Dioleoylphosphatidylethanolamine Research Articles

Enhancement of Skin Penetration of Hydrophilic and Lipophilic Compounds by pH-sensitive Liposomes.

Enhance skin penetration of hydrophilic and lipophilic compounds using liposomes that are responsible to the pH of the skin surface. pH-sensitive liposomes were prepared by a thin layer and freeze-thaw method with dioleoyl phosphatidyl ethanolamine and cholesteryl hemisuccinate. Liposomal fusion with stratum corneum lipid liposomes was measured using fluorescence resonance energy transfer. Particle diameter and zeta potential of the liposomes after fusion were measured by dynamic light scattering and electrophoresis. Under neutral pH conditions, the diameter of the pH-sensitive liposomes was 130 nm and their zeta potential was -70 mV. In weakly acidic conditions, the diameter was larger than 3,000 nm and the zeta potential was -50 mV. In contrast, the particle diameter and the zeta potential of the non-pH-sensitive liposomes remained constant under various pH conditions. A skin penetration study was performed on hairless mice skin using vertical diffusion cells, showing that the fusion ability of pH-sensitive liposomes was higher than that of non-pH-sensitive liposomes. In the skin penetration study was carried out using hydrophilic (calcein) and lipophilic (N-(7-nitrobenz- 2-oxa-1,3-diazol-4yl)-PE) (NBD-PE) model compounds which were applied to the skin with pH-sensitive liposomes as carrier. The fluorescent compounds contained within the pH-sensitive liposomes permeated the skin more effectively than those within non-pH-sensitive liposomes, and this ability was further enhanced with the lipophilic compound. These studies suggest that pH-sensitive liposomes have potential as an important tool for delivery of compounds into the skin.

Hydrophobic Surfactant Proteins Strongly Induce Negative Curvature

The hydrophobic surfactant proteins SP-B and SP-C greatly accelerate the adsorption of vesicles containing the surfactant lipids to form a film that lowers the surface tension of the air/water interface in the lungs. Pulmonary surfactant enters the interface by a process analogous to the fusion of two vesicles. As with fusion, several factors affect adsorption according to how they alter the curvature of lipid leaflets, suggesting that adsorption proceeds via a rate-limiting structure with negative curvature, in which the hydrophilic face of the phospholipid leaflets is concave. In the studies reported here, we tested whether the surfactant proteins might promote adsorption by inducing lipids to adopt a more negative curvature, closer to the configuration of the hypothetical intermediate. Our experiments used x-ray diffraction to determine how the proteins in their physiological ratio affect the radius of cylindrical monolayers in the negatively curved, inverse hexagonal phase. With binary mixtures of dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC), the proteins produced a dose-related effect on curvature that depended on the phospholipid composition. With DOPE alone, the proteins produced no change. With an increasing mol fraction of DOPC, the response to the proteins increased, reaching a maximum 50% reduction in cylindrical radius at 5% (w/w) protein. This change represented a doubling of curvature at the outer cylindrical surface. The change in spontaneous curvature, defined at approximately the level of the glycerol group, would be greater. Analysis of the results in terms of a Langmuir model for binding to a surface suggests that the effect of the lipids is consistent with a change in the maximum binding capacity. Our findings show that surfactant proteins can promote negative curvature, and support the possibility that they facilitate adsorption by that mechanism.

DOTAP/DOPE ratio and cell type determine transfection efficiency with DOTAP-liposomes

The effects of lipid compositions on their physicochemical properties and transfection efficiencies were investigated. Four liposome formulations with different 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) to dioleoylphosphatidylethanolamine (DOPE) weight ratios were investigated, that is, weight ratios 1:0 (T1P0), 3:1 (T3P1), 1:1 (T1P1), and 1:3 (T1P3). Mean sizes of liposomes were influenced by their lipid composition and the preparation concentration at the time of sonication. Zeta potentials of liposomes were inversely correlated with their liposome sizes. However, neither liposome sizes nor zeta potentials were correlated with transfection efficiency. The optimum composition of liposomes was cell-line dependent (T1P0 and T3P1 for Huh7 and AGS, T3P1 and T1P1 for COS7, and T1P1 and T1P3 for A549). The shape of lipoplexes was changed from lamellar to inverted hexagonal structure according to the increased ratio of DOPE, but there was no definite advantage of specific structure in transfection efficiency throughout all used cell lines. However, cellular internalization was consistently faster in T1P0, T3P1, T1P1 compared to T1P3 in all cell lines, suggesting the importance of endosomal escape. Our findings show that the transfection efficiency of DOTAP liposomes is mainly influenced by lipid composition and cell type, and not by size or zeta potential.

The Hydrophobic Surfactant Proteins Strongly Induce Lipid Curvature

The hydrophobic surfactant proteins, SP‐B and SP‐C, greatly accelerate adsorption by vesicles of the surfactant lipids to form a film that lowers the surface tension in the lungs. Pulmonary surfactant enters the interface by a process analogous to the fusion of two vesicles. Like fusion, several factors affect adsorption according to how they alter the curvature of lipid leaflets, and suggest that adsorption proceeds via a rate‐limiting structure with negative curvature, in which the hydrophilic face of the phospholipid leaflets is concave. The studies reported here tested whether the surfactant proteins might promote adsorption by inducing the lipids to adopt more negative curvature, closer to the configuration of the hypothetical intermediate. We used X‐ray diffraction to determine how the proteins in their physiological ratio affect the radius of cylindrical monolayers in the negatively curved, inverse hexagonal phase. With binary mixtures of dioleoyl phosphatidylethanolamine (DOPE) and dioleoyl phosphatidylcholine (DOPC), the proteins produced a dose‐related effect on curvature that depended on the phospholipid composition. With DOPE alone, the proteins produced no change. With increasing amounts of DOPC, the response to the proteins increased, reaching a maximum 50% reduction in cylindrical radius at 5% (w:w) protein. This change represented a doubling of curvature at the outer cylindrical surface. The change in spontaneous curvature, defined at approximately the level of the glycerol group, would be greater. Our findings show that the surfactant proteins can promote negative curvature, and support the possibility that they facilitate adsorption by that mechanism.

Impact of phosphatidylethanolamine on the antioxidant activity of α-tocopherol and trolox in bulk oil.

The amphiphilic phospholipids dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE), can form reverse micelles in bulk oils, which affects lipid oxidation chemistry. Previous studies showed that reverse micelles formed by DOPC and DOPE shorten the oxidation lag phase of stripped soybean oil. This study examined how these reverse micelles influence the activity of primary antioxidants such as the nonpolar α-tocopherol and the polar trolox in stripped and commercial soybean oils. The results showed that DOPC reverse micelles decreased the activity of 100 μM α-tocopherol or trolox. On the other hand, DOPE increased the antioxidant activity of both α-tocopherol and trolox. The polar trolox exhibited better antioxidant activity than the nonpolar α-tocopherol in the presence of both DOPC and DOPE reverse micelles because trolox partitioned more at the interfaces, which was confirmed by a fluorescence steady state study. Different ratios of DOPE to DOPC were added to oil containing 100 μM α-tocopherol, and antioxidant activity increased with increasing DOPE/DOPC ratio. Addition of DOPE to commercial oil inhibited lipid oxidation, whetrsd DOPC was ineffective. HPLC showed that DOPE regenerated α-tocopherol. This study indicates that the antioxidant activity of tocopherols could be improved by utilizing phosphatidylethanolamine (PE) to engineer the properties of reverse micelles in bulk oil.

Buffer-induced swelling and vesicle budding in binary lipid mixtures of dioleoylphosphatidylcholine:dioleoylphosphatidylethanolamine and dioleoylphosphatidylcholine:lysophosphatidylcholine using small-angle X-ray scattering and ³¹P static NMR.

A large variety of data exists on lipid phase behavior; however, it is mostly in nonbuffered systems over nonbiological temperature ranges. We present biophysical data on lipid mixtures of dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), and lysophosphatidylcholine (LysoPC) examining their behaviors in excess water and buffer systems over the temperature range 4-34 °C. These mixtures are commonly used to investigate the effects of spontaneous curvature on integral membrane proteins. Using small-angle X-ray scattering (SAXS) and (31)P NMR, we observed lamellar and vesicle phases, with the buffer causing an increase in the layer spacing. Increasing amounts of DOPE in a DOPC bilayer decreased the layer spacing of the mesophase, while the opposite trend was observed for increasing amounts of LysoPC. (31)P static NMR was used to analyze the DOPC:LysoPC samples to investigate the vesicle sizes present, with evidence of vesicle budding observed at LysoPC concentrations above 30 mol %. NMR line shapes were fitted using an adapted program accounting for the distortion of the lipids within the magnetic field. The distortion of the vesicle, because of magnetic susceptibility, varied with LysoPC content, and a discontinuity was found in both the water and buffer samples. Generally, the distortion increased with LysoPC content; however, at a ratio of DOPC:LysoPC 60:40, the sample showed a level of distortion of the vesicle similar to that of pure DOPC. This implies an increased flexibility in the membrane at this point. Commonly, the assumption is that for increasing LysoPC concentration there is a reduction in membrane tension, implying that estimations of membrane tension based on spontaneous curvature assumptions may not be accurate.

Anionic Lipid, pH-Sensitive Liposome-Gold Nanoparticle Hybrids for Gene Delivery - Quantitative Research of the Mechanism.

Gene therapy is a potential method for treating a large range of diseases. Gene vectors are widely used in gene therapy for promoting the gene delivery efficiency to the target cells. Here, gold nanoparticles (AuNPs) coated with dimethyldioctadecylammonium bromide (DODAB)/dioleoylphosphatidylethanolamine (DOPE) are synthesized using a facile method for a new gene vector (DODAB/DOPE-AuNPs), which possess 3- and 1.5-fold higher transfection efficiency than those of DODAB-AuNPs and a commercial transfection agent, respectively. Meanwhile, it is nontoxic with concentrations required for effective gene delivery. Imaging and quantification studies of cellular uptake reveal that DOPE increases gene copies in cells, which may be attributed to the smaller size of AuNPs/DNA complexes. The dissociation efficiency of DNA from the endocytic pathway is quantified by incubating with different buffers and investigated directly in the cells. The results suggest that DOPE increases the internalization of AuNPs/DNA complexes and promotes DNA release from early endosomes for the vector is sensitive to the anionic lipid membrane and the decreasing pH along the endocytic pathway. The new vector contains the potential to be the new alternative as gene delivery vector for biomedical applications.

Surface modification of tri-calcium phosphate nanoparticles by DOPE and/or anti-E6 antibody to enhance uptake of antisense of E6 mRNA

The main aim of this study was to evaluate the uptake of E6 mRNA antisense into cervical cancer cells, induced by human papilloma virus (HPV). In this study, the carrier of the antisense was tri-calcium phosphate nanoparticles (TCP NPs) conjugated with dioleoyl phosphatidyl ethanolamine (DOPE) and/or anti-E6 antibody. At first, TCP NPs were synthesized, coated with carboxy-polyethylene glycol, and then conjugated with anti-E6 antibody and/or DOPE by carbodiimide cross-linker. Then, a single stranded DNA, which was complementary (antisense) of E6 mRNA, was attached to each one. Finally, the uptake of conjugated and unconjugated TCP NPs into HelaS3 cells was separately evaluated by Fourier transform infrared spectroscopy, optical microscopy, and fluorescent microscopy. Also, the cytotoxicity of these carriers was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Overall, 4 types of TCP NPs were used in this study, including 1) TCP NPs conjugated with DOPE (TCP NPs/DOPE), 2) TCP NPs conjugated with DOPE and antibody (TCP NPs/DOPE/Anti-E6 Ab), 3) TCP NPs conjugated with antibody (TCP NPs/Anti-E6 Ab), and 4) TCP NPs which not conjugated with DOPE and antibody (unconjugated TCP NPs). Uptake tests showed that although all types of TCP NPs could transfer antisense of E6 mRNA into HelaS3 cells, TCP NPs/DOPE and TCP NPs/DOPE/Anti-E6 Ab had more uptake than TCP NPs/Anti-E6 Ab and unconjugated TCP NPs. Moreover, MTT assay showed that TCP NPs/DOPE was more toxic than TCP NPs/DOPE/Anti-E6 Ab, TCP NPs/Anti-E6 Ab, and unconjugated TCP NPs. It can be concluded that TCP NPs/DOPE/Anti-E6 Ab is a good choice for oligonucleotide delivery, because of higher uptake and less toxicity, compared with other formulations.

Hydrophobic nanoparticles promote lamellar to inverted hexagonal transition in phospholipid mesophases.

This study focuses on how the mesophase transition behaviour of the phospholipid dioleoyl phosphatidylethanolamine (DOPE) is altered by the presence of 10 nm hydrophobic and 14 nm hydrophilic silica nanoparticles (NPs) at different concentrations. The lamellar to inverted hexagonal phase transition (Lα-HII) of phospholipids is energetically analogous to the membrane fusion process, therefore understanding the Lα-HII transition with nanoparticulate additives is relevant to how membrane fusion may be affected by these additives, in this case the silica NPs. The overriding observation is that the HII/Lα boundaries in the DOPE p-T phase diagram were shifted by the presence of NPs: the hydrophobic NPs enlarged the HII phase region and thus encouraged the inverted hexagonal (HII) phase to occur at lower temperatures, whilst hydrophilic NPs appeared to stabilise the Lα phase region. This effect was also NP-concentration dependent, with a more pronounced effect for higher concentration of the hydrophobic NPs, but the trend was less clear cut for the hydrophilic NPs. There was no evidence that the NPs were intercalated into the mesophases, and as such it was likely that they might have undergone microphase separation and resided at the mesophase domain boundaries. Whilst the loci and exact roles of the NPs invite further investigation, we tentatively discuss these results in terms of both the surface chemistry of the NPs and the effect of their curvature on the elastic bending energy considerations during the mesophase transition.

Liposome-based Formulation for Intracellular Delivery of Functional Proteins.

The intracellular delivery of biologically active protein represents an important emerging strategy for both fundamental and therapeutic applications. Here, we optimized in vitro delivery of two functional proteins, the β-galactosidase (β-gal) enzyme and the anti-cytokeratin8 (K8) antibody, using liposome-based formulation. The guanidinium-cholesterol cationic lipid bis (guanidinium)-tren-cholesterol (BGTC) (bis (guanidinium)-tren-cholesterol) combined to the colipid dioleoyl phosphatidylethanolamine (DOPE) (dioleoyl phosphatidylethanolamine) was shown to efficiently deliver the β-gal intracellularly without compromising its activity. The lipid/protein molar ratio, protein amount, and culture medium were demonstrated to be key parameters affecting delivery efficiency. The protein itself is an essential factor requiring selection of the appropriate cationic lipid as illustrated by low K8 binding activity of the anti-K8 antibody using guanidinium-based liposome. Optimization of various lipids led to the identification of the aminoglycoside lipid dioleyl succinyl paromomycin (DOSP) associated with the imidazole-based helper lipid MM27 as a potent delivery system for K8 antibody, achieving delivery in 67% of HeLa cells. Cryo-transmission electron microscopy showed that the structure of supramolecular assemblies BGTC:DOPE/β-gal and DOSP:MM27/K8 were different depending on liposome types and lipid/protein molar ratio. Finally, we observed that K8 treatment with DOSP:MM27/K8 rescues the cyclic adenosine monophosphate (cAMP)-dependent chloride efflux in F508del-CFTR expressing cells, providing a new tool for the study of channelopathies.

Determining Material Elastic Properties of Arbitrarily-Shaped Membranes using Molecular Dynamics Simulations with Application to the Inverted Hexagonal Phase

Accumulating evidence indicates that diverse physiological processes are influenced by the lipid composition of the membrane and by its material properties. This has notably been shown for the function of diverse proteins and their oligomerization, and processes on larger scales such as membrane reshaping and fusion. Determination of the elastic properties of lipidic membranes is therefore of great importance to our understanding of these processes. Experimental approaches to determine the material properties of lipids remain challenging and usually rely on their study in a relaxed environment or in flat bilayers, although it is widely accepted that cell membranes can be under considerable stress and frustration as well as high local curvature. Whether this impacts the measured properties is a matter of debate so that studying membranes under more realistic conditions is key for our understanding how these material properties impact different physiological processes. In this context, we propose a computational method to determine the elastic properties of lipid assemblies of arbitrarily shaped interfaces and use it to study the impact of the curvature of a membrane on its elastic properties. Specifically, we apply the methodology to mixtures of DOPE (dioleoylphosphatidylethanolamine) lipids and cholesterol in the inverted-hexagonal and lamellar phases and find that the bending rigidity for a particular lipid composition critically depends on the geometry of the lipidic system. This dependence correlates on the molecular level to the changes in lipid chain order imposed by the membrane curvature, implying that these results should pertain to other situations where the membrane is deformed, stressed or frustrated that notably emerge around integral membrane proteins or during membrane remodeling processes such as budding.

Efficacious gene silencing in serum and significant apoptotic activity induction by survivin downregulation mediated by new cationic gemini tocopheryl lipids.

Nonviral gene delivery offers cationic liposomes as promising instruments for the delivery of double-stranded RNA (ds RNA) molecules for successful sequence-specific gene silencing (RNA interference). The efficient delivery of siRNA (small interfering RNA) to cells while avoiding unexpected side effects is an important prerequisite for the exploitation of the power of this excellent tool. We present here six new tocopherol based cationic gemini lipids, which induce substantial gene knockdown without any obvious cytotoxicity. All the efficient coliposomal formulations derived from each of these geminis and a helper lipid, dioleoylphosphatidylethanolamine (DOPE), were well characterized using physical methods such as atomic force microscopy (AFM) and dynamic light scattering (DLS). Zeta potential measurements were conducted to estimate the surface charge of these formulations. Flow cytometric analysis showed that the optimized coliposomal formulations could transfect anti-GFP siRNA efficiently in three different GFP expressing cell lines, viz., HEK 293T, HeLa, and Caco-2, significantly better than a potent commercial standard Lipofectamine 2000 (L2K) both in the absence and in the presence of serum (FBS). Notably, the knockdown activity of coliposomes of gemini lipids was not affected even in the presence of serum (10% and 50% FBS) while it dropped down for L2K significantly. Observations under a fluorescence microscope, RT-PCR, and Western blot analysis substantiated the flow cytometry results. The efficient cellular entry of labeled siRNA in GFP expressing cells as evidenced from confocal microscopy put forward these gemini lipids among the potent lipidic carriers for siRNA. The efficient transfection capabilities were also profiled in a more relevant fashion while performing siRNA transfections against survivin (an anti-apoptotic protein) which induced substantial apoptosis. Furthermore, the survivin downregulation improved the therapeutic efficacy levels of an anticancer drug, doxorubicin, significantly. In short, the new tocopherol based gemini lipids appear to be highly promising for achieving siRNA mediated gene knockdown in various cell lines.

Co-delivery of plasmid DNA and antisense oligodeoxyribonucleotide into human carcinoma cells by cationic liposomes.

This study aimed to evaluate the co-delivery of cationic liposome/plasmid DNA complexes and cationic liposome/antisense oligodeoxyribonucleotide (AS ODN) complexes in HeLa human cervical carcinoma cells. Dimethyldioctadecyl ammonium bromide (DDAB): dioleoyl phosphatidylethanolamine (DOPE) liposome/plasmid DNA complexes, and DDAB:DOPE liposome/AS ODN complexes were formulated and characterized in terms of agarose gel electrophoretic mobility, particle size and zeta potential. The complexes were evaluated for delivery of pEGFP plasmid DNA and AS ODN in HeLa cells. Cell growth inhibition was evaluated using p53 plasmid DNA and bcl-2 AS ODN, by codelivery of DDAB:DOPE liposome/p53 plasmid DNA and DDAB:DOPE liposome/bcl-2 AS ODN complexes. The particle size of DDAB:DOPE liposome/plasmid DNA complexes, and DDAB:DOPE liposome/AS ODN complexes were 180.6±2.0 to 372.3±2.4 nm, and zeta potentials were -26.7±1.2 to +6.8±0.4 mV, respectively. The AS ODN uptake and green fluorescent protein (GFP) expression upon their co-delivery by DDAB:DOPE liposomes were both high. Treatment of the cells with the co-delivery of DDAB:DOPE liposome/p53 plasmid DNA complexes and DDAB:DOPE liposome/ bcl-2 AS ODN complexes inhibited cell growth to a greater degree than that with either DDAB:DOPE liposome/p53 plasmid DNA complexes or DDAB:DOPE liposome/bcl-2 AS ODN complexes alone. These data suggest that co-delivery of cationic liposome/p53 plasmid DNA and cationic liposome/bcl-2 AS ODN complexes is an effective strategy to achieve enhanced therapeutic activities.

Fusogenic liposomal formulation of sirolimus: improvement of drug anti-proliferative effect on human T-cells

Context: Fusogenic liposomes are unique delivery vehicles capable of introducing their contents directly and efficiently into the cytoplasm.Objective: The objective of this study was to evaluate the potential of fusogenic liposomes containing Sirolimus to improve its anti-proliferative effect on T-lymphocyte cells.Materials and methods: Conventional liposomes containing Sirolimus were prepared from Dipalmitoylphosphatidylcholine (DPPC) and cholesterol using the modified ethanol injection method. To prepare fusogenic liposomes, dioleoylphosphatidylethanolamine (DOPE) was added to the conventional liposome formulation. The liposomes were characterized by their size, zeta potential, encapsulation efficiency percent (EE%) and chemical stability during 6 months. The in vitro release of liposomes, anti-proliferative effect and liposome uptake of both types of liposomes with optimized formulations were studied on human T-lymphocyte cells employing the MTT assay and fluorescein isothiocyanate-loaded liposomes.Results and discussion: The particle size of the liposomes was evaluated between 138 and 650 nm and mean zeta potential was in the range of −32.95 to −45.60 mV. The average EE% of the prepared conventional and fusogenic liposomes were 76.9% and 80.5%, respectively. Liposomal formulations released only 10–20% of encapsulated drug without any burst effect. In vitro immunosuppressive evaluation on T-cells showed that fusogenic liposomes have the best anti-proliferative effects and uptake on T-lymphocyte cell compared to the conventional liposomes.Conclusion: Our results indicated that fusogenic liposomes can be useful carriers for improving the inhibition of T-cell proliferation.

The Transmembrane Domain Peptide of Vesicular Stomatitis Virus Promotes Both Intermediate and Pore Formation during PEG-Mediated Vesicle Fusion

We propose mechanisms by which the transmembrane domain of vesicular stomatitis virus (VSV-TMD) promotes both initiation of fusion and formation of a fusion pore. Time courses of polyethyleneglycol (PEG)-mediated fusion of 25 nm small unilamellar vesicles composed of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), bovine brain sphingomyelin, and cholesterol (35:30:15:20 molar ratio) were recorded at pH 7.4 at five different temperatures (from 17°C to 37°C) and compared with time courses obtained with the same vesicles containing the fusion-active TMD of the G protein of VSV. Multiple time courses were fitted globally to a one-intermediate ensemble kinetic model to estimate the rate constants for conversion of the aggregated state to an intermediate hemifused state (k1, stalk, or I1) that rapidly transits to an unstable intermediate (I2 state) that converts to a final fusion pore state with a combined rate k3. The probabilities of lipid mixing, contents mixing, and contents leakage in the three states were also obtained from this analysis. The activation thermodynamics for each step were consistent with previously published models of lipid rearrangements during intermediate and pore formation. The influences of VSV-TMD, hexadecane, and VSV-TMD + hexadecane on the kinetics, activation thermodynamics, and membrane structure support the hypothesis that these two agents do not catalyze fusion by a common mechanism, except possibly at the lowest temperatures examined. VSV-TMD primarily catalyzed initial intermediate formation, although it substantially increased the probability of contents mixing in the intermediate state. Our results support the hypothesis that the catalytic influence of VSV-TMD on the initial-intermediate- and pore-forming steps of PEG-mediated fusion derives from its ability to impose a positive intrinsic curvature and thereby stress small unilamellar vesicle outer leaflets as well as the periphery of intermediate microstructures.

Effective induction of anti-tumor immunity using p5 HER-2/neu derived peptide encapsulated in fusogenic DOTAP cationic liposomes co-administrated with CpG-ODN

Cationic liposomes have been used as efficient antigen delivery systems for cancer vaccination. The current study has investigated whether the incorporation of the helper-fusogenic lipid dioleoylphosphatidylethanolamine (DOPE) in cationic liposomes composed of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP)–cholesterol enhances the cytosolic delivery of p5 HER-2/neu derived peptide (p5) and promotes cytotoxic T lymphocytes (CTL) response. The p5, which is a very hydrophobic peptide, was encapsulated into liposomes by using three different methods and characterized for their colloidal properties. A chaotropic loading method using 7M urea provided the highest encapsulation yields. Mice were first immunized with encapsulated p5 in liposomes composed of either DOTAP–cholesterol or DOTAP–cholesterol–DOPE, alone or co-administered with CpG-ODN, as an immunoadjuvant, then, inoculated with a subcutaneous injection of TUBO tumor cells. Results obtained from enzyme-linked immunospot, cytotoxicity and intracellular cytokine assays as well as tumor sizes and animal survival analysis demonstrated that p5 encapsulated in DOTAP–cholesterol–DOPE liposomes co-administered with CpG-ODN greatly enhanced the cytotoxic T lymphocytes response and highly inhibited the tumor progression. The outperformance of DOTAP–cholesterol–DOPE liposomes+CpG-ODN was found to be attributed to its capability in induction of both CD8+ and CD4+ responses. This formulation could be a suitable vaccine candidate against Her2 positive cancers and merits further investigations.

PEGylated galactosylated cationic liposomes for hepatocytic gene delivery

The efficiency of liposome-mediated gene delivery is greatly enhanced by appropriate decoration of vehicles with cell-specific targeting ligands. However, liposome–DNA complexes may still be opsonized in serum thus ablating any advantage gained. A stealth aspect may therefore be conferred on complexes by poly(ethylene glycol) (PEG) grafting. Here, we examined the effect that degree of PEGylation has on physicochemical properties, cytotoxicity and transfection activity of lipoplexes containing the cytofectin 3β-[N-(N′, N′-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T), the neutral co-lipid dioleoylphosphatidylethanolamine (DOPE), the asialoglycoprotein receptor (ASGP-R) targeted cholesteryl-β-d-galactopyranoside (Chol-β-Gal) ligand, and plasmid DNA in ASGP-R-negative (HEK293) and receptor-positive (HepG2) human cell lines. Lipoplexes were characterized by hydrodynamic sizing, electron microscopy, band shift, ethidium bromide (EtBr) intercalation and nuclease digestion assays. Cryo-TEM and DLS studies revealed that PEGylation generated smaller and more densely aggregated lipoplexes than their non-PEGylated counterparts. MTT and AB reduction studies showed that the lipoplexes elicited a dose-dependent cytotoxic effect in both cell lines, with cell viability remaining above 65% (MTT) and 50% (AB). The Ricinus communis (RCA120) agglutination test confirmed that the galactosyl residues on the targeted lipoplexes were well exposed and accessible. Transgene activity increased by 63% and 77% when HepG2 was confronted by the 2 and 5mole% PEGylated lipoplexes, respectively, compared to their non-PEGylated counterparts. Furthermore, Chol-T Chol-β-Gal 5% PEG complexes were able to achieve a 164% increase in transfection level in the ASGP-R positive cell line (HepG2) compared to HEK293 (ASGP-R negative). Results strongly indicate that PEGylation potentiates the activity of ASGP-R-targeted lipoplexes, highlighting their gene delivery potential.

Association of lipids with milk α- and β-caseins

We report the molecular interaction and the binding sites of cholesterol (CHOL), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioctadecyldimethyl-ammoniumbromide (DDAB), and dioleoylphosphatidylethanolamine (DOPE) with milk α- and β-caseins in aquous solution at physiological conditions. Fourier transform infrared (FTIR), fluorescence spectroscopic methods and molecular modeling were used to determine the binding sites of lipid–protein complexes and the effect of lipid interaction on the stability and conformation of α- and β-caseins. Structural analysis showed that lipids bind casein via mainly hydrophobic contact with association constants of KCHOL-α-casein=1.0 (±0.1)×104M−1, KDOPE-α-casein=5.0 (±0.07)×103M−1, KDDAB-α-casein=2.0 (±0.06)×104M−1, KDOTAP-α-casein=1.5 (±0.6)×104M−1, KCHOL-β-casein=1.0 (± 0.3)×104M−1, KDOPE-β-casein=1.5 (±0.06)×103M−1, KDDAB-β-casein=1.7 (±0.3)×104M−1 and KDOTAP-β-casein=2.1 (±0.5)×104M−1. The average number of binding sites occupied by lipid molecules on protein (n) were from 0.7 to 1.1. Docking showed different binding sites for α- and β-caseins toward lipid complexation with the free binding energies from −10 to −13kcal/mol. Casein conformation was altered by lipid interaction with a reduction of α-helix and β-sheet and an increase of random coil and turn structure suggesting a partial protein unfolding.Abbreviations: Cascasein; CHOLcholesterol; DOTAP1,2-dioleoyl-3-trimethylammonium-propane; DDABdioctadecyldimethylammonium bromide; DOPEdioleoylphosphatidylethanolamine; FTIRFourier transform infrared spectroscopy; CDcircular dichroism

Perifosine induced release of contents of trans cell-barrier transport efficient liposomes

Perifosine (OPP) containing liposomal formulation was previously found to deliver almost half of liposome encapsulated content through a tight cellular barrier in vitro. In order to understand the role of different liposome components, especially perifosine, in transendothelial transport the physical characteristics of liposome membranes composed of phosphatidylcholine, and cholesterol, as a main lipid constituents, and variable amount of helper lipids: dioleoyl phosphatidylethanolamine (DOPE), and alkylphospholipid perifosine.For this purpose, electron paramagnetic resonance (EPR) with computer aided EPR spectra simulation and fluorescence polarization spectroscopy were used to investigate how different membrane components influence membrane characteristics and the release of liposome entrapped substances. Beside methylester of palmitic acid with nitroxide group at different position on acyl chain usually used for such studies, the spin labeled and fluorescent labeled analog of perifosine were introduced.OPP increases membrane fluidity of liposomes as well as the release of liposome encapsulated content. The release of neutral molecules increases with OPP concentration, while the release of charged molecules is about an order of magnitude slower. Optimal OPP concentration, for release of charged molecules, is about 15mol%.These results are one step further toward the conclusion that the lysolipid-containing liposomes could be promising trans endothelial delivery system, since lysolipids, such as OPP, open tight cellular barriers, as was published before, and in the same time induce the release of liposome encapsulated content at physiological temperature, as shown here. Since many drug delivery systems are being developed, which mainly exploit the transcellular route of delivery through barrier-forming cells, we hope that the uniqueness of lysolipid-containing liposomes, exploiting the paracellular route, and thus avoiding efflux transporters, will foster further research in formulating other lysolipid-containing liposomes as drug delivery systems.

Novel serum-tolerant lipoplexes target the folate receptor efficiently

Gene transfer using non-viral vectors is a promising approach for the safe delivery of nucleic acid therapeutics. In this study, we investigate a lipid-based system for targeted gene delivery to malignant cells overexpressing the folate receptor (FR). Cationic liposomes were formulated with and without the targeting ligand folate conjugated to distearoylphosphatidyl ethanolamine polyethylene glycol 2000 (DSPE-PEG2000), the novel cytofectin 3β[N(N1,N1-dimethlaminopropylsuccinamidoethane)-carbamoyl]cholesterol (SGO4), which contains a 13atom, 15Å spacer element, and the helper lipid, dioleoylphosphatidylethanolamine (DOPE). Physicochemical parameters of the liposomes and lipoplexes were obtained by zeta sizing, zeta potential measurement and cryo-TEM. DNA-binding and protection capabilities of liposomes were confirmed by gel retardation assays, EtBr intercalation and nuclease protection assays. The complexes were assessed in an in vitro system for their effect on cell viability using the MTT assay, and gene transfection activity using the luciferase assay in three cell lines; HEK293 (FR-negative), HeLa (FR+-positive), KB (FR++-positive). Low cytotoxicities were observed in all cell lines, while transgene activity promoted by folate-tagged lipoplexes in FR-positive lines was tenfold greater than that by untargeted constructs and cell entry by folate complexes was demonstrably by FR mediation. These liposome formulations have the design capacity for in vivo application and may therefore be promising candidates for further development.