Biomedical subjects
Publications and source records attributed to Marie-Laure Miramon.
A short preparation of polyoxygenated macrocycles can be carried out by combining the formation of an acetal linkage, to introduce long alkyl chains, with a ring closure metathesis. As an example, this methodology was used to synthesize a new polyamino lipid.
During the preparation of 3-C-(6-O-acetyl-2,3,4-tri-O-benzyl-alpha-D-mannopyranosyl)-1-propene, using a published Sakurai-type reaction on the parent methyl glycoside, some observations were made on the sensitivity to reaction conditions that were not previously reported. This Note presents the study of this allylation reaction followed by acetolysis, which ultimately led to the best conditions to obtain the C-glycoside, and on further transformations to yield the corresponding aldehydic and acidic derivatives.
BACKGROUND: The low efficiency and toxicity of transfection in a primary culture of hepatocytes using cationic lipids remains a limiting step to the study of gene function and the setting up of non-viral gene therapy. METHODS: A novel class of cationic lipids (GBs) derived from natural glycine betaine compounds covalently linked to acyl chains by enzymatically hydrolysable peptide and ester bonds, a structure designed to reduce cytotoxicity, was used to improve transfection efficiency in a primary culture of rat hepatocytes. The relationship between lipid structure, lipoplex formulation and transfection efficiency was studied using six GBs (12-14-16, 22-24-26) varying in their spacer and acyl chains. RESULTS: GB12, characterized by short [(CH(2))(10)] acyl chains and spacer, allowed plasmid uptake in all cells and reporter gene expression in up to 40% of hepatocytes with a low cytotoxicity, a much higher efficiency compared with transfections using other reagents including Fugene6 and Lipofectin. We also showed that numerous cells accumulated high amounts of plasmids demonstrating that GB12 promoted a very efficient DNA transfer through plasma membrane leading to an increase in nuclear plasmid translocation, allowing a much higher gene expression. Moreover, GB12-transfected hepatocytes survived to injection in normal livers and were found to express the LacZ reporter gene. CONCLUSIONS: The non-toxic GB12 formulation is a powerful vehicle for plasmid delivery in cultured hepatocytes with relevance in liver gene therapy.
To date, no clear and constant relationship has been established between the chemical structure and the efficiency of non-viral transfection reagents. Despite the improvement of synthetic transfection systems, the capacity to transfect a target cell in a specific way is still a major challenge that gene therapy needs to overcome to be successful. Consequently, we developed a strategy aimed specifically at improving transfection of targeted human epithelial cells and to examine the possible effects of electrostatic interactions. Our attention therefore focused on the development of novel glycosylated formulations, based upon the introduction of one or two different carbohydrate ligands into (i) cationic lipid structures and (ii) synthetic neutral lipids incorporated into DNA and lipoplexes. Then, these formulations were tested in vitro on two human cell lines [HeLa and 16HBE14o(-)]. We report here that one of those formulations (CG 1/DOPE) is more efficient than DOTAP/DOPE. We determined that this non-viral transfection process is partially due to an endocytotic phenomenon mediated by targeting specific receptors directed toward specific carbohydrate elements. This was shown on 16HBE14o(-) cells where we observed a 43% and a 69% decrease in transfection when we blocked these receptors by the addition of free lactose and mannose, respectively. These results highlight the large adaptability of such monocationic glycolipids in the context of targeting and gene delivery.
The need for fast and efficient purification methods that can be easily handled and moreover automated is considerably increasing with the new techniques of high-throughput chemical synthesis. Following our previous work on the use of simple polymeric scavengers in fast reactions and purifications of organic substances, this article presents the results found during the development of a new method for the purification of phenolic substances. The purification method was found to be regulated by the interaction of acidic phenol groups with a basic polystyrene resin. Furthermore, the scavenging of phenolic isomers proved to be very selective for a given isomer. But the most interesting aspect of this method is that it is based on a simple contact in situ with the resin and that the adsorption/desorption process of the phenol was found to be solvent-dependent. The phenols can thus be freed from impurities, or other isomers, by a simple and fast contact with the resin in the first solvent, filtration, and washings, followed by liberation of the purified phenol by a last soaking in another solvent for desorption. The method was successfully applied to the purification of a crude reaction mixture issued from the Fries rearrangement of phenyl acetate, as well as to small libraries of phenolic derivatives.