Double-tailed lipid modification as a promising candidate for oligonucleotide delivery in mammalian cells

Background The potential use of nucleic acids as therapeutic drugs has triggered the quest for oligonucleotide conjugates with enhanced cellular permeability. To this end, the biophysical aspects of previously reported potential lipid oligodeoxyribonucleotide conjugates were studied including its membrane-binding properties and cellular uptake. Methods These conjugates were fully characterized by MALDI-TOF mass spectrometry and HPLC chromatography. Their ability to insert into lipid model membrane systems was evaluated by Langmuir balance and confocal microscopy followed by the study of the internalization of a lipid oligodeoxyribonucleotide conjugate bearing a double-tail lipid modification (C28) into different cell lines by confocal microscopy and flow cytometry. This compound was also compared with other lipid containing conjugates and with the classical lipoplex formulation using Transfectin as transfection reagent. Results This double-tail lipid modification showed better incorporation into both lipid model membranes and cell systems. Indeed, this lipid conjugation was capable of inserting the oligodeoxyribonucleotide into both liquid-disordered and liquid-ordered domains of model lipid bilayer systems and produced an enhancement of oligodeoxyribonucleotide uptake in cells, even better than the effect caused by lipoplexes. In addition, in β2 integrin (CR3) expressing cells this receptor was directly involved in the enhanced internalization of this compound. Conclusions All these features confirm that the dual lipid modification (C28) is an excellent modification for enhancing nucleic acid delivery without altering their binding properties. General significance Compared to the commercial lipoplex approach, oligodeoxyribonucleotide conjugation with C28 dual lipid modification seems to be promising to improve oligonucleotide delivery in mammalian cells.This work was supported with funds from the Spanish Ministry of Economy [Grant BFU2007-62062], the Basque Government [GIV06-42], the Spanish Ministry of Education [Grant CTQ2010-20541], the Generalitat de Catalunya [2009/SGR/208], the Instituto de Salud Carlos III [CB06_01_0019] and Fundación Biofísica Bizkaia. CIBER-BBN is an iniciative funded by the VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development. B.U-U. was supported by Universidad de País Vasco-UPV/EHU pre-doctoral fellowship and Fundación Biofísica Bizkaia. J.V.B. was a postdoctoral scientist supported by Universidad de País Vasco-UPV/EHU postdoctoral fellowship. The authors acknowledge the Servicio General de Microscopía Analítica y de Alta Resolución en Biomedicina at the University of Basque Country for assistance with confocal microscopy, Prof. A. Gómez-Muñoz for flow cytometry facilities and Eneritz Bilbao for excellent technical assistance.Peer reviewe

Binding of Triton X-100 to bovine serum albumin as studied by surface tension measurements

A previously published computerized drop-weight technique for surface tension measurements, not involving the use of radioactively labelled compounds, has been applied to the study of detergent binding to proteins. The procedure is based on the observation that the protein-surfactant complex is no longer surface-active. As an example, the binding of Triton X-100 to bovine serum albumin has been studied, and the results were found to be in good agreement with those obtained through established but less convenient methods. Our procedure should be useful for measurements of detergent binding to biomembranes. © 1991.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Kinetics of purple membrane dark-adaptation in the presence of Triton X-100

The kinetics of purple membrane dark adaptation were studied at pH 5 and 7, in the presence and absence of the nonionic detergent Triton X-100. The effect of both sublytic and lytic surfactant concentrations has been considered. Our results show that: (a) dark adaptation is faster at pH 5 than at pH 7, (b) dark adaptation is slower, and of smaller amplitude, in the presence than in the absence of Triton X-100. The data may be interpreted in terms of a simple first-order kinetic model, according to which light-dark adaptation would depend basically on the equilibrium between the 13-cis-and the all-trans-isomers. The experiments also suggest that at pH 5, but not at pH 7, solubilizing surfactant concentrations produce a considerable increase in the velocity of the dark adaptation reaction, perhaps through changes in the microenvironment of a protonable group. © 1990.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Solubilization of phospholipid bilayers by surfactants belonging to the Triton X series: Effect of polar group size

Various effects of Triton X-114, Triton X-100, Triton X-102, Triton X-165, and Triton X-305 (written in increasing order of cmc, hydrophile/lipophile balance, and number of ethylene oxide units, i.e. polar group size) have been tested on large unilamellar vesicles made of egg phosphatidylcholine. For both lytic and sublytic effects, Triton X-102 appears to be the most efficient member of the series. Leakage and solubilization parameters confirm the previous suggestion that these are two independent surfactant-induced processes.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Unexpected wide substrate specificity of C. perfringens α-toxin phospholipase C.

Clostridium perfringens phospholipase C (CpPLC), also called α-toxin, is the main virulence factor for gas gangrene in humans. The lipase activity serves the bacterium to generate lipid signals in the host eukaryotic cell, and ultimately to degrade the host cell membranes. Several previous reports indicated that CpPLC was specific for phosphatidylcholine and sphingomyelin. Molecular docking studies described in this paper predict favorable interactions of the CpPLC active site with other phospholipids, e.g. phosphatidylethanolamine, phosphatidylinositol and, to a lesser extent, phosphatidylglycerol. On the basis of these predictions, we have performed experimental studies showing α-toxin to degrade all the phospholipids mentioned above. The molecular docking data also provide an explanation for the observed lower activity of CpPCL on sphingomyelin as compared to the glycerophospholipids.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe