Matrix Stiffness Affects Endocytic Uptake of MK2-Inhibitor Peptides

Jamie L Brugnano,Alyssa Panitch

doi:10.1371/journal.pone.0084821

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.

Highlights

Matrix stiffness is an important regulator of cell behavior [1]
When comparing between data obtained from in vitro cell and in vivo animal models, we observed an unusual effect: concentrations of the YARA MK2 inhibitor peptide required for efficacy in cells ranged from 1000–3000 mM [24,26]; the concentration required for efficacy in animal models was ten to one hundred-fold less, in the range of 10–100 mM [25,28–31]
The two polyacrylamide gels that were used for further experimentation were the 0.03% bis-acrylamide for the ‘‘soft’’ gel and the 0.5% bisacrylamide for the ‘‘stiff’’ gel, which equate to a stiffness of approximately 4 and 22 kPa respectively

Summary

Introduction

Matrix stiffness is an important regulator of cell behavior [1]. Stiffness has been shown to affect cell morphology and spreading [2,3], proliferation [4], migration [5], apoptosis rate [4,6], and differentiation [7,8]. When comparing between data obtained from in vitro cell and in vivo animal models, we observed an unusual effect: concentrations of the YARA MK2 inhibitor peptide required for efficacy in cells ranged from 1000–3000 mM [24,26]; the concentration required for efficacy in animal models was ten to one hundred-fold less, in the range of 10–100 mM [25,28–31]. This phenomenon opposes what is normally observed in the pharmaceutical industry, as drug concentrations must usually increase to demonstrate efficacy when moving from cell culture to animal models due to metabolism and non-uniform distribution within the body. We hypothesized that the discrepancy observed in peptide concentration required to achieve efficacy in studies in vitro as compared to studies in vivo was due to the unrealistic stiffness of tissue culture polystyrene

Methods

Results

Conclusion