Silica nanoparticles with enlarged nanopore size for the loading and release of biological proteins Mohamed Eltohamy a, d , Ueon Sang Shin a, b , Hae-Won Kim a, b, c, ⁎ a Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Republic of Korea b Biomaterials and Tissue Engineering Lab., Department of Nanobiomedical Science and WCU Research Center, Dankook University, Republic of Korea c Department of Biomaterials Science, College of Dentistry, Dankook University, Republic of Korea d Glass Research Department, National Research Center, Egypt abstract article info Article history: Received 1 July 2011 Accepted 29 July 2011 Available online 3 August 2011 Keywords: Ceramics Nanoparticles Bioceramics Silica nanoparticles (SNs) with a nanoporous structure are attractive for the delivery of biomolecules. This study developed a SNs-based protein delivery system with nanopore sizes large enough to uptake protein molecules. The use of trioctylmethylammonium bromide (TOMAB) as an auxiliary chemical facilitated a dramatic increase in pore size from 2.6 nm to 17.4 nm. The surface was highly negatively-charged with a zeta potential of approximately -35 at pH 7. Positively-charged protein cytochrome C was encapsulated ef- fectively within the large pore spaces of the SNs, with a protein loading capacity of almost 2-fold increase due to the pore size increase. The loaded protein exhibited sustained release for approximately one week with an initial burst in a day, suggesting the SNs tailored with enlarged nanopores should be useful for the delivery of large protein molecules for tissue regeneration. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Silica nanoparticles (SNs) containing nanopores within the structure, which are more often known as mesoporous SNs, have been widely accepted as a potential delivery system for drugs, such as antibiotics, anticancer drugs, enzymes and nucleic acids [1–5]. Because of the nanopores inside the structure, SNs generally have an extremely high surface area (N 1000 m 2 /g) and surface volume (0.5–1 cm 3 /g) to allow a high degree of surface reaction and to be capable of loading a large amount of drugs [5]. The chemically and structurally stable inorganic oxide framework of the SNs protects the drug molecules from exposure to a degrading environment and species, such as proteases and denaturation chemicals [3–5]. Furthermore, the surface of the SNs can be functionalized with different chemicals to adopt specific molecules and induce relevant biological reactions [5]. Therefore, the nanopore structure and surface properties of the SNs should be considered importantly in the design for drug delivery systems. MCM-41 and SBA-15 are the most widely studied nanoporous SNs, both of which are based on the precursor tetraethyl orthosilicate (TEOS) and are prepared using polymer templates, such as cetyltrimethylammonium bromide (CTAB) for the generation of pore arrays inside the nanoparticles [5]. In the conventional approach of using TEOS and CTAB in a water-based ammonia solution, the pore sizes of the SNs were at most a few nano- meters. This range of pore sizes is sufficient only for encapsulating small- sized drugs, such as antibiotics, anticancers and proteins with a low molecular weight, limiting the applications for large-sized biomolecules, such as growth factors. Some methodologies have increased the nanopore sizes of SNs [5–11]. Mostly, alkane groups, such as decane, octane and hexane, have been used to enlarge the pore size of the silica nanoparticles, where the hydrophobic alkane chain is placed at the core of the micelle and swells the structure [8,9]. Organic auxiliary chemicals, such as 3,5-trimethylbenzene and 1,3,5- triisopropylbenzene, have also been used to control the pore size of SNs [10,11]. In this case, the interaction of the surfactant with the auxiliary molecules determines the pore size. Here we use trioctylmethylammonium bromide (TOMAB), which is considered to exert an auxiliary function for increasing the pore sizes, helping CTAB in the generation of a nanoporous structure within SNs. The processing routes to prepare SNs with enlarged pores are described, and the pore structure was examined. Moreover, the feasibility of the SNs in encapsulating proteins and subsequently releasing them were addressed using a model protein, cytochrome C. This study is considered to provide useful information on further developing biological protein delivery systems using inorganic nanoparticles. 2. Experimental The preparation of SNs with small-sized nanopores was based on the method applied conventionally [2–4]. Typically, 6.123 g of cetyltrimethylammonium bromide (CTAB) used as a template was dissolved in 34.617 g of water, and 10.514 g of a 32% NH 4 OH solution was added, which was followed by the addition of 3.5 g of tetraethyl Materials Letters 65 (2011) 3570–3573 ⁎ Corresponding author at: Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Republic of Korea. Tel.: +82 41 550 3081; fax: +82 41 550 3085. E-mail address: kimhw@dku.edu (H.-W. Kim). 0167-577X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.matlet.2011.07.112 Contents lists available at ScienceDirect Materials Letters journal homepage: www.elsevier.com/locate/matlet