Aggregation induced emission-active two-photon absorption zwitterionic chromophore for bioimaging application

Article abstract of DOI:10.1016/j.saa.2019.117571

The fabrication of two-photon absorption material is a versatile approach to achieve high resolution bioimaging with low phototoxicity yet remain sophisticated. Herein, a zwitterionic chromophore, MF, with D-π-A configuration has been rational designed and synthesized. Remarkably, MF exhibited enhanced one- and two-photon fluorescent in the aggregation states. Additionally, the obtained MF NPs encapsulated by Pluronic F-127, could be employed as a two-photon fluorescent probe for bioimaging. The results reveal that MF NPs could target mitochondria by using two-photon confocal microscopy and stimulated emission depletion nanoscopy methods.

Full text of DOI:10.1016/j.saa.2019.117571

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 226 (2020) 117571
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Aggregation induced emission-active two-photon absorption
zwitterionic chromophore for bioimaging application
,
b
,
,
,
, **
Fei Meng a 1, Chengkai Zhang a 1, Dandan Li a *, Yupeng Tian a
a Institute of Physics Science and Information Technology, College of Chemistry and Chemical Engineering, Key Laboratory of Functional Inorganic Materials
Chemistry of Anhui Province, Anhui Province Key Laboratory of Chemistry for Inorganic/Organic Hybrid Functionalized Materials, Key Laboratory of
Structure and Functional Regulation of Hybrid Materials of Ministry of Education, Anhui University, Hefei, 230601, PR China
b Department of Food and Environmental Engineering, Chuzhou Vocational and Technical College, Chuzhou, 239000, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
The fabrication of two-photon absorption material is a versatile approach to achieve high resolution
Received 15 August 2019
Received in revised form
18 September 2019
Accepted 27 September 2019
Available online 10 October 2019
bioimaging with low phototoxicity yet remain sophisticated. Herein, a zwitterionic chromophore, MF,
with D-p-A configuration has been rational designed and synthesized. Remarkably, MF exhibited
enhanced one- and two-photon fluorescent in the aggregation states. Additionally, the obtained MF NPs
encapsulated by Pluronic F-127, could be employed as a two-photon fluorescent probe for bioimaging.
The results reveal that MF NPs could target mitochondria by using two-photon confocal microscopy and
stimulated emission depletion nanoscopy methods.
Keywords:
Two-photon
© 2019 Elsevier B.V. All rights reserved.
Aggregation induced emission
Mitochondria
Bioimaging
1. Introduction
with optimized biocompatibility has been found to be a critical
determination for bioimaging, and which exhibit excellent fluo-
As the powerhouses of the cell, mitochondria are highly dy-
namic organelles regulated by coordinated fission and fusion
events [1]. Previous works have revealed that damaged or un-
wanted mitochondria can be selectively cleared away during the
mitophagy process to maintain a healthy population of mitochon-
dria [2e5]. Furthermore, amounts of studies unveiled that mito-
chondrial dynamics play crucial roles in controlling antitumor
immune responses [6]. In this sense, visualizing mitochondrial
dynamic changes is vital important in the fields of physiology, pa-
thology and pharmacology.
Thanks to its remarkable sensitivity, excellent spatial resolution
and noninvasive operation, fluorescence microscopy has become a
powerful tool for medical and biological applications [7e16].
Thereinto, benefit from the near infrared excitation light, two-
photon confocal microscopy has been exploited as an optimized
approach for bioimaging with deeper tissue penetration, higher
spacial resolution and weaker specimen photodamage [17e24].
Therefore, the search for novel two-photon absorbing materials
rescence under physiological condition is a highly desired target.
Bearing these considerations in mind, herein, we deployed a
systematic protocol to fabricate an ionic character or a complete
charge separation (i.e., zwitterionic) 2 PA chromophore within a D-
p-A model, MF (Scheme 1). The results show that MF displays
enhanced charge mobility and unveils obviously two-photon ac-
tivity in near infrared region because of its zwitterionic feature
[25,26]. In addition, the cationic properties drive it into mito-
chondria via electrostatic interaction [27] to visualize mitochon-
drial dynamic changes under two-photon confocal microscopy and
stimulated emission depletion nanoscopy methods.
2. Experiments
The synthetic routes of intermediate materials and target mo-
lecular (MF) in this work were shown in supporting information
(Scheme S1). As for MF, the synthetic procedure and characteriza-
tion data was displayed as follows: M2 (1 g, 3 mmol) was dissolved
in ethyl alcohol completely, then F1 (1.26 g, 5 mmol) was added
successively. The reaction was stirred at 80 ꢀC for 5 h. The mixture
was cooled to room temperature. Filtration with red product, yield
* Corresponding author.
** Corresponding author.
81.9%. 1H NMR (400 MHz, DMSO-d6)
J ¼ 16.1 Hz, 1H), 8.45 (d, J ¼ 8.6 Hz, 1H), 8.38 (d, J ¼ 8.6 Hz, 1H), 8.33
d 9.11 (s, 1H), 8.73 (d,
E-mail addresses: chemlidd@163.com (D. Li), yptian@ahu.edu.cn (Y. Tian).
These authors contributed equally to this work.
1
https://doi.org/10.1016/j.saa.2019.117571
1386-1425/© 2019 Elsevier B.V. All rights reserved.

2
F. Meng et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 226 (2020) 117571
emission spectra in different solvents) of MF (Fig. 1). As shown in
Fig. 1a, MF features one intense absorption band (about 500 nm),
which originates from the
p* or intramolecular charge transfer
p
ꢁ
(ICT) transition. With the aid of time-dependent density functional
theory (TD-DFT) calculations (Fig. S1), the obviously ICT can be
found within MF molecule. Interestingly, the solvability variation of
MF in different solutions lead to obviously difference of fluorescent
intensity (Fig. 1b). It motivated us to study its emission behavior in
aggregation state in detail.
As displayed in Fig. 2a, the emission spectra of MF in water/
ethanol mixtures with different ethanol fractions (fe, the volume
percentage of ethanol in water mixtures) were collected. The result
unambiguously reflect that the fluorescence intensity of MF
increased with increasing fe and accompanied with a small red-
shift emission, revealing the aggregation induced emission (AIE)
activity of MF. Considering the structure feature of MF, the above
phenomenon can be ascribed to the fact that the restriction of
intramolecular rotations (RIR) of the methyne within MF in the
aggregation state could enhance light emission [24]. Notably, AIE-
active molecule MF can effectively avoid the unwanted
aggregation-caused quenching due to the s high tendency of self-
aggregate under physiological conditions [28,29].
Scheme 1. Structure of MF.
(m, 2H), 8.22 (d, J ¼ 8.1 Hz,1H), 8.10 (s, 1H), 7.83 (dd, J ¼ 20.5, 8.7 Hz,
2H), 7.78 (m, 3H), 7.58 (t, J ¼ 7.6 Hz, 1H), 7.37 (t, J ¼ 7.2 Hz, 1H), 4.51
(s, 2H), 4.28 (s, 2H), 2.07 (s, 6H), 1.99 (s, 2H), 1.86 (d, J ¼ 36.2 Hz, 3H),
1.23 (s, 6H), 0.82 (d, J ¼ 6.9 Hz, 3H). 13C NMR (100 MHz, DMSO-d6)
d
(ppm): 205.18, 154.91, 143.88, 141.56, 137.64, 133.68, 131.06,
130.36,128.90,128.25,128.23,127.36,126.88,125.85,125.06,121.39,
112.68, 110.18, 110.07, 108.81, 53.79, 42.99, 35.00, 31.41, 28.87, 26.45,
25.53, 22.26, 13.27. ESI-MS: calculated for [M]þ: 485.3, found 485.3.
Encouraged by the above results, the AIE-active nanoparticles
(NPs) of MF for biological experiments were prepared via a re-
ported nano-precipitation method using Pluronic F-127 (F127) as
the matrix in our recent work [30]. For size distribution analysis,
MF NPs revealed an average size of 30 30 nm (Fig. 2b) evaluating
3. Results and discussion
Several techniques have been adopted to investigate the optical
properties of the target molecule MF. Firstly, we set out to inves-
tigate the linear optical photophysical properties (absorption and
Fig. 1. UV-vis absorption spectra (a) and one photon fluorescence spectra (b) of MF in different solvents (c ¼ 10-5 M).
Fig. 2. (a) Fluorescence spectra changes of MF in water with different ethanol fractions. (b) DLS of MF in water/ethanol (10/90, v/v) and SEM images (inset) of MF in ethanol fraction
90%.

F. Meng et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 226 (2020) 117571
3
Fig. 3. UV-vis absorption spectra (a) and one-photon fluorescence emission spectra (b) of in water and aggregation state.
Fig. 4. Time-resolved fluorescent spectra of MF-H2O (a) and MF NPs (b).
Fig. 5. (a) Effective two-photon absorption cross sections of MF-H2O and MF NPs in different excitation wavelengths with identical energy of 500 mW. (b) Cytotoxicity data results
of MF NPs at different concentrations for 24 h.
by DLS and SEM measurements. It is worth noting that the AIE-
active MF NPs showed enhanced emission intensity (Fig. 3) and
prolongedfluorescence time (Fig. 4). It is reasonable to expect that
MF NPs would show good uptake efficiency by living cells and
giving bright imaging results.
for two-photon bioimaging withhigher spacial resolution and
weaker specimen photodamage [30]. Fortunately, MF NPs exhibi-
ted low toxicity evaluating via MTT assay (Fig. 5b). The compre-
hensive merits of MF NPs motivated us to explore its two-photon
biological imaging application.
In addition, MF NPs exhibited considerable two-photon action
cross section within the wavelength range from 680 nm to 900 nm
(Fig. 5a), corroborating that it could serve as a judicious candidate
Using HeLa cells as a model, cell-staining experiments using
two-photon confocal microscopy (2PM) were carried out. The re-
sults showed that MF NPs (10 mM, 20 min) could readily enter HeLa

4
F. Meng et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 226 (2020) 117571
21871003), Doctor Startup Fund (S020118002/026), Hefei National
Laboratory for Physical Sciences at the Microscale (KF2019002),
Natural science research project of universities in Anhui Province
(KJ20191127).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.saa.2019.117571.
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