The Role of Organ Vascularization and Lipoplex-Serum Initial Contact in Intravenous Murine Lipofection

Abstract

Following intravenous administration of cationic lipid-DNA complexes (lipoplexes) into mice, transfection (lipofection) occurs predominantly in the lungs. This was attributed to high entrapment of lipoplexes in the extended lung vascular tree. To determine whether lipofection in other organs could be enhanced by increasing the degree of vascularization, we used a transgenic mouse model with tissue-specific angiogenesis in liver. Tail vein injection of N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP)/cholesterol lipoplexes resulted in increased lipoplex entrapment in hypervascularized liver but did not boost luciferase expression, suggesting that lipoplex delivery is not a sufficient condition for efficient organ lipofection. Because the intravenously injected lipoplexes migrated within seconds to lungs, we checked whether the effects of immediate contact with serum correlate with lung lipofection efficiency of different DOTAP-based formulations. Under conditions mimicking the injection environment, the lipoplex-serum interaction was strongly dependent on helper lipid and ionic strength: lipoplexes prepared in 150 mM NaCl or lipoplexes with high (>33 mol%) cholesterol were found to aggregate immediately. This aggregation process was irreversible and was inversely correlated with the percentage of lung cells that took up lipoplexes and with the efficiency of lipofection. No other structural changes in serum were observed for cholesterol-based lipoplexes. Dioleoyl phosphatidylethanolamine-based lipoplexes were found to give low expression, apparently because of an immediate loss of integrity in serum, without lipid-DNA dissociation. Our study suggests that efficient in vivo lipofection is the result of cross-talk between lipoplex composition, interaction with serum, hemodynamics, and target tissue "susceptibility" to transfection.Cationic lipids, which are extensively employed as in vitro transfection agents, are promising agents for in vivo delivery of nucleic acids for a variety of applications from functional proteomics (1) to therapeutics (2). Obviously, the applications of cationic lipid-DNA complexes (lipoplexes) as in vivo delivery vehicles are useful only when the lipoplex-mediated transfection (lipofection) is high enough to modify the function of the protein/gene of interest.The most challenging route of in vivo administration of lipoplexes is the intravenous (i.v.) 1 route. Following mouse tail vein injection, most of the transgene expression occurs in the lungs, producing more than 80% of the total expressed protein in the animal (3-5). Despite significant efforts to optimize lipoplex performance following i.v. administration (4, 6), the "built-in" biological factors determining the efficiency of lung lipofection remain unclear.It has been shown that within minutes after injection, 60 -80% of the lipoplexes are entrapped in the lungs (4,7,8). This "first-pass" entrapment was ascribed to the highly extended lung capillary bed and was proposed to explain the predominant expression in...

Targeted cationic poly(D,L-lactic-co-glycolic acid) nanoparticles for gene delivery to cultured cells

“…However, this is not the explanation here, given that in our nanoparticles, no aggregation or increased sized was observed in the presence of serum. Other authors stated that the bigger particle size in the presence of serum decreased transfection [34,35]. The lower zeta potential due to the binding to anionic serum proteins can also contribute to this enhancement [36][37][38], but here, the charge was positive enough that the particles were not converted to negatively charged particles in the presence of serum, and the systems became serum-resistant.…”

Novel dextran–spermine conjugates as transfecting agents: comparing water-soluble and micellar polymers

“…The lyophilized 'cake' was hydrated with 20 mM Hepes, pH 7.4, and vortexed for several minutes to form UHV, mostly uni-and oligolamellar vesicles of B 500 nm. 35 Plasmids pEGFP-C1 plasmid under the control of the CMV promoter, pLNCluc plasmid containing the firefly luciferase gene, pLacZ under the control of the SV40 or CMV promoter, and pCMVhGH were used for quantitative and qualitative assays. Plasmids were purified using the Nucleobond AX 500 column (Machery-Nagel, Duren, Germany) or using the Qiagen Plasmid Mega kit (Hilden, Germany).…”

Non-Viral Gene Delivery Systems Based on Cholesterol Cationic Lipids: Structure-Activity Relationships

“…Cholesterol initially used as a helper lipid form more stable but less efficient complexes than those containing DOPE in vitro. However, cholesterol containing lipoplexes have shown a higher rate of biological activity when compared to lipoplexes with DOPE, when these complexes were utilized in vivo Sternberg et al, 1998;Smith et al, 1998;Simberg et al, 2003). The significant transfection activity attained was attributed to an improved cell binding and uptake of the lipoplexes promoted by the presence of cholesterol (Crook et al, 1998) and/or better stability of the lipoplex in serum (Simberg et al, 2003).…”

“…However, cholesterol containing lipoplexes have shown a higher rate of biological activity when compared to lipoplexes with DOPE, when these complexes were utilized in vivo Sternberg et al, 1998;Smith et al, 1998;Simberg et al, 2003). The significant transfection activity attained was attributed to an improved cell binding and uptake of the lipoplexes promoted by the presence of cholesterol (Crook et al, 1998) and/or better stability of the lipoplex in serum (Simberg et al, 2003). In 1991, Gao et al reported the synthesis and application of the cholesterol-based cationic lipid 3-β-[N-(N′,N′-dimethylaminoethyl)carbamoyl]-cholesterol (DC-Chol, 1a), which was combined with DOPE to transfect mammalian cells (Gao & Huang, 1991 (Zhdanov et al, 1998;Gao & Hui, 2001;Geall et al, 2000;Percot et al, 2004;Ding et al, 2008;Medvedeva et al, 2009;Maslov et al, 2010).…”