Toward a Unified Description of the Electrostatic Assembly of Microgels and Nanoparticles.

The interplay of soft responsive particles, such as microgels, with nanoparticles (NPs) yields highly versatile complexes that show great potential for applications, ranging from plasmonic sensing to catalysis and drug delivery. However, the microgel-NP assembly process has not been investigated so far at the microscopic level, thus hindering the possibility of designing such hybrid systems a priori. In this work, we combine state-of-the-art numerical simulations with experiments to elucidate the fundamental mechanisms taking place when microgel-NP assembly is controlled by electrostatic interactions and the associated effects on the structure of the resulting complexes. We find a general behavior where, by increasing the number of interacting NPs, the microgel deswells up to a minimum size after which a plateau behavior occurs. This occurs either when NPs are mainly adsorbed to the microgel corona via the folding of the more external chains or when NPs penetrate inside the microgel, thereby inducing a collective reorganization of the polymer network. By varying microgel properties, such as fraction of cross-linkers or charge, as well as NP size and charge, we further show that the microgel deswelling curves can be rescaled onto a single master curve, for both experiments and simulations, demonstrating that the process is entirely controlled by the charge of the whole microgel-NP complex. Our results thus have a direct relevance in fundamental materials science and offer novel tools to tailor the nanofabrication of hybrid devices of technological interest.

Molecular origin of the two-step mechanism of gellan aggregation.

Among hydrocolloids, gellan is one of the most studied polysaccharides due to its ability to form mechanically stable gels. Despite its long-standing use, the gellan aggregation mechanism is still not understood because of the lack of atomistic information. Here, we fill this gap by developing a new gellan force field. Our simulations offer the first microscopic overview of gellan aggregation, detecting the coil to single-helix transition at dilute conditions and the formation of higher-order aggregates at high concentration through a two-step process: first, the formation of double helices and then their assembly into superstructures. For both steps, we also assess the role of monovalent and divalent cations, complementing simulations with rheology and atomic force microscopy experiments and highlighting the leading role of divalent cations. These results pave the way for future use of gellan-based systems in a variety of applications, from food science to art restoration.

Responsivity of Fractal Nanoparticle Assemblies to Multiple Stimuli: Structural Insights on the Modulation of the Optical Properties.

Multi-responsive nanomaterials based on the self-limited assembly of plasmonic nanoparticles are of great interest due to their widespread employment in sensing applications. We present a thorough investigation of a hybrid nanomaterial based on the protein-mediated aggregation of gold nanoparticles at varying protein concentration, pH and temperature. By combining Small Angle X-ray Scattering with extinction spectroscopy, we are able to frame the morphological features of the formed fractal aggregates in a theoretical model based on patchy interactions. Based on this, we established the main factors that determine the assembly process and their strong correlation with the optical properties of the assemblies. Moreover, the calibration curves that we obtained for each parameter investigated based on the extinction spectra point out to the notable flexibility of this nanomaterial, enabling the selection of different working ranges with high sensitivity. Our study opens for the rational tuning of the morphology and the optical properties of plasmonic assemblies to design colorimetric sensors with improved performances.

Unveiling the interaction of protein fibrils with gold nanoparticles by plasmon enhanced nano-spectroscopy.

The development of various degenerative diseases is suggested to be triggered by the uncontrolled organisation and aggregation of proteins into amyloid fibrils. For this reason, there are ongoing efforts to develop novel agents and approaches, including metal nanoparticle-based colloids, that dissolve amyloid structures and prevent pathogenic protein aggregation. In this contribution, the role of gold nanoparticles (AuNPs) in degrading amyloid fibrils of the model protein lysozyme is investigated. The amino acid composition of fibril surfaces before and after the incubation with AuNPs is determined at the single fibril level by exploiting the high spatial resolution and sensitivity provided by tip-enhanced and surface-enhanced Raman spectroscopies. This combined spectroscopic approach allows to reveal the molecular mechanisms driving the interaction between fibrils and AuNPs. Our results provide an important input for the understanding of amyloid fibrils and could have a potential translational impact on the development of strategies for the prevention and treatment of amyloid-related diseases.

Effect of 1-MHz ultrasound on the proinflammatory interleukin-6 secretion in human keratinocytes.

Keratinocytes, the main cell type of the skin, are one of the most exposed cells to environmental factors, providing a first defence barrier for the host and actively participating in immune response. In fact, keratinocytes express pattern recognition receptors that interact with pathogen associated molecular patterns and damage associated molecular patterns, leading to the production of cytokines and chemokines, including interleukin (IL)-6. Herein, we investigated whether mechanical energy transported by low intensity ultrasound (US) could generate a mechanical stress able to induce the release of inflammatory cytokine such IL-6 in the human keratinocyte cell line, HaCaT. The extensive clinical application of US in both diagnosis and therapy suggests the need to better understand the related biological effects. Our results point out that US promotes the overexpression and secretion of IL-6, associated with the activation of nuclear factor-κB (NF-κB). Furthermore, we observed a reduced cell viability dependent on exposure parameters together with alterations in membrane permeability, paving the way for further investigating the molecular mechanisms related to US exposure.

Assembling patchy plasmonic nanoparticles with aggregation-dependent antibacterial activity.

We realise an antibacterial nanomaterial based on the self-limited assembly of patchy plasmonic colloids, obtained by adsorption of lysozyme to gold nanoparticles. The possibility of selecting the size of the assemblies within several hundred nanometres allows for tuning their optical response in a wide range of frequencies from visible to near infrared. We also demonstrate an aggregation-dependent modulation of the catalytic activity, which results in an enhancement of the antibacterial performances for assemblies of the proper size. The gained overall control on structure, optical properties and biological activity of such nanomaterial paves the way for the development of novel antibacterial nanozymes with promising applications in treating multi drug resistant bacteria.

In vitro analysis of the trajectories of adhesive microbubbles approaching endothelial cells.

Adhesion is a key process when ultrasound contrast agents, i.e. microbubbles, approach pathological tissues. A way to accomplish tumour targeting is to tether surface engineered microbubbles to endothelial cells of the up-regulated vascularization of cancer tissues. This can be achieved by coupling the microbubbles surface with the Arginine-Glycine-Aspartate, RGD, sequence. Such molecule interacts with the integrin receptors placed on the endothelial cells. Stability and trajectories of RGD modified lipid shelled MBs have been analysed in vitro using microchannels coated with human umbilical vein endothelial cells, HUVEC. In the microchannels realistic conditions, close to the physiological ones, were reproduced replicating shear rate, roughness comparable to the endothelium and channel size mimicking the postcapillary venules. In these conditions, the analysis of the trajectories close to the walls highlights a substantial difference between the modified MBs and the plain ones. Moreover, MBs adhesion has dynamic features recalling the motion of neutrophils engaged near the substrate such as rolling, translations and transient detachments. These findings are useful for the optimization of in vivo imaging and targeting functions.

Exploring the Potentiality of a SERS-Active pH Nano-Biosensor.

The merging of the molecular specificity of Raman spectroscopy with the extraordinary optical properties of metallic nanoarchitectures is at the heart of Surface Enhanced Raman Spectroscopy (SERS), which in the last few decades proved its worth as powerful analytical tool with detection limits pushed to the single molecule recognition. Within this frame, SERS-based nanosensors for localized pH measurements have been developed and employed for a wide range of applications. Nevertheless, to improve the performances of such nanosensors, many key issues concerning their assembling, calibration and stability, that could significantly impact on the outcome of the pH measurements, need to be clarified. Here, we report on the detailed characterization of a case study SERS-active pH nanosensor, based on the conjugation of gold nanoparticles with the pH-sensitive molecular probe 4-mercaptobenzoic acid (4MBA). We analyzed and optimized all the aspects of the synthesis procedure and of the operating conditions to preserve the sensor stability and provide the highest responsiveness to pH. Exploiting the dependence of the SERS spectrum on the protonation degree of the carboxylic group at the edge of the 4MBA molecules, we derived a calibration curve for the nanosensor. The extrapolated working point, i.e., the pH value corresponding to the highest sensitivity, falls at pH 5.6, which corresponds to the pKa value of the molecule confined at the nanoparticle surface. A shift of the pKa of 4MBA, observed on the molecules confined at the nanostructured interface respect to the bulk counterpart, unveils the opportunity to assembly a SERS-based pH nanosensor with the ability to select its working point in the sensitivity region of interest, by acting on the nanostructured surface on which the molecular probe is confined. As a proof-of-concept, the nanosensor was successfully employed to measure the extracellular pH of normal and cancer cells, demonstrating the capability to discriminate between them.

PLGA based particles as "drug reservoir" for antitumor drug delivery: characterization and cytotoxicity studies.

Doxorubicin (DOX) is commonly used to treat several tumor types, but its severe side effects, primarily cardiotoxicity, represent a major limitation for its use in clinical settings. In this study we developed and characterized biodegradable and stable poly(D,L-lactic-co-glycolic) acid (PLGA) submicrocarriers employing an osmosis-based patented methodology, which allowed to optimize the drug loading efficiency up to 99%. Proceeding from this, we evaluated on MCF-7, a human breast cancer cell line, the ability of PLGA to promote the internalization of DOX and to improve its cytotoxicity in vitro. We found that the in vitro uptake efficiency is dramatically increased when DOX is loaded within PLGA colloidal carriers, which adhere to the cell membrane behaving as an efficient drug reservoir. In fact, the particles provide a diffusion-driven, sustained release of DOX across the cell membrane, resulting in high drug concentration. Accordingly, the cytotoxic analysis clearly showed that DOX-loaded PLGA exhibit a lower 50% inhibitory concentration than free DOX. The decay time of cell viability was successfully compared with DOX diffusion time constant from PLGA. The overall in vitro results highlight the potential of DOX-loaded PLGA particles to be employed as vectors with improved antitumor efficacy.

Phase Change Ultrasound Contrast Agents with a Photopolymerized Diacetylene Shell.

Phase change contrast agents for ultrasound (US) imaging consist of nanodroplets (NDs) with a perfluorocarbon (PFC) liquid core stabilized with a lipid or a polymer shell. Liquid ↔ gas transition, occurring in the core, can be triggered by US to produce acoustically active microbubbles (MBs) in a process named acoustic droplet vaporization (ADV). MB shells containing polymerized diacetylene moiety were considered as a good trade off between the lipid MBs, showing optimal attenuation, and the polymeric ones, displaying enhanced stability. This work reports on novel perfluoropentane and perfluorobutane NDs stabilized with a monolayer of an amphiphilic fatty acid, i.e. 10,12-pentacosadiynoic acid (PCDA), cured with ultraviolet (UV) irradiation. The photopolymerization of the diacetylene groups, evidenced by the appearance of a blue color due to the conjugation of ene-yne sequences, exhibits a chromatic transition from the nonfluorescent blue color to a fluorescent red color when the NDs are heated or the pH of the suspension is basic. An estimate of the molecular weights reached by the polymerized PCDA in the shell, poly(PCDA), has been obtained using gel permeation chromatography and MALDI-TOF mass spectrometry. The poly(PCDA)/PFC NDs show good biocompatibility with fibroblast cells. ADV efficiency and acoustic properties before and after the transition were tested using a 1 MHz probe, revealing a resonance frequency between 1 and 2 MHz similar to other lipidic MBs. The surface of PCDA shelled NDs can be easily modified without influencing the stability and the acoustic performances of droplets. As a proof of concept we report on the conjugation of cyclic RGD and PEG chains of the particles to support targeting ability toward endothelial cells.

Complex interfaces in "phase-change" contrast agents.

In this paper we report on the study of the interface of hybrid shell droplets encapsulating decafluoropentane (DFP), which exhibit interesting potentialities for ultrasound (US) imaging. The fabrication of the droplets is based on the deposition of a dextran methacrylate layer onto the surface of surfactants. The droplets have been stabilized against coalescence by UV curing, introducing crosslinks in the polymer layer and transforming the shell into an elastomeric membrane with a thickness of about 300 nm with viscoelastic behaviour. US irradiation induces the evaporation of the DFP core of the droplets transforming the particles into microbubbles (MBs). The presence of a robust crosslinked polymer shell introduces an unusual stability of the droplets also during the core phase transition and allows the recovery of the initial droplet state after a few minutes from switching off US. The interfacial tension of the droplets has been investigated by two approaches, the pendant drop method and an indirect method, based on the determination of the liquid ↔ gas transition point of DFP confined in the droplet core. The re-condensation process has been followed by capturing images of single MBs by confocal microscopy. The time evolution of MB relaxation to droplets was analysed in terms of a modified Church model to account for the structural complexity of the MB shell, i.e. a crosslinked polymer layer over a layer of surfactants. In this way the microrheology parameters of the shell were determined. In a previous paper (Chem. Commun., 2013, 49, 5763-5765) we showed that these systems could be used as ultrasound contrast agents (UCAs). In this work we substantiate this view assessing some key features offered by the viscoelastic nature of the droplet shell.

Monosialoganglioside-GM1 triggers binding of the amyloid-protein salmon calcitonin to a Langmuir membrane model mimicking the occurrence of lipid-rafts.

GM1 ganglioside is known to be involved in the amyloid-associated diseases and it is a crucial factor for the assembly of amyloid proteins on lipid-rafts, which are lipid structures located on the synaptic plasma membranes. Due to its slow aggregation rate, we employed salmon calcitonin (sCT) as a suitable probe representative of amyloid proteins, to study the interaction between this class of proteins and a membrane model. Here, we prepared a neuronal membrane model by depositing onto mica two Langmuir-Blodgett films in liquid-condensed phase: the outer monolayer was characterized by high content of GM1 (50%) and minority parts of cholesterol and POPC (25-25%), while the inner one by plain POPC. To deeply investigate the interaction of sCT with this model and the role-played by GM1, we prepared the outer leaflet adding sCT at a concentration such that the number of proteins equals that of GM1. Atomic Force Microscopy revealed the occurrence of two distinct kinds of flat surfaces, with globular aggregates localized exclusively on top of the highest one. To unravel the nature of the interaction, we studied by ζ-potential technique liposomes composed as the outer leaflet of the model. Results demonstrated that an electrostatic interaction sCT-GM1 occurred. Finally, to investigate the interaction thermodynamics between sCT and the outer leaflet, Langmuir films as the outer monolayer and containing increasing content of sCT were studied by compression isotherms and Brewster Angle Microscopy experiments. Based on the all body of results we propose an interaction model where GM1 plays a pivotal role.