Fabrication of aggregation induced emission active luminescent chitosan nanoparticles via a “one-pot” multicomponent reaction

Article abstract of DOI:10.1016/j.carbpol.2016.07.026

Chitosan based nanomaterials have been extensively examined for biomedical applications for their biodegradability, low toxicity, biological activity and low cost. In this work, a novel strategy for fabrication of luminescent polymeric nanoparticles (LPNs

Full text of DOI:10.1016/j.carbpol.2016.07.026

Accepted Manuscript
Title: Fabrication of aggregation induced emission active
Luminescent Chitosan Nanoparticles via a “one-pot”
Multicomponent Reaction
Author: Qing Wan Meiying Liu Dazhuang Xu Liucheng Mao
Jianwen Tian Hongye Huang Peng Gao Fengjie Deng
Xiaoyong Zhang Yen Wei
PII:
S0144-8617(16)30815-3
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.carbpol.2016.07.026
CARP 11325
To appear in:
Received date:
Revised date:
Accepted date:
8-4-2016
5-7-2016
7-7-2016
Please cite this article as: Wan, Qing., Liu, Meiying., Xu, Dazhuang., Mao, Liucheng.,
Tian, Jianwen., Huang, Hongye., Gao, Peng., Deng, Fengjie., Zhang, Xiaoyong., &
Wei, Yen., Fabrication of aggregation induced emission active Luminescent Chitosan
Nanoparticles via a “one-pot” Multicomponent Reaction.Carbohydrate Polymers
http://dx.doi.org/10.1016/j.carbpol.2016.07.026
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Fabrication of aggregation induced emission active Luminescent Chitosan Nanoparticles via a
one-pot” Multicomponent Reaction
“
a,#
a,#
a
a
a
a
Qing Wan , Meiying Liu , Dazhuang Xu , Liucheng Mao , Jianwen Tian , Hongye Huang , Peng
a
a,*
a,*
b,*
Gao , Fengjie Deng , Xiaoyong Zhang , Yen Wei
a Department of Chemistry and Jiangxi Provincial Key Laboratory of New Energy Chemistry,
Nanchang University, 999 Xuefu Avenue, Nanchang 330031, China.
b Department of Chemistry and the Tsinghua Center for Frontier Polymer Research, Tsinghua
University, Beijing, 100084, P. R. China.
#
These authors contributed equally to this work
Highlights

Fabrication of aggregation induced emission active chitosan through a multicomponent
reaction



The multicomponent reaction can occur under rather mild experimental conditions
The WS-Chitosan@An-CHO LPNs are promising for biological imaging applications
The multicomponent reaction can also be utilized for fabrication of many other LPNs
Abstract
Chitosan based nanomaterials have been extensively examined for biomedical applications for their
biodegradability, low toxicity, biological activity and low cost. In this work, a novel strategy for
fabrication of luminescent polymeric nanoparticles (LPNs) based on aggregation induced emission
(
AIE) dye and water soluble chitosan (WS-Chitosan) were firstly developed via a highly efficient
mercaptoacetic acid (MA) locking imine reaction. In this multicomponent reaction (MCR), MA serves
as “lock” to connect 9,10-Bis(aldehydephenl)anthracene dye (An-CHO) and amino-containing
WS-Chitosan under mild reaction conditions. The obtained WS-Chitosan@An-CHO LPNs show strong
yellow emission and great water dispersibility. Biological evaluation results demonstrated that
synthetic luminescent polymeric nanoparticles possess desirable cytocompatibility and distinct imaging
properties. Therefore, we have developed a facile and useful method to fabricate AIE active nanoprobes
with desirable properties for various biomedical applications. This strategy should be a general and

easy handling tool to fabricate many other AIE dye based materials.
Keywords: aggregation induced emission, water solube chitosan, multicomponent reaction,
luminescent polymeric nanoparticles, biomedical applications.
1
. Introduction
In recent years, growing attentions have been paid to develop bioprobes based on luminescent
polymeric nanoparticles (LPNs) for wide biomedical applications.(Liu et al., 2016; Liu et al., 2015; Nie,
Xing, Kim & Simons, 2007; Wang, Yuan, Guo, Xu & Chen, 2014) Fluorescent imaging has been
regarded as one of the useful tools to monitor the biological behavior process existed in complicated
intracellular system with strong sensitivity and absolute inviolability,(Kang, Ha, Yun, Yu & Chang,
2
011) and have been successfully applied as a versatile way for disease diagnosis,(Liu, Tu, Zhu, Ma &
Chen, 2013) tracking and treatment.(Shi, Liu, Geng, Tang & Liu, 2012) So far, traditional luminescent
probes were prepared based on some luminescent materials such as inorganic quantum dots(Wu et al.,
2
003; Zhang et al., 2013c) organic dyes,(Wang et al., 2005) organic dye-loaded silica nanoparticles and
fluorescent proteins,(Heilemann et al., 2008) which were extensively applied in labeling cells and
tissue specimens. Furthermore, the unique features such as high photostability and sensitivity of these
luminescent materials make them allow real-time imaging of important intracellular molecules.
Unfortunately, some vicious drawbacks existed in above-mentioned luminescent materials have
become obstacle for their progress in cell labeling application. For example, inorganic quantum dots
(
Qdots) suffer from high toxicity when they were accumulated in cellular environment owing to the
existence of heavy metal ions,(Derfus, Chan & Bhatia, 2004) which would cause terrible influence for
human healthy. While dye-loaded silica nanoparticles possess poor biodegradability although they are
highly luminescent and homogeneous.(Roy et al., 2005) The green fluorescent protein also encounter
high cost, easy enzyme degradation, small stokes shifts, terrible photostability and tedious transfection
process and transfection efficiency,(Romantschuk et al., 2000) limiting their potential use in biomedical
imaging application in spite of overcoming poor degradable problems bring from Qdots and fluorescent
silica nanoparticles. Meanwhile, traditional organic dyes such as fluoresceins and rhodamines often
suffer from luminescence quenching phenomena when presented in solution with high concentration or
aggregated state, which was also called aggregation caused quenching (ACQ) effect. Therefore, in

order to conquer these problems of poor biodegradability, toxicity and ACQ effect, novel fluorescent
materials with better optical and biological properties should be developed.
Aggregation induced emission (AIE) dyes were firstly reported by Tang et al in 2001.(Luo et al.,
2
001) AIE dyes emitted strong fluorescence in their aggregated state while non-fluorescence or weak
fluorescence in dispersed state, which could effectively solve ACQ effect of conventional organic dyes.
Since discovery, a number of AIE fluorogens such as siloles,(Wang, Zhang, Zhang, Zhu & Tang, 2010)
cyano-substituted diarylethene,(Zhang et al., 2013a; Zhang, Ma, Liu, Zhang, Jia & Wei, 2013)
tetraphenylethene,(Chi et al., 2012; Wan et al., 2016; Wan et al., 2015b; Wan et al., 2015c; Wu et al.,
2
012) tripheneylethene,(Chen et al., 2012; Li et al., 2011; Zhang et al., 2011; Zhang et al., 2010; Zhang
et al., 2014b) distyrylanthracene derivatives(Wang et al., 2013; Zhang et al., 2013f) have been
synthesized and drastically investigated for their potential applications in fields such as bio/chem
sensors, cell imaging and ion probes due to their excellent AIE characteristics, biodegradability and
cytocompatibility. Up to now, some strategies for fabricating luminescent probes based on AIE dyes
have drawn increasing attentions for their potential medical applications. Generally, two major methods
for constructing LPNs based on AIE dyes have been developed, which contained non-covalent methods
and covalent methods.(Huang et al., 2015; Liu et al., 2014a; Liu et al., 2014b; Zhang et al., 2014c;
Zhang et al., 2013e) The non-covalent methods for preparing LPNs were that embedding the AIE dyes
into the biocompatible amphiphilic polymers to obtain water dispersible luminescent nanoparticles.
Other silica nanoparticles encapsulation and bovine serum albumin (BSA) implantation methods were
also used to construct AIE active LPNs.(Qin et al., 2012; Zhang et al., 2013d; Zhang et al., 2014d)
Nevertheless, the easy leakage of AIE dyes and separation of surface coating from LPNs have become
serious restriction of non-covalent systems. On the other hand, covalent strategies to prepare LPNs
could be summarized that reversible addition fragmentation chain transfer polymerization,(Zhang et al.,
2
2
2
014f) cross-linked Schiff based dynamic bonds,(Wan et al., 2015a; Yang et al., 2013; Zhang et al.,
013e) emulsion polymerization(Zhang et al., 2014e) and anhydride ring-opening reaction.(Zhang et al.,
014a) Based on these techniques, numerous AIE active LPNs with uniform size, great
biocompatibility, high water dispersibility and strong luminescence have been fabricated and utilized
for cell imaging. Although the impressive progress was made for fabrication of AIE active LPNs in
methodology, more fabricated strategies with great stability, high-efficiency, short reaction time and
environmentally friendly (catalyst free, room temperature condition and air atmosphere) and

multifunctional potential will receive more pursuit from scientists.
The water-soluble chitosan (named as WS-Chitosan) based luminescent nanoparticles have been
intensively researched for biomedical applications due to their favorable features such as
biocompatibility, non-toxicity and degradable potential.(Qi, Xu, Jiang, Hu & Zou, 2004) In this
contribution, we report
a robust synthesis protocol to prepare ultra-small cross-linked
WS-Chitosan@An-CHO LPNs with uniform size, high water dispersibility, great biocompatibility and
strong luminescent property via a “one-pot” multicomponent reaction (MCR).
2
2
. Experimental
.1 Materials and methods
All chemical agents were of analytical grade and used as received without any further purification.
All the chemicals such as chitosan (deacetylation ≥ 95%, viscosity: 100-200 mpa.s), hydrogen peroxide
30 wt.%), ammonium persulphate (98%, 228.2 Da), anthracene (98%, 178.3 Da) and
(
terephthalaldehyde (98%, 134.13 Da) were purchased from Aladdin Chemistry Co., Ltd (Shanghai,
China). The dialysis bags (Size: 5 M, cut-off: 7000 Da) are purchased from Biotopped Co., Ltd
(
Beijing, China). All the other commercially available reagents were analytical grade and used without
further purification.
1H NMR spectra were recorded on Bruker Avance-400 spectrometer with D
2
3
O and CDCl as the
solvents. The synthetic polymers and materials were characterized by Fourier transform infrared
spectroscopy (FT-IR) using KBr pellets. The Fourier transform infrared (FT-IR) spectra were supplied
from Nicolet5700 (Thermo Nicolet corporation). Transmission electron microscopy (TEM) images
were recorded on a Hitachi 7650B microscope operated at 80 kV. The TEM specimens were got by
putting a drop of the nanoparticle ethanol suspension on a carbon-coated copper grid. The size
distribution of WS-Chitosan@An-CHO LNPs in phosphate buffered saline (PBS) was determined by
dynamic laser scattering using a Zeta Plus particle size analyzer (Zeta Plus, Brookhaven Instruments,
Holtsville, NY). The fluorescence data were obtained from the Fluorescence spectrophotometer (FSP,
model: C11367-11), which purchased from Hamamatsu (Japanese).
2
.2 Synthesis of 9, 10-Bis(bromomethyl)anthracene (Br-An-Br)
The synthesis route of An-CHO was displayed in Scheme S1. At first, the intermediate products of 9,
0-Bis(bromomethyl)anthracene and 9,10-bis(diethylphosphorylmethyl)anthracene were synthesized
1
by previous report.(Zhang et al., 2013d) For the preparation of 9,10-Bis(bromomethyl)anthracene: the

2 3
mixture of anthracene (7.6 g, 40 mmol), (CH O)n (4.8 g), AlCl (5.0 g) and solution of 33% HBr in
acetic acid (60 mL) was stirring at 60 °C for 6 h. The obtained solid was put into water and stirring 30
min to remove residual reactants, filtration and dry in vacuum one night. The residue product was
1
recrystallized in toluene three times to obtain yellow solid. Yield: 7.8 g (54%). H NMR (300 MHz,
CDCl
.3 Synthesis of 9,10-bis(diethylphosphorylmethyl)anthracene ( (OEt)
The 9, 10-Bis(bromomethyl)anthracene (1.2 g, 3.3 mmol) was mixed with triethyl phosphate (10
3
): δ 8.38 (m, 4H), δ 7.65 (m, 4H), δ 5.54 ppm (m, 4H).
2
2
(O)P-An-P(O)(OEt) )
2
mL). Resulting mixture was refluxed at 180 °C for 8 h in order to obtain the pure product. The solvent
was then removed at vacuum and the residue product was purified by a column chromatography on
1
silica gel using ethyl acetate/CH
Cl
2 2
as the eluent. Yield: 1.05g (68%). H NMR (300 MHz, CDCl
3
): δ
8
.38 (m, 4H), δ 7.6 (m, 4H), 4.25 (d, 4H), 3.8 (m, 8H), 1.06 ppm (t, 12H).
.4 synthesis of 9,10-Bis(aldehydephenl)anthracene (CHO-An-CHO)
The 9, 10-Bis(aldehydephenl)anthracene with unique AIE feature was synthesized by follows:
2
synthetic 9,10-Bis(diethylphosphorylmethyl)anthracene (1.37 g, 3 mmol), terephthalaldehyde (1 g, 7.5
mmol) and t-BuOK (896 mg, 8 mmol) were mixed and stirring in dry tetrahydrofuran (THF) solution
for 6 h at room temperature. The THF solution was removed by rotary evaporation and the residual
solid was wash with water and extract with ethyl acetate for three times. The obtained organic layer
was dried with anhydrous magnesium sulfate and purified by by a column chromatography on silica gel
using ethyl acetate/petroleum ether (1/10) as the eluent. The result solid was faint yellow. Yield: 0.8 g
1
(
60.8%). H NMR (300 MHz, CDCl
3
): δ 10.09 (d, 2H), δ 8.39 (m, 4H), δ 8.09 (m, 8H), δ 7.57 (m, 4H),
7
.00 ppm (m, 4H).
2
.5 preparation of water soluble chitosan (WS-Chitosan)
According to the previous report,(Tian, Liu, Hu & Zhao, 2004) the WS-Chitosan was prepared via
depolymerisation of insoluble chitosan using H
2
O
2
as oxidant (Scheme S2). The purchased chitosan
(2.0 M)
(
4.8 g) was dissolved in the hydrochloric acid solution (100 mL, 0.5%) and then 12 mL H O
2 2
was added. The mixed solution was stirring at 50 ºC for 4 h. After reaction, the mixed solution was
filtrated. The filtrate was adjusted to pH = 7.0 using NaOH solution and WS-Chitosan could be
obtained after adding excess ethanol. Thus obtained WS-Chitosan was dried in vacuum.
2
.6 Preparation of cross-linked WS-Chitosan@An-CHO LPNs
The WS-Chitosan and An-CHO with strong yellow fluorescence were locked by mercaptoacetic acid

(
MA) via a one-pot MCR reaction. Thus-obtained amphiphilic luminescent materials would
self-assemble into nanopaticles and have promising application prospects for cell imaging. On the other
hand, the synthetic route for preparation of WS-Chitosan@An-CHO LPNs via a one-pot MCR was
shown in Scheme 1. WS-Chitosan (500 mg) in 2 mL distilled water was blended with An-CHO (75.4
mg, 0.17 mmol) in 5 mL THF and charged into a dry Schlenk tube. After then, Schlenk tube was placed
in an oil bath at 30 °C for 30 min. Then, MA (156.4 mg, 1.7 mmol) was dropped gradually into the
Schlenk tube and stirring for other 4 h. Finally, the resulting reactive raw products were purified by
dialysis to remove residual reaction agents. The resultant products WS-Chitosan@An-CHO LPNs were
dried at vacuum for further characterization.
2
.7 Cytotoxicity evaluation of WS-Chitosan@An-CHO LPNs
In order to evaluate the harmful of WS-Chitosan@An-CHO LPNs for living cells, the cell viability
of WS-Chitosan@An-CHO LPNs with strong yellow fluorescence on HeLa cells was evaluated by cell
counting kit-8 (CCK-8) assay.(Zhang, Hu, Li, Tao & Wei, 2012; Zhang et al., 2015; Zhang et al., 2013b)
The experimental procedure could be demonstrated by follows. Cells were put into 96-well microplates
4
–1
at a density of 5×10 cells mL in 160 μL of respective medium containing 10% fetal bovine serum
(
(
FBS). After 24 h of cell attachment, WS-Chitosan@An-CHO LPNs with different final concentrations
–
1
10-100 µg mL ) were added and incubated with cells for 12 and 24 h. Then cells were repeatedly
washed with PBS three times to remove noninternalized nanoparticles. 10 μL of CCK-8 dye and 100
μL of Dulbecco’s modified eagle medium (DMEM) were added to each well and incubated for another
2
h. Afterward, plates were analyzed using a microplate reader (Victor Ш, Perkin-Elmer).
Measurements of formazan dye absorbance were carried out at 450 nm, with the reference wavelength
at 620 nm. The values were proportional to the number of live cells. The percent reduction of CCK-8
dye was compared to controls (cells without incubation with LPNs), which represented 100% CCK-8
reduction. Three replicate wells were used per microplate, and the experiment was operated for three
times. Cell survival was expressed as absorbance relative to that of untreated controls. Results are
presented as mean ± standard deviation (SD).
2
.8 Confocal microscopic imaging
HeLa cells were cultured in DMEM, supplemented with 10% heat-inactivated FBS, 2 mM glutamine,
00 U mL–1 penicillin, and 100 μg mL–1 of streptomycin. Cell culture was controlled at 37 °C in a
1

2
similar humidified condition of 95% air and 5% CO in culture medium. Culture medium should be
updated every three days for maintaining the exponential growth of the cells. Before treatment, cells
5
were seeded in a glass bottom dish with a density of 1×10 cells per dish. On the day of treatment, the
cells were incubated with WS-Chitosan@An-CHO LPNs at a final concentration of 20 µg mL–1 for 3 h
at 37 °C. Afterward, the cells were washed three times with PBS to remove the Chitosan@An-CHO
LPNs and then fixed with 4% paraformaldehyde for 10 min at room temperature. Cell images were
obtained using a confocal laser scanning microscope (CLSM) Zesis 710 3-channel (Zesis, Germany)
with the excitation wavelength of 458 nm.
3
. Results and discussion
Fabrication of ultra-small AIE active LPNs with biodegradability and great biocompatibility is
extremely important for intra-cellular bioimaging applications. As is well known, LPNs have been
applied as effective and multifunctional nanoparticles capable of enhancing the diagnostic and
therapeutic efficacy of imaging probes and targeted agents in cancer treatment due to the accumulation
at solid tumor site of polymeric nanoparticles by the enhanced permeation and retention effect. In this
work, we provide a robust method for the produce ultra-small cross-linked WS-Chitosan@An-CHO
LPNs with well-dispersed in water via one-pot process under mild reaction conditions. The AIE
fluorogen An-CHO was synthesized following prepared route described in afore-mentioned
1
information. The successful synthesis of AIE dye could be confirmed by the H NMR (Fig. S1). For
fabrication of cross-linked WS-Chitosan@An-CHO LPNs, the hydrophobic AIE dyes (An-CHO) were
conjugated with a hydrophilic and biodegradable biopolymer WS-Chitosan to form amphiphilic
fluorescent materials via a one-pot MCR using MA as the lock. Thus obtained amphiphilic
WS-Chitosan@An-CHO copolymers would self-assemble into nanoparticles of great stability in
aqueous solution because of their cross-linked structure and excellent water dispersibility.
The successful synthesis of An-CHO and WS-Chitosan@An-CHO LPNs could be confirmed from
1
the H NMR spectra using CDCl
3
and D
2
O as solvents (Fig. S1 and Fig. 1). As shown in Fig. S1(b), the
2 3
new peaks of chemical shift at 3.7-4.3 ppm represent the hydrogen atom of –CH and –CH . Compared
1
with the H NMR spectra of Br-An-Br, the appearance of new peak demonstrated that successful
1
synthesis of (OEt)
2
(O)P-An-P(O)(OEt)
2
. The H NMR spectrum of An-CHO was displayed in Fig.
S1(c). It suggested successful preparation of An-CHO dyes due to the appearance of new peak at 10.09

ppm, which is belonged to the aldehyde group. The chemical shift of each synthetic intermediate was
described in the synthetic route. On the other hand, in order to prove successful formation of
1
WS-Chitosan@An-CHO LPNs via conjugation of WS-Chitosan with An-CHO, two H NMR spectra
of WS-Chitosan and WS-Chitosan@An-CHO LPNs are compared. As seen in Fig. 1, the multiplet
peaks between 2.91 and 3.18 ppm for the WS-Chitosan@An-CHO sample are contributed to the –CH
in the cross-linked heterocycle, which provide powerful evidence for successful formation of
cross-linked structure. The peak of hydrogen atom of –NH existed in WS-Chitosan located at 5.01
2
2
ppm. However, peak intensity at the 5.01 ppm in WS-Chitosan@An-CHO was decreased as compared
with WS-Chitosan (Fig. 1). This comparison indicated that partial amino groups have reacted with
An-CHO. More importantly, some new peaks were found between 7.68 and 8.42 ppm, which could be
ascribed to the peaks of aromatic rings of An-CHO. The above results demonstrated that successful
fabrication of WS-Chitosan@An-CHO LPNs through a one-pot MCR.
1
Other than H NMR spectra, FT-IR spectra could also serve as powerful tool to confirm the
cross-linked reaction between WS-Chitosan and An-CHO. As shown in Fig. 2, the FT-IR spectra are
used to infer the functional groups in WS-Chitosan and WS-Chitosan@An-CHO LPNs. In general, the
O-H stretching vibration is overlapped to the N-H stretching. The IR peak of amino and hydroxyl
-
1
groups were appeared at ranging from 3200 to 3500 cm .(Wang, Wang, Jia, Li, Yu & Ding, 2014) After
conjugating with An-CHO to prepare the WS-Chitosan@An-CHO LPNs, the absorption peak of amino
groups is still existed. This could explain that amino groups of WS-Chitosan were partially reacted with
An-CHO using MA as cross-linked reagent. Furthermore, the new peaks at 1650 and 1290 cm -1 were
assigned to the C-C(O)-N of heterocycle and C=C groups, respectively. These peaks are not found in
the IR spectrum of WS-Chitosan, indicating that An-CHO has conjugated with chitosan successfully
via MCR. Therefore, we could draw a conclusion that the fabricated method described in this work is
capable for the preparation of AIE active LPNs through a facile one-pot MCR.
As well known, self-assembly of amphiphilic polymers to form nanoparticles is a facile and
promising strategy for various biomedical applications. Therefore, the size and morphology of
WS-Chitosan@An-CHO copolymers were characterized by TEM. As shown in Fig. 3, many uniform
spherical nanoparticles with diameter ranging from 30-100 nm could be clearly observed, providing us

direct evidence that successfully self-assemble into nanoparticles of amphiphilic
WS-Chitosan@An-CHO copolymers in aqueous solution. The size distribution of
WS-Chitosan@An-CHO LNPs in PBS was determined using a ZetaPlus particle size analyzer. Due to
their amphiphilic property, WS-Chitosan@An-CHO copolymers were self-assembled into
nanoparticles in PBS. We found that hydrodynamic size distribution of WS-Chitosan@An-CHO LNPs
was 128.0 ± 14.4 nm (Fig. S2). The size distribution from DLS measurement is greater than the size
from TEM characterization. This reason could be considered as the shrinkage of amphiphilic
fluorescent polysaccharide under drying procedure. Therefore, according to the results of TEM image
and DLS, we confirmed that successful preparation of AIE active luminescent nanoparticles based on
the combination of WS-Chitosan and An-CHO by a MCR method. During the self-assembly procedure,
the hydrophobic AIE dye An-CHO is expected to aggregate in the core of nanoparticles, which will
result in enhanced luminescence emission due to the AIE feature of An-CHO. Herein, the UV
absorption spectrum and fluorescent spectra are used to evaluate their water dispersibility and
fluorescent property. From Fig. 4A, the strong absorption peak appears at 210 nm can be ascribed to K
absorption band, suggesting that contained conjugated system of two double bonds (-C=C-C=C-) in
WS-Chitosan@An-CHO LPNs. Moreover, two strong absorption peaks at 273 and 281 nm were also
detected by UV-Vis spectrum of WS-Chitosan@An-CHO LPNs. These peaks can be attributed to
–
C=C- substituent groups on aromatic rings. The adsorption peak at 432 nm is possibly ascribed to the
redshift of n-π* transition, which implied that hetero atoms such as O, N or S were linked with the
aromatic rings. Furthermore, WS-Chitosan@An-CHO copolymers showed great dispersibility in water.
For example, the background of “AIE” word could be clearly observed in inset of Fig. 4A. These
results clearly confirm the successful conjugation of WS-Chitosan with An-CHO via a simple one-pot
MCR. The fluorescent spectra of WS-Chitosan@An-CHO LPNs were shown in Fig. 4B. The
maximum emission wavelength of WS-Chitosan@An-CHO LPNs was centered at 558 nm, while the
optimal fluorescent excitation wavelength was located at 436 nm. Meanwhile, the strong yellow
luminescence could be observed under irradiating by UV lamp at 365 nm (inset of Fig. 4B). This
suggested that the AIE feature of WS-Chitosan@An-CHO LPNs could efficiently overcome the ACQ
effect of LPNs based on conventional organic dyes.
The fluorescent properties such as intensity and photostability of LPNs play an important role in

their biomedical applications. Fig. S3 shows the photoluminescence (PL) spectra of
WS-Chitosan@An-CHO LPNs before and after continuously irradiating with UV lamp at 365 nm for 2
h. As compared with the PL intensity before irradiating, the fluorescent intensity was decreased in
some extent (Fig. S3). The above comparison suggested that the WS-Chitosan@An-CHO LPNs
excellent photostability, making them for more suitable for biological imaging especially for long time
tracing. In order to evaluate the potential biomedical application of WS-Chitosan@An-CHO LPNs, the
cell viability of WS-Chitosan@An-CHO LPNs towards HeLa cells was determined CCK-8 assay. Cells
were incubated with different concentrations of WS-Chitosan@An-CHO LPNs for 12 and 24 h. As
shown in Fig. 5, the cell viability values have not obviously changed under the experimental conditions.
The cell viability values are still greater than 95% even the particle concentration is greater than 100 µg
mL-1 for 24 h incubation. The half maximal inhibitory concentration (IC50) values of
WS-Chitosan@An-CHO LPNs at 12 and 24 h incubation were calculated to be 1630.3 and 422.3 µg
-
1
mL , respectively. The cell viability results indicated that WS-Chitosan@An-CHO LPNs possess good
biocompatibility. On the other hand, WS-Chitosan has been extensively examined for various
biomedical applications such as drug and gene delivery, antibacterial, tissue engineering and cell
encapsulation for its good biocompatibility, degradable and low cost and some specific biological
activity. Therefore, the WS-Chitosan@An-CHO LPNs should also be used for fabrication of
multifunctional biomaterials for various biomedical applications.
Considered the properties such as good water dispersibility, strong yellow fluorescence, uniform
morphology and facile fabrication procedure, the possible biomedical applications of
WS-Chitosan@An-CHO LPNs were evaluated based on their cell uptake behavior. Their cell uptake
behavior was evaluated by CLSM observation (Fig. 6). Fig. 6A shows the fluorescent image of HeLa
-
1
cells, which were incubated with 20 µg mL WS-Chitosan@An-CHO LPNs for 3 h. It can be seen that
strong fluorescent signals spread over cells could be clearly observed. However, in the centre of cells,
relative weak fluorescent signals are found. These areas should be ascribed to the location of cell
nucleus. The above results implied that cell uptakes of WS-Chitosan@An-CHO LPNs may be mainly
caused by typical endocytosis route. In addition, we can find that cells still adhered to cell plates very
well and kept their normal morphology (Fig. 6B). This also confirmed the good biocompatibility of
WS-Chitosan@An-CHO LPNs. Fig. 6C showed the emerged image of Fig. 6A and Fig. 6B. It can be

clearly observed that the fluorescent areas are overlapped with the location of cells, and the cell nucleus
is surrounded by the fluorescent signals, implied that WS-Chitosan@An-CHO LPNs are mainly
distributed in the cytoplasm after endocytosis. As compared with the IC50 values of
WS-Chitosan@An-CHO LPNs, the dosage used for cell imaging applications is much less. Therefore,
considered the good biocompatibility, efficient cell uptake and imaging performance of
WS-Chitosan@An-CHO LPNs, we could conclude that WS-Chitosan@An-CHO LPNs are promising
candidates for biological imaging applications.
4
. Conclusion
In summary, luminescent chitosan nanoparticles with AIE feature were hastily prepared for the first
time through a one-pot MCR, which relied on the MA as the lock to form covalent linkage between the
amino groups of WS-Chitosan and aldehyde groups of AIE dye. The obtained amphiphilic
WS-Chitosan@An-CHO copolymers could self-assemble into ultrabright LPNs in aqueous solution
because of hydrophobic AIE dyes as core while hydrophilic chitosan as shell, the biocompatibility and
cell uptake behavior of WS-Chitosan@An-CHO LPNs suggested their potential application prospect in
biomedical fields. In addition, the MCR can occur under rather mild experimental conditions such as
catalyst-free, efficient, atom-economical and multifunctional potential. On the other hand, many other
AIE active nanoparticles with different functional components and properties can also be fabricated
based on the similar MCR reaction where the properties of AIE dyes and polymers can be adjusted and
designed according to our demand. Furthermore, numerous functional groups were existed in
WS-Chitosan@An-CHO LPNs, which could be utilized for conjugation with other functional
components such as drugs, target agents and other imaging probes. Last but not least, many other
components such as antibodies and functional polymers could also be introduced into these AIE active
polymeric systems through adding them into the reaction system in one-pot. Therefore, we could
conclude that WS-Chitosan@An-CHO LPNs to be promising biomaterials for various biomedical
applications.
Acknowledgements
This research was supported by the National Science Foundation of China (Nos. 51363016, 21474057,
2
1564006, 21561022), and the National 973 Project (Nos. 2011CB935700).

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g
a
b
g
a
b
d
d
1
Fig.1 The H NMR spectra of WS-Chitosan and WS-Chitosan@An-CHO LPNs.
C=C
C=O
-
OH, -NH2
Fig. 2 Representative FT-IR spectra of WS-Chitosan and WS-Chitosan@An-CHO LPNs.
5
0 nm
2
00 nm
Fig. 3 TEM images of WS-Chitosan@An-CHO LPNs.

A
B
Fig. 4 (A) The UV-Vis absorption spectrum of WS-Chitosan@An-CHO LPNs dispersed in water, the
inset picture using “AIE” as background demonstrate that great water dispersibility of
WS-Chitosan@An-CHO LPNs. (B) The excitation (Ex) and emission (Em) fluorescent spectra of
WS-Chitosan@An-CHO LPNs. The inset picture suggests strong fluorescence emission of
WS-Chitosan@An-CHO LPNs.
Fig. 5 Cell viability evaluation of WS-Chitosan@An-CHO LPNs towards HeLa cells based on CCK-8
-
1
assay. The HeLa cells were incubated with 10-100 µg mL LPNs for 12 and 24 h.
2
0 μm
20 μm
20 μm
-
1
Fig. 6 CLSM images of HeLa cells incubated with 20 µg mL WS-Chitosan@An-CHO LPNs for 3 h.
(
A) excited with 405 nm, (B) bright field, (C) merged image of A and B. Scale bar = 20 µm.

Scheme 1 The synthetic route of WS-Chitosan@An-CHO LPNs using WS-Chitosan, An-CHO and MA
as the reaction agents.