Comparative transport analysis of cell penetrating peptides and Lysosomal sequences for selective tropism towards RPE cells.

Cell penetrating peptides are typically nonspecific, targeting multiple cell types without discrimination. However, subsets of Cell penetrating peptides (CPP) have been found, which show a 'homing' capacity or increased likelihood of internalizing into specific cell types and subcellular locations. Therapeutics intended to be delivered to tissues with a high degree of cellular diversity, such as the intraocular space, would benefit from delivery using CPP that can discriminate across multiple cell types. Lysosomal storage diseases in the retinal pigment epithelium (RPE) can impair cargo clearance, leading to RPE atrophy and blindness. Characterizing CPP for their capacity to effectively deliver cargo to the lysosomes of different cell types may expand treatment options for lysosomal storage disorders. We developed a combinatorial library of CPP and lysosomal sorting signals, applied to ARPE19 and B3 corneal lens cells, for the purpose of determining cell line specificity and internal targeting. Several candidate classes of CPP were found to have as much as 4 times the internalization efficiency in ARPE19 compared to B3. Follow-up cargo transport studies were also performed, which demonstrate effective internalization and lysosomal targeting in ARPE19 cells.

Fluorescence-Based Detection of Ferrous Iron in Senescent Cells.

A major limitation in aging research is the lack of reliable biomarkers to assess phenotypic changes with age or monitor response to antiaging interventions. This study investigates the role of intracellular ferrous iron (Fe2+) as a potential biomarker of senescence. Iron is known to accumulate in various tissues with age and recent studies have demonstrated that its level increases dramatically in senescent cells. The current techniques used to measure the accumulation of iron are cumbersome and only measure total iron not specific isotopes such as the redox reactive Fe2+. It is still to be determined whether the damaging form of iron (Fe2+) is specifically elevated in senescent cells. In this study, we assessed the potential use of a newly discovered Fe2+ reactive probe (SiRhoNox-1) for selective labeling of senescent cells in vitro. For this we have generated various senescent cell models and subjected them to SiRhoNox-1 labeling. Our results indicate that SiRhoNox-1 selectivity labels live senescent cells and was more specific and faster than current staining such as SA-ßGal or a derived fluorescent probe C12FDG. Together these findings suggest that SiRhoNox-1 may serve as a convenient tool to detect senescent cells based on their ferrous iron level.

High-Throughput Screening of Protein-Detergent Complexes Using Fluorescence Polarization Spectroscopy.

This article provides detailed protocols for a high-throughput fluorescence polarization (FP) spectroscopy approach to disentangle the interactions of membrane proteins with solubilizing detergents. Existing techniques for examining the membrane protein-detergent complex (PDC) interactions are low throughput and require high amounts of proteins. Here, we describe a 96-well analytical approach, which facilitates a scalable analysis of the PDC interactions at low-nanomolar concentrations of membrane proteins in native solutions. At detergent concentrations much greater than the equilibrium dissociation constant of the PDC, Kd , the FP anisotropy reaches a saturated value, so it is independent of the detergent concentration. On the contrary, at detergent concentrations comparable with or lower than the Kd , the FP anisotropy readout undergoes a time-dependent decrease, exhibiting a sensitive and specific detergent-dissociation signature. Our approach can also be used for determining the kinetic rate constants of association and dissociation. With further development, these protocols might be used in various arenas of membrane protein research that pertain to extraction, solubilization, and stabilization. © 2019 by John Wiley & Sons, Inc.