Left panels, nuclei stained with Syto-9; Middle panels, tdTomato expression; Right panels, merged. NRP-1 poorly [4, 5]. Because H2K peptides share a common sequence of CKXXK- with peptides that target the neuropilin-1 receptor (NRP-1) and activate this pathway [3], H2K peptides with reduced binding to DNA (compared to H2K4b) may more readily be enzymatically processed, enabling the transport of the polyplex through the tumor endothelium. Although NRP-1 mediated uptake of macromolecules is by a macropinocytosis-like mechanism, the energy-dependent and rapid trans-tissue penetration of these molecules is not well understood [2, 6]. Nevertheless, at least part of the NRP-mediated transport of macromolecules appears to be due to transcytosis [2, 7]. It has also been postulated that endothelial NRP-1 mediated transcytosis is a mechanism to transport nutrients in bulk to calorie-restricted tissues and that tumors have usurped this transport system to enhance their survival [2]. Indeed, inhibitors of the glucose transport system LCK (phospho-Ser59) antibody injected intratumorally markedly increased fluorescently-labeled molecules through the tumor endothelium [2]. Because H2K has a repeating sequence pattern of CKHHK-, transcytosis of the polyplex through the tumor endothelium provides a rationale for its enhanced tumor targeting and accumulation. Partial disruption of the polyplex, together with the associated binding and release of peptide from DNA, may also have an important role in endosomal lysis. If the endosomolytic peptide binds too tightly to DNA and stabilizes the polyplexes within acidic endosomes, the peptide will not be released and may not be particularly effective in lysing endosomes [8, 9]. Thus, with agents or endosomolytic peptides that do not alter significantly the biophysical properties of the H2K polyplex, transfection and should increase. When such endosomolytic agents are incorporated within polyplexes, transfection results may be more predictive of results. In this report, we examined both endosomolytic and NRP-1 mediated transport mechanisms and further investigated whether modifications of HK peptides based on these mechanisms augment transfection of HK polyplexes transfection and luciferase reporter assay Initially, 1105 cells were plated in 24-well plates in the presence of 500 l of DMEM with 10% serum; after 24 h, the cells reached 70% confluency. Unless otherwise indicated, increasing amounts of the peptide(s) (0.5 to 12.0 g) in 50 l were mixed with 1.0 g of the plasmid (CPG-Luciferase (Luc) in 42 l of Opti-MEM or water) and the mixture was allowed Sodium Tauroursodeoxycholate to stand for 30 min (1/1, w/w ratio for HK/DNA is approximately equal to 1.3/1, N/P ratio). The transfection protocol was performed as described previously with few modifications [15]. In brief, 50 l of the transfection complex were added to cells for 24 h. The cells were then lysed with 100 l of 1x passive lysis buffer (Promega Corp., Madison, WI, US). Protein concentration was measured by using the BCA protein assay kit (Pierce ThermoFisher Scientific, Waltham, MA, US). Luciferase activity was measured and expressed as relative light units (RLU) by the direct current Turner 20/20 luminometer (Turner Design, Sunnyvale, CA, US) as described previously [15] with 3 measurements performed for each concentration. Inhibition of Endocytosis Inhibition of endocytosis pathways was done as previously described [16, 17]. In brief, HUVEC (1 105 cells) Sodium Tauroursodeoxycholate were placed in 24-well plates 24 h before transfection. After the cells were pretreated with a polyclonal clathrin antibody (Sigma-Aldrich, St. Louis, MO, US) (1:100 vol/vol. dilution in cell culture medium), filipin (2.0 g/ml) (Sigma-Aldrich), polyclonal NRP-1 antibody (50 g/ ml) (R & D, Minneapolis, MN), or cytochalasin D (1 g/ml) (Sigma-Aldrich) for 30 min, the Sodium Tauroursodeoxycholate medium was changed to fresh EGM-2 (Lonza; Basel CH) Sodium Tauroursodeoxycholate /2% serum. As discussed previously, the HK polyplexes were then added to each well and luciferase activity was measured approximately 24 h later. Flow Cytometry Cells (2 106) (MDA-MB-435, HUVEC) were harvested, washed with PBS, and then fixed with 4% formaldehyde for 5 min. Goat anti mouse Neuropilin-1 antibody (R&D company, Minneapolis,.

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