Organization and dynamics of NBD-labeled lipids in lipid bilayer analyzed by FRET using the small membrane fluorescent probe AHBA as donor.

Fluorescent probes are employed to investigate natural and model membranes. It is important to know probe location and extent of perturbations they cause into the lipid bilayer. Förster Resonance Energy Transfer (FRET) is a useful tool to investigate phenomena involving plasma membranes, and reports in literature used relatively large fluorophores like 1,6-diphenylhexatriene, located at the center of the hydrophobic region, 4-aminophthalimide-based molecules located at lipid/water interfaces and BODIPY-labeled phosphatidylcholine. In this work we explored FRET process in 1,2-dimyristoyl-L-α-GPC large unilamellar vesicles, in gel and fluid phase, using as donor the very small group o-Abz bound to hexadecyl chain (2-amino-N-hexadecyl-benzamide - AHBA) and 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD) labeled lipids as acceptor. From the intensity decay of donor in presence of acceptors, the FRET efficiency was calculated, and used to fit the model proposed by Fung and Stryer to that efficiency. Using lipid bilayer structural data, the procedure allowed the determination of Förster distance for each donor-acceptor pair in vesicles, without imposing any value for the orientational factor κ2. From distance distributions between o-Abz in AHBA and NBD in lipid bilayer obtained using the program CONTIN, we obtained donor-acceptor populations having different separation distances. The populations reflect the occurrence of FRET involving probes in the same or in opposite leaflet. A dynamic picture emerged showing how relative position of the probes is dependent on the structural thermal phase of the DMPC bilayer. The results emphasize the need of careful analysis in order to understand processes involving fluorescent probes in model membranes.

Effects of nerolidol and limonene on stratum corneum membranes: A probe EPR and fluorescence spectroscopy study.

The sesquiterpene nerolidol and the monoterpene limonene are potent skin-permeation enhancers that have also been shown to have antitumor, antibacterial, antifungal and antiparasitic activities. Because terpenes are membrane-active compounds, we used electron paramagnetic resonance (EPR) spectroscopy of three membrane spin labels combined with the fluorescence spectroscopy of three lipid probes to study the interactions of these terpenes with stratum corneum (SC) intercellular membranes. An experimental apparatus was developed to assess the lipid fluidity of hydrated SC membranes via the fluorescence anisotropy of extrinsic membrane probes. Both EPR and fluorescence probes indicated that the intercellular membranes of neonatal SC rats undergo a main phase transition at approximately 50°C. Taken together, the results indicated that treatment with 1% nerolidol (v/v) caused large fluidity increases in the more ordered phases of SC membranes and that these effects gradually decreased with increasing temperature. Additionally, compared with (+)-limonene, nerolidol was better able to change the SC membrane dynamics. EPR and fluorescence data suggest that these terpenes act as spacers in lipid packaging and create increased lipid disorder in the more ordered regions and phases of SC membranes, notably leading to a population of probes with less restricted motion.

Transmittance and Autofluorescence of Neonatal Rat Stratum Corneum: Nerolidol Increases the Dynamics and Partitioning of Protoporphyrin IX into Intercellular Membranes.

In this work, we developed an experimental apparatus to directly measure transmittance and fluorescence in the stratum corneum (SC) ex vivo. The SC transmittance varied from ~6 to ~52 % in the wavelength range of 280-850 nm. For 260-300 nm excitation, the SC autofluorescence showed a strong emission band between 290 and 425 nm, which is associated with tryptophan, and another in the 600-670 nm range, which we attributed to a process involving resonance energy transfer to very hydrophobic keratin filaments. Weaker emission associated with less hydrophobic keratin filaments was also observed in the wavelength range of 350-480 nm. Protoporphyrin IX (PpIX) was incorporated into SC membranes, and its penetration was further increased by the addition of nerolidol to the treatment suspension. Both PpIX and the endogenous porphyrins showed fluorescence anisotropy consistent with their localization in SC membranes, and their molecular dynamics increased significantly in the presence of 1 % nerolidol. The emission and excitation spectra of PpIX and the endogenous SC porphyrins showed similar alterations during the photobleaching induced by 405-nm irradiation. This work also highlights the SC contribution to skin autofluorescence, which could be useful for fluorescence spectroscopy applications in the early diagnosis of skin diseases.

Interaction of miltefosine with intercellular membranes of stratum corneum and biomimetic lipid vesicles.

Miltefosine (MT) is an alkylphospholipid approved for breast cancer metastasis and visceral leishmaniasis treatments, although the respective action mechanisms at the molecular level remain poorly understood. In this work, the interaction of miltefosine with the lipid component of stratum corneum (SC), the uppermost skin layer, was studied by electron paramagnetic resonance (EPR) spectroscopy of several fatty acid spin-labels. In addition, the effect of miltefosine on (i) spherical lipid vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and (ii) lipids extracted from SC was also investigated, by EPR and time-resolved polarized fluorescence methods. In SC of neonatal Wistar rats, 4% (w/w) miltefosine give rise to a large increase of the fluidity of the intercellular membranes, in the temperature range from 6 to about 50°C. This effect becomes negligible at temperatures higher that ca. 60°C. In large unilamelar vesicles of DPPC no significant changes could be observed with a miltefosine concentration 25% molar, in close analogy with the behavior of biomimetic vesicles prepared with bovine brain ceramide, behenic acid and cholesterol. In these last samples, a 25 mol% molar concentration of miltefosine produced only a modest decrease in the bilayer fluidity. Although miltefosine is not a feasible skin permeation enhancer due to its toxicity, the information provided in this work could be of utility in the development of a MT topical treatment of cutaneous leishmaniasis.

Fluorescence spectroscopy of small peptides interacting with microheterogeneous micelles.

Many peptides containing tryptophan have therapeutic uses and can be studied by their fluorescent properties. The biological activity of these peptides involves interactions with many cellular components and micelles can function as carriers inside organisms. We report results from the interaction of small peptides containing tryptophan with several microheterogeneous systems: sodium dodecyl sulphate (SDS) micelles; sodium dodecyl sulphate-poly(ethylene oxide) (SDS-PEO) aggregates; and neutral polymeric micelles. We observed that specific parameters, such as wavelength of maximum emission and fluorescence anisotropy, could be used to ascertain the occurrence of interactions. Affinity constants were determined from changes in the intensity of emission while structural modifications in rotameric conformations were verified from time-resolved measurements. Information about the location and diffusion of peptides in the microheterogeneous systems were obtained from tryptophan emission quenching experiments using N-alkylpyridinium ions. The results show the importance of electrostatic and hydrophobic effects, and of the ionization state of charged residues, in the presence of anionic and amphiphilic SDS in the microheterogeneous systems. Conformational stability of peptides is best preserved in the interaction with the neutral polymeric micelles.

Spectroscopic characterization of 2-amino-N-hexadecyl-benzamide (AHBA), a new fluorescence probe for membranes.

We report the results of investigation on the spectroscopic properties of a new fluorescent lipophylic probe. The fluorophore o-aminobenzoic acid was covalently bound to the acyl chain hexadecylamine, producing the compound 2-amino-N-hexadecyl-benzamide. The behavior of the probe was dependent on the polarity of the medium: absorption and emission spectral position, quantum yield and lifetime decay indicate distinct behavior in water compared to ethanol and cyclohexane. The probe dissolves in the organic solvents, as indicated by the very low value of steady state fluorescence anisotropy and the short rotational correlation times obtained from fluorescence anisotropy decay measurements. On the other hand, the probe has low solubility in water, leading to the formation of aggregates in aqueous medium. The complex absorption spectrum in water was interpreted as originating from different forms of aggregation, as deduced from the wavelength dependence of anisotropy parameters. The probe interacts with surfactants in pre-micellar and micellar forms, as observed in experiments in the presence of sodium n-dodecylsulphate (SDS), n-cetyltrimethylammonium bromide (CTAB); 3-(dodecyl-dimethylammonium) propane-1-sulphonate (DPS) and 3-(hexadecyl-dimethylammonium) propane-1-sulphonate (HPS), and with vesicles of the phospholipid dimiristoyl-phosphatidylcholine (DMPC). The results demonstrate that AHBA is able to monitor properties like surface electric potential and phase transition of micelles and vesicles.

Study of the interaction between Apis mellifera venom and micro-heterogeneous systems.

The bee venom, used in treatment of inflammatory and articular diseases, is a complex mixture of peptides and enzymes and the presence of tryptophan allows the investigation by fluorescence techniques. Steady state and time-resolved fluorescence spectroscopy were used to study the interaction between bee venom extracted from Apis mellifera and three micro heterogeneous systems: sodium dodecylsulphate (SDS) micelles, sodium dodecylsulphate-poly(ethylene oxide) (SDS-PEO) aggregates, and the polymeric micelles LUTROL F127, formed by poly(ethylene oxide)-poly(propylene oxide)- poly(ethylene oxide). Fluorescence parameters in buffer solution were typical of peptides containing tryptophan exposed to the aqueous medium, and they gradually changed upon the addition of surfactant and polymeric micelles, demonstrating the interaction of the peptides with the micro heterogeneous systems. Quenching experiments were carried out using the N-alkylpyridinium ions (ethyl, hexyl, and dodecyl) as quenchers. In buffer solution the quenching has low efficiency and is independent of the alkyl chain length of the quencher. In the presence of the micro heterogeneous systems the extent of static and dynamic quenching enhanced, showing that both fluorophore and quenchers reside in the microvolume of the aggregates. The more hydrophobic quencher (dodecyl pyridinium ion) provides higher values for K (SV) and dynamic quenching constants, and SDS-PEO aggregates are most efficient to promote interaction between peptides and alkyl pyridinium ions. The results proved that bee venom interacts with drug delivery micelles of the copolymer LUTROL F127.

Tryptophan as a probe for acid-base equilibria in peptides.

We present results of time resolved fluorescence measurements performed in Tryptophan (Trp) derivatives and Trp-containing peptides in the pH range 3.0-11.0. For each compound a set of decay profiles measured in a given range of pH values was examined as a whole, using the global analysis technique. The data were fitted to two or three lifetime components and the analysis allowed the monitoring of the changes in the concentration of the different species contributing to the total fluorescence in that pH interval. The decay components were sensitive to the ionization state of groups neighboring the indol ring, and pK values for the equilibrium between protonated and deprotonated species were obtained from the preexponential factor of the lifetime components. In Trp, protonation of the amino terminal of the rotamer having electron transfer rate comparable to fluorescence decay rates was responsible for the interconvertion of a long lifetime component, to the 2.9 ns component usually observed in neutral pH. Trpbond;X peptides also have a single rotamer dominating the decay that is quenched by NH(3) (+). X-Trp peptides seem to be conformationally less restricted, and it is possible that rotamers interconvertion occur in high pH, increasing the population of nonquenched rotamers. Interconvertion between rotameric conformations of Trp are also present in the titration of ionizable groups in the side chain of peptides like His-Trp and Glu-Trp and control of pH is essential to the correct interpretation of fluorescence data in the study of peptides having such groups near to the Trp residue.