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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session C

Poster I5 - Mechanistic Study of Structured Cell Penetrating Peptides (CPPs) Uptake by Inhibition of Endocytosis Pathways

Friday, October 25, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Cell Penetrating Peptides, or CPPs, are an ideal method to deliver cargoes such as drugs, biosensors, or nanoparticles to intact cells without disrupting the plasma membrane. CPPs are short (~10-15 residues) sequences that are rich in positively charged amino acids that allows for electrostatic interactions between negatively charged head groups of the phospholipid membrane and the cationic peptide leading to the association of the peptide to the cell surface. While numerous CPPs have been identified and shown to deliver cargoes into cells, the mechanism of entry is still unknown. This is due to wide variations of CPP structural, chemical, and physical properties which can potentially alter the uptake mechanism. Our prior work has demonstrated a tradeoff between the uptake kinetics and intracellular stability of structured vs. unstructured CPPs; however, it is unclear how the different types of CPPs are internalized into intact cells. The goal of this study was to investigate this to provide new insight on the uptake mechanism in structured vs. unstructured CPPs using select inhibitors of endocytosis pathways such as clathrin-mediated, caveolae-mediated, and macropinocytosis. We hypothesize that inhibiting these endocytic pathways will provide new evidence on the mechanism of uptake in the different types of CPPs and will show how the secondary structure of CPPs play a role in their ability to be internalized into intact cells. These findings will provide new insight on how specific aspects of CPPs can alter their uptake which will translate to their ability to deliver select cargoes to intact cells.

Materials and

Methods:  Two different structured CPPs were studied that exhibit a β-hairpin sequence motif. The sequences were XWVRVpGO(FAM)WIRQ, where “p” denotes D-proline and ‘X’ is either an ornithine or arginine residue. Both peptides were synthesized using solid phase peptide synthesis and include a FAM (5,6, carboxyfluorescein) tag conjugated to an ornithine residue to allow for visualization and quantification using fluorescence microscopy and fluorometry. An unstructured, commercially available CPP (polyarginine) was also used. Select inhibitors of macropinocytosis, caveolae-mediated endocytosis, and energy dependence were used including: 5-(N-ethyl-N-isopropyl)-Amiloride (EIPA) at 100 µM, methyl-β-cyclodextrin (MꞵCD) at 5000 µM, and sodium azide (SA) at 10 mM. For each experiment, HeLa cells were plated at a density of 1x104 in 12-well plates and allowed to grow and spread for three days prior to interrogation. For each experiment, cells were incubated with one inhibitor for one hour and then the peptide for two hours at concentrations of 5, 10, and 20 µM at 37°C. After incubation, the cells were washed with extracellular buffer twice and treated with trypsin-EDTA to detach the cells and remove any residual extracellular peptide. The cells were then lysed with 0.1 M NaOH and clarified lysates were evaluated using fluorometry. Fluorescence signals were normalized using the equation: Normalized Fluorescence = 1000 x ((F) / (P – B)) where F denotes the fluorescent signal of cells incubated with the peptide, P denotes the average fluorescent signal of the peptide, and B denotes the average ECB signal.

Results, Conclusions, and Discussions: Concentration-dependent uptake studies yielded interesting result on the uptake of structured CPPs. We found that the uptake of CPPs with a β-hairpin sequence motif was significantly impaired using inhibitors of an energy dependent pathway (SA) and caveolae-mediated endocytosis (MβCD) while it was not significantly impaired through the macropinocytosis pathway (EIPA) (Figure 1). Because caveolae-mediated pathways require cholesterol and the MβCD inhibitor has a high affinity for cholesterol, it can deplete the amount of cholesterol from the cell membrane which could disrupt the lipid composition and leads to the dissolution of this pathway. Energy dependent pathways all require some form of energy, usually utilizing adenosine triphosphate (ATP). Sodium azide is known to decrease the amount of ATP, and this can have a negative impact on different energy dependent endocytic pathways, such as clathrin and caveolae-mediated endocytosis and macropinocytosis. Several studies have suggested that CPPs enter cells via a macropinocytosis pathway; however, this was found to not be the case for the structured CPP. This could be explained by the fact that only a single dose was used to investigate this pathway. EIPA primarily inhibits macropinocytosis by blocking the Na+/H+ pump which leads to disruption of ion homeostasis. This inhibition affects actin cytoskeleton remodeling and membrane ruffling, both of which are critical steps in this process. Currently work aims to explore this by performing additional biological replicates. With an increasing use of peptide-based reporters in addition to coupling CPPs with small molecule drugs, there is a need to explain and understand the different mechanistic uptake of structured CPPs. Ongoing future work will continue to explore additional variables like temperature, incubation time, and inhibitor concentration in both structured and unstructured CPPs, with and without cargoes, to further elucidate the differences in uptake mechanism. These findings will provide new insight into how CPPs gain entry into intact cells and provide a framework for how to select an ideal CPP to deliver a cargo.

Acknowledgements (Optional):