Suchergebnisse

© 2021 The Author(s). ; Revealing the intracellular location of novel therapeutic agents is paramount for the understanding of their effect at the cell ultrastructure level. Here, we apply a novel correlative cryo 3D imaging approach to determine the intracellular fate of a designed protein–nanomaterial hybrid with antifibrotic properties that shows great promise in mitigating myocardial fibrosis. Cryo 3D structured illumination microscopy (cryo-3D-SIM) pinpoints the location and cryo soft X-ray tomography (cryo-SXT) reveals the ultrastructural environment and subcellular localization of this nanomaterial with spatial correlation accuracy down to 70 nm in whole cells. This novel high resolution 3D cryo correlative approach unambiguously locates the nanomaterial after overnight treatment within multivesicular bodies which have been associated with endosomal trafficking events by confocal microscopy. Moreover, this approach allows assessing the cellular response towards the treatment by evaluating the morphological changes induced. This is especially relevant for the future usage of nanoformulations in clinical practices. This correlative super-resolution and X-ray imaging strategy joins high specificity, by the use of fluorescence, with high spatial resolution at 30 nm (half pitch) provided by cryo-SXT in whole cells, without the need of staining or fixation, and can be of particular benefit to locate specific molecules in the native cellular environment in bio-nanomedicine. ; This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 754397. This work was partially supported by the European Research Council ERC-CoG-648071-ProNANO, ERC-PoC-841063-NIMM; Spanish State Research Agency, Spain; (BIO2015-72124-EXP), (MDM-2017-0720), (PID2019-111649RB-I00) and (RTI2018-095214-B-I00); the Basque Government (Elkartek KK-2017/00008; RIS3-2019222005); and the Next Generation and Innovation Valdecilla (PREVAL19/05), (IDIVAL INNVAL17/22), (INNVAL20/34), (NVAL18/11) and (NextVal 18/14). This work was performed under the Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency – Grant No. MDM-2017-0720 (CIC biomaGUNE).