Properties and cellular uptake of photo-triggered mixed metallosurfactant vesicles intended for controlled CO delivery in gas therapy.

The scientific relevance of carbon monoxide has increased since it was discovered that it is a gasotransmitter involved in several biological processes. This fact stimulated research to find a secure and targeted delivery and lead to the synthesis of CO-releasing molecules. In this paper we present a vesicular CO delivery system triggered by light composed of a synthetized metallosurfactant (TCOL10) with two long carbon chains and a molybdenum-carbonyl complex. We studied the characteristics of mixed TCOL10/phosphatidylcholine metallosomes of different sizes. Vesicles from 80 to 800 nm in diameter are mainly unilamellar, do not disaggregate upon dilution, in the dark are physically and chemically stable at 4 °C for at least one month, and exhibit a lag phase of about 4 days before they show a spontaneous CO release at 37 °C. Internalization of metallosomes by cells was studied as function of the incubation time, and vesicle concentration and size. Results show that large vesicles are more efficiently internalized than the smaller ones in terms of the percentage of cells that show TCOL10 and the amount of drug that they take up. On balance, TCOL10 metallosomes constitute a promising and viable approach for efficient delivery of CO to biological systems.

New Bioconjugated Technetium and Rhenium Folates Synthesized by Transmetallation Reaction with Zinc Derivatives.

The zinc dithiocarbamates functionalized with folic acid 2Zn and 3Zn were synthesized with a simple straightforward method, using an appropriated folic acid derivative and a functionalized zinc dithiocarbamate (1Zn). Zinc complexes 2Zn and 3Zn show very low solubilities in water, making them useful for preparing Tc-99m radiopharmaceuticals with a potentially high molar activity. Thus, the transmetallation reaction in water medium between the zinc complexes 2Zn or 3Zn and the cation fac-[99mTc(H2O)3(CO)3]+, in the presence of the monodentate ligand TPPTS, leads to the formation of the 2 + 1 complexes fac-[99mTc(CO)3(SS)(P)] bioconjugated to folic acid (2Tc and 3Tc). In spite of the low solubility of 2Zn and 3Zn in water, the reaction yield is higher than 95%, and the excess zinc reagent is easily removed by centrifugation. The Tc-99m complexes were characterized by comparing their HPLC with those of the homologous rhenium complexes (2Re and 3Re) previously synthesized and characterized by standard methods. Preliminary in vivo studies with 2Tc and 3Tc indicate low specific binding to folate receptors. In summary, Tc-99m folates 2Tc and 3Tc were prepared in high yields, using a one-pot transmetallation reaction with low soluble zinc dithiocarbamates (>1 ppm), at moderate temperature, without needing a subsequent purification step.

Direct Synthesis of Rhenium and Technetium-99m Metallosurfactants by a Transmetallation Reaction of Lipophilic Groups: Potential Applications in the Radiolabeling of Liposomes.

A new zinc dithiocarbamate functionalized with palmitoyl groups is described as a useful tool for the preparation of metallosurfactants through a transmetallation reaction with the transition metals rhenium and technetium. An amphiphilic rhenium complex is synthesized by a transmetallation reaction with the zinc complex in presence of the polar phosphine sodium triphenylphosphine trisulfonate, which leads to a rhenium complex with a lipophilic dithiocarbamate and a polar phosphine ligand. The study of this rhenium complex has shown that it self-aggregates, leading to the formation of aggregates that have been analyzed by dynamic light scattering and cryotransmission electron microscopy (cryo-TEM). In addition, this amphiphilic rhenium complex is incorporated into soy phosphatidylcholine liposomes, whether liposomes are prepared by mixing phospholipid and the rhenium complex or by the incorporation of the rhenium complex into preformed liposomes. The one-pot reaction of the radiocompound [99mTc(H2O)3(CO)3]+ with the above-mentioned zinc dithiocarbamate, the phosphine sodium triphenylphosphine trisulfonate and the phospholipid soy phosphatidylcholine, leads to liposomes labeled with a Tc-99m homologous complex of the rhenium complex, in accordance with the high-performance liquid chromatography (HPLC) data.

Peptide Assembly on the Membrane Determines the HIV-1 Inhibitory Activity of Dual-Targeting Fusion Inhibitor Peptides.

Novel strategies in the design of HIV-1 fusion/entry inhibitors are based on the construction of dual-targeting fusion proteins and peptides with synergistic antiviral effects. In this work we describe the design of dual-targeting peptides composed of peptide domains of E2 and E1 envelope proteins from Human Pegivirus with the aim of targeting both the loop region and the fusion peptide domains of HIV-1 gp41. In a previous work, we described the inhibitory role of a highly conserved fragment of the E1 protein (domain 139-156) which interacts with the HIV-1 fusion peptide at the membrane level. Here, two different dual-targeting peptides, where this E1 peptide is located on the N- or the C-terminus respectively, have been chemically synthesized and their antiviral activities have been evaluated with HIV pseudotyped viruses from different clades. The study of the functional behaviour of peptides in a membranous environment attending to the peptide recognition of the target sites on gp41, the peptide conformation as well as the peptide affinity to the membrane, demonstrate that antiviral activity of the dual-targeting peptides is directly related to the peptide affinity and its subsequent assembly into the model membrane. The overall results point out to the necessity that fusion inhibitor peptides that specifically interfere with the N-terminal region of gp41 are embedded within the membrane in order to properly interact with their viral target.

Fourier transform infrared spectroscopy (FTIR) characterization of the interaction of anti-cancer photosensitizers with dendrimers.

The systemic or local administration of a photosensitizer for photodynamic therapy is highly limited by poor selectivity, rapid deactivation and long-lasting skin toxicity due to unfavorable biodistribution. Drug delivery systems based on nanocarriers may help specific and effective delivery of photosensitizers. In the present paper, the interaction of two photosensitizers, methylene blue and rose bengal, with phosphorous cationic and anionic dendrimers as potential nanocarriers, has been characterized. A novel method is presented based on the analysis of the infrared spectra of mixtures of photosensitizer and dendrimer. The capacity of dendrimers to bind the photosensitizers has been evaluated by obtaining the corresponding binding curves. It is shown that methylene blue interacts with both cationic and anionic dendrimers, whereas rose bengal only binds to the cationic ones. Dendrimers are shown to be potential nanocarriers for a specific delivery of both photosensitizers.

Steroidal Surfactants: Detection of Premicellar Aggregation, Secondary Aggregation Changes in Micelles, and Hosting of a Highly Charged Negative Substance.

CHAPSO and CHAPS are zwitterionic surfactants derived from bile salts which are usually employed in protein purification and for the preparation of liposomes and bicelles. Despite their spread use, there are significant discrepancies on the critical concentrations that determine their aggregation behavior. In this work, we study the interaction between these surfactants with the negative fluorescent dye pyranine (HPTS) by absorbance, fluorescence, and infrared spectrometry to establish their concentration-dependent aggregation. For the studied surfactants, we detect three critical concentrations showing their concentration-dependent presence as a monomeric form, premicellar aggregates, micelles, and a second type of micelle in aqueous medium. The nature of the interaction of HPTS with the surfactants was studied using analogues of their tails and the negative bile salt taurocholate (TC) as reference for the sterol ring. The results indicate that the chemical groups involved are the hydroxyl groups of the polar face of the sterol ring and the sulfonate groups of the dye. This interaction causes not only the incorporation of the negative dye in CHAPSO and CHAPS micelles but also its association with their premicellar aggregates. Surprisingly, this hosting behavior for a negative charged molecule was also detected for the negative bile salt TC, bypassing, in this way, the electrostatic repulsion between the guest and the host.

Characteristics and behaviour of liposomes when incubated with natural bile salt extract: implications for their use as oral drug delivery systems.

The use of liposomes for oral administration of drugs and for food applications is based on their ability to preserve entrapped substances and to increase their bioavailability. Bile salts are one of the agents that affect the liposome structure during intestinal digestion and the main reported studies on liposome/bile salt systems used only one bile salt. The aim of this work is to characterise the interaction of liposomes with a natural bile salt extract (BSE) at physiological pH and temperature. Three types of liposomes (fluid, gel-state and liquid-ordered bilayers) were studied. Phase diagrams were obtained and a very different behaviour was found. Fluid bilayers were completely permeable to an entrapped dye with partial or complete disruption of vesicles (final size 10 nm). Gel-state bilayers released their content but BSE led to the formation of large mixed structures (2000 nm). Liquid-ordered bilayers formed mixed vesicles (1000 nm) and, surprisingly, retained a high percentage of their aqueous content (about 50%). As a consequence, each type of liposome offers singular features to be used in oral applications due to their specific interaction with bile salts.

Preparation and characterization of iron-containing liposomes: their effect on soluble iron uptake by Caco-2 cells.

The aim of this work was to study the iron uptake of Caco-2 cells incubated with five different formulations of liposomes containing iron. The vesicles were also characterized before, during, and after in vitro digestion. Caco-2 cells were incubated with digested and nondigested liposomes, and soluble iron uptake was determined. Nondigested liposomes made with chitosan (CHI) or the cationic lipid, DC-Cholesterol (DC-CHOL), generated the highest iron uptake. However, these two formulations were highly unstable under in vitro digestion, resulting in nonmeasurable iron uptake. Digested conventional liposomes composed of soybean phosphatidylcholine (SPC), hydrogentated phosphatidylcholine (HSPC), or HSPC and cholesterol (CHOL) presented the highest iron-uptake values. These liposomal formulations protected iron from oxidation and improved iron uptake from intestinal cells, compared to an aqueous solution of ferrous sulphate.

Suspensions of repulsive colloidal particles near the glass transition: Time and frequency domain descriptions.

We study the relaxation of both spontaneous and shear-induced fluctuations in suspensions of charged-stabilized colloidal particles near the glass transition by dynamic light scattering and rheology. Both observations are here understood in terms of a common structural relaxation process under a hard-sphere mode-coupling formalism. For ergodic systems, we show that the descriptions of the relaxation dynamics in time and frequency domains are governed by a common set of dynamic parameters. It is further shown that the microscopic ergodicity break-up induces the emergence of the macroscopic glass elasticity.

Nondiffusive Brownian motion of deformable particles: breakdown of the "long-time tail".

We study the nondiffusive Brownian motion of both rigid and deformable mesoscopic particles by cross-correlated dynamic light scattering with microsecond temporal resolution. Whereas rigid particles show the classical long-time tail prediction, the transition to diffusive motion of deformable particles presents a striking behavior not explained by the existing hydrodynamic treatments. This observation can be interpreted in terms of a damped oscillatory deformational motion on time scales of the order of the Brownian time. Finally, we show that the nondiffusive Brownian motion depends on the specific flexibility of the particles.

Combined strategies for liposome characterization during in vitro digestion.

Three types of pyranine (HPTS)-containing liposomes were prepared by high-pressure homogenization under optimized conditions. At 37 degrees C, they were 1) fluid-state vesicles made from soybean phosphatidylcholine (SPC), 2) gel-state liposomes made from hydrogenated SPC (HSPC), and 3) solid-disordered membranes obtained from HSPC and cholesterol (HSPC-Chol). These liposome formulations were characterized before, during, and after in vitro digestion, which involved the presence of pH gradients, enzymes, and bile salts. Mean sizes and size distributions of the vesicles were determined by DLS; (31)P-NMR (nuclear magnetic resonance) was used to quantify lyso-PC forms; internal pH was monitored throughout digestion with two different fluorescent pH probes; and changes in bilayer permeability and HPTS encapsulation were determined by size-exclusion chromatography and fluorimetry. Differential scanning calorimetry analysis was also performed in order to study the effect of digestion on HSPC vesicles. SPC liposomes were physically stable during digestion; they presented 8% lyso-forms and an HPTS encapsulation around 85% after in vitro digestion. However, they were extremely permeable to ions, so that the internal pH immediately equilibrated with the bulk pH. HSPC liposomes were the most affected by the digestive process. Even though they were chemically stable, as inferred from the low lyso-PC content, very important changes in their size distribution were observed. A final 50% HPTS leakage was quantified after in vitro digestion. Nevertheless, they were the least permeable to protons under pH gradients. HSPC-Chol vesicles presented intermediate permeability to protons, having their internal pH decreased from approximately 6.8 to 4.6 after 1 hour of incubation at pH 2. This was the most chemically stable formulation and showed the highest encapsulation, even after in vitro digestion. Therefore, HSPC-Chol liposomes would be the most adequate choice for the design of lipid products for oral administration.

Surface fractals in liposome aggregation.

In this work, the aggregation of charged liposomes induced by magnesium is investigated. Static and dynamic light scattering, Fourier-transform infrared spectroscopy, and cryotransmission electron microscopy are used as experimental techniques. In particular, multiple intracluster scattering is reduced to a negligible amount using a cross-correlation light scattering scheme. The analysis of the cluster structure, probed by means of static light scattering, reveals an evolution from surface fractals to mass fractals with increasing magnesium concentration. Cryotransmission electron microscopy micrographs of the aggregates are consistent with this interpretation. In addition, a comparative analysis of these results with those previously reported in the presence of calcium suggests that the different hydration energy between lipid vesicles when these divalent cations are present plays a fundamental role in the cluster morphology. This suggestion is also supported by infrared spectroscopy data. The kinetics of the aggregation processes is also analyzed through the time evolution of the mean diffusion coefficient of the aggregates.

Photophysical changes of pyranine induced by surfactants: evidence of premicellar aggregates.

We studied photophysical changes of pyranine in different surfactant environments using spectrophotometry, steady-state fluorescence, and time-resolved fluorescence lifetime. The effect of surfactants on such properties varied as a function of the surfactant charge. Whereas anionic surfactants did not show any kind of interaction, the nonionic surfactant Triton X-100 produced spectral changes in the dye, as a consequence of the shift of equilibrium between its excited species. In the case of fluorescence, these changes allowed the critical micellar concentration of the surfactant to be determined. However, the most important features were obtained from the interaction with cationic surfactants of the n-alkyl trimethylammonium bromides. Such interactions enabled the formation of premicellar aggregates to be determined. In addition, three or four critical concentrations could be defined, which were dependent on the length of the hydrocarbon chain. One of these was the critical micellar concentration of the surfactant. The remaining two or three were in the very dilute concentration domain from which several types of premicellar aggregates are formed. However, the interaction with dodecyltrimethylammonium bromide at submicellar concentrations evidenced a quenching effect of a different nature (either static or combined), depending on the surfactant concentration. By deconvoluting the overall pyranine fluorescence emission spectrum into a sum of overlapping Lorentzian-Gaussian functions, the principal microenvironments of the pyranine molecules can be ascertained. Fluorescence data are consistent with the location of pyranine in a variety of sites, including partitioning that may be influenced by electrostatic and pi-cation interactions in aqueous micelles.

Growth of lipid vesicle structures: from surface fractals to mass fractals.

We study fractal vesicle aggregates whose morphology is conditioned by the interaction between the lipid vesicle membranes and calcium and magnesium ions. These morphologies are probed by means of static light scattering using a cross-correlation scheme that avoids the multiple intracluster scattering. In contrast to the branched structures induced by calcium, we report a singular surface- to mass-fractal transition controlled by the magnesium concentration. From infrared spectroscopy data we conclude that the specific dehydration of the lipid membranes due to these cations plays an essential role in short-range intervesicle interactions.

Preparation and characterization of extruded magnetoliposomes.

Phospholipid vesicles encapsulating magnetic nanoparticles (here after called magnetoliposomes) have been prepared for targeting a drug to a specific organ using a magnetic force, as well as for local hyperthermia therapy. Magnetoliposomes are also an ideal platform for use as contrast agents. We describe the preparation and characterization of liposomes containing magnetite, a ferrimagnetic material. These liposomes were obtained by extrusion. To prevent the aggregation of particles, the magnetite was treated--prior to encapsulation--with a surfactant, resulting in a stable ferrofluid suspension. Once the ferrofluid had been obtained, it was used to hydrate the phospholipid layers. Magnetoliposomes had a diameter of around 200 nm, the same pore size as the membranes used for the extrusion. The encapsulation efficiency was dependent on the initial amount of ferrofluid present at the encapsulation stage, and in the worst case was 19%. This value corresponded to 82.06 mmol of magnetite per mole of phospholipid. Although we have used a determined membrane pore to obtain the magnetoliposomes, the method described here allows to prepare magnetoliposomes of different sizes as well as of different magnetite content.

Aggregation of liposomes induced by calcium: a structural and kinetic study.

In this work, the calcium-induced aggregation of phosphatidylserine liposomes is probed by means of the analysis of the kinetics of such process as well as the aggregate morphology. This novel characterization of liposome aggregation involves the use of static and dynamic light-scattering techniques to obtain kinetic exponents and fractal dimensions. For salt concentrations larger than 5mM, a diffusion-limited aggregation regime is observed and the Brownian kernel properly describes the time evolution of the diffusion coefficient. For slow kinetics, a slightly modified multiple contact kernel is required. In any case, a time evolution model based on the numerical resolution of Smoluchowski's equation is proposed in order to establish a theoretical description for the aggregating system. Such a model provides an alternative procedure to determine the dimerization constant, which might supply valuable information about interaction mechanisms between phospholipid vesicles.