Glycosylated aggregation induced emission dye based fluorescent organic nanoparticles: preparation and bioimaging applications

Abstract

Glycosylated fluorescent organic nanoparticles (FONs) based on aggregation induced emission (AIE) dyes were fabricated for the first time through one-pot ring-opening reaction. These glycosylated AIE dye based FONs showed uniform morphology, high water dispersibility, strong red fluorescence and excellent biocompatibility, making them promising candidates for various biomedical applications.

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The development of fluorescent organic nanoparticles (FONs) for optical imaging applications has attracted great research attention in recent years.^1,2 As compared to small organic dyes and fluorescent inorganic nanoparticles, FONs show numerous advantages for biological applications, which include the designability of small organic dyes, ease of surface functionalization, biodegradable potential, and more importantly biocompatibility.^3–8 To date, a series of FONs based on the small organic dyes, conductor polymers, aggregation induced emission (AIE) dyes and dopamine et al. have been fabricated through various strategies.^9–17 The basic principle for the fabrication of FONs is incorporation of hydrophobic dyes into hydrophilic polymers, thus forming dyes containing amphiphilic copolymers. These amphiphilic copolymers can be further self-assembled into FONs, in which the hydrophobic dyes are encapsulated in the core of FONs, and the hydrophilic components are exposed on the surface of hydrophobic core to render the FONs water solubility. Although significant progress has been made, the fabrication of highly luminescent FONs is still a challenge because of the notorious aggregation-caused quenching (ACQ) effect.¹⁸ It is well known that the fluorescent intensity of FONs obtained from conventional organic dyes will be significantly decreased when they are self-assembled into FONs. Decrease the concentration of dyes may alleviate the ACQ effect while the luminous intensity may also be weakened.

Significant different from conventional organic dyes, some organic dyes emitted much stronger fluorescence in aggregate state than dispersed state.^19,20 These “abnormal” fluorescence phenomenon was first reported by Tang et al. in 2001 and was named as AIE.²¹ Since then, a variety of dyes with AIE properties include tetraphenylethene,^22–24 siloles,^25,26 triphenylethene,^27,28 distyrylanthracene^29–32 cyano-substituted diarylethene,^33–35 have been discovered. Because of their unique AIE properties, which could elegantly avoid the fluorescence quenching of conventional organic dyes, making AIE dyes promise for fabrication high luminescent FONs. In recent years, the fabrication of AIE dyes based fluorescent nanoprobes have attracted increasing research interest and AIE dyes based FONs have been extensively explored for various biomedical applications.^36–38 It has been demonstrated that the tetraphenylethene derivates could be self-assembled into FONs in the mixture solution of H₂O and tetrahydrofuran, the tetraphenylethene based FONs emitted strong fluorescence and could be further utilized for cell imaging applications.³⁹ However, organic solvent such as tetraphenylethene should be included to maintain the dispersion of such FONs in aqueous solution. It is well known that the existence of organic medium is harmful to living organisms. To overcome this problem, the encapsulation of AIE dyes (named An18) into a commercially available surfactant (named F127) and synthetic copolymers were also reported by our group.^40,41 More recently, some novel strategies have also be developed by our group, which including incorporation of AIE dyes into hydrophilic polymers via reversible addition–fragmentation chain transfer (RAFT) polymerization, conjugation of AIE dyes with biomacromolecules (chitosan, polylysine) through Shiff-base condensation reaction and synthesis of PEGylated AIE dyes based FONs using ring-opening (RO) reaction.^42–45 However, to the best of our knowledge, few attention have been focused on the fabrication of carbohydrate molecules functionalized AIE dyes based FONs.⁴⁶

Glycopolymers, also called the synthetic polymers with pendant carbohydrate groups, have recently attracted great attention due to potential for biomimicry and various biomedical applications ranged from drug delivery vehicles, therapeutic drugs, cell culture matrices to bioactive hydrogels and models of biological systems.⁴⁷ Because of their advantages over conventional synthetic polymers, the preparation of glycopolymers has been the subject of much recent interest.⁴⁷ In the present work, glycosylated AIE dyes based FONs were prepared via one-pot RO reaction for the first time. As shown in Scheme 1, AIE dye (named as PhNH₂) was first reacted with 4,4′-oxydiphthalic anhydride (OA) through RO polymerization. And then Glu was added to further reacted with PhNH₂–OA via RO condensation reaction. Because of their amphiphilic properties, PhNH₂–OA–Glu can be self-assembled into FONs (named as PhNH₂–OA–Glu FONs) in pure aqueous solution. To further explore their potential biomedical applications, biocompatibility and cell uptake behavior of PhNH₂–OA–Glu FONs were further evaluated. As compared with many other method for preparation of AIE dyes based FONs, RO reaction is rather simple and scalable, which can be occurred under room temperature and air atmosphere without needing catalysts and initiators. Therefore, the glycosylated FONs prepared via RO reaction are expected highly potential for various biomedical applications.

Scheme 1 Schematic showing the preparation of glycosylated PhNH₂–OA–Glu FONs through ring-opening polymerization (ROP) and cell imaging applications of PhNH₂–OA–Glu FONs.

PhNH₂ was prepared following the synthetic route as described in our previous report.⁴⁴ The successful synthesis of PhNH₂ was confirmed by standard spectroscopic methods. For preparation of the PhNH₂–OA–Glu, PhNH₂ with two amino end groups was first reacted with OA through RO polymerization at room temperature for 2 h. The designed degree of polymerization of PhNH₂–OA was 5 with anhydride end functional groups according to the feed molar ratio. As indicated by the gel permeation chromatography (GPC) results, the number of average molecular weights of PhNH₂–OA was 9616 Da according to our previous report.⁴⁴ To synthesis the glycosylated FONs, Glu beared with amino group was further reacted with anhydride end groups of PhNH₂–OA, thus glycosylated AIE dyes based FONs (PhNH₂–OA–Glu FONs) were obtained. Due to hydrophilic properties of Glu carboxyl groups, which were generated on FONs in RO reaction procedure, PhNH₂–OA–Glu copolymers are readily to self-assemble into organic nanoparticles in aqueous solution and show high dispersibility in aqueous solution (left bottle of Fig. S1†). In the PhNH₂–OA–Glu FONs, hydrophobic segments (such as PhNH₂) were wrapped in the interior and served as core of FONs. However, the Glu and carboxyl groups were covered on the surface of PhNH₂–OA–Glu FONs (Scheme 1). More importantly, due to AIE properties of PhNH₂, PhNH₂–OA–Glu FONs are expected to emit strong fluorescence in aqueous solution. As compared with previous methods, the method described in this work for fabrication of glycosylated AIE dyes based FONs has many other advantages for biomedical applications.^30,40 First, the RO reaction is rather simple, effective and scalable. It can be occured at room temperature and atmosphere without needing catalysts and initiators. Therefore it should be a very promise strategy for fabrication of glycosylated AIE dyes based FONs. on the other hand, a number of carboxyl groups were existed on the FONs, which could be further linked with other functional components, thus multifunctional imaging and therapeutic systems can be fabricated based the glycosylated AIE dyes based FONs. More importantly, apart from Glu, other carbohydrate molecules can be also utilized for fabrication of glycosylated AIE dyes based systems through RO condensation reaction.

The successful formation of PhNH₂–OA–Glu FONs was characterized by a series of characterization techniques such as infrared (IR) spectroscopy, UV-Vis spectroscopy, transmission electron microscopy (TEM) and fluorescent spectroscopy. Fig. 1A showed the IR spectra of PhNH₂, OA, Glu and PhNH₂–OA–Glu. It can be seen that two peaks located at 2845 and 2920 cm⁻¹ with strong absorbance intensity was observed.⁴⁸ These absorbance signals could be attributed to the stretching vibration of C–H band, evidencing the present of alkyl chain in PhNH₂. Furthermore, a series of absorbance bands located between 1450–1600 cm⁻¹ in PhNH₂ were also indentified from the IR spectra, which are possible ascribed to the stretching vibration of polycyclic aromatic rings of PhNH₂. However, a small absorbance peak located at 3400 cm⁻¹ can be assigned to the stretching vibration of N–H band of amino groups in PhNH₂. As compared with the spectrum of PhNH₂, a shoulder absorbance peak located at 1732 cm⁻¹ was emerged in the sample of PhNH₂–OA–Glu. The shoulder peak could be ascribed to the stretching vibration of C=O bond of carboxyl group, suggesting the successful formation of AIE dyes based FONs.^48–52 On the other hand, a new absorption peak located at 1108 cm⁻¹ was appeared, indicating the carboxyl group was existed in PhNH₂–OA–Glu. Finally, a very broad absorbance band was emerged at around 3400 cm⁻¹, which could be ascribed to the stretching vibration band of –OH, which was originated from the Glu or carboxyl groups. The IR spectra demonstrated that PhNH₂–OA–Glu FONs can be facilely fabricated via RO reaction.

Fig. 1 Characterization of PhNH₂ and PhNH₂–OA–Glu FONs, (A) normalized IR spectra of PhNH₂, OA, Glu and PhNH₂–OA–Glu FONs, strong stretching vibration bands of C=O which located at 1732 cm⁻¹ and C–O stretching vibration bands which located at 1108 cm⁻¹ were observed in the sample of PhNH₂–OA–Glu FONs, suggesting PhNH₂–OA–Glu FONs were successfully fabricated. (B) UV-Vis spectrum of PhNH₂–OA–Glu FONs. (C) TEM images of PhNH₂–OA–Glu FONs; images showed that the diameters of PhNH₂–OA–Glu FONs are about 100–200 nm. (D) PL spectra of PhNH₂–OA–Glu (in water), the emission wavelength of PhNH₂–OA–Glu FONs is 600 nm. The excitation spectra showed that the excitation wavelength is very broad.

UV-Vis spectrum of PhNH₂–OA–Glu FONs was displayed in Fig. 1B. It shows that the maximum absorption wavelength of PhNH₂–OA–Glu FONs was located at 470 nm. From UV-Vis spectrum, we also found that the entire spectrum started to increase in absorption from 800 nm, indicating that PhNH₂–OA–Glu copolymers were self-assembled into nanoparticles in pure aqueous solution (Fig. 1B). TEM and dynamic laser scattering (DLS) analysis were further examined to confirm the successful formation of nanoparticles via self-assemble of PhNH₂–OA–Glu in pure aqueous solution. As shown in Fig. 1C and S2,† uniform spherical nanoparticles with diameter ranged from 100–200 nm can be clearly observed. The TEM observation further confirmed the successful formation of nanoparticles when PhNH₂–OA–Glu copolymers were dispersed in aqueous solution. The average size and size distribution of PhNH₂–OA–Glu was determined DLS analysis, which showed that the hydrodynamic size of PhNH₂–OA–Glu in water is 295.9 ± 8.3 nm (polydisperse index is 0.012) in water and 436.5 ± 10.7 nm (polydisperse index is 0.005) in phosphate buffer solution (PBS). As compared with size determined by TEM observation, the hydrodynamic size determined by DLS analysis was somewhat larger. The possible reasons are the shrinkage of PhNH₂–OA–Glu FONs during the preparation of TEM samples and the swelling of PhNH₂–OA–Glu FONs in water. Due to their amphiphilic properties, PhNH₂–OA–Glu FONs can be well dispersed in pure aqueous solution. No obvious precipitation was observed when they were deposited for one week (left bottle of Fig. S1†). Because of the AIE properties of PhNH₂, PhNH₂–OA–Glu FONs showed strong red fluorescence in water when they were irradiated with UV lamp (λ = 365 nm) (right bottle of Fig. S1†). The fluorescent property of PhNH₂–OA–Glu FONs was further characterized by fluorescent spectroscopy. As shown in Fig. 1D spectra the emission peak of PhNH₂–OA–Glu was located at 600 nm when they were excited with 490 nm wavelength (Fig. 1D), which is well consistent with the optical image. More importantly, PhNH₂–OA–Glu FONs can be excited with broad wavelength region from 325 to 590 nm, which is very important for their biological imaging applications.

The biocompatibility of PhNH₂–OA–Glu FONs to human lung adenocarcinoma epithelial (A549) cells was also determined to evaluate their potential biomedical applications.^53–58 Fig. 2A–C showed the optical images of A549 cells after they were incubated with different concentrations (10–120 μg mL⁻¹) of PhNH₂–OA–Glu FONs for 24 h. It can be seen that cells still adhered to the cell plate very well after they were incubated with PhNH₂–OA–Glu FONs. As compared with the control cells (Fig. 2A), no obvious cell morphology change was observed through optical images even the concentration of PhNH₂–OA–Glu FONs is as high as 80 μg mL⁻¹ (Fig. 2C). On the other hand, no cell number decrease was found as compared with the control cells and cells incubated with different concentrations of PhNH₂–OA–Glu FONs (Fig. 2B and C). These optical microscopy observation results implied that PhNH₂–OA–Glu FONs are biocompatible with A549 cells. Furthermore, the CCK-8 assay was carried out to quantitatively evaluate the effect of PhNH₂–OA–Glu FONs to A549 cells.^59–64 As shown in Fig. 2D, no cell viability decrease was observed when cells were incubated with 10–120 μg mL⁻¹ of PhNH₂–OA–Glu FONs for 24 h. Even the concentration of PhNH₂–OA–Glu FONs is as high as 120 μg mL⁻¹, the cell viability values of PhNH₂–OA–Glu FONs are still greater than 85%. Cell viability measurement further confirmed the excellent biocompatibility of PhNH₂–OA–Glu FONs. Combination of their unique AIE fluorescence, high water dispersibility, excellent biocompatibility, advantages of glycosylated polymers and RO reaction, thus obtained glycosylated AIE dyes based FONs are expected highly desirable for biomedical applications.

Fig. 2 Biocompatibility evaluation of PhNH₂–OA–Glu FONs. (A–C) Optical microscopy images of A549 cells incubated with different concentrations of PhNH₂–OA–Glu FONs for 24 h, (A) control cells, (B) 10 μg mL⁻¹, (C) 80 μg mL⁻¹, (D) cell viability of PhNH₂–OA–Glu FONs with A549 cells (the concentrations of PhNH₂–OA–Glu FONs are ranged from 10–120 μg mL⁻¹).

To examine the potential biomedical applications of PhNH₂–OA–Glu FONs, their cell uptake behavior was evaluated by Confocal Laser Scanning Microscope (CLSM).^65,66 As showed in Fig. 3, the cell uptake of PhNH₂–OA–Glu FONs was clearly observed after cells were incubated with 10 μg mL⁻¹ of PhNH₂–OA–Glu FONs for 3 h (samples were irradiated with 543 nm laser). The successful dying of cells with FONs implying the potential biological imaging applications of PhNH₂–OA–Glu FONs. Many dark areas with relative weak fluorescence intensity can also be observed from CLSM images (Fig. 3B). These dark areas are possible the location of cell nucleus. Because the size of PhNH₂–OA–Glu FONs is obvious larger than that of cell nucleus pore, we believe that PhNH₂–OA–Glu FONs can not enter the cell nucleus directly. More importantly, due to strong fluorescent intensity of PhNH₂–OA–Glu FONs, the cell uptake of PhNH₂–OA–Glu FONs can be clearly observed even the concentration of FONs is as low as 10 μg mL⁻¹. Furthermore, if the surface of PhNH₂–OA–Glu FONs was further linked with targeting agents, the dosage used for cell imaging could be further decreased. Therefore, we believe that PhNH₂–OA–Glu FONs are biocompatible enough for practical biological imaging applications.

Fig. 3 CLSM images of A549 cells when they were incubated with 10 μg mL⁻¹ of PhNH₂–OA–Glu FONs for 3 h. (A) Bright field, (B) excited with 543 nm laser, (C) merge image of A and B. Scale bar = 20 μm.

In summary, the glycosylated AIE dyes based FONs (named as PhNH₂–OA–Glu FONs) were facilely prepared through one-pot RO reaction for the first time. This reaction can be occured under room temperature and air atmosphere without needing catalysts and initiators, which is rather simple and scalable. On the other hand, due to the existence of hydrophilic Glu and carboxyl groups on the surface of PhNH₂–OA–Glu FONs, thus obtained AIE dyes based FONs are readily self assembled into FONs in pure aqueous solution. These FONs possess numerous excellent properties such as uniform morphology, high water dispersibility, strong red fluorescence and excellent biocompatibility, making them highly potential for various biomedical applications. More importantly, due to the advantages of glycosylated polymers, PhNH₂–OA–Glu FONs are expected exhibited different properties for biomedical applications. Finally, a large number of carboxyl groups were existed on PhNH₂–OA–Glu FONs, many other functional components such as drugs, targeting agents and imaging agents can be further integrated into PhNH₂–OA–Glu FONs, thus multifunctional theranostic platforms based on PhNH₂–OA–Glu FONs can be fabricated. Combined these advantages of PhNH₂–OA–Glu FONs, we believe that such glycosylated AIE dyes based FONs should be of great research interest for various biomedical applications.

Acknowledgements

This research was supported by the National Science Foundation of China (no. 21134004, 21201108, 51363016, 21376190), and the National 973 Project (no. 2011CB935700), China Postdoctoral Science Foundation (no. 2012M520388, 2012M520243, 2013T60100, 2013T60178).

References

1. C. Wu, T. Schneider, M. Zeigler, J. Yu, P. G. Schiro, D. R. Burnham, J. D. McNeill and D. T. Chiu, J. Am. Chem. Soc., 2010, 132, 15410–15417.
2. F. Ye, C. Wu, Y. Jin, Y.-H. Chan, X. Zhang and D. T. Chiu, J. Am. Chem. Soc., 2011, 133, 8146–8149.
3. L. Feng, C. Zhu, H. Yuan, L. Liu, F. Lv and S. Wang, Chem. Soc. Rev., 2013, 42, 6620–6633.
4. J. Hui, X. Zhang, Z. Zhang, S. Wang, L. Tao, Y. Wei and X. Wang, Nanoscale, 2012, 4, 6967–6970.
5. X. Zhang, J. Hui, B. Yang, Y. Yang, D. Fan, M. Liu, L. Tao and Y. Wei, Polym. Chem., 2013, 4, 4120–4125.
6. X. Zhang, S. Wang, M. Liu, B. Yang, L. Feng, Y. Ji, L. Tao and Y. Wei, Phys. Chem. Chem. Phys., 2013, 15, 19013–19018.
7. H. Xie, F. Zeng, C. Yu and S. Wu, Polym. Chem., 2013, 4, 5416–5424.
8. P. Zhang, J. Chen, F. Huang, Z. Zeng, J. Hu, P. Yi, F. Zeng and S. Wu, Polym. Chem., 2013, 4, 2325–2332.
9. C. W. T. Leung, Y. Hong, S. Chen, E. Zhao, J. W. Lam and B. Z. Tang, J. Am. Chem. Soc., 2013, 135, 62–65.
10. H. Shi, J. Liu, J. Geng, B. Z. Tang and B. Liu, J. Am. Chem. Soc., 2012, 134, 9569–9572.
11. R. Zhan, A. J. H. Tan and B. Liu, Polym. Chem., 2011, 2, 417–421.
12. K. Li, J. Pan, S. S. Feng, A. W. Wu, K. Y. Pu, Y. Liu and B. Liu, Adv. Funct. Mater., 2009, 19, 3535–3542.
13. J. Liu, D. Ding, J. Geng and B. Liu, Polym. Chem., 2012, 3, 1567–1575.
14. D. Li, J. Yu and R. Xu, Chem. Commun., 2011, 47, 11077–11079.
15. D. Li, Z. Liang, J. Chen, J. Yu and R. Xu, Dalton Trans., 2013, 42, 9877–9883.
16. Z. Wang, B. Xu, L. Zhang, J. Zhang, T. Ma, J. Zhang, X. Fu and W. Tian, Nanoscale, 2013, 5, 2065–2072.
17. H. Lu, B. Xu, Y. Dong, F. Chen, Y. Li, Z. Li, J. He, H. Li and W. Tian, Langmuir, 2010, 26, 6838–6844.
18. K. Li, W. Qin, D. Ding, N. Tomczak, J. Geng, R. Liu, J. Liu, X. Zhang, H. Liu and B. Liu, Sci. Rep., 2013, 3, 1150.
19. Y. Liu, X. Feng, J.-g. Zhi and B. Tong, Chin. J. Polym. Sci., 2012, 30, 443–450.
20. X. Zhang, X. Zhang, B. Yang, M. Liu and Y. Wei, Colloids Surf., B, 2014, 116, 739–744.
21. J. Luo, Z. Xie, J. W. Y. Lam, L. Cheng, H. Chen, C. Qiu, H. S. Kwok, X. Zhan, Y. Liu, D. Zhu and B. Z. Tang, Chem. Commun., 2001, 1740–1741.
22. X. Zhang, Z. Chi, H. Li, B. Xu, X. Li, W. Zhou, S. Liu, Y. Zhang and J. Xu, Chem.–Asian J., 2011, 6, 808–811.
23. X. Zhang, Z. Chi, H. Li, B. Xu, X. Li, S. Liu, Y. Zhang and J. Xu, J. Mater. Chem., 2011, 21, 1788–1796.
24. X. Zhang, M. Liu, B. Yang, X. Zhang and Y. Wei, Colloids Surf., B, 2013, 112, 81–86.
25. Y. Yu, C. Feng, Y. Hong, J. Liu, S. Chen, K. M. Ng, K. Q. Luo and B. Z. Tang, Adv. Mater., 2011, 23, 3298–3302.
26. Z. Li, Y. Q. Dong, J. W. Lam, J. Sun, A. Qin, M. Häußler, Y. P. Dong, H. H. Sung, I. D. Williams and H. S. Kwok, Adv. Funct. Mater., 2009, 19, 905–917.
27. X. Zhang, Z. Chi, B. Xu, C. Chen, X. Zhou, Y. Zhang, S. Liu and J. Xu, J. Mater. Chem., 2012, 22, 18505–18513.
28. X. Zhang, X. Zhang, B. Yang, L. Liu, J. Hui, M. Liu, Y. Chen and Y. Wei, RSC Adv., 2014, 4, 10060–10066.
29. X. Zhang, Z. Chi, J. Zhang, H. Li, B. Xu, X. Li, S. Liu, Y. Zhang and J. Xu, J. Phys. Chem. B, 2011, 115, 7606–7611.
30. X. Zhang, X. Zhang, B. Yang, S. Wang, M. Liu, Y. Zhang and L. Tao, RSC Adv., 2013, 3, 9633–9636.
31. X. Zhang, X. Zhang, S. Wang, M. Liu, Y. Zhang, L. Tao and Y. Wei, ACS Appl. Mater. Interfaces, 2013, 5, 1943–1947.
32. X. Zhang, Z. Ma, Y. Yang, X. Zhang, Z. Chi, S. Liu, J. Xu, X. Jia and Y. Wei, Tetrahedron, 2014, 70, 924–929.
33. B. K. An, S. K. Kwon, S. D. Jung and S. Y. Park, J. Am. Chem. Soc., 2002, 124, 14410–14415.
34. X. Zhang, X. Zhang, B. Yang, M. Liu, W. Liu, Y. Chen and Y. Wei, Polym. Chem., 2014, 5, 356–360.
35. X. Zhang, X. Zhang, B. Yang, Y. Zhang and Y. Wei, ACS Appl. Mater. Interfaces, 2014, 6, 3600–3606.
36. A. Qin, J. W. Lam and B. Z. Tang, Prog. Polym. Sci., 2012, 37, 182–209.
37. Y. Hong, J. W. Lam and B. Z. Tang, Chem. Commun., 2009, 4332–4353.
38. X. Zhang, X. Zhang, B. Yang, J. Hui, M. Liu, Z. Chi, S. Liu and J. Xu, J. Mater. Chem. C, 2014, 2, 816–820.
39. R. Hu, C. A. Gómez-Durán, J. W. Lam, J. L. Belmonte-Vázquez, C. Deng, S. Chen, R. Ye, E. Peña-Cabrera, Y. Zhong and K. S. Wong, Chem. Commun., 2012, 48, 10099–10101.
40. X. Zhang, X. Zhang, S. Wang, M. Liu, L. Tao and Y. Wei, Nanoscale, 2013, 5, 147–150.
41. X. Zhang, X. Zhang, B. Yang, Y. Zhang, M. Liu, W. Liu, Y. Chen and Y. Wei, Colloids Surf., B, 2014, 1, 435–441.
42. X. Zhang, X. Zhang, B. Yang, M. Liu, W. Liu, Y. Chen and Y. Wei, Polym. Chem., 2013, 4, 4317–4321.
43. X. Zhang, M. Liu, B. Yang, X. Zhang, Z. Chi, S. Liu, J. Xu and Y. Wei, Polym. Chem., 2013, 4, 5060–5064.
44. X. Zhang, X. Zhang, B. Yang, J. Hui, M. Liu, Z. Chi, S. Liu, J. Xu and Y. Wei, Polym. Chem., 2014, 5, 318–322.
45. X. Zhang, X. Zhang, B. Yang, J. Hui, M. Liu, W. Liu, Y. Chen and Y. Wei, Polym. Chem., 2014, 5, 689–693.
46. M. Li, Y. Hong, Z. Wang, S. Chen, M. Gao, R. T. Kwok, W. Qin, J. W. Lam, Q. Zheng and B. Z. Tang, Macromol. Rapid Commun., 2013, 34, 767–771.
47. J. R. Kramer and T. J. Deming, Polym. Chem., 2014, 5, 671–682.
48. X. Zhang, M. Liu, Y. Zhang, B. Yang, Y. Ji, L. Feng, L. Tao, S. Li and Y. Wei, RSC Adv., 2012, 2, 12153–12155.
49. X. Zhang, J. Ji, X. Zhang, B. Yang, M. Liu, W. Liu, L. Tao, Y. Chen and Y. WeI, RSC Adv., 2013, 3, 21817–21823.
50. Y. Cao, X. Zhang, L. Tao, K. Li, Z. Xue, L. Feng and Y. Wei, ACS Appl. Mater. Interfaces, 2013, 5, 4438–4442.
51. X. Zhang, C. Fu, L. Feng, Y. Ji, L. Tao, Q. Huang, S. Li and Y. Wei, Polymer, 2012, 53, 3178–3184.
52. X. Zhang, J. Li, Y. Zhu, Y. Qi, Z. Zhu, W. Li and Q. Huang, Nucl. Sci. Tech., 2011, 22, 99–104.
53. Y. Zhu, W. Li, Q. Li, Y. Li, X. Zhang and Q. Huang, Carbon, 2009, 47, 1351–1358.
54. X. Zhang, Y. Zhu, J. Li, Z. Zhu, W. Li and Q. Huang, J. Nanopart. Res., 2011, 13, 6941–6952.
55. X. Zhang, J. Yin, C. Peng, W. Hu, Z. Zhu, W. Li, C. Fan and Q. Huang, Carbon, 2011, 49, 986–995.
56. X. Zhang, J. Yin, C. Kang, J. Li, Y. Zhu, W. Li, Q. Huang and Z. Zhu, Toxicol. Lett., 2010, 198, 237–243.
57. J. Li, Y. Zhu, W. Li, X. Zhang, Y. Peng and Q. Huang, Biomaterials, 2010, 31, 8410–8418.
58. Z. Li, Y. Geng, X. Zhang, W. Qi, Q. Fan, Y. Li, Z. Jiao, J. Wang, Y. Tang and X. Duan, J. Nanopart. Res., 2011, 13, 2939–2947.
59. X. Zhang, H. Qi, S. Wang, L. Feng, Y. Ji, L. Tao, S. Li and Y. Wei, Toxicol. Res., 2012, 1, 201–205.
60. X. Zhang, W. Hu, J. Li, L. Tao and Y. Wei, Toxicol. Res., 2012, 1, 62–68.
61. H. Qi, M. Liu, L. Xu, L. Feng, L. Tao, Y. Ji, X. Zhang and Y. Wei, Toxicol. Res., 2013, 2, 427–433.
62. X. Zhang, S. Wang, M. Liu, J. Hui, B. Yang, L. Tao and Y. Wei, Toxicol. Res., 2013, 2, 335–346.
63. Y. Zhu, X. Zhang, J. Zhu, Q. Zhao, Y. Li, W. Li, C. Fan and Q. Huang, Int. J. Mol. Sci., 2012, 13, 12336–12348.
64. L. Xu, X. Zhang, C. Zhu, Y. Zhang, C. Fu, B. Yang, L. Tao and Y. Wei, J. Biomater. Sci., Polym. Ed., 2013, 24, 1564–1574.
65. B. Yang, Y. Zhang, X. Zhang, L. Tao, S. Li and Y. Wei, Polym. Chem., 2012, 3, 3235–3238.
66. X. Zhang, S. Wang, C. Zhu, M. Liu, Y. Ji, L. Feng, L. Tao and Y. Wei, J. Colloid Interface Sci., 2013, 397, 39–44.

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Footnote

† Electronic supplementary information (ESI) available: Optical images of PhNH₂–OA–Glu FONs water dispersion, TEM image of PhNH₂–OA–Glu FONs, et al. See DOI: 10.1039/c4ra01176g

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