One-pot synthesis of monodispersed silica nanoparticles for diarylethene-based reversible fluorescence photoswitching in living cells

Article abstract of DOI:10.1039/c3cc42852d

A small 29 nm monodispersed silica nanoparticle 1a was synthesized as a diarylethene-based reversible fluorescence photoswitch by copolymerizing silane precursors in one-pot including 3a and 4. Reversible photoswitching of nanoparticle 1a was successfully achieved in living cells to show its potential as a highly distinguishable and safe fluorescence probe for cell tracking.

Full text of DOI:10.1039/c3cc42852d

ChemComm
View Article Online
View Journal | View Issue
COMMUNICATION
One-pot synthesis of monodispersed silica
nanoparticles for diarylethene-based reversible
fluorescence photoswitching in living cells†
Cite this: Chem. Commun., 2013,
49, 7528
Received 17th April 2013,
Accepted 27th June 2013
ab
a
a
Hye-youn Jung,z Suyeon You,z Chaewoon Lee,z Seungkwon Youb and
Yoonkyung Kim*a
DOI: 10.1039/c3cc42852d
www.rsc.org/chemcomm
A small 29 nm monodispersed silica nanoparticle 1a was synthe- surface-modification—inside two different types of living cells by
sized as a diarylethene-based reversible fluorescence photoswitch alternate irradiation with UV and visible light.
by copolymerizing silane precursors in one-pot including 3a and 4.
Several reports have described the preparation of DAE-based
Reversible photoswitching of nanoparticle 1a was successfully reversible fluorescence photoswitch systems which were built on
achieved in living cells to show its potential as a highly distinguish- nanostructures.7a,e,8a,b Despite the many known advantages,9
able and safe fluorescence probe for cell tracking.
practical use of these nanometer-sized photoswitch systems,
particularly for biomedical applications, can be yet limited due
Photochromic compounds (PCs) can undergo reversible photo- to their difficult preparation methods, heterogeneity, and low
switching between two isomeric forms of different absorption switching efficiency, as well as inherent toxicity and poor water-
spectra.1 Such a property has enabled PCs to be used as key solubility. Silica NPs10 are one of the most favored nanomaterials
functional moieties for erasable data storage media,2 photo- in biomedicine due to their relative safety and possibility to tune
chromic eye glasses, and fluorescence switches.3 Fluorescence their size during the preparation with a low polydispersity index.11
imaging4 is the most common technique to visualize cells, pro- Further, recent advances have allowed for the incorporation of
teins, and oligonucleotides in various biological studies. Recently, most of the small organic molecular fragments into the main
to facilitate detection of the fluorescence signal emitted from a silica framework by forming covalent bonds.12 Here, our strategies
particular probe in a complex biological environment, fluorescence to synthesize silica NPs for reversible fluorescence switching in
probes have been made switchable in response to specific stimuli.5 biological systems (Fig. 1a) were the following: (1) functionaliza-
Diarylethene (DAE) derivatives,6 which are known to be one of the tion of the FRET donor (a Cy3 derivative) and FRET acceptor
most efficient PCs for photoswitching, have been employed to (a DAE derivative), individually, with triethoxysilane (TES) groups
build the reversible fluorescence switch for biological imaging.7 (Fig. 1c) for their direct covalent incorporation into the silica
Although DAE7b or its aggregates7d can be fluorescent, it may also network during the formation of NPs; (2) addition of a large excess
serve as a nonfluorescent molecular switch to turn on or off the of DAE (3a) in comparison to Cy3 (4) to achieve effective quench-
fluorescence of a neighboring fluorophore through fluorescence ing of Cy3 fluorescence considering somewhat low photochromic
resonance energy transfer (FRET).7a,e,8 As our continued effort to conversion efficiency of DAE (using a carboxylic acid analogue 10,
develop an efficient and safe reversible fluorescence switch for Scheme S2, ESI†) in water under photoswitching conditions as
1
biological applications, here we report the synthesis of a size- estimated by H NMR (Fig. S2, ESI†); (3) adoption of a synthetic
controlled silica nanoparticle (NP) by copolymerizing all individual method12b that would produce highly monodispersed silica NPs
components in one-pot as silane precursors including those of DAE of preferably 20–25 nm in size (i.e., diameter) for facile cellular
(FRET acceptor) and a fluorophore (FRET donor). Moreover, we uptake and to exhibit homogeneous biological effects; (4) post-
show the efficient cellular uptake and reversible fluorescence modification of the amine functionalities of silica NPs into
photoswitching of these silica NPs—after the biocompatible carboxylate groups for enhanced biocompatibility.
The synthesis of our photoswitchable silica NPs was carried out
a Korea Research Institute of Bioscience and Biotechnology, Daejeon, 305-806,
in a medium containing the oil-in-water emulsion (Scheme S5,
ESI†).12b Specifically, each silane-functionalized precursor of DAE
(3a) and Cy3 (4), together with vinyltriethoxysilane (VTES) as the
bulk component, was added to the microemulsion made from
Tween-80, and the mixture was allowed to equilibrate with stirring
before polymerization. Here the most hydrophobic VTES is
expected to accumulate predominantly in the interior core region
Korea. E-mail: ykim@kribb.re.kr; Tel: +82 42 879 8443
b College of Life Sciences and Biotechnology, Korea University, Seoul, 136-713,
Korea
† Electronic supplementary information (ESI) available: Synthetic schemes,
detailed experimental section, mass/UV-Vis/fluorescence spectra, cytotoxicity
assay results, flow cytometry histograms, etc. See DOI: 10.1039/c3cc42852d
‡ These authors contributed equally to this work.
c
7528 Chem. Commun., 2013, 49, 7528--7530
This journal is The Royal Society of Chemistry 2013

View Article Online
Communication
ChemComm
broad band was exhibited around 550–650 nm for silica NP 1a
(Fig. S6a, ESI†). Indeed, this broad absorption band diminished
upon irradiation with visible light (590 nm, 30 min), suggesting its
correlation with the ring-closed isomer of DAE moieties. Here the
absorption band of the Cy3 derivative that would normally appear
around 550 nm was not explicit possibly due to the relatively low
degree of Cy3 substitution on the silica NP 1a (based on the molar
feed ratio). In contrast, the reversible switching was unambiguously
demonstrated in the fluorescence spectra of silica NP 1a upon
alternate irradiation with UV and visible light (Fig. 2 and Fig. S6b,
ESI†). For both silica NPs 1a and 1b, the Cy3 emission maximum
was observed at 568.5 nm (Fig. S6b and d, ESI†), which was slightly
red-shifted compared with that of a free dye at 563.0 nm (Fig. S5d,
ESI†). The degree of fluorescence quenching of 1a was found to be
54% on average over five cycles of photoswitching in PBS (Table S2,
ESI†). Evidently, no fluorescence quenching was observed upon
irradiation with UV light under the same photoswitching condi-
tions for neither the Cy3 dye nor the Cy3-derivatized silica NP 1b
without DAE (Fig. 2a, Fig. S5d, and S6d, ESI†). Rather, a slight drop
in fluorescence intensity was consistently noticed for Cy3 and 1b
upon irradiation with visible light in repeated experiments, which
appeared to be partially recovered in the subsequent irradiation
with UV light. Interestingly, for all three samples—1a, 1b, and
Fig. 1 (a) Silica NP 1a for reversible fluorescence photoswitching and (b) its non-
switchable control 1b. (c) Structures of silane derivatives 3a, 3b, and 4 incorpo-
rated covalently into the silica network during the formation of 1a or 1b by
copolymerization. (d) TEM images of silica NPs 1a and 1b and their amine-
terminated precursors 2a and 2b, respectively (for structures, see Scheme S5,
ESI†). Scale bars, 50 nm.
of the Tween-80 micelle, and 3a and 4 with extended aromatic Cy3—a minor degree of reduction in fluorescence intensity was
segments may likely as well localize in the interior. Upon initiation noticed, with increasing number of photoswitching cycles, perhaps
of the copolymerization reaction, N0-[3-(trimethoxysilyl)propyl]- due to photobleaching (Fig. 2a).
diethylenetriamine (DETA) was added to impart amine function-
Stem cells have been increasingly attracting attention as
alities to the NPs. It should be noted that unlike other DAE-based therapeutic agents, and indeed the precise tracking of these cells
photoswitchable nanostructures which were prepared by attaching inside the living system is imperative for effective treatment of
the premade 1 : 1 conjugate of FRET donor and FRET acceptor dyes diseases.13 Accordingly, we envisioned that if fluorescently labeled
to their surface,7e,8b our structure was prepared by adding indivi- cells in the biological system can be reversibly switched, they can
dual FRET dyes as reactive silane precursors simultaneously in the be explicitly distinguished among many other neighboring cells
copolymerization step to make the NPs. This strategy not only where detection of the fluorescence itself can be even more
simplifies the preparation method by reducing synthetic steps, but encumbered by the absorbance, light scattering, and autofluores-
also may allow for the free modulation of molar feed ratios, cence of the surrounding media. The photoswitching potential of
particularly between the FRET donor and acceptor dyes to poten- silica NP 1a was investigated in two different types of living cell
tially enhance the efficiency of fluorescence quenching.7a The cultures—human epithelial cervical cancer (HeLa) cells and
amino groups on the resulting silica NP 2a (Scheme S5, ESI†) human adipose-derived stem (hADS) cells (Fig. 3). To determine
could be converted into various functionalities by treating with the appropriate concentration and incubation time for effective
amine-reactive species, and here they were reacted with succinic internalization of silica NP 1a into cells for photoswitching, we
anhydride to enhance the biocompatibility of the NPs. The silica first evaluated the cytotoxicity and the cellular uptake efficiency of
NP 1a with carboxylate end groups indeed had a negatively charged 1a against each cell. Relatively high cell survival rates of 85% and
surface (zeta potential value of ꢀ29 mV, Table S1, ESI†), dispersed
well in aqueous media, and was highly monodispersed with
ca. 29 nm in mean diameter as determined by transmission
electron microscopy (TEM; Fig. 1d, Table S1, ESI†). Additionally,
as a non-switchable control, a fluorescent silica NP 1b with a
carboxylate surface (ca. 23 nm by TEM, zeta potential value of ꢀ31 mV)
was made in a similar manner using methoxy tetra(ethylene
glycol) (mTEG) derivative 3b instead of 3a (Fig. 1b–d).
Fig. 2 Photoswitching experiments using silica NPs (100 mg mLꢀ1) 1a and 1b, and
Next, to assess the feasibility of using our silica NP as a
reversible fluorescence switch under physiological conditions, the Cy3 (1 mM) in PBS (pH 7.4). Samples were irradiated alternately with UV (365 nm,
2 min, 2.60 mW cmꢀ2) and visible (590 nm, 30 min, 90 ꢁ 5 mW) light. (a)
photoswitching experiment was performed using sample solutions
in phosphate buffered saline (PBS). While an absorption band
centered at 586.0 nm (ring-closed isomer, Fig. S5a, ESI†) appeared
Fluorescence intensity at each emission maximum (irradiation at 510 nm). Photo-
switching was repeated for up to five cycles, beginning with UV light irradiation. (b)
Fluorescence spectra of 1a obtained at selected time points by brief interruption of
clearly for the carboxylic acid derivative of DAE 10 (Scheme S2,
irradiation starting from the off-state during the visible light irradiation of the third
ESI†) upon irradiation with UV light (365 nm, 2 min), only a weak photoswitching cycle and (c) the subsequent UV light irradiation of the fourth cycle.
c
This journal is The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 7528--7530 7529

View Article Online
ChemComm
Communication
The carboxylate-terminated silica NP 1a effectively internalized
into living cells—both cancerous (HeLa) and stem (hADS) cells—
to successfully undergo 10 cycles of photoswitching with no
apparent morphological damage. Interestingly, the fluorescence
on–off contrast of 1a determined inside the living cells was
much higher (5.4–6.2) than that obtained using PBS solution
in a cuvette. We envision that our biocompatible silica NP-based
photoswitch may find versatile utility in various applications,
such as tracking therapeutic stem cells in living systems, as a
highly distinguishable and relatively safe fluorescence probe.
This work was supported by the National Research Founda-
tion of Korea, funded by the Ministry of Science, ICT, and
Future Planning (NRF-2012M3A9B2028334 and WISET-2012-
0027) and Ministry of Education (NRF-2010-0008056), and the
KRIBB Research Initiative Program.
Fig. 3 Photoswitching experiments performed on living (a and c) HeLa or (b and d)
hADS cells with internalized silica NP 1a. Cells were irradiated alternately with UV
(365 nm, 2 min) and visible (590 nm, 30 min) light for up to 10 cycles. The average
fluorescence intensity values (mean ꢁ SD) measured in the region of interest (dotted
circles, 36 data points each) after each light application are plotted (c and d).
A sequential irradiation with UV (loss of fluorescence) and visible (fluorescence
recovery) light constitutes one full cycle of photoswitching (integers in the x-axis).
RD-TR-PE: red fluorescence. Scale bars, 20 mm.
Notes and references
1 (a) J. Zhang, Q. Zou and H. Tian, Adv. Mater., 2013, 25, 378;
(b) M.-M. Russew and S. Hecht, Adv. Mater., 2010, 22, 3348;
(c) F. M. Raymo and M. Tomasulo, Chem.–Eur. J., 2006, 12, 3186.
2 S. Kawata and Y. Kawata, Chem. Rev., 2000, 100, 1777.
3 (a) J. Cusido, E. Deniz and F. M. Raymo, Eur. J. Org. Chem., 2009,
2031; (b) T. Fukaminato, T. Sasaki, T. Kawai, N. Tamai and M. Irie,
J. Am. Chem. Soc., 2004, 126, 14843; (c) M. Irie, T. Fukaminato,
T. Sasaki, N. Tamai and T. Kawai, Nature, 2002, 420, 759;
(d) L. Giordano, T. M. Jovin, M. Irie and E. A. Jares-Erijman, J. Am.
Chem. Soc., 2002, 124, 7481.
4 (a) M. S. T. Gonçalves, Chem. Rev., 2009, 109, 190; (b) E. Kim and
S. B. Park, Chem.–Asian J., 2009, 4, 1646.
5 (a) H. Kobayashi and P. L. Choyke, Acc. Chem. Res., 2011, 44, 83;
(b) R. Weissleder, C.-H. Tung, U. Mahmood and A. Bogdanov Jr,
Nat. Biotechnol., 1999, 17, 375.
91% were achieved for HeLa and hADS cells, respectively, when
cells were incubated at 50 mg mLꢀ1 for 24 h (Fig. S7, ESI†). When
analyzed by flow cytometry, the internalization of silica NP 1a into
either cell type became more effective (i.e., higher fluorescence
intensity) with increasing incubation time, and generally the
uptake efficiency was somewhat higher for HeLa cells than for
hADS cells (Fig. S8, ESI†). Also, similar to the previously reported
anionic photoswitch with carboxylate surface groups,7a the inter-
nalized silica NP 1a was mostly found in the cytosolic region and
did not appear to enter the nuclei for either cell type when
visualized under a microscope. Next, the photoswitching experi-
ments were conducted for up to 10 cycles using living cells with
internalized silica NP 1a by irradiating alternately with UV
(365 nm, 2 min) and visible (590 nm, 30 min) light (Fig. S9, ESI†).
When Cy3 fluorescence was measured at the selected cytosolic
area of each cell type upon photoswitching (Fig. 3a and b), the
average quenching efficiencies of 84% and 81% were found for
HeLa and hADS cells, respectively (Table S2, ESI†). These values
corresponded to the fluorescence on–off contrast of 6.2 for HeLa
cells and 5.4 for hADS cells, which were higher than that
measured in a cuvette (on–off contrast of 2.2).7a Here, the
morphologies of HeLa and hADS cells, which underwent 10 cycles
of photoswitching, apparently remained intact. While the wave-
length of light for fluorescence quenching can be shifted to a less
harmful visible range by modifying the DAE structure,6 the effect
of UV light irradiation on cell viability under current photoswitch-
ing conditions was examined (Fig. S10, ESI†). Indeed, survival
rates of HeLa cells with internalized 1a that underwent up to five
cycles of photoswitching were similar (within the error range) to
those of the cells which were not irradiated.
6 M. Irie, Chem. Rev., 2000, 100, 1685.
7 (a) Y. Kim, H.-y. Jung, Y. H. Choe, C. Lee, S.-K. Ko, S. Koun, Y. Choi,
B. H. Chung, B. C. Park, T.-L. Huh, I. Shin and E. Kim, Angew. Chem.,
Int. Ed., 2012, 51, 2878; (b) U. Al-Atar, R. Fernandes, B. Johnsen,
D. Baillie and N. R. Branda, J. Am. Chem. Soc., 2009, 131, 15966;
(c) C. Yun, J. You, J. Kim, J. Huh and E. Kim, J. Photochem.
Photobiol., C, 2009, 10, 111; (d) Y. Zou, T. Yi, S. Xiao, F. Li, C. Li,
X. Gao, J. Wu, M. Yu and C. Huang, J. Am. Chem. Soc., 2008,
130, 15750; (e) N. Soh, K. Yoshida, H. Nakajima, K. Nakano,
T. Imato, T. Fukaminato and M. Irie, Chem. Commun., 2007, 5206.
´
´
8 (a) S. A. Dıaz, G. O. Menendez, M. H. Etchehon, L. Giordano,
T. M. Jovin and E. A. Jares-Erijman, ACS Nano, 2011, 5, 2795;
¨
(b) J. Folling, S. Polyakova, V. Belov, A. van Blaaderen, M. L. Bossi
and S. W. Hell, Small, 2008, 4, 134; (c) M. Bossi, V. Belov,
S. Polyakova and S. W. Hell, Angew. Chem., Int. Ed., 2006, 45, 7462.
9 (a) F. Alexis, E. Pridgen, L. K. Molnar and O. C. Farokhzad, Mol.
Pharmaceutics, 2008, 5, 505; (b) D. Peer, J. M. Karp, S. Hong,
O. C. Farokhzad, R. Margalit and R. Langer, Nat. Nanotechnol.,
2007, 2, 751; (c) R. Haag and F. Kratz, Angew. Chem., Int. Ed., 2006,
45, 1198; (d) V. P. Torchilin, Adv. Drug Delivery Rev., 2006, 58, 1532.
10 (a) F. Tang, L. Lim and D. Chen, Adv. Mater., 2012, 24, 1504;
(b) Y. Piao, A. Burns, J. Kim, U. Wiesner and T. Hyeon, Adv. Funct.
Mater., 2008, 18, 3745.
11 (a) K. Ma, H. Sai and U. Wiesner, J. Am. Chem. Soc., 2012, 134, 13180;
(b) Y. Jin, S. Lohstreter, D. T. Pierce, J. Parisien, M. Wu, C. Hall III
and J. X. Zhao, Chem. Mater., 2008, 20, 4411.
12 (a) R. Ciriminna, M. Sciortino, G. Alonzo, A. de Schrijver and
M. Pagliaro, Chem. Rev., 2011, 111, 765; (b) R. Kumar, I. Roy,
T. Y. Ohulchanskyy, L. N. Goswami, A. C. Bonoiu, E. J. Bergey,
K. M. Tramposch, A. Maitra and P. N. Prasad, ACS Nano, 2008,
2, 449.
In summary, we reported the one-pot multicomponent copoly-
merization strategy to prepare the monodispersed silica NP 1a
with a mean diameter of 29 nm for reversible fluorescence
photoswitching in living cells. This silica NP was synthesized
in the oil-in-water emulsion using silane precursors including
those of DAE (FRET acceptor) and Cy3 (FRET donor).
13 (a) T. Kim, E. Momin, J. Choi, K. Yuan, H. Zaidi, J. Kim, M. Park,
N. Lee, M. T. McMahon, A. Quinones-Hinojosa, J. W. M. Bulte,
T. Hyeon and A. A. Gilad, J. Am. Chem. Soc., 2011, 133, 2955;
(b) L. Ferreira, J. M. Karp, L. Nobre and R. Langer, Cell Stem Cell,
2008, 3, 136; (c) C.-W. Lu, Y. Hung, J.-K. Hsiao, M. Yao, T.-H. Chung,
Y.-S. Lin, S.-H. Wu, S.-C. Hsu, H.-M. Liu, C.-Y. Mou, C.-S. Yang,
D.-M. Huang and Y.-C. Chen, Nano Lett., 2007, 7, 149.
c
7530 Chem. Commun., 2013, 49, 7528--7530
This journal is The Royal Society of Chemistry 2013