The high impermeability and selectivity of the BBB prevent the transport of many drugs into the brain, making them ineffective for the treatment of central nervous system diseases. The cell-penetrating peptides (CPPs) represent a new strategy to functionalize carriers in order to deliver therapeutic molecules to the brain. Several studies have demonstrated that gH625, a peptide derived from the glycoprotein H of herpes simplex virus 1, is able to cross the membrane bilayer and represents an ideal CPP for the delivery BBB, because of its ability to escape from the endocytic pathways1. In this study we evaluated the internalization of gH625 in human neuroblastoma (SH-SY5Y) and astrocytoma (U87-MG) cells and in rat brain. Fluorescence and spectrofluorimetric in vitro analyses show a good rate of uptake in both cell lines after 2h of treatment with gH625 labeled with 4-chloro-7-nitrobenz-2-oxa-1,3-diazole (gH625-NBD) (Fig.1). The internalization is almost complete if higher concentration was used (5μM) and the signal is prevalently found within the cytoplasm. Immunofluorescence studies, using anti-GFAP and anti-BBB antibodies, were performed after intravenous administration (3h) of gH625-NBD (160μg/100 g bw) in rats. Five images for each experimental class were analyzed with ImageJ 1.48 software; the deconvolutionlab plugin was used to deconvolve image channels through the Tikhonov-Miller’s algorithm; the Co-localization Colormap plugin was then used to evaluate the degree of correlation between pair of pixels in the red and green channels, resulting in the distribution of the values of the normalized mean deviation product (nMDP) and the index of correlation as the fraction of positively correlated pixels in the image2. Co-localization studies produced a color scale map (from -1 to 1) where negative indexes (cold colors) represent no co-localization and indexes above 0 (hot colors) represent co-localization. The study reveals a high co-localization score with BBB and low co-localization score with GFAP protein of astrocytes; interestingly few neurons were labeled for gH625-NBD, indicating the passage through the BBB (Fig.2). The index of correlation shows poor positive correlation in gH625/anti-GFAP and high positive correlation in the gH625/anti-BBB. These data show that gH625 is up taken by neuronal cells and reaches the rat brain. Taken together, our results can be considered as a preliminary data to develop a liposome-based systems which involves the use of gH625 as an efficient drugs delivery through the BBB. 1Guarnieri D et al. 2013 Drug delivery: shuttle-mediated nanoparticle delivery to the BBB. Small 9(6): 806 2Jaskolski F et al. 2005 An automated method to quantify and visualize colocalized fluorescent signals. J of Neur Meth 146(1):42-49
A cell-penetrating peptide as a tool for delivery to blood brain barrier (BBB) / Iachetta, Giuseppina; Forte, Maurizio; Falanga, Annarita; Galdiero, Stefania; Valiante, Salvatore. - (2014).
A cell-penetrating peptide as a tool for delivery to blood brain barrier (BBB)
IACHETTA, GIUSEPPINA;FORTE, MAURIZIO;FALANGA, ANNARITA;GALDIERO, STEFANIA;VALIANTE, Salvatore
2014
Abstract
The high impermeability and selectivity of the BBB prevent the transport of many drugs into the brain, making them ineffective for the treatment of central nervous system diseases. The cell-penetrating peptides (CPPs) represent a new strategy to functionalize carriers in order to deliver therapeutic molecules to the brain. Several studies have demonstrated that gH625, a peptide derived from the glycoprotein H of herpes simplex virus 1, is able to cross the membrane bilayer and represents an ideal CPP for the delivery BBB, because of its ability to escape from the endocytic pathways1. In this study we evaluated the internalization of gH625 in human neuroblastoma (SH-SY5Y) and astrocytoma (U87-MG) cells and in rat brain. Fluorescence and spectrofluorimetric in vitro analyses show a good rate of uptake in both cell lines after 2h of treatment with gH625 labeled with 4-chloro-7-nitrobenz-2-oxa-1,3-diazole (gH625-NBD) (Fig.1). The internalization is almost complete if higher concentration was used (5μM) and the signal is prevalently found within the cytoplasm. Immunofluorescence studies, using anti-GFAP and anti-BBB antibodies, were performed after intravenous administration (3h) of gH625-NBD (160μg/100 g bw) in rats. Five images for each experimental class were analyzed with ImageJ 1.48 software; the deconvolutionlab plugin was used to deconvolve image channels through the Tikhonov-Miller’s algorithm; the Co-localization Colormap plugin was then used to evaluate the degree of correlation between pair of pixels in the red and green channels, resulting in the distribution of the values of the normalized mean deviation product (nMDP) and the index of correlation as the fraction of positively correlated pixels in the image2. Co-localization studies produced a color scale map (from -1 to 1) where negative indexes (cold colors) represent no co-localization and indexes above 0 (hot colors) represent co-localization. The study reveals a high co-localization score with BBB and low co-localization score with GFAP protein of astrocytes; interestingly few neurons were labeled for gH625-NBD, indicating the passage through the BBB (Fig.2). The index of correlation shows poor positive correlation in gH625/anti-GFAP and high positive correlation in the gH625/anti-BBB. These data show that gH625 is up taken by neuronal cells and reaches the rat brain. Taken together, our results can be considered as a preliminary data to develop a liposome-based systems which involves the use of gH625 as an efficient drugs delivery through the BBB. 1Guarnieri D et al. 2013 Drug delivery: shuttle-mediated nanoparticle delivery to the BBB. Small 9(6): 806 2Jaskolski F et al. 2005 An automated method to quantify and visualize colocalized fluorescent signals. J of Neur Meth 146(1):42-49I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.