Anna Salvati

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.

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.

Protein-nanoparticle interactions: What does the cell see?

Fluorescence correlation spectroscopy is used as a quantitative method to understand the binding and exchange behaviour of proteins on the surfaces of nanoparticles.

Role of cell cycle on the cellular uptake and dilution of nanoparticles in a cell population

Nanoparticles are considered a primary vehicle for targeted therapies because they can pass biological barriers and enter and distribute within cells by energy-dependent pathways. So far, most studies have shown that nanoparticle properties, such as size and surface, can influence how cells internalize nanoparticles. Here, we show that uptake of nanoparticles by cells is also influenced by their cell cycle phase. Although cells in different phases of the cell cycle were found to internalize nanoparticles at similar rates, after 24 h the concentration of nanoparticles in the cells could be ranked according to the different phases: G2/M > S > G0/G1. Nanoparticles that are internalized by cells are not exported from cells but are split between daughter cells when the parent cell divides. Our results suggest that future studies on nanoparticle uptake should consider the cell cycle, because, in a cell population, the dose of internalized nanoparticles in each cell varies as the cell advances through the cell cycle.

Nanoparticle Adhesion to the Cell Membrane and Its Effect on Nanoparticle Uptake Efficiency

The interactions between nanosized particles and living systems are commonly mediated by what adsorbs to the nanoparticle in the biological environment, its biomolecular corona, rather than the pristine surface. Here, we characterize the adhesion toward the cell membrane of nanoparticles of different material and size and study how this is modulated by the presence or absence of a corona on the nanoparticle surface. The results are corroborated with adsorption to simple model supported lipid bilayers using a quartz crystal microbalance. We conclude that the adsorption of proteins on the nanoparticle surface strongly reduces nanoparticle adhesion in comparison to what is observed for the bare material. Nanoparticle uptake is described as a two-step process, where the nanoparticles initially adhere to the cell membrane and subsequently are internalized by the cells via energy-dependent pathways. The lowered adhesion in the presence of proteins thereby causes a concomitant decrease in nanoparticle uptake efficiency. The presence of a biomolecular corona may confer specific interactions between the nanoparticle-corona complex and the cell surface including triggering of regulated cell uptake. An important effect of the corona is, however, a reduction in the purely unspecific interactions between the bare material and the cell membrane, which in itself disregarding specific interactions, causes a decrease in cellular uptake. We suggest that future nanoparticle-cell studies include, together with characterization of size, charge, and dispersion stability, an evaluation of the adhesion properties of the material to relevant membranes.

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.

Effects of Transport Inhibitors on the Cellular Uptake of Carboxylated Polystyrene Nanoparticles in Different Cell Lines

Nanotechnology is expected to play a vital role in the rapidly developing field of nanomedicine, creating innovative solutions and therapies for currently untreatable diseases, and providing new tools for various biomedical applications, such as drug delivery and gene therapy. In order to optimize the efficacy of nanoparticle (NP) delivery to cells, it is necessary to understand the mechanisms by which NPs are internalized by cells, as this will likely determine their ultimate sub-cellular fate and localisation. Here we have used pharmacological inhibitors of some of the major endocytic pathways to investigate nanoparticle uptake mechanisms in a range of representative human cell lines, including HeLa (cervical cancer), A549 (lung carcinoma) and 1321N1 (brain astrocytoma). Chlorpromazine and genistein were used to inhibit clathrin and caveolin mediated endocytosis, respectively. Cytochalasin A and nocodazole were used to inhibit, respectively, the polymerisation of actin and microtubule cytoskeleton. Uptake experiments were performed systematically across the different cell lines, using carboxylated polystyrene NPs of 40 nm and 200 nm diameters, as model NPs of sizes comparable to typical endocytic cargoes. The results clearly indicated that, in all cases and cell types, NPs entered cells via active energy dependent processes. NP uptake in HeLa and 1321N1 cells was strongly affected by actin depolymerisation, while A549 cells showed a stronger inhibition of NP uptake (in comparison to the other cell types) after microtubule disruption and treatment with genistein. A strong reduction of NP uptake was observed after chlorpromazine treatment only in the case of 1321N1 cells. These outcomes suggested that the same NP might exploit different uptake mechanisms to enter different cell types.

Reproducible Comet Assay of Amorphous Silica Nanoparticles Detects No Genotoxicity

Genotoxicity of commercial colloidal and laboratory-synthesized silica nanoparticles was tested using the single cell gel electrophoresis or Comet assay. By using a carefully developed protocol and careful characterization of the nanoparticle dispersions, Comet assays were performed on 3T3-L1 fibroblasts with 3, 6, and 24 h incubations and 4 or 40 microg/ml of silica nanoparticles. No significant genotoxicity was observed for the nanoparticles tested under the conditions described, and results were independently validated in two separate laboratories, showing that in vitro toxicity testing can be quantitatively reproducible.

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

UNLABELLED 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.

Quantitative Assessment of the Comparative Nanoparticle-Uptake Efficiency of a Range of Cell Lines

The mechanism(s) of nanoparticle-cell interactions are still not understood. At present there is little knowledge of the relevant length- and timescales for nanoparticle intracellular entry and localization within cells, or the cell-specificity of nanoparticle uptake and localisation. Here, the effect of particle size on the in-vitro intracellular uptake of model fluorescent carboxyl-modified polystyrene nanoparticles is investigated in various cell lines. A range of micro- and nanoparticles of defined sizes (40 nm to 2 μm) are incubated with a series of cell types, including HeLa and A549 epithelial cells, 1321N1 astrocytes, HCMEC D3 endothelial cells, and murine RAW 264.7 macrophages. Techniques such as confocal microscopy and flow cytometry are used to study particle uptake and subcellular localisation, making significant efforts to ensure reproducibility in a semiquantitative approach. The results indicate that internalization of (nano)particles is highly size-dependent for all cell lines studied, and the kinetics of uptake for the same type of nanoparticle varies in the different cell types. Interestingly, even cells not specialized for phagocytosis are able to internalize the larger nanoparticles. Intracellular uptake of all sizes of particles is observed to be highest in RAW 264.7 cells (a specialized phagocytic cell line) and the lowest in the HeLa cells. These results suggest that (nano)particle uptake might not follow commonly defined size limits for uptake processes, and highlight the variability of uptake kinetics for the same material in different cell types. These conclusions have important implications for the assessment of the safety of nanomaterials and for the potential biomedical applications of nanoparticles.