Stable biocompatible cross-linked fluorescent polymeric nanoparticles based on AIE dye and itaconic anhydride

Colloids and Surfaces B: Biointerfaces 121 (2014) 347–353

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Stable biocompatible cross-linked fluorescent polymeric nanoparticles based on AIE dye and itaconic anhydride Haiyin Li a , Xiqi Zhang b,∗ , Xiaoyong Zhang b , Bin Yang b , Yen Wei b a b

College of Chemistry and Pharmaceutical Sciences, Qingdao Agriculture University, Qingdao 266109, PR China Department of Chemistry and the Tsinghua Center for Frontier Polymer Research, Tsinghua University, Beijing 100084, PR China

a r t i c l e

i n f o

Article history: Received 19 March 2014 Received in revised form 8 May 2014 Accepted 5 June 2014 Available online 12 June 2014 Keywords: Itaconic anhydride Aggregation-induced emission Critical micelle concentration Cross-linked polymeric fluorescent nanoparticles Cell imaging

a b s t r a c t Self-assembly of polymeric materials to form nanoparticles is a particularly promising strategy for various biomedical applications, however, these self-assembling systems often encounter the critical micelle concentration (CMC) issue, as the nanoparticles is usually unstable at low concentration. Therefore, stable cross-linked fluorescent polymeric nanoparticles (FPNs) were covalently constructed from an aggregation induced emission (AIE) dye, itaconic anhydride, poly(ethylene glycol) monomethyl ether methacylate and polyethylenimine. These obtained PhE-ITA-20%(80%) FPNs were fully characterized by a series of techniques including 1 H NMR spectra, UV–vis absorption spectra, fluorescence spectra, FT-IR spectra, transmission electron microscopy, gel permeation chromatography, and dynamic light scattering. Such FPNs emitted intense fluorescence due to the introduction of aggregation induced emission dye. More importantly, the FPNs were found extremely stable in physiological solution even below the CMC owing to their cross-linked architectures. Biocompatibility evaluation and cell uptake behavior of the FPNs were further investigated to explore their potential biomedical applications, the demonstrated excellent biocompatibility made them promising for cell imaging. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Cellular fluorescence imaging has attracted much attention as a versatile visualization way for medical diagnosis, drug development, and clinical study [1,2]. Various fluorescent bioprobes have been reported and extensively studied for bioimaging applications over the past decades, among them are green fluorescent protein, semiconductor quantum dots, carbon dots, and organic dye based fluorescent nanoparticles [3–6]. However, green fluorescent protein often suffers from small stokes shifts, poor photostability, and tedious transfection process [7]. While semiconductor quantum dots are usually easy to aggregate in cellular environments leading to high cytotoxicity owing to the containing heavy metal ions [8]. Carbon dots show weak luminescence and nonfunctionalized hydrophobic feature, which limit their biomedical application [9,10]. For most organic dyes, hydrophobic planar structures will induce strong intermolecular ␲–␲ interactions, resulting in fluorescence quenching and photobleaching when aggregated in aqueous solution [11,12]. To conquer the aggregating

∗ Corresponding author. Tel.: +86 10 6279 2604. E-mail addresses: [email protected] (X. Zhang), [email protected] (Y. Wei). http://dx.doi.org/10.1016/j.colsurfb.2014.06.015 0927-7765/© 2014 Elsevier B.V. All rights reserved.

fluorescence quenching problem, another type of unique organic dyes emerges, firstly named aggregation-induced emission (AIE) dyes by Tang et al. [13]. The subsequent development of AIE dyes have given birth to a wide spectrum of molecular architectures, such as siloles [14], cyano-substituted diarylethylene [15], distyrylanthracene [16], tetraphenylethene [17], triphenylethene [18], and so on. Meanwhile, progressively investigation of these AIE materials for potential chemosensors and bioimaging applications has been proceeding [19]. Recently, various strategies for fabricating AIE dye based fluorescent nanoparticles have been developed. Prasad et al. [20] reported organically modified silica nanoparticles encapsulating a varying amount of AIE dye (BDSA), which provided a promising pathway to achieve a significant breakthrough in developing two-photon fluorescent probes for cell imaging. Jen et al. [21] presented a simple and novel method of utilizing AIE molecules as fluorescent probes for bioimaging via employing amphiphilic block copolymers to form polymeric micelles and function as nanocarriers to disperse hydrophobic AIE dyes. Tang and Liu et al. [22] utilized bovine serum albumin as the polymer matrix to encapsulate an AIE red fluorogen, which showed high brightness and low cytotoxicity for in vitro and in vivo far-red/near-infrared bioimaging. Wei’s group developed surfactant modification of AIE dye with commercialized non-ionic surfactant Pluronic F127 to afford biocompatible

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Scheme 1. Self-assembly of PhE-ITA-20%(80%) to form FPNs and their cell imaging applications.

fluorescent nanoparticles with good water solubility and excellent biocompatibility for cell imaging [23,24]. Although the above encapsulating strategies show good biological imaging capability, these non-covalent tactics will lead to dye leakage from the matrix, resulting in cytotoxicity when applied in bioimaging [25]. For biological applications, robust water-soluble AIE based nanoparticles are required, and the covalent strategy is a promising one. Therefore, AIE molecules grafted amphiphilic biopolymer chitosan was introduced by Tang et al. [26] to construct covalent AIE dye based amphiphilic polymer for cell imaging. Moreover, Wei’ group has reported several covalent strategies to fabricate AIE dye based polymer, including Schiff base condensation [27], emulsion polymerization [28], reversible addition fragmentation chain transfer (RAFT) polymerization [29], and anhydride ring-opening polymerization [30]. Despite many impressive advances in fabricating AIE based macromolecules, more versatile and robust strategies are still highly demanded. As many previously reported AIE based fluorescent polymers are linear polymers, which are not stable on dilute solution below the CMC, and will limit their real biomedical application [31]. In this case, cross-linked polymeric nanoparticles have been expected more stable than those non-crosslinked ones. However, the reported example of cross-linked polymeric nanoparticles for cell imaging is still rare, and the related construction methodology is also limited [32–34]. Thus, development of robust synthetic routes to prepare novel stable cross-linked fluorescent polymeric nanoparticles (FPNs) is of great scientific interest. Biobased materials have attracted much attention due to the ever-growing environmental problems, such as, consuming fossil fuel resources increases the net amount of carbon dioxide in the atmosphere and affects the global warming eventually [35]. Thus, it is considered very important to do research on polymer synthesis using renewable resources as starting materials. Itaconic acid is produced in a large scale by fermentation process and itaconic anhydride (ITA) is also regarded as one of key platform chemicals derived from biomass, which belongs to a renewable resource [36]. In this work, stable cross-linked copolymers PhE-ITA-20%(80%) were covalently constructed from renewable monomer ITA, an AIE dye (PhE), poly(ethylene glycol) monomethyl ether methacylate (PEGMMA), and polyethylenimine. The obtained amphiphilic cross-linked copolymers were prone to self-assembly into stable FPNs and could be highly dispersed in physiological solution (Scheme 1). Then, a series of techniques including 1 H NMR spectra, UV–vis absorption spectra, fluorescence spectra, FTIR spectra, transmission electron microscopy, gel permeation

chromatography, and dynamic light scattering were conducted to thoroughly characterize these FPNs. Meanwhile, the biocompatibility and cell uptake behavior of PhE-ITA-20%(80%) FPNs were studied to evaluate their potential applications for cell imaging.

2. Experimental procedure 2.1. Materials and characterization Phosphoryl chloride, N,N-dimethylformamide, 2-(4-bromophenyl)acetonitrile, tetrabutyl ammonium bromide, tetrakis(triphenylphosphine) palladium(0), Aliquat 336, 1,2-dichloroethane, 4-vinylphenylboronic acid, terabutyl ammonium hydroxide, itaconic anhydride, poly(ethylene glycol) monomethyl ether methacylate (Mn = 950), polyethylenimine (Mn = 600) were purchased from J&K Scientific Ltd. and used as received. All other agents and solvents were purchased from commercial sources and used directly without further purification. Ultra-pure water was used in the experiments. 1 H NMR spectra were measured on a Mercury-Plus 300 MHz spectrometer [d6 -DMSO as solvent and tetramethylsilane (TMS) as the internal standard]. The FT-IR spectra were obtained in a transmission mode on a Shimadzu Spectrum 8400 spectrometer (Japan). Typically, 8 scans at a resolution of 1 cm−1 were accumulated to obtain one spectrum. UV–vis absorption spectra were recorded on UV/Vis/NIR 2600 spectrometer (Shimadzu, Japan) using quartz cuvettes of 1 cm path length. Fluorescence spectra were measured on an F-4600 spectrometer with a slit width of 3 nm for both excitation and emission. Transmission electron microscopy (TEM) images were recorded on a HT7700 microscope (Hitachi, Japan) operated at 100 kV, the TEM specimens were made by placing a drop of the nanoparticles suspension on a carbon-coated copper grid. The size distributions and zeta potential measurements of PhE-ITA-20%(80%) FPNs in phosphate buffer solution (PBS), in cell culture medium, and in serum were determined using a zeta Plus apparatus (ZetaPlus, Brookhaven Instruments, Holtsville, NY). Gel permeation chromatography (GPC) analyses of polymers were performed using DMF as the eluent. The GPC system was a Shimadzu LC-20AD pump system comprising of an auto injector, a MZ-Gel ˚ followed by a SDplus 10.0 mm guard column (50 × 8.0 mm, 102 A) ˚ linear) and MZ-Gel SDplus 5.0 ␮m bead-size columns (50–106 A, a Shimadzu RID-10A refractive index detector. The system was calibrated with narrow molecular weight distribution polystyrene standards ranging from 200 to 106 g mol−1 .

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Scheme 2. Synthetic route of PhE-ITA-20%(80%).

2.2. Preparation of PhE-ITA-20%(80%) The AIE monomer PhE was prepared according to the literature methods [37]. The synthetic route of PhE-ITA-20%(80%) is shown in Scheme 2. For synthesis of PhE-ITA-20% FPNs, PhE (34 mg, 0.050 mmol), ITA (22 mg, 0.20 mmol), PEGMMA (760 mg, 0.80 mmol), AIBN (5.0 mg), and ethyl acetate (6 mL) were introduced in schlenk tube and purged by nitrogen flow for 30 min. The above mixture was put into an oil bath maintained at 80 ◦ C for 12 h. Then polyethylenimine (30 mg, 0.050 mmol) was added into the above mixture and stirred for another 2 h at room temperature. Afterwards, the crosslinking reaction was stopped, and dialyzed against tap water for 24 h and ethanol for 6 h using 7000 Da Mw cutoff dialysis membranes. Finally, the solution in dialysis bag was carried out by freeze-drying to obtain PhE-ITA-20% FPNs. The synthesis of PhEITA-80% FPNs was similar to that of PhE-ITA-20% FPNs, while the amounts of ITA, PEGMMA, and polyethylenimine were adjusted to 90 mg (0.80 mmol), 190 mg (0.20 mmol), and 120 mg (0.20 mmol), respectively. 2.3. Cytotoxicity of PhE-ITA-20%(80%) FPNs Cell morphology was observed to examine the effects of PhEITA-20%(80%) FPNs to A549 cells (origin from American Type Culture Collection). Briefly, cells were seeded in 6-well microplates at a density of 1 × 105 cells mL−1 in 2 mL of respective media containing 10% fetal bovine serum (FBS). After cell attachment, plates were washed with PBS and cells were treated with complete cell culture medium, or different concentrations of PhE-ITA-20%(80%) FPNs prepared in 10% FBS containing media for 24 h. Then all

samples were washed with PBS three times to remove the uninternalized nanoparticles. The morphology of cells was observed by using an optical microscopy (Leica, Germany), the overall magnification was ×100. The cell viability of PhE-ITA-20%(80%) FPNs on A549 cells was evaluated by cell counting kit-8 (CCK-8) assay. Briefly, cells were seeded in 96-well microplates at a density of 5 × 104 cells mL−1 in 160 ␮L of respective media containing 10% FBS. After 24 h of cell attachment, the cells were incubated with 10, 20, 40, 80, 120 ␮g mL−1 PhE-ITA-20%(80%) FPNs for 8 and 24 h. Then nanoparticles were removed and cells were washed with PBS three times. 10 ␮L of CCK-8 dye and 100 ␮L of DMEM cell culture medium were added to each well and incubated for 2 h at 37 ◦ C. Plates were then analyzed with 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 values were proportional to the number of live cells. The percent reduction of CCK-8 dye was compared to controls (cells not exposure to PhEITA-20%(80%) FPNs), which represented 100% CCK-8 reduction. Three replicate wells were used per microplate, and the experiment was repeated three times. Cell survival was expressed as absorbance relative to that of untreated controls. Results are presented as mean ± standard deviation (SD). 2.4. Confocal microscopic imaging of cells incubated with PhE-ITA-20%(80%) FPNs The cell uptakes of PhE-ITA-20%(80%) FPNs were evaluated by confocal microscopic imaging. Briefly, cells were seeded in a glass bottom dish with a density of 1 × 105 cells per dish. On the day of treatment, the cells were incubated with PhE-ITA-20%(80%) FPNs

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Fig. 2. FT-IR spectra of PhE, ITA, PEI, PhE-ITA-20%, and PhE-ITA-80%. Fig. 1.

1

H NMR spectra of PhE, PhE-ITA-20%, and PhE-ITA-80%.

at a final concentration of 10 ␮g mL−1 for 3 h at 37 ◦ C. Afterward, the cells were washed three times with PBS to remove the PhEITA-20%(80%) FPNs and then fixed with 4% paraformaldehyde for 10 min at room temperature. Cell images were taken with a Confocal Laser Scanning Microscope (CLSM) Zesis 710 3-channels (Zesis, Germany) with the excitation wavelength of 543 nm.

bending vibration band of N H. Furthermore, the stretching vibration bands of C O and C N were also observed in PhE-ITA20%(80%), which located at 1095 and 1031 cm−1 , respectively. All these results confirmed the successful formation of PhE-ITA20%(80%). Due to the amphiphilic properties of PhE-ITA-20%(80%), when they were dispersed in aqueous solution, the polymers were tended to self-assemble into nanoparticles with surfaces covered with

3. Results and discussion 3.1. Characterization of PhE-ITA-20%(80%) FPNs To prepare PhE-ITA-20%, the hydrophobic AIE monomer PhE and ITA were copolymerized with hydrophilic monomer PEGMMA via radical polymerization to obtain a linear amphiphilic polymer (PhE-ITA) (Scheme 2). The feed molar ratio of three monomers was 1:4:16 for PhE, ITA, and PEGMMA, respectively. Then PhE-ITA was facilely cross-linked by polyethylenimine via room temperature ring-opening polymerization under air atmosphere to afford the resulting cross-linked amphiphilic copolymers (PhE-ITA-20%), while the molar amount of polyethylenimine was equal to that of PhE. As for PhE-ITA-80%, the feed molar ratio of PhE, ITA, PEGMMA, and polyethylenimine was 1:16:4:4, respectively. The GPC result of PhE-ITA-20% showed that the number average molecular weight (Mn ) value was 52200, while the Mn of PhE-ITA-80% was determined as 24,500. The 1 H NMR spectra of PhE-ITA-20%(80%) in d6 -DMSO were conducted and shown in Fig. 1. The chemical shifts of aromatic hydrogens were observed changing from sharp peaks in PhE to attenuated or broader peaks in PhE-ITA-20%(80%), indicating the successful introduction of PhE into the copolymers. Successful syntheses of PhE-ITA-20%(80%) were further confirmed by FT-IR spectra. As shown in Fig. 2, two characteristic peaks located at 2913 and 2850 cm−1 were observed in the sample of PhE, which evidenced the stretching vibration of CH2 group. While two characteristic peaks located at 1844 and 1770 cm−1 could be clearly observed in ITA, which were ascribed to the stretching vibration of C O band. And the broad peak at 3286 cm−1 was the stretching vibration of N H band for polyethylenimine. After the radical copolymerization of PhE, ITA, PEGMMA, and crosslinking with polyethylenimine, the resulting cross-linked copolymers PhE-ITA-20%(80%) were formed. The stretching vibrations of COOH and N H bands were found in the copolymers laying at 3540 and 3292 cm−1 ; The C H band was obviously found at 2865 cm−1 ; The peak located at 1695 cm−1 could be ascribed to the C O stretching vibration band, while another peak located at 1650 cm−1 could be assigned to the

Fig. 3. (A) The UV absorption spectra of PhE-ITA-20%(80%) FPNs dispersed in water. Inset: visible images of PhE-ITA-20%(80%) FPNs in water. (B) The fluorescent spectra of PhE-ITA-20%(80%) FPNs dispersed in water. Inset: fluorescent images of PhEITA-20%(80%) FPNs dispersed in water taken at 365 nm UV light.

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Fig. 4. TEM images of PhE-ITA-20% FPNs (A) and PhE-ITA-80% FPNs (B) dispersed in water, scale bar = 200 nm. Intensity of the aggregate emission vs the logarithm of the concentration of PhE-ITA-20% FPNs (C) and PhE-ITA-80% FPNs (D) (ex = 543 nm, em = 580 nm).

hydrophilic carboxyl, amino, and poly(ethylene glycol) groups, while the conjugated aromatic groups were aggregated into the hydrophobic cores. Therefore, the resulting FPNs are expected with high dispersibility in aqueous environment. At the same time, along with the aggregation of AIE components into the cores, these obtained FPNs are envisaged to emit intense fluorescence. Herein, the UV absorption spectra and fluorescence spectra of PhE-ITA20%(80%) FPNs were studied to determine the water dispersibility and fluorescent properties of these FPNs. The UV spectra were shown in Fig. 3A, it could be found that there were two absorption peaks located at 330 and 430 nm. The spectra were also found starting to increase from the very beginning, indicating the existence of nanoparticles in the solution, which is ascribed to the Mie effect [38]. The insets of Fig. 3A gave us direct evidence of the high water dispersibility as the FPNs were readily dispersed in water with high transparency. The PhE-ITA-20%(80%) FPNs showed strong yellow fluorescence in water (inset of Fig. 3B), which was owing to the aggregation of the AIE components. The fluorescence spectra of PhE-ITA-20%(80%) FPNs in water were shown in Fig. 3B. The maximum emission wavelength was located at 580 nm, while the fluorescence excitation wavelength was around 438 nm. The intense fluorescence is greatly benefited for the potential cell imaging applications. Self-assembly of polymeric materials to form nanoparticles is a particularly promising strategy for various biomedical applications. Thus, the transmission electron microscopy (TEM) images were utilized to confirm the formation of the PhE-ITA-20%(80%) FPNs (Fig. 4A and B). Many spherical nanoparticles with diameters ranged from 50 to 100 nm could be clearly identified, which gave us direct evidence that the resulting amphiphilic cross-linked copolymers were self-assembled into nanoparticles in aqueous solution.

Meanwhile, the size distribution of PhE-ITA-20%(80%) FPNs in phosphate buffer solution (PBS) was determined using a zeta Plus particle size analyzer, showing that the size distribution was 231 ± 1 nm, with a polydispersity index (PDI) of 0.125 for PhE-ITA20% FPNs, and 190 ± 2 nm of size distribution and 0.235 of PDI for PhE-ITA-80% FPNs. Moreover, the zeta potential measurement of these FPNs in PBS were also studied and showed the zeta-potential value of −15.4 ± 2.2 mV for PhE-ITA-20% FPNs and −23.4 ± 1.9 mV for PhE-ITA-80% FPNs, respectively. The zeta potential results further confirmed the stable dispersibility of these FPNs in physiological solution. The size distributions of PhE-ITA-20%(80%) FPNs in cell culture medium and in serum have been determined. The size distributions of PhE-ITA-20% FPNs in cell culture medium and in serum were 315 ± 14 nm and 277 ± 26 nm, respectively. While the values of PhE-ITA-80% FPNs in cell culture medium and in serum were 237 ± 1 nm and 217 ± 2 nm, respectively. As compared with the above TEM images, the size characterized by TEM was somewhat smaller, which might be due to the drying-causing shrinkage of the amphiphilic copolymers. Moreover, the leakage study of the dye from PhE-ITA-20%(80%) FPNs was conducted with dialysis against PBS solution or THF for three days using 7000 Da Mw cutoff dialysis membranes, the results showed that no dye leakage occurred, which was due to the covalent connection of the AIE dye with the polymer. Self-assembly of polymeric materials to form nanoparticles often encounters the CMC issue, as the nanoparticles is usually unstable at low concentration [31]. Therefore, the cross-linking strategy has been employed into this work to solve the problem of CMC. Thus, the intensity of the aggregate emission vs the logarithm of the concentration of PhE-ITA-20%(80%) was carried out to evaluate the CMC, the values of CMC were 0.093 and 0.050 mg mL−1

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Fig. 5. Biocompatibility evaluation of PhE-ITA-80% FPNs. (A-C) optical microscopy images of A549 cells incubated with different concentrations of PhE-ITA-80% FPNs for 24 h, (A) control cells, (B) 40 ␮g mL−1 , (C) 120 ␮g mL−1 , (D) cell viability of PhE-ITA-80% FPNs with A549 cells for 8 and 24 h, respectively.

for PhE-ITA-20% and PhE-ITA-80% FPNs, respectively (Fig. 4C and D). What surprised us here was that when we determined the size distribution of PhE-ITA-20%(80%) FPNs below CMC, the FPNs still showed high stability in the solution, even when the concentration of the FPNs was as low as 0.1 ␮g mL−1 . Therefore, this cross-linking method is considered to play a significant role of solving the problem of CMC.

A549 cells was determined by cell counting kit-8 (CCK-8) assay. As shown in Fig. 5D, no cell viability decrease was found when the cells were incubated with 10–120 ␮g mL−1 of PhE-ITA-80% FPNs for 8 h and 24 h, even when the concentration of these FPNs was up to 120 ␮g mL−1 , the cell viability value was still greater than 90%. The biocompatibility of PhE-ITA-20% FPNs also showed a good result (Fig. S1). All the above results suggested that the fabricated FPNs were highly potential for cell imaging.

3.2. Biocompatibility of PhE-ITA-20%(80%) FPNs 3.3. Cell imaging applications of PhE-ITA-20%(80%) FPNs The biocompatibility was carried out to evaluate the potential biomedical applications of PhE-ITA-20%(80%) FPNs. The influences of PhE-ITA-80% FPNs to A549 cells were examined by optical microscopy after the cells were incubated with different concentrations of PhE-ITA-80% FPNs for 24 h (Fig. 5A–C). The result showed that the cells grew well when incubated with 40 and 120 ␮g mL−1 of PhE-ITA-80% FPNs, suggesting that the FPNs were biocompatible with cells. To quantitatively evaluate the cytocompatibility of PhE-ITA-80% FPNs, cell viability of the FPNs to

Based on the above presented results of biocompatibility, we started our investigations of further cell imaging applications of PhE-ITA-20%(80%) FPNs. The cell uptake behavior of PhE-ITA-80% FPNs was evaluated by Confocal Laser Scanning Microscope (CLSM) observation, which was shown in Fig. 6. Strong orange fluorescence could be observed within the cells after they were only incubated with 10 ␮g mL−1 of these FPNs, further, many areas with relative weak fluorescence intensity were found in the cells, which might

Fig. 6. CLSM images of A549 cells incubated with 10 ␮g mL−1 of PhE-ITA-80% FPNs for 3 h. (A) bright field, (B) fluorescent image which were excited with 405 nm laser, (C) merged image. Scale bar = 20 ␮m.

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be the possible location of cell nucleus (Fig. 6B), suggesting the PhE-ITA-80% FPNs could be facilely uptaken by cells with most of them located at cytoplasm, and these FPNs are considered uptaken by endocytosis of the cells. The cell imaging results of PhE-ITA20% FPNs were shown in Fig. S2, also indicating excellent staining performance. Although these anionic particles determined by zeta potential measurement may have repulsion with cell membrane, it has been previously demonstrated that these negative charges do not have negative effect for cell uptake [39]. Moreover, the hydrophobic groups of polymers and the PEG component may have some positive effect for cell uptake. Therefore, we could expect the PhE-ITA-20%(80%) FPNs should be promising candidates for cell imaging with combined advantages such as stable morphology in dilute physiological solution, intense fluorescence, good water dispersibility, and excellent biocompatibility. Moreover, in this work, itaconic anhydride was used as an alternative monomer for the construction of fluorescent polymer; its highly reactive anhydride group was greatly beneficial to connect with various functional components for diverse biomedical applications. In short, this work also provided a new construction methodology to afford stable fluorescent polymeric nanoparticles for cell imaging. 4. Conclusions

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Stable cross-linked copolymers (PhE-ITA-20%(80%)) were covalently constructed from renewable monomer (ITA), an AIE monomer (PhE), PEGMMA, and polyethylenimine. Such obtained amphiphilic cross-linked copolymers were prone to self-assemble into uniform FPNs with diameters ranging from 50 to 100 nm. These FPNs showed high water dispersibility and intense fluorescence in aqueous solution owing to the hydrophilic carboxyl, amino, and poly(ethylene glycol) groups at the surfaces and the AIE components in the cores. More importantly, the FPNs were found extremely stable in physiological solution even below the CMC, which derived from their cross-linked architectures. Biocompatibility evaluation and cell imaging results suggested that these FPNs were biocompatible for bioimaging applications.

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Acknowledgements This research was supported by the National Science Foundation of China (Nos. 21134004, 21201108, 51363016), and the National 973 Project (No. 2011CB935700), China Postdoctoral Science Foundation (2012M520243, 2013T60100), High-level Science Foundation of Qingdao Agriculture University (6631334). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2014.06.015.

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