Cu(I)-Glutathione Assembly Supported on ZIF-8 as Robust and Efficient Catalyst for Mild CO2 Conversions.

The Cu-glutathione (GSH) redox system, essential in biology, is designed here as a supramacromolecular assembly in which the tetrahedral 18e Cu(I) center loses a thiol ligand upon adsorption onto ZIF-8, as shown by EXAFS and DFT calculation, to generate a very robust 16e planar trigonal single-atom Cu(I) catalyst. Synergy between Cu(I) and ZIF-8, revealed by catalytic experiments and DFT, affords CO2 conversion into high-value-added chemicals with a wide scope of substrates by reaction with terminal alkynes or propargyl amines in excellent yields under mild conditions and reuse at least 10 times without significant decrease in catalytic efficiency.

Covalent functionalisation controlled by molecular design for the aptameric recognition of serotonin in graphene-based field-effect transistors.

In the last decade, solution-gated graphene field effect transistors (GFETs) showed their versatility in the development of a miniaturized multiplexed platform for electrophysiological recordings and sensing. Due to their working mechanism, the surface functionalisation and immobilisation of receptors are pivotal to ensure the proper functioning of devices. Herein, we present a controlled covalent functionalisation strategy based on molecular design and electrochemical triggering, which provide a monolayer-like functionalisation of micro-GFET arrays retaining the electronic properties of graphenes. The functionalisation layer as a receptor was then employed as the linker for serotonin aptamer conjugation. The micro-GFET arrays display sensitivity toward the target analyte in the micromolar range in a physiological buffer (PBS 10 mM). The sensor allows the in-flow real-time monitoring of serotonin transient concentrations with fast and reversible responses.

A Comparative Study of Ultrasmall Calcium Carbonate Nanoparticles for Targeting and Imaging Atherosclerotic Plaque.

Atherosclerosis is a complex disease that can lead to life-threatening events, such as myocardial infarction and ischemic stroke. Despite the severity of this disease, diagnosing plaque vulnerability remains challenging due to the lack of effective diagnostic tools. Conventional diagnostic protocols lack specificity and fail to predict the type of atherosclerotic lesion and the risk of plaque rupture. To address this issue, technologies are emerging, such as noninvasive medical imaging of atherosclerotic plaque with customized nanotechnological solutions. Modulating the biological interactions and contrast of nanoparticles in various imaging techniques, including magnetic resonance imaging, is possible through the careful design of their physicochemical properties. However, few examples of comparative studies between nanoparticles targeting different hallmarks of atherosclerosis exist to provide information about the plaque development stage. Our work demonstrates that Gd (III)-doped amorphous calcium carbonate nanoparticles are an effective tool for these comparative studies due to their high magnetic resonance contrast and physicochemical properties. In an animal model of atherosclerosis, we compare the imaging performance of three types of nanoparticles: bare amorphous calcium carbonate and those functionalized with the ligands alendronate (for microcalcification targeting) and trimannose (for inflammation targeting). Our study provides useful insights into ligand-mediated targeted imaging of atherosclerosis through a combination of in vivo imaging, ex vivo tissue analysis, and in vitro targeting experiments.

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.

Patterning of Self-Assembled Monolayers of Amphiphilic Multisegment Ligands on Nanoparticles and Design Parameters for Protein Interactions.

Functionalization of nanoparticles with specific ligands is helpful to control specific diagnostic and therapeutic responses such as protein adsorption, cell targeting, and circulation. Precision delivery critically depends on a fundamental understanding of the interplay between surface chemistry, ligand dynamics, and interaction with the biochemical environment. Due to limited atomic-scale insights into the structure and dynamics of nanoparticle-bound ligands from experiments, relationships of grafting density and ligand chemistry to observable properties such as hydrophilicity and protein interactions remain largely unknown. In this work, we uncover how self-assembled monolayers (SAMs) composed of multisegment ligands such as thioalkyl-PEG-(N-alkyl)amides on gold nanoparticles can mimic mixed hydrophobic and hydrophilic ligand coatings, including control of patterns, hydrophilicity, and specific recognition properties. Our results are derived from molecular dynamics simulations with the INTERFACE-CHARMM36 force field at picometer resolution and comparisons to experiments. Small changes in ligand hydrophobicity, via adjusting the length of the N-terminal alkyl groups, tune water penetration by multiples and control superficial ordering of alkyl chains from 0 to 70% regularity. Further parameters include the grafting density of the ligands, curvature of the nanoparticle surfaces, type of solvent, and overall ligand length, which were examined in detail. We explain the thermodynamic origin of the formation of heterogeneous patterns of multisegment ligand SAMs and illustrate how different degrees of ligand order on the nanoparticle surface affect interactions with bovine serum albumin. The resulting design principles can be applied to a variety of ligand chemistries to customize the behavior of functionalized nanoparticles in biological media and enhance therapeutic efficiency.

Intraparticle Kinetics Unveil Crowding and Enzyme Distribution Effects on the Performance of Cofactor-Dependent Heterogeneous Biocatalysts.

Multidimensional kinetic analysis of immobilized enzymes is essential to understand the enzyme functionality at the interface with solid materials. However, spatiotemporal kinetic characterization of heterogeneous biocatalysts on a microscopic level and under operando conditions has been rarely approached. As a case study, we selected self-sufficient heterogeneous biocatalysts where His-tagged cofactor-dependent enzymes (dehydrogenases, transaminases, and oxidases) are co-immobilized with their corresponding phosphorylated cofactors [nicotinamide adenine dinucleotide phosphate (NAD(P)H), pyridoxal phosphate (PLP), and flavin adenine dinucleotide (FAD)] on porous agarose microbeads coated with cationic polymers. These self-sufficient systems do not require the addition of exogenous cofactors to function, thus avoiding the extensive use of expensive cofactors. To comprehend the microscopic kinetics and thermodynamics of self-sufficient systems, we performed fluorescence recovery after photobleaching measurements, time-lapse fluorescence microscopy, and image analytics at both single-particle and intraparticle levels. These studies reveal a thermodynamic equilibrium that rules out the reversible interactions between the adsorbed phosphorylated cofactors and the polycations within the pores of the carriers, enabling the confined cofactors to access the active sites of the immobilized enzymes. Furthermore, this work unveils the relationship between the apparent Michaelis-Menten kinetic parameters and the enzyme density in the confined space, eliciting a negative effect of molecular crowding on the performance of some enzymes. Finally, we demonstrate that the intraparticle apparent enzyme kinetics are significantly affected by the enzyme spatial organization. Hence, multiscale characterization of immobilized enzymes serves as an instrumental tool to better understand the in operando functionality of enzymes within confined spaces.

In Vivo Evaluation of Multifunctional Gold Nanorods for Boron Neutron Capture and Photothermal Therapies.

The incidence and mortality of cancer demand more innovative approaches and combination therapies to increase treatment efficacy and decrease off-target side effects. We describe a boron-rich nanoparticle composite with potential applications in both boron neutron capture therapy (BNCT) and photothermal therapy (PTT). Our strategy is based on gold nanorods (AuNRs) stabilized with polyethylene glycol and functionalized with the water-soluble complex cobalt bis(dicarbollide) ([3,3'-Co(1,2-C2B9H11)2]-), commonly known as COSAN. Radiolabeling with the positron emitter copper-64 (64Cu) enabled in vivo tracking using positron emission tomography imaging. 64Cu-labeled multifunctionalized AuNRs proved to be radiochemically stable and capable of being accumulated in the tumor after intravenous administration in a mouse xenograft model of gastrointestinal cancer. The resulting multifunctional AuNRs showed high biocompatibility and the capacity to induce local heating under external stimulation and trigger cell death in heterogeneous cancer spheroids as well as the capacity to decrease cell viability under neutron irradiation in cancer cells. These results position our nanoconjugates as suitable candidates for combined BNCT/PTT therapies.

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

Although 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 °C, and FcMTPD-supported PdNPs are found to be the best nanocatalysts with a rate constant kapp = 7.8 × 10-2 s-1. Excellent catalytic results are also obtained in this reaction for FcMTPD-supported AuNPs with a rate constant kapp = 5.6 × 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.

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.

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 densities greater than 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 characterizing the cross-aggregation phase diagram of all the mixtures. Dynamic light scattering experiments were performed to evaluate the aggregation of two proteins, bovine serum albumin (BSA) and consensus tetratricopeptide repeat (CTPR), in solutions of one or both proteins. The experiments confirm our hypothesis and the simulations. These findings elucidate critical aspects of the cross-regulation of expression and aggregation of proteins exerted by the cell and on the evolutionary selection of folding and non-aggregating protein sequences, paving the way for new experimental tests.

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.

Mechanistic study of the nucleation and conformational changes of polyamines in presence of phosphate ions.

Polyamine Phosphate Nanoparticles (PANs) have great potential for the delivery of large therapeutics, such as plasmids and/or siRNAs. The formation of PANs by complexation of Poly(allylamine hydrochloride) (PAH) and phosphate ions from Phosphate Buffer (PB) was studied here, and how it is affected by the presence of phosphate ions from PB and ionic strength. From Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (DLS) the critical PB concentration for PANs formation was determined. Below this critical point, Small Angle X-ray Scattering (SAXS) studies revealed that small PAH-phosphate aggregates coexist with not complexed or weakly complexed polymer chains in solution and that the presence of the phosphate ions increases the Kuhn length of the polymer chains until that only spherical aggregates are present in solution. TEM, DLS and SAXS showed the increase of PANs size with ionic strength up to 250 mM NaCl. At higher NaCl concentrations, PANs disassemble into smaller aggregates. Isothermal Titration Calorimetry (ITC) showed that PAN formation is an exothermic process and the association of phosphates below the critical PB concentration is entropically controlled.

Impact of ConcanavalinA affinity in the intracellular fate of Protein Corona on Glucosamine Au nanoparticles.

Biological fate and toxicity of nanoparticles (NPs) are connected to the interaction between NPs and the protein corona (PC) spontaneously forming around NPs in biological matrixes. PC is a dynamic entity that confers biological identity to NPs. In this work, fluorescence cross-correlation spectroscopy (FCCS) is used to study the impact of specific interactions between the NP surface and proteins on the intracellular fate of PC. The stability of the PC formed around glucosamide-functionalized Au-NPs from ConcanavalinA (ConA) or Bovine Serum Albumin (BSA) is characterized by FCCS. The NPs show higher affinity for ConA and competitive assays show that ConA easily exchanges BSA. A549 cells are exposed to glucosamide-functionalized Au-NPs with preformed ConA and BSA PCs. Intracellularly the frequency of cross-correlation for Au NPs with ConA PC remains constant to a 70% value until 24 h while for BSA it decreases to a 15% during the same period. FCCS measurements in several locations in the cell point out a different level of aggregation for the NPs with either ConA or BSA PCs. Our results show that the affinity of NPs functionalized with a ligand with affinity for a specific protein in bulk is retained intracellularly influencing NP fate and translocation.

Application of polymersomes engineered to target p32 protein for detection of small breast tumors in mice.

Triple negative breast cancer (TNBC) is the deadliest form of breast cancer and its successful treatment critically depends on early diagnosis and therapy. The multi-compartment protein p32 is overexpressed and present at cell surfaces in a variety of tumors, including TNBC, specifically in the malignant cells and endothelial cells, and in macrophages localized in hypoxic areas of the tumor. Herein we used polyethylene glycol-polycaprolactone polymersomes that were affinity targeted with the p32-binding tumor penetrating peptide LinTT1 (AKRGARSTA) for imaging of TNBC lesions. A tyrosine residue was added to the peptide to allow for 124I labeling and PET imaging. In a TNBC model in mice, systemic LinTT1-targeted polymersomes accumulated in early tumor lesions more than twice as efficiently as untargeted polymersomes with up to 20% ID/cc at 24 h after administration. The PET-imaging was very sensitive, allowing detection of tumors as small as ∼20 mm3. Confocal imaging of tumor tissue sections revealed a high degree of vascular exit and stromal penetration of LinTT1-targeted polymersomes and co-localization with tumor-associated macrophages. Our studies show that systemic LinTT1-targeted polymersomes can be potentially used for precision-guided tumor imaging and treatment of TNBC.

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

HYPOTHESIS: It is known that nanoparticles (NPs) in a biological fluid are immediately coated by a protein corona (PC), composed of a hard (strongly bounded) and a soft (loosely associated) layers, which represents the real nano-interface interacting with the cellular membrane in vivo. In this regard, supported lipid bilayers (SLB) have extensively been used as relevant model systems for elucidating the interaction between biomembranes and NPs. Herein we show how the presence of a PC on the NP surface changes the interaction between NPs and lipid bilayers with particular care on the effects induced by the NPs on the bilayer structure. EXPERIMENTS: In the present work we combined Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) and Neutron Reflectometry (NR) experimental techniques to elucidate how the NP-membrane interaction is modulated by the presence of proteins in the environment and their effect on the lipid bilayer. FINDINGS: Our study showed that the NP-membrane interaction is significantly affected by the presence of proteins and in particular we observed an important role of the soft corona in this phenomenon.

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

AIM: With the rise in production of nanoparticles (NPs) for an ever-increasing number of applications, there is an urgent need to efficiently assess their potential toxicity. We propose a NP 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 NPs, Xenopus phenotypic scoring showed a strong correlation with cell based in vitro assays. Magnetite-cored NPs, negative for toxicity in vitro and Xenopus, were further confirmed as nontoxic in mice. CONCLUSION: The results highlight the potential of Xenopus embryo analysis as a fast screening approach for toxicity assessment of NPs, which could be introduced for the routine testing of nanomaterials.

Effect of protein corona magnetite nanoparticles derived from bread in vitro digestion on Caco-2 cells morphology and uptake.

Nanoparticles (NPs) in biological fluids immediately interact with proteins forming a biomolecular corona (PC) that imparts their biological identity. While several studies on the formation of the PC in human plasma have been reported, the PC of orally administrated NPs has been less investigated, mostly in the presence of a food matrix. In fact, food matrixes when digested are subject of several dynamic changes that will certainly affect the PC formed on the NPs. The lack of studies on this topic is clearly related to the difficulty in isolating representative PC NPs from such a complex environment. In this work magnetite NPs were added to in vitro simulated digestion simultaneously with bread and PC NPs were isolated after gastric and duodenal phases by sucrose gradient ultracentrifugation (UC). The PC NPs were characterized in terms of size and protein composition. Translocation studies were then performed on Caco-2 monolayers in a serum free environment and cell morphology was characterized by confocal microscopy. PC NPs isolated from gastric and duodenal phases were different in size, surface charge and protein corona composition. NP cellular uptake was enhanced by the digestive PC inducing morphology changes in the cell monolayer. Overall, in this work we were able to isolate PC NPs from digested fluids in the presence of a food matrix and study their biological response on Caco-2 cells.

Technical tip: high-resolution isolation of nanoparticle-protein corona complexes from physiological fluids.

Nanoparticles (NPs) in contact with biological fluids are generally coated with environmental proteins, forming a stronger layer of proteins around the NP surface called the hard corona. Protein corona complexes provide the biological identity of the NPs and their isolation and characterization are essential to understand their in vitro and in vivo behaviour. Here we present a one-step methodology to recover NPs from complex biological media in a stable non-aggregated form without affecting the structure or composition of the corona. This method allows NPs to be separated from complex fluids containing biological particulates and in a form suitable for use in further experiments. The study has been performed systematically comparing the new proposed methodology to standard approaches for a wide panel of NPs. NPs were first incubated in the biological fluid and successively recovered by sucrose gradient ultracentrifugation in order to separate the NPs and their protein corona from the loosely bound proteins. The isolated NP-protein complexes were characterized by size and protein composition through Dynamic Light Scattering, Nanoparticle Tracking Analysis, SDS-PAGE and LC-MS. The protocol described is versatile and can be applied to diverse nanomaterials and complex fluids. It is shown to have higher resolution in separating the multiple protein corona complexes from a biological environment with a much lower impact on their in situ structure compared to conventional centrifugal approaches.