Immobilization of aloin encapsulated into liposomes in Layer-by-layer films for transdermal drug delivery

Layer-by-layer (LbL) films have been exploited in drug delivery systems that may be used in the form of patches, but the encapsulation of poor water soluble drugs and their release with a controlled rate are still major challenges to be faced. In this paper, we demonstrate the controlled release of aloin (barbaloin), an important component of the widely used Aloe vera, encapsulated into liposomes and immobilized in LbL films with a polyelectrolyte. With a systematic study using fluorescence spectroscopy of aloin release from solutions and from LbL films with different phospholipid liposomes, we inferred that optimized release was achieved with aloin incorporated into palmitoyl oleyl phosphatidyl glycerol (POPG) or dipalmitoyl phosphatidyl glycerol (DPPG) liposomes immobilized in LbL films. Significantly, with this optimized system aloin was almost completely released within 30 h, with a small release rate at the end, which followed a sharp release in the first 5 h. Upon comparing the rates of the distinct systems, we conclude that the main factors controlling the release are the electrostatic interactions involving the negatively charged phospholipids. Because these interactions can be tuned in LbL films, the approach used here opens the way for new drug delivery systems to be developed with fine control of the drug release.     

Multiresidue determination of pesticides in honey samples by gas chromatography–mass spectrometry and application in environmental contamination

A simple and fast multiresidue method has been developed to determine 48 pesticides within the major groups of pesticides (organohalogen, organophosphorous, pyrethroids and organonitrogen) in representative samples of locally produced honey, in Bauru (State of São Paulo, Brazil) during 2003–2004. The recovery results found ranged from 76% to 95% and the limits of detection were lower than 0.01 mg/kg for gas chromatography with electron impact mass spectrometric detection in the selected ion monitoring mode (GC–MS-SIM). The results indicated that most pesticides found in the samples belonged to the organohalogen and organophosphorous groups and lower levels of residues of some organonitrogen and pyretroids were also detected. Malathion residues were detected in all the samples, in a high concentration, owing to its applications to control dengue mosquitoes in the area studied.     

Immobilization of liposomes in nanostructured layer-by-layer films containing dendrimers

Artificial vesicles or liposomes composed of lipid bilayers have been widely exploited as building blocks for artificial membranes, in attempts to mimic membrane interaction with drugs and proteins and to investigate drug delivery processes. In this study we report on the immobilization of liposomes of 1,2-dipalmitoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (Sodium Salt) (DPPG) in layer-by-layer (LbL) films, alternated with poly(amidoamine) G4 (PAMAM) dendrimer layers. The average size of the liposomes in solution was 120 nm as determined by dynamic light scattering, with their spherical shape being inferred from scanning electron microscopy (SEM) in cast films. LbL films containing up to 20 PAMAM/DPPG bilayers were assembled onto glass and/or silicon wafer substrates. The growth of the multilayers was achieved by alternately immersing the substrates into the PAMAM and DPPG solutions for 5 and 10 min, respectively. The formation of PAMAM/DPPG liposome multilayers and its ability to interact with BSA were confirmed by Fourier transform infrared spectroscopy (FTIR). The structural features and film thickness were obtained using X-ray diffraction and surface plasmon resonance (SPR).     

Probing Ambiguous Base‐Pairs by Genetic Transformation with XNA Templates

The templating potential of anhydrohexitol oligonucleotides bearing ambiguous bases was studied in vivo, by using a selection screen for mosaic heteroduplex plasmids in Escherichia coli. 1,5‐Anhydro‐2,3‐dideoxy‐2‐(5‐nitroindazol‐1‐yl)‐D ‐arabino‐hexitol showed the greatest ambiguity among the three nucleosides tested. At most two successive ambiguous bases could be tolerated on hexitol templates read in bacterial cells. Hexitol nucleosides bearing simplified heterocycles thus stand as promising monomers for generating random DNA sequences in vivo from defined synthetic oligonucleotides.     

On the Enzymatic Formation of Metal Base Pairs with Thiolated and pKa-Perturbed Nucleotides

The formation of artificial metal base pairs is an alluring and versatile method for the functionalization of nucleic acids. Access to DNA functionalized with metal base pairs is granted mainly by solid-phase synthesis. An alternative, yet underexplored method, envisions the installation of metal base pairs through the polymerization of modified nucleoside triphosphates. Herein, we have explored the possibility of using thiolated and pK_a-perturbed nucleotides for the enzymatic construction of artificial metal base pairs. The thiolated nucleotides S2C, S6G, and S4T as well as the fluorinated analogue 5FU are readily incorporated opposite a templating S4T nucleotide through the guidance of metal cations. Multiple incorporation of the modified nucleotides along with polymerase bypass of the unnatural base pairs are also possible under certain conditions. The thiolated nucleotides S4T, S4T, S2C, and S6G were also shown to be compatible with the synthesis of modified, high molecular weight single-stranded (ss)DNA products through TdT-mediated tailing reactions. Thus, sulfur-substitution and pK_a perturbation represent alternative strategies for the design of modified nucleotides compatible with the enzymatic construction of metal base pairs.     

Characterization of nitrogen-fixing cyanobacteria in the Brazilian Amazon floodplain

The diversity of the free-living nitrogen-fixing cyanobacterial community in the floodplain sediments along the Solimões and Amazon Rivers and some of their tributaries (Japurá, Negro and Madeira) was investigated. Five cyanobacterial genera were morphologically identified, four of which (Nostoc, Calothrix, Cylindrospermum and Fischerella) have not previously been isolated from the Brazilian Amazon floodplain. Nostoc strains were the most commonly found heterocyst-forming cyanobacteria. Five strains (N. muscorum CENA18 and CENA61, N. piscinale CENA21, Cylindrospermum sp. CENA33 and Fischerella sp. CENA19) were selected for growth measurement, ability to fix N₂ and phylogenetic analysis, based on their widespread distribution and morphological distinction. Molecular analyses employing 16S rRNA sequences indicated that some of the isolates may represent novel cyanobacterial species. Dinitrogen fixed by these strains was measured indirectly as acetylene reduction activity and ranged from 11.5 to 22.2 nmol C₂H₄ μg Chl a⁻¹ h⁻¹. These results provide evidence of widespread and importance of nitrogen-fixing cyanobacteria as a source of N inputs in the Amazonian ecosystem.     

Toward preserving the structure of the antigenic peptide p17-1 from the HIV-1 p17 protein in nanostructured films

Antigenic peptides may be immobilized in nanostructured films in order to build highly specific immunosensors and other devices that require molecular recognition, with no need to use complex molecules. A major challenge for such endeavors, however, is to preserve the secondary structure of the peptides after immobilization. In this study, we show that the peptide p17-1 (LSGGELDRWEKIRLRPGG), derived from the HIV-1 p17 protein, may be immobilized in Layer-by-Layer (LbL) films made with polyelectrolytes. Its structure was preserved only if incorporated into phospholipid liposomes, according to fluorescence and circular dichroism (CD) spectroscopy. The lack of secondary structure for the peptide in the LbL film may be associated with the film-forming procedure in which p17-1 was adsorbed from an aqueous solution, where it does not form alpha helices. The importance of structure preservation was clear in the attempts to produce electrochemical immunosensors with the p17-1 peptide without being protected in liposomes in an LbL film. There was no detectable influence of the presence of anti-p17 antibodies, though some molecular interaction could be inferred from the voltammograms. In contrast, for p17-1 incorporated in liposomes electrochemical immunosensors could be obtained with the voltamogramms showing strong molecular recognition with the antibodies. These results indicated that phospholipids serve as a suitable matrix for immobilization of peptides, and confirmed the importance of structure preservation in electrochemical immunosensors.     

Immobilization of cholesterol oxidase in LbL films and detection of cholesterol using ac measurements

The preserved activity of immobilized biomolecules in layer-by-layer (LbL) films can be exploited in various applications, including biosensing. In this study, cholesterol oxidase (COX) layers were alternated with layers of poly(allylamine hydrochloride) (PAH) in LbL films whose morphology was investigated with atomic force microscopy (AFM). The adsorption kinetics of COX layers comprised two regimes, a fast, first-order kinetics process followed by a slow process fitted with a Johnson–Mehl–Avrami (JMA) function, with exponent ~ 2 characteristic of aggregates growing as disks. The concept based on the use of sensor arrays to increase sensitivity, widely employed in electronic tongues, was extended to biosensing with impedance spectroscopy measurements. Using three sensing units, made of LbL films of PAH/COX and PAH/PVS (polyvinyl sulfonic acid) and a bare gold interdigitated electrode, we were able to detect cholesterol in aqueous solutions down to the 10^? 6 M level. This high sensitivity is attributed to the molecular-recognition interaction between COX and cholesterol, and opens the way for clinical tests to be made with low cost, fast experimental procedures.