Dynamic tracing using ultra-bright labeling and multi-photon microscopy identifies endothelial uptake of poloxamer 188 coated poly(lactic-co-glycolic acid) nano-carriers in vivo.

The potential of poly(lactic-co-glycolic acid) (PLGA) to design nanoparticles (NPs) and target the central nervous system remains to be exploited. In the current study we designed fluorescent 70-nm PLGA NPs, loaded with bulky fluorophores, thereby making them significantly brighter than quantum dots in single-particle fluorescence measurements. The high brightness of NPs enabled their visualization by intravital real-time 2-photon microscopy. Subsequently, we found that PLGA NPs coated with pluronic F-68 circulated in the blood substantially longer than uncoated NPs and were taken up by cerebro-vascular endothelial cells. Additionally, confocal microscopy revealed that coated PLGA NPs were present in late endothelial endosomes of cerebral vessels within 1 h after systemic injection and were more readily taken up by endothelial cells in peripheral organs. The combination of ultra-bright NPs and in vivo imaging may thus represent a promising approach to reduce the gap between development and clinical application of nanoparticle-based drug carriers.

CAR+ extracellular vesicles predict ICANS in patients with B cell lymphomas treated with CD19-directed CAR T cells.

BACKGROUND: Predicting Immune-effector Cell Associated Neurotoxicity Syndrome (ICANS) in patients infused with Chimeric Antigen Receptor T cells (CAR-T) is still a conundrum. This complication, thought to be consequent to CAR-T cell activation, arises a few days after infusion, when circulating CAR-T cells are scarce and specific CAR-T cell-derived biomarkers are lacking. METHODS: Human CD19.CAR-T cells were generated to gain insight into CAR+ extracellular vesicle (CAR+EV) release upon target engagement. A prospective cohort of 100 B-cell lymphoma patients infused with approved CD19.CAR-T cell products (axi-cel, brexu-cel and tisa-cel) was assessed for plasma CAR+EVs as potential biomarkers of in vivo CD19.CAR-T cell activation and predictors of ICANS. Human induced pluripotent stem cells (iPSCs)-derived neural cells were used as a model for CAR+EV-induced neurotoxicity. RESULTS: In vitro, exosome-like CAR+EVs were released by CD19.CAR-T cells upon target engagement. In vivo, CAR+EVs were detectable as early as 1 hour in the plasma of patients. A concentration > 132.8 CAR+EVs/µl at hour +1 or > 224.5 CAR+EVs/µl at day +1 predicted ICANS in advance of 4 days, with a sensitivity up to 96.55% and a specificity up to 80.36%, outperforming other potential ICANS predictors. Enolase 2 (ENO2+) nanoparticles were released by iPSCs-derived neural cells upon CAR+EVs exposure and were increased in the plasma of ICANS patients. CONCLUSIONS: These results convey that plasma CAR+EVs are an immediate signal of CD19.CAR-T cell activation, are suitable predictors of neurotoxicity, and may be involved in ICANS pathogenesis. TRIAL REGISTRATION: NCT04892433, NCT05807789.

Smartphone-assisted detection of nucleic acids by light-harvesting FRET-based nanoprobe.

Point-of-care assays for optical detection of biomolecular markers attract growing attention, because of their capacity to provide rapid and inexpensive diagnostics of cancer and infectious diseases. Here, we designed a nanoprobe compatible with a smartphone RGB camera for detection of nucleic acids. It is based on light-harvesting polymeric nanoparticles (NPs) encapsulating green fluorescent donor dyes that undergo efficient Förster Resonance Energy Transfer (FRET) to red fluorescent acceptor hybridized at the particle surface. Green-emitting NPs are based on rhodamine 110 and 6G dyes paired with bulky hydrophobic counterions, which prevent dye self-quenching and ensure efficient energy transfer. Their surface is functionalized with a capture DNA sequence for cancer marker survivin, hybridized with a short oligonucleotide bearing FRET acceptor ATTO647N. Obtained 40-nm poly(methyl methacrylate)-based NP probe, encapsulating octadecyl rhodamine 6G dyes with tetrakis(perfluoro-tert-butoxy)aluminate counterions (~6000 dyes per NP), and bearing 65 acceptors, shows efficient FRET with >20% quantum yield and a signal amplification (antenna effect) of 25. It exhibits ratiometric response to the target DNA by FRET acceptor displacement and enables DNA detection in solution by fluorescence spectroscopy (limit of detection 3 pM) and on surfaces at the single-particle level using two-color fluorescence microscopy. Using a smartphone RGB camera, the nanoprobe response can be readily detected at 10 pM target in true color and in red-to-green ratio images. Thus, our FRET-based nanoparticle biosensor enables detection of nucleic acid targets using a smartphone coupled to an appropriate optical setup, opening the way to simple and inexpensive point-of-care assays.