Metal contaminants promote degradation of lipid/DNA complexes during lyophilization

Oxidation reactions represent an important degradation pathway of nucleic acid-based pharmaceuticals. To evaluate the role of metal contamination and chelating agents in the formation of reactive oxygen species (ROS) during lyophilization, ROS generation and the stability of lipid/DNA complexes were investigated. Trehalose-containing formulations were lyophilized with different levels of transition metals. ROS generation was examined by adding proxyl fluorescamine to the formulations prior to freeze-drying. Results show that ROS were generated during lyophilization, and both supercoil content and transfection rates decreased as the levels of metal-induced ROS increased. The experiments incorporating chelators demonstrated that some of these agents (e.g., DTPA, desferal) clearly suppress ROS generation, while others (e.g., EDTA) enhance ROS. Surprisingly, there was not a strong correlation of ROS generated in the presence of chelators with the maintenance of supercoil content. In this study, we demonstrated the adverse effects of the presence of metals (especially Fe(2+)) in nonviral vector formulations. While some chelators attenuate ROS generation and preserve DNA integrity, the effects of these additives on vector stability during lyophilization are difficult to predict. Further study is needed to develop potent formulation strategies that inhibit ROS generation and DNA degradation during lyophilization and storage.     

Metal contaminants promote degradation of lipid/DNA complexes during lyophilization

Oxidation reactions represent an important degradation pathway of nucleic acid-based pharmaceuticals. To evaluate the role of metal contamination and chelating agents in the formation of reactive oxygen species (ROS) during lyophilization, ROS generation and the stability of lipid/DNA complexes were investigated. Trehalose-containing formulations were lyophilized with different levels of transition metals. ROS generation was examined by adding proxyl fluorescamine to the formulations prior to freeze-drying. Results show that ROS were generated during lyophilization, and both supercoil content and transfection rates decreased as the levels of metal-induced ROS increased. The experiments incorporating chelators demonstrated that some of these agents (e.g., DTPA, desferal) clearly suppress ROS generation, while others (e.g., EDTA) enhance ROS. Surprisingly, there was not a strong correlation of ROS generated in the presence of chelators with the maintenance of supercoil content. In this study, we demonstrated the adverse effects of the presence of metals (especially Fe²⁺) in nonviral vector formulations. While some chelators attenuate ROS generation and preserve DNA integrity, the effects of these additives on vector stability during lyophilization are difficult to predict. Further study is needed to develop potent formulation strategies that inhibit ROS generation and DNA degradation during lyophilization and storage.     

Seminal plasma reduces exogenous oxidative damage to human sperm, determined by the measurement of DNA strand breaks and lipid peroxidation

Exposure of spermatozoa to reactive oxygen species (ROS) has been associated with cellular injury, that includes DNA damage and lipid peroxidation. In addition, sperm preparation techniques such as centrifugation, commonly used prior to in vitro fertilization and scientific studies, are associated with the generation of ROS and an increase in the level of DNA damage. The preservation, therefore, of sperm in vitro that might decrease the potential for oxidative DNA damage to arise and allow for an improvement in semen quality used for artificial insemination, is of importance. Seminal plasma is a rich source of antioxidants, which, potentially, safeguards sperm from oxidative attack during storage and once ejaculated. We have investigated the protection of human spermatozoa from ROS afforded by seminal plasma. Sperm were exposed to exogenous ROS by incubating the cells with hydrogen peroxide in the presence of ferrous sulfate and ADP. Aliquots of seminal plasma were added to the incubation mixture in differing amounts, and the generation of DNA strand breaks and thiobarbituric acid reactive species (TBARS), indicative of lipid peroxidation, determined. Incubation of sperm with exogenous ROS resulted in a significant generation of DNA strand breaks and lipid peroxidation compared to basal levels of damage (P<0.05). Addition of seminal plasma to the incubation media produced a significant decrease in DNA strand breaks and TBARS (P<0. 05), when the amount of plasma added exceeded 60% v/v. The results indicate that spermatozoal oxidative damage induced by exogenous ROS, specifically DNA damage and lipid peroxidation, is reduced by the presence of seminal plasma.     

The NADPH- and iron-dependent lipid peroxidation in human placental microsomes

In pregnant females, placenta is the most important source of lipid hydroperoxides and other reactive oxygen species (ROS). The increased production of lipid peroxides is often linked to preeclampsia. In our study, we revealed that NADPH- and iron-dependent lipid peroxidation in human placental microsomes (HPM) occurred. In the presence of Fe²⁺ ion, HPM produced small amounts of thiobarbituric acid-reactive substances (TBARS) – a final product of lipid peroxidation. NADPH caused a strong increase of iron stimulated TBARS formation. TBARS formation was inhibited by superoxide dismutase, butylated hydroxytoluene and α-tocopherol but not by mannitol or catalase. TBARS and superoxide radical production was inhibited in similar manner by cytochrome P450 inhibitors. The results obtained led us to the following conclusions: (1) microsomal lipid peroxidation next to mitochondrial lipid peroxidation may by an important source of lipid hydroperoxides in blood during pregnancy and (2) superoxide radical released by microsomal cytochrome P450 is an important factor in NADPH- and iron-dependent lipid peroxidation in HPM.     

Activity of glutathione peroxidase, superoxide dismutase and catalase and lipid peroxidation intensity in stallion semen during storage at 5 degrees C

Sperm cell membranes are susceptible to peroxidative damage by an excess of reactive oxygen species (ROS). Antioxidative defence systems consisting of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) physiologically control the balance between ROS production and neutralization. In the present study the hypothesis was tested that lipid peroxidation occurs during storage of semen at 5 degrees C and that semen extender has positive effects on the antioxidative potential of equine semen. The aim of the study was to determine the activity of GSH-Px, SOD and CAT and the concentration of thiobarbituric acid reactive substances (TBARS) as an indicator of lipid peroxidation in native semen and after addition of extender, cooling and storage. Semen was collected from fertile Shetland stallions. In experiment 1, activity of antioxidative enzymes was determined immediately after semen collection and after 24 h storage at 5 degrees C. Enzyme activities were measured in native semen, semen diluted with semen extender, spermatozoa resuspended after centrifugation in extender and 0.9% NaCl as well as in undiluted and extender-diluted seminal plasma. In experiment 2, TBARS concentrations were analysed during storage of semen at 5 degrees C for 24 h. Semen storage for 24 h at 5 degrees C did not change activity of the examined enzymes. Antioxidative activity was significantly higher in extended than in native semen as well as in extended plasma than in undiluted plasma. In conclusion, the addition of semen extender increases the antioxidative activity in seminal plasma of stallions. Basal antioxidative activity in native semen as well as increased activity in extended semen are maintained over 24 h storage at 5 degrees C. TBARS content did not increase during semen storage. In conclusion, lipid peroxidation does not increase substantially during semen storage. The enzymatic antioxidative activity in semen apparently prevents ROS formation and is further increased by addition of semen extender.     

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.     

Effects of di- and polysaccharide formulations and storage conditions on survival of freeze-dried Sphingobium sp.

In this study we have compared the ability of the organic polymers Ficoll and hydroxyethylcellulose (HEC) and the disaccharides sucrose and trehalose to support cell survival during freeze-drying and subsequent storage of a gram-negative Sphingobium sp. In addition to determination of viability rates, cell integrity was evaluated using lipid peroxidation and RNA quality assays for the different storage conditions and formulation compositions. All formulations resulted in high initial cell survival rates after freeze-drying. However, the disaccharide formulations were superior to the polymer-based formulations in supporting cell survival during storage with the exception of Ficoll that upon storage under vacuum yielded bacterial survival rates equal to that of sucrose. Storage in the presence of both oxygen and moisture was detrimental for bacterial survival in all formulations tested, however, lipid peroxidation or RNA damages were not the controlling mechanisms for cell death in this system. The ability of Ficoll and HEC to support cell survival during freeze-drying show that organic polymers, expected to lack the water replacing capability of e.g. disaccharides, can successfully be used as lyoprotectants. For storage under vacuum conditions we suggest that the intracellular amount of sugars (i.e. trehalose), or other protective native cell components, is sufficient for a basic protection inside the bacteria cell and that the amorphous state is the most important aspect of the formulation excipient. However, when exposed to oxygen and moisture during storage this protection is not sufficient to prevent cell degeneration.     

Effect of ursolic acid, a triterpenoid antioxidant, on ultraviolet-B radiation-induced cytotoxicity, lipid peroxidation and DNA damage in human lymphocytes

Exposure to ultraviolet B (UVB, 280-320) radiation induces the formation of reactive oxygen species (ROS) in the biological system. In this study, we examined the protective effect of ursolic acid on UVB-induced lipid peroxidation and oxidative DNA damage with reference to alterations in cellular antioxidant status in human lymphocytes. Series of in vitro tests (hydroxyl radical, superoxide, nitric oxide, DPPH and ABTS radical scavenging assays) demonstrates antioxidant property of ursolic acid in our study. Treatment of lymphocytes with ursolic acid alone (at 10 microg/mL) gave no significant change in cell viability, thiobarbituric acid reactive substances (TBARSs), lipid hydroperoxides (LHPs), % tail DNA and tail moment when compared with normal lymphocytes. UVB-exposure significantly increased TBARS, LHP and % tail DNA, tail moment; decreased % cell viability and antioxidant status in irradiated lymphocytes. Treatment with ursolic acid 30 min prior to UVB-exposure resulted in a significant decline of TBARS, LHP, % tail DNA and tail moment and increased % cell viability as ursolic acid concentration increased. Based on our results we conclude that ursolic acid, a dietary antioxidant, mediates its protective effect through modulation of UVB-induced reactive oxygen species.     

Roles of lipid peroxidation and cytoplasmic droplets on in vitro fertilization capacity of sperm collected from bovine epididymides stored at 4 and 34 degrees C

Sperm recovery from the cauda epididymis can be very advantageous, for example, in case of the unexpected death of a genetically highly valuable animal, for preserving endangered species, or when the collection of sperm by other means becomes impossible. Studies indicate that epididymides stored at cooler temperatures result in better quality sperm. One of the factors that could negatively affect sperm viability during storage is lipid peroxidation, in which the sperm membrane's ability to resist attacks by reactive oxygen species (ROS) plays an important role. Another factor is the presence of cytoplasmic droplets, which appear in high numbers in epididymal sperm, and are known to influence oxidative stress. The objectives of this study were: to determine whether the post-slaughter storage temperature of the epididymis would effect the sperm membrane's resistance to lipid peroxidation and/or the sperm cell's fertilizing capacity in vitro and to elucidate the role played by the cytoplasmic droplets. Forty-eight testicles with epididymides (24 bulls) were collected following slaughter, and divided into two groups. One testicle from each pair was stored at 4 degrees C, and the other at 34 degrees C, for 2h, after which sperm was collected from the caudae epididymides. Sperm concentration was measured, and an aliquot containing 10(8)sperm was subjected to induced lipid peroxidation with ferrous sulphate and ascorbate (37 degrees C, 2h). Subsequently, thiobarbituric acid reactive substances (TBARS), as an index of lipid peroxidation, were measured. A second aliquot of the same sample was used in a routine in vitro fertilization performed in duplicate. Sperm from caudae epididymides stored at 34 degrees C resulted in lower rates of total blastocyst formation and had a higher percentage of distal droplets, when compared to sperm from epididymides stored at 4 degrees C (21.2+/-2.42 and 71.8+/-4.7% versus 33.5+/-1.8 and 23.7+/-4.7%, respectively, P<0.05). Storage temperature had no effect on TBARS levels. For samples stored at 4 degrees C, TBARS were negatively correlated with distal droplets (r=-0.63, P<0.05) and positively correlated with proximal droplets (r=0.42, P<0.05). In conclusion, our results show that short-term storage of epididymides at 4 degrees C provided sperm of higher quality and in vitro fertilizing capacity than storage at 34 degrees C. Although resistance to oxidative stress could not be shown to directly influence these results, distal sperm droplets that appeared in high numbers in the cooled epididymal sperm samples, may have exerted an antioxidant effect. We hypothesize that this protection against ROS is one of the functions of distal sperm droplets in the epididymis.     

New effects in polynucleotide release from cationic lipid carriers revealed by confocal imaging, fluorescence cross-correlation spectroscopy and single particle tracking

We report on new insights into the mechanisms of short single and double stranded oligonucleotide release from cationic lipid complexes (lipoplexes), used in gene therapy. Specifically, we modeled endosomal membranes using giant unilamellar vesicles and investigated the roles of various individual cellular phospholipids in interaction with lipoplexes. Our approach uses a combination of confocal imaging, fluorescence cross-correlation spectroscopy and single particle tracking, revealing several new aspects of the release: (a) phosphatidylserine and phosphatidylethanolamine are equally active in disassembling lipoplexes, while phosphatidylcholine and sphingomyelin are inert; (b) in contrast to earlier findings, phosphatidylethanolamine alone, in the absence of anionic phosphatidylserine triggers extensive release; (c) a double-stranded DNA structure remains well preserved after release; (d) lipoplexes exhibited preferential binding to transient lipid domains, which appear at the onset of lipoplex attachment to originally uniform membranes and vanish after initiation of polynucleotide release. The latter effect is likely related to phosphatidyleserine redistribution in membranes due to lipoplex binding. Real time tracking of single DOTAP/DOPE and DOTAP/DOPC lipoplexes showed that both particles remained compact and associated with membranes up to 1-2 min before fusion, indicating that a more complex mechanism, different from suggested earlier rapid fusion, promotes more efficient transfection by DOTAP/DOPE complexes.     

Optimization of low-cost drying methods to minimize lipid peroxidation in <Emphasis Type="Italic">Spirulina platensis</Emphasis> grown in the Philippines

Three low-cost drying methods (sun, solar, and draft oven) were optimized to produce Spirulina powder of optimal quality. Optimization in the pre-dehydration stage included the use of two antioxidants, α-tocopherol and tertiary-butyl hydroquinone (TBHQ), and two blanching methods, microwave and water bath, to inactivate enzymes. The efficiency of the pre-dehydration treatments at minimizing lipid peroxidation were evaluated in terms of the product’s oxidative stability using the thiobarbituric acid (TBA) test. The sample with the lowest TBA reactive substance (TBARS) value was considered the most stable. TBHQ was found to be significantly better than α-tocopherol in minimizing lipid peroxidation in blanched samples while α-tocopherol was better than TBHQ in unblanched samples. Microwave blanching exerted a greater stabilizing effect than water bath blanching. The combined effect of TBHQ and microwave blanching was found to be the most effective pre-dehydration treatment for minimizing lipid peroxidation in drying Spirulina. Among the three low-cost optimized drying methods, sun-drying produced a dried product with the lowest TBARS value (0.472 mg malondialdehyde.kg⁻¹), which was closest to that of the spray-dried (control) sample (0.434 mg MDA.kg⁻¹). Draft oven and solar drying produced dried products with the same average TBARS value (0.56 mg MDA.kg⁻¹). Sun-drying, when optimized, produced a dried product that was almost as stable as the spray-dried product. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines     

New effects in polynucleotide release from cationic lipid carriers revealed by confocal imaging, fluorescence cross-correlation spectroscopy and single particle tracking

We report on new insights into the mechanisms of short single and double stranded oligonucleotide release from cationic lipid complexes (lipoplexes), used in gene therapy. Specifically, we modeled endosomal membranes using giant unilamellar vesicles and investigated the roles of various individual cellular phospholipids in interaction with lipoplexes. Our approach uses a combination of confocal imaging, fluorescence cross-correlation spectroscopy and single particle tracking, revealing several new aspects of the release: (a) phosphatidylserine and phosphatidylethanolamine are equally active in disassembling lipoplexes, while phosphatidylcholine and sphingomyelin are inert; (b) in contrast to earlier findings, phosphatidylethanolamine alone, in the absence of anionic phosphatidylserine triggers extensive release; (c) a double-stranded DNA structure remains well preserved after release; (d) lipoplexes exhibited preferential binding to transient lipid domains, which appear at the onset of lipoplex attachment to originally uniform membranes and vanish after initiation of polynucleotide release. The latter effect is likely related to phosphatidyleserine redistribution in membranes due to lipoplex binding. Real time tracking of single DOTAP/DOPE and DOTAP/DOPC lipoplexes showed that both particles remained compact and associated with membranes up to 1-2 min before fusion, indicating that a more complex mechanism, different from suggested earlier rapid fusion, promotes more efficient transfection by DOTAP/DOPE complexes.     

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.     

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.     

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.     

Protective Effect of Carvacrol on Oxidative Stress and Cellular DNA Damage Induced by UVB Irradiation in Human Peripheral Lymphocytes

Exposure to ultraviolet B (UVB; 280‐320 nm) radiation induces the formation of reactive oxygen species (ROS) in the biological system. In this study, we examined the protective effect of carvacrol on UVB‐induced lipid peroxidation and oxidative DNA damage with reference to alterations in cellular an‐tioxidant status in human lymphocytes. A series of in vitro assays (hydroxyl radical, superoxide, nitric oxide, DPPH (2,2‐Diphenyl‐1‐picryl hydrazyl), and ABTS (2,2‐azino‐bis‐3‐ethylbenzothiazoline‐6‐sulfonic acid) radical scavenging assays) demonstrate antioxidant property of carvacrol in our study. UVB exposure significantly increased thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides (LHPs), % tail DNA and tail moment; decreased % cell viability and antioxidant status in UVB‐irradiated lymphocytes. Treatment with carvacrol 30 min prior to UVB‐exposure resulted in a significant decline of TBARS, LHP, % tail DNA, and tail moment and increased % cell viability as carvacrol concentration increased. UVB irradiated lymphocytes with carvacrol alone (at 10 μg/mL) gave no significant change in cell viability, TBARS, LHP, % tail DNA, and tail moment when compared with normal lymphocytes. On the basis of our results, we conclude that carvacrol, a dietary antioxidant, mediates its protective effect through modulation of UVB‐induced ROS.     

DNA release from lipoplexes by anionic lipids: correlation with lipid mesomorphism, interfacial curvature, and membrane fusion

DNA release from lipoplexes is an essential step during lipofection and is probably a result of charge neutralization by cellular anionic lipids. As a model system to test this possibility, fluorescence resonance energy transfer between DNA and lipid covalently labeled with Cy3 and BODIPY, respectively, was used to monitor the release of DNA from lipid surfaces induced by anionic liposomes. The separation of DNA from lipid measured this way was considerably slower and less complete than that estimated with noncovalently labeled DNA, and depends on the lipid composition of both lipoplexes and anionic liposomes. This result was confirmed by centrifugal separation of released DNA and lipid. X-ray diffraction revealed a clear correlation of the DNA release capacity of the anionic lipids with the interfacial curvature of the mesomorphic structures developed when the anionic and cationic liposomes were mixed. DNA release also correlated with the rate of fusion of anionic liposomes with lipoplexes. It is concluded that the tendency to fuse and the phase preference of the mixed lipid membranes are key factors for the rate and extent of DNA release. The approach presented emphasizes the importance of the lipid composition of both lipoplexes and target membranes and suggests optimal transfection may be obtained by tailoring lipoplex composition to the lipid composition of target cells.     

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.     

Lipid mixing between lipoplexes and plasma lipoproteins is a major barrier for intravenous transfection mediated by cationic lipids

It has been previously shown that transfection activity of cationic liposome/DNA lipoplexes delivered systemically is drastically inhibited by lipoproteins (Tandia, B. M., Vandenbranden, M., Wattiez, R., Lakhdar, Z., Ruysschaert, J. M., and Elouahabi, A. (2003) Mol Ther. 8, 264-273). In this work, we have compared the binding/uptake and transfection activities of DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride) and diC14-amidine (3-tetradecylamino-N-tert-butyl-N'-tetra-decylpropionamidine)-containing lipoplexes in the presence or absence of purified low density lipoproteins and high density lipoprotein. Binding/uptake of both lipoplexes by the mouse lung endothelial cell line was inhibited to a similar extent in the presence of lipoproteins. In contrast, transfection activity of diC14-amidine-containing lipoplexes was almost completely inhibited (approximately by 95%), whereas approximately 40% transfection activity of DOTAP-containing lipoplexes was preserved in the presence of lipoproteins. Interestingly, the ability of lipoproteins to inhibit the transfection efficiency of lipoplexes was well correlated with their ability to undergo lipid mixing with the cationic lipid bilayer as revealed by fluorescence resonance energy transfer assay. Incubation of lipoplexes with increased doses of lipoproteins resulted in enhanced lipid mixing and reduced transfection activity of the lipoplexes in mouse lung endothelial cells. The role of lipid mixing in transfection was further demonstrated using lipid-mixing inhibitor, lyso-phosphatidylcholine, or activator (dioleoylphosphatidylethanolamine). Incorporation of Lyso-PC into diC14-amidine-containing lipoplexes completely abolished their capacity to undergo lipid mixing with lipoproteins and allowed them to reach a high transfection efficiency in the presence of lipoproteins. On the other hand, the incorporation of dioleoylphosphatidylethanolamine into DOTAP/DNA lipoplex activated lipid mixing with the lipoproteins and was shown to be detrimental toward the transfection activity of these lipoplexes. Taken together, these results indicate that fusion of lipoplexes with lipoproteins is a limiting factor for in vivo transfection.     

Effect of "helper lipid" on lipoplex electrostatics

Lipoplexes, which are complexes between cationic liposomes (L+) and nucleic acids, are commonly used as a nucleic acid delivery system in vitro and in vivo. This study aimed to better characterize cationic liposome and lipoplex electrostatics, which seems to play a major role in the formation and the performance of lipoplexes in vitro and in vivo. We characterized lipoplexes based on two commonly used monocationic lipids, DOTAP and DMRIE, and one polycationic lipid, DOSPA--each with and without helper lipid (cholesterol or DOPE). Electrical surface potential (Psi0) and surface pH were determined using several surface pH-sensitive fluorophores attached either to a one-chain lipid (4-heptadecyl hydroxycoumarin (C17HC)) or to the primary amino group of the two-chain lipids (1,2-dioleyl-sn-glycero-3-phosphoethanolamine-N-carboxyfluorescein (CFPE) and 1,2-dioleyl-sn-glycero-3-phosphoethanolamine-N-7-hydroxycoumarin) (HC-DOPE). Zeta potentials of the DOTAP-based cationic liposomes and lipoplexes were compared with Psi0 determined using C17HC. The location and relatively low sensitivity of fluorescein to pH changes explains why CFPE is the least efficient in quantifying the differences between the various cationic liposomes and lipoplexes used in this study. The fact that, for all cationic liposomes studied, those containing DOPE as helper lipid have the least positive Psi0 indicates neutralization of the cationic charge by the negatively-charged phosphodiester of the DOPE. Zeta potential is much less positively charged than Psi0 determined by C17HC. The electrostatics affects size changes that occurred to the cationic liposomes upon lipoplex formation. The largest size increase (based on static light scattering measurements) for all formulations occurred at DNA-/L+ charge ratios 0.5-1. Comparing the use of the one-chain C17HC and the two-chain HC-DOPE for monitoring lipoplex electrostatics reveals that both are suitable, as long as there is no serum (or other lipidic assemblies) present in the medium; in the latter case, only the two-chain HC-DOPE gives reliable results. Increasing NaCl concentrations decrease surface potential. Neutralization by DNA is reduced in a NaCl-concentration-dependent manner.