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A nano-stopper approach to selectively engineer the surfaces of mesoporous silicon Wujun Xu, Jussi Rytkönen, Seppo Rönkkö, Tuomo Nissinen, Tuure Kinnunen, Mika Suvanto, Ale Närvänen, and Vesa-Pekka Lehto Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/cm503004b • Publication Date (Web): 11 Nov 2014 Downloaded from http://pubs.acs.org on November 23, 2014

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Chemistry of Materials

A nano-stopper approach to selectively engineer the surfaces of mesoporous silicon Wujun Xu,1 Jussi Rytkönen,2 Seppo Rönkkö,1 Tuomo Nissinen,1 Tuure Kinnunen,3 Mika Suvanto,4 Ale Närvänen2 and Vesa-Pekka Lehto*,1 1

Department of Applied Physics, University of Eastern Finland, POB 1627, 70211 Kuopio, Finland

2

School of Pharmacy, University of Eastern Finland, 70211 Kuopio, Finland

3

Institute of Clinical Medicine/Clinical Microbiology, University of Eastern Finland, 70210 Kuopio, Finland

4

Department of Chemistry, University of Eastern Finland, 80101 Joensuu, Finland

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ABSTRACT Successful applications of mesoporous materials often require different surface properties of internal pore walls and external surfaces. The different functional moieties on the different surfaces enable them to fulfill multiple application demands. In this study, we introduce a nano-stopper approach to selectively functionalize the different surfaces of porous silicon (PSi). The external surface was functionalized with amine groups to further graft with folic acid (FA) and fluorescein isothiocyanate (FITC) for targeting and imaging, respectively. The pore walls were functionalized with carboxyl groups to obtain higher loading degree of doxorubicin and realize a pH-triggered drug release. The engineered PSi drug carrier showed specific targeting against cancer cells and improved cell internalization due to the FA functionalization. Moreover, the PSi carrier presented an intracellular drug delivery with pH-triggered functionality. With the selective modification, the loading degree of the drug was increased fourfold without any compromise in the toxicity of the plain carrier.


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Table of Contents (TOC) Graphic



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1. INTRODUCTION Surface modifications of mesoporous materials have attracted considerable attention because various useful functionalities can be engineered onto the material to meet the different demands in drug delivery, catalysis, and separation applications.1-4 Surface modifications can be classified into nonselective and selective modification based on the distribution of the functional groups in mesoporous materials. Co-condensation synthesis of organic modified mesoporous silica is a typical example for the former non-selective modification, in which the functional groups are distributed on all of the surfaces of mesoporous materials.5,6 In contrast, in a selective modification, the grafting of functional groups is controlled on either the external surface or the internal pore walls.7 Mesoporous materials with different functional groups on external surface and pore walls can have several profound advantages and consequently expand their applications.8 The functional groups on the external surface are able to change the interactions between mesoporous materials and their application surroundings. For example, PEG molecules on the external surface can improve the biocompatibility of nanocarriers.9 On the other hand, the functional groups on the internal pore walls can have other desirable properties e.g. increasing load capacity of cargos, achieving environmental-triggered cargo release, etc. Until now, different approaches have been used to selectively modify the external surface and internal pore walls of mesoporous materials. With respect to the mesoporous materials synthesized by surfactant template such as mesoporous silica, the mesopores have been filled with surfactant micelles after material preparation. This property induces a ‘‘template-assisted’ method to selectively modify its external surface. The internal pore walls can be further modified with different organic groups after template extraction.10 As an alternative, Chen et al

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reported a diffusion-based deprotection strategy

for differential modification of mesoporous silica. At first, Fmoc-protected organosilanes were modified on the whole surface of the mesoporous materials. Because the deprotection reaction by the 4 ACS Paragon Plus Environment

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cleavage of Fmoc groups was much slower within the pores than that on the external surface, the differential modifications could be achieved by controlling the deprotection time. Another feasible approach for selective modification is to utilize the ‘molecule sieve effect’ of mesoporous materials,7 which determines the spatial selectivity of mesopores and restrict large molecules from gaining access into the mesopores. Organosilanes with long chain length cannot penetrate into the mesopores and thus they selectively modified the external surface, while the small molecule reactants such as 3aminopropyltriethoxysilane (APTES) were attached on the whole surface of mesoporous silica. Growing interest has been focused on the potential applications of mesoporous silicon (PSi) in the field of drug delivery due to its good biocompatibility, high surface area and large pore volume.12 Similar to other mesoporous materials, the surface modification of PSi is an important topic.13-15 However, few strategies have been published in the literatures describing selective modification of PSi. The inherent properties of PSi determine that it is a major challenge to modify PSi selectively. Initially, PSi is generally prepared with the method of chemical etching and thus there are no template micelles in mesopores. The reported method to utilize the property “template-filled” mesopores is not feasible for PSi. Secondly, the pore size of PSi materials was generally around or even larger than 10 nm. It is difficult to selectively modify the surface through ‘molecule sieve effect’ except by using large polymer and protein molecules. Recently, Kilian et al.16 demonstrated a method which relied on the hydrophobicity/hydrophilicity of the PSi Rugate Filters to introduce different surface functionalities. Since the hydrophobicity of the mesopores terminated with Si-H groups prevents the ingress of water, the external surface of PSi was modified with aqueous solutions while leaving the unmodified internal mesopores of the PSi filter. This study is a significant advance but the organic reactants in aqueous solution can be gradually adsorbed into mesopores with the prolonged reaction time due to the intense capillary action of mesopores, resulting in the partial modification of the pore walls. Therefore, the 5 ACS Paragon Plus Environment


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presence of the problems motivated us to develop a more effective and universal method for selective surface engineering of PSi. In this study, a smart strategy of nano-stopper, based on the "like dissolves like" principle, was proposed for the selective modifications of the external surface and internal pore walls of PSi. The nonpolar organic solvent of n-hexane was used as a “stopper” to fill the mesopore and prevent the penetration of reactants while functionalizing the external surface. Through this tailored modifications, the functional groups of -COOH and –NH2 were successfully grafted onto the internal pore surface and external surface of PSi, respectively. The –COOH groups on the pore surface was utilized to enhance the loading degree and control the release of anticancer drug doxorubicin·HCl (DOX). Subsequently, folic acid (FA) and fluorescein isothiocyanate (FITC) were conjugated on the external surface through further functionalization of –NH2 groups, achieving the purposes of active targeting and imaging, respectively. 2. EXPERIMENTAL SECTION 2.1 Materials Silicon wafers were provided by Okmetic. FA, FITC, 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC), H2O2, and APTES were purchased from Sigma-Aldrich. N-hexane and dimethyl sulfoxide (DMSO) was supplied by J.T.Baker. N-hydroxysuccinimide (NHS) was bought from Alfa Aesar. Undecylenic acid was provided by Merck KGaA. DOX was bought from Euroasian Chemicals Pvt Ltd. All chemicals were used as received. 2.2 PSi particles preparation


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PSi films were prepared by anodizing p+ silicon wafer with the resistivity of 0.01-0.02 Ωcm in the mixture of HF (38%)–ethanol (1:1).17 The dried PSi films were ball-milled with the rotation speed of 1000 rpm and then the milled PSi particles were sieved to obtain the desired size diameter below 25 µm. 2.3 Surface modifications of PSi 1). Oxidation of PSi external surface. The incipient-wetness impregnation method

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was applied to

construct the nano-stopper of n-hexane in the selective oxidation of PSi external surface. After filling the mesopores with n-hexane as nano-stoppers, the PSi particles were transferred into the oxidant solution of NH3·H2O/H2O2/H2O (1/1/5, vol/vol) and subsequently into HCl/H2O2/H2O (Scheme 1). After 30 min at ambient temperature, the sample was rinsed twice with deionized H2O and then once more with absolute ethanol. Since the mesopores were filled with a non-polar and highly hydrophobic solvent n-hexane, the aqueous solution containing inorganic oxidants cannot penetrate into the mesopores. The Si-H groups on the internal pore walls were protected from the oxidation reaction by the nano-stoppers in the mesopores. As a result, the Si-H groups on the external surface were selectively oxidized. The obtained PSi sample was dried at 65 ºC for 2 h and it was designated as PSiOHex. 2). Modification of PSi internal pore surface with carboxyl groups. Undecylenic acid (10 ml) was degassed under N2 gas for 30 min at ambient temperature. Afterwards 100 mg of PSi-OHex was added into the solution of undercylenic acid. The mixture was reacted at 120 ºC overnight in a sealed vessel. The sample was rinsed three times using absolute ethanol. Sonication treatment was applied in each rinsing to remove the residual undecylenic acid. The active groups of Si-H for the reaction located only



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on the internal pore surface of PSi-OHex and thus the carboxyl groups were selectively modified on the internal pore surface of PSi. The prepared sample was designated as PSi-OHex-COin.

Scheme 1. Illustration of the procedure for selective modifications of PSi

3). Modification of PSi external surface with FA. Initially, n-hexane was adsorbed into the mesopores of PSi-OHex-COin as the nano-stopper. The pore-filled PSi-OHex-COin particles were then transferred into the DMSO solution with 4.0 wt% of APTES. The mixture was reacted at an ambient temperature for 20 min. The APTES modified PSi sample was recovered by centrifuge and rinsed four times with ethanol. Subsequently the sample was treated in an oven at 65 ºC for 2 h and it was designated as PSiCOin-NHex. In this step, the nano-stopper was utilized as follows: DMSO is a high polar solvent and it is insoluble in n-hexane. Therefore, it cannot diffuse into the mesopores, which were full filled with the nano-stopper of n-hexane. Moreover, the internal pore walls were pre-modified with undecylenic acid before the external surface modification. Therefore, APTES did not gain access to modify the internal


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pore walls. The nano-stopper in the mesopores ensured that APTES was modified only on the external surface of PSi-OHex-COin. The conjugation of FA to the sample of PSi-COin-NHex was carried out via the EDC/NHS chemistry.19 Briefly, 44 mg of FA was added in 10 ml of anhydrous DMSO and stirred under nitrogen atmosphere for 2-3 h. Then 155 mg of EDC was added and the mixture reacted for additional 2 h under stirring. Subsequently, 110 mg of NHS was added and the reaction was continued for overnight at an ambient temperature under a nitrogen atmosphere in the dark. The activated FA was precipitated and recovered by an excess of CH2Cl2. Subsequently, it was mixed with the sample of PSi-COin-NHex in 5.0 ml DMSO solution. The mixture was stirred at ambient temperature in the dark overnight. The obtained FA modified PSi sample was rinsed three times using absolute ethanol and it was designated as PSiCOin-FAex. To recover nanoparticles (NPs) for following cell tests, the selectively modified PSi-COinFAex (

A Nanostopper Approach To Selectively Engineer ... - ACS Publications

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