Publications

Nanoparticles (NPs) are used to encapsulate therapeutic cargos and deliver them specifically to the target site. The intracellular trafficking of NPs dictates the NP-cargo distribution within different cellular compartments, and thus governs their efficacy and safety. Knowledge in this field is crucial to understand their biological fate and improve their rational design. However, there is a lack of methods that allow precise localization and quantification of individual NPs within distinct cellular compartments simultaneously. Here, we address this issue by proposing a correlative light and electron microscopy (CLEM) method combining direct stochastic optical reconstruction microscopy (dSTORM) and transmission electron microscopy (TEM). We aim at combining the advantages of both techniques to precisely address NP localization in the context of the cell ultrastructure. Individual fluorescently-labelled poly(lactide-co-glycolide)-poly(ethylene glycol) (PLGA-PEG) NPs were directly visualized by dSTORM and assigned to cellular compartments by TEM. We first tracked NPs along the endo-lysosomal pathway at different time points, then demonstrated the effect of chloroquine on their intracellular distribution (i.e. endosomal escape). The proposed protocol can be applied to fluorescently labelled NPs and/or cargo, including those not detectable by TEM alone. Our studies are of great relevance to obtain important information on NP trafficking, and crucial for the design of more complex nanomaterials aimed at cytoplasmic/nucleic drug delivery.

JTD Keywords: chemistry, delivery, endocytosis, endosomal escape, exocytosis, fluorescence, light, size, tomography, Cellular uptake, Chemistry, Delivery, Endocytosis, Endosomal escape, Exocytosis, Fluorescence, Light, Size, Tomography

Abstract Nerves are a component of the tumor microenvironment contributing to cancer progression, but the role of cells from nerves in facilitating cancer invasion remains poorly understood. Here we show that Schwann cells (SCs) activated by cancer cells collectively function as Tumor Activated Schwann cell Tracks (TASTs) that promote cancer cell migration and invasion. Non-myelinating SCs form TASTs and have cell gene expression signatures that correlate with diminished survival in patients with pancreatic ductal adenocarcinoma. In TASTs, dynamic SCs form tracks that serve as cancer pathways and apply forces on cancer cells to enhance cancer motility. These SCs are activated by c-Jun, analogous to their reprogramming during nerve repair. This study reveals a mechanism of cancer cell invasion that co-opts a wound repair process and exploits the ability of SCs to collectively organize into tracks. These findings establish a novel paradigm of how cancer cells spread and reveal therapeutic opportunities.

JTD Keywords: dissemination, escape, mechanisms, progression, Perineural invasion

© 2021 by the authors. Licensee MDPI, Basel, Switzerland. Achieving intracellular delivery of protein therapeutics within cells remains a significant challenge. Although custom formulations are available for some protein therapeutics, the development of non‐toxic delivery systems that can incorporate a variety of active protein cargo and maintain their stability, is a topic of great relevance. This study utilized ionic polyphosphazenes (PZ) that can assemble into supramolecular complexes through non‐covalent interactions with different types of protein cargo. We tested a PEGylated graft copolymer (PZ‐PEG) and a pyrrolidone containing linear derivative (PZ‐PYR) for their ability to intracellularly deliver FITC‐avidin, a model protein. In endothelial cells, PZ‐PYR/protein exhibited both faster internalization and higher uptake levels than PZ‐PEG/protein, while in cancer cells both polymers achieved similar uptake levels over time, although the internalization rate was slower for PZ‐PYR/protein. Uptake was mediated by endocytosis through multiple mechanisms, PZ‐PEG/avidin colocalized more profusely with endo-lysosomes, and PZ‐PYR/avidin achieved greater cytosolic delivery. Consequently, a PZ‐PYR-delivered anti‐F‐actin antibody was able to bind to cytosolic actin filaments without needing cell permeabilization. Similarly, a cell‐impermeable Bax‐BH3 peptide known to induce apoptosis, decreased cell viability when complexed with PZ‐PYR, demonstrating endo‐lysosomal escape. These biodegradable PZs were non‐toxic to cells and represent a promising platform for drug delivery of protein therapeutics.

JTD Keywords: cytosolic delivery, cytotoxicity, delivery of apoptotic peptides, endosomal escape, intracellular delivery of antibody, intracellular protein delivery, Cytosolic delivery, Cytotoxicity, Delivery of apoptotic peptides, Endosomal escape, Intracellular delivery of antibody, Intracellular protein delivery, Polyphosphazene polymers