Impact of nanosilver surface electronic distributions on serum protein interactions and hemocompatibility.

The translation of silver-based nanotechnology 'from bench to bedside' requires a deep understanding of the molecular aspects of its biological action, which remains controversial at low concentrations and non-spherical morphologies. Here, we present a hemocompatibility approach based on the effect of the distinctive electronic charge distribution in silver nanoparticles (nanosilver) on blood components. According to spectroscopic, volumetric, microscopic, dynamic light scattering measurements, pro-coagulant activity tests, and cellular inspection, we determine that at extremely low nanosilver concentrations (0.125-2.5µg ml-1), there is a relevant interaction effect on the serum albumin and red blood cells (RBCs). This explanation has its origin in the surface charge distribution of nanosilver particles and their electron-mediated energy transfer mechanism. Prism-shaped nanoparticles, with anisotropic charge distributions, act at the surface level, generating a compaction of the native protein molecule. In contrast, the spherical nanosilver particle, by exhibiting isotropic surface charge, generates a polar environment comparable to the solvent. Both morphologies induce aggregation at NPs/bovine serum albumin ≈ 0.044 molar ratio values without altering the coagulation cascade tests; however, the spherical-shaped nanosilver exerts a negative impact on RBCs. Overall, our results suggest that the electron distributions of nanosilver particles, even at extremely low concentrations, are a critical factor influencing the molecular structure of blood proteins' and RBCs' membranes. Isotropic forms of nanosilver should be considered with caution, as they are not always the least harmful.

Advanced Protein Drugs and Formulations.

Previously, the application of proteins was uncommon as therapeutically active molecules. Some of the first applications of proteins as drugs have been insulin and vaccines for overcoming a physiological deficiency and the prevention of diseases, respectively. Nowadays, proteins have many applications, not only as drugs but also as drug delivery systems to be administered by different routes. Due to their nature, the behavior of proteins varies while the conditions of the environment are modified. For this reason, it is necessary to study their behavior for predicting the correct manufacturing, storing, or combination with other possible molecules in a formulation or into the body. The application of techniques for predicting the behavior of proteins in different environments has led to associating this type of behavior into the body with the occurrence of diseases, such as celiac disease or Alzheimer's disease. Thus, this work shows an overview of the main types of proteins applied as active therapeutically molecules, proteins-based drug delivery systems, and techniques for predicting their stability into the primary storing container and the body.

Antibacterial alginate/nano-hydroxyapatite composites for bone tissue engineering: Assessment of their bioactivity, biocompatibility, and antibacterial activity.

Bone substitute materials based on bioceramics and polymers have evolved shifting from a passive role where they are merely accepted by the body; to an active role, where they respond to particular environmental conditions or to different types of cues generating suitable integration (osseointegration for this case) inside the host tissue. In this work, two types of composite materials based on a bioceramic (synthetic nano-hydroxyapatite, HA) and a biopolymer (sodium alginate, ALG) have been designed and assessed for promoting the bone regeneration. These materials were loaded with ciprofloxacin (CIP) for obtaining, not only a suitable material for a filling but with antibacterial properties. Therefore, their main features were studied through Fourier transformed-infrared spectroscopy (FT-IR), X-ray diffraction (XRD) and transmission electron microscopy (TEM). Ultraviolet-Visible (UV-Vis) spectroscopy was used for obtaining the released concentrations of CIP and Zeta-potential (ζ-potential) was used for characterizing the adsorption of CIP onto nanoparticles. The release profile of this drug has been fit with the Ritger-Peppas model, used for studying the release kinetics of hydrogel-based systems. The bioactivity of these composites was also evaluated after 30 days of incubation in a simulated body fluid solution (SBF). Then, the assessment of antibacterial capability against the three main strains cause osteomyelitis was performed. Finally, the cell viability study and the cellular morphology assay were also carried out. These last assays have shown encouraging results and, gathered with their other properties, such as their bioactivity and antibacterial properties; they could lead to propose these materials as new bone filler antibiotic devices.

Adsorption/desorption study of antibiotic and anti-inflammatory drugs onto bioactive hydroxyapatite nano-rods.

The use of high doses of antibacterial and anti-inflammatory drugs for patients with bone diseases, associated to implants or bone filling, can develop adverse effects; and consequently, it promotes to think new strategies to avoid this problem. In this work, it has been described the adsorption/release (or desorption) behavior of two drugs, ciprofloxacin (CIP) and ibuprofen (IBU), onto hydroxyapatite (nano-HA) at 37 °C. Through Ultraviolet-Visible (UV-Vis) spectroscopy, the concentrations of both drugs in adsorption, kinetic and desorption processes were obtained. The Fourier Transformed-Infrared (FT-IR) spectroscopy, Zeta-potential (ζ-potential), High-Resolution Transmission Electron Microscopy (H-TEM) and x-Ray Diffraction (xRD) were also used to characterize bared nanoparticles and those with adsorbed drugs. Five adsorption models (Langmuir, Freundlich, Sips, Temkin and Dubinin-Radushkevich) were used for describing the behavior of both active compounds. The adsorption processes (CIP/nano-HA and IBU/nano-HA) were better predicted by the Sips model than by the others. The kinetic adsorption data were processed, for both active agents, by application of Avrami's model. Desorption/release process (of both drugs) was evaluated though Korsmeyer-Peppas (K-P) model. Owing to the predictability of these systems, we propose the use of these active ceramics as potential bone filler for improving the treatment against bacterial bone infections and to avoid its associated inflammatory process.

Multi-drug delivery system based on lipid membrane mimetic coated nano-hydroxyapatite formulations.

Local delivery systems from an osteoconductive biomaterial are suggested as a promising strategy to avoid simultaneously peri-implant traumas and to induce tissue regeneration. In this work, it is detailed the design and construction of a multi-drug delivery formulation based on lipid membrane mimetic coated nano-hydroxyapatite, LMm/nano-HA, as a bone-specific drug delivery approach. The optimal LMm/nano-HA formulation was selected after analysing the lipid/nano-HA interaction by dynamic light scattering (DLS), ζ-potential, transmission electron microscopy (TEM), polarized optical microscopy (POM), differential scanning calorimetry (DSC) and UV-vis spectroscopy. After the initial screening, Ciprofloxacin and Ibuprofen simultaneous -load and -release efficiency from selected LMm/nano-HA was assessed. pH-responsive kinetic profiles of local drug distribution were characterized and compared with currently applied systemic doses. Finally, the systems' biocompatibility and drug released activity were positively validated. The obtained results demonstrated that LMm/nano-HA formulations can represent a valuable multi-modal platform in bone tissue therapies.

Celiac Disease: Historical Standpoint, New Perspectives of Treatments and Contemporary Research Techniques.

Celiac disease (CD) is an inflammatory syndrome that affects mainly the intestine, but also other organs. This ailment is also affected by the physicochemical behavior of gluten as such. From the medical standpoint, this pathology results from a combination of genetic and environmental factors. At the same time, gliadin (the alcohol-soluble fraction of gluten) along with other related oligomers, such as 33-gliadin, present high immunogenicity and are responsible for triggering of this disease. Within CD characterization, there are mainly two different approaches to carry out this study; one focuses on its chronic phase, while the other deals with its initial stages. Although the chronic phase of CD has been well characterized, the initiation of the inflammatory process is still unclear. As this process is apparently related to the aggregation of the oligomers involved in CD, the initiation of the disease could be explained by means of clarifying their self-assembly behavior. Thus, this work addresses the clinical explanation, within the chronic approach, attempting to combine it with the physicochemical techniques used for characterization of proteins aggregates as well.

Biomimetic fiber mesh scaffolds based on gelatin and hydroxyapatite nano-rods: Designing intrinsic skills to attain bone reparation abilities.

Intrinsic material skills have a deep effect on the mechanical and biological performance of bone substitutes, as well as on its associated biodegradation properties. In this work we have manipulated the preparation of collagenous derived fiber mesh frameworks to display a specific composition, morphology, open macroporosity, surface roughness and permeability characteristics. Next, the effect of the induced physicochemical attributes on the scaffold's mechanical behavior, bone bonding potential and biodegradability were evaluated. It was found that the scaffold microstructure, their inherent surface roughness, and the compression strength of the gelatin scaffolds can be modulated by the effect of the cross-linking agent and, essentially, by mimicking the nano-scale size of hydroxyapatite in natural bone. A clear effect of bioactive hydroxyapatite nano-rods on the scaffolds skills can be appreciated and it is greater than the effect of the cross-linking agent, offering a huge perspective for the upcoming progress of bone implant technology.

Development of a Nonionic Azobenzene Amphiphile for Remote Photocontrol of a Model Biomembrane.

We report the synthesis and characterization of a simple nonionic azoamphiphile, C12OazoE3OH, which behaves as an optically controlled molecule alone and in a biomembrane environment. First, Langmuir monolayer and Brewster angle microscopy (BAM) experiments showed that pure C12OazoE3OH enriched in the (E) isomer was able to form solidlike mesophase even at low surface pressure associated with supramolecular organization of the azobenzene derivative at the interface. On the other hand, pure C12OazoE3OH enriched in the (Z) isomer formed a less solidlike monolayer due to the bent geometry around the azobenzene moiety. Second, C12OazoE3OH is well-mixed in a biological membrane model, Lipoid s75 (up to 20%mol), and photoisomerization among the lipids proceeded smoothly depending on light conditions. It is proposed that the cross-sectional area of the hydroxyl triethylenglycol head of C12OazoE3OH inhibits azobenzenes H-aggregation in the model membrane; thus, the tails conformation change due to photoisomerization is transferred efficiently to the lipid membrane. We showed that the lipid membrane effectively senses the azobenzene geometrical change photomodulating some properties, like compressibility modulus, transition temperature, and morphology. In addition, photomodulation proceeds with a color change from yellow to orange, providing the possibility to externally monitor the system. Finally, Gibbs monolayers showed that C12OazoE3OH is able to penetrate the highly packing biomembrane model; thus, C12OazoE3OH might be used as photoswitchable molecular probe in real systems.

Study of the influence of ascorbyl palmitate and amiodarone in the stability of unilamellar liposomes.

Amiodarone (AMI) is a low water-solubility drug, which is very useful in the treatment of severe cardiac disease. Its adverse effects are associated with toxicity in different tissues. Several antioxidants have been shown to reduce, and prevent AMI toxicity. The aim of this work was to develop and characterize Dimyristoylphosphatidylcholine (DMPC) liposomal carriers doped with ascorbyl palmitate (Asc16) as antioxidant, in order to either minimize or avoid the adverse effects produced by AMI. The employment of liposomes would avoid the use of cosolvents in AMI formulations, and Asc16 could minimize the adverse effects of AMI. To evaluate the partition and integration of AMI and Asc16 in lipid membranes, penetration studies into DMPC monolayers were carried out. The disturbance of the liposomes membranes was studied by generalized polarization (GP). The stability of liposomes was evaluated experimentally and by means of the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. The size particle and zeta potential (ζ) values of the liposomes were used for application in calculations for attractive and repulsive forces in DLVO theory. In experimental conditions all of these vesicles showed stability at time 0, but only DMPC + Asc16 10% + AMI 10% liposomes kept their size stable and ζ during 28 days. These results are encouraging and suggest that such systems could be suitable for AMI delivery formulations.

Ascorbyl palmitate interaction with phospholipid monolayers: electrostatic and rheological preponderancy.

Ascorbyl palmitate (ASC16) is an anionic amphiphilic molecule of pharmacological interest due to its antioxidant properties. We found that ASC16 strongly interacted with model membranes. ASC16 penetrated phospholipid monolayers, with a cutoff near the theoretical surface pressure limit. The presence of a lipid film at the interface favored ASC16 insertion compared with a bare air/water surface. The adsorption and penetration time curves showed a biphasic behavior: the first rapid peak evidenced a fast adsorption of charged ASC16 molecules to the interface that promoted a lowering of surface pH, thus partially neutralizing and compacting the film. The second rise represented an approach to the equilibrium between the ASC16 molecules in the subphase and the surface monolayer, whose kinetics depended on the ionization state of the film. Based on the Langmuir dimiristoylphosphatidylcholine+ASC16 monolayer data, we estimated an ASC16 partition coefficient to dimiristoylphosphatidylcholine monolayers of 1.5×10(5) and a ΔGp=-6.7kcal·mol(-1). The rheological properties of the host membrane were determinant for ASC16 penetration kinetics: a fluid membrane, as provided by cholesterol, disrupted the liquid-condensed ASC16-enriched domains and favored ASC16 penetration. Subphase pH conditions affected ASC16 aggregation in bulk: the smaller structures at acidic pHs showed a faster equilibrium with the surface film than large lamellar ones. Our results revealed that the ASC16 interaction with model membranes has a highly complex regulation. The polymorphism in the ASC16 bulk aggregation added complexity to the equilibrium between the surface and subphase form of ASC16, whose understanding may shed light on the pharmacological function of this drug.

The ascorbyl palmitate-polyethyleneglycol 400-water system phase behavior.

Ascorbyl palmitate (Asc16) in polyethyleneglycol 400 (PEG 400)-water mixtures at weight fractions (w/w) between 0.05 and 1.0 were studied by differential scanning calorimetry (DSC) and polarizing microscopy (PM) at different temperatures. The employed PEG 400-water proportions were: 0-25-50% and 75% of polymer. A complete phase diagram was determined for each PEG 400-water mixture. A cubic mesophase and two (probably three) lamellar mesophases were detected in different regions of the phase diagrams. The addition of PEG 400 to the Asc16-water system shifts the limits of the liquid crystalline domains to lower temperature and surfactant concentration. At weight fraction of PEG 400≥50%, the limits of the domain of existence of cubic mesophase shift to low surfactant concentration compared with water-rich systems. The hydrated crystals are Asc16.2·6H(2)O. If the proportion of water is lower than that value, a mixture of hydrated and anhydrous crystals appears. Heating these crystals produce waxy crystals having melted hydrocarbon bilayers retaining their crystalline structure because the polar bilayers are still rigid.

Surface phase behavior and domain topography of ascorbyl palmitate monolayers.

Ascorbyl palmitate (ASC(16)) is a molecule of potential pharmacological interest due to its antioxidant properties and amphiphilic nature. The surface behavior of ASC(16) was studied using Langmuir monolayers and Brewster angle microscopy. This molecule formed stable monolayers at room temperature that showed phase transition from a liquid-expanded to liquid-condensed or crystalline phase, depending on the subphase conditions. Using a theoretical approach, we were able to explain the behavior of the ASC(16) film at different bulk pH values and salt conditions based on the surface pH and the dissociation fraction of the film. Both condensed phases corresponded to highly packed conditions with the crystalline phase occurring at a low charge density, showing molecular tilting and preferential growth at characteristic angles, while the liquid-condensed phase formed in highly charged surfaces revealed small flowerlike domains probably as a consequence of internal dipole repulsion. A smaller perpendicular dipole moment was observed for the crystalline than the liquid-condensed phase which may explain the domain features. In conclusion, ASC(16) showed a complex surface behavior that was highly sensitive to subphase conditions.

Colloidal properties of amiodarone in water at low concentration.

Amiodarone aqueous systems at concentrations C