Dipalmitoylphosphatidylglycerol Vesicles Research Articles

Binding of cationic peptides (KX)4K to DPPG bilayers. Increasing the hydrophobicity of the uncharged amino acid X drives formation of membrane bound β-sheets: A DSC and FT-IR study

The binding of cationic peptides of the sequence (KX)4K to lipid vesicles of negatively charged dipalmitoyl-phosphatidylglycerol (DPPG) was investigated by differential scanning calorimetry (DSC) and temperature dependent Fourier-transformed infrared (FT-IR) spectroscopy. The hydrophobicity of the uncharged amino acid X was changed from G (glycine) over A (alanine), Abu (α-aminobutyric acid), V (valine) to L (leucine). The binding of the peptides caused an increase of the phase transition temperature (Tm) of DPPG by up to 20°C. The shift depended on the charge ratio and on the hydrophobicity of the amino acid X. Unexpectedly, the upward shift of Tm increased with increasing hydrophobicity of X. FT-IR spectroscopy showed a shift of the CH2 stretching vibrations of DPPG to lower frequency, particularly for bilayers in the liquid-crystalline phase, indicating an ordering of the hydrocarbon chains when the peptides were bound. Changes in the lipid C=O vibrational band indicated a dehydration of the lipid headgroup region after peptide binding. (KG)4K was bound in an unordered structure at all temperatures. All other peptides formed intermolecular antiparallel β-sheets, when bound to gel phase DPPG. However, for (KA)4K and (KAbu)4K, the β-sheets converted into an unordered structure above Tm. In contrast, the β-sheet structures of (KV)4K and (KL)4K remained stable even at 80°C when bound to the liquid-crystalline phase of DPPG. Strong aggregation of DPPG vesicles occurred after peptide binding. For the aggregates, we suggest a structure, where aggregated single β-sheets are sandwiched between opposing DPPG bilayers with a dehydrated interfacial region.

Interaction of antitumoral fluorescent heteroaromatic compounds, a benzothienopyrrole and two thienoindoles, with DNA and lipid membranes

► The fluorescence properties of a benzothienopyrrole and two thienoindoles were studied. ► The compounds were incorporated in lipid membranes of DPPC, DPPG, egg lecithin and DODAB. ► All compounds exhibited a growth inhibitory effect on three human tumor cell lines. ► The benzothienopyrrole showed a higher affinity to DNA than the two thienoindoles. Three tricyclic compounds, a benzothieno[2,3- b ]pyrrole 1 , a thieno[3,2- f ]indole 2 and a thieno[2,3- e ]indole 3 , early synthesized by us, were evaluated for their capacity to inhibit the in vitro growth of three human tumor cell lines, MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer) and SF-268 (CNS cancer) and it was found that compound 1 is the most potent presenting the lowest GI 50 values (7.9–14.1 μM). The photophysical properties of the three compounds (absorption and fluorescence) were studied in different solvents and in lipid membranes of dipalmitoyl phosphatidylcholine (DPPC), dioctadecyldimethylammonium bromide (DODAB), phosphatidylcholine from egg yolk (egg lecithin, Egg-PC) and dipalmitoyl phosphatidylglycerol (DPPG), considering future drug delivery applications using liposomes. Fluorescence measurements indicate that all the compounds incorporated in lipid membranes are mainly located in the lipid bilayer. Compounds 1 and 3 clearly detect the gel to liquid-crystalline phase transition, while compound 2 feels a more hydrated environment in DPPG and DODAB vesicles. Due to the antitumoral properties of the three fluorescent compounds, interactions with salmon sperm DNA were investigated using fluorescence techniques which allowed the determination of binding constants ( K i ) and binding site sizes ( n ) for each compound. When compared with the thienoindoles 2 and 3 , the benzothienopyrrole 1 showed the highest affinity to DNA. Quenching experiments with iodide ion showed that intercalation is the preferred mode of binding of the three compounds to the DNA molecule.

Spray layer-by-layer films based on phospholipid vesicles aiming sensing application via e-tongue system

The Layer-by-Layer (LbL) technique via spraying (spray-LbL) has been applied as new and alternative methodology to fabricate ultrathin films due to its versatility in relation to the conventional dipping-LbL method, mainly in terms of faster layer deposition and larger coated area. In this work, the possibility of immobilizing vesicles of dipalmitoyl phosphatidyl glycerol (DPPG) phospholipid onto alternating layers of the polyelectrolyte poly(allylamine hydrochloride) (PAH) using the spray-LbL method was investigated, being the results compared to the conventional dipping-LbL method. The growth of (PAH/DPPG)n spray-LbL films was systematically monitored by quartz crystal microbalance (QCM) and ultraviolet–visible (UV–vis) absorption spectroscopy, revealing a linear increase of the absorbance vs deposited layers. In relation to a possible electrostatic interaction between the groups PO4− (DPPG) and NH3+ (PAH), it was observed through Fourier transform infrared (FTIR) absorption spectroscopy that the spectrum recorded for the spray-LbL film is basically a simple superposition of the FTIR spectra from PAH and DPPG casting films. The latter indicates a weak interaction between both materials, differently of the trend observed for (PAH/DPPG)n grown via dipping-LbL method. Atomic force microscopy (AFM) images of spray-LbL films showed evidences that the DPPG vesicles present in the aqueous dispersion are not destroyed when submitted to pressure conditions during the spray deposition. However, comparing to dipping-LbL, the DPPG vesicles do not cover completely the PAH layer for the spray-LbL film, which was further confirmed by surface-enhanced Raman scattering (SERS) measurements. Moreover, the AFM analysis showed that the spray-LbL deposition led to thicker PAH/DPPG bilayers in average than via dipping-LbL for the same concentrations of PAH solution and DPPG dispersion, which is consistent with QCM and UV–vis absorption results. PAH/DPPG films deposited by dipping- and spray-LbL techniques and also by Langmuir–Blodgett (LB) onto Pt interdigitated electrodes composing an array of sensing units (e-tongue) were applied in the detection of a xanthene derivate (eosin) in diluted solutions (10−9, 10−7 and 10−6M). Despite the LB and LbL films are formed by the same materials (PAH and DPPG), it was found that their different molecular architectures play an important role on the electrical response of Pt interdigitated electrodes in impedance spectroscopy measurements. The high sensitivity reached by these sensing units was intimately related to changes in the film morphology caused by the adsorption of the eosin molecules onto the film surfaces during electrical measurements, as evidenced by micro-Raman technique.

A Drug-Lipid Interaction Model: Atropine Induces Interdigitated Bilayer Structure

X-ray diffraction and fluorescence methods have been carried out to examine the effects of a drug, atropine, on the structure of model membranes: dipalmitoylphosphatidylglycerol(DPPG) multilamella vesicles(MLVs). A structural analysis by small angle x-ray diffraction shows that, with incorporation of atropine, the lamella repeating period of DPPG MLV is decreased from 5.89nm to 4.52nm. Using fluorescence probe 1,6-diphenyl-1,3,5-hexatriene(DPH) we find that the fluorescence intensity of DPH reduces largely at a narrow scope of atropine concentration. The experiments of fluorescence polarization of n-(9-anthroyloxyl)-stearic acid (nAS) and 16-(9-anthroyloxyl)-palmiticacid (16AP) discover that the normal polarization gradient almost disappears in DPPG/atropine system. By colligating the above results, we can draw a conclusion that atropine induces the transition from non-interdigitated to interdigitated stricture of DPPG vesicles in gel phase, which provides a sound model of drug-lipid interaction.

Fluorescence energy transfer in lipid vesicles. A time-resolved analysis using stretched exponentials

Fluorescence energy transfer in lipid vesicles between N-(7-nitrobenz-2-oxa-1,3,-diazol-4-yl-labelled phosphatidylethanolamine (acting as donor) and N-(lissamine-rhodamin B)-labelled phosphotidylethanolamine (acting as acceptor) was studied by steady state and time-resolved fluorescence quenching analysis. Both fluorescent phospholipids were incorporated as minor components in four different types of lipid vesicle: dipalmitoylphosphatidylglycerol vesicles in their L β gel phase at 20 °C and in their L α liquid crystalline phase at 50 °C, and egg yolk phosphatidylethanolamine vesicles at 40 °C in their L α liquid crystalline phase at pH 9.5 and in their H II inverted hexagonal phase at pH 5.0. The quenching of the donor fluorescence by energy transfer is diffusion controlled in all cases, except in the L β gel phase. The dimensionality and type of constraints imposed on diffusion are different in each case, with the most efficient diffusion-controlled quenching in the hexagonal phase.

The steric stabilization of small unilamellar vesicles

Steric stabilization of small unilamellar vesicles composed of dipalmitoyl phosphatidylglycerol (DPPG) is achieved by the incorporation of nonylphenol poly(ethylene oxide) ethers at varying mol%. Nonylphenol poly(ethylene oxide) ethers with an average of either 10.8 (NP 10 or 50 (NP 50 ethylene oxide groups are incorporated into vesicles at between 2.5 and 10 mol%. Vesicle aggregation is induced by the addition of either NaCl or CaCl 2 with the effective diameter of the vesicle dispersion measured as a function of time using photon correlation spectroscopy. The addition of between 5 and 20 mol% NP 10 to DPPG vesicles increases dispersion stability 10-fold in the presence of NaCl but only 2-fold in the presence of CaCl 2. Dispersion stability is increased 100-fold with the addition of 10–20 mol% NP 50 in NaCl and 1000-fold in the presence of CaCl 2. Vesicle aggregation is found to be reversible with 5 mol% added NP 50 in NaCl but only partially reversible in the presence of CaCl 2. The hydrophobic nonylphenol moiety of NP 50 is found to be critical for imparting dispersion stability when compared to the addition of pure poly(ethylene oxide) to vesicles.

Effects of poly( l-lysine) on the structural and thermotropic properties of dipalmitoylphosphatidylglycerol bilayers

The effects of poly( l-lysine) on the structural and thermotropic properties of dipalmitoylphosphatidylglycerol (DPPG) bilayers were studied with differential scanning calorimetry (DSC), X-ray diffraction and freeze-fracture electron microscopy. For thermal behavior, in the DPPG/poly( l-lysine) system the main transition temperature rises to 45.7°C and the pretransition disappears in opposition to pure DPPG vesicles. An additional transition appears approximately at 36°C for the DPPG/poly( l-lysine) system after incubation at 4°C for two months. The incubated sample gives a X-ray diffraction pattern having several additional reflections in the range of 0.2–0.9 nm at 15°C. These results suggest that even in the presence of poly( l-lysine) the DPPG bilayers form the subgel (L c) phase after the long incubation at a low temperature. The X-ray diffraction measurements indicate that the structure of the L c phase for DPPG/poly( l-lysine) system is different from that of pure DPPG bilayers. On the other hand, in the gel (L β′) phase, the wide-angle X-ray diffraction pattern suggests that the presence of poly( l-lysine) hardly affects the packing of hydrocarbon chains in the DPPG bilayers. The small-angle X-ray diffraction and freeze-fracture electron microscopy exhibit that the DPPG/poly( l-lysine) system forms a tightly packed multilamellar structure in which the poly( l-lysine) is intercalated between the subsequent DPPG bilayers.

Effects of random copolymers of lysine on the thermotropic behaviour of dipalmitoylphosphatidylglycerol vesicles. A fluorescence anisotropy study.

The effects of some random copolymers of lysine on the thermotropic behaviour of phospholipid vesicles, mainly dipalmitoylphosphatidylglycerol (DPPG), were studied by monitoring the steady-state fluorescence anisotropy with 1,6-diphenyl-1,3,5-hexatriene (DPH) as a membrane probe. A characteristic effect of the polylysine-tyrosine 4:1 copolymer was observed: DPPG vesicles are strongly stabilized, the gel-to-fluid transition going from 40 degrees C in the absence to 52 degrees C with an excess of copolymer (corresponding to a lipid/amino-acid ratio R = 0.5). For R greater than 0.5, the gel-to-fluid transition shows a three-step profile. This triphasic transition, which appears with a much better resolution than with polylysine, demonstrates the formation of stable distinct domains of reasonable size, coexisting from 41 to 51 degrees C. Such stability, over a temperature range of 10 degrees C, is quite unusual and unexpected for a phase separation of mixed lipid phases. The peculiarity of this copolymer was proved by investigating the interactions of DPPG vesicles with polylysine-tryptophan 4:1, polyornithine-lysine 4:1 and polylysine-tyrosine 1:1. We hypothesize that the observed effects are correlated with the ability of these copolymers to adopt/not adopt an alpha-helical or beta-sheet conformation upon interacting with anionic vesicles.