Sleeping Beauty mRNA-LNP enables stable rAAV transgene expression in mouse and NHP hepatocytes and improves vector potency.

Recombinant adeno-associated virus (rAAV) vector gene delivery systems have demonstrated great promise in clinical trials but continue to face durability and dose-related challenges. Unlike rAAV gene therapy, integrating gene addition approaches can provide curative expression in mitotically active cells and pediatric populations. We explored a novel in vivo delivery approach based on an engineered transposase, Sleeping Beauty (SB100X), delivered as an mRNA within a lipid nanoparticle (LNP), in combination with an rAAV-delivered transposable transgene. This combinatorial approach achieved correction of ornithine transcarbamylase deficiency in the neonatal Spfash mouse model following a single delivery to dividing hepatocytes in the newborn liver. Correction remained stable into adulthood, while a conventional rAAV approach resulted in a return to the disease state. In non-human primates, integration by transposition, mediated by this technology, improved gene expression 10-fold over conventional rAAV-mediated gene transfer while requiring 5-fold less vector. Additionally, integration site analysis confirmed a random profile while specifically targeting TA dinucleotides across the genome. Together, these findings demonstrate that transposable elements can improve rAAV-delivered therapies by lowering the vector dose requirement and associated toxicity while expanding target cell types.

Water-Dispersible and Biocompatible Iron Carbide Nanoparticles with High Specific Absorption Rate.

Magnetic nanoparticles are important tools for biomedicine, where they serve as versatile multifunctional instruments for a wide range of applications. Among these applications, magnetic hyperthermia is of special interest for the destruction of tumors and triggering of drug delivery. However, many applications of magnetic nanoparticles require high-quality magnetic nanoparticles displaying high specific absorption rates (SARs), which remains a challenge today. We report here the functionalization and stabilization in aqueous media of highly magnetic 15 nm iron carbide nanoparticles featuring excellent heating power through magnetic induction. The challenge of achieving water solubility and colloidal stability was addressed by designing and using specific dopamine-based ligands. The resulting nanoparticles were completely stable for several months in water, phosphate, phosphate-buffered saline, and serum-containing media. Iron carbide nanoparticles displayed high SARs in water and viscous media (water/glycerol mixtures), even after extended exposition to water and oxygen (SAR up to 1000 W·g-1 in water at 100 kHz, 47 mT). The cytotoxicity and cellular uptake of iron carbide nanoparticles could be easily tuned and were highly dependent on the chemical structure of the ligands used.

Rapid phenotyping of cancer stem cells using multichannel nanosensor arrays.

Cancer stem cells (CSCs) contribute to multidrug resistance, tumor recurrence and metastasis, making them prime therapeutic targets. Their ability to differentiate and lose stem cell properties makes them challenging to study. Currently, there is no simple assay that can quickly capture and trace the dynamic phenotypic changes on the CSC surface. Here, we report rapid discrimination of breast CSCs from non-CSCs using a nanoparticle-fluorescent-protein based sensor. This nanosensor was employed to discriminate CSCs from non-CSCs, as well as CSCs that had differentiated in vitro in two breast cancer models. Importantly, the sensor platform could also discriminate CSCs from the bulk population of cells in patient-derived xenografts of human breast cancer. Taken together, the results obtained demonstrate the feasibility of using the nanosensor to phenotype CSCs and monitor their fate. Furthermore, this approach provides a novel area for therapeutic interventions against these challenging targets.

Modulating the Catalytic Activity of Enzyme-like Nanoparticles Through their Surface Functionalization.

The inclusion of transition metal catalysts into nanoparticle scaffolds permits the creation of catalytic nanosystems (nanozymes) able to imitate the behaviour of natural enzymes. Here we report the fabrication of a family of nanozymes comprised of bioorthogonal ruthenium catalysts inserted in the protective monolayer of gold nanoparticles. By introducing simple modifications to the functional groups at the surface of the nanozymes, we have demonstrated control over the kinetic mechanism of our system. Cationic nanozymes with hydrophobic surface functionalities tend to replicate the classical Michaelis Menten model, while those with polar groups display substrate inhibition behaviour, a key mechanism present in 20 % of natural enzymes. The structural parameters described herein can be used for creating artificial nanosystems that mimic the complexity observed in cell machinery.

Cellular imaging of endosome entrapped small gold nanoparticles.

Small gold nanoparticles (sAuNPs, <10 nm in a core diameter) have been used for drug delivery and cancer therapy due to their high payload to carrier ratio. Information about the amount and location of sAuNPs in cells and tissues is critical to many applications. However, the current detection method (i.e., transmission electron microscopy) for such sAuNPs is limited due to the extensive sample preparation and the limited field of view. Here we use confocal laser scanning microscopy to provide endosome-entrapped sAuNP distributions and to quantify particle uptake into cells. The quantitative capabilities of the system were confirmed by inductively coupled plasma-mass spectrometry, with an observed linear relation between scattering intensity and the initial cellular uptake of sAuNPs using 4 nm and 6 nm core particles. The summary of the method is: •This non-invasive imaging strategy provides a tool for label-free real-time tracking and quantification of sAuNPs using a commercially available confocal laser scanning microscope.•Scattering intensity depends on particle size.•The linear relation established between scattering intensity and uptaken gold amount enables simultaneous quantitative assessment through simple image analysis.

Fabrication of functional nanofibers through post-nanoparticle functionalization.

A facile method is developed to functionalize nanofiber surfaces with nanoparticles (NPs) through dithiocarbamate chemistry. Gold nanoparticles (AuNPs) and quantum dots (QDs) are immobilized on the nanofiber surface. These surfaces provide scaffolds for further supramolecular functionalization, as demonstrated through the Förster resonance energy transfer (FRET) pairing of QD-decorated fibers and fluorescent proteins.

Engineering the nanoscale morphology of a quantum dot-fullerene assembly via complementary hydrogen bonding interactions.

We have demonstrated controlled assembly between CdSe quantum dots (QDs) and a fullerene (C60) derivative via complementary three-point hydrogen bonding interactions. The recognition-mediated assembly facilitated an interpenetrated network morphology and hence efficient charge transfer from QD to C60.