A Diruthenium Metallodrug as a Potent Inhibitor of Amyloid-ß Aggregation: Synergism of Mechanisms of Action.

The physical and chemical properties of paddlewheel diruthenium compounds are highly dependent on the nature of the ligands surrounding the bimetallic core. Herein, we compare the ability of two diruthenium compounds, [Ru2Cl(D-p-FPhF)(O2CCH3)3]·H2O (1) (D-p-FPhF- = N,N'-bis(4-fluorophenyl)formamidinate) and K3[Ru2(O2CO)4]·3H2O (2), to act as inhibitors of amyloid aggregation of the Aß1-42 peptide and its peculiar fragments, Aß1-16 and Aß21-40. A wide range of biophysical techniques has been used to determine the inhibition capacity against aggregation and the possible mechanism of action of these compounds (Thioflavin T fluorescence and autofluorescence assays, UV-vis absorption spectroscopy, circular dichroism, nuclear magnetic resonance, mass spectrometry, and electron scanning microscopy). Data show that the most effective inhibitory effect is shown for compound 1. This compound inhibits fiber formation and completely abolishes the cytotoxicity of Aß1-42. The antiaggregatory capacity of this complex can be explained by a binding mechanism of the dimetallic units to the peptide chain along with π-π interactions between the formamidinate ligand and the aromatic side chains. The results suggest the potential use of paddlewheel diruthenium complexes as neurodrugs and confirm the importance of the steric and charge effects on the properties of diruthenium compounds.

Comparative Analysis of the Inhibitory Mechanism of Aß1-42 Aggregation by Diruthenium Complexes.

There is a growing interest in the search for metal-based therapeutics for protein misfolding disorders such as Alzheimer's disease (AD). A novel and largely unexplored class of metallodrugs is constituted by paddlewheel diruthenium complexes, which exhibit unusual water solubility and stability and unique coordination modes to proteins. Here, we investigate the ability of the complexes [Ru2Cl(DPhF)(O2CCH3)3]·H2O (1), [Ru2Cl(DPhF)2(O2CCH3)2]·H2O (2), and K2[Ru2(DPhF)(CO3)3]·3H2O (3) (DPhF- = N,N'-diphenylformamidinate) to interfere with the amyloid aggregation of the Aß1-42 peptide. These compounds differ in charge and steric hindrance due to the coordination of a different number of bulky ligands. The mechanisms of action of the three complexes were studied by employing a plethora of physicochemical and biophysical techniques as well as cellular assays. All these studies converge on different mechanisms of inhibition of amyloid fibrillation: complexes 1 and 2 show a clear inhibitory effect due to an exchange ligand process in the Ru2 unit aided by aromatic interactions. Complex 3 shows no inhibition of aggregation, probably due to its negative charge in solution. This study demonstrates that slight variations in the ligands surrounding the bimetallic core can modulate the amyloid aggregation inhibition and supports the use of paddlewheel diruthenium complexes as promising therapeutics for Alzheimer's disease.

Cell mechanosensing is regulated by substrate strain energy rather than stiffness.

The ability of cells to perceive the mechanical identity of extracellular matrix, generally known as mechanosensing, is generally depicted as a consequence of an intricate balance between pulling forces actuated by the actin fibers on the adhesion plaques and the mechanical reaction of the supporting material. However, whether the cell is sensitive to the stiffness or to the energy required to deform the material remains unclear. To address this important issue, here the cytoskeleton mechanics of BALB/3T3 and MC3T3 cells seeded on linearly elastic substrates under different levels of deformation were studied. In particular, the effect of prestrain on cell mechanics was evaluated by seeding cells both on substrates with no prestrain and on substrates with different levels of prestrain. Results indicated that cells recognize the existence of prestrain, exhibiting a stiffer cytoskeleton on stretched material compared to cells seeded on unstretched substrate. Cytoskeleton mechanics of cells seeded on stretched material were, in addition, comparable to those measured after the stretching of the substrate and cells together to the same level of deformation. This observation clearly suggests that cell mechanosensing is not mediated only by the stiffness of the substrate, as widely assumed in the literature, but also by the deformation energy associated with the substrate. Indeed, the clutch model, based on the exclusive dependence of cell mechanics upon substrate stiffness, fails to describe our experimental results. By modifying the clutch model equations to incorporate the dependence on the strain energy, we were able to correctly interpret the experimental evidence.

Characterization of Hyaluronic Acid-Coated PLGA Nanoparticles by Surface-Enhanced Raman Spectroscopy.

Nanoparticles (NPs) coated with hyaluronic acid (HA) seem to be increasingly promising for targeted therapy due to HA chemical versatility, which allows them to bind drugs of different natures, and their affinity with the transmembrane receptor CD-44, overexpressed in tumor cells. However, an essential aspect for clinical use of NPs is formulation stability over time. For these reasons, analytical techniques capable of characterizing their physico-chemical properties are needed. In this work, poly(lactide-co-glycolide) (PLGA) NPs with an average diameter of 100-150 nm, coated with a few 10 s of nm of HA, were synthesized. For stability characterization, two complementary investigative techniques were used: Dynamic Light Scattering (DLS) and Surface-Enhanced Raman Scattering (SERS) spectroscopy. The first technique provided information on size, polidispersity index, and zeta-potential, and the second provided a deeper insight on the NP surface chemicals, allowing distinguishing of HA-coated NPs from uncoated ones. Furthermore, in order to estimate formulation stability over time, NPs were measured and monitored for two weeks. SERS results showed a progressive decrease in the signal associated with HA, which, however, is not detectable by the DLS measurements.

Conformational consequences of NPM1 rare mutations: An aggregation perspective in Acute Myeloid Leukemia.

Often proteins association is a physiological process used by cells to regulate their growth and to adapt to different stress conditions, including mutations. In the case of a subtype of Acute Myeloid Leukemia (AML), mutations of nucleophosmin 1 (NPM1) protein cause its aberrant cytoplasmatic mislocalization (NPMc+). We recently pointed out an amyloidogenic propensity of protein regions including the most common mutations of NPMc+ located in the C-terminal domain (CTD): they were able to form, in vitro, amyloid cytotoxic aggregates with fibrillar morphology. Herein, we analyzed the conformational characteristics of several peptides including rare AML mutations of NPMc+. By means of different spectroscopic, microscopic and cellular assays we evaluated the importance of amino acid composition, among rare AML mutations, to determine amyloidogenic propensity. This study could add a piece of knowledge to the structural consequences of mutations in cytoplasmatic NPM1c+.

Dynamic Manipulation of Cell Membrane Curvature by Light-Driven Reshaping of Azopolymer.

Local curvatures on the cell membrane serve as signaling hubs that promote curvature-dependent protein interactions and modulate a variety of cellular processes including endocytosis, exocytosis, and the actin cytoskeleton. However, precisely controlling the location and the degree of membrane curvature in live cells has not been possible until recently, where studies show that nanofabricated vertical structures on a substrate can imprint their shapes on the cell membrane to induce well-defined curvatures in adherent cells. Nevertheless, the intrinsic static nature of these engineered nanostructures prevents dynamic modulation of membrane curvatures. In this work, we engineer light-responsive polymer structures whose shape can be dynamically modulated by light and thus change the induced-membrane curvatures on-demand. Specifically, we fabricate three-dimensional azobenzene-based polymer structures that change from a vertical pillar to an elongated vertical bar shape upon green light illumination. We observe that U2OS cells cultured on azopolymer nanostructures rapidly respond to the topographical change of the substrate underneath. The dynamically induced high membrane curvatures at bar ends promote local accumulation of actin fibers and actin nucleator Arp2/3 complex. The ability to dynamically manipulate the membrane curvature and analyze protein response in real-time provides a new way to study curvature-dependent processes in live cells.

Intestine-on-chip device increases ECM remodeling inducing faster epithelial cell differentiation.

An intestine-on-chip has been developed to study intestinal physiology and pathophysiology as well as intestinal transport absorption and toxicity studies in a controlled and human similar environment. Here, we report that dynamic culture of an intestine-on-chip enhances extracellular matrix (ECM) remodeling of the stroma, basement membrane production and speeds up epithelial differentiation. We developed a three-dimensional human intestinal stromal equivalent composed of human intestinal subepithelial myofibroblasts embedded in their own ECM. Then, we cultured human colon carcinoma-derived cells in both static and dynamic conditions in the opportunely designed microfluidic system until the formation of a well-oriented epithelium. This low cost and handy microfluidic device allows to qualitatively and quantitatively detect epithelial polarization and mucus production as well as monitor barrier function and ECM remodeling after nutraceutical treatment.

A three-dimensional microfluidized liver system to assess hepatic drug metabolism and hepatotoxicity.

In this study, we propose the design and fabrication of a liver system on a chip. We first chose the most suitable three-dimensional liver-like model between cell spheroids and microtissue precursors, both based on the use of hepatocellular carcinoma cells (HepG2) to provide proof-of-concept data. Spheroids displayed high cell density but low expression of the typical hepatic biomarkers, whereas microtissue precursors showed stable viability and function over the entire culture time. The two liver-like models were compared in terms of cell viability, function, metabolism, and the P-glycoprotein 1 (P-gp) transport-protein expression with the microtissue precursors showing the best performance. Thus, we cultured them into a microfluidic biochip featured with three parallel channels shaped to mimic the hepatic sinusoids. To assess the detoxification potential of the microtissue-loaded biochip we challenged it with a model molecule (ethanol) at different concentrations and time points. Ethanol cytotoxicity was detected by a noninvasive measurement of cell viability based on cell autofluorescence. As expected, a dose-dependent decrease of albumin and urea secretion was observed in the ethanol-treated samples. We believe that the described totally human-derived platform, suitable for integration into a multiorgan microfluidic system, can provide a consistent innovative platform for drug development and toxicity studies.

3D stromal tissue equivalent affects intestinal epithelium morphogenesis in vitro.

Current in vitro models of human intestine commonly fail to mimic the complex intestinal functions and features required for drug development and disease research. Here, we deeply investigate the interaction existing between epithelium and the underneath stroma, and its role in the epithelium morphogenesis. We cultured human intestinal subepithelial myofibroblasts (ISEMFs) in two different 3D configurations: 3D-collagen gel equivalent (3D-CGE) and 3D cell-synthetized stromal equivalent (3D-CSSE). The 3D-CGEs were obtained by means of the traditional collagen-based cell technique and the 3D-CSSE were obtained by bottom-up tissue engineering strategy. The biophysical properties of both 3D models with regard to cell growth and composition (via histological analysis, immunofluorescence, and multiphoton imaging) were assessed. Then, human colorectal adenocarcinoma cell line (CaCo-2) was cultured on both the 3D constructs in order to produce the intestinal model. We identified higher levels of matrix-associated proteins from ISEMFs cultured in 3D-CSSE compared to 3D-CGE. Furthermore, multiphoton investigation revealed differences in the collagen network architecture in both models. At last, the more physiologically relevant stromal environment of the 3D-CSSE drove the CaCo-2 cell differentiation toward the four different type of intestinal epithelial cells (absorptive, mucus-secretory, enteroendocrine, and Paneth) phenotype and promotes, in contrast to the 3D-CGE, the production of the basement membrane. Taken together, these results highlight a fundamental role of the 3D stromal environment in addressing a correct epithelium morphogenesis as well as epithelial-stromal interface establishment.

Design, Synthesis and Characterization of Novel Co-Polymers Decorated with Peptides for the Selective Nanoparticle Transport across the Cerebral Endothelium.

The development of new strategies for enhancing drug delivery to the brain represents a major challenge in treating cerebral diseases. In this paper, we report on the synthesis and structural characterization of a biocompatible nanoparticle (NP) made up of poly(lactic-co-glycolic acid) (PLGA)-polyethylene glycol (PEG) co-polymer (namely PELGA) functionalized with the membranotropic peptide gH625 (gH) and the iron-mimicking peptide CRTIGPSVC (CRT) for transport across the blood-brain barrier (BBB). gH possesses a high translocation potency of the cell membrane. Conversely, CRT selectively recognizes the brain endothelium, which interacts with transferrin (Tf) and its receptor (TfR) through a non-canonical ligand-directed mechanism. We hypothesize that the delivery across the BBB of PELGA NPs should be efficiently enhanced by the NP functionalization with both gH and CRT. Synthesis of peptides and their conjugation to the PLGA as well as NP physical-chemical characterization are performed. Moreover, NP uptake, co-localization, adhesion under dynamic conditions, and permeation across in vitro BBB model are evaluated as a function of gH/CRT functionalization ratio. Results establish that the cooperative effect of CRT and gH may change the intra-cellular distribution of NPs and strengthen NP delivery across the BBB at the functionalization ratio 33% gH⁻66% CRT.

Shuttle-mediated nanoparticle transport across an in vitro brain endothelium under flow conditions.

The blood brain barrier (BBB) represents a challenge in the development of new nano-delivery systems able to reach the central nervous system (CNS). In order to test the efficacy of these nanocarriers, it is fundamental to use in vitro models that resemble the in vivo cell culture conditions. Here, we demonstrate for the first time the ability of a membranotropic peptide, namely gH625, to transport a cargo-acting as a shuttle-across the BBB layer under flow conditions that mimic the blood flow rate. To this aim, a BBB microfluidic device was designed based on a transparent polyester porous membrane sandwiched between a top and a bottom overlying channel made of poly(methyl methacrylate) (PMMA). Our data clearly indicate that this microfluidic system allows the growth of brain endothelial bEnd.3 cells and the formation of a confluent layer at 7 days of culture that hinders the passage of nanoparticles compared to porous membrane alone. The device was validated at a 5 µL/min working flow rate, where the capability of the model to remain intact after nanoparticle passage was shown. Very interestingly, the decoration with the gH625 peptide enhances the adhesion of nanoparticles to the endothelial layer and the BBB crossing in flow conditions, thus confirming the efficacy of the gH625 as a delivery platform to the brain. Biotechnol. Bioeng. 2017;114: 1087-1095. © 2016 Wiley Periodicals, Inc.

Ultrastable Liquid-Liquid Interface as Viable Route for Controlled Deposition of Biodegradable Polymer Nanocapsules.

Liquid-liquid interfaces are highly dynamic and characterized by an elevated interfacial tension as compared to solid-liquid interfaces. Therefore, they are gaining an increasing interest as viable templates for ordered assembly of molecules and nanoparticles. However, liquid-liquid interfaces are more difficult to handle compared to solid-liquid interfaces; their intrinsic instability may affect the assembly process, especially in the case of multiple deposition. Indeed, some attempts have been made in the deposition of polymer multilayers at liquid-liquid interfaces, but with limited control over size and stability. This study reports on the preparation of an ultrastable liquid-liquid interface based on an O/W secondary miniemulsion and its possible use as a template for the self-assembly of polymeric multilayer nanocapsules. Such polymer nanocapsules are made of entirely biodegradable materials, with highly controlled size-well under 200 nm-and multi-compartment and multifunctional features enriching their field of application in drug delivery, as well as in other bionanotechnology fields.

Energy independent uptake and release of polystyrene nanoparticles in primary mammalian cell cultures.

Nanoparticle (NPs) delivery systems in vivo promises to overcome many obstacles associated with the administration of drugs, vaccines, plasmid DNA and RNA materials, making the study of their cellular uptake a central issue in nanomedicine. The uptake of NPs may be influenced by the cell culture stage and the NPs physical-chemical properties. So far, controversial data on NPs uptake have been derived owing to the heterogeneity of NPs and the general use of immortalized cancer cell lines that often behave differently from each other and from primary mammalian cell cultures. Main aims of the present study were to investigate the uptake, endocytosis pathways, intracellular fate and release of well standardized model particles, i.e. fluorescent 44 nm polystyrene NPs (PS-NPs), on two primary mammalian cell cultures, i.e. bovine oviductal epithelial cells (BOEC) and human colon fibroblasts (HCF) by confocal microscopy and spectrofluorimetric analysis. Different drugs and conditions that inhibit specific internalization routes were used to understand the mechanisms that mediate PS-NP uptake. Our data showed that PS-NPs are rapidly internalized by both cell types 1) with similar saturation kinetics; 2) through ATP-independent processes, and 3) quickly released in the culture medium. Our results suggest that PS-NPs are able to rapidly cross the cell membrane through passive translocation during both uptake and release, and emphasize the need to carefully design NPs for drug delivery, to ensure their selective uptake and to optimize their retainment in the targeted cells.

Nanoengineered surfaces for focal adhesion guidance trigger mesenchymal stem cell self-organization and tenogenesis.

The initial conditions for morphogenesis trigger a cascade of events that ultimately dictate structure and functions of tissues and organs. Here we report that surface nanopatterning can control the initial assembly of focal adhesions, hence guiding human mesenchymal stem cells (hMSCs) through the process of self-organization and differentiation. This process self-sustains, leading to the development of macroscopic tissues with molecular profiles and microarchitecture reminiscent of embryonic tendons. Therefore, material surfaces can be in principle engineered to set off the hMSC program toward tissuegenesis in a deterministic manner by providing adequate sets of initial environmental conditions.

Supramolecular spectrally encoded microgels with double strand probes for absolute and direct miRNA fluorescence detection at high sensitivity.

We present novel microgels as a particle-based suspension array for direct and absolute microRNA (miRNA) detection. The microgels feature a flexible molecular architecture, antifouling properties, and enhanced sensitivity with a large dynamic range of detection. Specifically, they possess a core-shell molecular architecture with two different fluorescent dyes for multiplex spectral analyses and are endowed with a fluorescent probe for miRNA detection. Encoding and detection fluorescence signals are distinguishable by nonoverlapping emission spectra. Tunable fluorescence probe conjugation and emission confinement on single microgels allow for ultrasensitive miRNA detection. Indeed, the suspension array has high selectivity and sensitivity with absolute quantification, a detection limit of 10(-15) M, a dynamic range from 10(-9) to 10(-15) M, and higher accuracy than qRT-PCR. The antifouling properties of the microgels also permit the direct measurement of miRNAs in serum, without sample pretreatment or target amplification. A multiplexed assay has been tested for a set of miRNAs chosen as cancer biomarkers.

Tumor-activated prodrug (TAP)-conjugated nanoparticles with cleavable domains for safe doxorubicin delivery.

A major issue in chemotherapy is the lack of specificity of many antitumor drugs, which cause severe side effects and an impaired therapeutic response. Here we report on the design and characterization of model tumor activated prodrug-conjugated polystyrene (PS) nanoparticles (TAP-NPs) for the release of doxorubicin (Dox) triggered by matrix metalloprotease-2 (MMP2) enzyme, which is overexpressed in the extracellular matrix of tumors. In particular, TAP-NPs were produced by attaching Dox to poly(ethylene glycol) (PEG) through two MMP2-cleavable enzymes. The resulting adduct was then tethered to PS NPs. Results showed that Dox release was actually triggered by MMP2 cleavage and was dependent on enzyme concentration, with a plateau around 20 nM. Furthermore, significant cell cytotoxicity was observed towards three cell lines only in the presence of MMP2, but not in cells without enzyme pre-treatment, even after NP internalization by cells. These findings indicate the potential of TAP-NPs as suitable nanocarriers for an on demand, tumor--specific delivery of antitumor drugs after the response to an endogenous stimulus. Further advancements will focus on the translation of this production technology to biodegradable systems for the safe transport of cytotoxic drug to tumor tissues.

Encoding multiple holograms for speckle-noise reduction in optical display.

In digital holography (DH) a mixture of speckle and incoherent additive noise, which appears in numerical as well as in optical reconstruction, typically degrades the information of the object wavefront. Several methods have been proposed in order to suppress the noise contributions during recording or even during the reconstruction steps. Many of them are based on the incoherent combination of multiple holographic reconstructions achieving remarkable improvement, but only in the numerical reconstruction i.e. visualization on a pc monitor. So far, it has not been shown the direct synthesis of a digital hologram which provides the denoised optical reconstruction. Here, we propose a new effective method for encoding in a single complex wavefront the contribution of multiple incoherent reconstructions, thus allowing to obtain a single synthetic digital hologram that show significant speckle-reduction when optically projected by a Spatial Light Modulator (SLM).

Refocusing criterion via sparsity measurements in digital holography.

Several automatic approaches have been proposed in the past to compute the refocus distance in digital holography (DH). However most of them are based on a maximization or minimization of a suitable amplitude image contrast measure, regarded as a function of the reconstruction distance parameter. Here we show that, by using the sparsity measure coefficient regarded as a refocusing criterion in the holographic reconstruction, it is possible to recover the focus plane and, at the same time, establish the degree of sparsity of digital holograms, when samples of the diffraction Fresnel propagation integral are used as a sparse signal representation. We employ a sparsity measurement coefficient known as Gini's index thus showing for the first time, to the best of our knowledge, its application in DH, as an effective refocusing criterion. Demonstration is provided for different holographic configurations (i.e., lens and lensless apparatus) and for completely different objects (i.e., a thin pure phase microscopic object as an in vitro cell, and macroscopic puppets) preparation.

Holographic tracking of living cells by three-dimensional reconstructed complex wavefronts alignment.

We propose here a new three-dimensional (3D) holographic tracking method capable to track, simultaneously and in a single step, all the spatial coordinates of micro-objects. The approach is based on the enhanced correlation coefficient (ECC) maximization method but applied, for the first time to the best of our knowledge, directly on the holographic reconstructed complex wave fields. The key novelty of the proposed strategy is its ability to calculate simultaneously the 3D coordinates of cells, without decoupling the contribution of amplitude and phase. The proposed strategy is tested on living cells (i.e., NIH 3T3 mouse fibroblast) flowing into a microfluidic channel and compared with classical holographic tracking approach. Theoretical description and experimental validation of the proposed strategy are reported.

Highly efficient surface-enhanced Raman scattering substrate formulation by self-assembled gold nanoparticles physisorbed on poly(N-isopropylacrylamide) thermoresponsive hydrogels.

Employing thermoresponsive hydrogels as scaffolding material for noble metal surface loading might be useful for the fabrication of surface-enhanced Raman scattering (SERS) surfaces. Here, we report on a new, reproducible, and simple approach to engineer poly(N-isopropylacrylamide) (PNIPAAm) hydrogel surfaces optimized for physisorption of gold nanoparticles (AuNPs). The advantage of this approach consists of the simple mechanism by which AuNPs are adsorbed on hydrogel templates, without sophisticated chemical treatments for their conjugation with the hydrogel. The resulting PNIPAAm-40 nm AuNP modes demonstrate that this approach gives the capability to tune the interparticle distance and, therefore, to control and modulate SERS affinity upon temperature changing.