottom layer). (b)
Schematic illustration of the functions of “intracellular molecular
beacon delivery” and “cell harvest” of this microdevice, where the
“engineered” cells were harvested due to reverse-directional pressure.
Adapted with permission from ref 49. Copyright 2015 John Wiley and
Sons.
transfected plasmid encoding red fluorescence protein (pRFP),
confirming the potential for spatial control of gene trans-
fection.29 In another work, Melosh et al. developed Al2O3
nanostraw arrays that established continuous fluidic access
into cytoplasm for the delivery of genetic materials, proteins, and
small molecules (Figure 6a). The nanostraws could sponta-
neously penetrate the cell membrane (Figure 6b and c),
providing permanent pipelines between exogenous molecules
stored in fluidic chamber/microfluidic channel underneath the
nanostraw arrays and interior of cells adhered on surface of
nanostraw arrays.77 Furthermore, they delivered second
membrane-impermeable dyes (red color) 12 h after delivering
the first membrane-impermeable dyes (green color) to the cells.
As shown in Figure 6d−f, the two dyes colocalized in most of the
cells, confirming that the nanostraw−cytosol connection
remained open over extended periods after initial penetration.
In addition, green fluorescence protein (GFP)-expressing CHO
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Figure 6. (a) Schematic illustration of a cross-section view of a
representative nanostraw arrays-incorporated device for intracellular
delivery. (b, c) Representative SEM images of cells cultured on
nanostraw arrays with diameter of 100 nm, height of 1−2 μm and
density of 108 nanostraws/cm2. (d−f) Typical fluorescence images of
sequential delivery of two different dyes: (d) Alexa-Fluor 488-hydrazide
dye (green color), (e) Alexa-Fluor 568-hydrazide dye (red color), and
(f) a merged image indicating colocalization of dyes to CHO cells using
nanostraw arrays. Adapted with permission from ref 77. Copyright 2012
American Chemical Society.
cells on nanostraw arrays surface were delivered with Co2+ and
ethylenediamine-tetra-acetic acid (EDTA) for quenching and
dequenching GFP fluorescence, respectively. Introduction of
Co2+ in the microfluidic channel for one minute led to significant
reduction in fluorescence of the cells adhered on nanostraws,
while no changes in fluorescence of the cells existed in the
culture medium were observed. Subsequent delivery of EDTA
enabled 75% of quenched cells to recover fluorescence after 4
min. This fluorescent “blinking” demonstrated that it is possible
to directly manipulate intracellular contents using nanostraw
arrays with precise spatiotemporal control.77
The delivery of exogenous molecules into various cell types
may require nanostraw arrays with different geometry, hence a
simple method for preparation of nanostraw arrays with different
diameters, lengths, and densities is needed. Conventionally,
nanostraw arrays are prepared using nanoporous polycarbonate
(PC) membranes as starting templates.47,78,79,98 However,
nanoporous PC membranes with custom-designed diameters
and densities are often expensive or commercially unavailable.
Regarding this issue, Xie et al. introduced a novel method
applying O2 plasma to fabricate a series of membrane template
to prepare nanostraw arrays, in which the structural parameters
of nanostraws could be adjusted simply by controlling the
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Figure 7. Schematic illustration of (a) a microfluidic device integrated with anti-EpCAM coated HS-MSAs for capture of cancer cells and intracellular
delivery, the illustration of conjugating anti-EpCAM biomolecules onto surface of HS-MSAs by biotin−streptavidin interaction was shown in right
image. (b) Fabrication process of HS-MSAs combining sequential atomic layer deposition, mechanical polishing, O2 etching, hydrothermal growth,
and Al2O3 deposition processes. (c) Integration of HS-MSAs with a microfluidic device. Reproduced with permission from ref 81. Copyright 2019
John Wiley and Sons.
etching power and etching time.80 Furthermore, they applied
Al2O3 nanostraw arrays with differing structures alone or in
combination with external force for delivery of pRFP to CHO
cells. When using nanostraw arrays alone, only the nanostraws
with small diameter and length could transfect pRFP to CHO
cells but with an extremely low efficiency (1.5%). In contrast, the
transfection efficiency was significantly enhanced with the
assistance of squeezing force to promote the membrane
penetration.80 In the following work, they fabricated a
hierarchical spiky microstraw arrays (HS-MSAs) by hydro-
thermal growth method to increase the surface-to-volume ratio.
After conjugated with specific antibodies, antiepithelial cell
adhesion molecules (anti-EpCAM), the HS-MSAs could
capture cancer cells efficiently and selectively because of the
synergistic effect of hierarchical structures of HS-MSAs and the
specific recognition of anti-EpCAM (Figure 7a-b). Delivery of
staurosporine to the captured cancer cells was realized with
precise dose and spatiotemporal control using the HS-MSAs in
combination with microfluidic device (Figure 7c), following
with detection of the cancer cells in situ and promotion of cell
apoptosis. Because the capture, delivery and detection of cells
were realized in a single device, side effects related to releasing
captured cells were avoided, which were inevitable for traditional
cancer cells detection methods.81 In addition to the above-
mentioned Al2O3 nanostraw arrays, carbon nanostraw arrays,75
and Au nanostraw arrays82 were also developed as intracellular
molecular delivery platforms.
3. SURFACE-MEDIATED INTRACELLULAR DELIVERY
BY ELECTROPORATION
In addition to mechanical penetration, other external forces,
such as electric field, can also be used for intracellular delivery.26
Under a certain electric field, a potential difference across the cell
membrane forms. When the potential difference reaches a
critical magnitude of voltage, the formation of transient pores in
cell membrane occurs, thus exogenous cargoes in the
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surrounding environment are effectively delivered into cells
through electrophoresis or promotion of diffusion effect.20,99 In
general, electroporation can be classically grouped into three
categories: bulk electroporation (BEP), microelectroporation
(MEP), and nanoelectroporation (NEP).20 BEP is generally a
kind of solution-mediated electroporation, and MEP/NEP
includes both solution-mediated electroporation and surface-
mediated electroporation. For the purposes of this Review, we
only discuss those techniques that involve using a surface to
focus the electric field locally on the cell membrane. Although
BEP has been widely applied as a commercial technique for gene
transfection, it still has some concerns and limitations. The
traditional BEP requires a high voltage (>1000 V) for effective
transfection, which however, always lead to severe cell damage.
Moreover, the typical BEP usually treats plenty of cells at the
same time, and it is difficult for electric field to be evenly
distributed at the single cell level, resulting in randomness of cell
transfection.24,100,101 Alternatively, surface-mediated electro-
poration can enhance the local electric field strength around cells
because of contact between cells and surfaces, reducing the total
applied voltage to alleviate cell damage during delivery
process.102−105 In this section, we will introduce the develop-
ment of surface-mediated electroporation approaches for
intracellular delivery that are divided into four categories
based on the devices used: (i) microfluidics (Scheme 3a), (ii)
microelectrodes (Scheme 3b), (iii) micro/nanopore mem-
branes (Scheme 3c), and (iv) nanostraw arrays (Scheme 3d) as
summarized in Table 2.
3.1. Microfluidics. Surface-mediated microfluidic electro-
poration is a method for delivery of molecules to the cells by
applying a certain voltage at both ends or both sides of the
microfluidic channel after cell attachment.133,134 Lu et al.
developed a microfluidic device integrated with multifunctions
including intracellular protein delivery, cell culture, and
fluorescence imaging (Figure 8a). Effective delivery of a
fluorescence resonance energy transfer (FRET) biosensor in
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Scheme 3. Schematic Illustration of Different Surface-
Mediated Electroporation Approachesa
a
(a) Microfluidics electroporation, (b) microelectrodes electro-
poration, (c) micro/nanopores membrane electroporation, and (d)
nanostraw arrays electroporation.
its protein form to mEFs was achieved by applying a voltage
across the microchannel. The FRET signal was then recorded in
situ to reflect the intracellular molecular activity with high
spatiotemporal resolution.106 Moreover, they also used this
microfluidic electroporation system to deliver quantum dots to
cells for live-cell imaging and cell-molecule tracking.107
However, this electroporation system is unable to control the
dose of delivered molecules precisely. Lee et al. developed a
microfluidic device containing a unique microchannel-nano-
channel-microchannel structure by a DNA combing and
imprinting (DCI) method,135 achieving delivery of precise
amounts of molecules into cells without compromising cell
viability.108 The target cells and delivered molecules were
located in two microchannels separately. When an electrical
pulse was delivered between the two microchannels, a large
electric field was generated in the nanochannel to drive the
molecules through the cell membrane adjacent to the nano-
channel by electrophoresis. The dose of delivered molecules
could be precisely controlled by modulating the duration and
number of pulses. However, the yield of channels prepared using
manual DCI method was relatively low. In addition, the brittle
resin printed on the channel surface often formed small
microcracks, which might lead to leakage within the device to
reduce the service life. To this end, they improved the DCI
process by replacing manual peeling method with dip-combing
and using a softer resin, increasing the preparation speed and
reducing the product defects. Moreover, they shortened the
length of microchannel from 3000 to 50 μm, reducing the
obstacles for cells to move from the microchannel to the
nanochannel to improve cell loading efficiency.109
Intracellular delivery to rare cells, such as MSCs, is crucial for
both fundamental research and patient-derived-patient-used
transplantations.136 Valero et al. developed a microfluidic device
containing two channels, which are connected by microholes for
trapping living cells (Figure 8b and c). Because of the pressure
difference between the two channels, the target cells were
trapped to the microholes. The enhanced local electric field led
to delivery of pDNA to MSCs with transfection efficiency of 75%
and cell viability of about 100% at a low voltage (<4 V).110 In
another work, Hur et al. developed a robust, efficient, and
versatile on-chip vortex-assisted microfluidic electroporation
system, enabling simultaneous enrichment of target cells and
sequential delivery of diverse molecules into different cell types
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with precisely controlled dosage (Figure 8d). Moreover, this
system enabled on-chip optimization of electroporation
parameters for cells with changing properties.111 It should be
noted that most of the microfluidic electroporation systems
mentioned above usually handle a small amount of target cells
(less than 100) at one time, which is more suitable for single-cell
research but not for the clinical applications where a large
amount of cells are required.
3.2. Microelectrodes. In addition to microfluidics, electro-
poration based on microelectrodes has also been developed for
surface-mediated intracellular delivery, where the targeted cells
come in contact to surface of microelectrodes that produce
localized electric field. For example, Lin et al. developed an
electroporation-based system including interdigitated electrodes
at bottom and a flat plate electrode on top. Because of the
electrostatic force generated between the two electrodes, the
negatively charged pDNA in the solution accumulated on the
surface of interdigitated electrodes, facilitating their delivery to
the cells come in contact on the interdigitated electrodes under
electric field.114 This interdigitated electrodes was fabricated by
traditional microfabrication technology that was time-consum-
ing and required special equipment. To solve this problem, Xing
et al. fabricated a 3D interdigitated electrode microchip using a
simpler and more economical standard printed circuit board
technology, using which to deliver pDNA into suspension Jurkat
cells.115 Liang et al. developed a novel electroporation microchip
that was compatible with the standard multiwell plates used in
biological research (Figure 9a). Tens to hundreds of cells were
loaded on surface of the novel annular interdigitated electrode
underneath the microchip and were transfected with pDNA with
90% of transfection efficiency and 80% of cell viability. The
results of numerical simulations proved that the application of a
voltage of 150 V was enough to generate a uniform electric field,
which was significantly lower than the voltage used in traditional
BEP.112 However, random transfection is inevitable in the
process of microelectrode electroporation. To overcome this
limitation, instead of a single microelectrode, microelectrode
arrays consisting of conductive nanospikes with uniform
dimensions were developed for electroporation (Figure 9b).
Because of the high aspect ratio, these conductive nanospikes
could generate a highly concentrated electric field under a low
voltage (<5 V) to deliver molecules to single cell or multiple cells
with high delivery efficiency and good cell viability.117
3.3. Micro/Nanopore Membranes. Precise intracellular
delivery of molecules with well-defined amounts is crucial for a
variety of biological researches and practical applications, such
as cell reprogramming and gene therapy.3,88,91 To this end,
micro/nanopore membranes with multiple pores were devel-
oped for electroporation, in which only the small areas of cell
membrane adjacent to the pores were exposed to a high localized
electric field intensity. Therefore, delivered molecules are
electrophoretically driven into the cytoplasm directly during
electroporation and precise dosage control can be achieved
without compromising cell viability.
Lee et al. developed a series of sandwich-structured
electroporation systems based on micropore membranes.118,119
The system was composed of two poly(ethylene terephthalate)
(PET) nuclear pore membranes with an average pore diameter
of 0.4 and 3 μm as the bottom layer and top layer, respectively,
between which were the target cells. The transfected pDNA was
added from the top membrane, enriched on or around the
exposed cell membrane by electrophoresis under low voltage,
and then delivered to the cytoplasm under a relatively large
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Table 2. Summary of Surface-Mediated Intracellular Delivery by Electroporationa

electric field
style parameters delivered molecules cell type delivery efficiency cell viability ref
microfluidics 800 V/cm; 15 ms FRET mEFs 55% 106
microfluidics 400 V/cm; 15 ms quantum dots CHO 60 quantum dots/cell 71% 107
microchannel-nano- 220 V/2 mm ODN-Cy3/siRNA/GAPDH- Jurkat/K562 12% (ODN-Cy3 to Jurkat) 108
channel-microchan- MB
nel
microchannel-nano- pGFP/OSKM mEFs 90% 109
channel-microchan-
nel
microfluidics <4 V PI/pDNA C2C12/MSCs 75% (pDNA to C2C12) 100% 110
65% (pDNAto MSCs)
microfluidics <20 V proteins/siRNA/miRNA HEK293/MCF7 26%-71% (single protein to HEK293) 111
microelectrodes 40−150 V; siRNA/pDNA HEK293/HUVECs/ 90% 80% 112,
0.1−1 ms MDCK/HL-60 113
microelectrodes 150−424 V/cm; pDNA HEK293/HepG2/HU- 114
20 ms VECs/MC3T3 × 10−1
microelectrodes 60−70 V pEGFP-N1 HeLa/MCF7/COS7/Ju- 75% (HeLa)/65% (MCF7)/75% >60% 115
rkat/3T3-L1 (COS7)/55% (Jurkat)/40%
(3T3-L1)
microelectrode arrays 66−82 V calcein/FITC-BSA/pGFP hPCs 50% (calcein) >80% 116
microelectrode arrays 2V PI/EB HeLa >93% >93% 117
micropore membranes 35 V/cm; 500 ms pEGFP/pSEAP NIH3T3 90% 118
micropore membranes 150 V/cm pmaxGFP/gWiz SEAP mESCs 75% 119
micropore membranes 4−10 V; 10 ms GATA2 molecular beacon leukemia cells 83.4% 89−96% 120
micropore membranes 150 V/cm pSEAP mESCs 85% 121
micropore membranes 3−15 V lucifer yellow/pDNA HeLa >80% 122,
123
micropore membranes 20 V PI/pGFP HeLa/HT1080/neurons 95% (PI to HeLa) >90% 124
50% (pGFP to HT1080)
micropore membranes 25 V; 20 ms oligonucleotides/dacarba- myofibroblasts/Human >90% 95.6% 125
zine/CRISPR-Cas9 plas- A375 melanoma cells
mids
nanopore membranes 10−15 V; 5 ms PI/BSA/pmCherry HT1080/MDA-MB 231 95% (PI to HT 1080) 95% (PI to 126
cells 70% (pmCherry) HT 1080)
nanopore membranes 100−140 V FAM-ODN/PI/pGFP/ H9C2 cells/mEFs 10% (OSKM to mEFs) >90% 127
OSKM
nanopore membranes 20 V/30 V mCherry RNA/pGFP/Cas9 HeLa/HEK293/3T3/Ju- >80% (HeLa) 95% 128
RNP rkat 75% (RNA to Jurkat)
50% (pGFP to Jurkat)
nanostraw arrays 30 V; 500 μs pRFP CHO 57.6% 90% 80
nanostraw arrays 5−20 V; 20−200 PI/pRFP/pGFP CHO/HEK293T 67−95% >98% 129
μs
nanostraw arrays 20 V; 20 s GFP/mCherry-mRNA HEK293T/hiPSC-CMs/ 60−90% >90% 130
ESCs/hFs/mGs/mNs
nanostraw arrays 15 V; 200 μs PI/pDNA MCF-7 64.2% (pDNA) >95% 131
83.8% (PI)
nanostraw arrays 5 V; 200 μs PI HeLa 80% >95% 132
a
Abbreviation: fluorescence resonance energy transfer (FRET); 18-mer oligodeoxynucleotide conjugated with Cy3 (ODN-Cy3); GAPDH
molecular beacon (GAPDH-MB); Ethidium bromide (EB); plasmid gWiz GFP (5757 bp) (pEGFP); plasmid secreted alkaline phosphatase
(pSEAP, 6569 bp); FAM labeled oligonucleotides (FAM-ODN); Cas9 ribonucleoproteins (Cas9 RNP); fluorescein isothiocyanate-labeled bovine
serum albumin (FITC-BSA); bovine serum albumin (BSA); quantum dots (15−20 nm in diameter); oligonucleotides (molecular beacons, 22 bp),
pDNA (CRISPR-Cas9 expression vectors, > 9 kbp); OSKM (13 kbp); GFP/mCherry-mRNA (∼922 nt); human umbilical vein endothelial cells
(HUVECs); human prostate cancer cells (hPC); mouse embryonic stem cells (mESCs); human iPSC-derived cardiomyocytes (hiPSC-CMs);
mouse primary glia cells (mGs); mouse primary neuron cells (mNs).

voltage. In this process, cells come in contact to the underlying
membrane by applying a vacuum force, which might cause
certain damages to the cells. Alternatively, Sooryakumar and Lee
et al. combined cells and magnetic microspheres and applied low
magnetic field to bind the cells on the micropore membrane,
alleviating the cell damage. Using this system, they delivered
signal molecules (GATA2 molecular beacon) to leukemia cells
with a high throughput of 40 000 cells/cm2.120 Genetically
modified primary ESCs have been widely used for biological
research, which are commonly prepared via BEP in suspension.
However, most ESCs suffer the problems to reform colonies
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when they are removed from culture plates for BEP.137 To
overcome these challenges, Lee et al. presented a nanofiber-
based sandwich electroporation device, which could deliver
nucleic acid to ESCs in culture without removing them from the
culture substrates. As shown in Figure 10a, a nanofibrous
electrospun nanofiber of poly(ε-caprolactone)/gelatin was used
to provide optimum conditions for cell culture, and a
nanoporous membrane was placed on the top of target cells.
Under a highly focused electric field, plasmids were delivered
through the nanopores to ESCs with high efficiency. Moreover, a
spacer could be integrated to this system for modulation the
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Figure 8. (a) Setup of microfluidic electroporation for delivery of FRET biosensor to cells integrating electroporation delivery, cell culture, and
fluorescence imaging, where the microfluidic channel was coated with fibronectin to facilitate cell adhesion and culture. Reproduced with permission
from ref 106. Copyright 2014 The Royal Society of Chemistry. (b, c) Setup for electroporation-based molecular delivery to 9 cells using a microfluidic
device including 9 traps: (b) 3D scheme of trapped cells and (c) microfluidic chip layout. Reproduced with permission from ref 110. Copyright 2008
The Royal Society of Chemistry. (d) Schematic illustration of the structure, operational principle of a vortex-assisted electroporation system and its
applications on sequential delivery, gene knockdown and phenotype alteration. Reproduced with permission from ref 111. Copyright 2017 Springer
Nature.

shape and size of the cell colonies to enhance the focused electric
field on cells.121
In most sandwich-structured micropore membranes electro-
poration systems, the target cells come in contact to the bottom
membrane, leaving a gap between cells and the top nanoporous
membrane, through which the delivered molecules were added.
Therefore, it is difficult to deliver the same or different molecules
to one cell repeatedly. To solve this limitation, a series of
electroporation systems were developed by integration of
nanoporous aluminum membranes or nanoporous polycarbon-
ate membranes with an underlying PDMS chamber as a
molecule storage device. In these systems, the molecules could
be delivered continuously into the same cells through the
nanopores.122−124,126 For example, Lee et al. fabricated a 3D
nanopore membrane electroporation chip with uniform and
parallel nanopores (650 nm for pore diameter) by combining
projection photolithography and deep reactive ion-etching
(Figure 10b). When delivering cargo with large molecular
weight (13 kbp transcriptional plasmid), this system showed
significant higher efficiency as compared to the BEP system
(∼10% vs <1%).127
Many cell types, such as lymphocytes and circulating tumor
cells, exist in suspension. Successful delivery of functional
molecules to such suspension cells is of considerable importance
in immune therapy and cancer diagnostics,138,139 however, is less
investigated but more challenging compared with the delivery to
adherent cells. One main limitation of the above-mentioned
systems is that they are not suitable for delivery to suspension
cells because of the low adhesion and insufficient duration of
cell-surface contact.140 Therefore, it is crucial to develop a
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micro/nanopore membrane electroporation system capable of
delivering molecules to adherent cells as well as to suspension
cells. Recently, Yang et al. presented a novel nanopore
membrane device consisting of two flat titanium electrodes
and a PDMS holder with a track-etched PC nanoporous
membrane. To improve cell adhesion property, the membrane
was precoated with fibronectin or poly-L-lysine. Diverse
molecules including nucleic acids, functional proteins, and
Cas9 ribonucleoproteins were delivered to various cell types
including adherent cells (HeLa cells) and suspension cells
(Jurkat cells) using this device (Figure 11). The permeability of
a small area of the cell membrane could be enhanced under low
electric field due to the intimate contact between cells and
nanopores, making it easier for entry of exogenous molecules to
cells through electrophoresis. Especially, the integrity of the cells
was retained well. In addition, this system did not require
complicated fabrication processes, expensive materials, speci-
alized buffers, or cell manipulation, providing a flexible and
effective method for intracellular delivery.128
3.4. Nanostraw Arrays. Nanostraw arrays electroporation
is a novel electroporation method, in which a flat electrode is
located at the bottom of nanostraw arrays and the other
electrode is inserted into the upper cell medium. The unique
structure of nanostraws provide localized electric field to
enhance the membrane permeability transiently only over a
small area. More importantly, unlike other electroporation
systems in which the delivered molecules are randomly
suspended in the fresh opened pores, in nanostraw arrays the
molecules are directly drew into the cells, thereby improving the
speed and accuracy of delivery process.132
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Figure 9. (a) Multiple-well microelectrodes electroporation device consisting of multiple microchips. Left: Impression of microchip consisting of two
juxtaposed chrome and gold layers sputtered on a glass substrate. Scale bar: 5 μm. Reproduced with permission from ref 112. Copyright 2011 The
Royal Society of Chemistry. (b) Schematic illustration of an electroporation chip containing nanospike arrays. The electric field at nanospike arrays was
enhanced due to its high aspect ratio. Reproduced with permission from ref 117. Copyright 2017 Elsevier.

Xie et al. developed an Al2O3 nanostraw arrays electro-
poration system to deliver exogenous molecules (propidium
iodide (PI) and pRFP) to HEK293T cells with high delivery
efficiency (95% for PI delivery, 81% for pRFP transfection) and
98% cell viability (Figure 12a).129 The nanostraws with small
diameter (250 nm) were unable to penetrate the cell membrane,
thereby avoiding the leakage of intracellular proteins and ions
from the persistently opened pores of cell membrane. They also
investigated the mechanism of electroporation in this system.
Exogenous PI was added into the microfluidic channel at
intervals of 10 s and 10 min after electroporation. A few cells
showed red fluorescence after 10 s, and almost no cells showed
red fluorescence after 10 min (Figure 12b), indicating that the
cell membrane resealed within 10 min after electroporation.
Moreover, simultaneous delivery or sequential delivery of two
different pDNA (pGFP and pRFP) could be realized (Figure
12c). This system was not only suitable for a small amount of
rare cells (about 5 cells) but also for a large amount of cells
(greater than 100 000 cells) at one time.129 The dosage of
delivered molecules could also be precisely control, which is very
important for the efficiency of cell reprogramming that are
highly affected by the relative ratio of different functional
molecules.
The Al2O3 nanostraw arrays were fabricated by using atomic
layer deposition and Cl2/BCl3 reactive ion etching approaches,
which involved sophisticated equipment and complex proce-
dures, and failed to produce conductive nanostraw arrays. To
solve these limitations, Xie et al. developed an economical and
commonly accessible electrodeposition method for fabrication
of a series of conductive nanostraw arrays based on metals (e.g.,
Pt and Au) or conductive polymers (e.g., poly(3,4-ethyl-
enedioxythiophene), PEDOT). These conductive nanostraw
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arrays were biocompatible and could serve as a plat electrode at
the bottom without setting up another plat electrode. As a
demonstration, Pt nanostraw arrays were integrated with a low-
voltage nanoelectroporation system for delivery of drug and
sensing of intracellular enzymes (Figure 13).132 Effective
delivery of molecules in living cells with spatial control was
realized at ∼5 V, while nonconductive nanostraw arrays usually
require approximately 20 V for cell poration.129,130
4. SURFACE-MEDIATED INTRACELLULAR DELIVERY
BY PHOTOTHERMAL PORATION
In addition to mechanical penetration and electroporation,
photothermal poration based on the photothermal agents
(PTAs) is another effective physical approach for intracellular
delivery. Under irradiation of laser with suitable wavelength and
intensity, the PTAs convert the absorbed light energy into
thermal energy to increase the membrane permeabilization or
produce transient pores in cell membrane, facilitating entry of
exogenous molecules into cell interior. The possible photo-
thermal poration mechanisms are that the increase in local
temperature might (i) lead to the local dissociation of
constituent lipid molecules that make up the bilayer or
denaturation of integral glycoproteins141−143 and (ii) cause
the evaporation of water molecules around the PTAs to form
vapor nanobubbles (VNBs), the collapse of which would create
transient pores in the cell membrane.144−147 Compared with the
above-mentioned mechanical penetration and electroporation,
photothermal poration has better spatiotemporal control of
delivery. For example, efficient delivery of macromolecules
suspended in the surrounding cell medium was achieved by
allowing gold nanoparticles (GNPs) in solution to bind to the
cell membrane to create transient pores upon near-infrared
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Figure 10. (a) Schematic illustration of an electroporation system
based on a sandwich-structured micropore membrane. The top layer is
PET membrane and the bottom layer is Al2O3 membrane coated with
electrospun nanofiber. Reproduced with permission from ref 121.
Copyright 2013 American Chemical Society. (b) Schematic illustration
of a 3D nano electroporation (NEP) system consisting of a 3D NEP
chip, support platform, PDMS holders and electrodes. The exogenous
molecules stored in bottom chamber were delivered into cells came in
contact surface of chip under electric pulse. Reproduced with
permission from ref 127. Copyright 2016 The Royal Society of
Chemistry.
Figure 11. Schematic illustration of a nanopore membrane-based
electroporation system for intracellular delivery to both adherent cells
and suspension cells, where suspension cells came in contact bottom
surface under effect of centrifuge force and adherent cells were cultured
overnight before delivery procedure. The cargoes were delivered into
cell interior through electrophoresis. Adapted with permission from ref
128. Copyright 2019 National Academy of Sciences.
(NIR) irradiation.25,148 However, there are some drawbacks of
such solution-mediated photothermal poration such as possible
cytotoxicity caused by the PTAs that enter the cells together
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with the delivered molecules during irradiation, and limited
delivery efficiency because the amount of PTAs attached to the
cell membrane is limited. Alternatively, surface-mediated
photothermal poration approaches based on the surfaces on
which the PTAs are firmly immobilized show their own
advantages including low cytotoxicity by avoiding the
endocytosis of suspended PTAs, and improved delivery
efficiency due to the enhanced interaction between the
surface-immobilized PTAs and the surface-attached cells, and/
or increased chance of cell-to-molecule contact.149−151 In this
section, we will introduce the recent development of surface-
mediated photothermal poration approaches that are divided
into three categories based on the PTAs: gold nanomaterials,
PDA, and other photothermal materials (as summarized in
Table 3).
4.1. Gold Nanomaterials. Gold nanomaterials (such as
GNPs and gold nanorods (GNRs)) are typical PTAs with
excellent photothermal effects based on the localized surface
plasmon resonance (LSPR) and good biocompatibility,165
making them suitable candidates for fabrication of surfaces
with photothermal poration capability. In 2010, Chiou et al.
immobilized GNPs on the cell-culture Petri dishes. Under 532
nm visible light irradiation, the GNPs converted the absorbed
light energy into heat to generate explosive VNBs, promoting
the delivery of fluorescent dye (calcein) to attached HEK293T
cells.149 After this pioneering work, a series of photothermal
surfaces were developed to deliver diverse molecules into
various cell types. For example, Mazur et al. developed a
plasmonic nanostructured substrate with gold-coated tipless
pyramids arrays. Under NIR irradiation, the surface produced
heat to create nanobubbles and pressure wave, increasing the
permeability of cell membrane transiently and thus facilitating
the delivery of fluorescence molecules (calcein) in HeLa cells
with a delivery efficiency of 80%.159 In the following work, they
further developed a gold-coated thermoplasmonic substrate and
achieved delivery of molecules with different molecular weights
(0.6−2000 kDa) into HeLa CCL-2 cells with high delivery
efficiency (up to 95%) and high cell viability (up to 98%). The
throughput was as high as 50 000 cells/min, and could be further
enlarged by scaling up the substrate.160 However, it should be
noted that the thermoplasmonic-based substrates are relatively
expensive with complicated fabrication process, limiting their
broad applications.
Gold nanoparticle layer (GNPL) refer to a layer of dense
immobilized GNPs on a substrate, which has been recognized as
a versatile nanostructured platform for biomedical applications
due to the excellent properties of GNPs and unique two-
dimensional surface topography.166,167 In general, the fabrica-
tion of GNPL is simple, facile and does not need sophisticated
equipment. Compared with dispersed GNPs in solution, the
aggregated GNPs on GNPL exhibit a more efficient light-to-heat
conversion. In addition, GNPL exhibits unique micronanoto-
pography to provide multiple sites for cell contacting and
enhance local temperature induced by irradiation.168,169 On the
basis of these properties of GNPL, we developed a high-
throughput platform for intracellular delivery with broad
applicability for both delivered molecules and target cell
types.150 As illustrated in Figure 14a, under NIR irradiation,
the increase in surface temperature resulted in the enhanced
membrane transient permeability for the diffusion of molecules
into cells from the surrounding medium. The molecules were
demonstrated to be delivered to the interior of cells rather than
merely attached to the cell membrane (Figure 14b). Using
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Figure 12. (a) Schematic illustration of Al2O3 nanostraw arrays electroporation for intracellular delivery. The electric field was localized inducing
transient membrane permeability over a small area and decreasing cell damage. (b) Study of cell membrane recovery conditions associated with Al2O3
nanostraw arrays electroporation. Cells cultured on nanostraw arrays under electric field were recovered for 0 s, 10 s, and 10 min, respectively. Then PI
were added in the microfluidic channel at each time point. Scale bar: 50 μm. (c) Simultaneous transfection and sequential transfection with a 24-h
interval of pRFP and pGFP to cells under electroporation. Scale bar: 50 μm. Adapted with permission from ref 129. Copyright 2013 American
Chemical Society.

Figure 13. Schematic illustration of a device based on conductive nanostraw arrays achieving intracellular delivery, extraction of cytosol contents from
live cells, and detection of cell number. The conductive nanostraw arrays could be served as a planar electrode without setting up another planar
electrode at the bottom. Reproduced with permission from ref 132. Copyright 2019 American Chemical Society.

GNPL, high transfection efficiency (∼100%) of pGFP to easy-
to-transfect cells (e.g., HeLa cells) was obtained with negligible
cytotoxicity, however, the transfection efficiencies to hard-to-
transfect cells (e.g., 53% for mEFs and 9% for HUVECs) were
still limited, which might be due to the possible degradation of
naked pDNA by endonucleases. To improve the transfection
efficiency of such cells, we incorporated a chemical carrier
(polyethylenimine of low molecular weight (LPEI, 2 kDa)) into
the GNPL-mediated photothermal poration system to combine
their respective advantages together.151 In this upgraded
approach, the pDNA was first protected by LPEI to avoid the
degradation during the transfection process and the resulted
LPEI/pDNA complex was delivered into cytosol by GNPL
under NIR irradiation with dramatically improved delivery
efficiencies of recalcitrant cells (88.5 ± 9.2% for mEFs and 94.0
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± 6.3% for HUVECs). Moreover, besides model pDNA (e.g.,
pGFP), we also delivered a functional pDNA encoding ZNF580
gene (pZNF580) to HUVECs using this system; the transfected
cells exhibited improvement in both initial attachment and
proliferation for long-term culture.
GNRs are also gold nanomaterials with high photothermal
conversion capability resulting from their distinctive LSPR
effect. In addition, because of their adjustable aspect ratios,
GNRs can absorb light energy in a wider range (from visible to
NIR region).170 Utilizing the photothermal effect of GNRs, we
developed a NIR activated core−sheath structure electrospun
nanofiber for in situ controllable gene transfection.152 The
nanofiber was composed of a core layer containing complex of
fibroblast growth factor-encoding pDNA (pFGF) and LPEI and
a sheath layer of biocompatible and biodegradable gelatin/L-
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Table 3. Summary of Surface-Mediated Intracellular Delivery by Photothermal Porationa

PTAs delivered molecules cell types delivery efficiency cell viability ref
GNPs fluorescent dye HEK293T 149
GNPL TRITC-dextran/pGFP HeLa/mEFs/HUVECs/ 100% (HeLa) near 100% 150
mESCs 53% (pGFP to mEFs)
44% (pGFP to HUVECs)
pGFP mEFs/HUVECs 88.5% (pGFP to mEFs) 94% (pGFP 80% 151
to HUVECs)
GNRs plasmid bFGF NIH3T3 >85% >90% 152
GNRs DOX A498 cells 153
GNTs PI NIH3T3 ≥95% 154
PDA pDNA HUVECs 85% 155
pDNA HUVECs/HEK293T 40% 156
dextran/BSA/pGFP/pZNF580 HeLa/mEFs/HUVECs/ >99% >90% (HeLa/mEFs/ 157
hDFs/DCs HUVECs) 60%(DCs)
P-MNPs dextran/pDNA HeLa 67% >92.6% 158
TPS-Au FITC-dextran/Calcein HeLa CCL-2 up to 95% up to 98% 159,
160
TPS-TiN calcein dye HeLa CCL-2 >80% >90% 161
Ti thin FITC-dextran/polystyrene beads/ HeLa/hDFs/PB- 58% (bacteria to HeLa) >90% 162
films enzyme/bacteria MDMs/RPTECs 90% (FITC-dextran to hDFs and
RPTECs)
60%(FITC-dextran to PB-MDMs)
Ti thin calcein/FITC-dextran/bacterial enzyme β- Ramos suspension B cells 84% (calcein)/45% (FITC- >89% (pGFP) 163
films lactamase/pGFP dextran)/ 58% (pGFP)
SiNWAs RBITC-BSA/pGFP HeLa/Ramos/T cells 100% (HeLa)/83% (Ramos)/80% >95% 164
(T cells)
a
Abbreviations: Gold nanoparticles (GNPs); gold nanoparticle layer (GNPL); gold nanorods (GNRs); gold nanotubes (GNTs); porous magnetic
iron oxide nanoparticles (P-MNPs); gold-coated thermoplasmonic substrates (TPS-Au); TiN-coated thermoplasmonic substrates (TPS-TiN);
Calcein (0.6 kDa); Tetramethylrhodamine isothiocyanate-labeled dextran (TRITC-dextran, 4.4 kDa); fluorescein isothiocyanate-labeled dextran
(FITC-dextran, 0.6, 4, 40, 2000 kDa); pDNA encoding fibroblast growth factor/ZNF580 gene (pbFGF, 6825 bp/pZNF580); polystyrene beads
(20 nm to 1 μm); bacterial enzyme β-lactamase (29 kDa); doxorubicin (DOX); rhodamine B isothiocyanate-labeled bovine serum albumin
(RBITC-BSA, 66 kDa); peripheral blood monocyte-derived macrophages (PB-MDMs); renal proximal tubule epithelial cells (RPTECs).

poly(lactic acid) with uniformly distributed GNRs (Figure 15a).
Under NIR irradiation, the heat generated by the encapsulated
GNRs could (i) increase the permeability of the nanofibers to
achieve the controlled release of pFGF from the core layer and
(ii) generate transient holes in the membranes of NIH3T3
fibroblasts on the nanofibers to enhance the delivery of pFGF
(Figure 15b). The proliferation and migration of pFGF
transfected fibroblasts were effectively improved, indicating
great potential of such nanofibers with localized surface-
mediated gene transfection capability.
4.2. Polydopamine. Polydopamine (PDA) is well-known as
a mussel-inspired material, which has been extensively studied in
surface chemistry. Besides strong adhesive property, good
biocompatibility, easy functionalization, PDA also has excellent
photothermal property to effectively absorb and transfer NIR
light energy to heat.171−173 Taking advantages of these
properties, Ji and Ren et al. reported a photothermal poration
surface based on poly(D,L-lactide-co-glycolide) (PLGA) poly-
meric spongy film modified with PDA coating (Figure 16a).155
Because of the microporous structure, the PEI/pDNA
complexes could be loaded by a simple wicking effect and the
amount of loaded complexes could be varied by changing the
deposition time or solution concentration. Under NIR
irradiation, the primary endothelial cells came in contact to
the spongy film were delivered with loaded pDNA with high
efficiency (85%) and low cytotoxicity. In addition, the spatial
control delivery could be achieved via NIR irradiation in the
defined area. In the following work, they simplified the
fabrication process by codeposition of PDA and PEI on the
substrate and successfully transfected pGFP to endothelial
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cells.156 The potential delivery mechanism was examined by
Sytox (a membrane-impermeable dye) staining assay and lactate
dehydrogenase (LDH, a cytosolic enzyme present inside the
cell) assay, both of which were commonly used methods to test
the membrane integrity. As shown in Figure 16b, without NIR
irradiation, the attached cells did not show any fluorescence,
indicating the intact healthy cell membranes. In contrast, the
cells emitted bright red fluorescence under NIR irradiation and
the number of cells representing fluorescence increased with
increasing irradiation time. Moreover, quantification of the
released LDH showed that NIR irradiation resulted in an
obvious increase in LDH release. Taken together, it is suggested
that the intracellular delivery of this system was due to the
disturbance and destabilization of cell membrane resulted from
the localized hyperthermia generated by PDA.156
Besides intracellular delivery of functional molecules with
high efficiency, efficient and nontraumatic cell harvest is also
important and plays a critical role in many applications, in
particular ex vivo cell-based therapy, where the engineered cells
with delivered molecules are to be injected to a patient.1,3
However, the cell harvest capability is rarely mentioned in above
surface-mediated photothermal poration systems. Recently, we
developed a “two-in-one” platform integrated with intracellular
delivery and cell harvest functions (Figure 17).157 This surface
was fabricated by deposition of a vinyl group-containing PDA
layer, followed by polymerization to graft a thermoresponsive
polymer poly (N-isopropylacrylamide) (PNIPAAm). The
underlying PDA layer provided good photothermal effect for
effective delivery of a variety of exogenous molecules (e.g.,
polysaccharide, protein, and pDNA) to diverse cell types
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Figure 14. (a) Schematic illustration of intracellular delivery using the GNPL-mediated photothermal poration system. Left: Complete experimental
procedure of GNPL-mediated intracellular delivery. Right: Permeability of cell membrane was enhanced to facilitate entry of exogenous
macromolecules into cell interior. (b) Representative confocal microscopy images of HeLa cells delivered with TRITC-dextran using GNPL under
NIR irradiation measured using the Z-stack scan mode of confocal microscopy. Adapted with permission from ref 150. Copyright 2016 John Wiley and
Sons.

including three typical hard-to-transfect cells (mEFs, HUVECs,
and DCs). The grafted PNIPAAm did not influence the
photothermal properties of the PDA layer, but allowed the
surface to release the “engineered” cells by decreasing the
temperature of cell medium to induce the transition of surface
property from cell-adhesive to cell-repellent.174 The harvested
cells maintained their viability for subculture and the functions
of delivered molecules were conserved. Using HUVECs and
pZNF580 as a pair of model cell line and functional molecule,
the feasibility and effectiveness of this PDA−PNIPAAm
platform was demonstrated as the harvested “engineered”
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HUVECs exhibited significantly increased proliferation and
migration capabilities compared to unmodified cells.
4.3. Other Photothermal Materials. In addition to gold
nanomaterials and PDA, other photothermal materials have also
been exploited for photothermal poration intracellular delivery.
For example, Chiou et al. developed a massively parallel
photothermal biophotonic laser-assisted surgery tool (BLAST)
enabling the entry of large micrometer-sized cargoes into
cells.162 The BLAST platform consisted of a silicon chip with a
thin porous SiO2 membrane on top, providing an array of
micrometer-wide, trans-film holes whose side walls were
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Figure 15. (a) Schematic illustration of fabrication of the core−shell structured electrospun fiber composed of pbFGF/PEI complex as core layer and
GNRs/gelatin/L-poly(lactic acid) as shell layer. (b) Intracellular delivery of pbFGF to NIH3T3 fibroblasts using the core−shell structured electrospun
fiber under NIR irradiation. Left: Experimental procedure of electrospun fiber-mediated intracellular delivery. Right: Amplified images of cell
membrane with/without NIR irradiation. Reproduced with permission from ref 152. Copyright 2020 American Chemical Society.

Figure 16. (a) Schematic illustration of a PDA coated microporous spongy film platform for intracellular delivery via photothermal poration, where the
pDNA was first protected by bPEI to form a pDNA/bPEI complex, then the complex was loaded onto surface of PDA-coated microporous spongy film
by simple wicking effect. Reproduced with permission from ref 155. Copyright 2019 American Chemical Society. (b) Representative fluorescence
images of cells on PDA−PEI films without irradiation, with irradiation for 70 or 140 s, respectively. Red fluorescence is from delivered Sytox.
Reproduced with permission from ref 156. Copyright 2019 The Royal Society of Chemistry.

asymmetrically coated with crescent-shaped titanium thin films.
This BLAST platform harvested the light energy to induce rapid
heating and vaporization of adjacent water molecules to trigger
cavitation bubbles. These cavitation bubbles grew, coalesced,
and collapsed within 110 ns. The rapid bubble “explosion” led to
strong fluid flows to disrupt the nearby cell membrane,
facilitating the entry of the exogenous large cargoes including
proteins, bacteria, and nanoparticles to various cell types.
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Unfortunately, the above-mentioned surface-mediated photo-
thermal poration delivery systems are not suitable for suspension
cells due to the poor cell-surface contact.140 To solve this
limitation, Chiou et al. developed a photothermal 3D microwells
with sharp nanoscale metal-coated tips, permitting self-position
of suspension cells by synergetic effect of gravity with each
microwell and size effect of microwells in direct contact with
eight sharp tips (Figure 18).163 Under NIR irradiation, a range of
cargoes were delivered into suspension Ramos B cells stayed in
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Figure 17. Schematic illustration of the universal “two-in-one” platform
integrated with intracellular delivery and cell harvest functions based on
a PDA layer grafted with PNIPAAm. The cells were first delivered with
exogenous molecules under NIR irradiation, and then, the “engineered”
cells could be harvested from the surface by decreasing cell medium
temperature, finally, the harvested cells could be recultured for next use
or research. Reproduced with permission from ref 157. Copyright 2019
American Chemical Society.
microwells with a high throughput of >100 000 cells delivered
per minute.
Figure 18. Schematic illustration of intracellular photothermal delivery
for suspension cells using sharp nanoscale tips in microwells. Adapted
with permission from ref 163. Copyright 2019 American Chemical
Society.
Although the photothermal microwell-based system achieved
macromolecular delivery to suspension cells, the fabrication
process was complicated and no cell harvest function involved.
Ideally, a surface-mediated photothermal poration system
should have high delivery efficiency, high cell viability, and
high cell harvest capability for both adherent cells and
suspension cells. In addition, it is pointed out that low-
temperature treatment for cell harvest used in our PDA−
PNIPAAm system may decrease cell viability.175 Therefore, a
gentle way to trigger cell release is more promising. To this end,
we developed a smart photothermal poration platform that
combined three sequential functions including cell capture,
intracellular delivery, and cell harvest together to engineer living
cells (Figure 19).164 This platform was fabricated by the
modification of SiNWAs possessing photothermal effect with a
polymer containing phenylboronic acid (PBA) groups with
sugar-responsive property. The unique 3D nanostructure of
SiNWAs84,176 could increase the local density of PBA groups
that can form phenylboronate ester bonds with sialic acid177 and
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enhance the interactions between the surface of SiNWAs and
cell membranes, leading to a high capture capacity for both
adherent cells (e.g., HeLa cells) and suspension cells (e.g.,
Ramos cells and T cells) that overexpressed sialic acid on the
membrane. Under NIR irradiation, the local heat generated by
SiNWAs efficiently enhanced membrane permeability of
captured cells transiently for delivery of diverse macromolecules
(protein and pDNA). The transfection efficiency of pDNA to
the hard-to-transfect suspension immune T cells was more than
80%, significantly higher than that using the commercial
transfection reagents and other reported methods.178,179
Moreover, because of the sugar-responsiveness of phenyl-
boronate ester bonds,180,181 the “engineered” cells could be
released from the surface by sugar treatment. As sugars were
natural biomolecules and are generally nontoxic, the harvested
“engineered” cells maintained high viability for normal growth
and proliferation, suggesting the potentials for further
fundamental research and clinical application.164
5. SUMMARY AND PERSPECTIVES
The decade just passed has witnessed rapid developments in
surface-mediated PMD-based methods for the intracellular
delivery of exogenous molecules and functional materials. In this
Review, we systematically summarize achievements in intra-
cellular delivery systems based on mechanical penetration,
electroporation, and photothermal poration and discuss their
respective characteristics. PMD-based methods allow “crossing”
the outer cell membrane “barrier”, thus facilitating direct
delivery of diverse types of exogenous molecule into target
cells. Compared with traditional carrier-mediated methods and
solution-mediated PMD-based methods, surface-mediated
PMD-based methods exhibit high delivery efficiency, low
cytotoxicity, and broad applicability to diverse molecules and
cell types.
Although considerable progress has been made, many
challenges remain and these should be the focus of future
research. From fundamental point of view, a greater under-
standing of the mechanisms of physical membrane disruption,
such as whether the transient pores are produced in cell
membrane, the size of pores, the resealing of pores, and the
possible leakage of cell contents is increasingly important for this
field, but little detailed research has been done in this area
related to intracellular delivery, hence these factors require in-
depth investigation. Assessing the safety and effectiveness of
engineered cells is of importance for ex vivo cell therapy,
however, most of current researches merely focused on
investigating cell viability, but ignoring the further detection
such as the DNA damage, changes of cell fate and cell functions,
which requires engineering technology researchers to develop
more accurate detection tools and techniques to detect changes
of cell interior. From practical point of view, most of the
researches to date have investigated the delivery of model
molecules (such as pGFP) into model cell types, while the
delivery of functional molecules to edit cells (e.g., CRISPR/
Cas9-based genome editing for disease modeling and therapy)
to prepare therapeutic cells for in vitro or ex vivo cell-therapy
(e.g., chimeric antigen receptor (CAR)-T cells immunotherapy
for human cancer) or molecular probes and nanomaterial
sensors to analyze cell behaviors (e.g., live-cell imaging) remains
largely unexplored. Moreover, many platforms now only
involved intracellular delivery, while the upstream cell capture
or downstream cell detection and cell harvest are ignored.
Therefore, it is urgent to develop multifunctional platforms that
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Figure 19. Schematic illustration of a universal platform based on SiNWAs modified with sugar-responsive polymers for engineering cells. Left: Cells
including adherent and suspension cells were captured due to the synergistic effects of “nano-enhancement” of SiNWAs and chemical recognition of
PHB. Middle: Cells came in contact surface of SN-PHB were delivered with exogenous molecules under NIR irradiation. Right: “Engineered” cells
were harvested from surface by fructose treatment. Reproduced with permission from ref 164. Copyright 2020 John Wiley and Sons.
can conduct multiple cell manipulation processes, which can be
better applied to the in situ visualization of single cell analysis, in
situ chemical manipulation, and analysis of captured circulating
tumor cells and ex vivo cell-based therapies.
Finally, it is hoped that this Review will be of benefit to the
broad community engaged in engineering manufacture,
automatic research, intracellular molecular delivery, immuno-
therapy, regenerative medicine, tissue engineering, surface
chemistry, and other fields. For engineers, we expect to provide
them with inspirations to develop more compact and portable
devices integrating all cell manipulation processes and a robot
matching array-type and flow-through systems to achieve high
level of automation. With the development of surface-mediated
intracellular delivery methods by physical membrane disruption,
researchers need to develop precise toolboxes to study the
mechanisms of physical membrane disruption from the micro/
nano scale. It is hoped that future research will be able to regulate
cell function and detect the intracellular environment through
intracellular delivery strategies, thereby providing effective
strategies for drug delivery, cell therapy, gene editing, stem cell
transfection, reprogramming of induced pluripotent stem cells,
and neuronal analysis to contribute to various cancer treatments
and gene-related diseases.
■
AUTHOR INFORMATION
Corresponding Author
Qian Yu − State and Local Joint Engineering Laboratory for Novel
Functional Polymeric Materials, College of Chemistry, Chemical
Engineering and Materials Science, Soochow University, Suzhou
215123, P. R. China; orcid.org/0000-0003-3612-6951;
Email: yuqian@suda.edu.cn
Authors
Yangcui Qu − State and Local Joint Engineering Laboratory for
Novel Functional Polymeric Materials, College of Chemistry,
Chemical Engineering and Materials Science, Soochow University,
Suzhou 215123, P. R. China
Yanxia Zhang − Institute for Cardiovascular Science and
Department of Cardiovascular Surgery of the First Affiliated
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www.acsami.org Review
Hospital, Soochow University, Suzhou 215007, P. R. China;
orcid.org/0000-0002-1920-6709
Hong Chen − State and Local Joint Engineering Laboratory for
Novel Functional Polymeric Materials, College of Chemistry,
Chemical Engineering and Materials Science, Soochow University,
Suzhou 215123, P. R. China; orcid.org/0000-0001-7799-
4961
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsami.0c06978
Author Contributions
Contributions to this Review were made by all authors, and all
have seen and approved the submitted manuscript.
Author Contributions
§
Y.Q. and Y.Z. contributed equally.
Funding
The National Natural Science Foundation of China (21774086
and 21935008), the Natural Science Foundation of Jiangsu
Province (BK20180093), the Suzhou Municipal Science and
Technology Foundation (SYS2018026), and the Priority
Academic Program Development of Jiangsu Higher Education
Institutions (PAPD).
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work was supported by the National Natural Science
Foundation of China (21774086 and 21935008), the Natural
Science Foundation of Jiangsu Province (BK20180093), the
Suzhou Municipal Science and Technology Foundation
(SYS2018026), and the Priority Academic Program Develop-
ment of Jiangsu Higher Education Institutions (PAPD). The
authors thank Prof. John L. Brash for his helpful discussions and
Mr. Yang Zhou for his assistance on graphic works. The authors
also sincerely appreciated the anonymous reviewer for his/her
constructive comments, which are very helpful to improve the
thoroughness and presentation of this paper.
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