Fluorescent organic nanoparticle formation in lysosomes for cancer cell recognition

Article abstract of DOI:10.1039/b902399b

We present a convenient method to prepare fluorophores 3- or 3,7-divinyl substituted 10H-phenothiazines and their protonation forms can aggregate to become fluorescent organic nanoparticles (FONs) with bright fluorescent spots in the lysosomes of cancer cells but not normal cells.

Full text of DOI:10.1039/b902399b

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Fluorescent organic nanoparticle formation in lysosomes for cancer cell
recognition†
Hsin-Hung Lin, Sheng-Yuan Su and Cheng-Chung Chang*
Received 5th February 2009, Accepted 27th March 2009
First published as an Advance Article on the web 7th April 2009
DOI: 10.1039/b902399b
We present a convenient method to prepare fluorophores
3- or 3,7-divinyl substituted 10H-phenothiazines and their
protonation forms can aggregate to become fluorescent
organic nanoparticles (FONs) with bright fluorescent spots
in the lysosomes of cancer cells but not normal cells.
In this manuscript, we present a convenient method to prepare 3-
or 3,7-divinyl substituted 10H-phenothiazines (PVP and BPVP)
and build a donor–acceptor model as shown in Scheme 1. The
basicities of PVP and BPVP are evaluated since both fluorophores
can be partially protonated by lysosomes in the cells; the proto-
nated molecules, PVPH+ and BPVPH+, can then aggregate to form
fluorescent organic nanoparticles (FONs) with bright fluorescent
spots, which are clearly observed under a confocal microscope.
We developed optimized conditions for the aggregation-induced
enhanced emission (AIEE) properties of these PTZ derivatives,
and their related nanoparticles were observed by transmission
electron microscopy (TEM) images in vitro. Most importantly,
the bright fluorescent spots in lysosomes of cancer cells, which
were formed from the FONs, can be used in recognizing cancer
cells due to their multiple fluorescence characteristics.
The basic spectra of these compounds in DMSO solutions
are provided in Fig. 1. PVP and BPVP present well-defined
extinction coefficients and quantum yields in the visible region.
Once protonation is completed, the absorption bands of PVP and
BPVP show bathochromic shifts and the intensities of emission
peaks decrease in their quantum yields but with no apparent peak
shifts. We suggest that electron transfer from the phenothiazine
donor to the pyridinium acceptor and, subsequently, ground state
intramolecular charge transfer (ICT) is enhanced.10 However, the
excited state photo-induced electron transfer (PET) is weakened.11
Since the intensities of emissions are almost quenched in excess
acid, protonation spectra in Fig. 1 are presented with the end
points of protonation titration curves (Figure S1) of PVP and
BPVP in DMSO solutions. In order to estimate pKa values,
spectral changes as a function of pH for compounds PVP and
BPVP were measured in DMSO/H2O (5/95, v/v) solutions,
and their titration fitting curves are shown in Fig. 2. The pKa
values (also listed in Scheme 1) for the first protonation of
Cancer imaging using an optical technique is a sensitive, non-
invasive, and relatively inexpensive technique that has strong
potential for sensitive cancer diagnosis. In recent years, fluores-
cent organic nanoparticles (FONs) have received considerable
attention.1,2 Different from quantum dots and polymer nanopar-
ticles, FONs are expected to play various roles in a wide variety
of applications such as optoelectronic devices due to the flexibility
of synthetic approaches to such organic small compounds.2 The
switching of emission properties of FONs is often size-dependent
and related to the effects of intramolecular planarization or spe-
cific intermolecular aggregation conformation. However, there are
not many relevant topics on the application of FONs for cellular
diagnosis. The phenothiazine (PTZ) core has been considered to
be an important moiety in heterocyclic chemistry after it was first
reported in 1883.3 The well-defined electron-donating properties4
of PTZ can be partially associated with electrophores as it was
widely used in organic light-emitting diodes (OLEDs),5 acid–base
dyes and pigments,6 near-IR dyes,7 semiconductors,4,8 or chemical
sensors9 in material science.
Department of Chemistry, National Chung Hsing University, 250, Kuo
Kuang Road, Taichung, 402, Taiwan, R.O.C. E-mail: ccchang555@dragon.
nchu.edu.tw; Fax: +886-4-22862547
† Electronic supplementary information (ESI) available: Experimental ma-
terials, spectra and quantum yields measurements, cell culture incubation,
confocal cellular localizations, protonation spectra and solvent effect. See
DOI: 10.1039/b902399b
Scheme 1 Synthesis and protonation illustrations of PVP and BPVP. Reaction reagents and conditions: (i) NBS/THF, rt, 6 h; (ii) Pd(OAc)2/(o-tol)3P,
4-vinylpyridine, Et3N/MeCN, N2, 48 h.
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Fig. 1 (a) Absorption and (b) emission spectra for neutral and protonated PVP and BPVP in DMSO solutions. e: extinction coefficients (M-1 cm-1), U:
quantum yields (using Rhodamine Green as a standard). Protonation spectra were measured from the end points of the protonation titration curves of
PVP and BPVP in DMSO solutions, respectively.
Fig. 2 Absorption spectra of 25 mM (a) PVP and (b) BPVP in
DMSO/H2O (5/95, v/v) as a function of pH. The insets show the
absorption intensity plots (443 nm for PVP, 505 nm for BPVP) and their
pKa fitting curves (pKa values are shown as Scheme 1).
the compounds PVP (pKa ~ 3.64) and BPVP (pKa ~ 4.18) are
lower with respect to pyridine (~5.2), 2-vinyl pyridine (~4.8),
R
and the fluorescent pH indicator Oregon Greenꢀ 488 (~4.7)12;
while, in accord with distyryl-pyridine derivatives.13 The expected
stabilization of the protonated species (PVPH+ and BPVPH+)
Fig. 3 Confocal microscopy images of co-cultured CL1-0 lung cancer
and MRC-5 normal lung fibroblast cell lines. Cells were incubated with
show a lower pKa, indicating that a resonance effect between PTZ
6 mM BPVP for 4 h, and a 405 nm diode laser was used as the light
source. (a) Bright field image; (b) spectra recorded at 420–530 nm in PMT
1; (c) 530–650 nm in PMT 2; (d) merger of (b) with (c); (e) l scan from
470 to 650 nm with 5 nm integration. The spectra were obtained from the
average of 50 individual cancer and normal cells. (f)–(h): Magnified images
of 4 mM PVP co-staining with LysoTracker in a CL1-0 lung cancer cell.
The excitation source was a 405 nm diode laser for PVP compounds and
a 561 nm DPSS green laser for markers. Fluorescence photographs were
taken through related 420–530 nm (f) and 580–700 nm (g) ranges.
and pyridine moieties decreases the basicity of the first protonation
site. Obviously, further protonation at another pyridine ring of
BPVP occurs at pKa ~ 3.14 as compared to PVP. Furthermore,
excess acids can lead to the protonation of the 10 position N atom
on the PTZ moiety of PVP (pKa ~ 0.71) and BPVP (pKa ~ 0.22).
However, we have not provided a more detailed discussion as this
acidic process is not involved in the application of cellular staining.
The cellular staining results of compounds PVP and BPVP in
co-cultured MRC-5 normal lung fibroblast cells and CL1-0 lung
cancer cells are noteworthy. In contrast with the results observed in
normal cells, cancer cells show bright spots by confocal microscopy
(Fig. 3a–d) and the l scanning spectra suggests a different
emission wavelength of BPVP between normal and cancer cell lines
(Fig. 3e). Similar results are also reproduced in other cancer cell
lines (human osteosarcoma MG63, human lung adenocarcinoma
cell line A549 and a transformed murine fibroblast L929 cancer
line). Hence, we examined the immunofluorescence staining for the
intracellular localization of PVP and BPVP using commercially
available organelle probes (Figure S2). CL1-0 cancer cells were
incubated first with a compound for 4 h and then with the
organelle probes (MitoTracker Red CM-H2XRos for 30 min at
37 ◦C; LysoTracker Red DND-99 for 30 min at 37 ◦C). The
fluorescent bright spots were observed to colocalize with the
lysosomal markers, and the residues barely colocalized with the
mitochondrial markers. These results indicate that PVP and BPVP
molecules generally spread in the cytoplasm of living cells and
accumulate in lysosomes to result in the bright spots. The bright
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spots of lysosomes from PVP and BPVP both show stronger
emission bands at ~515 nm than residual remains in the cytoplasm
(560~580 nm), which is very similar to the emission peak positions
of free or protonated compounds in bulk solutions. Similar l
scanning spectra between PVP and BPVP are observed as expected
since their emission spectra centers and intensities are also similar
either in aqueous or DMSO solutions. Owing to these distinct
cellular staining patterns, we believe that both PVP and BPVP can
be used to differentiate cancer cells from normal cells once suitable
filters (510–530 nm) are constructed The image is shown in Fig. 3b
in which only cancer cells brighten up in the cell staining image.
In fact, after carefully examining the immunostaining images,
we observed that the bright spots do not fully merge with
LysoTracker but stay inside the lysosomal membrane (Fig. 3f–h).
Based on our knowledge, the enzymes contained in lysosomes
act optimally at a low pH; thus, lysosomes maintain a low pH
(~4.8) via proton-pumping vacuolar ATPases.14 According to
our pKa studies, the pyridine moieties of compounds PVP and
BPVP should be easily protonated or partially protonated in
fractions of the acidic water (as the insets in Fig. 4a and 4b) and
their relative TEM images (insets of Figs. 4c and Figs. 4d) clearly
show that PVPH+ and BPVPH+ nanoparticles are very fine spheres
with a mean diameter of 50–100 nm. Based on previous studies,2,3
it is known that the self-assembly of acidic PTZ compounds
leading to the formation of spherical nanoparticles is accompanied
by dramatic fluorescence changes. Alternatively, in the neutral
water/THF mixed solutions, PVP and BPVP do not exhibit AIEE
under the same experimental conditions. Thus, we conclude a
FONs model in which FONs were formed with aggregations of
PVPH+ or BPVPH+, but not PVP and BPVP, inside the vacuole of
lysosomes of cancer cells. That is, even though we incubated cell
lines with the neutral PVP or BPVP, the diversity of cellular images
comes from the outstanding behavior of PVPH+ or BPVPH+.
We also investigated the FON formation conditions of PTZ
derivatives under variable pH values and concentrations of
compounds in solutions. The nanoparticles are easily observed
in TEM images with varying concentrations from 5 to 100 mM
in acidic aqueous/THF mixed solutions. In addition, the distinct
AIEE are reproducible under the appropriate pH range (Figs. 4c
and Figs. 4d). Thus, we evaluate the AIEE areas for PVPH+
and BPVPH+ with their demarcation between pH values of 3.2–
4.7 and 3.5–5.2, respectively. When considering the dissociation
ability of HCl in the THF solvent, the actual pH values of these
mixed solutions should be higher than those marked in Fig. 4.
Thus, AIEE areas in Figs. 4c and Figs. 4d should cover the acidic
condition of lysosomes of living cells. On the other hand, either
in organic or aqueous solutions, the fluorescent intensities of PTZ
derivatives are decayed in the course of protonation. In addition,
R
lysosomes, like the fluorescent pH indicator Oregon Greenꢀ 488,
to become PVPH+ and BPVPH+. Then, the intensities of emissions
should be quenched with peak locations remaining unchanged (as
shown in Fig. 1). Therefore, the simple aggregation or solvent
effect does not seem effective in fully explaining the dramatic
increase in fluorescence in lysosomes of cancer cells. Thus, to
elucidate the formation of bright spots, we studied the emission
spectra of compounds PVP and BPVP under volume mixing
ratios of solutions containing certain pH acidic aqueous solutions
(pH = 3.95 for PVP, 4.21 for BPVP) and tetrahydrofuran (THF).
Figs. 4a and 4b show a drastic change in the fluorescence intensity
of PVP and BPVP in mixtures of acidic solution/THF.15 The
AIEE16 properties reach maximum quantum yields in 25% volume
the spectral properties of BPVPH2 are very close to BPVPH+,
+2
and the first protonation of BPVP is indistinguishable for the
di-substituted pyridine. Consequently, we observe that the AIEE
area of BPVPH+ is wider than PVPH+. In all, we suggest that
the protonated species PVPH+ or BPVPH+ serve as surfactant-
like molecules, which will further self-assemble to form micelle-
like nanoparticles due to their amphiphilic characteristics (their
critical micelle concentrations, CMC values, were determined in
Figure S3). Thus, it is reasonable to build a FON model based on
our studies, especially in the case of water/aprotic solvent mixed
solutions. However, at present, we have no evidence to elucidate
+2
+2
whether BPVPH2 produced in the cell or BPVPH2 can form
FONs. Nevertheless, we evaluate the AIEE formation conditions
and build a FON model for our PTZ derivatives. On the other
hand, based on our confocal studies, we believe that neutral PTZ
derivatives can locate in the lysosmes of cancer and normal cells,
while only protonated PTZ derivatives which stay in the lysosome
of cancer cells can form FONs and show the bright spot images.
According to the discussion above, the pH difference of lysosomes
between cancer and normal cells should be one of reasons to
achieve the cellular image difference between cancer and normal
cells.
Finally, we also explore the peak location of the l scanning
spectra in Fig. 3e. The solvent effect of PVPH+ and BPVPH+
instead of neutral compounds PVP and BPVP were measured
as shown in Figure S4. Both the emission bands showed hyp-
sochromic shifts along with hyperchromism in less polar solvents,
especially in toluene, ethyl acetate, and THF. Notably, these spectra
are collected under the first end point of the protonation titration
process in every solvent. They suggest that PVP or BPVP penetrate
Fig. 4 Illustrations of FONs for PVP (a, c) and BPVP (b, d). Emission
spectral variations under different mixing ratios of acidic aqueous solu-
tions at pH ~ 3.95 for (a) PVP and ~4.21 for (b) BPVP in THF. Insets
show the photographs obtained with the corresponding mixed solutions.
(c) The intensity plots of emissions of 0, 25 and 100% of H3O+/THF
mixed solutions containing PVP and (d) BPVP as functions of pH values,
respectively. Insets show the TEM images of nanoparticles obtained with
the corresponding mixed solution acidic solution/THF = 25/75 v/v (as
inset images of (a) and (b)). Excitation wavelength for each solution is
400 nm for PV and 440 nm for BPVP.
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into the cell and are then protonated in lysosomes. Eventually,
acidic compounds PVPH+ or BPVPH+ aggregate in the less polar
vacuoles to form FONs (Figs. 3f–h). This finding confirms the
AIEE results shown in Fig. 4—FONs are formed under the
acidic solution/THF (75/25, v/v) condition but not THF/acidic
solution (80/20, v/v) as described in literature.2b,2d Nevertheless,
we conclude that fluorescence is enhanced when compounds stay
in lysosomes but decreases in the cytoplasm. Further, these two
compounds can be used as cancer cell recognizers due to their
significant contrast in emission between cancer cells and normal
cells.
In summary, this manuscript describes the synthesis and eval-
uation of a set of 10H-phenothiazine derivatives that have been
modified in the 3 or 3,7- positions with electron-deficient pyridine
groups via ethenyl linkers. Their AIEE properties were observed
for both in vitro spectral studies and cellular staining. Finally, we
developed a vacuole model using FONs to elucidate the formation
of fluorescent bright spots, which are considered to form in the
lysosomes of cancer cells. With appropriate tuning of the spectral
characteristics of these compounds with regard to cellular staining,
they can be used as biomarkers in cell biology.
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Acknowledgements
This work was supported financially by the National Science
Council (NSC 97-2113-M-005-001) of Taiwan. Thanks Ta-Chau
Chang (Academia Sinica) for supporting cell lines and a number
of helpful discussions.
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