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.     

Lipid-DNA complex formation: reorganization and rupture of lipid vesicles in the presence of DNA as observed by cryoelectron microscopy.

Cryoelectron microscopy has been used to study the reorganization of unilamellar cationic lipid vesicles upon the addition of DNA. Unilamellar DNA-coated vesicles, as well as multilamellar DNA lipid complexes, could be observed. Also, DNA induced fusion of unilamellar vesicles was found. DNA appears to adsorb to the oppositely charged lipid bilayer in a monolayer of parallel helices and can act as a molecular "glue" enforcing close apposition of neighboring vesicle membranes. In samples with relatively high DNA content, there is evidence for DNA-induced aggregation and flattening of unilamellar vesicles. In these samples, multilamellar complexes are rare and contain only a small number of lamellae. At lower DNA contents, large multilamellar CL-DNA complexes, often with >10 bilayers, are formed. The multilamellar complexes in both types of sample frequently exhibit partially open bilayer segments on their outside surfaces. DNA seems to accumulate or coil near the edges of such unusually terminated membranes. Multilamellar lipid-DNA complexes appear to form by a mechanism that involves the rupture of an approaching vesicle and subsequent adsorption of its membrane to a "template" vesicle or a lipid-DNA complex.     

Characterization of DNA/lipid complexes by fluorescence resonance energy transfer

Fluorescence resonance energy transfer (FRET) is a potential method for the characterization of DNA-cationic lipid complexes (lipoplexes). In this work, we used FRET models assuming a multilamellar lipoplex arrangement. The application of these models allows the determination of the distance between the fluorescent intercalator on the DNA and a membrane dye on the lipid, and/or the evaluation of encapsulation efficiencies of this liposomal vehicle. The experiments were carried out in 1,2-dioleoyl-3-trimethylammonium-propane/pUC19 complexes with different charge ratios. We used 2-(3-(diphenylhexatrienyl)propanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (DPH-PC) and 2-(4,4-difluoro-5-octyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (BODIPY-PC) as membrane dyes, and ethidium bromide (EtBr) and BOBO-1 as DNA intercalators. In cationic complexes (charge ratios (+/-) >or= 2), we verified that BOBO-1 remains bound to DNA, and FRET occurs to the membrane dye. This was also confirmed by anisotropy and lifetime measurements. In complexes with all DNA bound to the lipid (charge ratio (+/-) = 4), we determined 27 A as the distance between the donor and acceptor planes (half the repeat distance for a multilamellar arrangement). In complexes with DNA unbound to the lipids (charge ratio (+/-) = 0.5 and 2), we calculated the encapsulation efficiencies. The presented FRET methodology is, to our knowledge, the first procedure allowing quantification of lipid-DNA contact.     

Liposome complexation efficiency monitored by FRET: effect of charge ratio,helper lipid and plasmid size

Cationic lipid/DNA complexes (lipoplexes) are promising vehicles for DNA vaccines or gene therapy. In these systems, transfection efficiency is highly related to lipoplex charge ratio, since lipoplexes with charge ratios (±) lower than electroneutrality have most DNA uncovered by the liposomes, and thus are unprotected from enzyme degradation. However, a large excess of cationic lipids is undesirable because of eventual cytotoxicity. The aim of this work was to determine the minimum charge ratio from which all DNA molecules are complexed by the liposomes varying the lipid formulation and plasmid size, using a new FRET (fluorescence resonance energy transfer) methodology. The similarity of FRET results, fluorescence intensity data and fluorescence decays of several charge ratios above (±) ≥ 4 or 5 confirmed that once all DNA is covered by the liposomes, additional lipid molecules do not affect the lipoplex multilamellar repeat distance. It was also verified by FRET that the presence of helper lipid reduces the amount of cationic lipid required for DNA protection but does not affect the lipoplex multilamellar repeat distance. This distance varies with the plasmid size when supercoiled plasmid is used, being apparently larger when longer plasmids are used. Our study indicates that, despite the complexity of these systems not being totally described by our model, FRET is an informative technique in lipoplex characterization.     

Lamellar cationic lipid‐DNA complexes from lipids with a strong preference for planar geometry: A Minimal Electrostatic Model

We formulate and analyze a minimal model, based on condensation theory, of the lamellar cationic lipid (CL)‐DNA complex of alternately charged lipid bilayers and DNA monolayers in a salt solution. Each lipid bilayer, composed by a random mixture of cationic and neutral lipids, is assumed to be a rigid uniformly charged plane. Each DNA monolayer, located between two lipid bilayers, is formed by the same number of parallel DNAs with a uniform separation distance. For the electrostatic calculation, the model lipoplex is collapsed to a single plane with charge density equal to the net lipid and DNA charge. The free energy difference between the lamellar lipoplex and a reference state of the same number of free lipid bilayers and free DNAs, is calculated as a function of the fraction of CLs, of the ratio of the number of CL charges to the number of negative charges of the DNA phosphates, and of the total number of planes. At the isoelectric point the free energy difference is minimal. The complex formation, already favoured by the decrease of the electrostatic charging free energy, is driven further by the free energy gain due to the release of counterions from the DNAs and from the lipid bilayers, if strongly charged. This minimal model compares well with experiment for lipids having a strong preference for planar geometry and with major features of more detailed models of the lipoplex. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 1114–1128, 2014.     

Phase diagram, stability, and overcharging of lamellar cationic lipid-DNA self-assembled complexes.

Cationic lipid-DNA (CL-DNA) complexes comprise a promising new class of synthetic nonviral gene delivery systems. When positively charged, they attach to the anionic cell surface and transfer DNA into the cell cytoplasm. We report a comprehensive x-ray diffraction study of the lamellar CL-DNA self-assemblies as a function of lipid composition and lipid/DNA ratio, aimed at elucidating the interactions determining their structure, charge, and thermodynamic stability. The driving force for the formation of charge-neutral complexes is the release of DNA and lipid counterions. Negatively charged complexes have a higher DNA packing density than isoelectric complexes, whereas positively charged ones have a lower packing density. This indicates that the overcharging of the complex away from its isoelectric point is caused by changes of the bulk structure with absorption of excess DNA or cationic lipid. The degree of overcharging is dependent on the membrane charge density, which is controlled by the ratio of neutral to cationic lipid in the bilayers. Importantly, overcharged complexes are observed to move toward their isoelectric charge-neutral point at higher concentration of salt co-ions, with positively overcharged complexes expelling cationic lipid and negatively overcharged complexes expelling DNA. Our observations should apply universally to the formation and structure of self-assemblies between oppositely charged macromolecules.     

NMR study of the interaction of retinoids with phospholipid bilayers

The interaction of three vitamin A derivatives or retinoids: all-trans-retinoic acid, 13-cis-retinoic acid and retinol with multilamellar phospholipid bilayers was studied using a combination of 2H- and 31P-NMR measurements. The following model membrane systems were used: (1) dipalmitoylphosphatidylcholine (DPPC) bilayers; (2) bilayers composed of a mixture of DPPC and bovine heart phosphatidylcholine (PC); (3) mixed PC/phosphatidylethanolamine (PE) bilayers. Only a weak interaction was observed between 13-cis-retinoic acid and DPPC membranes. Addition of all-trans-retinoic acid at a molar ratio of 1:2 to the lipid causes a small decrease (5 C degrees) in the gel to liquid crystalline phase-transition temperature of DPPC, a small increase in the order parameters of the lipid side-chains of single component bilayers and no measurable effect in the other lipid systems studied. Considerably larger perturbation in the lipid bilayer structure is introduced by addition of retinol which, at a molar ratio of 1:2 to the lipid, lowered the gel to liquid crystalline phase-transition temperature of DPPC by 21 C degrees and caused a decrease of order parameters of the lipid side-chains in all three lipid bilayer systems. These effects are consistent with intercalation of retinol molecules into the bilayer interior. The results for the mixed PC/PE bilayers indicate that the presence of retinol caused lateral separation of PE- and retinol-enriched regions.     

Structure, stability, and thermodynamics of lamellar DNA-lipid complexes.

We develop a statistical thermodynamic model for the phase evolution of DNA-cationic lipid complexes in aqueous solution, as a function of the ratios of charged to neutral lipid and charged lipid to DNA. The complexes consist of parallel strands of DNA intercalated in the water layers of lamellar stacks of mixed lipid bilayers, as determined by recent synchrotron x-ray measurements. Elastic deformations of the DNA and the lipid bilayers are neglected, but DNA-induced spatial inhomogeneities in the bilayer charge densities are included. The relevant nonlinear Poisson-Boltzmann equation is solved numerically, including self-consistent treatment of the boundary conditions at the polarized membrane surfaces. For a wide range of lipid compositions, the phase evolution is characterized by three regions of lipid to DNA charge ratio, rho: 1) for low rho, the complexes coexist with excess DNA, and the DNA-DNA spacing in the complex, d, is constant; 2) for intermediate rho, including the isoelectric point rho = 1, all of the lipid and DNA in solution is incorporated into the complex, whose inter-DNA distance d increases linearly with rho; and 3) for high rho, the complexes coexist with excess liposomes (whose lipid composition is different from that in the complex), and their spacing d is nearly, but not completely, independent of rho. These results can be understood in terms of a simple charging model that reflects the competition between counterion entropy and inter-DNA (rho < 1) and interbilayer (rho > 1) repulsions. Finally, our approach and conclusions are compared with theoretical work by others, and with relevant experiments.     

Natural lipid extracts and biomembrane-mimicking lipid compositions are disposed to form nonlamellar phases, and they release DNA from lipoplexes most efficiently

A viewpoint now emerging is that a critical factor in lipid-mediated transfection (lipofection) is the structural evolution of lipoplexes upon interacting and mixing with cellular lipids. Here we report our finding that lipid mixtures mimicking biomembrane lipid compositions are superior to pure anionic liposomes in their ability to release DNA from lipoplexes (cationic lipid/DNA complexes), even though they have a much lower negative charge density (and thus lower capacity to neutralize the positive charge of the lipoplex lipids). Flow fluorometry revealed that the portion of DNA released after a 30-min incubation of the cationic O-ethylphosphatidylcholine lipoplexes with the anionic phosphatidylserine or phosphatidylglycerol was 19% and 37%, respectively, whereas a mixture mimicking biomembranes (MM: phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine /cholesterol 45:20:20:15 w/w) and polar lipid extract from bovine liver released 62% and 74%, respectively, of the DNA content. A possible reason for this superior power in releasing DNA by the natural lipid mixtures was suggested by structural experiments: while pure anionic lipids typically form lamellae, the natural lipid mixtures exhibited a surprising predilection to form nonlamellar phases. Thus, the MM mixture arranged into lamellar arrays at physiological temperature, but began to convert to the hexagonal phase at a slightly higher temperature, ∼ 40-45 °C. A propensity to form nonlamellar phases (hexagonal, cubic, micellar) at close to physiological temperatures was also found with the lipid extracts from natural tissues (from bovine liver, brain, and heart). This result reveals that electrostatic interactions are only one of the factors involved in lipid-mediated DNA delivery. The tendency of lipid bilayers to form nonlamellar phases has been described in terms of bilayer “frustration” which imposes a nonzero intrinsic curvature of the two opposing monolayers. Because the stored curvature elastic energy in a “frustrated” bilayer seems to be comparable to the binding energy between cationic lipid and DNA, the balance between these two energies could play a significant role in the lipoplex-membrane interactions and DNA release energetics.     

Solutes in small amounts provide for lipid-bilayer softness: cholesterol,short-chain lipids,and bola lipids

The effect of the incorporation of small amounts (∼1 mole%) of amphiphilic solutes, such as cholesterol, a short-chain lipid (DC₁₀PC), and a bola lipid, into multilamellar DMPC bilayers is studied by small-angle neutron scattering and differential-scanning calorimetry. The anomalous swelling behavior observed in the transition region of pure DMPC bilayers is interpreted as an indication of bilayer softening and thermally reduced bending rigidity. Small amounts of the solutes are found to maintain or even enhance the bilayer softness. In the case of cholesterol, a systematic study shows that the well-known rigidification effect is observed only for cholesterol concentrations above 3–4 mole%. The results are discussed in relation to the physical properties of internal cell membranes. Received: 28 May 1996 / Accepted: 27 July 1996     

Natural lipid extracts and biomembrane-mimicking lipid compositions are disposed to form nonlamellar phases, and they release DNA from lipoplexes most efficiently

A viewpoint now emerging is that a critical factor in lipid-mediated transfection (lipofection) is the structural evolution of lipoplexes upon interacting and mixing with cellular lipids. Here we report our finding that lipid mixtures mimicking biomembrane lipid compositions are superior to pure anionic liposomes in their ability to release DNA from lipoplexes (cationic lipid/DNA complexes), even though they have a much lower negative charge density (and thus lower capacity to neutralize the positive charge of the lipoplex lipids). Flow fluorometry revealed that the portion of DNA released after a 30-min incubation of the cationic O-ethylphosphatidylcholine lipoplexes with the anionic phosphatidylserine or phosphatidylglycerol was 19% and 37%, respectively, whereas a mixture mimicking biomembranes (MM: phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine /cholesterol 45:20:20:15 w/w) and polar lipid extract from bovine liver released 62% and 74%, respectively, of the DNA content. A possible reason for this superior power in releasing DNA by the natural lipid mixtures was suggested by structural experiments: while pure anionic lipids typically form lamellae, the natural lipid mixtures exhibited a surprising predilection to form nonlamellar phases. Thus, the MM mixture arranged into lamellar arrays at physiological temperature, but began to convert to the hexagonal phase at a slightly higher temperature, approximately 40-45 degrees C. A propensity to form nonlamellar phases (hexagonal, cubic, micellar) at close to physiological temperatures was also found with the lipid extracts from natural tissues (from bovine liver, brain, and heart). This result reveals that electrostatic interactions are only one of the factors involved in lipid-mediated DNA delivery. The tendency of lipid bilayers to form nonlamellar phases has been described in terms of bilayer "frustration" which imposes a nonzero intrinsic curvature of the two opposing monolayers. Because the stored curvature elastic energy in a "frustrated" bilayer seems to be comparable to the binding energy between cationic lipid and DNA, the balance between these two energies could play a significant role in the lipoplex-membrane interactions and DNA release energetics.     

Plasmid DNA size does not affect the physicochemical properties of lipoplexes but modulates gene transfer efficiency.

Clinical applications of gene therapy mainly depend on the development of efficient gene transfer vectors. Large DNA molecules can only be transfected into cells by using synthetic vectors such as cationic lipids and polymers. The present investigation was therefore designed to explore the physicochemical properties of cationic lipid-DNA particles, with plasmids ranging from 900 to 52 500 bp. The colloidal stability of the lipoplexes formed by complexing lipopolyamine micelles with plasmid DNA of various lengths, depending on the charge ratio, resulted in the formation of three domains, respectively corresponding to negatively, neutrally and positively charged lipoplexes. Lipoplex morphology and structure were determined by the physicochemical characteristics of the DNA and of the cationic lipid. Thus, the lamellar spacing of the structure was determined by the cationic lipid and its spherical morphology by the DNA. The main result of this study was that the morphological and structural features of the lipopolyamine-DNA complexes did not depend on plasmid DNA length. On the other hand, their gene transfer capacity was affected by the size of plasmid DNA molecules which were sandwiched between the lipid bilayers. The most effective lipopolyamine-DNA complexes for gene transfer were those containing the shortest plasmid DNA.     

Fourier transform infrared spectroscopic studies of the interaction of the antimicrobial peptide gramicidin S with lipid micelles and with lipid monolayer and bilayer membranes

We have utilized Fourier transform infrared spectroscopy to study the interaction of the antimicrobial peptide gramicidin S (GS) with lipid micelles and with lipid monolayer and bilayer membranes as a function of temperature and of the phase state of the lipid. Since the conformation of GS does not change under the experimental conditions employed in this study, we could utilize the dependence of the frequency of the amide I band of the central beta-sheet region of this peptide on the polarity and hydrogen-bonding potential of its environment to probe GS interaction with and location in these lipid model membrane systems. We find that the GS is completely or partially excluded from the gel states of all of the lipid bilayers examined in this study but strongly partitions into lipid micelles, monolayers, or bilayers in the liquid-crystalline state. Moreover, in general, the penetration of GS into zwitterionic and uncharged lipid bilayer coincides closely with the gel to liquid-crystalline phase transition of the lipid. However, GS begins to penetrate into the gel-state bilayers of anionic phospholipids prior to the actual chain-melting phase transition, while in cationic lipid bilayers, GS does not partition strongly into the liquid-crystalline bilayer until temperatures well above the chain-melting phase transition are reached. In the liquid-crystalline state, the polarity of the environment of GS indicates that this peptide is located primarily at the polar/apolar interfacial region of the bilayer near the glycerol backbone region of the lipid molecule. However, the depth of GS penetration into this interfacial region can vary somewhat depending on the structure and charge of the lipid molecule. In general, GS associates most strongly with and penetrates most deeply into more disordered bilayers with a negative surface charge, although the detailed chemical structure of the lipid molecule and physical organization of the lipid aggregate (micelle versus monolayer versus bilayer) also have minor effects on these processes.     

Multilamellar structures of DNA complexes with cationic liposomes.

Studies of DNA complexes with cationic liposomes are prompted by the search for nonviral DNA carriers for gene therapy. Recent experiments have identified a stable multilamellar phase in which ordered smectic layers of DNA alternate with cationic bilayers. In this paper we identify the forces governing DNA adsorption on cationic lamellae, including a membrane-induced attraction between the adsorbed DNA. Calculating the DNA interhelical spacing as a function of system composition, the model successfully explains recent surprising observations.     

Factors governing the assembly of cationic phospholipid-DNA complexes

The interaction of DNA with a novel cationic phospholipid transfection reagent, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC), was investigated by monitoring thermal effects, particle size, vesicle rupture, and lipid mixing. By isothermal titration calorimetry, the heat of interaction between large unilamellar EDOPC vesicles and plasmid DNA was endothermic at both physiological and low ionic strength, although the heat absorbed was slightly larger at the higher ionic strength. The energetic driving force for DNA-EDOPC association is thus an increase in entropy, presumably due to release of counterions and water. The estimated minimum entropy gain per released counterion was 1.4 cal/mole- degrees K (about 0.7 kT), consistent with previous theoretical predictions. All experimental approaches revealed significant differences in the DNA-lipid particle, depending upon whether complexes were formed by the addition of DNA to lipid or vice versa. When EDOPC vesicles were titrated with DNA at physiological ionic strength, particle size increased, vesicles ruptured, and membrane lipids became mixed as the amount of DNA was added up to a 1.6:1 (+:-) charge ratio. This charge ratio also corresponded to the calorimetric end point. In contrast, when lipid was added to DNA, vesicles remained separate and intact until a charge ratio of 1:1 (+:-) was exceeded. Under such conditions, the calorimetric end point was 3:1 (+:-). Thus it is clear that fundamental differences in DNA-cationic lipid complexes exist, depending upon their mode of formation. A model is proposed to explain the major differences between these two situations. Significant effects of ionic strength were observed; these are rationalized in terms of the model. The implications of the analysis are that considerable control can be exerted over the structure of the complex by exploiting vectorial preparation methods and manipulating ionic strength.     

DNA-lipid complexes: stability of honeycomb-like and spaghetti-like structures.

A molecular level theory is presented for the thermodynamic stability of two (similar) types of structural complexes formed by (either single strand or supercoiled) DNA and cationic liposomes, both involving a monolayer-coated DNA as the central structural unit. In the "spaghetti" complex the central unit is surrounded by another, oppositely curved, monolayer, thus forming a bilayer mantle. The "honeycomb" complex is a bundle of hexagonally packed DNA-monolayer units. The formation free energy of these complexes, starting from a planar cationic/neutral lipid bilayer and bare DNA, is expressed as a sum of electrostatic, bending, mixing, and (for the honeycomb) chain frustration contributions. The electrostatic free energy is calculated using the Poisson-Boltzmann equation. The bending energy of the mixed lipid layers is treated in the quadratic curvature approximation with composition-dependent bending rigidity and spontaneous curvature. Ideal lipid mixing is assumed within each lipid monolayer. We found that the most stable monolayer-coated DNA units are formed when the charged/neutral lipid composition corresponds (nearly) to charge neutralization; the optimal monolayer radius corresponds to close DNA-monolayer contact. These conclusions are also valid for the honeycomb complex, as the chain frustration energy is found to be negligible. Typically, the stabilization energies for these structures are on the order of 1 k(B)T/A of DNA length, reflecting mainly the balance between the electrostatic and bending energies. The spaghetti complexes are less stable due to the additional bending energy of the external monolayer. A thermodynamic analysis is presented for calculating the equilibrium lipid compositions when the complexes coexist with excess bilayer.     

Refined structure of 1,2-diacyl-P-O-ethylphosphatidylcholine bilayer membranes

1,2-diacyl-P-O-ethylphosphatidylcholines are synthetic cationic lipids that show some promising properties as nonviral DNA transfection agents. To gain further insight in the effects of the additional ethyl group in the headgroup region on the bilayer structure we systematically investigated a homologous series of fully hydrated ethylphosphatidylcholines with linear saturated chains (C14:0, C16:0, and C18:0) by small- and wide-angle X-ray diffraction. Our data show that all of them form multilamellar vesicles with chain interdigitated gel phases. Paying regard to the very importance of the liquid-crystalline phase in gene transfection, we applied the novel MCG method on high resolution X-ray diffraction data of the C16:0 derivative to be able to gain structural information on this phase. Comparison of this ethylphosphatidylcholine with its parent compound, the unesterified phosphatidylcholine, revealed that the major difference in the liquid-crystalline phase is the significantly reduced water layer between the bilayers for the cationic lipid. This may be one factor that contributes to the comparatively good DNA transfection efficiency of ethylphosphatidylcholines.     

Evidence from deuterium nuclear magnetic resonance for the temperature-dependent reversible self-association of erythrocyte band 3 in dimyristoylphosphatidylcholine bilayers

Band 3, isolated from human erythrocytes, has been reconstituted into bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) deuterated in the terminal methyl groups of the choline head group. By use of Triton X-100 for selective extraction and purification of band 3 and then cholate for subsequent solubilization with the lipid, a number of reconstituted complexes were produced by exhaustive detergent dialysis with protein:lipid weight ratios of between 0.32:1 and 1.25:1. Electron micrographs of negatively stained complexes showed that this method produced large vesicles of greater than 300-nm diameter. Deuterium nuclear magnetic resonance (NMR) spectra from the choline methyl deuterons in bilayer lipid above the liquid-crystal-gel phase transition temperature were shown to change systematically with increasing concentrations of band 3 in the bilayers. The measured quadrupole splittings, taken as the separation of the turning points in the recorded spectra, decreased from a value of 1.28 kHz for pure lipid to 0.98 kHz for bilayers with a protein:lipid ratio of 1.25:1 at 26 degrees C. At 35 degrees C, a more pronounced decrease in the quadrupole splittings was measured. The data from the complexes with protein:lipid ratios up to 0.7:1 (w/w) obey the mathematical treatment for a rapid two-site exchange between lipids at the protein-lipid interface and the bulk lipid phase. The temperature dependence of the measured quadrupole splitting with respect to the protein:lipid ratio indicates that the amount of lipid at the protein-lipid interface increases with increasing temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of lipid-based reagents to mediate intracellular gene delivery

We characterized different cationic lipid-based gene delivery systems consisting of both liposomes and nonliposomal structures, in terms of their in vitro transfection activity, resistance to the presence of serum, protective effect against nuclease degradation and stability under different storage conditions. The effect of lipid/DNA charge ratio of the resulting complexes on these properties was also evaluated. Our results indicate that the highest levels of transfection activity were observed for complexes prepared from nonliposomal structures composed of FuGENE 6. However, their DNA protective effect was shown to be lower than that observed for cationic liposome formulations when prepared at the optimal (+/-) charge ratio. Our results suggest that lipoplexes are resistant to serum up to 30% when prepared at a 2:1 lipid/DNA charge ratio. However, when they were prepared at higher (+/-) charge ratios, they become sensitive to serum for even lower concentrations (10%). Replacement of dioleoyl-phosphatidylethanolamine (DOPE) by cholesterol enhanced the resistance of the complexes to the inhibitory effect of serum. This different biological activity in the presence of serum was attributed to different extents of binding of serum proteins to the complexes, as evaluated by the immunoblotting assay. Studies on the stability under storage show that lipoplexes maintain most of their biological activity when stored at -80 degrees C, following their fast freezing in liquid nitrogen.