Monitoring Biological Processes and Interactions at Lipid Membranes using Ion Channel-Based Sensors and Membrane Microarrays.

Many cellular processes involve molecular interactions at the cell membrane. Due to the complexity of living cells, these interactions are usually studied on model membranes. This thesis introduces two platforms based on model membranes for studying biological interactions and processes on cell membranes. In the first part of this thesis, we employed planar lipid bilayers to develop a novel, label-free, and sensitive assay for monitoring the activity of phospholipases D and C that are critical for cell signaling. The activities of these enzymes typically change the surface charge of the membrane. The present assay employs the ion channel-forming peptide gramicidin A to probe these changes and to monitor the activity of these phospholipases in situ and in real-time. Quantitative results from this assay, allowed us to investigate the kinetics of the heterogeneous catalysis of these enzymes. In addition we applied this gramicidin-based sensor to monitor the binding of two therapeutic drugs to various bilayers. Quinine, an anti-malaria agent, and imipramine, an anti-depressant, are positively-charged under physiological conditions and, once bound to a membrane, alter the membrane surface charge. The present assay probes these changes and makes it possible to quantify these binding events. In the second part of this work, we developed a technique that employs topographically-patterned hydrogel stamps to fabricate arrays of membranes and membrane proteins for screening of membrane interactions. This method takes advantage of the porous, hydrated, and biocompatible nature of hydrogels to print spatially-addressable arrays of membranes in a rapid and parallel fashion. We employed this method for two distinct approaches; one approach takes advantage of the storage capability of agarose stamps and minimizes the required time and amount of membrane preparations by generating multiple copies of a membrane array. The other approach takes advantage of on-stamp preconcentration of cellular membrane fragments to generate arrays of multilayered-membranes with high contents of proteins and enhances detection sensitivity. We used these arrays for screening the interactions of a protein (annexin V) and an anti-inflammatory drug (nimesulide) with various bilayers. We also carried out ligand-binding assays on these arrays and showed that the stamped proteins retained their binding activity.Ph.D.Biomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/64664/1/sheereen_1.pd

On fusogenicity of positively charged phased-separated lipid vesicles: experiments and computational simulations

This paper studies the fusogenicity of cationic liposomes in relation to their surface distribution of cationic lipids and utilizes membrane phase separation to control this surface distribution. It is found that concentrating the cationic lipids into small surface patches on liposomes, through phase-separation, can enhance liposome's fusogenicity. Further concentrating these lipids into smaller patches on the surface of liposomes led to an increased level of fusogenicity. These experimental findings are supported by numerical simulations using a mathematical model for phase-separated charged liposomes. Findings of this study may be used for design and development of highly fusogenic liposomes with minimal level of toxicity

Evaluation of the transport rate constant (<i>k</i>) for Pgp-mediated transport of Rho123 under different conditions.

<p>(A) Transport rate constant as a function of ATP concentration. Mean and SEM are presented with n = 3–11 protroteoliposomes (from independent experiments) per condition tested. Data were fitted to Michaelis-Menten equation (<i>K</i>_<i>m</i> = 0.53 ± 0.66 mM). (B-D) Transport rate constants as a function of (B) verapamil concentration, (C) colchicine concentration, and (D) cyclosporin A concentration. Mean and SEM are presented with n = 4–12 from independent electroformations per condition tested. Data were fitted to a one-phase exponential decay model to estimate <i>IC</i>_<i>50</i> values of 25.2 ± 5.0 μM for verapamil, 61.8 ± 34.8 μM for colchicine, and 0.23 ± 0.09 μM for cyclosporin A.</p

ATP dependent transport rate of Rho123 into giant liposomes by reconstituted hamster Pgp.

<p>(A) Rho123 transport rate in the presence and absence of ATP and in the absence of Pgp (–Pgp) in giant liposomes with PL-B formulation. Columns represent the mean and error bars show the SEM with n = 6–11 from independent electroformation events. * indicates p < 0.05 from non-paired t-test against 1 mM ATP transport. (B) Rho123 transport rate in giant liposomes as a function of ATP concentration. Data points represent the mean transport rate, and error bars represent SEM. The solid curve represents the best fit of the data to Michaelis-Menton model, with an estimated value of 0.42 ± 0.75 mM for <i>K</i>_<i>m</i> of ATP. Data points represent the mean transport rate, and error bars represent SEM from 3–11 independent electroformation events.</p

Preparation and characterization of size-controlled glioma spheroids using agarose hydrogel microwells.

Treatment of glioblastoma, the most common and aggressive type of primary brain tumors, is a major medical challenge and the development of new alternatives requires simple yet realistic models for these tumors. In vitro spheroid models offer attractive platforms to mimic the tumor behavior in vivo and have thus, been increasingly applied for assessment of drug efficacy in various tumors. The aim of this study was to produce and characterize size-controlled U251 glioma spheroids towards application in glioma drug evaluation studies. To this end, we fabricated agarose hydrogel microwells with cylindrical shape and diameters of 70-700 μm and applied these wells without any surface modification for glioma spheroid formation. The resultant spheroids were homogeneous in size and shape, exhibited high cell viability (> 90%), and had a similar growth rate to that of natural brain tumors. The final size of spheroids depended on cell seeding density and microwell size. The spheroids' volume increased linearly with the cell seeding density and the rate of this change increased with the well size. Lastly, we tested the therapeutic effect of an anti-cancer drug, Di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) on the resultant glioma spheroids and demonstrated the applicability of this spheroid model for drug efficacy studies

Lipid composition of the examined liposomes.

<p>Lipid composition of the examined liposomes.</p

Immunostaining and ATPase activity of Pgp on giant liposomes.

<p>(A) Confocal images of an electroformed giant PL-A. Green signal is from NBD-PE in the membrane and red signal is from RITC-Dextran, encapsulated in the liposomal lumen. (B) Immunostaining of a giant PL-C against anti-Pgp antibody. Positive staining (bottom panels) against anti-Pgp is shown with green signal with the secondary antibody, Alexa Fluor® 488 anti-mouse, and liposome membrane was labeled with rho-PE in red. The negative control (top panels) was performed on similar liposomes in the absence of the primary antibody incubation. (C) ATPase specific activity of Pgp in similarly formulated small PL-B and giant PL-B. The bars represent the mean and error bars show standard error of mean (SEM) with n = 3–6, from independent electroformation events.</p

Modulation of Pgp transport activity in giant liposomes using inhibitors.

<p>(A) Representative plots of the normalized Rho123 concentration inside the giant PLs over the course of the active transport activity with increasing verapamil concentration from 12.5 μM to 50 μM. (B-D) Rate of Rho123 transport into giant PL lumen as a function of (B) verapamil concentration, (C) colchicine concentration, (D) cyclosporin A concentration. Data points represent the mean and error bars show SEM with n = 3–12 from distinct electroformation events. Data were fitted into a one-phase exponential decay model (solid curves) to estimate <i>IC</i>_<i>50</i> of 26.6 ± 6.1 μM for verapamil, 94.6 ± 47.6 μM for colchicine, and 0.21 ± 0.07 <b>μM</b> for cyclosporine A.</p