Unveiling Liquid-Phase Exfoliation of Graphite and Boron Nitride Using Fluorescent Dyes Through Combined Experiments and Simulations.

Liquid-phase exfoliation (LPE) in aqueous solutions provides a simple, scalable, and green approach to produce 2D materials. By combining atomistic simulations with exfoliation experiments, the interaction between a surfactant and a 2D layer at the molecular scale can be better understood. In this work, two different dyes, corresponding to rhodamine B base (Rbb) and to a phenylboronic acid BODIPY (PBA-BODIPY) derivative, are employed as dispersants to exfoliate graphene and hexagonal boron nitride (hBN) through sonication-assisted LPE. The exfoliated 2D sheets, mostly as few-layers, exhibit good quality and high loading of dyes. Using molecular dynamics (MD) simulations, the binding free energies are calculated and the arrangement of both dyes on the layers are predicted. It has been found that the dyes show a higher affinity toward hBN than graphene, which is consistent with the higher yields of exfoliated hBN. Furthermore, it is demonstrated that the adsorption behavior of Rbb molecules on graphene and hBN is quite different compared to PBA-BODIPY.

Spotting Local Environments in Self-Assembled Monolayer-Protected Gold Nanoparticles.

Organic-inorganic (O-I) nanomaterials are versatile platforms for an incredible high number of applications, ranging from heterogeneous catalysis to molecular sensing, cell targeting, imaging, and cancer diagnosis and therapy, just to name a few. Much of their potential stems from the unique control of organic environments around inorganic sites within a single O-I nanomaterial, which allows for new properties that were inaccessible using purely organic or inorganic materials. Structural and mechanistic characterization plays a key role in understanding and rationally designing such hybrid nanoconstructs. Here, we introduce a general methodology to identify and classify local (supra)molecular environments in an archetypal class of O-I nanomaterials, i.e., self-assembled monolayer-protected gold nanoparticles (SAM-AuNPs). By using an atomistic machine-learning guided workflow based on the Smooth Overlap of Atomic Positions (SOAP) descriptor, we analyze a collection of chemically different SAM-AuNPs and detect and compare local environments in a way that is agnostic and automated, i.e., with no need of a priori information and minimal user intervention. In addition, the computational results coupled with experimental electron spin resonance measurements prove that is possible to have more than one local environment inside SAMs, being the thickness of the organic shell and solvation primary factors in the determining number and nature of multiple coexisting environments. These indications are extended to complex mixed hydrophilic-hydrophobic SAMs. This work demonstrates that it is possible to spot and compare local molecular environments in SAM-AuNPs exploiting atomistic machine-learning approaches, establishes ground rules to control them, and holds the potential for the rational design of O-I nanomaterials instructed from data.

DNA-Directed Protein Anchoring on Oligo/Alkanethiol-Coated Gold Nanoparticles: A Versatile Platform for Biosensing Applications.

Herein, we report on a smart biosensing platform that exploits gold nanoparticles (AuNPs) functionalized through ssDNA self-assembled monolayers (SAM) and the DNA-directed immobilization (DDI) of DNA-protein conjugates; a novel, high-sensitivity optical characterization technique based on a miniaturized gel electrophoresis chip integrated with online thermal lens spectrometry (MGEC-TLS), for the high-sensitivity detection of antigen binding events. Specifically, we characterized the physicochemical properties of 20 nm AuNPs covered with mixed SAMs of thiolated single-stranded DNA and bio-repellent molecules, referred to as top-terminated oligo-ethylene glycol (TOEG6), demonstrating high colloidal stability, optimal binder surface density, and proper hybridization capacity. Further, to explore the design in the frame of cancer-associated antigen detection, complementary ssDNA fragments conjugated with a nanobody, called C8, were loaded on the particles and employed to detect the presence of the HER2-ECD antigen in liquid. At variance with conventional surface plasmon resonance detection, MGEC-TLS characterization confirmed the capability of the assay to titrate the HER2-ECD antigen down to concentrations of 440 ng/mL. The high versatility of the directed protein-DNA conjugates immobilization through DNA hybridization on plasmonic scaffolds and coupled with the high sensitivity of the MGEC-TLS detection qualifies the proposed assay as a potential, easily operated biosensing strategy for the fast and label-free detection of disease-relevant antigens.

Thiolate end-group regulates ligand arrangement, hydration and affinity for small compounds in monolayer-protected gold nanoparticles.

The ability to control the properties of monolayer protected gold nanoparticles (MPNPs) discloses unrevealed features stemming from collective properties of the ligands forming the monolayer and presents opportunities to design new materials. To date, the influence of ligand end-group size and capacity to form hydrogen bonds on structure and hydration of small MPNPs (<5 nm) has been poorly studied. Here, we show that both features determine ligands order, solvent accessibility, capacity to host hydrophobic compounds and interfacial properties of MPNPs. The polarity perceived by a radical probe and its binding constant with the monolayer investigated by electron spin resonance is rationalized by molecular dynamics simulations, which suggest that larger space-filling groups - trimethylammonium, zwitterionic and short polyethylene glycol - favor a radial organization of the thiolates, whereas smaller groups - as sulfonate - promote the formation of bundles. Zwitterionic ligands create a surface network of hydrogen bonds, which affects nanoparticle hydrophobicity and maximize the partition equilibrium constant of the probe. This study discloses the role of the chemistry of the end-group on monolayer features with effects that span from molecular- to nano-scale and opens the door to a shift in the conception of new MPNPs exploiting the end-group as a novel design motif.

Label-Free, Rapid and Facile Gold-Nanoparticles-Based Assay as a Potential Spectroscopic Tool for Trastuzumab Quantification.

Monoclonal antibody-based immunotherapy is one of the pillars of cancer treatment. However, for an efficient and personalized approach to the therapy, a quantitative evaluation of the right dose for each patient is required. In this study, we developed a simple, label-free, and rapid approach to quantify Trastuzumab, a humanized IgG1 monoclonal antibody used against human epidermal growth factor receptor 2 (HER2), overexpressed in breast cancer patients, based on localized surface plasmon resonance (LSPR). The central idea of this work was to use gold nanoparticles (AuNPs) as plasmonic scaffolds, decorated with HER2 binders mixed with oligo-ethylene glycol (OEG) molecules, to tune the surface density of the attached macromolecules and to minimize nonspecific binding events. Specifically, we characterized and optimized a self-assembled monolayer of mixed alkylthiols terminated with nitrilotriacetic acid (NTA), and OEG3 as a spacing ligand to achieve both excellent dispersibility and high reliability in protein immobilization. The successful immobilization of histidine-tagged HER2 (His-tagged HER2) on NTA via cobalt (II) chelates was demonstrated, confirming the fully functional attachment of the proteins to the AuNP surface. The proposed design demonstrates the capability of producing a clear readout that enables the transduction of a Trastuzumab/HER2 binding event into optical signals based on the wavelength shifts in LSPR, which allowed for detecting clinically relevant concentrations of Trastuzumab down to 300 ng/mL in the buffer and 2 µg/mL in the diluted serum. This strategy was found to be fast and highly specific to Trastuzumab. These findings make the present platform an auspicious tool for developing affordable bio-nanosensors.

Probing Multiscale Factors Affecting the Reactivity of Nanoparticle-Bound Molecules.

The structures and physicochemical properties of surface-stabilizing molecules play a critical role in defining the properties, interactions, and functionality of hybrid nanomaterials such as monolayer-stabilized nanoparticles. Concurrently, the distinct surface-bound interfacial environment imposes very specific conditions on molecular reactivity and behavior in this setting. Our ability to probe hybrid nanoscale systems experimentally remains limited, yet understanding the consequences of surface confinement on molecular reactivity is crucial for enabling predictive nanoparticle synthon approaches for postsynthesis engineering of nanoparticle surface chemistry and construction of devices and materials from nanoparticle components. Here, we have undertaken an integrated experimental and computational study of the reaction kinetics for nanoparticle-bound hydrazones, which provide a prototypical platform for understanding chemical reactivity in a nanoconfined setting. Systematic variation of just one molecular-scale structural parameter-the distance between reactive site and nanoparticle surface-showed that the surface-bound reactivity is influenced by multiscale effects. Nanoparticle-bound reactions were tracked in situ using 19F NMR spectroscopy, allowing direct comparison to the reactions of analogous substrates in bulk solution. The surface-confined reactions proceed more slowly than their solution-phase counterparts, and kinetic inhibition becomes more significant for reactive sites positioned closer to the nanoparticle surface. Molecular dynamics simulations allowed us to identify distinct supramolecular architectures and unexpected dynamic features of the surface-bound molecules that underpin the experimentally observed trends in reactivity. This study allows us to draw general conclusions regarding interlinked structural and dynamical features across several length scales that influence interfacial reactivity in monolayer-confined environments.

Phase Behavior of Gradient Copolymer Melts with Different Gradient Strengths Revealed by Mesoscale Simulations.

Design and preparation of functional nanomaterials with specific properties requires precise control over their microscopic structure. A prototypical example is the self-assembly of diblock copolymers, which generate highly ordered structures controlled by three parameters: the chemical incompatibility between blocks, block size ratio and chain length. Recent advances in polymer synthesis have allowed for the preparation of gradient copolymers with controlled sequence chemistry, thus providing additional parameters to tailor their assembly. These are polydisperse monomer sequence, block size distribution and gradient strength. Here, we employ dissipative particle dynamics to describe the self-assembly of gradient copolymer melts with strong, intermediate, and weak gradient strength and compare their phase behavior to that of corresponding diblock copolymers. Gradient melts behave similarly when copolymers with a strong gradient are considered. Decreasing the gradient strength leads to the widening of the gyroid phase window, at the expense of cylindrical domains, and a remarkable extension of the lamellar phase. Finally, we show that weak gradient strength enhances chain packing in gyroid structures much more than in lamellar and cylindrical morphologies. Importantly, this work also provides a link between gradient copolymers morphology and parameters such as chemical incompatibility, chain length and monomer sequence as support for the rational design of these nanomaterials.

Fluorescent Imprinted Nanoparticles for the Effective Monitoring of Irinotecan in Human Plasma.

Fluorescent, imprinted nanosized polymers for the detection of irinotecan have been synthesised using a napthalimide polymerisable derivative (2-allyl-6-[2-(aminoethyl)-amino] napthalimide) as functional monomer. The imprinted polymers contain ethylene glycol dimethacrylate (EGDMA) as a cross-linker and were prepared by high dilution radical polymerisation in dimethylsulphoxide (DMSO). The material was able to rebind irinotecan up to 18 nmol/mg with good specificity. Fluorescence emission at 525 nm (excitation at 448 nm) was quenched by increasing concentrations of irinotecan via a static mechanism and also in analytically useful environments as mixtures of human plasma and organic solvents. This allowed the direct detection of irinotecan (in the 10 nM-30 µM range) in human plasma treated with acetonitrile; the limit of detection (LOD) was 9.4 nM, with within-run variability of 10% and day-to-day variability of 13%.

Molecular Features for Probing Small Amphiphilic Molecules with Self-Assembled Monolayer-Protected Nanoparticles.

The sensing of small molecules poses the challenge of developing devices able to discriminate between compounds that may be structurally very similar. Here, attention has been paid to the use of self-assembled monolayer (SAM)-protected gold nanoparticles since they enable a modular approach to tune single-molecule affinity and selectivity simply by changing functional moieties (i.e., covering ligands), along with multivalent molecular recognition. To date, the discovery of monolayers suitable for a specific molecular target has relied on trial-and-error approaches, with ligand chemistry being the main criterion used to modulate selectivity and sensitivity. By using molecular dynamics, we showcase that either individual molecular characteristics and/or collective features such as ligand flexibility, monolayer organization, ligand local ordering, and interfacial solvent properties can also be exploited conveniently. The knowledge of the molecular mechanisms that drive the recognition of small molecules on SAM-covered nanoparticles will critically expand our ability to manipulate and control such supramolecular systems.

Iron-mediated interaction of alpha synuclein with lipid raft model membranes.

The aberrant misfolding and aggregation of alpha synuclein (αS) into toxic oligomeric species is one of the key features associated with the pathogenesis of Parkinson's disease (PD). It involves different biochemical and biophysical factors as plasma membrane binding and interaction with heavy metal ions. In the present work, atomic force microscopy (AFM) is combined with Fourier Transform Infrared Spectroscopy (FTIR) measurements to investigate the interaction of wild-type (WT) and A53T mutated alpha synuclein with artificial lipid bilayers mimicking lipid raft (LR) domains, before and after ferrous cations (Fe2+) treatment. In the absence of iron, protein monomers produce a thinning of the membrane, targeting the non-raft phase of the bilayer preferentially. On the contrary, iron actively promotes the formation of globular protein aggregates, resembling oligomers, targeted to LR domains. In both aggregation states, monomer and oligomer, the mutated A53T protein exhibits a greater and faster membrane-interaction. These results underlie a new mechanism of membrane-protein interaction in PD. The targeting of Fe2+-promoted αS oligomers to LRs might be functional for the disease and be helpful for the development of new therapeutic strategies.

Effects of primary amine-based coatings on microglia internalization of nanogels.

Nanogels represent a pivotal class of biomaterials in the therapeutic intracellular treatment of many diseases, especially those involving the central nervous system (CNS). Their biocompatibility and synergy with the biological environment encourage their cellular uptake, releasing the curative cargo in the desired area. As a main drawback, microglia are generally able to phagocytize any foreign element overcoming the blood brain barrier (BBB), including these materials, drastically limiting their bioavailability for the target cells. In this work, we investigated the opportunity to tune and therefore reduce nanogel internalization in microglia cultures, exploiting the orthogonal chemical functionalization with primary amine groups, as a surface coating strategy. Nanogels are designed by following two methods: the direct grafting of aliphatic primary amines and the linkage of -NH2 modified PEG on the nanogel surface. The latter synthesis was proposed to evaluate the combination of PEGylation with the basic nitrogen atom. The achieved results indicate the possibility of effectively modulating the uptake of nanogels, in particular limiting their internalization using the PEG-NH2 coating. This outcome could be considered a promising strategy for the development of carriers for drugs or gene delivery that could overcome microglia scavenging.

Tuning the Properties of Nanogel Surfaces by Grafting Charged Alkylamine Brushes.

Nanogels are chemically crosslinked polymeric nanoparticles endowed with high encapsulation ability, tunable size, ease of preparation, and responsiveness to external stimuli. The presence of specific functional groups on their surfaces provides an opportunity to tune their surface properties and direct their behavior. In this work, we used mesoscale modeling to describe conformational and mechanical properties of nanogel surfaces formed by crosslinked polyethylene glycol and polyethyleneimine, and grafted by charged alkylamine brushes of different lengths. Simulations show that both number of chains per area and chain length can be used to tune the properties of the coating. Properly selecting these two parameters allows switching from a hydrated, responsive coating to a dried, highly charged layer. The results also suggest that the scaling behavior of alkylamine brushes, e.g., the transition from a mushroom to semi-dilute brush, is only weakly coupled with the shielding ability of the coating and much more with its compressibility.

Graphene oxide as a 2D platform for complexation and intracellular delivery of siRNA.

The development of efficient and safe nucleic acid delivery vectors remains an unmet need holding back translation of gene therapy approaches to the bedside. Graphene oxide (GO) could help bypass such bottlenecks, thanks to its large surface area, versatile chemistry and biocompatibility, which could overall enhance transfection efficiency while abolishing some of the limitations linked to the use of viral vectors. Here, we aimed to assess the capacity of bare GO, without any further surface modification, to complex a short double-stranded nucleic acid of biological relevance (siRNA) and mediate its intracellular delivery. GO formed stable complexes with siRNA at 10 : 1, 20 : 1 and 50 : 1 GO : siRNA mass ratios. Complexation was further corroborated by atomistic molecular dynamics simulations. GO : siRNA complexes were promptly internalized in a primary mouse cell culture, as early as 4 h after exposure. At this time point, intracellular siRNA levels were comparable to those provided by a lipid-based transfection reagent that achieved significant gene silencing. The time-lapse tracking of internalized GO and siRNA evidenced a sharp decrease of intracellular siRNA from 4 to 12 h, while GO was sequestered in large vesicles, which may explain the lack of biological effects (i.e. gene silencing) achieved by GO : siRNA complexes. This study underlines the potential of non-surface modified GO flakes to act as 2D siRNA delivery platforms, without the need for cationic functionalization, but warrants further vector optimization to allow the effective release of the nucleic acid and achieve efficient gene silencing.

Mixed Fluorinated/Hydrogenated Self-Assembled Monolayer-Protected Gold Nanoparticles: In Silico and In Vitro Behavior.

Gold nanoparticles (AuNPs) covered with mixtures of immiscible ligands present potentially anisotropic surfaces that can modulate their interactions at complex nano-bio interfaces. Mixed, self-assembled, monolayer (SAM)-protected AuNPs, prepared with incompatible hydrocarbon and fluorocarbon amphiphilic ligands, are used here to probe the molecular basis of surface phase separation and disclose the role of fluorinated ligands on the interaction with lipid model membranes and cells, by integrating in silico and experimental approaches. These results indicate that the presence of fluorinated amphiphilic ligands enhances the membrane binding ability and cellular uptake of gold nanoparticles with respect to those coated only with hydrogenated amphiphilic ligands. For mixed monolayers, computational results suggest that ligand phase separation occurs on the gold surface, and the resulting anisotropy affects the number of contacts and adhesion energies with a membrane bilayer. This reflects in a diverse membrane interaction for NPs with different surface morphologies, as determined by surface plasmon resonance, as well as differential effects on cells, as observed by flow cytometry and confocal microscopy. Overall, limited changes in monolayer features can significantly affect NP surface interfacial properties, which, in turn, affect the interaction of SAM-AuNPs with cellular membranes and subsequent effects on cells.

Theoretical Importance of PVP-Alginate Hydrogels Structure on Drug Release Kinetics.

BACKGROUND: The new concepts of personalized and precision medicine require the design of more and more refined delivery systems. In this frame, hydrogels can play a very important role as they represent the best surrogate of soft living tissues for what concerns rheological properties. Thus, this paper focusses on a global theoretical approach able to describe how hydrogel polymeric networks can affect the release kinetics of drugs characterized by different sizes. The attention is focused on a case study dealing with an interpenetrated hydrogel made up by alginate and poly(N-vinyl-2-pyrrolidone). METHODS: Information about polymeric network characteristics (mesh size distribution and polymer volume fraction) is deduced from the theoretical interpretation of the rheological and the low field Nuclear Magnetic Resonance (NMR) characterization of hydrogels. This information is then, embodied in the mass balance equation whose resolution provides the release kinetics. RESULTS: Our simulations indicate the influence of network characteristics on release kinetics. In addition, the reliability of the proposed approach is supported by the comparison of the model outcome with experimental release data. CONCLUSIONS: This study underlines the necessity of a global theoretical approach in order to design reliable delivery systems based on hydrogels.

Supramolecular Tripeptide Hydrogel Assembly with 5-Fluorouracil.

In this work, we present Thioflavin T fluorescence, transmission electron microscopy (TEM), circular dichroism (CD), Fourier-transformed infrared (FT-IR), and oscillatory rheometry studies applied to an antineoplastic drug, 5-fluorouracil (5-FU), embedded in a heterochiral tripeptide hydrogel to obtain a drug delivery supramolecular system. The release of 5-fluorouracil was monitored over time by reverse-phase high-performance liquid chromatography (HPLC) and its interaction with the tripeptide assemblies was probed by all-atom molecular dynamics simulations.

Quantitative 3D determination of self-assembled structures on nanoparticles using small angle neutron scattering.

The ligand shell (LS) determines a number of nanoparticles' properties. Nanoparticles' cores can be accurately characterized; yet the structure of the LS, when composed of mixture of molecules, can be described only qualitatively (e.g., patchy, Janus, and random). Here we show that quantitative description of the LS' morphology of monodisperse nanoparticles can be obtained using small-angle neutron scattering (SANS), measured at multiple contrasts, achieved by either ligand or solvent deuteration. Three-dimensional models of the nanoparticles' core and LS are generated using an ab initio reconstruction method. Characteristic length scales extracted from the models are compared with simulations. We also characterize the evolution of the LS upon thermal annealing, and investigate the LS morphology of mixed-ligand copper and silver nanoparticles as well as gold nanoparticles coated with ternary mixtures. Our results suggest that SANS combined with multiphase modeling is a versatile approach for the characterization of nanoparticles' LS.

The interaction of ß2-microglobulin with gold nanoparticles: impact of coating, charge and size.

Gold nanoparticles (AuNPs) have been proved to be ideal scaffolds to build nanodevices whose performance can be tuned by changing their coating. In particular, the interaction of AuNPs with proteins was revealed to be highly dependent on the physico-chemical properties of the gold cluster protecting monolayer. In this work we studied the behavior of three different alkanethiolate-coated AuNPs (AT-AuNPs) when they are incubated with a model amyloidogenic protein, ß2-microglobulin (ß2m), whose clinical relevance in dialysis-related amyloidosis (DRA) and structural properties are well known. To the aim we synthesized 6-mercaptohexanoic acid-coated AuNPs (MHA-AuNPs) and (11-mercaptoundecyl)-N,N,N-trimethylammonium bromide-coated AuNPs (MUTAB-AuNPs) of 7.5 nm diameter and 3-mercaptopropionic acid-coated AuNPs (MPA-AuNPs) of 3.6 nm diameter. To study the effects of the incubation with ß2m of these NPs that differ in charge and dimension, we employed NMR, UV-vis and fluorescence spectroscopy, along with transmission electron microscopy (TEM). The three tested AuNP systems gave different results. We found that MHA-AuNPs precipitate with the protein into large agglomerates inducing ß2m unfolding, MUTAB-AuNP precipitation is triggered by the protein that remains unchanged in solution, at least at the higher considered protein/NP ratio, and MPA-AuNPs interact preferentially with a localized region of the protein that stays essentially stably dissolved. These results stress the complexity of the bio-nano interface and the relevance and viability of the fine control of NP properties to master protein-NP interactions.

Gold nanoparticles with patterned surface monolayers for nanomedicine: current perspectives.

Molecular self-assembly is a topic attracting intense scientific interest. Various strategies have been developed for construction of molecular aggregates with rationally designed properties, geometries, and dimensions that promise to provide solutions to both theoretical and practical problems in areas such as drug delivery, medical diagnostics, and biosensors, to name but a few. In this respect, gold nanoparticles covered with self-assembled monolayers presenting nanoscale surface patterns-typically patched, striped or Janus-like domains-represent an emerging field. These systems are particularly intriguing for use in bio-nanotechnology applications, as presence of such monolayers with three-dimensional (3D) morphology provides nanoparticles with surface-dependent properties that, in turn, affect their biological behavior. Comprehensive understanding of the physicochemical interactions occurring at the interface between these versatile nanomaterials and biological systems is therefore crucial to fully exploit their potential. This review aims to explore the current state of development of such patterned, self-assembled monolayer-protected gold nanoparticles, through step-by-step analysis of their conceptual design, synthetic procedures, predicted and determined surface characteristics, interactions with and performance in biological environments, and experimental and computational methods currently employed for their investigation.

Morphological control of self-assembled multivalent (SAMul) heparin binding in highly competitive media.

Tuning molecular structures of self-assembling multivalent (SAMul) dendritic cationic lipopeptides controls the self-assembled morphology. In buffer, spherical micelles formed by higher generation systems bind polyanionic heparin better than worm-like micelles formed by lower generation systems. In human serum, the binding of spherical micelles to heparin is adversely affected, while worm-like micelles maintain their relative binding ability.