The Impact of Virgin and Aged Microstructured Plastics on Proteins: The Case of Hemoglobin Adsorption and Oxygenation.

Plastic particles, particularly micro- and nanoparticles, are emerging pollutants due to the ever-growing amount of plastics produced across a wide variety of sectors. When plastic particles enter a biological medium, they become surrounded by a corona, giving them their biological identity and determining their interactions in the living environment and their biological effects. Here, we studied the interactions of microstructured plastics with hemoglobin (Hb). Virgin polyethylene microparticles (PEMPs) and polypropylene microparticles (PPMPs) as well as heat- or irradiation-aged microparticles (ag-PEMPs and ag-PPMPs) were used to quantify Hb adsorption. Polypropylene filters (PP-filters) were used to measure the oxygenation of adsorbed Hb. Microstructured plastics were characterized using optical microscopy, SAXS, ATR-FTIR, XPS, and Raman spectroscopy. Adsorption isotherms showed that the Hb corona thickness is larger on PPMPs than on PEMPs and Hb has a higher affinity for PPMPs than for PEMPs. Hb had a lower affinity for ag-PEMPs and ag-PPMPs, but they can be adsorbed in larger amounts. The presence of partial charges on the plastic surface and the oxidation rate of microplastics may explain these differences. Tonometry experiments using an original method, the diffuse reflection of light, showed that adsorbed Hb on PP-filters retains its cooperativity, but its affinity for O2 decreases significantly.

Human Serum Albumin in the Presence of Small Platinum Nanoparticles.

Noble metal materials, especially platinum nanoparticles (Pt NPs), have immense potential in nanomedicine as therapeutic agents on account of their high electron density and their high surface area. Intravenous injection is proposed as the best mode to deliver the product to patients. However, our understanding of the reaction of nanoparticles with blood components, especially proteins, is far behind the explosive development of these agents. Using synchrotron radiation circular dichroism (SRCD), we investigated the structural and stability changes of human serum albumin (HSA) upon interaction with PEG-OH coated Pt NPs at nanomolar concentrations, conditions potentially encountered for intravenous injection. There is no strong complexation found between HSA and Pt NPs. However, for the highest molar ratio of NP:HSA of 1:1, an increase of 18 °C in the thermal unfolding of HSA was observed, which is attributed to increased thermal stability of HSA generated by preferential hydration. This work proposes a new and fast method to probe the potential toxicity of nanoparticles intended for clinical use with intravenous injection.

Radiation-Responsive Benzothiazolines as Potential Cleavable Fluorogenic Linkers for Drug Delivery.

Radiosensitive compounds can be useful for the detection of radiations and also as prodrugs that can be activated during a radiotherapy. Herein we describe the use of benzothiazolines, which upon treatment with 137 Cs produced γ-irradiation in water give rise to fluorescent benzothiazoles and concomitant release of amines or carboxylic acids. In a proof of concept study, we showed that benzothiazolines may be used as new cleavable linkers that can be triggered upon irradiation.

Role of the Protein Corona in the Colloidal Behavior of Microplastics.

Microparticles of polyethylene and polypropylene are largely found in aquatic environments because they are the most produced and persistent plastic materials. Once in biological media, they are covered by a layer of molecules, the so-called corona, mostly composed of proteins. A yeast protein extract from Saccharomyces cerevisiae was used as a protein system to observe interactions in complex biological media. Proteins, acting as surfactants and providing hydrophilic surfaces, allow the dispersion of highly hydrophobic particles in water and stabilize them. After 24 h, the microplastic quantity was up to 1 × 1011 particles per liter, whereas without protein, no particles remained in solution. Label-free imaging of the protein corona by synchrotron radiation deep UV fluorescence microscopy (SR-DUV) was performed. In situ images of the protein corona were obtained, and the adsorbed protein quantity, the coverage rate, and the corona heterogeneity were determined. The stability kinetics of the microplastic suspensions were measured by light transmission using a Turbiscan analyzer. Together, the microscopic and kinetics results demonstrate that the protein corona can very efficiently stabilize microplastics in solution provided that the protein corona quality is sufficient. Microplastic stability depends on different parameters such as the particle's intrinsic properties (size, density, hydrophobicity) and the protein corona formation that changes the particle wettability, electrostatic charge, and steric hindrance. By controlling these parameters with proteins, it becomes possible to keep microplastics in and out of solution, paving the way for applications in the field of microplastic pollution control and remediation.

How Nanoparticles Modify Adsorbed Proteins: Impact of Silica Nanoparticles on the Hemoglobin Active Site.

The adsorption of proteins on surfaces has been studied for a long time, but the relationship between the structural and functional properties of the adsorbed protein and the adsorption mechanism remains unclear. Using hemoglobin adsorbed on silica nanoparticles, we have previously shown that hemoglobin's affinity towards oxygen increases with adsorption. Nevertheless, it was also shown that there were no significant changes in the quaternary and secondary structures. In order to understand the change in activity, we decided in this work to focus on the active sites of hemoglobin, the heme and its iron. After measuring adsorption isotherms of porcine hemoglobin on Ludox silica nanoparticles, we analyzed the structural modifications of adsorbed hemoglobin by X-ray absorption spectroscopy and circular dichroism spectra in the Soret region. It was found that upon adsorption, there were modifications in the heme pocket environment due to changes in the angles of the heme vinyl functions. These alterations can explain the greater affinity observed.

A proteome scale study reveals how plastic surfaces and agitation promote protein aggregation.

Protein aggregation in biotherapeutics can reduce their activity and effectiveness. It may also promote immune reactions responsible for severe adverse effects. The impact of plastic materials on protein destabilization is not totally understood. Here, we propose to deconvolve the effects of material surface, air/liquid interface, and agitation to decipher their respective role in protein destabilization and aggregation. We analyzed the effect of polypropylene, TEFLON, glass and LOBIND surfaces on the stability of purified proteins (bovine serum albumin, hemoglobin and α-synuclein) and on a cell extract composed of 6000 soluble proteins during agitation (P = 0.1-1.2 W/kg). Proteomic analysis revealed that chaperonins, intrinsically disordered proteins and ribosomes were more sensitive to the combined effects of material surfaces and agitation while small metabolic oligomers could be protected in the same conditions. Protein loss observations coupled to Raman microscopy, dynamic light scattering and proteomic allowed us to propose a mechanistic model of protein destabilization by plastics. Our results suggest that protein loss is not primarily due to the nucleation of small aggregates in solution, but to the destabilization of proteins exposed to material surfaces and their subsequent aggregation at the sheared air/liquid interface, an effect that cannot be prevented by using LOBIND tubes. A guidance can be established on how to minimize these adverse effects. Remove one of the components of this combined stress - material, air (even partially), or agitation - and proteins will be preserved.

From soil to cacao bean: Unravelling the pathways of cadmium translocation in a high Cd accumulating cultivar of Theobroma cacao L.

The research on strategies to reduce cadmium (Cd) accumulation in cacao beans is currently limited by a lack of understanding of the Cd transfer pathways within the cacao tree. Here, we elucidated the transfer of Cd from soil to the nib (seed) in a high Cd accumulating cacao cultivar. Here, we elucidated the transfer of Cd from soil to the nib (seed) in a high Cd accumulating cacao cultivar through Cd stable isotope fractionation, speciation (X-Ray Absorption Spectroscopy), and localization (Laser Ablation Inductively Coupled Plasma Mass Spectrometry). The plant Cd concentrations were 10-28 higher than the topsoil Cd concentrations and increased as placenta< nib< testa< pod husk< root< leaf< branch. The retention of Cd in the roots was low. Light Cd isotopes were retained in the roots whilst heavier Cd isotopes were transported to the shoots (Δ 114/110 Cd shoot-root = 0.27 ± 0.02 ‰ (weighted average ± standard deviation)). Leaf Cd isotopes were heavier than Cd in the branches (Δ 114/110 Cd IF3 leaves-branch = 0.18 ± 0.01 ‰), confirming typical trends observed in annual crops. Nibs and branches were statistically not distinguishable (Δ 114/110 Cd nib-branch = -0.08‰ ± 0.06 ‰), contrary to the leaves and nibs (Δ 114/110 Cd nib-IF3 leaves = -0.25‰ ± 0.05 ‰). These isotope fractionation patterns alluded to a more direct transfer from branches to nibs rather than from leaves to nibs. The largest fraction (57%) of total plant Cd was present in the branches where it was primarily bound to carboxyl-ligands (60-100%) and mainly localized in the phloem rays and phelloderm of the bark. Cadmium in the nibs was mainly bound to oxygen ligands (60-90%), with phytate as the most plausible ligand. The weight of evidence suggested that Cd was transferred like other nutrients from root to shoot and accumulated in the phloem rays and phelloderm of the branches to reduce the transfer to foliage. Finally, the data indicated that the main contribution of nib Cd was from the phloem tissues of the branch rather than from leaf remobilization. This study extended the limited knowledge on Cd accumulation in perennial, woody crops and revealed that the Cd pathways in cacao are markedly different than in annual crops.

Human Serum Albumin in the Presence of AGuIX Nanoagents: Structure Stabilisation without Direct Interaction.

The gadolinium-based nanoagent named AGuIX® is a unique radiosensitizer and contrast agent which improves the performance of radiotherapy and medical imaging. Currently tested in clinical trials, AGuIX® is administrated to patients via intravenous injection. The presence of nanoparticles in the blood stream may induce harmful effects due to undesired interactions with blood components. Thus, there is an emerging need to understand the impact of these nanoagents when meeting blood proteins. In this work, the influence of nanoagents on the structure and stability of the most abundant blood protein, human serum albumin, is presented. Synchrotron radiation circular dichroism showed that AGuIX® does not bind to the protein, even at the high ratio of 45 nanoparticles per protein at 3 mg/L. However, it increases the stability of the albumin. Isothermal thermodynamic calorimetry and fluorescence emission spectroscopy demonstrated that the effect is due to preferential hydration processes. Thus, this study confirms that intravenous injection of AGuIX® presents limited risks of perturbing the blood stream. In a wider view, the methodology developed in this work may be applied to rapidly evaluate the impact and risk of other nano-products that could come into contact with the bloodstream.

In Situ Analysis of Weakly Bound Proteins Reveals Molecular Basis of Soft Corona Formation.

Few experimental techniques allow the analysis of the protein corona in situ. As a result, little is known on the effects of nanoparticles on weakly bound proteins that form the soft corona. Despite its biological importance, our understanding of the molecular bases driving its formation is limited. Here, we show that hemoglobin can form either a hard or a soft corona on silica nanoparticles depending on the pH conditions. Using cryoTEM and synchrotron-radiation circular dichroism, we show that nanoparticles alter the structure and the stability of weakly bound proteins in situ. Molecular dynamics simulation identified the structural elements driving protein-nanoparticle interaction. Based on thermodynamic analysis, we show that nanoparticles stabilize partially unfolded protein conformations by enthalpy-driven molecular interactions. We suggest that nanoparticles alter weakly bound proteins by shifting the equilibrium toward the unfolded states at physiological temperature. We show that the classical approach based on nanoparticle separation from the biological medium fails to detect destabilization of weakly bound proteins, and therefore cannot be used to fully predict the biological effects of nanomaterials in situ.

From Protein Corona to Colloidal Self-Assembly: The Importance of Protein Size in Protein-Nanoparticle Interactions.

Protein adsorption on nanoparticles is an important field of study, particularly with regard to nanomedicine and nanotoxicology. Many factors can influence the composition and structure of the layer(s) of adsorbed proteins, the so-called protein corona. However, the role of protein size has not been specifically investigated, although some evidence has indicated its potential important role in corona composition and structure. To assess the role of protein size, we studied the interactions of hemoproteins (spanning a large size range) with monodisperse silica nanoparticles. We combined various techniques-adsorption isotherms, isothermal titration calorimetry, circular dichroism, and transmission electron cryomicroscopy-to address this issue. Overall, the results show that small proteins behaved as typical model proteins, forming homogeneous monolayers on the nanoparticle surface (protein corona). Their adsorption is purely enthalpy-driven, with subtle structural changes. In contrast, large proteins interact with nanoparticles via entropy-driven mechanisms. Their structure is completely preserved during adsorption, and any given protein can directly bind to several nanoparticles, forming bridges in these newly formed protein-nanoparticle assemblies. Protein size is clearly an overlooked factor that should be integrated into proteomics and toxicological studies.