Synthesis and biological imaging of fluorescent polymeric nanoparticles with AIE feature via the combination of RAFT polymerization and post-polymerization modification

Article abstract of DOI:10.1016/j.dyepig.2018.05.032

In recent years, florescent polymeric nanoparticles (FPNs) containing aggregation-induced emission (AIE) fluorogens have received great intention for their potential applications in biological imaging and theranostic nanomedicine. Herein, we have developed AIE-active FPNs through a combination of reversible addition-fragmentation chain transfer (RAFT) polymerization and post-polymerization modification strategies. The salicylaldehyde (SA) containing zwitterionic copolymers are fabricated via RAFT polymerization and further modified by benzophenone hydrazone (BPH) via Schiff base reaction. The obtained AIE-active amphiphilic copolymers BPH-poly(FHMA-co-MPC) can self-assemble in aqueous solution with the hydrophobic fluorogens aggregating together to form the core and the hydrophilic chains to form the protective shell. BPH-poly(FHMA-co-MPC) and the resulting FPNs are characterized by ¹H nuclear magnetic resonance spectroscopy, Fourier transform infrared spectroscopy, transmission electron microscopy and fluorescence spectroscopy. Results demonstrate that BPH-poly(FHMA-co-MPC) are successfully synthesized and as-prepared BPH-poly(FHMA-co-MPC) FPNs exhibit desirable morphology and size, good dispersibility, high brightness, remarkable photostability and large Stokes shifts. More importantly, through cytotoxicity test and cell uptake behavior, these BPH-poly(FHMA-co-MPC) FPNs show low toxicity and excellent cell dyeing behavior. Taken together, we have developed a facile and effective method for the fabrication of AIE-active FPNs, which display great potential for biomedical applications.

Full text of DOI:10.1016/j.dyepig.2018.05.032

Accepted Manuscript
Synthesis and biological imaging of fluorescent polymeric nanoparticles with
AIE feature via the combination of RAFT polymerization and post-polymerization
modification
Yanzhu Liu, Liucheng Mao, Saijiao Yang, Meiying Liu, Hongye Huang, Yuanqing
Wen, Fengjie Deng, Yongxiu Li, Xiaoyong Zhang, Yen Wei
PII:
S0143-7208(17)32404-X
10.1016/j.dyepig.2018.05.032
DYPI 6761
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 21 November 2017
Revised Date: 15 May 2018
Accepted Date: 15 May 2018
Please cite this article as: Liu Y, Mao L, Yang S, Liu M, Huang H, Wen Y, Deng F, Li Y, Zhang X, Wei
Y, Synthesis and biological imaging of fluorescent polymeric nanoparticles with AIE feature via the
combination of RAFT polymerization and post-polymerization modification, Dyes and Pigments (2018),
doi: 10.1016/j.dyepig.2018.05.032.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
A novel strategy based on the combination of RAFT polymerization and post-polymerization has been
developed for the synthesis of fluorescent polymeric nanoparticles with ESIPT feature

ACCEPTED MANUSCRIPT
Synthesis and biological imaging of fluorescent polymeric nanoparticles with AIE feature via the
combination of RAFT polymerization and post-polymerization modification
Yanzhu Liua,#, Liucheng Maoa,#, Saijiao Yanga, Meiying Liua, Hongye Huanga, Yuanqing Wena, Fengjie
Denga, Yongxiu Lia,*, Xiaoyong Zhanga,*, Yen Weib,c,*
a Department of 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.
c Department of Chemistry and Center for Nanotechnology and Institute of Biomedical Technology,
Chung-Yuan Christian University, Chung-Li 32023, Taiwan
# These authors contributed equally to this work

ACCEPTED MANUSCRIPT
Abstract
In recent years, florescent polymeric nanoparticles (FPNs) containing aggregation-induced emission
(AIE) fluorogens have received great intention for their potential applications in biological imaging and
theranostic nanomedicine. Herein, we have developed AIE-active FPNs through a combination of
reversible addition-fragmentation chain transfer (RAFT) polymerization and post-polymerization
modification strategies. The salicylaldehyde (SA) containing zwitterionic copolymers are fabricated via
RAFT polymerization and further modified by benzophenone hydrazone (BPH) via Schiff base
reaction. The obtained AIE-active amphiphilic copolymers BPH-poly(FHMA-co-MPC) can
self-assemble in aqueous solution with the hydrophobic fluorogens aggregating together to form the
core and the hydrophilic chains to form the protective shell. BPH-poly(FHMA-co-MPC) and the
1
resulting FPNs are characterized by H nuclear magnetic resonance spectroscopy, Fourier transform
infrared spectroscopy, transmission electron microscopy and fluorescence spectroscopy. Results
demonstrate that BPH-poly(FHMA-co-MPC) are successfully synthesized and as-prepared
BPH-poly(FHMA-co-MPC) FPNs exhibit desirable morphology and size, good dispersibility, high
brightness, remarkable photostability and large Stokes shifts. More importantly, through cytotoxicity
test and cell uptake behavior, these BPH-poly(FHMA-co-MPC) FPNs show low toxicity and excellent
cell dyeing behavior. Taken together, we have developed a facile and effective method for the
fabrication of AIE-active FPNs, which display great potential for biomedical applications.
Key words: Aggregation-induced emission, post-polymerization, fluorescent polymeric nanoparticles,
biological imaging

ACCEPTED MANUSCRIPT
1 Introduction
Luminescent nanomaterials have attracted great attention of scientific community because of their
widespread applications in optics, electronics, memory systems, biotechnology, etc.[1, 2] Among them,
the development of fluorescent polymeric nanoparticles (FPNs) has aroused tremendous scientific
interests because fluorescence techniques are highly sensitive and easy to operate.[3, 4] Unfortunately,
most of the traditional organic molecules possess strong emission in their solution state but very weak
even non emission at high concentrations or in the aggregated states. Such quenching of emission
intensity could be attributed to notorious aggregation-caused quenching (ACQ) effect, which is an
intractable problem for lots of practical applications such as optoelectronic devices and biomedical
research.[5] Therefore, The ACQ effect has become a huge obstacle, which impels researchers to seek
anti-ACQ materials with higher luminescence efficiency in the aggregated state than in the dissolved
state. In 2001, Tang and co-workers discovered an unusual anti-ACQ photophysical phenomenon
which is known as aggregation-induced emission (AIE).[6] Subsequently, AIE-active materials
stimulate intense research interest owing to the fundamental importance and promising practical
applications, and the mechanisms and technological applications of AIE materials are unceasingly
developed and perfected.[1, 7, 8] For instance, restriction of intramolecular motions (RIM) as the
mechanism behind AIE has been generally accepted, and various practical applications of AIE
materials including optoelectronic devices, biological/chemical sensors, biological imaging and
theranostics have been exploited.[9-16]
In particular, the construction of FPNs based on AIE-active dyes through both physical and chemical
methods has made remarkable achievements.[17-20] At first, a class of biocompatible amphiphilic
commercialized or synthesized block copolymers were utilized to construct AIE-active FPNs with good
water dispersibility for various biomedical applications through a physical encapsulation strategy.[21,
22] On the other hand, a series of more robust and versatile chemical processes were employed for
fabrication of AIE-active FPNs. These fall into two categories: (1) Polymerizable AIE-active dyes
directly incorporate into polymers for fabrication of FNPs via different polymerization methods such as
emulsion polymerization, reversible addition fragmentation chain transfer (RAFT) polymerization, and
ring-opening polymerization;[23-26] (2) Functionalized AIE molecules conjugated with biocompatible
macromolecules (natural carbohydrate polymers and synthetic polymers) for construction of FPNs via
multifarious chemical reactions such as Schiff base condensation and multicomponent reaction.[13,
27-30] Despite many impressive advances in construction methodologies for the fabrication of
AIE-active FNPs, more facile preparation strategies are still highly desirable.
Post-polymerization means is a useful methodology for the functionalization of polymers and

ACCEPTED MANUSCRIPT
endowing their new properties for wide ranging applications.[31-34] The backbone of copolymers
contains a moiety with latent reactivity, which can be functionalized directly by the functional group
precursor.[35] In addition, Schiff base compounds have drawn extensive research attention and have
been widely put into use in the fields of medicine, catalysis, analytical chemistry, etc.[36, 37] Due to
enjoying the advantages of mild reaction conditions and high reaction rates, the Schiff base reaction is
used for protecting various functional groups and synthesizing a series of organic ligands.[38]
Moreover, the Schiff base ligands are derived by the condensation of aldehyde/ketone with a primary
amine, which plays a vital role in human welfare, modern coordination and medicinal chemistry.[39]
More importantly, integration of post-polymerization methods and Schiff base reaction, which can offer
a rather straightforward synthesis method and readily-modified structures to fabricate some functional
polymers.
In this contribution, the existing strategy for fabrication of AIE-active FPNs based on RAFT
polymerization and post-polymerization modification was used to prepare salicylaldehyde (SA)
containing zwitterionic copolymers were synthesized via RAFT polymerization followed by reaction
with benzophenone hydrazone (BPH) by a Schiff base reaction (Scheme 1). Thus obtained
BPH-poly(FHMA-co-MPC) copolymers tend to form nanoparticles in aqueous solution as a result of
their amphiphilic structure. Furthermore, the structure features, photophysics properties and
microscopic morphological characteristics of BPH-poly(FHMA-co-MPC) FPNs were determined by a
variety of characterization means. The cell cytotoxicity and cell uptake behavior of thus obtained
BPH-poly(FHMA-co-MPC) FPNs were further determined to evaluate their potential for cell imaging
applications.

ACCEPTED MANUSCRIPT
Scheme 1 Schematic illustration of the preparation procedure of BPH-poly(FHMA-co-MPC). (A)
Synthetic route to BPH, (I) hydrazine monohydrate, 40 ºC, 24 h; (B) Synthetic route to FHMA, (II)
(CHO)n, rt, 24 h, (III) methacrylic acid, 50 ºC, 24 h; (C) Synthetic route to BPH-poly(FHMA-co-MPC),
(IV) CTA, 70 ºC, 12 h, (V) BPH, 70 ºC, 4 h.
2 Experimental sections
2.1 Materials and instruments
Benzophenone (BP, Mw: 182.22 Da, 99%), salicylaldehyde (SA, Mw: 122.12 Da, 99%), methacrylic
acid (Mw: 86.09 Da, 99%) and absolute N,N-dimethylformamide (DMF) were purchased from Heowns
Biochemical Technology Co., Ltd. (Tianjin, China). Paraformaldehyde (Mw: 30.03, AR) and
2,2'-azobis(2-methylpropionitrile) (AIBN, Mw: 164.21 Da, 98%) were supplied by Aladdin Reagent
Co., Ltd. (Shanghai, China). 2-Methacryloyloxyethyl phosphorylcholine (MPC, Mw: 295.27 Da, 96%)
was purchased from Joy-Nature Science and Technology Development Institute. (Nanjing, China).
Hydrazine monohydrate (Mw: 32.05 Da, 99%) was supplied by TCI (Shanghai) Development Co., Ltd.
Other chemicals not mentioned here were of analytical grade, and were used as received. Deionized
water was used in throughout the experiments. The chain transfer agent (CTA) of

ACCEPTED MANUSCRIPT
4-cyano-4-(ethylthiocarbonothioylthio) pentanoic acid was synthesized by the literature methods.[40]
1H nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance-400 spectrometer
with tetramethylsilane (TMS) as the internal standard and CDCl3 or d6-DMSO as solvents. The Fourier
transform infrared (FT-IR) spectra were obtained in a transmission mode on a Nicolet 5700
spectrometer (Thermo Nicolet Corporation), using powdered samples diluted in KBr pellets. The
micromorphology of poly(FHMA-co-MPC) FPNs was investigated using a Hitachi 7650B transmission
electron microscopy (TEM) operating at 80 kV. The sample for TEM measurements was prepared by
dropping the solution onto a carbon-coated copper grid. The particle size of poly(FHMA-co-MPC)
FPNs was taken on a Zeta potential analyzer (Malvern, Zetasizer nano zs90). UV−visible absorption
spectra were recorded on the UV spectrometer (TU-1810, Persee) using quartz cuvettes of 1 cm path
length. Fluorescence spectra were tested by a Hitachi F-4500 spectrophotometer with a slit width of 10
nm for both excitation and emission. The critical micelle concentration (CMC) was determined by the
tangent method, in which ten different concentrations BPH-poly(FHMA-co-MPC) FPNs (0.5, 0.4, 0.3,
0.25, 0.15, 0.1, 0.05, 0.01, 0.001, 0.0001 mg mL-1) were prepared for scanning fluorescent spectra (the
fluorescent excitation wavelength and emission wavelength were set as 405 nm and 546 nm,
respectively.).
2.2 Synthesis of benzophenone hydrazone (BPH)
BPH was synthesized via Schiff base reaction. BP (2.0 g, 11.1 mmol) was dissolved in ethanol (80
mL) at 40 ºC, followed by addition of excess hydrazine monohydrate (3.6 mL). Adding one drop of
aceticacid as a catalyst before the mixture was stirred and refluxed for 24 h. After cooling to room
temperature, the crude product was concentrated and then dissolved in ethyl acetate. The product was
purified by washing the solution with deionized water thrice. After the organic solvent was evaporated
1
under reduced pressure, white power of BPH was obtained in 91% yield. H NMR (400 MHz, CDCl3)
+
δ (ppm): δ7.53-7.24 (10H, aromatic ring), 5.41 (s, 2H, -NH2). HR-MS (ESI) calculated for C13H13N2
(M+H)+ 197.1073, found 197.1079. Elemental analysis calculated (%) for C13H12N2 : C 79.56, H 6.16,
N 14.27, found C 78.52, H 6.40, N 14.12.
2.3 Synthesis of 3-formyl-4-hydroxybenzyl methacrylate (FHMA)
CMSA as intermediate product was prepared according to previous literature.[41] In addition, the
typical experimental process for the synthesis of FHMA as following: mixture of methacrylic acid
(1.72 g, 0.02 mol), NaOH (0.8 g, 0.02 mol) and deionized water (30 mL) in a 100 mL reaction bottle
and the mixture were stirred at room temperature for 30 min. Subsequently, CMSA (3.41 g, 0.02 mol),
KI (1.0 g, 0.002 mol) and toluene (5 mL) were added, and the solution stirred at 50 ºC for 24 h. After
cooling to room temperature, the mixture was extracted with ethyl acetate (20 mL × 3), and the organic

ACCEPTED MANUSCRIPT
extract was dried over anhydrous Na2SO4. After filtration, the solvent was removed by using a rotary
evaporator. The product was purified by silica gel column chromatography with ethyl acetate:
petroleum ether (v: v, 1 : 10) to give FHMA as a white crystal. Yield: 68%. 1H NMR (400 MHz, CDCl3)
δ (ppm): δ 11.05 (s, 1H, OH), 9.91 (s, 1H, CHO), 7.61-7.55 (2H, aromatic ring), 7.00 (1H, aromatic
ring), 6.14 (s, 1H, trans-CH2), 5.60 (s, 1H, cis-CH2), 5.16 (s, 2H, COOCH2), 1.96 (s, 3H, CH3C=CH2).
HR-MS (ESI) calculated for C12H11O4 (M-H)- 219.0663, found 219.0660. Elemental analysis
-
calculated (%) for C12H12O4 : C 65.45, H 5.49, O 29.06, found C 62.57, H 5.89, O 30.90.
2.4 Synthesis of BPH-poly(FHMA-co-MPC)
The BPH-poly(FHMA-co-MPC) was prepared by combining RAFT polymerization and
post-polymerization method. Specifically, FHMA (110 mg, 0.5 mmol), MPC (295 mg, 1.0 mmol), CTA
(28.9 mg, 0.2 mmol), AIBN (5 mg, 0.03 mmol) and absolute DMF (10 mL) were introduced into a
Schlenk tube and bubbled by nitrogen flow for 30 min. The reaction mixture was stirred at 70 ºC for 12
h. After the polymerization was completed, BPH (98 mg, 0.5 mmol) was added to the reaction system.
The new reaction mixture was further stirred at 70 ºC for 4 h. After cooling to room temperature, the
mixture was dialyzed against water for 24 h then ethanol for 24 h using 3500 Da Mw cutoff dialysis
membranes. Finally, the resulting product was carried out by freeze-drying.
2.5 Cytotoxicity of BPH-poly(FHMA-co-MPC) FPNs
The cytotoxicity of as-prepared BPH-poly(FHMA-co-MPC) FPNs was evaluated by cell counting
kit-8 (CCK-8) assay.[42] Briefly, HeLa cells were seeded in 96-well microplates at a density of 5 × 104
cells mL−1 in 160 µL of cell culture medium containing 10% fetal bovine serum (FBS). After overnight
culture, cells were incubated with different doses of BPH-poly(FHMA-co-MPC) FPNs (10-120 µg
mL-1) for 12 and 24 h, respectively. Then cells were washed with phosphate-buffered saline (PBS) for
three times. 10 µL of CCK-8 solution and 100 µL of Dulbecco's modified eagle medium (DMEM) cell
culture medium were added to each well and incubated for another 2 h at 37 °C. Finally, plates were
analyzed using a microplate reader (VictorШ, PerkinElmer). 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 not exposure to BPH-poly(FHMA-co-MPC) FPNs), 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).
2.6 Confocal microscopic imaging of BPH-poly(FHMA-co-MPC) FPNs
The cell uptake behavior of BPH-poly(FHMA-co-MPC) FPNs was investigated by a confocal laser

ACCEPTED MANUSCRIPT
scanning microscope (CLSM, Zeiss 710 3-channel, Germany) using HeLa cells. The excitation
wavelength was set as 405 nm. HeLa cells were set in a glass bottom dish with a density of 1 × 105
cells per dish. On the day of treatment, BPH-poly(FHMA-co-MPC) FPNs with a suitable concentration
of 40 µg mL−1 was incubated with cells for 3 h at 37 °C. Afterward, the cells were washed three times
with PBS to remove the redundant BPH-poly(FHMA-co-MPC) FPNs and then fixed with 4%
paraformaldehyde for 10 min at room temperature.
3 Results and discussion
1
The synthetic products of each step were characterized and confirmed by H NMR spectra. To be
specific, the characteristic chemical shift of BPH located at 5.41 ppm is attributed to -NH2 group,
demonstrating the successful synthesis of BPH via Schiff base reaction (Fig. S1). Besides, the
emerging chemical shifts of FHMA located at 6.14, 5.60 and 1.96 ppm are assigned to C=CH2 and
1
-CH3 groups after CMSA reacted with methacrylic acid (Fig. S2). More importantly, the H NMR
spectra of monomer MPC and the purified copolymer BPH-poly(FHMA-co-MPC) is shown in Fig. 1.
Obviously, the characteristic chemical shifts of the fluorescence moiety of BPH-poly(FHMA-co-MPC)
including the protons of OH (10.18 ppm, b), CH=N (8.91 ppm, c), and aromatic ring (7.66-6.72 ppm)
can be clearly observed, and the hydrophilic MPC group including the protons of N+-CH3 (3.31 ppm, f)
and C-CH3 (1.20 ppm, g) can also be clearly identified while the chemical shifts of 6.11 and 5.56 ppm
representing the ethylene group of MPC were disappeared in BPH-poly(FHMA-co-MPC),
1
demonstrating the successful RAFT polymerization and post-polymerization modification. The H
NMR results suggested the successful fabrication of the amphiphilic copolymer
BPH-poly(FHMA-co-MPC), which was also affirmed by other techniques.

ACCEPTED MANUSCRIPT
Fig. 1 1H NMR spectra of MPC and BPH-poly(FHMA-co-MPC). The specific NMR information about
MPC, C=N and aromatic ring has been clearly indentified by 1H NMR spectrum.
Furthermore, the successful synthesis of BPH, FHMA and BPH-poly(FHMA-co-MPC) was
confirmed by FT-IR spectroscopy. Typically, two characteristic peaks of 3426 and 1629 cm-1 are
assigned to the stretching vibration of NH2 and C=N group, respectively, indicating the successful
synthesis of BPH via Schiff base condensation (Fig. S3). Moreover, the strong stretching vibration
bands of C=C located at 1661 cm-1 and C=O located at 1717 cm-1 were found in FHMA when CMSA
reacted with methacrylic acid, which suggested its successful synthesis (Fig. 2A). More importantly,
compared with the FT-IR spectrum of FHMA, a new characteristic peak of 1590 cm-1 attributed to the

ACCEPTED MANUSCRIPT
stretching vibration of C=N is highlighted and the stretching vibration of C=C is disappeared in
as-prepared BPH-poly(FHMA-co-MPC), indicating the successful polymerization of CMSA and MPC
and further post-polymerization modification via Schiff base condensation (Fig. 2B). Combined the
results of 1H NMR spectra and FT-IR spectra, we can draw a conclusion that the zwitterionic
copolymer BPH-poly(FHMA-co-MPC) was synthesized successfully.
Fig. 2 FT-IR spectra of FHMA and BPH-poly(FHMA-co-MPC).
According to the previous reports, amphiphilic copolymers tend to form nanoparticles with a
hydrophobic core and a hydrophilic shell in aqueous environments through self-assembly strategy.[43]
Therefore, the morphology and size of BPH-poly(FHMA-co-MPC) FPNs were characterized by TEM
and DLS. As shown in Fig. 3A, lots of spherical nanoaggregates with diameters ranging about 100 nm
are observed, indicating the formation of nanoparticles via self-assembly when the as-prepared
amphiphilic copolymer BPH-poly(FHMA-co-MPC) dispersed in water. Furthermore, the
hydrodynamic size distribution of BPH-poly(FHMA-co-MPC) FPNs suggested that the size of these
FPNs in water is in the range of 50–200 nm (Fig. 3B). It is noteworthy that the size of
BPH-poly(FHMA-co-MPC) FPNs characterized by TEM is somewhat smaller than the hydrodynamic
size, which might be due to the shrinkage of micelle for TEM observation. Hence, the above results
demonstrated that BPH-poly(FHMA-co-MPC) FPNs possess suitable size and excellent water
dispersibility, implying their potential for biomedical applications.

ACCEPTED MANUSCRIPT
Fig. 3 (A) TEM image of BPH-poly(FHMA-co-MPC) FPNs dispersed in water, revealing spherical
nanoparticle morphology; (B) Size distribution of BPH-poly(FHMA-co-MPC) FPNs dispersed in water
determined by DLS.
The chemical composition of BPH-poly(FHMA-co-MPC) was analyzed by XPS spectrum. Carbon
(C), oxygen (O), nitrogen (N), phosphorus (P) and sulfur (S) elements are presented in low-resolution
XPS spectrum of BPH-poly(FHMA-co-MPC) (Fig. 4). Table S1 shows that the overall wt % of these
elements (C: O: N: P: S) as 71.74: 19.39: 5.89: 2.49: 0.49, respectively. The element S should be
derived from the CTA, while the element P should be originated from the MPC. The element N should
be derived both from the MPC and Schiff base. Moreover, based on the element ratio values, we could
clearly find that the content of N is obviously greater than that of P. This implied that the final
copolymers contain both MPC and Schiff base. And the molar ratio of Schiff base to MPC is 0.683:1.
All of the above results indicated that we have successfully synthesized the AIE-active polymers
through the combination of RAFT polymerization and post-polymerization modification (formation of
Schiff base).
Fig. 4 XPS spectrum of BPH-poly(FHMA-co-MPC) with different components of carbon (C), oxygen
(O), nitrogen (N), phosphorus (P) and sulfur (S) elements.

ACCEPTED MANUSCRIPT
High-resolution spectra of C 1s of BPH-poly(FHMA-co-MPC) is split into four main components:
-CH group at ~285 eV, C-O or C-N groups at 286 eV, and C=O group at 287 eV, respectively (Fig. 5A).
Several peaks that centered at 532.4 and 530.6 eV are found in the high-resolution spectrum of O 1s.
These peaks indicated that the atom O is conjugated with different elements (P, C). More importantly,
binding energies of C-N+ and C=N bond of the region N 1s in BPH-poly(FHMA-co-MPC) are 402.7
eV and 399.3 eV, respectively, which suggested BPH was conjugated with salicylaldehyde
(SA)-functional preliminary copolymer via Schiff base condensation (Fig. S5).[44] Final, the existence
of P and S also suggested that the copolymers were obtained through RAFT polymerization.
Fig. 5 High-resolution spectra of BPH-poly(FHMA-co-MPC). (A) The region of C1s, (B) The region
of O 1s, (C) The region of N 1s, (D) The region of P 2p.
Due to the amphiphilic properties, BPH-poly(FHMA-co-MPC) copolymers self-assemble in aqueous
solution with the hydrophobic fluorescent moieties aggregating together to form the core and the
hydrophilic MPC chains extended towards the aqueous solution. Thus obtained
BPH-poly(FHMA-co-MPC) FPNs are endowed with good water dispersibility. It can be seen that no
obvious precipitation and the background character “Nanchang University” could be observed when
BPH-poly(FHMA-co-MPC) FPNs were dispersed in water (inset of Fig. 6A). The UV absorption
spectrum of BPH-poly(FHMA-co-MPC) FPNs in water is shown in Fig. 6A, two peaks located at 306
and 352 nm could be found. The strong absorption peak at 306 nm could be attributed to π→π*
transition, which demonstrated the existence of conjugated chemical structure of

ACCEPTED MANUSCRIPT
BPH-poly(FHMA-co-MPC) FPNs. Furthermore, it can be concluded that aromatic rings have been
conjugated with hetero atom such as N of BPH-poly(FHMA-co-MPC) FPNs because a shoulder peak
at 352 nm could be ascribed to the R band of n→π* transition. Intense yellow fluorescence can be
observed from the BPH-poly(FHMA-co-MPC) FPNs after they were irradiated by a UV-lamp at 365
nm (inset of Fig. 6B). Fluorescence (FL) spectra shown in Fig. 6B indicated that the maximum
emission wavelength is located at 546 nm when BPH-poly(FHMA-co-MPC) FPNs in water were
excited with 405 nm wavelength. More importantly, the fluorescence stability of was
BPH-poly(FHMA-co-MPC) FPNs was also examined. As displayed in Fig. 6C, even though
BPH-poly(FHMA-co-MPC) FPNs suspension were irradiated at 365 nm for 1 h, no obvious decrease in
FL intensity was observed. It can be seen that the fluorescence intensity after irradiation is still greater
than 95% as compared with the value before irradiation. It suggests that BPH-poly(FHMA-co-MPC)
FPNs possess desirable photo-bleaching resistance. The AIE properties of BPH-poly(FHMA-co-MPC)
FPNs could be determined by good and poor solvents because the obtain BPH-poly(FHMA-co-MPC)
copolymers could dissolve well in some organic solvents such as DMF while just dispersed in aqueous
solution. As shown in Fig. 6D, no fluorescence could be observed when BPH-poly(FHMA-co-MPC)
copolymers were dissolved in DMF while bright yellow fluorescent could be observed with a
maximum emission peak at 546 nm, indicating obvious AIE properties. The mechanism of the AIE
properties for BPH-poly(FHMA-co-MPC) copolymers could be explained in a similar manner to
previous literatures.[45-47] In solution state, nonradiative relaxation of the excited states was activated
by the free intermolecular rotation around the N-N bond, resulting in fluorescence quenching. However,
after BPH-poly(FHMA-co-MPC) copolymers self-assemble in aqueous solution, the rotation was
inhibited in the aggregate state, which demonstrate their AIE attributes and the excited state
intramolecular proton transfer (ESIPT) fluorescence.

ACCEPTED MANUSCRIPT
Fig. 6 (A) The UV absorption spectrum of BPH-poly(FHMA-co-MPC) FPNs in water. Inset: visible
photograph of BPH-poly(FHMA-co-MPC) FPNs in water using the logo of “Nanchang University” as
background; (B) The FL spectra of BPH-poly(FHMA-co-MPC) FPNs in water. Inset: FL image of
BPH-poly(FHMA-co-MPC) FPNs in water taken at 365 nm UV light; (C) FL stability of
BPH-poly(PEG-co-FHMA) FPNs under continuous UV light exposure of 365 nm for 1 h; (D) The FL
emission spectra of BPH-poly(FHMA-co-MPC) FPNs in DMF and water. Inset: FL images of
BPH-poly(FHMA-co-MPC) FPNs in DMF (left) and water (right) taken at 365 nm UV light.
The critical micelle concentration (CMC) is the minimum concentration of surfactant or amphiphilic
polymers required for micelles to form, which is highly important for the construction of stable
micelles in low concentration of solution. Thus, the CMC value of the BPH-poly(FHMA-co-MPC)
FPNs was determined by conducting the FL spectra of BPH-poly(FHMA-co-MPC) FPNs in water at
different concentrations, with excitation wavelength was 405 nm (Fig. 7A). Furthermore, the intensity
of the fluorescence emission vs. the logarithm of the concentration of BPH-poly(FHMA-co-MPC)
FPNs was plotted to determine the CMC by the tangent method (Fig. 7B). The results suggested that
the CMC value of BPH-poly(FHMA-co-MPC) FNPs is 0.033 mg mL-1. Therefore, the
BPH-poly(FHMA-co-MPC) FNPs with low CMC value would be stable under diluent physiological
conditions and useful for practical applications.

ACCEPTED MANUSCRIPT
Fig. 7 (A) FL spectra of BPH-poly(FHMA-co-MPC) FPNs in water at different concentrations
(0.0001-0.5 mg mL-1), excitation wavelength was 402 nm. (B) The relationship of the FL intensity and
the logarithm of BPH-poly(FHMA-co-MPC) FPNs concentration in water.
All above characterization results suggested BPH-poly(FHMA-co-MPC) FPNs possess good water
dispersibility, desirable particle size and unique photophysical properties, which seem to suitable for
bioimaging applications. However, biocompatibility evaluation is a necessary step before evaluating
the potential biomedical applications of BPH-poly(FHMA-co-MPC) FPNs. As shown in Fig. 8, it is
worth mentioning that only little decrease of cell viability value after the cells were incubated with
BPH-poly(FHMA-co-MPC) FPNs at different concentrations for 12 h and 24 h. Moreover, the cell
viability value is still greater than 95% after 24 h incubation even the concentrations of
BPH-poly(FHMA-co-MPC) FPNs as high as 120 µg mL-1, suggesting that excellent biocompatibility
of BPH-poly(FHMA-co-MPC) FPNs. It could draw
a
preliminary conclusion that
BPH-poly(FHMA-co-MPC) FPNs have great potential in cell imaging application.
Fig. 8 Cell viability of BPH-poly(FHMA-co-MPC) FPNs with HeLa cells for 12 h and 24 h,
respectively.

ACCEPTED MANUSCRIPT
On the basis of excellent biocompatibility, the cellular uptake of BPH-poly(FHMA-co-MPC) FPNs
was investigated by CLSM. Bright field of CLSM image shown in Fig. 9A revealed that cells still keep
their normal morphology after they were incubated with 40 µg mL−1 of BPH-poly(FHMA-co-MPC)
FPNs for 3 h, further demonstrating their good biocompatibility. Moreover, many dark areas are
surrounded by intense yellow fluorescence, which suggested that BPH-poly(FHMA-co-MPC) FPNs are
internalized by HeLa cells via phagocytosis and mainly distributed in the whole cytoplasm (Fig. 9B).
These dark areas should be the locations of cell nuclei because the size of BPH-poly(FHMA-co-MPC)
FPNs is obvious larger than that of nucleus pores and therefore could not enter into cell nuclei. Fig. 9C
shows the merged image of bright field and fluorescent images. It can be seen that the fluorescence
signals are almost overlaid with the cell locations. It further indicated that the
BPH-poly(FHMA-co-MPC) FPNs are internalized through non-specific route. Combine the above cell
viability results so we can draw a concluded that BPH-poly(FHMA-co-MPC) FPNs have great potential
for practical biological imaging.
Fig. 9 CLSM images of HeLa cells incubated with 40 µg mL−1 of BPH-poly(FHMA-co-MPC) FPNs
for 3 h. (A) Bright field, (B) excited with 405 nm laser, (C) merge image of A and B. Scale bar = 20
µm.
4 Conclusions
In summary, we have developed a novel strategy for preparation of BPH-poly(FHMA-co-MPC)
FPNs through the combination of RAFT polymerization and post-polymerization modification. The
resultant BPH-poly(FHMA-co-MPC) FPNs showed numerous excellent properties such as high water
dispersibility, uniform morphology, intense yellow fluorescence, large Stokes shifts, remarkable
photostability and excellent biocompatibility, making them promising for biological imaging
applications. Compared with the construction of AIE-active FPNs based on polymerizable or
functionalized AIE molecules, the novel method provided by this contribution has many advantages.
First, it can avoid the complex process for the synthesis of AIE dyes. Secondly, the chemical
composition and fluorescence properties and the physicochemical properties of copolymers can be
facilely adjusted via the RAFT polymerization and Schiff base reaction. More importantly, this work

ACCEPTED MANUSCRIPT
provides novel ideas for the fabrication of AIE-active FPNs through the post-polymerization
modification.
Acknowledgements
This research was supported by the National Natural Science Foundation of China (Nos. 51363016,
21474057, 21564006, 21561022, 21644014), Natural Science Foundation of Jiangxi Province in China
(Nos. 20161BAB203072, 20161BAB213066).
References
[1] Mei J, Leung NL, Kwok RT, Lam JW, Tang BZ. Aggregation-induced emission: together we shine,
united we soar! Chem Rev. 2015;115(21):11718-940.
[2] Yao J, Yang M, Duan Y. Chemistry, biology, and medicine of fluorescent nanomaterials and related
systems: new insights into biosensing, bioimaging, genomics, diagnostics, and therapy. Chem Rev.
2014;114(12):6130-78.
[3] Li K, Liu B. Polymer-encapsulated organic nanoparticles for fluorescence and photoacoustic
imaging. Chem Soc Rev. 2014;43(18):6570-97.
[4] Liu M, Zeng G, Wang K, Wan Q, Tao L, Zhang X, et al. Recent developments in polydopamine: an
emerging soft matter for surface modification and biomedical applications. Nanoscale.
2016;8(38):16819-40.
[5] Hong Y, Lam JW, Tang BZ. Aggregation-induced emission. Chem Soc Rev. 2011;40(11):5361-88.
[6] Luo J, Xie Z, Lam JW, Cheng L, Chen H, Qiu C, et al. Aggregation-induced emission of 1-methyl-1, 2,
3, 4, 5-pentaphenylsilole. Chem Commun. 2001(18):1740-1.
[7] Mei J, Hong Y, Lam JW, Qin A, Tang Y, Tang BZ. Aggregation‐induced emission: the whole is more
brilliant than the parts. Adv Mater. 2014;26(31):5429-79.
[8] Nie H, Hu K, Cai Y, Peng Q, Zhao Z, Hu R, et al. Tetraphenylfuran: aggregation-induced emission or
aggregation-caused quenching? Mater Chem Front. 2017;1(6):1125-9.
[9] Ding D, Li K, Liu B, Tang BZ. Bioprobes based on AIE fluorogens. Accounts Chem Res.
2013;46(11):2441-53.
[10] Zhan C, You X, Zhang G, Zhang D. Bio‐/Chemosensors and Imaging with Aggregation‐Induced
Emission Luminogens. Chem Rec. 2016;16(4):2142-60.
[11] Hu R, Leung NL, Tang BZ. AIE macromolecules: syntheses, structures and functionalities. Chem Soc
Rev. 2014;43(13):4494-562.
[12] Leung CWT, Hong Y, Chen S, Zhao E, Lam JWY, Tang BZ. A photostable AIE luminogen for specific
mitochondrial imaging and tracking. J Am Chem Soc. 2012;135(1):62-5.
[13] Wang Z, Chen S, Lam JW, Qin W, Kwok RT, Xie N, et al. Long-term fluorescent cellular tracing by
the aggregates of AIE bioconjugates. J Am Chem Soc. 2013;135(22):8238-45.
[14] Shi H, Kwok RT, Liu J, Xing B, Tang BZ, Liu B. Real-time monitoring of cell apoptosis and drug
screening using fluorescent light-up probe with aggregation-induced emission characteristics. J Am
Chem Soc. 2012;134(43):17972-81.
[15] Shi H, Liu J, Geng J, Tang BZ, Liu B. Specific detection of integrin αvβ3 by light-up bioprobe with
aggregation-induced emission characteristics. J Am Chem Soc. 2012;134(23):9569-72.
[16] Feng L, Wang C, Deng X, Miao X, Wang J, Wang Y, et al. New application of AIEgens realized in
photodetectors: reduced work function of transparent electrodes and much improved performance.
Mater Chem Front. 2018.

ACCEPTED MANUSCRIPT
[17] Zhang X, Wang K, Liu M, Zhang X, Tao L, Chen Y, et al. Polymeric AIE-based nanoprobes for
biomedical applications: recent advances and perspectives. Nanoscale. 2015;7(27):11486-508.
[18] Zhan R, Pan Y, Manghnani PN, Liu B. AIE Polymers: Synthesis, Properties, and Biological
Applications. Macromol Biosci. 2017;17(5).
[19] Cao Z, Liang X, Chen H, Gao M, Zhao Z, Chen X, et al. Bright and biocompatible AIE polymeric
nanoparticles prepared from miniemulsion for fluorescence cell imaging. Polym Chem.
2016;7(35):5571-8.
[20] Li C, Liu X, He S, Huang Y, Cui D. Synthesis and AIE properties of PEG–PLA–PMPC based triblock
amphiphilic biodegradable polymers. Polym Chem. 2016;7(5):1121-8.
[21] Zhang X, Zhang X, Wang S, Liu M, Tao L, Wei Y. Surfactant modification of aggregation-induced
emission material as biocompatible nanoparticles: Facile preparation and cell imaging. Nanoscale.
2013;5(1):147-50.
[22] Zhang X, Zhang X, Yang B, Zhang Y, Liu M, Liu W, et al. Fabrication of water-dispersible and
biocompatible red fluorescent organic nanoparticles via PEGylation of aggregate induced emission
enhancement dye and their cell imaging applications. Colloid Surfaces B. 2014;113:435-41.
[23] Shen X, Shi Y, Peng B, Li K, Xiang J, Zhang G, et al. Fluorescent Polymeric Micelles with
Tetraphenylethylene Moieties and Their Application for the Selective Detection of Glucose. Macromol
Biosci. 2012;12(11):1583-90.
[24] Yuan Y, Xu S, Zhang C-J, Liu B. Light-responsive AIE nanoparticles with cytosolic drug release to
overcome drug resistance in cancer cells. Polym Chem. 2016;7(21):3530-9.
[25] Zhang J, Zhu J, Lu C, Gu Z, He T, Yang A, et al. A hyperbranched fluorescent supramolecular
polymer with aggregation induced emission (AIE) properties. Polym Chem. 2016;7(26):4317-21.
[26] Tian J, Jiang R, Gao P, Xu D, Mao L, Zeng G, et al. Synthesis and cell imaging applications of
amphiphilic AIE-active poly (amino acid) s. Mater Sci Eng C-Mater. 2017;79:563-9.
[27] Ma C, Xie G, Zhang X, Yang L, Li Y, Liu H, et al. Biocompatible fluorescent polymers from
PEGylation of an aggregation-induced emission dye. Dyes Pigments. 2017;139:672-80.
[28] Ma C, Zhang X, Yang L, Li Y, Liu H, Yang Y, et al. Alkyl length dependent mechanofluorochromism of
AIE-based phenothiazinyl fluorophenyl acrylonitrile derivatives. Dyes Pigments. 2017;136:85-91.
[29] Xie G, Ma C, Zhang X, Liu H, Yang L, Li Y, et al. Chitosan-based cross-linked fluorescent polymer
containing aggregation-induced emission fluorogen for cell imaging. Dyes Pigments. 2017;143:276-83.
[30] Wan Q, Jiang R, Mao L, Xu D, Zeng G, Shi Y, et al. A powerful “one-pot” tool for fabrication of
AIE-active luminescent organic nanoparticles through the combination of RAFT polymerization and
multicomponent reactions. Mater Chem Front. 2017;1(6):1051-8.
[31] Gauthier MA, Gibson MI, Klok HA. Synthesis of Functional Polymers by Post‐Polymerization
Modification. Angew Chem. 2009;48(1):48-58.
[32] Ghosh S, Basu S, Thayumanavan S. Simultaneous and reversible functionalization of copolymers
for biological applications. Macromolecules. 2006;39(17):5595-7.
[33] Zhang J, Wang L, Li C, Li Y, Liu J, Tu Y, et al. Preparation and characterization of solution
processable phthalocyanine‐containing polymers via a combination of RAFT polymerization and post
‐polymerization modification techniques. J Polym Sci Pol Chem. 2014;52(5):691-8.
[34] Fokina A, Lee Y, Chang JH, Braun L, Bae WK, Char K, et al. Side-chain conjugated polymers for use
in the active layers of hybrid semiconducting polymer/quantum dot light emitting diodes. Polym
Chem. 2016;7(1):101-12.
[35] Boaen NK, Hillmyer MA. Post-polymerization functionalization of polyolefins. Chem Soc Rev.

ACCEPTED MANUSCRIPT
2005;34(3):267-75.
[36] Gupta K, Sutar AK. Catalytic activities of Schiff base transition metal complexes. Coordin Chem
Rev. 2008;252(12-14):1420-50.
[37] Katsuki T. Unique asymmetric catalysis of cis-β metal complexes of salen and its related Schiff-base
ligands. Chem Soc Rev. 2004;33(7):437-44.
[38] Cozzi PG. Metal–Salen Schiff base complexes in catalysis: practical aspects. Chem Soc Rev.
2004;33(7):410-21.
[39] Adsule S, Barve V, Chen D, Ahmed F, Dou QP, Padhye S, et al. Novel Schiff base copper complexes
of quinoline-2 carboxaldehyde as proteasome inhibitors in human prostate cancer cells. J Med Chem.
2006;49(24):7242-6.
[40] Tao L, Liu J, Davis TP. Branched Polymer− Protein Conjugates Made From Mid-Chain-Functional P
(HPMA). Biomacromolecules. 2009;10(10):2847-51.
[41] Bagherzadeh M, Zare M. Synthesis, characterization, and catalysis of recyclable new
piperazine-bridged Mo (VI) polymers [MoO2 (Salen)(piperazine)] n in highly selective oxygenation of
alkenes and sulfides. J Coord Chem. 2013;66(16):2885-900.
[42] Zhang X, Hu W, Li J, Tao L, Wei Y. A comparative study of cellular uptake and cytotoxicity of
multi-walled carbon nanotubes, graphene oxide, and nanodiamond. Toxicol Res. 2012;1(1):62-8.
[43] Zhang X, Zhang X, Tao L, Chi Z, Xu J, Wei Y. Aggregation induced emission-based fluorescent
nanoparticles: fabrication methodologies and biomedical applications.
2014;2(28):4398-414.
J Mater Chem B.
[44] He W, Jiang C, Wang J, Lu L. High‐Rate Oxygen Electroreduction over Graphitic‐N Species
Exposed on 3D Hierarchically Porous Nitrogen ‐ Doped Carbons. Angew Chem Int Edit.
2014;53(36):9503-7.
[45] Peng L, Wei R, Li K, Zhou Z, Song P, Tong A. A ratiometric fluorescent probe for hydrophobic
proteins in aqueous solution based on aggregation-induced emission. Analyst. 2013;138(7):2068-72.
[46] Wei R, Song P, Tong A. Reversible thermochromism of aggregation-induced emission-active
benzophenone azine based on polymorph-dependent excited-state intramolecular proton transfer
fluorescence. J Phys Chem C. 2013;117(7):3467-74.
[47] Wang Z, Gui C, Zhao E, Wang J, Li X, Qin A, et al. Specific fluorescence probes for lipid droplets
based on simple AIEgens. ACS Appl Mater Inter. 2016;8(16):10193-200.

ACCEPTED MANUSCRIPT
► Fluorescent polymeric nanoparticles with aggregation-induced emission feature
► AIE-active FPNs through formation of Schiff base
► The method for preparation of AIE-active FPNs is rather simple and effective
► These AIE-active FPNs are promising for biomedical applications