BackgroundEvidence indicates that Bax functions as a “lipidic” pore to regulate mitochondrial outer membrane permeabilization (MOMP), the apoptosis commitment step, through unknown membrane elements. Here we show mitochondrial ceramide elevation facilitates MOMP-mediated cytochrome c release in HeLa cells by generating a previously-unrecognized mitochondrial ceramide-rich macrodomain (MCRM), which we visualize and isolate, into which Bax integrates.Methodology/Principal FindingsMCRMs, virtually non-existent in resting cells, form upon irradiation coupled to ceramide synthase-mediated ceramide elevation, optimizing Bax insertion/oligomerization and MOMP. MCRMs are detected by confocal microscopy in intact HeLa cells and isolated biophysically as a light membrane fraction from HeLa cell lysates. Inhibiting ceramide generation using a well-defined natural ceramide synthase inhibitor, Fumonisin B1, prevented radiation-induced Bax insertion, oligomerization and MOMP. MCRM deconstruction using purified mouse hepatic mitochondria revealed ceramide alone is non-apoptogenic. Rather Bax integrates into MCRMs, oligomerizing therein, conferring 1–2 log enhanced cytochrome c release. Consistent with this mechanism, MCRM Bax isolates as high molecular weight “pore-forming” oligomers, while non-MCRM membrane contains exclusively MOMP-incompatible monomeric Bax.Conclusions/SignificanceOur recent studies in the C. elegans germline indicate that mitochondrial ceramide generation is obligate for radiation-induced apoptosis, although a mechanism for ceramide action was not delineated. Here we demonstrate that ceramide, generated in the mitochondrial outer membrane of mammalian cells upon irradiation, forms a platform into which Bax inserts, oligomerizes and functionalizes as a pore. We posit conceptualization of ceramide as a membrane-based stress calibrator, driving membrane macrodomain organization, which in mitochondria regulates intensity of Bax-induced MOMP, and is pharmacologically tractable in vitro and in vivo.

Few reports quantitatively compare the performance of photoacoustic tomography (PAT) versus fluorescence molecular tomography (FMT) in vivo . We compared both modalities for the detection of signals from injected ICG liposomes in the tibial medullary space of 10 BALB/c mice in vivo and ex vivo . Signals significantly correlated between modalities (R² = 0.69) and within each modality in vivo versus ex vivo (PAT: R² = 0.70, FMT: R² = 0.76). Phantom studies showed that signals at 4 mm depth are detected down to 3.3 ng ICG by PAT and 33 ng by FMT, with a nominal spatial resolution below 0.5 mm in PAT and limited to 1 mm in FMT. Our study demonstrates comparable in vivo sensitivity, but superior ex vivo sensitivity and in vivo resolution for our ICG liposomes of the VevoLAZR versus the FMT2500. PAT provides a useful new tool for the high-resolution imaging of bone marrow signals, for example for monitoring drug delivery.

Background: The purpose of our study was to find a novel targeted imaging and drug delivery vehicle for inflammatory bowel disease (IBD). IBD is a common and troublesome disease which still lacks effective therapy and imaging options. As an attempt to improve the disease treatment, we tested αMSH for the targeting of nanoliposomes to IBD sites. αMSH, an endogenous tridecapeptide, binds to the melanocortin-1 receptor (MC1-R) and has antiinflammatory and immunomodulating effects. MC1-R are found on macrophages, neutrophils and the renal tubule system. We have formulated and tested a liposomal nanoparticle involving αMSH in order to achieve a specific targeting to the inflamed intestines. Methods: NDP-αMSH peptide conjugated to Alexa Fluor™ 680 was linked to the liposomal membrane via N-Succinyl PE and additionally loaded into the lumen of the liposomes. Liposomes without the αMSH-conjugate and free NDP- αMSH were used as a control. The liposomes were also loaded with ICG to track them. The liposomes were tested in DSS treated mice, which had received DSS via drinking order to develop a model IBD. Inflammation severity was assessed by the Disease Activity Index (DAI) score and ex vivo histological CD68 staining of samples taken from different parts of the intestine. The liposome targeting was analyzed by analyzing the ICG and ALEXA 680 fluorescence in the intestine compared to the biodistribution. Results: NPD-αMSH was successful labelled with Alexa and retained its biological activity. Liposomes located to expected destinations in the inflamed bowel regions and in the kidneys, where MC1-R are abundant. In vivo liposome targeting correlated with the macrophage concentration at the site of the inflammation supporting the active targeting of the liposomes through αMSH. The liposomal αMSH was well tolerated by animals. Conclusions: This study opens up the possibility to further develop an αMSH targeted theranostic delivery towards clinically relevant different applications in IBD inflammation but also opens possibilities in other inflammations like lung inflammation in Covid 19.

Background: Nanoparticle imaging and the imaging of the release of the loaded material from a nanoparticle system have attracted great attention in recent years. If the release of loaded molecules from delivery vehicles could be monitored reliably in vivo, it would speed up the development of drug delivery systems remarkably. Methods: Here we test a system that uses indocyanine green (ICG) as a fluorescent agent for studying release kinetics in vitro and in vivo of a lipid iron nanoparticle delivery system. The ICG spectral properties like its concentration, sensitivity and its fluctuations of absorption and emission wavelengths of the fluorescence can be utilized for gathering information about the change of the ICG surroundings. Results: We have found that the absorption, fluorescence, and photoacoustic spectra of the ICG in lipid iron nanoparticles differ from the spectra of ICG in the pure water and in plasma. We followed the ICG containing liposomal nanoparticle uptake into the squamous carcinoma cells (SCC) under a fluorescence microscopy and into SCC tumor in the nude mice orthotopic xenografts model under a surgical microscope. Conclusion: Absorption and emission properties of ICG in different solvent environment, like in plasma and human serum albumin, differ from those in aqueous solution. Photoacoustic spectral imaging confirmed a peak shift towards longer wavelengths and an intensity increase of ICG when bound to lipid. The SCC cells showed that the ICG containing liposomes bind to cell surface but are not internalized in the SCC-9 cells in 30 minutes’ incubation. We also showed here that ICG containing liposomal nanoparticles can be traced under a surgical camera in vivo in orthotopic SCC xenografts in mice.