Understanding How Endosomal Membrane Permeabilizing Agents Can Be Improved for Efficient Intracellular Delivery
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Date
2021-06-08
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Abstract
The ability to manipulate cells from within opens countless opportunities in the fields of fundamental life science research as well as medicine and therapeutics. Unfortunately, most molecular machines in a cell are sequestered behind the plasma membrane and are not easily accessible. Cell penetrating peptides (CPPs) and their ability to deliver biologically relevant cargos inside of cells have become promising tools for circumventing this issue. Currently, many CPPs that have shown the ability to deliver cargo into cells also suffer from a low endosomal escape activity. This results in most of the material being degraded and rendered useless.
In this study, I investigate how the problem of endosomal escape can be improved upon using a lysosomotropic hydrophobic weak base, UNC7938. This molecule sensitizes endosomes within the cell to enhance endosomolysis by several polycationic CPPs. Through this approach I show that we can improve the utility of such CPPs for delivery at lower concentrations and in the presence of serum. This combination can deliver DNA into cells refractory to transfection and a short chain variable fragment antibody. This suggests a unique chemical space to investigate for improving CPP-based delivery.
It is also possible to improve the endosomal escape activity of CPPs by modifying the structure of the CPP. Herein, I also investigate how modulating the hydrophobicity of an efficient multivalent TAT construct allows for the engineering of even more active CPPs. These constructs can be completely traceless (non-fluorescent and fully degradable) and enable delivery of functional gene editing machinery into the brain of mice.
Finally, I investigate the relationship between peptide hydrophobicity and activity and find that there is a positive correlation between hydrophobicity and peptide activity with an exception for when the hydrophobic moiety contains additional charges. Strikingly, this activity also comes at the cost of increased toxicity. While this is still being investigated, it appears that toxicity originates from both inside and outside the cell. Cells seem to be quickly permeabilized and initiate a downstream apoptotic response to peptide penetration.
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Cell penetrating peptide, Structure activity relationship, hydrophobicity, Tat, in vivo, cell delivery, molecular tools, lysosomotropic, late endosome