Drug delivery with nanocarriers relies on the interaction of individual nanocarriers with the cell surface. For lipid-based NCs, this interaction uniquely involves a process of membrane fusion between the lipid bilayer that makes up the NC and the cell membrane. Cubosomes have emerged as promising fusogenic NCs, however their individual interactions had not yet been directly observed due to difficulties in achieving adequate resolution or disentangling multiple interactions with common characterization techniques. Moreover, many studies on these interactions have been performed under static conditions which may not mimic the actual transport of NCs. Herein we have observed fusion of lipid cubosome NCs with lipid bilayers under flow. Total internal reflection microscopy has allowed visualisation of the fusion event which was sensitive to the lipid compositions and rationalized by lipid diffusion. The fusion event in supported lipid bilayers has been compared with those in cells, revealing a distinct similarity in kinetics.

Herein, we demonstrate a method for the functionalization of cubic phase lipid nanoparticles (cubosomes) with a series of magnetite (Fe3O4), copper oxide (Cu2O), and silver (Ag) nanocrystals, with prospective applications across a wide range of fields, including antimicrobial treatments. The resulting cubosomes are characterized using small-angle X-ray scattering and dynamic light scattering, demonstrating the retention of a typical cubic phase structure and particle size following nanocrystal encapsulation at concentrations up to 20% w/w. Cryogenic transmission electron microscopy reveals significant loading and association of each nanocrystal type with both monoolein- and phytantriol-based cubosomes. The antibiotic potential of these hybrid nanoparticles is demonstrated for the first time; cubosomes with embedded silver nanocrystals display a high level of antimicrobial activity against both Gram-positive and Gram-negative bacteria, with observed minimum inhibitory concentration values ranging from 15.6–250 μg/mL. Lastly, total internal reflection fluorescence microscopy is used to visualize cubosome–bacteria interactions, suggesting the involvement of particle interactions as a delivery mechanism.

Antibiotic-resistant bacteria pose a significant threat to humanity. Gram-negative strains have demonstrated resistance to last resort antibiotics, partially due to their outer membrane, which hinders transport of antimicrobials into the bacterium. Nanocarrier (NC)-mediated drug delivery is one proposed strategy for combating this emerging issue. Here, the uptake of self-assembled lipid nanocarriers of cubic symmetry (cubosomes) into bacteria revealed fundamental differences in the uptake mechanism between Gram-positive and Gram-negative bacteria. For Gram-positive bacteria, the NCs adhere to the outer peptidoglycan layers and slowly internalize to the bacterium. For Gram-negative bacteria, the NCs interact in two stages, fusion with the outer lipid membrane and then diffusion through the inner wall. The self-assembled nature of the cubosomes imparts a unique ability to transfer payloads via membrane fusion. Remarkably, the fusion uptake mechanism allowed rapid NC internalization by the Gram-negative bacteria, overcoming the outer membrane responsible for their heightened resilience. Here this is demonstrated by the marked reduction in the minimal inhibition concentration required for antibiotics against a pathogenic strain of Gram-negative bacteria, Escherichia coli. These results provide mechanistic insight for the development of lipid NCs as a new tool to combat bacteria.

No abstract

The recent development of X-ray free electron lasers (XFELs) has spurred the development of serial femtosecond nanocrystallography (SFX) which, for the first time, is enabling structure retrieval from sub-micron protein crystals. Although there are already a growing number of structures published using SFX, the technology is still very new and presents a number of unique challenges as well as opportunities for structural biologists. One of the biggest barriers to the success of SFX experiments is the preparation and selection of suitable protein crystal samples. Here we outline a protocol for preparing and screening for suitable XFEL targets.

No abstract

No abstract

No abstract