Membrane-modification effects, induced by ultraviolet (UV) irradiation in diacetylenic liposomes, were analyzed upon contact with cells, biological membranes, and proteins. Liposomes formulated with mixtures of unsaturated 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine and saturated 1,2-dimyristoyl-sn-glycero-3-phosphocholine, in a 1:1 molar ratio, were compared with those that were UV-irradiated and analyzed in several aspects. Membrane polymerization inherence on size stability was studied as well as its impact on mitochondrial and microsomal membrane peroxidation induction, hemolytic activity, and cell viability. Moreover, in order to gain insight about the possible irradiation effect on interfacial membrane properties, interaction with bovine serum albumin (BSA), lysozyme (Lyso), and apolipoprotein (apoA-I) was studied. Improved size stability was found for polymerized liposomes after a period of 30 days at 4°C. In addition, membrane irradiation had no marked effect on cell viability, hemolysis, or induction of microsomal and mitochondrial membrane peroxidation. Interfacial membrane characteristics were found to be altered after polymerization, since a differential protein binding for polymerized or nonpolymerized membranes was observed for BSA and Lyso, but not for apoA-I. The substantial contribution of this work is the finding that even when maintaining the same lipid composition, changes induced by UV irradiation are sufficient to increase size stability and establish differences in protein binding, in particular, reducing the amount of bound Lyso and BSA, without increasing formulation cytotoxicity. This work aimed at showing that the usage of diacetylenic lipids and UV modification of membrane interfacial properties should be strategies to be taken into consideration when designing new delivery systems.
Different viral and non-viral vectors have been designed to allow the delivery of nucleic acids in gene therapy. In general, non-viral vectors have been associated with increased safety for in vivo use; however, issues regarding their efficacy, toxicity and stability continue to drive further research. Thus, the aim of this study was to evaluate the potential use of the polymerizable diacetylenic lipid 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC8,9PC) as a strategy to formulate stable cationic lipopolymers in the delivery and protection of plasmid DNA. Cationic lipopolymers were prepared following two different methodologies by using DC8,9PC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and the cationic lipids (CL) 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), stearylamine (SA), and myristoylcholine chloride (MCL), in a molar ratio of 1:1:0.2 (DMPC:DC8,9PC:CL). The copolymerization methodology allowed obtaining cationic lipopolymers which were smaller in size than those obtained by the cationic addition methodology although both techniques presented high size stability over a 166-day incubation period at 4°C. Cationic lipopolymers containing DOTAP or MCL were more efficient in complexing DNA than those containing SA. Moreover, lipopolymers containing DOTAP were found to form highly stable complexes with DNA, able to resist serum DNAses degradation. Furthermore, neither of the cationic lipopolymers (with or without DNA) induced red blood cell hemolysis, although metabolic activity determined on the L-929 and Vero cell lines was found to be dependent on the cell line, the formulation and the presence of DNA. The high stability and DNA protection capacity as well as the reduced toxicity determined for the cationic lipopolymer containing DOTAP highlight the potential advantage of using lipopolymers when designing novel non-viral carrier systems for use in in vivo gene therapy. Thus, this work represents the first steps toward developing a cationic lipopolymer-based gene delivery system using polymerizable and cationic lipids.
Arsenic-binding proteins are under continuous research. Their identification and the elucidation of arsenic/protein interaction mechanisms are important because the biological effects of these complexes may be related not only to arsenic but also to the arsenic/protein structure. Although many proteins bearing a CXXC motif have been found to bind arsenic in vivo, new tools are necessary to identify new arsenic targets and allow research on protein/arsenic complexes. In this work, we analyzed the performance of the fluorescent compound APAO-FITC (synthesized from p-aminophenylarsenoxide, APAO, and fluorescein isothiocyanate, FITC) in arsenic/protein binding assays using thioredoxin 1 (Trx) as an arsenic-binding protein model. The Trx-APAO-FITC complex was studied through different spectroscopic techniques involving UV-Vis, fluorescence, atomic absorption, infrared and circular dichroism. Our results show that APAO-FITC binds efficiently and specifically to the Trx binding site, labeling the protein fluorescently, without altering its structure and activity. In summary, we were able to study a protein/arsenic complex model, using APAO-FITC as a labeling probe. The use of APAO-FITC in the identification of different protein and cell targets, as well as in in vivo biodistribution studies, conformational studies of arsenic-binding proteins, and studies for the design of drug delivery systems for arsenic anti-cancer therapies, is highly promising.
Arsenic compounds have shown medical usefulness since they proved to be effective in causing complete remission of acute promyelocytic leukemia. In this work we obtained a fluorescently labeled arsenic compound that can be used with current fluorescence techniques for basic and applied research, focused on arsenic-induced apoptosis studies. This compound is an arsanilic acid bearing a covalently linked FITC that was chemically synthesized and characterized by fluorescence, UV-Vis, mass and FTIR spectrometry. In addition, we assessed its apoptotic activity as well as its fluorescent labeling properties in HL60 cell line as a leukemia cell model through flow cytometry. We obtained a compound with a 1:1 FITC:arsenic ratio and a 595 m/z, confirming its structure by FTIR. This compound proved to be useful at inducing apoptosis in the leukemia cell model and labeling this apoptotic cell population, in such a way that the highest FITC fluorescence correlated with the highest arsenic amount.
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