Preparation of AIE-active fluorescent polymeric nanoparticles through a catalyst-free thiol-yne click reaction for bioimaging applications

Accepted Manuscript Preparation of AIE-active fluorescent polymeric nanoparticles through a catalyst-free thiol-yne click reaction for bioimaging applications

Qian-yong Cao, Ruming Jiang, Meiying Liu, Qing Wan, Dazhuang Xu, Jianwen Tian, Hongye Huang, Yuanqing Wen, Xiaoyong Zhang, Yen Wei PII: DOI: Reference:

S0928-4931(17)31859-3 doi: 10.1016/j.msec.2017.06.008 MSC 8135

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

15 May 2017 26 May 2017 15 June 2017

Please cite this article as: Qian-yong Cao, Ruming Jiang, Meiying Liu, Qing Wan, Dazhuang Xu, Jianwen Tian, Hongye Huang, Yuanqing Wen, Xiaoyong Zhang, Yen Wei , Preparation of AIE-active fluorescent polymeric nanoparticles through a catalyst-free thiol-yne click reaction for bioimaging applications, Materials Science & Engineering C (2017), doi: 10.1016/j.msec.2017.06.008

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ACCEPTED MANUSCRIPT Preparation of AIE-active fluorescent polymeric nanoparticles through a catalyst-free thiol-yne click reaction for bioimaging applications Qian-yong Caoa,#, Ruming Jianga,#, Meiying Liua, Qing Wana, Dazhuang Xua, Jianwen Tiana, Hongye Huanga, Yuanqing Wena,*, Xiaoyong Zhanga,*, Yen Weib,*

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Department of Chemistry, Nanchang University, 999 Xuefu Avenue, Nanchang 330031, China. Department of Chemistry and the Tsinghua Center for Frontier Polymer Research, Tsinghua

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University, Beijing, 100084, P. R. China.

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# These authors contributed equally to this work

ACCEPTED MANUSCRIPT Abstract Fluorescent polymeric nanoparticles (FPNs) with aggregation-induced emission (AIE) characteristics have attracted much attention for biomedical applications due to their remarkable AIE feature, high water dispersity and desirable biocompatibility. The development of facile and effective strategies for fabrication of these AIE-active FPNs therefore should be of great importance for their biomedical

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applications. In this work, we reported that a catalyst-free thiol-yne click reaction can be utilized for fabrication of AIE-active FPNs in short reaction time and even without protection of inert gas. The

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results indicated that the obtained AIE-active amphiphilic copolymers (PEGMA-PhE) can readily self-assemble into luminescent nanoparticles (PEGMA-PhE FPNs) with high water dispersity, uniform

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size and morphology, red fluorescence. Cell viability examination and cell uptake behavior of PEGMA-PhE FPNs confirmed that these AIE-active FPNs possess low toxicity towards cells and can

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be easily internalized by cells through non-specific route. Therefore the remarkable properties of PEGMA-PhE FPNs such as high water dispersity, AIE-active fluorescence and nanoscale size as well

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as excellent biocompatibility make them promising for biomedical applications.

Key words: Aggregation-induced emission, a catalyst-free thiol-yne click reaction, fluorescent polymeric nanoparticles, biological imaging

ACCEPTED MANUSCRIPT 1. Introduction Fluorescent nanoprobes have received enormously attention because of their highly potential for bioscience and biotechnology applications in the past few decades. Since the first report that using semiconductor quantum dots as fluorescent nanoprobes in diagnostics and biological imaging in 1998,[1] various fluorescent nanoprobes have been extensively explored based on different

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fluorescent materials such as fluorescent inorganic nanoparticles (FINs) and fluorescent proteins.[2-8] Nevertheless, numerous fluorescent nanoparticles have been reported diversified inherent

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disadvantages accompanying with deepening of the research. For example, FINs possess some advantages contained strong luminescence and great photostability, however, semiconductor quantum

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dots show high toxicity for human health owing to their poor biodegrability and long-term aggregation in vivo.[9] Moreover, on account of their high cost and easy photobleaching, the employ

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of green fluorescent proteins has been seriously restricted for biomedical applications.[10-16] Compared with the above fluorescent nanoparticles, fluorescent polymeric nanoparticles (FPNs)

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possess obvious advantages such as facile fabrication, low cytotoxicity, excellent biodegradability, desirable photostability and fluorescent quantum yields, which could imply a huge potential in

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sensing and biological imaging applications.[17] However, the fluorescent intensity of FPNs based on

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traditional organic dyes often suffer from a bad effect, that show fluorescence quenching phenomenon when at aggregated state or in solution of high concentrations. This fluorescence quenching phenomenon was named as aggregation caused quenching (ACQ) effect. Although reducing the

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concentrations of organic dyes in polymeric core could attenuate the ACQ effect, the fluorescent intensity of the final FPNs is still weak because of low content of fluorophores. Therefore, the

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development of novel fluorophores with strong luminescence at aggregated state or high concentrations should be of great importance for overcoming the ACQ effect. Since the unique aggregation induced emission (AIE) phenomenon was accidentally discovered and proposed by Tang and co-workers in 2001.[18, 19] The biological imaging applications of FPNs with AIE feature have become one of the most important research directions because the AIE-active dyes could effectively avoid the ACQ effect of traditional organic dyes and make the FPNs with intensive fluorescence possible.[20-24] Over the past years, various novel AIE-active organic dyes including silole,[25, 26] tetraphenylethene (TPE),[27-29] anthracene[30-32] and their derivatives have been synthesized and widely examined for applications in biological imaging, protein fibrillation

ACCEPTED MANUSCRIPT detection and fingerprint visualization and so on.[33-37] But most of these organic dyes have poor dissolving capacity in physiological solution, which largely limit their applications for biomedical and bioimaging to a great extent. Therefore, great attention has been focused on the fabrication of water dispersible AIE-active probes relied on the formation of amphiphilic copolymers containing AIE-active dyes.[38] A number of fabrication strategies such as non-covalent encapsulation,

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controlled living radical polymerization, ring-opening reaction, multicomponent reactions, formation of dynamic bonds and supramolecular interaction etc have also been developed to fabricate

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AIE-active copolymers in recent years.[39-42] The catalyst-free thiol-yne click reaction is a simple and effective method for click the thiol group and yne owing to its distinguished superiorities, such as

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mild reaction conditions, high atom economy, toleration and high efficiency etc.[43-45] Nevertheless, to the best of our knowledge, the fabrication of AIE-active FPNs using thiol-yne click reaction has not

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been reported thus far.

In this contribution, the catalyst-free thiol-yne click reaction was utilized for preparation of

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AIE-active FPNs for the first time. The thiol groups containing polymers (PEGMA-IA-SH) were obtained by the combination of free radical polymerization and ring opening reaction (Scheme S1).

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AIE-active dye with two alkynyl end groups (named as PhE-OE) was conjugated with

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PEGMA-IA-SH via the catalyst-free thiol-yne click reaction to obtain the AIE-active amphiphilic copolymers (PEGMA-PhE) in a short time (Scheme S1). The resultant PEGMA-PhE copolymers containing hydrophilic PPEGMA and hydrophobic AIE-active dye could self-assemble into polymeric

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nanostructure, in which the hydrophobic PhE-OE was aggregated in the core while the hydrophilic components were covered on these FPNs. Therefore, the final AIE-active FPNs will emit intense

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fluorescence and well dispersed in aqueous solution. The AIE-active FPNs were characterized by various characterization techniques, including 1H nuclear magnetic resonance (NMR) spectroscopy, fluorescence spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, transmission electron microscopy (TEM) etc. To evaluate their potential for biological imaging, the cell viability as well as cell uptake behavior of AIE-active PEGMA-PhE FPNs were also examined. 2. Experiment sections 2.1. Materials and characterization The PEGMA-IA and 10-hexadecyl-10H-phenothiazinewere synthesized and characterized according to previous report.[46, 47] Phenothiazine (Mw: 199.28 Da, 98%), sodium hydride (Mw: 24.00 Da, 60%

ACCEPTED MANUSCRIPT dispersion in mineral oil), 1-bromohexadecane (Mw: 305.34 Da, 97%), N,N-dimethylformamide (DMF, Mw: 73.09 Da), 1,2-dichloroethane (Mw: 98.96 Da, 99.8%), phosphoryl chloride (POCl 3), 4-ethynylphenylacetonitrile (Mw: 141.17 Da, 95%), tetrabutylammonium hydroxide (TBAOH, 0.8 M in methanol), poly(ethylene glycol) monomethyl ether methacrylate (PEGMA, Mw: 950 Da, 98%), itaconic anhydride (IA, Mw: 112.19 Da, 96%), 2,2-azodiisobutyronitrile (AIBN, Mw: 164.21 Da, 98%),

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cysteamine hydrochloride (Mw: 113.61 Da, 98%) were obtained from Aladdin (Shanghai, China) and were used directly without any further purification. Redistilled water was employed in the experiments.

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Anhydrous alcohol and ethyl acetate were purchased from Heowns (Tianjin, China). All other chemical medicines and dry reagents commercial available were used as received without any purification.

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Fluorescence spectra were collected in the fluorescence spectrophotometer (FSP, model: C11367-11). H NMR spectra were obtained from Bruker Avance-400 spectrometer using D2O and CDCl3 as the

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solvents. FT-IR spectra acquired on Nicolet5700 (Thermo Nicolet corporation) with KBr as the pellets. UV-Vis spectra were recorded on Perkin Elmer LAMBDA 35 UV/Vis system. Hitachi 7650B

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microscope was used to observe TEM images. The hydrodynamic size distribution of PEGMA-PhE FPNs in aqueous solution was determined by dynamic laser scattering (DLS) using a ZetaPlus

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2.2 Synthesis of PhE-OE

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apparatus (Brookhaven Instruments, Holtsville, NY).

The synthesis route of PhE-OE was displayed in Scheme S2. The PhE-CHO was synthesized via Vilsmeier-Haack reaction. In an oven-dried 500 mL flask，POCl3 (23.0 g, 0.15 mol) was added

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dropwise to anhydrous DMF (11.0g ,0.15 mol) at 0 °C with stirring. After 30 min, the 10-hexadecyl-10H-phenothiazine (12.7 g, 0.03 mol) in 50 mL of CH2ClCH2Cl was added dropwise.

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Then the reaction system was stirred at 90 °C in oil bath. After 24 h, the mixture was poured into cool water and extracted with ethyl acetate. The organic layer was dried with magnesium sulfate overnight. Then the pure yellow solid PhE-CHO was obtained via column chromatography (5.1 g. yield 35%). The successful synthesis of PhE-CHO was characterized by 1H NMR (Fig. S1). PhE-OE was synthesized based on PhE-CHO. The detailed experimental procedure was displayed below. In brief, PhE-CHO (0.96 g, 2 mmol), 4-ethynylphenylacetonitrile (0.67 g, 4.8 mmol) and TBAOH (0.8 M, 10 drops) in 10 mL of alcohol were stirred at 90 °C for 4 h. Then the reaction mixture was cooled to room temperature, precipitating dark red oil and washed with dried alcohol for several times, finally we can obtain a dark red solid PhE-OE (1.2 g, 83%). The successful synthesis of PhE-OE

ACCEPTED MANUSCRIPT was characterized by 1H NMR (Fig. S1).

2.3 Synthesis of PEGMA-IA-SH The PEGMA-IA-SH was obtained via free radical polymerization using AIBN as chain initiator (Scheme S3). PEGMA (950 mg, 1 mmol), IA (112 mg, 1mmol) and AIBN (16.4 mg, 0.1 mmol), DMF

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(5mL) were put in a 10 mL Schlenk tube and then sealed. The oxygen was evacuated and refilled with dry N2 for three times. The mixture was heated to 65 °C with stirring. After 24 h, the cysteamine

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hydrochloride (114 mg, 1 mmol) in DMF was injected into sealed system, was stirring for 2 h. Then PEGMA-IA-SH was obtained via using 3500 Da Mw cutoff dialysis membranes for dialyzed against

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tap water for 48 h and ethanol for 8 h, respectively. Finally, the obtained polymers were dried in vacuum drying oven at 40 °C for 48 h.

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2.4 Preparation of PEGMA-PhE FPNs

The PEGMA-PhE copolymers were synthesized via a catalyst-free thiol-yne click reaction. The

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reaction process was depicted as follows in details. In a dried 10 mL flask, PEGMA-IA-SH (200 mg) and PhE-OE (50 mg) was dissolved in 2 mL tetrahydrofuran (THF) and stirred at 30 °C for 2 h under

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air environment. Then the reaction was stopped and poured into cool diethyl ether precipitating a red

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solid. The solid was dissolved in THF and poured into cool diethyl ether for five times. Ultimately, orange red solid was dried at 40 °C for 48 h. 2.5 Cell viability evaluation of PEGMA-PhE FPNs

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The cell viability of PEGMA-PhE FPNs was determined to evaluate their potential biomedical application. In brief, human lung adenocarcinoma epithelial (A549) cells were cultured into 96-well

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microplates at a density of 5 × 104 cells mL–1 in 160 μL of respective media containing 10% fetal bovine serum (FBS). After the cell attachment for 24 h, the PEGMA-PhE FPNs with different concentrations (10-120 µg mL–1) were incubated with cells for 12 and 24 h. After then, the residual luminescent materials outside of cells were removed and cells were washed with phosphate buffer saline (PBS) five times. The cell viability values of PEGMA-PhE FPNs were determined using cell counting kit-8 (CCK-8).[48-51] 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 3 h. Afterward, plates were analyzed using a microplate reader (VictorШ, Perkin-Elmer). Measurements of formazan dye absorbance were carried out at 450 nm, with the reference wavelength at 620 nm. The

ACCEPTED MANUSCRIPT values were proportional to the number of live cells. The percent reduction of CCK-8 dye was compared to controls, 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 Cell uptake of PEGMA-PhE FPNs

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The cell uptake behavior of PEGMA-PhE FPNs was conducted by the confocal laser scanning microscope (CLSM) Zeiss 710 3-channel (Zeiss, Germany) using A549 cells. The excitation

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wavelength was set as 458 nm. A549 cells were set in a glass bottom dish with a density of 1×105 cells per dish. On the day of treatment, PEGMA-PhE FPNs with a suitable concentration of 40 μg mL-1 was

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incubated with cells for 3 h at 37 ºC. Afterward, the cells were washed three times with PBS to remove

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the PEGMA-PhE FPNs and then fixed with 4% paraformaldehyde for 10 min at room temperature.

ACCEPTED MANUSCRIPT 3. Results and discussion 3.1 Characterization of PEGMA-PhE FPNs The PEGMA-PhE copolymers were synthesized by free radical living polymerization using PEGMA and IA as monomers and AIBN as chain initiator. To introduce the thiol groups on PEGMA-PhE copolymers, cysteamine hydrochloride was conjugated with IA through a facile ring-opening reaction.

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The PEGMA-IA-SH was final covalently conjugated with PhE-OE via a novel catalyst-free thiol-yne click reaction in THF to obtain amphiphilic polymers PEGMA-PhE at moderate temperature within 2 h. 1

H NMR spectra of

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In order to confirm the successful fabrication of PEGMA-PhE FPNs,

PEGMA-IA-SH and PEGMA-PhE were recorded for comparison. As shown in Fig. 1, after thiol-yne

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click reaction of PEGMA-IA-SH and PhE-OE, the 1H NMR spectrum of PEGMA-PhE can find the typical chemical shifts from PEGMA-IA-SH and PhE-OE. In the 1H NMR spectra, a series of

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characteristic peaks at δ 8.6-7.0 and 4.0-2.0 ppm were found in amphiphilic PEGMA-PhE copolymers, which were attributed to the resonance of protons on the aromatic rings (related to PhE-OE) and

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methylene and methyl group (related to the hydrogen PEGMA), respectively. Furthermore, the signals of hydrogen from the aromatic rings between 8.6 and 7.0 ppm were disappeared when 1H NMR spectra

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of PEGMA-PhE using D2O as the solvent, which could be ascribed to PEGMA-PhE self-assembly into

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nanoparticles in D2O (Fig. S2). The above results indicated that the successful formation of

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PEGMA-PhE copolymers through the catalyst-free thiol-yne click reaction.

Fig. 1 1H NMR spectra of PEGMA-IA-SH and PEGMA-PhE use D2O and d6-DMSO as solvents.

In addition, the FT-IR spectra of PhE-OE, PEGMA-IA-SH and PEGMA-PhE are shown in Fig. 2. It can be seen that two characteristic peaks at 2100 and 2400 cm-1 were discovered in the samples of PhE-OE, which could be attributed to the stretching vibration of C≡C and C≡N. On the other hand, a

ACCEPTED MANUSCRIPT series of peaks at 1600-1450 cm-1 were observed, which can be explained by the stretching vibration of various benzyls. After covalent conjugation with PEGMA-IA-SH, a typical peak at 1400 cm-1 suggested the existence of double bond (-C=C-). More importantly, compared with PhE-OE, the typical single peak of triple bond (-C≡C-) about 2100 cm-1 was disappeared in the samples of PEGMA-PhE. Hence, we can further confirm that PEGMA-PhE copolymers were successfully fabricated based on the

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FT-IR spectra.

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Fig. 2 The FT-IR spectra of AIE dye PhE-OE and PEGMA-PhE FPNs, which is used to evidence

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successful preparation of PEGMA-PhE FPNs.

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PEGMA-PhE copolymers display amphiphilic properties because hydrophobic AIE-active PhE-OE is conjugated with hydrophilic PEGMA-IA-SH. Therefore, PEGMA-PhE copolymers will self-assemble into nanoparticles when they were dispersed in pure aqueous solution. Meanwhile, the

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cores of these nanoparticles were encapsulated with conjugated aromatic groups, while the hydrophilic carboxyl groups and PEG segments can coat on the surfaces. Thus, the TEM images were also used to

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the size and morphology of the final self-assemblies PEGMA-PhE FPNs. As shown in Fig. 3, many spherical nano-granules with diameter between 100-200 nm can be clearly observed. The results demonstrated that the preparation of PEGMA-PhE copolymer could self-assemble to form PEGMA-PhE in aqueous environment, indicating PEG segments was covered on the surface of PhE-OE successfully. Meanwhile, the size distribution of PEGMA-PhE FPNs is 100 ± 20 nm based on TEM images. Moreover, the hydrodynamic size distribution of PEGMA-PhE FPNs was demonstrated to be 240 ± 173 nm (Fig. S3). This result is comparable with that of TEM images. The small difference between the two characterization techniques is likely due to the shrinkage of PEGMA-PhE FPNs for TEM characterization. All the above results clearly confirmed the successful for preparation of

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amphiphilic luminescent copolymers, which can self-assemble into nanoparticles with small size.

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Fig. 3 TEM image of PEGMA-PhE FPNs. Many spherical nano-granule with diameter

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between100-200 nm can be clearly found.

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Owing to the formation of AIE-active FPNs, these nanoparticles can not only overcome the ACQ effect, but also emit intensive fluorescence in aqueous solution for the AIE feature of PhE-OE.

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Therefore, the optical properties of PEGMA-PhE FPNs were further investigated by UV-Vis and fluorescence spectroscopy. As shown in Fig. 4A, two obvious peaks can be discovered at 279 and 403 nm, which can be originated from the electron transition of conjugated rings and substitute groups of

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PhE-OE. On the other hand, we could find that the absorption of PEGMA-PhE was started to rise from

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800 to 200 nm, demonstrating that the self-assembly of PEGMA-PhE into nanoparticles successful in aqueous solution. This can be explained by the Mie effect and were also found by previous reports.[47,

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52] Moreover, the PEGMA-PhE FPNs are well dispersed in water and the logo of “Nanchang University” can be clearly observed (inset image of Fig. 4A). This further indicated the successful

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fabrication of amphiphilic PEGMA-PhE copolymers. More importantly, due to the aggregation of PhE-OE in the core of PEGMA-PhE FPNs, strong and uniform red fluorescence was observed after PEGMA-PhE suspensions were irradiated with UV lamp at 365 nm (inset image of Fig. 4B). The strong and uniform fluorescence also indicated that successful conjugation of PhE-OE and PEGMA-IA-SH through the catalyst-free thiol-yne click reaction. The fluorescent properties of PEGMA-PhE were investigated by fluorescent spectroscopy in detailed. As shown in Fig. 4B, the maximum emission wavelength of PEGMA-PhE was located at 602 nm, while the maximum excitation wavelength was located at 454 nm when the maximum emission wavelength was set at 602 nm. It’s worth mentioning that the excitation band wavelength of FPNs in water is rather broad ranging from

ACCEPTED MANUSCRIPT 300 to 500 nm, which is beneficial for bioimaging and biomedical applications. The fluorescence stability of fluorescent probes is very important for their bioimaging application. Therefore, the photostability of PEGMA-PhE FPNs was further determined by fluorescence spectra in aqueous solution. As displayed in Fig. S4, the fluorescent intensity value of PEGMA-PhE is 631.1 a.u.. After UV lamp irradiation for 1 h at 365 nm, the fluorescent intensity decreased to 601.3 a.u.. This

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demonstrated that PEGMA-PhE FPNs possess high photostability. Moreover, many factors that effected on the fluorescent properties of PEGMA-PhE FPNs were further evaluated. For example, it

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can be seen that when the concentrations of PEGMA-PhE FPNs were increased from 0.1 mg mL–1 to 0.5 mg mL–1. The locations of emission peaks have not obviously changed while the fluorescent

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intensities were gradually increased correspondingly (Fig. S5). The pH could also influence the fluorescent properties of PEGMA-PhE FPNs. It can be seen that the emission peaks of PEGMA-PhE

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FPNs were only a few shift in pH range from 1-14. However, the fluorescent intensities showed irregular changes under different pH values (Fig. S6). Furthermore, the effects of viscosity and solvents

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on the fluorescent properties of PEGMA-PhE FPNs were displayed in Fig. S7 and Fig. S8, respectively. It can be seen that the fluorescent intensities were increased with the increase of viscosity, while the

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fluorescent emission peaks were blue-shift (Fig. S7). This also indicated the AIE feature of

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PEGMA-PhE FPNs because the rotation of PhE was restricted in solvents with high viscosity. Similar to the effect of viscosity, the solvents could also effect on the fluorescent properties of PEGMA-PhE FPNs. It can be seen that the emission peak of PEGMA-PhE FPNs was blue-shift in dichloromethane

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with largest fluorescent intensity (Fig. 8).

Fig. 4 (A) UV-Vis spectrum of PEGMA-PhE FPNs; (B) fluorescence excitation (Ex) and emission (Em) spectra of PEGMA-PhE FPNs.

3.2. Biocompatibility of PEGMA-PhE FPNs

ACCEPTED MANUSCRIPT The excellent biocompatibility of fluorescent nanomaterials is essential for wide application in biomedical fields. Herein, the cell viability of PEGMA-PhE FPNs was adopted to evaluate their potential for biomedical applications prospects using A549 cells. As shown in Fig. 5, it can be seen that the cell viability values did not decline noticeably after cells were incubated with different concentrations of PEGMA-PhE FPNs for 12 and 24 h. Moreover, it is noteworthy that the cell viability values are obviously greater than 90% even the concentrations of PEGMA-PhE FPNs were up to 100

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μg mL-1. The cell viability results suggested that PEGMA-PhE FPNs possess desirable biocompatibility

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and are promising for biomedical applications.

Fig. 5 Cell viability values of PEGMA-PhE FPNs determined by CCK-8 assay. The A549 cells were

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incubated with 10-100 μg mL–1 of PEGMA-PhE FPNs for 12 and 24 h.

3.3 Cell imaging applications of PEGMA-PhE FPNs

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Based on the above advantages, the cell uptake behavior of PEGMA-FPNs was further determined by CLSM. As shown in Fig. 6B, the A549 cells still adhered to the cell plates very well and kept their shuttle morphology. It is well consistent with the cell viability results. One the other hand, the strong fluorescent signals can be observed when cells were excited with 458 nm laser (Fig. 6A). Meanwhile, many areas with relatively weak fluorescent intensity were found in the center of cells. These areas should be the locations of cell nuclei. The merged image in Fig. 6C shows that the locations with fluorescent signals are just covered on the cell locations, while their center is absent of fluorescent signals. This further confirmed that PEG-PhE FPNs can be internalized by A549 cells and mainly distributed in the cytoplasm. The effective cell uptake of PEG-PhE FPNs suggested that their potential

ACCEPTED MANUSCRIPT for biological imaging. Considered the high efficient of the click reaction and remarkable feature of these AIE-active FPNs, this work should be of great interest for fabrication of many multifunctional

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polymer composites for various biomedical applications.[53-78]

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Fig. 6 Cell imaging of PEGMA-PhE FPNs using CSLM, the concentration of PEGMA-PhE FPNs is 40

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µg mL−1. (A) Excited at 405 nm; (B) Bright field; (C) Combined images of A and B. Scale bar = 20 µm.

ACCEPTED MANUSCRIPT 4. Conclusions In summary, a high efficient method for the preparation of AIE-active FPNs with strong red fluorescence and desirable biocompatibility was developed via a novel catalyst-free thiol-yne click reaction for the first time. The reaction conditions are rather simple, rapid and efficient in the absent of catalysts. On the other hand, the final PEGMA-PhE copolymers show amphiphilic properties and can

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self-assemble into fluorescent nanoparticles with intensive fluorescence and high water dispersity. More importantly, the biological evaluation results suggested that PEGMA-PhE FPNs show almost

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non-toxicity and can be effectively internalized by cells. Combined with the above advantages, the

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PEGMA-PhE FPNs are of great potential for biomedical applications.

Acknowledgements

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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

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China (Nos. 20161BAB203072, 20161BAB213066). References

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Graphical abstract

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The catalyst-free thiol-yne click reaction was developed for preparation of fluorescent polymeric

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nanoparticles with aggregation-induced emission feature and applied for cell imaging

ACCEPTED MANUSCRIPT Highlights ►Aggregation-induced emission fluorescent polymeric nanoparticles ►One-pot catalyst-free thiol-yne click reaction ►Self-assembly of amphiphilic copolymers into AIE-active FPNs

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►PEGMA-PhE FPNs possess desirable properties for biomedical applications