Compaction process of calf thymus DNA by mixed cationic-zwitterionic liposomes: a physicochemical study

The compaction of calf thymus DNA (CT-DNA) by cationic liposomes constituted by a 1:1 mixture of a cationic lipid, 1,2-distearoyl-3-(trimethylammonio)propane chloride (DSTAP), and a zwitterionic lipid, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE, null net charge at pH = 7.4), has been evaluated in aqueous buffered solution at 298.15 K by means of conductometry, electrophoretic mobility, cryo-TEM, and fluorescence spectroscopy techniques. The results reveal that DSTAP/DOPE liposomes are mostly spherical and unilamelar, with a mean diameter of around 77 +/- 20 nm and a positively charged surface with a charge density of sigmazeta = (21 +/- 1) x 10(-3) C m(-2). When CT-DNA is present, the genosomes DSTAP/DOPE/CT-DNA, formed by means of a surface electrostatic interaction, are generally smaller than the liposomes. Furthermore, they show a tendency to fuse forming cluster-type structures when approaching isoneutrality, which has been determined by the electrochemical methods at around (L/D)phi = 5.6. The analysis of the decrease on the fluorescence emission of the fluorophore ethidium bromide, EtBr, initially intercalated between DNA base pairs, as long as the genosomes are formed has permitted us to confirm the electrostatic character of the DNA-liposome interaction.     

Multicomponent cationic lipid-DNA complex formation: role of lipid mixing

Multicomponent cationic lipid-DNA complexes (lipoplexes) were prepared by adding linear DNA to mixed lipid dispersions containing two populations of binary cationic liposomes and characterized by means of small angle X-ray scattering (SAXS). Four kinds of cationic liposomes were used. The first binary lipid mixture was made of the cationic lipid (3'[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol) and the neutral helper lipid dioleoylphosphocholine (DOPC) (DC-Chol/DOPC liposomes), the second one of the cationic 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the neutral dioleoylphosphatidylethanolamine (DOPE) (DOTAP/DOPE liposomes), the third one of DC-Chol and DOPE (DC-Chol/DOPE liposomes), and the fourth one of DOTAP and DOPC (DOTAP/DOPC liposomes). Upon DNA-induced fusion of liposomes, large lipid mixing at the molecular level occurs. As a result, highly organized mixed lipoplexes spontaneously form with membrane properties intermediate between those of starting liposomes. By varying the composition of lipid dispersions, different DNA packing density regimes can also be achieved. Furthermore, occurring lipid mixing was found to induce hexagonal to lamellar phase transition in DOTAP/DOPE membranes. Molecular mechanisms underlying experimental findings are discussed.     

The analysis of serum effects on structure, size and toxicity of DDAB-DOPE and DC-Chol-DOPE lipoplexes contributes to explain their different transfection efficiency

The effect of serum on structural properties of dimethyl-dioctadecyl-ammonium bromide (DDAB)–1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) liposomes and DDAB–DOPE/DNA lipoplexes has been investigated by energy dispersive X-ray diffraction (EDXD) technique, at different cationic lipid/DNA weight ratios (ρ). The role of serum on the size of lipoplexes has also been studied by dynamic light scattering. Lipoplex transfection efficiency (TE) as a function of ρ, and lipoplex toxicity to C6 rat glioma cells have been evaluated in Dulbecco's Modified Eagle Medium (DMEM) with and without serum. A multi-parametric analysis concerning the role of size, structure and cytotoxicity on transfection efficiency contributes to explain the experimental observation that 3β-[N-(N′,N′-dimethylaminoethane)carbamoyl]-cholesterol (DC-Chol)–DOPE/DNA transfect C6 cells better than DDAB–DOPE/DNA lipoplexes.     

The analysis of serum effects on structure,size and toxicity of DDAB–DOPE and DC-Chol–DOPE lipoplexes contributes to explain their different transfection efficiency

The effect of serum on structural properties of dimethyl-dioctadecyl-ammonium bromide (DDAB)–1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) liposomes and DDAB–DOPE/DNA lipoplexes has been investigated by energy dispersive X-ray diffraction (EDXD) technique, at different cationic lipid/DNA weight ratios (ρ). The role of serum on the size of lipoplexes has also been studied by dynamic light scattering. Lipoplex transfection efficiency (TE) as a function of ρ, and lipoplex toxicity to C6 rat glioma cells have been evaluated in Dulbecco's Modified Eagle Medium (DMEM) with and without serum. A multi-parametric analysis concerning the role of size, structure and cytotoxicity on transfection efficiency contributes to explain the experimental observation that 3β-[N-(N′,N′-dimethylaminoethane)carbamoyl]-cholesterol (DC-Chol)–DOPE/DNA transfect C6 cells better than DDAB–DOPE/DNA lipoplexes.     

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.     

The synergy between structural stability and DNA-binding controls the antibody production in EPC/DOTAP/DOPE liposomes and DOTAP/DOPE lipoplexes

We present a comparative study of the physico-chemical properties, in vitro cytotoxicity and in vivo antibody production of surface-complexed DNA in EPC/DOTAP/DOPE (50/25/25% molar) liposomes and DOTAP/DOPE (50/50% molar) lipoplexes. The study aims to correlate the biological behavior and structural properties of the lipid carriers. We used DNA-hsp65, whose naked action as a gene vaccine against tuberculosis has already been demonstrated. Additionally, surface-complexed DNA-hsp65 in EPC/DOTAP/DOPE (50/25/25% molar) liposomes was effective as a single-dose tuberculosis vaccine. The results obtained showed that the EPC inclusion stabilized the DOTAP/DOPE structure, producing higher melting temperature and lower zeta potential despite a close mean hydrodynamic diameter. Resemblances in morphologies were identified in both structures, although a higher fraction of loaded DNA was not electrostatically bound in EPC/DOTAP/DOPE. EPC also induced a striking reduction in cytotoxicity, similar to naked DNA-hsp65. The proper immune response lead to a polarized antibody production of the IgG2a isotype, even for the cytotoxic DOTAP/DOPE. However, the antibody production was detected at 15 and 30 days for DOTAP/DOPE and EPC/DOTAP/DOPE, respectively. Therefore, the in vivo antibody production neither correlates with the in vitro cytotoxicity, nor with the structural stability alone. The synergistic effect of the structural stability and DNA electrostatic binding upon the surface of structures account for the immunological effects. By adjusting the composition to generate proper packing and cationic lipid/DNA interaction, we allow for the optimization of liposome formulations for required immunization or gene therapy. In a specific manner, our results contribute to studies on the tuberculosis therapy and vaccination.     

Brewster angle microscopy and PMIRRAS study of DNA interactions with BGTC, a cationic lipid used for gene transfer

The lipid bis(guanidinium)-tris(2-aminoethyl)amine-cholesterol (BGTC) is a cationic cholesterol derivative bearing guanidinium polar headgroups which displays high transfection efficiency in vitro and in vivo when used alone or formulated as liposomes with the neutral colipid 1,2-di-[ cis-9-octadecenoyl]- sn-glycero-3-phosphoethanolamine (DOPE). Since transfection may be related to the structural and physicochemical properties of the self-assembled supramolecular lipid-DNA complexes, we used the Langmuir monolayer technique coupled with Brewster angle microscopy (BAM) and polarization modulation infrared reflection absorption spectroscopy (PMIRRAS) to investigate DNA-BGTC and DNA-BGTC/DOPE interactions at the air/water interface. We herein show that BGTC forms stable monolayers at the air/water interface. When DNA is injected into the subphase, it adsorbs to BGTC at 20 mN/m. Whatever the (+/-) charge ratio of the complexes used, defined as the ratio of positive charges of BGTC in the monolayer versus negative charges of DNA injected in the subphase, the DNA interacts with the cationic lipid and forms either an incomplete (no constituent in excess) or a complete (DNA in excess) monolayer of oriented double strands parallel to the lipid monolayer plan. We also show that, under a homogeneous BGTC/DOPE (3/2) monolayer at 20 mN/m, DNA adsorbs homogeneously to form an organized but incomplete layer whatever the charge ratio used (DNA in default or in excess). Compression beyond the collapse of these mixed DNA-BGTC/DOPE systems leads to the formation of dense DNA monolayers under an asymmetric lipid bilayer with a bottom layer of BGTC in contact with DNA and a top layer mainly constituted of DOPE. These results allow a better understanding of the mechanisms underlying the formation of the supramolecular BGTC-DNA complexes efficient for gene transfection.     

Formation of interfacial milk protein complexation to stabilize oil-in-water emulsions against calcium

The interactions between cationic liposomes doped with the anionic nucleolipid 1,2-dipalmitoyl-sn-glycero-3-cytidine diphosphate (DP-Cyt) and deoxyribonucleic acid (DNA) were investigated. Toward this goal, new liposomal and lipoplex formulations characterized by the presence of the anionic amphiphile DP-Cyt were proposed. The effects of incorporation of the cytosine functionalized lipid DP-Cyt into the cationic bilayers were analyzed by means of electrophoretic mobility, dynamic light scattering (DLS) and fluorescence spectroscopy techniques. These approaches allowed us to follow the DNA condensation process and to identify specific electrokinetic characteristics of liposome and DNA-liposome complexes formation. Specifically, DP-Cyt liposomes and DNA were shown to form electrically stable or unstable complexes depending on the charge ratio between the phosphate group of DNA and the cationic lipid. Remarkably, a prominent role for DP-Cyt in enhancing the DNA binding capacity on liposomes was demonstrated. Zeta potential experiments performed on systems with different liposomes/DNA ratio showed that the value of the charge neutralization point is a function of the content of the incorporated DP-Cyt. As a whole, our data demonstrate that the association of cationic DP-Cyt doped liposomes with DNA is driven by both electrostatic interaction and additional specific interactions at the polar head level based on the cytidine nucleobase.     

Polyadenylic acid binding on cationic liposomes doped with the non-ionic nucleolipid Lauroyl Uridine

In this work unilamellar liposomes doped with a novel non-ionic 5′-Uridine-head nucleolipid, Lauroyl Uridine (LU), were prepared and characterized for their ability to interact with the polynucleotide polyadenylic acid (poly-A). Vesicles, were made up of the cationic lipid DOTAP (1,2-Dioleoyl-3-Trimethylammonium-Propane), the zwitterionic lipid DOPE (1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine), and the novel amphiphile Lauroyl Uridine. The influence of the non-ionic nucleolipid on essential liposomes properties, such as the structure and net charge was first investigated by a comparative analysis performed on the different lipoplex preparations by means of ζ-potential and size measurements. Both structure and net charge of liposomes were shown to be not modified by the presence of the non-ionic nucleolipid.The role of the synthetic lipid inserted as anchor in the liposome bilayer in the condensation process between vesicles and the polynucleotide poly-A was then analyzed by UV–vis, Circular Dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. The data presented comparative UV–vis analyses that evidenced the occurrence of staking interactions in the poly-A only in LU containing lipoplexes. CD and NMR studies indicated the presence of H-bonding interaction between Lauroyl Uridine containing vesicles and the polynucleotide poly-A. The results presented in this work support a role for Lauroyl Uridine in A-U molecular recognition, thus, suggesting that cationic liposomes doped with the non-ionic nucleolipid Lauroyl Uridine could represent a model system to study molecular interactions among single stranded polynucleotides and lipid anchor bearing the complementary bases.     

Two-dimensional lipid mixing entropy regulates the formation of multicomponent lipoplexes

The mechanism of formation of multicomponent lipoplexes was investigated by means of synchrotron Small-Angle X-ray Diffraction (SAXD). Mixed lipid dispersions were prepared by mixing different populations of binary cationic liposomes. When adding DNA to mixed lipid dispersions, multicomponent lipoplexes spontaneously formed exhibiting structural properties, i.e., membrane thickness, surface charge density, and one-dimensional DNA packing density, intermediate between those of binary lipoplexes. These results suggested that DNA lets liposomes come into contact and fuse and that a complete lipid mixing at the molecular level occurs. The equilibrium structure of multicomponent lipoplexes was found to be unique and did not depend on the number and kind of populations composing lipid dispersion but only on the lipid species involved and on their relative molar ratio. According to recent theoretical models we identified two-dimensional lipid mixing entropy as the key factor regulating the existence of only multicomponent lipoplexes with ideally mixed lipid species.     

DNA–DNA electrostatic interactions within cationic lipid/DNA lamellar complexes

We present a simple theoretical analysis of the DNA–DNA electrostatic interactions within charge-neutral lamellar cationic lipid/DNA complexes (lipoplexes). Although always repulsive as a function of the DNA–DNA interaxial distance, the calculated electrostatic force shows a deep minimum for each value of lipid composition corresponding to an equilibrium distance of the system. The excellent agreement between the equilibrium distances predicted by the model and that experimentally observed in charge-neutral complexes as revealed by synchrotron X-ray diffraction, shows that the spatial dimensionality of both the lipids and the DNA may not be a crucial point to capture the essence of the DNA–DNA interactions within charge-neutral lipoplexes.     

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.     

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.     

Photophysical behavior of a dimeric cyanine dye (BOBO-1) within cationic liposomes

This study is aimed at establishing optimal conditions for the use of 2,2'-[1,3-propanediylbis[(dimethyliminio)-3,1-propanediyl-1(4H)-pyridinyl-4-ylidenemethy-lidyne]]bis[3-methyl]-tetraiodide (BOBO-1) as a fluorescent probe in the characterization of lipid/DNA complexes (lipoplexes). The fluorescence spectra, anisotropy, fluorescence lifetimes and fluorescence quantum yields of this dimeric cyanine dye in plasmid DNA (2694 base pairs) with and without cationic liposomes (1,2-dioleoyl-3-trimethylammonium-propane [DOTAP]), are reported. The photophysical behavior of the dye in the absence of lipid was studied for several dye/DNA ratios using both supercoiled and relaxed plasmid. At dye/DNA ratios (d/b) below 0.01 the fluorescence intensity increases linearly, whereas lifetime and anisotropy values of the dye are constant (tau approximately 2.5 ns and = 0.20). By agarose gel electrophoresis it was verified that up to d/b = 0.01 DNA conformation is not considerably modified, whereas for d/b = 0.05-0.06 a single heavy band appears on the gel. For these and higher dye/DNA ratios the fluorescence intensity, anisotropy and average lifetime values decrease with an increase in BOBO-1 concentration. When cationic liposomes are added to the BOBO-1/DNA complex, an additional effect is noticed: The difference in the environment probed by BOBO-1 bound to DNA leads to a decrease in quantum yield and average lifetime values, and a redshift is apparent in the emission spectrum. For fluorescence measurements including energy transfer (FRET), a d/b ratio of 0.01 seems to be adequate because no considerable change on DNA conformation is detected, a considerable fluorescent signal is still measured after lipoplex formation, and energy migration is not efficient.     

Label-free quantitative analysis for studying the interactions between nanoparticles and plasma proteins

A shotgun proteomics approach was used to compare human plasma protein binding capability with cationic liposomes, DNA–cationic lipid complexes (lipoplexes), and lipid–polycation–DNA (LPD) complexes. Nano-high-performance liquid chromatography coupled with a high-resolution LTQ Orbitrap XL mass spectrometer was used to characterize and compare their protein corona. Spectral counting and area under curve methods were used to perform label-free quantification. Substantial qualitative and quantitative differences were found among proteins bound to the three different systems investigated. Protein variety found on lipoplexes and LPD complexes was richer than that found on cationic liposomes. There were also significant differences between the amounts of protein. Such results could help in the design of gene-delivery systems, because some proteins could be more selectively bound rather than others, and their bio-distribution could be driven in vivo for more efficient and effective gene therapy.     

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.     

Effect of lipid headgroup composition on the interaction between melittin and lipid bilayers

The effect of the lipid polar headgroup on melittin-phospholipid interaction was investigated by cryo-TEM, fluorescence spectroscopy, ellipsometry, circular dichroism, electrophoresis and photon correlation spectroscopy. In particular, focus was placed on the effect of the lipid polar headgroup on peptide adsorption to, and penetration into, the lipid bilayer, as well as on resulting colloidal stability effects for large unilamellar liposomes. The effect of phospholipid headgroup properties on melittin-bilayer interaction was addressed by comparing liposomes containing phosphatidylcholine, -acid, and -inositol at varying ionic strength. Increasing the bilayer negative charge leads to an increased liposome tolerance toward melittin which is due to an electrostatic arrest of melittin at the membrane interface. Balancing the electrostatic attraction between the melittin positive charges and the phospholipid negative charges through a hydration repulsion, caused by inositol, reduced this surface arrest and increased liposome susceptibility to the disruptive actions of melittin. Furthermore, melittin was demonstrated to induce liposome structural destabilization on a colloidal scale which coincided with leakage induction for both anionic and zwitterionic systems. The latter findings thus clearly show that coalescence, aggregation, and fragmentation contribute to melittin-induced liposome leakage, and that detailed molecular analyses of melittin pore formation are incomplete without considering also these colloidal aspects.     

Syntheses, transfection efficacy and cell toxicity properties of novel cholesterol-based gemini lipids having hydroxyethyl head group

We have synthesized five new cholesterol based gemini cationic lipids possessing hydroxyethyl (-CH(2)CH(2)OH) function on each head group, which differ in the length of the polymethylene spacer chain. These gemini lipids are important for gene delivery processes as they possess pre-optimized molecular features, e.g., cholesterol backbone, ether linkage and a variable spacer chain between both the headgroups of the gemini lipids. Cationic liposomes were prepared from each of these lipids individually and as a mixture of individual cationic gemini lipid and 1,2-dioleoyl phosphatidylethanolamine (DOPE). Each gemini lipid based formulation induced better transfection activity than that of their monomeric counterpart. One such gemini lipid with a -(CH(2))(12)- spacer, HG-12, showed dramatic increase in the mean fluorescence intensity due to the expression of green-fluorescence protein (GFP) in the presence of 10% FBS compared to the conditions where there was no serum. Other gemini lipids retained their gene transfection efficiency without any marked decrease in the presence of serum. The only exception was seen with the gemini with a -(CH(2))(3)- spacer, HG-3, which on gene transfection in the presence of 10% FBS lost ~70% of its transfection efficiency. Overall the gemini lipid with a -(CH(2))(5)- spacer, HG-5, showed the highest transfection activity at N/P (lipid/DNA) ratio of 0.5 and lipid : DOPE molar ratio of 2. Upon comparison of the relevant parameters, e.g., %-transfected cells, the amount of DNA transfected to each cell and %-cell viability all together against Lipofectamine 2000, one of the best commercial transfecting agents, the optimized lipid formulation based on DOPE/HG-5 was found to be comparable. In terms of its ability to induce gene-transfer in the presence of serum and shelf-life DOPE/HG-5 liposome was found to be superior to its commercial counterpart. Confocal imaging analysis confirmed that in the presence of 10% serum using a Lipid : DOPE of 1 : 4 and N/P charge ratio of 0.75 with 1.2 μg DNA per well, HG-5 is better than Lipofectamine 2000.     

Synthesis and preliminary investigations of the siRNA delivery potential of novel, single-chain rigid cationic carotenoid lipids

The success of nucleic acid delivery requires the development of safe and efficient delivery vectors that overcome cellular barriers for effective transport. Herein we describe the synthesis of a series of novel, single-chain rigid cationic carotenoid lipids and a study of their preliminary in vitro siRNA delivery effectiveness and cellular toxicity. The efficiency of siRNA delivery by the single-chain lipid series was compared with that of known cationic lipid vectors, 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]-cholesterol (DC-Chol) and 1,2-dimyristoyl-sn-glyceryl-3-phosphoethanolamine (EPC) as positive controls. All cationic lipids (controls and single-chain lipids) were co-formulated into liposomes with the neutral co-lipid, 1,2-dioleolyl-sn-glycerol-3-phosphoethanolamine (DOPE). Cationic lipid-siRNA complexes of varying (+/-) molar charge ratios were formulated for delivery into HR5-CL11 cells. Of the five single-chain carotenoid lipids investigated, lipids 1, 2, 3 and 5 displayed significant knockdown efficiency with HR5-CL11 cells. In addition, lipid 1 exhibited the lowest levels of cytotoxicity with cell viability greater than 80% at all (+/-) molar charge ratios studied. This novel, single-chain rigid carotenoid-based cationic lipid represents a new class of transfection vector with excellent cell tolerance, accompanied with encouraging siRNA delivery efficiency.     

Polyelectrolyte-coated liposomes: stabilization of the interfacial complexes

Anionic liposomes, composed of egg lecithin (EL) or dipalmitoylphosphatidylcholine (DPPC) with 20 mol% of cardiolipin (CL(2-)), were mixed with cationic polymers, poly(4-vinylpyridine) fully quaternized with ethyl bromide (P2) or poly-l-lysine (PL). Polymer/liposome binding studies were carried out using electrophoretic mobility (EPM), fluorescence, and conductometry as the main analytical tools. Binding was also examined in the presence of added salt and polyacrylic acid (PAA). The following generalizations arose from the experiments: (a) Binding of P2 and PL to small EL/CL(2-) liposomes (60-80 nm in diameter) is electrostatic in nature and completely reversed by addition of salt or PAA. (b) Binding can be enhanced by hydrophobization of the polymer with cetyl groups. (c) Binding can also be enhanced by changing the phase state of the lipid bilayer from liquid to solid (i.e. going from EL to DPPC) or by increasing the size of the liposomes (i.e. going from 60-80 to 300 nm). By far the most promising systems, from the point of view of constructing polyelectrolyte multilayers on liposome cores without disruption of liposome integrity, involve small, liquid, anionic liposomes coated initially with polycations carrying pendant alkyl groups.