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Cationic Liposomes As Carriers for Gene Delivery: Physico-Chemical Characterization and Mechanism of Cell Transfection

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Cationic Liposomes As Carriers for Gene Delivery: Physico-Chemical Characterization and Mechanism of Cell Transfection African Journal of Biotechnology Vol. 11(11), pp. 2763-2773, 7 February, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.3019 ISSN 1684–5315 © 2012 Academic Journals Full Length Research Paper Cationic liposomes as carriers for gene delivery: Physico-chemical characterization and mechanism of cell transfection Bing Wang1,2, Jiti Zhou1, Shaohui Cui2, Baoling Yang2, Yinan Zhao2, Budiao Zhao2, Yan Duan3 and Shubiao Zhang2* 1College of Environmental and Biological Science, Dalian University of Technology, Dalian 116024, Liaoning, China. 2SEAC-ME Key Laboratory of Biotechnology and Bioresources Utilization, Dalian Nationalities University, Dalian 116600, Liaoning, China. 3Department of Pharmacy, National University of Singapore, Singapore 117543. Accepted 13 December, 2011 The inter-correlations among the parameters that influence the transfection efficiency of liposome-DNA complexes (lipoplexes) as carriers in gene therapy, optimum procedures for transfection and mechanism of cell transfection were investigated. The morphology, size and ζ-potential of liposomes and lipoplexes were measured by atomic force microscopy (AFM) and dynamic light scattering techniques. The lipoplexes formation was tested by using gel electrophoresis. Transfection efficiency was assessed using luciferase and green fluorescence protein reporter plasmids, respectively. It was found that transfection efficiency markedly depended on liposome to DNA weight ratio, lipoplexes morphology and size. The intracellular trafficking of exogenous DNAs was observed by confocal laser microscopy. After 4 h incubation, DNAs delivered by Lipofectamine 2000 reached the nucleus with a much higher frequency than 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). It was concluded that lipoplexes with filaments contributing more than globular obtained higher transfection efficiency with the maximum expression of 500000 RLU/mg luciferase in HeLa cells, for they are easy to release pDNA after endocytosis. These results implied that by further modifying the chemical features of the lipid vectors and controlling their biological behaviors, more efficient lipid delivery vectors may be achieved. Key words: Cationic liposomes, gene delivery, transfection efficiency, AFM, intracellular trafficking. INTRODUCTION As a promising strategy for the treatment of many exogenous genes into target cells are urgently awaited inherited and acquired diseases, gene therapy has (Zhang et al., 2007). Though viral systems usually give attracted many researchers during the past several high transfection efficiency, safety concerns from decades (Ma et al., 2007). The aim of gene therapy is to potential mutation, recombination, oncogenic effect, and deliver healthy exogenous genetic drugs such as plasmid high cost, however, greatly limit their therapeutic appli- DNA and single-strand oligonucleotides to replace a cations. In contrast, non-viral transfection agents, mainly missing gene so as to cure genetic diseases, for including cationic lipids and cationic polymers, are instance, cystic fibrosis, malignant melanoma, and believed to cause less safety problems although non- Gaucher's disease (Zhang et al., 2004). Based on these specific cytotoxicity associated with cationic liposomes factors, reliable and efficient vectors delivering has been observed (Matsui et al., 2006), thereby making it easier to manufacture in larger scale and to deliver large pieces of DNA (Laporte et al., 2006), and also to modulate chemically for improvement of transfection *Corresponding author. E-mail: [email protected]. Tel: +86 411 efficiency (Wasungu and Hoekstra, 2006). 876 56141. Fax: +86 411 876 44496. There are great deals of structural variations in 2764 Afr. J. Biotechnol. lipid molecules that are able to mediate transfection. (Ruponen et al., 2009). They were used as models in Since the cationic liposome vector was first introduced by order to correlate physico-chemical parameters affecting Felgner et al. (1987) who used N-[1-(2,3-dioleyloxy) transfection efficiency in this paper. The aim of this propyl]-N,N,N-trimethylammonium chloride (DOTMA) to present study was to examine the correlation of lipoplex carry out a DNA-transfection protocol, many cationic morphology, size, cell density, DNA amount and lipids and polycations used for DNA transfer have been liposome/pDNA weight ratio, lipoplex formation and widely investigated (Martin et al., 2005). For transfection transfection time, serum and cell lines with transfection application, cationic lipids are often mixed with helper efficiency and toxicity. To study the mechanism of lipids like dioleoylphosphatidylethanolamine (DOPE) or cationic lipid-mediated gene delivery with higher cholesterol, potentially promoting conversion of the transfection efficiency, we visualized intracellular lamellar lipoplex phase into a non-lamellar structure, trafficking with fluorescently labeled DNA molecules. which presumably rationalizes their ability to improve cationic lipid mediated transfection efficiency (Wasungu and Hoekstra, 2006). Moreover, optimization of MATERIALS AND METHODS transfection efficiency may be attempted by using a trial and error approach consisting of synthesizing and testing Reagents and materials a large number of derivatives. On the other hand, rational Commercial reagents, Lipofectamine 2000 and DOTAP, were design of highly efficient cationic lipids and polymers purchased from Invitrogen and Roche-Diagnostics, respectively as requires a deeper understanding of the interaction aqueous suspensions at concentrations of 1 mg/ml. The pGL3- between vectors and DNAs, as well as the cellular control plasmid (Promega) contained a modified coding region for pathways and mechanisms involved in DNA entry into firefly (Photinus pyralis) luciferase and the pGFP-N2 plasmid (Clontech) containing a coding region for green fluorescence cells and ultimately nucleus (Niculescu-Duvaz et al., protein. Plasmid DNAs were extracted and purified using the Endo- 2003; Elouahabi and Ruysschaert, 2005; Bally et al., Free Plasmid Purification Kit (Qiagen). λ DNA/EcoRI+ HindIII 1999). Marker was purchased from Fermentas. Cells were purchased from However, despite the recent clinical success, the basic Institute of Biochemistry and Cell Biology, SIBS, CAS, China. knowledge of particle structure–activity relationship is still Growth media, RPMI1640 and DMEM, were purchased from Gibco, unsatisfactory and the optimization of these systems and the other chemicals used were of reagent-grade. remains largely a result of trial and error (Masotti et al., 2009). In order to better understand the role of different Chemical structure and composition parameters in improving the clinical performances of these systems, a systematic in vitro study of the physico- The commercial liposomal formulations have different chemical chemical and biological properties of both liposomes and compositions and were generally mixtures of neutral and cationic lipoplexes is crucial. Liposome-mediated gene delivery lipids. The chemical structures of the lipids are shown in Figure 1. DOTAP liposome contains DOTAP (N-[1-(2,3-dioleoyloxy)propyl]- transfection processes, which ultimately govern the N,N,N-trimethylammonium chloride) and DOPE at 1:1 ratio. transfection efficiency, are influenced by a variety of Lipofectamine 2000 may be mixtures of lipofectin and lipofectamine physico-chemical parameters such as the cationic contain DOSPA (2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]- liposome/DNA charge ratio, the particle size of N,dimethyl-1-propanaminium trifluoroacetate), DOTMA and DOPE. lipoplexes, the superficial charge and the morphology. The difference between DOTAP and Lipofectamine 2000 may reside on the different head groups that in the latter case, consist of The details of the intracellular trafficking of exogenous a polyamine moiety. DNA should be very important for effective transgene expression. In the current, generally accepted model, exogenous DNA is incorporated by cells mainly via an Preparation of plasmid DNA endocytosis pathway (Wrobel and Collins, 1995; Huth et al., 2006). The enhanced released DNA as “naked” DNA The pGL3-control and pGFP-N2 plasmids were amplified in Escherichia coli (strain JM109 and DH5-α, respectively) cells and into the cytoplasm (Kamiya et al., 2002) and entry into purified by column chromatography (Sigma). The purity of the the nucleus is a very important obstacle to high plasmid was measured by OD260/OD280 (1.85-1.90; OD, optical transgene expression (Hashimoto et al., 2006). density), as well as by electrophoresis in a 0.8% agarose gel. Lipofectamine 2000 can efficiently transfect post-mitotic Plasmid DNA was labeled with Rhodamine (Rh) using Label It ® neurons (Farrow et al., 2006) and rat primary CX-Rhodamine Labeling Kit (Mirus). hepatocytes as well (Green et al., 2006), thus suggesting that it may promote penetration of DNA (Okumura et al., Electrophoresis 2009) through intact nuclear envelopes (Lonez et al., 2008) in these cell types, further demonstrating its utility. Cationic liposome/pDNA complexes were formed using pGL3- 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) control at a final DNA concentration of 0.5 μg/μL and at weight liposome was used to transfect cells with DNA and siRNA ratios from 0.5 to 8. The complexes were then loaded onto 0.8% agarose gels containing ethidium bromide (0.5 mg/ml). The gel (Kleivel et al., 2008) and to quantify intracellular DNA running buffer was composed of 40 mM Tris acetate and 1 mM release
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