Víctor Mosquera

Physicochemical characteristics of protein-NP bioconjugates: the role of particle curvature and solution conditions on human serum albumin conformation and fibrillogenesis inhibition.

Gold nanoparticles (Au NPs) from 5 to 100 nm in size synthesized with HAuCl(4) and sodium citrate were complexed with the plasma protein human serum albumin (HSA). Size, surface charge, and surface plasmon bands of the Au NPs are largely modified by the formation of a protein corona via electrostatic interactions and hydrogen bonding as revealed by thermodynamic data. Negative values of the entropy of binding suggested a restriction in the biomolecule mobility upon adsorption. The structure of the adsorbed protein molecules is slightly affected by the interaction with the metal surface, but this effect is enhanced as the NP curvature decreases. Also, it is observed that the protein molecules adsorbed onto the NP surface are more resistant to complete thermal denaturation than free protein ones as deduced from the increases in the melting temperature of the adsorbed protein. Differences in the conformations of the adsorbed protein molecules onto small (<40 nm) and large NPs were observed on the basis of ζ-potential data and FTIR spectroscopy, also suggesting a better resistance of adsorbed protein molecules to thermal denaturing conditions. We think this enhanced protein stability is responsible for a reduced formation of HSA amyloid-like fibrils in the presence of small Au NPs under HSA fibrillation conditions.

Existence of Different Structural Intermediates on the Fibrillation Pathway of Human Serum Albumin

The fibrillation propensity of the multidomain protein human serum albumin (HSA) was analyzed under different solution conditions. The aggregation kinetics, protein conformational changes upon self-assembly, and structure of the different intermediates on the fibrillation pathway were determined by means of thioflavin T (ThT) fluorescence and Congo Red absorbance; far- and near-ultraviolet circular dichroism; tryptophan fluorescence; Fourier transform infrared spectroscopy; x-ray diffraction; and transmission electron, scanning electron, atomic force, and microscopies. HSA fibrillation extends over several days of incubation without the presence of a lag phase, except for HSA samples incubated at acidic pH and room temperature in the absence of electrolyte. The absence of a lag phase occurs if the initial aggregation is a downhill process that does not require a highly organized and unstable nucleus. The fibrillation process is accompanied by a progressive increase in the beta-sheet (up to 26%) and unordered conformation at the expense of alpha-helical conformation, as revealed by ThT fluorescence and circular dichroism and Fourier transform infrared spectroscopies, but changes in the secondary structure contents depend on solution conditions. These changes also involve the presence of different structural intermediates in the aggregation pathway, such as oligomeric clusters (globules), bead-like structures, and ring-shaped aggregates. We suggest that fibril formation may take place through the role of association-competent oligomeric intermediates, resulting in a kinetic pathway via clustering of these oligomeric species to yield protofibrils and then fibrils. The resultant fibrils are elongated but curly, and differ in length depending on solution conditions. Under acidic conditions, circular fibrils are commonly observed if the fibrils are sufficiently flexible and long enough for the ends to find themselves regularly in close proximity to each other. These fibrils can be formed by an antiparallel arrangement of beta-strands forming the beta-sheet structure of the HSA fibrils as the most probable configuration. Very long incubation times lead to a more complex morphological variability of amyloid mature fibrils (i.e., long straight fibrils, flat-ribbon structures, laterally connected fibers, etc.). We also observed that mature straight fibrils can also grow by protein oligomers tending to align within the immediate vicinity of the fibers. This filament + monomers/oligomers scenario is an alternative pathway to the otherwise dominant filament + filament manner of the protein fibrils lateral growth. Conformational preferences for a certain pathway to become active may exist, and the influence of environmental conditions such as pH, temperature, and salt must be considered.

Prediction of Mortality and Major Cardiac Events by Exercise Echocardiography in Patients With Normal Exercise Electrocardiographic Testing

OBJECTIVES We sought to assess the value of exercise echocardiography (EE) for predicting outcome in patients with known or suspected coronary artery disease and normal exercise electrocardiogram (ECG) testing. BACKGROUND The prognostic value of EE in patients with normal exercise ECG testing has not been characterized. METHODS We studied 4,004 consecutive patients (2,358 men, mean age [+/- SD] 59.6 +/- 12.5 years) with interpretable ECG who underwent treadmill EE and did not develop chest pain or ischemic ECG abnormalities during the tests. Wall motion score index (WMSI) was evaluated at rest and with exercise, and the difference (DeltaWMSI) was calculated. Ischemia was defined as the development of new or worsening wall motion abnormalities with exercise. End points were all-cause mortality and major cardiac events (MACE). RESULTS Overall, 669 patients (16.7%) developed ischemia with exercise. During a mean follow-up of 4.5 +/- 3.4 years, 313 patients died, and 183 patients had a MACE before any revascularization procedure. The 5-year mortality and MACE rates were 6.4% and 4.2% in patients without ischemia versus 12.1% and 10.1% in those with ischemia, respectively (p < 0.001). In the multivariate analysis, DeltaWMSI remained an independent predictor of mortality (hazard ratio [HR]: 2.73, 95% confidence interval [CI]: 1.40 to 5.32, p = 0.003) and MACE (HR: 3.59, 95% CI: 1.42 to 9.07, p = 0.007). The addition of the EE results to the clinical, resting echocardiographic and exercise hemodynamic data significantly increased the global chi-square of the models for the prediction of mortality (p = 0.005) and MACE (p = 0.009). CONCLUSIONS The use of EE provides significant prognostic information for predicting mortality and MACE in patients with interpretable ECG and normal exercise ECG testing.

Fluorescent drug-loaded, polymeric-based, branched gold nanoshells for localized multimodal therapy and imaging of tumoral cells.

Here we report the synthesis of PLGA/DOXO-core Au-branched shell nanostructures (BGNSHs) functionalized with a human serum albumin/indocyanine green/folic acid complex (HSA-ICG-FA) to configure a multifunctional nanotheranostic platform. First, branched gold nanoshells (BGNSHs) were obtained through a seeded-growth surfactant-less method. These BGNSHs were loaded during the synthetic process with the chemotherapeutic drug doxorubicin, a DNA intercalating agent and topoisomerase II inhibitior. In parallel, the fluorescent near-infrared (NIR) dye indocyanine green (ICG) was conjugated to the protein human serum albumin (HSA) by electrostatic and hydrophobic interactions. Subsequently, folic acid was covalently attached to the HSA-ICG complex. In this way, we created a protein complex with targeting specificity and fluorescent imaging capability. The resulting HSA-ICG-FA complex was adsorbed to the gold nanostructures surface (BGNSH-HSA-ICG-FA) in a straightforward incubation process thanks to the high affinity of HSA to gold surface. In this manner, BGNSH-HSA-ICG-FA platforms were featured with multifunctional abilities: the possibility of fluorescence imaging for diagnosis and therapy monitoring by exploiting the inherent fluorescence of the dye, and a multimodal therapy approach consisting of the simultaneous combination of chemotherapy, provided by the loaded drug, and the potential cytotoxic effect of photodynamic and photothermal therapies provided by the dye and the gold nanolayer of the hybrid structure, respectively, upon NIR light irradiation of suitable wavelength. The combination of this trimodal approach was observed to exert a synergistic effect on the cytotoxicity of tumoral cells in vitro. Furthermore, FA was proved to enhance the internalization of nanoplatform. The ability of the nanoplatforms as fluorescence imaging contrast agents was tested by preliminary analyzing their biodistribution in vivo in a tumor-bearing mice model.

Influence of Electrostatic Interactions on the Fibrillation Process of Human Serum Albumin

The fibrillation propensity of the multidomain protein human serum albumin (HSA) has been analyzed under physiological and acidic conditions at room and elevated temperatures with varying ionic strengths by different spectroscopic techniques. The kinetics of fibril formation under the different solution conditions and the structures of resulting fibrillar aggregates were also determined. In this way, we have observed that fibril formation is largely affected by electrostatic shielding: at physiological pH, fibrillation is progressively more efficient and faster in the presence of up to 50 mM NaCl; meanwhile, at larger salt concentrations, excessive shielding and further enhancement of the solution hydrophobicity might involve a change in the energy landscape of the aggregation process, which makes the fibrillation process difficult. In contrast, under acidic conditions, a continuous progressive enhancement of HSA fibrillation is observed as the electrolyte concentration in solution increases. Both the distinct ionization and initial structural states of the protein before incubation may be the origin of this behavior. CD, FT-IR, and tryptophan fluorescence spectra seem to confirm this picture by monitoring the structural changes in both protein tertiary and secondary structures along the fibrillation process. On the other hand, the fibrillation of HSA does not show a lag phase except at pH 3.0 in the absence of added salt. Finally, differences in the structure of the intermediates and resulting fibrils under the different conditions are also elucidated by TEM and FT-IR.

Surface properties of some amphiphilic antidepressant drugs

Critical micelle concentrations and surface properties of the amphiphilic antidepressant drugs amitriptyline, nortriptyline and desipramine hydrochlorides in aqueous solution at 298 K were determined by surface tension measurements. Combining the surface parameters with the standard Gibbs energy of aggregation DG m calculated in a previous paper, the standard Gibbs energy of adsorption, DG ads , was obtained.

Left atrial size and risk for all-cause mortality and ischemic stroke

Background: Limited data are available on the relation between left atrial size and outcome among patients referred for clinically indicated echocardiograms. Our aim was to assess the association of left atrial size with all-cause mortality and ischemic stroke in a large cohort of patients referred for echocardiography. Methods: Left atrial diameter was measured in 52 639 patients aged 18 years or older (mean age 61.8 [standard deviation (SD) 16.3] years; 52.9% men) who underwent a first transthoracic echocardiogram for clinical reasons at our institution between April 1990 and March 2008. The outcomes were all-cause mortality and nonfatal ischemic stroke. Results: Based on the criteria of the American Society of Echocardiography, 50.4% of the patients had no left atrial enlargement, whereas 24.5% had mild, 13.3% had moderate and 11.7% had severe left atrial enlargement. Over a mean follow-up period of 5.5 (SD 4.1) years, 12 527 patients died, and 2314 patients had a nonfatal ischemic stroke. Cumulative 10-year survival was 73.7% among patients with normal left atrial size, 62.5% among those with mild enlargement, 54.8% among those with moderate enlargement and 45% among those with severe enlargement (p < 0.001). After adjustment in multivariable Cox proportional hazard analysis, left atrial diameter remained a predictor of all-cause mortality in both sexes (hazard ratio [HR] per 1-cm increment in left atrial size 1.17, 95% confidence interval [CI] 1.12–1.22, p < 0.001 in women, and HR 1.09, 95% CI 1.05–1.13, p < 0.001 in men) and of ischemic stroke in women (HR 1.25, 95% CI 1.14–1.37, p < 0.001). Interpretation: Left atrial diameter has a graded and independent association with all-cause mortality in both sexes and with ischemic stroke in women.

Effect of alcohols on the micellar properties in aqueous solution of alkyltrimethylammonium bromides

The effect ofn-butanol,n-propanol, andn-hexanol on the critical micelle concentration (CMC) and degree of ionisation of the micelles of dodecyl-, tetradecyl- and hexadecyltrimethylammonium bromides in aqueous solution has been determined by conductimetric techniques. Increase of the molality of added alcohol over the concentration ranges examined (up to 0.3 mol kg−1 butanol, 0.07 mol kg−1 pentanol and 0.025 mol kg−1 hexanol) caused a progressive decrease of CMC and increase of the degree of ionisation for each surfactant-alcohol system. At a constant molality of added alcohol the degree of ionisation increased with a) an increase of the chain length of the surfactant for each alcohol and b) an increase of the chain length of the alcohol for each surfactant. The distribution of each alcohol between the aqueous and micellar phases and the free energy of solubilization were determined from the change of CMC with molality of added alcohol.

Static and dynamic light scattering study on the association of some antidepressants in aqueous electrolyte solutions

The self-association of the antidepressant drugs amitriptyline, nortriptyline, doxepin and desipramine hydrochlorides in aqueous solution has been studied by static and dynamic light scattering at 298.15 K in the presence of added electrolyte (0.0–0.4 mol kg−1 NaCl). Self-assembly was by a closed association process, commencing at a well-defined critical concentration for all systems. Micelle properties determined from static and dynamic light scattering techniques as a function of electrolyte concentration were related to differences in the structure of the hydrophobe. The pair interaction potential between aggregates has been discussed in terms of the DLVO theory of colloidal stability.

Exact mean-field theory of ionic solutions: non-Debye screening

Abstract The main aim of this report is to analyze the equilibrium properties of primitive model (PM) ionic solutions in the formally exact mean-field formalism. Previously, we review the main theoretical and numerical results reported throughout the last century for homogeneous (electrolytes) and inhomogeneous (electric double layer, edl) ionic systems, starting with the classical mean-field theory of electrolytes due to Debye and Huckel (DH). In this formalism, the effective potential is derived from the Poisson–Boltzmann (PB) equation and its asymptotic behavior analyzed in the classical Debye theory of screening. The thermodynamic properties of electrolyte solutions are briefly reviewed in the DH formalism. The main analytical and numerical extensions of DH formalism are revised, ranging from the earliest extensions that overcome the linearization of the PB equation to the more sophisticated integral equation techniques introduced after the late 1960s. Some Monte Carlo and molecular dynamic simulations are also reviewed. The potential distributions in an inhomogeneous ionic system are studied in the classical PB framework, presenting the classical Gouy–Chapman (GC) theory of the electric double layer (edl) in a brief manner. The mean-field theory is adequately contextualized using field theoretic (FT) results and it is proven that the classical PB theory is recovered at the Gaussian or one-loop level of the exact FT, and a systematic way to obtain the corrections to the DH theory is derived. Particularly, it is proven following Kholodenko and Beyerlein that corrections to DH theory effectively lead to a renormalization of charges and Debye screening length. The main analytical and numerical results for this non-Debye screening length are reviewed, ranging from asymptotic expansions, self-consistent theory, nonlinear DH results and hypernetted chain (HNC) calculations. Finally, we study the exact mean-field theory of ionic solutions, the so-called dressed-ion theory (DIT). An analysis of its statistical foundations is reported together with a detailed study of its linear response function, α (k) , that generalizes the concept of screening length and contains all the information about the effective quantities. The relation of this quantity to the structure factor of the fluid is explicitly analyzed and the renormalized charges and screening length for a one component charged spheres (OCCS) system derived in the modified mean spherical approximation (MMSA), and a comparison of the DIT/MMSA predictions for the effective magnitudes to HNC results included. Besides, the predicted DIT/MMSA thermodynamic properties are studied for the RPM electrolyte and extensions of this formalism to asymmetric electrolyte solutions presented. The main DIT results for the edl due to Ennis et al. are introduced and, finally, we analyze the main features of the application of the new equilibrium formalism to the calculation of transport coefficients, the so termed dressed ion transport theory (DITT). In this framework, the relaxation and electrophoretic corrections to the ionic mobility are interpreted in terms of the existence of new kinetic entities in the bulk solution: the effective or dressed particles.