Protein-induced leakage and membrane destabilization of phosphatidylcholine and phosphatidylserine liposomes

By using spectroscopic and colloidal chemistry methods we studied the interactions of globular proteins with phospholipid membranes in relation to protein-promoted membrane fusion. We considered the effect of protein sorption on the destabilization of phosphatidylcholine and phosphatidylserine liposome membranes. Experimentally, we injected the proteins into fluorophore-quencher embedded liposome dispersions and recorded the leakage of fluorophore-quencher from the liposomes' inner compartment, which is due to the protein-induced destabilization of the phospholipid membranes. The release of fluorophore-quencher strongly depends on the protein concentration. The existence of monovalent and polyvalent cations also influences the protein-induced membrane destabilization by affecting the hydrophobic and electrostatic interactions.     

Fluorophore-linked zinc(II)dipicolylamine coordination complexes as sensors for phosphatidylserine-containing membranes

A series of Zn²⁺-2,2′-dipicolylamine (Zn²⁺-DPA) coordination complexes with an attached NBD fluorophore are synthesized and evaluated as fluorescent sensors. The sensors do not respond to vesicles composed of zwitterionic phosphatidylcholine, but the NBD fluorescence emission is enhanced in the presence of anionic vesicles. A sensor with two Zn²⁺-DPA units and a hydrophilic tris(ethyleneoxy) linker produced a larger emission enhancement than an analogue with a butyl linker, and titration with 1:1 POPC:POPS vesicles lead to an apparent phospholipid association constant of 5.3×10⁴ M⁻¹. The sensor can detect the presence of vesicles containing as little as 5% phosphatidylserine. The sensing effect apparently requires a membrane surface because the sensors do not respond to a phosphatidylserine derivative that is monodispersed in aqueous solution.     

Effects of liposomal phophatidylserine on phagocytic uptake of liposomes by macrophage-like HL-60RG cells

The purpose of this work is to know the effect of surface properties of liposomes on their phagocytic uptake by macrophages. For this, liposomes were prepared by the Bangham technique from the mixture of phosphatidylcholine (PC) and cholesterol (Chol) incorporated either with phosphatidylserine (PS), phosphatidylethanolamine (PE) or phosphatidic acid (PA). The liposomes thus prepared had diameters in the range between 150 and 260 nm. Electric surface properties of the liposomes and the macrophages differentiated from HL-60RG cells were determined by measuring their electrophoretic mobilities. The phagocytic uptake of liposomes with different contents of PS, PE and PA by macrophage-like HL-60RG cells was investigated by measuring oxygen consumption associated with phagocytic uptake. The phagocytic activity was found to be the highest with the PC–Chol liposomes containing 7 mol% PS, but no significant effects were observed with PA- and PE-containing PC–Chol liposomes. As the uptake was independent of the electric surface property of liposomes, PS was concluded to be specifically important for phagocytic activity of macrophages.     

Temperature optimization for improved determination of phosphatidylserine species by micro liquid chromatography with electrospray tandem mass spectrometric detection

A sensitive method for determination of disaturated phosphatidylserine species in the presence of their monounsaturated analogs has been developed, using micro liquid chromatography coupled to electrospray ionization tandem mass spectrometry. The hydrophobic nature of the phosphatidylserine species required a combination of low-eluting sample solvents and sub-ambient temperatures in order to focus large sample volumes up to 20 microL. The samples were dissolved in 2-propanol:hexane:water (20:10:4, v/v/v) prior to 1:9 dilution with ammonium formate buffer:2-propanol:tetrahydrofuran (30:55:15, v/v/v) and final 1:4 dilution with ammonium formate buffer (10 mM):2-propanol: tetrahydrofuran (55:37.5:7.5, v/v/v). The analytical column was a 0.5 x 150 mm stainless steel column packed with 5 microm C30 particles, while the mobile phase contained ammonium formate buffer (10 mM): 2-propanol:tetrahydrofuran (30:55:15, v/v/v). A temperature program from 5 degrees C (hold for 3 minutes) to 75 degrees C at 8 K/min provided separation of the disaturated phosphatidylserine species from their monounsaturated analogs, making available a sensitive determination of the isobaric species. The mass limit of detection for dipalmitoyl phosphatidylserine was 100 pg, corresponding to a concentration limit of detection of 5 pg/microL when using an injection volume of 20 microL. This is an improvement by a factor of 20 as compared to previously reported numbers obtained with conventional LC columns. The within-assay precision of dipalmitoyl phosphatidylserine was 11.9% RSD (n = 3), while the retention time precision was 4.1% RSD (n = 6).     

Programmable release of 2-O-d-glucopyranosyl-l-ascorbic acid and heparin from PCL-based nanofiber scaffold for reduction of inflammation and thrombosis

Reduction of inflammation and thrombosis caused by implanted devices is critical for clinical success. To this end, the strategy based on programmable release of anti-inflammatory and anti-thrombotic agents from the widely-used polycaprolactone (PCL)/gelatin nanofiber scaffold is developed. The release of 2-O-d-Glucopyranosyl-l-ascorbic Acid (AA-2G) and heparin are controlled by reactive oxygen species (ROS)-responsive poly(ethylene glycol)-based β-thioether ester copolymer (PEGDA-EDT) and mesoporous silica nanoparticles (MSN) in the nanofiber, respectively. The in vitro assay demonstrate that the scaffolds are hemocompatible with the resistance of platelet adhesion; the control release of AA-2G prevents initial inflammation and oxidation of the blood cells, and the subsequent release of heparin entitles nanofibers with long-term anti-thrombotic capability. In addition, rapid endothelialization is obtained on the surface of nanofiber scaffolds for the further enhancement of the hemocompatibility. In vivo implant evaluation convinces that the nanofiber scaffolds possess high biocompatibility with the substantial resistance for inflammation and thrombosis. Hence, our work paves a new way to develop the anti-inflammatory and anti-thrombotic tissue-engineering substrates through programmable delivery of two or multiple drugs.     

Thermodynamics of monolayers formed by mixtures of phosphatidylcholine/phosphatidylserine

In this work we obtain the thermodynamic properties of mixed (1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine) PC and (1-stearoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (sodium salt)) PS monolayers. Measurements of compressibility (isotherms, bulk modulus, and excess area per molecule) and surface potential show that the properties of monolayers at the air-water interface depend on the concentration of ions (Na(+) and K(+)) and the proportion of PS in the mixture. The dependence on PS arises because the molecule is originally bound to a Na(+) counterion; by increasing the concentration of ions the entropy changes, creating a favorable system for the bound counterions of PS to join the bulk, leaving a negatively charged molecule. This change leads to an increase in electrostatic repulsions which is reflected by the increase in area per molecule versus surface pressure and a higher surface potential. The results lead to the conclusion that this mixture of phospholipids follows a non ideal behavior and can help to understand the thermodynamic behavior of membranes made of binary mixtures of a zwitterionic and an anionic phospholipid with a bound counterion.     

Understanding lipid recognition by protein-mimicking cyclic peptides

This paper describes our investigation of the structural determinants of a designed cyclic peptide (cLac, cyclic peptide mimicking lactadherin) (Zheng, H.; Wang, F.; Wang, Q.; Gao, J. J. Am. Chem. Soc.2011, 133, 15280–15283) for phosphatidylserine (PS) recognition. A highly efficient strategy that takes advantage of the native chemical ligation (NCL) chemistry has been developed for the synthesis and labeling of cyclic peptides in general. Ala scanning of the cLac peptide revealed a sophisticated model for PS binding, in which the peptide scaffold assembles multiple polar residues to balance the desolvation and electrostatic interactions (salt bridge and hydrogen bonding) to achieve lipid selectivity. The results suggest that cLac effectively mimics the membrane binding mechanism of the parent protein lactadherin.     

Adsorption behavior of phospholipid vesicles at oil/water interfaces

By measuring the change in interfacial tension after adding phospholipid vesicles to an aqueous solution of electrolyte, we studied the adsorption behavior of phospholipid vesicles at oil/water interfaces. The effects of concentration of three kinds of electrolyte (NaCl, MgCl₂, LaCl₃) and of the mixing ratio of two kinds of phospholipids (phosphatidylcholine and phosphatidylserine), on the adsorption behavior at an oil/water interface were examined. The results were interpreted using the DLVO theory.     

Application of matrix-assisted laser desorption and ionization time-of-flight mass spectrometry for the characterization of the substrate specificity of neutrophil phospholipase A2

Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) was successfully applied for the analysis of various lipid classes. It can also be used for monitoring the digestion of phosphatidylcholine (PC) with phospholipase A₂ (PLA₂) and it was shown that MALDI-TOF MS possesses a number of advantages over well established methods for this purpose. In this work, we use MALDI-TOF MS for determination of the substrate specificity of neutrophil PLA₂. For the comparison of the selectivity of the enzyme to various phospholipid (PL) classes, the intensities of the signals arising from the product of the reaction (Sp) and the signal intensity of the residual substrate (Ss) were compared and the resulting Sp/Ss ratio was used as the measure. This approach was first tested with a model system pancreatic PLA₂ and afterwards two sources of the neutrophil PLA₂—the enzyme extracted from the neutrophils and the enzyme released from these cells—were tested for their substrate specificity. We will show that the neutrophil-secreted PLA₂ possesses high preferences for digestion of phosphatidic acid (PA) over other phospholipids. The method applied here is simple and much information can be obtained from a single mass spectrum. Moreover, this approach works well also with a crude biological systems, i.e. no prior purification of the enzyme is required for means of characterisation.     

A Versatile Method for Determining the Molar Ligand-Membrane Partition Coefficient

A novel method for the quantitative assessment of the membrane partitioning of a ligand from the aqueous phase is described, demonstrated here with the thoroughly studied antipsychotic chlorpromazine (CPZ). More specifically, collisional quenching of the fluorescence of a pyrene labeled fluorescent lipid analog 1-palmitoyl-2[10-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC) by CPZ was utilized, using 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine and -serine (POPC and POPS) liposomes as model membranes. The molar partition coefficient is obtained from two series of titrations, one with constant [phospholipid] and increasing [drug] and the other with constant [drug] and varying total [phospholipid], the latter further comprising of large unilamellar vesicles (LUVs) of POPC/POPS/PPDPC at a constant concentration of 10 μM and indicated concentrations of POPC/POPS LUVs. Notably, the approach described is generic and can be employed in screening for the membrane partitioning of compounds, providing that a suitable fluorescence parameter can be incorporated into one population of liposomes utilized as model membranes.