The therapeutic and research potentials of oligonucleotides (ONs) have been hampered in part by their inability to effectively escape endosomal compartments to reach their cytosolic and nuclear targets. Splice-switching ONs (SSOs) can be used with endosomolytic small molecule compounds to increase functional delivery. So far, development of these compounds has been hindered by a lack of high-resolution methods that can correlate SSO trafficking with SSO activity. Here we present in-depth characterization of two novel endosomolytic compounds by using a combination of microscopic and functional assays with high spatiotemporal resolution. This system allows the visualization of SSO trafficking, evaluation of endosomal membrane rupture, and quantitates SSO functional activity on a protein level in the presence of endosomolytic compounds. We confirm that the leakage of SSO into the cytosol occurs in parallel with the physical engorgement of LAMP1-positive late endosomes and lysosomes. We conclude that the new compounds interfere with SSO trafficking to the LAMP1-positive endosomal compartments while inducing endosomal membrane rupture and concurrent ON escape into the cytosol. The efficacy of these compounds advocates their use as novel, potent, and quick-acting transfection reagents for antisense ONs.

Extracellular vesicles (EVs) are nanosized cell-derived vesicles produced by all cells, which provide a route of intercellular communication by transmitting biological cargo. While EVs offer promise as therapeutic agents, the molecular mechanisms of EV biogenesis are not yet fully elucidated, in part due to the concurrence of numerous interwoven pathways which give rise to heterogenous EV populations in vitro. The equilibrium between the EV-producing pathways is heavily influenced by factors in the extracellular environment, in such a way that can be taken advantage of to boost production of engineered EVs. In this study, a quantifiable EV-engineering approach is used to investigate how different cell media conditions alter EV production. The presence of serum, exogenous EVs, and other signaling factors in cell media alters EV production at the physical, molecular, and transcriptional levels. Further, it is demonstrated that the ceramide-dependent EV biogenesis route is the major pathway to production of engineered EVs during optimized EV-production. These findings suggest a novel understanding to the mechanisms underlying EV production in cell culture which can be applied to develop advanced EV production methods.

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Histone deacetylase (HDAC) proteins are promising targets for cancer treatment, with several HDAC inhibitors used clinically as anticancer drugs. Most HDAC inhibitors nonspecifically interact with all or many of the 11 HDAC isoforms. Isoform-selective HDAC inhibitors would be useful tools to dissect the individual functions of HDAC proteins in cancer formation, in addition to potentially displaying effective anticancer properties. We report here a robust HDAC activity assay for screening selective HDAC inhibitors, which is inspired by the traditional enzyme-linked immunosorbent assay (ELISA). The key feature of the ELISA-based HDAC activity assay is use of mammalian cell-derived HDAC isoforms instead of recombinant proteins. Importantly, the assay was validated with several known HDAC inhibitors. The ELISA-based HDAC activity assay will facilitate the characterization of isoform-selective HDAC inhibitors against mammalian cell-derived HDAC proteins, which will enhance HDAC-centered cancer research and provide a foundation for anticancer drug development.

A new ferrofluidic droplet generator is demonstrated using an electropermanent magnet to change the viscosity of the ferrofluid oil phase in a flow-focusing junction, which enables control of the aqueous droplet size. Electropermanent magnets are capable of fast switching (< 100 $$\upmu$$μs) and have magnetic field strengths comparable to those of permanent magnets. When switched on, the magnetoviscous effect increases the viscosity of the ferrofluid, thus increasing the channel’s fluidic resistance, slowing the flow rate of the oil-based phase and changing the droplet diameter. We have demonstrated on-demand droplet size modulation as large as 44% at speeds fast enough to modulate the size of a single droplet in a stream. Due to compliance in the supply tubing and Polydimethylsiloxane (PDMS) microfluidic channels, the change in droplet size relaxes with a time scale of seconds. An application of droplet size modulation was demonstrated by integrating a passive size-based magnetic droplet sorting stage.