The use of a complex tetra-culture alveolar model to study the biological effects induced by gold nanoparticles with different physicochemical properties.

A substantial increase in engineered nanoparticles in consumer products has been observed, heightening human and environmental exposure. Inhalation represents the primary route of human exposure, necessitating a focus on lung toxicity studies. However, to avoid ethical concerns the use of in vitro models is an efficient alternative to in vivo models. This study utilized an in vitro human alveolar barrier model at air-liquid-interface with four cell lines, for evaluating the biological effects of different gold nanoparticles. Exposure to PEGylated gold nanospheres, nanorods, and nanostars did not significantly impact viability after 24 h, yet all AuNPs induced cytotoxicity in the form of membrane integrity impairment. Gold quantification revealed cellular uptake and transport. Transcriptomic analysis identified gene expression changes, particularly related to the enhancement of immune cells. Despite limited impact, distinct effects were observed, emphasizing the influence of nanoparticles physicochemical parameters while demonstrating the model's efficacy in investigating particle biological effects.

Development of a fast and simple method for the isolation of superparamagnetic iron oxide nanoparticles protein corona from protein-rich matrices.

The adsorption of proteins onto the surface of nanoparticle (NP) leads to the formation of the so-called "protein corona" as consisting both loosely and tightly bound proteins. It is well established that the biological identity of NPs that may be acquired after exposure to a biological matrix is mostly provided by the components of the hard corona as the pristine surface is generally less accessible for binding. For that reason, the isolation and the characterisation of the NP-corona complexes and identification of the associated biomolecules can help in understanding its biological behaviour. Established methods for the isolation of the NP-HC complexes are time-demanding and can lead to different results based on the isolation method applied. Herein, we have developed a fast and simple method using ferromagnetic beads isolated from commercial MACS column and used for the isolation of superparamagnetic NP following exposure to different types of biological milieu. We first demonstrated the ability to easily isolate superparamagnetic iron oxide NPs (IONPs) from different concentrations of human blood plasma, and also tested the method on the corona isolation using more complex biological matrices, such as culture medium containing pulmonary mucus where the ordinary corona methods cannot be applied. Our developed method showed less than 20% difference in plasma corona composition when compared with centrifugation. It also showed effective isolation of NP-HC complexes from mucus-containing culture media upon comparing with centrifugation and MACS columns, which failed to wash out the unbound proteins. Our study was supported with a full characterisation profile including dynamic light scattering, nanoparticle tracking analysis, analytical disk centrifuge, and zeta potentials. The biomolecules/ proteins composing the HC were separated by vertical gel electrophoresis and subsequently analysed by liquid chromatography-tandem mass spectrometry. In addition to our achievements in comparing different isolation methods to separate IONPs with corona from human plasma, this is the first study that provides a complete characterisation profile of particle protein corona after exposure in vitro to pulmonary mucus-containing culture media.

Changes of physico-chemical properties of nano-biomaterials by digestion fluids affect the physiological properties of epithelial intestinal cells and barrier models.

BACKGROUND: The widespread use of nano-biomaterials (NBMs) has increased the chance of human exposure. Although ingestion is one of the major routes of exposure to NBMs, it is not thoroughly studied to date. NBMs are expected to be dramatically modified following the transit into the oral-gastric-intestinal (OGI) tract. How these transformations affect their interaction with intestinal cells is still poorly understood. NBMs of different chemical nature-lipid-surfactant nanoparticles (LSNPs), carbon nanoparticles (CNPs), surface modified Fe3O4 nanoparticles (FNPs) and hydroxyapatite nanoparticles (HNPs)-were treated in a simulated human digestive system (SHDS) and then characterised. The biological effects of SHDS-treated and untreated NBMs were evaluated on primary (HCoEpiC) and immortalised (Caco-2, HCT116) epithelial intestinal cells and on an intestinal barrier model. RESULTS: The application of the in vitro SDHS modified the biocompatibility of NBMs on gastrointestinal cells. The differences between SHDS-treated and untreated NBMs could be attributed to the irreversible modification of the NBMs in the SHDS. Aggregation was detected for all NBMs regardless of their chemical nature, while pH- or enzyme-mediated partial degradation was detected for hydroxyapatite or polymer-coated iron oxide nanoparticles and lipid nanoparticles, respectively. The formation of a bio-corona, which contains proteases, was also demonstrated on all the analysed NBMs. In viability assays, undifferentiated primary cells were more sensitive than immortalised cells to digested NBMs, but neither pristine nor treated NBMs affected the intestinal barrier viability and permeability. SHDS-treated NBMs up-regulated the tight junction genes (claudin 3 and 5, occludin, zonula occludens 1) in intestinal barrier, with different patterns between each NBM, and increase the expression of both pro- and anti-inflammatory cytokines (IL-1ß, TNF-α, IL-22, IL-10). Notably, none of these NBMs showed any significant genotoxic effect. CONCLUSIONS: Overall, the results add a piece of evidence on the importance of applying validated in vitro SHDS models for the assessment of NBM intestinal toxicity/biocompatibility. We propose the association of chemical and microscopic characterization, SHDS and in vitro tests on both immortalised and primary cells as a robust screening pipeline useful to monitor the changes in the physico-chemical properties of ingested NBMs and their effects on intestinal cells.

Pathways Related to NLRP3 Inflammasome Activation Induced by Gold Nanorods.

The widespread and increasing use of engineered nanomaterials (ENM) increases the risk of human exposure, generating concern that ENM may provoke adverse health effects. In this respect, their physicochemical characteristics are critical. The immune system may respond to ENM through inflammatory reactions. The NLRP3 inflammasome responds to a wide range of ENM, and its activation is associated with various inflammatory diseases. Recently, anisotropic ENM have become of increasing interest, but knowledge of their effects on the immune system is still limited. The objective of the study was to compare the effects of gold ENM of different shapes on NLRP3 inflammasome activation and related signalling pathways. Differentiated THP-1 cells (wildtype, ASC- or NLRP3-deficient), were exposed to PEGylated gold nanorods, nanostars, and nanospheres, and, thus, also different surface chemistries, to assess NLRP3 inflammasome activation. Next, the exposed cells were subjected to gene expression analysis. Nanorods, but not nanostars or nanospheres, showed NLRP3 inflammasome activation. ASC- or NLRP3-deficient cells did not show this effect. Gene Set Enrichment Analysis revealed that gold nanorod-induced NLRP3 inflammasome activation was accompanied by downregulated sterol/cholesterol biosynthesis, oxidative phosphorylation, and purinergic receptor signalling. At the level of individual genes, downregulation of Paraoxonase-2, a protein that controls oxidative stress, was most notable. In conclusion, the shape and surface chemistry of gold nanoparticles determine NLRP3 inflammasome activation. Future studies should include particle uptake and intracellular localization.

Nanoparticle Biomolecular Corona-Based Enrichment of Plasma Glycoproteins for N-Glycan Profiling and Application in Biomarker Discovery.

Biomolecular corona formation has emerged as a recurring and important phenomenon in nanomedicine that has been investigated for potential applications in disease diagnosis. In this study, we have combined the "personalized protein corona" with the N-glycosylation profiling that has recently gained considerable interest in human plasma biomarker discovery as a powerful early warning diagnostic and patient stratification tool. We envisioned that the protein corona formation could be exploited as an enrichment step that is critically important in both proteomic and proteoglycomic workflows. By using silica nanoparticles, plasma fibrinogen was enriched to a level in which its proteomic and glycomic "fingerprints" could be traced with confidence. Despite being a more simplified glycan profile compared to full plasma, the corona glycan profile revealed a fibrinogen-derived glycan peak that was found to potentially distinguish lung cancer patients from controls in a pilot study.

Development of amyloid beta gold nanorod aggregates as optoacoustic probes.

Propagation of small amyloid beta (Aß) aggregates (or seeds) has been suggested as a potential mechanism of Alzheimer's disease progression. Monitoring the propagation of Aß seeds in an organism would enable testing of this hypothesis and, if confirmed, provide mechanistic insights. This requires a contrast agent for long-term tracking of the seeds. Gold nanorods combine several attractive features for this challenging task, in particular, their strong absorbance in the infrared (enabling optoacoustic imaging) and the availability of several established protocols for surface functionalisation. In this work, polymer-coated gold nanorods were conjugated with anti-Aß antibodies and attached to pre-formed Aß seeds. The resulting complexes were characterised for their optical properties by UV/Vis spectroscopy and multispectral optoacoustic tomography. The complexes retained their biophysical properties, i.e. their ability to seed Aß fibril formation. They remained stable in biological media for at least 2 days and showed no toxicity to SH-SY5Y neuroblastoma cells up to 1.5 nM and 6 µM of gold nanorods and Aß seeds, respectively. Taken together, this study describes the first steps in the development of probes for monitoring the spread of Aß seeds in animal models.

Probing the glycans accessibility in the nanoparticle biomolecular corona.

HYPOTHESIS: Following blood administration, the pristine surface of nanoparticles (NPs) associates with biomolecules from the surrounding environment forming the so-called "biomolecular corona". It is well accepted that the biomolecular corona dramatically affects the NP fate in the biological medium while the pristine surface is no longer available for binding. Recent studies have shown that the glycans associated with the proteins forming the corona have a role in the NP interaction with macrophages, but the glycan identities remain unknown. We aim here to identify the glycan composition of the biomolecular corona and to assess the role of these glycans in the interaction of the proteins from the corona with glycan binding biomolecules, such as lectins. EXPERIMENTS: In this study, we have characterized the biomolecular corona of citrate stabilised gold NPs after exposure of the NPs to blood plasma at two different plasma concentrations, mimicking the in vitro and in vivo conditions. We have extensively characterized the biomolecular corona using HILIC chromatography and shotgun proteomics. Following this, a lectin binding assay was carried out using Dynamic Light Scattering (DLS) and Fluorescence Correlation Spectroscopy (FCS) to assess whether proteins with known affinity towards specific glycans would bind to the corona. FINDINGS: Our findings highlighted that the protein corona composition is dependent on the exposing conditions. However, under both plasma concentrations, the biantennary sialylated glycans (A2G2S2) are enriched. DLS and FCS confirmed that the glycans are accessible for binding as the corona interacts with lectins with known affinity towards terminal sialic acids and the enzymatic removal of the glycans leads to a decrease in lectin affinity. This study shows for the first time that the glycans are present in the corona and that they could potentially be responsible for the modulation of NP biological processes as they can directly engage with glycan binding receptors that are highly expressed in an organism.

Hyperspectral-enhanced dark field analysis of individual and collective photo-responsive gold-copper sulfide nanoparticles.

We used hyperspectral-enhanced dark field microscopy for studying physicochemical changes in biomaterials by tracking their unique spectral signatures along their pathway through different biological environments typically found in any biomedical application. We correlate these spectral signatures with discrete environmental features causing changes in nanoparticles' physicochemical properties. We use this correlation to track the nanoparticles intracellularly and to assess the impact of these changes on their functionality. We focus on one example of a photothermal nanocomposite, i.e., polymer-coated gold/copper sulfide nanoparticles, because their performance depends on their localized surface plasmon peak, which is highly sensitive to environmental changes. We found spectral differences both in the dependence of time and discrete environmental factors, affecting the range of illumination wavelengths that can be used to activate the functionality of these types of nanoparticles. The presence of proteins (protein corona) and the increase in ionic strength induce a spectral broadening towards the NIR region which we associated with nanoparticles' agglomeration. In acidic environments, such as that of the lysosome, a red shift was also observed in addition to a decrease in the scattering intensity probably associated with a destabilization of the proteins and/or the change in the net charge of the polymer around the nanoparticles. We observed a loss of the photo-excitation potential of those nanoparticles exposed to acidic conditions in the <600 nm spectral rage. In a similar manner, ageing induces a transitioning from a broad multipeak spectrum to a distinct shoulder with time (up to 8 months) with the loss of spectral contribution in the 450-600 nm range. Hence, a fresh preparation of nanoparticles before their application would be recommended for an optimal performance. We highlight the impact of ageing and the acidic environment on the responsiveness of this type of plasmonic nanoparticle. Regardless of the spectral differences found, polymer-coated gold/copper sulfide nanoparticles retained their photothermal response as demonstrated in vitro upon two-photon irradiation. This could be ascribed to their robust geometry provided by the polymer coating. These results should be useful to rationally design plasmonic photothermal probes.

In depth characterisation of the biomolecular coronas of polymer coated inorganic nanoparticles with differential centrifugal sedimentation.

Advances in nanofabrication methods have enabled the tailoring of new strategies towards the controlled production of nanoparticles with attractive applications in healthcare. In many cases, their characterisation remains a big challenge, particularly for small-sized functional nanoparticles of 5 nm diameter or smaller, where current particle sizing techniques struggle to provide the required sensitivity and accuracy. There is a clear need for the development of new reliable characterisation approaches for the physico-chemical characterisation of nanoparticles with significant accuracy, particularly for the analysis of the particles in the presence of complex biological fluids. Herein, we show that the Differential Centrifugal Sedimentation can be utilised as a high-precision tool for the reliable characterisation of functional nanoparticles of different materials. We report a method to correlate the sedimentation shift with the polymer and biomolecule adsorption on the nanoparticle surface, validating the developed core-shell model. We also highlight its limit when measuring nanoparticles of smaller size and the need to use several complementary methods when characterising nanoparticle corona complexes.

Aqueous Stable Gold Nanostar/ZIF-8 Nanocomposites for Light-Triggered Release of Active Cargo Inside Living Cells.

A plasmonic core-shell gold nanostar/zeolitic-imidazolate-framework-8 (ZIF-8) nanocomposite was developed for the thermoplasmonic-driven release of encapsulated active molecules inside living cells. The nanocomposites were loaded, as a proof of concept, with bisbenzimide molecules as functional cargo and wrapped with an amphiphilic polymer that prevents ZIF-8 degradation and bisbenzimide leaking in aqueous media or inside living cells. The demonstrated molecule-release mechanism relies on the use of near-IR light coupled to the plasmonic absorption of the core gold nanostars, which creates local temperature gradients and thus, bisbenzimide thermodiffusion. Confocal microscopy and surface-enhanced Raman spectroscopy (SERS) were used to demonstrate bisbenzimide loading/leaking and near-IR-triggered cargo release inside cells, thereby leading to DNA staining.

How Entanglement of Different Physicochemical Properties Complicates the Prediction of in Vitro and in Vivo Interactions of Gold Nanoparticles.

The physicochemical properties of a set of 21 different gold nanoparticles (spherical and rod-shaped nanoparticles (NPs) of different diameters with three different surface coatings) were studied. Protein corona formation, in vitro uptake, effect on cell viability and proliferation, and in vivo biodistribution of these NPs were determined. The relation of the results of the different NPs was analyzed by hierarchical cluster analysis, which will tell which NPs have the most similar physicochemical properties and biological effects, without having to specify individual physicochemical parameters. The results show that the physicochemical properties of gold nanoparticles (Au NPs) are mainly accounted for by their hydrodynamic diameter and their zeta-potential. The formation of the protein corona is determined by the pH-dependence of their zeta-potential. While several reports found that in vitro uptake and in vivo biodistribution of NPs are correlated to individual physicochemical parameters, e. g., size, shape, or surface chemistry, such direct dependence in the investigated multidimensional set of NPs was not found in our study. This most likely is due to entanglement of the different parameters, which complicates the prediction of the biological effect of NPs in case multiple physicochemical properties are simultaneously varied. The in vitro uptake and in vivo biodistribution of NPs seem to be not directly driven by the protein corona, but the physicochemical properties determine as well the corona as they influence in vitro/ in vivo behaviors, and thus the effect of the protein corona would be rather indirect.

Laterally and Temporally Controlled Intracellular Staining by Light-Triggered Release of Encapsulated Fluorescent Markers.

Fluorescent molecular markers were encapsulated. The capsules were additionally modified with plasmonic nanoparticles. The encapsulated markers were endocytosed by cells. Upon light stimulation the plasmonic nanoparticles generated heat, which opened the encapsulation and transiently perforated the endosomal/lysosomal membrane surrounding the capsule, thus allowing for release of the marker into the cytosol. Fluorescence labeling of different intracellular compartments was demonstrated in this way. Most important, the cells do not need to be fixed and perforated, as the molecular markers are introduced into cells by endocytosis and subsequent light-induced release. Thus this technique allows for intracellular fluorescence labeling of living cells.

Gold-Based Nanomaterials for Applications in Nanomedicine.

In this review, an overview of the current state-of-the-art of gold-based nanomaterials (Au NPs) in medical applications is given. The unique properties of Au NPs, such as their tunable size, shape, and surface characteristics, optical properties, biocompatibility, low cytotoxicity, high stability, and multifunctionality potential, among others, make them highly attractive in many aspects of medicine. First, the preparation methods for various Au NPs including functionalization strategies for selective targeting are summarized. Second, recent progresses on their applications, ranging from the diagnostics to therapeutics are highlighted. Finally, the rapidly growing and promising field of gold-based theranostic nano-platforms is discussed. Considering the great body of existing information and the high speed of its renewal, we chose in this review to generalize the data that have been accumulated during the past few years for the most promising directions in the use of Au NPs in current medical research.

Conjugation of Polymer-Coated Gold Nanoparticles with Antibodies-Synthesis and Characterization.

The synthesis of polymer-coated gold nanoparticles with high colloidal stability is described, together with appropriate characterization techniques concerning the colloidal properties of the nanoparticles. Antibodies against vascular endothelial growth factor (VEGF) are conjugated to the surface of the nanoparticles. Antibody attachment is probed by different techniques, giving a guideline about the characterization of such conjugates. The effect of the nanoparticles on human adenocarcinoma alveolar basal epithelial cells (A549) and human umbilical vein endothelial cells (HUVECs) is probed in terms of internalization and viability assays.

Fluorescence-based ion-sensing with colloidal particles.

Particle-based fluorescence sensors for the quantification of specific ions can be made by coupling ion-sensitive fluorophores to carrier particles, or by using intrinsically fluorescent particles whose fluorescence properties depend on the concentration of the ions. Despite the advantages of such particle-based sensors for the quantitative detection of ions, such as the possibility to tune the surface chemistry and thus entry portal of the sensor particles to cells, they have also some associated problems. Problems involve for example crosstalk of the ion-sensitive fluorescence read-out with pH, or spectral overlap of the emission spectra of different fluorescent particles in multiplexing formats. Here the benefits of using particle-based fluorescence sensors, their limitations and strategies to overcome these limitations will be described and exemplified with selected examples.

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

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.