Efficacious redox-responsive gene delivery in serum by ferrocenylated monomeric and dimeric cationic cholesterols

Herein, we present the design and synthesis of new redox-active monomeric and dimeric (gemini) cationic lipids based on ferrocenylated cholesterol derivatives for gene delivery. The cationic cholesterols are shown to be transfection efficient after being formulated with the neutral helper lipid DOPE in the presence of serum (FBS). The redox activity of the resulting co-liposomes and their lipoplexes could be regulated using the alkanyl ferrocene moiety attached to the ammonium head groups of the cationic cholesterols. Atomic force microscopy (AFM), dynamic light scattering (DLS) and zeta potential measurements were performed to characterize the co-liposomal aggregates and their complexes with pDNA. The transfection efficiency of lipoplexes could be tuned by changing the oxidation state of the ferrocene moiety. The gene transfection capability was assayed in terms of green fluorescence protein (GFP) expression using pEGFP-C3 plasmid DNA in three cell lines of different origins, namely Caco-2, HEK293T and HeLa, in the presence of serum. The vesicles possessing ferrocene in the reduced state induced an efficient transfection, even better than a commercial reagent Lipofectamine 2000 (Lipo 2000) as evidenced by flow cytometry and fluorescence microscopy. All the co-liposomes containing the oxidized ferrocene displayed diminished levels of gene expression. Gene transfection events from the oxidized co-liposomes were further potentiated by introducing ascorbic acid (AA) as a reducing agent during lipoplex incubation with cells, leading to the resumption of transfection activity. Assessment of transfection capability of both reduced and oxidized co-liposomes was also undertaken following cellular internalization of labelled pDNA using confocal microscopy and flow cytometry. Overall, we demonstrate here controlled gene transfection activities using redox-driven, transfection efficient cationic monomeric and dimeric cholesterol lipids. Such systems could be used in gene delivery applications where transfection needs to be performed spatially or temporally.

Glioblastoma-derived Macrophage Colony-stimulating Factor (MCSF) Induces Microglial Release of Insulin-like Growth Factor-binding Protein 1 (IGFBP1) to Promote Angiogenesis

Glioblastoma (grade IV glioma/GBM) is the most common primary adult malignant brain tumor with poor prognosis. To characterize molecular determinants of tumor-stroma interaction in GBM, we profiled 48 serum cytokines and identified macrophage colony-stimulating factor (MCSF) as one of the elevated cytokines in sera from GBM patients. Both MCSF transcript and protein were up-regulated in GBM tissue samples through a spleen tyrosine kinase (SYK)-dependent activation of the PI3K-NF kappa B pathway. Ectopic overexpression and silencing experiments revealed that glioma-secreted MCSF has no role in autocrine functions and M2 polarization of macrophages. In contrast, silencing expression of MCSF in glioma cells prevented tube formation of human umbilical vein endothelial cells elicited by the supernatant from monocytes/microglial cells treated with conditioned medium from glioma cells. Quantitative proteomics based on stable isotope labeling by amino acids in cell culture showed that glioma-derived MCSF induces changes in microglial secretome and identified insulin-like growth factor-binding protein 1 (IGFBP1) as one of the MCSF-regulated proteins secreted by microglia. Silencing IGFBP1 expression in microglial cells or its neutralization by an antibody reduced the ability of supernatants derived from microglial cells treated with glioma cell-conditioned medium to induce angiogenesis. In conclusion, this study shows up-regulation of MCSF in GBM via a SYK-PI3K-NF kappa B-dependent mechanism and identifies IGFBP1 released by microglial cells as a novel mediator of MCSF-induced angiogenesis, of potential interest for developing targeted therapy to prevent GBM progression.

Mononuclear five-coordinate cobalt(II) complexes with N-4-coordinate pyrazole based ligand and pseudohalogens: synthesis, structures, DNA and protein binding study

A series of mononuclear five-coordinate cobalt(II) complexes, Co(dbdmp)(X)]Y, where dbdmp=N,N-diethyl-N,N-bis((3,5-dimethyl-1H-pyrazol-1-yl)methyl)ethane-1, 2-diamine, X=N-3(-)/NCO-/NCS- and Y=PF6-/BF4-/ClO4-, have been synthesized and characterized by microanalyses and spectroscopic techniques. Crystal structures of Co(N-3)(dbdmp)]PF6 (1), Co(N-3)(dbdmp)]ClO4 (3), Co(NCO)(dbdmp)]PF6 (4), Co(NCO)(dbdmp)]ClO4 (6), and Co(NCS)(dbdmp)]ClO4 (9) have been solved by single-crystal X-ray diffraction studies and showed that all the complexes have distorted trigonal bipyramidal geometry; PF6- counter anion containing complexes Co(N-3)(dbdmp)]PF6 and Co(NCO)(dbdmp)]PF6 have chiral space groups. The binding ability of synthesized complexes with CT-DNA and bovine serum albumin (BSA) has been studied by spectroscopic methods and viscosity measurements. The experimental results of absorption titration of cobalt(II) complexes with CT-DNA indicate that the complexes have ability to form adducts and they can stabilize the DNA helix. The cobalt(II) complexes exhibit good binding propensity to BSA protein.