Unsaturated Cationic Ortho Esters for Endosome Permeation in Gene Delivery

Abstract

Two cleavable cationic lipids were designed to trigger the fusogenicity and membrane permeation of their lipoplexes in endosomes via the formation of inverted hexagonal phases (HII). Both lipids contain a cationic head group and an unsaturated hydrophobic dioleylglycerol moiety joined together by a linear or a cyclic ortho ester linker. At pH 7.4, the lipids formed stable complexes with plasmid DNA together with the conelike helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). The decrease of pH enhanced the hydrolysis of the ortho ester linkers, which removed the cationic head groups and caused the aggregation of the lipoplexes. At pH 5.5, the cationic lipid N-[2-methyl-2-(1',2'-dioleylglyceroxy)dioxolan-4-yl]methyl-N,N,N-trimethylammonium iodide (2) with a cyclic ortho ester linker showed exceptional pH-sensitivity and triggered its lipoplex to permeate model biomembranes within the time span of endosome processing prior to lysosomal degradation. Lipid 2 significantly improved gene transfection in cultured cells compared to the pH-insensitive control lipid 1,2-dioleoyl-3-trimethylammoniopropane (DOTAP).

<em>Ex Vivo</em> Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

“…The absorbance spectrum of Triton X-100 does not include peaks in the 400-600 nm range recommended for this assay, and therefore, should not interfere with the normalization of experimental data. 20 Furthermore, for negative control samples, one should not observe significant hemolysis after the 1 hr incubation in any of the buffers used (i.e. even the most acidic buffer does not typically generate hemolysis on this timeframe).…”

Nature as a source of inspiration for cationic lipid synthesis

“…The cyclic ortho ester lipid 61 (Fig. 11) also showed a significantly higher transfection efficiency than DOTAP in CV-1 and HTB-129 cells (Chen et al 2007). In this case, two concomitant effects are thought to promptly destabilize the endosome membrane, the acid-catalyzed cleavage of the cationic head groups that caused the aggregation of the lipoplexes and the generated conelike lipid dioleylglycerol (DOG) that favored the formation of the fusogenic hexagonal phase.…”

“…Several ortho ester-containing cationic lipids have been synthesized and characterized for their susceptibility to undergo hydrolysis under mild acid conditions (Zhu et al 2000;By and Nantz 2004;Chen et al 2007). For instance, ortho ester lipid 54 (Fig.…”

trans-2-Aminocyclohexanol-based amphiphiles as highly efficient helper lipids for gene delivery by lipoplexes

“…The gene transfection procedure is similar to a reported method [33,34]. B16F1 cells (a mouse melanoma cell line, CRL-6323) and HeLa cells (a human cervical adenocarcinoma cell line, CCL-2) were cultured in DMEM containing 10% FBS, 4 mM L-glutamine, 1500 mg/L sodium bicarbonate and antibiotics (100 IU/mL penicillin and 100 μg/mL streptomycin) according to ATCC recommendations.…”