Date of Award
Master of Science in Integrative Biology (MSIB)
Dr. Jonathan McMurry
First Committee Member
Dr. Jonathan McMurry
Second Committee Member
Dr. Carol Chrestensen
Third Committee Member
Dr. Scott Nowak
Cell-penetrating peptides (CPPs) are small peptides capable of transporting molecules across the membrane. In most cases, the membrane is a blockade that prevents molecules from entering the cell, therefore CPPs have attracted much attention. Although CPPs are effective, this system is still hindered by many issues. The endosomal entrapment problem is the main rate-limiting step for cytosolic delivery and is a result of endocytosis. To combat this issue, a new CPP, TAT-CaM, that can reach the cytosol has been developed. Conventional methods involve observing these peptides in a static manner, which has hindered a complete understanding of CPPs. Therefore, a real-time imaging assay was developed to characterize CPP-mediated delivery. With this assay, delivery was monitored in real-time and quantified. Additionally, experiments were performed in a static manner to gain insight into endosomal escape and the mechanism of protein uptake. Quantification of protein delivery showed a linear increase in protein uptake and a delivery time of 5-10 minutes. This analysis also showed a small decrease of fluorescence at later time-points. This was expected as proteins can be recycled out the cell or can be degraded. Inhibition of endocytic pathways did not provide insight into the uptake mechanism of this system, therefore future mechanistic studies must be completed. Lastly, this CPP significantly increased cytosolic delivery of cargo as protein appeared more diffuse across the cytosol of cells. The knowledge gained here will be vital in understanding CPP delivery and will put CPPs one step closer to becoming a viable therapeutic tool.
Gentry, Schuyler, "Development of a Real-Time Cell Imaging Assay to Characterize the Delivery of a Cell-Penetrating Peptide Adaptor and its Associated Cargo" (2021). Master of Science in Integrative Biology Theses. 60.
Available for download on Wednesday, May 06, 2026