Listeriolysin O (LLO) is a pore-forming hemolysin secreted by the foodborne pathogen Listeria monocytogenes and is required for bacterial virulence. Current detection methods for L. monocytogenes are time-consuming, labor-intensive, and expensive, which is impractical considering the limitations of food storage. To overcome these problems, we developed a liposome-doped silica nanocomposite as a simple, inexpensive, and highly stable biosensor material that mimics existing whole-cell assays for LLO. Small unilamellar liposomes containing fluorescent dyes were immobilized within porous silica using alcohol-free sol-gel synthesis methods. The immobilized liposomes served as cellular surrogates for membrane insertion and pore formation by LLO. The integrity of liposomes in the solid-state sol-gel glass was investigated by fluorescence quenching and leaching assays. The materials were stable for at least 5 months in ambient conditions. Both free and immobilized liposomes responded to LLO at pH 6.0 with concentration dependent kinetics. The pore formation of LLO in liposome-doped silica composites displayed similar kinetic curves as free liposomes but with slower rates. LLO insertion into the immobilized liposomes was pH dependent. No increase in membrane permeability was observed at pH 7.4 for the liposome-doped composites in the presence of LLO. Immobilized liposomes can detect LLO in approximately 1.5 h using a steady state calibration and within 30 min using a kinetic calibration. These liposome silica composites potentially could be used for the detection of hemolysin producing L. monocytogenes as well as the many other bacteria that produce pore-forming toxins.

Nitric oxide (NO) is an important cell-signaling molecule whose role in a variety of cellular processes such as differentiation and apoptosis depends strongly on its concentration and flux levels. This work describes and characterizes a novel nitric oxide releasing nanocomposite, capable of photostimulated NO flux that can by dynamically modulated in within a range of biological levels. This material mimics the common compartmentalization strategies used by living cells to achieve its novel features. The material is constructed by encapsulating a photosensitive nitric oxide donor within lipid vesicles with an average diameter of 150 nm. The vesicles are then doped into the interstitial liquid phase of a solid porous silica matrix, which has previously demonstrated biological compatibility and capabilities as a growth surface for mammalian cells. Stimulation by a light source produces a step increase in NO concentration within seconds. The NO flux at the surface of the material is measured to be 14 pmol-cm(-2) sec(-1) using a NO selective self-referencing amperometric microsensor. The NO concentration profile decreases with distance perpendicular to the surface as expected for diffusion from a surface through an aqueous environment. A pattern of one minute light pulses produced uniform pulses of increased NO concentration of one minute duration. A linear relationship exists between NO surface concentration and photon flux, and this relationship can be used to tune the material response.

The occurrence of glass delamination is a serious concern for parenteral drug products. Over the past several years, there has been a series of product recalls involving glass delamination in parenteral drugs stored in vials which has led to heightened industry and regulatory scrutiny. In this study, a two-pronged approach was employed to assess the inner surface durability of vials and pre-filled syringes. Non-siliconized syringes were used in order to directly compare glass to glass performance between vials and syringes. The vial and syringe performance was screened with pharmaceutically relevant formulation conditions. The influence of pH, buffer type, ionic strength, and glass type and source was evaluated. In addition, an aggressive but discriminating formulation condition (glutaric acid, pH 11) was used to ascertain the impact of syringe processing. Advanced analytical tools including inductively coupled plasma/mass spectrometry, scanning electron microscopy, atomic force microscopy, and dynamic secondary ion mass spectroscopy showed significant differences in glass performance between vials and syringes. Pre-filled syringes outperform vials for most tests and conditions. The manufacturing conditions for vials lead to glass defects, not found in pre-filled syringes, which result in a less chemically resistant surface. The screening methodology presented in this work can be applied to assess suitability of primary containers for specific drug applications.

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