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Cytosolic delivery remains a major bottleneck for siRNA therapeutics. To facilitate delivery, siRNAs are often enclosed in nanoparticles (NPs). However, upon endocytosis such NPs are mainly trafficked towards lysosomes. To avoid degradation, cytosolic release of siRNA should occur prior to fusion of endosomes with lysosomes, but current endosomal escape strategies remain inefficient. In contrast to this paradigm, we aim to exploit lysosomal accumulation by treating NP-transfected cells with low molecular weight drugs that release the siRNA from the lysosomes into the cytosol. We show that FDA-approved cationic amphiphilic drugs (CADs) significantly improved gene silencing by siRNA-loaded nanogels in cancer cells through simple sequential incubation. CADs induced lysosomal phospholipidosis, leading to transient lysosomal membrane permeabilization and improved siRNA release without cytotoxicity. Of note, the lysosomes could be applied as an intracellular depot for triggered siRNA release by multiple CAD treatments.

Long-term in vivo imaging of cells is crucial for the understanding of cellular fate in biological processes in cancer research, immunology or in cell-based therapies such as beta cell transplantation in type I diabetes or stem cell therapy. Traditionally, cell labelling with the desired contrast agent occurs ex vivo via spontaneous endocytosis, which is a variable and slow process that requires optimization for each particular label-cell type combination. Following endocytic uptake, the contrast agents mostly remain entrapped in the endolysosomal compartment, which leads to signal instability, cytotoxicity and asymmetric inheritance of the labels upon cell division. Here, we demonstrate that these disadvantages can be circumvented by delivering contrast agents directly into the cytoplasm via vapour nanobubble photoporation. Compared to classic endocytic uptake, photoporation resulted in 50 and 3 times higher loading of fluorescent dextrans and quantum dots, respectively, with improved signal stability and reduced cytotoxicity. Most interestingly, cytosolic delivery by photoporation prevented asymmetric inheritance of labels by daughter cells over subsequent cell generations. Instead, unequal inheritance of endocytosed labels resulted in a dramatic increase in polydispersity of the amount of labels per cell with each cell division, hindering accurate quantification of cell numbers in vivo over time. The combined benefits of cell labelling by photoporation resulted in a marked improvement in long-term cell visibility in vivo where an insulin producing cell line (INS-1E cell line) labelled with fluorescent dextrans could be tracked for up to two months in Swiss Nude mice compared to two weeks for cells labelled by endocytosis.

Small nucleic acid (NA) therapeutics, such as small interfering RNA (siRNA), are generally formulated in nanoparticles (NPs) to overcome the multiple extra- and intracellular barriers upon in vivo administration. Interaction with target cells typically triggers endocytosis and sequesters the NPs in endosomes, thus hampering the pharmacological activity of the encapsulated siRNAs that occurs in the cytosol. Unfortunately, for most state-of-the-art NPs, endosomal escape is largely inefficient. As a result, the bulk of the endocytosed NA drug is rapidly trafficked toward the degradative lysosomes that are considered as a dead end for siRNA nanomedicines. In contrast to this paradigm, we recently reported that cationic amphiphilic drugs (CADs) could strongly promote functional siRNA delivery from the endolysosomal compartment via transient induction of lysosomal membrane permeabilization. However, many questions still remain regarding the broader applicability of such a CAD adjuvant effect on NA delivery. Here, we report a drug repurposing screen (National Institutes of Health Clinical Collection) that allowed identification of 56 CAD adjuvants. We furthermore demonstrate that the CAD adjuvant effect is dependent on the type of nanocarrier, with NPs that generate an appropriate pool of decomplexed siRNA in the endolysosomal compartment being most susceptible to CAD-promoted gene silencing. Finally, the CAD adjuvant effect was verified on human ovarian cancer cells and for antisense oligonucleotides. In conclusion, this study strongly expands our current knowledge on how CADs increase the cytosolic release of small NAs, providing relevant insights to more rationally combine CAD adjuvants with NA-loaded NPs for future therapeutic applications.