1-2-dielaidoylphosphatidylethanolamine and dioleoyl-phosphatidylethanolamine

The aim of this study is to develop targeted nanoliposome formulations to provide efficient treatment for breast cancer. In this study, peptide 18-modified poly(2-ethyl-2-oxazoline)-dioleoylphosphatidylethanolamine (P18-PEtOx-DOPE), was synthesised to construct nanoliposomes.. Doxorubicin (DOX) was encapsulated into the nanoliposomes by ethanol injection method. Particle size and polydispersity index were measured by dynamic light scattering. Zeta potential was determined by electrophoretic laser Doppler anemometry. The shape of the nanoliposomes was examined by transmission electron microscope. Specific bindings of P18-PEtOx-DOPE nanoliposomes were demonstrated on AU565 cells by confocal microscopy and flow cytometry studies.. DOX-loaded nanoliposomes with particle diameter of 150.00 ± 2.84 nm and PDI of 0.212 ± 0.013 were obtained. PEtOx-DOPE and PEtOx-DOPE nanoliposomes are non-toxic on HUVEC, HEK293 and hMSC cells for 48 h. Furthermore, P18-PEtOx-DOPE nanoliposomes demonstrated specificity towards AU565 cells with high binding affinity.. As a result, DOX-loaded P18-PEtOx-DOPE nanoliposomes can serve as favourable candidates in breast cancer targeted therapy.

Topics: Antibiotics, Antineoplastic; Breast Neoplasms; Cell Line, Tumor; Doxorubicin; Drug Delivery Systems; Drug Design; Drug Screening Assays, Antitumor; Drug Stability; Female; HEK293 Cells; Human Umbilical Vein Endothelial Cells; Humans; Liposomes; Myocytes, Smooth Muscle; Nanoparticles; Oxazoles; Particle Size; Peptides; Phosphatidylethanolamines; Scattering, Radiation

Mechanosensitive (MS) channels have an intimate relationship with membrane lipids that underlie their mechanosensitivity. Membrane lipids may influence channel activity by directly interacting with MS channels or by influencing the global properties of the membrane such as elastic area expansion modulus or bending rigidity. Previous work has implicated membrane stiffness as a potential determinant of the mechanosensitivity of E. coli (Ec)MscS. Here we systematically tested this hypothesis using patch fluorometry of azolectin liposomes doped with lipids of increasing elastic area expansion modulus. Increasing dioleoylphosphatidylethanolamine (DOPE) content of azolectin liposomes made it more difficult to activate EcMscS by membrane tension (i.e. increased gating threshold). This effect was exacerbated by stiffer forms of phosphatidylethanolamine such as the branched chain lipid diphytanoylphosphoethanolamine (DPhPE) or the fully saturated lipid distearoyl-sn-glycero-3-phosphoethanolamine (DSPE). Furthermore, a comparison of the branched chain lipid diphytanoylphosphocholine (DPhPC) to the stiffer DPhPE indicated again that it was harder to activate EcMscS in the presence of the stiffer DPhPE. We show that these effects are not due to changes in membrane bending rigidity as the membrane tension threshold of EcMscS in membranes doped with PC18:1 and PC18:3 remained the same, despite a two-fold difference in their bending rigidity. We also show that after prolonged pressure application sudden removal of force in softer membranes caused a rebound reactivation of EcMscS and we discuss the relevance of this phenomenon to bacterial osmoregulation. Collectively, our data suggests that membrane stiffness (elastic area expansion modulus) is one of the key determinants of the mechanosensitivity of EcMscS.

Topics: Biological Transport; Biomechanical Phenomena; Escherichia coli; Escherichia coli Proteins; Ion Channel Gating; Ion Channels; Lipid Bilayers; Liposomes; Mechanotransduction, Cellular; Membrane Lipids; Membranes; Patch-Clamp Techniques; Phosphatidylcholines; Phosphatidylethanolamines; Spheroplasts

The development of polymer-based nanoparticulate delivery systems for siRNA is important for the clinical success of gene therapy. However, there are some major drawbacks that need to be overcome. Short interfering RNA (siRNA) has been investigated as a potential therapeutic drug to silence disease-associated genes, but its usage is limited due to the lack of effective and safe nanocarriers. In this study, DOPE-PEI, a nanoparticle consisting of the fusogenic lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) conjugated with low-molecular-weight, 600 Da, branched polyethylenimine (PEI) was produced and optimized for siRNA delivery. This delivery system was modified with other components such as 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)2000] (DOPE-PEG2K), DOPE-PEG3.4K-bombesin and 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine/1,2-dioleoyl-3-trimethylammonium-propane (DOPE/DOTAP) and tested on PC-3 cells. The conjugation of DOPE to PEI polymer (DOPE-PEI) improved the efficiency of PEI to deliver siRNA into the cytosol and knockdown genes, but demonstrated high toxicity. The addition of DOPE-PEG2K reduced cellular toxicity by masking the surface positive charge of the DOPE-PEI/siRNA complex, with the incorporation of a gastrin-releasing peptide receptor (GRPR) targeting peptide and DOPE/DOTAP components improving the cellular uptake of siRNA into targeted cells and the siRNA knockdown efficiency.

Topics: Cell Line, Tumor; Drug Carriers; Fatty Acids, Monounsaturated; Gene Knockdown Techniques; Gene Transfer Techniques; Genetic Therapy; Humans; Imines; Lipids; Nanoparticles; PC-3 Cells; Peptides; Phosphatidylethanolamines; Polyethylene Glycols; Polyethylenes; Polymers; Quaternary Ammonium Compounds; Receptors, Bombesin; RNA, Small Interfering

The present study aim to design a liposomal electrochemical sensor using 1, 2-dioleoyl-3-trimethylammoniumpropane (DOTAP) and dioleoylphosphatidylethanolamine(DOPE), chimeric probes and p19, it has been considered as a caliper molecule as well. Also the competitor structural hybrid (RNA) was used to detect three types of miRs in one screen printed electrode modified by gold nanoparticle (SCPE/GNP). In this purpose, the sensor signal stabilized when the cationic DOTAP-DOPE with hybrids of the chimeric probes (Stem, M-linear) sandwiched in order to detect 221-124a miRs. Given the lack of accessibility to RNA-miRs segments of chimeric probes, p19 inhibited the electrochemical reaction and shifted signal to off. After that p19 connected with the free hybrid of T-linear/21miR (just RNA) as competing for structure and the signal was shifted to ON, again. In this study, the electrochemical measurements were performed between the potentials at -0.4 V and +0.4 V with 1 mM [Fe(CN)6]-3-/4 which DOTAP-DOPE acted as an enhancer layer in the electrostatically reaction. This sensor determines as low as 0.4 fM of miRNA with high selectivity and specificity for sequential analysis of 124a-221-21 miRs in just 2 h.

Topics: Biosensing Techniques; Electrochemical Techniques; Gold; Humans; Limit of Detection; Liposomes; Metal Nanoparticles; MicroRNAs; Phosphatidylethanolamines; Recombinant Proteins

Photodynamic therapy of cancer comprises the activation of photosensitizer molecules delivered to cancer cells, to generate reactive oxygen species that mediate cytotoxicity. In this study, previously synthesized dendritic magnesium tribenzoporphyrazines were incorporated into four types of liposomes containing either 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as the zwitterionic lipids. The addition of either l-α-phosphatidyl-dl-glycerol (PG) or 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) imparted a negative or positive charge, respectively. Novel formulations were tested in oral squamous cell carcinoma cell lines (CAL 27, HSC-3) as well as cervical adenocarcinoma cells (HeLa). Positively charged DOTAP:POPC liposomes were the most effective carriers for all tested tribenzoporphyrazines. Calculated IC

Topics: Cell Line, Tumor; Fatty Acids, Monounsaturated; HeLa Cells; Humans; Liposomes; Phosphatidylethanolamines; Photochemotherapy; Photosensitizing Agents; Quaternary Ammonium Compounds

Lipid-based nanoparticles as gene vectors have attracted considerable attention for their high gene transfection efficiency and low cytotoxicity. In our previous work, we synthesized gold nanoparticles/dimethyldioctadecylammonium bromide (DODAB)/dioleoylphosphatidylethanolamine (DOPE) (GDD) as anionic lipid- and pH-sensitive gene vectors. To further realize targeted gene transfection, a series of gold nanoparticles/DODAB/DOPE/DOPE-folic acid (DOPE-FA) with various ratios of DOPE-FA were prepared and termed as GFn (for which n=1.0, 2.5, 5.0, 7.5, or 10.0 %). The gene transfection efficiency mediated by GF2.5 can reach about 85 % for MCF-7 (FA-receptor-positive cells), higher than those of the negative control (GDD, 35 %) and positive control (Lipofectamine 2000, 65 %). However, GF2.5 does not further promote gene transfection into A549 (FA-receptor-negative cells). The higher gene transfection efficiency for MCF-7 cells can be attributed to enhanced cellular uptake efficiency mediated by the FA targeting ability. Furthermore, GF2.5 was also found to accumulate at the specific tumor site and showed enhanced in vivo gene delivery ability. In addition, no significant harm was observed for the main tissues of the mice after treatment with GF2.5. Therefore, GF2.5, with the targeting ability and improved transfection efficiency, shows promise for its utility in gene therapy for tumor cells that overexpress FA receptors. We believe the results of this study will find more broad applications in gene therapy.

Topics: A549 Cells; Animals; Cell Survival; Folic Acid; Genetic Vectors; Gold; Humans; MCF-7 Cells; Metal Nanoparticles; Mice; Microscopy, Confocal; Neoplasms; Phosphatidylethanolamines; Rhodamines; Spectrophotometry, Ultraviolet; Transfection

Cationic liposomes can be designed and developed in order to be an efficient gene delivery system for mammalian cells. Dendritic cell (DC) vaccines can be used to treat cancer, as cationic liposomes can deliver tumor antigens to cells while cells remain active. However, most methods used for liposome production are not able to reproduce in large scale the physicochemical and biological properties of liposomes produced in laboratory scale. In this context, ethanol injection method achieved promising results, although requiring post-treatment for size reduction and/or to remove residual ethanol. Thus, the purpose of this study was to generate cationic liposomes suitable for gene therapies via ethanol injection method in only one step (VEI) and compared to those submitted to a size reduction processes by microfluidization (MFV). For this, the method to produce cationic liposomes composed of egg phosphatidylcholine (EPC), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and 1,2-dioleoylphosphatidylethanolamine (DOPE) was optimized using a statistical design approach. As a result, the size of VEI decreased from 290 nm to 110 nm and the polydispersity from 0.54 to 0.17. In the case of MFV, size decreased from 128 nm to 107 nm and polydispersity from 0.40 to 0.18. ST and MFV before and after optimization were also characterized in terms of morphology by transmission electron microscopy (TEM) and structure by differential scanning calorimetry (DSC). Finally, to show their potential in gene/immune therapies applications, DCs were stimulated by such liposomes. Cells internalized liposomes, increasing expression of the costimulatory molecule CD86 and inducing T lymphocyte proliferation.

Topics: Animals; B7-2 Antigen; Cations; Cell Proliferation; Dendritic Cells; Ethanol; Fatty Acids, Monounsaturated; Gene Transfer Techniques; Genetic Therapy; Humans; Immunotherapy; Liposomes; Particle Size; Phosphatidylcholines; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Surface Properties; T-Lymphocytes

siRNA may be delivered as electrostatic complexes with cationic lipids (lipoplexes) or polycations (polyplexes). The purpose of this project was to determine the effect of cellular internalization mechanism(s) on siRNA-mediated gene silencing efficiency.. Lipoplexes were formed comprising siRNA and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP), cholesterol and dioleoyl phosphatidylethanolamine (DOPE), and polyplexes comprised siRNA with polyethylenimine (PEI). During transfections, specific uptake mechanisms were inhibited by pharmacological agents and RNAi-mediated knockdown of proteins involved in various endocytosis pathways. Confocal fluorescence microscopy further elucidated the predominant endocytic pathways of siRNA delivery via colocalization of vectors with endocytic vesicle markers.. Inhibition of macropinocytosis (MP), caveolin-mediated endocytosis (CvME), flotillin-mediated endocytosis (FME) and knockdown of ARF6 significantly decreased PEI/siRNA-mediated gene silencing. Inhibition of endocytosis pathways, however, had negligible effect on lipoplex uptake and gene silencing mediated by lipoplexes. Rather, internalization of lipoplexes and subsequent siRNA-mediated gene silencing occurred via an energy-independent process.. MP, CvME and FME, but not the acidified clathrin-mediated pathway, lead to effective gene silencing by PEI/siRNA polyplexes. Lipoplexes, in contrast, deliver siRNA primarily by direct fusion of the liposomal and cellular membranes. These results provide a new understanding of the mechanisms of siRNA delivery materials in HeLa cells and may aid in design of more effective RNAi strategies.

Topics: Cell Culture Techniques; Cell Survival; Cholesterol; Drug Delivery Systems; Drug Liberation; Endocytosis; Fatty Acids, Monounsaturated; Gene Silencing; HeLa Cells; Humans; Liposomes; Phosphatidylethanolamines; Polyethyleneimine; Quaternary Ammonium Compounds; RNA, Small Interfering

A series of novel 1,4,7,10-tetraazacyclododecane (cyclen)-based cationic lipids with asymmetric double hydrophobic tails (cholesteryl and long aliphatic chains) were designed and synthesized. Lysine was chosen as a linking moiety in the molecular backbone. The liposomes formed from 8 and dioleoylphosphatidylethanolamine (DOPE) could bind and condense plasmid DNA into nanoparticles under a low N/P ratio. These nano-scaled lipoplexes have low cytotoxicity, and might efficiently transfect A549 cells. In vitro transfection results revealed that all cationic lipids showed a comparable or better transfection efficiency (TE) than commercially available Lipofectamine 2000. The length and saturation degree of the aliphatic chain would affect their gene transfection performance, and the linoleic acid-containing 8e could give the best TE.

Topics: Acylation; Cations; Cell Death; Cell Line, Tumor; Cholesterol; Cyclams; DNA; Electrophoretic Mobility Shift Assay; Ethidium; Fluorescence; Heterocyclic Compounds; Humans; Hydrophobic and Hydrophilic Interactions; Lipids; Liposomes; Particle Size; Phosphatidylethanolamines; Static Electricity; Transfection

Nonviral gene delivery vectors now show good therapeutic potential: however, detailed characterization of the composition and macromolecular organization of such particles remains a challenge. This paper describes experiments to elucidate the structure of a ternary, targeted, lipopolyplex synthetic vector, the LID complex. This consists of a lipid component, Lipofectin (L) (1:1 DOTMA:DOPE), plasmid DNA (D), and a dual-function, cationic peptide component (I) containing DNA condensation and integrin-targeting sequences. Fluorophore-labeled lipid, peptide, and DNA components were used to formulate the vector, and the stoichiometry of the particles was established by fluorescence correlation spectroscopy (FCS). The size of the complex was measured by FCS, and the sizes of LID, L, LD, and ID complexes were measured by dynamic light scattering (DLS). Fluorescence quenching experiments and freeze-fracture electron microscopy were then used to demonstrate the arrangement of the lipid, peptide, and DNA components within the complex. These experiments showed that the cationic portion of the peptide, I, interacts with the plasmid DNA, resulting in a tightly condensed DNA-peptide inner core; this is surrounded by a disordered lipid layer, from which the integrin-targeting sequence of the peptide partially protrudes.

Topics: Biophysical Phenomena; Biophysics; Diffusion; DNA; Freeze Fracturing; Genetic Vectors; Integrins; Light; Liposomes; Microscopy, Electron; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Scattering, Radiation; Spectrometry, Fluorescence

Nonviral vectors consisting of integrin-targeting peptide/DNA (ID) complexes have the potential for widespread application in gene therapy. The transfection efficiency of this vector, however, has been limited by endosomal degradation. We now report that lipofectin (L) incorporated into the ID complexes enhances integrin-mediated transfection, increasing luciferase expression by more than 100-fold. The transfection efficiency of Lipofectin/Integrin-binding peptide/DNA (LID) complexes, assessed by beta-galactosidase reporter gene expression and X-gal staining, was improved from 1% to 10% to over 50% for three different cell lines, and from 0% to approximately 25% in corneal endothelium in vitro. Transfection complexes have been optimized with respect to their transfection efficiency and we have investigated their structure, function, and mode of transfection. Both ID and LID complexes formed particles, unlike the fibrous network formed by lipofectin/DNA complexes (LD). Integrin-mediated transfection by LID complexes was demonstrated by the substantially lower transfection efficiency of LKD complexes in which the integrin-biding peptide was substituted for K16 (K). Furthermore, the transfection efficiency of complexes was shown to be dependent on the amount of integrin-targeting ligand in the complex. Finally, a 34% reduction in integrin-mediated transfection efficiency by LID complexes was achieved with a competing monoclonal antibody. The role of lipofectin in LID complexes appears, therefore, to be that of a co-factor, enhancing the efficiency of integrin-mediated transfection. The mechanism of enhancement is likely to involve a reduction in the extent of endosomal degradation of DNA.

Topics: Amino Acid Sequence; Animals; Binding, Competitive; Cell Line; Cornea; Drug Carriers; Genetic Vectors; Humans; Ligands; Liposomes; Microscopy, Atomic Force; Molecular Sequence Data; Oligopeptides; Peptides; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Rabbits; Receptors, Fibronectin; Recombinant Fusion Proteins; Transfection

A promising class of compounds for DNA transfection have been designed by conjugating various polyamines to bile-acid-based amphiphiles. Formulations containing these compounds were tested for their ability to facilitate the uptake of a beta-galactosidase reporter plasmid into COS-7 cells. Dioleoyl phosphatidyl ethanolamine (DOPE) formulations of some of the compounds were several times better than Lipofectin at promoting DNA uptake. The most active compounds contained the most hydrophilic bile acid components. The activity is clearly not related to affinity for DNA: the hydrophobic bile acid conjugates were found to form stable complexes with DNA at lower charge ratios than the hydrophilic conjugates. We suggest that the high activity of the best compounds is related to their facial amphiphilicity, which may confer an ability to destabilize membranes. The success of these unusual cationic transfection agents may inspire the design of even more effective gene delivery agents.

Topics: Animals; Bile Acids and Salts; Cations; Cell Line, Transformed; Cell Membrane Permeability; Chemical Phenomena; Chemistry, Physical; Chlorocebus aethiops; DNA, Recombinant; Drug Design; Genes, Reporter; Molecular Structure; Particle Size; Phosphatidylethanolamines; Polyamines; Quaternary Ammonium Compounds; Transfection

Conformational disorder in liquid alkenes and in the L alpha and Hparallel phases of some unsaturated phospholipids has been monitored by FTIR spectroscopy. The CH2 wagging region (1330-1390 cm-1) in saturated chains contains vibrations of particular 2- and 3-bond conformational states as follows: 1341 cm-1, end-gauche (eg); 1352 cm-1, double gauche (gg); 1368 cm-1, the sum of kink and gtg states. In unsaturated chains, this spectral region revealed an additional band at 1362 cm-1 and (occasionally) a feature near 1348 cm-1. The 1362 cm-1 band is tentatively assigned to the wagging of CH2 groups adjacent to the C = C bond. Substantial populations of both gg and (kink+gtg) states are evident in the L alpha phases of unsaturated phosphatidylcholines (PC's). Unsaturated phosphatidylethanolamines (PE's) are more ordered than their PC counterparts, and possess fewer gg and eg states. Chain disorder in the Hparallel phase of PE's approaches that in L alpha phases of unsaturated PC's. Changes in conformer distributions during the L alpha-->Hparallel transition in 1,2-dioleoylphosphatidylethanolamine (DOPE), 1-palmitoyl,2-oleoylphosphatidylethanolamine (POPE), 1,2-dielaidoylphosphatidylethanolamine (DEPE), and N-methyl-DOPE(N-MeDOPE) were semi-quantitatively estimated. For DOPE and DEPE, slight cooperative increases in both gg and (kink+gtg) states occur, for POPE only the gg population increases and for N-MeDOPE only the kink+gtg populations increase. These disorder increases are consistent with the small calorimetric delta H for this transition. Difficulties in quantitative determination of conformational disorder in unsaturated chains are discussed.

Topics: Alkenes; Gas Chromatography-Mass Spectrometry; Magnetic Resonance Spectroscopy; Molecular Conformation; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Spectroscopy, Fourier Transform Infrared; Thermodynamics