CNS Delivery Via Adsorptive Transcytosis

Adsorptive-mediated transcytosis (AMT) provides a means for brain delivery of medicines across the blood-brain barrier (BBB). The BBB is readily equipped for the AMT process: it provides both the potential for binding and uptake of cationic molecules to the luminal surface of endothelial cells, and then for exocytosis at the abluminal surface. The transcytotic pathways present at the BBB and its morphological and enzymatic properties provide the means for movement of the molecules through the endothelial cytoplasm. AMT-based drug delivery to the brain was performed using cationic proteins and cell-penetrating peptides (CPPs). Protein cationization using either synthetic or natural polyamines is discussed and some examples of diamine/polyamine modified proteins that cross BBB are described. Two main families of CPPs belonging to the Tat-derived peptides and Syn-B vectors have been extensively used in CPP vector-mediated strategies allowing delivery of a large variety of small molecules as well as proteins across cell membranes in vitro and the BBB in vivo. CPP strategy suffers from several limitations such as toxicity and immunogenicity—like the cationization strategy—as well as the instability of peptide vectors in biological media. The review concludes by stressing the need to improve the understanding of AMT mechanisms at BBB and the effectiveness of cationized proteins and CPP-vectorized proteins as neurotherapeutics

A doubly labeled penetratin analogue as a ratiometric sensor for intracellular proteolytic stability.

Item does not contain fulltextEndocytosis has been shown to play a major role in the cellular import of cationic cell-penetrating peptides (CPPs) and CPP conjugates. Considering the presence of proteolytic activities inside the endolysosomal compartment, it is necessary to assess the consequences of the import mechanism on the intracellular integrity of the vector and the cargo. In this work, a penetratin analogue terminally labeled with two different fluorophores was synthesized and used as a sensor to quantitatively dissect the contribution of intracellular proteolytic activities on the breakdown of this specific CPP. Using a panel of lysosomal protease inhibitors, the endocytic compartment was identified as the major site of degradation. In contrast, an inhibitor of the proteasome had little effect on intracellular peptide integrity. Very remarkably, inhibitors of endolysosomal proteolysis also affected the intracellular distribution of fluorescence, leading to a reduction of fluorescein fluorescence in the cytoplasm. This change in fluorescence distribution was very similar to the one observed after incubation of cells with inhibitors of endosomal acidification. These results indicate that cytoplasmic fluorescence, typically interpreted as CPP entering the cytosol, may originate from proteolytic breakdown products.1 januari 201

Metabolic Cleavage and Translocation Efficiency of Selected Cell Penetrating Peptides: A Comparative Study with Epithelial Cell Cultures

We investigated the metabolic stability of four cell penetrating peptides (CPPs), namely SAP, hCT(9-32)-br, [Pα] and [Pβ], when in contact with either subconfluent HeLa, confluent MDCK or Calu-3 epithelial cell cultures. Additionally, through analysis of their cellular translocation efficiency, we evaluated possible relations between metabolic stability and translocation efficiency. Metabolic degradation kinetics and resulting metabolites were assessed using RP-HPLC and MALDI-TOF mass spectrometry. Translocation efficiencies were determined using fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM). Between HeLa, MDCK and Calu-3 we found the levels of proteolytic activities to be highly variable. However, for each peptide, the individual degradation patterns were quite similar. The metabolic stability of the investigated CPPs was in the order of CF-SAP = CF-hCT(9-32)-br > [Pβ]−IAF > [Pα] and we identified specific cleavage sites for each of the four peptides. Throughout, we observed higher translocation efficiencies into HeLa cells as compared to MDCK and Calu-3, corresponding to the lower state of differentiation of HeLa cell cultures. No direct relation between metabolic stability and translocation efficiency was found, indicating that metabolic stability in general is not a main limiting factor for efficient cellular translocation. Nevertheless, translocation of individual CPPs may be improved by structural modifications aiming at increased metabolic stability