multifunctional-micropipette-based-method-for-incorporation

Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers

MscL, a large conductance mechanosensitive channel (MSC), is a ubiquitous osmolyte release valve that helps bacteria survive abrupt hypo-osmotic shocks. ...

University of Georgia

Dendrimers have been proposed as therapeutic gene delivery platforms. Their superior transfection efficiency is attributed to their ability to buffer the acidification of the endosome and attach to the nucleic acids. For effective transfection, the strategy is to synthesize novel dendrimers that optimize both of these traits, but the prediction of the buffering behavior in the endosome remains elusive. It is suggested that buffering dendrimers induce an osmotic pressure sufficient to rupture the endosome and release nucleic acids, which forms to sequestrate most internalized exogenous materials. Presented here are the results of a computational study modeling osmotically driven endosome burst or the 'proton sponge effect.' The approach builds on previous cellular simulation efforts by linking the previous model with a sponge protonation model, then observing the impact on endosomal swelling and acidification. Calibrated and validated using reported experimental data, the simulations offer insights into defining the properties of suitable dendrimers for enhancing gene delivery as a function of polymer structure.

Modeling the proton sponge hypothesis: examining proton sponge effectiveness for enhancing intracellular gene delivery through multiscale modeling.

MscL, a stretch-activated channel, saves bacteria experiencing hypo-osmotic shocks from lysis. Its high conductance and controllable activation makes it a strong candidate to serve as a transducer in stimuli-responsive biomolecular materials. Droplet interface bilayers (DIBs), flexible insulating scaffolds for such materials, can be used as a new platform for incorporation and activation of MscL. Here, we report the first reconstitution and activation of the low-threshold V23T mutant of MscL in a DIB as a response to axial compressions of the droplets. Gating occurs near maximum compression of both droplets where tension in the membrane is maximal. The observed 0.1-3 nS conductance levels correspond to the V23T-MscL sub-conductive and fully open states recorded in native bacterial membranes or liposomes. Geometrical analysis of droplets during compression indicates that both contact angle and total area of the water-oil interfaces contribute to the generation of tension in the bilayer. The measured expansion of the interfaces by 2.5% is predicted to generate a 4-6 mN/m tension in the bilayer, just sufficient for gating. This work clarifies the principles of interconversion between bulk and surface forces in the DIB, facilitates the measurements of fundamental membrane properties, and improves our understanding of MscL response to membrane tension.

Article	Year
Modeling the proton sponge hypothesis: examining proton sponge effectiveness for enhancing intracellular gene delivery through multiscale modeling. Journal of biomaterials science. Polymer edition\| PubMed ID: 23565683	2013
Activation of bacterial channel MscL in mechanically stimulated droplet interface bilayers. Scientific reports\| PubMed ID: 26348441	2015

Eric C. Freeman

Biography

JoVE Journal Publications

Other Publications