-Lysines Improve Cationic Lipid-Mediated Gene Transfer in Vascular Cells in vitro and in vivo

The potential of two small polyL -lysines (sPLLs), low molecular weight sPLL (LMW-L) containing 7–30 lysine residues and L18 with 18 lysine repeats, to enhance the efficiency of liposome-mediated gene transfer (GT) with cationic lipid DOCSPER [1,3-dioleoyloxy-2-(N 5 -carbamoyl-spermine)-propane] in vascular smooth muscle cells (SMCs) was investigated. Dynamic light scattering was used for determination of particle size. Confocal microscopy was applied for colocalization studies of sPLLs and plasmid DNA inside cells. GT was performed in proliferating and quiescent primary porcine SMCs in vitro and in vivo in porcine femoral arteries. At low ionic strength, sPLLs formed small complexes with DNA (50– 100 nm). At high ionic strength, large complexes ( 1 1 m) were observed without any significant differences in particle size between lipoplexes (DOCSPER/DNA) and lipopolyplexes (DOCSPER/sPLL/DNA). Both sPLLs were colocalized with DNA inside cells 24 h after transfection, protecting DNA against degradation. DOCSPER/sPLL/DNA formulations enhanced GT in vitro up to 5-fold, in a porcine model using loReceived: December 1, 2006 Accepted after revision: February 7, 2007 Published online: March 30, 2007 Prof. Dr. Sigrid Nikol Department of Cardiology and Angiology Medizinische Klinik und Poliklinik C, University of Münster Albert-Schweitzer-Strasse 33, DE–48129 Münster (Germany) Tel. +49 251 834 8501, Fax +49 251 834 5101, E-Mail [email protected] © 2007 S. Karger AG, Basel 1018–1172/07/0444–0273$23.50/0 Accessible online at: www.karger.com/jvr Golda /Pelisek /Klocke /Engelmann / Rolland /Mekkaoui /Nikol J Vasc Res 2007;44:273–282 274 the local deposition of active agents [6] . It has been shown that perivascular events significantly affect neointima: adventitial irritation results in focal intimal thickening and markers applied to the adventitia can be visualized in the intima [7, 8] . Therefore, vascular SMCs may be a suitable target using adventitial deposition of gene therapy vectors [3, 9] . Viral and non-viral vector systems have been developed for gene transfer (GT). Viral vectors are powerful vehicles for GT into somatic cells [10] . Adenoviral vectors have been widely used in vascular GT. However, such gene carriers based on viral vectors are highly immunogenic, relatively difficult to handle compared with plasmids and require enhanced biosafety levels. In contrast, non-viral vectors are cost-efficient, easy to handle and hardly induce any immune response. Furthermore, no specific infrastructure is necessary. Non-viral vector systems are usually composed of either ‘naked’ DNA or DNA complexed with amphipathic complexing agents to achieve higher transfer efficiencies [11] . During the past years, liposome-mediated, non-viral GT has been considerably improved by the introduction of new cationic lipid formulations [12] . Cationic lipids condense DNA through ionic interactions, thereby forming lipoplexes [13] . Despite such improvements, non-viral GT, in particular transfections of stationary or low proliferating cells, remained rather inefficient [13] . Nevertheless, optimization of GT conditions using cationic lipids led to significantly improved transfection rates in cultures of proliferating or stationary primary vascular cells as well as in vivo in a porcine restenosis model [14] . Yet, GT efficiency still needs further improvement to achieve high levels of gene expression for clinical use. One of the limitations for sufficient levels of gene expression is the relatively inefficient release of DNA from endosomes into the cytoplasm and the limited protection of DNA against cytoplasmic nucleases allowing for high amounts of intact DNA to enter the nucleus. Promising DNA-condensing agents are cationic polymers like polylysines, polyarginines, spermine, spermidine, dendrimers and polyethylenimine [15] . These polymers are believed to still adhere to DNA after endosomal release, thus allowing for enhanced protection of the DNA against cytoplasmic nucleases. PolyL -lysines were already used to condense DNA into a virus-like structure [16] . However, they alone are rather inefficient in promoting GT. Furthermore, their toxicity significantly increases with molecular weight [17] . Promising alternatives are lipopolyplexes, i.e. the combination of cationic lipids and cationic polymers, e.g., polyL -lysine. Plasmid DNA complexed with polyL lysine of 23.8 kDa (163 lysine residues) and liposomes allowed for significantly improved GT [18] . However, short polyL -lysines (sPLLs) of 13–18 lysine residues are characterized by already sufficient DNA binding capacity but low toxicity [19] . Therefore, we investigated the capability of two sPLLs – L18 with 18 L -lysine residues and low molecular weight polyL -lysine (LMW-L) which is a mixture of polyL -lysines containing 7–30 L -lysine residues – in enhancing liposome-mediated transfection. Furthermore, GT experiments were performed in primary porcine SMCs representing more suitable cell culture models better mimicking the in vivo conditions. Materials and Methods DNA Plasmid, Liposomes and Adjuvants A  -galactosidase reporter gene vector pCMV (7.2 kb, cytomegalovirus-driven galactosidase gene; Clontech, Heidelberg, Germany) and the cationic lipid DOCSPER [1,3-dioleoyloxy-2(N 5 -carbamoyl-spermine)-propane] [20] were used for transfections. LMW-L (1–4 kDa) was purchased at Sigma-Aldrich (Munich, Germany) and L18 (2.6 kDa) was synthesized to a purity 1 95% by Dr. Arnold (Gene Center, Munich, Germany). Both are referred to as sPLLs. Complex Formation Lipopolexes or polyplexes were prepared as follows: first, plasmid DNA (pCMV ) and DOCSPER or polyL -lysine were diluted in separate tubes using either OptiMEM (Gibco, Eggenstein, Germany; high ionic strength solution) or deionised water (low ionic strength solution). Cationic lipid or polymer dilution was then added to the DNA tube at different ratios (DOCSPER/DNA 1/1– 12/1; sPLL/DNA 1/1–50/1; w/w), mixed and incubated at room temperature for 30–40 min to allow for complex formation. In the case of lipopolyplexes, DNA was first pre-complexed with sPLLs by adding the polymer at different ratios directly into the tube with plasmid DNA, mixed and incubated for 5–15 min prior to the addition of the diluted DOCSPER again at various ratios and incubated at room temperature for additional 30–40 min. Particle Size Measurement Particle size was determined using dynamic light scattering with Zetasizer 3000HS (Malvern Instruments, Malvern, UK) using 10 subruns for each measurement in a 400l complex solution volume. Complexes were prepared as for GT and diluted either in low or high ionic strength solution (deionized water or OptiMEM). Formulations were analysed by measuring particle size in 10-min intervals for up to 60 min following the complex formation. Resistance against DNase I Degradation Lipoplexes and lipopolyplexes were prepared in 400 l OptiMEM as described above using 2.5
g/ml of plasmid DNA. Following complex formation, 400 l DNase I solution, i.e. 60 U/ml