Dioleoyl-sn-glycero-3-phosphocholine Research Articles

Preparation, Characterization, Wound Healing, and Cytotoxicity Assay of PEGylated Nanophytosomes Loaded with 6-Gingerol

Nutrients are widely used for treating illnesses in traditional medicine. Ginger has long been used in folk medicine to treat motion sickness and other minor health disorders. Chronic non-healing wounds might elicit an inflammation response and cancerous mutation. Few clinical studies have investigated 6-gingerol's wound-healing activity due to its poor pharmacokinetic properties. However, nanotechnology can deliver 6-gingerol while possibly enhancing these properties. Our study aimed to develop a nanophytosome system loaded with 6-gingerol molecules to investigate the delivery system's influence on wound healing and anti-cancer activities. We adopted the thin-film hydration method to synthesize nanophytosomes. We used lipids in a ratio of 70:25:5 for DOPC(dioleoyl-sn-glycero-3-phosphocholine): cholesterol: DSPE/PEG2000, respectively. We loaded the 6-gingerol molecules in a concentration of 1.67 mg/mL and achieved size reduction via the extrusion technique. We determined cytotoxicity using lung, breast, and pancreatic cancer cell lines. We performed gene expression of inflammation markers and cytokines according to international protocols. The synthesized nanophytosome particle sizes were 150.16 ± 1.65, the total charge was -13.36 ± 1.266, and the polydispersity index was 0.060 ± 0.050. Transmission electron microscopy determined the synthesized particles' spherical shape and uniform size. The encapsulation efficiency was 34.54% ± 0.035. Our biological tests showed that 6-gingerol nanophytosomes displayed selective antiproliferative activity, considerable downregulation of inflammatory markers and cytokines, and an enhanced wound-healing process. Our results confirm the anti-cancer activity of PEGylated nanophytosome 6-gingerol, with superior activity exhibited in accelerating wound healing.

Fluidity-Guided Assembly of Au@Pt on Liposomes as a Catalase-Powered Nanomotor for Effective Cell Uptake in Cancer Cells and Plant Leaves.

The fluidity of the liposomes is essential to nanoparticle-membrane interactions. We herein report a liposomal nanomotor system by controlling the self-assembly behavior of gold core-platinum shell nanoparticles (Au@Pt) on liposomes. Au@Pt can aggregate immediately on fluid-phase dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes, forming an uneven distribution. By control of the lipid phase and fluidity, either using pure 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) above its phase transition temperature or adding cholesterol as an adjuvant to DPPC lipids, we precisely control the assembly of Au@Pt on liposomes. Au@Pt maintained high catalase-like activity on the liposomal surface, promoting the decomposition of H2O2 and the movement of the liposomal nanomotors. Finally, we demonstrate that liposomal nanomotors are biocompatible and they can speed up the cellular uptake in mammalian HepG2 cancer cells and Nicotiana tabacum (Nb) plant leaves. This liposomal nanomotor system is expected to be further investigated in biomedicine and plant nanotechnology.

Temperature induced lipid membrane restructuring and changes in nanomechanics

The naturally occurring milk sphingomyelin is of particular interest owing to its complex composition and involvement in the formation of the milk fat globule membrane (MFGM). Knowledge of membrane organization and nanomechanical stability has proved to be crucial in understanding their properties and functions. In this work, two model membrane systems composed of 1, 2 dioleoyl-sn-glycero-3-phosphocholine (DOPC), egg sphingomyelin (egg-SM) and cholesterol, and DOPC, milk sphingomyelin (milk-SM) and cholesterol were exposed to both RT and 10°C. The morphological and nanomechanical changes were investigated using atomic force microscopy (AFM) imaging and force mapping below RT using a designed liquid cell with temperature-control. In both systems, the size and shape of SM/Chol-enriched liquid ordered domains (Lo) and DOPC-enriched liquid disordered phase (Ld) were monitored at controlled temperatures. AFM based force-mapping showed that rupture forces were consistently higher for Lo domains than Ld phases and were decreased for Ld with decreasing temperature while an increase in breakthrough force was observed in Lo domains. More interestingly, dynamic changes and defect formations in the hydrated lipid bilayers were mostly detected at low temperature, suggesting a rearrangement of lipid molecules to relieve additional tension introduced upon cooling. Noteworthy, in these model membrane systems, tension-driven defects generally heal on reheating the sample. The results of this work bring new insights to low temperature induced membrane structural reorganization and mechanical stability changes which will bring us one step closer to understand more complex systems such as the MFGM.

Effect of cell membrane fatty acid composition of Escherichia coli on the resistance to pulsed electric field (PEF) treatment

The underlying relationships between growth temperature, membrane fatty acid composition (mFAC) and PEF resistance of Escherichia coli (E. coli) were explored. Results indicated the PEF resistance of E. coli at stationary stage was directly related to mFAC. For the cells at stationary stage, as growth temperature decreased from 37 °C to 15 °C, the PEF (23.3 kV/cm, 1.2 ms) lethality of E. coli was increased 1.5 log10, reached 3.2 log10. Meanwhile, the proportion of unsaturated fatty acid (UFA) was increased from 20.07 to 47.65%. And the fluidity index (FI) of the cell membrane was also increased from 0.42 to 1.42. It revealed that the PEF resistance of E. coli at stationary stage was determined by the mFAC and fluidity of the cell membrane. Those conclusions were also confirmed by cytomembrane fluidity determined by fluorescence probe (DPH) and the different PEF-induced electro-permeabilization of dioleoyl-sn-glycero-3-phosphocholine (DOPC) and Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) vesicles. On the other hand, for the cell at exponential stage, the PEF resistance of E. coli was rarely affected by mFAC. The PEF (23.3 kV/cm, 1.2 ms) lethality of cells was almost the same (3.2 log10) at different growth temperature.

Effects of vesicle components on the electro-permeability of lipid bilayers of vesicles induced by pulsed electric fields (PEF) treatment

In this study, the effects of pulsed electric field (PEF) treatments on the electro-permeability of vesicles made from dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), dioleoyl-sn-glycero-3-phosphocholine (DOPC), Soya phosphatidylcholine (soya PC), cholesterol and a binary mixture of them encapsulated 5(6)-Carboxyfluorescein (5(6)-CF) were investigated, based on the electric field strength of 0–40 kV/cm and treatment time of 0–4.8 ms. Results indicated that the electro-permeability of lipid membrane was directly related to the composition of lipid bilayers membrane of vesicles. As compared to DPPC vesicles, DOPC vesicles were much more sensitive to PEF treatment. For the DOPC vesicles, the 5(6)-CF release percentage (R) was 9.24%, 20.12%, and 30.62% after PEF treatment at 20, 30, and 40 kV/cm for 4.8 ms treatment time, respectively. However, for the DPPC vesicles, it was only 1.84%, 4.02%, and 6.51%, respectively. On the other hand, the electro-permeability of DOPC vesicles was greatly affected by the addition of DPPC. The electro-permeability of DOPC vesicles was decreased sharply with the increase of DPPC molar ratio. Under the PEF treatment with 40 kV/cm and 1.2 ms, it was observed that the R of DOPC vesicles decreased markedly from 27.56% to 9.80% with the molar ratio of DPPC increased from 0 to 40%.For the soya PC vesicles, the R was dropped sharply from 48.13% to 24.11% with the molar ratio of cholesterol increased from 0 to 50%. Raman spectroscopy analysis revealed that the decrease of electro-permeability of soya PC vesicles with the addition of cholesterol was responsible for the incorporating of cholesterol into hydrocarbon partition of lipid bilayers, leading to the order arrangement of hydrocarbon chain of phospholipids, which was resulted an increase in the thickness of bilayers membrane and decrease in the fluidity of bilayers membrane. However, for the mixture of DOPC and DPPC (DOPC/DPPC) vesicles, there has no effect on the conformation of hydrocarbon chain of phospholipids and the fluidity of bilayer membrane of DOPC/DPPC vesicles with the addition of DPPC.

Coexistence of native-like and non-native partially unfolded ferricytochrome c on the surface of cardiolipin-containing liposomes.

Cytochrome c, in spite of adopting a rather rigid structure around its prosthetic heme group, is rather diverse with regard to its function and structural variability. On the surface of the inner membrane of mitochondria it serves as an electron transfer carrier. However, at conditions which have not yet been unambiguously identified, cytochrome c can adopt a variety of non-native conformations, some of which exhibit peroxidase activity. Cardiolipin-containing liposomes have served as ideal model system to investigate the various modes of interaction between cytochrome c and the inner mitochrondrial membrane. We probed the binding of horse heart ferricytochrome c to liposomes formed with 20% tetraoleoyl cardiolipin (TOCL) and 80% dioleoyl-sn-glycero-3-phosphocholine (DOPC) as a function of lipid/protein ratio by fluorescence and visible circular dichroism spectroscopy. The obtained binding isotherms suggest that they reflect reversible binding processes, which excludes the possibility of significant protein insertion into the membrane. A global analysis of our data revealed the existence of two binding sites on the protein which causes rather different degrees of protein unfolding. We found that these two modes of interaction between protein and liposome led to conformational changes. While site 1 is relatively unaffected by NaCl, site 2 shows a more native-like state or a higher population of the native state in the presence of NaCl. At the highest utilized concentration of NaCl, there is only a 40% inhibition of the binding to site 2. We interpret our finding for this binding site as reflecting an equilibrium between electrostatically bound proteins with a high degree of unfolding and less unfolded proteins which bind either via H-bonding between lysine side chains and PO2(-) or hydrophobic interactions. With regard to site 2 binding, our results are reminiscent of the two-state equilibrium between a compact C and an extended E-state proposed by Pletneva and co-workers (Hanske et al. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 125-230). We conjecture that the nonelectrostatically bound proteins should have higher abilities to maintain the redox potential that is required for the function as an electron transfer protein.

Coexistence of Native-Like and Non-Native Misfolded Ferricytochrome C on the Surfac of Cardiolipin Containing Liposomes

Cytochrome c, in spite of adopting a rather rigid structure around its prosthetic heme group, is rather diverse with regard to its function and structural variability. On the surface of the inner membrane of mitochondria it serves as an electron transfer carrier. However, at conditions which have not yet been unambiguously identified, cytochrome c can adopt a variety of non-native conformations, some of which exhibit peroxidase activity. Cardiolipin-containing liposomes have served as ideal model system to investigate the various modes of interaction between cytochrome c and the inner mitochrondrial membrane. We probed the binding of horse heart ferricytochrome c to liposomes formed with 20% tetraoleoyl cardiolipin (TOCL) and 80% dioleoyl-sn-glycero-3-phosphocholine (DOPC) as a function of lipid/protein ratio by fluorescence and visible circular dichroism spectroscopy. The obtained binding isotherms suggest that they reflect reversible binding processes, which excludes the possibility of significant prote...

General model of phospholipid bilayers in fluid phase within the single chain mean field theory.

Coarse-grained model for saturated phospholipids: 1,2-didecanoyl-sn-glycero-3-phosphocholine (DCPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and unsaturated phospholipids: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC) is introduced within the single chain mean field theory. A single set of parameters adjusted for DMPC bilayers gives an adequate description of equilibrium and mechanical properties of a range of saturated lipid molecules that differ only in length of their hydrophobic tails and unsaturated (POPC, DOPC) phospholipids which have double bonds in the tails. A double bond is modeled with a fixed angle of 120°, while the rest of the parameters are kept the same as saturated lipids. The thickness of the bilayer and its hydrophobic core, the compressibility, and the equilibrium area per lipid correspond to experimentally measured values for each lipid, changing linearly with the length of the tail. The model for unsaturated phospholipids also fetches main thermodynamical properties of the bilayers. This model is used for an accurate estimation of the free energies of the compressed or stretched bilayers in stacks or multilayers and gives reasonable estimates for free energies. The proposed model may further be used for studies of mixtures of lipids, small molecule inclusions, interactions of bilayers with embedded proteins.

Physical Damage on Giant Vesicles Membrane as a Result of Methylene Blue Photoirradiation

In this study we pursue a closer analysis of the photodamage promoted on giant unilamellar vesicles membranes made of dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), by irradiating methylene blue present in the giant unilamellar vesicles solution. By means of optical microscopy and electro-deformation experiments, the physical damage on the vesicle membrane was followed and the phospholipids oxidation was evaluated in terms of changes in the membrane surface area and permeability. As expected, oxidation modifies structural characteristics of the phospholipids that lead to remarkable membrane alterations. By comparing DOPC- with POPC-made membranes, we observed that the rate of pore formation and vesicle degradation as a function of methylene blue concentration follows a diffusion law in the case of DOPC and a linear variation in the case of POPC. We attributed this scenario to the nucleation process of oxidized species following a diffusion-limited growth regime for DOPC and in the case of POPC a homogeneous nucleation process. On the basis of these premises, we constructed models based on reaction-diffusion equations that fit well with the experimental data. This information shows that the outcome of the photosensitization reactions is critically dependent on the type of lipid present in the membrane.

Orthogonal Adsorption Onto Nano‐Graphene Oxide Using Different Intermolecular Forces for Multiplexed Delivery

Nano-graphene oxide can adsorb both doxorubicin and zwitterionic dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes in an orthogonal and non-competing manner with high capacities based on different surface and intermolecular forces taking place on the heterogeneous surface of the graphene oxide. The system forms stable colloids, allowing co-delivery of both cargos to cancer cells.

Phase behavior and domain size in sphingomyelin-containing lipid bilayers

Membrane raft size measurements are crucial to understanding the stability and functionality of rafts in cells. The challenge of accurately measuring raft size is evidenced by the disparate reports of domain sizes, which range from nanometers to microns for the ternary model membrane system sphingomyelin (SM)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/cholesterol (Chol). Using Förster resonance energy transfer (FRET) and differential scanning calorimetry (DSC), we established phase diagrams for porcine brain SM (bSM)/dioleoyl-sn-glycero-3-phosphocholine (DOPC)/Chol and bSM/POPC/Chol at 15 and 25°C. By combining two techniques with different spatial sensitivities, namely FRET and small-angle neutron scattering (SANS), we have significantly narrowed the uncertainty in domain size estimates for bSM/POPC/Chol mixtures. Compositional trends in FRET data revealed coexisting domains at 15 and 25°C for both mixtures, while SANS measurements detected no domain formation for bSM/POPC/Chol. Together these results indicate that liquid domains in bSM/POPC/Chol are between 2 and 7nm in radius at 25°C: that is, domains must be on the order of the 2–6nm Förster distance of the FRET probes, but smaller than the ~7nm minimum cluster size detectable with SANS. However, for palmitoyl SM (PSM)/POPC/Chol at a similar composition, SANS detected coexisting liquid domains. This increase in domain size upon replacing the natural SM component (which consists of a mixture of chain lengths) with synthetic PSM, suggests a role for SM chain length in modulating raft size in vivo.

“End-Only” Functionalized Oligo(phenylene ethynylene)s: Synthesis, Photophysical and Biocidal Activity

It is essential to develop alternative strategies to treat infections, especially those infections caused by Staphylococcus aureus, which is responsible for most skin infections. Among those strategies, light-induced inactivation of pathogens appears to be a promising candidate. We present four novel “end only” oligo(phenylene ethynylene)s (EO-OPE-1s) that have the ends functionalized with cationic groups and are powerful light-activated biocides against Escherichia coli, Staphylococcus epidermidis, and S. aureus. We have correlated the light-induced biocidal activities with singlet oxygen quantum yields Φ (1O2) of EO-OPE-1s, and a higher Φ (1O2) correlates with a better light-induced biocidal activity. Coupled with our previous work on the interactions of EO-OPE-1s with dioleoyl-sn-glycero-3-phosphocholine (DOPC)/cholesterol vesicles, we believe the biocidal process involves the following: (1) EO-OPE-1s penetrate the bacterial membrane, (2) EO-OPE-1s photosensitize the generation of singlet oxygen and/or...

Identification of Surface Epitopes of Human Coagulation Factor Va That Are Important for Interaction with Activated Protein C and Heparin

Inactivation of factor Va (FVa) by activated protein C (APC) is a key reaction in the down-regulation of thrombin formation. FVa inactivation by APC is correlated with a loss of FXa cofactor activity as a result of three proteolytic cleavages in the FVa heavy chain at Arg306, Arg506, and Arg679. Recently, we have shown that heparin specifically inhibits the APC-mediated cleavage at Arg506 and stimulates cleavage at Arg306. Three-dimensional molecular models of APC docked at the Arg306 and Arg506 cleavage sites in FVa have identified several FVa amino acids that may be important for FVa inactivation by APC in the absence and presence of heparin. Mutagenesis of Lys320, Arg321, and Arg400 to Ala resulted in an increased inactivation rate by APC at Arg306, which indicates the importance of these residues in the FVa-APC interaction. No heparin-mediated stimulation of Arg306 cleavage was observed for these mutants, and stimulation by protein S was similar to that of wild type FVa. With this, we have now demonstrated that a cluster of basic residues in FVa comprising Lys320, Arg321, and Arg400 is required for the heparin-mediated stimulation of cleavage at Arg306 by APC. Furthermore, mutations that were introduced near the Arg506 cleavage site had a significant but modest effect on the rate of APC-catalyzed FVa inactivation, suggesting an extended interaction surface between the FVa Arg506 site and APC.

Motionally restricted tryptophan environments at the peptide-lipid interface of gramicidin channels.

The tryptophans in the gramicidin channel play a crucial role in the organization and function of the channel. The localization and dynamics of these tryptophans have been studied using fluorescence spectroscopy, especially utilizing environment-induced effects on the rates of solvent relaxation around these residues in membranes. When incorporated into model membranes of dioleoyl-sn-glycero-3-phosphocholine (DOPC), the tryptophans in the gramicidin channel exhibit a red edge excitation shift (REES) of 6 nm. In addition, fluorescence polarization shows both excitation and emission wavelength dependence. Fluorescence lifetime analysis shows a biexponential decay, corresponding to a short- and a long-lifetime component. The mean lifetime was found to be dependent on both excitation and emission wavelengths. Analysis of time-resolved emission spectra (TRES) shows a heterogeneous environment for the tryptophans consistent with the lifetime information. Taken together, these observations point out the motional restriction experienced by the tryptophans in the gramicidin channel. This is consistent with other studies in which such restrictions are thought to be imposed due to hydrogen bonding between the indole rings of the tryptophans and the neighboring lipid carbonyls. The significance of such organization in terms of functioning of the channel is brought out by the fact that substitution, photodamage, or chemical modification of these tryptophans is known to give rise to channels with conformation and reduced conductivity.

Restricted mobility of the sole tryptophan in membrane-bound melittin

In spite of numerous studies, there appears to be no consensus regarding the orientation and aggregation state of membrane-bound melittin. We report here the restricted environment of the sole tryptophan residue in membrane-bound melittin using environment-induced effects on the rates of solvent relaxation. When incorporated into unilamellar vesicles of dioleoyl- sn-glycero-3-phosphocholine (DOPC), melittin exhibits a red edge excitation shift (REES) of 5 nm. In addition, fluorescence polarization of melittin m the membrane shows both excitation and emission wavelength dependence. Taken together, these observations indicate that the tryptophan residue of melittin is located in a motionally restricted region in the membrane.

Fluorophore environments in membrane-bound probes: a red edge excitation shift study.

A shift in the wavelength of maximum fluorescence emission toward higher wavelengths, caused by a shift in the excitation wavelength toward the red edge of the absorption band, is termed the Red Edge Excitation Shift (REES). This effect is mostly observed with polar fluorophores in motionally restricted media such as very viscous solutions or condensed phases. In this paper, we report the red edge excitation shift of a membrane-bound phospholipid molecule whose headgroup is covalently labeled with a 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) moiety. When incorporated into model membranes of dioleoyl-sn-glycero-3-phosphocholine (DOPC), the NBD-labeled phospholipid (NBD-PE), exhibits a red edge excitation shift of 10 nm. In addition, fluorescence polarization of NBD-PE in membranes shows both excitation and emission wavelength dependence. The nonpolar membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH) does not show red edge excitation shift in model membranes. The lifetime of NBD-PE in DOPC vesicles was found to be dependent on both excitation and emission wavelengths. These wavelength-dependent lifetimes are correlated to the reorientation of solvent dipoles around the excited-state dipole of the NBD moiety in the membrane. The magnitude of the red shift in the emission maximum for NBD-PE was found to be independent of temperature, between 12 and 54 degrees C, and of the physical state (gel or fluid) of the membrane. Taken together, these observations are indicative of the motional restriction experienced by this fluorophore in the membrane. Red edge excitation shift promises to be a powerful tool in probing membrane organization and dynamics.