Abstract
Calcein leakage assays are a standard experimental set-up for probing the extent of damage induced by external agents on synthetic lipid vesicles. The fluorescence signal associated with calcein release from liposomes is the signature of vesicle disruption, transient pore formation or vesicle fusion. This type of assay is widely used to test the membrane disruptive effect of biological macromolecules, such as proteins, antimicrobial peptides and RNA and is also used on synthetic nanoparticles with a polymer, metal or oxide core. Little is known about the effect that calcein and other fluorescent dyes may have on the properties of lipid bilayers, potentially altering their structure and permeability. Here we develop a coarse-grained model of calcein that is compatible with the Martini force field for lipids. We validate the model by comparing its dimerization free energy, aggregation behavior at different concentrations and interaction with a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane to those obtained at atomistic resolution. Our coarse-grained description of calcein makes it suitable for the simulation of large calcein-filled liposomes and of their interactions with external agents, allowing for a direct comparison between simulations and experimental liposome leakage assays.
Highlights
Calcein is a slightly water soluble, green fluorescent dye that is widely used to study cell viability and as an indicator of lipid vesicle leakage by fluorescence microscopy imaging [1,2,3]
In the methods section we provide all the details about the force field parameters, molecular dynamics (MD) run set-up and system composition; in the results section we present the development of the CG Martini model of calcein, and its validation in terms of dimerization free energy, aggregation behavior at different concentrations and interaction with a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane
The parameterization of the atomistic model of calcein is based on the OPLS unitedatom (OPLS-UA) force field developed by Jorgensen et al [22]
Summary
Calcein is a slightly water soluble, green fluorescent dye that is widely used to study cell viability and as an indicator of lipid vesicle leakage by fluorescence microscopy imaging [1,2,3]. Calcein leakage assays are based on calcein self-quenching: at a concentration above 70 mM the fluorescence of the dye is quenched. In a typical leakage experiment, calcein is trapped into lipid vesicles or liposomes at a concentration above the self-quenching threshold. As calcein has a small permeability coefficient across a lipid bilayer [4], as long as the vesicles are intact the only fluorescence intensity that can be recorded is due to small leakage fluctuations.
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