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 fibril's 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.
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.
Conductivities and dielectric constant measurements in water at 25 °C have been made on the amphiphilics sodium n-dodecyl sulfate, n-dodecyltrimethylammonium bromide, and chlorpromazine hydrochloride. By using the conductivity/concentration data, critical micelle concentrations (cmc) have been determined by applying the Williams definition and two forms of the Phillips method. This first Phillips form consists of an approximation to Gaussians of the second derivative of the conductivity/concentration data followed by two consecutive integrations. The second form, which is proposed here, consists of the application of a combination of the Runge-Kutta numerical integrations method and the Levenberg-Marquardt leastsquares fitting algorithm. The proposed method permits the determination of the cmc in systems with low aggregation numbers and with slow variations of physical property/concentration curves allowing the determination of the so-called second cmc. A comparative study with results obtained by dielectric constant measurements has been carried out. With this new technique, the cmc's (first and second) are directly obtained as singular points in the dielectric constant/concentration curves, and thus, this technique is an alternative to the determination of cmc's from conductivities.
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.
Under in vitro solution conditions where the native state is destabilized, many proteins present an abnormal structure and metabolism associated with a strong tendency to self-aggregation into a polymeric amyloid fibril structure, suggesting that this ability is a generic feature of the polypeptide chains. Such structures play a key role in different pathogenesis of neurodegenerative diseases such as Alzheimer, Parkinson, or Creutzfeldt-Jakob. Here, we report the formation of amyloid fibrils in the plasma protein human serum albumin under different in vitro conditions monitored using a combination of spectrophotometric and microscopic techniques. Amyloid fibril formation, therefore, is also allowed in a protein with a high degree of structural complexity. We also infer from experimental data the existence of other protein aggregated species than fibrils, some of which seem to be formed by a structural rearrangement of the proper fibrils.
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