1-2-oleoylphosphatidylcholine and 1-2-dioleoyloxy-3-(trimethylammonium)propane

1-2-oleoylphosphatidylcholine has been researched along with 1-2-dioleoyloxy-3-(trimethylammonium)propane* in 53 studies

Reviews

1 review(s) available for 1-2-oleoylphosphatidylcholine and 1-2-dioleoyloxy-3-(trimethylammonium)propane

ArticleYear
Structures of lipid-DNA complexes: supramolecular assembly and gene delivery.
    Current opinion in structural biology, 2001, Volume: 11, Issue:4

    Recently, there has been a flurry of experimental work on understanding the supramolecular assemblies that are formed when cationic liposomes (CLs) are mixed with DNA. From a biomedical point of view, CLs (vesicles) are empirically known to be carriers of genes (sections of DNA) in nonviral gene delivery applications. Although viral-based carriers of DNA are presently the most common method of gene delivery, nonviral synthetic methods are rapidly emerging as alternative carriers, because of their ease of production and nonimmunogenicity (viral carriers very often evoke an undesirable and potentially lethal immune response). At the moment, cationic-lipid-based carriers have emerged as the most popular nonviral method to deliver genes in therapeutic applications, for example, CL carriers are used extensively in clinical trials worldwide. However, because the mechanism of transfection (the transfer of DNA into cells by CL carriers, followed by expression) of CL--DNA complexes remains largely unknown, the measured efficiencies are, at present, very low. The low transfection efficiencies of current nonviral gene delivery methods are the result of poorly understood transfection-related mechanisms at the molecular and self-assembled levels. Recently, work has been carried out on determining the supramolecular structures of CL--DNA complexes by the quantitative technique of synchrotron X-ray diffraction. When DNA is mixed with CLs (composed of mixtures of cationic DOTAP and neutral DOPC lipids), the resulting CL--DNA complex consists of a multilamellar structure (L(alpha)(C)) comprising DNA monolayers sandwiched between lipid bilayers. The existence of a different columnar inverted hexagonal (H(II)(C)) phase in CL--DNA complexes was also demonstrated using synchrotron X-ray diffraction. Ongoing functional studies and optical imaging of cells are expected to clarify the relationship between the supramolecular structures of CL--DNA complexes and transfection efficiency.

    Topics: Animals; DNA; Fatty Acids, Monounsaturated; Gene Transfer Techniques; Glycerophospholipids; Humans; Lipid Metabolism; Liposomes; Macromolecular Substances; Microscopy, Atomic Force; Models, Molecular; Phosphatidylcholines; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Transfection; X-Ray Diffraction

2001

Other Studies

52 other study(ies) available for 1-2-oleoylphosphatidylcholine and 1-2-dioleoyloxy-3-(trimethylammonium)propane

ArticleYear
Paclitaxel loading in cationic liposome vectors is enhanced by replacement of oleoyl with linoleoyl tails with distinct lipid shapes.
    Scientific reports, 2021, 03-31, Volume: 11, Issue:1

    Lipid carriers of hydrophobic paclitaxel (PTX) are used in clinical trials for cancer chemotherapy. Improving their loading capacity requires enhanced PTX solubilization. We compared the time-dependence of PTX membrane solubility as a function of PTX content in cationic liposomes (CLs) with lipid tails containing one (oleoyl; DOPC/DOTAP) or two (linoleoyl; DLinPC/newly synthesized DLinTAP) cis double bonds by using microscopy to generate kinetic phase diagrams. The DLin lipids displayed significantly increased PTX membrane solubility over DO lipids. Remarkably, 8 mol% PTX in DLinTAP/DLinPC CLs remained soluble for approximately as long as 3 mol% PTX (the solubility limit, which has been the focus of most previous studies and clinical trials) in DOTAP/DOPC CLs. The increase in solubility is likely caused by enhanced molecular affinity between lipid tails and PTX, rather than by the transition in membrane structure from bilayers to inverse cylindrical micelles observed with small-angle X-ray scattering. Importantly, the efficacy of PTX-loaded CLs against prostate cancer cells (their IC50 of PTX cytotoxicity) was unaffected by changing the lipid tails, and toxicity of the CL carrier was negligible. Moreover, efficacy was approximately doubled against melanoma cells for PTX-loaded DLinTAP/DLinPC over DOTAP/DOPC CLs. Our findings demonstrate the potential of chemical modifications of the lipid tails to increase the PTX membrane loading while maintaining (and in some cases even increasing) the efficacy of CLs. The increased PTX solubility will aid the development of liposomal PTX carriers that require significantly less lipid to deliver a given amount of PTX, reducing side effects and costs.

    Topics: Antineoplastic Agents; Fatty Acids, Monounsaturated; Humans; Linoleic Acids; Liposomes; Oleic Acid; Paclitaxel; PC-3 Cells; Phosphatidylcholines; Quaternary Ammonium Compounds

2021
Tuning liposome composition to modulate corona formation in human serum and cellular uptake.
    Acta biomaterialia, 2020, 04-01, Volume: 106

    Nano-sized objects such as liposomes are modified by adsorption of biomolecules in biological fluids. The resulting corona critically changes nanoparticle behavior at cellular level. A better control of corona composition could allow to modulate uptake by cells. Within this context, in this work, liposomes of different charge were prepared by mixing negatively charged and zwitterionic lipids to different ratios. The series obtained was used as a model system with tailored surface properties to modulate corona composition and determine the effects on liposome interactions with cells. Uptake efficiency and uptake kinetics of the different liposomes were determined by flow cytometry and fluorescence imaging. Particular care was taken in optimizing the methods to isolate the corona forming in human serum to prevent liposome agglomeration and to exclude residual free proteins, which could confuse the results. Thanks to the optimized methods, mass spectrometry of replicate corona isolations showed excellent reproducibility and this allowed semi-quantitative analysis to determine for each formulation the most abundant proteins in the corona. The results showed that by changing the fraction of zwitterionic and charged lipids in the bilayer, the amount and identity of the most abundant proteins adsorbed from serum differed. Interestingly, the formulations also showed very different uptake kinetics. Similar approaches can be used to tune lipid composition in a systematic way in order to obtain formulations with the desired corona and cell uptake behavior. STATEMENT OF SIGNIFICANCE: Liposomes and other nano-sized objects when introduced in biological fluids are known to adsorb biomolecules forming the so-called nanoparticle corona. This layer strongly affects the subsequent interactions of liposomes with cells. Here, by tuning lipid composition in a systematic way, a series of liposomes with tailored surface properties has been prepared to modulate the corona forming in human serum. Liposomes with very different cellular uptake kinetics have been obtained and their corona was identified in order to determine the most enriched proteins on the different formulations. By combining corona composition and uptake kinetics candidate corona proteins associated with reduced or increased uptake by cells can be identified and the liposome formulation can be tuned to obtain the desired uptake behavior.

    Topics: Adsorption; Animals; Blood Proteins; Cattle; Fatty Acids, Monounsaturated; Humans; Liposomes; Phosphatidylcholines; Phosphatidylethanolamines; Protein Corona; Quaternary Ammonium Compounds

2020
Enveloped artificial viral capsids self-assembled from anionic β-annulus peptide and cationic lipid bilayer.
    Chemical communications (Cambridge, England), 2020, Jul-04, Volume: 56, Issue:52

    Anionic artificial viral capsids were self-assembled from β-annulus-EE peptide, then complexed with lipid-bilayer-containing cationic lipids via electrostatic interaction to form enveloped artificial viral capsids. The critical aggregation concentration of the enveloped artificial viral capsid was significantly lower than that of the uncomplexed artificial viral capsid, indicating that the lipid bilayer stabilised the capsid structure.

    Topics: Anions; Capsid; Cations; Fatty Acids, Monounsaturated; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Humans; Lipid Bilayers; Nanoparticles; Peptides; Phosphatidylcholines; Quaternary Ammonium Compounds; Static Electricity; Viral Envelope Proteins; Virus Assembly; Viruses

2020
Improved Atomic Force Microscopy Stiffness Measurements of Nanoscale Liposomes by Cantilever Tip Shape Evaluation.
    Analytical chemistry, 2019, 08-20, Volume: 91, Issue:16

    The stiffness of nanoscale liposomes, as measured by atomic force microscopy (AFM), was investigated as a function of temperature, immobilization on solid substrates, and cantilever tip shape. The liposomes were composed of saturated lipids and cholesterol, and the stiffness values did not change over the temperature range of 25-37 °C and were independent of immobilization methods. However, the stiffness varied with the tip shape of the cantilever. Therefore, 24 cantilevers were evaluated in terms of tip shape and aspect ratio (length/width) via a nonblind tip reconstruction (NBTR) method that used a tip characterizer with isolated line structures having specified dimensions. A standard for screening the tip geometry was established. A 24-fold improvement in stiffness precision in terms of relative standard deviation was demonstrated by using at least three cantilevers that meet the criteria of having a tip aspect ratio greater than 2.5 and a quadratic tip shape function. A significant difference in stiffness was subsequently revealed between dipalmitoylphosphatidylcholine-cholesterol (1:1 molar ratio) and egg yolk phosphatidylcholine-cholesterol (1:1 molar ratio) liposomes. Tip analysis using NBTR improved the precision of AFM stiffness measurements, which will enable the control of mechanical properties of nanoscale liposomes for various applications.

    Topics: 1,2-Dipalmitoylphosphatidylcholine; Biotin; Cholesterol; Fatty Acids, Monounsaturated; Glass; Liposomes; Microscopy, Atomic Force; Phosphatidylcholines; Phosphatidylglycerols; Quaternary Ammonium Compounds; Streptavidin; Temperature; Water

2019
Effect of surface charge on the size-dependent cellular internalization of liposomes.
    Chemistry and physics of lipids, 2019, Volume: 224

    Here we report that the size dependence of cellular internalization of liposomes differs depending on the surface charge. We prepared liposomes of various lipid compositions ranging from 100 to 200 nm size. It was found that cationic liposomes composed of 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) were most effectively internalized into cells when their mean particle sizes were around 180 nm. When their size was reduced to around 90 nm, the level of internalization reduced six-fold. Conversely, hydrogenated soy phosphatidylcholine (HSPC)/N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (PEG2000-DSPE)/cholesterol(Chol) liposomes, HSPC/PEG2000-DSPE liposomes, and HSPC/Chol liposomes were most readily internalized when they were around 110 to 130 nm in mean particle size. Unlike DOPC/DOTAP liposomes the difference between the maximum and minimum levels of internalization was less than two-fold. It has been suggested that strong electrostatic interactions between cationic liposomes and the negatively charged plasma membrane affect the size dependence and optimal size range for internalization of liposomes. Size dependence of internalization should be carefully monitored for effective formulation development and quality control of liposome drug products.

    Topics: Cations; Cell Membrane Permeability; Cholesterol; Fatty Acids, Monounsaturated; Hep G2 Cells; Humans; Liposomes; Molecular Structure; Particle Size; Phosphatidylcholines; Phosphatidylethanolamines; Polyethylene Glycols; Quaternary Ammonium Compounds; Static Electricity; Structure-Activity Relationship; Surface Properties

2019
Control of Membrane Binding and Diffusion of Cholesteryl-Modified DNA Origami Nanostructures by DNA Spacers.
    Langmuir : the ACS journal of surfaces and colloids, 2018, 12-11, Volume: 34, Issue:49

    DNA origami nanotechnology is being increasingly used to mimic membrane-associated biophysical phenomena. Although a variety of DNA origami nanostructures has already been produced to target lipid membranes, the requirements for membrane binding have so far not been systematically assessed. Here, we used a set of elongated DNA origami structures with varying placement and number of cholesteryl-based membrane anchors to compare different strategies for their incorporation. Single and multiple cholesteryl anchors were attached to DNA nanostructures using single- and double-stranded DNA spacers of varying length. The produced DNA nanostructures were studied in terms of their membrane binding and diffusion. Our results show that the location and number of anchoring moieties play a crucial role for membrane binding of DNA nanostructures mainly if the cholesteryl anchors are in close proximity to the bulky DNA nanostructures. Moreover, the use of DNA spacers largely overcomes local steric hindrances and thus enhances membrane binding. Fluorescence correlation spectroscopy measurements demonstrate that the distinct physical properties of single- and double-stranded DNA spacers control the interaction of the amphipathic DNA nanostructures with lipid membranes. Thus, we provide a rational basis for the design of amphipathic DNA origami nanostructures to efficiently bind lipid membranes in various environments.

    Topics: Cholesterol; Diffusion; DNA, Single-Stranded; Fatty Acids, Monounsaturated; Molecular Structure; Nanostructures; Nucleic Acid Conformation; Phosphatidylcholines; Phosphatidylserines; Polyethylene Glycols; Quaternary Ammonium Compounds; Unilamellar Liposomes

2018
Salt-induced reentrant stability of polyion-decorated particles with tunable surface charge density.
    Colloids and surfaces. B, Biointerfaces, 2016, Jan-01, Volume: 137

    The electrostatic complexation between DOTAP/DOPC unilamellar liposomes and an oppositely charged polyelectrolyte (NaPA) has been investigated in a wide range of the liposome surface charge density. We systematically characterized the reentrant condensation and the charge inversion of polyelectrolyte-decorated liposomes by means of dynamic light scattering and electrophoresis. We explored the stability of this model polyelectrolyte/colloid system at different values of the surface charge of the bare liposomes and by changing two independent control parameters of the suspensions: the polyelectrolyte/colloid charge ratio and the ionic strength of the aqueous suspending medium. The progressive addition of neutral DOPC lipid within the liposome membrane gave rise to an interesting phenomenon which has not been observed previously: the stability diagram of the suspensions showed a novel reentrance due to the crossing of the desorption threshold of the polyelectrolyte. Indeed, at fixed charge density of the bare DOTAP/DOPC liposomes and for a wide range of polyion concentrations, we showed that the simple electrolyte addition first (low salt regime) destabilizes the suspensions because of the enhanced screening of the residual repulsion between the complexes, and then (high salt regime) determines the onset of a new stable phase, originated by the absence of polyelectrolyte adsorption on the particle surfaces. We show that the observed phenomenology can be rationalized within the modified Velegol-Thwar model for heterogeneously charged particles and that the polyelectrolyte desorption fits well the predictions of the adsorption theory of Winkler and Cherstvy [1]. Our findings unambiguously support the picture of the reentrant condensation as driven by the correlated adsorption of the polyelectrolyte chains on the particle surface, providing interesting insights into possible mechanisms for tailoring complex colloids via salt-induced effects.

    Topics: Fatty Acids, Monounsaturated; Ions; Liposomes; Phosphatidylcholines; Quaternary Ammonium Compounds; Salts; Surface Properties

2016
What the cell surface does not see: The gene vector under the protein corona.
    Colloids and surfaces. B, Biointerfaces, 2016, May-01, Volume: 141

    The fate of lipid-based nanovectors, used in genetic targeting inside cells, depends on their behavior in biological media. In fact, during both in vitro and in vivo transfection, nanovectors come in contact with proteins that compete for their surface and build the protein corona, their true biological identity while engaging the cell membrane. Nonetheless, after cell internalization, the efficacy of transfection may depend also on structural modifications that occurred under the protein cover, following interaction with biological fluids. Here, based on previous in vivo experiments, two widely used lipid mixtures, namely DOTAP/DOPC and DC-Chol/DOPE, were identified as paradigms to investigate the impact of the inner structure of nanovectors on the transfection efficiency, all being proficiently internalized. The evolution of the inner structure of cationic lipoplexes and nanoparticles based on such lipid mixtures, following interaction with human plasma, could be unraveled. Particles were investigated in high dilution, approaching the biosimilar conditions. Data have demonstrated that the modulation of their inner structure depends on their lipid composition and the plasma concentration, still preserving the genetic payload. Interestingly, protein contact induces a variety of inner structures with different perviousness, including reshaping into cubic phases of different porosity, sometimes observed upon interaction between carrier-lipids and cell-lipids. Cubic reshaping is of biological relevance, as lipid cubic phases have been recently associated to both fusogenicity and to the readiness in releasing the payload to the final target via endosomal escape.

    Topics: Cations; Cell Membrane; Cholesterol; DNA; Fatty Acids, Monounsaturated; Genetic Vectors; Humans; Lipids; Liposomes; Nanoparticles; Phosphatidylcholines; Phosphatidylethanolamines; Plasma; Protein Corona; Quaternary Ammonium Compounds; Scattering, Small Angle; Transfection; X-Ray Diffraction

2016
Quantitative Monitoring of Microphase Separation Behaviors in Cationic Liposomes Using HHC, DPH, and Laurdan: Estimation of the Local Electrostatic Potentials in Microdomains.
    Langmuir : the ACS journal of surfaces and colloids, 2016, Apr-19, Volume: 32, Issue:15

    Microphase separation behaviors of cationic liposomes have been investigated using a pH-sensitive fluorescent probe with 4-heptadecyl-7-hydroxycoumarin (HHC), 1,6-diphenyl-1,3,5-hexatriene, and 6-lauroyl-2-dimethylaminonaphthalene, and to estimate localized electrostatic potentials. Shifts of the apparent pKa values of HHC were observed in cationic liposomes in proportion to the amount of cationic lipids. Two pKa values were obtained with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/3β-[N(N',N'-dimethylaminoethane)-carbamoyl] cholesterol hydrochloride (DC-Ch) liposomes, while only one pKa value was generated with either DOPC/1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or DOPC/dimethyldioctadecylammonium-bromide (DODAB) liposomes. The physicochemical membrane property analyses, focusing on membrane fluidity and membrane polarity, revealed heterogeneity among DOPC/DC-Ch liposomes. By analyzing the pH titration curves using sigmoidal fitting, the localized electrostatic potentials were estimated. For DOPC/DOTAP = (7/3), the membrane was in the liquid-disordered phase and the density of cationic molecules was 0.41 cation/nm(2). For DOPC/DC-Ch = (7/3), the membrane was heterogeneous and the densities of cationic molecules in liquid-disordered and liquid-ordered phases were 0.25 and 1.24 cation/nm(2), respectively. We thereby conclude that the DC-Ch molecules can form nanodomains when these molecules are concentrated to 59%.

    Topics: 2-Naphthylamine; Cholesterol; Diphenylhexatriene; Fatty Acids, Monounsaturated; Fluorescent Dyes; Hydrogen-Ion Concentration; Laurates; Liposomes; Membrane Fluidity; Phosphatidylcholines; Quaternary Ammonium Compounds; Spectrometry, Fluorescence; Umbelliferones

2016
Self-assembly of size-controlled liposomes on DNA nanotemplates.
    Nature chemistry, 2016, Volume: 8, Issue:5

    Artificial lipid-bilayer membranes are valuable tools for the study of membrane structure and dynamics. For applications such as the study of vesicular transport and drug delivery, there is a pressing need for artificial vesicles with controlled size. However, controlling vesicle size and shape with nanometre precision is challenging, and approaches to achieve this can be heavily affected by lipid composition. Here, we present a bio-inspired templating method to generate highly monodispersed sub-100-nm unilamellar vesicles, where liposome self-assembly was nucleated and confined inside rigid DNA nanotemplates. Using this method, we produce homogeneous liposomes with four distinct predefined sizes. We also show that the method can be used with a variety of lipid compositions and probe the mechanism of templated liposome formation by capturing key intermediates during membrane self-assembly. The DNA nanotemplating strategy represents a conceptually novel way to guide lipid bilayer formation and could be generalized to engineer complex membrane/protein structures with nanoscale precision.

    Topics: DNA; Fatty Acids, Monounsaturated; Lipid Bilayers; Nanostructures; Particle Size; Phosphatidylcholines; Phosphatidylserines; Polyethylene Glycols; Quaternary Ammonium Compounds; Unilamellar Liposomes

2016
DMSO induces dehydration near lipid membrane surfaces.
    Biophysical journal, 2015, Jul-21, Volume: 109, Issue:2

    Dimethyl sulfoxide (DMSO) has been broadly used in biology as a cosolvent, a cryoprotectant, and an enhancer of membrane permeability, leading to the general assumption that DMSO-induced structural changes in cell membranes and their hydration water play important functional roles. Although the effects of DMSO on the membrane structure and the headgroup dehydration have been extensively studied, the mechanism by which DMSO invokes its effect on lipid membranes and the direct role of water in this process are unresolved. By directly probing the translational water diffusivity near unconfined lipid vesicle surfaces, the lipid headgroup mobility, and the repeat distances in multilamellar vesicles, we found that DMSO exclusively weakens the surface water network near the lipid membrane at a bulk DMSO mole fraction (XDMSO) of <0.1, regardless of the lipid composition and the lipid phase. Specifically, DMSO was found to effectively destabilize the hydration water structure at the lipid membrane surface at XDMSO <0.1, lower the energetic barrier to dehydrate this surface water, whose displacement otherwise requires a higher activation energy, consequently yielding compressed interbilayer distances in multilamellar vesicles at equilibrium with unaltered bilayer thicknesses. At XDMSO >0.1, DMSO enters the lipid interface and restricts the lipid headgroup motion. We postulate that DMSO acts as an efficient cryoprotectant even at low concentrations by exclusively disrupting the water network near the lipid membrane surface, weakening the cohesion between water and adhesion of water to the lipid headgroups, and so mitigating the stress induced by the volume change of water during freeze-thaw.

    Topics: 1,2-Dipalmitoylphosphatidylcholine; Diffusion; Dimethyl Sulfoxide; Fatty Acids, Monounsaturated; Magnetic Resonance Spectroscopy; Membranes, Artificial; Phosphatidylcholines; Phosphatidylglycerols; Quaternary Ammonium Compounds; Scattering, Small Angle; Water; X-Ray Diffraction

2015
Changes in Interfacial Tension of a Lipid Membrane Formed at the Water/Chloroform Interface upon DNA Complex Formation.
    Analytical sciences : the international journal of the Japan Society for Analytical Chemistry, 2015, Volume: 31, Issue:10

    Changes in the interfacial tension of a lipid monolayer membrane formed at the water/chloroform interface upon DNA addition were measured using the quasi-elastic laser scattering (QELS) method. A cationic lipid, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP), as well as zwitterionic lipids, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), were used to form lipid monolayer membranes at different calcium ion concentrations. A rapid decrease of the interfacial tension resulting from electrostatic interactions between DOTAP and DNA was observed within 10 s. However, such rapid decreases were not observed for DOPE or DOPC. A decrease in the interfacial tension was exhibited by DOPE after 1000 s from the addition of DNA, which may be due to an overall structural change in the DOPE membrane. A DOTAP/DOPE complex system showed behaviors attributable to both DOTAP and DOPE, whereas the behavior of the DOTAP/DOPC system resembled that of DOPC alone. The current results provide a model for the so-called lipoplex carriers used in gene therapy.

    Topics: Calcium Chloride; Cell Membrane; Chloroform; DNA; Fatty Acids, Monounsaturated; Phosphatidylcholines; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Surface Tension; Water

2015
PEGylated cationic liposome-DNA complexation in brine is pathway-dependent.
    Biochimica et biophysica acta, 2014, Volume: 1838, Issue:1 Pt B

    Cationic liposome-DNA (CL-DNA) complexes, are regarded as promising materials for safe and efficient delivery of genes for therapeutical applications. In order to be used in vivo, these complexes may be coated with a hydrophilic polymer (e.g. polyethylene-glycol, PEG) that provides steric stabilization towards adhesion of proteins and removal by the immune system. In this work we study the influence of the initial salt concentration (Cs) - which modulates the electrostatic interaction between oppositely charged vesicles and DNA - on the structure and stability of PEGylated CL-DNA particles. Previous small-angle X-ray scattering has shown that if non-PEGylated or PEGylated CL-DNA lamellar complexes are prepared in water, their structure is well defined with a high number of lipid membrane-DNA layers (larger than 20). Here we show that if these complexes are transferred to saline media (150mM NaCl or DMEM, both near physiological conditions), this structure remains nearly unchanged. Conversely, if PEGylated complexes are prepared in saline media, their lamellar structure is much looser, with fewer number of layers. This pathway dependent behavior of PEGylated complex formation in brine is modulated by the liposome membrane charge density and the mole fraction of PEG 2000 in the membranes, with the average number of layers decreasing with increasing Cs and in going from 5mol% to 10mol% PEG-lipid. Each of these structures (high and low number of layers) is stable with time, suggesting that despite complex formation being thermodynamically favored, the complexation process in PEGylated membranes, which determines the number of layers per particle, is kinetically controlled. In the extreme case (when polymer repulsions from 10mol% PEG-lipid are maximized and electrostatic attraction between PEGylated CLs and DNA are minimized at low membrane charge density) complex formation is suppressed at high Cs=150mM.

    Topics: Animals; Cations; Cattle; DNA; Fatty Acids, Monounsaturated; Gene Transfer Techniques; Hydrophobic and Hydrophilic Interactions; Liposomes; Phosphatidylcholines; Polyethylene Glycols; Quaternary Ammonium Compounds; Salts; Scattering, Small Angle; Static Electricity; Thermodynamics; X-Ray Diffraction

2014
Intra-lymph node injection of biodegradable polymer particles.
    Journal of visualized experiments : JoVE, 2014, Jan-02, Issue:83

    Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these foreign molecules to T and B lymphocytes. Lymph nodes have thus become critical targets for new vaccines and immunotherapies. A recent strategy for targeting these tissues is direct lymph node injection of soluble vaccine components, and clinical trials involving this technique have been promising. Several biomaterial strategies have also been investigated to improve lymph node targeting, for example, tuning particle size for optimal drainage of biomaterial vaccine particles. In this paper we present a new method that combines direct lymph node injection with biodegradable polymer particles that can be laden with antigen, adjuvant, or other vaccine components. In this method polymeric microparticles or nanoparticles are synthesized by a modified double emulsion protocol incorporating lipid stabilizers. Particle properties (e.g. size, cargo loading) are confirmed by laser diffraction and fluorescent microscopy, respectively. Mouse lymph nodes are then identified by peripheral injection of a nontoxic tracer dye that allows visualization of the target injection site and subsequent deposition of polymer particles in lymph nodes. This technique allows direct control over the doses and combinations of biomaterials and vaccine components delivered to lymph nodes and could be harnessed in the development of new biomaterial-based vaccines.

    Topics: Animals; Biocompatible Materials; Drug Delivery Systems; Fatty Acids, Monounsaturated; Lymph Nodes; Mice; Particle Size; Phosphatidylcholines; Phosphatidylethanolamines; Polyethylene Glycols; Quaternary Ammonium Compounds

2014
Intracellular trafficking mechanism of cationic phospholipids including cationic liposomes in HeLa cells.
    Die Pharmazie, 2014, Volume: 69, Issue:7

    The development of gene delivery methods is essential for the achievement of effective gene therapy. Elucidation of the intracellular transfer mechanism for cationic carriers is in progress, but there are few reports regarding the intracellular trafficking processes of the cationic phospholipids taken up into cells. In the present work, the trafficking processes of a cationic phospholipid (1,2-dioleoyl-3-trimethylammonium-propane, DOTAP) were investigated from intracellular uptake to extracellular efflux using cationic liposomes in vitro. Following intracellular transport of liposomes via endocytosis, DOTAP was localized in the endoplasmic reticulum, Golgi apparatus, and mitochondria. Moreover, the proteins involved in DOTAP intracellular trafficking and extracellular efflux were identified. In addition, helper lipids of cationic liposomes were found to partially affect this intracellulartrafficking. These findings might provide valuable information for designing cationic carriers and avoiding unexpected toxic side effects derived from cationic liposomal components.

    Topics: ATP-Binding Cassette Transporters; Cations; Electrochemistry; Endocytosis; Endoplasmic Reticulum; Fatty Acids, Monounsaturated; Gene Transfer Techniques; Golgi Apparatus; HeLa Cells; Humans; Intracellular Space; Liposomes; Microscopy, Confocal; Mitochondria; Nucleic Acids; Particle Size; Phosphatidylcholines; Phospholipids; Quaternary Ammonium Compounds; RNA, Small Interfering

2014
The role of the helper lipid on the DNA transfection efficiency of lipopolyplex formulations.
    Scientific reports, 2014, Nov-19, Volume: 4

    Multifunctional, lipopolyplex formulations comprising a mixture of cationic liposomes and cationic, receptor-targeting peptides have potential use in gene therapy applications. Lipopolyplex formulations described here are typically far more efficient transfection agents than binary lipoplex or polyplex formulations. It has been shown previously that the peptide component mediates both DNA packaging and targeting of the nanoparticle while in this report we investigate the contribution of the lipid component. We hypothesised that the lipid components synergise with the peptides in the transfection process by promoting endosomal escape after lipid bilayer fusion. Lipopolyplexes were prepared with cationic liposomes comprising DOTAP with either neutral lipid DOPE or DOPC. DOPE promotes fusogenic, inverted hexagonal lipid structures while DOPC promotes more stable laminar structures. Lipopolyplexes containing DOPE showed substantially higher transfection efficiency than those formulated with DOPC, both in vitro and in vivo. DOPE-containing lipopolyplexes showed rapid endosomal trafficking and nuclear accumulation of DNA while DOPC-containing formulations remained within the late endo-lysosomal compartments. These findings are consistent with previous finding for the role of DOPE in lipoplexes and support the hypothesis regarding the function of the lipid components in lipopolyplexes. These findings will help to inform future lipopolyplex design, strategies and clinical development processes.

    Topics: Animals; Cell Line; Cell Nucleus; DNA; Endosomes; Fatty Acids, Monounsaturated; Female; Humans; Lipid Bilayers; Liposomes; Lung; Membrane Fusion; Mice; Peptides; Phosphatidylcholines; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Respiratory Mucosa; Transfection

2014
Does fluoride disrupt hydrogen bond network in cationic lipid bilayer? Time-dependent fluorescence shift of Laurdan and molecular dynamics simulations.
    The Journal of chemical physics, 2014, Dec-14, Volume: 141, Issue:22

    Time-dependent fluorescence shift (TDFS) of Laurdan embedded in phospholipid bilayers reports on hydration and mobility of the phospholipid acylgroups. Exchange of H2O with D2O prolongs the lifetime of lipid-water and lipid-water-lipid interactions, which is reflected in a significantly slower TDFS kinetics. Combining TDFS measurements in H2O and D2O hydrated bilayers with atomistic molecular dynamics (MD) simulations provides a unique tool for characterization of the hydrogen bonding at the acylgroup level of lipid bilayers. In this work, we use this approach to study the influence of fluoride anions on the properties of cationic bilayers composed of trimethylammonium-propane (DOTAP). The results obtained for DOTAP are confronted with those for neutral phosphatidylcholine (DOPC) bilayers. Both in DOTAP and DOPC H2O/D2O exchange prolongs hydrogen-bonding lifetime and does not disturb bilayer structure. These results are confirmed by MD simulations. TDFS experiments show, however, that for DOTAP this effect is cancelled in the presence of fluoride ions. We interpret these results as evidence that strongly hydrated fluoride is able to steal water molecules that bridge lipid carbonyls. Consequently, when attracted to DOTAP bilayer, fluoride disrupts the local hydrogen-bonding network, and the differences in TDFS kinetics between H2O and D2O hydrated bilayers are no longer observed. A distinct behavior of fluoride is also evidenced by MD simulations, which show different lipid-ion binding for Cl(-) and F(-).

    Topics: 2-Naphthylamine; Fatty Acids, Monounsaturated; Fluorescent Dyes; Fluorides; Hydrogen Bonding; Laurates; Lipid Bilayers; Molecular Dynamics Simulation; Phosphatidylcholines; Quaternary Ammonium Compounds; Water

2014
Mesoporous silica-supported lipid bilayers (protocells) for DNA cargo delivery to the spinal cord.
    Journal of controlled release : official journal of the Controlled Release Society, 2013, Jun-10, Volume: 168, Issue:2

    Amorphous mesoporous silica nanoparticles ('protocells') that support surface lipid bilayers recently characterized in vitro as carrier constructs for small drug and DNA delivery are reported here as highly biocompatible both in vitro and in vivo, involving the brain and spinal cord following spinal delivery into the lumbosacral subarachnoid space (intrathecal; i.t.). Specifically, positively charged, 1, 2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP)-cholesterol (DOTAP:Chol) liposome-formulated protocells revealed stable in vitro cargo release kinetics and cellular interleukin-10 (IL-10) transgene transfection. Recent approaches using synthetic non-viral vector platforms to deliver the pain-suppressive therapeutic transgene, IL-10, to the spinal subarachnoid space have yielded promising results in animal models of peripheral neuropathy, a condition involving aberrant neuronal communication within sensory pathways in the nervous system. Non-viral drug and gene delivery protocell platforms offer potential flexibility because cargo release-rates can be pH-dependent. We report here that i.t. delivery of protocells, with modified chemistry supporting a surface coating of DOTAP:Chol liposomes and containing the IL-10 transgene, results in functional suppression of pain-related behavior in rats for extended periods. This study is the first demonstration that protocell vectors offer amenable and enduring in vivo biological characteristics that can be applied to spinal gene delivery.

    Topics: Animals; Artificial Cells; Cell Line; Cell Survival; Cholesterol; DNA; Fatty Acids, Monounsaturated; Gene Transfer Techniques; HEK293 Cells; Humans; Injections, Spinal; Interleukin-10; Lipid Bilayers; Liposomes; Male; Mice; Nitric Oxide; Phosphatidylcholines; Plasmids; Quaternary Ammonium Compounds; Rats; Rats, Sprague-Dawley; Sciatic Nerve; Silicon Dioxide; Spinal Cord

2013
Influence of charge density on bilayer bending rigidity in lipid vesicles: a combined dynamic light scattering and neutron spin-echo study.
    The European physical journal. E, Soft matter, 2013, Volume: 36, Issue:7

    We report a combined dynamic light scattering and neutron spin-echo study on vesicles composed of the uncharged stabilizing lipid 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) and the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). Mechanical properties of a model membrane and thus the corresponding bilayer undulation dynamics can be specifically tuned by changing its composition through lipid headgroup or acyl chain properties. We compare the undulation dynamics in lipid vesicles composed of DMPC/DOTAP to vesicles composed of a mixture of the uncharged helper lipid DMPC with the also uncharged reference lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). We have performed dynamic light scattering on the lipid mixtures to investigate changes in lipid vesicle size and the corresponding center-of-mass diffusion. We study lipid translational diffusion in the membrane plane and local bilayer undulations using neutron spin-echo spectroscopy, on two distinct time scales, namely around 25 ns and around 150 ns. Finally, we calculate the respective bilayer bending rigidities κ for both types of lipid vesicles. We find that on the local length scale inserting lipid headgroup charge into the membrane influences the bilayer undulation dynamics and bilayer bending rigidity κ less than inserting lipid acyl chain unsaturation: We observe a bilayer softening with increasing inhomogenity of the lipid mixture, which could be caused by a hydrophobic mismatch between the acyl chains of the respective lipid components, causing a lateral phase segregation (domain formation) in the membrane plane.

    Topics: Diffusion; Dimyristoylphosphatidylcholine; Fatty Acids, Monounsaturated; Glycerylphosphorylcholine; Lipid Bilayers; Liposomes; Neutron Diffraction; Phosphatidylcholines; Quaternary Ammonium Compounds; Spectrum Analysis, Raman; Static Electricity

2013
Mechanistic understanding of gene delivery mediated by highly efficient multicomponent envelope-type nanoparticle systems.
    Molecular pharmaceutics, 2013, Dec-02, Volume: 10, Issue:12

    We packaged condensed DNA/protamine particles in multicomponent envelope-type nanoparticle systems (MENS) combining different molar fractions of the cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic lipids dioleoylphosphocholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE). Dynamic light scattering (DLS) and microelectrophoresis allowed us to identify the cationic lipid/DNA charge ratio at which MENS are small sized and positively charged, while synchrotron small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM) revealed that MENS are well-shaped DNA/protamine particles covered by a lipid monobilayer. Transfection efficiency (TE) experiments indicate that a nanoparticle formulation, termed MENS-3, was not cytotoxic and highly efficient to transfect Chinese hamster ovary (CHO) cells. To rationalize TE, we performed a quantitative investigation of cell uptake, intracellular trafficking, endosomal escape, and final fate by laser scanning confocal microscopy (LSCM). We found that fluid-phase macropinocytosis is the only endocytosis pathway used by MENS-3. Once taken up by the cell, complexes that are actively transported by microtubules frequently fuse with lysosomes, while purely diffusing systems do not. Indeed, spatiotemporal image correlation spectroscopy (STICS) clarified that MENS-3 mostly exploit diffusion to move in the cytosol of CHO cells, thus explaining the high TE levels observed. Also, MENS-3 exhibited a marked endosomal rupture ability resulting in extraordinary DNA release. The lipid-dependent and structure-dependent TE boost suggests that efficient transfection requires both the membrane-fusogenic activity of the nanocarrier envelope and the employment of lipid species with intrinsic endosomal rupture ability.

    Topics: Animals; CHO Cells; Cholesterol; Cricetulus; DNA; Endocytosis; Endosomes; Fatty Acids, Monounsaturated; Gene Transfer Techniques; Lipids; Liposomes; Nanoparticles; Phosphatidylcholines; Phosphatidylethanolamines; Protamines; Quaternary Ammonium Compounds; Transfection

2013
Calcium phosphate nanoparticles with an asymmetric lipid bilayer coating for siRNA delivery to the tumor.
    Journal of controlled release : official journal of the Controlled Release Society, 2012, Feb-28, Volume: 158, Issue:1

    Calcium phosphate (CaP) nanoparticles (NP) with an asymmetric lipid bilayer coating have been designed for targeted delivery of siRNA to the tumor. An anionic lipid, dioleoylphosphatydic acid (DOPA), was employed as the inner leaflet lipid to coat the nano-size CaP cores, which entrap the siRNA, such that the coated cores were soluble in organic solvent. A suitable neutral or cationic lipid was used as the outer leaflet lipid to form an asymmetric lipid bilayer structure verified by the measurement of NP zeta potential. The resulting NP was named LCP-II with a size of about 25 to 30nm in diameter and contained a hollow core as revealed by TEM imaging. PEGylation of NP was done by including a PEG-phospholipid conjugate, with or without a targeting ligand anisamide, in the outer leaflet lipid mixture. The sub-cellular distribution studied in the sigma receptor positive human H460 lung cancer cells indicated that LCP-II could release more cargo to the cytoplasm than our previous lipid/protamine/DNA (LPD) formulation, leading to a significant (~40 fold in vitro and ~4 fold in vivo) improvement in siRNA delivery. Bio-distribution study showed that LCP-II required more PEGylation for MPS evasion than the previous LPD, probably due to increased surface curvature in LCP-II.

    Topics: Animals; Calcium Phosphates; Cell Line, Tumor; Fatty Acids, Monounsaturated; Female; Gene Silencing; Gene Transfer Techniques; Humans; Lipid Bilayers; Mice; Mice, Nude; Nanoparticles; Neoplasms; Phosphatidic Acids; Phosphatidylcholines; Quaternary Ammonium Compounds; RNA, Small Interfering

2012
Cholesterol-dependent macropinocytosis and endosomal escape control the transfection efficiency of lipoplexes in CHO living cells.
    Molecular pharmaceutics, 2012, Feb-06, Volume: 9, Issue:2

    Here we investigate the cellular uptake mechanism and final intracellular fate of two cationic liposome formulations characterized by similar physicochemical properties but very different lipid composition and efficiency for intracellular delivery of DNA. The first formulation is made of cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic helper dioleoylphosphocholine (DOPC), while the second one is made of the cationic 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic lipid dioleoylphosphatidylethanolamine (DOPE). Combining pharmacological and imaging approaches we show that both DOTAP-DOPC/DNA and DC-Chol-DOPE/DNA lipoplexes are taken up in Chinese hamster ovary (CHO) living cells mainly through fluid-phase macropinocytosis. Our results also indicate that lipoplex macropinocytosis is a cholesterol-sensitive uptake mechanism. On the other side, both clathrin-mediated and caveolae-mediated endocytosis play a minor role, if any, in the cell uptake. Colocalization of fluorescently tagged lipoplexes and Lysosensor, a primary lysosome marker, reveals that poorly efficient DOTAP-DOPC/DNA lipoplexes are largely degraded in the lysosomes, while efficient DC-Chol-DOPE/DNA systems can efficiently escape from endosomal compartments.

    Topics: Animals; CHO Cells; Cholesterol; Cricetinae; Cricetulus; DNA; Endosomes; Fatty Acids, Monounsaturated; Fluorescent Dyes; Liposomes; Microscopy, Confocal; Phosphatidylcholines; Phosphatidylethanolamines; Pinocytosis; Quaternary Ammonium Compounds; Transfection

2012
Transport of actin-decorated liposomes along myosin molecules in vitro.
    Biochemical and biophysical research communications, 2012, May-25, Volume: 422, Issue:1

    We examined whether actin filaments bound to positively charged liposomes could interact with myosin molecules and induce liposome motility. When liposomes were constructed from the mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cationic N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium (DOTAP), actin filaments bound to the liposomes. The actin-bound liposomes exhibited movement on myosin molecules in the presence of adenosine-5'-triphosphate (ATP). The displacement was almost linearly increased with time and the behavior differed from that of Brownian motion. Furthermore, the presence of 30% DOTAP in liposomes was most effective for transport. These data show that the actomyosin system was successfully integrated into the liposomes and possesses the ability to actively transport useful agents enclosed within the liposomes.

    Topics: Actin Cytoskeleton; Actins; Actomyosin; Adenosine Triphosphate; Fatty Acids, Monounsaturated; Hydrolysis; Liposomes; Myosin Subfragments; Myosins; Phosphatidylcholines; Quaternary Ammonium Compounds

2012
Supramolecular organization in self-assembly of chromatin and cationic lipid bilayers is controlled by membrane charge density.
    Biomacromolecules, 2012, Dec-10, Volume: 13, Issue:12

    In this work we have investigated the structures of aggregates formed in model systems of dilute aqueous mixtures of "model chromatin" consisting of either recombinant nucleosome core particles (NCPs) or nucleosome arrays consisting of 12 NCPs connected with 30 bp linker DNA, and liposomes made from different mixtures of cationic and zwitterionic lipids, 1,2-dioleoyl-3-trimethylammonium-propane chloride salt (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). The aggregates formed were characterized using different optical microscopy methods and small-angle X-ray scattering (SAXS), and the results are discussed in terms of the competing intermolecular interactions among the components. For a majority of the samples, the presence of lamellar structures could be identified. In samples with high fractions of DOTAP in the liposomes, well-defined lamellar structures very similar to those formed by the corresponding lipid mixtures and DNA alone (i.e., without histone proteins) were observed; in these aggregates, the histones are expelled from the model chromatin. The findings suggest that, with liposomes containing large fractions of cationic lipid, the dominating driving force for aggregation is the increase in translational entropy from the release of counterions, whereas with lower fractions of the cationic lipid, the entropy of mixing of the lipids within the bilayers results in a decreased DNA-lipid attraction.

    Topics: Cations; Chromatin; Chromatin Assembly and Disassembly; DNA; Fatty Acids, Monounsaturated; Histones; Lipid Bilayers; Liposomes; Microscopy, Confocal; Microscopy, Fluorescence; Nanotechnology; Nucleosomes; Phosphatidylcholines; Quaternary Ammonium Compounds; Recombinant Proteins; Scattering, Small Angle; X-Rays

2012
Dynamics and state of lipid bilayer-internal water unraveled with solution state 1H dynamic nuclear polarization.
    Physical chemistry chemical physics : PCCP, 2011, May-07, Volume: 13, Issue:17

    The dynamics and state of lipid bilayer-internal hydration water of unilamellar lipid vesicles dispersed in solutions is characterized. This study was enabled by a recently developed technique based on Overhauser dynamic nuclear polarization (DNP)-driven amplification of (1)H nuclear magnetic resonance (NMR) signal of hydration water. This technique can, in the full presence of bulk water, selectively quantify the translational dynamics of hydration water within ∼10 Å around spin labels that are specifically introduced to the local volume of interest within the lipid bilayer. With this approach, the local apparent diffusion coefficients of internal water at different depths of the lipid bilayer were determined. The modulation of these values as a response to external stimuli, such as the addition of sodium chloride or ethanol and the lipid phase transitions, that alter the fluctuations of bilayer interfaces together with the activation energy values of water diffusivity shows that water is not individually and homogeneously solvating lipid's hydrocarbon tails in the lipid bilayer. We provide experimental evidence that instead, water and the lipid membrane comprise a heterogeneous system whose constituents include transient hydrophobic water pores or water structures traversing the lipid bilayer. We show how these transient pore structures, as key vehicles for passive water transport can better reconcile our experimental data with existing literature data on lipid bilayer hydration and dynamics.

    Topics: 1,2-Dipalmitoylphosphatidylcholine; Diffusion; Fatty Acids, Monounsaturated; Kinetics; Lipid Bilayers; Magnetic Resonance Spectroscopy; Models, Chemical; Phosphatidylcholines; Phosphatidylglycerols; Quaternary Ammonium Compounds; Surface Properties; Water

2011
Amyloid-β aggregation on model lipid membranes: an atomic force microscopy study.
    Journal of Alzheimer's disease : JAD, 2011, Volume: 26, Issue:3

    Amyloid fibril formation is generally associated with many neurodegenerative disorders, including Alzheimer's disease (AD). Although fibril plaque formation is associated with biological membranes in vivo, the role of the cell surfaces in amyloid fibril formation and the molecular mechanism of amyloid toxicity are not well understood. Understanding the details of amyloid interaction with lipid membrane may shed light on the mechanism of amyloid toxicity. Using atomic force microscopy, we investigated aggregation of amyloid-β1-42 (Aβ1-42) on model phospholipid membranes as a function of time and membrane composition. Neutral, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), anionic - 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (DOPG), and cationic - 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), were used to study the effect of lipid type on amyloid binding. We showed that both the charge on the lipid head group and lipid phase affect the interaction of amyloid oligomers with the membrane surface changing the rate of adsorption and causing changes in membrane structure and structure of amyloid deposits. We observed that amyloid aggregates progressively accumulate in a similar manner on the surface of neutral DPPC gel phase membrane and on the surface of fluid phase negatively charged DOPG membrane. In contrast to DPPC and DOPG, positively charged fluid DOTAP membrane and neutral fluid phase DOPC membrane contain amyloid deposits with reduced height, which suggests fusing of Aβ1-42 into the lipid membrane surface.

    Topics: 1,2-Dipalmitoylphosphatidylcholine; Amyloid beta-Peptides; Fatty Acids, Monounsaturated; Gels; Image Processing, Computer-Assisted; Lipid Bilayers; Membrane Lipids; Membranes, Artificial; Microscopy, Atomic Force; Peptide Fragments; Phosphatidylcholines; Phosphatidylglycerols; Quaternary Ammonium Compounds; Software

2011
Universality of DNA adsorption behavior on the cationic membranes of nanolipoplexes.
    The journal of physical chemistry. B, 2010, Feb-11, Volume: 114, Issue:5

    Nanolipoplexes have emerged worldwide as the most prevalent synthetic gene delivery system. Nowadays, it is accepted that complete DNA protection and a precise control of the physical attributes of emerging complexes are major steps toward rational design of efficient nanocarriers. Here we revise the mechanism of DNA adsorption to the cationic membranes of lipid nanovectors. Here we show that both the DNA-binding ability of cationic membranes and the one-dimensional DNA packing density inside the complex depend on the cationic lipid/anionic DNA charge ratio. Remarkably, both these distributions are rescaled on universal curves when plotted against gamma, a dimensionless quantity expressing the ratio between the area of cationic membranes and that occupied by DNA molecules. As a result, the DNA condensation on the surface of lipid nanocarriers can be regarded as a two-step process. Our findings indicate a successful way to the rational design of next-generation drug delivery nanocarriers.

    Topics: Adsorption; Cations; DNA; Fatty Acids, Monounsaturated; Lipid Bilayers; Liposomes; Nanoparticles; Phosphatidylcholines; Quaternary Ammonium Compounds

2010
The role of cholesterol and structurally related molecules in enhancing transfection of cationic liposome-DNA complexes.
    The journal of physical chemistry. B, 2009, Apr-16, Volume: 113, Issue:15

    Motivated by its important role in gene delivery, we have studied the effect of cholesterol and analogs on the transfection efficiency (TE) of lamellar cationic liposome-DNA (CL-DNA) complexes in vitro. Addition of cholesterol to low-transfecting DOTAP/DOPC-DNA complexes increases TE, with 15 mol % cholesterol already yielding 10-fold improvement. Steroids lacking the alkyl tail only modestly enhance TE, while molecules retaining it strongly enhance TE. All steroid-containing CL-DNA complexes exhibit the lamellar structure. The increase in experimentally determined membrane charge density (a universal parameter governing the TE of lamellar CL-DNA complexes) with cholesterol content alone cannot account for the rapid increase of TE. Instead, the reduction of the hydration repulsion layer of the membrane, caused by replacement of DOPC by cholesterol, promotes fusion between cationic membranes of CL-DNA complexes and anionic endosomal membranes, thus facilitating release of complexes and enhancing TE.

    Topics: Cations; Cholesterol; DNA; Fatty Acids, Monounsaturated; Molecular Structure; Phosphatidylcholines; Quaternary Ammonium Compounds; Scattering, Small Angle; Transfection; X-Ray Diffraction

2009
Reactive oxygen species play a central role in the activity of cationic liposome based cancer vaccine.
    Journal of controlled release : official journal of the Controlled Release Society, 2008, Aug-25, Volume: 130, Issue:1

    Recently, we developed a simple and potent therapeutic liposome cancer vaccine consisting of a peptide antigen and a cationic lipid. The molecular mechanism of the adjuvanticity of cationic liposome was studied and described in the current report. First, cationic DOTAP liposome, but not the neutral liposome DOPC, was shown to generate reactive oxygen species (ROS) in mouse bone marrow-derived dendritic cells (BMDC). ROS generation by DOTAP was required for ERK and p38 activation and downstream chemokine/cytokine induction. Furthermore, ROS were shown to be involved in the expression of the co-stimulatory molecules CD86/CD80 induced by DOTAP. However, as the DOTAP concentration increased from 50 to 800 microM, the apoptotic marker Annexin V and ROS double positive cells increased, suggesting that high dose of DOTAP-generated ROS causes cell apoptosis. In vivo, optimal amount of ROS in the draining lymph nodes (DLN) and anti-tumor (HPV positive TC-1 tumor) activity induced by E7 peptide (antigen derived from E7 oncoprotein of human papillomavirus (HPV) type 16) formulated in 100 nmol DOTAP were attenuated by incorporating DOPC in the formulation, suggesting that ROS are essential for the vaccine induced anti-tumor activity. Moreover, 600 nmol DOTAP/E7 generated huge amount of ROS in the DLN and showed no activity of tumor regression. Interestingly, 600 nmol DOTAP/E7-induced ROS were tuned down to the same level induced by 100 nmol DOTAP/E7 by adding DOPC in the formulation and this formulation showed tumor regression activity. In conclusion, DOTAP is an active DC stimulator resulting in the activation of ERK and p38 and induction of chemokines, cytokines and co-stimulatory molecules mediated by appropriate amount of ROS. Our data elucidated an important mechanism of adjuvant activity of cationic liposome and could facilitate rational design of synthetic lipid based adjuvants and vaccine formulation.

    Topics: Adjuvants, Immunologic; Animals; Annexin A5; Blotting, Western; Cancer Vaccines; Cations; Cytokines; Extracellular Signal-Regulated MAP Kinases; Fatty Acids, Monounsaturated; Female; Flow Cytometry; Liposomes; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Neoplasms; Oncogene Proteins, Viral; p38 Mitogen-Activated Protein Kinases; Papillomavirus E7 Proteins; Phosphatidylcholines; Quaternary Ammonium Compounds; Reactive Oxygen Species

2008
Detection of supported lipid bilayers using their electric charge.
    Langmuir : the ACS journal of surfaces and colloids, 2008, Sep-02, Volume: 24, Issue:17

    We describe an electronic detection method for charged lipid bilayers supported on a Si 3N 4/SiO 2/Si substrate. The flat-band voltage was used to monitor the charge of the bilayers. We show that the flat-band voltage varies with lipid adsorption depending on the polarity and mole ratio of the charged lipids, the salt concentration, and the surface coverage. Cationic and anionic bilayers produced a decrease and an increase in the flat-band voltage, respectively. The voltage change increased as the percentage of charged lipid components was elevated in the planar bilayers with full surface coverage. In addition, the voltage variation increased when the salt concentration was decreased or when the surface coverage of planar bilayer patches was increased. These results demonstrate that charged bilayers can be detected from the field effect that they exert on a solid support.

    Topics: Anions; Biophysics; Cations; Electric Capacitance; Electric Conductivity; Electrochemistry; Fatty Acids, Monounsaturated; Lipid Bilayers; Lipids; Microscopy, Atomic Force; Models, Chemical; Models, Statistical; Phosphatidylcholines; Quaternary Ammonium Compounds; Salts; Surface Properties

2008
A new helper phospholipid for gene delivery.
    Chemical communications (Cambridge, England), 2008, Apr-07, Issue:13

    Enhanced gene transfection activity is observed when using a new helper lipid with DOTAP, compared to DOPE.

    Topics: Animals; beta-Galactosidase; Cell Line; CHO Cells; Cricetinae; Cricetulus; DNA; Fatty Acids, Monounsaturated; Gene Transfer Techniques; Humans; Molecular Structure; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Quaternary Ammonium Compounds; Transfection

2008
Small angle scattering and zeta potential of liposomes loaded with octa(carboranyl)porphyrazine.
    The journal of physical chemistry. B, 2007, Sep-06, Volume: 111, Issue:35

    In this work, the physicochemical characterization of liposomes loaded with a newly synthesized carboranyl porphyrazine (H2HECASPz) is described. This molecule represents a potential drug for different anticancer therapies, such as boron neutron capture therapy and for photodynamic therapy or photothermal therapy. Different loading methods and different lipid mixtures were tested. The corresponding loaded vectors were studied by small angle scattering, light scattering, and zeta potential. The combined analysis of structural data at various lengths of scales and the measurement of the surface charge allowed us to obtain a detailed characterization of the investigated systems. The mechanisms underlying the onset of differences in relevant physicochemical parameters (size, polydispersity, and charge) were also critically discussed.

    Topics: Fatty Acids, Monounsaturated; Liposomes; Phosphatidylcholines; Pyrazines; Quaternary Ammonium Compounds; Scattering, Small Angle; X-Ray Diffraction

2007
Charge dependence of a nanoscale supercrystal phase in a supported lipid bilayer.
    Journal of the American Chemical Society, 2007, Oct-03, Volume: 129, Issue:39

    Topics: Fatty Acids, Monounsaturated; Lipid Bilayers; Microscopy, Atomic Force; Nanoparticles; Phosphatidylcholines; Quaternary Ammonium Compounds

2007
Structure and mobility of lipid membranes on a thermoplastic substrate.
    Langmuir : the ACS journal of surfaces and colloids, 2006, Jan-17, Volume: 22, Issue:2

    Supported lipid membranes constitute one of the most important model systems for cell membranes. The properties of lipid membranes supported by the hydrophobic solid polymer cyclic olefin copolymer (COC) were investigated. Lipid layers consisting of varying amounts of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP, cationic) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, neutral) prepared by vesicle fusion and solvent exchange were compared. All lipid mixtures coated the COC surface homogeneously forming a fluid membrane as verified by fluorescence microscopy and fluorescence recovery after photobleaching (FRAP). The exact structure of the supported membranes was determined by synchrotron reflectivity experiments using a microfluidic chamber. The X-ray data are in agreement with a compressed (head-to-head distance = 29 angstroms) and less densely packed bilayer.

    Topics: Cations; Cycloparaffins; Fatty Acids, Monounsaturated; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Membrane Fluidity; Membrane Fusion; Membranes, Artificial; Microfluidic Analytical Techniques; Microscopy, Fluorescence; Phase Transition; Phosphatidylcholines; Photobleaching; Polymers; Quaternary Ammonium Compounds; Solvents; Surface Properties; X-Ray Diffraction

2006
One-dimensional thermotropic dilatation area of lipid headgroups within lamellar lipid/DNA complexes.
    Langmuir : the ACS journal of surfaces and colloids, 2006, Apr-25, Volume: 22, Issue:9

    Using simultaneous synchrotron small- and wide-angle X-ray diffraction (SWAXD), we investigated the thermotropic behavior of a cationic lipid mixture of DOTAP-DOPC (1,2-dioleoyl-3-trimethylammonium-propane-dioleoylphosphatidylcholine) liposomes complexed with calf thymus DNA. The DOTAP-DOPC/DNA complex reacts to temperature change by a bilayer compression normal to its surface and an expansion of the DNA in the plane of the rod lattice. By applying two independent recently developed models, we show here for the first time that the thermotropic dilatation area of lipid headgroups within the complexes is not isotropic but occurs parallel to the 1D DNA lattice (i.e., along the direction perpendicular to the DNA axis). Our results shed light on the role of spatial dimensionality in the DNA packing density within lamellar lipoplexes and provide experimental evidence that the interaction between DNA molecules confined between lipid bilayers can be regarded as a 1D problem.

    Topics: Animals; Cattle; DNA; Drug Delivery Systems; Fatty Acids, Monounsaturated; Gene Transfer Techniques; In Vitro Techniques; Lipid Bilayers; Lipids; Macromolecular Substances; Models, Molecular; Phosphatidylcholines; Quaternary Ammonium Compounds; Static Electricity; Synchrotrons; Thermodynamics; X-Ray Diffraction

2006
Headgroup hydration and mobility of DOTAP/DOPC bilayers: a fluorescence solvent relaxation study.
    Langmuir : the ACS journal of surfaces and colloids, 2006, Oct-10, Volume: 22, Issue:21

    The biophysical properties of liposome surfaces are critical for interactions between lipid aggregates and macromolecules. Liposomes formed from cationic lipids, commonly used to deliver genes into cells in vitro and in vivo, are an example of such a system. We apply the fluorescence solvent relaxation technique to study the structure and dynamics of fully hydrated liquid crystalline lipid bilayers composed of mixtures of cationic dioleoyltrimethylammoniumpropane (DOTAP) and neutral dioleoylphosphatidylcholine (DOPC). Using three different naphthalene derivatives as fluorescent dyes (Patman, Laurdan and Prodan) allowed different parts of the headgroup region to be probed. Wavelength-dependent parallax quenching measurements resulted in the precise determination of Laurdan and Patman locations within the DOPC bilayer. Acrylamide quenching experiments were used to examine DOTAP-induced dye relocalization. The nonmonotonic dependence of dipolar relaxation kinetics (occurring exclusively on the nanosecond time scale) on DOTAP content in the membrane was found to exhibit a maximum mean solvent relaxation time at 30 mol % of DOTAP. Up to 30 mol %, addition of DOTAP does not influence the amount of bound water at the level of the sn(1) carbonyls, but leads to an increased packing of phospholipid headgroups. Above this concentration, elevated lipid bilayer water penetration was observed.

    Topics: Acrylamide; Fatty Acids, Monounsaturated; Fluorescence; Lipid Bilayers; Molecular Structure; Phosphatidylcholines; Quaternary Ammonium Compounds; Solvents; Temperature; Time Factors; Water

2006
Following the formation of supported lipid bilayers on mica: a study combining AFM, QCM-D, and ellipsometry.
    Biophysical journal, 2005, Volume: 88, Issue:5

    Supported lipid bilayers (SLBs) are popular models of cell membranes with potential biotechnological applications and an understanding of the mechanisms of SLB formation is now emerging. Here we characterize, by combining atomic force microscopy, quartz crystal microbalance with dissipation monitoring, and ellipsometry, the formation of SLBs on mica from sonicated unilamellar vesicles using mixtures of zwitterionic, negatively and positively charged lipids. The results are compared with those we reported previously on silica. As on silica, electrostatic interactions were found to determine the pathway of lipid deposition. However, fundamental differences in the stability of surface-bound vesicles and the mobility of SLB patches were observed, and point out the determining role of the solid support in the SLB-formation process. The presence of calcium was found to have a much more pronounced influence on the lipid deposition process on mica than on silica. Our results indicate a specific calcium-mediated interaction between dioleoylphosphatidylserine molecules and mica. In addition, we show that the use of PLL-g-PEG modified tips considerably improves the AFM imaging of surface-bound vesicles and bilayer patches and evaluate the effects of the AFM tip on the apparent size and shape of these soft structures.

    Topics: Aluminum Silicates; Biophysics; Biotechnology; Calcium; Cell Membrane; Edetic Acid; Fatty Acids, Monounsaturated; Lipid Bilayers; Lipids; Microscopy, Atomic Force; Phosphatidylcholines; Phosphatidylserines; Polyethylene Glycols; Quaternary Ammonium Compounds; Silicon; Silicon Dioxide; Time Factors

2005
AFM characterization of solid-supported lipid multilayers prepared by spin-coating.
    Biochimica et biophysica acta, 2005, Jun-15, Volume: 1712, Issue:1

    Lipids are the principal components of biologically relevant structures as cellular membranes. They have been the subject of many studies due to their biological relevance and their potential applications. Different techniques, such as Langmuir-Blodgett and vesicle-fusion deposition, are available to deposit ordered lipid films on etched surfaces. Recently, a new technique of lipid film deposition has been proposed in which stacks of a small and well-controlled number of bilayers are prepared on a suitable substrate using a spin-coater. We studied the morphological properties of multi-layers made of cationic and neutral lipids (DOTAP and DOPC) and mixtures of them using dynamic mode atomic force microscopy (AFM). After adapting and optimizing, the spin-coating technique to deposit lipids on a chemically etched Silicon (1,0,0) substrate, a morphological nanometer-scale characterization of the aforementioned samples has been provided. The AFM study showed that an initial layer of ordered vesicles is formed and, afterward, depending on details of the spin-coating preparation protocol and to the dimension of the silicon substrate, vesicle fusion and structural rearrangements of the lipid layers may occur. The present data disclose the possibility to control the lipid's structures by acting on spin-coating parameters with promising perspectives for novel applications of lipid films.

    Topics: Biophysical Phenomena; Biophysics; Fatty Acids, Monounsaturated; Lipid Bilayers; Lipids; Microscopy, Atomic Force; Phosphatidylcholines; Quaternary Ammonium Compounds; Silicon; Surface Properties

2005
The interaction of water with the phospholipid head group and its relationship to the lipid electrical conductivity.
    Chemistry and physics of lipids, 2004, Volume: 131, Issue:2

    We have studied the interaction of water with the lipid head group by gravimetrically measuring the lipid water adsorption and the lateral dc electrical conductivity increase resulting from this hydration. We have done this for dimyristoyl phosphatidylcholine (DMPC) having protonated or deuterated hydrocarbon chains. These studies were also done for two cationic lipids having rather different polar head groups. All three lipids behave as strong water adsorbers and all three display a steep, logarithmic increase in the conductivity as the first 1-3 waters per lipid are adsorbed. This increase is usually 5-6 orders of magnitude. After the initial 1-3 waters are adsorbed, the conductivity increases much more gradually, upon additional water adsorption. This electrical behavior is also found for weak water adsorbers and appears to be independent of the head group composition. The conductivity behavior suggests two types of water interacting with the head group. Our studies also indicate that a choline-like component is responsible for the strong water binding nature of the lipids, although, both phosphate and choline make significant contributions to the total amount of adsorbed water. The conductivity behavior, however, does not depend on the presence of both these head group components.

    Topics: Deuterium; Dimyristoylphosphatidylcholine; Electric Conductivity; Fatty Acids, Monounsaturated; Lipid Bilayers; Models, Chemical; Phosphatidylcholines; Phospholipids; Quaternary Ammonium Compounds; Thermodynamics; Water

2004
Roles of anionic and cationic template components in biomineralization of CdS nanorods using self-assembled DNA-membrane complexes.
    Journal of the American Chemical Society, 2004, Nov-03, Volume: 126, Issue:43

    Complexes of anionic DNA and cationic liposomes self-assemble into a multilamellar structure where two-dimensional lipid sheets confine a periodic one-dimensional lattice of parallel DNA chains, between which Cd(2+) ions can condense, and be subsequently reacted with H(2)S to form CdS nanorods. In this work, we identify the synergistic roles of the anionic and cationic components within the DNA-membrane template; DNA is highly anionic and condenses the Cd(2+) ions, while the cationic membrane modulates the concentration of condensed Cd(2+) ions to control the final CdS nanorod dimensions. Due to the strong electrostatic interactions between the DNA sugar-phosphate backbone and the Cd(2+) ions, crystallographic control of CdS nanostructures is possible using these simple DNA-membrane templates, which we demonstrate using nanobeam electron diffraction experiments on individual templated CdS nanorods.

    Topics: Animals; Anions; Bacteriophage lambda; Cadmium Compounds; Cations; Cattle; Crystallization; DNA; DNA, Viral; Fatty Acids, Monounsaturated; Lipids; Liposomes; Nanostructures; Phosphatidylcholines; Quaternary Ammonium Compounds; Sulfides

2004
Modulation of cellular immune response against hepatitis C virus nonstructural protein 3 by cationic liposome encapsulated DNA immunization.
    Hepatology (Baltimore, Md.), 2003, Volume: 37, Issue:2

    A vaccine strategy directed to increase Th1 cellular immune responses, particularly to hepatitis C virus (HCV) nonstructural protein 3 (NS3), has considerable potential to overcome the infection with HCV. DNA vaccination can induce both humoral and cellular immune responses, but it became apparent that the cellular uptake of naked DNA injected into muscle was not very efficient, as much of the DNA is degraded by interstitial nucleases before it reaches the nucleus for transcription. In this paper, cationic liposomes composed of different cationic lipids, such as dimethyl-dioctadecylammonium bromide (DDAB), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), or 1,2-dioleoyl-sn-glycerol-3-ethylphosphocholine (DOEPC), were used to improve DNA immunization in mice, and their efficiencies were compared. It was found that cationic liposome-mediated DNA immunization induced stronger HCV NS3-specific immune responses than immunization with naked DNA alone. Cationic liposomes composed of DDAB and equimolar of a neutral lipid, egg yolk phosphatidylcholine (EPC), induced the strongest antigen-specific Th1 type immune responses among the cationic liposome investigated, whereas the liposomes composed of 2 cationic lipids, DDAB and DOEPC, induced an antigen-specific Th2 type immune response. All cationic liposomes used in this study triggered high-level, nonspecific IL-12 production in mice, a feature important for the development of maximum Th1 immune responses. In conclusion, the cationic liposome-mediated gene delivery is a viable HCV vaccine strategy that should be further tested in the chimpanzee model.

    Topics: Animals; Antibody Formation; Capsules; Cations; CD4-Positive T-Lymphocytes; Cell Line; Fatty Acids, Monounsaturated; Gene Transfer Techniques; Immunization; Interleukin-12; Liposomes; Mice; Phosphatidylcholines; Plasmids; Quaternary Ammonium Compounds; Th1 Cells; Transfection; Vaccines, DNA; Viral Nonstructural Proteins

2003
A close encounter with DNA.
    European biophysics journal : EBJ, 2003, Volume: 32, Issue:6

    Atomic force microscopy, associated with surface science, has the potential to resolve the secondary structure of DNA in liquid form with unusually high resolution and with unprecedented accuracy.

    Topics: Adsorption; Bacteriophage lambda; DNA, Viral; Fatty Acids, Monounsaturated; Lipid Bilayers; Liposomes; Microscopy, Atomic Force; Nucleic Acid Conformation; Phosphatidylcholines; Quaternary Ammonium Compounds

2003
Pathways of lipid vesicle deposition on solid surfaces: a combined QCM-D and AFM study.
    Biophysical journal, 2003, Volume: 85, Issue:5

    Supported lipid bilayers (SLBs) are popular models of cell membranes with potential biotechnological applications, yet the mechanism of SLB formation is only partially understood. In this study, the adsorption and subsequent conformational changes of sonicated unilamellar vesicles on silica supports were investigated by quartz crystal microbalance with dissipation monitoring and atomic force microscopy, using mixtures of zwitterionic, negatively charged, and positively charged lipids, both in the presence and in the absence of Ca(2+) ions. Four different pathways of vesicle deposition could be distinguished. Depending on their charge, vesicles i). did not adsorb; ii). formed a stable vesicular layer; or iii). decomposed into an SLB after adsorption at high critical coverage or iv). at low coverage. Calcium was shown to enhance the tendency of SLB formation for negatively charged and zwitterionic vesicles. The role of vesicle-support, interbilayer, and intrabilayer interactions in the formation of SLBs is discussed.

    Topics: Adsorption; Calcium; Edetic Acid; Fatty Acids, Monounsaturated; Lipid Bilayers; Liposomes; Macromolecular Substances; Membrane Fluidity; Membrane Fusion; Microscopy, Atomic Force; Molecular Conformation; Phosphatidylcholines; Phosphatidylserines; Quaternary Ammonium Compounds; Surface Properties; Transducers

2003
Efficient synthesis and cell-transfection properties of a new multivalent cationic lipid for nonviral gene delivery.
    Journal of medicinal chemistry, 2002, Nov-07, Volume: 45, Issue:23

    Lipid-mediated delivery of DNA into cells holds great promise both for gene therapy and basic research applications. This paper describes the efficient and facile synthesis and the characterization of a new multivalent cationic lipid with a double-branched headgroup structure for gene delivery applications. The synthetic scheme can be extended to give cationic lipids of different charge, spacer, or lipid chain length. The chemical and physical properties of self-assembled complexes of the cationic liposomes (CLs) with DNA give indications of why multivalent cationic lipids possess superior transfection properties. The lipid bears a headgroup with five charges in the fully protonated state, which is attached to an unsaturated double-chain hydrophobic moiety based on 3,4-dihydroxybenzoic acid. Liposomes consisting of the new multivalent lipid and the neutral lipid 1,2-dioleoyl-sn-glycerophosphatidylcholine (DOPC) were used to prepare complexes with DNA. Investigations of the structures of these complexes by optical microscopy and small-angle X-ray scattering reveal a lamellar L(alpha)(C) phase of CL-DNA complexes with the DNA molecules sandwiched between bilayers of the lipids. Experiments using plasmid DNA containing the firefly luciferase reporter gene show that these complexes efficiently transfect mammalian cells. When compared to the monovalent cationic lipid 2,3-dioleyloxypropyltrimethylammonium chloride (DOTAP), the higher charge density of the membranes of CL-DNA complexes achievable with the new multivalent lipid greatly increases transfection efficiency in the regime of small molar ratios of cationic to neutral lipid. This is desired to minimize the known toxicity effects of cationic lipids.

    Topics: Animals; Benzamides; Cations; Coleoptera; DNA; Fatty Acids, Monounsaturated; Genes, Reporter; L Cells; Liposomes; Luciferases; Mice; Phosphatidylcholines; Quaternary Ammonium Compounds; Scattering, Radiation; Spermine; Transfection; X-Rays

2002
Phase behavior, DNA ordering, and size instability of cationic lipoplexes. Relevance to optimal transfection activity.
    The Journal of biological chemistry, 2001, Dec-14, Volume: 276, Issue:50

    Mechanisms of cationic lipid-based nucleic acid delivery are receiving increasing attention, but despite this the factors that determine high or low activity of lipoplexes are poorly understood. This study is focused on the fine structure of cationic lipid-DNA complexes (lipoplexes) and its relevance to transfection efficiency. Monocationic (N-(1-(2,3-dioleoyloxy)propyl),N,N,N-trimethylammonium chloride, N-(1-(2,3-dimyristyloxypropyl)-N,N-dimethyl-(2-hydroxyethyl)ammonium bromide) and polycationic (2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanammonium trifluoroacetate) lipid-based assemblies, with or without neutral lipid (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine, cholesterol) were used to prepare lipoplexes of different L(+)/DNA(-) charge ratios. Circular dichroism, cryogenic-transmission electron microscopy, and static light scattering were used for lipoplex characterization, whereas expression of human growth hormone or green fluorescent protein was used to quantify transfection efficiency. All monocationic lipids in the presence of inverted hexagonal phase-promoting helper lipids (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine, cholesterol) induced appearance of Psi(-) DNA, a chiral tertiary DNA structure. The formation of Psi(-) DNA was also dependent on cationic lipid-DNA charge ratio. On the other hand, monocationic lipids either alone or with 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine as helper lipid, or polycationic 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanammonium trifluoroacetate-based assemblies, neither of which promotes a lipid-DNA hexagonal phase, did not induce the formation of Psi(-) DNA. Parallel transfection studies reveal that the size and phase instability of the lipoplexes, and not the formation of Psi(-) DNA structure, correlate with optimal transfection.

    Topics: 3T3 Cells; Animals; Cations; Circular Dichroism; DNA; Fatty Acids, Monounsaturated; Fluorescent Dyes; Glycerophospholipids; Green Fluorescent Proteins; Humans; Lipid Metabolism; Lipids; Liposomes; Luminescent Proteins; Mice; Microscopy, Electron; Models, Molecular; Phosphatidylcholines; Phosphatidylethanolamines; Plasmids; Quaternary Ammonium Compounds; Spermine; Time Factors; Transfection; Ultraviolet Rays

2001
DNA condensation in two dimensions.
    Proceedings of the National Academy of Sciences of the United States of America, 2000, Dec-19, Volume: 97, Issue:26

    We have found that divalent electrolyte counterions common in biological cells (Ca(2+), Mg(2+), and Mn(2+) ) can condense anionic DNA molecules confined to two-dimensional cationic surfaces. DNA-condensing agents in vivo include cationic histones and polyamines spermidine and spermine with sufficiently high valence (Z) 3 or larger. In vitro studies show that electrostatic forces between DNA chains in bulk aqueous solution containing divalent counterions remain purely repulsive, and DNA condensation requires counterion valence Z >/= 3. In striking contrast to bulk behavior, synchrotron x-ray diffraction and optical absorption experiments show that above a critical divalent counterion concentration the electrostatic forces between DNA chains adsorbed on surfaces of cationic membranes reverse from repulsive to attractive and lead to a chain collapse transition into a condensed phase of DNA tethered by divalent counterions. This demonstrates the importance of spatial dimensionality to intermolecular interactions where nonspecific counterion-induced electrostatic attractions between the like-charged polyelectrolytes overwhelm the electrostatic repulsions on a surface for Z = 2. This new phase, with a one-dimensional counterion liquid trapped between DNA chains at a density of 0.63 counterions per DNA bp, represents the most compact state of DNA on a surface in vitro and suggests applications in high-density storage of genetic information and organo-metallic materials processing.

    Topics: Bacteriophage lambda; Cations, Divalent; Chlorides; Cobalt; DNA; DNA, Viral; Fatty Acids, Monounsaturated; Lipid Bilayers; Magnesium Chloride; Manganese Compounds; Models, Molecular; Nucleic Acid Conformation; Phosphatidylcholines; Quaternary Ammonium Compounds; Spermidine; Spermine

2000
An inverted hexagonal phase of cationic liposome-DNA complexes related to DNA release and delivery.
    Science (New York, N.Y.), 1998, Jul-03, Volume: 281, Issue:5373

    A two-dimensional columnar phase in mixtures of DNA complexed with cationic liposomes has been found in the lipid composition regime known to be significantly more efficient at transfecting mammalian cells in culture compared to the lamellar (LalphaC) structure of cationic liposome-DNA complexes. The structure, derived from synchrotron x-ray diffraction, consists of DNA coated by cationic lipid monolayers and arranged on a two-dimensional hexagonal lattice (HIIC). Two membrane-altering pathways induce the LalphaC --> HIIC transition: one where the spontaneous curvature of the lipid monolayer is driven negative, and another where the membrane bending rigidity is lowered with a new class of helper-lipids. Optical microscopy revealed that the LalphaC complexes bind stably to anionic vesicles (models of cellular membranes), whereas the more transfectant HIIC complexes are unstable and rapidly fuse and release DNA upon adhering to anionic vesicles.

    Topics: Birefringence; Cations; DNA; Endocytosis; Endosomes; Fatty Acids, Monounsaturated; Hexanols; Lipid Bilayers; Liposomes; Membrane Fusion; Phosphatidylcholines; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Transfection; X-Ray Diffraction

1998
Liposome-cell interactions in vitro: effect of liposome surface charge on the binding and endocytosis of conventional and sterically stabilized liposomes.
    Biochemistry, 1998, Sep-15, Volume: 37, Issue:37

    The cellular uptake of liposomes is generally believed to be mediated by adsorption of liposomes onto the cell surface and subsequent endocytosis. This report examines the effect of liposome surface charge on liposomal binding and endocytosis in two different cell lines: a human ovarian carcinoma cell line (HeLa) and a murine derived mononuclear macrophage cell line (J774). The large unilamellar liposomes were composed of 1, 2-dioleolyl-sn-glycero-3-phosphatidylcholine with and without the addition of either a positively charged lipid, 1, 2-dioleoyl-3-dimethylammonium propanediol (DODAP), or a negatively charged lipid, 1,2-dioleolyl-sn-glycero-3-phosphatidylserine. In some experiments 5 mol % of the anionic PEG2000-PE or a neutral PEG lipid of the same molecular weight was added. HeLa cells were found to endocytose positively charged liposomes to a greater extent than either neutral or negatively charged liposomes. This preference was not lipid-specific since inclusion of a cationic cyanine dye, DiIC18(3), to impart positive charge in place of DODAP resulted in a similar extent of endocytosis. In contrast the extent of liposome interaction with J774 cells was greater for both cationic and anionic liposomes than for neutral liposomes. The greater uptake of positively charged liposomes by HeLa cells was also observed with sterically stabilized liposomes (PEG liposomes). Although the overall amount of endocytosis for all the PEG liposomes examined was attenuated relative to conventional liposomes, the extent of endocytosis was greatest for positively charged PEG liposomes, whereas negatively charged PEG2000-PE liposomes were hardly endocytosed by the HeLa cells. Incorporation of a neutral PEG lipid into liposomes permits the independent variation of liposome steric and electrostatic effects in a manner that may allow interactions with cells of the reticuloendothelial system to be minimized, yet permit strong interactions between liposomes and proliferating cells.

    Topics: Animals; Arylsulfonates; Binding Sites; Cations; Cell Line; Endocytosis; Fatty Acids, Monounsaturated; Fluorescent Dyes; HeLa Cells; Humans; Hydrogen-Ion Concentration; Liposomes; Macrophages; Mice; Microscopy, Fluorescence; Oleic Acids; Phosphatidylcholines; Phosphatidylethanolamines; Polyethylene Glycols; Quaternary Ammonium Compounds; Rhodamines; Spectrometry, Fluorescence; Surface Properties

1998
Structure of DNA-cationic liposome complexes: DNA intercalation in multilamellar membranes in distinct interhelical packing regimes.
    Science (New York, N.Y.), 1997, Feb-07, Volume: 275, Issue:5301

    Cationic liposomes complexed with DNA (CL-DNA) are promising synthetically based nonviral carriers of DNA vectors for gene therapy. The solution structure of CL-DNA complexes was probed on length scales from subnanometer to micrometer by synchrotron x-ray diffraction and optical microscopy. The addition of either linear lambda-phage or plasmid DNA to CLs resulted in an unexpected topological transition from liposomes to optically birefringent liquid-crystalline condensed globules. X-ray diffraction of the globules revealed a novel multilamellar structure with alternating lipid bilayer and DNA monolayers. The lambda-DNA chains form a one-dimensional lattice with distinct interhelical packing regimes. Remarkably, in the isoelectric point regime, the lambda-DNA interaxial spacing expands between 24.5 and 57.1 angstroms upon lipid dilution and is indicative of a long-range electrostatic-induced repulsion that is possibly enhanced by chain undulations.

    Topics: Bacteriophage lambda; Cations; Chemical Phenomena; Chemistry, Physical; DNA; DNA, Viral; Fatty Acids, Monounsaturated; Isoelectric Point; Light; Lipid Bilayers; Liposomes; Microscopy, Fluorescence; Microscopy, Interference; Nucleic Acid Conformation; Phosphatidylcholines; Phosphatidylethanolamines; Plasmids; Quaternary Ammonium Compounds; Scattering, Radiation; X-Ray Diffraction

1997
Electrostatic parameters of cationic liposomes commonly used for gene delivery as determined by 4-heptadecyl-7-hydroxycoumarin.
    Biochimica et biophysica acta, 1997, Oct-23, Volume: 1329, Issue:2

    Cationic liposomes are used to deliver genes into cells in vitro and in vivo. The present study is aimed to characterize the electrostatic parameters of cationic, large unilamellar vesicles, 110 +/- 20 nm in size, composed of DOTAP/DOPE (mole ratio 1/1), DOTAP/DOPC (mole ratio 1/1), 100% DOTAP, DMRIE/DOPE 1/1, or DC-CHOL/DOPE (mole ratio 1/1). {. DOTAP, N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine; DMRIE, 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide; DC-CHOL, 3beta[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol}. The cationic liposomes had a large positive surface potential and a high pH at the liposomal surface in 20 mM Hepes buffer (pH 7.4) as monitored by the pH-sensitive fluorophore 4-heptadecyl-7-hydroxycoumarin. In contrast to DOTAP and DMRIE which were 100% charged, DC-CHOL in DC-CHOL/DOPE (1/1) liposomes was only about 50% charged in 20 mM Hepes buffer (pH 7.4). This might result in an easier dissociation of bilayers containing DC-CHOL from the plasmid DNA (which is necessary to enable transcription), in a decrease of the charge on the external surfaces of the liposomes or DNA-lipid complexes, and in an increase in release of the DNA-lipid complex into the cytosol from the endosomes. Other electrostatic characteristics found were that the primary amine group of DOPE in cationic liposomes dissociated at high (> 7.9) pHbulk and that a salt bridge was likely between the quaternary amine of DOTAP or DMRIE and the phosphate group of DOPE or DOPC, but not between the tertiary amine of DC-CHOL and the phosphate group of DOPE. The liposomes containing DOTAP were unstable upon dilution, probably due to the high critical aggregation concentration of DOTAP, 7 X 10(-5) M. This might also be a mechanism of the dissociation of bilayers containing DOTAP from the plasmid DNA.

    Topics: Cholesterol; Chromatography, High Pressure Liquid; Fatty Acids, Monounsaturated; Fluorescent Dyes; Gene Transfer Techniques; Hydrogen-Ion Concentration; Kinetics; Lipids; Liposomes; Models, Structural; Molecular Conformation; Phosphatidylcholines; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Static Electricity; Structure-Activity Relationship; Surface Properties; Umbelliferones

1997
The role of helper lipids in cationic liposome-mediated gene transfer.
    Biophysical journal, 1996, Volume: 71, Issue:2

    In the procedure for cationic liposome-mediated transfection, the cationic lipid is usually mixed with a "helper lipid" to increase its transfection potency. The importance of helper lipids, including dioleoylphosphatidylcholine (DOPC) and phosphatidylethanolamine (dioleoyl PE), DO was examined. Freeze-fracture electron microscopy of DNA:cationic complexes containing the pSV-beta-GAL plasmid DNA, the cationic lipid dioleoyl trimethylammonium propane, and these helper lipids showed that the most efficient mixtures were aggregates of ensheathed DNA and fused liposomes. PE-containing complexes aggregated rapidly when added to culture media containing polyanions, whereas PC-containing complexes did not. However, more granules of PC-containing complexes were formed on cell surfaces after the complexes were added to Chinese hamster ovary (CHO) cells in transfection media. Pronase treatment inhibited transfection, whereas dilute poly-L-lysine enhanced transfection, indicating that the attachment of DNA:liposome complexes to cell surfaces was mediated by electrostatic interaction. Fluorescence spectroscopy studies confirmed that more PC-containing complexes than PE-containing complexes were associated with CHO cells, and that more PC-containing complexes were located in a low pH environment (likely to be within endosomes) with time. Cytochalasin-B had a stronger inhibitory effect on PC-containing liposome-mediated than on PE-containing liposome-mediated transfection. Confocal microscopic recording of the fluorescently label lipid and DNA uptake process indicated that many granules of DNA:cationic liposome complexes were internalized as a whole, whereas some DNA aggregates were left out on the cell surfaces after liposomes of the complexes fused with the plasma membranes. For CHO cells, endocytosis seems to be the main uptake pathway of DNA:cationic liposome complexes. More PC-containing granules than PE-containing granules were formed on cell surfaces by cytoskeleton-directed membrane motion, after their respective DNA:liposome complexes attached to cell surfaces by electrostatic means. Formation of granules on the cell surface facilitated and/or triggered endocytosis. Fusion between cationic liposomes and the cell membrane played a secondary role in determining transfection efficiency.

    Topics: Animals; beta-Galactosidase; CHO Cells; Cricetinae; Drug Carriers; Elasticity; Escherichia coli; Fatty Acids, Monounsaturated; Freeze Fracturing; Light; Liposomes; Microscopy, Confocal; Microscopy, Electron; Phosphatidylcholines; Phosphatidylethanolamines; Plasmids; Quaternary Ammonium Compounds; Recombinant Proteins; Scattering, Radiation; Transfection

1996
Fusion of cationic liposomes with mammalian cells occurs after endocytosis.
    Biochimica et biophysica acta, 1995, May-04, Volume: 1235, Issue:2

    The interaction of cationic liposomes prepared using either dioleoyltrimethylammonium propane (DOTAP) or 3 beta-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol (DC-CHOL) with model membranes and with cultured mammalian cells was examined using an assay developed for monitoring virus-cell fusion (Stegmann et al. (1993) Biochemistry 32, 11330-11337). Lipid mixing between cationic liposomes and liposomes composed of DOPE/dioleoylphosphatidylglycerol (DOPG) or dioleoylphosphatidylcholine (DOPC)/DOPG was insensitive to pH in the range of pH 4.5-7.0 and was not affected by sodium chloride concentration in the range of 0-150 mM. Lipid mixing was dependent on dioleoylphosphatidylethanolamine (DOPE), since cationic liposomes prepared using dioleoylphosphatidylcholine (DOPC) were incapable of lipid mixing with DOPC/DOPG liposomes. The interaction of cationic liposomes with Hep G-2 and CHO D- cells was also studied. For both cell types, liposome-cell lipid mixing was rapid at 37 degrees C, beginning within minutes and continuing for up to 1 hour after uptake. The extent of lipid mixing was decreased at 15 degrees C, especially at later (> or = 20 min) time points. This suggests that at least part of the observed lipid mixing occurred after reaching cellular lysosomes. No lipid mixing was seen at 4 degrees C. Monensin inhibited lipid mixing between cationic liposomes and the cells, despite having no effect on liposome uptake. Inhibition of endocytic uptake of liposomes, either by incubation in hypertonic media or by depletion of cellular ATP with sodium azide and 2-deoxyglucose abolished liposome-cell fusion in both cell types. These data demonstrate that binding to the cell surface is insufficient for cationic liposome-cell fusion and that uptake into the endocytic pathway is required for fusion to occur.

    Topics: Animals; Azides; Cations; CHO Cells; Cholesterol; Cricetinae; Deoxyglucose; Endocytosis; Fatty Acids, Monounsaturated; Hydrogen-Ion Concentration; Liposomes; Membrane Fusion; Microscopy, Fluorescence; Monensin; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Quaternary Ammonium Compounds; Saline Solution, Hypertonic; Sodium Azide

1995