Biology subjects

Brealey, J.

Publications and source records attributed to Brealey, J..

3 recordsLinked to original sources

bioRxiv2026

Lipid nanoparticle shape and morphology determine the magnitude and duration of mRNA transfection

Lipid nanoparticles (LNP) are widely used for nucleic acid delivery, with current optimization efforts mainly focusing on refining formulation features, enabling extrahepatic targeting, and improving therapeutic efficacy. Particle shape and internal architecture are variables often overlooked in LNP design, although they critically influence particle stability, biodistribution, cellular uptake, and transfection. Here, we investigate how formulation and process parameters impact LNP shape, morphology, and internal structure, and how these structural features, in turn, influence mRNA loading, cell uptake, and expression. We demonstrate that modulation of lipid composition by increasing DSPC content drives a transition from compact spherical to vesicular non-spherical morphologies, with altered RNA loading at the single-particle level. While non-spherical LNP exhibited higher cellular uptake than spherical LNP, it was identified that this increase was relatively modest compared with the substantially higher and sustained protein expression for non-spherical structures. This suggests that increased uptake alone cannot explain the enhanced transfection performance. The trend was conserved across LNP formulations comprising clinically relevant ionizable lipids SM-102, ALC-0315 and DLin-MC3-DMA. Taken together, these findings establish LNP shape and internal morphology as functionally relevant design variables that should be considered in the design of next-generation LNP with improved mRNA delivery and expression.

pharmacology and toxicology↗

bioRxiv2024

Quantitative fluorescent nanoparticle tracking analysis and nano-flow cytometry enable advanced characterization of single extracellular vesicles

Current state-of-the-art tools for analyzing extracellular vesicles (EVs) offer either highly sensitive but unidimensional bulk measurements of EV components, or high-resolution multiparametric single particle analyses which lack standardization and appropriate reference materials. This limits the accuracy of assessment of marker abundance and overall marker distribution among individual EVs, and finally, the understanding of true EV heterogeneity. In this study, we aimed to define the standardized operating procedures and reference material for fluorescent characterization of EVs with two commonly used EV analytical platforms - nanoparticle tracking analysis (NTA) and nano-flow cytometry (nFCM). For the first time, we achieved quantitative fluorescence analyses on ZetaView NTA and NanoAnalyzer nFCM instruments, by utilizing yellow-green FluoSpheres (FS) with assigned ERF (equivalent reference fluorophore) values. This standardization technique allowed for fluorescent EV signal to be expressed in ERF units (indicative of bound fluorescent antibodies per EV), thus enabling measurement of target protein marker abundance on individual EVs, and in the whole EV population. The NTAs and nFCMs limits of quantification (LoQ) were evaluated at 115 and 75 Alexa Fluor 488 (AF488) molecules, respectively. To complement these shortcomings, in-line bulk fluorescence measurements in a plate reader were performed. This provided absolute marker quantification, and more insightful analyses of EV heterogeneity and marker stoichiometry. The standardization method outlined in this work unlocks the full analytical potential of NTA and nFCM, enabling cross-platform data comparison. At the same time, it highlights some of the technical challenges and considerations, and thus contributes to the ongoing efforts towards development of EV analytical tools.

cell biology↗

bioRxiv2018

Characterising the microbiome from host shotgun sequencing data: bacterial and diatom community dynamics derived from killer whale skin

Recent exploration into the interactions and relationship between hosts and their microbiota has revealed a connection between many aspects of the hosts biology, health and associated microorganisms. Whereas amplicon sequencing has traditionally been used to characterise the microbiome, the increasing number of published population genomics datasets offer an underexploited opportunity to study microbial profiles from the host shotgun sequencing data. Here, we use sequence data originally generated from killer whale Orcinus orca skin biopsies for population genomics, to characterise the skin microbiome and investigate how host social and geographic factors influence the microbial community composition. Having identified 845 microbial taxa from 2.4 million reads that did not map to the killer whale reference genome, we found that both ecotypic and geographic factors influence community composition of killer whale skin microbiomes. Furthermore, we uncovered key taxa that drive the microbiome community composition and showed that they are embedded in unique networks, one of which is tentatively linked to diatom presence and poor skin condition. Community composition differed between Antarctic killer whales with and without diatom coverage, suggesting that the previously reported episodic migrations of Antarctic killer whales to warmer waters associated with skin turnover may control the effects of potentially pathogenic bacteria such as Tenacibaculum dicentrarchi. Our work demonstrates the feasibility of microbiome studies from host shotgun sequencing data and highlights the importance of metagenomics in understanding the relationship between host and microbial ecology.

evolutionary biology↗