Electrical signal guided click coating of chitosan hydrogel on conductive surface

Abstract

We report the coating of a chitosan hydrogel on a conductive surface by the “electro-click” method. Specifically, chitosan is functionalized with either azide or alkyne groups and a cathodic potential is applied to a gold chip which reduces Cu²⁺ to Cu⁺ and thereby catalyzes the in situ Huisgen 1,3-dipolar cycloadditions between alkynylated chitosan and azidated chitosan.

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The coating on an implant surface can enhance tissue healing, improve cell attachment and strengthen the integration of the implant to the bone.^1–3 While there are numerous techniques for implant coating,^4,5 electrodeposition proves to be an easy and reliable method for coating. Great efforts have been made to coat implant surfaces with biopolymers, due to their good biocompatibility, ease of processing and osteoinductivity. Until now, various biopolymers, especially natural polymers, have been adopted for implant coating,⁶ including gelatin,⁷ chitosan² and silk protein.

Chitosan, a product derived from chitin, is an aminopolysaccharide with appealing intrinsic properties (i.e. it is biocompatible, biodegradable, and non-toxic).^8–10 In addition, chitosan is pH responsive and forms films easily. Biological components and antibiotics can be entrapped in the chitosan matrix for controlled release.^11–13 Thus, chitosan is regarded as a bioactive material to generate a multifunctional coating on implants. Traditional methods for obtaining a chitosan coating mainly include chemical conjugation and physical bonding. Swanson et al. loaded a chitosan–vancomycin coating on the surface of a titanium alloy rod via a dip-coating process.¹⁴ Norowski et al. covalently bonded chitosan to a titanium surface via silane reactions.¹⁵ Lieder et al. studied the effects of the degree of deacetylation (DD) of chitosan on cell attachment, proliferation and osteogenic differentiation on titanium.¹⁶ In another study, Krastev et al. reported localized delivery of siRNA nanoplexes by hyaluronic acid/chitosan multilayer coatings on neuronal implants.¹⁷ Recently, Boccaccini et al. proposed that the electrophoretic deposition of chitosan is an attractive technique for bioactive coating construction on a metal surface.^18–20

In our previous work, we have shown that chitosan can be electrodeposited on titanium and the release of a drug embedded in a chitosan hydrogel can be controlled by electrical potentials.¹³ The use of electrical potentials suggests a simple and easy way for chitosan coating, which utilizes the pH responsive and film forming properties of chitosan.^21–23 In this study, we demonstrate an in situ generation of a chitosan hydrogel on a conductive surface by the “electro-click” method. Electrochemical click chemistry has been reported as a facile and efficient way for surface patterning and modification,^24–27 film assembly,²⁸ and sensor biofabrication.²⁹ The procedure for this study is shown in Scheme 1. First, chitosan with either alkyne groups or azide groups was synthesized through substitution of amine groups. Second, a gold chip was partially immersed in a solution containing azidated chitosan, alkynylated chitosan and Cu²⁺. Cyclic voltammetry (CV) was performed between 500 and −300 mV to initiate the click reaction between azidated chitosan and alkynylated chitosan, thus generating a conjugate chitosan hydrogel adhered to the gold surface. Finally, the antibiotic vancomycin, was co-deposited by the “electro-click” method and the antibacterial activity of the coated gold chip was evaluated by a disk diffusion method. The results demonstrate new possibilities for implant coating by coupling “electro-click” and chitosan deposition.

Scheme 1 Schematic illustrating the “electro-click” chitosan hydrogel on a gold chip triggered by a negative potential that reduces Cu²⁺ to Cu⁺.

We first functionalized chitosan with either alkyne or azide moieties. Alkynylated chitosan and azidated chitosan are precursors for the “electro-click” chitosan hydrogel. In one experiment, chitosan powder was dispersed in a mixture of NaOH and isopropyl alcohol containing propargyl bromide at 60 °C for 5 h. Then the alkynylated chitosan was filtered out and rinsed extensively by ethanol. The substitution of alkynylated chitosan is increased with an increased amount of propargyl bromide. However, high substitution will make the alkynylated chitosan insoluble in acid. In this study, alkynylated chitosan with a degree of substitution (DS) of 39.7% was used. In another experiment, chitosan was azidated by reacting with sodium azide at 4 °C for 40 min. Azidated chitosan with a DS of 12.3% was obtained by base precipitation and a freeze drying process. The reaction scheme is shown in Scheme 2.

Scheme 2 The reaction schemes for alkynylated chitosan and azidated chitosan.

Next we demonstrate that the click coating of the chitosan hydrogel on a conductive surface can be triggered by electrical signals. For this, a gold-coated silicon chip was chosen as a platform for chitosan coating. Cu²⁺ was added to a solution containing azidated chitosan and alkynylated chitosan to a final concentration of 30 mmol L⁻¹. The gold chip, as the working electrode, was partially immersed in the above solution and connected to a power supply with an Ag/AgCl wire used as a reference electrode and a platinum wire used as a counter electrode. The click reaction happened between azidated chitosan and alkynylated chitosan when cyclic voltammetry (CV) was performed in the voltage range of 500 to −300 mV for 10 min, which reduced Cu²⁺ to Cu⁺ adjacent to the gold chip that catalyzed the Huisgen 1,3-dipolar cycloadditions between alkynylated chitosan and azidated chitosan. In order to facilitate the observation of the formation of the chitosan hydrogel on the gold chip, a fluorescent probe (FITC) was added to the above solution to track the click coating process. Fig. 1a shows the fluorescent image of the deposited hydrogel on the gold chip after the CV scan for 10 min. It exhibits a strong fluorescence, suggesting in situ formation of the hydrogel. Without the CV scan, there is no fluorescence on the gold chip, as shown in Fig. 1b. By comparison, “electro-click” was also performed in solo azidated chitosan and alkynylated chitosan solutions, and, as expected, little fluorescence can be seen on the gold chip (Fig. 1c and d). In addition, the click coated chitosan hydrogel was observed by SEM. Fig. 1e and f show the morphologies of the surface and the cross-section, respectively. It should be noted that the hydrogel had a smooth and compact surface without noticeable phase separation. The thickness of the hydrogel is estimated to be 800 nm (Fig. 1f). The results in Fig. 1 indicate that the chitosan hydrogel can be “electro-clicked” on a conductive surface.

Fig. 1 The fluorescent images (a–d) and SEM images for the surface (e) and cross-section (f) of the click coated chitosan hydrogel on the gold chip. FITC was co-deposited during the “electro click” process, showing obvious fluorescence (a) and the control experiments without CV scan (b) and “electro click” in solo azidated chitosan (c) and alkynylated chitosan solution (d) showing little fluorescence.

The click coated chitosan film was further characterized by FT-IR and XPS. Fig. 2a shows the FT-IR spectrum of the “electro-clicked” chitosan hydrogel compared to chitosan, alkynylated chitosan and azidated chitosan. Compared to the spectrum of the initial chitosan, a new peak appears at 2100 cm⁻¹ in alkynylated chitosan, which is attributed to the alkyne groups.³⁰ For azidated chitosan, the absorption band at 2050 cm⁻¹ can be attributed to the azide moieties.^30,31 While for the click coated chitosan hydrogel, the peaks at 2100 cm⁻¹ and 2050 cm⁻¹ disappear, which indicates that the azide groups and alkyne groups react through the click reaction. We should note that the characteristic bands (1090 cm⁻¹ for hydroxyl groups, 1155 cm⁻¹ for pyranose rings and 1650 cm⁻¹ for acetyl amino groups) for chitosan remained through the click coating procedure, which suggests that the “electro-click” is a mild process for biopolymer coating.

Fig. 2 FT-IR (a) and XPS (b) spectra of the click coated chitosan hydrogel on the gold chip (the colored curves are fitted peaks for N 1s).

To obtain further chemical information, we performed XPS measurements. Fig. 2b shows the narrow scanned N 1s spectrum. Because of the presence of amine groups on chitosan, the spectrum shows two peaks for –NH₂ and –NH₃⁺ at 399.4 eV and 401.4 eV, respectively. In addition, there are two peaks at 400.5 eV and 402.0 eV with an intensity ratio of 2 : 1, which correspond to N atoms on the triazole ring. The presence of the peaks is in agreement with reported spectra for triazole moieties from click reactions.^32,33

The “electro-clicked” hydrogel can be co-deposited with other bioactive components. We demonstrate that the antibiotic vancomycin can be entrapped in the chitosan hydrogel during the “electro-click” process. The antimicrobial activities of click coated chitosan hydrogels with and without vancomycin were assessed using the disk diffusion method. Fig. 3 shows the inhibition zones for E. coli (Fig. 3, left image) and S. aureus (Fig. 3, right image). We can observe large inhibition zones around the vancomycin containing chips for both S. aureus and E. coli, and the average inhibition zone for S. aureus is 2.5 ± 0.5 cm and for E. coli is 2.5 ± 0.1 cm. By contrast, small inhibition zones can be seen for the chitosan hydrogel. The results demonstrate that “electro-click” can co-deposit and generate a bioactive coating on a conductive surface.

Fig. 3 Antimicrobial activities of the chitosan hydrogel on the gold chip against E. coli (left image) and S. aureus (right image). Gold chips with a click coated chitosan hydrogel containing vancomycin (a and c) and without vancomycin (b and d) are pasted on the agar plate.

Conclusions

In this work, we have explored the capability of “electro-click” to generate a chitosan coating on a conductive surface. The initiation of the click reaction was triggered by an on site generation of active copper(I) at the surface of a gold chip. The 1,3-dipolar cycloaddition reaction between alkynylated and azidated chitosan led to the formation of a chitosan hydrogel on the gold surface. The generation of the chitosan hydrogel by “electro-click” provides new possibilities to coat a conductive surface and will certainly expand the application of the chitosan hydrogel in implant coating.

Notes and references

1. G. Helary, F. Noirclere, J. Mayingi and V. Migonney, Acta Biomater., 2009, 5, 124–133.
2. S. Y. Seo, S. H. Park, H. J. Lee, Y. Heo, H. N. Na, K. I. Kim, J. H. Han, Y. Ito and T. I. Son, J. Appl. Polym. Sci., 2013, 128, 4322–4326.
3. N. J. Shah, M. N. Hyder, J. S. Moskowitz, M. A. Quadir, S. W. Morton, H. J. Seeherman, R. F. Padera, M. Spector and P. T. Hammond, Sci. Transl. Med., 2013, 5, 191ra83.
4. A. Abdal-hay, M. G. Hwang and J. K. Lim, J. Sol-Gel Sci. Technol., 2012, 64, 756–764.
5. T. A. Petrie, J. E. Raynor, C. D. Reyes, K. L. Burns, D. M. Collard and A. J. Garcia, Biomaterials, 2008, 29, 2849–2857.
6. E. Vanderleyden, S. Mullens, J. Luyten and P. Dubruel, Curr. Pharm. Des., 2012, 18, 2576–2590.
7. Z.-M. Huang, Y.-Y. Qi, S.-H. Du, G. Feng, H. Unuma and W.-Q. Yan, Sci. Technol. Adv. Mater., 2013, 14, 055001.
8. S. Senel and S. J. McClure, Adv. Drug Delivery Rev., 2004, 56, 1467–1480.
9. S. Hirano, Polym. Int., 1999, 48, 732–734.
10. H. M. Yi, L. Q. Wu, W. E. Bentley, R. Ghodssi, G. W. Rubloff, J. N. Culver and G. F. Payne, Biomacromolecules, 2005, 6, 2881–2894.
11. Q. Wang, N. Zhang, X. W. Hu, J. H. Yang and Y. M. Du, J. Biomed. Mater. Res., Part A, 2008, 85A, 881–887.
12. Q. Wang, Z. F. Dong, Y. M. Du and J. F. Kennedy, Carbohydr. Polym., 2007, 69, 336–343.
13. X. W. Shi, H. P. Wu, Y. Y. Li, X. Q. Wei and Y. M. Du, J. Biomed. Mater. Res., Part A, 2013, 101A, 1373–1378.
14. T. E. Swanson, X. Cheng and C. Friedrich, J. Biomed. Mater. Res., Part A, 2011, 97A, 167–176.
15. P. A. Norowski, H. S. Courtney, J. Babu, W. O. Haggard and J. D. Bumgardner, Implant Dent., 2011, 20, 56–67.
16. R. Lieder, M. Darai, M. B. Thor, C. H. Ng, J. M. Einarsson, S. Gudmundsson, B. Helgason, V. S. Gaware, M. Masson, J. Gislason, G. Orlygsson and O. E. Sigurjonsson, J. Biomed. Mater. Res., Part A, 2012, 100A, 3392–3399.
17. H. Hartmann, S. Hossfeld, B. Schlosshauer, U. Mittnacht, A. P. Pego, M. Dauner, M. Doser, D. Stoll and R. Krastev, J. Controlled Release, 2013, 168, 289–297.
18. A. R. Boccaccini, S. Keim, R. Ma, Y. Li and I. Zhitomirsky, J. R. Soc. Interface, 2010, 7, S581–S613.
19. L. Cordero-Arias, S. Cabanas-Polo, H. X. Gao, J. Gilabert, E. Sanchez, J. A. Roether, D. W. Schubert, S. Virtanen and A. R. Boccaccini, RSC Adv., 2013, 3, 11247–11254.
20. F. Gebhardt, S. Seuss, M. C. Turhan, H. Hornberger, S. Virtanen and A. R. Boccaccini, Mater. Lett., 2012, 66, 302–304.
21. X. Q. Wei, G. F. Payne, X. W. Shi and Y. M. Du, Soft Matter, 2013, 9, 2131–2135.
22. Y. Cheng, K. M. Gray, L. David, I. Royaud, G. F. Payne and G. W. Rubloff, Mater. Lett., 2012, 87, 97–100.
23. G. F. Payne, E. Kim, Y. Cheng, H. C. Wu, R. Ghodssi, G. W. Rubloff, S. R. Raghavan, J. N. Culver and W. E. Bentley, Soft Matter, 2013, 9, 6019–6032.
24. T. S. Hansen, A. E. Daugaard, S. Hvilsted and N. B. Larsen, Adv. Mater., 2009, 21, 4483–4486.
25. N. K. Devaraj, P. H. Dinolfo, C. E. D. Chidsey and J. P. Collman, J. Am. Chem. Soc., 2006, 128, 1794–1795.
26. X.-W. Shi, L. Qiu, Z. Nie, L. Xiao, G. F. Payne and Y. Du, Biofabrication, 2013, 5, 041001.
27. N. Shida, Y. Ishiguro, M. Atobe, T. Fuchigami and S. Inagi, ACS Macro Lett., 2012, 1, 656–659.
28. G. Rydzek, L. Jierry, A. Parat, J. S. Thomann, J. C. Voegel, B. Senger, J. Hemmerle, A. Ponche, B. Frisch, P. Schaaf and F. Boulmedais, Angew. Chem., Int. Ed., 2011, 50, 4374–4377.
29. S. J. P. Canete and R. Y. Lai, Chem. Commun., 2010, 46, 3941–3943.
30. H. Liao, R. Qi, M. Shen, X. Cao, R. Guo, Y. Zhang and X. Shi, Colloids Surf., B, 2011, 84, 528–535.
31. R. Kulbokaite, G. Ciuta, M. Netopilik and R. Makuska, React. Funct. Polym., 2009, 69, 771–778.
32. B. Guan, S. Ciampi, G. Le Saux, K. Gaus, P. J. Reece and J. J. Gooding, Langmuir, 2011, 27, 328–334.
33. F. Bebensee, C. Bombis, S. R. Vadapoo, J. R. Cramer, F. Besenbacher, K. V. Gothelf and T. R. Linderoth, J. Am. Chem. Soc., 2013, 135, 2136–2139.

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