NanoPARCEL: A method for controlling cellular behavior with external light

Article abstract of DOI:10.1039/c2cc32718j

We developed a simple preparation procedure for the protein encapsulated nanoparticle and used the nanoparticle for spatiotemporal activity control of various proteins. We succeeded in the local protein activation within cells by light using the nanoparti

Full text of DOI:10.1039/c2cc32718j

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Cite this: Chem. Commun., 2012, 48, 8380–8382
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COMMUNICATION
NanoPARCEL: a method for controlling cellular behavior with
external lightw
Shuhei Murayama,a Baowei Su,a Kohki Okabe,a Akihiro Kishimura,b Kensuke Osada,b
Masayuki Miura,a Takashi Funatsu,a Kazunori Kataokabc and Masaru Kato*a
Received 17th April 2012, Accepted 27th June 2012
DOI: 10.1039/c2cc32718j
We developed a simple preparation procedure for the protein
encapsulated nanoparticle and used the nanoparticle for spatio-
temporal activity control of various proteins. We succeeded in the
local protein activation within cells by light using the nanoparticle.
large (millimeter scale) for introduction into cells and because
the gels contain acrylamide, a suspected neurotoxin and
carcinogen.
In this study, we succeeded controlling protein activity
within cells by biocompatible 150 nm nanoparticles, which
have been successfully used as biomolecule carriers.13–17 Three
different nanoparticles containing different proteins were pre-
pared and used to carry the proteins into two different cell
lines. Protein release and activation were controlled by irra-
diation at 365 and 405 nm.18 Cell damage at these wavelengths
is negligible,19 and the timing, location, and intensity of the
light could be controlled. We also experimented with control-
ling cellular processes by changing the timing and location of
protein activation within living cells. We refer to this method
as nanoscale PARCEL (nanoPARCEL).
The biological functions of proteins are precisely controlled so
as to sustain life. The latest proteome analysis methods permit
profiling of all intracellular proteins expressed during a sampling
period and have revealed many new important roles of proteins.1,2
However, understanding cellular processes (e.g., proliferation,
differentiation, migration, and death) induced by changes in
intracellular protein levels simply by analyzing the levels at a
given time is difficult. The ability to reproduce cellular processes by
controlling protein levels can be expected to improve our under-
standing of the mechanisms of cellular processes. Noninvasive
methods for controlling intracellular protein levels, such as the use
of knockout mice, small interfering RNA, and light-activated
protein, have been reported,3–10 but a general method for
spatiotemporal control of protein activity within cells is lacking.
Here, we describe a general, noninvasive method for spatio-
temporal control of the activity of various proteins in cells.
We previously reported a method for photocontrol of protein
activity using a photodegradable hydrogel (Protein Activation
and Release from Cage by External Light; PARCEL).11,12
Proteins are encapsulated in a cross-linked gel network with a
mesh size smaller than the protein diameter; the encapsulated
protein is inactive because the network restricts protein inter-
actions with other compounds. The encapsulated protein is
released under physiological conditions by stimulation with external
light, whereupon protein activity is restored. This method can
be used for various proteins because no chemical modification
of the proteins is required. However, PARCEL cannot be used
to control protein activity within cells, because the gels are too
First, we synthesized a novel four-arm photocleavable linker
PEG-Photo-MA (Scheme 1) from polyethylene glycol and a
photocleavable unit. Each arm bears a photocleavable group
and a terminal methacryloyl unit.20 The linker was cleaved by
UV irradiation (356 and 405 nm).18 Polymerization of the linker
afforded an acrylamide-free gel. Surprisingly, we obtained 150 nm
nanoparticles by vortexing the photocleavable linker for 20 min at
4 1C. The size distribution of the protein-containing nanoparticles
was narrow (average diameter B150 nm, as indicated by
transmission electron microscopy (TEM); Fig. 1 and dynamic
light scattering (DLS); Fig. S1, ESIw). Although trypsin
concentration did not affect the nanoparticle size, the ammonium
persulfate (APS) concentration regulated the nanoparticle size
a Graduate School of Pharmaceutical Sciences and Global COE
Program, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo, Japan. E-mail: masaru-kato@umin.ac.jp;
Fax: +81-3-5841-1841; Tel: +81-3-5841-1840
b Department of Materials Engineering, Graduate School of
Engineering, The University of Tokyo, Tokyo, Japan
c Center for Disease Biology and Integrative Medicine,
Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c2cc32718j
Scheme 1 Chemical structure of PEG-Photo-MA.
c
8380 Chem. Commun., 2012, 48, 8380–8382
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the cytosol appeared red, indicating distribution of the injected
solution throughout the cytosol (right panels). No green
fluorescence was observed before irradiation, when the labeled
casein was encapsulated within the nanoparticles (upper left
panel). In contrast, green fluorescence was observed after the
labeled casein was released and then digested by intracellular
proteins (lower left panel). This result indicates that the
nanoparticles were stable in the cells before irradiation and
that the encapsulated protein was released by irradiation.
Control of regulatory protein activation in cells can be
expected to permit regulation of various cellular processes.
To achieve such regulation, we injected HuH-7 cells with a
mixture of dextran-Texas Red and nanoparticles containing
activated caspase-3, which plays a role in cell death. Then we
irradiated half the cells with UV light for 10 s. In the merged
phase contrast microscopy (PCM) and CLSM images of the
cells before irradiation and 24 h later (Fig. S6, ESIw), the
fluorescence of Texas Red was visible just after injection and
even after 24 h. Although the irradiated noninjected cells
remained undamaged, all the irradiated cells with nanoparticles
disappeared within 24 h, indicating that all the cells were dead
and that the released caspase-3 induced cell death within 24 h.
To confirm the release of the active caspase-3, we used the cells
which were treated with a fluorogenic substrate (CR(DEVD)2).
The red signal of the CR(DEVD)2 was observed from the cells
on the process of the cell death (Fig. S7, ESIw).
Finally, we demonstrated the regulation of cellular pro-
cesses (cell death) by releasing activated caspase-3 at a specific
location in a living cell. Nanoparticles containing activated
caspase-3 were introduced into a cell that had been transfected
with an end-binding 1 (EB1)-green fluorescence protein (GFP)
plasmid to label the microtubules with GFP in advance for
visualizing dynamic changes in the cytoskeleton (Fig. S8,
ESIw). When a spot of the cell (red circle, Fig. 4) was irradiated
with laser, protein was released and activated only at the
irradiated spot, and the released protein then diffused through-
out the cell. The movie in ESIw shows two cells: one was with
Fig. 1 Transmission electron micrographs of photodegradable
nanoparticle-encapsulated trypsin with and without UV irradiation.
All scale bars are 200 nm.
(Fig. S2, ESIw). No method for nanoparticle preparation at
high APS concentrations and low temperature has previously
been reported.21,22 The high APS concentration favored seed
formation; many seeds grew simultaneously. Nanoparticle
growth stopped when the monomer supply became insuffi-
cient. At high APS concentrations, growth stopped quickly,
because many nanoparticles grew simultaneously.
TEM (Fig. 1 and Fig. S3, ESIw) and DLS (Fig. S1, ESIw) results
suggested that the UV irradiation (365 nm 20 s 0.57 W cmÀ2
)
reduce the size of the nanoparticle (o60 nm). It is supposed that
the nanoparticles had collapsed owing to cleavage of the gel
network.
To evaluate the nanoparticles as protein carriers in vitro, we
encapsulated trypsin within the nanoparticles and evaluated its
stability and release efficiency by comparing trypsin activities
using BODIPY-casein, a fluorogenic substrate. Trypsin activity
was negligible without UV irradiation but strong with UV
irradiation (Fig. 2 and Fig. S4, ESIw). The activity of the released
trypsin was about 20% when compared with that of the original
trypsin (Fig. S5, ESIw). It is suggested that irradiation cleaved
the gel network, and the released trypsin hydrolyzed BODIPY-
casein. The reproducibility of trypsin activity after irradiation
was an acceptable value (RSD = 15.5%) for cell assay.
To verify the effectiveness of these nanoparticles as protein
containers within cells, we microinjected a mixture of dextran-
Texas Red (an indicator of injection) and nanoparticles con-
taining BODIPY-casein into living Cos-7 monkey kidney cells.
When BODIPY-casein is hydrolyzed by intercellular enzymes, it
fluoresces (data not shown). After injection, half the cells were
irradiated with UV light for 10 s (0.25 W cmÀ2), and the
remaining half were not subjected to any external stimulus. Many
cells were alive and unchanged in morphology after injection and
irradiation. In confocal laser scanning microscopy (CLSM)
images of the cells obtained 2 h after irradiation (Fig. 3),
Fig. 3 Photo induced release of BODIPY-casein within cells. CLSM
images of Cos-7 cells injected with nanoparticle-encapsulated BODIPY-
casein with and without UV observed at 2 h after irradiation. Green,
hydrolyzed BODIPY casein; red, dextran-Texas Red. All scale bars
are 20 mm.
Fig. 2 Regulation of trypsin activity by means of UV irradiation of
trypsin-containing nanoparticles in vitro. All samples were measured
in quintuplet, and error bars indicate standard deviations.
c
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processes should be nearly simultaneous. The nanoPARCEL
method could be used to activate proteins at a specific area of
a cell, and the triggered cellular processes could be observed
immediately after protein activation using a commonly used
microscopy technique.
In summary, proteins were effectively released and activated
within cells upon irradiation of protein-containing photo-
degradable nanoparticles with external light. Because chemical
modification is not necessary for activity control, nano-
PARCEL should be amenable to a variety of proteins. This
low invasive method for spatiotemporal control of protein
activity in cells should permit the regulation of cellular processes,
and we expect that research on protein function will be acceler-
ated by application of the method and that it will be a powerful
tool for characterization of complex cellular mechanisms.
This work was supported by a grant from the New Energy
and Industrial Technology Development Organization of
Japan and the Ministry of Education, Culture, Sports, Science
and Technology of Japan. B.S. was supported by the CMSI
Summer Internship Program of the University of Tokyo. We
thank S. Ikeda, T.A. Theofilus, Dr S. Lee, Dr Y. Mimori-
Kiyosue, Dr Y. Yamaguchi, and M. Sasaki for assistance.
Fig. 4 Local activation of caspase-3 by focused light. PCM images
show the progress of cell death induced by focused light: before
irradiation and 2, 4, and 10 min after irradiation. The red circle
indicates the irradiation point. All scale bars are 20 mm.
Notes and references
1 L. M. F. de Godoy, J. V. Olsen, J. Cox, M. L. Nielsen,
N. C. Hubner, F. Frohlich, T. C. Walther and M. Mann, Nature,
¨
2008, 455, 1251–1254.
no nanoparticles without irradiation (upper side of the movie),
whereas the other contained nanoparticles with irradiation
and underwent cell death induced by the focused light (lower
side of the movie). Fig. 4 and Fig. S8 (ESIw) show magnified
images of this cell. Although the lamellipodia at the cell edge
distant from the irradiation point did not change (Fig. 4, blue
arrows, upper edge of cell), the lamellipodia at the irradiated
edge retracted at about 2 min after irradiation (red arrows,
lower edge of cell). Four minutes after irradiation, the cell
started to expand at the irradiated edge while the lamellipodia
at the opposite edge maintained their original shape. The
irradiation point coincided with the point at which the
lamellipodia change started, suggesting that the change was
initiated by local activation of caspase-3. After approximately
10 min, the cell ruptured. The morphological change in the cell
nucleus (Fig. 4) and dilution of the GFP fluorescence indicated
cell death (Fig. S8, ESIw). Although the time required for cell
death depended on the cell, all the cells died within 20 min.
Cell death was not observed when the same experiment was
performed with nanoparticles that did not contain activated
caspase-3 (Fig. S9, ESIw). It is supposed that the nanoparticle
and its degradates were less invasive to cells.
2 J. Rappsilber, M. Mann and Y. Ishihama, Nat. Protocols, 2007, 2,
1896–1906.
3 K. R. Thomas, K. R. Folger and M. R. Capecchi, Cell
(Cambridge, Mass.), 1986, 44, 419–428.
4 O. Smithies, R. G. Gregg, S. S. Boggs, M. A. Doralewski and
R. S. Kucherlapati, Nature, 1985, 317, 230–234.
5 M. J. Evans and M. H. Kaufman, Nature, 1981, 292, 154–156.
6 A. Fire, S. Xu, M. K. Montgomery, S. A. Kostas, S. E. Driver and
C. C. Mello, Nature, 1998, 391, 806–811.
7 C. Chang, T. Fernandez, R. Panchal and H. Bayley, J. Am. Chem.
Soc., 1998, 120, 7661–7662.
8 E. M. Hahn and W. T. Muir, Angew. Chem., Int. Ed., 2004, 43,
5800–5803.
9 M. Endo, K. Nakayama, Y. Kaida and T. Majima, Angew. Chem.,
Int. Ed., 2004, 116, 5761–5763.
10 M. Yazawa, M. A. Sadaghiani, B. Hsueh and E. R. Dolmetsch,
Nat. Biotechnol., 2009, 27, 941–947.
11 S. Murayama and M. Kato, Anal. Chem., 2010, 82, 2186–2191.
12 S. Murayama, F. Ishizuka, K. Takagi, H. Inoda, A. Sano, T. Santa
and M. Kato, Anal. Chem., 2012, 84, 1374–1379.
13 D. A. Rothenfluh, H. Bermudez, C. P. O’Neil and J. A. Hubbell,
Nat. Mater., 2008, 7, 248–254.
14 M. Ferrari, Nat. Rev. Cancer, 2005, 5, 161–171.
15 M. E. Davis, Z. Chen and D. M. Shin, Nat. Rev. Drug Discovery,
2008, 7, 771–782.
16 N. Nishiyama, A. Iriyama, W. D. Jang, K. Miyata, K. Itaka,
Y. Inoue, H. Takahashi, Y. Yanagi, Y. Tamaki, H. Koyama and
K. Kataoka, Nat. Mater., 2005, 4, 934–941.
It was reported that protein function is changed by the activa-
tion area and timing, for example, caspases have various other
functions in addition to inducing apoptosis, and many researchers
are interested in the local cellular functions of caspases.23 Investi-
gation of local protein activity within cells is difficult because
methods, other than nanoPARCEL, for spatiotemporal control of
the activity of a variety of proteins within cells are limited.
Although we could control the local activity of proteins,
protein diffusion was fast, and the protein concentration
gradient disappeared after about 10 min. To circumvent this
problem, activation of local protein and monitoring of cellular
17 M. Murakami, H. Cabral, Y. Matsumoto, S. Wu, M. R. Kano,
T. Yamori, N. Nishiyama and K. Kataoka, Sci. Transl. Med.,
2011, 3, 64ra2.
18 A. M. Kloxin, M. W. Tibbitt and K. S. Anseth, Nat. Protocols,
2010, 5, 1867–1887.
19 A. M. Kloxin, A. M. Kaskko, C. N. Salinas and K. S. Anseth,
Science, 2009, 324, 59–63.
20 The acute toxicities (LD50 oral) of acrolein and methacrolein are 26
and 140 mg kgÀ1, respectively.
21 S. Nayak and L. A. Lyon, Angew. Chem., Int. Ed., 2005, 117, 7862–7886.
22 A. V. Kabanov and S. V. Vinogradov, Angew. Chem., Int. Ed.,
2009, 121, 5524–5536.
23 E. Kuranaga and M. Miura, Trends Cell Biol., 2007, 17, 135–144.
c
8382 Chem. Commun., 2012, 48, 8380–8382
This journal is The Royal Society of Chemistry 2012