ABSTRACT
Elastin-like polypeptide (ELP) nanoparticles are a versatile platform for targeted drug delivery. As for any type of nanocarrier system, an important challenge remains the ability of deep (tumor) tissue penetration. In this study, ELP particles with controlled surface density of the cell-penetrating peptide (CPP) octa-arginine (R8) were created by temperature-induced co-assembly. ELPs formed micellar nanoparticles with a diameter of around 60â¯nm. Cellular uptake in human skin fibroblasts was directly dependent on the surface density of R8 as confirmed by flow cytometry and confocal laser scanning microscopy. Remarkably, next to promoting cellular uptake, the presence of the CPP also enhanced penetration into spheroids generated from human glioblastoma U-87 cells. After 24â¯h, uptake into cells was observed in multiple layers towards the spheroid core. ELP particles not carrying any CPP did not penetrate. Clearly, a high CPP density exerted a dual benefit on cellular uptake and tissue penetration. At low nanoparticle concentration, there was evidence of a binding site barrier as observed for the penetration of molecules binding with high affinity to cell surface receptors. In conclusion, R8-functionalized ELP nanoparticles form an excellent delivery vehicle that combines tunability of surface characteristics with small and well-defined size.
Subject(s)
Drug Delivery Systems , Elastin/chemistry , Glioblastoma/metabolism , Nanoparticles , Oligopeptides/chemistry , Binding Sites , Cell Line, Tumor , Cell-Penetrating Peptides/chemistry , Chemistry, Pharmaceutical/methods , Flow Cytometry , Humans , Microscopy, Confocal/methods , Spheroids, Cellular/metabolism , Time FactorsABSTRACT
At low levels, reactive oxygen species (ROS) can act as signaling molecules within cells. When ROS production greatly exceeds the capacity of endogenous antioxidant systems, or antioxidant levels are reduced, ROS levels increase further. The latter is associated with induction of oxidative stress and associated signal transduction and characterized by ROS-induced changes in cellular redox homeostasis and/or damaging effects on biomolecules (e.g. DNA, proteins and lipids). Given the complex mechanisms involved in ROS production and removal, in combination with the lack of reporter molecules that are truly specific for a particular type of ROS, quantification of (sub)cellular ROS levels is a challenging task. In this chapter we describe two strategies to measure ROS: one approach to assess general oxidant levels using the chemical reporter CM-H2DCFDA (5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate), and a second approach allowing more specific analysis of cytosolic hydrogen peroxide (H2O2) levels using protein-based sensors (HyPer and SypHer).
