Multi-color cell imaging under identical excitation conditions with salicylideneaniline analogue-based fluorescent nanoparticles

Article abstract of DOI:10.1039/c4ra10021b

Six salicylideneaniline (SA) derivatives are synthesized through a condensation reaction. Benefiting from their coplanar molecular conformation and intramolecular hydrogen bonds, three of the compounds are found to exhibit aggregation-induced emission (AI

Full text of DOI:10.1039/c4ra10021b

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Multi-color cell imaging under identical excitation
conditions with salicylideneaniline analogue-based
fluorescent nanoparticles†
Cite this: RSC Adv., 2014, 4, 62021
a
b
c
b
a
c
*
*
Hongping Deng,‡ Bing Liu,‡ Chao Yang, Guolin Li, Yuanyuan Zhuang, Bo Li
and Xinyuan Zhu
a
*
Six salicylideneaniline (SA) derivatives are synthesized through a condensation reaction. Benefiting from
their coplanar molecular conformation and intramolecular hydrogen bonds, three of the compounds are
found to exhibit aggregation-induced emission (AIE) or aggregation-induced emission enhancement
(AIEE) behavior after self-assembling into nanoparticles that have a diameter of about 50 nm. Based on
the excited-state intramolecular proton transfer (ESIPT) properties, these fluorescent nanoparticles
(FNPs) display green, yellow or orange colors due to the formation of H- or J-aggregates. Interestingly,
FNPs derived from BMSpP show green to green-yellow fluorescence because of the partial
transformation of H-aggregates to J-aggregates. Under neutral conditions (pH ¼ 7.4), these FNPs are
stable, with the fluorescence intensity decreasing by less than 20% after 120 min, compared to a rapid
reduction at pH 5.5. Importantly, a two-photon fluorescence property of FNPs originating from
salicylideneaniline or its derivatives is reported for the first time. The two-photon absorption cross-
sections of the green, yellow and orange FNPs are found to be 7, 38 and 27 GM, respectively. After
conjugation with phospholipids, these FNPs show good water solubility and low cytotoxicity, which
make them potential candidates for cell imaging applications. Finally, multi-color cell imaging under
identical excitation conditions with single-emissive and multi-emissive FNPs has been achieved. These
results are significant for the control of the one- or two-photon fluorescent properties of such
derivatives, and provide a promising platform for multi-color cell imaging applications.
Received 8th September 2014
Accepted 10th November 2014
DOI: 10.1039/c4ra10021b
www.rsc.org/advances
limits their further bioapplications.5,7 On the other hand, organic
dyes are potential candidates for fabricating FNPs due to their
excellent optical properties and low toxicity.8 Unfortunately, the
Introduction
In recent years, uorescent nanoparticles (FNPs) have drawn a
great deal of attention for biomedical applications, such as cell-
specic targeting,1 bioanalysis,2 and detection and identication
of proteins.3 However, FNPs derived from uorescent tagging
methods oen suffer from some drawbacks.4,5 Self-luminescent
quantum dots (QDs) are widely applied to imaging and drug
delivery because of their long-term stability and the possibility of
simultaneous detection of multiple signals.6 Although QDs are
highly uorescent and photostable, their intrinsic cytotoxicity
aggregation of many organic dyes greatly quenches their light
emissions based on the aggregation-caused quenching (ACQ)
effect, which prevents the development of emissive nano-
particles.9 In 2001, Tang and his coworkers revealed a type of
highly emissive molecule in the aggregated state and dened this
phenomenon as aggregation-induced emission (AIE), providing
an example for obtaining highly emissive FNPs with organic
molecules.10 Since then, multi-color AIE uorophores have been
developed and widely used in biosensors and cell imaging appli-
cations.11,12 They also proposed the mechanism of the AIE effect to
be a process named restriction of intramolecular rotations (RIR)
and revealed that the E–Z isomerisation was not involved in this
process.13 However, the synthesis of AIE uorophores with large
Stokes shis in a convenient way is still a great challenge. An
efficient method to achieve large Stokes shis relies on excited-
state intramolecular proton transfer (ESIPT) molecules, which
have been reported as near infrared (NIR) or multi-color uo-
rophores, sensors and white luminescence materials.14–16
aSchool of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix
Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240,
P. R. China. E-mail: xyzhu@sjtu.edu.cn; Fax: +86-21-54741297; Tel: +86-21-34203400
bDepartment of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Harbin
Medical University, 23 Youzheng Street, Nangang District, Harbin 150001, P. R. China.
E-mail: liguolin@126.com; Fax: +86-21-54741297; Tel: +86-21-34203400
cKey Laboratory of Polar Materials and Devices, Ministry of Education, East China
Normal University, Shanghai 200241, P. R. China. E-mail: bli@ee.ecnu.edu.cn; Fax:
+86-21-54741297; Tel: +86-21-34203400
† Electronic Supplementary Information (ESI) available: 13C NMR spectra, TEM
data, MTT assays and other characterizations. See DOI: 10.1039/c4ra10021b
‡ These authors contributed equally to this work.
One of the best-known molecules demonstrating the ESIPT
phenomenon is salicylideneaniline (SA), which is the most
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 62021–62029 | 62021

View Article Online
RSC Advances
Paper
widely studied photochromic and thermochromic Schiff shis were referenced to residual peaks of deuterated solvents:
base.17–19 Also, many derivatives of SA have been synthesized in CDCl3 (7.26 ppm), DMSO-d6 (2.48 ppm).
order to study their spectroscopic and photophysical properties.
Fourier transform infrared (FTIR) spectroscopy. FTIR
In 2003, Grabowska and coworkers provided the pico- and spectra were measured between 4000 and 450 cmꢀ1 using a
femtosecond kinetics of N,N0-bis(salicylidene)-p-phenylenedi- Perkin Elmer Paragon 1000 spectrophotometer. All sample
amine (BSP), which is a famous symmetric derivative of SA.20 pellets were prepared by grinding the solid sample with dry
Usually, SA and BSP show very dim uorescence in solution and potassium bromide (KBr) under high pressure.
little research has focused on their AIE or AIEE properties. In
High resolution mass spectrometry (HRMS). HRMS was
2004, Yang and coworkers reported the uorescence enhance- performed on a Waters Micromass Q-TOF Premier Mass Spec-
ment of N,N0-bis(salicylidene)-p-phenylenediamine (BSP) in trometer. HRMS data were acquired for each sample from 50 to
nanoparticles compared to that in solution and also proposed a 1000 Da with a 0.10 s scan time and a 0.01 s interscan delay over
mechanism for the enhanced emission.21 Very recently, Ouyang a 10 min analysis time.
and coworkers controlled the color and morphology of four
Ultraviolet-visible (UV-vis) absorption spectra. The UV-vis
electron-donor-substituted AIE compounds.22 However, these absorption spectra of sample solutions were measured at
self-assembled structures are generally very large and lack water room temperature in the range of 265–550 nm on a Perkin
solubility and stability, precluding cell imaging applications. Elmer Lambda 20 UV-vis spectrometer.
Previously, we developed
a
macromolecular nanoparticle
Fluorescence and quantum yield measurements. The uo-
approach to obtain FNPs based on RIR of the uorophore.23,24 rescence emission measurements were carried out on a PTI-
Therefore, we believe that it will be very convenient to fabricate QM/TM/IM steady-state & time-resolved uorescence spectro-
multi-color FNPs with SA derivatives based on RIR and ESIPT.
uorometer (USA/CAN Photon Technology International Int.).
In the present work, multi-color FNPs are prepared and The excitation wavelength was lex ¼ 400 nm. Fluorescence
applied to multi-color cell imaging using SA derivatives. In quantum yields were determined using Rhodamine 6G as the
detail, six SA analogues are easily synthesized through standard (lex ¼ 488 nm, F ¼ 0.95). Quantum yields were
condensation reactions between amino and aldehyde groups. calculated using the same equation and method as given in the
Three of them show bright uorescence aer self-assembly into literature.25
H- or J-aggregates, owing to their coplanar molecular confor-
Two-photon uorescence spectra. The two-photon uores-
mation and intramolecular hydrogen bonds. The morphology cence was excited with fs pulses of different intensities at 800
of these aggregates is spherical with a diameter of about 50 nm, nm using a spectrophotometer (iHR550, HORIBA). The two-
conrmed by transmission electron microscopy (TEM). More- photon absorption cross sections were measured with Rhoda-
over, all the FNPs have good photostability under neutral mine B as the standard.
conditions and pH-responsive properties in acidic environ-
Transmission electron microscopy (TEM). TEM images were
ments. Importantly, the two-photon uorescence of these SA- obtained using a JEOL JEM-100CX-II instrument at a voltage of
derived FNPs is proven for the rst time. To obtain low cyto- 200 kV. Samples were prepared by drop-casting nano-aggregate
toxicity and good water solubility, phospholipids are used to solutions onto carbon-coated copper grids and then freeze-
encapsulate these FNPs. Finally, multi-color cell imaging under drying under vacuum before measurements.
identical excitation conditions is achieved using single-emissive
and multi-emissive FNPs.
Sample preparation
Preparation of nano-aggregates. Aer all the compounds
Experimental section
Materials
were dissolved in N,N0-dimethylformamide (DMF), deionized
water was added to the DMF solution via a syringe or washing
bottle to form a mixed system. Finally, the mixtures were
vibrated using an oscillator or just by hand. Then, the mixtures
were dialyzed using a dialysis bag (MWCO ¼ 3500 Da) for 12 h
before measurements, if necessary. For pH stability and pho-
tostability experiments, the mixture was dialyzed against PBS
solution (pH ¼ 7.4) and then dilute hydrochloric acid (HCl) was
added to tune the pH to 5.5 when necessary.
p-Phenylenediamine, m-phenylenediamine, o-phenylenediamine,
4-hydroxy benzaldehyde, 3,5-di-tert-butyl-2-hydroxybenzaldehyde,
salicylaldehyde and 2-hydroxy-4-methoxybenzaldehyde were
purchased from Aladdin and 1,2-dioleoyl-sn-glycero-3-
phosphocholine (DOPC) was purchased from Sigma Aldrich.
Ethanol (EtOH) was heated at reux with calcium oxide and then
distilled prior to use. Other reagents and solvents were purchased
from Shanghai Sinopharm reagent Co. Ltd., Shanghai, and used
without further purication unless otherwise mentioned.
Fabrication of single-emissive aggregates. Due to its excel-
lent biocompatibility and water solubility,26 DOPC was chosen
to encapsulate the different nano-aggregates. Firstly, DOPC was
dissolved in a methanol–chloroform (v/v ¼ 1 : 1) mixture and
the solvents were removed under reduced pressure to form a
Characterization
Nuclear magnetic resonance (NMR). 1H and 13C NMR thin lm. Then, one type of single nano-aggregate solution was
spectra were measured using a Varian MERCURY plus 400 NMR added and kept under ultrasound for 20 min.
spectrometer at 298 K with deuterated chloroform (CDCl3) or
Fabrication of multi-emissive aggregates. Briey, DOPC was
dimethyl sulfoxide-d6 (DMSO-d6) as the solvent. The chemical dissolved in a methanol–chloroform (v/v ¼ 1 : 1) solvent
62022 | RSC Adv., 2014, 4, 62021–62029
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
RSC Advances
mixture to form a thin lm aer removal of solvents. Then,
three types of single aggregate solution were added in a ratio of
3 : 5 : 12 (v/v/v) for 4, 5 and 6, respectively. Aer 20 min soni-
cation, the sample was successfully prepared.
Cell culture and internalization
L929, A549 or MCF-7 cells were seeded in exiPERM reusable
cell culture chambers in combination with glass coverslips and
cultured in DMEM (Dulbecco’s modied Eagle’s medium) (F-
12K was used for A549 cells) supplied with 10% FBS (fetal
bovine serum) and antibiotics (50 units per mL penicillin and
50 units per mL streptomycin) at 37 ꢁC under a humidied
atmosphere containing 5% CO2. Aer 24 h of culture, the
phospholipid-loaded single- or multi-emissive aggregates were
added to the culture wells, and the cells were incubated at 37 ꢁC
for 15 min or 30 min. Aer washing with PBS 3 times, the cells
were xed with 4% formaldehyde for 15 min at room temper-
ature, and then the slides were mounted and observed using a
DMI6000B. The excitation wavelength for all samples was 405
nm.
Scheme
1
The synthetic route and chemical structures of SA
derivatives N,N0-bis(4-hydroxyphenylene)-p-phenylenediamine
(BHPpP) (1), N,N0-bis(salicylidene)-m-phenylenediamine (BSmP) (2),
N,N0-bis(salicylidene)-o-phenylenediamine (BSoP) (3), N,N0-bis(sali-
cylidene)-p-phenylenediamine (BSpP) (4), N,N0-bis(4-methoxy-sali-
cylidene)-p-phenylenediamine (BMSpP) (5) and N,N0-bis(3,5-
bis(tertiarybutyl)-salicylidene)-p-phenylenediamine (BTBSpP) (6).
bands at about 293 nm and 361 nm, ascribed to the electron
transitions of p–p* and n–p*, respectively. Compared to
BHPpP, the n–p* transition absorption of BSpP (4) red-shis to
371 nm, while that of the p–p* blue-shis to 276 nm, which can
be attributed to the six-membered-ring intramolecular
hydrogen bonds. However, the n–p* transition absorptions of
compounds BSmP (2) and BSoP (3) present a blue-shi
phenomenon with the formation of intramolecular hydrogen
bonds, which is ascribed to the destruction of the coplanar
conformation of the molecules. As a matter of course, the
increased electron donating abilities of BMSpP (5) and BTBSpP
(6) result in red-shis in both the p–p* and n–p* absorptions.
Surprisingly, the DMF solutions of all the compounds show very
weak or even undetectable uorescence at room temperature.
Considering that SA compounds demonstrate AIE or AIEE
characteristics,21,22 we believe that these SA analogues also have
great potential as AIE or AIEE uorophores by RIR.
The AIE or AIEE characteristics of compounds 1–3 were
investigated in DMF–H2O solutions. Fig. 2B presents the UV
absorbance spectra of BHPpP in different DMF–H2O mixtures.
BHPpP has two absorption peaks in pure DMF as mentioned
above. However, the second absorption peak, which belongs to
the n–p* transition, gradually disappears with the increase in
water content, strongly indicating the breakage of the C]N
bonds. Indeed, the UV absorption spectrum of BHPpP in DMF–
H2O (1 : 99) is the same as that of the reaction starting mate-
rials. The lack of six-membered-ring intramolecular hydrogen
bonds leads to the fast breakage of the C]N bonds in BHPpP.
For compounds BSmP and BSoP, the destruction of the
coplanar conformation results in the heterogeneous formation
of J- or H-aggregates, which can be conrmed by the UV
absorbance spectra having both red- and blue-shi character-
istics (Fig. 2C and D).27–29 Thus, the nano-aggregates of both
BSmP and BSoP show undetectable uorescence in all DMF–
H2O solutions. In principle, compounds 1–3 act as references to
emphasize the importance of intramolecular hydrogen bonds
Cytotoxicity measurements of nano-aggregates
The cytotoxicity of the single-emissive aggregates was estimated
using an MTT viability assay against L929 cells. Firstly, L929
cells were placed into 96-well plates at a density of 1 ꢂ 104 cells
per well in 200 mL medium. Aer 24 h, the culture medium was
replaced with 200 mL of serial dilutions of aggregate. The cells
were cultured for another 48 h. Then, 20 mL of 5 mg mLꢀ1 MTT
assay stock solution in PBS was added to each well. Aer 4 h, the
medium was carefully replaced with 200 mL of DMSO and the
absorbance was measured using a BioTek® Synergy H4 at a
wavelength of 490 nm.
Results and discussion
SA derivatives were facilely prepared using the synthetic route in
Scheme 1. By changing the molecular structures of the different
reagents, six compounds were synthesized through a conven-
tional condensation of phenylenediamine and phenyl-
enealdehyde with good yields (>95%).21,22 Details of synthetic
procedures and identication are shown in the ESI.† SA deriv-
atives generally form six-membered-ring intramolecular
hydrogen bonds between the –OH groups and the N atoms,
1
which can be conrmed from the H NMR spectra (Fig. 1A).14
Compound BHPpP (1) exhibits a phenol signal at 10.10 ppm
due to its incapacity to form intramolecular hydrogen bonds.
Compared to BHPpP, the other compounds show signicantly
downeld phenol signals from 12.93 ppm to 13.93 ppm, giving
an obvious indication of the formation of strong hydrogen
bonds. It is clear that stronger electron donating abilities result
in a greater downeld shi in the phenol signals. Their struc-
ture was further characterized by FTIR and 13C NMR (Fig. 1B
and S1†).
To explore the potential optical properties of the six
compounds, UV absorbance spectra were measured and are
shown in Fig. 2A. Compound BHPpP (1) has two absorption
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 62021–62029 | 62023

View Article Online
RSC Advances
Paper
Fig. 1 1H NMR (A) and FTIR spectra (B) of salicylideneaniline derivatives BHPpP (1), BSmP (2), BSoP (3), BSpP (4), BMSpP (5) and BTBSpP (6).
Fig. 2 UV spectra of salicylideneaniline derivatives: (A) BHPpP (1), BSmP (2), BSoP (3), BSpP (4), BMSpP (5) and BTBSpP (6) in DMF; (B) BHPpP (1) in
DMF–H2O mixtures; (C) BSmP (2) in DMF–H2O mixtures; (D) BSoP (3) in DMF–H2O mixtures. Water contents (%) are listed.
and coplanar molecular conformation on the AIE or AIEE that shows an obvious AIE yellow uorescence aer self-
properties.
assembling into nano-aggregates. Under lower water content
In addition, the AIE or AIEE characteristics of compounds 4– (<75%), the UV peak displays both blue- and red-shi potential
6 were investigated in detail. BSpP was an accidental discovery compared to that with DMF, which can be regarded as a non-
62024 | RSC Adv., 2014, 4, 62021–62029
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
RSC Advances
equilibrium state. However, a clearly red-shied UV peak (by content (Fig. 3C), which gives a clear conrmation of the
about 70 nm) under higher water content indicates the forma- formation of H-aggregates.29 Moreover, the DMF solution of
tion of J-aggregates, which is more optimum under 90% water BMSpP shows weak green uorescence with an emission peak
content with a better buffer capacity compared to that with 99% at about 511 nm. With an increase in water content to 90%, the
water (Fig. 3A). Moreover, BSpP has undetectable uorescence uorescence intensity increases greatly and the emission peak
in DMF at room temperature. When water (a poor solvent for displays a 6 nm red-shi, while with 99% water content, the
BSpP) is added to the DMF solution, the emission intensity uorescence intensity decreases drastically and the emission
grows drastically with increasing water content, displaying a peak shows a further 4 nm red-shi (Fig. 3D). These experi-
clear AIE active feature (Fig. 3B).10–12 The emission peak is at 547 mental results indicate that BMSpP has an AIEE character and
nm, displaying a large Stokes shi (147 nm) owing to the ESIPT higher (99%) water content is not preferable for the formation
effect.14,16 With 99% water content, the emission intensity of ordered H-aggregates. It is amazing that the nano-aggregates
decreases due to the formation of less ordered J-aggregates. of BMSpP show a green-yellow uorescence with an emission
Based on these results, BMSpP and BTBSpP were designed peak at about 525 nm when adding water at a much faster rate
and prepared using different salicylaldehyde substituents and (Fig. S2†). However, the green-yellow uorescence rapidly
their optical properties were also investigated. Compared to vanishes within hours, indicating the formation of unstable
BSpP in mixed DMF–H2O solvents, the UV absorption peak of aggregates. The UV absorption spectra of BTBSpP in DMF and
BMSpP blue-shis by about 55 nm with an increase in water under low water content (below 80%) are almost identical and
Fig. 3 Normalized UV absorbance and fluorescence emission spectra of SA derivatives BSpP (4), BMSpP (5) and BTBSpP (6) in DMF–H2O
mixtures (10 mM, lex ¼ 400 nm) with varying volumetric fractions of water: BSpP (A and B), BMSpP (C and D), and BTBSpP (E and F).
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 62021–62029 | 62025

View Article Online
RSC Advances
Paper
only show a red-shi of about 10 nm with high water content photon properties along with sizes of all three FNPs are listed
(above 90%), demonstrating the formation of weak J-aggregates and presented in Table S1.†
due to the steric hindrance from the tert-butyl groups (Fig. 3E).
To conrm the formation of H- or J-aggregates of these
The uorescence emission spectrum of BTBSpP in DMF shows a compounds, concentration-dependent UV-vis absorption and
clear emission peak at about 574 nm and the emission intensity steady state uorescence spectra were measured in different
increases with increasing water content, which conrms the mixed solvents. Fig. S5† gives the corresponding results in a
AIEE feature.10–12 Just as with BSpP and BMSpP, the emission DMF–H2O solvent mixture. At low concentration, the absorp-
intensity of BTBSpP decreases under the highest water content , tion peak of the BSpP nanoparticles is at about 362 nm. With an
but without an obvious emission peak shi (Fig. 3F). To illus- increase in concentration, an obvious red-shi in the absorp-
trate the generality of the AIE or AIEE properties of compounds tion peak is clearly seen with a Stokes shi of about 68 nm,
4–6, the uorescence enhancement features for the corre- conrming the formation of J-aggregates. However, the uo-
sponding FNPs in THF–H2O and DMSO–H2O mixtures were also rescence can not be seen at low concentration. For the BMSpP
measured and are shown in Fig. S4.† From the results, the nanoparticles, a clear blue-shi in the absorption peak with
uorescence enhancement behavior is obvious in all the mixed increasing concentration affirms the formation of H-aggregates.
solvents. Thus, the highest uorescence quantum yields (QYs) In accord with the absorption, the uorescence peak also shows
of the green, yellow and orange FNPs were found to be 0.10, 0.26 a blue-shi of about 3 nm with higher concentration, con-
and 0.02, respectively (Table S1†). Subsequently, transmission rming the formation of H-aggregates. However, the absorption
electron microscopy (TEM) was applied to illustrate the and uorescence peaks of the BTBSpP nanoparticles also
morphology of the FNPs with the highest QYs (Fig. S3†). All of display a weak bathochromic-shi with increasing concentra-
the aggregates present a spherical structure with diameters of tion, which indicates the formation of weak J-aggregates.
about 57 nm, 52 nm, and 48 nm for BSpP, BMSpP and BTBSpP, Finally, concentration-dependent absorption and uorescence
respectively. For clarity, the UV-vis, uorescence and two- spectra were detected in DMSO–H2O and THF–H2O solvent
Fig. 4 Fluorescence stability studies under neutral (pH ¼ 7.4) and acidic (pH ¼ 5.5) conditions for BSpP, 4 (A), BMSpP, 5 (B) and BTBSpP, 6 (C), and
photostability of nano-aggregates (D, pH ¼ 7.4).
62026 | RSC Adv., 2014, 4, 62021–62029
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
RSC Advances
mixtures (Fig. S6 and S7†), and almost the same phenomena Fig. 4, all three nano-aggregates are very stable at pH ¼ 7.4 with
were observed for these systems, which further illustrates the the uorescence intensity decreasing less than 20% aer 120
formation of H- or J-aggregates of these compounds.
min, in contrast with the rapid degradation of BHPpP (1).
Considering that the SA analogues were connected by imine However, the uorescence intensity fades rapidly under acidic
bonds, the pH-responsive properties of the nano-aggregates of conditions at pH ¼ 5.5, especially during the rst 25 min.
different colors were studied in PBS solutions. As shown in However, the rate of fade of the uorescence intensity decreases
Fig. 5 Two-photon fluorescence emission spectra (A, C and E) and the corresponding linear fitting (B, D and F) of BSpP, BMSpP, and BTBSpP
under different excitation powers: BSpP (A and B), BMSpP (C and D), and BTBSpP (E and F).
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 62021–62029 | 62027

View Article Online
RSC Advances
Paper
between BSpP and BTBSpP, indicating the effect of steric same excitation. As depicted in Fig. 6, green, yellow and red
hindrance of substituent groups toward the migration of uorescence is very obvious in A549 and MCF-7 cells aer
protons. Subsequently, the photostability of the three nano- culturing for 15 min and 30 min. Indeed, the uorescence
aggregates in PBS solution at pH ¼ 7.4 was explored and is signals are from multi-emissive nanoparticles with the same
shown in Fig. 4D. In general, the three nano-aggregates show excitation conditions, which can raise the analysis reliability
good photostability with more than 80% uorescence intensity and accuracy.30
remaining aer 60 min irradiation with a 365 nm lamp.
To our surprise, all three SA-derived FNPs exhibit two-photon
uorescence properties, which have never been reported for SA
Conclusions
and its derivatives. In Fig. 5, the two-photon emission spectra In conclusion, six SA derivatives are easily synthesized with high
recorded under different radiation powers are shown. With an yields using traditional condensation reactions. Due to the
increase in radiation power, the emission intensity increases. ESIPT phenomenon, three chromophores with more coplanar
The good linear correlation between intensity (area) and the structures show an AIE or AIEE feature aer self-assembling
square of pump power conrms the two-photon emission into H- or J-aggregates. Moreover, the two-photon uorescence
properties of the three FNPs. In addition, the two-photon of the three SA aggregates is conrmed for the rst time. Finally,
absorption cross-sections are found to be 38, 7 and 27 GM for multi-color uorescence imaging with multi-emissive nano-
BSpP, BMSpP, and BTBSpP nanoparticles, respectively.
aggregates is successfully achieved under identical excitation
To benet from their bright and multi-colored uorescence, conditions using A549 and MCF-7 cells.
we intended to use the three types of SA FNP for cell imaging.
However, all three FNPs were unstable and aggregated in the
cell culture medium, which inhibited their cell applications. To
Acknowledgements
solve this problem, commercial phospholipids were employed This work was nancially supported by the National Basic
to regulate the cell entry of the nanoparticles, due to their good Research
Program
(2015CB931801,
2012CB821500,
biocompatibility and water solubility.26 As shown in Fig. S8,† 2013CB834506), and National Natural Science Foundation of
the phospholipid-encapsulated FNPs present little cytotoxicity China (51473093).
towards L929 cells even at the highest concentration (10 mM).
Subsequently, A549 and MCF-7 cells were cultured with single-
emissive nanoparticles and bright uorescence could be
Notes and references
observed aer 15 min or 30 min (Fig. S9†). The uorescence is
mostly in the cytoplasm of both types of cell. Subsequently,
multi-emissive nanoparticles were prepared by mixing the
green, yellow and orange FNPs, and these were then encapsu-
lated with phospholipids. Thus, the multi-emissive nano-
particles were applied in multi-color cell imaging under the
1 R. Weissleder, K. Kelly, E. Y. Sun, T. Shtatland and
L. Josephson, Nat. Biotechnol., 2005, 23, 1418.
2 R. Gill, M. Zayats and I. Willner, Angew. Chem., Int. Ed., 2008,
47, 7602.
3 C.-C. You, O. R. Miranda, B. Gider, P. S. Ghosh, I.-B. Kim,
B. Erdogan, S. A. Krovi, U. H. F. Bunz and V. M. Rotello,
Nat. Nanotechnol., 2007, 2, 318.
4 C. R. Jing and V. W. Cornish, Acc. Chem. Res., 2011, 44, 784.
5 X. D. Xue, Y. Y. Zhao, L. L. Dai, X. Zhang, X. H. Hao,
C. Q. Zhang, S. D. Huo, J. Liu, C. Liu, A. Kumar,
W.-Q. Chen, G. Z. Zou and X.-J. Liang, Adv. Mater., 2014,
26, 712.
6 I. L. Medints, H. T. Uyeda, E. R. Goldman and H. Mattoussi,
Nat. Mater., 2005, 4, 435.
7 K. T. Yong, W. C. Law, R. Hu, L. Ye, L. W. Liu, M. T. Swihart
and P. N. Prasad, Chem. Soc. Rev., 2013, 42, 1236.
8 L. Yuan, W. Y. Lin, K. B. Zheng, L. W. He and W. M. Huang,
Chem. Soc. Rev., 2013, 42, 622.
9 W. Z. Yuan, P. Lu, S. Chen, J. W. Y. Lam, Z. Wang, Y. Liu,
H. S. Kwok, Y. Ma and B. Z. Tang, Adv. Mater., 2010, 22, 2159.
10 J. Luo, Z. Xie, J. W. Y. Lam, L. Cheng, H. Chen, C. Qiu,
H. S. Kwok, X. Zhan, Y. Liu, D. Zhu and B. Z. Tang, Chem.
Commun., 2001, 1740.
11 Y. Liu, C. M. Deng, L. Tang, A. J. Qin, R. R. Hu, J. Z. Sun and
B. Z. Tang, J. Am. Chem. Soc., 2011, 133, 660.
12 C. W. T. Leung, Y. N. Hong, S. J. Chen, E. G. Zhao,
J. W. Y. Lam and B. T. Tang, J. Am. Chem. Soc., 2013, 135, 62.
13 N. W. Tseng, J. Z. Liu, J. C. Y. Ng, J. W. Y. Lam, H. H. Y. Sung,
I. D. Williams and B. Z. Tang, Chem. Sci., 2012, 3, 493.
Fig. 6 Multi-color fluorescence imaging of A549 (A and B) and MCF-7
(C and D) cells using multi-emission FNPs for 15 min (A and C) and 30
min (B and D) with green, yellow and red channels under identical
excitation conditions. Scale bar ¼ 20 mm. Concentration ¼ 2 mM, lex
¼
405 nm.
62028 | RSC Adv., 2014, 4, 62021–62029
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
RSC Advances
14 K. C. Tang, M. J. Chang, T. Y. Lin, H. A. Pan, T. C. Fang, 23 H. P. Deng, Q. Zhu, D. L. Wang, C. L. Tu, B. S. Zhu and
K. Y. Chen, W. Y. Huang, Y. H. Hsu and P. T. Chou, J. Am.
X. Y. Zhu, Polym. Chem., 2012, 3, 1975.
Chem. Soc., 2011, 133, 17738.
15 D. Maity, V. Kumar and T. Govindaraju, Org. Lett., 2012, 14,
24 Y. J. Zheng, G. L. Li, H. P. Deng, Y. Su, J. H. Liu and X. Y. Zhu,
Polym. Chem., 2014, 5, 2521.
6008.
25 A. Baldridge, S. H. Feng, Y.-T. Chang and L. M. Tolbert, ACS
Comb. Sci., 2011, 13, 214.
16 S. Park, J. E. Kwon and S. Y. Park, Phys. Chem. Chem. Phys.,
2012, 14, 8878.
26 K. Raemdonck, K. Braechmans, J. Demeester and
S. C. D. Smedt, Chem. Soc. Rev., 2014, 43, 444.
17 E. Hadjoudis and I. M. Mavridis, Chem. Soc. Rev., 2004, 33,
¨
579.
27 T. E. Kaiser, V. Stepanenko and F. Wurthner, J. Am. Chem.
18 M. Z. Zgierski and A. Grabowska, J. Chem. Phys., 2000, 112,
6329.
Soc., 2009, 131, 6719.
¨
¨
28 F. Wurthner, T. E. Kaiser and C. R. Saha-Moller, Angew.
19 V. Vargas, J. Phys. Chem. A, 2004, 108, 281.
Chem., Int. Ed., 2011, 50, 3376.
´
20 M. Zołek, J. Kubicki, A. Maciejewski, R. Naskr˛ecki and 29 R. J. Dong, B. S. Zhu, Y. F. Zhou, D. Y. Yan and X. Y. Zhu,
A. Grabowska, Chem. Phys. Lett., 2003, 369, 80. Angew. Chem., Int. Ed., 2012, 51, 11633.
21 S. Y. Li, L. M. He, F. Xiong, Y. Li and G. Q. Yang, J. Phys. 30 F. C. Alexandra, M. S. Chad, N. Iuliia, P. Stephane and
´
Chem. B, 2004, 108, 10887.
V. E. Svetlana, Angew. Chem., Int. Ed., 2014, 53, 2927.
22 C. X. Niu, L. Zhao, T. Fang, X. B. Deng, H. Ma, J. X. Zhang,
N. Na, J. S. Han and J. Ouyang, Langmuir, 2014, 30, 2351.
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 62021–62029 | 62029