Biomedical subjects

Daniel J Estes

Publications and source records attributed to Daniel J Estes.

3 recordsLinked to original sources

PubMed2006

Triggering and visualizing the aggregation and fusion of lipid membranes in microfluidic chambers.

We present a method that makes it possible to trigger, observe, and quantify membrane aggregation and fusion of giant liposomes in microfluidic chambers. Using electroformation from spin-coated films of lipids on transparent indium tin oxide electrodes, we formed two-dimensional networks of closely packed, surface-attached giant liposomes. We investigated the effects of fusogenic agents by simply flowing these molecules into the chambers and analyzing the resulting shape changes of more than 100 liposomes in parallel. We used this setup to quantify membrane fusion by several well-studied mechanisms, including fusion triggered by Ca2+, polyethylene glycol, and biospecific tethering. Directly observing many liposomes simultaneously proved particularly useful for studying fusion events in the presence of low concentrations of fusogenic agents, when fusion was rare and probabilistic. We applied this microfluidic fusion assay to investigate a novel 30-mer peptide derived from a recently identified human receptor protein, B5, that is important for membrane fusion during the entry of herpes simplex virus into host cells. This peptide triggered fusion of liposomes at an approximately 6 times higher probability than control peptides and caused irreversible interactions between adjacent membranes; it was, however, less fusogenic than Ca2+ at comparable concentrations. Closely packed, surface-attached giant liposomes in microfluidic chambers offer a method to observe membrane aggregation and fusion in parallel without requiring the use of micromanipulators. This technique makes it possible to characterize rapidly novel fusogenic agents under well-defined conditions.

Electrochemistry↗

PubMed2005

Electroformation of giant liposomes from spin-coated films of lipids.

This paper describes spin-coating of solutions of lipids and using the resulting thin films for electroformation of giant liposomes. Spin-coating made it possible to generate uniform films of lipids with controllable thickness over large surfaces (>25 cm(2)) of indium tin oxide. Establishing a range of thicknesses optimal for electroformation (25-50 nm), we demonstrate formation of giant liposomes from lipids (such as asolectin, phosphatidylserine, and phosphatidylglycerol) that do not readily form giant liposomes from traditional, droplet-derived films. We compared liposomes from a spin-coated film of lipids to liposomes formed from traditional droplet-derived films and found that spin-coated films produced larger (by factor of 2-5) and more abundant liposomes than droplet-derived films of lipids. Electroformation from spin-coated, homogenous lipid films of optimal thickness provided a reproducible way to obtain liposomes with diameters that are predominantly larger than 30 microm over the entire surface of formation.

Biotechnology↗

PubMed2005

Giant liposomes in physiological buffer using electroformation in a flow chamber.

We describe a method to obtain giant liposomes (diameter 10-100 microm) in solutions of high ionic strength to perform a membrane-binding assay under physiological conditions. Using electroformation on ITO electrodes, we formed surface-attached giant liposomes in solutions of glycerol in a flow chamber and then introduced solutions of high ionic strength (up to 2 M KCl) into this chamber. The ionic solution exchanged with the isoosmolar glycerol solution inside and outside the liposomes. An initial mismatch in index of refraction between the inside and outside of liposomes allowed for the observation of solution replacement. Ions and small polar molecules exchanged into and out of surface-attached liposomes within minutes. In contrast, liposomes formed in solutions of macromolecules retained molecules larger than 4 kDa, allowing for encapsulation of these molecules for hours or days even if the solution outside the liposomes was exchanged. We propose that solutes entered liposomes through lipid tubules that attach liposomes to the film of lipids on the surface of the ITO electrode. The method presented here makes it straightforward to perform flow-through binding assays on giant liposomes under conditions of physiological ionic strength. We performed a membrane-binding assay for annexin V, a calcium-dependent protein that binds to phosphatidylserine (PS). The binding of annexin V depended on the concentration of PS and decreased as ionic strength increased to physiological levels.

Animals↗