Physicochemical properties affecting lipofection potency of a new series of 1,2-dialkoylamidopropane-based cationic lipids

The in vitro transfection activity of a novel series of N,N'-diacyl-1,2-diaminopropyl-3-carbamoyl-(aminoethane) derivatives was evaluated against a mouse melanoma cell line at different +/- charge ratios, in the presence and absence of helper lipids. Only the unsaturated derivative N,N'-dioleoyl-1,2-diaminopropyl-3-carbamoyl-(aminoethane), (1,2lmp[5]) mediated significant increase in the reporter gene level which was significantly boosted in the presence of DOPE peaking at +/- charge ratio of 2. The electrostatic interactions between the cationic liposomes and plasmid DNA were investigated by gel electrophoresis, fluorescence spectroscopy, dynamic light scattering and electrophoretic mobility techniques. In agreement with the transfection results, 1,2lmp[5]/DOPE formulation was most efficient in associating with and retarding DNA migration. The improved association between the dioleoyl derivative and DNA was further confirmed by ethidium bromide displacement assay and particle size distribution analysis of the lipoplexes. Differential scanning calorimetry studies showed that 1,2lmp[5] was the only lipid that exhibited a main phase transition below 37 degrees C. Likewise, 1,2lmp[5] was the only lipid found to form all liquid expanded monolayers at 23 degrees C. In conclusion, the current findings suggest that high in vitro transfection activity is mediated by cationic lipids characterized by increased acyl chain fluidity and high interfacial elasticity.     

Phosphonodithioester–Amine Coupling as a Key Reaction Step for the Design of Cationic Amphiphiles Used for Gene Delivery

A convergent synthesis of cationic amphiphilic compounds is reported here with the use of the phosphonodithioester–amine coupling (PAC) reaction. This versatile reaction occurs at room temperature without any catalyst, allowing binding of the lipid moiety to a polar head group. This strategy is illustrated with the use of two lipid units featuring either two oleyl chains or two-branched saturated lipid chains. The final cationic amphiphiles were evaluated as carriers for plasmid DNA delivery in four cell lines (A549, Calu3, CFBE and 16HBE) and were compared to standards (BSV36 and KLN47). These new amphiphilic derivatives, which were formulated with DOPE or DOPE-cholesterol as helper lipids, feature high transfection efficacies when associated with DOPE. The highest transfection efficacies were observed in the four cell lines at low charge ratios (CR = 0.7, 1 or 2). At these CRs, no toxic effects were detected. Altogether, this new synthesis scheme using the PAC reaction opens up new possibilities for investigating the effects of lipid or polar head groups on transfection efficacies.     

Bis-Thioether-Containing Lipid Chains in Cationic Amphiphiles: Physicochemical Properties and Applications in Gene Delivery

Cationic amphiphiles featuring two thioether functions in each lipid chain of bicatenar cationic amphiphiles are reported here for the first time. The physicochemical properties and transfection abilities of these new amphiphiles were compared with those of already reported analogues featuring either (i) saturated, (ii) unsaturated or (iii) mono-thioether containing lipid chains. The homogeneity of the series of new compounds allowed to clearly underscore the effect of bis-thioether containing lipid chains. This study shows that besides previous strategies based on unsaturation or ramification, the incorporation of two thioether functions per lipid chain constitutes an original complementary alternative to tune the supramolecular properties of amphiphilic compounds. The potential of this strategy was evaluated in the context of gene delivery and report that two cationic amphiphiles (i. e. 4 a and 4 b) can be proposed as new efficient transfection reagents.     

Dramatic influence of the orientation of linker between hydrophilic and hydrophobic lipid moiety in liposomal gene delivery

A number of prior studies have demonstrated that the DNA-binding and gene transfection efficacies of cationic amphiphiles crucially depend on their various structural parameters including hydrophobic chain lengths, headgroup functionalities, and the nature of the linker-functionality used in tethering the polar headgroup and hydrophobic tails. However, to date addressing the issue of linker orientation remains unexplored in liposomal gene delivery. Toward probing the influence of linker orientation in cationic lipid mediated gene delivery, we have designed and synthesized two structurally isomeric remarkably similar cationic amphiphiles 1 and 2 bearing the same hydrophobic tails and the same polar headgroups connected by the same ester linker group. The only structural difference between the cationic amphiphiles 1 and 2 is the orientation of their linker ester functionality. While lipid 1 showed high gene transfer efficacies in multiple cultured animal cells, lipid 2 was essentially transfection incompetent. Findings in both transmission electron microscopic and dynamic laser light scattering studies revealed no significant size difference between the lipoplexes of lipids 1 and 2. Findings in confocal microscopic and fluorescence resonance energy transfer (FRET) experiments, taken together, support the notion that the remarkably higher gene transfer efficacies of lipid 1 compared to those of lipid 2 presumably originate from higher biomembrane fusogenicity of lipid 1 liposomes. Differential scanning calorimetry (DSC) and fluorescence anisotropy studies revealed a significantly higher gel-to-liquid crystalline temperature for the lipid 2 liposomes than that for lipid 1 liposomes. Findings in the dye entrapment experiment were also consistent with the higher rigidity of lipid 2/cholesterol (1:1 mole ratio) liposomes. Thus, the higher biomembrane fusibility of lipid 1 liposomes than that of lipid 2 liposomes presumably originates from the more rigid nature of lipid 2 cationic liposomes. Taken together, the present findings demonstrate for the first time that even as minor a structural variation as linker orientation reversal in cationic amphiphiles can profoundly influence DNA-binding characteristics, membrane rigidity, membrane fusibility, cellular uptake, and consequently gene delivery efficacies of cationic liposomes.     

Enhanced transfection efficiency of multicomponent lipoplexes in the regime of optimal membrane charge density

Recently, membrane charge density of lipid membranes, sigma M, has been recognized as a universal parameter that controls the transfection efficiency of complexes made of binary cationic liposomes and DNA (binary lipoplexes). Three distinct regimes, most likely related to interactions between complexes and cells, have also been identified. The purpose of this work was to investigate the transfection efficiency behavior of multicomponent lipoplexes in the regime of optimal membrane charge density (1< sigma M < 2 x 10 (-2) e/A (2)) and compare their performance with that of binary lipoplexes usually employed for gene delivery purposes. We found remarkable differences in transfection efficiency due to lipid composition, with maximum in efficiency being obtained when multicomponent lipoplexes were used to transfect NIH 3T3 cells, while binary lipoplexes were definitely less efficient. These findings suggested that multicomponent systems are especially promising lipoplex candidates. With the aim of providing new insights into the mechanism of transfection, we investigated the structural evolution of lipoplexes when interacting with anionic (cellular) lipids by means of synchrotron small-angle X-ray diffraction (SAXD), while the extent of DNA release upon interaction with anionic lipids was measured by electrophoresis on agarose gels. Interestingly, a clear trend was found that the transfection activity increased with the number of lipid components. These results highlight the compositional properties of carrier lipid/cellular lipid mixtures as decisive factors for transfection and suggest a strategy for the rational design of superior cationic lipid carriers.     

Structural stability against disintegration by anionic lipids rationalizes the efficiency of cationic liposome/DNA complexes

Reported here is the correlation between the transfection efficiency of cationic liposome/DNA complexes (lipoplexes) and the structural evolution that they undergo when interacting with anionic membrane lipids. Multicomponent lipoplexes, incorporating from three to six lipid species simultaneously, presented a much higher transfection efficiency than binary lipoplexes, which are more commonly used for gene-delivery purposes. The discovery that a high transfection efficiency can be achieved by employing multicomponent complexes at a lower-than-ever-before membrane charge density of lipoplexes was of primary significance. Synchrotron small-angle X-ray diffraction (SAXD) experiments showed that anionic liposomes made of dioleoylphosphatidylglycerol (DOPG) disintegrated the lamellar phase of lipoplexes. DNA unbinding was measured by electrophoresis on agarose gels. Most importantly, structural changes induced by anionic lipids strictly depended on the lipid composition of lipoplexes. We found evidence of the existence of three different regimes of stability related to the interaction between complexes and anionic membranes. Both unstable (with low membrane charge density, sigmaM) and highly stable lipoplexes (with high sigmaM) exhibited low transfection efficiency whereas highly efficient multicomponent lipoplexes exhibited an "optimal stability". This intermediate regime reflects a compromise between two opposing constraints: protection of DNA in the cytosol and endosomal escape. Here we advance the concept that structural stability, upon interaction with cellular anionic lipids, is a key factor governing the transfection efficiency of lipoplexes. Possible molecular mechanisms underlying experimental observations are also discussed.     

Structural properties of docosahexaenoyl phospholipid bilayers investigated by solid-state 2H NMR spectroscopy.

Polyunsaturated lipids in cellular membranes are known to play key roles in such diverse biological processes as vision, neuronal signaling, and apoptosis. One hypothesis is that polyunsaturated lipids are involved in second messenger functions in biological signaling. Another current hypothesis affirms that the functional role of polyunsaturated lipids relies on their ability to modulate physical properties of the lipid bilayer. The present research has employed solid-state 2H NMR spectroscopy to acquire knowledge of the molecular organization and material properties of polyunsaturated lipid bilayers. We report measurements for a homologous series of mixed-chain phosphatidylcholines containing a perdeuterated, saturated acyl chain (n:0) at the sn-1 position, adjacent to docosahexaenoic acid (DHA, 22:6omega3) at the sn-2 position. Measurements have been performed on fluid (L(alpha))-state multilamellar dispersions as a function of temperature for saturated acyl chain lengths of n = 12, 14, 16, and 18 carbons. The saturated sn-1 chains are therefore used as an intrinsic probe with site-specific resolution of the polyunsaturated bilayer structure. The 2H NMR order parameters as a function of acyl position (order profiles) have been analyzed using a mean-torque potential model for the chain segments, and the results are discussed in comparison with the homologous series of disaturated lipid bilayers. At a given absolute temperature, as the sn-1 acyl length adjacent to the sn-2 DHA chain is greater, the order of the initial chain segments increases, whereas that of the end segments decreases, in marked contrast with the corresponding disaturated series. For the latter, the order of the end segments is practically constant with acyl length, thus revealing a universal chain packing profile. We find that the DHA-containing series, while more complex, is still characterized by a universal chain packing profile, which is shifted relative to the homologous saturated series. Moreover, we show how introduction of DHA chains translates the order profile along the saturated chains, making more disordered states accessible within the bilayer central region. As a result, the area per lipid headgroup is increased as compared to disaturated bilayers. The systematic analysis of the 2H NMR data provides a basis for studies of lipid interactions with integral membrane proteins, for instance in relation to characteristic biological functions of highly unsaturated lipid membranes.     

Synthesis of a polyamidoamine dendron-bearing lipid having sugar moieties and its use for preparation of nonviral gene vectors

We have so far developed a new type of cationic lipid that consists of an amine-terminated poly(amidoamine) dendron and two long alkyl chains. In this study, we designed a dendron-bearing lipid (DL) modified with sugar moieties as the ligand at the terminal groups of the dendron to improve the transfection activities. We combined lactobionic acid at the end of every dendritic branch of the DL to produce galactose moieties-attached DL (Gal-DL). The lipoplexes containing Gal-DL exhibited enhancement of transfection of HepG2 cells. Moreover, Gal-DL significantly promoted the transfection activity of the lipoplexes including poly(ethylene glycol)-attached dendron-bearing lipid (PEG-DL).     

Influence of biological media on the structure and behavior of ferrocene-containing cationic lipid/DNA complexes used for DNA delivery

Biological media affect the physicochemical properties of cationic lipid-DNA complexes (lipoplexes) and can influence their ability to transfect cells. To develop new lipids for efficient DNA delivery, the influence of serum-containing media on the structures and properties of the resulting lipoplexes must be understood. To date, however, a clear and general picture of how serum-containing media influences the structures of lipoplexes has not been established. Some studies suggest that serum can disintegrate lipoplexes formed using certain types of cationic lipids, resulting in the inhibition of transfection. Other studies have demonstrated that lipoplexes formulated from other lipids are stable in the presence of serum and are able to transfect cells efficiently. In this article, we describe the influence of serum-containing media on lipoplexes formed using the redox-active cationic lipid bis(n-ferrocenylundecyl)dimethylammonium bromide (BFDMA). This lipoplex system promotes markedly decreased levels of transgene expression in COS-7 cells as serum concentrations are increased from 0 to 2, 5, 10, and 50% (v/v). To understand the cause of this decrease in transfection efficiency, we used cryogenic transmission electron microscopy (cryo-TEM) and measurements of zeta potential to characterize lipoplexes in cell culture media supplemented with 0, 2, 5, 10, and 50% serum. Cryo-TEM revealed that in serum-free media BFDMA lipoplexes form onionlike, multilamellar nanostructures. However, the presence of serum in the media caused disassociation of the intact multilamellar lipoplexes. At low serum concentrations (2 and 5%), DNA threads appeared to separate from the complex, leaving the nanostructure of the lipoplexes disrupted. At higher serum concentration (10%), disassociation increased and bundles of multilamellae were discharged from the main multilamellar complex. In contrast, lipoplexes characterized in serum-free aqueous salt (Li(2)SO(4)) medium and in OptiMEM cell culture medium (no serum) did not exhibit significant structural changes. The zeta potentials of lipoplexes in serum-free media (salt medium and cell culture medium) were similar (e.g., approximately -35 mV). Interestingly, the presence of serum caused the zeta potentials to become less negative (about -20 mV in OptiMEM and -10 mV in Li(2)SO(4)), even though serum contains negatively charged entities that have been demonstrated to lead to more negative zeta potentials in other lipoplex systems. The combined measurements of zeta potential and cryo-TEM are consistent with the proposition that DNA threads separate from the lipoplex in the presence of serum, resulting in a decrease in the net negative charge of the surface of the lipoplex.     

Ferrocene-containing cationic lipids: influence of redox state on cell transfection

A ferrocene-containing, redox-active cationic lipid that can be transformed using electrochemical methods yields large differences in cell transfection depending on the oxidation state of the lipid. Expression of enhanced green fluorescent protein and firefly luciferase occurs at very high levels when DNA lipoplexes are formulated using the lipid in the reduced state. In contrast, transfection is negligible when oxidized lipid is used. These observations suggest the basis of a general method that could be used to transform inactive lipoplex formulations to an active form through the application of externally applied electrical potentials. The ability to activate lipoplexes toward transfection electrochemically and "on demand" could create new opportunities to deliver DNA in vitro and in vivo with both spatial and temporal control.     

Interaction of lipoplexes with anionic lipids resulting in DNA release is a two-stage process

We propose a mechanism for DNA release from lipoplexes in cells that accounts for various observations of lipoplex-anionic lipid interactions. We examined the structural evolution of lipoplexes upon interaction with cellular lipids by synchrotron small-angle X-ray diffraction (SAXD), and the extent of DNA release from lipoplexes was determined by gel electrophoresis. We find that the interaction of lipoplexes with anionic cellular lipids is a two-stage process. In the first step, anionic lipids laterally diffuse into the complex and neutralize the cationic lipids. As a result, the membrane charge density of lipoplexes decreases and interactions between cationic lipids and DNA become weaker, but DNA is extremely poorly released. Only after the cationic charge of lipoplex membranes is completely neutralized by anionic lipids does DNA starts to be released significantly.     

Interaction of cationic lipid vesicles with model cell membranes--as determined by neutron reflectivity

Transfection of cells by DNA (for the purposes of gene therapy) can be effectively engineered through the use of cationic lipid/DNA "lipoplexes", although the transfection efficiency of these lipoplexes is sensitive to the neutral "helper" lipid included. Here, neutron reflectivity has been used to investigate the role of the helper lipid present during the interaction of cationic lipid vesicles with model cell membranes. Dimethyldioctadecylammonium bromide (DDAB) vesicles were formed with two different helper lipids, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) and cholesterol, and the interaction of these vesicles with a supported phospholipid bilayer was determined. DOPE-containing vesicles were found to interact faster with the membrane than those containing cholesterol, and vesicles containing either of the neutral helper lipids were found to interact faster than when DDAB alone was present. The interaction between the vesicles and the membrane was characterized by an exchange of lipid between the membrane and the lipid aggregates in solution; the deposition of vesicle bilayers on the surface of the membrane was not apparent.     

Effects of Headgroups and Serum on Gene Transfection of Alkaline Amino Acid Based Cationic Lipids

Three cationic lipids with lysylated(l), histidylated(2), and arginylated(3) headgroups and cholesterol hy-drophobic moiety were synthesized. The average sizes of liposomes and lipoplexes were around 100 and 160 nm, re-spectively. The gene transfection efficiency of the three lipoplexes loaded with pGL3 or pORF-LacZ was compared on 293T cells in the presence or the absence of serum. The transfection efficiency of the three lipoplexes in a se-rum-free medium was 2 to 3-fold higher than that of dioleoyl-trimethylammonium propane(DOTAP). In the presence of serum, however, most of the lipoplexes showed lower transfection activities; only lipoplex 3 retained its high transfection efficiency.     

Engineered lipids that cross-link the inner and outer leaflets of lipid bilayers

The application of supported lipid bilayer systems as molecular sensors, diagnostic devices, and medical implants is limited by their lack of stability. In an effort to enhance the stability of supported lipid bilayers, three pairs of phosphatidylcholine lipids were designed to cross-link at the termini of their 2-position acyl chain upon the formation of lipid bilayers. The cross-linked lipids span the lipid bilayer, resembling naturally occurring bolaamphiphiles that stabilize archaebacterial membranes against high temperatures. The three reactions investigated here include the acyl chain cross-linking between thiol and bromine groups, thiol and acryloyl groups, and cyclopentadiene and acryloyl groups. All three reactive lipid pairs were found to cross-link in liposomal membranes, as determined by thin-layer chromatography, ion-spray mass spectrometry, and 1H NMR. The monolayer film properties of the reactive amphiphiles were characterized by surface pressure-area isotherms and showed that stable monolayers formed at the air-water interface with limiting molecular areas comparable to that of pure saturated phosphatidylcholine lipids. Langmuir-Blodgett bilayers of dimyristoylphosphatidylcholine incorporating 15 mol % of the reactive thiol and acryloyl lipids had diffusion coefficients comparable with pure dimyristoylphosphatidylcholine, while bilayers with more than 25 mol % of the reactive lipids were immobile, suggesting that interleaflet cross-linking of the lipids inhibited membrane diffusion. Our results show that the reactive lipids can cross-link within a lipid bilayer and are suitable for assembling supported lipid bilayers using Langmuir-Blodgett deposition. By using terminally reactive amphiphiles to build up supported lipid bilayers with cross-linked leaflets, bolaamphiphiles can be incorporated into asymmetric solid supported membranes to increase their stability in biosensor and medical implant applications.     

On the gene delivery efficacies of pH-sensitive cationic lipids via endosomal protonation: a chemical biology investigation

In an effort to probe the importance of endosomal protonation in pH-sensitive, cationic, lipid-mediated, non-viral gene delivery, we have designed and synthesized a novel cholesterol-based, endosomal pH-sensitive, histidylated, cationic amphiphile (lipid 1), its less pH-sensitive counterpart with an electron-deficient, tosylated histidine head group (lipid 2) as well as a third new cholesterol-based, cationic lipid containing no histidine head group (lipid 3). For all the novel liposomes and lipoplexes, we evaluated hysicochemical characteristics, including lipid:DNA interactions, global surface charge, and sizes. As anticipated, lipid 2 showed lower efficacies than lipid 1 for the transfection of 293T7 cells with the cytoplasmic gene expression vector pT7Luc at lipid:DNA mole ratios of 3.6:1 and 1.8:1; both lipids were greatly inhibited in the presence of Bafilomycin A1. This demonstrates the involvement of imidazole ring protonation in the endosomal escape of DNA. Conversely, endosome escape of DNA with lipid 3 seemed to be independent of endosome acidification. However, with nuclear gene expression systems in 293T7, HepG2, and HeLa cells, the transfection efficacies of lipid 2 at a lipid:DNA mole ratio of 3.6:1 were found to be either equal to or somewhat lower than those of lipids 1 and 3. Interestingly, at a lipid:DNA mole ratio of 1.8:1, lipids 2 and 3 were remarkably more transfection efficient than lipid 1 in both HepG2 and HeLa cells. Mechanistic implications of such contrasting relative transfection profiles are delineated.     

Cationic lipo-thiophosphoramidates for gene delivery: synthesis, physico-chemical characterization and gene transfection activity--comparison with lipo-phosphoramidates

The synthesis of cationic lipo-thiophosphoramidates, a new family of cationic lipids designed for gene delivery, is reported herein. This new class of lipids is less polar than its oxygenated equivalent the lipo-phosphoramidates. Fluorescence anisotropy and FRET were used to determine the fluidity and fusogenicity of the lipo-phosphoramidates 3a-b and lipo-thiophosphoramidates 7a-b. The determination of both the size and the zeta potential of the nano-objects (liposomes and lipoplexes) and the determination of the DNA binding ability of the liposomes have completed the physico-chemical characterizations of the cationic lipids studied. Finally, the cationic lipids 3a-b and 7a-c have been evaluated as synthetic vectors for gene transfection into a variety of mammalian cell lines. The lipo-thiophosphoramidate 7a proved to be an efficient and low toxicity synthetic vector even when used at low lipid to DNA charge ratios.     

Determination of lipid phase transition temperatures in hybrid bilayer membranes

The main gel-to-liquid-crystal (LC) phase transition temperature, T(m), of the lipid monolayer in hybrid bilayer membranes (HBMs) was investigated using vibrational sum frequency spectroscopy (VSFS). In the gel phase, the acyl chains of the lipid molecules assume an ordered, all-trans configuration, whereas in the LC phase, the acyl chains exhibit a significant number of disordered gauche conformers. VSFS has unique sensitivity to the order/disorder transitions in the acyl chains and was used to determine T(m) for a series of saturated phosphatidylcholine lipids on octadecanethiolate self-assembled monolayers (SAMs). The values obtained for T(m) for all lipids studied are significantly higher than for the corresponding lipids in vesicles in solution. Additionally, the transition widths are broader for the lipids in HBMs. The underlying SAM clearly influences the phase behavior of the overlying lipid monolayer.     

Folate-equipped pegylated archaeal lipid derivatives: synthesis and transfection properties

We have previously shown that synthetic archaeal lipid analogues are useful vectors for drug/gene delivery. We report herein the synthesis and gene transfer properties of a series of novel di- and tetraether-type archaeal derivatives with a poly(ethylene glycol) (PEG) chain and further equipped with a folic acid (FA) group. The synthetic strategy and the purification by dialysis ensured complete removal of free FA. The lipids were mixed with a conventional glycine betaine-based cationic lipid and the resulting formulations were tested in transfection assays after complexation with plasmid DNA. All four novel co-lipids afforded efficient in vitro gene transfection. Moreover, the FA-equipped derivatives permitted ligand/receptor-based targeted transfection; their activity was inhibited when free FA was added to the transfection medium. These novel archaeal derivatives equipped with FA-PEG moieties may thus be of great interest for targeted in vivo transfection.     

Enhanced intravenous transgene expression in mouse lung using cyclic-head cationic lipids

Herein, we report enhanced intravenous mouse lung transfection using novel cyclic-head-group analogs of usually open-head cationic transfection lipids. Design and synthesis of the new cyclic-head lipid N,N-di-n-tetradecyl-3,4-dihydroxy-pyrrolidinium chloride (lipid 1) and its higher alkyl-chain analogs (lipids 2-4) and relative in vitro and in vivo gene transfer efficacies of cyclic-head lipids 1-4 to their corresponding open-head analogs [lipid 5, namely N,N-di-n-tetradecyl-N,N-(2-hydroxyethyl)ammonium chloride and its higher alkyl-chain analogs, lipids 6-8] have been described. In stark contrast to comparable in vitro transfection efficacies of both the cyclic- and open-head lipids, lipids 1-4 with cyclic heads were found to be significantly more efficient (by 5- to 11-fold) in transfecting mouse lung than their corresponding open-head analogs (5-8) upon intravenous administration. The cyclic-head lipid 3 with di-stearyl hydrophobic tail was found to be the most promising for future applications.     

Cationic vesicles consisting of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and phosphatidylcholines and their interaction with erythrocyte membrane

We studied the formation and stability of vesicles consisting of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and phosphatidylcholines by electron spin resonance (ESR) analysis and observation of their hemolytic activities. In contrast with previous findings on dimethyldialkylammoniums, DOTAP formed vesicles at 37 degrees C with phosphatidylcholines containing either saturated acyl chains such as dimyristoylphosphatidylcholine (DMPC) or unsaturated acyl chains such as dilinoleoylphosphatidylcholine (DLPC). Phosphatidylcholines made the bilayer more rigid and significantly reduced the hemolytic activity of DOTAP. In the presence of equimolar concentration of DOTAP and phosphatidylcholines, formation of tightly aggregated structures of several erythrocytes was observed, as previously reported for the vesicles containing dimethyldipalmitylammonium. These findings indicate that DOTAP vesicles were stabilized by phosphatidylcholines with either saturated acyl chains or unsaturated acyl chains, and the interaction with the lipid bilayer of biological membranes as cationic vesicles became prominent with minimal membrane damage by DOTAP monomers.