Browsing by Subject "Lipid membranes"
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Item Bioavailability of fullerene nanoparticles : factors affecting membrane partitioning and cellular uptake(2014-12) Ha, Yeonjeong; Liljestrand, Howard M. (Howard Michael); Katz, Lynn EllenInteractions of engineered nanomaterials (ENMs) with environmental interfaces have become a critical aspect of environmental health and safety evaluations. Carbon fullerene (C₆₀) has emerged at the forefront of nanoscale research and applications due to its unique properties. Although there are concerns associated with the harmful effects of fullerene towards living organisms, the mechanisms of fullerene toxicity are still under debate. A first step toward assessing these mechanisms requires evaluation of the bio-accumulation and bio-uptake of fullerene through lipid membranes which serve as biological barriers in cells. In this dissertation, partitioning of fullerene between water and lipid membranes and cellular uptake of fullerene were investigated to assess bioavailability of this nanoparticle. Traditional methods to estimate the equilibrium partitioning of molecular level chemicals between water and lipid membranes (K[subscript lipw]) cannot be applied to measure K[subscript lipw] of nanoparticles due to the large size of nanoparticle aggregates. In this study, we developed an in vitro method to estimate K[subscript lipw] of fullerene using solid supported lipid membranes (SSLMs) with various membrane compositions. K[subscript lipw] of fullerene increased with increasing acyl chain length and K[subscript lipw] values were higher after creating phase separation in ternary lipid membranes compared to pre-phase separation. In addition, the partitioning values (K[subscript lipw]) were found to depend on the lipid head charges. These results suggest that the lipid membrane composition can be a critical factor for assessing bioaccumulation of fullerene. Evaluation of the partitioning thermodynamics of fullerene demonstrated that the partitioning mechanism of fullerene is different from that of molecular level chemicals. It is generally acknowledged that molecular level chemicals partition into the hydrophobic center of lipid membranes (i.e., absorption), however, the partitioning mechanism of fullerene is a combination of adsorption on the lipid membrane surface and absorption. Caco-2 cellular uptake of fullerene nanoparticles was investigated using an in vitro method developed in this study to distinguish between active and passive transport across cell membranes. Energy dependent endocytosis is hypothesized to be the main cellular transport mechanism based on an observed temperature dependence of cellular uptake and evidence for saturation of the active sites of transport during cellular uptake of fullerene. Metabolic inhibitors decreased the mass of fullerene taken up by the cells, which supports an active transport mechanism of fullerene through the cell membranes. To evaluate bioavilability of fullerene under environmentally relevant conditions, the effects of humic acid and fetal bovine serum (FBS) on the lipid accumulation and cellular uptake were also investigated. Humic acid and FBS changed the surface characteristics of fullerene. The presence of FBS significantly decreased lipid accumulation of fullerene presumably due to higher steric hinderance of FBS coated fullerene as well as the changes in surface energy, water solubility, and lipid solubility of charged FBS coated fullerene relative to that of bare fullerene. Both humic acid and FBS also effectively lowered the cellular uptake of fullerene. These results imply that natural organic matter and biomolecules in natural aquatic and biological environments have significant effects on the bioavilability of fullerene nanoparticlesItem Pattern formation in cell-sized membranes(2017-05-04) Shindell, Orrin Abraham; Gordon, Vernita Diane; Florin, Ernst-Ludwig; Marder, Michael; Stachowiak, Jeanne; Bonnecaze, RogerThe research presented in this dissertation follows in the tradition of experimental membrane biophysics. Our goal is to study the physical mechanisms underlying organization in the plasma membrane of living cells by using model systems. The central result from our experiments is that mixed-lipid membrane vesicles that are adhered by proteins to a solid-supported lipid membrane can dynamically form long-lived holes at the adhesion interface between the membranes. The first set of experiments we discuss exhibit the stable persistence of static patterns. The patterns are formed by adhering ternary-lipid vesicle membranes to a planar membrane supported on a solid, glass substrate \textit{via} biotin-avidin binding. The membrane and avidin are marked with spectrally distinct fluorescent dyes. We use fluorescence microscopy to acquire data. Adhesion causes half of adhered vesicles to form rough annular patterns with a central region that is devoid of membrane dye and protein binders. The peripheral region is dense in proteins and enriched in dye compared to the free, non-adhered portion of the same membrane. We measure the volume V and surface area A of adhered membranes. Using the measure 6[square root of pi]V/A[superscript 3/2] we find 0.84 for patterned and 0.98 for non-patterned membranes. Thus, adhered vesicles have two equilibrium states, one with annular patterns and one without, and the transition between them involves a loss of internal volume. Collectively our results suggest the annular patterns are holes. Finally, we report on a dynamic pattern that occurs in binary-lipid membranes adhered to a supported lipid bilayer. The pattern consists of finger-shaped holes that invade the protein-bound region. We show the characteristics of the fingers depend on the density [rho] of the protein binders in the adhered region: the width of static fingers [lambda] scales as [lambda] \sim\ [rho] and the rate of finger formation r, defined as the number of fingers that branch off from a boundary per unit time, scales as ln [r] \sim\ [rho]. Theoretically, we treat the formation of a finger as a thermally activated event occurring in a tense elastic film. The activation energy required to form a finger is approximately 3.5 kT, a biologically relevant energy scale.