Advancing In Situ Analysis of Biomolecular Corona: Opportunities and Challenges in Utilizing Field-Flow Fractionation.

The biomolecular corona, a complex layer of biological molecules, envelops nanoparticles (NPs) upon exposure to biological fluids including blood. This dynamic interface is pivotal for the advancement of nanomedicine, particularly in areas of therapy and diagnostics. In situ analysis of the biomolecular corona is crucial, as it can substantially improve our ability to accurately predict the biological fate of nanomedicine and, therefore, enable development of more effective, safe, and precisely targeted nanomedicines. Despite its importance, the repertoire of techniques available for in situ analysis of the biomolecular corona is surprisingly limited. This tutorial review provides an overview of the available techniques for in situ analysis of biomolecular corona with a particular focus on exploring both the advantages and the limitations inherent in the use of field-flow fractionation (FFF) for in situ analysis of the biomolecular corona. It delves into how FFF can unravel the complexities of the corona, enhancing our understanding and guiding the design of next-generation nanomedicines for medical use.

Protein Corona Composition of Gold Nanocatalysts.

The interaction between nanoparticles (NPs) and biological environments is profoundly influenced by a stable, strongly adsorbed "hard" protein corona. This corona significantly determines the NPs' pharmacokinetics and biological destiny. Our study delves into the mechanisms by which colloidal Au nanocrystals that are synthesized electrochemically without surface-capping organic ligands, known as CNM-Au8, traverse the blood-brain barrier (BBB) and target human brain tissue for treating neurodegenerative disorders. We discovered that upon interaction with human plasma, CNM-Au8 gold nanocrystals (AuNCs) effectively attract a variety of crucial apolipoproteins, notably apolipoproteins E, to their surfaces. This interaction likely facilitates their passage through the BBB. Furthermore, the coronas of these AuNCs exhibit a substantial presence of albumin and a notable absence of opsonin-based proteins, contributing to prolonged blood circulation. These characteristics align well with the clinical performance observed for the CNM-Au8 NCs. This study highlights that AuNCs with intentionally engineered structures and surfactant-free surfaces can create a distinct protein corona composition. This finding holds significant promise for the development of advanced therapeutic agents aimed at combating neurodegenerative diseases.

Development and Application of Nanoparticle-Nanopolymer Composite Spheres for the Study of Environmental Processes.

Plastics have long been an environmental contaminant of concern as both large-scale plastic debris and as micro- and nano-plastics with demonstrated wide-scale ubiquity. Research in the past decade has focused on the potential toxicological risks posed by microplastics, as well as their unique fate and transport brought on by their colloidal nature. These efforts have been slowed by the lack of analytical techniques with sufficient sensitivity and selectivity to adequately detect and characterize these contaminants in environmental and biological matrices. To improve analytical analyses, microplastic tracers are developed with recognizable isotopic, metallic, or fluorescent signatures capable of being identified amidst a complex background. Here we describe the synthesis, characterization, and application of a novel synthetic copolymer nanoplastic based on polystyrene (PS) and poly(2-vinylpyridine) (P2VP) intercalated with gold, platinum or palladium nanoparticles that can be capped with different polymeric shells meant to mimic the intended microplastic. In this work, particles with PS and polymethylmethacrylate (PMMA) shells are used to examine the behavior of microplastic particles in estuarine sediment and coastal waters. The micro- and nanoplastic tracers, with sizes between 300 and 500 nm in diameter, were characterized using multiple physical, chemical, and colloidal analysis techniques. The metallic signatures of the tracers allow for quantification by both bulk and single-particle inductively-coupled plasma mass spectrometry (ICP-MS and spICP-MS, respectively). As a demonstration of environmental applicability, the tracers were equilibrated with sediment collected from Bellingham Bay, WA, United States to determine the degree to which microplastics bind and sink in an estuary based of grain size and organic carbon parameters. In these experiments, between 80 and 95% of particles were found to associate with the sediment, demonstrative of estuaries being a major anticipated sink for these contaminants. These materials show considerable promise in their versatility, potential for multiplexing, and utility in studying micro- and nano-plastic transport in real-world environments.

Experimental verification of the steric-entropic mode of retention in centrifugal field-flow fractionation using illite clay plates.

The commonly used theory to describe the normal Brownian mode of field-flow fractionation (FFF) assumes the particles to be point masses and hence the shape is ignored. Beckett and Giddings extended this theory to include the effect of thin rods and discs being forced very close to the accumulation wall. By including the decrease in the entropy this causes, they derived new expressions for the retention of such nonspherical particles in FFF. The steric-entropic theory predicts that when the sample cloud thickness is less than the major dimension of the rods or discs then particles elute earlier than predicted by the Brownian mode theory. This leads to an underestimation of the buoyant mass and equivalent spherical diameter calculated from FFF data. In this paper we report for the first time experimental data for the retention of thin illite particles in centrifugal FFF that agrees well with these steric-entropic predictions. Not only do the size distributions calculated using the Brownian mode theory shift to lower size when the field is increased but the shift in the retention ratio of the peak maxima of the FFF fractograms could be predicted fairly accurately by the steric-entropic equations.

Measurement of the Density of Engineered Silver Nanoparticles Using Centrifugal FFF-TEM and Single Particle ICP-MS.

A methodology has been developed to measure nanoparticle mass and density, by combining centrifugal field-flow fractionation (CeFFF; more commonly called sedimentation FFF or SdFFF) and transmission electron microscopy (TEM). Particle effective mass obtained from CeFFF retention data and particle size obtained from the TEM images were used to calculate the nanoparticle density. The method was initially applied to measure the density of monodispersed polystyrene latex nanoparticles. Measured densities for latex nanoparticles of 160-300 nm in diameter were in the range of 1041-1063 kg m-3 with standard deviations of 0.6-1.1%. Densities of engineered silver nanoparticles with nominal diameters of 30, 60, 75, and 100 nm were measured using this methodology. For all four silver nanoparticle samples, the measured densities were 18-24% lower than the nominal density of metallic silver, with an overall mean value of 7900 ± 675 kg m-3. Density values calculated using nanoparticle mass values obtained from single particle inductively coupled plasma-mass spectrometry (spICP-MS) measurements, corroborated the CeFFF-TEM results. The difference in the density of the silver nanoparticles compared to that of bulk silver suggests that the synthesis process could impart 20-37% porosity in silver nanoparticles. The data has important implications in the fields of nanomaterial, nanomedicine and nanotoxicology, where assumption of the bulk density for nanoparticles can result in erroneous estimation of parameters such as mass, size, porosity, and dosage. The presented methodology provides a straightforward and reproducible means for measurement of the density and porosity of engineered nanoparticles with a wide range of density and size.

Detection and Quantification of Silver Nanoparticles at Environmentally Relevant Concentrations Using Asymmetric Flow Field-Flow Fractionation Online with Single Particle Inductively Coupled Plasma Mass Spectrometry.

The presence of silver nanoparticles (AgNPs) in aquatic environments could potentially cause adverse impacts on ecosystems and human health. However, current understanding of the environmental fate and transport of AgNPs is still limited because their properties in complex environmental samples cannot be accurately determined. In this study, the feasibility of using asymmetric flow field-flow fractionation (AF4) connected online with single particle inductively coupled plasma mass spectrometry (spICPMS) to detect and quantify AgNPs at environmentally relevant concentrations was investigated. The AF4 channel had a thickness of 350 µm and its accumulation wall was a 10 kDa regenerated cellulose membrane. A 0.02% FL-70 surfactant solution was used as an AF4 carrier. With 1.2 mL/min AF4 cross-flow rate, 1.5 mL/min AF4 channel flow rate, and 5 ms spICPMS dwell time, the AF4-spICPMS can detect and quantify 40-80 nm AgNPs, as well as Ag-SiO2 core-shell nanoparticles (51.0 nm diameter Ag core and 21.6 nm SiO2 shell), with good recovery within 30 min. This system was not only effective in differentiating and quantifying different types of AgNPs with similar hydrodynamic diameters, such as in mixtures containing Ag-SiO2 core-shell nanoparticles and 40-80 nm AgNPs, but also suitable for differentiating between 40 nm AgNPs and elevated Ag(+) content. The study results indicate that AF4-spICPMS is capable of detecting and quantifying AgNPs and other engineered metal nanomaterials in environmental samples. Nevertheless, further studies are needed before AF4-spICPMS can become a routine analytical technique.

Cationic PAMAM dendrimers aggressively initiate blood clot formation.

Poly(amidoamine) (PAMAM) dendrimers are increasingly studied as model nanoparticles for a variety of biomedical applications, notably in systemic administrations. However, with respect to blood-contacting applications, amine-terminated dendrimers have recently been shown to activate platelets and cause a fatal, disseminated intravascular coagulation (DIC)-like condition in mice and rats. We here demonstrate that, upon addition to blood, cationic G7 PAMAM dendrimers induce fibrinogen aggregation, which may contribute to the in vivo DIC-like phenomenon. We demonstrate that amine-terminated dendrimers act directly on fibrinogen in a thrombin-independent manner to generate dense, high-molecular-weight fibrinogen aggregates with minimal fibrin fibril formation. In addition, we hypothesize this clot-like behavior is likely mediated by electrostatic interactions between the densely charged cationic dendrimer surface and negatively charged fibrinogen domains. Interestingly, cationic dendrimers also induced aggregation of albumin, suggesting that many negatively charged blood proteins may be affected by cationic dendrimers. To investigate this further, zebrafish embryos were employed to more specifically determine the speed of this phenomenon and the pathway- and dose-dependency of the resulting vascular occlusion phenotype. These novel findings show that G7 PAMAM dendrimers significantly and adversely impact many blood components to produce rapid coagulation and strongly suggest that these effects are independent of classic coagulation mechanisms. These results also strongly suggest the need to fully characterize amine-terminated PAMAM dendrimers in regard to their adverse effects on both coagulation and platelets, which may contribute to blood toxicity.

Aggregation of fullerol C60(OH)24 nanoparticles as revealed using flow field-flow fractionation and atomic force microscopy.

The effects of solution pH and 1:1 electrolyte concentration on the aggregation behavior of fullerol C(60)(OH)(24) nanoparticles were investigated using flow field-flow fractionation (FlFFF). Particle separations were confirmed by examining FFF fractions using atomic force microscopy (AFM). Results showed that fullerol C(60)(OH)(24) nanoparticles remain stable at low salt concentration (0.001 M NaCl) and basic pH (pH 10). Changing the pH did not affect the size significantly, but increasing the salt concentration promoted some aggregation. Fullerol C(60)(OH)(24) nanoparticles did not form large clusters and reached a maximum size of at most several nanometers. Particle interaction analysis using the colloid interaction theory as described by the energetics of electrostatic repulsion and van der Waals attraction explained the differences in the colloidal stability of the fullerol C(60)(OH)(24) nanoparticles under different solution conditions.

A novel method to detect unlabeled inorganic nanoparticles and submicron particles in tissue by sedimentation field-flow fractionation.

UNLABELLED: A novel methodology to detect unlabeled inorganic nanoparticles was experimentally demonstrated using a mixture of nano-sized (70 nm) and submicron (250 nm) silicon dioxide particles added to mammalian tissue. The size and concentration of environmentally relevant inorganic particles in a tissue sample can be determined by a procedure consisting of matrix digestion, particle recovery by centrifugation, size separation by sedimentation field-flow fractionation (SdFFF), and detection by light scattering. BACKGROUND: Laboratory nanoparticles that have been labeled by fluorescence, radioactivity, or rare elements have provided important information regarding nanoparticle uptake and translocation, but most nanomaterials that are commercially produced for industrial and consumer applications do not contain a specific label. METHODS: Both nitric acid digestion and enzyme digestion were tested with liver and lung tissue as well as with cultured cells. Tissue processing with a mixture of protease enzymes is preferred because it is applicable to a wide range of particle compositions. Samples were visualized via fluorescence microscopy and transmission electron microscopy to validate the SdFFF results. We describe in detail the tissue preparation procedures and discuss method sensitivity compared to reported levels of nanoparticles in vivo. CONCLUSION: Tissue digestion and SdFFF complement existing techniques by precisely identifying unlabeled metal oxide nanoparticles and unambiguously distinguishing nanoparticles (diameter<100 nm) from both soluble compounds and from larger particles of the same nominal elemental composition. This is an exciting capability that can facilitate epidemiological and toxicological research on natural and manufactured nanomaterials.