Fungal Enzyme-Responsive Hydrogel Drug Delivery Platform for Triggered Antifungal Release.

Fungal infections can lead to debilitating consequences if they are not treated effectively. Antifungal drugs used to treat these infections can be toxic and overuse contributes to growing antifungal resistance. Candida spp., particularly C. albicans, are implicated in a majority of these infections. Virulent C. albicans produce secreted aspartic proteases (Saps) that aid in pathogen tissue invasion and proliferation at an infected site. Here, fungi-responsive hydrogels are developed that degrade in the presence of Saps to provide a triggered release of encapsulated liposomal antifungals. The hydrogel backbone incorporates a Sap-cleavable peptide sequence enabling Sap-responsive degradation. Hydrogels are found to effectively degrade in the presence of Saps extracted from C. albicans. Encapsulated liposomal antifungals show similar release kinetics as hydrogel degradation products in the presence of Saps, supporting a degradation-dependent release mechanism. Antifungal liposome-loaded responsive hydrogels exhibit successful eradication of C. albicans cultures and remain stable in sterile murine wound fluid. Finally, no significant cytotoxicity is observed for murine fibroblast cells and red blood cells exposed to hydrogel degradation products. These fungi-responsive hydrogels have the potential to be used for local, on-demand delivery of antifungal drugs, for effective treatment of fungal infections while helping to limit unnecessary exposure to these therapeutics.

Antifungal liposomes: Lipid saturation and cholesterol concentration impact interaction with fungal and mammalian cells.

Liposomes are lipid-based nanoparticles that have been used to deliver encapsulated drugs for a variety of applications, including treatment of life-threatening fungal infections. By understanding the effect of composition on liposome interactions with both fungal and mammalian cells, new effective antifungal liposomes can be developed. In this study, we investigated the impact of lipid saturation and cholesterol content on fungal and mammalian cell interactions with liposomes. We used three phospholipids with different saturation levels (saturated hydrogenated soy phosphatidylcholine (HSPC), mono-unsaturated 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), and di-unsaturated 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC)) and cholesterol concentrations ranging from 15% to 40% (w/w) in our liposome formulations. Using flow cytometry, >80% of Candida albicans SC5314 cells were found to interact with all liposome formulations developed, while >50% of clinical isolates tested exhibited interaction with these liposomes. In contrast, POPC-containing formulations exhibited low levels of interaction with murine fibroblasts and human umbilical vein endothelial cells (<30%), while HSPC and PLPC formulations had >50% and >80% interaction, respectively. Further, PLPC formulations caused a significant decrease in mammalian cell viability. Formulations that resulted in low levels of mammalian cell interaction, minimal cytotoxicity, and high levels of fungal cell interaction were then used to encapsulate the antifungal drug, amphotericin B. These liposomes eradicated planktonic C. albicans at drug concentrations lower than free drug, potentially due to the high levels of liposome-C. albicans interaction. Overall, this study provides new insights into the design of liposome formulations towards the development of new antifungal therapeutics.

Assembly of Cell Mimicking Supported and Suspended Lipid Bilayer Models for the Study of Molecular Interactions.

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial protective barrier for all cell types, separating the internal cellular components from the extracellular environment. These membranes are composed largely of a lipid bilayer, which contains outer hydrophilic head groups and inner hydrophobic tail groups, along with various proteins and cholesterol. The composition and structure of the lipids themselves play a crucial role in regulating biological function, including interactions between cells and the cellular microenvironment, which may contain pharmaceuticals, biological toxins, and environmental toxicants. In this study, methods to formulate uni-lipid and multi-lipid supported and suspended cell mimicking lipid bilayers are described. Previously, uni-lipid phosphatidylcholine (PC) lipid bilayers as well as multi-lipid placental trophoblast-inspired lipid bilayers were developed for use in understanding molecular interactions. Here, methods for achieving both types of bilayer models will be presented. For cell mimicking multi-lipid bilayers, the desired lipid composition is first determined via lipid extraction from primary cells or cell lines followed by liquid chromatography-mass spectrometry (LC-MS). Using this composition, lipid vesicles are fabricated using a thin-film hydration and extrusion method and their hydrodynamic diameter and zeta potential are characterized. Supported and suspended lipid bilayers can then be formed using quartz crystal microbalance with dissipation monitoring (QCM-D) and on a porous membrane for use in a parallel artificial membrane permeability assay (PAMPA), respectively. The representative results highlight the reproducibility and versatility of in vitro cell membrane lipid bilayer models. The methods presented can aid in rapid, facile assessment of the interaction mechanisms, such as permeation, adsorption, and embedment, of various molecules and macromolecules with a cell membrane, helping in the screening of drug candidates and prediction of potential cellular toxicity.