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Layered silicate nanoparticles (LSN) are widely used in industrial applications and consumer products. They also have potential benefits in biomedical applications such as implantable devices and for drug delivery. To study how nanomaterials interact with cells and tissues, techniques to track and quantify their movement through different biological compartments are essential. While radiolabels can be very sensitive, particularly for in vivo studies, fluorescent labeling has been preferred in recent years because of the array of methods available to image and quantify fluorescent nanoparticles. However, labeling can be problematic, especially if it alters the physical properties of the nanomaterial. Herein is described a novel non-covalent labeling technique for LSN using readily available fluorescent dimeric cyanine dyes without the need to use excess amounts of dye to achieve labeling, or the need for removal of unbound dye. The approach utilizes the cationic binding properties of layered silicate clays and the multiple quaternary nitrogens associated with the dyes. Preparation of YOYO-1 labeled LSN with optimal dispersion in aqueous media is presented. The utilization of the labeled particles is then demonstrated in cell binding and uptake studies using flow cytometry and confocal microscopy. The labeled LSN are highly fluorescent, stable and exhibit identical physical properties with respect to the unlabeled nanoparticles. The general approach described here is applicable to other cyanine dyes and may be utilized more widely for labeling nanoparticles that comprise a crystalline plate structure with a high binding capacity.

Nicolin Tee,1 Yingdong Zhu,2 GysellM Mortimer,1 Darren J Martin,2 Rodney F Minchin11School of Biomedical Science, University of Queensland, Brisbane, QLD, Australia; 2Australian Institute ofBioengineering and Nanotechnology, University of Queensland, Brisbane, QLD, AustraliaAbstract: Polyurethanes are widely used in biomedical devices such as heart valves, pacemaker leads, catheters, vascular devices, and surgical dressings because of their excellent mechanical properties and good biocompatibility. Layered silicate nanoparticles can significantly increase tensile strength and breaking strain of polyurethanes potentially increasing the life span of biomedical devices that suffer from wear in vivo. However, very little is known about how these nanoparticles interact with proteins and cells and how they might exert unwanted effects. A series of fluoromica nanoparticles ranging in platelet size from 90 to over 600 nm in diameter were generated from the same base material ME100 by high energy milling and differential centrifugation. The cytotoxicity of the resulting particles was dependent on platelet size but in a manner that is opposite to many other types of nanomaterials. For the fluoromicas, the smaller the platelet size, the less toxicity was observed. The small fluoromica nanoparticles (

Toxicity elicited by nanoparticles has been extensively studied, especially for those nanomaterials designed for human use. Safety concerns with nanomedicines have led to a better understanding of the molecular and cellular events that occur when nanoparticles enter biological systems. The key adverse effects include oxidative stress, inflammatory reactions, and genotoxicity. There are now a number of clinical trials using various nanoparticle formulations that show trends in the type and severity of unwanted effects that might be expected with nanomedicines. One of the major concern is the propensity for nanoparticles to activate the immune system, resulting in minor side effects such as local inflammation and more serious side effects including complement activation-related pseudoallergy to life-threatening side effects such as hypercytokinemia. Many adverse effects are observed in only a subgroup of patients, which raises the important question of how these individuals may be identified before treatment. For nanovaccines there is the potential of guiding the development of new treatments by understanding how adverse effects are elicited and how they may be avoided.

While plasma proteins can influence the physicochemical properties of nanoparticles, the adsorption of protein to the surface of nanomaterials can also alter the structure and function of the protein. Here, we show that plasma proteins form a hard corona around synthetic layered silicate nanoparticles (LSN) and that one of the principle proteins is serum albumin. The protein corona was required for recognition of the nanoparticles by scavenger receptors, a major receptor family associated with the mononuclear phagocyte system (MPS). Albumin alone could direct nanoparticle uptake by human macrophages, which involved class A but not class B scavenger receptors. Upon binding to LSN, albumin unfolded to reveal a cryptic epitope that could also be exposed by heat denaturation. This work provides an understanding of how albumin, and possibly other proteins, can promote nanomaterial recognition by the MPS without albumin requiring chemical modification for scavenger receptor recognition. These findings also demonstrate an additional function for albumin in vivo.

Humans are exposed to nanoparticles in the environment as well as those in nanomaterials developed for biomedical applications. However, the safety and biologic effects of many nanoparticles remain to be elucidated. Over the past decade, our understanding of the interaction of proteins with various nanomaterials has grown. The protein corona can determine not only how nanoparticles interact with cells but also their biologic effects and toxicity. In this study, we describe the effects that several different classes of nanoparticles exert on the enzymatic activity of the cytosolic protein human arylamine N-acetyltransferase 1 (NAT1), a drug-metabolizing enzyme widely distributed in the body that is also responsible for the activation and detoxification of known carcinogens. We investigated three metal oxides (zinc oxide, titanium dioxide, and silicon dioxide), two synthetic clay nanoparticles (layered double hydroxide and layered silicate nanoparticles), and a self-assembling thermo-responsive polymeric nanoparticle that differ in size and surface characteristics. We found that the different nanoparticles induced very different responses, ranging from inhibition to marked enhancement of enzyme activity. The layered silicates did not directly inactivate NAT1, but was found to enhance substrate-dependent inhibition. These differing effects demonstrate the multiplicity of nanoparticle-protein interactions and suggest that enzyme activity may be compromised in organs exposed to nanoparticles, such as the lungs or reticulo-endothelial system.

The functional diversity of proteins is a major factor determining the complexity of cells and tissues. Both translational and post-translational modifications contribute to this diversity. Recently, protein unfolding and refolding has been recognised as another mechanism for diversity by unmasking buried or cryptic sequences (epitopes) that possess physiological functions. In the current review, we focus on extracellular proteins where folding dynamics can be influenced by mechanical forces, protein-protein interactions and denaturation. Many cryptic epitopes in these proteins are exposed following proteolytic cleavage, but recent data indicate that unfolding/refolding play an important role in regulating the physiological behaviour of extracellular proteins. By understanding how and when hidden sequences are exposed, novel techniques for manipulating the function of these proteins may be uncovered.

In biological fluids nanoparticles bind a range of molecules, particularly proteins, on their surface. The resulting protein corona influences biological activity and fate of nanoparticle in vivo. Corona composition is often determined by the biological milieu encountered at the entry portal into the body, and, can therefore, depend on the route of exposure to the nanoparticle. For environmental nanoparticles where exposure is by inhalation, this will be lung lining fluid. This study examined plasma and bronchoalveolar fluid (BALF) protein binding to engineered and environmental nanoparticles. We hypothesized that protein corona on nanoparticles would influence nanoparticle uptake and subsequent pro-inflammatory biological response in macrophages. All nanoparticles bound plasma and BALF proteins, but the profile of bound proteins varied between nanoparticles. Focusing on diesel exhaust nanoparticles (DENP), we identified proteins bound from plasma to include fibrinogen, and those bound from BALF to include albumin and surfactant proteins A and D. The presence on DENP of a plasma-derived corona or one of purified fibrinogen failed to evoke an inflammatory response in macrophages. However, coronae formed in BALF increased DENP uptake into macrophages two fold, and increased nanoparticulate carbon black (NanoCB) uptake fivefold. Furthermore, a BALF-derived corona increased IL-8 release from macrophages in response to DENP from 1720 ± 850 pg/mL to 5560 ± 1380 pg/mL (p = 0.014). These results demonstrate that the unique protein corona formed on nanoparticles plays an important role in determining biological reactivity and fate of nanoparticle in vivo. Importantly, these findings have implications for the mechanism of detrimental properties of environmental nanoparticles since the principle route of exposure to such particles is via the lung.

Layered double hydroxide (LDH) nanoparticles have the potential to benefit a myriad of medical, consumer and industrial products if intricate control of the nanoscale architecture and understanding of the biological interactions and potential toxicity of the LDH nanomaterials are achieved. Here, we report the synthesis and fractionation of LDH nanomaterials of well defined spatial characteristics through the careful control of synthesis conditions and the use of ultracentrifugation. The appropriateness of a range of fluorescent dyes, for labelling LDH nanoparticles to enable biological tracking, has also been investigated. These dyes include fluorescein, fluorescein isothiocyanate (FITC), 8-aminonaphthelene-1,3,6-trisulfonic acid (ANTS) and 8-aminopyrene-1,3,6-trisulfonic acid (APTS). Thermal degradation of the popular FITC dye during nanoparticle synthesis was revealed and suitable alternative fluorescent molecules established. Furthermore, we report for the first time the application of size exclusion chromatography on LDH nanoparticles as a fast, reliable and efficient method to achieve purification without causing nanoparticle agglomeration.

Properties of nanoparticles (NPs) are responsible for their interaction with various biomolecules such as proteins in biological environments. Amount and composition of the proteins associated with NPs, i.e. protein corona, are strongly dependent on physicochemical characteristics of the particles, as well as incubation parameters including temperature and protein concentration. More importantly, the protein corona can define the biological fate of the NPs. Here, we demonstrate that variations in the concentration of plasma protein led to significant changes in the composition of the hard corona adsorbed on the surface of different NPs including hydrophilic amorphous silica (SiO2), hydrophilic crystalline zeolite (EMT), and hydrophobic sulfonated-modified polystyrene. Alteration in the corona composition of the NPs is a result of the plasma concentration, i.e. it affects the release of inflammatory cytokines in a plasma concentration-dependent manner. The amorphous silica nanoparticles with hydrophilic surfaces induced the release of the inflammatory cytokines interleukin-8 (IL-8) and tumor necrosis factor (TNFα) in 10 % plasma concentration, but not at higher concentrations. A reverse trend was observed for the hydrophobic, sulfonated-modified polystyrene NPs. Remarkably the hydrophilic highly porous EMT NPs exhibited no cellular toxicity regardless to the plasma concentration. The results obtained in this study can be used to define optimal pathways for nanoparticles administration in vivo. These findings can assist researchers to better understand how NPs with different surface properties may interact with various proteins in vivo, and elucidate safety considerations for their biomedical applications.

Poly(ethylene glycol) helps nanomaterials evade the immune system by modifying the composition of proteins that are adsorbed on the surface of the materials.

Nanoparticles rapidly interact with the proteins present in biological fluids, such as blood. The proteins that are adsorbed onto the surface potentially dictate the biokinetics of the nanomaterials and their fate in vivo. Using nanoparticles with different sizes and surface characteristics, studies have reported the effects of physicochemical properties on the composition of adsorbed plasma proteins. However, to date, few studies have been conducted focusing on the nanoparticles that are commonly exposed to the general public, such as the metal oxides. Using previously established ultracentrifugation approaches, two-dimensional gel electrophoresis and mass spectrometry, the current study investigated the binding of human plasma proteins to commercially available titanium dioxide, silicon dioxide and zinc oxide nanoparticles. We found that, despite these particles having similar surface charges in buffer, they bound different plasma proteins. For TiO2, the shape of the nanoparticles was also an important determinant of protein binding. Agglomeration in water was observed for all of the nanoparticles and both TiO2 and ZnO further agglomerated in biological media. This led to an increase in the amount and number of different proteins bound to these nanoparticles. Proteins with important biological functions were identified, including immunoglobulins, lipoproteins, acute-phase proteins and proteins involved in complement pathways and coagulation. These results provide important insights into which human plasma proteins bind to particular metal oxide nanoparticles. Because protein absorption to nanoparticles may determine their interaction with cells and tissues in vivo, understanding how and why plasma proteins are adsorbed to these particles may be important for understanding their biological responses.