A study of complexation and biological fate of polyethyleneimine-siRNA polyplexes in vitro and in vivo by fluorescence correlation spectroscopy and positron emission tomography imaging.

A deeper knowledge on the formation and biological fate of polymer based gene vectors is needed for their translation into therapy. Here, polyplexes of polyethyleneimine (PEI) and silencing RNA (siRNA) are formed with theoretical N/P ratios of 2, 4 and 12. Fluorescence correlation spectroscopy (FCS) is used to study the formation of polyplexes from fluorescently labelled PEI and siRNA. FCS proves the presence of free PEI. From the analysis of the autocorrelation functions it was possible to determine the actual stoichiometry of polyplexes. FCS and fluorescence cross correlation spectroscopy (FCCS) are used to follow the fate of the polyplexes intracellularly. Polyplexes disassemble after 1 day inside cells. Positron emission tomography (PET) studies are conducted with radiolabelled polyplexes prepared with siRNA or PEI labelled with 2,3,5,6-tetrafluorophenyl 6-[18F]-fluoronicotinate ([18F]F-PyTFP). PET studies in healthy mice show that [18F]siRNA/PEI and siRNA/[18F]PEI polyplexes show similar biodistribution patterns with limited circulation in the bloodstream and accumulation in the liver. Higher activity for [18F]PEI in the kidney and bladder suggests the presence of free PEI.

Luminescent Carbon Nanodots Doped with Gadolinium (III): Purification Criteria, Chemical and Biological Characterization of a New Dual Fluorescence/MR Imaging Agent.

Carbon Dots (CDs) are luminescent quasi-spherical nanoparticles, possessing water solubility, high biocompatibility, and tunable chemical and physical properties for a wide range of applications, including nanomedicine and theranostics. The evaluation of new purification criteria, useful to achieve more reliable CDs, free from the interference of artifacts, is currently an object of debate in the field. Here, new CDs doped with gadolinium (Gd (III)), named Gd@CNDs, are presented as multifunctional probes for Magnetic Resonance Imaging (MRI). This new system is a case of study, to evaluate and/or combine different purification strategies, as a crucial approach to generate CDs with a better performance. Indeed, these new amorphous Gd@CNDs display good homogeneity, and they are free from emissive side products. Gd@CNDs (7-10 nm) contain 7% of Gd (III) w/w, display suitable and stable longitudinal relaxivity (r1 ) and with emissive behavior, therefore potentially useful for both MR and fluorescence imaging. They show good biocompatibility in both cellular and in vivo studies, cell permeability, and the ability to generate contrast in cellular pellets. Finally, MRI recording T1 -weighted images on mice after intravenous injection of Gd@CNDs, show signal enhancement in the liver, spleen, and kidney 30 min postinjection.

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.

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.

A Complete In Vitro Toxicological Assessment of the Biological Effects of Cerium Oxide Nanoparticles: From Acute Toxicity to Multi-Dose Subchronic Cytotoxicity Study.

Engineered nanomaterials (ENMs) are of significant relevance due to their unique properties, which have been exploited for widespread applications. Cerium oxide nanoparticles (CeO2-NPs) are one of most exploited ENM in the industry due to their excellent catalytic and multi-enzyme mimetic properties. Thus, the toxicological effects of these ENMs should be further studied. In this study, the acute and subchronic toxicity of CeO2-NPs were assessed. First, an in vitro multi-dose short-term (24 h) toxicological assessment was performed in three different cell lines: A549 and Calu3 were used to represented lung tissue and 3T3 was used as an interstitial tissue model. After that, a sub-chronic toxicity assessment (90 days) of these NPs was carried out on a realistic and well-established reconstituted primary human airway epithelial model (MucilAir™), cultured at the Air-Liquid Interface (ALI), to study the long-term effects of these particles. Results showed minor toxicity of CeO2-NPs in acute exposures. However, in subchronic exposures, cytotoxic and inflammatory responses were observed in the human airway epithelial model after 60 days of exposure to CeO2-NPs. These results suggest that acute toxicity approaches may underestimate the toxicological effect of some ENMs, highlighting the need for subchronic toxicological studies in order to accurately assess the toxicity of ENM and their cumulative effects in organisms.

Kinetics of the thermal response of poly(N-isopropylacrylamide co methacrylic acid) hydrogel microparticles under different environmental stimuli: A time-lapse NMR study.

HYPOTHESIS: Hydrogels of N-isopropylacrylamide and methacrylic acid (P(NIPAm-co-MAA)) display pH sensitivity and complex positively charged molecules through carboxylate groups, while having a critical solution temperature at which they reduce in volume and dehydrate. We aimed to elucidate how the responsiveness of MAA to environmental changes alters PNIPAm hydrogels at the molecular level using nuclear magnetic resonance (NMR). Time-lapse NMR allows us to follow the evolution of NMR signal under a temperature stimulus, providing unique information on conformational freedom of the hydrogel polymers. EXPERIMENTS: We used time-lapse NMR to follow the evolution of the NMR signal with time over a temperature change from 25 to 40°C and to study the swelling/deswelling kinetics of P(NIPAm-co-MAA) microgels at different pH values and ionic strengths, and in the presence of positively charged molecules complexing carboxylate groups. FINDINGS: At acid pH, hydrogel collapse is favored over neutral pH, and at basic pH the carboxylates remain steadily hydrated during temperature increase. Increasing ionic strength results in a faster, more effective collapse than decreasing pH. Complexation of medium-sized molecules with several charges (spermine, spermidine) causes a faster collapse than complexation with large molecular weight poly(allylamine) hydrochloride, but similar to the collapse effected by large poly(diallyldimethylammonium) chloride. This work opens new perspectives to using time-lapse NMR to study thermoresponsive systems that respond to multiple stimuli, with particular relevance in designing hydrogels for drug delivery.

A study of the complex interaction between poly allylamine hydrochloride and negatively charged poly(N-isopropylacrylamide-co-methacrylic acid) microgels.

Negatively charged poly(N-isopropylacrylamide-co-methacrylic acid) (P(NIPAm-co-MAA)) microgels undergo size changes in response to changes in temperature and pH. Complexation of these microgels with positively charged polyelectrolytes can greatly affect their physical properties and their capacity for encapsulating active molecules. Here we study the interaction between (P(NIPAm-co-MAA)) microgels and a model positively charged polyelectrolyte, poly allylamine hydrochloride (PAH), with different molecular weights. Experiments were conducted at temperatures below and above the lower critical solution temperature (LCST) of the microgel (30-32 °C), at 20 and 40 °C, respectively, and for PAH at molecular weights of 15, 50, and 140 kDa. Below the LCST, dynamic light scattering and zeta potential measurements with molecular simulation show that for the 15 kDa PAH there is preferential accumulation of PAH inside the microgel, whereas for the higher molecular weight PAH, the polyelectrolyte deposits mainly on the microgel surface. Above the LCST, PAH is preferentially located on the surface of the microgels for all molecular weights studied as a result of charge segregation in the hydrogels. Confocal scanning laser microscopy and flow cytometry were used to quantify rhodamine labelled PAH associated with the microgel. Isothermal titration calorimetry studies give insight into the thermodynamics of the interaction of PAH with the hydrogels, and how this interaction is affected by the molecular weight of PAH. Finally, microgels with encapsulated doxorubicin were exposed to PAH, revealing that the drug is displaced from the microgel by the PAH chains.

Self-assembly of poly(allylamine)/siRNA nanoparticles, their intracellular fate and siRNA delivery.

Silencing RNA (siRNA) technologies attract significant interest as a therapeutic tool for a large number of diseases. However, the medical translation of this technology is hampered by the lack of effective delivery vehicles for siRNAs in cytosol that prevent their degradation in the bloodstream. The use of molecular complexes based on polyamines have great potential for siRNA delivery as polyamines can protect the siRNA during circulation and at the same time favor siRNA translocation in cytosol. Here, nanoparticles are prepared by complexation of poly(allylamine hydrochloride) (PAH) and siRNA varying the ratio of nitrogen groups from PAH to phosphate groups from siRNA (N/P ratio). Nanoparticles are characterized by transmission electron microscopy and dynamic light scattering. The stability of complexes of green rhodamine labelled PAH (G-PAH) and Cy5 labelled siRNA (R-siRNA) at different pHs and in cell media is studied by fluorescence cross-correlation spectroscopy (FCCS). FCCS studies show that the nanoparticles are stable at physiological pH and in cell media but they disassemble at acidic pH. An optimal N/P ratio of 2 is identified in terms of stability in media, degradation at endosomal pH and toxicity. The intracellular fate of the complexes is studied following uptake in A549 cells. The cross-correlation between G-PAH and R-siRNA decreases substantially 24 h after uptake, while diffusion times of siRNA decrease indicating that the complexes disassemble, liberating the siRNAs. The release of siRNAs into the cytosol is confirmed with parallel confocal laser scanning microscopy. Flow cytometry studies show that PAH/siRNA nanoparticles are effective at silencing green fluorescent protein expression at low N/P ratios at which polyethylenimine/siRNA shows no significant silencing.

Fluorescence correlation spectroscopy as a tool for the study of the intracellular dynamics and biological fate of protein corona.

In biological fluids, nanoparticles (NPs) are in contact with proteins and other biomolecules. Proteins adsorb to NPs and form a coating called a protein corona (PC). The PC is known to greatly affect the interaction of NPs with biological systems. A comprehensive knowledge of the protein nanoparticle interaction is essential to understand the biological fate of NPs and for the design of NPs for biomedicine. Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) are sensitive spectroscopy techniques that measure fluorescence intensity fluctuations of single molecules inside a femtoliter confocal volume. Both techniques are suitable for studying the formation of protein corona around NPs and for examining corona stability in situ in biological matrixes. In this review we provide a short description of FCS/FCCS and their application in PC studies, highlighting results from our work about the impact of surface chemistry of NPs on corona formation and NP intracellular fate.