Competitive protein adsorption on charge regulating silica-like surfaces: the role of protonation equilibrium.

We develop a molecular thermodynamic theory to study the interaction of some proteins with a charge regulating silica-like surface under a wide range of conditions, including pH, salt concentration and protein concentration. Proteins are modeled using their three dimensional structure from crystallographic data and the average experimental pKa of amino acid residues. As model systems, we study single-protein and binary solutions of cytochrome c, green fluorescent protein, lysozyme and myoglobin. Our results show that protonation equilibrium plays a critical role in the interactions of proteins with these type of surfaces. The terminal hydroxyl groups on the surface display considerable extent of charge regulation; protein residues with titratable side chains increase protonation according to changes in the local environment and the drop in pH near the surface. This behavior defines protein-surface interactions and leads to the emergence of several phenomena: (i) a complex non-ideal surface charge behavior; (ii) a non-monotonic adsorption of proteins as a function of pH; and (iii) the presence of two spatial regions, a protein-rich and a protein-depleted layer, that occur simultaneously at different distances from the surface when pH is slightly above the isoelectric point of the protein. In binary mixtures, protein adsorption and surface-protein interactions cannot be predicted from single-protein solution considerations.

Inside the Protein Corona: From Binding Parameters to Unstained Hard and Soft Coronas Visualization.

Proteins spontaneously adsorb on nanoparticle surfaces when injected into the bloodstream. It drastically modifies the nanoparticle's fate and how they interact with organs and cells. Although this protein layer (protein corona) has been widely studied, the robustness of the most employed characterization methods and the visualization of its unstained fractions remain open questions. Here, synchrotron-based small-angle X-ray scattering was used to follow the corona formation and estimate binding parameters. At the same time, transmission electron microscopy under cryogenic conditions associated with cross-correlation image processing and energy-filtered transmission electron microscopy allowed to determine protein corona morphology and thickness together with the visualization of its unstained hard and soft fractions. The above-presented strategy shows tremendous potential for deciphering fundamental protein corona aspects and can contribute to rational medical nanoparticle engineering.

Biocompatible gum arabic-gold nanorod composite as an effective therapy for mistreated melanomas.

Advanced melanoma patients that are not included in common genetic classificatory groups lack effective and safe therapeutic options. Chemotherapy and immunotherapy show unsatisfactory results and devastating adverse effects for these called triple wild-type patients. New approaches exploring the intrinsic antitumor properties of gold nanoparticles might reverse this scenario as a safer and more effective alternative. Therefore, we investigated the efficacy and safety of a composite made of gum arabic-functionalized gold nanorods (GA-AuNRs) against triple wild-type melanoma. The natural polymer gum arabic successfully stabilized the nanorods in the biological environment and was essential to improve their biocompatibility. In vivo results obtained from treating triple wild-type melanoma-bearing mice showed that GA-AuNRs remarkably reduced primary tumor growth by 45%. Furthermore, GA-AuNRs induced tumor histological features associated with better prognosis while also reducing superficial lung metastasis depth and the incidence of intrapulmonary metastasis. GA-AuNRs' efficacy comes from their capacity to reduce melanoma cells ability to invade the extracellular matrix and grow into colonies, in addition to a likely immunomodulatory effect induced by gum arabic. Additionally, a broad safety investigation found no evidence of adverse effects after GA-AuNRs treatment. Therefore, this study unprecedentedly reports GA-AuNRs as a potential nanomedicine for advanced triple wild-type melanomas.

Degradable and colloidally stable zwitterionic-functionalized silica nanoparticles.

Aim: This work is focused on obtaining degradable mesoporous silica nanoparticles (DMSNs) which are able to maintain their colloidal stability in complex biological media. Materials & methods: DMSNs were synthesized using different ratios of disulfide organosilane (degradable structural moiety) and further functionalized with sulfobetaine silane (SBS) to enhance colloidal stability and improve biological compatibility. Results: There was a clear trade-off between nanoparticle degradability and colloidal stability, since full optimization of the degradation process generated unstable particles, while enhancing colloidal stability resulted in poor DMSNs degradation. It was also shown that acidic pH improved particle degradation which is commonly triggered by reduction stimulus. Conclusion: A chemical composition window was found where DMSNs presented satisfactory colloidal stability in biologically relevant medium, meaningful degradation profiles and high biocompatibility.

Shielding and stealth effects of zwitterion moieties in double-functionalized silica nanoparticles.

Surface functionalization of silica nanoparticles (SiO2NPs) has been considered as a promising strategy to develop target-specific nanostructures. However, finding a chemical functionalization that can be used as an active targeting moiety while preserving the nanoparticles colloidal stability in biological fluids is still challenging. We present here a dual surface modification strategy for SiO2NPs where a zwitterion (ZW) and a biologically active group (BAG) (amino, mercapto or carboxylic functionalities) are simultaneously grafted on the nanoparticles' surface. The rationale behind this strategy is to generate colloidally stable nanoparticles and avoid the nonspecific protein adsorption due to ZW groups insertion, while the effective interaction with biosystems is guaranteed by the BAGs presence. The biological efficacy was tested against VERO cells, E. coli bacteria and Zika viruses and a similar trend was observed for all tested particles. The desirable "stealth property" to prevent nonspecific protein adhesion also generated a ZW shielding effect of the BAG functionality hindering their proper interaction and activity in cells, bacteria and viruses.

Tailoring the Antimicrobial Response of Cationic Nanocellulose-Based Foams through Cryo-Templating.

To shed light on novel sustainable materials with antimicrobial functionality, in this contribution, we describe the use of cationic nanocellulose to produce foams featuring antibacterial activity against the powerful human pathogen Escherichia coli. Dialdehyde cellulose was cationized with Girard's reagent T (GRT), mechanically disintegrated into nanofibrillated cellulose (NFC), and shaped into foams through different protocols. All steps were carried out in aqueous media and in the absence of hazardous chemicals. While evaporative drying led to compact films (density of 1.3 g cm-3), freeze-casting (i.e., freezing and freeze-drying) produced monolithic cryogels with low densities (<50 mg cm-3) and porosities of ca. 98%. Although highly porous, the cryogels obtained through rapid freezing remarkably presented smaller pores than those that were previously frozen in a slow fashion. The quaternary ammonium groups of GRT-cationized NFC removed E. coli to different extents depending upon sample morphology. We demonstrated in an innovative manner that porosity, which is directly associated with surface area, and pore size play an essential role on the antimicrobial performance. This outcome arises from the inaccessibility of bacterial cells to cationic surfaces inside monoliths composed of small pores. We herein present an uncomplicated, environmentally friendly protocol for fine-tuning the porosity and pore size of all-cellulose materials through cryo-templating. Controlling these morphometric parameters allowed us to achieve a ca. 85% higher anti-E. coli activity when comparing samples made up of the very same material (i.e., the same NFC concentration and degree of substitution) but presented as dense films. These findings bear clear implications for the pursuit of sustainable materials presenting multifunctionality.

Monitoring the Surface Chemistry of Functionalized Nanomaterials with a Microfluidic Electronic Tongue.

Advances in nanomaterials have led to tremendous progress in different areas with the development of high performance and multifunctional platforms. However, a relevant gap remains in providing the mass-production of these nanomaterials with reproducible surfaces. Accordingly, the monitoring of such materials across their entire life cycle becomes mandatory to both industry and academy. In this paper, we use a microfluidic electronic tongue (e-tongue) as a user-friendly and cost-effective method to classify nanomaterials according to their surface chemistry. The chip relies on a new single response e-tongue with association of capacitors in parallel, which consisted of stainless steel microwires coated with SiO2, NiO2, Al2O3, and Fe2O3 thin films. Utilizing impedance spectroscopy and a multidimensional projection technique, the chip was sufficiently sensitive to distinguish silica nanoparticles and multiwalled carbon nanotubes dispersed in water in spite of the very small surface modifications induced by distinct functionalization and oxidation extents, respectively. Flow analyses were made acquiring the analytical readouts in a label-free mode. The device also allowed for multiplex monitoring in an unprecedented way to speed up the tests. Our goal is not to replace the traditional techniques of surface analysis, but rather propose the use of libraries from e-tongue data as benchmark for routine screening of modified nanomaterials in industry and academy.

Freeze-drying of silica nanoparticles: redispersibility toward nanomedicine applications.

AIM: To study freeze-drying of silica nanoparticles (SiO2NPs) in order to find suitable conditions to produce lyophilized powders with no aggregation after resuspension and storage. METHODS: SiO2NPs were synthesized using a Stöber-based procedure, and characterized by scanning electron microscopy, dynamic light scattering and nitrogen adsorption/desorption isotherms. SiO2NPs hydrodynamic diameters were compared prior and after freeze-drying in the presence/absence of carbohydrate protectants. RESULTS: Glucose was found to be the most suitable protectant against the detrimental effects of lyophilization. The minimum concentration of carbohydrate required to effectively protect SiO2NPs from aggregation during freeze-drying is influenced by the nanoparticle's size and texture. Negligible aggregation was observed during storage. CONCLUSION: Carbohydrates can be used during SiO2NPs freeze-drying process to obtain redispersable solids that maintain original sizes without residual aggregation.

Shape Tailored Magnetic Nanorings for Intracellular Hyperthermia Cancer Therapy.

ABSTARCT: This work explores a new class of vortex/magnetite/iron oxide nanoparticles designed for magnetic hyperthermia applications. These nanoparticles, named Vortex Iron oxide Particles (VIPs), are an alternative to the traditional Superparamagnetic Iron Oxide Nanoparticles (SPIONs), since VIPs present superior heating power while fulfilling the main requirements for biomedical applications (low cytotoxicity and nonremanent state). In addition, the present work demonstrates that the synthesized VIPs also promote an internalization and aggregation of the particles inside the cell, resulting in a highly localized hyperthermia in the presence of an alternating magnetic field. Thereby, we demonstrate a new and efficient magnetic hyperthermia strategy in which a small, but well localized, concentration of VIPs can promote an intracellular hyperthermia process.

Three-dimensional non-destructive soft-tissue visualization with X-ray staining micro-tomography.

Low inherent contrast in soft tissues has been limiting the use of X-ray absorption micro-computed tomography (micro-CT) to access high-resolution structural information of animal organs. The staining agents used in micro-CT to improve the contrast fail in providing high-quality images of whole organs of animals due to diffusion problems of the staining agent into the sample. We demonstrate a staining protocol that incorporates a biochemical conditioning step prior to exposure to the staining agent that succeeds in overcoming the diffusion problems, thus quickly providing high-quality micro-CT images of whole organs of mammals. Besides of yielding non-distorted three-dimensional information at the same spatial resolution accessible in histological sections, micro-CT images of whole organs stained by our method enable easy screening of slices along any direction of the volume thus demonstrating new possibilities of structural analysis in biomedical science.

Correlating the Morphological Properties and Structural Organization of Monodisperse Spherical Silica Nanoparticles Grown on a Commercial Silica Surface.

A variety of nanosilicas have been widely used to fabricate rough surfaces with superhydrophobic and superhydrophilic properties. In this context, we prepared mixed silica and mixed nanosilica that were generated by the growth and self-assembly of synthesized monodisperse silica nanospheres (11-30 nm, 363 m(2) g(-1) ) on the surface of Sylopol-948 and Dispercoll S3030 by using a base-catalyzed sol-gel route. Using this process, the interactions and hierarchical structure between the nano- and microsized synthesized silica particles were studied by changing the amount of tetraethoxysilane. The resulting materials were characterized by BET analysis, small-angle X-ray scattering (SAXS), dynamic light scattering, FTIR spectroscopy, and SEM. The mixed silica presented a higher specific surface area (326 m(2) g(-1) ), a six-fold higher percentage of (SiO)6 (44-68 %), and a higher amount of silanol groups (14.0-30.7 %) than Sylopol-948 (271 m(2) g(-1), 42.6 %, and 12.5 %, respectively). The morphological and hierarchical structural differences in the silica nanoparticles synthesized on the surface of commercial silica (micrometric or nanometric) were identified by SAXS. Mixed micrometric silica exhibited a higher degree of structural organization between particles than mixed nanosilica.

Nanometric organisation in blends of gellan/xyloglucan hydrogels.

Mixtures of gellan gum (GL) and a xyloglucan (XGJ) extracted from Hymenaea courbaril seeds were prepared in a solution of 0.15 mol L(-1) NaCl. Rheology measurements revealed that 2.4 g L(-1) pure GL formed a brittle hydrogel, and GL-XGJ blends showed improved pseudoplastic character with higher XGJ contents. SAXS analyses showed that the Rg dimensions ranged from 1.3 to 4.9 nm, with larger values occurring as the amount of XGJ increased, and diffusion tests indicated that better diffusion of methylene blue dye was obtained in the network with a higher XGJ content. AFM topographic images of the films deposited onto mica revealed fewer heterogeneous surfaces with increased XGJ contents. The water contact angle revealed more hydrophobic character on all of the films, and the wettability decreased with increasing amounts of XGJ. Therefore, the demonstrated benefit of using XGJ blends is the production of a soft material with improved interface properties.

The cold storage of green bananas affects the starch degradation during ripening at higher temperature.

The aim of this work was to investigate the starch degradation of bananas stored at low temperature (13°C, cold-stored group) and bananas stored at 19°C (control group) during ripening. The starch granules were isolated during different stages of banana ripening, and their structure was investigated using different techniques. The activities of α-amylase and ß-amylase associated to the starch granules were determined, and their presence was confirmed using immunolocalization assays. The increased molecular mobility likely facilitated the intake and action of α-amylase on the granule surface, where it was the prevalent enzyme in bananas stored at low temperature. The 10 days of storage at low temperature also influenced the sizes and shapes of the granules, with a predominance of rounded granules and pits on the surface along with superior amylose content, the higher amounts of amylopectin A-chains and the subtle increase in the A-type allomorph content.

Echinococcus granulosus antigen B structure: subunit composition and oligomeric states.

BACKGROUND: Antigen B (AgB) is the major protein secreted by the Echinococcus granulosus metacestode and is involved in key host-parasite interactions during infection. The full comprehension of AgB functions depends on the elucidation of several structural aspects that remain unknown, such as its subunit composition and oligomeric states. METHODOLOGY/PRINCIPAL FINDINGS: The subunit composition of E. granulosus AgB oligomers from individual bovine and human cysts was assessed by mass spectrometry associated with electrophoretic analysis. AgB8/1, AgB8/2, AgB8/3 and AgB8/4 subunits were identified in all samples analyzed, and an AgB8/2 variant (AgB8/2v8) was found in one bovine sample. The exponentially modified protein abundance index (emPAI) was used to estimate the relative abundance of the AgB subunits, revealing that AgB8/1 subunit was relatively overrepresented in all samples. The abundance of AgB8/3 subunit varied between bovine and human cysts. The oligomeric states formed by E. granulosus AgB and recombinant subunits available, rAgB8/1, rAgB8/2 and rAgB8/3, were characterized by native PAGE, light scattering and microscopy. Recombinant subunits showed markedly distinct oligomerization behaviors, forming oligomers with a maximum size relation of rAgB8/3>rAgB8/2>rAgB8/1. Moreover, the oligomeric states formed by rAgB8/3 subunit were more similar to those observed for AgB purified from hydatid fluid. Pressure-induced dissociation experiments demonstrated that the molecular assemblies formed by the more aggregative subunits, rAgB8/2 and rAgB8/3, also display higher structural stability. CONCLUSIONS/SIGNIFICANCE: For the first time, AgB subunit composition was analyzed in samples from single hydatid cysts, revealing qualitative and quantitative differences between samples. We showed that AgB oligomers are formed by different subunits, which have distinct abundances and oligomerization properties. Overall, our findings have significantly contributed to increase the current knowledge on AgB expression and structure, highlighting issues that may help to understand the parasite adaptive response during chronic infection.

Silica-maltose composites: obtaining drug carrier systems through tailored ultrastructural nanoparticles.

The formation of tailored silica-maltose composites through a simple and direct sol-gel chemistry approach is demonstrated. The ultrastructural organization of the composite associated with their tailorability allows envisaging a future application in drug delivery field. Ultraviolet-visible spectroscopy is used to follow the maltose encapsulation yield, whereas a combination of characterization techniques is employed to reconstruct the multilevel hierarchical structure of composites. Scanning electron microscopy shows that the overall size of spherical composites can be tuned from 250 to 750 nm by changing the amount of maltose within the structure. Composite size distribution indicates that this synthesis approach produces structures with low polydispersity as required for drug delivery purposes. Small-angle X-ray scattering and nitrogen adsorption-desorption techniques show evidence that the composite is elementarily formed by fundamental silica spheres with size ranging from approximately 4 to approximately 7 nm. Surface area of composites is reduced when maltose concentration is increased, which indicates that carbohydrate molecules are preferentially located into the interstitial space between fundamental silica spheres. Through an ultrastructural control over the synthesis process, it has been shown that sol-gel method employed here presents considerable potential for producing efficient drug carrier systems.

Insight into the structure of light-harvesting complex II and its stabilization in detergent solution.

The structure of spinach light-harvesting complex II (LHC II), stabilized in a solution of the detergent n-octyl-beta-D-glucoside (BOG), was investigated by small-angle neutron scattering (SANS). Physicochemical characterization of the isolated complex indicated that it was pure (>95%) and also in its native trimeric state. SANS with contrast variation was used to investigate the properties of the protein-detergent complex at three different H(2)O/D(2)O contrast match points, enabling the scattering properties of the protein and detergent to be investigated independently. The topological shape of LHC II, determined using ab initio shape restoration methods from the SANS data at the contrast match point of BOG, was consistent with the X-ray crystallographic structure of LHC II (Liu et al. Nature 2004 428, 287-292). The interactions of the protein and detergent were investigated at the contrast match point for the protein and also in 100% D(2)O. The data suggested that BOG micelle structure was altered by its interaction with LHC II, but large aggregate structures were not formed. Indirect Fourier transform analysis of the LHC II/BOG scattering curves showed that the increase in the maximum dimension of the protein-detergent complex was consistent with the presence of a monolayer of detergent surrounding the protein. A model of the LHC II/BOG complex was generated to interpret the measurements made in 100% D(2)O. This model adequately reproduced the overall size of the LHC II/BOG complex, but demonstrated that the detergent does not have a highly regular shape that surrounds the hydrophobic periphery of LHC II. In addition to demonstrating that natively structured LHC II can be produced for functional characterization and for use in artificial solar energy applications, the analysis and modeling approaches described here can be used for characterizing detergent-associated alpha-helical transmembrane proteins.

Size control of highly ordered HfO2 nanotube arrays and a possible growth mechanism.

Highly ordered HfO2 nanotube arrays were prepared through an electrochemical anodization in the presence of NH4F and ethylene glycol. The voltage-dependent pore size, wall thickness and porosity were studied using scanning electron microscopy and a wall thickness to pore size ratio was proposed on the basis of the results to serve as a boundary condition additional to the 10% porosity rule introduced by the Gosele group. The average distributions of the tube sizes and wall thicknesses of the nanotubes prepared at 20 V were determined from the small-angle x-ray scattering data using a simple polydisperse core-shell cylinder model fit. Temperature-dependent x-ray diffraction measurements show that the as-grown amorphous nanotube arrays can be converted into crystalline nanotube arrays at a temperature above 500 degrees C. Transmission electron microscopy study of the dimple layer under the as-grown nanotube arrays reveals the presence of a layer of ordered HfO2 nanocrystals. Further microscopic investigation of the nanotube root region indicates that the nanotubes develop from bulbs produced during anodization. A possible gas bubble initiated growth mechanism based on these observations was proposed.

Effect of the alkaline treatment on the ultrastructure of C-type starch granules.

The effect of alkaline treatment on the ultrastructure of C-type starch granules was investigated during the alkaline extraction of Araucaria angustifolia (pinhao) starch. The efficiency in protein removal was evaluated using intrinsic fluorescence and Kjeldahl's method. In parallel, morphological changes of starch granules were observed using scanning electron microscopy and atomic force microscopy. The starch crystallinity was monitored by wide-angle X-ray scattering and the lamellar structure was studied by small-angle X-ray scattering (SAXS). The paracrystalline model was employed to interpret the SAXS curves. It was found that the granular organization was significantly altered when alkaline solutions were used during the extraction. A partial degradation of B-type allomorph of starch and a significant compression of semicrystalline growth rings were observed.

Single crystals of V-amylose complexed with alpha-naphthol.

Lamellar square single crystals of V-amylose were obtained by adding alpha-naphthol to metastable dilute aqueous solutions of synthetic amylose chains with an average degree of polymerization of 100. The morphology and structure of the crystals were studied using low-dose transmission electron microscopy including high-resolution imaging, as well as electron and X-ray diffraction. The crystals are crystallized in a tetragonal P4(1)2(1)2 or P4(3)2(1)2 space group with unit cell parameters, calculated from X-ray diffraction data, a = b = 2.2844 nm (+/-0.0005) and c = 0.7806 nm (+/-0.001), implying the presence of two amylose chains per unit cell. High-resolution lattice images of the crystals confirmed that the amylose chains were crystallized as 8-fold helices corresponding to the repeat of four maltosyl units.

Self-assembly and structural characterization of Echinococcus granulosus antigen B recombinant subunit oligomers.

Echinococcus granulosus antigen B is an oligomeric protein of 120-160 kDa composed by 8-kDa (AgB8) subunits. Here, we demonstrated that the AgB8 recombinant subunits AgB8/1, AgB8/2 and AgB8/3 are able to self-associate into high order homo-oligomers, showing similar properties to that of parasite-produced AgB, making them valuable tools to study AgB structure. Dynamic light scattering, size exclusion chromatography and cross-linking assays revealed approximately 120- to 160-kDa recombinant oligomers, with a tendency to form populations with different aggregation states. Recombinant oligomers showed helical circular dichroism spectra and thermostability similar to those of purified AgB. Cross-linking and limited proteolysis experiments indicated different degrees of stability and compactness between the recombinant oligomers, with the AgB8/3 one showing a more stable and compact structure. We have also built AgB8 subunit structural models in order to predict the surfaces possibly involved in electrostatic and hydrophobic interactions during oligomerization.