Electrochemical and Spectroscopic Studies Performed for Indomethacin and 1-Naphthol Incorporated in 1-Butyl-3-methyl-imidazolium Bis(2-ethylhexyl) Sulfosuccinate Vesicles to Investigate Them as a Potentially pH-Sensitive Nanocarrier.

Characterization studies of 1-butyl-3-methyl-imidazolium bis(2-ethylhexyl) sulfosuccinate vesicles at different pH values have been carried out by using liquid surface tension, transmission electron microscopy, and dynamic light scattering. The results show that there are no vesicle changes in its size and negative Z potential at pH 3, 6, and 10. Furthermore, indomethacin and 1-naphthol, both pH-dependent, electroactive, and fluorescence probes, were used to further characterize the bilayer employing electrochemical and emission techniques. The partition of indomethacin and 1-naphthol between the water and bilayer pseudophases only occurs for the neutral species and does not happen for the anionic species because the highly negative Z bilayer potential prevents incorporation due to negative repulsion. For the neutral species, the partition constant values were evaluated by square wave voltammetry and emission spectroscopy. Finally, for the indomethacin incorporated into the vesicle bilayer at pH 3, the release profile was monitored over time at pH 6. It was found that a change in the pH values causes the complete release of indomethacin after 25 min, which led us to think that the vesicles presented in this work can be used as a pH-sensitive nanocarrier for neutral pH-sensitive drugs.

PRODAN Photophysics as a Tool to Determine the Bilayer Properties of Different Unilamellar Vesicles Composed of Phospholipids.

Vesicles formed by phospholipids are promising candidates for drug delivery. It is known that the lipid composition affects properties such as the rigidity-fluidity of the membrane and that it influences the bilayer permeability, but sometimes sophisticated techniques are selected to monitor them. In this work, we study the bilayer of different unilamellar vesicles composed of different lipids (1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC, and lecithin) and diverse techniques such as extruder and electrospun templates and using 6-propionyl-2-(N,N-dimethyl) aminonaphthalene (PRODAN) and its photophysics. Moreover, we were able to monitor the influence of cholesterol on the bilayers. We demonstrate that the bilayer properties can be evaluated using the emission feature of the molecular probe PRODAN. This fluorescent probe gives relevant information on the polarity and fluidity of the microenvironment for unilamellar vesicles formed by two different methods. The PRODAN emission at 434 nm suggests that the bilayer properties significantly change if DOPC or lecithin is used in the vesicle preparation especially in their fluidity. Moreover, cholesterol induces alterations in the bilayer's structural and microenvironmental properties to a greater or lesser degree in both vesicles. Thus, we propose an easy and elegant way to evaluate physicochemical properties, which is fundamental for manufacturing vesicles as a drug delivery system, simply by monitoring the molecular probe emission band centered at 434 nm, which corresponds to the PRODAN species deep inside the bilayer.

Green AOT reverse micelles as nanoreactors for alkaline phosphatase. The hydrogen bond "dances" between water and the enzyme, the reaction product, and the reverse micelles interface.

In this work, we present an investigation of the influence of water encapsulated in 1,4-bis-2-ethylhexylsulfosuccinate/methyl laurate and 1,4-bis-2-ethylhexylsulfosuccinate/isopropyl myristate reverse micelles on the enzymatic hydrolysis of 1-naphthyl phosphate by alkaline phosphatase. Our results show that the enzyme is active in the biocompatible reverse micelles studied and that the Michaelis-Menten kinetic model is valid in all systems. We found that both micellar systems studied have a particular behavior toward pH and that the penetration of external solvents into the interfaces is crucial to understanding the effect. Methyl laurate does not disrupt the interface and is not necessary to control the pH value since alkaline phosphatase in the center of the micelles is always solvated similarly. In contrast, isopropyl myristate disrupts the interfaces so that the water and 1-naphthol molecules cannot form hydrogen bond interactions with the polar head of the surfactant. Then, when the water is at pH = 7, the 1-naphthol moves away to the interfaces inhibiting alkaline phosphatase which is not observable when the water is at pH = 10. Our study shows that the concept of pH cannot be used directly in a confined environment. In addition, our research is of great importance in the field of reactions that occur in reverse micelles, catalyzed by enzymes.

Temperature thresholds of ecosystem respiration at a global scale.

Ecosystem respiration is a major component of the global terrestrial carbon cycle and is strongly influenced by temperature. The global extent of the temperature-ecosystem respiration relationship, however, has not been fully explored. Here, we test linear and threshold models of ecosystem respiration across 210 globally distributed eddy covariance sites over an extensive temperature range. We find thresholds to the global temperature-ecosystem respiration relationship at high and low air temperatures and mid soil temperatures, which represent transitions in the temperature dependence and sensitivity of ecosystem respiration. Annual ecosystem respiration rates show a markedly reduced temperature dependence and sensitivity compared to half-hourly rates, and a single mid-temperature threshold for both air and soil temperature. Our study indicates a distinction in the influence of environmental factors, including temperature, on ecosystem respiration between latitudinal and climate gradients at short (half-hourly) and long (annual) timescales. Such climatological differences in the temperature sensitivity of ecosystem respiration have important consequences for the terrestrial net carbon sink under ongoing climate change.

Ensemble modelling, uncertainty and robust predictions of organic carbon in long-term bare-fallow soils.

Simulation models represent soil organic carbon (SOC) dynamics in global carbon (C) cycle scenarios to support climate-change studies. It is imperative to increase confidence in long-term predictions of SOC dynamics by reducing the uncertainty in model estimates. We evaluated SOC simulated from an ensemble of 26 process-based C models by comparing simulations to experimental data from seven long-term bare-fallow (vegetation-free) plots at six sites: Denmark (two sites), France, Russia, Sweden and the United Kingdom. The decay of SOC in these plots has been monitored for decades since the last inputs of plant material, providing the opportunity to test decomposition without the continuous input of new organic material. The models were run independently over multi-year simulation periods (from 28 to 80 years) in a blind test with no calibration (Bln) and with the following three calibration scenarios, each providing different levels of information and/or allowing different levels of model fitting: (a) calibrating decomposition parameters separately at each experimental site (Spe); (b) using a generic, knowledge-based, parameterization applicable in the Central European region (Gen); and (c) using a combination of both (a) and (b) strategies (Mix). We addressed uncertainties from different modelling approaches with or without spin-up initialization of SOC. Changes in the multi-model median (MMM) of SOC were used as descriptors of the ensemble performance. On average across sites, Gen proved adequate in describing changes in SOC, with MMM equal to average SOC (and standard deviation) of 39.2 (±15.5) Mg C/ha compared to the observed mean of 36.0 (±19.7) Mg C/ha (last observed year), indicating sufficiently reliable SOC estimates. Moving to Mix (37.5 ± 16.7 Mg C/ha) and Spe (36.8 ± 19.8 Mg C/ha) provided only marginal gains in accuracy, but modellers would need to apply more knowledge and a greater calibration effort than in Gen, thereby limiting the wider applicability of models.

Water-soluble gold nanoparticles: recyclable catalysts for the reduction of aromatic nitro compounds in water.

A structure/catalytic activity study of water-soluble gold nanoparticles, stabilized by zwitterionic ligands derived from imidazolium salts, in the reduction of aromatic nitro compounds in pure water at different temperature, as well as their recyclability, was performed. Our studies indicate that the nanoparticles synthesized by an easy, fast and reproducible process, need a short characteristic induction time to restructure the surfaces and make them active. The differences observed in the catalytic activity of the nanoparticles, determined by using the typical Langmuir-Hinshelwood model, are strongly based on the degree of coverage and spatial arrangement of the imidazolium salts on them. Finally, we demonstrate that gold nanoparticles stabilized by non-traditional ligands can be an excellent choice for nitro compound degradation.

Generic parameters of first-order kinetics accurately describe soil organic matter decay in bare fallow soils over a wide edaphic and climatic range.

The conventional soil organic matter (SOM) decay paradigm considers the intrinsic quality of SOM as the dominant decay limitation with the result that it is modelled using simple first-order decay kinetics. This view and modelling approach is often criticized for being too simplistic and unreliable for predictive purposes. It is still under debate if first-order models can correctly capture the variability in temporal SOM decay observed between different agroecosystems and climates. To address this question, we calibrated a first-order model (Q) on six long-term bare fallow field experiments across Europe. Following conventional SOM decay theory, we assumed that parameters directly describing SOC decay (rate of SOM quality change and decomposer metabolism) are thermodynamically constrained and therefore valid for all sites. Initial litter input quality and edaphic interactions (both local by definition) and microbial efficiency (possibly affected by nutrient stoichiometry) were instead considered site-specific. Initial litter input quality explained most observed kinetics variability, and the model predicted a convergence toward a common kinetics over time. Site-specific variables played no detectable role. The decay of decades-old SOM seemed mostly influenced by OM chemistry and was well described by first order kinetics and a single set of general kinetics parameters.

Study of lipid peroxidation and ascorbic acid protective role in large unilamellar vesicles from a new electrochemical performance.

In this contribution an electrochemical study is described for the first time of lipid peroxidation and the role of antioxidant on lipid protection using large unilamellar vesicles (LUVs). In order to simulate the cell membrane, LUVs composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were used. A vesicle-modified electrode was constructed by immobilizing DOPC LUVs onto carbon paste electrodes (CPEs). Lipid peroxidation was studied electrochemically by incubating the vesicle-modified electrodes with hydroxyl (HO) radicals generated via the Fenton reaction. Oxidative damage induced by HO was verified by using square wave voltammetry (SWV) and was indirectly measured by the increase of electrochemical peak current to [Fe(CN)6]4- which was used as the electrochemical label. Ascorbic acid (AA) was used as the antioxidant model in order to study its efficacy on free radical scavenging. The decrease of the electrochemical signal confirms the protective key role promoted by AA in the prevention of lipid peroxidation in vesicles. Through microscopy, it was possible to observe morphologic modification on vesicle structures after lipid peroxidation in the presence or absence of AA.

Gold nanoparticles stabilized with sulphonated imidazolium salts in water and reverse micelles.

Herein we describe the synthesis of gold nanoparticles (Au-NPs) in presence of sulphonated imidazolium salts [1,3-bis(2,6-diisopropyl-4-sodiumsulfonatophenyl)imidazolium (L1), 1-mesityl-3-(3-sulfonatopropyl)imidazolium (L2) and 1-(3-sulfonatopropyl)imidazolium (L3)] in water and in a confinement environment created by reverse micelles (RMs). The Au-NPs were characterized-with an excellent agreement between different techniques-by UV-vis spectroscopy, transmission electron microscopy (TEM), dynamic light scattering (DLS) and zeta potential. In homogeneous media, the Au-NPs interact with the imidazolium ring and the sulphonate groups were directed away from the NPs' surface. This fact is responsible for the Au-NPs' stability-over three months-in water. Based on the obtained zeta potential values we assume the degree of coverage of the Au-NPs by the imidazolium salts. In n-heptane/sodium 1,4-bis (2-ethylhexyl) sulfosuccinate (AOT)/water RMs, the Au-NPs formed in presence of sulphonated imidazolium salts present different patterns depending on the ligand used as stabilizer. Interestingly, the Au-NPs are more stable in time when the salts are present in AOT RMs (three weeks) in comparison with the same RMs system but in absence of ligands (less than an hour). Clearly, the sulphonated imidazolium salts are very effective Au-NPs stabilizers in a different medium and this generates a plus to be able to use them for multiple purposes.

Effect of climate warming on the annual terrestrial net ecosystem CO2 exchange globally in the boreal and temperate regions.

The net ecosystem CO2 exchange is the result of the imbalance between the assimilation process (gross primary production, GPP) and ecosystem respiration (RE). The aim of this study was to investigate temperature sensitivities of these processes and the effect of climate warming on the annual terrestrial net ecosystem CO2 exchange globally in the boreal and temperate regions. A database of 403 site-years of ecosystem flux data at 101 sites in the world was collected and analyzed. Temperature sensitivities of rates of RE and GPP were quantified with Q 10, defined as the increase of RE (or GPP) rates with a temperature rise of 10 °C. Results showed that on the annual time scale, the intrinsic temperature sensitivity of GPP (Q 10sG ) was higher than or equivalent to the intrinsic temperature sensitivity of RE (Q 10sR ). Q 10sG was negatively correlated to the mean annual temperature (MAT), whereas Q 10sR was independent of MAT. The analysis of the current temperature sensitivities and net ecosystem production suggested that temperature rise might enhance the CO2 sink of terrestrial ecosystems both in the boreal and temperate regions. In addition, ecosystems in these regions with different plant functional types should sequester more CO2 with climate warming.

Gold nanoparticles covalently assembled onto vesicle structures as possible biosensing platform.

In this contribution a strategy is shown to covalently immobilize gold nanoparticles (AuNPs) onto vesicle bilayers with the aim of using this nanomaterial as platform for the future design of immunosensors. A novel methodology for the self-assembly of AuNPs onto large unilamellar vesicle structures is described. The vesicles were formed with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-undecanethiol (SH). After, the AuNPs photochemically synthesized in pure glycerol were mixed and anchored onto SH-DOPC vesicles. The data provided by voltammetry, spectrometry and microscopy techniques indicated that the AuNPs were successfully covalently anchored onto the vesicle bilayer and decorated vesicles exhibit a spherical shape with a size of 190 ± 10 nm. The developed procedure is easy, rapid and reproducible to start designing a possible immunosensor by using environmentally friendly procedures.

Singularities in the physicochemical properties of spontaneous AOT-BHD unilamellar vesicles in comparison with DOPC vesicles.

In the present work, we study different physicochemical properties of the spontaneous unilamellar vesicles created by the catanionic ionic liquid-like surfactant benzyl-n-hexadecyldimethylammonium 1,4-bis-2-ethylhexylsulfosuccinate (AOT-BHD), using two different fluorescent probes: 6-propionyl-2-(dimethylaminonaphthalene), PRODAN and trans-4-[4-(dimethylamino)-styryl]-1-methylpyridinium iodide, HC. Steady-state and time resolved fluorescence emission spectroscopy allowed us to find the unique properties of the AOT-BHD bilayer in comparison with vesicles formed using the traditional phospholipid 1,2-di-oleoyl-sn-glycero-3-phosphatidylcholine, DOPC. From the emission results, we observed that the region of the bilayer close to the polar head of AOT-BHD is a powerful electron donor environment, even larger than DOPC. Additionally, the AOT-BHD bilayer offers a less polar and slightly more viscous zone than DOPC. Thus, this particular bilayer is able to produce large incorporation of ionic and nonionic molecules and is very promising to be used as a nanocarrier in pharmacological, cosmetic and food fields.

On the characterization of NaDEHP/n-heptane nonaqueous reverse micelles: the effect of the polar solvent.

The behavior of two polar solvents, ethylene glycol (EG) and dimethylformamide (DMF), entrapped in sodium bis-(2-ethylhexyl) phosphate (NaDEHP)/n-heptane reverse micelles (RMs) was investigated using dynamic light scattering (DLS), molecular probe absorption and FT-IR spectroscopy. DLS results reveal the formation of RMs containing EG and DMF as a polar component. To the best of our knowledge this is the first report where both polar solvents are entrapped by the NaDEHP surfactant to effectively create RMs. We use the solvatochromism behavior of the molecular probe, 1-methyl-8-oxyquinolinum betaine (QB), and FT-IR spectroscopy to investigate the physicochemical properties of the non-aqueous RMs. Our results demonstrate that the NaDEHP surfactant interacts through hydrogen bonds with EG at the EG/NaDEHP interface and this interaction is responsible for destroying the bulk structure of pure solvent EG when entrapped in NaDEHP RMs. On the other hand, when DMF is incorporated inside the RMs the bulk structure of DMF is destroyed upon encapsulation by the Na-DMF interaction at the DMF/NaDEHP interface. Our results are completely different than the one observed for DMF/n-heptane/AOT. Our results show how the physicochemical properties, such as micropolarity, microviscosity and hydrogen bond interaction, of nonaqueous NaDEHP/n-heptane RMs interfaces can be dramatically changed by simply using different non-aqueous polar solvents. Thus, these results can be very useful to employ these novel RMs as nanoreactors since the dimensions of the RMs are around 10 to 20 nm.

Higher temperature sensitivity for stable than for labile soil organic carbon--evidence from incubations of long-term bare fallow soils.

The impact of climate change on the stability of soil organic carbon (SOC) remains a major source of uncertainty in predicting future changes in atmospheric CO2 levels. One unsettled issue is whether the mineralization response to temperature depends on SOC mineralization rate. Long-term (>25 years) bare fallow experiments (LTBF) in which the soil is kept free of any vegetation and organic inputs, and their associated archives of soil samples represent a unique research platform to examine this issue as with increasing duration of fallow, the lability of remaining total SOC decreases. We retrieved soils from LTBF experiments situated at Askov (Denmark), Grignon (France), Ultuna (Sweden), and Versailles (France) and sampled at the start of the experiments and after 25, 50, 52, and 79 years of bare fallow, respectively. Soils were incubated at 4, 12, 20, and 35 °C and the evolved CO2 monitored. The apparent activation energy (Ea) of SOC was then calculated for similar loss of CO2 at the different temperatures. The Ea was always higher for samples taken at the end of the bare-fallow period, implying a higher temperature sensitivity of stable C than of labile C. Our results provide strong evidence for a general relationship between temperature sensitivity and SOC stability upon which significant improvements in predictive models could be based.

Enzymatic hydrolysis of N-benzoyl-L-tyrosine p-nitroanilide by α-chymotrypsin in DMSO-water/AOT/n-heptane reverse micelles. A unique interfacial effect on the enzymatic activity.

The reverse micelle (RM) media are very good as nanoreactors because they can create a unique microenvironment for carrying out a variety of chemical and biochemical reactions. The aim of the present work is to determine the influence of different water-dimethyl sulfoxide (DMSO) mixtures encapsulated in 1,4-bis-2-ethylhexylsulfosuccinate (AOT)/n-heptane RMs on the enzymatic hydrolysis of N-benzoyl-L-tyrosine p-nitroanilide (Bz-Try-pNA) by α-chymotrypsin (α-CT). The reaction was first studied in homogeneous media at different DMSO-water mixture compositions and in DMSO-water/AOT/n-heptane RMs. The hydrolysis rates of Bz-Try-pNA catalyzed by α-CT were determined by UV-vis spectroscopy. The reaction follows the Michaelis-Menten mechanism and the kinetic parameters: kcat, KM, and kcat/KM were evaluated under different conditions. In this homogeneous media, DMSO plays an important role in the solubilization process of the peptide which is almost insoluble in water, but it has a tremendous impact on the inactivation of α-CT. It is shown that the enzyme dissolved in a 20% molar ratio of the DMSO-water mixture does not present enzymatic activity. Dynamic light scattering has been used to assess the formation of DMSO-water/AOT/heptane RMs at different DMSO compositions. The results also show that there is preferential solvation of the AOT RM interface by water molecules. To test the use of these RMs as nanoreactors, the kinetic parameters for the enzymatic reaction in these systems have been evaluated. The parameters were determined at fixed W(S) {W(S) = ([water] + [DMSO])/[AOT] = 20} at different DMSO-water compositions. The results show that the Michaelis-Menten mechanism is valid for α-CT in all the RM systems studied and that the reaction takes place at the RM interface. Surprisingly, it was observed that the enzyme encapsulated by the RMs show catalytic effects with similar kcat/KM values at any DMSO composition investigated, which evidence that DMSO molecules are localized far from the RM interface.

A unique ionic liquid with amphiphilic properties that can form reverse micelles and spontaneous unilamellar vesicles.

Catanionic surfactants: the synthesis of a new surfactant ionic liquid with unique properties is described. The formation of reverse micelles in benzene and large unilamellar vesicles, formed spontaneously without the help of any mechanical of chemistry methods, in water is demonstrated by using dynamic light scattering and small-angle X-ray scattering techniques.

Cationic reverse micelles create water with super hydrogen-bond-donor capacity for enzymatic catalysis: hydrolysis of 2-naphthyl acetate by alpha-chymotrypsin.

Reverse micelles (RMs) are very good nanoreactors because they can create a unique microenvironment for carrying out a variety of chemical and biochemical reactions. The aim of the present work is to determine the influence of different RM interfaces on the hydrolysis of 2-naphthyl acetate (2-NA) by alpha-chymotrypsin (alpha-CT). The reaction was studied in water/benzyl-n-hexadecyldimethylammonium chloride (BHDC)/benzene RMs and, its efficiency compared with that observed in pure water and in sodium 1,4-bis-2-ethylhexylsulfosuccinate (AOT) RMs. Thus, the hydrolysis rates of 2-NA catalyzed by alpha-CT were determined by spectroscopic measurements. In addition, the method used allows the joint evaluation of the substrate partition constant K(p) between the organic and the micellar pseudophase and the kinetic parameters: catalytic rate constant k(cat), and the Michaelis constant K(M) of the enzymatic reaction. The effect of the surfactant concentration on the kinetics parameters was determined at constant W(0)=[H(2)O]/[surfactant], and the variation of W(0) with surfactant constant concentration was investigated. The results show that the classical Michaelis-Menten mechanism is valid for alpha-CT in all of the RMs systems studied and that the reaction takes place at both RM interfaces. Moreover, the catalytic efficiency values k(cat)/K(M) obtained in the RMs systems are higher than that reported in water. Furthermore, there is a remarkable increase in alpha-CT efficiency in the cationic RMs in comparison with the anionic system, presumably due to the unique water properties found in these confined media. The results show that in cationic RMs the hydrogen-bond donor capacity of water is enhanced due to its interaction with the cationic interface. Hence, entrapped water can be converted into "super-water" for the enzymatic reaction studied in this work.

An alternative approach to quantify partition processes in confined environments: the electrochemical behavior of PRODAN in unilamellar vesicles.

Herein, we investigate the behavior of the electroactive molecular probe 6-propionyl-2-dimethyl amino naphthalene (PRODAN) in large unilamellar vesicles (LUV) formed with the phospholipid 1,2-di-oleoyl-sn-glycero-3-phosphatidylcholine (DOPC) by using cyclic voltammetry (CV). The CV studies in pure water confirm our previous spectroscopic results that PRODAN self-aggregates due to its low water solubility. Moreover, the electrochemical results also reveal that the PRODAN aggregated species are non-electroactive within the studied electrochemical potential region. In DOPC LUV media, the redox behavior of PRODAN shows how the LUV bilayer interacts with PRODAN aggregated species to form PRODAN monomer species. Moreover, the electrochemical response of PRODAN allows us to propose a model for explaining the electrochemical experimental results and--in conjunction with our measurements--for calculating the value of the partition constant (K(p)) of PRODAN between the water and LUV bilayer pseudophases. This value coincides with that obtained through an independent technique. Moreover, our electrochemical model allows us to calculate the diffusion coefficient (D) for the DOPC LUV, which coincides with the D value obtained through dynamic light scattering (DLS). Thus, our data clearly show that electrochemical measurements could be a powerful alternative approach to investigate the behavior of nonionic electroactive molecules embed in a confined environment such as the LUV bilayer. Moreover, we believe that this approach can be used to investigate the behavior of non-optical molecular drugs embedded in bilayer media.

Characterization of multifunctional reverse micelles' interfaces using hemicyanines as molecular probes. II: Effect of the surfactant.

In this work, we have investigated the behavior of the cationic hemicyanine trans-4-[4-(dimethylamino)-styryl]-N-methylpyridinium iodide (HC) in benzene/benzyl-n-hexadecyl dimethylammonium chloride (BHDC)/water reverse micelle media using absorption and emission spectroscopy in addition to the steady-state and time-resolved fluorescence emission techniques and compare the results to those obtained in benzene/sodium 1,4-bis-2-ethylhexylsulfosuccinate (AOT)/water reverse micelle media (Moyano, F.; et al. J. Phys. Chem. B 2009, 113, 4284.) in order to gain more insight about reverse micelle interface properties. Our results show that HC spectroscopic behavior is completely different when dissolved in AOT or in BHDC reverse micelle media. While the dye experiences an intramolecular charge-transfer process upon excitation in the former media, in BHDC, this process is inhibited because of the cationic nature of the surfactant. Interestingly, we also show that the water properties are different for water molecules sequestrated inside of an anionic and cationic reverse micelle system. This come out because the water molecules entrapped inside of the BHDC reverse micelle media appear to be non-electron-donating because of its interaction with the cationic surfactant polar head group. On the other hand, the water molecules sequestrated inside of the AOT reverse micelle systems show its electron-donor ability enhanced in comparison with its water bulk structure. These results could also explain the lack of nucleophilicity shown by the water molecules entrapped in BHDC reverse micelle media reported in previous kinetic studies.