Introducing Micrometer-Sized Artificial Objects into Live Cells: A Method for Cell–Giant Unilamellar Vesicle Electrofusion
Nomura S-iM (2014) Introducing Micrometer-Sized Artificial Objects into Live Cells: A Method for Cell-Giant
Unilamellar Vesicle Electrofusion. PLoS ONE 9(9): e106853. doi:10.1371/journal.pone.0106853
Introducing Micrometer-Sized Artificial Objects into Live Cells: A Method for Cell-Giant Unilamellar Vesicle Electrofusion
Akira C. Saito 0
Toshihiko Ogura 0
Kei Fujiwara 0
Satoshi Murata 0
Shin-ichiro M. Nomura 0
Arum Han, Texas A&M University, United States of America
0 1 Department of Bioengineering and Robotics, Tohoku University, Aoba-ku, Sendai, Japan, 2 Department of Developmental Neurobiology, Institute of Development, Aging and Cancer (IDAC), Tohoku University, Aoba-ku, Sendai, Japan , 3 CREST, JST , Tohoku University , Sendai, Miyagi , Japan , 4 Department of Biosciences and Informatics, Keio University , Hiyoshi, Kohokuku, Yokohama , Japan , 5 JSPS Research Fellow, Japan Society for the Promotion of Science , Chiyoda-ku, Tokyo , Japan
Here, we report a method for introducing large objects of up to a micrometer in diameter into cultured mammalian cells by electrofusion of giant unilamellar vesicles. We prepared GUVs containing various artificial objects using a water-in-oil (w/o) emulsion centrifugation method. GUVs and dispersed HeLa cells were exposed to an alternating current (AC) field to induce a linear cell-GUV alignment, and then a direct current (DC) pulse was applied to facilitate transient electrofusion. With uniformly sized fluorescent beads as size indexes, we successfully and efficiently introduced beads of 1 mm in diameter into living cells along with a plasmid mammalian expression vector. Our electrofusion did not affect cell viability. After the electrofusion, cells proliferated normally until confluence was reached, and the introduced fluorescent beads were inherited during cell division. Analysis by both confocal microscopy and flow cytometry supported these findings. As an alternative approach, we also introduced a designed nanostructure (DNA origami) into live cells. The results we report here represent a milestone for designing artificial symbiosis of functionally active objects (such as micro-machines) in living cells. Moreover, our technique can be used for drug delivery, tissue engineering, and cell manipulation.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its
Supporting Information files.
Funding: This work was supported by Grants-in-Aid for Scientific Research (#24104004, #22220001 for Satoshi Murata and Shin-ichiro M. Nomura, #25610117
for Shin-ichiro M. Nomura, and #23.3718 for Kei Fujiwara), and Core Research for Evolutional Science and Technology for Toshihiko Ogura. The funders had no
role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Direct introduction of functional objects into living cells is a
major topic in biology, medicine, and engineering studies, since
such techniques facilitate manipulation of cells and allows one to
change their functional properties arbitrarily. In order to introduce
various objects into cells, several methods have been developed,
for example, endocytosis and macropinocytosis .
Nonetheless, the sizes of introducible objects are largely limited: up to
several hundred nanometers and a few micrometers in diameter.
In addition, the uptake of objects is dependent on cell type, and
neither endocytosis nor macropinocytosis occur, for example, in
lymphocytes. Even after successful endocytosis, incorporated
objects are transported to the endosomes; they are then eventually
transferred to the lysosome, in which acidic hydrolases degrade the
materials. Hence, these two systems are not particularly suitable
for introduction of functionally active molecules and objects. To
overcome these obstacles, novel delivery systems have been
contrived, such as cationic liposomes and nanomicelles, that are
used for gene transfer; yet, only nucleic acids that are limited to a
few hundred nanometers in size can be introduced . By
employing peptide vectors, comparatively larger materials can be
introduced into cells, although the size limit of peptides and beads
is approximately 50 nm , which is again insufficient for delivery
of objects, such as DNA origami [10,11] and larger functional
On the other hand, several methods have been established that
allow penetration of the cell membrane; these include
microinjection, electroporation, and electrofusion. Cellcell electrofusion
is a traditional technology for generating hybridomas and involves
fusing adjoining cell membranes. Based on this classical technique,
Shirakashi et al. proposed cell and giant unilamellar vesicle (GUV)
fusion, by which GUVs loaded with low-molecular weight
oligosaccharides were fused with Jurkat cells . Nonetheless,
the efficiency of transfer of the GUV contents (trehalose, raffinose,
and KCL in this case) and cell viability were not measured in that
study, and only cellGUV electrofusion geometry was evaluated
microscopically and confirmed theoretically by a
Finite-ElementMethod analysis of the electric field around the fused cells. The
GUVs were prepared by a conventional electroformation
technique, which is quite a limited method for enclosing large-sized
objects into GUVs.
To date, standard methods have been used for preparation of
giant vesicles [13,14], that encapsulate substances; however, few
reports have described the encapsulation of micrometer-sized
substances into giant vesicles at high volume fractions .
Recently, the water-in-oil (w/o) emulsion centrifugation method
has been developed. With this new technique, it is now possible to
prepare GUVs that contain artificial materials larger than 1 mm in
Here we adopt the w/o emulsion centrifugation method to
entrap various large artificial objects (up to 1 mm in diameter) in
GUVs. After cellGUV electrofusion, the objects were transferred
into live cells, which retained high viability, and, more
importantly, underwent several rounds of normal cell division. Based upon
these observations, this method can be used in various
experimental situations, namely, simultaneous transfer of multiple
genes, proteins, and small molecules for generation of induced
pluripotent stem (iPS) cells, and even for creation of artificial cells
that bear molecular robots (e.g., DNA nanostructures and DNA
devices) in the cytosol.
Materials and Methods
Artificial objects for transfer
In general, negatively charged materials do not adhere well to
cell surfaces. To avoid non-specific absorption to the cell surface,
we used negatively charged lipids and materials for this
experiment; i.e., dioleoylphosphatidylglycerol (DOPG),
carboxylated beads, plasmid DNA, and DNA origami. Fluorescent
microbeads (FluoSpheres, carboxylate modified; 0.2, 0.5, 1.0,
and 2.0 mm in diameter, 2 mM surface azide group; lEx/
lEx = 505/515 nm) were purchased from Invitrogen. The initial
bead concentration for forming GUVs was 40 mM. An EGFP and
mCherry expression vector (pEGFP-C1, pmCherry) were
prepared using a NucleoBond Xtra Midi plus kit (Macherey-Nagel
GmbH & Co., Du ren, Germany), according to the manufacturers
instructions. The calculated concentration of the EGFP and
mCherry plasmid entrapped in GUVs was 220 and 230 ng/ml,
DNA origami with a chipped rectangular shape (60690 nm;
Figure S1 in File S1) was designed using caDNAno software
(http://cadnano.org). Table S1 in File S1 shows the complete
sequence of the DNA origami. The assembly of the structure was
checked by electrophoresis and atomic force microscopy (Figure
S2 in File S1). DNA origami was loaded into GUVs at a final
concentration of 3.36 nM.
GUV preparation by the w/o emulsion centrifugation
GUVs were prepared using the water-in-oil (w/o) emulsion
centrifugation method, with modifications .
Dioleoylphosphatidylcholine (DOPC, NOF, Japan), DOPG (NOF, Japan), and
cholesterol (Wako, Japan), at a weight ratio of 18:2:1 (total:
105 mg), were dissolved in 1050 ml chloroform. This solution was
poured into a glass tube (10 mm ), then first dried under argon
gas and subsequently under vacuum, and was then mixed with
500 ml of liquid paraffin (Wako, Japan). The mixture was treated
by ultrasonication at 60uC for 60 min. Artificial objects
(fluorescent microbeads, DNA origami, or plasmid DNA) were mixed
with the inner solution (consisting of 90 mM sucrose, 210 mM
mannitol, 0.1 mM CaCl2, 0.1 mM MgCl2, and target solution),
and 50 ml of the inner solution was then added to the lipid
mixture. Then, the tube was vortexed for 1 min to create a
micrometer-sized W/O emulsion. The emulsion was poured
gently onto the outer solution (consisting of 300 mM mannitol,
0.1 mM CaCl2, 0.1 mM MgCl2). After centrifugation at 18,0006g
for 30 min at 4uC, the emulsion was passed through the w/o
interface saturated with lipids to form a bilayer membrane. To
avoid mixing between oil and water, GUVs were extracted from
the bottom of the tube through a hole made using a syringe needle
(G25, Terumo, Japan). The average diameter of the GUVs was
calculated from microscopic images to be 37613 mm (see Figure
S3 in File S1). The number of beads entrapped in each GUV was
calculated to be in the order of 101104 from fluorescent
microscopic images. To confirm that the efficiency of introduction
of foreign objects is dependent on size, we prepared GUVs by
entrapping several sets of microbeads (0.2, 0.5, 1, and 2 mm). The
efficiency of entrapment of the beads in GUVs was estimated by
flow cytometry. The values were 99.1, 91.6, 81.9, and 67.3% for
beads of 0.2, 0.5, 1, and 2 mm in diameter, respectively. The
microscopic images obtained immediately prior to electrofusion
are shown in Figure S4 in File S1.
HeLa cells (obtained from ATCC, CCL-2) were cultured in
DMEM buffer (Gibco Invitrogen, Grand Island, NY, USA)
supplemented with 10% fetal bovine serum (Biowest, France) and
1% antibioticantimycotic (Gibco Invitrogen, Grand Island, NY,
USA). Cells were seeded onto plastic- or glass-based dishes
(diameter: 35 mm) and maintained in a 5% CO2 incubator (Astek
SCA-80D, Japan) at 37uC for 1, 2, 3, or 5 days.
Flow cytometric analysis
Flow cytometric analysis was performed to measure the quantity
of fluorescent microbeads introduced into the cells by the cell2
GUV electrofusion method. After electrofusion, HeLa cells were
seeded in 35-mm dishes and cultured for two days. The cells were
removed by adding 0.25% trypsinEDTA solution, and then
counted using flow cytometer (Cell Lab Quanta SC MPL,
Beckman Coulter, USA) equipped with a blue laser (488 nm). A
screening gate was set on the electronic cell volume vs. SSC plot,
to allow analysis only of cells of a size with a diameter in the range
of 1116 mm.
Figure 1. Schematic diagram of the cellGUV electrofusion process. Cells and GUVs are exposed to an alternative current (AC) field to induce
cell alignment (chain-of-pearls-like structure), and are then pulsed with direct current (DC) voltage to create breaks in the contact region between the
cell membrane and GUV surfaces. Representative images are shown to demonstrate the appearance of cellGUVs that had been exposed to the AC
field and DC pulse.
Microscopy and image acquisition
For fluorescence observation, cells were fixed with 1%
paraformaldehyde (Wako, Japan) for 30 min, washed with PBS
(Gibco Invitrogen, Grand Island, NY, USA), incubated with the
10-mM cell tracker Red CMTPX (Life Technologies, USA) in
DMSO for 15 min at 25uC. Thereafter, cells were washed with
PBS, and then incubated with 0.7 mg/ml Hoechst 33342
(Invitrogen, USA) for 15 min. Fluorescence images and phase
contrast images were acquired using a highly sensitive color
camera (DP-73, Olympus, Japan) attached to an inverted
fluorescent microscope (IX-71, Olympus, Japan). Cross sectional
images of cells were obtained using a confocal microscope
(FV1200, Olympus, Japan) with a set of lasers (405, 473, and 543 nm).
We first introduced fluorescent microbeads into HeLa cells by
electrofusion with GUVs. After application of an AC field and a
DC pulse, the suspension of cells was cultured. Figure 2A shows
the microscopic images of the HeLa cells with the introduced
0.2mm microbeads. Prior to observation, these cells were thoroughly
washed with PBS at 3 h after cell fusion. The treated cells survived
for at least 5 days and proliferated until confluence was reached.
The number of trials for each experiment was greater than five.
The treated cells were cultured for 3 days after fusion with
GUVs containing the beads. Figure 2B (left) shows a series of
confocal microscope images; in these images, 0.2, 0.5, and 1-mm
microbeads can be observed inside the cells. However, 2-mm
microbeads were not observed in the cells. The Z-stack images for
each size of microbeads introduced into HeLa cells are shown in
Figure S5 in File S1.
Flow cytometric quantification of the microbeads
introduced into cells
The percentage of HeLa cells containing the introduced
microbeads was quantified by flow cytometry. The cells were
cultured for 2 days after electrofusion. Figure 2B (right) shows
histograms of the cells emitting a green fluorescent signal. The
values, shown in the inset, for 0.2, 0.5, 1, and 2-mm microbeads,
and no microbeads, were 73, 50, 40, 0.38, and 0.31% respectively.
These values were defined as the ratio of cells that demonstrated a
fluorescence intensity of more than 10. These data revealed a
threshold between 1 and 2 mm for the size of beads introduced
into live cells by using cell2GUV electrofusion.
Introduction of plasmids and DNA origami into the cell
We also investigated the introduction of an EGFP-encoding
plasmid (pEGFP) and DNA origami into the cells by cellGUV
electrofusion (Figure 3). In Figure 3A, we show phase contrast and
fluorescent microscopy images of HeLa cells into which pEGFP
had been introduced and which had then been cultured for 1 and
5 days prior to observation. The number of EGFP-expressing cells
was counted from the fluorescent microscopic images. The transfer
efficiency of pEGFP was estimated to be approximately 20%. No
EGFP signal was observed for cells that had not been fused with
GUVs or had not been exposed to DC pulses (Figure 3A).
We also introduced fluorescence-labeled DNA origami into
HeLa cells. The origami structure was designed to allocate 282
fluorescent FITC-labels onto an area of 60690 nm2. The cell
image in Figure 3B was obtained by fluorescence microscopy
immediately after the electrofusion treatment; the green
fluorescent spot indicates the position of the FITC-tagged DNA origami.
When we used a bare fluorescently modified oligonucleotide,
without DNA origami, no fluorescent signal was observed in the
live cells. We noted that the fluorescent signal of the introduced
origami disappeared after overnight culturing.
Introduction of multiple artificial objects into the cells
We then confirmed whether it is possible to introduce multiple
artificial objects simultaneously into the live cell using our method.
We prepared GUVs entrapping both the pmCherry (red
fluorescent protein-encoding plasmid) and fluorescent microbeads
of different sizes (0.2, 0.5, and 1 mm in diameter). After
the treated HeLa cells. These images show HeLa cells into which (from the top) no beads, or beads of 0.2 mm, 0.5 mm, 1 mm, and 2 mm diameter
(green) had been introduced. The cytoplasm is shown in red and nuclei in blue, and merged images are shown in the right column. Scale bar =
10 mm. Right: Flow cytometric detection of microbeads introduced into HeLa cells. Single parameter histograms of the cell number versus log
fluorescence intensity are shown. The histograms represent a total of 7,00015,000 cells counted for each measurement.
electrofusion, the treated HeLa cells were then been cultured for 2
days prior to observation. Confocal microscopic images show the
results of cellGUV electrofusion experiments (Figure 4). In these
images, 0.2, 0.5, and 1-mm microbeads were observed inside the
Hela cells, which showed red fluorescence derived from the
mCherry expression plasmid that was introduced into the cells
along with the beads.
In this study, we demonstrated that the cellGUV electrofusion
method can be used to introduce artificial objects of up to at least
1-mm in size into cells, and that the hybrid cells had a viability
similar to that of normal cells.
Shirakashi et al. proposed that application of a high-voltage DC
pulse induces breakdown of the contact zones of cell and GUV
membranes , creating a small passage between them. In their
report, they concluded that an oligosaccharide solution could be
transferred into the cells in this way , although neither the
transfer efficiency nor the cell viability after electrofusion was
demonstrated. Furthermore, solid objects, such as fluorescent
beads and DNA origami, were not used as GUV contents, and
only water-soluble substances (trehalose, raffinose, and KCL) were
investigated. Hence, the possibility of using solid objects remained
unanswered. Here, we showed that large solid materials can be
introduced into cells safely and efficiently using our modification of
From our results, it appears that objects in the GUVs can pass
through the pores formed in the contact zones of cell and GUV
membranes, and then move into the cytosol. Osmotic pressures in
the cells and GUVs are similar (298 vs. 300 mOsm/kg,
respectively); hence, osmotic pressure would not be a force that
drives the movement of the GUV contents, although balance of
the pressures between cells and GUVs could prevent
unpredictable cytosolic flow and maintain high cell viability.
The live cell membrane is lined with a cortex consisting of a
mesh-like protein structure, called the membrane cytoskeleton
. Before entering into the cytosol, moving objects pass
through the pore formed in the cell membrane, and then collide
with the meshwork, which is an approximately 70-nm mesh
cytoskeletal protein network in the case of HeLa cells . Since
our results showed that electrofusion can introduce plastic
microbeads up to 1-mm in diameter, which then moved into the
cytosol, the meshwork could be stretched and dragged open,
transiently, along with expansion of the electrically formed pore.
In this study, plasmid DNA was also transferred into live cells,
and these expressed green fluorescence. We could demonstrate
that the plasmids were delivered into cytosol. This method does
not require potentially toxic chemicals, such as the positively
charged lipids that are required for gene transfection. Although
further optimization of the process will be investigated, the low
transfer efficiency of plasmids (approximately 20%) is attributable
to the low density of both the dispersed cells and the GUVs used
Many types of molecular devices based on DNA
nanotechnology have been designed to be functional within the cellular cytosol
. The fates of artificial DNA nanostructures in cells remain
unclear. To perform its function continuously in live cells, it seems
that the introduced DNA nanostructure must be stabilized and
protected from degradation by chemical modification. With our
cell2GUV electrofusion technique, one can introduce a spherical
object of up to 1 mm in diameter. This size allowance enables
direct introduction of DNA with some protective DNA-binding
compounds, such as biocompatible polycations, and even artificial
chromosomal structures. In our trial, the adopted DNA origami
was too large to pass through the nuclear pore (210 nm in
diameter). However, if the stability of the DNA origami in the cell
is high enough to survive several rounds of cell division, it can be
incorporated into the nucleus and act as an alternative source of
Constructing a cytoskeletal mesh that lines the newly enhanced
membrane region is time-consuming (requiring about 1520 min.)
. Resealing the cell membrane pore caused by the 200 V/mm
electric pulse requires several hundred seconds . Under our
experimental conditions, the membrane surface tension on the
cell-side, which is reinforced by the cytoskeleton, is greater than
that on the GUV-side. It is unlikely that the fused membrane can
maintain this tension difference until the cytoskeleton has been
reconstructed totally. In addition, the duration of pulsing and the
post fusion time (about 10 s) is much less than the time needed for
reconstruction of the cytoskeleton. Thus, we conclude that the
membrane fusion in our experiment seems to be quite a limited
and temporary event. After the electrical treatment, the GUVs
must have been separated from the cell again during the washing
stage. Although the word fusion does not adequately express the
entire phenomenon, contents of the GUV are transferred into live
cells via the electrical treatment. The detailed mechanisms
underlying the GUV content transfer should be investigated from
a cell scientific point of view. For now, we have termed this
method cellGUV electrotransfer.
Our method reported here could contribute to efficient
introduction of artificial structures and materials, such as large
magnetic beads, Yamanaka four factors, in either the form of
DNA or protein, for the production of iPS cells, and chemically
modified beads, into live cells. Moreover, in future, direct
introduction of a systematic molecular device complex , a
type of molecular robot, into the cellular cytosol should be tested.
These bioengineered hybrid cells are likely to be useful for drug
delivery, tissue engineering, and elucidation of cell mechanisms in
File S1 Figure S1: Schematic diagram of the square
DNA origami structure. M13mp18 ssDNA, its complementary
ssDNA sets (called staples), and green fluorescent
59-TGCCAGGATCTACTCATTGC-39) were purchased from Operon Technologies
(Japan) and Takara Bio (Japan), respectively. These DNAs were
mixed (M13:staples:FITC = 4 nM:20 nM:60 nM) and annealed in
a buffer (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM
DTT, pH 7.9, 25uC) for 3.5 h across a temperature range from 95
to 25uC at a rate of -1uC/3 min. Figure S2: Identification of
the DNA origami structure. (A) Electrophoresis analysis. Left,
middle, and right lanes contain the marker, bare plate-like DNA
origami structure, and fluorescently-tagged DNA origami,
respectively. The samples were analyzed by 1% agarose gel
electrophoresis (100V, 1 hour). The DNA origami structure, with or without
fluorescent (FITC) tag, was electrophoresed in a 1% agarose gel
that was exposed to DC 100 V for 1 h. FITC fluorescence was
detected using a ChemiDoc MP system (BioRad, Japan). A band
showing FITC-tagged origami was clearly observed under blue
light. (B) AFM images for the DNA origami. The AFM image was
obtained on an AFM system (Nano Live Vision, RIBM, Tsukuba,
Japan) using a silicon nitride cantilever (resonant frequency =
1.5 MHz, spring constant = 0.1 Nm-1, EBDTip radius = 24 nm,
Olympus BL-AC10DS-A2). The sample (2 mL) was adsorbed onto
a freshly cleaved mica plate for 5 min at room temperature, and
then washed twice with the same buffer solution. Scanning was
performed in the same buffer solution using a tapping mode. The
final concentration of the DNA (M13mp18) was 100 nM dissolved
in buffer (Tris/Tris-HCl 20 mM, Mg2+ 12.5 mM (pH 7.4)). Scale
bar = 100 nm. Figure S3: Size distribution of the formed
GUVs. To confirm the size distribution of the GUVs, we
prepared GUVs with the inner buffer of 40 mM Lucifer yellow
(SIGMA, Japan), 300 mM mannitol, 0.1 mM CaCl2, 0.1 mM
MgCl2. (A) Representative image of Lucifer yellow contained
GUVs. Scale bar = 50 mm. The fluorescent microscopic images of
GUVs obtained immediately after water-in-oil emulsion transfer
method. (B) The binarized image from (A). The size distribution
was calculated from the images by using Image J software (NIH).
(C) Size distribution of the Lucifer yellow contained GUVs
diameter (n = 471). The average diameter is 37613 mm, as mean
6 standard deviation. Figure S4: Representative
Pearlchain form of GUVs and HeLa cells suspension. Each
vesicle contained fluorescent beads of a particular size, e.g., 200
nm, 500 nm, 1 mm, and 2 mm in diameter. The coupled
micrographs of phase contrast and fluorescence image show the
same position of the sample immediately before the electrofusion.
Scale bar = 50 mm. Figure S5: Confocal microscopic
images of HeLa cells containing the introduced
fluorescent beads. (A) Microscopic images showing the cross-section of
the treated HeLa cells. These images show HeLa cells into which
(from the top) no beads, or beads of 0.2 mm, 0.5 mm, 1 mm, or
2 mm diameter (green) had been introduced. Z-stack acquisitions
were performed to detect the position of the beads from the dorsal
(left column) to the ventral (right column) cross-section of the cell.
Cells were stained with cell tracker and Hoechst to reveal the
cytoplasm (red) and nucleus (blue). Scale bar = 10 mm. (B) 3-D
reconstruction of images of HeLa cells containing 1-mm
microbeads, obtained from confocal microscopic image stacks of a
birdseye view (upper left), side view (lower left), and top view (right). Sky
blue, white line, and green dotted line indicate the bottom of the
cells, slice position of the top view, and side view, respectively.
Table S1: Sequences for the plate-like DNA origami
The authors express their gratitude to Mr. Ban Okabayashi (Tohoku
University) for his contribution to the DNA origami work.
Conceived and designed the experiments: ACS SMN TO. Performed the
experiments: ACS SMN TO. Analyzed the data: ACS SMN TO.
Contributed reagents/materials/analysis tools: TO KF SM SMN.
Contributed to the writing of the manuscript: ACS TO KF SM SMN.
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