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.     

Hydration of lipoplexes commonly used in gene delivery: follow-up by laurdan fluorescence changes and quantification by differential scanning calorimetry.

Lipoplexes, which are formed spontaneously between cationic liposomes and negatively charged nucleic acids, are commonly used for gene and oligonucleotide delivery in vitro and in vivo. Being assemblies, lipoplexes can be characterized by various physicochemical parameters, including size distribution, shape, physical state (lamellar, hexagonal type II and/or other phases), sign and magnitude of electrical surface potential, and level of hydration at the lipid-DNA interface. Only after all these variables will be characterized for lipoplexes with a broad spectrum of lipid compositions and DNA/cationic lipid (L(+)) mole (or charge) ratios can their relevance to transfection efficiency be understood. Of all these physicochemical parameters, hydration is the most neglected, and therefore the focus of this study. Cationic liposomes composed of DOTAP without and with helper lipids (DOPC, DOPE, or cholesterol) or of DC-Chol/DOPE were complexed with pDNA (S16 human growth hormone) at various DNA(-)/L(+) charge ratios (0.1-3.2). (DOTAP=N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride; DC-Chol=(3beta-[N-(N',N'-dimethylaminoethane)-carbamoyl]-cholester ol; DOPC=1, 2-dioleoyl-sn-glycero-3-phosphocholine; DOPE=1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine). The hydration levels of the different cationic liposomes and the DNA separately are compared with the hydration levels of the lipoplexes. Two independent approaches were applied to study hydration. First, we used a semi-quantitative approach of determining changes in the 'generalized polarization' (GP) of laurdan (6-dodecanoyl-2-dimethylaminonaphthalene). This method was recently used extensively and successfully to characterize changes of hydration at lipid-water interfaces. Laurdan excitation GP at 340 nm (GP(340)DOTAP. The GP(340) of lipoplexes of all lipid compositions (except those based on DC-Chol/DOPE) was higher than the GP(340) of the cationic liposomes alone and increased with increasing DNA(-)/L(+) charge ratio, reaching a plateau at a charge ratio of 1. 0, suggesting an increase in dehydration at the lipid-water interface with increasing DNA(-)/L(+) charge ratio. Confirmation was obtained from the second method, differential scanning calorimetry (DSC). DOTAP/DOPE lipoplexes with charge ratio 0.44 had 16.5% dehydration and with charge ratio 1.5, 46.4% dehydration. For DOTAP/Chol lipoplexes with these charge ratios, there was 17.9% and 49% dehydration, respectively. These data are in good agreement with the laurdan data described above. They suggest that the dehydration occurs during lipoplex formation and that this is a prerequisite for the intimate contact between cationic lipids and DNA.     

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.     

Interaction of cationic liposomes and their DNA complexes with monocytic leukemia cells

Cationic liposomes complexed with DNA have been used extensively as non-viral vectors for the intracellular delivery of reporter or therapeutic genes in culture and in vivo. We examined the relationship between the characteristics of the lipoplexes, their mode of interaction with monocytic THP-1 cells and their ability to transfect these cells. We determined the size and zeta potential of cationic liposomes (composed of 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) and its mixtures with neutral lipids), and lipoplexes at different (+/-) charge ratios. As the (+/-) charge ratio of the lipoplexes decreased to (1/1), a significant reduction in zeta potential and an increase in size was observed. The increase in size resulted from fusion between liposomes promoted by DNA, as demonstrated by a lipid mixing assay, and from aggregation of the complexes. Interaction of liposomes and lipoplexes with THP-1 cells was assessed by monitoring lipid mixing ('fusion') as well as binding and cell association. While no lipid mixing was observed with the 1/2 (+/-) lipid/DNA complexes, lipoplexes with higher (+/-) charge ratios underwent significant fusion in conjunction with extensive cell binding. Liposome binding to cells was dependent on the positive charge of the liposomes, and their fusion could be modulated by the co-lipid. DOTAP/phosphatidylethanolamine (1:1) liposomes fused with THP-1 cells, unlike DOTAP/phosphatidylcholine (1:1) liposomes, although both liposome types bound to the cells to a similar extent. The use of inhibitors of endocytosis indicated that fusion of the cationic liposomes with cells occurred mainly at the plasma membrane level. The presence of serum increased the size of the cationic liposomes, but not that of the lipoplexes. Low concentrations of serum (3%) completely inhibited the fusion of cationic liposomes with cells, while inhibiting binding by only 20%. Our results suggest that binding of cationic liposomes and lipoplexes to cells is governed primarily by electrostatic interactions, whereas their fusion is regulated by the lipid composition and sterically favorable interactions with cell surface molecules. In addition our results indicate no correlation between fusion of the lipoplexes with the plasma membrane and the levels of transfection.     

Structure and internal organization of overcharged cationic-lipid/peptide/DNA self-assembly complexes

The combination of cationic lipids with cationic peptides and DNA vectors can produce synergistic effects in gene delivery to eukaryotic cells. Binary complexes of cationic lipids with DNA are well-studied whereas little information is available about the structure of the ternary lipid/peptide/DNA (LPD) complexes and mechanisms defining DNA protection and delivery. Here we use synchrotron small angle X-ray scattering and dynamic light scattering zeta-potential measurements to determine structure and the net charge of supramolecular aggregates of complexes in mixtures of plasmid DNA, cationic liposomes formed from DOTAP, plus a linear cationic ε-oligolysine with the pendant α-amino acids Leu-Tyr-Arg (LYR), ε-(LYR)K10. These ternary complexes display multilamellar structures with relatively constant separation between DOTAP bilayers, accommodating a hydrated monolayer of parallel DNA rods. The DNA-DNA distance in the complexes varies as a function of the net positive to negative (lipid+peptide)/DNA charge ratio. An explanation for the observed dependence of DNA-DNA distance on charge ratio was proposed based on general polyelectrolyte properties of non-stoichiometric polycation-DNA mixtures.     

Isothermal titration calorimetric analysis of the interaction between cationic lipids and plasmid DNA

The effects of buffer and ionic strength upon the enthalpy of binding between plasmid DNA and a variety of cationic lipids used to enhance cellular transfection were studied using isothermal titration calorimetry at 25.0 degrees C and pH 7.4. The cationic lipids DOTAP (1,2-dioleoyl-3-trimethyl ammonium propane), DDAB (dimethyl dioctadecyl ammonium bromide), DOTAP:cholesterol (1:1), and DDAB:cholesterol (1:1) bound endothermally to plasmid DNA with a negligible proton exchange with buffer. In contrast, DOTAP: DOPE (L-alpha-dioleoyl phosphatidyl ethanolamine) (1:1) and DDAB:DOPE (1:1) liposomes displayed a negative enthalpy and a significant uptake of protons upon binding to plasmid DNA at neutral pH. These findings are most easily explained by a change in the apparent pKa of the amino group of DOPE upon binding. Complexes formed by reverse addition methods (DNA into lipid) produced different thermograms, sizes, zeta potentials, and aggregation behavior, suggesting that structurally different complexes were formed in each titration direction. Titrations performed in both directions in the presence of increasing ionic strength revealed a progressive decrease in the heat of binding and an increase in the lipid to DNA charge ratio at which aggregation occurred. The unfavorable binding enthalpy for the cationic lipids alone and with cholesterol implies an entropy-driven interaction, while the negative enthalpies observed with DOPE-containing lipid mixtures suggest an additional contribution from changes in protonation of DOPE.     

DOTAP cationic liposomes prefer relaxed over supercoiled plasmids

Cationic liposomes and DNA interact electrostatically to form complexes called lipoplexes. The amounts of unbound (free) DNA in a mixture of cationic liposomes and DNA at different cationic lipid:DNA molar ratios can be used to describe DNA binding isotherms; these provide a measure of the binding efficiency of DNA to different cationic lipid formulations at various medium conditions. In order to quantify the ratio between the various forms of naked DNA and supercoiled, relaxed and single-stranded DNA, and the ratio between cationic lipid bound and unbound DNA of various forms we developed a simple, sensitive quantitative assay using agarose gel electrophoresis, followed by staining with the fluorescent cyanine DNA dyes SYBR Green I or SYBR Gold. This assay was compared with that based on the use of ethidium bromide (the most commonly used nucleic acid stain). Unlike ethidium bromide, SYBR Green I DNA sensitivity and concentration-dependent fluorescence intensity were identical for supercoiled and nicked-relaxed forms. DNA detection by SYBR Green I in solution is approximately 40-fold more sensitive than by ethidium bromide for double-stranded DNA and approximately 10-fold for single-stranded DNA, and in agarose gel it is 16-fold more sensitive for double-stranded DNA compared with ethidium bromide. SYBR Gold performs similarly to SYBR Green I. This study shows that: (a) there is no significant difference in DNA binding isotherms to the monocationic DOTAP (DOTAP/DOPE) liposomes and to the polycationic DOSPA (DOSPA/DOPE) liposomes, even when four DOSPA positive charges are involved in the electrostatic interaction with DNA; (b) the helper lipids affect DNA binding, as DOTAP/DOPE liposomes bind more DNA than DOTAP/cholesterol; (c) in the process of lipoplex formation, when the DNA is a mixture of two forms, supercoiled and nicked-relaxed (open circular), there is a preference for the binding to the cationic liposomes of plasmid DNA in the nicked-relaxed over the supercoiled form. This preference is much more pronounced when the cationic liposome formulation is based on the monocationic lipid DOTAP than on the polycationic lipid DOSPA. The preference of DOTAP formulations to bind to the relaxed DNA plasmid suggests that the binding of supercoiled DNA is weaker and easier to dissociate from the complex.     

Physicochemical characterization and purification of cationic lipoplexes.

Cationic lipid-nucleic acid complexes (lipoplexes) consisting of dioleoyltrimethylammoniumpropane (DOTAP) liposomes and plasmid DNA were prepared at various charge ratios (cationic group to nucleotide phosphate), and the excess component was separated from the lipoplex. We measured the stoichiometry of the lipoplex, noted its colloidal properties, and observed its morphology and structure by electron microscopy. The colloidal properties of the lipoplexes were principally determined by the cationic lipid/DNA charge ratio and were independent of the lipid composition. In lipoplexes, the lipid membranes as observed in freeze-fracture electron microscopy were deformed into high-radius-of-curvature features whose characteristics depended on the lipid composition. Lipoplexes prepared at a threefold or greater excess of either DOTAP or DNA could be resolved into complexes with a defined stoichiometry and the excess component by sedimentation to equilibrium on sucrose gradients. The separated, positively charged complex retained high transfection activity and had reduced toxicity. The negatively charged lipoplex showed increased transfection activity compared to the starting mixture. In cryoelectron micrographs the positively charged complex was spherical and contained a condensed but indistinct interior structure. In contrast, the separated negatively charged lipoplexes had a prominent internal 5.9 +/- 0.1-nm periodic feature with material projecting as spikes from the spherical structure into the solution. It is likely that these two lipoplexes represent structures with different lipid and DNA packing.     

Interaction of oligonucleotides with cationic lipids: the relationship between electrostatics, hydration and state of aggregation

Lipoplexes, which are spontaneously formed complexes between oligonucleotide (ODN) and cationic lipid, can be used to deliver ODNs into cells, both in vitro and in vivo. The present study was aimed at characterizing the interactions associated with the formation of lipoplexes, specifically in terms of electrostatics, hydration and particle size. Large unilamellar vesicles (approximately 100 nm diameter), composed of either DOTAP, DOTAP/cholesterol (mole ratio 1:1) or DOTAP/DOPE (mole ratio 1:1) were employed as a model of cationic liposomes. Neutral vesicles ( approximately 100 nm diameter), composed of DOPC/DOPE (mole ratio 1:1), were employed as control liposomes. After ODN addition to vesicles, at different mole ratios, changes in pH and electrical surface potential at the lipid-water interface were analyzed by using the fluorophore heptadecyl-7-hydroxycoumarin. In separate 'mirror image' experiments, liposomes were added at different mole ratios to fluorescein isothiocyanate-labeled ODNs, thus yielding data about changes in the pH near the ODN molecules induced by the complexation with the cationic lipid. Particle size distribution and turbidity fluctuations were analyzed by the use of photon correlation spectroscopy and static light-scattering, respectively. In additional fluorescent probe studies, TMADPH was used to quantify membrane defects while laurdan was used to measure the level of hydration at the water-lipid interface. The results indicate that mutual neutralization of cationic lipids by ODNs and vice versa is a spontaneous reaction and that this neutralization is the main driving force for lipoplex generation. When lipid neutralization is partial, induced membrane defects cause the lipoplexes to exhibit increased size instability.     

Transfection using synthetic peptides: comparison of three DNA-compacting peptides and effect of centrifugation

Positively charged peptides have been shown to allow efficient transfection in vitro, especially when mixed with lipids. We have compared the ability of three positively charged peptides both to compact DNA and to increase the transfection efficiency of the cationic lipid DOTAP. The peptides are: a polymer of 17 lysines (pK17), YKAWK8WK (peptide K8) and SPKRSPKRSPKR (peptide P2). Peptides pK17 and K8 compact DNA efficiently in a gel retardation assay and protect DNA efficiently against DNase I degradation. Peptide P2, on the other hand, interacts weakly with DNA and provides poor protection. In order to compare their transfection efficiency, the three peptides were mixed with DNA (plasmid pEGFP-N1) at different charge ratios (+/-) and DOTAP (at a charge ratio of 2). The transfection efficiency was measured by FACS analysis at different times post-transfection. With NIH-3T3 cells, peptide P2 provides the highest transfection efficiency (about 40%), when compared with peptides pK17 (29%) and K8 (31%) and DOTAP alone (21%) under optimal conditions. Finally, we showed that centrifugation of the complexes onto the cells increased the transfection efficiency by a factor 1.5 to 2 with the various cell lines tested (ECV, primary human keratinocyte, CFT-2, NT-1).     

Factors affecting DNA binding and stability of association to cationic liposomes

Lipoplexes are complexes formed between cationic liposomes (L(+)) and polyanionic nucleic acids (P(-)). They are commonly used in vitro and in vivo as a nucleic acid delivery system. Our study aims are to investigate how DOTAP-based cationic liposomes, which vary in their helper lipid (cholesterol or DOPE) and in media of different ionic strengths affect the degree, mode of association and degree of condensation of pDNA. This was determined by ultracentrifugation and gel electrophoresis, methods based on different physical principles. In addition, the degree of pDNA condensation was also determined using the ethidium bromide (EtBr) intercalation assay. The results suggest that for cationic lipid compositions (DOTAP/DOPE and DOTAP/cholesterol), 1.5 M NaCl, but not 0.15 M NaCl, both prevent lipoplex formation and/or induce partial dissociation between lipid and DNA of preformed lipoplexes. The higher the salt concentration the greater is the similarity of DNA condensation (monitored by EtBr intercalation) between lipoplex DNA and free DNA. As determined by ultracentrifugation and agarose gel electrophoresis, 30-90% of the DNA is uncondensed. SDS below its critical micellar concentration (CMC) induced "de-condensation" of DNA without its physical release (assessed by ultracentrifugation) for both DOTAP/DOPE and DOTAP/cholesterol lipoplexes. As was assessed by agarose gel electrophoresis SDS induced release of 50-60% of DNA from the DOTAP/cholesterol lipoplex but not from the DOTAP/DOPE lipoplex. This study shows that there are conditions under which DNA is still physically associated with the cationic lipids but undergoes unwinding to become less condensed. We also proved that the helper lipid affects level and strength of the L(+) and DNA(-) electrostatic association; these interactions are weaker for DOTAP/cholesterol than for DOTAP/DOPE, despite the fact that the positive charge and surface pH of DOTAP/cholesterol and DOTAP/DOPE are similar.     

DNA pre-condensation with an amino acid-based cationic amphiphile. A viable approach for liposome-based gene delivery

A study related to the development and characterization of a new gene delivery system was performed. The approach consists in both the pre-condensation of plasmid DNA with an arginine-based cationic surfactant, arginine–N-lauroyl amide dihydrochloride (ALA), which was found not to be toxic, and the incorporation of the blood protein transferrin (Tf) into the formulations.Two cationic liposome formulations were used, one composed of a mixture of dioleoyl trimethylammoniopropane and cholesterol (DOTAP:Chol) and the other a pH sensitive formulation constituted of DOTAP, Chol, Dioleoyl phosphatidylethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS).Particles with different ALA/DNA and cationic lipid/DNA charge ratios were produced and a physicochemical characterization of the systems developed was performed. DNA conformational changes in the presence of ALA were studied by Circular Dichroism (CD) and the ALA binding to DNA was followed by surface tension measurements. Insight into the structure and morphology of the various ALA-complexes (complexes composed of ALA, DNA, Tf and liposomes) was obtained by cryogenic-Transmission Electron Microscopy (cryo-TEM) and the sizes of the ALA-complexes were determined through Photon Correlation Spectroscopy (PCS). We found that the transfection capacity of these systems is directly related with the presence of ALA and the lipidic composition. Complexes based on the pH sensitive liposome formulation present better transfection profiles. The correlation between the inner structure, density and size of the ALA-complexes and their biological activity is discussed. Overall, we demonstrate that the presence of ALA improves the transfection efficiency when conjugated with cationic liposome systems.     

The role of lipid charge density in the serum stability of cationic lipid/DNA complexes

To evaluate the role of lipid charge density in the serum stability of DOTAP-Chol/DNA complexes (lipoplexes), lipid-DNA interactions, extent of aggregation, supercoil content, and in vitro transfection efficiency of lipoplexes were investigated. In general, higher serum concentration destabilized, and increasing molar charge ratio of DOTAP to negatively charged phosphates in the DNA (DOTAP(+)/DNA(-)) stabilized lipoplexes in serum as assessed by the criteria used in this study. The increase of cholesterol content led to increased serum stability, and DOTAP:Chol (mol/mol 1:4)/DNA lipoplex with DOTAP(+)/DNA(-) ratio 4 was the most serum stable formulation of all the formulations examined, and maintained lipid-DNA interactions, did not aggregate and exhibited high in vitro transfection efficiency in 50% (v/v) serum. The increased stability of this formulation could not be explained by the decreased charge density of the lipid component. Furthermore, no single parameter examined in the study could be used to consistently predict the in vitro transfection efficiency of lipoplexes in serum. Surprisingly, no correlation between the maintenance of supercoiled DNA content and in vitro transfection efficiency was found in the study.     

DNA pre-condensation with an amino acid-based cationic amphiphile. A viable approach for liposome-based gene delivery

A study related to the development and characterization of a new gene delivery system was performed. The approach consists in both the pre-condensation of plasmid DNA with an arginine-based cationic surfactant, arginine-N-lauroyl amide dihydrochloride (ALA), which was found not to be toxic, and the incorporation of the blood protein transferrin (Tf) into the formulations.Two cationic liposome formulations were used, one composed of a mixture of dioleoyl trimethylammoniopropane and cholesterol (DOTAP:Chol) and the other a pH sensitive formulation constituted of DOTAP, Chol, Dioleoyl phosphatidylethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS).Particles with different ALA/DNA and cationic lipid/DNA charge ratios were produced and a physicochemical characterization of the systems developed was performed. DNA conformational changes in the presence of ALA were studied by Circular Dichroism (CD) and the ALA binding to DNA was followed by surface tension measurements. Insight into the structure and morphology of the various ALA-complexes (complexes composed of ALA, DNA, Tf and liposomes) was obtained by cryogenic-Transmission Electron Microscopy (cryo-TEM) and the sizes of the ALA-complexes were determined through Photon Correlation Spectroscopy (PCS). We found that the transfection capacity of these systems is directly related with the presence of ALA and the lipidic composition. Complexes based on the pH sensitive liposome formulation present better transfection profiles. The correlation between the inner structure, density and size of the ALA-complexes and their biological activity is discussed. Overall, we demonstrate that the presence of ALA improves the transfection efficiency when conjugated with cationic liposome systems.     

A simple but effective cancer vaccine consisting of an antigen and a cationic lipid

Developing a cancer vaccine with a potent adjuvant, which is safe for human use, remains to be an unmet need. In this study, we developed a simple, safe, yet efficient, peptide-based therapeutic cancer vaccine, DOTAP/E7 complex, which comprises only two molecules: a DOTAP cationic lipid and a peptide antigen derived from E7 oncoprotein of human papillomavirus (HPV) type 16. The anti-cancer activity of DOTAP/E7 against existing HPV positive TC-1 tumor was compared to that of our previous LPD/E7 formulation, which contains bacterial DNA CpG motifs. Tumor-bearing mice showed significant tumor inhibition following a single vaccination of either formulation at the optimal lipid dose, suggesting that DOTAP liposome alone can provide a potent adjuvant activity without plasmid DNA. E7 peptide formulated with DOTAP induced migration of activated dendritic cells (DC) to the draining lymph node (DLN) and efficiently generated functional antigen-specific CD8⁺ T lymphocyte responses. Accumulation of CD8⁺ tumor infiltrating T cells and apoptosis at tumor sites were observed after treatment with DOTAP/E7 complexes, which was also associated with a decreased amount of CD25⁺Foxp3⁺ regulatory T cells in treated animals. Reactive oxygen species (ROS) induced by DOTAP cationic lipid in DLN revealed a plausible mechanism of the initial interaction between DC and DOTAP. An adequate amount of ROS generation was apparently required for the initiation of the vaccine mechanism; however, an overdose of DOTAP induced massive ROS production and apoptosis of DC in DLN, which led to diminished anti-cancer immunity. Overall, these results indicate that cationic lipid DOTAP alone serves as an efficient vaccine adjuvant for the induction of a therapeutic, antigen-specific anti-cancer activity.     

Proteolipidic vectors for gene transfer to the lung

In order to develop improved synthetic gene transfer vectors, we have synthesized bifunctional peptides composed of a DNA binding peptide (P2) and ligand peptides selected by the phage display technique on tracheal epithelial cells. We have evaluated the capacity of these peptides to enhance the gene transfer efficiency of the cationic lipid DOTAP to the mouse lung. To optimize the in vivo transfection efficiency, we first compared the efficiency of DOTAP to transfect the lung by either intravenous injection or aerosolization. We then tested DNA/Peptide/DOTAP complexes formed at different Peptide/DNA and DOTAP/DNA charge ratios. Under optimal conditions, precompaction of DNA by peptide P2 gave a higher expression in the mouse lung using the luciferase reporter gene than DOTAP/DNA complexes. A further increase of transfection efficiency was obtained with the bifunctional peptide P2-9. Experiments performed with the GFP reporter gene showed expression in the alveolar parenchyme.     

Calorimetric study of the interaction of binary DMTAP/DOTAP cationic liposomes with plasmid DNA

Cationic liposomes have been suggested as possible agents for nonviral gene transfer. The interaction of plasmid DNA (pDNA) with dispersions of stable unilamellar cationic liposomes based on the binary lipid system 1,2-dimyristoyl-3-trimethyl-ammonium-propane (DMTAP):1,2-dioleoyl-3-trimethyl-ammonium-propane (DOTAP) has been studied by using isothermal titration calorimetry (ITC), high-precision differential scanning calorimetry (DSC), dynamic light scattering (DLS), and circular dichroism (CD). Systematic calorimetric and DLS exploration of the DMTAP:DOTAP binary system reveals that single-bilayer liposomes are stable at the 4:1 molar ratio, exhibiting the main lipid-phase transition temperature at ~25.3°C, and a total enthalpy change δH = 8.5 ± 0.4 kcal/mol. The interaction of pDNA with unilamellar DMTAP:DOTAP vesicles was investigated by ITC experiments, which clearly distinguished endothermic binding between the phosphate and the ammonium groups from exothermic processes, driven by slow kinetics, corresponding to interliposomal, DNA-triggered aggregation that leads to the formation of large multilamellar liposome/pDNA assemblies. Lipid-added-to-pDNA and pDNA-added-to-lipid experiments have been carried out in order to systematically explore the interaction mechanisms. Complex ITC profiles are revealed, which may be linked to packing rearrangements of the pDNA molecules bound at the outer liposomal surface, possibly due to binding to more than one liposome or due to p-DNA-enhanced heterogeneity in the local lipid concentration. DNA-mediated aggregation effects are detected at high [ammonium]/[phosphate] molar ratios in the case of lipid-added-to-pDNA interactions and at relatively low [phosphate]/[ammonium] molar ratios in the case of pDNA-added-to-lipid.     

Novel carbamate-linked quaternary ammonium lipids containing unsaturated hydrophobic chains for gene delivery

In this paper, two novel carbamate-linked quaternary ammonium lipids (MU18: a lipid with a mono-ammonium head; GU18: a lipid with a Gemini-ammonium head) containing unsaturated hydrophobic chains were designed and synthesized. The chemical structures of the synthetic lipids were characterized by infrared spectrum, ESI-MS, ¹H NMR, ¹³C NMR, and HPLC. For investigating the effect of unsaturation on gene delivery, the previous reported saturated cationic liposomes (MS18 and GS18) were used as comparison. Cationic liposomes were prepared by using these cationic lipids and neutral lipid DOPE at the molar ratio of 1:1. Particle sizes and zeta potentials of the cationic liposomes were studied to show that they were suitable for gene transfection. The binding abilities of the cationic liposomes were investigated by gel electrophoresis at various N/P ratios from 0.5/1 to 8/1. The results indicated that the binding ability of GU18 was much better than MU18 and the saturated cationic liposomes (MS18 and GS18). DNA transfection of these liposomes comparable to commercially available reagent (DOTAP) was achieved in vitro against Hela, HepG-2 and NCI-H460 cell lines. GU18 showed higher transfection at the N/P ratio of 3/1 than other cationic liposomes and the positive control, DOTAP. All of the liposomes presented a relatively low cytotoxicity, which was measured by MTT. Therefore, the synthetic lipids bearing unsaturated hydrophobic chains and Gemini-head could be promising candidates for gene delivery.     

The use of fluorescence resonance energy transfer to monitor dynamic changes of lipid-DNA interactions during lipoplex formation

Fluorescence resonance energy transfer (FRET) was used to monitor interactions between Cy3-labeled plasmid DNA and NBD-labeled cationic liposomes. FRET data show that binding of cationic liposomes to DNA occurs immediately upon mixing (within 1 min), but FRET efficiencies do not stabilize for 1-5 h. The time allowed for complex formation has effects on in vitro luciferase transfection efficiencies of DOPE-based lipoplexes; i.e., lipoplexes prepared with a 1-h incubation have much higher transfection efficiencies than samples with 1-min or 5-h incubations. The molar charge ratio of DOTAP to negatively charged phosphates in the DNA (DOTAP+/DNA-) also affected the interaction between liposomes and plasmid DNA, and interactions stabilized more rapidly at higher charge ratios. Lipoplexes formulated with DOPE were more resistant to high ionic strength than complexes formulated with cholesterol. Taken together, our data demonstrate that lipid-DNA interactions and in vitro transfection efficiencies are strongly affected by the time allowed for complex formation. This effect is especially evident in DOPE-based lipoplexes, and suggests that the time allowed for lipoplex formation is a parameter that should be carefully controlled in future studies.     

Transfection property of a new cholesterol-based cationic lipid containing tri-2-hydroxyethylamine as gene delivery vehicle

A novel cholesterol-based cationic lipid containing a tri-2- hydroxyethylamine head group and ether linker (Chol- THEA) was synthesized and examined as a potent gene delivery vehicle. In the preparation of cationic liposome, the addition of DOPE as helper lipid significantly increased the transfection efficiency. To find the optimum transfection efficiency, we screened various weight ratios of DOPE and liposome/DNA (N/P). The best transfection efficiency was found at the Chol-THEA:DOPE weight ratio of 1:1 and N/P weight ratio of 10~15. Most of the plasmid DNA was retarded by this liposome at the optimum N/P weight ratio of 10. The transfection efficiency of Chol-THEA liposome was compared with DOTAP, Lipofectamine, and DMRIE-C using the luciferase assay and GFP expression. Chol-THEA liposome with low toxicity had better or similar potency of gene delivery compared with commercial liposomes in COS-7, Huh-7, and MCF-7 cells. Therefore, Chol-THEA could be a useful non-viral vector for gene delivery.