Stability and dispersibility of microplastics in experimental exposure medium and their dimensional characterization by SMLS, SAXS, Raman microscopy, and SEM.

The plastic production that contributes to the global plastic reservoir presents a major challenge for society in managing plastic waste and mitigating the environmental damage of microplastic (MP) pollution. In the environment, the formation of biomolecular corona around MPs enhance the stability of MP suspensions, influencing the bioavailability and toxicity of MPs. Essential physical properties including MP stability, dispersibility, agglomeration, and dimensional size must be precisely defined and measured in complex media taking into account the formation of a protein corona. Using static multiple light scattering (SMLS), small angle X-ray scattering (SAXS), Raman microscopy, and scanning electron microscopy (SEM), we measured the particle size, density, stability, and agglomeration state of polyethylene and polypropylene MPs stabilized in aqueous suspension by BSA. SEM analysis revealed the formation of nanoplastic debris as MP suspensions aged. Our results suggest that protein adsorption favors the formation of secondary nanoplastics, potentially posing an additional threat to ecosystems. This approach provides analytical methodologies by integrating SEM, SMLS, and SAXS, for characterizing MP suspensions and highlights the effect of the protein corona on size measurements of micro/nanoplastics. Our analysis demonstrates the detectability of secondary nanoplastics by SEM, paving the way for monitoring and controlling human exposure.

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.

Superoxide Production under Soft X-ray Irradiation of Liquid Water.

Soft X-rays behave like particles with high linear energy transfer, as they deposit a large amount of their energy in the nanometric range, triggered by inner-shell ionization. In water, this can lead to the formation of a doubly ionized water molecule (H2O2+) and the emission of two secondary electrons (photoelectron and Auger electron). Our focus lies on detecting and quantifying the superoxide (HO2°) production via the direct pathway, i.e., from the reaction between the dissociation product of H2O2+, i.e., the oxygen atom (∼4 fs), and the °OH radicals present in the secondary electron tracks. The HO2° yield for 1620 eV photons, via this reaction pathway, was found to be 0.005 (±0.0007) µmol/J (formed within the ∼ps range). Experiments were also performed to determine the yield of HO2° production via another (indirect) pathway, involving solvated electrons. The indirect HO2° yield, measured experimentally as a function of photon energy (from 1700 to 350 eV), resulted in a steep decrease at around 1280 eV and a minimum close to zero at 800 eV. This behavior in contradiction with the theoretical prediction reveals the complexity hidden in the intratrack reactions.

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.

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.

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.

Electronic Structure and Solvation Effects from Core and Valence Photoelectron Spectroscopy of Serum Albumin.

X-ray photoelectron spectroscopy of bovine serum albumin (BSA) in a liquid jet is used to investigate the electronic structure of a solvated protein, yielding insight into charge transfer mechanisms in biological systems in their natural environment. No structural damage was observed in BSA following X-ray photoelectron spectroscopy in a liquid jet sample environment. Carbon and nitrogen atoms in different chemical environments were resolved in the X-ray photoelectron spectra of both solid and solvated BSA. The calculations of charge distributions demonstrate the difficulty of assigning chemical contributions in complex systems in an aqueous environment. The high-resolution X-ray core electron spectra recorded are unchanged upon solvation. A comparison of the valence bands of BSA in both phases is also presented. These bands display a higher sensitivity to solvation effects. The ionization energy of the solvated BSA is determined at 5.7 ± 0.3 eV. Experimental results are compared with theoretical calculations to distinguish the contributions of various molecular components to the electronic structure. This comparison points towards the role of water in hole delocalization in proteins.

Experimental determination of the curvature-induced intra-wall polarization of inorganic nanotubes.

Inspired by a natural nano-mineral known as imogolite, aluminosilicate inorganic nanotubes are appealing systems for photocatalysis. Here, we studied two types of synthetic imogolites: one is completely hydrophilic (IMO-OH), while the other has a hydrophilic exterior and a hydrophobic interior (IMO-CH3), enabling the encapsulation of organic molecules. We combined UV-Vis diffuse reflectance spectroscopy of imogolite powders and X-ray photoelectron spectroscopy of deposited imogolite films and isolated nanotubes agglomerates to obtain not only the band structure, but also the quantitative intra-wall polarization of both synthetic imogolites for the first time. The potential difference across the imogolite wall was determined to be 0.7 V for IMO-OH and around 0.2 V for IMO-CH3. The high curvature of the nanotubes, together with the thinness of their wall, favors efficient spontaneous charge separation and electron exchange reactions on both the internal and external nanotube surfaces. In addition, the positions of their valence and conduction band edges make them interesting candidates for co-catalysts or doped catalysts for water splitting, among other possible photocatalytic reactions relevant to energy and the environment.

A microfluidic dosimetry cell to irradiate solutions with poorly penetrating radiations: a step towards online dosimetry for synchrotron beamlines.

Synchrotron radiation can induce sample damage, whether intended or not. In the case of sensitive samples, such as biological ones, modifications can be significant. To understand and predict the effects due to exposure, it is necessary to know the ionizing radiation dose deposited in the sample. In the case of aqueous samples, deleterious effects are mostly induced by the production of reactive oxygen species via water radiolysis. These species are therefore good indicators of the dose. Here the application of a microfluidic cell specifically optimized for low penetrating soft X-ray radiation is reported. Sodium benzoate was used as a fluorescent dosimeter thanks to its specific detection of hydroxyl radicals, a radiolytic product of water. Measurements at 1.28 keV led to the determination of a hydroxyl production yield, G(HO.), of 0.025 ±â€…0.004 µmol J-1. This result is in agreement with the literature and confirms the high linear energy transfer behavior of soft X-rays. An analysis of the important parameters of the microfluidic dosimetry cell, as well as their influences over dosimetry, is also reported.

How Do Surface Properties of Nanoparticles Influence Their Diffusion in the Extracellular Matrix? A Model Study in Matrigel Using Polymer-Grafted Nanoparticles.

Diffusion of nanomedicines inside the extracellular matrix (ECM) has been identified as a key factor to achieve homogeneous distribution and therefore therapeutic efficacy. Here, we sought to determine the impact of nanoparticles' (NPs) surface properties on their ability to diffuse in the ECM. As model nano-objects, we used a library of gold nanoparticles grafted with a versatile polymethacrylate corona, which enabled the surface properties to be modified. To accurately recreate the features of the native ECM, diffusion studies were carried out in a tumor-derived gel (Matrigel). We developed two methods to evaluate the diffusion ability of NPs inside this model gel: an easy-to-implement one based on optical monitoring and another one using small-angle X-ray scattering (SAXS) measurements. Both enabled the determination of the diffusion coefficients of NPs and comparison of the influence of their various surface properties, while the SAXS technique also allowed to monitor the NPs' structure as they diffused inside the gel. Positive charges and hydrophobicity were found to particularly hinder diffusion, and the different results suggested on the whole the presence of NPs-matrix interactions, therefore underlying the importance of the ECM model. The accuracy of the tumor-derived gels used in this study was evidenced by in vivo experiments involving intratumoral injections of NPs on mice, which showed that diffusion patterns in the peripheral tumor tissues were quite similar to the ones obtained within the chosen ECM model.

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.

Combining surface chemistry modification and in situ small-angle scattering characterization to understand and optimize the biological behavior of nanomedicines.

Nanomedicines are considered as promising therapeutics for cancer treatment. However, clinical translation is still scarce, partly because their biological behavior is not well understood. Extracting general guidelines from the great variety of nanoparticles and conditions studied is indeed difficult, and relevant techniques are lacking to obtain in situ information. Here, both issues are solved by combining versatile model nanoparticles with in situ tools based on small-angle scattering techniques (SAS). The strategy was to develop a library of nanoparticles and perform systematic study of their interactions with biological systems. Considering the promising properties of gold nanoparticles as cancer therapeutics, polymethacrylate-grafted gold nanoparticles were chosen as models. Modulation of polymer chemistry was shown to change the surface properties while keeping the same structure for all nanoparticles. This unity allowed reliable comparison to extract general principles, while the synthesis versatility enabled to fine-tune the nanoparticles surface properties, especially through copolymerization, and thus to optimize their biological behavior. Two specific aspects were particularly examined: colloidal stability and cell uptake. Positive charges and hydrophobicity were identified as key parameters influencing toxicity and internalization. In situ SAS gave valuable information about nanoparticles evolution in biologically relevant environments. Good colloidal stability was thereby shown in cell culture media, while intracellular transformation and quantity of nanoparticles were monitored, highlighting the potential of these techniques for nanomedicines studies.

Soft X-ray Radiation and Monte Carlo Simulations: Good Tools to Describe the Radiation Chemistry of Sub-keV Electrons.

The description of the biological effects of ionizing radiation requires a good knowledge of the dose deposition processes at both the cellular and molecular scales. However, experimental studies on the energy deposition specificity of sub-keV electrons, produced by most radiations, including high-energy photons and heavy ions, are scarce. Soft X-rays (0.2-2 keV) are here used to probe the physical and physico-chemical events occurring upon exposure of liquid water to sub-keV electrons. Liquid water samples were irradiated with a monochromatic photon beam at the SOLEIL synchrotron. Hydroxyl radical quantification was conducted through HO• scavenging using benzoate to form fluorescent hydroxybenzoate. The yields of HO• radicals exhibit a minimum around 1.5 keV, in good agreement with indirect observation. Moreover, they are relatively independent of the benzoate concentration in the range investigated, which corresponds to scavenging times of 170 ns to 170 ps. These results provide evidence that sub-keV electrons behave as high linear energy transfer particles, since they are able to deposit tens to hundreds of electronvolts in nanometric volumes.

Time-resolved cathodoluminescence of DNA triggered by picosecond electron bunches.

Despite the tremendous importance of so-called ionizing radiations (X-rays, accelerated electrons and ions) in cancer treatment, most studies on their effects have focused on the ionization process itself, and neglect the excitation events the radiations can induce. Here, we show that the excited states of DNA exposed to accelerated electrons can be studied in the picosecond time domain using a recently developed cathodoluminescence system with high temporal resolution. Our study uses a table-top ultrafast, UV laser-triggered electron gun delivering picosecond electron bunches of keV energy. This scheme makes it possible to directly compare time-resolved cathodoluminescence with photoluminescence measurements. This comparison revealed qualitative differences, as well as quantitative similarities between excited states of DNA upon exposure to electrons or photons.

Protein Corona Composition of Silica Nanoparticles in Complex Media: Nanoparticle Size does not Matter.

Biomolecules, and particularly proteins, bind on nanoparticle (NP) surfaces to form the so-called protein corona. It is accepted that the corona drives the biological distribution and toxicity of NPs. Here, the corona composition and structure were studied using silica nanoparticles (SiNPs) of different sizes interacting with soluble yeast protein extracts. Adsorption isotherms showed that the amount of adsorbed proteins varied greatly upon NP size with large NPs having more adsorbed proteins per surface unit. The protein corona composition was studied using a large-scale label-free proteomic approach, combined with statistical and regression analyses. Most of the proteins adsorbed on the NPs were the same, regardless of the size of the NPs. To go beyond, the protein physicochemical parameters relevant for the adsorption were studied: electrostatic interactions and disordered regions are the main driving forces for the adsorption on SiNPs but polypeptide sequence length seems to be an important factor as well. This article demonstrates that curvature effects exhibited using model proteins are not determining factors for the corona composition on SiNPs, when dealing with complex biological media.

Albumin-driven disassembly of lipidic nanoparticles: the specific case of the squalene-adenosine nanodrug.

In the field of nanomedicine, nanostructured nanoparticles (NPs) made of self-assembling prodrugs emerged in the recent years with promising properties. In particular, squalene-based drug nanoparticles have already shown their efficiency through in vivo experiments. However, a complete pattern of their stability and interactions in the blood stream is still lacking. In this work we assess the behavior of squalene-adenosine (SQAd) nanoparticles - whose neuroprotective effect has already been demonstrated in murine models - in the presence of fetal bovine serum (FBS) and of bovine serum albumin (BSA), the main protein of blood plasma. Extensive physicochemical characterizations were performed using Small Angle Neutron Scattering (SANS), cryogenic transmission electron microscopy (Cryo-TEM), circular dichroism (CD), steady-state fluorescence spectroscopy (SSFS) and isothermal titration calorimetry (ITC) as well as in silico by means of ensemble docking simulations with human serum albumin (HSA). Significant changes in the colloidal stability of the nanoparticles in the presence of serum albumin were observed. SANS, CD and SSFS analyses demonstrated an interaction between SQAd and BSA, with a partial disassembly of the nanoparticles in the presence of BSA and the formation of a complex between SQAd and BSA. The interaction free energy of SQAd nanoparticles with BSA derived from ITC experiments, is about -8 kcal mol-1 which is further supported in silico by ensemble docking simulations. Overall, our results show that serum albumin partially disassembles SQAd nanoparticles by extracting individual SQAd monomers from them. As a consequence, the SQAd nanoparticles would act as a circulating reservoir in the blood stream. The approach developed in this study could be extended to other soft organic nanoparticles.

Improving 131I Radioiodine Therapy By Hybrid Polymer-Grafted Gold Nanoparticles.

BACKGROUND: Human trials combining external radiotherapy (RT) and metallic nanoparticles are currently underway in cancer patients. For internal RT, in which a radioisotope such as radioiodine is systemically administered into patients, there is also a need for enhancing treatment efficacy, decreasing radiation-induced side effects and overcoming radio-resistance. However, if strategies vectorising radioiodine through nanocarriers have been documented, sensitizing the neoplasm through the use of nanotherapeutics easily translatable to the clinic in combination with the standard systemic radioiodine treatment has not been assessed yet. METHOD AND MATERIALS: The present study explored the potential of hybrid poly(methacrylic acid)-grafted gold nanoparticles to improve the performances of systemic 131I-mediated RT on cancer cells and in tumor-bearing mice. Such nanoparticles were chosen based on their ability previously described by our group to safely withstand irradiation doses while exhibiting good biocompatibility and enhanced cellular uptake. RESULTS: In vitro clonogenic assays performed on melanoma and colorectal cancer cells showed that poly(methacrylic acid)-grafted gold nanoparticles (PMAA-AuNPs) could efficiently lead to a marked tumor cell mortality when combined to a low activity of radioiodine, which alone appeared to be essentially ineffective on tumor cells. In vivo, tumor enrichment with PMAA-AuNPs significantly enhanced the killing potential of a systemic radioiodine treatment. CONCLUSION: This is the first report of a simple and reliable nanomedicine-based approach to reduce the dose of radioiodine required to reach curability. In addition, these results open up novel perspectives for using high-Z metallic NPs in additional molecular radiation therapy demonstrating heterogeneous dose distributions.