Physical-chemical characterization of nanoparticles in relevant biological environments and their interactions with the cell surface

Nanoparticles (NPs) are versatile tools for nanomedicine and tuning features such as material, size and charge, imaging and targeting can be accomplished. However, NPs behaviour in vivo is modified upon interaction with the biological matter and formation of a protein corona (PC) coating the NP. The PC determines the NP biological identity and it is the ultimate interface with the surrounding environment. Therefore, a deep characterization of the NPs in biological media is important to predict adverse effects and improve NPs design. The aim of this thesis was to understand the effect of the PC formation from different biological fluids on NP- membranes interactions. For this purpose, core-shell gold and magnetite NPs coated by poly-maleic anhydride and pegylated were characterized my means of scattering, microscopic and spectroscopic techniques. Such NPs were characterized in serum and PC complexes were isolated. Sucrose-gradient ultracentrifugation (UC) was used guaranteeing quantitative recovery of homogeneous NP PC populations, simultaneously present in situ, and a lower impact on the in situ structures compared to conventional centrifugation protocols. NP interactions with supported lipid bilayers (SLB) were investigated by QCM-D and neutron reflectometry allowing resolving at the sub-nanometer scale any structural reorganization of the SLB upon NP application. Carboxylated NPs generally caused lipid hydration with different mechanisms, while HC NPs compared to in situ NPs and pure FBS had a lower impact on the bilayers possibly indicating a major impact of the soft corona. The last part of the project was focused on the PC evolution during simulated in vitro digestion with NPs. UC was suitable to isolate PC complexes from gastric and intestinal phases and SDS-PAGE and LC-MS suggested a PC ability to protect peptides from digestion degradation. The biological impact of the PC complexes was studied by confocal microscopy on Caco-2 cells revealing cells morphological alterations

Proteins Are Solitary! Pathways of Protein Folding and Aggregation in Protein Mixtures

We present a computational and experimental study on the folding and aggregation in solutions of multiple protein mixtures at different concentrations. We show how in protein mixtures, each component is capable of maintaining its folded state at desensitises higher then the one at which they would precipitate in single species solutions. We demonstrate the generality of our observation over many different proteins using computer simulations capable of fully characterising the cross-aggregation phase diagram of all the mixtures. Dynamic light Scattering experiments were performed to evaluate the aggregation of two proteins, the bovine serum albumin (BSA) and the consensus tetratricopeptide repeat (CTPR), in solutions of one or both proteins. The experiment confirm our hypothesis and the simulations. These findings elucidate critical aspects on the cross-regulation of expression and aggregation of proteins exerted by the cell and on the evolutionary selection of folding and not-aggregating protein sequences, paving the way for new experimental tests

An early developmental vertebrate model for nanomaterial safety:Bridging cell-based and mammalian toxicity assessment

Background. With the rise in production of nanoparticles for an ever-increasing number of applications, there is an urgent need to efficiently assess their potential toxicity. We propose a nanoparticle hazard assessment protocol that combines mammalian cytotoxicity data with embryonic vertebrate abnormality scoring to determine an overall toxicity index. Results. We observed that, after exposure to a range of nanoparticles, Xenopus phenotypic scoring showed a strong correlation with cell based in vitro assays. Magnetite-cored nanoparticles, negative for toxicity in vitro and Xenopus, were further confirmed as non-toxic in mice. Conclusion. The results highlight the potential of Xenopus embryo analysis as a fast screening approach for toxicity assessment of nanoparticles, which could be introduced for the routine testing of nanomaterials

Nanoscopic Agents in a Physiological Environment: The Importance of Understanding Their Characteristics

The application of nanotechnology in medicine signifies one of the most exciting developments in science over the last decade. Even though advancement has been made in nanoparticle engineering in terms of size, shape and surface functionalisation, the behaviour in vivo remains poorly characterised and understood. The potential impact of engineered nanomaterials on human health is strictly related to their behaviour in the biological environment. When in contact with biological fluids, nanoparticles spontaneously interact and adsorb proteins to dramatically change their surface properties. Thus, the nanoparticle surface acquires a new biological identity that will influence its stability and interaction with the cellular machinery, thereby affecting the nanoparticle biodistribution in vivo. This protein coating ‘expressed’ at the nanoparticle surface is what is ‘read’ by the cells. Consequently, methods to effectively study the structure and composition of this bio-nano interface have been emerging as key objectives in nanoscience. In this chapter, we discuss the state-of-the-art techniques for the physico-chemical characterisation of nanoparticle-protein complexes in the biological environment with particular emphasis on their impact on the efficiency and safety of a new generation of nanomedicines. We also highlight the barriers faced by nanomedicines for effective targeting and delivery in vivo

The effect of the protein corona on the interaction between nanoparticles and lipid bilayers

<p>Characteristics of included studies (Maxilla & Mandible).</p

Click dendrimer-Pd nanoparticle assemblies as enzyme mimics: catalytic o-phenylenediamine oxidation and application in colorimetric H2O2 detection

International audienceDendrimers have already been successfully used in nanoparticle (NP) catalysis for many years, particularly for Pd NP protection towards carbon-carbon coupling reactions. In this paper, assemblies between Pd nanoparticles and two generations of &quot;click&quot; dendrimers, with 27 (dendrimer-1) and 81 (dendrimer-2) triethylene glycol (TEG) termini, respectively, are examined for catalytic peroxidase-like oxidative activity. This catalysis is investigated with o-phenylenediamine (OPD) and H2O2 as the substrates in water, displaying different colours. The dendritic effect is negative upon increasing generation, i.e., dendrimer 1-Pd nanoparticles show the best results, providing values with a V-max of 1.49 x 10(-9) M s(-1) and K-m of 3.02 mM as obtained with the Michaelis-Menten model. The detection limit is 0.5 mu M of H2O2 detection with dendrimer-1-PdNPs, and both dendrimer-Pd nanocatalysts exhibit excellent robustness of catalytic activity, with a water-dispersive state being stable for at least 2 months, thus showing promise as a mimic for peroxidase catalysts

Effect of hybrid SiO2@Ag nanoparticles with raspberry-like morphology on the excited states of the photosensitizers Rose Bengal and riboflavin

Metal nanoparticles (NPs) can strongly affect the photophysics of organic molecules through different mechanisms. To investigate the effect of silver nanomaterials on the triplet state dynamics of the photosensitizers riboflavin (Rf) and Rose Bengal (RB2−), we have here synthesized core–shell silica silver nanoparticles with raspberry-like morphology (SiO2@Ag NPs). For the synthesis of SiO2@Ag NPs from SiO2 nanoparticles a new combination of reported strategies was employed. The synthetic methodology involves in a first step SnCl2 as a precursor to obtain a homogeneous deposition of silver nuclei on colloidal silica spheres. In a second step, the growth of the silver nanoparticles is mediated by the photochemically generated ketyl radical of the substituted benzoin Irgacure-2959 (I-2959). Both Rf and RB2− dyes are adsorbed on the nanoparticles. Transient absorption spectroscopy experiments showed that there is a charge transfer process from the excited state of the adsorbed Rf to the silver nanoparticles. However, no similar reaction is observed for RB2−. These results are explained in terms of the expected equilibrium constants of the electron transfer for both dyes.Fil: Martinez Porcel, Joaquin Emiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; Argentina. Centro de Investigacion Cooperativa En Biomateriales.; EspañaFil: Rivas Aiello, Maria Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; ArgentinaFil: Arce, Valeria Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Ópticas. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigaciones Ópticas. Universidad Nacional de La Plata. Centro de Investigaciones Ópticas; ArgentinaFil: Di Silvio, Desire. Centro de Investigacion Cooperativa En Biomateriales.; EspañaFil: Moya, Sergio Eduardo. Centro de Investigacion Cooperativa En Biomateriales.; España. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Martire, Daniel Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; Argentin

Ferrocenyl-terminated polyphenylene-type "click" dendrimers as supports for efficient gold and palladium nanocatalysis

International audienceAlthough dendrimer supports have been known as key parts of nanocatalysts, the capability of rigid dendrimers for this function has not yet been reported. Here, the study is focused on ferrocenylmethylenetriazolyl-terminated dendrimers (FcMTPD) as supports of remarkably efficient nanogold and nanopalladium catalysts. A biphasic system is elaborated to evaluate the catalytic activity of FcMTPD-supported Au and Pd nanoparticles (NPs) for the reduction of 4-nitrophenol to 4-aminophenol by NaBH4 at 20 degrees C, and FcMTPD-supported PdNPs are found to be the best nanocatalysts with a rate constant k(app) = 7.8 x 10(-2) s(-1). Excellent catalytic results are also obtained in this reaction for FcMTPD-supported AuNPs with a rate constant k(app) = 5.6 x 10(-2) s(-1). For both Pd NPs and AuNPs, the kinetic results are shown to strongly depend on the method of preparation of these NPs that influences the NP size and thus their catalytic efficiency. The FcMTPD-stabilized PdNPs are easily recovered and reused at least 13 times, and their catalytic performance displays only a slight decrease during the first seven runs

Ultrasensitive detection of SARS-CoV-2 spike protein by graphene field-effect transistors

COVID-19, caused by the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), originated a global health crisis, causing over 2 million casualties and altering human daily life all over the world. This pandemic emergency revealed the limitations of current diagnostic tests, highlighting the urgency to develop faster, more precise and sensitive sensors. Graphene field effect transistors (GFET) are analytical platforms that enclose all these requirements. However, the design of a sensitive and robust GFET is not a straightforward objective. In this work, we report a GFET array biosensor for the detection of SARS-CoV-2 spike protein using the human membrane protein involved in the virus internalisation: angiotensin-converting enzyme 2 (ACE2). By finely controlling the graphene functionalisation, by tuning the Debye length, and by deeply characterising the ACE2-spike protein interactions, we have been able to detect the target protein with an extremely low limit of detection (2.94 aM). This work set the basis for a new class of analytical platforms, based on human membrane proteins, with the potential to detect a broad variety of pathogens, even before their isolation, being a powerful tool in the fight against future pandemics