Utilization of photoinduced charge-separated state of donor-acceptor-linked molecules for regulation of cell membrane potential and ion transport

Article abstract of DOI:10.1021/ja3007275

The control of ion transport across cell membranes by light is an attractive strategy that allows targeted, fast control of precisely defined events in the biological membrane. Here we report a novel general strategy for the control of membrane potential

Full text of DOI:10.1021/ja3007275

Communication
pubs.acs.org/JACS
Utilization of Photoinduced Charge-Separated State of Donor−
Acceptor-Linked Molecules for Regulation of Cell Membrane
Potential and Ion Transport
Tomohiro Numata,†,# Tatsuya Murakami,‡,# Fumiaki Kawashima,§ Nobuhiro Morone,‡ John E. Heuser,‡
Yuta Takano,‡ Kei Ohkubo,∥ Shunichi Fukuzumi,∥,⊥ Yasuo Mori,*,† and Hiroshi Imahori*,‡,§
†Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Nishikyo-ku,
Kyoto 615-8510, Japan
‡Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
§Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Nishikyo-ku, Kyoto 615-8510, Japan
∥Department of Material and Life Science, Graduate School of Engineering, Osaka University, and ALCA, Japan Science and
Technology Agency (JST), Suita, Osaka 565-0871, Japan
⊥Department of Bioinspired Science, Ewha Womans University, Seoul 120-750, Korea
S
* Supporting Information
engineered to allow them to respond directly to light or to
ABSTRACT: The control of ion transport across cell
membranes by light is an attractive strategy that allows
targeted, fast control of precisely defined events in the
biological membrane. Here we report a novel general
strategy for the control of membrane potential and ion
transport by using charge-separation molecules and light.
Delivery of charge-separation molecules to the plasma
membrane of PC12 cells by a membranous nanocarrier
and subsequent light irradiation led to depolarization of
the membrane potential as well as inhibition of the
potassium ion flow across the membrane. Photoregulation
of the cell membrane potential and ion transport by using
charge-separation molecules is highly promising for
control of cell functions.
accommodate chemical modification with light-sensitive
molecules. Thus, examples of the photoregulation of ion
transport across biological membranes are still limited.
We focused on the nanoscale electric field of a photoinduced
charge-separated (CS) state of a donor−acceptor (D−A)-
linked molecule. If this linked molecule could be arranged on or
in the intact biological membrane and then the CS state could
be generated by photoinduced electron transfer (ET), the
extremely large electric field (∼106 V cm−1)11 of the
photoinduced CS state would affect electrophysiological
activities in cell membranes, such as gating of voltage-gated
ion channels and switching of ion transport across the
membrane. Here we report the first example of photoinhibition
of ion transport and subsequent depolarization in intact cells by
using charge-separation molecules.
In this study, we chose ferrocene (Fc)−porphyrin (P)−C60
linked triads as charge-separation molecules (Figure 1) because
photoexcitation results in photoinduced ET from the porphyrin
excited singlet state (1P*) to the C60 followed by a second ET
from the ferrocene to the porphyrin radical cation (P•+),
yielding Fc+−P−C60•−, which has a long lifetime (τ) of ∼0.01
ms and a high charge-separation efficiency of 0.25−0.99.12,13
To increase the affinity of the hydrophobic Fc−P−C60
molecule for the phospholipid bilayer membrane, a hydrophilic
cationic moiety (i.e., a tetramethylammonium group) was
tethered to the ferrocene moiety of the Fc−P−C60 molecule.10
The triads Fc−H2P−C60 and Fc−ZnP−C60 were synthesized
according to reported procedures [Figures S1 and S2 in the
Supporting Information (SI)]. Porphyrin reference compounds
with the cationic moiety but without the Fc and C60 moieties
(H2P-ref, ZnP-ref) were also prepared (Figures S3 and S4).
Synthetic procedures and characterization and optical proper-
ties are provided in the SI. Both the triads and porphyrin
reference compounds were prone to precipitate from aqueous
he integration of chemistry and biology has paved the way
T
for new interdisciplinary science and technology that
enable the control and manipulation of functions of biological
systems. This methodology has been extended to explore the
fusion between tailored materials and biological systems as well
as protein machines and artificial systems.1,2 Representative
examples involve the control of ion channels by photochemical
switching3 and the use of biomolecular motors and pumps
interfaced with synthetic systems.4,5
One of the challenges in controlling biological systems at the
molecular level is transporting functional molecules across
barriers such as biological membranes, as such control could
result in fascinating applications including sensing, detection,
and drug delivery.1,2 In particular, the control of ion transport
across cell membranes by light is fascinating because of its
targeted, fast control of precisely defined events in the
biological membrane. For instance, the optical control of
neuronal activity6,7 encompasses various strategies, including
photorelease of caged neurotransmitters,8 engineering of light-
gated receptors and channels,9 and naturally light-sensitive ion
channels.10 In these strategies, cells must be genetically
Received: January 22, 2012
Published: March 26, 2012
© 2012 American Chemical Society
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Figure 2. Cell membrane localization of Fc−H2P−C60. (a) Confocal
microscopy images of PC12 cells treated with H2P-ref-loaded cpHDL
and WGA−Alexa Fluor 488 conjugate. (b) Negatively stained electron
microscopy image of Fc−H2P−C60-loaded cpHDL. (c) Mica-flake
electron microscopy image of Fc−H2P−C60-loaded cpHDL. (d)
Freeze-fracture electron microscopy image of the outer surface of the
PC12 cell membrane after the treatment with Fc−H2P−C60-loaded
cpHDL. Clusters of discs similar to those shown in (b) are highlighted
in gray.
Figure 1. Molecular structures of charge-separation molecules and
reference compounds used in this study.
solution, which prevented the delivery of detectable amounts of
them to the cell membrane. Thus, cell-penetrating high-density
lipoprotein14 (cpHDL) or a membrane fusogenic liposome15
was used as a nanocarrier for delivery to the cell membrane.
HDL is believed to adopt a discoidal phospholipid bilayer
circumscribed by apoA-I proteins and can incorporate hydro-
phobic molecules in the phospholipid bilayer. CpHDL, which
was recently developed by our group as an intracellular drug
delivery carrier, was prepared by mixing palmitoyloleoylphos-
phatidylcholine (POPC) and a human apoA-I mutant fused to
a TAT sequence (YGRKKRRQRRR) in a 300:1 molar ratio.14
The membrane fusogenic liposome was prepared from 1-
octadecanoyl-2-((9Z)-octadecenoyl)-sn-glycero-3-phosphocho-
line (SOPC) and 1,2-di-((9Z)-octadecenoyl)-sn-glycero-3-
phosphoethanolamine (DOPE) in a 3:1 ratio.15
First, Fc−H2P−C60, Fc−ZnP−C60, H2P-ref, or ZnP-ref was
incorporated into cpHDL by simply mixing them for 1 h at 37
°C. The UV−vis absorption spectra revealed a characteristic
absorption arising from the porphyrin moiety, demonstrating
the incorporation of the dyes into cpHDL (Figures S5 and S6).
It is noteworthy that the Soret bands of the Fc−H2P−C60- and
Fc−ZnP−C60-loaded cpHDL in saline were red-shifted by ca.
10 nm relative to those of the triads in dimethyl sulfoxide
(DMSO), whereas the Soret bands of H2P-ref- and ZnP-ref-
loaded cpHDL revealed slight red and blue shifts, respectively,
relative to those of the porphyrin references in DMSO (Figure
S5). During the incorporation into cpHDL, the degree of
decrease in the fluorescence of the triads was much larger for
ZnP-ref than H2P-ref, indicating intense aggregation of ZnP-ref
compared with H2P-ref in cpHDL (Figure S6). Taking into
account the solubility of the triads and the references in
DMSO, these results show that the degree of aggregation
increases in the order H2P-ref < ZnP-ref < Fc−H2P−C60 < Fc−
ZnP−C60.
obtained for the PC12 cells treated with the ZnP-ref-loaded
cpHDL, but it was weaker because of the self-quenching of
ZnP-ref arising from the aggregation described above16 (Figure
S8c). In contrast, no fluorescence was observed for the PC12
cells treated with the Fc−H2P−C60 or Fc−ZnP−C60-loaded
cpHDL as a result of the intense quenching by the C60 via
ET12,13 as well as the aggregation quenching (Figures S7b and
S8b).16 The amount of the dye incorporated into PC12 cells,
which was spectrophotometrically determined after extraction
of the dye from the cells, was virtually the same for all the
systems [(2−3) × 107 molecules/cell]. These data indicate that
the triad molecules were similarly delivered to the cell
membrane.
To examine the structure of the triad-loaded cpHDL in the
cell membrane, transmission electron microscopy (TEM)
studies of the PC12 cells treated with the triad-loaded
cpHDL were performed. As shown in Figure 2b,c, Fc−H2P−
C60-loaded cpHDL was found to have a discoidal shape with a
thickness of ∼5 nm, analogous to that of empty HDLs, and
some of the discs were stacked to create lamellar structures.
The disks ranged from 60 to 80 nm in diameter, which was
much larger than that of physiologically relevant discoidal
HDLs (∼10 nm). Our previous report showed that discoidal
HDL can be enlarged more than 10-fold by loading with
hydrophobic molecules when prepared with an increased
amount of POPC (250:1 molar ratio of POPC to apoA-I).17
As the cpHDL used in this study was prepared at a 300:1 molar
ratio, this enlargement could be explained by Fc−H2P−C60
loading.
Next, freeze-fracture electron microscopy was applied to
visualize Fc−H2P−C60-loaded cpHDL in the cell membrane.
This technique does not need chemical fixation of cells, which
affects the cellular location of TAT peptide conjugates. Figure
2d shows a fractured surface in the vicinity of the outer cell
membrane of the PC12 cells treated with Fc−H2P−C60-loaded
cpHDL. The 60−80 nm discs were observed as planar clusters
located on the outer cell membrane. This clustering is
consistent with the observation of the dotted fluorescence of
Next, PC12 cells were treated with the dye-loaded cpHDL
for 5 min at 37 °C. Confocal microscopy analysis of the PC12
cells treated with the H2P-ref-loaded cpHDL revealed that the
fluorescence from the porphyrin moiety was colocalized with
that from wheat germ agglutinin (WGA)−Alexa Fluor 488
conjugate that recognizes cell-surface glycans, showing the
localization of H2P-ref in the vicinity of the cell membrane
(Figure 2a and Figure S7c). A similar fluorescence image was
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H2P-ref in Figure 2a as well as the intense aggregation behavior
of Fc−H2P−C60. Thus, it could be concluded that Fc−H2P−
C
60 was delivered to the surface of the outer cell membrane by
cpHDL. On the other hand, we did not obtain images
indicating incorporation of Fc−H2P−C60 inside the cell
membrane.
Photoinitiated changes in the membrane potential at 25 °C
were examined using patch clamp technique. After 3 min of
treatment of PC12 cells with the dye-loaded cpHDL, visible-
light illumination (400−450 nm) was started under the current
clamp. When the PC12 cells were treated with the Fc−H2P−
C60-loaded cpHDL, depolarization of the membrane potential
occurred gradually with time, reaching a constant membrane
potential (depolarization = 13 mV) in 1 min (Figure 3a). The
Figure 4. Photoinduced change in membrane current of PC12 cells
containing Fc−H2P−C60. Representative traces of time-dependent
changes in the membrane current (a, c, e) and I−V curve (b, d, f)
under illumination (400−450 nm, input power 5 mW cm−2) are
shown for PC12 cells treated with Fc−H2P−C60-loaded cpHDL. The
black and red curves in (b), (d), and (f) were collected at the time
points indicated by x and y, respectively, in (a), (c), and (e). The
insets in (b), (d), and (f) show magnified views around the V-axis
intercept.
Figure 3. Photoinduced depolarization of PC12 cells containing Fc−
H2P−C60. Representative traces of time-dependent changes in the
membrane potential under illumination (400−450 nm, input power 5
mW cm−2) are shown for PC12 cells treated for 3 min with (a) Fc−
H2P−C60-loaded cpHDL, (b) H2P-ref-loaded cpHDL, or (c) medium
only and then washed with phosphate-buffered saline.
degree of depolarization did not alter in PC12 cells treated with
a larger amount of Fc−H2P−C60-loaded cpHDL. On the other
hand, under the illumination neither PC12 cells themselves nor
the cells treated with cpHDL loaded with H2P-ref, which was
unable to undergo charge separation, showed change in the
membrane potential (Figure 3b,c). Therefore, the photo-
induced changes in membrane potential observed here clearly
occurred through the CS state of Fc−H2P−C60 under
illumination (Figures S9 and S10).18
Like cpHDL, the well-established SOPC−DOPE fusogenic
liposome,15 the diameter of which was determined to be 60 nm
with a second population of 200 nm (Figure S11), delivered
Fc−H2P−C60 to the PC12 cell membrane (Figure S7d). In fact,
it led the analogous photoinduced depolarization (Figure
S12a). These results support the delivery of Fc−H2P−C60 to
the cell membrane by cpHDL.
Next, photoinitiated changes in membrane current were
evaluated using the voltage clamp method. Under illumination
(Figure 4a, bar) the membrane current of PC12 cells treated
with the Fc−H2P−C60-loaded cpHDL decreased gradually to
reach a plateau (Figure 4a). The current−voltage profile
(Figure 4b, x) and the reversal potential of −70 mV19 (Figure
4b inset, x) are typical characteristics of voltage-gated
potassium channels.20 Under the illumination, the reversal
potential was significantly increased (Figure 4b inset, y),
suggesting inhibition of potassium ion transport. In addition,
this effect of illuminated Fc−H2P−C60 was ablated by
cotreatment with a potassium channel inhibitor, tetraethylam-
monium bromide (TEA) (Figure S13). Given no rectification
under the conditions of y in Figure 4b, the depolarization
observed in Figure 3a may result from the deactivation of the
voltage-gated potassium channels.
Some types of porphyrins have been clinically used for
photodynamic therapy, in which singlet oxygen (1O2) is
generated via energy transfer from the porphyrin excited triplet
state to O2 and attacks cancer cells.21 In the process of cell
killing, 1O2 produces oxidative cell membrane damage followed
by the depolarization of the membrane potential.22 Thus, the
possible involvement of 1O2 in the photoinduced depolarization
observed here was assessed using Singlet Oxygen Sensor Green
Reagent (SOSG) (Invitrogen)23 for Fc−H2P−C60 and H2P-ref
in DMSO (Figure S14). After the light illumination at 400−450
nm, Fc−H2P−C60 did not enhance the SOSG fluorescence,
whereas H2P-ref enhanced the SOSG fluorescence significantly,
1
implying that the O2 generation efficiency of H2P-ref is much
higher than that of Fc−H2P−C60. In agreement with these data,
phototoxicity of Fc−H2P−C60 delivered to the PC12 cell
membrane was not detected after 4.5 min illumination, whereas
all the cells loaded with H2P-ref were propidium iodide (PI)-
positive (Figure S15), which suggests that the cell membrane
was damaged heavily enough to allow PI influx across it. This
high phototoxicity of H2P-ref would be due to its photo-
dynamic effect. Taking into account the fact that the
depolarization was observed only in the treatment with the
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1
Notes
Fc−H2P−C60-loaded cpHDL, we could conclude that O2-
induced membrane damage is not involved in the depolariza-
tion observed in this study.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
Similar depolarization behavior was noted for the PC12 cells
treated with the Fc−ZnP−C60-loaded cpHDL (Figure S16),
but the degree of the depolarization (9 mV) was smaller than
for Fc−H2P−C60-loaded cpHDL. This result appears to
contradict the consideration that the depolarization occurred
through the CS state of Fc−H2P−C60 under illumination, as
the charge-separation efficiency of Fc−ZnP−C60 (0.99) is
higher than that of Fc−H2P−C60 (0.25) in benzonitrile
solution.12,13 One explanation for this discrepancy is that the
charge-separation efficiency is largely attenuated by aggregation
of the charge-separation molecules. As described above, Fc−
ZnP−C60 has an increased tendency to aggregate in cpHDL
and DMSO in comparison with Fc−H2P−C60. Thus, the
intense aggregation of Fc−ZnP−C60 on the membrane surface
■
This work was supported by the WPI Initiative of MEXT,
Japan, and in part by a Health Labor Sciences Research Grant
(nano-001 to N.M.) and by Innovative Areas, MEXT (N.M.),
and NRF/MEST of Korea through WCU(R31-2008-000-
10010-0) and GRL (2010-00353) Programs (S.F.).
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ASSOCIATED CONTENT
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S
* Supporting Information
(24) Conforti, L.; Millhorn, D. E. J. Physiol. 1997, 502, 293.
Experimental details and additional figures. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
mori@sbchem.kyoto-u.ac.jp; imahori@scl.kyoto-u.ac.jp
Author Contributions
#These authors contributed equally.
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