peroxisome-staining-mammalian-cells-using-peroxisome-specific

Peroxisome Staining in Mammalian Cells Using Peroxisome-Specific Probes

Peroxisomes are ubiquitous organelles with essential roles in human health, and their dysfunction leads to a spectrum of genetic disorders. Visualization ...

Ecole Polytechnique Fédérale de Lausanne (EPFL)

Peroxisomes are eukaryotic organelles that are essential for multiple metabolic pathways, including fatty acid oxidation, degradation of amino acids, and biosynthesis of ether lipids. Consequently, peroxisome dysfunction leads to pediatric-onset neurodegenerative conditions, including Peroxisome Biogenesis Disorders (PBD). Due to the dynamic, tissue-specific, and context-dependent nature of their biogenesis and function, live cell imaging of peroxisomes is essential for studying peroxisome regulation, as well as for the diagnosis of PBD-linked abnormalities. However, the peroxisomal imaging toolkit is lacking in many respects, with no reporters for substrate import, nor cell-permeable probes that could stain dysfunctional peroxisomes. Here we report that the BODIPY-C12 fluorescent fatty acid probe stains functional and dysfunctional peroxisomes in live mammalian cells. We then go on to improve BODIPY-C12, generating peroxisome-specific reagents, PeroxiSPY650 and PeroxiSPY555. These probes combine high peroxisome specificity, bright fluorescence in the red and far-red spectrum, and fast non-cytotoxic staining, making them ideal tools for live cell, whole organism, or tissue imaging of peroxisomes. Finally, we demonstrate that PeroxiSPY enables diagnosis of peroxisome abnormalities in the PBD CRISPR/Cas9 cell models and patient-derived cell lines.

Fluorescent fatty acid conjugates for live cell imaging of peroxisomes.

Lipid nanoparticles (LNPs) are among the most promising drug delivery carriers in research and development, with one major clinical application being messenger RNA (mRNA) vaccine. Current LNP production methods have the limit of generating low polydispersity index (PDI; PDI < 0.1) only for relatively small particles (<100 nm). It is known that larger LNPs have desirable properties, for example, particles with diameters in the 100 to 200 nm range have good immunogenicity. Yet, these larger particles' large PDI limits their clinical translation because of concerns about manufacturing reproducibility and possible side effects. We report here a facile approach to produce large and monodisperse (100-200 nm, PDI < 0.1) LNPs. The approach is based on adding 10 kDa polyethylene glycol (PEG) to a solution containing smaller LNPs. We show that PEG-induced depletion forces lead to the fusion of LNPs to form particles of approximately double the original size while keeping the same starting PDI. We discuss the fusion mechanism and show the parameters it depends on. In particular, we show that the fusion leads to a decrease in the fraction of empty LNPs. We show that the purification for PEG after fusion is simple and complete, thus, we believe that this is a method for the production of large LNP with low PDI that has a lot of potential to find industrial use.

Article	Year
Fluorescent fatty acid conjugates for live cell imaging of peroxisomes. Nature communications\| PubMed ID: 38773129	2024
Facile lipid nanoparticle size engineering approach via controllable fusion induced by depletion forces. Journal of colloid and interface science\| PubMed ID: 40147373	2025

Milena Schuhmacher

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