Aggregation-induced emission enhancement characteristics of naphthalimide derivatives and their applications in cell imaging

Article abstract of DOI:10.1039/c0jm02942d

Novel naphthalimide derivatives (NIM) with donor-acceptor architecture were successfully constructed and their unique fluorescent properties were investigated. In particular, the fluorescence intensities of 4-methoxystyrene substituted NIM-1 were solvent independent and its quantum yields varied from 0.4 in toluene to 0.52 in MeOH. However, 4-(N,N-diphenylamino) styrene substituted NIM-2 and 4-nitrostyrene substituted NIM-3 exhibited low luminescence in dilute solutions but were efficiently fluorescent under conditions of molecular aggregation. NIM-2 showed strong solid fluorescence with a longer wavelength emission and larger Stokes shift. In addition, NIM-3 showed unexpected blue-green fluorescence due to the formation of fluorescent organic nanoparticles (FONs) under mixed solution. These two cases were ascribed to the aggregation-induced enhanced emission (AIEE) effects and which can also reasonably explain the bright cellular images observed when cells were incubated with NIM-2 and NIM-3. Furthermore, compound NIM-2 can be used to recognize cancer cells owing to its subcellular behaviour. The Royal Society of Chemistry 2011.

Full text of DOI:10.1039/c0jm02942d

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PAPER
Aggregation-induced emission enhancement characteristics of naphthalimide
derivatives and their applications in cell imaging†
a
Hsin-Hung Lin, Yung-Chieh Chan, Jyun-Wei Chen and Cheng-Chung Chang
b
a
a
*
Received 4th September 2010, Accepted 3rd December 2010
DOI: 10.1039/c0jm02942d
Novel naphthalimide derivatives (NIM) with donor–acceptor architecture were successfully
constructed and their unique fluorescent properties were investigated. In particular, the fluorescence
intensities of 4-methoxystyrene substituted NIM-1 were solvent independent and its quantum yields
varied from 0.4 in toluene to 0.52 in MeOH. However, 4-(N,N-diphenylamino) styrene substituted
NIM-2 and 4-nitrostyrene substituted NIM-3 exhibited low luminescence in dilute solutions but were
efficiently fluorescent under conditions of molecular aggregation. NIM-2 showed strong solid
fluorescence with a longer wavelength emission and larger Stokes shift. In addition, NIM-3 showed
unexpected blue-green fluorescence due to the formation of fluorescent organic nanoparticles (FONs)
under mixed solution. These two cases were ascribed to the aggregation-induced enhanced emission
(AIEE) effects and which can also reasonably explain the bright cellular images observed when cells
were incubated with NIM-2 and NIM-3. Furthermore, compound NIM-2 can be used to recognize
cancer cells owing to its subcellular behaviour.
(AIEE) characteristics which promote great application potential
Introduction
as well as challenge the current knowledge of the photo-
luminescence process. Thus, to extend our understanding of
fluorescence mechanisms, exploration of new AIEE fluorophores
is of great interest.
Fluorescent dyes have specialized applications in biodiagnostic
assays.1 Thus, the development of high-efficiency fluorophores is
essential for the field of molecular sensors.2 Most conventional
fluorophores have the disadvantage of very small Stokes shifts,
which can lead to self-quenching and measurement error caused
by the excitation light and scattering. High levels of self-
quenching can also result in weak solid fluorescence, which might
partly explain why aggregation of fluorescent probe is unfavor-
able for cellular staining. Furthermore, most organic fluorescent
dyes exhibit fluorescence quenching in the aggregate or solid
states, and this results in the nonradiative deactivation of the
excited state becoming dominant because of the formation of
delocalized excitons or excimers.3 Recently, organic dyes that
exhibit strong fluorescent emission in their aggregate or solid
state have attracted increasing attention,4 such as siloles,5
1-cyano-trans-1,2-bis(40-methylbiphenyl)ethylene,6 thienyl-azu-
lene,7 arylethene derivatives,8 1,4-di[(E)-2-phenyl-1-prope-
nyl]benzene,9 and salicylaldehyde azine derivatives.10 These
fluorophores show aggregation-induced emission enhancement
1,8-Naphthalimide derivatives are a well-known class of
compounds that have a wide range of applications. Because of
their high fluorescence quantum yields and photostability, these
derivatives are considered to be good candidates for fluorescent
dichroic dyes,11 fluorescent polymeric paints or plastics12 and
fluorescent markers in medicine and biology.13 Moreover, the use
of 1,8-naphthalimide derivatives as DNA intercalators14 and
functionalized naphthalimide chromophores have been under
examination in solution as sensor dyes for metal ions15 and for
pH-changes.16 However, a review of previous investigations
about the synthesis and properties of 1,8-naphthalimide deriva-
tives revealed that most derivatives have been functionally
modified on the basis of the 1,8-naphthalimide structure con-
taining various alkylamino groups in the 4-position or
N-substitutions of the naphthalimide ring. In addition, their
spectral properties result in absorbance at short wavelengths
(below 400 nm); this is particularly problematic for biological
experiments because operations in the visible range of wave-
lengths are common-place for biological applications involving
aqueous systems. As a result, the choice of functional naph-
thalimide dyes is much more limited for confocal microscopy
systems. In the present study, our intention was to extend the
scope of available naphthalimide dyes by elongating the
p-conjugate resonance of rings and to describe in more detail
about their spectroscopic properties. When electron donor
aGraduate Institute of Biomedical Engineering, National Chung Hsing
University, 250 Kuo Kuang Road, Taichung, 402, Taiwan, R.O.C.
E-mail: ccchang555@dragon.nchu.edu.tw; Fax: (+886)-4-228 52422;
Tel: (+886)-4-22840734 ext. 24
bDepartment of Chemistry, National Chung Hsing University, 250 Kuo
Kuang Road, Taichung, 402, Taiwan, R.O.C
† Electronic supplementary information (ESI) available: Details of
materials synthetic procedures, immunofluorescence staining of NIM-1,
NIM-2 and NIM-3. See DOI: 10.1039/c0jm02942d
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groups were linked to the electron-acceptor naphthalimide core,
an intramolecular charge transfer occurred. Furthermore, the
new D–A structures were fabricated and the vinyl group worked
as a p spacer to link the donor and the acceptor; this extended the
conjugation systems to favor absorption and emission.
4. Cell culture conditions and compound incubation
The human embryonal lung MRC-5 normal fibroblast cells and
MCF7 human breast cancer cell were grown in Dulbecco’s
modified Eagle’s medium (DMEM) with non-essential amino
acids supplemented with 10% fetal calf serum (FCS). The human
lung adenocarcinoma cell lines CL1-0 cancer line were grown in
RPMI 1640 (Gibco/Invitrogenꢀ, Cat. no. 22400-089) medium
containing 10% fetal bovine serum (FBS). Cell cultures were
maintained at 37 ꢁC in a humidified atmosphere of 95% air and
5% CO2. The cells seeded in culture plates or dishes were incu-
bated with different concentrations of NIM derivatives, whose
DMSO stock solutions were diluted in serum-free medium before
use (1/100 v/v).
In previous studies, we described the AIEE properties and
developed fluorescent organic nanoparticle (FON) models of
anionic and cationic 10H-phenothiazine derivates.17,18 In
particular, the AIEE properties of protonated phenothiazine
were observed for both in vitro spectral studies and cellular
staining. Then, we developed a vacuole model using FONs to
elucidate the formation of fluorescent bright spots that are
considered to form in the lysosomes of cancer cells and can be
used in cancer cell recognizer fluorophores.18 In the present
study, we present the absorption and photoluminescence features
of naphthalimide chromophores substituted with 4-methoxy-
styrene, 4-(N,N-diphenylamino)styrene, 4-nitrostyrene and
4-hydroxystyrene moiety groups. Solvatochromic studies of the
different chromophores were performed to determine the
changes in dipole moments under excitation and to compare
the results with the observed photoluminescence efficiency. Then,
the AIEE properties were investigated and these phenomena
were applied to illustrate the cellular imaging. Finally, we tested
the ability of the chromophores to be used as cellular probes.
5. Cellular imaging
Before the observation of cellular localization, cells were seeded
into coverslips and incubated for 24 h. On the next day, cells were
incubated with 10 mM of NIM for 4 h, and then the fluorescence
images were taken under Leica AF6000 confocal fluorescence
microscopy with DFC310 FX Digital color camera; or the l
scanning and fluorescence images were taken under Leica TCS
SP5 confocal fluorescence microscopy. The excitation source was
a 405 nm diode laser for compounds. Fluorescence photographs
were taken through related ranges by photomultiplier tubes
(PMT).
Experimental section
1. Materials
6. General procedure for the synthesis of naphthalimide
derivatives
The general chemicals employed in this study were of the best
grade available and were obtained from Acros Organic Co.,
Merck Ltd., or Aldrich Chemical Co. and used without further
purification. All solvents were of spectrometric grade.
Synthesis of the naphthalimide derivatives is shown in Scheme 1.
The first stage of the reaction, in which commercial starting
material 4-bromo-1,8-naphthalic anhydride was reacted with the
RNH2, was performed conveniently in ethanol at room
temperature. Next, the samples were subjected to the Heck
coupling reaction with 4-methoxystyrene, 4-N,N-diphenylami-
nostyrene, 4-nitrostyrene or 4-acetoxystyrene under catalyst Pd
(OAc)2.21 Details of materials synthetic procedures and identifi-
cations are shown in the Supporting Information.† NIM-1:
2-(6-(4-methoxystyryl)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-
2. Apparatus
Absorption spectra were generated using a Thermo Genesys 6
UV-visible spectrophotometer, and fluorescence spectra were
recorded using
a
HORIBA JOBIN-YVON Fluoromas-4
spectrofluorometer with a 1 nm band-pass and a 1 cm cell length
at room temperature. Transmission electron microscopy (TEM)
was conducted on a Zeiss EM 902A operated at 80 kV. A mixed
solution (a mixed solvent of THF and deionized water, 75/25 v/v;
ref. 26) containing compound NIM-3 was deposited onto
carbon-coated copper grid.
N,N-dimethylethanamine.
NIM-2:
2-(6-(N,N-diphenyl-4-
3. Determination of quantum yields
The quantum yields of NIM derivatives were determined as
previously described using the following equation:19
2
Fu ¼ Fs ꢀ [Afu ꢀ As(lexs) ꢀ hu ]/[Afs ꢀ Au(lexu) ꢀ hs2] where
Fu is the quantum yield of unknown; Af is the integrated area
under the corrected emission spectra; A(lex) is the absorbance
area at the excitation wavelength; h is the refractive index of the
solution; the subscripts u and s refer to the unknown and
the standard, respectively. For the same lex, we chose 3,6-bis-
(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC) as the
standard, which has a quantum yield of 0.25 in glycerol and 0.02
in DMSO.20
Scheme
1 Reagents and conditions: (i) N,N-dimethylethane-1,2-
diamine, EtOH, r.t. 24 h; (ii) Pd(OAc)2/(o-tol)3P, 4-methoxystyrene
(NIM-1), 4-(N,N-diphenylamino)styrene (NIM-2), 4-nitrostyrene (NIM-
3), or 4-acetoxystyrene (compound 3), Et3N/MeCN, N2, reflux, 48 h. (iii)
KOH/MeOH, reflux, 2 h; then HCl.
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aminostyryl)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-N,N-dim-
ethylethanamine. NIM-3: 2-(6-(4-nitrostyryl)-1,3-dioxo-1H-ben-
zo[de]isoquinolin-2(3H)-yl)-N,N-dimethylethanamine. NIM-4:
2-(6-(4-hydroxystyryl)-1,3-dioxo-1H-benzo[de]isoquinolin-
2(3H)-yl)-N,N-dimethylethanamine.
states, these chromophores are not significantly stabilized by
solvation due to a relatively small dipole moment. Conversely, as
shown in Fig. 1, strong solvatochromic red shifts of the emission
spectra were observed for compounds NIM-1, NIM-2 and NIM-
4 with increasing solvent polarity. Therefore, the maximum red
shifts observed in the emission spectra when increasing the
polarity were 88 nm, 122 nm, 52 nm and 90 nm for NIM-1, NIM-
2, NIM-3 and NIM-4, respectively. When comparing the fluo-
rescence properties of these chromophores, NIM-2 showed
longer emission wavelengths in all investigated solvents, as well
as the largest Stokes shifts. This was likely because the auxiliary
electron-donating ability of triphenylamine units offered good
effective conjugation in the p-linked donor compounds and
increased fluorescent solvatochromism. Alternatively, the
absorption bands of the NIM-3 were blue shifted and lower than
400 nm which can be attributed to the electron withdrawing
group of nitrobenzyl at the 4-position of naphthalimide core.
Further information regarding the solvent sensitivity of the
absorption and emission spectra of these dyes was obtained from
evaluation of the Stokes shifts of each of the NIM dyes in terms
of Lippert plots.23 The Lippert-Mataga equation describes the
dependence of the energy difference between the ground state
and the excited state (in cmꢂ1) on the refractive index (n) and the
dielectric constant (3) of the solvent:
Molecular design and synthesis
Ethylene acts as the p-linker at the centre of the D–p–A type
chromophore structure. In this investigation, the naphthalimide
moiety was selected as the p-electron acceptor because of its
good chemical stability and the commercial availability of
various useful molecular building blocks. The 4-methoxyphenyl,
triphenylamine, and 4-hydroxyphenyl moieties were chosen as
the electron-donating unit (molecules NIM-1, 2, and 4). Alter-
natively, the 4-nitrophenyl moiety was used as electron-acceptor
in molecule NIM-3 to construct an A–p–A type chromophore
and its fluorescence properties with respect to other naph-
thalimide derivatives were investigated. Next, to avoid decom-
position of the naphthalic anhydride upon Heck reaction,
4-bromo-1,8-naphthalic anhydride, the starting material, was
reacted with N,N-dimethylethylenediamine in ethanol as the
initial reaction step. We also examined this imidation reaction
under other reaction conditions such as 1,4-dioxane, tetrahy-
drofuran, ethanol and dimethylformamide for an appropriate
refluxing time period.22 However, the expected amine containing
imide substituent was not obtained because amination also
occurred on the 4-position with Br. Moreover, we propose that
the spacer N,N-dimethylethylenediamine should be more suit-
able than other aliphatic chains in cellular staining. Finally, the
naphthalimide derivative was coupled with aryl olefins. That is,
the vinyl aryl substituents—either electron donor or acceptor—
were substituted at the 4-positions of naphthalimide and the
molecular structures of the compounds were shown in Scheme 1.
nA ꢂ nF ¼ 2=hc ꢀ Dm2 ꢀ Df ꢀ ,aꢂ3 þ constant
Df ¼ ð3 ꢂ 1Þ=ð23 þ 1Þ - ðn2 - 1Þ=ð2n2 þ 1Þ; Dm ¼ mE ꢂ mG
ðnA ꢂ nFÞ=Df ¼ 11307:6 Dm2 ꢀ ,aꢂ3 þ constant
where nA and nF are the wavenumbers (cmꢂ1) of the absorp-
tion and emission maxima, respectively. h ¼ 6.6256 ꢀ 10ꢂ27
erg s is Planck’s constant; c ¼ 2.9979 ꢀ 1010 cm sꢂ1 is the
speed of light; and a is the radius of the cavity in which the
fluorophore resides; mG and mE refer to the ground state and
excited state dipole moments, respectively; and the term Of is
the orientation polarizability. The slope of the Lippert plot
reflects the solvent sensitivity of a fluorophore. The calculated
values of Of for each solvent are presented in Table 1 and the
plot of On (nA ꢂ nF) vs. Of, for these solvents we examined,
is presented in Fig. 2a. Positive solvatochromism was found
for all chromophores, indicating that the involvement of
solvent polarity dependent intramolecular charge transfer
(ICT) emissive states.24 In addition, a degree of deviation from
Basic spectroscopic properties
The wavelengths of maximum absorption and emission, molar
absorptivity, quantum yields and Stokes shifts of the all new
NIM compounds in different organic solvents comparing a wide
polarity range are listed in Table 1. No significant solvent
polarity dependent spectral shifts in the absorption spectra were
observed for any of the dyes. This indicates that, in their ground
Table 1 Photophysical data of NIM-1 to NIM-4 in solvents of different polarities.a
NIM-1
NIM-2
NIM-3
NIM-4
Solvent (Of)
labs (3)
lem (F)
On labs (3)
lem (F)
On labs (3)
lem (F)
On labs (3)
lem (F)
On
Methanol (0.309)
Ethanol (0.289)
Acetonitrile (0.305)
DMSO (0.263)
417 (2.58) 575 (0.52) 6589 456 (2.04) 663 (<0.01) 6846 391 (1.95) 469 (0.11) 4253 404 (1.08) 596 (0.13) 7169
417 (2.08) 564 (0.57) 6250 455 (0.83) 659 (0.016) 6803 391 (2.03) 465 (0.12) 4070 398 (1.08) 586 (0.30) 7234
398 (1.42) 552 (0.50) 7009 448 (1.67) 668 (0.02) 7351 391 (4.40) 473 (0.06) 4433 398 (1.17) 560 (0.49) 7411
425 (1.96) 564 (0.60) 5798 461 (2.88) 674 (0.015) 6855 402 (2.10) 503 (0.14) 4994 413 (1.30) 589 (0.26) 6344
Acetone (0.284)
Chloroform (0.148)
Ethyl Acetate (0.200) 396 (1.18) 513 (0.41) 5759 446 (1.17) 604 (0.77)
400 (1.58) 542 (0.48) 6549 448 (1.58) 658 (0.06)
408 (1.65) 521 (0.44) 5315 461 (1.63) 624 (0.36)
7123 392 (3.06) 466 (0.06) 4050 392 (1.15) 554 (0.52) 6975
5666 387 (1.76) 463 (0.15) 4241 405 (1.29) 543 (0.42) 6354
5865 388 (2.81) 453 (0.04) 3698 393 (0.79) 527 (0.39) 6260
5747 391 (2.45) 451 (0.03) 3402 397 (1.10) 530 (0.57) 6119
4205 387 (2.61) 456 (0.03) 3909 399 (1.30) 506 (0.40) 5617
THF (0.210)
Toluene (0.013)
405 (1.29) 516 (0.55) 5311 450 (1.86) 607 (0.54)
406 (1.33) 487 (0.40) 4096 448 (1.83) 552 (0.81)
a
labs: absorption maximum; 3: molar absorptivity (104); lEm: emission maximum; F: Quantum yield; On: Stokes shift (cmꢂ1); Of: orientation
polarizability of Lippert equation.
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Fig. 1 Normalized fluorescence emission spectra of 20 mM NIM compounds in different solvents. Excited wavelengths are absorption maxima. The
emission peaks show red shift with increasing polarity of solvents.
the linear correlation in the polar protic solvents such as
ethanol and methanol which can potentially undergo hydrogen
bonding interactions with dipolar dyes. We found that the
Stokes shift of NIM-3 was weakly dependent on the polarity
with two slopes, but showed an appreciable change in aprotic
ꢀ
solvents. Using a value of ꢃ7.0 A for the radius of the Ons-
ager cavity of the naphthalimide derivatives (7.17, 7.89, 7.20
ꢀ
and 7.09 A for NIM-1, NIM-2, NIM-3, and NIM-4, respec-
tively), Dm values of ꢃ16.8 D for NIM-1, ꢃ20.9 D for NIM-2,
and ꢃ16.5 D for NIM-4 were obtained from the slopes.
However, the Dm value was relatively smaller in NIM-4 (ꢃ11.8
and ꢃ15.2 D), which bears an electron withdrawing group,
compared to those compounds bearing electron donating
groups. The large values of Dm indicate a substantial contri-
bution of intramolecular charge transfer (ICT) in the excited
states of these NIM derivatives. In general, the ICT states of
NIM-1, NIM-2, and NIM-4 have larger dipole moments in the
excited state than in the ground state as a result of the charge
separation between the donor and acceptor. Thus, we can
support colorful fluorophores based on NIM derivatives which
can form an ICT state that is normally sensitive to the solvent
polarity.
The fluorescence quantum yields of the NIM-1–NIM-4 dyes
showed no direct correlation with the solvent polarity parame-
ters, as shown in Fig. 2b. Compound NIM-2 produced very high
quantum yields (>0.8) under nonpolar solvents while NIM-3
exhibited low photoluminescence ability (<0.2) in all solvents.
The quantum yield values of NIM-1 and NIM-4 in the evaluated
non-protic solvents were found to be between about 0.4 and 0.6
and their quantum yields also remained high in polar non-protic
solvents like acetonitrile. However, in the polar protic methanol
and ethanol solutions, the fluorescence emission of the NIM-2–
NIM-4 compounds were more strongly quenched, especially in
Fig. 2 (a) the Lippert-Mataga plot of the Stokes shift Dn, (b) quantum
the case of NIM-2 and NIM-3 (FF < 0.01), while NIM-1 was still
yields of NIM versus the solvent polarity Of (3, n).
significantly fluorescent (FF ꢃ 0.52).
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that can be successfully applied were based on a single type of
fluorophore structure, common to all integrated sensor mole-
cules. The fluorescent optical property of NIM-1 makes this
proposal become possible when we successfully designed and
synthesized compound NIM-4. Indeed, the phenol moiety can be
easily modified or labeled using suitable spacers to become
optical sensors, non-doping OLEDS, or laser dyes. Furthermore,
we expect that this type of fluorophore will be useful as a cellular
probe due to its unique optical property.
Unique optical properties of NIM derivatives
When compared with other NIM derivatives, compound NIM-1
exhibited the most stable luminescence properties in most
organic solvents, with quantum yields ranging from 0.4 to 0.6
(Table 1 and Fig. 2b). Based on our knowledge, fluorophores
On the other hand, as shown in Fig. 3, NIM-2 showed strong
fluorescence in the solid state with an emission maxima at 597 nm
(fwhm ¼ 68 nm), which was excitation wavelength independent,
and Stokes shift of ꢃ5207 cmꢂ1. When compared with the solu-
tion spectrum, the narrower solid-state emission band is
intriguing because spectral broadening is a very common
phenomenon for solid emitting materials. Thus, we propose that
there are few intermolecular interactions between molecules, and
together with a large Stokes shift, this provides favorable factors
that eliminate self-quenching and enhance their solid fluores-
cence.25 In this case, the introduction of a triphenylamine ring
increases the steric hindrance, which prevents the molecules from
packing compactly, thereby avoiding the spectral broadening. As
discussed above, other molecules of NIM have better planarity
and are regularly parallel to each other, with partial p–p
stacking over the naphthalimide–styrene core scaffold. There-
fore, the large red-shift in the solid emission maximum in NIM-2
can be attributed to the increased intermolecular interactions in
the solid state when compared to those in the solutions. This
strong fluorescent emission in its solid state has broadened the
family of AIEE fluorophores.
Fig. 3 Normalized absorption, fluorescence spectra and a photo of
NIM-2 in the solid state and in chloroform.
Another phenomenon of AIEE is presented as a form of fluo-
rescent organic nanoparticles. When we studied the emission
spectra of compound NIM-3 under mixing volume ratios of
Fig. 4 Illustrations of FONs for NIM-3; a) plots of the fluorescent
intensities of variable volumes of mixed solutions versus concentration of
[H+] values. The preparation of mixed solution was described in ref. 26. b)
Emission spectral variations for NIM-3 under 0, 25, 50, 75 and 100
percentages of 10 mM HCl aqueous solutions mixing ratios with THF.
(Excitation wavelength for each solution is 400 nm.) Insets show the
photographs and TEM images of nanoparticles obtained under 10 mM
HCl aqueous solutions mixed with THF, 25/75 v/v, and scale bar: 50 nm.
Fig. 5 The bright field image (left) and fluorescence images (right, light
path through a 380/10 nm bp filter and emission was collected and filtered
through a 450-nm lp filter) of MCF-7 breast cancer cells incubated with
10 mM NIM compounds for 4 h.
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solutions containing acidic aqueous and tetrahydrogenfuran
(THF),26 the fluorescence intensities of NIM-3 showed an inter-
esting change. As shown in Fig. 4, the emission intensity of NIM-3
increased drastically when [H+] was added. The quantum yields
reached a maximum value in 75% volume fractions of the THF,
and the AIEE properties eventually became saturated in the
presence of 1.5 mM concentration of acid (Fig. 4a). Under these
conditions, the relative TEM images (insets of Fig. 4b) clearly
showed nanoparticles that were very fine spheres with a mean
diameter of 5–20 nm. Based on previous studies, we considered
that protonated NIM-3 (NIM-3H+) should serve as a surfactant-
like molecule that can further self-assemble to form micelle-like
nanoparticles due to its amphiphilic characteristic.18 Accordingly,
a FON model was developed based on water/aprotic solvent pair
solutions. Overall, we propose that the NIM-3H+ molecule can
aggregate to form micelles and that AIEE phenomenon is the
cause of FONs. Alternatively, in neutral water/THF mixed solu-
tions, compounds do not exhibit AIEE under the same experi-
mental conditions. Thus, we conclude that a FONs model which
was formed with aggregations of NIM-3H+, but not NIM-3.
derivatives can enter cells and undergo intracellular localization
to serve as a fluorescence probe, cells were incubated with NIM
derivatives and then subjected to fluorescence microscopy
imaging. Fig. 5 showed the fluorescence microscopy cellular
images of MCF7 (Human breast adenocarcinoma cell line)
treated with NIM-1, NIM-2 and NIM-3 for 4 h. Surprisingly,
fluorescent images were observed in all three compounds with
unique bright patterns (threads or bright spots). When we
checked their cellular localization, the majority of distribution of
NIM-1 was merged with MitoTracker while that of NIM-2 was
merged with LysoTracker. And the NIM-3 was partially located
in the Endoplasmic Reticulum (ER) and lysosome. (Fig. S, ESI†)
However, there is no question that these three compounds can
penetrate the cellular membrane and stay in the cytoplasm.
Based on our results, the emission abilities of NIM-2 and NIM-3
in most solutions were very low. These fluorescent images are
worthy of investigation not only to determine the subcellular
localizations, but also to resolve the luminance mechanisms.
The cellular staining results of compounds NIM-1 and NIM-2
in co-cultured MRC-5 normal lung fibroblast cells and CL1-
0 lung cancer cells were further examined by fluorescence
confocal microscopy. The molecular imaging of NIM-1 under
confocal microscopy (Fig. 6a–d) and the l (wavelength) scanning
spectra did not differ between cancer and normal cells, and their
emission spectra were located in the reasonable range as shown in
Cell staining
Cell permeability allows the compounds to enter living cells
enabling intracellular applications. To examine if NIM
Fig. 6 Confocal microscopy images of co-cultured CL1-0 lung cancer and MRC-5 normal lung fibroblast cell lines. Cells were incubated with 10 mM
compound for 4 h, and a 405 nm diode laser was used as the light source. Compound NIM-1: a. Bright field image; b. spectra recorded at 450–560 nm in
PMT 1; c. 530–650 nm in PMT 2; d. merger of b with c; i. l scanning from 460 to 650 nm with 5 nm integration. Compound NIM-2: e. Bright field image;
f. spectra recorded at 450–590 nm in PMT 1; g. 560–740 nm in PMT 2; h. merger of f with g; j. l scanning from 450 to 780 nm with 10 nm integration,
insert photo represents the zoom in of h but filter cut at 560 nm. The spectra were obtained from the average of 50 individual cancer and normal cells.
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Fig. 6i. However, the cellular staining result obtained using
NIM-2 was noteworthy. In contrast to the results observed in
normal cells, cancer cells showed bright spots by confocal
microscopy (Fig. 6e–h) when the spectra were recorded at
450–590 nm in PMT 1 and 550–740 nm in PMT 2. Additionally,
the merge gallery revealed bright spots on cancer cells but not
normal cells. Evaluation of the l scanning spectra suggests
a different emission wavelength of NIM-2 between bright spots
(ꢃ595 nm) and others cancer cell lines as well as normal cells
(Fig. 6j). Similar results were also observed in other cancer cell
lines. These results indicate that NIM-2 molecules generally
spread in the cytoplasm of living cells and accumulate in cancer
cells, which results in the generation of bright spots. These
distinct cellular staining patterns indicate that NIM-2 can be
used to differentiate cancer cells from normal cells once suitable
filters (550–620 nm) are constructed—as shown in the inset image
in Fig. 6j, in which only cancer cells presented bright spots in the
cell staining. Unfortunately, observation of NIM-3 under
confocal microscopy was not successful owing to its higher
absorption energy.
showed good permeability and were well dispersed in the cyto-
plasm. First, investigation of NIM-1 revealed that NIM-4 may
serve as a precursor of DNA or protein labelling markers, for
example, conjugate to isothiocyanate and succinimidyl ester
functional groups with suitable spacers.28 Second, the weak
luminescence of NIM-2 and NIM-3 can achieve bright cellular
imaging owing to their AIEE properties. More importantly,
NIM-2 can be used in cancer cell recognition. Finally, we have
developed color fluorescent probes, with varying emission
wavelengths at a fixed excitation wavelength, for application in
cellular imaging. Eventually, the fluorescence-environment
dependence, large Stokes shift, long emission wavelength,
permanent fluorescence quantum yields, and AIEE of these
naphthalimide derivatives fluorophores can be used to develop
ultrasensitive fluorescent molecular probes to study a variety of
biological events and processes.
Acknowledgements
This work was supported financially by the National Science
Council (NSC 99-2113-M-005 -011-MY2) of Taiwan.
Investigation of the brightness of NIM derivatives in
cells
Notes and references
Based on the above optical results, the apparent fluorescence
emission of NIM-1 was observed, while NIM-2 and NIM-3
posses AIEE properties. It is reasonable that NIM-1 presents
cellular brightness because its emission ability is strong in most of
solvents or environments. However, the cellular staining results
of compounds NIM-2 and NIM-3 are interesting. Indeed, the
quantum yields of NIM-2 and NIM-3 in most solvents were very
low while strong fluorescence only occurred when NIM-2 dis-
solved in very low ET30 solvents. Thus, the bright cellular image
of NIM-2 can be explained by two possibilities. First, the cellular
environment in which the NIM-2 molecule was located should
have a lower polarity condition. Second, molecules aggregated
with very high concentration; that is, molecular imaging of NIM-
2 is the representation of AIEE. Both of these possibilities would
result in a strong emission phenomenon of this compound in
cells. Moreover, the bright cellular image of NIM-3 should be
attributed to one more possibility; fluorescence enhancement
arose from FONs phenomenon under acidic condition. It is
known that cellular cytoplasm is acidic and lysosomes maintain
at lower pH.27 Therefore, it is possible that neutral NIM-3 can
permeate the cell membrane and be protonated in the cytoplasm
to form FONs, and consequently show bright cellular images.
From this cursory study, we suggest that both the NIM-2 and
NIM-3 cases of molecular imaging are due to the presentation of
AIEE, but that they function through different mechanisms.
Nevertheless, we support colorful cellular probes with similar
exciting light sources (under fluorescent microscopy, Fig. 5) for
the application of molecular imaging.
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Conclusions
The results of this study indicate that colorful fluorophores were
developed from NIM derivatives and their large Stokes shifts can
prevent self-quenching and measurement errors caused excita-
tion light and scattering. NIM-1, NIM-2, and NIM-3 clearly
€
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