Osmotic Shock-Triggered Assembly of Highly Charged, Nanoparticle-Supported Membranes

Abstract

Spherical nanoparticle-supported lipid bilayers (SSLBs) combine precision nanoparticle engineering with biocompatible interfaces for various applications, ranging from drug delivery platforms to structural probes for membrane proteins. Although the bulk, spontaneous assembly of vesicles and larger silica nanoparticles (>100 nm) robustly yields SSLBs, it will only occur with low charge density vesicles for smaller nanoparticles (<100 nm), a fundamental barrier in increasing SSLB utility and efficacy. Here, through whole mount and cryogenic transmission electron microscopy, we demonstrate that mixing osmotically loaded vesicles with smaller nanoparticles robustly drives the formation of SSLBs with high membrane charge density (up to 60% anionic lipid or 50% cationic lipid). We show that the osmolyte load necessary for SSLB formation is primarily a function of absolute membrane charge density and is not lipid headgroup-dependent, providing a generalizable, tunable approach toward bulk production of highly curved and charged SSLBs with various membrane compositions.

α-Synuclein Sterically Stabilizes Spherical Nanoparticle-Supported Lipid Bilayers

“…10 To simultaneously address membrane monodispersity for precision measurements and make feasible high-resolution synchrotron methods, we applied a recently developed technique to create spherical nanoparticle-supported lipid bilayers (SSLBs). 11 By using osmotic shock to coat highly monodisperse silica nanoparticles (60 nm diameter) with a lipid membrane of our choosing, we can utilize nanoparticlesupported, highly charged membranes with a curvature and charge in the physiological limit of synaptic vesicles. Furthermore, the larger X-ray scattering cross section of the silica nanoparticle core relative to membranes enables smallangle X-ray scattering (SAXS) and state-of-the-art X-ray photon correlation spectroscopy (XPCS) to probe colloidal structure and dynamics, respectively, while minimizing ionizing radiation damage to proteins and membranes.…”

“…For the spherical nanoparticle-supported lipid bilayer (SSLB) preparation, first, lipid vesicles were prepared, as described previously. 11 Briefly, lipids were suspended in HPLC-grade chloroform and aliquoted into another preweighed vial to the desired ratio at an overall lipid number concentration corresponding to three times the number of vesicles to nanoparticles. Chloroform was again evaporated via nitrogen flow and placed under a vacuum for >1 h, with the lipid mixture mass checked via analytical balance.…”

“…To simultaneously address membrane monodispersity for precision measurements and make feasible high-resolution synchrotron methods, we applied a recently developed technique to create spherical nanoparticle-supported lipid bilayers (SSLBs) . By using osmotic shock to coat highly monodisperse silica nanoparticles (60 nm diameter) with a lipid membrane of our choosing, we can utilize nanoparticle-supported, highly charged membranes with a curvature and charge in the physiological limit of synaptic vesicles.…”

The C‐Terminal Domain of α‐Synuclein Confers Steric Stabilization on Synaptic Vesicle‐Like Surfaces

“…SSLBs were prepared by rupturing lipid vesicles on nanoparticles via osmotic stress, as previously described. [8] The aminefunctionalized silica nanoparticles of ~60 nm diameter were purchased in ethanol (Lot # SCM0049) from nanoComposix (San Diego, CA, USA) and dialyzed against 1-L of MilliQ water overnight to exchange out ethanol with water. Nanoparticles were resuspended into assembly buffer (10 mM citrate pH 6.0, 150 mM NaCl) and was subsequently mixed with an equal volume of large unilamellar vesicles.…”

“…To directly probe the force response of S bound to membrane surfaces, we utilized novel spherical-nanoparticle supported lipid bilayers (SSLBs) and small-angle X-ray scattering (SAXS) to measure the interaction forces between SSLBs with bound S as a function of depletion attraction. [8][9][10] The nanoparticle core of SSLBs offers unique advantages; not only does the core enforce monodisperse, highly-charged membrane curvature (in the physiological limit of synaptic vesicles) but allows SAXS to selectively probe the position of the strongly-scattering core and bypass the comparatively weaker scattering from lipids, proteins, and polymers. A previous study by our group used the high scattering cross-section of SSLBs to demonstrate via X-ray photon correlation spectroscopy that S dispersed SSLB aggregates, direct evidence that S sterically stabilized membrane surfaces.…”