Imaging small molecule-induced endosomal escape of siRNA

Abstract

Small interfering RNAs (siRNAs) are a new class of promising therapeutic molecules that can be used for sequence-specific downregulation of disease-causing genes. However, endosomal entrapment of siRNA is a key hurdle for most delivery strategies, limiting the therapeutic effect. Here, we use live-cell microscopy and cytosolic galectin-9 as a sensor of membrane damage, to probe fundamental properties of endosomal escape of cholesterol-conjugated siRNA induced by endosome-disrupting compounds. We demonstrate efficient release of ligand-conjugated siRNA from vesicles damaged by small molecules, enhancing target knockdown up to ∼47-fold in tumor cells. Still, mismatch between siRNA-containing and drug-targeted endolysosomal compartments limits siRNA activity improvement. We also show widespread endosomal damage in macroscopic tumor spheroids after small molecule treatment, substantially improving siRNA delivery and knockdown throughout the spheroid. We believe the strategy to characterize endosomal escape presented here will be widely applicable, facilitating efforts to improve delivery of siRNA and other nucleic acid-based therapeutics.

A high-throughput Galectin-9 imaging assay for quantifying nanoparticle uptake, endosomal escape and functional RNA delivery

“…Not all delivery vehicles induce Galectin recruitment responses. Recently, extracellular vehicles were demonstrated to act in a GAL3-independent manner, however, it should be noted that GAL3 is a weaker identifier of endosomal damage than GAL9 22,43 . It will be interesting to examine whether other emerging delivery vectors for oligonucleotides such as dendrimers, particle modifications with cell-penetrating peptides or the use of viral-like particles invoke GAL9 recruitment responses that are similar to those induced by LNPs.…”

“…2a). We first used chloroquine, a cationic amphiphilic drug (CAD), which accumulates in late endosomal compartments (Maxfield 1982) and that was recently used by Du Rietz et al to study endosome rupture in GFP-GAL9 expressing HeLa cells 22,26 . Following administration of 0-80 µM chloroquine, we observed a clear, dose-dependent induction of large and bright mCherry-GAL9 positive structures with a maximum response after 8-12 h of treatment and at ~60-80 µM, consistent with the positive response obtained by Du Rietz et al (Fig.…”

“…Endosomal escape induced localisation of GAL8 was shown to recruit autophagy adaptors as part of a clearance response against Salmonella and therefore forms an important part of cellular host-defence 20 . GAL9 recruitment has no similarly reported perturbing effects on normal cellular function and was recently reported to evoke a greater recruitment response to endolysosome damage than GAL3 or GAL8 22 .…”

“Repair Me if You Can”: Membrane Damage, Response, and Control from the Viral Perspective

“…Endosomal membrane damage can also be caused via a mechanical/physical trigger, especially in the context of intracellular drug delivery, where it is important to achieve efficient endosomal escape. Some lipid formulations used in transfection of siRNAs were shown to be particularly efficient in causing endosomal membrane damage [ 84 , 85 ]. Gold nanoparticles and nanodiamonds were also employed to physically disrupt the endosomal membrane [ 86 , 87 ].…”

Development of New Strategies Using Extracellular Vesicles Loaded with Exogenous Nucleic Acid

“…Nucleic acid-based therapies are rapidly evolving in preclinical and clinical trials for several genetic diseases [1][2][3]. Viral vectors have been the primary carriers of nucleic acids used in gene therapy; due to their high transduction efficiency and absence of integration into the host genome, adenovirus (AV) vectors initially offered the promise of highly efficient and therapeutic in vivo gene delivery [4][5][6].…”