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Protein corona formation around nanoparticles – from the past to the future

The protein adsorption layer (a.k.a. the “protein corona”) that forms on the surface of colloidal nanoparticles plays an important role in their interaction with living matter. Thus, characterization of the protein corona is of utmost importance for understanding how exposure to nanoparticles affects the biological responses of cells and organisms. Although a lot of experimental studies have been reported in this direction, a comprehensive picture is still missing, in particular due to the multitude of different scenarios under which experiments have been performed. In this review an analysis of existing experimental data about the protein corona, and an outline for required future work will be given. In particular we review how existing simple analytical models such as the adopted Hill model may help to extract quantitative data from such experiments such as equilibrium dissociation and kinetic coefficients. Careful quantitative assessment of equilibrium and kinetic properties would allow for a comparison of protein binding data from the vast array of engineered nanoparticles, so that basic principles could be revealed. This review outlines that the field is in dire need of more quantitative studies to further our understanding of protein corona formation and its biological consequences.

Surface Functionalization of Nanoparticles with Polyethylene Glycol: Effects on Protein Adsorption and Cellular Uptake

Here we have investigated the effect of enshrouding polymer-coated nanoparticles (NPs) with polyethylene glycol (PEG) on the adsorption of proteins and uptake by cultured cells. PEG was covalently linked to the polymer surface to the maximal grafting density achievable under our experimental conditions. Changes in the effective hydrodynamic radius of the NPs upon adsorption of human serum albumin (HSA) and fibrinogen (FIB) were measured in situ using fluorescence correlation spectroscopy. For NPs without a PEG shell, a thickness increase of around 3 nm, corresponding to HSA monolayer adsorption, was measured at high HSA concentration. Only 50% of this value was found for NPs with PEGylated surfaces. While the size increase clearly reveals formation of a protein corona also for PEGylated NPs, fluorescence lifetime measurements and quenching experiments suggest that the adsorbed HSA molecules are buried within the PEG shell. For FIB adsorption onto PEGylated NPs, even less change in NP diameter was observed. In vitro uptake of the NPs by 3T3 fibroblasts was reduced to around 10% upon PEGylation with PEG chains of 10 kDa. Thus, even though the PEG coatings did not completely prevent protein adsorption, the PEGylated NPs still displayed a pronounced reduction of cellular uptake with respect to bare NPs, which is to be expected if the adsorbed proteins are not exposed on the NP surface.

Interfacing engineered nanoparticles with biological systems: anticipating adverse nano-bio interactions.

The innovative use of engineered nanomaterials in medicine, be it in therapy or diagnosis, is growing dramatically. This is motivated by the current extraordinary control over the synthesis of complex nanomaterials with a variety of biological functions (e.g. contrast agents, drug-delivery systems, transducers, amplifiers, etc.). Engineered nanomaterials are found in the bio-context with a variety of applications in fields such as sensing, imaging, therapy or diagnosis. As the degree of control to fabricate customized novel and/or enhanced nanomaterials evolves, often new applications, devices with enhanced performance or unprecedented sensing limits can be achieved. Of course, interfacing any novel material with biological systems has to be critically analyzed as many undesirable adverse effects can be triggered (e.g. toxicity, allergy, genotoxicity, etc.) and/or the performance of the nanomaterial can be compromised due to the unexpected phenomena in physiological environments (e.g. corrosion, aggregation, unspecific absorption of biomolecules, etc.). Despite the need for standard protocols for assessing the toxicity and bio-performance of each new functional nanomaterial, these are still scarce or currently under development. Nonetheless, nanotoxicology and relating adverse effects to the physico-chemical properties of nanomaterials are emerging areas of the utmost importance which have to be continuously revisited as any new material emerges. This review highlights recent progress concerning the interaction of nanomaterials with biological systems and following adverse effects.

Nanoparticle-modified polyelectrolyte capsules

The concept of polyelectrolyte capsules as multifunctional carrier systems is described. The walls of a capsule can be functionalized with fluorescent, magnetic, and heatable colloidal nanoparticles and also biological macromolecules, while its cavity can be loaded with cargo molecules. Potential applications of this carrier system for delivery and sensing in cells are discussed.

Gold nanoprisms as optoacoustic signal nanoamplifiers for in vivo bioimaging of gastrointestinal cancers.

Early detection of cancer greatly increases the chances of a simpler and more effective treatment. Traditional imaging techniques are often limited by shallow penetration, low sensitivity, low specificity, poor spatial resolution or the use of ionizing radiation. Hybrid modalities, like optoacoustic imaging, an emerging molecular imaging modality, contribute to improving most of these limitations. However, this imaging method is hindered by relatively low signal contrast. Here, gold nanoprisms (AuNPrs) are used as signal amplifiers in multispectral optoacoustic tomography (MSOT) to visualize gastrointestinal cancer. PEGylated AuNPrs are successfully internalized by HT-29 gastrointestinal cancer cells in vitro. Moreover, the particles show good biocompatibility and exhibit a surface plasmon band centered at 830 nm, a suitable wavelength for optoacoustic imaging purposes. These findings extend well to an in vivo setting, in which mice are injected with PEGylated AuNPrs in order to visualize tumor angiogenesis in gastrointestinal cancer cells. Overall, both our in vitro and in vivo results show that PEGylated AuNPrs have the capacity to penetrate tumors and provide a high-resolution signal amplifier for optoacoustic imaging. The combination of PEGylated AuNPrs and MSOT represents a significant advance for the in vivo imaging of cancers.

Tailoring the synthesis and heating ability of gold nanoprisms for bioapplications.

The paper describes a novel and straightforward wet-chemical synthetic route to produce biocompatible single-crystalline gold tabular nanoparticles, herein called nanoprisms (NPRs) due to their characteristic shape. Besides the novelty of the method to produce NPRs with an unprecedented high yield, the synthesis avoids the use of highly toxic cetyltrimethylammonium bromide (CTAB), the most widely used surfactant for the synthesis of gold anisotropic nanoparticles such as nanorods or nanoprisms. The method presented here allows for tuning the edge length of NPRs in the range of 100-170 nm by adjusting the final concentration/molar ratio of gold salt and reducing agent (thiosulfate), while the thickness of NPRs remained constant (9 nm). Thus, the surface plasmon band of NPRs can be set along the near-infrared (NIR) range. The resulting NPRs were derivatized with heterobifunctional polyethylene glycol (PEG) and 4-aminophenyl β-D-glucopyranoside (glucose) chains to improve their stability and cellular uptake, respectively. The heating properties of colloidal solutions of NPRs upon 1064 nm light illumination were evaluated. As a proof of concept, the biocompatibility and suitability of functional NPRs as photothermal agents were studied in cell cultures. Due to their biocompatibility (avoiding CTAB), ease of production, ease of functionalization, and remarkable heating features, the NPRs discussed herein represent a significant advance in the biocompatibility of nanoparticles and serve as an attractive alternative to those currently in use as plasmonic photothermal agents.

In vitro interaction of colloidal nanoparticles with mammalian cells: What have we learned thus far?

Summary The interfacing of colloidal nanoparticles with mammalian cells is now well into its second decade. In this review our goal is to highlight the more generally accepted concepts that we have gleaned from nearly twenty years of research. While details of these complex interactions strongly depend, amongst others, upon the specific properties of the nanoparticles used, the cell type, and their environmental conditions, a number of fundamental principles exist, which are outlined in this review.

Taking Advantage of Unspecific Interactions to Produce Highly Active Magnetic Nanoparticle−Antibody Conjugates

Several strategies for linking antibodies (Abs) through their Fc region in an oriented manner have been proposed at the present time. By using these strategies, the Fab region of the Ab is available for antigen molecular recognition, leading to a more efficient interaction. Most of these strategies are complex processes optimized mainly for the functionalization of surfaces or microbeads. These methodologies imply though the Ab modification through several steps of purification or the use of expensive immobilized proteins. Besides, the functionalization of magnetic nanoparticles (MNPs) turned out to be much more complex than expected due to the lack of stability of most MNPs at high ionic strength and non-neutral pH values. Therefore, there is still missing an efficient, easy and universal methodology for the immobilization of nonmodified Abs onto MNPs without involving their Fab regions during the immobilization process. Herein, we propose the functionalization of MNPs via a two-steps strategy that takes advantage of the ionic reversible interactions between the Ab and the MNP. These interactions make possible the orientation of the Ab on the MNP surface before being attached in an irreversible way via covalent bonds. Three Abs (Immunoglobulin G class) with very different isoelectric points (against peroxidase, carcinoembryonic antigen, and human chorionic gonadotropin hormone) were used to prove the general applicability of the strategy here proposed and its utility for the development of more bioactive NPs.

Gene Silencing Mediated by Magnetic Lipospheres Tagged with Small Interfering RNA

Lipospheres made from soy bean oil and a combination of the cationic lipid Metafectene and the helper lipid dioleoylphosphatidyl-ethanolamine were functionalized with magnetic nanoparticles (NPs) and small interfering RNA (siRNA). The resulting magnetic lipospheres loaded with siRNA are proven here as efficient nonviral vectors for gene silencing. Embedding magnetic NPs in the shell of lipospheres allows for magnetic force-assisted transfection (magnetofection) as well as magnetic targeting in both static and fluidic conditions mimicking the bloodstream.

Basic Physicochemical Properties of Polyethylene Glycol Coated Gold Nanoparticles that Determine Their Interaction with Cells

A homologous nanoparticle library was synthesized in which gold nanoparticles were coated with polyethylene glycol, whereby the diameter of the gold cores, as well as the thickness of the shell of polyethylene glycol, was varied. Basic physicochemical parameters of this two-dimensional nanoparticle library, such as size, ζ-potential, hydrophilicity, elasticity, and catalytic activity ,were determined. Cell uptake of selected nanoparticles with equal size yet varying thickness of the polymer shell and their effect on basic structural and functional cell parameters was determined. Data indicates that thinner, more hydrophilic coatings, combined with the partial functionalization with quaternary ammonium cations, result in a more efficient uptake, which relates to significant effects on structural and functional cell parameters.