Rossetti, F. | bioRxiv Science

bioRxiv2021

Quantifying nanotherapeutics penetration using hydrogel based microsystem as a new 3D in-vitro platform

The huge gap between 2D in vitro assays used for drug screening, and the in vivo 3D-physiological environment hampered reliable predictions for the route and accumulation of nanotherapeutics in vivo. For such nanotherapeutics, Multi-Cellular Tumour Spheroids (MCTS) is emerging as a good alternative in vitro model. However, the classical approaches to produce MCTS suffer from low yield, slow process, difficulties in MCTS manipulation and compatibility with high-magnification fluorescent optical microscopy. On the other hand, spheroid-on-chip set-ups developed so far require a microfluidic practical knowledge difficult to transfer to a cell biology laboratory. We present here a simple yet highly flexible 3D-model microsystem consisting of agarose-based microwells. Fully compatible with the multi-well plates format conventionally used in cell biology, our simple process enables the formation of hundreds of reproducible spheroids in a single pipetting. Immunostaining and fluorescent imaging including live high-resolution optical microscopy can be performed in-situ, with no manipulation of spheroids. As a proof-of-principle of the relevance of such in vitro platform for nanotherapeutics evaluation, this study investigates the kinetic and localization of nanoparticles within colorectal cancer MCTS cells (HCT-116). The nanoparticles chosen are sub-5 nm ultrasmall nanoparticles made of polysiloxane and gadolinium chelates that can be visualized in MRI (AGuIX(R), currently implicated in clinical trials as effective radiosensitizers for radiotherapy) and confocal microscopy after addition of Cy 5.5. We show that the amount of AGuIX(R) nanoparticles within cells is largely different in 2D and 3D. Using our flexible agarose-based microsystems, we are able to resolve spatially and temporally the penetration and distribution of AGuIX(R) nanoparticles within MCTS. The nanoparticles are first found in both extracellular and intracellular space of MCTS. While the extracellular part is washed away after few days, we evidenced intracellular localisation of AGuIX(R), mainly within lysosomes compartment, but also occasionally within mitochondria. Our agarose-based microsystem appears hence as a promising 3D in vitro user-friendly platform for investigation of nanotherapeutics transport, ahead of in vivo studies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/427020v2_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@bd48adorg.highwire.dtl.DTLVardef@ee7502org.highwire.dtl.DTLVardef@143ee60org.highwire.dtl.DTLVardef@d6f5ca_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

bioengineering↗

bioRxiv2018

Altered Fecal Microbiota and Urine Metabolome as Signatures of Soman Poisoning

The experimental pathophysiology of organophosphorus (OP) chemical exposure has been extensively reported. Here, we describe an altered fecal microbiota and urine metabolome that follows intoxication with soman, a lipophilic G class chemical warfare nerve agent. Non-anaesthetized Sprague-Dawley male rats were subcutaneously administered soman at 0.8 - 1.0 of the median lethal dose (LD50) and evaluated for signs of toxicity. Animals were stratified based on seizing activity to evaluate effects of soman exposure on fecal bacterial biota and urine metabolites. Soman exposure reshaped fecal bacterial biota by preferentially expanding Facklamia, Agrobacterium, Bilophila, Enterobacter, and Morganella genera of the Firmicutes and Proteobacteria phyla, some of which are known to hydrolyze OPs. However, analogous changes were not observed in the bacterial biota of the ileum, which remained the same irrespective of dose or seizing status of animals after exposure. Interestingly, when considering just the seizing status of animals, we found that the urine metabolome was markedly altered. Leukotriene C4, kynurenic acid, 5-hydroxyindoleacetic acid, norepinephrine, and aldosterone were excreted at much higher rates at 72 hrs in seizing animals, consistent with early multi-organ involvement during soman poisoning. However, at 75 days post soman exposure, bacterial biota stabilized and no differences were observed. These findings demonstrate the feasibility of using the dysbiosis of fecal bacterial biota in combination with urine metabolome alterations as forensic evidence for OP exposure temporally.\n\nImportanceThe paucity of assays to determine physiologically relevant OP exposure presents an opportunity to explore the use bacterial sentinels in combination with urine to assess changes in the exposed host. Recent advances in technologies and computational approaches have enabled researches to survey large community level changes of gut bacterial biota and metabolomic changes in various biospecimens. Here, we profile combined changes in bacterial biota and urine metabolome due to chemical warfare OP exposure. The significance of our work is to reveal that monitoring bacterial biota and urine metabolites as surrogates of OP exposure in biospecimens suitable for existing clinical laboratory workflows is plausible without the need for the development of new technology, invasive procedures, or complicated analytical approaches. The larger value of such an approach is that any setting with a moderate clinical chemistry and microbiology capability can determine pre-symptomatic exposure to enhance current triage standards in case of mass exposures, refugee movements, humanitarian missions, and training settings once an algorithm has been validated. In the event of \"potential\" exposures by time or distance, this assay can be further developed to estimate affected radius or time dimension for health monitoring and treatment interventions.

physiology↗