Accumulation of supramolecular nanoparticles self-assembled from a bola-shaped cytidylic acid-appended fluorescein dye in cell nuclei

Article abstract of DOI:10.1039/c4cc03733b

We examined the cellular uptake of the nanoparticles self-assembled from a bola-shaped cytidylic acid-appended fluorescein derivative (C-FLU-C). The accumulation of fluorescence in the Caco-2 cell nucleus was observed mainly after the plateau phase of cell growth, indicating that C-FLU-C permeated the nuclear envelope without nuclear-localizing tags.

Full text of DOI:10.1039/c4cc03733b

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Accumulation of supramolecular nanoparticles
self-assembled from a bola-shaped cytidylic
acid-appended fluorescein dye in cell nuclei†
Cite this: Chem. Commun., 2014,
50, 9295
Received 16th May 2014,
Accepted 24th June 2014
Rika Iwaura,*a Mutsumi Shirai,a Kaname Yoshidab and Mayumi Ohnishi-Kameyamaa
DOI: 10.1039/c4cc03733b
www.rsc.org/chemcomm
We examined the cellular uptake of the nanoparticles self-assembled
It is widely accepted that a bottom-up approach based on self-
from a bola-shaped cytidylic acid-appended fluorescein derivative assembling building blocks is a good strategy for the production of
(C-FLU-C). The accumulation of fluorescence in the Caco-2 cell well-defined, nanostructured materials.13 In aqueous solution,
nucleus was observed mainly after the plateau phase of cell growth, amphiphilic molecules self-assemble into nanostructures such as
indicating that C-FLU-C permeated the nuclear envelope without micelles, vesicles, fibers, and tubes, and these nanostructures are
nuclear-localizing tags.
expected to be used in many fields as, for example, sensor mem-
branes,14 hydrogelators,15 or templates for inorganic materials.16–19
The development of techniques for intracellular delivery of In particular, nanostructures formed from self-assembling amphi-
materials has attracted the attention of many researchers owing philes have attracted a lot of attention as promising vehicles for
to the potential application of these techniques in biomedical and intracellular delivery of materials because of their affinity with the
pharmaceutical uses such as bioanalysis or in vivo imaging.1–6 cell membrane.3,20,21 Previously, we studied the self-assembly of
Particularly, the delivery of materials to the nucleus is considered nucleotide-appended bolaamphiphiles, which are amphiphilic
to be an important technique because that is where genetic molecules with a hydrophilic nucleotide moiety at both ends of a
information is stored; however, most nanomaterials reported so long hydrophobic chain, and we found that these bolaamphiphiles
far remain trapped in the cytoplasm after penetrating the cell form stable nanostructures, such as nanosheets and helical and no
membrane. One of the main reasons for this is that the nuclear helical nanofibers, in aqueous solution.22–25 Cytidylic acid-appended
envelope acts as a barrier to protect the nucleus. Pores in the bolaamphiphiles spontaneously form stable nanoparticles with a
nuclear envelope regulate the exchange of molecules between relatively homogeneous diameter and size distribution of r100 nm
the nucleus and the cytoplasm by restricting the physical size of in aqueous solutions due to weaker stacking interactions between
molecules that can enter the nucleus by passive diffusion and cytosine moieties.23 In the present study, we synthesized fluorescent
signal-mediated transport mechanisms to around 9 and 39 nm, nanoparticles self-assembled from a cytidylic acid-appended
respectively.7 To overcome this problem, nanomaterials conju- fluorescent amphiphilic molecule, C-FLU-C, to perform cellular
gated with peptide-based nuclear-localizing signal (NLS) tags have uptake experiments of nanomaterials. We observed that the self-
been developed that successfully translocate into cell nuclei.8–11 assembling fluorescent nanoparticles were taken up into cells
Interestingly, Cheng et al. have reported the accumulation of and that cell-dependent accumulation occurred in the nuclei of
PEGylated SWCNTs in cell nuclei without modification with NLS two mammalian (Caco-2 and BALB/3T3) cell lines.
tags.12 Although the translocation of nanomaterials without
We designed and synthesized cytidylic acid-appended fluorescein
NLS tags into the nucleus must provide new insight into the (bis[5-(30-cytidylyloxy)pentyloxy]fluorescein; C-FLU-C) according to
use of nanomaterials for the targeting of cell nuclei, there still procedures described previously (Fig. 1a and Fig. S1, details in
remain unclear factors such as design of nanomaterials or uptake ESI†). Owing to the hydrophilic cytidylic acid moieties at each end
mechanisms into cell nuclei.
of the fluorescein molecule, C-FLU-C showed good solubility in
water and aqueous buffer solutions and spontaneously formed
fluorescent nanoparticles. We used transmission electron microscopy,
atomic force microscopy and size distribution measurement to verify
the morphology of the nanoparticles, and we confirmed that the
C-FLU-C nanoparticles in aqueous solution had a mean diameter of
70 nm and a relatively narrow size distribution (Fig. 1b; Fig. S2 and S3,
ESI†). In addition, TEM images (Fig. 1b and Fig. S2, ESI†)
a National Food Research Institute, National Agriculture and Food Research
Organization, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan.
E-mail: riwaura@affrc.go.jp
b Japan Fine Ceramics Center, 2-4-1 Mutsuno, Atsuta, Nagoya, 456-8587, Japan
† Electronic supplementary information (ESI) available: Synthesis, characteriza-
tion of the nanoparticles, cell culture methods, flow cytometric analysis, and
microscopic observations. See DOI: 10.1039/c4cc03733b
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Fig. 1 (a) Structure of cytidylic acid-appended fluorescein (C-FLU-C).
(b) Transmission electron microscopic image of self-assembled C-FLU-C
nanoparticles. Arrows indicate nanoparticles.
displayed no internal space in the nanoparticles, indicating that the
nanoparticles are not vesicles as observed in the self-assembly from
cytidylic acid-appended bolaamphiphiles.23 Emission spectra showed
maxima at around l 530 nm for both C-FLU-C and fluorescein in Tris-
HCl aqueous buffer solution (pH 8, ex = 480 nm, Fig. S4, ESI†). The
fluorescein with a lactone structure does not show fluorescence and
absorbance in the visible region;26 therefore, the fluorescence
of C-FLU-C should originate from the zwitterionic structure of
the fluorescein skeleton (Fig. 1a, lower). The yellow color of the
aqueous solution containing the self-assembled C-FLU-C is
explainable on the basis of the formation of zwitterionic
structure of the fluorescein moiety.27
Fig. 3 Light (left) and fluorescence (right) microscopic images of Caco-2
cells + C-FLU-C cultured for (a) 1 day, (b) 4 days, (c) 5 days, (d) 7 days, and
(e) 12 days, and of BALB/3T3 + C-FLU-C cultured for (f) 2 days, (g) 5 days,
(h) 6 days, (i) 7 days, and (j) 13 days. Arrows indicate nuclei.
Fluorescence from C-FLU-C was localized in the nuclei upon
incubation with Caco-2 cells (human colon carcinoma cell line).
Flow cytometric analysis (Fig. 2a and b) showed that fluores-
cence intensity increased after C-FLU-C was incubated with the
Caco-2 or BALB/3T3 (contact-inhibited semi-normal cell line:
mouse embryo cell line) cell line, implying that C-FLU-C either
entered the cells or was adsorbed on the cell surface. The
uptake efficiency28 curve of C-FLU-C into Caco-2 cells dramati-
cally increased up to day 5, after which it gradually increased
until it was 480% at day 14 of culture (Fig. 2c, K). However, in
BALB/3T3 cells, uptake efficiency remained constant after day 5
at approximately 55% (Fig. 2c, m). We did not observe any
intracellular uptake of C-FLU-C by the Caco-2 cell line cultured
at 8 1C to suppress cellular activity. This observation suggests that
C-FLU-C enters Caco-2 cells via an energy-dependent mechanism.
Growth curves of Caco-2 and BALB/3T3 cells in the presence or
absence of C-FLU-C gave similar standard sigmoidal curves
that became confluent at day 6 (Fig. S5, ESI†). C-FLU-C was
therefore not toxic to either cell line. Together the results for
cellular uptake efficiency and cell growth show that the uptake
efficiency of C-FLU-C nanoparticles into BALB/3T3 cells increased
most during cell division, whereas that of Caco-2 cells increased
most after cell growth reached confluence after 6 days of culture.
This observation suggests that the uptake of C-FLU-C nano-
particles by Caco-2 cells depends on mechanisms involved in
cell division or on other mechanisms such as those involved
in endocytosis. Fluorescence microscopic analysis was used to
confirm the intracellular uptake of C-FLU-C. A green fluorescence
signal was observed in the cytoplasm of both cell lines (Fig. 3). We
further confirmed the entry of C-FLU-C into Caco-2 cells by means
of laser-scanning confocal microscopy of the cell cytoplasm
(Fig. S6, ESI†). Interestingly, Caco-2 cells cultured in the presence
of C-FLU-C for 7 days showed the translocation of fluorescence
into the cell nuclei (Fig. 3d), and the fluorescence signal remained
after 12 days of culture (Fig. 3e); whereas, no fluorescence was
observed in the nuclei of BALB/3T3 cells (Fig. 3f–j).
To further investigate the accumulation of C-FLU-C in the nuclei
of Caco-2 cells, we carried out cell culture experiments using both
cell lines and fluorescein. Although we obtained images of fluores-
cence in the cytoplasm after culturing the Caco-2 or BALB/3T3 cell
line with fluorescein for 11 to 13 days (Fig. 4), we did not observe
the accumulation of fluorescence in the nuclei of either cell line.
This result suggests that the accumulation of C-FLU-C in the cell
nucleus is specific to Caco-2 cells and that the cytidylic acid moiety
in C-FLU-C is crucial for its transportation into the nucleus. In fact,
after colcemid treatment (to arrest cell growth at the M phase,
during which the nuclear envelope disassembles), C-FLU-C was
adsorbed on the chromatin of both Caco-2 and BALB/3T3 cells
(Fig. S7, ESI†). This result strongly indicates that the nuclear
envelope prevents the transportation of C-FLU-C into the nucleus
of BALB/3T3 cells. Although large molecules may enter the nucleus
Fig. 2 Flow cytometric analysis of (a) Caco-2 cells + C-FLU-C and
(b) BALB/3T3 cells + C-FLU-C. (c) Time-dependent uptake efficiency of
C-FLU-C into Caco-2 (K) and BALB/3T3 (m) cells.
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Scientific Research B (no. 23310084) from the Ministry of Educa-
tion, Culture, Sports, Science and Technology, Japan.
Notes and references
Fig. 4 Light (left) and fluorescence (right) microscopic images of Caco-2
cells + fluorescein cultured for (a) 11 days and of BALB/3T3 + fluorescein
cultured for (b) 13 days.
1 V. Biju, T. Itoh and M. Ishikawa, Chem. Soc. Rev., 2010, 39, 3031.
2 H. Hillaireau and P. Couvreur, Cell. Mol. Life Sci., 2009, 66, 2873.
3 A. H. Faraji and P. Wipf, Bioorg. Med. Chem., 2009, 17, 2950.
4 B. G. Trewyn, I. I. Slowing, S. Giri, H.-T. Chen and V. S. Y. Lin, Acc.
Chem. Res., 2007, 40, 846.
5 Z. Ge and S. Liu, Chem. Soc. Rev., 2013, 42, 7289.
6 K. Ariga, Y. Yamauchi, G. Pydzek, Q. Ji, Y. Yonamine, K. C.-W. Wu
and J. P. Hill, Chem. Lett., 2014, 43, 36.
during mitosis when the nuclear envelope breaks down, they are
generally excluded when the nucleus reassembles after mitosis.29
Unlike most large molecules, Cheng et al. reported that PEGylated
SWCNTs that are not conjugated to nuclear-localizing signal tags
localize not only in the nuclei of parent HeLa cells (human
epithelial carcinoma cell line) but also in the nuclei of daughter
HeLa cells.12 Cheng et al. suggest that PEGylated SWCNTs tether to
proteins or RNAs that are reimported into the nucleus after
mitosis.12 In the present study, marked accumulation of fluores-
cence from C-FLU-C in Caco-2 cell nuclei was seen after the cell
growth curve reached a plateau (after around 7 days). This
observation differs from that seen with PEGylated SWCNTs,12
indicating that in Caco-2 cells, C-FLU-C did not enter the nucleus
during mitosis, but instead permeated the cell envelope after the
nucleus was reassembled and then translocated into the nucleus.
Nuclear pores should not allow the transportation of C-FLU-C
nanoparticles (mean diameter, 70 nm) into the nucleus, because
the nanoparticles are too large.7 Therefore, we hypothesize that
C-FLU-C nanoparticles attach to or near nuclear pores in Caco-2
cells, dissociate, and are then transported into the nucleus as
C-FLU-C molecules or clusters. Interestingly, the adenovirus capsid
(d = 80 nm) dissociates and imports the capsid contents (DNA) into
the cell nucleus after docking at nuclear pores.30 We hypothesize
that the mechanism responsible for the translocation of C-FLU-C
into Caco-2 cell nuclei could be similar to that responsible for the
transport of the adenovirus capsid contents.
´
7 N. Pante and M. Kann, Mol. Biol. Cell, 2002, 13, 425.
8 L. Pan, Q. He, J. Liu, Y. Chen, M. Ma, L. Zhang and J. Shi, J. Am.
Chem. Soc., 2012, 134, 5722.
9 K. C. Weng, C. O. Noble, B. Papahadjopoulos-Sternberg, F. F. Chen,
D. C. Drummond, D. B. Kirpotin, D. Wang, Y. K. Hom, B. Hann and
J. W. Park, Nano Lett., 2008, 8, 2851.
10 A. K. Oyelere, P. C. Chen, X. Huang, I. H. El-Sayed and M. A. El-Sayed,
Bioconjugate Chem., 2007, 18, 1490.
11 A. G. Tkachenko, H. Xie, D. Coleman, W. Glomm, J. Ryan,
M. F. Anderson, S. Franzen and D. L. Feldheim, J. Am. Chem. Soc.,
2003, 125, 4700.
12 J. Cheng, K. A. S. Fernando, L. M. Veca, Y.-P. Sun, A. I. Lamond,
Y. W. Lam and S. H. Cheng, ACS Nano, 2008, 2, 2085.
13 (a) S. Lena, S. Masiero, S. Pieraccini and G. P. Spada, Chem. – Eur. J.,
2009, 15, 7792; (b) K. Ariga, Q. M. Ji, J. P. Hill, N. Kawazoe and
G. P. Chen, Expert Opin. Biol. Ther., 2009, 9, 307; (c) M. Brucale,
G. Zuccheri and B. Samori, Trends Biotechnol., 2006, 24, 235;
(d) M. Ramanathan, L. K. Shrestha, T. Mori, Q. Ji, J. P. Hill and
K. Ariga, Phys. Chem. Chem. Phys., 2013, 15, 10580; (e) R. Tian,
J. Chen and R. Niu, Nanoscale, 2014, 6, 3474.
14 D. C. Daila, L. T. Banner, E. J. Karimova, L. Tsurkan, X. Wang and
E. Pinkhassik, Angew. Chem., Int. Ed., 2008, 47, 7036.
15 M. d. Loos, B. L. Feringa and J. H. v. Esch, Eur. J. Org. Chem., 2005,
3615.
16 E. E. Sone and S. I. Stupp, Chem. Mater., 2011, 23, 2005.
17 H. Qiu and S. Che, Chem. Soc. Rev., 2011, 40, 1259.
18 J. E. Lofgreen and G. A. Ozin, Chem. Soc. Rev., 2014, 43, 911.
19 K. Ariga, A. Vinn, Y. Yamauchi, Q. Ji and J. P. Hill, Bull. Chem. Soc.
Jpn., 2012, 85, 1.
20 C. Bombelli, A. Stringaro, S. Borocci, G. Bozzuto, M. Colone,
L. Giansanti, R. Sgambato, L. Toccaceli, G. Mancini and
A. Molinari, Mol. Pharmaceutics, 2010, 7, 130.
21 M. C. Branco and J. P. Schneider, Acta Biomater., 2009, 5, 817.
22 R. Iwaura, F. J. M. Hoeben, M. Masuda, A. Schenning, E. W. Meijer
and T. Shimizu, J. Am. Chem. Soc., 2006, 128, 13298.
23 R. Iwaura, T. Iizawa, H. Minamikawa, M. Ohnishi-Kameyama and
T. Shimizu, Small, 2010, 6, 1131.
24 R. Iwaura, M. Ohnishi-Kameyama and T. Iizawa, Chem. – Eur. J.,
2009, 15, 3729.
25 R. Iwaura, K. Yoshida, M. Masuda, M. Ohnishi-Kameyama, M. Yoshida
and T. Shimizu, Angew. Chem., Int. Ed., 2003, 42, 1009.
26 S.-C. Chen, H. Nakamura and Z. Tamura, Chem. Pharm. Bull., 1979,
27, 475.
27 R. Markuszewski and H. Diehl, Talanta, 1980, 27, 937.
28 The uptake efficiency is determined as follows. Efficiency (%) =
100 ꢀ Cf/Ct. Cf: cell count containing fluorescent dyes. To remove
the effect of autofluorescence from the cells, each overlapped area of
flowcytometry histograms with that of 0 day was subtracted from
that of 1 to 14 days, and then we obtained cell count containing
dyes. Ct: total cell count.
In summary, fluorescein, which is widely used for the imaging
and labeling of biomolecules in cells,31 was used to synthesize the
molecular building block C-FLU-C, which self-assembles into
nanoparticles. C-FLU-C nanoparticles were efficiently taken up
by Caco-2 and BALB/3T3 cells without the need for additional
physical or chemical treatments. Uptake of C-FLU-C nanoparticles
did not affect the cell cycle under experimental conditions. We
demonstrated the cell-dependent transfer of C-FLU-C into the
nuclei of Caco-2 cells. Together these findings provide insights
into the cell-selective translocation of materials into the cell
nucleus that will be helpful in the development of biomedical
and pharmaceutical applications using nanoparticles such as cell-
selective drug delivery and imaging.
We thank Ms Hiroko Kato and Mr Satoshi Ijuin (Leica Micro-
systems K. K.) for laser-scanning confocal microscope observations.
This work was partially supported by KAKENHI Grants-in Aid for
29 J. A. Swanson and P. L. McNeil, Science, 1987, 238, 548.
30 A. Zlotnick, Virology, 2003, 315, 269.
31 Y. Urano, M. Kamiya, K. Kanda, T. Ueno, K. Hirose and T. Nagano,
J. Am. Chem. Soc., 2005, 127, 4888.
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 9295--9297 | 9297