Biology subjects

Du Rietz, H.

Publications and source records attributed to Du Rietz, H..

3 recordsLinked to original sources

bioRxiv2026

Nutritional Access Modulates Activity of a Small Molecule Enhancer of Endosomal Escape

For siRNA drugs to be relevant in tumors, poor endosomal escape of these drugs needs to be addressed. Endosomal escape can occur when the endolysosomal membrane is damaged and can be visualized by endogenously expressed fluorescent galectin-9 functioning as damage sensors. Tumor cells have unstable membranes and central parts of tumors have low nutrient levels contributing to reactive oxygen species which can induce membrane damage. We show that nutrient depletion alone does not induce endolysosomal membrane damage in HeLa and MCF7 cells or in HeLa spheroids. Serum depletion, however, enhanced endolysosomal membrane damage in HeLa cells when combined with the membrane destabilizing drug chloroquine, a cationic amphiphilic drug. This effect was almost completely abolished when depleting the cells of glucose, even when serum was present. This phenomenon could not be seen with other cationic amphiphilic drugs like siramesine and loperamide. In a functional experiment, co-treatment with chloroquine and siGFP significantly improved knockdown of GFP in the presence but not in the absence of glucose. Our results have implications for the development of chloroquine as an endosomal escape enhancer of RNA therapeutics in tumor contexts and stresses the importance of considering nutrient levels and tumor size in future screenings.

cancer biology↗

bioRxiv2024

Cellular and biophysical barriers to lipid nanoparticle mediated delivery of RNA to the cytosol

Lipid nanoparticle (LNP) formulation was the first approved delivery strategy for liver-targeted siRNA delivery and currently represent the most advanced platform for delivery of therapeutic mRNA in clinical use, after the approval of mRNA-based vaccines against the SARS-CoV-2 coronavirus. Still, poor efficiency of LNPs to promote cytosolic delivery of both mRNA and siRNA after internalization by target cells limits their potency and thus clinical application as RNA delivery vehicles - especially in extrahepatic tissues and tumors. It is still unclear how endosomal escape of LNP-delivered RNA cargo occurs, and why only a minute proportion of all therapeutic molecules reach the cytoplasm. Here, we explored the intracellular sorting, integrity and endosomal escape of both mRNA and siRNA loaded LNPs to characterize the nature of RNA release from endosomal compartments and pinpoint current bottlenecks. Using live-cell imaging and super-resolution microscopy of LNPs with fluorescently labeled RNA payload, we identify multiple distinct steps of substantial inefficiency in the cytosolic delivery of nucleic acid cargoes. We demonstrate that membrane damages marked by recruitment of galectins are conducive to cytosolic RNA delivery, while membrane perturbations recruiting the ESCRT machinery do not permit endosomal escape. By quantitative single-vesicle analysis, we show that only a small fraction of the nucleic acid cargo contained in the endosome is released to the cytosol upon LNP-triggered membrane damage detected by galectins. Unexpectedly, we also observe that only a subset of damaged endosomes contain RNA payload. Through FRET and super-resolution microscopy, using LNPs formulated with both fluorescently labeled ionizable lipid (MC3-BODIPY) and RNA, we find that RNA cargo and ionizable lipid segregate during endosomal sorting - both within single endosomes and to different endosomal compartments. Finally, we visualize localized MC3-BODIPY enrichment in endosomal membranes and membrane damage in direct proximity to siRNA-LNPs tethered to the luminal vesicle membrane. Taken together, this work has identified multiple mechanistically distinct barriers limiting intracellular RNA delivery by LNPs.

bioengineering↗

bioRxiv2021

Absolute quantification and single-cell dose-response of cytosolic siRNA delivery

Endosomal escape and subsequent cytosolic delivery of small inhibitory RNA (siRNA) therapeutics is believed to be highly inefficient. Since, it has not been possible to quantify cytosolic amounts of delivered siRNA at therapeutic doses, determination of delivery bottlenecks and total efficiency has been difficult. Here, we present a confocal microscopy-based method to detect cytosolic delivery of fluorescently labelled siRNA during lipid-mediated delivery. This method enables detection and quantification of sub-nanomolar cytosolic siRNA release amounts from individual release events with measures of quantitation confidence for each event. Single-cell kinetics of siRNA-mediated knockdown in cells expressing destabilized eGFP unveiled a dose-response relationship with respect to knockdown induction, depth and duration in the range from several hundred to thousands of cytosolic siRNA molecules. Accurate quantification of cytosolic siRNA, and the establishment of the intracellular dose-response relationships, will aid the development and characterization of novel delivery strategies for nucleic acid therapeutics.

cell biology↗