In vitro cytotoxicity of porous silicon microparticles: Effect of the particle concentration, surface chemistry and size

Abstract

We report here the in vitro cytotoxicity of mesoporous silicon (PSi) microparticles on the Caco-2 cells as a function of particle size fractions (1.2–75 μm), particle concentration (0.2–4 mg ml⁻¹) and incubation times (3, 11 and 24 h). The particle size (smaller PSi particles showed higher cytotoxicity) and the surface chemistry treatment of the PSi microparticles were considered to be the key factors regarding the toxicity aspects. These effects were significant after the 11 and 24 h exposure times, and were explained by cell–particle interactions involving mitochondrial disruption resulting from ATP depletion and reactive oxygen species production induced by the PSi surface. These events further induced an increase in cell apoptosis and consequent cell damage and cell death in a dose-dependent manner and as a function of the PSi particle size. These effects were, however, less pronounced with thermally oxidized PSi particles. Under the experimental conditions tested and at particle sizes >25 μm, the non-toxic threshold concentration for thermally hydrocarbonized and carbonized PSi particles was <2 mg ml⁻¹, and for thermally oxidized PSi microparticles was <4 mg ml⁻¹.

Introduction

Mesoporous silicon (PSi) microparticles have become one of the most promising materials for applications in human health care and have impacted tremendously on current biomedical materials science research [1], [2], [3], [4]. PSi microparticles have several advantages over the materials currently considered for oral drug delivery, starting from their top–down production method [1]. They can also be fabricated to resist the harsh conditions of the stomach and gastrointestinal (GI) lumen, maintaining their physicochemical properties, with the possibility of loading drugs into their pores to promote, for example, improved drug dissolution and fast release kinetics [2], [5], [6], [7], [8].

Recent studies on PSi have shown the great potential of these materials to overcome many of the problems associated with the oral delivery of peptides [9], [10] and poorly soluble drug molecules [1], [5], [6], [7]. The biocompatibility of PSi discs [11] and immunogenicity of flat, nanochannelled and nanoporous Si [12] towards human monocytes was demonstrated to be equivalent to tissue culture polystyrene [12]. When implanted into the rat eye, thermally oxidized PSi (TOPSi) membranes provoked only a very mild host reaction and showed slow erosion up to 9 weeks [3]. Interestingly, the internalization of TOPSi microparticles (1.6 and 3.2 μm) by endothelial cells was found to take place via phagocytosis and macropinocytosis, and was shown to be enhanced by the presence of inflammatory cytokines [4]. In further studies, thermally hydrocarbonized PSi microparticles (38–53 μm) have been shown to cause no significant increase in cytokine activity when administrated subcutaneously to mice [10], whereas PSi microparticles of 3 μm and smaller have been found to increase the cytotoxicity and inflammatory responses in macrophagial cells at concentrations higher than 200 μg ml⁻¹ [13].

Another important aspect is the possible formation of reactive oxygen species (ROS) in the presence of PSi microparticles at smaller particle sizes. A recent report addressed the production of ROS when hamster ovary and mutant cells were cultured on soaked crystalline-Si membranes for 60 days, inducing mitochondrial injury and DNA breaking [14]. Mitochondrial oxidative stress can originate from the loss of mitochondrial potential or by an increase in the antioxidant defense mechanism of the cells by producing ROS [15], [16]. ROS act as signalling molecules and regulate cell proliferation, differentiation and apoptosis, but they are toxic at high concentrations [16], [17], [18]. The ROS include the singlet oxygen, the extremely reactive superoxide anion radical, associated with the activation of apoptosis [16], [17] and hydrogen peroxide, which can be reduced partially to the reactive hydroxyl radical, which in turn can attack and damage DNA and proteins [16], [17], [19]. Intracellular ROS can also induce lipid peroxidation and protein oxidation [16], [17]. ROS can also originate at the particles’ surface, owing to their semiconductor and electronic properties, active electron configurations, UV activation, dissolution of material, hydrophobic/philic interactions and reduction–oxidation cycling, which will perturb the transfer processes in the cells [20]. Recent studies have suggested that PSi can generate ROS on its surface [21], but ROS have also been shown to degrade material surfaces [22] and to trigger the release of drug molecules attached to the surface of PSi microparticles [2].

To our knowledge, the in vitro quantification of large amounts of PSi particles relevant for oral delivery and their cytotoxic analysis in intestinal-based cells, especially in terms of ROS production, has not been previously explored. Suitable and complementary in vitro methods to study the PSi cytotoxicity are of utmost importance in order to avoid erroneous results [23], [24]. In this work, the in vitro cytotoxicity of PSi microparticles was evaluated using Caco-2 (a human colon carcinoma cell line) cells as a model for intestinal epithelial cells [25]. To cover different conditions of cell–particle interactions, several concentrations of the PSi materials with different surface chemistries (namely, thermally carbonized PSi, thermally oxidized PSi and thermally hydrocarbonized PSi) and size fractions were tested up to 24 h. This paper reports the effects of the particle size, surface chemical treatment and particle concentration thresholds of PSi microparticles on cytotoxicity, as well as their effect on both cellular ATP content and ROS production.