Mechanical activation opens a lipid-lined pore in OSCA ion channels.

OSCA/TMEM63 channels are the largest known family of mechanosensitive channels1-3, playing critical roles in plant4-7 and mammalian8,9 mechanotransduction. Here we determined 44 cryogenic electron microscopy structures of OSCA/TMEM63 channels in different environments to investigate the molecular basis of OSCA/TMEM63 channel mechanosensitivity. In nanodiscs, we mimicked increased membrane tension and observed a dilated pore with membrane access in one of the OSCA1.2 subunits. In liposomes, we captured the fully open structure of OSCA1.2 in the inside-in orientation, in which the pore shows a large lateral opening to the membrane. Unusually for ion channels, structural, functional and computational evidence supports the existence of a 'proteo-lipidic pore' in which lipids act as a wall of the ion permeation pathway. In the less tension-sensitive homologue OSCA3.1, we identified an 'interlocking' lipid tightly bound in the central cleft, keeping the channel closed. Mutation of the lipid-coordinating residues induced OSCA3.1 activation, revealing a conserved open conformation of OSCA channels. Our structures provide a global picture of the OSCA channel gating cycle, uncover the importance of bound lipids and show that each subunit can open independently. This expands both our understanding of channel-mediated mechanotransduction and channel pore formation, with important mechanistic implications for the TMEM16 and TMC protein families.

Phosphorothioated DNA Engineered Liposomes as a General Platform for Stimuli-Responsive Cell-Specific Intracellular Delivery and Genome Editing.

Intracellular protein delivery is highly desirable for protein drug-based cell therapy. Established technologies suffer from poor cell-specific cytosolic protein delivery, which hampers the targeting therapy of specific cell populations. A fusogenic liposome system enables cytosolic delivery, but its ability of cell-specific and controllable delivery is quite limited. Inspired by the kinetics of viral fusion, we designed a phosphorothioated DNA coatings-modified fusogenic liposome to mimic the function of viral hemagglutinin. The macromolecular fusion machine docks cargo-loaded liposomes at the membrane of target cells, triggers membrane fusion upon pH or UV light stimuli, and facilitates cytosolic protein delivery. Our results showed efficient cell-targeted delivery of proteins of various sizes and charges, indicating the phosphorothioated DNA plug-in unit on liposomes could be a general strategy for spatial-temporally controllable protein delivery both in vitro and in vivo.

RNA nanotherapeutics with fibrosis overexpression and retention for MASH treatment.

Metabolic dysfunction-associated steatohepatitis (MASH) poses challenges for targeted delivery and retention of therapeutic proteins due to excess extracellular matrix (ECM). Here we present a new approach to treat MASH, termed "Fibrosis overexpression and retention (FORT)". In this strategy, we design (1) retinoid-derivative lipid nanoparticle (LNP) to enable enhanced mRNA overexpression in fibrotic regions, and (2) mRNA modifications which facilitate anchoring of therapeutic proteins in ECM. LNPs containing carboxyl-retinoids, rather than alcohol- or ester-retinoids, effectively deliver mRNA with over 10-fold enhancement of protein expression in fibrotic livers. The carboxyl-retinoid rearrangement on the LNP surface improves protein binding and membrane fusion. Therapeutic proteins are then engineered with an endogenous collagen-binding domain. These fusion proteins exhibit increased retention in fibrotic lesions and reduced systemic toxicity. In vivo, fibrosis-targeting LNPs encoding fusion proteins demonstrate superior therapeutic efficacy in three clinically relevant male-animal MASH models. This approach holds promise in fibrotic diseases unsuited for protein injection.

Solid lipid nanoparticles for transdermal delivery of diclofenac sodium: preparation, characterization and in vitro studies.

The aim of this study was to prepare diclofenac sodium (DNa) solid lipid nanoparticles (SLNs) by a modified emulsion/solvent evaporation method for transdermal delivery. Five independent processing parameters including the lipid matrix, emulsifiers, co-emulsifiers, water-dispersed phase and organic phase were assessed systematically to enhance the entrapment of DNa. The SLNs produced by optimal formulation were submicrometre size with low polydispersity index, the entrapment efficiency was about 89% and the drug loading was about 9.5%. Shape and surface morphology were determined by transmission electron microscopy, which revealed the fairly spherical and core-shell shapes of the SLNs. The in vitro release of SLNs showed a two-step release pattern: one initial burst release followed by a second slow-release phase. In the in vitro cutaneous permeation studies, value of flux obtained for DNa solution was higher than that of SLNs suspension. SLNs had also been shown to improve the dermal localization of DNa.

A Model Membrane Platform for Reconstituting Mitochondrial Membrane Dynamics.

Mitochondrial dynamics is essential for the organelle's diverse functions and cellular responses. The crowded, spatially complex, mitochondrial membrane is a challenging environment to distinguish regulatory factors. Experimental control of protein and lipid components can help answer specific questions of regulation. Yet, quantitative manipulation of these factors is challenging in cellular assays. To investigate the molecular mechanism of mitochondria inner-membrane fusion, we introduced an in vitro reconstitution platform that mimics the lipid environment of the mitochondrial inner-membrane. Here we describe detailed steps for preparing lipid bilayers and reconstituting mitochondrial membrane proteins. The platform allowed analysis of intermediates in mitochondrial inner-membrane fusion, and the kinetics for individual transitions, in a quantitative manner. This protocol describes the fabrication of bilayers with asymmetric lipid composition and describes general considerations for reconstituting transmembrane proteins into a cushioned bilayer. The method may be applied to study other membrane systems.

Synergistic lipid compositions for albumin receptor mediated delivery of mRNA to the liver.

Lipid-like nanoparticles (LNPs) have potential as non-viral delivery systems for mRNA therapies. However, repeated administrations of LNPs may lead to accumulation of delivery materials and associated toxicity. To address this challenge, we have developed biodegradable lipids which improve LNPs clearance and reduce toxicity. We modify the backbone structure of Dlin-MC3-DMA by introducing alkyne and ester groups into the lipid tails. We evaluate the performance of these lipids when co-formulated with other amine containing lipid-like materials. We demonstrate that these formulations synergistically facilitate robust mRNA delivery with improved tolerability after single and repeated administrations. We further identify albumin-associated macropinocytosis and endocytosis as an ApoE-independent LNP cellular uptake pathway in the liver. Separately, the inclusion of alkyne lipids significantly increases membrane fusion to enhance mRNA release, leading to synergistic improvement of mRNA delivery. We believe that the rational design of LNPs with multiple amine-lipids increases the material space for mRNA delivery.