Fig. 3

Mechanism of action for PPAA-mediated CPP uptake. a Inhibition of rhodamine labeled PPAA polymer uptake in HCAVSMCs. b The effect of adding inhibitors during PPAA pretreatment on subsequent uptake of Alexa-488 labeled YARA-MK2i peptide in HCAVSMCs (inhibitors applied during PPAA treatment only). c Representative Z-stack images (images have x and y projections of 3-dimensional z-stack images below and to the right of each image, respectively) of the dose-dependent effects of dynasore during PPAA pretreatment on fluorescently labeled polymer and peptide uptake. d The effects of adding inhibitors throughout the entire treatment for both co-delivery and sequential delivery of PPAA and Alexa-488 labeled YARA-MK2i peptide in HCAVSMCs. For a, b, d, *p < 0.05, **p < 0.01, ***p < 0.001 vs. no inhibitor. e Zeta potential measurement of PPAA and YARA-MK2i peptide mediated changes in extracellular surface charge of HCAVSMCs, *p < 0.05. f Schematic representation of the proposed mechanism of action for PPAA-mediated CPP-peptide uptake and intracellular delivery. Poly(propylacrylic acid) (PPAA) interacts with the cell membrane via hydrophobic interactions with the propyl moiety of PPAA. This interaction increases the net negative charge of the cell membrane due to the carboxylate anion present on the polymer. This increase in net negative charge enhances cell surface electrostatic interactions with cationic CPPs, leading to enhanced peptide uptake through both macropinocytic and endocytic pathways. Following uptake, the decrease in endosomal pH results in protonation of the PPAA polymer. This pH change triggers the polymer to dissociate from the CPP-peptide cargo and interact with and destabilize the endosomal membrane, resulting in cytosolic peptide delivery. Statistical analyses were performed with a one-way ANOVA followed by Tukey’s post hoc test. Data are presented as means ± SEM graphically