Copyright 2008 American Chemical Society
AUGUST 05, 2008 VOLUME 24, NUMBER 15
Letters PEGylation of Porous Silicon Using Click Chemistry Leanne Britcher, Timothy J. Barnes,* Hans J. Griesser, and Clive A. Prestidge Ian Wark Research Institute, Special Research Centre for Particle and Material Interfaces, UniVersity of South Australia, Mawson Lakes BlVd, Mawson Lakes, South Australia, Australia 5095 ReceiVed May 26, 2008. ReVised Manuscript ReceiVed June 17, 2008 Porous silicon has received considerable interest in recent years in a range of biomedical applications, with its performance determined by surface chemistry. In this work, we investigate the PEGylation of porous silicon wafers using click chemistry. The porous silicon wafer surface chemistry was monitored at each stage of the reaction via photoacoustic Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, whereas sessile drop contact angle and model protein adsorption measurements were used to characterize the final PEGylated surface. This work highlights the simplicity of click-chemistry-based functionalization in tailoring the porous silicon surface chemistry and controlling protein-porous silicon interactions.
Porous silicon (pSi) has received considerable attention over recent years for use in a range of applications, including biosensing,1,2 oncology,3 and drug delivery.4–7 Recent interest in pSi has focused on its use as a delivery vehicle for therapeutic agents such as ibuprofen6 and insulin.7 Central to the use of pSi as an effective drug delivery system is the need to find balance between passivation of the highly reactive native Si-Hx surface species to minimize damage to the loaded molecule and controlling the release of the drug by erosion of the pSi matrix. * Corresponding author. Tel: +61 8 8302 5146. Fax: +61 8 8302 3683. E-mail:
[email protected]. (1) Bessueille, F.; Dugas, V.; Vikulov, V.; Cloarec, J. P.; Soutey, E.; Martin, J. R. Biosens. Bioelectron. 2005, 21, 908–916. (2) Kilian, K. A.; Bo¨cking, T. K.; King-Lacroix, G. J.; Gal, M.; Gooding, J. J. Chem. Commun. 2007, 19, 1936–1938. (3) Goh, A. S.-W.; Chung, A. Y.-F.; Lo, R. H.-G.; Lau, T.-N.; Yu, S. W.-K.; Cheng, M.; Satchithanantham, S.; Loong, S. L.-E.; Ng, D. C.-E.; Lim, B.-C.; Connor, S.; Chow, P. K.-H. Int. J. Radiat. Oncol. 2007, 67, 786–792. (4) Prestidge, C. A.; Barnes, T. J.; Lau, C.-H.; Barnett, C.; Loni, A.; Canham, L. Expert Opin. Drug DeliVery 2007, 4, 101–110. (5) Prestidge, C. A.; Barnes, T. J.; Mierczynska-Vasilev, A.; Skinner, W.; Peddie, F.; Barnett, C. Phys. Status Solidi A 2007, 204, 3361–3366. (6) Salonen, J.; Laitinen, L.; Kaukonen, A. M.; Tuura, J.; Bjorkqvist, M.; Heikkila, T.; Vaha-Heikkila, K.; Hirvonen, J.; Lehto, V. P. J. Controlled Release 2005, 108, 362–374. (7) Foraker, A. B.; Walczak, R. J.; Cohen, M. H.; Boiarski, T. A.; Grove, C. F.; Swaan, P. W. Pharm. Res. 2003, 20, 110–116.
To date, pSi surface modification has been accomplished via methods such as thermal oxidation,1,8 silane coupling,1,9 and the formation of Si-C bonds for which a range of chemistry has been proposed in the literature.10,11 Similarly, the functionalization of surfaces with poly(ethylene glycol) (PEG) to minimize nonspecific protein binding12 is well established, with numerous methodologies available for the PEGylation of pSi, including the silylation of thermally oxidized pSi via N-(triethoxysilylpropyl)-O-polyethylene glycol urethane (in toluene)9 or the thermal hydrosilation of pSi (undecylenic acid), followed by reaction with amino-tetra(ethylene glycol)-t-butyl ester in methylene chloride and N,N′-dicyclohexylcarboiimide.11 In this work, we report on the two-step PEGylation of pSi wafers based on click chemistry13,14 (as depicted schematically (8) Jarvis, K. L.; Barnes, T. J.; Badalyan, A.; Pendleton, P.; Prestidge, C. A. J. Phys. Chem. C, published online June 11, http://dx.doi.org/10.1021/jp800950j. (9) Low, S. P.; Williams, K. A.; Canham, L. T.; Voelcker, N. H. Biomaterials 2006, 27, 4538–4546. (10) Xia, B.; Xiao, S.-J.; Wang, J.; Guo, D.-J. Thin Solid Films 2005, 474, 306–309. (11) Schwartz, M. P.; Cunin, F.; Cheung, R. W.; Sailor, M. J. Phys. Status Solidi A 2005, 202, 1380–1384. (12) Kingshott, P.; Thissen, H.; Griesser, H. J. Biomaterials 2002, 23, 2043– 2056. (13) Evans, R. A. Aust. J. Chem. 2007, 60, 384–395.
10.1021/la801619v CCC: $40.75 2008 American Chemical Society Published on Web 07/01/2008
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Scheme 1. Click Chemistry Reaction Scheme for the PEGylation of pSi
Figure 1. High-resolution C 1s XPS spectra of (a) pSi, (b) pSi after the hydrosilation of 1,6-heptadiyne, and (c) pSi grafted with PEG.
in Scheme 1), which has previously been utilized to PEGylate both silicon and silica substrates.14–18 However, to date this methodology has not been applied to the PEGylation of pSi substrates. The pSi wafer (p+ silicon, 70% porosity, 30 µm porous layer, 10 nm average pore diameter, specific surface area ∼350 m2 g-1) was used as supplied (pSiMedica, Malvern, U.K.), and all chemicals were used as supplied without further purification. Initially, the pSi wafer was functionalized with acetylene groups via hydrosilation with 1,6-heptadiyne at room temperature under room-light illumination for 18 h as outlined by Meng et al.16 The acetylenylated pSi wafer was rinsed several times with anhydrous toluene and ethanol and then dried in a stream of nitrogen. The wafer was placed in a reaction vessel and then aqueous solutions of PEG-azide (0.4 mM, 1 mL) copper(II) sulfate pentahydrate (70 µM, 0.5 mL), and ascorbic acid (0.1 mM, 0.5 mL) were added, and the reaction was allowed to proceed for either 4 or 18 h. The wafers were washed for 16 h in 0.05 wt % ethylenediacetic acid (EDTA) at pH 8, followed by high-purity water and drying in a stream of nitrogen. The two-step modification procedure was monitored by X-ray photoelectron spectroscopy (XPS). After the hydrosilation of pSi with the heptadiyne, the C-H/Si atomic ratio increased from 0.14 to 0.33, indicating successful acetylenylation. Deconvolution of the high-resolution C 1s multiplex spectrum indicated the presence of three components at 284.1 eV (Si-C), 285.0 eV (C-H, CdC, or CtC) and 286.5 eV (C-O) (Figure 1). The C-O component (17% of the total carbon) is thought to occur from the reaction of ethanol with unreacted SiH groups. Upon reaction of the PEG-azide with the acetenylated pSi surface, an increase in the proportion of C-O/C-N species (286.5 eV) was observed in the high-resolution XPS C 1s spectrum (Figure 1), indicating the grafting of the PEG to the surface. The (14) Such, G. K.; Quinn, J. F.; Quinn, A.; Tjipto, E.; Caruso, F. J. Am. Chem. Soc. 2006, 128, 9318–9319. (15) Prakash, S.; Long, T. M.; Selby, J. C.; Moore, J. S.; Shannon, M. A. Anal. Chem. 2007, 79, 1661–1667. (16) Meng, J.; Averbuj, C.; Lewis, W. G.; Siuzdak, G.; Finn, M. G. Angew. Chem., Int. Ed. 2004, 43, 1255–1260. (17) Ciampi, S.; Bocking, T.; Kilian, K.; James, M.; Harper, J. B.; Gooding, J. Langmuir 2007, 23, 9320–9329. (18) Ostaci, R. V.; Damiron, D.; Capponi, S.; Vignaud, G.; Leger, L.; Grohens, Y.; Drockenmuller, E. Langmuir 2008, 24, 2732–2739.
Figure 2. High-resolution Si 2p XPS spectra of (a) pSi, (b) pSi after the hydrosilation of 1,6-heptadiyne, and (c) pSi grafted with PEG.
C-N component from the triazole was not resolved in the spectra because its contribution would be very small given that the C-O/ C-N ratio in the PEG-azide was 87/1. Triazole formation was also confirmed by the N 1s peak located at 400 eV14 and the absence of unreacted physisorbed species azide at 404.2 eV. The C-H component was also evident throughout the reaction, indicating that the carbon layer was not removed during reaction and subsequent EDTA wash. There did appear to be an inflection in the C 1s spectra above 286.5 eV, indicating low levels of CdO or O-CdO carbon species on the surface of the pSi (Figure 1). Only a small amount (2% of the total silicon) of Si-O-Si species were observed at 101-104 eV in the high-resolution Si 2p spectrum, indicating significant passivation of the pSi surface (Figure 2). However, our results have shown that the coupling reaction still occurred even if the pSi was not fully passivated. For drug release properties, a fully passivated layer is not desired because the oxidation of pSi is required to release the active product. Copper cations are known to react with SiH groups, forming a copper-plated surface.19 However, from the XPS survey,20 we observe no evidence of residual copper, indicating that the EDTA wash has removed the copper from the surface. (19) Hamm, D.; Sakka, T.; Ogata, Y. H. Electrochim. Acta 2004, 49, 4949– 4955. (20) See Supporting Information.
Letters
The heptyne layer would also provide a barrier to reaction of the copper ions with unreacted Si-H groups. Complimentary PA-FTIR spectra21 of the pSi wafer initially, after hydrosilation and PEG grafting, with characteristic Si-Hx and Oy-Si-Hx stretching modes observed at ∼2100 and 2200 cm-1, respectively, indicate that the PEG coating did not completely cover the internal pSi surface. The PA-FTIR experiment obtained data to a depth of ∼22 µm, in contrast to the XPS measurements that probe approximately four to five atomic layers of the pSi surface. This suggests that functionalization did not occur throughout the entire 30 µm porous layer. Further work with XPS depth profiling will be carried out to determine the depth and extent of functionalization. The PEGylated pSi’s resistance to protein adsorption was determined by the immersion of a PEGylated pSi wafer sample in a solution of 0.5 mg mL-1 human serum albumin (HSA) and phosphate-buffered saline (0.15 M) for 1 h; HSA has previously been shown to exhibit high affinity adsorption on porous silicon.22 The pSi wafer was then removed from the HSA solution and thoroughly rinsed with copious fresh PBS before finally being dried in a desiccator prior to the determination of the nitrogen atomic concentration via XPS. The initial nitrogen concentration of the PEGylated pSi was 0.3%, which increased to ∼1.7% after exposure to HSA. In contrast, after the exposure of bare pSi to HSA, a nitrogen concentration of 6.6% (equivalent to approximate monolayer surface coverage) was observed. The adsorption of HSA onto pSi will be discussed in more detail in a future paper; however, the hydrodynamic diameter of HSA (∼6 nm) was less than the pore diameter of pSi (∼10 nm), hence this did not impact its adsorption into the pores. Specific tailoring of PEG (21) See Supporting Information. (22) Tay, L.; Rowell, N. L.; Lockwood, D. J.; Boukherroub, R. J. Vac. Sci. Technol., A 2006, 24, 747–751.
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grafting density could be used to minimize protein denaturation while maintaining pSi biocompatibility. Finally, complimentary sessile drop static contact angle measurements of the pSi wafer before and after PEGylation have confirmed the presence of a stable grafted hydrophilic PEG layer, with a decrease in static θ from ∼115° for the bare pSi wafer to ∼3° for the PEGylated wafer, which is significantly lower than previously observed (∼62°) for the PEGylation of a silicon wafer.18 In contrast, upon oxidation of the pSi wafer at 800 °C, θ ≈ 20° was typically observed, highlighting the significant increase in the wettability of the PEGylated pSi wafer. In summary, pSi has been successfully modified with a PEG layer in a two-step process using the click chemistry approach. Partial passivation of the porous layer was achieved by the hydrosilation reaction, and the final PEG layer was stable, even after extensive EDTA washing. The presence of the PEG layer may assist in protecting entrapped protein molecules from denaturation, making the functionalized pSi useful for targeted protein delivery applications. Acknowledgment. We thank Karyn Jarvis for the contact angle and infrared measurements. pSiMedica Limited (a pSivida group company) is gratefully acknowledged for supplying pSi wafers. Funding from pSivida, an Australian Research Council (ARC) Linkage grant (LP0562379) and the ARC Special Research Centre for Particle and Material Interfaces (S00001491) is also acknowledged. Supporting Information Available: XPS survey spectrum of pSi wafer after PEGylation showing the absence of residual copper and characteristic PA-FTIR spectra of pSi during hydrosilation and PEGylation. This material is available free of charge via the Internet at http://pubs.acs.org. LA801619V
