Influence of charge ratio of liposome/DNA complexes on their size after extrusion and transfection efficiency

Marija Brgles, Maja Šantak, Beata Halassy, Dubravko Forcic, Jelka TomašicInstitute of Immunology, Research and Development Department, Zagreb, CroatiaBackground: Physicochemical characteristics of liposome/DNA complexes influence transfection efficiency and affect each other in a very intricate way. The result of this is discrepancies in conclusions drawn about the individual influence of each one.Methods: Aiming to elucidate the influence of liposome/DNA charge ratio and size on transfection efficiency and on each other, we used liposome/DNA complexes with charge ratio (+/-) in the range of 1–50 and extruded through membranes of 400, 200, and 100 nm. Plasmid DNA encoding green fluorescent protein was used to measure transfection efficiency by flow cytometry. Sizes of liposome/DNA complexes were measured by dynamic light scattering.Results: Liposome size was reduced after extrusion but this was mainly driven by the charge ratio and not by the size of the membrane pores. Reduction of complex size at each charge ratio positively correlated with transfection efficiency. When the size of the complexes was approximately constant, increasing the charge ratio was found to promote transfection efficiency. Cationic lipid N-(1-(2,3-dioleoyloxy)propyl)N,N,N trimethylammonium chloride was used for modulation of positive charge and a cytotoxicity test showed that increasing its amount increases cytotoxicity.Conclusion: It can be concluded that charge ratio dictates the size of the complex whereas overall size reduction and higher charge ratios promote transfection efficiency in vitro.Keywords: transfection efficiency, liposome charge, liposome siz

Syntheses and Enzymatic Hydrolyses of Acylated Methyl α-D-Mannopyranosides

Selective pivaloylations of methyl a-D-mannopyranoside under various reaction conditions have been studied. The structures of the products were established by 1H-NMR spectroscopy and acetylation of partially pivaloylated compounds. The order of reactivity of hydroxyl groups was established and found to be 6-OH>3-OH>2-OH>4-OH. The newly synthesized acylated monosaccharides were subjected to the hydrolysis by rabbit serum and esterases isolated from rabbit serum. Various degrees of regioselectivity were observed in these reactions, depending on the sugar structure

Intermolecular Aminolyses of 1-Thioglycosyl Esters of N-Acylamino Acids

Fully acetylated 1-thioglycopyranosyl esters of AT-acylaxnino acids (1-3), comprising different 1-thio sugars, undergo aminolysis with glycine methyl ester in dichloromethane at 40° to form the corresponding IV-acyldipeptide methyl esters. The relative reactivity of the C-l thioester bond towards aminolysis depends inter alia on the structure of the sugar moiety. Acylating efficiency of the 1-thio- esters was additionally demonstrated by aminolysis of 2,3,4,6-tetra- 0-aeetyl-l-S-(Af-fert-butyloxycmbonyl-L-tyrosyl)-l-thio-p-D-glucopyranose (3h) with peptidoglycan monomer (PGM, a disaccharide-pentapep- tide) in iV,)V-dimethylformamide at room temperature to give the corresponding disaccharide-hexapeptide

Modified glycopeptides related to cell wall peptidoglycan: conformational studies by NMR and molecular modelling

Polymeric peptidoglycans of bacterial cell walls, and smaller glycopeptides derived from them, exhibit versatile biological activities including immunomodulating properties. Peptidoglycan monomer (PGM) was isolated from Brevibacterium divaricatum and novel lipophilic derivatives of PGM bearing either (adamantyl-1-yl)-acetyl or Boc-Tyr substituents (Ad-PGM and BocTyr-PGM respectively) have recently been synthesized. We have obtained full assignments of the 1H and 13C spectra, using 2D NMR techniques, for all three compounds in DMSO solutions. NOESY/ROESY experiments have provided interproton distance restraints that were used in distance geometry modelling calculations to derive conformational preferences for each of these molecules. These data were supplemented with information available from chemical shifts, temperature dependence of amide proton shifts and proton?proton scalar couplings. Analysis of the results suggest that the lipophilic substituents attached to the Dap3-e amino group of the parent PGM molecule introduce changes to the conformational preferences of the peptide moiety. In PGM electrostatic interactions between charged end groups apparently promote folded conformations with participation of the long Dap side chain. Derivatives wherein such interactions are suppressed by acylation of the Dap3-e amino group are characterized by more extended conformations of the peptide chain. The new synthetic derivatives exhibit biological properties similar to those of the parent PGM. This may indicate that peripheral parts of the peptide chain such as the C-terminal and end groups of the long Dap side chain do not significantly contribute to the binding to receptors or enzymes participating in the biochemical interactions referred to abov