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Immobilization and characterization of giant unilamellar vesicles (GUVs) within porous silica glasses

Immobilization of cells or artificial liposomes has interesting applications in protein biology, membrane biophysics, biomedicine, biosensor technology and new materials development. In this work for the first time we have entrapped giant unilamellar vesicles (GUVs) in silica glasses prepared by the sol–gel process. Results show that GUVs are successfully confined in the porous matrix retaining their structural integrity for at least fifteen days, allowing single-vesicle studies to be performed. Using different fluorescence microscopy approaches, we have studied the effect of the encapsulation on membrane properties, such as their size and shape, hydration degree, domain coexistence and lipid lateral mobility. Results reveal that these properties are altered to a more or less degree after immobilization, but most of vesicles are affected in a similar fashion and no different populations are distinguished. Such effects are attributed to the increase in lateral packing induced by changes in the hydrostatic and/or osmotic pressure occurring during the sol–gel process, as well as to the establishment of interactions between the polar head of the phospholipids and the negatively charged silica surface of the porous matrix.

Biophysical and functional characterization of an ion channel peptide confined in a sol-gel matrix.

Immobilization of zwitterionic lipid membranes in sol-gel matrices induces irreversible alterations of the bilayer fluidity, which can limit the use of these systems for practical applications. Recently, we have reported that electrostatic interactions between phospholipids polar heads and the negative-charged silica surface of the porous matrix should be the cause of such behavior. In the present work, we analyze the effect of these interactions on the biophysical and functional properties of the ion-channel peptide gramicidin, entrapped in a sol-gel matrix, to get more insight on the ability of these inorganic materials to immobilize ion channels and other membrane-bound proteins. Gramicidin was reconstituted in anionic and zwitterionic liposomes and the effects of sol-gel immobilization on the biophysical properties of gramicidin were determined from changes in the photophysical properties of its tryptophan residues. In addition, the physical state of the immobilized lipid membrane containing gramicidin was analyzed by measuring the spectral shift of the fluorescent probe Laurdan. Finally, the ion-channel activity of the peptide was monitored upon sol-gel immobilization through a fluorescence quenching assay using the fluorescent dye pyrene-1,3,6,8-tetrasulfonic acid (PTSA). Results show that the channel properties of the immobilized gramicidin are preserved in both zwitterionic and anionic liposomes, even though the zwitterionic polar heads interact with the porous surface of the host matrix.

Selective Interaction of a Cationic Polyfluorene with Model Lipid Membranes: Anionic versus Zwitterionic Lipids

This paper explores the interaction mechanism between the conjugated polyelectrolyte {[9,9-bis(6’-N,N,N-trimethylammonium)hexyl]fluorene-phenylene}bromide (HTMA-PFP) and model lipid membranes. The study was carried out using different biophysical techniques, mainly fluorescence spectroscopy and microscopy. Results show that despite the preferential interaction of HTMA-PFP with anionic lipids, HTMA-PFP shows affinity for zwitterionic lipids; although the interaction mechanism is different as well as HTMA-PFP’s final membrane location. Whilst the polyelectrolyte is embedded within the lipid bilayer in the anionic membrane, it remains close to the surface, forming aggregates that are sensitive to the physical state of the lipid bilayer in the zwitterionic system. The different interaction mechanism is reflected in the polyelectrolyte fluorescence spectrum, since the maximum shifts to longer wavelengths in the zwitterionic system. The intrinsic fluorescence of HTMA-PFP was used to visualize the interaction between polymer and vesicles via fluorescence microscopy, thanks to its high quantum yield and photostability. This technique allows the selectivity of the polyelectrolyte and higher affinity for anionic membranes to be observed. The results confirmed the appropriateness of using HTMA-PFP as a membrane fluorescent marker and suggest that, given its different behaviour towards anionic and zwitterionic membranes, HTMA-PFP could be used for selective recognition and imaging of bacteria over mammalian cells.

Formation and Characterization of Stable Fluorescent Complexes Between Neutral Conjugated Polymers and Cyclodextrins

Solubilisation and stabilization of conjugated polymers, CPs, in aqueous media remains a challenge for many researches trying to extend the biological and environmental applications of this kind of polymers. A number of different alternatives have been considered to address this problem, which are mostly based on the enhancement of the macromolecule polarity, by appending hydrophilic side chains on the polymer backbone. In this work we have investigated a new strategy in which water solubilization is reached by external addition of classical cyclodextrins (α-, β- and γ-CDs) to a solution of non-polar CPs. This strategy allows working with such polymers eliminating the need to synthesize new water-soluble species. The polymer selected for the study was poly-[9,9-bis(6′-bromohexyl-2,7-fluoren-dyil)-co-alt-(benzene-1,4-diy)], PFPBr2, a polyfluorene previously synthesized in our laboratory. Results show that PFPBr2 forms fluorescent complexes in aqueous media with β-CD and γ-CD, and much less efficiently with α-CD, probably due to the small size of its cavity. The new PFPBr2/CD complexes are stable in time and in a large range of pH, however, at high concentration and temperature, they tend to aggregate and precipitate. In order to increase stabilization and minimize polymer aggregation, complexes were encapsulated inside the pores of silica glasses fabricated using the sol-gel process, obtaining transparent and fluorescent hybrid matrices which were stable in time and temperature. In addition, immobilization of the complexes allows an easy manipulation of the material, thus offering promising applications in the development of biological and chemical sensors.

Immobilization and characterization of the transmembrane ion channel peptide gramicidin in a sol-gel matrix

Immobilization of ion channels requires of a methodology able to retain the physical properties of the lipid bilayer where their activity is performed. However, most of lipid membrane immobilization methods have been observed to alter the structural properties of the bilayers. Use of sol-gel routes seems to be an interesting alternative, although unstable liposomes were obtained when conventional sol-gel methodology was employed for immobilizing. Recently, we have suggested that use of alcohol-free sol-gel routes combined with negatively charged lipids could minimize effects exerted by host matrix on liposome structure, increasing its stability. Here we confirm this assumption by analysing the physical properties of a series of zwitterionic and anionic liposomes entrapped in a sol-gel matrix and we develop a methodology able to retain the physical properties of the lipid bilayer. This methodology has been successfully used to immobilize the transmembrane ion channel peptide gramicidin. Gramicidin was reconstituted in anionic liposomes and its immobilization was confirmed from changes observed in the photophysical properties of the tryptophan residues. Ion channel activity was determined using the fluorescent dye pyrene-1,3,6,8-tetrasulphonic acid (PTSA) and long term stability of the immobilized system was checked from steady-state fluorescence anisotropy measurements.