The use of cell penetrating peptides to treat inflammatory responses in uterine cells

Abstract

A lack of effective therapies continues to impair attempts to prevent preterm birth associated with inflammation. This study aimed to broaden the scope of candidate agents available to address this clinical problem. Using live cell confocal microscopy, Western immunoblotting, cell toxicity assay, and quantitative polymerase chain reaction techniques this study investigated the ability of a novel class of peptide vectors, termed Cell Penetrating Peptides (CPPs), to deliver cargo to human uterine and placental cells. It examined the ability of a CPP-linked peptide cargo: the Nemo Binding Domain (NBD) peptide, to inhibit inflammatory Nuclear Factor Kappa B (NFκB) signalling in uterine cells; comparing this response to a group of small molecule inhibitors of inflammatory pathways. Three CPP derived vectors were able to deliver fluorescent cargo to uterine myometrial and placental amnion cells within one hour over a concentration range of 1-10μM. Similar uptake kinetics in uterine cells were observed with the use of a fluorescently-tagged CPP conjugated to NBD. The NBD peptide, conjugated to a CPP derived from antennopaedia protein (Pen-NBD), was able to inhibit cytokine-stimulated cyclooxygenase-2 (COX2) protein induction at four hours; an effect that was not seen with other CPP-NBD conjugations, nor with NBD-mutant or unconjugated peptide controls. Data derived from both Western blots and gene arrays indicated that the anti-inflammatory effects of Pen-NBD were comparable to non-peptidic small molecule inhibitors of NFκB. However, Pen-NBD peptide did not prevent the cytokine-induced degradation of Inhibitor of Kappa B Alpha (IκBα) protein; nor did it inhibit the cytokine-induced expression of NFκB pathway genes, thus the precise targeting of NBD peptide within uterine cells remains uncertain and may be distinct to the canonical NFκB pathway. This research demonstrates the proof of concept that CPPs can enter human utero-placental cells and can deliver bioactive cargo to exert an anti-inflammatory effect. It provides a framework by which future research can examine CPP mediated delivery of a broad variety of potential cargo into uterine cells and thus offers a novel approach for the development of treatments aimed at preventing preterm birth.