Interactions of cationic polystyrene nanoparticles with marine bivalve hemocytes in a physiological environment: Role of soluble hemolymph proteins.

The bivalve Mytilus galloprovincialis has proven as a suitable model invertebrate for evaluating the potential impact of nanoparticles (NPs) in the marine environment. In particular, in mussels, the immune system represents a sensitive target for different types of NPs. In environmental conditions, both NP intrinsic properties and those of the receiving medium will affect particle behavior and consequent bioavailability/uptake/toxicity. However, the evaluation of the biological effects of NPs requires additional understanding of how, once within the organism, NPs interact at the molecular level with cells in a physiological environment. In mammalian systems, different NPs associate with serum soluble components, organized into a "protein corona", which affects particle interactions with target cells. However, no information is available so far on the interactions of NPs with biological fluids of aquatic organisms. In this work, the influence of hemolymph serum (HS) on the in vitro effects of amino modified polystyrene NPs (PS-NH2) on Mytilus hemocytes was investigated. Hemocytes were incubated with PS-NH2 suspensions in HS (1, 5 and 50µg/mL) and the results were compared with those obtained in ASW medium. Cell functional parameters (lysosomal membrane stability, oxyradical production, phagocytosis) were evaluated, and morphological changes were investigated by TEM. The activation state of the signalling components involved in Mytilus immune response (p38 MAPK and PKC) was determined. The results show that in the presence of HS, PS-NH2 increased cellular damage and ROS production with respect to ASW medium. The effects were apparently mediated by disregulation of p38 MAPK signalling. The formation of a PS-NH2-protein corona in HS was investigated by centrifugation, and 1D- gel electrophoresis and nano-HPLC-ESI-MS/MS. The results identified the Putative C1q domain containing protein (MgC1q6) as the only component of the PS-NH2 hard protein corona in Mytilus hemolymph. These data represent the first evidence for the formation of a NP bio-corona in aquatic organisms and underline the importance of the recognizable biological identity of NPs in physiological exposure medium when testing their potential impact environmental model organisms. Although the results obtained in vitro do not entirely reflect a realistic exposure scenario and the more complex formation of a bio-corona that is likely to occur in vivo, these data will contribute to a better understanding of the effects of NPs in marine invertebrates.

Nano-sized polystyrene affects feeding, behavior and physiology of brine shrimp Artemia franciscana larvae.

Nano-sized polymers as polystyrene (PS) constitute one of the main challenges for marine ecosystems, since they can distribute along the whole water column affecting planktonic species and consequently disrupting the energy flow of marine ecosystems. Nowadays very little knowledge is available on the impact of nano-sized plastics on marine organisms. Therefore, the present study aims to evaluate the effects of 40nm anionic carboxylated (PS-COOH) and 50nm cationic amino (PS-NH2) polystyrene nanoparticles (PS NPs) on brine shrimp Artemia franciscana larvae. No signs of mortality were observed at 48h of exposure for both PS NPs at naplius stage but several sub-lethal effects were evident. PS-COOH (5-100µg/ml) resulted massively sequestered inside the gut lumen of larvae (48h) probably limiting food intake. Some of them were lately excreted as fecal pellets but not a full release was observed. Likewise, PS-NH2 (5-100µg/ml) accumulated in larvae (48h) but also adsorbed at the surface of sensorial antennules and appendages probably hampering larvae motility. In addition, larvae exposed to PS-NH2 undergo multiple molting events during 48h of exposure compared to controls. The activation of a defense mechanism based on a physiological process able to release toxic cationic NPs (PS-NH2) from the body can be hypothesized. The general observed accumulation of PS NPs within the gut during the 48h of exposure indicates a continuous bioavailability of nano-sized PS for planktonic species as well as a potential transfer along the trophic web. Therefore, nano-sized PS might be able to impair food uptake (feeding), behavior (motility) and physiology (multiple molting) of brine shrimp larvae with consequences not only at organism and population level but on the overall ecosystem based on the key role of zooplankton on marine food webs.

Trajectory-based co-localization measures for nanoparticle-cell interaction studies.

High-resolution live cell microscopy will soon have a fundamental role in understanding bio-nano interactions, providing material that can be exploited using single particle tracking techniques. The present work uses 3D timelapse images obtained with confocal microscopy, to temporally resolve the co-localization between polystyrene nanoparticles and lysosomes in live cells through object-based measurements.

Paracrine signalling of inflammatory cytokines from an in vitro blood brain barrier model upon exposure to polymeric nanoparticles.

Nanoparticle properties, such as small size relative to large highly modifiable surface area, offer great promise for neuro-therapeutics and nanodiagnostics. A fundamental understanding and control of how nanoparticles interact with the blood-brain barrier (BBB) could enable major developments in nanomedical treatment of previously intractable neurological disorders, and help ensure that nanoparticles not intended to reach the brain do not cause adverse effects. Nanosafety is of utmost importance to this field. However, a distinct lack of knowledge exists regarding nanoparticle accumulation within the BBB and the biological effects this may induce on neighbouring cells of the Central Nervous System (CNS), particularly in the long-term. This study focussed on the exposure of an in vitro BBB model to model carboxylated polystyrene nanoparticles (PS COOH NPs), as these nanoparticles are well characterised for in vitro experimentation and have been reported as non-toxic in many biological settings. TEM imaging showed accumulation but not degradation of 100 nm PS COOH NPs within the lysosomes of the in vitro BBB over time. Cytokine secretion analysis from the in vitro BBB post 24 h 100 nm PS COOH NP exposure showed a low level of pro-inflammatory RANTES protein secretion compared to control. In contrast, 24 h exposure of the in vitro BBB endothelium to 100 nm PS COOH NPs in the presence of underlying astrocytes caused a significant increase in pro-survival signalling. In conclusion, the tantalising possibilities of nanomedicine must be balanced by cautious studies into the possible long-term toxicity caused by accumulation of known 'toxic' and 'non-toxic' nanoparticles, as general toxicity assays may be disguising significant signalling regulation during long-term accumulation.

The protein corona mediates the impact of nanomaterials and slows amyloid beta fibrillation.

Put your coat on: It is well recognized that the surfaces of nanomaterials in biological media are covered by various biomolecules (e.g., proteins). A) The protein corona creates a shell over different nanomaterials, regardless of their physicochemical properties (e.g., composition and shape), resulting in reduced levels of amyloid beta fibril formation. B) Pristine nanomaterials might have acceleratory effects on the fibrillation of amyloid beta.

Surface coatings shape the protein corona of SPIONs with relevance to their application in vivo.

Superparamagnetic iron oxide nanoparticles (SPIONs) have proved their use in many biomedical applications, such as drug delivery, hyperthermia, and MRI (magnetic resonance imaging) contrast agents. Due to their instability in fluids, several surface coatings have been used to both stabilize and tune the properties of these nanoparticles (NPs) according to their applications. These coatings will strongly modify their surface properties and influence their interaction with the environment proteins in a relevant biological medium with a clear impact on their function. It is well-accepted that a protein corona is immediately formed when nanoparticles come in contact with a biological milieu, and the emergent bionano interface represents the biological identity of the particles. Here, we investigate how a different coating on the same magnetic core can influence the protein corona composition and structure with clear relevance to application of these NPs in medicine. In particular, we have studied the structure and composition of the protein corona-SPION complexes of magnetite nanoparticles stabilized with citric acid, poly(acrylic acid), or double layer oleic acid by a range of approaches, including dynamic light scattering, nanoparticle tracking analysis, differential centrifugal sedimentation, infrared spectroscopy, 1-D SDS gel electrophoresis, and mass spectroscopy.

Quantifying size-dependent interactions between fluorescently labeled polystyrene nanoparticles and mammalian cells.

BACKGROUND: Nanoparticles (NPs) are currently used in a wide variety of fields such as technology, medicine and industry. Due to the novelty of these applications and to ensure their success, a precise characterization of the interactions between NPs and cells is essential. FINDINGS: The current study explores the uptake of polystyrene NPs by 1321N1 human astrocytoma and A549 human lung carcinoma cell lines. In this work we show for the first time a comparison of the uptake rates of fluorescently labeled carboxylated polystyrene (PS) NPs of different sizes (20, 40 and 100 nm) in two different cell types, keeping the number of NPs per unit volume constant for all sizes. We propose a reliable methodology to control the dose of fluorescently labeled NPs, by counting individual NPs using automated particle detection from 3D confocal microscopy images. The possibility of detecting individual NPs also allowed us to calculate the size of each nanoparticle and compare the fluorescence of single NPs across different sizes, thereby providing a robust platform for normalization of NP internalization experiments as measured by flow cytometry. CONCLUSIONS: Our findings show that 40 nm NPs are internalized faster than 20 nm or 100 nm particles in both cell lines studied, suggesting that there is a privileged size gap in which the internalization of NPs is higher.

Physical-chemical aspects of protein corona: relevance to in vitro and in vivo biological impacts of nanoparticles.

It is now clearly emerging that besides size and shape, the other primary defining element of nanoscale objects in biological media is their long-lived protein ("hard") corona. This corona may be expressed as a durable, stabilizing coating of the bare surface of nanoparticle (NP) monomers, or it may be reflected in different subpopulations of particle assemblies, each presenting a durable protein coating. Using the approach and concepts of physical chemistry, we relate studies on the composition of the protein corona at different plasma concentrations with structural data on the complexes both in situ and free from excess plasma. This enables a high degree of confidence in the meaning of the hard protein corona in a biological context. Here, we present the protein adsorption for two compositionally different NPs, namely sulfonated polystyrene and silica NPs. NP-protein complexes are characterized by differential centrifugal sedimentation, dynamic light scattering, and zeta-potential both in situ and once isolated from plasma as a function of the protein/NP surface area ratio. We then introduce a semiquantitative determination of their hard corona composition using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrospray liquid chromatography mass spectrometry, which allows us to follow the total binding isotherms for the particles, identifying simultaneously the nature and amount of the most relevant proteins as a function of the plasma concentration. We find that the hard corona can evolve quite significantly as one passes from protein concentrations appropriate to in vitro cell studies to those present in in vivo studies, which has deep implications for in vitro-in vivo extrapolations and will require some consideration in the future.

Experimental and theoretical comparison of intracellular import of polymeric nanoparticles and small molecules: toward models of uptake kinetics.

Central to understanding how nanoscale objects interact with living matter is the need for reproducible and verifiable data that can be interpreted with confidence. Likely this will be the basis of durable advances in nanomedicine and nanomedical safety. To develop these fields, there is also considerable interest in advancing the first generation of theoretical models of nanoparticle (NP) uptake into cells, and NP biodistribution in general. Here we present an uptake study comparing the outcomes for free molecular dye and NPs labeled with the same dye. A simple flux-based approach is presented to model NP uptake. We find that the intracellular NP concentration grows linearly in time, and that the uptake is essentially irreversible, with the particles accumulating in lysosomes. A wide range of practical challenges, from labile dye release to NP aggregation and the need to account for cell division, are addressed to ensure that these studies yield meaningful kinetic information. FROM THE CLINICAL EDITOR: The authors present an uptake study comparing the outcomes for free molecular dye and NPs labeled with the same dye. A wide range of practical challenges are addressed including labile dye release, NP aggregation and the need to account for cell division with the goal that these studies yield meaningful kinetic information.

Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts.

Nanoparticles in a biological fluid (plasma, or otherwise) associate with a range of biopolymers, especially proteins, organized into the "protein corona" that is associated with the nanoparticle and continuously exchanging with the proteins in the environment. Methodologies to determine the corona and to understand its dependence on nanomaterial properties are likely to become important in bionanoscience. Here, we study the long-lived ("hard") protein corona formed from human plasma for a range of nanoparticles that differ in surface properties and size. Six different polystyrene nanoparticles were studied: three different surface chemistries (plain PS, carboxyl-modified, and amine-modified) and two sizes of each (50 and 100 nm), enabling us to perform systematic studies of the effect of surface properties and size on the detailed protein coronas. Proteins in the corona that are conserved and unique across the nanoparticle types were identified and classified according to the protein functional properties. Remarkably, both size and surface properties were found to play a very significant role in determining the nanoparticle coronas on the different particles of identical materials. We comment on the future need for scientific understanding, characterization, and possibly some additional emphasis on standards for the surfaces of nanoparticles.

Impact of dynamic sub-populations within grafted chains on the protein binding and colloidal stability of PEGylated nanoparticles.

Polyethylene glycol grafting has played a central role in preparing the surfaces of nano-probes for biological interaction, to extend blood circulation times and to modulate protein recognition and cellular uptake. However, the role of PEG graft dynamics and conformation in determining surface recognition processes is poorly understood primarily due to the absence of a microscopic picture of the surface presentation of the polymer. Here a detailed NMR analysis reveals three types of dynamic ethylene glycol units on PEG-grafted SiO2 nanoparticles (NPs) of the type commonly evaluated as long-circulating theranostic nano-probes; a narrow fraction with fast dynamics associated with the chain ends; a broadened fraction spectrally overlapped with the former arising from those parts of the chain experiencing some dynamic restriction; and a fraction too broad to be observed in the spectrum arising from units closer to the surface/graft which undergo slow motion on the NMR timescale. We demonstrate that ethylene glycol units transition between fractions as a function of temperature, core size, PEG chain length and surface coverage and demonstrate how this distribution affects colloidal stability and protein uptake. The implications of the findings for biological application of grafted nanoparticles are discussed in the context of accepted models for surface ligand conformation.

A Three-Dimensional Cell Culture Platform for Long Time-Scale Observations of Bio-Nano Interactions.

We know surprisingly little about the long-term outcomes for nanomaterials interacting with organisms. To date, most of what we know is derived from in vivo studies that limit the range of materials studied and the scope of advanced molecular biology tools applied. Long-term in vitro nanoparticle studies are hampered by a lack of suitable models, as standard cell culture techniques present several drawbacks, while technical limitations render current three-dimensional (3D) cellular spheroid models less suited. Now, by controlling the kinetic processes of cell assembly and division in a non-Newtonian culture medium, we engineer reproducible cell clusters of controlled size and phenotype, leading to a convenient and flexible long-term 3D culture that allows nanoparticle studies over many weeks in an in vitro setting. We present applications of this model for the assessment of intracellular polymeric and silica nanoparticle persistence and found that hydrocarbon-based polymeric nanoparticles undergo no apparent degradation over long time periods with no obvious biological impact, while amorphous silica nanoparticles degrade at different rates over several weeks, depending on their synthesis method.

Inhibition of amyloid beta protein fibrillation by polymeric nanoparticles.

Copolymeric NiPAM:BAM nanoparticles of varying hydrophobicity were found to retard fibrillation of the Alzheimer's disease-associated amyloid beta protein (Abeta). We found that these nanoparticles affect mainly the nucleation step of Abeta fibrillation. The elongation step is largely unaffected by the particles, and once the Abeta is nucleated, the fibrillation process occurs with the same rate as in the absence of nanoparticles. The extension of the lag phase for fibrillation of Abeta is strongly dependent on both the amount and surface character of the nanoparticles. Surface plasmon resonance studies show that Abeta binds to the nanoparticles and provide rate and equilibrium constants for the interaction. Numerical analysis of the kinetic data for fibrillation suggests that binding of monomeric Abeta and prefibrillar oligomers to the nanoparticles prevents fibrillation. Moreover, we find that fibrillation of Abeta initiated in the absence of nanoparticles can be reversed by addition of nanoparticles up to a particular time point before mature fibrils appear.

Detecting cryptic epitopes created by nanoparticles.

As potential applications of nanotechnology and nanoparticles increase, so too does the likelihood of human exposure to nanoparticles. Because of their small size, nanoparticles are easily taken up into cells (by receptor-mediated endocytosis), whereupon they have essentially free access to all cellular compartments. Similarly to macroscopic biomaterial surfaces (that is, implants), nanoparticles become coated with a layer of adsorbed proteins immediately upon contact with physiological solutions (unless special efforts are taken to prevent this). The process of adsorption often results in conformational changes of the adsorbed protein, which may be affected by the larger curvature of nanoparticles compared with implant surfaces. Protein adsorption may result in the exposure at the surface of amino acid residues that are normally buried in the core of the native protein, which are recognized by the cells as "cryptic epitopes." These cryptic epitopes may trigger inappropriate cellular signaling events (as opposed to being rejected by the cells as foreign bodies). However, identification of such surface-exposed epitopes is nontrivial, and the molecular nature of the adsorbed proteins should be investigated using biological and physical science methods in parallel with systems biology studies of the induced alterations in cell signaling.

Impact of cationic polystyrene nanoparticles (PS-NH2) on early embryo development of Mytilus galloprovincialis: Effects on shell formation.

The potential release of nanoparticles (NPs) into aquatic environments represents a growing concern for their possible impact on aquatic organisms. In this light, exposure studies during early life stages, which can be highly sensitive to environmental perturbations, would greatly help identifying potential adverse effects of NPs. Although in the marine bivalve Mytilus spp. the effects of different types of NPs have been widely investigated, little is known on the effects of NPs on the developing embryo. In M. galloprovincialis, emerging contaminants were shown to affect gene expression profiles during early embryo development (from trocophorae-24 hpf to D-veligers-48 hpf). In this work, the effects of amino-modified polystyrene NPs (PS-NH2) on mussel embryos were investigated. PS-NH2 affected the development of normal D-shaped larvae at 48 hpf (EC50 = 0.142 mg/L). Higher concentrations (5-20 mg/L) resulted in high embryotoxicity/developmental arrest. At concentrations ≅ EC50, PS-NH2 affected shell formation, as shown by optical and polarized light microscopy. In these conditions, transcription of 12 genes involved in different biological processes were evaluated. PS-NH2 induced dysregulation of transcription of genes involved in early shell formation (Chitin synthase, Carbonic anhydrase, Extrapallial Protein) at both 24 and 48 hpf. Decreased mRNA levels for ABC transporter p-glycoprotein-ABCB and Lysozyme were also observed at 48 hpf. SEM observations confirmed developmental toxicity at higher concentrations (5 mg/L). These data underline the sensitivity of Mytilus early embryos to PS-NH2 and support the hypothesis that calcifying larvae of marine species are particularly vulnerable to abiotic stressors, including exposure to selected types of NPs.

Amino-modified polystyrene nanoparticles affect signalling pathways of the sea urchin (Paracentrotus lividus) embryos.

Polystyrene nanoparticles have been shown to pose serious risk to marine organisms including sea urchin embryos based on their surface properties and consequently behaviour in natural sea water. The aim of this study is to investigate the toxicity pathways of amino polystyrene nanoparticles (PS-NH2, 50 nm) in Paracentrotus lividus embryos in terms of development and signalling at both protein and gene levels. Two sub-lethal concentrations of 3 and 4 µg/mL of PS-NH2 were used to expose sea urchin embryos in natural sea water (PS-NH2 as aggregates of 143 ± 5 nm). At 24 and 48 h post-fertilisation (hpf) embryonic development was monitored and variations in the levels of key proteins involved in stress response and development (Hsp70, Hsp60, MnSOD, Phospho-p38 Mapk) as well as the modulation of target genes (Pl-Hsp70, Pl-Hsp60, Pl-Cytochrome b, Pl-p38 Mapk, Pl-Caspase 8, Pl-Univin) were measured. At 48 hpf various striking teratogenic effects were observed such as the occurrence of cells/masses randomly distributed, severe skeletal defects and delayed development. At 24 hpf a significant up-regulation of Pl-Hsp70, Pl-p38 Mapk, Pl-Univin and Pl-Cas8 genes was found, while at 48 hpf only for Pl-Univin was observed. Protein profile showed different patterns as a significant increase of Hsp70 and Hsp60 only after 48 hpf compared to controls. Conversely, P-p38 Mapk protein significantly increased at 24 hpf and decreased at 48 hpf. Our findings highlight that PS-NH2 are able to disrupt sea urchin embryos development by modulating protein and gene profile providing new understandings into the signalling pathways involved.

Molecular basis of cell-biomaterial interaction: insights gained from transcriptomic and proteomic studies.

With the growing interest in clinical interventions that involve medical devices, the role for new biomaterials in modern medicine is currently expanding at a phenomenal rate. Failure of most implant materials stems from an inability to predict and control biological phenomena, such as protein adsorption and cell interaction, resulting in an inappropriate host response to the materials. Contemporary advances in biological investigation are starting to shift focus in the biomaterials field, in particular with the advent of high-throughput methodologies for gene and protein expression profiling. Here, we examine the role that emerging transcriptomic and proteomic technologies could play in relation to biomaterial development and usage. Moreover, a number of studies are highlighted which have utilized such approaches in order to try to create a deeper understanding of cell-biomaterial interactions and, hence, improve our ability to predict and control the biocompatibility of new materials.

Surface-induced changes in protein adsorption and implications for cellular phenotypic responses to surface interaction.

Understanding external factors that determine cellular phenotypic responses is of key interest in the field of biomaterials. Currently, material surface characteristics, protein adsorption and cellular phenotypic responses are all considered to be interrelated and ultimately determine the biocompatibility of materials. The exact nature of the relationship between these distinct, yet related, phenomena still remains to be elucidated. Through the use of a series of thermoresponsive N-isopropylacrylamide-based co-polymer films, we aimed to shed light on the relationship between surface hydrophobicity, protein adsorption and subsequent cellular response. Despite changes in co-polymer hydrophobicity mediated by altered ratios of constituent monomers, differential cellular response was only apparent in the presence of serum. Co-polymer films displayed alterations with respect to the amount of protein adsorbed on the surface, with individual serum proteins (albumin and fibronectin) displaying contrasting adsorption characteristics. Changes in protein adsorption corresponded to changes in cell adhesion, cytoskeletal organisation and cell morphology, as well as to changes in cell movement and intracellular signalling events. Examination of focal adhesion kinase (FAK), and extracellular signal-regulated kinase (ERK 1/2), important mediators of adhesion and growth factor-related signalling events, revealed a comparative reduction in phosphorylation of these signalling proteins in cells grown on co-polymers in comparison to those cultured on tissue culture polystyrene (TCP; used as a control surface). We also associated surface-mediated phenotypic alterations of cells grown on TCP and co-polymer films with particular changes in gene expression. These results indicate that cellular response to interaction with our series of co-polymer films is determined by the surface-adsorbed protein layer, which in turn is determined by the changing surface chemistry as the ratio of the co-monomers is altered.

Novel method to prepare morphologically rich polymeric surfaces for biomedical applications via phase separation and arrest of microgel particles.

We outline here a simple method to prepare polymeric surfaces of controlled surface topography on the micrometer scale, via assembly and arrest of microgel particles, for use in a range of biological applications to modify cell adhesion and spreading. In previous work by other groups, it has transpired that topography on the nanoscale is unlikely to be useful for this purpose, as roughness on this scale is often covered or coated by serum derived proteins during the early stages of cell adhesion and cells can easily bridge nanoscale roughness. Therefore, in our work, we have focused on roughness or topographic variations on the micrometer length scale. The basic idea is to modify the interactions between particles, thereby causing the microgel particles to phase separate into particle-dense and particle-dilute domains and to arrest these domains on the surface. The result is the creation of surfaces with controlled topography. By changing the particle size, it is possible to alter the size of the pores formed and their distribution in the film. Preliminary results show that the system can readily be arrested into a homologous series of such structures (formed from microgel particles of the same size and same chemical structure) with biological implications. At the extremes of this series, large phenotypic differences are observed between cells, ranging (at one end) from localization of the cells in the pores to (at the other end) cells that avoid such localization, and remain extended, growing along the ridges between the pores. This constitutes a sort of cell localization transition on a surface with identical chemical components, where only the morphology has been adjusted.

Biological in situ characterization of polymeric microbubble contrast agents.

Polymeric microbubbles (MBs) are gas filled particles composed of a thin stabilized polymer shell that have been recently developed as valid contrast agents for the combined use of ultrasonography (US), magnetic resonance imaging (MRI) and single photon emission computer tomography (SPECT) imaging. Due to their buoyancy, the commonly available approaches to study their behaviour in complex media are not easily applicable and their use in modern medicine requires such behaviour to be fully elucidated. Here we have used for the first time flow cytometry as a new high throughput approach that allows characterisation of the MB dispersion, prior to and after exposure in different biological media and we have additionally developed a method that allows characterisation of the strongly bound proteins adsorbed on the MBs, to fully predict their biological behaviour in biological milieu.