Biocompatibility evaluation of aniline oligomers with different end-functional groups

Abstract

With the ever increasing interest in biomedical applications of aniline oligomers, carefully and systematically evaluating their biocompatibility is of significant importance. In this paper, aniline oligomers with different end-functional groups (tetramer terminated with gluocose (T–G), pentamer terminated with amino group (P–NH₂) and pentamer terminated with carboxyl group (P–COOH)) were synthesized. Their cellular responses to adenocarcinomic human alveolar basal epithelial (A549) cells and human cervical carcinoma (HeLa) cells were investigated for the first time. To verify their potential for in vivo biomedical applications, the interactions between aniline oligomers and red blood cells (RBCs) were further examined. Results indicated that end groups of oligomers affected their cell response. Among all tested molecules, T–G showed the greatest cytotoxicity to both types of cells. Compared with HeLa cells, all oligomers exhibited better biocompatibility with A549 cells. Furthermore, the cell membrane of RBCs was still kept intact after incubation with aniline oligomers for over 4 h, indicating that the cytotoxicity of aniline oligomers was not due to the rupture of the cell membrane. We expect that these results could be helpful to understand the cytotoxicity of aniline oligomers and may give valuable instructions to design biocompatible aniline oligomers for biomedical applications.

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Introduction

Polyaniline (PA) is an important conducting polymer (CP) and has been extensively studied over the past several decades.^1–6 Because of its excellent electrical and optical properties, PA and PA based materials have been investigated for various applications including electronic nanodevices, catalysis and electrocatalysis, antifouling coatings, microwave absorption, electromagnetic interference shielding, electrorheological fluids, chemical and biological sensors, radical scavengers and antimicrobials, etc.^7–24 In particular, the biomedical applications of PA and other CPs, such as tissue engineering and neural probes, have recently attracted more and more attention, because cell activities such as cell adhesion, cell differentiation and cell growth could be tuned by electrical stimulus.^25–31 However, the main challenges in biomedical applications of PA and other CPs lie in their insolubility and non-degradability, which may induce adverse effects for long term in vivo applications.

Aniline oligomers and their derivates are expected to overcome these limitations. Because of easy synthesis, similar electroactivity and improved solubility, aniline oligomers and their derivates have been regarded as alternative electroactive materials and are more suitable for biomedical applications, compared with polyaniline. In the past, various strategies for the synthesis of aniline oligomers with different amine units and substituent groups have been developed.^32,33 The incorporation of these aniline oligomers into biodegradable and biocompatible polymers for biomedical applications has also been reported.^7,33–37 For example, Albertsson et al. reported that the reaction between epoxy groups in the network and the amine group of aniline oligomers resulted in polymers that could form biodegradable and conductive hydrogels.^38–40 Our previous reports have also suggested that the incorporation of aniline oligomers into biodegradable polymers could promote the growth and differentiation of the rat adrenal pheochromocytoma cell line (PC12), implying their potential biomedical applications in tissue engineering.^41–45 Although the biomedical applications of aniline oligomers have attracted increasing interest over the past few decades, their biocompatibility with living organisms is largely unknown. To the best of our knowledge, only one report has investigated the cytotoxicity of aniline oligomers.⁴⁶ The effects of end-functional groups of aniline oligomers on their biocompatibility have not been reported so far.

In the current work, cellular responses of aniline oligomers with different end-functional groups to adenocarcinomic human alveolar basal epithelial (A549) cells and human cervical carcinoma (HeLa) cells were evaluated and compared for the first time. To assess their suitability for biomedical applications, the interactions between the aniline oligomers and red blood cells (RBCs) were further investigated. We observed significant differences in the cellular responses of these aniline oligomers. Among them, T–G exhibited the highest cytotoxicity to both types of cells, implying that the end-functional groups play an important role in the cytotoxicity of aniline oligomers. However, the cell membrane of RBCs was still kept intact after incubation with aniline oligomers, indicating that the cytotoxicity of aniline oligomers was not due to the rupture of the cell membrane. We speculate that the significantly different cellular responses may be ascribed to the end-functional groups, which result in a different solubility.⁴⁶ As the first report focused on the influence of end-functional groups on the cytotoxicity and hemocompatibility of aniline oligomers, we expect that the results could provide a fundamental understanding of the cytotoxicity of aniline oligomers and crucial criteria for the design of more biocompatible aniline oligomers for biomedical applications.

2. Materials and methods

2.1 Materials and characterization

4,4′-Diaminodiphenylamine sulfate was purchased from Tokyo Chemical Industry Co., Ltd (TCI). Succinic anhydride, δ-gluconolactone and other chemicals were purchased from Alfa Aesar, China. All chemicals were used directly without further purification. Aniline tetramer and pentamer with different end-functional groups were synthesized according to our previous reports.^32,33 MALDI-TOF-MS spectra (AXIMA-CFR, SHIMADZU) of aniline oligomers (P–NH₂, T–G, P–COOH) were shown in Fig. 1, which demonstrated the successful synthesis of aniline oligomers.

Fig. 1 MALDI-TOF-MS spectra of aniline oligomers. The aniline oligomers possess different end-functional groups, which are denoted as P–NH₂, T–G, and P–COOH, respectively.

2.2 Cytotoxicity of aniline oligomers

2.2.1 Cell morphology observations. A549 cells and HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, 100 U mL⁻¹ penicillin, and 100 μg mL⁻¹ of streptomycin. Cell culture was maintained at 37 °C under humidified conditions of 95% air and 5% CO₂ in culture medium. The culture medium was changed every two or three days to maintain the exponential growth of the cells.

The cell morphology changes of cells exposed to aniline oligomers were examined by optical microscopy (DMI3000B, Leica, Germany). Briefly, cells were seeded on 6-well microplates at a density of 1 × 10⁵ cells mL⁻¹ in 2 mL of respective media containing 10% FBS. After cell attachment, the plates were washed with PBS and the cells were treated with complete cell culture medium which was prepared by adding 80 μg mL⁻¹ of P–NH₂, T–G and P–COOH to 10% FBS medium respectively. The morphology of cells was observed by optical microscopy.

2.2.2 Cell viability assay. The cell viability of aniline oligomers to A549 cells and HeLa cells was evaluated using the cell count kit-8 (CCK-8) assay according to our previous reports.^47–51 Briefly, cells were seeded on 96-well microplates at a density of 1 × 10⁵ cells mL⁻¹ in 160 μL of respective media containing 10% FBS. After 24 h of cell attachment, the cells were incubated with 10, 20, 40, 80 and 160 μg mL⁻¹ of aniline oligomers for 12 and 24 h. Then aniline oligomers were removed and cells were washed with PBS three times. 10 μL of CCK-8 dye and 100 μL 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 (VictorIII, 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 percentage reduction of CCK-8 dye was compared to controls (cells not exposed to aniline oligomers), which represented 100% CCK-8 dye reduction. Three replicate wells were used for each control and test concentration per microplate, and the experiment was repeated three times. Cell survival was expressed as the absorbance relative to that of untreated controls. Results are presented as means ± standard deviation (SD). The half maximal inhibitory concentration 50% (IC₅₀) values of aniline oligomers were calculated using SPSS 15.0 and were used to compare their cytotoxicity.

2.2.3 RBC morphology observation. Male Sprague–Dawley rats (200–250 g) were obtained from the Laboratory Animal Center, the Chinese Academy of Military Medical Sciences, China. The animals were individually housed in plastic cages, fed a commercial diet, and given water ad libitum. Absence of infection was checked for one week prior to the experiment. The permission of the local ethics committee was obtained, and all animal experiments were performed according to the Chinese law and accepted international standards on biomedical research.

Fresh blood was obtained from Sprague–Dawley rats and anticoagulated with heparin, and then the anticoagulated blood was centrifuged at 4000 rpm for 5 min.^52,53 The plasma and buffy coat were removed by aspiration. The separated erythrocytes were washed three times by centrifugation (4000 rpm, 5 min) in 10 volumes of PBS. The supernatant and buffy coat of white cells were carefully removed with each washing. During the last wash, the erythrocytes were obtained by centrifugation (4000 rpm, 5 min). Washed erythrocytes were finally re-suspended to the desired hematocrit level using the same buffer and stored at 4 °C and used within 6 h of sample preparation.

The effects of aniline oligomers on the morphology of RBCs were investigated by optical microscopy and scanning electron microscopy (SEM). The erythrocyte suspension (10% hematocrit) was incubated with 20 and 80 μg mL⁻¹ of aniline oligomer suspensions. At 1 and 4 h, 100 μL of these reaction mixtures were dropped onto a glass slide and observed by optical microscopy directly. The observation of the erythrocyte shape changes was carried out at 37 °C using an optical microscope (Leica, DMI3000B, Germany).

The detailed morphology of RBCs was further observed using a Phenom G2 Pro Desktop SEM. For SEM observations, RBCs were treated with aniline oligomers as described above. At 1 and 4 h, RBC suspensions were fixed with a glutaraldehyde solution (3%), followed by centrifugation (4000 rpm, 5 min), and then washed with gradient ethanol. Afterwards, the samples were vacuum-dried for SEM characterization.

3. Results and discussion

3.1 Cell morphology observation

Optical microscopy observation was first used to evaluate the biocompatibility of aniline oligomers.^54–56 Fig. 2 shows optical images of A549 cells exposed to aniline oligomers for 12 h. It can be seen that cells kept their normal morphology when they were incubated with 80 μg mL⁻¹ of P–NH₂ (Fig. 2B) and P–COOH (Fig. 2D). However, cell swelling was observed when they were exposed to the same concentration of T–G (Fig. 2C). The optical images gave us the impression that T–G is more toxic than P–NH₂ and P–COOH. When the incubation time was extended to 24 h, the changes of cell morphology and the decrease in cell number were clearly observed, especially for T–G (SFig. 1C†), further confirming the highest toxicity of T–G.

Fig. 2 Optical microscopy images of A549 cells which were exposed to aniline oligomers for 12 h. (A) Control cells, (B–D) cells treated with 80 μg mL⁻¹ of P–NH₂ (B), T–G (C), and P–COOH (D).

3.2 Cell viability of aniline oligomers

The cell viability of aniline oligomers to A549 cells and HeLa cells was then determined to compare the biocompatibility of aniline oligomers.^57–63 As shown in Fig. 3, time- and concentration-dependent cytotoxicity of aniline oligomers to A549 cells was confirmed. It can be seen that the cell viability decreased with increasing aniline oligomer concentrations. Furthermore, when the incubation time was increased from 12 to 24 h, the cell viability also decreased. More importantly, the cellular responses of aniline oligomers could also be influenced by their properties. As shown in Fig. 3A, when cells were incubated with 160 μg mL⁻¹ of T–G for 12 h, the cell viability value was about 67% (Fig. 3A). However, the cell viability values were greater than 80% when cells were incubated with the same concentration of P–NH₂ and P–COOH. The cell viability results were in good agreement with that of optical microscopy observation, further confirming the different cellular responses of aniline oligomers. On the basis of the cell viability results, IC₅₀ values were calculated and were used to compare the cytotoxicity of aniline oligomers. The IC₅₀ values of P–NH₂, T–G and P–COOH are 1595.5, 238.7 and 1316.2 μg mL⁻¹, respectively, for 12 h incubation. When the incubation time was extended to 24 h, the IC₅₀ values are 484.4, 170.0 and 329.8 μg mL⁻¹ for P–NH₂, T–G and P–COOH, respectively. These results further demonstrated the different cytotoxicity of aniline oligomers. In our previous report, the cytotoxicity of aniline oligomers (including the aniline dimer, trimer and tetramer) to A549 cells has also been investigated. We demonstrated that the aniline trimer with two amino groups showed significantly greater toxicity than the aniline dimer and tetramer.⁴⁶ We speculated that the different cellular responses of aniline oligomers are likely to be due to their different water solubilities. Compared with a previous report, linkage of glucose to the aniline tetramer showed a slightly improved toxicity to A549 as compared with that of the amino-terminal tetramer. These results implied that the cytotoxicity of aniline oligomers could be tailored by linkage of different functional groups, which is very important for the design of biocompatible aniline oligomers for biomedical applications. Furthermore, the aniline pentamer is obviously more biocompatible than the aniline trimer regardless of its terminated functional groups (P–NH₂/P–COOH), indicating that the molecular weight of aniline oligomers could also influence their cellular responses.

Fig. 3 Time- and concentration-dependent cell cytotoxicity of aniline oligomers to A549 cells. (A) The incubation time is 12 h; (B) the incubation time is 24 h. The concentration of aniline oligomers range from 10 to 160 μg mL⁻¹. The IC₅₀ values are 1595.5, 238.7 and 1316.2 μg mL⁻¹ for P–NH₂, T–G and P–COOH after they were incubated with HeLa cells for 12 h. When the incubation time was extended to 24 h, the IC₅₀ values for P–NH₂, T–G and P–COOH are 484.4, 170.0 and 329.8 μg mL⁻¹, respectively.

The cell viability of aniline oligomers to HeLa cells was also determined. As shown in Fig. 4, different cellular responses of aniline oligomers with different end-functional groups to HeLa cells were also observed, especially at high concentrations. It can be seen that the cell viability value of T–G to HeLa is about 34.7% for 24 h incubation, which is lower than that of P–NH₂ (61.9%) and P–COOH (56.2%). To compare the cytotoxicity of aniline oligomers and different cells, the IC₅₀ values of aniline oligomers to HeLa cells were also calculated. The IC₅₀ values of P–NH₂, T–G and P–COOH to HeLa cells are 428.2, 179.9 and 366.7 μg mL⁻¹, respectively, for 12 h incubation. When the incubation time was extended to 24 h, the IC₅₀ values of P–NH₂, T–G and P–COOH were changed to 311.9, 110.2 and 301.3 μg mL⁻¹, respectively. The IC₅₀ values of HeLa cells are much lower than that of A549 cells, indicating that cell type could also influence the cellular responses of aniline oligomers.^64–66 With the increasing interest in utilization of aniline oligomers for biomedical applications, a systematic evaluation of their biocompatibility is of significant importance. Combining the results presented here and our previous report, we demonstrated that both the properties of aniline oligomers (molecular weight or end-functional groups) and cell type could influence the cellular responses of aniline oligomers. Compared to the aniline trimer, the aniline pentamer (P–NH₂) with a high molecular weight showed a much better biocompatibility with cancer cells. We believe that the relatively good biocompatibility of P–NH₂ can also be attributed to their relatively poor water solubility for their higher molecular weight.⁴⁶ Although the mechanisms of different cellular responses of aniline oligomers are still unclear, the end-functional groups of aniline oligomers play a vital role. Furthermore, based on the above results, we suggest that the biocompatibility of aniline oligomers could be tailored via adjusting their end-functional groups and molecular weight, which could be useful to design biocompatible aniline oligomers for biomedical applications.^67–72

Fig. 4 Time- and concentration-dependent cell cytotoxicity of aniline oligomers to HeLa cells. (A) The incubation time is 12 h; (B) the incubation time is 24 h. The concentration of aniline oligomers range from 10 to 160 μg mL⁻¹. The IC₅₀ values of aniline oligomers to HeLa cells are 428.2, 179.9 and 366.7 μg mL⁻¹ for P–NH₂, T–G and P–COOH after they were incubated with A549 cells for 12 h. When the incubation time was extended to 24 h, the IC₅₀ values for P–NH₂, T–G and P–COOH are 311.9, 110.2 and 301.3 μg mL⁻¹, respectively.

3.3 Hemocompatibility of aniline oligomers

Hemocompatibility evaluation is a crucial step for materials before they could be used for biomedical applications. Herein, the hemocompatibility of aniline oligomers to RBCs was first evaluated by optical microscopy observation. Our results showed that the morphology of RBCs remain unchanged after they were incubated with 20 and 80 μg mL⁻¹ of aniline oligomers for 1 and 4 h (Fig. 5 and SFig. 2–4†), implying their excellent hemocompatibility. A hemolysis assay has also been done to evaluate the hemocompatibility of aniline oligomers; however, it is difficult to obtain credible data due to the interference of aniline oligomers, which cannot be completely precipitated under the centrifugation conditions (4000 rpm, 5 min). On the other hand, although the cell viability of aniline oligomers exhibits significant difference, little difference was found between aniline oligomers with different end-functional groups, implying that the cytotoxicity of aniline oligomers may not be due to the rupture of the cell membrane.

Fig. 5 Optical images of red blood cells incubated with aniline oligomers for 4 h. The concentration of aniline oligomers is 80 μg mL⁻¹. (A) PBS, (B) P–NH₂, (C) T–G, (D) P–COOH.

Furthermore, the influence of aniline oligomers on RBCs was investigated using SEM. As shown in Fig. 6, SEM images also demonstrated that the cell membranes of RBCs were intact after incubation with 20 μg mL⁻¹ of aniline oligomers. When the incubation time was further increased to 4 h, no significant cell rupture was observed when compared with the control cells (RBCs were incubated with PBS), further evidencing the good hemocompatibility of aniline oligomers (SFig. 5†).

Fig. 6 SEM images of RBCs incubated with aniline oligomers for 1 h. The concentration of aniline oligomers is 20 μg mL⁻¹. (A) PBS, (B) P–NH₂, (C) T–G, (D) P–COOH.

Although the biocompatibility of polyaniline with different functional groups and aniline oligomer based polymer composites has been demonstrated previously,^24,30,31,42 to the best of our knowledge, no reports have investigated the effects of end functional groups on the biocompatibility of aniline oligomers. On the other hand, because the physicochemical properties (e.g. molecular weight, water dispersion and charges etc.) of aniline oligomers are significantly different from those of polyaniline and aniline oligomer based polymer composites, we believe that their biocompatibility results could not represent the results of aniline oligomers. Therefore, it is still of great significance to carefully investigate the biocompatibility of aniline oligomers considering the rapid development of biomedical applications of aniline oligomer based materials.

4. Conclusion

In summary, the cellular responses of aniline oligomers with different end-functional groups (P–NH₂, T–G, P–COOH) to A549 cells and HeLa cells were evaluated and compared for the first time. Time- and concentration-dependent cytotoxicity of aniline oligomers has been confirmed. In addition, the properties of aniline oligomers could also influence their cytotoxicity. Among them, T–G, terminated with glucose, showed the greatest toxicity to cells, implying the importance of end-functional groups for their biocompatibility. Besides the properties of aniline oligomers, cell type could also influence their cellular responses. Compared with HeLa cells, A549 cells exhibited better biocompatibility to all the aniline oligomers. Furthermore, the cell membrane of RBCs were kept intact after they were incubated with aniline oligomers, indicating that their cytotoxicity may not be due to the rupture of the cell membrane. Although the underlying mechanisms of the different cellular responses of aniline oligomers are still unknown, the end-functional groups and molecular weight, which lead to different solubility, play an important role. Furthermore, our results demonstrated that aniline pentamers (P–NH₂, P–COOH) show good biocompatibility with cancer cell lines and RBCs, which could potentially be used for biomedical applications. This is the first report, focusing on the end-functional groups, on the biocompatibility of aniline oligomers, and we expect that the results presented in this work are valuable in the design of more biocompatible materials for biomedical applications.

Acknowledgements

This research was supported by the National Science Foundation of China (no. 21134004, 21104039, and 21201108), and the National 973 Project (no. 2011CB935700), the China Postdoctoral Science Foundation (no. 2012M520388).

Notes and references

1. H. Shirakawa, E. J. Louis, A. G. MacDiarmid, C. K. Chiang and A. J. Heeger, J. Chem. Soc., Chem. Commun., 1977, 578–580.
2. J. D. Stenger-Smith, Prog. Polym. Sci., 1998, 23, 57–79.
3. J. Stejskal, I. Sapurina and M. Trchova, Prog. Polym. Sci., 2010, 35, 1420–1481.
4. X. Lu, W. Zhang, C. Wang, T. C. Wen and Y. Wei, Prog. Polym. Sci., 2011, 36, 671–712.
5. B. Somboonsub, M. A. Invernale, S. Thongyai, P. Praserthdam, D. A. Scola and G. A. Sotzing, Polymer, 2010, 51, 1231–1236.
6. S. Bose, R. A. Khare and P. Moldenaers, Polymer, 2010, 51, 975–993.
7. Y. Wei, J. Wang, X. Jia, J. M. Yeh and P. Spellane, Polymer, 1995, 36, 4535–4537.
8. S. S. Jeon, H. H. An, C. S. Yoon and S. S. Im, Polymer, 2011, 52, 652–657.
9. H. Y. Huang, T. C. Huang, T. C. Yeh, C. Y. Tsai, C. L. Lai, M. H. Tsai, J. M. Yeh and Y. C. Chou, Polymer, 2011, 52, 2391–2400.
10. H. Korri-Youssoufi and A. Yassar, Biomacromolecules, 2001, 2, 58–64.
11. J. W. Lee, F. Serna, J. Nickels and C. E. Schmidt, Biomacromolecules, 2006, 7, 1692–1695.
12. D. Goldman and J. P. Lellouche, Carbon, 2010, 48, 4170–4177.
13. N. Gomez and C. E. Schmidt, J. Biomed. Mater. Res., Part A, 2007, 81, 135–149.
14. P. Humpolicek, V. Kasparkova, P. Saha and J. Stejskal, Synth. Met., 2012, 162, 722–727.
15. E. Abouzari-Lotf, H. Ghassemi, A. Shockravi, T. Zawodzinski and D. Schiraldi, Polymer, 2011, 52, 4709–4717.
16. B. Somboonsub, S. Srisuwan, M. A. Invernale, S. Thongyai, P. Praserthdam, D. A. Scola and G. A. Sotzing, Polymer, 2010, 51, 4472–4476.
17. S. U. Celik and A. Bozkurt, Polymer, 2011, 52, 4670–4675.
18. A. Madani, B. Nessark, R. Brayner, H. Elaissari, M. Jouini, C. Mangeney and M. M. Chehimi, Polymer, 2010, 51, 2825–2835.
19. M. Joubert, M. Bouhadid, D. Bégué, P. Iratçabal, N. Redon, J. Desbriéres and S. Reynaud, Polymer, 2010, 51, 1716–1722.
20. S. Ye, C. Shen, H. Pang, J. Wang and Y. Lu, Polymer, 2011, 52, 2542–2549.
21. M. Gizdavic-Nikolaidis, J. Travas-Sejdic, G. A. Bowmaker, R. P. Cooney and P. A. Kilmartin, Synth. Met., 2004, 140, 225–232.
22. M. Gizdavic-Nikolaidis, J. Travas-Sejdic, G. A. Bowmaker, R. P. Cooney, C. Thompson and P. A. Kilmartin, Curr. Appl. Phys., 2004, 4, 347–350.
23. M. R. Gizdavic-Nikolaidis, J. Bennett, Z. Zujovic, S. Swift and G. A. Bowmaker, Synth. Met., 2012, 162, 1114–1119.
24. M. R. Gizdavic-Nikolaidis, J. R. Bennett, S. Swift, A. J. Easteal and M. Ambrose, Acta Biomater., 2011, 7, 4204–4209.
25. T. J. Rivers, T. W. Hudson and C. E. Schmidt, Adv. Funct. Mater., 2002, 12, 33–37.
26. D. Beattie, K. H. Wong, C. Williams, L. A. Poole-Warren, T. P. Davis, C. Barner-Kowollik and M. H. Stenzel, Biomacromolecules, 2006, 7, 1072–1082.
27. B. Guo, Y. Sun, A. Finne-Wistrand, K. Mustafa and A. C. Albertsson, Acta Biomater., 2012, 8, 144–153.
28. E. Smela, Adv. Mater., 2003, 15, 481–494.
29. M. R. Abidian, D. H. Kim and D. C. Martin, Adv. Mater., 2006, 18, 405–409.
30. M. Gizdavic-Nikolaidis, S. Ray, J. R. Bennett, A. J. Easteal and R. P. Cooney, Macromol. Biosci., 2010, 10, 1424–1431.
31. M. Gizdavic-Nikolaidis, S. Ray, J. Bennett, S. Swift, G. Bowmaker and A. Easteal, J. Polym. Sci., Part A: Polym. Chem., 2011, 49, 4902–4910.
32. Y. Wei, C. Yang, G. Wei and G. Feng, Synth. Met., 1997, 84, 289–291.
33. Z. Y. Wang, C. Yang, J. P. Gao, J. Lin, X. Meng, Y. Wei and S. Li, Macromolecules, 1998, 31, 2702–2704.
34. W. Zhang, J. Feng, A. MacDiarmid and A. Epstein, Synth. Met., 1997, 84, 119–120.
35. B. Guo, A. Finne-Wistrand and A. C. Albertsson, Macromolecules, 2010, 43, 4472–4480.
36. Y. Wei, W. W. Focke, G. E. Wnek, A. Ray and A. G. MacDiarmid, J. Phys. Chem., 1989, 93, 495–499.
37. B. Guo, A. Finne-Wistrand and A. C. Albertsson, Chem. Mater., 2011, 23, 1254–1262.
38. B. Guo, A. Finne-Wistrand and A. C. Albertsson, Biomacromolecules, 2010, 11, 855–863.
39. B. Guo, A. Finne-Wistrand and A. C. Albertsson, J. Polym. Sci., Polym. Chem., 2011, 49, 2097–2105.
40. B. Guo, A. Finne-Wistrand and A. C. Albertsson, Biomacromolecules, 2011, 12, 2601–2609.
41. L. Huang, J. Hu, L. Lang, X. Wang, P. Zhang, X. Jing, X. Chen, P. I. Lelkes and A. G. MacDiarmid, Biomaterials, 2007, 28, 1741–1751.
42. M. Li, Y. Guo, Y. Wei, A. G. MacDiarmid and P. I. Lelkes, Biomaterials, 2006, 27, 2705–2715.
43. J. Hu, L. Huang, X. Zhuang, P. Zhang, L. Lang, X. Chen, Y. Wei and X. Jing, Biomacromolecules, 2008, 9, 2637–2644.
44. L. Huang, X. Zhuang, J. Hu, L. Lang, P. Zhang, Y. Wang, X. Chen, Y. Wei and X. Jing, Biomacromolecules, 2008, 9, 850–858.
45. Y. Liu, J. Hu, X. Zhuang, P. Zhang, Y. Wei, X. Wang and X. Chen, Macromol. Biosci., 2012, 12, 245–250.
46. X. Zhang, H. Qi, S. Wang, L. Feng, Y. Ji, L. Tao, S. Li and Y. Wei, Toxicol. Res., 2012, 1, 201–205.
47. Y. Zhu, W. Li, Q. Li, Y. Li, X. Zhang and Q. Huang, Carbon, 2009, 47, 1351–1358.
48. X. Zhang, S. Wang, L. Xu, L. Feng, Y. Ji, L. Tao, S. Li and Y. Wei, Nanoscale, 2012, 4, 5581–5584.
49. X. Zhang, X. Zhang, S. Wang, M. Liu, Y. Zhang, L. Tao and Y. Wei, ACS Appl. Mater. Interfaces, 2013, 5, 1943–1947.
50. Y. Zhang, B. Yang, X. Zhang, L. Xu, L. Tao, S. Li and Y. Wei, Chem. Commun., 2012, 48, 9305–9307.
51. X. Zhang, S. Wang, M. Liu, J. Hui, B. Yang, L. Tao and Y. Wei, Toxicol. Res., 2013, 2, 335–346.
52. X. Zhang, J. Yin, C. Peng, W. Hu, Z. Zhu, W. Li, C. Fan and Q. Huang, Carbon, 2011, 49, 986–995.
53. 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.
54. X. Zhang, X. Zhang, S. Wang, M. Liu, L. Tao and Y. Wei, Nanoscale, 2013, 5, 147–150.
55. X. Zhang, W. Hu, J. Li, L. Tao and Y. Wei, Toxicol. Res., 2012, 1, 62–68.
56. X. Zhang, X. Zhang, B. Yang, S. Wang, M. Liu, Y. Zhang and L. Tao, RSC Adv., 2013, 3, 9633–9636.
57. B. Yang, Y. Zhang, X. Zhang, L. Tao, S. Li and Y. Wei, Polym. Chem., 2012, 3, 2716–2719.
58. J. Hui, X. Zhang, Z. Zhang, S. Wang, L. Tao, Y. Wei and X. Wang, Nanoscale, 2012, 4, 6967–6970.
59. X. Zhang, S. Wang, C. Fu, L. Feng, Y. Ji, L. Tao, S. Li and Y. Wei, Polym. Chem., 2012, 3, 2716–2719.
60. X. Zhang, J. Hui, B. Yang, Y. Yang, D. Fan, M. Liu, L. Tao and Y. Wei, Polym. Chem., 2013, 4, 4120–4125.
61. B. Yang, Y. Zhang, X. Zhang, L. Tao, S. Li and Y. Wei, Polym. Chem., 2012, 3, 3235–3238.
62. X. Zhang, M. Liu, B. Yang, X. Zhang, Z. Chi, S. Liu and J. Xu, Polym. Chem., 2013, 4, 5060–5064.
63. X. Zhang, M. Liu, B. Yang, X. Zhang and Y. Wei, Colloids Surf., B Biointerfaces, 2013, 112, 81–86.
64. X. Zhang, Y. Zhu, J. Li, Z. Zhu, W. Li and Q. Huang, J. Nanopart. Res., 2011, 13, 6941–6952.
65. J. Li, Y. Zhu, W. Li, X. Zhang, Y. Peng and Q. Huang, Biomaterials, 2011, 31, 8410–8418.
66. 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.
67. X. Zhang, J. Yin, C. Kang, J. Li, Y. Zhu, W. Li, Q. Huang and Z. Zhu, Toxicol. Lett., 2010, 198, 237–243.
68. X. Zhang, J. Li, Y. Zhu, Y. Qi, Z. Zhu, W. Li and Q. Huang, Nucl. Sci. Technol., 2011, 22, 99–104.
69. L. Zhan, G. Yanxia, Z. Xiaoyong, Q. Wei, F. Qiaohui, L. Yan, J. Zongxian, W. Jianjun, T. Yuqin and D. Xiaojiang, J. Nanopart. Res., 2011, 13, 2939–2947.
70. X. Cai, J. Hao, X. Zhang, B. Yu, J. Ren, C. Luo, Q. Li, Q. Huang, X. Shi and W. Li, Toxicol. Appl. Pharmacol., 2010, 243, 27–34.
71. Y. Liang, X. Zhang, W. Luo, Z. Zhong, M. Lv, H. Feng, Y. Zhao and Q. Huang, Prep. Biochem. Biotechnol., 2011, 41, 243–251.
72. X. Zhang, C. Fu, L. Feng, Y. Ji, L. Tao, Q. Huang, S. Li and Y. Wei, Polymer, 2012, 53, 3178–3184.

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Footnote

† Electronic supplementary information (ESI) available: Optical images, TGA, BET etc. See DOI: 10.1039/c3tx50060h

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