Nanoparticles (NPs) are often functionalized with reactive groups such as amines and thiols for the subsequent conjugation of further molecules, e.g., stabilizing polymers, drugs, and proteins for targeting cells or specific diseases. In addition to the quantitative estimation of the reactive conjugation sites, their molecular positioning and nanoscale arrangement on single nanoparticles become more and more important for the tailored engineering and design of functional nanomaterials. Here, we use maleimide or sulfo-succinimidyl ester-modified 1.4 nm gold nanoclusters (AuNCs) to specifically label reactive thiol and amine groups with sub-2-nm precision on metal oxide and polymeric nanostructures. We confirm the binding of AuNCs by measuring and modeling sedimentation properties using analytical centrifugation, imaging their surface distribution and surface distances by transmission electron microscopy (TEM), and comparing the results to ensemble measurements of numbers of reactive surface groups obtained by common photometric assays. We map thiol and amine groups introduced on silica NPs (SiNPs), titania stars (Ti), silica inverse opals (SiOps), and polystyrene NPs (PS NPs). We show that the method is suitable for mapping local, clustered inhomogeneities of the reactive sites on single SiNPs introduced by masking certain areas during surface functionalization. Mapping precise positions of reactive surface groups is essential to the design and tailored ligation of multifunctional nanomaterials.

The transport and the delivery of drugs through nanocarriers is a great challenge of pharmacology. Since the production of liposomes to reduce the toxicity of doxorubicin in patients, a plethora of nanomaterials have been produced and characterized. Although it is widely known that elementary properties of nanomaterials influence their in vivo kinetics, such interaction is often poorly investigated in many preclinical studies. The present study aims to evaluate the actual effect of size and shape on the biodistribution of a set of gold nanoparticles (GNPs) after intravenous administration in mice. To this goal, quantitative data achieved by inductively coupled plasma mass spectrometry and observational results emerging from histochemistry (autometallography and enhanced dark-field hyperspectral microscopy) were combined. Since the immune system plays a role in bionano-interaction we used healthy immune-competent mice. To keep the immune surveillance on the physiological levels we synthesized endotoxin-free GNPs to be tested in specific pathogen-free animals. Our study mainly reveals that (a) the size and the shape greatly influence the kinetics of accumulation and excretion of GNPs in filter organs; (b) spherical and star-like GNPs showed the same percentage of accumulation, but a different localization in liver; (c) only star-like GNPs are able to accumulate in lung; (d) changes in the geometry did not improve the passage of the blood brain barrier. Overall, this study can be considered as a reliable starting point to drive the synthesis and the functionalization of potential candidates for theranostic purposes in many fields of research.Keywords: accumulation; biodistribution; biological barriers; gold nanoparticles; particle shape; particle size;

Biomolecules adsorbed on nanoparticles are known to confer a biological identity to nanoparticles, mediating the interactions with cells and biological barriers. However, how these molecules are presented on the particle surface in biological milieu remains unclear. The central aim of this study is to identify key protein recognition motifs and link them to specific cell-receptor interactions. Here, we employed an immuno-mapping technique to quantify epitope presentations of two major proteins in the serum corona, low-density lipoprotein and immunoglobulin G. Combining with a purpose-built receptor expression system, we show that both proteins present functional motifs to allow simultaneous recognition by low-density lipoprotein receptor and Fc-gamma receptor I of the corona. Our results suggest that the “labeling” of nanoparticles by biomolecular adsorption processes allows for multiple pathways in biological processes in which they may be “mistaken” for endogenous objects, such as lipoproteins, and exogenous ones, such as viral infections.Keywords: biomolecular corona; epitope; internalization; low-density lipoprotein; nanoparticle; receptor;

The shape and size of nanoparticles are important parameters affecting their biodistribution, bioactivity, and toxicity. The high-throughput characterisation of the nanoparticle shape in dispersion is a fundamental prerequisite for realistic in vitro and in vivo evaluation, however, with routinely available bench-top optical characterisation techniques, it remains a challenging task. Herein, we demonstrate the efficacy of a single particle extinction and scattering (SPES) technique for the in situ detection of the shape of nanoparticles in dispersion, applied to a small library of anisotropic gold particles, with a potential development for in-line detection. The use of SPES paves the way to the routine quantitative analysis of nanoparticles dispersed in biologically relevant fluids, which is of importance for the nanosafety assessment and any in vitro and in vivo administration of nanomaterials.

Nanoparticles have great potential as drug delivery vehicles or as imaging agents for treatment and diagnosis of various diseases. It is therefore crucial to understand how nanoparticles are taken up by cells, both phagocytic and non-phagocytic. Small interference RNA has previously been used to isolate the effect of particular receptors in nanoparticle uptake by silencing their expression. Here we show that, when it comes to receptors with overlapping function, interpretation of such data has to be done with caution. We followed the uptake of silica nanoparticles by scavenger receptors in A549 lung epithelial cells. While we successfully knocked-down gene expression of several different receptors within the scavenger receptor family (SR-A1, MARCO, SR-BI, LOX-1 and LDLR) this caused reciprocal up and down regulation of the other scavenger receptors. Subsequent nanoparticle uptake experiments in silenced cells exhibit a complex behaviour, which could easily be misinterpreted if reciprocal regulation is not considered. Preliminary identification of the actual scavenger receptors involved can be found by disentangling the effects mathematically. Finally, we show that the effects are still present under more realistic biological conditions, namely at higher serum concentrations.

Comprehensive characterization of nanomaterials for medical applications is a challenging and complex task due to the multitude of parameters which need to be taken into consideration in a broad range of conditions. Routine methods such as dynamic light scattering or nanoparticle tracking analysis provide some insight into the physicochemical properties of particle dispersions. For nanomedicine applications the information they supply can be of limited use. For this reason, there is a need for new methodologies and instruments that can provide additional data on nanoparticle properties such as their interactions with surfaces. Nanophotonic force microscopy has been shown as a viable method for measuring the force between surfaces and individual particles in the nano-size range. Here we outline a further application of this technique to measure the size of single particles and based on these measurement build the distribution of a sample. We demonstrate its efficacy by comparing the size distribution obtained with nanophotonic force microscopy to established instruments, such as dynamic light scattering and differential centrifugal sedimentation. Our results were in good agreement to those observed with all other instruments. Furthermore, we demonstrate that the methodology developed in this work can be used to study complex particle mixtures and the surface alteration of materials. For all cases studied, we were able to obtain both the size and the interaction potential of the particles with a surface in a single measurement.

In biological fluids, proteins and other biomolecules bind to the surface of nanoparticles to form a coating known as the protein corona which in turn becomes primary determinant of the nanoparticles’ fate and behaviour. Here we develop a QCM-based platform and methodology to obtain data from real-time interactions of nanoparticles with selected human plasma proteins. Polystyrene particles coated with transferrin are immobilized on QCM sensor chips and by means of a ‘sandwich’ format binding assay, specific epitopes on the particles can be quantified as measured by the increase of the sensor's resonant frequency. Cell binding experiments where adherent cells are directly grown on the sensor surface are also performed. Interaction of nanoparticles injected over the cell surface is observed only in the case of particle-transferrin complexes demonstrating that it is the nanoparticle-corona complex, rather than the native nanoparticle, “what the cell sees”, with the corona being the interface between the nanoparticle and the cellular system. Our data highlight the potential of the proposed QCM-based platform and methodology for characterization of the bio-nano-interface and tracking the interaction of nanoparticles with biological cells in the presence of a realistic milieu.

The range of possible nanostructures is so large and continuously growing, that collating and unifying the knowledge connected to them, including their biological activity, is a major challenge. Here we discuss a concept that is based on the connection of microscopic features of the nanomaterials to their biological impacts. We also consider what would be necessary to identify the features that control their biological interactions, and make them resemble each other in a biological context.

Characterizing the orientation of covalently conjugated proteins on nanoparticles, produced for in vitro and in vivo targeting, though an important feature of such a system, has proved challenging. Although extensive physicochemical characterization of targeting nanoparticles can be addressed in detail, relevant biological characterization of the nanointerface is crucial in order to select suitable nanomaterials for further in vitro or in vivo experiments. In this work, we adopt a methodology using antibody fragments (Fab) conjugated to gold nanoparticles (immunogold) to map the available epitopes on a transferrin grafted silica particle (SiO2–PEG8–Tf) as a proxy methodology to predict nanoparticle biological function, and therefore cellular receptor engagement. Data from the adopted method suggest that, on average, only ∼3.5% of proteins grafted on the SiO2–PEG8–Tf nanoparticle surface have a favorable orientation for recognition by the cellular receptor.

Observing structural integrity of nanoparticles is essential in bionanotechnology but not always straightforward to measure in situ and in real-time. Fluorescent labels used for tracking intrinsically nonfluorescent nanomaterials generally do not allow simultaneous observation of integrity. Consequently, structural changes like degradation and disassembly cannot easily be followed in situ using fluorescence signals. We show that thioflavin T (ThT), a fluorophore and molecular rotor known to tag specific fibril structures in amyloids, can “label” the structural integrity of widely used and intrinsically nonfluorescent, silica nanoparticles (SiNPs). Entrapment of ThT in SiNPs controls the fluorohphore’s relaxation pathway and leads to a red-shifted fluorescence spectrum providing real time information on SiNP integrity. The dynamic change of ThT fluorescence during degradation of doped SiNPs is found much higher than that of common labels fluorescein and rhodamine. Degradation kinetics of core–shell structures recorded by ThT fluorescence and light scattering prove the capability to clearly distinguish structural features during SiNPs degradation and allow obtaining degradation kinetics in vitro, in biological media, in serum, and in cells. The effect is transferable to different types of materials, here shown for ThT incorporated SiNPs with tightly tailorable sizes (9–100 nm), poly(lactic-co-glycolic acid) (PLGA) nanoparticles, poly(9-vinylcarbazole) (PVK) nanoparticles, and iron-doped-SiNPs (FeSiNPs). We thus suggest molecular rotors such as ThT as additional labels to effectively and easily sense nanoparticle structural status in situ and to enhance understanding and development of programmed nanoparticle disassembly in bionanotechnology.Keywords: biodegradation; bionano interactions; core−shell nanoparticles; differential centrifugal sedimentation; fluorescence labeling; molecular rotors; silica

It has been well established that the early stages of nanoparticle–cell interactions are governed, at least in part, by the layer of proteins and other biomolecules adsorbed and slowly exchanged with the surrounding biological media (biomolecular corona). Subsequent to membrane interactions, nanoparticles are typically internalized into the cell and trafficked along defined pathways such as, in many cases, the endolysosomal pathway. Indeed, if the original corona is partially retained on the nanoparticle surface, the biomolecules in this layer may play an important role in determining subsequent cellular processing. In this work, using a combination of organelle separation and fluorescence labeling of the initial extracellular corona, we clarify its intracellular evolution as nanoparticles travel within the cell. We show that specific proteins present in the original protein corona are retained on the nanoparticles until they accumulate in lysosomes, and, once there, they are degraded. We also report on how different bare surfaces (amino and carboxyl modified) affect the details of this evolution. One overarching discovery is that the same serum proteins can exhibit different intracellular processing when carried inside cells by nanoparticles, as components of their corona, compared to what is observed when they are transported freely from the extracellular medium.Keywords: biomolecular corona; degradation; lysosomes; nanoparticle cell interactions

Nanoparticles in physiological environments are known to selectively adsorb proteins and other biomolecules forming a tightly bound biomolecular ‘corona’ on their surface. Where the exchange times of the proteins are sufficiently long, it is believed that the protein corona constitutes the particle identity in biological milieu. Here we show that proteins in the corona retain their functional characteristics and can specifically bind to cognate proteins on arrays of thousands of immobilised human proteins. The biological identity of the nanomaterial is seen to be specific to the blood plasma concentration in which they are exposed. We show that the resulting in situ nanoparticle interactome is dependent on the protein concentration in plasma, with the emergence of a small number of dominant protein–protein interactions. These interactions are those driven by proteins that are adsorbed onto the particle surface and whose binding epitopes are subsequently expressed or presented suitably on the particle surface. We suggest that, since specific tailored protein arrays for target systems and organs can be designed, their use may be an important element in an overall study of the biomolecular corona.

Subcellular location of nanoparticles has been widely investigated with fluorescence microscopy, via fluorescently labeled antibodies to visualise target antigens in cells. However, fluorescence microscopy, such as confocal or live cell imaging, has generally limited 3D spatial resolution. Conventional electron microscopy can be useful in bridging resolution gap, but still not ideal in resolving subcellular organelle identities. Using the pre-embedding immunogold electron microscopic imaging, we performed accurate examination of the intracellular trafficking and gathered further evidence of transport mechanisms of silica nanoparticles across a human in vitro blood–brain barrier model. Our approach can effectively immunolocalise a variety of intracellular compartments and provide new insights into the uptake and subcellular transport of nanoparticles.

We describe the control of size and homogeneity in silica nanoparticle dispersions, prepared by a two-phase arginine catalysed aqueous method, through varying the upper organic solvent phase. The final particle dispersion characteristics can be controlled by varying features including solvent type and interfacial area, related to the rate of monomer transfer at the TEOS/water interface.

Nanomaterials challenge paradigms of in vitro testing because unlike molecular species, biomolecules in the dispersion medium modulate their interactions with cells. Exposing cells to nanoparticles known to cause cell death, we observed cytotoxicity suppression by increasing the amount of serum in the dispersion medium towards in vivo-relevant conditions.

For effective localization of functionalized nanoparticles at diseased tissues such as solid tumours or metastases through biorecognition, appropriate targeting vectors directed against selected tumour biomarkers are a key prerequisite. The diversity of such vector molecules ranges from proteins, including antibodies and fragments thereof, through aptamers and glycans to short peptides and small molecules. Here, we analyse the specific nanoparticle targeting capabilities of two previously suggested peptides (D4 and GE11) and a small camelid single-domain antibody (sdAb), representing potential recognition agents for the epidermal growth factor receptor (EGFR). We investigate specificity by way of receptor RNA silencing techniques and look at increasing complexity in vitro by introducing increasing concentrations of human or bovine serum. Peptides D4 and GE11 proved problematic to employ and conjugation resulted in non-receptor specific uptake into cells. Our results show that sdAb-functionalized particles can effectively target the EGFR, even in more complex bovine and human serum conditions where targeting specificity is largely conserved for increasing serum concentration. In human serum however, an inhibition of overall nanoparticle uptake is observed with increasing protein concentration. For highly affine targeting ligands such as sdAbs, targeting a receptor such as EGFR with low serum competitor abundance, receptor recognition function can still be partially realised in complex conditions. Here, we stress the value of evaluating the targeting efficiency of nanoparticle constructs in realistic biological milieu, prior to more extensive in vivo studies.

Nanoparticles (NP), when exposed to biological fluids, are coated by specific proteins that form the so-called protein corona. While some adsorbing proteins exchange with the surroundings on a short time scale, described as a “dynamic” corona, others with higher affinity and long-lived interaction with the NP surface form a “hard” corona (HC), which is believed to mediate NP interaction with cellular machineries. In-depth NP protein corona characterization is therefore a necessary step in understanding the relationship between surface layer structure and biological outcomes. In the present work, we evaluate the protein composition and stability over time and we systematically challenge the formed complexes with surfactants. Each challenge is characterized through different physicochemical measurements (dynamic light scattering, ζ-potential, and differential centrifugal sedimentation) alongside proteomic evaluation in titration type experiments (surfactant titration). 100 nm silicon oxide (Si) and 100 nm carboxylated polystyrene (PS-COOH) NPs cloaked by human plasma HC were titrated with 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS, zwitterionic), Triton X-100 (nonionic), sodium dodecyl sulfate (SDS, anionic), and dodecyltrimethylammonium bromide (DTAB, cationic) surfactants. Composition and density of HC together with size and ζ-potential of NP–HC complexes were tracked at each step after surfactant titration. Results on Si NP–HC complexes showed that SDS removes most of the HC, while DTAB induces NP agglomeration. Analogous results were obtained for PS NP–HC complexes. Interestingly, CHAPS and Triton X-100, thanks to similar surface binding preferences, enable selective extraction of apolipoprotein AI (ApoAI) from Si NP hard coronas, leaving unaltered the dispersion physicochemical properties. These findings indicate that surfactant titration can enable the study of NP–HC stability through surfactant variation and also selective separation of certain proteins from the HC. This approach thus has an immediate analytical value as well as potential applications in HC engineering.

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.

Understanding nanoparticle interactions with the central nervous system, in particular the blood–brain barrier, is key to advances in therapeutics, as well as assessing the safety of nanoparticles. Challenges in achieving insights have been significant, even for relatively simple models. Here we use a combination of live cell imaging and computational analysis to directly study nanoparticle translocation across a human in vitro blood–brain barrier model. This approach allows us to identify and avoid problems in more conventional inferential in vitro measurements by identifying the catalogue of events of barrier internalization and translocation as they occur. Potentially this approach opens up the window of applicability of in vitro models, thereby enabling in depth mechanistic studies in the future. Model nanoparticles are used to illustrate the method. For those, we find that translocation, though rare, appears to take place. On the other hand, barrier uptake is efficient, and since barrier export is small, there is significant accumulation within the barrier.Keywords: blood−brain barrier; confocal microscopy; live-cell imaging; lysosome; nanoparticle; TIRFM; transcytosis

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.

The blood–brain barrier (BBB) is a selective barrier, which controls and limits access to the central nervous system (CNS). The selectivity of the BBB relies on specialized characteristics of the endothelial cells that line the microvasculature, including the expression of intercellular tight junctions, which limit paracellular permeability. Several reports suggest that nanoparticles have a unique capacity to cross the BBB. However, direct evidence of nanoparticle transcytosis is difficult to obtain, and we found that typical transport studies present several limitations when applied to nanoparticles. In order to investigate the capacity of nanoparticles to access and transport across the BBB, several different nanomaterials, including silica, titania and albumin- or transferrin-conjugated gold nanoparticles of different sizes, were exposed to a human in vitro BBB model of endothelial hCMEC/D3 cells. Extensive transmission electron microscopy imaging was applied in order to describe nanoparticle endocytosis and typical intracellular localisation, as well as to look for evidence of eventual transcytosis. Our results show that all of the nanoparticles were internalised, to different extents, by the BBB model and accumulated along the endo–lysosomal pathway. Rare events suggestive of nanoparticle transcytosis were also observed for several of the tested materials.

Positively charged polymers and nanoparticles (NPs) can be toxic to cells in various systems. Using human astrocytoma cells, we have previously shown that 50 nm amine-modified polystyrene NPs damage mitochondria and induce cell death by apoptosis. Here we provide comprehensive details of the cellular events occurring after exposure to the NPs in a time-resolved manner. We demonstrate that the accumulation of NPs in lysosomes plays a central role in the observed cell death, leading to swelling of the lysosomes and release of cathepsins into the cytosol, which ultimately propagates the damage to the mitochondria with subsequent activation of apoptosis. This is accompanied and sustained by other events, such as increasing ROS levels and autophagy. Using various inhibitors, we also show the interplay between apoptosis and autophagy as a response to NP accumulation in lysosomes.

The interaction of nanoscaled materials with biological systems is currently the focus of a fast-growing area of investigation. Though many nanoparticles interact with cells without acute toxic responses, amino-modified polystyrene nanoparticles are known to induce cell death. We have found that by lowering their dose, cell death remains low for several days while, interestingly, cell cycle progression is arrested. In this scenario, nanoparticle uptake, which we have recently shown to be affected by cell cycle progression, develops differently over time due to the absence of cell division. This suggests that the same nanoparticles can trigger different pathways depending on exposure conditions and the dose accumulated.Keywords: cationic nanoparticles; cell cycle arrest; cell division; lysosome; nanoparticle; nanoparticle uptake; nanotoxicity

Nanoparticles have unique capacities of interacting with the cellular machinery and entering cells. To be able to exploit this potential, it is essential to understand what controls the interactions at the interface between nanoparticles and cells: it is now established that nanoparticles in biological media are covered by proteins and other biomolecules forming a “corona” on the nanoparticle surface, which confers a new identity to the nanoparticles. By labelling the proteins of the serum, using positively-charged polystyrene, we now show that this adsorbed layer is strong enough to be retained on the nanoparticles as they enter cells and is trafficked to the lysosomes on the nanoparticles. There, the corona is degraded and this is followed by lysosomal damage, leading to cytosolic release of lysosomal content, and ultimately apoptosis. Thus the corona protects the cells from the damage induced by the bare nanoparticle surface until enzymatically cleared in the lysosomes.From the Clinical EditorThis study investigates the effects of protein corona that normally forms on the surface of nanoparticles during in vivo use, describing the steps of intracellular processing of such particles, to enhance our understanding of how these particles interact with the cellular machinery.Nanoparticles in biological fluids are covered by a biomolecular corona: using positively charged nanoparticles and labelled serum, we show that nanoparticles retain, at least in part, their corona as they enter cells and are trafficked to the lysosomes. Thanks to this layer, the cationic damage usually observed on the cell membrane for bare particles is absent and appears only later, once the corona is degraded in the lysosomes, leading to lysosomal membrane permeabilization and apoptosis. This suggests a general paradigm in which the corona protects the cells from the early interactions with the bare material.Figure optionsDownload full-size imageDownload high-quality image (106 K)Download as PowerPoint slide

Here we show that commonly employed Stöber type fluorescently labelled silica nanoprobes degrade by hydrolytic dissolution, accelerated under biological media conditions as compared to water alone. We have thus developed a method to greatly improve their stability under such conditions.

Dendrimers are polymers with a strong role in nanomedicine. In the current work we have developed a platform for mapping out the biomolecule corona for polyamidoamine (PAMAM) dendrimers. Complement proteins including C3 and C4b were found for the high generation dendrimers, suggesting high affinities to the dendrimers and most importantly complement activation.

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.Keywords: adhesion; nanoparticle; nanoparticle uptake; protein corona; serum free; silica

Despite the increased application of nanomaterials in diagnostics and therapeutics, methods to study the interactions of nanoparticles with subcellular structures in living cells remain relatively undeveloped. Here we describe a robust and quantitative method that allows for the precise tracking of all cell-associated nanoparticles as they pass through endocytic compartments in a living cell. Using rapid multicolor 3D live cell confocal fluorescence microscopy, combined with transient overexpression of small GTPases marking various endocytic membranes, our studies reveal the kinetics of nanoparticle trafficking through early endosomes to late endosomes and lysosomes. We show that, following internalization, 40 nm polystyrene nanoparticles first pass through an early endosome intermediate decorated with Rab5, but that these nanoparticles rapidly transfer to late endosomes and ultimately lysosomes labeled with Rab9 and Rab7, respectively. Larger nanoparticles of 100 nm diameter also reach acidic Rab9- and Rab7-positive compartments although at a slower rate compared to the smaller 40 nm nanoparticles. Our work also reveals that relatively few nanoparticles are able to access endocytic recycling pathways, as judged by lack of significant colocalization with Rab11. Finally, we demonstrate that this quantitative approach is sufficiently sensitive to be able to detect rare events in nanoparticle trafficking, specifically the presence of nanoparticles in Rab1A-labeled structures, thereby revealing the wide range of intracellular interactions between nanoparticles and the intracellular environment.Keywords: colocalization; intracellular trafficking; live cell imaging; membrane traffic; nanoparticles; polystyrene particles; Rab GTPases

Protein adsorption to nanoparticles (NPs) is a key prerequisite to understand NP–cell interactions. While the layer thickness of the protein corona has been well characterized in many cases, the absolute number of bound proteins and their exchange dynamics in body fluids is difficult to assess. Here we measure the number of molecules adsorbed to sulfonate (PSOSO3H) and carboxyl-(PSCOOH) polystyrene NPs using fluorescence correlation spectroscopy. We find that the fraction of molecules bound to NPs falls onto a single, universal adsorption curve, if plotted as a function of molar protein-to-NP ratio. The adsorption curve shows the build-up of a strongly bound monolayer up to the point of monolayer saturation (at a geometrically defined protein-to-NP ratio), beyond which a secondary, weakly bound layer is formed. While the first layer is irreversibly bound (hard corona), the secondary layer (soft corona) exhibits dynamic exchange, if competing unlabeled is added. In the presence of plasma proteins, the hard corona is stable, while the soft corona is almost completely removed. The existence of two distinct time scales in the protein off-kinetics, for both NP types studied here, indicates the possibility of an exposure memory effect in the NP corona.Keywords: bionano interface; fluorescence correlation spectroscopy (FCS); nanoparticles−proteins interaction; protein corona; Transferrin

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.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.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.

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.

Here we show that commonly employed Stöber type fluorescently labelled silica nanoprobes degrade by hydrolytic dissolution, accelerated under biological media conditions as compared to water alone. We have thus developed a method to greatly improve their stability under such conditions.