Facile preparation, through Schiff base formation, of luminescent amphiphilic carbohydrate polymers with aggregation-induced emission characteristics for biological imaging

Abstract

Novel fluorescent amphiphilic copolymers were prepared via Schiff base condensation between an aggregation-induced emission (AIE) dye (named as PHE) with one amino group and oxidation starch (DAS) with aldehyde groups. The size, morphology, composition as well as optical properties of resultant PHE–DAS were characterized in detail by means of a number of techniques. To evaluate their biomedical application potential, their biocompatibility as well as cell uptake behavior were also determined. We confirmed that the PHE–DAS can readily self-assemble into core shell nanostructures and display good water dispersibility and strong luminescent emission. Furthermore, these AIE active luminescent polymeric microparticles (LPMs) exhibited negative toxicity towards A549 cells and desirable biological imaging properties. More importantly, many carboxyl groups were generated on the PHE–DAS LPMs, making them useful for further surface functionalization and drug delivery applications. Taken together, we have developed a facile strategy for fabrication of AIE active carbohydrate polymers, which exhibited great potential for biological imaging and drug delivery applications.

---

1. Introduction

Luminescent polymeric microparticles (LPMs) are attracting more and more research attention because of their potential applications in biological imaging, medical diagnosis and biological sensors.^1–5 Different from luminescent inorganic nanoparticles such as semiconductor quantum dots, fluorescent carbon dots, photoluminescent silicon nanoparticles, metallic nanoclusters and polymeric luminescent nanoparticles, LPMs show some obvious advantages, including the facile designability of dyes' chemical structure, controllability of their optical properties, diverse strategies for fabrication of organic dye-based LPMs and, more importantly, their better biocompatibility and degradability potential.^6–17 The self-assembly of dyes containing amphiphilic copolymers into core shell LPMs is the underlying mechanism.^18–20 However the fluorescence intensity of these LPMs that are based on typical organic dyes through self-assembly will be significantly decreased or completely quenched.²¹ This notorious phenomenon is well known as the aggregation-caused quenching (ACQ) effect, and it has largely hampered the practical applications of LPMs.³ Over the past few decades, great effort has been devoted to overcoming the ACQ effect of conventional organic dye-based LPMs; however most of them have been fruitless. Aggregation-induced emission (AIE) is an abnormal fluorescence phenomenon, which was proposed first by Tang et al. in 2001.^21–30 Different from the ACQ effect of normal organic dyes, the AIE-active dyes could emit much stronger and enhanced luminescence intensity in their aggregation or solid state.^31–34 The unique AIE properties make the AIE-active dyes promising candidates for fabrication of LPMs because they could elegantly overcome the drawbacks of LPMs based on conventional organic dyes.

Since the first discovery by Tang et al., a great number of dyes with the AIE feature have been synthesized.^35–37 However, most AIE dyes have hydrophobic characteristics and are unstable in biological media. Most AIE dyes are difficult to dissolve in aqueous solution directly. Thus different strategies have been developed to fabricate AIE-active dye-based LPMs to overcome their insolubility in aqueous solution.^38–40 For example, the fabrication of AIE-active LPMs based on the noncovalent self-assembly of AIE dyes and amphiphilic synthetic or commercially available copolymers has been demonstrated by Tang and Zhang et al.^8,41–43 The integration of polymerizable AIE dyes has also been demonstrated in our previous reports.^44,45 Furthermore, our recent reports have also demonstrated that AIE-active LPMs can be fabricated via the formation of stimuli-responsive bonds and supramolecular interaction.^8,46–48 Carbohydrate polymers are naturally-derived polymers that possess good biocompatibility, biodegradability, water dispersibility and diverse biological activities.⁴⁹ Because of their outstanding properties, renewability and low cost, biomaterials based on carbohydrate polymers have been intensively investigated over the past several decades. However, to the best of our knowledge, only very few reports have demonstrated the fabrication of AIE-active carbohydrate polymers. This is likely due to the poor solubility of carbohydrate polymers in organic solvents.

In this contribution, a facile strategy has been developed to prepare novel AIE-active amphiphilic copolymers via Schiff-base condensation between an amino group-terminated AIE dye (named PHE) and dialdehyde starch (DAS) with aldehyde groups, in aqueous solution. Aldehyde and amino groups can form a Schiff base under an acidic environment.^50–52 To provide a stable linkage of PHE and DAS, the PHE and DAS therefore underwent a condensation reduction in the presence of acetic acid to form a Schiff base, and surplus aldehyde groups were transformed into carboxyl groups after reacting with sodium hydroxide. The final products were named as PHE–DAS copolymers, which are composed of hydrophobic PHE and hydrophilic DAS. The successful formation of PHE–DAS was confirmed by UV-visible absorption spectroscopy, photoluminescence (PL) spectroscopy, X-ray photoelectron spectroscopy (XPS), and transmission electron microscopy (TEM). The biological imaging applications of PHE–DAS were evaluated by cell viability tests and confocal laser scanning microscopy (CLSM). We demonstrated that this strategy is simple, effective and universal. It should be a general strategy for fabrication of AIE-active LPMs and thus of great importance for the development of AIE-active LPMs.

2. Experimental

2.1 Measurements and materials

Phenothiazine, 1-bromohexadecane, phosphoryl chloride, ethyl acetate, 4-aminobenzyl cyanide, 1,2-dichloroethane, anhydrous magnesium sulfate, corn starch (food-grade), dried at 105 °C before use, and sodium periodate were purchased from Aladdin (Shanghai, China). All chemicals were used as received without purification and were of analytical grade. FT-IR spectra were obtained using a Nicolet 380 Fourier transform spectrometer with a resolution of 2 cm⁻¹. UV-visible absorption spectra were obtained with a PerkinElmer LAMBDA35 UV/Vis spectrometer. TEM images were recorded with a Hitachi 7650B microscope operated at 80 kV. The test specimens were prepared by placing a drop of nanoparticle suspension on a carbon-coated copper grid. Fluorescence spectra (FL) were obtained using a PELS-55 spectrometer with a slit width of 3 nm for both excitation and emission. XPS spectra were measured with a VGESCALAB 220-IXL spectrometer using an Al Kα X-ray source (1486.6 eV).

2.2 Synthesis of PHE–DAS copolymers

The synthetic route to PHE is depicted in Scheme 1A, and was achieved following a previous report by Zhang et al. with slight alteration.⁵³ Intermediate 1 was synthesized on the basis of the work of Zhang et al.⁵⁴ Intermediate 2 was obtained by the following steps. Firstly, at a temperature of 0 °C, 6 mL of dry DMF was slowly added to 5.23 g of phosphoryl chloride (34.2 mmol), and then the mixture was stirred for 3 h at room temperature. Then, the mixture was added to a solution that contained 7.68 g of intermediate 1 (17.04 mmol) in 18 mL of 1,2-dichloroethane. The mixture was heated to 90 °C after 1 h and then stirred for about 15 h at a temperature of 90 °C. Finally, the cooled mixture was added into 120 mL of distilled water. After extracting with ethyl acetate three times, the organic layer was dried over anhydrous magnesium sulfate (MgSO₄). After evaporation under vacuum, the crude product was purified by silica gel using petroleum ether–dichloromethane (1 : 1, v/v) to obtain pure intermediate 2. Then, 0.17 g of intermediate 2 (0.35 mmol) and 0.09 g (0.68 mmol) of 4-aminobenzyl cyanide were put into 6 mol and stirred at room temperature. Three drops of tetrabutylammonium hydroxide solution were added and then the mixture was heated with refluxing for 2 h to obtain an orange precipitate.

Scheme 1 Synthesis route to PHE and fabrication of PHE–DAS LPMs through formation of Schiff base between the amino group-terminated AIE dye (PHE) and oxidation corn starch. Some of the aldehyde groups were utilized for Schiff base formation; the remaining aldehyde groups were transformed into carboxyl groups, which could enhance the water dispersibility of the final PHE–DAS LPMs and could also potentially be utilized for carrying drugs such as cisplatin.

Corn starch (9.72 g of dry base) was added in 30 mL of distilled water and mixed with 60 mL of sodium periodate solutions (0.5 mol L⁻¹). Then, sulfuric acid was added to adjust the pH to 4.0. The mixture was stirred at 20 °C for 5 h in a dark environment and then filtered. Finally, the mixture was put in a rotary evaporator at 50 °C under vacuum to obtain DAS. DAS (300 mg) dissolved in water was then mixed with 50 mg PHE dissolved in tetrahydrofuran. The mixture was stirred under air atmosphere at room temperature for 5 h; then, 5 drops of acetic acid were added and the mixture was dialyzed against ethanol for 6 h using 3500 Da MW cutoff dialysis membranes. The precursors of PHE–DAS were purified via dialysis. Finally, 0.12 g of the precursor of the PHE–DAS copolymers was transferred to an Erlenmeyer flask and 6 mL of sodium hydroxide (0.2 mol L⁻¹) was added at the same time. The copolymer precursors were swirled in a water bath at 70 °C for 5 min. They were then cooled down quickly with high-speed swirling for 1 min under running tap water. The solution was dialyzed against tap water for 24 h and ethanol for 8 h using 3500 Da MW cutoff dialysis membranes.

2.3 Cytotoxicity assays

The cell viability of PHE–DAS on human lung adenocarcinoma epithelial (A549) cells was determined by MTS assays (Promega, USA).^55–58 Briefly, A549 cells were seeded in 96-well tissue culture plates at a density of 5 × 10⁴ cells per well in a final volume of 100 μL. After 24 h of cell attachment, cells were incubated with 10, 20, 40, 80, 100 μg mL⁻¹ PHE–DAS for 8 and 24 h. Each sample was repeated three times. Plates were washed with phosphate-buffered saline (PBS) for three times after removing fluorescent nanoparticles. Then to each well were added 10 μL of MTS and 90 μL of Dulbecco's modified Eagle medium (DMEM) cell culture medium; the plates were then incubated for 3 h at 37 °C. Finally, the plates were analyzed using a microplate reader (Sunrise, TECAN, Mannedorf, Switzerland) at 490 nm. Cell survival was expressed as a percentage compared to the control cells.

2.4 Cell imaging

A549 cells were cultured with culture medium in a culture plate for 24 h at a density of 1 × 10⁵ cells per mL. The culture medium comprised DMEM, 10% heat-inactivated fetal bovine serum, 100 U mL⁻¹ penicillin, and 100 μg mL⁻¹ of streptomycin. The cells were incubated with PHE–DAS at a final concentration of 40 μg mL⁻¹ for 3 h at 37 °C in 5% CO₂. Then the cells were washed using PBS to remove nanoparticles that did not enter the cells and fixed using 4% paraformaldehyde for 10 minutes at room temperature. The cell uptake behavior of PHE–DAS FONs was evaluated by CLSM observation, and digital monochromatic images were acquired using Leica Confocal software.

3. Results and discussion

3.1 Characterization of PHE–DAS

The fluorescent PHE–DAS nanoparticles were prepared following the synthetic route indicated in Scheme 1. First, DAS was obtained from oxidation corn starch using sodium periodate. DAS and PHE can form a Schiff base between the amino groups of PHE and the aldehyde groups of oxidation starch. The resultant copolymers were further treated with sodium hydroxide to obtain PHE–DAS with carboxyl groups. First, hydrophobic PHE was obtained using phenothiazine and 1-bromohexadecane. The structure was determined by ¹H NMR analysis and is shown in Fig. S1.† Peaks at about 7.23–7.07 ppm were observed, attributable to benzyl protons. At the same time, a peak at about 6.65 ppm ascribed to protons of NH₂ was also identified, which suggested the successful synthesis of PHE. The ¹H NMR spectrum of PHE–DAS in CDCl₃ is shown in Fig. S2.† The peaks at 2.96–2.81 ppm were attributable to the methylene protons of PHE–DAS. A new peak appeared at 7.25 ppm ascribed to the protons of carbon–nitrogen double bonds. All the above results suggested the successful synthesis of PHE–DAS. The successful synthesis of PHE–DAS copolymers was confirmed by FT-IR spectroscopy (Fig. 1). A characteristic peak located at 1607 cm⁻¹ was observed in the spectrum of PHE–DAS, evidencing the existence of the stretching vibration of C=O. Furthermore, a characteristic peak centered at 1041 cm⁻¹ provided evidence that a C–O bond was introduced into the copolymers. The C–N stretching vibration band is located at 1252 cm⁻¹. The stretching vibrations of CH₃ and CH₂ appeared in the PHE–DAS spectrum located at 2920 and 2856 cm⁻¹. Furthermore, the characteristic peaks of N–H and O–H were located at 3384 cm⁻¹. Meanwhile, the stretching vibrations of C–N located at 2205 cm⁻¹ disappeared in the spectrum of PHE–DAS. The characteristic peak of –CH₂– in the spectrum of copolymers at 734 cm⁻¹ was observed. The results of FT-IR spectra confirmed the successful synthesis of PHE–DAS. The formation of core–shell AIE-active LPMs was directly supported by TEM images. As shown in Fig. S3,† many spherical nanoparticles with diameters ranging from 200 to 500 nm could be clearly observed. The TEM images provide direct evidence that these AIE-active amphiphiles were synthesized successfully in aqueous solutions and that their shape and size were uniform. The successful formation of core shell nanostructures is mainly ascribed to the self-assembly of these AIE-active amphiphiles, in which the hydrophobic components (PHE) were encapsulated in the core while the hydrophilic components (hydroxyl and carboxyl groups) covered the surface. Therefore, we could expect that these self-assembled nanoparticles should emit strong luminescence due to the partial aggregation of AIE dye. On the other hand, these nanoparticles would display excellent water dispersibility, due to their surface being covered by hydrophilic starch.

Fig. 1 FT-IR spectra of DAS and PHE–DAS copolymers.

The chemical compositions of the PHE–DAS were measured by XPS in order to further prove the successful synthesis of the AIE-active LPMs. The survey scan XPS spectrum of PHE–DAS is shown in Fig. S4.† It can be seen that the chemical elements carbon (C), oxygen (O), nitrogen (N), and sulfur (S) were found in the sample of PHE–DAS. The existence of N and S in the sample of PHE–DAS indicated that the AIE dye PHE had successfully conjugated with DAS through the formation of a Schiff base because DAS theoretically lacks N and S. The C 1s, N 1s, O 1s and S 2p XPS spectra of PHE–DAS are displayed in Fig. 2. It can be seen that the N 1s and S 2p spectra were centered at 401 and 169 eV, respectively (Fig. 2B and D). This clearly evidences the presence of AIE dye in PHE–DAS. Furthermore, the overall percentages of C, N, O, and S were calculated based on XPS measurements. We found that the percentages of C, N, O, and S were 57.28%, 1.1%, 38.45%, and 3.16%, respectively (Table S1†). As compared with the theoretical value of N (1.1%) in PHE, the weight percentage of PHE to DAS should be 1 : 6.3. The calculated value is well consistent with the experimental data for the mass ratio of the synthesized PHE–DAS. Furthermore, we also found that the O content of PHE–DAS (38.45%) was obviously smaller than that of corn starch (52.33%). The decrease of O content in PHE–DAS also implied that PHE had been combined with DAS successfully. Therefore, according to the XPS analysis, we could conclude that the PHE–DAS copolymers had been formed via Schiff base formation.

Fig. 2 C 1s, N 1s, O 1s, and S 2p XPS spectra of PHE–DAS.

3.2 Optical properties of PHE–DAS

UV-visible and PL spectra were examined to characterize the optical properties of as-prepared copolymers. The UV absorption spectrum of PHE–DAS dispersed in water is shown in Fig. 3A, which indicated that the maximum absorption peak was located at 213 nm. Two other absorption peaks at 272 and 420 nm were also found in the UV-visible spectrum of PHE–DAS. These peaks could be attributed to the π → π* transition of aromatic rings. The UV-visible spectrum also implied that the number of conjugated double bonds was greater than 8 and that heteroatoms (N, S and O) had conjugated with these aromatic rings. The absorption spectrum could indicate the successful conjugation of PHE and DAS, due to the absence of conjugation structure in starch. The formation of a uniform water dispersion could also be adduced from the optical image in the inset of Fig. 3A. Meanwhile, intense orange fluorescence in water could be observed after the PHE–DAS water suspension had been irradiated by a UV lamp (λ = 365 nm) (inset of Fig. 3B). Consistent with the optical image, FL spectra of PHE–DAS showed that the maximum emission wavelength of PHE–DAS was located at 570 nm, while the optimal excitation wavelength was located at 467 nm and the excitation wavelength region was at 305–550 nm. The broad excitation wavelength and large Stokes shift could be beneficial for bioimaging applications. It is well known that phenothiazine is a type of AIE-active organic dye, which will emit more effectively in the aggregated or solid state. In the present work, the amphiphilic properties of PHE–DAS copolymers resulted in the self-assembly of copolymers. The hydrophobic organic dye (PHE) was encapsulated in the core and the hydrophilic starch derivative (DAS) covered the hydrophobic core. Therefore, the final PHE–DAS showed intense emission and good water dispersibility. All of these properties imply that PHE–DAS has great potential for biomedical applications.

Fig. 3 (A) UV-visible spectrum of PHE–DAS; inset is a visible image of such an AIE dye in water. (B) Fluorescence excitation (Ex) and emission (Em) spectra of PHE–DAS.

3.3 Biocompatibility of PHE–DAS

Evaluation of the biocompatibility of PHE–DAS is important in terms of potential biomedical applications. The effect of PHE–DAS LPMs on the cell viability of A549 cells was investigated by MTS assay. A549 cells were treated with a range of different concentrations of PHE–DAS for 8 and 24 h, and the samples without any treatment were defined as control samples. As indicated in Fig. 4, no cell viability decrease was observed when cells were incubated with 10–100 μg mL⁻¹ of PHE–DAS. At a concentration of 100 μg mL⁻¹, the cell viability value was still greater than 90%; and even when the incubation time was 24 h, no obvious cell viability changes were found. These results implied that PHE–DAS showed a high potential for biomedical applications. It is worth noting that the cell viability values of PHE–DAS appeared to be even greater than 100% after cells had been incubated with PHE–DAS for 8 and 24 h. A possible reason is that PHE–DAS was internalized by A549 cells. The internalized luminescent copolymers can confuse the determination of cell viability. On the other hand, when the incubation time was further increased to 36 and 48 h, the cell viability values were still greater than 90% (Fig. S5†). All of the above results demonstrated that PHE–DAS LPMs possess excellent biocompatibility and are promising for biomedical applications.

Fig. 4 Cell viability after incubation for 8 h and 24 h with different concentration of PHE–DAS (10–100 μg mL⁻¹).

The PHE–DAS LPMs were incubated with A549 cells in order to examine their bioimaging capability using CLSM imaging. As shown in Fig. 5, strong orange luminescence was observed inside the cells, indicating that the PHE–DAS LPMs had been internalized by A549 cells after the cells had been incubated with 40 μg mL⁻¹ of PHE–DAS for 3 h. The central regions of the cells, corresponding largely to the cell nucleus, showed relatively low fluorescent signals. Furthermore, the cells strongly adhered to the cell plates after being incubated with PHE–DAS LPMs (Fig. 5B). This also suggested that PHE–DAS LPMs possess good biocompatibility. Finally, the merged image of Fig. 5A and B is shown in Fig. 5C. It can be seen that the locations of the fluorescent signal and of the cells are largely overlapping, evidencing that PHE–DAS LPMs were truly internalized by cells and mainly distributed in the cytoplasm. The CLSM images clearly confirmed that PHE–DAS LPMs have potential biomedical applications. Taken together, we have developed a rather facile and novel method to fabricate AIE active starch through Schiff base formation. The resultant PHE–DAS LPMs contained a large number of hydroxyl and carboxyl groups on their surfaces, which made them highly dispersible in aqueous solution and with the potential for further conjugation reactions that could be used to endow the PHE–DAS LPMs with additional functions. Furthermore, the PHE–DAS LPMs showed desirable optical properties and good biocompatibility, making them suitable candidates for biological imaging applications. Combined with the advantages of starch and PHE, we believe the PHE–DAS LPMs should be of great potential for various biomedical applications.

Fig. 5 CLSM images of PHE–DAS LPMs with A549 cells. (A) Fluorescent image. (B) Bright field image. (C) Merged image of (A) and (B). The concentration of PHE–DAS LPMs is 40 μg mL⁻¹. The excitation wavelength of laser is 458 nm.

4. Conclusions

In summary, we report the preparation of amphiphilic copolymers named PHE–DAS by using a facile approach in aqueous solution. The diameter of the resultant AIE-active nanoparticles based on TEM images was in the range of 200–500 nm with uniform size. Because of hydrophilic carboxyl groups covering the surface, the PHE–DAS LPMs showed excellent dispersibility in physiological solution. Meanwhile, the PHE–DAS LPMs showed good fluorescent features, including broad excitation region, strong and long-wavelength excitation (with increasing intensity up to 467 nm) and intense yellow emission (570 nm). In addition, biocompatibility evaluation and cell imaging results suggested that the PHE–DAS LPMs were biocompatible enough for bioimaging applications. Because of their numerous excellent properties, PHE–DAS are expected to have a high potential for various biomedical applications.

Acknowledgements

This research was supported by the National Science Foundation of China (no. 51363016, 21474057, 21564006, 21561022), and the National 973 Project (no. 2011CB935700).

Notes and references

1. H. Xu, Q. Li, L. Wang, Y. He, J. Shi, B. Tang and C. Fan, Chem. Soc. Rev., 2014, 43, 2650–2661.
2. V. Wagner, A. Dullaart, A.-K. Bock and A. Zweck, Nat. Biotechnol., 2006, 24, 1211–1217.
3. J. Mei, N. L. Leung, R. T. Kwok, J. W. Lam and B. Z. Tang, Chem. Rev., 2015, 115, 11718–11940.
4. M. Chen and M. Yin, Prog. Polym. Sci., 2014, 39, 365–395.
5. S. A. Papadimitriou, Y. Salinas and M. Resmini, Chem.–Eur. J., 2016, 22, 3612–3620.
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. X. Zheng, M. Liu, J. Hui, D. Fan, H. Ma, X. Zhang, Y. Wang and Y. Wei, Phys. Chem. Chem. Phys., 2015, 17, 20301–20307.
8. X. Zhang, K. Wang, M. Liu, X. Zhang, L. Tao, Y. Chen and Y. Wei, Nanoscale, 2015, 7, 11486–11508.
9. X. Zhang, S. Wang, L. Xu, Y. Ji, L. Feng, L. Tao, S. Li and Y. Wei, Nanoscale, 2012, 4, 5581–5584.
10. J. Hui, X. Zhang, Z. Zhang, S. Wang, L. Tao, Y. Wei and X. Wang, Nanoscale, 2012, 4, 6967–6970.
11. 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.
12. X. Zhang, J. Hui, B. Yang, Y. Yang, D. Fan, M. Liu, L. Tao and Y. Wei, Polym. Chem., 2013, 4, 4120–4125.
13. X. Zhang, X. Zhang, B. Yang, J. Hui, M. Liu, W. Liu, Y. Chen and Y. Wei, Polym. Chem., 2014, 5, 689–693.
14. X. Zhang, X. Zhang, B. Yang, M. Liu, W. Liu, Y. Chen and Y. Wei, Polym. Chem., 2014, 5, 356–360.
15. X. Zhang, X. Zhang, B. Yang, M. Liu, W. Liu, Y. Chen and Y. Wei, Polym. Chem., 2014, 5, 399–404.
16. M. Liu, J. Ji, X. Zhang, X. Zhang, B. Yang, F. Deng, Z. Li, K. Wang, Y. Yang and Y. Wei, J. Mater. Chem. B, 2015, 3, 3476–3482.
17. X. Wang, H. Liu, J. Li, K. Ding, Z. Lv, Y. Yang, H. Chen and X. Li, Chem.–Asian J., 2014, 9, 784–789.
18. X. Zhang, M. Liu, B. Yang, X. Zhang and Y. Wei, Colloids Surf., B, 2013, 112, 81–86.
19. X. Zhang, X. Zhang, B. Yang, M. Liu and Y. Wei, Colloids Surf., B, 2014, 116, 739–744.
20. X. Zhang, X. Zhang, B. Yang, Y. Zhang, M. Liu, W. Liu, Y. Chen and Y. Wei, Colloids Surf., B, 2014, 1, 435–441.
21. Z. Wang, S. Chen, J. W. Lam, W. Qin, R. T. Kwok, N. Xie, Q. Hu and B. Z. Tang, J. Am. Chem. Soc., 2013, 135, 8238–8245.
22. J. Luo, Z. Xie, J. W. Lam, L. Cheng, H. Chen, C. Qiu, H. S. Kwok, X. Zhan, Y. Liu and D. Zhu, Chem. Commun., 2001, 1740–1741.
23. K. Wang, X. Zhang, X. Zhang, B. Yang, Z. Li, Q. Zhang, Z. Huang and Y. Wei, Macromol. Chem. Phys., 2015, 216, 678–684.
24. X. Zhang, X. Zhang, B. Yang, L. Liu, F. Deng, J. Hui, M. Liu, Y. Chen and Y. Wei, RSC Adv., 2014, 4, 24189–24193.
25. A. Shao, Y. Xie, S. Zhu, Z. Guo, S. Zhu, J. Guo, P. Shi, T. D. James, H. Tian and W.-H. Zhu, Angew. Chem., Int. Ed., 2015, 127, 7383–7388.
26. W. Guan, W. Zhou, C. Lu and B. Z. Tang, Angew. Chem., Int. Ed., 2015, 54, 15160–15164.
27. X. Zhang, X. Zhang, S. Wang, M. Liu, Y. Zhang, L. Tao and Y. Wei, ACS Appl. Mater. Interfaces, 2013, 5, 1943–1947.
28. H. Shi, R. T. Kwok, J. Liu, B. Xing, B. Z. Tang and B. Liu, J. Am. Chem. Soc., 2012, 134, 17972–17981.
29. H. Shi, J. Liu, J. Geng, B. Z. Tang and B. Liu, J. Am. Chem. Soc., 2012, 134, 9569–9572.
30. Y. Zhang, K. Chang, B. Xu, J. Chen, L. Yan, S. Ma, C. Wu and W. Tian, RSC Adv., 2015, 5, 36837–36844.
31. M. Faisal, Y. Hong, J. Liu, Y. Yu, J. W. Lam, A. Qin, P. Lu and B. Z. Tang, Chem.–Eur. J., 2010, 16, 4266–4272.
32. Y. Liu, A. Qin, X. Chen, X. Y. Shen, L. Tong, R. Hu, J. Z. Sun and B. Z. Tang, Chem.–Eur. J., 2011, 17, 14736–14740.
33. E. Pusztai, S. Jang, I. S. Toulokhonova, I. A. Guzei, R. West, R. Hu and B. Z. Tang, Chem.–Eur. J., 2013, 19, 8742–8745.
34. Z. Fan, D. Li, X. Yu, Y. Zhang, Y. Cai, J. Jin and J. Yu, Chem.–Eur. J., 2016, 22, 3681–3685.
35. 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.
36. J. Geng, K. Li, W. Qin, L. Ma, G. G. Gurzadyan, B. Z. Tang and B. Liu, Small, 2013, 9, 2012–2019.
37. X. Zhang, X. Zhang, B. Yang, J. Hui, M. Liu, Z. Chi, S. Liu, J. Xu and Y. Wei, Polym. Chem., 2014, 5, 683–688.
38. X. Zhang, X. Zhang, L. Tao, Z. Chi, J. Xu and Y. Wei, J. Mater. Chem. B, 2014, 2, 4398–4414.
39. S. Kim, H. E. Pudavar, A. Bonoiu and P. N. Prasad, Adv. Mater., 2007, 19, 3791–3795.
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, S. Wang, M. Liu, Y. Zhang and L. Tao, RSC Adv., 2013, 3, 9633–9636.
42. F. Mahtab, J. W. Lam, Y. Yu, J. Liu, W. Yuan, P. Lu and B. Z. Tang, Small, 2011, 7, 1448–1455.
43. X. Zhang, X. Zhang, L. tao, Z. Chi, J. Xu and Y. Wei, J. Mater. Chem. B, 2014, 2, 4398–4414.
44. X. Zhang, X. Zhang, B. Yang, L. Liu, F. Deng, J. Hui, M. Liu, Y. Chen and Y. Wei, RSC Adv., 2014, 4, 24189–24193.
45. X. Zhang, X. Zhang, B. Yang, L. Liu, J. Hui, M. Liu, Y. Chen and Y. Wei, RSC Adv., 2014, 4, 10060–10066.
46. X. Zhang, X. Zhang, B. Yang, J. Hui, M. Liu, Z. Chi, S. Liu, J. Xu and Y. Wei, J. Mater. Chem. C, 2014, 2, 816–820.
47. X. Zhang, M. Liu, B. Yang, X. Zhang and Y. Wei, Colloids Surf., B, 2013, 112, 81–86.
48. Q. Wan, C. He, K. Wang, M. Liu, H. Huang, Q. Huang, F. Deng, X. Zhang and Y. Wei, Tetrahedron, 2015, 71, 8791–8797.
49. K. Petersen, P. V. Nielsen, G. Bertelsen, M. Lawther, M. B. Olsen, N. H. Nilsson and G. Mortensen, Trends Food Sci. Technol., 1999, 10, 52–68.
50. X. Zhang, X. Zhang, B. Yang, M. Liu, W. Liu, Y. Chen and Y. Wei, Polym. Chem., 2013, 4, 4317–4321.
51. B. Yang, Y. Zhang, X. Zhang, L. Tao, S. Li and Y. Wei, Polym. Chem., 2012, 3, 3235–3238.
52. Y. Xin and J. Yuan, Polym. Chem., 2012, 3, 3045–3055.
53. X. Zhang, X. Zhang, B. Yang, M. Liu, W. Liu, Y. Chen and Y. Wei, Polym. Chem., 2014, 5, 399–404.
54. H. Li, X. Zhang, Z. Chi, B. Xu, W. Zhou, S. Liu, Y. Zhang and J. Xu, Org. Lett., 2011, 13, 556–559.
55. X. Zhang, W. Hu, J. Li, L. Tao and Y. Wei, Toxicol. Res., 2012, 1, 62–68.
56. X. Zhang, M. Liu, X. Zhang, F. Deng, C. Zhou, J. Hui, W. Liu and Y. Wei, Toxicol. Res., 2015, 4, 160–168.
57. X. Zhang, H. Qi, S. Wang, L. Feng, Y. Ji, L. Tao, S. Li and Y. Wei, Toxicol. Res., 2012, 1, 201–205.
58. X. Zhang, S. Wang, M. Liu, J. Hui, B. Yang, L. Tao and Y. Wei, Toxicol. Res., 2013, 2, 335–346.

---

Footnotes

† Electronic supplementary information (ESI) available: The ¹H NMR, TEM and XPS spectra of materials. See DOI: 10.1039/c6ra13714h

‡ These authors contributed equally to this work.

---