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Facile fabrication of AIE-based stable cross-linked fluorescent organic nanoparticles for cell imaging
Colloids and Surfaces B: Biointerfaces 116 (2014) 739–744
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Facile fabrication of AIE-based stable cross-linked fluorescent organic nanoparticles for cell imaging Xiqi Zhang b , Xiaoyong Zhang a,b,∗ , Bin Yang b , Junfeng Hui b , Meiying Liu c , Yen Wei b,∗∗ a
Department of Chemistry, Institute of Polymers, Nanchang University, 999 Xuefu Avenue, Nanchang 330031, China Department of Chemistry and Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Tsinghua University, Beijing 100084, China c Beijing National Laboratory for Molecular Sciences (BNLMS), Key Laboratory of Organic Solids, Laboratory of New Materials, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China b
a r t i c l e
i n f o
Article history: Received 25 September 2013 Received in revised form 28 November 2013 Accepted 5 December 2013 Available online 25 December 2013 Keywords: Aggregation-induced emission Polyethylene polyamine Anhydride ring-opening polymerization Cross-linked fluorescent organic nanoparticles Cell imaging
a b s t r a c t An aggregation induced emission dye (RNH2 ) with two amino end-groups was facilely incorporated into stable cross-linked fluorescent organic nanoparticles via room temperature anhydride ring-opening polymerization and consecutive cross-linking with polyethylene polyamine. Thus obtained RO-OA-PEPA FONs were characterized by a series of techniques including 1 H nuclear magnetic resonance, Fourier transform infrared spectroscopy, UV–vis absorption spectrum, fluorescent spectroscopy and transmission electron microscopy. Biocompatibility evaluation and cell uptake behavior of RO-OA-PEPA FONs were further investigated to explore their potential biomedical application. We demonstrated that such FONs showed high-water dispersibility, strong red fluorescence, stable uniform morphology (100–200 nm) and excellent biocompatibility, making them promising for cell imaging application. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Fluorescent organic nanoparticles (FONs) receive increasing interest for their high potential in sensing, imaging and biomedical applications [1–3]. Compared with the conventional fluorescent inorganic nanoparticles such as semiconductor quantum dots, carbon dots and fluorescent silicon nanoparticles, FONs offer an attractive alternative method with many advantages including non-toxicity, biodegradability, biocompatibility and appropriate functionalities [4–10]. However, most of the organic fluorophores are not soluble in physiological solution due to their inherent hydrophobic features, thus should be modified with hydrophilic groups but generally at the expense of the fluorescence intensity because when organic dyes are concentrated in a confined space like nanoparticle, luminescence quenching may simultaneously emerge owing to the notorious aggregation-caused
∗ Corresponding author at: Department of Chemistry, Institute of Polymers, Nanchang University, 999 Xuefu Avenue, Nanchang 330031, China. Tel.: +86 01062792604. ∗∗ Corresponding author. E-mail addresses: [email protected] (X. Zhang), [email protected] (Y. Wei). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.12.010
fluorescent quenching (ACQ) effect [11,12]. Therefore, to overcome this problem, unique aggregation induced emission (AIE) materials appeared with anti-ACQ effect, which emit stronger fluorescence in the aggregation state [13,14]. Hereafter, various AIE fluorogens such as siloles [15–18], tetraphenylethene [19–21], triphenylethylene [22,23], cyano-substituted diarylethene [24–26] and distyrylanthracene [27–32] derivatives have been fabricated and widely investigated for potential biomedical applications due to their attractive AIE feature, biodegradability and biocompatibility. Self-assembly of fluorescent dyes to form nanoparticles such as spherical micelles is a particularly promising strategy for various technological applications such as bioimaging and drug delivery [33–35]. Recently, some strategies including bare AIE nano-assembly, non-covalent strategy and covalent strategy for fabricating AIE based FONs have been developed with intense luminescence, uniform size, high water dispersibility and excellent biocompatibility, making them promising for cell imaging applications [36–44]. However, one of the main problems of bare AIE nano-assembly and non-covalent strategy is that the fluorophores can leak out of the particles with time [11]. Although the covalent strategy can form stable covalently linking between the fluorophore and polymer backbone, another trouble appears that such self-assembled structures are often unstable on dilute solution
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below the critical micelle concentration (CMC) [45,46]. Therefore, although many obvious advances have been achieved in the fabrication of AIE based FONs, more robust fabrication strategies are still highly desirable, including (a) stable dispersion in physiological solution below the CMC and (b) introducing reactive functional groups into the FONs to integrate drugs and targeting agents for multifunctional biomedical applications. Thus development of versatile synthetic strategies for novel stable cross-linked FONs is of significant scientific interest. Herein, a novel facile method of preparing AIE based stable cross-linked FONs was developed via room temperature anhydride ring-opening polymerization (ROP) based on an AIE dye (RNH2 ) with two amino end-groups and 4,4 -oxydiphthalic anhydride (OA) under an air atmosphere, and subsequent cross-linking with polyethylene polyamine (PEPA) (Scheme 1). Such obtained amphiphilic cross-linked copolymers (RO-OA-PEPA) are tended to self-assemble into stable FONs and show high dispersibility in aqueous solution. These obtained FONs were characterized by a series of techniques including nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FT-IR), UV–vis absorption spectrum (UV), fluorescent spectroscopy and transmission electron microscopy (TEM). Owing to the AIE feature of RNH2 , the resulting cross-linked FONs emitted strong red fluorescence in aqueous environment [47]. Finally, the biocompatibility and cell uptake behavior of RO-OA-PEPA FONs were further conducted to evaluate their potential for cell imaging applications. 2. Experimental procedure 2.1. Materials and characterization Phenothiazine, 3-bromoprop-1-ene, N,N-dimethylformamide (DMF), 1,2-dichloroethane, phosphoryl chloride, 4-aminobenzyl cyanide, tetrabutylammonium hydroxide (0.8 M in methanol), N,N-dimethylacetamide (DMAc), 4,4 -Oxydiphthalic anhydride purchased from Alfa Aesar were used as received. Polyethylene polyamine (C2n H5n Nn , MP 250 ◦ C, Density (20 ◦ C) 1.04 g/mL) was purchased from Aladdin Industrial Inc. All other agents and solvents were purchased from commercial sources and used directly without further purification. 1 H NMR spectrum was measured on a JEOL 400 MHz spectrometer [d6 -DMSO as solvent and tetramethylsilane (TMS) as the internal standard]. UV–visible absorption spectrum was recorded on UV/vis/NIR Perkin–Elmer lambda 750 spectrometer (Waltham, MA, USA) using quartz cuvettes of 1 cm path length. Fluorescence spectra were measured on a PE LS-55 spectrometer with a slit width of 3 nm for both excitation and emission. The FT-IR spectra were obtained in a transmission mode on a Perkin–Elmer Spectrum 100 spectrometer (Waltham, MA, USA). Typically, 8 scans at a resolution of 1 cm−1 were accumulated to obtain the spectrum. Transmission electron microscopy (TEM) images were recorded on a JEM-1200EX microscope 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 distribution of RO-OA-PEPA FONs in phosphate buffer solution (PBS) were determined using a zeta Plus apparatus (ZetaPlus, Brookhaven Instruments, Holtsville, NY).
atmosphere at room temperature for 2 h. Followed by adding PEPA (3.0 mg), which was dissolved in 3 mL DMAc. The above mixture was stirred at room temperature for another 0.5 h. Then the crosslinking reaction was stopped by adding 30 mL water, and then the RO-OA-PEPA water dispersion was treated by repeated centrifugal washing process for thrice. Finally, the resulting centrifuged solid was carried out by freeze-drying to obtain RO-OA-PEPA FONs. 2.3. Cytotoxicity of RO-OA-PEPA FONs Cell morphology was observed to examine the effects of ROOA-PEPA FONs to A549 cells. 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 ROOA-PEPA FONs 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 RO-OA-PEPA FONs 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 RO-OAPEPA FONs 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 (Victor III, 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 RO-OA-PEPA FONs), 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 using RO-OA-PEPA FONs A549 cells were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% heat-inactivated FBS, 2 mM glutamine, 100 U mL−1 penicillin, and 100 g mL−1 of streptomycin. Cell culture was maintained at 37 ◦ C in a humidified condition of 95% air and 5% CO2 in culture medium. Culture medium was changed every three days for maintaining the exponential growth of the cells. On the day prior to treatment, 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 RO-OA-PEPA FONs 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 RO-OAPEPA FONs 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-channel (Zesis, Germany) with the excitation wavelength of 543 nm.
2.2. Preparation of RO-OA-PEPA FONs
3. Results and discussion
The synthetic route of RO-OA-PEPA is showed in Scheme 2. RNH2 was synthesized according to our previous literature [26]. In the synthesis of RO-OA-PEPA, RNH2 (37 mg, 0.05 mmol) and 4,4 -oxydiphthalic anhydride (19 mg, 0.06 mmol) were dissolved in 10 mL DMAc. The above mixture was stirred under an air
3.1. Characterization of RO-OA-PEPA FONs 3.1.1. 1 H NMR and IR spectra To prepare RO-OA-PEPA FONs, the hydrophobic AIE dye (RNH2 ) was copolymerized with 4,4 -oxydiphthalic anhydride (OA) via
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Scheme 1. Schematic showing the preparation of RO-OA-PEPA FONs through room temperature anhydride ROP and consecutive cross-linking with PEPA based on an AIE dye (RNH2 ) and subsequent cell imaging applications of these obtained FONs.
Scheme 2. Synthetic route of RO-OA-PEPA.
room temperature ROP [48] under an air atmosphere for 2 h, based on the feed molar ratio (OA: RNH2 ) was 1.2, to obtain ROOA with the designed degree of polymerization of RO-OA was 5. Then these copolymers were facilely cross-linked with PEPA at room temperature for another 0.5 h to obtain the cross-linked amphiphilic copolymers RO-OA-PEPA. 1 H NMR spectrum of ROOA-PEPA in d6 -DMSO was conducted and shown in Fig. 1A. The appearance of the chemical shift around 8.0 ppm was observed, indicating the successful synthesis of RO-OA. Furthermore, the emergence of chemical shift ranged from 2.7 to 2.9 ppm was also found, further confirming the cross-linking of PEPA to afford ROOA-PEPA. The successful synthesis of RO-OA-PEPA copolymers was also confirmed by FT-IR. As shown in Fig. 1B, two characteristic peaks located at 2920 and 2844 cm−1 were observed in the sample of RNH2 , evidencing the stretching vibration of CH2 and CH3 groups. Meanwhile, a series of peaks distributed between 1450 and 1600 cm−1 can be ascribed to the stretching vibration of various aromatic rings. After anhydride ROP and consecutive cross-linking with PEPA, strong bending vibration of N H band of amide and amine groups located at 1594 cm−1 were observed in RO-OA-PEPA FONs [49,50]. On the other hand, a characteristic peak centered at 766 cm−1 was also observed, evidencing that the
wagging vibration of primary and secondary amines bands in the copolymers, and another characteristic peak located at 1084 cm−1 was also observed in copolymers, suggesting that C O band was introduced into the copolymers, meanwhile, the strong stretching vibrations of COOH and NH2 also appeared in the RO-OA-PEPA FONs located ranged from 3250 to 3400 cm−1 , all confirming successful formation of the cross-linked copolymers. Owing to the formation of hydrophilic carboxyl and amino groups in RO-OAPEPA, such obtained cross-linked copolymers showed amphiphilic properties. When dispersed in aqueous solution, these copolymers are tended to self-assemble into polymeric nanoparticles containing the aromatic groups as hydrophobic cores, along with the hydrophilic carboxyl and amino groups covered onto the surfaces as shells. As the AIE fluorophore RNH2 and other hydrophobic groups were aggregated into the nanoparticle cores, thus RO-OAPEPA FONs are expected to possess strong fluorescence and high dispersibility in aqueous solution. 3.1.2. Optical property The UV absorption spectrum of RO-OA-PEPA FONs dispersed in water indicated that the maximum absorption peak located at 468 nm (Fig. 2A). It is noteworthy that the entire spectrum started
Fig. 1. (A) 1 H NMR spectrum of RO-OA-PEPA dissolved in d6 -DMSO; (B) FT-IR spectra of RNH2 , OA, PEPA and RO-OA-PEPA.
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Fig. 2. (A) UV–vis spectrum of RO-OA-PEPA FONs, inset is the visible image of such FONs in water; (B) fluorescence excitation (Ex) and emission (Em) spectra of RO-OA-PEPA FONs, inset is the fluorescent image taken at 365 nm UV light.
to rise in absorption from 800 nm due to the Mie effect, suggesting the presence of nanoparticles [23]. The inset of Fig. 2A intuitively showed the high water dispersibility of RO-OA-PEPA cross-linked FONs. It was worth mentioning that no visible precipitation was found even after the above RO-OA-PEPA FONs were deposited for more than one month. The size distribution of RO-OA-PEPA FONs in phosphate buffer solution (PBS) was tested using a zeta Plus particle size analyzer, indicating that the size distribution of these FONs was 292.3 ± 2.7 nm with a polydispersity index of 0.149. Due to the AIE properties of RNH2 and self-assembly of the amphiphilic cross-linked copolymers, RO-OA-PEPA FONs showed strong red fluorescence dispersed in pure water (inset of Fig. 2B). The fluorescence excitation and emission spectra of RO-OA-PEPA FONs in water were shown in Fig. 2B. The maximum emission wavelength located at 604 nm, while the fluorescence excitation spectrum in water exhibited broad strong excitation wavelength from 350 to 560 nm. Meanwhile, fluorescence quantum yields (F) were estimated using Rhodamine 6G in ethanol as the standard (F = 95%), the absorbance of the solutions was kept around 0.05 to avoid internal filter effect. We demonstrated that the F of RO-OA-PEPA FONs in pure water is about 3.02%. These excellent fluorescent features including intense red emission, broad strong excitation and long wavelength excitation are greatly benefited for their potential cell imaging applications. 3.1.3. TEM The TEM images further confirmed the formation of the conjugated RO-OA-PEPA FONs (Fig. 3). Many spherical nanoparticles with diameters ranged from 100 to 200 nm can be clearly identified. As compared with the size distribution in PBS, the size characterized by TEM was somewhat smaller possibly due to shrinkage of micelle during the drying process. The TEM images given us evidence directly that the amphiphilic cross-linked copolymers were self-assembled into nanoparticles in aqueous solution. In our previous reports, biocompatible AIE FONs were fabricated through covalent strategy including Schiff-base reaction [42,44], reversible addition fragmentation chain transfer polymerization [25], emulsion polymerization [43] and anhydride ROP [26]. Compared with previous synthesized FONs, the FONs fabricated via anhydride ROP and consecutive cross-linking with PEPA in this work has many obvious advantages. First, many other components including drugs (e.g. cisplatin) and targeting agents could be further integrated into the AIE based FONs due to the existing reactive carboxyl groups. Second, the prepared cross-linked FONs showed stable dispersibility in physiological solution even below the CMC. Moreover, such FONs could be used as a polymeric transfection agent for building siRNA carriers ascribed to the surplus amino groups in the PEPA. In short, thus multifunctional
imaging platform of biocompatible AIE based cross-linked FONs can be facilely fabricated via room temperature anhydride ROP and consecutive cross-linking with PEPA. 3.2. Biocompatibility of RO-OA-PEPA FONs The biocompatibility was conducted to evaluate the potential biomedical applications of RO-OA-PEPA FONs [51]. First, the influences of RO-OA-PEPA FONs to human lung adenocarcinoma epithelial (A549) cells were examined by optical microscopy after cells were incubated with different concentrations of these FONs for 24 h (Fig. 4A–C). The result showed that no significant differences were found between the control cells and cells incubated with 10 and 80 g mL−1 of RO-OA-PEPA FONs, suggesting that these FONs are biocompatible with cells. To further confirm the cytocompatibility of RO-OA-PEPA FONs, cell viability of such FONs to A549 cells was determined by cell counting kit-8 (CCK-8) assay as described in our previous reports [52]. As shown in Fig. 4D, no cell viability decrease was observed when cells were incubated with 10–120 g mL−1 of these cross-linked FONs for 8 h and 24 h. The cell viability value is still higher than 90% even the concentration is up to 120 g mL−1 for 24 h, and no significant difference was observed. These results suggested that these FONs were highly potential for biomedical applications. 3.3. Cell imaging applications of RO-OA-PEPA FONs Based on the biocompatibility results, cell-imaging applications of RO-OA-PEPA FONs were further explored [53]. The cell uptake behavior of these FONs was evaluated by confocal laser scanning microscope (CLSM) observation. As shown in Fig. 5, strong red fluorescence could be observed at the cell location after they were incubated with only 10 g mL−1 of RO-OA-PEPA FONs. Furthermore, many areas with relative weak fluorescence intensity were found in the cells, indicating the possible location of cell nucleus (Fig. 5B), which was shown in previous reports [39,54]. These results suggested that RO-OA-PEPA FONs could be facilely uptaken by cells and mainly located at cytoplasm [42–44]. These FONs are considered that could not enter the cell nucleus directly as comparing with the size of FONs and nucleus pore (9 nm × 15 nm), therefore, the cellular internalization mechanism may be due to cellular endocytosis. More importantly, ascribed to the intense red fluorescence of these cross-linked FONs, strong signal could be detected, proving the good biocompatibility of RO-OA-PEPA FONs. Based on the cell viability results, RO-OAPEPA FONs are considered biocompatible enough for bioimaging applications.
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Fig. 3. TEM image of RO-OA-PEPA FONs dispersed in water, scale bar = 500 nm (A) and 200 nm (B).
Fig. 4. Biocompatibility evaluations of RO-OA-PEPA FONs. (A–C) Optical microscopy images of A549 cells incubated with different concentrations of RO-OA-PEPA FONs for 24 h: (A) control cells, (B) 10 g mL−1 , (C) 80 g mL−1 , (D) cell viability of RO-OA-PEPA FONs with A549 cells for 8 h and 24 h.
Fig. 5. CLSM images of A549 cells incubated with 10 g mL−1 of RO-OA-PEPA FONs for 3 h. (A) Bright field, (B) excited with 543 nm laser, (C) merge image of A and B. Scale bar = 20 m.
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4. Conclusions In summary, stable RO-OA-PEPA FONs were prepared through room temperature anhydride ROP based on an AIE dye (RNH2 ) with two amino end-groups and 4,4 -oxydiphthalic anhydride, then consecutive cross-linking with PEPA. Such obtained amphiphilic copolymers were tended to self-assemble into uniform spherical FONs with diameters ranged from 100 to 200 nm. These RO-OA-PEPA FONs showed high dispersibility and strong red fluorescence in physiological solution since their surfaces were covered with many hydrophilic carboxyl and amino groups. Biocompatibility evaluation and cell imaging results suggested that these FONs were biocompatible enough for bioimaging applications. More importantly, various AIE based cross-linked FONs could be facilely fabricated via room temperature anhydride ROP and subsequent cross-linking, taking advantage of different AIE based amino end-groups dyes, various multianhydrides and other polyamine derivatives, and can be further integrated with other components like drugs and targeting agents into the system using their own various functional groups to afford multifunctional platforms. Considered their various excellent performances, these obtained FONs are expected highly potential for various biomedical applications. 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, 2012M520388, 2013T60178). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18]
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Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Facile fabrication of AIE-based stable cross-linked fluorescent organic nanoparticles for cell imaging Xiqi Zhang b , Xiaoyong Zhang a,b,∗ , Bin Yang b , Junfeng Hui b , Meiying Liu c , Yen Wei b,∗∗ a
Department of Chemistry, Institute of Polymers, Nanchang University, 999 Xuefu Avenue, Nanchang 330031, China Department of Chemistry and Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Tsinghua University, Beijing 100084, China c Beijing National Laboratory for Molecular Sciences (BNLMS), Key Laboratory of Organic Solids, Laboratory of New Materials, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China b
a r t i c l e
i n f o
Article history: Received 25 September 2013 Received in revised form 28 November 2013 Accepted 5 December 2013 Available online 25 December 2013 Keywords: Aggregation-induced emission Polyethylene polyamine Anhydride ring-opening polymerization Cross-linked fluorescent organic nanoparticles Cell imaging
a b s t r a c t An aggregation induced emission dye (RNH2 ) with two amino end-groups was facilely incorporated into stable cross-linked fluorescent organic nanoparticles via room temperature anhydride ring-opening polymerization and consecutive cross-linking with polyethylene polyamine. Thus obtained RO-OA-PEPA FONs were characterized by a series of techniques including 1 H nuclear magnetic resonance, Fourier transform infrared spectroscopy, UV–vis absorption spectrum, fluorescent spectroscopy and transmission electron microscopy. Biocompatibility evaluation and cell uptake behavior of RO-OA-PEPA FONs were further investigated to explore their potential biomedical application. We demonstrated that such FONs showed high-water dispersibility, strong red fluorescence, stable uniform morphology (100–200 nm) and excellent biocompatibility, making them promising for cell imaging application. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Fluorescent organic nanoparticles (FONs) receive increasing interest for their high potential in sensing, imaging and biomedical applications [1–3]. Compared with the conventional fluorescent inorganic nanoparticles such as semiconductor quantum dots, carbon dots and fluorescent silicon nanoparticles, FONs offer an attractive alternative method with many advantages including non-toxicity, biodegradability, biocompatibility and appropriate functionalities [4–10]. However, most of the organic fluorophores are not soluble in physiological solution due to their inherent hydrophobic features, thus should be modified with hydrophilic groups but generally at the expense of the fluorescence intensity because when organic dyes are concentrated in a confined space like nanoparticle, luminescence quenching may simultaneously emerge owing to the notorious aggregation-caused
∗ Corresponding author at: Department of Chemistry, Institute of Polymers, Nanchang University, 999 Xuefu Avenue, Nanchang 330031, China. Tel.: +86 01062792604. ∗∗ Corresponding author. E-mail addresses: [email protected] (X. Zhang), [email protected] (Y. Wei). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.12.010
fluorescent quenching (ACQ) effect [11,12]. Therefore, to overcome this problem, unique aggregation induced emission (AIE) materials appeared with anti-ACQ effect, which emit stronger fluorescence in the aggregation state [13,14]. Hereafter, various AIE fluorogens such as siloles [15–18], tetraphenylethene [19–21], triphenylethylene [22,23], cyano-substituted diarylethene [24–26] and distyrylanthracene [27–32] derivatives have been fabricated and widely investigated for potential biomedical applications due to their attractive AIE feature, biodegradability and biocompatibility. Self-assembly of fluorescent dyes to form nanoparticles such as spherical micelles is a particularly promising strategy for various technological applications such as bioimaging and drug delivery [33–35]. Recently, some strategies including bare AIE nano-assembly, non-covalent strategy and covalent strategy for fabricating AIE based FONs have been developed with intense luminescence, uniform size, high water dispersibility and excellent biocompatibility, making them promising for cell imaging applications [36–44]. However, one of the main problems of bare AIE nano-assembly and non-covalent strategy is that the fluorophores can leak out of the particles with time [11]. Although the covalent strategy can form stable covalently linking between the fluorophore and polymer backbone, another trouble appears that such self-assembled structures are often unstable on dilute solution
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below the critical micelle concentration (CMC) [45,46]. Therefore, although many obvious advances have been achieved in the fabrication of AIE based FONs, more robust fabrication strategies are still highly desirable, including (a) stable dispersion in physiological solution below the CMC and (b) introducing reactive functional groups into the FONs to integrate drugs and targeting agents for multifunctional biomedical applications. Thus development of versatile synthetic strategies for novel stable cross-linked FONs is of significant scientific interest. Herein, a novel facile method of preparing AIE based stable cross-linked FONs was developed via room temperature anhydride ring-opening polymerization (ROP) based on an AIE dye (RNH2 ) with two amino end-groups and 4,4 -oxydiphthalic anhydride (OA) under an air atmosphere, and subsequent cross-linking with polyethylene polyamine (PEPA) (Scheme 1). Such obtained amphiphilic cross-linked copolymers (RO-OA-PEPA) are tended to self-assemble into stable FONs and show high dispersibility in aqueous solution. These obtained FONs were characterized by a series of techniques including nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FT-IR), UV–vis absorption spectrum (UV), fluorescent spectroscopy and transmission electron microscopy (TEM). Owing to the AIE feature of RNH2 , the resulting cross-linked FONs emitted strong red fluorescence in aqueous environment [47]. Finally, the biocompatibility and cell uptake behavior of RO-OA-PEPA FONs were further conducted to evaluate their potential for cell imaging applications. 2. Experimental procedure 2.1. Materials and characterization Phenothiazine, 3-bromoprop-1-ene, N,N-dimethylformamide (DMF), 1,2-dichloroethane, phosphoryl chloride, 4-aminobenzyl cyanide, tetrabutylammonium hydroxide (0.8 M in methanol), N,N-dimethylacetamide (DMAc), 4,4 -Oxydiphthalic anhydride purchased from Alfa Aesar were used as received. Polyethylene polyamine (C2n H5n Nn , MP 250 ◦ C, Density (20 ◦ C) 1.04 g/mL) was purchased from Aladdin Industrial Inc. All other agents and solvents were purchased from commercial sources and used directly without further purification. 1 H NMR spectrum was measured on a JEOL 400 MHz spectrometer [d6 -DMSO as solvent and tetramethylsilane (TMS) as the internal standard]. UV–visible absorption spectrum was recorded on UV/vis/NIR Perkin–Elmer lambda 750 spectrometer (Waltham, MA, USA) using quartz cuvettes of 1 cm path length. Fluorescence spectra were measured on a PE LS-55 spectrometer with a slit width of 3 nm for both excitation and emission. The FT-IR spectra were obtained in a transmission mode on a Perkin–Elmer Spectrum 100 spectrometer (Waltham, MA, USA). Typically, 8 scans at a resolution of 1 cm−1 were accumulated to obtain the spectrum. Transmission electron microscopy (TEM) images were recorded on a JEM-1200EX microscope 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 distribution of RO-OA-PEPA FONs in phosphate buffer solution (PBS) were determined using a zeta Plus apparatus (ZetaPlus, Brookhaven Instruments, Holtsville, NY).
atmosphere at room temperature for 2 h. Followed by adding PEPA (3.0 mg), which was dissolved in 3 mL DMAc. The above mixture was stirred at room temperature for another 0.5 h. Then the crosslinking reaction was stopped by adding 30 mL water, and then the RO-OA-PEPA water dispersion was treated by repeated centrifugal washing process for thrice. Finally, the resulting centrifuged solid was carried out by freeze-drying to obtain RO-OA-PEPA FONs. 2.3. Cytotoxicity of RO-OA-PEPA FONs Cell morphology was observed to examine the effects of ROOA-PEPA FONs to A549 cells. 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 ROOA-PEPA FONs 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 RO-OA-PEPA FONs 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 RO-OAPEPA FONs 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 (Victor III, 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 RO-OA-PEPA FONs), 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 using RO-OA-PEPA FONs A549 cells were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% heat-inactivated FBS, 2 mM glutamine, 100 U mL−1 penicillin, and 100 g mL−1 of streptomycin. Cell culture was maintained at 37 ◦ C in a humidified condition of 95% air and 5% CO2 in culture medium. Culture medium was changed every three days for maintaining the exponential growth of the cells. On the day prior to treatment, 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 RO-OA-PEPA FONs 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 RO-OAPEPA FONs 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-channel (Zesis, Germany) with the excitation wavelength of 543 nm.
2.2. Preparation of RO-OA-PEPA FONs
3. Results and discussion
The synthetic route of RO-OA-PEPA is showed in Scheme 2. RNH2 was synthesized according to our previous literature [26]. In the synthesis of RO-OA-PEPA, RNH2 (37 mg, 0.05 mmol) and 4,4 -oxydiphthalic anhydride (19 mg, 0.06 mmol) were dissolved in 10 mL DMAc. The above mixture was stirred under an air
3.1. Characterization of RO-OA-PEPA FONs 3.1.1. 1 H NMR and IR spectra To prepare RO-OA-PEPA FONs, the hydrophobic AIE dye (RNH2 ) was copolymerized with 4,4 -oxydiphthalic anhydride (OA) via
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Scheme 1. Schematic showing the preparation of RO-OA-PEPA FONs through room temperature anhydride ROP and consecutive cross-linking with PEPA based on an AIE dye (RNH2 ) and subsequent cell imaging applications of these obtained FONs.
Scheme 2. Synthetic route of RO-OA-PEPA.
room temperature ROP [48] under an air atmosphere for 2 h, based on the feed molar ratio (OA: RNH2 ) was 1.2, to obtain ROOA with the designed degree of polymerization of RO-OA was 5. Then these copolymers were facilely cross-linked with PEPA at room temperature for another 0.5 h to obtain the cross-linked amphiphilic copolymers RO-OA-PEPA. 1 H NMR spectrum of ROOA-PEPA in d6 -DMSO was conducted and shown in Fig. 1A. The appearance of the chemical shift around 8.0 ppm was observed, indicating the successful synthesis of RO-OA. Furthermore, the emergence of chemical shift ranged from 2.7 to 2.9 ppm was also found, further confirming the cross-linking of PEPA to afford ROOA-PEPA. The successful synthesis of RO-OA-PEPA copolymers was also confirmed by FT-IR. As shown in Fig. 1B, two characteristic peaks located at 2920 and 2844 cm−1 were observed in the sample of RNH2 , evidencing the stretching vibration of CH2 and CH3 groups. Meanwhile, a series of peaks distributed between 1450 and 1600 cm−1 can be ascribed to the stretching vibration of various aromatic rings. After anhydride ROP and consecutive cross-linking with PEPA, strong bending vibration of N H band of amide and amine groups located at 1594 cm−1 were observed in RO-OA-PEPA FONs [49,50]. On the other hand, a characteristic peak centered at 766 cm−1 was also observed, evidencing that the
wagging vibration of primary and secondary amines bands in the copolymers, and another characteristic peak located at 1084 cm−1 was also observed in copolymers, suggesting that C O band was introduced into the copolymers, meanwhile, the strong stretching vibrations of COOH and NH2 also appeared in the RO-OA-PEPA FONs located ranged from 3250 to 3400 cm−1 , all confirming successful formation of the cross-linked copolymers. Owing to the formation of hydrophilic carboxyl and amino groups in RO-OAPEPA, such obtained cross-linked copolymers showed amphiphilic properties. When dispersed in aqueous solution, these copolymers are tended to self-assemble into polymeric nanoparticles containing the aromatic groups as hydrophobic cores, along with the hydrophilic carboxyl and amino groups covered onto the surfaces as shells. As the AIE fluorophore RNH2 and other hydrophobic groups were aggregated into the nanoparticle cores, thus RO-OAPEPA FONs are expected to possess strong fluorescence and high dispersibility in aqueous solution. 3.1.2. Optical property The UV absorption spectrum of RO-OA-PEPA FONs dispersed in water indicated that the maximum absorption peak located at 468 nm (Fig. 2A). It is noteworthy that the entire spectrum started
Fig. 1. (A) 1 H NMR spectrum of RO-OA-PEPA dissolved in d6 -DMSO; (B) FT-IR spectra of RNH2 , OA, PEPA and RO-OA-PEPA.
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Fig. 2. (A) UV–vis spectrum of RO-OA-PEPA FONs, inset is the visible image of such FONs in water; (B) fluorescence excitation (Ex) and emission (Em) spectra of RO-OA-PEPA FONs, inset is the fluorescent image taken at 365 nm UV light.
to rise in absorption from 800 nm due to the Mie effect, suggesting the presence of nanoparticles [23]. The inset of Fig. 2A intuitively showed the high water dispersibility of RO-OA-PEPA cross-linked FONs. It was worth mentioning that no visible precipitation was found even after the above RO-OA-PEPA FONs were deposited for more than one month. The size distribution of RO-OA-PEPA FONs in phosphate buffer solution (PBS) was tested using a zeta Plus particle size analyzer, indicating that the size distribution of these FONs was 292.3 ± 2.7 nm with a polydispersity index of 0.149. Due to the AIE properties of RNH2 and self-assembly of the amphiphilic cross-linked copolymers, RO-OA-PEPA FONs showed strong red fluorescence dispersed in pure water (inset of Fig. 2B). The fluorescence excitation and emission spectra of RO-OA-PEPA FONs in water were shown in Fig. 2B. The maximum emission wavelength located at 604 nm, while the fluorescence excitation spectrum in water exhibited broad strong excitation wavelength from 350 to 560 nm. Meanwhile, fluorescence quantum yields (F) were estimated using Rhodamine 6G in ethanol as the standard (F = 95%), the absorbance of the solutions was kept around 0.05 to avoid internal filter effect. We demonstrated that the F of RO-OA-PEPA FONs in pure water is about 3.02%. These excellent fluorescent features including intense red emission, broad strong excitation and long wavelength excitation are greatly benefited for their potential cell imaging applications. 3.1.3. TEM The TEM images further confirmed the formation of the conjugated RO-OA-PEPA FONs (Fig. 3). Many spherical nanoparticles with diameters ranged from 100 to 200 nm can be clearly identified. As compared with the size distribution in PBS, the size characterized by TEM was somewhat smaller possibly due to shrinkage of micelle during the drying process. The TEM images given us evidence directly that the amphiphilic cross-linked copolymers were self-assembled into nanoparticles in aqueous solution. In our previous reports, biocompatible AIE FONs were fabricated through covalent strategy including Schiff-base reaction [42,44], reversible addition fragmentation chain transfer polymerization [25], emulsion polymerization [43] and anhydride ROP [26]. Compared with previous synthesized FONs, the FONs fabricated via anhydride ROP and consecutive cross-linking with PEPA in this work has many obvious advantages. First, many other components including drugs (e.g. cisplatin) and targeting agents could be further integrated into the AIE based FONs due to the existing reactive carboxyl groups. Second, the prepared cross-linked FONs showed stable dispersibility in physiological solution even below the CMC. Moreover, such FONs could be used as a polymeric transfection agent for building siRNA carriers ascribed to the surplus amino groups in the PEPA. In short, thus multifunctional
imaging platform of biocompatible AIE based cross-linked FONs can be facilely fabricated via room temperature anhydride ROP and consecutive cross-linking with PEPA. 3.2. Biocompatibility of RO-OA-PEPA FONs The biocompatibility was conducted to evaluate the potential biomedical applications of RO-OA-PEPA FONs [51]. First, the influences of RO-OA-PEPA FONs to human lung adenocarcinoma epithelial (A549) cells were examined by optical microscopy after cells were incubated with different concentrations of these FONs for 24 h (Fig. 4A–C). The result showed that no significant differences were found between the control cells and cells incubated with 10 and 80 g mL−1 of RO-OA-PEPA FONs, suggesting that these FONs are biocompatible with cells. To further confirm the cytocompatibility of RO-OA-PEPA FONs, cell viability of such FONs to A549 cells was determined by cell counting kit-8 (CCK-8) assay as described in our previous reports [52]. As shown in Fig. 4D, no cell viability decrease was observed when cells were incubated with 10–120 g mL−1 of these cross-linked FONs for 8 h and 24 h. The cell viability value is still higher than 90% even the concentration is up to 120 g mL−1 for 24 h, and no significant difference was observed. These results suggested that these FONs were highly potential for biomedical applications. 3.3. Cell imaging applications of RO-OA-PEPA FONs Based on the biocompatibility results, cell-imaging applications of RO-OA-PEPA FONs were further explored [53]. The cell uptake behavior of these FONs was evaluated by confocal laser scanning microscope (CLSM) observation. As shown in Fig. 5, strong red fluorescence could be observed at the cell location after they were incubated with only 10 g mL−1 of RO-OA-PEPA FONs. Furthermore, many areas with relative weak fluorescence intensity were found in the cells, indicating the possible location of cell nucleus (Fig. 5B), which was shown in previous reports [39,54]. These results suggested that RO-OA-PEPA FONs could be facilely uptaken by cells and mainly located at cytoplasm [42–44]. These FONs are considered that could not enter the cell nucleus directly as comparing with the size of FONs and nucleus pore (9 nm × 15 nm), therefore, the cellular internalization mechanism may be due to cellular endocytosis. More importantly, ascribed to the intense red fluorescence of these cross-linked FONs, strong signal could be detected, proving the good biocompatibility of RO-OA-PEPA FONs. Based on the cell viability results, RO-OAPEPA FONs are considered biocompatible enough for bioimaging applications.
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Fig. 3. TEM image of RO-OA-PEPA FONs dispersed in water, scale bar = 500 nm (A) and 200 nm (B).
Fig. 4. Biocompatibility evaluations of RO-OA-PEPA FONs. (A–C) Optical microscopy images of A549 cells incubated with different concentrations of RO-OA-PEPA FONs for 24 h: (A) control cells, (B) 10 g mL−1 , (C) 80 g mL−1 , (D) cell viability of RO-OA-PEPA FONs with A549 cells for 8 h and 24 h.
Fig. 5. CLSM images of A549 cells incubated with 10 g mL−1 of RO-OA-PEPA FONs for 3 h. (A) Bright field, (B) excited with 543 nm laser, (C) merge image of A and B. Scale bar = 20 m.
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4. Conclusions In summary, stable RO-OA-PEPA FONs were prepared through room temperature anhydride ROP based on an AIE dye (RNH2 ) with two amino end-groups and 4,4 -oxydiphthalic anhydride, then consecutive cross-linking with PEPA. Such obtained amphiphilic copolymers were tended to self-assemble into uniform spherical FONs with diameters ranged from 100 to 200 nm. These RO-OA-PEPA FONs showed high dispersibility and strong red fluorescence in physiological solution since their surfaces were covered with many hydrophilic carboxyl and amino groups. Biocompatibility evaluation and cell imaging results suggested that these FONs were biocompatible enough for bioimaging applications. More importantly, various AIE based cross-linked FONs could be facilely fabricated via room temperature anhydride ROP and subsequent cross-linking, taking advantage of different AIE based amino end-groups dyes, various multianhydrides and other polyamine derivatives, and can be further integrated with other components like drugs and targeting agents into the system using their own various functional groups to afford multifunctional platforms. Considered their various excellent performances, these obtained FONs are expected highly potential for various biomedical applications. 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, 2012M520388, 2013T60178). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18]
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