title: Matrix stiffness affects endocytic uptake of MK2-inhibitor peptides.
abstract: In this study, the role of substrate stiffness on the endocytic uptake of a cell-penetrating peptide was investigated. The cell-penetrating peptide, an inhibitor of mitogen-activated protein kinase activated protein kinase II (MK2), enters a primary mesothelial cell line predominantly through caveolae. Using tissue culture polystyrene and polyacrylamide gels of varying stiffness for cell culture, and flow cytometry quantification and enzyme-linked immunoassays (ELISA) for uptake assays, we showed that the amount of uptake of the peptide is increased on soft substrates. Further, peptide uptake per cell increased at lower cell density. The improved uptake seen on soft substrates in vitro better correlates with in vivo functional studies where 10-100 µM concentrations of the MK2 inhibitor cell penetrating peptide demonstrated functional activity in several disease models. Additional characterization showed actin polymerization did not affect uptake, while microtubule polymerization had a profound effect on uptake. This work demonstrates that cell culture substrate stiffness can play a role in endocytic uptake, and may be an important consideration to improve correlations between in vitro and in vivo drug efficacy.
authors.affiliation: Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana, United States of America.
authors.forename: Jamie L
authors.initials: JL
authors.lastname: Brugnano
authors.affiliation: Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana, United States of America.
authors.forename: Alyssa
authors.initials: A
authors.lastname: Panitch
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D057846:Cell-Penetrating Peptides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D016207:Cytokines / Q000096:biosynthesis; D004705:Endocytosis / Q000187:drug effects*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D055503:Protein Multimerization; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000099190:MAP-Kinase-Activated Kinase 2
chemical_list: D057846:Cell-Penetrating Peptides; D016207:Cytokines; D047908:Intracellular Signaling Peptides and Proteins; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D000099190:MAP-Kinase-Activated Kinase 2