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Abstract: Gene therapy provides powerful new approaches to curing a large variety of diseases, which are being explored in ongoing worldwide clinical trials. To overcome the limitations of viral gene delivery systems, synthetic nonviral vectors such as cationic liposomes (CLs) are desirable. However, improvements of their efficiency at reduced toxicity and a better understanding of their mechanism of action are required. We present the efficient synthesis of a series of degradable multivalent cationic lipids (CMVLn, n=2 to 5) containing a disulfide bond spacer between headgroup and lipophilic tails. This spacer is designed to be cleaved in the reducing milieu of the cytoplasm and thus decrease lipid toxicity. Small angle X-ray scattering demonstrates that the initially formed lamellar phase of CMVLn–DNA complexes completely disappears when reducing agents such as DTT or the biologically relevant reducing peptide glutathione are added to mimic the intracellular milieu. The CMVLs (n=3 to 5) exhibit reduced cytotoxicity and transfect mammalian cells with efficiencies comparable to those of highly efficient non-degradable analogs and benchmark commercial reagents such as Lipofectamine 2000. Thus, our results demonstrate that degradable disulfide spacers may be used to reduce the cytotoxicity of synthetic nonviral gene delivery carriers without compromising their transfection efficiency. [Copyright &y& Elsevier]

Materials with hierarchical architectures that combine soft and hard material domains with coalesced interfaces possess superior properties compared with their homogeneous counterparts 1-4 . These architectures in synthetic materials have been achieved through deterministic manufacturing strategies such as 3D printing, which require an a priori design and active intervention throughout the process to achieve architectures spanning multiple length scales 5-9 . Here we harness frontal polymerization spin mode dynamics to autonomously fabricate patterned crystalline domains in poly(cyclooctadiene) with multiscale organization. This rapid, dissipative processing method leads to the formation of amorphous and semi-crystalline domains emerging from the internal interfaces generated between the solid polymer and the propagating cure front. The size, spacing and arrangement of the domains are controlled by the interplay between the reaction kinetics, thermochemistry and boundary conditions. Small perturbations in the fabrication conditions reproducibly lead to remarkable changes in the patterned microstructure and the resulting strength, elastic modulus and toughness of the polymer. This ability to control mechanical properties and performance solely through the initial conditions and the mode of front propagation represents a marked advancement in the design and manufacturing of advanced multiscale materials., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

RNA therapeutics have the potential to resolve a myriad of genetic diseases. Lipid nanoparticles (LNPs) are among the most successful RNA delivery systems. Expanding their use for the treatment of more genetic diseases hinges on our ability to continuously evolve the design of LNPs with high potency, cellular-specific targeting, and low side effects. Overcoming the difficulty of releasing cargo from endocytosed LNPs remains a significant hurdle. Here, we investigate the fundamental properties of nonviral RNA nanoparticles pertaining to the activation of topological transformations of endosomal membranes and RNA translocation into the cytosol. We show that, beyond composition, LNP fusogenicity can be prescribed by designing LNP nanostructures that lower the energetic cost of fusion and fusion-pore formation with a target membrane. The inclusion of structurally active lipids leads to enhanced LNP endosomal fusion, fast evasion of endosomal entrapment, and efficacious RNA delivery. For example, conserving the lipid make-up, RNA-LNPs having cuboplex nanostructures are significantly more efficacious at endosomal escape than traditional lipoplex constructs.

Lipid-protein complexes are the basis of pulmonary surfactants covering the respiratory surface and mediating gas exchange in lungs. Cardiolipin is a mitochondrial lipid overexpressed in mammalian lungs infected by bacterial pneumonia. In addition, increased oxygen supply (hyperoxia) is a pathological factor also critical in bacterial pneumonia. In this paper we fabricate a micrometer-size graphene-based sensor to measure oxygen permeation through pulmonary membranes. Combining oxygen sensing, X-ray scattering, and Atomic Force Microscopy, we show that mammalian pulmonary membranes suffer a structural transformation induced by cardiolipin. We observe that cardiolipin promotes the formation of periodic protein-free inter-membrane contacts with rhombohedral symmetry. Membrane contacts, or stalks, promote a significant increase in oxygen gas permeation which may bear significance for alveoli gas exchange imbalance in pneumonia.

Materials that can be switched between low and high thermal conductivity states would advance the control and conversion of thermal energy. Employing in situ time-domain thermoreflectance (TDTR) and in situ synchrotron X-ray scattering, we report a reversible, light-responsive azobenzene polymer that switches between high (0.35 W m -1 K -1 ) and low thermal conductivity (0.10 W m -1 K -1 ) states. This threefold change in the thermal conductivity is achieved by modulation of chain alignment resulted from the conformational transition between planar ( trans ) and nonplanar ( cis ) azobenzene groups under UV and green light illumination. This conformational transition leads to changes in the π-π stacking geometry and drives the crystal-to-liquid transition, which is fully reversible and occurs on a time scale of tens of seconds at room temperature. This result demonstrates an effective control of the thermophysical properties of polymers by modulating interchain π-π networks by light., Competing Interests: The authors declare no conflict of interest.

Lipids self-assemble into diverse supramolecular structures that exhibit thermotropic and/or lyotropic behavior. Lyotropic mesophases, where membranes conform to periodic minimal surfaces dividing two nonpenetrating aqueous subspaces, are arguably one of the most intriguing phases of lipid materials. Traditional 3D bicontinuous cubic lipid materials appear as a polycrystal of varying degrees of order. When exposed to water, the properties of the molecular building blocks of the membrane determine specific swelling limits setting the lattice dimensions at about 15 nm. This limited swelling severely impairs their application as delivery vehicles of large drugs or as matrices for guiding protein crystallization. We report the discovery of self-assembly strategies leading to the emergence of lipid bicontinuous single crystals with unprecedented swelling capacity. The conventional strategy to increase unit cell size is tweaking membrane composition to include charged building blocks, a process to achieve electrostatic-driven swelling. In this paper, we demonstrate that controlling self-assembly external conditions when coupled to membrane composition yields 3D bicontinuous cubic phases that swell up to lattice dimensions of 68 nm. Importantly, and contrary to what is perceived for soft lyotropic materials in general, the self-assembly methodology enables the development of large super-swelled monocrystals. Utilizing small-angle X-ray scattering and cryoelectron microscopy, we underpin three crucial factors dictating the stabilization of super-swelled lipid bicontinuous cubic single crystals: ( i ) organic solvent drying speed, ( ii ) membrane charge density, and ( iii ) polyethylene glycol-conjugated lipids amount., Competing Interests: The authors declare no conflict of interest.

Lipids and nucleic acids (NAs) can hierarchically self-organize into a variety of nanostructures of increasingly complex geometries such as the 1D lamellar, 2D hexagonal, and 3D bicontinuous cubic phases. The diversity and complexity of those lipid-NA assemblies are interesting from a fundamental perspective as well as being relevant to the performance in gene delivery and gene silencing applications. The finding that not only the chemical make of the lipid-NA constructs, but their actual supramolecular organization, affects their gene transfection and silencing efficiencies has inspired physicists, chemists, and engineers to this field of research. At the moment it remains an open question how exactly the different lipid-NA structures interact with cells and organelles in order to output an optimal response. This article reviews our current understanding of the structures of different lipid-NA complexes and the corresponding cellular interaction mechanisms. The recent advances in designing optimal lipid-based NA carriers will be introduced with an emphasis on the structure-function relations.

In this paper, which is part of a collection in honor of Noel Clark's remarkable career on liquid crystal and soft matter research, we present examples of biologically inspired systems, which form liquid crystal (LC) phases with their LC nature impacting biological function in cells or being important in biomedical applications. One area focuses on understanding network and bundle formation of cytoskeletal polyampholytes (filamentous-actin, microtubules, and neurofilaments). Here, we describe studies on neurofilaments (NFs), the intermediate filaments of neurons, which form open network nematic liquid crystal hydrogels in axons. Synchrotron small-angle-x-ray scattering studies of NF-protein dilution experiments and NF hydrogels subjected to osmotic stress show that neurofilament networks are stabilized by competing long-range repulsion and attractions mediated by the neurofilament's polyampholytic sidearms. The attractions are present both at very large interfilament spacings, in the weak sidearm-interpenetrating regime , and at smaller interfilament spacings, in the strong sidearm-interpenetrating regime . A second series of experiments will describe the structure and properties of cationic liposomes (CLs) complexed with nucleic acids (NAs). CL-NA complexes form liquid crystalline phases, which interact in a structure-dependent manner with cellular membranes enabling the design of complexes for efficient delivery of nucleic acid (DNA, RNA) in therapeutic applications.

Environmentally responsive materials (i.e., materials that respond to changes in their environment with a change in their properties or structure) are attracting increasing amounts of interest. We recently designed and synthesized a series of cleavable multivalent lipids (CMVLn, with n = 2-5 being the number of positive headgroup charges at full protonation) with a disulfide bond in the linker between their cationic headgroup and hydrophobic tails. The self-assembled complexes of the CMVLs and DNA are a prototypical environmentally responsive material, undergoing extensive structural rearrangement when exposed to reducing agents. We investigated the structural evolution of CMVL-DNA complexes at varied complex composition, temperature, and incubation time using small-angle X-ray scattering (SAXS) and wide-angle X-ray scattering (WAXS). A related lipid with a stable linker, TMVL4, was used as a control. In a nonreducing environment, CMVL-DNA complexes form the lamellar (L(α)(C)) phase, with DNA rods sandwiched between lipid bilayers. However, new self-assembled phases form when the disulfide linker is cleaved by dithiothreitol or the biologically relevant reducing agent glutathione. The released DNA and cleaved CMVL headgroups form a loosely organized phase, giving rise to a characteristic broad SAXS correlation profile. CMVLs with high headgroup charge also form condensed DNA bundles. Intriguingly, the cleaved hydrophobic tails of the CMVLs reassemble into tilted chain-ordered L(β') phases upon incubation at physiological temperature (37 °C), as indicated by characteristic WAXS peaks. X-ray scattering further reveals that two of the three phases (L(βF), L(βL), and L(βI)) constituting the L(β') phase coexist in these samples. The described system may have applications in lipid-based nanotechnologies.