Biofunctionalized nanoneedles for the direct and site-selective delivery of probes into living cells.

Abstract

Accessing the interior of live cells with minimal intrusiveness for visualizing, probing, and interrogating biological processes has been the ultimate goal of much of the biological experimental development.
The recent development and use of the biofunctionalized nanoneedles for local and spatially controlled intracellular delivery brings in exciting new opportunities in accessing the interior of living cells. Here we review the technical aspect of this relatively new intracellular delivery method and the related demonstrations and studies and provide our perspectives on the potential wide applications of this new nanotechnology-based tool in the biological field, especially on its use for high-resolution studies of biological processes in living cells.
Different from the traditional micropipette-based needles for intracellular injection, a nanoneedle deploys a sub-100-nm-diameter solid nanowire as a needle to penetrate a cell membrane and to transfer and deliver the biological cargo conjugated onto its surface to the target regions inside a cell. Although the traditional micropipette-based needles can be more efficient in delivery biological cargoes, a nanoneedle-based delivery system offers an efficient introduction of biomolecules into living cells with high spatiotemporal resolution but minimal intrusion and damage. It offers a potential solution to quantitatively address biological processes at the nanoscale.
The nanoneedle-based cell delivery system provides new possibilities for efficient, specific, and precise introduction of biomolecules into living cells for high-resolution studies of biological processes, and it has potential application in addressing broad biological questions. This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.

Full text

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Review
Biofunctionalized nanoneedles for the direct and site-selective delivery of probes into
living cells☆
Kyungsuk Yuma, Min-Feng Yua, Ning Wanga, Yang K. Xiangb,⁎
aDepartment of Mechanical Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
bDepartment of Molecular and Integrative Physiology and Neuroscience Program, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
a b s t r a c ta r t i c l e i n f o
Article history:
Received 15 December 2009
Received in revised form 4 May 2010
Accepted 17 May 2010
Available online 24 May 2010
Keywords:
Nanoneedle
Cargo delivery into living cell
Imaging
Single-molecule study
Subcellular
Compartment
Nucleus
Cytoplasm
Background: Accessing the interior of live cells with minimal intrusiveness for visualizing, probing, and
interrogating biological processes has been the ultimate goal of much of the biological experimental
development.
Scope of review: The recent development and use of the biofunctionalized nanoneedles for local and spatially
controlled intracellular delivery brings in exciting new opportunities in accessing the interior of living cells.
Here we review the technical aspect of this relatively new intracellular delivery method and the related
demonstrations and studies and provide our perspectives on the potential wide applications of this new
nanotechnology-based tool in the biological field, especially on its use for high-resolution studies of
biological processes in living cells.
Major conclusions: Different from the traditional micropipette-based needles for intracellular injection, a
nanoneedle deploys a sub-100-nm-diameter solid nanowire as a needle to penetrate a cell membrane and to
transfer and deliver the biological cargo conjugated onto its surface to the target regions inside a cell.
Although the traditional micropipette-based needles can be more efficient in delivery biological cargoes, a
nanoneedle-based delivery system offers an efficient introduction of biomolecules into living cells with high
spatiotemporal resolution but minimal intrusion and damage. It offers a potential solution to quantitatively
address biological processes at the nanoscale.
General significance: The nanoneedle-based cell delivery system provides new possibilities for efficient,
specific, and precise introduction of biomolecules into living cells for high-resolution studies of biological
processes, and it has potential application in addressing broad biological questions.
This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
Nanotechnology has recently found increasing applications in
biology by providing new nanotechnology-based tools and materials
to probe and manipulate biological processes at the nanoscale (∼1 to
100 nm) [1], which is the length scale where many fundamental
biological processes occur. For instance, fluorescent semiconductor
nanoparticles, or quantum dots [2,3], have been used as probes to
visualize dynamic processes in living cells, including the dynamic
movement of singe membrane receptors [4–8], motor proteins [9],
nerve growth factors [10], and synaptic vesicles [11,12]; and magnetic
nanoparticles have been used to manipulate individual membrane
receptors to control signal transduction in living cells [13].
One-dimensional nanomaterials, such as nanotubes and nano-
wires,havealsobeenusedasintracellularbiosensors,deliverycarriers,
and imaging agents [14–21]. In particular, with their unique physical
and chemical properties distinct from both individual molecules and
bulk materials, chemically synthesized nanomaterials have presented
new opportunities and applications in biology and medicine, from
basic biophysical studies at the single-molecule level to the diagnosis
and treatment of diseases [22–24]. In addition, with their needle-like
nanoscale geometry and excellent mechanical and electrical proper-
ties, these high-aspect ratio nanostructures have been explored as
membrane-penetrating nanoneedles that can manipulate and sense
biologicalprocessesinsidecellwithminimalintrusivenessandtoxicity
[24–31]. For example, surface-functionalized nanotubes have been
used to deliver biomolecular species into living cells with high spatial
and temporal precision [27,30,31]. Conductive nanotubes have also
been envisioned as an electrochemical nanoprobe to measures
electrochemical events, redox environments, and signaling processes
occurring inside cells or between neighboring cells [31,32].
The transfer of biomolecules into living cells is a general practice
used to monitor or modify molecule-specific intracellular processes. It
Biochimica et Biophysica Acta 1810 (2011) 330–338
☆ This article is part of a Special Issue entitled Nanotechnologies - Emerging
Applications in Biomedicine.
⁎ Corresponding author. Department of Molecular and Integrative Physiology,
University of Illinois at Urbana Champaign, 407 S Goodwin Ave, Urbana, IL 61801,
USA. Tel.: +1 217 265 9448; fax: +1 217 333 1133.
E-mail address: kevinyx@illinois.edu (Y.K. Xiang).
0304-4165/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbagen.2010.05.005
Contents lists available at ScienceDirect
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providesanefficientwaytostudythetemporalandspatialregulationof
protein systems that underlie basic cellular functions [33]. Many
methods have been developed for this purpose [33–41]. Each of them
has its characteristic advantages and disadvantages with respect to cell
viability,transferefficiency,generalapplicability,andtechnicalrequire-
ments [33]. In this review, we discuss a new type of nanotechnology-
basedmethodologyfortheintroductionofbiomoleculesintolivingcells
and its potential implications in addressing biological questions.
2.Generaldescriptionofthenanoneedle-basedintracellulardelivery
Similar to a micropipette-based injection system, a nanoneedle-
basedintracellulardeliverysystemcomprisesananoneedle(ananotube
or a nanowire) on a macroscopic handle (an etched metallic wire or
simply a pulled glass micropipette) and a manipulator (a standard
piezoelectric micromanipulator) integrated with an inverted optical
microscope[26,27,30,31].Similaralsoinpracticetotheuseofastandard
micropipette-based injection system, the nanoneedle is manipulated
with the micromanipulator to penetrate into a target cell under the
observation of an optical microscope. The major difference between
the nanoneedle-based system and the micropipette-based injection
system is that in thenanoneedle-based system a sub-100-nm-diameter
nanowireisused(comparedtothemicrometer-sizedmicropipetteused
in the injection system) to penetrate the cell membrane, which intro-
ducesminimaldamagetothecellmembraneandminimaldisruptionto
the interior environment of a cell, and the materialstobedelivered into
the cell are carried by the nanoneedle surface and released through a
predesigned surface chemistry [27,30,42–44] and not through a
pressure-driven injection flow.
Such a configuration also allows the direct visual monitoring of the
whole nanoneedle-based delivery process (Fig 1A) and requires no
additional setup beyond what a biological science laboratory typical
has. The drawback is that its operation is limited by the resolution of
the optical microscope; thus, only nanoneedles with relatively large
diameter and length (diameter larger than ∼30 nm and length larger
than ∼3 μm) can be visually monitored and thus used. Other
configurations have used a nanoneedle mounted on an atomic force
microscope (AFM) probe and manipulated by an AFM. The advantage
of such an AFM-based nanoneedle system is that the force–
displacement dependence behavior when the nanoneedle approaches
towards and breaks through the cell membrane, and advances into
the cell interior can be quantitatively monitored with very high
resolution (Figs. 1B and C) [26–28]. However, the limitation is that the
direct visualization of the nanoneedle is difficult as the AFM cantilever
blocks the direct view of the nanoneedle, and the nanoneedle motion
is restricted along the vertical direction. Some AFM-based systems
have also been integrated with a confocal microscope; this allows the
three-dimensionalimagingofthenanoneedleandthetargetcell (Figs.
1B and C) [26,43]. However, because of the long acquisition time
needed in confocal imaging, real-time monitoring of the nanoneedle
operation and the dynamic cellular processes is difficult [45].
Overall, an ideal nanoneedle-based delivery system that can
control the nanoneedle at the nanoscale resolution, directly visualize
the nanoneedle and the target cell, and monitor the force exerted on
the nanoneedle would be desirable for the wider use of nanoneedles
for biological studies in living cells [45,46].
In the following, we discuss the fabrication and functionalization
of a typical nanoneedle for the intracellular delivery application.
3. Fabrication of nanoneedles for intracellular delivery
The most critical component in the nanoneedle-based system is
the nanoneedle,which is often attached to a macroscopic structure for
the ease of manual handling. For the intracellular delivery purpose,
the nanoneedle, in general, needs to have a needle-like structure
with nanoscale diameter (less than ∼100 nm) and microscale length
(∼1 to 20 μm, long enough to reach the target area inside a cell), be
mechanically rigid to survive the operation in an aqueous media and
to penetrate through the cell membrane [25,28], and have a surface
that can be chemically functionalized to attach the cargo on its surface
or to convey other functionalities to the nanoneedle [28].
Such a nanoneedle can be typically made by the following two
methods. First, chemically synthesized one-dimensional nanostruc-
tures, such as nanotubes and nanowires, can be directly used as
nanoneedles [27,28,30]. For example, chemically synthesized nano-
tubes (carbon nanotubes or boron nitride nanotubes) have ideal
properties as nanoneedles for intracellular delivery: they have the
needle-like structure with nanoscale diameter (∼1 to 100 nm) and
microscale length(∼1 to 100 μm)[47], large Young'smodulus (∼1 TPa)
and high tensile strength [48–51], while in the meantime, are resilient
[48,49,52,53]. There are well-developed methods to synthesize such
one-dimensional nanostructures with controlled sizes and shapes and
theyaremostlycommerciallyavailable.However,theprecisealignment
and stable assembly of such nanostructures into useful individual
nanoneedles are still challenging [25]. Reported methods for the
assembly of the nanostructures into needle-like structures include
direct attachment of nanotubes by using a manipulator [27,28,30,52–
55], catalyst patterning and direct growth of nanotubes by chemical
vapordeposition[56–58],alignmentandassemblyusingdielectrophor-
esisormagneticfields[25,59–66],andtransplantingofsinglenanotubes
encapsulatedinpolymerblocks[67,68].However,thesemethodsdonot
reproducibly produce nanoneedles in large quantity or produce high-
aspect ratio “water-stable” nanoneedles (that can survive the meniscus
forces involved in the intracellular delivery operation) [25,28,54].
Second, a nanoneedle can be fabricated by nanofabrication, such as
focusedionbeam(FIB)machining[26]anddirect-writenanofabrication
techniques [69–71]. For example, Nakamura et al. have fabricated Si
nanoneedlesbysharpeningSiAFMtipswithFIB[26,42–44,72–76].They
fabricated Si nanoneedles with diameters of ∼200 to 800 nm and
lengths of ∼5 to 10 μm and demonstrated the capability of these Si
nanoneedles to penetrate through both the cellular and nuclear
membranes of living cells. However, in general, nanofabrication
methods produce nanoneedles with relatively large diameters (in
most cases, larger than 100 nm) and short lengths.
4. Functionalization of nanoneedles for intracellular delivery
Several research groups have developed the nanoneedle-based
delivery system that uses the outer surface of the nanoneedle for
carryingthecargo forintracellulardelivery(Fig. 2) [27,30,42–44]. This
requires that the cargo is conjugated on the surface of the nanoneedle
and is able to be released from the surface of the nanoneedle once
transferred inside cells. There are various surface chemistry and
bioconjugation methods to functionalize the surface of the nanonee-
dle and conjugate the cargo on it. For example, the surface of the
nanoneedle can be directly functionalized (Fig. 2A) [27,43,66] or can
be first coated with other materials (e.g., gold) and then functiona-
lized (Fig. 2B) [28,30].
In the case of a carbon nanotube-based nanoneedle, it can be
functionalizedbyeithercovalentmethodsornoncovalentmethods.The
covalent methods use chemical reactions to chemically bond functional
groups directly on the surface of the CNT (e.g., carboxyl groups by
oxidation) [20,21,66,77]. The noncovalent methods use hydrophobic
and π–π interactions [20,21,27,77] to attach functional groups on the
CNT surface. For example, Chen et al. [27] used a linker molecule that
contains a pyrene moiety and a biotin moiety to functionalize the CNT
nanoneedle with nanoparticles: the pyrene moiety binds to the CNT
surfacethroughπ–πstackingandthestreptavidin-coatednanoparticles
are attached to the biotin moiety (Fig. 2A).
A Si nanowire-based nanoneedle can be functionalized by forming
self-assembly monolayers (SAMs) of alkylsilanes on the Si surface
through the silane coupling reaction. For instance, Nakamura et al.
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[42–44,73,75] used (3-aminopropyl)triethoxysilane and (3-mercap-
topropyl)trimethoxysilane to functionalize the surface of Si nano-
needles with SAMs with amine and thiol terminal groups and used
these functional groups to conjugate proteins, DNA, and polymers.
A more general approach is to coat the nanoneedle with a common
material, such as gold, and subsequently functionalize the coated
nanoneedle [28,30]. This approach is nonspecific to the type of
nanoneedles; thus, a “standard” surface chemistry method can be
used for various nanoneedles rather than synthesizing linker
molecules for a specific type of nanoneedles. The surface coating
also increases the mechanical integrity of the nanoneedle struc-
ture [28,30,78]. A well-studied method is to coat the nanoneedle with
a thin layer of gold (∼1 to 20 nm in thickness) and form a SAM
through the chemisorption of thiols on gold surfaces [28,30]. For
example, Yum et al. [30] developed a stepwise procedure to
functionalize gold-coated nanotube nanoneedles: they first formed
an amine-terminated SAM on the gold-coated nanoneedle, then
conjugated a linker molecule containing a disulfide bond within its
spacer and a biotin moiety, and finally attached streptavidin-coated
nanoparticles on the biotinylated nanoneedle by the binding of
streptavidin and biotin (Fig. 2B). Vakarelski et al. [28] also conjugated
gold nanoparticles on gold coated CNT nanoneedles by forming an
amine-terminated SAM and subsequently attaching gold nanoparti-
cles by electrostatic interactions.
Fig. 1. Nanoneedle manipulation with a micromanipulator and an AFM system. (A) Optical microscope images of a nanoneedle functionalized with quantum dots when the
nanoneedle was manipulated by a micromanipulator to penetrate into the cytoplasm of a living HeLa cell. The whole process was monitored in situ in the bright field (left), the
fluorescence (middle), or the combined bright field and fluorescence (right) image mode. The quantum dots attached on the nanoneedle are shown in red (middle) and bright white
(right). The unfocused dark shade on the right side is the tip of the macroscopic needle on which the nanoneedle is attached. (B) Cross-sectional laser-scanning confocal microscope
images of a living human mesenchymal stem cell (MSC) expressing red-fluorescent protein (DsRed2-NES) (top) and a Si nanoneedle conjugated with fluorescein isothiocyanate
(FITC) with green emission when the nanoneedle was manipulated by an AFM system to penetrate into the nucleus of the MSC cell (middle), and a force–distance curve obtained
from the AFM during the nanoneedle operation (bottom). (C) The same observation for a human embryonic kidney cell (HEK293). The nanoneedle contacted the cell membrane
(point I), began to feel the repulsive force as it indented the cell (point II), penetrated though the cell membrane (point III), and contacted the substrate (point IV).
(A) From Ref. [30] (Copyright 2009 American Chemical Society), (B) and (C) reproduced with permission from Ref. [43] (Copyright 2008 Elsevier).
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Comparing to the ready availability of numerous surface functio-
nalization methods for conjugating various materials on the nano-
needle, a more challenging task is to design a conjugation strategy
that can allow the controlled release of the conjugated materials from
the nanoneedle once transferred inside cells (Fig. 3). A simple
approach is to rely on the passive desorption of the nonchemically
attached cargo from the nanoneedle. For example, Nakamura et al.
[42–44] attached DNA on the functionalized Si nanoneedle (∼200 nm
in diameter) through electrostatic interactions and demonstrated the
release of the DNA from the nanoneedle through the passive
desorption of DNA inside cells (Fig 3A). They demonstrated the
delivery of plasmid DNA into the nucleus of single human mesen-
chymal stem cells (MSCs), human embryonic kidney cells (HEK293),
and breast cancer cells (MCF-7), using an AFM-based nanoneedle
system. In particular, the direct delivery of green fluorescent protein
(GFP) plasmid DNA into the nucleus of single target cells not only
selectively expressed GFP in the single target cells but also expressed
GFP at a high successful rate (∼70% for primary cultured human
MSCs), compared favorably with other nonviral gene delivery
methods (lipofection ∼50% and microinjection ∼10% for human
MSCs) (Fig 4A) [43]. However, because it is nonspecifically adsorbed
on the nanoneedle, the cargo (i.e., DNA in the study) is unselectively
desorbed from the nanoneedle both in the media and inside the target
cell in this approach. Thus, the delivery process must be completed
within several minutes after inserting the nanoneedle into the cell
media (before the complete desorption of cargo from the nanoneedle
into the media).
A more sophisticated method to release cargo specifically inside
cells is to use the reductive cleavage of the disulfide bond in the
reducing environment of the interior of cells; cells have the regulatory
mechanism that maintains the redox equilibrium of the intracellular
environment, wherethe disulfide bond is reductivelycleavedintotwo
thiol groups (Fig. 3B) [27,30]. Using this strategy, Chen et al. [27] and
Yum et al. [30] demonstrated the delivery of a discrete, small number
of protein-coated fluorescent quantum dots into living HeLa cells:
they attached the quantum dots on the nanoneedle through a linker
molecule containing a disulfide bond (Fig. 2B) and demonstrated the
release of the quantum dots inside cells by incubating the nanoneedle
inside cells for ∼15 to 30 minutes (a time required for the reduction of
the disulfide bond) (Fig. 3B). Furthermore, Yum et al. [30] demon-
strated the selective delivery of well-dispersed single quantum dots
into the cytoplasm and nucleus and the tracking of the delivered
quantum dots inside the cells (Figs. 4 and 5).
5. Features of the nanoneedle-based intracellular delivery
The intracellular delivery using a cell membrane-penetrating
nanoneedle has several unique features that may allow new strategies
forbiologicalexperimentsinsidelivingcells.Thenanoneedlecandeliver
adiscrete,smallnumberofmoleculesintoatargetareaofasingletarget
cell at desirable times without any apparent damage to the cell. The
general advantages and disadvantages of the nanoneedle-based
delivery method in comparison to other common delivery methods
are summarized in the Table 1 [33].
The nanoneedle can deliver cargo into living cells with spatial and
temporal precision. The ability of the nanoneedle to reach target areas
inside cells allows the direct delivery of cargo into target areas or
compartments inside cells, not readily achievable by conventional
delivery methods. For instance, it is demonstrated that the nanonee-
dle-based method can selectively deliver quantum dots into either the
cytoplasm or the nucleus of living HeLa cells (Figs. 4B and C) [30]. This
capability may potentially allow spatially resolved experiments inside
living cells (e.g., inside the nucleus). Because the cargo is released from
thenanoneedleinsidecells,thespatialresolutionofthedeliveryismostly
determinedbythesizeofthenanoneedleinserted intoacell(b∼100 nm
in diameter and ∼1 to 4 μm in length) and the spatial precision of the
Fig. 2. Surfacefunctionalizationofnanoneedlesandloadingofcargo.(A)SurfacefunctionalizationofaCNTnanoneedlewithstreptavidin-conjugatedquantumdots(QDs)throughalinker
molecule containing a disulfide bond. (B) Surface functionalization of a gold-coated nanoneedle with streptavidin-conjugated quantum dots (QDs) through a stepwise procedure.
(A) Reproduced with permission from Ref. [27] (Copyright 2007 National Academy of Sciences, USA). (B) From Ref. [30] (Copyright 2009 American Chemical Society).
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manipulator (e.g., a nanoscale resolution of ∼1 nm is achievable with a
piezoelectricmanipulator,suchasanAFMsystem).Thedeliverycanalso
bedonewithhightemporalprecision(e.g.,atdesirabletimesthroughout
the cell cycle); the temporal resolution of ∼15 to 30 minutes is
achievable, for example, when the reductive cleavage of the disulfide
bond is used as a release mechanism (Fig. 3B). Currently, we are also
workingonanelectrochemicaldeliveryapproach,inwhichthereleaseof
the attached cargo can be achieved by applying a pulse of a small
electrical potential to the nanoneedle. Such a delivery strategy can
significantly enhance the temporal resolution down to the several
seconds range. The delivery with such a high temporal precision will
allow the manipulation of cellular processes with short time scales, such
as signaling transduction and protein transportation inside of cells.
The nanoneedle can deliver a discrete, small number of molecules
into cells. For example, the nanoneedle-based method can uniquely
deliver well-dispersed single quantum dots into cells [30]; this
capability may potentially allow the use of the delivered quantum
dots for molecular imaging inside living cells (Figs. 4B and C). Despite
their bright and stable fluorescence, ideal for single-molecule
imaging, the lack of methods that can deliver singly dispersed
quantum dots into cells has hindered their use for molecular imaging
inside living cells, one of the most promising applications of quantum
dots [2,6]. The unique capability of the nanoneedle to precisely deliver
only a small number of nanoprobes can minimize the interference of
the delivered nanoprobes (e.g., quantum dots and magnetic nano-
particles) with intended experiments in living cells and the effect of
such nanoprobes on cellular physiology. For instance, the delivery of a
small number of quantum dots significantly reduced the background
signalfromtheout-of-focus quantumdots,enablingthedetection and
tracking of single quantum dots inside living cells even with a simple
epifluorescence microscope (Fig. 5A) [30]. The direct tracking showed
that the quantum dots diffused in the cytoplasm of HeLa cells with
varying diffusion coefficient D of ∼0.1 to 4 μm2/s, indicating the high
heterogeneity of physical properties and the molecular crowding of
the intracellular environment (Figs. 5B and C) [30]. In contrast, a
recent study also showed that endosomal accumulation of quantum
dots, introduced into cells via the endocytic pathways, can have
several effects on cell physiology [79]. In addition, spatially resolved
delivery of one or a traceable number of force probes (e.g., magnetic
nanoparticles) would be desirable for some cellular and molecular
mechanics experiments inside living cells, in which one needs to
know where the force is applied [80].
How the nanoneedle affects cellular function and viability is
important in any living cell experiments. Most studies showed that
the penetration of a nanoneedle into a living cell does not impair the
cell viability or membrane integrity. For example, the cell viability
tests, using the trypan blue exclusion assay, the Calcein AM assay, and
the Annexin V-FITC/propidium iodide assay, or the monitoring of the
cell proliferation with nanoneedles with diameter less than 100 nm
showed that mammalian cells, including HeLa cells, mouse embryonic
Fig. 3. Schematics of nanoneedle-based intracellular delivery. (A) Gene delivery using a DNA-adsorbed Si nanoneedle. The DAN is passively desorbed from the nanoneedle in the
nucleus of the cell. (B) Intracellular delivery though the reductive cleavage of the disulfide bond. The nanoneedle functionalized with cargo via a disulfide bond penetrates the cell
membrane. The cargo is released from the nanoneedle through the reduction of the disulfide bond in the cytosol of the cell. The nanoneedle is then retracted.
(A) Reproduced with permission from Ref. [43] (Copyright 2008 Elsevier). (B) From Ref. [27] (Copyright 2007 National Academy of Sciences, USA).
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