Determination of Potential CPP-Receptor Interactions for Improved Efficacy and Cell-Specificity

Objectives: To establish the role of glycosaminoglycan (GAG)-containing proteins and integrin beta-1 (ITGB1) in the binding and uptake of cell-penetrating peptides 599, RD3AD, RD3AL, and RL3AL.
Methods: Using chemical inhibitors, transient gene silencing via siRNAs, and CRISPR-Cas9 knock-out, the removal of GAGs (xylosyltransferase-1 (XYLT1)-KO) and ITGB1 and the effects this had on 599, RD3AD, RD3AL, and RL3AL’s interaction with various oral cancer cell lines was accessed. The peptide variants were complexed with various nucleic acid containing cargos and cell uptake, silencing efficiencies, and protein expression were subsequently characterized via quantitative uptake assays, confocal fluorescence microscopy, western blot, and qPCR analyses.
Results: Removal of GAGs, whether transient or through gene knock-out, had significant effects on cellular uptake and gene-silencing. CAL27 oral cancer cells showed significantly greater gene silencing compared to XYLT1-KO CAL27. Modification of heparan sulfate proteoglycans, GAG-containing proteins and potential receptors for CPPs, through the removal of sulfate groups, resulted in significant decreases in cellular uptake and gene-silencing. Pull-down assays revealed a mass that corresponds to syndecan-1, a heparan sulfate proteoglycan and known receptor of peptides. Additionally, confocal fluorescence microscopy has demonstrated colocalization of our CPPs with ITGB1, suggesting their potential interaction. Interestingly, gene silencing of ITGB1 prior to cell treatment with CPPs resulted in significant increases in cellular uptake, indicating the interaction of our CPPs with ITGB1 is potentially inhibitory.
Conclusions: Together, these data demonstrate that both GAG-containing proteins and ITGB1 may have a role in the binding and uptake of 599, RD3AD, RD3AL, and RL3AL, with GAG-containing proteins, potentially syndecan-1, promoting uptake and ITGB1 inhibiting uptake.