Lipid Structure Determines the Differential Impact of Single Metal Additions and Binary Mixtures of Manganese, Calcium and Magnesium on Membrane Fluidity and Liposome Size.

Unilamellar vesicles of the biologically relevant lipids phosphatidic acid (PA) and phosphatidylserine (PS) with fully saturated (DM-) or partly unsaturated (PO-) acyl side chains were exposed to Ca, Mn and Mg in single metal additions; in equimolar mixtures or by sequential additions of one metal at a time. Laurdan generalized polarization measured the membrane fluidity, while dynamic light scattering reported liposome size changes complemented by zeta potential. All metals induced membrane rigidity and increased liposome sizes across all systems. Mn had the strongest effect overall, but Mg was comparable for DMPS. Lipid side chain architecture was important as GP values for binary mixtures were higher than expected from the sum of values for single additions added to POPS but smaller for DMPS. Sequential additions were predominantly different for Ca:Mg mixtures. Mn induced the strongest increase of liposome size in saturated lipids whereas Ca effects dominated unsaturated matrices. Binary additions induced larger sizes than the sum of single additions for POPS, but much lower changes in DMPA. The order of addition was relevant for PS systems. Thus, lipid structure determines metal effects, but their impact is modulated by other ions. Thus, metal effects may differ with the local lipid architecture and metal concentrations within cells.

A Simple Method for Synthesis of Chitosan Nanoparticles with Ionic Gelation and Homogenization.

Chitosan nanoparticles (CNPs) are known to have great utility in many fields (pharmaceutical, agricultural, food industry, wastewater treatment, etc.). In this study we aimed to synthesize sub-100 nm CNPs as a precursor of new biopolymer-based virus surrogates for water applications. We present a simple yet efficient synthesis procedure for obtaining high yield, monodisperse CNPs with size 68-77 nm. The CNPs were synthesized by ionic gelation using low molecular weight chitosan (deacetylation 75-85%) and tripolyphosphate as crosslinker, under rigorous homogenization to decrease size and increase uniformity, and purified by passing through 0.1 µm polyethersulfone syringe filters. The CNPs were characterized using dynamic light scattering, tunable resistive pulse sensing, and scanning electron microscopy. We demonstrate reproducibility of this method at two separate facilities. The effects of pH, ionic strength and three different purification methods on the size and polydispersity of CNP formation were examined. Larger CNPs (95-219) were produced under ionic strength and pH controls, and when purified using ultracentrifugation or size exclusion chromatography. Smaller CNPs (68-77 nm) were formulated using homogenization and filtration, and could readily interact with negatively charge proteins and DNA, making them an ideal precursor for the development of DNA-labelled, protein-coated virus surrogates for environmental water applications.

Collagen - a newly discovered major player in protein corona formation on nanoparticles.

Tracking protein corona (PC) formation on the surface of nanoparticles (NPs) is a prerequisite for successful design of next generation nanocarriers with predictable fate and behavior. However, PC formation has mostly been investigated for plasma proteins without considering potential competition with the extravascular proteins either when the NPs exit the blood circulation or when they are injected extravascularly. This study investigates the deposition of collagen, an extravascular protein that is the most abundant in the body, and albumin, the most abundant vascular protein, on the surface of gold (Au) NPs using UV-Vis and fluorescence spectroscopy with the support of mathematical modeling. Moreover, a novel spectroscopic approach to determining the protein-NP binding constants and surface occupancy is presented. We show that albumin and collagen have drastically different affinities for Au NPs. Our data demonstrates that the surface bound albumin can be exchanged with collagen confirming the dynamic nature of PC in the extravascular milieu. We propose that future PC investigations in the framework of drug delivery should rely on understanding of the NP transit in the body, and include competition experiments with relevant vascular and extravascular proteins. Furthermore, our results that reveal very strong binding of collagen to AuNPs may lay the foundation for designing long circulating collagen-coated NPs with minimal surface adsorption of plasma proteins and, thus, reduced immune recognition.

Benefits and Detriments of Gadolinium from Medical Advances to Health and Ecological Risks.

Gadolinium (Gd)-containing chelates have been established as diagnostics tools. However, extensive use in magnetic resonance imaging has led to increased Gd levels in industrialized parts of the world, adding to natural occurrence and causing environmental and health concerns. A vast amount of data shows that metal may accumulate in the human body and its deposition has been detected in organs such as brain and liver. Moreover, the disease nephrogenic systemic fibrosis has been linked to increased Gd3+ levels. Investigation of Gd3+ effects at the cellular and molecular levels mostly revolves around calcium-dependent proteins, since Gd3+ competes with calcium due to their similar size; other reports focus on interaction of Gd3+ with nucleic acids and carbohydrates. However, little is known about Gd3+ effects on membranes; yet some results suggest that Gd3+ interacts strongly with biologically-relevant lipids (e.g., brain membrane constituents) and causes serious structural changes including enhanced membrane rigidity and propensity for lipid fusion and aggregation at much lower concentrations than other ions, both toxic and essential. This review surveys the impact of the anthropogenic use of Gd emphasizing health risks and discussing debilitating effects of Gd3+ on cell membrane organization that may lead to deleterious health consequences.

Rhodococcus aetherivorans BCP1 as cell factory for the production of intracellular tellurium nanorods under aerobic conditions.

BACKGROUND: Tellurite (TeO32-) is recognized as a toxic oxyanion to living organisms. However, mainly anaerobic or facultative-anaerobic microorganisms are able to tolerate and convert TeO32- into the less toxic and available form of elemental Tellurium (Te0), producing Te-deposits or Te-nanostructures. The use of TeO32--reducing bacteria can lead to the decontamination of polluted environments and the development of "green-synthesis" methods for the production of nanomaterials. In this study, the tolerance and the consumption of TeO32- have been investigated, along with the production and characterization of Te-nanorods by Rhodococcus aetherivorans BCP1 grown under aerobic conditions. RESULTS: Aerobically grown BCP1 cells showed high tolerance towards TeO32- with a minimal inhibitory concentration (MIC) of 2800 µg/mL (11.2 mM). TeO32- consumption has been evaluated exposing the BCP1 strain to either 100 or 500 µg/mL of K2TeO3 (unconditioned growth) or after re-inoculation in fresh medium with new addition of K2TeO3 (conditioned growth). A complete consumption of TeO32- at 100 µg/mL was observed under both growth conditions, although conditioned cells showed higher consumption rate. Unconditioned and conditioned BCP1 cells partially consumed TeO32- at 500 µg/mL. However, a greater TeO32- consumption was observed with conditioned cells. The production of intracellular, not aggregated and rod-shaped Te-nanostructures (TeNRs) was observed as a consequence of TeO32- reduction. Extracted TeNRs appear to be embedded in an organic surrounding material, as suggested by the chemical-physical characterization. Moreover, we observed longer TeNRs depending on either the concentration of precursor (100 or 500 µg/mL of K2TeO3) or the growth conditions (unconditioned or conditioned grown cells). CONCLUSIONS: Rhodococcus aetherivorans BCP1 is able to tolerate high concentrations of TeO32- during its growth under aerobic conditions. Moreover, compared to unconditioned BCP1 cells, TeO32- conditioned cells showed a higher oxyanion consumption rate (for 100 µg/mL of K2TeO3) or to consume greater amount of TeO32- (for 500 µg/mL of K2TeO3). TeO32- consumption by BCP1 cells led to the production of intracellular and not aggregated TeNRs embedded in an organic surrounding material. The high resistance of BCP1 to TeO32- along with its ability to produce Te-nanostructures supports the application of this microorganism as a possible eco-friendly nanofactory.

Phthalocyanine tetrasulfonates bind to multiple sites on natively-folded prion protein.

The phthalocyanine tetrasulfonates (PcTS), a class of cyclic tetrapyrroles, bind to the mammalian prion protein, PrP. Remarkably, they can act as anti-scrapie agents to prevent the formation and spread of infectious, misfolded PrP. While the effects of phthalocyanines on the diseased state have been investigated, the interaction between PcTS and PrP has not yet been extensively characterized. Here we use multiple, complementary assays (surface plasmon resonance, isothermal titration calorimetry, fluorescence correlation spectroscopy, and tryptophan fluorescence quenching) to characterize the binding of PcTS to natively-folded hamster PrP(90-232), in order to determine binding constants, ligand stoichiometry, influence of buffer ionic strength, and the effects of chelated metal ions. We found that binding strength depends strongly on chelated metal ions, with Al(3+)-PcTS binding the weakest and free-base PcTS the strongest of the three types tested (Al(3+), Zn(2+), and free-base). Buffer ionic strength also affected the binding, with K(d) increasing along with salt concentration. The binding isotherms indicated the presence of at least two different binding sites with micromolar affinities and a total stoichiometry of ~4-5 PcTS molecules per PrP molecule.

Profiling H2O2 from single COS-7 cells by means of scanning electrochemical microscopy.

We report quantitative determination of extracellular H2O2 released from single COS-7 cells with high spatial resolution, using scanning electrochemical microscopy (SECM). Our strategy of depth scan imaging in vertical x-z plane was conveniently utilized to a single cell for obtaining probe approach curves (PACs) to any positions on the membrane of a live cell by simply drawing a vertical line on one depth SECM image. This SECM mode provides an efficient way to record a batch of PACs, and visualize cell topography simultaneously. The H2O2 concentration at the membrane surface in the center of an intact COS-7 cell was deconvoluted from apparent O2, and determined to be 0.020 mM by overlapping the experimental PAC with the simulated one having a known H2O2 release value. The H2O2 profile determined in this way gives insight into physiological activity of single live cells. In addition, intracellular H2O2 profile was demonstrated using confocal microscopy by labelling the cells with a luminomphore, 2',7'-dichlorodihydrofluorescein diacetate. The two methodologies have illustrated complementary experimental results of H2O2 detection, indicating that H2O2 generation is centered at endoplasmic reticula.

Synthesis of Alginate Nanoparticles Using Hydrolyzed and Enzyme-Digested Alginate Using the Ionic Gelation and Water-in-Oil Emulsion Method.

Alginate nanoparticles (AlgNPs) are attracting increasing interest for a range of applications because of their good biocompatibility and their ability to be functionalized. Alginate is an easily accessible biopolymer which is readily gelled by the addition of cations such as calcium, facilitating a cost-effective and efficient production of nanoparticles. In this study, AlgNPs based on acid hydrolyzed and enzyme-digested alginate were synthesized by using ionic gelation and water-in-oil emulsification, with the goal to optimize key parameters to produce small uniform (<200 nm) AlgNPs. By the ionic gelation method, such AlgNPs were obtained when sample concentrations were 0.095 mg/mL for alginate and CaCl2 in the range of 0.03-0.10 mg/mL. Alginate and CaCl2 concentrations > 0.10 mg/mL resulted in sizes > 200 nm with relatively high dispersity. Sonication in lieu of magnetic stirring proved to further reduce size and increase homogeneity of the nanoparticles. In the water-in-oil emulsification method, nanoparticle growth was confined to inverse micelles in an oil phase, resulting in lower dispersity. Both the ionic gelation and water-in-oil emulsification methods were suitable for producing small uniform AlgNPs that can be further functionalized as required for various applications.

Biosynthesis of selenium-nanoparticles and -nanorods as a product of selenite bioconversion by the aerobic bacterium Rhodococcus aetherivorans BCP1.

The wide anthropogenic use of selenium compounds represents the major source of selenium pollution worldwide, causing environmental issues and health concerns. Microbe-based strategies for metal removal/recovery have received increasing interest thanks to the association of the microbial ability to detoxify toxic metal/metalloid polluted environments with the production of nanomaterials. This study investigates the tolerance and the bioconversion of selenite (SeO32-) by the aerobically grown Actinomycete Rhodococcus aetherivorans BCP1 in association with its ability to produce selenium nanoparticles and nanorods (SeNPs and SeNRs). The BCP1 strain showed high tolerance towards SeO32- with a Minimal Inhibitory Concentration (MIC) of 500mM. The bioconversion of SeO32- was evaluated considering two different physiological states of the BCP1 strain, i.e. unconditioned and/or conditioned cells, which correspond to cells exposed for the first time or after re-inoculation in fresh medium to either 0.5 or 2mM of Na2SeO3, respectively. SeO32- bioconversion was higher for conditioned grown cells compared to the unconditioned ones. Selenium nanostructures appeared polydisperse and not aggregated, as detected by electron microscopy, being embedded in an organic coating likely responsible for their stability, as suggested by the physical-chemical characterization. The production of smaller and/or larger SeNPs was influenced by the initial concentration of provided precursor, which resulted in the growth of longer and/or shorter SeNRs, respectively. The strong ability to tolerate high SeO32- concentrations coupled with SeNP and SeNR biosynthesis highlights promising new applications of Rhodococcus aetherivorans BCP1 as cell factory to produce stable Se-nanostructures, whose suitability might be exploited for biotechnology purposes.

Assembly, growth and conductive properties of tellurium nanorods produced by Rhodococcus aetherivorans BCP1.

Tellurite (TeO32-) is a hazardous and toxic oxyanion for living organisms. However, several microorganisms can bioconvert TeO32- into the less toxic form of elemental tellurium (Te0). Here, Rhodococcus aetherivorans BCP1 resting (non-growing) cells showed the proficiency to produce tellurium-based nanoparticles (NPs) and nanorods (NRs) through the bioconversion of TeO32-, depending on the oxyanion initial concentration and time of cellular incubation. Te-nanostructures initially appeared in the cytoplasm of BCP1 cells as spherical NPs, which, as the exposure time increased, were converted into NRs. This observation suggested the existence of an intracellular mechanism of TeNRs assembly and growth that resembled the chemical surfactant-assisted process for NRs synthesis. The TeNRs produced by the BCP1 strain showed an average length (>700 nm) almost doubled compared to those observed in other studies. Further, the biogenic TeNRs displayed a regular single-crystalline structure typically obtained for those chemically synthesized. The chemical-physical characterization of the biogenic TeNRs reflected their thermodynamic stability that is likely derived from amphiphilic biomolecules present in the organic layer surrounding the NRs. Finally, the biogenic TeNRs extract showed good electrical conductivity. Thus, these findings support the suitability of this strain as eco-friendly biocatalyst to produce high quality tellurium-based nanomaterials exploitable for technological purposes.

Binding Affinity of Inorganic Mercury and Cadmium to Biomimetic Erythrocyte Membranes.

Inorganic mercury and cadmium are becoming increasingly prevalent due to industrial activity and have been linked to cardiovascular disease and diabetes. The binding affinity of Hg, Cd, and their mixtures to biomimetic erythrocyte membranes was investigated by isothermal titration calorimetry in physiologically relevant media (100 mM NaCl, pH 7.4, 37 °C). The thermodynamic parameters were not expressed per mole of lipid but as metals binding to liposomes. To our knowledge, this method is novel and provides a more intuitive approach to understand such interactions. The results demonstrated that Hg interacted with membranes in the following order: PC (phosphatidylcholine) > 85:15 PC/PE (phosphatidylethanolamine) > 85:15 PC/PS (phosphatidylserine), with the binding constants ranging from 10 to 233 M-1. In contrast, Cd interacted most readily with negatively charged PC/PS membranes but not with the remaining systems. Metal mixtures bind less to PC/PE membranes than the individual constituents. The large entropy contribution from these interactions suggests possible water release and/or reorganization upon Hg and Cd binding to membranes. ζ-Potential data indicate that the process may be electrostatically driven. It is imperative to consider the chemical speciation of these metals in the presence of chloride to better understand metal-lipid interactions and their impact on biomembranes.

Differential interactions of gelatin nanoparticles with the major lipids of model lung surfactant: changes in the lateral membrane organization.

There has been an increasing interest in the potential of nanomedicine, particularly in the use of nanoparticles between 10 nm and 1 µm in diameter as drug delivery vehicles. For pulmonary drug delivery, it is important to understand the effect of polymeric nanoparticles on the lung surfactant in order to optimize the carriers by reducing their potential toxicological effects. This work presents a biophysical study of the impact of gelatin nanoparticles on packing and lateral organization of simple and complex lipid layers containing the major components of lung surfactant. Zwitterionic phosphatidylcholines, negatively charged phosphatidylglycerols, and the sterol cholesterol were employed in the models. In addition, the impact of acyl chain length was investigated. Packing was determined by surface pressure-area isotherms, whereas direct imaging of the surfactant at the air-water interface was performed using Brewster angle microscopy. Our results indicate minor changes in the surface pressure-area isotherms but concomitantly significant effects on the lateral organization of the monolayers upon nanoparticle addition. The data also suggest differential interactions of nanoparticles with the major lipid classes. Gelatin nanoparticles interact stronger with negatively charged phosphatidyl-glycerols compared to zwitterionic phosphatidyl-cholines. Furthermore, charge distribution depending on the molar lipid ratio and acyl chain saturation is important as well. Even cholesterol, whose concentration is low compared to other components, plays an important role in nanoparticle interactions.