In this report, we designed and synthesized a novel fluorescent single tailed surfactant (termed FEDS), which can disrupt endosomes, complex lipofectamine, and can also identify cells that have been transfected. FEDS was able to increase the gene editing efficiency of lipofectamine/Cas9 ribonucleoprotein by 300% via a combination of fluorescent based enrichment and endosomal disruption.

Protein therapeutics based on transcription factors, gene editing enzymes, signaling proteins and protein antigens, have the potential to provide cures for a wide number of untreatable diseases, but cannot be developed into therapeutics due to challenges in delivering them into the cytoplasm. There is therefore great interest in developing strategies that can enable proteins to enter the cytoplasm of cells. In this review article we will discuss recent progress in intracellular protein therapeutics, which are focused on the following four classes of therapeutics, Firstly, vaccine development, secondly, transcription factor therapies, thirdly, gene editing and finally, cancer therapeutics. These exciting new advances raise the prospect of developing cures for several un-treatable diseases.

We present an autocatalytic system for the detection and amplification of thiols termed the Methionase Chain Reaction (MCR). MCR is based on the reversible modification of the thiol producing enzyme Methionine Gamma-Lyase (MGL). MCR was able to amplify the concentration of thiols by a factor of 560 and was able to visually detect thiols at concentrations as low as 50 nM.

Reactive oxygen species (ROS) play a pivotal role in many cellular processes and can be either beneficial or harmful. The design of ROS-sensitive fluorophores has allowed for imaging of specific activity and has helped elucidate mechanisms of action for ROS. Understanding the oxidative role of ROS in the many roles it plays allows us to understand the human body. This review provides a concise overview of modern advances in the field of ROS imaging. Indeed, much has been learned about the role of ROS throughout the years; however, it has recently been shown that using nanoparticles, rather than individual small organic fluorophores, for ROS imaging can further our understanding of ROS.

Reduction sensitive linkers (RSLs) have the potential to transform the field of drug delivery due to their ease of use and selective cleavage in intracellular environments. However, despite their compelling attributes, developing reduction sensitive self-immolative linkers for aliphatic amines has been challenging due to their poor leaving group ability and high pK _a values. Here a traceless self-immolative linker composed of a dithiol-ethyl carbonate connected to a benzyl carbamate (DEC) is presented, which can modify aliphatic amines and release them rapidly and quantitatively after disulfide reduction. DEC was able to reversibly modify the lysine residues on CRISPR-Cas9 with either PEG, the cell penetrating peptide Arg₁₀, or donor DNA, and generated Cas9 conjugates with significantly improved biological properties. In particular, Cas9-DEC-PEG was able to diffuse through brain tissue significantly better than unmodified Cas9, making it a more suitable candidate for genome editing in animals. Furthermore, conjugation of Arg₁₀ to Cas9 with DEC was able to generate a self-delivering Cas9 RNP that could edit cells without transfection reagents. Finally, conjugation of donor DNA to Cas9 with DEC increased the homology directed DNA repair (HDR) rate of the Cas9 RNP by 50% in HEK 293T cell line. We anticipate that DEC will have numerous applications in biotechnology, given the ubiquitous presence of aliphatic amines on small molecule and protein therapeutics.