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.     

Evaluation of cationic assemblies constructed with amino acid based lipids for plasmid DNA delivery

We synthesized cationic lipids bearing lysine, histidine, or arginine as a cationic headgroup for use in gene transfer studies. The cationic assemblies formed from lysine- or arginine-type lipids gave unilamellar vesicles (approximately 100 nm diameter), whereas the morphology of the histidine-type lipids was tube-like. The competences of the cationic assemblies were sufficient to form lipoplexes, and the resulting lipoplexes were evaluated in terms of gene expression efficiencies with COS-7 cells. The lysine- or arginine-type lipids exhibited higher gene expression efficiencies than that of Lipofectamine2000, a conventional transgenic reagent, indicating that stable lipoplexes could be prepared between spherical cationic assemblies and plasmid DNA. The gene expression efficiency in relation to the cationic headgroup of the lipids was as follows: lysine > or = arginine > histidine. In addition, gene expression efficiency was enhanced by decreasing the length of the alkyl chain of the hydrophobic moiety. Unlike Lipofectamine2000, no reduction in transfection efficiency in the presence of fetal bovine serum was observed for the lipoplexes formed using synthetic cationic lipids. Moreover, the synthetic cationic lipids revealed remarkably low cytotoxicity compared with Lipofectamine2000. In conclusion, cationic assemblies formed from 1,5-ditetradecyl-N-lysyl-L-glutamate or 1,5-ditetradecyl-N-arginyl-L-glutamate can be used as an effective plasmid DNA delivery system.     

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.     

Effect of the head-group geometry of amino acid-based cationic surfactants on interaction with plasmid DNA

The interaction between DNA and different types of amino acid-based cationic surfactants was investigated. Particular attention was directed to determine the extent of influence of surfactant head-group geometry toward tuning the interaction behavior of these surfactants with DNA. An overview is obtained by gel retardation assay, isothermal titration calorimetry, fluorescence spectroscopy, and circular dichroism at different mole ratios of surfactant/DNA; also, cell viability was assessed. The studies show that the surfactants with more complex/bulkier hydrophobic head group interact more strongly with DNA but exclude ethidium bromide less efficiently; thus, the accessibility of DNA to small molecules is preserved to a certain extent. The presence of more hydrophobic groups surrounding the positive amino charge also gave rise to a significantly lower cytotoxicity. The surfactant self-assembly pattern is quite different without and with DNA, illustrating the roles of electrostatic and steric effects in determining the effective shape of a surfactant molecule.     

Physicochemical optimisation of plasmid delivery by cationic lipids

Non-viral gene therapy is based on the use of plasmid expression vectors and chemical or physical plasmid DNA delivery systems. This review discusses the roles of cationic lipids as vectors for gene transfection, reviews different strategies employed to improve cationic lipids for in vivo use, and provides original results on the physicochemistry of lipoplexes. Cationic lipid/DNA delivery vehicles have evolved considerably since their initial gene transfection experiments. Much work has been carried out to investigate their structure/activity relationships, methods of formulation and physicochemical properties. Further work has also focused on enhancing and prolonging their stability in a physiological environment as well as increasing their site-specific and tissue-specific interactions. Original data presented in this report confirm that cationic lipids associated to DNA form supramolecular lamellar structures, which protect DNA from serum DNAse degradation. The effect of formulation (and hence the size of the particles) on lipoplex in vivo circulation half-life and biodistribution is also discussed. A list of abbreviations can be found at the end of the review.     

Structural analysis of DNA complexation with cationic lipids

Complexes of cationic liposomes with DNA are promising tools to deliver genetic information into cells for gene therapy and vaccines. Electrostatic interaction is thought to be the major force in lipid–DNA interaction, while lipid-base binding and the stability of cationic lipid–DNA complexes have been the subject of more debate in recent years. The aim of this study was to examine the complexation of calf-thymus DNA with cholesterol (Chol), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioctadecyldimethylammoniumbromide (DDAB) and dioleoylphosphatidylethanolamine (DOPE), at physiological condition, using constant DNA concentration and various lipid contents. Fourier transform infrared (FTIR), UV-visible, circular dichroism spectroscopic methods and atomic force microscopy were used to analyse lipid-binding site, the binding constant and the effects of lipid interaction on DNA stability and conformation. Structural analysis showed a strong lipid–DNA interaction via major and minor grooves and the backbone phosphate group with overall binding constants of K_Chol = 1.4 (±0.5) × 10⁴ M⁻¹, K_DDAB = 2.4 (±0.80) × 10⁴ M⁻¹, K_DOTAP = 3.1 (±0.90) × 10⁴ M⁻¹ and K_DOPE = 1.45 (± 0.60) × 10⁴ M⁻¹. The order of stability of lipid–DNA complexation is DOTAP>DDAB>DOPE>Chol. Hydrophobic interactions between lipid aliphatic tails and DNA were observed. Chol and DOPE induced a partial B to A-DNA conformational transition, while a partial B to C-DNA alteration occurred for DDAB and DOTAP at high lipid concentrations. DNA aggregation was observed at high lipid content.     

Infrared spectroscopic characterization of the interaction of lipid bilayers with phenol, salicylic acid and o-acetylsalicylic acid

The interaction of phenol (PHE), salicylic acid (SA) and o-acetylsalicylic acid (ASA) with bilayers of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was investigated by infrared spectrometry. The temperature of the main gel to liquid crystal phase transition of DPPC is markedly depressed in the presence of the three guest molecules. The temperature depression depends on the nature and concentration of the additives. The temperature of the pretransition is also affected by these guest molecules and the depression in temperature is even more pronounced than that of the main transition temperature. Possible modes of interaction of these guest molecules with the lipid bilayers are discussed.     

Effect of helper lipids on the interaction of DNA with cationic lipid monolayers studied by specular neutron reflection

The interaction of DNA with monolayers of the cationic lipid dimethyldioctadecylammonium bromide, with/without 50 mol % of a neutral "helper" lipid, either dioleoylphosphatidylethanolamine or cholesterol, has been studied using specular neutron reflection, surface pressure-area isotherms, and Brewster angle microscopy. The amount of DNA bound to the lipid head groups has been comprehensively quantified in the range of 8-39 vol% of DNA with respect to the monolayer composition (monolayers composed of dimethyldioctadecylammonium bromide binding the most DNA and monolayers containing dioleoylphosphatidylethanolamine binding the least) and surface pressure (DNA binding being greatest at highest surface pressures). Surprisingly, regardless of these variables, the thickness of the DNA-containing layer remained approximately constant between 18 and 25 ?. This systematic study is the first direct quantification of the binding of DNA with two different helper-lipid-containing multicomponent monolayers, an important step toward understanding interaction parameters in more realistic models of gene delivery systems.     

Characterisation of three novel cationic lipids as liposomal complexes with DNA

Cationic lipids (CLs) are being increasingly exploited as transfection vectors for the delivery of DNA into eukaryotic cells. To obtain further insight to the complex formation and interactions between cationic liposomes and DNA, we characterised three novel cationic lipids, viz. bis[2-(11-phenoxyundecanoate)ethyl]-dimethylammonium bromide, N-hexadecyl-N-?10-[O-(4-acetoxy)-phenylundecanoate]ethyl?- dimethylammonium bromide, and bis[2-(11-butyloxyundecanoate)ethyl]dimethylammonium bromide. These lipids bear the same charged headgroup yet have different hydrophobic parts. Accordingly, we may anticipate their electrostatic interactions with DNA to be similar while differing in both thermal phase behaviour and physicochemical properties of their complexes with DNA. In keeping with the above all three lipids formed complexes with DNA as evidenced by light scattering, fluorescence spectroscopy and Langmuir film balance. Differential scanning calorimetry revealed very different phase behaviours for the binary mixtures of the three CLs with dimyristoylphosphatidylcholine and also provided evidence for DNA-induced lipid phase separation. These data were confirmed by compression isotherms and fluorescence microscopy of monolayers residing on an aqueous buffer, recorded both in the presence and absence of DNA. Importantly, binding to cationic liposomes appears to prevent thermal denaturation of DNA upon heating of the complexes. Likewise, renaturation of heat-treated DNA complexed with the cationic liposomes appears to be abolished as well.     

Chiral multinuclear macrocyclic polyamine complexes: synthesis, characterization and their interaction with plasmid DNA

A series of multinuclear macrocyclic polyamine metal (Zn(2+), Cu(2+), Co(2+)) complexes containing chiral dipeptide linkage were synthesized and used as artificial nuclease enzyme model. The interaction between the complexes and plasmid DNA (pUC19) was studied, and the results revealed that these complexes could act as powerful catalysts for the cleavage of plasmid DNA under physiological conditions.     

Synthesis and characterization of betaine-like diacyl lipids: zwitterionic lipids with the cationic amine at the bilayer interface

We synthesized and characterized a series of zwitterionic, acetate-terminated, quaternized amine diacyl lipids (AQ). These lipids have an inverted headgroup orientation as compared to naturally occurring phosphatidylcholine (PC) lipids; the cationic group is anchored at the membrane interface, while the anionic group extends into the aqueous phase. AQ lipids preferentially interact with highly polarizable anions (ClO(4)(-)) over less polarizable ions (Cl(-)), in accord with the Hofmeister series, as measured by the change in zeta potential of AQ liposomes. Conversely, AQ lipids have a weaker association with calcium than do PC lipids. The transition temperatures (Tm) of the AQ lipids are similar to the Tm observed with phosphatidylethanolamine (PE) lipids of the same chain length. AQ lipids form large lipid sheets after heating and sonication; however, in the presence of cholesterol (Chol), these lipids form stable liposomes that encapsulate carboxyfluorescein. The AQ:Chol liposomes retain their contents in the presence of serum at 37°C, and when injected intravenously into mice, their organ biodistribution is similar to that observed with PC:Chol liposomes. AQ lipids demonstrate that modulating the headgroup charge orientation significantly alters the biophysical properties of liposomes. For the drug carrier field, these new materials provide a non-phosphate containing zwitterlipid for the production of lipid vesicles.     

The counterion influence on cationic lipid-mediated transfection of plasmid DNA

A panel of DOTAP analogs was prepared by altering the anionic counterion that accompanies the trimethylammonium polar domain. The transfection of plasmid DNA into NIH3T3 cells and mouse lung was examined using the counterion analogs. The in vitro transfection activity decreased as follows: DOTAP · bisulfate > trifluoromethanesulfonate ∼ iodide ∼ bromide > dihydrogenphosphate ∼ chloride ∼ acetate > sulfate. A similar activity trend was observed in vivo.     

Physico-chemical characterization and transfection efficacy of cationic liposomes containing the pEGFP plasmid

Cationic liposomes-DNA complexes (lipoplexes) are largely used in gene delivery. Deciphering specific chemical and physical properties of lipoplexes is a necessary step to unravel the mechanisms underlying transfection and to improve transfection efficacy in each experimental model. In the present paper we investigated the physico-chemical features of lipoplexes containing a plasmid encoding for the GFP protein, in order to correlate these results with transfection efficacy. Cationic unilamellar vesicles (mean diameter 100 nm) were prepared, from the cationic DC-Chol lipid and the zwitterionic phospholipid DOPE. The two components of the liposome bilayer were used at molar ratio close to unity. ESR spectra were recorded and zeta potential zeta was measured on liposomes complexed with the plasmid. One of the main points of interest in this paper resided in the fact that both kinds of measurements were carried out in the same conditions (i.e. lipid concentration, medium composition, and pH) employed for cell transfection experiments. Transfection was performed on CHO cells; the percentage of fluorescent cells was evaluated and compared with the above physico-chemical features. It emerged that the composition and pH of the medium, the lipoplex/cell ratio, as well as the amount of lipoplex added to the cell culture were critical parameters for transfection efficacy. Finally, lipoplex surface charge played a fundamental role to achieve a high transfection level.     

Mixtures of cationic lipid O-ethylphosphatidylcholine with membrane lipids and DNA: phase diagrams

Ethylphosphatidylcholines are positively charged membrane lipid derivatives, which effectively transfect DNA into cells and are metabolized by the cells. For this reason, they are promising nonviral transfection agents. With the aim of revealing the kinds of lipid phases that may arise when lipoplexes interact with cellular lipids during DNA transfection, temperature-composition phase diagrams of mixtures of the O-ethyldipalmitoylphosphatidylcholine with representatives of the major lipid classes (phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, cholesterol) were constructed. Phase boundaries were determined using differential scanning calorimetry and synchrotron x-ray diffraction. The effects of ionic strength and of DNA presence were examined. A large variety of polymorphic and mesomorphic structures were observed. Surprisingly, marked enhancement of the affinity for nonlamellar phases was observed in mixtures with phosphatidylethanolamine and cholesterol as well as with phosphatidylglycerol (previously reported). Because of the potential relevance to transfection, it is noteworthy that such phases form at close to physiological conditions, and in the presence of DNA. All four mixtures exhibit a tendency to molecular clustering in the gel phase, presumably due to the specific interdigitated molecular arrangement of the O-ethyldipalmitoylphosphatidylcholine gel bilayers. It is evident that a remarkably broad array of lipid phases could arise in transfected cells and that these could have significant effects on transfection efficiency. The data may be particularly useful for selecting possible "helper" lipids in the lipoplex formulations, and in searches for correlations between lipoplex structure and transfection activity.     

Isolation and characterization of plasmid DNA fromRuminococcus

A method has been developed, utilizing mutanolysin and proteinase K, for the rapid lysis of strains of the rumen cellulolytic bacteriumRuminococcus. This has enabled bacterial chromosomal and plasmid DNA to be isolated. A small cryptic plasmid has been identified inRuminococcus flavefaciens strain 186. It is 5.2 kb long, contains a singleBamHI site, and two sites forBglII,EcoRI, andHindIII. This plasmid has potential in the development of genetic vectors for rumen bacteria.     

LacO-LacI interaction in affinity adsorption of plasmid DNA

Current approaches for purifying plasmids from bacterial production systems exploit the physiochemical properties of nucleic acids in non-specific capture systems. In this study, an affinity system for plasmid DNA (pDNA) purification has been developed utilizing the interaction between the lac operon (lacO) sequence contained in the pDNA and a 64mer synthetic peptide representing the DNA-binding domain of the lac repressor protein, LacI. Two plasmids were evaluated, the native pUC19 and pUC19 with dual lacO3/lacOs operators (pUC19(lacO3/lacOs)), where the lacOs operator is perfectly symmetrical. The DNA-protein affinity interaction was evaluated by surface plasmon resonance using a Biacore system. The affinity capture of DNA in a chromatography system was evaluated using LacI peptide that had been immobilized to Streamline adsorbent. The KD-values for double stranded DNA (dsDNA) fragments containing lacO1 and lacO3 and lacOS and lacO3 were 5.7 +/- 0.3 x 10(-11) M and 4.1 +/- 0.2 x 10(-11) M respectively, which compare favorably with literature reports of 5 x 10(-10)-1 x 10(-9) M for native lacO1 and 1-1.2 x 10(-10) M for lacO1 in a saline buffer. Densitometric analysis of the gel bands from the affinity chromatography run clearly showed a significant preference for capture of the supercoiled fraction from the feed pDNA sample. The results indicate the feasibility of the affinity approach for pDNA capture and purification using native protein-DNA interaction.     

F plasmid partition depends on interaction of SopA with non-specific DNA

Bacterial ATPases belonging to the ParA family assure partition of their replicons by forming dynamic assemblies which move replicon copies into the new cell-halves. The mechanism underlying partition is not understood for the Walker-box ATPase class, which includes most plasmid and all chromosomal ParAs. The ATPases studied both polymerize and interact with non-specific DNA in an ATP-dependent manner. Previous work showed that in vitro, polymerization of one such ATPase, SopA of plasmid F, is inhibited by DNA, suggesting that interaction of SopA with the host nucleoid could regulate partition. In an attempt to identify amino acids in SopA that are needed for interaction with non-specific DNA, we have found that mutation of codon 340 (lysine to alanine) reduces ATP-dependent DNA binding > 100-fold and correspondingly diminishes SopA activities that depend on it: inhibition of polymer formation and persistence, stimulation of basal-level ATP hydrolysis and localization over the nucleoid. The K340A mutant retained all other SopA properties tested except plasmid stabilization; substitution of the mutant SopA for wild-type nearly abolished mini-F partition. The behaviour of this mutant indicates a causal link between interaction with the cell's non-specific DNA and promotion of the dynamic behaviour that ensures F plasmid partition.     

Use of dithiodiglycolic acid as a tether for cationic lipids decreases the cytotoxicity and increases transgene expression of plasmid DNA in vitro.

Two major barriers that limit cationic lipids in gene delivery are low transfection efficiency and toxicity. In the present studies, we used dithiodiglycolic acid as a new tether for the polar and hydrophobic domains of a cationic lipid, cholesteryl hemidithiodiglycolyl tris(aminoethyl)amine (CHDTAEA). We compared the transfection activity and toxicity of CHDTAEA with its nondisulfide analogue and cholesteryl N-(dimethylaminoethyl) carbamate (DC-Chol). The liposomes of CHDTAEA had more than 2 orders of magnitude greater transfection activity than DC-Chol in CHO cells and 7 times greater transfection activity in SKnSH cells. CHDTAEA also demonstrated much less toxicity than the other two lipids. Dithiodiglycolic acid may act as an excellent linker in the application of cationic lipid syntheses.     

Intracellular visualization of BrdU-labeled plasmid DNA/cationic liposome complexes.

Difficulties in specific detection of transfected DNA in cells represent an important limitation in the study of the gene transfer process. We studied the cellular entry and fate of a plasmid DNA complexed with a cationic lipid, Vectamidine (3-tetradecylamino-N-tert-butyl-N'-tetradecylpropionamidine) in BHK21 cells. To facilitate its detection inside the cells, bromodeoxyuridine (BrdU) was incorporated into plasmid DNA under conditions that minimize plasmid alteration. BrdU was localized in cells incubated with Vectamidine/BrdU-labeled plasmid DNA complexes by immunogold labeling and electron microscopy (EM). Labeling was predominantly associated with aggregated liposome structures at the surface of and inside the cells. EM observations of cells transfected with Vectamidine/DNA complexes showed that the liposome/DNA aggregates accumulate in large vesicles in the cell cytosol. On the other hand, using rhodamine-labeled Vectamidine and revealing BrdU with FITC-conjugated antibodies permitted simultaneous detection in the cells of both components of the complexes with confocal laser scanning microscopy. The DNA and lipids co-localized at the surface of and inside the cells, indicating that the complex is internalized as a whole. Our results show that the BrdU-labeled plasmid DNA detection system can be a useful tool to visualize exogenous DNA entry into cells by a combination of electron and confocal microscopy.