Marco P. Monopoli

Biomolecular coronas provide the biological identity of nanosized materials

The search for understanding the interactions of nanosized materials with living organisms is leading to the rapid development of key applications, including improved drug delivery by targeting nanoparticles, and resolution of the potential threat of nanotechnological devices to organisms and the environment. Unless they are specifically designed to avoid it, nanoparticles in contact with biological fluids are rapidly covered by a selected group of biomolecules to form a corona that interacts with biological systems. Here we review the basic concept of the nanoparticle corona and its structure and composition, and highlight how the properties of the corona may be linked to its biological impacts. We conclude with a critical assessment of the key problems that need to be resolved in the near future.

Protein−Nanoparticle Interactions: Opportunities and Challenges

Protein Nanoparticle Interactions: Opportunities and Challenges Morteza Mahmoudi,* Iseult Lynch, Mohammad Reza Ejtehadi, Marco P. Monopoli, Francesca Baldelli Bombelli, and Sophie Laurent National Cell Bank, Pasteur Institute of Iran, Tehran, Iran Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran School of Chemistry and Chemical Biology & Conway Institute for Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland Department of Physics, Sharif University of Technology, Tehran, Iran School of Pharmacy, UEA, Norwich Research Park, Norwich,U.K. Department of General, Organic, and Biomedical Chemistry, NMR and Molecular Imaging Laboratory, University of Mons, Avenue Maistriau 19, B-7000 Mons, Belgium

Effects of the presence or absence of a protein corona on silica nanoparticle uptake and impact on cells

Nanoparticles enter cells through active processes, thanks to their capability of interacting with the cellular machinery. The protein layer (corona) that forms on their surface once nanoparticles are in contact with biological fluids, such as the cell serum, mediates the interactions with cells in situ. As a consequence of this, here we show that the same nanomaterial can lead to very different biological outcomes, when exposed to cells in the presence or absence of a preformed corona. In particular, silica nanoparticles exposed to cells in the absence of serum have a stronger adhesion to the cell membrane and higher internalization efficiency, in comparison to what is observed in medium containing serum, when a preformed corona is present on their surface. The different exposure conditions not only affect the uptake levels but also result in differences in the intracellular nanoparticle location and impact on cells. Interestingly, we also show that after only one hour of exposure, a corona of very different nature forms on the nanoparticles exposed to cells in the absence of serum. Evidence suggests that these different outcomes can all be connected to the different adhesion and surface properties in the two conditions.

Serum heat inactivation affects protein corona composition and nanoparticle uptake.

Nanoparticles are of an appropriate size to interact with cells, and are likely to use a range of cellular machinery for internalisation and trafficking to various sub-cellular compartments. It is now understood that once in contact with biological fluids, the nanoparticle surface gets covered by a highly specific layer of proteins, forming the nanoparticle protein corona. This protein layer is stable for times longer than the typical time scale of nanoparticle import, and thus can impact on particle uptake and trafficking inside the cells. In this work, the effect of the corona composition on nanoparticle uptake has been investigated, by studying the impact of serum heat inactivation and complement depletion on the load of nanoparticles accumulated inside the cell. For the same material and nanoparticle size, cellular uptake was found to be significantly different when the nanoparticles were dispersed in medium where the serum was heat inactivated or not heat inactivated, even for non-specialized cells, suggesting that different sera can lead to different nanoparticle doses. The fact that uptake was correlated with the amount of protein bound into the nanoparticle corona suggests the need for commonly agreed dispersion protocols for in vitro nanoparticle-cell studies.

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.

The "sweet" side of the protein corona: effects of glycosylation on nanoparticle-cell interactions.

The significance of a protein corona on nanoparticles in modulating particle properties and their biological interactions has been widely acknowledged. The protein corona is derived from proteins in biological fluids, many of which are glycosylated. To date, the glycans on the proteins have been largely overlooked in studies of nanoparticle-cell interactions. In this study, we demonstrate that glycosylation of the protein corona plays an important role in maintaining the colloidal stability of nanoparticles and influences nanoparticle-cell interactions. The removal of glycans from the protein corona enhances cell membrane adhesion and cell uptake of nanoparticles in comparison with the fully glycosylated form, resulting in the generation of a pro-inflammatory milieu by macrophages. This study highlights that the post-translational modification of proteins can significantly impact nanoparticle-cell interactions by modulating the protein corona properties.

Transferrin Coated Nanoparticles: Study of the Bionano Interface in Human Plasma

It is now well established that the surface of nanoparticles (NPs) in a biological environment is immediately modified by the adsorption of biomolecules with the formation of a protein corona and it is also accepted that the protein corona, rather than the original nanoparticle surface, defines a new biological identity. Consequently, a methodology to effectively study the interaction between nanomaterials and the biological corona encountered within an organism is a key objective in nanoscience for understanding the impact of the nanoparticle-protein interactions on the biological response in vitro and in vivo. Here, we outline an integrated methodology to address the different aspects governing the formation and the function of the protein corona of polystyrene nanoparticles coated with Transferrin by different strategies. Protein-NP complexes are studied both in situ (in human plasma, full corona FC) and after washing (hard corona, HC) in terms of structural properties, composition and second-order interactions with protein microarrays. Human protein microarrays are used to effectively study NP-corona/proteins interactions addressing the growing demand to advance investigations of the extrinsic function of corona complexes. Our data highlight the importance of this methodology as an analysis to be used in advance of the application of engineered NPs in biological environments.

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.

Elution of Labile Fluorescent Dye from Nanoparticles during Biological Use

Cells act as extremely efficient filters for elution of unbound fluorescent tags or impurities associated with nanoparticles, including those that cannot be removed by extensive cleaning. This has consequences for quantification of nanoparticle uptake and sub-cellular localization in vitro and in vivo as a result of the presence of significant amount of labile dye even following extensive cleaning by dialysis. Polyacrylamide gel electrophoresis (PAGE) can be used to monitor the elution of unbound fluorescent probes from nanoparticles, either commercially available or synthesized in-house, and to ensure their complete purification for biological studies, including cellular uptake and sub-cellular localisation. Very different fluorescence distribution within cells is observed after short dialysis times versus following extensive dialysis against a solvent in which the free dye is more soluble, due to the contribution from free dye. In the absence of an understanding of the presence of residual free dye in (most) labeled nanoparticle solutions, the total fluorescence intensity in cells following exposure to nanoparticle solutions could be mis-ascribed to the presence of nanoparticles through the cell, rather than correctly assigned to either a combination of free-dye and nanoparticle-bound dye, or even entirely to free dye depending on the exposure conditions (i.e. aggregation of the particles etc). Where all of the dye is nanoparticle-bound, the particles are highly localized in sub-cellular organelles, likely lysosomes, whereas in a system containing significant amounts of free dye, the fluorescence is distributed through the cell due to the free diffusion of the molecule dye across all cellular barriers and into the cytoplasm.

Titanium dioxide nanoparticles modulate the toxicological response to cadmium in the gills of Mytilus galloprovincialis

We investigated the influence of titanium dioxide nanoparticles (nano-TiO2) on the response to cadmium in the gills of the marine mussel Mytilus galloprovincialis in terms of accumulation and toxicity. Mussels were in vivo exposed to nano-TiO2, CdCl2, alone and in combination. Several cellular biomarkers were investigated in gills: ABC transport proteins and metallothioneins at gene/protein (abcb1, abcc-like and mt-20) and functional level, GST activity, NO production and DNA damage (Comet assay). Accumulation of total Cd and titanium in gills as in whole soft tissue was also investigated. Significant responses to Cd exposure were observed in mussel gills as up-regulation of abcb1 and mt-20 gene transcription, increases in total MT content, P-gp efflux and GST activity, DNA damage and NO production. Nano-TiO2 alone increased P-gp efflux activity and NO production. When combined with Cd, nano-TiO2 reduced the metal-induced effects by significantly lowering abcb1 gene transcription, GST activity, and DNA damage, whereas, additive effects were observed on NO production. A lower concentration of Cd was observed in the gills upon co-exposure, whereas, Ti levels were unaffected. A competitive effect in uptake/accumulation of nano-TiO2 and Cd seems to occur in gills. A confirmation is given by the observed absence of adsorption of Cd onto nano-TiO2 in sea water media.