Danielle Hirsch-Lerner

Lamellarity of cationic liposomes and mode of preparation of lipoplexes affect transfection efficiency

Transfection of NIH-3T3 cells by a human growth hormone expression vector complexed with liposomes composed of N-(1-(2, 3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP) with or without helper lipids was studied. The transfection efficiency was dependent on the lamellarity of the liposomes used to prepare the lipoplexes. Multilamellar vesicles (MLV) were more effective than large unilamellar vesicles (LUV) of approximately 100 nm, irrespective of lipid composition. The optimal DNA/DOTAP mole ratio for transfection was </=0.5, at which only 10-30% of DOTAP in the lipoplex is neutralized. Prolonged incubation time of lipoplexes before addition to cells slightly decreased the level of transfection. A major influence on the lipofection level was found when the mode of lipoplex preparation was varied. Mixing plasmid DNA and DOTAP/DOPE (1:1) LUV in two steps instead of one step resulted in a higher lipofection when at the first step the DNA/DOTAP mole ratio was 0.5 than when it was 2.0. Only static light-scattering measurement, which is related to particle size and particle size instability, revealed differences between the lipoplexes as a function of lamellarity of the vesicles (MLV or LUV), mixing order, and number of mixing steps. Other physical properties of these lipoplexes were dependent only on the DNA/DOTAP mole ratio, i.e. the extent of DOTAP neutralization (as monitored by ionization of the fluorophore 4-heptadecyl-7-hydroxycoumarin) and the extent of defects in lipid organization (as monitored by level of exposure of the fluorophore 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3, 5-hexatriene to water). The secondary and tertiary structure of DNA in lipoplexes was evaluated by circular dichroism spectroscopy. The results of this study point out that the structure of lipoplexes should be physicochemically characterized at two different levels: the macro level, which relates to size and size instability, and the micro level, which relates to the properties described above which are involved in the intimate interaction between the plasmid DNA and the lipids. At the micro level, all parameters are reversible, history-independent and are determined by DNA/DOTAP mole ratio. On the other hand, the macro level (which is the most important for transfection efficiency) is history-dependent and not reversible.

Probing DNA–cationic lipid interactions with the fluorophore trimethylammonium diphenyl-hexatriene (TMADPH)

The aim of this study is to get a better understanding of DNA-cationic lipid complex formation and its characterization through the properties of the lipid assembly, using fluorescent probes known to have different locations in the vesicle bilayer, 1,6-diphenylhexa-1,3,5-triene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMADPH). The location of these two fluorescent probes in the membrane differs; the positive charge of TMADPH is localized close to the water/lipid interface and its fluorophore is present in the upper part of the acyl chain region while DPH (lacking polar group) is embedded deeper in the hydrophobic part of the bilayer. Unilamellar vesicles ( approximately 100 nm size) composed of N-(1-(2, 3-dioleoyloxy)-propyl)-N,N,N-trimethylammonium chloride (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as a helper lipid (at 1 : 1 mole ratio) were used as a model of cationic liposomes. Both linear and circular DNA gave almost identical results. DNA-/L+ (mole charge ratio of DNA negatively-charged phosphate to positively-charged lipid) ratios have large effects on the measured parameters. The effects monitored through TMADPH are much more striking than those obtained through the use of DPH, suggesting that the major DNA-lipid interaction occurs at the lipid/water interface. The fact that DNA induced much larger changes in TMADPH fluorescence intensity in H2O than in D2O suggests that the changes in the exposure of TMADPH to water and solvent relaxation effects are involved in the interaction. At DNA-/L+>/=1, fluorescence intensity increased concomitantly with a small increase in TMADPH fluorescence anisotropy without much affect in the size of the complex. At DNA-/L+<0.6, fluorescence quenching proportional to DNA-/L+ occurred, as well as a large increase in TMADPH fluorescence anisotropy and in complex size. These results suggest that at low DNA-/L+, negatively-charged DNA condenses positively-charged lipid headgroups, thereby inducing formation of lipid-ordered domains. This phase separation results in membrane defects at the lipid/water interface and increased exposure of the hydrophobic upper parts of the acyl chains to water, as indicated by the quenching of TMADPH. This leads to instability and aggregation/fusion of the DNA-lipid complexes. On the other hand, at DNA-/L+>/=1, the condensing effect is smaller, involving homogeneous lateral condensation of all the lipids, leading to a reduction in water content near the probe, and the DNA-lipid complexes are relatively small and stable.

Comparison of Different Commercially Available Cationic Lipid-Based Transfection Kits

Abstract Cationic lipid-containing nonviral vectors for intracellular DNA delivery (lipoplexes) are becoming an important tool in a number of applications in life sciences, biotechnology, agriculture, and medicine. They became a biotechnology industry product on their own, being marketed as transfection kits. Their number on the market, now exceeding 50, is growing steadily. Life science researchers could face a problem having such a large choice of products. In such a situation, a straightforward comparison of existing transfection kits is useful. Therefore, several new proprietary transfection kits (Effectene, FuGENE 6, GeneSHUTTLE-20, GeneSHUTTLE-40, and GenePORTER Transfection Reagents), as well as well-established ones of known composition (Lipofectin, LipofectAmine, DOSPER, and DOTAP Transfection Reagents) were chosen for simultaneous comparison of their in vitro activity and shelf stability. Using expression of human growth hormone in NIH 3T3 cells, it was shown that most of the kits show roughly the same order of magnitude of transfection, with FuGENE 6 and GeneSHUTTLE-40 being somewhat better. In parallel, stability tests were performed to assess the degree of lipid hydrolysis in different kits. It was shown that in all kits, level of non-esterified fatty acids increased upon storage at 4°C in aqueous dispersion, suggesting base-catalyzed hydrolysis of the ester lipids. The pattern of degradation was also clearly visible when lipid kit components were analyzed on TLC plates.

Interplay in lipoplexes between type of pDNA promoter and lipid composition determines transfection efficiency of human growth hormone in NIH3T3 cells in culture.

This study was aimed to investigate if and to what extent there is an interplay between lipoplex physicochemical properties and plasmid promoter type affecting transfection efficiency in vitro. To reduce the number of variables only one cell type (NIH3T3 cells), one gene (human growth hormone), one cationic lipid (DOTAP) in a plasmid >85% in supercoiled form, and the same medium conditions were used. The variables of the physicochemical properties included presence and type of helper lipid (DOPE, DOPC, or cholesterol, all in 1:1 mole ratio with DOTAP), size and lamellarity of the liposomes used for lipoplex preparation (large unilamellar vesicles, LUV, versus multilamellar vesicles, MLV), and DNA(-)/cationic lipid(+) charge ratio, all containing the same human growth hormone but differing in their promoter enhancer region. Two of the promoters were of viral origin: (a) SV40 promoter (simian virus early promoter) and (b) CMV promoter (cytomegalovirus early promoter); two were of mammalian cell origin: (c) PABP promoter (human poly(A)-binding protein promoter) and (d) S16 promoter (mouse ribosomal protein (rp) S16 promoter). Transfection studies showed that, irrespective of promoter type, large (> or =500 nm) MLV were superior to approximately 100 nm LUV; the extent of superiority was dependent on liposome lipid composition (larger for 100% DOTAP and DOTAP/DOPE than for DOTAP/DOPC and DOTAP/cholesterol). The optimal DNA(-)/DOTAP(+) charge ratio for all types of lipoplexes used was 0.2 or 0.5 (namely, when the lipoplexes were positively charged). Scoring the six best lipoplex formulations (out of 128 studied) revealed the following order: pCMV (DOTAP/DOPE) >> pSV (DOTAP/DOPE)=pCMV(DOTAP/cholesterol)=pS16 (100% DOTAP)=pS16 DOTAP/DOPE >> pCMV (DOTAP/DOPC). The lack of trivial consistency in the transfection efficiency score, the pattern of transfection efficiency, and statistical analysis of the data suggest that there is cross-talk between promoter type and lipoplex lipid composition, which may be related to the way the promoter is associated with the lipids.

Characterization of the interplay between the main factors contributing to lipoplex-mediated transfection in cell cultures.

Transfection efficiency of lipoplex‐mediated gene delivery is multifactorial. However, the mode of interaction between the factors which affect transfection is not fully understood. To help fill this deficiency we evaluated the effect of the interplay between several variables that affect transfection efficiency in cell cultures. For this, we applied the Analysis of Variance Model with Fixed Effects and Repeated Measures to assess the data. The variables studied include: two different genes, Luc, and human growth hormone (hGH), in three different plasmids (two of which contain the luciferase (Luc) gene, but different promoter‐enhancer regions (CMV and H19) and one plasmid coding hGH with a S16 promoter); three topoisoforms of pDNA (supercoiled (SC), open circular (OC), and closed circular (CC)); three cationic lipid compositions, all based on the monocationic lipid DOTAP (100% DOTAP, DOTAP/DOPE 1 : 1, and DOTAP/cholesterol 1 : 1, all ratios are mole ratios); two DNA−/L+ charge ratios (0.2 and 0.5); and two cell lines (NIH 3T3 and MBT‐2). Our statistical analysis confirmed that the cell type, the gene used for transfection, the promoter type, the type of helper lipid, and DNA−/DOTAP+ charge ratio, all affect transfection efficiency in a statistically significant manner. The most efficient lipoplex formulation in both cell lines was that based on DOTAP (without helper lipid), having CC plasmid DNA. We suggest that for obtaining the most transfection‐efficient lipoplex one should select the best topoisoform of pDNA for each particular cell type, and complex it with cationic liposomes having optimal lipid composition. Copyright

Cationic Lipid-Nucleic Acid Complexes (Lipoplexes): From Physicochemical Properties to In Vitro and In Vivo Transfection Kits

Lipoplexes are complexes formed spontaneously upon mixing of negatively charged nucleic acids (or other polyelectrolytes such as proteins) with positively-charged lipid assemblies [1]( for definitions see [2]). The relevant nucleic acids include plasmid DNA (pDNA), linear DNA, single chain DNA, oligonucleotide (ODN), messenger RNA, and silencing double stranded short RNA (siRNA) [1, 2, 3] The lipoplex-mediated nucleic acid delivery appears to be a promising system for a broad spectrum of both in vitro and in vivo applications in life sciences, biotechnology, medicine, and agriculture [1]. These applications are referred to as “lipofection”, namely, lipoplex- mediated transfection [2]. Among all currently available nonviral delivery systems, lipoplexes are one of the most popular and most versatile. This “popularity” is explained by the following:

Physicochemical Characterization of DOTAP-Containing Lipoplexes by Fluorescent Probes: Relevance to Lipofection

Delivering therapeutic genes to cells by their complexation with cationic liposomes to form lipoplexes has been shown to be efficient in vitro and in vivo (Behr, 1994; Ledley, 1995). The lipoplexes (plasmid DNA complexed with cationic liposomes) are safer than viral vectors (Mulligan, 1993; Crystal, 1995) for the following reasons: the absence of viral DNA, no constraint on DNA size, protection of DNA from degradation, and ability to target recombinant genes to specific cells. Therefore, lipoplexes might become the mainstream of research for gene therapy. The successful use of these lipoplexes will depend on their efficient delivery to cells and their ability to produce therapeutic levels of gene expression.