10.1002/anie.202004448
Angewandte Chemie International Edition
COMMUNICATION
irradiation time), and (c) PI influx experiment with S. epidermidis (5 µM PI per
1 OD600 of bacteria).
vesicles, which results in calcein release. An increase in
membrane fluidity was also shown by EPR spectroscopy.
DSAzB opens up a variety of applications in fields such as
biocatalysis or biotechnology, which is suggested by the
isomerization-mediated light-induced propidium iodide influx into
living cells.
DSAzB was compared to COE2-4C to prove that calcein release
is caused by light induced isomerization of the azo-moiety
(Figure 3b). The fluorescence intensity of the control samples
and the samples with COE2-4C decreased due to
photobleaching of calcein. On the other hand, vesicles stained
with DSAzB were permeabilized by light-irradiation and 40% of
the calcein was released by the first (40 min) and 10% by the
second light exposure (90 min). These calcein release
experiments support that model membranes stained with
DSAzB are permeabilized by a light stimulus that leads to the
release of calcein from the inside of the vesicles.
Acknowledgements
This work was supported by the Alexander von Humboldt
Foundation (Feodor Lynen Return Fellowship to D.L.) and the
Institute for Collaborative Biotechnologies (Grant W911NF-09-D-
0001)
Electron paramagnetic resonance (EPR) was used to probe any
disruption of lipid ordering upon intercalation and subsequent
photoisomerization of DSAzB, for a complete description the
reader is directed to the Supporting Information.[28]
DMPG/DMPC liposomes containing 1 mol% of the spin probe
16-SASL were prepared by extrusion, diluted to 20 mM total lipid,
and stained with 0.2 mM DSAzB (1 mol% final). Samples
containing DSAzB showed a lower correlation time relative to
control samples (1.57 ns vs. 1.84 ns), indicating that
intercalation of DSAzB leads to an increase in lipid motion.
Following irradiation of the sample, an immediate drop in
correlation time to 1.50 ns was observed. When irradiation was
stopped, the correlation time increased slightly from 1.48 to
1.51 ns, implying a partial recovery of lipid order. Surprisingly,
the effect of DSAzB isomerization was not fully reversible in the
15 minutes following irradiation (1.51-1.52 ns). The persistence
of lipid disruption indicates that increased bilayer fluidity is not
the only mechanism by which permeability is increased – a
cooperative effect is likely (for a suggested mechanism see SI).
To further highlight possible applications of molecules such as
DSAzB, a permeabilization experiment with living cells (S.
epidermidis) was conducted (Figure 3c). Cells were treated with
2.5 μM DSAzB or COE2-4C for 30 min, propidium iodide (PI)
was then added and the fluorescence at 620 nm was measured.
All three samples (DSAzB, COE2-4C, control) had similar
emission intensities before irradiation which indicates that there
was no significant cell permeabilization by COEs in the dark at
the given concentration of COEs. The cells were irradiated at
405 nm at 10 and 25 minutes after PI addition. Remarkably, only
the DSAzB trace shows an increase of fluorescence intensity
(from 200 to 800 a.u.) due to PI uptake. Following the second
irradiation, a subsequent increase in PI uptake was observed.
These results demonstrate that DSAzB is able to repeatedly
permeabilize living cells by light-actuation – a potential strategy
for use in a wide range of cell-based technologies, such as
whole-cell biocatalysis or DNA uptake of bacteria. Further the
minimum inhibitory concentration (MIC) was tested and for
DSAzB 1 µM was obtained (see Supporting Information for
details).
Keywords: conjugated oligoelectrolytes • photoswitch •
membrane permeabilization • actuators • biocatalysis
[1]
[2]
G. S. Hartley, Nature 1937, 140, 281.
X. Song, C. Geiger, S. Vaday, J. Perlstein, D. Whitten, J. Photochem.
Photobiol. A. 1996, 102, 39.
[3]
a) D. Wang, W. Zhao, Q. Wei, C. Zhao, Y. Zheng, ChemPhotoChem
2018, 2, 403; b) Y. Tu, F. Peng, J. M. Heuvelmans, S. Liu, R. J. M.
Nolte, D. A. Wilson, Angew. Chem. Int. Ed. 2019, 58, 8687; Angew.
Chem. 2019, 131, 8779; c) Y. Sun, J. Ma, F. Zhang, F. Zhu, Y. Mei, L.
Liu, D. Tian, H. Li, Nat. Commun. 2017, 8, 260; d) M. Lohse, K.
Nowosinski, N. L. Traulsen, A. J. Achazi, L. K. S. von Krbek, B. Paulus,
C. A. Schalley, S. Hecht, Chem. Commun. 2015, 51, 9777.
a) M.-H. Li, P. Keller, Soft Matter. 2009, 5, 927; b) Q. Jin, C. Luy, J. Ji,
S. Agarwal, J. Polym. Sci., Part A: Polym. Chem. 2012, 50, 451; c) S.
Shrivastava, H. Matsuoka, Langmuir 2014, 30, 3957-3966.
(a) D. Liu, S. Wang, S. Xu, H. Liu, Langmuir, 2017, 33, 1004; (b) Y.
Kaneda, Adv. Drug Delivery Rev. 2000, 43, 197.
[4]
[5]
[6]
a) C. Pernpeintner, J. A. Frank, P. Urban, C. R. Roeske, S. D. Pritzl, D.
Trauner, T. Lohmüller, Langmuir 2017, 33, 4083;b) P. Urban, S. D.
Pritzl, D. B. Konrad, J. A. Frank, C. Pernpeintner, C. R. Roeske, D.
Trauner, T. Lohmüller, Langmuir 2018, 34, 13368.
[7]
(a) A. Ikeda, T. Hida, T. Nakano, S. Hino, K. Nobusawa, M. Akiyama, K.
Sugikawa, Chem. Lett. 2014, 43, 1551; (b) T. Hamada, Y. T. Sato, K.
Yoshikawa, Langmuir 2005, 21, 7626.
[8]
[9]
for a light-induced micelle to vesicle transition using surfactants see: S.
Shi, T. Yin, X. Tao, W. Shen, RSC Adv. 2015, 5, 75806.
a) A. Diguet, M. Yanagisawa, Y.-J. Liu, E. Brun, S. Adadie, S. Rudiuk,
D. Baigl, J. Am. Chem. Soc. 2012, 134, 4898; b) A. Diguet, N. K. Mani,
M. Geoffroy, M. Sollogoub, D. Baigl, Chem. Eur. J. 2010, 16, 11890.
[10] Y. Suzuki, K. H. Nagai, A. Zinchenko, T. Hamada, Langmuir 2017, 33,
2671.
[11] V. N. Georgiev, A. Grafmüller, D. Bléger, S. Hecht, S. Kunstmann, S.
Barbirz, R. Lipowsky, R. Dimova, Adv. Sci. 2018, 5, 1800432.
[12] Y. Li, X. An, Colloids Surf. B. 2019, 178, 238.
[13] Z.-K. Cui, T. Phoeung, P.-A. Rousseau, G. Rydzek, Q. Zhang, C. G.
Bazuin, M. Lafleur, Langmuir 2014, 30, 10818.
[14] M. L. DiFrancesco, F. Lodola, E. Colombo, L. Maragliano, M. Bramini,
G. M. Paternò, P. Baldelli, M. D. Serra, L. Lunelli, M. Marchioretto, G.
Grasselli, S. Cimò, L. Colella, D. Fazzi, F. Ortica, V. Vurro, C. G.
Eleftheriou, D. Shmal, J. F. Maya-Vetencourt, C. Bertarelli, G. Lanzani,
F. Benfenati, Nat. Nanotechnol. 2020, 10.1038/s41565-019-0632-6.
[15] L. E. Garner, J. Park, S. M. Dyar, A. Chworos, J. J. Sumner, G. C.
Bazan, J. Am. Chem. Soc. 2010, 132, 10042.
In summary, we introduce DSAzB, a new COE containing an
azo-group in the center of the molecule. Irradiation at 405 nm
leads to photoisomerization from trans to cis around the azo
functionality. When embedded into vesicles loaded with calcein,
DSAzB can induce light-activated permeabilization of the
[16] a) P. Gwozdzinska, R. Pawlowska, J. Milczarek, L. E. Garner, A. W.
Thomas, G. C. Bazan, A. Chworos, Chem. Commun. 2014, 50, 14859;
b) J. Milczarek, R. Pawlowska, R. Zurawinski, B. Lukasik, L. E. Garner,
A. Chworos, J. Photochem. Photobiol. B, Biol 2017, 170, 40.
This article is protected by copyright. All rights reserved.