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Search results for: 5 FAM [5 Carboxyfluorescein] *Validated for labeling peptides*

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#27676095   2016/10/04 To Up

Design and Synthesis of New Cell Penetrating Peptides: Diffusion and Distribution Inside the Cornea.

The role of cell penetrating peptides (CPPs) has been challenged in recent years for drug delivery to ocular tissues for the targeting of both anterior and posterior segments. The enhancement of trans-corneal transport for anterior segment targeting is a very important issue possibly leading to important outcomes on efficacy and to the opportunity of topical administration of molecules with unfavorable penetration properties. The aim of the present work was the design and synthesis of new CPPs, deriving from the structure of PEP-1 peptide. Synthesized peptides were labeled with 5-carboxyfluorescein (5-FAM), and their diffusion behavior and distribution inside the cornea were evaluated by a validated ex vivo model and a confocal microscopy approach. Newly synthesized peptides showed similar corneal permeation profiles as PEP-1 (P = 0.75 ± 0.56 × 10 cm/s), about 2.6-fold higher than 5-FAM (P = 0.29 ± 0.08 × 10 cm/s) despite the higher molecular weight. Confocal microscopy experiments highlighted the tendency of PEP-1 and its derived peptides to localize in the intercellular space and/or in the plasma membrane. Noteworthy, using penetratin as positive control, a higher trans-corneal permeation (P = 6.18 ± 1.46 × 10 cm/s) was evidenced together with a diffusion by intracellular route and a different accumulation between wings and basal epithelial cells, probably depending on the stage of cell development. Finally, PEP-1 and pep-7 proved to be safe and well tolerated when tested on human conjuctival cell line.
Silvia Pescina, Marina Sala, Cristina Padula, Maria Carmina Scala, Antonia Spensiero, Silvana Belletti, Rita Gatti, Ettore Novellino, Pietro Campiglia, Patrizia Santi, Sara Nicoli, Carmine Ostacolo

1849 related Products with: Design and Synthesis of New Cell Penetrating Peptides: Diffusion and Distribution Inside the Cornea.

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#26454788   2015/10/01 To Up

Assaying human neutrophil elastase activity by capillary zone electrophoresis combined with laser-induced fluorescence.

Skin aging is a progressive process determining the ultimate skin appearance. Human neutrophil elastase (HNE) has been shown to play an important role in the degradation of the extracellular matrix. In order to assay HNE kinetics, a novel online capillary zone electrophoresis (CZE) assay has been developed in this study for the determination of the maximum velocity (Vmax) and of the Michaelis-Menten constant (Km) of HNE regarding several potential substrates. These assays are based on short-end injection to shorten analysis time, on transverse diffusion of laminar flow profiles (TDLFP) for in-capillary reactant mixing, and on UV or laser-induced fluorescence (LIF) detection. Kinetic constants for a referenced peptidic substrate were determined using not only online assays but also offline (pre-capillary) mode. The results obtained were cross compared and compared to the literature in order to validate the developed assays. The hydrolysis of three new potential fluorogenic substrates by HNE was also monitored. Two new peptidic substrates for HNE were identified through this study. Km values of these novel substrates were successfully determined using online CZE assay (Km ∼0.07mM). This value was in the same order of magnitude of that of the referenced substrate despite the presence of the labeling group 5-carboxyfluorescein (5-FAM). HNE activity has never been assessed using online CZE-based assay, neither with UV nor with LIF detection. The developed assay conducted with the new labeled substrates is particularly sensitive (LOQ of few nM), does not require the presence of micelles in the BGE (which is the case for the reference substrate) and only necessitates few nanoliters of reactants making it particularly adapted for screening studies.
Syntia Fayad, Reine Nehmé, Pierre Lafite, Philippe Morin

1507 related Products with: Assaying human neutrophil elastase activity by capillary zone electrophoresis combined with laser-induced fluorescence.

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