(197a) Sterol Chemistry, but Not Lipid Domains, Modulates the Disruptive Effects of Nanomaterials on Membrane Models

Introduction: The interactions of nanoparticles with cell membranes have been increasingly studied due to the development of nanoparticle-based applications across a wide range of industries. Disruption of the membrane by nanoparticles can have deleterious effects on cell viability. Most studies have focused on the properties of the nanoparticles and less attention has been given to the role of the membrane structure in regulating the interactions. Depending on the lipid composition of the cell membrane, the membrane may exhibit heterogeneous phase segregation of lipids into disordered and ordered domains (lipid rafts). Formation of domains is influenced by the number and type of sterol present in the membrane, providing a facile method to modulate domain formation. The current study utilizes this method to examine the role of membrane domains in regulating the disruptive effects of silica nanoparticles on model membranes. However, in modulating the sterol composition of the model membrane, it is also possible to observe the impact of individual sterol chemistry on particle-membrane interactions. We are particularly interested in how sterol hydroxyl group orientation, sterol ring double bond placement, and R group identity impact the membrane.

Materials and Methods: Unilamellar vesicles 100 nm in diameter were synthesized with equimolar concentrations of sphingomyelin (SM), dipalmitoylphosphotidylcholine (DOPC), and a sterol. Various sterols were chosen to either promote or hinder domain formation in vesicles. Vesicles containing either cholesterol, ergosterol, coprostanol, or androstanol were synthesized. It was expected that vesicles containing cholesterol or ergosterol would exhibit ordered domain formation and vesicles containing coprostanol or androstanol would have few or no ordered domains. Plain silica nanoparticles with diameter 39.0 ± 2.9 nm and zeta potential = -8.22 ± 0.41 mV were used to study nanoparticle-membrane interactions. Membrane models were exposed to nanoparticles at a particle concentration of 0.01 g/L. Fluorescence anisotropy of diphenylhexatriene (DPH) and GUV imaging using rhodamine-PE allowed for quantification of domain formation in vesicles with different sterols. Following this, the degree of vesicle disruption upon nanoparticle exposure was quantified by incorporating the self-quenching fluorescent dye carboxyfluorescein into the vesicles and measuring its leakage over time.

Results and Discussion: DPH anisotropy indicated that vesicles containing cholesterol or ergosterol have higher lipid order compared to vesicles containing coprostanol or androstanol, evidenced by greater anisotropy values for cholesterol (0.20 ± 0.02) and ergosterol (0.21 ± 0.02) than for coprostanol (0.15 ± 0.01) and androstanol (0.13 ± 0.01) vesicles at 25 °C. GUV imaging with confocal microscopy complemented anisotropy results and showed distinct segregated ordered domains in vesicles containing either cholesterol and ergosterol and no domains in vesicles containing coprostanol. Nanoparticle leakage assays yielded no discernable relationship between physical membrane order or membrane disruption by particles. Leakage for vesicles containing cholesterol was 20% ± 6% while for ergosterol it was 37% ± 1% (measurements taken at 25 °C). For coprostanol vesicles at the same temperature, leakage was 11% ± 2.5% and for androstenol leakage was 75% ± 9%. Currently, we are investigating the impact of sterol chemistry (i.e. presence and location of double bonds, stereochemistry, and presence of R groups) on the nanoparticle membrane interaction to determine if this has a more dominant impact on interactions as opposed to presence of physical domains. We intend to expand or vesicle characterization using FRET analysis and to perform nanoparticle binding assays using FRET and GUV imaging to determine nanoparticle localization at the membrane.

Conclusions: Altering the sterol composition of lipid membrane models alters the membrane structure and affects assembly of ordered/disordered domains. However, there does not seem to be a predictable relationship between the degree of ordered domain formation in the vesicle and the nanoparticle interaction. We are investigating the role of sterol chemistry in modulating nanoparticle-membrane interactions.