Development of a Real-Time Cell Imaging Assay to Characterize the Delivery of a Cell-Penetrating Peptide Adaptor and its Associated Cargo

<p>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.</p>

Name thesis:degree_name

Master of Science in Integrative Biology (MSIB)

Level thesis:degree_level

Thesis

Discipline thesis:degree_discipline

Biology

Year dc:date.available

2021

Author dc:creator

• Gentry, Schuyler

Contributors dc:contributor

• Dr. Jonathan McMurry
• Dr. Carol Chrestensen
• Dr. Scott Nowak

• Cell-Penetrating Peptides
• Endosomal Escape
• Endocytosis
• Cytosolic Delivery
• Real-Time Imaging
• TAT-CaM
• Integrative Biology

Repository record dc:identifier

https://digitalcommons.kennesaw.edu/integrbiol_etd/60

OAI identifier oai:identifier

oai:digitalcommons.kennesaw.edu:integrbiol_etd-1060